R853 .U5601 1966 NATIONAL INSTITUTES OF HEALTH Review of INTRAMURAL RESEARCH 1966 U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE Public Health Service ISSUED IN A LIMITED EDITION FOR ADMINISTRATIVE USE Uhrwj (/. J. NATIONAL INSTITUTES OF HEALTH^^ Review of INTRAMURAL RESEARCH 1966 U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE Public Health Service National Institutes of Health, Bethesda, Maryland 20014 mo/ INSTITUTE RESEARCH DIRECTORS NATIONAL CANCER INSTITUTE NATIONAL HEART INSTITUTE NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Eugene J. Van Scott, M.D. Scientific Director for General Laboratories and Clinics C. Gordon Zubrod,M.D. Scientific Director for Chemotherapy Paul Kotin, M.D. Scientific Director for Etiology Nathaniel I. Berlin, M.D. Scientific Director for Clinical Research Robert W. Berliner, M.D. Scientific Director Donald S. Frederickson, M.D. Clinical Director John R. Seal, M.D. Scientific Director Vernon Knight, M.D. Clinical Director NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES Joseph E. Rail, M.D., Ph.D. Scientific Director Robert S. Gordon, Jr., M.D. Clinical Director NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT Roy Hertz, M.D. Scientific Director NATIONAL INSTITUTE OF DENTAL RESEARCH Seymour J. Kreshover, D.D.S., M.D., Ph.D. Scientific Director Edward J. Driscoll, D.D.S. Clinical Director NATIONAL INSTITUTE OF MENTAL HEALTH John C. Eberhart, Ph.D. Scientific Director Robert A. Cohen, M.D. Clinical Director NATIONAL INSTITUTE OF NEUROLOGICAL DISEASES AND BLINDNESS Karl Frank, Ph.D. Acting Scientific Director Maitland Baldwin, M.D. Clinical Director DIVISION OF BIOLOGICS STANDARDS Roderick Murray, M.D. Scientific Director G. Burroughs Mider, M.D. Director of Laboratories and Clinics William C. Mohler, M.D. Assistant to Director of Laboratories and Clinics 111 FOREWORD As the title implies this volume summarizes work done in the NIH laboratories and clinics and in the offices closely connected to these. It does not cover the research supported throughout the United States and in some other nations by grants from the various Institutes. Nor does it include research done on contract for the NIH unless this was a direct extension of an intramural program. This story covers the year from July 1965 through June 1966. The philosophy behind the Review remains the same as in previous years. These are the words of the staff who followed very general guidelines in writing their reports. They have received only minimal editing. Thus we have tried to preserve for the reader as direct a view as possible into our diverse and extremely active scientific community as its members pursue their investigations under the missions of the several Institutes and Divi- sions. Together these missions unite as the common purpose of the NIH, which strives to extend fundamental knowledge about the health and diseases of mankind. \^tf\Uo (S.SUiw James A. Shannon, M.D. CONTENTS Foreword NATIONAL CANCER INSTITUTE General Laboratories and Clinics Introduction The Laboratories The Clinical Branches Immunologic Research Macromolecular Biology Section Laboratory of Biochemistry Cytochemistry Section Nutrition and Carcinogenesis Section Tumor-Host Relations Section Nucleic Acids Section Protein Chemistry Section Laboratory of Biology The Thymus in Immunology Tumor Viruses Ultrastructure Cytology Plasma Cell Tumors Biochemical Genetics Genetics of Tumors Hepatic Carcinogenesis Malignant Transformation In Vitro Methodology in Tissue Culture Biochemistry Laboratory of Pathology Introduction Experimental Research Cancer in Man, and Related Animal Studies Cancer in Animals Induction and Pathogenesis Modifications in Carcinogenesis Biologic Factors in Neoplasia Spontaneous Tumors Transplanted Tumors Viral Carcinogenesis, and Problems of Immunology Polyoma Virus SV40 and Adenovirus Leukemogenic Viruses Virus Replication Interferon Page V 1 1 1 1 6 8 9 11 11 12 13 17 21 26 26 27 27 27 28 29 29 29 30 31 31 31 31 32 34 34 35 36 36 36 36 36 37 37 37 37 vii V"i CONTENTS Page Accumulation of Data on Laboratory Animals 37 Phylogenetic Aspects of Neoplasia 38 Development of New Techniques 39 Collaborative Research 39 Laboratory of Physiology 40 Cancer Physiology Section 40 Energy Metabolism Section 41 Physical Biology Section 42 Radiation Biology Section 43 Office of the Chief 45 Dermatology Branch 46 Mycosis Fungoides Lymphoma 46 Epidermal Growth and Reactivity 47 Melanogenesis 47 Endocrinology Branch 47 Immunology Branch 48 Metabolism Branch 49 Amino Acid Transport 49 Calcium Metabolism 49 Porphyrin Metabolism 50 Nucleic Acid 51 Metabolism of Plasma Proteins 51 Erythropoiesis 53 Bilirubin Metabolism 54 Surgery Branch 55 Chemotherapy 63 Acute Leukemia Service 64 Solid Tumor Service 64 "Life Island" 65 Radiation Branch 65 Clinical Branch, Baltimore Public Health Service Hospital 65 Cell Separation 66 Conclusion 66 Laboratory of Chemical Pharmacology 66 Etiology 69 Biology Branch — 70 Chemistry Branch 73 Viral Oncology 75 Viral Biology Branch 77 Electron Microscopy Section 77 Microbiology Section 77 Virus Studies Section 78 Virus Leukemia Section 78 Viral Leukemia and Lymphoma 78 Introduction 78 Research and Activities in the Branch 79 Special Virus-Leukemia Program 82 NATIONAL HEART INSTITUTE 83 Cardiology Branch 83 CONTENTS IX Page Mechanics and Energetics of Miocardial Contraction: Isolated Heart Muscle 83 Application of myocardial mechanics to the Intact Heart 86 Contractile Properties of the Failing heart 89 Isolated Heart Muscle and Subcellular Fractions 89 Intact Dog Heart 90 Clinical Studies 92 iX* On the Mechanism of Action of Digitalis 95 On Circulatory Control of the Autonomic Nervous System 96 Improvement of Cardiac Diagnostic Methods 98 Clinical Studies 99 u^" Idiopathic Hypertrophic Subaortic Stenosis 99 Other Clinical Studies 101 Section of Clinical Biophysics 103 Vascular Mechanics 103 Myocardial Mechanics 106 Lung Mechanics 106 Surgery Branch 108 Clinic of Surgery 108 Laboratory of Clinical Biochemistry 112 Amine Biogenesis ad Metabolism 112 Collagen and Hydroxyproline 113 Actinomycin Biosynthesis 114 Cobamide Dependent Methionine Synthesis 114 Formation of Formyl-Methionine sRNA 114 Aromatic Hydroxylation 114 Serine Metabolism 114 Studies on Amino Acid Transport 115 Peptides and Peptide Linkages 115 Brain Nucleic Acid 115 Bacterial Phenylalanine Hydroxylase and Its Cofactor 115 Nitrogen Containing Lipids 115 Enzyme Mechanisms 115 Experimental Therapeutics Branch 116 Biochemistry and Pharmacology of Aromatic Amines 116 Studies of Selected Proteins 118 Miscellaneous 120 Laboratory of Technical Development 120 Fluorescence Methods 120 Ultramicro Methods 122 Automation of Bacteria Counts and Antibiotic Sensitivity Tests 123 Artificial Organs 123 Fast Reaction Methods 124 Chromatographic and Ultrasonic Methods 124 Miscellaneous 125 Laboratory of Cardiovascular Physiology 125 Studies on Myocardial Mechanics 125 Myocardial Oxygen Consumption 127 Influence of Drugs and Electrolytes on the Heart 127 CONTENTS Page Electrical Stimulation of the Heart 128 Water and Salt Regulation 128 Peripheral Circulation 128 Kallikrein-Kininogen-kinin System 129 Laboratory of Kidney and Electrolyte Metabolism 129 Micropuncture Studies in the Kidney 129 Isolated and Separated Tubules in Rabbits in Vitro 131 Red Cell Studies 132 Studies in Toad Bladder 133 Chemical Characterization and Physiologic Significance of a Cardioglobulin System Present in Mammalian Plasma 134 Renin-Angiotensin-Aldosterone System and Other Humoral Factors 134 Laboratory of Biochemistry 135 Sections on Enzymes 135 Regulation of Divergent Biosynthetic Pathways of Metabolism 135 Cumulative Feed-Back Inhibition of Glutamine Synthetase 135 Uptake of Amino Acids by Isolated Cytoplasmic Membrane Preparations 138 Metabolism of Amino Acids 139 Reductive Deamination 139 Lysine Fermentation 139 One Carbon Metabolism 140 Methane Fermentation 140 Synthesis of Acetate from C0 2 141 Cystathionine Biosynthesis and Trans-sulfuration 141 Mechanism of Action of Vitamin Bi 2 Coenzymes 142 Ethanolamine Diaminase 142 Metabolism of Heterocyclic Compounds 143 Hydroxylation of Nicotinic Acid 143 Reduction of 6-Hydroxynicotinic Acid to 6-oxo-l,4,5,6- tetrahydronicotinic acid 143 Chemical Synthesis of 6-Hydroxynicotinic Acid 143 Section on Cellular Physiology 144 Muscle Proteins: Myosin 144 Extraction of Actinomyosin from Rabbit Muscle 144 Radiation Damage in Proteins 145 Protein Biosynthesis 145 Nonphosphorylated Intermediates of Energy Transfer and Protein Biosynthesis 145 Biochemistry and Cytology of Cell Transport 145 Section on Comparative Biochemistry 146 ACP Involvement in Lipid Metabolism 146 Fatty Acid Biosynthesis 146 Complex Lipid Biosynthesis 147 Sterol Biosynthesis 147 Mammalian ACP 147 Structure of E. coli ACP 148 CONTENTS XI Page Concentrations of ACP and CoA in E. coli 148 ACP Hydrolase 149 Clinical Endocrinology Branch 149 Steroidogenesis by the Adrenal Cortex 149 Calcium and Phosphorus Metabolism 150 Renal Physiology 150 Neuroendocrine Relationships 153 Laboratory of Metabolism 153 Section on Metabolism 154 Mobilization and Utilization of Free Fatty Acids 154 Metabolism of Adipose Tissue Studies in Vitro 154 Utilization of FFA in Vivo and in Vitro 154 FFA Metabolism in Cell Suspension of Ehrlich Ascites Tumor 154 Studies on the Consequences of Rapid FFA Mobilization 155 Factors Controlling Plasma Lipoprotein Concentration 155 Metabolic Studies in Refsum's Syndrome, a New Lipid Storage Disease 156 Clinical Studies 156 Animal Studies 157 Lymphatic Absorption of Lipids 158 Section on Molecular Diseases 159 Hyperlipoproteinemia 159 Structure and Function of Lipoproteins 160 Tissue Lipidoses 160 Protein Structure and Function 160 Section on Chemistry 161 Laboratory of Biochemical Genetics 162 Nucleotide Sequences of RNA Codons 162 Mechanism of Codon Recognition 163 Characteristics of Synonym RNA Codons 163 Codon Recognition on 30 S Ribosomes 163 Attachment of mRNA to Ribosomes 163 Template Activity of Modified RNA Codons 163 Universality of the Code 164 Regulatory Mechanisms Dependent on Viral Infection 164 Laboratory of Chemical Pharmacology 165 Adrenergic Neurochemical Transducer 165 Application of Steady State Kinetics 165 Turnover Rates and Times of Catecholamines and 5HT 165 Results of Studies of Turnover Rates 166 Kinetics of Storage 166 Recapture Mechanism 166 Mechanism of NE Release by Nerve Stimulation 167 Electrolyte Requirements for NE Storage and Uptake 167 Kinetics of Reserpine-Induced Release of Biogenic Amines 168 Relationship of Pharmacologic Effects of Reserpine to Effects on 5HT Storage 169 False Adrenergic Transmitters 169 XU CONTENTS Page Relationship of Pharmacologic Effects of False Transmitters to Rate of NE Efflux 169 Effects of Desmethylimipramine (DMI) on Adrenergic Neurons 169 The Serotonergic Transducer 170 Role of Serotonin (5HT) 170 Sympathetic Target Sites 170 Adipose Tissue Transducer System 170 Interaction of Sympathetic and Hormonal Systems 171 Adrenergic Blocking Agents 171 Electrolyte Requirements 171 Studies on Chemical-Induced Shock 172 The Nonmast Cell Histamine Transducer 172 Selective Labeling of Nonmast Cell Histamine 172 Distribution and Fate of Injected HVHistamine 173 Release of HVBistamine 173 Synthesis of Histamine 173 Factors that Affect Drug Action 174 Passage of Substances Across Membranes 174 Enzymatic Mechanism of Membrane Transport 174 Enzymatic Mechanisms of Drug Metabolism 175 Mechanism of Teratogenesis of Thalidomide 175 Pharmacogenetics . 176 Clinical Studies with Desmethylimipramine 176 Development of New Methods of Analysis 177 NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 179 Introduction 179 Laboratory of Clinical Investigations 180 Transmission Dynamics in Respiratory Infections 180 Antibody in Nasal Secretions and Serum 181 Chilling and Respiratory Infection 181 Adenovirus Soluble Antigens 181 Influenza 181 Mechanisms of Fever and Host Responses 181 Systemic Fungus Infection 182 Leprosy 182 Laboratory of Infectious Diseases 182 Adenoviruses as Oncogenic Agents 182 Tumor and T Antigens as Possible Determinants of Adenovirus Oncogenesis 182 Adenovirus-SV40 Hybrids 183 Adenovirus-Associated Viruses (AAV's) 183 Leukemia 183 Vaccine Against Mycoplasma Pneumoniae 183 Mycoplasma Epidemiology 183 Mycoplasma — Fundamental Studies 183 Host Resistance to Respiratory Disease 184 Studies in Oceania 184 CONTENTS Xiii Page Rubella Virus 184 Bacterial Metabolism and Physiology 185 Histoplasmosis 185 Laboratory of Biology of Viruses 185 Viral Structure 186 Cell Response to Viral Infection 186 Viral Oncogenesis 186 Genetic Relatedness 186 Laboratory of Tropical Virology 186 Middle America Research Unit (MARU) 187 Hemorrhagic Fever 187 Arbovirus Studies 187 Laboratory of Germfree Animal Research 187 Immunoglobulins in Germfree Mice 188 Genetic Factors May Regulate Immune Tissue Destruction 188 Clostridium Perfringens Lethal to Germfree Guinea Pigs 188 Severity of Amoebic Infections Influenced by Associated Bacteria 188 Low Serum Lysozyme Levels in Germfree and Ex-Germfree Rats 189 Immunoglobulins Quantitated in Human Malaria 189 Laboratory of Parasitic Diseases 189 Schistosomiasis 189 Amebiasis and Trichomoniasis 190 Glucose and Glycerol Utilization in Parasites 191 Laboratory of Parasite Chemotherapy 191 Malaria — Human 191 Malaria — Simian 192 Immunological Studies 193 Laboratory of Immunology 193 Autoimmunity 193 Transplantation Immunology 194 Hypersensitivity 194 Mitogens 194 Rocky Mountain Laboratory 195 Selected Zoonoses of Regional Importance 195 Rickettsial Diseases 195 Transmission of Disease Agents by Vectors 196 Encephalitides and Tick-Borne Diseases 197 Psittacosis — Lymphogranuloma-Trachoma (PLT) 197 Chronic Progressive Viral Disease 198 Allergy and Immunology 198 Immunoprophylaxis Against Tuberculosis 199 Bordetella Pertussis Antigens 200 Endotoxins 200 Microbial Proteins and Nucleic Acids 200 Biology of Microbial Agents in Arthropods 201 Laboratory of Bacterial Diseases 201 Mycoplasma 201 Brucellosis 202 XIV CONTENTS Page NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 203 Introduction 203 Laboratory of Nutrition and Endocrinology 204 Studies in Folic Acid 204 Large-Scale Processing of Biological Materials 205 Studies in Experimental Nutrition 205 Study of Protein Hormones 205 Diabetes and Fat Metabolism 205 Action on Isolated Fat Cells 205 Fat Soluble Vitamins and Related Substances 206 Reversible Chemical Modification of Cytochrome 206 Purine-Pyrimidine Balance and Hepatic Fat Metabolism 206 Laboratory of Biochemistry and Metabolism 206 Polysaccharide Metabolism 206 Bacterial Systems 206 Animal Systems 207 Glycoprotein Studies 207 Antibodies 207 Liver, Oviduct and Mastocytoma Glucoproteins 208 Ribonucleic Acid 208 Studies on RNase II 208 Studies on RNase I 208 Thionucleotide Studies 209 Biochemical Studies of Lysogeny 209 Enzymes Near the Bacterial Surface 209 Surface Enzymes; Removal by Osmotic Shock 209 Enzymatic Utilization of Model Compounds in Bacterial Systems 210 Phenotypic Expression of Genetic Information 210 Galactose in E. coli 210 Hormone-induced Differentiation, Mouse Gland 211 Reduction of Protein Disulfide Bonds 211 Laboratory of Chemistry 212 Section on Biochemical Mechanisms 212 Electrolytic Cleavage of Tyrosyl-Peptide Bonds 212 Synthetic Models for Hydrolytic Enzymes 212 Oxidation Mechanisms in Metabolic Processes 212 Section on Carbohydrates 212 Studies on Presumed Ester Linkage in Glycoproteins 212 iV,N-Diacylhexosamines and Conformation of Amino Sugars 213 Studies Synthesis of Polyvinyl Glycosides 213 Higher-Carbon Sugars 213 Thio Sugars 214 Amino Sugars 214 Section on Medicinal Chemistry 214 Evaluation of Analgesics 214 In Vitro Screening for Anti-Inflammatory Properties 214 Optical Resolutions of Benzomorphans 214 CONTENTS XV Page Abnormal Stevens Rearrangement 215 9-Acetoxy and -hydroxy-benzomorphans 215 New Aromatization for Z>eta-tetralones 215 Schiff-Bases and Oxazolidines 215 Potential Aldolase Inhibitors 215 Immunologic Studies in Clinical Hematology 215 Dihydrocodeinone Isomers 215 Polyethylenimine Studies 216 Synthesis of 4-p-methoxy-l-vinyl-2-pyrrolidone 216 Napth- and Benz-3,4-dihydro-oxazines 216 Section on Microanalytical Services and Instrumentation 216 Section on Steroids 216 Study of Alkaloids from Solanum congestiflorum 216 Synthesis and Reactions of Heteroisteroids 216 Mass Spectrometry of Steroidal Glycosides 217 Sterols from Parasites 217 Photoreduction of fieta-Estradiol 217 Chemistry of Iresine Celosia 217 Sterol Biosynthesis in Plants 217 Metabolism of C 14 -labeled Steroids 217 Adrenal Metabolism of the Gerbil 217 Biosynthesis and Metabolism of Biogenic Amines 218 Section on Metabolites 218 Batrachotoxin, Strongest Venom Known 218 Pharmacology and Toxicology of Batrachotoxin 218 Venoms of Other Amphibians _, 218 Synthesis and Biosynthesis of Dehydrobufotenine 218 Reactivity toward N-Bromosuccinimide of Tryptophan 219 Novel Photocyclization of Tryptophan Derivatives ^ iy Photoreductions of Tryptophan " Novel Photocyclization in Tyrosine and Tyramine 9 Photoreduction of Histidine 219 Binding Characteristics of Polyuridylic Acid 219 Template Activity of Uridyllic Acid 220 Mechanism of Two-Step Reduction of Thymidine 220 Stereoisomerism in Photoreduction of Thymidine 220 Stereochemistry of 3-Methylproline 220 Proton Magnetic Resonance Spectra of 3,4-Dehydroproline Derivatives 220 Proline Analogs, Effects upon Biosynthesis of Actinomycins 221 Analogs and Homologs of Proline and Hydroxyproline 221 Synthesis of Erythro-gamma-Hydroxy-L-\ysme 221 Synthesis and Metabolism of 6-Hydroxycatecholamines 221 Chemorelease of Norepinephrine from Mouse Hearts 222 Drugs Affecting the Nervous Systems 222 Depletion of Norepinephrine- 3 H from Heart by Alpha- Methyl-m-Tyrosine 222 Preparation of Gramicidin A, B, and C 222 Release of Kinin Activity from Human Kininogens 222 XVI CONTENTS Page Selective Cleavage of Cysteine Peptide Bonds 223 Cleavage of Cysteine Peptide Bonds in Insulin 223 Antibiotic Nisin 223 Active Carboxy Group of Pepsin 223 Improvement in Methodology of Amino Acid Analysis 224 Laboratory of Experimental Pathology 224 Introduction 224 Pathology of Rheumatic Diseases 225 Animal Joint Lubrication 225 Cytogenetics 226 Experiments on Reutilization of Chromosomal DNA 226 Meiotic Chromosome Studies in Human Male 226 Characterization of Enzymes in Histochemical Systems 226 Histochemical Localization of ATPase Activity 226 Studies in Oxidative Phosphorylation 227 Mechanism of Protection by 5-HT in Tryptophan Deficiency 227 Fluorescein Studies 227 Localization of Antigens in Tissues 227 Localization of Prolactin in Rat Pituitary 228 Localization of Prolactin in Fundulus heteroclitus, L. 228 Studies on Nucleic Acids and Protein Synthesis 228 Studies on Soluble Ribonucleic Acid 229 Electron Microscope Studies 229 Amyloidosis 229 Fine Structure of Rabbit Marrow Cells 229 Myeloid Elements 229 Studies of Human Lymphocytes 229 Fine Structural Characterization of Neuromelanin 230 Neuromelanin 230 Experimental Porphyria 230 Cytochemistry of Bacterial Phosphatases 230 Temperature Effect on Lipids of Leishmania 230 Mouse Thyroid Gland Cytology 231 Thyroid Parafollicular cells 231 Mucosaccharide Histochemistry 231 Microspectrophotometric Studies of Basic Protein 232 Iron Staining of Acid Mucosubstances 232 Studies on Endocarditis • 232 Studies on Dimethyl Sulfoxide 232 Studies on High Altitude Hypoxia 233 Studies on Hamycin 233 Cycasin Research 233 Studies in Germfree Rats 233 Mutagenic Effect 234 Transplacental Induction of Tumors 234 Prevention of Acute Toxicity of Cycasin 234 Transplantation of Cycasin Induced Neoplasms 234 Laboratory of Chemical Biology 234 Structure-Function Relationships in Proteins 234 CONTENTS XVU Page Structure, Function, and Synthesis of a Nuclease from Staphlococcus aureus 236 Mechanism of Control of Biosynthesis of Histidine in Salmonella 237 Biochemical Studies Preliminary to Investigation of Genetic Defects in Higher Organisms, including Man 238 Laboratory of Biochemical Pharmacology 238 Studies with EDTA-treated E. coli 238 Inhibitory Effect on Actinomycin 238 Surface Changes in EDTA-treated E. coli 239 Bacteriophage Studies 239 EDTA-treated E. coli 239 Lysogenic Induction 239 Ribosome Structure and Function 239 Amine Studies 239 Polysaccharides and Glycoproteins 240 Control of Enzyme Levels in Mammalian Tissues 240 Burn-Shock Studies 240 Burns 240 Tourniquet Trauma 240 Sulfur-containing Compounds 240 Leprosy Studies 240 Laboratory of Physical Biology 241 Molecular Structure 241 Surface Phenomena 242 Physiology and Biochemistry 242 Excitation and Coupling 242 Biological Interactions 243 Biological Ultrastructure 243 Cellular Biology 244 Laboratory of Biophysical Chemistry 244 Laboratory of Molecular Biology 246 Structure and Function of Glutamate Dehydrogenase 246 Enzyme Induction in Bacteria 246 Cysteine Biosynthesis 246 Enzyme Induction by Steroid Hormones 246 Active Transport of Amino Acids in Salmonella typhimurium 247 Amino Acid Incorporation into Protein In Vitro 247 Investigations of Nucleic Acids and Related Substances 247 X-ray Diffraction studies on Proteins 247 A Search for DNA Recombination In Vitro 248 Structure of Polyadenylic Acid at pH 7 248 Optical Properties of Nucleic Acids 248 Interaction of DNA with Polycations 248 Internal Proteins of Bacteriophage T2 248 Glucosylation and Kinetics of Endonuclease Degradation of DNA 249 Viscosity Studies of DNA of Bacteriophage Mutants 249 Chemistry and Genetics of Hemoglobin and Other Proteins 249 Biochemical Control Mechanisms in Histidine Biosyntheses 250 XV111 CONTENTS Page Mechanism of Lambda Bacteriophage Induction 250 Mathematical Research Branch 251 Clinical Investigations 253 Arthritis and Rheumatism Branch 254 Studies of Natural History of Disease 254 Lymphocyte Function in Sjogren's Syndrome 254 Immunological Reactivity in the Elderly 255 Variations in Rheumatoid Factor Titer 255 Clinical and Metabolic Evaluation of Cystinosis 255 Therapeutic Studies 255 Prophylactic Synovectomy in Rheumatoid Arthritis 255 Melphalan in Systemic Amyloidosis 255 Lymph Drainage in Systemic Lupus Erythematosus 255 Mechanisms of Disease 255 Purine Biosynthetic Pathways 255 Induced Renal Retention of Uric Acid 255 Role of Infection in Rheumatic Disease 256 Experimental Amyloidosis 256 Ribosomes in Hormonal Control of Protein Synthesis 256 Mechanisms of Action of Adrenocorticotrophic Hormone Studies on Immunoglobulins 256 Antibody Synthesis in the Spleen ^6 Macroglobulin Subchains 2 ^6 Macroglobulin Catabolism 256 Studies on Canine Immunoglobulins 257 Miscellaneous 257 Carbon-Fluorine Bonds in Biology 257 Metabolic Diseases Branch 257 Research on Human Physiology and Nutrition 257 Total Energy Balance 257 Effects of Heat Stress on Potassium Balance 257 Mechanisms of Water and Electrolyte Secretion by Human Sweat Gland 257 Calcium Metabolism 257 Bone Cell Metabolism 258 Chemistry and Physiology of Parathyroid Hormone 258 Action of Thyrocalcitonin 258 Studies of the Small Intestine 258 Whipple's Intestinal Lipodystrophy 258 Other Diseases 259 Intermediary Metabolism of the Intestinal Mucosa 259 Ultrastructure of the Human Intestinal Mucosa 259 Inborn Errors of Metabolism 260 Homocystinuria 260 Cystathioninuria 260 A New Disorder 260 Intermediary Metabolism 260 Clinical Endocrinology Branch 261 Thyroid Biochemistry 261 CONTENTS XIX Page Iodide Transport 261 Iodination Reactions and Thyroxine Synthesis 261 Iodoproteins 262 Protein Synthesis in the Thyroid Gland 262 Proteolytic Enzymes in the Thyroid Gland 262 Measurement of Iodocompounds in Biological Fluids 262 Pituitary Hormones 263 Growth Hormone 263 Gonadotropins 263 Thyrotropin 263 Vasopressin 263 Insulin, Glucagon and Carbohydrate Metabolism 263 Regulation of Protein Metabolism 263 Regulation of hepatic carbohydrate metabolism 264 Glucose Transport 264 Galactose Metabolism and Galactosemia 264 Amino Acid Transport 264 Protein Structure 265 Pediatric Metabolism Branch 265 Immunological and Biochemical Investigations 265 Inborn Error of Mucopolysaccharide Metabolism in Cystic Fibrosis 265 Serologic Reactions in Patient with CF 266 Pseudomonas aeruginosa and its Slime in Relation to Pathogenesis of Cystic Fibrosis 266 Structure-Function Studies with Electron Microscope 267 Effect of Aldosterone on Sweat Gland and Renal Function in Normal Subjects, Patients with CF, and Carriers 267 Normal adults and children 267 Patients with Cystic Fibrosis 267 Other Investigations in Cystic Fibrosis 268 Pregnancy in CF 268 New Syndromes of Pancreatic Deficiency 268 Albumin Turnover Studies in CF 268 Growth, Development, and sexual maturation in Patients with Cystic Fibrosis 269 Clinical Hematology Branch 269 Immunology of Blood Cell Deficiencies 269 Idiopathic Thrombocytopenic Purpura 269 Reticuloendothelial Blockade in Sequestration of Immunologically Altered Cells 269 Etiology of Transfusion Anuria 269 Post-transfusion Purpura 270 Isoimmune Neonatal Purpura 270 Transplantation Immunity 270 Histocompatibility in Human Homograft pairs 271 Immunologic Therapy of Malignancies 271 Immunological Considerations Attending Platelet Transfusions 272 XX CONTENTS Page Blood Coagulation and Diseases of Hemorrhage and Thrombosis 272 Spleen Controlling Level of Anti-hemophilic Factor 272 Evaluation, new Plasma Fraction in treatment of Classical Hemophilia 273 Basis for Thrombocytopenia in Malaria 273 Diseases of Megakaryocyte dysfunction 274 Platelet Physiology 274 Unusual Hemorrhagic Disorders 274 NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT 275 Introduction 275 Reproduction Program 275 Growth and Development Program 276 Aging Program 276 Mental Retardation Program 277 Intramural Programs 277 Thyroid-stimulating Hormone (TSH) 278 Luteinizing Hormone (LH) 278 Melanocyte-stimulating Hormone (MSH) 278 Growth Hormone 279 Laboratory of Biology - 279 Growth and Development Program 280 Aging Program 281 Summary 281 Research at Bethesda 284 Gerontology Branch 284 Aging in the Human 285 Human Performance Section 285 Section on Experimental Psychology 285 Metabolism Section 286 Nutritional Biochemistry Section 287 Pulmonary Physiology Section 287 Renal Section 288 Endocrinology Section 289 Metabolism Section 290 Biology of Aging 290 Section on Animal Behavior 290 Cardiovascular Section 291 Section on Nutritional Biochemistry 291 Section on Molecular Biology 292 Section on Comparative Biochemistry 293 Section on Cellular and Comparative Physiology 294 Biophysics Section 296 Mental Retardation Program 297 Direct Research 297 Laboratory of Biomedical Sciences 298 Biochemical Investigations 298 Cytogenetic Investigations 298 Neurophysiology Investigation 298 CONTENTS XXI Page Children's Diagnostic and Study Unit 299 NATIONAL INSTITUTE OF DENTAL RESEARCH 301 Introduction 301 Dental Caries 302 Periodontal Disease 306 Growth and Development 308 Collagen, Elastin, and Calcification 311 Cell Biology and Enzyme Chemistry 312 Virology, Immunology, and Microbial Physiology 314 Oral Soft Tissue Lesions, Clinical Diagnosis, Treatment 317 Laboratory of Microbiology 319 Immunology 319 Dental Caries 321 Virology 322 Microbial Physiology 323 Systematic Microbiology 324 Laboratory of Histology and Pathology 324 Microstructural Characteristics of Enamel 324 Crystallographic Studies of Synthetic and Biological Phosphates 325 Histochemical and Chemical Studies of Connective Tissues 325 Experimental Pathology 326 Bacterial and Macromolecular Structure 326 Laboratory of Biochemistry , 326 Enzyme Chemistry 326 Enzyme Structure and Mechanism of Action 327 Methyl Groups of sRNA 327 Phosphate and Dental Caries 327 Prenatal and Fetal Development Factors Influencing Oral Disease 328 Protein Chemistry 328 Fluoride Metabolism 329 Salivary Biochemistry 329 Epidemiology and Biometry Branch 329 Nutrition Surveys 330 Dental Caries 330 Periodontal Diseases 331 Occlusal Anomalies 331 Collateral Projects 331 Clinical Studies 331 Dental Services Branch 333 Work with the National Cancer Institute 333 Work with the National Institute of Arthritis and Metabolic Diseases 334 Work with the National Heart Institute 334 Oral Medicine and Surgery Branch 334 Studies of the Human Dental Pulp 334 Anesthesia Studies 335 Dental Caries 336 Studies of Soft Tissue Lesions 336 XXii CONTENTS Page Clinical Periodontal Studies 337 Animal Periodontal Studies 337 Oral Pathology Investigations 338 Oral and Pharyngeal Development Section 339 Basic Anatomical Studies 339 Basics Physiological Studies 339 Human Genetics Branch 340 Congenital Malformations 340 Genetic Factors in Dental Caries 341 Biochemical Genetic Defects 341 Effects of Radiation Exposure . 341 Biochemistry of Normal Variation and Cellular Biology 341 Cellular Biology and Cytogenetics Section 342 Future Studies 342 NATIONAL INSTITUTE OF MENTAL HEALTH 343 Introduction 343 Clinical Investigations 344 Addiction Research Center 346 General 346 Administrative 347 New Analgesics and Pathological Processes 348 Acute and Chronic Intoxication with Drugs 349 Marihuana Group 349 Clinical Studies of Intoxication 349 Biochemistry of Addiction 350 Neurophysiology and Neuropharmacology of Chronic Intoxication 350 Effect of Chloropromazine on Barbiturate Withdrawal 350 Barbiturate Withdrawal in rats 350 Free Choice Between Tap Water and 5% Alcohol in rats 351 Sodium Barbital on Electroconvulsive Threshold Elevation 351 Psychological Studies on Addiction 351 Mode of Action of Central Nervous System Depressants 352 Conditioning Factors in Opiate Addiction and Habituation 352 Experimental Studies with Human Subjects and Studies on Learning 353 Social Science Section 354 Biochemical Pharmacology 355 Laboratory of Socio-Environmental Studies 355 Studies of Social Context of Personality Development 355 Social Class, Occupation, Parental Values 355 Values and Behavior 356 Maternal Care and Child Behavior in Japan 356 Children's Orientation to Health and Illness 357 Experimental Modification of Children's Behavior 357 Review of Research on Social Development 357 Population Genetics 357 Social Psychological Correlates of Occupation 358 Studies of Mental Disorder 358 CONTENTS XX111 Page Quantitative Studies of Psychology and Schizophrenia 358 Social Epidemiology of Schizophrenia 358 Studies of Methodology of the Social Sciences 359 Assessment of Methods of Developmental Psychology 359 Methodological Principles of Survey Analysis 360 Laboratory of Neurophysiology 361 Laboratory of Psychology 363 Section on Early Learning and Development 363 Section on Personality 363 Office of the Chief 363 Section on Perception 363 Section on Higher Thought Processes 363 Section on Neuropsychology 363 Development of Behavioral Function 364 Neonatal Learning 364 Social Behavior 364 Dr. Gewirtz' program, Research on Impact of Environment 365 Intellectual Development 367 Analysis of Developed Functions 367 Communication 369 Thought Process 369 Variables Affecting Biological Systems 370 Biological Substrate of Behavioral Functions 370 Cortical Mechanisms in Sensation and Perception 371 Vision 371 Audition 371 Somesthesis 371 Visuomotor Coordination 372 Cortical Mechanisms in Problem Solving 372 Cortical-subcortical Relations and Regulation of Behavior 372 Destroyers of Behavioral Function 373 Crowding 373 Schizophrenia 374 Enhancers of Behavioral Function 378 Theoretical, Methodological and Technical Advances 379 Administrative Aspects of Laboratory 380 Child Research Branch 382 Clinical Investigations 382 Introduction 382 Patterns of Middle Class Early Marriage 384 Early Parent-Infant Patterns 385 Studies of Individual Differences in Infants 385 Toward a Theory of Marital Experience 386 New Developments 386 Adult Psychiatry Branch 387 Family Studies 387 Intrafamilial Relationships and Transactions 387 Study of Psychotherapy, Especially Family Therapy 389 Cross-Cultural Family Studies 390 XXiv CONTENTS Page Family Development 390 Experimental Ego Psychology 390 Clinical and Conceptual Studies of Schizophrenia 390 Personality Development 391 Twin and Sibling Studies 392 Psychosomatic Studies 394 Psychophysiology of Sleep 398 Laboratory of General and Comparative Biochemistry 400 Section on Proteins 400 Section on Cellular Regulatory Mechanisms 401 Section on Alkaloid Biosynthesis 402 Section on Technical Development 402 Projects Completed 403 Projects in Process 403 Projects Abandoned 404 Section on Technical Development Projects 404 Projects Initiated within the Section 404 LINC Programs in Current Active Use 404 LINC Programs in Preparation 404 Laboratory of Neurochemistry 404 Laboratory of Clinical Science 406 Section on Cerebral Metabolism 408 Section on Pharmacology 409 Section on Medicine 411 Section on Biochemistry 412 Section on Physiology 414 Pyramidal Tract Activity and Voluntary Movement 414 Brain Mechanisms in Eye Movement Information 415 Significance for Mental Health Research 415 Section on Psychiatry 416 Unit on Psychosomatics 418 Nervous and Circulatory Systems 418 Diet and Catecholamine Excretion 418 General Health of Families 419 Observations in Psychiatric Illness 419 Clinical Neuropharmacology Research Center 419 Laboratory of Neurobiology 422 NATIONAL INSTITUTE OF NEUROLOGICAL DISEASES AND BLINDNESS 425 Introduction 425 Ophthalmology 425 Medical Neurology 426 Surgical Neurology 426 Medical Neurology Branch 426 Clinical Investigation Program 426 Myopathies 427 Episodic Weakness 427 Myasthenia Gravis 427 Amyotrophic Lateral Sclerosis 428 CONTENTS XXV Page Methodology 428 Neuroradiology Section 428 Radiographic Diagnosis 428 Radiation Dosimetry 429 Isotopic Diagnosis 429 Neuropharmacology Section 429 Branch of Surgical Neurology 430 Developmental Defects 430 Epilepsy 431 Involuntary Movements 432 Brain Tumor 433 Cerebral Edema 433 Cerebral Trauma 434 Language and Memory 435 Effects of Low Temperatures 435 Neurosurgical Monitoring 436 Microbial Analysis of Neurosurgical Environment 436 Summary 437 Branch of Ophthalmology 437 Laboratory Investigations 438 Retina 438 Cornea and Vitreous 439 Choroid 440 Lens 440 Intraocular Pressure 440 Clinical Investigations 441 Retina 441 Uvea 441 Glaucoma 441 Ocular Changes in Systemic Disorders 442 Branch of Electroencephalography and Clinical Neurophysiology 442 Clinical Diagnostic Service 442 Research Activity 443 Other Activities and Organizational Aspects 444 Program Considered for Near Future 445 Laboratory of Neuroanatomical Sciences 445 Introduction 445 Analyses of Structure 446 Cytology 446 Auditory and Vestibular Systems 446 Olfactory System 446 Automatic Nerve Endings 446 Pathways of Transport in Brain 447 Cytoarchitecture 447 Auditory System 447 Muscle Spindle 448 Interactions in and among Sensory, Central, and Motor Systems 448 Trophic Interactions 448 XXVI CONTENTS Page Nerve-Muscle Relationships 448 Visual System 449 Morphogenesis 450 Auditory System 450 Regeneration 450 Future of the Program 450 Laboratory of Neuropathology 450 Section on Experimental Neuropathology 450 Perfection of Method of Preservation 450 Identification of Artifactual Changes 451 Pathologic Neuronal Manifestations 451 Identification of Cell Types 451 Laboratory of Neurophysiology 451 Laboratory of Biophysics 452 Laboratory of Neurochemistry 453 Laboratory of Molecular Biology 455 Structure and Alteration of Nucleic Acids 455 Use of Transforming DNA 455 Effect of Hydrogen Peroxide on DNA 456 Effect of Combining Mutagenic and Inactivating DNA alter- ations 457 Genetics Analysis of Virus Included Mutants of E. coli 457 Studies on the Structure of Chromosomes 457 Control Mechanisms and Differentiation 457 Depression of Alanine Dehydrogenase in Bacillus subtilis by Internal Induction 457 Lack of Sporulation in Cytochrome alpha-Deficient mutants of B. subtilis 458 New Lysyl sRNA Produced during Sporulation 458 Initiation of Germination 458 Laboratory of Perinatal Physiology 458 DIVISION OF BIOLOGICS STANDARDS 467 Introduction 467 Research and Development 468 Section on Experimental Virology , 468 Laboratory of Bacterial Products 469 Cholera Vaccine and Related Studies 469 Pertussis Vaccine 469 Tetanus Toxoids 469 Pleuropneumonia-like Organisms and Mycoplasma 470 Mycoplasma orale 470 Tuberculin 470 Poison Ivy 470 Laboratory of Biophysics and Biochemistry 470 Microbial Biophysics 470 Analytical Chemistry 471 Virus Characterization 471 Laboratory of Blood and Blood Products 471 Laboratory of Pathology 472 CONTENTS XXVH Page Neurovirulence Testing 473 Oncogenesis and Pathogenesis of Infectious Agents 473 Neuro- Anatomy 473 Laboratory of Viral Immunology 473 Rubella 473 Viral Genetics 474 Mumps 474 Vaccinia 474 Laboratory of Virology and Rickettsiology 474 Arboviruses 475 Avian Leukosis 475 Adenoviruses 475 Influenza 476 Rickettsiae 476 Tissue Culture 476 Tissue Culture Section 476 Paolins 476 Hepatitus 477 Contract Operations 477 NATIONAL CANCER INSTITUTE GENERAL LABORATORIES AND CLINICS Introduction The reorganization of the National Cancer In- stitute during fiscal year 1966, dividing the Institute activities into 4 program areas, in- corporates within the General Laboratories and Clinics (GL&C) program most of the Insti- tute's intramural research, although the for- mer Laboratory of Viral Oncology is now within the Etiology program area, and the Radiation Branch and Medicine Branch are now within the Chemotherapy program area. Currently, the GL&C consists of 4 Laborator- ies (Biochemistry, Biology, Physiology, and Pathology), 6 Clinical Branches (Surgery, Me- tabolism, Immunology, Endocrinology, Derma- tology, and Pathologic Anatomy), and one section reporting directly to the Office of the Scientific Director (Macromolecular Biology Section), the latter having been within the Laboratry of Viral Oncology prior to reor- ganization. Although the major targeted research activi- ties in tumor virology, radiation biology, and chemotherapy are now pursued within the other program areas, there remains substan- tial representation of these activities within the GL&C, particularly tumor virology and radiation biology. There is comparatively lit- tle representation of chemotherapy per se. However, the clinical investigations of chemo- therapy are a direct operational activity of the Medicine Branch pursued in the Clinical Cen- ter, which geographically provides for easy collaborative and cooperative clinical investi- gations between the clinical branches of the GL&C and the Medicine Branch of the Chemo- therapy area. The organizational identity of the more tar- geted programs of the Institute in turn identi- fies the research program of the GL&C as now consisting of the conduct of less targeted basic research but oriented toward developing knowledge leading to the final understanding of the nature of cancer, its causes, its treat- ment and its prevention. Superimposed upon this are identifiable characteristics of the re- search program which have evolved over the years from direction exerted by the scientists within the GL&C and the particular program interests generated within the Institute. Some identifying characteristics include a high de- gree of emphasis and strength in biochemis- try, virology, carcinogenesis and immunology. Beyond these areas of emphasis there is, how- ever, a wide range of interests represented within the GL&C which includes significant research activity in electron micrographic anatomy, in vitro and in vivo tissue culture studies, endocrinology, mathematical biology, genetics, cellular kinetics and anticarcinogen- esis. It should be noted there has been little emphasis to date on developmental biology, membrane biology, mathematical and theoreti- cal biology, lipid chemistry and connective tis- sue chemistry. The staff of the GL&C now consists of ap- proximately 500 persons, of which approxi- mately 200 are professional level staff. The following is an abbreviated account of selective research activities within the constit- uent laboratories and branches of the GL&C during fiscal year 1966. More detailed descrip- tion can be found in the individual reports of each area. The Laboratories The MACROMOLECULAR BIOLOGY SECTION, within the Laboratory of Viral Oncology prior to the NCI reorganization during the past year, ANNUAL KEVIEW OF INTRAMURAL RESEARCH is now a separate research unit not organiza- tionally attached to a laboratory or branch. Past and present research interests of the Section have involved the interaction of large molecules, including polysaccharides, proteins and nucleic acid polymers. The research course projected has some additional orientation to the process of malignant transformation of cells and tissue recognition phenomenon oc- curring during the process of differentiation, where the techniques and knowledge of the Section seem to be applicable. During the past year the active enzyme, B- galactosidase, of E. coli has been shown to be a tetramer, with a molecular weight of 540,000 and a sedimentation coefficient of 16s. The in- active unit is found to be a dimer, molecular weight of 220,000 and sedimentation coemicient of 10s. Denaturation of the enzyme with 5-6 M urea has yielded a monomer unit with a sedimentation coefficient of 2s. The base sequence of DNA of normal rat liver and of rat hepatoma has not been found to possess significant differences, on the basis of optical properties identified in "Melting" profiles. Intact RNA was isolated from the Rauscher mouse leukemia virus and has been character- ized as a single-stranded molecule, with a mole- cular weight of 13x10°. The isolation of the molecule should permit studies to determine whether the isolated RNA possesses complete genetic information to effect malignant trans- formation in the absence of the intact virus particle. The electron microscope facility has been reactivated, following several months idleness, and will permit certain membrane studies con- sidered to be important in understanding transformation, cell recognition during contact inhibition, morphogenesis, etc. In the laboratory OF biochemistry research is highly oriented toward identifying biochem- ical changes associated with the cancer process. Most of the work, however, is applicable to areas and problems other than cancer. Metabolism of tumors, particularly in re- gard to glycolytic characteristics, received fo- cused, attention by several investigators. Re- cent work utilizing tumors in in vivo isolation has shown that tumors under such conditions utilize oxygen and glucose in proportionate amounts, that such tumors do not selectively utilize glucose in the absence of oxygen. An additional observation made on this isolated tumor system is that tumors metablizing high levels of both oxygen and glucose do not grow faster than when lower levels are utilized, but indeed may grow less rapidly. The destructive effects of hyperthermia on tumors in the in- tact animal seem to be greater than on normal tissue; this disparity is due in part to impair- ment of the tumor blood flow which secondar- ily may account for decreased glucose and oyxgen supply. Production of transplantable heptomas, pro- viding research material for some two dozen collaborative laboratories in the United States and abroad, is now accomplished .through a contract with a commercial laboratory (Mel- par). Considerable study of these tumors, however, continues at NCI and NIH. It has been found that many tumors, similar to nor- mal liver, are slow growing, have fewer tissue- specific antigens, and have a reduced capacity to incorporate thymidine into DNA. Some of the free polyribosomes will bind to membranes from normal liver but polyribosomes, originally bound to membranes of normal rat liver, do not bind to membranes from hepatomas. The chemical carcinogens used to produce hepatomas have also been used to induce blad- der tumors. The induction of such tumors re- quires low dietary vitamin B6. Therefore, an "anti-carcinogenic" effect of vitamin B6 may be identified and in this respect is similar to the anti-carcinogenic effect of vitamin A iden- tified in studies performed in the Pathologic Anatomy Branch. Controls of enzyme functions have been studied in rat mammary tumors, normal liv- ers and hepatomas. Primary mammary tumors from lactating hosts have been found to have a negligible capacity to produce lactose al- though it is known that mammary tumors possess the anzymes involved in lactose syn- thesis. This may be an example of the loss of ability of neoplastic cells to respond to influ- ence controlling functions of normal cells. NATIONAL CANCER INSTITUTE 3 Clones of hepatoma cell lines, containing either high catalase activity or low catalase activity, have been isolated from liver metas- tases when mixtures of tumor cell lines have been injected into the spleens of animals. A positive feedback mechanism has been identified in normal rat liver which involves the enzyme phosphoryl choline-cytidyl trans- ferase, a step in the synthesis of lecithin. It has been found that the enzyme is activated by degraded phospholipid, and inhibited by a poly- nucleotide. Studies on the site of synthesis of protein within the cell, and the intracellular distribu- tion of protein after synthesis, have shown that approximately half of the protein in plasma cell tumors is synthesized by the mi- cromosome fraction and mitochondria, and another half synthesized by the free polyribo- somes. The protein synthesized by the micro- somal fraction and mitochondria tends to re- main within these structures, whereas the greater portion of the protein synthesized by the free polyribosomes is distributed as soluble protein within the cytoplasm. The structural ribosomal proteins of plasma cell tumors of BALB/c mice have been found to consist of approximately 35 discrete protein bands elec- trophoretically. These bands have been found to be identical for 9 different tumors, even though each tumor secretes a different mye- loma protein. The ribosomal protein pattern of the tumor is the same as that of the normal liver of these mice. However, the ribosomal pro- tein patterns of mouse liver, rat liver, and rab- bit reticulocytes are distinctly different from each other. Cellular DNA has been found to reside both in the nucleus and in mitochondria. Although a relatively small portion of cellular DNA is found in mitochondria, recent studies have shown that in the resting liver cell there is a tenfold greater synthesis of mitochondrial DNA compared to nuclear NDA, and that mito- chondrial DNA has a rapid turnover rate with a half life of seven days. Synthesis of nuclear DNA is accelerated after partial hepatectomy whereas mitochondrial DNA synthesis is un- affected. Studies of enzymes degrading DNA have shown the existence of a group of DNases which consist of a mixture of at least 2 endo- nucleases and 1 phosphodiesterase. The latter enzyme hydrolyzes not only DNA but also RNA. Work carried out in collaboration with the Weissman Institute has revealed that deoxy- nucleotides, greater than or equal to 7 in chain length, have produced reactivation of the im- mune response in thymectomized, X-ray irra- diated adult mice. Other work on the biochem- istry of immune mechanisms has been pursued within the Laboratory of Biochemistry in which polylysine models have been examined for antigenic capability, that is, ability to pro- voke delayed and immediate skin sensitivity. Results from these studies suggest that devel- opment of delayed responses requires a more biochemically complex antigen than that re- quired for the development of immediate hy- persensitivity, at least as measured in guinea pigs. A concerted attempt to develop means for isolating specific cell types is being directed both intramurally and in contract collabora- tion with outside facilities. A technique cur- rently under exploration is one employing poly- urethane foam containing antibody against specific cell types to be passed through the foam. To date, red blood cells have been con- centrated within this system by absorption to specific anti-RBC gamma globulin. The research activities of the LABORATORY OF BIOLOGY, although generally broad in scope, in- clude distinct emphasis on work in immunology, the behavior of cells under in vitro environ- ments, genetics, and carcinogenesis. Research in the field of immunology, particularly immu- nologic aspects of tumorigenesis and tumor recognition, is approached via the plasma cell system and the thymus. A thymic humoral factor has been identified as being responsbile for initiating and maintaining homeostatis of the lymphoid cell population controlling immu- nologic competence. A new observation is that spleen cells from C57BL mice with thymus in- tact produce "runt disease" when injected into strain A mice because of the graft versus host reaction; but if the spleen cells are from a thy- ANNUAL REVIEW OF INTRAMURAL RESEARCH mectomized C57BL they lack the immunologic competence to induce this reaction. Syngeneic thymic grafts in thymectomized C57BL mice restore this competence of the spleen cells. An inbred strain of mice is being closely ob- served because these animals develop, by the ages of 12 to 24 months, a reticulum cell neo- plasm closely resembling Hodgkin's Disease as seen in man. This disease is fatal within a 1-2 month period after its development. The animals carrying the lesions are found to pro- duce abnormal immunoglobulins. The plasma cell tumor is used in intensive studies on the differentiation of the plasma cell and its synthesis of specific immunoglob- ulins. The functional relationships between gene structure and immunoglobulins produced indicates that only one gene is operative in pro- ducing the heavy chain of the immunoglobu- lin molecule and one gene operative in produc- ing the light chain; all other possible operative genes being repressed during that time. Identification of immunoglobulins of mice is done by means of chain specific antisera pre- pared in inbred strains. The genetic patterns of tumor development are being studied by biochemical means, the initial work identifying urinary proteins of the mouse by various chemical detection meth- ods. These studies have indicated that some urinary proteins are under genetic control. The effect of specific genes on the occurrence of tumors in mice continues to be studied along classical lines established in the Laboratory of Biology. The development of mammary plaques, a pre-malignant lesion, has been found to be dependent not only on the genotype of the mouse but also on the strain of mammary tumor viruses carried by the animal. Much of the current research on the behav- ior of cells in in vitro conditions have been directed toward the malignant transformation process and influences within the tissue cul- tural environment which modify this change. It has been observed that malignant transfor- mation of cells occurs more rapidly in the presence of tumor virus (e.g. polyoma). It is, however, felt that neoplastic transformation may occur in cells free of tumor viruses. Addi- tional work on tissue culture systems is heav- ily focused on the effects of changes in the culture medium on the behavior of cells in gen- eral, their growth responses and metabolic characteristics. The biologic identities of hepatomas induced by chemical carcinogens (e.g. carbon tetra- chloride and fluorenamine compounds) are being studied both by light mircroscopic and electron microscopic techniques. The identifica- tion of ultrastructural characteristics of var- ious tumor cells, as well as cells in tissue cul- ture, is only in its early stages of development in this laboratory. The LABORATORY OF pathology and the pathologic anatomy BRANCH, although orga- nizationally distinct units, maintain an intimate functional relationship. It is, however, recog- nized that the primary responsibility of the Pathologic Anatomy Branch is to provide diag- nostic pathologic service to the several Insti- tutes, as well as to pursue research related to human disease. On the other hand, the Labora- tory of Pathology is involved primarily with experimental cancer research. The research activities of the Pathology units collectively may be grouped into three major categories, namely cancer in man, can- cer in animals and viral carcinogenesis. A fur- ther breakdown reveals a range of research in- terests and includes ongoing research in the following: biochemistry, carcinogenesis (chemi- ical, parasitic, hormonal, viral), viral oncology, immunology, comparative oncology and geo- graphic pathology, in addition to usual studies in human pathology. Recent biochemical work on the mechani- isms of interferon action have shown that the synthesis of interferon is dependent upon pro- tein synthesis and is inhibited by such chemi- cals as puromycin or p-fluorophenylalanine. Interferon in turn blocks synthesis of viral DNA and RNA. A number of studies are concerned with car- cinogenesis in man and animals. Several proj- ects are pursued to determine the carcinogenic- ity of a large number of chemical agents on one or more organ systems in small laboratory animals and in sub-human primates. The lat- ter animals were used in carcinogenicity stud- ies as long as 17 years ago when 3 MCA was NATIONAL CANCER INSTITUTE instilled in the stomach wall of a group of monkeys; no neoplastic lesions have been de- tected to date in these animals which are still under observation. Currently infant monkeys are receiving, either in the diet or sub-q, sev- eral carcinogenic chemicals including methyl- cholantrene, benzpyrene, aso-dyes, fluorena- mine compounds, urethane, aflotoxin, and diethyl nitroso amine. The last named chemical has induced 13 hepatomas and the tumor has been propagated to date via intracerebral transplantation in monkeys. Another monkey has developed chronic myelocytic leukemia in response to MIH, a chemotherapeutic drug currently in use in man. The phenomenon of vitamin anti-carcinogenesis, mentioned ear- lier, has been identified with vitamin A which has been shown to prevent cervical cancer and stomach cancer in hamsters given dimethyl- benzanthracene. An important relationship between the state of cellular differentiation and susceptibility to carcinogenic influences has been illustrated by the finding that a certain stage of develop- ment of mouse salivary gland is necessary before it is susceptible to the tumorigenic influ- ences of polyoma virus. The presence of mesen- chyme in turn is required for this differentia- tion to occur. A number of studies continue to explore the relationships between several viruses and can- cer or cancer-like diseases, and reference is made to the review of this work contained in the Summary Statement of Pathology. A significant observation in the functional response of lymphoid cells in tissue culture has been made on cells, isolated from a patient with lymphoma and maintained under in vitro conditions. These cells have been found to syn- thesize immunoglobulins. It has not been ascer- tained whether these are produced in response to a specific antigen, viral or otherwise. The research interests of the scientific staff of the laboratory of physiology constitute a somewhat wide spectrum which would include the following subjects: metabolic aspects of can- cer, UVL carcinogenesis, thyroid biology and physiology, biochemistry (protein, nucleic acids), radiation biology, mathematical biology and programming, pharmacobiology and im- munology. Recent, but unpublished, work indicates the short ultraviolet wave lengths (2537A) may be carcinogenic. The usual carcinogenic wave length at the earth's surface is found at 2969A, the erythema producing wave length, and no VL of 2537A normally reaches the earth's surface. Whereas the shorter wave lengths may be an additive carcinogenic factor, it should be recalled that earlier work in the Laboratory of Physiology has shown that longer (visible) light is protective against UV car- cinogenesis. Studies of the biologic behavior of the thy- roid gland have revealed that the resorption of colloid occurs by a seeming combination of mechanical and enzymatic events in which col- loid is taken into the peripheral cells of the follicles via pseudopodic entrapment by the cells; actual resorption of the colloid seems to occur intracellular^, presumably by enzymatic digestion. Recent studies in this unique organ have dealt with the kinetics through which the follicles establish and maintain a concen- tration of iodide above that of the blood. Inhibition of RNA sysnthesis in Sarcoma 37 cells by actinomycin has been shown to re- sult in diminished synthesis of proteins, in- cluding histones, and sterols. The inhibitory effect is reversed by glucose and other gly- colyzable substrates. Another study of intracel- lular control mechanisms has involved the in- hibition of hemoglobulin synthesis by both iron and heme. It is felt that this inhibition occurs via effects on polyribosome assembly. Mechanisms of recovery from radiation damage are being studied from several direc- tions. In addition to the phenomenon of "spon- taneous" recovery it has been found that cer- tain compounds (e.g. colchicine, endotoxin) protect cells against radiation damage when given after or before exposure. Isolated macromolecules in a dry state are found to be protected against radiolysis by metallic ions. Studies of the possible mechanisms by which a cell repairs radiation damage, in which DNA synthesis is controlled or inhibited, have indi- cated that DNA synthesis is not necessary for ANNUAL REVIEW OF INTRAMURAL RESEARCH repair to occur, whereas RNA synthesis may- be required. Recovery from radiation affects, and pro- tection against effects, are further explored in studies on radiation inhibition of natural antibody synthesis and recovery thereof, and in studies involving the tolerance or rejection of allogeneic bone marrow grafts and graft versus host reactions. The immunologic behav- ior of allogeneic bone marrow in animals has been found to be greatly modified by various manipulations of the bone marrow cells prior to innoculation. It may be wondered whether the antigenic identity of the cells is in any way changed by the manipulations. The Clinical Branches The clinical and laboratory research program of the dermatology branch has continued along lines developed in recent years with the addition of two new laboratory activities, namely research in cell biology and electron microscopy. The major groups of the research interests are: (1) Mycosis fungoides lymphoma, (2) Epidermal growth and differentiation, (3) Biology of lymphoreticular cells, and (4) Mela- nogenesis. The clinical research program conducted during the past year within this Branch has almost entirely involved the immunologic re- activity of patients with lymphona, particu- larly in a comparison of patients with mycosis fungoides with Hodgkin's Disease and other disease conditions having alterations in im- mune responsiveness. Mycosis fungoides re- mains as a distinct lymphoma insofar as patients with this disorder retain intact immu- nologic capabilities. An incidental finding has been made in pa- tients with mycosis fungoides topically treated with HN 2 . Most patients receiving such treat- ment have developed cutaneous sensitivity to this drug. However, the topical sensitivity either disappears or is markedly diminished following intravenous administration of HN 2 . Reorganization within the past year resulted in the transfer of portions of the endocrinol- ogy branch to the NICHHD with the result that the Endocrinology Branch, NCI, was re- duced to approximately half its previous size in both physical facilities and personnel. The re- search program of the Endocrinology Branch has been reoriented toward studies of androgen and estrogen synthesis and metabolism. Specific laboratory projects, and the recruitment of sci- entific personnel to implement the Branch pro- gram, have been initiated. The Endocrinology Branch has further ex- tended its interests in the problem of breast cancer. It has initiated explorations into pos- sible means to enhance the development of knowledge that will lead to a more precise definition of the disease and its therapeutic control. With the recognition that eventual thera- peutic control of breast cancer will require identification of a number of biologic deter- minants operative in the genesis and course of the tumor, the Branch places heavy emphasis on studies of hormonal patterns that may be associated with breast cancer, the isolation of prolactin and its quantitative chemical assay, and biologic assay of prolactin as measured by its effects on the mouse mammary gland. The latter at this time is planned to be initi- ated by collaborative involvement of other re- search units within and outside of NCI. The immunology branch functions as the focal organizational unit within the GL&C en- tirely concerned with immunologic aspects of the cancer process. In addition to its own inten- sive research activities, it has provided labora- tory facilities and guidance for the development of immunologic research in other branches and for further training of scientific personnel in immunologic techniques. Evaluation of human sera for immunoglob- ulin constituency has been initiated through a contract with a commercial laboratory (Mel- par). Through this means it is planned that a large number of sera, representing a wide range of disease states, will be so evaluated. The re- sults will be reviewed for possibilities of iden- tifying immunodiagnostic criteria. Lymphoid cells from a human lymphoma have been established in continuous culture. In collaboration with scientists in the Pathologic Anatomy Branch, these cells have been found to produce immunoglobulins and constitute NATIONAL CANCER INSTITUTE the first possible model to study the produc- tion of immunoglobulins by lymphoid cells in in vitro conditions, with the goal of producing specific human antibody. Further research activities of this Branch are identified in the special review entitled "Evaluation of Immunologic Research in GL&C," a separate item in this report. The metabolism BRANCH primarily conducts research relating either secondarily to the can- cer process or to matters frequently directly consequent to cancer, e.g., anemia of cancer. Research currently active includes the follow- ing: nucleic acid metabolism, amino acid trans- port, metabolism of albumin and globulin, por- phyrin metabolism, erythrophoresis, calcium metabolism and hemaglobulin synthesis. Refer- ence is made to the review given these studies in the summary report of the Branch. Selected attention is drawn to the findings during the past year from studies on porphy- rin metabolism. Delta-aminolevulinic acid syn- thetase (ALA synthetase) has been partially purified from mitochondria. The kinetics of induction of hepatic ALA synthetase have been studied along with the effects of inhibi- tors of protein synthesis (puromycin, actino- mycin-D, 5-fluorouracil, p-fluorophenylalan- ine, etc.). Glucose blocks induction of this enzyme as effectively as actincmycin-D. It has been shown that heme, the end product of the pathway which ALA synthetase controls, will prevent induction of ALA synthetase when in- jected intravenously at certain times before induction is initiated. This is the first direct evidence of heme as a repressor of ALA syn- thetase in mammalian tissues and supplements the indirect evidence from the tryptophane pyrrolase work reported last year. When heme is injected intravenously, the level of hepatic ALA synthetase oscillates markedly for periods of 4 to 5 days after a single dose. The oscilla- tions can be greatly amplified by administra- tion of inducer 5 hours before sacrifice. This probably is the first demonstrated example of significant oscillations of the level of an en- zyme. The only other example is reduced nico- tinamide adenine dinucleotide oscillations in in vitro yeast extracts of glycolyzing systems. The oscillations of ALA synthetase in mammal- ian liver explain Watson's observations on var- iations in bilirubin excretion following heme administration and raise the question of the role of ALA synthetase in biologic clock mech- anisms. The effects of diet on preventing the chemical abnormalities of acute porphrin pre- viously demonstrated in the experimental ani- mal have been extended to patients. A de- creased caloric intake can initiate attacks of the disease and high carbohydrate diets can end these attacks. A small fraction of women whose attacks of acture porphyria recurred cyclically with their menstrual periods have been maintained free of disease by use of oral anovulatory hormone preparations. DNA-RNA hybridization techniques have been used to study the taxonomy of microor- ganisms, mycoplasma and streptococci. Myco- plasms have been found to represent a highly heterogeneous group of organisms by this technique. The reliability of the hybridization technique for the identification of microorgan- ism types is supported by the fact that the results correlate exceedingly well with those from usual serologic typing of these organisms. A major responsibility of the surgery branch is to provide consultative advice and service to the several Institutes in clinical prob- lems requiring judgment in general, urological, otolaryngological, gynecological or thoracic sur- gery. In addition to this, a clinical and labora- tory investigative program, broad in scope, is actively carried out. Much of the research is directly concerned with development and eval- uation of surgical procedures applied to control of cancer in man, and control of complications secondary to cancer. Examples include clinical evaluation of combined ionizing radiation and surgery as a cancer therapeutic modality; and evaluation of pre- and post-operative prophy- lactic antibiotic administration to control infec- tious complications of cancer surgery. A study of the latter has shown that chloramphenicol, given pre- and pcst-operatively over a ten-day period, has markedly diminshed infectious com- plications to the low frequency of 7 % compared to 22-30% in patients receiving no antibiotic therapy. The Surgery Branch has been provided two laser units with which exploration of the ap- 8 ANNUAL REVIEW OF INTRAMURAL RESEARCH plicability of this type of radiation to research and treatment of disease in man and animals has been initiated. Pursuit of investigations utilizing these instruments will be in accord with initial results obtained and the likelihood of deriving significant information with their continued use. Greater and greater emphasis is placed on research on immunologic processes involved in transplantation, cellular recognition, and con- trol of the cancer process. Several studies are in progress. A study of the immunologic rela- tionship between pregnant women, or women with a trophoblastic tumor, and their hus- bands has been carried out using the technique of lymphocyte transformation in mixed leuko- cyte cultures. Preliminary results show that lymphocytes from normally pregnant women do not transform when grown with their hus- band's leukocytes in as high a percentage as they do when grown with cells from an unre- lated male. Further studies are being carried out to evaluate serial changes in each trimester of pregnancy and the changes that take place during and after chemotherapy of tropho- blastic neoplasms. A chemically-induced gastric adenocarcinoma has been found to be antigenic eliciting an 'immunologic response which can be adop- tively transferred by isologous lymphoid cells, and which may be abrogated by splenectomy or large doses of tumor cells. Attempts to uti- lize sensitized lymphoid cells to alter the growth of established tumors have resulted in possible slowing of tumor growth; complete regression has not occurred. Immunologic Research As earlier pointed out, while the research in- terests and activities within the GL&C, NCI, extend over a broad range of subject inter- ests, there are several subjects in which there is an intensity of interest and work. These include biochemistry, carcinogenesis, virology and immunology. Because of increasing evi- dence of the importance of immunologic factors in the development of some neoplasms, and an awareness of possible applicability of im- munologic factors to control the course of neo- plasms, a summary assessment of immunologic research within the GL&C seems appropriate. A significant amount of research directly related to or involving cancer immunology is found in the Laboratories of Biology and Bio- chemistry; and in the Immunology, Surgery, Pathologic Anatomy, Metabolism and Derma- tology Branches. Immunologic research has been fostered and intensively pursued in the Laboratory of Biol- ogy for a number of years. Currently the work within this Laboratory is directed towad elicit- ing antigenic identities of cancer cells; deter- mining the rcle exerted by thymic and lymphoid cells in directing immunologic capa- bilities of the organism; and determining molec- ular specificities of immunoglobulins synthe- sized by the plasma cell. To date it has been shown that some cancers of animals, particu- larly viral cancers, have cell-related antigenic characteristics which can be utilized to pre- vent or modify the early development or course of these tumors. Lymphoid cells of thymic or- igin have been shown to be highly functional in determining and maintaining immunologic competence of the mouse. A number of im- munoglobulins, each molecularly specific in composition, have been identified as products of plasma cell tumors. It is found that each tumor continues to synthesize a specific globulin. The specificity of the stimulus accounting for this is under study. In this experimental system, no antibody function of the immunoglobulins has been discerned as yet. Some studies of molecular identities of im- munoglobulins produced by plasma cell tumors, and factors controlling their synthesis, are pursued within the Laboratory of Biochemistry in collaboration with scientists of the Labora- tory of Biology. Additional research within the Laboratory of Biochemistry on the chemi- cal basis of immunity has been concerned with molecular determinants of antigenicity. Re- sults to date, from studies utilizing polylysine models, indicate the induction of delayed hy- persensitivity requires an antigen with struc- ture more complex than that required to in- duce immediate hypersensitivity. Research of the Immunology Branch is pri- marily directed toward studies in man. Scien- NATIONAL CANCER INSTITUTE 9 tists within this branch have over the years provided leadership in research which has led to better understanding of the biochemical identities and specificities of immuno-proteins in man. Work in animals has yielded funda- mental information on the reactivities of the various components of complement and the applicability of complement fixation tests to determine antigenic identities of chemically- induced tumors. The screening analysis of hu- man sera now underway in the Branch is di- rected toward identification of immunoprotein patterns which might be diagnostic of certain types of cancer in man. The work initiated collaboratively with scientists of the Pathologic Anatomy Branch has provided the first in- stance of continual immunoglobulin synthesis by lymphoid cells in tissue culture. Inasmuch as these cells are of human origin, the possi- bility is now raised of in vitro synthesis in other culture systems of specific antibody for human use. Immunofiuorescent studies of possible tumor specific antigens in man and animals have been carried out in the Pathologic Anatomy Branch in recent years. Some of these studies have suggested that specific tumor antigens may exist in man, although a definite conclusion cannot be made at this time. Collaborative work along these lines is being continued with other units in GL&C and with units in the other program areas of NCI. Immunologic research of the Surgery Branch has to date yielded positive informa- tion on the role of the small lymphocyte in maintaining immunologic competence in man. Partial depletion of the lymphocyte population by thoracic duct drainage has resulted in sig- nificant diminishment of delayed immune re- activity as measured by skin tests and homo- grafts. Immunologic research capabilities are being actively encouraged within this branch. Studies of the Dermatology Branch have shown that the patients with mycosis fun- goides lymphoma remain immunologically com- petent throughout the disease, in distinction to patients with Hodgkin's Disease. Immuno- logic challenges, cutaneous and systemic, of pa- tients with mycosis fungoides have been ob- served to effect partial or complete remissions of the disease in some patients. Results of clinical and laboratory work to date suggest there is a likelihood of influenc- ing cancer in man by immunologic means. It is intended that early leads will be tested as quickly as possible in man, but at a pace and in a fashion commensurate with standards ap- propriate to research in man. MACROMOLECULAR BIOLOGY SECTION In the recent reorganization of the NCI, the Macromolecular Biology Section was retained in the intramural research area in the Office of the Scientific Director for General Labora- tories and Clinics, NCI. The Macromolecular Biology Section was previously under the Laboratory of Viral Oncology, which has been transferred to Field Studies, Etiology. The de- cision was made to retain the Macromolecular Biology Section in the general intramural re- search area since the basic research in the Macromolecular Biology Section pertains to cell growth and differentiation in general, as well as to viral oncogenesis. During the past few years there has been increasing recognition that the new techniques and findings of molecular biology may lead to significant clarification of cellular differentia- tion on the macromolecular level. Differential macromolecular synthesis and differential uti- lization of energy through processes controlled by macromolecules are crucial in the control mechanisms of histo-differentiation. In order to understand the control mechanisms of can- cerous transformation, greater understanding must be gained of the processes of differentia- tion, cell division, and contact inhibition. The combined disciplines of molecular biol- ogy (biophysics, genetics, virology, biochem- istry, polymer and physical chemistry) were fused in recent years into a unique research facility at NIH in the Section of Macromolec- ular Biology. The aim was exploration of the role of macromolecules in cellular control mechanisms. Thus the research emphasized the interaction of macromolecules in the cellular control processes which govern differential synthesis of macromolecules, utilization of en- ergy, and growth of cells. 10 ANNUAL REVIEW OF INTRAMURAL RESEARCH In the field of macromolecular interactions and protein structure pertaining to control processes in energy utilization, Dr. Mora, with associates, was studying the changes in cyto- chrome c structure associated with intercon- version between the oxidized and reduced states. Certain polycations, prepared by substi- tuting polyglucose with basic residues, and certain sugars, reduce cytochrome c in a unique, slow and controllable fashion, so that both the rate and the extent of reduction of the cytochrome c can be varied. This technique permits observations on the changes in the tertiary structure of this respiratory protein during its conversion from oxidized to reduced form, heretofore impossible. Greater under- standing of protein conformational changes in cytochrome c allows better understanding of the control processes of oxidative phos- phorylation in terminal electron transport. Cy- tochrome c plays a central role in terminal electron transport in the mitochondria and in energy utilization in all types of cells. Dr. Shifrin shifted his research interest to the study of cellular control processes in bac- teria. In collaboration with investigators in the Laboratory of Chemical Biology, NIAMD, he studied the aggregation of the subunits of /?-galactosidase isolated from wild-type E. coli and from a mutant which lacks /?-galac- tosidase activity. He found that a tetramer of the subunit is required for the enzymatic func- tion, and a point mutation in the lac operon yields a protein which is enzymatically inac- tive primarily because the site required for ag- gregation of the subunits of the /?-galactosi- dase to an active tetramer is mutated. In an- other collaborative work with scientists in the Laboratory of Molecular Biology, NIAMD, Dr. Shifrin studied the mechanisms of action of an amino acid analogue, the a-hydrazino ana- logue of histidine, which inhibits bacterial growth. The study utilized a mutant of S. ty- phimurium defective in histidine transport system. He found that this metabolic inhibitor is functioning not by virtue of its ability to be incorporated into the protein and thus make a defective enzyme, but by acting as a feed- back inhibitor. The site of action was found to be on the acetylornithine transaminase in the arginine biosynthetic pathway. Thus, an im- portant linkage between the bacterial cell wall or membrane where materials are transported, and the control of cellular growth is clearly demonstrated in this study. Other work pertained to macromolecular differences in higher organisms. In one study, Dr. Smith carefully compared the nucleic acids of rat liver and hepatoma from the point of view of base sequence homology. Refined and novel physical measurements were used. Ob- servation of the changes (upon heating) in the optical properties of the DNA at various wavelengths, and comparison of the so-called "melting" profiles revealed no difference in the DNA from the two tissues. In addition, various techniques were employed in hybridiz- ing denatured single-stranded nucleic acids from the two tissues. Currently attempts are being made to compare liver and hepatoma messenger RNA by various hybridization tech- niques designed to reveal differences in their base sequences. The study of differences in macromolecular pathways of normal cells and tumor cells can be conveniently accomplished by the compari- son of normal cells and tumor virus-trans- formed cells in tissue culture. Preliminary puri- fication and characterization of the virus and its nucleic acid are essential. With the help of a small contract at Mel- par, Inc. (which expands the limited tissue culture facilities of the MBS), Drs. Luborsky and Wood studied the incorporation of tritium into DNA in mouse cells infected by the poly- oma virus. Under specific conditions, in the presence of FUDR and tritiated thymidine, it was found that only viral DNA was synthe- sized in mouse embryo cells. In the absence of a very critical concentration of the metabolic inhibitor the major amountt of the newly syn- thesized DNA is induced cellular DNA. These findings were possible after first developing a fractionating technique so that the cellular DNA was fully separated from the viral DNA on methylated albumin column, and after studying the balance of the synthesis of these two DNA species in the cells under various con- ditions. Conditions were also found to in- crease the level of incorporation of the tritium NATIONAL CANCER INSTITUTE 11 into the virus DNA. The highly radioactive viral DNA is used currently in refined nucleic acid homology studies, following the pioneer observations of Axelrod, Habel and Bolton. The objective is to see what part of the viral ge- nome is incorporated into the transformed tumor cells in different types of polyoma- induced tumors. Another objective is to see if the viral DNA genome, or a fragment, incor- porates perferentially into the transformed cell's mitochondrial DNA or into its nuclear DNA. Further objectives pertain to changes in macromolecular synthetic pathways and to cell surface changes during transformation of cells to cancer cells (see below). A significant achievement in the Macramo- lecular Biology Section was the first isolation of an intact RNA from an animal leukemia virus. A 13 million molecular weight single- stranded RNA molecule was isolated from the Rauscher mouse leukemia virus. It was shown that this molecule accounts for the full genome of the virus. To our best knowledge this RNA is the highest molecular weight RNA ever ob- served. Our method of isolating it in good yield opens the way for molecular biological studies of the RNA-containing viruses. One important finding in this respect is that the RNA is sin- gle-stranded. Questions which are now under study include: whether and how transforma- tion or virus induction may occur with a pure and intact single-stranded viral RNA; does this RNA possess the full genetic information for transformation, or is other information necessary? A particularly important change during transformation of a cell from the normal to the malignant state must be on the cell sur- face regulating the growth of the cell vis-a-vis the other cells. Acid mucopolysaccharides have been reported to be qualitatively different after virus infection and transformation of several cellular systems. Chemical methods are being developed in the Macromolecular Biology Sec- tion for the isolation and characterization of these mucopolysaccharides from various nor- mal cells and from virus producing and virus transformed cells. (Dr. Wood) The possible changes in surface charge dur- ing transformation of cells, and the effect of added polyanions and polycations on cell growth and on contact inhibition of normal and malignant cells are also being studied in tissue culture. (Dr. Schutz) Techniques for measuring DNA synthesis, rate of mitosis, cell overgrowth, etc., is being refined to per- mit reproducible measurement of the changes induced in cell growth by charged polymers. Lipid components of normal cells and of cells producing certain viruses, especially the so- called membrane viruses were compared (Dr. Johnson). It was found that more unsaturated and short chain lipids were produced in cells which were infected by the Rauscher, Moloney or Rous viruses, as well as in Rous-transformed cells. Future research will continue to center around the molecular basis of differentiation. With the help of two additional investigators, research on protein biosynthesis in subcellular systems and electron microscopic investiga- tions on cell surface changes will be integrated into this effort. LABORATORY OF BIOCHEMISTRY Cytochemistry Section Dr. D. Burk and Dr. M. Woods have con- tinued their investigations of the effects and modes of action of anti-cancer agents on the metabolism, growth, and death of cancer cells. Cancer tissues and cells have been exposed in vitro and in vivo to chemical or physical (hyperthermy, laser, laser, light) agents, and effects on their metabolism, growth, and death have been studied. In vitro exposures of 30-90 minutes to temperatures of no more than 43- 44° C sufficed to produce extensive or total killing of animal cancer cells, results similar to those obtained clinically by von Ardenne and Kirsch in Dresden, Germany. The thermal death temperatures and exposure times in- volved varied somewhat with the cancer cell type, and were lowered by certain drugs, such as steroids and serotonin, and raised by insulin and glucose. In vivo exposures to water tem- peratures of 43-44° C (90% total body im- mersion of host mouse) indicate that the tu- mor cells are more readily killed under in vivo than in vitro conditions, due in part to im- 12 ANNUAL REVIEW OF INTRAMURAL RESEARCH pairment of the tumor blood flow and subse- quent decreased glucose supply and anoxia. Concommitant with loss of viability as meas- ured by transplantation is a greatly decreased respiratory capacity and Pasteur Effect (inhi- bition of fermentation by oxygen gas), fol- lowed later by greatly decreased fermentation capacity. The in vitro metabolism and in vivo growth of two different tissue culture sarcoma cells lines, both derived years before from the same single cell, have remained relatively con- stant to each other over the last eight years in tissue culture. Drs. Burk and Woods are the recipients of the 1965 Gerhard Domagk Prize for Cancer Research which was awarded for their paper showing the connection between glucose fer- mentation and growth rate in the spectrum of Morris hepatomas. Dr. W. H. Evans in his biochemical studies of normal and leukemic leukocytes has joined with Dr. M. Rechcigl, Jr. in an examination of the peroxidative metabolism of leukocytes. Little is known about the relative roles of cata- lase and peroxidase although elevated catalase activity of leukocytes has been reported in chronic myelogenous leukemia. The present studies indicate that myeloper- oxidase in the intact cell is active in the pres- ence of sufficient aminotriazole to inhibit cata- lase. With cells disrupted by homogenization, however, both enzymes are inhibited. New data indicate that the inhibition of myeloperoxi- dase by amiotriazole depends on the presence of a peroxide-generating system whereas cata- lase can be inhibited by amiotriazole but un- der conditions where a much lower peroxide concentration exists. Inhibition by aminotria- zole of both myeloperoxidase and catalase also occurs when leukocytes are caused by phago- cytize polystyrene particles, another condition leading to increased hydrogen peroxide for- mation in intact cells. These observations sug- gest that catalase and myeloperoxidase are lo- cated in different compartments within the cell. Purified myeloperoxidase has been shown to oxidize formate-C 14 to C 14 2 in the pres- ence of H 2 2 . Myeloperoxidase may account for the major part of formate-C 14 oxidation in leukocytes since catalase activity can ac- count for only about 30% of the peroxidative metabolism of leukocytes as measured by for- mate-C 14 oxidation. Nutrition and Carcinogenesis Section Transplantable hepatomas, originally de- rived by Dr. H. P. Morris from primary hepa- tomas induced by a variety of aromatic amines, provide a spectrum of tumors which are being used to examine the relationship be- tween biochemical and biological characteris- tics and degree of malignancy. A total of 24 cooperating laboratories in the United States and abroad are involved in this continuing study. The growth rate of these transplantable tumors differs many fold and, morphologically, they vary from poorly or undifferentiated to highly differentiated neoplasms. Many tumors with biochemical characteris- tics similar to those of normal liver are slow growing, have reduced tissue-specific antigens, are not completely devoid of the slow-moving, h 2 electrophoretic class of proteins, and have a reduced capacity to incorporate thymidine into DNA. A high correlation has been estab- lished in cell free systems between growth rates of these hepatomas and the activities of the replicative and terminal DNA nucleotidyl transferases, the faster growing tumors hav- ing the higher activities. Degradative enzymes and kinases in such cell free systems were not rate-limiting. The very slow growing hepato- mas appear to have more organized endoplas- mic reticulum and fewer free ribosomes. Some of the free polyribosomes will bind to mem- branes from normal liver but polyribosomes, originally bound to membranes of normal rat liver, do not bind to membranes from hepa- tomas. It has been amply demonstrated that this spectrum of hepatomas provide a series with graded biochemical alterations, with some bio- chemical parameters increasing with growth rate, others decreasing, while still others re- main unchanged. Biochemical characterization seems to be much more specific than morpho- logical characterization since, despite the large number of tumors with different growth rates but similar morphology that have been de v el- NATIONAL CANCER INSTITUTE 13 oped, hepatomas with identical biochemical patterns have not been noted. Glutamic-oxalacetic transaminase (GOT) activity in these transplantable rat hepatomas has been shown by Drs. T. T. Otani and H. P. Morris to consist of two isozymes. Studies of the distribution of these isozymes, their Michaelis constants, and the effect of hor- mones have been made in the hepatoma and the liver of tumor-bearing animals. The host liver GOT content and isozyme distribution was not affected by adrenalectomy, hydrocor- tisone treatment, a combination of both nor by the presence of tumor. With the 5123B hepatoma, however, while hydrocortisone treatment or adenalectomy alone showed no effect, there was a shift in the isozyme dis- tribution of the tumor in females, but not in males, when hydrocortisone was administered to an adenalectomized animal. Dr. Morris has also been studying the role of tryptophan and indole in the induction of bladder tumors in rats ingesting N-2-fluorenyl- acetamide (2-FAA) and the effects of the level of 2-FAA on the induction of tumors in jaun- diced rats. Although all details of these experi- ments have not been completed, as reported last year, it appears that bladder tumors can be produced in Fischer strain rats without the addition of tryptophan. Jaundiced rats ingest- ing 0.025% 2-FAA develop as many liver tu- mors as do non jaundiced animals of the same genetic background. Lower levels of 2-FAA are now being studied to determine whether the early results were due to an overwhelming ex- posure to the carcinogen. Animal experiments have been completed and histologic studies are in progress. A series of "unnatural" /?-hydroxyamino acids and their derivatives have been synthe- sized by Dr. T. T. Otani for use as growth in- hibitors and racemic mixtures of disasterom- ers were tested. Certain of these were shown to possess moderate growth inhibiting activity in microbial and solid tumor tests systems. Since only one out of the four diasteromers is presumed to be active, increased inhibitory activity should result from the use of the pure resolved isomer. Milligram quantities of each of the four isomers of /?-hydroxynorleucine and their N-chloroacetyl derivatives have been prepared and remain to be tested. Tumor-Host Relations Section The study of the in vivo environment of the neoplastic cell by Dr. P. Gullino has been di- rected toward an examination of the energy requirements of growing tumor tissue. By use of the "tissue isolated" tumor preparation into which has been implanted a chamber for har- vesting interstitial fluid, blood flow, glucose, and lactic acid levels, and 2 and C0 2 ex- change levels can be measured continuously in interstitial fluid and in efferent and affer- ent blood. In three solid tumors, chosen because of dif- ferences in structure and biological behavior, it was found that about 50% of the available blood glucose was removed from the blood and utilized in different degree by each tumor. The limiting factor in oxygen utilization seems to be the supply since, within the limits of the 50% removal ratio, the three tumors used all the 2 that was received. It was not possible to saturate the ability of the neoplastic tissue to use 2 . Conversely, neoplastic tissues appar- ently were unable to remove a larger propor- tion of 2 from the blood when less is supplied. A decrease of arterial 2 from 20 to 5 vol- umes percent resulted in a decrease of 2 used. Furthermore, during acute in vivo lack of 2 , the tumors failed to respond with increased glucose utilization of lactate production. Glu- cose utilization and lactate production were di- rectly related to 2 consumption; no "spar- ing" effect (Pasteur effect) of 2 on glucose utilization could be demonstrated in vivo. In normoglycemic animals a sharp gradient of glucose concentration was found between plasma and interstitial fluid (from 150 mg/ 100 ml plasma to 0-8 mg/100 ml interstitial fluid). The tumors utilized all the glucose which passed through the capillary wall. The gradient was lost in hyperglycemia and restored by nor- moglycemia. Several hours were needed for the two phenomena to occur, suggesting that a glucose transfer system is operating across the capillary wall of tumors. Hyperglycemia (350 mg/100 ml plasma) lasting several days in- creased glucose utilization to a maximum level 14 ANNUAL REVIEW OF INTRAMURAL RESEARCH of about 30% above normal. Higher blood glu- cose levels did not induce greater glucose utili- zation. Tumors utilizing glucose at the highest possible level did not grow any faster; indeed, growth was poor in many of them. During nor- moglycemia the 3 tumors transformed about 35% of the glucose utilized into lactate. Dur- ing hyperglycemia more glucose was utilized and more lactate was eliminated by the 35% proportion was maintained. However, in tu- mors starved of glucose by severe hypogly- cemia (30 mg/100 ml plasma), the amount of lactate eliminated increased sharply in propor- tion to the amount of glucose utilized. More- over, in hyperglycemia produced immediately after glucose starvation, the elimination of lac- tate in tumors was decreased to negligible amounts; some even utilized lactate from the afferent blood. These observations imply the existence, in vivo, of several compartments in the neoplastic cell and that only in some of them does glycolysis occur with a special pri- ority. In an attempt to investigate the relation- ship between differentiation and the growth of neoplastic cell populations, Dr. P. Gullino and Dr. S. Roskes (NICHHD) have begun to em- ploy primary mammary tumors in rats. Pre- liminary results have shown that the majority of primary tumors from lactating hosts have a negligible capacity to produce lactose al- though it is known that mammary tumors pos- sess the enzymes involved in lactose synthesis and an appreciable level of activity can be demonstrated in vitro. It is Dr. E. Shelton's purpose to examine the normal and malignant transformation of cells. The free cells found in the peritoneal fluid of the mouse represent the "wandering cells" of the tissue spaces of the body (i.e., the extra- vascular wandering cells). These cells function by protecting the mouse from adverse effects of foreign materials or debris resulting from metabolism or death of host cells. In per- forming this function, the cells undergo changes in number and metabolism. However, little is known about the normal development of this cell population which must serve as the basis for a comparison of malignant cell be- havior. A study of the population dynamics of nor- mal free peritoneal cells has shown that the total number of peritoneal cells in the mouse rises from birth to a stable plateau at 4 months of age. The macrophage population reaches a stable plateau of between 2-3 x 10 6 cells at 30 days. The continued increase in total cells is due to the continued increase in total lym- phocytes. It is suggested that the increase in number of lymphocytes may be related to maturation of the immune system in the mouse. Major emphasis will be placed upon elu- cidating the fine structure of the peritoneal cells with reference to the age of the animal. Attention will then be turned to the study of the alterations in fine structure which are at- tendant upon growth in the diffusion chamber. As an example of another differentiating cell system, whole thymuses from new-born mice were grown in diffusion chambers implanted in thymectomized or intact, syngeneic or allo- geneic hosts of various ages. Whole mount preparations were made of the thymus tissue after it had grown for from 9-86 days. Thy- mic lymphocytes, granulocytes, macrophages and fibroblasts migrated rapidly from the ex- plant, the latter forming a sheet that covered the entire chamber. Viable lymphocytes were present in chambers removed from the host after 86 days. The striking feature of the cul- tures was the differentiation of the epithelium into sheets, clusters, and cords of cells remi- niscent of glandular epithelium. Whorls of cells resembling Hassall's bodies were occasionally seen. These epithelial cells were distinguished by the presence of large lipid-filled vacuoles and neutral mucopolysaccharide in the cyto- plasm, suggesting that the epithelial cells were engaged in secretory activity. In collaboration with Drs. E. L. Kuff and W. C. Hymer, Dr. Sheton has examined the fine structure of the free and bound ribosomes of mammalian cells, principally the RPC 20 plasma cell tumor of the mouse. Utilizing neg- ative and positive staining, the structure of these organelles has been visualized by electron microscopy in greater detail than hitherto pos- sible. The individual mammalian ribosome appears as a dual structure consisting of a sphere approximately 220 A in diameter NATIONAL CANCER INSTITUTE 15 (probably corresponding to a 50S ribosomal subunit) to which is attached a smaller struc- ture of variable morphology (probably corre- sponding to a 30S ribosomal subunit). The large 50S subunit is extremely stable but the morphology of the smaller subunit is variable. It has been seen as a flattened sphere, a cleft sphere, two small closely apposed spheres and various other shapes. The small subunit seems to be structurally unstable and instability may be related to the capacity of the smaller sub- unit to "accept" messenger RNA. Ribosomes are attached to membranes of the endoplasmic reticulum (ER) by the stable, 50S subunit. The unstable 30S subunit stands up and away from the membrane. The base of the 50S sub- unit appears to form an integral part of the ER membrane. Polyribosomes are formed by the joining together of individual ribosomes into rosettes or linear arrays. The patterns formed by the rosettes and linear polyribosomes as they set- tle out of suspension onto the membrane of the microscope grid have provided clues as to the way they are joined together. The evidence is very strong that the units of the polyribosome are joined together at the smaller subunit and that the polyribosome strand is flexible. Posi- tive stains of the polyribosomes provide evi- dence that the material holding the ribosomes together is a more substantial substance than the single strand of messenger RNA that has been postulated previously. Dr. M. Rechcigl, Jr. has embarked on stud- ies concerned with the selection and differen- tiation of cell populations of normal and tumor tissues. Understanding the interrelation- ships between cells requires systems with mini- mum heterogeneity of cell types. At present it is not known whether tumor preparations are mixed populations of tumor cells and whether variation in such populations influence results and interpretations by virtue of their varied biochemical characteristics. It had previously been shown (NCI-388, 1965) that the injection of pieces of tumor into the spleen of a compatible host gives rise to numerous metastases in the liver of the host. This technique has now been successfully used for separation of different cell lines. When pieces of tumor from HC-hepatomas (high catalase line) were injected into the spleen of OM/N rats, all liver metastases found showed high catalase activity (i.e., around 200 units). Pieces from the low line (LC hepatoma) had liver metastases with low catalase values (i.e., around 40 units). Freshly prepared mixtures from HC- and LC-tumor lines injected into spleen produced liver nodules possessing either low or high enzymatic activity. No intermedi- ate values were seen. Upon reinoculation into spleen, the nodules gave rise to liver metas- tases of either high or low activity, compar- able to the activity of the initial inoculated tissue. Similar results were obtained in stud- ies with mixtures of unrelated tumors and grown in different inbred or hybrid strains. Small pieces of a given liver nodule obtained from Fj hybrid experiments, transplanted sub- cutaneously into both original parent strains, would develop tumors only in one of the strains. Moreover, the developed tumors pos- sessed typical biochemical characteristics of the original tumors grown in these strains. All the evidence obtained so far implicates cloning as the selective process. On the other hand, it was established by Dr. Kuff that the small tumor nodules observed on the mesentary of mice early after intraper- itoneal transplantation of ascites plasma cell tumors (see NCI-366, 1965) do not exclusively represent clones derived from individual tu- mor cells. Transplantation of individual nodules, while feasible, does not presently appear to be a useful cloning technique for these tumors. Current studies by Dr. E. L. Kuff and coll- aborators are based upon the hypothesis that important regulatory mechanisms involved in cell reduplication and differentiation may have their basis not only in the molecular structure of the polyribosomal protein-syn- thetic units but also in the relationship of these units to the membranes of the endo- plasmic reticulum. Transplantable plasma-cell tumors, combining cellular proliferation with active synthesis of secretory protein, are par- ticularly suitable systems in which to study the functional activity of intracellular compo- nents. 16 ANNUAL REVIEW OF INTRAMURAL RESEARCH Cytoplasmic extracts of the RPC-20 plasma cell tumor, fractionated by sucrose density gradient centrifugation yield 4 major frac- tions: (a) a "membrane" fraction containing both microsomes with associated ribosomes and mitochondria, (b) membrane-free poly- ribosomes; (c) free monomeric ribosomes, and (d) soluble fraction. The particulate fractions have been examined by electron microscopy by Dr. Emma Shelton to verify their cytologi- cal composition. The kinetics of labelling ob- tained from in vivo incorporation studies with C 14 -leucine during incorporation times between 1.33 and 11 minutes suggested that protein synthesized on the free polyribosomes was rapidly transferred in vivo to the soluble fraction of the cell, while protein synthesized by the microsomes and mitochondria remained localized within these elements. In the RPC- 20 tumor, the free polyribosome fraction and the combined microsome-mitochondria frac- tion accounted for approximately equal pro- portions of the total cytoplasmic protein syn- thesis in vivo. These results indicated the existence of two biosynthetic compartments distinguishable in terms of the immediate distribution of their protein products. Accordingly, Drs. M. Wolf and L. Kedes have undertaken to determine whether a corresponding phenomenon could be observed during in vitro protein synthesis by the isolated fractions, and whether direct ex- perimental evidence could be obtained that the two compartments are concerned with the synthesis of a qualitatively different spectra of proteins. Tumors were pulse-labelled in vivo and the fractions isolated from them were in- cubated in vitro in the presence of soluble frac- ton, energy, and a complete C 12 -amino acid mixture. Under these conditions, 30-40% of the radioactive (nascent) protein of the free polyribosomes was transferred to the soluble fraction, the release phenomenon being time and energy dependent. In contrast, only 10% of the labelled protein of the membrane frac- tion (microsome plus mitochondria) appeared in the soluble fraction, and this transfer ap- peared to be non-enzymatic since it occurred equally well in the cold and the absence of energy. Control experiments have established that both fractions do, in fact, incorporate amino acid into protein under the in vitro conditions employed for the study of nascent protein release. The results are thus entirely in accord with those obtained in the in vivo ex- periments. The free polyribosome fraction of the plas- ma cell tumor has been studied in some detail by electron microscopy by Dr. Emma Shelton. The size distribution is generally larger than that reported for the polyribosomes of other types of tissue, with many polyribosomes com- posed of 15-20 monomeric ribosmal units and with no evidence that the polyribosomes con- tain any organized membrane structure. Sim- ilarly, chemical analysis has failed to reveal either phospholipid or neutral lipid. Since these polyribosomes are active in completion and release of nascent peptide chains, their protein synthetic activity may be independent of lipid components, in contradistinction to results reported with other cell types such as yeast and regenerating liver. Deoxycholate, a detergent in widespread use for isolation of polyribosomes from a variety of tissues, has a marked effect upon the free polyribosomes of the tumor, removing approximately one third of their total protein, without, however, removing the nascent protein. At the same time, the polyribosomes become more sensitive to ribonuclease action and less efficient in carry- ing out in vitro amino acid incorporation An analysis of the structural proteins of var- ious mammalian ribosomes by Dr. L. Kedes fol- lowed procedures established elsewhere but with plasma cell tumor ribosomes as the ini- tial objects of study. The complex acrylamide gel electrophoretic pattern of the ribosomal proteins (up to 35 discrete bands) was identi- cal in the case of 9 different tumors (each se- creting a different myeloma protein) growing in BALB/c mice. The tumor ribosomal pro- tein pattern was, in turn, the same as that given by the ribosomal proteins of normal BALB/c liver. However, the ribosomal pro- teins of mouse liver, rat liver, and rabbit retic- ulocytes could all be differentiated from one another on the basis of their disc electrophor- esis patterns. Comparison of the ribosomal pro- NATIONAL CANCER INSTITUTE 17 teins from inbred mouse strains has been ini- tiated. Efforts are being made by Dr. M. Wolf to test the biological activity of plasma cell tumor RNA upon intact cells in which endogenous messenger RNA synthesis had been blocked by treatment with actinomycin. Although ini- tial experiments seemed to indicate some ac- tivity (as judged by restoration of protein syn- thesis in the treated cells), reproducible results could not be obtained. The major problem ap- pears to be in finding suitable non-toxic substances which, upon complexing with the added RNA, will both protect it from enzy- matic degradation and facilitate its entrance into the target cells. Investigation by Mr. N. E. Roberts of the non-secretory subline of the RPC-20 plasma tumor has continued. It now appears (from immunological analysis of tissue fluids that were fortunately frozen during the time that conversion occurred) that the loss of the ca- pacity to produce specific secretory protein took place quite abruptly, possibly over a sin- gle transplant generation. Attempts to repro- duce the phenomenon by continued rapid transplantation of the producer line have been unsuccessful. Induction of the non-secretory subline by a viral infection of the cells has not been excluded. Dr. M. Rechcigl, Jr. has continued his in- vestigation of the physiological role of cata- lase and related enzymes and the mechanisms of their regulation in normal and neoplastic tissues. In collaboration with Drs. H. A. Hoffman and W. E. Heston, studies on the biochemical genetics of catalase have confirmed earlier findings that the level of liver catalase activ- ity in the mouse is controlled by a single pair of genes with low dominant to high. Ce has been suggested for the gene symbol. Present data indicate that the gene does not act di- rectly in the synthesis of the enzyme. The presence of the gene reduces catalase actitivy to one-half the normal amount rather than eliminating it completely and low activity is dominant to high in contrast to genetic con- trol over direct synthesis of enzymes. Further- more, the effect is apparently limited to the liver, implying that the gene must be provid- ing a regulator of the amount of liver catalase activity. This is in sharp contrast with acatala- semia in the Japanese, where the gene is ap- parently involved directly in the synthesis of catalase, for homozygous recessive individuals show hardly any catalase in all examined tis- sues. On the basis of preliminary data ob- tained by determining the rates and the kinet- ics of catalase synthesis and destruction in vivo, the "regulator gene" action causing the ob- served differences in the liver catalase activity in the investigated mouse strains is a result of the decreased rate of synthesis of the enzyme rather than the increased rate of its break- down. Experiments have been initiated to develop a histochemical technique which would permit a quantitative or semi-quantitative assessment of the relative amount of catalase present in individual cells. The general approach was to immobilize isolated cells in a thin agar sheet, lyse the cells in order to release the contents around the cells, and then specifically identify regions of high catalase by the ability of the enzyme to destroy peroxide, which serves as an oxidizing agent in the iodine reaction with starch. An assay capable of identifying cata- lase-containing cells as visible plaques has been developed but no quantitative information has yet been obtained. Preliminary studies using disc electrophor- esis indicate that a variety of tissues may contain several isozymes of catalase. The pos- sibility of having broken down the catalase protein into subunits cannot be overlooked though it seems rather unlikely. When the liver homogenate supernatants were heated with 3-amino-l,2,4-triazole, a drug known to destroy catalase, the electrophoretic pattern showed that one of the "catalases" was inactivated while the second catalase was still active. Nucleic Acids Section The objectves of Dr. W. Schneider's research are to isolate and identify the deoxyribose- containing compounds present in normal and cancer tissues, to determine the metabolic se- quences in which they are involved, and to study their relationship to DNA synthesis. 18 ANNUAL REVIEW OF INTRAMURAL RESEARCH Previous studies on normal liver and the Novi- koff hepatoma showed that the levels of dCDP- choline and dCDP-ethanolamine were 6 to 7 times as great in the tumor as in the liver. Studies on liver after partial hepatectomy now have shown that these compounds also increase in amount as early as 12 hours after the operation. A single determination on a pooled sample of hepatoma 5123, a minimal deviation tumor, showed that these compounds were also increased several-fold in this tissue. The fact that the concentration of these com- pounds increases in the liver shortly after par- tial hepatectomy, and is high in the minimal deviation hepatoma 5123 as well as in the highly malignant Novikoff hepatoma, provide further evidence for the importance of these compounds in growth, although an explana- tion for their increased concentration in grow- ing tissues remains obscure. The enxymatic synthesis of labeled phosphoryl choline, phos- choline, dCDP-ethanolamine, and OCDP-ethano- lamine has been completed and the results published. Previous work demonstrated that the phos- phoryl choline-cytidyl transferase of rat liver was activated by degraded phospholipids. This suggested to Drs. W. G. Fiscus and W. Schnei- der that a positive feedback mechanism was operative in lecithin biosynthesis and led to a study of the transferases. Experiments showed that a change in size of the enzyme occurred in the presence of activating phospholipid and indicated that an association of subunits was involved in the activation process. In addition, the activity of the transferase, in the pres- ence of increasing concentrations of CTP, showed a sigmoidal curve in the absence of activator. In the presence of activator the ac- tivity did not show this "lag" phase at low concentrations of CPT. Other experiments have shown that an inhibitor of the transfer- ase is present in rat liver particulate fractions and can be released into solution by boiling the particulate suspension. This inhibitory factor appears to be bucleotide in nature. If labeled CDP-choline is incubated with this fraction and a liver homogenate, the cytosine moiety of the CDP-choline is incorporated into a material which is also inhibitory to the trans- ferase and which may be a polynucleotide. These results suggest that CDP-choline is involved not only in phospholipid formation but may also be involved in the formation of polynucleotides. Of interest from this stand- point is the observation that dCDP-choline, but not CTP, CDP, or CMP, dilute the incorpo- ration of CDP-choline into the polynucleotide. Furthermore, the present studies indicate that the transferase belongs to the group of allosteric enzymes since its activity depended upon the presence of phospholipid activator, its conformation changed during activation possibly due to an association of subunits, its substrate concentration curve was sigmoidal in the absence of activator, and it was inhib- ited by a naturally occurring material which appears to be a polynucleotide. The work on DNA in isolated liver mito- chondria, begun last year by Dr. W. Schneider, was continued and some of the results have been published. A method was developed for isolating DNA from both nuclei and mitochon- dria in highly purified form. Tests of these DNA samples by buoyant density determina- tions in CsCl and by melting point determina- tions confirmed the results reported last year by direct base analyses and show that the guanine-cytosine content of the mitochondrial DNA was 2 to 3 percent lower than that of the nuclear DNA. In addition, these studies also showed that the mitochondrial DNA was of large size with a molecular weight of 8 to 9 million. The mitochondrial DNA also appeared to be more homogeneous than the nuclear DNA, as judged by the sharpness of the bands ob- tained in the CsCl centrifugations and the temperature range required to melt the nu- cleic acid samples. Another difference between the nuclear and mitochondrial DNA was in the rate at which labeled thymidine and de- oxycytidine were incorporated in vivo. Both of the precursors were found to be incorpo- rated to about 10 times as great an extent into liver mitochondrial DNA as into nuclear DNA in adult rats. More recent experiments have shown that although the incorporation into nuclear DNA was increased enormously 24 hours after partial hepatectomy, the incor- NATIONAL CANCER INSTITUTE 19 poration into mitochondrial DNA was unaf- fected. A study of the retention of label in the mitochondrial DNA showed that the specific activity of the mitochondrial DNA fell off rap- idly, with a half life of about 7 days. Preliminary experiments have also been car- ried out on the livers of rats fed the potent carcinogen, 3'-methyl-N,N-dimethylaminoazo- benzene (3'-MeDAB) or the non-carcinogen, 2-methyl-N,N-dimethylaminoazobenzene (2- MeDAB) for 14 and 28 days. The expected in- crease in liver mitochondria in the rats fed the 2-MeDAB and the expected decrease in liver mitochondria from rats fed the 3'-MeDAB were observed but the rate of incorporation of thymidine into the mitochondrial DNA did not seem to differ in these rats and in controls. The most significant finding of these experiments was a 2-fold increase in DNA in the 3'-Me- DAB liver mitochondria. It seems likely that the study of much shorter periods of feeding these dyes will be necessary to determine whether the mitochondrial DNA is affected by either dye. These studies on mitochondrial DNA pro- vide a new point of departure for experiments in cancer research. Since experiments per- formed here, as well as elsewhere, have shown that tumors contain fewer mitochondria and have decreased levels of mitochondrial en- zymes and that these changes in mitochondria occur in the liver in the earliest stages of chem- ical carcinogenesis, it seems logical to ask whether the point of attack of the carcinogen is at the mitochondrial DNA. Experiments on this point are in progress. During the past few months Dr. S. Lerner and Dr. W. Schneider have begun to examine by hybridization techniques the degree of com- plementarity that may exist between DNA and RNA isolated frcm rat liver mitochondria. Heretofore, efforts have been directed mainly toward the isolation and clean separation of DNA and RNA. If, however, mitochondrial RNA is found to hybridize with the DNA, this would suggest a transfer of information from the DNA, perhaps analogous to the RNA- mediated expression of information in protein synthesis, and would provide impetus for the investigation of possible autonomy for mito- chondrial biosynthesis which endogenous DNA might afford. Partial purification of TMP kinase free of TMP phosphatase activity was achieved by Dr. R. K. Kielley with successive steps involv- ing pH 4.8 precipitation, (NH 4 ) 2 S0 4 fraction- ation of soluble protein, and treatment with DEAE-Sephadex. TMP-phosphatase activity was totally ab- sent in the final product. The specific activity of the purified kinase is the highest reported from mammalian tissue, but the purified en- zyme is still contaminated with some ATPase. TdR kinase, diphosphokinase and myokinase activity, as determined by spectrophotometry, coupled enzyme assays. Other purification pro- cedures with Sephadex gel filtration and CM- Sephadex have been investigated and shown to be feasible. Removal of the protective sub- strate (2xlO~°mTMP) from the enzyme by passage through Sephadex G-25 led to in- activation, the degree depending on the time required for passage, suggesting a dissociable complex of the enzyme and protector. The kinetics of the time course of inactivation in- dicated a latent period lasting several minutes during which time an intermediate may be formed. It has been shown that TMP kinase, an enzyme which plays a key role in DNA synthesis, has a sulfhydryl requirement for stability, a requirement not detected in crude extracts of the enzyme. Pre-incubation expe- riments indicated that glutathione conferred increased stability to enzyme already pro- tected with TMP. It is noteworthy that stability in the presence of ATP occurred only when GSH was present. A tentative explanation might be that the ac- tual stabilizing nucleotide bound to the enzyme is the reaction product itself, TDP, rather than the substrate, TMP, which appeal's to be less effective. The GSH effect may serve to increase the binding strength of the enzyme for TDP to such an extent that the "bound TDP" is unavailable for further phosphorylation even though diphosphokinase and ATP are present to convert TDP to TTP. The nature and extent of the biochemical re- actions by which nucleic acid precursors are metabolized in cultured nonmalignant and neo- 20 ANNUAL REVIEW OF INTRAMURAL RESEARCH plastic cells are being studied by Dr. J. Rother- ham, particularly with respect to their possible significance in nucleic acid synthesis and neo- plastic transformation. It has been demonstrated that a strain of C3H mouse embryo cells (Tissue Culture Sec- tion, Laboratory of Biology) grown in chemi- cally-defined medium has a nutritional require- ment for thymidine. The thymidine can be replaced by doexycytidine or 5-methyldeoxycy- tidine but not by uridine, deoxyuridine or cyti- dine. Thus, it appears that the deoxynucleo- sides which were utilized have a significant metabolic function for this cell strain and that biochemical reactions by which the latter three nucleosides can be converted to the for- mer were not taking place. An explanation for these findings is being sought frcm tracer ex- periments in which the cells are exposed to C 14 -labeled nucleosides in the medium. From isolation of the nucleoside products found in the medium and in the DNA or RNA of the cells and from a determination of the approxi- mate extent of conversion of precursors to product it can be concluded that there is a rapid deamination of decxycytidine and/or phosphorylation followed by deamination and dephosphorylation — and that the deoxycyti- dine can also be phosphorylated and utilized for synthesis of DNA-deoxycytidine and/or thymidine. As part of a continuing study of the func- tion and relationships of the structure and se- quence of nucleic acids, the nucleotide distri- bution in nucleic acids, the development of new fractionation procedures, and the prepa- ration and characterization of nucleotide se- quences are under investigation by Drs. G. W. Rushizky and H. A. Sober. Various protein fractionation techniques are utilized for the isolation, from sources such as micro-organ- isms, of nucleases (both of RNases and DN- ses) with hydrolytic activities directed toward specific nucleotide linkages. Since DN- ases, in particular, are quite unstable, new techniques have to be developed. A group of DNases have been characterized and found to consist of a mixture of at least 2 endonucle- ases and 1 phosphodiesterase. The latter en- zyme hydrolyzes not only DNA but also RNA. Separation of the 2 endonucleases by standard protein fractionation techniques has not met with success since loss of the major portion of the enzymatic activity resulted. These DN- ases are obtained from the mold Aspergillus oryzae, which is also the source of the Very useful enzymes, RNases T 1 and T 2 . Other pre- viously described RNases have been prepared from B. subtilis, B. cereus, and various molds, and the possible presence of new DNases has been investigated. A RNase-free phosphatase from E. coli C90 has been prepared. There is always a need for new methods for the separation, and characterization of oligo- mers of mixed base ratios ranging in size from 10-100. To date, a supply of such large olig- omers is one of the limiting factors in design- ing such procedures. As a source of large oli- gonucleotides, MS2 phage RNA (chain length of 3,000 nucleotides) was purified in good yield. Octa- and decanucleotides from complete RNase T, digests of MS2 RNA were fraction- ated according to their (Ap + Cp): Up content by mapping or column chromatography at pH 2.7 in 7 M urea. Penta-, hexa-, and heptamer mixtures of identical Up (and Gp) composition were separated according to Ap:Cp ratios by the same procedures but at pH 4.3. Such oli- gomers have been examined for hyperchromy and optical rotatory dispersion. Even larger oligomers have been obtained by digestion of of MS2 RNA after it had been absorbed on DEAE-cellulcse, yielding groups of oligomers of chain length 14 to 400 with 51 to 84% re- covery of the starting material. Oligomers de- rived in this manner by Micrococcal nuclease digestion had base ratios very similar to those of the original MS2 RNA. On the other hand, oligonmers with Cp:Gp ratios of 2:1 were ob- tained when B. subtilis RNase was used as the enzyme (the ratio is approximately 1:1 in MS2 RNA). Preliminary results indicate that this procedure also is applicable to the isolation of large oligomers from DNA. These procedures have been used to prepare pure oligonucleotides of known chain length and nucleotide sequence and has permitted a study of their chemical, physical and biological properties. Oligomers so prepared have been used in studies of nuclease specificity, neigh- NATIONAL CANCER INSTITUTE 21 bor-neighbor interactions in single oligonucle- otide strands, and as test materials for com- putational analysis of nucleic acid spectra. Deoxynucleo tides, greater than or equal to 7 in chain length, have produced reactivation of the immune response in thymectomized, X-ray irradiated adult mice in exploratory ex- periments with Dr. M. Feldman (Weizmann Institute of Science). Attempts to duplicate the result in in vitro spleen cultures have so far been unsuccessful. Protein Chemistry Section The polylysine-polynucleotide system was chosen by Dr. H. A. Sober as a simple model of nucleoprotein interaction. This interaction is known to affect the biological function of the nucleic acid. A study of the nature of the binding forces, stoichiometry, and specificity of this interaction should shed some light on the mechanism by which a basic protein "rec- ognizes" a portion of the nucleic acid structure and affects its properties. Polylysines react on a molar basis with RNA to form insoluble complexes at low salt concen- trations and neutral pH. Soluble complexes are formed, however, at low polylysine-to-RNA ratios. The soluble complexes are susceptible to hydrolytic degradation by specific and non- specific ribonucleases with release of soluble nucleotides. Digestion continues until the for- mation of a precipitate with a nucleotide-to- lysine ratio of one. After dissociation of the pre- cipitated complex and separation of its com- ponents, the "protected" nucleotide chain is again susceptible to cleavage by the original enzyme. The protected nucleotide sequence is shorter than that of the original RNA. Thus, treatment with polylysine, n = 100, results in a protected RNA sequence of n = 99; the use of oligolysine (n = 13) results in a RNA seg- ment of n = 16. Oligolysines smaller than the heptamer do not form enzyme-resistant com- plexes. Present evidence indicates that the nucleo- tide composition of the protected sequence may be markedly different from that of the parent molecule and that overlapping nucleo- tide sequences may be obtained by this pro- cedure. While no base specificity has been found with heated or "denatured" RNA, with "na- tive" or unheated RNA, the protected RNA segments contain an altered proportion of gyanylic (G) and cytidylic (C) acid residues as much as 76% (G + C). Measurements by Dr. S. Latt have shown that the binding of oligolysine to MS2 RNA, Poly (I + C), and Poly (A + U) was reversible as long as the polynucleotide-oligolysine com- plex remained soluble. Preliminary data with the single homopolymers show precipitation even at very low free oligolysine concentra- tions. Binding of oligolysine to polyribonu- cleotide decreases markedly with increasing ionic stength, and is virtually nonexistent in 1.5 M NaCl, indicating that the major if not sole binding force is electrostatic. Binding strength increases markedly with oligolysine chain length. An equation for reversible bind- ing of a linear oligomer to a much larger lin- ear polymer was derived and experimental binding curves obtained were of the form pre- dicted. Another approach to nucleoprotein interac- tion is a new project by Dr. 0. W. McBride wherein the role of nucleic acid-bound proteins in the regulation of the transcription and translation of genetic information will be ex- amined. Studies have been initiated recently to determine the most suitable procedures for isolating soluble chromosomal nucleoproteins from rat liver in a physical condition closely re- sembling the native state of these nuclear components. A variety of modifications of two basically different procedures are being exam- ined. These methods involve either the direct isolation and purification of the chromosomal apparatus from aqueous solutions or isolation and extensive purification of whole nuclei prior to isolation of soluble nucleoproteins. The criteria which have been established for a satisfactory procedure are the isolation of a soluble nucleoprotein preparation without deg- radation or with minimal, controlled, and re- producible degradative methods and the ab- sence of dissociation, association or exchange of chromosomal components with either cyto- plasmic or nuclear non-chromosomal molecules. The possible exchange between soluble his- tones or non-histone proteins as well as be- 22 ANNUAL REVIEW OF INTRAMURAL RESEARCH tween soluble ribonucleic acids and nucleopro- teins will be examined under the specific ionic conditions of the isolation procedure with iso- topically-labeled material. Rat liver nucleopro- teins will be fractionated by a combination of several independent methods utilizing differ- ences in mass, buoyant density, electrostatic charge, and surface characteristics of these particles. The fractions will be examined for differences in ribonucleic acids, histones, non- histone proteins, and lipids associated with these DNA segments. Covalent and non-cova- lent associations between these various com- ponents will be investigated to obtain some in- sign concerning the particular component or components which may be responsible for a specific association with the nucleic acid. Attempts to purify the Moloney virus, which is known to produce a generalized lymphocytic neoplasm in mice and rats were made by Dr. C. W. Lees and Dr. H. A. Sober with the col- laboration of Dr. J. B. Moloney (Laboratory of Viral Biology). Fractionation of viral ac- tivity was attempted by ion-exchange chroma- tography, Sephadex chromatography, and the aqueous phase liquid-liquid partition system of Albertsson. While apparent purification of sev- eral-fold was obtained, reproducibility of the assays was inadequate to confirm these results or to permit the desired estimate of the re- covery of activity. These exploratory studies were designed to indicate the range within which fractionation procedures must remain in order to obtain good yields of active virus. Because of the large number of fractions which develop during fractionation, a "rapid" and reasonably quantitative assay procedure must be available. The 4-week bioassay used in these experiments does not satisfy these require- ments. Dr. R. W. Hartley, Jr. with the collaboration of Dr. C. W. Lees has been ivolved in the puri- fication and characterization of an extra cellu- lar ribonuclease of B. subtilis by physical and chemical techniques and the development of methods of culture and genetic analysis so that the organism and its enzyme product may be applied to a long range study of enzyme struc- ture, function, and synthesis. A strain of B. subtilis has been derived from strain H that will grow in a synthetic medium and produce ribonuclease on a continuous basis. This was achieved by an evolutionary process in a continuous fermenter. Experience with a small (100-200 ml per stage) two stage continuous fermenter has established the feas- ibility of producing crude enzyme in a contin- uous and semi-automatic fashion in such an apparatus. Production at a rate of at least 100 to 200 mg of crude enzyme per day is envis- aged with a 14 liter per stage set-up cur- rently available. Amino acid analysis of the purified enzyme has confirmed the absence of sulfur amino acids except for 0.2 moles per mole of methi- onine which is probably part of a contaminant. All of the weight and nitrogen of the enzyme was accounted for by the amino acid recov- ery. Dry wevht, nitrogen and U.V. absorp- tion determinations gave a nitrogen content of 17.7% and an E 1%/1 cm at 280 m/iof 20.9 The high extinction is in line with the high tryptophan content. Quantitative N-terminal analysis by dinitrophenylation in two experi- ments showed 0.7 and 0.9 mole of N-terminal alanine per mole of enzyme and no other N- terminal residues. Alanine alone was also found by the DNS ("dansylation" with di- methylnapthalene-sulfonyl chloride) method. The molecular weight of 10,700 was deter- mined by ultracentrifugal analysis. The enzyme is absorbed in considerable quan- tity by glass and other surfaces from water and many buffer systems. This is largely pre- vented by di- and tri-valent cations, especially iron, by amines such as choline and cetyltri- methylammonium bromide, and, fortunately, by 0.1 M ammonium bicarbonate which will quantitatively release enzyme adsorbed from 0.1 M Tris hydrochloride solution but not en- zyme adsorbed from water or washed with water. The dry enzyme is insensitive to heat (100°). In solution (0.1 M NH 4 HC0 3 ) how- ever, 90% of the activity is lost within an hour at 90° with significant loss of activity even at 60°. The protein remains in solution, but moves as a discrete band on disc electrophore- sis at pH 4.5 behind that of the active enzyme, suggesting that the loss of activity is due to NATIONAL CANCER INSTITUTE 23 hydrolysis of amide groups. The inactive en- zyme still reacts with a specific B. mbtilis in- tracellular ribonuclease inhibitor, preventing inhibition of added active enzymes. Activity remains constant in 1 2V HC1 at 0° up to 24 hours but is largely lost in one hour at room temperature. In 1 N NaOH at 0°, about 30% of the activity remains at 1 hour and very little remains at 4 hours. Several ribonuclease-negative mutants have been isolated as well as a larger number of protease- and amylase-negative mutants. Dr. E. A. Peterson and Dr. W. H. Evans have undertaken to develop methods for the frac- tionation of normal and leukemic blood and bone marrow leukocytes as a basis for the study of the fundamental biochemical changes that underlie the maturation of normal leu- kocytes and the factors that lead to the arrest of maturation in leukemia. Several stages of maturation in the ery- throid and myeloid series of guinea pig bone marrow cells have been partially resolved by sedimentation in a dilute density gradient at unit gravity in a simple apparatus. Besides the clear separation of erythrocytes from the my- eloid cells, a useful separation of blasts, neu- trophil myelocytes, and mature neutrophils was achieved, although the overlap of peaks left something to be desired. Lymphocytes emerged with the erythroid cells but were well separated from all other types except blasts. Peak enrichments of given cell types ranged from 3- to 15-fold. The time required for such resolution varied from 5 to 20 hours at 4°. However, the myeloid cells were 92 percent viable after 20 hours, as determined by the trypan blue method. Erythrocyte precursors were only 41 percent viable after this period and 67 percent viable after 5 hours. Although migration was almost linear with time, useful resolution did not increase in proportion to the time allowed for sedimentation under the same conditions, indicating an overlapping hetero- geneity of the cell classifications with respect to sedimentation rate. The medium employed was a Krebs-Ringer phosphate solution from which calcium and magnesium were omitted and to which 0.01 percent polyacrylate was added. These changes were necessary to pre- vent reaggregation of the cells after they were dispersed. The presence of polyacrylate did not affect the viability count. Studies of the behavior of guinea pig bone marrow cells on passage through slowly rotat- ing horizontal columns of open cell polyure- thane foam have been continued. Enzyme treatment of the cells in order to prevent non- specific retardation of a considerable portion of the myeloid cells in columns of foam having 100 pores per inch was mentioned in last year's report. A similar effect has now been achieved by dispersing the cells in a medium contain- ing polyacrylate and treating the foam with the same substance. Whether it is bound to the cell or merely cleans it (of, e.g., nucleopro- tein) is now known at present, but polyacry- late is bound to the foam. Similarly, these foams have been shown to bind polyethylene- imine and tetraethylene-pentamine. Moreover, foams treated with these polyamines strongly adsorb guinea pig bone marrow cells, includ- ing the red cells which pass unadsorbed through untreated foams under the same con- ditions. Thus, the foams can be converted readily into ion exchanges. Presumably, the polyacrylic acid chain is bound when one or more of its many carboxyl groups engages in an acid-exchange reaction with the polyester moiety of the particular polyurethane foams used in this work. The polyamine chains are similarly bound when one or more of the amino groups cleaves the polyester to form an amide bond. Theoretically, many substances can be attached to the foam in this way, pro- viding the possibility of readily tailoring the nature of the surface for a wide variety of pur- poses. Among others successfully bound were proteins such as histones, protamine, gamma- globulin, and albumin. The ability to bind gamma-globulin to the foam columns suggested the feasibility of uti- lizing immunological affinity for the separa- tion of cells, provided the bound gamma- globulin retained its activity. In collaboration with Dr. Michael Mage (NIDR) a study of such a possibility was undertaken, using the circu- lating red cells of the guinea pig as a model antigen. These experiments were carried out at room temperature (25°) in order to pro- 24 ANNUAL REVIEW OF INTRAMURAL RESEARCH mote a reasonably rapid immunological reac- tion. It was found that the circulating red cells were bound to untreated foam, presumably through the sialic acid on their surfaces, but they were readily dislodged by streams of li- quid, by very low-power sonication, or by squeezing the foam. Treatment of the foam with normal rabbit gamma-globulin prevented this binding of red cells, apparently by cover- ing the reactive sites. In contrast, foams treated with specific rabbit gamma-globulin (anti-guinea pig RBC) at the same protein concentration adsorbed the red cells entering the column and became bright red. Again, the bound red cells could be completely removed from the foam by the low shear applied by the moving liquid when the foam was squeezed. It is not known, as yet, whether the release of the cells involved dissociation of the antigen- antibody complex or cleavage of some other linkage, but the cleaned foam could be used re- peately to bind red cells. Similarly, the foam protected with normal gamma-globulin retained its protection through repeated uses. The spe- cificity of this interaction has not as yet been completely established, but sheep cells were not bound by the anti-guinea pig RBC foam. On the other hand, an anomalous low-inten- sity interaction between rabbit RBC's and nor- mal rabbit gamma-globulin and a strong in- teraction between rabbit RBC's and normal bovine gamma-globulin have been observed. The discovery that the polyester-based poly- urethane open-cell foams can readily be given a wide range of ion-exchange and immunolog- ical properties opens the way for the develop- ment of procedures for cell separation based on such principles. Studies of the nature of cell surfaces should also be aided, and appli- cations in basic immunological investigations are foreseen, particularly in view of the po- tential ability of this type of system to detect and measure monovalent as well as multivalent antibodies. Studies on the basis of heterogeneity of an- tibody formed to single, well-defined antigens have been performed by Dr. S. Schlossman (Beth Israel Hospital, Boston, Mass.) and Dr. H. A. Sober. A homologous series of com- pounds was used because the chemical defini- tion both in position of the hapten and in pep- tide chain length provide obvious advantages over less well-defined materials in studies deal- ing with the chemical basis of the immune response. Immunogenicity was observed in Hartley and strain 2 guinea pigs with a, N-DNP-hepta-, octa, and nona-L-lysine, whereas smaller a, N-DNP-L-lysines were not immunogenic. The L configuration and the presence of a hapten were also required for immunogenicity in this system. The same antigen, e.g., aiV-DNP-octa- L-lysine, induced the formation of both de- layed and immediate sensitivity. Using chem- ically defined «-DNP(Lys) n BuAm peptides, related peptides, and hapten-substituted pro- teins, only the immediate skin response (Ar- thus) could be elicited with hapten-substituted tetra-, penta-, or haxmers, whereas both imme- diate and delayed skin responses could be pro- voked by the octamer or nonamer. The hapten is an integral part of the determinant for both immediate and delayed skin reactivity, since poly-L-lysine was unable to elicit either reac- tion in sensitized animals. Immediate-type cross reactions occurred whenever the sensi- tizing and test antigen shared a common hap- tenic determinant. In contrast to this, in this system, delayed-type cross reactions occurred only when the test antigen and the sensitizing antigen contained both a large oligo-L-lysine carrier as well as the same haptenic determi- nant. These observations imply that the media- tion of the delayed response requires a larger determinant than is necessary to elicit the im- mediate response. High affinity antibody as the mediator of the delayed response is not con- sidered a satisfactory explanation of the ob- served phenomena. However, the fact that the ability to elicit the delayed response parallels the immunogenic capacity of these pepties suggests that the delayed response may re- quire the continued biosynthesis of antibody and may be analogous to a local in vivo sec- ondary response. The properties of poly-a-amino acids, model substances closely analogous to proteins, are being studied by Dr. H. A. Sober and M. C. Otey with the collaboration of Drs. A. Berger, NATIONAL CANCER INSTITUTE 25 A. Yaron and E. Katchalski (Weizmann In- stitute of Science, Rehovoth, Israel). Oligoly- syl peptides, in particular, are useful model compounds for the investigation of the inter- action and functions of the basic proteins. These histone analogs, now available in chain lengths ranging from 2 to 25 residues long have been used in a number of studies de- scribed above, namely: interaction with nu- cleic acids and polynucleotides to form nu- clease-resistant complexes, where the length of RNA segment is directly related to the length of oligolysine used; after mono- and di- hapten addition, provide a chemically-defmed series ranging from no- to full antigenicity which has been used to define the antigenic determinants of delayed and immediate skin sensitivity; optical rotatory dispersion studies with Dr. G. Fasman (Brandeis University), examining the effect of chain length on pro- tein conformation; and the variation in chemi- cal properties, such as the apparent pK of the e-amino group, the color yield with ninhydrin, and adsorption to polyanionic substances as the oligopeptide is extended. It has long been apparent that metals pres- ent in blood and tissues function in association with proteins, and whenever it has been pos- sible to examine this association, areas of ma- jor biochemical importance have been uncov- ered. By collaboration between the Laboratory of Biochemistry, NCI, and the Biophysics Lab- oratory (Peter Bent Brigham Hospital, Har- vard Medical School, Boston, Mass.), a com- bination of spectrographic analyses and protein fractionation has been developed which makes it possible to isolate and study the asso- ciation of metals and proteins in much greater detail. Several new zinc proteins have been recog- nized in serum by Dr. S. R. Himmelhoch using these procedures. In view of the abnormality of serum zinc concentration reported in Laen- nec's cirrhosis and in leukemia, the existence of these three zinc proteins is of special inter- est. In Laennec's cirrhosis at least, preliminary studies indicate that serum zinc alteration in this disease is a reflection of changes in the relative amounts of the firmly bound zinc moieties. Studies are in progress to complete the isolation of these proteins and discern their functional role. Earlier studies, using limited methods of protein fractionation had shown that a pro- tein could be prepared from human or horse renal cortex which contained about 30 resi- dues percent cysteine, and 5% cadmium and 2% zinc by weight. By application of the techniques developed from the above-men- tioned collaboration, Drs. S. R. Himmelhoch and N. H. R. Kagi and B. L. Valley (Harvard Medical School) have been able to show that this protein, metallothionein, is but one mem- ber of a family of at least four similar pro- teins of related amino acid composition, that these proteins occur in the supernatant frac- tion of renal cortical cells, and that they con- stitute 3% of the total protein in this source. Methods for preparation of 1 gram quantities of each of these components from both equine and human sources with quantitative recover- ies have been developed. Two additional cad- mium binding moieties, one of larger and the other of smaller molecular weight than the "metallothioneins" have been identified. To- gether these proteins account for 98% or more of renal cadmium content. Other metals of great interest in humanpathology, including lead and mercury, have been found in asso- ciation with seme of these moieties. The quan- titative amino acid composition of these pro- teins has been determined and demonstrates distinct differences in detail, but a common high cysteine content. The metallothioneins, a family of peculiar cadmium- and zinc-containing proteins, repre- sent an important portion of horse and human renal cortical cellular protein. Their unique structure, with every third amino acid pos- sessing a reactive sulfhydryl group, suggests a role in renal transport or detoxification pro- cesses. Their unusual metal content and small molecular weight provide a unique opportunity to study metal protein interaction in a defini- tive manner. Its role in human disease is under study. Fractionation of human serum on DEAE- cellulose had originally suggested that magne- sium might be associated with proteins of gamma-globulin mobility. However, further 26 ANNUAL REVIEW OP INTRAMURAL RESEARCH studies by Dr. N. A. Cummings point to a loose binding between the metal and proteins, i.e., the existence of a magnesium-protein complex rather than a metalloprotein per se. By com- bining analytic and preparative ultracentri- fugation, in collaboration with Dr. E. L. Kuff about 70% of the serum magnesium is found to be free, with the remaining 30% in a weak magnesium-albumin association. The informa- tion obtained indicates that magnesium in se- rum exists in a locsely-bound protein form, capable of being transported and easily re- leased by mild changes in salt concentration or pH. Cryoglobulins isolated by cold precipitation from sera have been further purified by gel filtration by Dr. N. A. Cummings and have been examined by physico-chemical methods in or- der to study the phenomenon of cryo-precipi- tation. The solubility characteristics of 7S-cry» oglobulins are similar to those of normal IgG globulins except for temperature effects. Cry- oglobulin solubility is a linear function of temperature. Although hydrogen-ion titrations failed to reveal significant differences in sur- face charges at different temperatures, meas- urements of viscosity, sedimentation, and sed- imentation equilibrium indicated a probable unfolding or anisotropic swelling of the mole- cule with rising temperature. Further studies to elucidate conformational changes will be undertaken, including optical rotatory disper- sion. LABORATORY OF BIOLOGY During the year the Laboratory of Biology has continued to make significant advances in the basic areas of biologic research as related to cancer. The program has been expanded particularly in the areas of protein chemistry, electron microscopy, experimental pathology, and cytogenetics. Progress is being made in bringing in new personnel to strengthen certain promising areas of research. We have also lost a few members. Dr. Morris K. Barrett retired in July and Dr. Margaret K. Barrett resigned in Au- gust. Dr. Charles Nicoll resigned in January to return to the University of California in Berke- ley, where he accepted a position in the De- partment of Physiology. Dr. William T. Hall, an electron microscop- ist, joined the Laboratory as a biologist in Sep- tember and now has his electron microscope and laboratory set up and has an active re- search program underway. Dr. Ettore Appella, a protein chemist, got his A.C.S. Fellowship renewed and joined the staff in the Carcino- genesis Section in September. He has been very actively engaged on the amino acid se- quences of the myeoloma immunoglobulins that Dr. Potter has been studying. Request for his appointment next September as a Visiting Associate has been submitted. Dr. Ram Par- shad, a cyto geneticist, joined the Tissue Cul- ture Section in November as an International Fellow. He is making significant contribution in studying the chromosomes of their cells in culture. Three new Research Associates joined the Laboratory in July, Dr. Hewes Agnew who is working with Dr. Law; Dr. Frederic Mushin- ski who is working with Dr. Potter; and Dr. Gerald Mackler who is working with Dr. Evans. Dr. John T. Mitchell, a developmental biolo- gist, is expected to join the staff of the Tissue Culture Section in May as a Staff Fellow, and appointment of Dr. Raymond Gantt, a bio- chemist, also as a Staff Fellow in Tissue Cul- ture has been approved for September. Dr. M. Ben-David from Hebrew University has been appointed to the staff of the Laboratory as a Visiting Scientist to report July 1st. He is an endocrinologist who is replacing Dr. Nicoll. His principal interest is prolactin and we hope to investigate the role of this hormone in mammary tumorigenesis. Two new Reserach Associates, Dr. Ruffner and Dr. Burstein, are joining the Laboratory July 1, replacing Dr. Granner and Dr. Lyon. This will make a total of 72 on the Staff of the Laboratory. No attempt is made to mention all findings in this summary, but examples are chosen to illustrate the scope of the program of the Laboratory under the following headings. The Thymus in Immunology Dr. Law is continuing the characterization of the thymic humoral factor that is respon- sible for initiating and maintaining lymphoid NATIONAL CANCER INSTITUTE 27 cell homeostasis and immunologic competence. This has been demonstrated in his studies on effect of neonatal thymectomy. One new ob- servation is that spleen cells from C57BL mice produce "runt disease" when injected into strain A mice because of the graft versus host reaction, but if the spleen cells are from a thymectomized C57BL they lack the immuno- logic competence to induce the graft versus host reaction. Syngenic thymic grafts in thy- mectomized C57BL mice restore this compe- tence of the spleen cells, but dissociated thy- mic grafts do not. The relation of this area of research to viral carcinogenesis is summar- ized in the next section. Tumor Viruses The observation that neonatal thymectomy can increase oncogenesis by certain viruses has been extended by Law, Ting, Agnew, and col- laborators. Strain A mice that have never been shown to develop tumors of the salivary glands when infected with polyoma virus do show such neoplasms as well as those of bone, mammary, hair follicle, and subcutaneous tis- sue when the infection with virus is preceded by neonatal thymectomy. Although the Mo- loney murine sarcomogenic virus induces tu- mors in mice it does not in newborn intact rats, but when the rats were neonatally thy- mectomized and then inoculated with the virus 30 to 50 percent of them did develop tumors at the site of inoculation. Neonatal thymec- tomy did not increase but actually reduced the occurrence of mammary tumors in C3H fe- male mice with the mammary tumor virus but made no difference in the occurrence of MGA induced sarcomas in C3H or C57BL mice. Furthermore, neonatal thymectomy has made no difference in the occurrence of MCA in- duced lung tumors in suceptible (C3HfxA)F! hybrid mide or in resistant (C3HxB)F 1 hy- brids, as shown by their collaborative study with Heston. These observations would indi- cate that the thymus is responsible for an im- munologic mechanism of the homograft type that causes target cells to resist oncogenesis by certain viruses such as polyoma and MSV, but with certain exceptions as the MTV. It is suggested that no virus is involved in MCA induced sarcomas and lung tumors and thus they are not influenced by thymectomy. Andervont has continued his observations of the activity of the mammary tumor virus MTV from strain RIII mice as compared with that of MTV from C3H mice when transmit- ted normally through the milk. The RIII MTV is a relatively weak virus that occasionally disappears or becomes inactive in RIII mice. When transmitted to agent free C3H mice the RIII MTV may also disappear, and when re- tained does not increase in activity. The high- ly active C3H virus has never been observed to disappear in C3H mice, but when intro- duced into RIII mice it can either remain ac- tive or disappear from them. Ultrastructure Cytology Dr. William Hall has his modern electron microscope laboratory set up and his research program underway. In studying the new strain DD with its premalignant, ormone responsive, plaques he has found that the malignant mam- mary tumors contain an abundance of both A and B mammary tumor virus particles, and the plaques also contain both A and B particles although not nearly so abundantly. In study- ing some of Dr. Evans' C3H mouse embryo cells in chemically defined media, he has ob- served many virus or virus-like particles which are as yet unclassified. Hepatomas in (C3HxY) Fj mice have not exhibited virus particles. They have, however, exhibited significant anomalies of the microbodies of the cells which represent the uricase component. Cells of spon- taneous and induced pulmonary tumors in our mice have failed to show any infectious vi- ruses with the E.M. Plasma Cell Tumors The plasma cell tumor is used extensively in the Laboratory by Potter and his group in studies on cell differentiation. Doctors Potter, Appella, and Mushinski have made consider- able progress in characterizing the gene-gene product relationships for the heavy chain locus in the mouse. In the mouse there have now been defined 7 immunoglobulin structural genes including 5 heavy chain genes: M, A, 28 ANNUAL REVIEW OF INTRAMURAL RESEARCH F, G, and H, and 2 light chain genes. A single plasma cell tumor utilizes only one heavy chain gene and one light chain gene, the others be- ing permanently "turned off". Structural var- iations resembling those in the light chains have been found in heavy chains. Iso-antisera have been produced that are specific for indi- vidual myeloma proteins and these provide val- uable tools in the search for antibody mole- cules that might resemble myeoloma proteins. Close linkage with no recombinants has been demonstrated between two heavy chain gene types. In the study of amino acid sequences the amino and carboxyl terminal sequences of five kappa light chains have been obtained. Structural analysis of the Fc fragments de- rived from mouse gamma and eta chains has shown the presence of twelve normal tryptic peptides indicating these genes are probably duplication products. This work has relevance to the understanding of the mechanism of the differentiation process and the mechanism of antibody formation. Mushinski is delving further into the pro- tein synthesis in these tumors using radio- active tracer techniques. He is investigating transfer RNA as the mediator of controlled amino acid ambiquities in these plasma cell tumors. Mclntire has been studying the plasma cell tumors and related reticulum cell tumors with more comparison to the multiple myeloma of man. In studying the macroglobulins of one tumor he observed the incorporation of a com- pletely different type of light chain into the immunoglobulin of the mouse, while formerly only one light chain had been shown. This is in line with human beings where two types of light chains have also been recognized. He has been studying renal disease in relation to the Bence Jones proteins produced by these tumors. Of 123 tested for Bence Jones pro- teins, 56 or 45 percent were positive, which is higher than the incidence cited for human beings. The kidneys associated with these tu- mors show cast formations in the convoluted tubules. Some reticulum cell neoplasms of the mouse other than plasma cell tumors produce these paraproteins and some of these are un- der study. Mclntire has studied plasma cell carcino- genesis in germ free mice with a minimum of antigenic stimulation, an undeveloped lym- phatic system, few plasma cells, and low level of immunoglobulins, and has observed in these mice a delay in plasma cell tumor develop- ment. Most of the tumors that arise are of other reticulum cell types. In this regard, thy- mectomy apparently has no influence on plas- ma cell carcinogenesis. Biochemical Genetics Hoffman has been studying the urinary pro- teins of the mouse by immunochemical meth- ods, agar gel double diffusion, and immuno- electrophoresis. Thirty-five inbred strains and substrains have been classified according to two different urinary protein types, Up-l a and Up-1\ In crosses between two prototype strains for type la (strain SWR) and type lb (strain C57BL/6) the F x shows a combi- nation of the two types and the F 2 segregates in a typical 1:2:1 ratio of the two parental types and the F a type. This indicates that these two urinary proteins are under the ge- netic control of a pair of co-dominant alleles at a single locus. Segregation ratios in first and second backcross generations have con- firmed this type of inheritance. By using sev- eral spearation techniques the la and the lb protein complexes have been isolated and purified. Hoffman has built up a linkage-testing stock of mice with 25 marker genes on 15 of the 20 chromosomes. With this stock he hopes to locate on specific chromosomes these genes for biochemical traits including low liver cata- lase, Ce, immunoglobulin-1, Ig-1, urinary protein, Up-1, and also a sparse coat texture gene, Ca De . Hoffman has also been identifying serum proteins of the rat. He has found that some of the rat serum proteins cross react with those of the mouse, whereas, others do not. All of this is directed towards building up a background of knowledge of biochemical genetics in the mouse that can later be related to the genetics of cancer in the mouse at the biochemical level. NATIONAL CANCER INSTITUTE 29 Genetics of Tumors Heston and collaborators have continued this study of effect of specific genes of the mouse on occurrence of tumors. The allelic genes lethal yellow, A y , and viable yellow, A Ty , have received greatest emphasis. Both increase occurrence of a number of tumors including mammary tumors, hepatomas, and lung tumors. It has been shown that the lethal yellow gene increases occurrence of mammary tumors independently of the mammary tumor virus, and studies of reciprocal transplantation of ovaries and of hypophyses show that its effect is not manifested through control of hormonal stimulation from either of these organs. It, therefore, appears that the gene is controlling the response of the mammary gland cell. In a study of hepatomas in reciprocal hy- brids between strain C3Hf and strain YBR with the A y gene it was observed that the A y gene, sex, and the strain of mother all could influence the occurrence of hepatomas, caus- ing the incidence to vary from zero in one group to 100 percent in one with intermediate inci- dences, depending upon the combination of these factors in the intermediate groups. How- ever, this was all closely correlated with the effect of these factors on normal growth. Progression of mammary tumors in mice is being studied through premalignant, hormone responsive, mammary plaques that occur in the unusual strain DD. The most significant observation on these plaques during the year is that whether or not they occur is dependent not only on the genotype of the mouse, but also on the strain of mammary tumor virus she carries. Hepatic Carcinogenesis Dr. Reuber has described the histology of 20 hepatomas of the rat, including those of Dr. Morris that have been used extensively in stud- ies of enzyme activity. There were classified as (1) highly differentiated heptocellular car- cinoma; )2) well-differentiated hepatocellular carcinoma; (3) poorly differentiated or undif- ferentiated carcinoma; and (4) cholangiohep- atomas. He has classified an unusual neo- plasm of the liver that has been observed in (C3HxY)F! mice as of bile duct origin. In histogenic studies of the livers of ham- sters given N-2-fluorenylacetamide and N- 2-fluorenyldiacetamide he has observed that cholangiofibrosis and cirrhosis preceded the development of well differentiated cholangio- carcinomas in both males and females. The lesions could be followed from (1) cholangio- fibrosis to (2) cholangiofibrosis with atypical cells to (3) small cholangiocarcinomas to (4) large cholangiocarcinomas with metastases to portoheptic lymph nodes. Dr. Reuber has described the development of carcinomas of the liver in rats following the administration of carbon tetrachloride. Although carbon tetrachloride has long been known to produce hepatomas in the mouse, it had not previously been observed to produce them in the rat. The older rats were more sus- ceptible to the induction of these tumors than were the younger rats, in contrast with hepa- toma induction with fluorenamine compounds where the younger rats are more susceptible. Older male rats also developed a higher inci- dence and more severe cirrhosis of the liver with administration of carbon tetrachloride than did the younger males. In the females the 12 and 24 week old animals had more severe cirrhosis than the 4 and 52 week old groups. The Malignant Transformation in Vitro Much of the effort of the Tissue Culture Section is directed toward the study of the malignant transformation in cells of various kinds in vitro, when and under what condi- tions it occurs, and what are the changes in the cell that can be associated with the trans- formation. One of the most interesting observations in this section is that of Evans and Andresen, later confirmed by Sanford, that the spontan- eous neoplastic conversion of C3Hf embryo cells in vitro is delayed with foetal calf serum as compared with horse serum. Chromosomes in neoplastic cells show structural rearrange- ments, but Sanford and Parshad have ob- served that the foetal calf serum appears to 30 ANNUAL REVIEW OF INTRAMURAL RESEARCH have a stabilizing influence on the chromo- somes. A number of changes in metabolic character- istics have been observed in the cells in vitro. Whether these are causally related to the ma- lignant transformation has not been ascer- tained. In some cases the changes may be merely owing to the cells growing in vitro. In one neoplastic strain Evans and cowork- ers have observed a change in metabolism of nucleic acid precursors. Some enzymes appear to be lacking or inactive. In another strain a change in lactate dehydrogenase behavior was noted that suggested a change in genetic con- trol rather than selection for cell type. In a line of cells on chemically defined me- dia studied by Westfall, lactate and malate dehydrogenase activities persisted at a high level but arginase and alkaline phosphatase showed changes in activity. In a line of liver cells arginase activity remained high, where- as there was a drop in production of urea, suggesting loss of critical liver function. The C3H mouse liver cells had twice the arginase activity as human skin cells in culture but both had high storage of glycogen. Sanford has observed in one clone of cells a great increase in glycolytic activity and at the same time an increase in tumor producing ca- pacity. Transformed lines showed very strik- ing histochemical variation in comparison with non-neoplastic lines. It is hoped to over- come this variation by cloning of the neo- plastic lines. In a study of a new strain of teratoma cells in culture that were diluted and inoculated into mice or culture it was found that even at the highest dilution the cells re- tained their differentiating capacity but the extent of differentiation varied inversely with the rate of tumor growth in vitro. With Dr. Rapp, Sanford has been studying Forssman antigen in hamster cells in vitro. Forssman antigen persisted in the cells for 12 months in vitro and since has been disappearing, al- though as yet the cells apparently have not become malignant. Sanford has demonstrated neoplastic trans- formation in hamster cells in vitro following treatment with polyoma virus. Conversion was not rapid and occurred at least one or two transplant generations following virus treat- ment. It appeared that transplantation anti- gens may be induced by the virus in cells already neoplastic. Morphologic alterations were noted but were not specific to virus in- fection. In looking for evidence of virus in her cell lines of both high- and low-tumor produc- ing capacity Sanford has found that some lines are positive for leukemia antigen. Dalton has found C particles in them but assays for biologic activity of the particles have always been negative. However, the neoplastic trans- formation may occur in cells free of the leu- kemia virus. Methodology in Tissue Culture Possibly the greatest advance in this area in recent years has been the development of the mass stirred fluid culture system with con- tinuous nutrient fluid renewal by Andresen, Bryant, and Evans. This system has been per- fected and now has been used for stain L cells in chemically defined media. Conceivably the cells could be maintained in this system indefi- nitely and examination of various aspects of growth could routinely be made through the malignant transformation. The system should also be of great value in the production of tis- sues for virus production. Commercial organ- izations will be very much interested in this system. Although the agitated fluid suspension cul- ture system developed in the Tissue Culture Section has been used for some time, Bryant and Evans continue to perfect it particularly in respect to growing cells in chemically de- fined media, and also to adapt and establish new lines of cells of various types including some mouse leukemia and mast cells, and a number of human cell lines. Five new lines of C3H mouse ambryonic fi- broblasts have been grown on the chemically defined media NCTC 135, demonstrating that this media is adequate for continuous growth of freshly explanted cultures. This, together with the fact that these cells can be main- tained for a long period of time, provides the system suitable for studying these cells through the malignant transformation and enhances NATIONAL CANCER INSTITUTE 31 the chances of causally relating observed changes to the transformation. A number of commercial establishments are now supplying media made according to the NCTC 135 formula and these are being tested for maintenance of cells in the Tissue Culture Section. Thus far none of these commercial media are wholly satisfactory, with the pos- sible exception of one powdered preparation of NCTC 135. Biochemistry Dr. Anderson has been summarizing her studies on glutamine antagonists and with Dr. Lyon has been continuing studies of mechan- isms of feedback control over "salvage" path- ways for pyrimidine nucleotide biosynthesis. Dr. Anderson and her program are transfer- ring to the Laboratory of Biochemistry that is expected to supply a better environment for her research. LABORATORY OF PATHOLOGY Introduction The work in Pathology is separated into two general areas, the Department of Pathologic Anatomy and the Laboratory of Pathology. The staff of Pathologic Anatomy performs the autopsies and examines the biopsies and sur- gical pathology and exfoliative cytology speci- mens for the Clinical Center. The annual re- ports on the number of these are attached. Another important operation maintained in the Laboratory of Pathology is the Patholog- ical Technology Section. A report (NCI 517) prepared by the head of this section, Mr. Joseph Albrecht, describes the work per- formed in this section. The primary aim of the staff of the Labora- tory of Pathology is to carry out experimental cancer research. For this a variety of tech- niques and experimental animals are used. Each senior staff member carries out his own experiments, but two or more members within the Laboratory may collaborate and individual members collaborate with many others in the National Cancer Institute, and in the National Institutes of Health. There is free exchange of information and assistance between and among all the pathol- ogists. While the Annual Report reflects in general the work of the two groups in pathol- ogy, it also shows that there is considerable interchange. Most of the members of the De- partment of Pathologic Anatomy have under study one or more problems involving cancer or some other disease in man which they carry out either independently or in collaboration with the clinicians. The autopsies and the sur- gical pathology specimens and biopsies are ex- amined with the greatest of care. They are initially studied by residents and their diag- noses are all reviewed by senior staff members. A notable achievement by the head of the Surgical Pathology and Postmortem Service of the Department of Pathologic Anatomy, Dr. Louis B. Thomas, is his work on a committee of the College of American Pathologists which has devised a nomenclature and code for pathological lesions. In collaboration with Dr. Arnold Pratt and the Biometrics Section of NIH this Systematized Nomenclature of Pathology is now being used in a computer retrieval sys- tem which permits the accurate and speedy retrieval of all autopsy and biopsy records in the National Cancer Institute. Most of the members of the Department of Pathologic Anatomy also carry out experi- mental research using animals, tissue culture and other special techniques. The residents in Pathologic Anatomy are particularly ambi- tious in carrying out research programs. Experimental Research The experimental work in the Laboratory of Pathology is not restricted to a single project, or to a group of closely related projects, but each pathologist follows his particular line of interest and training. Because of this diversity the projects in the Laboratory have been sep- arated into 6 rather loose categories for con- venience in preparing this summary. These are: Cancer in Man, and Related Animal Studies: Geographic pathology of cancer. Exfoliative cytology and cytogenetics. 32 ANNUAL REVIEW OF INTRAMURAL RESEARCH Possible etiologic factors in human envir- onment. African lymphoma. Cancer in Animals: Induction and pathogenesis. Modifications in carcinogenesis. Biologic factors in neoplasia. Spontaneous tumors. Transplanted tumors. Viral Carcinogenesis and Related Problems in Virology: Polyoma virus. SV40 and adenovirus. Leukemogenic viruses. Virus replication. Interferon. Accumulation of Data on Laboratory Animals and Other Species: Information on laboratory animals. Phylogenetic aspects of neoplasia and comparative oncology. Development of New Techniques and Their Application. Collaborative Research: Methods. Examples. Cancer in Man, and Related Animal Studies Since nearly all the members of the Profes- sional Staff of the Laboratory of Pathology and Department of Pathologic Anatomy are medically trained, many research activities are correlated with the problem of human cancer. Several projects are directly utilizing human material, and others are testing in animals substances in the human environment that may be carcinogenic. Geographic Pathology of Cancer — In- formation is accumulating that certain forms of cancer in human population groups occur in a higher incidence than would be expected. Intensive study of such groups, and the recog- nition of possible etiologic agents which can be tested on experimental animals may identify some environmental factors which could be eliminated or controlled and thereby prevent many cases of human cancer. The following studies have this aim. Maps of different geographic areas are in preparation to show the relative frequency of different types of cancer. When it is recog- nized that some type is especially common, a more intensive study of the population group is indicated. (Dunham and Bailar) The following types of cancer are being in- vestigated in particular geographic area: Bladder cancer in New Orleans: White males over 60 in this city are frequently affected. All histologic sections have been reviewed by the same pathologists (Stewart and Rabson) to establish uniformity in diagnosis. Question- aires on past history and possible exposure to carcinogens are being analyzed, and a report is now in preparation. (Dunham) Uterine cancer in New York City, Israel and Washington, D.C.: This type of cancer is notably frequent in Negro women and in- frequent in Jewish women. Histologic sections have been examined from patients with cancer, and over 3,500 women interviewed. The data are being coded and analyzed in an effort to detect an environmental factor that may ac- count for the racial difference. (Stewart, Dun- ham, Thomas, Edgcomb) A study of cancer in North American In- dians has been started. Cancers of the uterine cervix and biliary tract, and basal cell car- cinoma of the skin, are frequent in this ethnic group. The histologic diagnoses have been made on 700 specimens in the NIAMD. Correlations with age and sex will be made. A survey of the literature revealed that very little reliable information is now available on the Indians. (Dunham, Laqueur) Lung cancer in veterans of World War I: The medical history and biopsy and autopsy material from this group are gathered. All histological material has been reviewed by the same pathologist, and a uniform classification of histologic types has been made. Different histologic groups may have different signifi- cance and relationship to environmental fac- tors. This survey indicated the unreliability of random histologic diagnoses which have often been accepted uncritically by statis- ticians or epidemiologists. The results have been coded and a manuscript is in preparation. This study will supply important information NATIONAL CANCER INSTITUTE 33 on the influence of smoking on the incidence of lung cancer within this group. (Herrold) Gastric cancer in Japan: Cancer of the stom- ach is remarkably frequent in several unrelated human populations, and in Japan, this type of cancer is especially common. Migrant Japa- nese in Hawaii offer a good group of genetic- ally similar people in a different geographic area for comparison. The histologic sections are being reviewed by the same pathologist (Herrold), and any association with gastric ulcer, polyps or intestinal metaplasia is noted. A correlation will be made with data collected by a team of epidemiologists. An investigation is also made for viruses (Bryan); and elec- tron microscopy is done when possible. His- tochemical studies on the human material in- dicate a change in gastric mucins, and corre- lated studies of Syrian hamsters support this finding. This is a long term study that will re- quire many years, but a preliminary report will be given at the International Congress in Japan in 1966 (Herrold, Stewart, pathologists from Japan). EXFOLIATINE CYTOLOGY AND CYTOGENETIC. — A diagnostic service in exfoliative cytology is supplied to the Clinical Center, but in addi- tion to this, a number of research studies are in progress in collaboration with various clin- ical services. These relate to the presence of neoplastic or other cells on body surfaces or in fluids, and sex chromatin in exfoliated cells (Malmgren) . It has been determined: Number of neoplastic cells in the blood of cancer patients is not closely correlated with metastasis. Cancer cells are often recovered from wound washings after cancer surgery, but time will be required to determine whether the fre- quency is correlated with recurrence in the wound site. Leukemic cells in the spinal fluid provide a reliable indication of central nervous system involvement and in the assessment of therapy. Evaluation of intrathecal treatment can be made by observation of neoplastic cells in spinal fluid. An improved technique using a milipore fil- ter makes the identification of sex-chromatin in cells from the buccal mucosa more reliable. A cell line from a primary lymphoma of the ovary in an American woman has been studied using cytogenetic techniques. Ninety percent of cells in the primary tumor carried a marker chromosome which was not seen in the established continuous tissue culture line. This indicated that the marker chromosome was not an inseparable and necessary feature of the malignant cell. (Chu) A system for coding findings in exfoliative cytology has been prepared, and is now in operation. (Malmgren) Possible Etiologic Factors in Human Environment. — An important area in cancer research is the identification of environmental factors in a human population that may alter the expected incidence of cancer. Notable prog- ress in the prevention of cancer has come from recognition of the carcinogeni activity of soot, shale-oil, and radioactivity. Once an en- vironmental factor is suspected, tests on ani- mals are necessary to prove that it is carcino- genic and in a complex substance it is impor- tant to identify the most potent fraction. A number of experiments with this aim are now in progress in the Laboratory of Pathology. Absorbates from drinking water from New Orleans, where bladder cancer is high, and Birmingham, where it is low, are concentrated and given to mice in alcohol or chloroform. Lung tumors, a delicate indicator of carin- ogenicity were equally frequent in control and experimental groups. (Dunham) Betel quid chewing is associated with oral cancer in many areas of the world. Ingredi- ents of the quid have been tested in the hams- ter cheek pouch. Calcium hydroxide caused the most damage to the mucosa, and produced in- flammatory and hyperplastic lesions and epi- thelial atypia, but no cancer. Other sub- stances were ineffective. A paper describing these results has been accepted for publica- tion in the British Journal of Cancer. (Dun- ham) The increasing number of lung cancers in man is a cause for concern. No exact prototype of human lung cancer is known in animals, but a technique has been devised where a substance for testing can be incorporated in a beeswax pellet, and inserted into the lung 34 ANNUAL REVIEW OF INTRAMURAL RESEARCH tissue of a rat. When cancer develops it is epidermoid as in human lung - cancer. The for- mation of keratinizing cysts in the rat indi- cated a potentially cascinogenic substance. This technique will make the investigation of potentially carcinogenic substances for man more accurate. (Stanton) Attempts to produce bladder cancer in hamsters with schistosoma were unsuccessful, but since the Egyptian and the Gold Coast strain were each used, a comparison and search for histologic differences in the effects can be made. (Thomas) DMSO, a solvent suggested for therapy in man was tested in mice. No increase in tumors was noted. (Dunham) The long term effects of enovid (the anti- fertility pill) are under investigation in mice. It was found that the sterility dose is 4 or more times that required for women. All ani- mals are living after a year. One mouse that received enovid when newborn developed a granular cell myoblastoma of the cervix. Sim- ilar tumors were found in mice receiving es- trogens when newborn. (Dunn) Numerous other substances to which human beings are exposed have been tested, or are now being tested. Dihydrosafrol (DHSF) a food coloring proved to be toxic to OM rats on the usual laboratory chow, but when on an 80% corn meal diet the rats lived longer and some atypia of the esophageal mucosa was found. Esophageal cancer is high in Curacao, and natives use many decoctions of plants. Decoctions from 12 plants used by 11 patients with esophageal cancer have been given to ro- dents but it is too early to expect results. The quantity of zinc and copper in the soil has been correlated with the occurrence of certain human tumors. To test this in animals, squares of zinc, copper or iron were embedded in sub- cutaneous tissues of rats. Substances from heated fats were given to rats. Experiments begun by Dr. Wilhelm Hueper on the car- cinogenicity of heated fats are being con- tinued. The results are incomplete, but there is an indication of a weak carcinogenic ac- tion that is enhanced by repeated heating of the fat. (O'Gara) African Lymphoma. — The frequency of this form of cancer in children in Africa has aroused much speculation as to possible etio- logic factors. A series of experiments have been conducted in the Laboratory of Pathol- ogy, and comparisons made between a lym- phoma in an African child, and a tumor of similar cytology in an American woman. Each of the tumors has been grown in tissue cul- ture. Cells are similar and resemble trans- formed lymphocytes. In vitro studies of the cell line from the African lymphoma showed 40-77 chromosomes. Interferon was found in supernatant fluid. The cell line from the American woman produced immunoglobulins (Rabson). Herpes-like particles have been seen in both lymphoma lines and attempts to isolate a virus and to clarify the role of the particles in these tumors continue. The tissue culture cells could be infected with the herpes virus, but other viruses so far tried have not pro- liferated in the cultures. Twenty-one malig- nant lymphomas of various histologic types ob- tained from Clinical Center patients failed to show viruses or virus-like particles by elec- tron microscopy. (O'Conor) Cancer in Animals Many of the studies of cancer in animals are concerned with (a) induction and pathogene- sis, or the steps by which cancer develops after exposure to a carcinogen; (b) modifica- tions in the activity of carcinogens produced by altered environmental conditions or sub- stances; (c) biologic factors of neoplasia such as the behavior of tumors in tissue culture, and transfer by vectors; (d) spontaneous and unusual tumors; and (e) transplanted tumors. Introduction and Pathogenesis. — Devel- opment of gastric and skin appendage tumors produced by 2-7-FAA when given ,to preg- nant or lactating mothers, to newborns, and to adult rats is being studied. The distribution of skin appendage tumors produced by this chemical is being plotted on maps of the skin surface and a paper on histogenesis has been prepared. The tumor yield in the offspring is increased if 2-7-FAA is administered during both pregnancy and lactation. Carcinogenic NATIONAL CANCER INSTITUTE 35 action is less certain if given during- preg- nancy or lactation only. (Stewart, Snell) Monkeys were injected with MCA in the wall of the stomach 15-17 years ago. No can- cers have been found, but degenerative diseases such as arthritis have appeared. (Stewart, Snell) Several studies have been done with the ni- trosamines which have been found to be potent carcinogens. Nitrosamine compounds given by a variety of routes in hamsters produced tu- mors at similar sites, indicating that the dis- tribution, metabolic pathways, and excretion of the carcinogen were the critical factors. Tumors of the olfactory neuroepithelium in Syrian hamsters were of special interest, be- cause in previous reports these may have been mistaken for brain tumors by others. These studies emphasize that carcinogens may reach remote sites, and that when the site of tumor formation is in the lung, the carcinogen did not necessarily reach the lung by inhalation. (Herrold) Methylnitrosourea (MNU) was given by a number of routes to rats. Tumors histologically resembling neurilemmomas appeared at many different sites. Since use of this substance as a solvent has been proposed, awareness of its carcinogenic potency is important. (Stewart, Snell) Nitrosamine was given orally in various ve- hicles to general-purpose mice. Papillomas of the esophagus and forestomach were found in a few. (O'Gara) Syrian hamsters were given benzopyrene by intratracheal instillation and early changes in the bronchial epithelium were described. No tumors resulted from atmospheric pollutants or tobacco tar. An unanticipated finding from this experiment was the development of cryp- tococcus neoformans meningitis in one ham- ster. This disease occurs in man, where it has been presumed that the organism is blood- borne from a focus in the lung. Observations with the hamster suggest that the primary focus may be the nasal cavity and sinuses, from which sites the infection extends to the meninges. (Herrold) A prototype of human osteogenic sarcoma has been produced in rats by copper chelated N-OHAAF, introduced into the medullary cavity. Following this observation new experi- ments have been started. An improved tech- nique is used in inbred rats so that any tu- mors that develop can be transplanted. (Stan- ton) Hepatic cancer is being produced success- fully in primates after a short induction per- iod. Newborn and infant monkeys were used, and 13 hepatic neoplasms were produced by DENA. Successful intracerebral transplants have been made. This is the first time quick induction of cancer and transplantation has been accomplished in primates. The trans- planted tumors have now been used in thera- peutic trials. (O'Gara, Kelly) Chronic myelogenous leukemia developed in one monkey that received MIH, a drug used for treating Hodgkin's disease. Although leu- kemia has been induced in one monkey by irradiation this is the first time it has been induced in a primate by a chemical. (O'Gara, Kelly) Modifications in Carcinogenesis. — Vita- min A delayed the appearance of tumors of the forestomach in hamsters given DMBA. No inhibition of skin cancer was noted. (Chu, Malmgren) Hepatocarcinogenesis in the rat has been al- tered by dietary modifications: (a) Three times as much carcinogen N,N-Dimethyl-^>- phenylazoaniline is required to induce hepa- tomas on a complete diet, as it takes to induce cancer in rats on a deficient semi-synthetic diet. (6) n a complete Purina laboratory chow diet, the carcinogen N,N-Dimethyl-p- (m-toiylazo) aniline induced cysts and cho- lageofibrosis and a rare hepatoma, but the same dose of carcinogen gave 100% incidence of hepatoma on a deficient semi-synthetic diet, (c) An extract of U.V. irradiated fat in con- junction with subcarcinogenic dose of N,N- Dimethyl-p-phenylazoaniline had a tendency to induce hepatomas. The extract alone caused considerable liver damage but no neoplasms. (Mulay) Manipulation of copper and zinc concentra- tion in the rat diet failed to induce neoplastic lesions. Thus it fails to support the contention that a higher incidence of gastric cancer, in 36 ANNUAL REVIEW OP INTRAMURAL RESEARCH some areas, is related to use of food grown exclusively on soils with copper and zinc im- balance. (Mulay) Early alterations preceding the develop- ment of cancer are being investigated. Rats fed a hepatocarcinogenic diet showed a rise in adrenal steroid concentration, two weeks after the start of the diet and con- tinued high. The significance of this finding to azo dye carcinogenesis is discussed. (Mulay) Biologic Factors in Neoplasia. — Cell cul- tures have been made from a liver tumor in a rhesus monkey (see O'Gara) above. The cells grow slowly and the morphology remains epithelial with some attempt at differentiation, but show no evidence of function. Transplants back to the monkey brain have been successful. (Dawe) The ability of insect vectors to transmit neoplasms is under investigation. A reticulum cell sarcoma in the hamster has been under observation for several years. It was proved that it could be transmitted by feeding tumor tissue to susceptible hamsters and it has now been shown that it can be transmitted by the bite of the Aedes mosquito. Transmission is by cells. No multiplication of the cell or a virus within the mosquito could be noted. Attempts to transmit Rauscher cells or the virus have failed. Transfer of Moloney virus by the mos- quito was successful in 2 of 61 trials. (Ban- field) Spontaneous Tumors. — These are always under study in the Laboratory of Pathology and descriptions and classifications of them are published at intervals. A considerable number of histologic sections of retriculum cell sarcomas have been accumulated by Dr. Margaret Deringer. Since this group of tumors is especially confusing and complex the slides are being reviewed and a publication on the histological features is being prepared. (Dunn) Transplanted Tumors. — Observations on transplanted tumors and their behavior fur- nish valuable information on neoplasia. A number of these are carried and studied in the Laboratory of Pathology, and are made available to other investigators. Among many tumors of interest are an adrenal cortical tu- mor, gastric adenocarcinomas, a neurilem- moma, and a mesothelioma (Stewart and Snell). Others carried in mice are a myoepith- elioma with a leukemoid reaction, a kidney tumor, and a granular cell myoblastoma. (Dunn) Viral Carcinogenesis, and Problems of Immunology and Resistance The same interest in pathogenesis and the mechanisms of carcinogenesis will be found in the experiments carried out by pathologists with viruses as was noted in experiments with chemical carcinogens. Experiments now in progress are concerned with: Polyoma virus, SV40 and adenovirus 12, leukemogenic viruses, and Interferon. Polyoma Virus. — Studies on the interac- tion of mesenchymal and epithelial elements in organ culture, and the effect of the polyoma virus have continued. The specificity of the mesenchyme for the epithelium is critical cr-1 some combinations produce no neoplastic change, even in the presence of PV. Continued contact with natural mesenchymal tissue is re- quired for epithelial neoplasia, and neoplasia will not develop until some morphogenesis and differentiation take place. The salivary gland rudiments must reach a certain stage before neoplasia results. Viral oncogenesis is not sim- ply the result of interaction of the viral ge- nome with the cell genome — epigenetic fac- tors also operate and a proliferative stage is required. (Dawe) The behavior of a polyoma induced tumor in the hamster was compared with a spon- taneous and a carcinogen-induced tumor after intravenous injection of dissociated cells. The resulting nodules in the lung after injection of the polyoma tumor were interpreted as in- flammatory reactions rather than true neo- plasms. (Herrold) Thymectomy was shown to increase the on- cogenic effect of the polyoma virus. Of par- ticular interest was the observation that C57BL mice resistant to polyoma oncogenesis were made susceptible by thymectomy but sen- sitized spleen cells will inhibit this suscep- tibility even when given many weeks after the viral infection and thymectomy. A morphologic NATIONAL CANCER INSTITUTE 37 study of this effect is now in progress. (Stan- ton, Law) Two strains of the polyoma virus with dif- ferent oncogenic potentials but with a similar capacity for the induction of tumor antigens are available. These have been tested by the tumor rejection assay. Paradoxically, the more oncogenic virus inhibited the ability of the mouse to reject a non-polyoma tumor, possibly because of thymotrophic action. (Friedman) Sv40 and Adenovirus. — (a.) Newborn ham- sters were infected with SV40 and adeno 12 simultaneously and the progeny of the two viruses grown in mixed infection. The SV40 had previously been shown to induce tumors with large cells, while the adeno 12 tumor cells were small and hyperchromatic. The tu- mors that developed early were of a histologic type characteristic of adeno 12; tumors that developed later were of SV40 type, (b) A "T" antigen was shown by immunofluores- cence to be correlated with stippling seen by electron microscopy in monkey kidney cells with adenovirus 12. (c) The hybrid virus adeno 7-SV40 designated E46 produced SV40 type tumors, described as papillary ependy- omas when given intracerebrally. When given subcutaneously, it produced "mixed" tumors showing histologic areas like SV40 and other areas like adeno 12. (Rabson) Electron microscopic studies on mixed viral infections are continuing. Simultaneous repli- cation within the same nucleus of SV40 and herpes simplex virus has been demonstrated in green monkey kidney cells. Hybrid E46 has protein crystalline structures in the nucleus which are not found in the parent adeno 7 or SV40. EM studies on arbovirus infection are being correlated with biochemical studies in chicken fibroblasts infected with Semliki forest virus. (O'Conor) Leukemogenic Viruses. — Moloney virus in- fection in rats has disclosed previously in- apparent Bartonella organisms in the blood and encephalazoon lesions in the brain in the preleukemic state. This indicates an alteration in the defense mechanism during the preleu- kemic state, and raises the question whether an atypical response to infection may be found in a preleukemic state in man, and clinicians should be alert to this possibility. (Stanton) BALB/c mice infected with Rauscher virus and malaria die earlier. Malaria infection also causes an increase in macroglobulin. Changes in the globulins might be of diagnostic aid in malaria and leukemia. (Edgcomb) BALB/c mice with Rauscher virus devel- oped an extreme erythroblastic reaction but no leukemia. The erythroblastic reaction was inhibited (but not prevented) by feeding pro- phylthiouracil or by repeated blood trans- fusions (Dunn, Malmgren). Moloney virus in- fection was enhanced by DNA and RNA ob- tained from a commercial source. (Malmgren) Virus Replication. — Replication of an RNA virus is under study. A model cytopathic RNA virus infection with Semliki Forest Virus (arbovirus, group A) is used as a model in order to elucidate the mechanism of repli- cation of RNA viruses including possibly leu- kemia viruses. Studies have shown that in addition to the single standard RNA present in infectious virus, 2 other replicative forms are present in infected chick cells. One appears to be a double stranded viral forms. These forms are present in 3 distinct cytoplasmic particles. Protein synthesis by virus and the interrelationship between the 3 cytoplasmic particles are being investigated. Interferon. — Mechanisms of interaction of interferon and the possible role in carcino- genesis are under study. The action involves action protein synthesis. Interferon appears to limit production of viral DNA in vaccinia virus. (Friedman) Accumulation of Data on Laboratory Animals and Other Species Because the training of pathologists is not restricted to one organ system or type of dis- ease, they are often able to contribute to the general knowledge of the normal and patho- logic anatomy of many different animal spe- cies. This contribution is often incidental to the main purpose of cancer research, yet it is indispensable to the intelligent use of biologic material, because, unless the normal anatomy and spontaneous pathologic alterations are 38 ANNUAL REVIEW OP INTRAMURAL RESEARCH recognized, it is impossible to interpret the effects of experimental procedures. For ten years or more autopsies have been performed on old rats of 6 inbred strains. The incidence of tumors and other lesions has been accurately recorded. This information has become increasingly more complete and valuable and facts derived from this survey are often requested by pathologists within the NIH and from outside. A detailed review of renal lesions in these old rats, with a review of the literature was recently presented at a Conference on Spontaneous Diseases of Rats and Mice sponsored by the Nuffield Foundation in England. (Snell, Stewart) Mastomys have been introduced as a new laboratory species, and information on the nor- mal anatomy and spontaneous diseases is being accumulated by autopsies performed on 200 animals from the colony maintained at the NCI. These animals lived out the normal life- span. Tumors were frequent in the glandular stomach, the liver and the thymus. A kidney lesion resembling human glomerulonephritis was frequent. The female has a well developed prostate gland. (Stewart, Snell) Another species that has been used exten- sively as a laboratory animal for a decade or more is the hamster, yet comprehensive and reliable information on the normal anatomy and spontaneous disease of this useful rodent is still lacking. References describing the nor- mal anatomy are now being accumulated and information on the normal anatomy and spon- taneous diseases in hamsters used as controls in a variety of experiments is being compiled. (Herrold) Information from personal observations and the literature is being accumulated on the nor- mal and pathologic anatomy of the laboratory mouse. A paper on renal disease in mice and on amyloidosis in mice was given at the recent Conference in England. (Dunn) Complete autopsies are being performed on both control and experimental monkeys. At the present time information on the normal and pathologic anatomy of monkeys is deficient, scattered, and inaccurate, and reliable knowl- edge will be supplied only by competent pa- thologists who perform many autopsies. A form of vascular disease resulting from subcu- taneous injections of polycyclic hydrocarbons as been described. (O'Gara) An International Conference on Lung Tumor in Animals was held in Perugia, Italy, in June 1965. A principal address was delivered on comparison of histologic lung tumor types in fowls, lower animals and man. In preparation for this address, all previous descriptions on lung tumors in these animal species were re- viewed, and the histologic sections were exam- ined when available. Many of these came from the Philadelphia Zoo. This critical review re- vealed that many tumors previously accepted as primary were probably metastatic. Since captive wild animals and domestic are closely associated with man and share his environ- ment, any neoplasms found have special signi- ficance. (Stewart) Phylogenetic Aspects or Neoplasia. — An important project has been started on phylogenetic aspects of neoplasia in coopera- tion with the Smithsonian Institution and ma- rine biological laboratories. The collection and identification of neoplasms in invertebrates has been started, a literature survey now hav- ing over 300 entries will be continued and a registry of specimens will be made. Neoplasia in planaria and cockroaches is being investi- gated under laboratory conditions. The pla- naria have developed a curious spontaneous le- sion with inclusion bodies, but it is doubtfully neoplastic. Soft shell clams are being investi- gated for neoplastic changes. It is apparent that adequate criteria have not been used previ- ously to determine neoplasia in lower forms. (Dawe) Studies in comparative oncology include an effort to determine the neoplastic response of fish to chemical carcinogens. Cycasin was ad- ministered and severe liver damage produced which the fish survived. Hepatic tumors de- veloped in a restricted interval of time from 12-15 weeks. This fact together with the small size of the liver which permits serial section- ing should make this species a valuable indi- cator of potential carcinogenicity. (Stanton) NATIONAL CANCER INSTITUTE 39 Development of New Techniques and Their Application Since modern pathology is not static and is not restricted to anatomical dissection and ob- servations with the light microscope, new tech- niques must be introduced and old ones im- proved. New electron microscopic techniques are being developed especially. The electron microscope probe and the scan- ning electron microscope offer greater preci- sion in morphologic studies. Biological appli- cation of these new instruments will first be on an experimental basis. It is now possible to make a determination of the amount of phos- phorus in the nucleus. Three dimensional per- spective should become possible, and a better knowledge of the cell surface can be obtained. An analysis of tissue in situ can be accom- plished by these new methods. (Banfield) A new technique, the Transer, eliminates fixation and embedding of tissues for EM ex- amination, and offers new opportunities for analytical histological investigation. This will be especially valuable in continuing studies on collagenous connective tissue and elastin. (Banfield) Ferritin conjugated antibody studies are be- ing applied in viral oncology. An effort is be- ing made to improve the specificity of present methods while preserving cell ultrastructure. Findings are being correlated with immuno- fluorescence techniques. (O'Conor) Mice have been placed on a complete liquid diet, the commercially available Metrecal, and found to be healthy after 6 months. Develop- ment of an appropriate basic liquid food for mice would be desirable in many experiments, and possibly even for routine matintenance. (Dunn) Collaborative Research It is recognized that many research projects at the National Cancer Institute require the col- laboration of a pathologist, especially in the final evaluation of the effect of an experimen- tal procedure on laboratory animals. The Lab- oratory of Pathology has always tried to make this assistance available. Methods. — The experimental pathologist may take an active part in planning an ex- periment and in following it through; he may take the responsibility for all autopsies and histologic diagnoses in an experiment; he may review only the histologic sections in a given experiment; or may serve as a consultant to review selected material with no responsibility for the entire experiment and its publication. Finally, he may make use of material accu- mulated by other investigators for indepen- dent studies concerning pathologic alterations. It is emphasized that full collaboration of the pathologist at the time the experiment is planned is the most satisfactory arrangement for it insures the best and most economical se- lection of material for pathologic studies. The impressive amount of collaborative work carried on by the staff in Pathologic Anatomy with clinicians at the National Institutes of Health is shown in the report submitted by Dr. Louis Thomas (NCI-853). Pathologists also collaborate in experimental work on clinical problems which may not be directly related to cancer, but which could not be accomplished without pathology assistance to physicians of the Clinical Center staff. In addition to the use of the light micro- scope and standard autopsy procedures, indi- vidual members of the Laboratory of Pathology have become proficient in special techniques such as fluorescent antibody visualization, elec- tron microscopy, tissue culture, autoradiog- raphy, exfoliative cytology, special cytology, and histochemistry. These special skills are often made available in collaborative studies. Example:. — It would be tedious to consider all the collaborative work now in progress in the Laboratory of Pathology, and this should be unnecessary since it is covered in reports from other laboratories. However the following are noteworthy: With other scientists in cancer research: (a) Tumor cells were transplanted to day-old hamsters and the subsequent tumors were were treated with clam juice. Macrosis and regression was produced in the small tumors (Chu, C. P. Lee), (b) Variations in the estrone cycle were produced by continuous light and by interruption of the pathway to the pineal 40 ANNUAL REVIEW OF INTRAMURAL RESEARCH glad in rats (Chu, Wurtman). (c) Degenera- tive joint disease of Mastomys has been stud- ied (Snell, Sokoloff). (d) The histology of an- terior chamber transplants from tissue culture cells made to the eyes of mice has been de- scribed (Dunn, Evans). Research on clinical problems: (a) Emphy- sema is a crippling disease of increasing fre- quency. A probable factor in its pathogenesis is increased air pressure in airways. This has been produced by experimental procedures in rabbits. The amount of emphysema and tis- sue damage correlated with the air pressure, but spontaneous healing occurred, (b) Myo- cardial mechanics of the dog's heart were studied during systole. Apparently two groups of differently oriented muscle fibers determine dimensional changes during systole, (c) A se- ries of renal biopsies in lupus nephritis was studied in order to determine how uniform and widespread the lesions might be. (d) Endo- metrial changes and ovulation in non-fertile women after treatment with gonadotrophic hormones were studied. Induction of ovulation was produced in some cases. (Powell and others) Use of special techniques: Fluorescent anti- body studies. The following are some now in progress: antigens in patients with mycosis fungoides, antigens in human leukemia cells, monkey kidney cells with adena 12 and SV 40 virus, PV tumor antigen in lytic infection of mouse embryo cells, antigens to adenovirus 7- SV40 in induced neoplasms of the hamster kid- ney. (Malmgren and others) Electron microscopy of normal and patho- logic tissue. Application to epidemic diarrhea in mice, where virus particle so far have been found only in the lumen. (Banfield and others) LABORATORY OF PHYSIOLOGY Cancer Physiology Section Dr. S. H. Wollman and research associate Dr. J. E. Loewenstein are continuing their efforts in studying the iodide-concentrating mechanism of the thyroid gland. This investi- gation is concerned with (a) the mechanism by which the thyroid gland maintains a con- centration of iodide elevated above that in the blood, and (b) the mechanism by which the thyroid gland accumulates protein-bound io- dide. Kinetic studies on the exchange of radio- iodide between individual thyroid follicles and blood have indicated that their kinetic proper- ties are dependent on the size of the follicle. Experimental results support previous theoret- ical considerations made by Wollman and Dr. G. Andres (former research associate) that the average epithelial cell clears radioiodide from the blood and releases concentrated radioio- dide back to the blood to the same extent in follicles of all sizes and that the radioiodide transport properties of follicles depend on their surface to volume ratio. Kinetics of equilib- rium labeling of the protein-bound iodine in individual follicles reveal that at equilibrium the concentration of radioiodine in follicles, and ^therefore the stable iodine, is independent of follicle size. Follicles at the periphery of the thyroid lobe equilibrate slower than do the central follicles. The rate of equilibrium of central follicles (which parallels the rate of release of organic radioiodine) varies in- versely as follicle size. Dr. Rabinovitz and Dr. Honig and research associates Drs. Waxman and Freedman have have been doing extensive work on protein syn- thesis and its control in normal and tumor cells. This entails studies on the pathway (s) taken by amino acids in the synthesis of pro- tein and the controls exercised by the cells in regulating these processes. As information is obtained concerning protein synthesis in the normal cell, it is applied to the tumor-bearing animal with the view of selective inhibition of protein synthesis in cancer cells. With Dr. Honig, studies have been made of the require- ment of ribonucleic acid synthesis for some respiration dependent biosynthetic pathways in Sarcoma 37 ascites cells. Inhibitors of RNA synthesis in Sarcoma 37 cells, such as actino- mycin D, also inhibit the synthesis of proteins and sterols in these cells. They have found that the inhibition of the synthesis of protein and sterols can both be prevented and relieved by glucose or other glycolyzable substrates. The inhibition of protein synthesis has been dem- onstrated to occur at the step involving amino- acyl-transfer RNA formation. The level and ac- NATIONAL CANCER INSTITUTE 41 tivity of the enzyme catalyzing this reaction was identical in cells treated and not treated with actinomycin D. There appeared to be no difference in ATP level or in rate of ATP syn- thesis in cells with impaired biosynthetic ca- pabilities. They believe that there is present a short-lived RNA which is involved in the avail- ability of oxidative energy for biosynthetic processes. With Dr. Waxman, studies on the control of reticulocyte polyribosome content and hemoglobin synthesis by heme indicated that hemin enhances polyribosome stabilization and formation as well as globin synthesis. This ef- fect of hemin duplicates the results previously reported for iron salts. Cobaltous ion, deutero- hemin and zinc protoporphyrin were all capa- ble of producing the enhancing effects on poly- ribosomes and globin production. Nickel and zinc salts, as well as other cations, were in- effective. Lead salts, which lead to reticulocyte polysome disaggregation, apparently produce this effect by the inhibition of iron incorpora- tion into protoporphyrin. These data suggest that hemin is an intracellular mediator of reti- culocyte polyribosome assembly and maintains the functional integrity of the biosynthetic apparatus in these cells and precludes the for- mation of globin under conditions of iron de- ficiency. With Drs. Freedman and Honig, stud- ies on the control of nuclear-histone synthesis in chicken reticulocytes indicate that these re- ticulocytes in vitro can synthesize hemoglobin, nuclear ribonucleic acid and nuclear proteins including histones. If one adds actinomycin midazole, inhibition of nuclear ribonucleic acid D or 5, 6-dichloro-l-/3-D-ribofuransyl-benzi- synthesis occurs promptly. This also led to a rapid decrease in the rate of synthesis of cer- tain nuclear proteins, particularly histones. One histone fraction characterized by its solu- bility in 5% and insolubility in 20% trichloro- acetic acid was particularly sensitive to inhib- itors of RNA synthesis. These results suggest that the synthesis of specific histones may be under precise control in the reticulocyte nu- cleus through the mediation of newly-formed ribonucleic acid. Dr. Reid continues his studies on the deter- mination of the pattern of excretion of a broad group of metabolites by leukemic patients with special reference to nucleic acid congeners. The techniques involved are both chemical and mathematical. By employing chemical methods, urinary specimens are fractionated into a large group of mixed fractions of nu- cleic acid congeners. Mathematical techniques, primarily linear algebra, are employed to fur- ther resolve these mixed fractions by means of computational analysis of their ultraviolet ab- sorption spectra. Data-processing equipment is used to handle the larger volume of data generated. Continuing with the computational system for spectral file analysis (reported last year), further developments have been made. As many as 16 spectra can now be compared with the total file and those members which correspond at any one of three discrimination levels are obtained. Output by this procedure can be presented analyzed in various ways so as to identify groupings by clinical type and chromatographic characteristics and demon- strate any relationship within subgroups. Ap- plication of the computational system to analy- sis of chromatographic profiles has yielded helpful clarifications. Certain regions of the profiles are clearly common to the leukemia and controls; a few regions are present in the leukemia profiles which appear to have no counterpart in the controls; and certain regions are peculiar and do not correlate in a meaning- ful way with any others. Energy Metabolism Section Dr. Pratt and his associates, Drs. Morrison and Millar, have pursued their investigations of the patterns of heat production of rats with concomitant recording of feeding behavior and other activity. Their goal is to find the mode and extent to which feeding behavior (pat- terns) influences heat production and, in par- ticular, how the changes in feeding and gen- eral behavior pattern of the tumor-bearing rat influence its energy exchange. They are also in- vestigating the relative changes in water and material exchange in tissue compartments of the rat during imposed and induced changes in food intake and the effect on these of tumor induction and growth. They have found that the relative constancy of the size of the activ- ity compartment of total energy expenditure 42 ANNUAL REVIEW OF INTRAMURAL RESEARCH has been consistently confirmed in normal rats at normal temperatures. Energy expenditure of normal rats made aphagic by lesions in the lateral hypothalamus increases along with a disruption of normal patterns of differentiated activity. In rats in which the aphagia is sus- tained, the size of the activity compartment of total energy is greatly increased. Dr. Pratt, in collaboration with Mrs. Toal and Mr. William C. White, is continuing to de- velop mathematical and computer program- ming techniques for application in biomedical problems. Ultraviolet spectrophotometric anal- ysis of oligonucleotides has employed this tech- nique previously for the determination of the base composition and evaluation of sample purity of oligonucleotides. This has been ex- tended to determine nucleotide sequences. The 'technique involves obtaining spectroscopic data before and after enzymatic hydrolysis of mono-, di-, tri- and tetra-nucleotides of known sequence from which precise difference spec- tra are computed for each compound, utilizing mathematical (linear) programming solution methods. The difference spectra of oligoribonu- cleotides containing two or more adjacent adenylic acids were found to have a charac- teristic pattern, the magnitude of which was linearly related to the number of such adenylic acids present. Difference spectra sufficed to identify base composition and sequence for oligoadenylic acids. Dr. Pratt has been in charge of the com- puter operations of the Laboratory. The IBM 1620 Data Processing System with 40K digits of memory, 2 disk storage drives and a line printer have been operated with prime time being shared by Dr. Reid, Mr. White and Mrs. Toal, with occasional use by Dr. Draper and Mr. H. King. A major portion of the computer time available was occupied with information storage files and search and retrieval tech- niques. The organization of the file of NCI research grants was modified to achieve faster loading and more efficient retrieval and output procedures. About one-fourth of the active Cancer grants have been loaded and comple- tion of the file is awaiting the installation of an additional disk storage drive and the re- mainder of the punched paper tape input rec- ords. A storage and retrieval system for the abstracts of pharmacology papers presented at the 1965 FASEB meetings was organized and implemented. Programs were written to organize the file and supply output for several requests from Dr. Shannon. The information file of medical histories and clinical records from the chorio-carcinoma chemotherapy study has been initiated and the conversion and storage of data has been started. The or- ganization of the file is such that a search and retrieval on 121 lines of patient medical his- tory will be available and the identification and retrieval of specific clinical tests on any date for any patient from a list of more than 165 different tests and spanning several years and many admissions can be made. Physical Biology Section Dr. Shack continues his studies on the char- acterization of the nucleic acids and nucleopro- teins of normal and malignant tissues in terms of their physical, chemical, metabolic and bio- logical properties. These studies entail the de- velopment of additional procedures for isola- tion, fractionation and characterization of nucleic acids and nucleoproteins. Specific ob- jectives within this framework include the de- termination of whether (a) DNA of malignant cells, including those of viral origin differs from that of normal cells, such as deletion of a normal component, addition of an abnormal component or a possible detectable change of a normal component, and (&) does a "metasta- ble" DNA serve as a template for DNA synthe- sis. He has improved methodology so that sub- microgram amounts of DNA can be resolved by electrophoretic methods. These new procedures include (a) carrier systems containing unla- beled native and denatured DNA or (b) trace amounts of labelled native and denatured DNA as aids in the characterization of various DNA's. Using such procedures, partially rena- tured DNA was electrophoretically resolved from both the native and denatured forms. The type of product formed during renaturation of DNA depends on the conditions of original denaturation (temperature, salt concentra- tion). Renaturation of DNA, denatured at high salt concentration, gives a product con- NATIONAL CANCER INSTITUTE 43 sisting of both denatured and partially rena- tured DNA, while denaturation at low salt con- centration leads to a single species which ap- pears to be a partially renatured aggregate. Using infected tissue cultures as a source ma- terial, it has proved possible by such proce- dures to separate renatured polyoma DNA from the tissue DNA which remains denatured. Dr. Breitman and research associate Dr. Cannon are studying control mechanisms of purine and pyrimidine ribo- and deoxyribo- nucleotides. In collaboration with Dr. S. Perry and research associate Dr. R. A. Cooper, studies on pyrimidine metabo^sm in human leukocytes were conducted. It was observed: (1) The percent DNA-thymine derived from exogenous deoxythymidine increased from 13% to 87% over a range of deoxythymidine con- centrations from 0.03/x M to 30(V M. (2) De- oxythymidine caused expansion of the total deoxythymidine di- and tri-phosphate pool but did not influence the contribution of the path- way de novo to this pool. Hence, the increas- ing contribution of exogenous deoxythymi- dine to the formation of DNA-thymidine oc- curred because of a progressive dilution of deoxythymidine triphesphate synthesized de novo with deoxythymidine triphosphate de- rived from exogenous deoxythymidine. (3) In concentrations above 0.3 /jM, deoxythymidine inhibited DNA synthesis but not RNA synthe- sis. This inhibition was dependent on the continued presence of deoxythymidine in the medium and was reversed by the addition of deoxycytidine. (4) The incorporation of deoxy- thymidine- 3 H into deoxythymidine di- and tri- phosphate pools of intact leukocytes from nor- mal donors and patients with chronic myelo- genous leukemia reached a maximum within 5 minutes at 37° C. A steady state was main- tained during the subsequent 20 minutes at 37° C. The time to reach maximum incorpora- tion of deoxythymidine- 3 H into the deoxythy- midine monophosphate pool was slower than into the deoxythymidine di- or tri-phosphate pools. In leukemic cells, the rate of equilibrium of deoxythymidine monophosphate with deox- ythymidine- 3 H was related directly to the de- oxythymidine concentration and inversely to the temperature. (5) With increasing concen- trations of deoxythymidine, expansion of the deoxythymidine monophosphate pool derived from deoxythymidine was greater than expan- sion of the deoxythymidine di- and tri-phos- phate pools. In leukemic leukocytes the deoxy- thymidine di- and tri-phosphate pools were approximately equal in size and accounted for 65% of the thymine deoxynucleotides in the presence of 1 /*M of deoxythymidine but only 10% in the presence of 300 pM deoxythymi- dine. In normal leukocytes at these deoxythy- midine concentrations, the values were 35% and 5% respectively of the total thymine de- oxynucleotides. (6) Over the range of deoxy- thymidine concentrations studied, incorpora- tion of deoxythymidine- 3 !! into thymine deoxy- nucleoside-monophosphate pool was 2-5 fold greater and incorporation into thymine deoxy- nucleoside di- and tri-phosphate pool was 6-15 fold greater in chronic myelogenous leukemia than in normal leukocytes. Incorporation of 3 fiM. of deoxythymidine- 3 H into DNA was 30- fold greater in chronic myelogenous leukemia than in normal leukocytes. The results indi- cate that human leukocytes have the enzymat- ic capability to convert thymine to deoxythy- midine. This enzymatic capability is not ex- pressed in the absence of added deoxynucleo- sides because of rapid catabolism of deoxythy- midine and thymine and a limited supply of an available deoxyribose source. Radiation Biology Section Dr. Elkind and Dr. Francesco Mauro (Guest Worker) are studying the mechanism of the repair of X-ray damage of cells grown in tis- sue culture. Changes in protein, DNA and RNA synthesis and the effects on these syn- theses of drugs which affect repair of X-ray damage are under investigation. They have developed a model for the fractionated dose response of cultured mammalian cells which they have named a Repair-Progression model. The repair part starts promptly after the first dose. This also occurs at lowered temperature and in the absence of oxygen. The progression part also starts promptly but is temperature sensitive and can be retarded at 37° C by either inhibitors of protein synthesis, a defi- cient medium, or the absence of oxygen. Re- 44 ANNUAL REVIEW OF INTRAMURAL RESEARCH suits obtained with asynchronous as well as synchronous cells (synchronized by exposure to hydroxyurea) support the general features of the model and, hence, a study of the fractiona- tion responses to be divided into the repair and progression parts can be made. In addition, the influence of drugs on changes in repair and/or progression can be studied. DNA synthesis apparently is not required for repair. Inhibition of DNA synthesis can be produced by high concentration of thymidine (1-3 mM). Under these conditions, division stops after G 2 cells have divided, and from gross chemical measurements very little DNA is synthesized (less than 10%) in the ensuing 10 hours. The advantage of thymidine as a DNA inhibitor is that cell viability is not greatly affected. Using high concentrations of thymidine to inhibit DNA synthesis in synchronous and asynchro- nous cells, they observed that the "coupling" between DNA synthesis and X-ray response is stronger for first dose responses than for second dose responses (i.e., after sublethal damage repair). Thus it appears that even if X-ray response and DNA synthesis are tightly bound before a first dose, irradiation serves to uncouple this relationship. . Dr. Riesz has been continuing work on the effects of ionizing radiation on macromolecules of biological importance: In collaboration with Dr. F. H. White, Jr., of the Heart Institute, the distribution of free radicals in the gamma ra- diolysis of several dry proteins has been ex- amined. Gamma radiolysis of dry proteins in vacuo and subsequent exposure to tritiated hy- drogen sulfide, a radical interceptor, leads to the formation of carbon-tritium bonds. It has been shown that the observed tritium distribu- tion determined by amino acid analysis and scintillation counting, corresponds closely to the free radical distribution. Ribonuclease, car- boxymethylated reduced ribonuclease, lyso- zyme, chymotrypsinogen A, insulin and gelatin have been investigated. A comparison of all the proteins irradiated shows that in every in- stance methionine, proline and lysine have very high specific activities, while valine, iso- leucine and phenylalanine contain little trit- ium. However, each protein has, within this general pattern, its own characteristic radical distribution. The ratio of the highest to lowest specific activity for a given protein ranges from 20 for insulin to about 70 for ribonu- clease. Contrary to past interpretations of elec- tron spin resonance spectra, glycine radicals do not play a dominant role as free radical sites on carbon. Dr. Riesz has also studied the mechanism of the protective effect of certain metal ions in the y-radiolysis of dry metal- ribonuclease complexes. The reduction of cu- pric ions during the y-radiolysis of Cu 2+ -ribo- nuclease complexes was measured as a func- tion of dose and of Cu 2+ /ribonuclease ratio and compared with the survival of enzymatic activity. The results of experiments at rt^m and at liquid nitrogen temperatures support the proposal that metal ions protect because of their ability to act as interceptors of sub- excitation electrons and/or of hydrogen atoms. Dr. Smith is pursuing her studies on the kinetics of recovery of hemopoietic cells in irradiated animals. Employing the assay of the Till and McCulloch transplantation meth- od (counting colonies formed in the spleen), it has been possible to study the radio-protec- tive effects of such agents as /3-mercaptoethyl- amine, bacterial endotoxin, colchicine and other antimitotic agents. While colchicine seems to have a beneficial effect similar to that of endotoxin when given after irradiation, it has a moderate to pronounced effect on femoral colony forming units, marrow cellularity, per- ipheral leukocyte counts and survival. It ap- pears that the initial depressant effect is nec- essary for the subsequent early recovery ob- served when colchicine is given 1-3 days before irradiation. Studies on changes in peripheral blood and splenic colony formation using vin- blastine are being undertaken with tumors of low radiation sensitivity (lymphosarcoma 1798) and the Bruce lumphoma. Miss Uphoff is undertaking studies in the field of immunogenetics of tissue transplanta- tion. She is developing new methods of alter- ing the immune response as well as studying the possibility of modifying tissue antigenic- ity. In addition, a study of cell to cell or cell- antigen interaction in the initiation of the im- mune response is under investigation. In these investigations radiation is used chiefly as the NATIONAL CANCER INSTITUTE 45 immunosuppressant agent. Transplantation of hematopoietic tissue into irradiated mice is used to test donor and host response, since mechanism, sensitivity and specificity of tissue reactions can be demonstrated by appropriate selection of various inbred strains or hybrid combinations. Tolerance is demonstrated either by skin or tumor homograft test systems. Stud- ies on genetic factors influencing irradiation protection by bone marrow have indicated that the early deaths reported in two different strain combinations following mid-lethal X-ir- radiation and homologous bone marrow inocu- lation, were found to be unique to these two strain combinations (CBA vs C57BL and BALB/c or C 57 BL). If the inoculation of homologous bone marrow is delayed or if one pre-immunizes the X-irradiated host, early death patterns are altered. She has demon- strated that a simple gene difference at the H-2 locus between donor and host strains was found to be sufficient to account for the severe "secondary disease" following lethal X-irradi- ation and bone marrow therapy. Differences at H-l provide variable reactions, while differ- ences at H-3 produced no secondary disease. Immunologic tolerance to tumor homographs could be induced by pretreatment with ameth- opterin and antigen (viable cells from appro- priate strain). She has also demonstrated a chimera consisting of three genotypes. Dr. Maxwell is studying the effect of ioniz- ing radiation upon the mammalian cell both in vivo and in vitro and determining the role of RNA and DNA in radiation damage. He has shown that in the early stages the surviving ability of an irradiated suspension of rat liver ribosomal particles to synthesize (in vitro) acid-insoluble protein is approximately a log function of the radiation dose. Doses of Co 60 y-rays of the order of 150,000 rads reduce the synthesizing ability by a factor of two. This factor is approximately the same for both en- dogenous directed synthesis and artificial mes- senger (polyuridylic acid) directed synthesis. It also appears to be independent of the pres- ence or absence of dissolved oxygen in the irradiated solution. Dr. Draper has previously reported that nat- ural hemolysin production in rabbits is se- verely suppressed for about 10-14 days follow- ing a single exposure of 500 R whole-body irradiation. A subsequent recovery stage en- sues during which there is a renewal of natural antibody production at a relatively rapid rate. Enhanced serum titers are attained between the third and fourth postirradiation weeks. The recovery phase is usually accompan- ied by a change in the avidity of the antibody but the direction of the change is largely un- predictable. The production of highly avid natural hemolysin in some rabbits precludes the distinction between natural antibody and that induced specifically by immunization with sheep erythrocytes. In contrast to an active immune response, the production of natural hemolysin continues to be radiosensitive even during the rapid production stage during re- covery. This may be explained by the natural hemolysin titers representing the sum of a se- ries of short-term, weak, individual primary responses to the normally occurring hetero- phile antigens in small but constant supply from the rabbit's environment. This series of responses can be interrupted by moderate doses of X-rays. During the recovery stage it is likely that a larger population of cell is pro- ducing antibody. Such cells would be expected to be more radioresistant. Irradiation during the recovery phase often results in a delayed depression of titer indicating that the radio- sensitive cells are those exposed to antigen after irradiation. The spleen participates ac- tively in natural hemolysin formation. Studies are in progress concerning the production and nature of antibodies produced against differ- ent physical forms of the same antigen. Office of the Chief Dr. Blnm has pursued his studies in hyper- plasia induced in mouse epidermis by ultravio- let light. Methods of measuring hyperplasia are quite difficult. Illumination with visible light after irradiation with ultraviolet light increases the hyperplastic response. Hyperpla- sia occurs in the epidermis of the ventral, un- exposed surface of the mouse ear, concurrently with that, in the epidermis of the dorsal sur- face which is directly exposed to ultraviolet light. This suggests that the increase in cell 46 ANNUAL REVIEW OF INTRAMURAL RESEARCH proliferation is mediated by a diffusible sub- stance, since the ultraviolet light does not pen- etrate sufficiently to account for this effect. Injections of homogenates prepared from nor- mal and irradiated epidermis cause epidermal hyperplasia when injected into a local vein of the ear. Homogenates from irradiated ears pro- duce the greater hyperplasia, indicating that some specific substance is released by the ir- radiation. Dr. White together with Dr. Millar have been studying the role of a growing tumor as a stressing agent in protein metabolism. When pregnant rats are inoculated with the Walker 256 carcinosarcoma subcutaneously, effects ob- served are dependent on the time the tumor was inoculated. When female rats are inocu- lated with the tumor 7 days prior to evidence of pregnancy, the fetuses of all mothers are either resorbed or born dead. Rats with tumors less than 7 days of age when pregnant, deliver young which are normal in size. However, the inability of the mother to supply adequate milk results in animals which are stunted (about 50%) at weaning. These animals remain stunt- ed for 3 weeks before they appear the same weight as rats born from non-tumor-bearing animals. It appears that the rat with a 3-week tumor (prior to parturition) cannot consume enough food to satisfy the growth of the tu- mor and supply protein for the formation of milk. In this competition, the tumor obtains what it needs at the expense of the lactating gland. Pregnant rats with 2- or 1-week tumors, deliver normal litters and the offspring are essentially normal at weaning. With Dr. Bates, a study on the nitrogen excretion and oxygen consumption of Fischer rats bearing a mam- matropic tumor has been made. Tumors im- planted intramuscularly in these rates grow slowly. After 40 days, this tumor begins grow- ing. A tremendous quantity of protein is ex- creted by these rats and there is an increase in oxygen consumption. Although the total ni- trogen increases, urea values remain constant. Food intake of the tumor-bearing rats in- creases 2-3 fold. Paired feeding experiments are in progress to determine the effect of in- creased protein intake on protein excretion. The machine, glass blowing, and electronics shops, headed by Mr. Mencken, Mr. LeBrun, and Mr. Coffey, respectively, continue to make many research problems practical. The ability of these men to study the problems of the Lab- oratory and to design operational equipment not on the market has resulted in inestimable progress. In addition, their staffs have done an endless amount of emergency repair and modification jobs which not only have saved valuable time but have prevented the failure of many experiments. These unsung heroes have been an important arm in the structure and progress of the Laboratory. DERMATOLOGY BRANCH The clinical and laboratory research pro- gram of the Dermatology Branch has contin- ued along lines developed in recent years with the addition of two new laboratory activities, namely research in cell biology and electron microscopy. The major groups of the research interests are: (1) Mycosis Fungoides Lym- phoma, (2) Epidermal Growth and Differen- tiation, (3) Biology of Lymphoreticular Cells, and (4) Melanogenesis. Mycosis Fungoides Lymphoma Immunologic reactivity of patients with this disease remains normal throughout the course of the lymphoma. Whereas patients with other lymphomas, particularly Hodgkin's disease, have been found to be immunologically hypo- responsive, patients with Hodgkin's disease show no immunologic impairment, particu- larly demonstrable in studies of skin sensiti-. zation to materials such as dinitrochloroben- zene (DNCB) and the ability to reject homo- grafts. Diseases other than lymphoma have been studied for comparative immunologic re- sponsivenesses. These diseases have included leprosy and Sjogren's disease, both of which show marked impairment in capacity to react allergically to DNCB. Patients with leproma- tous leprosy have been found to be more aner- gic than patients with non-lepromatous dis- ease. Interesting phenomena has been observed in patients with Mycosis Fungoides who have NATIONAL CANCER INSTITUTE 47 developed cutaneous allergic sensitivity to topi- cally applied nitrogen mustard HN 2 . When such patients are given 1 mg. of HN 2 intra- venously, cutaneous sensitivity to HN 2 disap- pears. This requires further exploration, par- ticularly in regard to other materials since such a desensitization might have useful clini- cal applications. Epidermal Growth and Reactivity The hypothesis that basal cell tumors result from basal cells' inability to keratinize re- mains valid and is further supported by addi- tional studies. These studies have tested the ability of basal cell tumor cells to participate in healing processes, a process requiring a cell to keratinize. There has been no evidence that basal cell tumor cells have capabilities of re- epithelializing wounded skin. A futher dem- onstration of the keratinizing defect in basal cell tumor cells has been found when podophyl- lin is applied to cutaneous tumors. Whereas normal epithelium manifests a high order of keratinization in response to podophyllin man- ifests a high order of keratinization in re- sponse to podophyllin, basal cell tumor cells show no such response. Attention is called to the successful tissue culture of human skin, an achievement which has been difficult or impossible in the past by many investigators. When split thickness spec- imens of skin are put into culture, epithelium grows quite readily and remains morphologi- cally normal over periods of several weeks. The response of basal cell tumors to topi- cally applied 5-fluorouracil (5-FU) has been confirmed. The selective response of lesions, compared to uninvolved skin, seems to be due to the ease of penetration of drug through the damaged epidermis overlying lesions. Melangonesis The ability of a soil micro-organism to pro- duce a dark pigment by utilizing coumarin as a substrate seems to be due to the ability of the organism to convert coumarin to tyrosine, thence tyrosine to melanin. The series of syn- thetic and degradative steps involved in the conversion of coumarin to tyrosine have been studied. Of particular interest is the conver- sion of O-coumaric acid to melilotic acid, which utilizes an oxidoreductase enzyme requiring FAD as co-factor. The enzyme melilotic hy- droxylase, converting melilotic acid to dihy- droxyphenyl propionic acid, has been purified to the extent that it migrates homogenously on gel-electrophoresis. It has a molecular weight of about 65,000. ENDOCRINOLOGY BRANCH In the fall of 1965, the Endocrinology Branch was reorganized with somewhat less than half of its previous staff, laboratory space, and clinical facilities. This has necessi- tated some reductions in program. Several areas of the previous research program are being continued under other auspices and this report will summarize those projects only that have remained within the National Cancer In- stitute. The appreciation of the complexity of andro- gen secretion and metabolism has led to the use of better mathematical models and systems for analysis. In conjunction with the develop- ment of new methods for the measurement of plasma androstenedione and dehydroepiandro- sterone, it has become possible to describe com- pletely the multiple origins and relative im- portance of each in the maintenance of plasma androgen levels. Such methods have been ap- plied to the study of diseases involving the gonads and to the analysis of the metabolic activity of several steroid-producing cancers. Further definition of the hormonal milieu, particularly with respect to such cancers as those of the breast and uterus, awaits the de- velopment of more sensitive methods for meas- uring plasma estrogens and their metabolites. We are attempting two entirely different ap- proaches to this important problem. Our group has maintained an interest in the endocrine effects and therapy of pituitary tumors. The direct assay of pituitary trophic hormones and better methods of assessing neuro-endocrine relationships have resulted in some new concepts of hypothalamic-pituitary controls. It has become clear that gonadotro- pin activity is not necessarily the first func- tion of the pituitary to be lost with increas- 48 ANNUAL KEVIEW OF INTRAMURAL RESEARCH ing destruction of the gland. Further, it has been shown that these tumors may cause sig- nificant inhibition of pituitary function by in- terference with hypothalamic regulation rather than encroachment on the gland itself. Functional tumors of the endocrine glands have continued to interest our group. Pathways of biosynthesis in a luteoma were examined and the importance of the A 5 -pathway for synthesis and the role of the sulfoconjugates were delineated. The hypoglycemia produced by a functional adrenocortical cancer and the characteristics of a gonadotropin producing carcinoma of the bronchus were explored. Since endocrinology is concerned with the regulation of tissue growth and function, it is appropriate for the clinical endocrinologist to study regulatory mechanisms at the bio- chemical level. The initial studies have used a mammalian system, the chick oviduct, because a specific protein, avidin, is produced in re- sponse to a specific hormone, progesterone. Conditions for in vitro synthesis of avidin have been defined and methods for measuring small amounts of avidin have been devised. Our group has been strengthened this year by a visiting scientist from Australia. In the coming year, in addition to our normal com- plement of clinical associates, we shall have one third-year clinical Associate and a visiting scientist from Kyoto. Dr. Chrambach, a pro- tein chemist, has joined us recently and his skills should complement several ongoing pro- grams. We look forward to an expansion of the effort of the Branch during the coming year with increasing emphasis on the exploration of the hormonal milieu in relation to normal and abnormal tissue growth. IMMUNOLOGY BRANCH Two significant advances during the year profoundly influenced investigations in the Im- munology Branch. Development of the C'l Fixation and Trans- fer test by Drs. Borsos and Rapp in the Im- munochemistry Section has played a major role in antibody-cell antigen studies. The C'l Fixation and Transfer test has been applied in the Immunochemistry Section to theoretical and practical problems. It has made possible quantitative studies of cell antigens and of antibodies reacting with cell antigens, and has facilitated investigation of antibody: cell an- tigen reactions. One 18S IgM antibody mole- cule was shown to be sufficient to fix comple- ment and initiate cell lysis, but two 7S IgG antibody molecules must react with relatively closely spaced antigens to achieve the same effect. IgM and IgG, but not IgA, antibodies were shown to fix complement when reacting with human red cells. Differences in human Al and A2 erythrocyte antigens have been demonstrated and investigations of human leukocyte ( ?transplantation) antigens have begun. Viral tumor specific antigens were de- tected in studies of murine tumors induced by viruses. Applications of the C'l fixation and transfer technic should prove very valuable in studies of transplantation antigens, "tumor" antigens and genetic (and environmental) regulation of cell membrane composition. Immunoglobulin (gamma globulin) forma- tion in vitro in continuous culture of estab- lished human cell lines was demonstrated for the first time. IgG (7S immunoglobulin) and IgM (18S macroglobulin) formation has been accomplished. These cultures will be useful for basic biologic and biochemical study, but they may be most useful if antibody activity can be achieved. Specific human antibody synthe- sis in continuously cultured cells is now a goal of the Immunology Branch. The research activities of the Immunology Branch extended beyond the areas noted above. 29 publications for the four Senior Investi- gators are listed in the Annual Project Reports. Studies of cell, especially tumor, antigens have been extended. A program of transplantation studies was initiated. The mechanism of com- plement activation and cell damage by immune processes continue to be investigated. Immunodiagnostic facilities for multiple myeloma, macroglobulinemia and immunoglo- bulin deficiency syndromes were established through a contract with Melpar. Currently about 40 serums are tested each week. Studies of immunoglobulin structure and function have been extended. The Immunology Branch provides forums for continuous education and exchange of infor- NATIONAL CANCER INSTITUTE 49 mation for NCI investigators through two reg- ular weekly seminars — one on Molecular Im- munology, and one on Cellular Immunology. Also, tutorial seminars in Immunology and Immunochemistry were conducted by NCI Im- munology Branch staff in both Autumn and Spring terms. METABOLISM BRANCH Amino Acid Transport In an attempt to characterize further amino acid transport mechanisms in fetal rat cal- varia, the effect of puromycin dihydrochloride, an inhibitor of protein synthesis at the ribo- somal level, was studied. Inhibition of protein synthesis, measured as glycine incorporation into T.C.A. precipitable protein was approx- imately 90% at 10 ug/ml of puromycin hy- drochloride. The transport of alpha-amino isobutyric acid (AIB) was inhibited over a range of 10-600 ug/ml following a two-hour incubation in puromycin dihydrochloride. Ouabain and sodium cyanide have been shown previously to inhibit AIB uptake in fetal rat calvaria. If this inhibition is studied after varying incubation times in inhibitor followed by a standard 30 minute labeled amino acid uptake, there is initial inhibition of 35% for cyanide and 15% for ouabain when inhibitor and amino acid are added together. The rate of inhibition versus time slows on prolonged incubation. In contrast, when puromycin is used as inhibitor, there is small if any initial inhibition and the percent inhibition versus time forms a straight line on semi-log paper. A half-life for the transport process can be graphically determined at about 120 minutes. Active transport of lysine, a dibasic amino acid, was not inhibited by any concentration of puromycin dihydrochloride (10 ug/ml-600 ug/ml) and no inhibition could be seen over a 180-minute incubation period. Lysine uptake could be inhibited by cyanide in a manner analogous to that seen for AIB uptake. These findings suggest that puromycin can inhibit active transport of amino acids by inhibiting the synthesis of necessary proteins, and differ- ences in the catabolism of these proteins allow for further characterization of transport sys- tems. Investigations have been carried out evalu- ating the relationship between amino acid transport and collagen synthesis. A steady state model has been utilized where fetal rat calvaria are incubated in 0.14mM Proline for three hours and then carrier free Proline-C 14 is added for varying time intervals. It has been shown that AIB, hydroxy-proline and L-azetidine-2-carboxylic acid decrease the in- tracellular free amino acid proline pool, protein synthesis and collagen synthesis. In addition, lowering the proline concentration below 0.14mM results in decreased collagen synthe- sis. Dipeptides such as glycylproline, glycylhy- droxy-proline, hydroxyprolylglycine, and pro- lylglycine do not inhibit amino acid uptake. This suggests that although hormones may regulate the general level of cellular metab- olism, a secondary control of protein synthesis by the extracellular amino acid milieu is of importance. At present, efforts are underway to construct a multicompartmental mathemati- cal model for collagen synthesis so that quan- titation of effects of varying substances at specific steps in protein synthesis can be car- ried out. Calcium Metabolism A multicompartmental analysis for calcium kinetics has been developed. Metabolism bal- ance data combined with Ca 47 disappearance from blood, cumulative radioactivity in urine and feces, and surface counts from two sites are processed on an IBM 70904. By a least squares method of curve fitting, pool sizes and turnover rates may be calculated. Parameters which may be interpreted physiologically are bony accretion, bony resorption, urine and fecal turnover rates and gastrointestinal ab- sorption. During the past year, application of this pro- gram has concentrated on the mechanisms of calcium homeostasis. A series of 8 hypopara- thyroid patients were studied. Four of these patients were studied twice; once as a control, the second time at a new steady state pro- duced by injections of purified parathyroid hormone. In the hypoparathyroid patients, 50 ANNUAL REVIEW OF INTRAMURAL RESEARCH there was found a decrease in miscible cal- cium pool and a decrease in resorption of bone, but bony accretion, urine and fecal turnover rates were not significantly different from nor- mals. After being perturbed by parathyroid hormone, there were increases in bony resorp- tion as well as in urine and fecal turnovers. Other studies included three normal volun- teers on low and high calcium intakes. While the gastrointestinal tract was adequate in con- trolling the amount absorbed from high Ca + + intake, a low (200 mgm/day) calcium diet produced a negative calcium balance with in- creased bony resorption. The urinary and en- dogenous fecal turnovers were only slightly decreased. No significant change was seen in bony accretion. A patient with calcinosis universalis was also studied and found to have a large miscible calcium pool with slow exchange rates between compartments. The urinary and fecal turn- overs were also small. Bony accretion and re- sorption were not significantly different from normal. These changes were correlated with his- tochemical and crystallographic studies. The existing computer analysis is for a steady state system; a new computer pro- gram for analyzing the nonsteady state is being prepared. With this method, hormone depen- dent parameters for calcium control may be calculated. The feedback relationship between serum ionized calcium and parathyroid hor- mone production may be investigated in man. Porphyrin Metabolism 8-aminolevulinic acid synthetase (ALA synthetase) has been partially purified from mitochondria. Heme and other metallo-porphy- rins have been shown to produce inhibition of this enzyme in vitro only at concentrations above 5xlO _4 M, casting some doubt on the significance of heme as a functional inhibitor in vivo. The kinetics of induction of hepatic ALA synthetase have been studied alone with the effects of inhibitors of protein synthesis (pur- omycin, actinomycin D, 5-fluorouracil, p-fiuoro- phenylalanine, etc.). Glucose blocks induction of this enzyme as effectively as actinomycin D. A mathematical model of enzyme synthesis has been developed which allows the calcula- tion of the half life of the specific messenger RNA for a given enzyme. Using this model the half life of ALA synthetase is 67-74' and that of its messenger RNA is approximately the same, i.e., 40-70'. It has been shown that heme, the end prod- uct of the pathway which ALA synthetase controls, will prevent induction of ALA syn- thetase when injected intravenously at certain times before induction is initiated. This is the first direct evidence of heme as a repressor of ALA synthetase in mammalian tissues and supplements the indirect evidence from the tryptophane pyrrolase work reported last year. When heme is injected intravenously, the level of hepatic ALA synthetase oscillates markedly for periods of 4 to 5 days after a single dose. The oscillations can be greatly amplified by administration of inducer 5 hours before sacrifice. This probably is + he first dem- onstrated example of significant oscillations of the level of an enzyme. The only other exam- ple is reduced nicotinamide adenine dinucleo- tide oscillations in in vitro yeast extracts of glycolyzing systems. The oscillations of ALA synthetase in mammalian liver explain Wat- son's observations on variations in bilirubin excretion following heme administration and raise the question of the role of ALA syn- thetase in biologic clock mechanisms. The fact that ALA dehydrase (the enzyme following ALA synthetase in the porphyrin pathway) also oscillates after heme adminis- tration is evidence of a genetic operon con- trolling the two enzymes. A mathematical analysis of the kinetic factors controlling the amplitude and periodicity of the oscillations of these enzymes is in progress. The elevation of serum protein bound iodine in acute porphyria has been found to result from elevation of the serum level of thyroid binding globulin. Glucose loading tests in patients with acute porphyria have shown frequent abnormalities in the form of a decreased rate of glucose disappearance from the blood and abnormally increased blood pyruvate levels. A paradoxical response of blood levels of growth hormone has been demonstrated in NATIONAL CANCER INSTITUTE 51 seven patients with acute porphyria after glu- cose loading. The level of growth hormone rises rather than decreasing as seen in nor- mals. The syndrome of inappropriate ADH in acute porphyria has been demonstrated by di- rect assay of blood and urine for the hormone. These growth hormone and ADH abnormali- ties occur in most patients and indicate the frequency of abnormal hypothalamic physiol- ogy in acute porphyria. The effects of diet on preventing the chem- ical abnormalities of acute porphyria pre- viously demonstrated in the experimental ani- mal have been extended to patients. A de- creased caloric intake can initiate attacks of the disease and high carbohydrate diets can end these attacks. A small fraction of women whose attacks of acute porphyria recurred cyclically with their menstrual periods have been maintained free of disease by use of oral anovulatory hormone preparations. Nucleic Acid To estimate the amount of virus DNA in cells, radioactive RNA complementary to this DNA was prepared in vitro. This RNA was then reacted with DNA prepared from cells infected with or transformed by DNA viruses and the amount of DNA-RNA hybrid forma- tion was measured. DNA preparation from hamster cell lines transformed by SV40 virus had an increased amount of DNA complemen- tary to SV40 RNA, if and only if "T" anti- gen was demonstrable by immunofluorescence. This is evidence for correlation between "T" antigen and persistence of viral genome. AGMK cells infected with either adeno-7 virus alone, or adeno-7 and SV40 virus produced rather similar amounts of virus DNA, al- though the titer of infection virus was 100- 1000 higher in the infected cells. Thus, the "enhancement" by SV40 occurs at a level be- yond DNA synthesis. The same DNA-RNA hybridization-tech- niques have been applied to the taxonomy of mycoplasma and streptococci. By this tech- nique, the mycoplasma are a very heterogene- ous group of organisms. No relatedness was evident between M. pneumoniae and the other mycoplasmas tested. Other human mycoplasma fall into a number of species with only a low level of interspecific relatedness. Within some species, and of M. hominis I, there appeared to be much greater heterogeneity between dif- ferent isolates. Other groups such as M. pneu- moniae or the "FS" group were not detectably heterogeneous. Similar studies of the strepto- cocci showed excellent correlation between nu- cleic acid homology and serologic typing. Dif- ferent subgroups of streptococci were related and serotypes within a species were distin- guishable. Studies of adeno infected KB cells have dem- onstrated the appearance of an RNA com- ponent (VA-RNA) localized in the high speed supernate of the cell cytoplasma but distinct from transfer RNA. This has been partially characterized with regard to molecular prop- erties, metabolic properties, and base sequence in oligonucleotide maps. Somewhat similar RNA components are extractable from ribo- somes of uninfected cells. Preliminary studies by oligonucleotide analysis and nucleic acid hybridization techniques indicate these are not identical to VA-RNA. Metabolism of Plasma Proteins The known heterogeneity in the chemical nature of the antibody globulin molecules is paralleled by a markedly heterogeneous pat- tern of distribution synthesis and survival. In subjects without diseases affecting immuno- globulins, the survival half time of IgD was 2.8 days, of IgA 6.1 days, of IgM 5.1 days, and of IgG 24 days. Approximately 45 percent of the IgG and IgA molecules are distributed within the intravascular pool, while 70 to 80 percent of IgD and IgM molecules are within this pool. The catabolic pathway of each of these four major classes of immunoglobulins was shown to be completely independent of the other proteins and determined by the F c seg- ments of the immunoglobulin molecules, the part that does not contain the antibody-com- bining site. The immunoglobulin levels were studied in the sera of 70 patients with thymomas. Nine such patients were shown to have marked re- duction of IgG and IgA, with frequent recur- 52 ANNUAL REVIEW OP INTRAMURAL RESEARCH rent infections. Seven of these nine subjects had a marked reduction in IgM level. The two remaining subjects had extreme elevations of IgM comparable to that seen in the macro- globulinemia of Waldenstrom. These disorders of IgG, IgM and IgA concentration were shown to be predominantly disorders of immunoglo- bulin synthesis. In all but one of the patients with a reduced IgG concentration the IgG sur- vival was considerably prolonged. In the one remaining subject gastrointestinal protein loss was superimposed on the hypogammaglobu- linemia. One patient with Sjogren's syndrome and lymphoma was shown to have a pre- viously undescribed pathophysiological mecha- nism causing extreme reduction in the serum IgG concentration. This patient had a complex cryoglobulinemia that required the simultane- ous presence both in IgM and IgG molecules. The patient's own 19 S macroglobulin was re- quired for cryoprecipitation. The coprecipitant IgG required could be from any human source. Three factors contributed to the low estimate of the IgG levels. First, cryoprecipitation of serum IgG during quantitation of the levels in vitro at room temperature resulted in spur- iously low estimates for the serum concentra- tion. However, even when these studies were done under conditions that prevent cryoprecip- itation, the patient still had an exceedingly low serum concentration of IgG. This was shown to be due to a combination of defec- tive synthesis of IgG, and accelerated cata- bolism of IgG. The patient had a normal sur- vival of albumin and normal IgM, and thus had an isolated hypercatabolism of one pro- tein, IgG, presumably secondary to its forma- tion of aggregates with the cryomacroglobulin in vivo. Studies on the serum protein metabolism of patients with myotonia dystrophica were con- tinued. Patients with this disorder were shown to have a reduced concentration and total body pool size of IgG secondary to a unique heredi- tary disorder of the immunoglobulin catabo- lism and isolated hypercatabolism of IgG with normal IgG synthesis. Both the patients' and normal IgG were catabolized at the accelerated rate in the patients while the patients' IgG was catabolized at a normal rate in normal subjects. All other proteins studied, including ceruloplasmin, albumin, IgA, IgD and IgM, had a normal survival in these subjects. Studies on the protein metabolism of pa- tients with ataxia telangiectasia were con- tinued. These patients usually had a marked reduction or absence of serum IgA, but had a protein similar to IgA in the saliva. It was shown that the reduction in IgA seen in these patients was secondary to an extreme reduc- tion synthesis of this protein, and in some cases, hypercatabolism of this protein. It was shown that there is normally little or no trans- port of IgA from the serum into the salivary secretion, but that the very high levels of IgA normally seen in excretory secretions is prob- ably derived from specialized plasma cells in the mucosal and submucosal surfaces of the respiratory and gastrointestinal tract. The IgA is then coupled with a transfer piece in glan- dular structures and secreted as a first line of defense against infections entering the body. It should be noted that the patients with ataxia telangiectasia have a very high inci- dence of reticuloendothelial neoplasms. Subjects with the Wiscott-Aldrich Syn- drome, a disorder characterized by eczema, thrombocytopenia, a very high incidence of infections, and sex-linked recessive inheri- tance, have a markedly reduced level of IgM, a normal or increased level of IgG, and a mark- edly elevated serum concentration of IgA. Patients with the syndrome of intestinal lymphangiectasia were shown to have a short survival of all proteins studied, including IgG, IgA, IgM, ceruloplasmin albumin and fibrino- gen secondary to loss of these proteins into the gastrointestinal tract. The synthetic rate for each of the immunoglobulins was normal and the patients were able to produce antibodies to Vi and tularemia antigen. Marked lympho- cytopenia was noted in these patients pre- sumably due to loss of lymphocytes into the bowel secondary to the disorders of lymphatic channels. The patients with lymphopenia showed marked skin anergy to skin test anti- gen. Skin grafts from unrelated donors are sur- viving for over one year and second set grafts from the same donor are also intact at two months. NATIONAL CANCER INSTITUTE 53 A pattern of low immunoglobulins and skin anergy was also demonstrated in subjects with other causes of gastrointestinal protein loss associated with disorders of intestinal lymphat- ics, including Whipple's disease, constrictive pericarditis, and regional enteritis. Subjects with extreme gastrointestinal protein loss, without disorders of gastrointestinal lymphat- ics, such as subjects with sprue, allergic gas- troenteropathy, vascular diseases of the bowel, and ulcerative colitis, did not have lymphocy- topenia or anergy. Gastrointestinal protein loss was demonstrated for the first time in subjects with chronic vascular disease of the bowel, with the blind loop syndrome and in a patient with generalized myopathy and con- gestive heart failure. Studies on the role of the kidney in the metabolism of fractions of the immunoglobu- lins were extended. It was shown that the kid- ney plays a major role in the catabolism of human Bence-Jones proteins of either the lambda or kappa type, of normal human L chains, and of the L chain type of Bence- Jones protein formed secondary to multiple myeloma in mice. A number of other proteins that appear in the urine do not share this catabolic pathway. Specifically, the F ab and F c fragments of 7S gamma globulins, the Bence- Jones proteins of mice made up of both H chain and L chain, and those that are frag- ments of the H chain alone were not catabol- ized by uptake into the proximal convoluted tubule of the kidney. It is suggested that the H chain of immunoglobulin molecule protects the L chain from renal tubular metabolism. Previously it has been shown that the frac- tion of the circulating IgG catabolized per day is dependent on the concentration of this pro- tein, in contrast to the situation observed with IgA, IgM, ceruloplasmin and fibrinogen where the serum concentration of the protein does not affect the fractional catabolic rate. With IgG in man the fraction of the intravascular pool catabolized ranges from 2 percent in pa- tients with agammaglobulinemia to 16 percent in patients with multiple myeloma and a high level of IgG. Similar hypercatabolism of IgG has been demonstrated in mice following the infusion of IgG from a number of species or the F c fragments of the gamma globulin mole- cule. Similarly, the F c fragments survival is affected by the concentration of the intact IgG molecule. It was shown that enzyme induc- tion is not required for this hypercatabolism in response to infusion of the protein, since this phenomenon continues when the animals are given high doses of Actinomycin-D or cyclohexamide, materials that inhibit messen- ger RNA and protein synthesis, respectively. Mathematical analysis of these studies indi- cates that the best explanation for this phe- nomenon is the presence of a saturable protec- tion system for IgG molecules. In contrast to the formulation of Brambell these studies in- dicate that the absolute amount of IgG pro- tected, increases with increasing concentration of IgG, while the fraction of the total num- ber of molecules protected decreases sharply. The reciprocal of the quantity of molecules protected plotted against the reciprocal of the total circulating pool size gives a straight line function. From this graph, the total number of protection sites and the affinity of the mole- cule for the protection sites can be defined. It is postulated that the same protection sys- tem specific for IgG responsible for the con- centration effect is also responsible for the facilitated transport of IgG across the new- born gastrointestinal mucosa in animals and across the placenta in man. Erythropoiesis The studies of the relationship between metabolic rate and erythropoiesis in the hy- pothyroid dog have been completed. These studies show that in the dog made hypothyroid with large doses of iodine that there is a nor- mochromic normocytic anemia characterized by a decreased rate of synthesis of red cells that this anemia could be repaired with thy- roxin but not with dinitrophenol. It could not be repaired with iron, copper, cobalt, folic acid or vitamin B 12 . Dinitrophenol raised the meta- bolic rate to normal or even greater than nor- mal levels in both normal and hypothyroid dogs but neither in the hypothyroid dog nor in the normal dog did this result in a sig- nificant effect in erythropoiesis. This is then a demonstration of the uncoupling of erythro- 54 ANNUAL REVIEW OF INTRAMURAL RESEARCH poiesis and metabolic rate. In addition, the hypothyroid dog develops a marked lipidemia. This is corrected on the administration of thyroxine and dinitrophenol. Thus the altera- tions in blood lipids that accompany the hypo- thyroid state can be repaired by elevation of the metabolic rate to the normal by both thy- roxine and dinitrophenol. The initial stages in the development of a multicompartment (nine-pool compartment) model of iron metabolism utilizing the com- puter facilities of the Office of Mathematical Research of NIAMD have been completed. It is anticipated that this will yield better in- formation regarding the rate of formation of red cells than the simple mathematical models currently being used. Studies of hemoglobin synthesis by reticu- locytes from thalassemic patients demonstrated that overproduction of alpha chains occurs in thalassemia minor as well as thalassemia major. In contrast, there was slight or no de- tectable overproduction of alpha chains in hemoglobin Lepore trait. There was a recip- rocal relationship between the specific activity rates of alpha to beta chain and that of alpha to delta chains, suggesting that, as the chain production was depressed, there was a stimu- lation of delta chain production. Bilirubin Metabolism The bilirubin clearance method has been de- veloped and sufficient data has been acquired. The data shows that the rate of disappearance of bilirubin from the plasma in the sixteen patients studied can be described in terms of two rate components; the first having a half- time in the range of 20-30 minutes and the second a range from 100-200 minutes. In gen- eral, the rapid component is associated with the bulk of the removal. A three compartment model has been developed to quantitate the bilirubin production. This has yielded values ranging from 139 to 1530 mg per day. The principal abnormality seen in these two rate components has been in two patients with Gil- bert's disease where the second component was somewhat longer but, more particularly, it was a more significant fraction of the total turnover. With this model it is now thought possible to measure bilirubin production. Other studies have shown that in two pa- tients with a thoracic duct fistula that there was a small transfer to the order of one-half a percent of the bilirubin from plasma through hepatic lymph to the thoracic duct lymph. This did not appear to be a significant pathway anatomically for bilirubin. In the turkey, an animal with a nucleated red cell, there is a non-red cell source of bile pigment that is significant. The data have not yet been fully analyzed for its quantitative sig- nificance. Other studies of bilirubin physiology have been concerned with the development of an assay for the measurement of hepatic glu- curonyl transferase using bilirubin in physi- ological quantities as a substrate. The male rat shows higher values than the female rat. The assay has been also carried out in six patients with clinical evidence of inability to conjugate bilirubin and the amount of enzyme has shown to be decreased. The significance of this method lies in the hypothesis that bili- rubin glucuronyl transferase may be a different enzyme than other glucuronyl transferases. In the past measurements have used other sub- strate materials to determine the amount of glucuronyl transferase. The data may not be directly extrapolatable to the situation with bilirubin if another substrate is used. A series of biochemical studies have been undertaken to explain the mechanism of bili- rubin toxicity. The principal accomplishments have been a demonstration that unconjugated free bilirubin uncouples oxidative phosphory- lation. When unconjugated bilirubin is com- plexed with albumin this biochemical lesion is repaired in the liver and kidney but not in the brain. This is a possible explanation for the development of neurological lesions in kernicterus. The metabolism of bilirubin was studied in the kernicteric Gunn rat. This rat has an in- herited lack of hepatic glucuronyl transferase. This is manifest by a marked hyperbilirubi- nemia and in some animals by the neurological signs of kernicterus. On the kernicteric animal, bilirubin-C 14 could be demonstrated in the NATIONAL CANCER INSTITUTE 55 brain after intraperitoneal administration, while in equally jaundiced, but not neurologic- ally involved animals, bilirubin-C 11 could not be found in the brain. This animal and bili- rubin-C 14 made it possible to study the prob- lems of bilirubin and kernicterus. SURGERY BRANCH The Surgery Branch of the National Cancer Institute continues to act in a dual capacity carrying out its own specific clinical and lab- oratory investigations and providing consulta- tive surgical service to other branches of the National Cancer Institute and the clinical units of each of the other institutes of the National Institutes of Health. Because of the broad scope of investigative clinical studies being carried out by the various institutes, this service has included urological, Otolaryngol ogical, and gynecological as well as thoracic and general surgical consultations. During the previous twelve months, 1,240 surgical consultation requests were received and answered by the Surgery Branch Staff. Four hundred and twenty-nine of these were from the National Cancer Institute, while 811 were from clinical units of other institutes. These numbers were original requests and do not take into account the minimal five-fold increase that would exist if the repeated visits to each patient were recorded. Three hundred and eleven operative procedures were per- formed as a result of these consultations, 112 were from the medical units of the Cancer Insti- tute and 199 were performed on other institute patients. Forty-two percent of these procedures were major surgical endeavors. Consultants from the community saw 286 patients, 268 of whom were non-malignant otolaryngeal prob- lems. These consultants performed or assisted in only 12 surgical procedures. A total of 705 surgical operations were per- formed by the staff; 345 were major proce- dures and 360 were minor procedures. Surgery Branch admissions resulted in 394 surgical procedures, of which 63% were major opera- tions. The clinical interests of the Surgery Branch continue to be varied, and this is in part dic- tated by the type of patient referred. Since disease which can be controlled by standard surgical procedures is being handled in the community, 85% of the admissions were pa- tients with advanced but regionally localized disease not suitable for the usual surgical therapy. Sixty-eight percent of the admissions had received prior radiation, surgery or chemo- therapy and were treatment failures. Malignant disease of the head and neck con- tinues to be a prime interest. In past years head and neck cancer has constituted approx- imately one third of the Surgery Branch pa- tient care load. However, it has been gradually increasing and during this past year has ac- counted for 52% of the patient admissions. This increase might be in part due to the in- creased awareness of the dentist to malignant oral-pharyngeal disease and the apparent in- creased prevalence in heavy smokers, con- sumers of alcohol and "snuff dippers". During the past year, 15 patients have been direct referrals from the National Institute of Dental Research. The study of the effectiveness of pre- operative radiotherapy for oral, pharyngeal and laryngeal cancer continues. Radiotherapy is given 24 hours preoperatively on a random basis according to the double blind technique. The number of patients is low because of the limitation requiring previously untreated pa- tients. The 1000 roentgens given preopera- tively is based upon several of our laboratory studies which showed that metastases could be decreased significantly by this treatment in animal tumor systems by this therapy. Clinical experience to date indicates that 1000 roentgens in a single dose preoperatively is the maximum dosage which can be used and not have significant wound morbidity. Survival by conventional means of therapy from paranasal sinus cancer is veiy poor be- cause of frequent tumor extension into the ethmoid, sphenoid and pterygoid areas, result- ing in local recurrence in spite of the usual surgical or radiological techniques of treat- ment. Thirty-one patients have had a combined intracranial transfacial en bloc resection of the paranasal sinus area. Resection includes the cribriform plate and medial orbital walls and usually leaves the orbital contents intact. Operative mortality has been 7%. Follow-up 56 ANNUAL RFVIEW OF INTRAMURAL RESEARCH for from 4 months to 9 years indicates that 19 patients have remained disease free. The morbidity often associated with exten- sive head and neck surgery has become less of a problem. The National Institute of Dental Research continues to furnish these patients with meticulous day by day refitting - of well constructed prosthetic appliances. Through their cooperative efforts, we have been able to avoid the prolonged and repeated hospitaliza- tions so often associated with plastic recon- struction. In so doing, the operative defect may be carefully followed for local recurrence rather than have it hidden by an overlying skin pedicle. Morbidity has been further de- creased by the judicious use of antibiotics, cervical esophagostomy feeding intubation and better placed skin incisions. Even though ra- diation recurrent lesions constitute 71% of the head and neck material and surgery is in no sense less radical, there has not been a carotid hemorrhage in 28 months. We have been exploring the feasibility of full dose preoperative radiotherapy followed 6 weeks later with definitive surgery. Patients thus far chosen for this pilot venture have pre- sented large localized, malignant lesions and have been estimated to have an expected 5- year survival of less than 3 to 5%. Referred patients with carcinoma of the cer- vix continue to have had previous definitive surgery, supracervical hysterectomy, irradia- tion and histories of never having had a Papa- nicolaou smear. Our patients with advanced disease treated with extensive surgery show an operative mortality of less than 10% and an overall five-year survival of 43%. The patients who underwent radical hysterectomy have a 70% survival, anterior pelvic exenteration, 34% and total pelvic exenteration, 24%. With the development of better techniques for pa- tient selection the survival rate following radical surgery continues to improve. Unilat- eral leg edema, sciatic distribution of pain, bilateral renal involvement, lymphangiog- raphy, renal and surgical physiological studies are all clinical and diagnostic aids useful in determining feasibility of surgery. For se- lected advanced tumors of the pelvic organs, vagina, cervix, uterus bladder and rectum, surgery continues to offer the best chance of cure, but this is not done without, at times, considerable morbidity. Improved techniques of blood volume determination, red cell survival studies, bowel sterilization isotope renography and vital sign monitoring have provided an in- creased understanding of postoperative physi- ology and have permitted more knowledgeable postoperative care. All have combined to make radical surgery a practical means of bringing advanced pelvic disease under control. Experience with over 500 distal extremity lymphangiograms, indicate that this technique of demonstrating lymph nodes is an interesting research tool, but is of little real diagnostic sig- nificance in the surgical patient. Its value as seen by this surgical group is first, to draw the surgeon's attention to an area suspicious of lymph node involvement with tumor, and second to allow a more complete lymph node dissection for the surgeon infrequently in- volved with radical lymph node resection. The false positive and false negative interpre- tations greatly outnumber the incidences of ac- curate interpretations. In many patients this may be due to altered lymphatic drainage sec- ondary to previous radiotherapy. The ileal conduit continues to be the most satisfactory means of urinary diversion fol- lowing total pelvic exenteration. This technique results in few complications and preserves ex- cellent renal function in nearly all instances. However, attention must be directed toward continual antibacterial prophylaxis in order to prevent infectious complications. Of interest has been 13 patients who have undergone uri- nary diversion in spite of preoperative and op- erative evidence of unilateral ureteral occlu- sion. High transection and urinary diversion into an ileal conduit has resulted in all but one of the kidneys regaining function. In half the patients very satisfactory function re- turned, in the others improvement was noted but a variable degree of impairment remained. Radical cancer surgery is by its very nature extensive surgery and carries a relatively high mortality and morbidity. However, our experi- ence with over 100 radical hysterectomies, 50 anterior exenterations, 250 total pelvic exen- terations and 4 combined amputative proce- NATIONAL CANCER INSTITUTE 57 dures (2 of which are alive over 2 years) sug- gest that within the proper environment, this procedure should be offered to more patients with advanced pelvic disease. With intensive colostomy and urinary ileostomy instruction and guidance and vaginal reconstruction in se- lected instances, these patients who have had radical pelvic surgery can be restored to es- sentially a normal, pleasant and productive life. An increasing number of extremity sar- comas, most of which are recurrent following previous treatment are being seen by the Sur- gery Branch. Selected instances of amputation and particularly hemipelvectomy which allows removal of all extremity muscular and ten- donous insertions and origins have been per- formed with no mortality, a high degree of physical rehabilitation and an increased 3- year survival free of disease. A small, but significant number of patients are referred each year with malignant mela- noma. We are reviewing the perfusion studies and the surgical approach practiced in other centers. Our practice of wide local excision and lymph node dissection continues to be the treat- ment of choice with selected patients being referred for perfusion and surgery. When we have in, 41 patients, a 5-year survival of 16 patients and a 10-year survival of only 6 pa- tients, there is indication for incorporating well protocolled studies into the regime of this disease. With this consideration in mind, the Surgery Branch has developed a chemotherapy protocol in animals with S-91 melanoma. The limiting effects of methotrexate therapy are bone marrow depression and gastro-intestinal toxicity. Primate studies have been conducted to determine whether small amounts of citro- vorum factor given by hypogastric arterial in- fusion will selectively protect the pelvic bone marrow against the effects of intravenously administered methotrexate and yet not gen- erally circulated to interfere with the sys- temic anti-tumor effects. In addition, attempts to modify the gastro-intestinal tract toxicity by administration of non-abosrbabe intestinal antibiotics have been undertaken. At this time it appears that both of these premises are valid and that with their implementation much larger doses of methotrexate can be safely given. A long-term survey of infectious complica- tions following cancer surgery shows that the patient who carries a pathogenic organism with him into the operating room, whether it be from the skin, nose, or throat or parcularly within the tumor, runs a significantly in- creased risk of developing an infectious com- plication postoperatively. Staphylococcia in- fections predominate, but are more easily con- trolled and cause less patient morbidity than other organisms. Antibiotic therapy remains routine with all patients undergoing cancer surgery, chloramphenicol being the most sat- isfactory and frequently used drug. This drug, has been extensively used for 6 years by the Surgery Branch without evidence of hematolog- ical complications. Using high dosage for a limited ten day interval which includes the preoperative, operative, and postoperative pe- riods, infectious complications have decreased during the past year to 7% as compared to 10- 15% without preoperative coverage and 22- 30% without any antibiotic therapy. During 1965 staphylococcal infections accounted for only 36% of our infections and there were no deaths due to this organism. Staphylococcal infection certainly increases morbidity, but it is the pseudomonas, E. coli, Candida albicans and proteus organisms which have consist- ently been associated with the most resistant infections and the septic deaths. During the past year a fruitful cooperative clinical study has been carried on in associa- tion with the Experimental Therapeutics Branch, NHI. Eight patients with pheochro- mocytoma have undergone surgery following biochemical evaluation. From the care of this group of patients has emerged a program for relatively simple yet quite effective manage- ment of a clinical problem previously consid- ered formidable. Serial measurement of red cell volume, plas- ma volume and extracellular fluid volume has demonstrated that (1) following extensive sur- gery there is no evidence for a slowly circu- lating or non-circulating portion of the red cell volume; (2) post-operatively after exten- sive surgery there is a disproportionate plas- 58 ANNUAL REVIEW OF INTRAMURAL RESEARCH ma volume deficit present which has heretofore been unrecognized and is as yet unexplained; (3) post-operatively there is a deficit in the extravascular extracellular fluid volume sug- gesting the need for additional amounts of fluid at surgery. This laboratory has not been satisfied with the usual bio-assay methods of detecting anti- diuretic hormone and in the past 18 months has developed an immuno-assay technique ca- pable of measuring one hundred micromicro- grams of anti-diuretic hormone per ml. of solu- tion. Continued improvement in the sensitivity and precision of the technique is anticipated and then comprehensive physiological studies will be undertaken. During the past year, little attention has been directed toward further refinements in techniques of isolating tumor cells from the blood of cancer patients. It remains apparent that if enough blood is sampled from the can- cer-bearing patient, tumor cells will eventually be isolated. Surprisingly enough little correla- tion has been noted between patient survival and the presence or absence of circulating tumor cells. Tumor cell isolation and identifica- tion from wound washings and drainages has similarly been unrewarding in most respects. Possibly due to better techniques of filtering and certainly due to better criteria of identifi- cation the incidence of tumor cells has de- creased to such a low percentage as compared to previous years that validity of the overall study is questioned. A review of wound drain- age studies and survival in 170 patients shows no correlation as to the instance of local re- currence. This study has been unrewarding and suggests that an approach to local tumor re- currence through topical agents may not be successful no matter what agent is used. The cooperative, prospective study of pri- mary therapy of endometrial carcinoma should provide an answer to an old problem. The different types of therapy under study are sur- gery alone, preoperative radiation and surgery, and surgery and postoperative radiation. The long delay in starting this project continues, but patients will be assigned to therapy at random. Utilizing the Surgery Branch, as the Study Center, data from cooperating institu- tions will be collected and analyzed. The study is planned to include 400 patients. Ultimate analysis of the data after the completion of the five year follow-up will provide meaning- ful information concerning the relative effec- tiveness of the methods of therapy under study. A technique for transplanting endocrine glands in isologous animal systems has been used to study not only a state of endocrine hyperfunction but also the ability to regulate the animals endocrine activities through hor- mones produced by these organs. With these methods a physiological model has been used to study the inter-relationships between the parathyroid and thyroid glands in calcium homeostasis. Using hypercalcemic challenged, inbred rats and combining thyro-parathyroi- dectomy and parathyroid gland transplanta- tion at a remote site, further evidence has accumulated on the physiological role of a cal- cium lowering hormone. Preliminary evidence of a thyrocalcitonin-releasing factor from the parathyroid gland has also been demon- strated. In a similar experiment the function of the pineal gland and its relation to the men- strual cycle in normal females was shown. Ad- ditional studies using sympathetic cervical ganglia have demonstrated the histologic and biochemical survival of these tissues. A model was thus found which facilitates the study of the effects of pharmacological agents such as reserpine and tyramine on the release of nor- epinephrine. Observations on the effect of the VX 2 carcinoma in rabbits and on its ability to produce hypercalcemia are being made in an attempt to determine if the calcium imbalance is due to an excess of parathyroid hormone which may be produced by this tumor. Experiments are being carried out to evalu- ate the comparative abilities of oncogenic DNA viruses to induce neoplastic transformation in newborn hamster endocrine tissues in vitro. Transformation of pituitary, pineal thyroid, parathyroid, adrenal, testicle, and ovary tissue in explant cultures to malignant cells by Sim- ina virus 40 (SV40) and LEE 46 strain of adenovirus. These tumors are being evaluated for evidence of endocrine function and studies are being conducted for either a bio-assay or NATIONAL CANCER INSTITUTE 59 direct chemical assay of the tumors for specific hormones. A biological system for studying - tumor en- hancement with a specific fraction of the iso- logous immuno-globulins has been established. This consists of producing a potent antiserum in DBA strain of mice by repeated inoculations by EL4 ascites tumor from C57/BL mice. The antiserum is then fractionated by column chromatography and electrophoresis to see which of the specific immuno-globulins is re- sponsible for the enhancement phenomenon of these tumors across the H2 histocompatibility line. Further studies have been carried out in solid tumor patients subjected to thoracic duct lymph drainage to ascertain whether cel- lular or humoral factors in human lymph are responsible for suppression of the immune re- sponse. This has been done by taking the lymph from patients and subjecting it to sterile cen- trifugation to separate the lymphocytes from the protein fractions of lymph and then the protein fractions have been returned to the patients. The immune response was studied in these patients by small skin homografts and their ability to respond to a primary antigen. It has been shown that under these conditions the immune response is dependent more on the amount of protein and the depletion of immu- noglobins than it is the loss of small lympho- cytes in humans. Through collaborative studies with the Army Missile Command and Eastman Kodak the role of laser energy as an oncolytic tool continues to be intensively investigated. Pulsed energy from both ruby and neodymium sources and continuous wavelength studies from both Argon and C0 2 sources show tumoricidal ac- tivity in rodent, dog and primate liver. The use of the continuous wavelength laser as a "light knife" has been investigated. We are pessimis- tic about its ultimate clinical use either with skin or soft tissue. It is true that relatively bloodless hemihepatectomines can be safely performed with the CW laser and a massive amount of hepatic tissue may be destroyed with the pulsed laser without significant toxic effects to the host, but practicability is doubt- ful at the present stage of laser "refinement." At 1000 joule, semi-portable laser is being used daily in our laboratory and a 4 module, 1000 joule, neodymium unit will soon be installed. It is fair to say that we continue to look upon laser energy as the next innovation in cancer cure with "suppressed enthusiasm". Further studies in renal physiology have been performed which seek causes of renal vas- cular hypertension and better means of diag- nosis and surgical management. Further stud- ies of urinary oxygen tension (Up0 2 ) in dogs have been carried out before and had inversely related Up0 2 to medullary sodium reabsorp- tion under profound osmotic diuresis. Reduc- tion of glomerular filtration rate by clamping the reneal artery has a variable effect on Up0 2 suggesting the medullary blood flow (MBF) has a significant effect on Up0 2 under these conditions. Under the conditions of these stud- ies, however, medullary sodium transport ap- pears to be the determinant of Up0 2 . Until an accurate method of measuring MBF is devel- oped, this variable will remain uncontrolled and unknown. An attempt to validate 133 Xenon renal clear- ances as indicator of medullary blood flow is in progress. The medullary component of renal blood flow has been derived from clearance curves in a consistent manner and agree sub- stantially with data reported by others using autoradiographic techniques. Further study is indicated before accuracy can be determined. To test the hypothesis that angiotensin is inactivated by the liver, splenorenal venous shunt was performed in dogs made hyperten- sive by clamping the renal artery. None of the animals sustained relief of hypertension after shunting. Clinical trial of this procedure in hypertensive patients is not warranted. Epinephrine infusion into the renal artery to create temporaiy ischemia has been per- formed in one patient and several dogs. Anoxic tissue is known to be less sensitive to radia- tion effects. Marked deminution of renal blood flow during infusion of minute amounts of epinephrine occurred. We hope to show that there is protection against radiation damage to the body and thus allow larger doses of radio- therapy to be given to abdominal neoplasms. 60 ANNUAL REVIEW OF INTRAMURAL RESEARCH Clinical studies in renovascular hypertension have included differential studies of kidney- function and Up0 2 radioisotope renography and phonocatheter recording of renal artery pulse. Aortogram and differential function re- main the most reliable methods of diagnosing surgically curable renovascular disease. The value of phonocatheter and Up0 2 stud- ies await larger clinical experience. The isotope renogram is useful in following ureteral ob- struction and in management of patients fol- lowing renal artery surgery, but is too nonspe- cific to be the sole screening test for renal ar- tery stenosis. The renogram has also been help- ful in managing cancer patients after pelvic exenteration procedures requiring ileal con- duit diversion. It is a more accurate test of renal function than is the excretory urograms and can be used in patients allergic to uro- graphic contrast medium. Collaborative studies of patients with cysti- nuria have demonstrated the value of d-peni- cillamine in preventing recurrence of cystine calculi. Studies of the mechanism of increased cystein excretion by kidney biopsy and in vitro amino acid transport and renal arterial-venous amino acid differences suggest that increased turnover of cystine, lysine and arginine within renal parenchyma may be the basic defect in this disease. Urinary lactic dehydrogenase activity was studied in hamsters during induction of renal adenocarcinomas. In none of the experimental or control animals has total LDH excretion risen above upper limits of normal, even when both kidneys were almost entirely replaced by tumor. This study confirms previous reports from this department that urinary LDH is not a useful screening test for urinary tract neo- plasms. The effect of gonadotropins, testosterone and estradiol on surgically unilateral crypt- orchid rats has been studied. Gonadotropins and testosterone in moderately large dosage for 3 weeks did not appear to have had deleterious effects as judged by bilateral testis biopsy six weeks after the cryptorchid testis was returned to the scrotum. Intestinal perfusion may still be a method that permits repeated dialysis of uremic sub- jects over a long period. Emphasis is being placed on definition of the variables of the pro- cedure, including serum and perfusate electro- lyte concentrations, serum and perfusate osmo- lality, rates of intestinal exchange and transfer of electrolytes and other crystalloids and such factors as the flow rate and temperature of the perfusate solution. Demonstration of the physiologic events in surgically de-vascularized and re-vascularized ureters is in progress. Omental sleeves may pre- vent the high incidence of uretero-vaginal fis- tulae in women subjected to radical histerec- tomy for carcinoma of the cervix. The diagnostic significance of tetracycline fluorescence in early neoplasms accesible to ultraviolet endoscopy is controversial. It may be that tetracycline fluoresces only when che- lated to calcium in tissue fluid or within ma- crophages. This problem is currently being studied in New Zealand white rabbits with VX 2 carcinomas. The state of calcium metabo- lism is being varied with parathormone and with parathyroidectomy. Production of experimental and transitional cell carcinomas of the bladder with aniline dye products is well established. The similarity of these papillary carcinomas to tumors of vi- ral etiology is striking, yet, no experimental studies are on record to assess the role of viral "carcinogenesis" in papillary carcinoma of the bladder. Such a study is being conducted on neonatal hamster bladders and in tissue cul- ture employing polyoma, adenovirus 7 and 12, SV-40 and E46-M viruses. Serum leucine aminopeptidase values are be- ing studied in patients with normal pregnancy, multiple gestations and patients with tropho- blastic neoplasms. This is a follow-up of earlier work which suggested that the rise of values of this enzyme which takes place in normal pregnancy may be higher and/or earlier in twin pregnancies. Since it does not rise in patients with hydatidiform mole, further studies on its usefulness in making this differential diagno- sis are in order. The report that the increment in leucine aminopeptidase activity in the serum of women during pregnancy is sensitive to in- hibition with methionine is to be evaluated. NATIONAL CANCER INSTITUTE 61 A study of the immunologic relationship be- tween women with pregnancy or a trophoblas- tic tumor and their husbands cellular antigens has been carried out using the technique of lymphocyte transformation in mixed leuko- cyte cultures. Preliminary results show that lymphocytes from normally pregnant women do not transform when grown with their hus- band's leukocytes in as high a percentage as they do when grown with cells from an unre- lated male. This effect is not found in women with trophoblastic tumors. Further studies are being carried out to evaluate serial changes in each trimester of pregnancy and the changes that take place during and after chemother- apy of the trophoblastic neoplasms. The latter are being correlated with the response of the patients to chemotherapy. In order to evaluate the possibility of dam- age to the ovum in a resting Graafian follicle by tritiated thymidine given to study bone marrow cellular kinetics, radioautographs of the ovaries of previously treated Rhesus mon- keys were made and evaluated for evidence of tritium concentration in the ova or closely ad- jacent granulosa cells. These studies are in their initial phase. Immune suppression and its effect on homo- graft rejection and the growth pattern of transplanted human choriocarcinoma are be- ing studied in hamsters, mice and monkeys. Techniques used in the hamster to obtain this effect are the administration of drugs (Metho- trexate, Methotrexate and Citrovorum Factor, and Crotisone), a total body irradiation plus Cortisone and anti-thymocyte serum. In mice the effects of thymectomy alone and thymec- tomy followed by a course of anti-lymphocyte sera are being evaluated. Studies outlined in monkeys call for evaluating the effects of thy- mectomy alone, thymectomy plus anti-lympho- cyte sera treatment and also anti-thymocyte sera treatment alone. The evaluation and improvement of diag- nostic and operative culdoscopy has continued. The comparison of culdoscopy and X-ray gyne cography has resulted in the realization that either technique can provide satisfactory in- formation depending upon the availability of the techniques and the ability of the operators. The development of the intraperineal probe has converted the previously static, diagnostic culdoscopic examination to a dynamic one per- mitting the operator to palpate and manipu- late the visualized structures. The development of a simple and easy technique for positioning makes the procedure significantly shorter and easier on both the patient and the operating room personnel. The technique of ovarian bi- opsy performed under culdoscopic examination has been developed and perfected and provides a safe technique for obtaining ovarian tissue with entirely acceptable morbidity. The increased incidence of cystic ovarian enlargement and multiple pregnancy associ- ated with ovulation induction, using human menopausal gonadotropin (HMG or Pergonal) and human chorionic gonadotropin (HCG) suggest overdosage. To test this impression and to select the minimal ovulatory dose gradually increasing amount of HMG has been given to selected anovulatory or oligo-ovulatory patients and the dose response relationship noted. Re- sults indicate a sigmoid dose response relation- ship with a greater response being associated with larger doses. Minimal ovulatory doses have ranged from 450 to 1725 international units. Grouping of patients by diagnosis has resulted in a marked grouping of the minimal ovulatory doses. It would appear that continua- tion of the study would supply information which will permit Pergonal dosage to be se- lected for the individual patient with a mark- edly lessened chance of unwanted side effects. Ancillary studies which are underway as a part of the general Pergonal study include the measurement of human chorion gonadotropin in patients with Pergonal induced pregnancies, the early detection of plasma chorionic gona- dotropin in the first two weeks of gestation prior to the first missed menstrual period, de- termination of anti-Pergonal antibody in pa- tients treated with Pergonal and the determi- nation of the effects of Pergonal on plasma testosterone and androstenedione levels. The effects of pharmocologic doses of estro- gen on the reticuloendothelial system clearance rate of 131 I tagged aggregated human serum albumin and on the febrile response to endo- toxin and etiochloanolone is being studied in 62 ANNUAL REVIEW OF INTRAMURAL RESEARCH an attempt to elucidate the effects of this hor- mone on these physiolgic parameters. Ethiodol is a commonly used lymphangio- graphic radio-opaque medium. Preliminary carcinogenesis studies in mice suggested that this agent might have activity which would discourage its further use. These animal stud- ies made use of Ethiodol which was injected into three different sites. Subsequent animal investigations compared Ethicdol with the pop- py seed oil component of Ethiodol. The com- pleted data on these two oils shows that tu- mors appeared in mice at irregular intervals in all three injection sites. The response sug- gests that there may be some carcinogenic ac- tivity at the dosage level administered. Data on the pathological findings is now being assem- bled for statistical review. In the study of tumor metastases an accu- rate and reproducible method of identifying small tumor foci is necessary. Frequently the color of normal animal lung parenchyma and small experimental tumor implants is similar and it is difficult, if not impossible, to differen- tiate grossly between the two. Microscopic identification of tumor implants is often not practical because serial sections are expensive and the review is time-consuming. The accu- rate counting and sizing of small, lightly pig- mented tumor metastases in the lungs of ex- perimental mice have been facilitated by the development of a method whereby the lungs were insufflated with a 15% solution of India ink. After being washed with Feketes solution, which is a bleach preservative, these tiny im- plants, not detected in the unstained fresh lung, became discretely visible. The accuracy and time-saving features of this method aid in the verification of lung metastases and permit a much wider range of spontaneous and induced animal tumors in studies of experimental tu- mor metastases. A comparison study of survival, tumor growth, and metastases with transplanted, in- duced and spontaneous tumors in mice has shown that metastases observed in the three separate tumor systems were similar, in both amputated and intact tumor-bearing mice even though the growth rates of the tumor systems varied markedly. The similarity in the number of metastases observed is believed to be due to survival which was obtained by individual animal housing. On the basis of this study, it appears that the three tumor systems evalu- ated might be interchangeably used in metas- tases studies if recognition is given to their varied growth rates. The long term program on tumor metastases is in a transition period of orientation to the immunological approach to the understanding of local recurrence and metastases. Studies have been performed to investigate the nature of the immunity imparted by certain chemi- cally-induced tumors upon their hosts. Particu- lar emphasis has been focused on whether chemically induced tumor immunity exists in the autochthonous host, and whether the spe- cific immunity that we see in later transplant generations is due to antigenic alteration of the tumor in serial passages. The cellular lo- calization of this antigen seems to be in the nuclear fraction of the tumor tissue. With weakly antigenic tumors, i.e., S-91 melanoma, prior immunication with cell-free tumor ex- tracts had no significant effect on tumor growth. Enhancement occurred when this ex- tract was given at the time of tumor inocu- lation. Previously used methods for the study of tumor specific immunity have been extended to two new and unique tumor systems. A chem- cally-induced gastric adenocarcinoma has been found to be immunogenic, demonstrating an immunity which lasts at best, ten months, can be adoptively transferred by isologous lymphoid cells may be abrogated by splenec- tomy or large doses of tumor cells, and re- quires viable lymphoid cells for its effective- ness. Attempts to utilize immune lymphoid cells to alter the growth of established tumor have been only partially successful in that growth has been slowed but complete regres- sion has not been achieved. Similar immunologic phenomena have been demonstrated in another unusual system, a spontaneous fibrosarcoma. Immunotherapeutic experiments with this tumor of recent origin are in progress. Efforts to establish a number of new lines of visceral tumors in inbred rat strains are NATIONAL CANCER INSTITUTE 63 reaching fruition. Freely transplantable yet isologous tumors of visceral organs have not been studied in inbred rats, whose size is more appropriate to the evaluation of experimental operative surgery models than is the mouse. CHEMOTHERAPY The recent reorganization of the Chemother- apy Program has had a markedly unifying effect. The program plan (1), developed in last year's review of 1955-1966 experience, was approved by the National Institutes of Health and the National Advisory Cancer Council, and in September 1965 the new organization took form. The research strategy of the chemotherapy program is built around two objectives — the use of drugs to achieve selective killing of tu- mor cells and the search for new anti-tumor agents. Research on selective toxicity of drugs for the tumor cell advanced sharply during the past year. They key to this advance has been the development by Skipper of an animal model in which it is possible to estimate the number of tumor cells killed by drug and the number surviving. Drugs can be studied in the model to discover the optimal schedule for maximum tumor cell kill ("schedule sensitivity"). Bruce and his colleagues have studied the mitotic cy- cles of normal and of tumor cells in mice, and discovered differences that permit drug admin- istration to kill large numbers of tumor cells with slight effect on normal cells ("cycle sen- sitivity"). Merkle and colleagues have devel- oped a mathematical model that uses cycle and schedule sensitivity data in predicting curative drug regimens for a variety of human neo- plasms. The application of these new concepts to patients depends on ability to measure gen- eration times of tumor cells. Preliminary anal- ysis of those malignant diseases where drugs seemingly are curative, suggests that the bio- logical and mathematical models are predic- tive. The strategic objective of highest prior- ity at present is the study of selective toxicity of drugs in patients. Two correlative approaches support this ob- jective. One of these is a broad expansion of pharmacological studies of the known anti- tumor drugs. It is essential to know both the concentration of active drug at the target site and the time required at target for maximum tumor cell kill and minimal host toxicity. These studies are underway in both the Skipper model and in patients. Secondly, selective tox- icity can be enhanced by measures that pro- tect the host against the toxic effects of drugs. The most effective of these are platelet replace- ment and protection against the infections arising from drug induced host depletion. Re- search and development in these areas and in national resources have been sharply expanded. The second major objective is the discovery of new chemicals with anti-tumor activity. The method of search, now in its eleventh year at NCI has been thoroughly reviewed and sub- stantial changes invoked. There are over 30 drugs (counting analogues) with substantial effects in clinical cancers. These are active in 100 or more animal tumors. Examination of the screening data of 10 years shows that all but 2 of these drugs are selected by two animal tumors, L1210 leukemia in the mouse and Walker 256 carcinosarcoma implanted intra- muscularly in the rat. The conditions of these two animal tests can be adjusted to that few if any false positives will be selected. The pri- mary screen for the search has been rede- signed and at present is under a dry run with all known clinically active and a number of in- active drugs. The new screen permits the drop- ping of a number of less satisfactory animal tumors and will provide both drug schedule in- formation and dose response data. The amounts of drug required are about one-fifth those needed in the old screen so that much of tissue culture screening (used for the frequently in- adequate amounts of sample) can be dropped. While the new primary screen ensures that it can pick up signals of anti-tumor activity of a wide variety of synthetic and natural products, exploration must go forward on screens of other types. Until clinical activity is shown for drugs that are inactive in the primary screen, this question cannot be settled. Drugs highly active in other animal tumors but inactive in the primary screen are being sought for introduc- tion into clinical trial. 64 ANNUAL REVIEW OF INTRAMURAL RESEARCH The extent of the search for new agents has been under active debate. As long as the search is empirical, it would seem wise to allow con- siderable latitude. Since there are now a num- ber of effective synthetic agents, there is an opportunity to begin a study of structure activ- ity relationships. From this study it may be possible to concentrate synthesis efforts in the most rewarding areas. There have already been sharp reductions in studies of alkylating agents and anti-purines. At the same time, if the dif- ficulties of synthesis can be overcome, more needs to be done with the anti-folics and nu- cleosides. In the natural product areas there are a number of materials mostly of high mo- lecular weight highly active in the screen. The large size of these agents poses difficult prob- lems of purification and none of these materi- als has reached clinical trial. Since the known clinically active drugs are of relatively small molecular weight, perhaps these large mole- cules from plants may open a new era in can- cer chemotherapy. The new research on selective toxicity sug- gests the working hypothesis that large inter- mittent pulses of therapy are more damaging to tumor cells than to normal cells. If one ex- amines the best clinical results obtained with drugs, it is noted that these have in large part occurred with pulsed regimens. Remissions ap- proaching cure have been seen in choriocarci- noma, acute lymphocytic leukemia, Burkitt's lymphoma, childhood solid tumors and testicu- lar tumors. The drugs responsible include 2 anti-metabolies (methotrexate and 6 mercapto- purine), an alkylating agent (cyclophospha- mide), a bacterial fermentation product (actinomycin D), 2 plant products (vinca alka- loids), and an animal hormone (corticoster- oids). Cure is a word to be used with care in connection with disease, since in its strictest sense, it means that a large sample of treated patients subsequently die off at the same rate, appropriately corrected for age and sex, as the general population. While too little time has elapsed since the recognition of selective tox- icity of drugs for tumors, to make a judgment about cure, 5 year complete remissions are suf- ficiently common to generate a strong suspi- cion about the ultimate conclusion. A number of other tumors are quite sensitive to these and other drugs. The present tactical problem is to apply the concepts of selective toxicity in each instance. In addition the search for new agents gives evidence of new weapons. Streptonigrin, mi- thramycin, daunomycin, dibromomannitol and cytosine arabinoside have recently been found clinically active. All of these are strongly pos- itive in the new screen, as are an increasing number of natural products not yet in produc- tion for clinical trial. Acute Leukemia Service The intensive chemotherapeutic regimen, POMP, has been completed with an excellent complete remission rate (90%) and a low treatment mortality rate (3%). The median remission duration thus far is 16 months. Mor- bidity was kept at a moderate level by the vig- orous use of platelet transfusions and antibi- otics. Between 500 to 1000 platelet and leuko- cyte transfusions are given monthly. There is very little question that the use of platelet tran- fusions has been accompanied by a dramatic reduction in the incidence of hemorrhage in thrombocytopenic patients. On the other hand, the effectiveness of leukocyte transfusions (largely cells from donors with chronic myelo- cytic leukemia) is not as clear. However, their efficacy is for the first time being evaluated in a controlled study. The answer should be avail- able within the next year. The importance of this very high remission rate in acute leukemia is that it strongly indi- cates a shift in emphasis in the treatment of this disease. A major effort now needs to be directed toward the eradication of the leukemia leukocytes remaining in the patient in remis- sion. Accordingly more recent protocol studies have been designed with this thought in mind. Solid Tumor Service The major disease emphasis in the Solid Tumor Service continues to be the lymphomas and chronic myelocytic leukemia (CML). Based on the successful use of combination therapy in a small group of patients with Hodgkin's Disease (completed last year), a NATIONAL CANCER INSTITUTE 65 larger study is now under way using a some- what different combination. Thus far, 35 pa- tients have been treated and although it is too early for final evaluation, it appears that more complete remissions are possible, particularly in the advanced stages of this disease, with this program than has been achieved before. The Radiation Branch has been extremely active in the treatment with radiation alone of patients with localized Hodgkin's Disease and of those in Stage Ill-a. The early results are very en- couraging and suggest that cure in some of the late stages as well as in the early stages is quite likely. Along with the programs in therapy, vari- ous other aspects of the lymphomas are being investigated. These include the infectious com- plications, immune mechanisms and the effect of anti-tumor agents on delayed hypersensi- tivity, antibody production, and lymphocyte transformation. Surprisingly, the studies to date do not support the previous reports that the immune mechanism of untreated patients with Hodgkin's Disease is uniformly depressed. Studies are underway to clarify our knowl- edge of immunity in anergic individuals. With the availability of large numbers of normal lymphocytes through the use of the NCI cell separator, certain fundamental studies are pos- sible. In addition, the demonstration that thoracic duct lymphocytes can circulate for as long as 9 days raises the possibility that those cells may be of value in anergic patients under certain circumstances. In addition to the large program in the treatment of lymphomas, the Solid Tumor Service has been vitally interested in the ther- apy of the blastic transformation of CML. This has been an utterly hopeless phase of the disease in contrast to the earlier phase in which there are a number of agents which can at least offer palliation to the patient. My- leran remains the drug of choice but a new drug, dibrom-mannitol appears promising and is being evaluated. In the blastic phase, the application of the POMP regimen or modifica- tions of it has not been remarkably successful but new agents including cytosine arabinoside and daunomycin are currently under study. "Life Island" Additional experience was gained in the use of the patient protection unit, the "Life Is- land." Patient accrual has been slow even though a second unit was obtained during the year. Ten patients have been treated in these units and several points appear obvious to date: (1) It is possible to maintain a patient for long periods of time (at least 3 months) in a relatively germ-free environment. (2) Under such circumstances and in spite of se- vere leukopenia, infection is not a major prob- lem even with serious toxicity. (3) Whether drug toxicity is reduced remains to be seen but thus far the impression is that it is some- what less even though large doses of drugs being employed. (4) Finally, none of the pa- tients experienced significant remissions of any duration in spite of the super-intensive ther- apy. The use of these units poses difficult prob- lems in nursing and patient care. Accordingly, plans for laminar flow rooms are in the early stages of formulation. Whatever the ultimate role of the germ-free approach will be, there is no question that this technique has impor- tant applications in cancer chemotherapy. Radiation Branch In addition to its important role in the studies described above, the Radiation Branch has achieved very encouraging results in ap- plying fractionated spaced total body irradia- tion of patients with chronic lymphocytic leu- kemia and lymphosarcoma. With this ap- proach, the selective destruction of abnormal cells apparently occurs while normal hema- topoietic elements are relatively untouched. Clinical Branch, Baltimore Public Health Service Hospital As indicated there were many administra- tive problems which had to be resolved in the relationship of this Branch to the NCI. How- ever, in spite of this, the Baltimore Branch made a major contribution to experimental chemotherapy effort. It was particularly active in the leukemias, lymphomas and testicular tumors. 66 ANNUAL REVIEW OF INTRAMURAL RESEARCH Many studies were performed wholly or in part at the Baltimore Branch. It contributed a large number of patients both to the POMP protocol in the treatment of acute leukemia and the combination treatment (vincristine, nitrogen mustard, natulan, and prednisone) of Hodgkin's Disease. Cell Separation The joint effort with IBM to develop a ma- chine capable of separating granulocytes and platelets on a continuous flow in vivo basis saw some important progress during the year. With the development of a new model and a new centrifuge bowl design, it is now possible to separate 50% of both platelets and lympho- cytes passing through the machine. Attention is now being directed toward the concentra- tion of platelets which will permit their clinical use. A junior investigator who will work full time on the machine joined the program. Cur- rent studies are being done only on an in vivo basis using dogs primarily. Early results in the separation of granulocytes appear very promising in that yields are being obtained in the region of 35%. Conclusion It is apparent from the summary of the ac- tivities of the Clinical Trials area that the com- ing year should be a very exciting one. The organizational changes should permit a more intensive, efficient and intelligent effort in cancer chemotherapy and in the supporting programs. On the laboratory side important studies being carried out in leukocyte biochem- istry, leukocyte kinetics, cytogenetics, lympho- cyte transformation, immunity, electron mi- croscopy, tissue culture, etc., which are vital to the understanding of the malignant process. Generally, these have been undertaken because there were no similar studies elsewhere in the NIH and because the Clinical Trials area has the appropriate clinical material. The necessity for the diversity of studies within the Clinical Trials area makes it im- perative that additional senior personnel be acquired so that the various programs receive the direction of highly competent and trained individuals. A vigorous clinical program will be maintained. At the same time, in order to provide laboratory support for the clinical ac- tivities, at least two individuals with clinical interests as well as competence in such areas as biochemistry and immunology need to be recruited. Space, however, is a very serious problem, particularly in the Medicine Branch and makes it difficult to carry on existing pro- grams, let alone hire new investigators. With the studies now in progress, the com- ing year should bring significant advances in the treatment of acute leukemia and in Hodg- kin's Disease. There are strong indications that some of these patients will be cured. An important breakthrough appears imminent in the procurement of granulocytes and platelets in large numbers with the use of the NCI cell separator. Contracts with industrial firms will undoubtedly make it possible to provide these blood elements to other institutions on a wide scale. LABORATORY OF CHEMICAL PHARMACOLOGY The overall function of the OASDET in the search for chemical methods for the control of human neoplasms is the development of broad pharmacologic knowledge in laboratory ani- mals and in man of antitumor drugs. The de- gree of need for a comprehensive pharmacol- ogy program related to antitumor drugs de- pends in part upon the philosophy concerning the expected results of large scale screening programs. Extensive pharmacologic studies are not necessary if one subscribes to the notion that if enough compounds are screened long enough and hard enough a drug will be found, full blown, and fully effective which cures can- cer. The alternative, but not mutually exclusive notion, is that such a screening program will only provide leads to active antitumor com- pounds. Quantitative animal antitumor and host toxicity information concerning these compounds, and quantitative clinical toxicity and antitumor data, in conjunction with a thorough knowledge of the factors effecting the antineoplastic activity of such drugs (I NATIONAL CANCER INSTITUTE 67 consider all this pharmacology) is necessary to insure the development of chemical methods for the control of human neoplasms. While we are not involved in the primary- screening of compounds, we have responsibility in the field of experimental therapeutics, such as developing more accurate and reproducible systems for testing drugs, and in exploring more efficient means of treating tumor bear- ing animals by utilizing pharmacological in- formation. In the field of clinical chemother- apy, we are necessarily involved in the initial aspects at least of a new drug trial in terms of the clinical toxicology and pharmacology of a new drug. Information obtained from these clinical studies and studies in experimental an- imals is then used in the design and evalua- tion of the therapeutic trial. In the field of the pharmacology of chemo- therapeutic agents, we are responsible for de- veloping information on the factors, biological or chemical, which affect the antitumor ac- tivity of such drugs. The basic pharmacologic investigations in- clude a study of the absorption, tissue and cel- lular distribution, and excretion of these agents, and their metabolic fate. In addition, the biochemical mechanisms of action of anti- tumor drugs are investigated. Such data are of major importance in the design of the initial trial and the subsequent therapeutic trial in man. Another major area of research concerns the toxic effects of antitumor drugs. A thorough knowledge of the quantitative de- tails of the deleterious actions of antitumor drugs on normal cells and tissues is important in defining limitations in the therapeutic use of the drug. Of particular importance are quan- titative questions of extent or damage, rate of recovery and interval between dosing. In all these areas basic research is con- ducted, not only on the pharmacology of the specific drug, but also on the general princi- ples of pharmacology if these principles are not well known and understood. A recurring problem confronting us is that of comparative pharmacology. The development of any new drug from animal screening to clinical trial is an exercise in comparative pharmacology. The antitumor activity in the mouse and rat and the toxicity in mouse, rat and dog are used to attempt to predict activity and toxicity in man. The metabolism of a drug by mouse or rat tumor and normal cells is used as a basis for extrapolating to man. More information is needed comparing the pharmacology of such drugs in experimental animals and man. The following examples will illustrate cur- rent research activities relative to the afore- mentioned functions. The alkylating agents, for which the prototype is nitrogen mustard, are still important agents in the treatment of can- cer. Work this year has emphasized mecha- nisms of damage and repair of DNA by nitro- gen mustard with particular emphasis on cod ■ firming the hypothesis that crosslinking in tb eight position of the guanine moiety is a pr mary mechanism of action and further explor ing the possibility that nitrogen mustard mar also react with the phosphate groups. It was noted that DNA with at least two crosslinks between the chains, was still able to retain its transforming ability. This suggests that either a modest amount of alkylation does not impair the biological functions of this macromolecule, or that repair processes are available to neu- tralize the damage caused by crosslinking. A technique has been devised in which the alky- lated eight carbon in guanine may be specific- ally liberated as C0 2 under the proper condi- tions. This may allow a precise measure of the extent of crosslinking and for the development of firmer evidence that crosslinking in this position is critical to the action of mustard. The mechanism of action of hydroxyurea has yielded interesting and unexpected results. Hydroxyurea is degraded approximately 10- 25% in vivo to urea. Evidence has now been de- veloped that this degradation is accomplished by a direct reduction through one unit of the oxidative cytochrome chain. This is similar to the reductive drug metabolic pathway present in liver microsomes; however, this reaction occurs in mitrochondria. It is interesting that this reduction of hydroxyurea to urea occurs in vitro in the presence of ferric ion. These results suggest that the mechanism of action of hydroxyurea is to act as a biological oxidiz- ing agent. This is consistent with the sugges- tion that hydroxyurea may damage cells by in- 68 ANNUAL REVIEW OF INTRAMURAL RESEARCH hibition of ribonucleotide reductase and sub- sequent nucleic acid synthesis. Work continues on the overall pharmacology of MIH, the methylhydrazine derivative active in lymphoma and Hodgkin's Disease. In its me- tabolic degradation, it is clear now that this compound goes through methylhydrazine. This can react with pyridoxal to yield pyri- doxal methylhydrazone, a vitamin B 6 antag- onist. There is also evidence that MIH admin- istration can, through other metabolic prod- ucts, exert monoamine oxidase inhibition. There is experimental and clinical evidence of cen- tral nervous system activity of MIH which is consistent with monoamine oxidase inhibition. In terms of its metabolism in mice, methane is one of the excretory products. The exact origin of this methane from the MIH mole- cule is as yet to be determined. Work continues on the nitrosourea deriva- tives. One difference between the metabolism of 6fs-/?-chloroethylnitrosourea in man and lab- oratory animals appears to be that a much higher percentage of the BCNU remains in the body in man than in laboratory animals. In laboratory animals approximately 5% of the compound and its metabolites remain after 24 hours; in man, approximately 25% remain after one week. The implications of this in terms of human toxicology are as yet unclear. One of the interesting new derivatives of nitrosourea synthesized by the Southern Re- search Institute group is cyclohexylfluorethyl nitrosourea. This is a highly active compound which shows less delayed toxicity in mice than BCNU. Preliminary toxicology studies indi- cated that in the rat there were unexpected acute deaths after the administration of cyclo- hexylfluorethyl nitrosourea. It was suggested that these deaths might be related to fluoro- acetate toxicity, arising from the degradation of the fluorethyl moiety of the nitrosourea compound. One biochemical consequence of fluoroacetate toxicity is an increase in the citric acid content of a number of organs, par- ticularly liver and kidney. It was shown that after an acutely toxic dose of cyclohexyl- fluoroethyl nitrosourea, citric acid concentra- tion in rat kidneys rose strikingly. This supports the notion that the fluoroethyl de- rivatives of nitrosourea might cause unusual acute toxicity related to the metabolism to fluoroacetate. Work is continued on the folic acid antag- onists. In an interesting combination of bio- chemistry and experimental therapeutics, ef- forts are now being made, using the high di- hydrofolic reductase containing strains of L1210, to assay for number of leukemia cells by assaying for folic reductase. In certain of these strains the very high enzyme activity will allow an excellent comparison of the direct decrease in enzyme activity and the direct kill of the cells to be compared against indirect methods relying on increase in life span or number of survivors. In other studies the abil- ity of kidney and choroid plexus to concen- trate methotrexate in vitro has been demon- strated. There are two main areas concerning the toxicology of antitumor drugs. In terms of re- search toxicology, we are attempting to define optimum dose schedules for a variety of the antitumor drugs which will minimize host tox- icity. In attempts to determine an ideal sched- ule with benefit with optimum relationship be- tween tumor damage and host survival, one approach is to search for that schedule which gives the least amount of host damage in the presence of large amounts of drug. For this we use the "priming dose study" in which a sub- lethal dose of the drug is given one day to a large group of animals. On subsequent days, portions of this large group of animals are assayed for the LD50 of this agent. With a compound like methotrexate, it can be shown that the LD50 is very low on days 2-4 after the administration of a sublethal dose. Subse- quently, on days 7-9, the LD50 is very high and the possibility exists, in fact, that the animals may be able to tolerate a higher dose of the drug. With a compound like cyclophos- phamide, there is only a modest decrease in the LD50 on days 2, 3, and 4, and a return to normal on subsequent days without rebound. Cytosine arabinoside appears to be similar to methotrexate in this respect. It is interest- ing to note that this rebound previously de- scribed for methotrexate, also occurs for cytosine arabinoside. This rebound can be seen NATIONAL CANCER INSTITUTE 69 after a single dose or infusion, but not after a course exceeding four or five days. One can visualize the bone marrow reserve which might be available for mobilization after a single dose may be damaged after repeated doses of the drug. A number of new agents were evaluated for their preclinical toxicology this year. These studies, vital to the development of new drugs, are summarized briefly in the appendix of this report. The increasing importance of quantita- tive toxicological studies has encouraged us to begin a program attempting to relate host tox- icity and experimental tumor damage to plasma concentrations of antitumor drugs. As mentioned above, the development of metho- dology and the beginning study of the physi- ological disposition of a variety of important anticancer drugs has begun. A group has been formed including representative of intramural NIH and all of the contractors who are involved in physiological disposition studies. This group will exchange information on techniques and methods for studying physiological disposition and will begin to pull together all the available data on the anticancer drugs and will initiate new areas in which studies should be per- formed. The technique of ventricular perfusion in the animal and its use as a research tool have been reported in the last few years, as well as the adaptation of this technique for clinical use in the treatment of intracranial malig- nancies. This technique has been further de- veloped in this past year. Doses of methotrexate up to approximately V2 to 1 liter of a 1 mgm/ millileter solution of methotrexate have been used in ventricular perfusion. This concentra- tion of methotrexate seems to be the maximum tolerated because occasionally transient con- vulsive phenomina occur at this concentration. Such concentration, of course, should be very effective in its antitumor effects. We have also studied the toxicology of 8-azaguanine and have begun a cautious clinical trial of this agent in ventricular perfusion studies. 8-aza- guanine is of particular interest because brain tumors are thought not to have the enzyme, guanase, that detoxifies this compound, but normal brain and most of the normal body tissues to have guanase and thus the quan- tities of 8-azaguanine which return to the blood stream after perfusion will be rapidly detoxi- fied. Three patients with meningeal leukemia who were refractory to intrathecal aminopterin have had complete remissions of their menin- geal leukemia after ventriculo lumbar per- fusion. The success of this sort of study en- couraged us to move on into the area of pri- mary intracranial neoplasms such as gliomas. In considering the chemotherapy of gliomas and other primary intracranial neoplasms, the need for an appropriate screening and test system became apparent. This is not available and therefore we began the process of develop- ing such a system, preferably in an animal large enough to use for pharmacology studies. Currently we are testing the Rous sarcoma virus in newborn puppies. This has been re- ported by Rabotti to cause ependymomas or gliomas. We are attempting, so far success- fully, to maintain the Kelly-O'Gara rhesus monkey hepatic carcinoma in sequential intra- cranial transplant in young rhesus monkeys. Although it is not a primary but a metastatic type tumor, its usefulness for pharmacologic studies seems clear. As a third alternative, we are exploring the YABA virus tumor inocu- lated intracranially. Some success was reported in the past by workers at Roswell Park; with more purified and highly potent virus there is reason to hope that this might yield a satisfac- tory brain tumor model. ETIOLOGY The Office of the Associate Scientific Direc- tor for Carcinogenesis was created by the re- organization of the Institute to plan and co- ordinate programmed research in experimental carcinogenesis. The Chemistry Section of the former Carcinogenesis Studies Branch was ele- vated to branch status (Chemistry Branch) to support a more comprehensive approach to the chemical biology of cancer. The remainder of the former Carcinogenesis Studies Branch (namely the Carcinogen Screening Section, the Bioassay Section, and the Cytogenetics and Cytology Section) have been regrouped into a branch (Biology Branch). 70 ANNUAL REVIEW OF INTRAMURAL RESEARCH The work of the Chemistry Branch, which emphasizes the fundamental nature of the in- teraction of carcinogenic agents with living systems in the induction of cancer at the host, tissue, cellular, and molecular levels, has pro- gressed in a most satisfactor manner during the fiscal year. The progress along specific lines of endeavor has been succinctly presented in the summary report of the Branch Chief; no effort will be made to reiterate at this point. It is important, however, to point out that the fundamental nature of the research has in- fluenced the Branch's scientists in their de- cision to perform their research activities in their NCI laboratory facilities rather than through collaborative relationships supported by contracts. As a consequence, the size of the research program that can be implemented is intimately related to the amount of space that can be made available to the Branch. Un- fortunately, during this prelude to the occu- pancy of the new cancer building, this Branch works in difficult circumstances of crowding and inadequate facilities, about which little can be done; the staff is to be commended highly for its productivity during this fiscal year. The Biology Branch has continued to main- tain collaborative studies, supported by con- tracts, in addition to its in-house activities. Its program has been aimed principally at the identification and characterization of known and suspected chemical carcinogens, especially those commonly occurring in the human en- vironment; efforts to improve bioassay meth- ods employed in the identification and char- acterization of chemical carcinogens with the aim of improving the relevance of animal data to the human situation; and the study of effects of known and potential carcinogens at the cytologic and genetic levels. The major findings emanating from the work during this year are stated in the report of the Biology Branch Chief, and will not be reiterated here. BIOLOGY BRANCH The Biology Branch, created by the reor- ganization, is composed of a Carcinogen Screening Section, a Bioassay Section, and a Cytogenetics and Cytology Section. Dr. Falk is the Acting Branch Chief, pending recruit- ment of another scientist for this position. The common thread linking the programs of the various sections in the Branch is ex- perimental carcinogenesis. The purpose of the experimental carcinogenesis in the Carcinogen Screening Section is the identification of en- vironmental carcinogens and the characteriza- tion of the metabolism and biological action of these carcinogens in vivo. The Bioassay Sec- tion is concerned with improvement of the methodology of identifying and characterizing chemical and other carcinogens in living sys- tems to the end that screening in animals and in vitro may be made more efficient and rele- vant to the human situation. The cytogenetics and Cytology Section emphasizes the morpho- logic and biochemical interaction of carcino- gens with the host at the cellular level. As will be readily appreciated, these sectional di- visions are somewhat arbitrary, the activities of the three Sections truly representing a con- tinuum rather than discreet items. The Acting Chief of the Branch, in addition to fulfilling his responsibilities as the Asso- ciate Scientific Director for Carcinogenesis, has maintained active in-house research programs described in Individual Project Report Nos. NCI-4600 and NCI-4601 concerned with the contribution of pesticides to carcinogenic bur- den in rats and pesticides synergists, and has been the responsible Project Officer on a spec- trum of contract-supported projects (#PH43- 64-546, "Study of Air-borne Carcinogens by Means of Photodynamic Bioassay"; #PH43- 64-45, "The Co-Carcinogenicity of Aliphatic Hydrocarbons and Organic Disulfides"; #PH43-65-1036, "Carcinogenesis: Toxins in Foodstuffs"; # PH43-64-933, "Studies on Mucous Flow in the Intact Mammal Exposed to Tobacco Smoke"; #PH43-63-1165, "Oxi- dation Products of Fats"; #PH43-64-1158, "Piperonyl Butoxide as Carcinogen or Co- Carcinogen"; #PH43-64-567, "The Prepara- tion of Products Related to Dibenz (ah) An- thracene and Benzo(a)Pyrene"; #PH43-64- 938, "Tumor-Promoting Principles of Tobacco and Tobacco Smoke"; # PH43-64-865, "In- vestigation of Potential Anti-Radiation and anti-Neoplastic Compounds"; #PH43-64- NATIONAL CANCER INSTITUTE 71 504, "Biochemical and Morphological Compo- nents of Hepatic Carcinogenesis; #PH43-66- 508, "Potential Carcinogenicity of Cancer Che- otherapeutic Drugs"; #PH43-65-1000, "Re- search on Free Radicals and Alkylating Agents in Tobacco Smoke"; #PH43-65-1029, "Car- cinogenic Mycotoxins"). In addition, he has had a major share of the responsibility for the contract relationship with the Chicago Medical School (#PH43-65-67), under which a large- scale carcinogen screening program is main- tained and research on the fundamentals of cancer induction mechanisms is pursued. The pesticides carcinogenicity studies being conducted under contract by the Bionetics Re- search Laboratories, with close supervision by the Biology Branch scientists, has been progressing in a highly satisfactoiy manner. An exemplary number of valuable contribu- tions to the literature have been made with respect to pesticides chemistry; the long-term carcinogenicity experiments will be completed during the ensuing six months. The information to be obtained from these studies has been long awaited with great interest. The contract- supported research project on the carcinogeni- city of oxidized lipids, being conducted at Gen- eral Foods Corporation, has been augmented to include the determination in animals of car- cinogenicity of these lipid products. A new contract-supported research project has been initiated, in collaboration with the CCNSC, at the University of Pittsburgh for the determina- tion of the carcinogenicity of a number of chemo-therapeutic agents employed in clinical medicine. A complementary study of the car- cinogenicity of nitrofuran derivatives is being negotiated with the University of Wisconsin. The detailed descriptions of the established projects will be found in the contract narra- tives of this Branch. The Acting Branch Chief has been a prime force in the effort to obtain a collaborative relationship with Dr. Norton Nelson's group at New York University, for experimental car- cinogenesis involving asbestos products, poly- urethane foam products, peroxides, and epox- ides, and research into the carcinogenic and co-carcinogenic constituents of tobacco and to- bacco smoke. The Head of the Carcinogen Screening Sec- tion, while serving as an Assistant Project Officer on the Chicago Medical School contract, has been responsible for a number of other contract-supported carcinogen screening activi- ties and has maintained his own active in- house research program. These screening ac- tivities have resulted in the identification of about fifteen hitherto unrecognized carcino- gens. The Section's in-house research has made important contributions to the literature on the effects of androgens and estrogens on the susceptibility of rat liver to chemical carcino- gens; has developed a new system for the sepa- ration of urinary metabolites of carcinogenic aromatic amines; has described the forms in which carcinogenic amines and their metabo- lites circulate in the blood and transfer from one tissue compartment to another; has shown some of the effects of pituitary and adrenal hormones on the metabolism of N-hydroxy-N- 2-fluorenylacetamide to increase the level of the proximate carcinogen; has shown that some carcinogens combine with existing cel- lular and molecular targets rather than with the same targets during their periods of syn- thesis; and has demonstrated the competitive inhibition of binding of carcinogens at the target, using chloramphenicol. The utility of a standardized means of large-scale chemical car- cinogen screening and further progress on the synthesis and structural studies of naturally occurring carcinogens, such as aflatoxins, have been accomplished by contract-supported proj- ects. Members of the staff of this Section au- thored a number of review articles intended for wide circulation to alert chemists to the potential carcinogenic effects of the products of industry. The Bioassay Section has maintained con- tract-supported projects for the improvement of the methodology of carcinogen screening, while conducting its own research on the inhi- bition or anti-tumorigenic action of actinomy- cin-D. The screening methodology studies have emphasized the utilization of the mammary fat pad technique of DeOme and Faulkin, and the exploitation of the unusual susceptibility and promptness of response to carcinogenic 72 ANNUAL EEVIEW OF INTRAMURAL RESEARCH stimuli by animals in their neonatal period. Efforts are being made to evalute the useful- ness of animals born in particularly immature states, such as marsupials. More fundamental studies have demonstrated an anti-tumorigenic effect in skin carcinogenesis with 90/xg of actinomycin. In addition, actinomycin (1 to 90/xg) inhibited RNA synthesis in skin, whereas initiating amounts of DMBA produced a slight inhibition of RNA synthesis. This in- hibition occurred shortly after treatment with DMBA and was transitory. The inhibition of RNA synthesis by actinomycin occurred some- what later and was of a more lasting nature. The antitumorigenic effect of 90/xg of acti- nomycin was accompanied by an increase in protein synthesis, a change in mitotic index for interfollicular epidermis, and hyperplasia. Skin tumorigenesis was also inhibited by maleic anhydride, N-ethyl maleimide, and iodo- acetamide, agents which combine readily with -SH groups. These findings support the hypo- thesis that DMBA skin tumorigenesis may in- volve interaction with these -SH groups. Anti- tumorigenesis is not seen when DMBA is ap- plied 2 to 8 weeks after treatment with maleic anhydride, but is noted when this period is reduced to one week. Treatment with maleic anhydride for ten weeks produced inhibition, while treatment for one week was ineffective. Actinomycin was ineffective in inhibiting lung tumorigenesis even when administered at a high dose, despite a transient inhibition of RNA synthesis. Studies of DNA metabolism have been pursued, and interesting findings reported in the Individual Project Reports for this Section. A fascinating and important ob- servation has been the stronger carcinogenic stimulus by smooth-surfaced plastic implants than by those with rough surfaces. This find- ing may have some significance in the use of plastic prostheses in human beings. The transfer of Dr. Klein to Extramural Ac- tivities, NCI, will necessitate a recruiting ac- tion for the position of Head of the Bioassay Section. Dr. Klein's responsibilities as a Proj- ect Officer will be distributed among other members of the Branch. The Cytogenetics and Cytology Section is studying the quantitative effects of carcino- gens, employing models designed primarily to investigate somatic viruses. The systems in use include microbial, cell, and organ cultures and intact animals. Thus, this Section tends to bridge both the biological and chemical ap- proaches to carcinogenesis. A critical review of both the cancer and teratology literature made by the Section has resulted in the con- cept that the same environmental insult may be responsible for either cancer or malforma- tions or both. In this area, it has been demon- strated that important carcinogens, such as aflatoxins, uracil mustard, and urethan, all pro- duce malformations; the severity of the mal- formations being dependent upon the dose ad- ministered. The extent of the parallelism be- tween carcinogenic and teratogenic activity is being explored by the use of analogues and inhibitors of carcinogens. The effects of car- cinogens at the cellular level in terms of drug resistance and neoplastic transformation are under way using mouse and hamster material. Heart tissue, which is rarely the site of pri- mary malignancy, has been shown to undergo spontaneous neoplastic transformation result- ing in mesenchymal tumors. In addition, tissue culture cells underwent chromosomal changes consistent with an evolutionary pattern of genetic readjustment. Hamster embryo cells when exposed to carcinogenic polycyclic hydro- carbons underwent a series of morphological changes, including loss of contact inhibition, inhibition of mitosis, and death of many cells. Cells grown in the presence of the carcinogen resulted in permanent cell strains which pro- duce tumors when inoculated into hamsters. Transformed cells were resistant to the ex- posure of additional carcinogens while control cells exhibited toxicity. Current experiments are aimed at determining the possibility of selection of spontaneously transformed cells by the carcinogen, the acceleration of the car- cinogenesis process by the chemical or the ac- tual induction of the change by the carcinogen. Alkylating agents have been studied using spe- cific synthetic messenger ribonucleic acid (RNA) and using tissues of mice treated with uracil mustard. The synthetic messenger was inactivated by alkylating agents by either es- terification of chain terminal phosphate or NATIONAL CANCER INSTITUTE 73 cross-linking strains of RNA by forming di- guanyl derivatives. The administration of a high dose of uracil mustard to Strain A mice caused a marked inhibition of DNA synthesis in lung, liver, and kidney. Inhibition of total RNA synthesis and nuclear RNA synthesis was also observed at relatively high dosages of uracil mustard in the three tissues studied. Additional descriptions of these studies are contained in the Individual Project Reports of this Section and in the report on the Temple University contract (#PH43-66-68) with Dr. Daniel Swern. CHEMISTRY BRANCH The program of the Chemistry Branch is de- signed to clarify the fundamental nature of the interaction of carinogenic agents with living systems in the induction of cancer at the host, tissue, cellular, and molecular levels, and to in- vestigate the interaction of certain environ- mental agents with functioning biological sys- tems. The biochemical alterations in carcino- genesis and in the resultant tumor tissue are expressed as a number of phenotypic changes which include altered enzyme profile, altered responsiveness to environment, increased growth, loss of contact inhibition, and loss of specialized function. Since the phenotypic changes are heritable, they presumably reflect alterations in gene function, either at the ge- netic transcription level or at the translational level in the cytoplasm. The Chemistry Branch studies the biochemical nature of the factors controlling gene activity and messenger RNA translation, as well as the chemical nature of the genetic factors characterizing susceptibil- ity to carcinogenesis. Studies are made on the effects of carcinogens of various types, includ- ing chemical and viruses on geneaction sys- tems. The studies are performed on both the nature of the chemical and physical interaction of carcinogenic agents with various cellular components, and the effect of their interaction on the functional integrity of cellular control systems. Studies designed to elucidate the biochemi- cal nature and the time required for the initia- tion of skin tumorigenesis are in progress. One definitive result has shown that the time re- quired for the initiation of skin tumorigenesis is of short duration, of the order of 1 to 2 days. In addition, the initiation of skin tumori- genesis was found to depend on functioning gene activity and potent carcinogenic agents did not elicit initiation of tumorigenesis when the inhibitor of gene activity, actinomycin D was present. In related studies, actinomycin D was found to decrease the amount of carcinogen bound to skin DNA by 35% and not to affect the amount of carcinogen bound to total skin pro- tein. In other studies actinomycin D as well as the carcinogen, 7,12-dimethylbenzanthra- cene (DMBA), were both found to inhibit DNA and RNA synthesis. Investigations on the interaction of liver carcinogens with cellular macromolecules have demonstrated that three potent liver carcino- gens, N-hydroxy-AAF, 3'-methyl-DAB, and aflatoxin B 1( interact with DNA and cause a reduction in the RNA to DNA ratio of puri- fied liver nuclei. In addition, these agents were found to inhibit the incorporation of radioac- tive precursors into nuclear RNA. Thus these carcinogenic agents are altering gene activity at the level of RNA synthesis. Investigations on the mechanism of malig- nant transformation by Rous sarcoma virus (RSV) has shown that the initial stage of the transformation process requires DNA synthe- sis, and that subsequent stages allowing for the growth of RSV require the continuous partici- pation of cellurlar DNA. These important re- sults have clearly increased our understanding of the nature of malignant transformation by Rous sarcoma virus. As a corollary to these studies techniques have been developed for the growth of Rous sarcoma virus in liquid spin- ner cultures and in cells suspended in agar. The ability of cells to grow under these con- ditions requires their prior infection by Rous sarcoma virus. These techniques will permit the large scale production of Rous sarcoma virus. Investigation of vesicular stomatitis virus infection has shown that it induces in infected cells a new enzyme capable of synthesizing RNA. This enzyme did not appear in cells in- 74 ANNUAL REVIEW OF INTRAMURAL RESEARCH fected with Rous sarcoma virus or Rous-asso- ciated virus. Methods have been developed for the isola- tion of macromolecular components of liver chromatin under conditions in which the physiological relationship between these com- ponents is minimally disturbed. Using- some of these techniques, DNA molecules with a mole- cular weight of 3 times 10 8 daltons have been isolated from chick embryo. The distribution in DNA size was found to be unimodal, which is unlike the size distributions of metaphase chro- mosomes in this organism. An accurate deter- mination of DNA size will permit the investi- gation of the relationship between DNA size and carcinogenesis. In other studies DNA from mouse liver nuclei and mitochondria has been isolated and examined in respect to molecular weight and buoyant density. These studies have found that the minor component of mouse liver DNA, the satellite band, comprises about 10% of the nuclear DNA and is not found in mitochondrial DNA preparations. The molecular basis for the mutagenic action of hydroxylamine, nitrous acid, formaldehyde, and the carcinogen, N-methyl-N-nitroso-ure- than has been investigated. Hydroxylamine acts by altering the coding properties of de- oxycytidylic acid from guanylic acid incorpo- ration to adenylic acid incorporation. Formal- dehyde causes inactivation of the template properties of polymers, and the carcinogenic N-methyl-N-nitroso-urethan alters several parameters of template activity which are now being studied. Studies on the removal of messenger RNA- like activity from rat liver microsomes have found that this activity can be removed with- out disturbing the capability of microsomes to incporporate amino acids in the presence of synthetic messenger RNA. Indeed the removal of endogenous messenger RNA increases the sensitivity of microsomes to added synthetic messenger RNA in respect to both the ability to bind synthetic messenger RNA and the ac- tivity of the synthetic messenger RNA in di- recting amino acid incorporation. The effects of carcinogens, non-carcinogenic drugs, or environmental chemicals on the in- duction of specific drug metabolizing enzymes, and the mechanisms of this induction have been under study. The induced enzymes control to a significant extent the duration of drug ac- tion; their altered activity may be responsible for often-observed altered drug resistance and susceptibility. In addition, these hazards of drugs and environmental agents are capable of altering normal metabolism. Thus, pheno- barbital was found to increase the messenger RNA content of microsomes. It also increased the sensitivity of microsomes, but not ribo- somes, to the addition of synthetic messenger RNA. Phenobarbital also increases the endo- plasmic reticulum of the cell. Investigations have also been made of the effect of some of these agents on the genetic apparatus, and in particular, on RNA polymerase activity of the nucleus. Methods, utilizing the zonal centrifuge, are being developed for the preparation and iso- lation of macromolecules and cellular and sub- cellular fractions of biological systems. Separa- tions have been made of E. coli RNA, E. coll ribosomes, rat and mouse liver ribosomes, mi- crosomes, mitochondria, and whole cells. In ad- dition, preliminary work suggests the feasibil- ity of separating cell membranes and nerve endings. These techniques will greatly enhance the ability to isolate large amounts of cellular components needed for biochemical investiga- tions. An extensive electron microscope study of transformed tissue culture cells is in progress. This study has revealed that the so-called non- producer cells release morphologically complete but noninfectious viral particles. In addition, certain surface morphology appears altered in the transformed cells. In addition, many of the subcellular materials separated by the zonal centrifuge are being examined by electronc mi- croscopy. The electron microscopic pictures have contributed a semi-quantitative estimate of the number and kinds of riobosomes of vary- ing polymeric units. The distribution of these subcellular particles in the tumor and normal cells is being compared. The electron micro- scope has also been applied to the study of the epithelial cells of the bronchial tree in animals exposed to air pollutants. The electron micro- scopy study has provided evidence for a differ- NATIONAL CANCER INSTITUTE 75 ent degree of response and rate of recovery in the epithelial cells at different levels of the bronchial tree. Studies in the Section on Proteins have in the past year included the use of zone electro- phoresis in acrylamide gels as an analytical and preparative technique, studies of serum proteins in man and rodents, correlations be- tween serum haptoglobin type and disease, and studies on the preparation and properties of haptoglobin type 1-1 in human serum. A study of several hundred normal human serum specimens has confirmed the high reso- lution of "disc" electrophoresis in acrylamide gels. The major polymorphic systems demon- strated by such analyses are the group-specific component, transferrin and haptoglobin. Most variations between individuals could be attrib- uted to these systems. Other variations also exist, but could not be similarly explained. The high resolving power of such gels has been adapted to preparative purposes with ex- cellent results in the case of hemoglobin and haptoglobin type 1-1. Both these proteins have been isolated in highly purified form, with less than 1% detectable impurities. The interaction of haptoglobin and hemoglo- bin has been studied with this purified mate- rial. This interaction passes through an inter- mediate in which one-half molecule of hemo- globin reacts with one molecule of haptoglobin. This complex may add another one-half mole- cule of hemoglobin to form a fully saturated complex. A detailed analysis of this reaction was possible. An analysis of haptoglobin types in mice has shown that inbred strains differ with respect to whether they have haptoglobin. AKR and C 3 H mice, although normally without serum haptoglobin, are easily induced to produce it. Variations between mouse strains in this con- nection may be related to ease of "spontan- eous" tumor formation. Further data on haptoglobin types in hu- man keukemia confirm that such patients have an excess of the Hp 1 gene, over normal groups. Similar, but less marked, differences are found between control groups and female patients with carcinoma of the breast and male patients with carcinoma of the lung. A report showing an excess of Hp 1 in patients with juvenile rheumatoid arthritis could not be confirmed. An improved haptoglobin typing method was devised and used in these studies. VIRAL ONCOLOGY Activity in the tumor-virus program of NCI was further increased during the past fiscal year. An increase during the preceding year had brought about a split of the Laboratory of Viral Oncology and the creation of an addi- tional laboratory, the Laboratory of Viral Car- cinogenesis as well as of the Office of the Associate Scientific Director of Viral Oncology to coordinate tumor-virus research within the two laboratories. With the reorganization of NCI during the past fiscal year, this office and its constituent laboratories were transferred to the Office of the Scientific Director for Etiol- ogy, where they were consolidated with the tumor-virus program already in that Office within the branch previously designated as the Virology Research Resources Branch. The new designations of these laboratories and branches and their respective Branch Chiefs, are as follows: (1) Viral Biology Branch, Dr. A. J. Dalton, Chief; (2) Viral Carcinogenesis Branch, Dr. R. E. Stevenson, Chief; and (3) Viral Leukemia and Lymphoma Branch, Dr. F. J. Rauscher, Jr., Chief. The Viral Carcinogenesis Branch, which su- perseded the former Virology Research Re- sources Branch, continued its functions with respect to the development and management of resources essential to tumor-virus research, and assumed additional responsibility for di- rect program activities in the field of naked DNA tumor viruses. Pending completion of the new NCI building and the availability of lab- oratory space, the NCI, through the Viral Car- cinogenesis Branch, will embrace an intramu- ral research program on these DNA tumor viruses for the first time. Key staff scientists who will participate in this research as well as in other program activities have already been recruited. This Addition of scientific personnel and expertise has made possible the activation under contract of new major activities in sup- port of cancer- virus research, such as: (1) the 76 ANNUAL REVIEW OP INTRAMURAL RESEARCH viral diagnostic laboratory which will identify known agents and differentiate new agents turned up in the search for human cancer vi- ruses by scientists participating in special pro- grams of NCI; and (2) research on the devel- opment and production of simian virus re- agents, which will be critical for monitoring for and identification of contaminant simian vi- ruses if a human leukemia or other cancer virus is found which is transmissible in these pri- mate species. The oncogenic naked DNA viruses, which in- clude 5 of the human adeno-viruses, were dis- covered by virologists employing the tissue culture techniques of classical virology, and their intensive investigation has been pursued primarily in general virology laboratories. Up until recently the principal activity of the NCI in this area has resided in support of the com- munity of virologists engaged in oncological in- vestigations, through its virology research re- sources program. Developments during the past two years, and particularly during the last fis- cal year have revealed neoantigens in tumor cells of hamsters and other rodents induced by human adeno and other oncogenic naked DNA viruses, although infectious DNA virus is not reproduced in the oncogenic interactions. At the joint suggestion of Doctors Albert Sabin and Robert Huebner, an ad hoc group of leading virologists concerned with this problem was brought together for several discussions during the past year, under the sponsorship of the Viral Carcinogenesis Branch. The question was examined whether the time was ripe for a joint programmed activity in search of neoan- tigens in human cancers that are characteris- tic for certain known human viruses, including the adenoviruses. Although the time seems near when such studies might be initiated, it was concluded that refinements in existing tech- niques must be made before a satisfactory study can be launched. This burgeoning collec- tive effort may presage a future major program activity of NCI. The Viral Leukemia and Lymphoma Branch and the Viral Biology Branch have continued their singular as well as their joint activities involving the longer known membrane-bound tumor viruses of the types associated with nat- urally prevalent and transmissible neoplasms of animals. These include: (1) the C type RNA viruses associated with avian and murine leu- kemia and related neoplasias, and which bud from cytoplasmic membranes; and (2) "herpes- like" particles such as the Lucke frog kidney carcinoma virus which matures in the nucleus, and probably contains DNA. Viruses of both of these types are reproduced during, and remain associated with the overt neoplastic disease which they induce. In studies directed toward speeding the search for comparable agents in human leu- kemia and lymphoma, scientists of the Viral Leukemia and Lymphoma Branch have adapted two additional highly sensitive immunological techniques to the detection of murine leukemia virus antigen, and are now investigating appli- cations to the human problem along the same lines as previously reported for the fluorescent antibody technique (i.e., the detection of spe- cific, leukemia associated, antigens and/or anti- bodies which could be of viral derivation). The additional techniques, which have proven more sensitive than the fluorescent antibody tech- nique in murine systems, are: (a) the micro- Ouchterlony (double diffusion in gels) precip- itin reaction; and (b) specific hemagglutination of antigen-coated tanned erythrocytes. Consid- erable progress has also been made in the con- trolled degradation of murine leukemia viral particles into morphological subunits, and puri- fication of the subunits by density gradient centrifugation. The Viral Biology Branch continues to play a leading role in the search for viral etiological agents associated with human leukemia and lymphoma, using particularly the methods of electron mircroscopy and tissue culture. In col- laboration with clinical staff members 141 plasma specimens from 120 separate cases of leukemia were studied during the past year. Of these, about 13% were found to contain virus- like particles resembling the C type virus parti- cles of animal leukemias. This is approximately the same proportion of positive specimens ob- served in previously reported studies. The re- sults are consistent with those obtained in neo- plastic diseases of animals induced with low NATIONAL CANCER INSTITUTE 77 doses of RNA tumor viruses or in the naturally- occurring animal diseases. Membrane-bound virus particles of the "herpes" type have been observed by electron microscopy in 3 of 4 additional cell lines of Burkitt lymphoma sent to Dr. Manaker from Nigeria by Dr. Pulvertaft (University of Iba- dan). This brings to 9 the number of estab- lished cell lines of Burkitt lymphoma that have been investigated in several laboratories for the presence of virus-like particles. The "herpes-like" particle has been observed in 8 of the 9 cell lines. Similar particles were also ob- served in tissue culture cells from: (1) an in- duced myeloid leukemia of a monkey (original specimen supplied by Dr. Margaret Kelly of the Chemotherapy area of NCI); and (2) a human patient with chronic myeloid leukemia. The lat- ter observation brings to 5 the number of es- tablished cell lines of human leukemia that have shown "herpes-like" particles, out of a total of 12 investigated by participants in the Special Virus-Leukemia Program. Whether the "herpes-like" agent is a ubiqui- tous "passenger" virus, or whether it is a se- rious contender for an etiological role is not yet clear. It cannot be propagated in any cell line thus far tried, other than the explanted malignant cells in which it is found naturally. It shows no antigenic relationship to other members of the herpes group, or any other known virus thus far compared. In addition to basic research on membrane- bound tumor viruses, and experimental partici- pation in the search for counterpart human agents, members of the Viral Biological Branch are Chairmen of two major Segments and Vice Chairmen of two Segments of the Special Virus Leukemia Program. They also serve as Project Officers and supervisors of developmental re- search under contract amounting to approxi- mately $5 million. VIRAL BIOLOGY BRANCH Electron Microscopy Section Highlights of the results of collaborative studies on animals in which electron micro- scopy played an important role include the demonstration that the etiologic agent of Mo- loney sarcoma is indistinguishable morphologi- cally from the murine leukemia virus; the vis- ualization of the process of replication of avian-type viruses in brain tumors of the dog, induced by Rous sarcoma virus; and the dem- onstration of the presence of particles in tissue culture cells derived from the buffy coat of a monkey with chronic myelogenous leukemia having the same morphology as those associ- ated with tissue culture isolates from Burkitt lymphoma. In studies on human material, it has been found that the virus particles associated with Burkitt lymphoma differ from known herpes type viruses by the acquisition of a granular coat at some time during maturation. Also worth of note is the finding of Burkitt type particles in tissue culture cells derived from the buffy coat from a human case of chronic myeologenous leukemia. These findings may still be explained on the basis of passenger virus, but the presence of these particles in 8 out of 9 Burkitt lymphoma isolates and in iso- lates from 5 human cases of myeologenous leu- kemia (including the 4 Roswell Park isolates) suggests that something more than simple co- incidence is involved. In the screening program, 19 out of 141 plasmas from leukemic patients were found pos- itive for virus-like particles, an average of something more than 13%. A shift in emphasis is planned for this work with increased con- centration on lymph node biopsy material and less on plasma pellets. Microbiology Section Work in this Section has included the suc- cessful propagation of Burkitt lymphoma iso- lates, EB-1, 2, and 3 of Epstein and the Jiyoye, Kude, Ogun, and Ragi lines of Pulver- taft. Modification of the culture media and other experimental interventions were at- tempted with the aim of increasing virus yield. In addition the crossing of species barriers has been demonstrated by tumor induction fol- lowing intracranial inoculation of high titer Rous sarcoma virus into rabbits, guinea pigs, hamsters, cats and dogs. The common forms of tumors have been gliomas and meningiomas, 78 ANNUAL REVIEW OF INTRAMURAL RESEARCH although the tumors in guinea pigs have been difficult to classify. An experiment involving the feeding of mos- quitoes (Aedes egypti), first on Burkitt tissue culture cells, then on a monkey resulted even- tually in a modified peripheral blood picture and enlarged lymph nodes in the monkey. There has been no evidence of the development of the leukemic state, but this experiment should be followed up. It should be noted that the head of the Sec- tion on Microbiology spends fully two-thirds of his working hours on administrative matters connected with the SVLP. Virus Studies Section Extracts from the SL-1 isolate from Burkitt lymphoma suspended in 5% dimethyl sulphox- ide (DMSO) inoculated intracranially into newborn hamsters induced CNS symptoms, eventually as early as 5 or 6 days after inocu- lation. Members of the staff at the Pfizer Lab- oratories, Maywood, N.J., have confirmed these findings for material obtained from the Virus Studies Section but have been unable to confirm them starting with fresh homogenates of SL- 1 or EB-2 Burkitt isolates. To date the virus which undoubtedly is present has not been vis- ualized and it is important to determine whether it is of human origin or is a hamster virus activated by the treatment. Virus Leukemia Section Studies have continued on the virus induced murine sarcoma. Histopathological analysis in- dicates that it is a rhabdomyosarcoma with cross striation sometimes apparent in the larger of the two cell types. The large cells when prepared as whole mounts are very similar in appearance to cells present in human rhabdo- myosarcoma. Attempts to pass this virus into cortisone- treated rats have been successful with tumors similar to those induced in mice appearing within 1 to 2 months. Continued passage in rats has resulted in death within 24-36 hours after inoculation. This phenomenon is at pres- ent under study. An analysis of the results of the examination of human leukemic plasma pellets has demon- strated that there is a direct correlation be- tween high white cell counts and plasma pel- lets positive ofr virus-like particles. No other correlation has been found. It has been reported previously that an agent present in the plasma ceil tumor MPC-2 in- duces reticulum cell sarcomas. Since these tu- mors are very similar to Hodgkins sarcoma in man, it would be important to determine if they are consistently induced by a viral agent. Stud- ies under way suggest this as a possibility. In other experiments an agent has been demon- strated to be present in Sarcoma-37 which in- duces bone lesions in mice. These lesions are similar to the osteopetrosis induced in chick- ens by viruses of the avian leukosis complex. Evidence to date suggests that the agent from S-37 is the same as or closely related to the Moloney leukemia agent. This in turn suggests the existence of an important and fundamental similarity between the range of tumor types induced by viruses of the avian leukosis com- plex and viruses of the murine leukemia fam- ily. Improved transparent chambers were in- volved in the development of the information obtained from the last two studies. It should be noted that the head of the Virus Leukemia Sec- tion spends fully two-thirds of his working hours on administrative matters concerned with the SALE segment of the SVLP. VIRAL LEUKEMIA AND LYMPHOMA BRANCH Introduction The Viral Leukemia and Lymphoma Branch was organized and officially approved in De- cember of 1965 as a part of the overall reor- ganization of the National Cancer Institute. The personnel and intellectual nucleus of this Branch were derived almost entirely from the Laboratory of Viral Oncology. Dr. W. Ray Bryan, as Chief of this Laboratory, assembled and guided a group whose outstanding compe- tence and productivity became internationally acknowledged. Organizationally the Branch as proposed will include sections on Comparative Viral Oncology, Immunology, Molecular Virol- NATIONAL CANCER INSTITUTE 79 ogy, Ultrastructural Studies, and on Virus and Disease Modification. Laboratory personnel wh are currently functioning- as Section Heads are: Dr. Mary Fink, Immunology Section, with 8 support personnel; Dr. Timothy E. O'Connor, Molecular Virology Section, with 5 support personnel; Dr. Robert F. Zeigel, Ultrastruc- tural Studies Section, with 4 support person- nel. In addition, the transfer of Dr. Michael A. Chirigos and 4 support personnel from the Chemotherapy area to the Viral Leukemia and ymphoma Branch will be consummated with- in this fiscal year. Dr. Chirigos will head the Virus and Disease Modification Section. Re- cruitment actions of key scientists are cur- rently being processed. These and other actions and activities are being conducted in a manner that will take advantage of currently available space and equipment and in a manner that will allow a minimum of delay between the current level of activity and the expanded level which will be possible as soon as the new cancer building (Building 37) and the emergency vi- rus isolation building are completed (approxi- mately January 1968). The principal activities conducted within the Branch during fiscal year 1966 are discussed briefly according to the following categories: (a) research and other activities conducted within the Branch, and (b) Special Virus- Leukemia Program. Research and Other Activities Conducted Within the Branch Dr. Mary Fink in collaboration with other members of the Branch, Institute, and outside laboratories has continued and extended. her studies which are designed to evaluate exist- ing techniques and to devise new methods for the serological and immunological detection and characterization of viruses from ani- mal and human neoplasms. In particular, during the past year, these studies have been concerned with the techniques of immuno- fluorescence, micro-Ouchterlony (double diffu- sion in gels) and with specific hemagglutination of antigen coated tanned erythrocytes. In a lon- gitudinal study of the bone marrows from 26 cases of acute human leukemia, 79% of the bone marrow specimens from patients in re- lapse showed positive immunofluorescence. Only 25 % of the bone marrow specimens from patients in remission showed positive immuno- fluorescence. In 6 of 8 human leukemia patients, followed through several cycles of relapse- remission, there was no evidence of the pres- ence of immunofluorescent cells during remis- sion. Dr. Fink and co-workers continued to test for immunofluorescence many different mate- rials received from investigators throughout this country and abroad. In regard to the 21 different lines of human leukemia or lymphoma cells now maintained in tissue culture, the im- munofluorescent findings parallel the finding of herpes-like virus in these cultures. In collab- oration with Drs. Manaker and Dalton, several of these human lymphoma cell lines were ex- amined for the purpose of establishing the opti- mal time during tissue culture for demonstrat- ing immunofluorescence. These studies showed the fluorescence increases up to a maximum on the fourth or fifth day after fluid change. Vi- rus particles are also more numerous as de- tected by electron microscopy 4 or 5 days after fluid change. These and other studies designed to control the specificity of the immunofluores- cent reaction have been essentially confirmed in the laboratories of Drs. Yohn and Grace of the Roswell Park Memorial Institute and in lab- oratories of Dr. Zarafonetis at the University of Michign. A microdiffusion precipitation method using agar gels (Ouchterlony) was successfully adapted to the detection of several murine leukemia viruses. With the Rauscher strain, virus could be detected in the plasmas of viremic BALB/c mice as soon as 13 days post infection. Preliminary studies have indi- cated that the Rauscher and Friend viruses share at least 2 antigens but differ in at least 1 and that the Moloney leukemia virus shares at least 1 antigen with the Rauscher virus. This technique can be used not only to detect murine leukemia viruses but also to quantitate the amount of virus present in various systems and to differentiate the various murine leu- kemia viruses. A third technique which has been developed and applied to the detection and assay of murine leukemia virueses and their antiserums is that of hemagglutination of anti- gen coated tanned erythrocytes. This tech- 80 ANNUAL REVIEW OP INTRAMURAL RESEARCH nique is not only useful as a quantitative measurement of antibody against murine leu- kemia viruses but also, by blocking the reac- tion with antigen, the technique becomes an exquisitely sensitive test for the presence and amount of virus in materials of unknown po- tency. As little as 0.2 micrograms per ml of antigen protein can be detected with this tech- nique. Each of the 3 above mentioned techniques are being intensively applied to the detection and quantitation of viruses and/or virus anti- gen presumed to be present in selected mate- rials obtained from human leukemia and lym- phoma patients. These in vitro monitoring techniques are of paramount importance to the human leukemia problem because it has not yet been possible to develop a sensitive laboratory animal system (including primates) which will support replication of and/or disease induction by candidate human leukemia viruses. It be- comes necessary, therefore, to employ in vitro serological techniques not only to monitor for the presence of virus in laboratory systems but also to conduct sero-epidemiological surveys for the presence or absence of experience with selected viruses in different human population groups. Dr. Robert Zeigel and staff continued their numerous collaborative activities with scien- tists of this Branch as well as those associated with other areas of NIH. In collaboration with Dr. Mary Fink, they obtained electron micro- scopic evidence for the presence of significant amounts of virus in mice 5 days following in- fection with a murine leukemia virus (Rauscher strain). This finding is important because it shows that large quantities of virus are present in animals shortly after infection and long before the infection process culmi- nates in frank disease. Dr. Zeigel's studies have also shown that tissue cultured cells which support active repli- cation of murine leukemia viruses are also ca- pable of phagocytosing virus particles produced and liberated by these same cells. This finding suggests a mechanism for a cyclic reinfection of cells and/or a means for the clearance and potential inactivation of extracellular infec- tious virus. Studies performed in collaboration with Dr. Gordon Theilen, a visiting investigator of NCI during fiscal year 1966, established the ultrastructure of avian reticuloendotheliosis vi- rus. Although this virus produces a leukosis like disease, it appears to be morphologically different from other viruses of the avian leu- kosis complex. During the past year Dr. Zeigel confirmed a extended his findings that type "A" virus par- ticles are associated with all tumors induced in conventional and germfree BALB/c mice with tumor viruses and/or with chemical carci- nogens. It has been shown that the endoplasmic reticulum of cells "infected" with type "A" par- ticles is substantially modified. This finding is of potential importance in view of the possibil- ities that "A" type particles may (a) serve as biological precursors for the eventual appear- ance of leukemogenic "C" type particles, (b) interfere with the replication of virus or the induction of disease by "C" type particles, and (c) may serve as a helper virus for the repli- cation of and/or diease induction by "C" type particles. Studies conducted in the laboratory of Dr. Due Nguyen, a visiting investigator, who joined the VLLB in August of 1965, are aimed at the application, to tumor viruses, of classical detection and bio-assay methods developed and used for studies of the necrotizing or nontu- morigenic viruses. In particular, he has at- tempted to device an in vitro method for the bio-assay of murine leukemia viruses based on the production of interferon or the detection of interference of necrotizing viruses by the noncytopathogenic murine leukemia viruses. Studies are well under way in his laboratory to apply the ferritin labeled antibody tech- nique of electron microscopy, used so success- fully with influenze virus, for the detection and localization of murine leukemia viruses in in- fected cells. Dr. Nguyen was successful in es- tablishing a line of embryonic rat kidney cells in tissue culture. These cells were infected with a murine leukemia virus (Rauscher strain) and appear to support the replication of more recoverable murine leukemia virus than any other cell line available for this purpose. The goal of studies conducted by Dr. Tim- othy O'Connor as principal investigator is to NATIONAL CANCER INSTITUTE 81 provide an integrated background of physical, chemical and immunological data on animal leukemia viruses toward a determi nation of the possible association of a virus or viruses with human leukemia and lymphoma. The predomi- nant antigenic activity of murine leukemia virus (Rauscher strain) purified by density gradient centrifugation was shown by 3 differ- ent immunological techniques to be associated with the virus particle. It was also shown that both the Rauscher and Friend strains of murine leukemia virus after extensive purification on density gradients still showed a significant re- action with antiserum prepared against nor- mal BALB/c mouse tissue antigens. This was not entirely unexpected since collaborative studies with Dr. Guy de The showed by elec- tron microscopic techniques that purified mu- rine leukemia viruses were coated with low amounts of host-derived enzymes. Experiments are in progress to determine whether these virus-associated, host-derived antigens arise by adsorption or from true structural integration into the viron. Treatment of murine leukemia viruses with a combination of ether and the non-ionic deter- gent, Tween 80, resulted in emulsions which could be separated into ether and aqueous phases. Density gradient centrifugation of the ether extract yielded low density materials which contained considerable amounts of viral- specific antigen. It was not possible to definitely determine the morphological characteristics of these components. Density gradient centrifu- gation of the aqueous phases yielded materials having a density of 1.24 to 1.27. These mate- rials, therefore, are much more dense than the intact virus and in addition did not react with viral-specific antisera or with antiserums pre- pared to BALB/c mouse antigens. Electron microscopically, 2 types of "subviral" particles were seen. The most common type was a pol- ygonal structure, the center of which appeared to be occupied with a fibrillar material of low electron density. These particles may represent the naked nucleoids of the mature virus par- ticle. The second type of subviral structure appeared as a rigid circular structure with an electron lucent core and in some instances ap- peared to have a helical structure composed of morphological subunits. These structures may represent the nucleoids of immature virus par- ticles. These studies are important because they may make it possible to compare the chem- ical and physical characteristics of intact and degraded murine leukemia viruses to similar characteristics of viruses isolated from human leukemia and lymphoma materials. Dr. O'Con- nor's studies have made it possible to apply density gradient centrifugation to the routine quality control of virus production. This has been done in his own laboratory and, with his monitoring collaboration, is now being rou- tinely conducted in commercial laboratories engaged in the production of large quantities of murine leukemia viruses under contract to the National Cancer Institute. This test is sim- ple and rapid and provides an excellent esti- mate of the relative homogeneity and physical titer of different batches of murine leukemia viruses produced in different laboratories at different times. Members of the Branch served as Project Officers on 9 contracts, the total funding level for which was approximately $3.7 million. The workscopes of these contracts include funda- mental and applied studies on animal and hu- man leukemias, research services, and the pro- duction of large quantities of highly purified concentrates of infective murine leukemia vi- ruses. One of these contracts deserves special mention because it provides a critical resource and unique technical competence to the re- search activities of the Branch as well as to the entire Special Virus-Leukemia Program. The objectives of this contract (with Bionetics Research Laboratories, Inc.) are: (a) to deter- mine whether newborn, mother-deprived pri- mates of various species are susceptible to the oncogenic and/or leukemogenic effects of known viruses and of candidate viruses recovered di- rectly from man, and (6) to test available means and develop new means to enhance the susceptibility of primates to virus replication and/or disease induction. Pursuant to these ob- jectives, the contractor has provided, largely from his own breeding colony, over 700 new- born viable primates suitable for inoculation. These animals have been inoculated with high 82 ANNUAL REVIEW OF INTRAMURAL RESEARCH priority materials received from over 50 differ- ent investigators from 40 different laboratories throughout this country and abroad. Members of the Branch presented, by invi- tation, over 40 lectures on their studies and on the studies of others at various meetings and to many different research groups in this country and abroad. Considerable time was de- voted to training, advising and collaborating with many different scientists from intramu- ral and extramural laboratories. In particular, Dr. Mary Fink and staff have trained investi- gators in the theory and use of the immuno- fluorescent technique reported by Drs. Fink and Malmgren for the detection and monitoring of an antigen which appears to be associated predominantly with the cells of human leuke- mia and lymphoma patients. Similarly, Dr. Timothy O'Connor has trained visiting investi- gators in his own laboratory as well as in lab- oratories throughout this country in the use of density gradient centrifugation and other bio- chemical and biophysical techniques for the purification and characterization of viruses and of subviral products. During the past year members of the Branch have dispensed liter quantities of different types of murini leuke- mia viruses, and advise on effective utilization of these viruses to over 120 different investi- gators. Drs. O'Connor, Fink, and Rauscher served on several different panels and committees among which were the Field Studies Contract Review Committee, the NCI Primate Study Group, Program Segment Working Groups of the Special Virus-Leukemia Program, and as members of the Civil Service Microbiology and Chemistry Panels. Special Virus-Leukemia Program During past year overall management of the Special Virus-Leukemia Program, which was planned and implemented in September of 1964 through a special appropriation, was placed within the Office of the Chief, Viral Leukemia & Lymphoma Branch. The main objectives of this Program are: (1) to determine whether viruses comparable to those now known to be associated with avian and murine leukemia are etiological agents of human leukemia, and (2) to develop an effective vaccine or other means for the prevention and/or control of human leukemia and lymphoma if such etiolog- ical agents are found. The main assumption or working hypothesis on which the overall Program is based is that at least one virus is an indispensable element for the induction (directly or indirectly) of at least one kind of human leukemia (including lymphoma) and that the virus persists in the diseased indi- vidual. The overall Program was originally planned and modified during and following numerous discussions with key program lead- ers and research scientists of NCI as well as with university and industrial personnel expert in the general areas of virology, oncology, chemotherapy, etc. From the research stand- point the program is divided into 4 major areas of effort: Human Leukemia Etiology and Prevention, Special Animal Leukemia Ecology Studies, Biohazards Control and Containment, and Human Leukemia Therapy. Operationally the Program has been divided into seven pro- gram segments: Developmental Research, Test- ing and Monitoring, Resources and Logistics, Epidemiology, Special Animal Leukemia Ecol- ogy Studies, Human Leukemia Therapy, and Biohazards Control and Containment. These working groups, which with the exception of the Human Leukemia Therapy Segment, are chaired by senior scientists within the Etiology Area who are responsible for the development of research programs in their respective seg- ments within the context of the overall plan. Approximately 70 of 180 projects, which cur- rently make up the program plan, are being conducted by investigators in governmental laboratories and clinical facilities, in universi- ties, in nonprofit laboratories, and in commer- cial facilities. Selection and implementation of all projects are done on the basis of both sci- entific excellence and high priority relevance in terms of the integrated program. NATIONAL HEART INSTITUTE CARDIOLOGY BRANCH The fundamental objective of the research program of the Cardiology Branch, NHI is to provide an increased understanding - of the de- rangements in cardiac function which occurs in various forms of heart disease. In the pur- suit of this long-range objective it has become evident that the mechanics and energetics of the normal contractile process are poorly un- derstood and that gross clinical and hemody- namic studies on diseased hearts provide only superficial descriptions of disease processes. Accordingly, efforts have been underway for several years to analyze the behavior of the normal and diseased heart as a pump. More recently, these approaches have been broadened and cardiac performance is now also being analyzed from the point of view of the heart as a muscle. As in the past, investigations are simultaneously being carried out on isolated heart muscle, intact canine hearts, diseased canine hearts, as well as on patients with nor- mal and diseased cardiovascular systems. Mechanics and Energetics of Myocardial Contraction: Studies on Isolated Heart Muscle The active state of muscle has been defined as a mechanical measure of those processes in the contractile elements which generate force and are responsible for shortening. In a study designed to determine the manner in which the active state in heart muscle is established and dissipated it was found that: (1) in con- trast to skeletal muscle, the onset of maximum active state in heart muscle is delayed, de- veloping 100 to 150 msec, after the first evi- dence of active state; (2) the maximum active state is maintained for about 100 msec, and does not decline until just prior to the develop- ment of maximum active tension; and (3) inotropic interventions which alter the con- tractile state of the muscle, such as changing frequency of contractions, or norepinephrine, accelerate the onset, increase the intensity, and hasten the decline of the active state. There has long been controversy as to whether stimuli which exert a positive ino- tropic effect on the myocardium also induce a change in the true compliance of the heart muscle. Since these inotropic interventions are generally associated with substantial incre- ments in the force of contraction, the possi- bility was considered that the changes in com- pliance, were they to occur, might be mediated through increases in systolic force per se rather than through a direct effect of the ino- tropic intervention on the contractile system of the muscle. However, if changes in diastolic compliance of heart muscle are indeed due to alterations in the contractile system, they should be apparent whether a muscle is con- tracting under isotonic or isometric conditions. On the other hand, should changes in systolic force alone explain these findings, then no change in compliance should be observed under isotonic conditions, but should occur when- ever systolic force is increased, regardless of whether or not an inotropic influence is intro- duced. In a study on isolated papillary muscles it was observed that postextrasystolic poten- tiation, norepinephrine or calcium administra- tion produced no change in diastolic compli- ance in the isotonically contracting muscle or in the afterloaded muscle in which increased shortening occurred with no change in force. However, isometric contraction consistently in- duced a small fall in diastolic tension when the force of contraction rose. It was concluded that inotropic interventions per se do not in- duce a true change in myocardial compliance. However, diastolic compliance may increase 83 84 ANNUAL REVIEW OF INTRAMURAL RESEARCH when the force of contraction rises. These findings provide evidence for a series viscous component in heart muscle which is altered when changes in contractile force occur. A study has also been initiated to compare the magnitude of stress relaxation that occurs in series viscous elements of isolated cardiac muscle with that occurring in another com- ponent which had not been defined previously and which has been termed the "parallel vis- cous component." This parallel viscous com- ponent appears to be several times as extensi- ble as the series viscous component. It also appears that the interplay between effects due to these two viscous elements and their op- posite effects on the resting length of the con- tractile element may serve to explain many of the phenomena associated with homeometric autoregulation of the heart. The series elastic is one of the fundamental components of heart muscle and its properties must be characterized in order to allow an understanding of the behavior of the con- tractile elements. Accordingly, the series elas- ticity of cat papillary muscle was determined by a variety of techniques and revealed an extension of 5 to 10% of muscle length at a developed force of 10 gm. Inotropic interven- tions did not alter the series elastic compon- ent and calculation of contractile element ve- locity revealed that in auxotonic contractions contractile element velocity exhibits a secon- dary rise during the transition from the isometric to the isotonic phase. Data were also obtained to support the position that a portion of the series elastic is in series with both the contractile elements and the parallel elastic elements. Although ultrastructural and biochemical similarities are known to exist in the contrac- tile systems of cardiac and skeletal muscle fundamental differences in their force gener- ating processes have been postulated. This view is based on a ten-fold disparity in force generating capacity between the two types of muscle, which has previously been reported. However, an accurate comparison of the force generating capacity of cardiac and skeletal muscle depends upon establishing a maximum contractile state of cardiac muscle and utiliz- ing electronmicroscopic analyses to correct for its greater proportion of non-contractile ele- ments such as mitochondria, nuclei and inter- filamentous spaces. When this is applied to the cat papillary muscle preparation, the max- imum contractile force of cardiac muscle achieved during paired electrical stimulation was found to be comparable to that reported for skeletal muscle, suggesting that the force generating mechanisms are basically similar quantitatively. The mitral valve is generally thought to con- sist of elastic and fibrous tissue and its motion has been considered entirely passive, the re- sults of changes in ventricular and atrial pres- sures. However, this view was re-evaluated by assessing the mechanical properties of the mi- tral valve in vitro. The anterior leaflet of the mitral valve was placed in a myograph. With stimulation, it was found that the valve con- tracts actively and as with other contractile tissues, the force of contraction was found to be a direct function of length. Electronmi- croscopic study of the mitral valve has con- firmed the presence of heart muscle under- neath the valve. These studies have demon- strated the need for a total re-evaluation of the control of mitral valve motion both in nor- mal and pathological states. It is well known that stimulation of cardiac sympathetic nerves and the administration of norepinephrine (NE) increase the force of car- diac contraction. Thus, the sympathetic nervous system provides a mechanism for augmenting basal cardiac contractility, but it has not been known whether or not the cardiac stores of NE are necessary to establish normal baseline contractility and to maintain the potential for increasing contractility in response to posi- tive inotropic interventions other than sympa- thetic stimulation. This question is of particular interest in view of the depletion of myocardial NE stores associated with congestive heart failure. Accordingly, in order to assess the role played by endogenous norepinephrine (NE) stores in the intrinsic contractile state of car- diac muscle, the right ventricular papillary muscles from normal cats and cats with car- diac NE depletion produced by chronic car- diac denervation or reserpine pretreatment NATIONAL HEART INSTITUTE 85 were studied. Length-tension curves, force- velocity relations, the effects of alterations in frequency of contraction and the response to sustained postextrasystolic potentiation were determined. In addition, the electrical excita- bility and absolute refractory periods were measured. All of these properties were found to be normal in both groups of NE depleted muscles. It is concluded that cardiac stores of NE are not fundamental for maintaining the intrinsic contractile state of the myocardium, its absolute refractory period or electrical ex- citability. Further, release of endogenous NE from cardiac muscle does not appear to play an essential role in the mediation of the posi- tive inotropic effects of increasing frequency of contraction or of sustained postextrasystolic potentiation. Major interest has been directed to the ef- fects of sympathetic nervous system activity on cardiac contractility, but far less is known about the action of the parasympathetic ner- vous system. Acetylcholine (ACh), the para- sympathetic neurotransmitter, is said to exert a positive inotropic effect on the mammalian ventricle, an effect which has been attributed to the release of stored NE. In order to test this hypothesis, the effect of ACh on normal cat papillary muscles was compared to the re- sponse of muscles from hearts depleted of NE by chronic cardiac denervation and reserpine pretreatment. In the absence of NE the posi- tive response produced by ACh is unchanged from that observed in normal muscles. By ex- amining the response to ACh over a wide con- centration range and comparing it to that in isolated atrial tissue in the presence and ab- sence of atropine, the following hypothesis was developed to account for an otherwise be- wildering array of apparently contradictory data on parasympathetic control of the heart: There appear to be two types of receptor sites in the heart responsive to acetylcholine — Type I, intimately related to vagal nerve endings and whose stimulation is associated with a negative inotropic effect and Type II, unre- lated to the vagus nerve and whose stimula- tion results in a positive inotropic effect. This study thus emphasizes the need for a reexami- nation of the role of the parasympathetic ner- vous system in the control of myocardial con- tractility. The direct influence of the thyroid state on cardiac muscle has not been denned. While profound cardiovascular alterations are known to occur with hyper- and hypothyroidism, it is not clear from studies in the intact organism whether these result from fundamental changes in the heart muscle itself or merely reflect the heart's response to the systemic effects pro- duced by these endocrine disorders. The papil- lary muscles isolated from cats rendered hy- perthyroid and hypothyroid provides a prepa- ration in which it is possible to exclude as much as possible the role that neurohumoral and metabolic factors might play in indirectly altering the contractility. The level of thyroid state was found to have a profound influence on the intrinsic con- tractile state of isolated papillary muscles. Those parameters related to the speed of con- traction, i.e. the V m «, the time to peak ten- sion, the rate of tension development, and latency were particularly affected, in a manner indicating that the basic processes controlling the rate of force generation are accelerated by increased levels and slowed by decreased levels of thyroid hormone. These effects were found to be independent of temperature, fre- quency of contraction, and endogenous NE stores. The level of thyroid hormone was found to have less effect on contractile force than on the speed of its development, but the positive inotropic response to paired electrical stimula- tion, NE, and strophanthidin are dependent on the thyroid state. Thus, it appears that thy- roid hormone may directly affect the con- tractile state of heart muscle and condition the responsiveness of the heart to other agents that act upon it. In order to define the chemical energetics of cardiac muscle, the utilization of ATP and crea- tine phosphate (CP) in the papillary muscle of cat heart was measured. To accomplish this, cat right ventricular papillary muscles were poisoned with iodoacetic acid and nitrogen to prevent further production of ATP and CP. The basal consumption of high energy phos- phate was determined in resting muscles with- 86 ANNUAL REVIEW OF INTRAMURAL RESEARCH out tension by freezing muscles at various times after poisoning and assaying these muscles for CP and ATP. It was found that the utilization of high energy phosphates could be accounted for entirely by the disappearance of CP, indicating that creatine phosphokinase was intact. The basal rate of CP consumption was 0.71 /xmoles/g/min. Increasing resting tension (or muscle length) was found to increase basal CP consumption to a linear fashion over the physiologic range of muscle lengths. In addi- tion, CP utilization was determined in 49 iso- metrically contracting muscles stimulated to contract 10 to 50 times at the top of their length-tension curves. The utilization of CP by these muscles could be accounted for by a pre- diction equation with terms for the number of activations and the calculated contractile ele- ment work. The mechanochemical efficiency of cardiac muscle, defined as the work done di- vided by the energy cost of work plus activa- tion, averaged 39% in these studies. This is similar to the efficiency of skeletal muscle pre- viously determined by other investigators. The in vivo steady state levels of myocardial high energy phosphate stores (adenosine tri- phosphate-ATP and creatine phosphate-CP) may indicate the relative balance of energy production and utilization in the heart. Tech- niques developed in this laboratory have made it possible to determine in vivo stores of ATP and CP in rapidly frozen myocardial biopsies. Three groups of cats were studied in addition to normal controls: (1) cats with right ven- tricular hyertrophy and failure; (2) cats made hyperthyroid; and (3) cats made myxe- dematous. Right ventricular stores of ATP were not found to be significantly different in these groups. Right ventricular stores of CP were found to be depressed in hyperthyroid and pulmonary artery constricted cats. Right ventricular creatine stores were depressed in all three states. These findings indicate that the contractile state of the heart is not de- pendent on its high energy phosphate stores. In hyperthyroid cats, the absolute depression of CP stores may be related to a depression of total creatine stores. In pulmonary artery con- stricted cats, the low CP/total creatine ratio may indicate a relatively higher demand/ production ratio for chemical energy. Application of Myocardial Mechanics to the Intact Heart In order to increase understanding of the gross architecture and the ultrastructure of the left ventricle during various phases of the cardiac cycle, a technique was developed for the rapid fixation of the canine left ventricle during various phases of the cardiac cycle. A Gregg cannula was placed in the left main coronary artery, and at a selected time during diastole or systole, the fixing agent glutaral- dehyde, preceded by a bolus containing potas- sium chloride and acetylcholine, was injected with a power syringe. Silastic casts of the left ventricles were then prepared, and ven- tricular volumes measured directly. Portions of the myocardial wall were prepared for elec- tron microscopy, sectioned and analyzed for sarcomere lengths. Sarcomeres of the ven- tricular wall have averaged 2.10 /x during diastole and 1.84 n during systole. In the acutely dilated heart, sarcomeres of more than 2.25 jx have been observed with the forma- tion of H zones. Analyses of the gross dimen- sions of the fixed ventricle and of the silastic cast of its chamber have also been undertaken. Data have been obtained on ventricular wall thickness at the apex and at the waist of the left ventricle during systole and diastole, and on the base to apex and base to outflow tact dimensions. The relations between tension and the ve- locity of shortening in the intact left ventricle of the dog were examined in a manner ana- logous to that employed in isolated muscle, i.e. by serial, reproducible variations in the after- load alone, from a constant end-diastolic vol- ume. Sudden increases or decreases in the aortic pressure during diastole were produced, and ejection rate was measured with an electro- magnetic flowmeter; left ventricular wall ten- sion and the shortening velocities of the myo- cardial fibers and the contractile elements were then calculated. By analyzing isovolume points early in ejection, effects resulting from two NATIONAL HEART INSTITUTE 87 other determinants of shortening velocity, i.e. duration of the active state and the instan- taneous muscle length, were minimized. Shifts in the basic force-velocity relation with altera- tions in V m ax, obtained by extrapolation, and maximum tension were clearly demonstrated. Norepinephrine and paired electrical stimula- tion caused a shift in this relation to the right, with increases in velocity at any tension. Sim- ilar shifts were also apparent in curves relating tension to velocity, calculated during single isovolumic contractions. It was suggested that determination of these relations expands tra- ditional definitions of ventricular performance, and that estimation of changes in maximum velocity as well as maximum strength relative to muscle length provides direct information concerning alterations in the contractile state of the intact heart. The contractile state of the intact canine left ventricle was then studied by analyzing the instantaneous relations between force and contractile element velocity (V CE ) during the course of single isovolumic beats, and the sensi- tivity of this relation was compared to that of the ventricular function curve by exerting small inotropic influences. In 7 dogs, norepine- phrine always shifted the isovolumic force- velocity (FV) curve, increases occurring both in maximum V CE and maximum tension (Po); the ventricular function curve was unchanged in 4 of the 7 dogs. Moderate hypothermia (avg. 30.8° C) in 4 dogs increased P p , with no change or a fall in maximum V CE ; the ven- tricular function curve was shifted upward and to the left in 2 of these dogs and un- changed in 2. In 4 dogs moderate increases in heart rate (avg. 32 beats/min) increased the maximum V CE , with little change in P ; no shifts in the ventricular function curve oc- curred. Thus, the isovolumic FV curve was more sensitive than the ventricular function curve in detecting changes in myocardial con- tractile state, and provided more complete definition of alterations in left ventricular per- formance by allowing separation of effects due to changes in shortening velocity from those due to alterations in the strength of myocar- dial contraction. The effects of increasing heart rate on the relationship between contractile element ve- locity and myocardial wall tension were de- termined from single isovolumic contractions in 9 canine right heart bypass preparations. Increases in heart rate ranging from 10 to 70 beats per minute were produced by crushing the sino-atrial node and stimulating the right atrium. An increase in heart rate always pro- duced an increase in maximum calculated con- tractile element velocity and in other variables reflecting the speed of contraction, such as peak aortic flow rate, the peak first derivative of the left ventricular pressure pulse, peak con- tractile element power and the time to peak isovolumic tension. Maximum isovolumic ten- sion usually increased. It is concluded that in the intact canine left ventricle, increasing the frequency of contraction always increases the maximum contractile element velocity and maximum isovolumic force of contraction. The effect on contractile force of changing heart rate had not been determined directly in man, and accordingly a study was under- taken to provide definitive information con- cerning this relationship. At the time of cor- rective cardiac surgery a Walton-Brodie strain gauge arch was sutured to the right ventricle of 8 patients. In man, a "velocity staircase" i.e. an increase in the rate of tension develop- ment rather than a "force staircase" i.e. an increase in the peak tension development, oc- curs with changes in heart rate. This mecha- nism apparently permits the human ventricle to maintain its force of contraction and to pre- serve the duration of diastolic filling with in- creases in rate. It has been suggested that serotonin, known to act on vascular smooth muscle and known to be present in the heart, may play a role in controlling cardiac contractility. The direct ef- fects of serotonin on cardiac muscle have been examined in the isolated cat papillary muscle and in the intact dog heart, utilizing the right heart bypass isovolumic preparation described above. Preliminary results indicate that sero- tonin produces an increase in isometric ten- sion in the isolated muscle and a shift in the isovolumic force-velocity relationship to the 88 ANNUAL REVIEW OF INTRAMURAL RESEARCH right, both observations reflecting a positive inotropic effect. The physiologic significance of these findings is currently under investigation. An attempt has been made to determine whether the technique of sudden occlusion of left ventricular outflow could be applied to the study of force-velocity-length relationships in the intact unanesthetized dog. In addition, ef- forts have also been directed to deriving, for the first time, normalized values for the force- velocity-length relations in a group of normal intact dogs and to use these as a basis for com- parison with the relations obtained in dogs subjected to various chronic interventions. Several weeks before the experimental proce- dure a thoracotomy is performed. The peri- cardium is sutured to the left chest wall to facilitate later percutaneous left ventricular puncture. Isovolumic left ventricular contrac- tions are produced by balloon occlusion of the ascending aorta during diastole. Analysis of left ventricular pressure and the rate of de- velopment of pressure in isovolumic beats has been found to give meaningful force-velocity- length relationships in the lightly sedated, closed chest dog. Blood volume loading, by in- creasing cardiac filling and end-diastolic left ventricular fiber length, shifts the force-ve- locity curve to the right with an increase in maximum isovolumic tension development but produces no change in maximum velocity of contractile element shortening. Positive ino- tropic influences (digitalis glycosides, catecho- lamines, and paired electrical stimulation) produce changes in the force-velocity-length relationships similar to those seen in isolated cardiac muscle. Beta-adrenergic blockade in the intact dog depresses the left ventricular force- velocity curve, both in the presence and ab- sence of digitalis glycosides. In addition, the relationships between left ventricular tension and shortening in ejecting contractions, and maximum tension development in isovolumic beats promises to provide further insight into the mechanics of left ventricular function in the normal intact animal. As indicated above, it has been shown in studies in the intact dog heart that the rela- tion between instantaneous myocardial wall tension and the velocity of fiber shortening during systole provides a sensitive means of examining the contractile state of the left ven- tricle (LV). Previously, it has not been pos- sible to quantify this important relation in the human LV. A study was therefore undertaken to determine the course of tension development during systole and the velocity of circumferen- tial shortening of the human LV and to ex- amine the instantaneous tension-velocity rela- tion in patients with and without LV dys- function. In 15 patients, LV pressure was measured continuously with a cathetertip transducer, while radiographic contrast material was in- jected into the left atrium. The true radius (r) of the minor LV circumference (circ.) was then determined at 170 msec, intervals from cineangiograms, and this measurement correlated with instantaneous LV pressure (Pr), the tracing of which was recorded di- rectly on the cine film. The velocity of circum- ferential shortening (V CF ) was calculated as 2 n dr/dt. Wall tension in the corresponding slice of muscle was computed as: Prxr Wall thickness In patients without LV disease, the maxi- mum V CF averaged 2.05 circ./sec.; the cor- responding wall tensions ranged from 374 to 384 gm./cm 2 . In the patients who exhibited other hemodynamic evidence of LV disease, the maximum VCF's were consistently lower and averaged only 0.95 circ./sec, at levels of wall tension comparable to those observed in patients without evidence of LV dysfunction. These measurements provide the first de- scription of the tension-velocity relation in the intact human LV. In addition, the normalized measurements of VCF allow comparisons of the contractile state of the LV in patients with and without LV dysfunction. These preliminary re- sults indicate that the contractile state of the LV, as determined from the basic myocardial tension-velocity relation, can be assessed by the technique employed, and clearly serves to distinguish normal from abnormal LV func- tion. NATIONAL HEART INSTITUTE 89 Contractile Properties of the Failing Heart Studies on Isolated Heart Muscle and Sub- cellular Fractions It has long been appreciated that a quanti- tative definition of the contractile state of car- diac muscle obtained from hypertrophied and failing hearts is needed. The isolated cat papil- lary muscle preparation allows quantification of intrinsic cardiac contractility per unit of muscle, as well as determination of the re- sponses of heart muscle to inotropic interven- tions. The contractile properties of normal papillary muscles can be compared with cor- responding muscles from abnormal hearts. Ac- cordingly, techniques were developed for the production of right ventricular hypertrophy and right heart failure in cats by graded con- striction of the main pulmonary artery. Doubling of right ventricular weight occurs within several weeks after constriction. When the degree of pulmonary artery constriction is less severe, ventricular hypertrophy occurs with right ventricular systolic hypertension but without evidence of heart failure. In ani- mals in which the pulmonary artery is con- stricted more severely, hypertrophy is accompanied by heart failure with visceral congestion, ascites, pleural effusion, elevated right ventricular end-diastolic pressure and de- creased cardiac output. The muscles from cats with cardiac hyper- trophy and heart failure demonstrated pro- found depressions of the maximum isotonic velocity of shortening as well as of the max- imum isometric tension. It is concluded that congestive heart failure is associated with pro- found abnormalities in the contractile state per unit of heart muscle, with reduction of both the functional level of contractile state and its ceiling or maximum. These abnormali- ties do not appear to be dependent on an al- teration of the normal relation of sarcomere lengths to length-tension dynamics. Further it is tentatively proposed that ventricular hyper- trophy alone, in the absence of demonstrable heart failure, is associated with depression of contractile state per unit of cardiac muscle, although the absolute increase in total muscle mass may be sufficient to maintain cardiac compensation. It has been the general impression that the hypertrophied ventricle is less distensible than normal. This might be due either to an increase in the total muscle mass or to the decreased compliance of individual fibers. Accordingly the effect of hypertrophy on the resting length- tension relationship of isolated segments of normal and hypertrophied rat ventricles was determined. It was observed that although the hypertrophied ventricles exceeded the normal ventricles by an average of 20% in weight, there was no evidence that the hypertrophied myocardium is less distensible than normal, if proper corrections for differences in weight, length, and cross-sectional area were made. Collagen comprises approximately 4 percent of the dry weight of heart muscle and may con- tribute to the diastolic compliance of the ven- tricle. The few investigations of the connective tissue content in cardiac hypertrophy have not revealed a change in the collagen to muscle ratio in hypertrophied ventricles. However, only the central part of the ventricular wall, after removal of the epicardial and endocardial surfaces, has been studied. Since the surfaces also contribute to the hemodynamic charac- teristics of the entire ventricular wall it seemed important to determine if any area of the hypertrophied ventricle had altered amounts of collagen. Accordingly, tissue col- lagen content was determined in normal cat ventricles and in those with right ventricular hypertrophy due to banding of the main pul- monary artery. Each ventricle was split into epicardial and endocardial halves for determi- nation of collagen content and the ultrastruc- ture of these muscles was also examined. The hypertrophied right ventricles had markedly and significantly increased quantities of col- lagen per unit of muscle, while the left ventri- cle from the same hearts had normal collagen values. The epicardial half of the right ven- tricles had the highest collagen content but the content was also increased in the endocardial half. Large bundles of connective tissue were evident in the ultrastructural studies of the right ventricles. 90 ANNUAL REVIEW OF INTRAMURAL RESEARCH In vitro function of mitochondria from fail- ing hearts has been variously reported as nor- mal and abnormal. A re-evaluation of the ques- tion of mitochondrial integrity in heart failure has become necessary because of: (1) the re- cent development of polarographic techniques offering more accurate data than manometric techniques; and (2) the ability to determine precisely the physiologic function of failing heart muscle. Chronic heart failure was in- duced in cats by tight constriction of the pul- monary artery and in guinea pigs by tight constriction of the aorta. The presence of heart failure in the cats was identified by: (1) in vivo cardiac catheterization indicating high end-diastolic pressures; (2) pathologic find- ings such as ascites and massive right ven- tricular (RV) hypertrophy; (3) depressed mechanical performance of the isolated RV papillary muscle studied in vitro. After isola- tion of mitochondria from the right ventricle (cats) and left ventricle (guinea pigs), polaro- graphic determinations of oxygen consumption (Q0 2 ), respiratory control ratio (RC), and the ratio of phosphate esterified with ADP to the oxygen consumed (P/O ratio) were car- ried out. Determinations were performed at 26° C and 37° C with both pyruvate/malate and glutamate substrates. Initial studies have indicated no abnormalities of Q0 2 , RC or P/O ratio in mitochondria isolated from hearts in experimental heart failure. The possibility that there may be a bioen- ergetic defect in the myocardium involving an impairment of oxidative phosphorylation in heart failure in man was examined in myo- cardial tissue obtained from patients at the time of cardiac operations. Mitochondria were isolated from papillary muscles removed from the left ventricle during replacement of the mitral valve in 11 patients with left ven- tricular failure. Measurement of oxidative phosphorylation with pyruvate/malate as sub- strate gave P/O ratios averaging 2.8. Res- piratory control ratios, determined both mano- metrically and polarographically averaged 6.6 with pyruvate/malate and 5.9 with alpha- ketoglutarate. Endogenous ATPase activity av- eraged 0.14 /xmoles P liberated per minute per mg mitochondrial N; it averaged 0.36 with Mg ++ , 1.79 with 2,4-dinitrophenol, and was completely inhibited by oligomycin. These values are comparable to those reported for normal mitochondria obtained from experi- mental animals. Examination of myocardial tissue by elec- tronmicroscopy revealed no apparent abnor- mality of mitochondrial size or crystal pattern. Creatine phosphate was determined in rapidly frozen ventricular biopsies from 15 patients with heart failure undergoing valve replace- ment. The mean tissue concentration was 4.0 xmoles per g compared to 4.1/mioles per g in similar biopsies from 5 patients without heart failure. These biochemical studies indicate that electron transport and coupled phosphorylation appear to be normal in mitochondria isolated from failing human hearts and that there is no consistent reduction of the myocardial store of high energy phosphate. It is concluded that the formation of chemical energy is not im- paired in the failing heart, and it is suggested that the biochemical abnormality responsible for defective myocardial function involves utilization of energy in the contractile process. Studies on the Intact Dog Heart There is general agreement that both the high energy phosphate stores and the mechan- ical performance of the myocardium are de- pressed during severe hyposia. To determine whether a casual connection exists between these two phenomena, 18 dogs were respired with 6% 2 , 94% N 2 after sympathetic block- ade (hexamethonium and propranolol). Serial left ventricular (LV) biopsies were obtained as myocardial failure occurred, and the con- centrations of adenosine triphosphate (ATP) and creatine phosphate (CP) were measured. A transient increase in left ventricular stroke work during initial hypoxia was followed by a progressive rise in left ventricular end-dia- stolic pressure and fall in left ventricular stroke work. There was no change in mean ATP, even with severe heart failure. With early failure a significant fall in CP occurred in 10 of 18 dogs, and with late failure CP was always depressed. Since ATP, the final energy source for contraction, was unaffected in all dogs and NATIONAL HEART INSTITUTE 91 CP, the secondary energy store, was not de- pressed when failure first developed in some animals, it is concluded that hypoxic depres- sion of myocardial function is not initiated by a depression of the total myocardial high en- ergy phosphate stores. The levels of high energy phosphate stores and the status of anaerobic metabolism at the onset of ischemic heart failure induced by re- stricting flow to the left coronary artery were also studied. Left ventricular biopsies were ob- tained before ischemia and shortly after the onset of ischemic heart failure for measure- ment of concentrations of ATP and CP. Sam- ples of blood were drawn from the aorta and coronary sinus for determination of lactate and pyruvate concentrations. At the time of failure, arteriovenous lactate differences de- creased from an average of 1.29 to 0.60 mM and "excess lactate" increased by an average of 1.74 mM. There was no change in mean ATP (control, 6.45 vs. failure, 6.49 ^moles/g). Mean CP concentrations fell significantly (12.84 to 8.00 ^moles/g) but concentrations of CP were within 2 SD's of paired control samples in 5 of 19 observations. It is con- cluded that heart failure may be induced in the presence of relatively minor degrees of anaerobic metabolism, and that the total high energy phosphate stores are not necessarily compromised at this time. Depressions of cardiac norepinephrine stores have been described in this laboratory in chronic heart failure in man and in experi- mental heart failure in the dog, cat and guinea pig. Disturbances have been noted in both the uptake and storage of exogenous norepine- phrine, although the relative rate of norepine- phrine turnover is normal. These findings have suggested that there may be an absolute loss of sympathetic nerve endings in the heart in chronic heart failure. Tyrosine hydroxylase is the rate limiting enzyme in the synthesis of norepinephrine. It was thought, therefore, that the determination of cardiac tyrosine hydrox- ylase activity might provide insight into the mechanism of the depletion of nore- pinephrine stores in heart failure. Initial studies of myocardial tissue extracts have dem- onstrated the possibility of assaying tyrosine hydroxylase activity in canine right ventricle and have suggested that the activity of this enzyme is low in chronic right ventricular failure in dogs. As outlined in an earlier section of this re- port, other studies in this laboratory have shown that analysis of the tension-velocity re- lation throughout a single isovolumic beat pro- vides a sensitive index of the contractile state of the intact left ventricle of the dog. Accord- ingly, the effects of acute experimental heart failure on tension-velocity relations during iso- volumic contractions and auxotonic contrac- tions were studied. Cardiac failure was pro- duced by infusion of large doses of the beta- adrenergic blocking agent pronethalol, or by infusion of barbiturates. The right heart by- pass preparation was used to control cardiac output. Blood flow was measured with an electromagnetic blood flow transducer positioned about the aortic root. When veutricular end-diastolic volume was main- tained constant, the tension-velocity re- lation during isovolumic contraction was al- ways shifted markedly downward and to the left during acute failure of the left ventricle, with marked reductions both of max- imum measured contractile element velocity, the extrapolation to V m «, and the maximum isovolumic tension (P ). Acute failure also produced a decreased extent of fiber shorten- ing and an increase in the ratio of the ten- sion actually developed (P), to P . In some experiments, stroke volume was held constant so that end-diastolic pressure and volume were increased during acute cardiac failure. Al- though, under these circumstances, maximum measured velocity and V max are depressed, P„ remains unchanged or more often is ac- tually increased during acute failure. Further- more, it appears that while total contractile element work may not be greatly affected, the ratio of internal contractile element work to total work may be increased substantially. A marked augmentation of myocardial con- tractile force regularly accompanies paired electrical stimulation, a technique in which two stimuli are repetitively delivered to the ventri- cle with such timing that the second stimulus of each pair is introduced at the termination of 92 ANNUAL REVIEW OF INTRAMURAL RESEARCH the preceding refractory period. Profound pos- itive inotropic effects have been demonstrated in isolated heart muscle, in a variety of animal preparations and in conscious human subjects. However, the potential clinical usefulness of this technique has been sharply restricted by the finding that this augmentation of ventricu- lar contractility is not always translated into an elevation of cardiac output. Accordingly, an investigation was carried out in order to iden- tify circumstances in which the improvement of myocardial contractility produced by paired electrical stimulation would result in an eleva- tion of the cardiac output. The effects of paired stimulation were stud- ied in intact anesthetized dogs before and after barbiturate induced depression of myocardial performance. In the non-depressed state, paired electrical stimulation exerted a positive ino- tropic effect but tended to impair circulatory performance, lowering cardiac output and ar- terial pressure. On the other hand, when ap- plied during barbiturate-induced myocardial depression, paired electrical stimulation ele- vated cardiac output by an average of 129 per- cent, increased mean arterial pressure by 34 mm. Hg, and lowered mean right atrial pres- sure by 2.4 mm. Hg. The effects of paired elec- trical stimulation were compared to those of acetylstrophanthidin and were found to be quite similar both in the failing and non-fail- ing heart. It was concluded that the effects of paired electrical stimulation are dependent on the circulatory state which exists at the time it is applied. Clinical Studies The maximum cardiac output response to in- tense exercise might be limited either by the pumping ability of the heart, or by extracar- diac factors limiting ventricular filling at a time when the ventricles are still capable of augmenting the cardiac output. Previous stud- ies have suggested the latter mechanism, since a relationship between total blood volume and maximum exercise capacity has been demon- strated; the increased exercise capacity pro- duced by training is associated with an aug- mented blood volume, and an impaired circula- tory response to maximal exercise has been pro- duced by decreasing blood volume. However, it is not known if cardiac output during maximal exercise can be elevated by acute expansion of blood volume. Accordingly, six normal men were studied at rest and during maximal tread- mill exercise before and after an acute infusion of 1000-1200 ml. of the subject's own blood. Transfusion increased resting central venous pressure from an average of 0.0 to 1.9 mm. Hg and cardiac output from 5.34 to 6.81 L/min. (p<0.01). During exercise the augmented blood volume increased central venous pressure from an average of 1.2 to 8.6 mm. Hg, but cardiac output, which averaged 21.60 L/min. in the control studies, was not significantly changed following transfusion (21.62 L/min.); maxi- mum 2 uptake and the postexercise 2 debt were also unaltered. These results indicate that since total blood volume and ventricular filling pressure normally do not limit maximum car- diac output and 2 uptake, cardiac perform- ance during maximum exercise must be limited by the pumping ability of the heart. Left ventricular function has been analyzed in a variety of patients with and without car- diac dysfunction by assessing the cardiovascu- lar responses to supine muscular exercise on a bicycle ergometer. Left ventricular end-dias- tolic pressure was measured continuously and cardiac output and oxygen consumption were determined before and during exercise. A nor- mal pattern was established in 7 patients with- out left ventricular dysfunction and consisted of an exercise factor equal to or greater than 600 ml./ 100 ml. V0 2 , a left ventricular end- diastolic pressure (LVEDP) during exercise of less than 12 mm. Hg, and little change or a decrease in LVEDP, which was accompanied in most instances by an increase in the stroke volume. This response was then compared with that observed in 14 patients studied after ste- notic or regurgitant malformations of the aortic valve had been corrected by valve re- placement with a Starr-Edwards prosthesis, and with that seen in 31 patients with various other cardiac lesions. In the majority of patients with mitral stenosis the pattern of left ventricular func- tion during exercise was considered to be nor- mal, and in 5 patients with aortic valve pros- NATIONAL HEART INSTITUTE 93 theses, a normal pattern was also observed. Among the remaining patients, including those with prostheses, aortic stenosis, or left ventri- cular myocardial disease, two types of abnor- mal performance of the left ventricle were identified. In some patients, an increase in LVEDP was accompanied by an increase in the stroke volume, a response considered con- sistent with abnormal left ventricular dynam- ics in which the ventricle appeared to utilize primarily the Frank-Starling mechanism. In the remaining patients, an increase in LVEDP was accompanied by no change or a fall in the stroke volume, a response considered indicative of depressed left ventricular function. Thus, in the majority of these patients, determination of the LVEDP before and during exercise per- mitted definition of normal or abnormal left ventricular function. The method described thus appears to supply a practical and useful means of evaluating the functional status of the left ventricle in patients with and without myocardial dysfunction. Although a considerable body of information is available concerning the circulatory response of patients with heart disease to exercise in the supine position, little is known about the circulatory response of these patients to exer- cise in the upright posture. Since many of the symptoms of cardiac patients occur during exertion in the upright posture, it was consid- ered that a thorough understanding of the hemodynamic alterations occurring during mild to intense upright exercise would be of physiological and clinical importance. It was also hoped that a more reliable means of eval- uating the relative impairment of cardiac function could be obtained. Accordingly, pa- tients with various types of heart disease and normal subjects were studied during mild and intense levels of treadmill exercise. A Cournand catheter was introduced into the pulmonary artery in order to measure pulmonary arterial pressure and to obtain samples of mixed venous 2 content. Cardiac output was measured by the Fick principle, and oxygen consumption (V0 2 ), arterial blood pressure, and the elec- trocardiogram were continuously recorded. When cardiac output was plotted against V0 2 in the conventional manner, considerable over- lap was found between the two groups both in the absolute values of cardiac index and V0 2 , as well as in the slope of the line relating these two variables. Since the "exercise factor" de- scribes the slope of the line relating cardiac output to V0 2 , these results demonstrated that the "exercise factor" is an insensitive method for assessing the degree of cardiac impairment. On the other hand, if the cardiac index is plotted against the pulmonary artery (PA) 2 saturation, a clear difference in the response to exercise between normal subjects and pa- tients was evident. At lower levels of exercise (PA saturation > 40%) the cardiac output re- sponse of the two groups did not differ greatly. However, at levels of exercise which produced a PA 2 saturation of 35% or less, the circu- latory response of the patients contrasted dra- matically with that of normal subjects. It was found that at a PA 2 saturation of 30%, all normal subjects achieved a cardiac index greater than 7 liters/min, while no patient ex- ceeded a value of 5 liters/min. Thus, the level of the cardiac index at a PA 2 saturation of 30% provides a new and sensitive index of cardiac function which may prove to be of con- siderable clinical value. A comparison of the hemodynamic responses to exercise in the supine and upright positions was undertaken in patients with varying de- grees of impaired cardiac function. In patients with insignificant cardiac defects, cardiac out- put, mixed venous 2 saturation, and pulmo- nary arterial pressure were slightly but con- sistently higher at any given level of V0 2 in the supine position as compared to the upright. In contrast, in the patients with significant cardiac impairment, much higher pulmonary arterial pressures occurred in the supine posi- tion than during upright exercise. It also ap- peared that there was less of a difference be- tween the cardiac output during exercise in the supine and upright positions in the pa- tients as compared to the subjects with insig- nificant cardiac disease. Two of the more prominent manifestations of imparied cardiac function are an inability to augment the cardiac output appropriately in response to increased metabolic demands, and a diminished abiilty to excrete Na, with re- 94 ANNUAL REVIEW OF INTRAMURAL RESEARCH sultant fluid accumulation and edema. Since it has recently been observed that the cardiac response to maximal and submaximal levels of exercise in both normal subjects and patients with various forms of heart disease is reduced by blockade of the beta-adrenergic receptors, it became of interest to determine if this im- pairment of hemodynamic performance is as- sociated with changes in the patterns of Na excretion. Sixteen subjects, 13 men and 3 women, rang- ing in age from 21 to 57 years were studied. Six of the men were normal volunteers; the re- mainder of the subjects had heart disease of various types. Beta blockade altered the pat- tern of Na excretion in all subjects, and this impairment of Na excretion could be divided into three grades of severity. The mildest degree of impairment, observed in normal subjects and patients with reduced cardiovascular re- serve, consisted of an alteration in the diurnal pattern of Na excretion, the total 24-hour Na excretion remaining unchanged. An interme- diate degree of impairment, observed in some patients with heart disease, but not in normal subjects, was manifest by retardation of the rate of increase of Na excretion in response to progressive increases in Na intake, although Na balance was ultimately achieved. The most serious degree of impairment, observed in only one patient with heart failure, consisted of progressive Na retention, resulting in increas- ing fluid accumulation and edema. Although the results of this investigation suggest that beta-adrenergic blockade will not cause gross Na retention in most cardiac patients, it is stressed that inhibition of cardiac sympathetic nerve activity can occasionally precipitate dan- gerous cardiac decompensation unassociated with prior Na retention and weight gain. The results of this study further demonstrate the importance, particularly in patients with im- paired cardiac function, of the support which the sympathetic nervous system provides to myocardial performance. Although there has been extensive investi- gation of the central circulation in patients with heart failure, little information is avail- able concerning possible abnormalities of the peripheral vascular beds in these patients. Furthermore, only a few of the several varia- bles of the regional circulations have been correlated both among themselves and with the variables of the central circulation, even in normal subjects. At rest, forearm blood flow was found to vary directly with cardiac output, forearm resistance varied directly with total systemic resistance, venous tone varied direct- ly with forearm resistance and inversely with cardiac output. Patients with heart failure al- ways had lower values for forearm flow and higher values of forearm resistance and venous tone, providing complete separation of the two groups in the above comparisons. Cold stimula- tion and leg exercise resulted in an excessive elevation of forearm resistance and venous tone in patients with heart failure as opposed to normal subjects. It has been established that the elevation of the lower extremities in supine patients with congestive heart failure results in a decrease in forearm blood flow, while in normal subjects an increase in flow occurs. The mechanism of this paradoxical response in congestive heart failure has not been elucidated. In studies in which arteriolar and venous tone of the fore- arm were determined by plethysmography means, it was found that leg raising resulted in arteriolar and venous constriction in heart failure while the opposite effect was seen in normal subjects. In addition, it was observed that local nerve block in the forearm abolished the alteration of forearm blood flow in both groups of individuals, indicating that the vaso- constrictor response in heart failure and the vasodilator response in the normal subjects was mediated through a reflex. Since it is known that rapid changes in atrial pressure are important in initiating the abnormal vaso- constrictor response to leg raising in heart failure, it is postulated that a critical incre- ment in right atrial filling pressure reflexly initiates the response and these findings sug- gest that stretch receptors exist in these low pressure areas of the central circulation. The reactive hyperemia response, that is, the increase in blood flow immediately after resto- ration of the circulation after a period during which arterial inflow is occluded, was com- NATIONAL HEART INSTITUTE 95 pared in normal subjects and in a large group of patients with congestive heart failure, uti- lizing a plethysmographic technique. It was observed that the peak level of reactive hyper- emia as markedly reduced in the patients with heart failure compared to the normal sub- jects. The magnitude of the reduction of the hyperemic response was found to be a function of the duration of the period of circulatory ar- rest in normal subjects, progressive increases in postischemic blocd flow occurring as the du- ration of ischemia was prolonged, but this was not observed in the patients with heart failure. The mechanisms responsible for this phenom- enon have not yet been elucidated, but are cur- rently under investigation. It is unlikely that increased sympathetic vasoconstrictor activity can be held responsible, since local vasodilator metabolites are known to be capable of over- riding neurogenic effects. It would appear that the heart failure state in some manner in- creases the rigidity of the resistance vessels, and their altered mechanical properties do not allow them to respond in a normal fashion to the vasodilator metabolites that accumulate during circulatory arrest. Studies on the Mechanism of Action of Digitalis The digitalis glycosides are unquestionably the most important drugs used in the treat- ment of heart failure. Their mechanisms of ac- tion have been studied in this Branch since its establishment and these investigations were continued during the past year. Several investigators have proposed that the positive inotropic effect of digitalis on the heart is due, at least in part, to nore- pinephrine (NE) released from cardiac NE stores by the glycosides. Since this pro- posal is based on the finding of a decreased inotropic effect of digitalis after depletion of cardiac stores of NE by re- serpine or dichloroisoproterenol, the possibility was considered that this finding was due to a direct action of these antiadrenergic drugs rather than the depletion of NE. To examine this question of the responses of papillary mus- cles depleted of NE by chronic cardiac dener- vation and those in which the NE depletion was produced by reserpine pretreatment were studied. The muscles depleted of NE by chronic denervation respond normally to oaubain and strophanthidin while those equally depleted of NE by reserpine exhibited a reduced inotropic response to these glycosides. It is concluded that cardiac NE stores are not essential for the pos- itive inotropic effect of strophanthidin or oua- bain on the heart and that reserpine may in- terfere directly with the inotropic action of these cardiac glycosides. While digitalis glycosides improve the fail- ing myocardium, their effects on the normal heart remain controversial. Newer methods de- veloped in this laboratory permitted resolution of this controversey. The effects of ouabain (0.01 mg/kg) on ventricular force-velocity relations were studied in six patients following corrective cardiac operations. A beat-to-beat analysis of ventricular force-velocity relations was performed by relating the velocity of movement of roentgenopaque markers previ- ously sutured to external surfaces of the ven- tricles and intraventricular pressure at con- stant ventricular dimensions. It was observed that ouabain always augmented myocardial contractility as reflected in the force-velocity relation. Velocity of shortening increased an average of 77 ±5% above control while intra- ventricular pressure rose by an average of 23 ±6%. Despite this improvement in contrac- tility, no consistent changes in cardiac output were observed. Analogous changes in force- velocity curves were obtained when a cardiac glycoside was added to isolated papillary mus- cles removed from normal cats. It is concluded that the fundamental action of digitalis gly- cosides is to augment the contractile state of the human heart, whether normal or failing, but that in the absence of heart failure this improvement is not translated into an increase in cardiac output. There has been considerable dispute concern- ing the effects of digitalis on myocardial 2 consumption (MV0 2 ) and efficiency. Analyses of previous data suggested that the interpreta- tion of the results was complicated by the changes in circulatory dynamics induced by the drug. Accordingly, the effects of acetyl- strophanthidin (Avg. dose = 0.26 cat units/ 96 ANNUAL REVIEW OF INTRAMURAL RESEARCH kg) were studied in 6 non-failing, canine, right heart bypass preparations in which heart rate, stroke volume, and mean aortic pressure were held constant. MV0 2 increased in all experi- ments, by an average of 2.56 ml/min, while calculated cardiac efficiency declined by an av- erage of 24.4% of control. Even though mean arterial pressure was held constant, the glyco- side reduced the integrated systolic tension by an average of 40% and the peak systolic ten- sion by an average of 18%, chiefly as a conse- quence of a small decline in left ventricular end-diastolic volume. However, the velocity of myocardial fiber shortening increased consid- erably, the peak left ventricular ejection rate rising an average of 36% and the peak ven- tricular dp/dt increasing by an average of 82%. Acetylstrophanthidin did not alter MV0 2 in two hearts which were studied in an identi- cal manner, but in which left ventricular end- diastolic pressure was initially elevated. However in those experiments the large fall in end-diastolic volume resulted in a marked fall in systolic tension. In ad- dition, in one experiment, following acetyl- strophanthidin the left ventricular end- diastolic pressure and consequently myo- cardial wall tension were re-elevated to near control levels by increasing stroke volume. Under these circumstances, with no change in myocardial wall tension, oxygen consumption was increased with digitalis. It is concluded that digitalis tends to increase MV0 2 , but that its strongly positive inotropic effect frequently results in a reduction of ven- tricular wall tension which tends to oppose and to mask this effect. It is proposed that the in- crease in MV0 2 is related to the increased ve- locity of contraction induced by this agent. It is generally assumed that patients with mitral stenosis are, like patients with other cardiac disorders, benefited by digitalis. As a result, digitalis therapy was instituted in most patients with mitral stenosis, regardless of rhythm. However, the major impairment of cardiac performance in these patients results from mechanical obstruction to blood flow rather than from imparled myocardial func- tion. Based on this consideration, it was hy- pothesized that digitalis would not benefit pa- tients with mitral stenosis in normal sinus rhythm. On the other hand, relatively high mean left atrial pressures occur in patients with atrial fibrillation and rapid ventricular rates as a result of a greatly abbreviated dias- tolic filling time. In these subjects it would be expected that digitalis, by increasing the atrio- ventricular functional refractory period and thereby decreasing the ventricular response, would improve cardiac performance. Seven patients with pure mitral stenosis in normal sinus rhythm were studied at rest and at mild to intense levels of treadmill exercise. A Cournand catheter was placed in the pulmo- nary artery for measurement of pressure and withdrawal of mixed venous samples for car- diac output determination by the direct Fick method. Arterial pressure, oxygen consumption (V0 2 ), EKG, and heart rate were continu- ously recorded. After control values were ob- tained at rest and during exercise, ouabain, 0.01 mg/kg was administered intravenously and the study was repeated. Several patients were given digoxin orally, and the studies re- peated 4 to 7 days later. In 3 patients both the acute and chronic studies were performed. The hemodynamic response to exercise was as- sessed by comparing cardiac output, V0 2 , pul- monary arterial pressure, and mixed venous oxygen saturation before and after the admin- istration of digitalis. In patients with normal sinus rhythm all of the measured parameters of cardiac performance were essentially un- changed by either acute or chronic adminis- tration of digitalis. Thus, these findings do not support the position that digitalis favorably influences the circulatory dynamics of pa- tients with mitral stenosis and sinus rhythm. Studies on Circulatory Control of the Autonomic Nervous System The spcific role played by the autonomic nervous system in circulatory control has been of interest to investigators in this Branch for a number of years now. This interest has con- tinued, and studies carried out on the auto- nomic nervous system are included among the other headings of this report. In addition to the aforementioned investigations, four others were carried out during the past year. NATIONAL HEART INSTITUTE 97 It is generally agreed that norepinephrine is the mediator released at the sympathetic neu- roeffector junction. In addition, it is believed that the norepinephrine released from nerve terminals as a result of sympathetic nervous stimulation acts at the same sites as humorally transported norepinephrine. Furthermore, it is felt that blockade of neurally evoked vaso- constriction is as easily accomplished as block- ade of humorally mediated vasoconstriction. In order to study these aspects of neurotrans- mitter activity as they pertain to vasomotor control of the circulation, a canine preparation in which the skeletal muscle vascular beds of the hindlimb were perfused at a constant flow was utilized. Changes in perfusion pressure therefore reflected changes in vascular resist- ance of the limb. Reflex vasoconstriction in the limb was produced by bilateral carotid ar- tery occlusion and by hemorrhagic hypoten- sion. The changes produced by these reflex mechanisms were then compared with the changes in resistance brought about by the intra-arterial injection of norepinephrine. In the dogs that served as controls, both carotid sinus hypotension and intra-arterial norepinephrine caused large increases in vas- cular resistance. The other animals were pre- treated with phenoxybenzamine 15 mg/kg, a powerful alpha adrenergic blocking agent. In this group of animals, carotid sinus hypoten- sion once again produced a reflex vasoconstric- tion in the perfused hindlimb, although not to as great an extent as in the normal limb. In- tra-arterially administered norepinephrine, however, now produced an opposite effect, causing vasodilation. Thus, the pretreatment with phenoxybenzamine blocked the vasocon- striction normally produced by norepinephrine as a result of its alpha receptor stimulating properties and unmasked its beta receptor stim- ulating effects which produce vasodilation. This vasodilation could then be blocked by treat- ment with propranolol, a powerful beta-adren- ergic blocking agent, thereby confirming that it was indeed a beta effect which had been un- masked. These results indicate that humorally trans- ported norepinephrine and neurally released norepinephrine act to a large extent at spatially different sites. In addition, contrary to state- ments in the literature, it seems clear that the alpha receptors at the neuroeffector junction are not reached by blocking agents as effec- tively as are alpha receptors at other sites. Fi- nally, since reversal of reflex vasoconstriction was never seen, it seems reasonable to assume that the population of beta-receptors in the re- gion of the neuroeffector junction is smaller in comparison to the population of alpha re- ceptors than this ratio elsewhere. Since the demonstration by Marey in 1859, it has been known that heart rate varies in- versely with the arterial blood pressure. It is also firmly established that these changes in heart rate are mediated reflexly, the afferent arm of the reflex originating in the barorecep- tors located in the carotid sinus and aortic arch areas. It was felt that it would be of interest to define the relative roles of the two main baro- receptor areas in the control of heart rate. In nine dogs the carotid arterial circulation was isolated from the rest of the vascular bed and was perfused by a donor dog. Changes in arterial pressure could then be produced inde- pendently in the carotid sinus and in the aortic arch baroreceptor areas. It was found that both baroreceptor regions are important in the con- trol of heart rate, although the aortic arch areas often predominate. It was also shown that a positive input, produced by raising the perfusing blood pressure is a more adequate stimulus than the negative input of lowering perfusion pressure. Finally, as shown by large changes in blood volume in the trunk which oc- cur when carotid sinus pressure is raised, re- flexes originating in this area exert a profound influence on the volume of the peripheral vas- cular bed. The central nervous system controls heart rate by varying the impulse traffic in sympa- thetic and parasympathetic nerve fibers termi- nating in the sino-atrial node. Although there has been considerable interest in the mechan- isms by which the heart rate is altered in re- sponse to exercise, postural changes, and stim- ulation of the baroreceptors, the relative role of the two divisions of the autonomic nervous system in mediating these changes in rate in conscious man have not been clarified. Further- 98 ANNUAL REVIEW OP INTRAMURAL RESEARCH more, litle information is available concern- ing the manner in which the heart rate response to baroreceptor stimulation is modified dur- ing exercise. The relative roles of the efferent pathways whch mediate the heart rate response to su- pine exercise and tilting were investigated by observing the separate and combined effects of beta-adrenergic blockade and parasympathetic blockade on cardiac acceleration in normal sub- jects. From these studies it appears that in the supine resting state, parasympathetic restraint is the dominant influence on heart rate, and the accelerating effects of sympathetic stimu- lation are minor. The speeding of the heart in response to mild exercise appears to result largely from withdrawal of parasympathetic inhibition, since cardiac acceleration was found to be essentially unimpaired by sympa- thetic blockade but to be inhibited by para- sympathetic blockade and double blockade. At higher levels of exercise, however, cardiac ac- celeration must result in part from sympa- thetic stimulation, since sympathetic blockade reduces the augmentation of heart rate in com- parison to the control study. The finding that the increments in rate during heavy exercise are smaller after double blockade than after parasympathetic blockade alone further identi- fies the contribution of the sympathetic sys- tem. In contrast to light supine exercise, substantial speeding could still occur during tilting when the parasympathetic system was blocked. Thus cardiac acceleration in response to mild supine exercise appeared to depend pre- dominantly on parasympathetic withdrawal, whereas that produced by tilting involved a relatively greater degree of sympathetic stim- ulation. No alteration in baroreceptor sensitiv- ity was found on transition from rest to exercise, and at rest, baroreceptor induced alterations in heart rate are moderated by stimulation or withdrawal of either efferent system. However, during exercise, heart rate changes are primarily dependent on changes in the activity of the sympathetic nervous system. The presence of baroreceptors in the walls of the two atria has been postulated by many investigators. However, the importance of these proposed receptors in the reflex control of vascular resistance and venous tone in intact man has not been elucidated. An investigation was carried out which was designed to deter- mine the effects of stimulation of the atria, by electrically pacing the right atrium with a catheter electrode, on the vascular dynamics of the forearm, and on the total peripheral vascular resistance. In 8 patients with normal or near normal cardiovascular dynamics at rest, stimulating the atria at a rate above the basal rate resulted in a striking augmentation of forearm blood flow and reduction of calculated forearm vascular resistance, indicating that arterial dilatation had taken place. In addition, a decrease of venous tone was produced, indi- cating venodilatation. Thus, it appears that as postulated, there are receptors in the walls of the atria, the stimulation of which are capable of producing vasodilatation of the normal hu- man forearm. Improvement of Cardiac Diagnostic Methods The direct measurement of oxygen satura- tion of blood in the central circulation was made possible by the in vivo oximeter system utlizing fiber optics, as described in the 1965 annual report. Although that catheter system permitted the rapid continuous and accurate measurement of oxygen saturation, its clinical usefulness was markedly limited by the neces- sity of introducing a second catheter to meas- ure pressure, to inject indicator, or to withdraw blood. Accordingly, attention was di- rectly at encouraging and aiding in the devel- opment of a catheter which incorporates both fiber optics and a lumen. This catheter is now being utilized in the precise diagnosis of cer- tain forms of congenital heart disease and in studying the oxygen saturation and blood pres- sure in the pulmonary artery during and fol- lowing exercise in patients with varying de- grees of impairment of cardiac performance. In patients with congenital heart disease, the simultaneous measurement of oxygen satura- tion and pressure is making possible a more rapid exploration of these variables within the central circulation. It has been used to localize precisely and to quantify both left-to-right and right-to-left shunts by oxygen saturation NATIONAL HEART INSTITUTE 99 changes at these sites. The catheter lumen also permits the measurement of pressure, the cen- tral injection of indicator for peripheral arte- rial sampling and the withdrawal of blood for in vitro measurements. The execution of these maneuvers with a single catheter has proven useful and the simultaneous and direct meas- urement of oxygen saturation and pressure has been found particularly valuable in diagnostic procedures. In normal erect subjects pulmonary blood flow is greater to the dependent zones of the lung than to the apices, and this pattern may be reversed in mitral stenosis. External scin- tillation scanning of intravenously adminis- tered I 131 labeled macroaggregated of human serum albumin (MAA) was employed to eval- uate distribution of pulmonary blood flow in the erect posture in 13 normal subjects and 35 patients with mitral valve disease. The upper/ lower third ratio of blood flow (U/L) to the right lung averaged 0.43 in normals and was significantly greater (1.01) in patients with mitral valve disease (p< 0.001). In the absence of heart failire U/L did not exceed 0.65 until MLAP exceeded 14 mm. Hg and was less than 0.80 whenever MLAP was less than 15 mm. Hg. The correlation between U/L and MLAP was significant (r-Z 0.91, p< 0.001) and from the regression equation (MLAPZ = 14.0 U/L5.32), MLAP could be predicted to within 5 mm. Hg at the 95% confidence level. It is concluded that the close correlation be- tween the U/L ratio and the mean left atrial pressure indicated that it is possible to pre- dict the latter within reasonable limits in pa- tients with mitral valve disease by means of simple lung scanning. The method has been found especially useful in the screening of asymptomatic patients with the clinical find- ings of mitral valve disease, and, in the preop- erative study of patients so ill that left heart catheterization was considered to be unusually hazardous. Scanning is also of value in deter- mining whether the pulmonary venous pres- sure is elevated in patients with known severe pulmonary arterial hypertension, so that the presence of potentially correctible lesions such as mitral stenosis or cor triatriatum may be detected. The technique is safer, produces less discom- fort, and provides more quantitative data con- cerning the distribution of pulmonary blood flow than either pulmonary arteriography or differential bronchospirometry. The necessary equipment for the application of the scanning method is far simpler than that required for the radioactive gas techniques and it is readily available. Scintillation scanning of the lungs after ad- ministration of I 131 labeled macroaggregates of human serum albumin has also been em- ployed to delineate the patterns of pulmonary blood flow in patients with congenital heart disease. It was found that in patients with tet- ralogy of Fallot the blood flow through a sub- clavian-pulmonary arterial anastomosis is di- rected principally to the lung on the same side as the anastomosis and that the relative dis- parity in flows to each lung provides an in- dex of the efficacy of the anastomosis and of the development of pulmonary atresia. Also, the presence of anomalies that create pulmo- nary venous hypertension may be detected by demonstrating a reversal of the normal increase in perfusion from lung apex to base when the patient is in the erect posture. Avascular areas of the lung secondary to atresia or marked nar- rowing of the pulmonary arteries may also be detected by lung scanning. Right-to-left shunt- ing through a patent ductus arteriosus may be diagnosed if the accumulated radioactivity in the capillary bed of the feet exceeds that in the hands after intravenous injection of MAA. Clinical Studies Idiopathic Hypertrophic Subaortic Stenosis A detailed anatomic, hemodynamic, angio- graphic and clinical review of idopathic hyper- trophic subaortic stenosis (IHSS) was undertaken in relation to possible mechanisms responsible for the intraventricular pressure gradient that occurs in this disease. The fea- tures were considered in particular relation to the recent proposal that cavity obliteration, rather than obstruction, is responsible for the pressure gradient in these patients. Anatomic observations at necropsy and at operation have revealed "bars" or "bands" of hypertrophic 100 ANNUAL REVIEW OF INTRAMURAL RESEARCH muscle which appear to obstruct the left ven- tricular outflow tract; incision of these hyper- trophied areas of the interventricular septum appears to abolish a sphincter-like action of the ventricular outflow tract. Review of hemo- dynamic observations, using a flowmeter at the time of cardiac surgery, has revealed that ap- proximately 70% of the stroke volume is ejected during the time that a pressure gradi- ent exists. Angiograms were reviewed in an effort to clarify the location of the site of ob- struction, and it was concluded that a linear radiolucent area can frequently be identified, presumably at the point where the hypertro- phied septum impinges upon the anterior leaf- let of the mitral valve in late systole. Combined hemodynamic and angiographic measurements were made in 14 patients. It was shown that an abnormally elevated left ventri- cular systeolic pressure could be recorded in the inflow tract of the ventricle, while the tip of the recording catheter clearly lay within the pool of contrast medium. In addition, in many patients it was shown that when a catheter inserted into the left ventricle by the trans- septal route was withdrawn from the area of the inflow tract into the left atrium, it never traversed a zone of low systolic pressure, i.e., one in which left ventricular systolic pressure equaled aortic pressure. Indeed, the tracing al- ways reverted directly from an elevated left ventricular systolic pressure to left atrial pres- sure. These findings, together with clinical evidence, such as the presence of a systolic murmur in patients without mitral regurgita- tion and the marked reduction or elimination of the pressure gradient by operations de- signed to relieve obstruction, support the con- cept that obstruction plays a significant role in IHSS. It is now well established that the discrete forms of obstruction to left ventricular out- flow remain constant during hemodynamic in- terventions, and that striking variations in the severity of obstruction may be induced in IHSS by a variety of physiologic and pharma- cologic stimuli. The effects of changes in heart rate on the severity of obstruction in IHSS have not been clarified. However, since ventri- cular volume is reduced and the contractile state of the myocardium is improved when cardiac rate increases, the possibility was con- sidered that this intervention might intensify the obstruction in IHSS. The effects of increas- ing heart rate with atropine were determined in patients with IHSS and compared to the responses in patients with discrete forms of ob- struction to left ventricular outflow. It was ob- served that an increase in heart rate, presum- ably through a decrease in left ventricular end-systolic volume, increased the trans-left ventricular pressure gradient and the severity of obstruction in IHSS. In patients with dis- crete obstruction the gradient was reduced and the orifice size was unaltered by increased heart rate. Therefore, the importance of heart rate in IHSS has been defined and, in addition, a useful diagnostic test for the differentiation of dynamic and fixed obstruction to left ven- tricular outflow has been provided. Changes in body position have been shown to influence the degree of obstruction in IHSS. Tilting to the head-up position augments the left ventricular outflow tract gradient and tilt- ing to the head-down position has been found to ameliorate the obstruction. Although the resting apex cardiogram of patients with IHSS is often quite characteristic of this condition, frequently physiological and pharmacological stimuli must be used in order for the charac- teristic second systolic wave to appear. A study was undertaken to determine if the apex car- diogram recorded during tilting could be used as a simple diagnostic procedure to establish the diagnosis of IHSS. Eight of 17 of the pa- tients studied had a supine apex cardiogram characteristic of IHSS. Characteristic changes of IHSS could be elicited in 6 of the remain- ing 9 patients by use of the tilting maneuver. It is concluded that the use of the apex cardio- gram during tilting is a simple and valuable adjunct in the diagnosis of IHSS. Exercise is a potent stimulus to angina pec- toris in certain patients with IHSS. Exercise has been shown to activate cardiac adrenergic receptors and to increase both the exercise and post-exercise outflow tract gradients. Beta- adrenergic blockade has previously been shown to cause a decrease in the left ventricular out- NATIONAL HEART INSTITUTE 101 flow tract gradient during exercise when cam- pared with the pre-blockade exercise state. An investigation was undertaken to determine if beta-adrenergic blockade induced by oral pro- pranolol would be of benefit in preventing ex- ercise induced angina pectoris in patients with IHSS. Five of six patients with exercise-in- duced angina pectoris were improved on the days when propranolol was administered. These 5 patients walked an average of 10.5 minutes on the days when propranolol was ad- ministered in contrast to 6 minutes when placebo was administered. These differences are highly significant statistically, and pro- pranolol appears to be a valuable drug in the treatment of IHSS. Other Clinical Studies Only limited information is available con- cerning the symptomatic and hemodynamic results of operative correction of atrial septal defect (ASD) in older patients. Moreover, in patients with ASD beyond the fourth decade, in whom the lesion is commonly associated with significant symptomatic disability, the criteria for selection for operative treatment have not been well established. Accordingly, the clinical and hemodynamic findings in 48 patients with ASD over 40 years of age have been reviewed. The majority of patients (92%) reported sig- nificant symptomatic disability, and at cardiac catheterization they exhibited mild to moder- ate pulmonary arterial hypertension in the presence of a moderate to large left-to-right shunt. Thirty-four patients underwent opera- tive correction of the ASD, since 1960, how- ever, only one has died as the result of opera- tion. Following operation, all 12 Class III and IV patients, and 10 of 14 Class II patients were substantially improved. Postoperative cardiac catheterization revealed a substantial reduc- tion in pulmonary arterial pressures in 15 of the 16 patients in whom it was present pre- operatively. These results suggest that in the presence of a large left-to-right shunt, with or without moderate pulmonary arterial hyperten- sion, age should not contraindicate operative correction of an ASD when symptomatic dis- ability is present. Two studies have been carried out on the function of the left atrium in patients with mi- tral valve disease. In the first, the determi- nants of the height of the left atrial contrac- tion wave (a wave) were analyzed in 53 patients with pure mitral stenosis and sinus rhythm. Ratios of the height of the a wave to the v wave, the height of the a wave to the mean left atrial pressure, and the a wave pulse pressure to the mean left atrial pressure were related to a number of variables which are re- lated to the severity of the mitral stenosis, i.e., to the mean mitral valve pressure gradient, the mean left atrial pressure, the mean pulmo- nary artery pressure, and the left atrial size, estimated roentgenographically. It was ob- served that the relative height of the a wave varies inversely with the hemodynamic sever- ity of the mitral obstruction, indicating that as the disease process progresses, left atrial contraction may become weaker and make a smaller contribution to left ventricular filling. The occurrence of an atrial gallop sound is an unusual finding in patients with mitral re- gurgitation. The development of atrial fibrilla- tion in many patients with severe mitral re- gurgitation precludes the occurrence of an atrial sound and standard tests and reviews have not called attention to this finding even in patients with mitral regurgitation and sinus rhythm. The frequency of atrial gallop sounds was determined in a group of 51 patients with pure mitral regurgitation in whom the diag- nosis was established at open operation. Nine patients demonstrated atrial gallop sounds and all nine had ruptured chordae tendineae. None of six patients with primary rheumatic mitral valvular regurgitation and sinus rhythm dem- onstrated this finding. A history of rheumatic fever was uncommon in patients with ruptured chordae tendineae but a history of subacute bacterial endocarditis was common in both groups of patients. The duration of a history of a heart murmur and of symptoms were shorter in patients with ruptured chordae ten- dineae. The left atrial chamber was generally smaller in patients with ruptured chordae ten- dineae, and was evidently capable of contract- ing forcefully and causing a ventricular filling sound. 102 ANNUAL REVIEW OF INTRAMURAL RESEARCH From the clinical point of view it appears that in patients with mitral regurgitation whose cardiac disability is sufficiently severe to require mitral valve replacement, the find- ing of normal sinus rhythm should raise the suspicion of ruptured chordae tendineae. If, in addition, an atrial gallop sound is present the diagnosis of ruptured chordae tendineae is even more likely. Detailed preoperative and postoperative catheterization studies were carried out in 12 patients with advanced mitral and aortic valve disease in whom surgical replacement of both valves was performed utilizing ball-valve prostheses. Since the prostheses effectively re- lieve the mechanical burden on the left ven- tricle, any residual impairment of circulatory function observed postoperatively can be at- tributed to an impairment of myocardial func- tion. It was observed that replacement of both valves resulted in restoration of normal or nearly normal hemodynamics, recorded in the resting state. However, studies of left ventricu- lar function during exercise, as assessed by the relationship between cardiac output and oxygen consumption, between cardiac output and left ventricular end-diastolic pressure, and between stroke volume and left ventricular end- diastolic pressure, indicated that significant abnormalities of myocardial function and dy- namics exist that were not alleviated by com- bined valve replacement. Therefore, it appears likely that replacement fibrosis secondary to underlying myocardial disease and coronary artery disease, or myocardial dysfunction con- sequent to a chronic severe hemodynamic bur- den, contributes to the preoperative disability in these patients, and, in addition, may pre- vent complete symptomatic recovery following insertion of valvular prostheses. This postop- erative impairment of myocardial performance appears to be more pronounced than that ob- served in patients with isolated replacement of either the mitral or aortic valve. The study of phasic arterial blood flow pat- terns in intact, unanesthetized man has not previously been possible because of practical limitations in flow-measuring techniques. Recent improvements in the design of electro- magnetic flowmeter circuits and the miniatur- ization of flow transducers, suggested the pos- sibility that such instruments might prove suitable for these measurements. The charac- teristics of phasic and instantaneous mean brachial arterial (BA) blood flow, measured with the electromagnetic flowmeter, have been investigated in 35 patients undergoing cardiac catheterization. Phasic BA flow patterns were characteristically altered in the presence of valvular aortic stenosis, aortic regurgitation, and in hypertrophic subaortic stenosis, when compared with patterns recorded in patients without left heart disease. All patients exhib- ited spontaneous, often large variations in mean BA flow at rest. During forearm muscu- lar contraction, striking reductions in BA flow occurred, and, during maximal muscular con- traction, actual cessation of flow was observed. Reflex forearm vasoconstriction consistently occurred during the Valsalva maneuver, and with unilateral carotid occlusion, while fore- arm vasodilatation was demonstrated during carotid sinus stimulation. Thus this technique has permitted the first description of the nor- mal pattern of phasic BA flow in unanesthe- tized human subjects, as well as the altera- tions in phasic flow contour accompanying various cardiac lesions. In addition, it has al- lowed investigation of rapid, transient altera- tions in peripheral arterial blood flow not pre- viously accessible to measurement. Although the overproduction of serotonin remains the hallmark of the carcinoid syn- drome, recently attention has been focused on other biologically active substances elaborated by carcinoid tumors. The effects of brady- kinin, epinephrine, and reflex stimulation of the sympathetic nervous system, were com- pared on the arteries and veins of the forearm in carcinoid patients and normal subjects utilizing a plethysmographic technique. In con- trast to the normal subjects, in the carcinoid patients, epinephrine produced flushes, pro- found arteriolar dilatation, and resulted in a rise of brachial arterial bradykinin and the kinin-forming enzyme kallikrein; sympathetic stimulation produced flushes, attenuated vaso- constriction, and elevated plasma bradykinin and kallikrein. Therefore, this study provided NATIONAL HEART INSTITUTE 103 evidence to implicate the kallikrein-kinin sys- tem in the genesis of the carcinoid flush. Idiopathic infantile hypercalcemia, a disease probably related to deranged vitamin D me- tabolism, may be a feature of the syndrome consisting of supravalvar aortic stenosis, men- tal retardation, and a peculiar "elfin" facies. It is not known if the observed multiple-system involvement is genetically determined or if any or all of the features are related to deranged maternal vitamin D metabolism or fetal vita- min D metabolism, or to a combination of the two. Accordingly, a study was undertaken to determine if vitamin D crosses the placenta, and to explore the relationship between hyper- vitaminosis D in the mother and the develop- ment of supravalvular aortic stenosis in the offspring. Pregnant rabbits were divided into groups so that in addition to the stock diet some were given large amounts of vitamin D throughout gestation. Vitamin D bioassays revealed 7 and 8V2 times higher values of vitamin D in the mothers and the newborns, respectively, than in the controls, providing direct evidence of transplacental passage. A total of 14 anatomic abnormalities of the aorta was noted in 34 offspring whose mothers received 1.5 million units of vitamin D throughout pregnancy. Definite similarities were noted between the aortic lesions in 8 rabbits and supravalvular aortic stenosis in the human. Thirty-five con- trol offspring showed no abnormalities of the aorta. It is concluded that the vascular toxic effects of vitamin D cross the placenta. It seems reasonable to suggest that an in utero derangement in vitamin D metabolism on the part of mother and/or fetus may be responsi- ble for supravalvular aortic stenosis, especially when the latter is associated with infantile hypercalcemia. Section of Clinical Biophysics The major activities of this section are con- cerned with exploration of basic mechanisms in the physiology of the lung, heart and vascular system. These objectives require strong pro- grams in instrument development and develop- ment of computational procedures as well as a strong laboratory program. These activities will be described under three major headings: Vascular Mechanics, Myocardial Mechanics, and Pulmonary Mechanics. Vascula?- Mechanics Several moderately long term studies were concluded this year which gave us much bet- ter insight into the gross features of vascular dynamics. If we are to understand the mech- anisms determining circulatory behavior, it is necessary to gain a clear picture of the me- chanical behavior of both the vessel wall and the underlying hydrodynamics. A large num- ber of special devices and instruments have been devised which have permitted isolation of various vascular segments in the larger ar- teries, such that discrete measurements of pressure, forces, flows, displacements, and di- mensions could be recorded continuously on multi-channel F.M. tape. Rather formidable data processing programs have been developed which permit examination of these simultane- ous variables for unique relationships among them. The major findings to date can be sum- marized in the following listing: 1. The distensibility of the aorta decreases progressively along the aorta from the root to the bifurcation. 2. The "elastic" component of this visco- elastic system dominates the mechanical be- havior. 3. The vessel wall is incompressible. 4. The vessel wall demonstrates anisotropic properties, i.e. the elastic constants are differ- ent in the three different directions. 5. The longitudinal vascular tethering (the mechanical properties of the constraining tis- sues surrounding the vessel) have been meas- ured and can be represented by a linear mathe- matical model consisting of 1 elastic element, 1 visco-elastic element, 1 viscous element, and an inertance. 6. The harmonic content of both the pres- sure and the flow curves in the major arteries seldom exceeds about 10 cycles per second whereas the harmonic content of the pressure gradient and the time derivative of pressure usually exceeds 25 cps. 7. The hydraulic input impedance to most vascular beds, including the pulmonary artery 104 ANNUAL REVIEW OF INTRAMURAL RESEARCH and ascending aorta, are similar. The imped- ance spectrum is dominated by the constant term in each case. The time dependent terms decay rapidly with frequency. 8. The estimation of instantaneous blood flow, using the pressure gradient technique, has been validated in physical models as well as in the living pulsating vessel. The mean devia- tion of computed flow from monitored flow is of the order of 5% in the physical model, and of the order of 10% in the living pulsating vessel. 9. It has been established that the simple single degree of freedom model of pulsatile flow in a vessel segment proposed originally by this laboratory fits the experimental data as well as the more complicated and elaborate multidegree of freedom systems such as that suggested by Womersley. 10. The relationship between the time de- rivative of pressure and flow is a very indirect one which depends on the lumped properties of the entire vascular bed and, thus, contrary to the assertions appearing in the literature, it cannot be assumed to bear a unique relation- ship to the pulsatile flow at a point in the vascular bed. 11. Flow in the major arteries frequently can be shown to exceed the apparent critical Reynold's number, suggesting that arterial blood flow may be unstable or at the border- line between stable and turbulent flow during a significant portion of the cardiac cycle. 12. Pressure or flow disturbances are pro- pagated along the arterial tree at a velocity which is dependent on both frequency and on position along the tree. Pressure moduli appear to increase with distance along the aorta in a manner consistent with the observation noted above that the arterial tree becomes progres- sively stiffer peripherally. The lower harmonics of both the pressure and flow curve demon- strate strong reflections. The foregoing observations have clarified our picture of circulatory dynamics and have produced a number of useful mathematical models whose validity has been shown to hold at particular points along the vascular system. It remains to be seen whether an appropriate integration of these "segmented" models can predict over-all system behavior under widely varying conditions. This question can be ap- proached only by "driving" the entire vascular bed with known flow functions and observing the degree to which such an integrated model will permit us to predict the corresponding pressure functions, vessel displacements, etc. A special hydraulic flow generator is being designed for this purpose. With the foregoing major features of cir- culatory dynamics reasonably well defined, we are now in a position to turn to questions con- cerning the "fine-structure" of the pressure- flow relationships such as measurement of in- travascular velocity fields. These studies will allow us to probe more deeply into mechanisms associated with vessel wall "injury," i.e. the processes related to the "wear and tear" of aging and disease. To this end we began developing specialized measuring techniques about two years ago in conjunction with the Engineering and Mechan- ics Staff at Catholic University of America. If we are to gain insight into the fine structure of pulsatile flow, it is necessary to develop a ve- locity sensing probe which can be used to ex- plore the entire flow field in an artery. Dr. Ling of Catholic University is a specialist in the area of "hot-wire anemometry," and over the past year has developed a needle blood-flow probe which will permit us to map these flow fields. Studies validating the fidelity of this in- strument are currently being concluded. The device is linear and has a dynamic response extending beyond the range of our monitoring capabilities. Calculated extrapolations indicate that it is useful far into the kilocycle per sec- ond range. Therefore, it should be possible not only to map the "time smoothed" velocity pro- files in major arteries but also to estimate the frequency spectrum and energy content of the associated turbulence when present. Prelimi- nary studies in animals using this device indi- cates that the velocity profile is not symmetri- cal in the descending thoracic aorta. The rather consistent topography of athe- roma distribution in the human aorta sug- gests that hemodynamic events may be asso- ciated with the genesis of these lesions. The localization of atheroma does not appear to be NATIONAL HEART INSTITUTE 105 related to patterns of pressure distribution, but rather, to areas where one might expect high shearing stresses and/or turbulence. This ob- servation has suggested a "working hypothe- sis" which can be used as a scaffold upon which to direct future research in this pro- gram. It is hypothesized that a flux of lipoprotein molecules is driven toward the subendothelial tissue space from the blood phase by a con- centration gradient. This gradient may be in- creased by raising the blood concentration of fat. The "diffusivity" of the lipoproteins will depend primarily upon their molecular weight and their temperature in laminar flow and will depend in addition upon the "eddy" vis- cosity in turbulent flow. Factors assumed to act against this concen- tration gradient and diffusivity are the me- chanical resistance of the endothelial cell wall and perhaps also an electrical field of opposite polarity. This potential field is assumed to em- anate either from a fixed charge system in the endothelial wall or from selectively ab- sorbed materials at the blood-wall interface. To test this hypothesis two pilot studies have been completed. The first was designed to produce localized turbulence (to increase diffusivity) in dog aortas. The objective was to measure an associated increased deposition of fat and/or endothelial damage in the area of turbulence. Turbulence was produced by in- troducing a polished aluminum cylindrical plug into the descending thoracic aorta of dogs. The plug contained 2 patent longitudinal grooves placed axially along opposite sides of the outer surface. The plug size was chosen to occlude the unstretched aorta. Blood flow thus was di- verted into the two longitudinal channels formed on three sides by the walls of the groove and on the fourth side by that portion of the vessel wall covering the groove. Dimen- sions and pressure drops were such that the Reynold's number of the flow in the groove was about 1000 (critical). Intravenous infu- sions of emulsified fat were then given and the animal sacrificed after two hours. Serial sections were made of the longitudinal strips of vessel which had formed one wall of the high-velocity flow-channel. These were exam- ined for endothelial cell injury and fat deposi- tion. It was somewhat surprising to note that in nearly half of the dogs marked endothelial damage occurred in the area of high-shear flow. In all animals a very high correlation was demonstrated between the calculated hy- draulic shearing stress on the endothelial wall and the associated degree of endothelial dam- age. At times the shearing stress appeared to erode completely the endothelial cells down to the basement membrane. Evidence of fat in- filtration roughly parallels the degree of cel- lular damage. Clear cut evidence of fat infil- tration under the basement membrane was found in about V4 of the animals, strongly suggesting that fat was actually driven from the blood phase into the subendothelial region. These studies are being repeated employing a better controlled "shearing stress" so that both moderate degrees of exposure as well as heavy degrees of exposure may be compared. The second type of preliminary study was to explore the magnitude of any electrical bar- rier that might exist at the endothelial sur- face. A device was made which could be placed around the living pulsating blood ves- sel such that the position of electrodes intro- duced through the wall could be sensed electrically and recorded. Silver-silver chloride electrodes were used with high impedance am- plifiers to sense the voltages across the blood vessel lumen as well as along the lumen. The intravascular voltage distribution was found to be constant throughout the blood phase at about — 2 millivolts with respect to the uninjured adventitial tissue of the vessel. At the intimal surface the voltage suddenly drops about 10 millivolts. Histologic examina- tion of the electrode site in these instances showed no endothelial damage. The inference to be drawn is that this voltage represents some sort of "fixed charged" system on the endothelial surface and is not an "injury po- tential." As the electrode is advanced into the wall the voltage becomes initially somewhat more negative; however, then becomes pro- gressively more positive usually climbing to a value somewhat positive with respect to the 106 ANNUAL REVIEW OF INTRAMURAL RESEARCH voltage in the blood phase and occasionally positive even with respect to the adventitial surface of the blood vessel. The voltage on the ventral aspect of the aorta is about 4 mv more negative than that on the dorsal aspect of the aorta. Voltage differences also could be de- tected along the intimal surface of the aorta but no consistent trend could be established. Thus, it appears that there is, indeed, an elec- trical field probably associated with some "fixed charged" system at the endothelial sur- face. The significance of this as a barrier to passage of lipoproteins remains to be evalu- ated. Its significance in repelling the nega- tively charged blood cellular elements is ob- vious. Myocardial Mechanics The problem of myocardial function has been approached by three different avenues. The first has been study of cat papillary mus- cle preparation in an effort to devise empirical laws which will allow us to characterize the mechanical behavior of small units of muscle fibers. The second has been anatomical study of specially fixed total heart preparations from which dimensions, fiber distribution, and ori- entation have been determined microscopically. The third has been to study the sequence with which the various muscle masses are recruited into contraction under varying conditions of pressure and flow. The papillary muscle studies were greatly hindered by Dr. Feigl's move to the University of Pennsylvania where he has only recently been able to resume them. Moreover, efforts to derive empirical mathematical models for the data that we already have on hand have been hindered by the repeated delays in activating the hybrid computer unit. A three component model to describe myocardial fiber behavior has been devised and checked out on the large Honeywell 800 computer, however, use of this approach is extremely slow and expensive, therefore, we have deferred further analysis of the data until the high-speed hybrid facility is available. The anatomical studies are approaching com- pletion and have demonstrated that the canine heart may be considered to have a gross con- figuration somewhere between a cylinder and an ellipsoid revolution. Microscopically we have obtained clear cut evidence of consistent patterns of fiber orientation in the left ven- tricular wall. The inner 40% of the wall con- tains fibers running primarily in an axial di- rection. The middle 50% of the wall has fibers running in a circumferential direction and the outer 10% again in the axial direction at about 60° from the axis symmetry of the heart. Study of the sequence of contraction of the various muscle masses has indicated that a spectrum of behavior can be expected depend- ing upon the particular animal and the pres- sure presented to the ventricle. During systolic ejection about one-third of the hearts appear to contract primarily in a circumferential di- rection whereas one-third demonstrates pri- marily an apex to base contraction. The remain- ing third demonstrated various combinations of circumferential and longitudinal contrac- tion depending upon the phase of contraction. Attempts to correlate these modes of contrac- tion with the distribution of fibers and their orientation have been inconclusive primarily because of the small samples of anatomical data available. These studies will be extended such that a better sampling will be available and hopefully a relationship can be established between the sequence of contraction and the distribution and orientation of fibers in the muscle mass. Lung Mechanics The major activity over the past year in the area of pulmonary mechanics has been directed toward solution of two difficult practical prob- lems: automatic data processing techniques to handle the tremendous volumes of multi-chan- nel analog information necessary in defining the mechanical behavior of the lung, and sec- ondly, development of new instrumentation for the study of pulmonary mechanics in small animals, such as the rabbit. Although studies have continued in the area of mechanical auto- regulation of flow and flow through collapsible conduits, the pressing nature of the foregoing problems plus the continued unavailability of NATIONAL HEART INSTITUTE 107 the hybrid facility have made significant ad- vances in this latter area impossible. Analysis of the results of a study in pul- monary mechanics can present an overwhelm- ing problem of data reduction. In any mean- ingful or purposeful approach to these kinds of experiments, it is necessary to have the results analyzed from a particular experiment before proceeding to the next stage. Therefore, it is essential to streamline our methods of data analysis so that we may accomplish this objective. In a typical experiment 3, and us- ually more, variables are measured continu- ously with time as various prescribed respira- tory maneuvers are carried out. Although it is possible to record these data on a strip chart and then do a laborious graphic analy- sis, this frequently requires one man-week per experiment. This is obviously impractical, and, therefore, we have spent considerable effort developing processing techniques which will do these sorts of analyses automatically. General programs have been written and are now operational which will perform both edit- ing functions as well as data sorting routines. The editing routines compare the channels of data against certain editing criteria which may be specified, such as the absence of un- wanted noise, the adequacy of the prescribed maneuver, and so on. Once the data are edited to delete unwanted and redundant material, the data are then "sorted." "Sorting" consists of exploring the relation- ship between two variables at a time for se- lected constant values for all remaining vari- ables. If one has, in fact, captured all of the primary variables describing the system, then a meaningful relationship between the two se- lected variables will emerge. For example, in the case of pulmonary mechanics if one measures instantaneous flow, volume and pres- sure, one can ask for the relationship between pressure and flow for selected values of in- stantaneous volume. In this situation unique isovolume pressure flow curves emerge. At the experimenter's option, certain statistical fitting procedures then can be performed on the re- sulting data to allow the general form of the functional relationships to emerge immediately as smooth curves. Using these techniques it is now possible to perform an experiment one day and have the essence of the results displayed graphically the following day. The scientific economy of this is obvious. Standard techniques for measuring the var- iables for pulmonary mechanics in laboratory animals have not existed. This, therefore, has been our major effort in the area of pulmonary mechanics over the past year. A small body plethysmograph has been designed and fabri- cated in which instruments have been de- veloped for the accurate measurement of in- stantaneous respiratory flow, volume and pres- sure. The plethysmograph itself consists of a rigid plastic chamber in which it is possible to ventilate artifically an anesthetized and/or curarized animal. A positive-displacement elec- trical recording-spirometer continuously senses the volume changes of the animal. Instantane- ous flows are measured by a specially designed linear flow resistance device. The recording fidelity of these systems has been established in excess of 20 cps. Thus, adequate techniques are now available for the measurement of in- stantaneous flow and volume. The measurement of intrathoracic pressure in small animals however is quite another story. Detailed studies of the relationship of intrathoraic and intraesophageal pressure have been carried out in an effort to establish the conditions under which the latter could be used as an estimate of intrathoracic pressure. The obvious physiologic merit as well as sim- plicity of the esophageal approach makes con- siderable effort in this direction worthwhile. It has been found that over the upper two- thirds of the vital capacity the esophageal route is adequate for most purposes. However, there are a number of notable exceptions to this which must be evaluated. With the development of these better meas- uring techniques for small animals it is hoped that we can proceed to more detailed and bet- ter controlled studies of pulmonary mechanics in small animals as well as the development of suitable experimental animals for the study of the pathogenesis of chronic obstructive pul- monary disease such as emphysema. 108 ANNUAL REVIEW OF INTRAMURAL RESEARCH SURGERY BRANCH Clinic of Surgery The investigative efforts of the Surgery Branch have, as in past years, principally re- lated to studies of normal and abnormal cir- culatory physiology, particularly in relation to the methods and results of operative treatment in patients with congenital or acquired heart disease. A major proportion of patients referred to the Surgery Branch now require operations involving the use of prosthetic cardiac valves, and a number of clinical and laboratory in- vestigations have centered about the general problems associated with intracardiac prosthe- ses. The principal late complication which ac- companies the use of all presently available prosthetic valves is the formation of thrombus on them, and subsequent embolization. The anatomy of the calf heart and coagulation mechanisms of the calf closely resemble those of man, and various means of inhibiting thrombus formation on valves have been studied in this species. Standard Starr- Edwards valves universally collected thrombus in both the early and late postoperative period, while similar valves coated with graphite- benzalkonium-heparin seemed thrombus-re- sistant for one month, but later also clotted. In other calves with standard valves main- tained on warfarin, thrombus was prevented throughout the observation period of one year. The temporary thrombus-inhibiting effect of GBH coating is explained by other studies in which the coating was prepared with radio- active (H 3 ) heparin. With in vivo implanta- tion, 71% of the heparin was eluted from the surface within four hours, and more than 95% of it had disappeared within 36 days. The Melrose prosthesis in the tricuspid position has been used as a standard means of promoting thrombus. All such valves clotted within 48 hours in calves not given intravenous fluids postoperatively; animals given dextrose and saline survived for this period, but their valves revealed thrombus. In others given infusions of low molecular weight dextran during the first two days, 6 of 7 valves were thrombus- free at sacrifice. Studies are continuing to de- termine whether the effect of dextran in pre- venting thrombus formation is a specific one, or whether the effect is simply hemodilution. Initial observations indicate that the latter mechanism may be of principal importance. A controlled clinical study of the effectiveness of dextran after mitral valve replacement is un- derway, and most patients are treated with the agent for the 48-72 hours postoperatively. Seven patients with prosthetic mitral valves have died early with massive thrombosis of the prosthesis and atrium, and two others have died in the late postoperative period as the result of thrombus formation. A clinicopatho- logic study of the patients who died early indicated that the thrombosis probably re- sulted from a discrepancy between the size of the prosthetic valve and the size of the left ventricular cavity. In each, the muscular ven- tricular septum protruded into the cage of the valve, prevented full descent of the ball, and obstruction to left atrial emptying resulted. This observation has indicated the necessity for an unusually small Starr-Edwards valve, or a valve of the discoid type, in patients with mitral stenosis and small or normal left ven- tricular cavities. A new method for detecting intracardiac thrombus either pre- or postoperatively, has been studied in both animals and man. A rab- bit antibody specific for fibrinogen is labelled with I 131 and administered intravenously. The compound is concentrated where thrombus is in contract with blood. In dogs in which left atrial thrombi were produced experimentally, the radio-activity of the thrombus was suffi- cient to permit its detection by precordial scanning. In preliminary clinical trials, spe- cific labelling of left atrial thrombi was achieved in two patients with mitral valve disease, and in another with a thrombus in the left ventricle. The design and durability of the Starr-Ed- wards prosthetic valve has been proved in more than five years of clinical application but, as noted, thrombus formation on it is frequent. Past experience has proved that thrombus and emboli were never associated with the use of prostretic valves constructed of Teflon fabric, apparently because the valves quickly became NATIONAL HEART INSTITUTE 109 covered with host tissue. This principle has been applied to rigid Starr-Edwards valves, all metal parts of which were covered with a loosely woven fabric. Within six weeks of im- plantation all were covered with glistening tissue and none revealed thrombus. Continu- ing observations must be made to determine whether excessive tissue growth may result in dysfunction of the valve. Initial assessments have also been made of a new type of pros- thetic valve which may have advantages over those currently available. The valve consists of a ring-like body within which a stream- lined disc is held by an eccentric hinge. The opening angle of the valve can be controlled by the aerodynamic profile of the disc, and flow through it is largely laminar. Initial an- imal implantation is encouraging, and the valve will be subjected to detailed in vivo test- ing within the coming year. A major problem which may complicate the course of any open operation on the mitral valve is associated aortic regurgitation, and methods of detecting the aortic regurgitation before operation were assessed retrospectively in 156 patients with mitral valve disease. No troublesome regurgitation occurred when an aortic diastolic murmur was absent, but when such a murmur was present neither its in- tensity, the pulse pressure, nor the diastolic arterial pressure indicated the severity of the leak. The magnitude of regurgitant flow could be determined accurately only by cineaortog- raphy and this study is, therefore, indicated in every patient with mitral valve disease in whom a blowing diastolic murmur is heard. Abnormal left ventricular pressure pulses have previously been observed in patients with severe mitral regurgitation, indicating an ab- normal pattern of ventricular ejection and aortic flow. Instantaneous aortic flow was measured at operation in patients with severe mitral regurgitation before and after valve replacement, and in dogs in which mitral re- gurgitation of controlled magnitude was pro- duced. The pattern of aortic flow during mitral regurgitation was abnormal, and was charac- terized by an abbreviated systolic ejection per- iod and time to peak flow, an increase in peak flow and mean ejection rate, and increases in peak acceleration of flow and the volume ejected during the first half of systole. Normal flow patterns were recorded in the patients after mitral valve replacement. Another clinical study was made of the ef- fectiveness of operative treatment of atrial septal defect. Data were available in 175 pa- tients who were catheterized, operated upon, and followed thereafter. Six patients, four of whom were older than 40 years and were in congestive heart failure, died. Of 154 pa- tients studied postoperatively, all but 11 were proved to have complete abolition of the left- to-right shunt. A number of patients had se- vere pulmonary hypertension postoperatively, and with few exceptions elevated pulmonary arterial pressure and resistance persisted, even though the defect was closed. Residual pul- monary hypertension rarely caused symptoms, however. Approximately one-third of patients in have both clinical and hemodynamic evidences of severe tricuspid regurgitation, and in some clinics tricuspid valve replacement is frequently performed at the time of mitral replacement. Twenty-nine patients who had se- vere tricuspid regurgitation at the time of mi- tral valve replacement were managed conserva- tively; 24 had no operation on the tricuspid valve, and in five a tricuspid annuloplasty was performed. The operative mortality in this group (17%) did not differ from that in pa- tients without tricuspid regurgitation, and late clinical and hemodynamic studies re- vealed spontaneous regression of the tricuspid lesion as heart size and right ventricular pres- sure decreased. The experience indicates that tricuspid regurgitation which occurs in asso- ciation with mitral valve disease is usually of functional nature; the valve will become com- petent as the size of the heart decreases and tricuspid replacement is seldom indicated. The effects of tachycardia, induced by atro- pine, were assessed in patients with discrete aortic stenosis and in others with IHSS. With rate increases, the systolic pressure gradient was unchanged or fell in patients with fixed obstruction, while in those with IHSS the gradient always increased, and the calculated orifice area fell. It appears that the dimensions 110 ANNUAL REVIEW OF INTRAMURAL RESEARCH of the left ventricle decrease with tachycardia in patients with IHSS, and that the adminis- tration of atropine may prove a useful provoca- tive test in patients with this form of muscu- lar subaortic obstruction. The usefulness of the left atrial pressure pulse in assessing the severity of mitral ste- nosis was determined in 53 patients who were in regular sinus rhythm. It was observed that the height of the atrial a wave varied inversely with the hemodynamic severity of mitral ob- struction, suggesting that in advanced stages of mitral stenosis left atrial contraction is weaker and contributes relatively less to left ventricular filling. Certain patients, who are operated upon after long periods of congestive heart failure do poorly postoperatively, pos- sibly because myocardial norepinephrine stores are depleted. An attempt was made to identify such patients by preoperative studies of leg vascular resistance, determined after ipsilat- eral femoral arterial injections of tyramine or norepinephrine. Resistance was twice as high in patients who had been in failure as in asymptomatic ones, and the norepinephrine dose-response curve was distinctly steeper in the heart failure group. Each patient with heart failure was proved to have exceptionally low cardiac norepinephrine content. Several laboratory investigations have been related to cardiac arrhythmias and anti-ar- rhythmic drugs. It is recognized that patients in atrial fibrillation who are receiving digitalis may show evidence of digitalis toxicity follow- ing restoration of regular rhythm by counter- shock. This clinical observation suggested a study of the effects of atrial fibrillation on digitalis tolerance. In dogs, the toxic dose of acetylstrophanthidin was determined on suc- cessive occasions by titration. Later, and in the same animals, atrial fibrillation was pro- duced by atrial pacing and acetylstrophanthi- din titration repeated. The animals tolerated 10-15 percent more of the drug before develop- ing toxic manifestations during atrial fibrilla- tion. In other dogs some hemodynamic effects of atrial fibrillation were assessed. When fibril- lation was induced and ventricular rate was not controlled, systemic arterial pressure and cardiac output fell, and atrial pressure in- creased. In other animals measurements were made during atrial fibrillation and compared to those obtained during paced regular sinus rhythm at the same ventricular rate. Arterial pressure remained unchanged, but systemic flow was significantly lower during atrial fi- brillation than during regular rhythm at the same rate, an effect which can be attributed to impairment of atrial transport function. When ventricular tachycardia was induced, either by digitalis intoxication or by an ex- ogenous electrical pacemaker, the rate could be slowed in every animal by paired or cou- pled pacing. In these situations, ventricular tachycardia originated from a single ectopic focus. In other animals ventricular arrhyth- mias were produced by ligation of coronary artery branches, and multifocal ventricular contractions resulted. With this arrhyth- mia neither paired nor coupled pacing was effective, and either usually caused ventricu- lar fibrillation. Both lidocaine (Xylocaine) and procaine amide (Pronestyl) are widely used to control ventricular arrhythmias, but their effects on the contractile function of the heart have not been documented. In dogs in which heart rate, aortic pressure, and stroke volume were con- trolled, contractile force and left ventricular dp/dt were measured after these agents were administered. With lidocaine, either 1.5 or 3 mg./Kg., contractile force fell 18% after three minutes, but returned to control levels within 10 minutes. Either 6 or 12 mg./Kg. of pro- caine amide caused a progressive decrease in contractile force (maximum 15%) for 12 min- utes, and force remained depressed after 30 minutes. Diphenylhydantoin (Dilantin) was evaluated in a similar manner. Contractile force decreased 32%, and in other animals de- pression of ventricular function curves was always observed. The drug also decreased total peripheral vascular resistance in steady ar- terial bow preparations. A continuing laboratory and clinical inter- est of this unit has been the development, evaluation, and application of various biologic adhesives. On the basis of previous experi- mental studies, methyl-2-cyanoacrylate mono- mer has been found a satisfactory but not NATIONAL HEART INSTITUTE 111 ideal hemostatic agent. It is now undergoing systematic use in patients to reinforce closures of sutured incisions in the heart and great vessels, and to control bleeding from raw dis- sected areas. The courses of these patients, and the pathologic findings in those that die will be analyzed. A new adhesive system, con- sisting of gelatin, resorcinol and formaldehyde (GRF) has been developed. It permits a strong bond to be achieved in the presence of moisture, and has a high initial viscosity. Original prepa- rations were found to be toxic to tissue because of excess formaldehyde, a defect largely cor- rected in more recent formulations. In the dog, GRF was used to close incisions in the ileum, and all animals survived. In other dogs it ef- fectively controlled bleeding from the bisected spleen, the transsected kidney, and the incised bladder. It is contemplated that GRF will be found suitable for clinical application within the year. Previous reports have described studies of the effects of morphine on the heart and pe- ripheral circulation, and recent experiments evaluated the mechanism of action of this drug in the treatment of pulmonary edema. Pul- monary edema was produced in dogs by tech- niques which simulated the clinical disorders in which pulmonary edema commonly occurs. Morphine was administered and, as pulmonary edema subsided, strikingly and parallel de- creases in pulmonary arterial flow and pres- sure, and left atrial and left ventricular end- diastolic pressures were recorded. It appears that the principal beneficial effects of mor- phine in pulmonary edema result from an in- crease in vascular capacitance, and an asso- ciated decrease in systemic venous return. Mitral stenosis is one of the most frequent cardiovascular malformations encountered in patients, but no satisfactory experimental counterpart has been previously devised. In dogs, a suture was passed from the tip of the left atrial appendage, through the mitral valve, and out the posterolateral wall of the left ven- tricle. A cylinder of plastic sponge was at- tached to the atrial end of the suture, and the through the mitral valve ring. In dogs studied in both the early and late postoperative per- iods, left atrial and pulmonary arterial pres- sures were uniformly elevated, and diastolic gradients between the left atrium and left ventricle were present in all dogs. Long-term studies of the effects of mitral obstruction on the pulmonary vasculature can now be made. Other techniques for producing and studying the pulmonary vascular changes which result from various congenital cardiovascular malfor- mations have been devised. The end of a lobar pulmonary artery is anastromosed to the dis- tal end of an adjacent pulmonary vein; the lobe is then perfused with oxygenated blood at low pressure. In other similar preparations lobes are supplied with oxygenated blood under high pressure, or venous blood under high pres- sure. In this manner the relative effects of oxygen content and perfusion pressure on the pulmonary vascular bed can be assessed. The strain gauge arch is widely utilized to evaluate ventricular performance but the hem- odynamic variables which may influence the indicated contractile force have not been sys- tematically investigated. In dogs, recordings of the contractile forces of both ventricles were made as heart rate, stroke volume, and after- load were separately altered. Force increased with heart rate, but did not change over a wide range of stroke volumes. At constant rate and stroke volume, left ventricular force was a direct function of aortic pressure, whether the ventricle was empty or doing external work; this response was shown to result from changes in coronary flow. The experiment indicat that measurements of contractile force afford an accurate indication of ventricular performance only when rate and afterload are kept constant. Several clinical studies have originated from the pathology section of the Branch. The kid- neys of 132 patients who died of valvular heart disease were examined, and four were found to have extensive renal hemosiderosis. Each patient had a fixed calcified aortic valve which was both stenotic and regurgitant. Renal hemosiderosis was discovered in the kidneys of seven additional patients who had had prosthetic aortic valves inserted. These find- ings indicate that either a prosthetic valve or a diseased aortic valve can be responsible for intravascular hemolysis and, although renal 112 ANNUAL REVIEW OE INTRAMURAL RESEARCH hemosiderosis was often severe, there was no evidence that impairment of renal function resulted. The hearts of 24 patients who had the carcinoid syndrome were examined, and 16 were found to have carcinoid heart disease. In six, typical lesions were present in the left 3,ide of the heart as well as the right. The presence of cardinoid heart disease was evi- denced before death only by the presence of a murmur, unless detailed diagnostic studies were performed. Sections of the livers of 123 patients who died of valvular heart disease were examined and each was shown to have a brownish-yellow pigment in the centrolobu- lar hepatic cells. In none, however, was the pigment found to contain iron. In 24 patients of the group, however, selective stains were positive for hemosiderin and this finding was attributable to massive blood transfusions in the period immediately preceeding death. LABORATORY OF CLINICAL BIOCHEMISTRY Amine Biogenesis and Metabolism It is now generally accepted, as we originally proposed, that hydroxylation of tyrosine rep- resents the rate-limiting step in the production of norepinephrine. For this reason, inhibitors of this enzyme are far more effective in de- pleting tissues or norepinephrine than are in- hibitors of dopa decarboxylase or dpamine- iS-hydroxylase. Several classes of inhibitors of tyrosine hydroxylase have been found among tyrosine analogues and catechol derivatives. The former compete with tryosine the latter with the tetrahydropteridine cofactor. «- Methyl-tyrosine has proved to be a most po- tent inhibitor in vivo as well as in vitro. Using amethyl-tyrosine, it has been possible to show that when animals are subjected to stressful conditions such as severe exercise or exposure to cold, tissue levels of norepinephrine, even in brain, are maintained by virtue of a rapid re- synthesis. In other words stimulation of sym- pathetic tissues causes them not only to release norepinephrine but also to increase the rate of synthesis. These findings were corroborated by experiments with labeled tyrosine. It was shown that exercise and exposure to cold re- sult in the incorporation of 2 to 4 fold larger amounts of administered tyrosine- 14 C into norepinephrine. The procedures have no effect on the amount or specific activity of the free tyrosine- 14 C in the tissues. We have obtained some evidence of increased synthesis in isolated stimulated tissues. Other laboratories have obtained even more evidence for such an effect in isolated organ preparations. The mechan- ism for this increased synthesis is of interest. It appears that the increased synthesis is due to increased tyrosine hydroxylase activity. However, we have some evidence that neither exercise nor cold increase the absolute amount of the enzyme in tissues. It would appear, therefore, that the enzyme in vivo under nor- mal conditions is not operating at maximal capacity and that some factor or factors in- crease its activity. One possibility is that nor- epinephrine itself, which we have shown to be an inhibitor of tyrosine hydroxylase, controls the initial reaction by end-product inhibition. Several other laboratories have reported find- ings consistent with such a mechanism. We have obtained definite evidence that all three enzymes involved in norepinephrine syn- thesis are associated with a similar subcellu- lar fraction within tissues. We feel, however, that the homogenization and sedimentation techniques which were used are not adequate for unequivocal determination of intracellular localization. Because of this limitation, we have turned to fluorescent antibody methods. We have purified beef dopamine-/3-hydroxylase and prepared potent antibody to it in the rab- bit. A contract was awarded to Microbiological Associates to produce enzyme antibodies, pur- ify them and assist us in labeling them with fluorescent dye. Inhibition of tyrosine hydroxylase has been carried to the clinical level in joint studies with the Experimental Therapeutics Branch, a- Methyl-tyrosine was administered to patients and was shown to decrease the formation of norepinephrine. At doses of 1.5 to 3 gram per day about 70% inhibition was attained in a large number of patients with essential hyper- tension and pheochromocytoma. In the latter group, blood pressure was lowered to the nor- mal range as the excretion of norepinephrine and its metabolites fell to normal values. It NATIONAL HEART INSTITUTE 113 appears that a-methyl-tyrosine may become the treatment of choice for patients with ma- lignant pheochromocytoma. Another collaborative effort with the Exper- imental Therapeutics Branch concerns patients with phenylketonuria (PKU). Attempts will be made to answer two questions: (1) Is the small amount of conversion of phenylalanine to tyrosine which occurs in patients with PKU due to residual liver phenylalanine hydroxy- lase or is the latter enzyme completely lacking and the conversion carried out by the action of tyrosine hydroxylase in sympathetic nerves ? If the latter is the case, then a-methyl-tyro- sine, which specifically inhibits tyrosine hy- droxylase, should abolish conversion of phenyl- alanine to tyrosine in patients with PKU but have no effect on the conversion in normals. (2) Do the large amounts of phenylalanine in tissues of patients with PKU inhibit tyrosine hydroxylase sufficiently to disturb peripheral and central sympathetic activity? If so, then we may expect to find similarities between phe- nylalanine and a-methyl-tyrosine in their phar- macologic effects in patients without liver phe- nylalanine hydroxylase. Attempts are also be- ing made to produce animals deficient in liver phenylalanine hydroxylase activity by admin- istering p-chlorophenylalanine. Collagen and Hydroxypyroline Out laboratory was the first to demonstrate formation of collagen hydroxyproline in cell- free systems and to present evidence for hy- droxylation of peptidyl proline. Until last year several laboratories claimed that hydroxyla- tion occurred at the level of t-RNA-proline. In the last year these contrary claims have been answered and withdrawn and it is now clear from work in three other laboratories (Proc- kop, Lukens, Meister) that we were correct in the mechanism we proposed. Continuing these studies, we were able to show that all those tested of a number of tissues which form collagen are capable of accumulating a protein deficient in hypro- 14 C when incubated with proline- 14 C. This protein is formed when hy- droxylation is inhibited for any reason, e.g. lack of oxygen, chelation of Fe ++ , lack of as- corbic acid, destruction of proline hydroxylase. The protein can be extracted along with col- lagen by the use of neutral salt solutions or acetic acid and can be degraded to peptides by specific bacterial collagenase. More recently we showed that the solubilized newly formed hypro deficient "collagen" obtained from guin- ea pig granuloma, fetal rat skin and chick em- bryo can all be hydroxylated by chick embryo proline hydroxylase. The latter enzyme has now been demonstrated in soluble preparations ob- tained from rat liver, fetal rat skin, and guinea pig granuloma. Previously the chick embryo system was the only cell-free system in which proline hydroxylation could be demonstrated. Soluble proline hydroxylase, purified several fold, is stimulated by Fe ++ , ascorbic acid and an unknown dialyzable substance found in boiled tissue extracts. It dees not hydroxylate free proline or tripeptides containing proline. Proline, hydroxyproline, gly-pro-pro and gly- pro-hypro do not inhibit the hydroxylation of peptidyl-proline. This represents further evi- dence that smaller residues are not intermedi- ates in the hydroxylation. Two potent inhib- itors of peptidyl-proline hydroxylase are poly- proline-MW 10,000 and ( gly-pro-pro) n -MW 5,000. The former has been shown to inhibit competitively with solubilized protein sub- strate. Attempts are being made to produce in- hibitors which are not only specific, but small enough to penetrate into cells and inhibit the enzyme in vivo. Studies on peptidyl proline hydroxylase have been time consuming because the method of assay took about two days. Several years ago, we had considered using tritium labeled pro- line to follow proline hydroxylation by oxida- tive displacement of a tritium atom and we persuaded Dr. Bernhard Witkop to prepare 3,4- tritio-proline from 3,4-dehydroproline. V H COOH v H COOH T+ + H 2 0?±THO + H+ In the crude cell-free chick embryo systems, we could not use this procedure because of 114 ANNUAL REVIEW OF INTRAMURAL RESEARCH many interfering side reactions. However, Meister succeeded in using it in following hy- droxyproline formation in whole cell prepara- tions from guinea pig granuloma. Recently, we have been able to prepare soluble hydroxy- proline deficient substate, labeled with proline- 3 H, for studies of proline hydroxylase. This material was purified and shown to contain traces of hydroxyproline- 3 H (less than 1% of the proline). On incubation with proline hy- droxylase we have demonstrated a stoichio- metric displacement of tritium from substrate proline into water which is collected by a rapid distillation procedure. The method is extreme- ly rapid, the blanks are essentially zero and the labeled substrate is stable indefinitely. With this procedure, we hope to purify the en- zyme, characterize it and develop useful in- hibitors. In collaboration with Dr. Arieh Ber- ger, we are looking into syntehtic polypeptide substrates and inhibitors. Actinomycin Biosynthesis Although attempts to obtain synthesis of this peptide antibiotic in cell-free systems have failed, it has been possible to obtain more information on the nature of the synthetic process. 4-Methyl-3-hydroxyanthranilic acid (MHA) has been identified as a normal inter- mediate in the formation of actinomycin. Pre- sumably this compound adds on the amino acid present in the pentapeptide chain. It has also been shown that the addition of D-valine to the medium results in the accumulation of MHA. This D-amino acid known to be an in- hibitor of antibiotic production, inhibited either the formation or the attachment of the pentapeptide chain. Other studies have shown that actinomycin inhibits the organism that produces it, sug- gesting that the production of the antibiotic may be a way by which the organism regu- lates its metabolism. Highly- labeled actinomy- cin has been produced and its distribution in tumor-bearing mice was investigated. Cobamide Dependent Methionine Synthesis The cobamide-dependent enzyme catalyzing methyl transfer from N 5 -methyl-folate to ho- mocysteine has been purified 200-fold. It is salmon-red in color and studies are being initi- ated on the binding of radioactive substrates to the enzyme. The unique function of S-adeno- sylmethionine (AMe) in this reaction has been investigated. It appears that AMe methylates the enzyme since other methyl donors (methyl- iodide, etc.) can replace Ame in the catalytic reaction. Studies are continuing in the forma- tion of boloenzyme. apoenzyme + cobamide »holoenzyme A specific enzyme is necessary for this reac- tion if the cobamide employed is either hy- droxy or deoxyadenosyl-B 12 . Attempts to pu- rify this enzyme as well as apoenzyme are in progress. Formation of Formyl-methionine sNRA The synthesis of N-formyl-methionine sRNA may be an important reaction in the initiation of protein synthesis. The enzyme that cata- lyzes the reaction N 10 formyl — THF + methionine sRNA — >N — formyl methionine sRNA + THF has been purified 2000-fold. The characteris- tics of the reaction are under study. The spe- cificity for the substrate resides in the sRNA molecule. Ethionine and norleucine, when at- tached to the methionine sRNA, are also for- mylated. Aromatic Hydroxylation 5-Tritio-tryptophan has been synthesized and used to develop an assay for tryptophan hydroxylase. There is a marked isotope effect in a hydroxylase system from mast cells al- though none was observed in whole cells of Chromobacterium violaceum. Studies on the mechanism of ether cleavage by a microsomal system have shown that O 18 is not incorporated into the phenolic product. This type of reaction does not involve a single displacement of the methoxy group by molecu- lar oxygen. Serine Metabolism A simple and sensitive method for the assay of serine hydroxymethylase has been developed. NATIONAL HEART INSTITUTE 115 It is based on the isolation of formaldehyde released as the dimedon derivative. 2,3-Dihydroxybenzoylserine has been iso- lated as a metabolite of stationary cells of E. coli. It is synthesized by the following re- action: 2,3 — dihydroxybenzoic acid + serine + ATP — >2,3 — dihydroxybenzoylserine. The enzymatic reaction and the role of this compound in the metabolism of E. coli are un- der investigation. Studies on Amino Acid Transport Three aminonaphthylalanines were prepared and shown to have fluorescence characteristics which were discernible in the presence of ani- mal and bacterial cells. Two of these amino acids were found to be concentrated by Sar- coma-37 ascites cells in a manner comparable to that of naturally occurring aromatic amino acids. The fluorescence of the amino acid after its active transport into cells did not differ in excitation spectrum, emission spectrum, quan- tum yield and temperature coefficient. Exami- nation with the fluorescence microscope indi- cated uniform distribution through the cyto- plasm. Furthermore, rupture of the cells re- leased the amino acids completely. By contrast atabrine, eosin and anilinonaphthalenesulfonic acid, which are known to bind to macromole- cules, exhibited changes in many parameters and were not released from the cell on rup- ture. Since intracellular concentration of the aminonaphthylalanines does not alter fluores- cence characteristics which are normally changed when a small molecule binds to a large one it is reasonable to assume that the amino acid exists in a free form. These findings rep- resent direct evidence that actively trans- ported amino acids exist free in the cell. Peptides and Peptide Linkages The major kinin in wasp venom has been purified and subjected to sequence analysis. Preliminary data indicate that glutamic acid is at the N-terminal end. The sequence of two amino acids remain in doubt (in parenthesis) but the following structure is fairly certain: glu (asp, thr) lys lys leu arg lys gly arg pro pro gly phe ser pro phe arg bradykinin sequence The demonstration of /?-aspartyl linkages in fibrin has been established. Brain Nucleic Acid Studies with rats in which the liver is ligated have shown that simple precursors such as as- partic acid, orotic acid and glucose do not yield appreciable labeling in brain RNA. These results agree with studies on brain slices which indicate that brain requires preformed nucleo- sides for its synthesis of RNA and presumably obtains them via the blocd from the liver. Bacterial Phenylalanine Hydroxylase and Its Cofactor Induced phenylalanine hydroxylase of Pseu- domonas sp. has been purified to a high de- gree. It has been shown to have an absolute re- quirement for Fe ++ but can be activated by many other metals. The pteridine cofactor re- quired for activity is also synthesized by the organism and it has been shown that it is de- rived from guanosine phosphate. Some of the intermediate enzymes required for synthesis of the pteridine have been isolated and studied. Other aspects of control of aromatic amino acid biosynthesis in Pseudomonas have also been investigated. Nitrogen Containing Lipids The significance of palmitylethanolamide is still not clear. On the one hand it is normally made and found in tissues. On the other hand its synthesis in in vitro preparations seems to represent an artifact formed by the reaction of ethanolamine with some form of "activated" fatty acid. The nature of this activated fatty acid in tissues remains to be determined. Enzyme Mechanisms Glutamic dehydrogenase, glyceraldehyde-3- phosphate dehydrogenase, tyrosine hydroxyl- ase and dopamine-/?-hydroxylase have been sub- jected to careful kinetic studies. Antibodies 116 ANNUAL REVIEW OF INTRAMURAL RESEARCH have been prepared to some of these and the mechanism of interaction studied. EXPERIMENTAL THERAPEUTICS BRANCH A broad spectrum of research has been con- tinued over the past year with a major orienta- tion toward findings which may be of imme- diate or at least potential clinical significance. Data obtained will be considered under three headings: (1) Biochemistry and Pharmacology of Aromatic Amines, (2) Studies of Selected Proteins, and (3) Miscellaneous. Biochemistry and Pharmacology of Aromatic Amines It has been our impression that, with the exception of amines in urine collected follow- ing the ingestion of amine-containing foods, the urinary aromatic amines are of endogen- ous origin. Evidence has been presented in the literature, however, that the action of intesti- nal bacteria may contribute to urinary tyra- mine and tryptamine. Our studies on the effects of gluccse diets and intestinal sterilization have reconfirmed the conclusion that tyramine, tryptamine and metanephrine are of tissue ori- gin and reflect endogenous metabolism. Rather than a decrease in the excretion of tyramine and tryptamine during gut sterilization in pa- tients receiving a constant diet, a significant and consistent increase in excretion of these amines was observed. This latter finding has not been explained as yet. Previous work showed that a-hydrazino-his- tidine is a potent and selective inhibitor of his- tidine decarboxylase in animal systems both in vitro and in vivo. Clinical trials of the com- pound were initiated in three patients. It was quickly established that the drug is poorly tol- erated because of the development of emesis and further clinical use is not anticipated. Con- comitantly, a former member of the depart- ment has administered another histidine de- carboxylase inhibitor, 4-bromo-3-hydroxy-ben- zyloxyamine (NSD-1055), to patients with urticaria pigmentosa and has observed rapid and striking clinical improvement. Similar studies are planned in this department with NSD-1055 which is also a potent inhibitor of dopa decarboxylase and dopamine /3-oxidase. It was shown previously that up to 1.0 gm per day of this compound is well tolerated by hu- man subjects. A sensitive radioassay has been developed for measuring the activity of tryptophan hy- droxylase; the reaction catalyzed by this en- zyme appears to be the rate limiting step in the biosynthesis of serotonin. The assay was perfected using as a prototype the tryptophan hydroxylase from neoplastic murine mast cells. Preliminary attempts to study tryptophan hy- droxylase in mammalian brain tissue have in- dicated low levels of this enzyme in rabbit brain stem in some experiments. Unlike mast cell tryptophan hydroxylase, active prepara- tions of the brain stem were not stimulated by tetrahydropteridine or ferrous iron. The mast cell preparation is in use both here and else- where in screening programs for the discovery of the inhibitors of tryptophan hydroxylase. Such compounds should be useful in the treat- ment of patients with the carcinoid syndrome. First stage clinical testing is planned with one such agent, 3,4-dihydroxyphenyl-/3-propylacet- amide (dopacetamide), a potent inhibitor of tryptophan hydroxylase both in vitro and in vivo. Tryptophan deficiency has been suspected of causing African cardiomyopathy and serotonin of causing endomyocardial fibrosis. A number of studies of serotonin metabolism in trypto- phan deficient rats have been done with incon- clusive results. Peculiarly, such animals show a greater uptake of injected serotonin by the heart than do controls. Further studies are planned relative to reports in the literature that patients with African cardiomyopathy have elevated plasma serotonin concentrations and that tryptophan deficient rats have in- creased amounts of 5-hydroxyindole-acetic acid in the urine. Hydroxylation of tyrosine to dopa has been found to be the rate limiting step in the bio- synthesis of the catecholamines. In collabora- tion with members of LCB a number of stud- ies have been performed in laboratory animals. Rats, pretreated with the tyrosine hydroxylase inhibitor a-methyl-tyrosine («MPT) and then stressed by exercise on a treadmill or exposure NATIONAL HEART INSTITUTE 117 to cold (3° C), show a great reduction in brain, heart, spleen and adrenal catecholamines. There is little change with stress or aMPT alone. The findings are interpreted as indicat- ing increased rates of synthesis of norepine- phrine and epinephrine due to increased sympathetic nerve activity. The effect of sym- pathetic nerve activity on norepinephrine synthesis was evaluated more directly in a newly-designed, isolated nerve, perfused-heart preparation of the guinea pig. Nerve stimula- tion increased norepinephrine synthesis as judged by the difference in concentrations of the amine in the heart in the presence and ab- sence of «MPT. Thus far, however, it has not been possible to demonstrate an increased in- corporation of radioactivity into norepineph- rine from C 14 -tyrosine in the perfusing fluid. It may be necessary to add a monoamine oxi- dase inhibitor to prevent metabolism of the amine and to study the effect of varying con- centrations of tyrosine in the perfusing fluid. A number of tyrosine hydroxylase inhibitors have been compared for their catecholamine depleting potency in vivo in the hope that at least on compound in addition to aMPT might be available for clinical studies. One possibil- ity is L-3-iodo-a-methyl-tyrosine which is a more effective inhibitor than «MPT. Another is a-methyl-phenylalanine which is about as po- tent as aMPT and has the advantage of a much greater solubility in water. Clinical studies with aMPT have been concerned with its ab- sorption, metabolism, chemical effects and pharmacologic effects in patients with pheo- chromocytoma, and essential hypertension. About 30 patients have been studied, 17 of whom had pheochrcmocytoma. In oral doses of 400-400 mg/day of aMPT, inhibition of cate- cholamine synthesis was shown as indicated by the urinary excretion of catecholamines and their metabolites. Reductions of synthesis up to 60-90% were achieved by the larger doses in both types of patients. In cases of pheochro- mccytoma reductions of catecholamine synthe- sis were accompanied by marked clinical improvement; subjects with essential hyper- tension showed only a minimal blood pressure reduction while receiving the drug. It was shown that after oral administration of aMPT 45-90% is absorbed. After single oral doses of the drug maximum blood levels occur within two hours and 50% of the dose is excreted within 4 to 6 hours though detectable amounts of drug (radioactive label) are excreted for up to 3 days. Almost all the orally absorbed drug is excreted unaltered though small amounts appear as the decarboxylated product, amethyl- tyramine, and as catechol products of which a-methyl-dopa and a-methyl-dopamine have been identified. The drug appears to have defi- nite value in the management of cases of ma- lignant pheochromocytoma and in preparation of benign cases for surgery. In a few cases of the latter type the tumor has been rendered functionless by the drug. While a slight lower- ing of blood pressure is demonstrable in the patients with essential hypertension receiving large doses of the drug, it is doubtful that the hypotensive potency of the compound is suffi- cient to be therapeutically useful. Most pa- tients develop readily recognizable nervous system effects during treatment with aMPT. Typically the patients show sedation during the first two days of therapy; this may be pro- nounced but is usually greatly diminished with- in three days. At dosages of 3 gm or more per day, occasional patients develop tremor and agitation. Upon discontinuation of drug most patients show some degree of insomnia and heightened alertness and energy for several days before resuming their usual behavior pattern. Overt depression has not been ob- served. It is possible that aMPT would be therapeutically useful in other diseases such as thyrotoxicosis, Raynaud's disease, anxiety states, angina pectoris and endotoxin shock; further investigations are planned along these fines. The tyramine pressor test for pheochromocy- toma has now been evaluated in 20 patients with this tumor. The incidence of false nega- tive tests is in the range of 25-30% which is comparable to the incidence of false negatives with the histamine test. In contrast to the his- tamine test, however, there appears to be no hazard in the use of tryamine as a provoca- tive agent. A continuing interest in catechola- mine metabolism has resulted in extensive clin- ical experience in the medical and surgical 118 ANNUAL REVIEW OP INTRAMURAL RESEARCH management of patients with pheochromocy- toma. This has permitted a number of other studies to be performed. Contrary to data re- ported in the literature, no diminution in red cell mass or plasma volume has been found ex- cept in cases of severely ill patients with metastatic disease. In spite of the apparently normal blood volume it has been possible to obviate the typical severe hypotension follow- ing removal of tumor by vigorous transfusion with volume expanders (blood, plasma or al- bumin in saline solution). Experimental use of the /^-adrenergic blocking drug propranolol has shown it to be effective in the manage- ment of cardiac arrhythmias occurring during surgery. Several of the pheochromocytoma pa- tients studied during the past year are mem- bers of a single family and have exhibited the recently recognized triad of hyperparathyroid- ism, thyroid medullary carcinoma with amyloid deposits and bilateral adrenal pheochromocy- tomas. The huge increase in the work load of analyses of catecholamine metabolites has led us to set up a computer program for the calcu- lation of results. Studies on the turnover rates of radioactive dopamine, and norepinephrine and its metab- olites following the intravenous administration of H 3 dopa have been expanded considerably. The half time for turnover of dopamine, nor- epinephrine and vanilylmandelic acid averaged 6 hours, 8 hours and 14 hours respectively in normal subjects and hypertensive patients. No significant difference between normals and hy- pertensives was noted. During treatment with «MPT the specific activity of norepinephrine was increased, a finding consistent with a small dopa pool. Treatment with a monoamine oxidase inhibitor also increased the degree of labelling of norepinephrine and VMA but slowed their rates of decay. Reserpine therapy greatly increased the rate of decay. The latter finding is one of the most convincing demon- strations of an effect of reserpine in clinical doses on catecholamine metabolism in the human. Studies of Selected Proteins Measurements of the urinary excretion of hydroxyproline (HOPr) peptides as an index of endogenous collagen metabolism have been continued in a number of clinical circum- stances. Hydroxyproline excretion was found to be normal or slightly elevated in patients with scleroderma. When dimethylsulf oxide (DMSO) was applied topically to almost the entire body (in doses as high as 70 ml of 90% DMSO) of 5 patients with sclerodema, urin- ary hydroxyproline was unchanged. Healing of ischemic ulcers was noted in 2 patients con- comitant with DMSO therapy. Urinary hy- droxyproline excretion was also found to be unchanged in patients with scleroderma receiving up to 12 gm per day of potassium paraaminobenzoic acid or 2 gm daily of D-peni- cillamine. In one patient with severe sclero- derma who received /?-aminopropionitrile (BAPN) for 4 weeks at a dose of 3 gm daily, urinary HOPr increased about 50%. During the past year a number of techniques have been adapted to determination of a "col- lagen profile" in minced specimens of dermis obtained by punch biopsy. This profile consists in part of determination of water content, to- tal collagen, percent of collagen extractable into 0.5 molar acetic acid, and the degree of cross-linking in soluble collagen as estimated by disc electrophoresis on acrylamide gel. One hundred twenty biopsies from patients with various clinical disorders in soluble collagen was observed along with a decrease in cross- linking, associated with a high water content. Young normal scars had a similar collagen profile while scars older than 6 months resem- bled normal skin. D-penicillamine, used in the treatment of Wilson's disease, cystinuria and (by one investigator) rheumatoid arthritis, was found to produce an accumulation of sol- uble, poorly cross-linked precursors of insolu- ble collagen in the skin. This is the effect seen in animals given lathyrogenic agents such as BAPN. In several patients with Wilson's dis- ease treated with large doses of penicillamine for more than 4 years, levels of soluble collagen were found to be 5 times those in normals (pa- tients =20%; normals <3.5%). Possible thera- peutic use of penicillamine in scleroderma is suggested by the finding that in this disease soluble collagen in dermis is consistently less than normal. Heritable diseases of connective NATIONAL HEART INSTITUTE 119 tissue were also evaluated. Patients with Mar- fan's syndrome, Ehlers-Danlos syndrome and osteogenesis imperfecta were found to have normal collagen solubility and cross-linking. Patients with Ehlers-Danlos syndrome were found to have less total collagen expressed as ^g/HOPr/mg dry weight than normals. Sev- eral patients with homocystinuria — a condition with excess circulating and tissue levels of sulfhydryl-containing compounds similar to penicillamine — have had significantly in- creased solubility and decreased cross-linking in dermal collagen. Normal collagen profiles have been found in acromegaly and iatrogenic Cushing's disease. Adult patients with isolated growth hormone deficiency have less soluble collagen than normal. This is in contrast to the high levels of solubility with normal cross- linking in collagen of adolescents and children, reflecting phases of active growth and protein turnover. Earlier hypotheses that MarfanV syndrome and osteogenesis imperfecta repre sent abnormalities in the maturation of colla- gen thus were not supported by these studies. On the other hand penicillamine produces a true "collagen disorder" in man. The lathyrogenic agent BAPN has been ad- ministered carefully to five patients with advanced scleroderma. Maximum dose admin- istered was 3 gm per day and maximum dura- tion 4 weeks. A method was developed for measuring BAPN in biological materials using the automatic amino acid analyzer. The major metabolite of BAPN, cyanoacetic acid, has been measured by either gas or paper chromatog- raphy. After single or multiple oral dosage, 7- 15% of the drug can be recovered unchanged in the urine, and 50-75% as cyanoacetic acid. The half-life of BAPN in the blood is about 2.5 hours and no accumulation is observed on a 6 hour schedule of dosage. Mild allergic reactions developed in two of the patients, dis- appearing rapidly when the drug was discon- tinued. With the addition of the dermal colla- gen profile it is felt we can now proceed more rapidly and safely with trials of BAPN since the effect of the drug on collagen can be more accurately monitored and is thought to be the main basis of toxicity in experimental lathy- rism. The incidence of allergic reactions to the drug is unknown but thus far these have not been of sufficient severity to deter us from fur- ther investigations. Probably the BAPN stud- ies will be slowed somewhat by the recent find- ings that penicillamine, a current drug, also has powerful effects on collagen cross-linking. Since the cross-linking in collagen appears to involve the oxidative deamination of terminal amino groups of lysine residues, importance of a soluble monoamine oxidase is suggested. We have proceeded on the basis that plasma mono- amine oxidase may be the enzyme involved and accordingly have developed a method of radio- assay for this enzyme. Preliminary findings in- dicate that BAPN is a competitive inhibitor of plasma monoamine oxidase and that the mono- amine oxidase levels in plasma of patients re- ceiving pencillamine is markedly reduced. The conversion of radioactive proline to urinary peptide-bound hydroxyproline has been studied in young adult rats and in a sin- gle patient with scleroderma. The rats were studied several weeks after the administration of a single pulse label and at a time when the specific activity of urinary hydroxyproline was almost constant, reflecting metabolism of previously labeled and now insoluble collagen. When parathyroid hormone was administered to these rats, urinary hydroxyproline excre- tion increased to twice normal levels while hydroxyproline specific activity remained un- changed. This indicates that parathyroid hor- mone exerts its effects on the metabolism of insoluble collagen since the increase in urinary hydroxyproline originated from the labeled in- soluble collagen pool. Similar studies with thy- roid hormone indicate a mixed action, that is, both on synthesis and degradation. The at- tempt to label the collagen of a patient with scleroderma was successful. The specific activ- ity of his plasma hydroxyproline was found to have a half-life of about 6 days and the graph of his urinary hydroxyproline specific activity indicated contributions from at least two dif- ferent pools of collagen. The first had a half- life of 0.5 days and probably represents the sol- uble collagen pool while the second had a half-life of over 60 days, representing the in- soluble collagen pool. It will not be possible to do several studies on the effects of drugs on 120 ANNUAL REVIEW OF INTRAMURAL RESEARCH human collagen in this patient whose collagen pools have been labeled with C 14 in hydroxy- proline. Studies have continued on the small non- heme iron containing proteins which serve as electron carriers in low redox potential reac- tions in certain bacteria. These include the clostridial ferredoxins and a new protein termed "rubredoxin" which was first isolated and crystallized from C. pasteurianum in this laboratory. According to the literature ferre- doxin is a one electron carrier but conclusive evidence has now been obtained that clostri- dial ferredoxin is a two electron carrier. A search for ferredoxin-like proteins in mammal- ian systems has so far been unsuccessful. Stud- ies on rubredoxin indicate that it has a molec- ular weight of about 6,000, contains a single iron atom and has a redox potential (minus 0.05 v) considerably higher than ferredoxin (minus 0.42 v). Studies on the reactivity of the cysteines of the molecule as well as electron spin resonance studies indicate that the iron in ferredoxin is ionically bound in contrast to the apparent covalent iron-sulfur bonding in ferredoxin. Similarity of the protein to ferri- chrome A, transferrin and a number of ferric iron chelates is suggested. Although the meta- bolic significance of rubredoxin is unknown it would appear to be useful in the study of iron binding mechanisms. Miscellaneous The only study in this category which has potential of generating a new research pro- gram is that concerned with oxidative phos- phorylation in mitochondria. The work was an outgrowth of attempts to extend the ferre- doxin studies into mammalian systems. Of sev- eral different findings the most interesting were those indicating that administration of large depot doses of catecholamines in rats pro- duces an uncoupling of oxidative phosphoryla- tion in heart mitochondria studied in vitro. This catecholamine affect cannot be shown in vitro using mitochondria from untreated animals. The time course of catecholamine ac- cumulation by myocardium was found to be similar to the P/O ratio depression produced following catecholamine administration. Re- sults following pretreatment with adrenergic blocking agents provided further evidence of a correlation between uncoupling of oxidative phosphorylation and elevation of myocardial catecholamines. Although the mechanism of the catecholamine effect in vivo is not yet defined, the phenomenon itself may contribute to our understanding of oxygen "wasting" that can be produced by catecholamines in animals and in man. LABORATORY OF TECHNICAL DEVELOPMENT Fluorescence Methods Methods in current use for measuring the absolute quantum yield of fluorescence (ratio of quanta emitted to number absorbed) are unsatisfactory. The methods are either im- precise or require special equipment, thus mak- ing it difficult to check on claims of accuracy. It seemed to us that the Aminco-Bowman spec- trophotofluorometer could be used for this pur- pose, since our instrument was calibrated last year. To demonstrate that quantum yields could indeed be measured with this instrument, studies were undetraken to measure corrected spectra of compounds which had been reported on in the literature as well as compounds which had never been investigated with re- spect to fluorescence efficiency. By publishing our results, it was hoped that it could be estab- lished that commercially available instrumen- tation was capable of performing a determina- tion hitherto thought possible only with specially-built components. The spectrophotofluorometer was evaluated with regard to quantum yield determinations by making these determinations for some 30 solutions, for some of which there were litera- ture values. In general, the agreement with established values were good. Temperature de- pendence of quantum yield, and corrected emission spectra were determined. Sources of error, advantages, and disadvantages of the system were listed. Quantum yields of tyrosine, tryptophan, and phenylalanine were found to be about 33% lower than reported by Teale and Weber. Our NATIONAL HEART INSTITUTE 121 values of 0.15, 0.14, and 0.027 were recom- mended for use as standards in protein fluo- rescence work. Quantum yields of fluoro- phenylalanines (3 isomeric forms) were found to be about 0.19. Quantum yields and fluorescence polariza- tions were determined for a large number of dye-conjugates of bovine serum albumin. These are the first measurements of this type for such commonly used (in immunology, for ex- ample) dyes such as fluorescein and 1-di- methylaminonaphthalene-5-sulfonate. It was shown that dye-dye energy transfer occurs and causes a decrease in fluorescence yield with degree of labeling. Perrin plots showed that the depolarization was due to energy trans- fer in heavily labeled conjugates. Foster dis- tances were calculated for four types of dyes, and the results indicated that dipole dipole interaction was probable on a molecule of the size of BSA. The fluorescence quantum yield of 1-dimeth- ylaminonaphthalene-5-sulfonate was found to be lower than reported by Weber and Teale. In addition, the fluorescence was quenched by bicarbonate. It was pointed out that some workers had used the erroneously high figure of Weber and Teale in their calculations. Quantum yields of pyridoxamine-5-phos- phate, pyridoxamine, and pyridoxal were found to be 0.14, 0.11, and 0.048 at room temperature in neutral aqueous solutions. Fluorescence polarization spectrum of pyridoxamine-5-phos- phate and the temperature dependence of quantum yields of the three vitamin B 6 com- pounds were determined. The results clearly conflicted with a literature report that claimed the quantum yield of pyridoxamine-5-phos- phate was 0.55 under these conditions. While measuring the quantum yield of tyro- sine, it was noted that phosphate was a strong quencher of fluorescence. The quenching of tyrosine fluorescence was investigated in some detail and Stern-Volmer constants were de- termined. The tyrosine fluorescence of proteins was also found to be quenched by phosphate. Evidence from fluorescence polarization, tem- perature-dependence curves, the relative sizes of the quenching and association constants, and data from a larger quencher (propionate) showed that the quenching was mainly col- lisional. The use of phosphate as a probe of the accessibility of tyrosines in proteins was discussed. DPNH and TPNH fluorescence can be as- sayed in the Aminco-Bowman instrument at high concentrations (up to SxlO^M) by the use of a microcell and excitation at 395 mp. Under usual conditions of assay employed by many workers, the limit is no higher than 10 _5 M. It was shown that non-linearity of the fluorescence vs. concentration curves was due to absorption of the incident light rather than absorption of the emitted light. A single horizontally-oriented polarizer in the excitation beam was found to reduce dras- tically ,the amount of light scatter reaching the detector. It was shown that this was effec- tive for protein emission spectral work, where the intense scattering of the UV excitation is a problem. To demonstrate the effectiveness of hori- zontally polarized excitation in spectral work with proteins, the emission spectra of human serum albumin at pH's near 4 were deter- mined. It was discovered that there are marked changes over a narrow pH range, which had been established by other workers using a variety of physical techniques to coin- cide with an unfolding of HSA. Analysis of the spectra showed that acidification was ac- companied by a quenching of tryptophan fluo- rescence and an enhancement of tyrosine fluorescence. This can be understood in terms of a decrease in tyrosine-to-tryptophan energy transfer. In addition, a small number of experiments on the fluorescence of dye-labeled glutamic de- hydrogenase and polynucleotides were per- formed in continuation of projects started the previous year. These results still must be evalu- ated but they indicate that the extended sensi- tivity we have achieved in polarization meas- urements may increase the usefulness of the spectrophotofluorometer. All of the material above has either been published or submitted for publication. Emission spectra of bovine serum albumin and the interaction of this protein with a dye- 122 ANNUAL REVIEW OF INTRAMURAL RESEARCH anilinonaphthalene-sulfonate, are under inves- tigation. The use of such dyes as molecular probes of conformational change has been pos- tulated by others and confirmed by us. Protein chemists are interested in amino acids labeled with 1-dimethylaminonaphtha- lene-5-sulfonate ("dansyl amino-acids") since his dye locates amino acids on chromatograms with about 1000-fold more sensitivity than nin- hydrin. The fluorescence of these derivatives has never been characterized, but we have pre- pared about 23 dansyl amino acids and puri- fied them by thin layer chromatography. Quan- tum yields and corrected spectra have been determined. These results will be part of a paper in preparation. Ultramicro Methods The helium glow photometer for the simul- taneous analysis of Na and K at the picomole (10~ 12 moles) level has been further improved by the addition of operating convenience and proven by the application to several problems in the analysis of renal tubule micropuncture samples in cooperation with the LKEM. The extension of the method to calcium and magnesium was explored and preliminary re- sults indicated that sensitivity was more than adequate being in the 10 -13 mole range. Con- ditions for calcium measurements have been worked out to eliminate interference to the point that accuracy in the ±2% range is con- firmed for calcium at the picomole level with good possibility of extension to 10~ 13 moles. The magnesium method has not been explored sufficiently to predict performance. A fluorometric method for determining nan- ograms of inulin obtained by micropuncture of renal tubules has been developed. The re- action of fructose, the subunit of inulin, with dimedone in concentrated o-phosphoric acid produces a moderately fluorescent product which has its emission peak near 400 nm and excitation peak near 360 mm. A concentration of 1 fig/ml of fructose or inulin gives a sig- nal that is twice the blank signal. The method has been shown to be more sensi- tive than the colorimetric method commonly used. However, an additional order of magni- tude increase in sensitivity seems desirable. We will attempt to improve the optical arrange- ment to reduce the scattered light and to re- duce the volume of reagent required. A method of producing luminescence by treatment with ozone has been discovered to be a remarkedly convenient and highly sensitive method of analysis of a wide variety of com- pounds of biological interest. Although it has been known for many years that ozone can induce luminescence in a few compounds, it has never been explored as an analytical method for biochemical application. Our exploration has revealed that some com- pounds on suitable substrates emit enough light on exposure to ozone gas to permit clean assay at the level of 10 -14 grams and a large proportion of fluorescent substances can be excited to luminescence with ozone to permit assay at the 10~ 12 gram level. Several dozen compounds have been discov- ered to have useful luminescence by this method with some indication that several com- pounds with relatively poor sensitivity for ul- traviolet fluorescence excitation have high sensitivity by ozone excitation. For example, the dye Safranine is weakly fluorescent, but emits strongly when excited by ozone. Ozone chemiluminescence methods are being com- pared with fluorescence methods for specific problems to discover what advantages the method has over fluorescence or other analy- tical techniques. The spectrum of emission appears to be gen- erally related to that of fluorescence and pro- vides some specificity but the low level and evanescent character of the emission makes spectral measurements tedious. Excitation can be produced in solution or dry so that chromatographic plate spots can be reacted for quantitative assay. The compound is usually consumed in the process with the emission of a number of photons proportional to the quantity present. The apparatus is very simple, consisting of a photometric system and a suitable chamber for exposing the sample to ozone enriched oxygen provided by passing tank oxygen through an electric discharge pro- duced by a high voltage transformer. NATIONAL HEART INSTITUTE 123 Automation of Bacteria Counts and Antibiotic Sensitivity Tests Consideration of the burden of counting bacteria in body fluids and the determination of antibiotic sensitivity by the clinical lab- oratory indicated a need for a simplification of the methodology amenable to automation and reduction in time required before a defini- tive report can be rendered. A capillary tube method is based on the idea that nutrient agar in a capillary tube will permit the growth and multiplication of individual organisms to a multiplet of organisms in less time than it takes to form a recognizable colony and that a simple scanning and memory system can be used to recognize and count only those or- ganisms that have demonstrated their viabil- ity by beginning to multiply. The capillary tubes replace not only the petri dishes but serve as measuring pipettes and provide a linear array of organisms which can be counted with a much simpler system than a planar array. The capillary tube is exposed to a sharply defined plane of illumination provided by the light of a neon laser. The neon laser light is a continuous high intensity coherent source of simple design available at reasonable cost that can be focused so fine that the resolution of the system can be relatively independent of the radial position of the organism in the capil- lary. A simple magnetic tape recording of the scattered light from the initial population in- cluding debris is recorded, the capillary tube incubated and a new recording indexed to match the previous recording is made while the first signal is subtracted so that only those organisms that have multiplied are counted. Variation including repeated counts and incu- bations as well as histograms of growth rate per organism can be easily obtained to in- crease the information obtainable. Antibiotics incorporated at specific dilutions in the me- dium can easily be assayed for more specific information than can be obtained by clear zone methods. For example, it is apparent that the latent period before division can be meas- ured for a large number of individual organ- isms in a short time in contrast to the tedium of such observations by watching single or- ganisms for a first sign of division under the microscope. The apparatus has been constructed and tested for small particles and methods devel- oped for eliminating noise due to defects in the capillary tube and light scattering from the agar. Resolution and sensitivity seem ade- quate but application to actual counts and de- termination of accuracy have not been com- pleted. Artificial Organs The membrane oxygenator developed pre- viously is still not in production because of the manufacturer's delays in getting satisfac- tory production methods. Some experimental miniature oxygenators were constructed for isolated organ or tumor perfusion are now un- dergoing experimental evaluation in the NCI in a cooperative project. The biventricular cardiac assistor develop- ment has progressed through some 18 modifi- cations to delineate the important design parameters. In situ tests on fibrillating as well as normally beating dog hearts were performed. Normal blood pressure and nor- mal pulse wave forms were achieved. Defibril- lation promptly restored the normal beat after 4 hours of fibrillation in the assistor. The long- est duration of massage was 11 hours. A sys- tem to apply the assistance in synchrony with a normal beat has been developed to determine the ability of the assistor to augment the func- tion of a failing heart. It is presumed that maintenance of the integrity of the circula- tion will permit or accelerate healing of a damaged heart that would otherwise be unable to sustain the circulation. The assistor has been applied to dog hearts and the chest completely closed to confirm its potential for longer per- iods of support. Some improvements in the mechanical de- sign of the "artificial kidney" hemodialysis unit have improved the efficiency to permit high clearance at low dialysate flow. The ad- dition of adsorbants such as activated carbon further increases the effective concentration gradient for adsorbable substances. Most small 124 ANNUAL REVIEW OF INTRAMURAL RESEARCH molecule metabolites with the exception of urea are probably adsorbable. Hippuric acid dialysate capacity is increased 14 times. This represents a highly efficient compact disposa- ble unit not requiring - any priming blood, and using a minimum of dialysate. Fast Reaction Methods The developments in instruments and meth- ods for the study of fast chemical reactions in solution include the introduction of a new mixer, a fast pH detector system, and a new thermocouple amplifier. The new mixer per- mits 99% mixing to be achieved within 40 mi- croseconds or less even in the mixing of 92% glycerol and water. A small stopped flow apparatus has been constructed for us by the Biomedical Engi- neering Branch which permits the use of op- tical, fluorescence, pH, and thermal detectors. Time resolution for pH work is about 50 milli- seconds and sensitivity is 0.001 pH unit with the presently available glass electrodes. Ther- mal and optical time resolution is in the range of 2 to 5 milliseconds. Our instrument built by Science Products Corporation has improved time resolution with the new mixer system of 40 microseconds for a continuous flow thermal detector. Optical de- tection in stopped flow is 100 microseconds. Work has progressed on several fast thermal detectors to where a one millisecond response time has been achieved with glyptal coated copper-constantan thermocouples. Thin film sputtering techniques are being developed by Victory Engineering Corporation to coat the junctions with quartz in order to still further reduce the response time. The twin cell differential microcalorimeter has been under test for six months and is satis- factory for heats of reaction which produce 30 millicalories or more in 4 ml of solution. The Tris-HCl reaction is being developed as a solution calorimetry standard and a com- mercial version of the instrument has been manufactured by Science Products Corp. The data restoration program developed for the differential calorimeter has been put into operation and critically tested. It has been shown to be able to restore electrical heater data from the calorimeter to better than 2 accuracy regardless of the heat input function shape. Thus the adiabatic losses from the cal- orimeter can be corrected for, even when re- actions run for twenty minutes. Using the simulation of physical laws method of solving partial differential equations developed here, a theoretical treatment of the thermocouple response time measurements ob- tained using different thermoelectric materials and dimensions as well as different coating materials has been achieved which agrees within the experimental error of 2-5%. Chromatographic and Ultrasonic Methods Ultrasonic methods of measuring the content of sample in gas chromatography effluents de- veloped and applied last year have been suit- ably modified and tested for performance in liquid chromatographic effluent streams. The system tested measures the change in phase of a 20 MHz sound wave in a 1 ml cell to 0.01° due to a velocity change in response to a few micrograms of sample in 1 ml with the sensi- tivity limited by temperature fluctuations of the sample. An order of magnitude or two in- crease in sensitivity is available by better thermal stabilization. A variation of the method of sound velocity measurements has been applied to permit very accurate determination of sonic velocity in solutions. Here an interferometric technique based on standing wave measurements using a variable sound frequency which can be very accurately measured. The sound frequency is changed until the standing wave is re-estab- lished by accurately identifying the position of the standing wave peaks. Sonic velocity is com- puted electronically and is related to density and compressibility. As density is easily measured the method offers a very accurate measure of compressi- bility. Some preliminary measurements have been made to examine the possibility that changes in compressibility of a solution of large molecules would reflect the degree of dipole association and the influence of dielec- tric constant of the solvent on the association. The unfolding of albumin produced by low pH NATIONAL HEART INSTITUTE 125 values was disappointing but other systems may be more suitable. Miscellaneous A system of measuring - low gas flows (1 to 100 ml/min) where the soap bubble meter is not applicable due to solubility or permea- tion of the soap film was developed, applied and published. A microperfusion pump that delivers 25x10"* ml/min. by programmed controlled thermal expansion of the oil in a micropipette has been developed for micro puncture experiments. This pump eliminates the need to support a heavy mechanical piston drive mechanism on the micromanipulator to provide greater freedom of motion and versa- tility. An air bearing turbine-operated grindstone was devised and tested to provide a very sim- ple method of sharpening micropuncture pi- pettes. Recovery of microgram quantities of labeled fatty acids on T.L.C. films without solvent ex- traction and the current problems of recon- centrating the sample was demonstrated by vacuum distillation in a simple apparatus. The utility of activated carbon for the re- moval of residual fatty acids bound to pure samples of albumin was demonstrated. LABORATORY OF CARDIOVASCULAR PHYSIOLOGY This report represents the last to be sub- mitted by the Laboratory of Cardiovascular Physiology of the National Heart Institute. Since its inception in 1954 under the direction of Dr. S. J. Sarnoff until its inactivation on July 1 of this year, the laboratory has made many major contributions to cardiovascular physiology. Besides the high quality experi- mental work which was considered by many to characterize the laboratory, the laboratory provided a fertile field for the training of many young men, many of whom subsequently went on to senior positions in research teaching and medicine. The contributions of the laboratory which, to a large extent, represent the contri- butions of Dr. S. J. Sarnoff who retired as the result of illness in August of last year are best summarized in the citation which accom- panied the Gairdner Foundation Award pre- sented to Dr. Sarnoff in 1962, . . . "in recogni- tion of his contribution to the knowledge of cardiac physiology and especially for his dem- onstration of the interrelated roles of the ner- vous system, hormones, and heart size in the control of cardiac performance, thus estab- lishing physiological principles which have as- sisted medical scientists to better understand the action of the heart in normal and diseased states." Studies on Myocardial Mechanics The contribution of physical and chemical factors to the adaptation of the heart under varying conditions has continued to be of sub- stantial interest. Several years ago this labora- tory clearly denned the phenomenon of homeo- metric autoregulation, i.e., the increase in myocardial contractility associated with an elevation of ventricular outflow resistance. It was shown that homeometric autoregulation was associated with a loss of myocardial po- tassium. Since it was known that decreasing intramyocardial K + was associated with an increase in myocardial contractility in isolated cardiac muscle, we suggested that the appear- ance of homeometric autoregulation was causally related to the potassium loss. At the same time the possibility existed that homeo- metric autoregulation was caused by physical changes in the heart such as shape or com- pliance changes. The former possibility was of particular interest since we had observed that when aortic pressure was increased isovolumic systolic expansion often increased. This ob- servation suggested the possibility that a two- step Frank-Starling effect may occur when aortic pressure is increased; the first step oc- curring at end-diastole and the second step occurring with the expansion of the circum- ferential fibers. We had also observed, how- ever, that if heart size was increased the extent of isovolumic systolic expansion decreases. We therefore undertook experiments in which, when aortic pressure was increased, heart size also increased, in order to cancel the effects of the two interventions on isovolumic systolic 126 ANNUAL REVIEW OF INTRAMURAL RESEARCH expansion. Such experiments showed that an increase in isovolumic systolic expansion was not necessary for the appearance of homeo- metric autoregulation. In these same experi- ments we also observed that a change in ventri- cular compliance occurs when aortic pressure is increased. The compliance change however could not explain the changes in myocardial performance. We have concluded therefore that while physical factors may contribute to the adaptation of the heart when aortic pressure is elevated, they do not appear to be necessary for homeometric autoregulation. With respect to the possible contribution of compliance changes to ventricular adaptation, investigations were carried out to determine if ventricular compliance changes during single or paired ventricular pacing. In the ejecting heart in which aortic pressure and heart rate were maintained relatively constant, no dis- cernible influence of either type of stimulation on the relation between left ventricular end- diastolic pressure and circumference was ob- served. In contrast, paired ventricular stimu- lation was observed to increase the compliance of the isovolumic ventricle in which developed tension increases during stimulation. Also, the increase in compliance paralleled the increase in developed tension. These experiments taken in conjunction with the experiments in which homeometric autoregulation was induced sug- gest that paired stimulation has no direct effect on ventricular compliance but will alter com- pliance if developed tension is changed sig- nificantly. An understanding of the mechanisms whereby the output of the heart is controlled is, to a large extent, an understanding of the factors controlling the volume of blood in the heart at the end of diastole and at the end of systole. While it is appreciated that inotropic interventions modify the fraction of blood ejected by the ventricle at each beat the role of physical factors in controlling stroke vol- ume is not always appreciated. Studies were carried out therefore to determine the influ- ence of hemodynamic factors on ejected ven- tricular fraction. It was observed that increas- ing cardiac filling increased the fraction of blood ejected by the ventricle while an in- crease in aortic pressure decreased the ejected fraction. These experiments show therefore that hemodynamic variables can modify the ejected fraction and point out the inadequacy of this measurement as an index of myocardial con- tractility. Despite the large amount of published data describing the phenomenon in isolated tissue, it was reported recently that the classical Bow- ditch Staircase or "Treppe" could not be dem- onstrated in the intact dog atrium. This find- ing was unexpected, at least to us, for it would not seem appropriate for a change in fre- quency of stimulation to produce the well estab- lished increase in ventricular contractility without an accompanying increase in atrial contractility. The problem was studied there- fore in this laboratory with special emphasis on observing the influence of heart rate on atrial force and rate of development of force over a broad range (approximately 60-200/ min). The results of the studies have demon- strated conclusively that the contractility of the intact dog atrium is changed with respect to both force and velocity when heart rate is modified. Also, potentiation of contraction is produced by extrasystoles and by continuous post-extrasystolic potentiation. Thus, the in- crease in the force of atrial contraction pro- duced by increasing heart rate can signifi- cantly augment ventricular filling during tachycardia. When heart rate was increased to high levels in the above study pulsus alternans was oc- casionally observed. Since the experimental de- sign was such that force gauges were usually placed on both the atrium and ventricle it was possible to observe the influence of alternans on both chambers. Of interest has been the observations of what might be termed asynergistic alternans. This asyner- gism has been observed on the same side of the heart between atrium and ventricle as well as between the right and left side of the heart. Of further interest is the observation that dur- ing alternans the strong beat occurs from a lower tension than the weak beat suggesting that pulsus alternans may be the result of alternating diastolic compliance. NATIONAL HEART INSTITUTE 127 Myocardial Oxygen Consumption Studies concerning myocardial metabolism have been directed primarily towards de- termining those factors which can modify my- ocardial oxygen consumption. Previous studies using isolated cardiac muscle have been ex- tended to the whole heart in which it was found that developed tension is a major deter- minant of myocardial oxygen consumption. Studies in both the whole heart and isolated cardiac muscle have been the first to demon- strate conclusively that at a constant inotropic background the amount and/or rate of short- ening of cardiac muscle can influence oxygen consumption. In the working heart this in- fluence is small while in isolated muscle it is somewhat greater. When calcium is added to the bath containing isolated cardiac muscle while maintaining heart rate, preload and afterload constant, substantial increases in oxygen consumption are observed. Although the increase in oxygen consumption is asso- ciated with increases in the extent and rate of muscle shortening, it is also possible that a direct metabolic effect of the calcium is con- tributing to the oxygen consumption changes. When calcium is administered to the whole heart working from a high end-diastolic pres- sure, substantial increases in contractility occur without a consistent change in oxygen consumption. The failure to show a change in oxygen consumption under these conditions may be due to the cancelling effect of the de- crease in developed tension which would de- crease myocardial oxygen consumption and a direct metabolic effect which would increase oxygen consumption. The experiments men- tioned above in the whole heart would indicate that the extent or rate of shortening - would play only a small role. In any event, it is of interest that calcium, as we have shown also for acetyl strophanthidin and norepinephrine, can produce substantial increases in myocar- ial performance without a net oxygen cost. With respect to acetyl strophanthidin it is of interest that preliminary experiments suggest that this drug has an oxygen cost which does not necessarily correlate with changes in short- ening and/or rate of shortening. Influence of Drugs and Electrolytes on the Heart Previous studies from this laboratory have shown that the myocardial effects induced by various agents are associated with changes in myocardial potassium balance. These studies have been extended to determine the influence of calcium and sodium bicarbonate on myo- cardial performance and potassium balance. Like those of norepinephrine, the inotropic effects of calcium chloride have been found to be associated with a net gain of myocardial potassium and no consistent influence on myo- cardial oxygen consumption. The extent of the potassium change is directly related to the amount of calcium administered. The influence of calcium on K + balance is in contrast to the loss of myocardial K + associated with the ad- ministration of cardiac glycosides. These ex- periments have suggested the possibility that under inotropic influences the myocardial movements of K + and calcium occur in the same direction. This would then lead to the possibility that agents such as cardiac glyco- sides which cause a loss of myocardial K + may also produce an associated loss of myo- cardial calcium. Experiments have also been completed which characterize the myocardial response to so- dium bicarbonate infusion. This acid-base dis- turbance produces an initial decrease followed by an increase in myocardial performance. These changes are not dependent upon changes in myocardial potassium balance, coronary blood flow or changes in the effective catechol- amine background. Rather they appear to be the result of changes in intracellular hydro- gen ion concentration. It was demonstrated that during sodium bicarbonate infusion the bicarbonate ion concentration of cardiac mus- cle is increased. In view of the extensive use of Dicumarol and Coumadin as oral anticoagulants it was of interest to determine if these agents have a direct effect on myocardial performance when administered to the dog in therapeutic doses. Preliminary, although consistent, results indi- cate that over a wide dosage range Dicumarol depresses, while Coumadin appears to have little influence on the myocardium. 128 ANNUAL REVIEW OF INTRAMURAL RESEARCH Studies on Electrical Stimulation of the Heart Previous studies have shown a minimum energy threshold for cardiac stimulation in chronic and acute preparations at pulse dura- tions between 0.5 and 1.0 milliseconds, using a constant current stimulus source. Histological sections of cardiac tissue from these chronic preparation implantation sites have shown >0.2 mm fibrous ring thicknesses around the electrode. Thus, stimulation occurs in excitable tissue beyond this fibrous ring, i.e., beyond 0.2 mm from the electrode surface. Analysis of the electrochemical impedances at the electrode-electrolyte interface shows three components. Two of these impedances in- crease with time during the pulse, (activation and concentration impedances) while the third (ohmic) is constant. All vary with current density (not current) but only ohmic impe- dance is directly proportional to current den- sity. By studying the immediate stimulus elec- trode environment in electrolyte solutions and isolated heart tissue using glass microelec- trodes, it has been shown that activation and concentration impedances are distributed within 10 microns from the electrode surface. This is not the depolarizing region, and stimu- lus energy dissipated in this region does not contribute to stimulation. Only the ohmic im- pedance is distributed in the region where tis- sue is depolarized in chronic preparations. Since this impedance is directly proportional to the current density at the point of deploriza- tion, it is proposed that the electrical influence which leads to depolarization is also propor- tional to the current density in these prepara- tions. The distribution of ohmic impedance in my- ocardium is not uniform. It has been demon- strated in the isolated cat heart papillary mus- cle that this impedance is greater across fibers than along fibers. This provides a means of investigating "local disturbance" as a function of potential gradients, since for a constant current pulse the potential gradient is steeper across fibers than along fibers. Studies on Water and Salt Regulation Previous studies demonstrated a substantial contribution of intracardiac and intravascular receptors to the control of body water. The ex- tent to which non-cardiac vagal fibers contri- bute to the control of body water is not known. Experiments were carried out therefore to de- termine the influence of section of the vagal nerves on water diuresis in the dog in which cardiac vagal nerves had been ablated. In these animals vagal nerve section is always asso- ciated with a delayed antidiuresis which can be attributed to a decrease in free water ex- cretion. No consistent change in sodium excre- tion is seen. In some animals the antidiuresis is sustained while in others it is only transient. This variability appears to be related to the extent of water loading and thus the extent of the water diuresis. The findings suggest that extracardiac vagal pathways can modu- late free water excretion and thus possibly the circulating levels of ADH. At the same time, the failure of dogs given a heavy water load to show a sustained antidiuresis follow- ing vagotomy indicates that other controlling mechanisms are operating. The latter may re- side, at least in part, in the carotid sinus area. Studies on the Peripheral Circulation The contribution of potassium to the control of skeletal vasculature resistance has con- tinued to be of substantial interest. Previous studies demonstrated that the vasodilation of simulated muscle exercise or reactive hype- remia is associated with a loss of potassium from the muscle. When the resting muscle was perfused with K + so as to effect the same in- crease in venous K + as that which occurred during exercise or hyperemia, less vasodila- tion was observed. Studies were undertaken therefore to determine if changes in venous p0 2 can influence the vasodilator response to a given level of venous K + . The results of these experiments demonstrate that the influence of potassium on vascular resistance is deter- mined to a large extent by the oxygen supplied to the muscle. Thus, the lower the p0 2 of blood the greater the vasodilator effect of a given level of blood potassium. The experiments indicate that both oxygen and potassium con- tribute substantially to the control of skeletal muscle blood flow and that they can reinforce NATIONAL HEART INSTITUTE 129 each other in influencing vascular resistance in both active and resting skeletal muscle. The interrelationship between the vascular resistance changes induced in skeletal muscle by sympathetic stimulation and ionic balance has been studied. Substantial alterations in the total calcium concentration of the blood per- fusing the muscle does not influence the degree of vasoconstriction produced by the infusion of norepinephrine and epinephrine even though the calcium concentration was altered within broad extremes. The injection of CaCl 2 failed to increase or prolong the vasoconstriction in- duced by the single injection of these two catecholamines. It was found that these agents, norepinephrine and epinephrine, did substan- tially increase the venous p0 2 level (at con- stant arterial p0 2 and blood flow) when they produced vasoconstriction but exerted no effect on p0 2 when they produced vasodilation such as could be produced by epinephrine following an alpha receptor block. Of note, it was ob- served consistently that beta adrenergic stim- ulation resulted in an uptake of potassium by the muscle whether or not vasoconstriction or vasodilatation occurred. Chemically blocking the beta receptors prevented these potassium changes. Such findings indicate that these changes might be used in the study of adren- ergic receptors and could aid possibly in their chemical and morphological classification. It has been suggested by others that kinins act as physiological mediators of functional vasodilatation in glands. To test this proposal experiments were carried out in which the influence of carboxypeptidase B on the vasodi- latation of the cat salivary gland produced by stimulation of the chorda tympani nerve was determined. The direct infusion of kallidin into the artery of the salivary gland produced vaso- dilatation which was blocked by the simultane- ous administration of carboxypeptidase B. In contrast, Carboxypeptidase B did not block the vasodilatation associated with nerve stimula- tion. The vasodilatation was essentially blocked however, by a combination of atropine and a beta blocking agent. These experiments indicate therefore that the kinins play little or no role in the functional vasodilatation of the salivary gland and that the well known "atropine re- sistant" vasodilatation seen with nerve stimu- lation is the result of beta adrenergic stimula- tion. Studies on the Kallikrein-Kininogen-Kinin System, Investigations have been continued on the isolation and purification of the various com- ponents of this system. During the past year special emphasis was given to the isolation of kininogen, the substrate in plasma. Two dif- ferent kininogens have been isolated from human plasma. Both are glycoproteins con- taining hexose, sialic acid and glucosamine and have a molecular weight of approximately 50,- 000. Although immunologically identical they are clearly different by several physical, clin- ical and biological criteria. It appears that kininogen I has kallidin at the C-terminal se- quence and kininogen II has kallidin near the C-terminal end of the molecule. LABORATORY OF KD3NEY AND ELECTROLYTE METABOLISM The research activities of the Laboratory of Kidney and Electrolyte Metabolism encompass six general fields of interest: (1) The function of the intact kidney as determined by micro- puncture analyses of luminal fluid in dogs and smaller animals. (2) Water and electrolyte transport in isolated perfused portions of the rabbit nephron in vitro. (3) The character- ization of electrolyte transport in red cells and the associated biochemical processes. (4) Ana- logous studies in epithelial membranes such as toad bladder. (5) Characterization of a pro- tein-globulin complex in mammalian plasma. (6) The role of renin-angiotensin-aldosterone system. Micropuncture Studies in the Kidney Considerable progress has been made in the past year concerning the nature of the counter- current system for urinary concentration and dilution. Implicit in this theory has been the view that an osmotic gradient develops between the ascending and descending limbs of the loops of Henle in the deeper portions of the kid- ney by virtue of the transport of sodium chlor- 130 ANNUAL REVIEW OE INTRAMURAL RESEARCH ide out of the ascending limb without water (thereby reducing the osmotic pressure) and the loss of water without solute by osmotic flow from the descending limb (thereby in- creasing the osmotic pressure). Although most workers had subscribed to this view, no direct supportive evidence had been available, and in fact a number of previous attempts limited to portions of the loop very close to the turn had failed to demonstrate any significant differ- ence in the osmotic pressure of the two ad- jacent limbs. In the past year advantage has been taken of a partial nephrectomy technique developed in this laboratory which permits direct visualiza- tion of the deeper portions of the papilla in the rat (see last year's report), and thereby allows direct micropuncture of previously inaccessible portions of Henle's loop. Using this preparation, evidence has been obtained for the presence of an osmotic gradient between adjacent por- tions of the descending and ascending limbs consistent with the original countercurrent hypothesis. Thus, the osmotic pressure of fluid in the thin ascending limbs of loops of Henle is significantly lower than that of adjacent thin descending limbs. Similarly, the sodium con- centration is lower in fluid from the ascend- ing limb as compared with that of descending limb fluid. The difference in sodium chloride concentration between the two limbs accounts for most of the difference in osmolality, and is consistent with the idea that active transport of sodium chloride out of the ascending thin limb is the driving force for the creation of an osmotic gradient in the deeper portions of the medulla and papilla. These findings support the view that the thin loops of Henle participate actively as countercurrent multipliers in the production of a hypertonic renal medulla. In an earlier report it had been noted on the basis of micropuncture sampling and analysis of fluid from proximal tubules in the dog, that antidiuretic hormone appeared to stimulate so- dium reabsorption in this portion of the neph- ron. Although these results have been reported, we have remained skeptical about this inter- pretation since an effect of antidiuretic hor- mone in the proximal segment on sodium trans- port was inconsistent with other information regarding its site of action. Studies were there- fore carried out in dogs in which samples were collected from the same site (the recollection technique) in proximal segments first during water diuresis and again after administration of antidiuretic hormone. A new remarkably sensitive and accurate technique for inulin de- termination based on fluorescence that was developed in the Laboratory of Technical Devel- opment was used. The tubular fluid/plasma- inulin ratio, an index of water and sodium chloride reabsorption in the proximal neph- ron, did not change significantly when diuresis was interrupted by the injection of vasopres- sin. It was concluded that the hormone has no effect on proximal sodium transport, and that the earlier conclusions were probably erron- eous. The recollection technique is to be pursued in a variety of studies in the future in an effort to determine whether the maximal so- dium gradient developed in the proximal tu- bule during administration of osmotic diuret- ics can be altered by administration of diuretic agents thought to interfere with sodium trans- port in this segment of the nephron. No effect on sodium reabsorption as determined by changes in the tubular fluid/plasma ratio of inulin had been noted in earlier studies. How- ever, further analysis and study are required before final conclusions can be reached regard- ing the proximal sites of action of these drugs. In collaboration with the Department of Med- icine of Duke University, an investigation of electrolyte transport in the distal nephron of the dog is also in progress. The recognition of the presence of distal nephrons in the superfi- cial cortex of the dog has heretofore been a difficult problem. In the past year, however, it has been possible to detect the presence of an appreciable number of distal nephrons in the accessible portions of the superficial cortex by injecting a dye (lissamine green) which makes it relatively easy to locate these segments of the neprhon. In addition to the information be- ing acquired concerning the normal composi- tion of fluid in the distal tubule, it has been found that the administration of furosemide increases the tubular fluid/plasma ratio of sodium in the accessible portion of the distal NATIONAL HEART INSTITUTE 131 nephron and lowers the tubular fluid/plasma ratio of inulin. No effect on potassium concen- tration has been noted. These results support the view that furosemide limits sodium trans- port in the loops of Henle and permits the delivery of a greater volume of less dilute urine to the distal convolution. A number of other analogous studies, in progress in both dogs and rats, are directed at characterizing transport and homeostatic mechanisms in individual accessible segments of the kidney. Studies of Isolated and Separated Tubules in Rabbits In Vitro In earlier reports the results of kinetic analyses of electrolyte transport in slices of renal cortex and suspensions of renal tubules were discussed. On the basis of these studies it was concluded that a minimum of two so- dium and two potassium compartments are present within tubule cells. Thought the inter- pretation was reasonable on the basis of the available evidence, it was not possible to ex- clude with certainty that the pools of these electrolytes were located in different cells or tubule segments. Recently a technique for di- rect isolation and study of individual and vi- able segments of rabbit tubules has been per- fected. Kinetic studies of isotopic exchange have been repeated under a variety of condi- tions, and it has been concluded that the exist- ence of several potassium compartments repre- sents inhomogeneity of the tubule population in slices of cortex and in suspensions of corti- cal tissue, or in other words that each nephron segment contains only a single potassium com- partment. Similar analyses with respect to Na, however, revealed the presence of at least two compartments in individual segments. Portions of collecting tubules have also been dissected freehand from rabbit kidneys and perfused; unidirectional and net transport of water and solute have been measured under different experimental conditions. We had pre- viously proposed that the permeability changes induced by vasopressin in toad bladder and tu- bule are mediated by the intracellular forma- tion of cyclic 3' 5' AMP. This compound pro- duces all the effects of antidiuretic hormone in toad bladder and its concentration within the tissue is increased following incubation with hormone. No direct evidence of an effect of antidiuretic hormone on the permeability of the kidney tubule to water and solute had been developed in the past, although all investiga- tors had agreed that the effect must be similar to that in skin and bladder. Furthermore, no effect of cyclic AMP on H 2 permeability in the kidney in vivo had been obtained. Using the microperf usion technique with isolated collect- ing tubules it has now been demonstrated for the first time by direct measurement that anti- diuretic hormone does in fact increase the per- meability of the tubule membrane to water in fashion consistent with the idea that the hor- mone increases either the number or size of aqueous channels or pores within the limiting membrane. It has also been found that cyclic 3'5' AMP mimics antidiuretic hormone inso- far as permeability change is concerned in the collecting tubule, again suggesting that this compound may mediate the action of antidiu- retic hormone. No effect on urea permeability has been noted with either ADH or cyclic AMP, a finding which contrasts with observa- tions in toad bladder. This and other studies of electrolyte transport, the electrical charac- teristics of the membrane in proximal tubule and collecting tubule are in progress. The naturally occurring fatty acid, prosta- glandin (PGE), first discovered by Von Euler and subsequently characterized as to chemical composition by Bergstrom at the Karolinska Institute in Stockholm, appears to be a major component of "medullin," an extract made from renal medulla which lowers blood pres- sure in animals with reno-prival hypertension. Some time ago it was reported from this lab- oratory that PGE interfered with the permea- bility changes in toad bladder inuced by antidiuretic hormone and a physiological regula- tory role in the kidney was suggested. No effect on the changes in permeability induced with exogenous cyclic AMP was noted, a finding consistent with the view that PGE interferes with the mechanism by which antidiuretic hor- mone induces an increase in the concentration of cyclic AMP in the responsive tissues. PGE 132 ANNUAL REVIEW OF INTRAMURAL RESEARCH has now been shown to inhibit the effects of antidiuretic hormone on the permeability of the collecting tubule, adding credence to the view that this naturally occurring fatty acid may be physiologically significant in renal tissue. Red Cell Studies Studies of the characteristics of electrolyte transport across red cell membranes of a va- riety of species have been a continuing effort in this laboratory. Hemolyzed preparations of red cells (ghosts) as well as intact red cells have been studied. On the basis of earlier work, it had been assumed that transport of ions as measured by unidirectional fluxes can be sep- arated, operationally, into at least three dis- tinct components; active transport, that is, transport against an electrochemical gradient; passive diffusion; and so-called exchange diffu- sion. Exchange diffusion is assumed to involve a one-for-one exchange of an ion with a coun- terion of the same species, that is, sodium for sodium or potassium for potassium. Recent studies have cast considerable doubt on the op- erational definition of exchange diffusion. Ex- change diffusion is by definition symmetrical, and any manipulation which modifies one of the unidirectional fluxes must exert a similar effect on the reverse flux. This has not turned out to be the case in all instances of what had been assumed to be exchange diffusion. Further evidence has developed that essentially elimi- nates the possibility that true exchange diffu- sion, sodium for sodium, or potassium for po- tassium, does in fact occur in the human red cell. It now appears that sodium-sensitive so- dium efflux, previously considered to represent exchange diffusion, and potassium-sensitive potassium influx both are effected by a hitherto underscribed component of active transport. In the past year progress has been made in further characterizing this second active pump system. It has been established that sodium- sensitive sodium efflux is, in fact, active, but differs from the usual form of the potassium and sodium active transport mechanism (Pump I) since it is not inhibited by digitalis glyco- sides, does not utilize ATP as its proximate energy source, does not require external potas- sium, and is not stimulated by an increase in internal sodium concentration. Active sodium efflux of the second type (Pump II), though not inhibited by ouabain alone, is inhibited by a combination of ethacrynic acid and ouabain and is sensitive to external sodium. A compo- nent of active potassium influx has similar characteristics. This transport mechanism has been demonstrated both in ghost preparations and in intact human red cells. It appears that under normal conditions approximately 90% of the sodium movement out of the red cell is by active transport and approximately 90% of the potassium entering the cell moves by active transport. The classical active transport process moves approximately 60 to 70% of the sodium and potassium while the newly described mech- anism transports the other 20-30%. It is not clear whether the newly discovered mechanism should be interpreted as requiring a second carrier or whether an entirely new conceptual- ization of the transport process is required. The role of the active transport in the regu- lation of red cell metabolism has also been studied in both human red cells and ghost prep- arations. A successful attempt has been made to delineate the mechanism of interaction be- tween cation pump activity and the control of glycolysis in human red cells. The flux of K 42 and changes in enzyme activity in the glyco- lytic cycle were examined with and without ouabain and under a variety of other experi- mental conditions. It was observed that when the internal sodium of human red cells is ele- vated both potassium influx and lactate pro- duction rise, evidence of an increase in rate of glycolysis, and both become more sensitive to the inhibitory action of ouabain. This occurs with either glucose or adenosine as substrate. Fresh whole hemolysates enriched with sodium and magnesium will convert intermediates in the glycolytic chain above the triose-phosphate- dehydrogenase step to lactate at a rate which is slowed by ouabain. Metabolism of interme- diates below this step, that is beyond the phos- phoglycerate kinase step are not so affected by ouabain. The removal of membranes from the preparation eliminates the ouabain effect, evi- dence that the responsible enzyme is present in NATIONAL CANCER INSTITUTE 133 the membrane. Hemoglobin-free ghosts alone were shown to contain both triose phosphate dehydrogenase and phosphoglycerate kinase activity. The reaction rate of this two enzyme sequence was measured by following the con- version of DPN to DPNH and was found to be a function of the ADP concentration in the me- dium. On the basis of this and other studies it was concluded that the reaction catalyzed by membrane phosphoglycerate kinase is the step at which the sodium-potassium transport system influences the metabolic rate in red cells, and this action is possibly exerted via a compartmentalized ADP which is an immediate substrate for ghost phosphoglycerate kinase. The availability of the ADP is presumably de- pendent upon the activity of the sodium-potas- sium activated APTase system which is inhib- ited by ouabain, thereby accounting for the inhibition of electrolyte transport and metab- olism by this agent. Studies in Toad Bladder Vasopressin is known to have two character- istic effects on the urinary bladder of the toad: One, it increases the permeability to water, and two, it increases the rate of sodium transport. There is conflicting information concerning the energy requirement for each of these steps, and, in fact, as to whether metabolic energy was essential for the effect on permeability to water. As a first approach to the problem the effect of specific metabolic inhibitors on the responsiveness of the membrane to vasopressin was studied. The effect of these agents on the osmotic flow of water when stimulated by the hormone as well as on active sodium transport as estimated by short-circuit current were stud- ied under a variety of experimental conditions. It had been known that many of the metabolic inhibitors interfered with the active transport of sodium induced by vasopressin, but as im- plied above conflicting evidence was present concerning the effect of these agents on the permeability response insofar as water is con- cerned. It was observed in the present studies that a number of metabolic inhibitors inter- fered with the response of sodium transport to hormone and also depressed the basal rate of sodium transport of toad bladder. However, in contrast to findings of other investigators, a number of these agents under varying experi- mental conditions also interfered with the ac- tion of the hormone on osmotic flow of water. Although the effects of the individual metabolic inhibitors were specific, as indicated by studies in which changes in lactate production, CO» production from labelled glucose and pyruvate, and tissue citrate concentrations were meas- ured, it was not possible to pinpoint the pre- cise metabolic steps involved in the osmotic permeability response. However, it was pos- sible to conclude that metabolic energy is a necessary requirement for the alterations in- duced by ADH in the permeability of toad bladder to water. In addition to the above studies a major effort has been directed at examining the control of glycogenolysis in the toad bladder as changes are induced by anaerobiosis, vasopressin, oua- bain (an inhibitor of active sodium transport in this tissue) and the removal of sodium from the bathing medium. The crossover technique of Chance was used. In this instance, the tech- nique involves the measurement of the concen- tration of all of the glycolytic intermediates under control and experimental conditions. Rate controlling steps are detected as the site of reciprocal alterations in the concentration of substrate and product in experimental as com- pared to control situations. When glycogenoly- sis is inhibited by ouabain or the removal of sodium from the incubation solution, an in- crease in the concentration of glucoses-phos- phate and fructose-6-phosphate and a fall in the concentration of fructose diphosphate and tri- ose phosphate, pyruvate and lactate are ob- served, indicating inhibition of the conversion of f ructose-6-phosphate to fructose diphosphate, a step catalyzed by the enzyme phosphofructo- kinase. The opposite pattern was obtained when glycogenolysis was stimulated by anaerobiosis. In this instance the concentrations of glucose- 6-phosphate and fructose-6-phosphate were de- creased, and the concentrations of fructose di- phosphate, triose phosphate, pyruvate and lactate were elevated, indicating stimulation of phosphofructokinase. When vasopressin was 134 ANNUAL REVIEW OF INTRAMURAL RESEARCH used to stimulate glycogenolysis, a small but significant rise in the concentrations of glu- coses-phosphate and fructose-6-phosphate and a further increase in the concentration of fruc- tose diphosphate and triose phosphate were observed. This is evidence in support of the view that activation of both phosphorylase and phosphofructokinase is induced by vasopressin under the circumstances of these studies. Ana- logous studies using the crossover technique to pinpoint alterations in the rate controlling enzymes in the citric acid cycle are under way at the present time. It is also proposed to ex- amine the metabolic effects induced by aldo- sterone in this tissue in a similar fashion. Physiologic studies relating the permeability effects of antidiuretic hormone and its ana- logues to the concentration of divalent cations in the bathing medium are also in progress. It has been known for some time that an increase in the concentration of calcium in the bathing medium interferes with the water permeabil- ity response to antidiuretic hormone but does not eliminate the stimulation of sodium trans- port. No effect was observed on the action of cyclic AMP on either water or sodium move- ment. Presumably, the inhibitory step involv- ing calcium precedes the formation of cyclic AMP within the tissue. The dissociation of the sodium and water permeability response to the hormone suggests the presence of two separate steps mediated by cyclic AMP, one controlling water movement, the other that of sodium. Preliminary results in a further investigation of this problem indicate that although the re- sponse to cyclic 3'5' AMP is not affected by high concentrations of either calcium or mag- nesium, the increase in the rate of flow of water along an osmotic gradient in bladder in re- sponse to a variety of hormone analogues and to theophylline is inhibited by both of these divalent cations. The response to each hormone analogue apparently manifests a characteris- tic sensitivity pattern to one or the other ca- tion. Further studies are in progress to delin- eate the effects of high concentrations of cal- cium and magnesium on the diffusional permeability of the bladder to water and urea with and without hormone. The Chemical Characterization and Physiologic Significance of a Cardioglobulin System Present in Mammalian Plasma That Increases The Contractility of Isolated Frog Heart This system is thought to contain three globulin components, all of which are neces- sary for the contractility response. In the past year, further purification of the factors has been accomplished revealing that certain of the components from plasma of different species (rat and human) differ in characteris- tics. This has permitted improvement of an assay for the presence of the cardioglobulin system in plasma, and it has been reestablished that a small increase in the fractions referred to as cardioglobulin A and C occurs in patients with long-standing hypertension, and that a decrease in fraction C is present in about 30% of patients with idiopathic myocardial failure. The significance of these changes is at present unknown. The most striking new finding, how- ever, has been the uniform observation of a de- crease in at least one of the three components of the cardioglobulin system in plasma of 22 of 24 patients with lupus erythematosus. Nor- mal values, on the other hand, were observed in 17 other patients with other types of "con- nective tissue" disease. This aspect of the study will be pursued in an effort to determine the significance of the alterations in cardioglobu- lin composition in the plasma of patients with lupus erythematosus in the hope that it may yield information regarding the pathogenesis of the disease process. The Renin-Angiotensin-Aldosterone System and Other Humoral Factors in Homeostasis and Disease A major effort has been directed at examin- the renin-angiotensin-aldosterone system in normal, hypertensive animals and in those with experimentally induced heart failure and other causes of fluid retention. The major concern of this portion of the laboratory has been with establishing the mechanism of control of the secretion of aldosterone in normal and in dis- eased states. In addition, the nature and the function of certain extra-adrenal factors pro- moting sodium retention and excretion have NATIONAL HEART INSTITUTE 135 been investigated. Detailed background infor- mation concerning these problems has been presented in earlier reports. In experimental hypertension in the dog, the relation of plasma renin to sodium balance and arterial pressure was examined in a number of studies. The results revealed a striking corre- lation between plasma renin concentration and sodium balance, but no relationship of these factors to variations in arterial pressure. The secretion of desoxycorticosterone in experi- mental heart failure was studied in an attempt to determine if this hormone contributes sub- stantially to the total sodium retaining activi- ties in this physiologic derangement. It was observed that desoxycorticosterone secretion was normal in experimental heart failure. Studies were also initiated in an attempt to define whether or not a hepatic hormone may lead to renin release. Thus far the results have been negative. Of some significance was the observation that the renin-aldosterone system is present in lower vertebrates, such as the opossum and the American bullfrog. In both of these animals renin and ACTH increased aldosterone secretion and studies of the juxta- glomerular apparatus were interpreted as in- dicating hyperplasia and hypergranulation dur- ing sodium depletion. Evidence has been obtained in the past in this and other laboratories, on the basis of cross-circulation studies, that a humoral fac- tor is involved in the natriuresis of saline load- ing. If one infuses saline into a donor animal and cross-circulates its blood into a recipient, a significant augmentation in sodium excre- tion occurs in the recipient animal. Attempts to determine the locus of secretion of this un- known humoral factor by cross-circulation of blood from hepatectomized, decapitated or ne- phrectomized dogs have thus far yielded nega- ive results. Further studies, however, are in progress. Attempts have also been made to ob- tain information concerning the afferent input into the system involved in the natriuresis of saline loading. The natriuretic response to the infusion of blood was compared to the response to infusion of saline. It was noted that blood as well as saline produced substantial increases in sodium excretion. Additional studies, how- ever, are necessary to define adequately those factors which lead to certin differences in the responses to blood and saline which were ob- served in these studies and to determine whether or not the afferent input for the natri- uretic mechanism is related mainly to volume expansion or to other factors as well. LABORATORY OF BIOCHEMISTRY Section on Enzymes The Section on Enzymes continues to direct its attention to the following major areas of in- vestigation: (1) the intracellular regulation of enzyme activities, with particular emphasis on the regulation of divergent metabolic path- ways; (2) the uptake of amino acids by iso- lated cytoplasmic membrane preparations; (3) the metabolism of amino acids; (4) the metabolism of one-carbon compounds; (5) cystathionine synthesis and trans-sulfura- tion; (6) the mechanism of action of vitamin B 12 , and; (7) the dissimilation of heterocyclic compounds. These studies embrace broad areas of enzyme function in metabolism and have led inadvertently to the discovery of several biochemical processes in which vitamin B 12 derivatives and non-heme iron electron carrier proteins (ferredoxins) are of fundamental significance. These special processes have there- fore been the target of intensive study since they represent model systems whose investi- gation may help to elucidate the mechanism of action of vitamin B 12 compounds and non- heme iron proteins in intermediary metabolism. The Regulation of Divergent Biosynthetic Pathways of Metabolism Cumulative Feed-back Inhibition of Glu- tamine Synthetase. — Previous studies in this laboratory have shown that the glutamine syn- thetase of Escherichia coli is subject to feedback inhibition by eight different end-prod- ucts of glutamine metabolism; namely, tryp- tophan, histidine, AMP, CTP, carbamyl-P, glucosamine-6-P, glycine and alanine. On the basis of kinetic studies it is concluded that each of these compounds is independent in its action. When tested individually at saturating 136 ANNUAL REVIEW OF INTRAMURAL RESEARCH concentrations each product will cause only partial inhibition of the glutamine synthetase activity; however, these effects are cumulative, and when all eight compounds are present si- multaneously, over 95% of the enzyme activ- ity is inhibited. In efforts to understand the mechanism of this unique cumulative feed-back inhibition pattern, the glutamine synthetase of E. coli has been isolated as a homeogeneous, crystalline protein and some of its properties have been determined. On the basis of various physical and chemi- cal measurements including sedimentation analysis, light scattering measurements, disc gel electrophoresis, amino acid analysis and fingerprint analysis of tryptic digests, it has been established that the enzyme has a molec- ular weight of about 680,000 and that it is composed of 12-14 probably identical subunits. Complete disaggregation of the enzyme into its catalytically inactive subunits (MW = 49,- 000-53,000) is achieved by treatment either with 4 M guanidine or with 1.0 M urea plus 0.01 M EDTA. After complete disaggregation by the latter method (4°, pH 7.0), the addi- tion of 0.02 M MnCl 2 causes reaggregation of the subunits to form an oligomer that is in- distinguishable from the native enzyme with regard to sedimentation constant and disc gel electrophoretic mobility. This reaggregation is accompanied by restoration of up to 80% of the normal catalytic activity. The enzyme contains 4 cysteine residues per 53,000 molecular weight subunit. At high pro- tein concentrations, 300 /xg/ml, these sulfhy- dryl groups are all inaccessible to titration with alkylating agents or with Ellman's rea- gent. However, quantitative titration of the sulfhydryl groups with Ellman's reagent is possible following denaturation with dodecyl sulfate or 4 M guanidine. On the other hand, in dilute solutions, 250 ju.g/ml, the enzyme does react slowly with alkylating agents such as p-chlor-mercuriphenyl sulfate (PCMPS) in the absence of protein denaturants. This alky- lation is attended by dissociation into catalyti- cally inactive subunits of undetermined size. From considerations of the kinetics of gluta- mine synthetase inhibition and the diverse structures of the 8 different end-products of glutamine metabolism it was previously con- cluded that the enzyme (for that matter prob- ably each of the identical subunits) possesses separate binding sites for each of the 8 feed- back inhibitors. Efforts to achieve differential inactivation of these separate binding sites by means of selective denaturation through con- trolled proteolytic digestion were unsuccess- ful. On the other hand, the existence of sepa- rate binding sites for each inhibitor is supported by investigations of the kinetics of inhibition and also from studies on the effects of the various feed-back inhibitors on the in- activation of the enzyme by PCMPS. For example, the inhibition of glutamine synthe- tase by tryptophan, glycine and CTP is com- petitive with respect to the substrate gluta- mate, yet these three inhibitors apparently do not react at a common binding site since tryp- tophan protects the enzyme from inactivation by PCMPS, whereas CTP enhances the inac- tivation and glycine has no effect. It is evi- dent that histidine and glucosamine-6-P in- volve binding sites that are different from those that react with the other inhibitors since these two compounds are competitive only with respect to the substrate ammonia; yet histi- dine protects the enzyme from PCMPS inacti- vation, whereas glucosamine-6-P has little effect. Finally, the three end-products that are noncompetitive inhibitors of glutamine syn- thetase activity are differential in their effects on PCMPS inactivation. Thus, AMP protects against PCMPS inactivation, carbamyl-P en- hances the inactivation and alanine has little effect. These results are most compatible with the conclusion that the enzyme possesses sepa- rate binding sites for each of the 8 feed-back inhibitors. In order to determine the number of bind- ing sites for tryptophan, some preliminary studies have been carried out in collaboration with Dr. Gregorio Weber at the University of Illinois. For these studies advantage was taken of the fact that the tryptophan analog, 6-nitro- tryptophan (NT), will replace tryptophan as a feed-back inhibitor and protects the enzyme against PCMPS inactivation. Since NT is non- NATIONAL HEART INSTITUTE 137 fluorescent and possesses a strong light absorp- tion band above 300 m^, its binding to the enzyme is accompanied by a quenching of pro- tein fluorescence at 335 m/i. By measuring the degree of quenching as a function of NT con- centration, it has been estimated that there are about 33 moles of NT bound per mole of enzyme at saturation concentrations, and that the dissociation constant is 5x1 -5 M. From these data it is concluded that between 2 and 3 equivalents of NT are bound to each subunit. The further observation that the quenching of protein fluorescence by NT is not influenced by saturating concentrations of alanine, gly- cine or glutamate, support the conclusion that the various feedback inhibitors are independ- ent in their actions and that the binding of one inhibitor is not influenced by the binding of another. If the results of NT binding using the fluorescence technique can be confirmed by the more conventional method (i.e., by equi- librium dialysis) this fluorescence technique will be exploited further to study the interac- tions of the enzyme and the various end- products and substrates. Finally, a new highly sensitive method has been disclosed which permits more careful kinetic measurements of glutamine synthetase activity. In this method the ADP formed as a by-product of the glutamine reaction (1) is coupled with reactions catalyzed by pyruvate kinase (reaction 2) and lactate dehydrogenase (reaction 3). The oxidation of DPNH in the over-all reaction 4 is followed spectrophoto- metrically and under appropriate conditions is a measure of the glutamine synthetase activity. ATP + NH 3 + glutamate — > glutamine + Pi + ADP ADP + p-enolpyruvate — >ATP + Pyruvate (1) (2) Pyruvate + DPNH + H + — >DPN + lactate Sum: Glutamate + NH 3 + DPNH + p-enolpyruvate + + H+ — > glutamine + lactate + DPN (3) (4) Using this method it was discovered that glutamine synthesis is not a linear function of time but exhibits a short lag of 1-2 minutes before maximum velocity is reached. The lag can be eliminated if this enzyme is given a prior incubation with very high concentrations of glutamate at room temperature. The concen- tration of glutamate (0.4 M) required for this effect is considerably greater than that needed to saturate the enzyme as a substrate in gluta- mine synthesis. The results suggest that gluta- mate at high concentration induces a confor- mational change in the enzyme to a form that is catalytically more active. Removal of the glutamate by sephadex filtration results in the immediate return to a preparation that exhib- its a lag phase. In order to determine the generality of the cumulative feed-back control mechanism in the regulation of glutamine synthetase, the inhibi- tion patterns of the enzyme from various sources have been examined. Ten different mi- croorganisms were studied including 5 aerobic bacteria representing 4 different generic types, and one species each of an obligately anaerobic organism, a photosynthetic bacterium, a mold, a yeast and a green alga. Although significant differences were found in the regulation pat- terns with respect to the nature and extent in inhibition, in all instances the glutamine synthetase activity was subject to inhibition by several or all of the end-products of gluta- mine metabolism. In most cases, a cumulative feedback mechanism seems to operate for majority of the inhibitors; however, in some instances certain pairs of inhibitors are antag- onistic or synergestic in their action. Of spe- cial interest is the strongly synergistic effects of AMP and glutamine or of AMP and histi- dine that are observed with the glutamine synthetases of Bacillus cereus and Bascillus Licheniformis. The enzymes from these organ- isms promise to be of unique interest from the standpoint of allosteric interactions and puri- fication of the enzyme from B. licheniformis is in progress. 138 ANNUAL REVIEW OE INTRAMURAL RESEARCH The Uptake of Amino Acids by Isolated Cytoplasmic Membrane Preparations In an effort to determine the mechanism of carrier-mediated transport, the uptake of amino acids and sugars by isolated cytoplasmic membranes from Escherichia coli is under in- vestigation. The membranes are prepared from penicillin-induced spheroplasts by osmotic shock in hypotonic salt solution containing DNase, followed by the addition of EDTA, and are separated from trace amounts of intact cells and spheroplasts by sucrose density gra- dient sedimentation. Homogeneity of the prep- arations was established by electron and phase contrast microscopy, viability assays, chemi- cal and enzymatic assays, and disc gel electro- phoresis. The isolated membranes contain less than 5.0% of the DNA or RNA and less than 1% of the soluble cytoplasmic protein of the cell. Previous studies showed that such mem- brane preparations from wild-type E. coli W catalyze the energy dependent uptake of gly- cine, whereas similar preparations from a mu- tant strain (WS), having a defective glycine uptake capacity, failed to do so. Although these results indicated the glycine uptake by the membranes is the manifestation of a physiologically significant transport process, interpretation of the results was complicated by the discovery that the glycine taken up by the membranes was largely converted to phos- phatidyl ethanolamine, probably via serine and phosphatidyl serine. As a consequence, the in- tramembranal concentration of free glycine did not significantly exceed the external con- centration. It was therefore not possible to determine if the energy requirement for gly- cine uptake is concerned solely with a glycine specific carrier mediated concentration system or if it is related also to the conversion of gly- cine to phospholipids. In view of these considerations attention was turned to the uptake of proline whose poten- tial metabolic fate is more restricted. It has been found that membrane preparations from E. coli W6, a proline auxotroph, catalyze the energy dependent concentrative uptake of pro- line; whereas comparable preparations from a mutant strain E. coli W157, that is deficient in proline uptake and exchange capacity, do not catalyze concentrative uptake of proline. In contrast to the results with glycine, the mem- branes from the parental strain (W6) estab- lish an intramembranal concentration of pro- line strain (W6) establish an intramembranal concentration of proline that is 50 times greater than the external concentration. More- over, more than 80% of the proline taken up can be accounted for as free proline. Proline uptake by W6 membranes exhibits saturation kinetics at low external proline concentrations, is oxygen dependent and is stimulated by glucose; it is inhibited by DNP, iodoacetamide, amytal and by hydroxyproline but not by other amino acids or by Chloromy- cetin, or KCN. Fixed proline exchanges with external proline and hydroxyproline but not with other amino acids. Proline uptake by W157 membranes exhibits linear kinetics over a 100,000-fold concentration range and is not stimulated by glucose; it is not inhibited by DNP, iodoacetamide, or hydroxyproline in concentrations sufficient to produce maximum inhibition of proline uptake by W6 mem- branes. Proline taken up by W157 membranes does not exchange with external amino acids. When membranes are allowed to accumulate proline, sonicated or solublized with detergent and then passed over a Sephadex G-50 column, less than 5% of the radioactivity is associated with the large molecular weight components coming off the column at the front. This find- ing indicates that the intramembranal proline is not bound to a membrane component by a covalent bond. Finally, preliminary studies on thiomethyl- galactoside uptake by membrane preparations from constitutive and deletion mutants for the lactose operon have yielded results thus far similar to those reported above for the proline system. These results indicate that membrane prep- arations provide an excellent medium for stud- ies on the mechanism of carrier mediated transport. In future studies efforts will be made to determine the nature of the energy dependent process and to establish the mech- anism of permease action. NATIONAL HEART INSTITUTE 139 The Metabolism of Amino Acids Reductive Deamination. — Studies on the mechanism of the anaerobic phosphorylation associated with electron transfer during the reductive deamination of glycine by soluble enzyme preparations of Clostridium sticklandii have been continued. As noted earlier, the glycine reductase system is extremely complex, it has been resolved into at least eight separate protein components some or all of which are required for glycine reduction depending upon the nature of the electron donor system em- ployed. Two of the protein fractions, Fraction GR and Protein A are always needed irrespec- tive of the electron donor. When TPNH or DPNH are electron donors the respective TPNH or DPNH flavoprotein dehydrogenase are also required and in addition ferredoxin (the non- heme iron electron carrier protein), and a pro- tein fraction B, (obtained by precipitation between 0.55 and 0.9 saturated ammonium sul- fate), an arsenite sensitive protein fraction and an acetyl CoA dependent enzyme compon- ent are all needed. All of these protein com- ponents, with the possible exception of ferre- doxin and DPNH dehydrogenase, which have not been tested, are required also when re- duced methyl viologen is the electron donor. On the other hand, when a dimercaptan such as dimercaptothreitol is the electron donor only Fractions GR and Protein A and possibly the arsenite sensitive catalyst and the acetyl CoA dependent enzymes are needed. Thus, the arti- ficial dimercaptan electron donors appear to bypass a highly complex electron transport system needed in the normal metabolism. The partial purification of Protein A, an aci- dic low molecular weight protein component of the glycine reductase system, was described previously. Further studies show that this is a sulfhydryl protein that is inactivated by alkylation of its reduced form with iodoaceta- mide. The aggregated, less acidic, partially in- active form of protein A can be disaggre- gated and reactivated, to some extent, by prolonged treatment with dimercaptothreitol in 3 M guanidine. A possible phosphoryl group carrier function for protein A was suggested by the fact that orthophosphate partially pro- tects protein A from inactivation and aggre- gation. However, in crude extracts no phos- phorylation of protein A with P :t2 -labeled ATP or acetyl-P could be detected. On the other hand, it may be highly significant that under these conditions P 32 is readily incor- porated into the flavin nucleotide prosthetic groups of the TPNH and DPNH flavoprotein dehydrogenases whose isolation was previously reported. Lysine Fermentation. — The fermentation of lysine by cell-free extracts of C. sticklandii and Clostridium M-E leads to the formation of ammonia, acetate, butyrate and ATP ac- cording to the over-all equation: Lysine+2H.20 + Pi+ADP ATP + 2NH 3 + acetate + butyrate Previous studies have shown this to be a com- plicated process requiring the participation of several enzymes, and a number of cofactors in- cluding CoA, DPN, Fe ++ , «-ketoglutarate, pyr- uvate, acetyl CoA and vitamin B 12 coenzyme. From the standpoint of intermediary me- tabolism, the identification of /3-lysine and 3,5- diaminohexanoic acid as early intermediates in lysine degradation discloses a very unusual biochemical mechanism. The conversion of a-lysine to /3-lysine involves migration of the a-amino group to the /3-position. This reac- tion was first described by Barker et al. and has now been shown to be the first detectable step in the fermentation of lysine by Clos- tridium sticklandii and Clostridium-ME. The reaction is sensitive to sulfhydryl reagents and probably involves a cofactor since it is inhib- ited by treatment of enzyme preparation with either charcoal or Dowex-1-formate. The second detectable step is the conversion of /3-lysine to 3,5-diaminohexanoic acid. This unusual reaction involves migration of the amino group from the 6 to the 5 carbon atom. From the biochemical point of view this trans- formation is of special interest since it in- volves participation of vitamin B 12 coenzyme; in addition, ATP, Mg ++ and probably pyruvate are needed. When a-lysine or /?-lysine are fermented by cell-free extracts at pH 7.0 (one pH unit be- low the optimum for the over-all fermenta- 140 ANNUAL REVIEW OF INTRAMURAL RESEARCH tion), 3,5-diaminohexanoic acid accumulates in substantial amounts (30%) and has been isolated in good yield by chromatography on Dowex 50 and silicic acid columns. The enzymatic product was identified as 3,5- diaminohexanoic acid or the basis of the infra red and NMR spectra and by high resolution mass spectrometry of the enzymatic product and of its 8-lactam and N,N-dibenzoyl deriva- tives. Authentic samples of 3,5-diaminohexa- noic acid and its S-lactam derivative were pre- pared by reaction of ammonia with sorbic acid under high pressure. The synthetic products were shown to be identical with the enzymatic products by comparison of their chromatog- raphic behaviors and their NMR and mass spectra and of their abilities to be fermented to acetate, butyrate and ammonia by cell-free extracts of C. sticklandii. Future studies will be aimed at the purifica- tion of the enzymes involved in the synthesis of /Mysine and 3,5-diaminohexanoic acid. In particular the role of vitamin B ]2 in the con- version of /3-lysine to 3,5-diaminohexanoic acid will be studied in detail. One Carbon Metabolism Methane Fermentation. — A role of a methyl-B 12 derivative in the enzymatic synthe- sis of methane is suggested by the previous studies in this laboratory showing that crude extracts of Methanosarcina barkeri catalyze the synthesis of methyl-B 12 from methanol and vitamin B 12s , and in the presence of ap- propriate electron donors, they catalyze also the conversion of methyl-Bi 2 to methane. It has now been established that formaldehyde, the carboxyl group of pyruvate and unidenti- fied endogenous substrates present in crude extracts are also able to react enzymatically with B 1M to form methyl-B 12 . In order to elucidate the mechanism of methyl-Bi 2 synthesis, the enzymatic reac- tion between methanol and B 12s has been studied in detail. Methyl-B, 2 synthesis is stim- ulated by a reducing atmosphere (H 2 vs argon) and is inhibited by aminopterin, ar- senite, intrinsic factor and o-phenanthroline; all of which inhibit also the conversion of methanol to methane. The enzyme system that catalyzes the syn- thesis of methyl-B 12 has been resolved by DEAE-cellulose chromatography into three fractions, protein fractions R and A, and a frac- tion containing a heat stable cofactor, all of which are necessary in addition to ATP and Mg ++ in order to obtain methyl-B 12 synthe- sis. The active component of fraction R is probably a highly acidic red protein that has a spectrum typical of a B 12 -protein. This com- ponent is enriched throughout a 30-50 fold purification of the active protein by means of chromatography on Bio-gel P-100 or P-200 and TEAE-cellulose or DEAE-Sephadex. Fraction A contains at least two components which are required for optimal methyl-B 12 synthesis. One component has been identified as a ferredoxin- type protein by its spectrum in the oxidized and reduced states, and also by the fact that it will replace ferredoxin in the DPN-linked hydrogenase system of Clostridium kluyveri. Whether or not the ferredoxin-like activity of fraction A is identical with clostridial ferre- doxins remains to be established. The function of the other protein component in fraction A that is required for methyl-B 12 synthesis is not known. The heat stable cofactor obtained in the effluent fraction from DEAE-cellulose columns has not yet been identified, but it has been partially purified and some of its properties have been determined. It is stable in 2 N HC1 for 2 hours at 80° or in 0.1 N NaOH for 30 minutes at 100° C. It is anionic as shown by elution from Dowex-1-Cl with 0.1 N HC1. It is quantitatively adsorbed on charcoal and eluted with ammoniacal ethanol. The most highly purified preparations have no absorp- tion in the visible spectral region, but do ab- sorb at 260 m/x; it remains to be seen if the absorption is due to the active component. It has not been possible to resolve the en- yzme system catalyzing the over-all conversion of methanol to methane into its component parts, presumably because of the oxygen la- bility of the system. Nevertheless, when low concentrations of crude extract are used, sig- NATIONAL HEART INSTITUTE 141 nificant stimulation of the over-all conversion of methanol to methane is obtained by adding ATP, CoA and each of the resolved compon- ents (i.e., protein Fractions A and R and the heat stable cofactor) shown to be required for the synthesis of methyl-B 12 . This is further evidence that the enzyme system catalyzing the synthesis of methyl-B 12 is involved in the synthesis of methane. In addition to the puri- fied heat stable cofactor described above, the conversion of methanol to methane appears to require still another heat stable cofactor pres- ent in boiled cell extracts. Further studies will be made to characterize the heat stable cofactor and to establish the interrelationship of the various protein frac- tions involved in methyl-B 12 synthesis and in the synthesis of methane from methanol. Synthesis of Acetate from C0 2 . — Previous studies in this laboratory have shown that cell free extracts of Clostridium thermoaceticum catalyze the total synthesis of acetate from C0 2 . A role of a methyl-cobalamin derivative in this synthesis is indicated by the fact that the incorporation of C0 2 into acetate is par- tially inhibited by intrinsic factor and by the fact that cell-free extracts catalyze the de novo synthesis of Co-methyl-cobalamin from C0 2 , B 12s and pyruvate, and are able to utilize the methyl group of Co-methyl-cobalamin for the synthesis of the acetate methyl group. As reported last year the enzyme system that catalyzes the latter reaction has been re- solved into two protein fractions which to- gether with clostridial ferredoxin from other sources are required for the utilization of Co- methyl-cobalamin in acetate synthesis. In con- tinuing studies a third protein fraction has been obtained from crude extracts that will re- place the ferredoxin requirement. This fraction possesses ferredoxin-like activity since it will substitute for ferredoxin in the hydrogenase system of C. kluyveri; however, it has an atyp- ical chromatographic behavior on DEAE-cellu- lose columns that differentiate it from other ferredoxins. The possible relation of this ferre- doxin-like compound to the recently reported favodoxin is under investigation. In addition to the three protein fractions, the utilization of Co-methyl-cobalamin for acetate synthesis requires the presence of pyruvate and CoA; other «-ketoacids except a-ketobutyrate are not able to replace pyru- vate. The specific role of pyruvate and CoA could be (1) to serve as a source of acetyl-CoA ior acetyl-P; (2) to serve as a source of low potential electrons, as for example reduced fer- redoxin; or (3) to serve as a source of an active carboxyl group via a transcarboxyla- tion mechanism. Thus far attempts to demon- strate one or more of these functions have failed. The pyruvate requirement cannot be re- placed by acetyl-P or acetyl-CoA or systems generating these compounds; nor can it be re- placed by other sources of low potential elec- trons such as reduced methyl viologen nor by reduced ferredoxin generated by the hydro- genase system of C. kluyveri; moreover, a bio- tin dependent transcarboxylation function is contraindicated by the insensitivity to avidin of the over-all acetate synthesis or of the ex- change of C0 2 with the pyruvate carboxyl group, that is also catalyzed by cell-free ex- tracts. In future studies further efforts will be made to identify the individual steps in the metab- olism of Co-methyl-cobalamin and especially to establish the roles of pyruvate or of ferre- doxin in the synthesis of acetate. Cystathionine Biosynthesis and Trans- sulfuration The major findings of the past year are in 4 areas. The first concerns the reactions mediat- ing cystathionine synthesis from cysteine and homoserine (biosynthetic trans-sulfuration) in fungi. Two decades have elapsed since this process was first postulated to occur, and since 3 genes were defined which appeared to govern it in Neurospora. Mutation in one of these genes has now been found to result in: (a) failure to accumulate the acetyl ester of homo- serine; (6) ability to respond to this ester nutritionally; and (c) lack of an enzyme cat- lyzing an exchange between the ester and free homoserine. Second, one of the enzymes of bacterial bio- synthetic transsulfuration, cystathionine y- 142 ANNUAL REVIEW OF INTRAMURAL RESEARCH synthetase, has been obtained in pure form from Salmonella, and its physical properties and subunit structure have been characterized. Third, the cystathionine y-cleavage enzyme of Neurospora (reverse trans-sulfuration) has been found to be derepressed 60-fold by sulfur deprivation. Such striking derepression in this organism has previously been associated with enzymes that appear spontaneously only dur- ing sexual differentiation. However, in con- trast to the latter enzymes, cystathionine y-synthetase was not elevated after total star- vation. A non-sporulating yeast showed no re- sponse to sulfur deprivation. A unique kind of pyridoxal phosphate ca- talysis has been detected through the effect of added N-ethylmaleimide on y-elimination reactions. Instead of a-ketobutyrate, a new product is formed, identified last year as struc- ture I. CH 3 I CHCOCOOH R The structure has been confirmed by mass spec- troscopy. The major findings of the past year were concerned with the mechanism of for- mation of compound I. The discovery that /^-substituted 4-carbon amino acids also react to yield I has restricted the possible points of maleimide attack to intermediates in enamine- ketimine tautomerization. The possibility that free aminocrotonate was the reactive interme- diate was emphasized by the discovery of a slow non-enzymic formation of compound I from a-ketobutyrate and maleimide, which specifically required ammonia as catalyst. Re- cent work has been concerned with the latter reaction. A comparison of the effects of am- monia and of a series of amines on the rate of solvent exchange with the ^-hydrogen of a-ketobutyrate has made it unlikely that the non-enzymic reaction involves the formation of a-aminocrotonate. A possible clue to the cat- alytic action of ammonia has come from the finding that the rate of hydrolysis of malei- mides is decreased in its presence. The Mechanism of Action of Vitamin B lt Coenzymes Ethanolamine Diaminase. — It was pre- viously established in this laboratory that the conversion of ethanolamine to acetaldehyde and ammonia by a clostridial enzyme is a vitamin B 12 coenzyme dependent reaction. Pur- ification of the ethanolamine deaminase was therefore undertaken in order to obtain a sim- ple model system that could be used for inves- tigation of the mechanism of cobamide coenzyme action. A relatively simple three step procedure has been developed for the isolation of the enzyme from cell-free extracts of ethanolamine adapted cells. The isolated deaminase is homogeneous as judged by its sedimentation in the ultra- centrifuge and its mobility on disc gel electro- phoresis. The pure enzyme has an absolute requirement for cobamide coenzyme and for potassium ions. The pH optimum is from 6.8 to 8.2. Preincubation of the deaminase and coenzyme in the absence of substrate results in diminished activity within a few seconds and over 90% inactivation within five minutes. At low concentrations of the cobamide coen- zyme there is a significant lag period in the deamination reaction which decreases with in- creases in coenzyme concentration. The molecular weight of the deaminase is approximately 500,000 gm. as determined by the Yphantis sedimentation equilibrium method. The sedimentation coefficient is 14.5 Svedbergs. Preliminary studies indicate that 4 M guanidine causes the enzyme to dissociate into subunits of about 55,000 gm. molecular weight. The enzyme is stable indefinitely in liquid nitrogen and for several days at 4° C in concentrated solution. Amino acid analysis shows 3 half cystine residues per minimum molecular weight of 28,660 gm. Parachloro- mercuriphenylsulfonate is the only sulfhydryl reagent of several tested that inhibited the NATIONAL HEART INSTITUTE 143 enzyme. The enzyme is highly specific. Of a large number of structurally related compounds tested, ethanolamine is the only compound that will serve as a substrate. The purified deaminase is pink to orange in color due to the presence of 1.35-2.1 equiva- lents of tightly bound a-adenyl cobamide de- rivative. The cobamide cannot be removed from the enzyme by charcoal or by dialysis, but it is removed by treatment with acid ammonium sulfate. In the presence of added cobamide co- enzyme the resolved enzyme has a higher spe- cific catalytic activity than the unresolved en- zyme suggesting that the cobamide bound to the enzyme is a partially degraded, catalytic- ally inactive compound that occupies some of the coenzyme binding sites. Preincubation of the enzyme with 10 ~ 8 M cyano-cobalamin re- sults in significant noncompetitive inhibition. The number and nature of the cobamide binding sites, the nature of the enzyme-co- enzyme interaction, and the mechanism of the substrate-coenzyme interaction are subjects of future study. The Metabolism of Heterocyclic Compounds The hydroxylation of nicotinic acid. — The conversion of nicotinic acid to 6-hydroxy- nicotinic acid is the first step in the anaerobic decomposition of nicotinic acid by a clostridial species. Purification of the enzyme that cata- lyzes this step is being carried out with the ultimate objective of obtaining a model sys- tem to investigate the mechanism of anaerobic hydroxylation of aromatic compounds. This is a problem of unique biochemical interest since the hydroxylation of aromatic compounds in other systems so far investigated is an obli- gately aerobic process involving the incorpora- tion of molecular oxygen into the hydroxyl group. Previous reports have shown that TPN is a specific electron acceptor in the anaerobic hy- droxylation of nicotinic acid as catalyzed by partially purified enzyme preparations, and that the enzyme catalyzed reaction is stimu- lated by Fe + + and by glutathione. It has now been found that the Fe ++ and glutathione re- quirements can be replaced by dithiothreitol plus vitamin B v > derivative. These supplements are apparently needed only to remove trace amounts of oxygen since they can all be elim- inated if the reaction mixture is made strictly anaerobic by exhaustive gassing with helium. In contrast to aerobic hydroxylatioD reactions, anaerobic hydroxylation of nicotinic acid is a freely reversible process. When sup- plemented with an appropriate TPNH gener- ating system the clostridial enzyme catalyzes the reduction of 6-hydroxynicotinic acid t< nicotinic acid. The reduction of 6-hydroxynicotinic acid to 6-oxo-l, 4, 5, 6-tetrahydronicotinic acid. — The second step in the fermentation of nico- tinic acid is the reduction of the double bond between carbons 4 and 5. The enzyme catalyz- ing this reaction has been partially purified and some of its properties determined. Re- duced ferredoxin appears to be the immediate election donor. The previously reported re- quirement or pyruvate and CoA in the me- tabolism of 6-hydroxynicotinic acid is prob- ably associated with the fact that in crude extracts of the Clostridium, pyruvate serves as an electron donor for the reduction of fer- redoxin. This conclusion is supported by the fact that neither CoA nor pyruvate are needed for the reduction of 6-hydroxynicotinic acid when other sources of reduced ferredoxin are supplied, as for example, dithionite or molecu- lar hydrogen plus the C. kluyveri hydrogenase system. Finally, it has been shown that re- duced ferredoxin can be replaced by low po- tential dyes such as reduced methyl viologen, but not by the reduced forms of dyes with greater oxidation-reduction potentials. The chemical synthesis of 6-hydroxynico- tinic acid. — Elaboration of the mechanism of nicotinic acid dissimilation would be greatly facilitated by the use of isotopically labeled 6-hydroxynicotinic acid. Therefore, various methods for the chemical synthesis of this compound have been investigated in an effort to determine a practical procedure for the synthesis of C 14 -labeled derivatives. After sev- eral attempts, one reasonable method was developed. This method involves reaction of the 144 ANNUAL REVIEW OF INTRAMURAL RESEARCH ester of nicotinic acid with benzyl bromide to form the quarternary salt which is then oxi- dized by alkaline ferricyanide to the corre- sponding a-pyridone carboxylic acid. By treat- ment with POCl 5 the a-pyridone was con- verted to 6-chloronicotinic acid which was finally hydrolyzed by strong acid to yield 6- hydroxynicotinic acid. Section on Cellular Physiology The research program of the Cellular Physi- ology Section continues to be directed toward the structural basis of the biochemical activi- ties of proteins, their biosynthesis and func- tional relationships in the integrated activities of cell structure. This program is broadly di- vided into three main projects: Studies on protein structure: In this area re- search is primarily focused on the fibrous pro- teins involved in muscle contraction and blood clotting with major emphasis on substructure and the relationship of primary structure to biochemical activity. Protein biosynthesis: Investigations in this area are concerned with the role of cellular membranes in protein biosynthesis and the energetics of the process. Biochemistry and cytology of cell transport: Principal attention in this area is focused on the cell membrane and its role in phagocytosis and pinocytosis. Summaries of the individual projects follow. Muscle Proteins: Myosin Previous reports have summarized investi- gations which led to development of a three stranded rope model of the myosin molecule with a globular portion at one end. The three strands of this model are physically and chem- ically identical polypeptide chains all having the same sense in this rod shaped molecule with their amino terminal ends in the globular portion of the molecule. This portion also con- tains the enzymic sites. The latter are controlled by two cysteine residues in each polypeptide chain. Methods were developed in the past for individually labeling these two cysteine resi- dues with a radioactive sulfhydryl reagent. Peptide fragments resulting from tryptic and chymotryptic digestion of the labeled protein have been isolated and the sequences of amino acids in the peptides determined. These two cysteine residues appear to be associated in the following twelve amino acid sequence: Cys (Si) .Gly.Asn.Val.Leu.Glu.Gly.Ile.Arg.Ile. Cys(Si).Arg. While it is not clear in this sequence how Cys(S 2 ) affects the enzyme activity it seems possible that Cys (SO influences the binding of the polyanion substrate by the two posi- tively charged arginine residues closely asso- ciated with Si. Studies on the Extraction of Actomyosin from Rabbit Muscle Actomyosin, a complex of the two proteins — actinand myosin — appears to provide a model of the functional relationship of these two pro- teins in the contractile process. While actomyosin can be prepared from its components, it is also extractable from muscle, under some conditions, particularly in pro- longed extraction of the tissue. The present study was directed at the factors influencing the extraction of actomyosin from muscle and the nature of the low molecular weight pro- teins associated with the preparation. In con- firmation of earlier proposals, it was observed that extraction of actomyosin occurs subse- quent to breakdown of the intrinsic ATP of muscle. Extraction of actomyosin is strongly inhibited by phosphate ions. In the presence of 0.2 M phosphate no actomyosin appears in the extracts even after 24 hours extraction. This is not due to any suppression of ATP breakdown, which occurs at the same rate as in the absence of phosphate. It appears that high concentrations of phosphate ions may pro- duce the same effect as ATP in maintaining the structure of the myofilaments and thereby inhibiting actomyosin extraction. In the normal extraction of actomyosin low molecular weight components were detected in association with myosin before the extracted protein developed the characteristics of ac- tomyosin. These low molecular weight com- NATIONAL HEART INSTITUTE 145 ponents appeared to be primarily either G- actin or F-actin of a very low degree of poly- merization. Their presence in a myosin prepara- tion, though not detectable by the usual cri- teria of purity, suggests the need for develop- ing new means of evaluating homogenety in this protein. Radiation Damage in Proteins The previous report described an original technique for trapping and labeling free radi- cals formed in gamma-irradiation of protein using tritiated hydrogen sulfide. The amino acids residues susceptible to free radical for- mation can then be identified through the ra- dioactive label. This technique has been applied to a number of proteins — ribonuclease, lyso- zyme, chymotrypsinogen, insulin, myoglobin, and gelatin. Methionine and proline were the residues most heavily labeled in all proteins — alanine, isoleucine, leucine, phenylalanine and valine were the least labeled. From examina- tion of proteolytic digests of some of the pro- teins, it appears that the most heavily labeled residues are widely distributed in the amino acid sequence. Experiments with unfolded — denatured — proteins as compared to the native proteins indicate that conformation plays a role in free radical distribution. Repair of ra- diation damage by reaction of the tritiated hydrogen sulfide with free radical also occurs as judged by recovery of enzyme activity and other properties of the native protein. Protein Biosynthesis Previous reports have demonstrated the high metabolic activity of the lipid soluble amino acid and peptide fractions of hen oviduct. In the current work it was found that when water dispersions of radioactive lipo amino acids were incubated in an homogenate the amino acid moieties were efficiently incorporated into homogenate proteins. This incorporation was more than 10 times as efficient as incorporation of the free amino acid and its incorporation was not reduced by the presence of a large free amino acid pool indicating a direct trans- fer from the lipo amino acid moiety into homo- genate protein. The active lipoamino acid ma- terial can be purified by thin layer chroma- tography (T.L.C.); however, a single radioac- tive amino acid will produce at least 9 differ- ent components on T.L.C., though only one or two of these appear to be active for incorpora- tion into protein. Nonphosphorylated Intermediates of Energy Transfer and Protein Biosynthesis Recent work in a number of laboratories has indicated that the electron transport sys- tem gives rise to high energy intermediates that are available to a number of mitrochon- dial endergonic processes, including amino acid uptake, prior to their involvement of inor- ganic phosphate leading to ATP formation. In the present work, the generality of this non-phosphorylated intermediate concept has been examined in anaerobically grown yeast in which it was found that classical uhcouplers of oxidative phosphorylation, dinitrophenol and sodium azide inhibit protein synthesis without inhibiting glycolysis or disturbing intra-cellular ATP levels. The uncouplers also inhibited RNA formation under anaerobic con- ditions, although RNA precursor pools and the enzyme believed to be responsible for RK syn- thesis, DNA dependent RNA polymerase, were unaffected by those agents. Although not con- clusive, the studies suggest an effective link of the synthetic activities for RNA and protein in the cell with transient non-phosphorylated intermediates of energy transfer in mitochon- dria or other intracellular membrane systems. Biochemistry and Cytology of Cell Transport Continuing earlier studies on the uptake of fatty acids by amoebae, it has been found that unesterified fatty acids are taken up by en- ergy-independent processes to give two forms of bound fatty acids. One is removable by washing the cells with serum albumin and the other is not. The latter class is converted to glycerides and phospholipids in a subsequent energy dependent step. Extending earlier studies on the chemistry of some of the straining and fixation tech- niques used in electron microscopy, the struc- ture of the reaction product of oxmium tetrox- 146 ANNUAL REVIEW OF INTRAMURAL RESEARCH ide with oleic acid or methyl oleate has been determined as: CH 3 (CH 2 )7CH - CH(CH 2 ) 7 COOR V Qs CH 3 (CH 2 ) 7 CH - CH(CH 2 ) 7 COOR Demonstration that the osmic acid reacts with the double bonds in unsaturated fatty acids or their derivatives rather than with the polar portions of the molecules as widely as- sume, has many implications for the interpre- tation of electron micrographs. Phagocytosis by amoebae has been studied using polystyrene beads. Conditions were es- tablished in which it was possible to study some of the kinetics of the process, the appar- ent kinetic parameters being independent of the size of the beads, large beads being taken up individually and the smaller beads accumu- lating on the surface and being taken up in clusters. Phagocytosis appears to be a highly discriminative process, for C 14 -glucose and I 131 albumin present in the medium are not taken up with the beads. The over-all process is very efficient; 10'' cells (0.004 ml) can re- move essentially all the beads from one ml of medium in 15 min and at saturation the average amoeba has taken up about 40 beads of 2.6 jx diameter. The amount of membrane surrounding the internal beads is about equal to the mass of membrane surrounding the cell. During phagocytosis no change in cell volume occurs. These appear to be the first quantita- tive studies on the process of phagocytosis. Section on Comparative Biochemistry The activities of the Section on Comparative Biochemistry are centered upon the investiga- tion of mechanisms of lipid biosynthesis as well as a study of the control mechanisms in lipid biosynthesis. Previous work in this laboratory has established that the acyl carrier protein (ACP) plays an important function in fatty acid biosynthesis. Acyl groups involved in fatty acid synthesis are bound to ACP through thio- ester linkage with the sulfhydryl of the pros- thetic group, 4'-phosphopantetheine. The mech- anism of ACP involvement in this biosynthetic pathway has been further probed. Several of the reactions of this pathway have been stud- ied in purified systems in order to understand the enzymatic mechanism involved. The total structure of ACP, which appears to be ex- tremely important in determining the reactiv- ity of acyl-ACP derivatives with the enzymes of fatty acid synthesis, has been further stud- ied. The general occurrence of ACP in fatty acid biosynthesizing systems has been estab- lished by experiments which indicate its pres- ence in mammalian enzyme systems. The general biochemical significance of ACP has been further studied by a search for new reac- tions in which ACP may be involved. Experi- ments suggest that it may be involved in both complex lipid synthesis and sterol synthesis. Thus ACP is the acyl carrier in a number of biosynthetic reactions in which CoA was pre- viously thought to be involved. Therefore a study of the absolute concentrations of CoA and ACP was carried out in E. coli. In addi- tion studies have been initiated which should explain the regulation of ACP concentration in the cell. ACP Involvement in Lipid Metabolism Fatty Acid Biosynthesis. — ACP func- tions as the acyl carrier in the biosynthesis of long-chain fatty acids. The condensation reac- tion of this pathway is complex and requires three enzymes in E. coli: Malonyl-S-CoA + ACP-SH— Malonyl-S-ACP + CoA-SH Acetyl-S-CoA + ACP-SH— Acetyl-S-ACP + CoA-SH Acetyl-S-ACP + malonyl-S-ACP?±Acetoacetyl-S-ACP + CO, + ACP-SH (1) (2) (3) Sum: Malonyl-S-CoA + Acetyl-S-CoA + ACP-SH— Acetoacetyl-S-ACP + CO, + 2CoA-SH (4) NATIONAL HEART INSTITUTE 147 Reactions 1, 2 and 3 are catalyzed by malonyl transacylase, acetyl transacylase and (3-keto- acyl-ACP synthetase respectively. Malonyl transacylase was isolated as a homogeneous protein as shown by the presence of a single component upon disc-gel electrophoresis. Be- cause this enzyme catalyzes the transfer of a malonyl group from the sulfhydryl of CoA to the sulfhydryl group of ACP and because the enzyme is a sulfhydryl protein studies were carried out to determine whether the trans- acylase contains a prosthetic group such as phosphopantetheine. The amino acid composi- tion of the protein indicates that it does not contain this prosthetic group since there is no /?-ananine or mercaptoethylamine in this pro- tein. Thus, although the enzyme catalyzes the acyl transfer reaction, the sulfhydryl group of the enzyme involved in the transacylation is not a component of phosphopantetheine. /?-Ketoacyl-ACP synthetase was purified ap- proximately 400-fold, 2-fold over the previously reported value. This enzyme catalyzes the con- densation of acetyl-ACP with malonyl-ACP to form acetoacetyl-ACP, C0 2 and ACP-SH. This enzyme, like the acetyl and the malonyl transacylases, is a sulfhydryl protein. Since it had been shown previously that acetyl-ACP protects this enzyme against inactivation by N-ethylmaleimide, the hypothesis had been formulated that an acetyl-enzyme intermediate might occur during this over-all reaction. The 400-fold purified enzyme, although still con- taining 4 protein components upon disc-gel electrophoresis, was reacted with radioactive acetyl-ACP under various conditions. However all attempts to isolate an acetyl-enzyme or acetyl-ACP-enzyme intermediate failed. Al- though acetyl-ACP must bind to this enzyme, the binding may not be sufficiently stable to allow isolation of the substrate-enzyme com- plex. Acetyl transacylase as well as the other en- zymes of fatty acid synthesis in E. coli are being studied to determine whether other pro- tein-bound prosthetic groups are involved in this pathway. Complex Lipid Biosynthesis. — Membrane preparations of E. coli catalyze the synthesis of monopalmitin from ^-glycerophosphate and palmityl-ACP. The monopalmitin was identi- fied by column and thin layer chromatography. Since glycerol cannot substitute for ^glycero- phosphate in this system, it is postulated that the primary reaction product is lysophospha- tidic acid which undergoes secondary dephos- phorylation. This same membrane preparation catalyzes the synthesis of lysophosphatidic acid from a-glycerophosphate and palmityl-CoA. It is not yet known whether separate enzyme systems are involved in the esterification of a- glycerophosphate by palmityl-ACP and by pal- mityl-CoA. Sterol Biosynthesis. — A possible role for ACP in sterol synthesis through its involve- ment in /3-hydroxy-/?-methylglutarate (HMG) synthesis is suggested in experiments recently carried out with yeast condensing enzyme. In- cubation of yeast condensing enzyme with acetyl-CoA and acetoacetyl-CoA gave rise as expected to HMG-CoA, whereas a similar in- cubation substituting acetoacety-ACP for ace- toacetyl-CoA yielded HMG-ACP. Although the reaction rate was faster with acetoacetyl-CoA, the fact that acetoacetyl-ACP was metabolized is significant. E. coli ACP was utilized in these experiments and it is known that yeast en- zymes discriminate against bacterial ACP de- rivatives. The possibility that ACP is involved in this reaction awaits experiments in which the enzyme and the ACP utilized are from the same source. Mammalian ACP ACP has been identified in several bacteria in this laboratory and in several plants by P. Stumpf (University of California). Its identi- fication in more complex fatty acid synthesiz- ing systems such as those of yeast, pigeons, and mammals is difficult since the ACP is contained within a protein complex in those systems. In- jection of C 14 -pantothenate into pantothenate deficient rats leads to the formation of C 14 - ACP within the rat fatty acid synthetase com- plex. This labeled ACP can thus be studied and perhaps isolated from the complex. Using this technique of in vivo labeling of ACP, it was previously established that the rat fatty acid synthetase contains protein-bound C 14 phos- 148 ANNUAL REVIEW OF INTRAMURAL RESEARCH phopantetheine. Attempts to dissociate intact labeled ACP from this purified synthetase com- plex by treatment with deoxycholate, high pH, low ionic strength, urea, and guanidine have failed. Thus it is possible that ACP is part of the matrix of the enzyme complex in mam- mals. Further experiments to delineate mam- malian ACP will be pursued. Structure of E. coli ACP Previous studies have demonstrated that the E. coli enzymes of fatty acid synthesis utilize as substrates acyl thioesters of ACP rather than acyl thioesters of CoA. Since both ACP and CoA contain 4'-phosphopantetheine as the ter- minal component to which the acyl group is bound, it is clear that some structural feature other than the prosthetic group accounts for the fact that ACP derivatives are the pre- ferred substrates for these enzymes. Studies of the primary structure of ACP have indicated the following partial structure: (14-19) Ser-Thr-Ile Glu Glu Arg) (lie Glu Gly-Leu-Yal-Lys) I (Glu Gly I Ala Asp (24) ( ~&er- rAspartate peptide Ala-Gln-His-Gly— _Lys_ Lys-Ala-Met-Val-Leu Leu -= (56-61) -Tryptic peptide II 4'-phosphopantetheine As shown above the prosthetic group is attached to serine which is about 24 residues from the amino terminus of the molecule. Peptides have been obtained by treating ACP with dilute HC1, CNBr and trypsin. Treatment of ACP with dilute HC1 cleaves the protein at the as- partic acid residue which is adjacent to the se- rine to which phosphopantetheine is bound. The resulting peptide which contains the pros- thetic group and the C terminus of the mole- cule was isolated and found to be inactive as tested in the malonyl-CoA-C0 2 exchange reac- tion. Tryptic digestion of ACP gave rise to a large peptide containing the prosthetic group and comprising about one-half the ACP mole- cule. This peptide was inactive as tested in the malonyl-CoA-C0 2 exchange reaction and in the /?-ketoacyl-ACP synthetase reaction. How- ever an acetoacetyl derivative of this peptide was active in the /3-ketoacyl-ACP reductase re- action. Kinetic studies with this enzyme have indicated that acetoacetyl-ACP is the best sub- strate, but both acetoacetyl-peptide and ace- toacetyl-pantetheine are active in the reaction. It is of particular interest that the pantetheine derivative is more reactive than the peptide de- rivative. These studies indicate the importance of the ACP protein structure in conferring the reactivity with the various enzymes of fatty acid synthesis. The specific sites of ACP respon- sible for the activity of ACP derivatives with these enzymes will be further studied. Concentrations of ACP and CoA in E. coli Evidence is accumulating which indicates that ACP rather than CoA is the acyl carrier in a number of biosynthetic reactions. There- fore it is of interest to study the concentra- tions of these two compounds in some biologi- cal system. A convenient system for this is a pantothenate- requiring E. coli auxotrophy. When this organism is grown on ^-panto- thenate both CoA and ACP are labeled and therefore can be easily measured. This auxo- troph was grown on different concentrations of C 14 -pantothenate. Cells grown on limiting concentrations of pantothenate contained about 0.65 m^mole ACP/mg protein and 0.04 nut- mole of CoA/mg protein. When the bacteria were grown in an excess of pantothenate, ex- tracts contained 3.0 m^moles CoA/mg protein and 0.65 m^mole ACP/mg protein. Thus the concentration of ACP did not vary under these conditions whereas the concentration of CoA NATIONAL HEART INSTITUTE 149 was directly related to the concentration of pantothenate in the medium. When cells grown in an excess of pantothenate were transferred to pantothenate deficient medium, the CoA concentration decreased dramatically as the cells continued to grow normally. The ACP con- centration did not change under these condi- tions. Thus it is clear that the ACP concentra- tion of the cell is maintained at the expense of cellular CoA. These experiments also sug- gest that CoA can be the source of phospho- pantetheine for ACP synthesis. ACP Hydrolase Study of the biosynthesis of ACP and inves- tigation of additional reactions in which ACP is involved may be aided by the availability of ACP hydrolase (ACPase). This enzyme, puri- fied from E. colt, catalyzes the manganese- dependent cleavage of 4'-phosphopantetheine from ACP and thereby inactivates ACP. One product of this reaction is 4'-phosphopante- theine which has been identified by column chromatography. The protein product, apoACP, has been isolated and shown to have approxi- mately the same molecular weight and amino acid composition as ACP minus the prosthetic group. A hydrolytic cleavage is assumed in this reaction, by analogy to other phosphodiester- ase reactions, but a /3-elimination has not been ruled out. ACPase does not catalyze either the inactivation of any other protein that has been tested nor the cleavage of CoA. Its extreme specificity is demonstrated by its failure to catalyze the cleavage of 4'-phosphopantetheine from acetyl-ACP or from large peptides of ACP. It is also inactive against ACP of Clo- stridium butyricum, and it does not cleave phosphopantetheine from the mammalian fatty acid synthetase complex. This striking speci- ficity suggests that ACPase may be involved in the rigid control of cellular holoACP con- centration. CLINICAL ENDOCRINOLOGY BRANCH Studies in the Clinical Endocrinology Branch have included investigations of: A. Studies of steroidogenesis by the adrenal cortex, with especial reference to biogenetic control mechanisms, the adrenogenital syn- drome and the relationship of the renin-angio- tensin system to aldosterone metabolism. B. Studies of calcium and phosphorus me- tabolism, including in vitro studies of bone mineral and of solutions of calcium and phos- phorus, in vivo studies in rachitic dogs, and clinical studies in patients with metabolic bone diseases. C. Studies of renal physiology, with espe- cial reference to dynamic measurements and analysis of the renal circulation, studies of the amino acid clearance in cystinuria with and without treatment, and measurements of adre- nal factors affecting free water clearances. D. Studies of neuro-endocrine relationships, including measurements of factors controlling catecholamine excretion, studies of hormonal and nonhormonal disorders of taste, smell and hearing sensitivity, and studies of steroid ac- cumulation in the central nervous system. Studies of Steroidogenesis by the Adrenal Cortex Steroidogenesis was measured directly in vivo and in vitro in dogs. The rate of secre- tion of steroids into the adrenal vein as influ- enced by sodium deprivation, angiotensin in- fusion and potassium loading were measured over a two-day study period. The animals were then sacrificed and steroidogenesis was meas- ured in vitro with and without the addition of precursors. The effect of stimulation at a rate- limiting step was observed by measuring the production of steroids below and above such a step. In this way, it was shown that sodium deprivation stimulated steroidogenesis at at least two steps, one before the production of corticosterone, and another apparently between the corticosterone and aldosterone. The stim- ulus to the first of these may be angiotensin, but the second appears to be clearly separate from angiotensin. A similar technique is being applied to the measurement of effects of potas- sium and of other agents acting on steroid- ogenesis. Studies on the biogenetic defects for steroids in the adrenogenital syndrome were extended. Studies support the hypothesis that patients with the adrenogenital syndrome and congeni- 150 ANNUAL REVIEW OF INTRAMURAL RESEARCH tal adrenal hyperplasia may have one or two biogenetic defects in hydroxylation of proges- terone or of 17-hydroxyprogesterone as sub- strate. When a defect is present only in hy- droxylation of 17-hydroxyprogesterone, there is hypersecretion of corticosterone and of aldos- terone, suggesting an actual facilitation of hy- droxylation of progesterone. It was further shown that this step was not maximally active in the subject on average sodium intake, but could be strongly stimulated by sodium depri- vation and that it was dependent on ACTH. Patients with hypertension were studied for involvement of the renin-angiotensin system by measurement of circulating renin and of renin secretion, as influenced by sodium intake and by posture, and by measurement of a wide variety of clinical indices, the most important of which appear to be the aldosterone secretion and metabolic clearance rate and the response of the serum potassium to sodium loading. The aldosterone secretion rate reveals the presence of aldosteronism and the response to sodium loading distinguishes those patients in whom over-production of aldosterone is autonomous in that secretion is not decreased by the sodium loading, and produces hypokalemia. Adrenal hyperplasia, autonomous in these respects, was found to be as common a cause of primary aldosteronism as tumor. In patients with tu- mor, renin concentration in the plasma is nor- mal in recumbency with average sodium intake. The relative role of renin and of the pitui- tary in the control of aldosterone secretion was investigated in patients with hypopitui- tarism. Aldosterone secretion and metabolic clearance rate and plasma renin were measured in patients with hypopituitarism before and after sodium deprivation. Early results sug- gest that effect of sodium deprivation on al- dosterone secretion is normal in hypopituitar- ism (as opposed to reported results that aldos- terone excretion is subnormal), but that the role of renin in stimulating this response is relatively much greater than that in normal subjects. Patients with panhypopituitarism are also under study for the effect of growth hor- mone upon production of renin and upon the secretion of aldosterone. Preliminary results suggest that aldosterone secretion rate is stim- ulated by purified human growth hormone. The role of renin in steroidogenesis was further studied by measurement of steroido- genesis and of plasma angiotensin in normal subjects during infusion of angiotensin in sub- pressor quantities on the one hand, and during deprivation of sodium on the other. Results suggest that angiotensin stimulates production of aldosterone, corticosterone and of Cortisol and that sodium deprivation is an effective stimulus to the production of angiotensin. The routes of secretion of circulating renin were further explored in normal dogs and compared with those found after constriction of the in- ferior vena cava, a procedure which increases renin secretion. It was found that whereas renal lymph delivery of renin increased mark- edly with caval constriction, it nevertheless never represented more than a very small frac- tion of the total renin secretion via the renal vein. Studies of Calcium and Phosphorus Metabolism Ionic exchange with bone mineral was re- explored in dynamic studies in which the time course of entry of calcium and phosphorous ion into bone mineral was explored, and the effect thereon of various ions in solution was meas- ured. In this way, it was shown that uptake of calcium by the crystals is isoionic and can be characterized as a sum of four exponential curves; the same constants characterized loss of calcium from labelled crystals. The effect of ions upon the exchange was found to be closely related to the effect of these ions upon the or- ganization of water molecules, and thus pre- sumably upon exchange at the layer of hydra- tion. The observations for calcium could be re- peated with essentially identical results with phosphorus. These studies are continuing with observation of the effect of hormones and other agents upon exchange. Effects of calcium ions on stearylphosphate monolayers are being fur- ther explored. The observation that calcium ions contract the film area per mole provides a direct measure of calcium phosphate inter- action in the solution phase. This tool may thus be used to explore the agents which affect this interaction. NATIONAL HEART INSTITUTE 151 The effects of Vitamin D on parathyroid hormone were further explored. In rachitic dogs, it was found that a state of endogenous hyperparathyroidism exists and removal of the parathyroid had its usual effects, including that of decreasing phosphate clearance. Ad- ministration of parathyroid extract promptly returned phosphate clearance to previous val- ues, despite the continued absence of Vitamin D. Parathyroidectomized rachitic dogs were further treated with Vitamin D itself, and this was shown to have a parathyroid hormone-like action in increasing phosphate clearance. The role of the parathyroid and of Vitamin D on calcium clearance is being studied in a similar preparation. In such animals, parathyroidec- tomy increases calcium clearance, a finding consistent with the view that rickets repre- sents a state of hyperparathyroidism. Whereas parathyroid extract was shown to be clearly effective in the Vitamin D deficient dogs, its action was quantitatively greatly augmented by addition of small doses of Vitamin D. The effects of thyrocalcitonin on renal clearance of calcium and phosphorus were studied in dogs with separate renal artery cannulas. The infusion of thyrocalcitonin in one side had no renal artery cannulas. The infusion of thyro- calcitonin in one side had no effect on phosphate clearance on that side as compared to the op- posite side. Studies in patients involve the development of a more satisfactory model for analysis of calcium accretion rate in patients subjected to various procedures and in patients with var- ious metabolic bone diseases. Compartmental analysis for stable and isotopic calcium was laborated with the help of the department of Biomathematical Research. These methods show that our investigations obtain a great in- crease in resolution by the prolonged observa- tion of radioactivity in the intact arm with a Packard-Armac counter. Analysis of results ob- tained thus far suggests that calcium accre- tion is markedly augmented in acromegaly and this result is apparent by any method of anal- ysis. This finding probably has no relation- ship to the known increase in bone formation rate in acromegaly, because the locus of the bone formation is clearlv different from the locus of the calcium accretion and because in- fusion of calcium into normal subjects produces equally striking increases in accretion rate. Studies of patients with osteoporosis receiving calcium 47 intravenously reveal an exponential decay characterized by at least three rate con- stants as measured over a 21-day span. The analysis of the curve suggests that patients with idiopathic hypercalciuria have a larger calcium "pool" than patients with osteoporosis, but that the loss of isotope from bones is more rapid with hypercalciuria than osteoporosis. In two patients with pseudohypoparathyroid- ism, together with hypothyroidism, the effects of parathyroid extract were measured with and without replacement of thyroid. In this way, a quantitative dependence of the action of par- athyroid extract upon the status of thyroid function was shown. Preliminary results sug- gest that a similar dependence of the action of Vitamin D upon the presence or absence of thyroxin wall be found in these patients. Studies on the role of collagen metabolism in bone metabolism and of the possible use of urinary hydroxyproline as an index of bone metabolism continue. Human growth hormone was shown to increase hydroxyproline excre- tion in patients with hypopituitarism, as pre- viously reported from several centers. Effects of cortisone on the rate of growth induced by human growth hormone and on the rate of hy- droxyproline excretion are under study. Stud- ies of the effect of calcium loading in osteopo- rosis have been greatly expanded to include measurements of the rate of bone formation and destruction as measured by microradiog- raphy and the rate of bone formation as meas- ured by labelling with tetracycline as well as the effects on calcium dynamics and the effects on calcium and phosphorus balance. Control studies are completed in three patients and in two, the preliminary results from the calcium loading experiments are available, and suggest a clear effect on bone dynamics. The role of in- testinal hyperabsorption of calcium as a deter- minant of hypercalciuria is under study in a large number of patients, with direct measure- ment of accumulation of isotopes in the blood and the bone tissue, when oral isotope is given. The result suggests that a syndrome of hyper- 152 ANNUAL REVIEW OF INTRAMURAL RESEARCH absorption hypercalciuria may be clearly sep- arable from other causes of renal stone for- mation. Studies of Renal Physiology Studies on the hormonal control of sodium and water excretion were continued in experi- mental animals, in normal human volunteers and in patients with pathological retention of sodium. Quantitative retention of dietary so- dium was produced in adrenalectomized dogs receiving sodium-retaining steroids by the ap- plication of inferior vena caval cuff. The effect of adrenergic ganglionic blockade was then measured with infusion of pentolinium. The pentolinium produced decreases of blood pres- sure and less frequently marked decreases in filtered sodium load; it nevertheless consist- ently produced moderate to marked increases in sodium excretion. These results confirmed previous results indicating a role of the adren- ergic nervous system in the control of renal handling of sodium. This was further explored in normal subjects and in patients with abnor- mal retention of sodium by the use of agents producing adrenergic blockade. Parahydroxy- amphetamine was found to decrease blood pressure and to lower the post Valsalva over- shoot of arterial pressure, and the cold pressor response. These results indicate that parahy- droxyamphetamine can function as a "false transmitter", replacing norepinephrine at stor- age sites, and producing adrenergic blockade. It was further found that patients subjected to adrenergic blockade with hydroxyampheta- mine lost sodium more readily and showed a more rapid escape from the effect of sodium- retaining steroids than they did before the drug was given. The effect of beta adrenergic blockade with propranolol and of alpha adre- nergic blockade with dibenzyline was studied in a number of subjects with pathologic sodium retention and in other subjects who were re- ceiving sodium-retaining steroids. Both forms of blockade facilitated escape from sodium re- tention or decrease in the quantity of edema; beta blockade was somewhat more effective in this regard than alpha blockade. Whereas these results again suggest a function of the adrenergic nervous system in renal handling of sodium, they further suggest that the role of alpha and of beta receptors in this regard will require reevaluation. The mechanism of such actions was further explored with direct measurements of intra- renal hemodynamics as measured by Xenon- 133 . Dogs were prepared with exteriorized bladders and individual renal arterial cannulas and prepared for gamma ray counting above the kidney. Xenon- 133 was then injected rap- idly intra-arterially and the rate of decay of renal radioactivity measured as a function of time. The resultant curves were analyzed by computer and shown to fit well to a sum of four exponentials. The effects of caval con- striction on intrarenal blood flow were studied by this means and by the direct measurement of inulin and para animo hippurate clearances. The results suggest an effect of caval constric- tion upon intrarenal distribution of blood. The mechanism for the redistribution and the ef- fects of various vasoactive drugs on renal cir culation are under investigation. Studies with cystinuria were greatly ex- tended. The effects of penicillamine in systinu- ria were extended and compared with those of n-acetyl-penicillamine. Clinically, n-acetyl-pen- icillamine appears to induce less sensitivity reactions than penicillamine and to have less action as an anti-pyridoxine agent. Direct measurements of cystine excretion show that it has quantitatively as great or almost as great an effect in reducing urinary free cystine. Pre- vious unexplained results of penicillamine in decreasing total urinary half-cystine were con- cerned for n-acetyl-penicillamine and were ex- plored by (1) measurement of total excretion of cystine after institution of therapy, and (2) measurement of renal clearance of cystine, be- fore and after treatment with the penicilla- mines. In this way, it was shown that the decrease in total cystine excretion with the penicillamines does not depend upon a previ- ous increase in removal of the cystine by the urinary route and that the penicillamines con- sistently decrease serum cystine and have little effect on cystine clearance. Accordingly, the results establish a second role for penicillamine in cystinuria independent of that on the renal NATIONAL HEART INSTITUTE 153 tubules and on the sulfhydryl exchange in the renal tubules and in the urine. This effect man- ifested by a clear decrease in serum cystine with no change or decrease in renal clearance of cystine is under further investigation. Stud- ies in cystinosis with penicillamine have been instituted. The extension of these studies to more patients has been deferred pending the development of a reliable method of measuring cystine pool. The effects of adrenal factors on free water clearance have been studied. It was found that whereas small doses of sodium-retaining ster- oids have been shown to increase free water clearance while producing a corresponding de- crease in sodium and osmolar clearance, very large doses may decrease free water clearance while decreasing osmolar and sodium clearance even more markedly. These results could be ex- plained only as an effect of sodium-retaining steroids on proximal tubular sodium reabsorp- tion. This effect was further explored by meas- uring the effects of aldosterone antagonists in subjects under the influence of sodium-retain- ing steroids while maximally hydrated. In such subjects, (both adrenalectomized dogs and pa- tients with Addison's disease) blockade of the effect of sodium-retaining steroids with aldos- terone antagonists produced increases of free water clearance, the result which confirms a proximal site for some of the sodium retention induced by the steroid. Studies of Neuroendocrine Relationships The excretion of free and bound epinephrine and norepinephrine was measured in normal subjects as a function of the time of day. The effect of changes of sodium balance and of so- dium intake on catecholamine excretion was also measured. The results show a clear circa- dian pattern for catecholamine excretion in the normal with lowest values during the hours immediately after midnight. Acute changes in sodium balance induced by sodium loading or sodium deprivation did not produce concomi- tant changes in urinary bound or free epine- phrine or norepinephrine. These results differ from those reported elsewhere in which less specific methods for analysis were utilized. It is confirmed that epinephrine excretion may be normal in patients with adrenal insufficiency following adrenalectomy and that excretion of norepinephrine and bound norepinephrine may be markedly increased in Addison's disease. The effects of steroid hormones on nervous system activity were further studied. Adrenal insufficiency was found to produce marked augmentation of sensitivity of hearing which was represented approximately equally at all frequencies tested. Hearing was returned to normal in these patients with administration of carbohydrate active steroids. It was not af- fected by sodium-retaining steroids or by changes in sodium balance. The central nervous system of normal and of adrenalextomized cats was analyzed for the presence of corticosteroids. It was found that in the normal animals, corticosterone and Cor- tisol were found in brain, spinal cord, and pi- tuitary in concentrations at least ten times those in peripheral blood. Steroids in all these tissues were significantly less two weeks after adrenalectomy, but were still significantly higher than the concentrations in peripheral blood. Results indicate concentration of steroid by nervous tissue. The function of taste and smell was explored in patients with a wide variety of endocrine and non-endocrine disorders. In patients with extensive surgical resection of mandibular, maxillary and palatal areas, residual taste was measured and the results serve to extend exist- ing knowledge of the areas for normal taste of the various modalities. A number of patients with hypogonadism were studied and found to have hyposmia and an inability to taste sour and bitter. Patients in this group were chromo- somally of the XO type. A new syndrome was found in which facial hypoplasia, and retar- dation of gonadal and bone age were associ- ated with a decrease of recognition sensitivity for taste, for stereognosis of the mouth and for smell. LABORATORY OF METABOLISM The work of the Laboratory of Metabolism will be summarized here by Section. Members of the three Sections continue to collaborate productively on a number of problems, but the 154 ANNUAL KEVIEW OP INTRAMURAL RESEARCH bulk of the work in each group stands inde- pendently and is so reviewed. Section on Metabolism Mobilization and Utilization of Free Fatty Acids Metabolism of Adipose Tissue Studied in vitro. — An in vitro effect of thyroid hor- mone on adipose tissue has been demonstrated for the first time. Incubation of epididymal fat pads with triiodothyronine (1.25xl0~ 5 M) -significantly enhanced the response of the tis- sue to epinephrine, as measured by release of glycerol and free fatty acids (FFA). As little as 1 hour of incubation with hormone elicited a significant effect. Tissues from hypothyroid rats required longer exposure to the hormone, but after 2 hours the responsiveness to epi- nephrine, which is reduced in tissues of hypo- thyroid animals, was restored to normal. The thyroid hormone did not appear to influence the basal rate of glycerol release, but enhanced the responsiveness to epinephrine and also to ACTH and TSH. The responsiveness of the phosphorylase system to epinephrine was also enhanced in tissues incubated with triiodthy- ronine (T 3 ). These findings suggest that the effect of T 3 on adipose tissue is to "sensitize" the system that forms cyclic-3'5'-AMP. Previous studies in this laboratory estab- lished for the first time that prostaglandins inhibit the lipolytic action of epinephrine, ACTH and several other lipid-mobilizing hor- mones. Studies completed during the past year show that direct addition of cyclic-3'5'-AMP to the medium at high concentration can stimu- late lipolysis, compatible with the proposed key role of this compound in leading to activation of adipose tissue lipase. PGEi did not counter- act the effect of added cyclic-3'5'-AMP. In ad- dition it was shown that even high concen- trations of PGE! do not counteract the effects of high concentrations of theophyllin. The lat- ter compound inhibits a phosphodiesterase that normally breaks down cyclic-AMP. These results are interpreted to mean that under con- ditions in which the rate of cyclic-AMP for- mation is limiting (i.e., the low concentrations of theophyllin) PGE X can inhibit lipolysis, but that under conditions in which the further rate of formation is not limiting (i.e., at high theophyllin concentrations) PGEj becomes in- effective since it does not counteract the effects of cyclic-AMP once formed. Work in other laboratories has shown that insulin inhibits the lipolytic action of epineph- rine, ACTH and other lipid-mobilizing hor- mones, and that it acts by inhibiting produc- tion of cyclic-AMP. Thus, the effects of PGE X are closely analogous to those of insulin. For these reasons, the effects of PGEx on glucose metabolism were studied. It has been shown that PGEi stimulates glucose uptake by adipose tissue and that it enhances incorporations of glucose-C 14 into fatty acids. PGEi-217, a dehydrated derivative of PGEj, does not in- hibit epinephrine-induced lipolysis, and it did not stimulate glucose uptake. In contrast to its effects of glucose utilization by adipose tissue, PGE! did not affect glucose uptake by the rat diaphragm. Thus, PGEi is "insulin-like" in only a limited way. On the other hand, these findings are important in that they may clar- ify the fundamental basis of the action of in- sulin which may be related in some way to the adenyl cyclase system. The qualitative and quantitative responsiveness of this system in different tissues may vary, but the basic en- zymatic processes in the membrane may be closely related. Utilization of FFA inVivo and inVitro FFA Metabolism in Cell Suspension op Ehrlich Ascites tumor. — The convenient iso- lated cell suspensions obtainable by in vivo cul- ure of Ehrlich ascites tumors in the peritoneal cavity of the mouse continue to be valuable in studies of basic aspects of FFA uptake and utilization. It has been shown that under ap- propriate conditions ascites tumor cells can effect net release of FFA to the medium.To demonsrate release it is necessary to use very small concentrations of FFA in the medium (i.e. to treat the commercial albumin preparations exhaustively to remove the FFA normally at- tached to them). Isotopic studies show that this release actually occurs at all times, but under most conditions there is a net uptake even NATIONAL HEART INSTITUTE 155 while endogenous FFA are being released into the medium. This is the first example of net release of FFA from a tissue other than adi- pose tissue. The results suggest that there need not be any unique mechanism in adipose tissue for release of FFA. Instead release may be so striking in adipose tissue only because the ac- tivity" of FFA builds up to sufficiently high levels to permit net release in the face of physiologic FFA: albumin ratios in medium or plasma. The results, if they can be extrapolated, also may help to explain some paradoxical find- ings in isotopic studies of FFA metabolism. For example, the apparently greater uptake of C 14 - almitate over that of C 12 -palmitate in per- fused heart preparations may simply reflect exchange of intracellular unlabeled FFA for medium labeled FFA. The results also suggest that the intracellular FFA pools, low in terms microequivalents per gram, but highly signifi- cant in the total body mass, may influence the composition of plasma FFA and the behavior of labeled FFA introduced into the plasma. The utilization of endogenous fatty acid es- ters was studied by first introducing labeled FFA into the cells and then incubating in the cells in the absence of labeled FFA. Under these conditions, the cells break down endogenous lipids and a net decrease in stored lipid esters was demonstrated. By far the largest fraction utilized was drawn from stored phospholipids. Unexpectedly it was found that the presence of a high concentration of FFA in the medium did not inhibit the breakdown of labeled lipid esters. This continued at about the same rate as in the absence of medium FFA, a large frac- tion of the label being released into the medium as FFA. However, in the presence of glucose plus FFA, depletion of endogenous lipid was prevented and there was a net increase. Frac- tionation of the cell lipid into sub-classes showed that lecithin was the major source of fatty acids used in the absence of exogenous sub- strate. Furthermore it appears that the stored phospholipids do not constitute a kinetically homogenous pool since radioactivity decreased more rapidly than total phospholipid concen- tration. The presence of high FFA concentrations in the medium did not inhibit glucose utilization by ascites tumor cells. In this tissue there is no evidence to support the Randle hypothesis regarding competition between these major substrates. Studies on the Consequences of Rapid FFA Mobilization. — Previous studies from this laboratory implicate high FFA plasma concen- trations as relevant to a number of metabolic events: deposition of fat in the liver, increase of plasma lipoprotein concentrations, increase in plasma ketone body concentrations, and in- creases in metabolic rate of certain tissues. It has not been possible, however, to test the effects of high FFA concentration in isolation; i.e., it has been necessary to use hormone treat- ment or other devices to induce high FFA con- centrations. A method has been developed by which FFA can be infused in large amounts without the hemolytic and thrombotic implications ordi- narily encountered. Arterial blood from a dog is continuously introduced into a newly de- signed centrifuge (developed by IBM under contract with the National Cancer Institute). The centrifuge separates cells and plasma and allows each to be withdrawn separately from the head. This separation allows introduction of FFA at high concentration into the plasma line leading from the centrifuge. This allows the FFA to become bound to the albumin be- fore plasma and cells are recombined, thus avoiding hemolysis. With this method it has been possible to give as much as 5 grams of sodium oleate per hour without ill effects. Plasma FFA concentrations were increased 2- 3 fold. With the development of this technique, it should now be possible to answer directly a number of interesting questions concerning effects of FFA concentration on other aspects of body metabolism. Factors Controlling Plasma Lipoprotein Con- centration Previous work has led to the hypothesis that rapid uptake of FFA by the liver in some way stimulated lipoprotein formation and secre- tion. This problem has been further studied 156 ANNUAL REVIEW OP INTRAMURAL RESEARCH using intravenous infusions of triglyceride emulsions in rabbits. It has been shown that after a 5-hour infusion of olive oil (emulsified in a dilute albumin solution) during which a total of 2 g/kg was injected, there was a marked increase in the plasma concentration of very low density lipoproteins. That this rep- resented a true increase in lipoprotein concen- tration was established by preparative ultra- centrifugation and analysis of fractions and also by paper electrophoresis. It had been pre- viously postulated by other investigators that triglyceride emulsions induced changes in plasma lipid concentrations by "dissolving" lipids from tissue stores. If this were the mech- anism it should not be accompanied by in- creases in lipoprotein-protein but rather by a simple increase in the lipid content of the emulsion in the blood stream. The present stud- ies suggest that this is not the mechanism. The mechanism involved remains to be elucidated and could represent either an inhibition of the removal of lipoproteins or a stimulation of their secretion into the plasma. The latter interpre- tation would be consonant with the earlier studies mentioned above, but further work is needed before reaching a final conclusion. Metabolic Studies in Refsum's Syndrome, a new lipid storage disease Last year's progress report summarized pre- liminary clinical studies in an unusual neuro- logic disease, heredopathia atactica polyneuriti- formis (Refsum's syndrome), in which there is marked accumulation of a branched-chain fatty acid, phytanic acid (3,7,11,15-tetrameth- ylhexadecanoic acid). Continuing clinical stud- ies and animal studies (on the metabolism of phytanic acid and related branched-chain com- pounds) have led to the following tentative conclusions: 1. Patients with Refsum's disease do not syn- thesize phytanic acid endogenously at any sig- nificant rate. There is no significant synthesis of phytanic acid in normal rats either from ace- tate or mevalonate. 2. The metabolic error in Refsum's disease involves a degradative pathway for the oxida- tion and elimination of phytanic acid. How- ever, it appears that the block is incomplete and there is some turnover of phytanic acid. 3. Animal studies show that both dietary phytol and phytanic acid, absorbed primarily by the lymphatic route, are potential precurs- ors of body phytanic acid and lead to accumu- lation of the latter when fed at high doses. Both phytanic acid (3,7,11, 15-tetramethylhex- adec-2-enoic acid) and dihytrophytol (3,7,11,- 15-tetramethylhexadecanol) can be converted to phytanic acid in the rat, showing that there are two potential pathways for the conversion of phytol to phytanic acid. 4. Modification of the diet of two patients with Refsum's disease so as to reduce the intake of these potential precursors (phytol, in chlor- ophyll, and phytanic acid, in butter fat and other ruminant fats) has led to a delayed but highly significant fall in plasma phytanic acid concentration. After one year on the diet, con- centrations have decreased by more than 75%. In one patient there was an increase in nerve conduction velocity and some increase in mus- cle strength, but a final determination as to whether this dietary treatment is curative can- not be made at present. These studies taken together establish the nature of the metabolic error in this new lipid storage disease. Like phenylketonuria, the disease appears to be one in which the accumulation of a metabolite hinges on the nature of substrates presented in the diet. It will be important to elucidate the pathway of degradation and determine wheth- er phytanic acid itself or one of its metabolites induces the nervous system dysfunction. The results already obtained on a restricted diet strongly suggest that any patients showing abnormal accumulation of phytanic acid deserve a trial with such dietary restriction. Clinical Studies. — In the absence of iden- tified case material in this country, collabora- tion has continued with Professor Sigvald Ref- sum, Professor Lorentz Eldjarn and their coworkers in Oslo. Normal control subjects have been studied in the Clinical Center, and two patients with Refsum's disease have been stud- ied in Oslo. Labeled substrates prepared here are shipped to Oslo, and biological samples are collected and returned to us for analysis. NATIONAL HEART INSTITUTE 157 (a) When these studies were begun, it seemed highly likely that the phytanic acid accumulating in Refsum's disease might be en- dcgenously synthesized. The basic branched- chain skeleton could be derived by extension of farnesyl pyrophosphate, a normal interme- diate in cholesterol synthesis, by addition of a fourth isoprene unit. This reaction occurs nor- mally in plants on the pathway for carotene biosynthesis. As reported last year, when mevalonic acid-2-C 14 was injected intrave- nously into a patient with Refsum's disease, there was a normal rate of incorporation into plasma cholesterol, but virtually none into plasma phytanic acid. In order to rule out the possibility that biosynthesis might be going on, but at a very slow rate, this patient has been restudied using D 2 as precursor. A con- stant level of D 2 in body water was main- tained for a period of four months. Incorpora- tion of deuterium into cholesterol increased progressively, but incorporation into plasma phytanic acid was minimal, at the limits of detectability, and showed no tendency to in- crease with time. The small incorporation ob- served corresponds to replacement of at most 2 of the 40 hydrogen atoms in phytanic acid, and mass spectrometric results show that this in- corporation is limited to the carboxyl end of the molecule, compatible with non-enzymatic exchange at positions adjacent to the carboxyl carbon. On biogenetic grounds, one would ex- pect replacement of 14 hydrogen atoms if bio- synthesis occurred from acetate. The high- resolution mass spectrometer was invaluable in connection with these studies. (b) Studies in which phytol-U-C 14 was given orally to six normal subjects and to two subjects with Refsum's disease demonstrate a marked reduction in the capacity of the latter to oxidize it. Labeled phytanic acid was demon- strated in the plasma of both controls and pa- tients during the first day. However, in the normal subjects, this had virtually disappeared by 24-48 hours, whereas labeled phytanic acid persisted in the plasma of the patients and could still be demonstrated as late as 90 days after administration of the dose. The tracer dose was well absorbed (60-80%) and there was no difference between controls and pa- tients in this regard. In collaboration with Dr. Herbert Kayden at New York University, a patient with clinical manifestations virtually indistinguishable from those of Refsum's syndrome, but show- ing no phytanic acid in plasma or liver, was similarly studied. Results in this patient were comparable to those in normal control subjects. The possibility that this patient has a definite metabolic error, but one that involves a closely related or even identical pathway, is under in- vestigation. The conversion of tracer doses of phytol- C 14 to C 14 2 by the two patients with Ref- sum's disease was only 10-20% that of nor- mal subjects. Since only tracer doses were given, these results may underestimate the se- verity of the block. In fact, the prolonged per- sistence of labeled phytanic acid in the plasma would suggest that degradative capacity may be more severly limited. For these reasons, studies have been started using a "loading" dose of 1 gram of phytol. Normal subjects con- vert approximately the same fraction of this 1-gram dose to C0 2 as they do of a tracer dose. Studies in patients with Refsum's disease using the loading dose are now in progress. (c) Although routine analysis of human se- rum lipids by gas liquid chromatography does not reveal the presence of phytanic acid, it has now been possible to demonstrate that it is present normally, but at extremely low con- centrations (0.2 mg/100 ml). A number of closely related branched-chain compounds have also been demonstrated to be present in normal human serum at these trace levels. In view of the large capacity of the normal subject to metabolize phytol and phytanic acid, one would not expect significant concentrations since the intake must be very low. Analysis of the Clini- cal Center diet shows that daily intake of phy- tanic acid may be about 60 mg and of phytol probably less than 5 mg. Animal Studies. — As previously reported, phytol is readily converted to phytanic acid by the rat. Similar results have now been obtained in the mouse, rabbit and chinchilla. In vitro conversion of phytol to phytanic acid has been demonstrated in liver homogenates. 158 ANNUAL REVIEW OF INTRAMURAL RESEARCH Radioactive dihydrophytol and phytenic acid (the 2,3-unsaturated form of phytanic acid) have been prepared, and it has been shown that both can be converted to phytanic acid. Ani- mals fed phytol chronically show very signifi- cant tissue concentrations of phytenic acid, but little or no dihydrophytol. After a single oral dose of phytol, phytenic acid could be demon- strated in the lymph, but no dihydrophytol could be found. These results suggest that the major pathway may involve, first, oxidation of phytol to phytenic acid, and then, reduction of the double bond, but until kinetic studies can be done, this conclusion remains tentative. Intravenously injected C 14 -phytanic acid (in rats) was converted to C0 2 as rapidly as intra- venously injected C 14 -phytol, indicating that phytol oxidation may involve phytanic acid as an obligatory intermediate. The pathway for degradation of phytanic acid is being investi- gated in rats in vivo and in vitro. Preliminary results suggest that the first step in break- down is a decarboxylation reaction yielding pristanic acid. Seubert has studied the path- way for degradation of farnesoic acid in mi- croorganisms. Farnesoic acid has a 15-carbon branched-chain skeleton analogous to that of phytanic acid but includes three double bonds. His studies show that degradation involves a carboxylation reaction which is biotin-depend- ent. Rats were maintained on a biotin-deficient diet for extended periods, but their capacity to oxidize phytanic acid remained unimpaired. We have shown that the same microorganism used by Seubert can grow on phytanic acid as the sole carbon source, and studies are in progress to compare the degradation of phy- tanic acid with that of farnesoic acid. In an attempt to induce changes in the nerv- ous system like those seen in Refsum's disease, rats were maintained on phytol diets, which induce accumulation of phytanic acid in the blood and tissues. However, high dosages ar- rest growth and cause a high mortality within the first month or two; low dosages are toler- ated but as associated with only relatively small increases in tissue phytanic acid concen- tration. Rats have been maintained for up to a year on diets containing 0.5 by weight of phytol. In none of the animals thus far have there been changes in the retina, peripheral nerves, or central nervous system analogous to those seen in the clinical disease. However, because it is not possible to maintain high dose concentrations for extended periods of time, these negative results cannot be taken to rule out a cause-and-effect relationship between ac- cumulation of phytanic acid and nervous sys- tem changes in Refsum's disease. Lymphatic Absorption of Lipids 1. The absorption of phytol, phytanic acid and other related substrates has been studied in rats in which the thoracic lymph duct was cannulated. It was shown that phytol and phy- tanic acid are both readily absorbed largely by way of the lymph. Phytol appears in the lymph mostly in combined form, only a small fraction being present as free phytol. Most of the phytol was present in a fraction shown to be esters of phytol and long-chain fatty acids. It was shown that during the course of absorption as much as 15% of the absorbed phytol was converted to phytanic acid. Phy- tenic acid was also demonstrated in the lymph, but no demonstrable dihydrophytol or any aldehyde derivatives were present. The capacity for phytol absorption in the rat is large; after a 300-mg dose, as much as 60 mg was ab- sorbed in 24 hours. Under similar conditions, phytanic acid was absorbed somewhat more rapidly, and the total absorption exceeded 100 mg. Since a major source of phytol in the diet is that which is esterified to the porphyrin nucleus of chlorophyll, it was of interest to determine to what extent phytol is available for absorption. C 14 -labeled pheophytin a was prepared from tobacco leaves grown in C 14 2 . This was administered to rats by stomach tube and radioactivity determined in collected lymph, urine, C0 2 and body tissues. Less than 5% of the administered radioactivity was recovered in these fractions. Of the small amount of radioactivity present in the lymph, less than half migrated in TLC with phytol or phytanic acid. If these results can be ex- trapolated to man, it would appear that die- NATIONAL HEART INSTITUTE 159 tary chlorophyll is not likely to be a major source of phytol, a finding of great relevance in designing appropriate diets for patients with Refsum's disease. 2. The origin of endogenous lipid in thoracic duct lymph: Even on diets devoid of fat, the lymph continues to show significant concentra- tions of lipid, but the origin of this remains unclear. It has now been shown that the fatty acid composition of thoracic duct lipid is quite different with respect to long-chain fatty acids from that of the lipid in the bile. An out- standing difference was the presence in lymph of a 20-carbon mono-unsaturated fatty acid which was not present in biliary lipid. The structure of this fatty acid has been estab- lished as docoso-13-enoic acid. This acid was also present in intestinal mucosa and in the intestinal contents of germ-free rats. It thus appears that the bile is not a major source of endogenous lymph lipids. Further studies are in progress to determine the origin of endogenous lymph lipids. Section on Molecular Diseases Studies of Hyperlipoproteinemia In 1965 the Section introduced a system for identification of familial disorders of plasma lipid concentrations based on comparison of lipoprotein patterns with clinical and genetic information. Major effort was devoted during the past year to further development of this method and its application to study of more kindreds. The major accomplishments have been as follows: 1. An analytical sequence has been deter- mined that begins with a minimum examina- tion of the plasma by paper electrophoresis in albuminated buffer which segregates nearly all "normals" from "abnormals" and groups the latter in five types (I-V). When neces- sary to achieve firmer segregation of the ab- normal types, cholesterol or glyceride determi- nations, or a simplified quantitation of alpha and beta lipoproteins can be added seriatim. 2. The rational base for this analytical se- quence has been thoroughly tested by compari- sons with other pertinent methods of separat- ing lipoproteins including use of immunochem- ical methods for an absolute definition of lipoprotein content. The information provided by this approach is superior to that obtained by lipid determinations alone, is feasible and economical for adaptation by most clinical laboratories, epidemiologists and geneticists, and cannot be obtained by any other available single method for lipoprotein separations, with the possible exception of the new computer- adapted analytical ultracentrifuge. (A compar- ison is now in progress in collaboration with the Donner Laboratory.) 3. Electrophoretograms have now been ob- tained in more than 1500 subjects; the full analytical sequence has been performed in over 700 subjects. "Normal limits" of distribution for four key variables have been set using 450 normals, and about 3000 inpatient days have been devoted to intensive metabolic study of subjects with ramilial disease for correlation of clinical findings with abnormal lipoprotein patterns. 4. Through study of 120 affected individuals from 45 kindreds, "familial hypercholes- terolemia" has been decisively split into at least two genotypes, which as now redefined are: Type II, the more common form of hy- perbetalipoproteinemia, and Type III, a rare recessive syndrome with a peculiar anomaly in density of beta lipoprotein. Type III, or "broad beta disease", had never before been clearly segregated from among hypercholes- terolemic subjects. It is associated with severe atherosclerosis and is more responsive to therapy than Type II. 5. Increasing experience with Type IV (en- dogenous hyperlipemia) and Type V (mixed hyperlipemia) strongly suggests the presence of more than one mutation, some of fairly high frequency in Americans. For the first time we have detected these heritable abnormalities in children and have laid groundwork for bet- ter means of segregating the defects, including the establishment of normal limits for "carbo- hydrate induction" in terms of glyceride re- sponses to standard dietary loads. 6. Cooperative projects, including the analy- sis in Bethesda of ten samples per week from the "prediabetic" population (defined by max- imum genetic hazard for diabetes) from the 160 ANNUAL REVIEW OF INTRAMURAL RESEARCH Joslin Clinic and all subjects undergoing cor- onary angiography at the Peter Bent Brigham Hospital, have been established. Training in the techniques or other assistance has been provided to representatives of nearly 50 groups in America and abroad desiring to set up the same system. Offering, as it does, better stan- dardization of nomenclature and enhanced recognition of familial or sporadic disorders having a significant association with atheros- clerosis and diabetes, this project is being ad- justed to long-range goals. Studies of the Structure and Function of Lipoproteins The chemical definition of lipoproteins and the functional interrelationships are pertinent to the use of lipoprotein patterns to define clinical disorders of fat transport. The study of heritable deficiencies in either beta lipopro- tein (abetalipoprcteinemia) or alpha lipopro- teins (Tangier disease) has continued to be valuable in this regard. Six patients with abet- alipoproteinemia were shown conclusively to have less than 1/100,000 of the normal amount of plasma beta lipoprotein (probably none at all), and four were shown to be unable to mobi- lize endogenous glyceride as well as from chylomincrons. It was concluded that beta lipo- protein is essential for moving glyceride out of cells. We have previously shown that patients lacking normal alpha lipoprotein can transport glycerides, assigning another function, yet un- known, to these lipoproteins. Using immunochemical methods, it has now been demonstrated in seven patients with Tan- gier disease (from four different unrelated American families) that alpha lipoprotein an- tigenically different from the normal alpha lipoprotein is present. Parents of these patients have both normal and "Tangier" alpha lipo- protein, suggesting that this disease is due to a mutant structural gene. The homozygous ab- normal is thus able to elaborate only a small amount of aberrant lipoprotein that fails to prevent cholesterol ester deposition in tissues. The latter was shown this year to involve even the skin of these patients. Collaborative studies with Drs. Windmueller of NIAMD have provided strong evidence that the action of orotic acid in depressing blood lipids, an effect which he had shown previously, is due to specific inhibition of the elaboration of beta lipoprotein by the liver. Studies of Tissue Lipidoses The discovery by Drs. Brady and Kanfer (NINDB) of a sphingomy elm-cleaving en- zyme was extended to tissues from five pa- tients with Niemann-Pick disease. In all the enzyme was drastically reduced below control levels, strongly suggesting that this is the heritable defect in this disease. With the col- laboration of Dr. Uhlendorf in DBS, 24 cell lines in tissue culture from homozygous ab- normals and an equal number from heterogy- zotes are now ready for enzyme studies. It has been previously shown here that the chemical defect is perpetuated in culture. Protein Structure and Function 1. Efforts to determine the chemical prop- erties, relationship of structure to biological and immunological function, and mechanism of action of parathyroid hormone progressed sig- nificantly during the year. The tryptic peptides of parathyroid hormone were prepared and isolated and their amino acid composition determined. Polypeptide frag- ments from the hormone produced by other methods of cleavage, permitted the alignment of tryptic peptides in order, thereby providing a working model of the covalent structure of the hormonal polypeptide. By a variety of chemical and enzymic methods, limited cleavage of the polypeptide and selective mod- ification of individual amino acid residues were achieved; this permitted identification of an active fragment of the hormone representing one-fourth of the total sequence containing a minimum structure requisite for both biologi- cal and immunological activity. The radioimmunoassay technique has been extended to measurements of parathyroid hor- mone in the plasma of cows, goats, and sheep after stimulation or suppression of hormone production by changes in the concentration of plasma calcium and other ions. The rates of disappearance were estimated by the radioim- munoassay technique, for endogenous hormone NATIONAL HEART INSTITUTE 161 and for administered 131 I-labeled or unlabeled parathyroid hormone. These studies have per- mitted the calculation of the daily secretion rate of parathyroid hormone and the rate of synthesis necessary to maintain constant pro- duction of the hormone. It has been shown that plasma calcium controls parathyroid hormone secretion within narrow limits, hormone con- centration changing rapidly and markedly (changes of 10-15 fold) in response to physi- ologically or pharmacologically induced changes in plasma calcium. It was determined that parathyroid hormone secretion varies in- versely with serum calcium. From the calcu- lated regression line and other observations it is clear that hormone is secreted continuously at a basal level, with further increments in secretion caused by progressive degrees of hy- pocalcemia. It was shown that plasma phos- phate had no direct effect on parathyroid hor- mone secretion. Efforts have continued to develop the radio- immunoassay technique for routine measure- ment of parathyroid hormone concentration in human plasma. Although the technique is not yet satisfactory for this purpose a number of clinical applications have been possible. It has been shown that the parathyroid hormone is produced by nonparathyroid tumors, explain- ing the syndrome of "ectopic hyperpara- thyroidism" in patients with certain types of nonparathyroid malignancy. Further, the de- velopment of antibody causing resistance to the biological actions of the parathyroid hormone has been demonstrated in patients treated for prolonged periods of time with commercial parathyroid extract. As a possible aid in de- velopment of the assay for work in human sub- jects, human parathyroid hormone has been extensively purified after extraction from parathyroid adenoma tissue. It has been found that human parathyroid hormone is closely similar to bovine hormone in its chemical and biological properties. With highly purified human preparations available, the immunolog- ical reactivity of human and beef hormone can be compared. Use of the human hormone might improve the assay for work in human subjects through proper selection of antisera or use of human PTH as tracer. Studies have continued with evaluation of the effects of parathyroid hormone on ion transport in mitochondria. Although initially these studies suggested that the effect of para- thyroid hormone on mitochondria was highly selective and perhaps reflected the true mode of action of the hormone, more recent studies suggest that these effects may not be specific. In view of the great potential interest in the effects of parathyroid hormone on ion trans- location, studies are continuing to correlate the biological activity of derivatives of para- thyroid hormone in vivo with effects on the in vitro system. This should conclusively es- tablish the significance of the in vitro assay systems. 2. Studies of the conformation and immu- nological properties of ribonuclease have been continued in further efforts to understand the role of protein conformation in enzymic and antigenic activity. Derivatives of ribonuclease with minor changes in covalent structure have been shown to have widely divergent confor- mational stability detectable by marked changes in thermally induced denaturation and antigenic reactivity. 3. Collaborative studies of the treatment of cystinuria by use of penicillamine have been extended. Recent studies have further defined the mechanisms of action of the drug. Lack of change in clearance of cystine during treat- ment with the penicillamines and the appear- ance of the mixed disulfide in plasma indicate that the principle site of action is in plasma or gut, rather than in the kidney. The agent N-acetyl-penicillamine has been shown to be an even more promising therapeutic agent. The N-acetyl form of the drug appears to have a reduced potential for toxic reactions in long term use. Section on Chemistry The Section on Chemistiy is mainly con- cerned with structural analysis, synthesis and biosynthesis of compounds of biological origin. The Section also undertakes the development of new analytical techniques for compounds of biological interest. At the present, much of the activity of the group is centered around 162 ANNUAL REVIEW OP INTRAMURAL RESEARCH the mass spectrometer which appears to be generating a great deal of interest among bio- chemical investigators outside the Section. After some early difficulties, it can now be considered a reliable instrument around which biochemical experiments (e.g. isotope labeling) may be designed. Also, its utility in structural analysis is enormous (see below). In July the addition of a high-speed scanning system and tape recorder will add greatly to its value as a tool for structural analysis. Alone, or in collaboration with other groups, members of the Section have this year: 1. Continued synthesis of compounds re- lated to phytol for studies of Refsum's disease, (phytanic acid, phytenic acid, pristane and pristanic acid). 2. Identified pristane and phytane in rat liver. 3. Identified the metabolites of 2 fluoro- benzoic acid. 4. Studied the enzymatic conversion of GTP to 2'-deoxy-GTP and located the label incorporated. 5. Identified an unusual case of hydrogen bonding in a 10-membered ring and studied the conformation of the mole- cule (dihydrotazettine methine al- cohol). 6. Demonstrated the structure of the fla- vone milletine B from an Ethiopian fish poison. 7. Related the structure of a new alkaloid astrocasidine to astrocasine. 8. Proved the structure and absolute stereochemistry of astrophylline and synthesized a related compound. 9. Resolved plasma kininogen (kallidi- dinogen) into two immunologically identical forms, I and II, differentiated by chromatographic and chemical be- havior. Structural work on these com- pounds is in progress. 10. Developed an antiserum to kininogen I to assist in its purification and to an- swer the question whether kininogens I and II are present as such in human serum. 11. Developed a new method of long-range peak matching for mass measurement in the mass spectrometer. 12. Developed a solvent shift method for counting methoxyl groups using nmr spectroscopy. 13. Investigated the hydrogen bonding of ortho substituted benzoic acids. 14. Decided an important structural ques- tion regarding ureasterone. 15. Solved the structure and synthesized a homologue of the uropygiols. 16. Run the highest molecular weight com- pound ever analyzed by mass spec- trometry, C 72 H 24 8 F 28 N 4 P 4 (3628). 17. Developed a new blocking group for ketones. 18. Elucidated the structure of the "Sal- monella Resistance Factor". 19. Completed a mass spectrometric survey of estrogen trimethylsilyl ethers. 20. Elucidated the biosynthesis of a series of methylenebisphloroglucinols and found an in vitro enzyme system capa- ble of bringing about the critical step. LABORATORY OF BIOCHEMICAL GENETICS Nucleotide Sequences of RNA Codons During the past year nucleotide sequences of 17 RNA codons were determined and were assigned to amino acids or special functions. The sequences of virtually all codons have now been determined in this laboratory. The fol- lowing generalizations can be made concern- ing the nature of the code. (A) Amino acids, which are structurally or metabolically related correspond to structurally related RNA codons. (B) The code is logically degenerate. Recogni- tion of the 3'-terminal base in a trinucleotide is most variable and fit several synonym pat- terns. Alternate 3'-terminal bases of synonyms are as follows: U = C; G = A; U; G; U = C = A; and A = G (U). Degeneracy patterns were also found at the 5'-terminal position of trinucleo- tides. For example, U = C in the case of leu- cine and tryptophan, and U = C = A=(G) in the case of N-f ormyl-methionine sRNA. NATIONAL HEART INSTITUTE 163 Mechanism of Codon Recognition The patterns of synonym codons apparently define the characteristics of general codon recognition mechanisms. Purified sRNA frac- tions were used to gain further insight into mechanisms of codon recognition. Yeast Ala- sRNA of known base sequence and estimated to be greater than 95% pure was obtained from Dr. Robert Holley and was found to recog- nize the following synonym Ala-codons, GCU, GCC, GCA, and possibly also GCG. These re- sults demonstrate that one molecule of sRNA can recognize 3, possibly 4, synonym codons, and suggests that recognition occurs by anti- parallel, alternate base pairing between codons in mRNA and an IGC anticodon sequence in Ala-sRNA. Cells often contain multiple species sRNA for the same amino acid. Multiple species of E. coli, Val-, Ala-, and Met-sRNA were sepa- rated by counter-current distribution tech- niques and the response of each fraction to synonym codons was determined. The major peak of Val-sRNA recognized GUA, GUG, and GUU. This peak may represent a composite of two sRNA species, one responding to GUA, GUG and GUU, the other only to GUG. How- ever, a minor Val-sRNA species recognized only GUC and GUU. Ala-sRNAi responded preferentially to GCA and GCG; whereas Ala-sRNA 2 responded to CGU, GCC, GCA, and GCG. Met-sRNAj accepted formyl groups and re- sponded to UUG, CUG, AUG, and less well to GUG. Met-sRNA 2 did not accept formyl groups and responded principally to AUG. N- formyl-Met-sRNA apparently serves as an initiator of protein synthesis; selecting the first word to be read and phasing subsequent reading All of the available evidence suggests that the third (occasionally the first) base of a triplet can hydrogen bond with alternate bases in sRNA. The degeneracy patterns noted above result from alternate base pairing. Characteristics of Synonym RNA Codons Relative template activities and specificities of synonym codons were investigated by as- saying the formation of AA-sRNA-ribosome- codon complexes in reactions containing dif- ferent concentrations of Mg ++ and also in the presence of putrescine and spermidine. Syn- onym codons were found to differ markedly in both template activity and specificity. Also shifts in Mg ++ concentrations had a greater effect upon the template activity of some tri- nucleotides than others corresponding to the same amino acids. The biological consequences of these corresponding to the same amino acids. The biological consequences of these findings remain to be assessed. Some synonym codons may play special roles in protein syn- thesis, such as specifying the beginning or end of the message; others may be necessary for the synthesis of certain proteins or may selectively influence the rate of protein syn- thesis. Codon Recognition on 30 S Ribosomes During protein synthesis on 70 S ribosomes AA-sRNA may attach to both 30 S and 50 S ribosomal subunits. However, codon recogni- tion can occur on 30 S subunits only. Two binding sites for sRNA were found on 30 S subunits. AA-sRNA binding to one riobosmal site was dependent on K + , binding to the other ribosomal site did not require K + . The charac- teristics of each site were determined and com- pared to sRNA binding sites on 70 S ribosomes Attachment of mRNA to Ribosomes 3 H-oligonucleotide templates were synthe- sized and the interaction between such tem- plates and ribosomes were studied. The bind- ing of ^-oligonucleotides of chain length 3 to 9 was dependent upon sRNA. The use of la- beled oligonucleotides provides a highly sensi- tive technique for detecting codon recognition by deacylated sRNA or by a special function sRNA. Template Activty of Modified RNA Codons RNA and DNA contain three classes of codons differing in structure; 5'-terminal, 3'- terminal and internal codons. We previously 164 ANNUAL REVIEW OF INTRAMURAL RESEARCH showed that modification of terminal codons markedly affect template activity. Since such mechanisms may also regulate the rate of pro- tein synthesis in vivo, we have continued to explore the relation between codon modifica- tion and template activity. Trinucleoside diphosphate analogs were pre- pared to assess the effects of such modifications upon codon recognition. Substituting 5' = , 2' = , 3'— terminal or 2'— internal ribose hydroxyls of oligonucleotides markedly affected their tem- plate activity in directing the binding of AA- sRNA to ribosomes. The relative template ac- tivity of oligo U preparations was as follows: p = 5'= UpUpU > UpUpU > CH 3 = p = 5'- UpUpU > UpUpU-3'-p > UpUpU-3'-p-OCH 3 > UpUpU = 2', 3' = cyclic phosphate. Trimers with (2'-5') phosphodiester linkages, (2'-5')- UpUpU and also (2' = 5')-ApApA did not serve as templates for phenylalanine- or lysine-sRNA, respectively. The relative template efficiency of oligo A preparations was as follows: p-5'- ApApA > ApApA >ApApA-3'-p > ApApA- 2'-p. The hexamers, UpUpUpUpUpU and ApAp- ApApApA were considerably more active as templates than the corresponding pentamers. These data indicate that two adjacent triplets are recognized by two AA-sRNA molecules bound to nearby ribosomal sites. A doublet with 5'-terminal phosphate, pUpC served as a template for serine-sRNA whereas a doublet without terminal phosphate, UpC, did not. Al-through the template efficiency of pUpC was lower than that of the triplet. UpCpU the data show that serine-sRNA can recognize pUpC. Universality of the Code Base sequences of codons recognized by AA-sRNA from amphibian and mammalian liver (Xenopus laevis and guinea pig liver, respectively) were almost identical to those recognized by corresponding E. coli AA-sRNA preparations. Thus, synonym codon groups are largely universal. However, the following species-dependent differences in relative ac- tivity of synonym codons were detected: eRNA Codon Bacterial (E. coli) Amphibian (X. laevia liver ) Mammalian (Guinea Pig liver) ARG AGG CGG + + + + + + + + + + + + + + + + ALA GCC GCG + + + + + + + + + + + + + ILE AUA — + + + + LYS AAG + + + + + + + + + SER UCG AGU AGC + + + + + + + + + + + + + + + + + + + + + + + + + + THR ACG + + + + + + + + No differences found for additional synonyms corresponding to above amino acids and for all codons for: ASP, CYS, GLU, HIS, PHE, PRO, TYR, and VAL. It is possible that some species-dependent differences in codon recog- nition may selectively influence the rate of translation of messenger RNA. Regulatory Mechanisms Dependent on Viral Infection Infection of E. coli by T-2 bacteriophage results, within one minute, in the synthesis of a protein which apparently modifies one Leu-sRNA species present in the E. coli host. Simultaneously, host protein synthesis is in- hibited. In collaboration with N. and K. Sue- oka, who reported these phenomena, Leu- sRNA was prepared from phage infected and control E. coli cells and the response of each preparation to codons was determined. The modified Leu-sRNA produced after phage in- fection attached to ribosomes in response to poly UG but not to any triplet containing U or G, or to any other Leu-codon. The following hypothesis was advanced: The modified Leu- codon. The following hypothesis was ad- vanced: The modified Leu-sRNA fraction in- hibits E. coli, but not T-2 phage protien syn- thesis either by preventing the initiation of protein synthesis or by blocking two adjacent sRNA binding sites on ribosomes, and hence preventing further attachment of AA-sRNA to ribosomes. Studies are in progress to define possible consequences of selective modification of com- NATIONAL HEART INSTITUTE 165 ponents required for codon recognition. Par- ticular attention is being focused upon mech- anisms which may selectively control the rate of protein synthesis during viral infection and embryonic differentiation. LABORATORY OF CHEMICAL PHARMACOLOGY The Adrenergic Neurochemical Transducer Application of Steady State Kinetics It has not been generally appreciated that monoamines appear to diffuse from nerve end- ings continuously at a rate proportional to the amine concentration. This is not readily appar- ent since the amine level is maintained con- stant by continuous synthesis. Rates of syn- thesis and efflux are equal, hence K (rate of synthesis) = k Co where C is the amine concentration and k is the rate constant of edux. Proof that efflux of NE is proportional to NE concentration of the normal steady state is obtained from the decline in level after blockade of synthesis with a-methyltyrosine. In this case, d [NE] dt = -k[NE] and [NE] = [NE], exp (-kt) (1) Since synthesis is continuous (and zero or- der) then d [NE] dt K - k[NE] and the general expression for NE level be- comes [NE] = X" ~(x - [NE]„)exp (-kt) (2) After blockade of storage, the amine level does not decline to zero but to a new steady state concentration. The latter can be defined by the equation [NE] = 1~ provided k is assigned a value larger than that for normal organs. The application of kinetics to the drug- induced release of monoamines provides a val- uable tool in disclosing new facets of the behav- ior of nerve endings and the nature of drug action. In this approach, it is assumed that a change in amine level can result only from change in one of the parameters in equation (3), i.e., the rate either of synthesis or efflux is increased. With a releasing drug, the action is best considered as an increased rate constant of efflux. Furthermore, the pharmacologic ef- fects of a number of drugs bear a closer rela- tionship to the increase in rate constant of amine efflux than to the final steady state level. Turnover Rates and Times of Catecholamines and 5 HT ^ At the steady state, K = k C (equation 3). To calculate the rate of synthesis, the rate con- stant k must be determined. For NE, this may be determined: (1) From decline in label after tracer doses of H 3 -NE; (2) From decline in [NE] after blockade of synthesis by a-methyl- tyrosine; (3) Rate of refilling NE stores after depletion by tyramine; (4) From decline in H 3 -NE after tracer doses of H 3 -DOPA. The methods all give similar results. Synthesis of brain 5HT is not readily blocked nor can 5HT be readily labeled. However, at the steady state Rate of synthesis (K) — > 5HT ki 5HIAA loss of 5HIAA hence K = ki [5HT] = k 2 [5HIAA]o . k 2 , the rate constant of 5HIAA efflux id de- termined from the decline of its concentration after blockade of MAO and from the rise after blockade (with probenecid) of the process that transports it from brain. These methods, which yield results similar to those calculated from rise in [5HT] after blockade of monoamine oxi- dase, have the advantage that turnover rates can be determined after 5HT stores are de- pleted by reserpine, i.e., K = k r [5HIAA] r where k r is rate constant of 5HIAA efflux, and [5HIAA] r is the level of acid after reserpine In a simplified procedure for measuring 5HT 166 ANNUAL REVIEW OP INTRAMURAL RESEARCH turnover, each animal is used as its own con- trol by taking advantage of relationship [5HIAAlo ki [5HT] k 2 Provided k 2 is unchanged, an increase in this ratio (normally about 1) signifies an increased 5HT turnover rate. Results of Studies of Turnover Rates The turnover time of brain 5HT is about 1 hr, showing that all the amine in granules is rapidly replaced without use of drugs. In con- trast, the turnover time of brain NE is about 8 hr. The turnover time of NE (as well as 5HT) is similar in various brain regions despite dif- ferences in steady state levels of these amines in different parts of the brain. This implies that regional differences in concentration arise mainly from differences in the concentration of neurons that are essentially alike. The turn- over times of NE in peripheral tissues are also similar despite differing levels. In studies of the origin of NE nerve ending granules, the turnover time of NE in cervical sympathetic ganglion was found to be 6 times that in corresponding nerve endings. This might mean that granules in the nerve body are deficient in a binding component, perhaps ATP. The levels of biogenic amines are controlled in part by product inhibition. Thus after re- serpine, the formation of brain 5HT in rats is increased by about 30% as shown by in- creased steady state levels of 5HIAA. After blockade of MAO, when 5HT and NE levels rise to a plateau, rates of synthesis are almost zero. In view of these observations, it may be necessary to apply corrections to the values for K in the kinetic equations above. A problem arises in explaining why NE in brain of rat, rabbit and cat have about the same turnover time; yet after blockade of MAO, NE in the rat rises rapidly, in the rab- bit slowly, and in the cat not at all. Kinetics of Storage Releasable and resistant pools. Our observa- tions suggest that the common view that NE is stored in tyramine-releasable and tyramine- resistant pools is not valid. When the tyramine level is maintained about 3 [xg/g, NE stores in heart are depleted exponentially to levels too low to measure (T i/2-51 min) as though the amine were confined to a single compart- ment. Since the dissociation of NE from its complex in granules is not a rate limiting step in NE release (see later), the tyramine pre- sumably acts on the presynaptic membrane. Our results with tyramine are inconsistent with our previous studies with H 3 -NE from which it was concluded that the transfer of NE between granules and cytoplasm is the rate lim- iting step. From this it would be inferred that tyramine would deplete NE stores by 50% only after several hr. Improved techniques of H 3 counting made it possible to assay tissue H 3 - NE, after the injection of 100 ng/kg. The level of 1-H 3 -NE in heart now declines as a single exponental (T V2-13 hr), over a period from 2 min to 40 hr. On increasing the dose of H 3 -NE 10-fold, a diphasic curve is once more obtained. In support of these results, H 3 -NE formed in heart and brain after injection of H 3 -DOPA declines as a single exponential during 1 to 40 hr. Thus, H 3 -NE given in truly tracer amounts causes rapid and uniform labeling of endogen- ous NE. It may be concluded that NE rapidly equilibrates between granules and cytoplasm and that the loss of NE from the free pool is rapidly replaced by dissociation of amine from NE complex. The diphasic decline, after more than tracer amounts of H 3 -NE, is associated with an in- creased uptake of the label. The rapid half-life, about 2 hr, of this "excess" NE suggests that it gains access to some part of cytoplasm from which its disappearance is relatively rapid. The Recapture Mechanism Sympathetic stimulation (10/sec) of the colon from cats treated with phenoxybenzamine causes a 6- to 8-fold increase in NE over- flow in the venous effluent. Similar results with- out phenoxybenzamine are achieved by stimu- : lating at 30/sec. These results imply that the ; NE released on depolarization of the nerve ter- minal normally forms a complex with receptor sites, which acts as a brake to diffusion into the general circulation. When the integrity of NATIONAL HEART INSTITUTE 167 the axonal membrane is restored by repolari- zation, NE on receptors is recaptured through action of the pump. If receptor sites are occu- pied by phenoxybenzamine, they no longer hin- der the diffusion of NE, which escapes into the circulation during depolarization, and is no longer available on subsequent repolarization. Thus, the blocking agent augments overflow simply because it interferes with shuttling of NE between receptor and nerve ending. The venous overflow of NE after nerve stim- ulation in presence of phenoxybenzamine pre- sumably represents the amount of transmitter actually released and bound to receptors, and normally re-incorporated into storage sites. Our results show that each nerve impulse re- leases about 70 pg of NE/g of colon. At this rate, amine stores would be exhausted within 30 min, this suggests that the important func- tion of the re-uptake process is to insure that NE stores are not depleted. Mechanism of NE Release by Nerve Stimu- lation The distinctive action of phenoxybenzamine on the re-uptake process makes it a valuable tool in studies of the mechanism that liberates NE from nerve endings. The overflow of NE declines on repetitive sympathetic simulation (10/sec), and ceases within 15 min, though amine stores are reduced by less than 10%. Moreover, most of the release occurs within the first 3 min. A rest period of 1 hr is ade- quate to restore the response to nerve stimu- lation. Since depletion of NE stores by drugs (ty- ramine and reserpine) can be rapid and com- plete, depletion by nerve stimuli must be lim- ited by some step not involved in drug release. This raises the possibility that release of NE by nerve stimulation is limited to vesicles in the vicinity of the synaptic cleft. It is possible that nerve stimulation, which causes influx of free Ca ++ , causes granules to fuse with the neuronal membrane. By this view, nerve exci- tation at a relatively high frequency would re- strict vesicles to vicinity of membrane. In the presence of phenoxybenzamine, these vesicles would be depleted through constant overflow. When stimulation is stopped, the empty vesi- cles at the membrane could gradually be re- placed by Brownian movement of fully loaded ones. Electrolyte Requirements for NE Storage and Uptake Our studies show that Na + is an absolute requirement for NE storage. Thus the efflux of H 3 -NE, when heart slices from rats treated with H 3 -NE are incubated with Krebs' Ringer, similar to that of heart in vivo (T V2-13 hr). Storage is markedly decreased in Na + free media (isotonic sucrose, Li + or choline) as shown by the rapid efflux of H 3 -NE. The out- flow is markedly decreased when sucrose is replaced by various amounts of Na + , approach- ing an asymptote in media containing 40 mM of Na + , as though some Na + dependent system were reaching a maximum rate. These results indicate that Na + is an absolute requirement in storage of NE. K + inhibits the effects of Na + . Thus efflux of NE is rapid in isotonic K + and is reduced by replacing K + by Na + . However, much great- er amounts of Na + are required to reduce the efflux in the presence of K", than of sucrose; Thus about 100 mM of Na + in the presence of 45 mM of K + are needed before the rate of efflux is reduced to an asymptote. K + appears competitively to inhibit the process stimulated by Na + . Ca ++ is also an absolute requirement for NE storage since the omission of this cation ( + EDTA) results in a rapid rate of efflux. Preliminary studies show that heart slices fail to concentrate H 3 -NE in Na + -free media, containing isotonic K + or Li + . These results are of potential importance: (1) They suggest that the active transport of NE is possible be- cause the carrier mechanism has an enhanced affinity for NE in the presence of Na + and reduced affinity in the presence of K + . This view would explain how NE is transported from an outside medium high in Na + to an inside one, high in K + . (2) Many of the pro- posed effects of electrolytes on release or syn- thesis of neurohormones, might well be effects on storage. 168 ANNUAL REVIEW OF INTRAMURAL RESEARCH Kinetics of Reserpine-Induced Release of Bio- genic Amines The action of reserpine on monoamines is closely tied up with the problem of their stor- age. Current opinion proposes two specialized storage mechanisms; one in granules is im- paired by reserpine, the second a membrane pump, is insensitive to reserpine but inhibited by cocaine. As a result nerve ending models proposed by other workers generally include the following assumptions: (1) NE formation is normally controlled by the rate of utiliza- tion; (2) NE is stored in releasable and non- releasable compartments; (3) NE in granules is stored by a specialized energy-requiring process which is impaired by reserpine; (4) NE is still taken up by nerve endings and held for some time after depletion of stores by reser- pine; (5) NE is also taken up by a membrane pump that is blocked by cocaine, which how- ever does not release the amine; (6) Tyramine, metaraminol and other polar phenylethyl- amines release NE by stoichiometric displace- ment from granules. A kinetic analysis of amine release by reser- pine seemed essential. In studies supporting the view that reserpine acts directly on granules, the drug added to a granule suspension pre- vents the uptake of H 3 -NE, but does not release NE. To explain why the amine is not released from granules, the drug is postulated to act by blocking the uptake of dopamine into gran- ules. Depletion of NE is then attributed to fail- ure of synthesis to replenish the physiologic release of the amine. We have shown this veiw to be untenable by showing that the maximal rate at which heart NE is released by reserpine is about 40 times greater than that of synthesis. In applying kinetics to reserpine-induced re- lease of monoamines, we have treated the mem- brane carrier mechanism as a barrier to free flow of amine. After blockade of NE synthesis, even without reserpine, the amine levels de- cline exponentially to zero; reserpine merely increases the rate constant of efflux and in- creases the speed at which the level declines to zero. When synthesis of NE is not blocked, reser- pine decreases the amine level, not to zero, but to the new steady state value defined by [NE] = kC where k describes the slope of the exponential by which the amine declines to the new steady state value. The rate constant is maximal after a large dose of reserpine (5 mg/kg i.v.); the amine stores then decline exponentially almost to zero. The maximum efflux rates are rapid. Half lives are about 16 min for heart NE and about 7 min for brain 5HT, NE and dopamine. Calculation of free amine at nerve endings after complete blockade of storage is 2 to 3% of normal for brain NE and DA and about 12% for 5HT. The higher level of 5HT reflects the greater rate of synthesis of 5HT. The high level of 5HT represents that available to re- ceptors after reserpine administration. The fact that reserpine, given in a maxi- mally effective dose, depletes monoamines at a maximum rate provides a common frame of reference in comparing the effects of reserpine. Thus if a dose of reserpine releases heart NE at a rate less than that corresponding to a half life of 16 min, it may be concluded that stor- age mechanisms are incompletely inhibited. The extent to which reserpine blocks the monoamine pump may be calculated from the ration of the observed rate of amine efflux to that after a maximally effective dose of reser- pine. In testing whether or not reserpine blocks the membrane pump, the drug must be given in maximally effective doses. Few if any stud- ies have shown evidence of this. Our results demonstrate that when H 3 -NE is injected into rats 4 hr after a maximally effective dose of reserpine, little if any label is taken up and retained by adrenergic neurons. Kinetic stud- ies provide a more rigid proof that reserpine completely blocks the membrane pump. H 3 -NE is given to rats shortly after a maximally ef- fective dose of reserpine followed by a MAO inhibitor. A few minutes later, the traces of H 3 -NE in heart disappear with a half-life that j is much shorter than that of endogenous NE still formed in nerve endings, showing that the H 3 -NE was not taken up by nerve endings. NATIONAL HEART INSTITUTE 169 Relationship of Pharmacologic Effects of Res- erpine to Effects on 5HT Storage Since the effect of reserpine on amine efflux is a measure of the blockade of the storage process, the pharmacologic effects of the drug may be compared with the inhibition of stor- age. Since previous studies indicate that block- ade of NE storage does not account for the central effects of reserpine, we compared these effects with 5HT storage. The results show that the pharmacologic effects (including se- dation, blepharospasm, etc.) are closely related to the rate constant of 5 HT efflux and that the most profound effects are elicited by doses of reserpine that alicit a maximum release. Doses higher than this do not increase the rate of efflux or the intensity of drug action. Thus 0.9 and 5 mg/kg of drug both deplete 5HT by about 90%. The higher dose reduces the 5HT level by 90% in about 20 min; the decline after 0.9 mg/kg is much less steep and [5HT] levels off in 1 hr at 75% of normal. In 4 hr however, the loss increases to 90%. False Adrenergic Transmitters A number of phenylethylamine analogues including metaraminol, 1-methyl NE, and octo- pamine are taken up and "stored" by adrener- gic neurons by a saturable process and are re- leased by nerve stimuli and by reserpine. A current misconception holds that these com- pounds release NE by simple physical displace- ment. Our results show that two separate proc- esses are involved. Initially the drug is taken up and retained by neurons, where it shares occupancy with NE; the drug then enhances permeability of presynaptic terminals, possibly by eliciting persistent depolarization. Our re- sults indicate that tyramine and guanethidine are also false transmitters. Studies of the effects of these agents, both in vivo, and on heart slices have provided further insight into their action: (1) Guan- ethidine, tyramine, octopamine and metarami- nol all release NE at the same maximum rate, about one-third that elicited by reserpine. (2) Desmethylimipramine prevents the release of NE by these substances (in vivo experiments). In exploring the possibility that these drugs produce presynaptic depolarization, the effects of guanethidine and K + have been compared. High levels of K + , produce an efflux of NE almost as rapid as that produced by guanethi- dine. The effects of both substances are blocked by bretylium and by excess Ca ++ . Kinetics may also be applied to the release of NE by the false transmitters. In this case the false transmitter may well increase the rate constant of efflux by increasing the poros- ity of the neuronal membrane. The rate would then increase with dose, approaching a maxi- mum as the uptake process becomes saturated. At the new steady state [NE] = -f" In other words, these agents need not reduce the NE level to zero. Even after a maximally effective dose of bretylium or guanethidine, the steady state level is 3 times that after reserpine. Relationship of Pharmacologic Effects of False Transmitters to Rate of NE Efflux In previous studies we showed that the rate of NE efflux is directly proportional to the uptake of guanethidine by adrenergic neurons. From these results it was suggested that the rate of efflux and the sympatholytic effects were both related to the extent of presynaptic polarization. Preliminary results suggest that the rate of NE efflux is also proportional to uptake of metaraminol. Failure of this drug to lower blood pressure may be explained by the direct stimulatory effect of the compound on adrenergic receptors. Effects of Desmethylimipramine (DMI) on Adrenergic Neurons A number of substances, of which DMI is typical, exert little action of their own but are potent antidepressants, block the uptake of catecholamines, and potentiate the actions of NE and amphetamine. Last year we reported that DMI, like cocaine, has a selective action in changing the permeability of adrenergic neurons to NE. Furthermore it prevents the various false transmitters from releasing NE. In continuation of studies of the effects of DMI 170 ANNUAL REVIEW OF INTRAMURAL RESEARCH on neuronal membranes, we have shown that large doses of NE displace H 3 -NE taken up by nerve endings in rats. If animals are first given DMI, the NE is still taken up by nerve endings but fails to displace H 3 -NE. These re- sults suggest that DMI acts largely on granule membranes and interferes with the exchange of the amine between granules and cytoplasm (or neuronal membrane). DMI given to rats whose NE stores are la- beled, reduces the spontaneous efflux of H 3 - NE without lowering the endogenous level. These results suggest that DMI reduces the synthesis of NE. We postulate that DMI by reducing the efflux of NE from granules (which contain little or no free amine) results in accumulation of free NE which then blocks its own synthesis. A possible clue to the antidepressant action of DMI is the marked reduction in the maxi- mum rate of efflux after reserpine administra- tion. Finally DMI added in high concentration to heart slices in vitro causes the release of NE. This supports the view that DMI has changed the conformation of the neuronal membrane. The Serotonergic Transducers Role of Serotonin (5HT) Precise definition of the role of 5HT in brain still eludes us though 5HT and NE transducers can be described in almost the same terms. Few drugs have a selective action on the 5HT transducer. Although our results strongly sug- gest that the action of reserpine is mediated through the continuous release of unbound 5HT, clear proof is still lacking. Attempts are being made to find a role for brain 5HT by measuring the turnover rate of the amine in animals subjected to various physiologic states in the hope that this might disclose whether serotonergic pathways are involved. When rats are exposed to a tempera- ture of 38° C, brain 5HT formation is in- creased by 75%. This suggests that 5HT neu- rons in brain may be involved in control of temperature by dissipation of heat. Investigators at Pfizer & Co. have shown that p-chlorophenylalanine inhibits the syn- thesis of 5HT without eliciting pharmacologic effects nor counteracting the effects of reser- pine. In our hands, large repeated doses of the drug (methyl ester) depletes brain 5HT of rats by 80% and brain NE by 30%. The de- cline in 5HT is definitely related to blockade of synthesis since the 5HIAA level declines and that of 5HT fails to rise after blockade of MAO. The animals are hyperactive but it is not known whether this is correlated with the loss of 5HT. Attempts were made to establish the exist- ence of 5HT stores in peripheral tissues from the uptake of H 3 -5HT in thrombocytopenic rats. Preliminary results suggest that there may be small depots of endogenous 5HT in thyroid, heart, and other tissues. Sympathetic Target Sites Adipose Tissue Transducer System The organism makes constant adjustments to the environment by a unique type of adapta- tion in which the nervous system causes the almost instantaneous activation of enzyme systems. In this regard, adipose tissue cells may be considered as transducer systems in which the input is the sympathetic transmitter and the output is FFA. With these cells, we are particularly concerned with the way in which a physiological signal — a catecholamine — is converted to a biochemical trigger, e.g., cyclic AMP inside the cell. Last year, theophylline, which protects cyclic AMP from inactivation, was shown to be a powerful tool for studies of events in fat cells since its maximum lipolytic effect is 3 times that of NE. These studies showed that the activation of lipase by NE is normally limited by a ceiling in the steady state level of cyclic AMP and that theophylline, by blocking phos- phodiesterase, raise this ceiling causing cyclic AMP to accumulate to a concentration that produces complete activation of lipase. In sup- port of this view, the accumulation of cyclic AMP caused by theophylline was found to be closely associated with the lipolytic response and the blockade of phosphodiesterase. Before concluding that cyclic AMP is re- sponsible for the mobilization of FFA, it must NATIONAL HEART INSTITUTE 171 be shown that the nucleotide itself can actually increase lipolysis. Although the incubation of fat cells with cyclic AMP elicits only a slight lipolytic activity, the addition of theophylline in amounts having negligible activity by them- selves, causes lipolysis equal to that obtained by excess amounts of theophylline. Our studies indicate that cyclic AMP in adi- pose tissue is formed continuously, at a slow rate, in sympathectomized (SX) and adrenalec- tomized (ADX) rats. This indicates: (1) that NE is not necessary for adenyl cyclase activ- ity but merely increases it, (2) that adenalec- tomy does not affect adenyl cyclase, but only the process that activates it. Interaction of Sympathetic and Hormonal Systems An important accomplishment has been the development of a precise method for the assay of adenyl cyclase. With this method, changes in the actual amount of adenyl cyclase may be distinguished from activation of the enzyme produced by NE or ACTH. The enzyme is measured by the rate of production of IP- cyclic AMP 3 from H 3 -APT. Cyclic AMP is sep- arated from labeled contaminants by anion exchange chromatography and by treatment with a BaS0 4 .Zn(OH) 2 gel. The formation of cyclic AMP was shown to be proportional to time of incubation and to enzyme concentra- tion. The nucleotide AMP was identified by isotope dilution of the product itself and of the H 3 -5'-AMP formed by hydrolysis with puri- fied phosphodiesterase. Adipose tissue from hyperthyroid rats is hy- perresponsive to NE and the maximal response to the amine is more than doubled. This is re- lated to the fact that the amount of adenyl cyclase is more than doubled. In contrast, adi- pose tissue from thyroidectomized rats is poor- ly responsive to NE; after treatment of rats with triiodothyronine, the response of the adi- pose tissue is restored. These results indicate that the link between the metabolic effects of the sympathetic and thyroid systems is through adenyl cyclase. Since adipose tissue from euthyroid, hyperthyroid and hypothyroid rats give the same maximal response to theo- phylline, it is concluded that lipase itself is not affected by throid hormone. Adipose tissue from rats treated with corti- sone is also hyper responsive to NE due to an increase in adenyl cyclase. Again the amount of lipase is unaffected. However, the cortisone does not add to the maximum effect produced by thyroxin. The relationship between the per- missive and induction action of cortisone is not clear from these studies. After fasting for 48 hr, the adenyl cyclase is again increased without a corresponding in- crease in lipase. Cortisone, thyroxine, cold-exposure and fast- ing do not enhance phosphodiesterase, in fact the activity of this enzyme is increased by cold-exposure. Adrenergic Blocking Agents Our studies with adrenergic blocking agents in vitro have clarified some of the confusion in classifying receptor sites in adipose tissue. Dose-response relationships indicate that DCI, a beta blocking agent, competitively blocks NE-induced lipolysis but has little activity on theophylline-induced activity. Phentolamine, an* alpha blocking agent, acts at a different site since it blocks the effects of NE and theo- phylline to the same extent. Studies of adipose tissue homogenates indicate that this site is the lipase system itself. Electrolyte Requirements It is difficult to describe the effects of NE on fat cells in classical terms such as depolari- zation, since NE elicits almost as much lipoly- sis when the fat cells are incubated in isotonic sucrose as in Krebs' Ringer solution. The lipo- lytic activity of NE in isotonic sucrose is en- hanced by the action of small amounts of K + . On increasing the [K + ] the lipolytic activity of NE is progressively diminished, and is ab- sent at a [K + ] of 100 to 150 mM. This inhibi- tory effect of K + is not counteracted by Na + . The absence of Ca ++ reduces NE lipolysis by only about 50%. In considering the component parts of the adipose tissue transducer system, drugs may act on adirose tissue in a number of ways: (1) Displacement of NE from receptor sites, 172 ANNUAL REVIEW OF INTRAMURAL RESEARCH (DCI); (2) Interference with membrane de- polarization of cell by NE (adrenalectomy ?); (3) Inhibition of adenyl cyclase (no drug yet demonstrated); (4) Direct activation of adenyl cyclase (catecholamines); (5) Induction of adenyl cyclase (thyroid, cortisone); (6) Inhi- bition of cyclic AMP action; (7) Inhibition of phosphodiesterase (theophylline); (8) Activa- tion of phosphodiesterase (nicotinic acid); (9) Inhibition of lipase system (phentol- amine; (10) Shifts in K + — insulin. Studies on Chemical-Induced Shock Previous reports from this laboratory have shown that ADX rats and rats whose adren- ergic function is blocked fail to respond to ex- ternal stimuli that require an increased expenditure of energy. The failure of epi- nephrine to elicit sympathetic responses in ADX animals indicates that their incapacity to withstand cold or strenuous work results from failure of sympathetic target organs to respond to transmitted messages. Communications are re-established by aldosterone as well as gluco- corticoids, suggesting that the inexcitability of adrenergic receptors after adrenalectomy is related to changes in electrolytes. We are now studying substances, categori- cally classified by Selye as stressors, and known to be much more toxic in ADX than in normal animals. We hope that the study of shock, in- duced by drugs whose mechanism of action is known, might lead to a better understanding of clinical shock. We started this study with the limited objective of establishing whether the toxicity of these stressor agents is enhanced in SX animals as well as in ADX animals. Our results show histamine and endotoxin, in doses that are not lethal to intact rats, are almost 100% lethal to both ADX and SX rats. Treatment of ADX rats with a glucocorticoid or with epinephrine-in-oil provides partial pro- tection against the lethal effects of histamine and endotoxin; complete protection is provided by giving both substances. Treatment of SX rats with epinephrine, alone, provides complete protection against the lethal effects of histamine and endotoxin. Both histamine and endotoxin produce a blood pressure drop presumably by dilating the small blood vessels. The lethal effects of these substances in ADX and SX animals might stem from their action on the microcirculation. In normal animals, protection against hypo- tension is provided by signals sent to the brain via the baroreceptors, and the resultant adrenocortical discharge. In SX animals, there are no catecholamines to be discharged; in ADX animals, the catecholamines have little effect on the sympathetic system in the absence of glucocorticoids. The lethality of formalin and tourniquet trauma is also greatly increased in ADX rats. Thus doses of formalin or a degree of tourni- quet trauma that cause no deaths in control animals are 100% lethal in ADX rats but the toxicity is not enhanced in SX rats. Moreover, treatment of ADX rats with a glucocorticoid provides complete protection against the lethal effects of formalin and trauma. DOCA also affords considerable protection. Thus the lethal effects of formalin and tourniquet trauma are not mediated by the circulation. The results are consistent with the view that formalin and tourniquet trauma produce a toxic agent which is responsible for the toxic effects. Glucocorti- coids would act by preventing the formation of this agent rather than by overcoming its effects. In support of this view, is the well- known fact that the lethal effects of tourniquet trauma occur only after removal of the tourni- quet. The Nonmast Cell Histamine Transducer All tissues contain considerable amounts of histamine not in mast cells; in fact in some species including rabbit, cat (except for skin) and man, mast cell stores comprise only a small fraction of total histamine in the body. Last year we reported results which have led us to the provisional conclusion that nonmast cell histamine mediates exocrine secretions. Selective Labeling of Nonmast Cell Histamine The view that parenterally administered H 3 -histamine selectively labels nonmast cell stores has now been fortified by studies show- ing that the specific activities of histamine in rat gastric mucosa and cat salivary gland are NATIONAL HEART INSTITUTE 173 almost indentical with those of amine released by cholinergic agents into saliva and gastric juice. From these results it may be inferred that the decline in radioactivity accurately re- flects the endogenous turnover of histamine and that the release of histamine can be calcu- lated from the release of radioactivity. The assay of H 3 -histamine by isctopic dilu- tion has shown that the second part of the diphasic disappearance of radioactivity is arti- factual and results from tritiated water pres- ent as an impurity in the injected H 3 -hista- mine as well as from that formed by exchange in the bcdy. Corrected values now reveal that turnover of H 3 -histamine in various exocrine glands corresponds to a half-life of about 1 hr and in skeletal muscle of about 4 hr. Distribution and Fate of Injected H '-Histamine In rats, the highly polar metabolite present in various tissues has been identified as a con- jugate, presumably the riboside, of histamine itself and not of the acid as reported by others. In the cat, the uptake of H 3 -histamine is also high in exocrine glands and occurs to some extent in all tissues. In exocrine organs H 3 - histamine is rapidly converted to methylhista- mine which is also retained by tissues. H 3 - histamine in the cat is excreted in 2 phases; at first the urine contains considerable amounts of free histamine and methylhistamine. The rapid decline in the excretion of these amines coincides with their disappearance from plas- ma. At this time, the urinary excretion of the acid metabolites remains high. Since studies with tissue slices show that deamination oc- curs largely in kidney and liver, these results suggest that the bases are released into blood largely unchanged and are then deaminated by kidney and liver. Methylhistamine injected into the submaxil- lary gland, intra-arterially, elicits some saliva- tion; placed in lumen of cat stomach it elicits gastric secretion. These findings raised the question whether methylation of endogenous histamine leads to its inactivation or whether the substance is normally stored and has a physiological role. Various cat organs contain considerable amounts of a substance that, like methylhista- mine, is chloroform extratcable, deaminated by diamine oxidase, and reacts with dinitro- fluorobenzene to form a derivative with a similar R f value. However, mass spectrogra- ph^ (Dr. Highet, Lab. of Metabolism) of the substance indicates that it is not methylhista- mine. The distribution of the "apparent" methylhistamine is of interest since it is high in brain stem and certain exocrine glands but absent from cerebellum and skeletal muscle. The specificity of the histamine uptake mechanism was studied by measuring the uptake of H-methylhistamine in rats. The pat- tern of uptake and disappearance of methyl- histamine closely resembles that of H 3 -hista- mine. From current evidence we favor the view that methylhistamine is not normally formed and retained, and that exogenous methylhista- mine may act like a "false transmitter". Release of H '-Histamine Histamine appears to have an important role in exocrine glands. Thus stimulation of parasympathetic nerves or administration of acetylcholine or its analogues cause the release of histamine into the secretions of submaxil- lary gland, gastric mucosa, bile and pancreas. Pilocarpine also causes a marked efflux of H 3 - histamine into these secretions. Gastrin and a factor in saliva cause the efflux of H 3 -hista- mine into gastric juice. Secretin induces the efflux of H 3 -histamine into bile and pancreatic secretions. Finally preliminary results indicate that stimulation of skeletal muscle is associated with the release of H 3 -histamine. Synthesis of Histamine Last year we reported evidence that the syn- thesis of histamine in the salivary gland was regulated by utilization. Additional studies show that after ligation of the esophagus to deprive the rat stomach of the stimulatory ef- fects of saliva, gastric secretion is reduced by about 90% and the turnover of histamine by about 75%. 174 ANNUAL REVIEW OF INTRAMURAL RESEARCH Factors That Affect Drug Action Passage of Substances Across Membranes Central nervous system. Since the pharma- cologic effects of polar drugs are often studied after intraventricular injection, it is impor- tant to understand the nature of the boundary between ventricles and brain substance. After intraventricular injection, C 14 labeled inulin, sucrose and mannitol enter brain tissue at rates roughly proportional to their diffusion coefficients in agar gel of infinite thickness. Such studies might make it possible to esti- mate the fraction of the ependymal surface permeable to large lipid-insoluble molecules. Further studies have been made of the ac- tive transport system in rat brain which trans- fers 5HIAA directly from brain to blood. Last year it was reported that this process is com- pletely blocked by probenecid. The transport system is also blocked by dinitrophenol given in hyperthermic doses. In addition it is mark- edly impaired when rats are exposed to a tem- perature of 38° C for 16 hr. It is possible that such impairment is a sensitive indicator of brain damage. Intestinal absorption. Highly liposoluble sub- stances like DDT and dieldrin are absorbed in part via the lymphatic circulation of the in- testine. The substances are absorbed by the fat transport process of the intestinal epithelium, presumably due to their association with lipid micelles. Biliary excretion of drugs. Carboxylic acids, sulfonic acids and chlorothiazide are secreted into bile by the same transport mechanism. Probenecid, a carboxylic acid, which markedly inhibits the biliary secretion of organic acids, is itself excreted in bile and appears to act competitively. The choleretic activities of pro- benecid and chlorothiazide are indirect effects stemming from the large quantities of water they take into bile by their osmotic effects. Ouabain, an active inhibitor of many trans- port mechanisms, is itself secreted into bile by a process that differs from those which secrete organic acids and bases. In addition, ouabain is also taken up by liver slices by an active transport process. Thus the liver transports organic com- pounds by at least three general processes; one for acids, one for quarternary and per- haps tertiary amines, and one for ouabain and presumably other glycosides. Enzymatic Mechanism of Membrane Transport Research is directed toward a molecular- physiologic explanation of processes carried out by cell membranes. Two sorts of processes await explanation: (1) the active transport mechanisms which utilize energy to move ca- tions, glucose, amino acids, catecholamines and other substances across various cell mem- branes; (2) processes by which nerve stimuli induce transient changes in conducting mem- branes and neurohormones produce transient permeability changes at receptor sites. It is hoped that studies of the Na + , K + , ATPase from beef brain will provide a clue to the mechanism by which ATP energy is used for cation transport. Attention has been shifted to the active center of this enzyme. Previous work has identified as an acylphos- phate, a phosphorylated intermediate whose formation is catalyzed by Na + . We have at- tempted to determine the concentration of transport sites in the membrane by "trapping" the acylphosphate as a stable hydroxamate. The results suggest that Na + might cause two effects: (1) a reaction with ATP to yield the protein-bound acylphosphate and (2) a con- formational change that protects the acyl de- rivative from reacting with hydroxylamine. Attention is now focused mainly on the K + - stimulated phosphatase step as the point where most of the ATP energy is utilized for the transolocation of ions. We have sought artificial substrates which interact specifically with the K + -sensitive step. p-NPPase which catalyzes the K + -stimulated hydrolysis of p-nitrophenylphosphatase resembles ATPase in certain respects; after adrenalectomy the ac- tivities of both enzymes in kidney are de- creased and are restored by corticosterone. Differences in ouabain inhibition, however, show that the two enzymes are different. p-NPPase may provide a model for the study of Reactivated systems; more recent studies suggest that acetylphosphate may serve as a NATIONAL HEART INSTITUTE 175 substrate for the K + -sensitive step of the gen- uine transport ATPase. Studies have shown that aldosterone does not act by changing the synthesis of trans- port ATPase. Thus aldosterone exerts drug effects on transport in the toad bladder but has no effect on ATPase or on enzymes in- volved in ATP synthesis. It is possible that aldosterone induces the synthesis of a mem- brane component controlling the diffusibility of Na + . An attempt is underway to identify adrener- gic receptors by labeling with covalently fixed blocking agents. Experiments in connection with these studies have shown that reserpine produces a 5-fold increase in submaxillary glycoprotein in the denervated as well as in the innervated gland. The possibility that this action is mediated by the pituitary-adrenal system is under investigation. Enzymatic Mechanisms of Drug Metabolism Enzyme processes. Microsomes contain a cytochrome which in its reduced form com- bines with CO to form a complex; the cyto- chrome is called P-450 because the complex with CO has an absorption maximum at this wavelength. P-450 in microsomes is reduced by TPNH and then reacts with 2 to form an "active 2 " complex which transfers 2 to various drugs. In the absence of drug sub- strate, this complex breaks down to H 2 2 . P-450 also participates in the anerobic re- duction of nitro and azo compounds. These general reactions are illustrated below TPNH cytochrome C — reductase TPNH > P-450 > O2-P-450 — > Oxidation X I of Drug Reduction of H 2 2 nitro and azo drugs P-450 is a component of the TPNH-depen- dent enzymes in microsomes that are responsi- ble for the hydroxylation, dealkylation, deam- ination and desulfuration of drugs and for at least one enzyme that catalyzes sulfoxidation. TPNH cytochrome c reductase is now im- plicated as a component of P-^450 reductase by studies showing that the reactions which utilize drugs and TPNH-cytochrome c re- dictase have practically the same Km's with respect to TPNH. In addition, 2'-adenosine phosphate inhibits the reductase and the drug reactions to the same degree. Small but reproducible changes in the ab- sorption spectrum of liver microsomes are pro- duced by substrates and inhibitors of drug- metabolizing enzymes, even in the absence of TPNH. These changes are of two types. In presence of substrates, the dissociation con- stants for these changes closely agree with Km's (substrate) of the oxidation reactions, suggesting that they represent the formation of enzyme-substrate complexes. Substances that inhibit drug oxidation appear to act either by displacing substrate from the enzyme or by interfering with the flow of electrons between TPHN cytochrome c reductase and P^450. Induction of microsomal phospholipid by phenobarbital. The increased oxidation and reduction of drugs produced by phenobarbital and other foreign compounds is associated with increased amounts of protein and phospholipid in liver microsomes. The increased content of protein can be explained by an increased rate of protein synthesis. However, studies of the incorporation of P-32 suggest that the in- crease in phospholipid is caused by a decrease in phospholipid catagolism. Mechanism of Teratogenesis by Thalidomide The mechanism of thalidomide-induced tera- togenesis in laboratory animals, is still un- known. We have now shown that about 3% of the thalidomide in rabbit fetuses is bound to tissues irreversibly, mainly with nuclear RNA and DNA. This incorporation may be a characteristic of rapidly developing tissues, for 2 to 3 times as much labeled thalidomide is incorporated into nuclear RNA and DNA of rapidly growing rat liver (after partial hepatectomy) as in the liver of normal rats. These findings make it likely that thalidomide causes teratogenicity by acylation of RNA and DNA or its precursors and suggest that alky- lating agents and thalidomide cause tera- togenic effects through similar mechanisms. 176 ANNUAL REVIEW OF INTRAMURAL RESEARCH Such a reaction would explain our findings that differences in the embryo toxicity of thalidomide in the rabbit and the rat are minimized when the drug is administered in- travenously since the effects are likely to be related to a high level of drug action over a short time. By giving the drug intravenously, reproducible effects are obtained in rabbits with doses ranging from 2.5 to 10 mg/kg and in rats with doses of about 10 mg/kg. Pharmacogenetics The problem of individual variability in re- sponse to pharmacologic agents is receiving much attention because increasingly large populations are being exposed to drugs. Since each of the multiple enzymes that control drug metabolism is genetically controlled, mutations may lead to toxicity through accumulation of the drug (acatalasia, atypical cholinesterase and slow isoniazid inactivation). Our studies are focused on the basic ques- tion whether inbred animals respond more uniformly to a drug than outbred animals (Fi generation hybrids). The total variability of the strain is reduced, providing that genetic components of variation, such as color and antigenic variation predominate over environ- mental effects. Reports on responses to bar- biturates are conflicting. Some claim that the response of inbred mice is more variable than that of outbred mice, others that it is less. Our results show that hexobarbital elicits a sleeping time that, in some inbred strains of mice, is more variable than in outbred mice, whereas in others it is equal and in still others it is less. These variations are attributable largely to differences in rate of drug metab- olism. On recovery of righting reflex, the brain hexobarbital level is similar in all strains, indicating that the CNS sensitivity to the drug is remarkably uniform. Strain differences in hexobarbital metabo- lism may be related to participation of multiple forms or the isozymes of a given enzyme. Lac- tate dehydrogenase (LDH) which exists as 5 isozymes in most body cells, provides us with a model of how these forms result when 2 dis- similar subunits combine randomly to form active polymers. The synthesis of the sub- units may be controlled by gene activity, but in addition the catabolism of enzymes may be an important variable in determining distribution. The relative stabilities of Pure LDH 1 and 5 were studied as possible variables in their bio- logical regulation. Stability to heat was differ- entially altered by pH, ionic strength, NADH, oxaloacetate, malate, and fructose-1-6 diphos- phate. The results show that substances may be fixed to allosteric sites thereby protecting the molecule from inactivation or accelerating heat denaturation. Three distinct allosteric sites have been identified on LDH 1 and 5. Clinical Studies with Desmethylimipramine An NIH-sponsored collaborative study with the Karolinska Institute is providing a par- tial answer to an important question: Are in- dividual differences in metabolism of liposolu- ble drugs used in treatment of mental diseases large enough to account for individual differ- ences in side effects and efficacy. In this study, plasma levels of the antidepressant drug des- methylimipramine (DMI) are determined by the isotope derivative technique dveloped in this laboratory (see methods) by Dr. W. Ham- mer, a participant in this project. The results indicate: 1. Plasma levels of patients on the same dosage regimen differ by as much as a factor of 20. 2. Thus far, side-effects are seen only in those patients who metabolize the drug slowly and who have the highest plasma level. 3. In general therapeutic effects on depres- sion are associated with the concentra- tion of DMI in the plasma. 4. Patients shortly after treatment with bar- biturates have very low levels of DMI. 5. The side-effects in two patients were characterized as "extreme anxiety". No detectable DMI was found in the plasma of these subjects and subsequent studies have shown extreme anxiety to be a re- 6. The psychiatrists collaborating in this re- search now insist on plasma levels of flection of lack of treatment. NATIONAL HEART INSTITUTE 177 DMI; for the first time they feel that they can control therapy and toxicity. Development of New Methods of Analysis The ever increasing potency of modern drugs, especially those used in the treatment of mental disease, has created the need for more methods of higher sensitivity for the assay of organic compounds. The isotope derivative technique, described in last year's report has been applied to the routine assay of desmethylimipramine and has yielded particularly rewarding results. This method is based on the extraction from plasma of desmethylimipramine and its subsequent acetylation with H 3 -acetic anhydride of high specific activity. The method has been modified so that as little as 5 ng/ml of drug can be accurately measured. A potentially simpler approach for the rou- tine assay of drugs in ng amounts is the use of gas liquid chromatography with an electron capture detector. This sensitive device responds to organic compounds containing halogen or nitro groups at levels of about 10 ~ 15 M. If a drug does not contain these groups they may be introduced by forming a derivative of the compound after its extraction from plasma. A method for assay of amphetamine, the- oretically applicable to other aliphatic pri- mary amines, involves condensation with Dansyl chloride (the demethylamino analogue of naphthalenesulfonychloride) to yield a highly fluorescent derivative. A method has been developed for the assay of methylhistamine in biological tissues. In this procedure methylhistamine is assayed as the dinitro-fluorobenzene derivative. NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES Introduction The year began with Dr. Leon Jacobs con- tinuing as Acting Scientific Director. In late September Dr. John R. Seal joined NIAID as Associate Director for Intramural Research. Dr. Seal had retired from the United States Navy on September 1, 1965, after serving in various capacities in the Navy medical re- search program during the major part of his military career. Dr. Jacobs remained as a con- sultant to the Director, NIAID, until January 1, 1966, when he retired from the Commis- sioned Corps of the U.S. Public Health Service and joined the Division of Biologies Standards NIH. These transitions had little effect on the momentum of the broad-based intramural re- search program. More serious in prospect was the retirement or resignation of other pro- gram leaders, and as the year closed the In- stitute was recruiting for a new Clinical Di- rector with the need to restaff the Laboratory of Clinical Investigations to a large degree, facing needs to restructure and redirect its research effort related to tropical medicine in- volving changes in the programs in tropical virology and parasitology, and seeking leader- ship for the medical mycology research pro- gram. Despite these unsettling influences, the year was a remarkably productive one with a con- tinuing output of scientific publications re- flecting the leadership role the NIAID Intra- mural Research Program holds in many aspects of infectious diseases and immunology. Dr. Karl Habel was selected to give the An- nual Dyer Lecture at the National Institutes of Health. Drs. Habel, Leon Jacobs, Robert Hueb- ner, and Robert Coatney were awarded Dis- tinguished Service Medals by the United States Public Health Service for their scien- tific contributions. Dr. Maurice Landy received the DHEW Superior Service Award. Many other honors were afforded the scientific staff as are detailed in the reports of the individual laboratories. A new parameter was added to the responsi- bilities of the NIAID Intramural Research Program with the assignment of Chairmanship of the NIH Cholera Advisory Committee to Dr. Seal. This responsibility includes the sci- entific management of the SEATO Cholera Research Program and the Pakistan-SEATO Cholera Research Laboratory in Dacca, East Pakistan. The SEATO Cholera Research Pro- gram was developed as a result of the spread of cholers in Southeast Asia, the Pacific, and westward from its traditional endemic focus beginning in 1958, and includes the sponsorship of scientific symposia for exchange of infor- mation, grants, training contracts, and re- search in cholera in SEATO member countries with basic funding by the Agency for Inter- national Development (AID) and scientific management by NIH as a result of several AID-DHEW, AID-NIH interagency agree- ments. The Pakistan-SEATO Cholera Research Laboratory in East Pakistan is sponsored under the umbrella of the Program and a government of Pakistan-AID agreement but receives addi- tional funding and support through a NIH- PL 480 research grant and from several SEATO member countries for conduct of chol- era research. Among its more notable contri- butions during this year have been informa- tion on the high degree of effectiveness of United States manufactured cholera vaccine in the prevention of cholera, the long duration of vaccine induced immunity, particularly in 179 180 ANNUAL REVIEW OF INTRAMURAL RESEARCH adults in cholera endemic areas, added data on the value of tetracycline in the therapy of cholera along with comparative data on the lesser effectiveness of several other antibiotics, new information on non-cholera diarrheas and on a malabsorption syndrome which occurs in Americans subsisting on a local diet, and rec- ognition of hypoglycemia as a complication of cholera in infants. The Board of Scientific Counselors of NIAID was asked to undertake a review of the entire viral research program. Departing from the usual custom, three meetings were held dur- ing the year, one at the Middle America Re- search Unit (MARU), Canal Zone, Republic of Panama, and another meeting featuring a re- view of the "slow" or chronic virus disease research sponsored by the NINDB, the Ex- tramural Research Program of NIAID, and the Rocky Mountain Laboratory. One important area in which the advice of the Board had been sought was that of arbovirus research. The Board emphasized the National needs for NIAI intramural research to continue a strong pro- gram in arbovirus research and recommended that the excellent small program at the Mid- dle America Research Unit (MARU) be strengthened and extended both by the as- signment of a more broadly based mission and staff to MARU and by more active scientific collaboration with the Gorgas Memorial Lab- oratory and university-based research groups. The Board also recognized the leading role of the Rocky Mountain Laboratory in research on animal models of slowly developing, progres- sive viral infections causing neuropathies, pul- monary, vascular, and renal disease, the prob- able relevance of the work to certain human diseases, and the probable inability of aca- demic institutions to play any prominent role in this type of research during the next dec- ade. It recommended strengthening of the viral research competence of the Rocky Mountain Laboratory and installation of new facilities to permit inoculation of material of human ori- gin, particularly brain specimens from hu- mans dying of "Kuru," into the animal species being studied. The importance of the Intramural Research Program to the Collaborative Research Pro- gram of the NIAID was more evident as the demands on the intramural scientific staff in- creased. The Laboratories of Infectious Diseases and Tropical Virology undertook the testing of many of the reagents being developed in the Research Reference Reagents Branch, pro- vided seed stocks of viruses, developed new methodology and trained some contractor per- sonnel in their use, site-visited contractors' in- stallations, and provided informed members to advisory committees. The Laboratory of Infec- tious Diseases was especially involved in the Vaccine Development Branch with several of the staff making important contributions as project officers and participants in the field testing of new vaccines such as the oral adenovirus type 4 vaccine and parainfluenza vaccines, research for further definition of the oncogenic potential of various adenoviruses, initial testing of vaccine against mycoplasma pneumoniae, developments in methodology, and advisory services to the Vaccine Development Committee. The Laboratory of Immunology pro- vided very similar support to the Transplan- tation Immunology Branch. The variety and scope of the Intramural Re- search Program is illustrated in the selected examples of research which are summarized in the following pages. LABORATORY OF CLINICAL INVESTIGATIONS A major emphasis of the laboratory has been in the further study of respiratory virus dynamics in the infection of human volun- teers derived from the Federal prison system. Other research on the mechanisms of fever and host responses in various syndromes, in immunogenetics, in parasitology, and in lep- rosy has continued. Transmission Dynamics in Respiratory Infections Using Coxsackie A-21 virus as a model, ex- tensive studies were made in human volun- teers of transmission dynamics. Quantitative studies were carried out on the presence of virus in respiratory tract secretions, the role of breathing, speaking, coughing and sneezing NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 181 in release of viral particles to room air, the effect of particle size in creation of viral aero- sols which remain in suspension in room air for long periods of time and on the transmission of infections via the airborne route to other volunteers. Coughing- was shown to be the most important source of airborne viral particles and human-to-human transmission via the air- borne route conclusively demonstrated. Air- borne transmission of Coxsackie A-21 virus infections between artificially infected volun- teers and other non-immune volunteers under dormitory conditions was also demonstrated. Virus was recovered from the dormitory air by use of large volume air sampling tech- niques during these trials. Remarkably small doses of virus were infec- tive when aerosolized in small particles and this was particularly true in adenovirus in- fections. The human infectious dose (HID 50 ) was about 30 TCID 50 for Coxsackie A-21, 1 TCID, a for adenovirus type 4, and 0.68 TCID S „ for rhinovirus. Even smaller doses given by nasal drops caused similar illness in the case of Coxsackie virus and rhinovirus. A larger dose of adenoviris by nasal drops was neces- sary to infect and only the aerosol route regu- larly produced typical illness. Antibody in Nasal Secretions and Serum By challenge experiments in volunteers the apparent greater importance of 9S-14S anti- body in resistance to rhinovirus infection than 7S or 19S antibody was demonstrated. 11-14S antibody was shown to appear in nasal secre- tions, tears, and saliva after its first appear- ance in sera and was associated with only yA immunoglobulin in these secretions. This evi- dence strengthened the hypothesis that yA antibody in secretions results from an active selective transport mechanism between serum and secretion but does not rule out a possible local production of antibody. Chilling and Respiratory Infection Controlled experiments were made on the effects of chilling with and without an actual decrease in body temperature on the course of rhinovirus infections. No evidence could be obtained that chilling before, during, or sub- sequent to the clinical disease in any way al- tered its course. Adenovirus Soluble Antigens Purified hexon and fiber antigens from ade- noviruses types 1 and 4 were administered to volunteers with good neutralizing antibody re- sponse in a high percentage of subjects. The 7S antibody response was similar in degree and duration to that observed in human sub- jects given infectious virus. Influenza Influenza was produced in volunteers with three TCID- )0 doses of A2 virus when given as an aerosol. Low levels of homologous neutraliz- ing antibody did not protect against infection with this small dose nor did heterologous anti- body in higher titers. A/Equi 2 virus caused a high incidence of infection but infrequent illness in human volunteers. Serial passage of equine influenza virus in man did not increase its pathogenicity for man or decrease its path- ogenicity for equines. Their findings suggested that A/Equi 2 virus is rather host specific and that horse-to-man transfer under natural conditions is probably a rare event. Mechanisms of Fever and Host Responses Extensive studies are in progress relating to the pathogenesis of fever, reticuloendothelial function and immunological reactivity of hu- mans and experimental animals. Investigations are being carried out in patients with famil- ial Mediterranean fever, recurrent fever of un- known etiology, leprosy, a variety of neoplasms and normal volunteers. Through utilization of a specific and sensi- tive assay for etiocholanoalone, it has been shown that increased plasma levels of this steroid are not correlated with febrile episodes in a variety of patients. The administration of this pyrogenic steroid is proving useful as a tool for studies of bone marrow reserve, granu- locyte kinetics and fever. A bentonite flocculation test was developed for the demonstration of circulating anticryp- 182 ANNUAL KEVIEW OF INTRAMURAL RESEARCH tococcal antibodies in patients with cryptococ- cosis. Patients with Hodgkin's disease have an en- hanced phagocytic capacity. Likewise, patients with lepromatous leprosy have an increased reticuloendothelial clearing capacity in addi- tion to a variety of immunological defects. It was also shown that passively transferred endotoxin tolerance persists for at least 3 weeks despite rapid decay of passively trans- ferred antibody. Systemic Fungus Infection Attempts to improve chemotherapy of sys- temic mycoses met with mixed success. In vitro tests of Nocardia suggested two potentially useful antibiotics: ampicillin and capreomycin. A combination of amphotericin B and hy- droxystilbamidine appears promising in the treatment of mice infected with North Ameri- can blastomycosis. Intraventricular adminis- tration of amphotericin B by use of a new prosthesis has not been encouraging. Hamycin, a new antifungal antibiotic, was dropped from clinical trials because of poor chemotherapeu- tic effect. Factors affecting the cure of cryptococcosis with amphocericin have been the subject of continuing study. The role of immunologic paralysis, the occurrence of delayed hyper- sensitivity, and the means of adaptation by Cryptococcus to chemotherapy have been eval- uated with respect to their effect on prognosis. Leprosy The leprosy program continued with the evaluation of rimino compound B.663, in the treatment of lepromatous leprosy. The patient under study is of particular interest because he had received no prior or concurrent sul- fone therapy. Increasingly precise bac- teriologic techniques have confirmed the anti- biotic potency of B.663 against the leprosy bacillus. In addition, experience suggests that B.663 possesses an anti-inflammatory action against erythema nodosum leprosum reactions. These reactions constitute the most objectionable fea- ture of sulfone therapy, which is currently standard, and B.663 may prove to have wide application used in combination with the sul- fones. Laboratory of Infectious Diseases The efforts of this laboratory continue to be focused in several major programs: 1. Cancer and leukemia virus studies. 2. The viral and mycoplasma causes of acute respiratory diseases. 3. Rubella. 4. Epidemiology of picoranaviruses and eosinophilia meningitis in Oceania. 5. Bacterial metabolism and physiology. 6. Medical mycology. The virus research groups work in close re- lationship with the National Cancer Institute on the problems of oncogenic viruses and with the National Institute of Neurological Diseases and Blindness on Rubella and related problems. Adenoviruses as Oncogenic Agents Eight of 31 human adenoviruses have been shown to cause cancers in hamsters (types 3, 7, 12, 14, 16, 21, and 31). The first and chief indication that the tumors induced by adeno- virus inoculation were actually directed by the adenovirus genes was the discovery of non- virion complement fixing (CF) antigens in the virus-free tumor cells. Similar nonvirion anti- gens, now called T antigens or neoantigens occur in cells infected with adenoviruses. These antigens were shown to be new proteins which were synthesized by the input virus before new viral DNA appeared. The T antigens there- fore represent a revolutionary new category of virus induced antigens. Many different lines of research here and in other laboratories con- firmed these findings not only for adenoviruses but for other DNA viruses, such as SV40 and polyoma. Tumor and T Antigens as Possible Determinants of Adenovirus Oncogenesis The presence of virus-specific but non-virion T antigens in cells infected and/or trans- formed by human adenoviruses were confirmed for oncogenic strains of simian, canine and NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 183 bovine adenoviruses. Thus, the regular pres- ence of T antigens in adenovirus tumors sug- gested that such antigens may be determinants of oncogenesis, a hypothesis that was supported by remarkable correlations between groupings of the adenoviruses according to oncogenic po- tentials, Green's G + C DNA base composi- tions, Rosen's hemagglutinins (HA), and T antigens. Adenovrus-SV40 Hybrids Exciting new discoveries on the transfer of SV40 and adenovirus genetic information from the adenovirus type 7-SV40 hybrid to other adenoviruses were made. Not only was the SV40 genome transferred from type 7 to type 2, but the genome of type 7 responsible for stimulating production of the virus specific T antigen was transferred as well. These obser- vations proved without question that viral DNA was responsible for the synthesis of the non-virion T antigens. Adenovirus-Associated Viruses (AAV's) A new defective virus, dependent entirely on adenoviruses acting as helpers for replica- tion, was discovered almost simultaneously by workers at Baylor University, The University of Pittsburgh and the Laboratory of Infectious Diseases. The AAV virus was found to be the smallest of the known viruses, measuring no more than 15 m^ in diameter. Its DNA was shown to be double stranded and to have a base composition much different from that of the adenoviruses from which they were de- rived. Also, recently discovered were two addi- tional serologically distinct AAV's. The AAV's are quite unique among mammalian viruses; however, similar wholly dependent "satellite" viruses are found among the plant viruses. Leukemia Extensive studies with several animal models continued. It was shown that infectious virus was obtained when hamster tumor cells result- ing from Bryan Rous Sarcoma Virus (defec- tive virus) were grown in mixed culture with chick embryo fibroblasts. When such mixed cul- tures were injected into RIF-free chicks, non- infectious sarcomas resulted which had the karotype of chicken cells and CF antigens of the Rous Sarcoma cells. Tissue cultures de- rived from these sarcomas remained free of in- fectious virus until an avian leucosis virus was added, following which Rous Sarcoma Virus appeared. In other studies, a quantitative test for murine leukemia viruses was developed as was an in vitro assay system for mouse (Mo- loney) Sarcoma Virus. Vaccine Against Mycoplasma Pneumoniae A protective effect of a formalin inactivated M. pneumoniae vaccine was demonstrated in volunteers. Men who developed antibody fol- lowing inoculation of the vaccine resisted chal- lenge with a virulent suspension of the or- ganism, whereas illness occurred in 10 of 13 in unvaccinated controls. Mycoplasma Epidemiology Considerable advances were made in under- standing the epidemiology of Mycoplasma behavior of this organism a longitudinal study of seven training platoons was made at the Parris Island, S.C., Marine Recruit Training Center. Fifty to 74% of recruits possessed M. pneumoniae growth-inhibition antibody at the start of training. Among the antibody nega- tive (i.e., susceptible) recruits the infection rate for the 14-week training period was quite high — 60 to 80%. Growth-inhibition antibody correlated well with resistance to infection, but it was not completely protective since ap- proximately 20% of recruits with pre-existing anibody became infected during training. The latter finding provides the first evidence that reinfection with M. pneumoniae occurs under natural conditions. Mycoplasma — Fundamental Studies Three major advances were made in under- standing these organisms. First, the hemolysin of M. pneumoniae was found to be a peroxide. This finding has implications in the pathogene- sis of mycoplasma-induced disease as well as in autoimmune phenomena induced by myco- plasma. Second, the DNA homology technique 184 ANNUAL REVIEW OF INTRAMURAL RESEARCH revealed the genetic distinctness of the myco- plasmas from bacteria. This finding clearly es- tablished the mycoplasmas as a separate group of microorganisms, a group with its own iden- tity. Third, the protective antigen of M. pneu- moniae was defined as a lipoprotein. The lipid hapten of this complex was further defined as a low molecular weight phospholipid contain- ing glycerol, four amino acids and several fatty acids. The chemical structure of this protec- tive antigen is nearing definition. Host Resistance to Respiratory Disease In volunteers challenged with type I parain- fluenza virus, serum antibody did not correlate well with resistance to infection. In contrast, antibody in nasal secretions did correlate well with resistance to infection. Such antibody proved to be the decisive factor in determin- ing the outcome of experimental challenge with type I parainfluenza virus. An inactivated type I virus vaccine stimulated serum but not nasal secretion antibody. Such vaccinated individuals did not resist experimental challenge with the virus. However, antibodies developed in the nasal secretions of volunteers following infec- tion and such individuals resisted rechallenge. Studies in Oceania Picornaviruses have been found to be fre- quent causes of infection among adults and children in Honolulu. The exact nature of the problem remains to be defined since the Cox- sackie Group A related viruses recovered are not pathogenic for suckling mice and neutral- izing antibody cannot be studied. An outbreak of Dengue in French Polynesia druing 1964-65 has been further analyzed and indicates that this was the first introduction of an arthropodborne virus into the island in the life span of individuals studied. More evidence has been obtained on the re- lationship between A. cantonensis and eosino- philic meningitis in studies in Thailand and Oceania. The probable source through inges- tion of raw shellfish, fish and vegetables has been indicated. Recent outbreaks in French Polynesia apparently resulted from contami- nated lettuce. Rubella Virus These studies are supervised by Dr. John Sever of NINDB and Dr. Huebner of NIAID. In collaboration with Drs. A. Fabiyi, G. Git- nick, L. White, R. J. Hildebrandt, and D. A. Fuccillo, they are studying the role and be- havior of rubella and other viruses as causes of perinatal disease and defects. Rubella virus given to pregnant ferrets early in gestation produced runting and abortion of the newborn. Neutralizing antibodies de- veloped in all inoculated animals, showing that active infection had been produced. Newborn ferrets given rubella virus develop a chronic infection lasting 6 to 8 weeks. The newborn and pregnant ferret represents the first and as yet only animal study system available for rubella virus other than primates. Should the runting and abortion prove to be due to infec- tion of fetal tissues, this system may prove ex- tremely useful in the study of birth defects. Rubella virus syndrome is also being studied clinically, serologically and epidemiologically. Preliminary data derived from the NINDB Perinatal Study groups have provided a pro- file of the immune status to rubella among women of childbearing age. Considerable geo- graphical variations were observed; antibodies were found in 85% of the women in the east- ern part of the U.S., in 70% on the west coast and in less than 50% in Hawaii. A new rubella vaccine development program was initiated in collaboration with NIAID's Vaccine Development Branch with Dr. John Sever, NINDB, as the major project officer on a number of contracts. This program is only in the beginning stage; however, several types of vaccines produced on commercial contracts should be available for field testing early in FY 1967. Reference virus pools and antisera needed for evaluation of vaccines have been produced in this laboratory and distributed to the various contractors. Various methods for increasing the titer of rubella virus have been successful, and these have been utilized for producing complement fixation test antigens and also seed stocks for candidate rubella vaccines. NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 185 Bacterial Metabolism ami Physiology Hydrogenomonas eutropia has been utilized in model system study of electron transport mechanisms because of its ability to utilize molecular hydrogen. Further evidence was ob- tained on the role of cytochome c but the exact enzyme which mediates transfer of electrons has not as yet been defined. Solution of this problem will enable studies on the role of organic nutrients in metabolism in auto- trophic and heterotrophic cells. Studies on the role of iron and siderophilin in the growth and metabolism of Staphylo- coccus aureus continue. Marked physiological differences exist between iron-deficient and iron-rich cells of S. aureus and these are addi- tionally altered by growth of such cells in the presence or absence of glucose. Iron-deficient cells vary in the following characteritsics: poor growth, imparled respiration, increased gly- colytic activity, decreased catalase activity, a reduction in cytochromes and cytochrome oxi- dase, and decreased sensitivity to cyanide in- hibition. Iron seems to act as an inductor of electron transport and respiratory enzyme for- mation. There have also been further studies on the enzyme which releases teichoic acid from S. aureus cell walls. This is produced in varying amounts by nine different strains and has been purified 10-fold in one preparation. Na + , Mg + + and cobalt are required for activity. If further purification can be achieved and the linkage point of attack identified, removal of teichoic acid from the cell wall may permit identification of the role of this acid in cell- wall replication and in the binding of certain antiobiotics. Studies on the structure, replication and ge- netics of streptococci also continued. These in- clude characteristics of L-form colonies. By electron microscopy, small intravescular mas- ses of elementary bodies in the order of 0.05/j. are seen but filtration studies have failed to demonstrate colony forming capacities of units smaller than 0.22^. in size. The surface recep- tor site of Group C streptococci has been identi- fied but the phage receptor site in Group A streptococci remains unidentified despite inten- sive efforts to identify its location and nature. The coexistence of two M antigens in the cell wall of Group A streptococcus has been con- firmed and the occurrence of a type 12 M pro- tein in a Group C streptococcus observed. Ef- forts to achieve laboratory transduction of Group A streptococci have not as yet been suc- cessful but continue since this apparently oc- curs in nature and the ability to manipulate the organism in the laboratory will open path- ways to the genetic study of virulence and antigenic variability. Histoplasmosis Various lines of study have been pursued. Several nitrogenous suppdements to sterile soil enhanced the multiplication of H. capsulatum but in nonsterile soil inhibited growth. These studies along with a number of analyses of soils from other sites suggest that no single nutritional factor determines wheter H. cap- sulatum can grow at a particular site. Rather, there is indication that the distribution of bac- terial antagonists is the limiting factor. At the Middle America Research Unit, the role of bats in dissemination of histoplasmosis was further identified. An outbreak in persons following a visit to a bat cave was intensively studied with development of both clinical and serologic evidence that previous disease was relatively protective. The disease was also identified for the first time in Western Texas when found in free-tail bats. LABORATORY OF BIOLOGY OF VIRUSES Previously established experimental studies on the structure of viral nucleic acids and the molecular events involved in virus replication have continued during this fiscal year, but, in addition, there has been an increased effort aimed at determining the functional aspects of these nucleic acids. As in the recent past, these basic biochemical investigations have not been limited to phenomena occurring in virus-in- fected cells but of necessity have tested similar parameters in normal control cells. 186 ANNUAL REVIEW OF INTRAMURAL RESEARCH Viral Structure Findings on virus structure have varied from electron microscopic demonstration that polyoma virus may be made up of complex lamination of protein layers with a DNA core to the characterization of the physiochemical configuration of viral RNA and DNA mole- cules. Three projects have looked at the struc- ture of the nucleic acid from DNA viruses and two from RNA viruses. In both types of viruses the studies have included single and double-stranded forms of the two kinds of nu- cleic acids. In fact, in the case of the rat virus this work represents the first definitive demon- stration that there exists a mammalian virus containing single-stranded DNA. A very inter- esting "satellite" virus which is a small agent unable to replicate by itself and found multi- plying only in the same cell with adenoviruses has been shown to be a DNA virus whose nu- cleic acid is double-stranded. Further infor- mation on the structure of the DNA of SV 40 virus — a tumor inducing agent — has been ob- tained through studying the effect of radiation on its physical properties. Cell Response to Viral Infection The examination of the biochemical events taking place in the virus infected cell are in- separable from study of nucleic acid functions of both the cell and the virus. Since messenger RNA is required for the production of all spe- cific proteins and this function occurs in a physical unit made of ribosomes, one of the staff has been developing methods for isolating and purifying these cellular elements. The im- portance of the cellular polyribosomes for translating the virus information into viral protein products has been demonstrated in the vaccinia (DNA), Reo virus (RNA) and polio- virus (RNA) systems. Furthermore, in basic studies on the mode of action of the impor- ant antiviral substance, interferon, evidence has been obtained that the effect may be local- ized at the level of association of viral RNA with ribosomes. Viral Oncogenesis Polyoma virus DNA synthesis in lytic infec- tion has been found to be preceded by the early appearance of specific "tumor" antigen and an increased activity of enzymes required for DNA synthesis. Of special significance is the simultaneous increase in cell DNA synthe- sis since this may be a requirement for inte- gration of viral genome into cell genome during oncogenic transformation. Another experimen- tal finding having significance concerning the relationship of tumor virus DNA to cell DNA is the demonstration that a single cell can be transformed by two different DNA tumor vi- ruses — SV 40 and polyoma. Such doubly trans- formed cells contain two types of specific tumor antigens induced by both viruses and appear to have an increased growth potential. This finding raises the possibility that more than one site for integration of viral genome is available on the genome of the cell. Genetic Relatedness Several of the laboratory's projects have been aimed at nucleic acid functions in the nor- mal mammalian cell. Previous results had shown a degree of common structure in the DNAs of various animal species varying ac- cording to their evolutionary relatedness. Now by study of pieces of the cell genome, the com- monness has been shown to reside chiefly in those areas of the DNA highest in G-C base content. By manipulation of conditions during comparison of, for instance, the DNAs of man and chimpanzees, previously undemonstrable differences can now be shown. Another signifi- cant finding is evidence pointing to the exist- ence of repeating sequences of bases in cell DNA and the separation of these stretches of the genome into classes according to their fre- quency of occurrence. Preliminary experiments have been initiated on the separation and characterization of chromosomes from mam- malian cells. Already techniques have been de- veloped for obtaining large, workable, amounts of partially purified human chromosomes which can now be tested for a variety of biochemical functions. LABORATORY OF TROPICAL VIROLOGY Major efforts continued to be related to the study of the properties of South American NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 187 hemorrhagic fever viruses. Amapari virus, a new member of the Tacaribe group, was shown to be more adaptable to suckling hamsters than to suckling mice. The advantages of tissue cul- ture cell lines derived from newborn rabbits MA III) and green monkey kidney (Vero) were investigated for propagation of Tacaribe, Amapari and Junin viruses. Each cell line had definite advantages and disadvantages. Efforts to obtain cytopathogenic effects with Machupo virus in human embryonic kidney cell lines were essentially unrewarding. MIDDLE AMERICA RESEARCH UNIT (MARU) PANAMA CANAL ZONE Efforts were concentrated toward the fur- ther definition of South American hemorrhagic fever viruses and on the prevalence of arbovi- ruses in Central America. More than 11,000 sera have been placed on file for screening for antibody to arboviruses. The bulk of these sera were obtained in collaborative studies between MARU and INCAP in Costa Rica during ICNND sponsored nutrition surveys through- out Central American countries. IBM data processing has been utilized for data record- ing, retrieval, and analysis of the information which is accumulating on arbovirus infections. Hemorrhagic Fever Continued surveillance in San Joaquin re- sulted in diagnosis of 18 sporadic cases of dis- where trapping results indicated residual pop- ease during calendar year 1965. Most of these were concentrated in two areas of the town ulations of Calomys callosus. Field studies in Bolivia, Peru, Paraguay, and Brazil were successfully completed. Testing of human sera to date reveals no evidence of past HF infection. At least 5 candidate HF agents have been recovered from Calomys captured in Brazil and San Ignacio, Bolivia. Studies in experimental animals were ex- tended. Adult Rattus rattus, Proechimysguy- annensis and possibly Mus musculus do not develop chronic infections with viruria follow- ing Machupo virus inoculation. Other rodent species are being tested. Asymptomatic infection was induced in new- born Calomys by Junin, Tacaribe, and Ama- pari viruses. Antibodies were produced by all viruses. Analysis of animal tissues for evi- dence of chronic infection is in progress. Cross challenge tests in marmosets indicate that infection with Junin or Tacaribe viruses does not protect against subsequent lethal Ma- chupo infection. Attempts to attenuate Machu- po virus for this host are under way. Arbovirus Studies Continuous sentinel monitoring of virus ac- tivity was maintained at Gamboa in the Canal Zone. Eighteen strains of VEE virus, seven of EEE and one of Bussaquara were recovered between June and December 1965. As in pre- vious years activity declined below detectable levels from January through April. A study of dynamics of infection by VEE and related Pixuna and Mucambo viruses in reservoir rodents Proechimys semispinostis and Sigmodon hispidus was completed. Viremias ranged from 2-5 days duration, most animals survived, and virus was recovered from throat swabs, but not feces or urine. Animals devel- oped anitbodies to all three viruses following infection by any one of them. VEE infection conferred complete protection against Pixuna and Macambo viruses, but Pixuna infection failed to protect completely against subsequent VEE challenge. A study of antigenic variation among VEE strains was begun. It appears that donor host is not a significant variable, and that clear cut geographic differences are recognizable. LABORATORY OF GERMFREE ANIMAL RESEARCH The laboratory continues to take advantage of the germfree animal to study certain infec- tious processes and immune reactions under conditions where previous infection and im- mune responses have not altered the host's re- activity. The germ free animal has seemed a particularly promising model for the study of autoimmune phenomena. 188 ANNUAL REVIEW OF INTRAMURAL RESEARCH Immunoglobulins in Germfree Mice It has been shown that germfree mice usu- ally synthesize and contain in their sera only 7Syiand IgM globulins. These proteins are synthesized by spleen and lymph nodes. In con- ventional mice similar tissues form all immu- noglobulins. The ileum of conventional mice forms principally IgA globulin while that of germfree mice usually forms none. Levels of 7Syi and IgM globulins can be increased by immunization with ferritin and the somatic lipopolysaccharide of Escherichia coli. A sin- gle high dose of endotoxin and two appropri- ately spaced, but not a single injection, of fer- ritin result in the synthesis of 7Sy 2 globulin by the spleen. Formation of this protein shows a high correlation with the appearance of ger- minal centers in the spleen.The response to endotoxin is characterized by a prolonged in- crease in IgM globulin to levels between 1.5 and 2 times control levels, and this response is indepdendent of dose between 0.5 and 50 ^g. A single injection of ferritin produces a tran- sient increase of IgM globulin to 0.2-0.2 mg/ ml, about double the control levels, followed by a 30 percent increase in 7Sy x globulin. The secondary response to ferritin shows an in- crease in IgM globulin of a magnitude similar to that seen in the primary response, but the elevation is much more prolonged. In addition, the serum concentration of 7Syx globulin ap- proximately doubles. Much of the relatively large increases produced by these immuniza- tions cannot be accounted for as specific anti- body. Genetic Factors May Regulate Immune Tissue Destruction The incidence of autoallergic thyroiditis in two strains of guinea pigs was compared. The Hartley strain animal developed the disease more frequently and after lower antigenic im- munizing doses than did the Strain 2 guinea pig. Nevertheless, the antithyroid antibody titer and delayed skin reactivity in the two strains of guinea pigs were identical. These strain differences, in the face of an apparently equal gross immune response, may be due to quantitative differences in the classes of im munoglobulins produced, to variations in cell structure in which one type may be more easily damaged than the other, or to different amounts of reactive intermediates which are involved in immune tissue destruction. Clostridium Perfringens Lethal to Germfree Guinea Pigs A high mortality rate is associated with the transfer of germfree guinea pigs to an open animal room where they come into contact with a variety of bacterial species. In studies designed to investigate the nature of this in- fectious process, a strain of Clostridium 'per- fringens was isolated from the intestinal con- tents of moribund ex-germfree guinea pigs. In vitro cultivation of this organism led to the production of heat-labile exotoxin (s) which on intraperitoneal injection into mice produred death. Oral inoculations of germfree\ guinea pigs with this Clostridium produced death within 12-18 hours. Other investigations have shown that conventional guinea pigs often har- bor this organism in the intestinal tract yet are tolerant to heavy oral doses. These studies have provided new knowledge as to the nature of this increased susceptibility of germfree guinea pigs to bacterial exotoxins. Severity of Amoebic Infections Influenced by Associated Bacteria Conventional guinea pigs harboring a vari- ety of intestinal bacterial species often develop fatal amoebiasis after intracecal inoculation with Endamoeba histolytica whereas germfree guinea pigs do not develop severe disease. By monocontaminating groups of germfree ani- mals with known species of bacteria and in- fecting each group with the same strain of E. histolytica, differences in severity of the infections have been observed depending upon the bacterial species present in the intestine. When Bacillus subtilis was used as the bacterial associate, all animals succumbed to the infec- tion. However, the infection of guinea pigs with amoebae and a species of Micrococcus re- sulted in no mortality. Various gradations of severity were observed with other known spe- cies of bacteria and, in addition, differences NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 189 were observed in the intestinal lesions pro- duced. These preliminary observations sug- gest that the severity of experimental intesti- nal amoebiasis in guinea pigs may be influ- enced by the species of associated bacteria. Low Serum Lysozyme Levels in Germfree and Ex-Germfree Rats Levels of serum lysozyme in germfree and ex-germfree Fisher and Sprague-Dawley rats have been found to be significantly lower than in conventional animals of the same strains. However, no significant difference in enzyme free rats. Large amounts of lysozyme are pres- ent in leukocytes and a close correlation is known to exist between enzyme levels and total granulocyte counts. In the three animal groups studied, this granulocyte-lysozyme association has confirmed, i.e., the lowest levels of lysozyme and the lowest leukocyte counts being found in the germfree rats. Immunoglobulins Quantitated in Human Malaria The importance of the increases of the var- ious immunoglobulin classes formed in re- sponse to infection with the malaria Plasmodia has been emphazied by recent studies. Serial quantitation of the three immunoglobulins IgM, IgG and IgA during primary attacks of vivax and cynomolgi malaria have shown that a close association exists between increased immunoglobulin levels and the formation of specific malarial antibody. Large amounts of IgM and IgG globulin were formed during the course of the primary attack in all volunteers — the increase in IgA globulin was less strik- ing. Volunteers with the milder cynomolgi in- fections of lower parasitemia synthesized less of the three immunoglobulins than did those with vivax malaria. No significant increases were observed in IgD globulin during the pri- mary malarial infection. Fractionation of sera taken from a single vivax volunteer during the primary attack demonstrated that both the 19S and 7S fractions had specific antibody against the infecting strain. However, during the secondary response, when the patient re- lapsed, there was no specific malarial anti- body activity in the 19S fraction and antibody activity appeared to be confined to the 7S fraction. Laboratory of Parasitic Diseases A relatively broad approach to the problems of parasitism has been the primary concern of the laboratory, emphasizing basic research, coupled with field studies in various areas of the world. Five field projects have been conducted out- side the U.S. Dr. Allen W. Cheever has con- tinued his studies on schistosomiasis pathology in Salvador, Bahia, Brazil. Two other programs on schistosomiasis will reach completion soon; one supported by PL-480 funds in Egypt on mass treatment with sodium antimony dimer- capto succinate (Astiban); the other in Puerto Rico on continuous drug infusion. Two addi- tional PL-480 programs, one concerned with amebiasis in India, the other with toxoplas- mosis in Israel, have continued at a productive level. Schistosomiasis The release of cercariae from the snail host is not influenced by an innate circadian rhythm. Light can stimulate their release in the absence of temperature change. If the light intensity is at least 60 foot candle power, even a few seconds of light can stimulate cercariae release. In the absence of light, a temperature rise of 4° C. causes their release, but a 2° rise does not. Low temperatures inactivate cercariae; their sedimentation rate at 1-3° C. is 4.95 ± 0.09 cm per minute. At 11 to 13° C. cercariae become sufficiently inactive so that they cannot maintain their positional level in the water; these data suggest that human infection will not occur in water below this temperature range. The determination of cercarial concentra- tions in natural bodies of water is fundamental to an understanding of their population dy- namics and to the transmission of the infec- tion. A simple continuous flow centrifuge has been developed for the quantitative collection of cercariae. Seventy to ninety percent of the 190 ANNUAL REVIEW OF INTRAMURAL RESEARCH organisms were consistently recovered from one-liter water samples and over 50 percent from larger samples. The technique appears promising for field use. Laboratory studies indicate that miracidia infected snails 10 meters from their point of release, and were infective up to 8 hours after they hatched. These and other data suggest that miracidia have a capacity to infect snails in the field at considerable distances from their point of release from the schistosome egg. Highly important findings were made con- cerning the pathogenesis of huma schistosome infection based on quantitative data obtained from perfusion of cases autopsied at the Hos- pital Professor Edgard Santos in Bahia, Brazil. The study has revealed a number of parasito- logic differences between patients with and without disease, i.e., with or without Symmer's fibrosis. Patients with Symmer's fibrosis often have heavy infections, and many of those with lower worm burdens have definite or presump- tive evidence of previous treatment. Egg dis- tribution is distinctive in cases of Symmer's fibrosis. The majority are found in the small intestine, and rectal tissues contain very few eggs. The opposite is true in cases without evi- dent anatomic disease. In Symmer's cases, eggs in the liver are concentrated in the portal areas, whereas in the other cases they are dis- tributed randomly within the liver. Approxi- mately 8% of "asymptomatic" cases have worm burdens comparable to those of the more heavily infected Symmer's cases. The factors determining the presence or absence of dis- ease are not clear. Collaborative studies on the effect of single monthly injections of Astiban to village popu- lations in Egypt were continued. These stud- ies have shown that urinary shedding of ova of S. hematobium ceased in about 90 percent of recipients under such therapy and had not resumed in any significant number of victims during a six month period after therapy was stopped. These observations indicate that the drug in this dosage has both chemoprophylac- tic and chemotherapeutic properties and the method would seem to be suitable for mass treatment of populations in hyperendemic areas. In Puerto Rico it was shown that a slow continuous infusion or a series of closely spaced small injections of Stibophen was better tol- erated by the patient and therapeutically more effective than were larger, more widely spaced injections. Very similar observations were made with Astiban (sodium antimony dimer- capto succinate). Amebiasis and Trichomoniasis With the aid of a sensitive microfluorimeter and measuring techniques developed in this laboratory, it has been possible for the first time to demonstrate a spectrum of antigenicity in some 16 strains or lines of Entamoeba his- tolytica growing in association with a mixed bacterial flora. It has also been shown that antigenic differences exist between 3 strains of Trichomonas, two of them derived from a single source, but maintained under different conditions for 18 months. Fluorescent antibody serology has confirmed a high incidence of amebic liver abcess in pa- tients seen at the hospital in Hyderabad, India. In addition, about 55% of cases with the vague symptomatology of chronic intestinal or he- patic amebiasis" are positive in the FA test for amebiasis, compared to 30% of patients without such clinical signs. There is consider- able serologic evidence accumulating that the amebiasis picture in countries such as the United States cannot be compared in any real- istic manner to that in an endemic area such as India. There has been no correlation be- tween positive serologies and infection with E. hartmanni, so-called "small race E. histo- lytica," thus supporting the view that this is a separate species from E. histolytica. Continued improvement has been made in the medium used for the axenic cultivation of E. histolytica. Considerable variation was en- countered in the ability of different lots or horse serum and liver extract, respectively, to support growth. These axenic cultures have for the first time made available a system for the testing of drugs directly against amoebae. Prior tests conducted on amoebae grown in associa- tion with microbial flora were always incon- clusive because of the possibility that the drug NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 191 acted on the microbes which served as food for the amoebae. Humatin (Paromomycin sul- fate) killed axenic amoebae at levels of 8 and 16 micrograms/ml medium. In vitro synthesis of polyamino acids was demonstrated using - subcellular fractions of axenic E. histolytica under controlled condi- tions. Tetracycline, an antibiotic apparently without an effect on the mammalian protein syntehtic machinery, was markedly inhibitory. This provides the first direct evidence that the therapeutic action of tetracycline in amebiasis is specifically on the amebae and not on the associated bacteria, and affords a clue as to its mechanism of action. Polyribosomes from the amoebae, constitute the first identification of these biochemical structures in a protozoan cell. Glucose and Glycerol Utilization in Parasites Comparative studies of glucose and glycerol uptake by the larval and adult forms of Taenia teaeniaeformis, and by different species of try- panosomes under various conditions have re- vealed interesting similarities and differences. The larva absorbs more glucerol aerobically than anaerobically, a difference lacking in the adult worm. For the larva, an inverse relation exists between concentration of environmental glycerol and glucose leakage. In the absence of Na + , marked inhibition of glucerol and glu- cose uptake occurs, the effect being more pro- nounced on the latter. Phloridzin completely inhibits glucose uptake, but has no effect on that of glycerol. The data suggest that at very low environmental glycerol concentrations, ab- sorption involves active transport mechanisms, while at higher concentrations diffusion, prob- ably facilitated diffusion, appears to prevail. Of 8 species of trypanosomes studied, only T. gambiense and T. rhodesiense consumed large amounts of glucerol. When T. gambiense was kept in a mixture containing equal amounts of glycerol and glucose, definite mu- tual inhibition of absorption was observed. In addition, other differences between tapeworms and T. gambiense were found to exist in respect to extraneous influences. Laboratory of Parasite Chemotherapy The major research effort of the laboratory continued to be centered on problems in ma- laria. Resistance of strains of falciparum ma- laria to all commonly used synthetic antima- larial compounds remains a severe problem, particularly in Vietnam and other areas of Southeast Asia. The need for new and effec- tive drug regimens to combat this problem has dictated continuation and expansion of this area of research. Further studies on the many host-parasite-vector combinations now avail- able in the simian malarias have contributed much to our program of evaluation of antima- larial agents and to our understanding of the biology of the parasite in the primate host. Malaria — Human Studies continued in the clinical testing of antimalarial drugs in human volunteers, with emphasis on strains of parasites resistant to certain compounds and on possible methods for overcoming the resistance problem. Activity of cycloguanil pamoate (CI-501), the injectable long-acting antimalarial, and that of CI-564, a combination of CI-501 and DADDS (the acetylated diaminodiphenyl sulfone), was eval- uated against strains of vivax and falciparum malarias resistant to chlorguanide, the parent compound of CI-501. While CI-564 was effec- tive against the normal strain of Plasmodium vivax, when the strain had been made chlor- guanide-resistant the drug had only minimal antimalarial activity. Trails of CI-564 against multiresistant strains of P. falcipamm were encouraging. Although these strains were re- sistant to normal doses of chlorguanide or CI-501, in most cases volunteers were suc- cess fully protected from or cleared of infection when CI-564 was used as a suppressive or therapeutic agent. Four additional strains of drug-resistant falciparum malaria were isolated and studied in volunteers. These strains were all from Southeast Asia and included one from Thai- land (Thai II), one from Malaysia (Malayan IV) and two from South Vietnam (SV-1 and SV-II). The first three strains have been ex- tensively evaluated and all possess a wide spec- 192 ANNUAL REVIEW OF INTRAMURAL RESEARCH trum of resistance, being refractory to chloro- quine, chloroguanide and mepacrine. The SV- I strain appeared to be sensitive to 50 mg. of pyrimethamine, but the others were also re- sistant to this compound. While quinine sulfate was curative when given as a 5-day course of treatment in cases of Thai II and Malayan IV, THE SV-I strain appears to be resistant to a 10-day course of quinine, requiring 14 days of this drug for a cure. The SV-II strain is a more recent isolate, and was derived from an infec- tion which had recrudesced after extensive therapy with quinine (10 grains t.i.d. X 14 days, followed by 5 grains t.i.d. for 6 weeks). This strain is resistant to chloroquine and chlorguanide. In line with the urgent need for alternative methods of treatment of the resistant strains, studies have been done on the usefulness of sulfonamides, either alone or in combination with pyrimethamine. Forty volunteers have participated in the evaluation of thee sulfon- amides: sulfadiazine, sulfamethoxypyridazine (Midicel), and sulforthodimethoxine Fana- sil). The Malayan III strain of P. falciparum was used for the initial studies; it is resistant to all synthetic antimalarials tested, but sen- sitive to the 5-day treatment with quinine sul- fate. Sulfadiazine was given in a 5-day course of treatment, while the longer acting com- pounds. (Midicel and Franasil) were given as single doses. Results indicated varying degrees of antimalarial activity for all three of the sulfonamides when used alone, but that none was completely reliable for the termination of these malarias. When pyrimethamine (as a sin- gle dose) was given concurrently with the sulfonamides the curative effect was signifi- cantly enhanced. A pyrimethamine-Fanasil combination was evaluated against three addi- tional resistant strains: Thai II, Malayan IV and South Vietnam I. In a total of 24 volun- teers treated, this combination was successful in effecting a cure in 21. Studies have continued on the West African strain of P. ovale to characterize the clinical and parasitological patterns of this infection in volunteers. Contrary to prior descriptions, ovale malaria appears to present moderately severe clinical manifestations. The strain re- sponds readily to standard antimalarial regi- mens. A single dose of 10 grains of quinine sulfate successfully clears patent paraistemias in about four days. Observations on relapse patterns of quinine-treated, mosquito-inocu- lated cases indicate a wide range of intervals between initial and subsequent attacks. While relapse has been seen as early as 17 days after treatment of the primary attack, of particular interest are the extended latent periods some- times seen. In one case the interval between primary attack and first relapse was 255 days. Malaria — Simian Studies on simian malaria as a possible zo- onotic disease of man have been conducted on several fronts. A new strain of Plasmodium brasilianum, the South American quartan par- asite of monkeys, has Deen transmitted to vol- unteers through the bites of infected mosqui- toes. The infectionin man was extremely mild toes. The infection in man was extremely mild, man to man through subinoculation of infected blood. Similarly, the quartan parasite of mon- keys of Southeast Asia, P. inui, has been suc- cessfully transmitted to volunteers through the bites of infected mosquitoes. Clinical manifes- tations were mild. The infections were self- limiting and antimalarial intervention not re- quired. Similar exposure of volunteers to mos- quitoes infected with P. gonderi, P. Coatneyi, P. fieldi, and several additional strains of P. cynomolgi have not resulted in detectable par- asitemias. Of great interest has been the isolation of a strain of P. knowlesi from a person recently returned from Malaya. This strain has been studied in 8 volunteers and in rhesus mon- keys. The infection is characterized in man by moderately severe clinical manifestations. The fever pattern is quotidian, with fever as high as 104.8° F. The maximum parasite count has been about 20,000 per cmm of blood. The infection is generally self-limiting, but anti- malarial intervention has been deemed advis- able in three cases. It has been possible to infect monkeys through mosquitoes fed on human carriers. The course of infection in monkeys is generally very severe, almost in- NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 193 variably terminating- fatally in the absence of antimalarial intervention. This is the first doc- umented case of a human acquiring a monkey malaria under natural conditions and definitely establishes it as at least an occasional zoonosis. To follow this lead, studies were re-established in Malaysia, particularly in the area from which this case was known to have originated, to determine the extent to which this zoonosis might exist as a real problem. Results of this limited study are not yet complete; however, it seems evident that, although this zoonosis may occur occasionally, it is not a significant problem in the overall consideration of malaria in these areas at this point in time. Extensive studies on the exoerythrocytic stages of the simian malarias have been initi- ated, primarily to provide a reliable system for the evaluation of antimalarial compounds against these cryptic forms, but also to expand our knowledge of the entire life cycle of the primate malarias. Techniques have been de- vised for the direct massive intrahepatic inocu- lation of sporozoites, which enhances the loca- tion and identification of these stages on sub- sequent biopsy. Using this method it has been possible to demonstrate developmental exoery- throcytic stages of P. coatneyi, P. fieldi, P. knowlesie, P. brasilianum and several strains each of P. inui and P. cynomolgi. Preliminary studies are under way to determine the activ- ity of known antimalarial agents against these forms. Immunological Studies The availability of a large number of human, simian, avian and rodent malarias has made possible continuation of studies on the devel- opment and persistance of specific antibody, as determined by the fluorescent antibody (FA) method and methods of Immunoelectro- phoresis. Monkeys infected with various spe- cies of malaria normally develop antibody re- sponse to a very high level within three weeks after infection. These levels seldom increase, even though the parasitemia persists for ex- tended periods. FA tests have been conducted on several large series of serum samples from Nigeria, Upper Volta and Liberia (West Africa). These era were tested against five Plasmodium anti- gens and the responses, in general, indicate an increase in titer with age and higher titers in males than infemales. In all groups under 13 years of age, the highest response was to the P. falciparum antigen. Those over 13 years of age had equal mean titers to the P. falciparum and the P. brasilianium antigens, the latter species being an indicator of previous P. ma- la riae infection. Examination of sera from individuals in- fected more than 10 years previously with P. vivax (during the renowned Campfire Girls epidemic in California) indicated that, although the FA responses were weak, they could be distinguished in most cases from persons of the same age who had not experienced malaria. Antigenic analyses of the simian malarias, are being done preliminary to use of parasiten or their components in experiments on immu- nization. Electrophoretic analysis reveals a minimum of four or five distinct components. Immunoglobulin fractions have been collected from 10 strains or species and are being used, along with whole parasite extracts, for immu- nization of animals. LABORATORY OF IMMUNOLOGY Autoimmunity Immunofluorescence studies have established that serum immunoglobulins from patients with myasthenia gravis react in vitro with striated muscle of a wide variety of both ver- tebrate and no n- vertebrate species. The occur- rence of striated cells in the thymus of several vertebrate species has been confirmed; recip- rocal absorption studies with striated muscle and thymus have suggested common antigenic determinants in these tissues. By electron mi- croscopy it was established that there are thymic cells possessing the sarcomeric and myofilamentous structure of striated muscle. In collaboration with NINDB it was shown that the occurrence of these immunoglobulins in human sera is correlated with the occurrence of thymomas but that in the thymomas studied, there was no correlation with my- asthenia gravis. 194 ANNUAL KEVIEW OF INTRAMURAL RESEARCH Thyroid gland transplants have been made into deep intramuscular sites in inbred Strain 13 guinea pigs. A significant number of the Strain 13 isografts of normal thyroids showed evidence of mononuclear cell infiltrates some weeks later. This partial rejection of normal thyroid isografts constitutes an indication that skin grafts may not be a wholly adequate in- dicator of histocompatibility of other tissues or organs. Some of the findings on lymph node transfers given in a previous annual report have also been consistent with such a possi- bility. In view of the age and familial influences on certain human demyelinating diseases, and prior experiments on experimental autoim- mune encephalomyelitis (EAE) in guinea pigs, studies on EAE were extended to primates. Guinea pig spinal cord in complete Freund's adjuvant was administered to Rhesus monk- eys of various ages as a single injection. Ani- mals ranging in age from laboratory-bred new- borns, infants, and juveniles to fully mature adults proved susceptible to allergic encephalo- myelitis. Of 22 animals tested, all developed marked neurological disorders and only two survived the disease. Severe inflammatory les- ions were found in the central nervous system. Hemorrhagic retinopathy preceded or accom- panied the clinical and neurological symptoms in nearly all cases. Since in neonates and pre- matures the onset of clinical manifestations was delayed and took a more prolonged course and the pattern and distribution of brain lesions differed between newborn and older animals, there is an age-dependent factor in this disease in monkeys. Transplantation Immunology Pretreatment of donor mice with any of wide variety of antigens apparently unrelated to transplantation antigens leads to markedly diminished capacity of donor lymphoid cells to evoke in recipient mice fatal graft-vs-host reactions across H-2 histocompatibility bar- riers. Alterations in donor spleen cells were not a reflection of coexistent lymphoid hyper- plasia induced by some antigens, rather re- duced donor cell immunocompetence appeared to be related to some form of antigenic com- petition. Pretreatment of donors with a single antigen by no means pre-empted the available population of competent cells. Instead, it ap- pears to impose an overall reduction in their capacity to respond to a second unrelated an- tigenic stimulus. This kind of modification of donor immunocompetence represents a novel, unrestricted approach to the development of transplantation tolerance in higher mammals. Hypersensitivity The availability of inbred strains of guinea pigs, a species in which the different facets of the immune response are best known, has been utilized as a unique resource for analysis of the mechanisms and genetics of hypersensi- tivity. Use of chemically defined synthetic polypeptides for this purpose is advantageous in that the specificity of the resulting immune response is assured by the known chemical composition and the hereditarily determined capacity to respond against these structures. The presence of lysine residues in otherwise antigenic copolymer synthetic polypeptides con- sistently results in the lack of antigenicity for Strain 13 guinea pigs; immune response in Strain 2 pigs is normal. These findings open the way for investigation of issues such as whether a peptide need be antigenic in a given host in order to evoke tolerance and to affect the response to another antigen. Mitogens The plant mitogens from the red kidney bean Phaseolus vulgaris and from the root of the pokeweed Phytolacca americanus, each of which produce distinctive immunologic and bi- ochemical transformation of human peripheral lymphocytes, have been progressively purified and partially characterized. Saline extracts of these source materials display hemagglutinat- ing, leukagglutinating and mitogenic proper- ties. Mitogenic activity was found by different analytical methods to be associated with a sin- gle protein component. Purified mitogens proved to be glycoproteins — but displaying different biological characteristics. Distinctive chromatographic and electrophoretic mobilities for the kidney bean and the pokeweed mito- gens were indicative of separate and distinc- NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 195 tive structures. The transformation in vitro of peripheral blood lymphocytes by the pokeweed mitogens involves two transformed cell types: a large blast-like cell indistinguishable from that seen in in vitro transformation by the kidney bean mitogen and cell type at present seen in mitogenic transformation, with fea- tures resembling early antibody-forming cells. Histcqhemical and radioautographic studies on pokeweed-stimulated cultures revealed that 50-60 percent of the initial population of cells were transformed by the pokeweed mitogen and that prominent among these was a dis- tinctive cell type with cytologic characteris- tics resembling plasma cells. ROCKY MOUNTAIN LABORATORY HAMILTON, MONT. Selected Zoonoses of Regional Importance Rabies was isolated from five bats, four of which were collected in western Montana and one in Idaho. One isolate is particularly note- worthy since it was obtained from a sick bat collected in December. The discovery that mice frequently survive after rabies infection prompted an inquiry into the role of maternal antibodies on the resis- tance of offspring of immune white mice. Placental transfer of antibodies could not be shown, but lacteal secretions were found to account for acquired resistance and colostrum was not more important than milk. Immun- ity, which increased with continued suckling, existed for more than 35 days after birth. In other studies on immunity against rabies attempts were made to characterize a rabies- virus-inhibiting substance which develops in brains of animals that have recovered from rabies. This virus-neutralizing factor did not appear after vaccination, even after live virus vaccination. As the titer of the inhibiting sub- stance increased, the virus content of the brain tissue decreased. The ability to distinguish re- covered animals from other immunes by dem- onstration of this inhibitory substance enables a study of the role of abortive rabies in the natural epidemiology of the disease. Last year it was shown that an ether extract of F. tularenis was a reliable skin-test antigen for the detection of past infections with this organism. This test was applied to laboratory employees who had been vaccinated with Ft. Detrick live tularemia vaccine. All individuals developed agglutinins ranging in titer from 1:20 to 1:640 but mean titers were lower than those expected after natural infection. Most vaccinated individuals reacted positively to the skin-test antigen but the average size of reac- tion was smaller than that arising in people who have had the natural disease. Rickettsial Diseases Investigations on the role of domestic ani- mals and wildlife and their ectoparasites on the epidemiology of typhus have been contin- ued in Egypt and South America. In Egypt serologic evidence of infection of livestock was found again this year, but serum titers were not as high. Among wildlife tested, antibodies against Rickettsia proivazekii have been shown, with reasonable certainty, in only two specimens of Rattus rattus. So far, attempts to isolate typhus rickettsiae from 250 wild rodents and ticks collected from them or do- mestic animals have failed. In South Ameri- can studies, supported in part by the Pan Amer- ican Health Organization, serologic surveys were conducted in two known endemic areas in Chile. Typhus antibodies were not detected in serums taken from cattle, sheep, goats, llamas, or burros, but 12 of 80 human serums collected near La Quiaca were positive, three of which had dominant antibodies against epi- demic typhus. In contrast to observations in Egypt and Chile, findings in Ethiopia indicate that ty- phus is still prevalent. Of 99 serums submitted from patients thought to have had a rickett- sial infection, none reacted with R. conori, but 62 were positive for Q fever and 43 were positive for typhus; 19 of the 43 reacted prin- cipally against R. proivazekii, 7 against R. typhi and the remaining 17 had equal titers against both antigens. This year Dr. Reiss- Gutfreund submitted three strains of typhus to RML for confirmation of their identity. These strains, isolated in the same area from which previous isolations were made from live- 196 ANNUAL REVIEW OP INTRAMURAL RESEARCH stock and ticks, were identified as follows: R. prowazekii (JRS) isolated from Hyalomma truncation taken from cattle; R. typhi (ZH97) from H. truncatum taken from cattle; and R. typhi (L308) isolated from human body lice. A burro and two goats were inoculated with the ZRS strain of R. prowazekii to determine the susceptibility of these hosts and to evalu- ate the serologic response after infection. At- tempts to isolate rickettsiae from the blood of these animals failed but all developed higher antibody titers against epidemic than against endemic typhus. Transovarial passage of R. rickettsi in Der- macentor andersoni has been investigated in ticks fed on normal and spotted-fever-immune guinea pigs. For six generations, 12 lines of D. andersoni fed on normal nimals trans- mitted the virulent R type to 100 percent of their progeny. In the same number of genera- tions, 8 of 19 lines of D. andersoni fed on immune animals lost rickettsial infection. In the remaining 11 lines, 100 percent transo- varial passage occurred and virulence of the R type was not changed. Further studies were made on the immuno- logic and serologic responses to Q fever vac- cines. A single dose of phase I or II vaccine in guinea pigs was as effective as two doses, provided the same total quantity was given. Antibody of the 19S type was produced for the first 30 days, after which 7S appeared. A dim ethylsulf oxide extract of phase I antigen, which is a polysaccharide-fatty-acid complex, protected guinea pigs and induced the produc- tion of only phase I antibody. Field trials with human volunteers of phase I and phase II vaccines have been organized under the supervision of the AFEB Commis- sion on Rickettsial Diseases. The results so far indicate that the administration of 30 fig of Henzerling phase I vaccine in a single dose provided complete protection against 3000 GPID r „, aerosol doses. Similar results were ob- tained when 165 fig on Henzerling phase II vaccine was employed, but a four-fold increase in phase I agglutinins in three of the six men challenged later suggests that limited multi- plication of the challenge organisms may have occurred. Reference examination of serums by the ra- dioisotope precipitation (RIP) test is one of the services provided by this laboratory as WHO Regional Reference Center for Human Rickettsioses. Of all serologic tests for Q fever, the RIP test is considered to be most sensitive and specific. Among various groups tested, in- fected rates have varied from 2% to 66%, a rate found among veterinarians in large animal practice. Clinical Q fever has not been a factor in any of the groups studied. In other representative tests 4.3% of 208 patients in tuberculosis hospitals in the Midwest and 62% of 99 persons in Ethiopia, who were thought to have had typhus, were seropositive. Transmission of Disease Agents by Vectors In several collaborative efforts, chiefly with NAMRU-3 in Egypt, some major segments of classification of ticks from various parts of the world were completed. Some important taxo- nomic studies of the New World chiggers also were undertaken. During the year, 52 new species, of which six were assigned to new genera, were described in papers published or in press. Investigations have been continued on the genetics of a paralytigenic factor in Derma- centor andersoni; the 7th generation of an inbred line from British Columbia has shown the same remarkable ability to cause tick pa- ralysis as did previous generations. In view of the suspected relationship of Wolbachia in Argas arboreus to this tick's ability to par- alyze pigeons, the causal relationship of Wol- bachia recently isolated from D. andersoni to tick paralysis in mammals is being in- vestigated. Other research in this project has been quite diverse. Studies have been continued on three strains of the Hughes virus complex iso- lated from Ornithodoros denmarki and O. capensis. Eastern equine encephalitis (EEE) virus was shown to survive in Ornithonyssits bacoti for five to seven days. In other studies involving this virus, the common mealworm, Tenebrio moiitor, was evaluated as a research tool. The larvae, which were found to be as NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 197 susceptible as mosquitoes, yielded virus titers as high as 10 6 when held at 98° F. Encephalitides and Tick-Born Diseases In view of the reported isolation of WEE from naturally infected snakes, studies were conducted during an acute outbreak of WEE in northeastern Montana to determine whether snakes may acquire virus from C. tarsalis-bird cycle. Since 7 or 10 pools of C. tarsalis col- lected during the outbreak contained WEE virus, and 64% of 85 chickens in flocks in the immediate area had developed antibodies, the outbreak was characterized by intense virus, activity. Eighteen cases in man were con- firmed serologically and an estimated 300 cases occurred in horses. More than 300 snakes of five species and 110 leopard frogs were col- lected during the post-epidemic period and tested under various conditions supposedly con- ducive for the isolation of virus, but none was recovered. Studies were renewed on the immunologic response in human volunteers vaccinated with an experimental Colorado tick fever vaccine in 1961. In March 1965, 7 of 10 individuals still had neutralizing antibodies varying in index from 32 to as high as 4,470. After skin test with the vaccine all 10 persons developed a significant rise in titer, persumably from the small amount of antigen used in the skin test. PGittacosis-Lymphogranuloma-Trachoma ( PLT ) Research on the PLT group of agents has been conducted chiefly to develop and improve methods of isolation and purification, to de- velop methods whereby more luxuriant growth can be obtained, and to develop more sensitive and specific diagnostic reagents. Of two new cell lines established (mouse embryo and mouse placenta) the latter was more suitable for the propagation of PLT agents. The radioisotope precipitation (RIP) test continues to show promise as a highly sensi- tive technique for detecting antibodies against the PLT group. Several aspects of the test, however, require further study before it can be used as a practical tool. Since P 32 has a rela- tively short half life, a new batch of antigen has to be prepared about every two months. This difficulty has been partially overcome by labeling with C 14 -tagged adenine, but, un- fortunately, only a small part of the label was incorporated into the meningopneumonitis par- ticles. Further studies with other C 11 compon- ents are planned to find one with a higher rate of radioisotope incorporation. Screen tests have been conducted on various groups of human serums. Significant titers were demonstrated on the serums of 15% of 415 Montana fur trappers, 25% of 29 Dubois sheep station personnel, and 23% of 88 North- ern Cheyenne Indians. The RIP test con- firmed CF findings in a group of serums from 14 patients with "chronic carditis," all of whom had PLT group CF antibodies. The significance of these interesting results is as yet unknown. Five of six serums from clinical cases of lymphogranuloma venereum (LGV) had RIP antibodies. The RIP test ap- pears to be more sensitive than the CF test in detecting prior PLT infection. These find- ings suggest that PLT group infections are far more prevalent than they generally were thought to be. Further epidemiologic study of trachoma in two boarding schools on the Northern Cheyenne Indian Reservation were conducted during the year. In March 1965, 538 students were examined — 82 were thought to have trachoma. A single isolation was made from a student with stage I trachoma. Serums ob- tained from these students were tested by both the CF test and the RIP test. By the CF test, 25% of serums from possible cases had CF activity in dilutions of 1:4 or greater. None of these serums reacted to trachoma type- specific cell- wall antigens. In contrast, 58% of these serums reacted positively in the RIP test. The results of the RIP test also correlated well with the clinical classification of eye disease. Serologic tests of serums from a few adults with stage II or IV trachoma revealed the presence of high RIP titers in the absence of any CF activity. This finding suggests that the two tests measure different type of anti- body and that the RIP antibody response is more durable than the CF response. 198 ANNUAL REVIEW OF INTRAMURAL RESEARCH Chronic Progressive Viral Disease This year another animal disease, chronic interstitial pneumonitis of sheep, was shown to be caused by a slow-growing filterable agent. In the 15 months this project has been underway typical disease appeared in four of eig'ht sheep inoculated intravenously with fil- trates of a suspension of lungs from naturally infected sheep. Three of nine sheep which re- ceived similar material directly into the right lung also developed the disease. Sheep inocu- lated intracerebrally with similar material have remained healthy as have goats inocu- lated either intravenously or directly into the lung. The extent of lymphoreticular prolifera- tion in the lungs of sheep that died or were killed was particularly striking. Additional results of an experiment begun in August 1962, more clearly indicate the dif- ferences in the distribution of scrapie virus during advanced disease between goats inocu- lated intracerebrally and those inoculated sub- cutaneously. In the former, virus was virtually limited to the nervous system, whereas in those inoculated subcutaneously that became infected with scrapie 19 and 21 months after inocula- tion, virus was found in many extraneural sites, notably lymphocytic tissues. Such wide distribution outside the nervous system has not been found so far in a goat that did not develop disease until 33 months post inocula- tion. Results have again indicated the impor- tance of lymphocytic tissues in the pathogene- sis of scrapie. During the first 12 months after inoculation only one of 15 goats yielded virus, a small amount in the left prescapular lymph node that drained the site of inoculation. Dur- ing nine months of the second year, virus was detected in six of nine goats examined. Of 36 tissues regularly tested, only lymphocytic tis- sues contained detectable amounts. Twenty- nine months after inoculation, signs of clinical disease have not yet appeared in 15 remaining goats. Results obtained in this experiment are interpreted as indicating a long latent period, during which virus cannot be detected, fol- lowed by slow replication and spread of virus primarily in lymphocytic tissues. The agent which causes encephalopathy of mink was found to pass through both 100 imi millipore niters and 100 m^ gradacol mem- branes. Thus, the size of the infectious agent is well below that of known non-viral patho- gens. In Pastel mink severely affected with this disease the largest amount of virus was found in the brain, with lesser amounts in various other tissues, but it could not be recov- ered from blood or serum. When small amounts of virus were present in the inoculum, incuba- tion periods of over 400 days were observed. The viremia that occurs in Aleutian disease was studied in Sapphire (an Aleutian color phase) and Pastel (a non- Aleutian color phase) mink. In Sapphire mink the virus could be recovered at biweekly intervals until death which occurred about 12 to 22 weeks after inoculation. During this period the levels of serum gamma globulin rose by 40-45% with- out any apparent effect on the presence of virus in the blood. In Pastel mink virus was recovered at two weeks and 12 weeks post inoculation but not thereafter through 22 weeks. Pastel mink remained free of serum protein changes and are still healthy 32 weeks after inoculation. In another experiment virus was detected in the mesenteric lymph nodes of two apparently healthy Pastel mink 22 months after inoculation. These observations on viremia indicate a clear-cut difference in the mode of virus growth in the two color phases and further emphasizes the importance of genotype in the pathogenesis of the disease. Allergy and Immunology Further comparisons have been made of the Hart.ey strain guinea pigs and strain 13 guinea pigs regarding their suitability for research on delayed hypersensitivity. Previously, strain 13 guinea pigs were shown to produce anti- body less actively than did the Hartley strain. Strain 13 guinea pigs developed pure delayed hypersensitivity to egg albumin when sensi- tized with a conjugate of paraaminobenzoic acid (PABA) and hen egg albumin (HEA), whereas Hartley strain guinea pigs developed an immediate type of hypersensitivity. When both strains were inoculated with live BCG, they developed hypersensitivity to tuberculin but the reaction in strain 13 guinea pigs were much weaker. When these guinea pigs were NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 199 sensitized with killed Q fever phase I ricket- tsiae and tested several weeks later with phase I cells, reactions were similar in both strains. Immunoprophylaxis Against Tuberculosis Previous studies have shown conclusively that heat-treated cell walls of Mycobacterium tuberculosis when coated with light mineral oil are highly immunog-enic and protect mice against aerosol challenge with fully virulent tubercle bacilli. Over a two-year period numer- ous vaccines have been prepared from 12 differ- ent batches of cell walls and tested repeatedly. Except for a few formulated with an insuffi- cient quantity of oil, all vaccines have been highly potent in repetitive tests. Mice immun- ized with these cell-wall vaccines have har- bored up to five logs fewer virulent mycobac- teria in their lungs after aerosol challenge than did unvaccinated control mice. In contrast, mice vaccinated with the standard Rosenthal's BCG vaccine had about two logs fewer when they were cultured after aerosol challenge. None of several vegetable oils, which are metabolizable, served to enhance immunogen- icity of cell-wall preparations. Both heavy and light mineral oil were effective whereas kero- sene was ineffective in enhancing potency. Drakeol 6VR, which has been approved as an adjuvant in human vaccines, was found to be as satisfactory as light mineral oil. The syn- thetic hydrocarbon, 7-n-hexyloctadecane, was slightly superior to light mineral oil. The 7-n- hexyloctadecane had one advantage not shared by other oils, because mixtures of this sub- stance with Tween 80 and cell walls disperse instantly in saline to give stable emulsions. Preliminary tests were undertaken to de- termine whether the cell-wall vaccines con- tained any endotoxic factor which would be harmful if this product were used in man. Mice survived intraperitoneal inoculations with doses as high as 10 mg and 11-day-old-chick embryos were not killed by intravenously in- jected doses of 400 fig, the maximum dose em- ployed in contrast, 0.25 g of cell walls from several enteric Gram-negative bacteria con- stitutes as average median lethal dose for chick embryos. Likewise, 20 fig of Gram-nega- tive cell walls will evoke a typical endotoxic febrile response in rabbits whereas doses of mycobacterial cell walls as high as 1 mg did not produce significant fever. The potency of cell-wall vaccines as tested by the aerosol challenge method was shown to be dependent upon a specific cell-wall anti- gen which was shared in part by related species such as M. kansasii, M. avium, M. aquae, and M. butyricum but not by species such as Salmonella, Brucella, Listeria, and Corynebacterium pseudotuberculosis. The re- sults of these experiments also gave prelim- inary evidence that cell walls from H37Ra con- ferred a higher degree of immunity than any other preparations so far tested. Skin tests in rabbits and guinea pigs sensi- tized with living or killed whole cells or cell walls of M. tuberculosis, M. balnei, group I, II, III, or IV of the unclassified Mycobacteria were performed in continuing efforts to develop more specific antigens for use in detection and differentiation of mycobacterial infec- tions. As previously reported for M. tuber- culosis, M. phlei, and M. butyricum, proto- plasms and cell walls of these additional My- cobacteria give rise to delayed reactions when injected intradermally into sensitized rabbits or guinea pigs. The specificity of the reactions produced by cell walls and protoplasm differed. Minimal amounts of cell walls produced lesions in animals sensitized with either homologous or heterologous material. However, weaker dilu- tions of protoplasm which give rise to reac- tions in animals sensitized with homologous antigens usually would not elicit reactions in those sensitized with heterologous material. In collaboration with Dr. Leon Schmidt, safety tests of the standard cell-wall vaccinje enhanced with Drakeol have been initiated at the National Center for Primate Biology, Uni- versity of California at Davis, California. Be- cause of reactions at the site of inoculation, which were entirely unexpected, future po- tency tests of this product in monkeys have been delayed until further attempts are made to eliminate the factor responsible from the cell-wall vaccine. The safety test has been re- designed to provide preliminary information on the protective properties of this vaccine for monkeys. 200 ANNUAL REVIEW OP INTRAMURAL RESEARCH Bordetella Pertussis Antigens Further studies on the histamine sensitizing factor (HSF) of B. pertussis were directed chiefly toward finding a practical method for isolating and purifying large quantities of this substance. Reasonably pure preparations have been obtained by magnesium sulfate precipi- tation of HSF from alkaline saline extracts of acetone-dried cells. HSF in Dupanol sensitized mice to histamine, to serotonin, to passively and actively induced anaphylaxis, and it in- creased antibody production by adjuvant ac- tion. This material was also found to induce hyperacute experimental allergic encephalomy- elitis (EAE) in rats that had received guinea pig cord suspensions. Thus, these preparations of reasonably pure HSF have most of the ac- tivity found in whole B. pertussis cells and possibly a single substance is responsible for most of these biological activities. B. pertussis strains are known to vary tre- mendously in their antigenic composition, as judged by agglutination and agglutination ab- sorption tests. Six specific antigens for B. per- tussis have been described. No significant dif- ferences in protective or histamine sensitizing ability were found among strains containing antigens 1, 3; 1, 2, 4; 1, 2, 3, 4; or 1, 2, 3, 4, 5. If immunity can be transferred passively with antigen-specific antiserums it would be possi- ble to identify the antigen associated with his- tamine sensitizing production and immunity in mice. Endotoxins The native hapten extracted from proto- plasm of Escherichia coli was studied further to determine whether this substance was indeed a precursor of endotoxin. This substance was found in nine of 11 strains of E. coli representative of four serotypes but none could be extracted from strains of Citrobacter freun- dii, Salmonella enteritidis, Salmonella typhi, and Serratia marascens. The molecular weights of native haptens isolated from these strains of E. coli averaged about 150,000; their physi- cal dimensions averaged about 15 A by 1200 A. In spite of the close serologic and chemical identity (sugar composition) of native hapten with endotoxin, it is unlikely that this ma- terial is a direct precursor of endotoxin. The physical dimensions could not conceivably be those of a subunit of endotoxin, and it does not behave like subunits derived from bile salt- dissociated endotoxin. The results of a study of the effect of sur- factants on endotoxins have provided addi- tional data in support of the theory of a micel- lar structure of endotoxin. When fully active endotoxin was treated with sodium desoxycho- late, subunits formed, with a molecular weight of 20,000 and dimensions of 10.5 A by 250 A. The dissociated endotoxin was about 100- fold less potent in the pyrogenicity test in rabbits and it was no longer capable of stimu- lating an antibody response in rabbits. When the bile salts were removed by dialysis, the subunits reaggregated to form endotoxin par- ticles with a molecular weight of 500,000 and dimensions of 40 A and 1,000 A. The biologic activity of the dialyzed material was essen- tially restored. These results were consonant with the theory that the elements of endotoxin are composed of micellar aggregates of chain- like subunits. Also, it seems that the varying physical structures of isolated endotoxins re- flect only the end-to-end aggregation of basic elements produced by the extraction procedure or subsequent treatments. Microbial Proteins and Nucleic Acids Studies have been continued on the genetic relationships between various microorganisms as determined by the degree of polynucleotide similarity. Nucleotide base sequence homology between species was determined by mixing ra- dioisotope-labeled and sheared DNA fragments of one species with agar-embedded DNA of another species. Recently taxonomists have suggested that two new genera, Francisella and Yersinia, be added to the family Brucellaceae. Pasteurella tularensis and P. novicida would be placed in the genus Francisella and P. pestis and P. pseudotuberculosis in the genus Yersinia. P. tularensis and P. novicida were found to have a strong reciprocal cross relationship but to have no genetic relationship with P. pestis, P. pseudotuberculosis, or P. multocida. NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES 201 Biology of Microbial Agents in Arthropods This new project is concerned with the de- velopment of arthropod tissue cultures, their use for biologic studies of arthropod-trans- mitted pathogens, and with the development and behavior of pathogens in arthropod vec- tors. So far, attempts to develop a serially- propagating line of tick cells from metamor- phosing nymphal viscera have not been success- ful, but some explants have survived for 240 days. Tick hemocytes cultures survived for an average of three weeks, with some retaining living cells for four months. Colorado tick fever virus was successfully grown in primary cul- tures of nymphal Dermacentor andersoni. By 21-35 days after inoculation, the virus content of cell-free media had increased by as much as three to five logs, and in one instance virus was still detected for 126 days after inocula- tion. . Electron microscopic study of the subcellu- lar structure of Rickettsia rickettsi and R. prowazekii confirmed findings of previously reported studies that their internal structure closely parallels that of many bacteria. In in- fected ticks R. rickettsi were found in tissues of the hind gut, salivary glands, genital sys- tem and Malpighian tubules. Each organism was surrounded by a "halo" thought to be created by a tissue-digesting enzyme. LABORATORY OF BACTERIAL DISEASES Mycoplasma Mycoplasma organisms have been repeatedly isolated from the tissues of normal mice, par- ticularly from the brain and lung and in one instance, from blood. All of these isolates proved to be Mycoplasma neurolypticum. Sim- ilarly, M. neurolyticum also has been recov- ered from brain, lung, liver and enlarged lymph nodes of mice of several leukemia bear- ing strains, and from mice with transmissible leukemia atuopsied in the terminal stages of the disease. The possible role of M. neuroly- ticum and other mycoplasma in the origin of leukemia, and the role they may play in pro- ducing confusion and misinterpretation of ex- perimental results in leukemia research is being explored. Work on murine and other animal myco- plasma has led to the first identification of swine mycoplasma as tissue culture contam- inants. A mycoplasma isolated from murine leukemia cells in culture by a scientist at the Sloan-Kettering Institute was shown to be re- lated to strains of M. granularum, a serotype previously recovered only from swine. These studies also confirmed the work of other in- vestigators (LID) that some previously un- classified tissue culture contaminants were strains of M. hyorhinis, another serotype of swine origin. In a collaborative study with Dr. Leon Smith, St. Michaels Hospital, Newark, N.J., over 60 blood specimens from normal post- partum patients have been cultured. Only one recovery of mycoplasma was made in this series. This patient developed a low grade fever 24 hours after delivery and M. hominis, type 1, was recovered from the blood. There was a concomitant rise in antibody against M. hom- inis in the patient's serum. This finding pro- vides additional evidence of the pathogenicity of M. hominis, type 1, for humans, and indi- cates the necessity of a search for these organisms in fevers of unknown origin. In germfree guinea pigs inoculated with My- coplasma pneumoniae, the organisms were re- covered from the nasal passages with regu- larity over the six-week test period. Results with a high passage laboratory strain and with a recent human isolate were sim- ilar. There was no clinical disease nor gross pathological findings. Only low serum antibody titers developed. The germfree guinea pig does not appear to be a suitable host for the study of M. pneumoniae. In collaboration with the Perinatal Research Branch, NINDB, a study is under way to de- termine the normal mycoplasma flora of mon- keys. About 80% of the monkeys cultured have yielded mycoplasma. To date at least four types of mycoplasma have been isolated. Preliminary data indicate that at least one of these types may be similar to, or identical with, a type isolated from man. 202 ANNUAL REVIEW OF INTRAMURAL RESEARCH Brucellosis As the program for eradication of bovine brucellosis has progressed, new and unexpected problems have arisen which threaten to im- pede progress toward total eradication of the disease. The question has arisen whether the use of Bmcella abortus, Strain 19 vaccine may be responsible for foci of infection in cer- tain problem herds, and whether the vaccine strain may actually be responsible for some infections. This Laboratory is collaborating in the study of unusual strains of Brucella isolated from wild and domestic sources in an attempt to clarify their ecological relation- ships. Studies on DNA homology in the genus Brucella were extended and completed. By the technique employed, DNA derived from smooth cultures of the three classical species, and from Br, neotomae, and rough Br. suis, cannot be differentiated one from another. The genus is thus highly homogenous and the S to R change was not reflected in the homology studies. NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES INTRODUCTION The following reports give some indication of the work of the NIAMD in the past year. Several features may be noted. There is a con- tinuing increase in interest and work in the field now known as Molecular Biology. This leitmotif of biological sciences in the 1960's has expanded to embrace work in clinical de- partments, pathology and biochemistry, and has also influenced the direction of work in the more physical and chemical sciences. In the latter case, the ready availability and the acknowledged biological importance of syn- thetic or purified polynucleotides and proteins (or their monomers) has stimulated quite basic studies of their physical and chemical properties. It seems reasonably certain that we are in the midst of a revolution in biology. Part and parcel of this revolution is the in- creasing interest in biological materials shown by chemists, physical chemists and theoretical chemists. And with this interest and activity, there has occurred an increasingly close col- laboration between physical and biological scientists. The answers to many fundamental problems in biology can only be found in the details of the chemical and physical interac- tions of the complex molecules found in living systems. It seems evident, therefore, that at NIH a systematic effort must be made to pro- vide facilities for a larger number of physical scientists, including those mainly concerned with theory. Engineering efforts to find ad hoc solutions to specific diseases cannot, it would appear, play more than a minor role in the eventual understanding of biological processes and con- trol of their disorders. The trend in the work of the scientists in NIAMD is clearly in the direction of investigation of biological ma- terials and processes at their most fundamen- tal level. It is not always apparent how many of these highly detailed and complicated studies of what seems to be very minor problems may have practical and clinical applications. Bac- teriophage, for example, may seem an unusual material for scientists to work upon, as these organisms only infect bacteria. Their extreme simplicity, however, (some strains have as few as a dozen genetic units compared to a hun- dred thousand or more in mammalian cells) permit accurate studies on the biochemical mechanisms of transfer of genetic information. This in turn serves as a model for virus in- fection of animal cells. Indeed bacteriophage infection of bacteria may well have significant implications in human disease as witnessed by the fact that diphtheria bacteria only pro- duce diphtheria toxin when they are infected with a certain bacteriophage. Hence, work in NIAMD in circular forms of DNA whose pres- ent function is unknown may well have impli- cations far beyond bacteriophage. On another level, the extensive studies on induction of enzyme synthesis in bacteria in NIAMD may appear unrelated to human dis- ease. However, unless we can understand this relatively simple biological system it seems clear that we will never understand how the human organism can adapt to the extraordi- narily varied diets people live upon, nor the dramatic situations in which certain cells change their pattern of adaptation and become cancerous. In fact, the best hope for eventual control of cancer in man is much more likely to derive from studies of the forces involved in the interaction of nucleotides in DNA than from empiric testing of anti-tumor drugs. 203 204 ANNUAL REVIEW OF INTRAMURAL RESEARCH Another major problem in human disease involves congenital abnormalities. There is some worry nowadays that, with improved methods of maintaining individuals possessing deleterious genes, we may eventually approach the situation in which the population is di- vided only into physicians and patients. A ra- tional approach to this problem seems to in- volve two factors: (1) differentiation, since errors in this process may lead to non-genetic congenital malformations and (2) mechanisms of disease production from deleterious genes. The first aspect is currently under study under defined conditions as the differentiation of the mammary gland. The hormones required, some of the biochemical reactions involved and their morphologic expressions have been elucidated. The exact role of the hormones and their mode of action, if they can be uncovered, could lead to practical approaches for preventing errors in differentiation — namely congenital anom- alies. The mechanism of the production of disease from genetic errors has already been partially clarified in many cases. In sickle cell anemia, not only the nature of the amino acid substi- tution which is the primary defect, but also a sensible chemical rationale for how this substi- tution produces the clinical picture of sickling have been advanced — largely by scientists now at NIAMD. The precise biochemical lesions in galactosemia, xanthinuria, homocystinuria, cystinuria, cystathioninuria, and gout have been elucidated over the past few years at NIAMD and elsewhere. In most cases a sensi- ble and effective therapy has been devised after the precise metabolic defect has been discov- ered. Just as importantly, tests have been de- vised for many of these inherited defects so that the healthy carrier can be identified. With this information there is hope that a positive eugenics type approach may eventually be able to reduce the incidence of these diseases. Many times apparently esoteric chemical pro- cedures are developed which appear to be of only theoretical significance. Recently, however, NIAMD scientists, by separating the optical isomers of certain morphine derivatives, were able to show that one of the isomers actually antagonized the dependency effects of mor- phine but retained analgesic activity. The fund of basic knowledge in the biological sciences is still immeasurably small compared to the rich and marvelously complex organiza- tion of structures and compounds that result in living cells. We can therefore anticipate that rational therapy for the serious diseases that beset mankind will await a clearer knowledge of biological processes. LABORATORY OF NUTRITION AND ENDOCRINOLOGY Studies on Folic Acid The catalytic and kinetic properties of the conversion of folic acid and dihydrofolic acid to the metabolically active tetrahydro-form have been investigated utilizing the highly purified dihydrofolic reductase from liver. At neutral pH values with TPNH as the reducing agent, the enzyme is completely specific for dihydrofolate, whereas folic acid and DPNH are inert as substrates. However, in the acid pH range, 4-5, both folic and dihydrofolic acid serve as substrates and DPNH may serve as the reducing agent. Kinetic analysis of the reaction indicated that the apparent affinity of dihydrofolic re- ductase for dihydrofolate is remarkably high. In addition, the interaction of dihydrofolate with the enzyme does not follow the expected "normal" kinetic behavior with respect to the velocity of the reaction vs. dihydrofolate con- centration. On the other hand, the interaction of folic acid with the enzyme exhibits "nor- mal" kinetics and the analysis suggests that folic acid is bound to the enzyme some 50 times less strongly than dihydrofolic acid. The inter- action of TPNH with the enzyme exhibits "normal" kinetic behavior at neutral pH when dihydrofolate is used as substrate. However, at acid pH values "abnormal" kinetics are noted using either dihydrofolate or folate. In addition, TPN was observed to be a potent inhibitor of the reductase reaction. Kinetic analysis demonstrated that the apparent affin- ity of the enzyme for TPN was almost the same as for TPNH. This is in line with the NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 205 observation that TPN as well as TPNH is an effective stabilizing agent for the enzyme. Previously reported studies which described a marked activation of this enzyme by urea and organic mercurials suggested that dihy- drofolic reductase possesses many of the prop- erties of an "allosteric" enzyme. From this point of view, the "abnormal" behavior of the reaction with respect to dihydrofolate is not surprising since many of the "allosteric" en- zymes exhibit this type of "abnormal" kinetics. Thus the "normal" kinetic behavior observed when folic acid is used as the substrate correl- ates with the lack of significant stimulation of the enzyme by both urea and the organic mercurials in the presence of the fully oxi- dized folate, pteroylglutamic acid. The studies on the more complex and as yet unidentified forms of folic acid in tissues were directed to the stockpiling of a stable concen- trate and the development of chromatographic techniques to be used in further purification procedures to yield several components as pure compounds. (Drs. B. T. Kaufman and J. C. Keresztesy) Large-scale Processing of Biological Materials The large-scale laboratory continues to as- sist NIH investigators in procedures which re- quire processing and preparing materials in the volume too large for normal laboratory facilities. Fermentation operations were again this year in greatest demand. (D. L. Rogerson, Jr. and Dr. J. C. Keresztesy) Studies in Experimental Nutrition The studies on the development of liver ne- crosis in rats as influenced by various dietary components and state of "germfreeness" have been continued. It was found that many of the test animals may die without showing any, or very little, obvious damage to the liver cells. Other parameters for assessing liver damage are being explored. The possible role of the intestinal flora in the prevention of blacktongue in dogs was in- vestigated. The antibiotic, terramycin, was without effect. The administration of mixtures of antibiotics, together with the isolation tech- niques, are under study to influence intestinal flora in rats and dogs which will prove to be beneficial to the host. (Dr. F. S. Daft and E. G. McDaniel) Study of Protein Hormones Modifications of the structure of thyroid stimulating hormone (TSH), whether by en- zymic or chemical means destroy both biologi- cal and immunological activity. Physicochem- ical studies with bovine growth hormone have shown that in alkaline solution a monomeric form exists which again dimerizes upon acidi- fication. (Dr. P. G. Condliffe) Diabetes and Fat Metabolism The induction of diabetes in rats, by hor- mones from a transplantable pituitary tumor, was studied. Partial pancreatectomy was nec- essary to reduce the antidiabetic effect of en- dogenous insulin from the host's pancreas. Based on this finding partially pancreatecto- mized rats have been used to quantitatively study the hormonal interactions of ACTH and growth hormone upon the induction of dia- betes. A synergism was demonstrated. Immu- noassays of insulin in blood showed that the hormonally induced diabetes is not due to a fall in insulin level for actually the insulin level increased. (Dr. R. W. Bates) The metabolism of the diaphragm and fat tissue from rats after the induction of diabetes, by operative procedures, were investigated. The diaphragm muscle in vitro responded nor- mally to insulin but the adipose tissue was more insensitive to insulin. This latter partially accounts for the in vivo peripheral resistance to insulin found in some diabetics. (Dr. S. S. Chernick) Mechanism of Action on Isolated Fat Cells Using individual fat cells, it was demon- strated that many of the effects of hormones on the fat cells are at the level of the cell mem- brane. The hormones modify the permeability of the wall either directly or indirectly, e.g., lipolytic hormones release fatty acids which will alter cell permeability. The cell membrane may be altered sufficiently so that intracellu- 206 ANNUAL REVIEW OF INTRAMURAL RESEARCH lar enzymes are released to the medium. These effects are believed to be mediated by effects on the phospholipids in the cell wall. (Dr. M. Rodbell) Metabolism and Function of Fat Soluble Vitamins and Related Substances Lipid analyses of testes from eight species of animals including man showed characteristic fatty acid patterns, particularly in the polyun- saturated acids, for each species. These differ- ences could not be explained soley by the varying diets. Human testis had the highest content of the most unsaturated fatty acid (22 carbons, 6 double bonds) found in tissues. A comparison of the lipid changes which oc- cur in rat testes damaged by deficiencies of vitamins A and E and also of zinc revealed that the most marked loss of phospholipid and alteration of the proportions of polyunsatu- rated fatty acids occurred in vitamin E defi- ciency. These results suggest a special involve- ment of vitamin E in lipid metabolism in the testis. The biopotencies and metabolism of the amine and N-methyl amine analogs of tocopherols we studied in chicks, a-tocopheramine was equally as active as standard «-tocopherol whereas (3- tocopheramine (like /3-tocopherol) was about one-third as active. N-methyl-/3-tocopheramine, however, was fully as active as /?-tocopherol as was also N-methyl-y-tocopheramine. The toco- pheramines were recovered unchanged from the blood and liver and were not converted to their tocopherols or quinones. (Dr. J. C. Bieri) Reversible Chemical Modifications of Cytochrome Salicylaldehyde was found to react specifi- cally with the lysine residues of cytochrome c to form a complex with reduced activity in the NADH reductase system and in the oxidation of ascorbate. Decrease of activity and also of solubility was proportional to the number of lysine groups involved (up to a total of 19 per molecule). Dialysis of the complex removed all salicylaldehyde and restored solubility and the ability to oxidize ascorbate but did not restore NADH-reductase activity until treatment with acid or alkali. The latter observation suggested the existence of polymers and chromatographic evidence for at least eight molecular species was obtained. (Dr. J. N. Williams) Relationship Between Purine-Pyrimidine Balance and Hepatic Fat Metabolism Perfusion studies comparing livers from nor- mal and orotic acid-fed rats have shown that the normal liver synthesizes and secretes into the perfusate ample quantities of fatty acids. The liver from the orotic acid-fed rat has a re- duced rate of fatty acid synthesis but more strikingly there is an almost complete block in lipid secretion into the perfusate. Further evidence that orotic acid interferes with lipo- protein formation was obtained by immuno- and paper electrophoretic analysis, ^-lipopro- teins were absent and a-lipoproteins signifi- cantly reduced in plasma from rats fed orotic acid. The disappearance of plasma /?-lipopro- teins was complete one week after administer- ing orotic acid but the effect was reversed by feeding adenine for only one or two days. (Dr. H. G. Windmueller) LABORATORY OF BIOCHEMISTRY AND METABOLISM Polysaccharide Metabolism Bacterial Systems Work on the 3-amino sugar isolated from Xanthomonas campestris has been completed. The crystalline preparation has been identified unequivocally as 3-acetamido-3,6- 450 fold from rabbit serum, and character- 240 ANNUAL REVIEW OP INTRAMURAL RESEARCH ized. Of particular interest is the finding that protonated species of the amines interact with the enzyme (in contrast, the nuproton- ated form reacts with the human monoamine oxidase). (C. M. McEwen) Serum levels of monamine oxidase in 119 cases of liver disease indicated a positive cor- relation of elevated enzyme levels with hepatic fibrosis, portal hypertension, and portal-system collateral circulation. This may possibly offer promise as a simple screening test for cirrhosis. (C. M. McEwen with Dr. Donald 0. Castell, Dept. of Medicine, U.S. Naval Hospital, Be- thesda Md.) Polysaccharides and glycoproteins A new rhamnose nucleotide, UDP-L-rham- nose, has been isolated and characterized from a bacterial source. (V. Ginsburg) Tracer experiments have shown that the ribose in lipopolysaccharide (Salmonella ar- tis) is formed by the same pathway as RNA ribose. (R. Kaufman) Control of enzyme levels in mammalian tissues Animal tissues contain four distinct hexo- kinases, including a liver specific high K glucokinase and three different low K m hexo- kinases. The latter have been purified and characterized. Each purified enzyme type, re- gardless of tissue source, has certain unique properties that distinguish it from other hexo- kinase types. (R. T. Schimke, L. Grossbard and E. W. Sweeney) The inactivation of «-glucosidase of Sac- charomyces cerevisiae had been studied as a model of "deadaptation". This inactivation re- quires energy, and is prevented by a cyclohexi- mide, an inhibitor of protein synthesis. (R. T. Schimke and E. W. Sweeney) Burn — shock studies Burns Studies were continued on the role of bac- terial infection in the mortality observed after burns. E. coli monocontaminated mice or a-hemo- lytic streptococcus and pasteurella decontami- nated mice show no difference in mortality after burns from germ-free mice. (K. Markley and E. Smallman) Burned mice were 300-1000 times more sen- sitive to E. coli and S. typhosa endotoxin than non-burned mice. Mice tolerant to a lethal dose of endotoxin showed a lower shock mortality after burns. (K. Markley and E. Smallman) Simple methods were devised for standard- izing fatal pseudomonas infections in burned mice. Six drugs were found to be effective in treatment. In view of the importance of bac- terial infection in clinical burns, this method may be very important in the evaluation of new therapeutic agents. (S. M. Rosenthal) Tourniquet Trauma Somewhat surprisingly germ-free mice showed a significantly decreased shock mortal- ity compared to conventional mice. Both groups were very sensitive to E. coli endotoxin. Mice made tolerant to endotoxin were protected partially against trauma, and mice surviving tourniquet trauma were protected against en- dotoxin. These studies implicate endotoxin as an important factor in shock mortality, in ad- dition to the electrolyte and fluid factors pre- viously reported from this laboratory. (K. Markley and E. Smallman) Sulfur-containing compounds It has been previously reported that the en- zyme glutathione reductase has two reducible bonds per unit of its flavin content, only one of which is involved in its catalytic function. From recent experiments a major new concept has developed according to which the second reducible bond, which appears to be a disulfide, does function in a regulatory mechanism al- though it does not participate in the catalytic mechanism. When this bond is reduced the enzyme ceases to function. Its reduction is hin- dered by TPN which therefore may be classi- fied as an activator. (S. Black, S. Hopper and Blondel Hazel) Leprosy studies Growth of acid-fast organisms have been observed in macrophage cultures after inocu- NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 241 lation with biopsy material from human lep- rosy cases. A few serial passages were success- ful. (Y. Chang and R. Anderson in Dr. Knight's laboratory) Improvements have been made in the growth conditions of Mycobacterium lepraemurium in macrophages, permitting a generation time of 7 days, which is comparable to the growth rate of this organism in mice. (Y. Chang and R. Anderson) LABORATORY OF PHYSICAL BIOLOGY The Laboratory has experienced a productive year, with no major changes in general orien- tation. Research highlights for fiscal 1966 are summarized briefly below, grouped in one of several possible series of informal categories. Molecular Structure A major interest of LPB continues to be physicochemical studies of molecular structure and interaction at levels of organization rang- ing from interatomic forces in simple inor- ganic molecules to tertiary and quaternary structure of proteins and polymers. Last year's Report went into some detail about the prin- ciples of operation of NMR (nuclear magnetic resonance spectroscopy), EPR or ESR (elec- tron spin resonance spectroscopy), IR (infra- red spectroscopy), ORD (optical rotatory dis- persion), CD (circular dichroism) and other physical methods commonly used by LPB scientists: that material will not be repeated here. Mention should be made, however, of our rapidly growing use of electronic computer technology for collecting and analyzing data. In a continuing effort to specify unique in- tramolecular force fields for hydrides and fluorides of Sn, B and related Group IV-VI elements it was found that Coriolis coupling constants could be used to supplement rota- tional distortion and vibrational amplitude data. (Levin, Abramowitz, Ziffer, Berney, Comeford) Isotopically labeled oxyhalide complexes of Cr, Mo and W have been investigated by ESR in an attempt to obtain electron density meas- urements and in-plane bond parameters. (Kon, Sharpless) Free radicals of pyruvic acid de- rivatives were examined by ESR to identify isomers and ESR spectra of irradiated ribonu- clease, gelatin, myoglobin and lysozyme were studied to determine optimum exposure to tri- tiated H 2 S for y-ray radiolysis. (Kon, Cahn- mann, Matsuura, Riesz, White) NMR spectra of the iodo, methoxy, nitro and other analogs of DDT have been analyzed in a continuing study of the resonance of the aro- matic rings. (Sharpless) By studying the UV spectra and optical ac- tivity of various acylchymotrypsin derivatives and related model compounds it has been pos- sible to determine that the normal s-trans configuration of the C = bond, with respect to the C = C in acrylic substrates, is isomerized to the s-cris configuration. This explains why the more energetic cis configuration is favored when the substrate complexes at the active site of the native enzyme. (Charney, Bernhard) UV spectra of substituted ethylenes at var- ious temperatures have been recorded to pro- vide a basis for a quantum mechanical expla- nation of the so-called mystery spectral band of olefins. (McDiarmid, Charney) Instrumentation and theory have now been completed for electric field induction of dichro- ism and birefringence in DNA and polypep- tides, from which information on tertiary conformation may be expected. (Yamaoka, Charney) Similar plans for gaseous HC1 are also about to bear fruit. (Needham, Charney) NMR studies of base pairing of nucleosides have shown specific hydrogen bonding similar to that between polynucleotides and have pro- vided thermodynamic and kinetic data for elu- cidating internally hindered rotation of amino and methylamino groups in substituted cyto- sines. A new technique for IR spectroscopy at 20° K is affording much improved band resolu- tion. (Becker, Miles, Shoup, Gramstad, Brad- ley) The three photodimers of the cyclohexenone piperitone have been studied by NMR and identified as head to head dimers. The stereo- isomerism at the junctions of the 6- and 4- membered rings has been determined as both cis in two of the dimers and at least one trans 242 ANNUAL REVIEW OF INTRAMURAL RESEARCH in the third (Ziffer). A similar stereochemical analysis is now being applied to steroidal com- pounds. (Ziffer, Williams) ORD measurements of non-planer conju- gated transoid dienes confirm the validity of a rule, derived in this laboratory, that connects the sense of skewness of the chromophore with the sign of the Cotton effects. A heteroannular cisoid diene, however, has given a Cotton effect of sign opposite to that predicted, and will re- quire further investigation. (Weiss, Ziffer, Sharpless) An analysis of ORD curves of the combina- tion of the dye acridine orange with DNA and polyglutamic acid showed that four Cotton effects are required for unique specification of the band structure in both cases. (Yamaoka and Resnik). In the course of this work an im- portant source of artifact in certain ORD in- struments was discovered. (Resnik, Yamaoka) Surface Phenomena Besides work involving excitation and per- meability changes, much LPB research im- pinges on, implicates or implies controlled pas- sage of material across cellular and subcellular membranes. The Laboratory also supports in- vestigations aimed at providing defined and precisely controlled physicochemical models for certain properties of biological membranes. Work has continued on defined synthetic liquid ion-exchange membranes which exhibit high selectivity and transmissivity for most inorganic and organic ions. The behavior of such membranes has also been treated theoret- ically in relation to carrier transport and in comparison with biological membranes. (Soll- ner, Shean) Exploration has begun of long-range repul- sive forces, amounting to as much as 25 dynes/cm 2 over distances as great as three microns, which arise between charged glass surfaces in liquid media. Such forces are im- portant in colloidal dispersions and organized structures. (Sollner, Gottlieb) The forces which maintain structural integ- rity and reactivity of manomolecular layers have been studied with phosphate films spread on thin oil membranes. The role of hydrogen bonding in phosphate group aggregation with H + decrease has been defined, and a 10 cycle/ sec fluctuation in membrane resistance has been induced by low D.C. voltages. (Gershfeld) Physiology and Biochemistry Dimethyl sulfoxide, an industrial solvent with a remarkable spectrum of biomedical effects, was found to intensify the effects of exercise upon rats, as reflected in liberation of various tissue enzymes into the serum, and pathological changes. (Altland, Highman, Nel- son, Brubach, Barbus, and Thompson) Rat lysosomes were found to be ruptured by hypoxia before serum enzyme concentration increases suggesting that lysosomal enzymes are important in initiating hypozia pathology. (Nelson, Parker) In studying protein metabolism during in- sect metamorphosis it was found that nearly twenty distinct small peptides occur in larval blood, that blood protein concentration in- creases some 300-fold during larval growth and that larval blood proteins are probably trans- ferred intact into adult tissue. (Levenbook, Bauer, Chen) Excitation and Coupling Processes by which one type of energy is trans- duced into another or one cell structure trig- gers activity in another site have increased sev- eral LPB investigators. Previous work on the linkage between ab- sorption of light in the squid retina and the generation of electrical current in visual ele- ments is being supplemented by chemical stud- ies of the visual pigments. The results suggest that retinaldehyde links to the protein opsin via the e-amino group of lysine (F. Hagins, Gross, Bauer, W. Hagins). Changes in Cotton effects of rhodopsin with exposure to light sug- gest a three point association, with no change in secondary structure of the peptide part of the photopigment. (Hagins, Ziffer). A number of phospholipids associated with rhodopsin have been identified. (Adams) In striated muscle the electrical excitation that leads to contraction is thought to be con- NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 243 ducted into the muscle via infoldings of the surface membrane and to eventuate in release of Ca + + . It has now been found that the cal- cium release depends on depolarization of the "sarcoplasmic reticulum" (Podolsky, Costan- tin). By comparing the response of skinned slow and fast frog muscle fibers to Ca ++ , the 10X difference in contraction rates has been shown to be intrinsic to the fiber, not the acti- vation mechanism. (Costantin, Podolsky) A number of sophisticated optical methods localization of and measurement of molecular orientation of chlorophyll and protein in sub- cellular structures of green cells have been de- veloped. Preliminary work indicates that these techniques will provide new data for the eluci- dation of pathways of light-activated energy transfer. (Olson, Jennings) Biological Interactions The study of the dynamic influences of cell on cell in an organism (as opposed to a cul- ture) or of organism on organism in a com- munity (as opposed to a population) represents the opposite extreme to the biochemical or ultrastructural dissection of the cellular unit. Several examples have been investigated re- cently in LPB. The simple multicellular animal Hydra in- cludes species which are colorless and species in which many of the cells contain symbiotic green algae. By freeing green hydras of their algae the process of reinfection and the rela- tive infectivities of algae separated from host tissue, of several species of free-living green algae, and of algae migrating from grafted green tissue, have been studied. (Park, Ort- meyer, Greenblatt) The colonial marine soft coral Renilla is favorable for study of nervous conduction at its simplest level because the individual zooids are connected by a non-polarized slowly-con- ducting nerve net. By local electrical stimula- tion and recording of induced bioluminescence the kinetics of the responses of the two classes of individuals in the colony have been deter- mined and some details of the coordination of excitation established. (Hanson, Buck) A unique example of insect communal activ- ity, in which thousands of fireflies flash in rhythmic synchronism for long periods, was studied in Borneo and Thailand. Coordination was found to be precise within 30 msec in a 500 msec cycle, to depend on visual feedback, and to involve central nervous delay and ad- justment of endogenous excitation with respect to the preceding mass flash. (J. Buck and E. Buck) Biological Ultrastructure Our electron microscope facility has made possible a variety of findings, ranging from evidence supplementing or confirming results from other methods to full-dress cytological investigations. In studies of frog striated muscle an im- proved test for intracellular localization of so- dium was developed (Tice). Glucocoricoster- oids were found to destroy the mitochondria of both red and white muscle of the rat, but affect fibrillar structure only in red muscle. (Tice, Engel) A comparative study of the photogenic or- gans of certain American and Far Eastern fire- flies has revealed numerous systematic differ- ences, the most striking being the intracellular penetration of air tubules and direct nerve- photocyte connections in the oriental forms. (Peterson) Electron micrographs of human sickle cell anemia erythrocytes and of extracted S-hemo- globin suggest that the pigment molecules can stack in chains, the chains aggregating in par-, allel to form six-stranded hollow molecular cables. (Murayama) Ox liver catalase has been shown to consist of approximately spherical units which give a tetramolecular orthorhombic unit cell in dry crystals and a hexagonal bimolecular associa- tion in wet crystals. Thyroglobulin molecules do not form true crystals but long chains of linearly associated spherical units in random side-by-side array. (Labaw) Electron micrographs were supplied as illus- trations for several investigations described elsewhere. (Hanna) 244 ANNUAL REVIEW OF INTRAMURAL RESEARCH Cellular Biology Cells as functional units have received an unusual amount of attention from LPB inves- tigators this year. In a continuing attempt at exhaustive bio- chemical characterization of a particular cell type, a comprehensive analysis of carbon me- tabolism in the green flagellate Euglena has been completed. As part of a survey of extraor- dinary metabolic capabilities in microorgan- isms, Thiobacillus thiooxidans has been found able to grow in endogenous H 2 S0 4 and survive at an acidity of pH 0.0, and Halobacter salinar- ium, growing in a required concentration of 4 M NaCl, has been shown not to excrete large amounts of nucleic acid as had been reported in the literature. Kempner, Miller) In the luminous bacterium Achromobacter fischeri a "noise spectrum" analysis has shown that the cell does not emit photons in packets (Hanson, Hagins), and continuous recording of light emission during culture growth dem- onstrates a wide but systematic discrepancy between cell number and luminous output. (Hanson, Kempner) The rat hemoparasite Trypanosoma lewisi cannot ordinarily establish itself in mouse blood, but by blocking the mouse's immunological defenses by administering rat globulins the parasite is enabled to grow in the foreign host. (Greenblatt, Clipper) LABORATORY OF BIOPHYSICAL CHEMISTRY The main interest of this laboratory is the study of the correlation between structure and function of proteins with special interest cen- tered on those involved in blood clotting and muscular contraction. The main aspects of the clotting of fibrinogen have now been clarified. Fibrinogen can carry out its role in hemostasis and wound healing only if two enzymes act on it. Thrombin, the clot forming enzyme of plasma, splits off two peptides from fibrinogen, and as a result, the fibrin molecules form a network. The clot stabilizing enzyme (Laki-Lorand Factor) intro- duces bonds between amino groups of one fi- brin molecule and the asparagin and gluta- mine groups of another. Apparently, only those asparagin groups are involved that carry a sugar moiety. (Laki, Gladner, Chandrasek- har) From these basic foundations, we can now branch out to inspect the broader biological significance of this clotting process. It is now well established that the peptides released from fibrinogen have physiological ac- tivity. This could be conveniently followed by their action in protentiating bradykinin. It turned out that in a number of peptides, a region containing -Asp. Tyr- residues, in oth- ers, the region containing -Asp. Ser- residues, could be implicated in this activity. Using sym- pathectomized animals, a direct vasoconstrictor action of these peptides could also be demon- strated. (Gladner, Osbahr, Colman, Laki) Since our pioneering work, which estab- lished the amino acid sequence of the peptides released during clotting of bovine fibrinogen, the sequence of many other of these peptides originating from various animals is known. The similarities in any two of these peptides may be correlated to the time elapsed since these separated from a common ancestor. It was found that if the logarithm of the amount of correspondence (similarity) is used for plot- ting, a straight line resulted. In contrast to a similar plot of other proteins (cytochrome C, for example), the plot of these peptides cannot be extrapolated beyond the time of the appearance of the vertebrates. From this, we concluded that before the appearance of the vertebrates, a different clotting system may have operated. It is now an established fact that in Limulus (horseshoe crab), the clotting proteins are not freely distributed in the plas- ma, but are confined to cells circulating in the blood. These cells release the clotting material when they come into contact with endotoxin. Since these cells in the Limulus are the pro- totype of the platelets of vertebrate plasma, it is now understandable that a serious condition arises if the platelets become sensitized to en- dotoxin and release their clotting proteins. (Laki) It appears that clot formation is involved, not only in hemostasis and wound healing, but also in inflammation reactions. Under the in- NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 245 fluence of Cortisol, an enzyme can be isolated from the skin of a number of animals, which clots bovine fibrinogen by splitting off an elon- gated version of peptide B. (Gladner, Houck, Murtaugh) The molecular weight of the liver enzyme, transglutaminase, isolated from guinea pig liver by Folk's group, has been established. (Carroll, Mitchell) This enzyme acts on fibri- nogen as the clot stabilizing enzyme. It is now becoming increasingly evident that without a detailed knowledge of the struc- ture of the contractile proteins, the mechanism of muscular contraction cannot be understood. Therefore, our attention was centered on the molecular aspects of these proteins. Actin and tropomyosin were modified by enzymatically introducing the amine, putres- cine, into the glutamine residues of these pro- teins. The liver enzyme, transglutaminase, used in these experiments, could be demon- strated to occur in many other tissues. In addition to introducing amines into these pro- teins, this enzyme could bind actin and tropo- myosin together by using actin as the amine donor and tropomyosin as the acceptor. This enzyme may thus function as a last step in protein synthesis by connecting the subunits of a larger protein together. (Laki, Derrick) Methylated, SH-reduced and S-carboxyme- thylated tropomyosins were prepared to clarify the conformation of this highly charged and helical constituent of muscle. (Bodwell) Two years ago, it was shown in this labora- tory that methylation of glycerol-treated mus- cle completely inhibited its ATPase activity, but reduced the shortening response by only 40%. Pyrophosphate, adenosine diphosphate and other large cations became contracting agents for these methylated fibers. This find- ing prompted us to ascertain which end-groups or residues are methylated. We are investi- gating this problem by the use of C 14 -contain- ing dimethyl sulfate (methylating agent). (Bowen) We have demonstrated that actin and tropo- myosin contained an acetylated N-terminal group. These findings gave us further chem- ical means with which to establish minimum molecular weights for these proteins. (Laki, Alving) I would like to list now experiments that are not directly related to the topics discussed above, but knowledge gained from these stimu- lated our way of looking on some of the prob- lems. The molecular weight of a small, bacterial nuclease was determined by sedimentation and diffusion measurements and found to be con- sistent with the value calculated from the chemical composition. Using viscosity measure- ments, this protein, which requires calcium ion for its enzymatic activity, was found to under- go a thermal transition to a more unfolded form at about 50° C, but this transition is prevented or reversed by the presence of small amounts of Ca + + . (Carroll, Anfinsen, LCB) A relatively crude model of lysozyme, based on the assumption that charged groups occur in clusters, gave a chain configuration prac- tically identical with that determined from X-ray crystal analysis. (Saroff) The excretion of abnormal amounts of cys- tathionine in the urine occurs in patients which may have various congenital defects and mental retardation. The demonstration of an abnormally high concentration of cys- thathionine in tissue extracts of a case of cystathioninuria prompted the hypothesis that this syndrome resulted from a deficiency of the enzyme cystathionase. We tested this hy- pothesis and found that cystathionase activity was markedly reduced in the liver of a pa- tient suffering from cystathioninuria. (Mudd, NIMH; Finkelstein, VA; Laster, CI; Irre- verre) The discovery of the new amino acid, cis- and £rans-3-hydroxyproline both found occur- ing in an antibiotic polypeptide, Telomycin, and the trans-form, in vertebrate and inver- tebrate collagen, led to the investigation of the amino acid composition of the proteins and cell wall of the Telomycin producing strep- tomyces. Whereas the organism synthesizes the two diastereoisomers of 3-hydroxyproline in Telomycin, neither of these two amino acids is incorporated into the proteins or cell wall of the organism. 246 ANNUAL REVIEW OP INTRAMURAL RESEARCH An unusual amino acid was also isolated from the hydrolysates of the proteins and cell walls of the streptomyces by column and paper chromatography and identified as muramic acid. A very sensitive, modified Sakaguchi spray for the detection of arginine and monosub- stituted guanidine compounds was developed. Due to its sensitivity and stability of color, this spray has been found useful for locating arginine and arginine-containing peptides specially in fingerprinting or sequence studies in proteins. It was found that polystyrene sulfonic acid, a synthetic model for charged proteins, bound cupric ions quite strongly without causing pre- cipitation. The indication of binding comes from the change of the absorption spectrum of the copper upon the addition of polymer. Barium ion is able to desplace the bound cop- per and return the absorption characteristics of the free cupric ion. (Carroll, Eisenberg) LABORATORY OF MOLECULAR BIOLOGY Structure and Function of Glutamate Dehydrogenase from Bovine Liver Succinylation of about 4 lysines per chain of the bovine liver enzyme yields an electro- phoretically more rapidly moving but still en- zymatically active protein. Mixture of the succinylated protein with the native protein (each containing 6 subunits) should permit the detection of exchange of subunits between the two forms of the enzyme. However, electroph- oretic analysis of such a mixture showed no exchange even under conditions where allos- teric modification of the protein is going on. We conclude that the allosteric change in gluta- mate dehydrogenase mediated by GTP and DPNH does not involve dissociation of the in- dividual subunits (MW 50,000) from the na- tive enzymatically active molecule (MW 300 to 400,000). Reaction of the enzyme with fluorodinitro- benzene suggests that only a single lysine is reactive under the conditions tested. The pep- tide containing this lysine will be isolated and its composition and sequence investigated to provide further evidence for the conclusion presented last year that the subunits of this allosteric enzyme are chemically indentical. (G. Tomkins, J. Kallos, E. Appella) Enzyme Induction in Bacteria Continued studies on the lactrose (lac) op- eron have suggested that the enzymes are induced in sequence rather than simultaneously as a result of the sequential transcription of the lac genes. Delay in transcription of the A gene (for thiogalactoside transacetylase) re- sults in a slower production of this enzyme with respect to that of /3-glactosidase. A delay in transcription can be induced either by in- hibitors of protein synthesis or by nonsense mutations (amber or ochre) in the gene. The delay induced by the latter means can be cor- rected by suppression of the nonsense muta- tions. These findings can be rationalized by a model relating messenger RNA transcription in simultaneous enzyme synthesis. (G. Tom- kins, P. von Knippenberg, D. Alpers) Cysteine Biosynthesis Two enzymes have been identified in ex- tracts of E. coli and S. typhimurium which catalyze the conversion of L-serine and inor- ganic sulfide to L-cysteine. The first enzyme catalyzes the reaction: L serine + acetyl CoA to form O-acetyl L-serine. The second enzyme catalyzes the reaction O-acetyl L-serine + SH to give L-cysteine. The gene locus controlling the first enzyme is the Cys E region of the salmonella chromosome. The gene controlling the second enzyme is not yet known. The first reaction is inhibited by the end product L- cysteine. This is the first demonstration of the mechanism of cysteine biosynthesis in micro- organisms. (N. Kredich, G. Tomkins) Enzyme Induction by Steroid Hormones A newly established line of hepatoma cells growing in tissue culture has been developed. The cells grow exponentially with a doubling time of 24 to 30 hours. Dexamethasone 10 _5 m | or other glucocorticoids induce the synthesis of tyrosine-a-ketoglutarate transaminase from 5 to 20-fold within 6 to 8 hours after their NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 247 addition. Induction occurs in dividing or rest- ing cells and is inhibited by inhibitors of either protein or of RNA synthesis. Inhibition of RNA synthesis after maximal levels of the enzyme are reached causes a further stimula- tion of enzyme synthesis. It is thought that the inducer acts at two levels, the nuclear level and the cytoplasmic level to regulate the syn- thesis of the protein. The enzyme has been crystallized from rat liver and its physical and enzymological properties studied. An antibody prepared to it shows immunological identity with uninduced rat liver enzyme as well as induced and basal tyrosine transaminase in the hepatoma cells. Experiments involving ra- dioactive amino acid incorporation into im- munological precipitates suggests that the in- crease in enzyme level is due to an increase in the rate of synthesis of the protein. Experi- ments using C 1J and tritium labelled amino acids suggests that in addition to tyrosine transaminase several other proteins are also induced by the hormone. Studies on RNA syn- thesis show minimal if any increase in the rate of messenger RNA production. Other classes of RNA do not appear to be affected by the hormone. (G. Tomkins, E. B. Thompson, S. Hayashi, T. Gelehrter, B. Peterkofsky, D. Granner) Active Transport of Amino Acids in Salmonella Typhimurium Further work has been done on the trans- port systems for amino acids in Salmonella. Radioactive lipids have been obtained after ex- posure for one minute of whole cells to radio- active amino acid. At least four fractions could be distinguished by column chromatography. Two of these liberated the intact amino acid upon mild alkaline hydrolysis. The identifica- tion of these radioactive intermediates is under way. Involvement of this fraction into the process of transport is being investigated. The histidine permease mutant has been mapped (50% linkage with Pur F). The aro- matic permease mutant has been partially mapped (between pro A and gal). (Giovanna Ferro-Luzzi Ames) Amino Acid Incorporation into Protein in Vitro: Mechanisms and Possible Metabolic Controls Experiments in model systems showing that the rate of protein synthesis directed by syn- thetic polyribonucleotides can be controlled by reversible alterations in ordered structures of the polymers have been extended. Investiga- tions of the mechanisms of the final step in protein synthesis have been initiated. (E. S. Maxwell, S. Raebum, F. B. Howard, H. T. Miles, and L. Barnett) Chemical and Structural Investigations of Nucleic Acids and Related Substances A new polynucleotide, poly 2-amino- adenylic acid, has been prepared by chemical synthesis of the 5'-pyrophosphate and enzy- matic polymerization. The complex of this polymer with poly U provides the first exam- ple in addition to the natural G-C pair of a two-stranded helix stabilized by three inter- base hydrogen bonds. Physical studies show that the additional hydrogen bond increases the stability but that other specific attractive factors must be important in G-C affinity. GTP and ATP form helices with comple- mentary polynucleotides if and only if diva- lent cations are present. This Mg-dependent substrate template pairing is probably im- portant in nucleic acid synthesis. Dihydrouridine residues in copolymers of U and H 2 U do not bind to poly A and reduce the ability of the normal uridine residues to do so. Specific base pairing of monomeric nucleo- sides has been demonstrated in organic sol- vents by nuclear magnetic resonance. The G-C interaction is far stronger than any other, in- cluding other pairs which also form three hy- drogen bonds. (H. Todd Miles, F. B. Howard, R. V. Ravindranathan, E. D. Becker (LPB), Dr. R. R. Shoup (LPB), Dr. P. Cerutti (LC), Dr. M. Chamberlin (Univ. of Calif.), Mr. J. Frazier) X-ray Diffraction Studies on Proteins X-ray diffraction studies on A-chymotrypsin have been continued. A number of new heavy atom derivatives have been discovered. One of 248 ANNUAL REVIEW OP INTRAMURAL RESEARCH these, phenyl mercury acetate, has been lo- cated in two-dimensional projections and three-dimensional data is being collected to confirm this interpretation. Several heavy atom derivatives have been discovered for crystals of DIP trypsin and these are being examined in detail. (David R. Davies, Gerson Cohen, Enid Silverton, Hazel Braxton) A Search for DNA Recombination in Vitro Ultracentrifugation and standard enzymo- logical techniques are being used to find and characterize an enzyme which produces cova- lent links between DNA molecules. Assay sys- tems have been developed which involve the separation of two DNA's which can be differ- entially eluted from hydroxylapatite columns. Attempts to detect linkage using crude E. coli extracts have so far been equivocal. (Martin Gellert) Structure of Polyadenylic Acid at pH 7 We have carried out a study of some of the solution properties of poly A. Samples of the polymer have been fractionated according to molecular weight by a combined variation of salt concentration and temperature. An inves- tigation has been made of the molecular shape as a function of salt and temperature. Under ideal solvent conditions, poly A which is 80% in the ordered form behaves in a manner characteristic of a classical random coil. The finding is entirely consistent with the predic- tions of the earlier spectral analyses of poly A thermal denaturation. (Henryk Eisenberg, Gary Felsenfeld, Marc Leng) Optical Properties of Nucleic Acids The analytical spectral techniques developed in earlier years have been brought to a high degree of precision. As one byproduct of the investigation it has become possible to analyze any of the spectra of nucleic acid samples (native, denatured, or hyperchromic spectra) to determine the concentration and base com- position of the DNA. In the case of the hyperchromic spectrum and the spectrum of the high temperature form, it is only necessary to make observations at four wavelengths to determine concentration and composition with an accuracy comparable to that of any of the other techniques for de- termining base composition. The method has the advantage of being much more rapid than any other. We have also used the spectral methods to analyze in detail the denaturation of A. phage DNA. We find that it is possible to detect the internal heterogeneity of base sequence which was first revealed by other investigators using ultracentrifugal methods. It appears that the spectral analysis will permit rapid determina- tion of such internal heterogeneity, and of homologies of sequence between different but related DNA's. (S. Hirschman, G. Felsenfeld) Interaction of DNA with Polycations The interaction between DNA and synthetic basic polypeptides has been studied. Prelimi- nary results reveal that polylysine can be made to interact preferentially with A-T rich DNA, while polyarginine does not. The interaction of DNA with spermine has also been investi- gated. Contrary to reports of other investiga- tors, spermine exhibits no selectivity with re- gard to the base composition of binding sites. (Marc Leng, Gary Felsenfeld, Shalom Z. Hirschman) Internal Proteins of Bacteriophage T2 The internal proteins of T2 bacteriophage have been further purified. Current work indi- cates that the acid soluble fraction contains two components and the acid insoluble frac- tion, two or more components. The acid-soluble components are well resolved on DEAE. Based on P-60 and P-100 acrylamide columns, both are approximately 15,000 molecular weight. Pulse-labelling in- fected cells and then isolation of the proteins from the resulting phage indicates both com- ponents are synthesized early in the infection, one having roughly the kinetics of an "early enzyme" and the other being made even sooner in the infection. The kinetics have been essen- tially substantiated when the proteins were NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 249 isolated by direct T.G.A. extraction of infected cells. The acid-insoluble components are partially resolved by acrylamide columns. A minor peak appeal's in the size range corresponding to ca. 30-50,000 molecular weight, a major peak at about 15,000 mol. wt. and indication of a third smaller material. All the acid-insoluble ma- terials are made before or during the period of early enzyme synthesis. (Steven B. Zimmer- man, G. Felsenfeld, G. Sandeen) Effect of Glucosylation on the Kinetics of Endonuclease Degradation of DNA It has been demonstrated viscosimetrically that the initial rates of degradation of DNA by pancreatic DNAse and endonuclease I are in the order T*2 = T*4>T2>T4:=T6 with rela- tive rates of 4:2:1. Deoxyribonuclease II was found to degrate T*4 and T4 DNA at the same rate. These data show that the kinetics of en- zymatic degradation are sensitive to rather subtitle differences between the DNA's of the various bacteriophages. The most likely explanation of these results is that the different rates are due to the ex- tent of glucosylation although on account of the complex glucosylation patterns of the T- even bacteriophage DNA's it is not possible to determine whether the mode (stereochemistry) of glucosylation also influences the kinetics. (Philip D. Ross, William B. Uphold) Viscosity Studies of the DNA of Bacteriophage Mutants T5 — Differences in the intrinsic viscosity of intact DNA isolated from a series of T5 bac- teriophage mutants have been found. These dif- ferences would correspond to a difference in molecular weight of 9% between T5+ and the deletion mutant T5 st-0. This result is in ex- cellent agreement with a 7% MW difference calculated from sedimentation constant by Rubinstein. The fact that s and [??] vary in the same direction enables one to conclude from hydrodynamics alone that these differ- ences are a true size effect. A difference of 7% has been demonstrated by direct measure- ments of length in the electron microscope (McHattie & Thomas). This work establishes that the viscosity technique is capable of de- tecting small differences in the size of bac- teriophage DNA molecules. T4 — Small, reproducible differences have been found in the intrinsic viscosity of intact DNA isolated from various rll mutants of bacteriophage T4. When the phage are grown on E. coli B a correlation between the intrin- sic viscosity of the DNA and the genetic de- letion size is found, however the size differences calculated from the viscosity are much larger than estimates of the size by genetic tech- niques. When the phage are grown on other bacterial strains different sorts of results are obtained indicating that the viscosity is in- fluenced in part by the complex physiology of growth conditions which reciprocally may re- flect a property of the genetic mutation. (Philip D. Ross, Robert L. Scruggs) Chemistry and Genetics of Hemoglobin and Other Proteins 1,2-cyclohexanedione was found to react with the guanido group of arginine to form a stable derivative. Modification of the arginyl residues of proteins with this compound blocked the action of trypsin at the arginine positions but not the lysine positions. Factors affecting denaturation and dissocia- tion of hemoglobin were studied. It was found that alkali denaturation is inhibited by sulfhy- dryl blocking agents and by mercaptoethanol. Although human adult hemoglobin, fetal hemo- globin, and bovine adult hemoglobin differ greatly in their resistance to denaturation by alkali, they were found to be very similar with regard to dissociation into half-molecules at high pH. The amino acid substitutions of two abnor- mal hemoglobins were determined. Hemoglo- bin Hopkins-1 has an aspartyl residue in place of a lysyl residue in position 95 of the beta chain, and hemoglobin J has an aspartyl resi- due in place of glycyl residue in position 16 of the beta chain. (H. A. Itano, A. J. Gottlieb, S. Yamada, and E. R. Tudor) 250 ANNUAL REVIEW OP INTRAMURAL RESEARCH Biochemical Control Mechanisms in Histidine Biosynthesis The pathway of histidine biosynthesis in Salmonella typhimurium has been shown to be unbranched and to involve 10 enzymes. As- says for the ten enzymes have been developed. The genes for these enzymes are in a cluster on the Salmonella chromosome. Transfer-RNA and the control of the histi- dine operon. (J. R. Roth, D. Silbert, G. Fink, P. E. Hartman, and B. N. Ames) The control mechanism that regulates the rate of synthesis of the group of histidine biosynthetic enzymes made by this cluster of genes is being examined to try and under- stand the mechanisms of "repression." Four classes of regulatory mutants have been ob- tained which are derepressed for the enzymes of the histidine operon. Their properties sug- gest that histidine-tRNA is important in re- pression and not free histidine. One of these regulator genes is the gene for the histidine activating enzyme. The mutants in this gene have an enzyme with a decreased affinity for histidine. Another of the genes involves the histidine-tRNA. The translation of the histidine operon. (R. G. Martin, D. B. Berkowitz, and H. J. Whit- field, Jr.) We have continued the study of polarity (i.e. mutants in one gene which affect the other genes in an operon distal to the operator region) in an attempt to understand the bio- chemical basis for it. A random group of 65 mutants in the C gene (the aminotransferase) have been examined in this regard. These mu- tants have been divided into chain terminating types containing the UAG (amber) or UAA (ochre) triplets, missense and frameshift classes. A method has been developed for classifying mutants into these groups. All the amber and ochre and frameshift mutants are polar: none of the missense mutants are polar. The frameshift mutants behave as if they are polar not of themselves but rather because they produce nonsense condons beyond the point of the mutation. The effect of the frame- shift mutation does not extend beyond the lim- its of a given cistron i.e. the enzymes subse- quent to the block are present, albeit in reduced amounts. A model for translation based on the hy- pothesis that the degree of polarity is a func- tion of the type of proximal chain initiator has been developed. Frameshift mutagenesis in Salmonella. (B. N. Ames, H. J. Whitfield, Jr.) A frameshift mutation is one where bases are added or deleted from the DNA so that the translation of the genetic message which occurs in groups of 3 bases is out of frame. The agents that cause these frameshifts (acri- dines such as proflavin) do not work in bac- teria. Numerous polar mutants in the histi- dine operon are not ambers or ochres and appear to be frameshift mutants. A number of them are revertable by a class of aza-acridines, benzacridines, acridines, and quinolines with a polyamine side chain. Our evidence so far indicates that these mutagens specifically cause frameshifts and not transitions or transver- sions. We have been learning about the spe- cificity of mutagenic agents using these mu- tants. Purification and protein chemistry of two of the enzymes of histidine biosynthesis. (M. J. Vol! and R. G. Martin) The first enzyme and the aminotransferase have been purified and their protein chemis- try has been investigated. Mechanism of Lambda Bacteriophage Induction Lysogenic induction and curing constitute two processes in which a specific recombina- tional event occurs as a consequence of the expression of certain genes of a temperate bacteriophage. Studies are being undertaken to identify the genes involved and determine their roles in this event. Methods have been developed for readily determining (a) whether a reagent can cause lysogenic induction, (b) genetic requirements for the curing of one bacteriophage by an- other. A thermosensitive phage mutant (desig- nated 434hy C t ) is used in the latter method. Considerable attention has been devoted to the unusual characteristics of this phage. Some of the properties of 434 hy C t support the follow- ing conclusions: NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 251 The phage repressor is unstable even at low temperature, more unstable at high tem- perature. Derepression leads to a reversible phase of phage development which is then followed by an irreversible phase. A system responsi- ble for curin appears in the reversible phase. In addition, certain bizarre properties of 434hy C t are being investigated. (M. Yarmo- linsky) MATHEMATICAL RESEARCH BRANCH As of February 24, 1966, the Office of Mathe- matical Research has been designated the Mathematical Research Branch. Research and consultation have continued along the general lines set forth in the sum- mary of last year. Primarily, the work has consisted of generalization and extension in depth of previously initiated studies in mathe- matical biology and related mathematics and application of methods, mathematical models and theories previously developed to selected biological problems. New approaches and meth- ods have been brought into play and the de- mands of application have entailed modifica- tion of formulations and some more or less tangential studies. However there have been few significant departures from the general areas of research and the broad objectives out- lined in previous summaries. The main areas of research are: Mathemati- cal formulation and analysis of models for dendritic neurons; mathematical and compu- tational studies of visual systems; general theory of transport processes with special ref- erence to renal concentrating systems; mathe- matical description of the transport-diffusion- chemical kinetic problem for substances in the blood-capillary complex; mathematical prob- lems arising from the rate behavior of metabolic systems; mathematical and compu- tational methodology for compartmental and related systems. Several theoretical preductions concerning synaptic activity of dendrites have received support from recent anatomical and physio- logical studies. A theoretical analysis of elec- tric potentials in rabbit olfactory bulb led (Rail and Shepherd in 1964) to the hypothe- sis that the secondary dendrites of mitral cells deliver synaptic excitation to the dendrites of granule cells and that the depolarized granule cell dendrites then deliver synaptic inhibition to the mitral dendrites. This hypothesis met the requirements of the then existing physio- logical data for the mammalian olfactory bulb. However, dendro-dendritic synaptic interac- tions of this kind had not been previously ob- served or postulated for any nervous system, and the hypothesis remained tentative until the results of electron-micrographic studies of this region (external plexiform layer of olfac- tory bulb) should become available. Thus, it was exciting to leam from an independent electron-micrographic study (Reese and Brightman, NINDB) that there are synaptic contacts between granule and mitral dendrites, and moreover, that these contacts are unusual in that neighboring contacts (between the same pair of dendrites) have opposite synaptic polarity (presynaptic-to-postsynaptic) as judged by the locations of synaptic vesicles. This anatomical finding fit the requirements of the physiological hypothesis so well, that collaborative discussions were undertaken to explore and writeup the implications. What has emerged is the concept and evidence for a new kind of synaptic pathway for inhibition; it provides a negative feedback to the mitral cells; it can provide for the lateral inhibition that is needed for sensory discrimination; it can provide for adaptive inhibition which can adjust a sensory system to a wide range of input intensity. Also the granule cells of ol- factory bulb, like the amacrine cells of verte- brate retina, possess no axons; this new hy- pothesis provdes an interpretation of how such cells could perform an important task without generating a nerve impulse. Thus, the implications extend beyond the olfactory bulb, and are being explored. An entirely different set of computations has been carired out to provide theoretical predictions being tested by new experimental data on cat motoneurons obtained by colleagues in the spinal cord sec- tion (NINDB). Several cases of good agree- ment between theory and experiment have re- 252 ANNUAL REVIEW OF INTRAMURAL RESEARCH suited and are being written for publication. (Dr. W. Rail) The Fuortes-Hodgkin model for visual re- sponse of the Limulus eye, extended by the ad- dition of a second feedback loop was studied further. Suitable adjustment of the parame- ters of the second slow loop gives good agree- ment with responses to trains of pulses. A paper on the one loop model appeared in the Journal of Physiology. A simple set of instruc- tions for use of the SAAM program for models of the Fuortes-Hodgkin type was written. Some of the relations between graph theory and matrix theory which are particularly rele- vant to compartmental systems were explored, and some new theorems on reciprocity derived. A paper has been prepared. As an aid to embryological studies of the lenses of chicks, an equation was fitted to the lens profiles. This is useful in determining vol- umes, surface areas, and also as a compact way of describing the course of development. (Mrs. R. B. Marimont, NINDB, Associate Member MRB) Work has continued on various aspects of the renal concentrating system. One question has been whether a counter current multiplier with active sodium transport limited to the medulla can account for the sodium concen- tration profile in the medulla. It has been shown (Stephenson, Nature 206, 1215, 1965) that in a two-loop system (which included the loop of Henle and the medullary vessels) this is not possible. This work was extended to systems with any number of loops in which trans-tubular water as well as salt movement is allowed. This work is scheduled to appear in the Biophysical Journal. A study of the effect of diffusion and trans-tubular water movement on counter current multiplier effi- ciency has been begun. A thermodynamic analysis of certain features of counter cur- rent multipliers has also been initiated. Finally, preliminary analytical work on ran- dom walk models of counter current multipliers has been done with the objective of program- ming some of these models for machine com- putation. (Dr. J. Stephenson, NHI, Associate Member MRB) A numerical method and the corresponding computer program have been developed to solve for the oxygen concentration along a capillary and in the surrounding tissue. The consumption of oxygen in the tissue is assumed to obey non-linear (formal "Michaelis-Menten") chem- ical kinetics. By changing the kinetic parame- ters it is possible to explore the oxygen concen- tration profiles for the cases of zero, first and second order kinetics, all for a given oxygen consumption, arterial oxygen saturation and blood flow. Several other features of the sys- tem have been explored. In particular the in- fluence of the chemical kinetic parameters on the mean intercapillary distance, subject to a prescribed oxygen consumption which avoids oxygen debt, has been explored and, for the first time, it is now possible to study the influence of the kinetic parameter on the mean capillary length. The interplay of kinetic parameters, mean intercapillary distance, mean capillary length and cardiac output for a given oxygen consumption which avoids oxygen debt suggests an interesting and com- plex optimization problem. The numerical method consists, briefly, of writing the differ- ential equations as one non-linear system which is solved by a generalized n-dimensional Newton-Raphson technique, each linear step of which is solved by an iterative over-relaxa- tion method. Rigorous convergence proofs and error bounds for the method have been for- mulated, An effort was made in the above formula- tion to incorporate the best estimates of the minetic parameters the Ovhemoglobin kinet- ics in whole blood. A mathematical model for the Roughton-Gibson experimental set-up was developed which allows more information to be extracted from their data. A description of gas exchange at the lung alveolar-capillary level is being developed which will presumably eliminate some appar- ently incompatible results which have appeared in this field. (Dr. Jose Gonzalez-' Fernandez) The Moore-Penrose pseudoinverse for a ma- trix has, from some points of view, been well studied. Recently (the first in 1960, the last in 1964) other classes of generalized inverses NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 253 have been denned. As yet there has been little work on their properties and, with the excep- tion of the thesis of C. A. Rohde (May 1964) and a paper by Rohde scheduled to appear this year, nothing approaching a systematic comparative study of these various classes of inverses. Such a study was begun last year. Our terminology is: The matrix B is a /-in- verse of A if it obeys the first j of the equations (1) ABA=A, (2) BAB = B, (3) AB = E 1 = E 1 *, (4) BA=Et = E**. The 4-inverse, the Moore-Penrose inverse, is the only unique one but the others are far simpler to con- struct. It can be shown that for many pur- poses (in particular linear estimation, certain analytical properties, etc.) the character of the results is independent of the choice of generalized inverse. In other contexts one can get computational and algebraic simplicity at a small price (or for free) in terms of the breadth of conclusions if it can be shown that a j -inverse shares certain properties with the 4-inverse or the proper inverse of a non-sin- gular matrix. There are properties of a proper inverse which though well nigh trivial (the inverse of a hermitian matrix is hermetian; the inverse of the inverse is the matrix, transposition and inversion are commutative operations, etc.) do not necessarily hold for a generalized inverse and we must give up cer- tain conclusions and disallow certain opera- tions when these properties fail. In practice then it is extremely important to know what properties of A are inherited by B, when A and B commute, when they have common in- variant subspaces, when the (non-zero) roots are reciprocally related, and so on. Many such questions are settled by the following result: Let T,(AC) = T t (A)T(C), T 2 (AC) = T,(Q- T 2 (A) and let M have the properties of Ti and/or T 2 and additionally M(A*) = M(A)*. It has been shown for what value of j it is true that T^B) or M(B) is a j -inverse of Tj(A) or M(A) when B is a j-inverse of A. These transformations include conjugate- transpose, adjoint, compound matrices, com- pound ad jugate, and induced matrices. Sim- ilar results have been obtained for equivalence unless it includes similarity, congruence and multiplication by a non-singular matrix. The existence of each higher inverse has been shown to follow from that of a 1-inverse and the importance of this lies as much in compu- tational application (for the proof is con- structive and quite simple) as in theory and moreover is the basis of the theorem: The existence of a 1-inverse, B, with any of the properties (i)-(iv) implies the existence of a j-inverse, for every j, with that property where (i) B hermitian when A is hermitian (ii) B commutes with A, (iii) E! or E 2 nor- mal, (iv) B symmetrizable when A is symmet- rizable. Results too numerous to list here have been obtained on conditions under which A and B are commutative or quasi-commutative, have common eigenvectors have common principal vectors associated with non-zero roots and have reciprocally related non-zero roots. Fin- nally, an equality has been derived based on a j-inverse in which includes the famous Kan- torovitch inequality as a special case and which provides a novel and exceedingly simple discussion of the conditions for strict equality. (Dr. John Z. Hearon) A new addition to the SAAM program has been programmed. This is a PREREAD sec- tion containing about 50 subroutines and has new and more extensive data input capability. It also has compiler capability and permits the entering of mathematical equations to- gether with the data. This PREREAD is es- sential for the new extended capabilities being incorporated into SAAM for ON-LINE com- puter operations. As a result of new iodine kinetics studies, further structure in the thyroid system has been recognized and a new iodine kinetics model has been developed. The new model con- tains a delay phase that did not seem to be necessary in previous studies. The new model was also tested and found compatible with past studies. Collaborative studies on a metabolism and absorption of Ca have also continued. (Dr. Mones Berman) CLINICAL INVESTIGATIONS During this fiscal year, a number of organi- zational rearrangements have been completed within the branches of NIAMD Clinical Inves- 254 ANNUAL REVIEW OF INTRAMURAL RESEARCH tigations. At the outset of the year, Dr. Robert Gordon replaced Dr. G. Donald Whedon as Chief of the Metabolic Diseases Branch. Dr. Whedon continues his interest in clinical in- vestigation within the Branch, but is no longer required to divert time from his responsibili- ties as Institute Director to carry out the du- ties of Branch Chief. Within the branch, three sections were separately delineated. The Sec- tion on Mineral Metabolism is now headed by Dr. Gerald Aurbach, formerly an investigator in the Laboratory of Nutrition and Endocri- nology. In addition, the branch includes the Section on Gastroenterology, headed by Dr. Leonard Laster, and the Section on Physiology and Clinical Nutrition under Dr. Gordon. New sections have also been organized within the Clinical Endocrinology Branch. The Section on Endocrine Biochemistry is headed by Dr. Jacob Robbins, while the Section on Diabetes and Intermediary Metabolism is under the leadership of Dr. Stanton Segal. Finally, effec- tive March 1, 1966, separate sections within the Arthritis and Rheumatism Branch were recognized. Dr. Jarvis Seegmiller has become Chief of the Section on Human Biochemical Genetics, while Dr. John Decker heads both the Branch and the Section on Connective Tis- sue Disease. Dr. John Decker joined the staff of the In- stitute on September 19, 1965 to fill the va- cancy created by the death of Dr. Joseph Bunim. Before joining the Institute, Dr. Decker had been Associate Professor of Medi- cine at the University of Washington School of Medicine in Seattle, and head of the Division of Arthritis. Before his appointment to the medical faculty of the University of Washing- ton in 1958, Dr. Decker had been a Research Fellow of the Arthritis and Rheumatism Foun- dation at the Massachusetts General Hospital under Dr. Wlater Bauer. Dr. Decker had served the NIAMD previously as a member of the Arthritis Training Grant Committee, and is an active member of the American Rheuma- tism Association, for which he has edited the "Primer on the Rheumatic Diseases." At the Clinical Center, Dr. Decker will continue his interests in clinical and laboratory investiga- tions of rheumatoid arthritis, lupus erythema- tosus, and related rheumatic diseases. On July 1, 1965 a new group of 11 Clinical Associates joined the Institute, replacing 8 who had completed their two-year period of duty. These men have, as always, been the mainstay of patient care activities, and have made major contributions to clinical and lab- oratory investigations. The high caliber of the junior investigators who have carried out the function of Clinical Associate within the NIA- MD has been of the greatest importance in as- suring the excellence of the Institute's clinical research program. Other temporary members of the clinical investigations staff during this year include 12 visiting scientists and guest workers from overseas. The NIAMD has made two beds within its nursing units available to the National Insti- tute of Dental Research, in addition to accom- modating its own patients. During the period March 1, 1965 through February 29, 1966, 480 patients were admitted for a total of 17,066 hospital days. The average patient stay was 46 days, and the average census was 67% of capacity. During the same period, 1446 outpa- tients were seen for study and treatment. ARTHRITIS AND RHEUMATISM BRANCH The Branch has been divided into two sec- tions in the interest of more clearly delineating functions. Dr. Seegmiller now heads the Sec- tion on Human Biochemical Genetics; his inter- est in gout continues but, in addition, the sec- deals with a variety of other heritable metablic disorders. Dr. Decker directs the Section on Connective Tissue Disease which is concerned with rheumatoid arthritis, systemic lupus erythematosus, and related disorders, often grouped as the "collagen diseases." Clinical Studies of Natural History of Disease Lymphocyte Function in Sjogren's Syndrome Sjogren's syndrome continues to be a focus of activity and is heavily represented among patients admitted for study. A reduced lym- phoblastic response of patient lymphocytes cultured with phytohemagglutinin has been ob- served; the defect is most pronounced in pa- NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 255 tients with accompanying pseudolymphoma or rheumatoid arthritis. The finding has been substantiated and extended by the further ob- servation that skin sensitization to dinitro- chlorobenzene is not as readily or as often induced as in normals. (Drs. Talal, Barth, Levinthal, NCI, and Waldorf, NCI) Immunological Reactivity in the Elderly In the above studies it was noted that skin sensitization was more likely to occur in pa- tients without circulating "autoimmune" anti- bodies and vice versa. Elderly normal individu- als often show such antibodies and studies of circulating antibodies and skin sensitization are now in progress in a group of 150 such people. (Drs. Decker, Talal, Waldorf, NCI, and Willkens, University of Washington in Seattle) Variations in Rheumatoid Factor Titer Using data and sera assembled at the Clini- cal Center since 1957, an effort is being made to relate the clinical status of the patient to changes in the titer of rheumatoid factor over a mean time span of 5V£ years. The results will be pertinent to prognostic problems and to identification of possible antigens. (Dr. Decker) Climical and Metabolic Evaluation of Cystinosis Detailed clinical and metabolic studies of children with cystinosis continues. The identi- fication of a peripheral retinal lesion, present before the classical biochemical lesions are de- tectable, holds considerable promise in terms of preventing damage by early treatment. Two modes of therapy — with penicillamine and with a synthetic low cystine diet — are under evaluation. (Drs. Seegmiller, Schneider, Craw- hall, and Wong, NINDB) Therapeutic Studies Prophylactic Synovectomy in Rheumatoid Ar- thritis Surgical synovectomy is being carried out on one randomly selected joint of a pair of symmetrically diseased joints neither of which show erosive disease by X-ray. The results of this long-term study should provide informa- tion on the efficacy of the procedure in pre- venting damage as well as some data on appro- priate selection of cases of surgery. (Dr. Decker and Dr. Peterson, George Washington University) Melphalan in Systemic Amyloidosis This disease, commonly accompanied by a bone marrow picture suggestive of multiple myeoloma, is invariably fatal. Therapy with melphalan, perhaps the "best" agent for mye- loma, is under trial. The tendency of 131 iodide to localize in parencymal organs involved by amyloid is being studied. (Drs. Barth and Decker) Lymph Drainage in Systemic Lupus Erythe- matosus It is possible to cannulate the thoracic duct and exteriorize its entire flow. This makes pos- sible the removal of lymphocytes, immuno- globulins, or both. The procedure has been done in a single patient with severe systemic lupus erythematosus with nephritis. The subsequent favorable changes in renal function were suf- ficiently encouraging to conduct more exten- sive trials. (Drs. Decker, Cohen, and Irvin, NCI) Mechanisms of Disease Purine Biosynthetic Pathways The influence of a variety of naturally oc- curring metabolites on glycine incorporation into uric acid and on total uric acid synthesis is under study in an effort to explain the regu- latory defect which is considered to be the cause of overproduction of uric acid in certain individuals with gout. Adenine markedly in- hibits glycine incorporation into uric acid and the effect of orotic acid is to be tested. (Drs. Seegmiller, Rosenbloom, and Kelley) Induced Renal Retention of Uric Acid Acute renal function studies have shown marked renal retention of uric acid upon infu- sion of /3-hydroxybutyrate or sodium acetoace- tate while the third major "ketone body," ace- 256 ANNUAL REVIEW OF INTRAMURAL RESEARCH tone, had no effect. The finding probably explains the hyperuricemia which has been ob- served in patients who are totally fasted, on high fat diets, or in diabetic ketoacidosis. (Dr. Seegmiller) Role of Infection in Rheumatic Disease Efforts to isolate mycoplasma or bedsonia from the synovial fluids and tissues of patients with rheumatoid arthritis or Reiter's syn- drome, respectively, have not been fruitful but are continuing. Serological studies, on the other hand, indicate more reactivity to a spe- cific mycoplasma, M. salivarium in rheumatoid sera, and more complement fixing reactivity to a bedsonia antigen in Reiter's sera than in appropriately matched control sera. (Drs. Decker, Chanock (NIAID), Barile and Hopps (DBS)) Experimental Amyloidosis Casein-induced mouse amyloidosis has been successfully produced and the effect of various modifications of the immune apparatus, such as theymectomy, irradiation, and cytotoxic drugs, is under study. In addition, efforts are being made to study 131 iodide-amyloid inter- actions in the experimental disease. (Drs. Barth and Decker) Ribosomes in Hormonal Control of Protein Synthesis Studies of amino acid incorporation by ribo- somes from hypophysectomized or thyroidec- tomized rat livers demonstrated that the ribo- somal reading of a synthetic messenger RNA was defective. The results suggest that the hormonal control of protein synthesis occurs, in part, at least, at the ribosomal level. (Drs. Garren and Richardson) Mechanism of Action of Adrenocorticotropic Hormone The action of ACTH on the adrenal is blocked by inhibitors of protein synthesis. When protein synthesis was blocked it was found that the side chain cleavage of choles- terol, the first step in its conversion to corti- costerone, was inhibited. It was thus proposed that ACTH facilitates the synthesis of a pro- tein which is responsible for initiating the transformation of cholesterol to corticosterone. (Drs. Garren, Davis, NHI, and Ney, Vander- bilt Medical School) Studies on Immunoglobulins Antibody Synthesis in the Spleen Some noteworthy properties of membrane- bound ribosomes as compared to free ribo- somes of rat spleen have been found. In a cell- free system, protein synthesis by the bound ribosome is sharply inhibited with chloram- phenicol and is not readily induced by syn- thetic messenger. The reported effect of chlor- amphenicol on antibody production in vivo suggests that the observed effect in the cell- free system is, in fact, an antibody protein synthesis but this point continues under study. (Drs. Talal and Plotz) Macroglobulin Subchains The previously discovered probability that yM was composed of 5 rather than 6 subunits has been amply confirmed by (a) disulfide bond reduction and consequent chain separa- tion with dithiothreitol and by (b) the re- sults of partial tryptic digestion of the mole- cule. The latter procedure showed the molecule to be divalent and completely analogous to the structure of yG. Using depolarization of fluor- escence, the yM molecule was found to be com- posed of loosely bound, non-interacting sub- units, possibly explaining the uniquely high agglutinating activity of yM antibodies. (Drs. Metzger, Miller, Perlamn, Edelhoch, Stone and Bladen and Mage, NIDR) Macroglobulin Catabolism Radioiodine-labelled yM and subunits of yM produced with dithiothreitol, were given to animals and to patients and their catabolism was followed. The subunits showed a much! shorter half-life than did the whole molecule. In sharp contrast to the situation with yG and its subunits, there was no evidence of compe- tition between yM and subunits of yM for catabolism. (Dr. Cohen) NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 257 Studies on Canine Immunoglobulins A thorough study of canine immunoglobulins is being carried forward and has provided in- formation of six different moieties, one of them in the macroglobulin class. Canine colos- trum contains the same six but in different proportions. The isohemagglutinins of canine A 2 blood group are also being classified. The metabolism of these molecules is of importance because of the occurrence of several model dis- eases in dogs, particularly hemolytic anemia and systemic lupus erythematosus. (Dr. John- son, and Drs. Swisher and Trobold, University of Rochester) Miscellaneous Carbon-Fluorine Bonds in Biology Enrichment methods have produced a bac- terium which can break the carbon-fluorine bond, and can use fluorinated organic com- pounds as the sole source of carbon. Fluoride accumulates in the growth medium. Attempts to isolate the microbial enzyme responsible for cleavage are continuing. These studies are of interest in relation to the known effects of fluorine substituents in delaying the catabolism of a variety of organic compounds, such as the synthetic corticosteroids. (D. Goldman) METABOLIC DISEASES BRANCH Research on Human Physiology and Nutrition Clinical Studies of Total Energy Balance The NIAMD Metabolic Chamber, which has been used in the past for studies on total en- ergy metabolism by indirect calorimetry, is being re-instrumented to take advantage of newer electronic devices for data collection and processing. New plans also include provi- sions for measurement of carbon isotopes in expired air, so that a wide range of studies of oxidation of labeled precursors may be under- taken. (Drs. Thompson and Gordon) Effects of Heat Stress on Potassium Balance Investigation of the effect of a high sodium, low potassium rice diet (similar to diets com- mon in Southeast Asia) on the potassium bal- ance of normal volunteers has continued. On this diet, healthy men experience a 10 to 15% decrease in potassium content over 1 to 2 months. Loss of potassium is increased by heat exposure and sweating. However, none of the diseases of hot climates, some of which were considered possibly to be due to potassium de- pletion, were reproduced by the experiment. (Drs. Gordon, Thompson, Waller (NIAMD Ex- tramural), and Cage (NCI)) Mechanisms of Water and Electrolyte Secre- tion by the Human Siveat Gland In the course of the foregoing study, it was noted that as serum potassium levels fell, sweat potassium followed in proportion. This observation suggests that potassium enters sweat by passive diffusion from extracellular fluid. This information, together with a re- view of literature on the structure and func- tion of sweat glands, has led to a hypothesis which attributes to lactate ion the primary role in establishing the electrochemical gradi- ents that move water and ions from body fluids to the sweat duct. This hypothesis has been peepared for publication, and is being tested in current experimental work. (Drs. Gordon, Thompson, Waller (NIAMD Extramural), and Cage (NCI)) Clinical Investigation of Calcium Metabolism A kinetic model was devised to describe cal- cium absorption in man; in collaboration with Dr. Mones Berman a computer program was established to analyze this compartmental model. This kinetic analysis has been applied to study several clinical disorders of calcium metabolism. Both calcium and parathyroid hormone appear to influence calcium absorp- tion in man. Further preliminary observations suggest that the decreased urinary losses of calcium induced by high phosphate intake is mediated through an effort to decrease bone re- sorption. Patients with idiopathic osteoporosis were tested with regard to lactose tolerance and je- junal biopsy specimens were obtained; flat lac- tose tolerance tests were associated with intes- tinal lactase deficiency as measured by direct 258 ANNUAL REVIEW OF INTRAMURAL RESEARCH enzymatic analysis with the biopsy specimens. Eight percent of osteoporotic subjects over the age of 50 were deficient of the enzyme whereas subjects without evidence of bone demineraliza- tion in the same age group showed normal enzyme activity in the intestine. (Drs. Birge, Keutmann, Aurbach and Whedon) Bone Cell Metabolism Fetal bone cells have been isolated and cul- tured in vitro. Collagen synthesis was meas- ured by determining C 14 hydroxyproline in protein. An interesting observation has been made that ascorbic acid stimulates collagen synthesis in this preparation; the action of ascorbic acid is probably related to its pro- posed function in catalyzing hydroxylation of proline. (Dr. Birge) Chemistry and Physiology of Parathyroid Hor- mone Parathyroid hormone has been isolated in pure form and digested with trypsin. Analysis of tryptic peptides of the hormone as well as other peptide fragments produced by different chemical enzymatic means yielded information to assign each tryptic peptide to a position in the molecule; this assemblage of peptide sub- units thus placed in linear order has become a working model for the structure of the hor- monal polypeptide. A minimal area of the molecule requisite for biological and immuno- logical activity has been isolated from weak acid digests of the pure hormonal molecule. This fragment accounted for a sequence 20 amino acids long at the carboxyl-end of the polypeptide chain. Further studies showed that the methionine, tryptophan and tyrosine with- in the acid fragment region are each critically important for biological activity. Studies on the secretion of parathyroid hormone have been carried out by measuring parathyroid hormone in the blood of ruminants with a highly sensitive radioimmunoassay. From these experiments it can be concluded that blood cal- cium is the major if not only physiological regulator of parathyroid secretion. Some puri- fication of human parathyroid hormone has been accomplished after extracting human parathyriod adenomas with 8 molar urea. It has been shown that human parathyroid hor- mone behaves similarly throughout fractiona- tion, gel filtration on Sephadex and chroma- tography on cellulose ion exchanges. A further finding has been that the sedimentation rates of bovine and human parathyroid hormone are similar in the ultracentrifuge. (Drs. Aurbach, Keutmann, O'Riordan, Potts (NHI) and Sher- wood (NHI) Studies on the Action of Thyrocalcitonin The action of thryocalcitonin, a hypocal- cemic factor extracted from the thyroid gland, was studied by kinetic analysis of the distrib- ution of calcium-45 injected intravenously into rats. The effect was examined with a kinetic model set up through the computer program and developed by Dr. Mones Berman. The ex- perimental findings could be reproduced in the model system when the return of unlabeled cal- cium from a reservoir switched off. The most likely location of this reservoir is bone and it is apparent that thyrocalcitonin acts by sup- pressing bone resorption. (Drs. O'Riordan and Aurbach) Studies of the Small Intestine Whippple's Intestinal Lipodystrophy The long-term study of the pathophysiology of untreated and treated Whipple's disease continues. The female patient described in last year's report whose disease relapsed 3 years after she had been treated with antibiotics and steroids for 4 months responded favorably, but slowly, to reinstitution of the same treatment. Her relapse was unusual in that she developed hypoalbuminemia (due in part to exudative enteropathy), hypochloesterolemia and hypo- carotenemia but no steatorrhea. A new patient was added to the series bring- ing the total number studied to 8. He was treated only with antibiotics and his response was satisfactory. He is the second patient in the series to be treated without steroids. Of the remaining 6 patients, one has died of gastric cancer, and the others continue to re- main in remission. Dspite their general good health, they all continue to show PAS-positive macrophages in the lamina propria of the NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 259 small intestine. (Drs. Laster and Heizer, NI- AMD; Dr. Waldmann, NCI) Other Diseases It is generally accepted that extreme flat- tening of the mucosa of the small intestine is characteristic and diagnostic of a sensitivity to dietary gluten. During investigations of gluten-sensitive enteropathy in this laboratory several patients were studied who had a flat mucosa but proof of their sensitivity to gluten was lacking or indefinite. One patient with flat mucosa and hypogammaglobulinemia im- proved her intestinal function when she was fed a gluten-free diet. However, after restora- tion of a normal diet her intestinal function has remained relatively normal for more than one year. Another patient with malabsorption and a flat intestinal mucosa responded only slowly and partially to a gluten-free diet, but began to improve dramatically after treatment with antibiotics, a measure which should not improve a patient with a typical gluten-sensi- tive enteropathy. Another patient with malab- sorption and a flat intestinal mucosa responded well to a gluten-free diet but has also devel- oped wide-spread cancer which may be a re- ticulum cell sarcoma. These deviations of clini- cal behavior in patients with a flat small intes- tine mucosa are under study. Because of the finding of unsuspected mal- absorption in 2 patients with Hodgkins dis- ease, a study of the small intestine in Hodgkins disease has been started. To date, 3 of 6 addi- tional patients studied have had functional and/or structural abnormalities of the mucosa of the small intestine. Attempts are in progress to determine whether a patient of this type, with a flat intestinal mucosa, is gluten-sensi- tive. (Drs. Laster and Heizer, NIAMD; Drs. Carbone and Vietzke, NCI) Intermediary Metabolism of the Intestinal Mucosa Studies of the synthesis of 27-carbon sterols by scrapings of mucosa from the guinea pig small intestine were extended to show that pu- rified preparations of sodium taurocholate and sodium glycocholate, the predominant bile salts of guinea pig bile, are capable of inhibiting sterol biosynthesis in the mucosal scrapings in vitro. At 20 mM each bile salt all but oblit- erated sterol synthesis by mucosa of guinea pig ileum. At 4mM inhibition was still observed, but to a lesser extent. These findings suggest that bile salts, which are most actively ab- sorbed by the ileum, may contribute to the reg- ulation of intestinal sterol synthesis. (Drs. Ockner and Laster, and Mrs. Woodson, NIAMD) Ultrastructure of the Human Intestinal Mu- cosa A collaborative study of the ultrastructure and chemical composition at the subcellular level of the human small intestine mucosa has been initiated with Dr. A. J. Tousimis (Con- tract No. PH 43-66-10). The ultimate purpose of the collaboration is to apply electron probe microanalysis, electron microscopy, electron microscope histochemistry and electron micro- scope radioautography to studies of the human intestinal mucosa in health and disease, and in the presence of various forms of chemical and physical injury. Since the methods for electron microscopy used in this study represent partial modifica- tion of conventional procedures, an exploration of the normal human small intestine mucosa was undertaken with the new techniques. Epithelial cells of the intestinal mucosa adhere to each other at the apical region of their la- teral surfaces. This region is termed the junc- tional complex and it comprises the zonula occludens, zonula adherens and the desmosome. Biopsies from more than 50 subjects have been studied to determine variations in the confor- mation of the unit membrane in the zonula occludens of normal human intestinal mucosa. Effects of chemical injury on the zonula occlu- dens are also under study. In preparation for the application of elec- tron probe microanalysis to biological tissues and to the human intestinal mucosa in particu- lar, methods have been developed to render observations quantitative. Preliminary studies of the presence and distribution of sulfur and iron in the normal human intestinal columnar cell have been completed. (Drs. Tousimis, Bio- 260 ANNUAL REVIEW OF INTRAMURAL RESEARCH dynamics Research Corp.; and Laster, NIAMD) Inborn Errors of Metabolism Homocystinuria As new cases of homocystinuria continue to be discovered, it becomes evident that this in- born error is a relatively frequent cause of mental retardation, second only perhaps to phenylketonuria. The initial demonstration by this group that the underlying defect in homo- cystinuria is a deficiency of activity of cysta- thionine synthase was based on studies of 2 patients with the disease. The findings have now been extended to show that 3 additional patients with homocystinuria are markedly deficient in cystathionine synthase activity. In each of 2 families the parents of a patient with cystathionine synthase deficiency were shown to have a partial reduction in activity of this enzyme to about 60% of the mean control value. These heterozygous parents have neither clinical manifestations of the disease nor ab- normal amounts of homocystine in the urine. Additional studies indicate that the brain of a patient with homocystinuria is deficient in cystatnionine synthase activity. The demon- stration by this group that a patient with marked deficiency of cystathoinine synthase activity has an impaired capacity to convert the sulfur atom of L-methionine to urinary inorganic sulfate, has led to the development of a clinical test for marked deficiency of cystathionine synthase activity. (Dr. Laster, NIAMD; Dr. Mudd, NIMH; Drs. Finkelstein and Irreverre, NIAMD) Cystathioninuria Cystathioninuris was discovered in 1958. It appears to be a familial disorder, the clinical manifestations of which have not yet been fully defined because of a paucity of cases. Mental retardation is a feature of the disease. One such patient was studied by this group and it was shown that activity of the enzyme cysta- thionase was markedly reduced in an extract of liver from the patient. This enzyme catalyzes the hydrolytic cleavage of cystathionine to cysteine, a-ketobutyrate, and ammonia. The claim of Frimpter, that addition of pyridoxal phosphate in vitro stimulates cystathionase activity in extracts of liver tissue from patients with cystathioninuria, could not be confirmed in this case. Crystalline rat liver cystathioninase has been reported to catalyze deamination of homo- serine to a-ketobutyrate and ammonia. The ex- tract of the liver from the cystathioninuric patient was tested and a deficiency of this en- zymatic activity was found. Thus, the evidence suggests that in man a single protein catalyzes both cystathionase and homoserine dehydra- tase activities, and that there are at least 2 enzyme activity deficiencies in cystathioninu- ria. (Drs. Finkelstein, NIAMD; Mudd, NIMH; Laster and Irreverre, NIAMD) A New Disorder During the course of screening procedures for the detection of patients with homocysti- nuria and cystathioninuria a mentally retarded patient was found to excrete a compound in the urine which, to the knowledge of this group, has not been reported to occur in hu- man urine. The identification of the compound and of the enzymatic defect underlying the disorder are in progress. The patient's family history suggests the abnormality is a familial one. (Drs. Irreverre and Laster, NIAMD; Dr. Mudd, NIMH; and Dr. Heizer, NIAMD) Intermediary Metabolism In considering the possibility that metabo- lism of the small intestine mucosa is abnormal in patients with gluten-sensitive enteropathy because of an immune mechanism, the study of a model system was considered. The plan was to test whether the reaction between hu- man platelets and human antibodies to plate- lets alters platelet metabolism. Two parame- ters of platelet metabolism were tested and were found to be unaffected by the addition of antibodies. In the course of these studies it was observed, however, that the addition of thrombin to platelets in vitro results in marked and prolonged stimulation of glucose oxidation via the glycolic pathway. Additionl studies suggest that thrombin acts on one or more steps earlier in the pathway than the one in NATIONAL INSTITUTE OF AKTHRITIS AND METABOLIC DISEASES 261 which pyruvate appears. Thrombin may be acting on the hexokinase reaction or on the transport of substrate into platelets. As an associated or independent effect, thrombin also inhibits galactose oxidation. In testing whether thrombin stimulates oxidation of glucose by platelets by inducing the synthesis of new en- zymes, evidence was obtained to show that platelets are capable of protein synthesis. To the knowledge of this group, this has not been shown before. Thrombin did not appear to act on protein synthesis in platelets. (Drs. War- shaw, Laster, and Shulman, NIAMD) CLINICAL ENDOCRINOLOGY BRANCH The Branch underwent an administrative reorganization during the past year, largely as part of an effort to expand its activities in the field of diabetes research. Two sections were created: the Section on Diabetes and Interme- diary Metabolism, and the Section on Endo- crine Biochemistry. None of the permanent staff was on a prlonged leave-of-absence dur- ing 1965-1966, but one served as Visiting Professor at the University of Naples in the spring and early summer. A number of visitors from abroad joined the Branch: one from Bel- gium, two from England, three from Italy and two from Japan. Thyroid Biochemistry Iodide Transport In order to understand more fully the mat- ter of iodide transport, attention was turned to tissues which do not "trap" iodide. The Ehrlich ascites tumor of mice was chosen as a source of isolated cells of this type. These cells were found to exclude iodide and related anions (Br~, Re0 4 _ , WOr). The efflux of io- dide from the ascites cell was dependent on the metabolic integrity of the cell and was in- hibited by cardiac glycosides, quinidine and certain nucleotides. The relationship of iodide transport across the ascites cell membrane to that in the thyroid cells is not yet known. (Drs. Wolff and Salvatore) The mechanism of the antigoitrogenic effect of antithyroid anions (Re0 4 ~ and C10 4 ~), when given with prophylthiouracil. Although prophylthiouracil appeared to augment thyroid enlargement produced by TSH injection, this effect was not diminished by simultaneous feed- ing of the anions. The mechanism of their an- tigoitrogenic effect remains obscure. (Drs. Wolff and Alexander) The mechanism of iodide transport in the thyroid was investigated from the standpoint of the roll of the cell membrane. Phospholi- pases A and C inhibited iodide transport. Al- though certain phospholipids prevented this effect, they were not capable of restoring trans- port after the membrane had been damaged. Further studies of this subject are in progress. (Drs. Wolff and Larsen) Iodination Reactions and Thyroxine Synthesis In an attempt to clarify the process of iodi- nation and thyroxine synthesis in biological iodoproteins, studies have been undertaken with proteins of known structure which do not normally contain iodine. The rate of iodination of lysozyme with I 3 ~ at pH 8.5 was more rapid in 8M urea than in water. Only two of the three tyrosines were iodinated in water but all three in urea. Mono- iodotyrosine was produced at low iodine levels (<2 moles of I 2 /mole of lysozyme), but di- iodotyrosine was the major product at higher levels. Thyroxine was not found. Iodohistidine (mainly monoiodohistidine) was formed after on tyrosyl residue had been fully iodinated. The " quantitative importance of iodohistidine formation had not been appreciated previously. (Drs. Wolff and Covelli) Iodination of bovine pancreatic ribonuclease in water produced three iodotyrosyl residues and one iodohistidyl, whereas in urea two ad- ditional tyrosyl residues and one additional histodyl residue were iodinated. The histodyl residue iodinated in water was proven to be His-119 by studies in which His-119 was blocked by carboxy-methylation and also by hydrolysis with subtilisin. The initial loss of enzyme activity during iodination is probably due primarily to tyrosine iodination. (Drs. Wolff and Covelli) Iodination of human serum albumin was studied by difference spectrophotometry in 262 A.NNUAL REVIEW OP INTRAMURAL RESEARCH water and urea. The conversion of "buried" tyrosyl to "buried" iodotyrosyl groups was demonstrated. This indicated that groups nor- mally in the interior of the protein molecule can react,, presumably at the surface, under cer- tain conditions and then return to the interior. This finding may have relevance to the bio- logical iodination of thyroglobulin, since inte- rior iodotyrosyl residues occur in the native molecule. (Drs. Edelhoch and Perlman) Further clarification was obtained from the model reaction in which 4-hydroxy-3,5-diiodo- phenylpyruvic acid (DIHPPA) and 3,5-iodo- tyrosine (DIT) couple to form thyroxine. Preliminary studies on DIHPPA by UV- and NMR-spectoscopy defined the relation be- tween its keto and enol forms in various buf- fers and solvents. 2 is requried for the for- mation of thyroxine from DIHPPA and DIT. It was shown that the 2 is utilized to form a stable oxidized intermediate of DIHPPA, which is then able to react with DIT in the absence of 2 . This intermediate is not a free phenoxy radical such as that formed by the oxidation of DIHPPA with permanganate. The intermediate, however, gives rise to a dif- ferent free radical at a slightly alkaline pH or at neutral pH in certain organic solvents. In- vestigations on the nature of this free radical and of the oxidized intermediate are now under way. (Drs. Cahnmann, Nishinaga and Kon) Iodoproteins In a further search for abnormal thyro- lobulins, such as that found in South African cattle with congenital goiter, five lines of the Wollman transplantable rat thyroid tumor have been investigated. One of these (line 1-8) contains considerable amounts of abnormal thyroglobulin. This iodoprotein has a sedimen- tation coefficient of about 8S, is excluded from Sephadex G-200 and forms a soluble complex with antithyroglobulin antibody. A partial purification has been achieved by filtration through granulated 7% agar. Purification of a quantity suitable for molecular studies is underway. (Dr. Robbins) In the course of this work, a rapid and sim- ple method for gel filtration analysis of thy- roid extracts was devised. It employs a thin layer of G-100 or G-200 Sephadex on a glass plate, and provides a satisfactory separation of thyroglobulin, iodinated serum protein and particulate iodoprotein. (Drs. De Nayer and Robbins) Protein Synthesis in the Thyroid Gland The thyroid gland is a useful tissue in which to study protein synthesis since it produces in large quantities a unique protein, thyroglobu- lin, which is the site of thyroxine synthesis. Work is now under way to define the simplest subcellular system for thyroglobulin synthesis. Thyroid ribosomal fractions have been pre- pared which are capable of incorporating amino acids into protein in the presence of "pH 5 enzyme", an energy source, and either endogenous or synthetic messenger RNA. Im- munochemical identification of the product is under study. It is hoped that this simplified system will be useful in studying the poly- merization of thyroglobulin subunits. and in the investigation of control mechanisms for thyroglobulin synthesis. (Drs. Kondo, Robbins, and Rail) Proteolytic Enzymes in the Thyroid Gland Further study on the neutral protease in rat thyroid gland revealed that it resembled chymotrypsin in its specificity. On this basis it was considered possible that the activity originated in mast cells, which are known to contain a chymotrypsin-like protease. This was, indeed, found to be the case. The role of mast cells in the rat thyroid is not understood, but it is not considered possible that this enzyme can be involved in the hydrolysis of thyroglob- ulin in vivo. The enzyme has been partially purified, and has an approximate molecular weight of 23,000. (Drs. Pastan and Almqvist) Measurement of Iodocompounds in Biological Fluids Further progress has been made in the reso- lution of iodoamino acids by resin column chromatography. The failure to separate thy- ! roxine and triiodothyronine in serum has been i shown to be due to serum albumin, which ad- sorbs to the resin, precipitates in the presence < NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 263 of the organic solvent, and cause accretion of the resin granules. The problem may be avoided by hydrolysis of the serum prior to chroma- tography or the sequential use of two columns. (Dr. Lewallen) Pituitary Hormones Growth Hormone Studies on the molecular properties of bo- vine growth hormone have been continued. The molecule dissociates into halves in alkali, with a pK of 11.05. A major increase of fluo- rescence also occurs, with a higher pK, and indicates a molecular alteration in addition to dissociation. (Drs. Edelhoch and Condliffe) During the study of a series of patients with acromegaly, who are being evaluated with respect to response to radiation therapy, it was found that they tended to have de- pressed levels of PBI in serum. This has been traced to a depression of thyroxine-binding capacity of the thyroxine-binding globulin. A concomitant increase in thyroxine-binding prealbumin occurs. The mechanism of these alterations is not yet known. (Drs. Roth and Hollander) By the use of radioimmunoassay of growth hormone, two growth retarded children were shown to have isolated deficiency of growth hormone. Prior to the availability of immunoas- say, such isolated deficiencies of pituitary hor- mone were difficult to identify with precision. (Dr. Roth) Gonadotropins A fourth case of carcinoma of the lung with gynecomastia has been studied. Gonadotropic activity was recovered from the blood and the tumor. Since this tumor was clearly not a trophoblastic tumor, this case adds signifi- cantly to the syndrome of gonadotropin-secret- ing lung carcinoma. (Dr. Rosen) A radioimmunoassay for human follicle- stimulating hormone (FSH) has been devel- oped, using double antibody precipitation to separate free and bound hormone. The assay has a sensitivity which permits measurements on plasma, and physiological studies employ- ing the assay are now under way. (Drs. Rosen and Schlaff ) Thyrotropin In order to determine the initial site of in- teraction of thyrotropin (TSH) with its tar- get tissue, TSH was incubated briefly with thyroid slices. The hormonal response persisted despite extensive washing of the tissue, but could be abolished by anti-TSH antibodies. Therefore, the relatively intact hormone ap- pears to be bound extracellularly, at least in the early stages of its interaction. Analogous findings have been obtained with insulin, which becomes similarly bound to striated muscle. This type of binding may be a general property of peptide hormones. (Drs. Pastan, Roth and Macchia) Vasopresssin Progress has been made on the development of a radioimmunoassay for vasopressin. Spe- cific antibodies against pure arginine vasopres sin (AVP), lysine vasopressin and oxytocin have been produced by injecting these small peptides without prior coupling to larger mole- cules. 131 I-labeled pure peptide could be puri- fied to high specific activity since it is differ- entially absorbed to dextran gels compared to unlabeled peptide. The assay is highly spe- cific, and is able to detect blood levels of 50 /x/xg/m of AVP. It has been possible to measure the hormone in blood after hemor- rhage, severe dehydration, and other condi- tions causing elevated blood levels. Further im- provement in precision and sensitivity is under way, and the physiologic and diagnostic appli- cation of the assay is being explored. (Drs. Roth and Klein) Insulin, Glucagon and Carbohydrate Metabolism Regulation of protein metabolism by insulin and glucagon Studies have continued with the isolated, cyclically perfused rat liver. Perfusion with insulin at 2.4 /ig/hr. inhibits the release of L- valine- u C from prelabeled livers by 40%. Since valine is not metabolized or synthesized by the 264 ANNUAL REVIEW OF INTRAMURAL RESEARCH liver, its release represents protein degrada- tion. Glucagon at 10 jug/hr. increases valine release slightly, but in the presence of insulin causes a 60% increase in degradation. Valine entry into the liver is not affected. Protein breakdown is not affected by glucose concen- tration from 1 to 4 mg/ml, and is reduced by omission of red blood cells from the per- fusate. Similar insulin effects have been ob tained with the perfused rat hind limb, indi- cating that the effects are not confined to the liver. The alterations are large enough to sug- gest that these hormones significantly affect protein metabolism in vivo. (Drs. Mortimore and Glinsman) Regulation of hepatic carbohydrate metabo- lism Hepatic glycogen breaks down rapidly in vitro. In the perfused liver, glycogen break- down was nearly complete after 60 minutes of perfusion with oxygenated Krebs-Ringer bi- carbonate buffer. Addition of red blood cells to the perfusate markedly decreased glycogen breakdown, roughly in proportion to the oxy- gen uptake, and this effect was dependent on glucose concentration. The red cell effect is probably localized at an early step in glucose utilization. Since liver apparently lacks com- petitive glucose transport, as indicated by the lack of effect of 3-0-methy glucose, the red cell effect may be at glucose phosphorylation. In- sulin, on the other hand, inhibits glucose re- lease by a mechanism which is not dependent on glucose concentration in the perfusate, and hence is probably localized at a site or sites within the glycogen cycle. (Drs. Mortimore and Glinsman) Glucose Transport A study of the characteristics of glucose transport in intestinal mucosa has been ini- tiated in normal and diabetic humans. With the use of «-methyl glycoside as a model sugar, Na + dependent active transport has been shown in normal intestine. Lactrose transport in patients with lactase deficiency has also been examined. Lactose does not appear to be transported into the intes- tinal mucosa in this disorder. (Drs. London and Segal) Galactose Metabolism and Galactosemia The enzyme galactose-1-phosphate uridyl transferase in rat liver has been investigated with a new assay developed for this study. The enzyme is strongly inhibited by both sub- strates-galactose-1-phosphate and UDP-glu- cose-1-phosphate. Enzyme activity is present in the fetal liver at 18 days, increases pro- gressively to 10 days after birth, and then falls to the adult levels by 45 days. This ac- tivity parallels that of galactokinase. (Miss Bertoli and Dr. Segal) A new pathway for galactose metabolism has been found in mammalian liver. Galactose is oxidized to galactono-lactone which is con verted to galactonate. The latter is then oxi- dized to the 3-keto derivative, which is decar- boxylated to form D-Xylulose, a sugar capable of entering the pentose phosphate pathway of glucose metabolism. The enzyme galactose de- hydrogenase, which catalyzes the first step, has been purified 100 fold. Both oxidation steps require DPN. In rats, large doses of 14 C-galactonate are ex- tensively oxidized to 14 C0 2 . Although small doses are excreted in the urine unchanged, loading the animal with galactose causes even tracer doses of galactonate to be metabolized. This new pathway, therefore, appears to op- erate in vivo. (Drs. Cuatrecasas and Segal) Amino Acid Transport In a continuation of studies on the defect in cystinuria, a third variant of the disease has been found, based on amino acid trans port in intestinal mucosa. The transport of diabasic amino acids may be completely or par- tially defective, as may that of cystine. Three of the four possible combinations have been found. Furthermore, cysteine transport is normal ! in mucosa which is completely usable to trans- port cystine. This finding was made possible by the discovery that dithiothreitol could maintain cysteine in a reduced state under NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 265 aerobic conditions, and thus enable direct study of its transport. Studies with kidney slices indicate that cysteine and cystine may have separate transport mechanisms. Once trans- ported into the tissue, however, cystine is converted to cysteine. This has been demon- strated by forming the mixed disulfide with N-ethylmaleimide. (Drs. Crawhall and Segal) Investigation of the relationship between transport of the dibasic amino acids and cyst(e)ine in kidney cortex slices has shown that they have independent influx mechanisms. The dibasic amino acids and cysteine, how- ever, share a common efflux mechanism. (Drs. Schwartzman and Segal) The possible participation of a phosphatidyl peptide as the carrier in amino acid trans- port was investigated. The finding that the rate of labeling of the amino acid pool was greater than that of the lipid fraction is in- consistent with the stated hypothesis. (Drs. Schwartzman, Crawhall and Segal) Protein Structure The 19S immune globulin and its reduced and digested subunits has been studied by polorization of flourescence. The relaxation times of the various substructures were only slightly smaller than that of the native mole- cule. This indicates that the substructures have rotational independence in the native molecule. (Drs. Edelhochand Metzger) Further studies have been done on the frag- mentation of antibovine serum albumin rabbit immunoglobulin by cyanogen bromide. The 5.3S fragment was purified, and found to have a molecular weight of 94,800. A study of its immunological properties showed that, unlike the native antibody, it failed to produce pas- sive cutaneous anaphylaxis and had a reduced ability to fix complement. A comparison of its properties with those of the papain and pepsin fragments of immune globulin indicated that the cleavage with CNBr occur within that re- gion of the heavy chains which is between the critical points of cleavage by the two enzymes. (Dr. Cahnmann and Drs. Arnon and Sela- Weizmann Institute) PEDIATRIC METABOLISM BRANCH This branch has concentrated its activity primarily on efforts to elucidate the pathogene- sis of the inborn error of metabolism, cystic fibrosis of the pancreas (CF). Four principal areas of study have been followed: Establishing the nature of the sweat gland defect in CF by the use of structure-function studies with the electron-microscope. Attempts to define further the immunologi- cal and biochemical defect of mucopolysac- charide metabolism considered to be responsi- ble for the protean manifestations of the disease. Observations on the relationship between salt retaining steroids and the striking and unique abnormality of sweat and other body fluids in cystic fibrosis. Clinical and pathologic studies designed to improve the knowledge of the pathogenesis, course and complications of this disease, and its treatment. Immunological and Biochemical Investigations Evaluation of the inborn error of mucopoly- saccharide metabolism in cystic fibrosis The search for a unique antigenic mucopro- tein as a genetic marker for the generalized mucosubstance abnormality in cystic fibrosis has been continued. A systematic immunologic study comparing body fluids and tissue extracts of patients with CF and normal sub- jects was initiated in preceding years. Rabbit serum antibodies and donkey serum antibodies prepared by inoculating macromolecular pre- cipitates from urine, saliva, pancreatic cyst fluid, and tissue homogenates from lung, pan- creas, and salivary glands, have been used, as well as specific antisera to urinary mucopro- tein of Tamm and Horsfall. This year special attention was paid to the urinary glycoprotein of Tamm and Horsfall as it represented a separate fraction whose immunologic and chemical activity could be analyzed in detail. The following conclusions can be drawn from these observations: The present methods have not been able to detect any antigenic or component sugar con- 266 ANNUAL REVIEW OF INTRAMURAL RESEARCH tent differences between Tamm and Horsfall urinary mucoprotein or other glycoprotein from urine of patients with cystic fibrosis and that of normal subjects. The antigenic determinants of the Tamm and Horsfall urinary glycoprotein are not found in the organs and tissues which are involved in the pathology of cystic fibrosis, except for the kidney where it is also found in the normal subject. The glycoprotein fraction consists of at least two antigenic components and easily breaks down into subunits of different sizes. There is no evidence to indicate the glycoprotein is a single substance in vivo. From these investigations we can conclude that immunological and chemical differences between cystic fibrosis and normal urinary Tamm and Horsfall glycoprotein do not exist. However, one cannot make the statement that physical differences do not exist, and studies in this regard are being carried out. The possible genetic significance of this gly- coprotein was further pointed out by German investigators, who claimed the Tamm-Horsfall fraction was absent in urines of Negroes, in whom cystic fibrosis is rare. Urines obtained from American Negro children with and with- out cystic fibrosis as well as from native Afri- can Negroes shows no difference in the content of this urinary glycoprotein. (Drs. Schwartz, Pallavicini, and di Sant'Agnese) Serologic Reactions in Patients with CF The importance of this study lies in the evaluation of host defense and host response to antigenic challenges because of the chronic nature of the pulmonary disease in CF and because of the finding of Staphylococcus aureus in the sputum of almost every patient and the presence of Pseudomonas aeruginosa as well in approximately 70% of the patients with this disease. Investigations of the im- munoglobulin groups involved in serologic anti- body responses to bacterial insult and studies of antibodies secreted by the respiratory tract have given information regarding most defense of the respiratory epithelium. Using Immuno- electrophoresis and immunodiffusion tech- niques, serum immunoglobulin levels in pa- tients with this disease were found to be ele- vated. The hypergammaglobulinemia is reflecte primarily in elevations of IgG and IgA. IgM becomes elevated in the most severe cases of pulmonary disease, several months before sig- nificant changes appear in clinical or roent- genographic symptoms. Mixed saliva from these patients contains IgG and IgA. Serum IgD levels are in the normal range. (Dr. Schwartz) Study of Pseudomonas Aeruginosa and its Slime in Relation to the Pathogenesis of Cystic Fibrosis Pseudomonas aeroguinosa isolated from the nasopharyngeal flora of patients with cystic fibrosis produces usually large quantities of viscous slime. Because of the frequent and al- most unique association of cystic fibrosis and this mucoid type of organism, there was rea- son to believe that this change might be in- duced by a chemical compound produced by fibrocystic patients which acts as a substrate. In addition, the role, if any, of Pseudomonas aeruginosa in making worse the chronic in- flammatory and obstructive pulmonary disease of cystic fibrosis needed to be clarified. A variety of studies have been performed including the demonstration of precipitin anti- bodies in the serum of most patients with cys- tic fibrosis from whom Pseudomonas could be isolated from the nasopharynx. A large num- ber of mucoid and non-mucoid strains of this organism were typed, and many different strains were found to be present in the noses and throats of patients with cystic fibrosis. A change from a non-mucoid to a mucoid type of organism could be produced by varying the composition of the growth media. The major component of the mucoid material of Pseu- domonas aeruginosa was found by paper chro- matography to be mannuronic acid. Mannu- ronic acid, however, could not be detected in 25 sputum samples from fibrocystic patients carrying this organism. No deoxyribonuclease or collagenase activity could be demonstrated from isolates of Pseudomonas, but elastase ac- tivity was consistently found. Pseudomonas NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 267 elastase was shown to differ from elastase of pancreatic origin by its electrophoretic mo- bility in agar-gel and by its lack of inhibi- tion by human serum. (Drs. Schwartz, Palla- vicini, and di Sant'Agnese) Structure-Function Studies With the Electron-Microscope A morphologic abnormality which would ac- count for the functional defect of sweat glands in CF was looked for. Using skin punch bi- opsies from both normal subjects and patients with CF, numerous techniques were utilized for the characterization of mucopolysaccharide structures, both intra- and extra-cellular. Me- tabolic activity as reflected in sweat gland lysozomes were also studied, as well as sites of electrolyte transport. No distinct differences between the staining for mucopolysaccharide material could be found between CF patients and normal sub- jects using a variety of histochemical meth- ods. However, dense granules were present in the sweat glands of patients with CF, but not in sweat glands of normal patients when the preparations were stained with permanganate- aldehyde-fuchsin. Similar localization of stain was present when this material was studied with acid phosphatase stains; and, in the elec- tron microscope, staining of these sites also occurred with dialyzed iron. However, the as- sociation of acid mucosubstance and acid phosphatase has not been previously seen in lysozomes in any tissue and represents an im- portant finding for the evaluation of cell func- tion. In preliminary studies further electron mi- croscopic evaluation of transport sites in sweat glands revealed that patients with CF do not deposit as much reactive material at expected transport sites as do normal subjects. (Dr. Grand and Dr. Spicer, LEP) The Effect of Aldosterone on Sweat Gland and Renal Function in Normal Subjects, Patients with Cystic Fibrosis, and Carriers of the CF Gene Investigations of the effects of sodium re- taining steroids on sodium transport in sweat glands and kidneys have been continued and expanded. These studies originally were ini- tiated because of suggestions in the literature that the peculiar sweat abnormality in cystic fibrosis is due to alteration in the relationship between sodium retaining steroids and the sweat gland as an end organ. Patients in this study were selected either from the normal vol- unteer patient program or from the cystic fibrosis clinic with an age range of 6 to 26 years. Groups of patients below and above the age of 13 were divided as children and adults respectively. Similar study protocols were fol- lowed for all patients and included sweat tests by iontophoresis of pilocarpine nitrate twice daily, constant diet, environment, activity, and suitable control periods before and after ster- oid administration. In adults aldosterone was given intramuscularly at a dose of 1.0 mg./24 hours, and in children 0.5 mg/10 kg. of body weight per 24 hours. Normal Adults and Children These subjects had comparable sweat gland results with aldosterone producing sweat so- dium retention, sweat potassium excretion and with a fall in sodium to potassium ratio of approximately the same degree. Age differences were not seen. In adults the renal responses to aldosterone were as expected from the lit- erature. Normal children, however, showed a surprising inability to reduce urinary so- dium, did not exhibit the escape expected dur- ing the remainder of the treatment period, and reacted by only a small rebound when the steroid was stopped. The results suggested a peculiar lack of responsiveness of the juvenile kidney to the sodium retaining effects of exo- genous aldosterone. The oldest child gave an adult type response. The findings are quite dif- ferent from those seen in normal children sub- jected to sodium restriction with the produc- tion of endogenous aldosterone. Patients with Cystic Fib?-osis In this group, the responses of renal sodium and potassium excretion were in all ways com- parable to those of normal subjects. Sweat sodium retention was significantly less than in 268 ANNUAL REVIEW OF INTRAMURAL RESEARCH normals. The sweat potassium response in cys- tic fibrosis patients was in the normal range as was the fall of sodium to potassium ratio, there being no statistically significant differ- ence in the two groups for the latter two parameters. Mothers of CF patients showed kidney and sweat responses identical to those of normal adults. This ability of sweat glands of patients with CF to respond to large doses of sodium retain- ing steroids makes it impossible to define the sweat defect in this disease on the basis purely of an end-organ unresponsiveness to normally secreted steroids. (Drs. di Sant'Agnese, Grand, Schwartz, and Pallavicini) Other Investigations in Cystic Fibrosis Pregnance in Cystic Fibrosis Because of the longer survival and increas- ing life span of subjects with cystic fibrosis new problems arise; such patients present seri- ous and often unique problems in management, not readily comparable to those of other disease entities. This study represents the first attempt to collect information on pregnant women with cystic fibrosis and their offspring. Two patients were followed on the wards of this Institute and data on eight others was obtained from the literature and from a national survey. It was found that the toll of morbidity and mor- tality as well as the high risk of fetal prema- turity made the pregnant women with this dis- order a significant challenge in management. In addition, significant genetic information was obtained from this survey. The fact that all of the 11 children of mothers with cystic fibrosis who survived long enough for obser- vations to be made were normal is further evi- dence that cystic fibrosis is transmitted as an autosomal recessive disorder. It was calculated that, assuming the incidence of carriers for the cystic fibrosis gene in the United States to be approximately 2 to 5 percent of the general population, the overall risk of having an af- fected child in a random mating of a fibro- cystic mother with a male of unknown genetic status would be 1 to 2 percent. (Drs. di Sant'- Agnese, Grand, and Schartz) Neiv Syndromes of Pancreatic deficiency Sim- ulating Cystic Fibrosis The studies initiated last year on patients thought to have cystic fibrosis on the basis of pancreatic insufficiency and at times chronic pulmonary disease, but with normal sweat elec- trolytes, were continued and extended. Fur- ther metabolic, endocrinologic, and pathologic studies were performed on the 24-year-old white male with pancreatic deficiency, micro- cephaly, dwarfism, deafness, hypothyroidism, chronic lung disease and a chromosome ab- normality. Additional studies were also carried out on a 16-year-old white female with dwarf- ism, pancreatic insufficiency and bone marrow dysfunction who required splenectomy for hy- persplenism. Several other patients with similar condi- tions are being studied at the present time in the hope of elucidating these previously un- recognized, interesting new disease entities. Albumin Turnover Studies in Cystic Fibrosis Hypoproteinemia is a rare, but known com- plication of cystic fibrosis. Previous investiga- tors have suggested decreased synthesis on the basis of malabsorption and poor nutrition as the main etiological factor. However, loss of protein by gastrointestinal protein leakage or through the respiratory tract and hemodilu- tion are also possibilities. 125 I albumin turnover studies were performed in 10 patients with cystic fibrosis and 51 Cr albumin tests for gastrointestinal protein leak- age were performed on 10. Albumin survival was studied in the whole body counter using 131 I labeled human albumin in 5 patients with cystic fibrosis. The results indicated that the hypolbumine- mia in patients with cystic fibrosis may be due to a variety of causes. Examples of low serum albumin due to an expanded plasma vol- ume, but with normal total circulating albu- min were seen as well as patients in whom the hypoalbuminemia could be explained, at least in part, by some increased loss of albu- min into the gut. In one patient with cystic fibrosis and multilobular biliary cirrhosis sec- ondary to cystic fibrosis, failure of synthesis appeared to play a role. NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 269 These studies are being continued and it is expected that they will clarify the etiology of the hypoalbuminemia in this disorder and point to methods for more effective therapy. (Drs. Schwartz and di Sant'Agnese, and Dr. Strober, NCI) Studies of Growth, Development and Sexual Maturation in Patients with cystic fibrosis Retarded growth has been considered up to the present time as a consistent feature of the clinical picture of cystic fibrosis. It is now becoming apparent that the eventual height achieved by these patients as young adults is normal or close to normal for the age group, even though the adolescent growth spurt may at time be delayed. This appears to be espe- cially true of patients diagnosed later in life, presumably indicating that the lesser degree of pulmonary involvement has inter- fered only slightly or not at all with the growth process. A program of intensive clinical and metabolic studies on young adults with cystic fibrosis is being initiated to study further this question. Sexual maturation has proceeded normally in patients with cystic fibrosis in both males and females even in the presence of severe lung disease. Unpublished histologic observa- tions indicate extensive periurethral gland and prostatic concretions in post-pubertal males with cystic fibrosis. Evaluation of these various parameters is being undertaken. (Drs. 0. SantAgnese, Schwartz, and Grand) CLINICAL HEMATOLOGY BRANCH Study of the Immunology of Blood Cell Deficiencies Idiopathic Thrombocytopenic Purpura Following identification of the ITP factor as a 7S gamma globulin, a quantitative in vivo assay system, using passive transfer of ITP plasma, was developed to evaluate the effectiveness of different forms of therapy with respect to levels of circulating ITP factor. The titer of ITP factor has been compared with clinically demonstrable response to adrenocor- ticosteroid therapy and splenectomy in a total of 23 cases. Patients who benefited from therap all had very low plasma titers of ITP factor (less than 1:4), whereas all but two patients who did not respond to therapy had high titers of ITP factor (greater than 1:20). The two exceptions were in a patient who had re- ceived the antimetabolite 6-mercaptopurine in attempts to suppress immunolgobulin forma- tion and in a patient who had been exposed to benzene and other organic solvents over a period of years in the course of his work. The method of in vivo titration of ITP fac- tor used not only appears to be helpful in predicting the outcome of therapy, particularly splenectomy, but also helps differentiate in- stances of isolated megakaryocyte dysfunction from otherwise apparently classical ITP. (Drs. Shulman, Watkins, Libre and Cowan) The Effects of Reticuloendothelial Blockade in Sequestration of Immunologically Altered Cells Continued evaluation of the use of intra- venous red cell stroma as a reticuloendothelial blocking agent has confirmed the effectiveness of this material in preventing platelets sensi- tized by minimal concentrations of antibodies from being sequested. It was hoped that re- ticuloendothelial blockade would provide effec- tive therapy for patients with a variety of im- munologic blood cell deficiency states who were refractory to more conventional forms of treat- ment. Use of intravenous red cell stroma in two cases of severe ITP, however, proved to be ineffective, no doubt because cells highly sensitized by immunoglobulins compete effec- tively with stroma for sites of sequestration, whereas lightly sensitized cells do not. (Drs. Shulman, Cowan, Libre and Watkins) The Etiology of Transfusion Anuria There is a body of literature concerning the role of hemoglobin in production of anuria, the most serious consequence of mismatched trans- fusion. There has been no evalution of the ef- fects of stroma as a possible toxic agent in the hemolytic transfusion reaction. In evalu- ating intravenous red cell stroma as a possible 270 ANNUAL REVIEW OF INTRAMURAL RESEARCH therapeutic agent (see above), it was found that renal function, as measured by creatinine and PSP clearance, total urinary output, pH and specific gravity, etc., remained entirely normal when stroma from 9 to 10 units of blood per day were given for a week or more. However, on one occasion when a unit of stroma containing a minor blood group antigen was inadvertently given to a patient who had formed an antibody against it, the mismatched" stroma produced a mild febrile reaction and consequent anuria typical of that following a hemolytic transfusion reaction, de- spite the fact that no hemoglobin was involved. The evolution of the reneal lesion was typical of that following intravascular hemolysis and fortunately renal function returned completely to normal. Renal failure following intravascu- lar hemolysis is therefore clearly due to the antigen-antibody reaction, and if anything, only secondarily contributed to by precipi- tation of hemoglobin in renal tubules. There are no other known instances of antigen-anti- body reactions per se in which the kidney is the target organ, although much experimental work has been done in attempts to relate antigen-antibody reactions to lesions such as glomerulonephritis and nephrosis. Work on stroma infusions is continuing in association with Drs. Holland and Schmidt. (Drs. Shulman, Libre. Schmidt (CC) and Holland (CC)) Post-Transfusion Purpura Four additional cases of the rare syndrome of post-transfusion purpura were studied through referrals of patients from London, New Jersey, Baltimore and St. Louis. The syn- drome represents the only example of a self- destructive lesion induced by sensitization to an isoantigen (see previous summary). The ten cases that have now been observed are providing clues to factors leading to this type of sensitization. For instance, it is apparent that prior exposure to the antigen is neces- sary some years before the second exposure that intiates thrombocytopenia. All but one case were in women who had had children, and all of the children tested had the causa- tive Pl Al antigen on their platelets (inherited from the father). The one male patient had had a previous transfusion of antigenic blood ten years before the provocative transfusion. Response to therapy in all instances was that predictable from the experimental evaluation of therapy in all instances was that predictable from the experimental evaluation of thrombo- cytopenia induced by passively transferred antibody in normal individuals, splenectomized individuals and individuals receiving steroids. This syndrome provides insight into the types of sensitization to foreign antigens that may lead to apparent "autoimmune" states. (Dr. Shulman and Miss Hiller) Isoimmune neonatal purpura An additional 24 families have been studied in which neonatal purpura occurred as a result of isoimmunization of the mother by fetal platelets and leukocytes. The data on fre- quency of immunization to specific antigens in the accumulated 175 cases continues to pro- vide important information on the relative an- tigenicity of specific platelet and white cell antigens which are potentially important in transplantation immunity (see below). Effec- tiveness of steroid therapy to the mother an- tenatally, the child postnatally, and of exchange transfusion, continues to be evalu- ated. (Dr. Shulman and Miss Hiller) Transplantation Immunity Three additional platelet and leukocyte blood group antigens were identified and character- ized during the past year, to bring the total identified by C.H.B. to 12 specific platelet or leukocyte groups that can be used in unequivo- cal immunologic tests for phenotyping cells. These continue to be the only antisera available for clear-cut typing of individuals in platelet and leukocyte antigen systems by complement fixation and cytotoxicity techniques. Typing sera for leukocytes in other laboratories that are dependent on agglutination or antiglobulin consumption tests have been found to be to- tally inadequate for obtaining reproducible re- sults. Despite the indications in lower animals that some white cell, platelet, and even red cell NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 271 antigens are the same as general tissue trans- plantation antigens, the massive amount of work done in attempts to correlate blood cell antigens with transplantation immunity in man has not been conclusive. In attempts to find an animal system for directly evaluating human transplantation antigens, the antigenic content of blood cells from lower animals and non-human primates have been tested, using the monospecific antisera mentioned above, as well as multispecific sera derived from patients receiving numerous transfusions. The follow ing findings resulted from continued work done under a contract with Dr. Moor-Jankowski of New York University: Human isoantigens were present more frequently in primates as they ascend the evolutionary scale from mon- keys to anthropoid apes, Rhesus monkeys being- most dissimilar and chimpanzees most similar to man. Animal cells were found to be effec- tive in characterizing the specificity of human isoantibodies and in fractionating sera con- taining mixtures of isoantibodies. Chimpan- zees, baboons and Rhesus monkeys were read- ily immunized against human leukocytes and platelets, but only chimpanzees produced an- tisera that distinguished differences between individual human beings. Heteroimmunization of this type did not appear to be as practical as use of sera from multitransfused human beings, for the human sera are readily avail- able from patients who willingly donate the required amount, and the mixtures of anti- bodies formed in human beings are not as com- plex as those formed in non-human primates. In addition, the availability of chimpanzees is too limited to justify use of these animals simply to produce typing sera or to perform blind histocompatibility tests. Anthropoid apes appear to be best utilized in transplantation research for crucial experiments to evaluate some shared human antigens once analysis of the survival of human grafts indicates which antigens should be evaluated in this way (see below). (Dr. Shulman and Miss Hiller) Evaluation of Histocompatibility in Human Homograft pairs In collaboration with Dr. Marchioro, Dr. Starzel, Dr. Goldsmith and Dr. Haglin, who are surgeons performing renal homotransplanta- tion, C. H. B. has been able to collect 38 human transplant pairs and phenotype them with the large number of platelet and white cell anti- bodies available in the laboratory. The out- come of transplantation with respect to sur- vival, the nature of rejection if it occurs, and the histologic picture of biopsies taken at inter- vals up to two years after transplantation, has been correlated with the number of incompat- ibilities between recipient and donor in white cell and platelet antigen systems. Although the literature contains reports implicating white cell antigens as being histocompatibility an- tigens, and suggests that the number of incom- patibilities of white cell antigens is very significant with respect to renal transplant re- jections, C.H.B. has not found this to be the case. No one specific antigen can be impli- cated as a transplantation antigen and as a matter of fact, there appears to be no cor- relation between the number of mismatched white cell antigens as identified by comple- ment fixation and cell toxicity tests and graft survival. Since this series is as large as any studied, these data indicate the difficulties faced in attempting to evaluate transplantation an- tigens in patients, using white cell and platelet antigens as indicators of compatibility. Although cultured human blood and lymph cells were found to contain leukocyte isoanti- gens, cultured human somatic cells did not, by criteria of adsorption as well as direct com- plement fixation tests. This suggests that leu- kocyte antigens may not predict incompatili- ties in organ transplantation other than bone marrow. Mixed agglutination techniques have been used by other laboratories and reportedly detect antigens in common between white cells and other organs. Attempts to confirm this possibility, using numerous antisera, have so far suggested that the shared antigens do not exist. (Drs. Shulman and Libre and Miss Hiller) Immunologic Therapy of Malignancies The possibility that some somatic tissues share antigens with blood cells and that malig- nant blood cells or other tissues may be de- 272 ANNUAL REVIEW OF INTRAMURAL RESEARCH stroyed by isoantibodies against normal leuko- cytes or platelets provides a good starting point for evaluating possible immunologic therapy of tumors. In current work, cultured Burkitt lymphoma and leukemia cells have been tested for their antigenic content in col- laboration with Drs. Rabson and Shohet and found to contain the usual distribution of an- tigens that normal leukocytes contain. This, combined with the fact that cells from pa- tients with chronic and acute leukemia also contain the normal complement of laukocyte antigens, suggests that these antigens are a relatively invariable component of the cell and that antibodies against them will be effective in producing cytotoxicity. So far, in vivo tests have been carried out on only three laukemics, one chronic lymphocytic, one chronic myelo- cytic and one acute myelocytic form. The first two patients had a dramatic decrease in cir- culating leukemic cells with much less anti- body than ejected from in vivo tests with the same antibodies in individuals with normal cells. The patient with acute leukemia had a marked febrile reaction to relatively small amounts of antibody, but evidence of only relatively small numbers of peripheral cells destroyed. None of the patients had sustained effects from the infusions. Studies are continu- ing to elucidate reasons for increased suscep- tibility of some leukemic cells and to determine possible effects of these isoantibodies on mar- row precursors. The fact that cultured leu- kemic and lymphomatous cells contain all of the recognized isoantigens suggests that ho- mologous immunization for production of spe- cific anti-tumor antisera will be difficult, if not impossible, unless these antigenic factors are taken into account. (Drs. Shulman and Watkins) Immunological Considerations Attending Platelet Transfusions Platelet transfusions in aplastic anemia, idi- opathic thrombocytopenic purpura and leuke- mia were evaluated with respect to the effects of isoimmunization on the survival of platelets and the effects of circulating levels on thera- peutic effectiveness. It was found that isoim- munization against platelets occurs infre- quently with less than 10 transfusions, but that the frequency of immunization rises stead- ily with the number of transfusions until all patients eventually become immunized. Isoim- munization against common platelet antigens prevents transfused platelets from circulating and from being hemostatically effective. More than half of the anti-platelet isoantibodies that arose after transfusion were "incomplete" and could not be detected by conventional serologic tests. Some of these antibodies were identified by ' blocking" tests or by passive transfer. These findings explained why refrac- toriness to platelet transfusions has not always been accompanied by serologic evidence of im- munization. Isoimmune destruction of platelets was found to be a relatively innocuous occurrence and did not contraindicate further trials of platelet transfusions. Although platelets could be matched under some circumstances when isoimmunization took place, at the present time empirical selection of donors based on survival of transfused platelets appears to be the best approach to platelet transfusion ther- apy in sensitized individuals. (Dr. Shulman and Miss Hiller) Study of Blood Coagulation and Diseases of Hemorrhage and Thrombosis Identification of the Spleen as an Organ Con- trolling the Level of Anti-Hemophilic Factor Little is known about the physiologic varia- tions in Factor VIII and the site of produc- tion of this factor has not yet been identified. In recent years it has been found that Factor VIII is elevated in stressful situations and some pathologic conditions, but reasons for these changes have been obscure. Using adren- alin infusions as a means of studying changes in Factor VIII in stressful situations, it was found that Factor VIII levels could be ele- vated as much as two times normal judged by in vitro assays and that plasma obtained from individuals receiving adrenalin was as effec- tive in elevating the Factor VIII levels in NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES 273 hemophiliacs as indicated by in vitro tests. A surprising finding was that Factor VIII did not become elevated in asplenic individuals who were given adrenalin. The spleen is not the organ that produces Factor VIII, for as- plenic individuals have a normal concentra- tion of the factor. It appears that the spleen is able to function as a storehouse of Factor VIII and release it under stress, including the stress of severe exercise. Since platelet levels also increase under similar stimuli, there ap- peared to be a possible relationship between platelet count and Factor VIII level. There was, however, no correlation in the timing of the platelet and Factor VIII responses, and in chronic diseases, in contrast to acutely stress- ful situations, it was found that thrombocy- tosis was associated with relatively low levels of Factor VIII and thrombocytopenia with rel- atively high levels. The reason for the inverse relationship between platelet levels and Factor VIII in chronic diseases is not yet apparent, for numerous in vitro studies on the effects of platelets on Factor VIII have failed to repro- duce the in vivo phenomenon. (Drs. Libre, Shulman and Cowan) Evaluation of a new Plasma Fraction in the Treatment of Classical Hemophilia For many years it has been known that Fac- tor VIII travels with fibrinogen in most physi- cochemical fractionation procedures. A very simple method of precipitating fibrinogen from plasma that has been known for about 20 years, involves slow thawing to produce a cryoprecipitate. This fraction of plasma has been found to contain on the order of 60% of plasma Factor VIII. When resuspended in small volumes, it was effective in producing in vivo Factor VIII levels high enough to per- form major surgery in three patients without causing vascular overload or any other un- toward effect. It was superior to very expen- sive commercial preparations of Fraction I in that it contained a higher ratio of Factor VIII to fibrinogen and other proteins, contained less calcium binding agents which produce toxic symptomatology, and did not contain anti-red cell antibodies that are coprecipitated with fibrinogen in the commercial Fraction I prepa- ration. Of special importance is the fact that the cryoprecipitate is a by-product of other Blood Banking procedures. It can be removed from a unit of blood without contamination, using ap- propriate plastic equipment, and the unit can then be reconstituted for usual transfusion purposes. In the Clinical Center, a waste prod- uct of platelet transfusions, the supernatant plasma, has been found to be an excellent source of Factor VIII for treating our hemo- philiac patients undergoing major surgery. The cryofibrinogen precipatate appears to be the best product available for treatment of hemophiliacs at present and can be made in any small hospital that has minimum Blood Banking equipment, can be stored in the deep freeze at home by hemophiliacs, and can be ad- ministered without special equipment. (Drs. Cooke, Holland (CC), and Shulman) The Basis for Thrombocytopenia in Malaria Using malaria as a disease for studying the pathophysiology of thrombocytopenia associ- ated with infections, we had up until last year found that initiation of intravascular coagula- tion could not account for the phenomenon, despite numerous proponents of this mechan- ism in the literature. By using different strains of malaria parasites, and determining platelet survival and localization with chromium-la- beled cells, as well as quantitative measure- ments of all known clotting factors and anti- malaria antibodies and complement levels, it was found that thrombocytopenia developed independently of parasitemia, was not due to bone marrow inhibition, and was not associ- ated with significant changes in any of the clotting factors. It was always associated with development of anti-malarial antibodies and fall in plasma complement. This therefore ap- pears to be an immunologically induced throm- bocytopenia, probably a reflection of the gen- eral type of thrombocytopenic states docu- mented in human beings in association with circulating antigen-antibody complexes. (Drs. Shulman, Sheagren and Jeffrey) 274 ANNUAL REVIEW OP INTRAMURAL RESEARCH Diseases of Megakaryocyte Dysfunction Following last year's demonstration that the thrombocytopenia of Aldrich syndrome is due to a peculiarity in the maturation of mega- karyocytes rather than peripheral destruction of platelets as previously supposed, several pe- diatric and adult patients with thrombocyto- penia and adequate megakaryocytes have been found to have normal platelet survival. Gen- erally it has been assumed that adequate mega- karyocytes in the bone marrow indicate peri- pheral destruction. Continuing studies are aimed at differentiating abnormal from nor- mal megakaryocytes. (Drs. Shulman and Wat- kins) Platelet Physiology Very little is known about the nature of the physiologic control of platelet levels and the organs responsible for removing these cells normally. Using plasmapheresis and labeled cell techniques in normal volunteers, it has been found that human beings have no platelet re- serve that can be called forth under an acute thrombocytopenic stimulus and that newly re- leased platelets from the bone marrow do not appear directly in the circulation. By studying the splenic content of platelets in individuals splenectomized for a variety of diseases and comparing results obtained in normal individ- uals and splenectomized individuals, it appears that normally platelets reside in the spleen dur- ing a 2 to 3 day maturation phase after release from the bone marrow. This temporary seques- tration, combined in diseased states with any additional insult by such factors as isoantibod- ies or ITP plasma, acts synergistically to de- stroy platelets. Adrenalin infusions appear to be able to release physiologically sequestered platelets and have been a useful tool in evalu- ating normal platelet production. Continued use of the total body counter appropriately calibrated to take into account all body areas of platelet sequestration has provided addi- tional information on the sites of abnormal and normal platelet sequestration. (Drs. Shul- man, Watkins, Cowan and Libre) Studies of Unusual Hemorrhagic Disorders Studies of acquired hemophilia due to ab- normal gammaglobulins have continued in at- tempts to elucidate the nature of the immuni- zation leading to this unusual "autoimmune" manifestation. The number of patients now studied with this rare abnormality is nine. Studies also continue on an unusual form of painful purpura, autoerythrocyte sensitization, in attempts to establish the basis for abnormal hemorrhage in which all known vascular and coagulation factors are normal. (Ds. Shulman, Cowan and Watkins) NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT INTRODUCTION In fiscal year 1966, the NICHD was reor- ganized into three major operating arms: The Scientific Programs under a Scientific Direc- tor, Communications Activities under an As- sociate Director, and Program Services under an Associate Director. The Central concept of the reorganization was to place the principle scientific focus on four substantive program areas rather than on administrative mechan- isms or scientific disciplines. These program areas, Reproduction, Growth and Development, Aging, and Mental Retardation, are under the general direction of a Scientific Director. This organization enables the Institute to accomplish its mission more effectively by fo- cusing on the goals of research rather than the means. The substantive programs are respon- sible in the respective areas for both the con- duct and support of research and training. In furtherance of this concept, broad direction and planning of intramural and extramural efforts are appropriately interrelated by an echelon of senior staff. REPRODUCTION PROGRAM The Laboratory of Biology and the Endorcin- ology and Metabolism Branch were created in December 1965 as a result of a transfer of a number of former members of the Endocrin- ology Branch, NCI, to the newly formed Re- production Program of NICHD. The transi- tion was not difficult because of the avid interest in reproductive biology developed ear- lier under the leadership of Dr. Roy Hertz, who became NICHDN'S first Scientific Director. The research activities of the Laboratory of Biology concentrate on problems relating to con ception, gestation, interrelationships of the anterior pituitary-adrenal-reproductive sys- tems, and the role of biotin and related fac- tors in the physiology of reproduction. Primate studies by this group have been directed toward systematic, quantitative studies of hormone production and excretion through pregnancy and the menstrual cycle in the rhesus monkey. They are also conducting related studies of ovarian function in the pregnant animal. The staff of the clinical Endocrinology and Metabolism Branch have united as an effective team for the practice of clinical medicine and the conduct of clinical and laboratory research. Efforts begun by this group while affiliated with the Endocrinology Branch of the NCI have been continued, largely without interrup- tion. Areas of investigative interest include the biological and radioimmunological assays of protein and polypeptide hormones, cytogenetic aspects of endocrine disorders, hormonal in- duction of ovulation, and follow-up of patients successfully treated for gestational trophoblas- tic neoplasms. In collaboration with the NINDB, construc- tion of facilities for investigations on primates has been planned. Laboratories and a primate facility will be constructed at El Centro Med- ico and Sebana Seca in Puerto Rico. NICHD's activities, established in conjunction with the NINDB's program of research in the develop- ment of the central nervous system, will focus on the areas of basic reproductive biology and reproductive behavior. The Program reorganization within the In- stitute resulted in the creation of the Reproduc- tion Program by the combination of the former Reproductive Biology, Perinatal Biology, Con- genital Malformations, and Developmentl Phar- macology Programs. In terms of extramural research and research training, the new Pro- gram constitutes about one-half of the NICHD grants. During the year, there was a marked in- 275 276 ANNUAL REVIEW OP INTRAMURAL RESEARCH crease in interest in research on population problems. The number of such applications is increasing, but the total investment should be larger. With the advice and encouragement of of the Council, the Reproduction Program con- tinues its active programming of foreign re- search and training grants. GROWTH AND DEVELOPMENT PROGRAM The reorganization of the Institute created a Biological Sciences Branch and a Behavioral Sciences Branch in the Growth and Develop- ment Program. In the Nutrition Section of the Biological Sciences Branch, studies being con- ducted include investigations on the regulation of hepatic protein metabolism and the regula- tion of carbohydrate metabolism in the isolated rat liver. In the Personality Development Section of the Behavioral Sciences Branch, research has re- cently been initiated in certain Washington, D.C., Head Start populations. This project is designed to analyze the components of learning environments of preschool children; to delin- eate the characteristics of children from de- prived environments; to study relationships between specific environmental variables and changes in intellectual and personality charac- teristics; and to study the interrelationships between cognitive and personality functions of preschool children. The Program's interest in the sudden death syndrome has contined. The proceedings of the Conference on the subject, held in 1964, have been published and are being distributed. The MEDLARS printout on the sudden death syn- drome continues to go to interested investiga- tors. A review article prepared on contract is before the Editorial Board of a national journal. AGING PROGRAM During the Fiscal Year 1966, the Gerontol- ogy Branch in Batlimore, Md., was transferred from the National Heart Institute to the NI- CHD. This activity is currently being carried out in laboratories and wards in the Baltimore City hospitals. A building to house this Branch is currently being constructed and should be ready for occupancy about October 1967. The goals of the Gerontology Branch are to describe in quantitative terms the changes that take place with aging and to investigate the basic biological mechanisms of these changes with a view to reducing the impairments and disabilities of older people. Highlights of findings from the Gerontology Branch which have extended knowledge of ag- ing include the following: (1) Older subjects show an impaired response with respect to blood sugar levels to the intravenous adminis- tration of tolbutamide. (2) The impairment of glucoye tolerance in the aged cannot be scribed to an increase in free fatty acids in the plasma. (3) Total caloric, vitamin, and min- eral intakes diminish with age even in subjects whose diets are not limited by economic fac- tors. (4) A mathematical method has been de- vised which may make it possible to assess individual differences in non-uniformity of lung ventilation. (5) A double isotope derivative assay for angiotonin has been developed which permits the quantitative estimation of nano- gram amounts of the hormone. (6) In rats the ability to make fine discriminations in taste diminishes significantly with age. (7) Age changes in collagen may be due to a reoganiza- tion of existing intra-molecular crosslinks rather than the addition of new crosslinks. The Extramural Program supports research and training for research that bears on the aging process and the developmental and retro- gressive changes that occur during the adult years. As of March 31, 1966, there were 63 re- search grants, nine training grants, seven fel- lowships, four research career developmental awards, and one research career award being supported by the Aging Program. The total expenditure by the Aging Program was in ex- cess of $6 million. The Program supports a wide range of disciplines that are concerned with various aspects of aging. These diciplines can be divided into four major areas of inter- est: molecular and cellular biology, compara- tive and human biology, behavioral sciences, , and social gerontology. The Aging Program has collaborated with the Scientific Information Centers Branch inf developing a system that will permit rapid and comprehensive storage and retrieval of infor- NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT 277 mation bearing upon aging. This will be achieved by the use of both abstract journals and completely computerized techniques. A wide range of subjects, from cellular biology to social gerontology, will be covered. It is believed that the ability to retrieve information rapidly and comprehensively would be a major aid to scientists in the field of aging. MENTAL RETARDATION PROGRAM Three Branches and one Laboratory have been organized to carry out direct research activities in mental retardation. These include a Clinical Research Branch, a Laboratory of Biomedical Sciences, a Behavioral Sciences Branch, and an Epidemiology Branch. The Clinical Research Branch and the Laboratory of Biomedical Sciences are operational, but the Behavioral Sciences Branch will not be func- tional until fiscal year 1967, and the Epidemi- ology Branch in fiscal year 1968. The Children's Diagnostic and Study Unit is a Section in the Clinical Research Branch. This outpatient clinic is located at the National Naval Medical Center. The first patient was in December 1965, and by the end of the fiscal year, approximately 45 to 50 children will have been evaluated. The purpose of the Children's Diagnostic and Study Unit is to provide a pro- gram of clinical research and a source of case material for basic research in the biomedical and behavioral aspects of mental retardation. Studies are just being started and include investigations on improving attending behavior in the preschool ratardate, behaviorl assess- ment of neonates at risk for mental handicaps, and informational discrepancies by parents about their mentally retarded children. The mission of the Laboratory of Biomedi- cal Sciences is to contribute to the Program's multidisciplinary investigation of mental re- tardation. Initially, the Laboratory will empha- size biochemsitry, cytogenetics, and neuro- physiology. Projects conducted by personnel in the Labor- atory of Biomedical Sciences include pregnancy- associated plasma proteins, electrophoretic var- iants of leucocyte alkaline phosphatase, studies on hypercalcemia, effects of nutirition on mye- linization, effects of amino acids on myeliniza- tion, and evaluation of methods of therapy in homocystinuria. INTRAMURAL PROGRAMS The principal population research carried out by the Reproduction Program of NICHD is conducted by Dr. Roy Hertz and his asso- ciates who for a number of years have carried out important research on the endocrinological aspects of reproduction. Dr. Henry Vaillant, an NICHD Research As- sociate, was assigned through the Population Council to a research project designed to meas- ure the efficacy and safety of the intrauterine device with particular emphasis to its possible relationship to cervical neoplasia. This project is being conducted in Barbados, an area with a high population density and a government favorably disposed to family planning. Our interest in population research has be- come menifest through the use of contracts. The largest current contract supports the Third Growth of American Family Study. The princi- pal investigator, Dr. Charles Westoff of Prince- ton, is analyzing the data from the survey con- ducted last fall and will give a report of his findings in the near future. Another contract (reimbursable agreement) is held by Dr. Harold Hawk of the Department of Agriculture Research State in Beltsville. Dr. Hawk is carrying out an intensive study of the effect of intrauterine contraceptive devices on the reproductive function of various domestic animals. His findings are significant and ex- tremely interesting. The action of these de- vices appears to be markedly different in differ- ent species. The data from this study not only has direct relevance to fertility control but also indicates that intrauterine foreign bodies may prove to be a useful biological tool. There are several smaller contracts also un- der w r ay. Of particular interest is one (reim- bursable agreement) with the District of Co- lumbia Department of Public Health to deter- mine if it is possible to carry out in the District a long-term study of the safety and efficacy of various contraceptive methods. We hope that the District will prove to be one of several loca- tions where studies of this type may be car- ried out. 278 ANNUAL REVIEW OF INTRAMURAL RESEARCH The staff of the clinical Endrocrinology and Metabolism Branch, consisting of two senior physicians, four clinical associates, two bio- chemists, four technicians and one secretary have united as an effective team for the prac- tice of clinical medicine and the conduct of clinical and laboratory research. Efforts begun by this group while affiliated with the Endo- crinology Branch of NCI have been continued, largely without interruption. Areas of investigative interest include the following: Biological and radioimmunological assays of protein and polypeptide hormones Isolation and purification of protein and polypeptide hormones Cytogenic aspects of endocrine disorders Hormonal induction of ovulation Follow-up of patients successfully trated for gestational trophoblastic neoplasms Application of computer technology to problems in clinical and laboratory in- vestigation The inter-relations of our individual and joint efforts will be apparent from the discus- sion of results obtained as the several interest areas are considered in detail. This integration is the spontaneous derivative of a community of interests rather than the result of adminis- tratively-imposed, goal-oriented or directed re- search. Thyroid-stimuating Hormone (TSH) Estimations of secretion rates of TSH in euthyroid and hypothyroid human subjects have been completed. Reasonably reliable esti- mates of degradation time and secretion rates of TSH in man have been made for the first time. Results show that in hypothyroidism, se- cretion rates are increased and degradation time prolonged, accounting for the high levels of TSH observed in the plasma of patients with primary myxedema. Similar studies have been performed in cat- tle using a radioimmunoassay for bovine TSH. Luteinizing Hormone (LH) A radioimmunoassay for luteinizing hor- mone in man (HLH) has been developed. The method is capable of detecting and quantify- ing this hormone in volumes of 0.3 ml. of plasma or less and is about 500 times as sensi- tive as the most sensitive biologic assays. For the first time levels of luteinizing hor- mone have been followed in plasma of normal women daily throughout the menstrual cycle. A single sharp peak of activity has been found, occurring about mid-cycle, lasting for 24 hours, and associated with presumptive evidence of ovulation (elevation of basal body temperature and increased urinary pregnanediol excretion). The height of these peaks equals or exceeds levels regularly found in post- menopausal women. In women with ovaries and irregular menses, minor day-to-day variations have been observed for periods of up to 50 days of amen- orrhea. Levels in normal males show minor daily variations and no significant variation has been shown diurnally or in relationship to bed rest or ambulation. In contrast, levels are high in castrate males. Clomiphene, an agent used in attempts to induce ovulation in anovulatory or oligo-ovula- tory women, has been shown to cause an eleva- tion of plasma luteinizing hormone in both men and women. Studies are currently under way to determine the effects of the oral con- traceptive agents on plasma leuteinizing hor- mone activity. Parallel studies, including immunoassays of blood and biologic and immunologic assays in urine, are being carried out in a population of prepubertal children ranging in age from 6 to 9 years. These investigations, in addition to providing information of clinical usefulness in evaluating urinary gonadotropin excretion, should provide some information about the hor- monal changes incident to puberty. The assay methods offer promise in the clin- ical evaluation of disorders of pituitary and gonadal function. Melanocyte-stimulating Hormone (MSH) Considerable progress has been made in the development of a radioimmunassay for alpha melanocyte-stimulating hormone. The assay can be carried out with excellent sensitivity and precision in the absence of serum. So far, it has not been possible to demonstrate any NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT 279 alpha MSH in either blcod or pituitary tissue of humans but it appeal's to be present in pi- tuitary tissue of albino rats. The method is not sufficiently sensitive to permit detection of this hormone in the blood of normal rats. The half-time of disappearance of alpha MSH injected into hypophysectomized rats has been studied by three methods: Following disappearance by biologic assay using hypophysectomized frogs Following disappearance of radioimmuno- assay Following disappearance of tracer amounts of radioactively labeled alpha MSH Data obtained thus far show a half-life of 1.6 minutes by bioassay and of 2.1 minutes by radioimmunoassay. A much longer half-life of 7.8 minutes has been observed in following the disappearance of the radioactively labeled hor- mone. Studies of the basis for this disparity indicate that the process of labeling alpha MSH results in alterations in the molecule so that biologically it is handled differently than un- labeled hormone. This important observation has considerable pertinence to studies using tracer amounts of isotopically labled hormones in determination of plasma half-life, secretion rates, etc. Growth Hormone A clinical test has been devised for the evalu- ation of pituitary growth hormone secretory capacity by measuring plasma levels of this hormone before and after the administration of pyrogens. The test has the advantage that pituitary ACTH reserve can also be tested si- multaneously by measurment of plasma Corti- sol levels. This test has been utilized in evalu- ating patients suspected of having pituitary dysfunction in order to ascertain whether the dysfunction relates to one or more pituitary hormones. LABORATORY OF BIOLOGY The research activities of the Laboratory of Biology are being concentrated on problems relating to conception, gestation, interrelation- ships of the anterior pituitary-adrenal-repro- ductive systems and the role of biotin and related factors in the physiology of repro- duction. Primate studies by this group have been di- rected toward systematic, quantitative studies of hormone production and excretion through- out pregnancy and the menstrual cycle in the rhesus monkey (Macaca mulatta) as well as related studies of ovarian function in the preg- nant animal. In parallel studies in monkeys and rats, we have observed that chemical stim- uli for decidua formation in the rodent appear to have no effect in the rhesus monkey. In ear- lier experiments by this group, it was shown that ovarian hormones are not necessary for normal gestation after the third week of preg- nancy, a finding in keeping with observations in man. These marked differences in the repro- ductive physiology of rodents make it clear the more intensive primate research is neces- sary. Steroid hormone metabolism in pregnant monkeys is being actively investigated. As a result of improved analytical procedures, espe- cially isotopic tracer methods, thin-layer and gas chromotography, we are now beginning to obtain quantitatively reliable information on steroid hormone excretion. Estrone, estradiol 17-/3, and estriol have been identified in mon- key pregnancy urine and quantitative methods are now being applied to determine alterations in estrogen excretion during normal preg- nancy. It is expected that this data will prove useful in understanding normal and pathologi- cal changes during pregnancy. Studies on the nature and effects of fetal hor- mones are continuing. We have found that the fetal rabbit testis has the capacity to synthe- size hormones prior to the time of differentia- tion of the embryonal reproductive tract. Cur- rent efforts are directed at identification of hormones in the gonads of the fetal pig. Knowl- edge of the actions of fetal hormones should lead to a better understanding of the mechan- isms regulating fetal tissue differentiation in both normal and abnormal development. Our investigations on the effects of anterior pituitary hormones on sex steroid production mediated by the adrenal cortex have demon- strated that ACTH in high doses stimulates es- 280 ANNUAL REVIEW OF INTRAMURAL RESEARCH trogen production in the ovariectomized mon- key. Earlier findings on the adrenal-mediated androgenic action of exogenous ACTH and pro- lactin in the castrated male rat have been extended by means of hormone-producing pi- tuitary tumors which reproduce the androgenic effects reported earlier. Thus we can stimulate certain aspects of steroid function found in pathological states. The study of biotin distribution in maternal and fetal tissues has demonstrated a marked elevation of biotin in fetal muscle and brain in contrast to the corresponding maternal tis- sues. Study of cofactor metabolism opens new areas of research in reproduction physiology at the molecular level. It is to be anticipated that in the coming year we shall continue current research in ad- dition to initiating studies relating to implan- tation and placenta formation in monkeys and the basic processes of reproduction as our goal. It is a pleasure to express our appreciation for the moral and material support provided by the NICHD staff. GROWTH AND DEVELOPMENT PROGRAM The final approval of the organizational pat- tern for the entire Institute of Child Health and Human Development in December 1965 brought formal structure to the Growth and Development Program. This area represents one-fourth of the Institute's scientific program. One-third of the Institute's extramural activi- ties are in the Growth and Development Pro- gram. The Growth and Development staff is work- ing with the Information Center staff in the final phases of the preparation of a working vocabulary for indexing the literature in this field. One working conference was held with several of the same consultants used a year ago. The proceedings of the Conference on Sud- den Death in Infants held in Seattle, Washing- ton, September 1964, were published in May 1966. Distribution of the proceedings upon re- quest to physicians, investigators, and organi- zations supporting research on this subject has been initiated. The Institute staff has continued to make the MEDLARS sudden death bimonthly printout available to investigators working in the field. The Growth and Development Program has assumed administrative responsibility for six of the Institute's clinical or research associ- ates. Each physician has been placed with re- search laboratories outside NICHD. The activ- ities are briefly summarized as follows: Clinical nuerology combined with a research experience- study of various aspects of cortical maturation in the cat. Staining techniques for the immature cortex have been a major prob- lem in the development of the research. One immediate goal is to quantify the pattern of dendritic growth of the cortical neurons. In immunochemistry — a study to elaborate and refine a new method to quantitatively esti- mate immune cytolysis of nucleated cells; dif- ferentiation of various murine leukemias by complement fixation and application of this test to studies of rabbit antibody to mouse leu- kemia; the ontogeny of sheep complement; the evaluation of complement in neonatal cord blood and amniotic fluid and correlation with respect to hemolytic disease of the newborn. In a biochemistry laboratory — a study of the biological properties of rodent mammary tu- mors of varying functional differentiation. Tu- mors have been produced in rats by injection with nitrosourea. The initial problem has been the development of methods for the quantita- tion of milk production by tumors. In surgery, clinical care of patients; prepara- tion of a manuscript on hypernephromas; de- veloping plans to study the effect of Dextran infusions in reducing the incidence of metas- tases. In oro-pharyngeal development — Took part in a review and analysis of dysphagia in in- fants; then initiated an anatomical, physiologi- cal, and orthodontic study which includes cineradiography. A spirometer has been adapted to measure palatal-pharyngeal insuffi- ciency. Sucking pressure and sucking behavior is under study in cleft palate infants. In biochemistry — various mechanisms of growth, particularly recovery growth is under study in children with cardiac defects, endo- crine abnormalities and malnutrition along NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT 281 with their normal siblings. Also, rats are be- ing studied, observing the effect of starvation, hormonal deficiency and stress. Work on ca- loric expenditure and dietary intake under var- ious conditions for children has been under- taken. AGING PROGRAM Summary The Aging Program supports research and training for research that bears on the aging process and the developmental and retrogres- sive changes that occur during the adult years. It is thus concerned with problems of the en- tire adult lifespan as well as problems of the elderly. The program supports a wide range of disciplines that are concerned with various as- pects of aging. These disciplines can be divided into four major areas of interest; Molecular and Cellular Biology, Comparative and Human Biology, Bahvioral Sciences, and Social Ger- ontology. During the fiscal year 1966 the Gerontology branch in Baltimore, Maryland was trans- ferred from the National Heart Institute to the National Institute of Child Health and Hu- man Development. This activity is currently being carried on in laboratories and wards in the Baltimore City Hospitals. A building to house this activity is currently being con- structed and should be ready for occupancy about October 1967. One vital area of the research carried out under the auspices of the Aging Program deals with the basic biology of aging. In the past two decades there have been develop- ments that augur well for an increased under- standing of the basic biology of aging. There has been a transformation of our knowledge of the extremely minute structure of the cell brought about by electron microscopy and its ancillary techniques. Our knowledge of the ge- netic mechanisms through which the daily ac- tivities of cells are controlled has increased greatly. The entire body of information that deals with chemical changes in cells and their bases in cellular structure has expanded great- ly. It may be that an understanding of the basic biology of aging may be found in this area involving cellular biology and now under investigation by morphologists, geneticists, biochemists, and biophysicists. It is extremely important to an understanding of the basic biology of aging that work in these funda- mental areas continue and that the concepts and techniques of these fields be applied to the study of the aging. One possible mechanism of aging deals with the changes in macromolecular structures that are replaced veiy slowly. One such protein is collagen. Evidence has accumulated that chem- ical cross-links in collagen increase with in- creasing age. This may have some importance from the standpoint of the function of collagen. In addition similar changes may take place in proteins less accessible to study than collagen. The Aging Program is supporting the work of several investigators interested in collagen and in changes in collagen with age. One project has shown that an aldehyde forms one of the cross-links that occur in collagen. Work is be- ing carried on with several agents that re- duce cross-linking to see if such a reduction has any effect on other manifestations of aging. It is possible that during aging errors de- velop in the genetic deoxyribunucleic acid (DNA) which contains the information that directs the metabolic activities of the cell. One current theory is that this is the fundamental mechanism of aging. If errors do occur in DNA with increasing age, then one might expect these errors to lead to the formation of de- fective ribinucleic acid (RNA) which in turn would lead to defective enzyme function. These defective enzymes might not suppress the rate of formation of RNA as normal ones do. Thus there might be a greatly accelerated production of RNA. This possibility is being invesigated in several laboratories. As cells grow old they may fail to excrete all the metabolic products that they produce. One line of evidence suggesting that this is the case is the accumulation of insoluble, brown material in some of the cells of older animals. Woik by several investigators on these pig- ments is being supported. The pigments are being studied from the standpoint of their chemical composition and their mode of pro- duction. One theory that is emerging as a re- 282 ANNUAL REVIEW OF INTRAMURAL RESEARCH suit of these studies is that the cellular lyso- somes release enzymes that digest cellular con- stituents and leave an indigestible residue of lipids and other products which form the pig- ment. Still another approach to the basic biology of aging suggests that with increasing age there is an increased production of antibodies against the bodies own cells. It is known that the autoantibodies in the plasma increase with age and that the incidence of amyloidosis in- creases with age. The Aging Program is sup- porting research bearing on this approach. A study on the rats of oxidation by young and senescent rats of various compounds labeled with radioactive carbon has been supported. The overall results and conclusions from the study may be summarized as follows: Acetate- 2- 14 C is oxidized more rapidly in senescent rats than in adult rats. No significant age- associated differences in oxidative rats were observed with several other compounds includ- ing acetate-l- 14 C, octanote-l- 14 C, propionate- 2- 14 C, D-glucose-l- 14 C, succinate-2- 14 C. DL- alanine-3- 14 C, and DL-glutamate-3-4- 14 C. The results have suggested that there may be an alteration with advancing age in the metabolic control mechanisms involving mitochondrial respiration and the extra-mitochondrial bio- synthetic mechanisms. Studies on the regula- tory mechanisms, possibly involving changes in concentrations of intermediates, allosteric alterations of enzyme activity, and hormonal effects will be considered in future research. One of the recurrent theories in aging re- search deals with the alterations in tissue per- meability with age. There has been much spec- ulation but few direct data on this topic. Re- cent work under the auspices of the Aging Pro- gram have shown that aortic permeability in- creases greatly with age up to the age of about 45 years. Further studies on the permeability of connective tissue as a function of age will be carried out. The survival of labeled erythorcytes from young and old rats has been studied in young and old rats in work supported by the Aging Program. It has been found that neither the age of the donor nor the age of the recipient has any effect on the survival of the erythro- cytes. There are many biologic problems that arise as a result of the aging process and that mani- fest themselves at the level of the organ sys- tems that comprise the body and at the level of the intact organism. These problems in the human can be effectively investigated by cross- sectional and logitudinal studies. The Aging Program is supporting several such studies. One of these is the study being conducted by the Gerontology Branch in Baltimore. About 600 participants have been involved in this study. They have been examined from 1 to 6 times. Measurements of biochemical, physio- logical, and psychological variables have been made. One of the very interesting findings of this study is that carbohydrate tolerance de- creases progressively with age and that beyond the age of 45 years half of the subjects studied have glucose tolerance curves that by the standards applied to young adults would be considered diabetic. In the past year these subjects have been shown to have a smaller de- crease in plasma glucose following intravenous tolbutamide than young subjects. This repre- sents further evidence of impairment of car- bohydrate metabolism with age. In addition to the above and other studies which include cross-sectional and longitudinal components, some purely cross-sectional studies are being carried out in men and experimental animals. Purely cross-sectional studies have the advantage of permitting a relatively rapid accumulation of information though they will not answer certain types of questions. One of these studies has shown that older subjects are unable to correct an induced aci- dosis as rapidly as young subjects. This is due to an impairment of excretion of titratable acidity and ammonia in the urine. Studies on the ability of the isolated rat heart to perform work have unequivocally con- firmed the previous findings by the same inves- tigators of a significant decrease in cardiac capability with age. Studies to determine the mechanism of this decrease in capability are continuing. The Aging Program sponsors a number of analyses of age-related differences in behavior NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT 283 and of relationships between age and psycho- logical, sociological, physiological, and medical variables. Work is being carried out both on humans and on rodents. Work sponsored by the Aging Program and elsewhere shows a wel- come trend away from a mere cataloging of age-related differences in behavior and toward analyses of the nature of these differences and the development of techniques to optimize psy- chological capacities in the older organism. An abundant literature indicates that older humans usually learn verbal associations much less rapidly than young adult humans. Recent experiments supported by the Aging Program indicate that much of this apparent deficit is due to a reluctance to respond which in turn appears to be related to physiological over- arousal or "anxiety" as defined by autonomic measures. Present work on these variables in- dicates that verbal-associative learning in older humans can be improved greatly by allowing more time in which to respond, perhaps by blocking autonomic activity by drugs, and per- haps by manipulating the older learner's ex- pectancies of the time available for responding. Older people also have been reported to per- form more poorly than young on certain kinds of logical problems which require logical infer- ences and the discovery of concepts or rules and require a considerable short-term mem- ory load. Studies on the qualitative work pat- terns and "strategies" of young and old humans are being supported. Several investigations are dealing with the "information processing" rates and techniques of younger and older humans in a variety of laboratory tasks. Although older people are usually found to be slower, and sometimes poorer than the young in most such laboratory tasks, one of the Aging Program's grantees is testing the hypothesis that the human's ability to learn the patterns in a flow of information increases with age throughout most of the working life. Other investigators are dealing with a variety of other relevant variables, such as short-term memory load. Several projects concerned with psychologi- cal task performances and concurrent psycho- physiological indices of arousal and stress, such as electroencephalographic patterns, auto- nomic changes and free fatty acid mobilization are being supported. The effects on behavior of manipulating autonomic activity by chemi- cal means, in young and old humans are also being studied. Other longitudinal studies in humans relate changes in intelligence test scores and personality measures across time with changes in health status, electroencephalo- gram, chromosomal changes, and other physio- logical and biological measures. It has been demonstrated that a sudden change in environment — such as being moved from one institution to another — produces a substantial increase in death or the develop- ment of depression and other psychiatric con- ditions in elderly humans. A large group of elderly people scheduled to enter institutions will be studied from one year before to one year after institutionalization, with emphasis on personality structure and psychiatric status, in an effort to mitigate this unfortunate effect. A scale of "attitudes toward the elderly" is being developed in order to counsel those on whom the elderly person is dependent. A less well-developed area for research car- ried out under the auspices of the Aging Pro- gram is that of social gerontology. Investiga- tions in this area focus upon developmental or retrogressive reactions of whole persons rather than upon alterations in abstracted processes such as verbal association, visual acuity, or a particular motor response. Investigators tend to view persons within their existing environ- mental contexts. Of particular relevance are the effects of economic, physical, physiological and social background factors, and changes in them which are usually concomitant with ad- vancing chronological age in present-day soci- ety. Such investigations will help to clarify knowledge of aging through differentiating those changes determined by chronological age from those consequent upon alterations in en- vironmental influences, which are commonly concurrent with advancing age in our culture. One NICHD-supported investigator analyzed some effects of the well-documented low in- come level of older people. He studied the ways in which older persons used their more limited incomes, whether the amounts they spent for various categories of consumptions were ade- 284 ANNUAL REVIEW OF INTRAMURAL RESEARCH quate for their needs, and ways in which the patterns of expenditure of the aged differed from those of younger segments of the popu- lation. Though aging and illness are not synony- mous, the incidence of chronic ailments is much higher for people over 65 than for those younger. An investigator found that, while older people were generally aware of the in- creased likelihood of their incurring such ill- ness, only a small percent expressed concern. Concern about illness was not significantly re- lated to objective or subjective health evalua- tions, but probably was influenced by the se- curity of these old persons in family life, religion, a stable environment, and financial resources. Results of another study are consistent in showing that socioeconomic variables are re- lated to reported health status. Tendency to report chronic health conditions was related to labor force status, financial position, and edu- cation. More specifically, blue collar workers tended to report illness more frequently than white collar workers, the higher education groups reported less illness than persons at lower educational levels, and higher income groups report less than the lower income groups. Great emphasis has been placed on the train- ing area in the last year. The amount of money approved for training in aging by the National Advisory Council has tripled in the last year. The total number of training grants approved has increased from 5 to 15. The Aging Program has collaborated with the Information Center of the Communications Branch to help in developing a system that will permit the storage and retrieval by abstract journals and computerized techniques of infor- mation bearing on aging. All areas from cellu- lar biology to social gerontology will be cov- ered. It is believed that the ability to retrieve information rapidly and comprehensively will be a major aid to scientists in the field of aging. Research at Bethesda Two research projects are being conducted by the staff of the Aging Program at Bethesda. One of these is concerned with the changes in connective tissue with age. Studies carried out under this project have shown that there is a striking increase in the permeability of the aortic wall to albumin with increasing age. Studies directed toward detecting changes in the permeability of other connective tissues are being started. Such changes in permeability o: various connective tissues might have consider able significance with regard to changes in the functioning of those tissues with age. The second research project is concerned with the reaction of elderly people to their housing conditions. Previous studies by the in- vestigator concerned have shown that housing has a great psychological impact on the elderly. The present studies are designed to confirm and extend the previous studies. GERONTOLOGY BRANCH The goals of the Gerontology Branch are to describe in quantitative terms the changes that take place with aging and to investigate the basic biological mechanisms of these changes with a view to reducing the impair- ments and disabilities of older people. The re- search program falls into two major cate- gories: (a) the description of biochemical, physiological, and psychological changes that take place with aging, and (b) investigations of the mechanisms of age changes at the so- cial, psychological, clinical, physiological, bio- chemical, cellular, and molecular levels. During the past year, members of the pro- fessional staff have contributed considerabl< time and effort to working out the design oJ many special features of the Gerontology Re search Building to be constructed in Balti more. Construction of this 6.9 million dollai research building started in September 1965 Completion of the building is scheduled foi October 1967. Highlights of findings from the Gerontolog: Branch which have extended knowledge o: aging include the following. Age differences in interference effects ii learning are significantly reduced when antici pation time during the original learning i long. NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT 285 Older subjects show an impaired response with respect to blood sugar levels to the intra- venous administration of tolbutamide. These results offer further evidence of impairment of carbohydrate metabolism with advancing age. The impairment of glucose tolerance in the aged cannot be ascribed to an increase in free fatty acids in the plasma. Total caloric, vitamin, and mineral intakes diminish with age even in subjects whose diets are not limited by economic factors. In con- trast, the intake of vitamin C increased after age 70. In about half of the middle aged and elderly subjects the dietary intake of Ca was less than the NRC recommended intake. A mathematical method has been devised which may make it possible to assess individ- ual differences in non-uniformity of lung ven- tilation. The impairment in the ability of the aged kidney to form ammonia is not due to inade- quate amounts of substrate (glutamine) for its production. A double isotope derivative assay for angio- ' tonin has been developed which permits the quantitative estimation of nanogram amounts of the hormone. A method has been developed which permits the achievement of a steady state of blood glu- cose levels at any concentration desired by i the investigator. This method will be of great i value in investigating many problems of glu- cose metabolism in humans, such as the mech- : anisms of insulin synthesis and release in re- sponse to hyperglycemia. In rats the ability to make fine discrimina- tions in taste diminishes significantly with age. Although the experimental introduction of i "errors" by feeding ethionine to rats increases : protein and RNA metabolism, no age differ- ences in the response were observed. In rats, enzymes involved in neural trans- , mission and conduction failed to show sig- nificant differences between young and old rats. The reduction of food intake in maternal rats has been shown to produce young which grow at a slower rate than normal and fail i to attain normal adult size even when fed ad libitum after weaning. This method will be of great value in projected studies of the life lengthening effects of starvation in rats. Under appropriate conditions the degrada- tion of RNA by metal ions such as zinc can be made specific and this specificity can be changed by inhibition of the degradation by another metal ion. Thus non-enzymatic reac- tions may be utilized for base-sequence deter- minations of nucleic acids. An additional coupling factor associated with oxidative phosphorylation has been iso- lated from cardiac mitochondria. Age changes in collagen may be due to a reorientation of existing intra-molecular cross- links rather than the addition of new cross- links. Aging in the Human The longitudinal multi-disciplinary study of subjects aged 17 to 103 years has been suc- cessfully continued in the past year. The total subject group now numbers 587 self -recruited community-dwelling men. It is 8 years since the study began and we are thus just begin- ning to examine the earliest volunteers for the sixth time. Half of the group has now had at least 3 visits (at approximately 18-month intervals) and one fourth of the group has had 4 visits. Considering the life span of man and the anticipated rates of change in the various physiological and psychological func- tions being measured, the study is reaching a stage of longevity in which it will begin to be appropriate to analyze the data for longi- tudinal trends and for intercorrelations of various related functions. In anticipation of this complex task the rate of conversion of data to a punch card system is being increased and preliminary tests of the data retrieval sys- tem have been conducted by the Human Per- formance Section. These subjects, the Longitudinal Group, con- tinue to provide a valuable cross-sectional pop- ulation for a variety of studies by several sec- tions of the Gerontology Branch. Because of their high level of education, these subjects are of special value in studies of psychological functions. The Section on Ex- perimental Psychology has been especially in- terested in determining the factors which lie 286 ANNUAL REVIEW OF INTRAMURAL RESEARCH behind the impaired learning ability which has been observed even in these subjects. Al- though the elderly subjects can learn material to the same criterion as the young, it takes them longer to do so. It is now apparent that the extent of the impairment observed depends on the conditions imposed in the learning ex- periment. In fact, when old subjects are al- lowed as much time as they want to respond they can reach the criterion of learning with the same number of trials as the young, but the time will be longer. The number of errors (as well as the number of trials required) increase more with age when the time given to respond is short than when it is long. In fact, the rela- tionship between errors and age is curvilinear when the anticipation time is short, whereas it is linear with a smaller slope when the anti- cipation interval is long. It has also been shown that old subjects are more susceptible to interference (increase in errors) from ex- traneous material introduced between trials than are the young. However, it has now been found that interference is greater in old sub- jects than in young only if the time available to respond when learning the original task is short; if that t^ime is long, there is no age difference in the degree of interference from an interpolated task. These studies lead to the hypothesis that short-term memory storage may be a primary factor in the difficulties older people experi- ence in learning. The experiments have shown, however, that alterations in the condi- tions of learning can compensate for this dif- ficulty. It is further hypothesized that active responding is conducive to learning, particu- larly for an old person and lengthening the an- ticipation interval increases the probability of a correct response. It is also possible that re- enforcement by presentation of simultaneous cues from different sense modalities will en- hance learning in the adult. Further studies to test these hypotheses will be conducted. Unravelling of the complex relation between (1) the decline in performance of a physio- logical function with increasing age, and (2) the increasing prevalence with age of a disease associated with that function is one of the major areas of investigation in clinical geron- tology. A prototype of this problem is under study in the Metabolism Section: (1) the physiological decline with age in the ability to metabolize administered glucose loads, and (2) the age-associated increase in the preva- lence of overt diabetes mellitus. The immediate goals of this program have been (a) to de- lineate the underlying mechanism (s) of the decline in function with age, and (b) to de- velop the first realistic age-adjusted standards for the several diagnostic tests for diabetes mellitus used in clinical medicine. Studies on the Longitudinal Group had pre- viously shown that the standards in common use for interpretation of the intravenous glu- cose tolerance test and the cortisone glucose tolerance test were appropriate for young adults only since their application to middle- aged and older adults resulted in a diagnosis of diabetes in 50% of the group. In the past year an extensive study was done on the Intra- venous Tolbutamide Response Test as well. The test is an interesting addition to the diabetes diagnostic armamentarium of the physician since it is not simply a variant of glucose tol- erance testing and reputedly has higher spe- cificity than other diagnostic procedures. Re- sults show that just as diabetics respond de- ficiently in the test, the presumably nondia- betic middle-aged and older longitudinal sub- jects also have deficient responses when compared to young adults. The rigid applica- tion of the currently recommended standards for normality to subjects over age 40 results in the classification of almost half of them as "diabetic". These unrealistic standards are probably the result of an inappropriate tech- nique for selection of the subjects used to es- tablish the normal standards for the test. From the results on the Longitudinal Subjects new standards for the test have been developed. These are presented in the form of a nomo- gram which permits the determination of the percentile rank of each individual among his age peers. The Longitudinal Subjects have also been used as a group for the testing of one of the current hypotheses concerning, the cause of de- creased glucose tolerance. The hypothesis states that free fatty acids (FFA) in plasms NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT 287 inhibit glucose utilization. The decline in tol- erance to glucose with increasing age then could be explained by an increasing concen- tration in FFA with age. Results in Longi- tudinal Subjects who were given oral glucose tolerance tests, however, show no correlation between FFA concentration and age or be- tween FFA concentration and tolerance to glu- cose. Results of a computer analysis of detailed 7-day food intake diaries on 252 Longitudinal Subjects have been analyzed under the super- vision of the Nutritional Biochemistry Section. 1 This again is purely a cross-sectional study at this time. The conversion of dietary food por- tions of the nutrient elements (calories, car- bohydrates, protein, fat, minerals, and vita- mins) was accomplished with a computer pro- gram devised by members of the Heart Disease Control Program. At the moment, it is possi- ble only to characterize the nutrient intake of the Longitudinal Group as a function of age; important correlations with other parameters (anthropometrical, physiological, and biochem- ical) will be the next step in the analysis of the data. Thus far it is clear that in the upper middle-class highly educated group of men, total caloric intake, vitamin and mineral in- i take tend to decrease with age with the main i exception that intake of vitamin C does not , follow this trend and, in fact, its intake is increased in the diet after age 70. Of interest is the fact that National Research Council reco- mended allowances were exceeded for all nu- trients except Ca in the great majority of sub- jects. Calcium intake was below NRC allow- ances in about half of the middle-aged and older subjects. The pattern of food intake shows distinct differences with increasing age in that while the percentage of total calories derived from protein remains constant, more of the calories are derived from carbohydrate while fat plays a decreasing role as a source : of calories. Whether these differences in caloric : source in individual subjects on spontaneous diets will correlate with such parameters as serum lipid levels, carbohydrate tolerance, or the prevalence of atherosclerotic disease at 1 the time of the study or its incidence in the future are some of the problems that remain to be determined. Studies on the Longitudinal Group by the Pulmonaiy Physiology Section have begun to be examined for longitudinal trends in indi- vidual subjects. Preliminary analyses on 70 sub- jects who have had 4 visits show that with regard to vital capacity and maximal breath- ing capacity, only the older subjects show a downward trend in function while younger subjects show either no change or an increase in function. The ability to detect age differ- ences in time trends in the relatively brief period of 6 years (4 visits) is encouraging. A subsample of the Longitudinal Group, 20 subjects aged 39-83 years, has been studied in a set of experiments using techniques too complex to apply to the entire group. The char- acteristics of the barrier in the lungs across which gases must diffuse have been thought to change with increasing age; pulmonaiy dif- fusing capacity presumably decreases. How- ever, these measured changes could be arte- factual due to an increasing degree of non-uniformity of lung ventilation associated with aging. This hypothesis was tested using a technique for measuring pulmonary diffus- ing capacity by sequential steady state and washout methods. The extreme complexity of the equations generated by a multi-compart- ment system with non-uniform ventilation volume/diffusion was handled with the aid of a computer program (IBM 7094, University of Chicago). Thus more accurate meas- urements of diffusing capacity could be made in the non-uniform lung. These measurements show that part if not all of the age changes in diffusing capacity previously reported may be the result of an age differential in the non- uniformity error of the methods employed to make the measurement. Future studies from the Section on Pulmon- ary Physiology will capitalize further on the advantages of the high speed digital com- puter in terms of accuracy, ease, and rapidity of the necessarily complex calculations in this field. In addition, pulmonaiy evaluations will be continued with measurements of lung vol- umes, pulmonaiy gas distribution, diffusion and compliance measurements. The addition 288 ANNUAL REVIEW OF INTRAMURAL RESEARCH of a body plethysmograph will also permit more direct estimates of the effects of deep breathing on thoracic gas volume and distri- bution. New tests introduced into the longitudinal testing program in the past year included (1) detailed blood typing in order to characterize the participants immunogenetically, (2) "tap- ping-in-place" tests in order to investigate the possibility that the extra time required by older subjects in the two target tapping test is due to uncertainty in aim, and (3) a self- administered activity survey in place of the time-consuming interview. Renal function in the Longitudinal Group has been evaluated with the 24-hour creatinine clearance test. In the past year the number of subjects with 3 consecutive tests has increased to 192, a group large enough to permit a pre- liminary examination for cross-sectional data, individual longitudinal trends, and within-sub- ject variance. The cross-sectional data were best fitted with a second order regression; this de- velopment is interesting since, unlike most lin- ear physiological age differences, in this test the decrements with age are small in early adult life, but become much more pronounced as age advances. The within-subject coefficients of variation at all ages are about 11-13 per- cent. The importance of this type of analysis is that it permits us to plan the most efficient testing schedule in terms of number and fre- quency of re-tests for the detection of longi- tudinal trends. Such information should also prove of value to other prospective studies on man now in progress or planned for the fu- ture. We plan to use similar anlytical tech- niques for the other tested physiological vari- ables in our program. A number of clinical investigations in the Branch are being conducted on subjects other than the Longitudinal Group. In some of these studies the stimulus to the study again was the apparent similarity between changes occur- ring with advancing age on the one hand and in certain disease states on the other. The Renal Section has continued its studies on the factors controlling urinary cation ex- cretion. It had previously been noted that under conditions of a water diuresis during the basal state older subjects commonly (7 of 15 studied) had an inappropriately high loss of sodium in the urine. Unchecked, this defect could lead to hyponatremia. A detailed inves- tigation has now been made of the mechanism of this defect and, in a related study, of the mechanism of the development of hypo- natremia seen in certain acute and chronic illnesses (tuberculosis, malignancies, febrile states). For these comparative studies rela- tively healthy older subjects on the Gerontology Ward at the Baltimore City Hospitals composed the first group and all of the verified cases of hyponatremia (sodium concentration less than 125 mEq./L.) seen on the wards of the Balti- more City Hospitals during an 18-month period comprised the second group. The 16 hypona- tremics were supplemented by another equal sized group of patients with similar illnesses, patients who might be expected to develop hy- ponatremia but whose serum sodium concen- tration had not yet reached abnormal levels. Distinct differences in these groups were found. The defect in the old but healthy group with inappropriate urine sodium loss was cor- rectible by administration of glucose or re- lated metabolites. Since the renal medulla is totally dependent upon glycolysis, this result suggests that an overnight fast leads to inad- equate substrate supply to renal tubules with resultant inadequate sodium reabsorption; glu- cose availability then corrects this defect. In contrast, the acutely and chronically ill pa- tients and hyponatremics rarely showed this defect. In 4 carefully studied patients and syn- drome of inappropriate anti-diuretic hormone secretion could be shown to be responsible for the hyponatremia. These subjects had not only balance studies, but also measurements of aldo- sterone secretion rates, steroid levels, blood volume determinations, and erythrocyte elec- trolyte levels. In the past year another defect of the renal function in the aged has been reinvestigated. The observation had been made in the Geron- tology Branch a number of years ago that re- covery of serum pH following an administered acid load (ammonium chloride) was slower in older than in younger subjects. The mechanism of this defect has now been investigated by NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT 289 measuring the urinary excretion of "titratable acid" and of ammonium ion. Both of these avenues of H + loss in the urine were decreased in the older subjects. The decrease in titratable acidity was commensurate with the lowered glomerular filtration rate in the aged, but the diminished ability to excrete ammonium was proportionately greater than the GFR de- crease. A further study was therefore con- ducted to test the hypothesis that the defective ability to excrete ammonia is due either to inadequate substrate (glutamine) or enzyme (glutaminase) for its production. Glutamine ad- ministration to old and young subjects resulted in equal increases in NH :i production during an acid load and therefore the hypothesis seems unlikely. An extension of these acid-base studies con- ducted by the Renal Section concerned the regulation of hydrogen ion concentration with the mitochondria of rat liver. Mitochondrial preparations met the criteria of respiratory control and their pH was measured with the O DMO technique. The pH was found to be linearly related to the extramitochondrial pH when the latter was set at levels of 6.90 to 7.60, but intramitochondrial fluid always re- mained somewhat more alkaline by about 0.3 pH unit. The Endocrinology Section has also con- ducted studies in an area in which the change in a physiological variable (blood pressure) with increasing age resembles the change oc- curring in a disease syndrome (hypertension). Understanding of the relation between aging and disease in this instance again requires knowledge of the basic mechanisms underlying the changes. An intensive effort has therefore continued to develop a physico-chemical method for the measurement of plasma renin and an- giotensin. Progress in this area of research has been long hampered by lack of such methods; heretofore, all analytical techniques depended on bioassays, most of which are of question- able specificity and precision. Over the past year we have succeeded in developing a double isotope derivative assay for angiotensin. This assay permits quantitative determination of nanogram amounts of the hormone. This de- gree of sensitivity is required for the measure- ment of the small quantities of angiotensin generated during in vitro assays of plasma renin. It should soon be possible to apply this new technique to the determination of renin. The importance of this project is along both theoretical and practical lines. Along theore- tical lines, it is the first isotope derivative assay to be developed for a small polypeptide hor- mone and could lead to the development of other assays of similar important peptides. A practical renin assay, on the other hand, would be a major step in the everyday hospital differ- ential diagnosis of hypertensive disease, since it would almost certainly permit recognition of unilateral ("ischemic") renovascular hyper- tension. Recently the determination of plasma renin has assumed great importance in the diagnosis of primary aldosteronism, a disease which may account for as much as 10-25 per- cent of cases of hypertension heretofore con- sidered to be of the "essential" variety. The problem has, therefore, a place in the attack on hypertension, a major age related disease complex. In addition, renin and angiotensin are intimately related disease complex. In ad- dition, renin and angiotensin are intimately related to the secretion of the adrenal glands' mineralocorticoid hormone, aldosterone, and adequate methodology is badly needed in this area of physiology and pathophysiology. An additional aspect of the program is an inter- related study of renin-angiotensin-aldosterone in normal old persons. The data obtained in this study will establish normal standards for al- dosterone secretion rate under conditions of salt loading and salt depletion. This informa- tion is unavailable but is badly needed in eval- uating the role of aldosterone in the elderly hypertensive subject. Another area of major interest to the En- docrinology Section is the field of thyroid hor- mone metabolism. The control of the rate of thyroid hormone degradation is age-dependent, as we have shown previously, but factors which determine this rate are largely unknown. By studying, as we presently are doing, the situa- tion in acute febrile illness (bacterial infec- tion) where rapid changes in hormone me- tabolism are known to occur, we hope to learn more of the normal control mechanisms for 290 ANNUAL REVIEW OF INTRAMURAL RESEARCH thyroid hormone metabolism and ultimately those involved in the changes with age. Of cur- rent interest are the interrelationships of hor- mone degradation rate, plasma thyroxine-bind- ing proteins and plasma "free" (non-protein- bound) thyroxine. Preliminary results in hu- man subjects experimentally infected with the organism of tularemia (Pasteurella tularensis) indicate that in this particular febrile illness thyroxine degradation rate is not accelerated, in contrast to the situation in other bacterial pneumonias. Nonetheless, thyroxine binding pre-albumin falls to low levels and, presum- ably, "free" thyroxine rises as a consequence. These results would appear to exclude "free" thyroxine and febrile illness, per se, as im- portant physiological determinants of thyrox- ine degradation rate and will require exten- sive revision of current thinking in this area. The remarkable but unexplained decrease in glucose tolerance with increasing age has pro- vided the stimulus to the development of a new experimental technique for the study of the physiology of the glucose-insulin system by the Metabolism Section. The feed-back prin- ciple is used to adjust periodically the rate of infusion of glucose intravenously in order to maintain a predetermined new steady-state arterial glucose concentration. In these studies the hyperglycemic stimulus to the beta cells of the islets of Langerhans is constant and nearly identical in all subjects, in con- trast to the marked variability in glucose levels that occur during the usual glucose tolerance tests. The ability to achieve a steady state With this technique is shown by the low co- efficients of variation in glucose concentration over a two-hour period. In 32 subjects the mean CV was 3.9 percent. The time course of insulin release in response to sustained hyper- glucemia is complex and cannot be described by a simple dose-response curve; in young adult subjects plasma concentrations of insulin con- tinue to increase over the two-hour period. This changing insulin level is reflected by the pro- gressively increasing glucose infusion rate needed simply to maintain the arterial glucose concentration at its predetermined level. Older subjects differ from younger subjects in that (1) they require a lower glucose infusion rate and (2) they show less change in infusion- rate over the two-hour period. The implication of these age differences is that there is a di- minished ability to respond to hyperglycemic stress in old age. The ability to increase plasma insulin concentrations progressively over a two-hour period which young people possess seems to be deficient in older sub- jects. These age differences raise questions con- cerning the mechanism of insulin synthesis and release in response to hyperglycemia; the servo-infusion technique should help to answer these. Biology of Aging Although the quantitative description of age changes is of great importance, the develop- ment of methods for mimizing the deleterious ecects of these changes depends on under- standing the basic mechanisms of aging. This requires studies on an animal species in which genetic and environmental factors can be controlled and experimental techniques can be applied to isolated systems. Studies on basic cellular and molecular mechanisms must be pursued agressively. It is for this reason that part of the resources of the Branch have been devoted to studies of cellular biochemistry and the structural characteristics of molecules in- volved in biological processes. Recent gerontological theory has attempted to explain age differences in motivation on the basis of physiological factors and in terms of differences in response to various environ- mental factors. A general factor termed "rig- idity" has also been proposed as an explana- tory construct. The program of the Section on Animal Behavior is concerned with (a) de- termining basic behavioral age differences, and (b) finding environmental factors which reduce age differences. The use of lower organ- isms such as the rat is necessary for such a program. It has been found that environmental varia- bles such as systematic handling by the in- vestigator or testing during the dark portion of the dark-light cycle which reduced fear in young rats as evidenced by increased explora- tory behavior generally had no effect on old animals. However, restriction of food intake NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT 291 increased exploratory behavior in both young and old animals. Thus, an increase in motiva- tion (food deprivation) has resulted in be- haviorally less rigid groups of animals, which are similar to animals younger in age. An initial experimental study of taste discrimination was concerned with ingestion of very low concentrations of sucrose or quin- ine and the effects of past experience with quinine upon sucrose ingestion and vice versa for male rats 1, 5, 15, and 25 months old. Old animals did not discriminate the solutions as well as younger groups, and preceding inges- tion experience was important (e.g., rats initi- ally tested with sucrose drank more quinine and rats initially tested with quinine drank less sucrose). However the effects of past ex- perience were clearly not a function of age, but rather of reduced sensory discrimination with increasing age. Changes in the plasticity of the nervous system could be responsible for the re- duction of sensory discriminability with in- creasing age. A second experiment examined the ability of rats 1, 5, 15 and 25 months old to discrim- inate between two above threshold sucrose so- lutions. Normally rats prefer the more highly concentrated of two sucrose solutions. By mak- ing the discrimination more difficult, i.e., pair- ing very similar solutions, it was possible to examine age differences in the ability to de- tect the more highly concentrated solution. If the neural system of the aged rat was more rigid (e.g., reverberating cell assemblies were less flexible in form or resistant to change) than in the young rat, such discriminations should be more difficult for the older rats than for young. The data were consistent with such a hypothesis. In addition, when presented with palatable solutions young rats markedly in- creased their fluid intake, while senescent (25- month-old) rats were unable to alter ingestion rates. The fluid intake increase for young rats was greater as the discrimination became more difficult, suggesting that younger rats may also make more comparisons between tube pairs, i.e., have a higher general behavioral plasticity, than senescent rats. Other experiments are planned to test fur- ther the hypothesis that aging is associated with greater rigidity of behavior. In this con- nection conditioning studies will be carried out on old and young animals with various schedules of training and the introduction of multiple responses. Rats have also been used for studies on age differences in the performance of the isolated heart by the Cardiovasctdar Section. Addi- tional experiments have fully confirmed the preliminary report that the heart of the sene- scent rat shows a significant impairment in its capacity to do work. The left ventricular work index expressed in gm-M/100 mg dry heart weight for the isolated heart-lung prep- aration was 34 ± 4 for 24-month-old animals and 166 ± 14 for 12-month-old animals (P< 0.01). Similar differences (15 ± 5 and 77 ± 13) were found for the isolated heart. It has also been found that direct electrical stimula- tion of the sino-auricular node fails to revive hearts that have worked to exhaustion. Hence it is concluded that the failure to perform is related to biochemical factors in the myocar- dium. These studies will continue to explore the biochemical mechanism of failure of the senescent hearts to perform as well as adult hearts. Studies of specific enzyme systems and concentrations of substrates essential for en- ergy production will be conducted. One of the current theories of aging pro- poses that with senescence alterations occur in the structure of the DNA molecule. This error is transmitted to messenger RNA and ultimately to newly synthesized enzymes. These defective enzymes may be inactive and therefore an accumulation of substrates within the cell may take place. Data obtained during the past year in the Section on Nutritional Biochemistry have failed to support this theory of aging, which proposes an accumulation of structural errors in DNA which are reflected in RNA and pro- tein, as well as increased turnover of these compounds to compensate for such errors. For example, it may be postulated that structurally altered proteins should be detected by altera- tions in the mobility of enzymes during gel electrophoresis. However, no changes with age were found in the patterns of isoenzymes of seven dehydrogenases in various tissues of 292 ANNUAL REVIEW OF INTRAMURAL RESEARCH rats. Furthermore, although marked increases were found in the metabolism of RNA and pro- tein when animals were fed ethionine to ex- perimentally introduce errors into protein, a comparison of 40 old (24-31 months old) and 40 young- (12-month-old) rats failed to demon- strate differences in metabolism of RNA and protein between the two age groups. It has been proposed that age changes may be most easily detected in enzymes which con- trol specialized functions of highly differenti- ated cells. In order to test this proposal, two enzymes which are involved in neural conduc- tion and transmission were measured in cere- brum, cerebellum and brain stem of young and old rats. However, no age-associated al- terations were observed in the concentrations of DNA, RNA or protein or in the activities of acetyl cholinesterase or sodium-potassium activated ATPase. Efforts made to identify the factors respon- sible for increased longevity in rats subjected to dietary restriction have required time con- suming measurement of daily food allotments for large numbers of animals. During the past year, nutritional methods were sought which would avoid this necessity but would yield animals with similar characteristics and lon- gevity. Preliminary data indicate that restric- tion of nutrients prior to weaning by (1) re- duction of maternal intake or (2) employing, during lactation, mothers who had just nursed another litter, produced animals which ex- hibited low growth rates and which were un- able to attain body weights equal to normal animals in spite of unlimited access to food following weaning. Although data on lon- gevity are not yet available, these procedures may offer means of obtaining restricted ani- mals without the laborious task of daily food rationing. The experiments conducted thus far to test the error theory of aging based on the ex- perimental introduction of structural al- terations into protein were complicated by the influence of the ethyl group of ethionine on RNA and fat as well as protein metab- olism. Therefore, studies will be conducted in which a different analog, viz., p-fluorophenyla- lanine, will be fed to rats and changes in RNA and protein metabolism will be measured in liver slices. In addition, the in vivo incorpora- tion of radioactively labelled p-fluorophenyl- alanine and ethionine into liver proteins will be carried out to demonstrate that the analogs are incorporated into proteins. Finally, tech- niques are being perfected so that age-asso- ciated structural alterations in enzymes, as proposed by the error theory, can be detected by the quantitative determination of the iso- zymes of a variety of dehydrogenases in rat tissues. The studies in progress to test the proposal that age changes may be most easily detected in those enzymes which control specialized functions of highly differentiated cells will be continued. Similar data to that already re- ported for the rat will be obtained on mice, via., the activities of acetyl cholinesterase and sodium-potassium activated ATPase will be measured in cerebrum, cerebellum and brain stem and samples of these same sections will be examined for morphological alterations with age. Efforts will be continued to obtain restricted rats without the laborious task of daily food rationing by restriction of nutrients prior to weaning. Studies will be conducted to produce large numbers of animals in order that data on longevity as well as information on the biochemical characteristics of such animals throughout the life span will be obtained. In view of the key role of DNA and RNA in relation to "error" theories of aging the Ger- ontology Branch has placed special emphasis on studies of the molecular structure of these and related compounds. The principal activity in the Section on Molecular Biology in the past few years has been the elucidation of the interaction of metal ions with the nucleic acids. There have been two main objectives in these studies: (1) to provide chemical techniques for the determina- tion of the sequence of the nucleotides in the nucleic acid chains and (2) to understand the biological role of such metal ion-nucleic acid interaction. The importance of such fun- damental studies to an ultimate understand- ing of the aging phenomenon is obvious. NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT 293 During the past year the important discov- ery has been made that under appropriate con- ditions the degradation of RNA by metal ions such as zinc can be made specific, and this specificity can be changed by inhibition of the degradation by other metal ions. Thus the way has been paved for the use of nonenzy- matic reactions in the specific cleavage of nu- cleic acids for sequence determination. In the effort to determine the biological role of the metal-nucleic acid interaction we have carried out extensive investigations on the ef- fect of metals on the reaction of DNA with polysine, and the DNA-polylysine reaction it- self has been subjected to intensive study. Several physical chemical techniques have been pioneered for the study of nucleic acid-protein interaction; the lack of progress in this im- portant field has been due primarily to the absence of such techniques. Each nucleotide of DNA is bound to one amino acid in polylysine. The DNA-polylysine complex is severed by some metal ions and not by others. We believe that the unwinding and rewinding of DNA dur- ing replication involves such equilibria between nucleic acid, protein (histone) and metal ions. These studies do not prove this hypothesis, but represent the beginning of an attempt to unravel these complicated relation- ships. During the coming year we intend to exploit the findings on the specific cleavage of RNA by metals. The reaction of metals with nu- cleic acids and proteins will lead to studies on the DNA-histone interaction, and, hopefully, to an understanding of the role of metals and histones in DNA regulation and the implica- tion of these roles to the aging process. A new project on immunological studies on age-in- duced changes in tissue composition will be begun. Previous work from the Section on Compara- tive Biochemistry had demonstrated that the oxidation of acetate-2- 14 C to 14 C0 2 occurred more rapidly in senescent than in adult rats. The findings have been explained on the basis of a change with age in the metabolic rela- tionship between respiratory reactions of mito- chondria and the extra-mitochondrial biosyn- thetic reactions. In order to devise rational experiments to understand the molecular basis for the above observation, a detailed knowl- edge of cellular energy-linked reactions and their metabolic control is necessary. The studies on the mechanism of oxidative phosphorylation in beef heart mitochondria have been extended. The work has centered on the first site of phosphorylation; viz., that occurring during the oxidation of NADH by ubiquinone catalyzed by the ubiquinone re- ductase flavoprotein. The experimental ap- proach and findings are summarized in the fol- lowing formal scheme. ATP NADH Factor A Factor B ? Flavoprotein Ubiquinone A coupling factor, an enzyme tentatively called Factor A, has been purified further. Over 90 of the protein appears as a single band in electrophoresis on cellulose acetate or poly- acrylamide gel. It restores P/O and related partial reactions in urea-treated-phosphoryla- tion-deficient particles. These particles have only electron transport capacity in the absence of added Factor A. The purified factor has little ATPase activity, which can be stimulated over 20-fold by exposure to pH 8.5 and 43° C. The appearance of the ATPase activity is be- lieved to be due to distortion of the active site concerned with the normal phosphate transfer function. The distortion could make the enzymephosphate complex susceptible to hydrolysis by increasing the accessibility of water. The major accomplishment in the laboratory has been the isolation of a second coupling fac- tor. The new factor, tentatively designated Factor B, is another heat-labile, non-dialyzable protein. In particles depleted of phosphoryla- tion coupling factors by urea-treatment, Fac- 294 ANNUAL REVIEW OF INTRAMURAL RESEARCH tor B above gave little stimulation of energy- linked reactions. In the presence of saturation levels of Factor A, however, Factor B gave large additional stimulation. In ammonia- treated particles, which respond but little to Factor A, Factor B produced extensive stimula- tion of ATP-driven NAD reduction and P/O. The results clearly establish that the two factors are distinct from each other. Factor B did not affect oxidation rates in any of the particles. Its role, very likely in the energy transfer sequence, remains to be investigated. The flavoprotein in the above scheme is probably NADH-uniquinone reductase. This en- zyme has been isolated as two separate com- ponents with quite similar properties by chro- matography on hydroxylapatite. It is conceiv- able that the two forms are derived from the same original mitochondrial segment by cleav- age in two different ways. Both, reductaste preparations (QRl and QR2) catalyze the oxi- dation of NADH by suspensions of uniquinone- 10 at high rates. They appear essentialy as single, but separate, bands in electrophoresis on cellulose acetate strips and polyacrylamide gel. In sedimentation velocity experiments (carried out in collaboration with Dr. H. Edel- hoch, NIAMD) better than 90% of the protein migrated under one peak. The molecular weights of QRl and QR2 have been determined by sedimentation equilibrium to be 69,000 and 87,000 respectively. The reductases have bound FMN, nonheme iron and acid-labile sulfur which can be liberated as H 2 S. Thus, three of the components of the first site of mitochondrial oxidative phosphoryla- tion system have been isolated. Indirect evi- dence has suggested the involvement of an ad- ditional undiscovered factor, structural protein and phospholipid in the over-all reaction. Among the demonstrated changes occurring during aging is the loss of cells from a num- ber of tissues containing irreplacable cell types (e.g., nerve cells). However, the events lead- ing to cell death and loss during aging are not well understood and therefore during the past years fundamental studies on histochemical and chemical changes during cell death in model systems, under controlled conditions, have been carried out. Earlier histochemical studies on the ischemic death of cardiac cells carried out in the Section on Cellular and Com- parative Physiology had indicated that irre- versible injury occurs when heart muscle is kept under anaerobic conditions for sufficient periods to exhaust glycogen reserves and sug- gested that the irreversible phase is reached as the adenosine triphosphate concentration drops in consequence of the depletion of suit- able glycolytic substrates. This thesis was tested using the cellular level of adenosine tri- phosphate as a function of the duration of anaerobiosis. It was found that ATP concen- tration drops to 50% of the intial level within 7 minutes after the imposition of anaerobic conditions and to about 5% of the normal level by 15 minutes. This decrease in ATP level was paralleled, surprisingly, by the loss of ability by isolated mitochondria to carry out phos- phorylation reactions in vitro using the firefly continuous oxidative phosphorylation assay de- veloped in this laboratory as the monitoring system. A study of the properties of these mi- tochondria revealed that they, as well as all mammalian and avian mitochondria tested, are extremely sensitive to dilution. Dilution be- low about 0.1 mg. mitochondrial protein/ml completely abolishes the capacity to phosphory- late adenine nucleotides. However, oxidative phosphorylation can be completely and instan- taneously restored by the addition of about 0.5 mg/ml of serum albumin to the reaction mixture. With the exception of lactalbumin, which is about 15% as effective as serum albu- min, no other proteins tested show this recou- pling effect. This powerful influence of a serum protein on the efficiency of substrate utilization in the production of usable energy carrying intermediates suggests that these substances play a regulatory role in vivo. The level of se- rum albumin intracellularly, which in turn may be under hormonal influence (thyroxin, etc.), may thus moderate the oxygen consumption of tissue cells and the organism, and decreases during aging in the intrcellular concentration of serium albumin or analogous factors may lead to a decreased efficiency of production of usable energy intermediates such as ATP. The mechanisms of serum albumin action in vitro will be examined, particularly its effect on mi- NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT 295 tochondrial myokinase (which is preliminary studies appears to be stimulated by SA addi- tion). The intracellular level of serum albumin vs. age as well as other studies suggested by the above will be pursued further. A variety of lines of evidence indicate that cessation of cell division as well as related dif- ferentiation processes play a central role in the aging of higher forms of life. For example, animals with indeterminate life spans (e.g., plaice, Tcstiido); cells in tissue culture appear to have a limited growth potential (about 50 cell divisions); underfeeding of experimental animals slows down both the growth rate and the rate of aging as measured by age specific mortality; and finally, a general inhibition of cell division is observable when differentiation takes place which results in many cases in cells which can no longer be replaced (e.g., nerve, muscle), which cells are also the locus of many functional decreases during aging. In order to evaluate the role of cell division, arrest of mitosis and differentiation in the ag- ing process, a deeper understanding of related cell biology is desirable and the research activi- ties of cell biologists in the Branch and in the collaborating VA Aging RResearch Labora- tory in Baltimore are directed toward ques- tions bearing on the relationships between aging and these cellular properties. Cell division in metazoa is influenced both by intracellular and extracellular factors. The finding by cell culturists (e.g., Hayflick) that there is a limited division potential in human cells in artificial media suggests a model of aging in which important cell lines may ex- haust their division potential during the life of an individual and directly cause senescence. That this is a general property of cells capable of growing in culture has been shown during the past year. Chick myoblast cultures are ca- pable of undergoing about 20 divisions and the number of divisions obtained decreases with increasing age of the donor embryo. Using hu- man epidermis labeled with tritiated thymidine, supplied by Dr. Gerald Weinstein of the Uni- versity of Miami, work in this laboratory has shown that this tissue in vivo is not subject to the limitation of fibroblasts in culture. This was established by radioautographically fol- lowing the fate in serial sections of daughter cells of individual cells pulse labeled with tri- tiated thymidine. If the system follows the Hayflick rule, all daughter cells should move upward into the spinous layer coherently in groups of 2". Since single labeled spinous cells as well as triplets and other unpredicted groupings were observed in this human mate- rial, it appears that human epidermis is not limited to 50 divisions. Whether other impor- tant cell types are limited by this rule is the subject of present and projected studies. Environmental factors affecting cell division and differentiation under controlled conditions constitute another facet of the research of the Cell Biology Group within the Branch. It has been shown that slight variations in the pH of the medium inhibit cell division and promote differentiation into striated multinucleate mus- cle fibers. Below pH 7.2 cell division predomi- nates; above pH 7.4 differentiation is favored. Interestingly, another factor which affects cell division is the level of serum albumin in the medium. Although some albumin is necessary for growth, somewhat higher levels are inhib- itory. Related studies on the effect of sera of different ages, a la Carrell, are also under way. Finally, the hypothesis that the process of differentiation leads to senescence by shutting off the synthesis of certain important cell com- ponents as a by-product of the specialized syn- thesis implicit in differentiation is being tested. These limiting components are suppos- edly long lived and possess a low turnover rate or no turnover at all. The specific hypothesis being examined is that the pattern of synthesis is controlled by the species of s-RNA present and/or activated in specific differentiating cells. Since each amino acid is specified on the average by any one of 3 different codon triplets, the selection during differentiation of an appropriate group of triplets (codon set) for synthesis and/or amino acid activation would impose severe re- strictions on the kinds of messenger that can be translated, since only those messengers can be read for which the corresponding species of s-RNA are available. 296 ANNUAL REVIEW OF INTRAMURAL RESEARCH This proposition is being tested inter alia by comparing the pattern of s-RNA activation produced by activating enzymes from a variety of tissues from the same species. Identical s- RNA's are breifly labeled with tritiated amino acid in the presence of one enzyme and C 14 labeled amino acid by another enzyme. The labeled s/RNA's are combined, chromatograph- ically resolved and the ratio of tritium to C 14 in each fraction determined. Concurrently the concentration of various s-RNA species pres- ent in different tissues is being examined. Connective tissue is a fibrous matrix in which cells, water, mineral, vasculature, etc. are suspended. This idealized separation facili- tates measurement of certain biophysical prop- erties, viz., mechanics of deformation, diffusion- permeation and binding of water, the syntheses and depletion of macromolecular components in the matrix. Aging of connective tissue will be understood in terms of these properties, and others, as well as molecular and regulatory systems of fibroblasts and other tissue cells. Until there is a unified concept of the inte- grated connective tissue systems, this artificial separation serves a useful purpose. Present work in the Biophysics Section is concerned with the fibrous macromolecules of the connective tissue matrix, viz., collagen, elastin, and mucopolysaccharide. Since these substances perform a mechanical function in the physiological systems, the main research effort so far has been concerned with deforma- tion analysis. The mechanics of tissue deforma- tion was investigated in aging tendon, skin, and blood vessels. Experimental data on tendons suggests that longitudinal elasticity depends upon waviness of collagen fibers. A model of connective tissue elasticity was proposed in which the longitudinal extensibility is ex- pressed as a function of the number of curled fibers which are transformed into uncurled fibers. Mathematical representation of this model results in equations which are in agree- ment with experimental data. From this it ap- pears that stiffness of large fibers increases during growth-development, but there is no further increase throughout the later period of life. On the other hand, small tendons show a concomitant reduction in stiffness; at all ages there is a negative relationship between stiff- ness and size of tendon (cross-sectional area). Since the original data were corrected for cross-sectional area, this negative dependence is a second-order effect. Presumably it repre- sents an accumulation of imperfections dur- ing the build-up of large fibers. Aging, there- fore, reduces the imperfections in large ten- dons, but it increases them in small tendons. Data on aging dermis showed an initial lin- ear extensibility, but this became non-linear at a critical strain. Morphological evidence suggests that the initial linear portion is due to elastin, whereas the non-linear data result from collagen fibers which become uncurled with stretch. Mathematical description of these delineated segments yields parameters of elas- ticity for aging dermis. It was noted that in- dices changed rapidly only during the early growth-development period of life. Comparison of these measurements with data in the litera- ture also showed that composition of dermis, i.e., mucopolysaccharide, water, collagen, and elastin do not change rapidly during aging, but only during growth and development. When the elasticity data of blood vessels were treated like that of dermis, it was seen that human arterial segments changed mark- edly during aging, but only slightly during the period of growth and development. Rat tail arteries and veins, in contra-distinction to hu- man arteries, behaved more like refractile ten- dons, i.e., they did not change greatly during the later months of the life span. Thus, one sees that connective tissue has both sensitivity and selectivity toward fac- tors governing transformations during growth development, and aging. Small quantities of aged human skin can be obtained by dermatologic biopsy, but there are restrictions on the types of measurements pos- sible because of the small size of the sample. Compression tests can be done on these small samples (biopsy), and data can be used to study molecular transformations of the fibrous matrix. It was observed that compressibility (stress-strain) data are similar to extensibility (stress-strain) data for calf and rat skin. This testing procedure will be expanded and used to study the biophysical properties of connec- NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT 297 tive tissue in organs from which it might be possible to get only microscopic size tissue samples. The above summary minimizes the impor- tance of some observations which purport to show aging of connective tissue. This is true only to the extent that one recognizes the phys- ical state of collagen in these samples. All of the above studies pertain to non-melted connec- tive tissue, i.e., to collagen fibers which are in a state of high crystallinity. Biophysical analy- sis of elasticity of amorphous collagen in melted tendon or skin shows, in contradistinc- tion with the crystalline state, that extensive changes are introduced at the molecular level. It is generally concluded that melted collagen chains show an increase in the extent of inter- chain crosslinking, but recent evidence in the literature suggests a modification of this inter- pretation. It is now seen that perhaps these crosslinks are reoriented from intra- to inter- molecular tropocollagen, thus not requiring a priori the addition of many new crosslinks. This would provide a rational explanation for the limited increase in stiffness of crystalline collagen fibers in tendon and dermis. Vascular collagen, admittedly, would undergo more ex- tensive crosslinking than tendon or dermis col- lagen, a case difference which will be investi- gated more thoroughly. Endocrine systems appear to control or influ- ence some parts of the deposition-polymeriza- tion mechanism of connective tissue. The ex- tent to which this is modified by aging, or the possibility that it is the basis of changes in connective tissue with aging, now is the subject of speculation. A series of experiments now underway will delineate certain aspects of the control mechanism. Primary emphasis will be placed on the effect of age on the rate or re- sponse of connective tissue to an endocrine dis- turbance. This design is "longitudinal" in concept in that endorcine disturbances will be induced at various ages then followed by serial observation. The first series of measurements will determine the rate of response to hypo- physectomy of rats which are hypophysectom- ized at various ages. Tests will be conducted on elasticity of dermis, tendon, and blood vessels to establish the generality, or specificity, of their response. Various organs will be weighed while the femur length and whole animal weight will be used to follow growth. Ulti- mately, the project will explore the longitu- dinal response to disturbances of pituitary, thyroid, and adrenal functions. MENTAL RETARDATION PROGRAM The Mental Retardation Program has pri- mary responsibility for research in the biolog- ical, behavioral and social aspects of mental retardation within the context of child health and human development. This responsibility embraces all factors — biological, social and be- havioral — that influence subnormal develop- ment. Direct Research Three Branches and one Laboratory have been organized to carry out direct research ac- tivities in mental retardation. These include a Clinical Research Branch, a Laboratory of Biomedical Sciences, a Behavioral Sciences Branch, and an Epidemiology Branch. The last two Branches are not operational yet. Two Sections will be included in the Clinical Research Branch — the Children's Diagnostic and Study Unit, and an Inpatient Diagnostic and Study Unit. Plans are now being formu- lated for the Inpatient Unit. The Children's Diagnostic and Study Unit is operational and is located at the National Naval Medical Center. The purpose of this Outpatient Clinic is to provide a program of clinical research and source of case material for basic research in the biomedical and behavioral aspects of mental retardation. Currently two new patients are be- ing evaluated each week by a multidisciplinary team. The Clinic team is interested in studies on learning. A small nursery school is asso- ciated with the Clinic and will provide an ideal setting for studies of learning, motivation, and teaching procedures. Specific studies are being carried out on im- proving attending behavior in preschool re- tardates; behavioral assessment of neonates; in- formational discrepancies by parents; behav- 298 ANNUAL KEVIEW OF INTRAMURAL RESEARCH ioral assessment in a free-play situation; and operant conditioning audometric techniques. A Laboratory of Biomedical Sciences is us- ing borrowed space on and off the Reservation and has been collaborating with other Insti- tutes on specific projects. Architects are now de- veloping plans to renovate space for biomedi- cal and behavioral research laboratories at the National Naval Medical Center. These labora- tories will be located adjacent to the Children's Diagnostic and Study Unit to facilitate collab- oration between clinicians and laboratory in- vestigators. This facility will be ready for occupancy early in 1967. LABORATORY OF BIOMEDICAL SCIENCES The mission of the Laboratory of Biomedical Sciences is to contribute to the Program's mul- tidisciplinary investigation of mental retarda- tion. Initially the Laboratory will emphasize biochemistry, cytogenetics, and neurophysi- ology. To this end, investigators from each of these disciplines have been recruited. The staff of the Laboratory now consists of 15 individ- uals (9 professional, 4 technical, and 2 clerical). During the past fiscal year, some of the bio- chemical research has been located in Room 308, Building 4, NIAMD. This module has been occupied by Dr. Robinson since his trans- fer from NIAMD to NICHD in July, 1965, and will be vacated on July 1, 1966. The Cytoge- netics Unit, headed by Dr. Matti Al-Aish, is located in the REEL Building of the National Naval Medical Center (NMMC). Dr. Freder- ick Maire joined the Laboratory in March and has begun his research work in neurophysi- ology in the Children's Diagnostic and Study Unit. Plans for additional laboratory facilities are in the design phase. These laboratories will be located adjacent to the Children's Diagnostic and Study Unit at NMNC and will be ready for occupancy before the end of the next fiscal year. To avoid a delay in staffing after comple- tion of the laboratories, personnel have been recruited and are now engaged in collaborative investigation in other Institutes. This expe- dient also should help to increase our inter- Institute collaborative work in the future and thereby considerably augment our research program. General Review of the Activities of the Laboratory Projects in progress during the past year are enumerated below. They have been classified according to the major discipline involved, but many aspects of the work are interdisciplinary in nature. Biochemical Investigations 1. Pregnancy-associated plasma proteins. 2. Electrophoretic variants of leucocyte al- kaline phosphatase. 3. Inhibition studies on serum cholinesterase. 4. Development of a general method for de- detecting peroxide-producing enzymes on starch gels. 5. Hypercalcemia study. 6. Effects of nutrition on myelinization. 7. Effects of amino acids on myelinization. 8. Evaluation of methods of therapy in homo- cystinuria. Cytogenetic Investigations 1. Cytogenetic aspects of mental retarda- tion. 2. Studies on human chromosome abnormal- ities. 3. Alteration of metaphase chromosome mor- phology and DNA synthesis during mi- totic cycle. 4. The role of viruses in chromosome abnor- malities in spontaneous abortions. 5. Cytogenetic studies of viral-transformed hamster kidney cells. 6. Clinical cytogenetic studies. 7. Cytogenetic studies of human abortuses. 8. Chromosome morphology in the vitamin E deficient rat. Neurophysiology Investigation Serial neurological and neurophysiological testing in the diagnosis of childhood disorders: The thesis being advanced by the Laboratory, as is evident in the foregoing list of individual NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT 299 projects, is that an exhaustive effort should be made to describe as many of the mental retar- dation syndromes as possible in terms of mod- ern molecular biology. This approach leads naturally to an examination of the sequence of events through which the genetic information of the chromosomes is utilized for biosynthesis of enzymes and structural macromolecules. The effect of an abnormal enzyme is frequently manifest as an accumulation of its substrate resulting in profound derangement of the nor- mal pathways of intermediary metabolism. Two important aspects of these derangements must then be considered: (1) the effects of excess or deficiency of normal intermediary metabolites on cellular differentiation and or- ganogenesis (particularly of the brain); and (2) the pharmacologic consequences of these excesses or deficiencies on nervous and mental function. Pursuant to the objective of describ- ing mental retardation at the molecular, cel- lular, and organ levels of organization, the Laboratory is emphasizing genetics, develop- mental enzymology, intermediary metabolism, and neurophysiology. In addition to its research activities, the Cytogenetics Unit has been responsible for the training of four individuals in the latest cyto- genetic techniques during the past year. CHILDREN'S DIAGNOSTIC AND STUDY UNIT The Children's Diagnostic and Study Unit (CD&SU) is an outpatient facility where clin- ical research in biology, medicine, behavior, learning, speech and hearing, epidemiology, genetics as well as other types of investigations will be carried out on mentally retarded chil- dren. In addition, the Clinic will be used to train clinical and research associates as well as staff fellows. It will also be a source of clini- cal material for basic research. Collaborative studies will be carried out with the biochem- stry, neurophysiology and behavioral sciences laboratories of the Mental Retardation Pro- gram. A multidisciplinary team approach will be maintained in the clinical evaluation of pa- tients. The Clinic population will be composed of children, six years old or younger, who fall under one of the following categories: Those who appear or have been said to be developing slowly; Those who appear or have been diagnosed as mentally retarded; Those who appear or have been diagnosed as having learning handicaps or signifi- cant communication disorders; and Those who appear or have been said to have birth trauma, neurological impair- ments or other injuries or symptoms which may be related to learning diffi- culties. Normal controls, if indicated, will also be used in our studies. On November 29, 1965, the Clinic staff moved to its present location in Building 125 at the National Naval Medical Center. Although the Clinic was officially opened on January 19, 1966, the first patient was evaluated on Decem- ber 20, 1965. In order to determine the proportion of men- tally retarded children who are seen in the Pediatric Outpatient Clinic at the U.S. Naval Hospital, the Children's Diagnostic and Study Unit staff developed a case finding question- naire which was mailed to families whose chil- dren were seen in the Outpatient Clinic between January 1, 1965, and July 31, 1965. Children who have been labeled as mentally retarded or developing slowly constitute the main source of the CD&SU population. In addition, refer- rals are being received from the Pediatric De- partments of the different military hospitals in the area, including Fort Belvoir, Patuxent Naval Hospital, and Andrews Air Force Base. Each referral, including self-referral from par- ents, is made through the Department of Pedi- atrics of the U.S. Naval Hospital. Each child is seen by a social worker, public health nurse, clinical psychologist, speech and hearing pathologist, education specialist, pedi- atrician, child neurologist, child psychiatrist and cytogeneticist. Consultation, if necessary, is obtained from various specialists from the U.S. Naval Hospital. A minimum of two days is needed to evaluate a child. Consequently, the Clinic has been able 300 ANNUAL REVIEW OF INTRAMURAL RESEARCH to evaluate two new patients a week. Case con- have been evaluated. By the end of the current ferences and parent conferences are held on fiscal year, we expect to see 21 more patients, each case. Referrals, if indicated, are made to No individual research projects are being various community agencies since the Clinic is carried out as of April 13, 1966. However, we not designed, administratively and function- expect to initiate and pursue the attached de- ally, to provide service or therapeutic functions scriptions of research projects before the end to the patients. As of April 12, 1966, 25 patients of the current fiscal year. THE NATIONAL INSTITUTE OF DENTAL RESEARCH INTRODUCTION An essential underpinning- for effective attack on the major oral health problems of today is the interdisciplinary crossfeeding provided by the Institute's several laboratories and branches. Coupled with high visibility of dis- ease oriented mission is the maintenance of a research environment of excellence that con- tinues to attract young basic scientists of su- perior quality. During the past year the Institute's intra- mural professional staff of 84 members con- tributed over 100 papers to the scientific lit- erature. Other avenues of communication, such as abstracts and attendance at meetings, pro- vided further opportunity to contribute new knowledge and information to the dental and related sciences. Such scientific reports reflect a breadth of activity varying from develop- mental, clinical, and applied research to ob- servations and findings in fundamental sci- ences having no immediately identifiable or applicable relationship to problems of oral and dental health. During the year, for example, the isolation for the first time from gingival tissues of a collagen-destroying enzyme believed active in periodontal disease led to collaborative stud- ies which have demonstrated this cellular fac- tor in neuromuscular diseases. Similarly, the refinement of demineralization technics to con- serve enzymatic activity in tissue specimens prepared for cytochemical study has led to new knowledge of the pathogenesis of skeletal and tooth defects in ascorbic acid deficiency. Further illustrative of the Institute's broad- ening range of activities are its basic physio- logical studies on (1) elicitation of varied pat- terns of cry and related arousal response by electrodes placed stereotaxically in the brain stem of the cat, and the demonstration of ac- tion details by correlated pressure recording sound recording, laryngeal photography, and regional cineradiography; (2) neurophysiologi- cal patterns of representation of sensation in the trigeminal nucleus; (3) mechanisms of in- tegration of afferent information in the cere- bral cortex; and (4) patterns of respiratory motor response to stimulation in the pharyn- geal area. In cytological research, combined application of electron microscopy, antigenic fractiona- tion, labeled specific antibody, and pharmaco- logical testing have demonstrated recently that the endotoxic somatic lipopolysaccharide anti- gen in Veillonella is localized in a distinctive three-layered outer membrane. This can be stripped off by phenolic extraction, leaving the rest of the cell morphologically intact. An inner rigid wall, which maintains cellular in- tegrity was demonstrated by its digestion with lysozyme, a mucopolysaccharase. While the laboratory science base of the Institute's program has been enriched, there is also under way a concomitant acceleration of activity in the clinical component. For ex- ample, a new topical application procedure for caries control, utilizing mouth adaptors with a fluoride gel, has been tested for the second year on school children. As a result of promis- ing preliminary findings, the Peace Corps has adopted the procedure for use in the field. Also, community interest stimulated by this study has been largely responsible for a proposal to fluoridate the township's water supply. In order to further advance the Institute's programs, there was created this year an Im- munology Section with a view to coordination, more efficient planning and performance, and eventual extension of current immunologic projects. This move not only recognizes the natural place of immunology as a major com- ponent in the Institute's Laboratory of Micro- 301 302 ANNUAL REVIEW OF INTRAMURAL RESEARCH biology, but signalizes also the trend of basic investigations relating to periodontal disease. In similar fashion, there was constituted in the Laboratory of Biochemistry a new sec- tion on Pharmacology which seeks to bring greater emphasis and focus on the teratogenic mechanisms of drug action and other environ- mental factors in oral-facial malformations. Alert to the manpower needs of dental re- search and the opportunity to provide unique postdoctoral educational opportunities for promising young investigators, the Institute during the past year continued its support of trainees and fellows. These included four re- search associates, four clinical associates, one staff fellow, one visiting fellow, and one grad- uate student in out-of -service training. In its engagement in international activities, the Institute has adhered carefully to the im- portant purposes of advancing its categorical mission. Visiting associates are currently par- ticipating in studies with collaborators in our Laboratories of Microbiology, and Histology and Pathology. In reverse fashion, one of our own professional staff is on a one-year foreign assignment at the Royal Dental College in Malmo, Sweden, where he is pursuing contin- uing studies on microbiological factors in den- tal caries. A particularly rewarding association was again experienced during the past year between Institute staff and the Board of Scientific Counselors. Giving their attention to a wide range of issues including recruitment of key personnel, furtherance of our epidemiological activities, and advice on computer needs, the Board members were indeed a valuable part of Institute planning. In keeping with the format of previous an- nual presentations of the Scientific Director, the current report will again emphasize pro- gram areas of research rather than activities contained within the science discipline orienta- tion of laboratories and branches. DENTAL CARIES Rampant dental caries is a very severe form of disease in which practically all of the teeth are attacked by decay in a relatively short pe- riod of time. It is found chiefly in young chil- dren, but may develop in adults who previously had little or no caries experience. Under suit- able experimental conditions, comparable forms of rampant caries can be developed in laboratory animals such as rats and hamsters. From a research standpoint, rampant caries offers a most favorable opportunity to study the basic factors which activate or control the caries process because the usually prolonged time element in the development of carious le- sions is reduced to a minimum, and the deter- mination of caries activity can be much more certain than in caries of usual severity. In or- der to evaluate the many factors which may be important in rampant caries, a series of clinical and laboratory observations were made under well controlled conditions. Perhaps the most significant condition underlying the de- velopment of rampant caries in young patients was frequent nibbling of sweets and other fer- mentable foodstuffs. The animal experiments testing these foods showed that some are very cariogenic, many are moderately cariogenic, and a few are non- cariogenic to rats. During the past year, ex- periments also were conducted to determine the extent to which the very cariogenic foods would be made less cariogenic and the non- cariogenic foods made cariogenic by proce- dures such as cooking, adding water, grinding, adding sugar versus adding fluorides, and other modifications of the food. In one example it was found that certain noncariogenic foods such as "dog biscuit" were not made very car- iogenic by even large additions of sugar, and contrariwise, some highly cariogenic foods were not made non-cariogenic by the addition of fluoride. These experiments indicate that cariogen- icity is affected by several factors including chemical properties, physical properties, taste of the food, and the time it takes for it to be eaten. Previous experimental work on the etiology of dental caries and periodontal disease has clearly demonstrated the importance of bac- terial plaque deposits in both these infections. Consequently, this year's studies have concen- NATIONAL INSTITUTE OF DENTAL RESEARCH 303 trated on defining various factors involved in plaque formation and on testing the efficacy of different drugs and proprietary formulations in the control of plaque formation. The results indicate that many microorganisms, and among these several streptococci of human ori- gin, are capable of inducing plaque and active caries in hamsters. The influence of diet, es- pecially the effect of different sugars, has demonstrated that while sucrose in the diet is associated with rapid plaque formation, su- crose substitutes are much less plaque-con- ducive. The host factor has been investigated in studies aimed at determining the effect of age on tooth resistance to plaque associated lesions. The findings suggest that in the absence of fluoride, tooth age is not a factor in caries sus- ceptibility. While fluoride enhances tooth re- sistance, some fluoride-containing preparations also appear to be active in controlling plaque formation. None of the commercially available, fluoride-containing dentrifrices, however, have been effective in plaque control when tested in the hamster. The continued testing of anti- cariogenic drugs and methods in the experi- mental animal system and in in vitro studies will serve as an important step in the evalua- tion of agents suitable for human clinical trials. For a number of years considerable empha- sis has been placed by Institute scientists on the study of enamel structure, normal as well as pathological, and valuable new data accu- mulated which have advanced our understand- ing of the morphology of sound and carious human enamel, especially with regard to the prismatic structures and their participation in the spread of the carious process. While these earlier studies dealt with subsurface enamel, current efforts are being directed toward the surface layer. This layer is of particular inter- est because of its apparent greater resistance to decalcification as seen in early enamel car- ies, the so-called white spot lesions, where a seemingly intact surface layer covers an area of extensive subsurface demineralization. Al- though the differences in reactivity may be attributed to chemical differences such as high fluoride contents, the present study demon- strates clearly that morphological differences also exist. The lack of prism structure, the ori- entation of the crystals perpendicular to the surface, and the higher mineral content, cou- pled with a possible increase in crystal size, are all factors which might contribute to a decreased solubility. The influence and role of this "prismless" surface layer in the onset and spread of the carious lesion, especially relative to morphological differences from one tooth to another, are some of the important questions for which answers are being sought. A contributory factor to the success of these studies has been the new microradiographic equipment which was constructed and put into service last year. The availability of an X-ray tube with exchangeable targets to vary wave lengths now makes it possible to detect very minute differences in mineral content which could not be recorded by previous methods. Another related objective of this broad pro- gram in caries research has been to identify and classify human oral streptococci and to de- termine whether or not specific strains can be identified as etiologic agents. Utilizing sero- logic screening of plaque smears and fluores- cent antibody technique for identification of strains in histologic sections of carious teeth and in mixed cultures of dental plaque, it was found that certain serologic groups of strepto- cocci could be excluded as etiological factors. These observations are making it possible to fo- cus, and ultimately identify, the suspect sero- logic groups. Related experiments with gnotobiotic rats indicate that some adaption of the rodent mouth, sometimes involving antigenic shift, is necessary in order for these human oral strep- tococci to establish themselves and produce caries in the animal model system. Evidence continues to accumulate that the level of dental caries activity in experimental animals is determined by very intricately bal- anced relationships between host, a limited va- riety of oral microorganisms, and dietary fac- tors, notably sucrose. A considerable range of microbial species has now been tested in gnoto- biotic rats and in hamsters deficient in cario- genic flora. Except for a single atypical strain of Lactobacillus acidophilus, only certain 304 ANNUAL REVIEW OF INTRAMURAL RESEARCH strains of a particular cultural and immuno- logical type of oral streptococcus have been found capable of initiating caries in such ani- mals. Significantly, Institute scientists and col- laborators at the National Children's Cardiac Hospital, Miami, Florida, and the Royal Detanl School, Malmo, Sweden, have isolated during the past two years a number of "rat- type" and "hamster-type" streptococci from human caries lesions and saliva. Many of these have proved to be cariogenic in the respective species of ro- dent. Recently, streptococcal strains have been isolated from humans, which are cariogenic in both rats and hamsters. In view of the long history of unsuccessful attempts to transmit caries activity from humans to animals, and from one species of animal to another, these re- sults have been very surprising. Most impor- tantly, they have encouraged renewed search for a specific bacterial element in human car- ies. Sufficiently distinctive cultural character- istics of thesp types of streptococci have been ascertained 10 make possible beginning hu- man epidemiological studies. It would be espe- cially meaningful, for example, to determine whether these organisms are natural inhabi- ants of the oral cavity in population groups with low caries experience, or whether the or- ganisms must be transmitted to an individual before the disease can be established. The reasons why some strains of these strep- tococci are cariogenic, while others are not, seem to relate to their behavior with sucrose. Thus, it has been demonstrated that cariogenic strains produce far more dental plaque, in vivo and in vitro, in the presence of sucrose as com- pared to equivalent amounts of other carbo- hydrates. On the other hand, cariogenic and non-cariogenic streptococci do not differ in their rates of acid production in vitro from such sugars as glucose, fructose, maltose, su- crose or mixtures of glucose and fructose. Nev- ertheless, when any of these sugars or sorbitol, or a hydrogenated starch product are substi- tuted for sucrose in a caries-conducive diet, the incidence and severity of caries decrease markedly. These results are entirely consistent with the large amount of epidemiological evi- dence that dietary sucrose is a major determi- nant of caries in man. Recent evidence indicates that differences in the caries susceptibility of different strains of rats correlate with their ability to support a cariogenic flora. When different strains of rats are fed the same diet and given equal exposure to a source of cariogenic flora, the levels of caries activity and the animals' ability to trans- mit the flora differ approximately in parallel. On a different caries-conducive diet, however, the relative susceptibilities of two strains of rats can be reversed; that is, a normally "re- sistant" strain develops more caries than its counterpart "susceptible" strain when both are fed one of our standard caries-conducive diets. Other studies indicate that the numbers of car- iogenic streptococci in the mouths of rats and hamsters parallel the levels of caries activity. These results suggest that the support of a car- iogenic flora depends upon some host-diet rela- tionship, rather than on host and dietary fac- tors independently. In previous years the Institute has partici- pated actively in surveys conducted by the Interdepartmental Committee on Nutrition for 'National Defense (now the Nutrition Sec- tion, Office of International Research, NIH). Population groups represented an ethnic and geographic spread ranging from sites in Alas- ka, Central and South America, to Asia and Africa. During the past year data from the survey in Nigeria were analyzed and new sur- veys were initiated in Guatemala and El Sal- vador. Studies of dental caries within the United States included investigations with tube-fed children, clinical trials with a phosphate- fluoride dentifrice and a fluoride gel carried in a mouth applicator, and further analysis of findings in a long-term study carried out with children during the first ten years of use of a fluoridated community water. Currently, analysis is continuing with the objective of discovering specific population factors which are associated with relative sus- ceptibility to disease, on the general hypothesis that any causative factor should be found asso- ciated with disease in a consistent and predic- table manner, population after population. Such associations should have been demon- strable because the prevalence of oral disease NATIONAL INSTITUTE OF DENTAL RESEAKCH 305 was found to vary, area by area, by factors of 30- to 60-fold. For example, optimum, or even excessive, intakes of fluoride were invariably associated with an inhibition of dental caries, and there was a general relation between car- ies prevalence and per capita consumption of refined sugar. On the other hand, other widely- held theories were not supported. Very low caries prevalences were found in populations subsisting principally on such foods as rice, cassava, or yam, which are highly cariogenic in laboratory animals. No support could be ad- duced for the theory of a "protective" food or food element other than fluoride. Leads for further studies came from such findings as the observation in Viet Nam, Lebanon, and Nigeria of children with deciduous teeth attacked by caries alongside permanent teeth that were seemingly immune. One ominous note emerged; there was clear evidence that caries prevalence is rising, sometimes swiftly, in most populations now relatively free from this disease. Studies of dental caries in laboratory ani- mals fed by stomach tube have yielded much basic information about the relative roles of substrate and disease. Hitherto, there have been no parallel studies in human beings. Dur- ing the year an investigation was begun with tube-fed children in the Sunland Hospital, Or- lando, Fla. This study should establish or re- fute basic inferences about the baterial nature of dental caries which have not heretofore been studied directly in human beings. Other labora- tory investigations have demonstrated that den- tal caries activity can be controlled at will in hamsters through the application of fluoride gels or antibiotics, topically to the teeth, using a fitted vinyl mouthpiece as a carrier. A field trial of a fluoride gel applied in this manner is in its second year with children of Cheekto- waga, New York. Preliminary findings indi- cate that (a) much higher concentrations of fluoride appear in the outer layers of tooth enamel after such treatment than after con- ventional methods of application, and (b) that a single hygienist can supervise many more children when this technic is followed. An in- terim examination showed that caries had been inhibited in the study children, but that too little time had elapsed for determination of the magnitude of the effect. Since the mechanisms governing the deposi- tion of fluoride in calcifying structures are not understood, it is apparent that the role of fluoride in inhibition of caries and its possible inhibitoiy effect on bone loss in resorptive bone disease will be empirical until the mech- anisms by which these effects occur are eluci- dated. A number of studies, therefore, are being carried out on the effect of fluoride in resorptive bone disease in the human and in experimen- tally-induced bone loss in the rat. Most signifi- cant is that fluoride inhibits alveolar bone loss induced in the rat by either hydrocortisone or a low protein diet. However, maximum levels of fluoride tolerated by the rat do not reverse the decrease of bone protein and citrate syn- thesis as induced by hydrocortisone. In one in- dividual having multiple myeloma, the admin- istration of about 30 mgs of fluoride per day for about two years was found to reduce the mobilization of fluoride from the bones. Research pertinent to the cariostatic effect of phosphates proceeded during the year with attempts to define the mechanism of its action. Additional evidence was obtained in support of the hypothesis that this cariostatic action is localized in the oral cavity. Thus, NaH 2 Po 4 added to drinking water or diet of white rats for a period of two weeks prior to initiating a cariogenic diet reduced the ultimate cario- genic effect of the diet. Apparently the tooth surfaces and/or the oral milieu had been favorably conditioned against caries by this prior exposui*e to phosphate. To resolve the possible effect of phosphate on the chemistry of both permanent and decid- uous teeth, the Pitman-Moore miniature pig and standard Duroc swine were fed low- vs. high-phosphate diets for a period of one year, starting at weaning age. The animals were sacrificed when both permanent and deciduous teeth were erupted and still in situ. Ash, cal- cium phosphorus, magnesium, and carbon di- oxide were determined in enamel and dentin of permanent and deciduous teeth. The low phosphorus diet did not effect the content of the above major constituents of the teeth, as compared with the teeth of swine fed a normal 306 ANNUAL REVIEW OF INTRAMURAL RESEARCH and higher phosphate diet. Apparently the cariostatic effect of phosphate supplements as observed in experimental rats may not be re- lated to a change in major components of the teeth. Related to the demonstrated cariostatic effect of phosphates on dental caries in the rat and hamster is the reported increase of phosphate in the saliva of humans associated with a low caries experience. Since phosphate is involved in the glycolytic cycle, studies have been ini- tiated on the relation of various glycolytic en- zymes of saliva to dental caries. Some enzymes involved in the so-called "shunt" pathway have also been studied. Thus, hexokinase, aldolase, glucose-6-phosphate dehydrogenase, 6-phospho- gluconate dehydrogenase, phosphoglucose iso- merase, and phosphoglucomutase have been found in whole saliva, whereas in parotid saliva only hexokinase, aldolase, and phospho- glucose isomerase were present. Bacteria, cellu- lar debris, or perhaps the submaxillary saliva thus may serve as a source of enzymes not found in the pure parotid secretion. Study of the activity of these enzymes in caries-free, caries-normal, and caries-rampant individuals will be continued. It can be concluded that the problem of car- ies control assumes quite different aspects de- pending on the microbiological status of the subject population. If relatively caries-free populations, such as those found in several African nations, enjoy this status because they are not infected with cariogenic microflora, caries activity should remain low regardless of dietary composition, particularly the con- sumption of sucrose. On the other hand, if po- tentially cariogenic microorganisms are al- ready widely prevalent in such populations, then introduction of sucrose into the diet should automatically result in increased car- ies activity. In the United States and most Western nations, sucrose consumption and car- ies are so prevalent that we must assume that cariogenic microorganisms are ubiquitous. Probably the only way to eradicate dental caries would be to eliminate the causative mi- croorganisms from a population. Until this be- comes feasible, as by suitable use of antibiotics, other control measures of known efficacy such as fluoridation and dietary regulation should be rigorously applied. In the past, two major factors (fluoride and refined carbohydrates) have been shown to consistently influence the amount of dental car- ies in a population. A third consistent relation- ship has been established between the inherited ability to taste phenylthiocarbamide and den- tal caries, such that tasters for this substance have from 28 to 40 percent lower def (decayed- extracted-filled deciduous teeth) rates than nontasters. This finding was verified in a num- ber of studies. Investigation of the thiourea con- centration in saliva indicates this was not the basis for the caries difference observed. Thus, a genetic factor in dental caries is well demon- strated for the first time in clinical studies. PERIODONTAL DISEASE Within the past few years periodontal dis- ease has been recognized as a major public health problem in the United States, where it is a major cause of oral pain and tooth loss. Clinical management of the condition is diffi- cult, expensive, and time-consuming, and the number of available specialists is quite inade- quate to meet the need for treatment. On a world-wide basis, people of the United States are probably the most favored in this regard; elsewhere, the periodontal diseases tend to be more prevalent, more severe, and earlier in on- set, and professional personnel are even less able to cope with the problems. Studies, to date, have led to the clear infer- ence that prevention of this disease entity must depend upon control of deposits of dental calculus, with little benefit to be gained by nutritional therapy. Tooth loss in many of the populations studied by Dental Institute epidemi- ologists was high from periodontal disease, which leads to deterioration and loss of the structures which support the teeth in the mouth. In such areas as Lebanon, Trinidad, Viet Nam, Thailand, Burma, and Nigeria, the onset of disease was early, and advanced destruction was common even in young individuals. Noma, a disease which has virtually disappeared in western civilizations, was common in children in Nigeria. It was usually associated with pro- tein-calorie malnutrition, exposure to malaria, NATIONAL INSTITUTE OF DENTAL RESEARCH 307 recent history of rubella, and an overwhelm- ing infection with Vincent's organisms. As a general rule, high prevalences of perio- dontal disease were seen in those populations where children exhibited signs of malnutrition in calories, protein, and vitamin A and caro- tene. There was a consistent and positive re- lationship between deposits of oral debris and calculus and the severity of periodontal disease in every population studied. When these fac- tors, and age, were held constant, no consistent or strong relation could be demonstrated be- tween disease and nutritional status. Estimates of nutritional status were based on biochemi- chemical determinations, dietary studies, and the character of diets followed in the past. Fac- tors brought under study included such items as vitamin A, beta carotene, ascorbic acid, thi- amine, riboflavin, niacin, total proteins and protein fractions in serum, calcium, iodine, and iron. On the basis of the findings, the value of any of these agents in therapy may be ques- tioned. On the other hand, calculus formation in the rat was related to a high protein concentration in whole saliva and with an elevated acid and alkaline phosphatase activity. Also, rat-strain differences seemed to be of more importance than diet variables as a factor in calculus for- mation. It is pertinent to mention also the parallel demonstration of extracellular poly- saccharide (levan) production from sucrose by Odontomyces viscosus. A variety of other common sugars showed no such formation. This provides a reasonable explanation for the accumulation of this organism as a gelatinous plaque causing a form of periodontal disease in hamsters fed a high-sucrose diet. Very recently a study of autogenous reim- plantation of human teeth was initiated. It is hoped that by observing the response of the periodontal tissues to tooth reimplantation some knowledge relative to the factors permit- ting the differentiation of fibroblasts into ce- mentoblasts will be obtained. After reimplan- tation, the teeth become firmly attached with new alveolar bone formation. In the absence of continued cemental production, periodontal fibers lose their attachment to teeth with even- tual exfoliation. New functioning cementoblasts have not been found earlier than 29 days after reimplantation. In the treatment of the osseous defects in periodontosis, preliminary results indicate that the autogenous transplantation of developing third molars into the first molar sites can be an effective way of inducing healing of the al- veolar bony lesions and in restoring periodontal health. In another study, dental abnormalities were produced in Syrian hamsters by infections with H-l (isolated from human tumors by H. W. Toolan— Bull. N.Y. Acad. Med., 37:305: 1961), OLV (isolated from rats by G. Dall- dorf— Bull. N.Y. Acad. Med, 36:795:1960), Krisini (isolated from connective tissue of car- cinogen-treated rats by V. M. Zhdanov and Z. I. Merekalove — Veprosy Virusologii, 3:339: 1963), and Rat Virus (originally isolated from laboratory rats by L. Kilham and L. J. Olivier; Oral Surg., Oral Med., and Oral Path., 15:756:- 1962, 15:1302:1962, and 17:116:1964). The abnormalities produced were generally some- what similar, and consisted of root resorptions, misshapen roots, bony ankylosis of roots, and microdontia of the third molars. Differences were noted only between the OLV and H-l virus. These consisted of absence of mandibular third molars in the animals infected with OLV virus; and a severe inhibition of osteoblastic activity and bone formation of the mesial and mesiolingual aspects of both maxillary and mandibular first molars in animals infected with OLV and H-l virus, enabling Stenson's gland, which is a normal component of the sinus, to come into close apposition with the mesial root of the maxillary first molar. In a study of the effects of various viremias on healing wounds, including periodontium, Syrian hamsters were injected intravenously with OLV infected tissue culture fluid which was obtained from rat embryo. Inoculation was at time intervals of to 6 days following frac- ture of the left forearm bones (Group I) or extraction of the maxillary right second molar tooth (Group II). The intravenous injections of OLV strain of rat virus delayed fracture and alveolar socket healing when administered prior to the third postoperative day. The de- 308 ANNUAL REVIEW OF INTRAMURAL RESEARCH lay in healing was transitory; i.e., by 15 days postoperatively no alteration in healing could be discerned. Extensive investigations by Institute scien- ■ tists and others have established that the mem- bers of the oral microbiota most significantly implicated in the pathogenesis of chronic perio- dontitis possess sufficient pathogenic factors to account for the principal features of the perio- dontal lesion. These factors include lipopoly- saccharide endotoxins and histolytic enzymes (mucopolysaccharases, proteases, peptidases, collagenase, and other hydrolases). The obser- vation can now be added that ammonia and hydrogen sulfide, in concentrations that might reasonably be expected from bacterial activity in the gingival crevice, exert cytopathic effects on epithelium similar to that lining the gingi- val crevice. The toxicity of these agents was demonstrated in rabbits with istonic solution in neutral pH ranges similar to the ranges exhibited by human mouth fluids. Other directions of research are giving in- creased attention to the immunological relation- ships between the host and the microbial prod- ucts occurring in the gingival crevice and periodontal pocket, and to associated basic im- munologic studies. Thus, it has been shown that repeated deposition of antigen in the normal gingival pocket of the rabbit results in appear- ance of homologous antibody in the blood se- rum and in chronic allergic inflammation of the gingiva, histologically very similar to that seen in chronic human gingivitis. Paradoxi- cally, and despite many investigations, little evidence has been presented that humans form antibodies to their indigenous gingival crevice bacteria. This failure seems to be due, at least partially, to the use of insufficiently sensitive serological methods. Using indirect hemagglu- tination and bactericidal tests for antibody, significant levels have been found in human sera against Leptotrichia buccalis, Veillonella alcalescens, and Fusobacterium polymorphum. Subjects with advanced chronic periodontitis exhibit significantly higher titers to F. poly- morphum that subjects with a clinically unin- flamed periodontium. Parallel studies have shown that the concentrations of antibodies in parotid secretion are only about 1:1300 those of serum. Presumably the major part of anti- bodies in the oral cavity comes from the blood serium, via gingival crevice exudate. The histochemical and chemical studies of enzymes involved in connective tissue forma- tion and destruction alos have been continued during the past year. Special emphasis has been placed on determination of collagenase activity in a number of connective tissue sites under normal and pathological conditions fol- lowing the discovery last year of the enzyme system in human gingival tissues. Of the nor- mal tissues tested so far, only uterus, gingivae and, to a lesser extent, portions of the oral mucosa exhibit collagenolytic activity. Some of the most exciting results, however, have been the demonstration of the enzyme in cultures of dermis from individuals with certain neuro- muscular disorders and collagen diseases. Iso- lation and purification of the collagenase is being attempted and when accomplished will allow investigation of its action on the collagen structure. The cellular origin of the collagenase also is being studied. Results already obtained represent an important extension of our basic knowledge and, when fully exploited, will un- doubtedly contribute significantly to a better understanding of the metabolic processes in healthy and diseased connective tissues. GROWTH AND DEVELOPMENT A population of 4,200 Indians in North Caro- lina has been exhaustively examined for clefts of lip and palate, and 42 cases have been found which makes this frequency the highest known in man. Since a number of other congenital anomalies appear to be associated with this type of defect in the same population, some clue may be uncovered to help define the pathogene- sis of the oral defect. Studies of cleft lip and palate children and their families in selected population groups in the United States indicate that, among the rel- atives of children with oral clefts, 27 percent have a hearing loss of 20 db or more at 500, 1000, 2000, or 4000 cps, compared to only 13 percent among control proband relatives. Other parameters such as visual acuity and minor anomalies (possible indication of a cleft palate habitus) have not shown a significant differ- NATIONAL INSTITUTE OF DENTAL RESEARCH 309 ence between the two groups. This would indi- cate that there are familial factors (possibly genetic) operating to cause a local, first arch syndrome disturbance rather than a general disturbance in development. One of the major efforts of the Institute dur- ing the past year has been the establishment of a data retrieval system for research on con- genital malformations among all American In- dian neonates born in PHS facilities. To date, approximately 10,000 birth records have been examined of Indians bora during the last year and a half. These data have yielded some unex- pected results. Foe example: (1) only 5.0 per- cent of all Indian births are premature com- pared to 7.0 percent for whites and 9.7 percent for non- whites; (2) the incidence of Indian stillbirths was 4 in 1,000 births compared to 13.7 in 1,000 U.S. white births and 26.7 in 1,000 U.S. non- white births; (3) Indian neo- natal death rates are 13 per 1,000 births com- pared to 16.7 per 1,000 births for whites and 26.1 per 1,000 births for non-whites; (4) ma- jor congenital malformations were 15 per 1,000 births, and minor malformations were 34 per 1,000 births; (5) of the major malformations, 2.2 per 1,000 represented clefts of the lip or palate; (6) approximately 32 per 1,000 births or 3.2 percent Indian children either die in the neonatal period or have major malformations. When corrected for underreporting, this is about the same value obtained in Japanese in- fants (4.3 percent) ; and (7) the malformation pattern in American Indians is intermediate between Caucasian and Mongoloid patterns. Investigations of biochemical genetic defects in families and populations indicated that American Indian infants do not metabolize bilirubin in the same manner as Caucasian neo- nates; i.e., they have higher and more pro- longed neonatal levels of unconjugated biliru- bin, which apparently is normal for children in this ethnic group. Thus, indications for ex- change transfusion are different in Indian neo- nates than they are in Caucasian neonates. In interesting contrast to findings in Ameri- can Indians is the striking absence of maloc- clusion among primitive groups of Brazilian Indians. This study has shown a 5 percent in- cidence of such malformations compared with approximately 50 percent for a corresponding group derived from the same tribal stock who have been in contact with civilization for a period 20-30 years. Orthodontic care claims a considerable share of the funds spent to improve dental health, but the natural history of occlusion has re- ceived little emphasis in epidemiological study. Thus, a program of study is currently being designed to determine the sequence of events, and the causative factors, which lead to unde- sirable states of occlusion, in the hope that some of these factors may be amenable to sim- ple methods of control in early states of de- velopment. Principal focus is on a longitudinal study in which individual children are exam- ined and reexamined over a period which in- cludes critical periods of tooth emergence and jaw development. The Institute's Oral Pharyngeal Development Section has continued its studies during the past year of the structure and the motor per- formance of the mouth and pharynx. Basic anatomical studies have included (1) vital staining demonstration of differential skeletal growth patterns in face and cranium of rats, rabbits, pigs, and sheep; (2) detailed descrip- tion of intrinsic growth patterns of individual bones as observed when they are cultured sep- arately in vitro; and (3) sequence of histologi- cal demonstration of responses of bone to de- forming forces. In the human, changes have been described in spatial relation to nasal sep- tum and base of cranium during postnatal development. Analogous studies of facial and cranial skel- eton have continued in subjects distorted by anomalies and/or neurological impairments. These have included standard methods of ra- diological cephalometry, and adaptation of laminography which demonstrate the mid-line structures. In clinical application, evaluations of the basic orthodontic methods of tooth dis- placement have been performed, using preci- sion techniques of portrayal of spatial changes of teeth. Studies of upper respiratory and feeding functions have continued in an increasing num- ber and variety of subjects impaired by ano- maly, such as the Pierre Robin syndrome. The 310 ANNUAL REVIEW OF INTRAMURAL RESEARCH methods of cineradiography, cinephotography, respiratory displacement, sound recording, and spectrographs display have been utilized in the study of selected patients having cleft pal- ate and other oral and pharyngeal anomalies. Transducer methods of observation of tongue contacts and margin pressures have been de- veloped, and multiple-site inputs recorded in parallel with speech and swallow indications, by these methods, Institute investigators have been able to redefine disabilities in terms of their deficiences and compensations of phar- yngeal actions, rather than the overt deficiency of palate and related structures. Such demon- stration methods also have been extended to subjects with neurological impairments of the oral and pharyngeal area, and initial descrip- tions have been made of the particular distor- tions of feeding, pharyngeal airway mainte- nance, and vocalization in subjects having spastic dyskinesia, athetosis, lower motor neu- ron disorders, and regional kinesthetic sensory disorders. Clinical experimental methods have been devised to evaluate oral sensation and percep- tion, and the development of oral perception of form (oral stereognosis) has been calibrated in normal children. These approaches have made possible the description, to date, of syn- dromes of disability of perception in dysar- thric and orally dysphagic subjects. Analogously, studies of the chemosensations, smell and taste, have been adapted to children and to neurologically impaired persons; and first demonstrations have been made of disa- bilities of these special senses in facially de- formed children. In another study concerned with the inter- action of sensory stimuli in association areas of the cerebral cortex in cats, it has been found that when peripheral stimuli are em- ployed (light flash, auditory click, and forepaw shock), most cells in the anterior lateral gyrus exhibit preferential responsiveness to auditory click stimulation. This preferential responsive- ness, however, was noted to be dependent upon the types of stimuli employed; i.e., when cells are activated by electrical stimulation of optic and auditory nerves and the dorsal column of the spinal cord, equal responses to the stimuli are most prevalent. A comparison of response characteristics in anterior lateral and anterior middle suprasylvian gyri have revealed that re- sponses to sensory stimuli are not identical in these two association areas, irrespective of the types of stimuli. In a study of the functional organization of the trigeminal brainstem in the cat, it was found that the chief sensory nucleus of the trigeminal nerve contains cells with different response characteristics. Almost all cells stud- ied to date have had small receptive fields in the oro-facial area and exhibit a short latency (1-5 msec) response to electrical stimulation of this field. The modifying influences upon these cells by peripheral and central inputs can be separated into three groups: (1) cells whose activity is not modified by cortical, thala- mic, or light flash stimulation; (2) cells whose activity is modified by cortical stimulation and orthodromic thalamic stimulation; and (3) cells whose activity is modified by both periph- eral and central inputs. Very few cells were found to be activated antidromically by stimu- lation of the contralateral arcuate nucleus. Histological analysis of electrode track place- ments indicates a more medial location of cells whose activity is modified by peripheral or cen- tral input, or both. In determining the genetic mode of inheri- tance of the developmental anomaly, taurodon- tism, radiographic surveys and clinical histo- ries of four affected families were made. Al- though no parent possessing the taurodont tooth form was identified, the trait occurred in ratios of (1) one of three children, (2) one of two children, and (3) two of two children. Of further interest was that taurodontism is found in both deciduous and permanent teeth of males and females. Also, its mode of inheri- tance is apparently compatible with a recessive characteristic, and not a dominant one as sug- gested in the German literature. With respect to its histologic appearance, there is normal odontoblastic and periodontal membrane ac- tivity, thus lending further support to the orig- inal theory that the developmental site of the taurodont tooth form is Hertwig's epithelial sheath. NATIONAL INSTITUTE OF DENTAL RESEARCH 311 Among the major objectives of the Institute's experimental program in teratology have been (1) to study the occurrence of metabolites of chlorcyclizine in maternal and fetal tissue dur- ing organogenesis, (2) to specify the effects of certain drugs on implantation and fetal devel- opment, and (3) to compare the histology of benzhydrylpiperazine vs. Vitamin A-induced cleft palate. Studying principally the rat and mouse species, it was found that, qualitatively, chlorcyclizine and norchlorcyclizine are the only detectable metabolites with at least one tertiary amine, found to be present in maternal and fetal tissue. The ratio of norchlorcyclizine to chlorcyclizine in fetuses is about 15:1. When the demethylation inhibitor SKF 525 A is administered together with chlorcyclizine, the fetal norchlorcyclizine to chlorcyclizine ra- tio is approximately 2:1. However, under these conditions of inhibited demethylation, terato- genesis was still present at approximately the same incidence. In attempts to identify the specific and mini- mal chemical structure responsible for a tera- togenic drug effect, it was found that buclizine hydrochloride induces the same malformations as meclizine and chlorcyclizine. Three antihis- tamines; i.e., perphenazine, prochlorperazine, and trifluoroperazine, which do not have a pi- perazine moiety in their molecular structure, have demonstrated only a low (5.4%) terato- genic effect. Histological observations of palatal clefts produced by agents of the benzhydrylpiperaz- ine series of drugs have shown that the adhe- sion in the palatal area consists of a fusion of the overlying epithelium of the tongue with the palatal processes. Gross examination of rat fetuses whose mothers had been given exces- sive doses of Vitamin A disclosed microstomia, mandibular-maxillary ankylosis, and cleft pal- ate. Histologically, the palatal processes of these fetuses were hypoplastic, and there was must heterotopic cartilage located throughout the maxillary areas. Recently, studies of congenital malforma- mations in experimental animal model systems have been extended to species other than ro- dents. These have included the swine, in which oral-facial defects were produced with high dosages of chlorcyclizine hydrochloride, and the Rhesus monkey, in which metabolic path- ways of teratogenic drug actions are currently being observed. In another area of research, the effect of tetracycline hydrochloride on enamel develop- ment has been explored further, and a repro- ducible dose-response relationships has been established. Using these results as reference, the studies were extended to include the effect of comparable dosages of oxytetracycline. Al- though the latter antibiotic was capable of in- troducing mineralization disturbances at simi- lar dose levels, gross hypoplastic lesions were not seen, suggesting that oxytetracycline is somewhat less toxic than tetracycline hydro- chloride. The similarity of the tetracycline in- duced defects to those resulting from injec- tions of a number of other chemicals indicates a common, non-specific response. Localization and identification of the cytological sites af- fected by the various toxic agents promise to yield considerable data on enamel formation in general. COLLAGEN, ELASTIN, AND CALCIFICATION Collagen is the major structural protein of vertebrates and many invertebrates. The pro- tein, in the form of a rod-like monomer, under- goes aggregation into fibers which gain addi- tional stability by a maturation process involv- ing the introduction of covalent crosslinks. These are chemical links and are found both between polypeptide chains in the same mole- cule and between chains in adjacent molecules. Past studies have elucidated the chain struc- ture. Current studies are related to a more detailed examination of the chemistry and biosynthesis of crosslinks. This is being accom- plished by utilizing chemical and enzymatic cleavage to give peptides suitable for chemical studies that cannot be done on the whole mole- cule owing to its large size. An enzyme from the tadpole cleaves the mole- cule into two pieces of unequal size. Isolation and characterization of these pieces has shown that intramolecular crosslinks occur only in one, indicating that crosslinks are specific in location. Limited cleavage of native collagen with chymotrypsin, and characterization of 312 ANNUAL REVIEW OF INTRAMURAL RESEARCH the products, has demonstrated that this en- zyme splits a few peptide-bonds at the N-ter- minal end of the molecule. The crosslink is also lost, demonstrating that it must be in this re- gion of the molecule. Chemical cleavage with cyanogen bromide has permitted the isolation of peptides from the crosslink region. Studies of amino acid sequences in these peptides have shown that the crosslink is derived from a lysyl-residue, five amino acids from the N-ter- minal end. Preliminary to crosslinking, the lysyl side chain is converted to an aldehyde. Two of these aldehydes in adjacent chains form a crosslink probably by an aldol-type re- action. In protein chemistry studies, it has become apparent that although elastin is not as well characterized as collagen, it is known to be crosslinked, and lysine is its precursor. How- ever, rather than two lysyl residues forming a crosslink as in collagen, four are involved to give highly stable ring structures which are necessary for the elastic properties displayed by this protein. Chemical and histologic stud- ies have shown that aldehydes are intermedi- ates, again in parallel to the crosslinking in collagen. In the toxic condition known as lathyrism, crosslinking of both elastin and collagen is in- hibited. Recent findings suggest that this in- hibition is in the enzymatic conversion of the lysyl side chain to an aldehyde. A similar inhi- bition is produced in copper deficiency, sug- gesting that a copper-containing enzyme may be involved. Related studies of calcification of organic matrices have been under way using an in vitro system with elastin as the nucleating tissue. Results, to date, indicate that trace amounts of a heavy metal such as ferric ion are re- quired for calcification. The metal is apparently associated with sulfhydryl and perhaps imida- zole groups in the protein phase. The first stage of calcification may involve the deposition of octacalcium phosphate at nucleation sites containing the complexed metal, with a later conversion to hydroxyapatite. A series of calcium phosphates have been made synthetically and have been examined by X-ray diffraction and by infrared spectropho- tometry. Among the problems investigated have been definition of conditions under which large hydroxyapatite crystals may be grown routinely and a single crystal X-ray structure determination made of a selected, well-defined crystal. The availability of such crystalline ma- terial of known structural composition is of major importance to all investigators inter- ested in studying the chemical reactivity of hydroxyapatite in both synthetic and biologi- cal systems. Other studies have dealt with the characterization of the infrared absorption spectra of pure synthetic apatites and the iden- tification of band changes associated with the presence of fluoride and carbonate ions in the crystal lattice. The knowledge of the exact lo- cation of these ions within the lattice is impor- tant for our understanding of their effect on caries susceptibility. The relationship between fluoride content and crystallinity of biological mineral also has been under investigation using X-ray diffrac- tion techniques. One aspect of this project has been the de- velopment of new methods for assessing the degree of crystallinity directly from measure- ments taken from the instrument instead of using the more cumbersome template approach developed previously in this laboratory. The data compiled through the direct methods can be fed into a computer, thus facilitating more extensive studies of age effects on bone crystal- linity in high and low fluoride areas. A second facet of the work has involved assessing the effect of fluoride on crystallinity of enamel. As with bone it was found that the crystallin- ity of human enamel improved with increased fluoride. Yet in the enamel the changes in crys- tallinity were not restricted to the A-axes di- rections, but also included the C-axes. Since both size and strain factors influence the de- gree of cystallinity, the full meaning of the ob- served changes awaits a separation of the two factors. CELL BIOLOGY AND ENZYME CHEMISTRY Resolution of the mechanism of action and specificity of proteolytic enzymes can serve to advance our understanding of biochemical processes. Such knowledge also may be utilized NATIONAL INSTITUTE OF DENTAL RESEARCH 313 for protein and enzyme modification, for study of protein molecular structure, and to advance our knowledge of a particular function of a body tissue. At the present time, the immedi- ate objectives of this research relate to the specificity and mechanisms of the enzymatic action of chymotrypsin-C, as well as guinea pig liver transglutaminase. Whereas the amino acid sequence of an ap- proximately twenty-member tryptic peptide of the chymotrypsin-C active site has certain sim- ilarities to the analogous peptide from chymo- tiypsin-A, an outstanding difference is the fact that the peptide from the C-enzyme has only one methionine residue. The active peptide site of the A-enzyme contains two methionine resi- dues. Likewise, an enzyme inhibitor specific for chymotrypsin-A has little or no inhibiting activity toward the C-enzyme. Purified guinea pig transglutaminase was found to be essentially homogeneous. It con- tains 21 thiol-groups and no disulfide bridges. Only one thiol-group is involved in the active center. From tryptic and chymotryptic digests of C 14 -labeled transglutaminase, a labeled pep- tide was isolated and found to contain the fol- lowing amino acids: 1 tyrosine, 1 asparagine, 1 C 14 -labeled amide of carboxymethyl cysteine, 1 glycine, and 3 tryptophans. The tryptophan is present in the C-terminal position. Studies of enzyme structure and mechanism have related principally to the physical-chemi- cal properties of aldolase, and a correlation of its structure with catalytic function. Results, to date, have yielded significant information on the number and type of active sites pos- sessed by this enzyme. Thus, when native fructose 1,6-diphosphate (FDP) is reduced to native FDP-aldolase, it becomes evident that three highly organized active sites are involved, each of which contains two nonidentical phos- phate-binding sites. Protein synthesis involves the polymeriza- tion of individual amino acids into high molec- ular weight polypeptides. Soluble ribonucleic acid (sRNA) plays an essential role in this process as a specific carrier of amino acids to the site of polymerization. The Institute's pro- gram in this area is designed, therefore, to elucidate the role of methylated and thiolated constituents of sRNA in its biological activity and specificity. Results have indicated that while yeast amino-acid-activating enzymes can esterify amino acids to sRNA which contains methylated bases, these enzymes are unable to carry out this reaction with methyl-deficient sRNA. Continuing studies are attempting to resolve the role and significance of the methyl- ated base of sRNA in its acceptor function. Current results indicate that methylated bases of sRNA play a significant role (1) in the ini- tial attachment of a specific amino acid to sRNA, (2) in a reaction catalyzed by specific amino-acid-enzymes, and (3) in the capacity of the sRNA molecule carrying an amino acid, to find its proper alignment on the polymeriza- tion template. An investigation relative to thionucleotides (sulfur-containing) in sRNA have pertained to their functional significance. Thus, it has been shown that formation of minor bases in sRNA is controlled by the sulfur amino acids cysteine and methionine. This relationship forms a cyclic metabolic regulatory system. Evidence was obtained that under ordinary circumstances the leucine activating enzyme from yeast recognized E. coli sRNA about 75 percent of the extent recognized by the enzyme from ti. coli. Research concerned with the translation, transcription, and replication of genetic infor- mation at the cellular level also received appre- ciable attention during the past year. Particu- larly significant have been the studies of salivary enzyme polymorphism and the demon- stration of such polymorphism in the salivary amylase isoenzymes of humans. That the com- position of these isoenzymes is under general genetic control has been demonstrated by showing a generic difference in these isoamyl- ases. Studies relating to the transcription of ge- netic information and its regulation within the cell also have been advanced through the use of a lymphocyte-phytohemagglutinin system. In this system, non-dividing lymphocytes are stimulated to enlarge and divide in vitro by treatment with phytohemagglutinin extracted from kidney beans. 314 ANNUAL REVIEW OF INTRAMURAL RESEARCH Collaborative studies with the National Can- cer Institute and the National Institute of Child Health and Human Development of RNA me- tabolism of human lymphocytes stimulated with phytohemagglutinin (PHA) indicate that the effect of this extract is to produce a synthe- sis of non-ribosomal RNA, whereas stimulation by specific antigens (streptolysin-0 or tubercu- lin) results in cell growth with predominant synthesis of ribosomal RNA. The characteris- tics of non-ribosomal RNA are like those of messenger RNA. If this is messenger RNA, it implies that PHA has an important action on the cellular mechanisms which regulate the synthesis of messenger RNA. This system, therefore, may provide a tool for understand- ing the regulatory mechanisms which govern transcription of genetic information within the mammalian cell. Malignant lymphoma cells resemble antigen- stimulated lymphocytes more closely as regards RNA metabolism than they do PHA stimulated lymphocytes. This may reflect the fact that the lymphoma cells still have greater control over their messenger RNA synthesis than the PHA stimulated lymphocytes have. This infor- mation is important for an understanding not only of the control of cell growth but also of cell differentiation. VIROLOGY, IMMUNOLOGY, AND MICROBIAL PHYSIOLOGY Continuing study of persistent herpes sim- plex (HSV) infection in a cell culture system now warrants provisional conclusions as to its mechanism, and inferences as to recur- rent herpes infection in humans. The fact that persistent cyclic HSV infections can be initiated and maintained in the presence or absence of homologous antibody indicates that the underlying mechanism is not de- pendent on antibody directly or its ability to reduce extracellular HSV. Even when anti- body is present, virus transmission still oc- curs by cell-to-cell transfer and by the reat- tachment of infectious cellular material. The only effect of antibody that has been iden- tified is its ability to reduce the severity of the infections to a low grade, comparable to a subclinical stage in the human disease. The maintenance of these persistent infections appears to depend on the establishment of a dynamic cell-virus equilibrium which is char- acterized by the continual selection of cells resistant to the major fraction of virus being produced at any one time. Selection of such resistant cells should ultimately result in a cell population completely resistant to HSV infection. This does not occur, however, due to virus variations which parallel the cellu- lar changes. The result appears to be that the selection of resistant cells is offset by the production of either a new virus variant, to which the cells are still susceptible, or to changes in the virus population resulting in selection by the cells of a virus variant which was present as a minor fraction. Super- imposed on this continual selection mecha- nism is the probable production of factors that temporarily enhance the cells' resis- tance to HSV infection. Such factors, which thus far have been found in only low titers, are probably synthesized and maintained in localized areas of the cell cultures, thus al- lowing the few surviving cells to proliferate and replenish the cell sheet. It has been found, in fact, that replenishment of the cell sheet in localized areas must occur before a new cycle of virus multiplication and cyto- pathic effect is initiated. In the presence of antibody, the severity of the infections in cultures producing a non- proliferative virus variant is reduced to a low level. After varying lengths of time, a short- term exacerbation paralleled by the appearance of a syncytium-forming variant is evident, followed by the reestablishment of a low grade infection. The occurrence of these exacerbations in vitro is strikingly similar to the recurrent episodes seen in human herpes labialis and appears to be related to viral changes which temporarily alter the cell-virus equilibrium in favor of the virus. Ultimately, the cells are able to readjust to the new virus type being produced, and a subclinical type infection is reestablished. Previously, it had been reported that HSV could be cultured from the saliva of rabbits many months after intraperitoneal injection. It has now been found that the HSV-antibody NATIONAL INSTITUTE OF DENTAL RESEARCH 315 response in such animals is strong and lasting, with cyclic variations very similar to the re- sponse to HSV infection in man. In contrast, the antibody titer induced by inactivated HSV is low and transitory. These results indicate that HSV persists in the tissues of animals re- ceiving live virus, with intermittent immuniz- ing bursts. However, no HSV could be cultured from testes, kidney, liver, spleen, lung, lacri- mal glands, submaxillary glands, lymph nodes, and brain of such animals. Rather surprisingly, organ cultures of all these tissues from the immune animals, except lymph nodes and brain, were susceptible to infection with HSV. Pro- visionally, it has been concluded that HSV persists in some inapparent form in these ani- mals or, quite possibly, in tissues not yet tested for this virus. Since lactic dehydrogenase virus (LDV) in- duces no discernible cytopathic effect, its cel- lular location in infected mice has been a mystery. In collaborative studies with the Na- tional Cancer Institute, this has now been par- tially solved by the electronmicroscopic obser- vation of particles having the fine structure of LDV, in peritoneal macrophages. Presumably, this virus is either phagocytized by and/or replicated in the macrophages. Rat submaxillary gland (RSMG) virus, de- scribed in previous reports, has been shown to be associated with a specific cold hemaggluti- nin, whose titer in triturates of the glands is a simply determinable and sensitive indicator of the presence of the virus in its natural host. This indicator has shown that RMSG virus is absent or scant in both germfree and con- ventional rats until two months of age. After three months of age, it becomes ubiquitous, with high hemagglutinin titers. Concurrently, a specific immunoglobulin hemagglutination in- hibitor appears in the animals' sera. Its titer also was shown to be low in the younger ani- mals and to increase with age. In general, the inhibitor titers of serum parallel the titer of he- magglutinin in the glands. These tests will greatly facilitate study of the ecology of this virus. Basic studies aimed at relating bacterial and macromolecular structure to an experimental situation also were emphasized during the past year. The initial electron microscopic investi- gations on endotoxin formation and the char- acter of the endotoxin of one kind of organism have been extended to other endotoxin produc- ing gram-negative bacteria. While all the or- ganisms appear structurally similar, the iso- lated endotoxic particles vary in morphology. Work has also been directed at determining the bacterial killing due to complement de- pendent antigen-antibody reactions. The initial findings demonstrate a similarity in morphol- ogy between complement dependent bacterioly- sis and haemolysis. Studies on human amyloid have been con- tinued with special emphasis on purification of the pathological protein in order to charac- terize it chemically. The degree of purification is assessed electronmicroscopically as in the ef- fect of various chemical treatments. These in- vestigations constitute additional steps toward widening our knowledge of the morphology of this protein. Continuing studies of the relationships be- tween lactic dehydrogenase virus (LDV) and its host animal (mouse) have paid unexpected dividends in the immunological field. LDV is unusual in several respects: (1) infected ani- mals exhibit lifelong viremia but remain ap- parently healthy; (2) the only known conse- quence of LDV infection has been persistent elevation of a number of plasma enzyme activi- ties, due at least partially to impaired clear- ance mechanisms; and (3) until now no anti- body response to LDV has been discernible. That LDV does activate an immunological re- sponse to its host is indicated by the recent finding that infection of germfree mice results in an elevated level of plasma y-globulin (not due to impaired clearance) and a great in- crease of germinal centers in spleen and lymph nodes. Both these parameters normally are very low in germfree animals, and their in- crease is usually interpreted as an immunologi- cal response. How much of this y-globulin production represents antibody cannot be as- certained at this time. Extension of these experiments, however, has shown that LDV acts as an immunological adjuvant; that is, LDV-infected conventional mice have a greatly enhanced capacity to produce antibody to a 316 ANNUAL REVIEW OF INTRAMURAL RESEARCH foreign protein. Further investigation of these phenomena, including other viruses and hosts, should provide a new approach to some aspects of immunological response. Consistent with the foregoing results, it was possible to demonstrate neutralizing antibody in the sera of mice infected for three months or longer, after selectively inactivating the LDV in the sera. Such treated sera readily neutralize LDV obtained from early infections (early LDV), before antibody has been pro- duced; however, LDV obtained from long- standing infections (late LDV) is rather re- sistant. This result suggests that late LDV exists as an infectious virus-antibody complex. Consistent with this interpretation, late LDV could be "neutralized" by antimouse sera pre- pared in goats. This surprising discovery led to the concept of "sensitization" of virus by antiviral antibody, without neutralization, and opens the way to a fresh interpretation and in- vestigation of viral neutralization by anti- bodies, whose mechanism has never been satis- factorily elucidated. The chemistry of the labile, enzyme-sensi- tive region linking the three stable domains of rabbit yG immunoglobulin has been studied further by analyzing the amino-acid composi- tion of fragments prepared with pepsin, insol- uble papain, mild reduction, and alkylation. In this way it has been possible to narrow the locale of the important disulfide bond linking the heavy chains of rabbit yG to a region be- tween the respective peptide bonds hydrolyzed by pepsin and papain. These studies have been materially facilitated by improvements in the program for automatic digital data acquisition and computer calculation developed for the amino-acid analyzer. Further study during the past year also was made of the paradoxical "lactate dehydrogen- ase" of Butyribacterium rettgeri, which can- not convert lactate to pyruvate, as such en- zymes usually do. In effect, it functions as an irreversible pyruvate reductase. Using a 100- fold purified preparation of this enzyme, the reaction with pryuvate rate was a sigmoidal function of pyruvate concentration, not a hy- perbolic one. This indicates that the enzyme is allosteric, requiring reactions at two sites on the molecule, and therefore might function in a regulatory capacity. An associated observa- tion was that reduction of pyruvate is strongly inhibited by adenosine triphosphate (ATP) but not by adenosine monophosphate. Thus, under conditions where the cellular energy supply (ATP) derived from glycolysis is high, and the potential for biosynthesis is conse- quently great, the conversion of pyruvate to lactate might be inhibited. Pyruvate would then remain available for a variety of biosyn- thetic reaction sequences, such as amino acid and fatty acid synthesis. Such a regulatory mechanism would provide the cell with a novel means of modulating the carbon supply for biosynthesis through a linkage with the avail- able energy supply. It is known that the biochemical and nutri- tional process taking place in the oral micro- biota are dynamic and complex. The micro- biota must depend not only upon the exogenous supply of metabolites but also upon the nutri- tional climate contributed by the symbiotic nature of the oral microbiota. The latter im- plies that certain microorganisms are capable of synthesizing varieties of metabolites which may be favorable to other microorganisms. Hence, the knowledge of how microorganisms can synthesize these metabolites may contrib- ute to a better understanding of the microbiota which influence the many problems of oral health. Continued investigation of the biosyn- thesis of the vitamin folic acid, by enzymes extracted from lactobacilli, have shown that the pathway from guanosine monophosphate involves elimination of the number 8 carbon atom as formate, and subsequent closure to form the pteridine ring. A novel nonenzymatic reaction was discovered whereby guanosine monophosphate, ferrous ion, and mercaptoe- thanol yield an intermediate compound that releases formate when treated with lactobacil- lus extracts. Knowledge of the nature of these reactions should help elucidate the biosynthe- sis of folic acid. Evidence continues to accumulate that the rate of differentiation of the cellular slime mold, Dictyostelium discoideum, from ameboid to spore stage depends on intracellular accu- mulation of the monomeric constituents of NATIONAL INSTITUTE OF DENTAL RESEARCH 317 ribonucleic acid. Thus, the S^nucleotides of adenine, guanine, cytosine, and uracil stimu- late differentiation with equal efficacy, when supplied exogenously. The whole molecule is required. Respective purines, pyrimidines, and nucleosides are inhibitory. Pointing in the same direction is the fact that a number of sub- stances other than nucleotides can stimulate differentiation, while causing intracellular ac- cumulation of ultraviolet-absorbing materials. The latter have now been identified as a mix- ture of the mononucleotides and nucleosides of ribonucleic acid. This analysis was made pos- sible by development of an improved column chromatography procedure combining molecu- lar sieve and on exchange chromatography. ORAL SOFT TISSUE LESIONS AND CLINICAL DIAGNOSIS AND TREATMENT As the Oral Medicine and Surgery Branch of the Institute has grown, efforts have been made to give more adequate attention to the major problem areas of dentistry. Studies of the human dental pulp have em- phasized a continuing evaluation of response to changes induced by dental drilling proce- dures and by various restorative and related materials, such as cavity liners. These investi- gations have furnished the dental profession with some very practical information on oper- ative procedures, particularly in regard to opti- mal cutting speeds, the proper use of coolants, and modifications in technic necessary for the safe placement of experimental restorative ma- terials. Because of the reduced inflammatory re- sponse of the pulp following high speed cutting technics, the incidence of reparative dentin production has been greatly reduced. Thus, den- tinal tubules remain open and permit the toxic or irritating products of sterilizing agents, ce- ments and silicates to permeate to the pulp tis- sue and cause further damage. This lack of reparative dentin formation creates a formid- able problem in restorative dentistry, especially in the field of full mouth rehabilitation where often the entire coronal dentin is exposed. Ex- perimental drugs designed to produce sensitiv- ity of teeth (i.e., corticosteroid compounds) and to more effectively seal the dentinal tubules are being sought, as well as drugs and technics to increase the incidence of reparative dentin formation. When prepared cavities are washed with a steroid formula containing 1% prednisolone in a vehicle of parachlorophenol, cresatin and gum camphor, before restoration with zinc oxide and eugenol, it is found that the pupal response to the cavity preparation is mini- mized about 50%. When prednisolone is used without the vehicle, the inflammatory response is sustained only 12 days. Also, when the same formula is applied to the cavity preparation several days after the full potential of the re- sponse has occurred, the resolution period is still shortened. In a study comparing the healing of surgi- cally exposed dental pulps in germf ree and con- ventional rats, the latter group showed an im- mediate, severe inflammatory response which quickly led to total pulpal necorsis, whereas the germfree animals, without exception, showed minimal inflammatory response (in spite of food impaction) with subsequent den- tinal bridging. In another area of clinical investigations, a collaborative study with the Anesthesiology Department of the Clinical Center on general anesthesia in ambulatory dental patients has been developing important information con- cerning the physiological effects of various anesthetic agents and oral surgical procedures. Since in some localities there are almost as many general anesthetics administered in den- tal offices as in local hospitals, such informa- tion should prove particularly important for the specialty of oral surgery. Accumulated data are providing a continuing record of pulse, blood pressure, arterial 2 saturation, respiratory phenomena, cortical brain activity, and the electrical activity of the heart. Among the more significant findings have been (1) consistent hypertension in all ambulatory anesthesias, which directly paral- lels the intensity of the surgical stimulation; (2) preoperative and operative tachycardias in almost 100 percent of the anesthetics (the preoperative changes in rate being apprehen- sive in nature whereas the operative changes 318 ANNUAL REVIEW OF INTRAMURAL RESEARCH are due primarily to the pharmacologic action of the intravenous barbiturates and secondar- ily, to the surgical stimulation in extremely light anesthetic planes); and (3) depression of arterial oxygen saturation, which is a con- trollable factor related to anesthetic manage- ment and drug administration (i.e., avoidance of obstructions and drug overdosage). Studies of soft tissue lesions continued to re- ceive major attention during the past year. As described in earlier reports, a transitional L- form of an alpha steptococcus has been isolated from or allesions in patients with recurrent aphthae and periadenitis aphthae. This orga- nism has now been consistently recovered from lesions in numerous patients on repeated ex- aminations over a 12-month period. Blood ob- tained for culture during two exacerbations in one patient was found positive for the same organism, and a stable L-form was obtained from scar tissue at the site of previous lesions during a remission. The injection of a licensed intravenous vac- cine (Strep, indifferent, Lilly) into one patient with periadenitis aphthae, over a period of several months, reduced the severity of the ul- cers but did not prevent their recurrence com- pletely. Therapeutic investigation also is under way on these stubborn and resistant chronic debilitating diseases with a limited success observed with various forms of acromycin and topical steroids. Parallel animal studies have suggested that hypersensitivity to the antigens of the alpha streptococcus isolated from recurrent aphthae is an important factor in the development of these lesions. Positive skin tests (delayed type hypersensitivity) to these antigens were ob- tained in patients with aphthous stomatitis but not in control individuals. The degree of the skin test reaction was found to be directly proportionate to the severity of the disease in the patients tested. The injection of a vaccine intravenously for 5 days prior to skin challenge reduced the size and duration of the ulcers experimentally pro- duced in the hypersensitized and control guinea pigs. The greatest protective effect was noted in the non-sensitized (controls) guinea pigs. In studies of mucous membrane changes associated with age and certain diseases, it has become apparent that human buccal mucosa, although appearing clinically normal, may un- dergo various changes with advancing age. Since the buccal site is frequently biopsied, standards need to be established on normal mucosa to eliminate errors in diagnosis due to the age factor. Other work is being carried out on a pre- viously undescribed "focal epithelial hyperpla- sia" in Indian children. Special studies, in- cluding viral analyses and electromicroscopy, failed to reveal evidence of a viral agent. The present knowledge concerning this lesion indi- cates that either an environmental or a genetic factor is involved in its development. Preliminary studies, using tritiated thymi- dine, on oral mucosa involved in verrucous carcinoma have revealed epithelial turnover rates remarkably similar to those believed to be found in normal oral mucosa. Similar in- vestigations are currently under way to bet- ter define the oral mucosal alterations in pa- tients with Darier's disease. In a collaborative study with the Laboratory of Pathology, National Cancer Institute, the in- dividual and/or combined roles that calcium hydroxide, tobacco, and gambier might play in causing betal quid induced carcinomas of oral mucosal tissues has been under examina- tion. Principally utilizing the hamster cheek pouch as a model system, one or more of the following changes were noted: Deposits of cal- cium, inflammation, giant cell formation and fibroblastic proliferation in the lamina pro- pria; and inflammation, ulceration, atrophy, hyperplasia, hyperkeratosis, parakeratosis, acanthosis, and cellular atypia in the epithe- lium. In no instances were squamous cell car- cinomas produced, nor were any changes noted in those cheek pouches treated with snuff or starch powder alone. Another study designed to classify and deter- mine the tissue of origin of benign fibro- osseous lesions of the jaws drew heavily on material made available at the Armed Forces Institute of Pathology. Among the more sig- nificant observations were: (1) that ossifying fibroma, cementifying fibroma, and cemento- NATIONAL INSTITUTE OF DENTAL RESEARCH 319 ossifying fibroma appear to arise predomi- nantly from the periodontal membrane but can also arise from the medullary bone; (2) that oxytalan fibers may occur in most benign fibro-osseous lesions of the jaws, regardless of their tissue of origin, provided that mature collagen fibers are also present in the lesion; (3) that since oxytalan fibers and pre-elastic fibers cannot be distinguished with present histochemical methods, the demonstration of fibrous elements stained with the oxytalan fiber method does not constitute conclusive evidence of odontogenic origin of the tumor; and (4) that the birefringence pattern under polarized light serves as an excellent differential for diag- nosis. Fibrous dysplasia gives a random irreg- ular birefringence, indicative of woven bone, whereas the other fibro-osseous lesions mani- fest birefringence as parallel light and dark bands, indicative of the varying degrees of lamellar bone formation. In a related study to define the behavioral pat- terns of osteosarcoma and chondrosarcoma of the jaws, the following findings have been re- ported: (1) tumors in the mandibular symphy- sis area are most amenable to cure whereas those in the maxillary sinus are least amen- able; (2) the average age of occurrence for osteosarcomas of the jaws is about a decade older than for osteochondroma of the jaws, but there is a quite wide range, and the range is wider for the mandible than for the maxilla; and (3) early findings are quite variable and often non-specific, and diagnosis depends upon correlation of clinical behavior, histologic ap- pearance, and roentgenographic appearance, any one of which may be deceivingly benign appearing. An important early finding may be roentgenographic evidence of symmetrical widening of the periodontal membrane space, with maintenance of an undisturbed lamina dura radiopacity. Investigation of Gardner's syndrome, which consists of multiple odontomas, multiple osteo- mas of the jaw, multiple polyps of the intestine, and subcutaneous tumors which are inherited, revealed that these patients are refractive to the effects of parathyroid hormone. Thus, it is possible that a group of diseases, including pseudohypoparathyroidism, basal cell nevus- jaw cysts syndrome, and Gardner's syndrome, share some defect in parathyroid hormone utilization. Other diseases with similar signs and symptoms are being investigated for this biochemical abnormality. Genetic studies of inherited sensory traits indicate that the inability to smell potassium cyanide is a polymorphism and a recessively inherited trait. In related activities, saliva studies have yielded a major finding concerning amylase isoenzymes in humans. A polymorphism in man has been defined in that different forms of the enzyme are consistently produced in a particular individual, and a generic difference exists in parotid amylase between man and rat. A discrepancy in the classical method for de- termining secretor status was found and tested on 2,875 individuals, which indicates that ap- proximately 6V2 percent more aberrant secretor individuals can be detected by this new method. The foregoing account of intramural re- search reflects a rather purposeful approach to the delineated problems and objectives en- compassed in the Institute's broadly based pro- gram. Likewise, it makes abundantly clear the view that dentistry's attainments of tomorrow will be a direct consequence of the extension of the boundaries of knowledge, and that this can come about only coincident with the intimate bonding of the health professions and related sciences in the kind of environment exempli- fied at the National Institute of Health. LABORATORY OF MICROBIOLOGY Immunology The principal organizational event in the Laboratoiy of Microbiology this year was the creation of the Immunology Section, with a view to co-ordination, more efficient planning and performance, and eventual extension of current immunologic projects. This move not only recognizes formally the natural place of immunology as a major component in this Lab- oratory, but signalizes also the trend of our basic investigations relating to periodontal disease. Extensive investigations in this Lab- oratoiy and elsewhere have established that the members of the oral microbiota most sig- 320 ANNUAL KEVIEW OF INTRAMURAL RESEARCH nificantly implicated in the pathogenesis of chronic periodontitis possess sufficient patho- genic factors to account for the principal fea- tures of the periodontal lesion. These factors include lipopolysaccharide endotoxins and his- tolytic enzymes (mucopolysaccharases, prote- ases, peptidases, collagenase, and other hydrolases). We can now add the observation that neutral solutions of ammonium ion and hydrogen sulfide, in concentrations that might reasonably be expected from known bacterial actions in the gingival crevice, exert a marked cytopathic effect on unkeratinized epithelium such as that lining the critical contact of gin- giva with oral bacteria. With this background largely filled in, it seemed timely to increase attention to the im- munological relationships between the host and the microbial products occurring in the gin- gival crevice and periodontal pocket, and to associated basic immunologic studies. Thus, it was shown that repeated deposition of antigen in the normal gingival pocket of the rabbit resulted in appearance of homologous antibody in the blood serum and in chronic allergic in- flammation of the gingival histologically very similar to that seen in chronic human gingi- vitis. Paradoxically and despite many investi- gations, little evidence has been presented that humans form antibodies to their indigenous gingival crevice bacteria. This failure seems to be due at least partially to the use of insuf- ficiently sensitive serological methods. Using indirect hemagglutination and bactericidal tests for antibody, we have found regularly significant amounts of antibodies to Leptotri- chia buccalis, Veillonella alcalescens, and Fuso- bacterium polymorphum in human sera. Sub- jects with advanced chronic periodontitis ex- hibited significantly higher titers to F. poly- morphum than subjects with a clinically unin- flamed periodontium. Parallel studies showed that the concentrations of antibodies in paro- tid secretion were about 1:1300 those of serum. Presumably the major part of antibodies in the oral cavity comes from the blood serum, via gingival crevice exudate. Continuing studies of the relationships be- tween lactic dehydrogenase virus (LDV) and its host animal (mouse) have paid unexpected dividends in the immunological field. LDV is unusual in several respects: LDV-infected ani- mals exhibit lifelong viremia but remain ap- parently healthy; the only known consequence of LDV infection has been persistent elevation of a number of plasma enzyme activities, due at least partially to impaired clearance mech- anisms; and until now no antibody response to LDV has been discernible. That LDV does activate an immunological response of its host is indicated by our recent finding that LDV infection of germfree mice results in an ele- vated level of plasma y-globulin (not due to impaired clearance) and a great increase of germinal centers in spleen and lymph nodes. Both these parameters normally are very low in germfree animals, and their increase is usually interpreted as an immunological re- sponse. How much of this y-globulin produc- tion represents antibody cannot be ascertained at this time. Extension of these experiments, however, showed that LDV acts as an immuno- logical adjuvant; that is, LDV-infected con- ventional mice have greatly enhanced capacity to produce antibody to a foreign protein. Fur- ther investigation of these phenomena, includ- ing other viruses and hosts, should provide a new approach to some aspects of immunologi- cal response. Consistent with the foregoing results, it was possible to demonstrate neutralizing antibody in the sera of mice infected for three months or longer, after selectively inactivating the LDV in the sera. Such treated sera readily neutralize LDV obtained from early infections (early LDV), before antibody has been pro- duced; however, LDV obtained from long- standing infections (late LDV) is rather re- sistant. This result suggested that late LDV existed as an infectious virus-antibody com- plex. Consistent with this interpretation, late LDV could be "neutralized" by antimouse sera prepared in goats. This surprising discovery led to the concept of "sensitization" of virus by antiviral antibody, without neutralization. It opens the way to a fresh interpretation and investigation of viral neutralization by anti- bodies, whose mechanism has never been satis- factorily elucidated. NATIONAL INSTITUTE OF DENTAL RESEARCH 321 The chemistry of the labile, enzyme-sensi- tive region linking the three stable domains of rabbit yG immunoglobulin has been studied further by analyzing the amino-acid composi- tion of fragments prepared with pepsin, insol- uble papain, mild reduction, and alkylation. In this way it has been possible to narrow the locale of the important disulfide bond linking the heavy chains of rabbit yG to a region be- tween the respective peptide bonds hydrolyzed by pepsin and papain. These studies have been materially facilitated by improvements in the program for automatic digital data acquisition and computer calculation developed for the amino-acid analyzer. Dental Caries Evidence continues to accumulate that the level of dental caries activity in experimental animals is determined by veiy intricately bal- anced relationships between host, a limited variety of oral microorganisms, and dietary factors, notably sucrose. A considerable range of microbial species has now been tested in gnotobiotic rats and in hamsters deficient in cariogenic flora. Except for a single atypical strain of Lactobacillus acidoj)hilus , only cer- tain strains of a particular cultural and im- munological type of oral streptococcus have been found capable of initiating caries in such animals. Significantly, this Laboratory and col- laborators at National Children's Cardiac Hos- pital, Miami, Fla., and Royal Dental School, Malmo, Sweden, have isolated during the past two years a number of "rat-type" and "ham- ster-type" streptococci from human caries le- sions and saliva. Many of these have proved to be cariogenic in the respective species of rodent. Recently we have isolated streptococcal strains from humans, which are cariogenic in both rats and hamsters. In view of the long history of unsuccessful attempts to transmit caries activity from humans to animals, and from one species of animal to another, these results have been very surprising. Most im- portantly, they have encouarged renewed search for a specific bacterial element in human caries. Sufficiently distinctive cultural charac- teristics of these types of streptococci have been ascertained to make possible beginning human epidemiological studies. It would be es- pecially meaningful, for example, to determine whether these organisms are natural inhabit- ants of the oral cavity in population groups with low caries experience, or whether the or- ganisms must be transmitted to an individual before the disease can be established. The reasons why some strains of these strep- tococci are cariogenic, while others are not, seem to relate to their behavior with sucrose. Thus we have demonstrated that cariogenic strains produce far more dental plaque, in vivo and in vitro in the presence of sucrose as com- pared to equivalent amounts of other carbo- hydrates. On the other hand, cariogenic and noncariogenic streptococci did not differ in their rates of acid production, and fermented glucose, fructose, maltose, or mixtures of glu- cose and fructose as readily as sucrose. Never- theless, when any of these sugars, or sorbitol, or a hydrogenated starch product was substi- tuted for sucrose in a caries-conducive diet, the incidence and severity of caries decreased markedly. These results are of course entirely consistent with the large amount of epidemio- logical evidence that dietary sucrose is a major determinant of caries in man. (Concerning su- crose it is pertinent to mention also the parallel demonstration of extracellular polysaccharide (levan) production from sucrose, but not from a variety of other common sugars, by Odonto- myces viscosus. This provides a reasonable ex- planation for the accumulation of this organ- ism as a gelatinous plaque causing a form of periodontal disease in hamsters fed a high- sucrose diet.) Recent evidence indicates that differences in the caries susceptibility of different strains of rats correlate with their ability to support a cariogenic flora. When different strains of rats have been fed the same diet and given equal exposure to a source of cariogenic flora, the levels of caries activity and the animals' abil- ity to transmit the flora have differed, approxi- mately in parallel. On a different caries-con- ducive diet, however, the relative susceptibili- ties of two strains of rats can be reversed; that is, a normally "resistant" strains has de- veloped more caries than its counterpart "sus- 322 ANNUAL REVIEW OF INTRAMURAL RESEARCH ceptible" strain when both were fed one of our standard caries-conducive diets. Other studies indicate that the numbers of cariogenic strep- tococci in the mouths of rats and hamsters parallel the levels of caries activity. These re- sults suggest that the support of a cariogenic flora depends upon some host-diet relationship, rather than on host and dietary factors inde- pendently. We conclude that the problem of caries con- trol assumes quite different aspects depending on the microbiological status of the subject population. If relatively caries-free populations, such as those found in several African nations, enjoy this status because they are not infected with cariogenic microflora, caries activity should remain low regardless of dietary com- position, particularly the consumption of su- crose. On the other hand, if potentially cario- genic microorganisms are already widely prevalent in such populations, then introduction of sucrose into the diet should automatically result in increased caries activity. In the United States and most Western nations, su- crose consumption and caries are so prevalent that we must assume that cariogenic micro- organisms are ubiquitous. Probably the only way to eradicate dental caries would be to elim- inate the causative microorganisms from a pop- ulation. Until this becomes feasible, as by suitable use of antibiotics, other control meas- ures of known efficacy such as fluoridation and dietary regulation should be rigorously applied. Virology Continuing study of persistent herpes sim- plex virus (HSV) infection in a cell culture system now warrants provisional conclusions as to its mechanism and inferences as to re- current herpes infection in humans. The fact that persistent cyclic HSV infections can be initiated and maintained in the presence or absence of homologous antibody indicates that the underlying mechanism is not depend- ent on antibody directly or its ability to reduce extracellular HSV. Even when antibody is present, virus transmission still occurs by cell- to-cell transfer and by the reattachment of in- fectious cellular material. The only effect of antibody which we have been able to identify is its ability to reduce the severity of the in- fections to a low grade, comparable to a sub- clinical stage in the human disease. The maintenance of these persistent infections ap- pears to depend on the establishment of a dynamic cell-virus equilibrium which is char- acterized by the continual selection of cells resistant to the major fraction of virus being produced at any one time. Selection of such re- sistant cells should ultimately result in a cell population completely resistant to HSV infec- tion. This does not occur, however, due to virus variations which parallel the cellular changes. The result appears to be that the selection of resistant cells is offset by the production of either a new virus variant, to which the cells are still susceptible, or to changes in the virus population resulting in selection by the cells of a virus variant which was present as a minor fraction. Superimposed on this continual selec- tion mechanism is the probable production of factors that temporarily enhance the cells re- sistance to HSV infection. Such factors, which thus far have been found in only low titers, are probably synthesized and maintained in localized areas of the cell cultures, thus allow- ing the few surviving cells to proliferate and replenish the cell sheet. It has been found, in fact, that replenishment of the cell sheet in localized areas must occur before a new cycle of virus multiplication and cytopathic effect is initiated. In the presence of antibody the se- verity of the infections in cultures producing a non-proliferative virus variant is reduced to a low level. After varying lengths of time, a short-term exacerbation paralleled by the ap- pearance of a syncytium-forming variant is evident, followed by the re-establishment of a low grade infection. The occurrence of these exacerbations in vitro is strikingly similar to the recurrent episodes seen in human herpes labialis and appears to be related to viral changes which temporarily alter the cell-virus equilibrium in favor of the virus. Ultimately, the cells are able to readjust to the new virus type being produced, and a subclinical type in- fection is re-established. Previously we have reported that HSV could be cultured from the salivas of rabbits many months after intraperitoneal injection. We NATIONAL INSTITUTE OF DENTAL RESEAKCH 323 have now found that the HSV-antibody re- sponse in such animals is strong and lasting, with cyclic variations very similar to the re- sponse to HSV infection in man. In contrast, the antibody titer induced by inactivated HSV is low and transitory. These results indicate that HSV persists in the tissues of animals receiving live virus, with intermittent immu- nizing bursts. However, no HSV could be cul- tured from testes, kidney, liver, spleen, lung, lacrimal glands, submaxillary glands, lymph nodes, and brain of such animals. Rather sur- prisingly, organ cultures of all these tissues from the immune animals, except lymph nodes and brain, were susceptible to infection with HSV. Provisionally we conclude that HSV per- sists in some inapparent form in these ani- mals, or quite possibly in tissues not yet tested for this virus. Since lactic dehydrogenase virus (LDV, see above) induces no discernible cytopathic effect, its cellular location in infected mice has been a mystery. This has now been partially solved by the electron microscopic observation of par- ticles having the fine structure of LDV, in peri- toneal macrophages. Presumably this virus is either phagocytized by and/or replicates in the macrophages. Rat submaxillary gland (RSMG) virus, de- scribed in previous reports, is associated with a specific cold hemagglutinin, whose titer in triturates of the glands is a simply determi- nable and sensitive indicator of the presence of the virus in its natural host. This indicator showed that RSMG virus was absent or scant in both germfree and conventional rats until two months of age. After three months of age, it was ubiquitous, with high hemagglutinin titers. Concurrently, a specific immunoglobulin hemagglutination inhibitor appears in the ani- mals' sera. Its titer also was low in the younger animals and increased with age. On average, the inhibitor titers of serum paralleled the titers of hemagglutinin in the glands. These tests will greatly facilitate study of the ecology of this virus. Microbial Physiology Further study was made of the paradoxical "lactate dehydrogenase" of Butyribacterium rettgeri, which cannot convert lactate to py- ruvate, as such enzymes usually do. In effect, it functions as an irreversible pyruvate re- ductase. Using a 100-fold purified preparation of this enzyme, the reaction with pyruvate was found to deviate from classical enzyme kinetics, that is, the reaction rate was a sig- moidal function of pyruvate concentration, not a hyperbolic one. This indicates that the enzyme is allosteric, requiring reactions at two sites on the molecule, and therefore might function in a regulatory capacity. An associ- ated observation was that reduction of pyru- vate was strongly inhibited by adenosine tri- phosphate (ATP) but not by adenosine monophosphate. Thus, under conditions where the cellular energy supply (ATP) derived from glycolysis is high, and the potential for bio- synthesis is consequently great, the conversion of pyruvate to lactate might be inhibited. Py- ruvate would then remain available for a variety of biosynthetic reaction sequences, such as amino acid and fatty acid synthesis. Such a regulatory mechanism would provide the cell with a novel means of modulating the carbon supply for biosynthesis through a linkage with the available energy supply. Continued investigation of the biosynthesis of the vitamin folic acid, by enzymes extracted from lactobacilli, showed that the pathway from guanosine monophosphate involves elim- ination of the number 8 carbon atom as formate, and subsequent closure to form the pteridine ring. A novel nonenzymatic reaction was discovered, whereby guanosine mono- phosphate, ferrous ion, and mercaptoethanol yield an intermediate compound that releases formate when treated with lactobacillus ex- tracts. Knowledge of the nature of these re- actions should help elucidate the biosynthesis of folic acid. Evidence continues to accumulate that the rate of differentiation of the cellular slime mold, Dictyostelium discoideum, from ameboid to spore stage, depends on intracellular accum- ulation of the monomeric constituents of ribonucleic acid. Thus the 5'-nucleotides of adenine, guanine, cytosine, and uracil stim- ulate differentiation with equal efficacy, when supplied exogenously. The whole molecule is 324 ANNUAL REVIEW OF INTRAMURAL RESEARCH required. Respective purines, pyrimidines, and nucleosides are inhibitory. Pointing in the same direction is the fact that a number of substances other than nucleotides can stimu- late differentiation, while causing intracellu- lar accumulation of ultraviolet-absorbing ma- terials. The latter have now been identified as a mixture of the nononucleotides and by nucleosides of ribonucleic acid. This analysis was made possible by development of an im- proved column chromatography procedure combining molecular sieve and ion exchange chromatography. Systematic Microbiology Two members of the staff continued to de- vote much effort to accumulation of necessary data and their collation by respective interna- tional committees engaged in clarifying defini- tions of Lactobacilleae, Neisseriaceae, Pro- pionibacteriaceae, Actinomyces, and Bifido- bacterium. Definitive redescription of the genus Veillonella was completed and pub- lished. In the cytological area, combined appli- cation of electron microscopy, antigenic frac- tionation, labeled specific antibody, and pharmacological testing demonstrated that the endotoxic somatic lipopolysaccharide antigen in Veillonella is localized in a distinctive three- layered outer membrane. This can be stripped off by phenolic extraction, leaving the rest of the cell morphologically intact. An inner rigid wall, which maintains cellular integrity, was demonstrated by its digestion with lysozyme, a mucopolysaccharase. Basic studies of these kinds are of the first importance, particularly in the oral field, be- cause the usefulness of large parts of previous work is vitiated by lack of adequate cytologi- cal, immunological, biochemical, and pathoge- netic characterization of microorganisms. LABORATORY OF HISTOLOGY AND PATHOLOGY For the purposes of the present report the activities of the Laboratory of Histology and Pathology are summarized according to several areas of general interest. The proj- ects from which the results have been gathered together are carried on by staff members alone or jointly, and often in col- laboration with workers from other labora- tories. The specialized fields represented in- clude biophysics, histochemistry and experi- mental pathology. Microstructural Characteristics of Normal and Abnormally Developed Enamel For many years considerable emphasis has been placed on the study of enamel struc- tures, normal as well as pathological. Valuable new data have been accumulated which have advanced our understanding of the morpho- logy of sound and carious human enamel, especially with regard to the prismatic structures and their participation in the spread of the carious process. While the pre- vious studies dealt with subsurface enamel this year's efforts have been directed toward the surface layer. This layer is of particular interest because of its apparent greater re- sistance to decalcification as seen in early enamel caries, the so-called white spot lesions, where a seemingly intact surface layer covers an area of extensive subsurface demineral- ization. Although the differences in reac- tivity may be attributed to chemical differ- ences such as higher fluoride contents, the present study demonstrates clearly that mor- phological differences also exist. The lack of prism structure, the orientation of the crystals perpendicular to the surface, the higher mineral contents coupled with a pos- sible increase in crystal size are all factors which might contribute to an increased re- sistance. The influence and role of this "prismless" surface layer in the onset and spread of the carious lesions especially re- lative to morphological differences from one tooth to another are some of the important questions which may find an answer in the continuing research. A contributory factor to the success of these studies has been the new microradio- graphic equipment which was constructed and put into service last year. The availability of an X-ray tube with exchangeable tar- gets has made it possible by selecting X-rays of suitable wave lengths to detect very minute NATIONAL INSTITUTE OF DENTAL RESEARCH 325 differences in mineral contents which could not be recorded by previous methods. The effect of tetracycline hydrochloride on enamel development has been explored fur- ther and a reproducible dose-response rela- tionship has been established. Using these results as reference, the studies were extended to include the effect of comparable dosages of oxytetracycline. Although the latter an- tibiotic was capable of introducing mineral- ization disturbances at similar dose levels, gross hypoplastic lesions were not seen, sug- gesting that oxytetracycline is somewhat less toxic than tetracycline hydrochloride. The similarity of the tetracycline induced de- fects to those resulting from injections of a number of other chemicals indicates a com- mon non-specific response. Localization and identification of the cytological sites affected by the various toxic agents promise to yield considerable date on enamel formation in general. vestigated using X-ray diffraction techniques. One aspect of this project has been the develop- ment of new methods for assessing the degree of crystallinity directly from measurements taken from the instrument instead of using the more cumbersome template approach developed previously in this laboratory. The data com- piled through the direct methods can be fed into a computer, thus facilitating more exten- sive studies of age effects on bone crystallinity in high and low fluoride areas. A second facet of the work has involved assessing the effect of fluoride on crystallinity of enamel. As with bone it was found that the crystallinity of human enamel improved with increased fluor- ide. Yet in the enamel the changes in crystal- linity were not restricted to the a-axes directions, but also included the c-axis. Since both size and strain factors influence the de- gree of crystallinity, the full meaning of the observed changes awaits a separation of the two factors. Crystallographic Studies of Synthetic and Biological Phosphates A series of calcium phosphates have been made synthetically and have been examined by X-ray diffraction and by infrared spectro- photometry. Among the problems investigated have been definition of conditions under which large hydroxy-apatite crystals may be grown routinely and a single crystal X-ray structure determination of a selected, well-defined crys- tal. The availability of such crystalline material of known structural composition is of major importance to all investigators interested in studying the chemical reactivity of hydroxy- apatite in both synthetic and biological sys- tems. Other studies have dealt with the charac- terization of the infrared absorption spectra of pure synthetic apatites and the identification of band changes associated with the presence of fluoride and carbonate ions in the crystal latice. The knowledge of the exact location of these ions within the lattice is important for our understanding of their effect on caries sus- ceptibility. The relationship between fluoride contents and crystallinity of biological mineral was in- Histochemical and Chemical Studies of Connective Tissues The histochemical and chemical studies of enzymes involved in connective tissue forma- tion and destruction have been continued. Special emphasis has been placed on deter- mination of collagenase activity in a number of connective tissue sites under normal and pathological conditions following the dis- covery last year of the enzyme system in human gingival tissues. Of the normal tis- sues tested so far, only uterus, gingivae and to a lesser extent portions of the oral mucosa have exhibited collagenolytic activity. Some of the most exciting results, however, have been the demonstration of the enzyme in cultures of dermis from individuals with certain neuromuscular disorders and collagen diseases. Isolation and purification of the collagenase is being attempted and when accomplished will allow investigation of its action on the collagen structure. The cellular origin of the collagenase is also being studied. The results already obtained represent an important ex- tension of our basic knowledge and when fully exploited will undoubtedly contribute signifi- cantly to a better understanding of the met- 326 ANNUAL REVIEW OP INTRAMURAL RESEARCH abolic processes in healthy and diseased con- nective tissues. Experimental Pathology Previous experimental work on the etiology of dental caries and periodontal disease has clearly demonstrated the importance of bac- terial plaque deposits in both these infections. Consequently, this years studies have been concentrated in defining various factors in- volved in plaque formation and on testing the efficacy of different drugs and proprietary formulations in the control of plaque forma- tion. The results indicate that many micro- organisms and among these several strepto- cocci of human origin are capable of induc- ing plaque and active caries in hamsters. The influence of diet, especially the effect of different sugars, has demonstrated that while sucrose in the diet is associated with rapid plaque formation, sucrose substitutes are much less plaque conducive. The host factor has been investigated in studies aimed at determining the effect of maturation on tooth resistance to plaque associated lesions. The findings suggest that in the absence of fluoride, tooth age is not a factor in caries susceptibility. While fluoride enhances tooth resistance, some fluoride containing preparations also ap- pear to be active in controlling plaque forma- tion. None of the commercially available, fluoride containing dentifrices, however, have been effective in plaque control when tested in the experimental hamster model. The con- tinued testing of anticariogenic drugs and methods in the experimental animal system and in in vitro studies will serve as an im- portant step in the evaluation of agents suit- able for human clinical trials. Bacterial and M acromolecular Structure These are largely basic studies aimed at relating morphology to an experimental situa- tion. The initial electron microscopic invest- igations on endotoxin formation and the character of the endotoxin of one kind of organism have been extended to other entoxin producing gram-negative bacteria. While all the organisms appear structurally similar, the isolated endotoxin particles vary in morphol- ogy. Work has also been directed at determin- ing the bacterial killing due to complement dependent antigen-antibody reactions. The initial findings demonstrate a similarity in morphology between complement dependent bacteriolysis and hemolysis. Studies on human amyloid have been continued with special em- phasis on purification of the pathological pro- tein in order to characterize it chemically. The degree of purification is assessed electron microscopically as in the effect of various chemical treatments. These investigations con- stitute additional steps toward widening our knowledge of the morphology of this protein. LABORATORY OF BIOCHEMISTRY Research continued in special areas of biochemistry, pharmacology, calcification and the etiology of dental caries. The research in pharmacology now involved in the study of dental caries. The research in pharmacology now involved in the study of oral-facial mal- formations induced by teratogenic drugs was organized into a Pharmacology Section of the Laboratory. Enzyme Chemistry Resolution of the mechanism of action and specificity of proteolytic enzymes advances understanding of all biochemical processes. Likewise these may be utilized for protein and enzyme modification, for study of protein mo- lecuar structure, to advance our knowledge of a particular function of a body tissue. At this time, the immediate objectives of this research relate to the specificity and mechanisms of the enzymatic action of chymotrypsin C, as well as guinea pig liver transglutaminase. Whereas the amino acid sequence of an ap- proximately twenty-member tryptic peptide of the chymotrypsin C enzyme has only one methionine residue. The active peptide site of the A enzyme contains two methionine re- sidues. Likewise an enzyme inhibitor specific for chy-motrypsin A has little or no inhibiting activity toward the C enzyme. Pruified guinea pig transglutaminase was found to be essentially homogeneous. It con- NATIONAL INSTITUTE OF DENTAL RESEAECH 327 tains 21 thiol-groups and no disulfide bridges. Only one thiol-group is involved in the active center. From tryptic and chymotryptic digests of C ''-labeled transglutaminase, a labeled peptide was isolated and found to contain the following amino acids, one tyrosine, one as- paragine, one C "-labeled amide of carbox- ymethyl cysteine, one glycine and three tryp- tophans. The tryptophan is present in the C-terminal position. Enzyme Structure and Mechanism of Action The studies related to enzyme structure and mechanism pertained to the physical-chemical properties of aldolase, and a correlation of its structure with catalytic function. Results have yielded information on the number and type of active sites possessed by this enzyme. Thus when native fructose-1, 6-diphosphate (FDP) was reduced to native FDP-aldolase there was evidence that three highly organized active sites were involved, each of which contained two nonidentical phosphate-binding sites. Methyl Groups of sRNA Protein synthesis involves the polymeriza- tion of individual amino acids into high molec- ular weight polypeptides, and soluble ribonu- cleic acid (sRNA) plays an essential role in this process as a specific carrier of amino acids to the site of polymerization. Our program in this area is designed therefore, to elucidate the role of methylated and thiolated constituents of sRNA in its biological activity and specificity. Results have indicated that while yeast amino acid activating enzymes can esterify amino acids to sRNA which contains methylated bases, these enzymes were unable to carry out this reaction with methyl-deficient sRNA. Continuing studies attempt to resolve the role and significance of the methylated base of sRNA, in its acceptor function. The results in- dicate that methylated base of sRNA, in its acceptor function. The results indicate that methylated bases of sRNA play a significant role (a) in the initial attachment of a specific amino acid to sRNA, (6) in a reaction cata- lyzed by specific amino acid activating enzymes and (c) in the capacity of the sRNA molecule carrying an amino acid, to find its proper alignment on the polymerization template. The investigation relative to thionucleotides (sulfur-containing) in sRNA has pertained to their functional significance. It was shown that formation of minor bases in sRNA is con- trolled by the sulfur amino acids cysteine and methionine. This relationship forms a cyclic metabolic regulatory system. Evidence was ob- tained that under ordinary circumstances the leucine activating enzyme from yeast recog- nizes E. coli sRNA about 75% of the extent recognized by the enzyme from E. coli. Phosphate and Dental Caries The research pertinent to the cariostatic effect of phosphates proceeded with attempts to define the mechanism of its action. Addi- tional evidence was obtained in support of the hypothesis that this cariostatic action is lo- calized in the oral cavity. Thus, NaH 2 P0 4 added to drinking water or diet of white rats for a period of two weeks prior to initiating a cariogenic diet reduced the ultimate cario- genic effect of the diet. Apparently the tooth surfaces and/or the oral milieu, had been fav- orably conditioned against caries by this prior exposure to phosphate. To resolve the possible effect of phosphate on the chemistry of both permanent and de- ciduous teeth, the Pitman-Moore miniature pig and standard Duroc swine were fed low- vs. high-phosphate diets for a period of one year, starting at weaning age. The animals were sacrificed when both permanent and deciduous teeth were erupted and still in situ. Ash, cal- cium phosphorus, magnesium and carbon di- oxide were determined in enamel and dentin of permanent and deciduous teeth. The low phos- phorus diet did not affect the content of the above major constituents of the teeth, as com- pared with the teeth of swine fed a normal and higher phosphate diet. Apparently the cariostatic effect of phosphate supplements as observed in experimental rats, may not be re- lated to a change in major components of the teeth. 328 ANNUAL REVIEW OF INTRAMURAL RESEARCH Prenatal and Fetal Development Factors Influencing Oral Disease The current objectives in this program of research are as follows: (a) to study the oc- currence of metabolites of chlorcyelizine in maternal and fetal tissue during organogene- sis, (&) to specify the effects of certain drugs on implantation and fetal development and (c) to compare the histology of benzhy- drylpiperazine vs. Vitamin A induced cleft palate. Qualitatively chlorcyelizine and nor- chlorcyclizine were the only detectable meta- bolites with at least one tertiary amine, found to be present in maternal and fetal tissue. The ratio of norchlorcyclizine to chlorcyelizine in fetuses (normally about 15:1) could be re- versed by administration of a demethylation inhibitor to a ratio of about 2:1. Under these conditions of inhibited demethylation, how- ever, teratogenesis was still present at approx- imately the same incidence. In attempts to identify the specific and min- imal chemical structure responsible for a teratogenic drug effect, it was found that bucli- zine hydrochloride induced the same malfor- mations as meclizine and chlorcyelizine. Three antihistamines, i.e. perphenazine, prochlorper- azine and trifluoroperazine which do not have a piperazine moiety in their molecular struc- ture demonstrated only a low (5.4%) terato- genic effect. Histological observations on palatal clefts produced by agents of the benzhydrylpipera- zine series of drugs, showed that the adhesion in the palatal area consisted of a fusion of the overlying epithelium of the tongue and the palatal processes. Gross examination of rat fetuses resulting from excessive doses of Vita- min A disclosed microstomia, mandibular- maxillary ankylosis, and cleft palate. Histo- logically the palatal processes of these fetuses were variable and vestigial in nature. Asso- ciated with these hypoplastic palatal processes was intramembranous bone formation. A large amount of heterotopic cartilage was located throughout the maxillary areas. It is particularly important to explore ani- mal species, in addition to the mouse and rat, which may be susceptible to teratogenic drugs. Thus, it was found that at high levels, chlor- cyelizine hydrochloride induced abortion in swine. In reduced quantities this teratogen produced typical oral-facial malformations in the swine fetus. The Rhesus monkey is now under observation to determine the status of this species with respect to susceptibility to teratogenic drugs. Protein Chemistry Collagen is the major structural protein of vertebrates and many invertebrates. The pro- tein, in the form of a rod-like monomer, under- goes aggregation into fibers which gain addi- tional stability by a maturation process in- volving the introduction of covalent crosslinks. These are chemical links that are found both between polypeptide chains in the same mole- cule and between chains in adjacent molecules. Past studies have elucidated the chain struc- ture. Current studies are related to a more detailed examination of the chemistry and bio- synthesis of crosslinks. This is being accom- plished by utilizing chemical and enzymatic cleavage to give peptides suitable for chemical studies that cannot be done on the whole mole- cule owing to its large size. An enzyme from the tadpole cleaves the molecule into two pieces of unequal size. Isola- tion and characterization of these pieces have shown that intramolecular crosslinks occur only in one, indicating that crosslinks are specific in location. Limited cleavage of native collagen with chymotrypsin and characteriza- tion of the products have demonstrated that this enzyme splits a few peptide bonds at the N-terminal end of the molecule. The crosslink is also lost, demonstrating that it must be in this region of the molecule. Chemical cleavage with cyanogen bromide has permitted the iso- lation of peptides from the crosslink region. Studies of amino acid sequences in these pep- tides have shown that the crosslink is derived from a lysyl residue, five amino acids from the N-terminal end. Preliminary to crosslinking the lysyl side chain is converted to an alde- hyde. Two of these aldehydes in adjacent chains form a crosslink probably by an aldol- type reaction. NATIONAL INSTITUTE OF DENTAL RESEARCH 329 Elastin is not as well characterized as a protein as collagen; but this connective tissue protein is also known to be crosslinked and lysine is the precursor. Rather than two lysyl residues forming a crosslink as in collagen, four are involved to give highly stable ring structures which are necessary for the elastic properties displayed by this protein. Chemical and histologic studies have shown that alde- hydes are intermediates, again in parallel to crosslinking in collagen. In the toxic condition known as lathyrism crosslinking of both elastin and collagen is inhibited. The inhibition appears to be in the enzymatic conversion of the lysyl side chain to an aldehyde. A similar inhibition is pro- duced in copper deficiency, suggesting that a copper-containing enzyme may be involved. The calcification of organic matrices is being studied using an in vitro system with elastin as the nucleating tissue. It has been found that trace amounts of a heavy metal such as ferric ion are required for calcification. The metal is apparently associated with sulfhydryl and perhaps imidazole groups in the protein phase. The first stage of calcification may involve the deposition of octacalcium phosphate at nuclea- tion sites containing the complexed metal, with a later conversion to hydroxyapatite. Fluoride Metabolism The mechanisms governing the deposition of fluoride in calcifying structures are not un- derstood. The role of fluoride in the inhibition of caries and its possible inhibitory effect on bone loss in resorptive bone disease, require resolution of the mechanisms by which these effects occur. A number of studies, therefore, are being carried out on the effect of fluoride in resorptive bone disease in the human, and in experimentally induced bone loss in the rat. Fluoride inhibited alveolar bone loss induced in the rat by either hydrocortisone or a low protein diet. However, maximum levels of fluoride tolerated by the rat did not reverse the decrease of bone protein and citrate syn- thesis as induced by hydrocortisone. In one individual having multiple myeloma, the administration of about 30 mgs of fluoride per day for about two years, reduced the mo- bilization of fluoride from the bones. Salivary Biochemistry Phosphates have been shown to markedly reduce dental caries in the rat and hamster, and an increase of phosphate in the saliva of humans has been reported to be associated with a low caries experience. Since phosphate is involved in the glycolytic cycle, studies have been initiated on the relation of various glyco- lytic enzymes of saliva to dental caries. Some enzymes involved in the so-called "shunt" path- way have also been studied. Thus, hexokinase, aldolase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phospho- glucose isomerase and phosphoglucomutase have been found in whole saliva, whereas in parotid saliva, only hexokinase, aldolase and phosphoglucose isomerase were present. Bac- teria, cellular debris or perhaps the submaxil- lary saliva thus may serve as a source of en- zymes not found in the pure parotid secretion. The activity of these enzymes in caries-free, caries-normal and caries-rampant individuals will be studied. In collaboration with the Oral Medicine and Surgery Branch of NIDR, calculus formation in the rat was related to a high protein con- centration in whole saliva and with an ele- vated acid and alkaline phosphatase activity. Ratstrain differences seemed to be of more im- portance than diet variables, as a factor in cal- culus formation. EPIDEMIOLOGY AND BIOMETRY BRANCH During the year the Epidemiology and Bi- ometry Branch continued its studies of the epidemiological characteristics of oral diseases. Activities included: (1) nutrition surveys in cooperation with the Research Branch of the Office of International Research; (2) investi- gations of the epidemiology of dental caries, including studies of the fluoride-dental caries relationship, clinical trials of caries inhibitory agents, and the nature of the disease in chil- dren fed by intubation; (3) studies of perio- dontal disease; (4) dental arch dimensions and 330 ANNUAL REVIEW OP INTRAMURAL RESEARCH occlusal anomalies in populations; and (5) col- lateral activities. Nutrition Surveys In previous years the branch has partici- pated directly in surveys organized by the or- ganization then titled the Interdepartmental Committee on Nutrition for National Defense. Survey sites included locations in Alaska, Asia, Africa, and Central and South America. Data from previous surveys has been collated with findings from each new one as it is developed. During the past year data from the survey in Nigeria were analyzed and assistance in the field was given to survey teams working in Guatemala and El Salvador. Analysis is continuing with the objective of discovering specific population factors which are associated with relative susceptibility to disease, on the general hypothesis that any causative factor should be found associated with disease in a consistent and predictable manner, population after population. Such as- sociations should have been demonstrable be- cause the prevalence of oral disease was found to vary, area by area, by factors of 30 to 60- fold. Optimum (or even excessive) intakes of fluoride were invariably associated with an inhibition of dental caries and there was a general relation between caries prevalence and per capita consumption of refined sugar, but other widely-held theories were not supported. Very low caries prevalences were found in populations subsisting principally on such foods as rice, cassava, or yam, which are highly cariogenic in laboratory animals. No support could be adduced for the theory of a "protec- tive" food or food element other than fluoride. Leads for further studies came from such find- ings as the observation (in Viet Nam, Lebanon, and Nigeria) of children with deciduous teeth attacked by caries alongside permanent teeth seemingly immune. One ominous note emerged: there was clear evidence that caries prevalence is rising, sometimes swiftly, in most popula- tions now relatively free from this disease. Tooth loss in some of these populations was high from another disease — periodontal dis- ease, which leads to deterioration and loss of the structures which support the teeth in the mouth. In such areas as Lebanon, Trinidad, Viet Nam, Thailand, Burma, and Nigeria the onset of disease was early and advanced de- struction was common even in young indi- viduals. Noma, a disease which has virtually disappeared in western civilizations, was com- mon in children in Nigeria. It was usually associated with protein-calorie malnutrition, exposure to malaria, recent history of rubella, and an overwhelming infection with Vincent's organisms. As a general rule high prevalences of perio- dontal disease were seen in those populations where children exhibited signs of malnutri- tion in calories, protein, and vitamin A and carotene. There was a consistent and positive relationship between deposits of oral debris and calculus and the severity of periodontal disease in every population studied. When these factors, and age, were held constant no consistent or strong relation could be demon- strated between disease and nutritional status. Estimates of nutritional status were based on biochemical determinations, dietary studies, and the character of diets followed in the past. Factors brought under study included such items as vitamin A, beta carotene, as- corbic acid, thiamine, riboflavin, niacin, total proteins and protein fractions in serum, cal- cium, iodine, and iron. On the basis of the findings the value of any of these agents in therapy may be questioned. Dental Caries Studies of dental caries within the United States included investigations with tube-fed children, clinical trials with a phosphate- fluoride dentifrice and a fluoride gel carried in a mouth applicator, and further analysis of findings in a long-term study carried out with children during the first ten years of use of a fluoridated community water. Studies of dental caries in laboratory ani- mals fed by stomach tube have yielded much basic information about the relative roles of substrate and disease in these animals. Hitherto there have been no parallel studies in human beings. During the year an investiga- the bacterial nature of dental caries which NATIONAL INSTITUTE OF DENTAL RESEARCH 331 tion was begun with tube-fed children in the Sunland Hospital, Orlando, Florida. This study should establish or deny basic inferences about have not heretofore been studied directly in human beings. Other laboratory investigations have demon- strated that dental caries activity can be con- trolled at will in hamsters through the application of fluoride gels or antibiotics, topically to the teeth using a fitted vinyl mouthpiece as a carrier. A field trial of a fluoride gel applied in this manner is in its second year with children of Cheektowaga, N.Y. Preliminary findings indicate that (a) much higher concentrations of fluoride appear in the outer layers of tooth enamel after this than after conventional methods of applica- tion, and (6) that a single hygienist can su- pervise many more children when this technic is followed. An interim examination disclosed caries had been inhibited in the study children but too little time has elapsed for determina- tion of the magnitude of the effect. Principal findings in the longitudinal fluori- dation study have been published in previous years. This study involved the reexamination of individual children annually for a period of ten years. The basic data are being reentered to test such hypothesis as the possible beneficial effect of prenatal ingestion of a fluoride water. The population is being utilized further in studies of occlusion and hygiene. Periodontal Diseases Within the past few years periodontal dis- ease has been recognized as a major public health problem in the United States, where it is a major cause of oral pain and tooth loss. Clinical management of the conditions is dif- ficult, expensive and time consuming and the number of available specialists is quite inade- quate to meet the need for such treatment. On a world-wide basis, people of the United States are probably the most favored in this regard; elsewhere the periodontal diseases tend to be more prevalent, more severe, and earlier in onset, and professional personnel are even less able to cope with the problems. Studies con- ducted by the Branch have led to the clear inference that control of this disease entity must depend upon control of deposits of dental calculus, with little benefit to be gained by nutritional therapy. Occlusal Anomalies Orthodontic care claims a considerable share of the funds spent to improve dental health, but the natural history of occlusion has received little emphasis in epidemiological study. The present program of study in the Branch is designed to determine the sequence of events, and the causative factors, which lead to undesirable states of occlusion, in the hope that some of these factors may be susceptible to simple methods of control in early stages of development. Principal focus is on a longitudinal study in which individual children are examined and reexamined over a period which includes criti- cal periods of tooth emergence and jaw de- velopment. Collateral Projects In addition to direct research activities and biometric services to our professional staff, a considerable amount of time was devoted to consultation on the design and conduct of field studies being undertaken by others. In some instances this involved direct participation in the first stages of the field work. Principal bene- ficiaries were research grantees of the Dental Institute, the World Health Organization, the Pan American Health Organization, and la Instituto de Nutricion de Centro America y Panama. CLEVICAL STUDIES As Clinical Investigations have grown and appropriate staff has been recruited, every effort for the utilization of the Dental Services Branch for human research studies has been made. Where at one time, few joint studies were operating, now many are in progress with considerable productivity. It is our phi- losophy that what cannot be accomplished else- where should be attempted here. Emphasis is placed on urging and supporting to our max- imum, human research over animal studies. Because human research is very frustrating 332 ANNUAL REVIEW OF INTRAMURAL RESEARCH in terms of patient problems, delays, and ex- tended experimental periods, we permit our investigators to become involved in animal projects to the extent that it represents at least a back-up to some proposed study or a human project recently initiated. The summary that follows merely repre- sents a broad panoramic view of all our ac- tivities. It should be appreciated that with NIH facilities the depth of study of even one patient represents a research study and per- mits us to excel over many other institutions with greater census potential. In the human dental pulp studies, we have now collected over 4,000 human teeth that were normal to begin with but have received a known traumatic episode for microscopic in- terpretation. This number of teeth represents a collection larger than all the human teeth used for intentional experimental purposes in all the world's literature previously reported. This collection presents us to the field of den- tistry as an international leader for such studies. With the use of collaborative funds the program has every opportunity to expand in the future. The opportunity for an oral surgeon to work with general anesthesiologists in a dental op- eratory provides us with information that is sadly deficient throughout the dental profes- sion. Again the Clinical Center provides us with a unique opportunity. Since in some areas there are almost as many general anesthetics administered in dental offices as in local hos- pitals, the basic physiological data from this study should be invaluable to the specialty of oral surgery. In human dental caries, the importance of snack habits has become most enlightening. With back-up studies in rats and hamsters, it has been shown how destructive in terms of dental caries certain food substances can be when used individually and how protective others can be in other instances. This project is continuing with pertinent combinations to further our knowledge of controlling ram- pant caries through diet. The one disease in dentistry that is most neglected is aphthous stomatitis. Here is an area where patients truly suffer over long periods of time and often without let-up. This is a disease that is continually referred to the next dentist down the street. With Clinical Center facilities we have approximately 90 pa- tients suffering with this ailment under con- tinual observation. By calling on the skills and know-how of many disciplines, progress has been made in controlling the disease and aborting recurrent episodes of this painful dis- order. With antibiotics and vaccines there is developing a basic understanding of how sen- sitization of an individual to a very low order of an infecting organism can lead to an acute exacerbation of the disease. New diseases continue to be recognized. So- called Heck's disease (focal epithelial hyper- plasia), although first described in the Ameri- can Indian by members of our staff, has now been found in Central and South America, Alaska and in a recent migrant from Samoa. Although the viral etiology of this disease has been temporarily ruled out, the genetic aspect of it has yet to be dealt with. The use of radioactive isotopes has become an essential tool in many of our studies. Another bogged down area of dentistry has been the field of tooth transplantation. Al- though attempted for thousands of years, prog- ress to date has not greatly improved. Al- though many such studies are presently going on throughout the world, the simple knowledge regarding the significance of the regeneration of cementoblasts has been continually ne- glected. Yet without a complete knowledge of this important cell, any hope to encourage the true reattachment and function of a perio- dontal membrane is handicapped. We have been deficient in studying oral can- cer in terms of experimentation. Although we can thoroughly evaluate selected patients, here again is an instance where animal back-up is necessary. A hamster study involving tobacco, gambier and calcium hydroxide in causing betal quid oral carcinoma is in progress. Al- though cellular atypism has been observed, no outright cancer has yet developed. The Oral Pharyngeal Development Section continues to provide basic information to sta- bilize this area. Vital staining demonstrations of differential skeletal growth patterns in face NATIONAL INSTITUTE OF DENTAL RESEARCH 333 and cranium of four laboratory mammals is in progress. Analogous studies of facial and cranial skeleton continue in human subjects distorted by anomalies and/or accompanying neurological impairments. Transducer methods of observation of tongue contacts and margin pressures have been developed with multiple-site inputs being recorded in parallel with speech and swallow. The development of oral perception in terms of form (oral stereognosis) continues to be calibrated in normal and abnormal children. Syndromes of disability of perception are being described. Similar studies related to chemosensation (small and taste) have been adapted to chil- dren and to neurologically impaired persons. Related studies of the orofacial area of cats concerned with the interaction of sensory stimuli in association areas of the cerebral cortex are continuing. Epidemiologic studies related to congenital malformations have been very productive. It has been established where the incidence in American Indians fits in relation to Caucasian and mongoloid groups. Biochemical studies have elicited information regarding genetic background and potential for dental caries. Also neonates metabolize different elements in different manners according to certain ethnic groups. The relationship of the effects of para- thyroid hormone in various syndromes is being studied. Unexpectedly, no increase in dental anomalies occurred in children exposed to atomic fallout although an increased incidence of thyroid nodules did occur. In salivary studies it has been found that a polymorphism in human amylase isoenzymes exists, that different forms of the enzymes are consistently produced in particular individ- uals. In cytogenetics it has been shown that phy- tohemagglutinin has an important action on the cellular mechanisms which regulate the lymphocytes. This system may provide a test for understanding the regulatory mechanisms which govern transcription of genetic infor- mation within the mammalian cell. Considerable effort has been made to hold this summary within a summaiy down to a few pages. By being less specific in describing our studies, some brevity has been maintained but at the same time it is obvious that preju- dice exists and certain emphasis has been placed where the personal experience has been greatest. In the future, it might be recommended that every investigator provide a one-sentence sum- maiy of the meaningfulness of each of his projects. DENTAL SERVICES BRANCH During the past years the research programs of the Dental Institute have become more inte- grated with the activities of the dental clinic area. A close working relationship exists be- tween investigative staff and the patient care dental officers in many collaborative studies. Concomitantly, our major responsibilities to provide optimum dental care for the research beneficiaries of the various categorical insti- tutes has continued in an effective manner. The Dental Services Branch provides a com- plete oral examination, evaluation, consulta- tion and dental therapy for the patients of the Clinical Center. The above objectives are being met by the Branch's contributions to important National Institute of Dental Research programs in the various specialties in dentistry, such as: Oral surgery with general anesthesia methods, hu- man dental pulp studies for the biologic evalu- ation of restorative dental procedures, altera- tions in the maxillo-mandibular jaw relation in the transition from natural dentition to dentures, control of rampant caries, post sur- gical tissue healing of impacted third molars related to periodontal involvement. Commensurate with our responsibilities in performing optimum dental care for the 500- bed Clinical Center, the Dental Services Branch has actively collaborated with the total Na- tional Institute of Health research effort. The following examples may be cited: National Cancer Institute Participation in operations about the head and neck, including neck dissections. This has 334 ANNUAL REVIEW OP INTRAMURAL RESEARCH been of mutual benefit to our staff and the maxillofacial surgeons. Suggestions made by dentists during surgery are of major impor- tance to the ultimate success of the final prosthesis and rehabilitation of the oro-facial region. During the past year an increasing number of maxillo-facial prostheses have been con- structed for patients with cancer. An efficient working relationship continues between the dental clinic and the National Cancer Institute surgery staff in the management of cases sur- gically treated for neoplasm. Notable are those patients with paranasal sinus disease sur- gically approached by the combined intra- cranial facial procedure. A three-stage maxil- lofacial prosthesis is designed and fabricated for each patient. The Branch renders further important con- tributions to the Leukemia Service of the Na- tional Cancer Institute in handling a myriad of oral problems associated with this disease. National Institute of Arthritis and Metabolic Diseases In a study of Fraction 1 of Cohn, a fibrino- gen and AHG (anti-hemophilic globulin con- centrate), the Dental Institute's role has been to assess the response of hemophiliac patients to multiple dental extractions. The results of these collaborative studies have been prepared for publication. National Heart Institute Patients with congenital defects, who are to have heart surgery, pose problems of dental management in both the pre- and post-surgical periods. Special considerations also are neces- sary in the performance of dental operations on patients with hypertension where fear and anxiety present particular problems. In this project considerable experience and care is required for proper dental evaluation and treatment of patients being maintained on anti- hypertensive drugs. All patients with rheumatic heart disease present particular difficulties. As an example, in the absence of proper dental care and prep- aration, even such a simple procedure as oral prophylaxis can precipitate a fatal, acute bac- terial endocarditis. The same holds true for patients who are to undergo or have under- gone cardiac surgery, resulting in prosthetic heart valve replacement. During this fiscal year consolidation and re- vision of the medical and dental records was completed. This now permits a more efficient flow of records within the clinic as well as to and from the Medical Records Department, Clinical Center. This procedure satisfies the requirements of the Medical Board, Clinical Center; Medical Records Department, Clinical Center and Dental Services Branch, NIDR. Extensive renovation of the dental clinic was planned and instituted during this fiscal year. The major objective was to modernize the existing modules in order to provide a more efficient physical facility within the lim- ited space available. The installation of easily maintained, bright operatories allowing the dentist and dental assistant to be seated dur- ing the four-handed dental procedures results in performing more dentistry for chronically ill patients with less time and energy expended. The following areas have been completed or underway: 1B-04 Special Prosthetic Operatory 1B-06 Special Prosthetic Laboratory 1B-07 Oral Surgery 1B-09 Oral Surgery 1B-12 Dental Operatory ORAL MEDICINE AND SURGERY BRANCH As the Oral Medicine and Surgery Branch has grown, we have attempted to acquire staff that would give emphasis to all the main problem areas of dentistry. Although some areas are still understaffed because of space and recruitment problems we feel that we are finally on the way. A brief review of the activities in the broad program of the Oral Medicine and Surgery Branch follows: Studies of the Human Dental Pulp In this project there is a continuing evalua- tion of the response of the human dental pulp to changes induced by dental drilling proce- NATIONAL INSTITUTE OF DENTAL RESEAKCH 335 dures and by various restorative and related materials, such as cavity liners. This study has furnished the dental profession with some very practical information on operative pro- cedures, particularly in regard to optimal cut- ting speeds, the proper use of coolants, and modifications in technic necessary for the safe placement of experimental restorative ma- terials. Because of the reduced inflammatory re- sponse of the pulp following high speed cutting technics, the incidence of reparative dentin production has been greatly reduced. Thus den- tinal tubules remain open and permit the toxic or irritating products of sterilizing agents, cements and silicates to permeate to the pulp tissue and cause further damage. This lack of reparative dentin formation is creat- ing a formidable problem in restorative den- tistry, especially in the field of full mouth re- habilitation where often the entire coronal dentin is exposed. Experimental drugs de- signed to reduce sensitivity of teeth (i.e., cor- ticosteroid compounds) and to more effectively seal the dentinal tubules are being sought, as well as drugs and technics to increase the incidence of reparative dentin formation. When prepared cavities are washed with a steroid formula containing 1% prednisolone in a vehicle of parachlorophenol, cresatin and gum camphor, before restoration with zinc oxide and eugenol, it was found that the pulpal response to the cavity preparation was min- imized about 50%. When the prednisolone was used without the vehicle, the inflammatory response was sustained only 12 days. Also when the same formula was applied to the cavity preparation several days after the full potential of the response had occurred, the resolution period was still shortened. In an attempt to find more ideal restorative materials, collaborative research is being con- ducted with the Eastman Dental Center and with an industrial non-dental manufacturer who are providing adhesive filling materials which will mechanically and chemically bond with tooth structure. The biologic effects on the human dental pulp tissues of two such experi- mental materials are currently under investi- gation. Because the Clinical Center can supply only about 300 human teeth per year for such studies, it has been necessary to supplement our needs with contract arrangements with several universities and other government fa- cilities. Anesthesia Studies A collaborative study with the Anesthesi- ology Department of the Clinical Center on general anesthesia in ambulatory dental pa- tients is developing important information concerning the physiological effects of various anesthetic agents and oral surgical procedures. These data provide a continuing record of pulse, blood pressure, arterial 2 saturation, respiratory phenomena, cortical brain activity and the electrical activity of the heart. The accumulated data of over five years of study have been and will continue to be used as a baseline of comparison for the new anesthetic drugs which are being introduced for use in oral surgery. All of the agents and combina- tions of agents commonly used in oral sur- gery have been used and evaluated in this study including Fluothane, which drug is of special interest at the present time because of its alleged hepatoxicity. The incidence of this complication is extremely low, so that our failure to find any evidence of the association may be statistical rather than factual. Among the more significant findings from a practical standpoint are: Consistant hypertension in all ambula- tory anesthesias, which directly parallels the intensity of the surgical stimulation; Preoperative and operative tachycardias in almost 100 percent of the anesthetics (the pre-operative changes in rate being ap- prehensive in nature) whereas the operative changes are due primarily to the pharma- cologic action of the intravenous barbitu- rates and secondarily, to the surgical stimu- lation in extremely light anesthetic planes; and That depression of arterial oxygen sat- uration is a controllable factor related to anesthetic management and drug adminis- 336 ANNUAL REVIEW OF INTRAMURAL RESEARCH tration (i.e., avoidance of obstructions and drug overdosage). Since in some areas there are almost as many general anesthetics administered in den- tal offices as in the local hospitals, and since there are no other such studies being con- ducted, the basic physiological data from this study should prove important for the specialty of oral surgery. Dental Caries Rampant dental caries is a very severe form of disease in which practically all of the teeth are attacked by decay in a relatively short period of time. It is found chiefly in young children, but may develop in adults who pre- viously had little or no caries experience. Under suitable experimental conditions, com- parable forms of rampant caries can be developed in laboratory animals such as rats and hamsters. From a research standpoint, rampant car? s offers a most favorable oppor- tunity to study the basic factors which acti- vate or control the caries process because the lesions is reduced to a minimum, and the de- termination of caries activity can be much more certain than in caries of usual severity. The purpose of this study is to evaluate in clinical studies the many factors which may be important in different cases of rampant caries, and to study the more important of these factors in laboratory and animal experi- ments with the goal of establishing more effec- tive means for solution of the caries problem. The study of new patients with rampant caries during the past year has further ex- tended the evidence for the basic conditions underlying the development of rampant caries. For example, the social aspects of this problem were illustrated in a child with rampant caries which had developed after the family had moved to a new neighborhood where she was the only young child and the neighbors con- tinually gave her "sweets to eat." A consistant finding in rampant caries patients has been the uncovering of some source of fermentable foodstuff above that in the average diet. The animal experiments testing these foods shows that some are very cariogenic, many are moderately cariogenic and a few are noncario- genic to rats. During the past years experi- ments were also conducted to determine the extent to which the very cariogenic foods would be made less cariogenic and the non- cariogenic foods such as cookies, adding water, grinding, adding sugar versus adding fluo- rides, and other modifications of the food. It was found that certain noncariogenic foods such as "dog biscuit" were not made very cariogenic by even large additions of sugar, and contrariwise, some highly cario- genic foods were not made non-cariogenic by the addition of fluoride. These experiments in- dicate that cariogenicity is affected by several factors including chemical properties, physical properties, taste of the food, and the time it takes for it to be eaten. Several of these fac- tors seem to be favorable in "dog biscuits," and these are being studied. Another objective of this project is to identify and classify human oral streptococci and to determine whether or not specific strains of streptococci can be identified as etiologic agents in dental caries. The floures- cent antibody technique is employed to iden- tify these strains in histologic sections of carious teeth and in the mixed cultures of dental plaque smears. Continued serologic screening of plaque smears from patients has indicated that cer- tain serologic groups of streptococci are not involved in the caries process and has there- fore intensified the implications of certain other serologic groups of streptococci in the caries process. Experiments with gnotobiotic rats have in- dicated that some adaption of the rodent mouth, sometimes involving antigenic shift is necessary in order for these human oral strep- tococci to establish themselves and produce caries. Studies of Soft Tissue Lesions The transitional L-form of an alpha strep- tococcus has been isolated from oral lesions in patients with recurrent aphthae and peria- denitis aphthae. This organism was consist- ently recoered from lesions in numerous NATIONAL INSTITUTE OF DENTAL RESEARCH 337 patients on repeated examinations over a 12- month period. Blood obtained for culture dur- ing two exacerbations in one patient was found positive for the same organism. A stable L-form was obtained from scar tissues at the site of previous lesions during a remission. The injection of a licensed intravenous vac- cine (Strep, indifferent, Lilly) into one patient with periadenitis aphthae, over a period of several months reduced the severity of the ulcers but did not prevent their recurrence completely. Therapeutic investigation is also under way on all these stubborn and resistant chronic debilitating diseases with a limited success observed with various forms of acromy- cin and topical steroids. Animal studies have indicated that hypersen- sitivity to the antigens of the alpha strep- tococcus isolated from recurrent aphthae is an important factor in the development of these lesions. Positive skin tests (delayed type hyper- sensitivity) to these antigens are obtained in patients with aphthous stomatitis but not con- trol individuals. The degree of the skin test reaction is directly proportionate to the sever- ity of the disease in the patient tested. The injection of a vaccine intravenously for 5 days prior to skin challenge reduced the size and duration of the ulcers experimentally pro- duced in the hypersensitized and control guinea pigs. The greatest protective effect was noted in the non-sensitized (controls) guinea pigs. Mucous Membrane Changes Associated with Age and Certain Disease. It is apparent that human buccal mucosa, although appearing clin- ically normal, may undergo various changes with the increasing age of the patient. Since the buccal site is frequently biopsied, standards need to be established on normal mucosa to eliminate errors in diagnosis due to the age factor. Further work is being carried out regard- ing the previously undescribed "focal epithe- lial hyperplasia" in Indian children. Special studies including viral analyses and electron microscopy failed to reveal evidence of a viral agent. The present knowledge concerning this lesion indicates either an environmental or genetic factor is involved in the development of this lesion. Preliminary studies using tritiated thymi- dine on oral mucosa involved in verrucous car- cinoma have revealed epithelial turnover rates remarkably similar to those believed to be found in normal oral mucosa. Similar studies have been performed on the oral mucosa of patients with Darier's disease. Preliminary re- sults reveal a need for further utilization of this technique in the study of normal as well as disease states involving the oral mucosa. Clinical Periodontal Studies Very recently a protocol for autogenous re- implantation of human teeth was approved. It is hoped that by studying the response of the periodontal tissues to tooth reimplantation that some knowledge relative to the factors permitting the differentiation of fibroblasts into cementoblasts will be obtained. After re- implantation, the teeth became firmly attached with new alveolar bone formation. In the ab- sence of continued cemental production, perio- dontal fibers lose their attachment to teeth with eventual exfoliation. New functioning cementoblasts have not been found earlier than 29 days. In the treatment of the osseous defects in periodontosis, preliminary results indicate that the autogenous transplantation of developing third molars into the first molar sites can be an effective way of inducing healing of the alveolar bony lesions and in restoring perio- dontal health. Animal Periodontal Studies Dental abnormalities were produced in Sy- rian hamsters by infections with H-l, OLV, Krisini and Rat Virus. The abnormalities pro- duced were generally somewiiat similar and consisted of root resorptions, misshapen roots, bony ankylosis of roots and microdontia of the third molars. The differences noted be- tween these four viruses were: the absence of mandibular third molars in the animals in- fected with OLV virus, a severe inhibition of osteoblastic activity and bone formation of the mesial and mesio-lingual aspects of both maxil- 338 ANNUAL REVIEW OF INTRAMURAL RESEARCH lary and mandibular first molars in animals infected with OLV and Hl-virus, enabling Stenson's gland, which is a normal component of the sinus, to come into close apposition with the mesial root of the maxillary first molar. In studying the effects of various viremias on healing wounds in adult animals, thirty five-day old male Syrian hamsters were in- jected intravenously with OLV infected tissue culture fluid which was obtained from rat embryo tissue cultures, at time intervals of hours to 6 days following fracture of the left forearm bones (Group I) or extraction of the maxillary right second molar tooth (Group II). The intravenous injections of OLV healing when administered prior to the third post- operative day. The delay in healing was transitory, i.e., by 15 days postoperatively no alteration in healing could be discerned. In a study comparing the healing of sur- gically exposed dental pulps in germfree and conventional rats, conventional animals showed an immediate, severe inflammatory response which quickly led to total pulpal necrosis; while the germfree animals, without exception, showed minimal inflammatory re- sponse (in spite of food impaction) with sub- sequent dentinal bridging. Oral Pathology Investigations In a study of hamsters' pouches to determine the individual and/or combined roles that calcium hydroxide, tobacco and gambier might play in causing betel quid induced carci- nomas, it was found that the affected pouches treated with calcium hydroxide showed one or more of the following lesions: deposits of cal- cium, inflammation, giant cells, and fibro- blastic proliferation in the lamina propria; and inflammation, ulceration, atrophy, hyper- plasia, hyperkeratosis, parakeratosis, acantho- sis, and cellular atypia in the epithelium. No squamous cell carcinomas were produced in any of the groups, and no changes were noted in those cheek pouches treated with snuff or starch powder alone. Several ham- sters treated with gambier developed minute ulcers with inflammation. In determining the genetic mode of inheri- tance of taurodontism by means of radio- graphic surveys and clinical histories of the involved families, four families in which com- plete radiographic documentation was ob- tained, revealed no parent possessing the tau- rodont tooth form. The trait occurred in ratios of (a) one of three children, (b) one of two children, and (c) two of two children. Taurodontism was found in both deciduous and permanent teeth of both males and fe- males. The mode of inheritance of taurodontism is apparently compatible with a recessive char- acteristic, but our evidence is not definitive. It is not a dominant characteristic as sup- posed in the German literature. Histologic ex- amination revealed normal odontoblastic and periodontal membrane activity, lending fur- ther support to the original theory that the developmental site of the taurodont tooth form is Hertwig's epithelial sheath. In a study to classify and determine the tissue of origin of benign fibro-osseous lesions of the jaws from the vast wealth of case ma- terial in the files of the AFIP, the following in- formation has been derived: Ossifying fibroma, cementifying fibroma, and cementoossifying fibroma appear to arise predominantly from the periodontal membrane but they can also arise from the medullary bone. Oxytalan fibers may occur in most benign fibro-osseous lesions of the jaws, regardless of their tissue of origin, provided that mature collagen fibers are also present in the lesion. Inasmuch as oxytalan fibers and pre-elastic fibers cannot be distinguished with present histochemical methods, the demonstration of fibrous elements stained with the oxytalan fiber method does not constitute conclusive evidence of odontogenic origin of the tumor. The birefringence pattern under polarized light does serve as an excellent differential for diagnosis. Fibrous dysplasia gives a random irregular birefringence, indicative of woven bones, whereas, the other fibro-osseous lesions manifest birefringence as parallel light and dark bands, indicative of the varying degrees of lamellar bone formation. NATIONAL INSTITUTE OF DENTAL RESEARCH 339 In a study to define the behavioral patterns of osteosarcoma and chondrosarcoma of the jaws, the following findings have been derived: There is no apparent racial or sex predilec- tion but females appear far more likely to have mandibular tumors than maxillary ones, whereas males are about equally divided be- tween maxillary and mandibular tumors. Rad- ical resection appears to offer the best hope for cure. Tumors in the mandibular symphysis area are almost amenable to cure; those in the maxillary sinus are least amenable. The average age of occurrence for osteosarcomas of the jaw is about a decade older than for os- teosarcomas of the jaws, but there is a quite wide range, and the range is wider for the mandible than for the maxilla. Early findings are quite variable and often non-specific. Early diagnosis depends upon correlation of clinical behavior, histologic appearance, and roent- genographic appearance, any one of which may be deceivingly benign appearing. An impor- tant early finding may be roentgenographic evidence of symmetrical widening of the perio- dontal membrane space, with maintenance of an undisturbed lamina dura radiopacity. Oral and Pharyngeal Development Section The Section has continued in basic experi- mentation and clinical application studies of the structure and the motor performance of the mouth and pharynx. Basic anatomical studies include: (1) vital staining demonstra- tion of differential skeletal growth patterns in face and cranium of four laboratory mannals, (2) detailed development patterns, in separate items of skeleton cultured in vitro, (3) se- quence of histological demonstration of re- sponses of bone to deforming forces. In the human, changes have been described in spatial relation to nasal septem and base of cranium during postnatal development. Analogous studies of facial and cranial skeleton have continued in subjects distorted by anomalies and/or neurological impair- ments. These have included standard methods of radiological cephalometry and also adap- tations of laminography which demonstrate the mid-line structures. In clinical application, evaluations of the basic orthodontic methods of tooth displacement have been performed, using precision techniques of portrayal of spatial changes of teeth. Basic physiological studies include: (1) elicitation of varied patterns of cry and re- lated arousal response by electrodes placed stereotaxically in the brain stem of cat, and the demonstration of action details by cor- related pressure recording, sound recording, laryngeal photography and regional cine- radiography; (2) neurophysiological studies of patterns of representation of sensation in the trigeminal nucleus, mechanisms of integration of afferent information in the cerebral cortex, and (4) patterns of respiratory motor re- sponse to stimulation in the pharyngeal area. Studies of upper respiratory and feeding functions have continued in an increasing number and variety of subjects impaired by anomaly, such as the Pierre Robin syndrome. The methods of cineradiography, cinephotog- raphy, respiratory displacement, and sound re- cording and spectrographic display have been utilized in study of selected patients having cleft palate and other pharyngeal and oral anomalies. Transducer methods of observation of tongue contacts and margin pressures have been developed; multiple-site inputs are re- corded in parallel with speech and swallow indications. By these methods, we have come to re-definition of their disabilities in terms of their deficiencies and compensations of pharyngeal actions, rather than the overt de- ficiency of their palate and related structures. These demonstration methods have also been extended to subjects having neurological im- pairments of the oral and pharyngeal area, and initial descriptions have been made of the particular distortions of feeding, or pharyn- geal airway maintenance and of vocalization in subjects having spastic dyskinesia, athe- tosis, lower motor neuron disorders, and re- gional kinesthetic sensory disorders. Clinical experimental methods have been developed of evaluation of oral sensation and perception, and the development of oral per- ception of form (oral stereognosis) has been calibrated in normal children. Syndromes of disability of perception have now been de- 340 ANNUAL REVIEW OF INTRAMURAL RESEARCH scribed in dysarthric and orally dysphagia sub- jects. Analagously, studies of the chemosensa- tions: smell and taste, have been adapted to children and to neurologically impaired per- sons. First demonstrations have been made of disabilities of these special senses in facially deformed children. In a study on cats concerned with the inter- action of sensory stimuli in association areas of the cerebral cortex, it has been found when peripheral stimuli were employed (light flash, auditory click, and forepaw shock) most cells in anterior lateral gyrus exhibited pre- ferential responsiveness to auditory click stim- ulation. This preferential responsiveness, how- ever, was dependent upon the types of stimuli employed; when cells were activated by elec- trical stimulation of optic and auditory nerves and the dorsal column of the spinal cord, equal responses to the stimuli were most prevalent. A comparison of response characteristics in anterior lateral and anterior middle suprasyl- vian gyri revealed that responses to sensory stimuli were not identical in these two asso- ciation areas, irrespective of the types of stimuli. In a study on the functional organization of the trigeminal brainstem in the cat, it was found that the chief sensory nucleus of the tirgeminal nerve contains cells with different response characteristics. Almost all cells studied to this date had small receptive fields in the oro-facial area and exhibited a short latency (1-5 msec) response to electrical stim- ulation of this field. The modifying influences upon these cells by peripheral and central in- puts can be separated into three groups: (1) cells whose activity was not modified by cor- tical, thalamic, or light flash stimulation; (2) cells whose activity was modified by cortical stimulation and orthodromic thalamic stimu- lation; and (3) cells whose activity was modi- fied by both peripheral and central inputs. Very few cells were activated antidromically by stimulation of the contralateral arcuate nucleus. Histological analysis of electrode track placements indicates a more medial lo- cation of cells whose activity was modified by peripheral or central input, or both. HUMAN GENETICS BRANCH Congenital Malformations One of the major efforts of the Human Ge- netics Branch has been the establishment of a data retrieval system for research on con- genital malformations among all American In- dian neonates born in PHS facilities. To date, we have completed approximately 10,000 birth records of the Indians born during the last year and a half. These data have yielded some unexpected results: Only 5.1 percent of all Indian births are premature compared to 70 percent for white and 9.7 percent for non-whites. Indian stillbirths were 4/1000 births compared to 13.7/1000 U.S. whites and 26.7 1000 for non-whites. Indian neonatal death rates are 13/1000 births compared to 16.7/ 1000 for white and 26.1/1000 for non-whites. Major congenital malformations were 15/1000 births and minor malformations were 34/100 births. Approximately 32/1000 births or 3.2 per- cent Indian children either die in the neo- natal period or have major malformations. When corrected for underreporting, this is about the same value obtained in Japanese infants (4.3 percent). The data suggest that the malformation pattern is intermediate between Caucasian and mongoloid patterns. Study of specific malformations among Bra- zilian Indians indicates a striking absence of malocculusion in primitive groups, i.e., 5 per- cent compared to approximately 50 percent for a comparable group derived from the same tribal stock who have been in contact with civilization for only 20-30 years. A newly described benign oral lesion, focal epithelial hyperplasia, was encountered only among persons of Indian ancestry in North America, Guatemala, El Salvador and Brazil. Studies of cleft lip and palate children and their families indicate that, among the rela- tives of children with oral clefts, 27 percent have a hearing loss of 20 db or more at 500, 1000, 2000 or 4000 cps, compared to only 13 percent among control proband relatives. Other parameters such as visual acuity, minor NATIONAL INSTITUTE OF DENTAL RESEAKCH 341 anomalies (possibly indication of a cleft palate habitus) did not show a significant difference between the two groups. This would indicate that there are familial factors (possibly ge- netic) operating to cause a local (first arch syndrome) disturbance rather than a general disturbance in development as regards to oral clefts. A population of 4200 Indians in North Caro- lina have been exhaustively examined for clefts of lip and palate and 42 cases have been found which makes this frequency the highest known in man. A number of other congenital anomalies appear to be associated with this type of defect in this population which may help define a specific form of the disease. One of the major undertakings of the branch was the preparation of a chapter on malfor- mations of the head and neck for a Handbook of Congenital Malformations for the National Foundation. Genetic Factors in Dental Caries In the past, two major factors (fluoride and refined carbohydrates) have been shown to consistently influence the amount of dental caries in a population. A third consistent re- lationship has been established between the inherited ability to taste phenylthiocarbamide and dental caries, such that tasters for this substance have from 28 to 40 percent lower def rates than nontasters. This finding was verified in a number of studies. Investigation of the thiourea concentration in saliva indi- cates this was not the basis for the caries dif- ference observed. Biochemical Genetic Defects Investigations of biochemical genetic de- fects in families and populations indicated that Indian infants do not metabolize bilirubin in the same manner as Caucasian neonates. They have higher and more prolonged neonatal levels of unconjugated bilirubin, which apparently is normal for children in these ethnic groups. Thus indications for exchange transfusion are different in Indian neonates than they are in Caucasian neonates. Investigation of Gardner's syndrome which consists of multiple odentomas, multiple oste- omas of the jaw, multiple polyps of the intestine, and subcutaneous tumors which are inherited revealed that these patients are re- fractive to the effects of parathyroid hormone. Thus, it is possible that a group of diseases including pseudohypoparathyroidism, basal cell nevus-jaw cysts syndrome, and Gardner's syn- drome, share some defect in parathyroid hor- mone utilization. Other diseases with similar signs and symptoms are being investigated for this biochemical abnormality. Genetic studies of inherited sensory traits in- dicate that the inability to smell potassium cyanide is a polymorphism and a recessively inherited trait. Effects of Radiation Exposure Children in three Utah communities were investigated for possible effects of atomic fall- out. Study of dental hypoplasias, abnormalities of pigmentation, taste and smell ability, did not reveal any detectable difference between the exposed and unexposed communities. Sev- enty of 2,000 children in Washington County (the exposed community) and 25 of 14,000 children in Graham County (the control com- munity) were thought to have nodular growths by at least one examining physician. Studies of a sample of these children are being con- ducted by the University of Utah Medical Cen- ter. Biochemistry of Normal Variation and Cellular Biology Saliva studies have yielded a major finding concerning amylase isoenzjnnes in humans. A polymorphism in man has been defined such that different forms of the enzyme are con- sistently produced in a particular individual and that a generic difference exists in parotid amylase between man and rat. A discrepancy in the classical method for determining secretor status was found and tested on 2,875 individuals, which indicates that approximately 6V2 percent more aberrant secretor individuals can be detected by this new method. 342 ANNUAL REVIEW OF INTRAMURAL RESEARCH SECTION ON CELLULAR BIOLOGY AND CYTOGENETICS The section on cellular biology and cytoge- netics is conducting research into the transla- tion, transcription and replication of genetic information at the cellular level. The transla- tion of genetic information into cellular prod- ucts is being investigated through studies of salivary enzyme polymorphism. A polymorph- ism among humans has been defined in the salivary amylase isoenzymes. That the compo- sition of these isoenzymes is under general genetic control has been demonstrated by showing a generic difference in these isoamy- lases. Studies relating to the transcription of ge- netic information and its regulation within the cell are being conducted, using the lymphocyte- phytohemagglutinin system. In this system, non-dividing lymphocytes are stimulated to enlarge and divide in vitro by treatment with phytohemagglutinin extracted from kidney beans. Studies of RNA metabolism of human lym- phocytes stimulated with phytohemagglutinin indicate that the effect of this extract is to produce a synthesis of non-ribosomal RNA whereas stimulation by specific antigens (streptolysin or tuberculin) results in cell growth with predominant synthesis of riboso- mal RNA. The characteristics of non-riboso- mal RNA are like those of messenger RNA. If this is messenger RNA, it implies that PHA has an important action on the cellular mech- anisms which regulate the synthesis of mes- senger RNA. This system, therefore, may pro- vide a tool for understanding the regulatory mechanisms which govern transcription of ge- netic information within the mammalian cell. Malignant lymphoma cells resemble antigen- stimulated lymphocytes more closely as re- gards RNA metabolism than they do PHA stimulated lymphocytes. This may reflect the fact that the lymphoma cells still have greater control over their messenger RNA synthesis than the PHA stimulated lymphocytes have. This information is important for an under- standing not only of the control of cell growth but, also, of cell differentiation. Future Studies Present plans are to pursue the study of congenital malformations with particular ref- erence to oral and facial clefts, the genetics of various sensory mechanisms affecting taste, small and hearing, the biochemistry of saliva as it relates to PTC taste ability and caries immunity and the regulatory mechanisms of cell growth and differentiation. NATIONAL INSTITUTE OF MENTAL HEALTH INTRODUCTION During the year just ended the NIMH In- tramural program has had another very pro- ductive year, as evidenced by the summary reports prepared by the laboratory and branch chiefs in the pages which follow. Despite chronic crowding in every laboratory, the im- portant questions have been vigorously pur- sued, and there is every reason for pride in the accomplishments of the dedicated members of the Intramural staff. As has been true for several years, this year again saw our scien- tific staff remain intact. No major investiga- tors left, despite numerous invitations from universities at generally much higher salaries. It is a pleasure to mention the appointment of two new Laboratory Chiefs during the year. Dr. Gian C. Salmoiraghi was appointed Acting Chief of the CNRC (soon to be made per- manent), succeeding Dr. Joel Elkes, and Dr. Ichiji Tasaki was appointed Chief of the Lab- oratory of Neurobiology, succeeding Dr. Rob- ert Livingston. Both are old friends and long- term members of the staff. We are grateful to them for undertaking the greater responsibili- ties of chief of a laboratory. We expect our most serious crowding to be relieved by the new space that should be avail- able in fiscal 1968. Construction is ahead of schedule on Building 36, the basic re- search building which we are to share with the NINDB, and if the pace continues it may be completed in November, 1967. We antici- pate completion of Buildings 110 and 111, the two laboratory buildings at the NIH Farm, also in November, 1967, and have just let a contract (June, 1966) for construction of a temporary building to house Dr. Calhoun's rat population studies at the Farm. It should be completed by late fall of 1966. Further delays about site have dogged the progress of the new building for the Child Research Branch, and the prediction now is for completion in May, 1968. A site has finally been approved at the southwest corner of the NIH campus, with an access road for subject families directly into the community, while access to the rest of the campus is to be by another, non-connecting road. It promises to be a good location in most of the important ways. The year past saw a tentative conclusion to the process of planning for use of the new space to be made available by completion of Building 36. Existing plans called for moving to that building the Laboratories of Neuro- physiology, General and Comparative Biochem- istry, Neurochemistry, and Neurobiology, and the Section on Technical Development. To those units we have now added the Sections on Cerebral Metabolism and Biochemistry of the Laboratory of Clinical Science, a new sec- tion or laboratory on neuroanatomy, the office of the Associate Director, and an Associate Di- rector's reserve of some 3,000 square feet. The latter is intended for use by assignment to in- vestigators in amounts needed for specific projects over limited periods of time. Sceptics feel that once in occupancy an investigator can never be uprooted, and that such a reserve will soon be dissipated. I trust that this pes- simistic prediction will turn out to be in error. Building 10, after the above moves, will then house all of the research in Bethesda (except that of the Child Research Branch) which in- volves the study of patients or of other human subjects. Included will be the Adult Psychiatry Branch, the Socio-environmental Studies Lab- oratory, most of the Psychology Laboratory, the clinical half of the Clinical Science Lab- oratory, the Office of the Director of Clinical Investigations, and a proposed new section on psychopharmacology. The latter term is used to refer to research which relates pharmacol- 343 344 ANNUAL REVIEW OF INTRAMURAL RESEARCH ogy-biochemistry to behavior and mental process. We had hoped that the accession of 37,000 square feet of new space would make possible a greater variety of new programs than the above represents. That it did not is testimony to the crowding which now exists and to the too long put off provision of adequate facilities for the able senior investigators on our staff. Their needs must get priority over new pro- grams. During most of the past year, because of the difficulties in scheduling, no member of the staff was on work assignment abroad. The NIMH Board of Scientific Counselors met twice during the year. On November 4 and 5, 1965, two half -days were spent in a review of several of the major clinical research pro- grams of the adult Psychiatry Branch and the Laboratory of Clinical Science. From the for- mer, Dr. Wynne reviewed the Branch program and described his own work on schizophrenia and family interaction; Dr. Pollin reported on his study of schizophrenic twins and siblings; Dr. Shapiro described his work with the families of disturbed adolescents; and Dr. Bun- ney summarized his research on depression. From the Laboratory of Clinical Science, Dr. Durell outlined his study of families of psy- chotic patients in milieu therapy. On March 10 and 11, 1966, the first day was largely de- voted to a review by Dr. Dan Bradley and the members of his staff of the research program of the Section on Physical Chemistry of the Laboratory of Neurochemistry. In addition to Dr. Bradley, the others participating were Drs. DeVoe, Merril, Nash, Neville, Small, and Stone. On the second day the Board and Laboratory Chiefs traveled by bus to the NIH Farm at Poolesville to hear a review of plans for the research in brain and behavior scheduled for the Farm, and to see the terrain and present facilities of the Laboratory Aids Branch. Drs. Calhoun, Stanley, and Eberhart took part. Dr. MacLean was scheduled to participate but a death in his family prevented his attending. Dr. Robert Morison presided in the absence of the Chairman, Dr. S. Bernard Wortis. The Intramural staff were saddened in March to learn of the death of Dr. Heinrich Waelsch, a valued friend and adviser and a member of the Board of Scientific Counselors since 1964. His wisdom, warmth, and unfailing helpfulness will be very much missed. CLINICAL INVESTIGATIONS If one were to write a job description for a research director, there are three qualities which might be considered as among those which are essential: he should be able to recog- nize potential creativity in investigators; he should muster the supports which promote the actualization of this potential; and he should worry — by that I mean that he should have a continuing concern about the program as an organism greater than the sum of its indi- vidual parts. This latter area is obscure and ill-defined, yet I am certain that it exerts an important influence on the work carried out by the individual investigators. One cannot help but be impressed by the range and excellence of the studies described in the following pages. If one measures success by the esteem of professional colleagues, it is worth noting that each year has brought to members of our staff some of the highest awards offered by the various profssional as- sociations to which they belong — distinctions in which each branch and laboratory has shared; for example, of the six Salmon lectures which will have been given between 1961 and 1966, three of the lecturers were invited while they were members of the NIMH staff. It is not an exaggeration to say that our participa- tion is sought in every important national or international conference in those areas in which we are doing research. Staff members are repeatedly offered professorships in out- standing universities. It is doubly gratifying that much of this represents recognition for work conceived and carried out here by inves- tigators who established themselves as impor- tant figures in their respective fields after they came to the National Institute of Mental Health. I cannot look back to the early days of the program without some sense of chagrin, think- ing of how eagerly and persistently we sought to recruit a larger number of established and widely-recognized research authorities, and of how frustrated we felt at our failure to enlist NATIONAL INSTITUTE OF MENTAL HEALTH 345 their interest. It does those highly-respected men no discredit to say that this failure was the best thing that could have happened. It forced us to give more substantial and long- term support to a group of younger investiga- tors who have proved to be so outstandingly productive that they have made us look very good indeed, and much wiser than we, in fact, were. In the face of this undoubted success, is it at all reasonable to find that one does have some worry? I find that I do, and yet the fact that the current system works so well makes me diffident and hesitant about expressing any degree of concern. For whatever the ob- servation may be worth, however, it seems to me that our program specifically, and perhaps medical research generally, appears at times to be in a pressure-cooker. Everything is heated up and speeded up, there is a certain air of fierce determination and relentless for- ward movement which would make a slave- driver feel useless and unnecessary. I cannot say that I identify any bad effects from this; an amazingly large number of the staff want more space and more support so that they may do more work. The work that is done is neither shallow nor incomplete; conclusions are amply documented. When people leave it is often be- cause they are offered even more resources than we can make available to them. I realize that in coming to NIMH in 1952, I had in my mind's eye returning to the cloistered atmos- phere of the Department of Physiology at the University of Chicago twenty years earlier — it did not take long to realize that the dead past is indeed dead. It may exist in the indi- vidual laboratories, but I find myself some- what frustrated at the lack of any sense of leisure in the program as a whole, at the rela- tively infrequent opportunities for contact be- tween members of the various disciplines in which ideas may be casually exchanged, at the fact that it is necessary to make an appoint- ment days in advance to see almost anyone. I shall have to offer it as only an intuitive guess that if we could find a mechanism which would result in some slowing down and in pro- viding occasions for increased casual but in- terested contact between the several labora- tories and branches, our work would be no less thorough and just possibly more rewarding. I realize that many things seem to work against this; the sheer size of the program for one; the factory-like architecture of the Clinical Center with its lack of common rooms and its emphasis on what is utilitarian for an- other; and the understandable desire to par- ticipate in the increasingly large number of national and international meetings for yet another. But I believe that an effort to bring about some such change is worth making. In my opinion, the growth in scientific stat- ure of our relatively young staff is a bit of history which merits serious thought. The suc- cess the program has enjoyed is the result of the fact that these men and women were given career-type support and a level of research resources which they would not have been granted so soon at a university or by us if we had succeeded in recruiting more senior men. They were freed from the burden of developing a series of individual project applications which would have had to earn the approval of a variegated review group. Although there were some critical pressures on them from other in- vestigators (for the most part silent) and al- though their promotions but not their levels of support depended on some evidence of pro- ductivity, by far the major pressures were self-generated and led them to develop increas- ingly more significant and powerful studies. If our experience can be generalized, grant- ing agencies might seriously consider the ad- visability of decreasing project-type and in- creasing program-type support for young men and reversing that procedure for established figures. This, in effect, is what happens in the intramural program; whenever a branch or laboratory chief wishes to change his research without discontinuing his current studies, his request for new resources is by necessity rig- orously reviewed since he is always competing with others for very limited reserve funds. The past year has brought a major change in direction of the program of the Clinical Neuro- pharmacology Research Center, which had been established in collaboration with Saint Elizabeths Hospital in 1957. As originally con- ceived by its first Chief, Dr. Joel Elkes, and 346 ANNUAL KEVIEW OF INTRAMURAL RESEARCH as its name implies, its aim was to conduct both clinical and laboratory studies in a large mental hospital setting — studies which, it was hoped, would prove mutually enriching to the clinician concerned primarily with his pa- tient's behavior and to the laboratory scientist concerned with behavior at the segmental or even the cellular level. Several years ago Saint Elizabeths was granted direct research funds by the Congress for the establishment of a Clinical and Behavioral Studies Center which, to a degree, complemented the work of the Clinical Neuropharmacology Research Center. After considerable thought, it was decided that the total collaborative research program could best be developed if Saint Elizabeths Hospital were to assume all responsibility for clinical care, and if the energies of the Clinical Neuro- pharmacology Research Center staff were to be directed primarily toward the study of funda- mental neurophysiological and neurochemical processes involved in behavior. Dr. C. G. Sal- moiraghi, Chief of the Clinical Neurophar- macology Research Center, was appointed Di- rector of Research for Saint Elizabeths Hos- pital by Dr. Cameron; Dr. Francis N. Waldrop, Director of Training and also Director of the Clinical and Behavioral Studies Center of Saint Elizabeths Hospital was appointed Spec- ial Assistant for Research and Training to the Clinical Neuropharmacology Research Center, NIMH, by Dr. Yolles. This new plan places re- sponsibility where there is the greatest con- centration of competence, and we look forward to an even more productive and cooperative relationship than we have enjoyed in the past. There has been another development in NIMH indirectly related to the intramural pro- gram. This is the establishment of a Field Studies Program Branch under the' direction of Dr. James Osberg, formerly Chief of the Mental Health Study Center. The Study Cen- ter will be transferred to the new program, which will be concerned with operational re- search in the increasingly broad areas of responsibility with which the Institute has been charged. This new development is grati- fying on two counts: it recognizes once more the importance to the Institute of maintain- ing a strong program of basic research free from the many practical problems pressing urgently for investigation and solution. At the same time, it provides another research pro- gram within the Institute with which some of our investigators may collaborate from time to time. The possibility that staff might wish to move in either direction from one program to the other on occasion increases the richness of our research resources. ADDICTION RESEARCH CENTER General The past year has been an especially pro- ductive one at the Addiction Research Center, with real progress being made in several im- portant areas of drug abuse and drug de- pendence. Important advances have been made in the understanding of some of the basic pathological processes that occur in narcotic addiction, as well as advances in the mode of treatment of these defects. Additional evidence has been acquired indicating that signs of ab- stinence in the rat can be conditioned. Further, strong supporting evidence has been obtained concerning the existence of protracted absti- nence. Protracted abstinence can at least be partially characterized as an appetitive state in which there is an increased consumption of food, water, and narcotics. Preliminary studies have indicated that there may also be bio- chemical alterations in the brain. The brains of addict rats that have been withdrawn for six months have larger quantities of brain nore- pinephrine than comparable control animals. During abstinence in man the respiratory cen- ter becomes hypersensitive to carbon dioxide, and this observation may provide a precise and sensitive measure of a basic pathophysio- logical alteration produced by addiction. Studies showing that the effects of nar- cotics can be effectively blocked by chronic administration of the narcotic antagonist cy- clazocine have been concluded. Cyclazocine is currently undergoing clinical trial in the treat- ment of drug addiction in New York City, and the preliminary observations seem encourag- ing. They have confirmed the basic pharma- cologic observations made at the Addiction Research Center, namely, that the subjective NATIONAL INSTITUTE OF MENTAL HEALTH 347 and dependence producing effects of narcotics are diminished, and in most instances com- pletely antagonized when cyclazocine is taken daily. Studies of derivatives from cannabis are continuing. Several isomers of tetrahydrocan- nabinol isolated and furnished by Prof. Dr. F. Korte of the Organic Chemical Institute, Uni- versity of Bonn, Germany, have been studied. Studies on the measurement of the relative strength and duration of sedative-hypnotic agents have also been continued. It has been shown, for example, that both meprobamate and chlordiazepoxide, like pentobarbital, can prolong the duration and increase the fre- quency of postrotational nystagmus. It is hoped that this method will permit the quanti- tative study of these types of agents. Studies on the treatment of addiction to sed- ative-hypnotics have revealed that chlorproma- zine does not effectively antagonize the very serious abstinence sign, convulsions. A number of drug state and personality scales have been developed, using the Addic- tion Research Center Inventory, which are proving to be of great value. Since many of these scales were developed using prisoner subjects, an extensive effort has been made to standardize this inventory on other popula- tions. Thus far, studies have been made of over 100 mentally ill subjects (tested by R. Long and P. Philip, St. Peters State Hospital, St. Peters, Minn.), normal subjects (college students tested by S. Grupp, Illinois State Uni- versity, Normal, 111., and W. Johnson of Morn- ingside College, Sioux City, Iowa), alcoholics (G. Fuller, Willmar State Hospital, Willmar, Minn.), and criminals (J. Panton, Central Prison, Raleigh, N.C.). In this regard, an al- cohol withdrawal scale has been developed. In state dependent studies, the observation of Overton that rats make differential responses on the basis of prior injection of pentobarbital has been confirmed; however, under morphine there is almost complete response fixation. Fundamental to our understanding of prob- lems of addiction is the mode of action of addicting drugs, for drugs produce their ac- tions by interacting with other molecules of the body. It has recently been demonstrated that narcotics alter phospholipid metabolism. These results may be of great importance since there are indications that neurohumors may have their effects upon lipid membranes of ex- citable tissues. Further, it has been demon- strated that phenobarbital can alter phos- pholipid and microsomal membrane metabo- lism in vitro. Studies of the impact of addictive processes on the social functioning of the individual is one of our most important concerns, and yet one of the areas in which it is most difficult to conduct well-controlled experiments. Impor- tant advances in this problem have been made using follow-up studies of addict pa- tients released from the Public Health Service Hospital. One of these, the Kentucky follow-up study, is nearing completion and a most sig- nificant and important observation has been made — namely, the first documentation of the fact that addiction is followed by the deteri- oration in major social roles. Thus employment is less stable, most marriages end in divorce or separation and subjects become more deeply involved in criminal behavior. Administrative Surgeon D. R. Jasinski came on duty as medical officer on 1 July 1965. Surgeon C. W. Gorodetzky, medical officer, was assigned to the Department of Pharmacology, University of Kentucky, for out-of-Service training as of 1 September 1965. It is hoped that this will be the beginning of a joint training effort by the University of Kentucky College of Medi- cine and the Addiction Research Center which will, hopefully, lead to the development of both clinical and basic investigators who have a high level of interest in problems of di-ug abuse and who will develop expertise in this area. Surgeon D. C. Kay, a Mental Health Career Development officer, has spent two-fifths of his time during fiscal year 1966 in the Addic- tion Research Center and will be assigned to the ARC on a full-time basis as of 1 July 1966. Dr. C. M. Redman, chemist, joined the staff of the ARC as a staff fellow on 1 October 1965. Dr. W. M. Bates transferred from the ARC to the PHS Hospital, Fort Worth, Tex., where he accepted a position as director of research. 348 ANNUAL REVIEW OF INTRAMURAL RESEARCH For all intents and purposes the new Basic Research Laboratory is complete. It was ap- proved for beneficial occupancy 21 September 1965 and is now almost completely occupied. An occupancy inspection of the building has been made and, although there are a number of minor items to be corrected, it is hoped the contractor will have the building in shape for final inspection by May. Studies on Addictive Properties of New Analge- sics and Patho-Physiological Processes Associ- ated with Physical Dependence Studies of the ability of chronic adminis- tration of the narcotic antagonist cyclazocine to block the euphorogenic, lethal and physical dependence producing properties of narcotics have been completed. It has been clearly demonstrated that cyclazocine can be admin- istered in sufficient doses (4 to 8 mg/day) to protect subjects from narcotics without the production of undesirable side effects. Two clinical trials of cyclazocine for the treatment of narcotic addicts are currently being under- taken in New York City. At the present time the results of these clinical trials are definitely encouraging. Naloxone (N-allyl-noroxymorphone) has been studied for its abuse potentiality. This in- teresting antagonist appears to be entirely de- void of agonistic action. Thus, unlike nalorphine and cyclazocine, naloxone does not produce dysphoria, ataxia, uncontrolled thoughts or other subjective effects, nor does it produce pupillary constriction or respiratory depres- sion. On the other hand, studies of this agent's ability to precipitate abstinence indicate that it is probably at least six times more potent than nalorphine as an antagonist. It has been demonstrated that in patients physically dependent on 240 mg of morphine per day and who are mildly to moderately ab- stinent, the respiratory center becomes hyper- sensitive to carbon dioxide. Thus, the slope of the alveolar carbon dioxide-minute volume stimulus response curve is almost twice as steep in the abstinent physically-dependent subjects as it is in the same subjects before they were made dependent. It thus appears that the ab- stinence sign hyperpnea can be explained on the basis of increased sensitivity of the respi- ratory center. The finding that pentobarbital prolongs postrotational nystagmus has been confirmed in studies, using electro-oculography, and that this prolongation is proportional to the dose of pentobarbital. Further, it has been found that the frequency of postrotational nystagmus is also increased by pentobarbital and that this increase is also proportional to the dose administered. Studies concerned with the mechanism of prolongation of post-rotational nystagmus indicate that this is mediated ini part by decreasing the ability of subjects toi attend. Studies on the addiction liability of alpha d-2-acetoxy-l,2-diphenyl-3-methyl-4-pyrrolidino butane hydrochloride (Lilly 31518, ARC I-C-27) have been completed, and indicate that I-C-27 is a typically opiate-like drug that is between one-half and one-fifth as po- tent as morphine. The effects of intramuscularly administered codeine (90, 180 and 360 mg) and morphine (7.5, 15 and 30 mg) have been compared on pupillary diameter as well as subjective effects as assessed by the single dose questionnaires. By all measures, codeine appears to be approx- imately one-tenth to one-fifteenth as potent as morphine. Of fundamental importance is the observation that the effects of codeine increase up to the dose level of 360 mg. This observation confirms Houde's findings, but is in: disagreement with previous observations which have indicated that the effects of co- deine reach a plateau at a much lower dose level. Codeine may have a slightly more rapid onset of action and shorter duration of action than morphine, but these data are not clear- cut. Dihydrocodeinone - O - (carboxym ethyl) - oxime dihydrate (codoxime, ARC I-A-41, WSM-7051), an agent that is being consid- ered for use as an antitussive, has been as- sessed for abuse potentiality and compared with hydrocodone. The results indicate that both codoxime and hydrocodone possess typ- ically morphine-like properties. Codoxime is one-seventh to one-fifteenth as potent as mor- phine; whereas, hydrocodone is approximately NATIONAL INSTITUTE OF MENTAL HEALTH 349 equipotent. Both drug's have time-action courses that are similar to that of morphine. The effects of morphine (12 and 24 mg/70 kg) and pentobaibital (50, 150, 250, and 300 mg/70 kg) were compared, using single dose questionnaires and a recently designed ques- tionnaire containing ten items from the mor- phine-benzedrine group scale and ten items from the pentobarbital-alcohol-chlorpromazine scale. The scores obtained with 150 mg of pentobarbital were equal to those obtained by 24 mg of morphine; however, scores were not increased by larger doses of pentobarbital. Responses on this scale to morphine were, how- ever, dose-related. On the other hand, pento- barbital produced increasing responses on the chlorpromazine - alcohol - pentobarbital scale with increasing doses; whereas, responses under the morphine condition were only slightly greater than placebo responses. Acute and Chronic Intoxication with Drugs Other than Analgesics, Barbiturates and Alcohol Studies in the Marihuana Group We are now collaborating with two groups in the study of effects of compounds isolated from marihuana or of synthetic materials chemically resembling compounds found in marihuana. The two laboratories are the Or- ganic Chemical Institute of the University of Bonn, Germany (Dr. F. Korte), and the Psy- chopharmacology Service Center. We have pre- viously shown that tetrahydrocannabinol iso- lated from hashish is an active material which causes subjective symptoms recognized as being similar to those of marihuana by ex- perienced marihuana smokers. The activity of crude extracts of hashish or marihuana cor- relates only with the tetrahydrocannabinol content. The exact chemical structure of tetra- hydrocannabinol has, up until recently, not been known, but it seems likely that the exact structure of natural tetrahydrocannabinol has now been determined and supplies of syn- thetic materials will soon be available. Studies on two synthetic samples of tetrahydrocannab- inol showed that both are relatively inert, as was a sample of synthetic cannabidiol. Nat- ural tetrahydrocannabinol is about 2.5 times as active when smoked as when taken orally. In sufficient dose (200 to 250 mcg/kg smoked), natural tetrahydrocannabinol is a psychotomimetic drug. Smoking can be used as an assay method in man. It now seems quite likely that the exact chemical structure of natural tetrahydro- cannabinol has been determined and that a chemist will soon synthesize the active ma- terials in quantity. If this is achieved a co- operative detailed study of the general phar- macology, neuropharmacology and psycho- pharmacology of tetrahydrocannabinol will be- come possible. Since marihuana is one of the most widely used intoxicants of the world, and since the United States has a considerable problem of abuse of marihuana, the importance of such studies is evident. The role of the Ad- diction Research Center in these investiga- tions might include a study of chronic intoxication with the natural tetrahydrocan- nabinol, including determinations of the degree of tolerance developed, a more complete de- lineation of the psychopharmacological effects in man, and studies of possible potentiating agents other than tetrahydrocannabinol per se. Clinical Studies of Intoxication with Alcohol, Barbiturates and Related Drugs During the current year studies on the sub- jective changes associated with alcohol with- drawal were completed. Scales for alcohol withdrawal have been developed with the Addiction Research Center Inventory (AR- CI). These scales may be useful for following the course of withdrawal, evaluating the efficacy of treatment of withdrawal, and, fin- ally, from a methodological point of view, to determine when personality tests may be administered so that the results will not be contaminated by withdrawal. Severity of with- drawal symptoms as measured by the with- drawal scale is correlated with subjective need for alcohol or barbiturates, course of treat- ment, occurrence of confusion and halluci- nations during the last alcoholic spree, and time since the last alcoholic spree, but not with the use of specific alcoholic beverages such as beer, wine or whiskey during the last 350 ANNUAL REVIEW OF INTRAMURAL RESEARCH spree, length of alcoholism and amount drunk, or membership in AA. A psychopathic scale developed from the ARCI highly differentiates criminals and op- iate addicts from normal and mentally ill subjects, and is judged to be better than other available tests for this purpose. Alcoholics ob- tained intermediate scores on this scale. The scale is significantly correlated with several measures of psychopathic deviation on the MMPI and the California Psychological Inven- tory. Two editions of the 16 Personality Factor Questionnaire were studied to determine their equivalence. Certain scales on these versions of the questionnaire were found to be non- equivalent. One scale from the 1950 edition indicated that addicts are high in ego strength, whereas the equivalent scale on the 1956 edi- tion indicated that addicts are low in ego strength. This study clearly indicates that cau- tion should be exercised in generalizing about the true validity of scales that have face validity. Biochemistry of Addiction The effects of chronic morphine treatment, as well as primary and protracted abstinence from morphine, on urinary epinephrine, no- repinephrine and dopamine, food and water intake, urinary output, body weight, and tem- perature were studied in the rat. In addition, the weight and catecholamine levels of brain and adrenal glands were determined 143 days after the last injection of morphine. During addiction the mean excretion of urinary cate- cholamines was significantly higher in the ad- dicted animals than in the control animals. Following abrupt withdrawal of morphine, epinephrine excretion increased and reached the maximum within 48 hours, a time at which dopamine values were also at their peak. No- repinephrine excretion became pronounced on the third day of abstinence. By the end of two weeks, urinary catecholamine values were nor- mal or subnormal. Epinephrine and dopamine excretion in the abstinent rats did not vary significantly from control values throughout protracted abstinence; whereas, urinary nore- pinephrine levels fell below control levels and remained subnormal. During protracted abstinence rats ate and drank more than com- parable control groups. These and other physi- ological abnormalities were clearly evident during the first half of the protracted ab- stinence; however, all measures were normal at the time of sacrifice. No significant differ- ence was observed between brain and adrenal weights and brain epinephrine levels of the control and addict rats. However, the brain concentrations of norepinephrine were sig- nificantly higher and the concentrations of brain dopamine were significantly lower in ad- dict rats than in control rats. Adrenal epine- phrine and norepinephrine tended to increase but these changes were not statistically sig- nificant. Neurophysiology and Neuropharmacology of Chronic Intoxication with Barbiturates and Re- lated Drugs The Effects of Chlor promazine on Barbiturate Withdrawal Seizures Dogs chronically intoxicated with and ad- dicted to sodium barbital were given chlor- promazine following abrupt withdrawal of barbiturates. Both the chlorpromazine-treated and control dogs developed abstinence convul- sions, as well as other signs of barbiturate withdrawal. It is concluded that chlorproma- zine is not a useful therapeutic adjunct in the treatment of the serious sign of barbiturate withdrawal, major seizures. Barbiturate Withdraival in Adrenalectomized and Hypophysectomized Rats Wistar rats were adrenalectomized and maintained on 1 mg daily doses of deoxy- corticosterone during progressive intoxication with sodium barbital. Sprague-Dawley hypo- physectomized rats that were maintained on special rat chow were also chronically intox- icated with sodium barbital. Following abrupt withdrawal of sodium barbital the control, adrenalectomized, and hypophysectomized rats developed abstinence convulsions. These exper- iments suggest that the pituitary and adrenal glands probably do not play an important role NATIONAL INSTITUTE OF MENTAL HEALTH 351 in the mechanism underlying barbiturate con- vulsions in the rat. Free Choice Bettoeen Tap Water and 5% Alco- hol in Rats During Barbital Withdraival After drinking increasingly concentrated solutions of sodium barbital, a group of rats was given access to both tap water and 5% alcohol during withdrawal. There was no pre- liminary training or conditioning. During withdrawal there was no indication that a preference for alcohol developed. Perhaps the significant reduction in fluid consumption that developed in the rats during the first several days of barbiturate withdrawal interfered with the development for the preference for alcohol that might have been the consequence of an induced need. The Effect of Sodium Barbital on Electrocon- vulsive Threshold Elevation in Cats Daily induced electroconvulsions in cats re- sulted in a gradual tolerant-like elevation of the electrical threshold for seizures. The hy- pothesis that an anticonvulsant drug, such as barbital, might interfere with the develop- ment of electroconvulsive tolerance was tested. Fixed daily doses of sodium barbital were ad- ministered to a group of cats during a three- week period that were also subjected to daily electroconvulsions. The sodium barbital aug- mented rather than decreased the elevation of electroconvulsive threshold when compared to control cats. The increase was not entirely due to a direct threshold elevating effect that is a consequence of the anticonvulsant effects of sodium barbital. Further studies of this phe- nomenon will be undertaken. Psychological Studies on Addiction During the year the Addiction Research Cen- ter Inventory (ARCI) was administered to newly admitted addicts at the Public Health Service Hospital at Lexington, with the coop- eration of Mr. M. J. Meketon, with the object of defining subjective changes associated with opiate withdrawal, and to study the differences between newly admitted addicts and subjects who are accepted for research studies. Pre- liminary analysis of these data suggests that newly admitted subjects are more like the nonaddict clinical groups than are research addicts on the general drug effects scale, which may suggest that addicts on the research ward answer questions within a more restricted frame of reference, or, alternatively, that ad- dicts in general deny experiences which suggest change when they are not on drugs. The study comparing physician addicts and general hospital population addicts with a representative group of practicing physicians collected by Drs. W. G. Dahlstrom and R. S. Spain, University of North Carolina, using the MMPI has been completed. Further, a com- parison of a new group of addict physicians collected by Mr. Meketon have confirmed the observations made on the initial group of phy- sician addicts. As indicated previously, the representative physicians scored within the normal range on all scales, while, strikingly, the addict physicians produced significant ele- vations on all clinical scales except for hypo- mania and social introversion. Except for the masculine-feminine scale the mean scores of the general hospital addicts were much more ab- normal than these of addict physicians. In addition to psychopathy, neuroticism, depres- sion, and social maladjustment being shown by the addict physicians, the masculine-feminine scale indicated a considerable degree of sexual instability and deviation without showing homosexuality. Judging by the progressively increasing degree of personality abnormalities from the representative physician to the addict physician, and from the normal physician to the general hospitalized addict, abnormal per- sonality characteristics become increasingly in- fluential in the onset of addiction as legal and quasi-legal availability of narcotics becomes less. Studies on state dependency behavior of the rat in an escape from shock situation using two safe goal boxes have been initiated. These studies have been used to test the Overton hy- pothesis of state dependency, which may be stated as follows: A response learned while an animal is under the influence of a specific drug will occur more frequently when the animal is tested under the influence of that particular 352 ANNUAL KEVIEW OF INTRAMURAL RESEARCH drug than under any other condition. Results have indicated that rats trained under pento- barbital make differential response when again pentobarbital, confirming the observations of Overton. However, animals trained under the influence of morphine show almost complete stereotopy or response fixation under both mor- phine and saline conditions when subsequently tested. If such response fixation occurs in man, adaptability would be reduced by morphine. In addition, since the effects of morphine ap- parently generalize to the no-drug condition, readjustment would be more difficult in the drug-free state following the use of morphine. The Mode of Action of Central Nervous System Depressants The effects of graded doses of nalorphine, cy- clazocine, morphine and naloxone were stud- ied on the flexer reflex of the chronic spinal dog. It was first observed that naloxone did not depress the flexor reflex even in subconvulsant doses in the dog; whereas, nalorphine produced partial depression of the flexor reflex. The de- pression produced by nalorphine was depen- dent upon the strength of the stimulus used to evoke the flexor reflex (0.8 kg stimulus pro- duced an 84% depression, 1.6 kg stimulus, 75% depression, and 3.2 kg, 61% depression). On the other hand, morphine and cyclazocine pro- duced almost complete depression of the flexor reflex for all strengths of stimulation. These results can at least be interpreted as indicat- ing the narcotic antagonists can differ one from the other with regard to their intrinsic activity. Thus, naloxone has zero intrinsic ac- tivity. Nalorphine has less intrinsic activity than either cyclazocine or morphine. Further, it has been shown that the apparent intrinsic activity of the narcotic antagonists depend upon the strength of the nociceptive stimulus that evokes the suppressed reflex. Studies are being continued in an attempt to understand neurohumoral mechanisms that are responsible for the flexor reflex in the chronic spinal dog, as well as mediating the morphine abstinence syndrome in this prepa- ration. It has been observed that gamma hydroxy- butyric acid will suppress the running move- ments and hyperexcitability of the flexor and crossed extensor reflexes in the abstinent de- pendent spinal dog. However, since gamma hydroxybutyric acid will also suppress the flexor and crossed extensor reflexes in the non- dependent nonabstinent chronic spinal dog, it is felt that this represents a more general de- pressant action rather than a selective depres- sion of facilitatory influences caused by absti- nence. Conditioning Factors in Opiate Addiction and Habituation (relapse) The methods employed in the studies herein reported have been described in detail in previ- ous annual reports for fiscal years 1964 and 1965. However, the designations of the various subgroups of rats in the replicate study (see project report for 1965) were changed to the following: ADT (morphine-addicted, etonitazene-trained) CDT (saline-injected, etonitazine-trained) AUT (morphine-addicted, untrained) CUT (saline-injected, untrained) During the last year a total of sevel relapse tests have been completed, extending to 142 days after the termination of all injections (morphine in the cases of ADT and AUT and saline in the case of CDT and CUT). In addi- tion, extensive statistical analyses of the data have been made obtained in relapse tests I (9 days abstinent), II (23 days abstinent), III (44 days abstinent), and IV-VII combined (58-142 days abstinent), with regard to (a) "wet dog" shake frequencies in home cage and in linear maze, and (b) free choice drinking of water or etonitazene from 8 p.m. to 8 a.m. in the linear maze. Analysis of variance of "wet dog" shakes indi- cated that previous addiction, place of absti- nence, and an interaction (previous addiction x place of abstinence) all were significant on each of the three relapse tests. These results clearly indicate the abstinence phenomena had j become classically conditioned to the environ- I ment (linear maze or home cage) in which the : appropriate subgroup (ADT or AUT) had been i abstinent from morphine each night during the six week morphine injection prior to per- manent morphine withdrawal. However, such NATIONAL INSTITUTE OF MENTAL HEALTH 353 evidence of classical conditioning was not ob- tained from the analysis of data for relapse tests IV-VII, indicating that extinction of this conditioned response had occurred between the 44th and 58th day of abstinence. Analysis of the free choice data confirmed previous impressions, namely, that postaddict subgroups (ADT and AUT) drank signifi- cantly more etonitazene solution than their re- spective nonaddict controls (CDT and CUT) on every relapse test through 140 days after termination of injection, excepting only the first relapse test. There was, however, no sig- nificant difference between the trained and untrained postaddict subgroups for any of the relapse tests. From the data obtained in both the original and present studies, it is now clear that, in the rat, previous physical dependence on morphine per se is an important factor in generating a disposition to relapse long after morphine with- drawal and the subsidence of the primary morphine abstinence syndrome. For reasons mentioned in the individual project reports for 1964, it is considered unlikely that such a dis- position is due to residual tolerance to opiates, rather the nonaversiveness of etonitazene for postaddict rats relative to nonaddict rats may be due to persistence of homeostatic imbalance, as manifested by the long-enduring abstinence syndrome of which total wet dog counts (un- conditioned plus conditioned) may be an indi- cator in the present study. These findings and conclusions emphasize the need for intensive research on the possibility that secondary ab- stinence also occurs in man. Confirmation of the conditionability (clas- sical) of at least one morphine abstinence phenomenon (wet dog) in the rat supports the hypothesis that in man relapse may be due in part to the recurrence of the abstinence phe- nomena (as conditioned responses) long after morphine withdrawal. Theoretically, prior re- inforcement of opioid-acquisitory behavior by suppression of morphine abstinence phenom- ena during periods of active addiction to mor- phine should play a role in generating a dis- position to relapse, but the influence of this factor, as well as of conditioning of wet dogs on etonitazene drinking in relapse tests, could not be demonstrated by the techniques em- ployed in both the original and present studies in the rate. Currently, studies are underway to deter- mine if hustling will increase the probability of relapse. In addition, it is hoped to initiate a study of the effects of lesions in the limbic system (e.g., bilateral cingulumotomy) on both conditioned and unconditioned abstinence phe- nomena and on relapse tendencies of morphine addicted rats, provided that appropriate means are found for obtaining additional equipment and the services of an additional technician. Experimental Studies with Human Subjects and Studies on Learning A series of primary and supplemental experi- ments have been concluded which dealt with the effects of single doses of morphine upon conditioned and unconditioned electrodermal responses. These experiments have studied the effects of morphine (16 mg/70 kg) upon (a) the acquisition of a classically conditioned electrodermal response, (b) the retention of a previously established differentially conditioned electrodermal response, and (c) evocation of conditioned anticipatory electrodermal re- sponses. One of the major purposes of these ex- periments was to seek evidence in support of the hypothesis that the morphine produced clinical analgesia is related causally to the reduction by morphine of conditioned auto- nomic responses. The overall results of the experiments yielded little, if any, support for this hypothesis. With regard to the acquisition of conditioned electrodermal responses, we observed a mod- erate, though statistically significant (P<0.05), reduction in the level of conditioning compared with placebo. The reduction was not greater than that produced by 200 mg/70 kg of pento- barbital, however. Thus, though there was a reduction in the acquisition of conditioning, the size of the reduction in absolute terms was not remarkable and not likely to be of signifi- cance for the mechanism of morphine pro- duced analgesia. In the case of conditioned anticipatory elec- trodermal responses, in which subjects were able to anticipate a noxious stimulus, morphine 354 ANNUAL REVIEW OF INTRAMURAL RESEARCH did not produce any reduction in the frequency or magnitude of responses. Probably most pertinent for a test of our hypothesis was the study of the effect of mor- phine upon electrodermal responses which had been established before the morphine effect was tested. For this test a differential, or discrimi- native, conditioning procedure was used in which one particular tone was reinforced while another tone was not. The test for the effect of morphine was made only after the discrimina- tive response had been established during sev- eral weeks of training. In this situation morphine did not reduce the frequency or magnitude of conditioned responses. Mor- phine did, however, produce an increase in the frequency and magnitude of the partially adapted response to the nonreinforced tone. A new laboratory has been established in the Basic Research Laboratory to study aspects of the neuropharmacology of learning. The ini- tial aim of this study will be to study condi- tioned responses using direct cerebral stimu- lation for both the conditioned and uncondi- tioned stimuli. It is intended by using this model of learning to study the facilitatory and inhibitory roles of stimulating reinforcing sys- tems; the effects of single, multiple and tempo- rally and spatially summed stimuli; and the role of neurohumors in the conditioned re- sponse. It is further hoped that this -method can be employed for studying the role of nucleic acids and proteins in memory. Social Science Section Analysis of data from the Kentucky follow- up study has been completed and a final report prepared. The last annual report mentioned the high death rate and the relatively high rate of abstinence among subjects. Other findings in- clude clear signs of personality problems in subjects prior to the onset of addiction in poor employment records, poor military adjustment, and alcoholism. Although many subjects felt that their addiction was a consequence of med- ical treatment or treatment of alcoholism, more than half of the men and one-sixth of the women gave pleasure seeking as a reason. After addiction began, most subjects became in- volved in an addict subculture. The extent of this involvement was determined in part by the reason for becoming addicted, by age on onset, by decade in which addiction began, by sex, and by urban-rural status. The major de- terminant of involvement was the source of the narcotics. Those subjects who had a stable (medical) source of narcotics showed the least involvement. Addiction was followed by deterioration in major social roles. Employment became less stable, most marriages ended in divorce or sep- aration, and subjects began to acquire crimi- nal records or committed more crimes than before addiction. While characteristics of subjects as individ- uals were associated with later relapse and abstinence to a statistically significant degree, these associations do not seem to be efficient predictors of post-hospital drug status. The abstinence in the sample and the degree of prevalence in addiction in Kentucky seems to be a consequence of increasing difficulty in ob- taining narcotics. The unavailability of nar- cotics in turn seemed to be due to severl fac- tors, including the improvement in medical practice, more vigorous law enforcement ef- forts, growing public disapproval of drug use, and probably shipping restrictions that oc- curred during World War II. Subjects varied as to the means to which they would go to obtain narcotics. Thus, some refused to steal narcotics, to forge prescriptions, or to try to "make" physicians. The vast majority of abstinent ad- dicts did not take a step that was available to all, namely, moving to places such as Chi- cago or New York where drugs were available. Computer programs have been written and tested for analysis of hospital admission data (Lexington and Fort Worth) for the 1935- 1965 admissions. A study of the validity and reliability of field data obtained in the Puerto Rico study has been completed. Interview re- sponses have been checked against prior hospi- tal records, FBI arrest records, and the results of urine testing with the finding that addicts can and will recout their prior criminal hisory and current drug use with considerable accu- racy. Several small scale studies based on inter- NATIONAL INSTITUTE OF MENTAL HEALTH 355 views with Lexington patients were initiated during the year. Biochemical Pharmacology It was previously reported that morphine and nalorphine stimulated the incorporation of P 32 into phosphatidylinositol, phosphatide acid, lysophosphatidylinositol, phosphatidylse- rine, phosphatidylethanolamine and diphos- phoinositide in cerebral cortex slices while inhibiting the incorporation of P 32 into phos- phatidylcholine. These studies have been ex- tended into morphine-tolerant, abstinent, and dependent guinea pigs. No change in phospho- lipid metabolism was observed in cortical slices of chronically morphinized animals when in- cubated alone. However, the effects of mor- phine in stimulating and inhibiting phospho- lipid metabolism was less than that previously observed in nontolerant animals. Although variable effects have been observed in mor- phine-dependent guinea pigs during with- drawal, marked stimulation of the increased phosphate into phosphatidic acid and triphos- phoinositide have been observed. These findings provide additional support for the hypothesis that the narcotics may produce part of their effects in altering metabolism of phospholipids. Studies of the metabolism of tritium-labeled cyclazocine in nontolerant and tolerant dogs have been nearly completed. The egress of cyclazocine seems to be more rapid than egress of morphine from the brain of the non- tolerant dog. The effects of sodium phenobarbital and morphine on phospholipid metabolism in mi- crosomal membranes in vitro have been stud- ed. Both of these compounds inhibit the me- tabolism of the major lipid component phos- phatidylcholine. The kinetics of the inhibition of these two drugs on this compound differ. The metabolism of other phospholipid compo- nents has not been affected. The antibiotic puromycin was used to cause the release of incomplete proteins from their site of synthe- sis on ribosomes attached to membranes. The released peptides were found to pass through the membranes. The metabolism of components of the membrane during the transfer of newly synthesized proteins is being studied. LABORATORY OF SOCIO-ENVIRONMENTAL STUDIES The work of this Laboratory is focused on the study of social influences upon personality and behavior. It is convenient to group our current investigations into four content areas: the social context of personality development, the social psychological correlates of occupa- tion, social factors in mental disorder, and the methodology of social research. Studies of the Social Context of Personality Development This is the domain in which the largest por- tion of the Laboratory's work is concentrated. Social Class, Occupation, and Parental Values The first completed analysis to come from Dr. Leonard Pearlin's study of parent-child relationships in Turin, Italy, demonstrates the value of cress-cultural, comparative research. Pearlin's investigation was designed as a repli- cation and extension of a study of social class and parent-child relationships we had previ- ously conducted in Washington, D.C. The earlier study had shown a distinct difference in emphasis in middle- and working-glass par- ents' values for their children: middle-class parents value self-direction more highly than do working-class parents; working-class par- ents emphasize, instead, conformity to exter- nal proscription. The present analysis is ad- dressed to two questions: (1) Is social class related to parental values in Italy in the same way as in the United States? (2) If so, Ito what extent is this due to the characteristically different occupational experiences of middle- and working-class parents? Self-di recti on seems more possible and more necessary in middle-class occupations; working-class occu- pations allow much less room for, in fact might penalize, anything other than obedience to rules and directives set down by others. The cross-national comparison shows that Italian parental values are more adult-centered, American more child-centered. Despite this cultural difference, the relationship of social class to parental values is much the same in Italy as in the United States. There is some- 356 ANNUAL REVIEW OF INTRAMURAL RESEARCH thing intrinsic to social stratification that has strikingly similar effects in the two countries. To determine the degree to which differ- ences in the occupational circumstances of the middle and the working class might underlie class differences in parental values, fathers were questioned about three aspects of occupa- tional life which together define the limits of and demands for the exercise of self-direction at work. These are the closeness of supervision to which a man is subjected, the substance of the work he does — that is, whether he works primarily with things, with ideas, or with people — and the degree to which his work re- quires self-reliance. Each of the three is inde- pendently related to fathers' values for their children: fathers who are closely supervised, who work primarily with things, whose jobs do not require much self-reliance, are more likely to value the child's conforming to adult direction, fathers who are not closely super- vised, who work primarily with ideas or with people, whose jobs do require a large measure of self-reliance are more likely to value the child's self-direction. These three aspects of the fathers' occupational experience, which of course are highly correlated with social class, account for a very large part of the difference between middle- and working-class fathers' values and a smaller, but still substantial, part of the social class difference in their wives' values. The next step will be to analyze more fully the functioning of the family in Italy. Values and Behavior When Pearlin completed his survey in Turin, Dr. Marian Yarrow proceeded to study a sub- sample of the families experimentally. This provides an opportunity to study the relation- ship of expressed values to actual behavior. The analysis of these data (being carried out by Drs. Pearlin, Yarrow, and Harry Scarr) is less advanced, but some intriguing results have emerged. In the experimental situation, the child was given simple tasks, presented so as to arouse an effort to achieve the best possible perform- ance. During some of the experiments, the child's mother was present, during others his father. The parent was instructed not to do the child's tasks for him, but was otherwise free to act as he thought appropriate. Some parents actively intervened,, to direct and con- trol the child's actions. Those who did were disproportionately the ones whose aspirations for their children surpass their own achieve- ments. More than that, they were dispropor- ionately the ones who value the child's con- forming to adult authority — not those who value the child's self-direction. The twin con- ditions of having high aspirations for one's child and disvaluing his self-direction are highly predictive of whether or not a parent will intervene to direct his child's actions rather than allow him opportunity for self- direction. Maternal Care and Child Behavior in Japan Dr. William Caudill has been carrying out a systematic observational study of mother-child relationships in Japan, and comparing the results with those that he and Mrs. Helen Weinstein have obtained, using the same methods, in the United States. The analysis is in process, but far enough along for some conclusions to be clearly established. (These are based on a study of three-to-four month old infants. Data on these same children at age 2V2 have been collected but are still to be analyzed. ) There is, of course, a basic similarity between Japanese and American practices in that ma- ternal attention in both cultures is centered on the infant's needs for sleep, food, and clothing. Beyond this, however, the contrast between the two cultures is great. The American mother talks to her baby more, and seems more apt to encourage him to respond and be active. The American baby is more often alone, is more active in the manipulation of his body and in the use of objects, and vocalizes more. The Japanese mother rocks her baby more, and talks to him less. Her actions seem directed to soothing and quieting the baby rather than to encouraging response and activity. The Japa- nese baby is less often alone, and is both physi- cally and vocally quieter. These differences be- tween the cultures do not occur as isolated characteristics of behavior, but rather are in- NATIONAL INSTITUTE OF MENTAL HEALTH 357 terwoven into culturally dissimilar patterns. Even by the age of three or four months, a great deal of cultural "learning" has occurred. Among middle-class families in Japan, the husband's occupational locus is related to the behavior of his wife and of their infant. The baby in families of small independent business- men is less often alone, and more often awake and protesting. The mother in such a family talksmore to her baby, holds and rocks him more. In contrast, the infant in families of sal- aried employees seems more quiet and passive. The wives of salaried employees, in their move toward modernity, seem to have subtracted from traditional ways of caretaking rather than to have added anything new. Children's Orientations to Health and Illness Dr. John Campbell's study of how children develop orientations to health and illness is in the data-collection phase, but there are some intriguing preliminary findings from the data already in hand. One is that nine to twelve- year old children are more resistent to "giv- ing in" to illness — to immobilization and de- pendency — than are six to eight-year olds. Another is that children who, by their own and by their mothers' testimony, are more inclined to take risks, are less inclined to give in to illness. With more such information, Campbell hopes to ascertain the processes that go into children's developing conceptions of health and illness. The Experimental Modification of Children's Behavior Drs. Phyllis Scott, Roger Burton, and Marian Yarrow conducted an experiment (reported last year) in the modification of a child's un- desirable, aggressive behavior; the experiment was notable in its successful transplantation of the principles of social reinforcement from the laboratory to the natural situation of the nursery school classroom, with its many uncon- trolled variables. Further analyses of these data have clarified some of the issues involved, and have encouraged Drs. Scott and Yarrow to go one step further: to attempt to use modifi- cation procedures such as social reinforcement and modeling, again in the natural situation, to inculcate desirable, "non-egocentric" behav- ior in nursery school children. This study is presently being planned. A Review of Research on Social Development During this past year, Dr. Roger Burton had the opportunity to review and assess past stud- ies of social development for the International Encyclopedia of the Social Sciences. His prin- cipal conclusions are: (1) many of the find- ings derived from interviews about child- rearing practices can be organized along the same three semantic dimensions found in stud- ies of the meaning of words generally; it may be that the underlying dimensions of language, rather than dimensions of the behavior being spoken about, account for the relationships found; (2) the theoretical model underlying most research on socialization, that of a pas- sive child acted upon by the adult world, needs revision to take into account new evidence on variations in the constitutional characteristics and predispositional tendencies of the infant; (3) studies of the effects of stress on infants indicate that too little stimulation is deleteri- ous, that an extra amount of regular or "nor- mal" stimulation under appropriate conditions can promote desirable behavior, and it is even possible that stressful stimulation may cause some desired consequences; and (4) unambig- uous lableling by parents of what behavior they approve and what they disapprove in- creases the likelihood of the child's behaving appropriately in a variety of relevant situa- tions. Three major trends are foreseen. First is the application of learning principles from the experimental laboratory to more complex kinds of human behavior as they occur in natural set- tings. Second is an increase in self-consciously critical assessment of the method and data used in studies of social development; few of our theories can actually be tested with data now available. Third is increasing attention to studies of the newborn, and how his con- stitutional characteristics and predispositions interact with his social environment. Population Genetics Dr. Gordon Allen, the sole geneticist in this 358 ANNUAL REVIEW OF INTRAMURAL RESEARCH Laboratory, is now planning a study to be done in collaboration with Dr. Calvin Redekop of Earlham College, of Old Colony Mennonites in Mexico. The intent is to detect and describe variations in survival and reproduction in a long-isolated rural population. This population is unique in North America for its large size, combined with social isolation, cultural uni- formity, "natural" reproduction and preserva- tion of family structure. Under these condi- tions it seems likely that survival and repro- duction may depend to a significant extent on emasurable behavior differences. This would constitute natural selection of psychological variables as hitherto postulated but never docu- mented. The Social Psychological Correlates of Occupation We are processing the data from interviews with three thousand men, representative of all men employed in civilian occupations in the United States. The interviews were conducted for us by the National Opinion Research Cen- ter, using a schedule developed by Drs. Melvin Kohn, Carmi Schooler, and Morris Rosenberg. The intent here is to trace some of the major social psychological correlates of a number of presumably important dimensions of occupa- tional position and experience. (The analysis, from the Turin data, of the relationship of three aspects of occupational experience to parental values is a good example of what is intended, and in a sense is a happy pre-test of some of the ideas that motivate this study.) The year and a half since the interviews were conducted has been devoted to coding the data, checking reliability, carrying out elabo- rate checks on possible sources of error and inconsistency, and planning the analyses to come. Studies of Mental Disorder Work in this area, this year, was largely devoted to reviewing and assessing past re- search on schizophrenia. Quantitative Studies of the Psychology of Schizophrenia Dr. Carmi Schooler (in collaboration with Dr. Solomon Feldman of Northern Illinois University) undertook the massive job of ab- stracting and organizing all of the quantita- tive studies on the psychology of schizophrenia that had appeared in standard journals (or were mentioned in major review papers) since 1950. In reviewing these studies, Schooler found that a major portion of the research has centered on five hypothesized decrements in the functioning of some or all schizophrenics: an avoidance of intense or novel stimuli, a decrement in set and attention to stimulus in- put, a decrement in appropriateness of gener- alization and categorization from input, a dec- ment in motivation to undertake action, and an avoidance of interaction with and psycho- logical closeness to other people. Some studies of these hypothesized decrements have com- pared schizophrenics to normals, others have compared different types of schizophrenic pa- tients, for example, those with good versus those with bad premorbid histories, paranoids versus nonparanoids, acutely ill versus chroni- cally ill patients. At the present time, all the decrement hypotheses are incompletely proved but viable. Even if all were accepted as proved, however, grave theoretical problems would re- main. If any one, or some combination, is taken as basic, it is possible to predict the remaining decrements as likely outcomes. The number of alternative theories imaginable is staggering. The Social Epidemiology of Schizophrenia Dr. Melvin Kohn reviewed studies of the social epidemiology of schizophrenia, with ma- jor emphasis on assessing the evidence for the hypothesis that social class is related to the incidence of schizophrenia, and on considering the implications of this possibility for our un- derstanding of the dynamics of the disorders. A large number of complementary studies all seem to point to the same conclusion: that rates of mental disorder, particularly of schizo- phrenia, are correlated with various measures of socio-economic status, at least in large cities, and this probably is not just a matter of drift or duration of illness or who gets hospitalized or some other artifact of the methods we use. In all probability, more schizophrenia is ac- tually produced at lower socio-economic levels. At minimum, this seems to be a potentially useful working hypothesis. NATIONAL INSTITUTE OF MENTAL HEALTH 559 There is some evidence that the greater amounts of stress suffered by people at lower class levels enters into the apparently greater incidence of schizophrenia, but that this is not ,all that is involved. Class differences in pat- terns of family relationships may matter, too. To date, however, there has been no evidence of any difference between the family relation- ships of schizophrenics and those of normal families of the lower and working classes. It may be that the family patterns of the lower classes are in some way broadly conducive to schizophrenic personality development. Clear- ly, though, these patterns do not provide a sufficient explanation of schizophrenia. A review of this field necessarily forces one to be aware of the serious methodological defi- ciencies of past studies. It would be erroneous, however, to conclude that improvement in method is what is principally needed to ad- vance this field. Important as that is, it is not nearly so important as a new stance toward ideas. Rarely have investigators designed their studies to pursue some definite idea or to choose definitively between two alternative in- terpretations of past results. There has, in fact, been a fear of theory in this field of investiga- tion, with the predictable result that the most preposterous ad hoc theories have been dragged in to explain or explain away ambiguous find- ings. Perhaps the most important thing to be learned from an examination of past research is the desperate need for bringing theory in, in time, when designing our investigations. Studies of the Methodology of the Social Sciences One major empirical investigation and one important analysis of the logic of our methods of data analysis have been pursued this year. The first, by Drs. Marian Yarrow, John Camp- bell, and Roger Burton, is specifically concerned with the field of developmental psychology. The second, by Dr. Morris Rosenberg, is specifically concerned with the methodology of survey re- search. Both have ramifications that affect all social science research. Assessment of the Methods of Developmental Psychology Several coordinated studies are being pur- sued simultaneously. Essentially, they involve the systematic comparison of most of our major methods of doing research — compari- sons of interviews to observations, of retrospec- tive interviews to contemporaneously conduct- ed interviews, of various methods of observing and of recording and classifying observations — as well as the replication of significant past research, all to determine just how much cre- dence can be placed on the methods on which virtually all our research has been based. The results thus far are dramatic and disquieting. Here are some of the recent findings: A replication of one highly respected study, the Sears, Maccoby, Levin study of parental antecedents of children's aggression, depend- ency and conscience, produced many incon- sistent findings even though very similar techniques of measurement and analysis were used. A re-examination of research by many investigators extends this picture of little sys- tematic consistency of findings about antece- dent-consequent relationships. A close assess- ment of more favorable reviews of this field indicates that they are based in large measure on the selective inclusion of studies and occa- sionally even of specific findings. Response reliability of mothers' reports has been assessed by a comparison of mothers' answers to a self-administered questionnaire and to an interview, conducted about nine months apart. The two sets of data, on simi- lar dimensions of childrearing and children's behavior, show considerable inconsistency. For example, correlations between pairs of ques- tions regarding the child's dependent behav- iors ranged from +.17 to +.32; the correla- tions on mothers' levels of demands on the child range from —.03 to +.32. Such low cor- relations suggest that a considerable propor- tion of the variance in such measures has not been adequately accounted for. The utility of such items for systematic hypothesis testing- is considerably limited. Comparisons of three sets of data on identical dimensions of childrearing (derived from con- temporaneous records, mothers' later recall, and children's later recall) show correlations that range from + .04 on the use of particular disciplinary techniques to +.37 on ratings of 360 ANNUAL REVIEW OF INTRAMURAL RESEARCH maternal warmth. Children's reports of their earlier development and experiences corre- spond less well to contemporaneous records than do mothers' reports. Dimensions crucial to developmental theory (such as early envi- ronmental traumata, early relationship with mother, early signs of the child's inability to relate to others) yield correlations ranging from + .20 to + .38. A score was obtained for each mother on the degree of correspondence between the earlier contemporaneously obtained data and her later recall. Degree of correspondence was not re- lated to the sex or to the ordinal position of the child. It was slightly related to how much time had passed (the shorter the interval, the greater the correspondence). Mothers whose recall is similar to the baseline data tend to have had good relationships with their chil- dren (at the baseline period), and their chil- dren tend to have scored well on positively valued personality attributes. Where mothers' recollections differ from the contemporaneous accounts, either the mother-child relationship of the child's personality was more proble- matic. Examination of observational techniques shows that certain procedures for assessing re- liability have built-in inadequacies. For exam- ple, correspondence between profiles of a given person's behavior (i.e., the rank-ordering of categories of behavior) as appraised by two different observers has two major drawbacks as a measure of reliability: (a) since a profile is a summary of the frequency of interaction over a period of time, the extent of agreement on classification of specific acts within that sequence cannot be assessed, (b) The degree of correspondence between the behavior profiles of two randomly chosen children is nearly as high as that between two independent sets of observations of the same child. The correspond- ence presumedly indicative of reliability of ob- servation may merely reflect a more general common patterning in the observed behavior. Methodological Principles of Survey Analysis Dr. Morris Rosenberg has been systemati- cally re-examining just what it is we do in analyzing survey data. In a series of papers, he has specified the various analytic purposes served by the introduction of "third variables," reconsidered our assumptions about the rela- tionship of indices to concepts, further devel- oped his method of test factor standardization, and clarified the distinction between symmet- rical and asymmetrical relationships. No more than a skeleton picture of this work is pos- sible here. One major area of his work is in the specifi- cation of symmetrical, reciprocal, and asym- metrical relationships, and on the elaboration of three-variable relationships. Symmetrical relationships are those which are alternative indicaators of the same concept, consequences of a common cause, functionally interdependn or elements of a common complex. Asymmetri- cal relationships are those involving an asso- ciation between a stimulus and a response, disposition and a response, a property and a disposition, a necessary condition and an effect, an ends and a means, and a structure and its imminent outcome. When a third variable is introduced into a two-variable relationship, it may serve as a test factor, a specifying condition, or as an- other independent variable. Six types of test factors are distinguished and their theoretical implications elaborated: extraneous variables, component variables, intervening variables, suppressor variables, distorter variables, and antecedent variables. When the third variable represents a specifying condition, it may chal- lenge, confirm, refine, or radically revise a the- ory; it may enable one to select between alter- native hypotheses, reveal positive results in non-correlations, reveal trends or processes, and clarify the nature of the relationship, the independent variable, and the specifying condi- tion itself. When the third variable serves as another independent variable, it may reveal independent effects, relative effects, cumulative effects, and typological effects. The value of this is in the added power it affords us to know precisely what we are doing, and to be certain that we have not overlooked theoretically fruitful possibilities for analysis and interpretation. NATIONAL INSTITUTE OF MENTAL HEALTH 361 LABORATORY OF NEUROPHYSIOLOGY Section on General Neurophysiology is pro- ceeding- with analysis of shifts of electrical potential of the brain which appear to be primarily due to complex metabolic transac- tions across the blood-brain barrier. It has been shown that there is great species dif- ference between rabbit, rat and dog on the one hand and the cat and rhesus monkey (and presumably man) on the other hand. The brains of the first group shift positive with respiratory acidosis, and the brains of the second group shift negative. It has been found that some injury factor reverses this response in the latter animals. The injury factor has not been specified, but general deterioration of the preparation and hypoxia seem to be involved. Concussed monkey brains also show reversed reactions. It appears that the capillaryglia blood-brain transport sys- tems are involved in this reaction and that vasomotor disturbances are important. It is now probable that the voltage shifts occur- ring in the three stages of sleep, in arousal reactions and possibly the voltage shifts ob- served in certain kinds of conditioning and learning experiments are all of the same general nature. That is, not primarily neu- ronal but a complex metabolic phenomenon. These observations have considerable inter- est for all kinds of brain disease and injury including circulatory disturbances and cere- bral vascular accidents. Another project deals with the problem of data handling to retrieve not exactly syn- chronized evoked reactions which occur at the various stations of a physiological reflex re- sponse. The glabella reflex is the example under study. The technical procedures have been proven and the method will be applied to other systems. As part of general program on C0 2 and pH effects on neurons, a study of the nerve cells of the Aplysia is under way. Currently temperature functions of the pacemaker cells are under study. The instrumentation section is proceeding with numerous improvements in existing technics and development of new ones. The Section on Limbic Integration and Behavior has continued its investigations on two broad aspects of the functions of the limbic system, a basic part of the forebrain inherited from lower mammals and now re- cognized as playing an important role in emotional behavior and visceral functions. The first problem pertains to the relation- ship of the visual and limbic systems in the evolution of primate behavior, and the second concerns the central representation of pain and the role of the limbic system in modify- ing neural mechanisms responding to nox- ous stimuli. The importance of the first problem is em- phasized by a number of anatomical and clinical considerations. In the evolution of higher primates and man, the limbic cortical areas adjacent to the primary visual areas undergo a remarkable expansion. The fusi- form gyrus lying parallel with the hippo- campal gyrus develops as new structure in the brains of higher primates. The clinical observations of Penfield have revealed that irritative lesions in or near the medial tem- poral limbic cortex may result in a variety of visual illusions and emotional manifesta- tions. Patients may experience subjective feel- ings of deja vu, macropsia, micropsia, feel- ings of being in a dream, feelings of famil- iarity or strangeness, paranoid feelings, feel- ings of fear, loneliness, sorrow, disgust, and sometimes ecstasy. Recently Slater and Beard have analyzed 69 cases of temporal lobe epilepsy in which the clinical picture was indistinguishable from that of chronic schiz- ophrenia. Finally, the problem is relevant to the unanswered question of how the visual system establishes a working relationship with the hypothalamus. In neuroanatomical and microelectrode stud- ies on this problem the Section has gained basically new information indicating how the medial temporal convolutions are implicated in visual symptomatology and hypothalamic regulation. Heretofore it has been classically believed that no fibers from the optic radia- tions terminate in the medial temporal con- volutions. In a neuroanatomical study, how- ever, in which lesions have been placed in 17 squirrel monkeys, it has been found with a 362 ANNUAL REVIEW OF INTRAMURAL RESEARCH modification of the Nauta stain that follow- ing lesions placed in the lateral part of the lateral geniculate body, a continuous band of degeneration is traced not only into the cortex of this gyrus, but also into the adjacent parts of the fusiform and lingual gyri. The band of degenerating fibers would appear to cor- respond to that part of the optic radiation in man known as Meyer's temporal loop. Some degeneration can also be traced further caudally into the prestriate limbic cortex of the retrosplenial region. Another new and highly significant finding is that following a lesion placed in the inferior pulvinar (a nucleus generally regarded as a visual associa- tion nucleus) degeneration develops in an out- ermost band of fibers leading to the posterior hippocampal gyrus. Electrophysiological support for these find- ings has been obtained in microelectrode studies in which the representation of visually responding units has been mapped in the hippocampal gyrus and neighboring convolu- tions in waking squirrel monkeys. Recordings have been made from more than a 1000 lim- bic units. These experiments have confirmed findings of the initial exploratory study in anesthetized animals that visual stimulation activates units in the posterior hippocampal gyrus and adjacent parts of the fusiform and lingual gyri. Of particular interest has been the identification of a new class of cells in the posterior hippocampal gyrus that responds similarly to slowly adapting "on" units of the retina. In the retrosplenial region some cells have been found that respond only to stim- ulation of the contralateral eye, suggest- ing that they are activated by the primitive temporal monocular crescent. As both the posterior hippocampal gyrus and re- trosplenial cortex are a major source of con- nections to the hippocampus, which in turn projects to the hypothalamus, it is apparent how the present findings have significant im- plications in regard to the influence of the limbic system on circadian rhythms, neu- rovegetative and emotional mechanisms, and the dreaming and autonomic aspects of the rapid eye movement (REM) phase of sleep. The Section's current investigation of the interaction of the fornix and fifth nerve on units at the thalamic level was suggested by previous experiments in which it was found that hippocampal seizures propagating in the limbic system result in an elevation of the threshold of the behavioral response to noxious stimulation of the face and other parts of the body. The present microelectrode studies in awake squirrel monkeys take advantage of the finding that a stimulus to the fifth nerve of sufficient intensity to induce signs of pain if given repetitively does not appear to dis- turb the animal when administered singly. This provides an experimentally innocuous means of mapping the distribution of thalamic units responding to a potentially noxious stimulus. In addition to units in the classical relay nucleus (n. ventralis posteromedialis), fifth nerve stimulation has been found to ac- tivate units in the caudal intralaminar nuclei and in parts of the tegmental area. A prior shock to the fornix may inhibit the fifth nerve response of units in the caudal inter- laminar nuclei for a period lasting up to 200 msec, but has no effect on cells of the clas- sical relay nucleus. Following a hippocampal after-discharge induced by tetanic stimulation of the fornix the inhibitory effect on intra- laminar units may last as long as two minutes. This latter finding is of particular interest in regard to limbic seizures seen clinically in which patients may burn or otherwise injure themselves because of apparent unawareness of painful stimuli. One of the most interesting findings, to neurophysiologists, of the electro-microscopic studies of nerve and muscle cells has been the redefinition of the boundaries of the cells. Previously it was simply a matter of a cell having an inside and an outside, but now we find that there are channels which connect the extracellular space to the more internal portions of the cell. The channels are separat- ed from the cytoplasm by membranes. Ex- amples of these systems are the transverse tubular of striated muscle fibers and the in- vagination in the giant neurons of the snail, Aplysia. The Section on Membrane Physiology has been investigating the properties of both of these systems in order to define the mode NATIONAL INSTITUTE OF MENTAL HEALTH 363 of transfer of substances along them. The first step is to determine the electrical pro- perties of these pathways as a means of describing the relative ease of transfer along them. In the Aplysia neurons, it appears that the invaginations are the major pathway for transfer in and out of the cell and may be considered to be extension of the surface membrane. In muscle, the transfer across the walls of the transverse tubular system along the tubules has greater restrictions. These restrictions must be incorporated in the de- velopment of models which describe the mech- anism by which the electrical activity of a muscle fiber induces activity in the contrac- tile apparatus of the muscle fiber. LABORATORY OF PSYCHOLOGY Since I shall organize this report around major areas of investigation which cuts across Section boundaries, I am introducing the re- port with a statement of the general aims of the various Sections. The Section on Early Learning and Develop- ment is concerned primarily with learning and development in both human and animal sub- jects. Investigation in the human area is directed toward learning in social contexts (specifically the mechanisms of adaptation and social learning) in the early phase of life. The methods used are controlled obser- vation and experimental control. The overall research aim of the animal unit is to contri- bute to our understanding of learning by analyzing the action and correlates of rein- forcer presentation and omission. To determine which facts and principles discovered are more general and which are specific to a particu- lar class of reinforcers, to a particular class of behavior, or to a particular age of the animal subject, studies are being carried out on early ingestive behavior, early instrument- al behavior, and juvenile social behavior, as well as traditional adult instrumental and operant behavior. The effect of both homeostatic reinforcers, such as food, and nonhomeostatic reinforcers, such as a passive person for a juvenile puppy, are being in- vestigated. In the Section on Personality, two major research programs have developed within the overall goal of investigating factors which facilitate or interfere with the effective func- tioning of the individual: the identification of factors which influence personality devel- opment and change; and the study of factors affecting cognitive processes and development. In the Office of the Chief two major areas of research are represented, a study of the nature and etiology of schizophrenia, and a study of the process of communication. The Section on Perception is primarily in- terested in two major and separate areas: the nature of the perceptual process, its devel- opment, character and aberrations; and the environmental and genetic variables affecting biological systems. The Section on Higher Thought Processes studies the process of problem-solving. Its investigators are interested in the fundamen- tal nature of these processes as well as their operation in various normal and disturbed groups. The Section on N europsychology is con- cerned with the relationships of the brain to behavior in human and animal subjects. These relationships are studied through both abla- tion and stimulation techniques. In carrying out these programs some of the work is carried on entirely within the Sec- tion structure, some in cooperation with other Sections, some in cooperation with other Laboratories and Institutes, and some with outside agencies both here and abroad. Against this background I have organized the report into five major substantive areas, dealing with topics without regard for Sec- tion lines. I have also added some general material concerned with advances and pro- blems in the Laboratory. The five substan- tive areas are: (1) Development of behavior- al functions; (2) Analysis of developed be- havioral functions; (3) Biological substrate of behavioral functions; (4) Forces destruc- tive of behavioral functions; (5) Forces that enhance behavioral functions. The two ad- ditional areas are: (6) Theoretical, method- ological and technical advances; (7) Admin- istrative aspects. 364 ANNUAL REVIEW OF INTRAMURAL RESEARCH Development of Behavioral Function In recent years a substantial amount of the investigative effort in the Laboratory has concentrated on one aspect or another of early development — both in animal and man. Aside from some planned work of Dr. Rosvold's group with monkeys and Dr. Cal- houn with rats, the major attack in animals has been made by Drs. Stanley and Bacon at Poolesville. They have worked with social behavior in young dogs and are presently experimenting with early learning in neonatal puppies. Neonatal Learning Sucking behavior of neonatal mammals is a classic example of an innate action pattern present at birth in a remarkably organized form. When attached to a teat, the sucking puppy is likely to obtain milk correlated with waves of negative pressure generated in its mouth. From the point of view of reinforcer action, the components of sucking make up the only possibly operant responses that a new- born mammal can perform fast enough to provide a sufficiently large behavioral sample for analysis. Accordingly, puppies were trained to suck on an artificial nipple, followed by repeated sessions with a fixed time interval of milk reinforcement, the milk being delivered con- tingent upon the first suck 10 seconds after the preceding milk delivery. Two of six 2-week-old puppies displayed a pattern of sucking in accord with the temporal spacing of milk deliveries, indicating discriminative operant learning. The discriminative learning prowess of the infant puppy has been directly confirmed by Dr. Bacon who joined the staff this year and is investigating instrumental approach and avoidance behavior as distinguished from in- gestive behavior. He established that 1-week- old puppies can discriminate between the hard- ware cloth and the terry cloth sides of a discrimination box because they gravitate toward the side where they had received milk. These findings add to the growing body of data showing that the newborn puppy, which was thought to be devoid of any learn- ing ability less than 10 years ago, can learn, and apparently does so on the same basis as adult organisms. Social Behavior The young dog readily forms a strong emotional attachment to people. This fact sug- gested that such attachments could be mea- sured by the speed with which a dog runs repeatedly to a person. If the speed of run- ning improved with such opportunities, viewed as training trials analogous to food-reinforced training trials in alleyways for rats, it would follow that a person or some aspect of hand- ling the puppy was a reinforcer for the dog. Data, collected at another institution by Dr. Stanley and Dr. Bacon, showed not only that a person was a reinforcer for the dog, but that the most consistent reinforcer was sim- ply a passive, unreactive person. Additional data on passive person reinforcement was analyzed and reported this year. Basenji puppies given intermittent passive person re- inforcement learned and extinguished ap- proach behavior in the same fashion as pup- pies given continuous or 100% reinforce- ment. This finding is comparable to that found with small amounts of food reward in the rat. It suggests that the passive person functions as a weak reinforcer in the basenji. This work will be continued using other breeds. The other studies on early functioning are focused on similar problems in human devel- opment in different contexts and with dif- ferent degrees of analytic detail. They range from studies of perceptual and cognitive pro- cess, through a detailed study of the process of social learning, to a study of intellectual development in molar settings. The first is a study of object-perception in children conducted by Drs. Carlson and Rapoport. Generally accepted hypotheses con- cerning the development of object-perception often assume a progression from relatively greater perceptual dependence upon the visual angle subtended by an object to a more com- plete perceptual integration of angular size with various sensory cues to object-distance. Recent work done here and elsewhere on size- NATIONAL INSTITUTE OF MENTAL HEALTH 365 and distance-perception in adults has seri- ously questioned the theoretical basis for this assumption. In perceptual experiments adults can produce size-judgments which approach geometrically correct angular values and which deviate correspondingly from an ac- curate judgment of object-size. These devia- tions now appear to be due to a cognitive attitude about size-distance relationships rather than any basic capacity to perceive angular size as such. This possibility has im- portant implications for hypotheses of neural mechanisms underlying perception and for the interpretation of apparent differences in object-perception between normal adults and other populations such as children and psy- chologically deviant groups. Comparable pro- cedures for testing size-judgment in both chil- dren and adults have been worked out in preparation for a study of the age range from approximately 4 to 10 years. Dr. Caron, in his studies of conceptual development, has extended his investigation of the processes underlying the phenomenon of conceptual abstraction in the preverbal child. A widely held view of concept forma- tion and concept transfer may be charac- terized as the "verbal hypothesis." This view states that the ability to treat similar in- stances of a concept equivalently is dependent on the acquisition and application of common verbal labels for each instance of the concept. Dr. Caron has undertaken to test an alternate hypothesis, i.e. the "discrimination hypoth- esis," which holds that conceptual abstrac- tion and transfer are not dependent on the application of common verbal labels but rather on the individual's ability to dif- ferentiate the common sensory feature or property found in each instance of the con- cept. Dr. Caron and his associates have conducted a series of four studies testing the adequacy of the discrimination hypothesis to explain the development and transfer of concepts in preschool children. One study directly com- pared the relative effectiveness of verbal and discrimination training on the ability of children to transfer learned responses to concepCuaiiy more complex but related stim- uli. The evidence thus far obtained appears to support the discrimination hypothesis. The other three studies involve examination of the factors which appear to facilitate or impede discriminative abstraction. The re- sults of this series of studies demonstrate that children may be assisted to develop con- cepts at a very early age without reference to verbal training. The fact that man's abil- ity to think abstractly precedes the devel- opment of an appropriate verbal response system permits the inference that humans possess an elemental representational system to which language and other coding systems are later coordinated. A practical implication of these findings is that the current pro- grams which are concerned with the en- richment of the culturally marginal, handi- capped or retarded child should place greater emphasis on developing techniques to high- light the sensory features which define con- cepts rather than continuing to stress ver- balization of concepts as such. Dr. Gewirtz' program of Research on the Impact of Environmental Stimulation on Children's Learning and Development has Five Overlapping Foci The first general focus has been on be- havior, and has included many topics only a few of which we have the space to mention: the determination of infant behavior reper- toires in early life, with the attempt to separate unlearned from learned components of those behaviors; the acquisition of re- sponsiveness to the environment; the deter- mination of the ages at which different learn- ings can first occur; the acquisition of con- trol by infant and child behaviors of con- ditioned discriminative and reinforcing stim- uli (non-social and social) provided by the environment; the determination of the con- trol by the infant over behaviors of parents and caretakers; and the determination of the consequences in key infant behaviors of the stimuli provided through caretaking. The second general focus of this research program has been on environment, and has involved attempts to specify the nature of functional stimulus units for learning, in everyday social contexts. Attention has been 366 ANNUAL REVIEW OF INTRAMURAL RESEARCH directed to identifying critically and rigorous- ly useful dimensions and units of the environ- ment, implied by terms such as "richness," "privation," "deprivation," and "separation." During the past year several reports have been published from the project under these two research foci. Two of these reports, of monograph length, have dealt with the age course of infant smiling in the first 18 months of life in four Israeli child-rearing environments. The forms of the age curves for smiling differed among the enironmental groups. Attempts were made to understand these differences in terms of what we knew about the different conditions they provided in- fants in them. The possibility that the smil- ing habituation curves (to a constant stimlus across 12 minutes of time) for individual in- fants differ in the several Israeli environments is currently being explored. The form of decline of the habituation curves was found to be dramatically like the form of similar response curves in which responses were made to constant stimuli by lower animal species. Slow but steady progress has been made during the past year on the large companion study (being conducted in collaboration with Dr. H. B. Gewirtz) in which 108 infants in four child-rearing environments were studied for one entire day at four age points during the first year. Stimuli provided by the en- vironment to the infant, the infant's behaviors and the contingencies between stimuli and behaviors have been catalogued. After con- siderable preparatory work several computer programs were recently completed which have permitted the start of analyses of two major segments of data. A third focus of this program has been on conceptualizing the different processes that could be considered to represent learning and, more generally, adaptive behavior change; and, when required, on extending basic learning conceptions to the analysis of some of the complexities and aberrations of life in the early years (e.g., "privation," "deprivation," "separation"). In this attempt, a variety of possible processes of change are scrutinized, including the separate effects of stimulation, conditioning, performance, and setting-drive conditions. Further, these proc- esses have provided the bases for attempts to design environments that would enhance early dimensional and contextual learnings, as well as the possibilities for efficient adap- tive and social learning and development. Dr. Gewirtz has prepared a major theoretical analysis of these issues for the Committee for Re-examining Early Child Care. It is directed to various basic and applied fields interested in early child growth and development and is to be published this year. A fourth focus of the program has been on the strategies and tactics of caretaking of in- fants and older children in intact families, in day-care installations, and in residential institutions. This research has involved anal- yses of concepts like "tender loving care," which reflect aims of child rearing valued by society. Further, in terms of what is known of early learning, the approach has been involv- ing the design of effective patterns of care- taking to (1) maximize the possibilities of attaining socially desirable child-rearing out- comes in the behaviors of children, (2) fa- cilitate bringing out the full capabilities of the developing child, and (3) make unam- bivalent the caretaker's relationship to the child (which could result from the caretaker's behaviors coming under the child's control). For the above ends, a study was carried out on the instrumental crying (for attention) of infants as young as six weeks of life in a well-baby ward of a children's hospital. It was found that the crying of the infants was maintained, indeed strengthened, by the atten- tion readily provided by the well-intentioned caretakers to the infants when they would cry. A research carried out illustrated clearly that, by not responding to apparently un- warranted crying together with responding to socially approved behavior like smiling, the infants stopped crying unnecessarily and be- came "charming" companions for the care- takers. A fifth focus of the Section's research pro- gram has been on the setting conditions that determine stimulus efficacy for behavior on a particular occasion. These conditions may op- erate at one time point (e.g., "ground" for NATIONAL INSTITUTE OF MENTAL HEALTH 367 "stimulus figure") or across time points (e.g., deprivation or satiation contexts for a stimulus) to heighten or lower the efficacy of stimuli for behaviors. Such setting condi- tions are particularly important for an under- standing of child behavior systems in na- tural settings, given that the child's capacities are developing and limited. In a series of studies, Dr. Gewirtz and his associates have found that the more frequently social stimuli are presented to children ("satiation"), the less effective those stimuli are as reinforcing stimuli in a subsequent learning situation. Further, the more recovery time between the receipt of stimuli and the learning task, the greater the recovery was found to be. This type of relationship has been identified here- tofore primarily with organismically relevant appetitive stimuli with lower organisms. Two reports of this research have been submitted for publication this past year, and have been well received. As seen in some aspects of the work done by Dr. Gewirtz' Section such as Dr. Etzel's study on the crying child, and in Dr. Schae- fer's work with culturally deprived children, the implications of basic studies naturally lead to the testing out of the hypotheses de- veloped in the real situation. Intellectual Development Dr. Schaefer, on the basis of Dr. Bayley's findings in her normative study, is currently conducting a research program aimed at de- termining whether it is possible to raise the level of intellectual performance of Negro in- fants of lower socioeconomic status, through a program of intellectual stimulation. Part of the effort is to determine whether this training program, which does not in- volve removing the child from the home, can provide significant influence on the child's motivation, language development and functioning intelligence. If this pro- gram is successful, that is. if it turns out that lower functioning intellectual per- formance is not a result of matu rational factors, a new method of assisting the child •of lower socioeconomic status will become available. The research, which has been un- dertaken in collaboration with the Bureau of Social Research, Catholic University of America, includes an experimental group of 30 Negro infants of lower socioeconomic status and a control sample of 30 comparable infants. The experimental group is being tutored in the home beginning at 15 months. Tutoring sessions are offered five days a week, one hour a day. Both the experimental and the control groups will be tested at 21, 27, and 36 months to assess what specific abil- ities have been affected and what differences may be found between the tutored and un- tutored groups. A related study is being carried out with another group of Negro infants from a "more stable suburban Negro community, in association with the Home Study Program of Kensington, Md. Some of the infants will be tutored beginning at one year and others be- ginning at two years of age in order to deter- mine whether significant differences may be at- tributable to earlier intervention. Both of these groups as well as a "no-tutoring" control group will also be tested at three years of age to determine the effects of the tutor- ing program. Analysis of Developed Functions There are three areas of investigation of de- veloped, rather than developing, functions going on in the Laboratory, namely, communi- cation, thought, and environmental and genetic variables affecting biological systems. Communication The study of human communication involves two sections of the Laboratory, the Office of the Chief, represented by Dr. Allen T. Dittmann and the Section on Personality, represented by Dr. Donald S. Boomer. The principal research focus remains the study of the phonemic clause which takes into account the rhythmic charac- teristics of speech. A theoretical analysis of the processes involved in the communication of emotions or affects is also under way. Dr. Boomer has continued his research aimed at formulating a psycholinguistic theory of speech encoding and decoding. He has sys- tematically tested the hypothesis that the pho- 368 ANNUAL REVIEW OF INTRAMURAL RESEARCH nemic clause is the unit of speech which bears on speech formulation and comprehension. During the past year he published the first major statement regarding the phonemic clause and its role in the interrelationship between speech and thought. His current research has provided further evidence that the phonemic clause rather than the isolated word is the functional unit of speech and encoding. He has initiated a study aimed at testing the hypothe- sis that the phonemic clause is also related to the decoding process as represented by the com- prehension of orally presented material. Interest in the phonemic clause has in- creased because of evidence gathered in past years that this is the fundamental unit in which spontaneous speech is formulated and uttered by the speaker, and by which it is un- derstood by the listener. On the encoding side the psychological process is seen as successively assembling groups of words into syntactic pat- terns. On the decoding side the hypothesis states that temporal strings of phonetic signals are accumulated and held in abeyance until the end of the phonemic clause, at which time the entire assembly is decoded as a pattern and understood as a single meaning unit. Most of the evidence already at hand concerns the en- coding side. Examples of this type of research include the study of hesitations and filled pauses. Since these pauses are found to occur toward the beginning of the clause, it appears that decisions about the whole unit are made at the outset, after which the remainder can be spoken relatively fluently. Data for evidence like that from hesitations are also being collected from the study of re- traces and false starts in spontaneous speech. The hypothesis under test here is that ( 1 ) these phenomena are confined within phonemic clauses; (2) they do not cross clause bounda- ries; and (3) they occur at the outset of pho- nemic clauses. A parallel hypothesis about the phonemic clause from the decoding side has also been formulated and data are being gath- ered for its test. The hypothesis states that in conversational speech, the listener indicates his understanding by certain behaviors which occur only at the end of phonemic clauses, be- haviors like head nods and vocal insertions like "Mm-hmm," "Yes," and "I see." Since vocal behaviors lend themselves readily to study, a recording situation for brief conver- sations between pairs of normal control sub- jects has been set up so that the coding of the speaker's clauses and identification of the listener's vocalizations can be done completely independently. Preliminary examination of other interview material, collected for the body movement study to be reported below, indi- cates that the "Mm-hmm" is inserted almost exclusively at junctures between clauses. The response is sufficiently selective, however, that it may be possible to identify some larger unit, based on differentiating among the three types of juncture. Instrumental work is being done on one of the distinctive features of the phonemic clause — primary stress — which, by definition, can occur once and only once per clause. Primary stress is being studied because it is more amen- able to physical measurement than is the other distinctive feature — the boundary between clauses, or juncture. The identification of such boundaries depends on linguistic judgment and is not communicable in more objective terms. Measurements are being made of three acoustic aspects of a series of primary stresses, peak amplitude, integrated amplitude, and du- ration, with a view to specifying its charac- teristics well enough for test in speech syn- method of data collection, i.e., output from ac- One of uhe basic contributions of Dr. Boom- er's research extends beyond the field of psy- cholinguistics in that it provides a more pre- cise tool for a broad range of studies involving speech and comprehension. If future work con- tinues to support the hypothesis that the pho- nemic clause is the natural unit of syntactic processing, both in speech transmission and in reception, then a meaningful unit for speech analysis will have been provided mental health investigators concerned with such problems as psychotherapy, psychopathology, cognitive de- velopment, etc. In order to provide a direct test of Dr. Boomer's hypothesis that the phonemic clause is the basic unit in speech formulation and com- prehension, it is necessary to vary intonational features in otherwise identical utterances in NATIONAL INSTITUTE OF MENTAL HEALTH 369 order to assess the effects of these features on recognition and comprehension. A human speaker, however, cannot simultaneously con- trol his voice on all of the relevant dimensions; such stimuli can be produced only by an elec- tronic speech synthesizer. In order to gain experience with such an instrument it is planned that Dr. Boomer will spend a year, beginning June 1966, working at the Depart- ment of Phonetics, Edinburg University, Scot- land. Study of the relationship between body movements and speech rhythm patterns con- tinues this year with a replication of the study completed last year in which hand and head movements, like hesitation forms in speech, were found to occur toward the beginnings of phonemic clauses, while foot movements were found to occur independent of these speech rhythms. The replication uses a different method of data collection, i.e., output from ac- celerometers which are attached to head, hands, and shoes of the subjects. The signals are written out on an operations recorder along with an oscillogram of speech for location of pauses and collation of the phonemic clause coding, which is done from audio tape and typescript. Subjects engage in 15-minute con- versational sessions on two successive days during which the interviewer makes every ef- fort to reduce the stress which might result from being in this unusual situation. A great deal of development work was necessary, in collaboration with the Technical Development Section, to perfect the apparatus, and 10 nor- mal control subjects have been run in the experiment. The results are only partially analyzed. The theoretical work on emotional commu- nication is an outgrowth of a literature search begun by Dr. Dittmann during his assignment in Milan, Italy. Originally intended to cover only a small aspect of the topic, the work has broadened and is seen as an application of the mathematical theory of communication pro- posed by Shannon. In some areas the theoiy must be added to, because of the special prob- lems cf human communication: first, emotional information is more complex than that en- countered in telecommunication where the theory was developed; second, in social inter- action there is confusion of the functions of the communication system, such as encoding, choice of channel, and the like, which can be performed by independent components in the original applications of the theory; and third, the types of interference or noise in human communication are more varied and more com- plex than the random noise encountered in telecommunication. These differences do not seem, however, to mean that communication theory is irrelevant to problems of emotional communication. Rather, the theory is a gen- eral one, and details must be filled in wherever it is applied. Dr. Dittmann has been working up notes for a monograph which will present the details of this theoretical analysis. Thought Process In the area of thought process, which has emphasized problem-solving, extensive studies have been carried out. The ability to avoid or correct heuristically crude or detrimental methods of inquiry has been studied with more than 150 subjects using an elementary set of problem-solving tasks monitored by HEP, the Heuristic Evaluation Programmer I, which was designed and developed in our Laboratory. The following are, in quite general terms, the principal heuristic deficits identified thus far: (a) Sequential inquiries were poorly organized so that the cognitive strain associated with a solution effort was much greater than it needed to be. (6) The questions reflected by the sub- jects' operations were so specific that the in- formaticn elicited was near minimal, (c) Poor- ly adapted behaviors were allowed to persever- ate without exploring obvious alternatives. (d) Quite direct implications of information elicited were not used, (e) Failure to use nega- tive information prolonged the use of ineffi- cient procedures. In order to secure an opportunity to observe the interpersonal relations between individuals engaged in a cooperative solution effort, HEP has been redesigned to operate alternately from two display panels located in separate cubicles. The cooperating problem-solvers can communicate over a speaker system and are free to question, advise, or criticize one another 370 ANNUAL REVIEW OF INTRAMURAL RESEARCH during the solution effort. Pilot work has been carried out on 10 pairs of subjects to standard- ize procedures and instructions preliminary to a study of interaction and cooperation between schizophrenic patients and their mothers. The amount and type of verbal interaction which occurs in a structured problem-solving situa- tion and the effects of heuristic performance of the subjects will be studied. A group of college students has been tested on a set of very difficult, three-element HEPP problems. Using a procedure whereby displays are automatically read into the apparatus, ef- forts were made to teach strategies for solving these complex problems. Although the subjects were told that a number of solution strategies would be shown, only a few changed their pre- viously developed solution procedures. There was evidence of a surprising inability to in- vent techniques for reducing cognitive strain or to learn from the displays rather obvious methods for doing so. Enviromental and Genetic Variables Affecting Biological Systems Dr. Calhoun has been responsible for this area. His analysis has concentrated on two major areas: drinking and the development of indices for the assessment of behavior. His emphasis on the first, drinking behavior, rep- resents a strategy of gradually presenting, in published form, aspects of a new and compre- hensive theory of behavior. He is presently limiting himself to the lever-pressing-drinking behavior which is so familiar to many experi- mental psychologists. In fact, drinking behav- ior does mirror most of the processes typical of other behavioral states with regard to the control of their duration. Then, once this base- line publication has been completed, he plans to proceed with more concise treatments of the similar processes exhibited by sleep, loco- motion, eating, and grooming as major behav- ioral states of the rat. Only after each of these is treated separately will he engage in the ex- amination of the more complex processes of initiation and sequence of behavioral states, since these two topics can only be treated in the context of the entire repertoire of hehav- ioral states. Then, after the presentation of all of this separate material, he will present the broader theoretical formulation which he be- lieves to form the heuristic basis for a more precise understanding of emotion, mood, mo- tivation, drive, learning, conformity, and crea- tivity. With regard to the second major area, the development of indices for the assessment of various factors involved in behavioral states, Dr. Calhoun is now approaching a stage of com- puter analyis where he has available indices on: (1) growth, (2) vitamin A storage, (3) reproduction, (4) arthritis, (5) serum lipids, (6) blood clotting, (7) mortality, (8) social withdrawal, (9) social relations, and (10) spa- tial attachment. These indices will be utilized initially to explore the degree to which indi- viduals with similar histories (in part deter- mined by these indices themselves) do in fact resemble each other with regard to these in- dices. Although several strategies will have to be followed in this assessment, the final objec- tive will be to expand the conceptual framework of the stochastics of social systems with pri- mary emphasis on the index of social with- drawal. Biological Substrate of Behavioral Functions In this major category of research being carried out in the Laboratory the emphasis is on the organic structural factors which underlie behavioral functions, an area of investigation belonging almost exclusively to the Section on Neuropsychology. Important advances have been made in each of three major fields in which the Section has been involved. Since it would extend the report inordinately to sepa- rate out from the discussions of the biological substrates determined by experimental inter- vention from those interventions occurring naturally, such as epilepsy and Parkinson's dis- ease, the consideration of the latter has been included with the present discussion of the work of the Section. It is clear, however, that the methods conventionally used by the Sec- tion have relevance as well for the subsequent section "Destroyers of Behavioral Function." The three main areas of work of the Section may be considered under: cortical mechanisms in sensation and perception, cortical mechan- NATIONAL INSTITUTE OF MENTAL HEALTH 371 isms in problem solving, and cortical-subcor- tical relations and the regulation of behavior. Cortical Mechanisms in Sensation and Per- ception Vision. — The experiments on visual func- tions have been concerned largely with the way in which the cortical visual areas in- teract. Specifically, the aim is to test the pro- posal that the prestriate cortex is an essential relay in a pathway linking the striate area with the inferotemporal "visual" area. The implication of a series of studies is that the inferotemporal area is important for pattern perception not only in macular vision (i.e., the field of greatest visual acuity), but also in peripheral vision. This tentative conclusion has an important bearing on the visual studies be- ing carried out on neurosurgical patients, de- scribed next. The first experiment on the effects in man of unilateral temporal lobe excisions (performed by NINDB surgeons for the relief of epilepsy) indicated that letter recognition was impaired in the visual field contralateral to the removal. The finding is consistent with the view, first suggested by some work on monkeys, that each temporal lobe is more intimately related to the striate cortex of the same hemisphere (and hence to the contralateral visual field) than it is to the striate cortex of the opposite hemi- sphere. An alternative explanation of the con- tralateral impairment is possible, however. A temporal lobe removal in man is nearly always accompanied by damage to the genicule-striate radiations, resulting in a contralateral upper quadrantic field defect. Although care is taken to expose visual material in an intact part of the field (e.g., along the horizontal meridian or even in the ventral quadrant) the possibil- ity exists that the so-called intact field is in fact defective in subtle ways as a direct result of the radiation damage. To investigate this question, a second experiment was performed in which, in addition to pattern discrimination, basic sensory capacity in the "spared" fields was determined in 15 cases with left and 15 with right temporal lobe removals together with 15 normal control subjects matched for age, sex and education. As before, discrimina- tion of letters and patterns was found to be impaired in the field contralateral to the exci- sions. Thus, the pattern discrimination loss in man would seem to be explicable in the same way as the pattern discrimination loss in mon- keys, i.e., as a result of damage to the tempo- ral (and perhaps, specifically, the inferotempo- ral cortex). Audition. — The work this year in audition has failed to confirm an earlier suggestion that the superior temporal cortex serves functions in audition analogous to those served by the inferotemporal cortex in vision. Specifically, lesions oof the superior temporal convolution which spare the primary auditory projection area in the supratemporal plane do not seem to interfere with auditory discrimination per- formance. Since negative results were also ob- tained f ollowing lesions of the primary auditory area alone, an experiment has been undertaken which involves total lesions of the superior temporal gyrus, combining the two areas re- moved separately in the previous studies. Pre- liminary results indicate that the total re- moval does produce losses in the retnetion of an auditory frequency discrimination and in auditory frequency thresholds. The defect does not appear to be confined to audition, however, but extends to visual and spatial problems as well, thereby raising a puzzling problem for interpretation. Somesthesis. — The over-all aim of this pro- gram is to identify the cortical mechanisms serving somesthetic sensation-perception, learning, and memory. In the past year, the work has concentrated on contributions to dis- crimination learning and performance made by regions outside the classical sensorimotor area, located in the hemisphere contralateral to each hand. One such contribution, made by the sensorimotor area ipsilateral to the hand tested, was described in last year's report: monkeys with ipsilateral sensorimotor lesions showed deficits in learning difficult form dis- crimination, whereas they were unimpaired in learning preliminary, easier discrimination habits. The opposite pattern of deficit has now been found in monkeys with contralateral nonsensorimotor lesions: They have enormous difficulty in learning the pre- 372 ANNUAL EEVIEW OF INTRAMURAL RESEARCH liminary habits, whereas once this basic strat- egy has been acquired, they show the normal rate of learning on the difficult form discrimi- nations. With respect to thresholds for detec- tion of size or roughness differences, the two le- sions had similar effects: size thresholds were unaffected, whereas roughness thresholds were elevated. The experiments show that all the factors necessary for normal somesthetic learn- ing are not contained within the contralateral sensorimotor area. Instead, it seems likely that the deficit depends on afferents to or efferents from the ipsilateral area and in this sense should be regarded as a true sensory or sen- sorimotor impairment. By contrast, the unilateral nonsensorimotor lesion (which included section of the forebrain commissures) has effects which are strictly contralateral and which appear to depend cru- cially upon the loss of cortico-cortical connec- tions. The effects of cerebral lesions on somesthetic functions are also being studied in man. Pa- tients admitted to the Clinical Center for sur- gical therapy of Parkinson's disease (lesions of nucleus ventralis lateralis) are being studied pre- and post-operatively. This nucleus is the origin of the major afferent system to the motor cortex, a region which may serve soma- tosensory functions as well. Quantitative tests of punctate pressure sensitivity, two-point dis- crimination, and point localization are applied to the face, hands, trunk, and feet in order to determine the nature and distribution of the possible impairment. Only a few suitable pa- tients are obtainable each year, and more are needed before results can be reported. Visuomotor Coordination. — Observations made on the monkeys with the unilateral non- sensorimotor lesion including section of the forebrain commissures suggested that, in addi- tion to the somesthetic loss described earlier, there is a striking deficit in visual guidance of the limbs contralateral to the damaged hemi- sphere. Tentatively, it can be concluded that there is a loss of crossed integration between the visual input to one hemisphere and the motor output from the other. Crossed visuom- otor reactions in animals with section of the commissures alone, as described in the litera- ture, have not yielded as striking or as con- sistent effects as those described here. The role of a unilateral cortical removal in produc- ing this "disconnection syndrome" appears to be unexpectedly important and calls into ques- tion the adequacy of the current conception of the mechanism involved in such syndromes. Cortical Mechanisms in Problem Solving Further attempts have been made this year to specify the nature of the behavioral loss pro- duced by lesions of the principalis area in the monkey. It was established long ago that this was the focal lesion in the frontal lobes for yielding the classical delayed-response and de- layed-alternaticn deficits. More recently, evi- dence was obtained suggesting that the major source of the difficulty in these tests for ani- mals with pricipalis lesions was the spatial feature. To pursue this possibility further, animals have been trained on an object alter- nation problem which contains the same delay and reversal features as those of the classical tests but differs from them in eliminating spa- tiality as a relevant cue. It should be noted that eliminating the spatial cue renders the problem inordinately more difficult for normal animals. Despite this, preliminary results in- dicate that a principalis lesion which is con- fined to the banks and depths of the princi- palis sulcus, while seriously interfering with performance on spatial alternation, leaves per- formance on the more difficult, nonspatial ver- sion completely unimpaired. If this preliminary finding is confirmed it will not only provide a dramatic demonstration of the specificity of the defect produced by principalis lesions, but it will also pave the way for an intensive in- vestigation into the nature of the spatial im- pairment. Cortical-sub cortical Relations and the Regula- tion of Behavior The classical view that behavioral responses are produced by cortico-cortical influences playing upon the precentral motor cortex has received little support in recent years. On the other hand there is increasing evidence that there is considerable regulation of behavior through cortico-subcortical mechanisms. Be- NATIONAL INSTITUTE OF MENTAL HEALTH 373 havioral studies implicating the caudate nu- cleus in behavior known to be subserved by frontal cortex, together with the recent dis- covery of a topographically organized system of direct connections between cortex and cau- date nucleus has focused our attention on this subcortical structure. Our earlier work had demonstrated that the head of the caudate nucleus subserves many of the functions of the frontal cortex with which it is anatomically related. However, recent anatomical findings suggested that a more pre- cise relationship existed; namely, that on the one hand, the anterodorsal sector of the head of the caudate and dorsolateral frontal cortex would have similar functions, and that, on the other hand, the ventrolateral sector of the head of the caudate and orbitofrontal cortex would have similar functions. In a behavioral study investigating this possibility it was clearly evi- dent that the anterodorsal caudate and dorso- lateral frontal cortex did have similar func- tions, but the evidence for the ventrolateral and orbitofrontal cortical relationship was, at best, equivocal. Further analysis of the data this year, however, indicates that the equivo- cal result was an artefact of the scoring pro- cedure and that the ventrolateral caudate and orbitofrontal cortex do indeed have similar functions. This same study had demonstrated that the tail cf the caudate and inferotemporal cortex also have similar functions, both being concerned with visually-guided behavior. One implication of this close similarity in function between caudate and cortex is that in the absence of cortex the caudate may be able to subserve cortical functions. If this is proven to be the case, a solution wall have been found to one of the major problems in neuro- psychology; a mechanism of vicarious func- tioning will have been discovered. Two strate- gies are being followed to explore this prob- lem. Both involve placing lesions in infant monkeys since it is known that cortical lesions sustained in infancy are likely to have less se- vere effects than the identical lesions sustained later in life. The first strategy is to examine the effects of partial and total frontal lobe ablations in infancy (60 days of age) and at a later age (2 to 3 years of age) on a wide variety of tests in which frontal lobe function is implicated. The second strategy is more spe- cifically concerned with the major question as to whether or not the caudate nucleaus sub- sumes functions of the cortex in its absence. For this purpose both infant and adult mon- keys are being prepared with caudate nucleus lesions and with combined caudate and cortex lesions .They will be tested later on the bat- tery of tests used for the frontal animals. The Section has seme preliminary data which sug- gest that a pattern of deficits and sparing will be found as a function of prefrontal lesions which will provide a sound basis for demon- strating whether or not the caudate is a locus of vicarious functioning. Two ether studies have been concerned with an analysis of the function of the caudate nu- cleus. The one followed the same design that was so successful in dissociating the spatial factor from the reversal factor in a frontal animal's impairment. The other study, analyz- ing the function of the caudate, utilized the methods of prism-adaptation studies. This woik has now been completed. Of the many cortical and subcortical lesions sampled, those in frontal cortex and in the caudate nucleus im- paired the process of prism adaptation. These findings lend further support to the notion that the caudate and prefrontal cortex have similar functions. It suggests further that both may be concerned with processes involved in visu- omotor coordination. Destroyers of Behavioral Function The Laboratory of Psychology is presently engaged in studies of two major areas which concern themselves with forces having a nega- tive impact on the quality of behavioral func- tioning: the effects of crowding and the effects of schizophrenia. Another area cf this kind of effect — that of brain damage — has already been considered in the part dealing with neu- ropsychology. Crowding Dr. Calhoun has conducted a literature sur- vey of population in its relation to mental health, as background for an extensive empi- rical investigation of the effects of crowding 374 ANNUAL KEVIEW OP INTRAMURAL RESEARCH in rats which he expects to commence in the next fiscal year. A 1000-page anthology based upon 400 recent articles and books has been assembled. This is serving as a basis for as- sessing the influence of changes in the size of the human population, its geographic distribu- tion, and its composition on mental health, and to identify major problem areas justifying more intensive investigation. This anthology is also serving as the starting point from which to explore a theory of concept creativity aris- ing from his research on the duration and se- quence of behaviors in the rat. This theory provides a method for computer-aided identifi- cation of ideas that may be most profitably associated in developing a projection from con- sensus opinion. Furthermore, it provides a basis for associations between novel ideas and consensus ideas which have the greatest prob- ability of generating useful new conceptual relationships. Dr. Calhoun, in his survey of literature on population and mental health, has concen- trated on papers in which the author has at- tempted to develop a general conceptual frame- work, or ones which try to define major issues which are now apparent or will be in the fu- ture. He is also concerned with methods for dealing with those identified problems, and, while he has by-passed strictly experimental or clinical studies, the major gaps in communi- cation between the several disciplines which study the interrelationship of population and mental health are apparent in these papers. Each discipline seems to have particular con- cepts of which other disciplines are unaware or unappreciative. Dr. Calhoun hopes to pro- vide a tool which researchers could bring to bear on the problem of population and mental health. Schizophrenia The work on schizophrenia which are orig- inally concentrated in the Officie of the Chief has enlisted the efforts of several persons out- side this office. This work has encompassed studies in perception, behavioral deficits of var- ious kinds, emphasizing particularly deficits of attention and problem-solving, and the rela- tions between performance deficits and psycho- logical arousal. The heredity problem has been vigorously pursued, particularly the heredity response in the "susceptible" person, as has the further extension and development of the the- ory of "segmental set." Over the past several yers, Dr. Carlson to- gether with Dr. Feinberg have studied the extraordinary lengthening of response-time which schizophrenic patients manifest in judg- ing the velocity of a moving visual stimulus. This effect is not attributable to inattention, lack of motivation, or misunderstanding of th* 1 task. It appears to be a distortion in the func- tional use of relatively short time-intervals, but from the literature on the subject and from our own results it is clear that the matter can- not be explicated adequately without investi- gation of some basic methodological and con- ceptual issues involved in the study of time- judgment. One major problem is that logically equivalent methods of assessing time-percep- tion do not always yield logically consistent results, even with normal individuals. As long as the reasons for such discrepancies remain obscure, it is not possible to determine the va- lidity of any given substantive effect with suf- ficient generality to be useful. These investigators have, therefore, under- taken a study directed toward a number of problems involved in understanding time-per- ception itself, as well as toward determining the nature of the particular deviation which seems to occur with schizophrenics. This study requires the collection of a rather large amount of time- judgment data from normal groups and schizophrenic and neurological patients. Most of the preliminary work involved in de- termining suitable experimental procedures and methods of analysis have been completed this year. Dr. Zahn's investigations continue to focus on behavioral deficits in schizophrenia, with emphasis on deficits of attention and on the relations between performance deficits and physiological arousal. During the past year emphasis has been given to studies involving the concurrent re- cording of the responses of autonomic varia- bles such as skin resistance, heart rate, finger pulse volume, skin temperature and respiration NATIONAL INSTITUTE OF MENTAL HEALTH 375 to stimuli which may be considered as varying in meaning or in demandingness. Previous work had shown that, while acute and chronic schizophrenic subjects are respectively equal to or greater than normals in physiological responsivity to "meaningless" auditory stimuli, they are significantly lower in physiological responsivity to the more demanding stimuli of a simple reaction time experiment. Similar investigations are in progress with identical twins and their parents and with par- ents whose biological or adopted children are schizophrenic. Preliminary analyses of the data of 12 twin pairs who are discordant for schizophrenia show that the relationships of physiological responsivity to the demanding- ness of the stimuli for the non-schizophrenic and schizophrenic twins are much like those previously obtained for unrelated normal and schizophrenic subjects, respectively. Reaction time differences show a similar relationship. This tentatively suggests that these phenom- ena are related to the symptomatic presence of schizophrenic rather than to a genetic "sub- strate." Further and more detailed analyses of the physiological characteristics of these subje:ts are in progress. Another study, done with Dr. Schooler of LSES, NIMH, investigated the influence of having a patient cooperate in a block design task with another "patient" (a confederate) as opposed to doing the task with just the ex- perimenter present on performance and on the level of arousal. Contrary to expectation, the performance level was improved by coopera- tion with the other "patient" but in accord with expectation, the level of physiological arousal was significantly increased by working with the other "patient." The huge amount of data collected in these studies has necessitated the development of a system for automatically reducing it by com- puter directly from analog tape records. Such a system for the GSR has been in operation during the past year and data from over 300 experimental sessions have been processed. A similar system for reducing heart rate data is currently under development. The "set index," a reaction time method of proven effectiveness for discriminating be- tween chronic and acute schizophrenics, brain injured subjects, ami normal controls, is being used to test subjects with psychomotor epilepsy before and after surgical (usually temporal lobectomy) treatment of the disorder. Our in- terest in this group stems from the frequent reports of their schizophrenic-like behavior. Dr. Zahn hopes to be able to determine whether a temporal lobe disturbance might be involved in the attenticnal deficits in schizophrenia. Another reaction time study with chronic schizophrenics concerns the effects on reaction time of shock reinforcement for slow responses. Previously we had found that this technique increased the speed of response markedly in chronic schizophrenic patients. The purpose of the current study is to replicate this and to deteimine the possible roles of information and arousal level in the improvement, Dr. Jerome and Dr. Young have been study- ing the problem-solving ability of schizophren- ics on HEPP. When conditions are provided to enable schizophrenics to make a maximal effort to achieve an understanding of the instructions, they are usually capable of solving the simpler HEPP problems by some means. They do, how- ever, manifest various deficiencies which are of the kind that have been characterized else- where in this report under the discussion of higher thought processes. Schizophrenic per- formance has been compared with that of nor- mal subjects, normal twin siblings and parents. Normal control subjects (of college sophomore level), whose mental test scores are far super- ior to those of schizophrenics, solve all prob- lems more efficiently and use more sophisti- cated heuristics than schizophrenics. This is, of course, expected. The performance of the nor- mal twin sibling is superior to that of his schiz- ophrenic sibling only on the average; the in- stances in which the schizophrenic is superior to the normal being less frequent than the converse. It is very common for at least one of the parents of a schizophrenic to exhibit obvious heuristic deficits such as disorganiza- tion of inquiry and poor comprehension of implications which are characteristic of the schizophrenic himself. Such parents often fail to generalize their solution strategy when the problem size increases and evidence more dif- 376 ANNUAL REVIEW OF INTRAMURAL RESEARCH ficulty in changing set when problem type ne- cessitates such a change. The focus of Dr. Rosenthal's research has be- come more and more sharply outlined around two points: (1) The contribution of heredity to schizophrenia in the context of different early rearing environments. (2) The nature of the personality-behavioral configurations reflecting this heredity component in the non- schizophrenic but "susceptible" person. In carrying out research on these issues, he is now actively involved in six research proj- ects, but to a different extent in each. A continuing study of the Genain Quadru- plets. His collaborator in this study is Dr. Olive W. Quinn, Professor of Sociology at Goucher College, They are following not only the clini- cal course of the girls, but also their life ex- periences and their reactions to them. However, they are not limiting their concern to a nar- rative description of events and behaviors in the usual kind of follow-up study. They plan to capitalize on the genetic identity of the girls and the mass of detailed history available of their developmental years. They hope to eluci- date more precisely the role of various person- ality functions in the development and expres- sion of mental illness, emphasizing the kinds of experiences that determine the response pattern developed by each girl with respect to the particular function, the reaction of in- volved persons to that response, and the conse- quent developmental sequences (especially their adaptational character) that lead toward schizophrenic behavior. We will include such functions as sexuality, aggression, dependency, interpersonal attachments, and perhaps some others. Studies of discordant MZ twins and their parents. Dr. Rosenthal has reduced his partici- pation in this study for several reasons: (a) It does not help to elucidate the contribution of heredity to schizophrenia, as he thought it might when he first proposed the study. This earlier belief was based on differences that were found between discordant and concordant MZ twins in Slater's twin series, but subse- quent studies by Kringlen and Tienari did not show the same differences, (b) He disapproves of the method of sampling used in the study, and consequently he is not certain that the families brought in for study are representa- tive of discordant-twin families in general. (c) The value of studying psychological differ- ences in the twins themselves is limited since the differences by and large reflect the ravages of the illness, which can be studied incompari- sons of schizophrenics and normal controls. This is not a final judgment but he does not expect it to change. A study in Israel which is designed specifi- cally to get at the two main interests men- tioned above. His principal collaborator is Dr. Shmuel Nelga rof Kiryat Tivon, Israel. The study involves two presumed genotypes (schizo- phrenic vs. non-schizophrenic parents) and two different rearing environments: the usual nuclear family setting vs. the kibbutz way of life. The design permits the partitioning of variance into proportions associated with type of parent, type of rearing, and interaction be- tween parentage and rearing with respect to a variety of personality characteristics and test measures in children aged 9 to 12. The study did not get under way until September, 1965 because Dr. Nagler had to fulfill earlier com- mitments. The preliminary phase has involved discus- sions with several heads of hospitals, educa- tional and mental health officials to obtain per- mission to use hospital and school records for locating index and control families. This phase has not been easy or quick, but Dr. Nagler has managed to obtain cooperation at every point so far. Most cases have been selected and it looks as though we can meet our hoped for n of 20 Ss in each of the 4 cells in our design. A full staff of very able people has been assembled including, besides Dr. Nagler, a psychiatrist, a psychologist, two social workers, a secretary and a consultant who is Professor of Psychol- gy at Hebrew University. The staff has been holding meetings regularly to deal with many problems: the tests and procedures to be used, the approach to the kibbutzim, schools and families, the length of time set aside for the examination of the children, the location of the examination site — which will probably be Hebrew University, the timing of the testing (two half days), observational procedures in NATIONAL INSTITUTE OF MENTAL HEALTH 377 natural settings, the type of interviews to be used with the child and key informants, the selection of controls, etc. During this phase, Dr. Rosenthal has been in close contact with Dr. Nagler. A dry run evaluation of tests and procedures using non- subjects will be started in mid-April. Dr. Ro- senthal will visit Israel in mid-May to review the study and help the staff settle on a final selection of procedures. In all probability, the examinations of the actual subjects will begin in June or July. A study in Denmark to evaluate the inci- dence of schizophrenia in the biological and adoptive families of children who were adopted by non-family members and who subsequently became schizophrenic. Dr. Rosenthal's collabo- rators are Dr. S. S. Kety, Dr. P. Wender, and Dr. F. Schulsinger. At this stage, all index cases (schizophrenic and manic-depressive adoptees) have been selected as well as a num- ber of their controls. In the meantime, the group have begun the study of the families in those instances where selected controls have been obtained. This last phase of the study should take another year or more. A study in Denmark of children who had a schizophrenic parent but who were given up for adoption to unrelated persons at an early age. Dr. Rosenthal is carrying out this project with Dr. S. S. Kety, Dr. P. Wender, and Dr.F. Schulsinger. This study began in August, 1965 with actual examination of subjects begun in November, 1965. To date, about 25 subjects of each index and control, have been examined, and 20 index cases and 20 controls selected. At this point, it cannot be determined how many index cases the search will yield, but this phase of the study is progressing well and we may not be far off our original estimate of 100 index cases. If the yield of cases reaches that high, it will probably be impossible to test the origi- nal group of 100 in the two years for which they originally budgeted. The study will have to be extended or a number of subjects will have to be omitted from the study. Thus far, very few cases have been lost. A very prelimi- nary glance at some data gives the group rea- son to believe that they will find theoretically relevant differences between the index subjects and their controls on a number of aspects of personality functioning. A study of adoptive parents and natural par- ents of schizophrenic subjects, being done at the NIH. To date 10 families have been exam- ined in which the schizophrenic child was adopted and 3 families in which the schizo- phrenic subject was the biological child. The latter families are matched to the former with respect to SES, sex and age of the child, age of the parents, et al. The two groups of parents will be compared with respect to various as- pects of personality functioning. An addi- tional 7 control families have been selected and it is expected that all will be examined by the end of the summer. If the results of the study are promising, the investigators will plan to extend it to include a more systematic sam- pling and a larger battery of tests. During the year Dr. Shakow has continued his work on previous empirical studies of schiz- ophrenia and further analysis of the extensive data for its theoretical implications. His three major productions have been the preparation of papers on schizophrenic perceptual proces- ses, a consideration of the implications of the findings from the experimental psychological studies of schizophrenia for the understand- ing of normal function, and a detailed paper on the problems of nosology in psychopathol- ogy and their relationship to research in the field. The empirical study reported deals with auditory apperceptive reactions to the tauto- phone by schizophrenic and normal subjects. The vowel patterns which comprise the stimuli of the tautophone, the auditory apperceptive device used, were administered to 25 schizo- phrenic male patients (11 hebephrenic, 6 par- anoid, and 8 of other types) and to 20 normal male subjects. Quantitative, as well as quali- tative, analyses differentiated the groups. These analyses indicated that the normal sub- jects appeared to accept the tautophone as an apperceptive task, the hebephrenic subjects personalized the situation, and the paranoid subjects seemed to impersonalize the task. The profiles found in these groups are consistent with findings from other perceptual, appercep- tive and projective studies. The personality 378 ANNUAL REVIEW OF INTRAMURAL RESEARCH structure of schizophrenics and of schizo- phrenic subtypes are considered in relation to two aspects of attention — scanning - and articu- lation. The major finding is that schizophren- ics show extreme forms of behavior — either under- or over-scanning, or under-or over- articulation. In the consideration of the implications of schizoprenia research for the understanding of normal psychological function, Dr. Shakow first considered the general relationships of psychopathology to normality, then reviewed several decades of research in schizophrenia, giving the major findings and the inferences for psychological functioning in schizophrenia and normality. These generalizations were then seen in the context of various theories with regard to psychological functioning par- ticularly the different between the establish- ment of generalized and segmental set. In the discussion of the problem of nosology, Dr. Shakow considered first the general atti- tude the investigator should take in confront- ing the problem of classification of mental disorder. He viewed this in the context of his personal associations with the problem as pointed up by his own studies of schizophrenia. Various underlying issues of the classification problem were considered. Dr. Shakow con- cludes with a summary of what he learned dur- ing his own efforts in the categoraization of mental disorders, both the problems involved and the cautions to be kept in mind when ap- proaching these problems. These included the FOLLOWING POINTS: the use of multiple simple approaches, care in the use of controls with either direct or inferential studies, a prop- er balance between the holistic and the seg- mental approaches, stress on the nomethetic based on extensive idographic studies, an in- creased emphasis on metaphoric as opposed to literal approaches both in the use of generali- zations and in the use of structural/dynamic descriptions when they are based on earlier careful use of the empiric, and a concentration on the prior acquisition of context experience as background for the employment of method. Enhancers of Behavioral Function Some preliminary work in this broad area undertaken by Dr. Bergman in the context of psychotherapy was discontinued with his pre- mature death. The only investigations being currently conducted in this important area are those by Drs. Parloff and Datta of the Section on Personality on Westinghouse winners. In view of the fact that the more and less creative samples investigated by Drs. Parloff and Datta are clearly differentiated on per- sonality variables, a study has been under- taken to determine whether characteristic par- ent-child relationships are associated with demonstrated differences in subsequent crea- tive performance. Subject reports on their re- lationships with each parent during each of three age periods are currently being analyzed but no conclusions are available as yet. Further evidence revealed that the person- ality patterns which are associated with high creative performance in a group of adolescents studied by Dr. Datta and Dr. Parloff are con- sistent across vocations. This finding is of par- ticular interest since the data analyses reveal many highly significant personality differences which are attributable to vocational interest. A similar analysis of variance was performed on data obtained from four highly creative adult samples and their appropriate controls. As in the adolescent sample, personality fac- tors are clearly associated with creativity level independent of the particular vocation. A com- parison of the personality variables which dif- ferentiate both the adolescent and the adult creative samples from their control groups re- vealed the following similarities: high levels of ambition, activity, forcefulness, self-accep- tance, and spontaneity; ability to recognize and admit views which are unconventional; a high level of independence and self-reliance; and a broad range of interests. The person- ality features which characterize and differen- tiate the adult creative sample are very similar to those of the adolescent creative sample; how- ever, the members of the adult creative group appear to be even less concerned than the adolescent group with making a good impres- sion on others, are more dominant, have a greater sense of well-being, and are more flex- ible. A two-year follow-up of the adolescent sample reveals that although the personality NATIONAL INSTITUTE OF MENTAL HEALTH 379 characteristics remain fairly stable, the changes which occur in the "high" creative sample appear to be in the direction of moving closer to the personality pattern which has been identified as characterizing the adult cre- ative man. Most impressive, perhaps, is the fact that the adolescent creative sample be- came significantly more flexible than the con- trol group during the two years since high school graduation. The two-year follow-up re- testing of the total sample revealed that the "high" creative group continued to be differ- entiated from the "less" creative group on the same personality scales which had earlier dif- ferentiated them. Since the data analyses based on the ad- olescent sample indicate that a longitudinal study is warranted, periodic follow-up studies of this group and the replication sample will be undertaken. Another aspect of the crea- tivity research program will be concerned with experimentally investigating variables that seem to affect creative performance. In the view of the investigators, one of the factors which has been neglected in these studies is the role of the standards and values of the "creative man." It may not be sufficient simply to train individuals to report a number of "original" ideas, for the creative man must also be able to select from among his ideas those which are worth pursuing. On the basis of some preliminary work undertaken by Dr. Datta, a research project will be conducted to assess the relationship between the sub- ject's standards and his judged creativity level. The research will also investigate the subject's capacity to produce "creative" works in accordance with (a) the consensual stand- ards of judges, and (b) the subject's own standards for creative performance. Theoretical, Methodological and Technical Advances During the course of the year several ad- vances have been made along theoretical, methodological and technical lines. An important advance in the logic of meas- urement was proposed by Dr. Stanley. In- creasing evidence that neonatal dogs can learn, when correlated with similar evidence obtained from human infants by other in- vestigators, has suggested a new approach to behavioral quantification. Rather than taking behavioral theory as the basis for measure- ment, the approach starts with a general theory of measurement. The general theory is then applied to behavior as recorded in learning situations. Considerable work on specific equations remains to be done, but it is already clear that the mathematical man- ipulations of idealized "data" curves will gen- erate specific research. For example, mathe- matical analysis implies that continuous (100%) operant or instrumental reinforcer action can be viewed as functioning indirect- ly. That is, the idealized curves alone do not imply that the reinforced response is being strengthened. Rather, they imply only that nonreinforced responses habituate or extin- guish. This view of instrumental and operant reinforcer action. is similar to, but not iden- tical with, aspects of contiguity theory, statis- tical learning theory, and an evolutionary analogy of reinforcer action suggested by others. Methodological and technical advances have been made in various parts of the Laboratory. Dr. Schaefer has developed some new measurement techniques. One of the problems facing the investigator who is concerned with the antecedents of personality development is the fact that he must frequently depend upon retrospective accounts from the subject or patient regarding significant life experiences. Dr. Schaefer, in cooperation with Dr. Nancy Bayley, has undertaken a much needed study to test the validity of the adult subject's re- ports of his early relationships with his parents. By utilizing material obtained from a longitudinal study, the Berkeley Growth Study, it has been possible to compare the subject's ratings of his mother's behavior to- ward him during adolescence and infancy with the ratings made by psychologists who actual- ly observed the parent-child interaction at the time of the subject's infancy and adolescence. It was found that subjects at age 36 were able to report the behavior of their parents which had occurred 25 years earlier in a 380 ANNUAL REVIEW OF INTRAMURAL RESEARCH manner which agreed closely with the de- scription obtained at the time from the pro- fessional observer. The correlations between the subject's etrospective reports and the ob- server's reports regarding parent-child re- lationships during infancy were, however, much lower. This research supports the use of subject's recall of parent behaviors at least as early as age 11. Dr. Schaefer has also investigated a simple technique which could be devised to provide systematic in- formation concerning the individual child's behavior and adjustment from early childhood through late adolescence. He is attempting to determine whether school teachers, if properly trained and if provided with good methods, can be a source of such data. One of the ad- vantages of such systematic data collection would be the early detection of maladjusted behavior. Dr. Stanley, because of the nature of his research, has worked hard to achieve auto- mation of his apparatus, which would result in the improvement of the measurement of early sucking behavior, make better control over liquid stimuli possible, and reduce the need for arduous work schedules of personnel in the unit. Considerable time has been spent by this unit together with the Technical Development Section in designing and testing automated research equipment. The equip- ment has proved adequate for maintenance of healthy infant animals, but inadequate for research. It is hoped that the equipment will appreciatively reduce the need for around-the- clock work by personnel. Dr. Jerome and his group have also made technical advances in the development of a Sequential Relations Apparatus. The SRA, adapted from the Logical Analysis Device, presents a series of problems involving the combination of a number of elements into vari- ous causal relations. The subject's task is to analyze the relations that exist between a set of input buttons and a goal signal. When he has discovered how to produce the goal signal under the constraints of the problem, he has solved it. The whole procedure is controlled and recorded by an IBM 1620 computer; this allows a maximum flexibility for developing proce- dures and experimental controls which would be impossible with special purpose equipment. Five subjects have been run in the exploratory phase of this study;procedures and problems are still in the developmental stage. The 1311 Disk Drive which has been added to the NIMH computing system operated by the Laboratory has greatly expedited several phases of the data processing work. However, since more than half of the logged time on the computer is consumed by the preparation of copy, the efficiency of the facility could prob- ably be doubled by the addition of a moder- ately fast line printer. Mr. John James, the programmer associated with this facility, has written a machine-free macro-language implemented by the IBM 1620. It is anticipated that it will greatly reduce the confusion of tongues created by having access to several machines, each with its own vernac- ular. The system permits the programmer to write in a single computer language when us- ing an assembler for which Mr. James has developed macro instructions. He is reporting on this language to the Fall Joint Computer Conference of AFIPS (American Federation of Information Processing Societies). Dr. Rosvold's group has made progress with several new devices. Preliminary testing of a remote stimulation equipment has been com- pleted, and all units are satisfactory. A differ- ent switching mechanism will have to be developed, however, for reliable switching from electrode to electrode. General Electric is cur- rently working on this problem and appears to be ready to supply the more reliable switch. The device for locating structures within the brain by using changes in impedance from an electrode passing through tissue has been im- proved to the point where it is now a highly reliable instrument. It will be used in experi- ments requiring accurate locating of subcorti- cal structures. An ultrasonic device for making subcortical lesions deep in the brain without damaging intervening tissue should be deliv- ered soon. Administrative Aspects of Laboratory I should like finally to review some special aspects of the Laboratory's functioning during NATIONAL INSTITUTE OF MENTAL HEALTH 381 the last year. First let me consider several per- sistent problems still seeking solution before discussing the general condition of the Lab- oratory. Despite a generally favorable atmosphere, four major problems still trouble the Labora- tory: animal procurement, isolated units, equipment, and salary level. Difficulty in pro- curing appropriate animals for research in the behavioral sciences has long been a problem because NIH's system favors users of large numbers of animals whose only requirement is good physical care. Our investigators, however, require animals whose behavioral histories are known. Because of the inadequacies of the NIH system we have been forced, on the one hand, to sacrifice precious experimental space for the mere holding of animals and, on the other, to spend valuable time of experimenters on the mere procurement of appropriate animals. I trust that the work of Dr. Stanley's Com- mittee, together with the efforts being made with the Laboratory Aides Branch, will im- prove this handicapping situation. The second problem is that of equipment. A great deal of effort is being devoted to the construction of special apparatus systems. The systems are carefully designed, but their con- struction has been held up by numerous delays in obtaining funds, in contract negotiations, in delivery by a contractor, or in fabrication by NIH facilities. Professionals within a Sec- tion, who should be devoting their time to re- search activities, would have to do a major portion of this work to avoid delay. Even if this is sometimes possible, as it is generally not, without exception it is undesirable. The result is frequently that experiments which should have been completed last year are not undertaken at least until the next year. NIH's facilities for supplying suitable apparatus for experimental psychological research needs careful examination. The efficient handling of isolated facilities such as the Poolesville Farm is a third major problem. The administrative problems attend- ant upon the establishment of a new research unit in rather primitive housing have, during the past year, channelled time and effort away from productive research. The development of such facilities would be facilitated by the as- signment of special administrative assistance during the formative stages to help with such particularly difficult problems as the procure- ment of personnel. Perhaps an administrative officer should give a specified amount of time (say a day, a week, or whatever time seems appropriate) to such new operations until they are well established units with their own ad- ministrative force. Despite the problems at Poolesville, the refinement of unique research apparatus, the conduct of current research, and the planning of future research emphasis is gratifying. The morale of the staff remains high and is boosted still higher by the antici- pation of organizing a first class productive research laboratory when the prospect of the new Animal Behavior Laboratory building be- comes a reality in FY 1967. The fourth major problem is the super-grade restriction of behavioral scientists. Unless the present unfair discrimination in such promo- tions is ameliorated within the next few years, the continued employment, as well as recruit- ment, of promising and productive scientists in our area will be jeopardized. New developments hold much promise for the Laboratory. The development of the Pooles- ville Farm Operation and the new Child Re- search Building should enable us to extend our work in the respective areas involved. I have already said something about the Poolesville operation in this respect. Let me add a few words about the Child Research operation. The research of the Section on Early Learning and Development outlined earlier represents, es- sentially, the research themes which are to be emphasized in the reorganized Section when it moves in about a year to its new facility in the Child Research Center. Plans are being made to add additional personnel to partici- pate in experimental researches under the themes outlined. Until now, the lack of facili- ties has limited us considerably in the experi- mental work we could do. It is hoped that mcst of the present researches listed under the five foci will be completed and reports from them published before the Section moves to the new research facility where it will con- tinue with new studies in these areas. 382 ANNUAL REVIEW OF INTRAMURAL RESEARCH The possibility of establishing a Section on Behavioral Genetics should be considered in the context of other possible administrative changes. A separate section in the Laboratory of Psychology concerned with these and re- lated research problems seems administra- tively desirable because studies in this area are expensive and because they are important to the scientific development of abnormal psy- chology and psychiatry. The proposed section should remain small for the first few years, dedicated to the successful completion of the studies already begun and additional ones ad- dressed to the same basic issues. In conclusion let me say that both the morale and productivity of the Laboratory are good as evidenced by the body of this report and the publications which have come out of the Laboratory during this year. We have also had a group of visiting scientists and fellows of high quality, a condition we expect will continue in the coming years. It has also been gratifying to witness the recognition that many of the members of the Laboratory have received in the positions they have been offered by other institutions, as well as the consulta- tions they have been called upon to provide in areas directly connected with their fields of competence. CHILD RESEARCH BRANCH Clinical Investigations Introduction — Now completing its seventh year of longitudinal research on three inter- connected problems — the exploration of psy- chological patterns of marriage, of early par- ent-infant relationships and of stable indi- vidual differences between infants from birth to age three years — the Child Research Branch program is at a major transition point. The data from the early studies at three develop- mental stages (initial marriage relationship, initial parent-infant relationship and the pre- socialization period when the first infant is 21/2 years old) has not been nearly completely analyzed and reported. Plans for the next lon- gitudinal study are complete. By means of this new program to be initiated in September 19GG, we will investigate whether the patterns dis- covered on pilot samples ranging from 30 to 100 subjects can be replicated on samples ranging from 100 to over 1000 subjects. As always the research planning for the core lon- gitudinal study by three Sections of the Branch is carried out in a coordinated fash- ion, thus facilitating comparison within the same family of marital variables, parent- infant behavior variables, and infant behavior variables. At the same time, each Section re- mains free to pursue special interests of pri- mary concern within the particular area. Closely tied in with the new and expanded size of the program is its steadily growing requirements for personnel and for space. A number of new positions, not now available, are urgently needed. It was indeed disappoint- ing, though not totally unexpected on the basis of past experience, to have another six to nine months delay appear before the implementa- tion of our new Developmental Research Build- ing. This delay was occasioned by a dispute over the location of a new highway on the southern boundary of the National Institutes of Health reservation. Unfortunately the re- siting of the building will also necessitate an expensive re-traversing of the tentative stage in the Building planning process; hopefully this will not cut so deeply into available money for construction that either the space within the building itself will suffer or that new funds will have to be found. The plan is for an imaginative new building which will meet the best prognostications not only as to the needs of this program, but also to meet the general requirements of conceivable future basic re- search programs on early family development and on infant development. The unique and spacious "outdoor" play yard enclosed within the walls and ceiling of the building will pro- vide an unparalleled opportunity for control- ling the effects of weather on the behavior of preschool children the year around. Much improved facilities for observing and record- ing the behavior of adults and children will be provided throughout the building. Limited space will also be made available to investi- gators within the Laboratory of Psychology and the Adult Psychiatry Branch, National Institute of Mental Health, within the build- NATIONAL INSTITUTE OF MENTAL HEALTH 383 ing. There will also be, at long last, some space for Visiting Scientists and for other col- laborative studies which we have not been able to accommodate in the past. As now sched- uled, and unless further unforeseen delays occur, the building will be open in time for us to utilize the new nursery school laboratory for the next longitudinal study. That is to say, our present facilities in Wilson House will be used to gather early data on our 1000 or more recent marriages, but by the time their in- fants have reached age 2 1 /? and are ready for study in the nursery school, we should be moved into our new Building. One difficult problem in planning the new program is to estimate the readiness of the Branch to engage in collaborative studies with other intramural research groups. It has become obvious that the more diverse the in- formation included within an expensive longi- tudinal effort of this kind, the more opportu- nity would result for exploring a wider variety of hypotheses. On the other hand, we have dis- covered in some past studies that we have begun to pass the point of no return in de- manding a heavy worked of time and energy from our families, so that fatigue effects or shifts in willingness to cooperate begin to ap- pear. The program is somewhat different from much research going on both at NIH and in other developmental laboratories in Universi- ties because of its concentration on the effects of relatively concrete and simple variables, like sex of the child, birth order, whether bot- tle or breast fed, how much involvement the parents have with relatives, and the like. Other investigators have urged greater invest- ment in variables like cognitive or perceptual style — and other personality variables — but as a strategic matter, it does not seem unrea- sonable to try to define the effects of subcul- ture, birth order, sex of the child, and so forth before trying to incorporate variables which while perhaps more appealing theoret- ically in an abstract sense, may well have less predictive power when trying to create de- velopmental typologies. A somewhat similar problem has arisen this past year also having to do with the explora- tory and basic-naturalistic orientation of the program, somewhat at variance with the cur- rent ethos of mental health investigators. We tried and failed to use the foreign grant mechanism to gain support for a cross-cultural collaborative study with the Department of Neuropsychiatry in Taipei, Taiwan. While many factors contributed to this failure, a significant one was the attitude of Study Sec- tion members, an attitude which I have had occasion to observe in relation to other natu- ralistic and basic exploratory studies of be- havior. This is an attitude which places higher value upon preformed and limited hypotheses and upon narrow experimental design than upon an open matrix of alternative hypotheses and a design based primarily upon naturalistic observations, supplemented by more precise experiments only as the problem becomes clearer. Essentially, as for a number of years, I continue to feel that a major obstacle to the progress of mental health research is the un- derestimation of the complexity of the phe- nomena with which we are confronted and — much too often under the pressure for quick "clean" results — a too-great need for prema- ture definition of the nature of the problem under study. Particularly with the new mathe- matical tools and the increased computer re- sources now available, a more honest and com- plete confrontation of the problems of develop- mental research on children and on families is now increasingly possible. There is every expectation that this program may make a substantive contribution to better understanding of the interaction of congenital (possibly genetic) factors with family rela- tionship factors in shaping the behavior and experience of the infant as he grows to age three years. At this time we have clearcut variables in both these domains of interest which are gathered on all our longitudinal subjects. With this in prospect, it seems ap- propriate during the next year to lay more firm plans than in the past for cross-cultural collaborative studies. For there is always a considerable danger, no matter how well it appears that we have controlled for interven- ing variables, that our theory of the interac- tion of mother-father-and-infant across the early developmental stages of the infant's life 384 ANNUAL REVIEW OF INTRAMURAL RESEARCH has hidden biases deriving from the fact that all the data was gathered within one single culture. The identical behavior can have com- pletely different experiential meaning not only from family to family within the United States, but in a much more radical sense be- tween different national or sub-cultural envir- onments. Until these studies are carried out, we will not have tested the limits of our the- oretical conclusions about what is truly sig- nificant in the interaction of parental behavior with infant behavior. If similar results can be obtained crossculturally, and while controlling for the relevant congenital factors in the in- fant, we will have gone a long way toward demonstrating the validity of specific patterns of parent effects on children and specific child effects on parents. Patterns of Middle Class Early Marriage The intensive pilot study of 50 recently mar- ried couples strongly suggests that, within the middle class at least, four dimensions of the psychological relationship between husband and wife may in a rather critical fashion serve to shape a great variety of behavioral and attitudinal elements in the marriage. Perhaps the most prominent of these is the question of how involved and how attached the hus- band and wife are to their families of procrea- tion. We are concerned here with a variety of kinds of involvement, including the frequency of telephone contact, of meal times and of other social occasions together; included also is the participation of the older generation in daily decisions made by the couple, the involve- ment of the husband in a family business, and a variety of other meaningful week-by-week personal involvements. There is an apparent linkage between high involvement with the older generation during the newlywed phase of marriage and professing the Jewish faith; this linkage may be stronger with the less intellectual couples and with families where there is an investment on the part of the hus- band in a family business. Another dimension with considerable statis- tical power to separate patterns within the pilot sample is the wife's or husband's report of a variety of family difficulties during child- hood or adolescence. These include complaints about frequent arguments in the family, overly strict discipline and other forms of chronic disturbance, illness and unhappiness. The in- teresting finding emerged, which is consistent with the concept that a developmental stage during the adult period of life may have differ- ential impact on the two sexes, that husbands who make these complaints become over- invested in career ambition and less intimately involved with their wives, tending to select rather child-centered (as opposed to husband- centered) women to marry. With the wives who enter marriage with these complaints about their past, one finds a more open expres- sion of conflicts directly with the husband in the newlywed phase, interpersonal marital conflicts which are enacted in many social situations as well as in private with the hus- band. From these preliminary findings we have derived hypotheses to be tested in the next longitudinal study; should these hypotheses be later confirmed they will be clearly relevant to preventive psychiatric planning for interven- ing with young families in the middle class community. In addition the question of whether the young wife is, in early marriage, involved pri- marily with her marital relationship, or in using the marriage as an immediate jumping off place for producing a brood of children, also has turned out to differentiate many other behaviors and attitudes among our sample of couples. Finally, the direct observation of hus- band-wife communication during experimental situations has provided the dimension of ra- tionality vs. affectivity while solving a new problem. There is a puzzling but intuitively not surprising connection between logical com- munication style without laughter, without self-derogation and without hostility, and a close attachment by a couple with their rela- tives and parents. This too will be investigated in the next longitudinal study. It is an illus- tration of the fulfilment of one of the early aims of the Child Research Branch program, namely to develop new brief tests of family communication and problem-solving which would bear a dependable relationship to larger social role and family structure patterns. Here NATIONAL INSTITUTE OF MENTAL HEALTH 385 the color matching test and the form match- ing- test have considerable value, although as time goes on very likely there will be further technical improvements. At any rate these techniques — as well as some developed by other investigators elsewhere — do represent a break- through methodologically toward the day when brief methodology for studying the psychol- ogy of families can be built into large scale epidemiological and population research. In addition, considerable work is ongoing toward the improvement of paper and pencil instru- ments in our marriage research; indeed the large sample of 1000 couples will receive only paper and pencil instruments with the color matching test. A variety of new problems are upon us this coming year as a result of processing samples of subjects about ten times as large as we have heretofore dealt with. A new computer remote station is required to prevent immediate back- up of large amounts of unprocessed data and also to permit immediate data searching and analysis during the course of the project. In order to deal with this new sample three new staff fellows (psychiatrists) will join us July 1st. We will be selecting subjects from the entire Washington area, with the considerable aid of the Capital Beltway; whereas in pre- vious work we limited ourselves more to the Northwest Washington and Montgomery County areas. Early Parent-Infant Patterns Research on the early phases of develop- ment of the mother-infant relationship, and to a much less extent the father-infant relation- ship, has progressed now to the point where useful techniques and variables are in ongoing use during the first month of life, during the third month of life, and at a year and a half of age. During the earlier stages of development the data analyzed and reported so far in the literature from our program is in agreement with other investigators, in pointing to the salience of the sex of the infant and of the state of arousal of the infant as determinants of parent-infant behavior. Our group has been able to link these primary biological variables with pre-pregnancy attitudes of the mother-to- be as regards her desires for maternal contact with an infant. It also appears that when di- rectly observing the mother and infant to- gether in their home, the style of the mother's behavior when attempting to quiet an irritable baby or when attempting to provide the in- fant with pleasurable arousing stimulation, do systematically interact with the above infant variables. The past findings from this area of research are now being replicated on an inde- pendent sample carefully controlled for parity and with a wider range of subjects than has been true before with respect to maternal age, ethnicity and education. This does not repre- sent a serious excursion as yet by the Branch into studies of sub-cultural contrast beyond the white middle class within the urban com- munity but rather represents an attempt to get as great a range of psychological differences in our exploration of early parent-infant behav- iors. Studies of Individual Differences in Infants Work in this Section has continued to pur- sue the earlier discoveries of an infant leth- argy pattern, which appears to predict a de- pendency and contact-need pattern in the nursery school, as well as to pursue the rela- tionship between respiratory and feeding be- haviors in the newborn period to later nurs- ery school persistent task-orientation behav- ior, and avoidance of socialization. As a result of these interests, and in order to prepare for the next longitudinal study, there has been a concentrated effort to extend and improve our assessment of the neonate in the areas of sleep behavior and other psychophysiological parameters. As mentioned above, there are strong suggestions of a linkage between arousal and sleep variables in the neonate with mediation of maternal contact-comfort. The program has also discovered that an index of mild physical anomalies, an index obtained by the nursery school teacher, seems to have considerable value. To be specific, we are investigating the hypothesis that in the third year of life anomalous finger and toe construction, together with a high palate, re- lates to high scores on impulsivity, emotional lability, low tractibility and high levels of ag- 586 ANNUAL REVIEW OF INTRAMURAL RESEARCH gression. The interesting - thing about this is that these findings are all within a non-brain- damaged group of children from which — by all available criteria — children having even minimal likelihood of brain damage had ap- parently been eliminated. Thus, as has been increasingly evident from recent literature, the factor of very mild hidden morphological immaturity or very mild brain damage, is identifiably significant in determining psy- chological development. In the work being planned there will be a new focus on first born children to fit in with the total longitudinal study as well as on ex- panded studies of visual attention behavior, feeding behavior, birth weight, respiration and their connection with early style of so- cialization. Three new staff fellows will join the Section this year (a pediatrician and two psychologists) to assist in carrying forward these new directions. Toward A Theory of Marital Experience Until the last few years the focus of most developmental research has been on "hard variables" within the tradition of the natural sciences and of experimentation. This has been a recent historical advance as, with better con- trol over variables and with more resourceful data reduction techniques, many new concrete facts are emerging. Nevertheless there are re- strictions upon our understanding of the child's development if research is confined to the study of overt behavior without reference to inner experience. Ultimately the experience of the family constitutes the matrix which nurtures the child's personality; the behavior of family members constitutes only direct or indirect and partial expressions of actual ex- perience. From a theoretical point of view what is most needed now is the development of new models for understanding process variables within family relationships, models which are tied to operational criteria but which at the same time are concerned primarily with ex- perience. During the past year two investiga- tors on our staff have begun work on such a model, which is concerned with the experience of intentionality at any given moment during an actual ongoing relationship between a hus- band and a wife. Our interest here is to in- vestigate systematic relationships between sev- eral ways of experiencing intentionality and how these may be related to certain forms of conversation between the couple. A variety of techniques are being used: interviews, home visits, and movies. Preliminary theoretical pa- pers are in various stages of preparation, hav- ing to do with the concepts of play vs. game as forms of interaction and having to do with imaginative vs. unimaginative rigid modes of expressing intention. New Developments The Branch is beginning to put more effort into intensive clinical case evaluations of cou- ples who represent salient patterns with re- spect to theory or to frequency of occurrence. Completely adequate staff resources for carry- ing out such clinical investigations are not available but some studies of this kind are getting under way. Carrying out clinical in- vestigations, often involving a fairly intensive relationship between one or more of our staff and one or more members of the subject- family, does not precisely conform to previous administrative models for research on normal volunteers. Although these investigative studies may provide certain learning or insight to the subjects in the course of the study, their purpose is in an actual sense not aimed psychotherapy. In those small numbers of cases where the families do suffer from anx- iety, treatment-like issues may, however, be- come involved in the process of investigation. But this is of course true of transference issues which intrude into any intense continuing hu- man relationship. Without regard to whether the study is being carried on by a physician, psychologist or social scientist on our staff, these clinical investigations are not primarily being carried out in a medical treatment con- test but rather are being carried out in the context of social science research. With a large number of young couples being studied as to matters personal, and with the very brief contacts anticipated with many sub- jects with whom we do not have the oppor- tunity (unlike in our past work) to develop a NATIONAL INSTITUTE OF MENTAL HEALTH 387 meaningful relationship, it is inevitable that the future will bring an increase in episodes of community misunderstanding. These arise from disturbed families as well as from indi- viduals who do not comprehend the connection between scientific investigation of family life and improving public health practice. As noted above, the extension of certain studies in a very tentative fashion, to include a few lower socioeconomic subjects and to in- clude those who did not graduate from high school, or who have attained an advanced pro- fessional degree, and to include older couples beyond the early stages of family development, will give us a broader spectrum of pretest data to use in planning possible comparative studies with our core group of middle class young family subjects. The opportunity to be relieved during the coming year by my colleague, Richard Q. Bell, Ph.D., of the administrative work in order to reflect upon the scientific and health impli- cations of our findings, and to engage in needed theoretical work, will provide further perspective on the problems referred to in this Annual Report. ADULT PSYCHIATRY BRANCH The activities of Adult Psychiatry Branch staff fall into four main categories: (1) plan- ning and execution of specific research efforts, as spelled out in the Individual Project Re- ports and summarized here; (2) psychiatric clinical care and clinical training; (3) psychi- atric research training; and (4) consultative and collaborative work with colleagues and professional groups both at NIH and else- where. Research activities are divided into five sec- tions, as follows: (1) Family Studies, (2) Studies of Personality Development, (3) Twin and Sibling Studies, (4) Studies in Psychoso- matic Medicine, (5) Studies of the Psycho- physiology of Sleep. Thus, a broad range of interests are represented, from psychosocial to psychobiologic, in the different parts of the program. The in-patient clinical work is conducted within two 12-bed wards in the Clinical Cen- ter; in addition, the Family Studies and the Personality Development Sections work with a number of out-patient families. Also, when ap- propriate, the cooperation of hospitals and re- search organizations elsewhere is obtained in order to gain access to larger samples of re- search data. For example, we have been able to analyze psychological test data collected elsewhere in order to verify leads which have developed from data obtained in our own pro- gram. Although most of the research in the program is with human beings, it has become increasingly important for certain psychobio- logic hypotheses to be explored first in animal studies. Family Studies Three sections in the Adult Psychiatry Branch — the Section on Family Studies, un- der Dr. Wynne; the Section on Personality De- velopment, under Dr. Shapiro; and the Sec- tion on Twin and Sibling Studies, under Dr. Pollin — all regard the family as an area of concern, although, as will be indicated, they work with somewhat different kinds of fam- ilies and use different concepts and methods in their work. The Section on Family Studies is concerned with understanding and treating psycho- pathology, especially schizophrenia, in relation to the family context. In order to do justice to this complex subject, it is necessary to go both within and beyond the family as such: to study, on the one hand, certain dimensions of individual and psychophysiologic function- ing, both of schizophrenics and, for compari- son purposes, of nonschizophrenics; and to study, on the other hand, factors of the com- munity and broader social structure and cul- ture which help determine the meaning and impact of familial influences in individual de- velopment. At present, these research and treatment interests can conveniently be viewed as clustering into six distinct, although closely linked, categories: Intrafamilial Relationships and Transactions As reported in previous years, this research program has established that it is possible to differentiate or predict blindly what kind of psychiatric disorder the off-spring of a family 388 ANNUAL REVIEW OP INTRAMURAL RESEARCH have from the forms in which the other mem- bers of the family communicate and inter- personally relate. This work makes use of the sociologic concept of the family as a partially self-contained social organization or social sub- system and of a psychodynamic theory of nor- mal and schizophrenic ego development. In their conceptualization of "normal" develop- ment, which goes astray in particular ways with schizophrenics, Wynne and Singer have reasoned that there needs to be a reasonably appropriate "fit" between the innate charac- teristics of each child at successive stages of maturation and the characteristics of the en- vironment. The latter includes the pat- terns with which the parents focus attention and meaning and communicate with one an- other and with their children. This means that certain transactional conditions are necessary as framework for the child to learn, for example, culturally appropriate skills, such as "reality testing," a sense of self and self- boundaries, an interest and ability in estab- lishing interpersonal relationships, and an in- terest and ability in establishing directionality and goal-directedness in his life. The form in which the individual child's development proceeds is a resultant not only of the infant's emerging capacity to engage with and become oriented to significant caretakers from whom he can then learn, but of the care- takers' ability to engage the infant's and later the child's attention and to orient him to those aspects of speech, behavior, and other events which will be important in his expectable life experiences. Thus, it is at the stage of engage- ment, orientation, and what, broadly speak- ing, can be called attentional processes that the first difficulties can arise which may lead into schizophrenic forms of ego impairment. Wynne, Singer and colleagues have found that a fruitful concept for linking the difficul- ties of individual offspring is to deviances in family communicational patterns is to use a concept which labeled the "sharing of foci of attention." Such sharing of attention is re- garded as a pre-requisite for learning to relate to another person, to communicate with con- sensually understood meanings, and to learn the numerous and essential ways of interpre- ting physical and cultural reality. Thus, it is hypothesized that communication difficulties in parents which could affect the development of core ego functions that are in fact impaired in schizophrenics, include, most importantly, defects and deviances in the man- ner in which foci of attention are shared. These problems lead directly into a variety of other difficulties in such areas as use of lan- guage, task-orientation and interpersonal re- lations. During the past year scoring manuals have been derived from these principles which can be used reliably and quite simply by other investigators. A paper has been completed by Singer and Wynne describing scoring manuals for use with individual Rorschach and TAT tests obtained from parents. Also, during this year two papers have been published in which the Singer Scoring Manual has been ap- plied by Wild et al. at Yale to Object Sorting Test data obtained from parents of schizo- phrenics and "normals". Using a similar emphasis upon the transac- tional aspects of test behavior, rather than the intrapsychic, projective or symbolic aspects, Dr. Nathene Loveland has been working on scoring manuals for use with various forms of the Relation Rorschach technique. This tech- nique consists of asking two or more people with various relationships to each other, such as husband and wife, families, patient and therapist, etc., to see how many things they can find in a Rorschach inkblot on which they can agree. No tester is present during their discussion, which is tape recorded. This ap- proach lends itself to a comparison of each person's behavior in the different interper- sonal settings of, for example, individual Ror- schach, Spouse Rorschach, Family Rorschach, and Doctor-patient Rorschach. Material from some 80 Spouse Rorschachs is currently being scored, with comparisons of the couples along a variety of dimensions, including the diag- nosis of their offspring. As still another means of studying marital and family transactions, Wynne, Morris and colleagues have been extending their earlier studies. Dr. Morris had blindly predicted diag- NATIONAL INSTITUTE OF MENTAL HEALTH 389 nostic features of offspring from communica- tion styles of parents as found in excerpts of family therapy from which diagnostically rele- vant facts about the offspring were excluded. Morris has replicated this study on a new sample of excerpts frcm six families with three other raters also making predictions. Correct predictions of schizophrenic versus nonschizo- phrenic offspring were made in 21 out of 24 instances. By a conservative test of signifi- cance, p = 0.01. This indicates that the methods of the Morris- Wynne study are communicable and replicable. During this year a new interview format was devised for eliciting communication sam- ples from couples with an interviewer but no offspring present. Methods for evaluating and scoring these research interview protocols fol- low lines similar to those used with the other ways of sampling communication of family members. Each procedure provides a setting in which certain kinds of deviances are some- what more easily and frequently elicited. These differences between the procedures are a mat- ter of emphasis and degree, rather than that any of the procedures elicits a qualitatively unique set of behaviors. It is indeed striking that the patterns of these communication de- viances can be identified in a great variety of transactions. The patterns appear to be per- vasive, structured or "stylistic" features of the ways the family members engage in trans- actions and, it is hypothesized, have appeared in parental relationships with their growing offspring. In addition to these detailed studies of com- munication patterns, somewhat different con- ceptual vantage points are being used for look- ing at patterns of role relationships in families and at patterns of emotional closeness and dis- tance between family members. Research and therapeutic interviews, as well as selected psychological tests, are being used in the de- velopment of a series of rating scales appli- cable to these features. The Study of Psychotherapy, Especially Fam- ily Therapy Treatment indications, techniques, processes, and outcome. Family and marriage therapy have rapidly become recognized as important additions to the traditional psychiatric treat- ment repertroy. An approach especially em- phasized in the Family Studies Section and in the Section on Personality Development (Dr. Shapiro) is the technique of having one or two therapists, most commonly a psychiatrist and a psychiatric social worker, meet to- gether with all of the members of a family. This approach has been variously termed con- joint family therapy, family unit therapy, or family group therapy. The immediate, on- going transactions of family members with one another and with the therapist are re- garded as the most significant starting-point data to be explored, understood and treated in this approach. It has become increasingly apparent, once that therapists have broad- ened their vision to consider more than an individual patient, that many psychiatric problems cannot appropriately be located within individual family members but are problems of communication and relationships that often involve all members of a family or household. A special and growing interest in family therapy and family psychiatiy arises because it can be regarded as a foothold or starting point for the participation of clinical psychia- trists in broader comprehensive treatment pro- grams and preventive psychiatiy. Not only is the family the most immediate and most emo- tionally significant interpersonal influence in the psychological development and functioning of most individuals, but it also constitutes the most accessible naturally occurring group in society with whom psychiatrists can use the skills developed in ordinary clinical training. During the past few years the number of psychiatrists, social workers, and clinical psychologists who regard themselves as "fam- ily therapists" in all or part of their work has increased at a startling pace. As a member of the Group for the Advancement of Psychiatiy Committee on the Family, Dr. Wynne is help- ing plan a national survey of current practices and objectives of family therapists. Dr. C. C. Beels of this section is making a critical review of the literature on family therapy. Such re- views are especially necessary at this time 390 ANNUAL REVIEW OF INTRAMURAL RESEARCH before dogmas and doctrines take over the thinking of persons in this still fresh and promising field. For the same reason, it seems imperative that research on family therapy as a treat- ment technique be set up. During this year Drs. McCormack and Smith of this section have embarked upon an interesting pilot study in which two techniques of family therapy are being compared in a well-thought out research design that includes a series of new methods for evaluating the quality of intrafamilial com- munication and family-therapist communica- tion. For example in the Relation-Rorschach technique applied to this study by Dr. Love- land, the family therapist and the family meet together and try to reach a consensus about what they see in the Rorschach cards; the form in which they do so appears to provide valuable data about the quality of the thera- peutic relationship. In addition to the study of family therapy as a treatment technique in its own right, the use of family therapy has served two other research purposes: it has provided a rela- tively unstructured, free-wheeling source of research hypotheses and unanticipated ideas, and tape-recordings of the psychotherapeutic interviews with families have been extracted for special research purposes in work both of the Family Studies Section and the Section on Personality Development. Cross-cultural Family Studies One of the most important research means for advancing our knowledge both of individual psychopathology and of the social context in which it occurs is the cross-cul- tural approach. This provides a way of dis- tinguishing the conditions under which, for example, a particular symptomatic picture is and is not associated with a family or other social variable. This year Dr. Wynne has been involved in the planning of a cross-cultural International Pilot Study sponsored by the World Health Organization. In this study diag- nostic methods and criteria for the evaluation of schizophrenic patients will be worked out for the first time in such a way that diag- nostic comparability from one country to an- other may become possible. In another study Drs. Singer and Wynne are evaluating whether psychological test protocols obtained from families of schizophrenics and nonschizophrenics in Japan and Lebanon can be scored and assessed with the same criteria which were used in the American family sam- ples. Thus far, it does seem possible to predict diagnosis of offspring blindly from parental tests obtained in these culturally diverse set- tings. Because the patient-family links studied in this research are concerned with the form or structure of thinking and communicating, rather than the content of thinking, it was reasoned that the methods of scoring should be applicable regardless of cultural back- ground. Dr. Wynne is also continuing with the study of other kinds of cross-cultural data obtained previous field work in Lebanon, especially data on the value-orientations of five Lebanese subcultures obtained in collaboration with Dr. Herant Katchadourian. Studies of Family Development Collection of retrospective data about the sequences of family development through time have continued, particularly by Dr. Juliana Franz and Miss Carol Hoover. A pilot study was initiated by Dr. Loveland in which the Relation Rorschach technique was applied to the families with very young chil- dren. This may provide a useful means of studying family communication and relation- ship patterns on a longitudinal, prospective basis. Such work is important for the planning of future studies in this Section. Experimental Ego Psychology Studies of sensory, perceptual, and cogni- tive processes, especially in schizophrenics; and Clinical and Conceptual Studies of Schizo- phrenia In these areas some very intriguing and promising methodologic progress has been made during the past year. With the joining of the staff of Dr. Julian Silverman, experi- mental methods for the study of cognitive and related dimensions in schizophrenics and non- NATIONAL INSTITUTE OF MENTAL HEALTH 391 schizophrenics are being used. The dimensions studied experimentally are being conceptual- ized in terms that can be approached with clinical ratings as well. This rapprochement between careful clinical research and experi- mental studies of schizophrenics has long been an important need. As an example of these experimental meth- ods being introduced, the Mackworth eye- movement camera has provided a direct means of studying scanning behavior. Previous in- direct measures of scanning, as applied by Silverman to the study of schizophrenics, have suggested that acute reactive paranoid pa- tients scan more extensively than normal, whereas nonparanoid and chronic paranoid pa- tients scan minimally. Such work has impli- cations for reconceptualizing the heterogeneity and variability of schizophrenic functioning along dimensions that probably will have an improved rationale over those previously used. At the same time that this experimental work work is proceeding, Dr. Wynne and colleagues have been engaged in defining a series of rat- ing scales for use with individual research in- terviews and projective tests which will facili- tate comparisons of these approaches and, in turn, is related to the concepts derived from the study of family members. The family re- search has led to a consideration of continua along which clinically symptomatic family and nonsymptomalic but otherwise similar family members vary. Thus, the study of the dimensions in schizophrenic functioning em- erging from the experimental psychological studies and from the family studies has come to have significnat overlap. During this year the contributions to the understanding of schizophrenia and family relations stemming from this program have been recognized in the award to Dr. Wynne of the 1966 Frieda Fromm-Reichmann Prize for research on Schizophrenia by the Ameri- can Academy of Psychoanalysis and the award to Drs. Wynne and Singer of the 1966 Hof- heimer Prize for psychiatric research by the American Psychiatric Association. Personality Development The work in the Section on Personality De- velopment has been concerned with classifica- tion of psychological issues pertinent to ado- lescence and has been carired on in two re- search groups, one under Dr. Roger Shapiro and the other, terminated during this year, under Dr. Earle Silber. The therapeutic and research activities under Dr. Shapiro are intimately related to some of those taking place in the Family Studies Section. Both sections are greatly con- cerned with studying the usefulness of con- joint family therapy; the staff of the two sec- tions share the same clinical in-patient facili- ties, participate in clinical conferences to- gether, and evaluate some of the same families, from somewhat different vantage points, in their systematic research activities. In Dr. Shapiro's study, families are observed in which there is overt personality disturb- ance in an adolescent member who has been unable to adapt to living away from his fam- ily in a university setting, has developed psy- chiatric symptomatology, and has withdrawn from school with the suggestion that he get psychiatric treatment. A relation has been hypothesized between parental definition of the adolescent and the adolescent's personality disorder. This hypothesis has led to a research focus upon interactions between parents and adolescents in conjoint family sessions in which the parents' view or image of the adoles- cent can be directly inferred from their be- havior with him. This inferred image of the parents is called "delineation of the adoles- cent." The determinants of delineation in the individual psychology of the parent are analyzed; and the relation of parental delinea- tion to the identity problem of the adolescent is scrutinized both through study of psycho- therapeutic interviews and the use of research procedures, including projective tests, and self-concept interviews. In these ways an at- tempt is made to define the determinants of adolescent disturbance which are related to his family experience. The program of therapy includes conjoint family therapy, individual psychotherapy for the adolescent, and marital counseling of the parents. The work under Dr. Earle Silber, terminated in October, 1965, with his departure from the 392 ANNUAL REVIEW OF INTRAMURAL RESEARCH research staff, was mainly concerned with de- veloping and validating methods for the sys- tematic study of identity problems in late adolescence. Factors affecting the self-esteem of adolescents were studied in considerable detail. The concluding phase of this study in- volved an experimental procedure in which the self-esteem of a group of adolescents was as- sessed before and after they were given lower self-esteem ratings by adult authority figures. This experimental group showed significantly greater change toward lowered self-esteem than was found in a control group. Within the experimental group, those subjects who had higher self-esteem, less psychopathology and more autonomy in their relation with their parents responded more selectively with change on those dimensions on which they had obtained information contradictory to their self-image, but also preserved a more generalized high level of self-esteem. Twin and Sibling Studies During the past year the Section on Twin and Sibling Studies under Dr. William Pollin has continued to locate and admit for intensive multidisciplinary study additional families with twins discordant for schizophrenia. It has also made a beginning in similarly evalu- ating families with twins concordant for schizophrenia and with twins without notable psychopathology. A total of 20 families wth twins have now been admitted and studied, the additional families seen during this past year have for the most part demonstrated the same consistent patterns of interrelated bio- logical and psychological phenomena pre- viously defined. In addition, several new as- pects of the psychobiological interactions which consistently tend to differentiate the index from the control twins in the discor- dant pairs have become apparent. The pattern of consistent life history differ- ences that has been found to differentiate the schizophrenic from the nonschizophrenic twin in the series thus far studied here at NIMH includes the following: (a) the schizophrenic twin-to-be has in 12 of 13 cases weighed less at birth and now tends to demonstrate more "soft" neurological signs than the cotwin con- trol; (b) tended in childhood and adolescence to be less competent, organized, effective and show less sustained goal directed activity than his cotwin; and (c) tended to be the more sensitive, anxious and unhappy of the twins from an early age on. The data, though con- sistent thus far, do not as yet permit extensive conclusions to be drawn; it is still not clear whether they relate to schizophrenia per se, to a susceptibility to various forms of psy- chopathology in a more general sense, or de- rive in part or in whole from some as yet unrecognized sampling bias. Our current ten- tative formulation of these data is that in the group of families studied there existed initial, non-genetic, constitutional differences between the twins. These initial constitutional dif- ferences reflected in the different birth weights, involved most importantly a different level of biological maturity or competence, which resulted in less smooth and effective operation of various adaptational and internal environment regulators in the smaller twin. These sometimes slight biologic differences contributed to or determined the very early establishment of role difference within the family relationship patterns. The smaller twin, as a result of these relationship and role differ- ences, experienced a sequence of reinforcing events in childhood years which in toto ac- centuated rather than mitigated the initial minimal disparity in coping potential. The in- creasing intertwin differences in personality, particularly in ego structure, led to an in- creasingly unfavorable stresscoping ratio, in the index twin, with passing years. That is to say, there was an increasing tendency to generate stress in dealing with ongoing de- velopmental events and transitions, and a rela- tively decreased ability to cope with them. Drs. Pollin and Jades Stabenau are currently at- tempting to expand this formulation and to synthesize a multifactorial theory of the etiology of schizophrenia. Constitutional and biological differences be- tween the index and control twins thus far delineated included in summary the following: the index twins show a higher lactate/pyru- vate ration (Frohman; 9/10); have a lower PBI (11/12); more "soft" neurologic signs NATIONAL INSTITUTE OF MENTAL HEALTH 393 (9/11); weighed less at birth (12/13). These biological variables, and other where the data is not yet clearcut, have been clarified in particular by Drs. Stabenau and Loren Mosher, working collaboratively with a number of other investigators, here at NIH, and in other research centers. Dr. Fred Guggenheim has focused on organic factors in the twins' par- ents and has found a surprisingly high inci- dence of significant medical illness in them. He found that 7 of the first 13 mothers studied had well-documented thyroid disease, a preva- lence rate (58%) which is elevated to a statistically significant degree when compared to other prevalence and case finding studies. Additionally, there appeared to be an elevated incidence of malignant neoplastic disease and an overall pattern suggestive of excessive physical illness in these parents. These find- ings raise questions about the possible rela- tionship of thyroid function to the phenome- non of twinning, though it is not yet possible to exclude some type of as yet unrecognized sample bias in accounting for the results. It appears most likely, however, that they reflect the heightened degree of intra-familial ten- sion in the prepsychotic years within these families in a manner consistent with the dem- onstration by Hinkle and others, in large scale studies, of the relationship between emotional crisis and episodes of physical illness. In the continuing analysis of data obtained from three groups of families with young adult siblings discordant for schizophrenia, or juvenile delinquency, or where both siblings were well-adjusted, Dr. Stabenau has devised a new, additional objective indicator of a greater degree of familial stress during early critical developmental periods in the life of the index as compared to the control. In the S families 7 of 11 indexes as compared to only 2 of 11 controls had younger siblings born within 2 years of their birth; in the de- linquent families the corresponding ratios were 7 of 8 indexes and 2 of 8 controls; and in the well-adjusted control families the ratios were only 1 of 5 for both index and controls. Working with TAT material from these same three groups of S, D, and N families, Mrs. Martha Werner and Dr. Stabenau have noted an additional significant distinction be- tween the normal and psychopathology fami- lies. In keeping with their hypothesis con- cerning the role of repressed rage in the for- mation of schizophrenic symptoms and de- linquent behavior, it was found that on card 13 MF 28 of 39 stories told by the S families had themes of violence, murder or death; that 26 of 32 stories in the D families and only 5 of 20 responses by the N families had similar themes. In addition to the phenomenon of a differ- ential degree of stress in the family as a whole occurring during the first two years in the life of the schizophrenic or delinquent-to-be as compared to the life of the control a num- ber of other factors have been found which help explain the presence within a given fam- ily of psychopathology in one child and not the other. These include (1) a clearcut pat- tern of differential identification with the index-to-be much more closely identified and allied with the psychologically sicker of the two parents; (2) a psychological Gestalt which places one of the siblings in the role of being much more caught up in or used as a surrogate for some intense rivalry between the parents; (3) certain accidental historical or biological differences determine that the index-to-be to a much greater extent than the control restimu- lates some unresolved though previously dor- mant major conflict within one or the other of the parents; (4) the early establishment of role differences cause one of the siblings or twins to receive considerably less by way of psychological growth and ego-syntonic life ex- periences. Dr. Loren Mosher has studied the determi- nants of psychopathology in a twin population by exploring the relationship of such psy- chopathology to differences in identification and cognitive style. His findings thus far include the following: the index twins, when compared with their cotwin, regardless of sex, are most often identified with their mothers, and are rated and test out as being more "global" or amorphous (field dependent) in their cognitive style (or type of schizophrenic thought disorder); the identification variable appears to have less effect on cognitive style 394 ANNUAL REVIEW OP INTRAMURAL RESEARCH than does psychopathology) female twin pairs and mothers are more "global" (field depend- ent) than male pairs and fathers. The fact that females more often have "global" cogni- tive styles (or amorphous thought disorders) may be relevant to the higher concordance rate for schizophrenia in female twins and the larger number of "chronic" schizophrenic fe- males in mental institutions. Psychosomatic Studies The Section on Psychosomatic Medicine is a part of the Adult Psychiatry Branch which is more heavily concerned with relationships be- tween biology and behavior. Although specific projects range from laboratory research at a molecular level to clinical investigations at a molar level, the overriding and unifying theme of the Section's work is a furtherance of a meaningful understanding of the behav- ioral-biochemical paradigm. There is a good deal of inferential evidence from both clinical and animal studies that norepinephrine (NE) metabolism in human brain may markedly influence a variety of behaviors. However, due to several methodo- logical problems it has not been possible to obtain direct estimates of rates of metabolism of norepinephrine by brain in clinical inves- tigations. Because of the importance of such knowledge for research into the relationships between brain NE and behavior, much of the work under Dr. James Maas over the past three years has been devoted to the develop- ment of a technique whereby reliable, indi- vidual measures of norepinephrine metabolism in brain may be obtained. In the initial phases of this work it was found that (a) norepine- phrine given into the cisterna magna of dogs rapidly enters brain and by five hours is in a pool which is not freely accessible to the cere- brospinal fluid (CSF); (b) 50-70% of iso- topically labelled NE (H 3 NE) enters blood from CSF, but (c) when rather large amounts of H 3 NE are injected into blood essentially no radioactivity is found in brain or the DSF. These findings suggested that by giving an intravenous infusion of C 14 NE at the same time that H ! NE was injected into the dis- terna magna one could derive, from assays of metabolic products of H 3 and C 14 in urine, direct estimates of the amount of NE which enters brain and the rate at which NE in brain is changed to various degradation prod- ucts. Experimental data obtained from work with dogs indicate that such a double isotope technique does yield quantitative information as to rates of degradation of isotopically la- belled NE by brain. In addition, further ani- mal experimentation has been done over the past year to simplify the mathematics in- volved in this approach and to find methods whereby the fraction of H 3 NE entering brain might be increased. The results obtained thus far suggest that the reliability of the method is such that the application of this technique to clinical investigations is now feasible. Dur- ing the coming year it is planned to use this double isotope approach in studies of severely depressed patients. Other studies of norepinephrine metabolism at a more molecular level are also being done by the group under Dr. Maas. The enzymatic steps involved in synthesis and breakdown of NE are fairly well understood, but the mech- anisms by which the catecholamines may be bound or stored intracellular^ in brain are less clear. Because of the structure of the NE molecule, i.e. a catechol moiety with an ethan- nolamine side chain, a reasonable hypothesis is that the catecholamines form complexes with metals with a consequent change in physical properties which may explain the phenomenon of binding. Three lines of evi- dence support this hypothesis; (a) it has been found that synaptic vesicles isolated from rat brain have more than enough metal (Mg, Cu, Fe, Zn) to complex with the quantity of catecholamines present in this subcellular frac- tion of brain; (b) ethylenediamine, a chelating agent, prevents uptake of exogenous NE by synaptic vesicles; and (c) in vitro evidence has been found for the formation of ternary complexes between metal, NE and ATP. These findings as well as data reported by a variety of investigators are consistent with, and sup- portive of, the hypothesis that coordination between metals and NE are importantly re- lated to the intracellular binding of NE. Pres- ent work has been directed towards obtain- NATIONAL INSTITUTE OF MENTAL HEALTH 395 ing very pure preparations of synaptic vesi- cles as demonstrated by electron microscopy, and then following a variety of treatments, measuring rates of uptake and release of metals and NE. In the process of the above investigations it became apparent that metals might be also quite important for membrane function. Data obtained indicate that (a) metals coordinate with phospholipids and alter their solubility characteristics; (b) ternary complex forma- tion between metal, phospholipids, and ATP occurs; and (c) the solubility characteristics of the ternary complex is similar to that of the phospholipid alone. From these data a molecular model of neural membranes which can explain reversible transitions between two phases having different physical properties was constructed. To understand further the role that these transition metals might have in neural func- tioning, electron spin resonance spectroscopy of subcellular fractions of brain (myelin, mi- tochondria, and synaptosomes) is now being done in collaboration with Dr. Hideo Kon of NIAMD. Although there was some initial tech- nical problems with this approach, these now seem to be corrected and some interesting and potentially important data are emerging which may aid in our understanding of the molecular mechanisms involved in synaptic functioning. In addition to these molecular approaches, Dr. Maas and co-workers have been studying the relationships between biogenic amines and critical periods of development. There is a good deal of experimental evidence from a variety of animal studies which indicates that during development there are critical periods in which environmental influences are most important in determining the ability of the adult animal to respond to stress, to learn, and, in the cases of dogs, to form social bonds. In an earlier investigation of some of the biological proc- esses which might underlie this latter phe- nomenon it was found that during the develop- mental period critical for primary social for- mation in the dog there were marked changes in norepinephrine and serotonin levels as well as catechol-O-methyl transferase activity in- most areas of the brain examined. More spe- cifically, around the beginning of the period critical for primary social bond formation bio- genic amine levels and enzyme activity were high but markedly decreased over the follow- ing one to two weeks. As a beginning in the understanding of the possible significance of this finding in relation to behavioral corre- lates collaborative work with investigators at the Jackson Memorial Laboratories at Bar Har- bor, Maine was undertaken in which one group of pups at 5 weeks of age and another at 7 weeks were given Reserpine for a period of five to ten weeks, taken off the drug and then tested in a variety of ways at 26, 36, and 52 weeks of age. Control animals were littermates and were given Nembutal. Experimental and control animals were housed together. From this first study the data indicate that those animals which received Reserpine (a drug which lowers body stores of norepinephrine and serotonin) from 5 to 15 weeks of age are, when tested as adults, somewhat more fearful and do not learn maze problems as well as the control subjects. The dogs which were begun on Reserpine at 7 weeks (and con- tinued until 15 weeks of age) seen as adults to show test behavior somewhat intermediate between these two groups. Due to the small sample size, firm statements must await the outcome of studies now in progress which have as their goal the replication and extension of these earlier findings. In a somewhat different approach to the relationship of central nervous system amines and behavior, work is being continued in an attempt to obtain neurochemocal-behavioral correlations in strains of mice and rats which have been inbred for differences in emotion- ality or fearfulness as measured by the open- field test. Initially, in working with mice which differed in terms of this behavior, it was found that the more "emotional" strain of mouse (BALM/cJ) had a higher level of serotonin when compared with another less emotional strain. (C57BL10cJ) In addition, the work seemed to indicate that the serotonin differ- ential was due to brain stem and limbic struc- tures rather than other areas. Since this initial finding, work has proceeded along lines which attempt to relate this serotonin difference to 396 ANNUAL REVIEW OF INTRAMURAL RESEARCH behavior in other strains and species. It has been found that: (a) in three other strains of mice, one of whom was "emotional" and the other two not, serotonin differences in the pre- dicted direction were found, and (b) reactive and nonreactive strains of rats which were bred by Broadhurst for differences in open- field behavior also show brain serotonin differ- ences in the predicted direction. Correlations between ambulation scores in the open-field and serotonin levels in limbic structures in brain were done and there was found to be a significant negative correlation. Present work in progress deals with obtaining F a and F 2 animals from the reactive and nonreactive strains for behavioral testing and serotonin assays. The clinical portion of the section's work has been concerned chiefly with studies of depressive reactions and has been carried out under the direction of Dr. William E. Bunney, Jr. This program involves the investigation of biochemical and behavioral aspects of depres- sive reactions. In the last year six reports have been published which deal with a variety of psychological and biological factors in depres- sive illness. Issues and questions raised in these publications continue to be explored, and in addition four new areas of investigation have been initiated. The basic research tools used in this research are continuous, longitudinal methods of ob- taining behavioral data through the use of rating scales, coupled with an ongoing collec- tion of urine and plasma specimens. Research findings built on this data have been published during the past year and include the follow- ing: Urinary excretion of 17-hydroxyeorticoste- roids (17-OHCS), an index of anteriar pitui- tary-adrenacortical activity, is elevated in cer- tain subgroups of depressed patients. High positive correlations exist between longitudinal behavioral ratings of depression and fluctuations of 17-OHCS levels for a given patients. Changes in 17-OHCS levels accompany be- havioral changes rather than preceding or fol- lowing them. Changes in 17-OHCS levels can be used to study and locate specific days for analysis of precipitating events and to characterize the categories of precipitating events that seem relevant to depressive crisis. It was hypothesized that 17-OHCS levels re- flect what has here been termed psychological distress or pain and that this is particularly intense in acutely suicidal patients. The data suggest that elevated urinary 17-OHCS levels may offer a possible biochemical test for sui- cidal potential. A review of the current literature plus on- going investigations indicate that changes in norepinephrine metabolism may have etiolog- ical significance in depressive reactions. These findings have been described in detail in one of the papers published during the past year. Analysis of an additional body of data bearing on the relationship of norepinephrine metabolism to depression has been recently completed. Findings show high urinary no- repinephrine levels and different excretion pat- terns of breakdown products of norepinephrine in patients with acute psychotic depressions as contrasted with those having neurotic de- pressions. This investigation represents a three-year study of urinary norepinephrine and breakdown products in depressive reac- tions. Based on comprehensive clinical obser- vations, two members of the research group placed the sixteen patients in this study into one of two diagnostic groups, acute psychotic depression and neurotic depression. The ac- curacy of these judgements were validated by two independent raters. The acute psychotic depressive group showed marked elevations of norepinephrine levels in contrast with the neurotic depressive group. The difference be- tween the norepinephrine levels in the two groups was significant at the 0.002 level. All the individual means of acute psychotic de- pressive groups were higher than those in the neurotic depressive group. Norepinephrine lev- els in the neurotic depressive group are more stable than those in thepsychotic depressive group as shown by the ranges and the standard deviations. One possible abnormality in nore- pinephrine metabolism in depression may in- volve and inhibition of one of the enzymes involved in the breakdown of norepinephrine. NATIONAL INSTITUTE OF MENTAL HEALTH 397 The findings of this study offer evidence com- patible with this hypothesis. The analysis of additional data confirming the hypothesis that 17-hydroxycorticosteroids may offer a test for suicidal potential has been completed. Through research additional data on a number of suicide attempts have been collected. Steroid data on three additional suicides occurring in the other research cen- ters has also been reported here, which tends to support the original hypothesis. As our body of data concerning this problem increases, more sophisticated analysis and qualification of findings becomes possible. Two additional studies have been completed and are in the process of data analysis. The first involves a further attempt to investigate the role of norepinephrine in depression and involves infusions of precursors of norepine- phrine and serotonin in an attempt to change brain amine levels. Specific and interesting be- havioral changes have been observed. The sig- nificance of these are currently being analyzed; however, no long lasting improve- ment of the depressive mood was noted. The second area involves analysis of pre- cipitating factors occurring prior to the onset of depression. It is hypothesized that investi- gation of these factors should offer some clues concerning the core problems of depression plus giving us basic information about response patterns of the individual patients to stress. Forty patients and their relatives have been intensively studied to date. The factors have been broken down into external factors, con- trolled and uncontrolled by the patients, and internal factors. The temporal sequence of these events has been analyzed. Approximately 20 patients who fall according to all diagnostic criteria (except the absence of a precipitating event) into the category of endogenous depres- sion have been studied. In almost all of these individuals clear cut precipitating factors have been present. This finding clearly challenges the validity of the concept of the endogenous depression as an entity and has specific impli- cations for future research. From these data it is hypothesized that all cases of depression whether they fit the "endogenous or reactive" category are stress precipitated. For this rea- son studies of the biochemical stress reactions of the depressed patient are being actively continued. In addition to the work thus far described, which is at or near completion, data for other projects have been collected but are at present unanalyzed. The first of these studies involved an attempt to study the mechanisms by which depressed patients maintain extremely high levels of 17-OHCS. This is a particularly in- teresting phenomenon in that patients may maintain levels five times normal for six months and yet do not develop symptoms of Cushing's disease. The drugs dexametha- sone and metopirine have been used as re- search tools. Tentative evidence from a num- ber of severely depressed patients suggest that abnormalities may exist in steroid metabolism. Attempts are being made to develop a hy- pothesis which accounts for the observed changes in steroid, catecholamine and electro- lyte metabolism in depressed patients. It seems likely at this time that these factors are in- terrelated. A second area of work in progress has in- volved the use of lithium in the treatment of manic-depressive patients and in the treat- ment of depressed patients. For as yet unknown reasons, lithium seems to be a highly specific treatment for manic-depressive illness and can be used as a research tool for the study of the mechanism of action of lithium. A number of theoretical concepts concerning the mecha- nism of action of lithium have been developed and are currently being investigated. If suc- cessful, such studies could lead to a more basic understanding of psychopathology than pre- viously anticipated. A few patients whose psy- chopathology is extremely sensitive to changes in lithium levels have been studied and from this work and from a review of the literature it would appear that the use of lithium in this country is unrealistically conservative and lim- ited. A third area of work involves interest in studying the mechanism of action of electric shock therapy. During the past year a small study in dogs was initiated to investigate the effect of electric shock therapy on norepine- phrine metabolism. A large potential exists in 398 ANNUAL KEVIEW QF INTRAMURAL RESEARCH this area and currently awaits space and per- sonnel. A fourth area of work in progress involves a study of the thought patterns of depressive patients. During the past year a good deal of progress has been made in this area. Cur- rently, 20 depressd patients and 20 schizo- phrenic patients are being studied. These are being matched for depressive content so that it is impossible to differentiate them on this factor. Independent raters are now attempting to predict blindly which patient is psychotic- ally depressed and which is schizophrenic on the basis of categories developed from the analysis of their thinking disorders. A num- ber of previously undescribed characteristics of psychotic depressive thinking have been defined. Future plans involve a continuation of the above four projects and the initiation of three new areas of work. These involve, first, a col- laborative project with a number of hospitals to evaluate the usefulness of finding that 17- OHCS levels may offer an index of suicidal potential. The second area is concerned with a con- tinued study of lithium and a continued in- vestigation into the mechanism of action of lithium. One of the most relevant hypothesis concerning the mechanism of action of lithium and its effect on nerve transmission in the brain involves its possible action on the sodium this it is critical to study intracellular sodium. A method for this has been worked out in collaboration with the Bethesda Naval Re- search Medical Center and involves the use of neutron activation. The third area of future research involves a direct testing of the norepinephrine hy- pothesis through a study of the metabolism and turnover rates of norepinephrine in de- pressed patients. Psychophysiology of Sleep Longitudinal studies of electrographic sleep patterns in hospitalized depressive patients continue to be the principal focus of interest for the Section on Psychophysiology of Sleep. An innovation accomplished during the past year which promises to be widely adopted in clinical sleep studies was the installation of permanent recording cables from the sleep lab- oratory to the nearby psychiatric ward. This permits electroencephalographic and other polygraphic recording from patients while they remain in the accustomed setting of the ward, rather than requiring that they sleep in the unfamiliar setting of the laboratory. In retrospect it now appears as though this is the only way in which meaningful studies of sleep patterns in psychiatric patients could be accomplished, and, indeed, current sleep studies of depressive patients have been greatly facilitated. In addition to extending the series of patients for whom three night samples of sleep recordings have been studied at various stages of illness, nightly sleep re- cordings from selected patients are now being obtained over very extended periods, in one case amounting to 111 consecutive nights. For the first time this type of study provides de- tailed and precise knowledge about variations in sleep patterns of psychiatric patients in relation to day-to-day changes in clinical status or various therapeutic interventions. In the patient referred to, for example, this data re- veals marked variations in total sleep from night to night, yet an unrelenting and cumu- lative sleep deprivation over the months of the study. The degree of REMS (rapid eye move- ment state) deprivation is still more severe. Therapeutic trials of an experimental antide- pressant drug were associated with total abo- lition of REMS, and its withdrawal was fol- lowed by marked increases of REMS above normal levels, demonstrating that the experi- mental phenomenon of REMS deprivation can be a naturally occurring one under clin- ical circumstances. Although these studies have been handi- capped during the past year by the relative un- availability of suitable patients, it is intended that they will be extended to additional de- pressed patients and eventually to other diag- nostic categories as well. Many technical prob- lems remain to be solved, but it is foresee- able that nightly monitoring of sleep patterns may soon become a routine procedure in clin- cal evaluation and management of psychia- tric patients. NATIONAL INSTITUTE OF MENTAL HEALTH 399 The earlier findings of our sleep laboratory concerning profound variations in blood pres- sure and other vital functions during the REM state, as well as their probable implication in a number of important medical problems are now receiving ample confirmation. For exam- ple, evidence obtained here during the pre- vious year that nocturnal angina episodes are highly correlated with REMS periods has now been substantiated by work reported else- where, but in one further angina pa- tient studied here during the past year these episodes were not related to REMS periods. The anginal pain of this man generally oc- curred with the first few hours of the night when our previous studies have found the low- est diumal levels of blood pressure. Additional patients with this condition are also very much sought for study, but it now appears that the pathophysiology of nocturnal angina is probably not always the same, nor always related to the vegetative disturbances of REMS. The descriptive physiology of the REMS continues to be of great interest, as the physi- cal reverberations of this third state seem to extend wherever they are sought. One aspect of particular attention currently is the associ- ation of the REMS with penile erection. Since one of the pioneer workers in this area, Dr. Ismet Karacan, is visiting scientist in the Sec- tion this year, further systematic study of this relationship is being pursued using normal control subjects. To date this has focused upon the normative characteristics of erection during entire nights of uninterrupted sleep in order to clarify details of temporal rela- tionships, inter- and intra-REM period differ- ences, or intersubject variations in this mani- festation. After the normal courses of these events are better known, it is intended to use this data as a basis for testing the effects of a variety of experimental interventions, such as sleep deprivation, specific REMS deprivation, stress, etc. Comparative studies of sleep and REMS in primitive mammals and reptiles have been vir- tually set aside during the past year because of lack of suitable space or facilities for carrying them further. It is hoped that they can be resumed in the future when additional space becomes available and that a collaboration might be worked out with the National Zoo- logical Park for pursuing them more ex- tensively in a wider variety of species and under more naturalistic conditions. One com- parative question which has been answered is whether the penile erection element of REMS is entirely a human characteristic, or whether it occurs in the REMS of other mam- mals as well. It was demonstrated for the first time in our laboratory that penile erec- tion during REM periods occurs in the Rhesus monkey as it does in man. That this is so offers promise that the neurophysiological mechanism of erection during REMS might be clarified, but since the Section on Limbic In- tegration, of the Laboratory of Neurophysi- ology (Dr. Paul MacLean), has already done a great deal to elucidate the central nervous mechanisms of erection in the Squirrel mon- key, it would be particularly appropriate to study the mechanism of erection uuring REMS in that species. In collaboration with that sec- tion, therefore, present efforts are directed to study the sleep patterns of the Squirrel mon- key and to determine whether penile erection is associated with REMS period of that spec- ies. At the present stage, technical problems in making electrographic sleep recordings of this primate are still troubling, and a variety of approaches to the chronic restraint which is nee' •. ; e being attempted. I >ic lesearch in the physiology of the REM state hab now entered a distinctly biochemical phase. Many lines of inquiry point to the ex- istence of a neurohumoral mechanism respon- sible for the periodic triggering of this re- markable physiological condition. The effects of depriving animals of this state suggest that a hypothetical chemical substance accumulates and has the effect of producing generalized hyperexcitability within the nervous system. Direct biochemical substantiation for this hy- pothesis is still lacking, but evidence suggests that catecholamines or serotonin are likely involved. Data relevant to this hypothesis is being sought by the Section on Sleep on two levels in collaboration with other research units of 400 ANNUAL REVIEW OF INTRAMURAL RESEARCH NIMH and NHL Since the Section on Experi- mental Therapeutics of the NHI (Dr. David Horwitz) is making clinical trials on new and potent inhibitors of catechol amine synthesis, such as alpha-methyl-tyrosine, this provides an opportunity for studying the sleep patterns of medical patients receiving these drugs be- fore, during, and after their administration. The first two exploratory studies in this series are now nearing completion. Another approach to this > problem is being carried out in collaboration with the Section on Medicine, of the Laboratory of Clinical Sci- ence (Dr. Kopin). Rats are being selectively deprived of REMS over periods of four days, after which various structures of their brain tissue ae assayed for norepinephrine and serotonin levels in comparison with those of control groups which were not so treated. Re- sults of this analysis are still pending, but further studies of similar nature are contem- plated in which "turn-over" rates of the same substances will be studied by means of radio- isotope techniques. The challenge presented by the REM state is a rare opportunity for com- bined efforts of scientists concerned with molar aspects of behavior and those involved in molecular physiological mechanisms. That our Section on Psychophysiology of Sleep is now sharing in such collaborations is in keep- ing with the unique advantages and best tra- ditions of NIMH Intramural Research. LABORATORY OF GENERAL AND COMPARATIVE BIOCHEMISTRY During the last 12 months the research pro- gram of the Laboratory of General and Com- parative Biochemistry has continued along the lines that have been developing in the last few years and include now the following main areas of interest: (1) mechanism and path- ways of protein biosynthesis; (2) biological oxygenation; (3) the biochemistry of men- tal retardation; (4) biological methylation and alkaloid biosynthesis; and (5) structure-func- tion relationships in proteins. Work along these main lines tends to break- down the division into three sections and col- laboration between sections has been very suc- cessful and productive. We have had no major personnel changes and have enjoyed a year of productive and stimulating research work in our chosen fields. Section on Proteins The research efforts of the Section on Pro- teins were divided between two problems dur- ing the last year as during earlier years. The main effort of the Section, and in fact of the whole laboratory, dealt with the mole- cular biology of sRNA. A number of individual projects describe more in detail the multifacet approach to the structure of serine sRNA and its interaction with a number of biopolymers, such as messenger and ribosomal RNAs, the specific serine sRNA synthetase, the CMP AMP pyrophosphorylase, etc. The second area of investigation dealt with protein structure and the correlation of mole- cular structure to biological function. It is now clear that the key event in the trans- lation of the triplet code of messenger RNA into the polypeptide sequence of protein re- quires the interaction between sRNA and mRNA. SRNA's biological function is related to its ability to decode the information con- tained in the messenger RNA, to recognize the amino acid specific sRNA synthetases, and to interact with the ribosomes, where the as- sembly of polypeptide chains takes place, thus, bringing together the three components of the several needed for protein synthesis. The most significant advance in this area of work in our laboratory has been the progress in the purification of serine sRNA from yeast. We now have developed a technique for the purification of this material in reasonable quantity and in better than 90% purity. No other sRNA of comparable purity has been obtained elsewhere, to our knowledge. Im- proved techniques of nucleotide sequence, based on separation and characterization of oligonu- cleotide fragments, lead us to hope that de- termination of the base sequence of serine sRNA from yeast may be achieved in the next year or sooner. Elucidation of the primary structure of amino acid specific sRNA, how- ever, is only the first step in the develop- ment of our understanding of the molecular NATIONAL INSTITUTE OF MENTAL HEALTH 401 biology of sRNA. Much further work can be anticipated on comparative structural chem- istry of sRNAs, on physico-chemical studies and on nucleic acid protein interaction. Interest in the relationship between protein structure and biological function underlies many aspects of the work in the laboratory as it is indeed in the mainstream of biochemical thinking. Several projects represent our effort in this line of investigation. Thus, studies of the crystalline serine sRNA synthetase have disclosed that the enzyme protein only recog- nize the intact sRNA molecule, loss of only the terminal nucleotide from sRNA being suffi- cient to eliminate all interaction between se- rine sRNA and serine sRNA synthetase. Work on the thetin enzyme (thetin-homocysteine methylpherase), one of the favorite proteins (of this laboratory, has shown that many of the structural features of this enzyme are shared by other unrelated proteins. The studies of Klee on pancreatic ribonuclease shed new light on the conformation of this enzyme. Work 'with the highly purified dopamine ^-hydroxy- lase has revealed the role of protein bound copper in the hydroxylation reaction leading to the biosynthesis of norepinephrine. (This aspect of the Laboratory's effort is described below in more detail.) Section on Cellular Regulatory Mechanisms The research efforts of the Section were di- vided between two unrelated problems during the last year. The first area deals with the original, and continuing, interest in the prob- lem of reactions catalyzed by hydroxylating enzymes. The second problem is one that was only recently initiated in the Section and is still in the preliminary stages of investiga- tion: skeletal muscle hypertrophy. In the area of hydroxylating enzymes, there were several significant advances. Previous work in the Section led to the discoveiy of a new coenzyme, dihydrobiopterin, which is an essential component of the phenylalanine-hy- droxylating system. Subsequently, we demon- strated that this pteridine plays a similar role in the adrenal enzyme system that catalyzes the conversion of tyrosine to dopa. This step is the first one in the biosynthetic pathway for the neurohormones, norepinephrine and epine- phrine. Work in other laboratories has shown that dihydrobiopterin functions as a cofactor in a variety of oxygen-requiring enzyme sys- tems. We had previously shown that one other naturally occurring pteridine, sepiapterin, shows high cofactor activity in the phenyla- lanine-hydroxylating system. It has now been found that sepiapterin, itself, is not active, but must first be converted to dihydrobiop- terin before it can function as a cofactor. A new enzyme that catalyzes this conversion has been extensively purified from rat liver ex- tracts. This enzyme named sepiapterin re- ductase, catalyzes the following reaction: TPNH + H + + sepiapterin^TPN+ + dihydrobiopterin Since it has been shown that the reaction catalyzed by this new enzyme is leversible, the discovery of sepiapterin reductase raises the question of whether sepiapterin might not occur in mammalian tissue. Until now, this pteridine has been demonstrated only in lower animals such as insects and fishes, where it is believed to function as a pigment. In the dopamine /3-hydroxylase catalyzed re- action, it has previously been shown in this .Section that the enzyme is a copper protein. It was also demonstrated that the protein- bound copper undergoes oxidation and re- duction during the hydroxylation reaction. This conclusion has been fully confirmed by a electron-spin-resonance measurements. Furth- ermore, with the use of this technique, it has been found that fumarate, an activator of the enzyme, enhances the oxidation of the pro tein-bound copper. This observation may pi< vide an explanation for the activating effect «/" fumarate. During the last year, a new disease has teen described, which may be a variant of phenyl- ketonuria. This new disease, characterized by moderately elevated levels of serum phenyla- lanine, has been called phenylalanemia. As part of our long term interest in genetic diseases related to phenylalanine metabolism, we have recently initiated, in collaboration with Dr. Joseph Kennedy in Boston, a study of the phenylalanine hydroxylating system in liver 402 ANNUAL REVIEW OF INTRAMURAL RESEARCH biopsy samples from patients with phenyla- lanemia. Our preliminary findings indicate that the phenylalanine hydroxylating- system is not fully active in the liver samples from these patients. The enzymatic defect is almost as se- vere in this new disease as in classical PKU. In future studies we hope to be able to put on a quantitative basis the difference in phenylalanine activities in the two diseases. In the muscle hypertrophy problem, a tech- nique has been developed which for the first time leads to skeletal muscle hypertrophy in animals, under controlled laboratory condi- tions. The method gives measurable increases in muscle mass, as well as increases in muscle strength. Chemical analysis of a wide variety of mus- cle constituents has demonstrated that in the hypertrophied muscle, there is an increase in content of glycogen and RNA. Time-course studies are underway to determine if these chemical changes occur before or after hyper- trophy. Section on Alkaloid Biosynthesis The year 1965-66 has seen continued ad- vance in several areas of sulfur aminoacid metabolism. Studies have been initiated in collaboration with Dr. Uhlendorf of the transsulfuration pathway in mammalian cells growing in tissue culture. Factors affecting the levels of the per- tinent enzymes are being explored. The long- range goal of these studies is to make available a source of tissue which one could obtain from both control patients and patients with en- zyme deficiencies at the present time demon- strable only in liver tissue. This would make possible the study of a variety of genetic and metabolic problems now precluded because of lack of available material. The findings may be of general interest in relation to a great number of diseases. In addition, the findings of Eagle and his collaborators suggests that the transsulfuration pathway is modified as cells undergo "transformation" to malignancy. A more detailed understanding of this phe- nomenon will be of interest for the study of such transformation. Thermodynamic studies carried out in col- laboration with Dr. Klee have led to insight into the structural factors which help to con- fer their "high-energy" nature on sulfonium compounds. The biological methyl donor, S-ad- enosylmethionine is the most energy-rich such compound known. Detailed studies of its con- formation in solution have been performed and led not only to a picture of the actual shape of this molecule, but also to the suggestion that most adenosine and other purine ribo- sides in solution have a conformation differ- ent from that described to them until now. Since September, 1965, we have been for- tunate to have as a Visiting Scientist, Dr. John Giovanelli, an expert in plant biochemistry. The results are as yet preliminary, but already a number of interesting leads have developed which it is hoped will lead to many new dis- coveries as they are followed up. The human disease, cystathioninuria, a cause of mental retardation, has been proven to be due to a lack of the enzyme cystathi- onase. A few more detailed studies of the na- ture of the defect in this disease have been carried out with a view toward explaining the puzzling action of pyridoxine in these pa- tients. Lack of material has been a limiting factor. It was also shown that these patients lack homoserine dehydrated activity, opening a new area for metabolic investigation. Studies on homocystinuria have been con- tinued. One of the questions raised here last year has been answered. A homocystinuric pa- tient with an normal IQ has been found to have only 2-3% of the normal concentration of cystathionine synthase in his liver. This is a level of enzyme found in many homocystin- uric patients. This finding suggests that envir- onmental manipulation (i.e., dietary manage- ment) will almost certainly be successful in preventing the mental retardation which oc- curs in most patients who lack cystathionine synthase. Section on Technical Development During the 12 months since July 1965 the Section on Technical Development continued to fulfill its traditional role of support of NIMH and NINDB research efforts. Continu- NATIONAL INSTITUTE OF MENTAL HEALTH 403 ing assistance was made available in shop help, special purpose instruments, and some modest instrumentation systems. The section continues to grow toward assistance in com- puter oriented research and digital instru- mentation. Use of the section's LINC computer has al- most doubled since last year and has reached the saturation point — 105 hours per week. The ability to take on new projects is now seri- ously limited by production runs on existing projects. Plans to relieve this pressure in- clude: Transfer where possible of existing pro- duction runs to CDPB, while limiting LINC functions to A to D conversion and online data editing. Aquisition of additional computing equip- ment in fiscal 1967. Help to laboratories toward setting up their own facilities where the workload jus- tifies such a step. The policy toward repair and maintenance of equipment has continued the same as in the previous year, with the section handling emergency problems but referring routine and long-term problems to the Instrument Branch. The electronic component stocking service con- tinues with the occasional acquisition of gov- ernment surplus components being used to supplement substantially the purchased stock. Because of limitations in space, no signifi- cant expansion or change of plans is contem- plated within the section until the move to new quarters is made sometime in fiscal 1968. At that time, it is planned to move the section to Building 36 while maintaining a small ma- chine shop and electronic fabrication facility in Building 10. Staff will probably be expanded to include an additional technician for the Building 10 facility; and the additional space in Building 36 will probably permit the addi- tion of a technician, a mechanical engineer, and a physicist to the staff. Section personnel have contributed signifi- cantly to research projects in laboratories of the two Institutes. In all cases this work has been described in the reports of the labora- tories involved. A total of 164 projects were completed in ithe past year. A full review of all of them would be too lengthy; however, a summary list of the more important ones follows: Projects completed Project Requestor EEG amplifier and power supply Dr. K. Gaarder Pulse discriminator and counter Dr. P. Nelson Brain mapping impedance package _ . Dr. H. Rosvold Hydrogen electrode chambers Dr. M. Reivich Automatic timer and pulser unit Dr. H. Steinberg Adjustable head rest chair Dr. J. Silberman Camera optical system for oscilloscope photography Dr. D. Carpenter Lucite prism and electrode holder Dr. D. Carpenter Galvanic skin response meter Dr. P. Bergman Automatic blood sampler Dr. M. Reivich Skin temperature sensing system Dr. F. Snyder Electrophoresis chambers _ Dr. C. August Dr. M. Kies Dr. B. Rasmussen Digital coding units for animal observation Dr. D. Galin Dr. P. MacLean Revolving table with automatic nipple insertion Dr. W. Stanley Automatic tape recorder control Dr. D. Boomer Conceptual training apparatus Dr. A. Caron Modulated oscillators (2) . Dr. P. Nelson High impedance solid state amplifier^ Dr. L. Binstock Paper tape comparator Dr. E. Jerome Digital recording system Dr. V. Carlson Implanted heart-rate transmitter Dr. C. Nagel 1620 computer interface Dr. E. Jerome Soundproof chamber for use over operating table Dr. E. Evans Respiration sensor Dr. F. Snyder Electric shutter system for carousel projector Dr. A. Caron Projects in process Project Requestor Semiconductor strain gage for use with wrist flexion tester _ .-Dr. E. Evarts Motorized nipple assembly .. Dr. W. Stanley Constant current device _ Dr. F. Snyder Solid state pulse generators . Mr. L. Binstock Dr. K. Chandler Programmed volume control Dr. F. Snyder One second timer for oscilloscope camera Mr. L. Binstock Audio tape clock system Dr. R. Ryder Automatic projector system to facil- itate eye movement measurement Dr. J. Silberman Motorized sphere - Dr. P. MacLean Photocell controlled dual feeding chamber - Dr. W. Stanley Circular puppy feeding compart- ment Dr. W. Stanley Impedance mapping device _ Dr. F. Snyder 404 ANNUAL REVIEW OP INTRAMURAL RESEARCH Project Requestor Tissue section plotter Dr. A. Coulombre Puppy feeding nipple with vacuum sensor Dr. W. Stanley Programmed light intensity control Dr. F. Snyder Projects abandoned Project Requestor Thermistor blood-flow sensor Dr. W. Marshall Wrist flexion and extension tester Dr. E. Evarts Section on Technical Development Projects Projects initiated ivithin the section Project Status Digital tape transport system for LINC In process Active delay-line filter In process Neuron modeling system In process Solid state integrator Complete LINC low-pass filters Complete LINC sample and hold modification (2) Complete LINC rack no. 3 tape recorder Complete Bi-phasic constant current generator. Complete LINC programs in current active use Program Requestor Power spectrum analysis of the EEG_Dr. L. Speck Evoked response averaging for ran- dom blocks of optical stimulus pairs. LINC sorts stimulus pairs of equal duration, averages and computes standard error for mean^Dr. L. Speck Analysis for galvanic skin response data Dr. T. Zahn Preparation of programmed stimu- lus tapes for reaction timer Dr. T. Zahn Cumulative recording of food intake in a competitive situation for a social colony of squirrel monkeys Dr. P. MacLean Averaging of non-time-locked evoked responses by means of adaptive cross-correlation Dr. C. Woody Analyis of motor performance in subjects with Parkinson's tremor Dr. J. Van Buren On-line measures of single cell ac- tivity in auditory system of the cat Dr. E. Evans Dr. P. Nelson LINC pograms in preparation Program Requestor Display and filming of axon model based on van der Pol equations solved on 1MB 360/40 Dr. R. FitzHugh Digitization of oscillographic traces recorded on 35mm film with op- tical scanner Mr. L. Binstock Dr. R. Taylor Analysis of evoked responses Dr. G. Ojemann Dr. J. Van Buren Project Power spectra of EEG from elec- trode implanted in temporal lobe during a learning situation Utility programs to write and read magnetic tapes compatible with the IBM 360 Requestor Dr. P. Fedio Dr. G. Ojemann Mr. W. Sheriff . Mr. M. Bruce Mr. J. Bryan Mr. W. Sheriff Activity of cortical pyramidal cells in relation to flexor-extensor ac- tivity of the arm of a monkey during a period of trained behav- ior Dr. E. Evarts LABORATORY OF NEUROCHEMISTRY During fiscal year 1966 the Laboratory made a number of significant contributions to the general body of knowledge of how biological processes operate at the molecular level: A molecular-level model of ribosomes was proposed on the basis of studies with the dye- stacking method developed in this laboratory. In the model, ribosomal ribonucleic acid is stretched out on the ribosome surface in a sin- gle-strand, thereby providing a relatively non- specific matrix on which to bind messenger ribonucleic acid during protein synthesis. A computer program for simulating chemi- cal reactions between biochemicals was devel- oped and applied to the problem of complex formation between nucleotide bases. All of the known properties of such complexes become explicable in terms of simple electrostatic in- teraction between the bases. This work repre- sents the first successful attempt to understand how and why biological systems employ such complexes to store genetic information and to translate that information into action via pro- tein synthesis. Computer programs which reconstruct se- quences of biopolymers automatically, devel- oped in this Laboratory, were employed to test the consistency between proposed sequences and published fragment data as well as to de- sign optimum experimental conditions for de- termining sequences. A new sequence was proposed for alanine-transfer ribonucleic acid and an optimum schedule of procedures for determining the sequences of the other trans- fer ribonucleic acids was developed. NATIONAL INSTITUTE OF MENTAL HEALTH 405 A combined experimental and theoretical approach to the problem of the mode of action of non-competitive enzyme inhibitors was de- veloped. The binding constants for chlorpro- mazine and serotonin derivatives on histamine methyl transferase were measured and found to agree semi-quantitatively with the values predicted on the basis of the computed electron donating abilities of the inhibitors. Studies of the optical properties of biologi- cal polysaccharides, using optical theories de- veloped in part in this Laboratory, showed that at least some of these biopolymers exist in helical structures just as do polynucleotides and polypeptides. Work was begun on the isolation and charac- terization of cell membrane proteins, using a method of membrane isolation developed by one of the members of the Laboratory, as an initial step in determining how membranes control or otherwise affect cell differentiation and organization. At least fifteen different proteins were isolated from liver cell mem- branes and separated by disc/electrophoresis. Work was continued to elucidate the nature of a fascinating biological discovery, made in this Laboratory, that chloroplasts isolated from the unicellular plant euglena will survive and reproduce when injected into hydra. The new, classical theory of the interaction of light with molecular aggregates, developed in this Laboratory, was extended to include re- fraction, reflection, optical activity and circu- lar dichroism of crystals and solutions. Studies on the evolution and genetic con- trol of immunoglobulins were pursued with vigor, using techniques for determining their structure developed in the Laboratory. Of the two genes known to be related to yG-immuno- globulin synthesis in the rabbit, the b locus has been shown to control at least part of the amino acid sequence of the light chains while the a locus controls the structure of at least part of the heavy chain. The complexity of the lemon shark, a primitive vertebrate, was shown to be comparable to that of mammals although, unlike mammals, the lemon shark lacks the other major classes of immunoglob- ulins. The results of these studies have been pre- sented to the general scientific audience in the form of seventeen scientific papers which are either in press or were published in fiscal year 19G6. Fulfillment of less direct, but equally im- portant, responsibilities of the staff to the sci- entific community were met by editing jour- nals, refereeing manuscripts for journals, lecturing at universities and teaching courses in the graduate program at NIH and at local universities. Recognition of the scientific pro- ductivity of the group was indicated by offers to staff members of Chairmanships of Depart- ments and Professorships at universities of good standing, by invitations to give lectures and present papers at important meetings and at universities, by a constantly increasing num- ber of applications for positions in the labora- tory by well qualified scientists, and by a rap- idly increasing rate of requests for reprints of published articles. During fiscal year 1966 the Laboratory de- voted a considerable amount of time, energy and thought to the development of plans for the future. In order to give such plans tangible form, the staff members prepared, at no little cost in time and effort, the equivalent of re- search grant proposals which outlined in de- tail their research objectives and the means needed to cany them out over the next three to ten years. Critical to our attempts to create plans for the future has been an attempt to evaluate the current status and probable evolutionary path of biological research and our role in that evolutionary process. At the risk of oversimpli- fying a vast, heterogeneous field of science, it appears possible to divide biological research into analysis and synthesis. At present there is much effort being expended to analyze bio- logical events in terms of molecular events. Our role in this effort is clear: we are specialists in elucidating the how and why of molecular events which occur in biology in terms of the underlying physical forces involved. When one engages in the effort to analyze biological events in terms of molecular events, one is immediately impressed by the fact that there exists a logical progression in the types of biological processes that should be studied, i.e., those biological processes involving the 406 ANNUAL REVIEW OF INTRAMURAL RESEARCH least number of individual molecular events should be studied first. Whether by chance or conscious design, the field of molecular biology has followed this simple logical principle. En- zyme-substrate and antigen-antibody interac- tions, and the storage and translation of ge- netic information are current foci of interest and study for the molecular biologist since they involve smallish numbers of molecular events. Our Laboratory has concentrated on these same problems. Once a body of reliable knowledge on these simple processes has been accumulated, the field of molecular biology can move progressively and with confidence to- ward the analysis of biological events which, presumably, involve larger numbers of molec- ular events. Thus, one can envisage a natural evolution of the field toward cell differentia- tion, growth, organization, division, aging, in- fection and repair and then toward organ and organism behavior. Although it is clear that powerful drugs such as LSD or biopolymers such as RNA can affect very complex processes such as memory, learning, perception and con- sciousness, we feel that reliable knowledge of such processes and the effects chemicals have on them will come only by the long, arduous route now being followed by the molecular biologists. In the course of this evolution, anal- ysis will be supplemented by synthesis of indi- vidual molecular events into increasingly com- plex molecular systems. It is in the realm of syntheis where computers will become increas- ingly necessary in molecular biology, for only the computer can simultaneously retain the hard-won knowledge of each molecular event and organize large numbers of such events into an integrated system. In order to be able to con- tinue in the forefront of molecular biological research as it shifts emphasis from anlaysis to analysis-symthesis, our Laboratory is taking advantage of every available opportunity to assist in the development of computer facili- ties at NIH and to devise new methods for using them to study biological events. LABORATORY OF CLINICAL SCIENCE The Laboratory of Clinical Science, which is conserned with the broad area which extends from the basic biological sciences into the prob- lems of clinical psychiatry, is comprised of senior investigators each of whom is identified with a major biological discipline in addition to clinical training or interests. The research of the year now nearing completion has been concerned with an extension on the part of a number of the senior investigators of their research into areas to which they have in re- cent years contributed signifiant new concepts and information. Dr. Louis Sokoloff has made considerable progress toward identifying the soluble thy- roxine-mitochondrial product, which his ear- lier work showed to have a specific and im- portant effect on protein synthesis, which may probably represent a fundamental mechanism of the physiological action of thyroid hormone in the body. With Dr. Krause, he has demon- strated a thyroxine effect on the synthesis of a specific, naturally occurring protein — hemo- globin — in addition to the general effects on protein synthesis in brain and liver which he has previously demonstrated. Dr. Julius Axelrod has pursued many of the ramifications which his earlier work on cate- cholamine metabolism made possible. With Jacques Glowinski and Leslie Iversen, a number of studies were completed dealing with the ef- fects of important psychoactive drugs, such as reserpine, monoamine oxidase inhibitors, imi- pramine, and amphetamine, which appear capable of explaining the therapeutic effects of these agents in terms of their actions at adrenergic synapses in the brain. In coopera- tion with Dr. Richard Wurtman, an important regulatory action of adrenocortical steroids on epinephrine formation in the adjacent adrenal medulla has been discovered with clear indica- tions that this mechanism may be involved significantly in the adaptive response of the organism to various kinds of stress. Further studies on the endocrine function of the pineal indicated by previous work of these investi- gators have been concerned with the circadian rhythms of serotonin and catecholamine asso- ciated enzymes, and effects and pathways of environmental lighting in this rhythm. Dr. Irwin Kopin has extended his earlier studies of the factors mediating and attenuat- NATIONAL INSTITUTE OF MENTAL HEALTH 407 ing the action of norepinephrine at peripheral sympathetic nerve endings. Earlier with Mu- sacchio and currently with Schildkraut and Schanberg, studies have been undertaken which appear capable of defining some of the regula- tions involved in norepinephrine synthesis at these sites. With Baldessarini, attempts are be- ing made to develop the brain slice as a useful experimental model in factors affecting the re- lease of biogenic amines in the brain. These investigators have continued their basic con- tributions to the mechanism of transmethyla- tion in the brain. One current hypothesis has suggested an involvement of this process in schizophrenia. Dr. Marian Kies has continued her work on experimental autoimmune encephalitis, the best defined of all organ specific immune dis- eases, toward an understanding of autoimmune phenomena generally and, more specifically, in the brain, the encephalitogen which she has purified from brain and shown to be identical with the basic protein extracted from pure myelin has been used in the development of an extremely sensitive test for myelin proteins. With Dr. Borriss, an inhibitory effect on lym- phocyte protein synthesis has been demon- strated by purified encephalitogens. With Dr. August, a technique has been developed for detecting antigen-antibody combinations which does not require coupled biological assay and which may be useful in screening large numbers of sera for specific antibodies. Dr. Edward Evarts has extended his studies of individual cerebral neurons in freely moving animals which made possible his earlier im- portant contributions to the physiology of sleep. His current studies on the activity of pyramidal tract neurons in connection with voluntary movement have elucidated for the first time important temporal relationships be- tween the activity of different classes of such neurons and conditioned reaction times which promise to provide clarification of the physio- logical mechanisms of sensorimotor integra- tion. With Dr. Bizzi, an important regulation of visual sensory input has been found which appears to anticipate eye movement. This find- ing is the first demonstration of what appears to be an important general principle underly- ing central nervous organization. It is possible that whenever voluntary movements are car- ried out impulses go not only to the appropri- ate muscles but also to the sensory system, the information from which would be expected to be modified by the movement. Dr. Jack Durell has obtained some gratify- ing results in his program of study of the in- teraction between the biological organism and the social field in two therepautic milieux which he and a dedicated staff have established. In addition to certain clinical predictors of out- come which his pilot studies have suggested, a number of interesting biochemical findings have been obtained. His studies with Dr. Schildkraut on the excretion of catecholamine metabolites, in imipramine induced and spon- taneous remission in depression, have been ex- tended with corroboration of their additional findings of a rise in the secretion of norme- tanephrine in association with clinical improve- ment. Studies have been initiated in collabora- tion with other investigators of the Laboratory directed at an examination of sodium move- ments between intracellular and extracellular compartments of the brain in association with clinical change in the manic depressive syn- drome. The studies with Dr. Ryan on an alleged plasma factor in schizophrenia have been com- pleted. They were successful in demonstrating that the increased lactate production by eryth- rocytes incubated with certain human plasma was mediated through hemolysis, which in turn was caused by the action of a complement- requiring antibody present in the plasma of certain individuals. They were unable to demonstrate that this antibody is characteris- tically found in schizophrenics. With Dr. Frie- del, it has been possible to pursue earlier find- ings on the action of acetylcholine on phos- phatide acid metabolism in brain fractions containing synapatic elements. An hypothesis has been formulated which is compatible with many different observations in the literature and work has begun on the testing of its com- ponents. With Dr. Guggenheim, Dr. Philippe Cardon has been conducting collaborative clinical re- search in a number of different areas includ- ing an extension of his earlier studies on the 408 ANNUAL REVIEW OF INTRAMURAL RESEARCH differing response in various classes of indi- viduals to norepinephrine and, in collaboration with Dr. Pollin of the Laboratory of Adult Psychiatry, on the incidence of medical illness in the families of twins discordant for schizo- phrenia. With Drs. Reivich and Isaacs, the Labora- tory Chief has been extending his earlier in- terest in cerebral circulation to examination of the regional circulation under a number of physiological states. A productive collaboration with Drs. Evarts and Sokoloff has made pos- sible an approach to regional metabolism in various stages of sleep. The collaborative stud- ies with Drs. Rosenthal and Wender on nature- nurture interrelationships in schizophrenia ex- amined through study of adopted individuals is at its peak of data collection. It is hoped that results of certain of these studies will be avail- able at the time of the next annual report. More detailed summaries of the work of the individual sections prepared by the respective section chiefs follow. Section on Cerebral Metabolism The Section on Cerebral Metabolism has continued its research on the mechanism of action of the thyroid hormones, the relation of this mechanism of action to the different responses of mature and immature brain to the thyroid hormones, and the role of these hor- mones in the biochemical processes underlying development, maturation, and functional activ- ity of the central nervous system. It was work done in this project that first demonstrated that thyroid hormones stimulate the rate of protein biosynthesis. This effect of thyroxine was found in vivo and in vitro in cell-free preparations, but only in tissues which respond to thyroid hormones with increased oxygen consumption. Hypothyroidism is asso- ciated with a decrease in the rate of protein biosynthesis. Inhibition of protein biosynthesis in vivo by means of puromycin, a drug which inhibits protein biosynthesis at the same step or one preceding the step stimulated by thy- roxine, resulted in only slight decreases in total body metabolic rate of euthyroid animals but in marked reductions of the metabolic rate in hyperthyroid animals; in fact, puromycin acutely, and completely reduced the metablic rate of the thyrotoxic animals to the level of normal or puromycin-treated euthyroid ani- mals. These results indicated that a higher per- centage of the total body oxygen consumption in thyrotoxicosis is associated with the process of protein biosynthesis than in the normal state and that the increased metabolic rate of hy- perthyroidism is almost entirely secondary to the increased rate of protein biosynthesis. For the past two years the major efforts of the Section have been directed at the molecu- lar mechanism of the thyroxine stimulation of protein biosynthesis. Evidence has been accu- mulated that it is not thyroxine itself but a product of a thyroxine-mitochondrial reaction which is responsible for the stimulation. The stimulation has been localized to the step in- volving the transfer of sRNA-bound amino acid to ribosomal protein. It results in in- creased protein biosynthetic activity of the ribonucleoprotein particles, and this stimula- tion is independent of any effect on messenger RNA synthesis. In fact, it has been demon- strated to occur within 2 hours after the ad- ministration of thyroid hormones to an animal, a period of time far shorter than that reported to be required for RNA synthesis to be affected. Furthermore, thyroxine stimulates protein bio- synthesis in vitro in the absence of messenger RNA synthesis and, in fact, stimulates syn- thetic polyribonucleotide-directed amino acid incorporation into artificial polypeptides. Progress has been made on the isolation, purification and identification of the thyrox- ine-mitochondrial product responsible for the stimulation. It has been separated from the mitochondrial fraction and found to be soluble heat-stable, dialyzable, acid-labile, relatively alkali-stable, and destroyed by ashing. By means of substitution and kinetic studies, it has been distinguished from GTP, ATP, GSH, cyclic AMP, potassium, Mg ++ , or other sub- stances currently known or suspected to influ- ence the rate of protein biosynthesis. Recently it has been found to be possible to remove con- taminating nuceotides by charcoal treatment of the factor solution without serious loss of factor activity; this may be an important puri- NATIONAL INSTITUTE OF MENTAL HEALTH 409 fication step and may lead to increased progress in purifying and identifying the factor. In the previous year it was found that thy- roxine in vitro could stimulate the incorpora- tion of amino acids into the a and /? chains of hemoglobin being synthesized by cell-free rabbit reticulocyte lysates. This was the first demonstration of a thyroxine effect on the syn- thesis of a specific, naturally occurring protein. Together with the observed effects in liver and immature brain, this finding in regard to hemo- globin synthesis suggests that the thyroxine effect is not limited to one or a few specific proteins but is a generalized effect on the pro- tein biosynthetic machinery. Furthermore, both chains of hemoglobin have been well characterized, and it is known that the N-ter- minal amino acid (the initial amino acid laid down in the synthesis of the protein molecule) in both cases is L-valine. During the past year, N-terminal and interior amino acid analyses have been carried out, and though the studies are not yet complete, it is already apparent that thyroxine stimulates not only the comple- tion of the chains but also the initiation of the chain synthesis. This is an important finding in that it is the first demonstration that the thyroxine effect is on de novo protein synthesis. It was observed in earlier studies that amino acid incorporation into protein in immature brain preparations is more rapid than in ma- ture brain preparations. Also thyroxine stim- ulates amino acid incorporation in the imma- ture brain but not in mature brain. Both differ- ences are the results of some functional dissim- ilarities between mature and immature brain mitochondria. Another functional difference between mature and immature brain mito- chondria has now been observed. Immature brain mitochondria contain the enzyme, (3- hydroxybutyrate dehydrogenase, which re- mains active during the period of maturation of the brain and then rapidly declines to neg- ligible levels. Two such enzymes have been ob- served in other tissues, one which catalyzes the oxidation of the D-isomer of /?-hydroxybu- tyrate to acetoacetate and the other which catalyzes the oxidation of the L-isomer. Dur- ing the past year it has been clearly demon- strated that it is only the D-enzyme which is present and undergoing the changes in activ- ity during maturation of the brain. It has been solubilized and partially purified, and evi- dence of a heat-stable cofactor in the reac- tion has been obtained. This cofactor can be partially replaced by commercial lecithin, but the replacement is less effective than the nat- ural cofactor. The exact function of this en- zyme is not known, but it is believed to be involved in lipid synthetic or metabolic path- ways. Investigations are under way to deter- mine if this enzyme plays any part in the bio- chemical processes related to maturation of the brain. In the course of studying mitochondrial pro- tein biosynthesis in brain, it was observed that impure preparations of mitichondria from immature brain contaminated with myelin and nerve endings also incorporate amino acids in vitro into peptide linkage in the proteolipid of myelin. This finding offers the possibility of studying the mechanisms of myelin synthesis in vitro, and studies along these lines are be- ing carried on. The studies currently in progress and being planned for next year are along the lines indi- cated by the open questions and gaps in our knowledge pointed out in the discussion above. It is likely that the maximum efforts will be directed at the identification of the thyroxine- mitochondrial product. Section on Pharmacology The direction of the Section on Pharmacol- ogy still continues along four main lines of in- vestigations: the physiological dispositions of hormones and drugs, biochemical mechanisms of actions of drugs, enzymes involved in the metabolism of drugs and hormones and the function of the pineal glad. Mainly through the efforts of visiting scien- tists, Jacques Glowinski and Leslie Iversen, considerable new information concerning the physiological disposition of tP-noradrenaline in the rat brain has been obtained. In the past year, efforts were directed to studying the up- take, subcellular distribution and turnover of H -noradrenaline in different areas of the brain. IP-noradrenaline injected into the lat- eral ventricle of the brain was taken up in 410 ANNUAL REVIEW OF INTRAMURAL RESEARCH relatively large amounts in those areas having high endogenous concentrations of the cate- cholamine (hypothalamus, midbrain) and in small amounts in those areas having small amounts of the amine (cortex, cerebellum). Radioautographic studies also showed that H 3 -noradrenaline was highly localized in tracts of the limbic area of the brain. Various re- gions of the brain showed differences in the subcellular distribution and the turnover rate of noradrenaline. Although the cerebellum con- tains a low concentration of noradrenaline, it appears to utilize this amine at a greater rate than any other area of the brain. With the use of H 3 -dopamine and drugs in- hibiting uptake of H 3 -noradrenaline, it was demonstrated that the central adrenergic neu- rons release noradrenaline and then recapture the discharged neurotransmitter. Marked re- gional differences in the effect of amphetamine and imipramine on inhibiting the uptake of H 3 -noradrenaline were observed. There were also considerable differences in the ability to release the neurotransmitter by amphetamine and reserpine in various brain areas. In sub- cellular studies, amphetamine was found to act mainly at the nerve terminal while reserpine exerted its effects along the entire neuron and cell body. There was a correlation between the behavioral effects of reserpine and its ability to inhibit the accumulation of ^-noradrena- line in brain neurons. Methoxy metabolites of catecholamine (nor- metanephrine, metanephrine, and methoxy dopamine) enhanced the uptake of ^-nora- drenaline in certain tissues (salivary gland, vas deferens). Since normetanephrine is formed at the nerve terminal, it is suggested that this metabolite may serve a mediating role in the reuptake of H 3 -noradrenaline. Since the adrenergic receptor is the most important element in the adrenergic mechan- ism, an attempt to examine its biochemical properties was begun. The uptake of H 3 -nor- adrenaline in extraneural tissues (which in- cludes the receptor) and the effect of drugs were studied. After blocking neuronal uptake mechanisms with cocaine, it was found that a-adrenergic blocking agents had a profound effect inhibiting intracellular extraneural up- take of H 3 -noradrenaline into heart muscle. /3-Adrenergic blocking agents had a less marked effect. Preliminary studies suggest that up- take processes which are different from neu- ronal uptake are involved in extraneural intra- cellular accumulation of H 3 -noradrenaline in the heart muscle. This study is being carried out in collaboration with Drs. Eisenfeld and Krakoff. A study on the possible relationship between the development of hypertension and the mal- functioning of the sympathetic nervous system was undertaken with Drs. Jacques de Champ- lain and Lawrence Krakoff. Rats were made hy- pertensive with DOC and high sodium diet and the uptake and metabolism of H 3 -noradrena- line were studied. There was a clear and highly significant inverse relationship between the degree of hypertension and the ability to ac- cumulate H 3 -noradrenaline in heart and cer- tain tissues. Dr. Eisenfeld had continued to study the properties of the binding of H 3 -estradiol and target organs in the periphery and central nervous system. This uptake has been shown to be highly selective. A compartmental model for estradiol uptake by target tissues has been derived from the experimental data. In collaboration with Dr. Richard Wurtman, it was found that the adrenaline-forming en- zyme in the adrenal medulla is controlled by the secretion of corticoids in the adrenal cortex and by ACTH secreted by the pituitary gland. The corticoids act by increasing the synthesis of the protein that makes the adrenaline-form- ing enzyme. Two types of ectopic tumors were charac- terized by measuring two unique enzymes dis- covered inthis laboratory. One ectopic tumor contained a methanol-forming enzyme which is found only in the pituitary and another con- tained a melatonin-forming enzyme which is confined to the mammalian pineal gland. The melatonin-forming enzyme was found for the first time in species (amphibians, fish) where this hormone exerts its skin blanching effects. A new enzyme that O-methylates monophenol was found. It is present in the microsome of liver and other tissues. Studies with the pineal showed a 24-hour NATIONAL INSTITUTE OF MENTAL HEALTH 411 rhythm in the noradrenaline content of this gland. Unlike the serotonin rhythm, the cate- cholamine rhythm is completely controlled by environmental lighting. The circadian seroto- nin rhythm was found to be present at birth and for the first 12 days to be partly influ- enced by extra retinal receptors. The serotonin rhythm in the pineal appears to arise from a periodic release of the biogenic amine. The in- formation concerning environmental lighting reaches the pineal gland gonads via the medial forebrain bundle. Interruption of the classical visual tract in the brain has no influence on the effect of environmental lighting on pineal gland and gonads. Preliminary results indicated the messages concerning endogenous rhythm are first sent through the medial forebrain bundle. Section on Medicine The current investigations of the Section on Medicine concern the synthesis, storage, re- lease, metabolism and mode of action of the bio- genic amines and their modification by nerve impulses, aging, drug treatment and endocrine and electrolyte status. The rate of norepinephrine synthesis is ex- amined by studying its formation from various labeled catcholamine precursors or by study- ing the rate of decrease of its specific activity after labeling with norepinephrine-H 3 . Norepi- nephrine synthesis appears to depend on the occupation of norepinephrine storage sites in the synaptic vesicles of the neuron. Nerve stim- ulation releases norepinephrine from the nerve ending. A major fraction is taken back into the sympathetic nerve by an active transport system which is subject to interference by drugs such as cocaine or desmethylimipramine and by sympathomimetic amines. Portions of the remaining released norepinephrine react with the receptor, are destroyed by catechol-O- methyltransferase or enter the circulation. Such losses are replenished by synthsis. Thus, nerve stimulation or any environmental influ- ence which results in an increased number of impulses stimulates synthesis. Drugs which block reuptake of norepinephrine released by nerve stimulation further increase norepine- phrine synthesis just as tyramine depletion of norepinephrine stores stimulates synthesis of norepinephrine, as we previously observed. Brain or heart slices concentrate several tritium-labeled birogenic amines (norepine- phrine, sertonin, histamine, etc.), and elec- trical stimulation induces their release. Such release of norepinephrine is inhibited when calcium content is low or by drugs such as pentobarbital, chlorpromazine and desmethy- limipramine. The tissue-slice technique may provide a method for studying the action of drugs, ions and hormones on the process of transmitter-release coupling. The demonstration that labeled amines in- jected into the ventricles of rats may provide a valid tracer for endogenous amines stimu- lated the search for a simpler means of label- ing the amine stores of brain. Intracisternal injection appears to be as valid as intraven- tricular and is being routinely used in this laboratory for such studies. Turnover rates of norepinephrine in various physiological states (sleep, REM deprivation, cold exposure, etc.) and alterations of turnover in response to drugs are also being studied. Nembutal anes- thesia has been shown to elevate initial levels of several amines as well as the level of urea, but ether anesthesia does not appear to have the same effect. This may reflect cerebrospinal fluid circulatory changes or alterations in dif- fusion barriers in brain during nembutal an- esthesia. Studies of substances which can replace norepinephrine at its binding sites and can act as false neurochemical transmitters continue. Their possible role in disease states and drug action has been previously reported by this laboratory. The interaction of the false trans- mitters with bretylium, which interferes with axonal nerve impulse transduction to transmit- ter release, is being examined. Bretylium does not cause great release of norepinephrine, but it does very efficiently release some of the a- methylated false transmitters. This difference in action may provide a means for assessing avidity of amine binding to norepinephrine storage sites. Investigation of familial dysautonomia has led to observations of clinical abnormalities in several sensory systems: taste, hearing, per- 412 ANNUAL REVIEW OF INTRAMURAL RESEARCH ception of heat, cold and pain, and abnormali- ties in cardiovascular as well as peripheral reflexes. Such abnormalities are being investi- gated in collaboration with physicians of the National Heart Institute. Both the patients and their mothers appear to excrete excessive amounts of homovanillic acid, a metabolite of dopamine. The patients excrete lower amounts of VMA, the major norepinephrine metabolite, than do their mothers. Since no significant ab- normalities in metabolism of administered catecholamines have been found 1 , further in- vestigation in the project will be held in abey- ance until methods for assessment of receptor physiology in man can be developed. Methylation is a major pathway for metab- olism of the catechol- and other amines. The addition of a methyl group alters the activity of the compound and may result in activity increase or decrease, or it may impart entirely new properties to the molecule. S-Adenosyl- methionine is the major methyl donor in mam- mals, and a method of assaying this key inter- mediate has been developed. Methionine in- creases SAMe levels, while methyl acceptors diminish its concentration. Levels are higher in infant liver and brain than in correspond- ing adult organs. The levels are elevated in leukemic white blood cells and depressed in livers of animals with a portacaval shunt, pre- sumably because dietary methionine fails to reach the liver. The dynamics of activation of methionine are currently being studied. Section on Biochemistry The Section on Biochemistry has combined the techniques of immunology and biochemis- try to study the phenomenon of autoimmune pathology. Because of the responsibility of sci- entists in the Institute to extend and develop areas of knowledge pertaining specifically to mental health we have concentrated on the brain as the experimental model. The informa- tion gained, as well as the techniques developed, are generally applicable to autoimmune path- ology of other organs. Experimental autoimmune encphalitis is the best defined of all of the organ specific immune diseases. Current studies on the etiology of this disease are based almost entirely on results of investigations of the Section on Biochemistry over the last ten to twelve yea:- Our group was the first to isolate a purified basic protein fraction from brain and demonstrate that the major portion, if not all, of the encephalito- genic activity of whole tissue resided in this group of proteins. Additional studies have dem- onstrated the immunologic homogeneity of the basic proteins as CNS specific, species nonspe- cific antigens. Thus, purified myelin protein antigens will soon be available for testing hy- potheses of CNS autoimmunity in both clini- cal and experimental situations. Our initial observation that basic proteins constitute an important fraction of myelin proteins was proven by actual isolation of the protein from purified myelin. The basic protein extracted from pure myelin was shown to be identical to the purified encephalitogen pre- viously isolated from whole brain homogenate. An important consequence of this discovery was the fact that an extremely sensitive bio- logical test could be added to the various chem- ical techniques available for studying myelin proteins. As little as ly of encephalitogen can be detected by bioassay whereas most chemical analyses for specific proteins require much more, e.g., a single disk gel electrophoretic pat- tern requires 50-100A, at the very least 25\. With this added analytical tool, we obtained some of the earliest definitive data on purified myelin protein-lipid complexes: the identifica- tion of some of the lipid firmly bound to pro- teins. Fractionation of these protein-lipid com- plexes by solvent distribution coupled with bioassay of the fractions provided proof of the complexity of myelin proteins (as opposed to the widespread belief that "myelin protein" was a single molecular entity). During the past year, the work pioneered by this laboratory — demonstration that basic pro- teins comprised an important part of myelin — has been acknowledged by other major neuro- chemical groups by corroborating publications. In a study of the role of lymphoid cells in the development of experimental autoimmune encephalitis, we have investigated the effect of purified CNS basic proteins on in vitro protein synthesis by guinea pig lymph node cells. Dr. Elsa Borriss, a visiting scientist in the Bio- NATIONAL INSTITUTE OF MENTAL HEALTH 413 chemistry Section, has demonstrated that in vitro incorporation of leucine C-14 into guinea pig lymphocyte proteins is inhibited nonspe- cifically by purified encephalitogens from var- ious species, both homologous (guinea pig) and heterologous (human, bovine). Less pure heterologous encephalitogenic preparations ex- hibit the opposite phenomenon — i.e., specific stimulation of leucine C-14 incorporation. The stimulation, however, is related to the presence of species specific determinants on these bovine basic protein preparations. The demonstration that CNS basic proteins are able to affect metabolic activity of living cells in vitro suggests the possibility that these proteins may function as regulators of myelin synthesis in vivo analogous to the idea that histones are regulators of nuclear activity. (Preliminary data obtained with C. Klee sup- port this idea.) The long-term goal of this project is to study the role of lymph node cells in the development of delayed hypersensitivity. Experiments are in progress to icolate the newly synthesized (C-14 labeled) globulins. Immunoelectropho- resis coupled with radioautography will be used to define their location in the culture (cell bound or in the medium) and their immuno- logic specificity. As an adjunct to his other immunologic stud- ies, Dr. August has developed a technique for detecting antigen-antibody combination which may be used to study all types of binding phe- nomena. The prerequisite is that small mole- cules involved can be isotopically labeled. It possesses the great advantage that the method does not require a coupled biological phenom- enon such as skin reactivity, complement fixa- tion, hemagglutination, anaphylaxis, etc. for determination of antibody. Furthermore, many tests can be run simultaneously making it a useful method for screening large numbers of sera for specific antibodies. The technique depends on the differential migration of large and small molecules through synthetic gels. We have used 1-125 labeled an- tigen (the small molecule) to detect the pres- ence of specific antibody (large molecule) in various experimental sera. With proper choice of experimental conditions and controls, the type of antibody (19s-macroglobulin or 7s-y- globulin) can be determined, as well as infor- mation regarding the amount of antibody and the avidity of the antigen-antibody combina- tion. Not only is the method useful for detec- tion of antibody, it can be adapted for the study of other biological interactions such as occur in drug allergies. Another application is immunoassay of low molecular weight hor- mones at levels of sensitivity far below cur- rent techniques. Finally, the laboratory has continued its study of the molecular size of the encephalito- gen and characterization of its active site(s?). Fractions from serum digests of pure anti- gen are being investigated with regard to molecular size, encephalitogenic activity, anti- genic specificity, and amino acid composition. The major constituent of the bovine encephali- togen is a cathodic protein of moleculr size ^ 60,000 (estimated by gel filtration on Sepha- dex G-75). Serum digests contain small amounts of this component plus three other UV absorbing fractions. The latter are presum- ably peptides of smaller size — some less than 10,000 molecular weight. At least two of the three peptide fractions have encephalitogenic activity. The biological activity of the smallest of the group is still under test. Related studies have shown that different preparations of encephalitogenic basic protein may have widely different molecular sizes. For example, certain preparations consist predom- inantly of a basic protein which is one-half or one-fourth the size of the bovine preparation used for serum digestion experiments. In searching for an explanation for this variabil- ity in size, we have obtained data which sup- port the hypothesis that variable activity of brain cathespin and/or serum proteinase pres- ent in the original brain homogenate is respon- sible for the size of the final purified protein. Because of our long and varied experience in this field, we are frequently approached by other laboratories for collaboration, advice or assistance in carrying out studies on EAE. We are currently "collaborating" in this manner with Dr. M. Wolman in Tel Aviv, Dr. Marten- son in Surrey, England, Drs. Amaducci and Cazzula in Italy, Dr. Waksman at Yale, Drs. 414 ANNUAL REVIEW OF INTRAMURAL RESEARCH Campbell and Wolfgram in Los Angeles, Dr. Tourtellotte in Ann Arbor, Michigan, Dr. D. Heilman at the V.A. Hospital in the District of Columbia, etc. A particularly interesting collaborative study on experimental autoimmune encephali- tis is being carried out with Dr. Peter Lampert at the Armed Forces Institute of Pathology. He has studied the pathological lesions associated with the disease by electronmicroscopy. We hope to combine the techniques of the two groups in an attempt to demonstrate the pres- ence or lack of specific antibody in the area of the lesions. Section on Physiology Two major problems have been investigated in the Section on Physiology during the past year: 1) the relation of pyramidal tract ac- tivity to voluntary movement, and 2) mechan- isms whereby the brain coordinates and inte- grates eye movement information with input from visual stimulation of the retina. These two areas of investigation will be described sepa- rately. The Relation of Pyramidal Tract Activity to Voluntary Movement In the previous annual report, it was pointed out that it has now become possible to record the activity of individual cerebral neurons in moving animals. This new technique opens up an important area which had previously been uninvestigated: prior to the recent work car- ried out in the Section, there had been no stud- ies of the way in which pyramidal tract neu- rons (PTNs) control and initiate voluntary movement. In the past year investigations have been di- rected toward determining the precise time in a stimulus-response sequence at which the activity of PTNs comes into play. In order to determine the timing of this pyramidal tract discharge it was necessary that the monkeys make a specific hand movement in response to a stimulus. Monkeys were trained to depress a modified telegraph key until a light came on (the light being the conditioned stimulus) and then release (conditioned response) the key promptly following light onset. This stimulus- response sequence is analogous to simple reac- tion time tests used in man and, in fact, the reaction times of monkeys who have been thor- oughly trained are the same as the reaction times of highly motivated and well-trained human subjects. This experimental situation then, allowed investigation of the central events intervening between stimulus and re- sponse in a conditioned hand movement. The results of the study answered four ma- jor questions of pyramidal tract physiology. These questions and their answers follow. At what point in the interval between con- ditioned stimulus (light onset) and condi- tioned response (wrist extension) does modi- fication of discharge in PTNs occur? It was found that even in cases of minimum (180 msec.) reaction times by the monkey, the latency of the antecedant modification of pyra- midal tract discharge does not fall below 100 msec. This 100 msec, latency stands in sharp contrast to the 30 msec, latency with which PTNs in the motor cortex discharge in re- sponse to a photic stimulus in animals anesthe- tized with chloralose. It is clear then, that for this conditioned movement, the latency of re- sponse in PTNs is delayed at least 70 msec, beyond the minimum latency demanded by the anatomical connections between the retina and motor cortex. What sequence of neuronal events takes place during this 70 msec, delay? An an- swer to this question would provide useful clues as to mechanisms of sensorimotor inte- gration. Is the occurrence of the wrist movement tem- porally locked to the PTN response? There was a strong positive correlation between the reaction time of the monkey and the latency of response in PTNs. Thus, in the most proficient monkeys, PTN discharge might begin 100 msec, after the conditioned stimulus; the arm muscles might begin to show electromyo- graphic responses about 140 msec, following the conditioned stimulus; and the final re- sponse (opening the contact) might occur in 180 msec. For longer latencies of PTN response there were longer reaction times. Thus, it was found that not only did PTN activity precede the behavioral response, but also that length- ening of response latencies in PTNs was asso- NATIONAL INSTITUTE OF MENTAL HEALTH 415 ciated with lengthening of behavioral response latencies, i.e., reaction times. What is the relation of axonal conduction velocity of a PTN and the response which it shows in association with movement? In a pre- vious study described in the annual report one year ago, it was found that PTNs with high axonal conduction velocities tended to be silent in the absence of movement, but to become ex- tremely active during movement. PTNs with lower axonal conduction velocities were toni- cally active even in the absence of movement, The present study of a conditioned movement revealed an anlogous relationship between ax- onal conduction velocity and discharge proper- ties in PTNs. Thus, units which were silent while the monkey was maintaining wrist flex- ion but spring into intense activity prior to wrist extension had high axonal conduction velocities. Units with low axonal conduction velocities did not show such sharp transient responses and when they were related to the wrist extension showed either a reduction in discharge frequency or an increase of what had been tonic discharge persisting through- out wrist flexion. Is activity of PTNs related only to contralat- eral movements, or are there some PTNs whose discharge is related to ipsilateral movements as well? The great majority of PTNs exam- ined were related to movements of the contra- lateral wrist and were relatively inactive in relation to ipsilateral wrist movements. Some units were found, however, for which the re- verse was true, and this finding provides addi- tional evidence for the role of the pyramidal tract in control of ipsilateral movement. Mechanisms Whereby the Brain Coordinates and Integrates Eye Movement Information with Input from Visual Stimulation of the Retina This study of the integration of eye move- ment information with information from the visual input to the retina grew out of a previous study of activity in the lateral geniculate nu- cleus during the rapid eye movements (REMs) of sleep. This study showed that each REM of sleep is associated with a volley of impulses arising in the oculomotor centers and imping- ing on the lateral geniculate. It was reasoned that the occurrence of this "corollary" dis- charge in the lateral geniculate must have sig- nificance with respect to waking as well as to sleeping eye movements, and it was therefore decided to investigate this problem in the case of the eye movements of waking monkeys. The technique of single unit recording was em- ployed. It was found that certain neurons in the lateral geniculate nucleus discharge in relation to eye movements, some of these neurons dis- charging even before the eye movement has occurred. Such prior discharge cannot be the result of feedback from the eye muscles and therefore proves that there may be a discharge of neurons in a sensory pathway in anticipa- tion of a coming movement. This finding is the first demonstration of what will probably turn out to be a highly important general principle underlying CNS organization. It seems not un- likely that whenever intentional or voluntary movements are carried out, impulses go not only to the muscles whose contraction is neces- sary for the occurrence of the movement, but also to these sensory systems whose incoming information will be modified by the movement. In a sense, one may say that instead of having to wait to be told by input from the periphery that a particular movement has occurred, the sensory systems are told of the impending movement even before it takes place. These sensory systems may then interpret the input which comes to them in the light of this knowledge. The theoretical formulation proposed above is not a new one (it was first proposed by neu- rologists and psychologists many decades ago). The present experiment, however, provides the first clear proof of the theory and is also the first to show corollary discharge at the level of the single neuron in association with volun- tary movement. Significance for Mental Health Research The two projects described above involve analyses of the central events associated with voluntary movement. The study of hand move- ments is aimed at discovering how output in- formation is coded, while the study of eye movements is primarily concerned with the de- 416 ANNUAL REVIEW OF INTRAMURAL RESEARCH lineating events in sensory systems which are associated with voluntary movements. These two projects complement each other in that an ultimate general understanding of voluntary movement will require consideration of both of these classes of problems. The broad aim of these projects, then, is to achieve an understanding of the central mech- anisms underlying voluntary movement. At first glance it might seem that this problem bears little relevance to problems of mental health and disease. However, students of the mind have long been struck by the existence of an indissoluble relation between the movement output of the organism and the very essence of mind and thought. The greatest brain-research workers, ranging from Hughlings Jackson to Roger Sperry, have suggested that our under- standing of the mind and its disorders may be facilitated by working into the system "back- wards" from its output, rather than forward from its input. It therefore seems likely that the principles of nervous system organization revealed by our studies on voluntary movement may provide a deeper understanding of how the brain functions in its most complex as- pects. Such an understanding would clearly con- tribute to our knowledge of thought processes in both health and disease. Section on Psychiatry The past year has been a gratifying one. The new activities and new directions of research initiated during the previous two years have begun to solidify, creating the sense that the Section is now well on the way to establishing its long-range research course. As previously stated, it is the goal of the Section to increase our understanding of the mechanisms by which disturbed behavior is generated or dissipated through a greater appreciation of the interac- tion between the biological organism and the social field. The major fields of observation are the two therapeutic communities which have been devised in a way which permits much data collection both of a social interactional nature and a biological nature. The longitudinal de- scriptive clinical ratings have been institution- alized and it is envisioned that the information thus stored will prove most instructive when analyzed several years hence. The plans to ini- tiate controlled studies have been formalized an are now in effect. Because of the desirability of attempting to obtain a relatively homogen- eous group of patients for control studies, the rate of admission of such patients to the wards has been slow but there is reason to believe that a sufficient number will have been studied within the next five years to make the results quite meaningful. An extensive follow-up study of all of the patients admitted to the therapeu- tic community is now under way which will provide very valuable clinical dscriptive mate- rial from which various hypotheses can be gen- erated for more rigorous testing. In addition, the regular collection of urine and other bio- logical samples as well as patients' participa- tion in various physiologic procedures is now well institutionalized in both units. In addition to the sense of smooth institu- tionalization of the research, there is also clear evidence of a maturing of the therapeutic team with a corresponding growth of autonomy and creativity among the therapeutic staff mem- bers at lower echelons. The psychiatric staff, as well as the auxiliary and nursing staff are able to operate with much less supervision di- rectly from the Section Chief, while maintain- ing the clinical approach and philosophy which had originally been introduced by the Section Chief. In recent months this has allowed the Section Chief much greater time to employ in consolidation of the research activities and it is envisioned that the following year will be even more successful in that regard. In addition to clinically based studies, the Section endeavors to maintain a continued in- terest in basic neurochemical research. The Section Chief is well aware of the danger that biological psychiatry can become both poor psy- chiatry and poor biology. Therefore, in addition to the large emphasis placed upon the social interactional therapeutic milieu itself, it is felt necessary to maintain a contact with basic neurochemical research so that the biological concepts employed in the psychiatric research will remain in apropriate contact with the rap- idly advancing events in the neurochemical sciences. Since a major portion of the research effort NATIONAL INSTITUTE OF MENTAL HEALTH 417 of the Section represents long-range clinical research from which there are few results until after many years of data collection and analy- sis, the aforementioned sense of a consolidation in organization is a very important measure of the progress made during the year. This is not to say that there have not been a number of specific findings in the various areas and these shall now be elucidated: The analysis of the pilot follow-up study and the relation of status at follow-up to a number of initial social parameters has been virtually completed and will soon be ready for publica- tion. It was found, by and large, that the staff attitudes towards the patients during his first week of hospitalization could partially predict the clinical state at follow-up one year later. In addition, the discharge status was a good predictor of the status at follow up. These findings suggest various hypothesis regarding the dynamics of the therapeutic community and consideration of means of testing some of these hypotheses are now under way. The re- sults suggest that the attitudes of staff are in fact operators, influencing whether or not a patient's outcome will be favorable. Moreover, the results suggest further that the 4 East therapeutic milieu has, in fact, achieved cer- tain of its goals which were the carrying out of therapeutic gains into the community, thus facilitating the patient's social integration into the community-at-large. It has been shown in a series of schizo- phrenic patients with remitting psychoses that depression of mood characteristically occurred following the remission of psychosis. This is significant in the clinical sense it pro- vides a warning that the staff must be aware of possible suicidal potential particularly when psychotic symptoms first remit. In addition, the findings have provided the basis for theo- retical considerations regarding the psycho- logical and neurophysiological mechanisms con- cerned in psychosis and the relationship of these to possible genetic factors and to pheno- thiazine therapy. Studies are under way relat- ing changes in adrenal steroid secretion to the changes in affect and to the administration of phenothiazines. Observations such as these, though open to many interpretations, help to provide some of the documented empirical basis for hypothesis generation and thus serve a heuristic purpose. We have had difficulty finding patients with cyclical schizophreniform psychoses but have managed to study one such patient this year. There are indications that changes in excre- tion of catecholamine metabolites and thyroid radioiodine uptake accompanies his psychotic episodes. The results bear some similarity to results previously obtained on a patient with an alternating motility cycle while differing sharply from our findings and those of Gjess- ing on periodic catatonia. It appeals that we have isolated two problems of physiological change associated with two different clinical syndromes, but this conclusion requires corro- boration. Studies on the changes in catecholamine me- tabolism associated with the imipramine treat- ment of depression have continued. The earlier suggestion that a rise in the secretion of nor- metanephrine is associated with definitive clin- ical improvement with imipramine has been corroborated on additional patients. The rela- tionship between the excretion rates of the various catcholamine metabolites are now un- der study as are additional patients who are being treated with imipramine or amitripty- line or without antidepressant agents to deter- mine whether these findings are specific to imipramine. Methods have been developed to study elec- trolyte metabolism in manic depressive patients and the effects of lithium and imipramine on these variables. The methods including the use of whole-body counters and probes to detect head sodium appear to be satisfactoiy and the data collection period is just beginning. The clinical staff has treated several patients suc- cessfully with lithium carbonate. These pa- tients had previously been treated in a number of other ways without success and the results with lithium carbonate appear promising. With the departure of Dr. Ryan from the lab- oratory, studies on an alleged plasma factor in schizophrenia have virtually been termi- nated. The analysis of the data from a large population study at the Rockland State Hos- pital gave little evidence to support an associa- 418 ANNUAL REVIEW OF INTRAMURAL RESEARCH tion of the antibody affecting chicken erythro- cytes with schizophrenia. There are several interesting possibilities to follow up, however, especially in that some studies of the Section on Twin Studies indicate that psychosomatic fac- tors may operate to influence the level anti- body in certain schizophrenic individuals. This is not of primary interest to this Section and it is not clear whether these studies will be undertaken. Previous indications that there may be some elevation in the activity of the sodium pump ATPase in certain psychotic states could not be confirmed in a population study at D.C. Gen- eral Hospital. Unfortunately, it was difficult to control for the influence of a number of in- tervening variables at D.C. General Hospital and currently more controlled longitudinal study of patients whose clinical state under- goes change is under way in an effort to deter- mine whether there are any associated changes in the sodium pump ATPase. We have specu- lated that alterations in the activity of this enzyme may also be associated with the changes in electrolyte metabolism is manic depressive illness, and this problem is being approached with the use of laboratory and human physiology techniques. Basic studies on membrane function have narrowed down to the studies of the mechan- ism of acetylcholine effects on phospholipid metabolism. It has been shown that the acetyl- choline sensitive phosphatidic acid metabolism of brain homogenates is limited almsot com- pletely to the subcellular fractions containing "synaptasomes". Since the arrival of Dr. Rob- ert Friedel in our laboratory there has been an intensive interest in studying the mechanism of this process in brain synaptasomes. An hy- pothesis has been formulated which relates a number of observations in the literature and suggests that the primary action of acetylcho- line is upon a phosphatidylinositol phospho- diesterase. The testing of this hypothesis has required the tooling up in terms of the labor- atory's capacity to separate and purify phospholipids, particularly triphosphoinositide and to prepare triphosphoinositide phospho- diesterase from brain and other tisse. These methods have now been mastered but it is too early to state whether the results are confirm- ing our hypothesis or not. The hypothesis appears to be an important one since, if cor- rect, it may provide insights into the mechan- ism of acetylcholine mediated membrane depolarization and consequently synaptic trans- mission. Knowledge about the molecular mech- anism of this reaction could be of profound significance to studies of brain function and the effects of pharmacological agents upon the brain. Unit on Psychosomatics Members of the Unit have continued their activities in collaborative clinical research in several different areas. Nervous and Circulatory Systems The postulate that responsiveness to norepi- nephrine may be related to patterns of night- time catecholamine excretion could not be confirmed. The rate at which the forearm de- creases in circumference in late diastole cor- relates moderately well with forearm blood flow through the range of flow rates measured at rest, during epinephrine infusion, and the com- pressor procedure. Evaluation of the estimated isometric period of ventricular contraction as an index of cardiac sympathetic tone has given puzzling prelimary results: by this criterion, high "cardiac sympathetic tone" may be asso- ciated with loiv free NE excretion. Inciden- tally, females have been found to have longer isometric periods than males — of interest when related to the general impression that individuals with long periods may be less sub- ject to coronary artery disease. Diet and Catecholamine Excretion With Guggenheim, quantitative data on ef- fects of diet on catecholamines and their me- tabolites in urine have been collected. Differ- ences between very restricted and moderately restricted diets can be demonstrated (by paired comparison) in excretion of VMA, epineph- rine and metanephrine. These differences are small compared with the differences among individuals on controlled diets. Norepinephrine excretion could not be altered by very exces- sive intake of coffee, chocolate, bananas, fruits, NATIONAL INSTITUTE OF MENTAL HEALTH 419 vegetables, and vanilla. These data are very helpful for planning longitudinal studies of psychiatric patients. General Health of Families As a contribution to studies of families and schizoprenia, Guggenheim has expanded ob- servations on their medical health — a further thyroid disease and cancer in parents of dis- cordant identical twins. These observations may be pertinent to the possibility that schiz- ophrenia may be usefully viewed as one of many possible manifestations of a broader psychological disorganization and vulnerability in families. Observations in Psychiatric Illness With other units and sections, Guggenheim has continued studies of heart rate and blood pressure in depression, but results cannot yet be given. Baer is evaluating the response to ACTH of the adrenal glands of depressed pa- tients, and now is starting to follow total ex- changeable body sodium in depressed patients, by means of the whole body counter. This is very promising because accurate daily estima- tions are possible which are not subject to the cumulative errors of inaccurate urine col- lection. CLINICAL NEUROPHARMACOLOGY RESEARCH CENTER The Clinical Neuropharmacology Research Center (CNRC) is located in renovated por- tions of the William A. White Building on the campus of Saint Elizabeths Hospital (SEH), a large Federal mental hospital in Washington, D.C. Established by NIMH in the late fifties in accord with SEH, CNRC gradually evolved a back-bone basic laboratory program of neuro- logical research around which clinical research activities were fleshed out when desirable, to take advantage of the opportunities provided by the hospital's environment on the one hand and of the physical and intellectual support provided by the basic research program on the other. The experience gained during the past sev- eral years with this type of arrangement and the development by SEH since the early sixties of its own Behavioral and Clinical Studies Cen- ter (BCSC) permitted last year to more pre- cisely define the role of CNRC intramurally and vis a vis the SEH complex of research and training activities. Accordingly, steps were taken to bring about the strengthening of CNRC basic research activities, concomitantly with a reduction in the level of direct CNRC sponsorship of those clinical studies which ex- perience showed to be more expediently carried out by SEH staff or as a collaborative endeavor with other clinical NIMH laboratories. Additionally, closer ties were established be- tween the CNRC and the SEH's research and training components through courtesy appoint- ments extended to CNRC senior investigators by the Superintendent, SEH and by the Depart- ment of Psychiatry, George Washington Uni- versity Medical School, with which SEH has a close working relationship. Thus the Acting Chief, CNRC was appointed Director of Re- search, SEH with direct responsibility to the Superintendent, SEH for the coordination of all research activities at SEH, inclusive of those of the hospital's BCSC, now led by the former Director of Behavioral Studies, BCSC who is concurrently Director of Training, SEH. Voluntary participation of senior CN- RC staff in SEH training programs for resi- dents and medical students was facilitated by the appointment of Drs. G. C. Salmoira- ghi (neurophysiology-neuropharmacology,) H. Weil-Malherbe (neurochemistry) and S. Szara (psychopharmacology) as Associate Clinical Professors, Department of Psychiatry, George Washington University Medical School. As in previous years, much of our effort wa r directed toward the elucidation of the mode of action of endogenous brain substances and re lated pharmaca appearing to affect central synaptic transmission. Most of these studies were, by necessity, carried out in animals but some utilized selected groups of patients made available through collaborative programs with SEH and the Adult Psychiatry Branch, NIMH. Human subjects were also used in studies for the development of analytical tools to assist in the interpretation of EEG records and for the study of saccadic eye movements. Utilizing 5-barreled glass micropipette elec- 420 ANNUAL REVIEW OP INTRAMURAL RESEARCH trodes, which permit the controlled electro- phoretic administration of up to 3 drugs di- rectly at the site of extracellular unit recording, studies were carried out on the lumbar seg- ments of the cat spinal cord to determine the properties of pharmacological responsiveness of single spinal neurons to the suspected cen- tral transmitters acetylcholine (ACh), norepi- nephrine (NE) and serotonin (5-HT). The results of these studies, in addition to confirm- ing evidence for ACh-mediation of an excita- tory synaptic input on Renshaw cells, yielded data highly suggestive for adrenergic synapses on motoneurons as well as on Renshaw cells and other interneurons, presumably part of a bulbospinal pathway shown by others to be composed of NE-containing nerve fibers. These studies, moreover, showed each of the three suspected transmitters to be capable of produc- ing either facilitation or depression of cell ac- tivity, depending upon the type of cell studied. Taken together with all other evidence that we have thus far obtained from other CNS regions, the observations now made in the spinal cord leave little doubt that the direction of a unit's response to a suspected transmitter is not exclusively determined by the latter's chemical nature — as commonly assumed — but depends also upon characteristic properties of the effector cell's receptive membrane, deter- mining whether excitation or inhibition will occur. Hence our evidence begins to suggest that the same transmitter may be either exci- tatory or inhibitory for different neurons or even for different synapses of the same neuron. To eventually test this hypothesis as well as to obtain more persuasive evidence that ACh, NE and 5-HT are indeed transmitters in the mammalian CNS, intracellular recording is re- quired to compare the conductance changes produced by a naturally released transmitter with those produced by the extracellular ad- ministration of the suspected substance, and to investigate the effects of potentiating and blocking drugs. For this reason, a multibar- reled concentric micropipette electrode has been developed and is now being tested on moto- neurons. Additionally, a major effort was made to develop a preparation suitable for the study of the pharmacological properties of individual patches of a nerve cell membrane using the gastro-esophageal ganglion of a Nudibranchs which contains one very large and 2 medium- size neurons. Since these three cells form a miniature CNS, it is hoped that this preparation will be suitable for studying morphological and/or electrophysiological changes possible relatable to learning. In any event, such a preparation will be sought as part of a program of research in comparative neurophysiology likely to evolve from current studies by the Section on Neuro- pysiology and from those soon to be initiated on the fine structure and the cytochemistry of peripheral and central neurons. The metabolism of NE and other catechola- mines continues to be investigated by the Sec- tion on Neurochemistry. Pursuing earlier stud- ies by this Section on the binding and storage of catecholamines in tissues, it has not been shown that rapid freezing substantially in- creases the proportion of particle-bound to free NE in the brain, while incubation of brain homogenates causes rapid release of particle- bound NE. The distribution of dopamine be- tween particulate and soluble fractions was however little affected by these procedures and it was found that the ratio of particle- bound to free dopamine in different brain re- gions is less variable than for NE, suggesting that the mechanism of binding is different for the two brain catecholamines. The mechanism/s of NE binding, release and reuptake were also studied in a variety of other tissues, while the patterns of urinary excretion of catecholamines and their metabo- lites continued to be investigated as part of collaborative programs with NASA Manned Spacecraft Center and with the Adult Psychi- atry Branch, NIMH. The latter program deals with catecholamine metabolism in depressive disorders and emphasizes the longitudinal study of individual cases. It has been found that catecholamine excretion correlates with the type of illness as well as with the changing phases of mood. Related to these studies is the continuous scrutiny of the analytical methods available for the estimation of catecholamine metabo- lites with the view of improving both their NATIONAL INSTITUTE OF MENTAL HEALTH 421 sensitivity and specificity. Particular attention was given to the method for the estimation of metanephrine and normetanephrine which in our hands consistently yielded values consider- ably below those obtained in other laboratories. Using; labeled substrates, it was possible to pin- point the steps where losses occurred and to introduce corrective modifications. Neverthe- less, our present results are not substantially different from the earlier ones, suggesting that the difference with the values obtained by others is attributable to greater specificity of our method. The estimation of 3,4-dihydroxymandelic acid (DHMA) also claims our attention. Ear- lier hopes of using a bacterial mandelic dehy- drogenase for this purpose could not be real- ized since the enzyme was specific for DHMA having the L-configuration whereas the acid excreted in urine has the D-configuration. At- tempts were made to convert the D- to L-acid by adding mandalate racemase, another bac- terial enzyme. This enzyme readily reacts with D-mandelic acid; it also has some activity to- ward D-p-hydroxymandelic acid, but it was found to be completely inactive toward DHMA or 3-methoxy-4-hydroxymandelic acid (VMA). Therefore, another approach is now being sought for the estimation of DHMA. Arising from this work, the properties of mandalate racemase were further character- ized. It appears that this enzyme requires mag- nesium ions, or certain other metal ions, for activity. It was found that the enzyme is in- hibited by chelators of magnesium; other ani- onic inhibitors, namely fluoride and phosphate, were found to compete with the substrate for the enzyme-magnesium complex. The pH- optimum and the Miehaelis constant of this enzyme were also determined. An intriguing finding was the observation that a color- or fluorescence-producing reaction occurs in about 60% of blood samples from schizonphrenics but in only about 6% of normal controls. The reacting material appears to be a porphyrin, probably formed from a precursor in red blood cells but this precursor does not seem to be identical with hemoglobin. The metabolism and the psychodysleptic ef- fects of certain tryptamine derivatives were studied in a collaborative program of the Sec- tion on Psychopharmacology with SEH staff. Using a double blind design, three shortacting psychotropic tryptamine derivatives N.N.-di- ethyltryptamine (DET), N.N.-dipropyltrypta- mine (DPT) and 6-fluoro-N.N.-diethyltrypta- mine (6-FDET) were compared in a sample of chronic alcoholic patients. With DET and DPT, the patients experienced subjective ef- fects at the 0.7 mg/kg dose, although the observers were unable to detect any significant change. At the I mg/kg dose, however, sig- nificant objective and subjective changes could be demonstrated. These effects did not appear to be relatable to autonomic changes. Supporting this view was the finding that 6-FDET reproduced some of the autonomic effects of DET and DPT without the hallu- cinogenic or psychotomimetic components re- sulting from their administration. Hence, 6-FDET may be considered as an "active placebo", permitting a more objective assess- ment of the psychodysleptic action of other chemically related compounds. Parallel studies were carried out in rats trained to press a lever to obtain liquid food on a variable-interval schedule. Both DET and 6-FDET were effective but DET had a stronger action. Attempts are now being made to develop new fluorimetric methods for the estimation of 6-hydroxy monoalkyl indoleamines and other drug-related metabolites in urines and, more generally, to develop methods to objectively measure dysleptic drug effects. In the same vein, computer methodologies for the analysis of visual evoked responses, autocorrelation and power spectra of the EEG, are being developed in collaboration with the Section on Technical Development, IR, NIMH and a laboratory for the study of fine eye move- ments has been established. It has now been shown that saccadic eye movements are phasi- cally related to alpha rhythm. A study differ- entiating saccade-linked left and right eye movements and "on" and off" visual evoked responses is now in progress; a first study of fine eye movements in pathological population groups is about to begin. Utilizing some of the facilities previously devoted to clinical purposes, a laboratory will 422 ANNUAL REVIEW OF INTRAMURAL RESEARCH be established to be run jointly with the Labora- tories of Psychology and Socio-environmental Studies and the Adult Psychiatry Branch CI, NIMH for collaborative projects and to provide other NIMH scientists with the facilities to conduct studies at SEH. Other space will be renovated to provide badly needed basic science laboratories. It is clear, however, that only a new research building could properly accommo- date our program and a representation to this effect has been forwarded through channels. LABORATORY OF NEUROBIOLOGY The ultimate goal of the research program of the Laboratory of Neurobiology is to elucidate physico-chemical bases for various physiologi- cal and behavioral processes taking place in this laboratory can be divided into the follow- ing four general categories: (1) studies of ex- citable and artificial membranes, (2) investi- gations of physiological properties of neuro- glial and ependymal cells, (3) analysis of the electric activities of the cerebral cortex of waking animals, and (4) studies of sensory mechanisms. Diverse but well coordinated work is needed in order to be able to make progress toward achieving the ultimate goal. During the fiscal year 1966, considerable progress was made in physico-chemical analy- sis of artificial and excitable membranes by Drs. Tasaki, Singer, Watanabe and Kobatake. By perfusing the interior of the squid giant axon with various favorable solutions, it was shown that the protein molecules in the mem- brane play an essential role in the progress of action potential production. The effects of ex- racellular cations were studied, and it was found that excitation in sodium-free and so- dium-containing media were essentially the same. Further experimental evidence was ob- tained in support of the "two stable state hypothesis" of nerve excitation and a quanti- tative theory of excitation is being developed. It was suggested that transition of the mem- brane from the resting state to the active state represents a sudden change (first-order phase- transition) of the membrane macromolecules. The project on neuroglia and ependyma was pursued efficiently by Drs. I. Singer and S. Goodman. By using tissue culture material, it was shown that the activity of ependymal cilia was governed by the same physico-chemical parameters that determine excitation in squid axons. It was shown that neuroglial cells are highly sensitive to various neutral salts and pharmacological agents. Ths finding offers a new basis for interpreting the mechanism by which cilia beat, and demonstrate the wide applicability of these physico-chemical princi- ples to biological processes. The electrical activity of the cat cerebral cortex was studied by Dr. E. Podvoll with mul- tiple recording electrodes chronically im- planted at many levels of the auditory system. Integrated activity from the thalamus showed rises during activity was lowest during deep ("slow wave") sleep. In contrast to the E.E.G., there was no difficulty relating integrated ac- tivity to "transitional" behavioral states. The dynamic changes during sleep and waking in the thalamus was observed to a lesser degree in the reticular formation, and was not seen in other subcortical nuclei. Drs. Pcdvoll and Goodman are progressing in their attempt to construct a three dimensional current vector representation of electrical ac- tivity in the auditory cortex in response to acoustic stimulation. Unique current vector patterns are being mapped out in both deep and superficial layers of the cortex and in the underlying white matter. It was shown by this method that invasion of nerve impulses into the auditory cortex generated electric currents which change their direction, as well as their intensity, as functions of time. Dr. Goodman has also been pursuing the re- lationship of intrareticular evoked potentials to arousal. The data collected shows clearly that during all levels of arousal (from alert to sleeping to paradoxial sleep) there is a direct, linear relationship between the amplitude of evoked potential and the integrated activity recorded from the thalamus. Therefore, there is a functional relationship between the level of arousal of an animal and the size of the "sig- nal" (evoked potential) in the reticular for- mation. Mrs. R. Marimont continued her studies on NATIONAL INSTITUTE OF MENTAL HEALTH 423 the Fuortes-Hcdgkin model for visual percep- restores the shape of the curve to approxi- tion derived from Limulus eye. A second feed- mately that of the linear system. A pedagogi- back loop was added to the Fuortes-Hodgkin cal manual was written, "Simple SAAMing," model which greatly improves the agreement for the use of other XIH scientists in program- between model and system. This second loop ming SAAM with such models. NATIONAL INSTITUTE OF NEUROLOGICAL DISEASES AND BLINDNESS INTRODUCTION The Intramural Program of the NINDB has consisted of four Branches (dealing with hu- man patients) and six Laboratories for some years. Last year the Laboratory of Perinatal Physiology in Puerto Rico was transferred to the Intramural Program. During the 12 months since July 1965 the Section on Technical Development continued to fulfill its traditional role of support of NIMH and NINDB research efforts. Continuing as- sistance was made available in shop help, spe- cial purpose instruments, and some modest in- strumentation systems. The section continues to grow toward assistance in computer ori- ented research and digital instrumentation. Use of the section's LINC computer has almost doubled since last year and has reached the saturation point — 105 hours per week. The ability to take on new projects is now seriously limited by production runs on existing projects. Plans to relieve this pressure include: Transfer where possible of existing produc- tion runs to CDPB, while limiting LINC func- tions to A to D conversion and on-line data editing. Acquisition of additional computing equip- ment in fiscal 1967. Help to laboratories toward setting up their own facilities where the workload justifies such a step. Section personnel have contributed signifi- cantly to research projects in laboratories of the two Institutes. In all cases this work has been described in the reports of the laborator- ies involved. The number of research projects reported this year is 218, an increase of 26. Forty-nine projects were completed and/or terminated and 59 new projects were described. One- hundred and seven projects involve collabora- tion with other Laboratories and Branches within NINDB, within NIH and with outside organizations. Forty-two cross Institute lines and 49 show collaboration outside NIH. In lieu of a recapitulation of scientific re- sults reference is made to the summary re- ports by each Laboratory and Branch Chief. In accordance with the Institute policy, the Clinical Director's report reflects only the pa- tient care activities and not the program con- tent. As before, it is a pleasure to thank the Director and staff of the Clinical Center with- out whose skilled support we could not con- tinue to function. OPHTHALMOLOGY From 1 July 1965 to 20 April 1966, 146 pa- tients were admitted to the 13-West Nursing Unit accounting for 6,685 inpatient days. The outpatient census totalled 495 patients and 1,663 visits to the outpatient. Consultation re- quests from other Institutes totalled 1,279, exceeding by approximately 100 the figure re- ported for last year. The number of major operations rose from 26 to 63. Minor surgical interventions appeared to decrease because many of these are now performed in the treat- ment room. MEDICAL NEUROLOGY For the clinical investigations, 233 patients were admitted for a total of 5,761 patient days and there were 1,290 outpatient visits. There were 300 muscle and brain biopsies obtained. The clinical neurologists responded to 384 con- sultation requests from other departments and performed the required myelograms, pneumo- encephalograms and cerebral angiograms. 425 426 ANNUAL REVIEW OF INTRAMURAL RESEARCH SURGICAL NEUROLOGY During the period 16 April 1965 through 15 April 1966, 214 persons participated in the clinical investigations as inpatients totalling 7,047 patient days. 593 were examined as out- patients in a total of 754 visits. There were 154 major operative procedures, 15 minor sur- gical procedures and 129 physiological moni- toring procedures in the new surgical suite. MEDICAL NEUROLOGY BRANCH Clinical Investigation Program Introduction Our function is to apply the most promising basic research techniques to the clinical prob- lems of the patients. The essentiality of an in- ter-related multi-dimensional attack on the chosen target diseases is to be emphasized. Added to the techniques of histochemistry and tissue culture have been biochemistry and im- munology, but in quite modest forms due to limitation of space and personnel. The tech- niques of electron-microscopy and autoradi- ography were achieved only on a collaborative basis, due to acute lack of facilities for these important investigations. We are very appre- ciative of the collaboration received in these and other techniques. It is obvious that to have a balanced clinical investigative program, each of these six techniques must be provided for more adequately. For the clinical investigations, 233 patients were admitted for a total of 5,761 patients days, and there were 1,290 out-patient visits. There were 300 muscle and brain biopsies obtained. The clinical neurologists carried a considerable service responsibility. They provided 384 con- sultations to other departments, and performed the indicated myelograms, pneumoencephalo- grams, and cerebral angiograms on those pa- tients. The two-year approved residency training program in clinical neurology has continued; medical students and residents from Howard University were taught clinical neurology weekly; and investigators and technicians in neurology and especially the application of en- zyme histochemistry to human neuromuscular disease. The collaborative research program in neu- romuscular disease with the Department of Neurology, Warsaw Medical Academy has con- tinued under the P.L. 480 program. During the past year, 20 of our 40 papers published (or in press) represented collaboration between the Medical Neurology Branch and other units. Myopathies A monograph on Current Concepts of Myo- pathies has been published, containing the research results and opinions of our group. Six additional chapters in different symposia and monographs described aspects of our his- tochemical techniques and detailed results in various neuromuscular diseases. In myotonic dystrophy the following have been delineated — temporomandibular joint dysfunction; vir- tually diagnostic specific atrophy of histochem- ical type I muscle fibers; and a unique hyper- catabolism of serum gamma globulin (IgG) protein (with NCI). The cardiac lesion of this disease has been successfully treated by elec- tro-cardioversion. Two other multisystem dis- eases with known protein defects, ataxia-tel- angiectasia (/32A globulin deficiency) and acanthocytosis (/? lipoprotein deficiency) were shown to have a myopathic component, in- creasing the number of myopathies associated with known metabolic defects. A new disease, late onset progressive rod myopathy, has been described. Similar rods were produced experi- mentally in tenotomized cat soleus. Contrary to other investigators, abnormalities of lactate dehydrogenase isoenzyme 5, myoglobin, and total body potassium (K 40 method) were found to be not disease-specific in humans. In addi- tion to biochemical changes, subtle histochem- ical abnormalities were found in muscle biopsies of clinically normal carriers on Du- chenne dystrophy, but their interpretation must await histochemical studies of muscle of normal volunteers. Detailed correlation of electromyographic, repetitive stimulation, motor conduction velocity, and motor unit territory determinations with clinical and his- tochemical aspects of 500 completely studied patients having neuromuscular disease is in progress. Attempts are being made to obtain reproducible cultures of human muscle biop- NATIONAL INSTITUTE OF NEUROLOGICAL DISEASES AND BLINDNESS 427 sies in vitro by maximow slide and diffusion- chamber techniques. In many cancer patients, generalized muscle weakness is a major cause of disability. Histochemically, we have shown carcinomatous (or cachectic) muscle atrophy preferentially to involve type II muscle fibers. The pathogenesis and prevention of type II fiber atrophy is being pursued with the hope of symptomatically benefiting patients weak- ened by cancer. In collagen-vascular disease (a major cause of strokes in young adults) blood vessels in muscle biopsies are being studied by histochemistry and electron-micro- scopy to analyse the generalized vascular ab- normalities. Episodic Weakness Our new classification of the periodic pa- ralyses and non-dystrophic myotonias, based on the provocative and therapeutic effects of various ionic unbalancing tests, developed last year, has proved valuable in choosing the cor- rect therapy for these patients, most of whom have responded quite well. Histochemical studies, which now make it doubtful that structural changes in the muscle fibers are responsible for the weakness in the initial part of the paralytic attack, are being paral- lelled by electron-microscopic studies. Refrac- tory period measurements of single muscle fibers and muscle fiber conduction velocity are being done between and during attacks in these patients, and we hope will soon be com- bined with forearm perfusion studies. In a patient with succinylcholine-induced paralysis, the low serum cholinesterase was found by electrophoresis to be associated with a newly recognized selective absence of the fastest two cholinsterase bands, and new pharmacologi- cally atypical patterns of cholinesterase were described in her serum and the serum of her relatives. Myasthenia Gravis Histochemistry showed that every muscle biopsy from 45 myasthenia gravis patients was abnormal, with either denervation or pref- erential atrophy of type II fibers present in all. Histochemistry also demonstrated a new as- pect of lymphorrhages, namely that each was around one or more abnormal muscle fibers. A detailed combined immunofluorescent and his- tochemical study showed that the muscle-bind- ing factor (described by Strauss) was not bound to the neuromuscular junctions, con- trary to previous assumptions of others. In collaboration with Strauss and colleagues (NCI), a combined clinical (including prostig- mine and curare tests) and immunofluorescent study showed that 24% of patients with thy- moma but without associated myasthenia gravis had the muscle-binding factor in their serum. A new toxic effect of colistin methane- sulfonate (Coly-Mycin) consisting of neuro- muscular blockade at therapeutic blood levels of the drug has been described in a patient with Sjogren's syndrome. Amyotrophic Lateral Sclerosis (ALS) and Other Diseases Affecting the Lower Motor Neuron In the clinical and pathologic spectrum of familial anterior horn cell disease, it has been demonstrated that within individual families infantile spinal muscular atrophy blends im- perceptibly with the juvenile proximal form. In ALS, primary or associated biochemical and immunologic abnormalities are being sought. Distant cancer has been found in a few pa- tients, but its possible pathogenic role remains unknown. Immunologic abnormalities of the serum could not be found by gel diffusion or passive cutaneous anaphylaxis. With a tissue culture screening system, no toxic or infec- tious agents in serum and spinal fluid were demonstrated. An intravenous form of the ar- ginine tolerance test was developed and ap- pears to be more useful than the oral test — its results in ALS are under study. With Dr. Fullmer (NIDR), abnormal collagenase activ- ity has been demonstrated in skin from ALS patients and a few other neuromuscular diseases which, though probably abnormal, is not disease-specific. Electron-microscopic search of brain tissue for viral particles is in progress, and search for a transmittible agent is underway (these same studies are also being done in sub-acute inclusion body encephalitis) with NINDB-CFR and NCI). Contrasting with the assumptions of others, the carbohy- 428 ANNUAL REVIEW OF INTRAMURAL RESEARCH drate intolerance found in about 25% of ALS patients is not disease-specific, having been found in about the same percent of patients with myotonic dystrophy, progressive muscu- lar dystrophy, and chronic peripheral neuro- pathy. More detailed studies of pancreatic function are underway to further characterize these changes. The apparatus we have de- signed and built for quantitating muscle strength has been shown to give reproducible results in normal controls and in patients. It serves as one aspect of evaluating ALS patients who are participating in our double- blind placebo-controlled therapeutic trials. As yet no drug tested has been found of thera- peutic value. RNA metabolism has been studied with its tritiated specific precursor, uridine. The autoradiographic pattern of ap- pearance in the nucleus and cytoplasm of motor neurons has been established in rabbits. The first pilot application of this technique to human neurologic disease has indicated a normal pattern in one case of infantile spinal muscular atrophy; the patterns obtained in ALS patients are being evaluated. A histo- chemical study of denervated and tenotom- ized cat muscles has shown a number of unex- pected changes and emphasized the difficulties in relating experimental animal conditions to human neuromuscular diseases. Produced for the first time in animals were target fibers, rods, type I fiber atrophy, and type II fiber atrophy. Three new histochemical changes were described in patients — "type grouping", empirically considered a sign of chronic de- nervation; presence of target fibers to be nearly always in type I fibers; and occur- rence of central cores to be exclusively in type I fibers, which reemphasized our earlier suggestion that central core disease may be a neurogenic disorder instead of a myopathy. In ataxia-telangiectasia the following have been demonstrated — an unusual type of dia- betes mellitus with marked hyperinsulinism; unexpected presence of immunoglobulin A in bone marrow and parotid cells; a profound defect of IgA synthesis and in 2 of 5 patients concomitant IgA hypercatabolism (with NCI); and impaired in vitro lymphocyte transformation (with NCI). The first two patients having a new familial syndrome of recurrent peripheral neuropathy with a-lipoprotein deficiency (Langier dis- ease) have been discovered (with NHI). A new syndrome of familial hypertrophic inter- stitial neuropathy and unique cataracts has been described. Vincristine, an anti-metabolite, has been shown in patients to have a pref- erential effect on the fusimotor system and thus is being tried as an anti-spasticity or anti-rigidity agent (with NCI). Methodology The role and mechanisms of substantive tetrazolium compounds and of phenazine methosulfate in the false localization of his- tochemical reactions has been described. A new type of EDTA-activated myofibrillar ATPase activity in type I fibers has been found histochemically. A method for seeking the location of the pharmacologic effect (my- oclonic jerks) of d-tubocurarine perfused in cat ventricles has been achieved by use of the radioactive compound, and the penetration of the various nuclear masses has been studied. The rapid monitoring of tissue being processed for electron-microscopy by simultaneous his- tochemistry has been developed and proved to save considerable professional time. Neuroradiology Section Radiographic Diagnosis Selective arteriography in patients with spinal cord arteriovenous aneurysms has al- lowed the demonstration of arteries feeding the malformation in five cases. This in turn has permitted surgical ligation of the main feeders in four paraplegic patients with re- sulting improvement in all of them. An Atlas of Pathologic Pneumoencephalography Anat- omy is now in press. The "empty sella" is a condition in which the normal intrasellar sub- arachnoidal space fills a good part of the sella turcica. These sellae, generally borderline large or frankly large, contain a large amount of cerebrospinal fluid but normal or smaller than normal pituitary glands. The knowledge of the large empty sella is important for: a) differential diagnosis from intrasellar tumors; NATIONAL INSTITUTE OF NEUROLOGICAL DISEASES AND BLINDNESS 429 b) explanation of certain hypopituitaric syn- dromes and certain types of spontaneous cere- brospinal fluid rhinorrhea; and c) to avoid mistakes when contemplating transsphenoidal (surgical and 90a) or external radiation pi- tuitary treatment. Further experience has been gained with the useful refinement of pneumo- encephalography, "axial transverse encepha- lography". This is now an established routine diagnostic procedure. A cooperative project is now underway on the growth hormone effects in dwarfs. Repeated sella turcica measure- ments are being taken in the patients so treated. The reevaluation by computer tech- niques of the angiographic patterns of super- ficial cerebral veins in the two hemispheres in a large group of patients has been con- tinued. As another aspect of studying cere- brovascular disease, the prognostic significance of parasellar carotid artery calcifications will be evaluated at the end of 1966, 10 years after the beginning of the study of the par- ticular group of patients used for this project. Radiation Dosimetry The thermoluminescent LiF crystals have proven disappointing for evaluating secon- dary radiation from irradiated residual x-ray opaque material in the spinal canal. New at- tempts are being made with the recently in- troduced LiF-Tef Ion dosimeters. Isotopic Diagnosis The clinical comparison of isotopes for brain scanning has been expanded to now in- clude RISA, 9Sm Tc pertechnetate, RIAF (ra- dio-iodinated antifibrinogen) and 197 Hg Neo- hydrin. The points which have been established with the multiple isotope-multiple scan technique are: (a) most space-occupying intracranial lesions can be diagnosed by sev- eral isotopes, but in some cases a diagnosis can be reached only or much better with one tracer; (b) RISA has a "relative" specificity for metastatic lesions; (c) 197 Hg Neohydrin has a "relative" specificity for glial tumors; (d) RIAF may have specificity for sarcomas and certain large clots; and (e) " m Tc per- technetate is probably most useful as a quick screening tracer and it can be most effectively employed with an Anger-type camera. In over one year of extensive testing, the Tetrascanner has proven itself to be a very useful piece of equipment, combining the high resolution of the rectilinear scanners with a speed approach- ing that possible with stationary detecting machines (cameras). The Tetrascanner is the best available device for rapid high resolution three-dimensional brain scanning. The isotopic scanning of cerebrospinal fluid shunts is now an established procedure. Extensive additional experience has been gathered with the tech- niques of isotope-ventriculography and isotope-cisternography. These now may be considered as routine diagnostic procedures. The Rous sarcoma virus brain tumors in dogs, first induced in collaboration with Dr. Rabotti (NCI), are now being used by other investi- gators. Salivary gland scanning with " m Tc per- technetate has been shown to be a useful diagnostic test for detecting the presence of abnormalities in the area of the salivary glands and for differentiating among salivary lesions (tumors, inflammatory processes, and primary and post-radiation atrophy). Prepa- ration of the monograph Brain Scanning has continued. Neuropharmacology Section During the past year we have been able to clarify the ionic events which lead to contrac- ture of slow muscle. Myogenic contracture can be initiated in slow muscle by the withdrawal of external Ca ++ . Present evidence indicates that loss of membrane Ca ++ is the initial ionic event, followed by increased permeabil- ity to Na + which leads to internal sodium accumulation. Contracture and an inflow of Na ++ occur concurrently. Smooth muscle de- velops Ca ++ deprivation contractures without depolarization. We have been able to develop muscle models relating ionic movement to ten- sion development without consideration of depolarization. Further experimental verifica- tion of such models is needed. The present studies have furnished evidence that (1) Ca + + has a dual role in the function of slow skeletal muscle; (2) there exists a Na + cur- rent mechanism different from the one in fast muscle or nerves which is responsible for the 430 ANNUAL REVIEW OF INTRAMURAL RESEARCH action potential because (a) it occurs in slow muscle that is not capable of a propagated potential and, (b) it is not affected by pro- caine which inactivates the Na + current re- sponsible for the action potential; (3) there is a K + requirement for complete relaxation in slow muscle. This requirement indicates that part of the active transport of Na + in slow muscle is K + dependent. The studies with fast twitch-type rat skele- tal muscle have related to the mechanical prop- erties of both the series elastic elements and the contractile components. Load extension characteristics of the elastic elements per- mitted calculation of kinetic and potential energy during' isometric contraction. The ex- periments do not support the prevailing con- cept of a passive undamped series elasticity in fast muscle. A study has been made in the rate of the elastic and contractile properties of a rapidly (anterior tibial) and a slowly (soleus) contracting fast muscle after immo- bilization by joint fixation. Disuse resulted in changes in the rate of energy expenditure which produced a significant increase in the intrinsic speed of the slowly contracting mus- cles. The elastic properties of the more slowly contracting soleus were changed by immobili- zation so that the elasticity more nearly re- sembled that of the more rapidly contracting anterior tibial muscle. As part of a P.L. 480 project, a serpen- tarium has been established at the Physiology Department of Ein Shams University, Cairo, Egypt, to maintain adequate sources of snake venom. A polyvalent antivenom from immune horses has been prepared for human use. The venom of Cerastes Cerastes has been found to block nerve conduction and is not reversed by KCl or physostigminej it has no direct effect on muscle. Walterinessia Aegypta venom produces hypoglycemia apparently by stimulating insulin secretion from the islet /3-cells. BRANCH OF SURGICAL NEUROLOGY Since the last report, this Branch has conducted investigations under the following categorical titles: developmental defects, epi- lepsy, involuntary movements, brain tumor, cerebral edema, cerebral trauma, language an