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Full text of "Report of program activities : National Institute of Child Health and Human Development"

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ANNUAL REPORT 
OF 
PROGRAM ACTIVITIES 
US' NATIONAL INSTITUTE OF CHILD HEALTH AND HUMAN DEVELOPMENT 

Fiscal Year 1973 
Part II 



-U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE 
Public Health Service National Institutes of Health 



fir 



NICHD ANNUAL REPORT 

July 1, 1972 through June 30, 1973 

Office of the Scientific Director 

Growth and change of the NICHD intramural research program have continued in 
this fiscal year. The Laboratory of Cellular and Comparative Physiology, 
GRC, has been expanded and reorganized under a new Branch Chief, Dr. Takashi 
Makinodan, with the addition of a new section on Cellular Immunology added 
to the Branch. Dr. Makinodan and his staff are intensively pursuing new 
leads in the rapidly developing field of cellular immunology as related to 
human aging. The Section on Molecular Structure under Dr. Erhard Gross has 
been transferred from the Laboratory of Biomedical Sciences to the Reproduc- 
tion Research Branch to facilitate closer collaboration in synthesis and 
structural analysis of hormones and their analogs. The Children's Diagnostic 
and Study Branch has been abolished. Planning has been completed and a 
contract awarded for construction of a new three story obstetrics and nursery 
unit to be added to the G wing of the Clinical Center. This will house the 
research and clinical activities of the Pregnancy Research Branch and a 
perinatology research program. The Social and Behavioral Sciences Branch 
has moved from space at the National Naval Medical Center to renovated 
facilities in the Auburn Building. The Laboratory of Biomedical Sciences 
and the Laboratory of Molecular Genetics have consolidated programs in newly 
renovated space in Building 6. 

In fiscal year 1973 total budgeted positions for intramural research increased 
from 277 to 321. In addition a total of 106 scientists worked in NICHD 
laboratories as Guest Workers, Guest Scientists, and Visiting Fellows. 
Another 63 employees in part-time, temporary, WAE, stay-in-school, and work- 
study categories participated in the intramural research programs. 

Developmental InmiunoloRy Branch - Accomplishments 

1. Development of a means of immunization of infants against Hemophilus 
influenzae type b meningitis, the largest source of acquired mental retarda- 
tion in the United States, remains the major research effort of scientists 
In this Branch. In a pilot study this year the immunogenic ity of 

H. influenzae type b polysaccharide has been evaluated by studying the 
clinical condition and serum antibodies of 23 infants injected with 5-10 jug 
of polysaccharide when 2-3 months of age. On follow-up all injected infants 
had serum anti-type b antibodies well above the average level for their age, 
and none had levels in the nondetectable range, in contrast to 307o of unin- 
jected infants who had no detectable anti-type b antibody at the same age. 
Thus, infants so treated at 2-3 months of age respond with sustained anti- 
body levels. A controlled clinical trial of this means of inmunization will 
begin in Mecklenburg County, N.C. in the coming year. 

2. Another mechanism for inducing immunity to H. influenzae that may be 
simpler, more effective, and has far-reaching implications for the whole 
field of Immunology and infectious disease, is also under study in this 
Branch. Strains of non-pathogenic E. coll have been isolated which have 

an antigen that cross-reacts with the polysaccharide antigen of H. Influenzae 
type b. These strains of bacteria, when fed to newborn animals, produce no 



F-1 



illness, but do Induce the formation of antibodies which protect the animal 
against infections due to H. influenzae . These strains are ubiquitous in 
nature, and probably constitute the source of the "natural" immunity to 
H. influenzae which most humans develop by age 6. Active immunization of 
all children in infancy by this "natural" means would appear to be ideal. 
Evidence that this method is indeed the "natural" source of immunity in 
humans was provided by a survey of stool cultures of all normal newborn 
infants discharged from the Charlotte Memorial Hospital in Charlotte, North 
Carolina. Of these infants, 1.1% had cultures that contained the cross- 
reacting E. Coli strain in the neonatal period. When reexamined at 4-13 
months of age, these infants all had detectable anti-type b antibodies in 
the serum at levels above the average. Thus, natural acquisition of the 
organism in the newborn nursery, which caused neither symptoms in these 
infants nor interference with normal growth and development, resulted in 
acquisition of protective antibodies to H. influenzae type b. In infants 
uncolonized by these strains of E. Coli early in life antibody titres to 
H. influenzae at later dates were significantly lower than it was in those 
who were colonized. 

3. Scientists in this laboratory have also identified non-pathogenic 
organisms with cross-reacting antigens to D. pneumoniae and N. meningitidis , 
raising the possibility of applying this method of immunization to diseases 
caused by these and other organisms as well. More extensive animal testing 
of this immunization technique will be carried out in the coming year, in 
preparation for later clinical trials. 

Social and Behavioral Sciences Branch - Accomplishments 

1. Study of early environmental influences on child development has demon- 
strated effects of father absence during infancy. The amount of father 
interaction with male infants was positively related to general developmental 
status as measured by the Bayley Mental Development Index. It was also re- 
lated to the child's social responsiveness, efforts to elicit feedback from 
the environment, and exploratory behavior. There were no measurable sig- 
nificant effects of father interaction on female infants. 

2. Comparison of infant caretaking patterns of mothers and surrogates showed 
that on the whole mothers provided higher levels of stimulation. They were 
more demonstrative in expressing positive affect, spent more time in non- 
caretaking activities with their infants, played with their infants more, 

and provided them with a greater variety of play objects. When assessed for 
general developmental status or on specific aspects of development, however, 
infants in surrogate care did not differ from Infants cared for by their own 
mothers. With the growing number of working mothers, this type of study 
gains added importance. 

Reproduction Research Branch - Accomplishments 

1. Sub-units of HCG have been prepared and characterized chemically, 
immunologically, and biologically. Specific antlsera have been prepared 
fiir each sub-unit . The sub-units were shown to be free of biologic activity. 
Free HCC«Cwas found in plasma throughout pregnancy, a finding which accounts 

F-2 



for previously noted discrepancies between biologic and immunologic measure- 
ments of HCG. The individual sub-units have been detected and levels 
measured in tissue, serum, and urine of patients with HCG-secreting tumors, 
providing evidence of cloning of cancer cells in human metastatic lesions. 

2. Clinical studies of testicular regulatory mechanisms are continuing in 
Infertile males. Serum FSH levels are high when germ cells are absent, but 
It has been difficult to decide whether the germ cell or Sertoli cell is 
responsible for regulation of FSH levels. Study of three men with a normal 
sperm count but only dead sperm exhibited high FSH levels, providing strong 
evidence that the Sertoli cell regulates FSH secretion. Additional evidence 
that this is the case was provided by a study in rats. When Vitamin A 
deficiency was used to induce reversible germ cell depletion, the FSH level 
did not rise, thus implicating the Sertoli cell in FSH regulation. A 
clinical trial has been initiated to test the effect of large doses of FSH 
in various types of male infertility. 

3. Chromaffin granule membranes are being intensively studied as a model 
secretory cell membrane system. The molecular parameters of the membrane 
proteins have been studied by a novel system of discontinuous buffer 
electrophoresis in sodium dodecyl sulphur at neutral pH. X-ray diffraction 
studies of these membranes revealed the existence of protein particulates 
distributed symmetrically on the area bounded by the membrane. 

4. Prolactin has been isolated from amniotic fluid, and a practical method 
has been devised for preparation of the hormone. The identity of serum, 
urine, and amniotic fluid prolactin was proved by physico-chemical, biologic, 
and immunologic criteria. 

5. For the first time techniques have been perfected permitting Isolation 
and numerical quantitation of lysosomes from human placentas obtained at 
various times during pregnancy. 

Behavioral Biology Branch - Accomplishments 

Section on Brain and Behavior 

1. Several studies of primate vocalization have been completed. Sound 
spectrographlc analysis of the vocalization of one pair of squirrel monkeys 
indicates that each animal has a distinctive fine structure In its vocal 
productions. Call duration, number of syllables, and complexity of syllable 
structure all seem to differentiate and make unique an individual monkey's 
vocal repertoire. Responses of single neurons in the primate auditory cortex 
to complex biologically significant acoustic stimuli are little affected by 
wide fluctuation in arousal level of the animal. The complex synaptic 
connections responsible for the relatively specific responses to such 
stimuli as species-specific vocalizations thus represent securely organized 
sensory channels, rather than a diffuse system related to attention and 
arousal. 



F-3 



Section on Neurobiology 

1. Biochemical analytical methods developed in the Branch have been used to 
study the chemical secretory patterns of individual neurons of the molluscan 
nervous system. Synaptic inhibition was found to reduce the rate of incor- 
poration by neurons of radioactive leucine into two low molecular weight 
neurosecretory proteins by 50%. Dopamine, the presumed transmitter mediating 
the synaptic inhibition, produced a similar reduction in synthesis of the 
neurosecretory protein. Synthesis rates of other proteins in the cell studied 
were not significantly reduced by either synaptic inhibition or dopamine. 
'^oth treatments increase membrane potential and permeability to potassium. 
High potassium concentrations increase permeability to potassium but decrease 
membrane potential, and result in a doubling of the synthesis rate of the 
neurosecretory protein. Thus, increase in potassium permeability is probably 
not the control mechanism involved; present studies are focusing on analysis 
of the Increased membrane potential as the mechanism responsible for control 
of neurosecretory protein synthesis. 

2. Scientists have succeeded in dissociating and establishing in tissue 
culture mouse cerebellar neurons, and demonstrated that these neurons develop 
complex patterns of spontaneous and synaptically mediated electrical activity. 
Clearly distinguishable morphologic types and a range of electrophysiologic 
characteristics were found in the surviving neuronal networks, so that some 
substantial representation of the relatively simple normal cerebellar cir- 
cuitry appears in the culture systems. Studies are proceeding on cells 

from a variety of genetic mutants with well-established neuropathologic 
abnormalities, to attempt to ascertain at the cellular level the basis for 
the abnormalities. 

Laboratory of Molecular Genetics - Accomplishments 

1. Studies directed toward understanding the regulated expression of the 
globin gene have indicated that the reverse transcript of purified globin 
message can be used as a highly specific and sensitive probe for quantitating 
specific globin genes and mRNA. The number of genes corresponding to globin 
in several different avian and mammalian tissues, as demonstrated by this 
technique, is rather small, consisting of 3 to 5 globin genes per diploid 
genome. The number of globin genes is not amplified during the process of 
differentiation, so at least for globin, the gene amplification hypothesis 
appears excluded. Application of this technique to studying the process of 
differentiation in tissue culture has provided evidence that transcription 

of the globin genes represents a rate limiting step in differentiation, and 
that globin message rises from an undetectable amount to 3000-6000 molecules 
per cell in the fully differentiated state. 

2. Continued studies of the integration and excision of phage X into bacteria 
have sho;-m that host chromosomal material can be incorporated into the phage 
genome in a non-random fashion. This observation provides the initial 
evidence that phage \ can be used as a generalized transducing probe. 
Fxnmination of thi! genes necessary for the formation of the viral particle 
;;howK lluit thest? proteins are produced in a coordinate fashion, and suggests 
Ihat tliiTc are controls at some post-transcript ional level. These results 

K-4 



provide a clearer picture of the process of viral insertion and excision and 
the elements needed for packaging the viral chromosome into immature virus 
particles. 

Laboratory of Biomedical Sciences - Accomplishments 

Section on Developmental Enzymology 

1. Optimal assay conditions were ascertained and specific activities deter- 
mined for a large number of placental enzymes, and zymogram techniques devised 
for detecting isoenzyme variation among them. Qualitative and quantitative 
comparisons were made of these enzyme activities in normal term placenta and 
malignant trophoblast in tissue culture. The tissue in culture was found to 
he virtually devoid of heat stable (placental) alkaline phosphatase, and had 
an unusual lactate dehydrogenase enzyme, differing strikingly from that of 
the term placenta. 

Section on Intermediary Metabolism 

1. Investigations in this laboratory on the biochemistry of brain transfer 
RNA are designed to identify aspects that are unique to brain and appear to 
be related to brain function. Brain mRNA has been purified and described in 
terms of size, poly A sequences, and ability to be translated by a protein 
synthesizing system. Differences have been found between the messenger 
fraction from the brains of immature and of adult animals with respect to the 
length of the poly A sequences. 

Section on Physiological Controls 

1. All of the enzymes known to participate in glycogen cycle phosphorylations 
have been partially or completely purified. Glycogen synthetase phosphatase 
was found to be a general phosphoprotein phosphatase that dephosphorylates 
phosphorylase - b kinase and other protein substrates of the cyclic AMP - 
dependent protein kinase. The development of fetal rat liver glycogen syn- 
thesis was reproduced in a completely defined organ culture system and shown 
to be related to the development of glycogen synthetase. 

2. A new method for measuring tryptophan hydroxy labe was developed and used 
to show that this enzyme is rate-limiting in serotonin synthesis in the 
pineal gland and is controlled by tryptophan levels in the circulation. 
Norepinephrine was shown to decrease pineal serotonin levels by a sequence of 
reactions involving a specific receptor, cyclic AMP, and the induction of 
serotonin N-acetyltransferase. Antigonadotrophic effects of the pineal gland 
have been further delineated. A potent antigonadotrophic compound produced 
bv the pineal gland was purified and shown to be a small peptide. 

Section on Developmental Pharmacology 

1. Using cells in tissue culture, which offer a considerable advantage over 
using the intact animal, scientists in this section have determined that in- 
duction of activity of mono-oxygenase and other drug-metabolizing enzymes by 



F-5 



phenobarbital, polycyclic hydrocarbons, or biogenic amines is controlled at 
two levels: the level of transcription involving gene activation, and a post- 
Tanscrlptional level in which the normal rate of decay of induced enzyme 
activity is retarded. The induction process effected by phenobarbital is more 
dependent on ribosomal RNA synthesis than that effected by either aromatic 
h.vdrocarbons or biogenic amines. 

Pregnancy Research Branch - Accomplishments 

1. Scientists in this Branch have continued to evaluate the pregnant baboon 
as a primate model for studying the mechanisms regulating steroid synthesis 
and metabolism during pregnancy. Numerous studies indicate that the approx- 
imation of the human condition is quite close, and that the model will be a 
useful one. For example, placental utilization of estrogen precursors from 
the maternal circulation seems impeded in both the human and the baboon. 
Likewise both species demonstrate that maternal and fetal metabolism of 
Cortisol differs from that in the non-pregnant state. Efforts are presently 
being made to develop and maintain a chronic preparation of the pregnant 
baboon, restraining the animal in a chair over a long period of time with 
Tiaternal and fetal catheters in place. 

2. Comparison has been made of the relative usefulness of maternal serum 
and urine estrogens for assessment of f eto-placental status in high risk 
human pregnancy. The study showed that serial 24-hour urinary estrogen 
determinations provide the most reliable data to monitor estrogen metabolism 
as a criterion of fetal well-being. 

3. A new accurate and sensitive radioimmunoassay for monkey glucagon has 
been applied to the primate model diabetic pregnancy. Glucagon levels in the 
mother and fetus are not affected by fasting or induced hyperglycemia in 
either the normal or the streptozotocin-induced glucose intolerant animal. 
Glucagon administered intravenously in physiologic amounts does not cross 
the monkey placenta in either direction. In the newborn there is a linear 
relation between plasma glucose and glucagon levels that is not present in 
the fetus. 

Gerontology Research Center - Accomplishments 

Laboratory of Behavioral Sciences 

1. Studies of logical reasoning in elderly men in the Baltimore Longitudinal 
Study of Aging have shown that men over age 60 make more errors than younger 
men. Analysis of the errors indicates that older men are more likely to make 
premature attempts to synthesize information than are younger men, resulting 
in repeating solution attempts that have failed. These results correspond 
to a related study in rats, wherein older rats had much more difficulty in 
solving a maze than young rats, due to perseverative errors. However, when 
the older rats were prevented from developing the perseverative error pattern 
In early trials, they learned more rapidly than the young rats. Because of 
the similarity in type of errors, it may be possible to improve performance 
of the elderly humans by a training procedure similar to the one effective in 
the rats. 

F-6 



2. Work on operant conditioning of autonomic functions has continued in this 
laboratory, A patient with mild essential hypertension has been able to 
maintain her systolic blood pressure 30 mg below initial baseline levels 
after training. Monkeys have been able to markedly reduce their heart rates 
by conditioning. Several human subjects have demonstrated long-term capability 
of eliminating cardiac arrhythmias after conditioning. Extending the condi- 
tioning procedure to bowel control, subjects with prolonged intractable 
fecal incontinence have been able to achieve complete continence after 
operant conditioning. These studies demonstrate that conscious control over 
autonomic function is possible, and may have applicability in numerous areas 
of medicine. 

Clinical Physiology Branch 

1. The Baltimore Londitudinal Study of Aging continues to be a major 
research resource. Particular efforts this year have been directed to 
development of a statistical method for predicting the requisite duration of 
a longitudinal study, the required frequency of data acquisition, and the 
number of subjects needed, in order to define the rates of aging changes in 
selected functions with specified accuracy. These techniques greatly aid 
continued planning for the Baltimore study, and have application for longi- 
tudinal studies in general. 

2. The incidence of cutaneous malignancies increases with age. Studies of 
chronic actinic damage as a precursor of these malignancies have assumed that 
the damaging radiation spectrum is confined to the 290-320 nm wavelengths, 
and that longer wavelength UV radiation (320-400 nm) protects against sun 
damage to skin by inducing tanning. Study in this Branch of the effect of 
age on sensitivity to UV light of different wavelengths has shown that, on 
the contrary, the longer UV wavelengths have an augmentative effect rather 
than a protective one. These findings indicate that present preparations to 
prevent sun damage should be modified so that they protect against a broader 
spectrum of UV light. 

3. A new labeled polymeric substrate has been developed to assay renin and 
to aid in study of this kidney enzyme involved in the regulation of blood 
pressure. With this assay it has been found that renin is inhibited by a 
variety of proteins and peptides. The techniques and concepts developed in 
this laboratory should permit development of renin inhibitors of potential 
usefulness in clinical treatment of hypertension. 

Laboratory of Cellular and Comparative Physiology 

1, Observing that associated with aging and immunosenescence there is an 
increase in incidence of immunodeficiency diseases including autoimmunity, 
cancer, and infection, researchers in this Branch have been intensively 
studying changes in the immune system with aging. They have found, for 
example, that of the three cell types (T-cells, B-cells, and A-cells) in- 
volved in the initiation of a humoral immune response, the T and B cells are 
altered in immunologically deficient old mice, but A cells appear normal. 
Proliferative capacity of T and B cells decreases as much as 10-fold with age, 
a finding of key significance because the number of functional effector cells 

F-7 



generated in an immune response depends on the efficiency of their ability to 
divide. In addition, evidence was found that there is an age-associated 
increase in either the relative number or the efficiency of regulator cells 
which suppress the immune response, to account in another way for the reduction 
in immune response with aging. 

2. Study of mice with an exceptionally long life span showed an age-related 
decline in cell-mediated immune activity and in resistance to allogeneic 
tumor cells. This decline had previously been observed in mice with short 
life spans, so that critics claimed that life-shortening diseases imposing 
on the immune system were responsible for the decline. The new evidence 
should allay this criticism. 

3. A new research program in biomembrane physiology, emphasizing age effects 
on hormone binding, has yielded evidence that membranes from adipose tissue, 
skeletal muscle, brain, and prostate gland of aging rats have a progressive 
decline in steroid hormone binding. This decline is due in part to a re- 
duction in the number of macromolecular binding sites per unit of tissue. 

Laboratory of Molecular Aging 

1. Active transport of glucose in the kidney has been studied in isolated 
renal brush border membranes and found to consist of at least two processes, 
a high-affinity binding system and a carrier-mediated transport system. 
Kinetic studies of the binding have characterized time course, reversibility, 
saturability, alterability by phlorizin, and the influence of Na , Ca , and 
Mg ions. A particulate fraction derived from detergent-extracted brush 
border membranes has the same affinity and capacity as intact membranes, but 
has lost about 907o of its protein content and shows no evidence of vesicular 
structures. The mechanism of uptake of high concentration of glucose by 
intact brush border membranes shows both influx and efflux components and is 
consistent with the hypothesis that there is a carrier-mediated transport 
into and out of vesicles. The rate of glucose transport by the membrane is 
greatly enhanced by a Na gradient from the outside to the inside of the 
membrane . 

2. Relationship of the mechanism of infection to age of the cell has been 
studied extensively in tissue culture. Viral replication was unaffected by 
age of the cell, until just before cell death, when there was a decrease. 
Induction of interferon, the viral defense mechanism of the cell, was possible 
throughout the life span, but older cells required a larger stimulus to pro- 
duce a given amount of interferon, indicating that the sensitivity and/or 
efficiency of the process declines with age. It was also found that in 
senescent cells the antiviral state is produced more rapidly by stimulation 
with the interferon inducer poly I:C than with interferon itself, suggesting 
either another antiviral protection mechanism in senescent cells or a change 
in the relative rates of uptake of poly I:C and interferon in the senescent 
cell. These studies may help to explain the increased susceptibility of the 
elderly to viral infections. 



F-8 



Collaborative Guest Scientist Program 

1. The hypothesis that protein synthesis is impaired as age advances was 
examined by studying incorporation of C-leucine or ^C-labeled amino acids 
by liver microsomes from adult (12 month) and aged (20-31 month) female rats, 
Amino acid incorporation into protein was decreased by 12-437o in the cell- 
free system from senescent rats. Furthermore, the cytosol (105,000 g super- 
natant) from senescent animals inhibited protein synthesis by microsomes 
from the young rats, while the cytosol from the young rats combined with 
senescent microsomes functioned as a completely senescent system. The 
results support the concept that protein synthesis is impaired in senescent 
cells, though the inhibitory effect of cytosol from aged cells is a new 
finding of unknown significance. 

2. Effects of age on isolated cardiac muscle function were studied in rats. 
Contractility of the muscle was increased by norepinephrine and paired 
pacing, and decreased by hypoxia. No age differences were found in response 
to hypoxia or paired pacing, indicating that there are no major alterations 
in anaerobic metabolism with aging. By contrast, at higher dose levels of 
norepinephrine the inotropic response was less in the old than in the young, 
a finding that is probably reflected in decreased "cardiac reserve", the 
ability to respond to increased stress, in the older animals. 



F-9 



Serial No. HD DB - 7 

1. Office of the Scientific Director 

2. 

3. Bethesda, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Specific Cellular Immunity in Recovery from Viral Infection 

Previous Serial Number: Same 

Principle Investigator: Lon R. White, M.D. 

Other Investigator: Harrie Anne Sutton, Biologist 

Cooperating Unit: None 

Man Years : 



Total: 


0.0 


Professional: 


0.0 


Other: 


0.0 



Project Description: 

Objectives : To determine the role and mechanism of acquired, specific, 
cell-mediated immune responses in recovery from viral infection in vitro and 
in vivo . 

Methods employed : The initial studies have utilized infection of mice 
with MVM (minute virus of mice) and vaccinia virus. Spleen cells from 
control or immunized animals have been put into culture with cells previously 
infected with the appropriate agent. The following variables are: a) virus 
replication, b) virus-induced cell injury, c) cell-induced cell injury 
(lymphocyte cytotoxicity) . 

Major findings : No evidence of such immunity has yet been demonstrated. 

Significance to biomedical research and the program of the Institute : 
It appears likely that specific cellular immunity may play a major role in 
termination of viral infections in vivo and in the host cell injury seen in 
certain viral infections. It is further hypothesized that an incompetence 
or abnormality of this mechanism may occur when the animal is first exposed 
to the virus during immunologic immaturity. The in vitro demonstration of 
such phenomena would represent a major advancement in our understanding of 
basic immune phenomena and the pathogenesis of virus-induced diseases. 

Proposed course : This project has been terminated. 

Honors and Awards : None Publications : None 



F-10 



Serial No. HD DB - 5 

1. Office of the Scientific Director 

2. 

3. Bethesda, Maryland 

PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Immune mechanisms in chronic and congenital viral infections 

Previous Serial Number: Same 

Principle Investigator: Lon R. White, M. D. , DB , NICHD 

Other Investigators: George Nerao , Ph.D., 

Jacob A. Brody, M. D. 

Harrie Anne Sutton, biologist, DB, NICHD 

Samuel Baron, M.D., LVD, NIAID 

Cooperating Units: Epidemiology Branch, C&FR, NINDS [NDS (CF) - 70 E 1833] 

Man Years : 

Total: 0.1 
Professional: 0.1 
Other: 0.0 

Project Description: 

Objectives : 1. To establish models of chronic, congenital, and/or 
latent viral infection in animals and in tissue culture. 2. In infected 
animals, to define the development of immunity to the infecting and to 
heterologous antigens. 3. In tissue culture, to define the effect of 
immunologic factors (antibodies, immune lymphocytes, interferon) on the 
evolution of acute and chronic infections. 

Methods employed : Neonatal and pregnant mice are injected with either 
a reovirus type 1 or the minute virus of mice (MVM) . The persistence of 
virus in animals is determined by isolation and fluorescent and iramuno- 
fluorescent techniques. Serum levels of hemagglutination inhibition 
antibodies to both agents are followed. Lymphocyte transformation and 
cytotoxic activity is studied in spleen cell cultures in the presence of 
phy tohemagglutinin on specific antigens. Interferon production is studied 
by assay of media from tissue culture preparations of cells infected with 
MVM, and by the ability of MVM infected cells to support the growth of 
other viruses. Sensitivity of MVM to interferon is tested by determining 
the yield of virus following exposure of cells in culture to known amounts 
of interferon. 



F-r 



Serial No. HD DB - 5 

Major findings : MVM does not appear to induce interferon elaboration 
in vitro . Pretreatment of L cells with interferon produces a mild to 
moderate diminution in virus yield. 

Significance to biomedical research and the program of the Institute : 
Infection of the human embryo or fetus with rubella or cytomegalovirus is 
associated not only with developmental abnormalities and mental retardation, 
but also with infection persisting for months or years after, despite the 
presence of neutralizing antibody in the serum. There are several examples 
of related phenomena in experimental animals infected during prenatal or 
neonatal life. Previous studies have suggested that an impairment of the 
immune function of lymphocytes may be associated with such persistent 
infection. These phenomena provide insight into the question of how viral 
infection is normally terminated, and represent a challenge to older ideas 
on the role of specific cellular immunity in the natural history of virus 
infections. The investigation described represents a direct experimental 
approach to elucidating the cause and course of chronic infection following 
initial exposure to the agent during immunologic immaturity. The results of 
this investigation will be of immediate relevance to our understanding of 
other types of illness known or suspected to be associated with chronic viral 
infection. In addition, they may suggest new approaches to research in the 
role of congenital viral infection as a cause of diseases of unknown etiology 
such as prematurity, idiopathic growth failure, malignancy, mental retardation, 
developmental malformation, and autoimmune diseases. 

Proposed course : To be continued. 

Honors and Awards : None 

Publications: Manuscript in preparation. 

NOTE: Notice of Research Project (Form PHS-166) filed under the NDS Project 
Number. (vide supra) 



F-12 



Serial No. HD DB - 15 

1. Office of the Scientific Director 

2. 

3. Bethesda, Maryland 

PHS-NIH 

Individual Project Report 

July 1, 19 72 through June 30, 1973 

Project Title: Search for a negative DNA strand in cells infected with the 
minute virus of mice (MVM) 

Previous Serial Number: Same 

Principle Investigator: Lon R. White, M.D., DB, NICHD 

Other Investigators: Harrie Anne Sutton, Biologist, DB, NICHD 

George Nemo, Ph.D. 
Jacob A. Brody, M.D. 

Cooperating Unit: Epidemiology Branch, C&FR, NINDS [NDS (CF) - 71 E 1922] 

Man Years : 

Total: 0.4 

Professional: 0.4 
Other: 0.0 

Project Description: 

Objectives : a. To demonstrate a "template" DNA Cnegative strand) 
complementary to the single-stranded DNA (positive strand) of the MVM virion, 
b. To determine if the negative strand is associated with the cell's chromo- 
somal DNA. c. To investigate the kinetics of negative strand synthesis 
relative to cellular DNA replication, cellular division, virus infection, and 
virus production. d. To utilize this method to detect a latent or cryptic 
viral infection in the cells of experimental animals and in tissue culture, 
e. To characterize the virion DNA, the "negative strand," and replicative 
forms of MVM DNA. 

Methods employed : These studies utilize radioisotope "tagged" viral 
DNA. Annealing with "cold" DNA from virus infected cells is demonstrated by 
liquid scintillation detection of double- and single-stranded DNA in fractions 
eluted from a hydroxyapatite column. 

Major findings : Contrary to expectations, DNA extracted from purified, 
concentrated virus preparations is inhoraogeneous and partially double- 
stranded. A significant amount of additional duplex formation results when 
the initially single-stranded DNA is allowed to "self-anneal." That these 
sequences are truly viral and not from the host cell is shown by the failure 
of added mouse or rat DNA to influence the rate or extent of annealing. 
Denaturation and reannealing kinetics of the initially double-stranded 



F-13 



Serial No, HD DB - 15 

suggests the presence of one or more loop or hairpin molecular configurations. 
Nucleic acid hybridization reactions using total cell DNA and a "stripped" 
(all double-stranded parts removed) viral DNA "probe," has now been used to 
detect complementary sequences in DNA from both acutely infected L cells 
(tissue culture) and newborn mice. Negative results were found with normal 
mouse (3 strains), rat, and guinea pig DNA, as well as with DNA from 2 
tissue culture lines. Unexpectedly, positive results were found with DNA 
from a leukemic rat. Current work is focused on the utilization of this 
method to detect a latent or persistent parvovirus infection in animals and 
tissue culture. 

Significance to biomedical research and the program of the Institute : 
Characterization of virus-cell interactions using a single— stranded DNA virus 
(parvovirus group, of which MVM is representative) is expected to advance our 
understanding of the mechanisms by which a virus infection might alter the 
genetic potential of the host cell (either a somatic or germ cell) as well as 
of mechanisms involved with the establishment of latent and chronic viral 
infections. Congenital, chronic, or latent virus infections may be involved 
with the etiologies of certain reproductive, developmental, and chronic 
diseases whose causes are now unknown. This experimental approach may prove 
to be directly applicable to a search for a parvovirus DNA in the cells in 
animals and persons with such diseases. 

Proposed course ; To be continued at an increased level of effort. 

Honors and Awards : None 

Publications: Manuscript in preparation. 

NOTE: Notice of Research Project (Form PHS-166) filed under NDS PROJECT 
NUMBER. (vide supra) 



F-14 



Serial No. HD DB - 20 

1. Office of the Scientific Director 

2. 

3. Bethesda, Maryland 

PHS-NIH 

Individual Project Report 

July 1, 19 72 through June 30, 1973 

Project Title: Studies on the consequences of viral infection in early life 

Previous Serial Number: Same 

Principal Investigator: Lon R. White, M.D., DB , NICHD 

Other Investigators: Dwayne Reed, M.D. 

Harrie Anne Sutton, Biologist, DB, NICHD 
Jacob A. Brody, M.D. 

Cooperating Unit: Epidemiology Branch, C&FR, NINDS [NDS (CF) - 72 E 1979] 

Man Years : 

Total: 

Professional: 
Other: 

Project Description: 

Objectives : To investigate immune responses and neurologic development 
in children known or suspected of having contracted certain virus infections 
during infancy or early childhood. The initial studies have focused on 
infection with "natural" measles during the first year of life. 

Methods employed : A population of approximately 100 children in and 
around Little Rock, Arkansas, known to have had measles before one year of 
ag5 were identified with the assistance of a local hospital physician and 
puislic health officers. To test the feasibility of an intensive, longitudinal 
study of this cohort and control children, an attempt was made to contact, 
evaluate and obtain serum from several "case" and "control" children. Sera 
were tested for anti-measles antibody by hemagglutination inhibition. 

Major findings : No "control" children could be identified. Five "cases" 
were examined and were considered neurologically normal although their anti- 
body titers were slightly elevated above expected levels. 

Significance to biomedical research and the program of the Institute : 
Arbovirus infections involving the CNS and occurring in infancy or early 
childhood are known to be associated with signs of minimal brain damage- as 
the children near school age. This occurs even though the original Illness 
was mild and followed by apparently complete recovery. Subclinical CNS 
involvement with the common childhood illnesses (measles, mumps, herpes 



F-15 



Serial No. HD DB - 20 

simplex, varicella, roseola infantum) is frequent; the neurologic sequelae of 
these illnesses are undefined but may be of considerable importance. lliis 
study represents an attempt to define the role of such early infections in 
the causation of minimal brain damage and related neurologic abnormalities. 

Proposed course : This project has been terminated. 

Honors and Awards: None 

Publications : None 



F-16 



Serial No. HD DB - 16 

1, Office of the Scientific Director 

2. 

3. Bethesda, Maryland 

PHS-NIH 

Individual Project Report 

July 1, 19 72 through June 30, 19 73 

Project Title: Development and application of methods for the study of the 
minute virus of mice (MVM) 

Previous Serial Number: Same 

Principal Investigator: Lon R. White, M.D. , DB, NICHD 

Other Investigators: Harrie Anne Sutton, Biologist, DB, NICHD 

George Nemo, Ph.D. 
M.D. Hoggan, Ph.D., LVD, NIAID 

Cooperating Units: Epidemiology Branch, C&FR, NINDS [NDS (CF) - 72 E 1980] 

Laboratory of Viral Diseases, NIAID 

Man Years : 

Total: 1.8 

Professional: 1.0 
Other: 0.8 

Project Description: 

Objectives : To develop improved methods for the propagation, purifi- 
cation, and quantitation of the virus and its nuclei acid. To investigate 
basic biological, biochemical, and structural properties of the virus and 
its DNA in order to develop techniques to investigate its persistence in 
congeni tally and chronically infected experimental animals. 

Methods employed : The agent is propagated in tissue culture and detected 
by cytopathic effect or antigen production. Its DNA is radioisotope "tagged" 
during replication. It is purified by differential centrifugation and 
by chemical means. The virus, its proteins, and its DNA are characterized 
by velocity sedimentation in sucrose gradients, electron micrographic 
appearance, salt and thermal elution characteristics from hydroxy apatite 
columns, and poly aery lamide gel electrophoresis. 

Major findings ; Previous reports have described the growth of MVM in 
only two cell lines, both primary, with CPE in only one of these. We have 
extended this to 4 continuous cell lines, 2 of which show CPE. We have 
shown that whereas maximum virus production demands that the host cell divide, 
infection can occur in non-dividing cells and the virus can remain "latent" 
without apparent injury to the cell until it divides. In addition, wc have 
established chronic infection in four cell lines, two of which h.ivi- been 

F-17 



Serial No. HD DB - 16 

producing virus in high concentration continuously for 2 years. We have 
accomplished a 100-1000 x improvement in yield of virus and hemagglutinating 
antigen and have shown that purification by Isopycnic ultracentrif ugation 
is associated with moderate virus disruption and loss of infectivity. 
Additional studies have revealed no significant loss of hemagglutination 
or infectivity titers with 3 cycles of f reeze-thawing or with heating at 
56°C for up to 90 minutes. UV inactivation progresses in approximately one 
log-j^Q decrements per second under standard conditions. Ihe in vitro 
infectivity titration method has been improved from a rather unreliable 
one-month procedure (which works poorly with "wild" strains of MVM) to a 
sharply reproducible, reliable one-week procedure usable with all strains. 
The nuclei acid, extracted by NaOH treatment and by a pronase-phenol- 
chloroform method, has been found to be extremely labile and "sticky," 
adsorbing to plastics, millipore filters, and dialysis tubing. Results 
of hydroxyapatite chromatography suggest that it may have a "snap back" 
area where it is double-stranded, that some virions may contain some 
"negative" strand DNA, and that the DNA may be packaged in segments. A 
method has been developed for the demonstration of viral DNA in infected 
cells and are being used to study the course of MVM infection in vi tro and 
in vivo . The virus has at least 3 proteins, whose molecular weights are 
between 50,000 and 80,000, and whose relative concentrations roost closely 
resemble the pattern observed with a bovine parvovirus. Differences in 
relative concentration have been observed with cultivation in different 
cell lines; these are currently under study. 

Significance to biomedical research and the program of the Institute : 
MVM is representative of a group of small, single— stranded DNA viruses (the 
parvoviruses) which are characterized by ubiquity, generally low virulence 
and cy topathogenicity , an apparent preference for dividing cells, and a high 
frequency of transplacental infections. The study of this agent is expected 
to lead to a better understanding of mechanisms and the possible role of 
viruses in the causation of certain genetic, reproductive, and developmental 
abnormalities. MVM has been relatively unstudied, presumable because of 
technical problems. The improved methods which we have developed, and the 
basic information obtained by the application of these methods now make such 
studies feasible. 

Proposed course : To be continued at present level of effort. 

Honors and Awards : None 

Publications: Manuscript in preparation. 

NOTE: Notice of Research Project (Form PHS-166) filed under NDS Project 
Number (vide supra) . 



F-18 



NICHD Annual Report 

July 1, 1972 through June 30, 1973 

Developmental Innnunology Branch 

Summary 

The immunogenicity of the Haemophilus Influenzae type b polysaccharide in- 
jected into 2-3 month old infants has been evaluated by a study of the serum 
antibodies and clinical condition of 23 injected at 2-3 months of age. All 
infants have serum anti type b antibodies well above the average level for 
this age. None of the infants had serum antibody levels in the non-detectable 
range as contrasted to 30% of infants of this age who did not have anti 
type b antibodies detectable. Therefore, our conclusion is that 2-3 month 
old infants respond with sustained antibody levels following immunization 
with doses of 5-10 /ug of the H. influenzae type b polysaccharide. A survey 
of stool cultures taken from normal infants discharged from the nursery 
at Charlotte Memorial Hospital reveal that 1.1% have an E^. coli with a cross 
reactive antigen. In one study at ages 4-13 months, these infants all had 
detectable serum anti type b antibodies above the average level, indicating 
that natural acquisition of this organism, shown to have caused no symptoms 
in these infants or interference with their normal development, results 
in the acquisition of antibodies to H. influenzae type b. 

A noncapsular antigen isolated from the JH. influenzae taken from otitis 
media has been studied. Several protein fractions containing material re- 
active with H. influenzae typing serum of all six encapsulated varieties 
have been isolated. Immunization with this antigenic preparation induced 
the formation of serum antibodies that were bactericidal for H. influenzae 
type b and whose biologic activity could not be removed by a previous 
absorption with the purified capsule. The noncapsular antigen from the 
H. influenzae strain "Hope" absorbed bactericidal activity from several 
H. influenzae type b sera but could not remove this activity from antiserum 
prepared by injecting the cross reactive E. coll. These data indicate that 
a noncapsular antigen, shared by several encapsulated 11. Influenzae strains, 
can be isolated. The antigen induced the formation of serum bactericidal 
antibodies when the encapsulated strain was injected and, by itself, could 
induce the formation of serum bactericidal antibodies toward the encapsulated 
strain. 

A protein polysaccharide conjugate of human serum albumin and the capsular 
polysaccharide of H. influenzae type b has been prepared. The conjugation 
was effected by marking an isocyanate derivative of the purified capsular 
polysaccharide with cyanogen bromide. The purified conjugate is currently 
under evaluation as an Immunogen following its injection into primates. 

The cellular response to H. influenzae type b capsular polysaccharide is 
being evaluated by the Jerne plaque technique in newborn animals fed two 
strains of E, coli at birth. In addition, the cellular reactivity of these 
fed and non-fed animals is being evaluated after a challenge dose of live 
H. Influenzae type b. Evaluation of the cells reactive with the type b 
capsular polysaccharide indicates the greater sensitization in the fed 
animals. Further, challenge with a non-lethal dose of H. influenzae type b 

ir-'-jces the formation of a greater proportion of cells that are reacting 

FAl 



with the polysaccharide In the plaque technique. These studies indicate 
that the cellular basis for natural Immunity may be a more precise and 
earlier method of detection of sensitization than by simple examination 
of the serum anticapsular antibodies. 

Two cross reacting E. coll were fed to adult primates. At a dosage level 
of lO-'--'-, no symptoms were observed in any of the fed animals. A A-8 fold 
rise in serum antl type b antibodies was observed one to two weeks after 
feeding. Colonization with the fed organism was transient, lasting only 
ore nonth, indicating that in an adult animal, the feeding of a cross 
reactive antigen will induce the formation of serum antibodies to H. 
Influenzae type b. Nine newborn primates have been fed strains Easter 
c.nd 89, all animals were colonized for a variable duration period, and 
no symptoms or Influence in growth and development were observed in the 
fed animals. Currently, their serum antl type b antibodies are being 
measured. 

In collaboration with Dr. George McCracken, the University of Texas, Dallas, 
Texas, the Combined Study Group concerned with the antimicrobial chemotherapy 
of neonatal meningitis, Dr. Frits Orskov, International Escherichia Centre 
(V!HO) , Copenhagen, Denmark, and Dr. Emll Gotschllch, Rockefeller University, 
a survey of E. coll strains Isolated from neonatal meningitis has been con- 
ducted. This survey was prompted by the observations that E.. coll "K" 
antigens of the acidic polysaccharide capsular type was the structure most 
directly related with virulence rather than the more studied somatic or 
"0" antigen. It was found that of 44 isolates from the CSF, 38 (86%) of 
these E^. coli have the "Kl" antigen. This E. coll antigen is of great 
interest to the Immunochemist and the biologist because of its unusual 
cher.lcal and immunologic properties. This substance is serochemically 
identical to the capsular polysaccharide of meningococcus group B and 
consists of a homopolymer of sialic acid. This sialic acid, in contrast 
to the sialic acid polymer of the meningococcal group C polysaccharide, is 
neuramlnidase-sensltlve, resistant to HCl-catalyzed methanolysls, and is 
non- immunogenic when injected in the purified form. As a component of 
the bacteria, it is poorly immunogenic when present in the nasopharynx, 
injected intravenously as whole formaldehyde- treated bacteria. Thus, it 
would seem that this polymer, which Is related to the invasive properties 
of Neisseria, may also confer virulence to another organism bacteria. 
The relation of this bacterial antigen to the immune status of the infant 
and mother is currently under investigation. 

Currently, E. coll possessing the cross reacting antigen to the H. influenzae 
type b polysaccharide Isolated from the stool of normal and asymptomatic 
infants are being collected and studied for their Immunogenicity. All 
organisms are of the 075 :H5 serotype and none has been associated with 
d:!sease symptoms In human infants. 

A symposium was conducted in collaboration with the National Institute of 
Allergy and Infectious Diseases to review mechanisms for the induction of 
Immunity to polysaccharides by rendering these polysaccharides immunogenic. 
One of the more pressing objectives of the conference was to reach an under- 
standing of the immunogenicity of polysaccharides and a characterization 
of the biosynthesis of antibodies to these polysaccharides in infants and 

FA-2 



children. Publication of the symposium should provide a collection of 
information related to the field of preventive immunization for invasive 
diseases, especially meningitis, for the first time and should serve as 
a source of information and inspiration for young people entering the 
field of science. 



FA3 



Serial No. HD-I-4 (c) 

1. Developmental Immunology Branch 

2. Bethesda, Maryland 

PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Identification of non-pathogenic bacterial antigens cross 
reactive with meningococcal and pneumococcal capsular 
structures. 

irrlnciple Investigator: John B. Robbins, M.D. 

Other Investigators: Rachel Schneerson, M.D. 

Emil C. Gotschlich, M.D. 
Teh-Yung Liu, Ph.D. 

Cooperating Units: Rockefeller University, New York, N.Y. 

Brookhaven National Laboratory, Long Island, N.Y. 

Man Years: Total: 24/12 

Professional: 24/12 
Other : None 

Project Description: 

Objectives: "Natural" and protective serum antibodies to the capsular 
polysaccharide of N. meningitidis Group A have been detected in about 70% 
of children and young adults despite the scarcity of nasopharyngeal isolates 
or disease caused by this organism in the United States for the past 
20 years. Further, the wide prevalence of anti capsular antibodies to 
N. meningitidis Group C, D. pneumoniae types I and III, and H. influenzae type 
b is not readily explained by asymptomatic carriage of the Tiomolcgous organ- 
Isn. Accordingly, a search was conducted for non-pathogenic bacteria with 
similar polysaccharide structures as the capsular polysaccharides for these 
pvogenic organisms to provide an explanation for the "natural" antibodies 
to these antigens as well as to consider such cross reacting bacteria as 
possible immunogens or vaccines. 

Methods Employed: Approximately 5,000 bacteria from serotyplcally defined 
collections from the CDC, Atlanta, Ga. , WHO Escherichia Reference 
Centre, Copenhagen, Denmark, and the collection of defined bacilli of Dr. 
Ruth Gordon, Rutgers University, New Brunswick, N.J., have been analyzed for 
cross reacting antigens by immunodiffusion with appropriate antlsera. 
C^oss immunogenic ity has been analyzed by intravenous injection of the 
bacteria and analysis of the resultant serum by quantitative and biological 
assays such as protection tests. 

Major Findings: Several naturally occurlng Bacillus ptimilis as well as 7 of 
the 20 strains of the collection of Dr. Ruth Gordon were found to possess a 
cross reacting antigen to the meningococcal Group A polysaccharide. The 
cross reacting from all these Z, pumilis strains was immunologically identical 
and the chemical structure was shown to consist of N-acetyl-mannosamine 
phosphate, mucopeptlde and glycerol phosphate. The immunodominate antigen 

FM 



was found to be N-acetyl-D-Mannosamine phosphate. 

Four E. coll with a cross-reacting antigen to the Group C meningococcal 
polysaccharide have been found. The cross-reacting antigen Is a homopolymer 
of sialic acid that Is susceptible to neuraminidase and Is cleaved by HCl- 
catalyzed methanolysls Indicating Its resemblance to both the Group B and 
Group C meningococcal polysaccharides. Immunization of animals with both 
the Group A and Group C cross reactants raised precipitating antisera to 
the appropriate antigen and this precipitating antiserum has specific group 
protective activity. 

HE. coll and one strain of Bacillus cereus (ATCC No. 10201) were detected 
with cross reacting antigen to the pneumococcal type III polysaccharide. 
Both cross reacting antigens contained glucuronic acid and both the E. coll 
and B. cereus elicited the formation of anti t3rpe III antibodies when inject- 
ed Into rabbits. 

Significance of this research - Structural analogs that are Immunogenic 
have been detected to the capsular polysaccharides of pyogenic bacteria. 
In many cases, these enteric organisms are responsible fcr the age-related 
development of "natural" and protective antibodies to these bacteria. 

Honors and Awards: None 

Publications: 

Robbins, J.B., Myerowltz, R.L. , Whlsnant, J.K. , Argaman, M. , Schneerson, R. , 
Handzel, Z.T., and Gotschllch, E.G.: Enteric Bacteria Cross-Reactive with 
Neisseria meningitidis Groups A and C and Dlplococcus pneumoniae Types I 
and III. Infect . Immun . 6:651-656, 1972. 

Robbins, J.B., Gotschllch, E.G., Liu, T.Y. , Schneerson, R. , Handzel, Z.T., 
Argaman, M. , Parke, Jr., J.C., and Myerowltz, R.L. : Bacterial Antigens 
Cross Reactive with the Capsular Polysaccharides of Haemophilus influenzae 
type b. Neisseria meningitidis Groups A and C and Dlplococcus pneumoniae 
types I and III. Proc. Symposium on New Approaches for Inducing Natural 
Immunity to Pyogenic Organisms, March 1973 (in press). 



FA5 



Serial No. HD-I-8 

1. Developmental Immunology Branch 

2. Bethesda, Maryland 

PHS-NIH 
Individxial Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Identification of the immune response to Haemophilus 
Influenzae type b capsular polysaccharide Induced by 
neonatal feeding of bacteria with cross reacting 

antigens. 

Previous Serial Number: Same 

Principle Investigators: Zeev Handzel, M.D. 

Richard Myerowitz, M.D. 
Rachel Schneerson, M.D. 
John B. Robbins, M.D. 

Other Investigators: None 

Man Years: Total: 12/12 

Professional: 12/12 
Others: None 

Project Description: 

Objectives: To identify the effect upon "natural" immunity to Haemophilus 
Influenzae type "b by feeding neonatal rabbits and adult and neonatal primates 
with bacteria containing cross reacting antigens to the capsular polysaccharide 
of Haemophilus Influenzae type b, and to follow human newborns with 
"natural" infections of cross reacting bacteria with respect to their growth 
and development and H. influenzae type b antibody formation. 

Methods Employed: Neonatal animals have been fed two E. coll strains 
designated 075 :H5 which contain a newly described cross reacting antigen 
to the type b polysaccharide. Colonization with these organisms was 
determined by culturing the stool and nasopharyngeal area with antiserum 
agar plates. Measurement of serum antibodies to the type b capsular poly- 
saccharide as well as to E^. coll antigens was done by radioimmunoassay and 
a passive hemagglutenation test. 

Significance of Work: Induction of "natural" Immunity to Haemophilus 
influenzae tjrpe b by neonatal feeding of a cross reacting, non-pathogenic 
bacteria, might provide an alternate method for Immunization against 
diseases caused by this organism. In addition, since analogs to the 
capsular polysaccharides toward other pyogenic bacteria including menlngo- 
ccccl and pneumococcl have been found, this method, if proved to be safe 
and immunogenic, might provide a general method for the artificial and 
accelerated induction of "natural" immunity toward bacteria that cause 
ir.enlngltis. Such an immunization program would offer a simple method, 
independent of the inhibition effect of maternal serum antibody, of early 
and widespread immunization against bacterial meningitis. 

F>6 



Honors and Awards: None 

Publications: 

Robbins, J.B., Schneerson, R. , Argaman, M. , and Handzel, Z.T.: Haemophilus 
influenzae type b: Disease and Immunity in humans. Ann . Intern . Med . 
78:259-269, 1973. 

Myerowltz, R.L., Handzel, Z.T. , Schneerson, R. , and Robbins, J.B.: Induction 
of Haemophilus influenzae type b capsular antibody in neonatal rabbits by 
gastrointestinal colonization with cross-reacting Escherichia coll. Infect . 
Immun. 7:137-140, 1973. 

Robbins, J.B., Handzel, Z.T., Schneerson, R. , Parke, J.C., Jr., Argaman, M. 
and Robbins, J.B.: Heterolmmunization of primates to H. influenzae type b 
capsular polysaccharide (HITB) by gastrointestinal colonization with a non- 
cnteropathogenic E^. coli (075:K147:H5 strains Easter and "89") possessing 
a cross reacting capsular antigen (CRA) to HITB. Society for Pediatric 
Research (in press). 



FA7 



Serial No. HD-1-9 

1. Developmental Immunology Branch 

2. Bethesda, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Studies of Escherichia coll Isolated from Cases of 
Neonatal Meningitis. 

Previous Serial Number: None 

Principle Investigators: John B. Robbins, M.D. 

George H. McCracken, M.D. 
Emll Gotschlich, M.D. 
Frits Orskov, M.D. 
Lars Hanson, M.D. 

Cooperating Units: The University of Texas Southwestern Medical School 

At Dallas; 

The Rockefeller University, New York City; 
The Escherichia Reference Centre (WHO) , Statenserum- 
institut, Copenhagen, Denmark; 

University of Goteborg, Department of Immunology, 
Goteborg, Sweden. 

Man Years: Total: 5/12 

Professional: 5/12 
Others: None 

Project Description: 

Objectives: To study the "K" or capsular polysaccharide antigens of E^. coll 
isolated from cases of neonatal meningitis with antlsera to known pyogenic 
organisms. To observe if the serologic properties of capsular polysaccharides 
of known meningitis organisms such as Haemophilus Influenzae type b, 
Neisseria meningitidis Groups A,B, and C and Diplococcus pneumoniae types 
I and III. 

Methods Employed: Dr. George McCracken Is the Director of a Combined Study 
Group concerned with evaluation of the antimicrobial chemotherapy of neo- 
natal meningitis. E^. coll Isolated from these cases is studied by immuno- 
diffusion with antlsera to the above mentioned pyogens. Mouse virulence 
assays and counterimmunoelectrophoresls for capsular antigen in blood and 
CSF. Serotyping for H and antigens are done by described methods using 
reagents at the E. coll Reference Centre. 

Major Findings: Of 67 E^. coll isolates from neonatal meningitis, 58 Isolates 
have come directly from the CSF. 50 of these E^. coll have a capsular poly- 
saccharide that is scrochemlcally identical to the Group B meningococcal 
polysaccharide. This E. coll structure is a homopolymer of sialic acid 
that is susceptible to hydrolysis with neuraminidase. The purified poly- 
saccharide In contrast to the other capsular polysaccharides of meningococci, 

FAS 



pneumococci, and H. Influenzae type b Is non- immunogenic . The E^. coli 
capsular antigen was found to be independent of somatic or flagella antigens. 

Significance of this Research: A virulence factor has been found for neo- 
natal meningitis due to E. coli . Such questions as the role of placentally 
transmitted antibody, colostral antibody, maternal or surrounding flora, may 
be investigated to understand the pathogenesis of this disease and, perhaps, 
to design preventive measures. 

Honors and Awards: None 
Publications: None 



FA9 



NICHD Annual Report 
July 1, 1972 through June 30, 1973 

Developmental Immunology Branch 
Contract and Collaborative Research 

Contract Title: Development and Testing of Capsular Polysaccharide of 
Haemophilus influenzae type b 

Contractor: Charlotte Memorial Hospital 
P.O. Box 2554 
Charlotte, North Carolina 

Money Allocated: $38,000 

Objectives: To confirm the immunogenic ity of the type b polysaccharide in 
infants and children. To study the optimum dose (serum antibody response) 
in infants and children. To determine the relationship between maternal 
serum antibody and the immune response to the type b capsular polysaccharide. 
To determine the effect upon the immune response to the type b capsular 
polysaccharide of the presence of bacterial antigens with cross reactive 
moieties to the type b capsular polysaccharide. To determine the overall 
incidence, the age distribution, and other factors such as economic and 
racial makeup of infants who have contracted Haemophilus influenzae type b 
diseases in Mecklenburg County. 

Methods: To determine the optimum immunogenic dose of type b polysaccharide 
for Infants as well as to study the effect of the immune response of the 
type b polysaccharide conferred by bacterial antigens with cross reacting 
antigens to the Haemophilus influenzae type b polysaccharide, all Infants 
who leave the newborn nursery at Charlotte Memorial Hospital will have 
their stool cultures examined for cross reacting antigens by the anti serum 
agar technique and their level of anti type b antibodies as studied in the 
cord serum and maternal serum will be measured by the radioassay. When 
these infants return to Charlotte Memorial Hospital to participate in their 
continuing care program, the parents of two to three month old infants ap- 
pearing for their first DPT injection will be Interviewed and requested to 
have their infants particpate in the immunization program. Blood samples, 
nasopharyngeal cultures, and rectal swabs will be obtained at one, two, 
three and six months following injection of the polysaccharide. Untoward 
reactions following the injection are to be studied by phoning the parents 
of the child within 24 hours following Injection. 

A survey of the prevalence of cross reacting antigens and the effect of 
non-typable Haemophilus Influenzae upon the immune response to the type b 
polysaccharide is also being studied. Four-day old infants with cross 
reacting bacterial antigens isolated from their stool will be studied for 
the sequential appearance of anti type b antibodies "natural" immunity, as 
coirpared to controlled infants and children without demonstrable cross 
reacting bacteria. 



FMO 



Honors and Awards: None 

Publications: 

Parke, J.C, Jr., Schneerson, R. , Robbins, J.B.: The attack rate, age 
incidence, racial distribution, and case fatality rate of Haemophilus 
influenzae type b meningitis in Mecklenburg County, North Carolina: 
J. Pediat . 81: 765-769, 1972. 

Robbins, J.B., Parke, J.C, Jr., Schneerson, R. , and Whisnant, J.K.: 
Quantitative Measurement of "Natural" and Immunization-induced Haemophilus 
influenzae type b Capsular Polysaccharide Antibodies. Pediat . Res . 7:103- 
110, 1973. 



FAil 



NICHD Annual Report 
July 1, 1972 through June 30, 1973 
Social and Behavioral Sciences Branch 



Summary of Research Activities 

In progress are several related studies concerned with fundamental issues 
in early development: the measurement of temperament in early infancy; 
the analysis of the early environment and its influence on development; the 
role of the father in infant care and his influence on the child's 
development; the development of focused relationships during the first year; 
the infant's selective impact on his environment, how his characteristics 
determine the effective environment by selectively eliciting and responding 
to stimulation. Many of these studies are using the same sample at different 
developmental periods. 

In the study of temperamental characteristics, we are assessing individual 
differences at three days in irritability, consolability, alertness, 
responsiveness to visual and to auditory stimuli. We are interested in 
determining whether there is any consistency in these characteristics between 
three days and four weeks and whether there is any predictability to character- 
istics assessed at six months. The main instrument being used is the Brazelton 
Neonatal Assessment Scale. In a study of the clinical usefulness of the 
Scale we found that it distinguishes infants of mothers on methadone from 
normal infants. Methadone infants at three days were found to be more 
irritable, less alert and less responsive to visual stimuli. These findings 
were presented on a symposium on the Brazelton Neonatal Assessment Scale at 
the meetings of the Society for Research in Child Development, March 1973. 

A major aspect of this investigation of infant temperament is concerned with 
analyzing the infant and his early environment as a dynamic interactional 
system. We are not simply concerned with the effects of the environment on 
the infant, but are interested in how the infant influences the patterns of 
stimulation he receives from his caretakers. For example, we are testing 
the hypothesis that the amount of stimulation an infant receives may be 
dependent on his level of alertness and responsiveness. We are looking 
closely at patterns of maternal behavior with the infant at four weeks in 
an effort to determine the extent to which maternal behavior towards the 
infant is influenced by his characteristics. We are investigating whether 
discrepancies between mother and infant in such characteristics as activity 
level are associated with disturbances in mother-infant adaptation. For 
this study we have extended our mother-infant interaction observation 
schedule downward and have developed an observational technique for studying 
mothers and four week infants. This schedule includes a more elaborate 
approach for studying interaction contingencies, and has more differentiated 
categories for measuring tactile and kinesthetic stimulation. Another 
Important addition to the scale is a set of categories to take into consid- 
eration the infant's state during his interactions with his mother. 



FB-1 



A third study with this sample is focusing on the role of the father with 
the young infant, his direct interactions with the infant, his emotional 
support to the mother, and his influence as mediated by the mother. 
Observations of father-infant interaction are being made in the home, and 
interviews are being conducted with the father. This study's significance 
lies in its concern with an area largely neglected by other investigators. 

The infants assessed neonatally are being studied again at six months. 
There are several aspects to this study. One is to determine whether there 
is any predictability from neonatal temperamental characteristics to cognitive 
and cognitive-motivational characteristics assessed at six months. Another 
issue we are looking at is the Impact of the very early mother-infant 
lelationship and patterns of stimulation on development at six months. 

With the same middle-class sample, we are replicating an investigation of 
early environmental influences on development that was cairried out with 
infants from lower socioeconomic homes. All too frequently important 
implications for theory and for practice are drawn from data based on a few 
studies in restricted experimental or natural situations or in which the 
sample is limited to one cultural group. In order to be able to make 
meaningful generalizations about the significance of specific aspects of 
early experience for development, it seems important to examine the 
relationships in a variety of environmental contexts. 

Also in progress is an investigation of "attachment" at one year of age. 
Although the study of attachment is central to understanding the development 
of the capacity for meaningful interpersonal relationships, the major indices 
that have been used in studies of attachment are fear of strangers and 
protest on being separated from the mother. Most of the research on 
attachment has been carried out in contrived situations of a rather stressful 
nature. There is as yet little understanding of the factors that influence 
the quality of the reciprocal affectional relationship. In this study we 
arc trying to assess the factors in the mother and in the infant which affect 
this relationship. We are also comparing indices of attachment in experi- 
mental situations with observations of infants' reactions to similar 
situations occurring naturally in the home. Underlying this methodological 
question is the conceptual issue of what are the most appropriate behavioral 
measures of this relationship. A theoretical paper on some issues in the 
conceptualization of attachment and the choice of criteria for measuring 
attachment was published this year. 

The impact of compensatory educational programs such as Headstart or the 
effects of day care programs on the development of young children are 
problems of great practical import. The measures that have been used to 
evaluate these programs have been largely restricted to tests of intellectual 
development; very little has been done to evaluate the impact of these 
programs on the child's social and motivational development. This year we 
have been part of a collaborative study with the University of Florida, 
Temple University and Syracuse University to develop and test out moti- 
vitional and social measures, such as persistence and trust, in young children 
from six months to three years of age. 



FB-2 



In preparation is a book on the methodology and substantive findings of the 
investigation of early environmental influences on personality and cognitive 
development of infants from homes of lower socioeconomic status. This 
comprehensive report presents the differential effects on infant development 
of components of maternal stimulation and of specific aspects of the 
inanimate environment. Several theoretically meaningful relationships have 
been found between aspects of stimulation and infant development. For 
instance, kinesthetic stimulation had significant relationships with many 
aspects of development, while responsiveness of inanimate objects had a 
more selective effect; it was most highly related to secondary circular 
reactions, the infant's efforts to elicit feedback from the environment. 
The relevance of these findings for several important theoretical formu- 
lations are being discussed in detail. In addition to the significance of 
these findings for theories of early development, we expect that the 
techniques developed for home observations will be useful contributions to 
methodology. Several investigators have already adapted these techniques 
for a variety of research problems. 

Analyses of data from this study on other aspects of early environmental 
influences have yielded some interesting findings with regard to the effects 
of father absence during infancy and substitute caretaking by surrogates 
in families where the mother is working. With regard to father influences 
it was found that the amount of father interaction with boy infants was 
significantly related to general developmental status as measured by the 
Bayley Mental Developmental Index; it was also significantly related to 
social responsiveness, to an index of secondary circular reactions and to 
measures of the infant's exploratory behavior with novel objects. There 
were no significant effects on the functioning of girl infants. These data 
were presented in a paper at the meetings of the Society for Research in 
Child Development. 

With the ever growing number of working mothers, questions regarding the 
effects of surrogate caretaking are becoming Increasingly important. In 
a paper presented at the meetings of the Society for Research in Child 
Development in Philadelphia, data comparing the caretaking patterns of 
mothers and surrogates were given. On the whole, mothers provided higher 
levels of stimulation than the surrogates. They were more demonstrative in 
expressing positive affect, they spent more time in non-caretaking activities 
with their infants, they played with their infants more and provided them 
ivith a greater variety of play objects. Infants in surrogate care, however, 
did not differ from infants cared for by their own mothers in general 
developmental status or on specific aspects of development. 

Members of the staff have continued to serve on committees of professional 
societies, on the editorial boards of journals and have provided consultation 
to a variety of organizations. 

Dr. Frank Pedersen has been on a committee of the Society for Research in 
Child Development which has developed a code of ethical principles for 



FB-3 



research with young children' and their parents. This statement of principles 
has been accepted by the Governing Council of the Society and will be 
published in the Directory of the Society, 

Dr. Pedersen has given consultation to Station WGBH-TV in Boston regarding 
the content of a program which will focus on the role of the father in 
psychological development. This program will be moderated by Dr. T. Berry 

Brazelton. 

Dr. Robert Klein and Dr. Frank Pedersen have continued to provide consultation 
to the Bureau of Standards which is assisting in setting industry standards 
^'or toy safety. 

Dr. Frank Pedersen participated in a workshop on "Research in Early Childhood" 
sponsored by the District of Columbia Psychological Association at 
Williamsburg, Virginia, November 11-12, 1972. He discussed environmental 
factors affecting early development and presented some of the data from our 
study, "Environmental Influences on Cognitive and Personality Development 
in Infancy." 

Dr. Robert Klein is serving as a consultant on statistical design to the 
Center for Policy Review of Catholic University. 

Dr. Leon Yarrow has just completed a two year term as President of the 
Society for Research in Child Development, an interdisciplinary society 
composed of behavioral, social and biological scientists concerned with human 
development. Dr. Yarrow organized a Presidential Sjmiposium for the biennial 
meetings of the Society in March 1973 that dealt with issues of basic and 
applied research in child development and the mutually beneficial inter- 
actions between theory and application. 

Dr. Yarrow continues to serve on the Governing Council of the Society for 
Research in Child Development. He is currently a member of a joint 
committee of the American Academy of Pediatrics, the Academy of Child 
Psychiatry and the Society for Research in Child Development on planning 
Interdisciplinary sjmiposia and conferences for the meetings of these 
societies. He is a member of the editorial boards of the Merrill-Palmer 
Quarterly Journal of Human Development and an editorial consultant to the 
Monographs of the Society for Research in Child Development. 

Dr. Yarrow presented lectures to the Psychology Department of the University 
of Virginia in Charlottesville and to the Child Psychiatry Division of 
Children's Hospital Washington, D. C. on the differentiation of the early 
environment and its selective impact on infant development. Dr. Yarrow was 
an invited discussant in a conference on "The Origins of Behavior" sponsored 
by the Educational Testing Service in Princeton, New Jersey. At the 
midwinter meetings of the American Psychoanalytic Association in New York 
he was a discussant on a workshop, "Psychoanalytic Perspectives on Programs 
f r Young Disadvantaged Children." He is also an invited participant in 



FB-A 



a conference on "Cultural and Social Influences in Infancy and Early 
Childhood" sponsored by the Wenner-Gren Foundation for Anthropological 
Research at Burg Wartenstein, Austria, June 18-26, 1973. 

There have been several changes in staff during the past year. Dr. George 
Morgan joined the staff of the Social and Behavioral Sciences Branch as 
a senior staff fellow in February. Dr. Michael Duchowny became a staff 
member in July 1972 as a Research Associate. Dr. William Davidson completed 
his assignment as Research Associate in June 1972. Ms. Florine Carpenter 
resigned as secretary. Ms. Alice Frances came on duty as secretary, July 
1972, 

Papers Presented at Professional Meetings : 

Copans, S. A. "Clinical Impressions of Communal Childrearing." Annual 
Meeting of the American Psychological Association, Honolulu, August 1972. 

>'organ, G. A. "Determinants of Infants' Reactions to Strangers: Effects 
of Age and Situation." Biennial Meeting of the Society for Research in 
Child Development, Philadelphia, March 1973. 

Pedersen, F., Rubenstein, J. and Yarrow, L. "Father Absence in Infancy." 
Biennial Meeting of the Society for Research in Child Development, 
Philadelphia, March 1973. 

Rubenstein, J. , Pedersen, F. and Yarrow, L. "A Comparison of Maternal and 
Surrogate Caretaking Behavior of Five-Month-Old Infants." Biennial Meeting 
of the Society for Research in Child Development, Philadelphia, March 1973. 

Soule, A. B., Standley, K. , Copans, S. and Davis, M. "Clinical Implications 
of the Brazelton Scale." Biennial Meetings of the Society for Research in 
Child Development, Philadelphia, March 1973. 

Standley, K. and Soule, A. B. "Women in Professions: Historic Antecedents 
and Current Lifestyles." Annual Meeting of the American Psychological 
Association, Honolulu, September 1972. 

Yarrow, L. "Cognitive and Motivational Development in Early Childhood." 
.Annual Meeting of the American Psychological Association, Honolulu, 
September 1972. 

Yarrow, L. Pedersen, F. and Rubenstein, J. "Mother-Infant Interaction and 
Development in Infancy." An invited paper to be presented at the Wenner- 
Gren Conference on Cultural and Social Influences in Infancy and Early 
Childhood at Burg-Wartenstein in Austria, June 1973. 



FB-5 



Publications : 

Brown, W„ : A prospective study of post partum psychiatric disorders. In 
Morris, N. (ed.): Psychosomatic Medicine in Obstetrics and Gynaecology . Basel, 
Switzerland, S, Karger, 1972, pp. 350-354. 

Brown, W. , Manning, T. and Grodin, J.: Prenatal psychological state and 
the use of drugs in labor. American Journal of Obstetrics and Gynecology . 
113: 598-601, 1972. 

'Jrown, W. , Manning, T. and Grodin, J.: The relationship of antenatal and 
rerinatal psychological variables to the use of drugs in labor. Psycho - 
somatic Medicine . 34: 119-127, 1972. 

Copans, S. A.: Human prenatal effects: Methodological problems and some 
suggested solutions. Merrill-Palmer Quarterly . In press. 

Morgan, G. A. and Ricciuti, H. N. : The Morgan-Rlcciuti method. In Simon, A., 
Boyer, E. G. and Karafin, G. R. (eds,): Anthology of Early Childhood 
Observation Techniques . Philadelphia, Research for Better Schools. In Press. 

Morgan, G. A.: Fear of strangers in human infants. In Brockman, L. M. , 
!^iteley, J. H. and Zubek, J. P. (eds.): Child Development: Selected 
Readings . Toronto, McClelland and Stewart Ltd. In press. 

Soule, A. B. and Standley, K,: Lawyers' perceptions of sex discrimination 
in their profession. Journal of American Bar Association . In press. 

Standley, K. and Soule, A. B.: Women and architecture. Journal of 
Architectural Education . In press. 

Standley, K. and Soule A. B.: Women in male-dominated professions: Contrasts 
in their personal and vocational histories. Journal of Vocational Behavior . 
In press. 

Yarrow, L. J.: Attachment and dependency: A developmental perspective. 
In Gewritz, J. L. (ed.): Attachment and Dependency . Washington, D. C, , 
V. H. Winston & Sons, Inc., 1972, pp. 81-95. 

Yarrow, L. J.: Enrichment and deprivation: Towards a conceptual and 
empirical differentiation of the early environment. In Monks, F. J., 
DeWitt, J. and Hartup, W. (eds.): Determinants of Behavioral Development . 
New York, Academic Press, Inc., 1972, pp. 313-329. 

Yarrow, L. J. and Klein, R. P.: Summary of research with the Inventory of 
Children's Preschool Experiences. In Simon, A., Boyer, E. G. and Karafin, 
G. R. (eds.): Anthology of Early Childhood Observation Techniques . 
Philadelphia, Research for Better Schools. In press. 



•fe-6 



Yarrow, L. , Sklar, S., Pedersen, F. , Lomonaco, S. and Fox, D.: Inventory 
of Children's Preschool Experiences. In Simon, A., Boyer, E. G. and 
Karafin, G. R. (eds.): Anthology of Early Childhood Observation Techniques . 
Philadelphia, Research for Better Schools. In press. 



FB-7 



Serial No. HD-GD3(c) 

1. Social and Behavioral Sciences Branch 

2. 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Effects of Preschool Experiences on the Intellectual and 
Personality Development of Children from Culturally 
Deprived Backgrounds 

Previous Serial Number: Same 

Principal Investigator: Leon J. Yarrow, Ph. D. 

Other Investigators: Robert P. Klein, Ph. D. , Frank A. Pedersen, Ph. D. , 

Kay Standley, Ph. D. , Sandra Sklar, M.A. , 
Bette Marcus, M.A. 

Cooperating Units: Office of Economic Opportunity, Project Headstart, 

National Capital Day Care Association 

Man Years: 

Total: .75 
Professional: .45 
Other: .30 

Project Description: 

Objectives : This study has been concerned with the impact of early preschool 
experiences on the development of cognitive skills, behavioral controls and 
self-concept in four-to-five-year-old children from culturally disadvantaged 
backgrounds. A major goal has been to develop methods for differentiated 
analyses of preschool learning environments, utilizing concepts from social 
learning theory, cognitive developmental theories and operant learning 
theory. 

Methods Employed : The sample consisted of 58 children in 12 preschool class- 
rooms in Headstart and day care programs. Time-sampling observational 
techniques were used to analyze the preschool environment. To provide a 
sample of different classroom activities and minimize effects of day-to-day 
changes, each child was observed for 20 observational units on each of three 
days in a balanced order. These observations were made on each child at 
tlircc different points in the school year — in the fall, shortly after the 
bii;f lining of scliool; in the middle of the term; and in the early summer, 



FB-8 



toward the end of the term. A comprehensive observational system was 
developed. This system is organized in terms of the following major 
categories: teacher behavior, which include dimensions of teacher control, 
cognitive input dimensions, the affective qualities of the interaction, and 
evaluative feedback; child-behavior variables, focusing on self-initiated 
or self-directed activities; and background and setting variables. 

Measures of the dependent variables consisted of several standard tests and 
others developed especially for this investigation. For studying cognitive 
f'-.nctions, the following techniques were used: the Wechsler Preschool Scale 
of Intelligence, several measures of language functions, the Johns Hopkins 
Perceptual Test, selected items from the Frostig Developmental Test of 
Visual Perception, and a specially designed object categorization test for 
assessing concept development. As measures of "behavioral controls," we 
have adapted the Maccoby motor inhibition measures, and have developed 
techniques to assess capacity to delay gratification, a measure of focused 
attention, and a situational test of response to a difficult task. In 
addition, we have developed a self-concept test designed to tap various j 
aspects of the child's feelings about himself, his competencies and 
deficiencies, and his perception of how he is evaluated by other people. 

Major Findings : Analyses of the data have been completed. To assess the 
relationships between the child's experiences in the classroom and changes 
in general intellectual level, in specific cognitive functions, and on his 
self-concept, simple correlational analyses and multiple regression analyses 
were carried out. 

The major findings have been reported in previous annual reviews. Essentially 
there were few significant relationships between specific experiences in 
these preschool programs and cognitive and personal-social characteristics. 

Significance to Biomedical Research and the Program of the Institute : 
Current controversy about the relative merits of various approaches to 
preschool programs emphasizes the need for greater conceptual clarity about 
the preschool environment. The concepts and techniques developed in this 
research should contribute to more adequate methodologies for studying 
complex environmental inputs in relation to the cognitive and personal- 
social development of preschool children. 

Proposed Course of Project : All analyses have been completed. It is hoped 
that current data collection activities on other projects will allow time 
to complete the final report by the middle of next fiscal year. 

Honors and Awards: None 



PB-9 



Publications ; 

Yarrow, L. J. and Klein, R. P.: Summary of research with the Inventory of 
Children's Preschool Experiences. In Simon, A., Boyer, E. G. and Karafin, 
G. R. (eds.): Anthology of Early Childhood Observation Techniques . 
Philadelphia, Research for Better Schools. In press. 

Yarrow, L., Sklar, S. , Pedersen, F., Lomonaco, S. and Fox, D.: Inventory 
of Children's Preschool Experiences. In Simon, A., Boyer, E. G. and 
Karafin, G. R. (eds.): Anthology of Early Childhood Observation Techniques . 
Philadelphia, Research for Better Schools. In press. 



FBIO 



Serial No, HD-GD4 (c) 

1. Social and Behavioral Sciences Branch 

2. 

3. Bethesda, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Environmental Influences on Cognitive and Personality 
Development in Infancy 

Previous Serial Number: Same 

Principal Investigators: Leon J. Yarrow, Ph. D. , Judith L. Rubenstein, Ph.D., 

Frank A. Pedersen, Ph. D. 

Other Investigators: Myrna Fivel, M.A., Joan Durfee, M.A. , Richard Cain, M.Ed. 

Cooperating Units: D. C. Department of Public Health, Children's Hospital 

of the District of Columbia, Group Health Association 
of Washington, D. C. 

Man Years: 

Total: 1.10 
Professional: 1.00 
Other: .10 

Project Description: 

Objectives : In this investigation of early environmental influences on 
cognitive and personality development during the first year of life, one 
major goal has been to develop an observational instrument to analyze the 
components of the early environment. A second objective was to study 
relationships between specific parameters of the environment and specific 
aspects of development during the first six months. 

Methods Employed : The original sample consisted of 70 infants and their 
caretakers obtained through two well-baby clinics in the District of 
Columbia and through a private medical care program. Pediatric and 
neurological examinations were conducted to exclude infants with abnormalities 
that might be associated with developmental retardation. For the major 
analyses we selected from the original sample 41 cases in which we felt 
there was a representative sample of the infant's early experiences. These 
were cases in which there was a caretaker who had been responsible for the 
baby for three months or longer, and in which there had been no abrupt 
rnvironmental changes around the observation period. 



FMl 



Data on environmental variables were obtained through time-sampling 
observations in the home when the infants were five months of age. Measures 
of infant characteristics were obtained by means of the Bayley Scales of 
Infant Development and a situational measure of exploratory behavior and 
preference for novel stimuli. 

Major Findings : The major findings have been summarized in previous annual 
reports. A number of significant relationships were found between specific 
parameters of the environment and differentiated aspects of infant 
functioning. During the past year we have concentrated on the preparation 
of a detailed report on the findings and their theoretical and methodological 
implications for publication as a book. In addition two papers on discrete 
aspects of the study have been presented at the meetings of the Society 
for Research in Child Development. A paper, "Mother-Infant Interaction 
and Development in Infancy," has been prepared for presentation at a 
conference on Social and Cultural Influences in Infancy and Early Childhood 
sponsored by the Wenner-Gren Foundation to be held June 18-26, 1973 at 
Burg Wartenstein, Austria. 

The first paper, "A Comparison of Maternal and Surrogate Caretaking Behavior 
of Five-Month-Old Infants," presented at the Society for Research in Child 
Development meetings by Judith Rubenstein has important implications for 
the ever increasing number of working mothers whose children have substitute 
caretakers for part or all of the day. The mothers and surrogates were 
compared on eleven measures of the environment which previous analyses had 
indicated were significantly related to important aspects of infant 
development. Mothers provided more stimulation than the surrogates on all 
eleven measures; five of these differences were statistically significant. 
Mothers were more demonstrative than surrogate caretakers in the expression 
of positive affect, in the amount of time they spent playing with their 
infants and in providing them with play objects; they gave their infants 
more varied social experiences and a greater variety of inanimate objects. 
These differences in patterns of stimulation did not, however, seem to 
affect infant functioning. Infants in surrogate care, on the whole, did 
not differ from infants cared for by their own mothers in general 
developmental status or on specific sectors of development. 

The second paper, "Father Absence in Infancy," presented by Frank Pedersen 
at the meetings of the Society for Research in Child Development compared 
infants reared in father absent homes with infants reared in homes in 
which the father was present. Forty-nine per cent of the sample recruited 
from public well-baby clinics were from father absent homes. Infants from 
father absent and father present homes were compared on si::teen measures 
of cognitive, social, motor and motivational development. We also 
obtained from the mother an estimate of the amount of the father's inter- 
action with the infant. 

A number of interesting findings emerged. Although father absence has no 
relationship to functioning in female infants, several significant 
relationships were found in the male infants. Amount of father interaction 



FB-12 



with boy infants is significantly related to the Bayley Mental Developmental 
Index, to the Bayley clusters. Social Responsiveness, and Secondary Circular 
Reactions, and two measures of exploration of novel objects. We checked 
carefully the possibility that these findings might be artifactual, that 
they might be systematically related to other factors, i.e., differences 
in maternal behavior or socioeconomic status—and eliminated these 
possibilities. These data do not support the prevalent assumption that 
maternal variables alone are the only important influence on infant 
development. 

Significance to Biomedical Research and the Program of the Institute ; 
This research has significance for theories of early development and* has 
implications for early child care programs. In addition, the techniques 
developed for home obsGrvations will be useful contributions to methodology 
of studying the early environment. 

Proposed Course of Proiect: It is planned to report the major findings in 
book form and terminate this study early in the next fiscal year. 

Honors and Awards : None 

Papers Presented at Professional Meetings: 

Pcdersen, F. , Rubenstein, J. and Yarrow, L.: Father absence in infancy. 
Presented at the Biennial Meeting of the Society for Research in Child 
Development, Philadelphia, Pa., March 1973. 

Rubenstein, J., Pedersen, F. and Yarrow, L. : A comparison of maternal and 

surrogate caretaking behavior of five-month-old infants. Presented at 

the Biennial Meeting of the Society for Research in Child Development, 
Philadelphia, Pa., March 1973. 

Yarrow, L. : Cognitive and motivational development in early childhood. 
Presented at the annual meeting of the American Psychological Association, 
Honolulu, September 1972. 

Yarrow, L. , Pedersen, F. and Rubenstein, J.: Mother-infant interaction and 
development in infancy. An invited paper to be presented at the Wenner- 
Gren Conference on Cultural and Social Influences in Infancy and Early 
Childhood in Austria, June 1973. 

Publications ; 

Yarrow, L. J.: Enrichment and deprivation: Towards a conceptual and 
empirical differentiation of the early environment. In Monks, F. J., 
r)eWitt, J. and Hartup, W. (eds.): Determinants of Behavioral Development . 
New York, Academic Press, Inc., 1972, pp. 313-239. 



FM3 



Serial No. HD-SBl(c) 



1. Social and Behavioral Sciences Branch 

2. 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: An Observational Approach to the Measurement of Preschool 
Environments 

Previous Serial Number: Same 

Principal Investigator: Leon J. Yarrow, Ph. D. 

Other Investigators: Robert P. Klein, Ph. D. , Sandra Sklar, M.A., 

Alice Abramson, M. A. 

Cooperating Units: Montessori Society of Chevy Chase, Bank Street College 

of Education 

Man Years: 

Total: .50 

Professional: .50 
Other: 

Project Description: 

Objectives : This research is concerned with refinement and further 
development of a time-sampling observational technique for describing 
children's experiences in preschool settings. The observational technique 
was developed in an earlier study to assess the impact of experiences in 
Headstart programs. In this study two model preschool programs with 
differences in educational philosophies were compared. We were interested 
in determining the extent to which the observational instrument was able to 
distinguish unique characteristics of different kinds of preschool programs. 
A second and related purpose of fhe study is to describe differences in 
children's experiences in programs based on different philosophies. Although 
there has been a proliferation of models for preschool intervention during 
the past several years, there is as yet no observational research describing 
the ways in which these programs vary in terms of children's experiences. 

Methods Employed : The sample consisted of 48 children in six preschool 
classrooms, three based on the Montessori 'prepared environment' model and 
three on the Bank Street College 'child-centered' model. To provide a 
representative sample of the different classroom experiences of children in 
the two models, each child was observed for ten observational units of two 

FB-14 



minutes duration on each of six days. Our previously developed observational 
technique was refined for use in this study. The observational scheme 
describes the following dimensions: child-behavior variables, focusing on 
the nature of a child's cognitive, perceptual-motor, language, social and 
affective experiences; teacher-behavior variables, including components of 
teacher control, cognitive input, affective interaction with children, and 
evaluative feedback; and structural components of the environment, including 
the degree of structure of classroom materials and the extent of freedom 
given the child in choice of activities. 

Major Findings : Statistical comparisons of the programs have been completed 
using analysis of variance. The comparisons show that the scale is 
differentiating between the programs on predicted dimensions. For example, 
there is a significantly greater amount of fantasy play and self-expressive 
behavior of the children in the Bank Street Schools. In the Montessori 
classrooms there is significantly more time spent on pre-academic activities, 
such as, reading, writing, quantitative skills and in visual discrimination. 

In addition to the univariate analyses performed last year several 
multivariate analyses were performed this year. These were of two types: 
between program (multivariate analysis of variance and discriminant analysis) 
and within program (factor analysis). The between program analyses indicated 
that the major dimension differentiating the programs was whether 
or not the child's activities allowed for many or few response possibilities 
(unstructured vs. structured activities). In addition, the amount of fantasy 
play the child participated In was an important distinguishing variable. 
The factor analysis for each program separately gave a somewhat different 
picture. For each program there were two independent dimensions which 
accounted for significant portions of the variance, but the structure of 
these dimensions did not correspond to that obtained by the between program 
analyses. This is not particularly surprising, but it does point up the 
fact that the variables necessary to describe a program will depend on the 
particular purpose of the description. 

Significance to Biomedical Research and the Progrcun of the Institute : 
Controversies about the relative merits of various approaches to preschool 
education and intervention have existed for several years. A major factor 
preventing resolution of these controversies Is the lack of descriptive data 
based on direct observation of these different programs. The instrument 
refined in this project should provide a tool for further observational 
research. The substantive findings from the project should provide 
important data about the differing experiences of children in various programs. 

Proposed Course of Project ; The report from this project will be published 

along with the reports of the Head Start project. 

I 

Honors and Awards: None 



FBI 5 



Papers Presented at Professional Meetings : 

Dr. Robert Klein has organized a symposium "Multivariate Comparison of Two 
Model Preschool Programs" as part of the 1973 meetings of the International 
Society for the Study of Behavioral Development in Ann Arbor, Michigan. 
In addition to chairing the symposium he will give a paper on the results 
of this project, particularly the multivariate analyses. 



FW6 



Serial No, HD-SB2 (c) 

1. Social and Behavioral Sciences Branch 

2. 

3. Bethesda, Maryland 



PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: An Observational Study of Father-Infant Interaction 

Previous Serial Number: Same 

Principal Investigators: Frank A. Pedersen, Ph. D. , David Schachter, M.D. 

Cooperating Units: Pediatrics & Obstetrical Service, National Naval 

Medical Center 

Man Years: 

Total: 

Professional: 

Other: 

Project Description: 

Objectives : To obtain descriptive data on the early father-infant 
relationship in natural settings. A methodological component of the 
study will compare the types of information obtained from interviews 
of the fathers with data obtained in direct observation of father-infant 
interaction. The study will also attempt to determine whether there are 
mediated paternal effects, i.e., whether the father affects the mother's 
relationship with the infant. A major goal of the study will be to develop 
a conceptual model for understanding paternal influences as a part of the 
early environment. 

Methods Employed : The sample will consist of approximately 40 middle- 
class fathers and their first-born infants (the same sample used in 
Project HD-SB4(c). At age four weeks the father is interviewed regarding 
the early postnatal period, his current relationship with the infant, and 
aspects of the husband-wife relationship which may bear on how each parent 
relates to the baby. At age five weeks an observation is conducted in 
the home, usually in the early evening, focusing on the father's inter- 
action with the baby. The observational system is an adaptation of the 
method described in Project HD-SB4(c). Data from that project on the infant 
and the mother-infant relationship will be utilized in certain analyses. 



FB47 



Major Findings : In focusing on the earliest periods of the father's 
contact with the baby, we have attempted to conceptualize the role of the 
father in terms of three areas: (1) his behavior in direct interaction 
with the baby; (2) his behavior which is instrumental to the mother, and 
(3) his emotional support given to the mother during her early adaptation 
to the baby. We have developed an interview that covers these areas. 
Inter-rater reliability has been established on variables relevant to these 
areas, and we have coded approximately 25 interviews. Observations have 
been completed on these cases as well, although we have not begun formal 
tabulations of these results. We are impressed with the wide range of 
differences in fathers in all areas, but it is too early in the data 
collection process to give more than impressionistic results. 

Significance to Biomedical Research and the Program of the Institute : Most 
of our knowledge of early parental behavior is limited to the mother-infant 
unit. The apparent assumption that this is a closed system, relatively 
unaffected by the father, and that early paternal behavior is of no 
consequence is at least open to question. The observational data of father- 
infant interaction will be unique in their own right, and it is hoped that 
this study will contribute to our understanding of the infant's early 
environment. 

Proposed Course of Proiect : We expect to complete the data collection in 
this fiscal year. Data analyses will be carried out in the coming year. 

Honors and Awards: None 



FM8 



Serial No. HD-SB4(c) 

1. Social and Behavioral Sciences Branch 

2. 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 



Project Title: Effects of Infant Temperamental Characteristics on Mother- 
Infant Adaptation 

Previous Serial Number: 

Principal Investigator: Leon J. Yarrow, Ph. D. 

Other Investigators: Stuart Copans, M.D., Michael Duchowny, M.D., 

Robert Klein, Ph. D. , A. Bradley Soule, M.D,, 
Kay Standley, Ph. D. , Richard Cain, M.Ed., 
Myrna Fivel, M.A. 



Cooperating Units: 



Pediatrics and Obstetrical Services of National Naval 
Medical Center; Childbirth Education Association 



Man Years: 




Total: 


5.75 


Professional: 


5.50 


Other: 


0.25 



Project Description: 

Objectives : To study the effects of infant temperamental characteristics 
on patterns of maternal care and attitudes towards the infant. Within this 
overall objective there are a number of specific methodological and 
substantive questions: 

1. Is there a basic consistency between infant characteristics 
assessed in the neonatal period and infant characteristics at four weeks? 

2. What are the relationships between mother-infant adaptation at 
four weeks and certain prenatal variables: her attitudes and 
expectations regarding the infant; the husband-wife relationship during 
pregnancy; the woman's physical and psychological adaptation to the 
pregnancy experience? 



FB'19 



3. To what extent do infant temperamental characteristics influence 
maternal caretaking patterns and mother-infant adaptation? Does a 
discrepancy in activity level between mother and infant lead to disturbed 
adaptation? Is there a relationship between the infant's visual alertness 
and amount of visual stimulation provided by the mother? 

Methods Employed : The sample consists of 40 parents and their first-born 
infants, approximately 20 boys and 20 girls. Only normal infants, free 
from complications during pregnancy and delivery have been included in the 
study. 

There are five phases of data collection: 

1. Prenatal interview with prospective parents in which basic 
demographic data are obtained. Both parents are interviewed about their 
expectations and feelings about the unborn child, the degree of solidarity 
between husband and wife and general adaptation to pregnancy. 

2. Neonatal assessment of infant in newborn nursery. Infant 
characteristics such as visual alertness, auditory responsiveness, 
irritability, consolability are evaluated using the Brazelton Neonatal 
Assessment Scale. 

3. Four week assessment of infant using the Brazelton Scale. 

4. Four week time-sampling observation of mother-infant interaction 
in the home. 

5. Interview with the mother at four weeks regarding her feelings 
towards the infant and her perception of infant characteristics. 

Major Findings : Data collection has been completed on 35 cases. It is 
planned to complete data collection by July 1973. 

Some preliminary analyses have been carried out on the reliability and 
clinical usefulness of the Brazelton Scale. To assess the clinical value 
of the Scale comparisons were made between normal infants and babies born 
to mothers on methadone maintenance. Significant differences were found 
at three days between the normal and methadone infants on several dimensions, 
e.g., sensory responsiveness and irritability, indicating that the scale is 
sensitive to differences in the newborn period. These findings were 
reported on a symposium on the Brazelton Neonatal Assessment Scale at the 
Biennial meetings of the Society for Research in Child Development by 
Bradley Soule. 

Significance to Biomedical Research and the Program of the Institute ; 
Most research on early development has concentrated on environmental 
influences on the young infant conceptualized in essentially unidirectional 
terms, i.e., the effects of environmental events on infant development. 
This model is being replaced by an interactional one in which the child is 
seen as an active agent who influences the behavior of other people towards 
him. This investigation should begin to give some understanding of 
reciprocal interactions between infant and mother. 



FB-20 



Proposed Course of Proiect : Collection of data will be continued during 
this fiscal year. Analyses of data will be made during FY 1974. 

Honors and Awards : None 

Publications : 

Copans, S. A.: Human prenatal effects: A suggested methodology for future 
study. Merrill-Palmer Quarterly Journal of Human Development , in press. 

Papers Presented at Professional Meetings : 

Soule, B., Standley, K. , Copans, S. and Davis, M. : Clinical implications of 
the Brazelton Scale, Paper presented at the Biennial Meetings of the 
Society for research in Child Development, Philadelphia, Pa., March 1973. 



F&21 



Serial No. HD-SB5 



1. Social and Behavioral Sciences Branch 

2. 

3. Bethesda, Maryland 



PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 



Project Title: Development of Measures of Social and Motivational Functions 
in Young Children 

Previous Serial Number: None 

Principal Investigator: Leon J. Yarrow, Ph. D. 

Other Investigators: Colleen Rand, Ph. D. , Kay Jennings, M.A. , Sarah 

Friedman, M. A. , Martha Weiss, Leonie Black 

Cooperating Units: Institute for Development of Human Resources, University 

of Florida; Developmental Research Laboratory, Temple 
University; Syracuse University Children's Center 

Man Years: 

Total: 2.0 
Professional: 2.0 
Other: 

Project Description: 

Objectives : Although there are many tests of cognitive functioning for 
young children, there are few standardized instruments for measuring other 
important aspects of development. In this study we have worked on the 
development of measures of social and motivational functions, i.e., 
persistence and trust, for use with children between six months and three 
years. This has been a collaborative undertaking with the University of 
Florida, Syracuse University and Temple University. 

Methods Employed : Subjects were white middle-class infants tested within a 
week of their six and twelve month birthdays and within three weeks of 
their second and third birthdays. 

We have concentrated on developing a series of measures for six and twelve 
month infants. Measures developed for the two and three year olds at the 
other centers have also been tried out in our laboratory. 



FB-22 



Major Findings : Measures of trust in strangers and persistence with objects 
have been developed for all age groups. In Bethesda 41 infants have been 
studied with these measures. 

Significance to Biomedical Research and the Program of the Institute : 
Measures that have been used to evaluate compensatory educational programs 
or the effects of day care programs on the development of young children 
have for the most part been limited to cognitive development. The measures 
developed in this project will provide means of assessing other important 
aspects of development that may be influenced by these programs. 

Proposed Course of Project : Data collection will be completed early in the 
next fiscal year. 

Honors and Awards: None 



FB23 



Serial No. HD-SB6 



1. Social and Behavioral Sciences Branch 

2. 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1972 

Project Title: Factors Associated with the Development of Mother-Infant 
Attachment During the First Year 

Previous Serial Number: None 

Principal Investigators: Leon J, Yarrow, Ph. D. , Robert Klein, Ph. D. 

Other Investigators: Joan Durfee, M.A. , Myrna Fivel, M.A. , George Morgan, Ph.D. 

Cooperating Units: None 

Man Years: 

Total: .60 

Professional: .50 
Other: .10 

Project Description: 

Objectives : Although there have been a number of studies on the development 
of the distinctive tie between mother and infant during the first years of 
life, there is as yet little understanding of the factors that influence the 
development of this unique focused relationship. The major objective of 
this study is to assess the factors in the mother and infant, and in the 
early mother-infant interaction which affect the nature of this relationship. 
The second objective is methodological. The studies that have been done on 
attachment have used a variety of techniques — interview with the mother, 
structured situations in the laboratory--and a variety of indices to measure 
attachment. In this study, we plan to compare data obtained from observations 
in the home, from an interview with the mother and from observations of 
structured situations in the laboratory. Underlying this methodological 
question is the conceptual issue of what are the most appropriate behavioral 
measures of this relationship. 

Methods Employed : The sample will consist of forty mothers and their first 
born twelve-month-old infants. Some of these infants will be part of the 
sample first studied prenatally (Project No. HD-SB4(c). The remainder of 
the sample will have entered the study at six months. 

Two home observations of approximately three hours duration are conducted 
within two weeks of the baby's first birthday. These observations code 

FB24 



selected mother and infant behaviors in a number of critical situations 
which are comparable to measures that have been used in laboratory studies 
of attachment behavior. The infant's reactions are coded to such events 
as the mother entering the room, leaving the room, the appearance of the 
mother when the infant is crying. The infant's vocalizations, smiles, 
approach behaviors, cessation of crying are recorded. 

Following the completion of the two home observations, the mother and infant 
are brought to the Social and Behavioral Sciences' playroom for a series of 
observations. 

1. The infant's initial response to the female experimenter/ 
interviewer is observed from behind a one-way mirror. The experimenter 
approaches the infant in a pre-determined , structured but natural manner. 

2. The mother is interviewed, using a semi-structured interview 
schedule regarding selected aspects of her baby's social functioning in 
relation to herself, the baby's father, other familiar people, and strangers. 

3. During the interview, the infant is provided with toys and his 
exploratory behavior and social initiative and responses (vis a vis mother 
and interviewer) are observed from behind the one-way mirror. 

4. At the completion of the interview proper, the mother is asked 
to leave the room and the infant's reaction to being left with the inter- 
viewer is recorded. 

Major Findings : Methods for observation of critical situations in the home 
have been developed and pretested. Observer reliability has been established, 
Data collection has just begun. Five infant-mother home and laboratory 
observations have been made. 

Significance to Biomedical Research and the Program of the Institute : The 
study of "attachment" is central to understanding the development of 
affectional relationships. Most of the research on attachment has been 
carried out in contrived situations of a rather stressful nature. There 
has been little comparison of reactions of infants in these experimental 
situations with behavior in natural settings. Also very limited is our 
knowledge of the contribution of temperamental factors in the infant on the 
developing mother-infant affectional relationship. 

Proposed Course of Project : Data collection will be carried on during the 
next fiscal year. 

Honors and Awards : None 

Publications : 

Yarrow, L. J.: Attachment and dependency: A developmental perspective. In 
Gewirtz, J. L. (ed.): Attachment and Dependency . Washington, D.C., V. H. 
Winston & Sons, Inc., 1972, pp. 81-95. 



FB-25 



Serial No. Hn-.SR7 



1. Social and Behavioral Sciences Branch 

2. 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Early Environmental Influences on Cognitive and Personality 
Development During Infancy: A Replication with a Middle- 
Class Sample 

Previous Serial Number: None 

Principal Investigator: Leon J. Yarrow, Ph. D. 

Other Investigators: Robert P. Klein, Ph. D. , Frank A. Pedersen, Ph. D., 

Joan Durfee, M.A. , Myrna Fivel, M.A. , Stuart Copans, M.D., 
A. Bradley Soule III, M.D. 

Cooperating Units: None 

Man Years: 

Total: 1.75 

Professional: 1.50 
Other: .25 

Project Description: 

Objectives : In a previous study with a sample composed largely of infants 
and mothers of low socioeconomic status some significant relationships 
were found between a number of early environmental variables and infant 
development. These findings had important implications for developmental 
theory. Because of their theoretical significance, it seemed important 
to test the generality of these findings to determine whether similar 
relationships would be found in other environmental contexts with different 
levels and patterns of stimulation. Therefore, we are replicating the 
previous study with a middle-class sample. 

Methods Employed : The sample will consist of 40 infants, 20 boys and 20 
girls and their mothers. It is planned to study at five to six months of 
age the infants first studied as neonates in the reciprocal interaction 
project (See Project No. HD-SB4). 

Data on the environmental variables are being obtained by time-sampling 
observations in the home using the mother-infant interaction schedule 



FBi.6 



developed in the previous study. Measures of infant characteristics are 
derived from the Bayley Scales of Infant Development and through a 
situational test designed to measure exploratory behavior and preference 
for novel stimuli. 

Mai or Findings : Data have been collected on 19 subjects. 

Significance to Biomedical Research and the Program of the Institute : 
All too frequently theoretical derivations in the behavioral sciences are 
based on a few studies in limited experimental or natural situations in 
which the sample is limited to one cultural group. If we are to make 
meaningful generalizations about the significance of specific aspects of 
early experience for development, it is necessary to examine these 
relationships in a variety of environmental contexts. 

Proposed Course of Proiect : Data collection will continue during the next 
fiscal year. 

Honors and Awards: None 



Fae? 



Serial No. HD-SB8 



1. Social and Behavioral Sciences Branch 

2. 

3. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Factors Influencing Career Choice in Women 

Previous Serial Number: None 

Principal Investigators: Kay Standley, Ph. D. , A. Bradley Soule, III, M.D. 

Cooperating Units: None 

Man Years: 

Total: ,40 
Professional: ,40 
Other: 

Project Description: 

Objectives : To study facilitators and barriers to women's entry into elite 
occupations by examining the family backgrounds and early life histories of 
some women who made innovative career decisions. Another area of major 
interest is the women's perceptions of sex discrimination in their professions. 

Methods Employed : The sample consisted of 71 women in law, medicine, and 
architecture who for the most part resided in the Washington area. Each 
was interviewed concerning her family and personal history as well as her 
vocational decisions and current position. 

Major Findings : In the analyses completed to date, it has been possible to 
describe the population as one emerging from urban, affluent, educationally 
advantaged and professional backgrounds. Parents seem to have been very 
involved and invested in the academic achievement of these women. In terms 
of their current lifestyles, most of the women noted a disturbing discontinuity- 
in some instances conflict — between their careers and their personal (non- 
occupational) lives. The women commonly reported "dual personnae" themes-- 
instances in which social expectations of them at work and at home were in 
difficult contradiction. 

Significance to Biomedical Research and the Program of the Institute : 
There is increasing social and practical concern about the relatively few 
women in elite occupational categories. Study of the vocational 



FB£8 



development of successful women should aid our understanding of the obstacles 
encountered by women seeking vocational fulfillment. 

Proposed Course of Project : The project has been completed. 

Honors and Awards : None 

Publications : 

Soule, A. B. and Standley, K.: Lawyers' perceptions of sex discrimination in 
their profession. Journal of American Bar Association , in press. 

Standley, K. and Soule, A. B.: Women and architecture. Journal of 
Architectural Education , in press. 

Standley, K. and Soule, A. B.: Women in male-dominated professions: Contrasts 
in their personal and vocational histories. Journal of Vocational Behavior , 
in press. 

\t 
Papers Presented at Professional Meetings : |; 

Standley, K. and Soule, A. B.: Women in professions: Historic antecedents and , 

current lifestyles. Presented at the annual meeting of the American j" 

Psychological Association, Honolulu, September 1972. 



c 



FM9 



NICHD ANNUAL REPORT 
July 1, 1972 through June 30, 1973 
Reproduction Research Branch 

SUMMARY 

The mission of the Reproduction Research Branch is the development of scient- 
ific data and theory basic to reproductive biology. Research efforts there- 
fore extend from the chemistry of the protein and steroid hormones to physio- 
logic and clinical studies in man. To prosecute these investigations success- 
fully, a variety of tools and skills are necessary: physical chemistry of 
macromolecules, protein isolation and analysis, tissue and organ culture, 
ultrastructure, enzymology, radioligand assays of protein and steroid 
hormones, mathematical analyses of immunoassays and the kinetics of protein 
hormone binding, characterization of in vitro steroid biosynthetic pathways, 
dynamic analysis of steroid hormone metabolism in man. A continuing interest 
and involvement in clinical problems are not only desirable but necessary 
since the results of study in man are the final arbiter of the relevance of 
results in the laboratory. 

There have been several important contributions from those investigators work- 
ing in the broad area of protein chemistry. Prolactin was isolated from 
amniotic fluid and a practical method was developed for preparation of the 
hormone. The identity of serum, urine, and amniotic fluid prolactin was 
proved by physico-chemical, biologic, and immunologic criteria. To define 
the molecular parameters of chromaffin granule membrane proteins, a novel 
system for discontinuous buffer electrophoresis in SDS at neutral pH was de- 
vised. Luteinizing hormone-releasing hormone (LHRH) was monoiodinated with 
125i with preservation of biologic activity, and methods were found to 
separate labeled from unlabeled species to obtain a tracer of maximum 
specific activity. The analysis and synthesis of several membrane-active 
peptides of the gramicidin and nisin series were reported. 

Studies in the mechanism of hormone action have taken several directions. A 
mathematical theory of drug-receptor interaction has been adapted and extended 
to apply to current problems and validation is being sought from experimental 
studies and by computer simulation. X-ray diffraction studies of the chro- 
maffin granule membrane revealed the existence of sub-membrane particles dis- 
tributed symmetrically and composed of proteins. This is the first step in 
the analysis of membrane changes accompanying secretion. Solubilization and 
partial purification of soluble receptors of the testis for LH and HCG were 
achieved. The receptor had a MW of about 200,000 and retained its high 
affinity and specificity for HCG. Stimulation of steroidogenesis by HCG 
occurred without detectable increase in cAMP production suggesting that cAMP 
may not be an essential intermediate in LH action. Specific receptor sites 
for angiotensin II were demonstrated in adrenal cortex homogenates, probably 
on microsomal membrane fraction. 

the techniques of the cell biologist have proved useful. A clone of cells has 
been isolated from a hamster ductus deferens tumor that contains a specific 
•0Totej.Li receptor for both testosterone and estradiol. Proceeding from last 



FC-1 



year's observations of the effect of estrogen on follicular development, it 
has been shown that porcine granulosa cells contain an estrone receptor. 

The inception of embryogenesis on the sea urchin is accompanied by net syn- 
thesis of polyadenylic acid, a probable requisite step before translation of 
preexisting genetic messages. The embryo was shown to contain DNA polymerases 
capable of using RNA templates for DNA synthesis, thereby providing an alter- 
native mechanism for achieving new informational content. The lysosome of 
the human term placenta was examined and two populations identified. The first 
trimester placenta is 6 to 8 times richer in lysosomes than term placenta. 
Conventional lysosomal enzyme markers are not satisfactory for placental lyso- 
somes . 

The gonadotropins remain a major focus of interest. In collaboration with 
Dr. R. Canfield, Columbia University, sub-units of HCG have been prepared, 
characterized chemically, immunologically and biologically and distributed 
to investigators around the world. Specific antisera were prepared for each 
sub-unit and made available to the National Pituitary Agency for distribution. 
The sub-units were shown to be free of biologic activity. Free HCGa was found 
in plasma throughout pregnancy thereby accounting for previously noted dis- 
crepancies between biologic and immunologic measurements of HCG. The sub- 
units of HCG have been measured in tissue, serum, and urine of patients with 
HCG-secreting tumors and the finding that individual metastases had differing 
contents of sub-units has been interpreted as evidence that cloning of cancer 
cells occurs in man. 

A new technique for a simple solid-phase radioimmunoassay of steroids has 
been reported. Measurement of Cortisol and estradiol in plasma can be made 
in two hours. A method was developed for measuring free estradiol in urine 
since it is the best measure of total estrogenic load. The filtration of 
estradiol by the kidney was estimated using measurements of non-protein bound 
plasma estradiol. 

In the clinical area, the time devoted previously to development of methods 
has facilitated several new studies. A group of women with secondary amenor- 
rhea and weight loss was identified in whom the mechanism of the amenorrhea 
could be attributed to hypothalamic dysfunction on the basis of testing of 
pituitary and hypothalamic function. The increased sensitivity of an improved 
human prolactin assay facilitated investigation of the hypothalamic-pituitary 
unit. High plasma prolactin concentrations were noted in some patients with 
pituitary tumors but without lactation. 

The male infertility clinic continues to serve to identify "experiments of 
nature" that illuminate testicular regulatory mechanisms. Serum FSH is high 
when germ cells are absent. It has been difficult to decide whether the 
germ cell or Sertoli cell is responsible for regulation of FSH levels. The 
finding in three men that a normal sperm count with death of all sperm assoc- 
iated with high FSH is strong evidence that the Sertoli cell is the regulator 
of FSH secretion. A clinical trial has been initiated for the use of high 
doses of FSH in various types of male infertility. 

The clinical studies have been complemented by related work in the rat. Vitamin 

FC-2 



A deficiency has been used to achieve reversible germ cell depletion. The 
FSH did not rise again implicating the Sertoli cell as the source of FSH 
regulatory control. A study of enzyme markers in the testis suggests that 
Y-glutamyl-transpeptidase may be a marker for the mature Sertoli cell. 

Research utilizing the primate colony has been carried out by collaborations 
among many members of the Branch. Pregnancy in the rhesus monkey is not 
characterized by continued excretion of chorionic gonadotropins as in man. 
A radioimmunoassay for monkey CG was developed and is useful in purification 
and clinical studies. The antigenicity of gonadotropins of primates was 
examined. Antisera to hCGB recognized common antigenic determinants in man, 
gorilla, and chimpanzee but not in the rhesus monkey. Electron microscopy 
of the rhesus corpus luteum throughout pregnancy was performed and the 
morphologic features are consistent with continuing steroidogenesis. Ovari- 
ectomy in the rhesus after day 24 caused no abnormalities in sex steroid 
concentrations in the blood and peak estradiol levels were attained at day 
35 as in the normal pregnant monkey. Thus, as in man, corpus luteum function 
during pregnancy is essential only during the early weeks of pregnancy. 

The Branch will again undergo change. Dr. W. Tullner is retiring after 
many years of productive research and leadership. Dr. Gary Hodgen will be 
Acting Head, Endocrinology Section. Dr. Peter 0. Kohler is moving to Baylor 
University. His contributions in the laboratory and ward will be difficult 
to replace. Dr. D. L. Loriaux will serve as Acting Head, Clinical Service. 
Dr. Kevin Catt will become Head of a new section. Hormonal Regulation, and 
expand his efforts in several aspects of regulatory mechanisms. The Section 
of Molecular Structure, headed by Dr. E. Gross has joined the Branch. 

Our labors this year have again been aided greatly by visiting scientists, 
fellows, and guest workers. These scientists and our Clinical Associates 
have been a continued source of stimulation. The collaborative efforts 
within the Branch and with other scientists in Bethesda and elsewhere have 
strengthened the research and multiplied our effectiveness. Finally, the 
support of the Institute for the use of service contracts has enabled us to 
shift some of the burden of routine work and has thereby freed our staff for 
more creative research. 



PC- 3 



Serial No. HD-RPLl 

1. Reproduction Research Branch 

2. Bethesda, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 



Project Title: Biology of Reproduction: Clinical and Experimental 
Principal Investigator: Dr. Mortimer B. Lipsett 



Other Investigators: 



Dr. G. T. Ross, Dr. W. W. Tullner, Dr. P. 0. Kohler, 
Dr. R. J. Sherins, Dr. D. Rodbard, Dr. A. Chrambach, 
Dr. H. Pollard, Dr. K. Catt, Dr. M. D. Catt, Dr. B. 
Gulyas, Dr. W. Nixon, Dr. G. Hodgen, Dr. D. Loriaux, 
Dr. C. Cargille, Dr. E. Gross, Dr. H. Chen, Dr. R. 
Becker, Dr. P. Krueger, Dr. B. Nisula, Dr. R. Mecklen- 
burg, Dr. L. Corash, Dr. W. Gerber, Dr. P. Fredlund, 
Dr. J. Hansen, Dr. R. Vigersky, Dr. S. M. Harman, 
Mr. P. E. Rayford, Mr. D. Ryan, Mr. G. Coffman, Mrs. M. 
Wyckoff, Mr. C. Turner, Mr. D. Barber, Mr. R. Reid, 
Mr. J. Brice, Mrs. M. Collins, Miss J. Brown, Mr. J. 
Morel 1. 



Cooperating Units: 



Veterans Administration Hospital, Palo Alto; Department 
of Applied Mathematics, Harvard University, Boston; De- 
partment of Anatomy, Harvard University, Boston; Univ. 
of Paris School of Medicine, Paris; Max Planck Inst., 
Gottingen; Columbia Univ. School of Medicine, New York; 
Georgetown Univ. School of Medicine, Washington, D.C. ; 
Emory Univ. School of Medicine, Atlanta; M. D, Anderson 
Inst., Houston; Physical Sciences Laboratory, DCRT; 
Laboratory of Biochemistry and Metabolism, NIAMD; Sec- 
tion of Mineral Metabolism, NIAMD: Laboratory of Experi- 
mental Pathology, NIAMD; Laboratory of Cell Biology, NCI; 
Laboratory of Biomedical Sciences, NICHD; Department of 
Obstetrics & Gynecology, National Naval Medical Center, 
Bethesda; Laboratory of Clinical Science, NIMH; Mass. 
Institute of Technology, Cambridge. 



Man Years: 



Total: 62 
Professional: 
Other: 24 



38 



■ FC-4 



Project Description: 
Objectives : 

1. To understand molecular mechanisms of hormone action; 

2. To define pituitary and gonadal secretion in health and disease; 

3. To explore the processes involved in maintenance of blood and tissue 
hormone levels; 

4. To develop methods for studying the interaction of the seminiferous 
tubule and the pituitary hypothalamic unit; 

5. To describe functionally the biology of germ cell maturation in the 
gonads. 

Methods Employed : 

A great variety of techniques are employed since the research encompasses such 
widely divergent areas as mathematical biology, protein hormone chemistry and 
physiology, tissue culture, steroid biochemistry, morphology, and clinical 
investigation. New methods that have been either developed or adapted during 
the year are: 

A new more efficient solid-phase method for peptide amide synthesis; 

The use of fluorescent techniques to identify lysosomes in tissue. This 
facilitates the preparation of lysosomes; 

New chemical non-enzymatic degradative techniques for structural eluci- 
dation of membrane-active peptides with unusual structural features; 

The use of isoelectric focusing on sucrose columns as a preparative 
technique for protein purification; 

Adaptation and validation of a method for rapid extraction of gonado- 
tropins from urine for quantitation by immunoassay; 

Development of a simple fast radioligand assay for steroid hormones; 

An improved method for radioimmunoassay of Angiotensin II; 

The calculation of affinity constants and binding capacity with routine 
testing of four alternative models. 

Major Findings : 

I. Protein Chemistry 

Since amniotic fluid is a good source of prolactin, it was necessary to 
show that this prolactin was the same as pituitary and serum prolactin. This 
identity was proved by physico-chemical criteria, biologic assay, and immuno- 
logic cross-reactivity. The high isoelectric point of prolactin suggested 
that efficient separation from isohormones of growth hormone would be achieved 



FC-5 



by isoelectric focusing. Small amounts of homogeneous prolactin were prepared 
by gel filtration, isoelectric focusing and gel electrophoresis. From this 
experience, the procedures are now being adapted for preparative work. 

Two years ago it was reported that plasmln digestion of growth honnone yielded 
a growth hormone with a ten-fold increase in specific activity. Preparative 
methods have now been devised for isolation of milligram amounts of this sub- 
stance. 

The methodology of separating any charged species from any other under optimal 
conditions and with "quantitative" physical characterization of the species 
nas been published, bringing to completion a project of 10 years. The papers 
cover every major aspect of fractionation on polyacrylamide gel. The compre- 
hensive theory and discussion of the various aspects of fractionation on 
polyacrylamide contributes a strategy to fractionation that is at the same 
time universal and flexible. At each step of this strategy, we have provided 
apparatus, procedures and computer programs for the rapid and error-free 
evaluation of the data and their statistical limits. The programs take the 
measured electrophoretic mobility, or relative mobility, of a band and (1) 
convert it to measures of molecular size and net charge with their confidence 
limits; (2) calculate the gel concentration for optimal resolution between 
any set of 2 components; (3) define identity or non-identity between any 2 
components; (4) simulate the time course of preparative elution fractionation 
on PAGE and indicate the time, for any gel volume, required for the quantita- 
tive separation of any component from any other. This set of computer pro- 
grams has been made available to the public via commercial time sharing and 
by mailing out of tapes. 

Another program's package, developed by use of T.M. Jovin's theory and pro- 
gram for the analysis and generation of multiphasic buffer systems, extends 
the applicability of gel electrophoresis and "stacking" to the entire pH 
range. 

Apparatus and procedures have been developed for analytical PAGE and IFPA, 
transverse and longitudinal gel sectioning, autoradiography of gel slices, 
formation of pore gradient gels, preparative PAGE and IFPA, automatic elution 
flow rate deceleration, eluate concentration, eluent reservoirs of adjustable 
level, determination of polymerization efficiency, determination of boundary 
displacement, gel slab electrophoresis in discontinuous buffer systems, and 
numerous other minor items. We are in the process of developing a procedure 
for preparation and purification of iodinated proteins which will combine in 
one purification of the iodinated protein. This should also provide vastly 
improved radiation safety compared with present techniques. 

The importance of this body of theory and experience was recognized at the 
NIH by a request for a special course for NIH personnel. Drs. Chrambach and 
Rodbard taught a two-week course to 20 NIH sicentists. 

New methods for calculating affinity constants ("K") and binding capacities, 
("q") have been developed. In particular, the programs now routinely test 
four alternative models and then select the model with the minimum number of 



parameters required to give a satisfactory fit. Computations which formerly 
required a full day's work with a complicated "custom-fit" program are now 
done routinely in a few minutes. 

The vast majority of "K" and "q" values in the literature are calculated in- 
correctly (no weighting, correlation of errors in X and Y, incorrect models, 
improper "curve peeling"). A major attempt to address this problem was the 
writing of several review articles and chapters on this subject. The theory 
of protein-ligand binding will be useful in a variety of studies concerned 
with the concentration of "free" (unbound) steroids in plasma. 

A mathematical theory to describe the transient state of isoelectric focusing 
(pH gradient electrophoresis) was derived. This should be useful in connec- 
tion with experimental studies, especially those dealing with determination 
of physical-chemical constants (e.g., diffusion coefficients) from PAGE and 
isoelectric focusing. 

In order to prepare labeled LHRH for clinical studies, iodination was per- 
formed using the lacto e oxidase method. Under appropriate conditions, 
labeled LHRH was separated from unlabeled material, thus yielding a tracer of 
maximum specific activity. It was shown to be monoiodinated and to retain 
full biologic activity. 

Proposed course of project: The preparation of sufficient amounts of pure 
prolactin and growth hormone isohormones for structural studies will be under- 
taken. 

II. Mechanism of Honnone Action 

The mathematical theory for hormone-receptor and drug-receptor interaction, 
as developed by the pharmacologists, has been reviewed, revised, and extended 
to apply to current problems in mechanism of hormone action. This work is 
based on, and represents an extension of previous studies from this laboratory 
on protein-ligand interactions, antigen-antibody reactions, and the theory of 
radioimmunoassays . 

The paradoxical dissociation of "binding" curves and "response" curves in 
in vitro systems may be due to allosteric effects, heterogeneity of binding 
sites, or a quantal mechanism whereby individual cells in the target tissue 
respond in a quantal fashion when a threshold number of receptor sites are 
filled. Current studies are aimed at distinguishing among these possibilities, 
and involve both experimental design, computer simulation studies, and data 
analysis. 

A method was developed for treating membranes of bovine chromaffin granules in 
such a manner as to orient them on a planar surface. This allowed X-ray dif- 
fraction studies to be performed and structural information to be obtained. 
The specific result was that the membrane is chemically asymmetric and that 
objects of approximately 70 A in diameter are distributed in the plane of the 
r.ii-mbrnne In a symmetric fashion. The symmetry may be, in part, hexagonal. 

Electron microscopy of the membranes revealed that the product was vesicular, 

FC-7 



as was the original organelle. This was confirmed by both thin section and 
negative stain methods. Freeze-fracture electron microscopy confirmed: (1) 
existence of submembrane particles, approximately 70A in diameter; (2) asym- 
metric distribution of the particle with regard to membrane surfaces; and 
(3) ordered arrays of particles in the membrane plane, showing 5-fold axes 
and surrounded by 6-fold axes. 

Protein chemistry studies on these membranes revealed that there were at least 
8 major species by disc gel electrophoresis in SDS. All had molecular weights 
of less than 65,000. A novel system for doing discontinuous buffer electro- 
phoresis of membrane protein in SDS at neutral pH was developed. This system 
has been used to define the molecular parameters of the membrane protein more 
precisely and to make a tentative identification of dopamine-6-hydroxylase, 
relative to a sample of the purified enzyme. The membrane was also found to 
be up to 20% cytochrome br£2 and cytochrome b5g2 reductase, suggesting that 
electron transport is an important membrane function. 

Quantitative lipid analysis has corroborated our measurements of hydrated 
density from sucrose gradients (approximately 1.12 gcm~-^) and yielded an 
estimate that the membrane is 40-A5% protein. Thin-layer chromatography 
has revealed the presence of common phospholipids, but lysolecithin is present 
in only small quantity. Previously it had been reported that the membrane 
was unusual in that up to 20% of the phospholipids were lysolecithin. 

Release of cyclic AMP and testosterone by the incubated rat testis has been 
determined during further studies on gonadotropin action in vitro. Stimula- 
tion of steroidogenesis by low gonadotropin concentrations (<10~ M hCG) has 
been shown to occur without an accompanying rise in cyclic AMP production, 
suggesting that cyclic AMP may not be an essential intermediate in the mode 
of action of LH on the Leydig cell. This observation has also been made in 
isolated Leydig cells prepared by collagenase dispersion of the rat testis. 
Quantitative binding studies of dispersed interstitial cells have shown that 
the number of LH/hCG receptors per cell is about 50,000. 

A requirement for calcium in gonadotropin action has also been demonstrated 
in the rat testis. Removal of Ca+ by EGTA abolishes the release of cyclic 
AMP and testosterone in response to hCG, and also blocks the testosterone 
response to dibutyryl cyclic AMP. Binding of gonadotropin is not affected 
by such changes in calcium concentration, indicating that the requirement for 
calcium is at the level of receptor coupling to adenylate cyclase and also at 
later stages of steroidogenesis. 

Gonadotropin receptors for LH and hCG were extracted from cell fractions of 
the testis and ovary with the non- ionic detergent, Triton X-100. The solu- 
bilized receptors remained in solution after centrifugation at 360,000 g for 
2 hrs and comprised more than 90% of the original particulate binding sites. 

125 
For binding studies of the soluble receptors with I-labeled hCG, separa- 
tion of bound and free tracer was accomplished by precipitation of the bound 
complex with polyethylene glycol. This assay system has been extensively 
used to carry out binding and kinetic studies, as well as for monitoring 



receptor activity during physico-chemical studies. The binding was tempera- 
ture dependent, with higher rate of uptake at 37°C but lower binding capacity 
due to receptor degradation at higher temperature. The soluble receptor re- 
tained hormonal specificity for LH and hCG and displayed high affinity for 
hCG at 0° and 24° (Ka = 0.5-1 x 10 M"!) and low capacity lO'^^mole/gm. The 
association rate constant was 6.1 x 10 M min at 4°C. The dissociation 
occurred extremely slowly with a first order dissociation rate constant of 
1.2 X 10~^min . Comparison between testicular and ovarian binding data 
showed striking similarities. Combining data obtained from gel filtration 
studies on Sepharose 6B columns and from sucrose gradient centrifugation on 
the free receptor and receptor-hormone complex the size and shape of the 
molecules were calculated: 

Hydrodynamic radius 64A (for both forms) 

Sedimentation constant 

free receptor 6.5S 

hormone-receptor complex 7.5S 

Molecular weight j* 

it 
free receptor 194,000 " 

hormone receptor complex 224,000 J 



The axial ratios (prolate) of the two forms were 12 and 10.2 respectively. P 

The properties of the hCG/LH gonadotropin receptors were consistent with those T 

of a highly asymmetric molecule predominantly protein in nature, with relatively ; 
high conformational stability. 

Specific receptor sites for angiotensin II were demonstrated in the bovine 
adrenal cortex by binding studies with -'H- and I-angiotensin II. The re- , 
ceptor affinity for angiotensin II is higher than that for angiot«.ii3in I, and 
the binding sites in adrenal homogenates are situated in a microsomal mem- 
brane fraction with enzymic characteristics of cell membrane. Fragments 
analogues, and antagonists of angiotensin II have binding- inhibition potencies 
which are proportional to their biological activities in vivo. 

Proposed course of project - It is planned to develop isomorphous derivatives 
of the membrane in which electron or X-ray dense probes are attached to speci- 
fic enzymes in the membrane in order to locate them geometrically. These in- 
clude a ferritin-anti-dopamine-6-hydroxylase reagent suitable for both X-ray 
and electron microscopic analysis and a ferritin-SH reagent that might allow 
us to localize the -SH group on the active transport ATPase. These studies 
would tie in with functional studies on the chemistry of the transport process 
for catecholamines, and on the mechanism of release of the neurotransmitter 
from the cell. 

Purification of gonadotropin receptors will be continued to obtain enough pro- 
tein for structure-function studies. Stimulus-secretion coupling in testis 
and zona glomerulosa of the adrenal cortex will be a continuing focus of work. 
Angiotensin II receptors will be characterized and studied. 



FC-9 



III. Cell Biology 

The immunological studies with trophoblast have been extended. Previous 
work had indicated that lymphocytes from a variety of donors (normal people, 
pregnant women, women with moles or choriocarcinoma) did not recognize the 
JEG lines of choriocarcinoma in culture as foreign. This had suggested that 
histocompatibility antigens were masked or absent. However, another cultured 
cell line of choriocarcinoma cells, recently isolated in our laboratory, is 
not recognized as foreign by lymphocytes from some normal people. This sug- 
gests that the reason the placenta is not rejected during normal pregnancy 
and the reason choriocarcinoma spreads so vigorously is not an inability of 
the cell to express surface antigens. 

Of some interest are the findings that administration of antisera to hCG to 
hamsters bearing human choriocarcinoma results in significant reduction in 
tumor growth as well as inhibition of the peripheral manifestation of the 
tumor's hormonal activity. 

A clone of ductus deferens tumor cells was established. These cells show a 
specific uptake of testosterone and estradiol. The specific binding material 
has been isolated from cytosol and is presently being characterized. Testos- 
terone appears to increase the growth rate of these cells. This cell line 
will provide an jLn vitro model for sex steroid action. Since estradiol stimu- 
lates the growth of the granulosa cell, the steroid binding properties of 
porcine granulosa cells in culture were examined. These cells specifically 
bind estrone and estradiol, and the binding characteristics differ from those 
of uterine cytosol receptor. However, the binding material appears to be very 
fragile and a satisfactory preparation from cytosol is not yet available. 

Proposed course of project - Since the senior investigator is leaving, it seems 
likely that most of this work will move with him. 

IV. Gonadotropins 

In a continuing collaboration with Dr. R. Canfield and workers in New York, 
immunologic and biologic assays have been used to characterize preparations 
of human chorionic gonadotropin during purification and production of sub- 
units of human chorionic gonadotropin. Methods have been developed for large 
scale production of a highly purified reagent for use in structure-function 
studies. In addition to studies conducted jointly between the two labora- 
tories, these substances have been made available to a number of other investi- 
gators around the world. Results of recently completed studies justify the 
following conclusions: (1) Highly purified alpha and beta subunits have been 
shown to be free of biologic activity ±n vivo and Jji vitro; (2) Except for 
differences in carbohydrate content, the alpha subunits of hCG and hLH have 
identical, primary structures; (3) A structural basis has been established for 
antigenic differences in hLH and hCG. The hCG beta subunit contains about 30 
additional aminoacids added onto the C terminal subunit of beta unit of hLH. 
This terminal portion of hCG beta subunit has been isolated and studies of 
its antigenic behavior are in progress; (4) In collaborative studies with 
Dr. Harold Edelboch, NIAMD, it has been shown that subunits of hCG do not bind 



ANS, whereas native hormone does. The extent of ANS binding, and thus of re- 
combination, can be monitored by changes in fluorescence of aqueous solutions 
containing the dye and the subunits so that the kinetics of the recombination 
can be monitored. Preliminary studies indicate that this method is useful 
for following the course of reactions during hybridization of complementary 
subunits of glycoprotein hormones from different species. 

Placental tissue was obtained from elective abortions performed at several 
different hospitals in the area. The tissue was extracted and gel filtered. 
The eluates were radioimmunoassayed for hCG, hCGa and hCGB. In the first 
6-10 weeks of gestation, three different physical forms of hCGa and two dif- 
ferent physical forms of hCGB were evident. The additional forms of hCG sub- 
units found had a higher apparent molecular weight than did highly purified 
native subunits. These previously unrecognized forms of subunits could repre- 
sent intracellular subunits released before final processing. After the 10th 
week of gestation, the amount of hCGa exceeded hCG by several fold. Since the 
subunit is devoid of biologic activity, its presence could account for the 
lower biologic to immunologic activity ratio noted after the first trimester 
of pregnancy. 

Plasma, urine and tumor tissue extracts were studied for altered forms of 
hCG and its subunits. All subjects studied with nongestational hCG secreting 
tumors were found to have unbalanced synthesis, secretion or excretion of 
hCG and its subunits. Interestingly, hCG subunits were not detectable in 
plasma or urine of patients with molar pregnancies. 

100 ml of an hCG3 antiserum was given to the National Pituitary Agency for 
distribution to interested, qualified investigators. The antisera can be 
used for early diagnosis of pregnancy, following therapeutic course of 
patients with hCG secreting tumors and as a screening assay tumor marker 
since several different neoplasms have been shown to ectopically secrete hCG. 

The structure-function inter-relationships of the gonadotropins and various 
human thyroid stimulators were investigated. Results of these studies sup- 
port the current concept of an alpha subunit structure of the gonadotropins 
similar to that of the pituitary thyrotropin but dissimilar to that of the 
thyroid stimulating activity extracted from placenta. In contrast to the 
thyroid stimulator extracted from placenta, the thyroid stimulator which 
circulates during pregnancy is similar in structure to hCG. Immunologic and 
biologic evidence suggests that a thyroid stimulator distinct from hTSH and 
LATS circulates in the blood of some men with testicular tumors. This poten- 
tial tumor marker may be similar to the thyroid stimulator of pregnancy. 

Proposed Course of Project - Collaborative studies with the laboratories of 
Darrell Ward at Houston and Robert Canfield at Columbia are projected for 
next year. It is proposed to examine the antigenic and biologic properties 
of hybrids of fragments such as tryptic peptides of alpha subunits with beta 
subunits of hCG and ovine LH in attempts to define minimal amino acid sequences 
required for biologic activities. 

The "big" subunits found in placental tissue will be characterized and studied 



FC-11 



to determine whether hCG is synthesized initially in a "big" form as two other 
polypeptide hormones appear to be. 

V. Clinical Studies 

Applications of immunologic and biologic assays for gonadotropins to the study 
of physiology of human reproduction have continued. Drs. Reiter and Kulin 
have shown that iramunoreactive gonadotropins can be satisfactorily recovered 
from human urine by precipitation with acetone. This method represents a 
significant advance over the kaolin acetone method presently used for extract- 
ing biologically active gonadotropins. The acetone method is currently being 
used to compare patterns of urinary gonadotropin excretion in first morning 
voidings and 24-hour urine collections from normal women and from prepubertal 
boys and girls. 

Approximately 40 new patients with menstrual aberrations were studied over the 
past year. Testing with LH-RH was added to our protocol to better evaluate 
pituitary-hypothalamic function in these patients. A new sub-group was ident- 
ified using this approach. A significant population of patients with secondary 
amenorrhea presented with a history of voluntary weight loss accompanying 
cessation of spontaneous menses. None of this group of patients had a clini- 
cal diagnosis of anorexia nervosa. As a group, they (1) had low to barely 
detectable urinary and plasma gonadotropins; (2) failed to respond to clomi- 
phene in terms of a rise of plasma gonadotropin levels, and (3) all responded 
to LRF with a significant rise of plasma FSH and LH implying that hypothalamic 
dysfunction was the basis for their clinical syndrome. 

In women not responding to clomiphene and who did not have primary ovarian 
disease, ovulation was induced with sequential parenteral menopausal gonado- 
tropin and hCG. This form of therapy was carried out only in those women 
interested in conception. Plasma and urine were obtained at monthly intervals 
from those women who conceived. The plasma and urine were subjected to Sepha- 
dex G-lOO chromatography and each eluate assayed for hCG, hCGa and hCGB in 
homologous radioimmunoassays. In short, free hCGa was found in plasma through- 
out pregnancy. 

The sensitivity of a heterologous system for measurement of human prolactin 
has been improved by the use of -'■'^-'l labeled human prolactin. The new assay 
is precise and sufficiently sensitive for measurement of basal levels of the 
hormone in serum fro^a normal men and women. This assay has been coupled with 
pharmacological methods for perturbing pituitary prolactin secretion in 
diagnostic evaluation of patients suspected of having functional disorders of 
the hypothalamic-pituitary unit. It has been shown that serum prolactin con- 
centrations are high in most patients having pituitary tumors with or without 
associated galactorrhea. In addition to these clinical studies, this assay 
was used to establish the similarity of physical properties of prolactin in 
human amniotic fluid and pituitary extracts. 

The extensive use of radioimmunoassays in clinical investigation has required 
that data processing be handled efficiently. A new, improved computer program 
for RIA data processing has been developed and is being distributed by the 



FC-12 



National Technical Information Service (magnetic tapes and listings). More 
than 200 copies of the program have been distributed. Earlier versions of 
the program have been adapted to virtually all desk-top calculators and mini- 
computers, and many manufacturers and distributors of RIA reagents have 
adopted the "logit-log" method, so that it is probably the must widely used 
of any method developed to date. 

The new program provides: 

(1) Estimation of K values even for non-linear Scatchard plot; 

(2) Testing of linearity, testing of parallelism, and combined potency 
estimates; 

(3) An optimization program, advising on changes needed to make sub- 
sequent assays more sensitive; 

(4) New input-output options; and 

(5) New detailed statistical analyses, providing more efficient utili- 
zation of data. 

Assays for plasma renin activity (PRA) , renin concentration (PRC) and renin 
substrate (PRS) , employing radioimmunoassay of Angiotensin I, have been 
established and validated, and an improved method for radioimmunoassay of 
circulating angiotensin II has been developed. These assays are currently 
being applied to studies in various forms of hypertension, and during oral 
contraceptive therapy and pregnancy. The most significant result of the 
clinical studies has been the demonstration that "low renin" hypertension 
does not protect against heart attacks and strokes as claimed by Laragh. In 
a large series of hypertension patients (studied in collaboration with the 
Hypertension Unit of the D. C. General Hospital), those with low renin hyper- 
tension were found to have the same incidence of complications as those with 
normal or high renin levels. This finding has important implications for the 
management and prognosis of patients with 'low renin' essential hypertension. 

The male infertility clinic is providing a major clinical resource for the 
Washington area. The significance of a low sperm count is being defined and 
men with consistent oligospermia (total sperm count, 25 million) are able to 
impregnate their wives. Extensive study has revealed the large variability 
of the sperm count and emphasizes the necessity for long control periods in 
order to assess attempts at therapy. 

Plasma FSH levels are elevated in azoospermia and, in general, are inversely 
correlated with the sperm count. Administration of hCG suppresses FSH to 
very low levels yet spermatogenesis is unchanged. This suggests, in accoE^ 
with data in other species, that FSH may not be required for maintenance of 
spermatogenesis . 

During attempts at therapy with hCG, many men developed gynecomastia. Since 
plasma prolactin did not increase and estradiol concentration doubled, it is 
now appropriate to attribute this gynecomastia to the effect of estrogen. 

To facilitate measurement of steroid hormones in clinical studies, a technique 
of co-polymerization of antisera with polyacrylamide has been developed which 
renders the separation of bound from free, a time independent process, and 



FC-13 



thereby simplifies the method. The technique has adequate sensitivity to 

measure 4 picograms of estradiol in an unchromatographed extract of human 

plasma. It is currently being used in the routine measurement of plasma and 

urinary Cortisol and estradiol. 

A method has been developed for the measurement of free estradiol in human 
urine. Urinary free estradiol correlates directly with plasma estradiol 
through the menstrual cycle. To complement these data, the plasma binding 
of estradiol at 37° was examined and the association constants of estradiol 
with TeBG and albumin were measured. From these data and estimation of TeBG 
binding capacity, we estimate that most of the estradiol filterd at the 
glomerulus is not reabsorbed. 

Because of obvious abnormalities of gonadotropin secretion in women with 
anorexia nervosa, hypothalamic function in these patients was studied. 
Patients with anorexia nervosa were unable to maintain core body temperature 
in a cold environment. Two people, equally cachectic but without anorexia 
nervosa, could maintain body temperature. The defect persisted in one patient 
who had recovered from anorexia nervosa. Patients with anorexia nervosa had 
abnormal responses to a hot environment. Four of five patients had partial 
diabetes insipidus. Response to LHRH was normal in all but one patient. This 
patient was taking estrogen and thyroxine, both of which are said to block 
LHRH activity. Other parameters of anterior pituitary function were normal. 
These novel findings are interpreted as indicating disordered hypothalamic 
function in anorexia nervosa. 

Moniodinated LHRH was infused into normal volunteers. The initial ti /2 was 
estimated at between 3 and 4 min. The distribution volume approximated plasma 
volume. Similar data were obtained in the rat. 

Projected Course of Clinical Studies - The clinical study of men and women 
with problems of infertility will be continued since this population of 
patients is available for analysis of particular endocrine abnormalities as 
well as for new therapeutic interventions. The mathematical programs for 
radioimmunoassay will be improved by use of a four parameter logistic model 
that will provide improved curve fitting and a new "generation" of RIA pro- 
grams. Hypothalamic disease will be explored extensively by testing with LH- 
RH and thyrotropln-releasing hormone. 

Proposed Course for the Branch 

The general area of hormone action will be expanded with the creation of a 
section headed by Dr. K. J. Catt. Progress achieved in purification of the 
hCG receptor has been rapid and the stability of this receptor augurs well 
for future work. The development of techniques for studying protein structure 
of membranes will permit new meaningful exploration of membrane response to 
polypeptide hormones. 

The need for more biology in the Branch remains. The departure of Dr. P. 0. 
Kohler removes one of our productive biologists. Studies of follicular devel- 
opment and fertilization should constitue an important field of research in 
the Branch. When positions become available, it is planned to develop such 
a Section. 

FC-14 



Serial No. HD-RPL2 

1. Reproduction Research Branch 

2. Section of Endocrinology 

3. Bethesda, Maryland 



PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 



Project Title: Physiology of Testis 

Principal Investigator: Dr. William W. Tullner 

Other Investigators: Dr. Gary D. Hodgen and Dr. Philip M. Krueger 

Man Years: 

Total: 3 
Professional: 3 
Other: 

Project Description: 

Objectives : 

1. To develop methods for measuring function of specific tubular cells; 

2. To understand the regulation of FSH secretion. 

Major Findings : 

Having characterized the pattern of three enzymic markers during the ini- 
tiation of spermatogenesis in rats, we used these testis specific markers to 
monitor selective germinal cell depletion in vitamin A deficient rats. In 
130 day old vitamin A deficient rats, lactic dehydrogenase (LDH) was 62% of 
controls and LDH-X isozymes were undetectable, indicating the absence of 
pachytene spermatocytes, spermatids, and spermatozoa. Similarly, loss of 
these advanced germinal cells was marked by reduced sorbitol dehydrogenase 
levels to 19% of controls. These findings were confirmed histologically. 
Despite the virtual absence of germinal cells, y-glutamyl-transpeptidase (GTP) 
remained near control levels and Sertoli cells were histologically normal. 
These findings suggest that the vitamin A deficient rat is a useful model for 
accomplishing selective depletion of the germinal epithelium and supports 
earlier data suggesting that GTP may be an enzyme marker for Sertoli cells. 

Serum FSH, LH and testosterone levels in adult rats were measured after 
artificially induced unilateral and bilateral cryptorchidism and unilateral 
and bilateral castration. The data do not support the view that cryptorchidism 
is an acceptable model system to test the suppressive effects of an FSH 
inhibitory substance, said to be released from the seminiferous tubule, since 
bilateral cryptorchidism resulted in elevations of both FSH and LH. Further, 



FC-15 



neither unilateral nor bilateral cryptorchidism had any significant influence 
on testosterone levels. Removal of the epididymis did not affect LH and FSH 
concentrations . 

A vitamin A deficient colony of male rats has been established because 
it provides a model to study the reversible depletion of testicular germ 
cells. Earlier studies in humans demonstrated a selective increase in FSH 
concentration when germinal aplasia occurred as a consequence of chemo- 
therapy; which suggested that the seminiferous tubule produced a factor 
capable of inhibiting FSH secretion. In this rodent model, depletion of germ 
cells occurs down to the spermatogonial level, and yet no changes in plasma 
FSH have been demonstrated. This strongly suggests that release of the "FSH 
inhibitor" from the tubule likely occurs at the Sertoli cell and/or low 
spermatogonial site. This animal model would appear to be extremely valuable 
for further understanding of the hormonal regulation of spermatogenesis. 

Proposed Course of the Project : 

Emphasis will be placed in two areas. The exploitation of the vitamin A 
deficient rat system will be continued to identify the cell involved in FSH 
regulation. A model system will be devised for use in attempts to isolate 
tubular FSH-regulating factor. 



FC-16 



Serial No. HD-RPL3 

1. Reproduction Research Branch 

2. Section of Endocrinology 

3. Bethesda, Maryland 
PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Fertilization and Embryogenesis 

Principal Investigator: Dr. William W. Tullner 

Other Investigators: Dr. Bela J. Gulyas and Dr. Donald W. Slater 

Man Years : 

Total: 3 
Professional: 3 
Other: 

Project Description: 

Objectives ; 

1. To describe the ultrastructural features of early embryogenesis; 

2. To define the processes involved in processing and controlling of 
information at the molecular level during early embryogenesis. 

Major Findings : 

Earlier observations on the contractile ring and the mid-body in 
dividing rabbit zygotes were further pursued. In late anaphase of karyo- 
kinesis a shallow indentation appears in the vitelline membrane in a plane 
perpendicular to the spindle. Microfilaments form the contractile ring in 
the shallow indentation. Throughout furrow formation these 50 to 80 A 
filaments underlie the broad leading edge of the cleavage furrow. Also in 
late anaphase stem-bodies can be identified centrally in the egg. Each stem- 
body consists of several microtubules in an electron dense matrix. As 
division progresses the stem-bodies fuse into a mid-body which at several 
points fuses with the contractile ring. Primitive intermediate junctions 
develop between the resulting two blastomeres. 

During the course of examining more than 500 rabbit zygotes, which were 
obtained from normally mated females, approximately 1.1% of them were found 
to be spontaneously polyspermic. The presence of supernumerary (polyspermic) 
sperm with varying degrees of transformation into pronuclei, in zygotes of 
similar age, suggests that entry of the supernumerary sperm can occur at 
various times after fertilization. The early stages of transformation into 
pronucleus slightly diverge from that of the normal fertilization. Unlike 
normal fertilization, the nuclear envelope is formed around the condensed 



FC-17 



supernumerary spena head. Decondensation of the chromatin occurs in the 
confines of the nuclear envelope, rather than free in the cytoplasm. In 
nearly all of the polyspermlc zygotes, the male and female pronuclei were 
in one hemisphere of the zygote and the superniimerary male nucleus was 
located peripherally in the opposite hemisphere. The dislocation of the 
pronuclei is an abnormal feature since in the normal zygotes the male and 
female pronuclei migrate to the center of the zygote and remain there until 
first cleavage. 

Unfertilized sea urchin eggs and embryos have been shown to contain two 
classes of DNA polymerases functionally distinguishable via their potential 
to employ synthetic DNA.RNA hybrids as well as RNA.RNA duplexes as templates 
for DNA synthesis. This RNA-mediated DNA synthesis is dependent on the addi- 
tion of the pertinent polymerases, templates and cof actors and results in an 
asymmetrically transcribed product the molecular makeup of which is dictated 
by the RNA moiety of the template employed. Although partially purified 
polymerase preparations obtained from either unfertilized eggs or embryos 
contain a similar spectrum of DNA- and RNA- instructed potential polymerases, 
the divalent cation requirements and thermal labilities of the RNA-instructed 
holopolymerases are not identical. Examination of the respective polymerase 
saturation levels and competition experiments between appropriate templates 
further suggests that both the egg and embryo possess polymerases with 
differing template affinities. 

Molecular hybridization between H-poly(U) and unlabeled RNA from un- 
fertilized eggs and embryos has been employed to demonstrate that the incep- 
tion of embryogenesis is accompanied by a greater than two-fold net synthesis 
of polyadenylic acid [poly (A)], a species of RNA known to be a proprietary 
property of metazoan genetic messages. This synthesis was shown to be immune 
to any tandem transcriptive requirement but significantly stimulated by the 
inhibition of protein synthesis. Our findings indicate that the controlled 
adenylation of pre-existing genetic messages present in the ovum is modulated 
by a post-fertilization translational event other than the synthesis of the 
pertinent polymerase (s) per se . These results thereby represent the first 
documentation of a fertilization elicited net synthesis of an RNA species 
and, equally important, suggest that such synthesis is not only under negative 
genetic control, but that the RNA moiety in question is of a regulatory rather 
than codogenic nature. 

As the vigorous increment in protein synthesis known to follow fertiliza- 
tion precedes the above-noted net synthesis of poly (A) by approximately 60 
minutes, the biology of the system, coupled with the procedures employed, 
permitted a direct comparison of the disposition and characteristics of the 
preadenylated maternal RNA with those genetic messages adenylated in response 
to fertilization. The results obtained establish that preadenylated latent 
genetic messages of maternal origin are predominantly located in the ovum's 
subribosomal fraction and that fertilization elicits a rapid transposition of 
these transcripts into the zygote's ribosomal fraction. Thus the observed 
reallocation of adenylated RNA parallels the increase in protein synthesis 
which occurs after fertilization and thereby supports the interpretation that 
the adenylated RNA present in the unfertilized egg primarily represents 
anabolic intermediates rather than catabolic products of oogenic metabolism. 

FC-18 



Proposed Course of the Project : 

The molecular biology component will be transferred to the Gerontology 
Research Center. At the ultrastructural level, the mode of formation of the 
two types of nucleoli in rabbit embryos will be examined. Further studies on 
the genesis of the annulate lamellae in rabbit zygotes are planned. 



FC-19 



Serial No. HD-RPU 

1. Reproduction Research Branch 

2. Section of Endocrinology 

3. Bethesda, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: The Use of the Rhesus Monkey in Studies of Pregnancy 

Principal Investigator: Dr. William W. Tullner 

Other Investigators: Dr. Gary D. Hodgen and Dr. Wilbert E. Nixon 

Man Years: 

Total: 3 
Professional: 3 
Other: 

Project Description: 

Objectives : 

1. To develop methods for measurement of pituitary and placental hormones; 

2. To evaluate the function of the corpus luteum; 

3. To describe and define the sources of steroid hormones during pregnancy. 

Major Findings ; 

A radioimmunoassay system has been developed which is capable of measuring 
rhCG in biological materials and extracts of these materials. The RIA system 
utilizes a rat luteinizing hormone (rLH) tracer, an antiserum against human 
chorionic gonadotropin (hCG) and a house reference standard (HRP) obtained 
from the urine of a pregnant monkey. Urine extracts from pregnant animals 
dose out parallel to the standard, and sera from pregnant animals also can be 
assayed in this system. No response is obtained with urine or serum from 
normal non-pregnant animals or from castrate animals. The assay is 5 times 
more sensitive than present bioassays. In physiological studies, this system 
yields results comparable to those found by bioassay. However, the 
biological/immunological ratio approaches two. 

Previous efforts to purify rhCG from urinary kaolin-acetone extracts 
utilizing polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing 
in polyacrylamide (IFPA) have been replaced by gel permeation chromatography 
and by ion-exchange chromatography on sephadex polymers. Estimation of 
effectiveness of purification procedures by rhCG RIA has made it possible to 
recognize those eluates of increased potency. Kaolin-acetone extracts were 
subjected to Sephadex G-lOO gel filtration, cation-exchange Sephadex 



FC-20 



chromatography and Sephadex G-200 gel filtration with a 30-40 fold increase 
in potency of a small fraction using the standard preparation (HRP) as 
reference material. 

During the past three years, there has been disagreement as to whether 
the placenta of the rhesus monkey produces chorionic gonadotropin throughout 
pregnancy, and especially during the two to three weeks immediately preceding 
delivery. Another laboratory has published two studies showing increasing 
chorionic gonadotropin concentrations near the time of parturition. These 
studies have employed both bioassay and radioimmunoassay measurements. In 
contrast, we have been unable to detect any biological activity in placental 
extracts of term placentas either delivered naturally or obtained by C- 
section. Further, when kaolin-acetone concentrates of urine pools representing 
the entire week preceding natural delivery were tested in the mouse uterine 
weight assay (sensitive to 0.1 lU of hCG equivalents), no activity was 
detected. Currently, we are employing a radioimmunoassay for rhCG in these 
studies- 
Common antigenicity of primate gonadotropins was tested in homologous 
hCG-a and hCG-6 radioimmunoassay systems. The hCG-B system recognized common 
antigenic determinants in man, gorilla, and chimpanzee but not rhesus monkey. 
Except for the rhesus monkey, primate chorionic gonadotropins gave responses 
in the hCG-a system. However, evidence for non-identity was found. 

A series of pregnant rhesus monkeys was ovariectomized at 22 to 24 days 
gestation. VThen the patterns of plasma estrogens (estradiol-176 and estrone) 
and progesterone were compared with intact controls, it was found that at 
about gestation day 35, estradiol-176 and estrone levels, which had been un- 
detectable in ovariectomized monkeys and at basal levels in intact controls, 
rose to peak levels at about 80 days and maintained these levels until 
parturition in both groups. Throughout most of this period, the plasma 
concentration of estradiol-173 exceeded that of estrone by 4-5 fold. Like- 
wise, progesterone levels resembled those seen in intact animals. Thus, 
production of these hormones from approximately 25 days of gestation to 
parturition must be exclusively from sources other than the ovary (placenta, 
adrenals). With expulsion of the placenta at parturition, estrogen and 
progesterone concentration declined to basal levels indicating that the 
placenta was the major source of these hormones. 

From conception to day 25, peaks of estrogen were observed, one, just be- 
fore ovulation, the other at about day 20 of pregnancy. On the basis of 
earlier findings, the latter surge of estrogen may derive from the corpus 
luteum in response to increasing levels of chorionic gonadotropin. The ratio 
of estradiol-176 to estrone is approximately one in this early period of 
gestation. Since we had previously shown that pregnancy cannot be maintained 
in the absence of ovaries prior to gestation day 22, the evidence points to 
an ovarian source of these steroids. Levels of rhesus chorionic gonadotropin 
were similar in ovariectomized and intact monkeys throughout the brief period 
of its production. 

Light microscopic and fine structural observations were made on the corpus 
luteum of late pregnancy. Emphasis was placed on the granulosa lutein cells. 

FC-21 



These cells are 25 to 30 y in diameter and their plasma membranes are bordered 
by microvilli. The cytoplasm contains high concentrations of: 1) rod-shaped 
mitochondria with tubular cristae, 2) elaborate Golgi complexes and 
associated large tubular cisternae, 3) tubular as well as cisternal agranular 
endoplasmic reticulum, 4) well-developed parallel arrays of granular 
endoplasmic reticulum, 5) bundles of 50 A filaments and 6) dispersed lipid 
droplets. Large intracellular lacunae are present in the cytoplasm of most 
cells. It was concluded that the morphological features of the granulosa- 
lutein cells of late pregnancy are consistent with steroid synthesis. 

Proposed Course of the Project : 

FSH is being purified from extracts of monkey urine. This will be used 
to develop specific antibodies. Heterologous systems for immunoassay have 
been developed and are being validated. 

Purification of rhCG will be continued and the gestational events 
associated with CG secretion will be studied. The role of maternal FSH in 
follicular development of the fetus will be examined. 



FC-22 



Serial No, HD-LB3 

1. Reproduction Research Branch 

2. Section on Molecular Structure 

3. Bethesda, Maryland 



Project Title: 



PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 



Analytical Techniques and Instrumentation 

a. Amino Acid Analysis 

b. Automatic Monitoring of Chromatographic Experiments 



Previous Serial Nximber: Same 
Principal Investigator: E. Gross 
Other Investigators: 



M. Morishita 

J . Brown 

J. L. Morell 

Systems Maintenance Section DRS-BEI, NIH 
Division of Computer Research and Technology, NIH 



Cooperating Units: 



Man Years: 

Total: 5 
Professional: 3 
Others: 2 

Project Description: 

Objectives ; 

1. Determination of the amino acid composition of peptides and proteins; 

2. Improvement of existing amino acid analyzers and automation of 
techniques otherwise requiring many time-consuming operations; 

3. Adaptation of conventional amino acid analyzers to the separation and 
quantitation of unusual amino acids of microbial origin; 

4. Design, development, and operation of automated multiple sample 
applicators, the final instrument to offer adequate capacity for the 
continuous operation for prolonged periods of time without attendance. 

Methods Employed ; 

lonexchange chromatography on custom resins of spherical shape, 
colorimetry, and electronic data processing equipment. 



FC-23 



Major Findings : 

The second modified and improved sample applicator has been perfected to 
the point that it now operates on a 24-hour basis and performing 4-6 amino 
acid analyses depending upon the mode of operation. This facilitates 
greatly the handling of large numbers of amino acid analyses with amino 
acids of unusual structural features from membrane-active peptides, such as 
nisin, subtilin, cinnamycin, and duramycln. In the latter two peptides the 
presence of two new amino acids, 6-hydroxyaspartic acid and lysinoalanine, 
necessitated adaptation of the one-column program. These amino acids are 
now well separated and quantitatively determined. 

For the amino acid analysis of samples frequently available in only 
small quantities, one analyzer has been modified by electronic signal 
amplification for measurement of amino acids at the level of a fraction of a 
nanomole. 

Significance to Biomedical Research and the Program of the Institute ; 

The structural elucidation of peptides and proteins requires large 
numbers of amino acid analyses. The extensive use of the amino acid 
analyzers with automatic sample application has considerably facilitated 
the structural elucidation of nisin, subtilin, cinnamycin, and duramycin, all 
peptides with unusual amino acids. Unique degradation products of these 
peptides are also quickly determined by related approaches. Nisin and 
fragments of nisin are capable of inducing fetal resorptions in rats. The 
precise knowledge of the mode of action of these peptides will only be 
possible after characterization of the active peptides with the help of 
numerous amino acid analyses. 

Proposed Course of Project : 

(1) The refinement of the design features of the applicators will be 
continued. Amino acid analyzers equipped with automated sample applicators 
will be linked to small independent computers capable of feedback and 
process control in order to demonstrate advanced techniques of laboratory 
automation and their application to protein chemistry. (2) The hydrolyses 
of peptides and proteins will be automated, and samples directly advanced 
to the automatic sample applicator for column loading. (3) Other 
laboratory processes will be further automated in collaboration with the 
Division of Computer and Research Technology. (4) The sequence analysis of 
peptides and proteins will be automated (a) by utilizing solid supports and 
(b) employing mass spectroscopic techniques. (5) Sensitivity in amino 
acid analysis will be further increased by (a) colorimeter (modification in- 
clusive of constant temperature accomodation; (b) the use of ionexchange 
columns of lower diameter; and (c) modifications in reagents and buffers and 
their adequate accomodation. 



FC-24 



Serial No. HD-LB14 

1. Reproduction Research Branch 

2. Section on Molecular Structure 

3. Bethesda, Maryland 



PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 



Project Title: 



Cell Organelles from Human Placenta: Formation, Isolation, 
Characterization, and Function Under Normal and Abnormal 
Conditions 



Previous Serial Number: Same 



Principal Investigator: E. Gross 



Other Investigators: 



Cooperating Units; 



Man Years: 



L. Corash 
W. Gerber 

I. Boime, Washington University, School of Medicine, 
St. Louis, Missouri 



iiCi 

J 



Total: 2 
Professional: 2 
Others: 

Project Description: 

Objectives : 

1. Isolation and characterization of cell organelles, primarily of lysosomes; 

2. Study of the membrane of lysosomes: chemical composition, reactivity, 
permeability, structural enzymes; 

3. Constituents of lysosomes: isolation and characterization of enzymes, 
basic proteins, and other components; 

4. Correlation of lysosomal enzymes and their function with the life cycle 
of the cell under normal and pathological conditions; 

5. Analysis of lysosomal enzyme levels in the human placenta at various 
stages of gestation and the role they may play in human fetal develop- 
ment; 

6. Preparation of a cell-free system of protein synthesis using lysosomes 
isolated from first trimester human placenta. 



FC-25 



Methods Employed ; 

Ultracentrifugation, zonal centrifugation, carrier-free electrophoresis, 
techniques of enzyme analysis, fluorescence and electron microscopy, separa- 
tion techniques for biopolymers. 

Major Findings : 

The syncytium is the major source of lysosomes in human term placenta. 
There are at least two different populations of lysosomes, one of which is 
associated with the endoplasmic reticulum. Acid phosphatase is not a 
suitable marker for human term placental lysosomes. The glucose-6-phosphate 
r.ydrolyzing activity of human term placenta is not identical with the 
conventionally defined glucose-6-phosphatase. Peroxisomes are not present in 
human term placenta. The syncytium of first trimester human placenta is also 
rich in lysosomes and contains two populations of the organelle. The latency 
of arylsulfatase of first trimester hum^an placental lysosomes is different 
from that of the other acid hydrolases. First trimester human placenta is 
six to eight times richer in lysosomes than human term placenta. Vital 
staining of tissue from first trimester human placenta demonstrates the effect 
of nisin on lysosomes. Pretreatment with the peptide or its H2N-terminal 
fragment abolishes the uptake of the vital stain. 

Significance to Biomedical Research and the Program of the Institute : 

Lysosomes are of central significance throughout the life cycle of the 
cell. The large number of hydrolases present in the organelle perform 
numerous important functions under normal and abnormal conditions. Profound 
physiological events, such as those of reproduction, may depend upon the 
availability of lysosomal enzymes. Extensive investigations of lysosomes in 
cells from different organs of the maternal as well as the fetal organism 
will contribute to the better understanding of the role of this organelle 
during development. 

Proposed Course of Project : 

(1) Improvement and application of techniques for the isolation of 
lysosomes from different types of developing tissue; (2) Isolation and 
characterization of lysosomal enzymes and other protein constituents from the 
organelle; (3) Elucidation of the structure and function of the lysosomal 
membrane; (A) Study of lysosomes under normal and abnormal conditions; 
(5) Investigation of the effect of low molecular weight peptides and proteins 
on the lysosomal membrane; (6) Correlation of the structure of membrane-active 
compounds with structural features of the lysosomal membrane; (7) Investiga- 
tion of the enzyme content of human placental lysosomes at various stages of 
gestation. 



FC-26 



Serial No. HD-LB15 

1. Reproduction Research Branch 

2. Section on Molecular Structure 

3. Bethesda, Maryland 

PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Fetal Resorption 

Previous Serial Number : Same 

Principal Investigator: E. Gross 

Other Investigators: None 

Cooperating Units: K. C. Snell, Laboratory of Pathology, National 

Cancer Institute 

Man Years: 

Total: 1/12 
Professional: 1/12 
Others: 

Project Description: 

Objectives : 

1. Isolation, purification, and structural elucidation of substances 
capable of inducing fetal resorption; 

2. Correlation of the structure and mode of action of these compounds; 

3. Identification of the cellular constituents affected by and/or 
contributing to fetal resorption. 

Methods Employed : 

(1) Microbial culturing techniques to provide compounds with effects on 
the feto-placental unit, (2) chromatographic and counter-current distribution 
techniques in purification steps, (3) enzymatic and nonenzymatic methods in 
combination with amino acid analysis to determine the structure of peptides 
affecting fetal development, (4) animal experiments to test the activity of 
substances for which an effect on fetal development has been predicted, 
(5) characterization of tissue samples from the feto-placental unit, cell 
biological characterization to determine the mode of action of fetal 
resorption inducing agents after, for instance, radiolabeling by synthetic 
approaches. 



FC-27 



r 

I 



Major Findings : 

Nisin and HjN-tenninal fragments of nlsin Induce fetal resorption in rats 
when administered Intravenously. Via the same route, these two compounds are 
also effective in preventing implantation by an as yet unknown mechanism. 
The COOH-terminal fragment of nisin is not effective. Its enzymatic degrada- 
tion and inactlvation is a likely event following intravenous injection. 
This is apparently also the case for nisin Itself when it is administered 
orally. The gramicidins A, B, and C induce fetal resorption and prevent 
implantation when given orally. 

Significance to Biomedical Research and the Program of the Institute : 

The regulatory mechanisms operative at many stages of intrauterine 
development are not fully understood. Studies with substances of novel types 
of structures may serve to (1) identify physiological processes that contrib- 
ute to the normal course of pregnancy, (2) detect undesirable metabolites 
which affect gestation adversely, and (3) offer chemotherapeutic approaches 
to pregnancy regulation. 

Proposed Course of Project : 

The substances indicated are being tested in a contract research 
laboratory. After the completion of these screening procedures if warranted, 
the following studies will be conducted: (1) Structural elucidation of 
peptides which affect fetal development; (2) correlation of structure and 
function during this physiological event; (3) synthesis of peptides in which 
the structural elements of nisin and the physiological properties of the 
gramicidins are combined; (4) investigation of dose levels, routes of 
administration, and timing of administration of substances that affect fetal 
development; (5) study of the effect of previously encountered resorptions on 
subsequent pregnancies; and (6) extension of these studies to primates. 



FC-28 



Serial No. HD-LB5 

1. Reproduction Research Branch 

2. Section on Molecular Structure 

3. Bethesda, Maryland 

PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Synthesis of Biologically Active Peptides 

Previous Serial Number: Same 

Principal Investigator: E. Gross 

Other Investigators: K. Noda 

Cooperating Units: E. Blout, Harvard Medical School, Boston, Massachusetts nC 

Man Years: | 



Total: 14/12 
Professional: 14/12 
Others: 

Project Description: 

Objectives : 

1. Synthesis of peptides with biological activity, such as: 

a. effects on fetal development, and 

b. hormone releasing properties; 

2. Testing and evaluating the biological properties of synthetic peptides. 

Methods Employed : 

(1) Conventional techniques of peptide synthesis, (2) solid phase methods 
of peptide synthesis, (3) chromatographic and countercurrent distribution 
techniques for separation and purification, (4) animal experiments and bio- 
assays in the biological evaluation of the synthetic products, (5) radio- 
immunoassays in screening procedures; and (6) nuclear magnetic resonance and 
mass spectroscopic analysis of peptides. 

Major Findings: 

Synthesis via solid phase techniques of: Luteinizing hormone-follicle 
stimulating hormone releasing factor (LH/FSH-RF) ; H2N-termlnal trlpeptlde 
amide of LH/FSH-RF; the peptide has less than 0.1% of the activity of the 
natural releasing factor. A new solid phase technique, developed to 



FC-29 



I 



facilitate the synthesis of peptide amides, is based on the chemistry of a,6- 
unsaturated amino acids and utilized the experience gained during the 
structural elucidation of nisin and subtilin. 

Significance to Biomedical Research the Program of the Institute : 

The highly hydrophobic gramicidins A, B, and C constitute a unique group 
of peptides. Their alternating pattern of L and D-amino acids allows the 
formation of specific types of helices which, in turn, are capable of acting 
as ion-transporting transmembrane channels. The gramicidins A, B, and C 
affect fetal development in the rat. They also interfere with implantation. 
The hormone-releasing factors are of great interest as potential fertility 
regulating agents. 

Proposed Course of Project : 

(1) Synthesis of analogues of gramicidin A, B, and C; (2) synthesis of 
radio- labeled gramicidins; (3) synthesis of nisin, fragments of nisin, and 
analogs of these peptides with and without radioactive amino acids; (4) 
synthesis of hormone-releasing factors of peptide structure; (5) synthesis of 
subtilin, fragments of subtilin, and analogs of the parent molecule and 
partial structures; development of techniques for peptides synthesis. 



FC-30 



Serial No. HD-LB19 

1. Reproduction Research Branch 

2. Section on Molecular Structure 

3. Bethesda, Maryland 



PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 



Project Title: 



Chemical Modification of Amino Acids. Selective Nonenzymatic 
Cleavage of Peptide Bonds. Structure-Function Relationship 
of Biologically Active Peptides and Enzymes 



Previous Serial Number: Same 
Principal Investigator: E. Gross 
Other Investigators: 



Cooperating Units: 



J. L. Morell 

M. Morishita 

J. Brown 

E. Nebelin 

H. C. Chen 

L. C. Craig, Rockefeller University, New York, 

New York; F. H. White, Jr., Laboratory of Biochemistry 

National Heart and Lung Institute; E. D. Becker, 

Laboratory of Physical Biology, National Institute of 

Arthritis, Metabolism, and Digestive Diseases; 

E. Scoffone, University of Padova, Padova, Italy 



3 

) 

r 



Man Years: 

Total: ^ 
Professional: 4 
Others: 

Project Description: 

Objectives : 

1. Chemical modification of reactive groups in polyfunctional amino acids; 

2. Selective cleavage of peptide bonds (nonenzymatic fragmentation) by 
application of chemical rea 

3. Production, isolation, and purification of biologically active peptides 
and enzymes; 

4. Fragmentation of biologically active peptides and enzymes with the 
objective of studying residual activity; 

5. Synthesis of enzyme inhibitors and study of inhibitory effects, identifi- 
cation of the site of inhibition and of loci responsible for enzyme 
activity; 



FC-31 



6. Structural elucidation of biologically active peptides and correlation of 
structure and function. 

Methods Employed ; 

(1) Enzymes; (2) chemical reagents for nonenzjnnatic fragmentation; 
(3) high energy radiation techniques; (4) photochemical techniques; (5) 
advanced separation and analytical techniques: chromatography, electropho- 
resis, countercurrent and counter double current distribution, automatic 
sample application in the amino acid analysis, electronic equipment in data 
acquisition and processing; (6) nuclear magnetic resonance; (7) mass 
spectroscopy; and (8) culturing techniques and bioassays. 

Major Findings ; 

(1) Nisln : The ot-aminobutyric acid moieties of the 6-methyllanthionine 
residues and the alanine moiety of lanthlonine first in the linear sequence 
are of the D-configuration. This indicates a stereospeclflc step in the bio- 
synthesis of the peptide. (2) Subtllln : The structure of subtilin has been 
established as that of a heterodetic pentacyclic peptide of 32 amino acids. 
Nlsin and subtilin, thus, have in common the higher structural principle of 
five heterodetic peptide cycles all of which are Identical in their respective 
size. (3) Cinnamycin and duramycln are closely related peptides with 
lanthlonine and 6-methyllanthlonlne. Dehydroalanlne is present in these 
molecules in the masked state of lyslnoalanine. The two peptides resist 
enzymatic degradation and nonenzymatlc fragmentation methods are necessary for 
their structural elucidation. (A) High energy radiation of the COOH-terminal 
fragment of nisln in the presence of tritium Incorporates the label with 
preference in hlstidine and serine, a fact Indicating a distinct role of these 
two amino acid residues. (5) The synthesis of human chorionic gonadotrophin 
in trophoblast culture is affected by nisin. (6) Nisln and nisln fragments 
undergo photochemical changes which are likely to originate with the a,6- 
unsaturated amino acids. (7) Sizeable quantities of nisln and nisin fragments 
have been purified for studies of their effects on lysosomes and on fetal 
development. (8) Pepsinogen has been fragmented by consecutive application of 
the cyanogen bromide reaction at methionine and S-methylcysteine. The 
resulting fragments have been separated and purified to finally cleave the 
products of the pseudo-N-+0-acyl shift. (9) The bromate/bromide oxidation 
reaction is applicable to the fragmentation of peptides by cleavage of the 
tryptophyl peptide bond. (10) Esterification of terminal COOH-groups followed 
by reduction to the alcohol and N->0-acyl shift constitute a route to the 
stepwise removal of COOH-terminal amino acid residues. 

Significance to Biomedical Research and the Program of the Institute ; 

The detailed knowledge of the structure of nisin, and of other peptides 
with a,6-unsaturated amino acids is of great importance in view of the 
biological properties of these biopolymers. The implied working hypothesis 
of the interaction of a, 6-unsaturated amino acids with vital sulfhydryl groups 
may well be of broad biological significance. This reaction is reversible and 
linked to the formation of ketoacids and amides, frequently encountered 



FC-32 



constituents of many biological systems. These structural considerations 
form the basis for the study of the effect of nisin and related peptides on 
fetal development. 

Proposed Course of Project : 

(1) Continued exploration and application of nonenzymatic methods to the 
structural elucidation of peptides and proteins: (a) application of selective 
oxidants to the cleavage of tryptophyl peptide bonds; (b) study of the 
selective suppression of the cleavage of methionyl peptide bonds upon treat- 
ment with cyanogen bromide; (c) fragmentation of reduced and S-alkylated 
peptides and proteins; fragmentation of peptides and proteins subsequent to 
mercaptan addition to a,6-unsaturated amino acids; direct fragmentation at 
a, 3-unsaturated amino acids, at times preceded by 6-elimination; (d) the 
fragmentation at aspartic acid residues after esterif ication and reduction to 
be developed to a generally applicable method of nonenzymatic fragmentation. 
(2) Nisin : Continued structural elucidation of nlsin-like peptides from 
Streptococcus lactis . (3) Subtilin : Elucidation of the configuration of the 
6-carbon atoms of 6-methyllanthionine and determination of the isomerism in 
dehydrobutyrine study of phylogenetic aspects of the structure of peptides ,ij.; 

containing lanthionines and a, 6-unsaturated amino acids. (4) Cinnamycin and ^*'| 
Duramycin : Structural elucidation and structural correlation. (5) Prepara- Jj, 
tion of peptides with activities on fetal growth. i 

r 



FC-33 



Honors and Awards : 

Mortimer B. Lipsett 

Invited Lectures 

International Cancer Meeting, Yugoslavia, August 1972 

University of Paris, September 1972 

University of Copenhagen, September 1972 

Endocrine Society, Post-graduate Assembly, Birmingham, October 1972 

Annual Meeting, Brazilian Endocrine Society, Brazil, November 1972 

American College of Physicians, Brown University, Providence, November 1972 

College of Physicians and Surgeons, Columbia University, New York, February 

1973 
American College of Physicians, New York, March 1973 
New York Academy of Science, New York, March 1973 
Massachusetts Institute of Technology, Cambridge, April 1973 
Serono Foundation, Course in Endocrinology, Florence, April 1973 
American College of Physicians, Ann Arbor, May 1973 

Technical Review Committee, Male Reproduction, World Health Organization, 

1972- 
Chairman, Post-graduate Teachers Assembly, Endocrine Society, 1973- 
Committee on Endocrinology and Metabolism, Food and Drug Administration 1973- 
Elected Corresponding Member German Endocrine Society, 1973- 

G. T. Ross 

Invited Lectures 

John E. Fogarty International Center and League for the Fight Against Cancer 

of the Croatian Republic of Yugoslavia, Yugoslavia, August 1972 
College of Physicians and Surgeons, Columbia University, New York, September 

1972 
Airlie Foundation, Airlie, Virginia, October 1972 
Late Effects Workshop Meeting, Children's Cancer Research Foundation, Boston, 

October 1972 
Harold C. Mack Symposium, Wayne State University and Harper Hospital, Detroit, 

November 1972 
American College of Physicians Post Graduate Course, Johns Hopkins Medical 

Institutions, Baltimore, November 1972 

Awards Committee, Endocrine Society, 1972 

Endocrine Study Section, 1972 

Breast Cancer Task Force, Treatment Committee, 1972 

Scientific Advisor to the Medical Research Institute of Worcester, Inc., 

Worcester, 1972- 
Committee on Standards of the International Society of Endocrinology, 1973- 

Wm. W. Tullner 

Constiltant In Endocrinology and Metaboliem, The George Washington University 
(•rniliint C3 ndiooJ , Wnnli I tip.t nti, U. C. 

l''C-34 



Research Associate, Department of Zoology, Graduate School, Howard University, 
Washington, D.C. 

Erhard Gross 

Invited Lectures 



Third American Peptide Symposium, Boston, June 1972 

Ninth International Congress of Biochemistry, Stockholm, June 1973 

K. J. Catt 

Invited Lectures 

Vanderbilt University, Nashville, July 1972 

Worcester Foundation for Experimental Biology and Medicine, Shrewsbury, 

February 1973 
George Washington University, Washington, D. C., February 1973 
University of Florida, Gainesville, March 1973 
University of California, Los Angeles, March 1973 
Department of Nuclear Medicine, Johns Hopkins University, Baltimore, 

March 1973 
University of Milan, May 1973 

Member, American Society for Clinical Investigation (Young Turks), May 1973 

M, L. Dufau Catt 

Invited Lectures 

University of Milan, May 1973 

A. Chrambach 

Invited Lectures 

International Conference on Isoelectric Focusing and Isotachophoresis, 

New York, May 1972 
Gordon Research Conference, New Hampton, June 1972 
Conference on Electrophoresis and Isoelectric Focusing in Polyacrylamide 

Gel, Tubingen, Germany, October 1972 

G. D. Hodgen 

Invited Lectures 

Satellite Symposium of IV International Congress of Endocrinology, Washington, 
D.C. , June 1972 

P. 0. Kohler 

Fellow, American College of Physicians, October 1972 

Member, American Society for Clinical Investigation (Young Turks), May 1973 

FC-35 



H. B. Pollard 

Invited Lectures 

University of Chicago, November 1972 

Membrane Conference, Squaw Valley, March 1973 

University of California, Berkeley, March 1973 

University of California, San Francisco, March 1973 

California Institute of Technology, Pasadena, March 1973 

Johns Hopkins University, Baltimore, April 1973 

Oxford University, Laboratory of Molecular Biophysics, Oxford, May 1973 

International Catecholamine Congress, Strasbourg, May 1973 

.Washington Crystal Colloquim, June 1973 

D. Rodbard 

Invited Lectures 

Vanderbilt University, Nashville, July 1972 

University of Colorado and Veterans Administration Hospital, Denver, 

September 1972 
Max-Planck Institute, Tubingen, October 1972 
Stanford University Medical School, Palo Alto, December 1972 
University of California Medical Center, San Francisco, December 1972 
University of Washington Medical School and American Association of 

Clinical Chemists, Seattle, December 1972 
University of California Medical School, Los Angeles, December 1972 
Harbor General Hospital, Torrance, December 1972 

Wadsworth Veterans Administration Hospital, Los Angeles, December 1972 
Kroc Foundation for the Advancement of Sciences, Santa Barbara, April 1973 

R. J. Sherins 

Invited Lectures 

Columbia Presbyterian Hospital, New York, September 1972 

Hershey Medical Center, Hershey, October 1972 

Holy Cross Hospital, Silver Spring, January 1973 

George Washington University Medical School, Washington, D.C., February 1973 

Washington Pediatric Society, March 1973 

Fellow, American College of Physicians, 1973 

J. L. Vaitukaitis 

Invited Lectures 

University of Maryland School of Dentistry, Baltimore, February 1973 
American Fertility Society Postgraduate Course, San Francisco, April 1973 
George Washington School of Medicine, Washington, D.C., January & February 1973 
University of California, San Diego, November 1972 
Boston University School of Medicine, Boston, April 1973 



FC-36 



PUBLICATIONS 

Ben-David, M. , and Chrambach, A. : Electrophoretlc identity of biologically 
active pituitary and serum prolactins in the lactating and perphenazine- 
treated rat. Acta Endocrinol . 72: 654-662, 1973. 

Ben-David, M. , Rodbard, D. , Bates, R.W. , Bridson, W.E., and Chrambach, A.: 
Human prolactin in plasma, amniotic fluid and pituitary identity and 
characterization by criteria of electrophoresis and isoelectric focusing in 
polyacrylamide gel. J. Clin . Endocrinol . Metab. 36: 135-148, 1973. 

Bennett, W.I., Dufau, M.L., Catt, K.J., and Tullner, W.W. : Effect of human 
menopausal gonadotropin upon spermatogenesis and testosterone production in 
juvenile Rhesus monkeys. Endocrinology 92: 813-821, 1973. 

Berman, M.L., Hanson, K. , and Hellman, I.L.: Effect of breast-feeding on 
postpartum menstruation, ovulation, and pregnancy in Alaskan Eskimos. Am . 
J. Obstet . Gynecol . 114: 524-534, 1972. 

Braunstein, G.D., Bridson, W.E., Glass, A., Hull, E.W., and Mclntire, K.R. : 
In vivo and in vitro production of hCG and alpha-fetoprotein by a virilizing 
hepatoblastoma. J. Clin . Endocrinol . Metab . 35: 857-862, 1972. 

Braunstein, G.D., Grodin, J.M., Vaitukaitis, J., and Ross, G.T.: Secretory 
rates of hCG by normal trophoblast. Am . jJ. Obstet . Gynecol . 115: 447-450, 
1973. 

Braunstein, G.D., and Kohler, P.O.: Pituitary function in Hand-Schuller- 
Christian Disease. New Engl . J. Med. 286: 1225-1229, 1972. 

Braunstein, G.D., Vaitukaitis, J.L., Carbone, P.P., and Ross, G.T.: Ectopic 
production of hCG by neoplasma. Ann. Intern . Med. 78: 39-45, 1973. 

Braunstein, G.D., Vaitukaitis, J.L., and Ross, G.T.: The in vivo behavior 
of hCG after dissociation into subunits. Endocrinology 91: 1030-1036, 1972. 

Cain, M.D., Coghlan, J. P., and Catt, K.J.: Measurement of angiotensin II in 
blood by radioimmunoassay. Clin. Chim . Acta 39: 21-34, 1972. 

Cantz, M. , Chrambach, A., Bach, G. , and Neufeld, E.F.: The Hunter corrective 
factor. J. Biol . Chem. 247: 5456-5462, 1972. 

Cargille, CM., Vaitukaitis, J.L., Bermudez, J. A., and Ross, G.T. : A 
differential effect of ethinyl estradiol upon plasma FSH and LH relating to 
time of administration in the menstrual cycle. J^. Clin . Endocrinol . Metab. 
36: 87-94, 1973. 

Catt, K.J., Dufau, M.L., and Tsuruhara, T. : Absence of intrinsic biological 
activity in LH and hCG subunits. J. Clin. Endocrinol . Metab . 36: 73-80, 1973. 

Catt, K.J., Tsuruhara, T. , and Dufau, M.L.: Gonadotrophin binding sites of 
the rat testis. Biochim . Biophys . Acta 279: 194-201, 1972. 

Catt, K.J., Watanabe, K. , and Dufau, M.L.: Cyclic AMP released by rat testis 
during gonadotrophin stimulation in vitro. Nature 239: 280-281, 1972. 



FC-37 



Chrambach, A., Hearing, E. , Lunney, J., and Rodbard, D. : Experimental 
validation of the predicted properties of a multiphasic buffer system applied 
to polyacrylamlde gel electrophoresis. Separation Sci . 7: 725-745, 1972. 

Chrambach, A., Kapadia, G. , and Cantz, M. : Isotachophoresis on polyacrylamlde 
gel. Separation Sci . 7: 785-816, 1972. 

Chrambach, A., Pickett, J., Schlam, M.L., Kapadia, G., and Holtzman, N.A.: 
A partitioned slab apparatus for one-dimensional polyacrylamlde gel 
electrophoresis in multiphasic buffer systems under a wide range of condi- 
tions. Separation Sci . 7: 773-783, 1972. 

Chrambach, A., and Rodbard, D. : Polymerization of polyacrylamlde gels: 
efficiency and reproducibility as a function of catalyst concentration. 
Separation Sci . 7: 663-703, 1972. 

Cohen, J., Hagemeyer, H.P., Pollard, H.B., and Schechter, A.: Proton 
magnetic resonance study of the histiding residues of sperm whale and horse 
myoglobins. J. Mol. Biol . 71: 513-519, 1972. 

Corvol, P., Falk, R. , Freifeld, M. , and Hardin, C.W. : In vitro studies of 
progesterone binding proteins in guinea pig uterus. Endocrinology 90: 1464- 
1469, 1972. 

Dekaban, A., Parks, J., and Ross, G.T.: Laurence Moon Syndrome. Evaluation 
of endocrinological function and phenotypic concordance and report of cases. 
Med . Ann . D. C. 41: 687-694, 1972. 

Dirksen, M.L., and Chrambach, A.: Studies on the redox state in polyacry- 
lamlde gels. Separation Sci . 7: 747-772, 1972. 

Dufau, M.L., and Catt, K.J.: Extraction of soluble gonadotrophin receptors 
from rat testis. Nature New Biology 242: 246-248, 1973. 

Dufau, M.L., Catt, K.J., and Tsuruhara, T. : Biological activity of human 
chorionic gonadotropin released from testis binding sites. Proc . Natl . 
Acad. Sci. 69: 2414-2416, 1972. 

Dufau, M.L., Tsuruhara, T. , and Catt, K.J.: Interaction of glycoprotein 
hormones with agarose-concanavalin A. Biochim . Biophys . Acta 278: 281-292, 
1972. 

Dufau, M.L., Watanabe, K. , and Catt, K.J. : Stimulation of cyclic AMP produc- 
tion by the rat testis during incubation with hCG in vitro. Endocrinology 
92: 6-11, 1973. 

Edlow, J.B., Kohler, P.O., and Robinson, J.C.: Choriocarcinoma in vitro: 
Evidence of enzyme transfer between culture medium and cells. Proc . Soc . 
Exp . Biol . Med . 141: 798-801, 1972. 

Goldenberg, R.L., Bridson, W.E., and Kohler, P.O.: Estrogen stimulation of 
progesterone synthesis by porcine granulosa cells in culture. Biochem . 
Biophys . Res . Commun . 48: 101-107, 1972. 

FC-38 



Goldenberg, R.L., Grodin, J.M. , Vaitukaitis, J.L. , and Ross, G.T.: With- 
drawal bleeding and LH secretion following progesterone in women with 
amenorrhea. Am. J. Obstet . Gynecol . 115: 193-196, 1973. 

Goldenberg, R.L., Reiter, E.O., and Ross, G.T.: Follicle response to 
exogenous gonadotropins: an estrogen-mediated phenomenon. Fertil. Steril . 
24: 121-125, 1973. 

Goldenberg, R.L., Reiter, E.O., Vaitukaitis, J.L., and Ross, G.T. : Inter- 
action of FSH and hCG on follicle development in the ovarian augmentation 
reaction. Endocrinology 91: 533-536, 1972. 

Goldenberg, R.L., Vaitukaitis, J.L., and Ross, G.T.: Estrogen and follicle 
stimulating hormone interactions on follicle growth in rats. Endocrinology 
90: 1492-1498, 1972. 

Grodin, J.M. : Secondary amenorrhea in the adolescent. Pediatric Clinics of 
North America . 19: 619-630, 1972. 

Gross, H.A., Ruder, H.J., Brown, K.S., and Lipsett, M.B.: A radioimmuno- 
assay plasma corticosterone. Steroids 20: 681-695, 1972. 

Gulyas, B.J.: The rabbit zygote II. The fate of annulate lamellae during 
first cleavage. 1. Zellforsch . Mikrosk . Anat. 133: 187-200, 1972. 

Gulyas, B.J.: The rabbit zygote III. Formation of the blastomere nucleus. 
J. Cell Biol . 55: 533-541, 1972. 

Hodgen, G.D., Dufau, M.L., Catt, K.J., and Tullner, W.W. : Estrogens, 
progesterone and chorionic gonadotropin in pregnant rhesus monkeys. 
Endocrinology 91: 896-900, 1972. 

Hodgen, G.D., Nixon, W.E., Vaitukaitis, J.L., Tullner, W.W. , and Ross, G.T. : 
Neutralization of primate chorionic gonadotropin activities by antisera 
against the subunits of human chorionic gonadotropin in radioimmunoassay and 
bioassay. Endocrinology 92: 705-709, 1973. 

Kapadia, G., and Chrambach, A.: Recovery of protein in preparative poly- 
acrylamide gel electrophoresis. Anal . Biochem . 48: 90-102, 1972. 

Knazek, R.A., Gullino, P.M., Kohler, P.O., and Dedrick, R.L. : Cell culture 
on artificial capillaries. Science 178: 65-67 , 1972. 

Kulin, H.E., and Reiter, E.G.: Gonadotropin suppression by low dose estrogen 
in men: Evidence for differential effects upon FSH and LH. J. Clin . 
Endocrinol . Me tab . 35: 836-839, 1972. 

Kulin, H.E., and Reiter, E.G.: Ontogeny of the in vitro uptake of tritiated 
estradiol by the hypothalamus of the female rat. Endocrinology 90: 1371- 
1374, 1972. 



FC-39 



Loriaux, D.L., Guy, R.L., and Lipsett, M.B.: A simple, quick, solid-phase 
method for radioimmunoassay of plasma estradiol in late pregnancy and of 
plasma Cortisol. J_. Clin . Endocrinol . Metab . 36: 788-790, 1973. 

Loriaux, D.L., and Lipsett, M.B.: Radioligand assay for A^-SS-hydroxy- 
steroids II. 5-androstene-3e,176-diol and 3e,17a-dlhydroxy-5-pregnen-20-one. 
Steroids 19: 681-688, 1972. 

Loriaux, D.L., Ruder, H.J., Knab, D.R., and Lipsett, M.B.: Estrone sulfate, 
estrone, estradiol and estriol plasma levels in human pregnancy. J^. Clin . 
Endocrinol . Metab . 35: 887-891, 1972. 

Means, A.R., and Valtukaitls, J.L.: Peptide hormone "receptors" specific 
binding of ^H-FSH to testis. Endocrinology 90: 39-46, 1972. 

Miles, L.E.M., Simmons, J.E., and Chrambach, A.: Instability of pH gradients 
in isoelectric focusing on polyacrylamide gel. Anal . Blochem . 49: 109-117, 
1972. 

Mlyachi, Y., Chrambach, A., Mecklenburg, R.S., and Lipsett, M.B.: Preparation 
and properties of 125i-lhRH. Endocrinology 92: 151-156, 1973. 

Miyachi, Y., Nleschlag, and Lipsett, M.B.: The secretion of gonadotropins 
and testosterone by the neonatal male rat. Endocrinology 92: 1-5, 1973. 

Myerowtiz, R.L., Chrambach, A., Rodbard, D., and Robbins, J.B.: Isolation 
and characterization of mouse serum alpha- 1-antltrypslns. Anal . Blochem . 
48: 394-401, 1972. 

Nleschlag, E., Loriaux, D.L., and Lipsett, M.B.: Radioligand assay for A - 
36-hydroxysteroids 36-hydroxy-5-androstene-17-one and its 3-sulfate. 
Steroids 19: 669-679, 1972. 

Rayford, P.L., Valtukaitls, J.L., Ross, G.T., Morgan, F.J., and Canfleld, R.E.; 
Use of specific antlsera to characterize biologic activity of hCGB subunit 
preparations. Endocrinology 91: 144-146, 1972. 

Reiter, E.G., Braunstein, G.D., Hamwood, S.M., and Kulin, H.E.: Suppressed 
follicle-stimulating hormone (FSH) in women with gestational trophoblastic 
neoplasms. J_. Clin. Endocrinol . Metab. 36: 697-701, 1973. 

Reiter, E.G., Goldenberg, R.L., Valtukaitls, J.L., and Ross, G.T.: A role 
of endogenous estrogen in normal ovarian development in the neonatal rat. 
Endocrinology 91: 1537-1539, 1972. 

Reiter, E.G., Goldenberg, R.L., Valtukaitls, J.L., and Ross, G.T.: Evidence 
for a role of estrogen in the ovarian augmentation reaction. Endocrinology 
91: 1518-1522, 1972. 

Reiter, E.G., and Kulin, H.E.: Sexual maturation In the female: Normal 
development and precocious puberty. Pediatric Clinics of North America 19: 
581-603, 1972. 



FC-40 



Reiter, E.O., Kulin, H.E., and Hamwood, S.H.: Preparation of urine con- 
taining small amounts of FSH and LH for radioimmunoassay: Comparison of the 
kaolin-acetone and acetone extraction techniques. J^. Clin . Endocrinol . 
Metab, 36: 661-665, 1973. 

Reiter, E.O., Kulin, H.E., and Loriaux, D.L.: FSH suppression during short 
term hCG administration: A gonadally mediated process. J_. Clin. Endocrinol . 
Metab . 34: 1080-1084, 1972. 

Rodbard, D.: Standardization of radioimmunoassay procedures: Section 3.2.6 
Data presentation and analysis. International Atomic Energy Agency Panel 
Report, Vienna, 1972. 

Rodbard, D. : Standardization of radioimmunoassay procedures: Section 3.2.8 
Automation. International Atomic Energy Agency Panel Report, Vienna, 1972. 

Rodbard, D. , and Catt, K.J.: Mathematical theory of radioligand assays: The 
kinetics of separation of bound from free. J^. Ster . Biochem . 3: 255-273, 
1972. 

Rodbard, D. , Levitov, C. , and Chrambach, A.: Electrophoresis in highly cross- 
linked polyacrylamide gels. Separation Sci . 7: 705-723, 1972. 

Rodbard, D., and Weiss, G.H.: Mathematical theory of immunoradiometric 
(labeled antibody) assays. Anal . Biochem . 52: 10-44, 1973. 

Ruder, H.J., Guy, R.L. , and Lipsett, M.B.: A radioimmunoassay for Cortisol 
in plasma and urine. J. Clin . Endocrinol . Metab . 35: 219-224, 1972. 

Sherins, R.J., Vaitukaitis, J.L., and Chrambach, A.: Physical characteriza- 
tion of hFSH and its desialylation products by isoelectric focusing and 
electrophoresis in polyacrylamide gel. Endocrinology 92: 1135-1141, 1973. 

Slater, D.W., Slater, I., and Gillespie, D.: Post-fertilization synthesis 
of polyadenyllc acid In sea urchin embryos. Nature 240: 333-337, 1972. 

Slater, I., and Slater, D.W.: DNA polymerase potentials of sea urchin 
embryos. Nature New Biology 237: 81-85, 1972. 

Tsuruhara, T., Dufau, M.L., Hickman, J., and Catt, K.J.: Biological 
properties of hCG after removal of terminal sialic acid and galactose 
residues. Endocrinology 91: 296-301, 1972. 

Tsuruhara, T. , Van Hall, E.V. , Dufau, M.L., and Catt, K.J. : Ovarian binding 
of Intact and desialylated hCG in vivo and in vitro. Endocrinology 91: 463- 
469, 1972. 

Vaitukaitis, J.L., Braunstein, G.D., and Ross, G.T.: A radioimmunoassay 
which specifically measures human chorionic gonadotropin in the presence of 
human luteinizing hormone. Am. J_. Obstet . Gynecol . 113: 751-758, 1972. 



FC-41 



Vaitukaitis, J,L,, Ross, G.T., and Relchert, L.E.: Immunologic and biologic 
behavior of hCG and bovine LH subunit hybrids. Endocrinology 92: 411-416, 
1973. 

Vaitukaitis, J.L., Ross, G.T., Reichert, L.E., and Ward, D.N.: Immunologic 
basis for within and between species cross reactivity of luteinizing 
hormone. Endocrinology 91: 1337-1342, 1972. 

Van Thiel, D.H., Sherins, R.J., Myers, G.H., Jr., and De Vita, V.T., Jr.: 
Evidence for a specific seminiferous tubular factor affecting follicle- 
stimulating hormone secretion in man. J_. Clin . Invest . 51: 1009-1019, 1972. 

Weiss, G.H., and Rodbard, D. : Diffusion-dependent peak broadening in pore 
gradient electrophoresis. Separation Sci . 7: 217-232, 1972. 



REVIEWS AND CHAPTERS 

Canfield, R.E., Morgan, F.J., Kammerman, S., and Ross, G.T.: Studies of the 
structure and recombination of the subunits of hCG. In Margoulies, M. , 
and Greenwood, F.C. (eds.): Protein and Polypeptide Hormones . Amsterdam, 
The Netherlands, Excerpta Medica, Part II, 1972, pp. 341-343. 

Catt, K.J., Cain, M.D., and Menard, J.: Radioimmunoassay studies of the 
renin-angiotensin system in human hypertension and during estrogen treat- 
ment. In Genest, J., and Kiow, E. (eds.): Hypertension , Berlin, Germany, 
Springer- Verlag, 1972, pp. 591-604. 

Ross, G.T.: Pituitary-gonadal interactions in man before and after puberty. 
In Segal, S.J., Crozier, R. , Corfman, P. A., and Condliffe, P. (eds.): 
Regulation of Mammalian Reproduction . Springfield, 111., 1972. 

Ross, G.T.: HCG: Physiology and hormonal effects. In Berson, S.A, (ed.): 
Methods in Clinical Investigative and Diagnostic Endocrinology . Amsterdam, 
The Netherlands, No. Holland Publishing Co., Vol. 2B, Part 3, 1973, pp. 
743-748. 

Ross, G.T.: Bioassay for HCG: In Berson, S.A., and Yalow, R.S. (eds.): 
Methods in Clinical Investigative and Diagnostic Endocrinology . Amsterdam, 
The Netherlands, No. Holland Publishing Co., Vol. 2B, Part 3, 1973, 
pp. 749-756. 

Ross, G.T., Van Hall, E.V., Vaitukaitis, J.L., Braunstein, G.D., and 

Rayford, P.L.: Sialic acid and the immunologic and biologic activity of 

gonadotropins. In Saxena, B.B., Beling, C.G., and Gandy, H.M. (eds.): 

Gonadotropins . New York, New York, John Wiley and Sons, Inc., 1972, 
pp. 417-423. 

Tullner, W.W. : Chorionic gonadotrophin in non-human primates. In 
Diczfalusy, E. (ed.): Symposium on the Use of Non-human Primates on Problems 
of Human Reproduction, Sukhumi, USSR, 1971. Acta Endocr. Suppl . 166: 
1972, pp. 200-213. 

FC-42 



Vaitukaitls, J.L., and Ross, G.T.: Antigenic similarities among the human 
glycoprotein hormones and their subunits. In Saxena, B,B., Beling, C.G., 
and Gandy, H.M. (eds.): Gonadotropins . New York, New York, John Wiley and 
Sons, Inc., 1972, pp. 435-447. 

Van Hall, E.V. , Vaitukaitis, J.L., Ross, G.T., Hickman, J.W., and Ashwell, G. 
Sialic acid and the function of hCG. In Margoulies, M. , and Greenwood, F.C. 
(eds.): Protein and Polypeptide Hormones . Amsterdam, The Netherlands, 
Excerpta Medica, Part 2, 1972, pp. 335-340. 



"51 
1 



FC-43 



NICHD Annual Report 

Behavioral Biology Branch 

July 1, 1972 through June 30, 1973 

Section on Brain and Behavior 

The professionals in this section are Drs. David Symmes suid John Newman. Mrs. 
Garland Riggs has contributed through her duties on a professional services 
contract. 

1. Drs. Newmfioi and Symmes have shown that the responses of single neurons 
in the primate auditory cortex to complex biologically significant acoustic 
stimuli are little affected by wide fluctuation in arousal level of the 
animal. The complex synaptic connections responsible for the relatively 
specific responses to such stimuli as species-specific vocalizations, there- 
fore, represent securely organized sensory channels, rather than some diffuse 
system related to attention or arousal. 

2. Drs. Symmes and Newman completed two studies on the discrimination of 
vocalizations by squirrel monkeys in a behavioral situation. One group of 
monkeys performed quite well in differentially responding to two, quite simi- 
lar variants of natural isolation peeps. The discriminations were based on 
relatively subtle aspects of these natural calls, since simple variation of 
loudness or duration could be shown not to be involved in the discrimination. 
Another group of monkeys, while discriminating between natxiral and computer 
generated isolation peeps did not discriminate between dissimilair pairs of 
computer generated isolation peeps. These results strongly indicate that 
natural calls contain distinctive but subtle features to which the natural 
discriminative systems are peculiarly acutely attuned. 

3. Extensive sound spectrographic analysis of the vocalization of one pair 
of squirrel monkeys indicate that each animal has a distinctive fine structure 
in its vocal productions. Call duration, number of syllables and complexity 
of syllable structure all seem to differentiate and made unique an individual 
monkey's vocal repertoire. 

1*. Dr. Symmes has completed histological verification of his studies of brain 
electriceil activity in isolation reared monkeys. The histological evidence 
generally supports the conclusion that abnormal electriceil activity was re- 
corded from the vicinity of the frimbria and dentate gyrus of the hippocamptts . 
Some placements were slightly dorsal to the hippocampus, but close enough to 
be consistent with evidence that stimulation of these sites induced hippo- 
campal after-discharges. The higher placements riile out possible damage to 
the hippocampus itself emd therefore argue against an irritative lesion as a 
basis for the observed electricail abnormeilities . Computer analysis confirmed 
in an objective quantitative way the previously reported nystagmus related 
electrical abnormalities in the Isolates. The limbic sites on the side oppo- 
site the calorically stimulated vestibular apparatus were more involved than 
ipsilatereuL sites. 



FD-1 



Section on Nevirobiology 

The professionsds in this section include Drs. Phillip Nelson, Harold Gainer, 
Gerald Fischbach, Bruce Schrier, Earl Giller, Ronald Fisk, Anthony Breuer, 
John Whysner, Mary anna Henkart, Jeffery Barker, Stephen Cohen, Clifford 
Christian and Asher Shainberg. 

1. Drs. Harold Gainer and Jeffery Barker have used the biochemical analytical 
methods developed by Dr. Gainer in an extensive study of individual neuronal 
synthetic patterns in the molluscan nervous system. Individual neurons have 
distinctive patterns of protein synthesis and one class of neurons exhibited 
sufficiently high turnover rates to warrant a study of the effects of electri- 
cal emd synaptic activity on their synthesis patterns. Synaptic inhibition 
reduced the rate of incorporation of radioactive leucine into two low molec- 
ular weight neurosecretory proteins by 30%. Dopsunine, the presumed trans- 
mitter mediating the synaptic inhibition produced similar reduction in syn- 
thesis of the neurosecretory protein. The synthesis rates of other proteins 
in the cell studies were not significantly influenced by either synaptic in- 
hibition or dopamine. Both these treatments increase membrane potential and 
increase surface membrane permeability to potassium. High potassium solu- 
tions increase potassium permeability but lower membrane potential and this 
treatment produces a selective doubling of the synthesis rate of the low 
molecular weight proteins. This argues against the increase in potassivun 
permeability being the causative control event and the possibility that an 
increased membrane potential might be directly involved in control of protein 
synthesis is currently under investigation. Other possible factors under 
consideration are changes in intracellular calcium or sodium ions. 

Studies on protein synthesis in squid axons indicate that Schwann cells are 
the site for synthesis of a wide range of proteins which are subsequently 
found in the axon. The regulatory mechanisms governing this glial -neuronal 
interaction are now under study. 

Divalent cations have been shown to play a major regulatory role in both the 
bursting pacemaker activity of molluscan neurosecretory cells and the 
seasonal modulation of this pacemaking activity. Sterols also are involved 
in regulation of this behavior; 25-hydroxycholecalciferol (a vitamin D 
metabolite) reversibly inhibits the pacemaker activity of these cells. 
Naturally occurring sterols from the molluscan nervous system in normal and 
aestivated animals are being investigated to see whether these materials may 
play seme role in regulating neural behavior in these animals. Studies on 
the mechanisms of the bursting pacemaker potentials in the neurosecretory 
cells (in collaboration with Dr. T. Smith, Laboratory of Neurophysiology, 
NINDS) indicate that this activity is primarily due to two membrane properties 
1) a voltage dependent inward divalent and monovalent cation current closely 
coupled to 2) a voltage-dependent outward potassium current. 

These combined electrophysiologic and biochemical approaches have defined an 
important class of membr€uie properties and show a close coupling between mem- 
brane events and synthesis of specific proteins within the cell. 



FD-2 



2. These experiments involve Drs. Nelson, Peacock, Giller, Fisk and Shainberg. 
Mouse cerebellar neurons have been shown to siirvive dissociation and develop 
complex patterns of spontaneous and synaptically mediated electrical activity. 
Clearly distinguishable, morphologic types and a range of electrophysiologic 
characteristics vere found in the surviving neuronal networks so that some 
sub8t€intial representation of the relatively simple normal cerebellar 
circuitry is presumably present in the culture systems. Moiise cerebellxm is 

a favorable preparation for in^ vitro studies from the standpoint of the 
variety of genetic mutants with relatively well established neuropathologic 
abnormalities. 

Interactions between nerve and muscle have been studied in cultures derived 
from dissociation of tissues of dystrophic chick embryos. Less than 25? of 
muscle cells in normal combined spinal cord-muscle culture became innervated 
but well over 30% of dystrophic muscle gave evidence of innervation when com- 
bined with either normal or dystrophic spinaLL cord cells. The possibility 
that this represents some defect in the "trophic" mechanism whereby multiple 
innervation is prevented needs to be investigated. 

Calcivun ions play an important role in active electrical membrane responses 
in both neuroblsistoma cells and fibroblasts (L-cells). Action currents in 
the neural cells are partly carried by calcium ions and intracellularly in- 
jected calcium elicits the L-cell response with a very short latency. The 
veu-ious stimuli \rtiich can elicit this fibroblast response therefore probably 
work through the intracellular release of calcium. Since intracellulaur 
calcium release has been shown to be an important factor involved in the 
regulation of cell division and a variety of metabolic processes, these pre- 
parations are favorable for study of the coupling between s\irface membrane 
activity and intracellular metabolism. 

Four to five fold enrichment for cell surface membranes has been obtained by 
subcellular fractionation. The neuroblastoma and one L-cell clone have been 
studied, with disc electrophoretic separation of total cellular and surface 
membrane proteins. While the resolution of the system appears to be good, 
and clear differences in the protein composition of neuronal and nonneuronal 
cell membranes have been found, substantially more data are required in differ- 
ent clones €ind with different growth conditions in order to begin to make the 
correlations between membrane structure and function which is the purpose of 
this work. 

3. Drs. Fischbach and Cohen have studied factors important in the regulation 
and disposition of acetylcholine receptors on the surface of chick muscle 
fibers. Innervation of the muscle fibers does not change the general level 
of acetylcholine sensitivity, but muscle activity does have a pronounced 
effect on chemosensitivity. A high degree of ACh sensitivity all along the 
fiber sxirface characterizes muscle fibers in which €0.1 electrical and 
mechanical activity has been blocked by tetrodotoxin or lidocaine. Untreated 
spontaneoiisly active fibers have somewhat lower ACh sensitivity and electri- 
cally stimulated fibers are less than one-tenth as sensitive as the treated 
inactive fibers. These changes in sensitivity are nearly complete within 2k 
hours. Morphologic study (by electron microscopy and by phase contrast or 
Nomarski cinematography) show a number of features that might account for the 

FD-3 



variation in concentration of receptor molecules in the muscle surface. 
Coarse membrane folds, microTilli and an extensive tubtLLo-vesiculaa" network 
that communicates with the extracellular space are likely candidates. Time- 
lapse cine studies have shovn that muscle nuclei move up to 300 y within 
muscle fibers at rates as fast as 1*0 u/hr. This may explain some of the ex- 
ceptions to the general rxile that high ACh sensitivity is correlated with 
xmderlying muscle nuclei. Drs. Fischbach and Whysner have shown that ACh 
receptors of cultured muscle cells bind a-bungarotoxin in the same manner as 
receptors in other cells. Toxin binding sites increase as myoblasts fuse 
with one another and reach a plateau after U-6 days . The irreversible toxin- 
receptor interaction is not altered when the muscle membrane is depolarized 
by high potassium solutions. 

h. Dr. Schrier in collaboration with Dr. Alfred Oilman has continued a study 
of the accumulation of cyclic adenosine monophosphate stimulated by the 
addition of adenosine to cultxires of rat brain cells or to neuroblastoma- 
L cell hybrids. This adenosine-stimvilated cAMP accumulation, peciiliar to 
brain of all body tissues which have been studied, is paraxodically inhibited 
by theophylline in both brsdn slices emd brain cell cultures , but not in the 
hybrid cell lines. In the brain cell cultures, changes in plating density 
which increase choline ac ety It ransf erase activity also increase the magnitude 
of the adenosine response while decreasing the rise in cAMP produced by 
catecholamines. Since glial cells exhibit the catecholamine response, it 
would appear that the adenosine response is likely to be a neuronal marker. 
The rat brain cell cultures have been shown to contain a large population of 
cells \rtiich generate typical neuronal action potentials and are synaptically 
coupled as well. Some cells in the cultures were found to contain the glial 
fibrillary acidic protein characteristic of certain brain astrocytes in^ vivo . 
These preparations, therefore, exhibit a full spectrtun of neurobiologic 
phenomena. 

Methods have been developed for a study of the synthesis, uptake, storage 
and excretion of the putative neurotransmitters y-amlnobutyric acid (GABA), 
taurine, 3-alanine and of isethionic acid. Three glioma cell lines were 
studied and compared to a brain fibroblast cell line. GABA was synthesized 
by the glial cells from glutamic acid in the medium and GABA uptake occurred 
via a saturable component with an affinity constant of 10~5m and a non- 
saturable component that was responsible for about 90/J of total uptake at 
external concentration of l60 UM. The fibroblasts exhibited only the non- 
satvirable component. All four cell lines showed 2-component uptake for 
taurine with saturable component affinity constants of 10" ^M and V^^j^ vsdues 
nearly 20-fold higher than those for GABA. The non-satiirable component wsis 
relatively less significant for taurine than for GABA uptake reaching 30-U5J5 
for external taurine concentration of 200 yM. In experiments evaluating ex- 
cretion rates it was found that cell/medium concentration ratios of 1000-fold 
would be maintained with respect to taurine. The data siiggested that glial 
cells may modulate neuronal activity by regulating levels of neuroactive 
substances in the extracell\ilar fluid. 

5. Dr. Henkart has set up the electron microscopic facility in the Behavioral 
Biology Bramch and has extended her earlier studies utilizing the EM to 
demonstrate cellular sites of calcium binding or accumulation. These studies 

FD-U 



are designed to explore the possibility that certain physiologic, metabolic 
function in neurons and other cells are regulated in coordination with mem- 
brane activity via calcixan entry or release of calcium from intracellular 
stores. She has found that the fine structure of the endoplasmic reticulxom 
of the squid giant axon changes when the divalent cation composition of the 
external medixan is altered and when membrane potential is changed. The 
relationships between subsurface cisternae of the ER and the surface membrane 
is a function of the concentration and type of divalent cation in the ex- 
ternal medium. Similar effects have been seen in the ER of Aplysia neurons. 
The observations so far suggest an analogy between the ER of neuron and the 
sarcoplasmic reticulum of muscle, which is known to be a calcium-sequestering 
and releasing system. This system plays an important metabolic regulatory 
role in mviscle, and if the analogy to the neuronal ER holds, it will be an 
important area for study in neuroned auid other cell types. 



lid. 

1 

A ■ 



FD-5 



Serial No. HD-BB2 

1. Behavioral Biology Branch 

2. 

3. Bethesda, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Studies of CNS fiuictioning in isolation reared monkeys. 

Previous Serial Nvunber: Same 

PrincipsLL Investigator: David Symmes 

Other Investigators: None 

Cooperating Units: None 

Man Years: 

Total: 0.25 
Professional: 0.25 
Others: 0.0 

Project Description: 

Objectives ; To describe eaterations in CNS function, if any, which are 
associated vith the isolation-reared syndrome in monkeys. 

Major Findings : Data collection was completed at the end of FY 1972. The 
work done on this project during FY 1973 was limited to preparation and study 
of histological material from the brains of the implanted monkeys , and to 
computer analysis of the nystagmus related abnormality observed in the iso- 
lation reared group. 

The histological evidence generally supports the interpretation that the 
spontaneous abnormalities were recorded from the vicinity of the fimbria and 
dentate gyrus of the hippocampus. Some placements were slightly dorseLL to 
the hippocampus but close enough to Indicate that stimulation of these cells 
induced after discharges of the hippocampus. The higher placements rule out 
possible damage to the hippocampus itself and therefore eliminate an irri- 
tative lesion explanation of the EEC abnormalities observed. 

Computer analysis of the nystagmus related abnormality provided quantitative 
evidence in support of the previously reported difference between isolate 
and control monkeys. An additional unsuspected finding was an asymmetry of 
"nystagmus following" - the limbic sites on the side opposite to the irri- 
gated e&r were somewhat more involved theui those on the ipsilateral side. It 
is possible that these data point to an anatomical pathway from vestibular to 
limbic structures which is abnormal. 



FD-6 



Significance to Biomedical Research and the Program of the Institute : The 
severity and persistence of the behavioral abnormalities associated with iso- 
lation rearing are well documented, yet little or no data exists on the neuro- 
chemical or neurophysiological mechanisms which may be causally related. 
Some insight into these mechanisms could have wide applicability in the dia- 
gnosis and treatment of certain childhood disorders, particulturly autism. 

Proposed Course ; The project has been inactive diiring most of FY 1973 in part 
due to the long lead time involved in securing additioned monkeys and in part 
due to controversy related to interpretation of the findings, most of which 
were reported in last year's annual project report. 

Honors euid Awards: None 

Publications : None 



7 

1 



FD-7 



Serial No. HD-BB3 (l) 

1. Behavioral Biology Branch 

2. 

3. Bethesda, Maryland 

PHS-NIH 
Individvial Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Morphologic and biochemical studies of nervous system cells 
in culture. 



Previous Serial Number: Same 



Principal Investigator; 
Other Investigators: 

Cooperating Units: 



Bruce K. Schrier, M.D., Ph.D. 

Phillip G. Nelson, M.D., Ph.D. 
Earl L. Giller, M.D., Ph.D. 

Alfred G. Oilman, M.D. , Ph.D., Dept. of Pharmacology 

University of Virginia, School of Medicine, 

Chaurlottesville, Virginia 
Samuel H. Wilson, M.D. , Laboratory of Biochemistry, 

National Cancer Institute 
A. Bignami, M.D., Vetereois Administration Hospital, 

Pathology Research, Palo Alto, California 



Man Years: 

Total: 1.5 
Professionad: 1.0 
Others: 0.5 

Project Description: 

Objectives : 1) Elucidate the mechanism of the adenosine stimulated accumu- 
lation of cyclic AMP in, and peculiar to, brain. 2) Further study of the 
development of nervous system biochemical, and electrophysiological char- 
acteristics in the brain cell culture system. 3) Evaluate the importance of 
glial contributions to nervous system function. U) Continue a search for 
biochemical markers of cell types in the nervous system. 

Methods Employed : Methods for growing brain cells in culture and assaying 
for nervous system marker enzymes have been described previously. Assays 
for intracellular cyclic AMP were performed by the method of Oilman. Cells 
containing glial fibrillary acidic protein were identified by immunofluor- 
escence by the method of Bignami, et al . Methods for detecting taurine and 
g-alanine synthesis by cells auid for collecting and counting tritiated macro- 
molecules with high efficiency were developed. 

Major Findings : 1) Investigations of hormone-stimulated accumulations of 
cyclic AMP in brain have continued with a study of the response due to 



FD-8 



adenosine in fetal rat brain cell cultures . The adenosine response , peculiar 
to brain, is characterized by an unexplained inhibition by theophylline and 
by adenosine potentiation of the response to norepinephrine. The character- 
istics of the adenosine response in brain slices were reproduced in the 
brain cell cultures, indicating that the cultures are a suitable simplified 
system for studying brain cyclic AMP metabolism. Changes in plating density 
which resulted in increased magnitude of the adenosine response eLLso increased 
the activity of choline acetyltransf erase, a neuronal marker enzyme, and de- 
creased the magnitude of the catecholamine response. Since the latter has 
been shown to be, at least in part, due to glia, the data suggest that the 
adenosine response is a neuronal marker. In a survey of adenosine analogs, 
N*-isopentenyladenosine and 2-chloroadenosine were found to produce a re- 
sponse similEO* to that stimulated by adenosine . The halogenated anedog was 
more potent, produced a greater maximal effect, and was apparently operating 
by the same mechanism as adencdine. We are presently studying the metabolic 
fate of 2-chloroadenosine in brain cell cultures in order to determine whether 
the adenosine response is receptor-mediated and/or functions through pro- 
vision of substrate for formation of cyclic AMP via substrate-limited or sub- 
strate-regulated pathways . 

2) In related studies, a theophylline-stimulated adenosine response was ob- 
served in some established cultured cell lines (mouse neuroblastoma X L cell 
hybrids produced by Dr. J. Minna). These cells were similarly more re- 
sponsive to 2-chloroadenosine than to adenosine and both responses were po- 
tentiated by theophylline. The investigation of the adenosine response in 
these cells is continuing in conjunction with the brain cell cvilture studies. 

3) In order to determine the best method for assay of macromolecular synthesis 
by cultured cells, a study was made with Dr. Samuel H. Wilson of the optimal 
conditions for filtration collection and counting of acid-precipitated tri- 
tiated macromolecules . The following results were obtained using whole, 

high molecular weight (25 x 10° daltons) or sheeu-ed (mean, 185,000 daltons) 
bacteriophage Ty ['h]DNA. (a) Counting efficiences were much higher and more 
reliable when the DNA and co-precipitant protein were dissolved in the scin- 
tillation solution prior to counting. With two solubilization-counting 
techniques counting efficiencies were independent of the amount of co- 
precipitant protein, (b) Counting efficiencies depended markedly on protein 
content when solubilization was not effected, (c) DNA collected on, and 
solubilized from, glass fiber filters counted at much higher efficiencies 
than the highest efficiencies obtained with nitrocellulose filters, (d) With 
high molecular weight or sheared DNA, collection of DNA on nitrocellulose 
filters was complete without added protein; with glass fiber filters complete 
collection of all of the sheared DNA was not attained, regardless of the 
amount of co-precipitant protein, (e) The type of filter did not appear to 
affect the magnitude of the reaction "blank" (tritiated thymidine triphos- 
phate retained by the filter). (f) Addition of competing small ^molecules to 
the precipitant and additional washes were found to lower this "blank", 
although the additional washes removed additional sheared DNA from glass 
fiber filters. It was concluded that macromolecules of small or unknown 
molecular weight should be collected on nitrocellulose filters and counted 
only after solubilization. 



FD-9 



n 



h) In order to evaluate the possibility that glioma cells might be capable of 
synthesis of the putative neurotransmitters Y-an^inobutyrate (GABA), taurine, 
isethionic acid sind S-aJLanine, methods were developed for assay for synthesis 
of these compounds by cells in c\J.ture. High voltage electrophoretic systems 
vere devised which gave adequate separations of all the compovinds from their 
precursors and from each other, except taurine from cysteine and cystine. 
Treatment of samples with bromine water was fovind to convert cystine and 
cysteine to cysteic acid, thus allowing assay of taurine as well. 

5) In a continuation of the studies with glieLL tumor cell metabolism of cer- 
tain putative neurotransmitters, the following additionsG. observations were 
made, (a) C-6 cells in cult\ire synthesized GABA and glutamine from glutamate 
in the mediiom and glutamine, but not GABA or 3-alanine, from aspartate in the 
medium, (b) GABA uptake, identically for C-6, 0-2^^ and NT-1, consisted of a 
saturable component with an affinity constant of about lO'^M, and a non- 
saturable component which was responsible for 90/t of total uptake at externed. 
concentrations of l60 UM. (c) RBF cells did not apparently have the satiirable 
component of GABA uptake, (d) All four cell lines showed two-comjKsnent up- 
takes for taurine with saturable component affinities of about 10~5 and Vj^g^ 
values neeurly 20-fold higher than those for GABA. The non-satxirable component 
was relatively less significant for taurine than for GABA uptakes, reaching 
30? of total uptake for C-2i, NT-1 and RBF cells and k^% for C-6 cells at ex- 
ternal concentrations of 200 pM. (e) In experiments to evaluate excretion 
rates it was found that C-6 and C-2i cells could maintain cell/medium con- 

f centration ratios for taurine in excess of 1000- fold. These data, with those 
previously obtained, are consistent with the possibility that glial cells in 
the nervous system may modtilate neuronsul activity by control of the extra- 
cellular levels of some neuroactive substsinces. 

6) Fetal rat brain cell cultures, treated with media containing inactivated 
horse ser\m and FUdR were foxind to contain cells which, by electrophysiologic 
criteria, resembled neurons. Evidence for the presence of functional synapses 
was €lLso obtained. The cultures also contained cells \rtiich stained for the 
"glial fibrillary acidic protein" characteristic of certain brain astrocytes 
in vivo . Thus, each brain characteristic for which we have assayed has been 
found in the brain cell cultures. 

Significance to Biomedical Reseturch and the Program of the Institute : We 
are beginning to understeind the development of the neirvoxis system and some 
of its functions via studies with culture systems. These studies are 
significant to biomedical research in that they provide simplified systems 
with which such developmental events , in healthy and diseased cells , may be 
examined. Interactions between various cell types in the central and periph- 
eral nervous systems are of basic relevance to neurobiology. In addition, an 
understanding of the genetic and biochemical control mechanisms necessary to 
regulate and/or modify these interactions will contribute significantly to 
the diagnosis and treatment of nervous system diseaises. 

Proposed Course ; The following studies are actively continuing, l) Mechanism 
of the adenosine-mediated cyclic AMP response in brain cell cultures, 2) Bio- 
chemical and electrophysiologic events and controls associated with the de- 
velopment of neuromuscular Junctions in cultured cells. 3) A continued 

FD-10 



search for new markers of nervous system function and new cell cultixre systems 
in which marker control systems may be investigated, h) Further evaluation 
of biochemical and electrophysiologic developmental phenomena in cultured 
brain cells. 

Honors and Awards: None 

Publications: 

1. Schrier, B. K.: Surface cultvire of fetal mammalian brain cells: effect 
of subculture on morphology and choline acetyltransf erase activity. J. 
Neurobiol . k: 117-121* , 1973. 

2. Shapiro, D. L. and Schrier, B. K.: Cell cultures of fetal rat brain: 
growth euid marker enzyme development. Ejcptl. Cell Res . 77: 239-2U7, 
1973. 

3. Shapiro, D. L.: Morphological and biochemical alterations in fetal rat ; 
brain cells cultxired in the presence of monobutyryl cyclic AMP. Nattire iin|l 
2l+l: 203-20U, 1973. *: 

I 



FD-11 



Serial No. HD-BBU 

1. Behavioral Biology Branch 

2. 

3. Bethesda, Maryland 

PHS-NIH 

Individusd. Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Information processing in the centreLL auditory system of 

ni ATimiftI a . 

Previous Serial Number: Same 

Principal Investigator: John D. Nevman, Ph.D. 

Other Investigators: David Symmes, Ph.D. 

Cooperating Units: Mrs. Garland Riggs 

Man Years: 

Total: 2.25 
Professionail: 1.75 
Others: 0.5 

Project Description: 

Objectives : This project is concerned with the processing of complex sounds 
by the primate cerebral cortex and related brain structures. Special atten- 
tion is being padd to sounds used by primates in intra-specific communication, 
to determine which cues aire used by the brain and the animal to distinguish 
between the various elements of the commvinication repertoire. 

Methods Employed and Major Findings ; 1) Single unit recording ; Action po- 
tentials are recorded from single cells in the auditory cortex of unanaes- 
thetized squirrel monkeys. Neuronal discharges are analyzed for excitatory 
or inhibitory effects of vocal stimuli. This year we have investigated the 
role of arousal level in influencing response patterns to species-specific 
vocalizations. In this study we monitored surface EEG and altered arousal 
level via electrical stimulation of electrodes implanted in the midbrain 
reticuleir formation. The principsd finding was that despite widespread 
fluctuations in arousal (as Judged from computed analysis of EEG) the re- 
sponse properties of single auditory neurons remained relatively constant. 
Neither spontaneous discharge rate nor responsiveness to vocal stimuli corre- 
lated with arousal level in the great majority of units studied. A small 
number of units, however, were found to be influenced by reticxilar stimu- 
lation at current levels sufficient to produce behavioral and EEG arousal 
in drowsy monkeys. These included units which became responsive to pre- 
viously ineffective vocal stimuli, and others which were restricted or 
weakened in their responses. These findings suggest that the complex feature 
detection properties of auditory cortex neurons are not characterized by 

FD-12 



great plasticity during shifts in arousal level. 

2) Behavioral training : Two studies were completed relating to the capacity 
of squirrel monkeys to discriminate between naturally occvirring variations in 
one call type, the isolation peep. This call type was chosen because of its 
demonstrated semantic significance (restoration of troop cohesion diiring 
periods of obstructed visual contact), because of its relatively simple acous- 
tic structure, and because of our finding that auditory neurons can discrimi- 
nate between isolation peep variants. The first study attempted to test the 
hypothesis that natural variations of this call type are behaviorally meaning- 
ful to the monkey. Monkeys were trained via shock avoidance techniques to 
Jump from one perch to another when one isolation peep variant was presented, 
and to refrain from Jumping when a second vsuriant was presented (i.e., a go- 
no go shuttle task). Three monkeys mastered this task readily. The foiirth 
monkey did not attain the 90 percent correct performemce level of the other 
three, but did demonstrate better than chance discrimination. The hypothesis 
in question was tested and confirmed by continuing the training with new 
isolation peep variants, without differential reward (i.e., no punishment 

for incorrect responses). All three of the monkeys demonstrated spontaneous 
discrimination of several veuriants, some of which were nearly identical in 
several physiceJ. features. By varying loudness, and using calls of differing 
durations, we established that these spontaneous discriminations were not 
based on simple acoustic features shared with the original trsLtning pair, but 
represented complex feature recognition of subtle variations in acoustic 
structure of this call type. In a second study, models of isolation peeps 
were synthesized with a PDP-12 computer, permitting control of specific com- 
ponents of acoustic structure. Using a paradigm similar to the first study, 
four monkeys were trained to discriminate between a natural isolation peep 
and an artifically synthesized model. However, all four failed to reliably 
discriminate betveen two artificial models. These results are consistent 
with other studies showing that monkeys do not attend closely to aurtificial 
acoustic stimuli. 

3) Sound spectrographic analysis ; Using the Voiceprint sound spectrograph, 
analyses have continued to assess the transfer of information by vocalizations. 
Extensive analyses of one p«dr of squirrel monkeys has shown that the acoustic 
structure of certain call typis are distinctive for each individual. Dis- 
tinctive differences are reflected in several attributes of vocal structure: 
call duration, number of syllables, and complexity of syllable structure, as 
well £is in more subtle aspects of acoustic structure. 

These findings suggest that the physical nature of vocalizations serves as 
a "vocal signature" for the individuaJ. monkey. 

Significance to Biomedical Research and the Program of the Institute : These 
findings have provided further substantiation of earlier results in this 
project that squirrel monkeys possess a complex acoustic communication 
system, that natural variations in the acoustic structure of certain vocali- 
zations are differentiated by the animal, and that many neurons in the audi- 
tory cortex possess attributes necessary for the perception of these vocal 
differences. 



FD-13 



Proposed Course ; The work to date in this project has produced a substeintial 
description of the encoding of voced-izations by neurons in the primate audi- 
tory cortex, and suggested some behavioral correlates of this neural pro- 
cessing. Future work may now begin to deal with other related questions: 
1) the role of early experience euad inheritance in the establishment of neu- 
ronal specificity to vocalizations; 2) the functional relationships of other 
brain systems (especially the frontal -limbic system) to the auditory cortex; 
3) the relationship between chemical specificity (as defined by differentied 
sensitivity to various transmitter substances) and sensory specificity (as 
defined by differential specificity to vocalizations). 

Honors and Awards: None 

Publications: 

1. Newman, J. D. and Wollberg, Z. Multiple coding of species-specific 
vocalizations in the auditory cortex of squirrel monkeys. Brain Research 
5k: 287-30U, 1973. 

2. Funkenstein, H. and Winter, P.: Responses to acoustic stimuli of linits 
in the auditory cortex of awake squirrel monkeys. Exptl. Brain Res ., in 
press. 

3. Winter, P. and Funkenstein, H.: The effect of species-specific vocali- 
zation on the discharge of auditory corticad cells in the awake squirrel 
monkey ( Saimiri sciureiis ) . Exptl. Brain Res . , in press . 

k, Evans, £. F. and Nelson, P. G. : On the functional relationship between 
the dorsfLL and ventral divisions of the cochlear nucleus of the cat . 
Exptl. Brain Res ., in press. 

3. Evans, £. F. and Nelson, P. G. : The responses of single neurons in the 
cochlear nucleus of the cat as a function of their location and the 
anaesthetic state. Exptl . Brain Res . , in press. 



FD-li* 



Serial No. HD-BB5 

1. Behavioral Biology Branch 

2. 

3. Bethesda, Maryland 

PHS-NIH 
Individual Project Report 
JvOy 1, 1972 through June 30, 1973 

Project Title: Neurobiologic studies of neuronal and other cell types in 
cell culture. 

Previous Serial Number: Same 

Principal Investigators: Phillip G. Nelson, M.D., Ph.D. 

Earl L. Giller, M.D., Ph.D. 

Other Investigators: Harris R. Fisk, M.D., Ph.D. 

Cooperating Units: Department of Biology, Yale University, New Haven, <;! 

Connecticut ;i; 

Laboratory of Biochemical Genetics, National Heeu^ X 

and Lung Institute, NIH > 

Laboratory of Molecular Biology, The Weizmann "' 

Institute, Rehovot, Israel 

Man Years: 

Total: 7-5 
Professional: 3-5 
Others : U . 

Project Description: 

Objectives : To describe neurobiologically interesting properties of normal 
and continuous lines of cells in tissue cultiire, to elucidate the molecular 
basis for these properties and to anedyze the control mechanisms regulating 
their expression. In particular, the electrophysiologic euid metabolic inter- 
actions between different cell types are of great interest. 

Methods Employed : Intracellular microelectrode recording and iontophoretic 
application of materials to the exterior and interior of cultured cells are 
used in anadyzing cell function. Subcellular fractionation and gel electro- 
phoretic separation of the protein constituents of the various subcellular 
fractions, in psurticular the surface membremes have been continued. A 
variety of ctilttire media have been studied for their capacity to support 
neuronal survival, and differentiation. 

Major Findings ; Mouse cerebellum has been extensively studied in other 
laboratories from the standpoint of maturational timetable for various cell 
types and the characteristics of a number of neurologic mutants which involve 



FD-15 



the cerebellum. We have foiind that cell cultures prepared by dissociating 
the fetal mouse cerebellum produce networks of electrically active and 
synaptically interacting neurons. Both excitatory and inhibitory postsynaptic 
potentials have been recorded and vigorous rhythmic, synaptically mediated 
patterns of electrical impulses are generated within these networks. A 
variety of morphologically and electrophysiologically defined cell types were 
seen so that some substantial representation of the neuronal constituents of 
the cerebellum should be available for study in vitro . 

A detailed study of the culture conditions needed to optimize mouse spinsJ. 
cord and muscle culture has been made as a prerequisite for investigating the 
biochemical consequences of the establishment of synaptic interactions between 
these cells. Variation in the type of serum in the medium and the timing of 
the use of different antimetabolites have been shown to be important in pro- 
ducing the best survival and differentiation of both nerve and muscle. 

Since chick spinal cord and muscle have been shown to be favorable prepara- 
tions for study in cell cultures, we have used this material for an initial 
study of a genetically determined disease, that of muscular dystrophy. Both 
dystrophic muscle and nerve develop well in cell culture and both normal and 
dystrophic nerve establish plentiful neuromuscular Junctions with dystrophic 
muscle. The incidence of such synaptic connection appeared higher them in 
normal cultures as did the incidence of electrical coupling between cells in 
these pathological cultures. The significance of these findings remains to 
be investigated, but it is an attractive hypothesis that the disease is in 
part due to a defect in the trophic function that is involved in preventing 
multiple innervation of muscle. 

Calcium ions may play an importeuit role in coupling surface membrane electri- 
cal events to cytoplasmic metabolic processes (see projects of Drs. Gainer 
and Henkart). We have found (in collaboration with Dr. Ilan Spector) that 
in some clones of neuroblastoma cells a significant proportion (10-20/J) of 
the ionic currents which flow during the action potential are resistant to 
tetrodotoxin and are blocked by cobalt which indicates that they aj-e calcium 
currents. The active electrical response in fibroblasts (L-cells) elicited 
by mechanical, electrical emd chemical stimuli also involves calcium ions, 
for intracellxilar injection of calcium ions elicits the electrical response. 
Both these cell lines and products of their hybridization represent favora- 
ble material for the study of tremsmembrane and intreu:yloplasmic calcium 
movements and the metabolic and structural consequences of such calcium move- 
ment. 

Four to five fold enrichment for surface membranes as measured by radio- 
iodine labelling of these membranes has been achieved by differential centri- 
fugation. Distinctive patterns of membreine proteins in L-cells and one clone 
of neuroblastoma cells have been shown by disc gel electrophoresis. The 
resolution of the system is good and its application to a more extensive 
range of material is indicated. 

Significance to BiomedicsLL Research and the Program of the Institute : From 
a general standpoint, the use of well-defined tissue culture systems is 
playing an increasingly important role in analyzing neurobiologic problems in 

FD-16 



cellular and moleculax terms. Neuronal differentiation and the development 
and maintenance of complex cell-cell relationships axe crucial for normal 
system function emd these processes can be studied in detail in the culture 
systems. Specifically, the relationship of surface membrane structure 
(protein composition) emd function (action potential generation and Ca++ 
fluxes) to cell metabolism and intercellular interactions are of significance 
to these broader areas. The direct study of various neuropathologic states 
with tissue cxilture models is a promising approach but the more general under- 
standing of normal developmental mechanisms is of probably greater importance. 

Proposed Course; Biochemical and morphologiceil studies of the interactions 
betveen different classes of neural cells in culture has begun and will be 
continued. What are the biochemical consequences when synaptic connections 
sure formed in the different preparation? Are neural transmitter related 
enzyme or other cell constituents induced or repressed? If so what is the 
mecheuaism? Further histochemiceil and microchemical characterization of the 
cell types occxirring in the different normal cultures will be made. 

Clonal analysis of the control of surface membrane properties such as calcium 
permeability mechanisms and chemosensitivity will be continued. The relation- 
ship between these properties and cell metabolism will be studied in neuro- 
blastoma and L-cells. We will continue the anaJLysis of membrane composition 
on a broader range of cell lines and attempt to relate these analyses to 
physiologic function in these lines. 

Honors and Awards: None 

Publications : 

1. Peacock, J. H. and Nelson, P. G.: Chemosensitivity of mouse neuroblastoma 
cells in vitro. J. Neurobiol ., in press. 

2. Peacock, J. H., McMorris, F. A. and Nelson, P. G. : Electrical excita- 
bility and chemosensitivity of mouse neuroblastoma X mouse or human 
fibroblast hybrids. Exptl. Cell Res ., in press. 

3. Nelson, P. G. and Peacock, J. H.: Electrical activity in dissociated 
cell cultures from fetal mouse cerebellum. Brain Res . , in press. 

k. Peacock, J. H., Nelson, P. G. and Goldstone, M. W. : Electrophysiologic 
study of cultured neurons dissociated from spinal cords and dorsal root 
ganglia of fetal mice. Develop. Biol . 30: 137-152, 1973- 

5. Peacock, J. H. and Nelson, P. G.: Synaptogenesis in cell cultures of 
dystrophic chick neurons and norotubes . J. Neurol. Neurosurg. Psychiat. , 
in press. 

6. Nelson, P. G. and Peacock, J. H.: Acetylcholine responses in L-cells. 
Science 177: 1005-1C07, 1972. 

7. Nelson, P. G. and Pe8u:ock, J. H.: Transmission of an active electrical 
response between fibroblasts (L-cells) in cell culture. J. Gen. Physiol ., 
in press. 

FD-17 



Serial No. HD-BB6 

1. Behavioral Biology Branch 

2. 

3. Bethesda, Maryland 

PHS-NIH 
Individual. Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Synapse formation between nerve and muscle cells in tissue 
culture . 

Pievious Serial Nimiber: Same 

Principal Investigator: Gerald D. Fischbach, M.D. 

Other Investigators: Phillip Nelson, M.D., Ph.D. 

Maryanna Henkeo^i, Ph.D. 
Stephen A. Cohen, M.D. 
John Whysner, M.D. , Ph.D. 
Anthony C. Breuer, M.D. 

Cooperating Units: Drs. V. T. Marchesi and B. Hirani, National Institute 

of Arthritis and Metabolic Diseases. 

Man Years: 

Total: h.3 
Professional: h-O 
Others: 0.5 

Project Description: 

Objectives : Our long-term objective remains the study of neuron and muscle 
cell differentiation and synapse formation in cell culture. We plan to con- 
tinue study of the sequence of events during nerve-muscle Jvinction formation, 
of factors that regulate postsynaptic membrane chemoreceptors , of the degree 
and mechanism of specificity in intemeuroned synapse formation and of the 
development of different types of electrically excitable membranes. In each 
of these areas, ultrastructuraLL and biochemical correlates of electrophysio- 
logiceLLly defined phenomena will be sought. 

Methods Employed ; Conventional cell cultxire and intracellvilar microelectrode 
techniques remain the backbone of our experimental approach. Relatively 
purified cultures (muscle and nerve cells) have been obtained by the use of 
drugs that kill dividing cells and a "low growth" culture media. 

Autoradiographic and electron microscopic techniques have been tailored to 
the cell culture system. Time-lapse cine studies of growing neurites and 
miiscle cells sure iinderway. Histologic techniques include silver, acetyl- 
cholinesterase and methylene blue stains. With Drs. Marchesi and Hirani, we 
have experimented with immuno-ferritin techniques. 

FD-18 



Major Findings ; Innervation does not change the general level of muscle mem- 
TDrane ACh sensitivity but muscle activity does. Fibers chronically stimu- 
lated through large electrodes implanted in the culture dish are less sen- 
sitive than unstimulated fibers and less than one-tenth as sensitive as com- 
pletely Inactive fibers (grovn in the presence of tetrodotoxin or lidocaine). 
Changes are nearly complete within 2k hours . 

Several specializations of the surface membrane have been identified by thin 
section and scanning electron microscopy (by M. Henkart) ^Ich may explain 
areas of increased ACh sensitivity. Coaxse membrane folds, microvilli and 
an extensive tubulo-vesicular network that communicates with the extracellu- 
lar space are likely candidates. Attempts to directly visualize receptor 
sites with a-bungarotoxin-ferritin conjugates have met with some success. 
Small clusters of ferritin (clusters of receptors) have been Identified by 
freeze etch and transmission EM techniques. Time-lapse cine studies have 
shown that muscle nuclei move long distances (up to 300 \x) within muscle 
fibers at rates as fast as kO y/hr. This may explain some of the exceptions 
in the correlation between muscle nuclei and hot spots. 

ACh receptors of cultured muscle cells bind a-bungarotoxin in the same manner 
as receptors in other cells . Toxin binding sites Increase as myoblasts fuse 
with one another and reach a plateau after h-6 days. The Initial rate of 
binding cem be reduced to less than 5% of control by pre-incubation with d- 
Tubocurare but toxin eventually overcomes the curare competition. The 
binding, which is apparently irreversible, is not altered when the muscle 
membrane, is depolarized by high K* solutions. The time course of binding is 
closely paralleled by decline in ACh sensitivity. There is no "lag" in de- 
pression of sensitivity so it is tinlikely that a large pool of "spare" re- 
ceptors exist. 

Functions^, nerve-muscle contacts have been Identified as early as 60 hours 
after addition of spinal cord cells to muscle cultures. Attempts to demon- 
strate coupling at these early contacts by injection of the dye proscion 
yellow into muscle fibers have, so far, been negative. 

Significance to Biomedical Resefux:h and the Program of the Institute : The 
mechanism of synapse formation is a bsisic question in embryology. Formation 
and interuption of synaptic coupling between nerve and various nerve and 
muscle cells has profound structural and metabolic consequences. The tissue 
culture technique offers considerable hope for anedyzing this striking bio- 
logic phenomenon. Modification of synaptic function by changes in activity 
pattern would help in understanding mechemism of neuronal pleisticity or 
learning. Before asking the relevant biochemical questions, the basic 
electrophysiological phenomena must be understood. Tissue culture has pro- 
vided a convenient system in this regard. 

Proposed Covtrse : We plan to focus on early nerve-muscle synapses to determine 
the relation between nerve terminals, hot spots and muscle nuclei and to 
further search for direct electrical coupling. Toxin-ferritin conjugates 
will be used to determine the distribution of receptors over the various 
membrane specializations described above. The purity and stability of the 
conjxigates must be determined. In addition. Intracellular sites of receptor 

rD-19 



synthesis vlll be sought by applying conjugates to protein embedded sections 
of muscle cells. The relation between activity and surface ACh receptors 
will be further analyzed after uncoupling membrane excitation from subsequent 
contraction. Combined, D2O and cobsLlt treatment (to prevent contraction) has 
been the best means so far. The distribution of sensitivity over stimulated 
innervated fibers will be determined to see if nerve termined.s maintain hot 
spots. The super-sensitivity of inactive fibers will be studied to determine 
if it is due to new synthesis of receptors or to uncovering of pre-existing 
sites. 

Honors and Awards: None 

Publications: 

1. Fischbach, G. D. and Cohen, S. A.: The distribution of acetylcholine 
sensitivity over unlnnervated and innervated muscle fibers grown in cell 
culture. Develop. Biol ., in press. 

2. Cohen, S. A. and Fischbach, G. D.: Regulation of muscle acetylcholine 
sensitivity by muscle activity in cell culture. Science , in press. 

3. Fischbach, G. D., Fambrough, D. and Nelson, P. G.: A Syii^>oslum on neuron 
and muscle cell cultures. Fed. Proc , in press. 

k. Fischbach, G. D. , Henksirt, M. , Cohen, S. A., Whysner, J. A. and Breuer, 
A.: Neuromuscular Junction formation in culture. 26th Symposium of 
Society of General Physiologists, Woods Hole, Massachusetts, 1972, in 
press. 



FD-20 



Serial No. HD-BB8 

1. Behavioral Biology Branch 

2. 

3. Bethesda, Maryland 

PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Correlative neurochemical and electrophysiological analyses 
of individual, identifiable neurons in Molluscan nervous 
systems. 

Previous Serial Number: Same 

Principal Investigator: Harold Gainer, Ph.D. 



Other Investigators: 
Cooperating Units: 



Jeffery L. Bsu-ker, M.D. 

Thomas Smith, M.D., Laboratory of Neiirophysiology, 

NINDS 
Ichiji Tsisaki, M.D., Laboratory of Neurobiology, 

NIMH and Marine Biological Laboratory, Woods Hole, 

Mass. 
Maryanna Henkart, Ph.D., Behavioral Biology Branch, 

NICHD 
Milton Brightman, Ph.D. and Lisa Prescott, M.D. 

Laboratory of Neuropathology, NINDS 
Raymond Lasek, Ph.D., Case-Westera Reserve Univ., 

Department of Anatomy, School of Medicine and Marine 

Biological Laboratory, Woods Hole, Mass. 



I 



Man Years: 



Total: 

Professional: 
Others : 

Project Description: 



2.5 
2.0 
0.5 



Objectives : To study the mechanisms by which the physiological activities of 
identifiable neurons are coupled to their macromolecular synthesis patteins. 

Methods Employed; SeversuL methods for the separation and aneLLysis of proteins 
synthesized by single neurons have been developed in this laboratory. These 
include dissection of the individual cells from the gauiglion under a low- 
pover microscope with the use of fine needles as well as mechanical homo- 
genization of the cells and chemical solubilization of their proteins. The 
analytical tools include (l) genered electrophoresis of proteins in which 
their separation is based on molecular weight and charge, (2) poly acryl amide 
gel electrophoresis of proteins in sodium dodecyl sulfate (separation based 
on molecular weight only) and (3) isoelectric focusing of proteins 



FD-21 



(separation based on charge only). All of these separation techniques have 
been developed and xised on both the macro and the micro scale, and together 
with radioisotopes of amino-acids and other labeled precursors, considerable 
information has been gathered about the protein metabolism of single neurons . 
Other "single-cell" chemical methodologies being developed in our laboratory 
include (l) ultracentrifugation of membranes and cytoplasmic contents, 
(2) methods in lipid and liproprotein biochemistry, (3) nucleotide biochemi- 
cal techniques, euid {U) intracellular isotope injection techniques for axo- 
plasmic transport studies. 

Two experimental preparations are being utilized for these studies. The iso- 
lated brain of the land snail, Otala lac tea , auid the abdominal gajiglion of 
the sea hare, Aplysia califomica . Both nervous systems contain large, 
identifiable neurons which are appropriate for combined electrophysiological- 
neurochemical studies. Conventional electrophysiological recording and stimu- 
lating methods are also employed in these studies. Some of these methods are 
(1) intracellular recording from individual, identified cells in molluscan 
ganglia, (2) stimulation techniques for the purpose of studying synaptic 
transmission, (3) manipulation of the ionic environment for the purpose of 
studying membr£uie potentials and their ionic bases and {k) voltage-clcunp 
techniques for the purpose of studying the ionic currents involved in membrane 
potentied. changes. 

In addition, the giant axon of the squid has been intracellularly perfused 
with protein radiodination solutions (i.e., lactoperoxidase techniques) in a 
search for functionally significant nerve membrane proteins. 

Major Findings ; Extensive investigations of five identified neurons in Otala 
lac tea and eight identified neurons in Aplysia califomica reveaLLed that 
individual ne\u*ons had distinct and unique patterns of protein synthesis 
(Gainer and Wollberg, in prepeuration) . In particular, one class of neurons 
appeared to exhibit sufficient plasticity to warrant a study of the effects 
of electrical and synaptic activity on their synthesis patterns. Consequent- 
ly, we have investigated whether physiological input to a nerve cell can 
selectively and significantly influence the biochemiced. processes of that 
cell. For this purpose we have chosen to study the effects of synaptic stimu- 
lation on the regulation of protein synthesis in an identified neurosecretory 
cell found in the mollusca, Aplysia califomica . The bursting pacemaker 
neuron, ^15. in the abdomineLL ganglion of Aplysia califomica exhibits a 
specific pattern of protein synthesis ^en incubated in *H-Leucine. SDS- 
polyacrylamide gel electrophoresis shows that 20-25% of the de novo synthe- 
sized proteins of the cell is accounted for by a single molecular weight 
class (around 12,000 daltons). Stimulation of the branchial nerve for 5 
hours at a rate of 5/sec, hyperpolarized the cell and blocked slLI spontaineous 
spike activity. Comparisons of the protein synthesis patterns of synaptically 
inhibited neurons and spontaneously active control neurons showed that 
synaptic inhibition produced a selective 30? decrease in the synthesis of the 
12,000 dalton pesik. The putative inhibitory transmitter, dopamine, produced 
the same selective inhibition when added to the bathing medium. Depolari- 
zation of the cell by increasing the potassium ion concentration of the 
medium, selectively stimulated the synthesis of the 12,000 deilton protein by 
5O-TO/J. Thus, the rate of synthesis of the 12,000 dalton protein in R15 may 

FD-22 



be regulated by the membrane potential of the cell, which in turn is under 
control of a dopaminergic inhibitory synapse. Studies are presently under 
way to elucidate the mechanisms underlying this regulation phenomenon. 

Studies on the protein synthesis of the giant axon of the Squid (in collabo- 
ration with Dr. R. Lasek, at the Marine Biological Laboratory, Woods Hole, 
Massachusetts) have led to the observations that the axon's newly synthesized 
protein has its origin in the Schwann cell layer immediately surrounding the 
axon. We have considerable evidence for the intercellular transfer of these 
proteins (ranging from 0.5 million to < 12,000 daltons, in preparation) and 
now are studying the regxilation mechanisms underlying these glial -nevironal 
interactions. Other studies on the squid axon (with Dr. Tasaki) have revealed 
a small, labile protein on the inner surface of the axon membrane which is 
affected by dei>olajrization (see Gainer, et^ al , Biol . Bull . , 1972). 

Our electrophysiological studies of the above-mentioned neurosecretory cells 

have revealed that divalent cations play a major and important regulatory 

role in both the bursting pacemaker potential activity of these cells and •■ 

their seasonal modulation. As part of this project, we have concluded from ^ 

our intracellular studies that bursting pacemaker potential activity is due "P 

predomineuitly to a time- and voltage-dependent potassiiun conductance, modu- 

lated by divalent cations and superimposed upon a constant sodium conductance. i. 

In collaboration with Dr. T. Smith, we &re now applying the voltage clamp ^ 

technique to an investigation of the ionic currents involved at various phases ■" 

of the bursting pacemaker potential cycle. Our results indicate that the 

current flow during the depolsurizing phase of the cycle is predominantly 

inward, being predominantly dependent on both external sodium and divalent , 

cation concentration. This current (and the bursting pacemaker potential 

activity) can be antagonized by cobalt applied externally. The flow of 

current at the start of the hyperpolarizing phase of the cycle is mainly out- 

W£urd and can be antagonized by both barium and tetraethylammonium. From these 

initial findings we have concluded that bursting pacemaker potential activity 

is primarily dependent on two membrane properties: (1) a voltage dependent 

inward divalent (calcium and magnesium) - monovalent cation (sodium) current 

closely coi5)led to (2) a voltage-dependent outward potassium current. These 

studies have demonstrated an important regulatory role for divalent cations 

both in the seeisoneil modulation of the neurosecretory cell's rhythm and the 

bursting pacemaker potentieLL itself. We plan to study the effects of divalent 

cations on the protein synthesis patterns of the neurosecretory cell in an 

attempt to better understand how the snail controls neurosecretion dviring 

aestivation. 

In collaboration with Dr. M. Henkart (electron microscopy) and Drs. M. 
Brightman and Prescott (freeze fracture and etching), we have initiated a 
study correlating the morphological changes in these neurosecretory neurons 
with their electrical, metabolic, and secretory activity. 

Significance to Biomedical Research emd the Program of the Institute : The 
approach described above is specificad-ly designed to correlate relevant bio- 
chemical changes taking place in the nejrvous system with its specific 
functional activity. The heterogeneity of nervous tissue makes the most 



FD-23 



direct approach to this problem a desirable one. In this approach individvial 
identifiable neurons can be studied electrically and biochemically thereby 
providing a direct correlation between these events - and it is hoped 
ultimately an insight into what membrane phenomena in neurons lead to long- 
term intraneuronal biochemictLL chsmges . The molluscan nervous system repre- 
sents an excellent model for not only excitation-metabolic coupling studies, 
but also as a model of hypothalamic regulatory systems in mammals . The neuron 
upon which we are focusing our efforts, is modulated by dopaminergic synaptic 
input, and appears to receive long-term negative feedback via circulatory 
steroids, and positive feedback by small peptides. Thus, in this single 
neuron, there resides a wide range of phenomenology characteristic of hypo- 
thalamic nuclei, which are amenable for detailed study in this model system. 
We believe that such model systems are essential for the aneO-ysis of regula- 
tory mechanisms in neurons and developing nervous systems. 

Proposed Course : In view of our current findings that protein synthesis 
can be selectively modulated by synaptic input, we intend to study the mem- 
brane mechanisms which are coupled into the intracellular biochemical control 
systems. After investigation of the ionic mechanism(8) underlying the initial 
step at the membreme level, we plan to study the other steps involved in the 
transduction of the signal and regulation of protein synthesis. Does the 
initial signal act at a particular reaction in the protein synthesis pathway 
(e.g., at the transcriptionaLl or at the translational level or both)? Are 
there secondary or tertieuy messengers involved in tremsduction? A major 
effort will be made to determine which sub-cellular organelles are involved in 
the activity-metabolic coupling processes, and to characterize the fxinctional 
morphology of the cell's xiltrastiructure via a veiriety of techniques (e.g., 
electron microscopy and autoradiography, freeze fracture, etc.). 

Honors and Awards: None 



Publications 
1. 



Gainer, H.: Isoelectric focusing at the 10"-^^ to 10~9 gram level. Anal . 
Biochem. 51: 61*6-650, 1973- 



2. Gainer, H., Carbone, £., Singer, I., Sisco, K. euid Teisaki, I. Identifi- 
cation of some membrane protein subunits obtained from the squid giant 
axon. Biol. Bull . lU3: U62-U63, 1972. 

3. Colbum, T. R., Wollberg, Z. and Gedner, H.: A logarithmic display of 
interspike-interval for long-term, patterned neural activity. Medical 
and Biological Engineering , in press. 

U. Barker, J. L. and Levitan, H.: The antagonism between salicylate-induced 
and pH-induced changes in the membrane conductance of Molluscan neurons. 
Biochem. et Biophysica Acta 27^: 638-6U3, 1972. 

5. Levitsui, H. and Barker, J. L. : Salicylate: A structure activity study of 
its effects on membrane permeability. Science 176: ll*23-lU25, 1972. 



FD-2U 



6. Levitan, H. and Barker, J. L. : Membrane permeability: Cation selectivity 
reversibly eLLtered by salicylate. Science I78: 63-6h , 1972. 

7. Levitan, H. and Barker, J. L.: Effect of non-narcotic analgesics on 
membrane permeability of Molluscan neurons. Nature Nev Biology 239: 55- 
57, 1972. 



I 



r- 



FD-25 



Serial No. HD-BB9 

1. Behavioral Biology Branch 

2. 

3. Bethesda, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: A correlated study of the fine structure and the function of 
the endoplasmic reticulum (ER) of neurons. 

Previous Serial Number: New project. 

Principal Investigator: Maryanna Henkart , Ph.D. 

Other Investigators: None 

None 



Cooperating Units: 




Man Years: 




Total: 


1.8 


Professional: 


0.8 


Others : 


1.0 



Project Description: 

Objectives : To develop and apply methods for demonstration with the electron 
microscope of sites of Ca binding or accumulation in order to explore the 
possibility that certain physiological and/or metabolic functions in neurons 
are regulated in coordination with membrane activity via Ca entry or release 
of Ca from internal stores . 

Methods Employed : Two systems are being studied: 1) the squid giant axon 
and 2) identified neiirons of Aplysia (R2 and Ris)* The fine structure of the 
endoplasmic reticulum is being analysed in these preparations using serial 
sectioning and section tilting techniques. The identification of Ca- 
accumulation sites is being carried out with techniques involving fixation in 
the presence of various concentrations of divalent ions (Ca, Mg), substitu- 
tion of more electron-opaque divalent ions (Sr, Co, La), and fixation in 
buffer solutions that should precipitate divalent ions of interest (POj^). 
The tissues are fixed under veurious physiological conditions produced by 
alteration of the ionic compositions of the physiological saline bathing the 
living material (e.g. depolarized by increased external K*). The changes of 
morphology of the ER under these various conditions are €Uialyzed. 

Major Findings ; The fine structure of the endoplasmic reticulum of the squid 
giajit axon changes under conditions in which the divalent ion composition of 
the external solutions are altered. The percent of surface membrane forming 
Junctions with cisternae of the endoplasmic reticulum varies with the diva- 
lent ion concentration and the divalent ion species in the external solutions. 

FD-26 



Sr is visible as dense deposits in cistemae of the endoplasmic reticulvun. 
When the axon is depolarized the ER swells in the presence of increased ex- 
ternal divalent ion concentration. The swelling produced in the presence 
of a given concentration of Sr or Ca is greater than that produced in the 
presence of the same concentration of Mg. Some similar changes have been 
observed in the ER of Aplyaia neurons . 

Significtmce to BiomediciLL Research and the Program of the Institute : The 
observations so far suggest an an&logy between the endoplasmic reticulum of 
neurons and the sarcoplasmic reticulum of muscle which is known to function 
as a Ca-sequestering amd releasing system. In muscle the Ca release is 
coupled with membrane potential changes, and incresised internal free Ca 
triggers muscle contraction and controls the activity of at least one enzyme, 
phosphorylase b-kinase that results in the mobilization of glucose from 
glycogen, providing an increase in energy available to support the muscle 
activity. If the analogy holds for nerve cells it suggests that the ER may 
be involved in coupling the electrical activity of the svirface membrane to 
intracellular activities, the coupling being mediated by Ca released from the 
ER. If this is the case it could represent a critical control mechanism for 
a number of neuronal activities - possibly including growth and differentia- 
tion. 

Proposed Course ; The function of the ER in controlling the intracellular Ca 
concentration in neurons must be further substantiated. Changes in the ER 
in electrically and synaptically stimulated neurons must be studied. In 
collaboration with Drs. Gainer and Barker the possibility that activity is 
coupled to synthesis of specific proteins via Ca will be investigated. In 
collaboration with Dr. A. Breuer we intend to test the effect of altered di- 
valent ion compositions on intra-axonsLL movement of particles directly ob- 
served with Nomarski optics. If alterations of external divalent ion com- 
ixjsition or concentration alter the rate or direction of particle movement 
the material will be fixed and examined with the electron microscope to 
ascess changes in configuration of the ER or other organelles that may be in- 
volved in particle trajisport. 

Honors and Awards: None 

Publications: None 



FD-27 



NICHD ANNUAL REPORT 
July 1, 1972 through June 30, 1973 
Laboratory of Molecular Genetics 

The Laboratory of Molecular Genetics conducts research directed toward under- 
standing the molecular processes involved in the transmission of genetic infor- 
mation from generation to generation and in the regulated expression of this 
genetic information in cells during growth and differentiation. A variety of 
model systems are under investigation in the laboratory, ranging from bacterial 
and animal viruses to complex vertebrate regulatory responses involving the 
synthesis of specific antibodies and the development of specific organ systems. 
Taken together, the laboratory joins active workers in the areas of genetics, 
biochemistry, immunology, virology and cellular biology to bring their skills 
to bear on problems of mutual interest. During the past year, the Laboratory 
of Molecular Genetics has, for the first time, had an opportunity to join to- 
gether in a single building. At present, the laboratory is comprised of seven 
research groups. Their activities are described in the phylogenetic order of 
the biologic systems with which their research is primarily concerned. 

A, The process of integration and excision of the phage A has been shown to 
occur at a number of sites on the E^. coli chromosome when the normal attachment 
site from A is removed. This integration has been shown to occur at specific 
sites in the £. coli chromosome (integration is nonrandom) . The process of 
integration and excision from these sites requires the same phage gene neces- 
sary for normal integration and excision. This process is not influenced by the 
recombination systems controlled by the bacterial host chromosome. The process 
by which the A chromosome is organized into the phage particle has been investi- 
gated also. When the normal processing mechanism is interrupted, phage particles 
appear to accumulate a "headful" of A DNA, and processing, that is cutting, of 
viral genome occurs by alternative mechanisms. These investigators have also 
shown that host chromosomal material can be incorporated into the A genome. 

This observation provides the initial evidence that phage A can be used as a 
generalized transducing phage. In addition, the genes necessary for the forma- 
tion of the viral particle have been examined and evidence suggests that these 
proteins are produced in a coordinate fashion suggesting the existence of con- 
trols at some post-transcriptional level. Taken together, these results give 
a much clearer picture of the process of viral insertion and excision and the 
elements necessary for the packaging of the viral chromosome in immature virus 
particles. 

B. Factors which influence the initiation and termination of genetic transcrip- 
tion have been further investigated in terms of regulatory proteins and ribo- 
nucleases. Specifically, an extremely interesting ribonuclease, ribonuclease H, 
has been isolated from a number of sources. This enzyme specifically degrades 
RNA which forms a duplex with a strand of complementary DNA. Inasmuch as RNA 
transcription is thought to be intimately related to gene replication, this 



FF-1 



enzyme may have an important role in the process of chromosomal replication. 
Ribonuclease H has been shown to be an integral part of the avian myeloblasto- 
sis reverse transcriptase enzyme. This enzyme has also been isolated from £. 
coli , and a determined effort has been made to correlate it with one of the 
temperature sensitive mutants defective in DNA replication. None of these seem 
affected. In addition, ribonuclease H activity has been found in association 
with DNA polymerase I of £. col i , but not with the DNA polymerase which can be 
isolated after the infection of E_. coli with bacteriophage T4. In the course 
of this work, a factor has been identified from £. coli which depresses the 
level of RNA synthesis and which may be related to the physiologic termination 
of mRNA synthesis in E. coli . 

C. Studies on the integrative control of macromolecular synthesis have result- 
ed in the development of a number of interesting observations. Two unusual 
guanine nucleotides, ppGpp and pppGpp, have been thought to function in the 
regulation of protein and nucleic acid biosynthesis in bacterial cells. This 
year, purified cell-free systems have been devised which indicate that a por- 
tion of the protein synthetic apparatus of the cell is directly involved in 
the biosynthesis of these nucleotides. Specifically, the synthesis of ppGpp 
requires highly purified ribosomes, transfer and messenger RNA as well as ap- 
propriate phosphate accepting molecules. The fact that these nucleotides are 
synthesized by ribosomes "at rest" correlates well with the physiologic obser- 
vation that these nucleotides accumulate when protein synthesis is inhibited. 
Studies on the mechanism of phosphorylation suggest that this occurs by pyro- 
phosphorylation. In addition, the protein synthetic system can be used to 
prepare large amounts of these compounds for further study. 

These guanine nucleotides have been shown specifically to stimulate several 
bacterial operons which can be transcribed and translated in a cell -free system. 
Evidence suggests that this regulation is affecting the transcription reaction, 
namely RNA polymerase activity. Additional experiments have shown that the 
guanine nucleoside ppGpp can interact with the elongation factor Tu, but that 
it will not substitute for pppG in protein biosynthesis. In contrast, pppGpp 
can substitute for pppG in several of the protein biosynthesis reactions. 

Taken together, these results suggest a close metabolic connection between the 
processes of translation and transcription, possibly involving mediation by 
ppGpp or pppGpp. 

D. The process of regulated gene expression has been investigated in viral 
systems as well. The simplest of these has been the human adenovirus, a rela- 
tively small DNA-containing virus which serves as a model for the regulated ex- 
pression of a group of genes in higher organisms. Work with this system began 
in September, 1972, and results are, therefore, preliminary at this point. The 
basic strategy involved has been to isolate specific adenovirus mRNAs and to 
correlate these mRNAs with specific viral proteins using cell-free systems to 



FF-? 



identify products. The specific mRNAs will then be used to establish a genetic 
and regulatory map of the adenovirus genome. Thus far, adenovirus mRNA has been 
isolated using oligo-dT-cellulose affinity chromatography. This mRNA has been 
translated in the Krebs ascites II cell -free system, giving rise to a unique and 
reproducible pattern of bands in polyacryl amide gels. The analysis of these 
products and their correlation with specific viral proteins is underway. 

E. Certain aspects of the process of differentiation have been studied using 
embryonic chick lens fibers in vivo and in tissue culture. These studies have 
been directed towards the description of the process of normal differentiation 
of lens fibers and towards understanding these regulatory mechanisms at the 
molecular level. During the past year, these workers have shown that the ter- 
minal phases of lens fiber differentiation can occur in tissue culture. These 
results correlate well with previous studies of this group indicating that the 
initial stages of this process can also occur in this tissue culture system. 
This observation has been coupled with the finding that insulin can substitute 
for serum in the culture medium stimulating the assembly of microtubules and the 
elongation of the lens fiber cells. In contrast to serum, insulin does not 
promote the de novo synthesis of delta crystallin. Additional studies have been 
directed toward the isolation and translation of delta crystallin mRNA. mRNA 
has been isolated from purified lens fibers and eluted from oligo-dT-cellulose. 
This material has been shown to direct cell -free protein synthesis in the Krebs 
ascites system. Studies are currently underway to characterize the product and 
to identify it as chick lens delta crystallin. If these studies are successful, 
the mRNA will be further purified and transcribed using the avian myeloblastosis 
reverse transcriptase. This cDNA can then be used in studies viewing the regu- 
lated appearance of delta crystallin mRNA in tissue culture material. 

F. Studies directed towards understanding the regulated expression of the 
globin and immunoglobulin genes have permitted workers to draw several important 
conclusions. These studies have indicated that the reverse transcript of puri- 
fied globin message can be used as a highly specific and sensitive probe for 
quantitating specific globin genes and mRNA. These studies indicate that the 
number of genes corresponding to globin in a variety of avian and mammalian 
tissues is rather small, 3 to 5 globin genes per diploid genome, and is not 
amplified during the process of differentiation. With respect to globin genes, 
the gene amplification hypothesis appears to be ruled out. Further studies 
have indicated that the process of differentiation can be studied using tissue 
culture cells. These studies suggest that the transcription of the globin genes 
represents a rate limiting step in the process of differentiation of these cells. 
Globin message has been quantitated and rises from an undetectable amount to 
3,000 to 6,000 molecules per cell in the fully differentiated state. 

Further studies of the immunoglobulin light chain message suggest that it is a 
poly(A) containing mRNA which directs the synthesis of both the constant and 
variable regions of the immunoglobulin light chain. This mRNA can also be 



transcribed using the reverse transcriptase. Hybridization analyses using this 
cDNA suggest that the mRNA preparations, in contrast to those of the globin mRNA, 
are not yet pure. Further studies indicate that there is a sequence of cDNA 
present which corresponds to a highly reiterated component of the mouse genome. 
The implications of these data are being considered in the light of models 
which can be used to explain the diversity of antibody molecules. 

The techniques developed in the course of these studies have had direct impli- 
cations for examining the pathophysiology of thalassemia, an inherited human 
anemia. A number of laboratories have been using the reverse transcript of 
human alpha and beta globin mRNA to quantitate the respective globin mRNAs in 
reticulocytes of infected individuals. 

G. Antibody biosynthesis has also been studied at the cellular level . Experi- 
ments have been carried out to clarify the role of specific cell types in the 
induced immune response. These have demonstrated that a derivative of cyclic 
AMP can amplify the number of B type cells responding to specific immunization. 
In addition, studies which indicate certain antigens have an initial suppres- 
sive T cell which may regulate the activity and numbers of B cells involved in 
a specific immune response. One extremely important observation made by these 
workers relates to the in vitro production of antibodies by immunocytes. It 
seems that a number of plaques noted using the plaque-forming center assay with 
rabbit spleen cell cultures may be due to pre-formed background antibody which 
is present in homologous rabbit serum used in all these assays. This observa- 
tion has great importance for all studies of in vitro antibody formation. It 
will doubtless cause considerable re-thinking of experiments carried out in 
this area. 

Preliminary studies have suggested that the antibody required to develop and 
visualize 7S IgG secreting cells is itself a high affinity IgG antibody with 
major anti-Fc activity. In addition, an antiserum containing a strong sup- 
pressive activity directed towards 19S IgM placque-forming cells has also been 
discovered. The active agent appears to be a high affinity IgG with a major 
anti-Fab activity. These reagents are now being prepared using isolated immuno- 
globulin fractions as antigens. As such, they may have relevance to the mech- 
anism of initiation and suppression of the respective immune responses. 



The Laboratory of Molecular Genetics has past its first year in our new Build- 
ing 6 location. The new groups now located adjacent one another have created 
an impressive and exciting scientific atmosphere. A great deal of productive 
exchange between working groups has occurred. The organizational phase of the 
laboratory's activities having passed, we now look forward to the further de- 
velopment and expansion of the younger research groups. As a number of us are 
interested in taking advantage of the electron microscope for purposes of genetic 
mapping, we hope to attract an excellent scientist who is an expert in this 
area during the coming year. 

FE-4 



Serial No. HD-LB16 

1. Laboratory of Molecular Genetics 

2. 

3. Bethesda 

PHS-NIH 

Individual Peoject Report 

July 1, 1972 through June 30, 1973 

Project Title: Control Mechanism in Temperate Bacteriophage A 

I. The control of normal phage integration and excision 

II. Secondary pathways of phage integration and excision 

III. The physiology of abnormally excised phage 

IV. Packaging of viral and bacterial DNA by bacteriophage X. 

Previous Serial Number: Same 

Principal Investigator: R. Weisberg 

Other Investigators: N. Sternberg 

Cooperating Units: Laboratory of Molecular Biology, Section on 

Biochemical Genetics, NCI 

Dr. Ronald Luftig 

Department of Microbiology, Duke University, 

Durham, North Carolina 

Dr. Kazunori Shimada 

Department of Biological Sciences, Stanford 

University, Palo Alto, California 

Man Years 






Total: 24/12 
Professional: 24/12 
Others: 

Project Description: 

Objectives : To understand the mechanism of the insertion of virus DNA into 
and its excision from the chromosome of its host; to understand the control 
the phage exerts over the synthesis of proteins which are required for these 
processes; to study the nature of abnormal phage insertion and excision with 
the aims of understanding the normal process, on the one hand, and of deter- 
mining the feasibility of using abnormal insertion as a tool for biochemical 
analysis of bacterial gene products on the other; to analyze the mechanism of 

FE-5 



conversion of the virus nucleic acid to a mature, infectious virus particle. 

Methods Employed : The organisms of study are bacteriophage X, other genetically 
related phages, and their host, the bacterium £. coli . Genetic techniques are 
being used to obtain cells which carry virus DNA inserted at abnormal chromo- 
somal sites; from such abnormal lysogens, we obtain novel types of transducing 
phage lines (phage strains whose chromosomes contain DNA of bacterial origin). 
Phage mutants with specific chromosomal aberrations are being used to charac- 
terize the activity and stability of the factors which govern insertion and 
excision. Temperature sensitive control mutants are being used to study the 
regulation of the synthesis of these factors. Density gradient analysis and 
electron-microscopic visualization of DNA are being used to determine the struc- 
ture of the chromosome of a newly isolated phage variant. A bacterial mutant 
which is defective in one of the steps of virus morphogenesis is being used to 
characterize the cellular structures required for viral development. Likewise, 
morphogenesis-defective phage mutants are being used to determine the sequence of 
steps involved in the synthesis of the x head. 

Major Findings : (1) In the course of lysogenization by phage A, the viral 
DNA is inserted into the bacterial chromosome at a specific chromosomal site 
called the attachment site. We have shown that insertion still occurs, al- 
though at reduced frequency, in mutant bacteria which have lost the attachment 
site. Such abnormal insertion can occur at many different chromosomal loca- 
tions, although it otherwise resembles normal insertion. Ocassionally, ab- 
normal insertion of the virus DNA occurs within a bacterial gene, thereby 
inactivating the function of that gene. Not all genes are equally likely to 
be inactivated: the frequency of inactivation for different genes varies over 
a greater than 10^-fold range. This highly nonrandom pattern of inactivation 
suggests that abnormal insertion occurs preferentially at a limited number 
of sites. We have confirmed this hypothesis by precisely locating a repeatedly 
used insertion site within a particular gene. (2) We previously found that 
insertion of viral DNA into a bacterial gene is a precisely reversible process. 
We know this because loss (or excision) of the viral DNA lead to restoration 
of gene function (see last year's report). Excision from within a gene re- 
quires the same virus-specific proteins that are required for excision of x 
DNA from the normal attachment site: the int and xis proteins. By increasing 
the power of our selective technique, we have now shown that gene function is 
restored in fewer than one cell in 10^° when int or xis protein is lacking. 
This result shows that excision of the viral DNA is not promoted to any 
measurable extent by recombination systems controlled exclusively by bacterial 
genes. This finding has implications with respect to the structure of the 
attachment site. (3) It is known that x DNA replicating inside an infected 
cell exists as oligomers of several x chromosomes joined head to tail. Dur- 
ing morphogenesis these oligomers are cut into monomers and packaged by 
structural proteins of the phage. Cutting occurs at a unique site called the 
end-join. In circumstances where formation of oligomeric DNA is prevented, 
we believe that X DNA is cut and packaged in a different way. One cut is in- 

FE-6 



f 

i 

L 



deed made at the end-join, but the second cut, instead of occurring at a 
second end-join, seems to occur in such a way that the amount of DNA that is 
finally packaged corresponds to the maximum amount of DNA that can be accommo- 
dated inside a a head (a "headful"). We also have evidence suggesting that 
this method of packaging can be used, although with low efficiency, to form 
virus particles containing DNA derived entirely from the host chromosome. The 
headful mechanism of packaging is a normal feature of the life cycle of several 
groups of viruses not closely related to X but had not previously been observed 
for A. (4) There are at least seven phage genes (A, W, B, D, E, and F) and 
one host gene (groE) which are essential for the packaging of a DNA and the 
formation of the A head. We have shown that a mutation in the groE gene 
interferes with cell growth and drastically reduces stable RNA synthesis, 
in addition to its effect on phage head formation. The RNA synthesis defect, 
however, also requires a second as yet unidentified genetic determinant. 
Genetic analyses suggest that the head formation defect of groE mutants is 
the result of a faulty interaction between phage head proteins B and C. The 
faulty interaction can be partially compensated for by virus mutations which 
lower the level of gene £ protein. Consistent with this hypothesis are the 
following observations: (a) the low levels of active phage made in groE hosts 
may lack the phage gene C protein and (b) false revertants (genetic alterations 
reversing the affect of a mutation but which are located at sites distinct 
from that mutation) of mutations in genes B and C map in gene E and visa-versa. 
Thus, a host function can play a role in phage A head formation and we can be- 
gin to specify what that role is. (5) A "head" proteins are made in vastly 
differing amounts, but yet there appears to be no significant difference in 
the mRNA levels corresponding to these proteins. This argues for some sort 
of translational control mechanism. We have shown that the level of a particu- 
lar "head" protein (W) is controlled by the level of a second "head" protein 
(B) . This suggests a model, one protein controlling the translation of a second 
functionally related protein, which could account for some of the differences 
in the amount of different proteins. 

Significance to Biomedical Research and the Program of the Institute : The 
insertion and excision of viral (and other episomal) DNA is known to change 
the physiology of the host organism. In addition, these viruses (and other 
episomal elements) provide an important mechanism of genetic transfer and 
exchange in the microbial world. Viruses promote genetic transfer because 
of their ability to convert host genes to an infectious form by packaging them 
inside viral structural proteins. It is highly likely that such a successful 
symbiotic relationship has an analogy in the relationship between certain 
animal viruses and the cells of higher organisms. In fact it is known that 
animal viruses can package the DNA of their hosts although the biological 
significance of this phenomenon is unknown. Moreover, an understanding of 
the control mechanisms operative in these easily studied microorganisms has 
obvious relevance to an understanding of regulatory elements in higher organisms. 
The ability to direct the insertion of phage DNA to predetermined locations on 

FE-7 



the host chromosome should prove to be an extremely useful and widely appli- 
cable method for isolating gene products and studying the control of bacterial 
gene expression. 

Proposed Course of Project : We intend to further characterize the mechanism 
of phage DNA insertion into abnormal sites and its relation to normal insertion. 
We hope to construct new transducing phages for regions of the bacterial chromo- 
some that are of particular interest (see last year's report). We plan to 
further characterize the phage particles which we believe are formed by the 
"headful" mechanism with the aim of testing the headful model for a. We plan 
to continue studies initiated several years ago on the properties of a x 
variant unable to circularize its DNA. We plan to determine the nature of 
the cellular component affected by the groE mutation and how it can affect such 
diverse cellular functions as stable RNA synthesis and phage x head formation. 
We also intend to confirm and extend present findings of translational control 
for phage proteins using in vitro translation systems. 

Honors and Awards : None 

Publications: 

1. Shimada, K., Weisberg, R. and Gottesman, M. (1973). E. coli Mutants 
Produced by the Insertion of Bacteriophage A DNA. Genetics , in press. 

2. Sternberg, N. (1973). A bacterial mutant defective for phage A head 
formation ( groE ). I. Initial characterization. J. Mol . Biol . , in 
press. 

3. Sternberg, N. (1973). A bacterial mutant defective for phage A head 
formation ( groE ). II. The propagation of phage . J. Mol . Biol . , 
in press. 



FE-8 



Serial No. hd-lmg-2 

T . Laboratory of Molecular Genetics 

2. 

3. Bethesda 

PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Factors Influencing Genetic Transcription-Initiation and 
Termination 

Previous Serial Number: Same 

Principal Investigator: Robert J. Crouch, Ph.D. 

Other Investigators: None 

Cooperating Units: Cold Spring Harbor Laboratory, Cold Spring Harbor, S 

New York. Dr. Walter Keller Z 

Z 



Man Years 



Total : 1 ■Z 

Professional: 1 "*' 

Others: 



<!,}« 



Project Description: 

Objectives : The study of the coordinate expression of several genes during 
the development of the bacteriophages T4 and lambda has led to many current 
concepts of gene regulation. Several proteins have been identified as pos- 
sible candidates for controlling transcription-one of these, termed "rho", 
acts in vitro by terminating transcription- thereby inhibiting the expression 
of genes proximal to these stopping points. Recently a second protein, kappa, 
has been shown to have similar properties to rho- and as reported in last 
years annual report, I have isolated a separate protein with similar properties 
but not identical to rho or kappa. The objective of the research reported in 
this project is to discover how these proteins are related to in vivo regula- 
tion-specifically by determining how the proteins function in vTtro . A 
second objective reported in this project is to relate a protein, ribonuclease 
H (RNase H) to some cellular function. RNase H has several possible functions 
related to gene regulation. 

Methods Employed : Rho, kappa and what I call depression factor are all puri- 
fied by standard techniques of ion exchange chromatography and sizing. 
RNase H assays are performed by degradation of P labeled rA'dT and separa- 
tion of the degradative products by thin layer electrophoresis on polyethyl- 
eneimine sheets. This technique allow us to distinguish between RNase H and 
other enzymes capable of attacking the substrate. 

Major Findings : 

FE-9 



a). Progress in 1973 

1 . Demonstration that AMV reverse transcriptase also contains as an 
integral part of the protein a ribonuclease H activity. The RNase 
H of AMV is inhibited by antiserum against AMV reverse tran- 
criptase but a similar RNase H from chick embryos is not inhibited 
by the same antiserum indicating a separate origin for the viral 
RNase H activity. 

2. Demonstration of the presence of RNase H activity in £. coli and 
a search for the function of the enzyme. With the exception of 
one gene, all genes which now have DNA ts mutants of unknown 
function have been examined for altered RNase H's - with a complete 
negative result thus far. 

3. RNA degrading activity of E. coli DNA polymerase I and T4 DNA poly- 
merase have been studied. "DNA polymerase I of £. coli can degrade 
RNA of DNA- RNA hybrids and also the DNA of DNA-RNAliyB"rids. T4 DNA 
polymerase is unable to degrade the RNA of DNA-RNA hybrids. 

4. Ribonuclease III has been studied to separate it from RNase H of 
E. col i . Ribonuclease III can not degrade rA-dT. This latter 
Tinding with RNase III is either a contaminant or has some base 
specificity. 

5. A factor from E. coli which depresses the level of RNA synthesis 
in vitro has been partially purified and characterized. The 
protein differs from rho and a similar factor, kappa, in a number 
of ways. Several properties indicate a function, in vitro , similar 
to that ascribed to rho. 

b). Direction of Research in 1974 

T. Continuation of E. coli RNase H studies-changes after infection 
with T4, T7, and X. RFat happens to RNase H with ts DNA mutant 
shifts from permissive to nonpermissive and than back again? 

2. Ribonuclease III - can it degrade DNA-RNA of fix and what are pro- 
ducts of degradation? 

3. Further purification of depression factor and nature of action. 

Significance to Biomedical Research and the Program of the Institute : The 
regulation of transcription can be essential in controlling gene expression 
in most organisms. Certainly changes in gene activity medicated through regu- 
lation of transcription can affect normal cellular activity while failure to 
regulate properly or to reorient transcription can be the basis of many medical 
abnormalities. 

Honors and Av^ards : (Robert Crouch) 
r Invited Lectures : 

a. Cold Spring Harbor Laboratory, July 1972 

b. University of Washington, Seattle, Feb 1973 Genetics Department 

c. Genetics "Group" Michigan State University, East Lansing Michigan 
March 1973 

Publications : 

1. Article published in Periodical 

a. Keller, W. and Crouch, R., Degradation of DNA-RNA Hybrids by 
Ribonuclease H and DNA Polymerases of Cellular and Viral Origin, 

FE-10 



Proc . Nat . Acad . Sci , 69: 3360-3364, 1972 

2. Article published in book 

a. Keller, W. and Crouch, R. The Relationship of Viral to Cellular 
Ribonuclease H, 4th Lepetit Col log . 1n press. 









FE-ll 



Serial No. hd-lmg-i 

1. Laboratory of Molecular Genetics 

2. 

3. Bethesda 



PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Integrative Control of Macromolecular Synthesis 

Previous Serial Number: Same 

Principal Investigator: Michael Cashel , M.D., Ph.D. 



Other Investigators: 
Cooperating Units: 



H.A. Raue, Ph.D. 

Ernest Hamel , M.D., Ph.D. 

Department of Biological Sciences, Columbia 

University; 

Department of Chemistry, University of Minnesota; 

Department of Biochemistry, School of Medicine, 

University of Minnesota; 

Roche Institute of Molecular Biology 



Man Years 



Total 



3.6 

Professional: 2.6 
Other: 1.0 

Project Description 

Objectives : The broad objective of this project is to understand physiological 
cellular response to changes in nutritional and environmental conditions. The 
ensueing coordinate adjustments of cellular components and activities to allow an 
adaptive transition to renewed cellular growth and avoidance of cellular injury. 
Specifically we are continuing to explore the roles played by unusual guanine 
nucleotides ppGpp and pppGpp which apparently can function as "coarse tuning" 
regulators of major cecllular synthetic processes as well as also showing some 
highly specific regulatory functions. As before, our approach to the role of 
these nucleotides is subdivided into three areas: 1) biosynthesis mechanism; 
2) regulation of genetic activity and 3) regulation of metabolic activity. 

Methods Employed : In addition to classical biochemical methods, solubility 
gradient techniques have been further refined using solid matrix immobilization 



FE-12 



of insoluble precipitates. We are also exploring centrifugal gradient solu- 
bilization approaches which combine the solubilization gradient with a density 
gradient which limits diffusion of protein molecules to the point of solubiliza- 
tion. The permeable cell techniques discussed earlier have been optimized and 
now give access to new preparative routes of nucleotide synthesis as well as 
analytical questions. Methods for the study of the partial reactions in protein 
synthesis have been brought to the laboratory by Dr. Hamel . Through collabora- 
tions we have gained access to the particular expertise of other laboratories 
regarding carbon natural abundance nuclear magnetic resonance spectroscopy, 
coupled transcription-translation systems, phospholipid synthesis with insoluble 
membrane systems and further studies with particular homogeneous protein systems, 
such as EF-Tu and EF-G. Finally, knowledge of the ribosomal dependent synthesis 
reaction has been exploited for more efficient preparative synthesis of ppGpp as 
well as for synthesis of pppGpp and hydrolysis resistant derivatives, such as 
pcppGpp. 

I. Biosynthesis of ppGpp 

Early in the project year, the in vitro synthesis of ppGpp and pppGpp 
was reported by (Haseltine, Block, Gilbert and Webber, Harvard Univ.) to require 
ribosomes, GDP (or GTP) and ATP substrates, EF-G and a protein factor eluted 
from ribosomes with high salt. Using this starting point, we have confirmed 
this finding and established the identity of ppGpp produced in cells with the 
in vitro product. The protein factor catalyzing the ppGpp synthetic reaction 
has been purified to apparent homogenity as judged by SDS gel electrophoresis. 
The factor has a molecular weight of 80,000 in such gels and migrates as a mono- 
mer in Sephadex G-lOO columns equilibrated in 1 M NHi^Cl . Under optimal assay 
conditions, the protein factor has a specific activity of 25 moles of product 
synthesized/sec/mole factor. This rate is nearly equal to the rate of peptide 
bond formation per ribosome. The availability of pure factor enable a deter- 
mination of reaction requirements in a relatively pure system. 

1. Reaction requirements have so far been determined as follows: 

A) High salt washed ribosomes : A requirement for extensively (5X) 
washed ribosomes in a 1 M NH^Cl is readily demonstrable. Interestingly, the 
ribosomal requirement for ppGpp synthesis is less fastidious than the ribosomal 
requirement for protein synthesis. Ribosomes can be treated so as to abolish 
their ability to participate in protein synthesis without altering their ability 
to promote ppGpp synthesis. Stoichiometric studies reveal saturation of factor 
activity at ribosome: factor ratios of less than one suggesting either that the 
factor acts catalytically or that not all the ribosomes are active. The minimum 
ribosomal unit capable of acting in this regard is being determined. 

B) Transfer RNA and messenger RNA : With the purified system an absolute 
requirement for uncharged tRNA is observed and cannot be replaced by aminoacyl- 

FE-13 



ated tRNA. This differential effect of the two forms of tRNA verifies pre- 
dictions based on ppGpp synthesis in whole cells. A second RNA requirement 
exists which so far is not yet absolute; a requirement for messenger RNA codons. 
This requirement can be satisified with either polynucleotides or triplets. The 
codon requirement relates to the tRNA requirement in that the latter is codon- 
specific. Thus uncharged phe-tRNA will stimulate only in the presence of poly U 
but not in codons lacking UUU codons such as in (A,G,C) . The stoichiometry and 
structure activity relationships for the tRNA are being investigated. 

C) Substrate requirements : The phosphate-accepting substrates are limited 
to GDP, GTP and pcppG; no other purine ribo- or deoxyribo-nucleoside 5' di- or 
tri-phosphate will substitute. The phosphate donating substrate seems specific 
to ATP and no activity is seen with dATP, ADP, XTP, ITP, pcppA or pnppA. 

2. Complementation in a mutant system : Even low-salt washed ribosomes 
derived from mutant strains unable to synthesize ppGpp during amino acid star- 
vation also show no synthesis of ppGpp in vitro . Addition of apparently pure 
factor derived from stringent strains restores the mutant capacity to enable 
the synthesis of ppGpp. This complementation result seemingly accounts for 
observations that the rel allele is dominant over the rel allele. It seems 
very likely that the ppGpp synthetic factor is after all the product of the rel 
gene. 

3. The reaction is independent of EF-G : Although the Harvard group 
originally reported that EF-G was required for the ppGpp synthetic reaction, 
there are several criteria indicating this is not the case for the purified 
system. The purified system is judged to not be contaminated with EF-G for 
lack of i) GTPase activities, ii) an appropriate band after SDS gel electrophor- 
esis of 100 X the amounts of reaction constituents and iii) immunoprecipitation 
with authentic anti-G antibody under conditions which detect EF-G admixtures at 
ratios of 1 mole % of ribosomes. The reaction is i) not inhibited by concentra- 
tions of anti-G antibody which abolish 85% of GTPase activity of added EF-G, 

ii) neither stimulated nor inhibited by adding authentic EF-G to the reaction, 
iii) not inhibited by concentrations of fusidic acid which inhibit 90% of the GDP 
binding activity of EF-G and iv) not inhibited by removal of ribosomal proteins 
(L-7 and L-12) which are required for EF-G ribosomal dependent activity. 

4. ppGpp synthesis occurs by pyrophosphorylation : Studies on the mechanism 
of phosphorylation of GDP and GTP to produce ppGpp and pppGpp respectively in- 
dicates that the reaction proceeds as a pyrophosphate transfer reaction rather 
than two successive single phosphorylations by ATP. The relative uniqueness 

of pyrophosphate transfers in the construction of polymeric phosphates makes 
this reaction of some biochemical interest, particularly because the 6 phos- 
phage residue of ATP becomes inserted solely as the a residue on the ribose 3' 
position of the product. 



FE-14 



5. In vitro preparative synthesis of ppGpp, pppGpp and pcppGpp : The 

in vitro ribosomal reaction has been exploited as a preparative tool because 
of its very high specific activities. In a reaction which is linear for hours, 
1 mg of factor together with ribosomes catalyses the conversion of about 3-1/2 
g of GTP to product every 15 minutes. The reaction allows preferential synthesis 
of ppGpp from GTP by inclusion of EF-G, preferential synthesis of pppGpp by 
inclusion of pyruvic kinase and PEP, and the synthesis of pcppGpp by substituting 
pcppG for GTP as the phosphate accepting substrate. The preparative route has 
been modified from that used with earlier fermentor cultures so as to allow reuse 
of the ribosomal bound factors for repeated syntheses. In this manner it is 
relatively simple to produce gram amounts of these nucleotides and derivatives, 
thus facilitating studies of their effects. 

6. C13/C14 NMR structure studies : In collaboration with Drs. G. Gray 
and J. Bodley we have examined synthetic ppGpp under carbon natural abundance 
NMR spectra. Such measurements can detect the ribose positions phosphorylated 
as giving a broadening of a spectral peak corresponding to positions adjacent 
to those phosphorylated. Such spectra allow an unequivocal assignment of one 
pyrophosphate in ppGpp to the ribose 5' position and the other to the ribose 3' 
position. Thus the structure of the compound is ppG3'pp, confirming conjectures 
made earlier in these reports. 

7. The in vitro synthetic reaction relates well to cell synthesis : The in 
vitro ppGpp synthetic reaction, even at this early stage of characterization ap- 
parently can account for much of what was known of ppGpp synthesis from in vivo 
evidence. For example, the relatively enormous specific activities are expected 
as are requirements for messenger RNA and uncharged tRNA. Even the existence of 
a pyrophosphorylation reaction was deduced from cellular chase kinetics. Of 
great interest is the fact that the majority of the factor is ribosomal bound 

to low salt washed ribosomes and not found in S 100 fractions. Also that the 
reaction is inhibited by tetracycline (as in cells) but not by chloramphenicol, 
which does inhibit ppGpp synthesis in amino acid starved cells. These last two 
feactures are not predicted as is the intriguing dilemma posed by the apparent 
specificity of the reaction for guanine nucleotides coupled with the noninvolve- 
ment of the elongation factors in protein synthesis, which are thought to gen- 
erate this specificity of protein synthesis. Apparently there are alternative 
mechanisms of generating this specificity as well. 

II. Regulation of Genetic Activity 

Previous project reports have drawn parallels between the inverse relation 
between ppGpp levels in cells and the rates of RNA accumulation on one hand and 
the ability of ppGpp to specifically bind to the initiation site on RNA polymerase 
so as to inhibit RNA chain initiations starting with GTP on the other. We have 
collaborated with Dr. G. Zubay in order to explore the powerful in vitro analysis 
of genetic expression in crude extracts capable of coupled transcription and 

FE-15 



translation. Such extracts are capable of hydrolyzing pppGpp to ppGpp quite 
rapidly, while both ppGpp and pcppGpp are very stable. The addition of ppGpp 
(or pppGpp which is converted to ppGpp) to this system results in promoter 
specific stimulation of expression of the ara^, l_a£, and trp operons while at 
the same time giving inhibition of the expression of the arg operon. With the 
non-hydrolysable pcppGpp, no stimulations were observed, but inhibition of arg 
operon activity (75%) was indistinguishable from that given by ppGpp. For the 
lac operon no stimulation was also observed with GTP, GDP, pGp, pppGp and cGMP. 
These studies indicate that the stimulation of genetic expression is highly 
specific for the ppGpp structure, while inhibition of arg gene expression occurs 
with both ppGpp and pppGpp but not with GDP or GTP. 

It is possible to experimentally isolate the translational component of 
coupled system activities by substituting purified messenger RNA in place of 
DNA in the cell -free system. With 6-galactosidase message as well as MS 2 
RNA, ppGpp and pppGpp show only a slight inhibitory effect on message-directed 
protein synthesis. Since these nucleotides do affect coupled transcription 
and translation activities of the lac operon we surmise that these effects are 
exerted at the level of RNA polymerase activity. 

tvr 
Analogous studies of the DNA-directed synthesis of tRNA •' show no effect 

of moderate levels of ppGpp, pcppGpp or pppGpp. Even high concentrations of 

ppGpp, bordering on levels of any guanine nucleotide which gives non-specific 

inhibition, only slightly inhibits synthesis of this tRNA species. Rather sur- 

pringly it is apparent that neither ppGpp nor pppGpp act alone as negative 

effectors on the synthesis of this tRNA species. This is surprising since 

precisely this species of tRNA does have its synthesis regulated in 080 Su,t. 

infected, amino acid starved stringent cells. 

Continuing studies with essentially pure RNA polymerase interactions with 
nucleotides indicate that the enzyme overall has a means of minimizing binding 
of pppGpp to its elongation sites. If this were to occur, the compound should be 
an inhibitor of elongation with a potency similar to 3'deoxy ATP; however its 
inhibition characteristics are barely detectable on elongation. Thus there is 
further substantiation that both ppGpp and pppGpp act at the level of RNA chain 
initiation with the purified enzyme. 

III. Metabolic Activity Effects 

1. ppGpp binds to EF-Tu and interacts like GDP : Studies of nucleotide 
binding to protein synthetic elongation factor TU indicate that ppGpp can 
interact with this protein in essentially the same manner as GDP. Similarities 
in bdth binding and dissociation kinetics are observed. Bound GDP can exchange 
with pplipp und vice v«'rsa. In the presence of EF-Ts, ppGpp bound to Tu is re- 
ledsed and exchanged with appropriate concentrations of GTP. Again like GDP, 
ppGpp bound to Tu will not support the formation of a ppGpp, Tu, tRNA triplex. 

FE-16 



2. Ability of pppGpp to substitute for pppG in protein synthesis : The 
extent to which pppGpp and ppGpp can substitute for GTP has been examined. ppGpp 
does not substitute for GTP in any reactions, but behaves analogously with GDP. 
pppGpp can effectively substitute for GTP in reactions catalyzed by initiation 
factor 2 (ribosomal binding of fmet-tRNA and formation of N-formylmethionyl- 
puromycin) and by elongation factor T (ribosomal binding of phe-tRNA and di- 
peptidyl tRNA formation). Neither pppGpp nor ppGpp was able to support poly- 
phenylanine synthesis. With elongation factor G, pppGpp is almost completely 
unable to support the formation of N-acetylphe-phe-puromycin, yet ribosomal and 

G dependent hydrolysis of pppGpp occurs, yielding ppGpp, This hydrolysis is 
mediated by G factor since it is blocked by fusidic acid as well as by anti-G 
antibody. The rates of EF-G dependent binding of pppGpp to ribosomes in the 
presence of fusidic acid was nearly the same as GTP binding. Thus the failure 
of pppGpp to support overall protein synthesis and polyphenylalanine synthesis 
can be attributed to its failure to support a late step in the translocation 
reaction. We are currently attempting to pinpoint this impairment. 

3. Permeable cell studies : We have established that a combination of 
temperature shock and osmotic pressure shock renders preparations of E^. coli 
permeable, lacking in acid extractable pools, and capable of RNA, protein and 
ppGpp synthesis under appropriate supplementation conditions. In such prepara- 
tion, the regulatory characteristics of ppGpp synthesis mimic those of whole 
cells. The synthesis of ppGpp further can be closely correlated with the preser^ 
vation of the capacity to synthesize proteins. The protein synthesis observed 
can be coupled to transcriptional events but at efficiencies about 100 times 
higher than with cell-free systems. In this system mRNA decay kinetics can be 
demonstrated to be only slightly slower than in whole cells; ppGpp synthesis is 
dependent upon the presence of mRNA. 

The system can also be used to measure decay of ppGpp, an attribute of 
whole cells that so far is not obtained with in vitro systems. By allowing 
phosphate labeling of ppGpp, then chasing with cold phosphate the decay of 
radioactive ppGpp can be followed. If a complete array of amino acids is 
present and protein synthesis allowed by appropriate supplementation, only 
then does ppGpp breakdown at a rapid rate. A full complement of amino acids 
alone (without protein synthesis) does not lead to breakdown of ppGpp. The 
possible role of translocation in ppGpp decay is further suggested by the fact 
that fusidic acid (0.4 mM) almost completely inhibits ppGpp decay and similarly 
affects protein synthesis in this system. In marked contrast, S30 preparations 
as well as ribosomes + EF-G which are catalyzing translocation do not similarly 
lead to consumption of ppGpp. 

Since a variety of cell evidence (summarized earlier) rather strongly 
suggests that ppGpp might well be a normal intermediate in protein synthesis 
and consumed by protein synthesis, these observations are being pursued in 
hopes of resolving these discrepancies between permeable cell and whole cell 

FF_17 



c 



behavior as compared to the inability of these nucleotides to drive protein 
synthesis in classical synthetic systems. 

Significance of Biomedical Research and the Program of the Institute : Integra- 
tive control processes in general, and adaptive regulation to nutritional and 
environmental changes in particular are common to all growing cells. An under- 
standing of these processes at any level contributes to knowledge of how cells 
respond to their immediate environment and its changes. Such adaptations unques- 
tionably occur during differentiation and development and disturbance of these 
processes constitutes aspects of neoplastic cell transformations. The role of 
ppGpp has emerged as a master regulatory compound present in enormous amounts, 
controlled by perturbations in protein synthesis, capable of regulating genetic 
transcription and interacting with a variety of cellular functions that account 
for a sizeable part of cellular biosynthetic capacities. These include nucleo- 
tide synthesis, membrane transport, phospholipid synthesis, RNA synthesis, pro- 
tein synthesis, and glycolytic fermentation of sugars. 

Proposed Course of Project : We plan to pursue the mechanism of the observed 
specificity of the biosynthetic reaction for guanine nucleotides as well as the 
requirements for tRNA and mRNA codons. In addition, the ribosomal requirement 
for the reaction will be dissected to ascertain the minimal ribosomal components 
needed since ppGpp synthesis provides an alternative way of looking at ribosomal 
function. In addition, we shall continue our studies on the mechanism of inter- 
action of ppGpp with gene transcription by RNA polymerase. The discrepancies 
revealed by comparison of in vitro protein synthesis with in vivo process will 
be analyzed both from the point of view of ppGpp consumption and the functional 
basis of the involvement of the 3' hydroxyl group of GTP in protein synthesis. 

Honors and Awards: 

Member American Society of Biological Chemists 

Invited Speaker and Lectures 

Gordon Conference on Biological Regulation Mechanisms, July, 1972 

Cold Spring Harbor Symposium on Ribosomes, September, 1972 

Laboratory of Biochemistry, National Heart and Lung Institute, December, 

1972 
Molecular Disease Branch, National Heart and Lung Institute, December, 

1972 
Department of Biochemistry, Duke University Medical Center, Durham, North 

Carolina, January, 1973 
Department of Biochemistry, University of Minnesota Medical Center, 

Minneapolis, Minnesota, February, 1973 
NICHD Institute Seminar 



FE-18 



Publications: 

1. Miller, D.L., Cashel , M. and Weissbach, H.: "The Interaction of Guanosine 
5'-diphosphate,2'(3')-diphosphdte with the Bacterial Elongation Factor Tu.' 
Arch. Biochem Biophys . 154: 675-682, 1973. 

2. Raue, H.A. and Cashel, M.: "Regultion of RNA Synthesis in Escherichiji 
coji. I. Characterization of cells subjected to siiiiull.iiiooiis Iciiiperaluri- 
and osmotic shock." Accepted Biociiei)i. Biophys. Acta. 

3. Raue, H.A. and Cashel, M.: "Preparation of [''''P] n labeled Ribonucleoside 
5' triphosphates from Permeabil ized Cells." Accepted Analytical Biochem - 
istry . 



c 



3 

13 



FE-19 



Serial No. hd-lmg-4 

1. Laboratory of Molecular Genetics 

2. 

3. Bethesda 

PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: In vitro Translation of Adenovirus Messenger RNA's 

Previous Serial Number: None I 

Principal Investigator: Heiner Westphal 

Other Investigator: Lawerence Eron 

Collaborating Investigators: Robert Callahan 

Philip Leder 

Cooperating Units: None 

M an years 

Total: 2.5 
Professional: 1.667 
Other: 0.83 

Project Description: 

bjectives : To study the function of genetic elements of tumor viruses in 
lytically infected and in transformed cells. 

Methods Employed : Messenger RNA is extracted from cells containing genetic 
elements of trie tumor virus Adenovirus Type 2, and purified by adsorption to 
poly-dT-cellulose. Purified RNA preparations are characterized according to 
size, and to content and complexity of virus genetic information. The biolo- | 
gical activity of virus-specific messenger RNA's in directing protein synthesis 
in extracts derived from mammalian cells is tested in systems described else- 
where (see Dr. Leder 's report). 

Major Findings : The work was started in September 1972 with setting up the 
biochemical laboratory and the cell growth facilities. Our first experimental 
results were obtained a few months ago, and we can, therefore, only offer 
preliminary conclusions from the work done so far. 

Messenger RNA extracted at late time of productive infection from KB cell 
cultures exposed to Adenovirus Type 2, is very active in directing protein 
synthesis in mammalian cell -free protein-making systems. The polypeptide 
synthesized vn^ vitro display a unique and reproducible pattern of bands in 
acrylamide gel electrophoresis. The mRNA preparations contain about 75% of the « 
genetic complexity of Adenovirus Type 2 DNA. Contaminating host cell mRNA's I 
can, at least in part, be removed from the preparations by preparative f 

FE-20 I 



hybridization to and elution from viral DNA. 

Significance to Biomedical Research and Program of the Institute : With the 
establishment of suitably advanced mammalian cell -free protein making systems 
a characterization of the functions coded for by tumor virus genetic elements 
is now within experimental reach. There is strong hope that an analysis of 
these functions will ultimately aid the understanding of the oncogenic 
aciton of these viruses. 

Proposed Course of Project : Proteins obtained from mRNA-directed in vitro 
polypeptide synthesis will be characterized by acrylamide gel electrophoresis 
and by tryptic peptide analysis, using appropriate in vivo - virus proteins 
as a reference. Attempts will be made to map the structural genes coding 
for identifiable virus proteins. Nonstructural virus proteins may eventually 
be identified with the help of in vivo proteins, the synthesis of which is 
induced by virus infection. 

Honors and Awards: None 

Publications: None 



c 



•J 



FE-21 



serial No. hd-lmg-3 
1 . Laboratory of Molecular Genetics 
2. 
3. Bethesda 

PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Molecular Aspects of the Differentiation of Embryonic Chick 
Lens Fibers in vivo and in Tissue Culture. 

({ 

Previous Serial Number: Same 

Principal Investigator: Joram Piatigorsky 

Other Investigators: Peggy Zelenka 

Leonard M. Mil stone 
Sonia S. Rothschild 

Cooperating Units: None 

Man Years 

Total : 4 ' 

Professional: 3 
Other: 1 

Project Description: 

Objectives: The objective of this research is to describe in detail the se- 
quence of events leading to the normal differentiation of lens fibers and to 
understand the regulatory mechanisms at the molecular level. The lens fiber 
is chosen as a model system to study cell differentiation for 2 reasons. 
First, it involves simple, easily assayed morphological changes (cell elon- 
gation and nuclear degeneration) and considerable synthesis of lens specific 
protein (80% of the cell protein is delta crystal 1 in). Secondly, the process | 
of lens fiber differentiation takes place in tissue culture if the medium is 
supplemented with serum, and is thus amenable to experimentation. We have 
shown in previous annual reports that lens cell elongation in culutre only 
occurs in epithelial cells explanted on or before the fifteenth day of devel- 
opment, that the elongation is inversely related to the extent of DNA syn- 
thesis and cell division, and that the early phase of cell elogation is 
associated with an increase in the relative proportion of delta crystallin 
synthesis. Evidence indicated that the higher proportion of delta crystallin 
synthesis is due to an increase in RNA synthesis and a greater stability of 
delta crystallin mRNA relative to other mRNAs. The initial doubling of cell 
length occurs in the absence of RNA or protein synthesis, and is related to 
the assembly of longitudinally oriented microtubules. Finally, we showed 
that the early phase of cell elongation can be stimulated by insulin in a | 
chemically defined medium. The present report concerns our recent findings il 
on the effect of Insulin on the cultured cells, the extent of differentiation ! 
that will occur j[n vitro and the isolation and j[n vitro translation of mRNA 

FE-22 



3 



from embryonic lens fibers. 

Method Employed : Histology was performed by conventional procedures using 

Carnoy's fixation and paraffin embedding; electron microscopy was done 

using glutaraldehyde fixation and Epon embedding. Cell length was measured 

from histological sections using a calibrated ocular micrometer. Delta 

crystal 1 in mRNA was phenol extracted from fractionated homogenates of lens 

fibers of 15-day old chick embryos; it was purified by oligo-dT cellulose 

chromatography, analyzed by polyacryl amide gel electrophoresis and translated > 

in a Krebs ascites cell -free system. Proteins were analyzed by sodium 

dodecyl sulfate-polyacrylamide gel electrophoresis. 

Major Findings : (1) The terminal phases of lens fiber differentiation can 
occur in tissue cultured Central regions of expl anted lens epithelia from 
6-day-old chick embryos were maintained in tissue culture for 4 weeks to 
determine the extent to which lens fiber differentiation would progress in 
vitro . Cellular outgrowth from the expl ants created 3 distinct zones; name- 
ly, a thick central zone, a thicker annular zone and a flattened peripheral ^ 
zone. Cells of the central and annular zones underwent morphological and 2, 

biochemical changes which correspond to the differentiation of lens fibers j[n C 
vivo . The mean cell length increased a minimum of 25-fold. The nuclei in L 

the longer cells became pycnotic; DNA' remained in the nuclei but accumulated v' 
single-strand breaks. The cytoplasm became filled with a homogeneous granular 
matrix. Organelle density decreased, but microtubules persisted, mostly 
along surface membranes; free ribosomal clusters were present. There were 
occasional desmosomes and infoldings of cell membranes. The proportion of J 

ribosomal RNA synthesized decreased relative to the total RNA synthesized, " 

especially in the central zone. Finally, the proportion of delta crystallin ) 
synthesized increased to 40 to 50% of the newly synthesized protein. These ■' 
data suggest that the transformation of lens epithelial cells into fibers 
results from a programmed differentiation which can take place in tissue 
culture. (2) Insulin stimulates microtubule assembly and cell elongation , 
but not delta crystallin synthesis . One yg/ml of insulin, like serum, sti- 
mulated a doubling of lens epithelial cell length and an assembly of longi- 
tudinally oriented microtubules; colchicine treatment inhibited this cell 
elongation. In contrast to serum, insulin neither promoted further lens 
cell elongation nor appreciably stimulated the synthesis of bulk proteins or 
of delta crystallin under the present conditions. These data indicate that 
the early morphological events of lens fiber differentiation can be initiated 
by insulin in a chemically defined, serum-free environment without significant- 
ly affecting protein synthesis. (3) Delta crystallin mRNA has been extracted 
and translated in vitro . Purified lens fibers from 15-day old chicken embryos 
were used as a source to extract delta crystallin mRNA, because these are 
relatively big (about 3 mg per fiber mass), easily dissected, and synthesize 
large amounts of delta crystallin; very few other proteins are made. RNA 
was phenol extracted from the polysomes or from the post-nuclear supernatant 
fraction of homogenates of the lens fibers and was subsequently eluted 
from oligo-dT-cellulose. The RNA which adsorbed to the column at the time 
application was assumed to contain stretches of poly-A at the 3 '-end by 
dnalogy with similar studies of RNA extracted from other eukaryotic cells. 
The poly-A containing RNAs represented 3 to 5% of the RNA from polysomes 
and 0.3 to 0.5% of the total cellular RNA; 1 to 5 ug of poly-A containing RNA 

FE-23 



was extracted from the cytoplasm of TOO 15-day old embryonic chick lens 
fiber masses. The cytoplasmic poly-A containing RNAs migrated as a single 
band in polyacrylanide gels with a size corresponding to a molecular weight 
of approximately 700,000 daltons. By contrast with rRNA, the poly-A 
containing RNA stimulated the incorporation of radioactively labeled amino 
acids into protein in an ascites cell-free system; 0.5, 1 and 2 yg of the 
RNA stimulated proportionately greater amounts of incorporation. The in- 
corporation had a Mg"'"''" optimum of 3 mM. At least 50% of the new protein 
stimulated by the poly-A-containing RNA comigrated on sodium dodecyl 
sulfate polyacryl amide gels with delta crystallin. The other proteins 
whose synthesis were stimulated by the lens RNA were smaller than delta 
crystallin. These smaller proteins remained with the ribosomal pellet when 
the cell -free ascites system was centrifuged after incubation with the lens 
RNA and, therefore, may represent nascent delta crystallin polypeptides. 
Thus, mRNA can be purified from embryonic lens fibers on the basis of its 
poly-A content; in vitro experiments suggest that this mRNA translates 
mostly, perhaps entirely, delta crystallin. 

Significance to Biomedical Research and the Program of the Institute : One 
of the difficulties of studying cellular differentiation is that embryon i c 
systems have many unidentified variables and generally involve interaction 
of many cell types. The differentiation of lens fibers has the advantages 
that (1) it occurs synchronously in a single population of excised 
epithelial cells, (2) all processes examined in culture mimic those which 
take place in vivo during the normal development of lens fibers, (3) large 
amounts of easily identified lens specific proteins are involved and (4) 
simple morphogenetic changes can be related to biochemical events. The 
finding that insulin can stimulate lens cell elongation in a chemically de- 
fined medium should facilitate further analysis of the mechanism of cell 
elongation. These studies, then, give information on the mechanisms of 
cellular differentiation, which is relevant to the goals of this Institute. 

Proposed Course of Project : During the next year, the action of insulin on 
the lens epithelial cells will be studied in detail. It is planned to purify 
and characterize delta crystallin mRNA and then to make a radioactive DNA 
sequence complementary to it to use as a probe for titrating gene dosages and 
quantitating delta crystallin mRNA levels in the differentiating lens fiber 
in vivo and in vitro . Other mRNAs will be isolated from the lens epithelial 
cells; these will be examined with respect to their synthesis and stability. 
The transport of mRNA from nucleus to cytoplasm will be followed during 
differentiation of the lens cells. The rates of synthesis of bulk proteins, 
delta crystallin and microtubule protein will be compared at different stages 
of differentiation. Finally, the physical and chemical properties of delta 
crystallin will be studied. 

Honors and Awards : (Joram Piatigorsky) 
V. Invited lectures : 

a. Fertilization and Gamete Physiology Training Program, Woods Hole, 
Mass. Aug. 1972 

b. National Eye Institute Seminar, Oct. 1972 

c. National Heart and Lung Institute Seminar, Feb. 1973. 

d. National Cancer Institute Seminar, March, 1973. 



FF_9/I 



2. Co-Chairman and Speaker Microtubule Session, American Society of Cell 
Biology Meetings, St. Louis, Mo., Nov. 1972. 

3. Invited participant and speaker. Lens Differentiation Workshop, 
Rochester, Michigan, March 1973. 

4. Special reviewer, N.I.H. Study Section in Cell Biology, April 1973. 



Publications: 



^iatigorsky, J. and Rothschild, S.S.: Loss during development of i 
ability of chick embryonic lens cells to elongate in culture: Inv( 
relationship between cell division and elongation. Develop. Biol . 
?8: 382-389. 1972. 



2. Piatigorsky, J., Webster, H. deF., and Wollberg, M.: Cell elongation 
in the cultured embryonic chick lens epithelium with and without pro- 
tein synthesis: Involvement of microtubules. J. Cell Biol . 55: 
82-92, 1972. 



■» 



3. Piatigorsky, J.: Insulin initiation of lens fiber differentiation in _^ 
culture: elongation of embryonic lens epithelial cells. Develop . if- 
Biol . 30: 214-216, 1973. "« 

4. Piatigorsky, J., Rothschild, S.S., and Wollberg, M.: Stimulation by 

insulin of cell elongation and microtubule assembly in cultured embry- "j 
onic chick lens epithelia. Proc Nat. Acad. Sci., USA , 70 : •'•, 

1195-1198, 1973. "^ 

5. Craig, S.P. and Piatigorsky, J.: Cell elongation and delta crystallin !) 
synthesis without RNA synthesis in cultured early embryonic chick lens !' 
epithelia. Biochim. Biophys. Acta , in press. 

6. Piatigorsky, J., Rothschild, S.S., and Milstone, L. M.: Differenti- 
ation of lens fibers in explanted embryonic chick lens epithelia. 
Develop. Biol ., in press. 



FF-PR 



i 



Serial No. HD-LBC6 

1. Laboratory of Molecular Genetics 

2. 

3. Bethesda 



PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 



Project Title: 



Growth and 



Regulated Gene Expression During Cell 
Differentiation (Note change in Project Title) 

I. Gene expression during erythropoiesis 

II. Gene expression during development of immunocompetency 



Previous Serial Number: Same 

Principal Investigator: P. Leder 

Other Investigators: H. Aviv 

R. Callahan 

J. Gielen 

S. Krauss 

S. Packman 

J. Ross 

D. Swan 



Cooperating Units; 



Man Years 

Total : 

Professional : 
Other: 

Project Description: 



Dr. Yoji Ikawa 

Viral Leukemia and Lymphoma Branch, NCI 



9 

8 
1 



Objectives : To define in genetic and molecular terms the mechanisms which 
regulate the flow of genetic information during cell growth and development. 
Specific model systems involving the differentiation of red blood cell pre- 
cursors and immunocytes have been chosen for detailed studies. These serve 
as paradigms to view the process of differentiation as well as to investi- 
gate the organization of the chromosome and its relation to the immune pro- 
cess. 



Methods Employed : 
plinary approach. 



These investigations have involved a complex, inter-disci' 
The model systems that have been chosen involve the main- 



tenance in culture of a unique line of erythrocytic leukemia cells, T-3-C12. 
This cell, when appropriately treated, undergoes a form of erythroid dif- 
ferentiation. Initial studies involving the immune system have focussed 
on several myeloma tumors which produce specific immunoglobulin light chains. 

Our strategy has involved the isolation of specific mRNAs for globin and 
the immunoglobulin light chain using techniques of extraction and affinty 
chromatography developed in this laboratory. The purified mRNAs are converted 
into their DNA complements using the avian myeloblastosis reverse transcriptase, 
a procedure also developed in this laboratory. The DNA complements of these 
purified mRNAs are used in hybridization kinetic analyses to determine globin 
and immunoglobulin gene dosage and to quantitate respective mRNAs. Generally 
these procedures involve the use of tumors grown in animals, cloned lines 
grown in culture and the applications of sophisticated biochemical and physical 
chemical techniques in the study of these systems. In addition, studies have 
continued on the regulated expression of the bacterial protein synthetic 
elements. The methods involved have been described in previous progress 
reports. 

Major Findings : (1) DNA has been synthesized which is complementary to 
mouse, rabbit, human and duck globin mRNA. This material exhibits the specific 
hybridization properties expected of a true copy of the respective globin genes. 

(2) Using these radioactive cDNAs as probes, the evolutionary relationship 
among these organisms has been determined at the level of the globin genes. 

(3) Again using the cDNA probes, the number of genes corresponding to globin 
in duck and mammalian cells has been determined to be no more than 3 to 5 
globin gene copies per diploid genome. Thus, the amplification hypothesis 

of differentiation has been ruled out. (4) Using an in vitro culture system, 
the rate-limiting events in the expression of the globin genes has been identi- 
fied. Globin mRNA is absent in cells prior to the induction of differentiation. 
After induction, 3,000 to 6,000 molecules of globin mRNA can be detected in 
cell culture systems. (5) Further progress has been made in the purification 
of the immunoglobulin light chain mRNA. (6) Immunoglobulin light chain mRNA 
has been translated in a cell-free system. (7) A precursor of light chain, 
comprising 10 to 15 more amino acids, has been identified in the cell -free sys- 
tem. (8) The immunoglobulin light chain message has been shown to contain a 
poly(A) sequence and has been shown to direct the synthesis of both constant 
and variable regions of the light chain. A post-transcriptional joining of 
cariable and constant regions has therefore been ruled out. The message has 
been shown to be monocistronic. (9) DNA complementary to the myeloma mRNA 
has been synthesized and characterized. (10) The complementary DNA has been 
used to assess the purity of mRNA preparations. The mRNA has been found to be 
less than 10% pure. (11) Preparative hybridization has been used to prepare 
a purified light chain cDNA. This material has been used in hybridization 
nnnlysps of thr constant region genome in myeloma tumors. These studies, though 
In iixMjro-.-, , •.uu<jt".L Lh« |jr«v.t'ncH of highly reltoraLod scqurncr. (1?) The 



FE-27 



cell-free protein synthesizing system has been used to translate bacteriophage 
mRNAs, laying the groundwork for a test system for identifying suppressor mutants 
in mammalian cells, (13) The cell-free system has also been used in order to 
translate mRNA derived from adenovirus infected cells. 

Significance to Biomedical Research and the Program of the Institute : These 
studies are directed towards understanding the fundamental processes involved 
in the expression of genetic information. These processes is obviously relevant 
to the normal and abnormal embryonic differentiation, maturation and to the 
orderly development and function of the human organism. In addition to the 
relevance of these studies to inherited disorders, the work is also relevant 
to the process of oncogenesis as well as to disease processes which are poly- 
genic such as diabetes, hypertension and susceptibility to oncogenic disorders. 
Our current findings have provided a means for studying the regulated expres- 
sion of specific genes in higher organisms. These are particularly relevant to 
determining the pathophysiology of thalassemia. These techniques may also prove 
useful in the direct transmission of genetic characteristics from cell to cell 
in vitro 

Proposed Course of Project : Having identified the transcriptional reaction 
as essential to the regulated expression of globin genes, this process will 
be studied in detail. We hope to establish a cell-free system for the tran- 
scription of globin genes directly from chromatin. These will be used to de- 
termine and identify specific regulatory elements involved in the expression 
of these genes. Further studies will be directed toward identifying a coordin- 
ated set of genes, regulated together with globin. Specifically these will 
involve enzymes required for the biosynthesis of heme. We also hope to iso- 
late or transform mutant cell lines affected in their ability to undergo erythro- 
differentiation. 

Further studies will be required in order to purify completely immunoglobulin 
mRNA. A variety of techniques involving the isolation of nucleic acids will be 
employed. When immunoglobulin light chain mRNA of sufficient purity is obtain- 
ed, it will be transcribed into cDNA and studies will be undertaken to account 
for the constant and variable regions of irmiunoglobulin molecules in genetic 
terms. Work will also proceed directed toward identifying the products of 
adenovirus mRNAs, in collaboration with the laboratory of Dr. Heiner Westphal . 
These will generally involve the characterization of tryptic peptides derived 
from cell-free systems, and their comparison with authentic viral proteins. 

Honors and Awards: Invited Lectures 

Gordon Conference on Animal Cells and Viruses, August, 1972 
Department of Oncology, Columbia University, September, 1972 
Plenary Lecturer, German Bjiochemical Society, October, 1972 
Department of Biochemistry, Brandeis University, November, 1972 

/ 5 



Department of Biochemistry, Yale University, November, 1972 
Department of Embryology, Carneige Institution of Washington, 

Baltimore, Maryland, December, 1972 
Department of Cellular Biology, Albert Einstein College of Medicine, 

December, 1972 
Symposium Lecturer, American Association for the Advancement of Sciences, 

December, 1972 
Department of Biochemistry, Harvard Medical School, February, 1973 
Rockefeller University, February, 1973 

Department of Biochemistry, Princeton University, March, 1973 
Invited Lecture, Current Topics in Biochemistry Lecture Series, National 

Institutes of Health, March, 1973 
Invited Participant, Fogarty Center Conference on Cultured Cells, April, 

1973 
Invited Seminar Lecture, American Society of Immunologists, Atlantic City, 

April, 1973 
Department of Microbiology, Medical School, Washington University, St. Louis, 

May, 1973 
President, Foundation for Advanced Edcuation in the Sciences, Inc., 

Bethesda, Maryland 
Member, Study Section on Physiological Chemistry, National Institutes of 

Health, Bethesda, Maryland 
Invited Participant, Work Shop on Protein Biosynthesis, Aarhus, Denmark, 

June, 1973 
Chairman-Elect, Gordon Conference on Nucleic Acids, 1974 
Invited Lecturer, Cold Spring Harbor Symposium on Chromosome Organization, 

June, 1973 

Publications: 

1. Callahan, R. and Leder, P.: In Vitro Initiation of Coliphage T7mRNA. 
Arch, of Biochem. & Biophys ., 153: 802-813, 1972. 

2. Leder, P., Skogerson, L.S. and Callahan, R.: Translation Initiation: 
Defects Arising in £. coli Infected with Phage T7, x and Qg. Arch . 
of Biochem. & Biophys . 153: 814-822, 1972. 

3. Swan, D., Aviv, H. and Leder, P.: Purification and Properties of Bio- 
logically Active mRNA for a Myeloma Light Chain. Proc. Nat. Acad. Sci . 
U.S.A . 69: 1967-1971, 1972. 

4. Boime, I. and Leder, P. Protein Synthesis Directed by Encephalomyocardi- 
tis Virus mRNA. III. Discrete Polypeptides Arising from a Monocistronic 
Messenger In Vitro . Arch, of Biochem. & Biophys . 153: 706-713, 1972. 

5. Aviv, H., Boime, I., Loyd, B. and Leder, P.: Translation of Bacterio- 
phage QgmRNA in a Murine Krebs II Ascites Tumor Cell-Free System. 

FE-29 



Science 178: 1293-1295, 1972. 

6. Aviv, H., Packman, S., Swan, D., Ross, J. and Leder, P.: In Vitro 
Synthesis of DNA Complementary to mRNA Derived from a Light Chain- 
Producing Myeloma Tumor. Nature 241 : 174-176, 1973. 

7. Packman, S., Aviv, H., Ross, J. and Leder, P.: A Comparison of Globin 
Genes in Duck Reticulocytes and Liver Cells. Biochem. Biophys. Res. 
Commun. 49: 813-819, 1972. 

8. Ross, J., Ikawa, Y. and Leder, P.: Globin mRNA Induction During Eryth- 
roid Differentiation of Cultured Leukemia Cells. Proc. Nat. Acad. Sci ., 
U.S.A. 69: 3620-3623, 1972. 



iD 






\ 

VE-30 



Serial No. hd-lb20 

1 • Laboratory of Molecular Genetics 
2. 

3. Bethesda 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Regulation of Antibody Biosynthesis 

I. Mechanisms of consignment of cells to determinant- 
specific immunocompetency 

II. Promoters and amplifers of antibody biosynthesis 
in cell culture 

III. Initiation of determinant specific antibody biosynthesis 
in cell culture 

IV. Deviation of immunocompetent cells 

Previous Serial Number: Same 

Principal Investigator: E.E. Hanna 

Other Investigators: C. Lambert 

Cooperating Units: None 

Man Years 

Total: 24/12 
Professional: 24/12 
Other: 

Project Description 

Objectives^: To clarify postulated pathways of antibody biosynthesis and to ex- 
plore alternative pathways to cellular biosynthesis and secretion of antigen 
induced antibodies. This involves delineating the roles of non-antibody-secret- 
ing cells, such as the thymus influenced or derived lymphoid cell (T-cell) and 
the more remote non-lymphoid cell type, the macrophage. The latter is a cell 
known to make contact with antigen first during natural or artifical immuniza- 
tion. We hope to learn more about the nature of the active principle in homo- 
logous serum which seems to be a required supplement in rabbit lymphoid cell 
cultures. If small amounts of Ig or subunits of Ig are required to initiate 
antibody biosynthesis in culture and the synthesized product can be shown to be 
distinct from these, we will have essentially defined a new Ig primer. It will 
then be of interest to understand its mechanism of action and its origin. 
[E£. , it would be interesting to compare such an Ig to receptors at the surface 
of Ig-secreting cells (B-cells) and to the very rarely demonstrated receptor at 
the surface of T-cells]. It is possible that such a receptor Ig has its activity 
associated in some manner with the macrophage cell type. 

We wish to pursue further the role and the origin of certain cyclic nucleo- 
tides found to stimulate or amplify B-cells. We wish to determine relative 

FE-31 



i! 

■Ml 

'J 



efficacies of cAMP and cGMP to amplify B-cells (proliferation and secretion). 
This is important because of a ^ery current debate over which of the two com- 
pounds is the better modulator of lymphocyte activity. 

As solutions to the problem of how cells initiate antigen specific Ig secre- 
tion, we will turn our attention toward developing more direct means of sel- 
ectively regulating the antibody mechanism at the cellular level. 

Methods Employed : Antibody secreting cells (B-cells) are detected as discrete 
holes (plaques), in a confluent lawn of erythrocytes bearing native and chemical- 
ly linked haptenic determinants. B-cell precursors, with receptors at their sur- 
face, may be detected by the rosette test and other similar adherance tests. 
Detection of T-cells remains a problem since their presence is determined indi- 
rectly as a synergistic activity. Manipulation of T-cells is presumed when one 
has enhanced or suppressed this activity. We have prepared anti-theta antisera 
using both neonatal and adult thymus cells as antigen. We hope to obtain a more 
efficient T-cell inactivator by selective absorption and by pooling these anti- 
sera. Since concanavalin-A (Con-A) binds preferentially to T-cells, we have pre- 
pared very potent anti-Con-A antisera which in concert with a fluorescent label 
may be hefpful in identifying T-cell populations. Experiments will continue to be 
preformed both in vivo and in cell culture. 

Density gradients and antigen-specific, affinity columns are still the best 
available methods for isolating and harvesting cell populations, but they are 
by no means fully satisfactory. We will continue to seek better isolation and 
cloning methods. 

The replica-hapten-specific plaquing method developed by us is still the 
method of choice for assessing B-cell secretions with respect to their specific- 
ities and also for monitoring any potential cloning method. Agar-gel and inmuno- 
electrophoretic procedures will be employed as needed. 

The ability to recover irranunocompetent cells from liquid nitrogen storage 
with essentially the activity of fresh material greatly facilitates our studies 
since it allows us to repeat experiments on the same biological material. 

Major Findings : We have demonstrated that dibutyryl-cAMP can amplify the number 
of B-cells responding to immunization (in press). In these studies a T-cell de- 
pendent antigen was used in both thymectomized (Tx) mice and sham Tx mice. The 
result was enhancement of the number of secreting B-cells in each group. Be- 
cause of this it is important to ascertain whether cyclic nucleotides are 
mediators of T-cell function or whether they are associated in another fashion. 

We have interpreted some of our earlier findings from studies of immuno- 
suppression by a streptococcal toxin as a possible selective effect upon sup- 
pressor T-cells. B-cells are presumed to recover from the cytotoxin earlier 
than suppressor T-cell. This temporary lack of suppressor cells would allow 
for the observed enhancement. We will test this alternative using T-cell 
independent antigens, such as the purified pneumococcal capsular antigen 
in Tx animals and in congenically athymic animals. 

We have found an important artifact in the rabbit spleen cell culture 

FE-32 



procedure used to study antibody biosynthesis. Homologous rabbit serum is a 
reported nutritional requirement in such cultures. Yet, we found that all 
sera effective in this respect contained pre-formed, "background" anti-sheep 
erythrocyte (SE) antibody. This antibody was shown to sensitize SE added as 
antigen to immunize cultures. The sensitized SE form micro-aggregates which 
are 1ysed by complement in the assay procedure. This forms well-defined 
plaques which are artifacts and are eronously enumerated as antibody forming 
cells (Lambert and Hanna-manuscript in preparation). This artifact is 
probably present in the data of published reports. Publication of this result 
should stimulate reassessments of other reports on antibody biosynthesis which 
were quantitated in culture by a plaque procedure. Our future direction will 
be to determine if a small amount of transferred antibody (or subunits) may 
serve an initiator or primer functions as discussed above. 

We have found that the antibody required to develop and visualize 7S IgG 
secreting PFC populations is itself a high affinity IgG with major anti-Fc 
activity. The active component of an antiserum with strong suppressive activity 
for 19S IgM PFC Is a high affinity IgG with major anti-Fab activity. The 
amount of anti-light chain activity within the anti-Fab antibody is important 
in this respect (manuscript in preparation). In preparations to be made in the 
future, these target subunits of Ig will be isolated and used as antigen to 
produce the particular anti-Ig reagent desired. These findings may have re- 
levance also in initiation or suppression of certain Ig responses. 

Results of our study which suggested combining combining site overlap of IgG 
into adjacent native determinants of the carrier moiety of a hapten-carrier im- 
munogen were equivocal. We decided therefore to repeat the experiment with a 
more comprehensive immunization schedule, involving injections of immunogens con- 
sisting of reciprocal non-cross-reacting carriers. These procedures have been 
carried out, the cells have been harvested, and cryogenically stored. Assay of 
these cells should confirm or negate our original conclusions. 

Significance to Biomedical Research and the Program of the Institute : Ability 
to regulate or modulate specific antibody responses will have great advantage 
over current measures of generalized immunosuppression methods in current use. 
It should be possible to selectively suppress a response, e.g . , to a grafted tis- 
sue or organ, while maintaining desirable immunologic surveillance against dis- 
ease producing immunogens. Hopefully it should be possible to enhance this sur- 
veillance. Our findings should also complement and clarify scientific thought 
on cellular control mechanisms in general. 

Proposed Course of Project : I have chosen to point out the future direction of 
eacn facet as it was described for sake of clarity and conciseness. 

Honors and Awards 

Invited to present a research lecture at Howard University 

Elected to membership in the American Association of Immunologist (AAI). 

Represented the American Association of Immunologists in the ad hoc 

committee meeting with Black Scientist, sponsored by the Federation 
of America n Societies for Experimental Biology (FASEB) . 

August 1972, selected by the National Research Council to give advisory 

FE-33 



service representing scientist and engineers from minority 
backgrounds. 

Publications : 

1. Uzunova, Anelia, D. , and Hanna, Edgar, E.: B-cell Amplification by 
N°, 0^ -dibutyrl Adenosine-3' -5' Cyclic Posphate in Mice. Cellular 
Immunology , in press. 



2. Hanna, Edgar, and Watson, Dennis W.: Enhanced Immune Response Following 
Immunosuppression 
Immunity , in press 



Immunosuppression by Streptococcal Pyrogenic Exotoxin. Infection and | 



I 



'|t-34 

i 



NICHD Annual Report 
July 1, 1972 through June 30, 1973 
Laboratory of Biomedical Sciences 

The Laboratory of Biomedical Sciences conducts basic research in developmental 
enzymology, intermediary metabolism, physiological controls, and developmental 
pharmacology. The objective of these investigations is the elucidation of 
the molecular basis for selected types of mental and physical developmental 
pathology. ' 

In the Section on Developmental Enzymology interest centers on the separation, 
purification, and characterization of enzymes from the human placenta and 
from fetal tissues in both normal and abnormal pregnancies. A study of the 
properties of pregnancy-associated enzymes from normal and abnormal gestations 
may give information about the etiology of reproductive disorders such as 
spontaneous or habitual abortions, stillbirths, or molar pregnancies. 

Optimal assay conditions were ascertained and specific activities determined Jit' 

for the following placental enzymes: alkaline phosphatase, lactate dehydro- J|jJ 

genase, hexokinase, phosphoglucomutase, phosphoglucose isomerase, catechole- "•■ 

0-methyltransf erase, and two enzymes of the degradative pathway of lysine "", 

(saccharopine dehydrogenase and saccharopine reductase) . Zymogram techniques j., 

were devised for detecting isoenzyme variation among the first five enzymes. ^ 

These enzymes were qualitatively and quantitatively compared in normal "J 

term placenta and malignant trophoblast in tissue culture. Two striking -i 
differences were noted: the tissue in culture is virtually devoid of heat 

stable (placental) alkaline phosphatase, and it has an unusual lactate de- . 

hydrogenase isoenzyme. Studies designed to characterize these enzyme J 
differences are progressing. 

Investigators in the Section on Intermediary Metabolism develop laboratory 
analogs of mental retardation through the use of specific chemical agents and 
enzyme inhibitors, investigate the biosynthesis and degradation of macro- 
molecules in neural tissue, elucidate mechanisms of transport of small 
molecules into and out of the brain, and evaluate the effects of excesses or 
deficiencies of intermediary metabolites on cellular differentiation and 
organogenesis (particularly of the brain). The studies in the Section range 
from classical biochemical investigations of enzymes of neurochemical interest 
and pharmacological efforts to inhibit or stimulate these enzymes to behavioral 
experiments on animals in which the activity of such enzymes has been altered 
or inhibited. In this way we hope to provide information on the basic bio- 
chemistry of the brain as well as on the conditions which alter the normal 
brain functions such as various genetically-transmitted mental retardation 
syndromes . 

Among recent advances made by the Section are: (1) the further elucidation 
of the biochemical, pathological, developmental, and behavioral aspects of 
the rat model of phenylketonuria; (2) the purification and characterization 
of bacterial tryptophan hydroxylase and bacterial dihydropteridine reductase, 
two enzymes involved in bacterial hydroxylation; (3) the preparation and 
characterization of poly-A containing RNA's from rat brain and a study of 



FKL 



their function in information transfer vis-a-vis brain messenger RNA; (4) 
The demonstration and characterization of a new protein factor involved in 
stimulating pteridine biosynthesis in bacterial systems; (5) The development 
of new methodology for the study of purine phosphoribosyltransf erase, the 
enzyme involved in the Lesch-Nyhan syndrome, and the study of this enzyme in 
rat brain. 

The Section on Physiological Controls studies basic endocrine and neuroendo- 
crine mechanisms and factors controlling their development . A focus on 
synthesis and mode of action of cyclic AMP reflects the potential importance 
of this ubiquitous intracellular regulator to our understanding of metabolic 
regulation, cell growth, and differentiation. Most studies utilize two 
metabolic pathways as models for delineating interactions between hormones, 
neurotransmitters, and nutrients. Protein phosphorylations controlling 
glycogen metabolism provides us with a well-characterized model of cyclic AMP 
action; indole metabolism in the pineal gland has proved excellent for studying 
the mode of action and synthesis of neurotransmitters. 

During the past year, we have obtained partial or complete purification of all 
of the enzymes known to participate in glycogen cycle phosphorylations. Of 
major significance was the observation that glycogen synthetase phosphatase 
is a general phosphoprotein phosphatase that dephosphorylates phosphorylase-b 
kinase and other protein substrates of the cyclic AMP-dependent protein kinase. 
The development of fetal rat liver glycogen synthesis has been reproduced in 
a completely defined organ culture system and shown to be related to the 
development of glycogen synthetase. In these studies, separate and sequential 
roles for hydrocortisone and insulin have been characterized. 

A new method for measuring tryptophan hydroxylation was developed and used to 
show that this rate-limiting enzyme in serotonin synthesis in the pineal gland 
is controlled by circulating tyrptophan levels. Norepinephrine was shown to 
decrease pineal serotonin levels by a sequence of reactions involving a 
specific receptor, cyclic AMP, and the induction of serotonin N acetyltrans- 
f erase. Antigonadotrophic affects of the pineal gland have been further 
delineated. A potent antigonadotrophic compound produced by the pineal gland 
was purified and shown to be a small peptide. 

The Section on Developmental Pharmacology studies the subcellular processes 
involved in the "induction" of pharmacologically important enzymes by drugs, 
polycyclic hydrocarbons, or insecticides (i.e., xenobiotics) . The combination 
of these environmental stimuli and the host's genetic constitution therefore 
influence the levels of these mono-oxygenase activities in maternal tissues, 
in the placenta, and transplacentally in fetal tissues. Consequently, we are 
concerned with (1) the interplay between genetic expression and environmental 
stimuli, (2) possible effects of xenobiotics on normal fetal growth and 
development, (3) mechanisms of "pharmacological immaturity" of the premature 
and newborn, (4) mechanisms of disease of the fetus and newborn involving 
oxidative and conjugative metabolism of normal body substrates such as steroids 
and bilirubin, and (5) possible therapeutic and diagnostic procedures for 
aiding and predicting pharmacogenetic disorders and teratogenic effects in 
the fetus and neonate. 



FEB 



With fetal cell cultures derived from the entire hamster or mouse, from rat 
or mouse liver, and most recently from established cell lines of rat or mouse 
hepatoma or of normal liver origin, we have developed an experimental model 
having numerous advantages over studies in the intact animal. We have 
determined that the "induction," or stimulation, of mono-oxygenase activity 
such as aryl hydrocarbon hydroxylase and other drug-metabolizing enzymes by 
phenobarbital, polycyclic hydrocarbons, or biogenic amines is controlled at 
two levels: the level of transcription involving gene activation, and a 
posttranscriptional effect in which the normal rate of decay of induced 
enzyme activity is retarded. The induction process effected by phenobarbital 
is more dependent on ribosomal RNA synthesis than that effected by either 
aromatic hydrocarbons or biogenic amines. 

With the use of inbred strains of mice, we have determined that the "induction" 
of at least 7 drug-metabolizing enzyme activities can segregate as a single 
autosomal dominant trait, can be domlnantly repressed, or can be expressed as 
a codominant trait, depending on the inbred strains crossed. A new hemo- 
protein, cytochrome P-448, is concomitantly sjmthesized during the induction 
process. In mice in which the induction process and P-448 formation are 
relatively "nonresponsive" to aromatic hydrocarbons such as 3-methylcholan- 
threne or 5,6-benzoflavone, these parameters are fully expressed by admini- 
stration of a compound having extremely high affinity for all tissues, 
2,3,6, 7-tetrachlorodibenzo-£-dioxin; it thus appears that the Ah locus in 
"nonresponsive" mice can respond normally perhaps because the deficient 
"receptor site" is made to function by an inducing compound of sufficiently 
high affinity. The relative presence of "aromatic hydrocarbon responsiveness" 
in certain mice results in more 3-methylcholanthrene-initiated tumors, in a 
shortened sleeping time when given certain drugs, in a shortened survival time 
when dosed with large amounts of polycyclic hydrocarbons, and in an increased 
protection against the toxicity of certain insecticides. These genetically 
mediated dissimilarities in xenobiotic metabolism may be a useful probe in 
demonstrating differences in the rates of formation of certain drug-produced 
birth defects. 



FF3 



< 



Serial No. HD-LBl(c) 

1. Laboratory of Biomedical Sciences 

2. Section on Developmental Enzymology 

3. Bethesda 

PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Developmental Enzymology 

Previous Serial Number: Same " 

i 

Principal Investigator: J. C. Robinson 

Other Investigators: C. Chen 

J. B. Edlow 
T. Fjellstedt 
T. Ohta 
J. R. Relacion 

Cooperating Units: P. 0. Kohler, Reproduction Research Branch, NICHD 

Man Years ' 

Total: 48/12 
Professional: 48/12 
Other: 0/12 

Project Description: 

Objectives : The main objectives of this project are: (1) to study qualitative 
and quantitative changes in selected enzyme systems in the developing placenta 
and fetal organs in normal gestation; (2) to study qualitative and quantitative 
changes in these same enzyme systems in the developing placenta and fetal 
organs in reproductive disorders such as abortions, stillbirths, and fetal I 
malformations; (3) to isolate, purify, and characterize these enzymes from ' 
the normal placenta; and (4) to identify and characterize pregnancy-associated 
enzymes in maternal blood plasma. 

Methods Employed : The principal methods employed are: (1) the zymogram 
technique, by which electrophoretic variants of enzymes can be detected; 
(2) autoradiography, with which potential carrier function is evaluated (for 
example, by using I^^l-t^gged thryroxine) ; (3) enzyme assays, which are 
usually based on spectrophotometric or radiometric methods; (4) protein iso- 
lation techniques, including salt- and solvent-fractionation, gel-filtration, 
and ion-exchange chromotography; (5) paper and thin layer chromatography; 
(6) tissue culture; and (7) immunochemical procedures. 

Major Findings : (1) The specific activities and electrophoretic patterns jj 
of three carbohydrate-metabolizing enzymes — hexokinase, phosphoglucomutase. 



FP4 



and phosphoglucose isomerase — are essentially the same in cultured chorio- 
carcinoma cells as in term placenta. (2) In addition to the 5 lactate 
dehydrogenase (LDH) isoenzymes characteristic of term placenta, the chorio- 
carcinoma zymogram has an additional substrate-dependent band between LDH-2 
and LDH-3. (3) Heat-stable alkaline phosphatase is strikingly reduced in 
cultured choriocarcinoma cells as compared with term placenta. This low 
basal activity was increased approximately 20-fold by the inclusion of 5- 
bromodeoxyuridine in the culture medium. (4) We demonstrated that the enzymes 
involved in the initial steps of lysine metabolism are present in human 
placental tissue. Optimum stability and assay conditions were established 
for the enzymes involved in the first two sequential reactions (lysine to 
saccharopine and saccharopine to a amino adipic-6-semlaldehyde) , and Michaelis 
constants were determined for the substrates used in the spectrophotometric 
assay. Reaction products were confirmed by formation of chemical derivatives, 
where appropriate, and by thin-layer chromatography. Initial purification 
procedures of ammonium sulfate fractionation and DEAE-cellulose column 
chromatography resulted in approximately a 100-fold purification of these 
enzymes. (5) A quantity of crystalline saccaropine sufficient for a wide 
range of experiments was prepared biosynthetically and purified by ion 
exchange chromatography, paper chromatography, and repeated crystallization. 
(6) No phenylethanolomine-N-methyl-transferase activity was found either in 
human term placenta or rat term placenta. (7) The mean specific activity 
(n mole metanephrine formed/hr/mg wet weight) of catechol-0-methyl transferase 
(COMT) of seven human term placentas was 0.58. The range was 0.46 to 0.68. 
(8) In a preliminary study, COMT specific activity in rat placenta decreased 
significantly from 2.91 on gestational day 12 to 1.88 on day 14. The specific 
activity was still lower (1.62) on day 16. A slight rebound was noted from 
day 18 (1.91) through day 20 (2.11). 

Significance to Biomedical Research and the Program of the Institute : The 
study of placental enzymes in normal pregnancy will provide a suitable base- 
line with which one can compare the same enzyme systems in placental explants, 
in benign and malignant trophoblast cells in culture, and in placental tissue 
from reproductive disorders such as stillbirths and abortions. For example, 
consistent changes in enzymes in abortion material may reflect differences in 
placental function, which in turn could affect fetal viability. Hopefully, 
these studies will lead to increased understanding of both normal and abnormal 
biochemical processes involved in pregnancy and fetal development. 

Proposed Course of Pro.ject ; (1) Characterization of selected enzymes from 
placenta, maternal plasma, amniotic fluid, and trophoblast cells in culture 
will be undertaken. (2) Correlations between abnormal pregnancies and 
variations in fetal or maternal enzyme systems will be sought. 

Honors and Awards : None 

Publications : 

1. Edlow, J.B., Kohler, P.O. and Robinson, J.C: Choriocarcinoma 
in vitro: Evidence for exchange of enzymes between culture 
medium and cells. Proc. Soc. Exp. Biol. Med . 141: 798-801, 1972. 



FK5 



Serial No. HD-LB9 



1. Laboratory of Biomedical Sciences 

2. Section on Intermediary Metabolism 

3. Bethesda 



PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Models of Mental Retardation 

Previous Serial Number: Same 

Principal Investigator: G. Guroff 

Other Investigators: A. Andersen 

V . Rowe 

Cooperating Units: S. Zigler, Department of Pediatrics, Duke University 

Medical Center, Durham, North Carolina 
J. Sidbury, Department of Pediatrics, Duke University 
Medical Center, Durham, North Carolina 

Man Years 

Total: 27/12 
Professional: 27/12 
Other: 0/12 

Objectives : The objectives of this research are: (1) to produce biochemical 
models of mental retardation sjmdromes in laboratory animals by chemical 
means; (2) to investigate the pathological and behavioral aspects of the 
chemical treatments; (3) to study the mechanisms by which specific chemical 
alterations produce a change in normal neural functioning; (4) to attempt 
new methods for preventing such alterations hopefully pointing the way for 
new clinical approaches. 

Methods Employed : The methods employed include standard enzymatic assay 
techniques, histological examinations, and behavioral evaluation. 

Major Findings : (1) Treatment of newborn rats with £-chlorophenylalanine 
plus phenylalanine produces a biochemical picture similar to that seen in 
clinical phenylketonuria, e.g. high blood phenylalanine-to-tyrosine ratio, 
phenylketones in the urine, low phenylalanine hydroxylase levels, lowered 
brain serotonin. (2) Such treatment during the development of the rat 
(0-21 days) produces, in the adult animal, behavioral and pathological alter- 
ations analogous to clinical phenylketonuria, e.g. hyperactivity, deficiencies 
in problem solving, smaller brains. (3) Such treatment also produces a delay 
in developmental milestones, e.g., eye opening, histological changes in the 
brain, and heightened cerebral excitability in the infant, treated rat. 
(4) The treated rat metabolizes the phenylalanine load to abnormal products 
characteristic of the clinical state; in the 21-day old rat the major 



metabolite has been shown to be phenyllactic acid. (5) Cataracts appear in 
the adult rats several months after termination of the treatment; the pattern 
of free amino acids and of soluble proteins in the lenses of these cataractous 
animals is altered in a characteristic and reproducible way. 

Significance to Biomedical Research and the Program of the Institute : No 
adequate model of mental retardation has yet been produced in a laboratory 
animal. The need for such a disease analog is great, since only then can 
investigations be initiated toward understanding the biochemical basis for 
the retardation. The endeavor to produce a chemical model in this Laboratory 
centers around phenylketonuria because it is the best characterized from a 
biochemical and a behavioral aspect, and because a great deal is known about 
the enzyme which is deleted, phenylalanine hydroxylase. If such a model can 
be produced, substantial information should be available about the causes and 
perhaps the prevention of tlie retardation. 

Proposed Course of Project : (1) Attempts to exacerbate the lesion by prenatal 
administration of the p-chlorophenylalanine and phenylalanine. (2) Attempts 
to characterize chemically the metabolic lesion by administering one of the 
products of the abberant metabolism, e.g., phenyllactic acid, to see if it 
causes the same behavioral alterations in the adult rats. (3) Attempts to 
prevent the defect from developing by simultaneously administering other 
materials, e.g., 5-hydroxy tryptophan. (4) Attempts to alleviate at least the 
secondary effects of the disease, e.g., skin rash and irritability, in adult 
phenylketonuria by altering the amino acid balance of the blood. (5) Attempts 
to produce other behavioral anomalies in rats, e.g., the Lesch-Nyhan Syndrome, 
by a similar approach. 

Honors and Awards: None 

Publications: 

1. Andersen, A. and Guroff, G. : Enduring behavioral changes in rats 
with experimental phenylketonuria . Proc. Nat. Acad. Sci., U.S.A . , 
69: 863-867, 1972. 

2. Andersen, A., Abramowitz, A., and Guroff, G.: Biochemical and 
behavioral changes in rats with experimental phenylketonuria. 

In Barchas, J. (Ed.): Behavioral Effects of Changes in Level of 
Brain Serotonin . Palo Alto, Academic Press, in press. 

3. Andersen, A. and Guroff, G. : An animal model of phenylketonuria. 
Am. J. Pathol., in press. 



FK7 



Serial No. HD-LBIO 



1. Laboratory of Biomedical Sciences 

2. Section on Intermediary Metabolism 

3. Bethesda 



PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Mechanism of Aromatic Hydroxy lation 

Previous Serial Number: Same 

Principal Investigator: G. Guroff 

Other Investigators: C. Letendre 

C. Williams 
('. Pickens 

Cooperating Units: None 

Man Years: 

Total: 39/12 
Professional: 27/12 
Others: 12/12 

Project Description: 

Objectives : The objectives of this research are: (1) to provide information 
on the process of enzymatic hydroxy lation; and (2) to get detailed informa- 
tion on the nature of phenylalanine hydroxylase. 

Methods Employed : The methods employed include standard techniques of enzyme 
kinetics and purification, antibody detection, and organic synthesis and 
characterization. 

Major Findings : (1) At least four different bacterial species prodvici' an 
inducible phenylalanine hydroxylase; (2) Chromobacter will also produce a 
tryptophan hydroxylase which is not the same enzyme as its phenylalanine 
hydroxylase: (3) Chromobacter tryptophan hydroxylase is a pterldlne- 
requiring enzyme similar to the one found in mammals for the synthesis of 
Herotonin; tt had never been obtained from bacteria before; (A) dihydro- 
plerldlno reductase, an enzyme functioning in the coupled phenylalanine 
hydroxylase syHtcm, is also Inducible by phenylalanine In Comamonas ; It 
has been. purified and characterized from this organism. 

Significance to Biomedical Research and the Program of the Institute : 
Information about the phenylalanine and tryptophan hydroxylases and about 
ways to inhibit or stimulate them relates directly to the general problems 
of normal neural function and of mental retardation. The phenylalanine 



FS8 



hydroxylase is the missing enzyme in the best characterized of the genetic 
retardations, phenylketonuria. The tryptophan hydroxylase is the first and 
rate-limiting enzyme in the biosynthesis of the neurohumoral agent, serotonin. 
Detailed analysis on the structure and mechanism of these enzymes will pro- 
vide new and unique information since relatively little has been accomplished 
on hydroxylases in general and few, if any, pteridine enzymes have been 
reported as homogeneous preparations. 

Proposed Course of Project : (1) Continued study of the purification, mode of 
action, and structure of the bacterial enzymes phenylalanine hydroxylase, 
tryptophan hydroxylase, and dihydropteridine reductase. 

Honors and Awards : None 

Publications: None 



1. 



FR9 



Serial No. HD-LBll 



1. Laboratory of Biomedical Sciences 

2. Section on Intermediary Metabolism 

3. Bethesda 



PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Biosynthesis and Function of Pteridines 

Previous Serial Number: Same 

Principal Investigator: G. Guroff 



Other Investigators 


: J . Cone 




J. Plowman 


Cooperating Units: 


None 


Man Years: 




Total: 


27/12 


Professional: 


15/12 


Other: 


12/12 



Project Description: 

Objectives : The objectives of this research are: (1) to provide information 
on the enzymes in the blosynthetic pathway to the pteridlne ring; (2) to 
investigate the functions of pteridines in addition to their action as 
hydroxylase cof actors. 

Methods Employed : The methods used include standard techniques of enzyme 
purification, isotope measurement, high-voltage electrophoresis, and 
fluorescence detection. 

Major Findings : (1) The first enzyme in the pteridine blosynthetic pathway 
of Comamonas has been fractionated into two protein portions; one seems to 
have a stimulating effect on the other and may be involved in altering the 
stereochemistry of the product produced. 

Significance to Biomedical Research and the Program of the Institute : The 
pteridine cofactors are important in a number of hydroxylase reactions 
including those leading to the neurohormones serotonin and norepinephrine. 
Information about the pharmacology and biochemistry of these blosynthetic 
enzymes may lead to the ability to inhibit or stimulate these pathways and 
perhaps to new therapeutic tools for maladies involving these neurohormones. 

Proposed Course of Project : (1) Purification of the two portions of the first 
enzyme and elucidation of the function of each; (2) purification and char- 
acterization of other enzymes of pteridine metabolism; (3) attempts to link 

FKIO 



pteridines or pteridine derivatives to the enzymes they serve; (4) study of 
pteridine biosynthetic enzymes in photosynthetic organisms; (5) search for 
other functions of pteridines. 

Honors and Awards: None 

Publications: None 



1 



FKll 



Serial No. HD-LB12 



1. Laboratory of Biomedical Sciences 

2. Section on Intermediary Metabolism 
3 • Bethesda 



PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Biochemistry of Brain Macromolecules 

Previous Serial Number: Same 

Principal Investigator: G. Guroff 



Other Investigators 


: J. DeLarco 




A. Abramowitz 




S . Nakagawa 




K. Bromwell 


Cooperating Units: 


None 


Man Years: 




Total: 


51/12 


Professional: 


39/12 


Others: 


12/12 



Project Description: 

Objectives : The objectives of this research are: (1) to investigate the 
biochemistry of brain macromolecules; (2) to determine if any aspects exist 
that are unique to brain; and (3) to find out whether any unique aspects are 
related to the unique function of the brain. 

Methods Employed : The methods involved in this study include isotope 
techniques, high voltage electrophoresis, and tissue slice metabolic studies, 
as well as standard methods for purification and investigation of macro- 
molecules. 

Major Findings : (1) Brain "messenger" RNA's have been purified by their 
ability to bind to oligo(dT) -cellulose; (2) the RNA binding properties of 
various celluloses have been described and explained; (3) brain "messenger" 
RNA has been described in terms of its size, its poly (A) sequences, and its 
ability to be translated by a protein synthesis system; (4) synaptosomal 
RNA has been purified and its properties studied; its resemblance to mito- 
chondrial RNA has been noted; (5) differences between the messenger fraction 
from the brains of immature and of adult animals with respect to the length 
of the poly (A) sequences have been found. 

Significance to Biomedical Research and the Program of the Institute : 
Substantial evidence exists that brain macromolecules are involved in the 

FB12 



unique functions of the brain. A continuing question exists as to the 

directness of this involvement. These studies may shed light on this 

question by finding properties of brain macromolecules compatible with a 

unique function such as the storage of memory. Further, development of < 

such systems as the tissue slice for RNA synthesis may provide tools for 

the investigation of the biochemical alterations occurring in certain types 

of mental retardation. 

Proposed Course of Pro.lect : (1) Further characterization of rat brain 
messenger RNA; (2) attempts to understand the differences In the messenger 
fraction from young and from adult rats; (3) attempts to isolate messenger 
RNA's for brain-specific proteins; (4) separation of the various cell types 
from brain, and study of their messenger RNA's. 

Honors and Awards: None 

Publications: 

1. Guroff, G. : Biochemistry of the brain. In Mason, H.S. (Ed.): •** 
Functional Biochemistry . MacMillan Co., in press. "U 

2. Guroff, G. : Special aspects of amino acid metabolism. In Albers, « 
R.W., Slegel, G.J., Katzman, R. , and Agranoff, B.W. (Eds.): Basic '"■ 
Neurochemistry . Little, Brown and Co., 1972, pp. 191-206. ". 

3. Gutensohn, W. and Guroff, G. : A rapid assay for purine phosphorlbosyl J 
transferases. Anal. Blochem . 47: 132-138. 1972. h' 

s 

4. Gutensohn, W. and Guroff, G.: Hypoxanthlne-guanine phosphoribosyl- 
transf erase from rat brain (purification, kinetic properties, 
development, and distribution). J. Neurochem . 19: 2139-2150, 1972. 

5. DeLarco, J. and Guroff, G. : The synthesis of RNA which binds to 
oligo(dT)-cellulose by brain in vivo and in vitro . Blochem. Biophys . 
Res. Comm . 49: 1233-1240, 1972. 

6. Nakagawa, S., and Guroff, G. : The uptake of purines by rat brain - 
in vivo and in vitro . J. Neurochem . , in press . 

7. DeLarco, J. and Guroff, G. : The binding of RNA to various 
celluloses. Blochem. Biophys. Res. Comm . 50: 486-492, 1973. 



FBi3 



Serial No. HD-LB4 



1. Laboratory of Biomedical Sciences 

2. Section on Physiological Controls 

3. Bethesda 



PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Development of Mammalian Metabolic Regulation 

Previous Serial Number: Same 

Principal Investigator: W. H. Glinsmann 

Other Investigators: H. Eisen 

F. Zieve 

M. Mltsunaga 

F . Huang 

Cooperating Units: I. Goldfine, Clinical Endocrinology Branch, NIAMD 

K. Freude, Minneapolis Veterans Hospital, University 

of Minnesota 
L. Zieve, Minneapolis Veterans Hospital, University 

of Minnesota 
P. Sherline, Department of Medicine, Washington 

University School of Medicine, St. Louis, Mo. 

Man Years 

Total: 69/12 
Professional: 51/12 
Others: 18/12 

Project Description: 

Objectives : This project seeks to define in mammalian cells: (1) mechanisms 
underlying differential modulation of carbohydrate and lipid metabolism and 
adaptive protein synthesis by hormones and nutrients; and (2) factors control- 
ling development of these mechanisms. A general model is being examined 
based on control of protein phosphorylations which regulate rate-limiting 
enzymes. Current interest centers on a comprehensive description of glycogen 
cycle enzymes. 

Methods Employed : Isolated cell and organ culture techniques and isolated 
organ perfusion are used in studying liver glucose production and rate- 
limiting enzymes in glycogen metabolism. Enzymes and regulatory proteins 
controlling glycogen turnover are isolated; their mode of regulation is 
described. 

M ajor Findings : 1 . Phosphoproteins controlling glycogen turnover : Many 
<lp|aMB iiT liow t:v<''l<' AMT jnediaten thp pffecte of glnra^on nnd eplneplir Ine 

FK14 



on glycogenolysis are knovm: cyclic AMP-activatlon of a general protein 
kinase which phosphorylates regulatory proteins has been studied extensively. 
However, increasing evidence supports the concept that hormonal and substrate 
effects on protein phosphorylation are also mediated through the corresponding 
phosphatase reactions. We are developing a detailed model for studying these 
interactions based on separation of glycogen cycle enzymes. Partial purifi- 
cation of the major enzymes has been achieved. 

We postulated the existence of a general phosphoproteln phosphatase that 
reverses the effects of the cyclic AMP-dependent protein kinase and obtained 
initial evidence that glycogen synthetase phosphatase is this enzyme. Sub- 
strate specificity of the phosphatase, metal requirements, and effects of 
metabolic intermediates have been partially characterized. 

Phosphorylase phosphatase, about which little is known, has been partially 
purified. 

2. Development of hormonal and substrate regulation of glycogen turnover 
in isolated fetal liver : We have recently developed conditions for main- 
taining in defined media viable, hormonally-responsive explants of fetal 
and neonatal rat liver. The developmental pattern of glycogen synthesis has 
been duplicated in this system by the sequential administration of hydro- 
cortisone and insulin. Evidence has been obtained for the following: (1) 
direct regulation of glycogen synthetase and phosphorylase by glucose does 
not occur in fetal tissue; (2) in term fetal explants, insulin stimulates 
glycogen synthesis and activation of glycogen synthetase by a process 
requiring protein synthesis; (3) at an early gestational age (day 15-16) 
when total glycogen synthetase levels are low, insulin does not stimulate 
glycogen synthesis although specific insulin receptors are fully developed; 
(4) the initial induction of glycogen synthetase requires hydrocortisone 
(glucocorticoids) , and subsequent activation of this enzyme by insulin 
promotes glycogen sjmthesls. 

The organ culture system in which the development of glycogen synthesis can 
be duplicated is likely applicable to studies in development of other regula- 
tory systems. Additional studies with the system have shown that the fetal 
liver adrenergic receptor which controls glycogenolysis is of the beta type. 

3. Post-heparin lipolytic enzymes : The hormonal stimulation of the hydrolysis 
of adipose tissue triglycerides has been studied extensively, and has been 
shown to be mediated through the cyclic AMP-dependent protein kinase. The 
opposite physiological process, i.e., the hydrolysis of plasma glycerides and 
their subsequent uptake by tissues, is also under hormonal control, but the 
molecular basis of this regulation has not yet been elucidated. As an initial 
step in the study of such regulation, we have separated and characterized the 
lipolytic activities of post-heparin plasma. Post-heparin monoglyceride 
esterase has been purified 10,000-fold and has been shown to be identical to 
post-heparin phospholipase A^ , but distinct from post-heparin lipase. Pre- 
liminary studies of the acute regulation of lipoprotein lipase in tissue 
extracts have been carried out. A rapid and sensitive lipase assay has been 
devised. 



FKL5 



Significance to Biomedical Research and the Program of the Institute : 
Regulation of cellular metabolism, growth, and differentiation at many 
phylogenetic levels is in part mediated by a complex series of interactions 
between glucose and factors which alter cyclic AMP metabolism. Currently, 
the best characterized model for cyclic AMP action in mammalian tissue (where 
cyclic AMP acts as an intracellular mediator for many hormones and neuro- 
transmitters) is the cascade of glycogen cycle protein phosphorylations 
initiated by cyclic AMP. We and others have shown that these reactions are 
also regulated by glucose, insulin, and glucocorticoids. Therefore a compre- 
hensive decription of these reactions will hopefully allow us to develop a 
general model for cyclic AMP-glucose interactions in mammalian cells and 
thus provide a basis for detailed studies on factors affecting the development 
of the corresponding regulatory systems. The control of lipid metabolism is 
another system in which protein phosphorylations play an important regulatory 
role and in which interactions among hormones, substrates, and cyclic AMP can 
be studied. 

Proposed Course of Project : Separation and characterization of enzymes 
participating in glycogen cycle phosphorylations will continue with particular 
emphasis on the regulatory roles of a general phosphoprotein phosphatase and 
phosphorylase phosphatase. Possible mechanisms mediating the effects of 
glucose and insulin will be sought. 

Organ culture of fetal explants will be used to further define factors 
influencing the development of rate-limiting enzymes or hormone receptors 
that control glucose homeostasis, glycogen metabolism, gluconeogenesis, and 
cyclic AMP turnover. 

The purification and characterization of the enzymes invovlved in the 
transport of fats from plasma into tissues will be continued. Immunologic 
techniques will be used to study the specific roles of the various enzymes 
in the hydrolysis of lipoprotein glycerides. The study of the acute regulation 
of plasma and tissue lipolytic enzymes will be pursued, with emphasis on the 
role of protein phosphorylations and dephosphorylations. 

Honors and Awards: None 

Publication: 

1. Sherline, P., Lynch, A., and Gllnsmann, W. : Cyclic AMP and 
adrenergic receptor control of rat liver glycogen metabolism. 
Endocrinology 91: 680-690, 1972. 

2. Goldfine, I.D. and Sherline, P.: Insulin action in isolated rat 
thymocytes. II. independence of insulin and cyclic adenosine 
monophosphate. J. Biol. Chem . 247: 6927-6931, 1972. 

3. Eisen, H.J. and Glinsmann, W.H. : Effects of insulin on glycogen 
synthesis in fetal rat liver in organ culture. Endocrinology 92: 
584-588, 1973. 



FKa.6 



f 



4. Zieve, F.J. and Glinsmann, W.H. : Activation of glycogen synthetase 
and inactivation of phosphorylase kinase by the same phosphoproteln 
phosphatase. Biochem. Biophys. Res. Commun . 50: 872-878, 1973. 

5. Eisen, H.J., Sherline, P., and Glinsmann, W.H. : Activation of 
glycogen synthetase by insulin in fetal rat liver in organ culture. 
Endocrinology , in press. 



FBa7 



Serial No. HP LB18 

1. Laboratory of Biomedical Sciences 

2. Section on Physiological Controls 

3. Bethesda 



PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

rrolfcl "I'ltlf: Rfp.iilat 1(111 oi Neurot^iuliu r i nu Mefdlxil 1 dm 

TreviouB Serial Number: Same 



Principal Investigator: 
Other Investigators: 

Cooperating Units: 



D. C. Klein 

S. A. Binkley 
A. Parfitt 
M. A. Vaughan 

R. Y. Moore, Division of Neurology, University of 

Chicago Medical School, Chicago, Illinois 
R. Bensinger, Laboratory of Vision Research, NEI 
A. Yuwiler and A. Geller, Neurobiochemistry 

Laboratory, V.A. Brentwood Hospital, Los Angeles, 

California 
G. Jaim-Etchevery, Instituto de Anatomia General y 

Embriologia, Universidad de Buenos Aires, Argentina 
L. Wetterberg and M. Backstrom, Uppsala University, 

Uppsala, Sweden 
J. Pisano, Experimental Thrapeutic Branch, NHLI 
S. MacBride and C. Ralph, Biology Department, 

University of Pittsburg, Pennsylvania 



Man Years 



Total: 

Professional: 

Others: 

Project Description: 



60/12 
42/12 
18/12 



Objectives : (1) To describe the mechanism through which neuronal activity 
regulates biochemical activity in a neuroendocrine tissue, the pineal gland, 
and in the brain; (2) to describe factors regulating the development of 
enzyme systems and regulatory mechanisms in neural and neuroendocrine tissues; 
(3) to describe the role of N-acetylation of serotonin in the brain; (4) to 
describe the effects of drugs on indoleamine metabolism in developing and 
matiirp npuroendocrlne tissue; (5) to Isolate and characterize a pineal peptide 
whuli liililbltrt ovai'lctn functloM; (6) to «lt>»i;rib«< tito roRulntlon of l lio Ht— 
cretion and production of the antigonadotrophlc pineal peptide. 



FE18 



Methods Employed : (1) Organ culture is used to provide a well defined and 
easily controlled experimental system for studying neurochemical regulation. 
Rat, human, and avian pineal tissue is cultured and treated with drugs and 
neurochemicals. The effects of these treatments are determined by measuring: 
(a) the total amount of tryptophan hydroxylated; (b) the amount of cyclic 
AMP in the tissue; (c) the activity of the enzyme that degrades cyclic AMP, 
phosphodiesterase; (d) the activity of N-acetyltransf erase, the enzyme that 
converts serotonin to N-acetylserotonin; (e) the amount of serotonin in the 
gland and culture medium; and (f) the conversion of tryptophan to serotonin 
N-acetylserotonin, melatonin, hydroxyindoleacetic acid, hydroxtryptophol, and 
other serotonin derivatives. (2) The development of enzyme systems and 
regulatory mechanism in neural tissue is studied by describing the development 
of a system in the intact animal. Two systems of interest are the regulatory 
system controlling the activity of N-acetyltransferase and the activity of 
hydroxyindole-0-methyltransferase. The biochemically undifferentiated 
tissue is cultured and treated with hormones, transmitters, and drugs to study 
the factors regulating the biochemical differentiation of the tissue. (3) The 
pineal peptide is isolated by classical biochemical techniques including 
organic partitioning, gel filtration and thin-layer chromatography. The 
active material is monitored using the compensatory ovarian hypertrophy assay. 
The purified material is analyzed with an amino acid analyzer. 

Major Findings : 1. Neural regulation of biochemical activity : a. Adrenergic 
regulation of serotonin: The amount of serotonin in the pineal gland is regu- 
lated by the release of norepinephrine from nerve endings. The released trans- 
mitter interacts with highly specific receptors in postsynaptic membranes. 
This receptor responds to the L-form of norepinephrine but not the D-form, does 
not respond to dopamine, and can be blocked by a B-adrenergic antagonist but 
not by a-adrenergic antagonists. The 6-adrenergic protagonist, isoprel, mimicks 
the effects of norepinephrine, but the a-adrenergic antagonist, phenylephrine, 
is without effect. The effects of the interaction of norepinephrine with the 
receptor is to increase the activity of adenyl cyclase. This in turn causes 
an increase in the amount of cyclic AMP in the cells. The increase in the 
amount of cyclic AMP causes an increase in the activity of N-acetyltransferase, 
resulting in a decrease in the amount of serotonin in the cell and an increase 
in the amount of N-acetylserotonin and melatonin produced, b. Regulation of 
tryptophan hydroxy lat ion: The amount of tryptophan hydroxylated by a pineal 
gland is proportional to the concentration of external tryptophan up to 0.5 mM. 
At higher concentrations the rate of tryptophan hydroxylation gradually de- 
creases. This decrease may be due to an accumulation of hydroxytryptophan or 
serotonin in the tissue. Hydroxylation depends upon ongoing protein synthe- 
sis; in the absence of protein synthesis, hydroxylation ceases after eight 
hours, c. Regulation of indoleamlne metabolism in the pineal gland: The 
regulation of indoleamlne metabolism in the avian pineal gland resembles that 
in the mammalian pineal gland. At night the activity of N-acetyltransferase 
increases many fold. This causes an increase in the amount of melatonin in 
the gland. The activity of hydroxyindole-0-methyl transferase, in contrast, 
does not increase. Unlike the rat pineal gland, the rhythms in indoleamlne 
metabolism in the bird persist in the absence of the superior cervical ganglia. 

2. Pineal peptides ; A peptide has been isolated that has a molecular weight 
less than 2000. Analysis of thin-layer chromatography and by N-terminal 

FK19 



dansylation indicates the preparation purified is homogenous. Less than 1 
nanogram injected I .P. to a mouse 30 minutes after unilateral ovarectomy 
prevents the normal compensatory hypertrophy usually observed 6 days later. 

3. Regulation of biological rhythms : The circadian rhythm in N-acetyltrans- 
ferase appears to be driven by an endogenously generated "clock" in the 
suprachiasmatic nucleus. Destruction of all apparent neural input to this 
area does not block the circadian rhythm, whereas sectioning the neural output 
from this area does. 

Significance to Biomedical Research and the Program of the Institute : These 
studies have provided explanations of how neurochemical metabolism can be 
regulated. Such explanations may also apply to neural tissue in general and 
provide models for describing brain function. The studies with drugs will 
also explain some of the effects of drugs on a molecular basis and will allow 
for a better understanding of the action of drugs and the normal functioning 
of neural tissue and for the development of more efficient drugs in the future. 
The pineal peptide that has been isolated will provide the reproductive endo- 
crinologist with another valuable tool that he may use as a drug or as a 
diagnostic tool. This compound could be used effectively in the regulation 
of reproduction because it is so potent and long acting. 

Proposed Course of Project : (1) The regulation of neurochemical metabolism 
will have as its focus the regulation of the activity of N-acetyltransf erase. 
The nature of the neural control of gene expression will be determined. (2) 
The effects of hormones and drugs on the neural control of gene expression 
will be investigated. The nature of the regulation of biochemical differen- 
tiation will be studied. (3) The pineal peptide will be described as to 
amino acid composition and sequence. The peptide will be synthesized and 
the effects of the peptide studied. A radioimmunoassay for the peptide will 
be developed and the physiology of the production and secretion of the peptide 
will be described. 

Honors and Awards: Neurosciences Research Program Fellow, Intensive Study 
Program, 1972. 

Publications: 

32 

1. Berg, G.R. and Klein, D.C. : Norepinephrine stimulates P incor- 
poration into phosphatidyl inositol in membranes of cultured pineal 
glands. J. Neurochem . 19: 2519-2532, 1972. 

2. Ellison, N., Weller, J., and Klein, D.C: Development of a 
circadian rhythm in the activity of pineal serotonin N-acetyl- 
transf erase. J. Neurochem . 19: 1335-1341, 1972. 

3. Sllberateln, S.D., Lemberger, L. , Klein, D.C, Axelrod, J., and 
Kopln, T.J.: Induction of adrenal tyrosine hydroxylase activity 
111 ornnii culture. NeuropharmacoloRy 11: 721-726, 1972. 



[ 



FKO 



4. Klein, D.C.: Evidence for the placental transfer of melatonin 
[acetyl- H]. Nature 237: 117-118, 1972. 

5. Strada, S.J., Klein, D.C., Weller, J., and Weiss, B. : Effects of 
norepinephrine on the concentration of adenosine 3' ,5 '-monophosphate 
of rat pineal gland in organ culture. Endocrinology 90: 1470-1475, 
1972. 

6. Klein, D.C.: The role of serotonin N-acetyltransferase in the ' 
adrenergic regulation of indole metabolism in the pineal gland. 

In Usdin, E. and Barchas, J. (Eds.): Serotonin and Behavior . 
New York, Academic Press, in press. 

7. Klein, D.C. and Weller, J.L. : Rapid light-induced decrease in 
pineal serotonin N-acetyltransferase activity. Science 177: 532-533, 
1972. 

8. Klein, D.C: Melatonin metabolism. In Kopin, I. and Rail, E. (Eds.): 
Methods in Investigative and Diagnostic Endocrinology . Amsterdam, ^ 
North-Holland Publishing Co., 1972, pp. 550-568. « 

mi 

9. Klein, D.C. and Weller, J.: Adrenergic-adenosine 3' ,5 '-monophosphate 
regulation of serotonin N-acetyltransferase activity and the temporal 
relationship of serotonin N-acetyltransferase activity to synthesis I 
of 3H-N-acetylserotonin and ^ij-melatonin in the cultured rat pineal ' 
gland. J. Pharm. Therap . , in press. j 

,) 

10. Klein, D.C, Yuwiler, A., Weller, J.L., and Plotkin, S.: Post- J 
synaptic adrenergic-cyclic AMP control of the serotonin content •■ 
of cultured rat pineal glands. J. Neurochem . , in press. 

11. Klein, D.C: Circadian rhythms in indole metabolism in the rat 
pineal gland. In Parker, D.K.O. (Ed.): The Neurosclences : Third 
Study Volume . Cambridge, MIT Press, in press. 

12. Binkley, S., MacBride, S., Klein, D.C, and Ralph, C: Pineal 
enzymes: Regulation of avian melatonin synthesis. Science , in 
press. 

13. Coyle, J., Jacobowitz, S., Klein, D.C, and Axelrod, J.: Dopamine 
neurons in explants of substantia nigra in culture. J. Neurobiol . , 
in press. 



FMl 



i 



Serial No. HD-LB8(1) 

1. Laboratory of Biomedical Sciences 

2. Section on Developmental Pharmacology 

3. Bethesda 



PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Developmental Pharmacology 

Previous Serial Number: Same 



I 



Principal Investigator: 
Other Investigators: 



Cooperating Units: 



D. W. Nebert 

I. S. Owens 

J. R. Robinson 

A. Niwa 

J. S. Felton 

N. Considine 

W . Tenney 

R. M. Friedman, Laboratory of Pathology, NCI 

E. B. Thompson, Laboratory of Biochemistry, NCI 
J. W. Daly, Laboratory of Chemistry, NIAMD 

H. Kon, Laboratory of Chemical Physics, NIAMD 
M. J. Coon, University of Michigan, Ann Arbor 

A. P. Poland, University of Rochester Med. Sch. , N.Y. 
R. E. Kouri, Microbiological Associates, Inc., Bethesda 

B. Paul, Birth Defects Institute of New York State, 
Albany 



Man Years 

Total 56/12 
Professional 42/12 
Others: 14/12 

Project Description: 

Objectives : Membrane-bound mixed-function oxygenases are important in the 
metabolism of drugs, carcinogens, and insecticides, as well as many endogenous 
lipophilic substrates such as steroids, hemin, and bilirubin. We found that 
one such oxygenase, aryl hydrocarbon hydroxylase, is inducible in mammalian 
fetal liver cells grown in culture by polycyclic hydrocarbons and by pheno- 
barbital. Whereas it is well known that polycyclic hydrocarbons and pheno- 
barbital induce hepatic drug-metabolizing enzymes by different mechanisms, 
the precise effects evoked by these two classes of microsomal enzyme inducers 
are not understood. Cell culture thus provides an opportunity for studying 
the mechanisms of microsomal oxygenase induction in an experimental system 
which is more rigidly controlled than similar studies in the Intact animal. 



FK22 



Understanding the mechanisms by which drug-metabolizing enzymes are induced 
may elucidate important concepts in drug metabolism, developmental pharma- 
cology, steroid biochemistry, chemical carcinogenesis, and entomology. The 
main objectives of this project are: (a) to compare the rates of hydroj.ylase 
synthesis and degradation and the physical properties of the phenobarbital- 
induced enzyme with those of the hydroxylase induced by polycyclic hydro- 
carbons, (b) to determine the extent to which hormones and nutrition affect 
the hydroxylase induction by either compound, (c) to characterize specific 
changes in nuclear acidic proteins, cellular RNA, and microsomal membrane 
protein during the induction process by either compound, (d) to measure entry 
and binding of various inducers to specific receptor sites within the hepato- 
cytes, (e) to develop methods for inhibiting or stimulating the sequence of 
events involved during the oxygenase induction by either class of inducers, 
and (f) to use this experimental system for studying the control of specific 
gene expression in higher organisms. 

Methods Employed ; The principal methods employed are: (1) recording spectro 
photometry of turbid solutions, (2) enzyme assays involving radiometric, 
spectrophotometric, and spectrophotofluorometric determinations of product 
formation or substrate disappearance, (3) radioisotopic measurements using 
tritium and carbon-14, (4) cell culture techniques, (5) differential centri- 
fugation, (6) phase microscopy, (7) equilibrium dialysis, (8) polyacrylamide 
gel electrophoresis, (9) column and thin-layer chromatography, and (10) mag- 
netic resonance techniques. 

Major Findings ; (1) Mouse skin tumorigenesis initiated by 7,12-dimethyl- 
benz[a]anthracene or benzo[a]pyrene, with or without promotion by repeated 
applications of phorbol ester, is unrelated to genetic differences in the | 
extent of aryl hydrocarbon hydroxylase induction by polycyclic hydrocarbons 
in inbred or hybrid C57BL/6N and DBA/2N mice; on the contrary, mouse sarcomas 
Initiated by a single subcutaneous dose of 3-methylcholanthrene is related to 
the extent of the hydroxylase induction in these parent strains and their pro- 
geny from backcrosses and intercrosses. (2) Levels of hepatic epoxide hydrast 
activity were not closely associated with genetic differences in aryl hydro- 
carbon hydroxylase in these strains. (3) Aryl hydrocarbon hydroxylase 
activity is inducible in mouse 3T3 fibroblasts by benz [a] anthracene, whereas 
no detectable basal or inducible levels occur in hamster BHK cells or rat- 
hepatoma HTC cells and barely detectable inducible levels of this enzyme 
are found in human D98 cultures. Induction of the hydroxylase activity occurs 
to about the same degree as that in the mouse parent line — in 6 out of 9 
mouse-hamster hybrids and in 3 out of 3 mouse-htmian hybrids formed by somatic- 
cell fusion. (4) In mouse 3T3-rat HTC hybrids, levels of inducible hydroxylase 
activity range from the same as to more than 20-fold greater than that in the 
3T3 parent. (5) Tyrosine aminotransferase activity is inducible in HTC cells 
by dexamethasone, whereas only low noninducible levels of this enzyme exist 
in 3T3 cells; the aminotransferase levels remain very low and noninducible 
in all of these same fused somatic-cell hybrids, and there is immunological 
evidence indicating that all of the basal noninducible aminotransferase 
activity is derived from the mouse 3T3 parent. (6) Aryl liydrocarbon hydroxy- 
lase induction by aromatic hydrocarbons is expressed dominantly, codominantly, 
or repressed dominantly, depending on the inbred strains of mice crossed. 



FK23 



(7) Induction of the microsomal hydroxylase activity by aromatic hydrocarbons 
is associated with concomitant synthesis of a new CO-binding hemoprotein, 
"cytochrome P-448," as determined by (i) spectral changes in the binding of 
n-octylamine to the microsomes, (ii) preferential inhibition of benzo[a]pyrene 
hydroxylation in vitro by various lipophilic test compounds, and (iii) para- 
magnetic changes in oxidized microsomes measured by electron paramagnetic 
resonance spectroscopy below 10° K; these changes are quite similar in liver 
or kidney microsomes from mouse, rat, or rabbit. (8) The rate at which the 
hydroxylase activity accumulates in fetal rat liver primary cultures treated 
with 1 mM norepinephrine or other biogenic amines of similar structure is 
quite similar to that by phenobarbital or polycyclic hydrocarbons. (9) The 
effect of norepinephrine is greater than that of phenobarbital or 3-methyl- 
cholanthrene in retarding the normal rate of degradation of induced hydroxy- 
lase activity in the presence or the absence of protein synthesis. (10) Either 
phenobarbital or 3-methylcholanthrene or norepinephrine as a second inducer 
can direct at some posttranscriptional level a further rise in hydroxylase 
activity after treatment of fetal rat hepatocyte cultures with the first 
inducer. (11) A simple radiometric assay for determining aminopyrine N- 
demethylase activity was developed, using the principle of partitioning the 
polar l^C-formaldehyde metabolite complexed with semicarbazide from the 
lipophilic nonmetabolized substrate (methyl--'-^C-labeled aminopyrine) . This 
radioisotopic assay correlates well with the standard colorimetric method and 
is at least 100 times more sensitive. (12) In cell cultures previously 
treated with homologous interferon, the magnitude of antiviral activity and 
the degree of stimulation of aryl hydrocarbon hydroxylase induction appear to 
be directly related; both activities demonstrate species specificity, sensi- 
tivity to heat, and sensitivity to trypsin. (13) When mice are administered 
aromatic hydrocarbons, the induction of numerous enzyme "activities" — all 
membrane-bound mono-oxygenases having cytochrome P-450 associated with their 
active sites — is associated with the same genetic locus or with closely linked 
loci; these enzyme "activities" are: aryl hydrocarbon hydroxylase, p^-nitro- 
anisole 0-demethylase, 7-ethoxycoumarin 0-deethylase, 3-methyl-4-methylamino- 
azobenzene N-demethylase, zoxazolamine hydroxylase, acetanilide hydroxylase, 
naphthalene hydroxylase, and benzenesulfonanilide hydroxylase. (14) The 
expression of numerous other microsomal enzymes are not associated with this 
same genetic locus: aminopyrine N-demethylase, NADPH-cytochrome P-450 
reductase, NADPH-cytochrome c^ reductase, d-benzphetamine N-demethylase, 
chlorcyclizine N-demethylase, ethylmorphine N-demethylase, pentobarbital 
hydroxylase, and 6B-, 7a-, and 16a-testosterone hydroxylases. (15) The 
extent of aryl hydrocarbon hydroxylase induction in various tissue of 10 
inbred strains of mice by aromatic hydrocarbon administration in vivo is 
correlated with the magnitude of hydroxylase induction in fetal cell cultures 
derived from each of these strains. (16) Cytotoxicity in cell culture, 
caused by the metabolic potentiation of polycylic hydrocarbons by the inducible 
hydroxylase, is correlated with a decreased survival time when aromatic hydro- 
carbon-"responsive" mice are given large doses of polycyclic hydrocarbons. 
(17) Aryl hydrocarbon hydroxylase activity also is inducible in more than 5 
liver-derived established cell lines treated with polycyclic hydrocarbons, 
biogenic amines, or phenobarbital in the growth medium. 



FK24 



Significance to Biomedical Research and the Program of the Institute : Pheno- 
barbital is the most commonly used drug from a class of more than 200 drugs, 
chemicals, and insecticides that induce microsomal enzymes apparently by the 
same mechanism. Administration of any of these substances to the pregnant 
animal causes transplacental induction of these mixed-function oxygenases in 
the fetal liver. These enzyme systems metabolize steroids, hemin, cholesterol, 
fatty acids, indoles, sympathomimetic amines, thyroxine, and bilirubin, as 
well as lipophilic foreign substrates. Administration of polycyclic hydro- 
carbons to the pregnant animal produces induction of this enzyme activity in 
the placenta and also transplacental stimulation of the hydroxylase in sev- 
eral fetal tissues. Cigarette smoking is associated with increases in human 
placental aryl hydrocarbon hydroxylase activity. Consequently, studies in- 
volving the development and extent of induction of mixed-function oxygenases 
in fetal tissues and in the placenta during various periods of gestation, are 
relevant to the program of this Institute in the following areas: (1) possible 
effects of environmental pollutants (i.e., polycyclic hydrocarbons and insecti- 
cides) and various drugs on normal fetal growth and development and on the 
health of the pregnant woman, (2) mechanisms of "pharmacological immaturity" of 
the premature and newborn, (3) mechanisms of disease of the fetus and newborn 
concerning oxygenative and conjugative metabolism of normal body substrates such 
as steroids and bilirubin, and (4) possible therapeutic and diganostic proce- 
dures for aiding and predicting pharmacogenetic defects in the fetus and neonate. 

Proposed Course of Project : (1) Further delineation of differences between 
mixed-function oxygenase induction by phenobarbital and that by polycyclic 
hydrocarbons, (2) characterization of specific and nonspecific binding of 
these various inducers to subcellular sites, (3) examination of the effects 
of hormones and nutrition on hydroxylase induction by either type of inducers, 

(4) study of various inhibitors on the induction process in cell culture, 

(5) characterization of the various components of the membrane-bound "hydrox- 
ylase" system, (6) analysis of differences in product formation and biophysical 
properties between the control, phenobarbital, and polycyclic hydrocarbon- 
induced oxygenases, and (7) extrapolation of these studies to formulate a 
general theory for explaining the mechanisms by which gene expression is 
controlled in mammalian cells. 

Honors and Awards : 

Nominated for John J. Abel Award, American Society for Pharmacology and 
Experimental Therapeutics. Nominated for Outstanding Young Scientist of 
Maryland Award 

Publications: 

1. Nebert, D.W., Benedict, W.F., Gielen, J.E., Oesch, F. , and Daly, J.W.: 
Aryl hydrocarbon hydroxylase, epoxide hydrase, and 7,12-dimethyl- 
benz[a]anthracene-produced skin tumorigenesis in the mouse. Mol. 
Pharmacol . 8: 374-379, 1972. 

2. Nebert, D.W. , and Gielen, J.E.: Genetic regulation of aryl hydro- 
tarbon liydroxy 1 /iso induction. Fed. Proc . 31: 1315-1325, 1972. 



FK25 



i: 



3. Benedict, W.F., Paul, B., and Nebert , D.W. : Expression of benz[a]- 
anthracene-inducible aryl hydrocarbon hydroxylase activity in mouse- 
hamster and mouse-human somatic-cell hybrids. Biochem. Biophys . 
Res. Commun . 48: 293-298, 1972. 

4. Benedict, W.F., Nebert, D.W., and Thompson, E.B.: Expression of aryl 
hydrocarbon hydroxylase induction and suppression of tyrosine amino- 
transferase induction in somatic-cell hybrids. Proc . Nat. Acad. Sci . 
U.S.A . 69: 2179-2183, 1972. 

5. Nebert, D.W. , Gielen, J.E., and Goujon, P.M.: Genetic expression of 
aryl hydrocarbon hydroxylase induction. III. Changes in the binding 
of n-octylamine to cytochrome P-450. Mol . Pharmacol . 8: 651-666, 
1972. 

6. Goujon, P.M., Nebert, D.W., and Gielen, J.E.: Genetic expression of 
aryl hydrocarbon hydroxylase induction. IV. Interaction of various 
compounds with different forms of cytochrome P-450 and the effect 

on benzo[a]pyrene metabolism in vitro . Mol . Pharmacol . 8: 667-680, 
1972. 

7. Gielen, J.E. and Nebert, D.W. : Aryl hydrocarbon hydroxylase induction 
in mammalian liver cell culture. III. Effects of various sera, 
hormones, biogenic amines, and other endogenous compounds on the 
enzyme activity. J. Biol. Chem . 247: 7591-7602, 1972. 

8. Nebert, D.W. and Kon, H. : Genetic regulation of aryl hydrocarbon 
hydroxylase Induction. V. Specific changes in spin state of cyto- 
chrome P450 from genetically responsive animals . J. Biol. Chem . 
248: 169-178, 1973. 

9. Poland, A. P. and Nebert, D.W. : A sensitive radiometric assay of 
aminopyrine N-demethylation. J . Pharmacol . Exper . Therap . 184: 
269-277, 1973. 

10. Nebert, D.W. and Friedman, R.M. : Stimulation of aryl hydrocarbon 
hydroxylase induction in cell cultures by interferon. J. Virol . 
11: 193-197, 1973. 

11. Nebert, D.W., Considine, N. , and Kon, H. : Genetic differences in 
cytochrome P-450 during induction of mono-oxygenase activities. 
Drug Metab. Dispos ., in press. 

12. Benedict, W.F., Considine, N. , and Nebert, D.W. : Genetic differences 
in aryl hydrocarbon hydroxylase induction and benzo[a]pyrene-produced 
tumorigenesis In the mouse. Mol. Pharmacol ., in press. 

13. Nebert, D.W. : Use of fetal cell culture as an experimental system 
for predicting drug metabolism in the intact animal. Clin. Pharmacol . 
Therap . , in press. 



rifli'fi 



14. Nebert, D.W. , Considine, N. , and Owens, I.S.: Genetic expression 
of aryl hydrocarbon hydroxylase induction. VI. Control of other 
aromatic hydrocarbon-inducible mono-oxygenase activities at or 
near the same genetic locus. Arch. Biochem. Biophys ., in press. 

15. Benedict, W.F., Gielen, J.E., Owens, I.S., Niwa, A., and Nebert, D.W.: 
Aryl hydrocarbon hydroxylase induction in mammalian liver cell cul- 
ture. IV. Stimulation of the enzyme activity in established cell 
lines derived from rat or mouse hepatoma and from normal rat liver. 
Biochem. Pharmacol ., in press. 

16. Nebert, D.W. , Benedict, W.F. , and Kouri, R.E. : Aromatic hydrocarbon- 
produced tumorigenesis and the genetic differences in aryl hydro- 
carbon hydroxylase induction. In Ts'o, P.O. and Dipaolo, J. A. (Eds.): 
Model Studies in Chemical Carciongenesis , in press. 

17. Nebert, D.W. : Genetic and environmental factors influencing placental 
and fetal metabolism of drugs. In Moghissi, K.S. (Ed.): The 
Placenta: Biological and Clinical Aspects , in press. 



FB27 



i: 



NICHD Annual Report 
July 1, 1972 through June 30, 1973 
Pregnancy Research Branch 

SUMMARY 

The Pregnancy Research Branch has pursued its mission by investigating 
certain aspects of biochemistry and physiology in normal and abnormal 
mammalian pregnancy. The animal models used are the pregnant Rhesus monkey, 
(Macaca mulatta) , the pregnant baboon, (Papio papio) , and the pregnant Dorset 
sheep. In many instances data that has been obtained equates to that which 
has been available from human pregnancy; this fact strengthens and supports 
the belief that extrapolation from these models is pertinent to human 
pregnancy. 

There are two main groups in the branch. The group in steroid hormone bio- 
chemistry has continued to evaluate and use the baboon as a model for studies 
directed at the elucidation of the mechanisms regulating steroid hormone 
synthesis and metabolism during pregnancy. The studies have provided 
additional confirmation that this species is a useful model for estrogen 
synthesis during human pregnancy in several important respects. As is the 
case in human pregnancy, placental utilization of estrogen precursors from 
the maternal circulation appears to be impeded. Thus rate limiting steps in 
the placenta as well as variations in the supply of precursors may regulate 
estrogen production. Preliminary data indicate that maternal and fetal 
metabolism of Cortisol, particularly with respect to the extent of conjugation, 
differs from that of the nonpregnant state; this has also been found to be 
true for humans. Placental mechanisms for regulation of estrogen production 
have been studied by examination of placental activity of aromatase and 
sulfatase in normal and complicated human pregnancies. These two enzymes 
appear to be largely independent of maternal disease. It seems that 
variations in their total activities does not provide a general mechanism for 
regulation of estrogen production; however, competitive inhibition of the 
sulfatase by endogenous steroids and in particular by alternative substrates 
may provide such a mechanism. Studies with synthetic sulfatase inhibitors may 
provide an opportunity for experimental modulation of estrogen synthesis. 

Finally, a comparison has been made of the utility of estrogen determinations 
on maternal urine and serum for assessment of feto-placental status in the 
high risk human pregnancy. Despite certain practical and theoretical ad- 
vantages claimed for blood determinations, this study showed that in most 
high risk pregnancies serial 2^ hour urinary determinations provide the most 
reliable data to monitor estrogen metabolism as a criterion of feto-placental 
well being. An extension of thfs work has been the finding of a circadian 
rhythm in blood and urine estrogen concentrations in the third trimester. 
This has been attributed to changes in precursors known to be secreted 
rhythmically by the maternal adrenal. 

The other group in the branch has concerned itself with the endocrine physiol- 
ogy that pertains in pregnancy. The ongoing study of carbohydrate metabolism 
during primate pregnancy has been further expanded through the availability 
of an accurate and sensitive radioimmunoassay for monkey glucagon. Glucagon 

FG-1 



levels in the mother and fetus are not affected by fasting or induced hyper- 
glycemia in either the normal or the streptozotoci n induced glucose intolerant 
animal. Glucagon intravenously administered in physiologic amounts does not 
cross the monkey placenta in either direction. In the newborn, there is a 
linear relationship between plasma glucose and glucagon levels whereas it is 
not present in the fetus. 

A new protocol to examine renin-angiotensin relationships has been started in 
sheep pregnancies. This animal model allows the chronic implantation of both 
maternal and fetal catheters for periods of up to three weeks and thereby 
allows the measurement of blood components obtained in a nonstressed, non- 
anesthetized state. Preliminary evidence on plasma renin activity suggests 
that the baseline plasma renin activities of the mother are relatively 
constant on a day to day basis but those of the fetus are not. 

The other major approach of this group is examination of calcium homeostasis 
in the subhuman pregnant primate. The methodology has now been acquired to 
assay parathormone by radioimmunoassay, to measure ionized calcium with a flow 
through electrode, and to measure total calcium with atomic absorption 
spectrometry. Preliminary evidence indicates that parathormone cannot be 
detected in fetal plasma. When the fetal ionized calcium levels are decreased 
by the infusion of EDTA, barely detectable concentrations of parathormone are 
found . 

Studies on the amniotic fluid fat composition of primate pregnancy have been 
completed this year. The pattern of amniotic fluid fat concentration changes 
near term in both the baboon and the monkey in a manner similar to that found 
in the human. The leci thin/sphi ngomyel I n ratio abruptly reverses at 3k% 
gestational age. In the monkey this is associated with an increasing 
proportion of palmitic acid esters and relatively fewer myristic acid esters 
as it is in the human. In contrast, no change in lecithin fatty acid compos- 
ition occurs in the baboon at this time but rather both of the two pathways 
responsible for lecithin production increase activity in parallel. This 
indicates that the monkey is an acceptable model for examination of the 
physiology and pathophysiology of lung maturation in the human whereas the 
baboon is not. 

The fact that fetal experiments in the subhuman primate must be performed 
under anesthesia markedly limits the versatility of this animal model. This 
year considerable time and effort has been directed toward the maintenance of 
a chronic preparation in the baboon. The approach has been primarily directed 
toward technologic improvements in existing restraining devices plus methodol- 
ogy to maintain catheter patency over long periods of time. The stress of 
restraint appears to be associated with the premature induction of labor. 
However, there does appear to be a time interval in mid-pregnancy In which the 
animal can be introduced to the restraint without the complication of labor 
occurring. It also appears that catheter patency can be ttaintained in both 
the carotid artery and in the jugular vein for periods of up to one month. 
The next important step will be to place catheters into fetal vessels, 
ameliorate initial postoperative uterine irritability, maintain catheter 
patency for periods greater than five days, and then perform physiologic ex- 
periments without upsetting maternal homeostasis. There is every reason to 

FG-2 



believe that this will be an extremely difficult task but the reward deserves 
the effort. 

The branch has been considerably strengthened by the addition of two more 
professionals, a move of the laboratories to the Clinical Center in which ease 
of communication and consultation is much greater, and the presence of an 
active building program. An animal operating suite has been constructed and 
will be ready for utilization soon. The major problem in the animal work has 
been the acquisition of accurately timed gestations. The problem is most acute 
in sheep and least acute in the monkey. It is anticipated that many of the 
problems will be eliminated by the fall. 

The branch has been responsible for the care of three pregnant women who had 
concurrent medical diseases which were being managed by other groups in the 
Clinical Center. Two of these patients were unique in the medical literature. 
The care of the patients and their subsequent newborns have emphasized the 
appropriateness of acquiring additional facilities, personnel, and laboratory 
resources if a competent professional effort is to be expended for the care of 
pregnant humans. The beginning of construction of the clinical facility of 
this branch further emphasizes the need to attract competent clinicians with 
research interests oriented toward physiological and biochemical questions in 
human pregnancy. This will be a major activity of the branch this year. 



FG-3 



Serial No. HD-PRl 



1. Pregnancy Research Branch 

2. Bethesda, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Carbohydrate Metabolism during Primate Pregnancy 

Principal Investigator: Ronald A. Chez, M.D. 

Other Investigators: Alan R. Fleischman, M.D.; Daniel H. Mintz, M.D., 

University of Miami; Antoinette Schenk, University of 
Miami; Donald L. Hutchinson, M.D., University of 
Pittsburgh; I. Arthur Mirsky, M.D., VA Hospital, Los 
Angeles, California; Hernando Salazar, M.D., University 
of Pittsburgh; Rosemary Fox, University of Pittsburgh; 
Robert Miller; Sally Bowen , B.S. 

Cooperating Units: Experimental Surgery & Clinical Medicine Section, 

Laboratory Aids Branch, Division of Research Services, 
NIH. 



Man Years: 




Total: h. 
Professional : 2, 
Other: 2, 


5 
.0 
.5 


Project Description: 




Objectives: 





1. To examine the effect of oral glycemic stimuli on fetal pancreatic beta 
cell responsiveness. 

2. To determine if glucagon is transferred across the primate placenta 
and if hypergl ucagonemia exists in pregnancies associated with 
maternal carbohydrate intolerance. 

3. To compare a monkey pregnant model of induced diabetes mellitus with 
data presently available from human pregnancies associated with 
diabetes mel 1 i tus. 

^. To determine if there is limitation to maternal -fetal placental transfer 

of glucose. 
5. To determine if streptozotoci n is transferred to the fetus from the 

mother. 

Methods Employed : Accurately timed gestations in Rhesus monkeys are used. 
St reptozotocin , 'iG-'jS mg/kg, is administered intravenously to the adult 
female prior to or in the first trimester of pregnancy. This drug induces 
a glucose intolerant state secondary to pancreatic beta cell destruction. 
During the third trimester, acute experiments are performed under general 

FG-A 



anesthesia and aseptic surgical techniques. Cannulae are placed into both 
the maternal and fetal circulations to permit sequential sampling of blood. 
A catheter is placed transuterine into the fetal stomach via the mouth and 
glucose solution is injected into the stomach lumen. Blood aliquots are 
analyzed for glucose, free fatty acids, insulin growth hormone, and glucagon 
using biochemical and radioimmunoassay procedures. After performing the 
same experiment on the newborn in the first day of life, tissue is examined 
histologically from both mother and conceptus. 

Major Findings : The primary focus of this work has been in progress for 
several years. The results to date are summarized in four articles. J. Clin. 
Invest. 48:176, '69, J. Clin. Invest. '♦9:1517, '70, J. Clin. Invest. Metab. 
20:805, '71, J. Clin. Invest. 51:837, '72. The work done this year has 
demonstrated a failure of glucose orally administered in utero to enhance 
plasma insulin concentrations in either the normal or abnormal fetus. When 
the same stimulus is administered to the newborn, plasma insulin levels 
increase. Glucagon, intravenously administered in physiologic amounts, does 
not cross the monkey placenta in either direction. There is a lack of 
hypergl ucagonemia during fasting or induced hyperglycemia in the normal or 
streptozotoci n mother or fetus. There is a linear relationship between 
newborn plasma glucose and glucagon levels. Streptozotoci n crosses the 
monkey placenta in all stages of gestation. The rate of transfer and the 
dilution by maternal blood result in maximum fetal concentrations 1/lOth 
that of the mother. No histological or anthropometric or biochemical effect 
of this reduced dose can be measured in the newborn. 

Significance to Bio-Medical Research and to the Institute : Prior to this study, 
the interrelationships of fetal and maternal glucose, insulin, growth hormone, 
and glucagon could only be conjectured in primate pregnancy. The success 
of the streptozotocin diabetic model has validated the theory of fetal 
hyperi nsul ini sm in diabetic pregnancies. In question is the singular 
significance of maternal euglycemia in the measurement of diabetic pregnan- 
cies if maternal hyperami noaci demia coexists. The therapeutic implications 
for human pregnancies are primarily in the area of basic mechanisms for the 
early induction of fetal beta cell responsiveness to glycemic and amino 
acid stimuli prior to term delivery. 

Proposed Course : The absence of a chronic fetal preparation in the monkey 

limits the direction of this protocol. Until such time as one is developed, 
no new protocols in pregnancy are being planned. 

The effects of this induced carbohydrate intolerance state on adult monkey 
retina and muscle capillary basement membranes are now being investigated. 

Honors and Awards: Ronald A. Chez 

Appointed Clinical Professor, Department Ob-Gyn, Howard University 

School of Medicine, Washington, D.C., 1972. 
Member, Committee on Nutrition, American College of Obstetrics and 

Gynecologists, 1972. 
Member, Committee for Annual Clinical Meeting, American College of 

Obstetrics and Gynecologists, 1972. 



FG-5 



Member, Editorial Board, Contemporary Ob-Gyn , 1972. 

Member, Editorial Board, MedCom, 1972. 

Member, Editorial Board, Human Reproduction, 1973. 

Member, Ad Hoc Panel on Postcoital Estrogens (Center for Population, NICHD), 

January, 1973. 
Member, FIC Committee on Prevention of Fetal and Perinatal Diseases, 1973. 
Invited Speaker, The Institute for Comprehensive Medicine, Crystal City, 

Virginia, June, 1972. 
Invited Lecturer, Sir Joseph Barcroft Centenary Symposium, Cambridge, 

England, July, 1972. 
Invited Lecturer, New York University Graduate Program, New York, New York, 

September, 1972. 
Invited Lecturer, Family Planning Council of Southwestern Pa. £ the 

Commonwealth of Pennsylvania, Pittsburgh, Pa., October, 1972. 
Invited Lecturer, Howard University, Washington, D.C., November, 1972. 
Invited Lecturer, D.C. Society of Obstetricians and Gynecologists, Washing- 
ton, D.C., December, 1972. 
Invited Lecturer, Columbia Hospital for Women, Washington, D.C, January, 

1973- 
Invited Speaker, Ross Conference on "The Endocrine Milieu of Pregnancy, 

Puerperium and Childhood, St. Maarten, February, 1973. 
Invited Speaker, The National Foundation-March of Dimes, New York, New York, 

March, 1973- 
Invited Lecturer, Obstetrical Society of Northern Virginia, Seven Corners, 

Virginia, March, 1973- 
Invited Lecturer, Harvard University Medical School, Boston, Mass., March, 

1973. 
Invited Lecturer, Washington Adventist Hospital, Takoma Park, Maryland, 

April, 1973. 
Invited Lecturer, The University of Iowa Hospitals 6 Clinics, Iowa City, 

Iowa, April , 1973- 
Invited Lecturer, George Washington University Hospital, Washington, D.C, 

May, 1973. 
Visiting Professor, University of Hawaii, Honolulu, Hawaii, May, 1973- 
Invited Speaker, Fairfax Hospital, Falls Church, Virginia, June, 1973. 

Publ icat ions: 

Mintz, D.H., Chez, R.A. , Hutchinson, D.L.: Subhuman Primate Pregnancy 
Complicated By Streptozotocin- Induced Diabetes Mellitus., J. Clin. Invest . 
51:837, 1972. 

Chez, R.A., Mintz, D.H., Hutchinson, D.L., Horger, E.O. III.: Third 
Trimester Simian Fetal Carbohydrate Metabolism., In Physiological Bio- 
chemistry of the Fetus , Hodari , Charles C Thomas, Springfield, 1972. 

Chez, R.A., Josimovich, J.B., Schultz, S.G.: The Transfer of HPL Across 
Isolated Amnion-Chorion., Gynec. Invest . 1:312, 1972. 

Chez, R.A.: In Vitro Flux of Human Placental Lactogen Across Fetal 
Membranes., In 6th Rochester Trophoblast Conference , 1971, Lund and Choate, 
University of Rochester Press, 1972. 

FG-6 



Chez, R.A., Schlesselman, J.J., Salazar, H. , Fox, R. : Single Placentas in 
the Rhesus Monkey., J. Med. Prim . 1:230, 1972. 

Chez, R.A. , Mintz, D.H.: The Development and Function of the Human Endocrine 
Pancreas., In Symposium "The Endocrine Milieu of Pregnancy, Puerperium and 
Chi 1 dhood ." Ross Laboratories, Columbus, Ohio, 1973. 

Chez, R.A.: Fetal Factors Relating to Labor, In Uterine Contraction - Side 
Effects of Steroidal Contraceptives , Josimovich, Wi ley- Interscience , New 
York, 1973. 

Salazar, H. , Chez, R.A. , Pardo, M. : Absence of Ul trastructural Changes in 
the Basement Membrane of Monkey Muscle Capillaries in Streptozotoci n - 
Induced Carbohydrate Intolerance., Amer . J . Patho . , 1973i (In Press). 

Chez, R.A. , Mintz, D.H., Hutchinson, D.L.: Carbohydrate Metabolism in 
Primate Pregnancy., In Proc. ^th Int'l Congress on Endocrinology , Excerpta 
Medica , 1973, (in PressT 

Chez, R.A. : The Role of the Pharmacist in Family Planning: A Pennsylvania 
Survey., J. Amer. Pharm. Assoc . NS12: 464, 1972. 

Andrews, B.F., Apgar, V., Chez, R.A. , Freeman, M.G., Gluck, L., Kubarych, 
S. , Queenan, J.T. , Warrick, L.: New Management Concepts in High-Risk 
Pregnancy, Patient Care : 96-141, March 15, 1973. 

Andrews, B.F., Apgar, V., Chez, R.A. , Freeman, M.G., Gluck, L. , Queenan, 
J.T., Warrick, L.: Identifying High-Risk Pregnancy - In Time., Patient Ca re' 
18-50, September 30, 1972. 

Chez, R.A.: Prenatal Care - "First Do No Harm.", Con temp. Ob/Gyn 1:9, 1973- 

Chez, R.A. , Fleischman, A.R.: Fetal Therapeutics - Challenges and Responsi- 
bilities., Clin. Pharm. and Therapeutics , 1973 (In Press). 

Chez, R.A.: Drugs and the Unborn Patient., Contemp. Ob/Gyn , 1973 (in Press). 

Chez, R.A.: The Use of Learning Aids in a Human Sterilization Program., In 
Human Sterilization , Richart and Prager, Charles C. Thomas, Springfield, 
T97r 

Brackin, M.A. , Chez, R.A. : A Programmed Audiovisual Orientation to an Out- 
patient Clinic, J.Obstet. Gynec. S Neonatal Nursing , 1:33, 1972. 

Stenchever, M.A. , Chez, R.A. , et al.: The APGO Steering Committee for Coop- 
erative Teaching in Obstetrics and Gynecology-National Library of Medicine 
Project-A Progress Report., J. Rep rod. Med . 10:19, 1973. 



FG-7 



Serial No. HD-PR2 



1 . Pregnancy Research Branch 
2. Bethesda, Maryland 

PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Steroid Hormones in Primate Pregnancy 

Principal Investigator: John D. Townsley, Ph.D. 

Other Investigators: C. Deans Crystle, M.D., Gerald J. Pepe, Ph.D., Nancy M. 

Jensen, B.S., Elliece Smith, B.S., Linda J. Gartman, 
Norman Dubin, Ph.D., University of Maryland; George 
Grannis, Ph.D., Ohio State University. 

Cooperating Units: Experimental Surgery & Clinical Medicine Section, 

Laboratory Aids Branch, Division of Research Services. 

Man Years: 

Total: 5.2 
Professional : 3-2 
Other: 2.0 

Project Description: 

Object ives : 

1. To ellucidate the mechanisms regulating steroid hormone synthesis and 
metabolism during human pregnancy. 

2. To evaluate and use the baboon as a model for such studies. 

3. To compare the utility of estrogen determinations in maternal urine and 
serum for the assessment of feto-placental status in the high-risk 
pregnancy. 

Methods Employed : The project demands a variety of complementary approaches 
ranging from metabolic studies in the intact pregnant primate to determin- 
ation of activities of steroidogenic enzymes in sub-cellular fractions of 
placenta. Analytical methods include f 1 uorometr ic, color imetri c, and 
radioimmunoassays for steroids in body fluids and double isotope techniques 
for kinetic enzyme assays and the metabolic studies in vivo. 

Major Findings : Comparative evaluation of serum estradiol-1 7B and estriol 
and urinary total estrogens and estrogen-creatinine ratio as indices of 
fetal health has been completed. Despite certain practical and theoretical 
advantages claimed for blood determinations this study showed that in most 
high-risk pregnancies serial 2'* hr urinary determinations provide the most 
reliable data to monitor estrogen metabolism as one parameter of feto- 
placental well-being. The circadian rhythm in blood and urine estrogen 
concentrations, which we have shown in the third trimester and attributed 

FG-8 



to changes in precursors known to be secreted rhythmically by the maternal 
adrenal, has subsequently been confirmed in two other laboratories. 

Determination of human placental aromatase and sulfatase activities in 
normal and complicated pregnancies indicate that they are largely independ- 
ent of maternal disease and that variations in their activity does not 
provide a general mechanism for regulation of estrogen production. However, 
competitive inhibition of the sulfatase by endogenous steroids, in partic- 
ular alternative substrates, may well provide such a mechanism. Our studies 
indicate that synthetic inhibitors may be found which can be used for ex- 
perimental modulation of estrogen synthesis. Estrogen precursors adminis- 
trated i ntra-amn iot ical ly appear to be utilized more readily than those 
provided via the maternal circulation. This suggests that the importance 
of precursor availability in regulating placental estrogen production may 
depend on the origin of the precursor. 

Studies with pregnant baboons confirm our previous conclusion that this 
species provides a useful model for estrogen synthesis during human pregnancy 
in several important respects. As is the case in human pregnancy utilization 
of estrogen precursors from the maternal circulation appears to be impeded. 
Preliminary data indicate that maternal and fetal metabolism of Cortisol, 
particularly with respect to the extent of conjugation, differs from that 
of the nonpregnant animal. This is also true for humans. 

Significance to Bio-Medical Research and to the Institute : Because of the 
widespread utilization of steroid hormone assays to monitor feto-placental 
status and the apparent importance of such hormones in maintaining the 
pregnancy and in the onset of labor an understanding of the mechanisms 
controlling their synthesis and metabolism is essential for definitive 
resolution of many problems of reproductive failure. Our approaches provide 
fundamental knowledge of these processes and practical information of immed- 
iate use to the clinician. Further elucidation of controlling mechanisms 
will require manipulative procedures in both the mother and the fetus which 
are precluded in humans. An adequate animal model must be found in order 
to do this and our studies continue to indicate that the baboon is a 
promising candidate. 

Proposed Course : Our major thrust will involve the baboon as a model. Studies 
indicating impeded utilization of maternal circulating estrogen precursors 
will be extended and the reasons for this finding elucidated. The import- 
ance of variation in fetal circulating precursors in regulating estrogen 
production will be explored. The intrauterine and neonatal maturation of 
the fetal -newborn adrenal-pi tui tary-hypothalmi c axis, as judged by changing 
response to tropic stimuli and C]q and C21 steroid production, will be 
studied. Investigation of placental mechanisms regulating estrogen synthesis 
in the presence of excess precursors will be continued. 

Honors and Awards: John D. Towns ley 

Invited Lecturer, Fifty Fifth Annual Meeting of the Endocrine Society, 
Chicago, June, 1973. 



FG-9 



Publ ications: 

Townsley, J.D.: Steroid Hormones in pregnancy: The Baboon as a model for 
Studies on Estrogen Synthesis., Acta Endocr . Suppl 166:191, 1972. 

Townsley, J.D., Rubin, E.J.: Structure-Activity Correlations for Steroid 
Inhibition of Human Placental Steroid 3-Su1 fatase. , Research on Steroids , 
1972, (In Press). 

Townsley, J.D., Dubin, N.H., Grannis, G.F., Gartman, L.J., Crystle, CD.: 
Circadian Rhythms of Serum and Urinary Estrogens in Pregnancy., J . Clin . 
Endoc r. 36: 289. 1973- 

Dubin, N.H., Crystle, CD., Grannis, G.F., Townsley, J.D.: Comparison of 
Maternal Serum Estriol and Urinary Estrogen Determinations as Indices of 
Fetal Health., Am. J. Ob. Gyn. 115:835, 1973. 

Townsley, J.D., Gartman, L.J., Crystle, CD.: Maternal Serum 1 73-Estradiol 
Levels in Normal and Complicated Pregnancies: A Comparison with other 
Estrogen Indices of Fetal Health., Am. J. Ob. Gyn . 115:830, 1973. 

Townsley, J.D.: Further Studies on the Regulation of Human Placental Steroid 
3-Sulfatase Activity., Endocr . 1973, (In Press). 

Townsley, J.D., Rubin, E.J., Crystle, CD.: Evaluation of Placental Steroid 
3-Sulfatase and Aromatase Activities as Regulators of Estrogen Production 
in Human Pregnancy., Am. J. Ob. Gyn . 1973, (in Press). 

Honors and Awards: C Deans Crystle 

Appointed Assistant Clinical Professor, Department Obstetrics and Gynecology, 

Georgetown University, Washington, D.C, 1972. 
Invited Lecturer, Ohio State University, Department Obstetrics and Gynecology, 

November, 1972. 
Invited Lecturer, Good Samaritan Hospital, Department Obstetrics and 

Gynecology, Dayton, Ohio, November, 1972. 
Invited Lecturer, University of Connecticut School of Medicine, Department 

Obstetrics and Gynecology, April, 1973. 

Publ ications: 

Townsley, J. b.'j'Dubin, N.H., Grannis, G.F., Gartman, L.J., Crystle, CD.: 
Circadian Rhythms of Serum and Urinary Estrogen in Pregnancy., J . Clin . 
Endocr . 36:289, 1973- 

Townsley, J.D., Gartman, L.J., Crystle, CD.: Maternal Serum 1 7B-Estradiol 
Levels in Normal and Complicated Pregnancies: A Comparison with other 
Estrogen Indices of Fetal Health., Am. J. Ob. Gyn . 115:830, 1973- 

Dubin, N.H., Crystle, CD., Grannis, G.F., Townsley, J.D.: Comparison of 
Maternal Serum Estriol and Urinary Estrogen Determinations as Indices of 
Fetal Health., Am. J. Ob. Gyn . 115:835, 1973- 

FG-10 



Crystle, CD., Sawaya,.G., Stevens, V.C: Effects of Ethinyl Estradiol on 
Serum Gonadotropins in Postpartum Women., Am. J. Ob. Gyn . 1973, (in Press). 

Townsley, J.D., Rubin, E.J., Crystle, CD.: Evaluation of Placental 3- 
Sulfatase and Aromatase Activities as Regulators of Estrogen Production in 
Human Pregnancy., Am. J . Ob. .Gyn . 1973, (in Press). 

Honors and Awards: Gerald J. Pepe , None 

Publ i cat ions : 

Pepe, G.J., Rothchild, I.: The Effect of Hypophysectomy on Day 12 of 
Pregnancy on the Serum Progesterone Level and the Time of Parturition in the 
Rat., Endocr . 91:1380, 1972. 

Morishige, W.K., Pepe, G.J., Rothchild, I.: Serum Luteinizing Hormone, 
Prolactin, and Progesterone Levels During Pregnancy in the Rat., Endocr . 
1973, (In Press). 

Rothchild, I., Billiar, R.B., Kline, I.T., Pepe, G.J.: Progesterone 
Secretion in Rats Following Hypophysectomy and Hysterectomy; Pregnancy 
Compared with Pseudopregnancy , Pseudopregnancy plus Deciduomata, and 
Lactation., J. Endocr . 1973, (In Press). 

Pepe, G.J., Rothchild, I.: Serum Progesterone Levels in Ovariectomi zed Rats 
Injected with Progesterone and Estrone: Relation to Pregnancy Maintenance 
and Growth of Decidual Tissue., Endocr . 1973, (in Press). 

Pepe, G.J., Rothchild, 1.: Metabolic Clearance Rate of Progesterone: 
Comparison Between Ovariectomi zed , Pregnant, Pseudopregnant , and Deciduoma- 
Bearing Pseudopregnant Rats., Endocr. 1973, (in Press). 



FG-11 



Serial No. HD-PR3 



1 . Pregnancy Research Branch 

2. Bethesda, Maryland 

PHS-NJH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Amniotic Fluid Fat Concentrations in Primate Pregnancy 

Principal Investigator: Ronald A. Chez, M.D. 

Other Investigators: Louis Gluck, M.D., University of California, La Jolla; 

Sal ly Bowen , B. S . 

Cooperating Units: Experimental Surgery & Clinical Medicine Section, 

Laboratory Aids Branch, Division of Research Services. 

Man Years: 

Total: 0.25 
Professional: 0.10 
Other: 0.15 

Project Description: 

Objectives : 

1. To determine the patterns of amniotic fluid fat composition in monkey 
and baboon pregnancies. 

2. To examine the effect of fetal tracheal and/or esophageal ligation on 
amniotic fluid phospholipid and sphingomyelin ratios. 

Methods Employed : Rhesus monkeys (Macaca mulatta) and baboons (Papio papio) 
with accurately timed gestations are used. Transabdominal percutaneous 
amniocentesis is sequentially performed during pregnancy. Late in the 
second trimester, fetal tracheal and/or esophageal ligation is performed 
under aseptic surgical conditions. The fetus is delivered electively at 
term via cesarean section. 

Major Findings : The pattern of amniotic fluid fat concentrations changes 
near term in both subhuman primate species in a manner similar to that 
found in the human. The lecithin sphingomyelin (L/S) abruptly reverses at 
3k% gestational age. In the monkey, this is associated with an increasing 
proportion of palmitic acid esters and relatively fewer myristic acid esters. 
In contrast, no change in lecithin fatty acid composition occurs in the 
baboon. Tracheal ligation is associated with nondistended lungs (in contrast 
to the sheep), and relative oligohydramnios. After surgery, the fatty acid 
esters on the surface active lecithin in the amniotic fluid did not change 
near term. Examination of the lecithin in the ligated bronchial tree-lungs 
showed it to have surface active leithin of fatty acid composition consis- 
tent with maturity and therefore with that in amniotic fluid. For reasons 



FGH2 



that remain unclear, esophageal ligation is associated with early fetal 
den I se . 

Significance to Bio-Medical Research and to the Institute : The L/S in human 
amniotic fluid is a reliable diagnostic tool for the determination of fetal 
lung maturity. The effects of congenital anomalies, intrauterine stress, 
and maternal or fetal medication remain unknown.' The use of this animal 
model will permit research into the physiology and pathophysiology of these 
pathways . 

Proposed Course : An attempt to induce fetal lung maturation prior to its 

normal occurrence will be considered. There is a suggestion that intrauterine 
chronic asphyxia and fetal adrenal cortical activity are coregulators of 
fetal lung maturation. The effects of the administration of corticosteroids 
to the fetus and fetal adrenal -hypophysectomy may be examined. 

It is possible to examine the kinetics of the 5 enzymes involved in lecithin 
formation in the lungs using in vi tro lung slice incubation techniques. 
Fresh newborn lung is a byproduct of other experimental protocols. This 
protocol is presently being examined for possibility. 

Honors and Awards: None 

Publ i cations : None 



FG-13 



Serial No. HD-PR4 



1. Pregnancy Research Branch 

2, Bethesda, Maryland 

PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: The Effects of Induced Fetal Blood pH changes on Fetal Blood 
Flow 

Principal Investigator: Ronald A. Chez, M.D. 

Other Investigators: Alan R. Fleischman, M.D.; Andre Hellegers, M.D., George- 
town University; Robert Cefalo, M.D., Georgetown Univer- 
sity; Joan Simkovich, Ph.D., Georgetown University; Sally 
Bowen , B.S. 

;~ Cooperating Units: Division of Research Services, Veterinary Resources 
Y" Branch, Animal Center Section, Ungulate Unit, NIH 

Man Years: 

;5 Total: ^.k 
'-J Profess ional : 3-0 
"^ Other: 0.^ 

Project Description: 

Object i ves : 

1. To study the effects of induced fetal metabolic acidosis on fetal blood 
flow, fetal oxygen consumption, uterine blood flow, and fetal and 
maternal plasma oxygen tensions. 

2. To study the effects of induced fetal respiratory acidosis on the same 
parameters. 

3. To determine whether permeability characteristics of placental oxygen 
transfer can be modified. 

Methods Employed : Accurately timed gestations in Dorset sheep are utilized. 
Under anesthesia and aseptic surgical conditions, cannulae are placed into 
the maternal and fetal circulations. The latter is done without interrupt- 
ing the integrity of the amniotic sac. The experiment is then performed 
under this acute set of conditions on 3-7 days subsequently after the animal 
has recovered and is in a nonstressed nonanestheti zed state. Measurements 
are made with the autoanalyzer , Van Slyke apparatus, and blood gas-pH 
equipment . 

Hemi hysterectomy was performed in 16 sheep prior to conception; 12 of these 
conceived and are presently pregnant. Hemi hysterectomy immediately prior 
to the acute experiments cited above have been done in six instances. 



FG-»1 k 



Major Findings : The production of a metabolic alkalosis confined to the 
fetus has no appreciable effect on fetal oxygen consumption although it 
decreases the fetal PO2. The latter is produced by a leftward shift in the 
oxygen dissociation curve. Since the quantity of oxygen crossing the plac- 
enta is unaltered, the theoretical benefits to the fetus by the increase in 
its blood oxygen affinity do not in fact seem to operate. 

The infusion of diamox into the fetus is a useful tool in producing uni- 
lateral fetal respiratory acidosis by the trapping of carbon dioxide on the 
fetal side of the placenta. 

The removal of a portion of the placental implantation sites prior to preg- 
nancy, or of the placenta itself during pregnancy has no deleterious effects 
on fetal growth, providing it is not accompanied by shunting of blood through 
the ablated area. In this respect the placenta seems to bear some analogy 
to the lung. The data tend to confirm that blood flow, rather than placental 
diffusion-limitation is critical in maternal-fetal nutrient transfer. 

Halothane may be a hazardous anesthetic to the fetus since it decreases 
uterine blood flow, umbilical blood flow and fetal oxygen consumption. Of 
these, uterine blood flow probably caused secondarily by a decrease in cardiac 
output, is the greater hazard. 

Significance to Bio-Medical Research and to the Institute : The utility of fetal 
heart rate, acid base, and gas monitoring in human pregnancy continues to 
be debated. In addition to a lack of data on the significance of some of 
the changes observed, there is a lack of information as to fetal therapeutic 
implications. Data from the pregnant sheep model has been successfully ex- 
trapolated to human pregnancy in several areas. It is believed that this 
project will provide physiological and therapeutic information on fetal acid- 
base control mechanisms, fetal blood flow, and fetal oxygen consumption with 
and without anesthesia. 

Proposed Course : No new protocols are planned at this time. 

Honors and Awards: None 

Publications: Ph.D. Thesis, Joan Simkovich 



FG-15 



Serial No. HD-PR5 



1 . Pregnancy Research Branch 

2. Bethesda, Maryland 

PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Calcium Homeostasis in the Subhuman Pregnant Primate 

Principal Investigator: Ronald A. Chez, M.D. 

Other Investigators: Alan R. Fleischman, M.D.; Daniel H. Mintz, M.D., Univer- 
sity of Miami; Antoinette Schenk, University of Miami; 
Sal ly Bowen , B.S. 

Cooperating Units: Experimental Surgery S Clinical Medicine Section, 

Laboratory Aids Branch, Division of Research Services. 



Man Years: 




Total : 


1.5 


Professional : 


0.6 


Other: 


0.9 



Project Description: 
Objectives : 

1. To examine the bidirectional placental transfer of radioi sotopical ly 
labelled bovine parathyroid hormone and synthetic human calcitonin. 

2. To study the effects of perturbations of maternal and fetal serum 
calcium and phosphorous on the serum calcium, ionized calcium, and 
immunoreacti ve parathyroid hormone and calcitonin in the fetal and 
maternal circulations. 

3. To study the effects of the intravascular administration of PTH and CT 
on placental transport of radiolabel led calcium. 

A. To investigate the hormonal control of calcium homeostasis in the neonate 
and the pathophysiology associated with neonatal tetany. 

Methods Employed : Rhesus monkeys (Macaca mulatta and Macaca speciosa) with 
accurately timed gestations are used. Under aseptic surgical conditions, 
cannulae are placed into the fetal and maternal circulations. An attempt 
is made to maintain the integrity of the amniotic sac. Highly purified 
bovine PTH or synthetic human CT iodinated with radioactive iodine, calcium 
gluconate, or EDTA are intravenously administered to mother or fetus. Blood 
aliquots are sequentially obtained for appropriate measurements utilizing 
atomic absorption spectrometry, flow through electrodes, and radioimmunoassay. 

Major Findings : Preliminary data suggests that a limited bidirectional 
placental transfer of PTH occurs in the third trimester, and that CT does 
not cross the placenta. PTH cannot be detected in fetal plasma. When the 

FiG-16 



fetal ionized calcium levels are decreased by the infusion of EDTA, barely 
detectable concentrations are measured. 

Significance to Bio-Medical Research and to the Institute : Pregnancy elicits 
a physiologic adaptive response in the mother to protect a late pregnancy 
fetal claim on calcium for mineralization of the fetal skeleton. There is 
a paucity of decisive data in human pregnancy secondary to the inherent 
experimental difficulties encountered in studying healthy patients and the 
lack until recently of reliable radioimmunoassay techniques for the 
measurement of plasma levels of parathormone and thyroid calcitonin. The 
utilization of the subhuman primate permits investigation of this complex 
problem with techniques which permit study of the fetus in utero and assay 
procedures that can be performed on small al iquots of plasma. It is hoped 
that the data generated will provide the first definitive exploration in 
primates of the parathyroid-calci tonin releasing mechanism studied 
simultaneously in both fetus and mother. 

Proposed Course : All aspects of the project require exploration and a major 
commitment to this project is planned. The obtaining of data will depend 
upon the availability of pregnant monkeys now that the radioimmunoassay 
and ionized calcium techniques have been developed. 

Honors and Awards: None 

Publications: None 



FGH7 



Serial No. H0-PR6 



1. Pregnancy Research Branch 

2. Bethesda, Maryland 

PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Prolactin Metabolism During Primate Pregnancy 

Principal Investigator: Ronald A. Chez, M.D. 

Other Investigators: Alan R. Fleischman, M.D.; John E.A. Tyson, M.D., Johns 

Hopkins University; Janice Huth, Johns Hopkins University; 
Beverly Smith, Johns Hopkins University; Prudence Thomas, 
Johns Hopkins University; Sally Bowen, B.S. 

Cooperating Units: Experimental Surgery £ Clinical Medicine Section, 

Laboratory Aids Branch, Division of Research Services. 

Man Years: 

Total: 2.7 
Professional : 1.0 
Other: 1.7 

Project Description: 

Objectives : To study the bidirectional placental and amniotic fluid transfer 
of prolactin in primate pregnancy. 

Methods Employed : Ten Rhesus monkeys (Macaca mulatta and Macaca speciosa) 
with accurately timed gestations have been used. Under aseptic surgical 
conditions, cannulae are placed into the maternal and fetal circulations as 
well as the intact amniotic sac. Highly purified human prolactin iodinated 
with radioactive isotopes is injected into one of the three compartments. 
Sequential aliquots of blood and amniotic fluid are examined for hot and 
cold prolactin concentrations using radioimmunoassay and chromatography 
techniques . 

Major Findings : Prolactin does not cross the primate placenta in either 
direction in the third trimester. The prolactin in amniotic fluid is only 
partially of maternal origin and not of fetal origin. Prolactin in amniotic 
fluid exchanges minimally with fetal or maternal blood. 

Significance to Bio-Medical Research and to the Institute : The amniotic fluid 
concentration of prolactin is approximately ten times that of the levels 
found in maternal and fetal plasma. Prolactin has been shown to affect the 
transfer of sodium across the gills of fish and the renal tubules of dogs. 
The role of prolactin in the exchange of water and electrolytes across fetal 
membranes needs to be clarified. This study describes some of the fundamental 
physiology necessary as a foundation. 

FG-J8 



Proposed Course : Further use of the intact primate pregnancy is not planned. 
Under consideration is a protocol examining the effects of prolactin on 
sodium transfer across fetal membranes mounted in an Ussing chamber. 

Honors and Awards: None 

Publ i cat ions: None 



4 



FG-19 



Serial No. HD-PR-7 



c 



1 . Pregnancy Research Branch 

2. Bethesda, Maryland 

PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Renin-Angiotensin Relationships in Sheep Pregnancy 

Principal Investigator: Ronald A. Chez, M.D. 

Other Investigators: Alan R. Fleischman, M.D.; Gary K. Cakes, M.D.; Kevrn J. 

Catt, M.D., Reproduction Research Branch, NICHD; Martha 
Ashburn, Reproduction Research Branch, NICHD; Sally 
Bowen , B.S. 

Cooperating Units: NIH Animal Center, Poolesville, Maryland 

Man Years: 

Total: 5.5 
Profess ional : ^.0 
Other: 0.2 



? 

,3 Project Description: 

^ Objectives : 

1. To examine the bidirectional placental transfer of renin. 

2. To study the effects of perturbations of maternal and fetal plasma 
electrolytes and volume on serum renin activity. 

Methods Employed : Accurately timed gestations in Dorset sheep are utilized. 
Under anesthesia and aseptic surgical conditions, cannulae are placed into 
the maternal and fetal circulations. The latter is done without interrupt- 
ing the integrity of the amniotic sac. The experiment is then performed 
under this acute set of conditions on 3-21 days subsequently after the 
animal has recovered and is in a nonstressed nonanesthet i zed state. Measure- 
ments of renin activity are made utilizing radioimmunoassay techniques. 

Major Findings : The baseline plasma renin activity of the mother is constant 
from day to day in the third trimester; that of the fetus is not with 
marked daily fluctuations and a consistent positive gradient to the mother. 
Potent diuretics administered to the mother are associated with elevations 
in maternal plasma renin activity and a variable response in the fetus. 
Fetal nephrectomy at the beginning of the third trimester is accompanied by 
a high mortality for reasons that are not yet clear. 

Significance to Bio-Medical Research and to the Institute : The Renin-Angiotensin 
system is a major determinant of salt and water metabolism in pregnancy. The 
factors and variables that regulate and Influence the production and 

FG-20 



secretion in pregnancy of renin are not known. The possibility exists that 
the system in the fetus and mother are interdependent or independent. The 
implications of such a relationship may have import in human prenatal care 
and the management of disease states in pregnancy. 

Proposed Course: All aspects of the project require exploration and a major 
commitment to this project is planned. 

Honors and Awards: None 

Publ ications : None 



FG-^1 



PHS-NIH 
Pregnancy Research Branch 
Contract and Collaborative Research 
July 1, 1972 through June 30, 1973 

Contract Title: Human Fetal -Maternal Metabolism Studied Under Conditions of 
Acute Fasting 

Principle Investigator: Ronald A. Chez, M.D. 

Other Investigators: John E.A. Tyson, M. r. , Johns Hopkins University; Janice 

Huth, John Hopkins University; Beverly Smith, Johns 
Hopkins University; Prudence Thomas, Johns Hopkins 
Univers i ty . 

Contractor: Johns Hopkins University 

School of Medicine 

Department of Obstetrics £ Gynecology 
Baltimore, Maryland 21205 

Money A1 located: $103,305 

Man Years: 



Total : 


k.2 


Professional : 


1.2 


Other: 


3.0 



Contract Description: 
Object Ives : 

1. To study the effects of acute starvation induced in the first half of 
pregnancy on human maternal plasma placental lactogen concentration. 

2. To study the relationship between carbohydrate-insulin-growth hormone 
metabolism and human maternal plasma and amniotic fluid concentrations 
of prolactin. 

3. To measure the concentrations of the essential and glucogenic amino 
acids in human maternal plasma, fetal plasma, and amniotic fluid under 
conditions of acute maternal starvation and/or increments in maternal 
plasma placental lactogen concentration. 

Methods Employed : Twenty helathy female volunteers between the ages of I8 
and 30 will be considered for this study. When approval for interruption 
of pregnancy has been obtained, the patient will be interviewed and if 
found acceptable will be asked to participate in the study. The responsibil- 
ity for recommending interruption of pregnancy, for the choice of procedure 
used for interruption, and for the surgical management is that of physician 
other than the investigator. After receipt of an informed consent, the 
patient will be scheduled for admission to the Clinical Investigation Unit 
of the Johns Hopkins Hospital. The patients' activity is restricted to the 
ward. Sequential samples of maternal blood, maternal urine, and amniotic 
fluid are obtained. 

FG-22 



Major Findings : It appears that there is a selective response on behalf of 
the pituitary gland, depending upon the amino acid infused; thus the infus- 
ion of arginine in pregnant women, following a fast, in the first 20 weeks 
of gestation, is not associated with marked rises In growth hormone; whereas 
prolactin increments are significant. The infusion of alanine, on the other 
hand, is not associated with any significant increments in prolacting but 
with marked increases in the release of growth hormone. 

The laboratory is currently completing all the radioimmunoassays for growth 
hormone, insulin, placental lactogen and prolactin as well as the amino 
acid profiles on aliquots obtained from 80 fasts. 

Significance to Bio-Medical Research and to the Institute : The role and import 
of human placental lactogen (HPL, human chorionic somatomammotropin) and 
prolactin (HPr) in pregnancy remains undefined. There is preliminary evid- 
ence that acute starvation of the human mother, in the first half of pregnancy, 
is associated with moderate to high rises of maternal plasma HPL concentra- 
tions that then return to baseline with feeding. Furthermore, there is also 
preliminary evidence that maternal plasma HPr concentrations rise and amniotic 
fluid HPr concentrations fall in pregnancy. The relationship of maternal- 
fetal nutrition to these changes is not known. 

Proposed Course : The contract fiscal year ends April 30, 1973- A renewal is 
not necessary or planned. 

Honors and Awards: None 

Publ icat ions: None 



FG-23 



NICHD Annual Report 
July 1, 1972 through June 30, 1973 

Gerontology Research Center 
Office of the Chief 



Research Services 

The Technical Development Section has continued its maintenance and 
development services to the scientific programs of the Center. Several new 
instruments have been designed and constructed along with day-to-day main- 
tenance services and technical consultations. Advances have been made in 
the capabilities of the GRC computer system. 

A precision muscle suspension system with temperature controlled 
muscle chambers and specially designed muscle clips and electrodes was de- 
signed and constructed for studies on age differences in cardiac performance 
in the rat. 

An automated device for recording of reaction time was developed for 
the Longitudinal Study. Reliability of the test data will be increased by the 
automation of both the stimulus presentation and data recording. 

The Psychophysiology Section was assisted by the development of a 
blood flow integrator for use in its studies of long term conditioning of blood 
pressure and cardiac output in the monkey. A unique feature of this instru- 
ment is its capability to eliminate the baseline drift usually encountered in 
these measurements. 

Several instruments were designed and fabricated for the Section on 
Human Learning and Problem Solving. Visual stimuli are presented with the 
use of a new control timer, and data gathered and recorded on a new, more 
reliable printer for the programs concerned with serial and paired-associate 
learning. A stimulus programmer, utilizing paper tape, automatically se- 
quences visual stimuli for a study on short term retention. An interface 
with the GRC computer system has been completed which will, with the com- 
pletion of software, present complex stimuli in a concept -learning experiment. 

In the past year there has been a sizable increase in the usage of the 
GRC computer system. Approximately fifteen different programs regularly 
use the "background" capabilities of the system to reduce data and perform 
computations. The Technical Development Section supports these efforts by 
training programmers, aiding in the debugging of programs, and maintaining 
the computer system. The capabilities of the system have also been in- 
creased. An FM analog tape recorder has been added to allow the reduction 
of pulmonary and cardiovascular data. The required interface was designed 
and installed along with general purpose software by the TD Section. An in- 
terval timer was constructed and installed in support of the on-line pulmo- 
nary testing system being developed for the Longitudinal Study. General pur- 
pose software developed by the TD Section include programs which (1) fit 



FH4 



polynomials and non -linear functions to data, (2) allow easy digitization of 
signals from the analog tape system, (3) simplify the plotting of data, and 
(4) allow the punching of protocol tapes for laboratory stimulus controllers. 

The Photography and Arts Section provided drawings, photographic 
prints, and projection slides for support to 46 GRC scientists and 5 guest 
scientists. 

There has been a substantial increase in requests for projection 
slides. In order to meet this increased work load, a new procedure was de- 
veloped for direct positive slides to eliminate the need for intermediate film 
negatives. 

During the past year, the Animal Resources Facility has provided 
veterinary services, technical assistance, and animal care for the expanding 
research program of the GRC. The number of animals cared for has in- 
creased significantly this year. The production colony of aging, outbred 
Wistar rats has been maintained at a level of 10,000 animals. In addition, 
care has been provided for 9,000 mice, 900 rabbits, 600 rats, 82 dogs, and 
10 Rhesus monkeys acquired from outside sources. 

This year a total of 1,085 rats have been issued from the production 
colony to 19 investigators representing every branch of GRC and to five 
guest scientists from outside institutions. 

A small colony of 1,600 aging mice is being maintained for the Labo- 
ratory of Cellular and Comparative Physiology. Another colony was set up 
to breed five strains of mice acquired from the University of Edinburgh. 
Three strains consist of inbred mice and the other two strains are mutants 
which are maintained by a backcross with C57B1/6 males. This colony, 
which now holds approximately 500 mice, is being used in genetic studies. 

In collaboration with the Laboratory of Cellular and Comparative 
Physiology, a detailed survey of pathology present in rats in the production 
colony has been initiated. 

A laminar -air -flow system has been installed in one of the animal 
holding rooms. This system should be capable of holding up to 1,400 mice 
while also providing a flexible barrier against environmental stress and con- 
tagion. The practicality and effectiveness of this system will be tested 
during the coming year. 

Surgical facilities, equipment, and technical assistance were provided 
to investigators conducting experiments which required the use of surgical 
procedures. This year, the technique of implanting an indwelling, electro- 
magnetic, arterial flow sensor around the base of the aorta was applied and 
developed for use by the Laboratory of Behavioral Sciences. Rhesus mon- 
keys undergoing this procedure have survived for 7 weeks before being 
sacrificed for pathological examination. 

Information Services. GRC internal communication was further 
improved with the advent of a Center newsletter in January 1973. This 



FHfi 



newsletter, developed by the Public Information Officer after consultation 
with employees and administrative staff, is proving an effective vehicle for 
informing staff about official GRC operations and general activities of 
interest to everyone working in the facility. 

Media inquiries rose this year to 128 from the 118 reported last 
year. Stories dealing with the Center or its programs appeared in the New 
York Times, Saturday Review /The Sciences, the National Observer, the 
Washington Star-News, Newsweek, the Baltimore Sun, and National History 
Magazine. Additional contacts were made with Newsday, the New York 
Daily News, the Wall Street Journal, Time, Fortune, and two German maga- 
zines, Der Spiegel and Stern. Medical World News and Chemical and Engi- 
neering News also carried articles on the Center. 

Electronic media contacting GRC included CBS-TV, BBC-TV, 
WBAL-TV, and WCAO -Radio (Baltimore), the Voice of America, Radio 
Free Europe, World Health Organization Radio, Italian RAI-TV, and Ontario 
Educational TV. 

A total of 157 people participated in 42 tours of the facility, including 
visitors from Australia, Denmark, Germany, and Israel. 

The GRC Public Information Officer set up and operated a working 
Pressroom for the annual meeting of the Gerontological Society at that 
group's request. 

Library . In addition to providing the usual services to GRC investi- 
gators, the Library staff continued to search out and index the world litera- 
ture relating to gerontology and geriatrics. During the year over 3,600 
references were identified and classified. The reference lists were pub- 
lished quarterly in the Journal of Gerontology. 

Collaborative Guest Scientist Program 

During the year, 15 guest scientist programs were supported by the 
GRC. These programs were staffed by 35 investigators with 30 supporting 
personnel. Guest scientist programs included studies of cardiac perform- 
ance in the rat, skeletal muscle physiology, cell biology, gastrointestinal 
physiology, and endocrinology. 

Cardiac Performance in the Rat. Isolated heart muscle (left ventric- 
ular trabeculae carneae) from hearts of young and old rats was subjected to 
anoxia (95% N2 - 5% CO2), paired pacing (post extra systolic potentiation), 
and exposure to norepinephrine at varying concentrations. Contractility of 
the heart muscle was increased by norepinephrine and paired pacing and de- 
creased by hypoxia. Although there was no significant age difference in the 
threshold dosage of norepinephrine, at higher dose levels the inotropic re- 
sponse was less in the old than the young. No age differences were found in 
the response to hypoxia or paired stimulation. 

These preliminary results suggest that aging in the rat heart is 



FH-3 



associated with a decrement in the magnitude of the specific inotropic re- 
sponse to norepinephrine. The lack of an age related increase in hypoxic 
sensitivity suggests that there are no major alterations in anaerobic metabo- 
lism associated with aging. The decrease with age in the response of cardi- 
ac muscle to catecholamines would likely be reflected in decreased "cardiac 
reserve" — a decreased ability to meet increased stress. 

Aging of Skeletal Muscle. Previous studies have indicated that 
chronically denervated muscles in the rat had a lower collagen content (less 
total hydroxyproline) than the contra-lateral normal muscle. The current 
study was designed to isolate age effects, compensation effects, and dener- 
vation effects on the collagen metabolism of old and young animals. It was 
found that the hydroxyproline concentration in normal muscles increased 
with growth, leveled off at 16-22 months of age, but increased markedly at 
28 months. Following denervation there was a reduction in muscle weight. 
The hydroxyproline content of the muscle increased significantly in both 
young and old rats, but the increase was slightly greater in the old than in 
the young. Age of the animal does not seem to influence the effects of de- 
nervation significantly. 

Tumor Incidence in Senescent Rats . One of our guest scientists is 
examining the incidence of tumors and other pathology in pituitaries, thyroid 
glands, and kidneys in young and senescent rats. Preliminary results show 
a low incidence of thyroid tumors in both young and old rats, but adenomas 
of the pituitary gland have been found in 75% of the senescent rats (aged 24- 
26 months). None have been found in 12-18 month old animals. 

Cell Biologv. One hypothesis of cellular aging and death is that pro- 
tein synthesis is impaired with advancing age. To test this hypothesis a,^ 
guest scientist studied the incorporation of C leucine or a mixture of C 
labeled amino acids into cold TCA insoluble material by liver microsomal 
fractions from mature adult (12 month old) and old (20-31 month old) female 
rats. A decreased amino acid incorporation (ranging from 12 to 43% in in- 
dividual experiments) into protein occurred in the cell-free system from 
senescent rat livers. Also, cross-mixing experiments indicated that the 
cytosol (105,000 g supernatant) from senescent animals inhibited protein 
synthesis by microsomes from 12 month old rats. The combination of cyto- 
sol from 12 month old animals with microsomes from senescent animals 
functioned like the completely senescent systems. These results support the 
hypothesis that protein synthesis is impaired in senescent cells. The inhib- 
itory effect of cytosol from aged cells is a new finding of considerable 
interest. 

One of the current theories of aging at the cellular level is that cells 
can undergo only a finite number of divisions. This phenomenon has been 
demonstrated by Hayflick in some cells, namely, the WI-38. However, it is 
not known whether this phenomenon occurs in other types of cells. One of 
our guest scientists is utilizing regeneration in the earthworm to test this 
hypothesis. This model was chosen because cells from different segments 
of the worm have different regenerative potentials. Worms cut at the 90th 
segment will regenerate 10 segments, while those cut at the 50th segment 
will regenerate 40-50 segments. The experiments will test in tissue culture 



FH4 



the number of cell divisions that can be carried out by cells removed from 
different segments of the worm. New techniques have been worked out and, 
although the initiation of growth is delayed, blastema cells from the earth- 
worm can now be cultured in vivo. A variety of cell types are obtained, so 
that cloning will be necessary. It has also been found that the brain is not 
necessary for regeneration in the earthworm. 

In view of the Hayflick phenomenon, studies on age-related changes 
in normal cells in culture can provide in terms of model systems important 
information about the cellular bases of aging. Using the cell culture tech- 
nique, it has been found that DNA synthesis is exponentially related to age. 
Cell age can be correlated with certain ultrastructural changes in cultured 
cells, such as an increase in secondary lysosomes. Older cells have a re- 
duced capacity to take up exogenous proteins. Some of the age-related 
changes can be modulated by Cortisol and other similar hormones. As in 
the intact organism, the responsiveness of cultured cells to a hormone sig- 
nal declines as the cell ages. These cellular processes may provide the 
basis for understanding the age associated impairments in physiological 
control mechanisms. 

It was shown that when human fetal fibroblasts were transferred 
from in vivo to in vitro conditions, the cells undergo functional and ultra- 
structural changes that reflect possibly altered metabolic activities. Cells 
under in vitro conditions show marked hypertrophy of the rough endoplasmic 
reticulum, indicative of increased protein synthetic activity. The most 
striking change consists in a marked enhancement of pinocytosis and stimu- 
lation of the lysosomal system, which clearly reflects a change in the nutri- 
tional mechanisms in the cultured cells. Macromolecular uptake or bulk 
transport becomes an important means for internalization of materials from 
the external environment required for meeting the nutritional requirements 
under in vitro conditions. 

Possible alterations of the adenyl -cyclase, cyclic AMP, phosphodi- 
esterase system during in vitro aging might result in the impairment of key 
metabolic reactions in the cells mediated by this system, which could be re- 
sponsible for the deterioration of cell function and loss of reproductive capa- 
city that characterizes aging in cell culture conditions. The WI-38 cell line 
provides a model system for studying changes in the metabolism of cells as 
they "age". These changes in metabolism are an expression of control 
mechanisms. One such control mechanism of system involves cyclic AMP. 
The synthesis and degradation of this compound have been shown to be im- 
portant factors in the regulation of cell metabolism. At present, activity 
levels of adenyl -cyclase and P.D.E. are being determined as a function of 
passage levels in order to see whether there are changes with cellular "age" 
(low vs. high passage). If there are changes in the activity of these en- 
zymes, can changes in intracellular cyclic AMP concentrations be corre- 
lated with these changes? 

Preliminary studies on the activity levels of adenyl-cyclase sug- 
gested a marked decrease in enzyme activity in SV-40 transformed fibro- 
blasts when compared to normal cells. In addition, with the more sensitive 
adenyl-cyclase assay, it has been possible to demonstrate an increase in 

; 



adenyl-cyclase activity in older passage cells when compared to the younger 
passage cells. There does not seem to be any change, however, in the ac- 
tivity of P.D.E. when comparisons are made between young and old cell pas- 
sages. Further, work recently begun suggests that AMP may play an im- 
portant role in the regulation of growth in WI-38 cells as Tneasured by both 
absolute increases in cell population and the proportion of nuclei incorpor- 
ating radio -labeled thymidine. 

Results of other experiments comparing uptake and passage level 
(age) demonstrated marked differences in the absorptive capability of Phase 
I-II and Phase ni cells. Specifically, young (Phase I-n) cells took up about 
0. 5 nanograms media protein per microgram cell protein during a 4 -hour in- 
cubation, while old (Phase III) cells pinocytosed 0. 3 nanograms media pro- 
tein per microgram cell protein in the same period (P < .01). These pat- 
terns of uptake were found to be consistent regardless of the proportion of 
total protein in the pulsing medium. 

Accumulation of lipofuscin "wear and tear" or age "pigment" is one 
of the most striking cytological changes observed during aging. Male sex 
accessory organs have been used as a model for investigating in_vivo. deposi- 
tion of this pigment during aging. The rationale for using this model is as 
follows: The cytological mechanisms of formation of autophagic vacuoles 
after castration and the evolution of these bodies are similar to those ob- 
served in normal rats during aging, where many of the lipofuscin granules 
originate from autophagic vacuoles. In the castrate, the induction of the au- 
tophagic process can be controlled, and as it takes place rapidly and in abun- 
dance the various stages in the formation and evolution of autophagic vacuoles 
is readily detected. Furthermore, the fact that autophagia induced by castra- 
tion can be reversed by androgens, whereas spontaneously developed auto- 
phagia during aging is irreversible by any procedure known, offers a unique 
opportunity for investigating the pathologic factors responsible for the chemi- 
cal changes in the latter that result in the accumulation and occupancy of 
large portions of the cytoplasm by metabolically inactive residues. 

Studies of the three major components of the male sex accessory 
tract have shown clearly that lipofuscin originates from both autophagic vac- 
uoles and phagosomes concerned with autodegradation of obsolete cytoplas- 
mic organelles and absorbed exogenous materials respectively. In early 
stages, these digestive vacuoles contain abundant lysosomal enzymes, but as 
the intravacuolar digestive process proceeds, enzyme activity decreases and 
finally disappears. The following course of events can be postulated in rela- 
tion to subcellular mechanisms involved in the genesis of lipofuscin from 
autophagic vacuoles and phagosomes. Obsolete or denatured cell organelles 
and pinocytosed materials within digestive vacuoles undergo digestion of the 
protein component of lipoprotein complexes that enter into the formation of 
cell organelle membranes. The lipid part, probably due to poor lipolytic 
activity of lysosomal enzymes, remains undigested and precipitates in the 
form of membranous arrays — so-called myelin figures. In later stages 
these membranous arrays evolve into a homogeneous lipid-like material, 
which presumably represents lipofuscin or some other inert residue that can- 
not be further degraded by the enzymatic machinery of the cell. 



FH-e 



Since plasma membranes are involved in the cellular control of trans- 
port and metabolism, they represent potential sites for age changes. A pilot 
study of the protein composition of plasma membranes from rat kidney was 
carried out. Membranes were separated by sucrose gradient techniques. 
The membranes were then solubilized and their protein components were 
separated by poly acrilamide gel electrophoresis. Animals from three age 
groups (6-8 mo. , 12-14 mo. , and over 24 mo.) were studied. Preliminary 
results show two small peaks in the preparations from old kidneys which are 
absent in the young. Work is continuing to isolate and characterize the atyp- 
ical proteins which appear in plasma membranes from senescent rats. 

Physiology of the Gastrointestinal Tract. A number of scientists 
have carried out studies using the gastrointestinal tract as a model system. 
These studies range from investigations of cellular responses to toxins to in- 
vestigations of motility and absorption from the intestine. 

Using micropuncture of individual cells and recording voltage and re- 
sistance across the cell surface, it was found that theophylline causes a de- 
polarization of inter -villus cells of the intestine and an increase in resist- 
ance across villus cells. These findings are consistent with the known ef- 
fects of theophylline on electrolyte transport, that is, a stimulation of chlo- 
ride secretion and inhibition of sodium absorption. It is suggested that the 
inter-villus areas principally contribute to fluid excretion. This base-line 
information is necessary for projected studies of the mode of action of the 
effects of enterotoxins on the gut. 

The mechanisms of interaction of enterotoxins with cells is being 
studied in order to understand how toxins bind to cells and influence cellular 
metabolism. The goal is to discover ways to block the action of enterotoxins 
and to identify the relationships of toxin effects to those of physiologic 
stimuli. 

Epididymal fat pads from young and old rats were incubated with col- 
lagenase and a suspension of isolated cells was used for the experiments. 
It was found that monosialoganglioside, which specifically binds cholera tox- 
in, will enhance responsiveness of fat cells to toxin and raise the responsive- 
ness of cells from older rats to equal those of young rats. Cholera toxin ex- 
erts its effect at a site not blocked by A or B adrenergic blocking agents, al- 
though there is a slight effect by A blockers such as phentolamine or diben- 
zyline on response to toxin. Adenyl cyclase is stimulated by both E. coli 
and V. cholerae enterotoxins. Prior exposure to these toxins enhanced re- 
sponsiveness of membranes to subsequent challenge by epinephrine. 

Cellular responsiveness to enterotoxins in the case of rat epididymal 
fat cells is clearly age dependent. Discovery that ganglioside binds cholera 
toxin and enhances cell response to toxin if cells are pre -incubated with this 
substance allows experiments in which the ability to restore cells from older 
rats to responses characteristic of younger rats opens the door to specific 
experiments which could relate the numbers of a specific receptor to aging. 
Enhanced response by cell membranes to hormones after exposure to toxins 
could be an important parameter to study in cells from animals of different 
ages. 



FH-7 



Enterotoxins were also prepared from culture filtrates of E. coli 
sterilized by filtration and concentrated by lyophylization. Biological activ- 
ity of the enterotoxins was assayed in the infant and adult rabbit ileal loop 
model. Antitoxin assays were performed by measuring neutralization of en- 
terotoxin in the adult rabbit ileal loop. Immunization studies were done on 
adult rabbits using antigen mixed in incomplete Freund's adjuvant as the pri- 
mary immunization method. Challenges will be made of the ileal loops of 
the immunized rabbits. 

Major findings were: Enterotoxins from E. coli isolated from child- 
ren with diarrhea from Arizona and Alexandria, Egypt, have been identified. 
Neutralization of six different E. coli enterotoxin preparations (each from a 
different E. coli serotype) by the standard E. coli antiserum was demon- 
strated. These E. coli strains were isolated from India and the United 
States. Immunological similarities between enterotoxins of E. coli and V. 
cholerae were demonstrated by neutralization tests. Preliminary results 
suggest that children infected with enterotoxigenic E. coli strains develop 
antitoxin titers during convalescence. As of this report, the experiments 
involving challenge of immunized rabbits are still in progress. 

Diarrheal disease due to E. coli is primarily a childhood disease. 
Why the disease is seen less frequently in adults is not known, but may be 
related to immunologic processes. The finding of a common enterotoxin 
antigen suggests that immunization could be useful in control of the disease. 

Studies on the effects of enterotoxins and pharmacologic agents on 
gut secretion were carried out in dogs by measuring net secretion, mucosal 
adenyl catalase activity, time course of toxin effect and mucosal histology. 
The effect of desoxycorticosterone (DOCA) on small bowel electrolyte trans- 
port was studied in dogs with Thiry-Vella loops of duodenum or ileum. 
Electrolyte content and volume of secretions induced by cholera toxin were 
studied before and after 3 or 6 days of DOCA administration. 

There was no structural abnormality in mucosa during toxin -induced 
secretion. There was no increase in clearance of protein from serum to 
gut lumen. The E. coli toxin appears to be deactivated or consumed as a 
result of exposure to the gut, thus a sustained secretory response requires 
continuous application of toxin. Control "toxin" from a nontoxigenic strain 
of E. coli produced none of these effects. 

The time-courses of heat -stable (ST) and heat -labile (LT) E. coli 
toxins were different in rabbit small bowel. ST effect was immediate in on- 
set and of brief duration, the maximum cumulative secretory response being 
at 4-6 hours. By 18 hours ST effect was usually gone. LT effect was slight- 
ly slower in onset and its duration depended upon total dose of toxin. At low 
doses it peaked at 10 hours, at higher doses it peaked beyond 18 hours. The 
time of peak effect of cholera toxin was not related to dose, being beyond 18 
hours even at submaximal doses. LT was neutralized by antibody to whole 
E. coli toxin but ST was not. The optimum time to assay ST was 6 hours, 
while that for LT was 18 hours. 

DOCA has the previously unrecognized effect of stimulating potassium 

FH6 



excretion by the distal ileum, but not the duodenum, during gut secretion in- 
duced by cholera toxin. It had no effect upon secretory volume. 

Acute expansion of extracellular fluid volume in dogs caused marked 
secretion by the small bowel. This secretion was not associated with altera- 
tions in mucosal adenyl cyclase activity. 

Studies on the binding of E. coli and cholera enterotoxin to gut muco- 
sa indicated that ganglioside binds heat -labile but not heat -stable E. coli en- 
terotoxin. This is in contrast to the previous report that ganglioside did not 
bind E. coli toxin. The binding of E. coli LT by ganglioside appears much 
less efficient than that of cholera toxin. This observation is being pursued 
as a possible means of purifying E. coli toxin. Affinity chromatographic 
techniques are being used to attempt to bind LT to ganglioside and then elute 
it from the complex of ganglioside and agarose. 

In studies elsewhere further evidence is accumulating that ganglio- 
side (specifically the sialidase resistant monosialoganglioside) is the mem- 
brane component of mammalian cells which binds and is the receptor for 
cholera toxin. 

Antitoxic immunity to cholera has been further investigated to clarify 
the roles of serum or locally produced antitoxin in protection against cholera. 
Antitoxin titers have been measured in serum and jejunal washings after lo- 
cal immunization or active or passive parenteral immunization. 

It was found that both serum and locally derived antibody can protect 
against toxin challenge. Serum antibody is largely IgG whereas local anti- 
body is IgG and IgA. Passive immunization by intravenous serum adminis- 
tration yielded measurable intraluminal antibody and protected against toxin 
challenge. Intraluminal toxoid administration was not effective in boosting 
serum antitoxin titers. 

Dogs immunized parenterally or with parenteral plus oral glutaralde- 
hyde toxoid were protected for at least 1 month to vibrio challenge. Serum 
titers were low to toxoid alone but were markedly improved by use of an al- 
uminum adjuvant. 

Cholera antitoxin titered in rabbit gut segments gave results similar 
to those in rabbit skin. However, at low levels of activity the rabbit gut 
system appeared more sensitive than rabbit skin. 

Serum vibriocidal antibody induced by a subcellular vibrio vaccine 
was unprotective despite high serum vibriocidal antibody titers. This indi- 
cates that this antibody is not responsible for protection induced by whole 
cell vaccines. 

Serum titers of vibrio motility inhibiting antibody increased after 
parenteral immunization with whole cell vaccine. These titers appeared in- 
dependent of agglutinating antibody titer. They have not yet been measured 
in gut washings. 

The everted gut technique has been used to study age differences in 

FH-Q 



intestinal absorption of folate in the rat. No significant differences were 
found in the absorption of folate in 6, 12, and 24 month old animals. 

Motility of the Gastrointestinal Tract . Esophageal Studies: Biofeed- 
back control of lower esophageal sphincter contraction was achieved using 
tandem open tipped catheters to measure pressures from the esophagus, 
lower esophageal sphincter and stomach in normals and patients with eso- 
phageal reflux. Instantaneous biofeedback was provided to the subjects by 
means of a meter. Both normals and patients with reflux esophagitis were 
able to increase lower esophageal sphincter pressures without corresponding 
changes in intraesophageal, intragastric pressures or in respiration. This 
skill persisted after biofeedback was discontinued. These studies demon- 
strate the ability to condition autonomic gastrointestinal activity by biofeed- 
back techniques and open the possibility of operant control of disordered 
gastrointestinal functions, including reflux esophagitis, which occurs more 
commonly with increasing age. 

Anorectal: Adult patients with severe intractable fecal incontinence 
(continuous daily soiling) were tested to evaluate manometric and clinical 
effects of biofeedback training in the control of anal sphincter responses. 
Pressures were measured by triple balloon system from the rectum, inter- 
nal and external anal sphincters and sphincteric reflexes were initiated by 
transient distension of the rectal balloon (simulating arrival of stool in the 
rectum). All incontinent patients demonstrated normal internal sphincter 
reflexes but absent or markedly impaired external sphincter responses em- 
phasizing the role of the external sphincter in maintenance of continence. 
Instantaneous visual feedback was provided by permitting patients to observe 
the direct writing records. Patients were taught to contract the external 
sphincter in synchrony with internal sphincter relaxation. The success of 
this technique in achieving fecal continence even after visual feedback was 
discontinued indicates the importance of synchronized sphincter contraction 
for maintenance of continence and the potential value of these techniques as 
a simple means of treating fecal continence, a problem which increases in 
incidence in an aging population. 

Colon Motility and Compliance . Recordings of rectal compliance and 
colonic motility from the rectosigmoid, rectum, and internal anal sphincter 
in normals and in patients with irritable bowel syndrome demonstrated two 
types of colonic responses to rectosigmoid distension in normals and an ad- 
ditional "spastic" contraction in patients with irritable bowel syndrome. Ac- 
tive colonic contractions were usually associated with sensation of gas or an 
urge to defecate, depending on the location of the spasm. The aggravation of 
these contractions by feeding correlates with clinical observation that symp- 
toms of irritable bowel syndrome are aggravated by food intake. These 
studies are being applied to patients with irritable bowel syndrome along an 
aging continuum, in light of recent information that irritable bowel syndrome 
may be a precursor of diverticular disease and that diverticular disease in- 
creases appreciably with increasing age. Biofeedback techniques will be 
employed to attempt inhibition of exaggerated spastic responses as a means 
of treating this disorder. 

Osteoporosis . Although osteoporosis and loss of calcium from bone 

FH-10 



occurs with advancing age, precise measurements of bone density in intact 
human subjects has not been possible. Recent technical advances (photon 
absorbtiometry) now permit a precise assessment (±4%) of bone mineral con- 
tent in intact humans. A collaborative project to follow changes in bone min- 
eral content has been initiated, using subjects from the Baltimore Longitudi- 
nal Study. A preliminary analysis of observations on 107 subjects (age 
range 25-90) failed to demonstrate a significant correlation between bone 
mineral content and age. 

The double-blind clinical trial of salmon calcitonin in the treatment 
of symptomatic osteoporosis has been suspended because of recall of the 
therapeutic agent by the manufacturer because of its previously unapprecia- 
ted short shelf life. The design of the trials involved 90 days treatment 
which could not be carried out with a single preparation of calcitonin. 

Thyroid Studies. The hypothesis that one of the mechanisms of aging 
is a reduction in the effectiveness of physiological controls is also under in- 
vestigation. Since hormones play an important regulatory role, their action 
at the cellular level is being studied by one of our guest scientists. It has 
been found that there are specific intracellular proteins which bind thyroid 
hormones. Physical properties of these proteins have been characterized, 
including mobilities in several electrophoretic systems, molecular weight, 
isosonic point, affinity and behavior in various chromatographic systems. 
This S protein from dog liver and kidney has been purified 150 -fold. The 
cellular cytosol binding proteins are distinct from serum carriers of thyrox- 
ine and tri-iodothyronine and are presumed to modulate intracellular actions 
of these hormones. Current work involves quantitation of these proteins and 
studies of cytosol protein -nucleic interaction. 

Amyloidosis and Aging. Senile amyloidosis is a significant disease 
of the elderly. Cardiovascular amyloidosis in particular appears to be as- 
sociated with a significant morbidity. At present there are no accepted or 
proven forms of therapy for amyloidosis, and studies directed at possible 
mechanisms of amyloid resorption represent important steps toward this 
end. 

Recent studies suggest that amyloid fibrils may be phagocytosed under 
certain conditions. In tissue cultures, for instance, amyloid fibrils may be 
phagocytosed by blood macrophages provided antiserum to alkali -denatured 
amyloid fibrils is added. Recent observations suggest that, in the rabbit, 
spontaneous amyloid resorption in the spleen occurs within a defined time 
period following amyloid induction. 

The introduction of purified rabbit amyloid fibrils into the subcutane- 
ous tissues of BDF inbred mice resulted in resorption of the fibrils within 
5-9 days. This was preceded by an accute inflammatory response, but dur- 
ing resorption the cellular reaction was predominantly macrophagic. Acid 
phosphatase preparations showed that macrophages nearest the residual amy- 
loid deposits were particularly rich in lysosomes and light microscopy re- 
vealed intracytoplasmic inclusions of amyloid. Electron microscopic 
studies are currently underway. 



FH-11 



Intramural Research 

The Laboratory of Molecular Aging. Research projects, utilizing a 
variety of model systems, have examined questions related to regulatory 
processes in the transmission of genetic information, mechanisms of con- 
trol of enzymatic activity and cellular metabolism, and biochemical changes 
during aging that alter the responses and functions of subcellular organelles 
and cells. 

An extensive study of infection of human cells (WI-38) in culture by 
vesicular stomatitis virus and its prevention indicates: (1) Cellular viral 
defense, i.e., the induction of interferon, is functional throughout the life 
span of the cells up to the moribund state, but the amount of poly IC required 
for induction increases with age. (2) In senescent cells the polynucleotide 
inducer of interferon produces the antiviral state faster than treatment with 
interferon itself, suggesting either another antiviral protection mechanism 
in senescent cells or a change in the relative rates of uptake of poly IC and 
interferon in the senescent cell. 

Synthetic analogs of nucleic acids, representing modifications of the 
structure of native genetic material, have been shown to interfere with spe- 
cific biological processes and, therefore, can be used to study biochemical 
mechanisms. Thus, polyvinyl uracil and polyvinyl adenine both inhibit mu- 
rine leukemia virus infection. The inhibition of the leukemia virus appears 
to be related to the inhibition of a RNA -dependent DNA polymerase. 

Complexes of platinum have been used to explore the mechanism by 
which the replication of DNA and synthesis of RNA are impeded by metal 
ions. It is known that the cis-form of a platinum complex inhibits DNA rep- 
lication, cell division, and is an effective antitumor agent, whereas the 
trans -form of the complex is not active in these respects. It has now been 
found that the cis -isomer, but not the trans -isomer, forms complexes with 
DNA. The Pt-containing complex inhibits transcription in an in vitro system, 
and this inhibition is on the polymerization process, without affecting the in- 
itiation process. A technique has been developed to replace the zinc, the 
natural metal in RNA polymerase, with several metals including copper and 
to retain enzymatic activity. The substitution of copper, allowing electron 
spin resonance studies, may provide a valuable tool in elucidating the 
molecular action of the enzyme. 

Studies on the mechanism of active transport of sugar in the kidney 
have continued to make significant progress and several underlying princi- 
ples have been established. Glucose transport by isolated renal brush bor- 
der membranes is comprised of at least two processes, a high affinity bind- 
ing system and a carrier -mediated transport system. The presence of a 
stereo -specific high affinity binding system has been unequivocally demon- 
strated by equilibrium dialysis. Kinetic studies of the binding have charac- 
terized its time course, reversibility, saturability, susceptibility to the 
pro^ence of ^lorizin, and have determined the effects of Na+ as well as 
Ca"^"*" and Mg """. A particulate fraction derived from detergent-extracted 
brush border membranes has been prepared. This preparation has the 
same affinity and capacity as intact membranes but has lost about 90% of its 



FIH2 



protein content and shows no evidence of vesicular structure. Evidence for 
a glucose -bound protein has been obtained. The mechanism of uptake of 
high concentrations of glucose by the intact brush border membranes shows 
both influx and efflux components and is consistent with a hypothesis for a 
carrier -mediated transport into and out of vesicles. Prior loading experi- 
ments demonstrate the phenomenon of counter-transport or facilitated ex- 
change diffusion. Although Na+, per se, is not required for uptake of glu- 
cose by the brush borders, the rate of glucose transport by the membrane is 
greatly enhanced by a Na+ gradient from the outside to the inside of the 
membrane. 

Extensive studies on the control of pyruvate oxidation in mitochondria 
from blowfly flight muscle, and confirmed with mitochondria from rabbit 
heart muscle, have led to a novel hypothesis to explain the regulation of 
Krebs cycle activity. Both ADP and inorganic phosphate are required for 
the uncoupled (FCCP) oxidation of pyruvate as well as for the coupled oxida- 
tion. The Fr:CP -stimulated oxidation is markedly inhibited by physiological 
levels of Ca'^+. The apparent Kj = 10|jM. The electron transport chain of 
the mitochondria is fully functional in the presence of Ca^"*" since the oxida- 
tion of a -glycerolphosphate is not inhibited, indicating control at the dehy- 
drogenase level. Kinetic studies show that the Ca^+ inhibition is uncompeti- 
tive with respect to ADP and FCCP, but complex, and noncompetitive with 
respect to pyruvate. The results of these and other studies suggest that 
pyruvate oxidation and Krebs cycle activity is limited by NAD-dependent iso- 
citrate dehydrogenase activity and Ca2+ plays a role in its regulation. An 
additional mechanism for the control of pyruvate metabolism has been demon- 
strated in mitochondria for the first time. In analogy to glycogen phosphoryl- 
ase, this involves the phosphorylation and dephosphorylation of the pyruvate 
dehydrogenase complex by a kinase and phosphatase, respectively. In con- 
trast, however, with pyruvate dehydrogenase, the phosphorylated form is in- 
active whereas the dephosphorylated form is active. 

A correspondence between the decline in the ability of aged blowflies 
to fly and in the maximally stimulated respiratory rate (State 3) and respira- 
tory control ratio of flight muscle mitochondria was reported previously. 
The biochemical mechanism and locus of this decrement with senescence has 
been investigated further by examining the bioenertetic properties of mito- 
chondria from mature and aged flies for rates of oxidation during coupled 
oxidative phosphorylation, activities of the dehydrogenases, concentrations 
of components of the electron transport chain, phosphorylation of ADP, and 
the coupling mechanism itself. Evidence, to date, indicates that the specific 
activities of a-glycerolphosphate and pyruvate dehydrogenases are unaltered 
by age. Similarly, the concentrations of cytochromes a, as, b, and c+ci are 
the same for both age groups. ATP synthetase, measured as ATPase, is 
unchanged with senescence. In contrast, the uncoupled oxidation of sub- 
strates is significantly decreased with aging, showing the 30-40% decrement 
between mature (7-10 days) and aged (31-33 days) as seen previously for the 
maximally stimulated coupled rate of respiration. These observations nar- 
row the possible loci of the defect in aging to the bioenergetic system be- 
tween the primary dehydrogenase and the FCCP -sensitive uncoupling reaction. 

Hormonally (parathyroid, synthetic 1-34, and catecholamine) 

f FH43 



stimulated adenyl cyclase has been demonstrated in renal cortical mem- 
branes. Luminal brush border membranes bind c-AMP, as determined by 
equilibrium dialysis and Millipore filtration experiments. Kinetic parame- 
ters of the binding have been examined. It is anticipated that the binding of 
c-AMP may initiate kinase activation and potentially exert a controlling role 
in renal tubular function. 

Laboratory of Cellular and Comparative Physiolog y. FY'73 has been 
highlighted by a major change in the emphasis of program goals and in the 
accompanying research activities to acMeve these goals. 

The current goals are three -fold: (a) To conduct studies on the nature 
of age-related deteriorating functional changes of cells and tissue systems 
whose growth and differentiation events are reasonably well understood, (b) 
to determine the underlying mechanisms of these changes, and (c) to develop 
methods for early detection of the deterioration and, hopefully, methods to 
control or reverse the deterioration. 

To implement these goals emphasis will be on the use of cells and 
tissues of mammalian species, including man, in in vitro and in vivo cul- 
tures. Moreover only a few closely related model systems will be analyzed, 
but they will be studied systematically. 

The major thrust in research activity has been in immunology and 
aging, with the creation of the Immunology Section, for the following reasons. 
(1) Associated with, or as a consequence of immunosenescence, the incidence 
of immunodeficiency diseases including autoimmunity, cancer and infection 
increases with age. (2) Cellular, molecular and genetic knowledge of the 
ontogeny, phylogeny and differentiation process involved with the immune 
system is well understood, probably more so than any other functional sys- 
tem. (3) The system is amenable to elegant cellular and molecular analyses 
and, therefore, offers great promise for successful manipulation of the 
system. 

During the past year, members of the Immunology Section expanded 
their effort in the creation of the laboratories and initiation of research pro- 
jects. Hence fruition of their current research task should become apparent 
at the earliest during the next fiscal year. Nevertheless, basic findings, 
some of which were established just prior to their arrival at the GRC, are 
as follows. 

(1) Of the three cell types (thymus -derived T cells, bone marrow- 
derived B cells, and accessory A cells) involved in the initiation of a humor- 
al immune response, the T and B cells are altered in immunologically defi- 
cient old mice, but A cells appear normal. 

(2) The proliferative capacity of T and B cells is decreased as much 
as lO-fold with age. This is a very critical function because the number of 
functional effector cells generated in an immune response is dependent upon 
the ability of these cells to divide efficiently. 

(3) Limiting dilution, reconstitution, and young cell-old cell mixture 



FH-14 



• 
analyses offer indirect evidence that either the relative number or the effi- 
ciency of regulator cells that suppress an immune response increase with 
age. For example, it was found that the humoral immune activity of a mix- 
ture of spleen cells from young and old mice was lower than the sum of in- 
dividual responses, indicating that there are cells in old mice that can in- 
hibit young immunocompetent cells from responding to antigenic stimulation. 

(4) One of the deficiencies of radiation -induced allogeneic bone mar- 
row chimeras was shown to reside in the thymus gland, the seat of immuno- 
deficiency diseases in most cases. Previously it was shown that these chi- 
meras are suitable model animals to study immunosenescence. 

(5) Limiting numbers of lymphoid cells undergo maximum antibody 
response in vitro only when they are cultured under optimum cell concentra- 
tion, a variable which was not taken into consideration previously. This in- 
formation offers us the option of assessing quantitatively the activity of lim- 
iting numbers of lymphoid cells and, hopefully, one or two clones of compe- 
tent cells. 

(6) Mice with an exceptionally long life span show a decline with age 
in cell -mediated immune activity and resistance to allogeneic tumor cells. 
Previously the decline in activity was observed in mice with short life spans. 
Therefore critics have argued that life -shortening diseases imposing on the 
immune system were responsible for the decline in cell -mediated immunity. 
These results should minimize such criticisms. 

Based on these findings efforts are focused currently on the cellular 
and molecular definitions of immunosenescence with emphasis on (a) isola- 
tion and characterization of promotor and suppressor cells, (b) ability of 
stem cells to generate thymus -derived T and bone marrow -derived B cells, 
(c) life spans of memory cells, (d) mitogen receptor sites and the cyclic 
AMP system of proliferating T and B precursor cells, and (e) characteriza- 
tion and repair of induced and naturally occurring immunodeficiency states. 

Research has also been initiated on a small scale in biomembrane 
physiology, with emphasis on the effect of age on hormone bindings, for two 
reasons: (a) It will complement ongoing molecular studies in immunology 
and (b) the time is "ripe" to initiate cellular and molecular studies focused 
on the nature of inefficient response to hormones and drugs by the aged. 
Preliminary results showed that adipose tissue, skeletal muscle, brain and 
prostate gland of aging rats exhibit a progressive decUne in steroid hormone 
binding due, in part, to a reduction in the number of macromolecular bind- 
ing sites per unit tissue mass. 

Another area of research that can contribute to the program goals of 
LCCP is mammalian genetics. An understanding of the genetic mechanisms 
of age-related functional deterioration at the molecular level is obligatory. 
To this end, research is being promoted to understand, at the cellular level, 
human and animal disorders manifesting altered development and aging. 
Currently a laboratory is being created to carry out two studies: (a) The role 
of chromosomal aneusomy in the impaired proliferation of fibroblasts and 
immunocompetent cells in relation to aging and (b) the influence of gene 



FH-15 



dosage and maternal environment on the life span of embryos and individuals 
carrying lethal genes. The long-term goal of these studies is to "sort out" 
and elucidate those genetic alterations which can impair the proliferative 
capacity of somatic cells. Information derived from this program should 
have a tremendous impact on cellular biology of aging, for impairment with 
age of the proliferative capacity is characteristic of certain somatic cells. 

Two sections in existence prior to FY'73 are the Nutrition and Mor- 
phology Sections. The former is concerned with the influence of nutrition 
and antimetabolites on the life span and various tissue enzyme activities in 
rats. The latter is concerned with structural and cytochemical changes as- 
sociated with development and aging of myocardial and skeletogenic cells 
and with the growth patterns of coelenterates as influenced by temperature 
and endogenous rhythmic changes. 

During the past year, the Nutrition Section completed preliminary 
studies in two areas: (a) The effect of cycloheximide on the rate of develop- 
ment of chick embryos and (b) the effect of protein undernourishment on the 
life span of middle-aged rats. In the former study it was found that nontoxic 
levels of cycloheximide, an inhibitor of protein synthesis, when given to 
1 day old chick embryos delay their rate of development at least until day 17, 
as judged by morphological parameters, heart beat, and DNA content. How- 
ever, the activities of lysosomal and mitochondrial enzymes were not 
affected. 

The latter study revealed that reduction in the dietary protein intake 
in 16 month old rats from the standard 24% to 8 or 4% had no influence on 
their subsequent mean life expectancy. However, rats restricted to 12% 
casein diet lived on the average of two months longer. The life promoting 
effect of 12% protein intake was manifested during the first 3-4 months fol- 
lowing dietary restraint. In view of the profound implication this observa- 
tion offers, a more systematic study is being initiated. 

Ultrastructural cytochemical studies by the Morphology Section re- 
lating lysosomal activities and aging processes in the left ventricular cells 
of aging rats revealed that in mural myocardial cells there are (a) two dif- 
ferent pathways by which primary lysosomes are formed and (b) two differ- 
ent mechanisms of lysosomal degradation of mitochondria. The two path- 
ways of synthesis, transport and packaging of primary lysosomes are the 
nuclear pole zone route and the peripheral route. The latter, which bypasses 
the Golgi, provides the cells a more direct route to sites of action of the ly- 
sosomes. Lysosomal degradation of mitochondria occurs by the more com- 
mon matrix densification process and the less common matrix clarification 
process. Studies on papillary myocardial cells of 24 month old rats showed 
that there are two types of changes: (a) Cells that appear grossly normal in 
structural appearance but showing marked intracellular changes in compari- 
son to those of 6 and 12 month old rats, and (b) cells that appear grossly 
abnormal in structural appearance. Ultrastructural definition of age related 
deterioration of heart muscle cells should provide us a better insight how to 
resolve whether the changes observed are the cause or consequence of senes- 
cence of the organ. 



FH-16 



studies on endogenous circannual rhythms in growth, development 
and life spans in marine coelenterates revealed that they can persist for over 
3 years in the absence of periodic signals from the environment. The period 
for the cycles at three ambient temperatures, 10° , 17° , and 24° C, was about 
one year. The life span of hydranths during prolific growth of the colony was 
considerably longer than during repressed growth. These observations 
challenge the generally accepted concept that there is a specific age at which 
death is characteristic for a species. 

Clinical Physiology Branch . Research in the Clinical Physiology 
Branch continues to be geared toward a physiological and biochemical dis- 
section of mechanisms underlying both adaptive and the deteriorative changes 
of aging--and, when possible, to accomplish this goal in man. 

The invaluable resource for our study of human aging continues to be 
the Longitudinal Aging Study. This year has been one of continued develop- 
ment and refinement of unique techniques for longitudinal data analysis. 

To quantitate age changes in the lung, the forced expiratory volume 
in one second (FEV 1.0) has been measured. The mean rate of aging within 
individual subjects can be defined in young and middle-aged adults with less 
than 25% error and in old subjects with less than 10% error. 

Blood pressure increases with age show important trends not previ- 
ously realized. Blood pressure has been recorded under basal conditions 
(during basal metabolic rate measurements) and under "casual" conditions, 
that is, as recorded by a physician during a physical examination. The var- 
iance associated with the usual casual conditions of blood pressure record- 
ing makes it impossible to detect age changes in systolic pressure of indi- 
vidual subjects with adequate accuracy. On the other hand, measurements 
of systolic pressure made under basal conditions shows progressively great- 
er rates of increase with advancing age and these age changes within an indi- 
vidual can be defined with statistical confidence. 

Serum cholesterol and triglyceride levels show strikingly different 
aging trends. Statistically significant increases occurred in cholesterol (er- 
rors of less than 25%) in young and middle-aged adults, but leveled off after 
the age of 60-65 years. Triglyceride levels were constant in young adults 
and fell significantly (errors again less than 25%) in middle-aged and older 
men. Identification of the reasons underlying the differences in these changes 
await multivariate analysis of dietary, activity, and other metabolic variaDies 
in the coming year. 

The versatility of the new analytical technique for determining the de- 
sign of longitudinal studies is best seen when it is used predictively, i. e. , 
for future planning. It can be shown that most of the physiological variables 
of interest to gerontologists will require 15 to 30 years of study in order to 
achieve reasonably accurate estimates of rates of aging in individual subjects 
across the adult age span. This statistical approach in combination with the 
basic data provided by the Baltimore Longitudinal Study is an achievement 
which clearly has far-reaching implications for the future. 

The difficult problem of separating age changes from disease changes 

FH-17 



has not been dealt with adequately either theoretically or practically by other 
ongoing studies of human aging. We have devoted much effort the past year 
to the development of techniques to permit this separation. An automated 
chart review was the initial step which converted detailed data from medical 
histories, physical examinations, and laboratory data to an efficiently ar- 
ranged computerized summary. Comments written by the examining physi- 
cian are also included in the computerized summary. With these data now 
available on over 4,000 subject -visits, detailed criteria has been developed 
for the classification and identification of those disease states which could 
alter physiological functions independent of age. Detailed classification has 
been accomplished for pulmonary diseases, renal diseases, coronary heart 
disease, and cerebrovascular disease. These classifications should provide 
the standards which other longitudinal studies of human aging will be able to 
use. 

New studies have been introduced into the Longitudinal Study this 
year. Cutaneous malignancies are a serious problem, especially in older 
subjects. Studies of chronic actinic damage as a precursor of these malig- 
nancies have assumed that the damaging radiation spectrum is 290-320 nm 
and that longer wavelength ultraviolet radiation (320-400) causes "tanning" 
and may even protect against sun damage to the skin. A comprehensive 
study on the effect of age on sensitivity to UV light of different wavelengths 
and on the time course of the radiation effects is therefore underway. Re- 
sults to date show that the longer UV wavelengths actually have an augmenta- 
tive effect, not a protective effect. 

Observations on additional genetic markers have been introduced into 
the longitudinal study in order to assess further the role of genetics in aging. 
Measurements of haptoglobin, ceruloplasmin, and transferrin in plasma and 
dermatoglyphic analyses are now being made on all subjects in the longitudi- 
nal study. 

An interesting analysis has been made of various estimates in use for 
the determination of body fatness. Not only heights and weights, but also the 
detailed Behnke Index (which includes abdominal girth), and skin -fold thick- 
nesses have been determined. Many of the subjects in the longitudinal 
study underwent both gains and losses in body weight during the period of ob- 
servations. Since the changes in body weight were due almost entirely to 
spontaneous dietary decisions on the part of the subject, they largely reflect 
changes in fatness rather than changes in lean body mass. Changes in the 
individual indices of body fatness were correlated with changes in body weight. 
It was found that changes in skin folds correlated with loss or gain of body 
weight with an r of only 0.4; the complex Behnke Index correlated better, r 
values of 0.64-0.78 in subjects of different ages, while the best indicator of 
change in fatness was the simple measurement of abdominal girth which cor- 
related between 0. 64 and 0. 90 in subjects of different ages. It appears that 
abdominal girth is a distinctly better measurement of fatness than is skinfold 
thickness. 

Study of the chemistry of human renin, a kidney enzyme involved in 
the pathogenesis of hypertension, has progressed through the use of a new 
labeled polymeric substrate assay previously developed in the Section on 



Ftt48 



Endocrinology. This enzyme is inhibited by a variety of proteins and pep- 
tides. Studies of the mechanism of this inhibition with a model protein, 
hemoglobin, with hemoglobin chains, and with chemical and enzymatic cleav- 
age fragments of hemoglobin have shown that large peptides are the most 
potent inhibitors. The leucine -leucine bond, which is necessary for renin 
action on its substrates, is not necessary for inhibition by hemoglobin frag- 
ments. A number of small synthetic peptides are also potent inhibitors of 
renin. Renin preparations, free of peptidase activity, have now been shown 
to contain protease activity against hemoglobin. A new concept of renin 
emerges in which the previous unique characteristics of the enzyme are suc- 
ceeded by one in which renin shares many of the characteristics of only rela- 
tively specific acid proteases of wide biologic distribution. The techniques 
and concepts recently developed should permit the development of renin in- 
hibitors of potential clinical usefulness. 

The possible involvement of the adenyl cyclase ("second messenger") 
system in senescence is being explored. This membrane -bound enzyme is 
activated as the initial step in the action of many hormones. As a result, 
cyclic adenosine monophosphate (cyclic AMP) is formed; a variety of meta- 
bolic processes are then initiated by this compound. Although hints of in- 
volvement of this system in certain age-related alterations of hormone ac- 
tion can be extracted from the literature, no actual studies of the enzyme in 
senescence have been reported to date. Our preliminary data now indicate 
that, in rat liver, senescence is associated with a marked increase of acti- 
vation of adenyl cyclase by epinephrine. Although non-specific activation 
with fluoride is also somewhat enhanced, activation by another hormone, 
glucagon, is unaffected. These observations indicate the existence of hor- 
mone-specific effects of age on membrane structure and function. More- 
over, they suggest that altered cyclase responses may provide a molecular 
basis for the alterated sensitivity to hormones which has been seen with ag- 
ing. This line of investigation is being extended to include a variety of hor- 
mone-activated cyclase responses in a number of tissues. 

The nature of the age decrements in myocardial performance has 
been explored by studies using isolated trabeculae carneae of young, middle - 
age, and old rats. In these preparations, single and paired stimulation has 
been given under conditions of exposure to graded levels of norepinephrine 
and to hypoxia. Biophysical muscle responses have been quantitated. The 
oldest animals show a reduction in developed tension and in the maximal rate 
of tension development in response to catecholamines. No age differences 
occurred in the paired-stimulation and hypoxia studies. The successful de- 
velopment of this experimental procedure and the demonstration of age dif- 
ferences in response to catecholamines in these heart muscle preparations 
will lead to further pharmacologic studies on the role of the sympathetic 
nervous system in determining age differences in response of the heart to 
stress. 

Studies have also been carried out on the intact dog heart to test the 
hypothesis that there is a damaged but salvageable area of heart muscle after 
coronary artery ligation. The experimental variables in this complex, 
carefully controlled preparation are (1) myocardial damage as quantitated by 
ST segment surface mapping, (2) intramyocardial oxygenation using a 



FH49 



polarographic method, (3) coronary artery perfusion pressure. Results 
show a significant correlation between the ST segment map and myocardial 
oxygen tension on a minute -to -minute basis in each dog. Furthermore, in- 
creasing the coronary perfusion pressure significantly improved oxygena- 
tion and decreased the area of ST segment abnormality. There is thus now 
experimental evidence (1) to support the validity of using ST segment changes 
as quantitative indices of the ischemic zone and (2) to support the rationale 
of elevating coronary perfusion pressure to reduce infarct size. 

Studies carried out in collaboration with Baltimore City Hospitals 
and Johns Hopkins University scientists have increased our understanding of 
myeloma kidney. Thirty-six patients have now been studied prospectively. 
Changes in urine concentrating and acidifying ability preceded GFR changes. 
PAH clearance decreased out of proportion to GFR decreases. Only patients 
with free Bence -Jones protein demonstrated impaired tubular and glomeru- 
lar function. Renal biopsy showed that changes in renal function correlated 
closely with tubular atrophy and degeneration, but not with tubular casts. 
It appears then that Bence-Jones protein exerts a direct toxic tubular effect 
and that glomerular dysfunction occurs only after severe tubular damage has 
occurred. 

The previously developed model for insulin and glucose metabolism 
in man has been used to compute the changes in glucose content of two of the 
glucose compartments when changes in glucose concentration are induced in 
the third compartment, viz. , the blood volume. This computation is critical, 
especially when large changes in blood glucose concentration occur rapidly. 
Since one of the experimental variables computed in these studies is glucose 
utilization by the body, the glucose which has been infused but which has gone 
only into the extracellular glucose spaces must be subtracted from the total 
glucose infusion rate. This computation could not be made with any degree 
of accuracy until a successful glucose model was developed. 

The complex biphasic response of insulin secretion in response to 
hyperglycemia has now also been treated mathematically by the development 
of a mathematical model of the pancreas. The pancreas model behaves "cor- 
rectly" in terms of its release of insulin in response to hyperglycemia and it 
can also reproduce the different insulin responses in young and old subjects. 
Factors influencing insulin secretion other than glucose concentration and 
age can also be accounted for by the model. The next goal is the incorpora- 
tion of the pancreas model into the previously developed glucose and insulin 
models of the body to close the loop of these interrelated variables. 

The glucose -clamp technique (both the hyperglycemic clamp to study 
beta cell sensitivity and the insulin clamp to study sensitivity to insulin) has 
been used in a prospective study of the mechanisms underlying the glucose 
intolerance of uremia. The differentiation of uremic glucose intolerance 
from diabetic renal disease with uremia is important in terms of decisions 
concerning acceptance of patients into dialysis and transplantation programs; 
diabetics with uremia have such a poor prognosis that they have been given 
very low priority in these programs. Eight uremic patients have been stud- 
ied. Seven of them showed resistance to insulin, a mechanism for glucose 
intolerance which is clearly different from true diabetes mellitus. Four 



FH-20 



subjects have been studied both before and after chronic hemodialysis. All 
four showed marked improvement in glucose tolerance as their uremia was 
ameliorated. The improvement in tolerance could be accounted for by an 
increase in sensitivity to insulin in three patients and by an increased secre- 
tion of insulin in the fourth. Therefore, while both factors may play a role 
in uremic glucose intolerance, insensitivity to insulin appears to be the more 
important. Glucose intolerance in uremics thus should not per se make sub- 
jects ineligible for definitive therapeutic programs since it frequently is not 
indicative of true diabetes mellitus. 

Laboratory of Behavioral Sciences. During the year the animal be- 
havior program was transferred from the Psychophysiology Section to the 
Section on Human Learning and Problem Solving, and the name of the latter 
was changed to the Section on Learning and Problem Solving. The goal of 
this reorganization was to facilitate the scientific integration of these two 
programs, and this interaction has already begun to occur. 

Previous studies of logical reasoning in the Baltimore Longitudinal 
Study Program had shown that men over age 60 made significantly more er- 
rors than younger men in reaching solutions to complex logical problems 
when the task required them to integrate the problem solution with the task 
analysis. The errors were of two kinds: (a) requests for information which 
was inferable from existing information; (b) redundant requests for informa- 
tion already given. In an analysis designed to separate analytic behavior 
from synthetic behavior, it was observed that the tendency of the older men 
to make redundant requests was greatly reduced. This finding indicates that 
the older men are more likely to make premature attempts to synthesize in- 
formation than are the younger men. and that this tendency results in repeat- 
ing solution attempts which have failed. 

Problem solving behavior is also being investigated in rats. Prev- 
ious research in this project has shown that older rats frequently are unable 
to solve complex mazes which are easily mastered by younger animals. 
Analyses of the maze -running behavior of the older animals showed a strik- 
ing tendency for these animals to make perseverative errors, i.e. , a given 
animal tended to repeat its errors at the same point in the maze. However, 
the specific point in the maze where this occurred was idiosyncratic. Ex- 
periments completed this year have shown that it is possible to train all old 
rats in this task merely by preventing them from making errors on early 
trials. In fact, old rats so trained learn more proficiently (reach criterion 
in fewer trials) than do young rats. 

It seems possible that the forcing of behavior in early trials in the 
animal study may be conceptually similar to breaking down the problem solv- 
ing task into an analytical and a synthetic component. If so, the results from 
this study and the previously cited study in man are concordant. This inter- 
pretation is supported by the observation that both the rats and the men made 
perseverative or redundant errors. If these two findings are related, then it 
should be possible to investigate the mechanisms of perseverative errors in 
great detail using the rat model. 

A study of learning and memory in man which was completed this 

FH-21 



year also underscores the important, deleterious role played by persevera- 
tive errors. When subjects are required to recall information in a series 
of independent tests of recall, the older men show a tendency to make errors 
of intrusion, i.e., they are likely to recall information from a previous test 
and to intrude those data into the present test. This tendency for persevera- 
tive errors — which in exaggerated form occurs in severely brain -damaged 
patients — suggests that older subjects generally have a deficit in the ability 
to retrieve selectively newly acquired information. Nevertheless, as cited 
earlier, it is possible to devise learning procedures which effectively 
circumvent this problem. 

The results of longitudinal (six year) analyses of performance on the 
Benton Revised Visual Retention Test, a measure of short-term memory for 
geometric figures, shows that longitudinal changes parallel cross-sectional 
ones. Subjects 40 years or less at the time of first testing show small in- 
creases in errors over a six-year interval; subjects in their 50s or 60s at 
time of first testing show a greater increase in errors, and subjects in their 
70s at first testing show very large increases in errors of recall. These re- 
sults are especially interesting because longitudinal age changes in perform- 
ance on psychometric tests where time pressure is absent have rarely 
corresponded to results with cross -sectional tests. 

A study of the effect of activity on longevity in mice was completed 
this year. Twenty-three month old male hybrid mice which were allowed 
ad lib access to an activity wheel for one month lived 10% (3 months) longer 
than did a matched control group. These differences were not due to weight 
loss among the active animals. Females did not show any gain in survival 
as a result of activity. 

The clinical significance of visceral conditioning depends in part upon 
the ability of the subject to control his abnormal responses when he is away 
from the laboratory and in part upon his ability to maintain normal respons- 
es in the presence of external stresses. The evidence for control away from 
the laboratory comes from a number of studies done in this laboratory over 
the past several years in which it has been shown that patients with abnormal 
heart rhythms can reduce the prevalence of these rhythms as measured by 
continuous, 10-hour monitoring devices. 

The evidence for the ability of subjects to maintain cardiac control in 
the face of external stressors was obtained from a study carried out with mon- 
keys during this year. Each of four monkeys was trained to slow its heart 
rate, and each also was trained to appreciate a warning click which signaled 
an impending, inescapable electric shock and to discriminate this click from |l 
a neutral click not associated with shock. Training in these two procedures ■ 
was given independently. The average increase in heart rate to the warning 
click was about 6 beats/minute and to the neutral click was 1 beat/minute. 
Blood pressure responses to the warning click were 9/7 mm Hg and to the 
.neutral click were -2/-1 mm Hg. All of these responses were highly re- 
liable. In the critical test of this study the clicks were sounded at a time 
when the monkey had to maintain slow heart rates. The question at issue was, 
"Would the animals slow their rates or would the click-stress prevail?" The 
results were clear. During the warning click period, heart rates fell about 



Fl*22 



I 



19 beats/minute and during the neutral click period heart rates fell about 12 
beats/minute. Thus the animals slowed their heart rates despite the ines- 
capable stressor (shock). Interestingly, the monkeys continued to respond 
to the clicks with similar pressor responses (0/-1 mm Hg to the neutral 
click and 4/2 mm Hg to the warning click) even though they had reversed 
their cardiac responses. 

In addition to the studies of the application of training techniques to 
the control of cardiovascular activity, a study was completed this year to 
evaluate the application of such training to the control of bowel function. A 
series of five adult and one child with histories of severe, intractable fecal 
incontinence (continuous daily soiling) were studied to determine if they 
could be taught to control anal sphincter activity. Four of the adult patients 
have achieved complete continence for periods ranging from one to three 
years, the 6 -year -old child has remained continent for about two months, 
and the other patient has shown some improvement. Since the training was 
done on an outpatient basis and since patients could learn control quickly 
(two to three, one -hour sessions), it seems possible that the technique can 
be readily adapted as a simple means of treating some cases of fecal 
incontinence. 



FH-23 



i 



4 






J 



NICHD ANNUAL REPORT 
July 1, 1972 through June 30, 1973 
Gerontology Research Center 
Laboratory of Behavioral Sciences 

There has been a significant reorganization within the Laboratory this 
year. The animal behavior program was transferred from the Psychophysiology 
Section to The Section on Human Learning and Problem Solving, and the name of 
the latter was changed to The Section on Learning and Problem Solving. 
The goal of this reorganization was to facilitate the scientific integration 
of these two programs, and this interaction has already begun to occur. 

Previous studies of logical reasoning in the Baltimore Longitudinal Study 
Program had shown that men over age 60 made significantly more errors than 
younger men in reaching solutions to complex logical problems when the task 
required them to integrate the problem solution with the task analysis. The 
errors were of two kinds: (a) requests for information which was inferable 
from existing information; (b) redundant requests for information. In an 
analysis carried out this year on data obtained from participants in the 
program in a study designed to separate analytic behavior from synthetic 
behavior, it was observed that the tendency of the older men to make 
redundant requests was greatly reduced. This finding indicates that the 
older men are more likely to make premature attempts to synthesize information 
than are the younger men, and that this tendency results in repeating 
solution attempts which have failed. 

Problem solving behavior is also being investigated in rats. Previous 
research in this project has shown that older rats frequently are unable 
to solve complex mazes which are easily mastered by younger animals. Analyses 
of the maze-running behavior of the older animals showed a striking tendency 
for these animals to make perseverative errors: i.e., a given animal tended 
to repeat its errors at the same point in the maze, however, the specific 
point in the maze where this occurred was idiosyncratic. Experiments 
completed this year have shown that it is possible to train all old rats in 
this task merely by preventing them from making errors on early trials: In 
fact, old rats so trained learn more proficiently (reach criterion in fewer 
trials) than do young rats. 

It seems possible that the forcing of behavior in early trials in the animal 
study may be conceptually similar to breaking down the problem solving task 
into an analytical and a synthetic component. If so, the results from this 
study and the previously cited study in man are concordant. This interpre- 
tation is supported by the observation that the rats and the men both made 
perseverative or redundant errors. If these two findings are related, 
then it should be possible to investigate the mechanisms of perseverative 
errors in great detail using the rat model. 

A study of learning and memory in man which was completed this year also 
underscores the important, deleterious role played by perseverative errors. 



FI-1 



When subjects are required to recall information in a series of independent 
tests of recall, the older men show a significant tendency to make errors 
of intrusion: i.e., they are likely to recall information from a previous 
test and to intrude those data into the present test. This tendency for 
perseverative ■errors--which in exaggerated form occurs in severely brain- 
damaged patients--suggests that older subjects generally have a deficit in 
the ability to retrieve selectively, newly acquired information. Never- 
theless as cited earlier, it is possible to devise learning procedures 
which effectively circumvent this problem. 

The results of longitudinal (six year) analyses of performance on the 
Benton Revised Visual Retention Test, a measure of short-term memory for 
geometric figures, shows that longitudinal changes parallel cross-sectional 
ones: Subjects 40 years or less at time of first testing show small increases 
in errors; subjects in their 50s or 60s at time of first testing show a 
greater increase in errors; and subjects in their 70s at first testing show 
very large increases in errors of recall. These results are especially 
interesting because longitudinal age changes in performance on psychometric 
tests where time pressure is absent have rarely corresponded to results with 
cross-sectional tests. 

A study of the effect of activity on longevity in mice was completed this 
year. Twenty- three-month old, male hybrid mice which were allowed ad-lib 
access to an activity wheel for one month lived ten percent (three months) 
longer than did a matched control group. These differences were not due 
to weight loss among the active animals since there were no weight differences 
between the groups. Females did not show any gain in survival as a result 
of activity. 

Studies were initiated in the Psychophysiology Section this year to learn 
whether patients with essential hypertension could be taught to lower their 
blood pressures significantly. One patient has been studied this year. 
The patient was a 60 year old woman who had mild essential hypertention 
of at least 25 years duration. Her systolic blood pressure during the previous 
year was 1A7 mm Hg. (based on 15 clinic visits). After three weeks of 
training in the laboratory to control her blood pressure, the patient was 
instructed to take home blood pressures. Her average systolic blood 
pressure at home over the next three weeks (based on 25 determinations/day) 
was 127 mm Hg. Furthermore, this patient also was able to lower her systolic 
pressure voluntarily from this level to 110 mm Hg. (based on 25, self- 
administered trials/day over a three week period). These data are encouraging, 
however, with only one patient it is far too soon to draw any conclusions 
other than that the project is worth continuing. 

The clinical significance of visceral conditioning depends in part upon the 
ability of the subject to control his abnormal responses away from the 
laboratory, and in part upon his ability to maintain normal responses in 
presence of external stresses. The evidence for control away from the 
laboratory comes from a number of studies done in this laboratory over the 
past several years in which it has been shown that patients with abnormal 
liriirt rhythms can reduce the prevelance of these rhythms as measured by 

FI-2 



continuous, 10 hour monitoring devices. The evidence for the ability of 
subjects to maintain cardiac control in the face of external stressors was 
obtained from a study carried out with monkeys during this year. 

Each of four monkeys was trained to slow its heart rate, and each also 
was trained to appreciate a warning click which signalled an impending, 
inescapable electric shock and to discriminate this click from a neutral, 
unshocked click. Training in these two procedures was given independently. 
The average increase in heart rate to the warning click was about 6 beats/ 
minute and to the neutral click was 1 beat/minute. Blood pressure responses 
to the warning click were 9/7 mm Hg. and to the neutral click were -2/-1 
mm Hg. All of these responses were highly reliable. In the critical 
test of this study the clicks were sounded at a time when the monkeys had 
to maintain slow heart rates. The question at issue was, "Would the animals 
slow their rates or would the click-stress prevail?" The results were 
clear: During the warning click period heart rates fell about 19 beats/ 
minute and during the neutral click period heart rates fell about 12 beats/ 
minute. Thus the animals slowed their heart rates despite the inescapable 
stressor. Interestingly, the monkeys continued to respond to the clicks 
with similar pressor responses (0/-1 mm Hg. to the neutral click and 4/2 
mm Hg. to the warning click) even though they had reversed their cardiac 
responses. 

In addition to the studies of the application of training techniques to the 
control of cardiovascular activity, a study was completed this year to 
evaluate the application of such training to the control of bowel function. 
A series of five adult and one child patients with histories of severe, in- 
tractable fecal incontinence (continuous daily soiling) were studied to 
determine if they could be taught to control anal sphincter activity. Four 
of the adult patients have achieved complete continence for periods ranging 
from one to three years, the six year old child has remained continent 
for ab»ut two months, the other patient has shown some improvement. Since 
the training was done on an outpatient basis and since patients could 
learn control quickly (two to three, one hour sessions), it seems possible 
that the technique can be readily adapted as a simple means of treating 
some cases of fecal incontinence. 



FI - 3 



Serial No. HD-AG7 (c) 

1. Gerontology Research Center 

2. Laboratory of Behavioral Sciences 

3. Section on Learning and Problem 

Solving 

4. Baltimore, Maryland 

PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Verbal Learning and Age 

Previous Serial Number: Same 

Principal Investigators: David Arenberg, Ph.D. (50%) 

Elizabeth A. Robertson, Ph.D. (68%) 
(E.O.D. 9-1-72) 

Other Investigators: Phillip Thorne 

Richard Allen, Ph.D. 

Cooperating Units: Baltimore City Hospitals 

Man Years: 

Total: 2,58 
Professional: 1.18 
Others: 1.40 

Project Description: 

Obj ec tives : General objectives are to discover conditions which affect 
age differences in acquisition and retention of verbal material in man for 
two purposes: (1) an understanding of the factors which enable us to 
modify age differences experimentally should provide valuable information 
about aging processes and about psychological processes of learning; and 
(2) an understanding of the conditions which are particularly beneficial 
or deleterious to learning by the old may have implications for training 
old workers and education for the elderly. 

Method Employed : Experiment XV was an age study of free recall in which 
lists of words were presented under three different conditions: (1) visual 
only, (2) visual plus passive auditory augmentation (the experimenter read 
each word aloud as it was presented), and (3) visual plus active auditory 
augmentation (the subject said each word aloud as it was presented). In 
free recall, the task is to recall as many words as one can after one 
presentation of the list. A similar study of free recall and age was 
conducted by Dr. Robertson in another laboratory (Experiment XVa). In that 
study, the auditory and visual presentations were independent; i.e., each 
list was presented in one modality. 



FI -4 



Experiment XVII is a study of aging and encoding and decoding aspects of 
memory. Ten, three-letter items are presented under one of three conditions 
(decoding uncertainty) for each subject, and the task is to recall the 
three-letter items. The three letters in each item can be encoded into 
a word by three different rules. Each subject is instructed about one of 
the rules which determines a particular level of decoding uncertainty. For 
example, the three-letter item "UFN" can be encoded into the word "FUN" by 
the following three encoding rules: (1) reverse the first two letters; 
(2) place the vowel in the middle, or (3) rearrange the letters. These 
three encoding rules provide different amounts of information for decoding 
the word to recall the original three-letter item: (I) no decoding 
uncertainty--only one possible decoding according to the encoding rule; 
(2) low decoding uncertainty-- two decodings possible; and (3) high decoding 
uncertainty--f ive decodings possible. After the ten items are presented, 
subjects are required to recall the letter groups and then the words; the 
number of words recalled and items recalled are the dependent measures. The 
experiment was designed to study the effects of decoding uncertainty on 
the recall of the letter-items and the words for five age groups of educated 
men. 

Experiment XXI is another free recall study; it was designed to specify the 
source of interference discovered in Experiment XV. In that earlier study, 
it was found that recall of words presented at the beginning of a list is 
impaired by hearing or saying all the words in a list. Similar results 
were found in another study of free recall in which lists were presented 
by audio tape or by slides. Although auditory presentations resulted 
in more words recalled overall, fewer words were recalled from the beginning 
of the list than for visual presentations. In a pilot study using mixed 
lists, an attempt was made to determine for words near the beginning of a 
list whether saying interferes with recall of the spoken word or with 
recall of previous words in the list. It was found that when subjects were 
required to say only designated words (and Look at the others), the "say" 
words were so compelling that few "look" words could be recalled. As a 
result, the interference effect the study was designed to investigate was 
entirely masked. Therefore, the procedure was modified. In the new study, 
the experimenter says specified words to provide the systematic variation 
of "look" and "listen" words. If this procedure does not permit the original 
effect to emerge, it may be necessary to use mixed lists of auditory and 
visual presentations rather than auditory augmentation. 

Experiment XXIV is a new study of recognition memory and aging. In a 

recognition memory task, information is presented to an individual and he 

is subsequently required to distinguish items previously presented from a 

larger sample of items. Recent studies in the literature indicate that, 

under some conditions, age differences are not found for recognition memory. 

This study was designed to investigate two types of recognition procedures 

to determine whether age differences would be found. In addition, the 

results should provide information about whether there is an age deficit 

in temporal discrimination in a recognition memory task. Two recognitions 

tasks are used with auditory or visual presentation of the words at either 

a fast (1 sec. per word) or a slow (4 sec. per word) rate. In both tasks, 

three nine-word lists are presented, and then a print-out of these 27 

words as well as nine other "distractor" words is immediately given to a 

FI -5 



subject. In one task, (recognition), the subject is asked to underline the 
words which were presented. In the other task (list discrimination), the 
subject is asked to designate to which list a word "belongs" by writing a 
"1" beside the word if it was in the first list, a "2" if it was in the 
second, a "3" in the third, and a "0" if the word had not been presented. 
The primary dependent measure is the number of correct responses adjusted 
for guessing. Each individual performs both of these recognition memory 
tasks under both input modality conditions at one level of the presentation 
rate variable. 

Experiment XXV is a new study of short-term recognition memory using signal 
detection procedures. On the basis of recent empirical data using signal 
detection methods, a mathematical model has been developed which provides 
measures of acquisition and decay for individual subjects. One such study 
comparing young and old adults has been conducted in collaboration with 
Dr. Richard Allen of Baltimore City Hospitals; visual presentation of 
digits was used. in Experiment XXV, auditory as well as visual 
presentation will be used because under some conditions, the elderly benefit 
more than the young from auditory inputs in memory performance. Recent 
studies have also shown ear differences which have been attributed to 
hemispheric differences in the brain. An exploration of the acquisition 
and decay paremeters for the two ears for subjects of different ages may 
provide some information about relative age deficits for the two hemispheres. 
In addition, laterality differences in memory may be related to how subjects 
handle information dichotically, i.e., when different information is 
presented simultaneously to the two ears. An earlier study (Experiment 
XXII) in this project showed that some people handle such information ear 
by ear (sequential processing) whereas others handle it by pairs as presented 
(simultaneous processing). The approach (sequential or simultaneous) and 
the effectiveness (memory performance) may be related to the relative 
effectiveness of the two ears in short-term memory. In these recognition 
memory tasks, a series of items (digits, letters, words) are presented 
followed by a cue item. The task is to indicate whether the cue item had 
been presented in the series, and a certainty rating is made for each response. 
The acquisition and decay perimeters are obtained by plotting a signal 
detection measure of memory against time (presented position). The slope of 
the line provides a measure of decay, and the intercept estimates strength of 
acquisition at time zero (at input before decay begins). 

Major Findings ; Analyses of errors in Experiments XV and XVa, studies of free 
recall using auditory and visual presentations, showed a rather unexpected 
result. In this task, old subjects committed more errors than young adults. 
Just the opposite has been found in rote learning tasks (in which lists are 
presented many times); omission errors characterize the performance of the 
old learner in such tasks, and frequency of commission errors tends to be 
low. The difference between the rote and the free-recall procedures which 
probably accounts for the differences in frequency of commission errors is 
the feedback. In rote tasks, a subject's responses are immediately confirmed 
(or disconf irmed) and he knows immediately whether he is right or wrong. For 
the old learner, it is likely that an omission error is more acceptable than 
a commission error. In free recall, no feedback is provided, and the increase 

FI-6 



in commission errors with age probably represents a retrieval deficit. In 
Experiment XVa, the commission errors were further analyzed, and it was 
found, in all four modality-rate conditions, that the old made more 
intrusion errors (words from previous lists) than the young (See Table 1). 
Of particular interest are the intrusion errors of words which were not 
recalled at the appropriate time (when the list which included that 
word had been presented). This means that the word was available (in store) 
but not accessible at the time it was supposed to be recalled. The fact 
that, in all four modality-rate conditions, old subjects commit such errors 
more frequently than young adults is further evidence of a retrieval deficit 
with age and also suggests that the temporal cues associated with those 
words have been lost (See Table 2). 

Experiment XVII is the memory study in which letter groups are encoded into 
words and later decoded back to the original order of letters. Preliminary 
results reported last year indicated that the effect of the encoding rule 
(which produces variations in decoding uncertainty) affects performance in 
recall of the letter groups for all five age groups, as predicted; but the 
age effect was consistently high only for the seventy-year-old group. 
The sample is now sufficiently large to look at the performance measures 
for subjects who did not comply with the instructions. They were categorized 
into those who recalled the words first (letter groups are supposed to be 
recalled before the words) and those who did not form words to help them 
remember the letter groups (See Table 3). For those who recalled the words 
first, more words were recalled; but for the two conditions with some decoding 
uncertainty, fewer letter groups were recalled, as would be expected from the 
response interference of writing the words first. The data are sparse, but 
it looks as if the men over 60 are particularly susceptible fo the response 
interference when there is some decoding uncertainty. Age declines are 
emerging for all three conditions for those subjects who did not form words 
to help them remember the letter groups. It is also interesting to note that 
for those subjects, for zero decoding uncertainty, fewer letter groups were 
recalled than for those who used words as instructed; but the reverse was 
true for high decoding uncertainty. In other words, for the decoding 
condition which provides little help in remembering the letter-groups, fewer 
letter groups were recalled by those who used that approach than by those 
who used their own method; this was particularly evident for groups under 50. 

Experiment XXI is the most recent study of free recall and age using the new 
procedure for mixed lists. In the new procedure, some words are read aloud 
by the experimenter as they are displayed, and other words are only displayed 
visually. Date collection was initiated using the new "listen" procedure, 
but the samples are too small to look at preliminary results. 

Experiment XXIV is a new study of word recognition. Data collection has 
progressed sufficiently to show age differences emerging, but the effects 
of modality, rate, and type of recognition are not yet clear. (See Tabic A) 

Experiment XXV is a new study of recognition memory using signal detection 
procedures. Data collection is expected to begin by the end of the reporting 
year. 



FI- 



Significance to Bio-Medical Research and the Program of the Institute : 
Learning is more central to experimental psychology than any other behavior 
of higher organisms and some of the most striking and consistently reported 
behavioral age differences in the gerontological literature have been 
found in verbal learning performance. The experiments in this project are 
designed to identify basic mechanisms of learning and retention and to 
measure changes in these functions that occur with age. 

Aging, viewed as a natural experiment in which many changes occur, permits 
one to identify a variety of processes which may be important underlying 
mechanisms of learning. In addition, knowledge about experimental variables 
which affect age differences will be valuable in developing techniques for 
optimizing learning of the older person. 

Proposed Course of Project ; Two manuscripts are being prepared for 
publication for Experiments XV and XVa. The sample for Experiment XVII 
will be completed during the next reporting year, data will be analyzed, 
and a manuscript will be written. Data collection will continue for 
Experiments XXI, XXIV, and XXV. Longitudinal results for serial and 
paired-associate learning (Experiments I and II) and visual retention of 
geometric figures will also be reported. The relations between the changes 
in these measures and blood pressure will be analyzed and reported. New 
studies of dichotic listening and tachistoscopic presentations (visual) will 
be initiated to study memory and modes of processing (sequential vs. 
simultaneous processing and effects of modality). 

Honors and Awards : Drs. Arenberg and Robertson were invited to write the 

first chapter of a book entitled "Education for Aging" edited by Drs. 

Mason and Grabowski; the chapter is entitled "The Older Person as a Learner." 

Publications: None 



FI- 8 



TABLE 1. MEAN NUMBER OF TOTAL INTER-LIST INTRUSIONS 
(Experiment XVa) 







MODALITY 




AGE 


AUDITION 


VISION 






20-39 


4.10 


6.40 




FAST RATE 
(I'VWORD) 


40-59 
60-79 


6.20 
10.80 


12.50 
16.60 






20-39 


2.30 


3.50 




SLOW RATE 


40-59 


5.30 


9.80 




(4'7W0RD) 


60-79 


10.50 


12.00 





FI -9 



TABLE 3. DECODING MEANS - APRIL, 1973 (EXPERIMENT XVII) 



UNDER 40 
40 's 
50 "s 
60's 

70's 

NOUNS 
FIRST 

UNDER 50 
50's 
OVER 60 

ANOTHER 
SYSTEM 

UNDER 50 
50's 
OVER 60 



NO 


UNCERTAINTY 


LOW 


UNCERTAINTY 


HIGH 


UNCERTAINTY 


N 


ITEMS WORDS 


N 


ITEMS WORDS 


N 


ITEMS WORDS 


16 


8.5 8.5 


15 


5.5 7.5 


14 


5.1 7.8 


24 


7.5 8.1 


18 


5.1 6.8 


19 


4,3 6.9 


23 


7.3 7.6 


24 


5.3 7.2 


22 


4.9 7.3 


19 


7.3 7.5 


21 


5.1 6.9 


16 


3.7 6.3 


21 


6.2 6.9 


21 


3.8 6.0 


20 


3.4 6.6 


7 


8.0 8.1 


13 


6.0 8.4 


6 


2.7 6.7 


12 


7.6 8.8 


8 


3.9 7.6 


4 


5.5 8.0 


14 


7.5 8.2 


6 


2.7 8.0 


6 


3.0 8.3 


5 


6.6 


8 


6.1 


24 


6.1 


7 


5.3 


6 


5.0 


13 


5.6 


8 


4.7 


11 


4.5 


11 


3.8 



FI -10 



TABLE 4. MEAN NUMBER OF CORRECT RESPONSES PER BLOCK 
(EXPERIMENT XXIV) 

AGE RECOGNITION LIST DISCRIMINATION 

20-39 x= 21.96 x^ ^^jg 

^0-59 x- 21.50 X. 11.21 

60+ x= 19.10 x= 8.85 



FI. 11 



TABLE 2. MEAN NUMBER OF TOTAL INTER-LIST INTRUSIONS NOT PREVIOUSLY RECALLED 
("RETRIEVAL" ERRORS) (EXPERIMENT XVa) 





AGE 


MODALITY 1 




AUDITION 


VISION 




FAST RATE 


20-39 


0.80 


1.10 




(1"/W0RD) 


40-59 


1,30 


4.50 






60-79 


3.60 


5.00 






20-39 


0.40 


0.40 




SLOW RATE 


40-59 


0.60 


1.50 




(^"/WORD) 


60-79 


2.40 


3.80 





FI- 12 



Serial No. HD-AG8 (c) 

1. Gerontology Research Center 

2. Laboratory of Behavioral Sciences 

3. Section on Learning and Problem 

Solving 

4. Baltimore, Maryland 21224 



PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Perceptual Retention and Age 

Previous Serial Number: Same 

Principal Investigators: David Arenberg, Ph.D. 

Elizabeth A. Robertson, Ph.D. 
(EOD 9-1-72) 

Other Investigators: None 

Cooperating Units: Baltimore City Hospitals 

Man Years: 



(157J 



Total: 0.25 
Professional: 0.15 
Others: 0.10 

Project Description: 

Objectives : The general objectives are to investigate the effects of 
interference in perceptual retention and in perception: (1) to determine 
whether aging results in increased susceptibility to interference; (2) to 
explore conditions which affect age differences in interference; and (3) to 
hypothesize and develop procedures for testing mechanisms which may account 
for the empirical findings. 



Methods Employed : In Experimen 
project, the basic retention pa 
presentations: initial, interp 
a specific period of time is a 
required to identify different 
provided. The periods the ligh 
0.20, 0.35, 0.62, and 1.10 sec. 
"one," "two," "three," and "fou 
confirmed or corrected. This i 
set which is to identify 32 per 
correctness of judgment is prov 
of incorrectly identified stimu 
1.10, 1.6., 2.14, and 2.68 sec. 



t V, as in all previous experiments in this 
radigm consists of three sets of stimulus 
olated, and final. A light or sound on for 
stimulus presentation and the subject is 
durations by the labels that have been 
t can be on in the initial and final sets are 

The four periods are verbally labeled 
r" in order, and eight practice judgments are 
s followed by the primary task of the initial 
iods during which no feedback about the 
ided. Performance is measured by the number 
li. The periods for the interpolated set are 

These are labeled, practice with feedback 



FI- 13 



is provided, and then interpolated stimuli are presented without feedback. 
The final set consists of 32 presentations of the periods used in the 
initial set, but no practice is provided. The primary data consist of the 
change in performance (number of incorrectly identified stimuli) from the 
initial to the final sets. The conditions of the experiment are determined 
by the procedures during the interpolated set. Previous experiments 
(I, II, & III) have shown consistently that errors increase more for old 
than for young men when the interpolated set consists of visually presented 
stimuli labeled "one," "two," "three," and "four." In a more recent study 
(Experiment IV), it was shown that at least part of the age difference in 
interference was due to confusion introduced by the use of the same labels 
for the interpolated and the other sets of stimuli. When the labels for the 
interpolated set were "four," "five," "six," and "seven," the age difference 
in interference was substantially reduced. In Experiment V, the labels used 
for the interpolated set and the modality (visual or auditory) of those 
stimuli were included as independent variables. 

Experiment VI is a new age study of speed of information processing. Stimulus 
pairs are presented for brief fractions of a second and two matching tasks 
are included: one is based upon physical identity, the other nominal identity. 
Under the physical identity conditions, the task is to affirm pairs such as 
AA and aa, but reject pairs such as Aa. Under nominal identity conditions, 
the task is to affirm pairs such as Aa, but reject pairs such as Ab. It is 
known that response times for physical identity conditions are shorter 
than for nominal identity, and the additional time required for the latter 
is attributed to the additional information processing required to affirm 
or reject a nominal match. In Experiment VI, it is hypothesized not only 
that an old group will be slower on both tasks, but that the difference bet- 
ween performance on physical and nominal matching will be greater for an 
old group than for a group of young adults. In addition, the pairs are 
displayed in different visual fields to explore differences in laterality 
(hemishperes of the brain). 

Major Findings ; In Experiment V, interference is measured under four 
conditions for the interpolated task (and each subject performs under one 
condition): (1) visual--same labels; (2) visual--dif f erent labels; (3) 
auditory--same labels; and (4) audi tory--diff erent labels (See Table 1). 
The two visual conditions have been included in previous studies in this 
project (Experiments I, II, III, and IV), and preliminary results are similar 
to the earlier findings; i.e., the old show greater susceptibility to the 
interfering effects of the interpolated task, but the age difference is 
reduced when confusability is minimized by using different labels for the 
interpolated stimuli. The auditory conditions are included for the first 
time in Experiment V, and the results emerging are rather unusual. As 
expected, the young groups are slightly less affected by the auditory 
interference. It was predicted that auditory stimuli would interfere less 
with visual judgments than would visual stimuli. For the old, however, the 
opposite result is emerging. Under both auditory interpolated conditions, 
the old are showing more interference than under the visual interpolated 
conditions; and this modality difference is particularly striking for the 
high-confusability condition in which the set of interpolated stimuli have 



FI- 14 



the same labels as the set of primary stimuli. 

Significance to Bio-Medical Research and the Program of the Institute ; The 
general idea that, with age, a person becomes more susceptible to interference 
is well entrenched in gerontological thinking and is often used to "explain" 
age differences in performance. The evidence for this idea, however, is 
sparse. It is the purpose of this project to explore the generality of the 
age-interference hypothesis for both non-verbal memory and perception. It a 
is important to know, for both theoretical and applied reasons, under 
which conditions the older individual is especially interference prone. 

Proposed Course of Project ; Data collection will continue for Experiment V 

and will be initiated for Experiment VI. Experiments planned in this ,. 

project will explore age effects of more basic perceptual interference ' « 

phenomena such as masking and disinhibi tion. " ^ 

Honors and Awards ; None ' 

Publications: None 



FI - 15 



TABLE 1. MEAN ERRORS (EXPERIMENT V) 







VISUAL 






SAME 


DIFFERENT 




Young 


Old 


Young 


Old 


Initial Errors 
Final Errors 


6.32 
10.89 


10.13 
16.50 


6.48 
10.33 


7.25 
12.44 


Difference 

N 


4.57 
19 


6.37 
8 


3.85 
21 


5.19 
16 



AUDITORY 

SAME DIFFERENT 

Young Old Young Old 

8.22 7.00 8.22 8,38 

12.28 17.88 11.39 14.50 

4.06 10.88 3.17 6.12 

18 8 18 8 



FI- 16 



Serial No. HD-AG6 (c) 

1. Gerontology Research Center 

2. Laboratory of Behavioral Sciences 

3. Section on Learning and Problem 

Solving 

4. Baltimore, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Problem Solving and Age 

Previous Serial Number: Same 

Principal Investigator: David Arenberg, Ph.D. (50%) 

Leonard M. Giambra, Ph.D. (66% - E.O.D. 8-14-73) 

Other Investigators: Phillip Thorne 

Cooperating Units: Baltimore City Hospitals 

Miami University of Ohio 
College of Notre Dame of Maryland 

Man Years: 

Total: 2.46 

Professional: 1.16 
Others: 1.30 

Project Description: 

Objectives ; The general goals are to explore and identify reasoning processes 
in man, to determine in what ways these processes change with age, and to 
develop techniques for reducing age deficits in reasoning performance. In 
this project, reasoning is studied by using problem-solving procedures in 
which on-going solution behavior can be observed and quantified. Experiments 
are designed to answer such questions as: (1) Is effectiveness in acquiring 
relevant information affected by aging? (2) Is effectiveness in synthesizing 
available information affected by aging? (3) What kinds of solution 
strategies are used and in what ways are they related to age? (4) How does 
imposing a memory load affect solution strategies for young and old adults? 

Methods Employed ; Experiments I and IV use the same equipment and the same 
problems, but the procedures are different. The display board for all 
problems includes nine lights on the periphery of a circle and a goal light 
in the center. The peripheral lights can be turned on by switches. In 
Experiment I, a subject's task is to light the goal light in a prescribed 
way. For each problem, a disk is superimposed on the light display panel. 
On the disk are arrows connecting various lights. An arrow indicates that 
one of three logical relations (previously explained to the subject) exists 



FI -17 



between the lights that arrow connects, but the specific relation is not 
identified. The solution of each problem requires lighting the goal light 
by a sequence of inputs in which only the switches for a specified set of 
three (of the nine) peripheral lights can be used. Time between inputs is 
controlled by the subject. In addition to correctness of the solution, 
performance is evaluated by determining whether an input (at the time it is 
made: (1) provides new information, (2) provides inferable information, 
or (3) is overtly redundant. In Experiment I, subjects are not required 
explicitly to identify the relation (meaning) of each arrow on the problem 
disk. This is the procedure typically used in these problems. A subject 
may reach a solution with great difficulty when one (or more) of his 
implicit identifications of the relations is incorrect. This will sometimes 
appear to be a decrement in synthesis performance, when in fact the difficulty 
should be attributed to the mis-identification which is an analysis error. 
Experiment IV was designed to avoid the confounding of analysis and synthesis 
performance. What is a one-task problem in Experiment I was transformed 
into a two-task problem. The first task is to identify the relations (the 
meanings of the arrows between lights). After this is completed, the subject 
is provided with all the correct identifications, and these are reviewed 
before he is permitted to work on the solution to the problem. As a result, 
synthesis performance, i.e., putting the information together to reach a 
solution, is always based upon accurate and complete information, and is 
uncontaminated by poor performance during analysis (identification of arrow 
meanings). In addition to correctness in each task, effectiveness of 
performance can also be measured. For the analysis task, one index of 
effectiveness is the number of correct identifications. For the synthesis 
task, all inputs other than the minimum required to attain the solution are 
superfluous. The number of superfluous inputs is an index of ineffectiveness 
in synthesis performance. 

a. 

The term "input" refers to the situation in which one or more lights 

are on and a subject indicates he wants the results of those lights. Those 

lights are then extinguished, and according to the logical relations of the 

arrows pointing from those lights, the resultant lights are turned on. 

Experiment VII is the most recent in a series of experiments designed to 
explore an unustial (but highly replicable) finding from cross-sectional 
analyses of the longitudinal study of concept identification (Experiment V). 
Under conditions of high initial information it was found in Experiment V 
that effectiveness in identifying disjunctive concepts was superior to 
effectiveness in identifying logically equivalent conjunctive concepts. 
Superior performance for disjunctive over conjunctive concepts is virtually 
unknown in the literature, but it was found for four independent age groups 
under the conditions of the longitudinal study. Attempts have since been 
made to identify variables and procedures which contributed to this unusual 
finding. In Experiment VII, the primary variable is what we have termed 
"linguistic congruence." In Experiment V, the positive designation is 
always "Died" which has negative linguistic properties. In Experiment VII, 
designations for half the sample are also linguistically incongruent, wh«r«as 
for the other half the designations are congruent with their linguistic 
properties; e.g., "Well" is the positive designation and "Sick" is the 



FI- 18 



negative. Logically equivalent conjunctive and disjunctive concepts with 
high and low initial information gain are included. 

Experiment VIII is a new concept-identification study which departs sub- 
stantially from all the others in this project. The intent is to explore 
subjects' preferences between information expressed conjunctively and 
information expressed disjunctively when the choices are equivalent in 
information gain. It is possible to provide choices with equivalent (or 
non-equivalent) information gain by modifying the equivalence-of-information 
method developed in this Section. Two experiments have been designed in 
which each preference in a sequence is represented on two variables--conjunc- 
tive or disjunctive, and positive or negative; and the resultant vector of 
an entire sequence of preferences provides a preference summary. Each choice 
in the sequence to establish an overall preference is between two alternatives 
with equivalent information. In the first preference study, in addition to 
the preference problems, two types of performance problems were included. 
In one, four examples were presented simultaneously for each problem, with 
all designations consistent in one dimension, i.e., all four designations 
were conjunctive (two positive and two negative), all were disjunctive 
(two positive and two negative), all were positive (two conjunctive and two 
disjunctive), or all were negative (two conjunctive and two disjunctive). 
All four such problems are inf ormationally equivalent, but shorter times to 
reach a correct solution were predicted when the consistent element was 
prevalent in the choices during the preference problems. In other words, it 
was predicted that a preference for a particular type of designation would 
be reflected in faster performance on problems with designations like those 
preferred than on problems with non-preferred designations. In the second 
type of performance problem, only one example with its designation was 
given, and the task was to assign all Ik pairs (solution possibilities) into 
those that were viable (consistent with the example) and those that were 
not viable (inconsistent with the example). Again it was predicted that 
performance would be related to preference. In addition to the preference 
and performance tasks, subjects were asked to rate the designations on 
informativeness, ease in handling the information (difficulty), and ease 
in keeping track of the information (memory). 

Experiment IX consists of several new studies of concept identification in 
which the interval between examples is a variable. In all previous studies 
of concept identification in the project, the memory load was minimized by 
providing the subject with a written record of the examples and their 
designations throughout each problem (designations are feedback messages 
indicating whether an example is positive, i.e., exemplifies the concept, or 
negative, i.e., does no,t exemplify the concept). In Experiment IX, memory 
load is manipulated by including as variables time between examples, and 
the number of examples on display at any one time. For all of the studies, 
examples consist of displays with three "dimensions" and each dimension can 
assume one of three "values." The dimensions are number, color, and shape. 
The number can be one, two, or three; the color can be red, green, or black; 
the shape can be a circle, square, or plus sign. An example consists of 
a combination of a number, a color, and a shape. In the first study, only 
conjunctive concepts are used. Subjects are instructed that two dimensions 



FI - 19 



are relevant, but are not told that the concept rule is conjunction, i.e., 
two attributes must both be present for an example to be positive. The 
concept could be three and square, two and red, green and circle, or any of 
the 27 possible pairs of attributes. After each example, the subject 
responds with one of the two designations (positive or negative), and then 
the correct designation is presented. All 27 possible examples are presented 
sequentially, and the number of correct designations is the primary dependent 
measure. In the first study, the interval between examples is 1, 9, 17, or 
25 seconds, and three problems are presented to investigate the learning 
factor. The other independent variables are age and sex. 

Experiment X is another group of new cgncept studies, similar to Experiment 
IX, but instead of varying the interval between examples, the primary 
variable is the designation label. Instead of "positive" and "negative," 
or "yes" and "no," the feedback message can be "A" or "B,"or "A" or "not A," 
for example. Other independent variables are age, sex, concept rule, and 
the effect of experience (comparing first, second, and third problems). 

Major Findings ; Preliminary results of Experiment IV (N=173), which is a 
modification of Experiment I (a study of logical problem solving), were quite 
similar to the cross-sectional results of the earlier study (See Table 1). 
The proportion of successful solutions declined with age; and for those who 
were able to solve a problem, the number of noninformative inputs increased 
with age. In addition, due to the procedural changes, it was possible to 
look at the analysis and synthesis parts of the problems separately in 
Experiment IV. In the analysis parts, very few errors were made in identify- 
ing arrow meanings by any age group. Although the number of noninformative 
inputs increased with age, the differences were small in analysis. Noninform- 
ative inputs can be either inferrable or overtly redundant. In Experiment 
I, a substantial portion of the age differences in noninformative inputs were 
attributable to the increase in overtly redundant inputs with age. The 
results of Experiment IV indicate that those overtly redundant inputs in 
Experiment I were synthesis difficulties and most likely represented incorrect 
solution attempts which were repeated in whole or in part. 

Experiment VII is the concept-identification study in which linguistic 
congruence of the feedback messages were varied; i.e., the positive 
designation could have positive or negative linguistic properties. It was 
found that students from nearby high schools were inappropriate for this 
study, and data collection was initiated for a sample of college students. 

Experiment VIII, the first study of concept preference, found no strong 
preferences among highly intelligent college students. It is likely that- 
a less select sample of procedural changes requiring an additional memory 
load would yield preferences, especially if the performance was timed. 
Ratings on all three scales were found to divide examples quite consistently 
into high information and low information. Conjunctive positive examples 
and disjunctive negative examples are highly informative (permit the 
elimination of a large number of possible solutions), and they were rated 
not only more informative than conjunctive negative and disjunctive positive 



FI- 20 



examples, but easier to handle and easier to keep track of. No differences 
in ratings were found between the two highly informative examples or between 
the two low-information examples. 

Data collection is about to begin for Experiment IX, a concept study in 
which a memory load is required and the interval between examples is varied. 

In the first study in Experiment X (Xa), five pairs of designations were "A" 
and "B," "A" and "not A," "+" and "-•," "*" and "0," or "Died" and "Lived." 
The concept rule was conjunction, disjunction, conditional, or biconditional. 
Data have been collected for all the young subjects (N=160), but analyses 
have not been completed. 

Significance to Bio-Medical Research and the Program of the Institute ; 
Reasoning is among the most prized behaviors of man and among the most 
elusive for experimental study. In this project, methods have been and 
will be developed for obtaining quantifiable measures of step-by-step 
performance on reasoning problems. Some of these methods also provide 
patterns of responses which represent strategies in solving such problems. 

Measures are obtained in current experiments to study changes in reasoning 
processes with age. These studies, in addition to identifying basic 
reasoning processes, should indicate how pervasive reasoning deficits are 
with age, whether education and cognitive activity mitigate such deficits, 
and what techniques could be used to minimize decline in reasoning. 

Proposed Course of Project ; When the ten or twelve subjects who are due 
for a repeat session in Experiment I are seen this year, that longitudinal 
sample will be completed; and a manuscript describing that study will be 
prepared for publication. It will be the first such longitudinal study 
in the gerontological literature. Data collection will continue for 
Experiment IV, and that sample will be closed, at which time a manuscript 
will be prepared for publication. Data collection will continue for 
Experiments VII, IX, and X, and another concept-preference study will be 
designed to follow up Experiment VIII. 

Ho nors and Awards: Dr. Arenberg was appointed chairman of the Program 
Committee for the Division on Adult Development and Aging of the American 
Psychological Association for its meetings in Montreal in August, 1973. 

Publications: None 



FI -21 



TABLE 1. MEANS OF PROBLEM SOLVING MEASURES (EXPERIMENT IV) 







ANALYSIS 








SYNTHESIS 








NO. 


NONIN- 


REDUN- 


PROPORTION 


SUPER- 






N 


CORRECT 


FORMATIVE 


DANT 


SOLVED 




FLUOUS 


REDUNDANT 


PROBLEM I 


















Under 40 


21 


10.3 


1.95 


.05 


.95 




2.30 


1.00 


AG's 


46 


9.6 


3.31 


.24 


.85 




4.41 


2.15 


50's 


44 


10.2 


3.30 


.34 


.91 




5.48 


2.47 


60 's 


38 


9.7 


3.41 


.35 


.76 




4.17 


1.76 


70's 


38 


9.5 


4.36 


.81 


.68 




4.46 


1.88 


80's 


6 


7.7 


4.67 


1.33 


.00 











PROBLEM II 
















Under 40 


20 


9.8 


1.72 


.00 


.90 


1.72 


0.44 


40' s 


42 


9.7 


2.40 


.03 


.88 


3,95 


1.14 


50's 


40 


9.5 


2.60 


.03 


.88 


4.17 


1.51 


60's 


25 


9.5 


2.78 


.04 


.76 


10.24 


5.76 


70's 


26 


9.2 


3.95 


.45 


.54 


9.43 


3.71 


80 's 
























FI-22 



Serial No. HD-LBS-2 

1. Gerontology Research Center 

2. Laboratory of Behavioral Sciences 

3. Section on Learning and Problem 

Solving 

4. Baltimore, Maryland 

PHS-NIH 

Individual Project Report 

July 1, 1972 - June 30, 1973 

Project Title: Daydreaming and Aging 

Previous Serial Number: None 

Principal Investigator: Leonard M, Giarabra, Ph.D. (22%; E.O.D. 8-14-72) 

Other Investigators: None 

Cooperating Units: Baltimore City Hospitals 

Community College of Baltimore 
College of Notre Dame of Maryland 
Miami University of Ohio 

Man Years: 

Total: .45 

Professional: .22 
Others: .23 

Project Description: 

Obj ec tives : To daydream is to have spoataa •■> js thoughts that are relati- 
vely unrelated to the task at hand. Such thought processes are usually 
considered the preserve of the adolescent and young adult. The purpose 
of this study is to relate frequency and content of daydreams to age 
across the entire adult range. With changes of age come changes in 
attitudes, responsibilities, life style, experiences, potentialities, 
leisure time, demands on the individual's attention, and physiology. It 
is hypothesized that the examination of daydreaming tendencies of various 
ages will reveal the effect of these age related variables upon the 
thinking processes of individuals. Thus the primary objective of this 
project is to examine daydreaming tendencies to determine age differences 
and changes in this type of thinking process. 

Methods Employed : The daydreaming tendencies of every participant in 
this project is obtained through self report via the laiaglnal Processes 
Inventor/ developed and copyrighted by Jerome L. Singer and John S. Antrobus. 
The Tmaginal Processes laveiitory (IPI) is a 344 item questionnaire which 
has both specific aru] general items concerning daydreams, night dreams, 
fantasy, -itc. There are two groups that are contributing to the data pool. 



FI -23 



The first group consists of volunteers recruited in the community and at 
colleges and universities. The second group consists of the participants 
in the Baltimore Longitudinal Study of the GRC. The first group consists 
of individuals from a wide variety of ages, sex, income, race, religion, 
ethnic background, and socioeconomic class. The longitudinal group is all 
male and relatively homogeneous with regard to income, race, religion, 
ethnic background and socioeconomic class. Both groups allow for a cross- 
sectional comparison of age differences in daydreaming while only the 
longitudinal group allows for a truly longitudinal determination of age 
related change (if any). Since this project began in December of 1972, 
no longitudinal information is available. 

A total of 135 males were divided into five age groups: 17-22 
(n=88), 27-35 (n=8), 38-49 (n=ll), 51-62 (n=18), and 66-77 (n=9). The 
youngest age group was made up of students of Miami University. The 
remaining groups were composed of participants in the Longitudinal Study 
of the Gerontology Research Center of the National Institute of Child 
Health and Development and represent the first portion of a continuing 
effort. 

Major Findings ; Since this must be a brief report and since the number of 
S^s was relatively small, the results of the questionnaire will be portrayed 
by looking at only some selected items. Some of these items along with 
the proportion of each age group selecting each items' five alternatives 
are given in Table 1. 



Table 1 goes about here 



Attitudes toward daydreaming - Item 177 indicates that at least 727o of 
every age group considers daydreaming in a positive light; furthermore that 
the youngest group has the most negative feelings toward daydreaming. It 
would be reasonable to conclude that the Ss would be honestly answering 
the questionnaire since daydreaming appears to be a socially desirable 
behavior which one would not conceal. 

Daydreaming frequency/duration - Items 1, 2, and 3 are pertinent. Item 1 
indicates that daydream frequency is a maximum at ages 17-22 and slowly 
decreases to the 38-49 age where it levels off into the 51-62 age then 
slightly decreases for the 66-77 age. Note that even at age 66-77 
daydreaming occurs a few times or more a day for 667„ of the group. Item 
2 indicates that the percent of waking thoughts in daydreams or fantasies 
parallels frequency for the five age groups except that the 66-77 age 
group tends to devote a greater percentage of time than the 51-62 age group. 
Item 3 parallels item 2. One might conclude that daydreaming does steadily 
decline with age but that with retirement age there is a slight increase 
of daydreaming. Most importantly, however, one must conclude that for the 
niajoritv daydreaming continues throughout life. 

FI -24 



Time orientation of daydreams - Items 28, 68, 69, 99, 146, 239, and 295 are 
pertinent here. It is commonly believed that most daydreams are future 
oriented and with increasing age (and a decreasing future) that future 
oriented daydreams would diminish and past-oriented daydreams would increase. 
Daydreams about "different ways of finishing things I still have to do in 
my life" were highest for the two youngest groups and lowest for the 38-49 
group with a resurgence toward the higher levels for the two oldest age 
groups. Reliving "happy or exciting experiences in my daydreams" was 
highest for the youngest group and sharply declined for the 27-35 group 
followed by a monotonic decline for the remaining age groups. Daydreams 
"closely related to problems that come up in daily life" occurred most 
frequently with the youngest group and least often with the eldest group; 
the ages between showed a decline except for a resurgence at 38-49. 
Daydreams which are "so much like the things I do from day-to-day" occurred 
most often in the 38-49 age span and declined with decreases or increases 
of age except that the eldest group had a greater tendency than the second 
eldest. Events which happened over a year ago were most often in the 
daydreams of the eldest closely followed by the youngest group; this type 
of daydream occurred least often in the 27-35 and 51-62 age spans with the 
38-49 age span near minimum. The greatest concern with the present occurred 
in the daydreams of the youngest and eldest age groups with the 51-62 
group showing the least concern. These results were not completely consis- 
tent but it appears that a past orientation in daydreams predominates in 
both the youngest and oldest age groups with the middle age groups (especi- 
ally the 38-49) being present oriented in their daydreams. It is true 
however that the tendency to have daydreams about the less recent past was 
weakest in the youngest group with a gain in strength with increasing age 
until maximum with the eldest group. Finally one must conclude that 
looking toward the future in daydreams is an important and pervasive orien- 
tation of daydreaming in the elderly. 

Daydreams regarding personal expectations - Items 57, 115, and 199 are 
pertinent. Daydreams about exceeding parents' expectations were at a high 
level for the youngest group; for the other age groups such daydreams were 
at a relatively low level finally dropping to near zero for the 66-77 age 
group. Daydreaming about being promoted to a better position was highest 
for the 17-22 age span and lowest for the 66-77 age span with a steady 
decline between. The tendency to imagine oneself as attractive to the 
opposite sex was about equally strongest at 17-22 and 27-35 followed by a 
diminuation with succeeding ages to a minimum for the eldest. Essentially, 
then increasing age brings with it a decreasing frequency of daydreams 
which are achievement specific, perhaps brought about by the esteem deflating 
experiences which are likely to pile up with age. 

Daydreams about impossible and heroic things - Daydreaming about "utterly 
impossible situations" were uncommon at all ages with the 17-22 age span 
showing the greatest tendency and the 27-35 age span the least. Items 
270 and 317 are about heroic daydreams and parallel one another. The 
tendency was strongest in the youngest group with a sharp decline for the 
27- T) age group followed by a further decline to a minimum in the 38-49 
and 51-()2 age spans followed by a small increase with the 66-77 age span. 
It would seem that the post-school jump in responsibilities, a job, and 
family would account for this. 

FI . 25 



TABLE I 
The Proportion of Each Age Group Selecting Each Alternative of Selected Items 
Item Number and Item Age (in years) 

17-22 27-35 38-49 51-62 66-77 



Daydreams or fantasies make up: 

(a) No part of my waking thoughts 

(b) Less than 10% of my waking thoughts 27 

(c) At least 10% of my waking thoughts 39 

(d) At least 25% of my waking thoughts 26 

(e) At lest 50% of my waking thoughts 6 



3. I would describe myself as: 

(a) Someone who never daydreams 

(b) Someone who very rar ly daydreams 

(c) Someone who tends toward occasional 
daydreaming 

(d) Someone who tends toward moderate 
daydreaming 

(e) A habitual daydreamer 

57. In my daydreams, I exceed my parents' 
expectation: 

(a) Definitely not true 

(b) Usually not true 

(c) Usually true 

(d) True 

(e) Very true 

68. I often relive happy or exciting 
experiences in my daydreams: 

(a) Definitely not true 

(b) Usually not true 

(c) Usually true 

(d) True 

(e) Very true 

115.1 daydream about being promoted to a 
better position: 

(a) Definitely not true 

(b) Usually not true 
((.' ) Usual ly true 

((I) True 

(e) Very true 









11 


11 


37 


45 


61 


44 


37 


45 


22 


22 


25 


9 


5 


22 
























5 





4 


12 


18 


11 


22 


31 


50 


63 


50 


33 


48 


25 


18 


27 


33 


14 


12 





5 


11 



2 





36 


47 


50 


42 


62 


54 


23 


50 


29 


37 


9 


17 





18 








11 





7 















3 


12 





17 


12 


9 


25 


36 


35 


50 


23 


50 


54 


35 


25 


35 


12 


9 


11 


12 


28 















12 


12 


27 


41 


25 


44 


50 


54 


29 


62 


19 


25 


18 


23 


12 


20 


12 





5 





3 















FI - 26 



Item Number and Item 



146.1 often daydream about events that 
happened over a year ago: 

(a) Definitely not true 

(b) Usually not true 

(c) Usually true 

(d) True 

(e) Very true 

239.1 daydream more about events that have 
already happened than about things in 
the future: 

(a) Definitely not true 

(b) Usually not true 

(c) Usually true 

(d) True 

(e) Very true 

270.1 picture myself risking my life to 
save someone I love: 



Age (in years) 
17-22 27-35 38-49 51-62 66-77 



3 





9 


17 





32 


87 


45 


58 


25 


31 





45 


11 


50 


23 


12 





11 


25 


7 















5 


12 


9 


5 





47 


50 


54 


52 


62 


29 


25 


27 , 


35 





12 


12 


9 


5 


37 


4 















(a) Definitely not true 

(b) Usually not true 

(c) Usually true 

(d) True 

(e) Very true 



2 


12 


36 


33 


37 


22 


62 


45 


44 


25 


32 


25 


18 


16 


37 


28 








5 





13 















vr 



FI - 27 



Significance to Bio-Medical Research and Program of the Institute ; The 
study of daydreaming is fundamentally a study of thought processes. Further- 
more there has been little research in this area of thought processes 
especially across the entire adult age range. In order to understand fully 
the thought processes of man, the total spectrum of those processes need to 
be examined. In addition, one must know how this wide spectrum is affected 
by aging. Thus the study of daydreaming in adults, along with other 
variables such as differences in age, socioeconomic status, attitudes, etc. 
may help us understand the fundamental processes which underly them all. 

Proposed Course of Project; Data collection will be continued from the 
community to broaden the population base and from the longitudinal participants 
on their next and subsequent visits to obtain measures of changes with age. 

Honors and Awards ; Dr. Giambra was invited to lecture to the members of the 
Psychology Club of Loyola College (December, 1972), to the members of the 
Senior Citizens Group (Bykota House) in Towson, Maryland (February, 1972), 
and to the Students of Community College of Baltimore (April, 1973). 

Publications ; Giambra, L.M. Daydreaming in males from seventeen to seventy- 
seven; A preliminary report. Proceedings of the 1973 Convention of the 
American Psychological Association (in press). 



VI -28 



Serial No. HD-LBS-1 

1. Gerontology Research Center 

2. Laboratory of Behavioral Sciences 

3. Section on Learning and Problem 

Solving 
A. Baltimore, Maryland 



PHS-NIH 
Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Behavioral Genetics and Aging. 

Previous Serial Number: None 

Principal Investigator: Charles L. Goodrick, Ph.D. (407o) 

Other Investigators: None 

Cooperating Units: Baltimore City Hospitals 

Man Years: 



Total: 


.80 


Professional: 


.80 


Others 


.00 



Project Description: 

Objectives : The principal objectives of this project are (I) to determine 
group differences for behavioral traits and longevity among inbred strains 
of mice; (2) to determine: (a) heritability (degree of genetic determination 
vs. degree of environmental determination); (b) mode of inheritance (e.g. 
over-dominant, dominant, intermediate, or recessive); and (c) number of 
segregating units (gene blocks) controlling a particular trait (e.g. 
longevity); and (3) to examine relative behavioral differences among mouse 
strains as aging progresses. 

Other objectives include analyses of the influence of diet (e.g. protein 
available) on behavioral traits, growth, and longevity; and determination 
of single-gene influences upon behavioral traits, growth, and longevity. 

Methods Employed: Inbred mice(C57BL/6J and A/J)of a high degree of homozygosity 
are maintained under uniform environmental conditions. The animals are 
tested behaviorally during one period of their life span, e.g., when mature, 
mature-old, or aged. Old age is determined as the 50% mortality point for 
groups maintained throughout their life span. Statistically reliable 
techniques have been developed to determine behaviors relevant to natural 
selection such as exploration, general activity level, emotionality, simple 
or complex problem solving ability and taste preference. The use of 



FI-29 



segregating groups allows an estimate of the mode of inheritance, e.g., 
dominant or intermediate, and the number of gene blocks or segregating 
units controlling behavioral traits or life span. The diets used for 
studies in which protein intake is varied among groups of inbred and hybrid 
mice are isocaloric synthetic diets obtained from General Biochemicals, 
Chagrin Falls, Ohio. Deprived animals receive 47o casein in their diets 
whereas the control group receives a 267o casein diet. Numerous kinds of 
mutant mice are maintained on the C57BL/6J background at the Jackson Memorial 
Laboratories; Bar Harbor, Maine. Our work has concentrated on the albino, 
beige, yellow and obese mutations. 

Major Findings : 

A. An experiment has been completed to determine the effects of aging 
and genetic constitution upon behavior and longevity for A/J and C57BL/6J 
inbred mice. A/J mice are albino (white coat), short lived behaviorally 
inactive mice, while C57BL/6J mice are melanic (black coat), long lived, 
behaviorally active mice. Both groups die of nonspecific causes. Groups 
of A/J, C57BL/6J, Fi and F2 mice have been tested on a number of behavioral 
measures with an N at least 16 per group at 5, 14, and 23 months of age, 
and at the time of advanced old age for the four groups: A/J (23 mo.) 
C57BL/6J (26 mo.), F^ and F2 (29 mo.) at the approximate time of senility 
or 507o mortality. Behavioral data were collected using standard tests 
developed in our laboratory to determine: (1) sensory discrimination, (2) 
preference for ethyl alcohol, (3) performance and retention of a learned 
operant response, (4) exploration behavior, and (5) emotionality. These 
data are in the process of analysis. 

B. An experiment was initiated to examine the generality of mode of 
inheritance for the behavioral traits of exploration behavior, emotionality* 
and alcohol preference using mature-young mice of four inbred strains. The 
inbred strains were A/J, BALB/cJ, C57BL/6J, and DBA/2J, while the hybrid 
groups were AxB, AxC, AxD, BxC, BxD, and CxD, and both males and females were 
tested for each of the ten groups (total N=360). This experiment was 
necessary because one of the major drawbacks to our studies of aging and gene- 
tics using A/J and C57BL/6J mice was the possible lack of generality for any 
results obtained. For exploration behavior, it was hypothesized that 
exploration activity is inherited in the same manner for all six Fi hybrid 
groups, and the specific mode of inheritance is intermediate. A diallel 
genetic analysis of combining abilities has been completed for this measure. 
This analysis provides strong evidence that the general mode of inheritance 

is similar for all Fj^ groups because GCA (general combining ability) was 
statistically significant for both male and female groups, while SCA 
(specific combining ability, indicative of differing modes of inheritance), 
was not statistically significant for male or female groups. The mode 
of inheritance was intermediate as hypothesized. 

C. A number of groups of mice are being currently maintained to obtain 
mortality data. Our early studies used A/J, C57BL/6J, F, , and F2 groups for 
studies of behavioral differences for populations which differ with respect 



FI-30 



to genetic constitution and age. In addition to behavioral measures, 100 
animals for each group were used to determine average longevity. In order 
to obtain additional information and determine the possible generality of 
our earlier findings, we are maintaining populations of A/J, BALB/cJ, C57BL/ 
6J, and DBA/2J inbred mice, and the six possible Fj^ hybrid groups. This 
study will yield information with respect to longevity of these inbred 
strains, and additional information with respect to the mode of inheritance 
of longevity. Groups of four mouse mutations with littermate controls 
are also being maintained to determine longevity, all on the C57BL/6J 
background. These four mutations (Bg, A, Y, and ob) all differ from 
controls on the major dimension of body weight. Further data are also being 
obtained with regard to the body weight and longevity of A/J, C57BL/6J, 
and Fi hybrid groups fed isocaloric synthetic diets which differ in protein 
content. These longevity projects will continue for an additional 24 
months. 

D. A series of experiments is in progress concerned with growth, 
longevity, and behavior as a function of level of dietary protein. An 
experiment has been completed that was designed to determine the effect of 
environment and dietary protein upon growth and behavior of inbred and 
hybrid mice. The subjects were A/J, C57, and Fj^ hybrid mice (N=300, 100/ 
group). At 5-weeks of age the mice were weighed and placed on either a low 
or normal protein diet (N=50 low protein and N=50 normal protein for each 
group). The average weight for each group at this time did not deviate 
significantly from 15 grams. The test diets were synthetic, isocaloric 
diets which contained either 267o casein or WL casein as the source of 
protein. Each subgroup (N=50) was housed in small suspended cages (1 per 
unit, N=14; 2 per unit, N=16) or large clear plastic cages with 5 mice 
per cage. In addition to measures of body weight, after the age of 15 weeks 
each mouse was given 2 tests, one of forced exploration and emotionality, 
the other of free exploration and emotionality. The major results of 
this experiment were the following. Group differences in weight were highly 
statistically significant, F (2,234)= 41.95, £<.01. A/J mice were lowest 
in body weight, C57BL/6J mice were intermediate in body weight, and Fi 
mice had the highest body weight. Mice maintained on low protein diets had 
lower mean body weight than mice maintained on normal protein diets, F 
(1,234)= 840.18, £<.01. The Group x Diet interaction was statistically 
significant, F (2,234)= 5.63, £<.01, because differences between low and 
normal protein groups were less for A/J mice than for C57BL/6J or Fj^ mice. 
The interaction of Diet x Caging was also statistically significant, F 
(2,234)= 24.43, £<.01. This interaction was due to an effect which 
occurred for all three mouse groups. For normal protein groups, mean body 
weight was greater for mice which were group caged than for isolated mice, 
while for low protein groups, mean body weight was lower for mice which 
were group caged than for isolated mice. 

For forced exploration scores, group differences were highly statistically 
significant, F (2,200)= 637.54, £<.01, with A/J scores low, C57BL/6J scores 
high, and F]^ scores intermediate. Groups given low protein diets had higher 
scores than groups given normal protein diets, F ( 1 , 200)=4. 75, £<.05, and 
mice which were group caged engaged in less exploration than singly caged 
mice, F (2,200) = 17.97, £<.01. 

FI-31 



The Group x Diet interaction was statistically significant, F(2,200)= 23.08, 
£< .01, due to larger differences for A/J than for F]^ mice and a reversal 
for C57BL/6J groups as a function of diet. All low protein groups of A/J 
and F]^ mice had higher activity scores than normal protein groups, while 
C57BL/6J normal protein groups all had higher scores than low protein 
groups. An interaction was obtained between Group and Caging F (4,200)= 
4.56, £< .01. The decrease in exploration as a function of increasing 
number of mice per cage was greatest for the C57BL/6J group, smaller for 
the F]^ group, and not obtained for the A/J group. Scores for all F]^ 
groups were not significantly different from the midparental values (Pi + P2). 

^ 2 J 

For the analysis of scores of free exploration (C57 and F,), both main 
effects of Group and Diet and their interaction were statistically significant. 
Group X Diet F (1,132)= 28.33, £< .01, Table 1. Hybrid groups fed low 
protein diets had significantly higher free exploration scores than hybrid 
groups fed normal protein diets, but C57 dietary groups were not significantly 
different. Caging was not a significant variable for this test, and the 
other interactions were not statistically significant. Although the Fj^ 
normal protein groups all obtained mean scores intermediate in MI, the MI 
for all low protein groups was partial dominance, with means for groups 
caged 1 or 5 per cage significantly greater (£ < .05) than midparental 
values. 

Analyses of emotionality scores obtained during both exploration tests (free 
and forced, A/J and F, only) indicated that open field emotionality was 
greater with increasing numbers in the cage; for forced exploration, caging, 
F (2,132)= 13.78, £< .01; for free exploration, caging, F (2,132)= 11.61, 
£< .01. The same effect of caging was obtained for both C 57 groups fed 
normal protein. Diet was also a significant main effect for both tests. 
Mice maintained on low protein diets had lower scores of emotionality than 
mice maintained on normal protein diets. Diet (forced exploration), F (1,132)= 
13.77, £< .01, (free exploration) F (1,132) = 26.53, £< .01. 

During tests of C57 mice fed low protein diets, 65 of 72 scores were zero, 
while 31 of 72 scores were zero for C57 mice fed normal protein diets. 
For emotionality scores during the free exploration test, the second order 
interaction attained statistical significance because Fi scores for low 
protein groups housed 1 or 2 per cage were significantly lower than scores 
of A/J groups, while all other comparisons did not differ significantly. 
All F]^ groups, with the exception of the two above, scored significantly 
higher than the midparental values, but were not significantly different 
from the higher scoring parental value. This experiment supplements 
previous research which obtained the same basic behavioral differences between 
mice fed low or normal protein diets, and in addition examined the effects 
of environmental complexity or group caging. 

E. Water intake, alcohol intake, and alcohol preference also were 
determined for mice fed low (47.) or normal (26%) percentages of protein in 
isocaloric synthelii diets. A/I, (;57, and hybrid male mice 6-months of age 
were subjects (N=24()). Mice which are fed Purina rodent chow were also 
tested. The protein content of Purina chow is about 257., but the concentration 
of carbohydrate (in this case, sugar) is much less than that of the normal 

FI-32 



protein synthetic diet. When the mice given Purina chow were tested for 
alcohol preference, the A/J strain avoided the alcohol, the C57 strain 
preferred alcohol, and the hybrid groups were intermediate in preference 
for alcohol. Fluid intake was similar for all groups, from 4 to 5 ml. 
per day, and group differences were consistent for all alcohol concentrations. 
Mice given synthetic diets which differed in protein content (Normal 
protein = 267„, low protein = k°L) differed with respect to the above control 
data in both alcohol preference and total fluid intake. At a moderate 
alcohol concentration of 57o, normal protein groups obtained scores similar 
to those of groups fed Purina chow, while low protein groups all obtained 
similar preference mean scores of over 50%. The total alcohol intake was 
higher for all low protein groups than for normal protein groups because 
total fluid intake was greater for low protein than for normal protein 
groups. At the higher alcohol concentration of 10%, there was a reduced 
alcohol preference for both protein levels. This effect of an aversion to 
the high alcohol concentration was greater for low than for normal protein 
groups. The C57 group fed Purina chow obtained very high alcohol preference 
values at the 10% concentrations, compared with groups fed low or normal 
protein synthetic diets. There are two findings of major importance with 
respect to this research; (a) the reactivity to moderate levels of this 
drug was greater for mice fed low protein diets than for mice fed normal 
protein diets, (b) for C57 mice alcohol preference for the high concentration 
was greatly reduced for animals fed synthetic diets compared with the 
preference of controls fed Purina chow. Some feature of the synthetic 
diet resulted in a marked reduction in alcohol preference for animals which 
normally highly prefer alcohol solutions. 

Significance to Bio-Medical Research and Program for the Institute ; 
The study of the genetics of behavior and longevity allows an assessment of: 
(1) the mode of inheritance for the factor studied (i.e., dominant, interme- 
diate, etc.); (2) the relative importance of hereditary and environmental 
factors; and (3) the number of genes or gene blocks which control the factor 
studied. Lack of adequate dietary protein is a condition which affects a 
large proportion of the world population. Our program attempts to determine 
the effect of diet (such as different proportions of protein in the total 
diet), during particular stages of the life span upon behavior and 
longevity for animal populations which differ in genetic constitution. 

Studies of single-gene mutant animals are of importance because they allow 
the assessment of the importance of a specific genetic locus for physiological 
or behavioral factors. 

Proposed Course of Project; Further inbred strains and Fj^ hybrid groups are 
being studied to determine the generality of mode of inheritance for 
behavioral factors. Cross-sectional and longitudinal studies of mouse 
behavior will continue with mouse strains BALB/cJ and DBA/2J, in addition 
to the A/J and C57BL/6J mouse strains. The longevity of inbred and hybrid 
groups are also being determined. Experiments with low and normal protein 
diets should determine; (1) the relationships between growth rate and 
longevity, (2^ the effect of low, normal, or high protein diets upon behavio 
at maturity after access to these diets during various stages of development, 
and (3) the effects of a present diet of low, normal, or high protein upon 

behavior . 

FI-33 



r 



Honors and Awards : None 

Publications ; Goodrick, C. Exploration activity and emotionality of albino 
and pigmented mice: Inheritance and the effects of test 

illumination. Journal of Comparative and Physiological 

Psychology, In Press . 



TABLE 1 

Mean scores of free exploration as a function of group, diet, and number 
of mice per cage. 



NUMBER PER 



1 



5 



••• £ .05, F. vs. 



MP 



GROUP 



CAGE DIET C57 F (MP) A/J 



Low 39.8 28.3(20.1)* 0.3 

Normal 39.9 17.9(20.5) 1.0 



Low 
Normal 

Low 
Normal 



40.2 


27,2(23.5) 


6.8 


39.8 


22.2(20.2) 


6.0 



33.5 


31.5(18.2)* 


2.9 


40.9 


17.3(20.7) 


0.5 



FI-34 



Serial No. HD-AG 27 

1. Gerontology Research Center 

2. Laboratory of Behavioral Sciences 

3. Section on Learning and Problem 

Solving 

4. Baltimore, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Exercise, general activity level and aging 

Previous Serial Number: Same 

Principal Investigator: Charles L. Goodrick, Ph. D, (307.) 

Other Investigator: None 

Cooperating Units: Baltimore City Hospitals 

Man Years: 

Total: .60 
Professional: .60 
Others: .00 

Project Description: 

Objectives : The general objectives are: (1) to determine methods for 
increasing physical activity of lower animals, (2) to examine behavioral 
and longevity differences among animals which differ in physical activity 
level, and (3) to determine the physiological mechanisms underlying 
differences in activity. 

Methods Employed ; Wistar rats are placed in activity wheels and allowed 
limited access to food. The result is an increment in physical activity 
(running) compared with normally fed animals or control animals. Hungry 
animals may also be rewarded with food for running. Other studies utilize 
inbred, hybrid, and mutant mice or species which differ in activity level 
due to different genetic constitutions. (See project HD-LB 1 Behavioral 
genetics and aging). 

Major Findings : 

A. In the past the major research in this area in our laboratory was 
concerned with exploration behavior and variables which may act to reduce 
age differences in exploration. Having completed a series of experiments 
concerning exploration and aging, some of the present projects are directly 
examining the effects of exercise upon longevity and exploration behavior 
for mire and rats. At this time last year, the longevity data were incomplete 



FI-35 



for an experiment in which the effects of access to an activity wheel for a 
30-day period were determined for F2 hybrid mice. Male and Female F2 hybrid 
mice of black, brown, or white (albino) coat colors were studied. One-half 
of each of six groups (N=20/group) were allowed 30 days of free exercise at 
23-months of age and the other half were placed in individual cages during 
this period. The previously reported data clearly found an increment 
in exploration activity for groups allowed wheel exercise. Additional 
analyses are now complete for measures of longevity and behavior as a function 
of coat color, sex, and exercise condition. Mean scores of longevity, wheel 
activity, exploration activity, and emotionality are given in Table 1 as 
a function of exercise condition and sex. The only main effect that was 
statistically significant for the longevity score was that of Condition, 
F (1,108)=8.07, £< .01. This effect must be considered in relation to 
the Condition x Sex interaction, F (1,108)= 8.24, £< .01. Although the 
differences were significant for male mice, _t (28)= 2.56, £< .05, 
differences were not significant for female mice. Coat color did not 
affect longevity for this mouse population. Body weight for female mice 
was significantly lower than for male mice, F (1,108)= 4.05, £< .05, the 
only significant difference for this measure. Control and experimental mice 
had been paired on the basis of body weight prior to wheel tests. Significant 
differences in wheel activity were not obtained with respect to coat color 
or sex for these groups. Wheel activity averaged over 3,000 revolutions per 
day, much Less than mean scores obtained for mature-young mice (5 mo. old) 
of 10,000 revolutions per day. Mice allowed access to the activity wheels 
engaged in significantly more exploration activity, F (1,108)= 18.14, 
p <.01, and were significantly less emotional, £ (1,108)= 7.54, £< .01, 
than control mice (Table 1). Sex differences and condition differences 
were not obtained and interactions were not statistically significant for 
either measure. 

B. Recent studies of immature nursing rat pups by other investigators 
have suggested that immature 16-18 day old pups are highly active in a 
novel environment. The interpretation of these data was that there is a 
peak in exploration activity at this age. Our previous research found 
that recently weaned rats 31 days old scored very high on tests of exploration, 
and after 3-6 months of age exploration decreased as a function of increasing 
age. A series of experiments were completed to determine the tendency of 
nursing rat pups to leave the familiar nest area, and additional data were 
obtained to determine the amount of exploration of a novel environment 
for rats 16, 24, and 32 days old. Rat pups (N=73) did not consistently 
leave the nest area to explore a novel environment until 23 days of age 
when tested at 17, 19, or 23 days of age, although parents explored at 
a high level during all three tests. Tests of forced exploration (dependent 
upon locomotor ability) and free exploration (independent of locomotor 
ability) indicated that exploration activity was a monotonic increasing 
function of age for rat pups age 16, 24, and 32 days old. These data were 
in agreement with previous work from our laboratory which found that 
immature rats 31 days old are at a maximal stage of exploration activity, 
compared with exploration activity of mature-old or aged rats. 



FI-36 



C. An experiment has been completed to determine the adaptation to a 
novel environment during a single test of long duration by rats over the 
age range of 16 to 750 days of age. Groups included nursing rat pups 16-18 
days old, immature weaned rats 2A-32 days old, mature-young rats 150-180 
days old, and aged rats 750-800 days old (N=96, 24/group), Rats were 
tested individually in 2-bar operant test chambers for 2-hour intervals, with 
the number of bar presses recorded every 15-minutes during the 2 hours. 
Preliminary analyses indicate that rats 16-18 days old do not adapt to the 
environment over a 2-hour test interval. Bar pressing responses remain at 
moderate levels throughout the entire test period. Immature, mature-young, 
and aged groups adapt quickly to the novel environment, with most responses 
in the initial 15-minutes of the 2-hour test interval. It was hypothesized 
that the failure to adapt to the novel environment by 16-18 day old 
nursing rat pups was due to strong motivation to return to the home nest, 
rather than to a strong exploration drive, as other experimenters had 
suggested. An additional experiment was completed to test the hypothesis 
that immature nursing rats 16-18 days old would adapt to the novel 
environment if group tested with their littermates. MoLeover, it was 
expected that young immature rats 24-32 days old would adapt less quickly 
if tested in groups with their littermates. Rats at these two ages (16-18 
days and 24-32 days were tested individually, in pairs, or in groups of three. 
The results were in complete agreement with the hypotheses. Groups 16-18 
days old adapted more quickly to the novel environment as a function of 
increasing number of pups in the test environment. Rats 24-32 days old 
adapted less quickly to the novel environment as a function of increasing 
number of immature rats in the test environment. 

Significance to Bio-Medical Research and the Program of the Institute ; 
One of the consistent findings of gerontological research is the decline 
in general activity level of old animals compared with young animals. It 
is important to determine whether quantity of activity (e.g. wheel activity) 
and/or quality of activity (e.g. increased exploration behavior or greater 
response variability) may be increased for old and senile animals. It is 
also important to examine the role of heredity with respect to voluntary 
exercise throughout the entire lifespan, and the effect of exercise upon 
behavioral decrements associated with advanced old age. 

Proposed Course of the Project ; Mature-young, mature-old, and aged rats 
will be trained to increase base activity level for food rewards to 
determine: (1) maximum increases which may be obtained, (2) the durability 
of increases (i.e., if increments in activity are maintained after training 
ceases), and (3) if increased activity level results in transfer to other 
tests or conditions. Data will be analyzed to determine: (1) correlations 
between general activity level and longevity and (2) longevity for 
exercised and nonexercised groups of project ft. Projects B, C, and D 
will be continued. 

Honors and Awards : None 

Publications: None 



FI-37 



TABLE 1 

Mean longevity, body weight, wheel activity, exploration, and 
emotionality for male and female mice as a function of exercise 



EXERCISE 
MALE FEMALE 



Longevity 


30.^ 


29.8 


Body Weight 


29.5 


28.2 


Wheel Activity/1000 


32.5 


33.8 


Exploration 


50.9 


49.7 


Emotionality 


7.0 


6.6 



CONTROL 


MALE 


FEMALE 


27.5 


29.8 


29.4 


28.3 


37.0 


37.2 


9.9 


9.7 



[-38 



Serial No, HD-AG 25 

1. Gerontology Research Center 

2. Laboratory of Behavioral Sciences 

3. Section on Learning and Problem 

Solving 

4. Baltimore, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Operant performance, memory and aging 

Previous Serial Number: Same 

Principal Investigator: Charles L. Goodrick, Ph.D. (30%) 

Other Investigators: None 

Cooperating Units: Baltimore City Hospitals 

Man Years: 

Total: .60 
Professional: .60 
Others: .00 

Project Description: 

Objectives : Research on both human and animal subjects has shown that 
short-term memory deficits and motor performance deficits are major problems 
of aged individuals. An interpretation of maze learning differences between 
young and aged rats is that these differences are due to short-term memory 
deficits of the old animals. The general objectives of one phase of this 
project are: (1) to analyze complex maze learning of young and aged animals; 
and (2) to determine variables which may act to enhance or retard maze 
learning ability. The objectives of the other phase of this project are: 
(1) to study age differences in motor performance during operant responding; 
and (2) to develop operant techniques which improve the retention of learned 
responses in aged animals. 

Methods Employed: Operant conditioning performance and retention studies 
have used 2-bar test boxes in which hungry animals are trained to press one 
bar to obtain a food reward while the alternate bar remains neutral. By 
increasing the complexity of the task (using two bars rather than one), it 
is possible to make a finer analysis of performance and the retention process. 
We are studying performance and retention as a function of reward schedule, 
and we are particularly interested in the partial reinforcement effect. The 
retention of partially rewarded responses is vastly greater than for responses 
continuously rewarded; and analysis of this phenomenon will provide information 
regarding the general retention process. A complex lA-unit multiple-T maze 
Is also utilized. This maze has been shown to have a high degree of reliabi- 
lity and has been used in many major studies of aging and learning. Additional 

FI-39 



mazes of 6-units are being developed to study mastery of consecutive problems 
by young and aged rats. These mazes will be used to analyze aging effects 
in short-term and long-term memory, and determine aging effects in proactive 
and retroactive inhibition. 

Major Findings ; 

A. Studies in the last few years from this laboratory have utilized a 
complex maze problem to show clear age differences in learning ability for 
mature-young and aged rats. One test factor found to reduce age differences 
was distribution of practice. Aged rats given massed practice were able 

to reduce error scores (a measure independent of activity level) to the same 
level as that of young rats. During distributed practice trials, aged rats 
make perseverative errors within the maze, i.e., they tend to repeat errors 
over trials at the same culs-de-sac to a much greater degree than young rats. 
Further information is now available with respect to changes in perseverative 
error frequencies and percentages over a long series of trials for young and 
aged rats tested with distributed trials or moderately massed trials. The 
analysis was a 2X2 design (Practice Condition X Trials), using the percentage 
scores of just the aged rats. The aged distributed practice groups had a 
significantly higher percentage of perseverative errors than the aged massed 
practice groups, F (1,30)= 10.04, £< .01. This was due to an increase in 
the percentage of such errors over trials for the aged distributed practice 
groups, while a decrease in the percentage of such errors occurred over 
Trials for the aged massed practice group. Practice Condition X Trials, 
F (1,30)= 12.24, £< .01. Although the group mean differences in the 
percentage of perseverative errors were not statistically significant on 
trials 1-20, group mean differences were highly statistically significant 
for Trials 21-40, t_ (30)= 6.75, £< .01. Table 1 gives the mean number 
of culs-de-sac where six or more errors occurred, mean number of perseverative 
errors, and mean percentage of perseverative errors as a function of practice 
condition and age. On the basis of past research in this laboratory it was 
hypothesized that age differences in learning were due to short term memory 
deficits of aged animals. An analysis of the elimination of perseverative 
errors during training of aged rats utilizing moderately massed practice 
(4 trials/day) has given additional support to this hypothesis. Errors 
at specific culs-de-sac were normally obtained on initial trials during the 
daily 4-trial sequence. Correct responses were obtained on the later trials 
during the daily 4-trial sequence, until mastery of the cul-de-sac occurred 
with a correct turn occurring during all future trials (Table 2). This 
finding, based on a detailed analysis of individual records of the exact 
path of aged rats over a long series of learning trials, provides strong 
evidence that learning deficits of aged animals may be the result of short- 
term memory deficits. 

B. Another hypothesis, based on our earlier research, was that aged 
animals would benefit more from a forced correct sequence early in the 
training series than younger animals. Groups of mature-young and aged rats 
have been given initial tests in which barriers are placed at all culs-de-sac 
so that all animals must run the correct sequence of 14 turns after which 
they receive a food reward. After these preliminary tests, the rats were 
given additional learning trials with the barriers removed. During these 

learning trials the aged rats made fewer errors than the young rats and all 

FI-40 



J 



aged rats quickly mastered the maze problem. The implications of this 
finding are that learning may be facilitated more for slow learners than for 
fast learners through procedures which require active correct responses 
early in training. Further animals are to be tested and the results will 
be given in detail in the next annual report. 

C. An important aspect of the animal behavior program has been the 
systematic investigation of the retention of learned responses following 
training utilizing operant test chambers. During these tests the influence 
of complex schedules of reward upon performance during training and 
extinction after training may be determined. The use of two-bar test boxes 
with one bar reward contingent and the other bar neutral allows an 
assessment of performance (number of responses) which is independent of the 
learning measure (percentage of reward bar responses), an important feature 
for studies which may involve active young animals and inactive aged animals. 
A further study in this series has examined operant performance for young 
and aged rats on fixed interval schedules of reward. This type of schedule 
is time-contingent, i.e., a reward is given for a response following a fixed 
time interval, but the number of responses is unspecified. The major findings 
of this experiment were that during training number of responses per reward 
was in increasing function of the duration of the fixed interval, but 
number of responses per reward did not differ significantly for motivated 
mature-young and aged rats. This experiment provides further support for 
results obtained earlier in our laboratory that number of responses during 
operant testing failed to yield an expected result of lower levels of 
response for aged than young animals. Furthermore, the total level of 
responses was similar for young and aged rats during extinction trials, and 
the percentage of reward bar responses during training and extinction trials 
were also similar for young and aged rats. The importance of this 
essentially negative finding is being examined in relation to the very 
large age differences obtained for other tests with a high activity component 
such as wheel activity or free exploration activity. 

Significance to Bio-Medical Research and the Program of the Institute ; 
"Learning and/or memory deficits represent a major problem among the aged 
human population. Major behavioral or physiological techniques to reduce 
performance deficits obtained for aged, lower animals have been studied in 
our laboratory. This project may facilitate research with higher organisms 
such as man by determining the optimal conditions for learning and for 
retention of learned responses. 

Proposed Course of Project : Further studies are in progress to determine 
the nature of the partial reinforcement effect in relation to (a) time 
contingent vs. response contingent partial reinforcement and, (b) massed 
vs. distributed extinction trials. Other studies will examine age differences 
in operant performance as a function of response ef f ortfulness. Maze studies 
will concentrate upon the effects of central nervous system stimulants on 
behavioral rigidity within the maze for old rats. We will also initiate 
preliminary studies of rote learning and perceptual learning of human 
subj eC ts. 



FI-4L 



Honors and Awards: None 



Publications : 



Goodrick, C. Learning by mature-young and aged Wistar 
albino rats as a function of test complexity. Journal of 
Gerontology , 1972, 27_, 353-357. 

Goodrick, C. Error goal-gradients of mature-young and aged 
rats during training in a 14-unit spacial maze. Psychological 
Reports . 1973, 32, 359-362. 

Goodrick, C. Maze learning of mature-young and aged rats as a 
function of distribution of practice. Journal of Experimental 
Psychology , In press. 



TABLE 1 

Mean number of culs-de-sac where six or more errors occurred (a), mean 
number of perseverative errors (b), and mean percentage of perseverative 
errors (c) as a function of practice condition and age. 



Practice Condition 


Group 


Trials 1-20 Trials 21-40 


a b c a b c 








Distributed 
Massed 


Young 
Young 


5.4 0.9 14.3 0.4 0.0 0.0 

6.5 1.4 19.8 0.4 0.0 0.0 


Distributed 
Massed 


Aged 
Aged 


7.1 3.8 50.5 3.8 2.5 64.9 
6.9 3.4 49.1 2.9 1.3 34.2 



FI-42 



TABLE 2 

Errors at thirty-three perseverative choice points as a function of series 
and trials for aged rats given massed practice 



Trials 



Series 



A B C D E F G 



33 


31 


25 


18 


7 


5 





33 


23 


13 


10 


5 


1 





33 


17 


12 


5 





1 





33 


5 


U 


2 












33 33 26 21 8 5 
Total Perseverative Choice Points 



FI-43 



Serial No. HD-AG16 (8) (c) 

1. Gerontology Research Center 

2. Laboratory of Behavioral Sciences 

3. Physiological Psychology Section 

4. Baltimore, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Learned Modification of Visceral Function in Man 

Principal Investigator: Bernard T. Engel, Ph.D. (407.,) 

Other Investigators: Donald A. Kristt, M.D. (457.) 

Marvin G. Schuster, M.D. (57.) 

Cooperating Units: Baltimore City Hospitals 

Man Years: 



Total: 


2.20 


Professional: 


.90 


Other: 


1.30 



Project Description: 
Objectives : 

A. Learned control of blood pressure. 

1. To determine whether patients with essential hypertension can 

be taught to control their systolic blood pressure (SBP) through operant 
conditioning techniques. 

2. To determine whether patients so trained can achieve a significant 
and sustained lowering of their abnormally elevated blood pressure. 

B. Learned control of anal sphincter function. 

1. To determine whether patients with severe, intractable fecal 
incontinence (continuous daily soiling) can be trained to control anal 
sphincter responses. 

2. To determine whether responses of the internal as well as the 
external anal sphincter can be brought under voluntary control. 

3. To determine whether such control, once learned, will lead to 
clinically significant improvements in lower bowel function. 



FI-44 



Methods Employed ; 

A. Learned control of blood pressure. 

Volunteer patients are recruited from the Baltimore City Hospitals (BCH) 
Hypertension Clinic. During the study patients are hospitalized from 
one to three weeks on the research ward of BCH. Patients are trained 
in the laboratory to raise and lower median SEP. During a typical 
experimental session the patient lies semireclined in a hospital bed. 
Arranged in front of him are a vertical display of three lights. The 
top light is green, the middle light is yellow and the bottom light 
is red. In addition, there is a digital percent meter that provided 
the patient with a cumulative record of his success during a trial. 
A standard blood pressure cuff, covering a small crystal microphone, 
is applied over the ante-cubital fossa. The microphone detects the 
presence of Korotkoff's sounds. 

Each session is divided into three phases. Phase 1 is a determination 
of baseline SBP. Before going on to phase 2, the patient's blood 
pressure has to become stable over at least five trials. Each trial is 
50 heart beats in duration. Following the 50 beat trial the BP cuff 
is deflated and the patient is permitted to rest for 32 sec. before the 
next trial. 

Phase 2 consists of a battery of 18-24 training trials punctuated by 
several short pauses. During this phase the patient tries to control 
his blood pressure. Phase 3 is a post- training SBP baseline of 5 trials. 

If the patient is being trained to raise his blood pressure, the green 
light is turned on and remains lit throughout the training trial. Lights 
are off during baseline phases, and inter-trial rest periods. When 
the patient raises his SBP (i.e., produces more Korotkoff sounds (KS) 
at a fixed cuff pressure in a given trial), the yellow light goes on and 
remains on as long as Korotkoff sounds are produced. If the patient's 
SBP fell below cuff pressure for a given heart beat and no KS was 
produced, then the yellow light would go off. Each heart beat accompanied 
by a KS during "raising" training registers + 27, on the cumulative 
percent meter. Each patient is instructed to keep the yellow light on 
as much as possible and to accumulate as high a percent score as possible. 

If the patient is being trained to lower his SBP below cuff pressure, 
reinforcement (the yellow light) is provided when production of KS is 
suppressed. In this case the percent meter reflects the number of heart 
beats not accompanied by KS . 

After the patient has been trained separately to raise and to lower his 
SBP, he is further tested under differential training conditions. During 
the training phases of these sessions the patient is required to 
alternately raise and lower his SBP over 6-8 consecutive trials. The 
onset of each training condition is signaled by the appropriate cue 
lights, either green or red. 



FT-45 



B. Learned Control of anal sphincter function. 

Sphincteric activity is monitored by means of pressure recordings 
from a triple balloon system. One balloon is placed in the rectum, 
a second balloon is placed at the internal sphincter and the third 
balloon is placed at the external sphincter. In normal subjects 
distension of the rectum elicits reflex relaxation of the internal 
sphincter, and simultaneous contraction of the external sphincter. 

Each patient in this study was evaluated in three ways: 1) his clinical 
history was taken; 2) rectal examination was conducted to determine 
clinically the degree of sphincteric tone; 3) the rectal distension 
procedure described above was employed: a) to determine which sphincter 
was malfunctioning, and b) to determine the threshold of response 
of the sphincteric reflex. Thresholds were determined by systematically 
inflating the rectal balloon with a known volume of air and simultaneously 
observing the sphincteric responses. 

Once the patient's threshold response was determined, a series of 
baseline recordings was made to provide an objective basis for measuring 
change. After the baseline measurements were made, the patient was 
permitted to observe the tracings from the pressure manometer, and he 
was instructed to "make the responses look like normal ones." The patient 
had received instructions earlier on the nature of normal sphincteric 
activity. Depending upon the particular abnormality the patient 
manifested, he would be instructed to try to control either sphincter 
or the synchrony between the sphincters. 

Patients were seen in one to three training sessions separated by periods 
ranging from two to six weeks. Improvement was measured in terms of 
change in sphincteric activity in the laboratory, in terms of the 
physicians judgement of the change in anal sphincteric tone and in terms 
of the patient's report of his ability to remain continent. 

Seven patients were seen. Two were men and five were women. Age range 
was 6 years to 55 years. The patients had histories of fecal incontinence 
ranging from one year to several years. In three of the patients the 
tests indicated abnormal motor responses in both sphincters and in the 
other four patients only the external sphincter malfunctioned. 

Major Findings : 

A. Learned control of blood pressure. 

One patient completed three weeks of conditioning. She is 60 years old 
and has mild essential hypertension of at least 25 years duration. Her 
hypertension showed marked exacerbation during her only pregnancy in 
1954. SBP at this time was 260 mm Hg. Presently she is mildly 
hypertensive, well controlled on diuretic therapy. Her mean SBP during 
clinic visits over the past year was 147.5 mm Hg (n = 15 visits). During 
thr period of her training while she was hospitalized, diuretic therapy 
w.is discontinued and low salt diet instituted in its place. This was 
doni' U) obviato tho common hypotensive side effects of hospitalization 

FI-46 



of active, medically controlled hypertensives. 

This patient was hospitalized for three weeks. She was first trained to 
raise, then to lower and finally to alternate raising and lowering of 
SBP. 

The first stage of training consisted of nine raising sessions. The 
mean elevation over baseline SBP for these sessions was 10 mm Hg. In 
one session, toward the end of the block of "raising" sessions, an 
elevation of 20 mm Hg. was achieved. 

The initial and terminal baselines, phase 1 and phase 3, were generally 
quite similar differing by a mean of +0.7 mm Hg. in all but one session 
in which the terminal baseline rose 13 ram Hg. Cuff pressure was adjusted 
on a trial to trial basis so that 25-757. of Korotkoff's sounds were 
produced each trial: i.e., cuff pressure was adjusted to match median 
SBP throughout the session. 

The results during the subsequent 14 lowering sessions are less clearcut. 
In seven sessions lowering of SBP ranged from 4-19 mm Hg. (mean 8.6 mm 
Hg. ) below initial baseline. In the remaining seven sessions mean 
lowering was 0.7 mm Hg. However, the terminal baseline was raised over 
both initial baseline and training level. Mean baseline differences in 
these latter seven sessions was +5.0 mm Hg. as compared to the mean 
difference in the successful sessions of -1.2 mm Hg. This suggests 
that although lowering did not occur in these sessions elevations of SBP 
during training were suppressed. No trend relating level of initial SBP 
and degree of lowering was apparent. 

The final stage of training was 12 alternation sessions. In the same 
session the patient was asked to first lower SBP for six trials then 
raise it for six trials and finally to lower from this new level for a 
final eight trials. The patient had most difficulty lowering after 
raising SBP. The mean initial lowering was -4.8 mm Hg. ; mean raising 
+6.1 mm Hg. During final lowering SBP rose by a mean of 1.7 mm Hg. In 
the last four alternate sessions, terminal lowering was achieved: mean 
-2.3 mm Hg. In the sessions prior to discharge, the patient's alternate 
record was (L)-4, (R)+8, (L)-14 mm Hg. 

The patient returned one month following training for a follow-up 
alternate session. The patient was medicated in her usual fashion 
during this session. Her alternate record at this time was (L)-5, 
(R)+7, (L)-4 mm Hg. 

In addition to the laboratory studies this patient also was taught to 
take home blood pressures. The results from this phase of the study show 
a mean systolic BP of 127 mm Hg. (n=525 determinations over three weeks) 
as compared to clinic SBP of 147.5 mm Hg. during the previous year; the 
patient also is able to voluntarily lower her SBP from this average 
level by a mean of 17.6 mm Hg. using this device (this mean also was based 
on 25 readings/day for three weeks). 



FI-47 



B, Learned control of anal sphincter function. 

One of the patients who had a history of an anal fissure withdrew from 
the study after one session because the measurement procedure was painful. 
None of the other patients complained of pain. Five of the six patients 
have shown significant improvement (defined as no soiling according to 
patient's self report), which has persisted since the time of study. 
The periods of improvement are: 2 mos., 6 mos., 1 yr. , 2 yrs. and 3 yrs. 
One patient reported some improvement (defined as reduced soiling 
according to patient's report). He has sustained his control for about 
1 yr. to date. 

In addition to the changes in continence reported by the patients, there 
are objective signs of significant improvements in bowel function. 
Tracings taken from each patient while he was in the laboratory show 
systematic signs of improvement. This effect is true for the internal 
sphincter responses as well as for responses of the external sphincters. 

In addition to the scientific implications of these results, the clinical 
effects are especially interesting. The procedure utilized was adapted 
from the diagnostic procedures now used by many gastroenterologists. 
Thus, the technology for applying these techniques clinically is already 
available to practicing physicians. A second factor which adds to the 
clinical potential of this conditioning procedure is that the patients 
were able to learn to control their sphincters quickly. In no case was 
more than three sessions necessary. 

Significance ; This project is designed to increase our understanding of 
learned control of autonomic responses and the application of this 
knowledge to the control of various illnesses. These data add further 
credence to the conclusion that responses mediated by the autonomic 
nervous system can be brought under voluntary control, and they show 
that such control holds out the possibility of clinically useful 
applications. 

Proposed Course : At least five additional patients with high blood 
pressure will be studied. If these techniques prove efficacious with 
these patients, they may be extended to evaluate the clinical efficacy 
of blood pressure control in the regulation of pain associated with 
angina pectoris. 

Honors and Awards ; Dr. Engel presented a lecture (by invitation) to the 
joint meeting of the Maryland Psychiatric Association and the Maryland 
Psychological Association (December, 1972) 

Dr. Engel presented at the medical grand rounds of the Rhode Island 
Hospital (March', 1973) 

Dr. i:ng<'l presented at the cardiology grand rounds of the Johns Hopkins 
Hospital (March, 1^)73) 



FI-48 



Publications ; Bleecker, E.R. and Engel, B.T. Learned control of 
ventricular rate in patients with atrial fibrillation. Psychosomati c 
Medicine , 1973, 35, 161-175 

Bleecker, E.R. and Engel, B.T. Learned control of cardiac rate and 
cardiac conduction in the Wolff - Parkinson - White Syndrome. New 
England Journal of Medicine , 1973, 288, 560-562. 

Engel, B.T. Operant conditioning of cardiac function: Some implications 
for psychosomatic medicine. Behavior Therapy , in press. 

Engel, B.T. Clinical applications of operant conditioning techniques in 
the control of the cardia arrhythmias. Seminars in Psychiatry , in press. 



FI-^9 



Serial No. HD-AG2 (8) 

1. Gerontology Research Center 

2. Laboratory of Behavioral Sciences 

3. Physiological Psychology Section 

4. Baltimore, Maryland 
PHS-NIH 

Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Learned Modification of Visceral Function in Animals 

Previous Serial Number: Same 

Principal Investigator: Bernard T. Engel, Ph.D. (55%) 

Other Investigators: George W. Ainslie, Jr., M.D. (907o) 

Donald A. Kristt, M.D. (45%) 

Cooperating Units: Baltimore City Hospitals 

Man Years: 



Total: 


4.20 


Professional : 


1.80 


Other: 


2.30 



Project Description: 
Obj ectives ; 

A. Operant Conditioning of Heart Rate. 

To train monkeys to voluntarily control their cardiac functions, and to use 
this preparation to identify the physiological and psychological mechanisms 
associated with this control. 

B. Depressor activity of the rat hypothalamus. 

1. To locate and define vasodepressor areas in the rat diencephalon. 

2. To develop a chronic, awake preparation in the rat in which such 
an effect can be elicited and continuously monitored. 

3. To utilize this hypotensive effect to determine whether sponta- 
neously hypertensive rats would engage in intracranial self- 
stimulation to obtain this transient lowering of blood pressure. 

Methods Employed : 

A. Operant Conditioning of Heart Rate. 
Three studies have been performed during the last year: 



i [-50 



i 



1. Interaction of operant and respondent conditioning of cardiac rate- 
Two factor learning theory has long held that operant learning is greatly 
affected by respondent conditioning to cues in the experimental environment, 
however, the possibility that respondent conditioning can be altered by 
operant incentives has received little attention. This has probably been so 
because operant learning was believed until recently to take place only with 
skeletal muscle responses. Since evidence for respondent conditioning of 
these responses has been equivocal, there has been no experimental preparation 
in which operant modification of respondent conditioning could be studied. 
The recent discovery that visceral responses can be operantly conditioned 
makes such an experiment possible. 

Subjects were six monkeys of which five were naive and one had previously 

had extensive training in cardiac slowing as an operant to avoid electric 
shock. Each subject had its external iliac artery cannulated under sterile 

surgical conditions. The polyethylene catheters were kept open by continuous 

infusion of heparinized normal saline at the rate of about 20 units/hr. After 

surgery each subject lived in its own restraining chair in an individual 
booth which was sound-deadened when the door was closed. 

Each animal usually received four experimental sessions each weekday. The 
subject's door was closed and no stimuli were introduced for 20 min. to 
allow its heart rate and blood pressure to reach a stable level. These 
responses were then measured for 512 sec. by an on-line Raytheon 704 
computer, which then conducted a 2048 sec. experimental session. The sessions 
were one of three kinds: Operant rate control to avoid electric shock, 
respondent conditioning to a stimulus predicting an unavoidable electric 
shock, or a combination of both. 

Operant rate control was taught by means of feedback from a row of three 
lights facing the subject. Throughout each session one of the side lights 
was on, warning the subject that it must speed or slow its heart or receive 
a 0.5 sec, 10 ma shock to its tail. A green light on the subject's left 
commanded speeding, and a red light on its right slowing. The white light 
in the center was on whenever the last inter-beat interval had met a criterion 
based on baseline heart rate. A free running clock produced an electrical 
impulse every 8 sec, and if the white light was off when this impulse occurred 
the monkey received a shock. It was safe from shock whenever the white light 
was on, signalling that its rate was meeting the criterion. 

During respondent conditioning the cue lights were never on, but 128 sec. 
periods of clicking from a loudspeaker alternated with 128 sec. period of 
silence. The frequency of the click alternated in turn between two and 
20 clicks/sec, so the following pattern repeated itself every 512 sec: 
128 sec. of 2/sec. clicks, 128 sec. of silence, 128 sec. of 20/sec. clicks, 
128 sec. silence. For each monkey one of the click patterns (warning clicks) 
was always followed by four rapid 0.1 sec, 10 ma shocks to the tail; the 
other click pattern (neutral clicks) was never followed by shock. 



FI-51 



During the combined condition, both of the above schedules were in force 
simuLtaneously. 



Two subjects were initially conditioned to the clicks. When their cardiac 
responses to the clicks were stable, they were given rate control sessions 
in which they learned to slow their heart rate to avoid shock. Two other 
subjects including the experienced monkey began with rate control training. 
After each subject had learned to change its rate in the desired direction, 
it was given more respondent conditioning until its cardiac responses were 
again stable. It was then put in the combined situation, and then in the 
respondent-only situation again. This process was repeated with rate 
training in the opposite direction for two animals. In addition to these 
subjects two animals which were trained to speed their hearts first also 
are in this group. 

2. The effect of chronic, sustained, operantly conditioned cardiac 
changes on cardiovascular function. 

The purpose of this project is to learn whether monkeys can be made to 
develop abnormal cardiac responses through conditioning procedures. Two 
animals are now being studied. Each animal participates in a three-phase 
study. Phase 1 is a four week baseline period during which each animal's 
heart rate and blood pressure is continuously monitored for 18 hr/day, five 
days/wk. The monitoring period is from 6:00 PM until noon the following 
day. Phase 2 is a three week period during which each animal is taught to 
control its heart rate to avoid an electric shock. Animal 1 was trained 
to speed its rate and animal 2 was trained to slow its rate. 

Phase 3 is of indeterminate length, however, it will persist for at least 
6 mos. During phase 3 each animal is in a training situation 18 hr/day, 
five days/wk (from 6:00 PM until noon). As long as the animal maintains 
its heart rate above (animal 1) or below (animal 2) a criterion rate, the 
animal is "safe." Whenever its rate is outside of the criterion region, 
the animal accumulates "bad time." When the animal accumulates 64 sec. of 
bad time, the appropriate shock-avoidance contingency is turned on and 
remains on for 1024 sec. (about 17 min.). Thus, the animal must maintain 
a certain level of heart rate to avoid turning on its schedule, and it 
must avoid shock whenever the schedule is on. 

The criterion heart rate which determines the schedule is set by the 
experimenter, and it is made progressively more difficult as the study 
proceeds. Animal 1 which has been in phase 3 for two weeks at this time, 
and which must maintain a relative tachycardia, had its criterion set at 
100 beats/min. initially, and now has a criterion of 120 beats/min. Animal 
2 which has been studied for about six weeks now, and which has to maintain 
a relative bradycardia, had its criterion set at 200 beats/min. initially, 
and now has its criterion set at 160 beats/min. 



FI-52 



B. Depressor Activity of the Rat Hypothalamus. 

Twenty-two male Wistar rats were utilized. 13 were 6 to 11 mos. of age; 
the other 9 were 5 mos. of age. All rats were anesthetized with pentobarbital, 
40-59 mg/kg. administered intraperi toneally. During the deep phases of 
anesthesia an arterial catheter (either femoral or carotid) was placed and 
the animal was prepared and fixed in a sterotaxic apparatus. Blood pressure 
and heart rate were continuously monitored throughout the experiment. While 
the rat was in the stereotax, an electrode was placed unilaterally so that 
its exposed tip was in the region of the antero-lateral hypothalamus medial 
to median forebrain bundle. A zone 1.2 mm. lateral to the midline was 
explored from approx A. 2.0 to A 6.2, H 6.0 to 9.2 (Massopust, L.C. Jr., 
Diencephalon of the rat (in) Sheer, O.E., Electrical stimulation of the 
brain . , 1961, Univ. of Texas, 182-202). Exploration was first performed 
with monopolar electrodes since it seemed likely that vasodepresser effects 
might involve several structures. Stimulation consisted of trains of square- 
wave pulses. They were produced by a Grass square wave generator via an 
isolated constant current unit. Stimulation parameters were as follows: 
Train duration (TD) 2500-5000 ms; Train frequency (TPS), 0.1 Trains/sec; 
pulse duration (PD), 1-8 ms ; pulse frequency (PPS), 50-200/sec. Current 
levels ranged from 100 to 750 ua. 

For refinement of the localization and elucidation of the mechanism of 
action of the stimulation a concentric bipolar electrode was utilized. 
Stimulation paremeters were: TD, 50-5000 ms.; TPS, 0.1-1.0; PD, 5-8 ms ; 
PPS, 100. Current levels ranged from 50-400 ua. Two vasoactive drugs 
were employed to produce changes in blood pressure levels. Isoproterenol 
and epinephrine were used in doses of l-4^,ng/kg. body weight. 

All rats were decapitated and their brains fixed in 107o formalin for gross 
and/or microscopic evaluation of electrode localization. 

Major Findings ; 

A. Operant Conditioning of Heart Rate. 

1. Interaction of operant and respondent conditioning of heart rate. 

It often required several weeks for the animals' behavior to stabilize. 
Because of this, only the first half of the experiment has been completed 
and analyzed on the first four monkeys, despite the fact that they have 
been tested as long as ten months. This study will be completed by the 
end of this reporting period, however. The initial rate training for all 
four was in slowing, and clear data exist about this manipulation. The two 
monkeys which began with respondent conditioning (20 sessions) quickly 
learned to raise their heart rates significantly during the warning click 
(6 and 37 beats/min. during the last week of conditioning, t=3.29 and 9.07 
respectively). During rate training (about 30 sessions for the previously 
naive subjects) all four monkeys learned to slow their rates significantly. 
The average change in rate for all four was 14 beats/min. Three of them 
rarely received avoidable shocks after their initial training, but one had 
intermittent episodes of poor performance and high rate of shock. During 
subsequent respondent conditioning trials (30-90 sessions), all four speeded 
in the presence of warning clicks. The average heart rate during warning 

FI-53 



clicks for all four animals was 5 beats/rain higher than during neutral 
clicks, and 12.3 beats/min higher than in the absence of auditory stimuli. 

When operant and respondent schedules were in force simultaneously (80-130 
sessions), three of the four monkeys developed lower rates during the 
warning than during the neutral clicks; the exception was the monkey whose 
rate control was imperfect, and even it showed less tendency than it had 
previously to speed during the warning clicks. The subjects' mean rate 
during warning clicks was 7,0 beats/min lower than during actual clicks, 
13.4 beats/min lower than in the absence of auditory stimuli. These changes 
in the subjects' conditioned heart rates persisted after they returned to 
the respondent-only schedule. Although one monkey reverted to its original 
pattern of differential speeding after 40 sessions, the change of direction 
in the others was stable throughout more than 80 sessions. Systolic and 
diastolic blood pressure during rate training differed from subject to 
subject, however, they were generally consistent over time for each 
individual. During respondent conditioning blood pressures rose during the 
warning clicks for all subjects throughout the experiment regardless of 
the direction of rate change, and changed virtually not at all during the 
neutral clicks. 

Table 1 summarizes the data from all phases of this study. 

TABLE 1 

Average Changes in heart rates (beats/min) and blood pressures (mm. Hg.) 
during different phases of the study (Cond 1, respondent conditioning 
before pairing; Comb, simultaneous respondent and operant conditioning; 
Cond 2, respondent conditioning after pairing). 



Cond 1 



Comb 



Cond 2 



Heart Rate 



Warning clicks 
Neutral clicks 
No Clicks 



5.9 

0.9 

-6.4 



•18.7 

•11.7 

15.3 



-4.7 
-2.3 
-5.7 



Systolic Pressure 



Warning clicks 
Neutral clicks 
No clicks 



8.8 
-1.8 
■1.9 



4.2 


4.0 


0.4 


-2.6 


0.1 


-0.4 



Diastolic Pressure 



Warning clicks 
Neutral clicks 
No clicks 



6.9 
■0.7 
-1.2 



2.2 
-0.7 
-0.6 



2.7 
-1.7 
•0.2 



FI-54 



2. The effect of chronic, sustained, operantly conditioned cardiac 
changes on cardiovascular function. 

It is too soon to carry out a systematic analysis of the data in terms of 
such factors as diurnal variation, however, some general findings can now 
be reported. Animal 1 which was trained to speed its heart rate had average 
weekly heart rates before speeding training of 130, 137, 118 and 132 beats/ 
min. Blood pressures were 130/77, 123/67, 120/65 and 118/64 mm Hg. During 
two weeks of post- training study its heart rates were 145 and 143 beats/min 
and its blood pressures were 132/78 and 136/83 mm Hg. Thus, under conditions 
of 18 hr. avoidance training it has been possible to maintain a relative 
tachycardia over two weeks. Obviously, it is much too soon to speculate 
beyond these data at this time. 

Animal 2 was trained to slow its rate. Its average weekly rates before 
training were 147, 146, 146 and 140 beats/min. Blood pressures over this 
period were 146/107, 140/100, 125/82 and 118/76 mm Hg. After slowing 
training and during six weeks of continued avoidance conditioning its 
weekly average rates were 148, 152, 152, 139, 149 and 133 beats/min. Blood 
pressures were 124/80, 126/80, 117/74, 113/73, 120/78 and 111/72 mm Hg. 
Thus it appears that heart rate is beginning to fall below baseline levels 
while blood pressure remains stable. 

During periods when the shock schedules are operative animal I's heart 
rate is about 15 beats/min faster than when it is off schedule. Animal 
2's heart rate is about 15 beats/min. slower when it is on schedule. These 
data are evidence that the schedules are still effective, and that the 
animals are still actively avoiding shock by selectively modifying their 
heart rates appropriately. 

3. Autopsy findings. 

Four monkeys that had had indwelling distal abdominal aortic catheters 
were autopsied. The catheters were in place for 3 mos. to 18 mos. In the 
two monkeys with catheters present for approximately 3 mos., moderately 
advanced arteriosclerotic changes were apparent. In one case, the catheter 
had been completely walled off by a fibrous intimal lesion. Adventitial 
fibrosis was conspicuous and lymphadenopathy was present locally. In a 
second case, thinning and mild aneurysmal dilation of the wall was noted in 
one area. The adventitial fibrosis and lymphadenopathy were similar. The 
remaining two monkeys showed striking arteriosclerotic aneursymal changes 
of the distal abdominal aorta. The longest lived catheter, 18 mos, was 
found in a thin walled, blood filled, friable aneursymal sac. The sac 
measured 14 cm in length and 6 cm in width. It was located in the left, 
lower retroperitoneum and was adherent to the subjacent flank muscles. The 
findings in these four animals suggest that the presence of continual intimal 
irritation from an indwelling catheter recapitulates the usual arterioscle- 
rotic changes that eventuate in aneursym formation. 



FI-55 



Two monkeys that had had a periaortic flow probe implanted were euthenized 
and autopsied after 3 mos. A loose fitting probe was used in one monkey. 
Fifteen percent of its inner surface perimeter was covered with dacron 
mesh. Histologic examination of the subjacent aorta showed a generally 
intact wall where peri-adventi tial fibrosis involved the dacron mesh. The 
opposite wall (subjacent to bare probe) evidenced intra-medial hemorrhage 
and fibrosis with extensive loss and fragmentation of elastic tissue. In 
the second monkey the flow probe was 50% dacron covered and was a tighter 
fit. Here, there were no focal medial degenerative changes. However, there 
was a uniform band of sub-adventi tial fibrosis in both the dacron protected 
and non-protected areas. This suggests that greatest longevity is most 
likely to ensue from a dacron wrapped, loosely fitting probe. The prognosis 
must be guarded for periods greater than 3 months using any other procedure. 

B. Depressor Activity of the rat hypothalamus 

1. Monopolar electrodes: A3. 6, H7 . 7 , LI. 2 is a site where maximum 
monophasic depressor activity is produced: hereafter called SME. Generally, 
the effect is stable and r^producable with a mean fall in blood pressure of 
14.7/14.5 mm Hg. (N=6). Occasionally pressures will fall 30 to 40 mm Hg. , 
and sometimes a considerably smaller response is noted. Heart rates rarely 
changed during stimulation. Activity mapping in the L 1.2 plane showed 
that 1 mm anterior to the SME there was a silent zone. Further rostrad 
(A6.2), in the vicinity of the anterior commissure, vasodepressor activity 
was again observed. This is consistent with observations in the dog, cat 
and monkey. The effect was also noted caudal to the original site at A 3.2 
(i.e., 0.4 mm posterior to SME) but disappeared by A 2.5 (1.1 mm posterior 
to SME). Activity in the horizontal plane was also explored. From the 
cortex to H 6.7 no change was noted. A minimal change was noted at H 7.2 
of -6/-4 mm Hg. Moving down past SME the response becomes biphasic. The 
first phase is a brief response lasting approximately 500 ms or less. It 
is followed by a more prolonged depressor phase with a return to baseline 
in 3-7 min. The more ventral the stimulation the more conspicuous the 
positive phase. At the lowest point explored (H 9.2) the mean change in 
pressure for 2 consecutive stimulations for each phase was as follows 
14/11, -16/-13 mm Hg. where the signs indicate pressor and depressor effects 
respectively. An analysis of stimulation parameters indicated that the 
depressor effect was a function of TD, PPS, PD and current. In general, 
decreasing the current density produces a smaller effect on pressure, how- 
ever, this effect is not linear. Halving the current intensity produced a 
decrement of 1/3; lowering the current 4/5 halved the effect. 

Histologic examination of the brains showed the tip site to be bounded by 
lateral hypothalamic neurons, the fornix and the cerebral peduncle. This 
agrees well with expected electrode placement. This raises 3 major options 
for mediation of the effect: (1) Direct stimulation or inhibitions of 
vasoregulatory neurons in the hypothalamus; (2) Indirect effect of 
skeletal muscle circulatory dynamics via the cerebral peduncle: (3) Indirect 
effect on the limbic system via the fornix. 



FI-56 



2. Bipolar electrodes: Although evaluation of the data for this 
aspect of the work has not been completed, certain preliminary findings can 
be presented. 

The depressor effect and its dependence on current density recapitulates 
the trend noted above. However, much lower current densities were needed: 
e.g., TD 500 ms, TPS 1.0, PPS 100, PD 8 and current 100 ua. The early 
positive phase, though usually small, i.e. 2-4 mm Hg. was more constantly 
present. The depressor phase showed considerable variablity: i.e., from 
to A8 mm Hg. The variability was particularly interesting in that changes 
in the depressor phase were reciprocal with the pressor phase changes. 
That is, in the 5 rats evaluated to date in this regard, there were U 
episodes where the depressor phase was near zero. The pressor phase in 
three of these instances was a mean rise of 21.3 mm Hg. in diastolic pressure. 
Further evaluation revealed that the depressor effect was directly related 
to the instantaneous pre-stimulation pressure. The amplitude of the pressor 
phase was generally inversely proportional to the instantaneous pre-stimula- 
tion blood pressure, however, this latter relationship was less dependable 
than that noted for the depressor effect. 

Preliminary evaluation of the effects of isoproterenol and epinephrine on 
the production of these changes shows: (a) that the direction and magnitude 
of stimulation induced change in blood pressure was inversely related to 
the net direction of change in the several minutes before stimulation. 
For example, during the recovery phase from epinephrine stimulation a 
pressor effect is produced. The depressor effect is zero despite the 
markedly elevated pre-stimulation pressure levels. (b) that despite the 
unchanging heart rate during usual stimulation, cardiac autonomic efferent 
nerves were also stimulated since stimulation plus isoproterenol produced 
a sustained cardiac arrhythmia. Isoproterenol alone did not produce an 
arrhythmia. 

Significance to Biomedical Research and the Program of the Institute ; 

A. Operant Conditioning of Heart Rate. 

Experiment 1 provides clear evidence that the form of at least one conditioned 
response, always regarded in the past as innate, can be modified by operant 
learning. This modification may persist long after it ceases to be differ- 
entially rewarded. While nothing can be said as yet about the mechanism 
or generality of this phenomenon, it offers an experimental approach to 
the large set of psychosomatic diseases that are apparently mediated by 
maladaptive autonomic responses. Experiment 2 also is important to our 
understanding of the nature of psychosomatic disorders. Despite the 
plethora of theoretical articles on the role of behavioral stress in the 
etiology of such illnesses, definitive experimental evidence of this process 
does not exist. 

B. Depressor activity of the rat hypothalamus. 

The work performed to date can provide a basis for further exploration of 
hypothalamic cardiovascular regulation. The rat appears to offer the advantage 

FI-57 



that the spectrum of regulatory maneuvers are much more limited than those 
of higher mammals. 

Propose Course of Project . 

A. Operant Conditioning of Heart Rate. 

In Experiment 1 comparison of the results of interpolated slowing training 
with results of speeding training should discriminate between the two 
most likely mechanisms for the operant modification of these conditioned 
responses: "Superstitious" failure of an avoidance response to extinguish 
(analogous to the self-punishment that has been described for some skeletal 
muscle operants), or some intrinsic effect of voluntary slowing that 
mitigates the aversiveness of shock. Trials using other unconditioned 
stimuli (air puffs, frights, etc.,) should provide data about whether the 
new responses generalize to other situations. This project will be 
completed within this fiscal year. As already indicated, experiment 2 will 
continue for at least 6 mos. 

B. Depressor Activity of the Rat Hypothalamus. 

Once the depressor site in the hypothalamus has been specified, phase 2 
of the study will begin. In this phase rats will have chronically implanted 
electrodes in their hypothalamuses , and after they have been made hypertensive, 
they will be allowed to stimulate their brains electrically. Control 
animals which are normotensive also will be tested. 

Honors and Awards : Dr. Engel lectured to the staff and fellows of the Center 
for the Study of Aging and Human Development, Duke University School of 
Medicine (February, 1973). 

Dr. Engel presented a lecture at the Washington School of Psychiatry 
(March, 1973) 

Publications : Engel, B.T. and Bleecker, E.R. Application of Operant 
Conditioning Techniques to the Control of the Cardia Arrhythmias, (in) 
Christ, P. A., et al. Contemporary Trends in Cardiovascular Psychophysiology. 
Aldine-Atherton, Chicago, in press. 



FI-58 



Serial No. HD-AG14 (8) (c) 

1. Gerontology Research Center 

2. Laboratory of Behavioral Sciences 

3. Physiological Psychology Section 

4. Baltimore, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project title: Reaction Time and Age 

Previous Serial Number: Same 

Principal Investigator: Bernard T. Engel, Ph.D. (57„) 

Other Investigators: Phillip R. Thorne (57o) 

Cooperating Units: Baltimore City Hospitals 

Man Years: 



Total: 


.50 


Professional : 


.10 


Other: 


.40 



Project Description: 

O bj ectives ; The purpose of this project is to study the effect of delay 
time on human reaction time, and to evaluate the function of age on this 
effect. 

Method Employed : In the reaction time procedure used in this project, 
a subject is presented with an auditory signal (a tone to which he is 
supposed to depress a thumb switch as quickly as possible). The time 
between the onset of the tone and the switch press is the measure of 
his reaction time. Since the purpose of this project is to study the 
effect of time delay on reaction time, two time periods preceding each 
tone are systematically varied: 1) The time immediately preceding the 
onset of the tone--the f oreperiod--is signalled by a light which goes on 
either 3 sec, 6 sec. or 12 sec. before the tone, and which remains on 
until the tone sounds; 2) The time between the end of one trial and the 
onset of the light of the next trial--the intertrial interval--is also 
either 3 sec, 6 sec. or 12 sec. By means of an appropriate statistical 
design, the effects of each of these time periods on reaction time, 
and the effect of the interaction between them on reaction time can be 
systematically studied. 

Major Findings : Since this study has just begun, no results can be 
rt'ported now. It is likely that data collection will be completed within 
our month, howcvor. 



FI-59 



Significance ; It is well-known from a number of previous studies in 
this laboratory and elsewhere that older men have more difficulty coping 
with time pressure than younger men. This fact has been shown in 
studies of learning, problem solving, motor ability, and in intelligence 
tests. However, the mechanism underlying the aging effect is not 
known. The purpose of this project is to determine whether there are 
age differences in reaction time as a function of foreperiod duration 
and/or intertrial interval. If so, then it will be possible to 
utilize this model system to analyze the mechanisms which mediate the 
aging effect. 

Proposed course ; Two groups of 20 men each, one young (age 20-30) and 
one old (50-60) will be studied. Depending upon the results, a third, 
older group may also be studied. If an age effect is observed it will 
be possible, because of the experimental design chosen, to determine 
whether the effect occurs during the foreperiod or during the intertrial 
interval. Subsequent studies will then be designed to analyze the effect 
further. 

Honors and Awards ; None. 

Publications; Engel, B.T., Thorne, P.R. and Quilter, R.E. On the 
relationships among sex, age, response mode, cardiac 
cycle phase, breathing cycle phases, and simple reaction 
time. Journal of Gerontology . 1972, 11_, 456-460. 



FI-60 



NICHD Annual Report 

July 1, 1972 through June 30, 1973 

Laboratory of Behavioral Sciences 

Contract and Collaborative Research 



Contract Number: 
Contract Title: 

Contractor: 
Money Allocated: 



NIH 70-2296 

Twenty-five year follow-up analysis among former Army 
Air Force cadets. 

University of California (Los Angeles) 
None for FY 73 

$10,673 (supplement for FY 72 
36,251 (total for FY 71 and FY 72) 



Objectives : In 19AA Dr. M. A. Wenger carried out a series of psychological 
and physiological tests on a group of 2111 Army Air Force cadets. In 
1964 he sent health questionnaires to 1032 of these men and received 
725 responses. On the basis of an analysis of these results it appeared 
that the 1944 tests might predict the occurence of some diseases among 
these men. 

The purpose of this contract is to enable Wenger to carry out a survey 
of as many of the 2111 men as he can locate to determine the value 
of the 1944 test battery in predicting the subsequent development of 
certain psychological and physiological disorders. The reason this 
contract was initiated through this laboratory is that if the Wenger 
test battery does have predictive validity we would propose to include 
it in the longitudinal study program of the Gerontology Research Center. 

Major Findings : It was possible to locate 1624 of the original sample 
of 2111 men (777,.). Of these, 1444 (897o) were living and 180 were dead. 
Among the sample of 1624 men, 1248 who were physically fit at the time 
of testing in 1944 were still alive and 159 were dead. 

A detailed summary of findings is available with the contractor's final 
report. This report will summarize some of his major observations. 

1. Subjects who died of heart attacks classified as other than 
myocardial infarction had autonomic balance scores in 1944 which 
indicated relative sympathetic nervous system (SNS) dominance. 

2. Subjects who developed high blood pressure showed SNS dominance 
in 1944, whereas, subjects who were classified as having low blood 
pressure in 1971 had autonomic balance scores in 1944 which were 
indicative of relative parasympathetic (PNS) dominance. 

3. In addition to the results already cited, subjects who 
developed bronchitis, skin rash or hapatitis also showed PNS dominance, 
whereas, subjects who complained of eczema or periods of anxiety in 
1''71 showed SNS dominance in 1944. 



FI-61 



4. Most of the subjects who reported the presence of "psychosomatic" 
symptoms showed PNS dominance. Since most of the items checked indicated 
gastro-intestinal or respiratory complaints, the PNS effect seems 
reasonable. Interestingly, the only two "psychosomatic" items which 
showed a significant SNS effect were "severe itching" and "swollen ankles". 
The correlation between itching and SNS dominance is consistent with 
the previously reported relationship between SNS dominance and eczema. 

Significance ; The results of this survey indicate that tests of autonomic 
balance can predict the occurrence of symptoms or of diseases 25 years 
later. Although the predictions are not sufficient to be directly 
applicable, they are sufficient to indicate that such tests are worth 
considering in a longitudinal test battery. 

Proposed Course : As soon as the resources become available, pilot 
studies will be proposed for inclusion in the Baltimore Longitudinal 
Study Project. 



FI-62 



NICHD Annual Report 
July 1, 1972 through June 30, 1973 
Gerontology Research Center 
Clinical Physiology Branch 

Research in the Clinical Physiology Branch continues to be geared toward 
a physiological and biochemical dissection of mechanisms underlying both 
adaptive and the deteriorative changes of aging - and, when possible, to 
accomplish this goal in man. An understanding of the complex mechanisms 
underlying the processes of "normal aging" is an essential preliminary step 
to an understanding of the still more complex interrelations of aging and 
disease. As these complexities are clarified, preventive and therapeutic 
measures can logically be anticipated to follow, with the ultimate goal of 
achieving the physically and mentally vigorous old age that is now reached 
by only a small fraction of the population. 

The invaluable resource for our study of human aging continues to be 
the Longitudinal Aging Study. This year has been one of continued development 
and refinement of unique techniques of longitudinal data analysis. We previously 
described a new statistical technique which enables the gerontolog ist to fin (]) 
the required duration of a longitudinal study, (2) the required frequency 
of testing for data acquisitions, and (3) the required number of subjects 
needed in order to define the rates of aging changes in a variety of critical 
functions with specified accuracy. We can now report the degree of success 
achieved using the experimental strategies of the current Longitudinal Study. 
Several examples can illustrate this technique: 

(a) To quantitate age changes in the lung, the forced expiratory volume 
in one second (FEV 1.0) has been measured; the mean rate of aging can be 
defined in young and middle-aged adults with less than 25% error and in old 
subjects with less than 10% error. 

(b) Blood pressure increases with age show important trends not pre- 
viously realized. We have recorded blood pressure under truly basal conditions 
(during basal metabolic rate measurements) and under "casual" conditions, that 
is, as recorded by a physician during a physical examination. The variance 
attendant upon the usual casual conditions of blood pressure recording makes 

it impossible to detect true age changes in systolic pressure with adequate 
accuracy. Basal systolic pressure on the other hand shows progressively 
greater rates of increase with advancing age and these age changes can be 
defined with statistical confideqjce. 

(c) Serum cholesterol and triglyceride levels show strikingly different 
aging trends. Statistically significant increases occur in cholesterol 
(errors of less than 25%) in young and middle-aged adults, but level off in 
late life. Triglyceride levels are constant in young adults and fall signi- 
ficantly (errors again less than 25%) in middle-aged and older men. Reasons 
underlying the differences in these changes await multivariate analysis of 
dietary, activity, and other metabolic variables in the coming year. 

The versatility of the new analytical technique is best seen when it Is 
used predict ively, i.e. for future planning. Again an example can illustrate 
the principle. In order to define the rate of increase in basal systolic 
blood pressure in middle-aged adults with great accuracy (defined here as an 

FJ-1 



error of less than 25%) several experimental strategies could be followed: 
(1) i f an annual examination is performed, 23 years will be required to meet 
this accuracy goal; (2) if examinations are performed every 18 months, 27 
years will be required; (3) if biennial examinations are made, 30 years will 
be required. Thus for this particular variable , planners of longitudinal 
studies must think either in terms of a very long-term commitment or in terms 
of much more frequent examinations than are usually performed. Furthermore 
we can show that most of the physiological variables of interest to geronto- 
logists will require from 15 to 30 years of study in order to achieve reasonably 
accurate estimates of rates of aging across the adult age span. This statis- 
tical approach in combination with the basic data provided by the Baltimore 
Longitudinal Study is an achievement which clearly has far-reaching implications 
for the future. 

The difficult problem of separating age changes from disease changes has 
not been dealt with adequately, either theoretically or practically, by other 
ongoing studies of human aging. We have devoted much effort the past year to 
the development of techniques to permit this separation. An automated chart 
review was the initial step which converted detailed data from medical histo- 
ries, physical examinations, and laboratory data to an efficiently arranged 
computerized summary. Multiple-choice entries on histories have been found 
frequently to be misleading unless the "free field" comments of either the 
subject or the physician are also considered. These comments are also inclu- 
ded in the computerized summary. With these data now available on over UOOO 
subject-visits, detailed criteria have been developed for the classification 
and identification of those disease states which could alter physiological 
functions independent of age. Detailed classifications should provide the 
standards which other longitudinal studies of human aging will be able to use. 

New studies have been introduced into the Longitudinal Study this year. 
Cutaneous malignancies are a serious problem especially in older subjects. 
Studies of chronic actinic damage as a precursor of these malignancies have 
assumed that the damaging radiation spectrum is 290-320 nm and that longer 
wavelength ultraviolet radiation (320-^00) causes "tanning" and may even 
protect against sun damage to skin. A comprehensive study on the effect of 
age on sensitivity to UV light of different wavelengths and on the time 
course of these radiation effects is therefore underway. Results to date 
show that the longer UV wavelengths actually have an augmentative effect, 
not a protective effect; thus present preparations to prevent sun damage should 
be modified so that they protect against a fuller spectrum of UV light. 
Furthermore, the studies show that individual responses to radiation can be 
determined in a much shorter period of time than had previously been thought 
possible; this may have implications for future screening techniques. 

New genetic markers have been introduced into the longitudinal study in 
order to assess further the role of genetics in the aging process. We are 
now measuring haptoglobin, ceru loplasmi n, and transferrin in plasma and are 
obtaining dermatog lyph i c analysis on all subjects. 

An interesting analysis have been made of various estimates in use for 
the determination of body fatness. We have obtained not only heights and 



Fj-2 



weights, but also the detailed Behnke Index (which includes the abdominal 
girth), and skin-fold thicknesses. By taking advantage of the longitudinal 
nature of the data, we have available many instances of changes in body weight, 
both gains and losses, which in our individual subjects are due almost entirely 
to spontaneous changes in caloric intake on their part. Thus changes in body 
weight largely reflect changes in obesity rather than changes in lean body 
mass. Changes in the individual indices of body fatness were correlated 
with changes in body weight in order to test which of the measurements was 
the best index of changes in obesity tissue. The rather surprising result 
was that changes in skin fold thickness correlated rather poorly with changes 
in body weight with an r of only O.k; the complex Behnke Index correlated 
better with r values of 0.64 - O.78 in subjects in different age groups, while 
the best indicator was the simple measurement of abdominal girth which corre- 
lated between 0.64 and O.9O in subjects of different ages. The inadequacy of 
height-weight tables of judging obesity has been commented upon repeatedly 
and generally skin-fold thickness measurements have been recommended as a 
siimple addition to the measurement of height and weight in epidemiologic 
studies in order to improve the accuracy of the esimate of obesity. It would 
appear that abdominal girth is a distinctly better measurement of fatness 
than is skinfold thickness. 

The nature of the age decrements in myocardial performance has been 
explored by studies using the isolated trabeculae carneae of young, middle- 
aged, and old rats. In these perfused preparations, single and paired 
stimulation has been given under conditions of exposure to graded levels of 
norpinephr ine and to hypoxia. Biophysical muscle responses have been 
quantitated. The oldest animals show a reduction in developed tension and 
in the maximal rate of tension development in response to catecholamines. 
No age differences occurred in the paired-stimulation and hypoxia studies. 
The successful development of this experimental procedure and the demonstration 
of age differences in response to catecholamines in these heart muscle pre- 
parations will lead to further pharmacologic studies on the role of the 
sympathetic nervous system in determining age differences in responses of 
the heart to stress. 

Studies have also been carried out on the intact dog heart to test the 
hypothesis that there is a damaged but salvageable area of heart muscle after 
coronary ligation. The experimental variables in this complex, carefully 
controlled preparation are (1) myocardial damage as quantitated by ST seg- 
ment surface mapping, (2) i nt ramyocard ia 1 oxygenation using a polarographic 
method, (3) coronary artery perfusion pressure. Results show significant 
correlation between the ST segment map and myocardial oxygen tension on a 
minute-to-minute basis in each dog. Furthermore increasing the coronary 
perfusion pressure significantly improves oxygenation and decreases the area 
of ST segment abnormality. There is thus now experimental evidence (1) to 
support the validity of using ST segment changes as quantitative indices of 
the ischemic zone and (2) to support the rationale of elevating coronary 
perfusion pressure to reduce infarct size. 

Study of the chemistry of human renin, a kidney enzyme involved in the 
pathogenesis of hypertension, has progressed during the past year through 



FJ-3 



the use of a new labeled polymeric substrate assay previously developed in 
the Endocrinology Section. We have discovered that the enzyme is inhibited 
by a variety of proteins and peptides. Studies of the mechanism of this 
inhibition with a model protein, hemoglobin, with hemoglobin chains, and with 
chemical and enzymatic cleavage fragments of hemoglobin have shown that large 
peptides are the most potent inhibitors, but that peptide fragments are also 
effective. The leuci ne- leucine bond, which is necessary for renin action on 
its substrates, is not necessary for inhibition by hemoglobin fragments. A 
number of small synthetic peptides have also been discovered to be potent 
inhibitors of renin. Structural requirements for inhibition have been studied. 
Renin preparations, free of peptidase activity, have been shown to contain 
protease activity against hemoglobin. A new concept of renin emerges in which 
renin shares many of the characteristics of only relatively specific acid 
proteases of wide biologic distribution. The techniques and concepts recently 
developed in our laboratory should permit the development of renin inhibitors 
of potential clinical usefulness. They will also allow the development of 
affinity chromatography procedures which could result in the isolation of the 
enzyme in pure form. 

The possible involvement of the adenyl cyclase ("second messenger") 
system in senescence is being explored. This membrane-bound enzyme is 
activated as the initial step in the action of many hormones. As a result, 
cyclic adenosine monophosphate (cyclic AMP) is formed; a variety of metabolic 
processes are then initiated by this compound. Although hints of involvement 
of this system in certain age-related alterations of hormone action can be 
extracted from the literature, no actual studies of the enzyme in senescence 
have been reported to date. Our preliminary data now indicate that, in rat 
liver, senescence is associated with a marked increase of activation of 
adenyl cyclase by epinephrine. Although non-specific activation with fluoride 
is also somewhat enhanced, activation by another hormone, glucagon, is unaffected. 
These observations indicate the existence of hormone-specific effects of age 
on membrane structure and function. Moreover, they suggest that altered 
cyclase responses may provide a molecular basis for the altered sensitivity 
to hormones which has been seen with aging. This line of investigation is 
being extended to include a variety of hormone-activated cyclase responses 
in a number of tissues. 

The previously developed model for insulin and glucose metabolism in man 
has been used to compute changes in glucose content of two of the glucose 
compartments when changes in glucose concentration are induced in the third 
compartment (the blood volume). This computation is critical especially when 
large changes in blood glucose concentration occur rapidly, as in the hyper- 
glycemic clamp experiments. Since one of the experimental variables computed 
in these studies is glucose utilization by the body, the glucose which has 
been infused but which has gone only into the extracellular glucose spaces 
must be subtracted from the total glucose infusion rate. This computation 
could not be made with any degree of accuracy until a successful glucose 
model was developed. 

The complex biphasic response of insulin secretion in response to 
hyperglycemia has now also been treated mathematically by the development of 



FJ-4 



a model of the pancreas. The pancreas model behaves "correctly" in terms of 
its release of insulin in response to hyperglycemia and it can also reproduce 
the different insulin responses in young and old subjects. Factors influencing 
insulin secretion other than glucose concentration and age can also be accounted 
for by the model. The next goal is the incorporation of the pancreas model 
into the previously developed glucose and insulin models of the body to 
close the loop of these interrelated variables. 

The glucose-clamp technique (both the hyperglycemic clamp to study beta 
cell sensitivity and the euglycemic clamp during insulin infusion to study 
sensitivity to insulin) has been used in a prospective study of the mechanisms 
underlying the glucose intolerance of uremia. The differentiation of uremic 
glucose intolerance from diabetic renal disease with uremia is important in 
terms of decisions concerning acceptance of patients into dialysis and 
transplantation programs; diabetics with uremia have such poor prognosis 
that they have been given very low priority in these programs. Eight uremic 
patients have been studied. Seven of them showed resistance to insulin, a 
mechanism for glucose intolerance which is clearly different from true diabetes 
mellitus. Four subjects have been studied both before and after chronic 
hemodialysis. All four showed marked improvement in glucose tolerance as their 
uremia was ameliorated. The improvement in tolerance could be accounted for 
by an increase in sensitivity to insulin in three patients and by an increased 
secretion of insulin in the fourth. Therefore, while both factors may play 
a role in uremic glucose intolerance, insens i t i vi ty to insulin appears to 
be the more important. Glucose intolerance in uremics thus should not per 
se make subjects ineligible for definitive therapeutic programs since 
it frequently is not indicative of true diabetes mellitus. 

Studies carried out in collaboration with Baltimore City Hospitals and 
Johns Hopkins University scientists have increased our understanding of 
myeloma kidney. Thirty six patients have now been studied prospectively. 
Changes in urine concentrating and acidifying ability preceded GFR changes. 
PAH clearance decreased out of proportion to decreases in GFR. Only patients 
with free Bence-Jones protein demonstrated impaired tubular and glomerular 
function. Renal biopsy showed that changes in renal function correlated 
closely with tubular atrophy and degeneration, but not with tubular casts. 
It appears than that Bence-Jones protein exerts a direct toxic tubular effect 
and that glomerular dysfunction occurs only after severe tubular damage has 
occurred. 

An interesting patient has evolved from this study, the first successful 
renal transplant in a myeloma patient. A long-term remission of the myeloma 
was induced by cyclophosphamide therapy but renal failure persisted. After 
nine months of dialysis, cadaveric transplantation was accomplished now with 
successful function 14 weeks later. Myeloma is thus no longer necessarily to 
be considered an absolute contra-indicat ion to transplantation, if favorable 
responses to chemo-therapy result in what would otherwise be terminal renal 
fai lure. 



FJ-5 



Serial No. HD-AG4(c) 

1, Clinical Physiology Branch 

2, Human Performance Section 

3, Baltimore, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 



Project Title: Age Changes in Human Performance 
Previous Serial Number: Same 



Principal Investigators; 
Other Investigators: 
Cooperating Units: 



Ronald Fine 
Alan Zuckerman 

N. W. Shock 
A, H. Norris 

Baltimore City Hospitals 

Dr. A. T. Welford 

Department of Psychology 
The University of Adelaide 
Adelaide, South Australia 



n Years: 




Total: 


2.40 


Professional: 


1.40 


Other : 


1.00 



Project Description: 

Objectives: This project is designed to study the effects of aging on: 
(a) pulmonary and cardiovascular responses to exercise, (b) the rate of re- 
covery of physiologic equilibrium after exercise, (c) efficiency of mus- 
cular exercise and (d) work output and fatigue in man. Detailed evaluation 
of pulmonary function provides a basis for identifying a major factor in 
the limitation of work performance. In addition, neuromuscular and cardio- 
vascular limitations are assessed. 

Methods Employed : Measured amounts of physical work are obtained in sub- 
jects of varying ages by means of a calibrated arm ergometer and quantita- 
tive mechanical analysis of limb movement, A treadmill is used to induce 
higher levels of work. Measurements of oxygen uptake, CO2 elimination, 
pulmonary ventilation volume, heart rate, blood pressure, and electro- 
cardiogram are made before, during and after standardized amounts of exercis 
In .ulditiiMi, the volume of the lungs and functional capacities of the pul- 
1111)11,1 r\ system arc evaluated. Other studies include measurements of speed 
i>r iicrvf cmuliict ion, roi\Lex latency and muscle action potentials. 



FJ-6 



Major Findings : During the year, analysis in relation to this project has 
primarily focused upon pulmonary function measurements. 

Two physicians independently classified study participants with respect to 
diagnosed diseases and deformities which could modify performance on pul- 
monary function tests. Subjects falling into this category constituted 
about 17% of subjects in age groups 25-34, 35-44, 45-54, 55-64 and about 
35% of those in age groups 65-74, 75-84 and 85-94, On the average, subjects 
in the disease group had lower maximum breathing capacity (MBC) and lower 
vital capacity (VC) than subjects without evidence of disease. The identi- 
fication of subjects without significant disease thus permits an evaluation 
of the effects of aging processes per se on pulmonary function without con- 
founding effects due to disease and other non-aging factors. It also per- 
mits other variables which might influence the rate of pulmonary aging 
independent of recognizable disease to be evaluated. 

Smoking, for example, has been reported as having a significant effect on 
both MBC and VC. When comparisons between smokers and non-smokers were 
limited to the non-disease group, differences attributable to smoking were 
still present even in this clinically "pure" group. 

Thus, the deteriorative impact of cigarette smoking on pulmonary function 
is demonstrable in subjects who do not yet manifest evidence of broncho- 
pulmonary disease by history, physical examination, or chest x-ray. 

Maximum breathing capacity was found to be significantly related to max- 
imum working power of the arms and shoulders of participants in the longi- 
tudinal studies. Since both maximum work and MBC decrease with increasing 
age, a partial correlation with age held constant was computed (partial 
r = 0,37). The strength of this relationship suggeststhat a significant 
portion of the decline with age in MBC is attributable to a loss of muscle 
and loss of muscular coordination. Tlie remainder of the decline with age 
is attributable to other changes in lung and chest \i«ll. Consideration 
should, therefore, be given to muscle and motor status in the interpretation 
of the MBC test for pulmonary diagnostic purposes. 

An automated on-line data collection system has been developed to improve 
the accuracy of the pulmonary function measurements, eliminate transcription 
errors, provide immediate feedback to the technician, and facilitate new 
approaches to data analysis. All of the raw data will be preserved for sub- 
sequent reanalysis by new methods should they become of clinical interest 
in the future. Tests to be processed by the automated system include: 
Vital Capacity, Forced Vital Capacity, Maximum Ventilatory Volume, and Ni- 
trogen Washout, 

Significance to Bio-Mcdical Research and the Program of the Institute ; 
The decline of the ability of some older people to perform their day-to-day 
activities and to engage in pursuits which contribute to the economic and 
social strength of our society represents a national loss. Identification 
of the physiological, medical and social correlates of high levels of phys- 
ical strength and psycho-motor performance in middle and old age, as well 



FJ-7 



as declines in these abilities, should lead to techniques designed to 
reduce the rate of decline in performance capacities with age. 

Proposed Course : Appraisal of participants in the longitudinal studies 
will be based on measurements of muscle strength in maximum power generating 
ability during arm exercise. Cardiovascular, ventilatory and metabolic 
responses to standardized arm ergometer exercise and monitored treadmill 
exercise will be used to classify participants into fitness categories. 
Measurements of lung volumes and uniformity of pulmonary ventilation will 
be made to characterize the respiratory competence of the longitudinal 
studies participants and to evaluate the benefits of the cessation of 
smoking in this group. 

Honors and Awards: None 

Publications: None 



FJ-8 



Serial No. HD-AG3(c) 

1, Clinical Physiology Branch 

2, Human Performance Section 

3, Baltimore, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Longitudinal Studies of Human Physiology, Biochemistry 
and Psychology 

Previous Serial Number: Same 

Principal Investigators: N. W, Shock, Director 

R, Andres, Clinical Director 

Other Investigators: D. Arenberg 

B. D. Bricker 
M. Butler 

B. T. Engle 
R. Fine 

G. E. Martin 
A. H. Norris 

C. C. Plato 
J. D. Tobin 
A, Zuckerman 

Cooperating Units: Baltimore City Hospitals 

Dr. Bernice H. Cohen 

School of Hygiene and Public Health 

The Johns Hopkins University, Baltimore 

Dr, Gaylord Knox 
Department of Radiology 
Baltimore City Hospitals 

Mrs, Catherine Staneck 
Design Methods Section 
Data Management Branch 
DCRT-NIH, Bethesda 

Df. James Schlesselman and Dr. Samuel W. Greenhouse 
Epidemiology and Biometry Branch 
NICHD-NIH, Bethesda 



FJ-9 



>fcin Years: 

Total: 14.15 
Professional: 4.65 
Other: 9.50 

Project Description: 

Objectives: This project is designed to: (1) secure replicate measures of 
physiological, pathological, biochemical and psychological variables on 
longitudinal study participants at specified intervals; (2) summarize and 
compare the results of testing in relation to age according to cross-sec- 
tional and longitudinal formats; (3) identify factors V'jhich may be related 
to changes of function over time and to age at death; and to (4) determine 
whether the data obtained support one or another theory as to the mechanisms 
responsible for age-related functional decrements. 

Methods Employed : The Sample: Study participants are all male volunteers 
recruited by subjects already members of the program. Recruits agree to 
return to GRC in Baltimore for 2-l/2 days of testing at 12 or 18 month inter- 
vals, depending on age, for an indeterminate period of years. At entry into 
the program, 87% of subjects reported at least some college, 87% were identi- 
fied with professional, technical or managerial occupations, 90% were presently 
married, 81% described themselves as financially comfortable, and of the group 
who returned for the fifth visit, 90% had rated their health as good or ex- 
cellent on both first and fifth visits. 

Testing procedures: Since inception of the study, a major objective of data 
collection has been to secure uniformity in testing procedures. In the area 
of psychological testing, standardized tests of personality, intelligence 
and performance are employed in addition to especially-designed procedures 
to assess verbal-learning and problem-solving abilities. Systematic methods 
have guided inquiry into past smoking habits, the dietary history and areas 
of behavioral experience. Physical examinations and medical histories are 
performed with check lists to insure completeness, while the variables gen- 
erated by tests of kidney function, pulmonary function, neuromuscular func- 
tion, cardiovascular function, and tests of vision, hearing and reaction 
time follow well-defined and generally accepted procedures. 

Data management: In addition to an implied obligation to protect personal 
privacy and to submit reports of the physical examination to designated per- 
sonal physicians, primary emphasis is given to the derivation of 'best 
measures' which are posted for purposes of keypunching and verification. 
These data are then transferred into the computer file, but before being 
utilized, are reviewed for completeness and reasonableness. A further re- 
view is performed by summarizing test results by age groups and calendar 
years to be certain of the consistency of statistical parameters over the 
course of the study. 

Hijor Findings : By April 4, 1973, 960 male subjects had completed one or 
morn visits to GRC, accounting for a total of 4419 participant visits since 

FJ-10 



the beginning of data collection. As of this date, 65 7 subjects were class- 
ified as being of active status, 1 had requested inactive status, 106 had 
deceased, 82 had formally withdrawn from the study and 114 no longer respondpd 
to inquiry. 

In a tabulation designed to compare study subjects born in different decades 
according to social class, three socioeconomic indicators were employed: 
years of school completed, highest degree received and usual occupation. 
The percentage distributions over birth cohorts proved remarkably uniform, 
considering the fact that males in their twenties and thirties still have 
an opportunity to complete advanced degrees and to move into technical, pro- 
fessional and managerial occupational positions. Thus, the results help 
resolve the question of whether our recruiting procedures may have resulted 
in the inclusion of younger subjects with different socioeconomic character- 
istics. 

A small statistical program library is being developed for use on the on-site 
Raytheon 706 minicomputer to facilitate analysis of the large volume of data 
now on file. These programs should significantly reduce computing costs 
and improve turnaround time for many analyses. The fact that this system 
is compatible with existing programs at DGRT makes it feasible to transfer 
intermediate data tapes between the two systems. However, the primary mas- 
ter file will be maintained at DCRT to perform analyses which are too com- 
plex for the smaller computer to handle. 

The analysis of data from several segments of the Longitudinal Study is 
dependent upon appropriate clinical classification of the study participants. 
From the inception of the study, detailed histories and physical examinations 
have been recorded at the time of each visit using multiple choice question- 
naires supplemented by liberal use of free-field comments. During the past 
year computer programs have been developed to facilitate use of this data 
which has been previously keypunched, edited, and entered into the Master 
File of the Longitudinal Study, The computer-generated chart abstracts 
average two or three pages in length (as compared to two or three volumes 
for the original hand-written charts) and have been successfully used to 
complete clinical classification for a^rdiovascular, Renal, and Pulmonary 
diseases for each of nearly 1000 participants in the study. 

Only the positive findings on history or physical exam of particular interest 
to the investigator is included in the computerized summary along with the 
time course of the finding and any associated free-field comments. The 
availability of these summaries markedly reduce the time necessary to com- 
plete the classifications and also improved their accuracy. The classifi- 
cations themselves were assigned by the individual investigator who read 
the summaries and not by a computerized diagnosis algorythm. A computerized 
diagnostic program could not have dealt with the complexity of the free-field 
comments, which significantly improved the quality of the data gathered by 
multiple choice questionnaires. This detailed review of the clinical data, 
gatliered as part of the Longitudinal Study, has led to plans for improving 
the quality of those data during the next data collection cycle which will 
begin in July, ]973. Preliminary work for developing on-line automated 

FJ-11 



patient interviowing systems has also been undertaken as an outgrowth of 
the Automated Chart Review Project, 

Although creatinine clearance has been shown to decrease with age, it has 
been questioned ^'Thether this decrease represents time aging per se or the 
adverse effect of common diseases wliich become increasingly prevalent as 
age advances. These diseases include neplirolithiasis, prostatism, hyper- 
tension, urinary tract infections, diabetes, and arteriosclerotic cardio- 
vascular disease. We have, therefore, categorized each visit on each subject 
in the Baltimore Longitudinal Study with respect to those clinical disorders 
which are known to influence creatinine clearance, A group of 5 71 men remained 
who had none of these disorders. Despite this intensive "purification" of 
the population, a striking effect of age was present. There was a progressive 
decline in clearance, decade by decade, from a level of 139 ml/min/l,73 m^ 
surface area (± 22 SD) at age 30 to 96 ± 20 at age 80, Analysis of symptoms 
of prostatism and prostatic size showed no effect of these variables on 
creatinine clearance in this population. These data, from a large, intensive- 
ly studies group, define normative age-corrected standards for creatinine 
clearance in man. 

The Longitudinal Study has provided some unique information on blood pres- 
sure and aging. Blood pressure is measured in the usual fashion as part of 
the physician's physical examination, and in addition, basal blood pressures 
are obtained during standardized BMR conditions. These measurements are 
referred to as "casual" and "basal" blood pressures. Analyses of age dif- 
ferences between casual and basal pressure and in pulse pressures under 
these conditions have now been made. The clinical classification scheme 
used for creatinine clearance was applied to the blood pressure data to 
minimize the impact of age-related diseases on intrinsic aging processes. 
On 581 subjects v^o were defined as normal, a clear-cut "stress effect" of 
the first examination on blood pressure was shown on a longitudinal visit- 
to-visit analysis of pressure changes; the first visit pressures were clearly 
above the blood pressure slopes established by measurements on subsequent 
visits. True aging trends in blood pressure, therefore, must be computed 
omitting the first data point. 

Age-associated increase in systolic, diastolic, and pulse pressure was found 
as expected. Of importance \'ras the finding that casual pressures exceeded 
basal values by about 8 mm systolic and 5 mm diastolic from ages 30 to 60 
but this excess of pressure under casual conditions fell to about 6 and 2 mm 
respectively in the 70 year old age group. 

These data are of value in interpreting blood pressure data obtained under 
differing experimental conditions. Thus, the critical levels reported by 
the V. A. Cooperative Study Group Trial and measured under resting conditions 
m;iy be at least S/s mm lower than the casual values obtained in a physician's 
office. 

Till' i)launiiig of ](ingitu(liiial studies involves decisions concerning the total 
(liiratinn of' tlie study and the frequency of testing. Such decisions have 
usually dopcndod upon educated guesses rather than upon rational statistical 

FJ-12 



principles. An experimental strategy which will result in the accurate 
definition of the rate of change (slope) in a function depends upon (T ) 
the magnitude of the slope, and (2) the variance in the data points. Tlius, 
a function which changes strikingly with age and in which the data points 
comprising the individual slopes show little variance, can be defined with 
reasonable accuracy in a relatively short period of time with relatively 
infrequent observations. The obverse is equally true. Using statistical 
methods recently developed at NICHD, and using data from the Baltimore 
Longitudinal Study, we can now provide estimates of appropriate strategies 
for a variety of functions which are of interest to physiological and clin- 
ical gerontologists. The implications for the planning of ongoing and 
future longitudinal studies as well as for possible therapeutic intervention 
studies are clear: on the usual annual or biennial testing schedule, most 
functions will require about 15 years of testing in order to define aging 
rates with acceptable accuracy. 

Significance to Bio-Medical Research and the Program of the Institute : 
A major goal of the Longitudinal Program is a deeper understanding of age- 
related changes in the different organ systems, and their interrelation- 
ships. In this regard, the identification of coronary artery disease 
serves to characterize an age-related disease in the cardiovascular system. 
Numerous studies elsewhere have demonstrated that heart disease occurs with 
increased frequency in those who are hypertensive, inactive, obese or dia- 
betic and in those who smoke cigarettes or have elevated blood lipid levels. 
The potential contribution of our Longitudinal Study to this important pub- 
lic health problem lies in the intensity of study of a number of these 
variables in this study. 

Collection of this body of data will place us in a position to test theories 
of aging with the human population of the longitudinal studies. Results 
of comparisonsbetween old and young individuals are presently available, 
but these do not permit us to evaluate theories of aging adequately. Our 
ability to do so will depend in part upon a comparison of the integrated 
medical , physiological, psychological and biochemical profiles of the 
individual participant with various subsequent outcomes which we may be 
in a position to monitor in the same individual. Outcomes such as length 
of life, occurrence of disease, rate of change in a functional parameter, 
among other things, will be sought. Some theories assume that a few cells 
are lost during each unit of time so that in an individual or in an indi- 
vidual organ system changes of a gradual nature would be found. These 
theories are consistent with the summation of a series of distributed minor 
accidents (such as radiation hits) if only a few cells are involved each 
time. Other theories would relate large changes in function with the 
occurrence of a disease episode, or major accident resulting in n substan- 
tial loss of cells. These theories are consistent with abrupt changes in 
functional levels which would be maintained until another major accident 
results in a new level. In addition, these two widely different types of 
theories could be found in combination with sudden as we31 as s] ow changes 
operating simultaneously. 

Proposed Course : Data collection will be continued as in the past, except 
as new equipment may provide greater capacity for accurate detei-minations 
and for automating procedures. The fact that automated procedures for 

FJ-13 



testing auditory reaction time and for measuring numerous parameters of 
pulmonary function have now been inylemented will greatly aid the achieve- 
ment of the above objectives. 

Honors and Awards: Dr, Shock received the American Pioneers in Aging Medal 
from the Institute of Gerontology at the University of Michigan — Wayne State 
University, 1972, 

Dr, Shock received the First Annual Award for Outstanding Contributions to 
Bio-Medical Sciences and Aging from the Ethel Percy Andrus Gerontology Center 
at the University of Southern California, 1973, 

Publications: Shock, N. W, : Gerontology - past, present, and future. In: 
D.F. Chebotarev, V, V, Frolkis, and A, Ya, Mints (Editors), Proceedings of 
the 9th International Congress of Gerontology, Vol. 1, Plenary and Section 
Sessions^ iTie Congress, Kiev, U.S.S.R. , July, 1972, pp, 13-21. 

Andres, R, , and J. Tobin: Aging, carbohydrate metabolism and diabetes. In: 
D. F. Chebotarev, V. V. Frolkis, and A. Ya. Mints (Editors), Proceedings o? 
the 9th International Congress of Gerontology, Vol, 1, Plenary and Section 
Sessions . The Congress, Kiev, U.S.S.R., July 1972, pp. 276-280. 

Tobin, J. D., R, S. Sherwin, J. E. Liljenquist, R, A, Insel, and R. Andres: 
Insulin sensitivity and kinetics in man. (Abstract 435) in: D.F. Chebotarev, 
V. V. Frolkis, and A. Ya. Mints (Editors), Proceedings of the 9th International 
Congress of Gerontology, Vol. 3, Abstm cts . The Congress, Kiev, U.S.S.R. , 
July 1972, p. 155. 

Shock, N. W. : Energy metabolism, caloric intake and physical activity in the 
aging. In: L. A. Carlson (Editor), Nutrition in Old Age (X Symposia of the 
Swedish Nutrition Fdn.), Almqvist & Wiksell, Uppsala, 1972, pp. 12-23. 

Shock, N. W. : Gerontology and geriatrics. In: Encyclopaedia Britannica . 
Encyclopaedia Britannica, Inc., Chicago, 1973, pp. 363-365. 

Shaw, D. J., D. A. Rothbaum, C. S. Angell, and N, W, Shock: The effects of 
age and blood pressure upon the systolic time intervals in males aged 20-89 
years, J. Gerontology, 28: (2), 133-139, April 1973, 



FJ-14 



Serial Nu. lID-AG5(c) 

1. Clinical Physiology Branch 

2. Human Performance Section 

3. Baltimore, Maryland 

PHS-NIII 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Age relationships of body composition, nutrition and physical 
activity 

Previous Serial Number: Same 

Principal Investigators: Reubin Andres 

N. W. Shock 

Other Investigators: Marlene Butler 

Arthur H. Norris 

Cooperating Units: Baltimore City Hospitals 

Dr. Paul Gyorgy 
Philadelphia General Hospital 
Department of Public Health 
Pliiladolphia, Pennsylvania 

Dr. M^-ron Brin 

Assistant Director for Clinical Nutrition 

Hoffman-LaRoche, Inc, 

Nutley, New Jersey 

Professor George Vose 
Research Institute 
Texas Women's University 
Denton, Texas 

Dr. Harald Schraer 
Department of Biophysics 
Pennsylvania State University 
University Park, Pennsylvania 

Man Years: 

Total: 2.15 

Professional: .15 
Other: 2.00 



FJ-15 



Project Description: 

Objectives : This project is designed to describe differences in body com- 
position, nutrition, and physical activity between males of diverse age, 
examine these differences in relation to age, and to determine whether such 
differences may influence physiological and biochemical characteristics of 
individual subjects. 

Methods Employed : Height, weight, and body circumferences of longitudinal 
study participants are obtained by standard anthropometric methods, Roen- 
tgenographic and anthropometric estimates of skeletal mass are combined 
with height, weight, and body circumferences to provide an estimate of 
body fat. Other estimates of fat include skinfold thickness measurements 
and fat thickness measurements from X-rays. Estimates of lean body mass 
include: (1) basal metabolic rate determinations made with standard open 
circuit methods, (2) twenty-four hour urinary excretion of creatinine, and 
(3) total body water and extracellular i^ater determinations made from the 
distribution curves of injected antipyrine and sodium thiocyanate. Nu- 
trient intakes and activity calories are estimated from interviews and self- 
administered questionnaires. All such measurements are repeated according 
to schedule in the course of each subjects participation in the longitudi- 
nal program. 

In a collaborative study with nutritioniste at Philadelphia General Hospital 
and at Hof fman-LaRoche, Inc. , the status of three vitamins of the B complex 
are assessed. Excretion levels of thiamine (Bi) and riboflavin (B2) are 
obtained from a 24 hour urine collection. Transketolase activity and 
glutathione reductase activity in red blood cells are measured and serve 
as additional indicators for adequacy of thiamine and riboflavin respectively. 

Tests for pyridoxine (B5) status include determinations of pyridoxal phos- 
phate in plasma and glutamic oxalic transaminase in plasma and in erythro- 
cytes. Amino acid load tests using tryptophan and methionine are also 
given. One-half of the subjects receive tryptophan in an amount equal to 
25 mg per kg body weight. A subsequent urine collection is analyzed for 
xanthurenic acid. The rest of the subjects receive methionine, 50 mg per 
kg body weight. The urine collected after the methionine load test will 
be analyzed for cystathionine and other methionine metabolites. 

Major Findings: The collection of blood and urine samples for purposes of 
the B vitamin study has been completed for 620 men, 91% of the active 
longitudinal studies population. The tentative conclusions made last year 
have been strengthened and no further data collection is planned at this 
time. Manuscripts are in preparation. 

Interrelationships of measures of fat and lean body mass have been computed 
in the longitudinal studies population. Indices of fatness were skinfold 
thicknesses, abdominal circumferences and an estimate of fat based on the 
Behnko Anthropometric Index. Indices of lean mass were basal metabolic 
rate, 24 hour creatinine excretion, intracellular water, biceps circumfer- 
ence and the Bclinkc Index, Stature, arm strength and caloric expenditure 

FJ-16 



attributable to physical activity were also considered (1) in a correlation 
analysis and (2) as predictors of change in body weight. 

To avoid confounding of the relationship between paired variables because 
of a common relationship of the variables to age, partial correlation 
coefficients were computed with age held constant. Currelations among 
variables which estimated fat were significant (partial r = 0.46 - 0,61), 
Similarly, variables which estimated lean body mass were significant 
(partial r = 0,34 - 0.50). As expected, total body weight was strongly 
related to both fat and lean mass estimators (partial r = 0.46 - 0,84), 
In addition, abdominal circumference was strongly related to both fat 
and lean mass estimators (partial r =0,49 - 0.84). Stature, arm strength 
and activity calories were not significantly related to fet and lean mass 
estimators. Stature was related to total body weight (partial j* = 0.47), 

The group of estimators of fat were interrelated as were the group of 
estimators of lean body mass. There were no correlations between the 
estimators of lean mass and estimators of fat. Body weight and abdominal 
circumferences were related to both groups of estimators. 

The relative usefulness of these estimators of body mass for estimation 
of change in body fet was evaluated. It was assumed that weight changes 
between visits (12 to 18 month interval) represented gain or loss of fat. 
This is probably a valid assumption for this relatively sedentary popu- 
lation. Visit by vist changes in weight were compared to changes in 
estimator variables for corresponding visits. Correlations between 
A weight and ^estimator were calculated for young (15-44 years), middle 
aged (45-64 years), and old (65 or more years) subjects. The best estimator 
of change in weight was abdominal circumference, r = 0,90 in young subjects 
to r = 0,60 in old subjects. The second best estimator was the Behnke 
Anthropometric Index, r =0,78 in young to r = 0,64 old. 

Change in skinfold thickness and change in weight were correlated with an 
r of only 0.37 and there was no difference between young and old subjects 
in this relationship. The surprising finding that change in abdominal 
girth reflects change in body weight better than does skinfold thickness 
suggests that the addition of abdominal girth to height and weight measure- 
ments in epidemiological studies may yield a better index of fatness than 
will skinfold measurements. 

Significance to Bio-Medical Research and the Program of the Institute ; 
Nutritional deficiencies in the agalare known to be common and are gen- 
erally attributed more to the socio-economic deprivation of this group than 
to biological or physiological aging effects. The volunteers in the Longi- 
tudinal Study Group under investigation at the Gerontology Research Center 
are not a deprived group — it may be characterized as upper-middle' class 
and has a very high educational level. It, therefore, offers a unique 
opportunity to study nutritional status under very favorable conditions. 
The nutritional effects of biological aging per sc may therefore be sepa- 
rated from what miglit be called "social aging." 

Certain clianges in organ systems with advancing age and various entities 

FJ-17 



of disease are thought to be affected by diet, levels of physical activity 
and body composition. From the repeated assessment of these fectors over 
time, it may be possible to determine their relative contributions to 
longevity and the maintenance of health and vigor in later life. Difficul- 
ties associated with obtaining estimates of eating habits, activity and 
body composition in the past make a prospective approach necessary for the 
collection of reliable information. 

Proposed Course : Prediction equations for suggested estimators of fat and 
lean body mass will be developed. Interrelationships of changes in body 
composition and changes in physical activity and food intake will be 
explored. 

Future plans for the B vitamin study are to complete the laboratory and 
the statistical analyses on the 620 men. Repeat tests available on 160 
subjects afford an opportunity to study the variance of the measurements 
within individuals. 

Mean daily intakes of thiamine, riboflavin, and pyridoxine, as calculated 
from a seven-day food intake record, as well as amounts of these three 
vitamins ingested daily from vitamin supplements, will be examined for 
age differences and these intakes will be correlated with the biochemical 
estimates of B vitamin status. 

Correlations between urine tests and blood tests for the same vitamin will 
be done for each category of adequacy; low, normal, and high. 

Subjects will be classified according to the presence of several diseases 
which may be related to vitamin deficiency and each group will be examined 
for differences in vitamin status. 

Studies of diet, physical activity, and body composition will continue to 
be performed according to schedule. Correlations among the several esti- 
mates of lean body mass and of obesity will be further analyzed. The 
power of these estimates in predicting certain deleterious aging variables 
will provide evidence of the relative value of these measurements. 

Honors and Awards: None 

Publications: None 



rJ-18 



Serial No, HD-CP2(l)(c) 

1, Clinical Physiology Branch 

2, Human Performance Section 

3, Rillimore, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Identification and characterization of cerebrovascular 

disease in Longitudinal Participants and correlations with 
other parameters of physical and psychological function. 

Previous Serial Number: Same 

Principal Investigator: C. Paul Scott 

Other Investigators: Arthur H. Norris 

Reubin Andres 

Cooperating Units: Baltimore City Hospitals 

Man Years: 



Total: 


0.00 


Professional : 


0.00 


Other: 


0.00 



Project Description: This project has been incorporated into the project: 
Longitudinal Studies of Human Physiology, Biochemistry and Psychology, 
Serial No. HD-AG3(c). The classification system developed under this project 
is being continued for later evaluation as data accumulate. 

Honors and A\*irds: None 

Publications: None 



FJ-19 



Serial No. HD-CP4(9)(c) 

1. Clinical Physiology Branch 

2. Human Performance Section 

3. Baltimore, Nferyland 

PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Pulmonary Function Studies: Spectral analysis of cardio- 
pulmonary parameters as a means of quantifying cardio- 
pulmonary function 

Previous Serial Number: Same 

Principal Investigator: Ronald Fine 

Other Investigators: Phillip Thome 

Arthur H. Norris 

Cooperating Units: Baltimore City Hospitals 

Man Years: 

Total: 0,00 
Professional: 0.00 
Other: 0.00 

Project Description: This project has been discontinued. The technical and 
computational methods required could not be solved. 

Honors and Awards: None 

Publications: None 



rj-20 



Serial No. HD-AGl(c)7 

1, Clinical Physiology Branch 

2, Human Performance Section 

3, Baltimore, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1973 through June 30, 1973 

Project Title: Marital, Sexual and Social Factors in Aging 

Previous Serial Number: Same 

Principal Investigator: C. Martin 

Other Investigators: None 

Cooperating Units: Baltimore City Hospitals 

bfcin Years: 

Total: 0.20 
Professional: 0.20 
Other: 0.00 

Project Description: 

Objectives : Major goals are to: (1) describe the effects of age and gen- 
eration on marital adjustment, sexual reactivity and sexual performance, 
(2) determine viiether individual differences in sexual functioning may be 
related to some aspect of health, physiologic state or psychologic trait, 
and to (3) discover whether differences in marital, sexual and social 
background may be associated with specific diseases or longevity. 

Me th od s Empl oy ed : Each subject in the course of participating in the 
longitudinal study, is asked to contribute an interview dealing with his 
history of marriage and sexual activity. Subjects are further questioned 
with respect to parental-home experience, military service, occupational 
and educational history and with respect to indicators of emotional stress. 
In requesting the interview, the investigator outlines the objectives of 
the study, provides assurance regarding confidence and emphasizes the vol- 
untary nature of such a contribution. By now 609 subjects have completed 
interviews, another 16 have partially completed interviews while 14 declined 
to be interviewed. Resulting data are incorporated into the computer 
master file. 

Participants are also asked to complete the Activities and Attitudes 
Questionnaire on their first, fifth and ninth visits to ORG. The ques- 
tionnaire, initially designed to provide scores indicative of personal 
adjustment, contains many items concerning demographic, attitudinal and 

FJ-21 



behavioral kinds of information. Coded responses to these questions are 
regularly updated in the master file. 

Major Findings : Utilizing the contingency table computer program, vari- 
ables derived from the above sources of data were subjected to two types 
of analysis. In the first, or cross-sectional type, variables were tabu- 
lated according to age for evidence of changes with age. Illustrative 
data from the interview, shown below, indicate how current status of 
coital potency tends to decline with advancing age. This finding will 
hardly occasion surprise although the data are unique in that systematic 
information pertaining to current status of erotic reactivity, coital 
activity and erectibility were used to derive the set of mutually exclusive 
and e>diaustive categories. It should be noted that such data provide no 
evidence as to why some men become impotent or partly so with increasing 
age v^ile other men do not. 

Current Status of Coital Potency 



Current Status 



Number of subjects 



Age at Interview 
20-39 40-49 50-59 



60-69 



70-79 



75 



128 



151 



100 



83 



Never or incidental failure 
Occ/freq failure to obtain 
Occ/freq failure to maintain 
Occ/freq both types of failure 
Absent: eroticism or erectility 
Indeterminate: no recent coitus 
Total 





Percentages 






92.0 


89.1 80.8 


62,0 


44.6 


2.7 


2,3 4.0 


4,0 


6.0 




3.1 4.6 


8,0 


9.6 


1.3 


3.1 4.6 


6.0 


7.2 




1.3 


11.0 


15.7 


4.0 


2.3 4.6 


9,0 


16.9 



100.0 



99.9 



99.9 



100.0 



100.0 



The second type of analysis explored the statistical relationships between key 
variables when age was held constant. The example shown below describes how 
the two variables, age at first coitus and number of coital partners before 
age 40, are strongly correlated among subjects falling into two age groups. 
Since this relationship had not previously been noted, these data are again 
unique. Findings of this kind can contribute to a better understanding of male 
sexuality, and knowledge of this relationship may well prove useful for inter- 
pretation should either of these variables appear as an important correlate in 
subsequent analyses. 





Interviewed at 




Interviewed at 






Ages 40-59 






Ages 60-79 




No. 


Coital Partners 


No, 


Coital Partners 


Age First 


b. 


efore Age 40 




before Age 40 


Coitus 


0-1 


2-4 5-8 
8 21 


9+ 
40 


0-1 


2-4 5-8 9+ 


< 18 


2 2 22 


.1 S-2() 


4 


2') 22 


37 


3 


13 12 15 


21-24 


28 


.'Ul 3 3 


6 


21 


13 6 4 


25-49 


33 


6 3 


1 


61 


9 2 



Total 



65 



73 



59 84 



85 



37 



20 43 



FJ-22 



Significance to Bio-Medical Research and the Program of the Institute ; 
The findings reported above and many others involving non-sexual variables, 
generated by these several approaches to analysis are of descriptive and 
correlative interest. On the other hand, in the context of an investigation 
into the processes & vicissitudes of aging, such data are hardly of etiologic 
interest for they supply no clues to the mechanisms involved. Clearly, other 
analytic approaches are needed for etiologic objectives. 

Proposed Course : The third and next stage of anlysis calls for specifying 
those variables whose variation over individuals remains to be explained. 
Here, variables v<?hich represent the culmination of some process over time, 
are related to other variables that are concomittant with or antecedent to 
endpoint status, for evidence of association. Now that data on prostatic 
size and symptoms are nearly complete, plans are to relate the four end- 
point variables: (1) prostatic size, (2) prostatic symptoms, (3) current 
status of coital potency, and (4) level of recent sexual activity, to one 
another, to various physiologic variables and to individual differences in 
marital, sexual and social background characteristics. 

Honors and Awards: Dr, Martin was invited to present a paper on November 1-3, 
1973 to the Society for Life History Research in Psych opathology. 

Publications: None 



FJ-23 



Serial No. HD-CP 13 

1, Clinical Physiology Branch 

2, Human Performance Section 

3, Baltimore, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 



Project Title: Derinatoglyphics and Clinical Medicine 
Previous Serial Numbers 



Principal Investigator ; 
Other Investigators: 



Cooperating Units ; 



HD-CDl(c), HD-GD45(c), HD-CD48(c), HD-CD17(c), 
HD-CD49(c), HD-GD51(c), HD-CD52(c), HD-CD53(c), 
HD-CD43(c), HD-GD44(c) 

Chris C. Plato 

W. Wertelecki 

J, D. Niswander 

F. S. Steinberg 

J. Cereghino 

C, C, Fraumeni, Jr. 

Department of Pediatrics 

Medical University of South Carolina 

Department of Human Genetics, NIDR 

Program Statistics Branch, NICHD 

C & F Research, NINDS 



Man Years: 



Total: 



.50 

Professional: .50 
Other: .00 

Project Description: 

Objectives : To establish possible statistical associations between various 
clinical anomalies and dermatoglyphic features. 

Meth od s Empl oy ed : Finger and palmprints were collected from patients with 
cleft palate, congenital heart disease and leukemia, and from their parents 
and sibs where possible. Prints were also obtained from patients with Down's 
Syndrome and ntli^r types of mental retardation. Prints taken from sibs were 
tn sorvr as iiiliTna] controls in addition to panels of white and black Amor- 
ncans used as ciinlrii.l suli.ji'cts. Several methodologies were developed to 
J'.ic i;i .i talij il(.'l.i i J ('(1 fciiiiparisons. 



FJ-24 



H 



Major Findings : Statistical analysis revealed significant relationships 
between the disease entities of leukemia, Down's Syndrome and deletion of 
the long arm of the 18th chromosome, and certain dermatoglyphic characteris- 
tics. 

Significance to Bio-Modical Research and the Program of the Institute : 
The finding of important statistical associations between various diseases 
and dermatoglyphic markers may assist in the determination of their etiology. 

Proposed Course : To continue this method of investigation taking into 
. consideration other clinical anomalies. 

Honors and Awards: Mr. Plato will present a report on Down's Syndrome to the 
8th Joint Meeting of the U.S.P.H.S, Professional Associations, in Phoenix, 
Arizona. 

Mr. Plato gave three reports on methods and one on leukemia at the IV Inter- 
national Congress of Human Genetics (Paris), 

Publications: Plato, C. G, , Cereghino, J., and Steinberg, F.S. Palmar 
dermatoglyphics of Do\<m's Syndrome, Revisited, Pediatric Research 
7:111-118, 1973. 

IpPlato, C. C. , and Wertelccki, W, A method for subclassifying the inter- 
digital patterns: A comparative study of palmar configurations. Am, J. 
Physical Anthropology , 37:97-110, 1972. 

Wertelecki, W, , Plato, C. , Fraumeni, C, , and Niswander, J. Dermatoglyphics 
in leukemia. Pediatric Research. In press. 



FJ-25 



Serial No. HD-CP 14 

1, Clinical Physiology Branch 

2, Human Performance Section 

3, Baltimore, ^feryland 



PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 



Project Title: Twin Studies: I, Structure and Color of the Iris 

II. X Chromosome Inactivation 
III. Dermatoglyphics and Zygocity Determination 



Previous Serial Numbers; 
Principal Investigator: 
Other Investigators: 

Cooperating Units: 



HD-CD41(c), HD-CD35(c), HD-GD8(c) 

Chris C. Plato 

J. T. Schwartz 

J. D. Mann 

J. D. Niswander 

F & D Research, NEI 

Butterworth Hospital 
Grand Rapids, Michigan 

Department of Human Genetics, NIDR 

Man Years: 

Total: .10 
Professional: ,10 
Other : . 00 

Project Description: 

Objectives : 1, To determine the degree of variability and heritability 
of the structure of the human iris, 2, To test the Lyon Hypothesis re- 
garding X chromosome inactivation. 3. To develop, through dermatoglyphics, 
further criteria for twin zygocity diagnosis. 

Methods Employed : Thorough eye examinations, color slides of the irises 
and finger and palmprints were obtained from over 600 pairs of twins. 
Zygocity was established by elaborate blood group typing, perinatal infor- 
mation, and physical similarities. Fourteen pairs diagnosed as monozygotic 
but being discordant for the Xg sex linked blood group were involved with 
their parents and sibs, in the X chromosome inactivation study. 

Hij'oi' Findings: Thruu;;h our twin studies we demonstrated for the first 
time that mother- l"etus incompatibility, due to the Xg blood group, may 



FJ-26 



I 

result in fetal loss. Because of the sex-linked inheritance of the Xg^, 
the fetal loss is confined only to the female fetus. 

Significance to Bio-Medical Research and the Program of the Institute ; 
T] To establish possible associations between iris structure and eye dis- 
orders; 2. to provide supporting or rebutting evidence for the Lyon Hypoth- 
esis for X chromosome inactivation; 3, to offer additional means for deter- 
mining twin zygocity. 

Proposed Course of Project : To evaluate the data already collected and to 
^ perform the appropriate statistical tests. 

Honors and Awards: None 

Publications: None 



FJ-27 



Serial No. HD-CP 15 

1. Clinical Physiology Branch 

2. Human Performance Section 

3. Baltimore, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 



Project Title: Dermatoglyphics of Primitive and Developing Populations 

Previous Serial Numbers: HD-CDlO(c), HD-GDll(c), HD-CD36(c) 

HD-CD46(c), HD-CD50(c) 

Principal Investigator: Chris C. Plato 

Other Investigators: D. Carleton Gajdusek 

Ralph Garruto 
Robert McLennan 
Paul Brovm 



Cooperating Units: 



Special Chronic Disease Studies 
Department of Anthropology 
Pennsylvania State University 

C & F Research, NINDS 

International Agency for Research on Cancer 
WHO, Lyon, France 



Man Years: 



Total: 



.20 

Professional: ,20 
Other : . 00 

Project Description: 

Objectives: This project represents part of a multidisciplinary effort in 
conjunction with the World Health Organization to study various genetic, 
clinical and anthropological markers among the developing peoples of the 
South Pacific and South America. 



Methods Employed 

method. 



Dermatoglyphics were collected by the Faurot inkless 



Major Findings : Dcrmatoglyphic characteristics are for the most part 
governed by multiple goncs and complex typos of inheritance. Consequently, 
lliey arc not readily vulnerable to the influences of selection or genetic 
drift chic to isolation or migration. Thus, the incidence of dermatoglyphic 
cliaractoristics found in difi^erent populations may reveal the nature of the 



FJ-28 



relationships existing between populations in the remote as well as the 
more recent past. 

In Cyprus, similarities found with respect to derma toglyphics and to 
thalassemia between peoples in the highlands and the lowlands are indicative 
of an ancient relationship , while wide differences in blood groups suggest 
recent changes resulting from isolation and genetic drift among highland 
peoples, and from migration and admixture among the lowland people. 

In New Guinea, the Fore (a people affected with Kuru) had different dermat- 
oglyphics and also blood groups than their neighbors, the Anga, The findings 
indicate ancient and persistent genetic differences due to mechanisms 
affecting isolation. Among the Quechuas of Peru, dermatoglj/phic differences 
were evident while serological factors were quite alike. In contrast to 
the Cyprus situation, this latter finding is suggestive of ancient genetic 
differences which have largely disappeared because of more recent admixture. 

Significance to Biomedical Research and the Program of the Institute: 
The thorough evaluation of genetic and clinical markers of the few rema in ing 
primitive populations will allow scientists at a later time to ascertain 
the effects of "western" culture, infections and environmental pollution 
upon populations, thus far relatively unexposed to these kinds of influence. 

Honors and Awards: Mr. Plato delivered reports at the annual meetings of the 
American Society of Human Genetics and the American Association of Physical 
Anthropology, 

Mr, Plato also presented papers at the IV International Congress of Human 
Genetics in Paris, 

Publications: Plato, C, G, , Brown, P. and Gajdusek, D.C, : Dermatoglyphics 
of the Micronesians from the Outer Islands of Yap, Zeitscrift fur Morph 
und Anthrop, (Stutgart) 64:29 44, 1972. 

Plato, C. C. and Gajdusek, B.C.: Genetic studies in relation to Kuru: 

V. Dermatoglyphics of the Fore and Anga populations of the Eastern Highlands 

of New Guinea. American J, Hum. Genet. 2 4( supplement): S86-S94, 1972, 

Plato, C. C. and Gajdusek, D.C. Dermatoglyphics of the Natives of Western 
New Guinea, Human Biology in Oceania; l(4):255-258, 1972, 



FJ-29 



Serial No, HD-CP 16 

1, Clinical Physiology Branch 

2, Human Performance Section 

3, Baltimore, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Genetic Studies on Longitudinal Program Subjects 

Previous Serial Number: New Project 

Principal Investigator: Chris C. Plato 

Other Investigators : None 

Cooperating Units: None 

Man Years: 

Total: .20 

Professional: .20 
Other: .00 

Project Description: 

Objectives : To generate dermatoglyphic, haptoglobin, transferrin and 
ceruloplasmin data on Longitudinal Study subjects. These genetic markers 
will be examined with respect to differences in aging characteristics of 
the studied population. 

Methods Employed : Serum protein determinations are carried out by the use 
of vertical starch gel electrophoresis. Finger and palm prints are collected 
by the Faurot inkless method. 

Major Findings : Data collection was recently initiated. It will require 
18 months in order to characterize most of the active participants in the 
study. 

Significance to Bio-Medical Research and the Program of the Institute ; 
Will provide further genetic identification of longitudinal subjects. Re- 
peated evaluations of serum of the same individuals may indicate age changes 
in the total liaptoglobin or haptoglobin-subtype levels. The derma toglyphics 
of older subjects, compared to those of our seven-year-old controls may offer 
cviilcnce of selectiv(.' advantage for certain dermatoglyphic characters. 

Proposed Course : To continue data collection and analysis. 



FJ-30 



Honors and Awards: None 
Publications: None 



FJ-31 



Serial No. HD-cPl(c) 

1. Gerontology Research Center 

2. Clinical Physiology Branch 

3. Cardiovascular Section 

4. Baltimore, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 



Project Title: A programmed stress test for the electro- 
cardiographic diagnosis of coronary artery 
disease 

Previous Serial Number: Same 

Principal Investigators: Charles S. Angell 

Edward G. Lakatta 

Other Investigators: Nathan W. Shock 

Reubin Andres 

Cooperating Unit: Baltimore City Hospitals 

Man Years: 



Total: 


.20 


Professional: 


.20 


Others: 


.00 



Project Description: 

This test has been systematically administered to 
participants in the Longitudinal Study. Results are 
incorporated into the report on the Longitudinal Study. 



FJ-32 



Serial No, HD-CP6(c) 

1. Gerontology Research Center 

2. Clinical Physiology Branch 

3. Cardiovascular Section 

4. Baltimore, Maryland 



PHS-NIH 
Individual Projact Report 
July 1, 1972 through June 30, 1973 



Project Title: Non-invasive evaluation of ventricular 

function in man by measurement of systolic 
time intervals 

Previous Serial Number: Same 

Principal Investigators: Donald A. Rothbaum - resigned July 1, 

1972 
Charles S. Angell 

Other Investigator: Nathan W. Shock 

Cooperating Unit: Baltimore City Hospitals 

Man Years: None 

Project Description: This project has been completed. 

Honors and Awards: None 

Publication: 

Shaw, D. J., Rothbaum, D. A., Angell, C. S., and Shock, N.W. 
Effects of age and blood pressure upon the systolic time 
intervals in males aged 20-89 years. J. Geront,, 28: (2), 
133-139, 1973. 



FJ-33 



Serial No. PID-CP 9 

1. Gerontology Research Center 

2. Clinical Physiology Branch 

3. Cardiovascular Section 

4. Baltimore, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 



Project Title: Effects of coronary perfusion pressure on 

myocardial oxygen tension and epicardial 
ST segment changes in acute infarction in 
dogs. 

Previous Serial Number: Same 

Principal investigators: Charles S. Angell 

Edward G. Lakatta 

Other Investigators: Myron Weisfeldt 

Nathan W. Shock 

Cooperating Unit: None 

Man Years: 

Total: 1.0 
Professional: .6 
Others: .4 

Project Description: 

Objectives : The concept of a compromised yet potentially 
viable zone of myocardium in an acute infarct forms the 
basis of current attempts at myocardial salvage by the use 
of coronary artery bypass grafts, intraaortic counter- 
pulsation, and various pharmacologic interventions. Impli- 
cit in these therapeutic maneuvers is an attempt to limit 
infarct size by restoring to normal the altered balance 
between myocardial oxygenation and myocardial oxygen demand 
that is thought to prevail locally in ischemic areas. 

The purpose of this study is to evaluate the effects of 
coronary perfusion pressure on myocardial tissue oxygen 
tension and the epicardial ST segment map of ischemic 
injury during the first 15 minutes after coronary artery 
ligation under conditions of constant aortic pressure and 



FJ-34 



i 



heart rate. In so doing, the hypothesis that changes in ST 
segment levels in an ischemic zone reflect myocardial oxygen- 
ation on a minute to minute basis is tested as is the hypo- 
thesis that elevation of coronary perfusion pressure promotes 
reduction of infarct size. 

Methods : An open chest, intact canine heart preparation is 
used. Intramyocardial oxygenation is measured using a polar- 
ographic technique. The magnitude of ischemic injury is 
assessed by ST segment mapping. Appropriate catheters are 
placed for arterial pressure measurement, venous infusion, 
and arterial withdrawal. The left coronary artery is perfused 
from a reservoir of arterial blood the height of which deter- 
mines coronary perfusion pressure. 

Experimental observations are recorded under baseline condi- 
tions, after coronary artery ligation, after elevation of 
coronary perfusion pressure and after reduction of coronary 
perfusion pressure. Appropriate control experiments are 
performed to evaluate the i^ vivo stability of the oxygen 
electrode system and to study changes in experimental varia- 
bles that occur in the absence of changes in coronary per- 
fusion pressure. 



Major Findings ; The project is 90% comple 
correlation (p<.04) between intramyocardia 
and the ST segment map was obtained in eve 
indicating that changes in ST segment leve 
zone immediately after coronary artery lig 
reflect myocardial oxygenation on a minute 
The hypothesis that elevation of coronary 
promotes reduction in infarct size as asse 
mapping has also been confirmed as the fol 
cates . 



ted. Significant 
1 oxygen tension 
ry experiment 
Is in an ischemic 
ation do indeed 

to minute basis, 
perfusion pressure 
ssed by ST segment 
lowing table indi- 



Control 



p Ligation 





CPP 100 


CPP 100 


CPP 145 


CPP 70 


P^2 


15.76 ± 0.80 


3.91 ± 0.93 
(p<.001) 


10.42 ± 1.83 
(p<.001) 


4.00 ± 0.92 
(p<.001) 


St 


1.68 ± 0.3 


5.08 ± 1.0 
(p<.02) 


2.37 ± 0.99 
(p<.002) 


4.52 ± 1.13 
(p<.004) 



Significance to Biomedical Research and the Program of the 
Institute : The importance of infarct size in the etiology 
of cardiogenic shock as a sequel to acute myocardial infarc- 
tion has been well-documented, and it is recognized that a 
possible therapeutic approach to this problem is an attempt 
to limit infarct size by salvaging ischemic but potentially 



FJ-35 



viable myocardium. The significant correlation that we have 
demonstrated between myocardial oxygenation and the ST seg- 
ment map supports the validity of previous studies using 
the ST segment map as an index of infarct size. In addition, 
it appears that the ST segment levels reflect myocardial 
oxygenation and that coronary perfusion pressure changes 
produce changes in myocardial oxygenation that influence 
the viability of ischemic myocardium and ultimately infarct 
size , 

Proposed C"-urse of Project ; This project will be completed 
by June 30, 1973. 

Honors and Awards: 

Dr. Angell has been invited to present the results of this 
project to the Division of Cardiology, Johns Hopkins 
Hospital, Baltimore, at a Myocardial Infarction Research 
Unit Conference. 

Publications: None, 



FJ-36 



Serial No. HD-CP 10 

1. Gerontology Research Center 

2 . Clinical Physiology Branch 

3. Cardiovascular Section 

4. Baltimore, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 



Project Title: Age differences in cardiovascular performance 

and baroreceptor control mechanisms in intact 
unanesthetized rats. 

Previous Serial Number: Same 

Principal Investigators: Donald A. Rothbaum 

Charles S. Angell 

Other Investigator: Nathan W. Shock 

Cooperating Unit: Baltimore City Hospitals 

Man Years: None. 

Project Description: This project has been completed. 

Honors and Awards: None 

Publications : 

Rothbaum, D. A., Shaw, D. J., Angell, C. S., and Shock, N.W. 
Cardiac performance in the unanesthetized senescent male 
rat. Accepted for publication in Journal of Gerontology, 



FJ-37 



Serial No. HD-CP 17 

1. Gerontology Research Center 

2. Clinical Physiology Branch 

3. Cardiovascular Section 

4. Baltimore, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Effect of age on the response to inotropic inter- 
ventions and hypoxia of rat trabeculae carneae 

' Previous Serial Number: None 

J Principal investigators: Edward G. Lakatta 

Charles S. Angell 

Other Investigators: Nathan W. Shock 

Myron Wesifeldt 

Cooperating Units: Baltimore City Hospitals and 

Johns Hopkins Hospital, Division of 
Cardiology 

Man Years: 

Total: .6 

Professional: .6 
Others: .0 

Project Description: 

Objectives ; Recent observations in man and in intact animals 

have suggested that there are important relationships of 
catecholamine effect and age. The objectives of this project 
are (1) to determine if there is an age related difference in 
the intrinsic response of isolated heart muscle to the ino- 
tropic effects of catecholamines, in terms of dose-response 
relationships, maximal inotropic response, and minimal dosage 
necessary to produce an inotropic response, (2) to measure the 
effects of paired stimulation, a non-catechol intervention; 
these results will be used to determine whether a resulting 
age difference in the response to catechols might be attri- 
buted to the known lower intrinsic level of contractility in 
the senile rat heart muscle, or whether there is indeed a 
specific alteration in the response to catecholamines, (3) 
to determine the rate of decline in performance of cardiac 
muscle of young and old rat heart muscle following exposure 
to severe hypoxia and the rate and extent ^pf recovery follow- 
ing reoxygcnation . 

FJ-38 



Methods ; Trabeculae carneae were removed from the left 
ventricles of rat hearts of age 6-, 12-, and 24-27-month-old 
rats under ether anesthesia. The average cross sectional 
area of these muscle strips was less than 1 mm2 . The muscles 
were suspended between two clips and placed in a chamber 
which was perfused with Krebs-Ringer Bicarbonate solution; 
this solution was modified by reducing the calcium and magnes- 
ium by one half, and by adding dextrose in concentration of 
300 mg%. This perfusate was bubbled with 5% CO2 and 95% O2 
both in the reservoir and directly in the chamber. The temp- 
erature was kept constant at 28 ± .5* by a Hooke water circu- 
lator. After ninety minutes of equilibration at one gram of 
resting tension, the length which produced the greatest 
developed tension was determined (Lmax) ; after another 60 
minutes a baseline value was recorded for the following para- 
meters: Resting tension, developed tension, maximal rate of 
rise of tension, time to peak tension, and time for peak 
developed tension to fall 1/2 of its value. The muscle was 
stimulated at a rate of 24 per minute. 

After baseline measurements, the following interventions were 
made: Paired stimulation was produced by introducing a second 
stimulus at varying intervals after the initial stimulus over 
the range of 400 to 120 msec delay. Norepinephrine was 
infused in five different successive doses (range 1 x 10" to 
8.5 X 10~5) allowing 8 minutes equilibration at each dose. 

Acute hypoxia was created by withdrawing the oxygenated per- 
fusate from the chamber and replacing it with perfusate which 
had beenbubbled with 95% N2 - 5% CO2 ; the chamber was then 
also bubbled with this gas mixture. With this method, the PO2 
fell to 25 mm by 10 minutes; the muscles were kept hypoxic 
for an additional 10 minutes. For reoxygenation, the above 
sequence was reversed, the P02 reaching baseline (PO2 690 mm 
Hg) by the end of the first minute. 

Following the experiment, cross-section area was determined. 
The muscle was assumed to be a cylinder and by multiplying 
the weight by the density (1.063), then dividing the volume 
by the length cross section area in mm2 was estimated. The 
results were normalized to grams of tension and rate of 
tension development per mm2 . The results obtained during 
interventions were expressed as % of baseline values. 

Major Findings ; The experiment is approximately 90% completed. 
Preliminary results indicate that there is a highly signifi- 
cant age difference in developed tension and maximal rate of 
tension development in response to the maximal dosage of 
catecholamines, the youngest group performing better than the 
oldest group. In contrast, there seems to be no significant 
age difference in response to paired stimulation and exposure 
to hypoxia . 



FJ-39 



Significance to Biomedical Research and the Program of this 
Institute ; To date, there is no available data on the age 
differences in the response of isolated heart muscle to 
catecholamines paired stimulation and acute hypoxia. This 
project will contribute original observations in these areas 
forming the basis for an overall study of the role of the 
sympathetic nervous system in the cardiovascular response to 
stress of the aging animal. It also provides insight into 
age effects on the general responsiveness of heart muscle to 
react to inotropic stimuli and acute hypoxia. 

Proposed Course of the Project ; To complete the remaining 
10% of the experiments and to fully aialyze the data. In 
addition to control group, using the highest dose of 
norepinephrine as the initial dose must be examined; subse- 
quent to this, insight into the mechanisms for the age dif- 
ference in response to catechols will be sought by determin- 
ing tissue catecholamines, response after catecholamine 
depletion, and the response to isoproterenol. 

Honors and Awards: 

Preliminary results presented at the Research Seminar of 
the Division of Cardiology at Johns Hopkins University. 

Publications: None. 



FJ-40 ^ 



Serial No. HD-AG21(c) 

1. Gerontology Research Center 

2. Clinical Physiology Branch 

3. Endocrinology Section 

4. Baltimore, Maryland 
PHS-NIH 

Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: The renin -angiotensin system. I. Chemical -radiosotope deri- 
vative assay of plasma renin and angiotensin. II. Plasma 
renin-plasma aldosterone interrelationships during aging in 
man. III. Labeled polymeric substrates for renin and relat- 
ed enzymes. IV. Inhibitors of renin: peptides and proteins. 

Previous Serial Number: HD-AG21(c) 

Principal Investigators: Robert I. Gregerman 

Robert J, Worlonan 
J. Howard Lutz (Resigned 9/30/72) 

Other Investigators: None 

Cooperating Units: Baltimore City Hospitals 

Man Years : 
Total : 

Professional: 
Other: 

Project Description: 

Objectives : Renin is a protease, previously thought to have a high degree 
of specificity, which is released from the juxtaglomerular apparatus of the 
kidney. A variety of physiologic and pathophysiologic stimuli are responsi- 
ble for stimulation or inhibition of renin release. Once in the circula- 
tion the enzyme cleaves a decapeptide, angiotensin. I, from a circulating 
plasma globulin. This peptide is activated upon passage through the lung 
to form an octapeptide, angiotensin II, which pharmacologically is a power- 
ful blood pressure elevating material and physiologically appears to regu- 
late the production of the major adrenal steroid hormone, aldosterone, reg- 
ulating in turn mineral metabolism. Aldosterone is directly involved in 
certain forms of hypertension, while the excessive release of renin is dir- 
ectly involved in another type of hypertension, so-called renovascular hy- ' 
pertension. 

The chemical measurement of renin has an important place in clinical diag- 
nostic circumstances, while very precise measurement of the enzyme contin- 
ues to be a major problem area for research. For example, "low renin" hy- 
pcMtonsion li.is recently iiccn claimctl to be associated with decreased card- 
iovascular mortality, while a renin concentration ratio greater than 1.5 
between the two renal veins is said to herald a successful surgical result 



FJ-41 



in renovascular hypertension. Physiologic studies in aging man have been 
very limited, but a major age-related change of aldosterone secretory rate 
has suggested that altered renin physiology occurs at advanced age. 

Because of these considerations, our laboratory has been interested in the 
development of new approaches to the measurement of renin and angiotensin, 
in the chemistry of the peptide and the enzyme, and in the physiology of 
the renin-angiotensin-aldosterone system in normal and pathologic aging. 

Methods Enployed : Previous reports have given the details of a double iso- 
tope derivative assay for measurement of renin through assay of angiotensin 

I. We have in the past year used this technique to compare renin assays by 
our own technique and that of others. 

Another aspect of this phase of our work uses a labeled polymeric substrate 
assay for renin which we have developed and previously described. With 
this technique we have in the past year studied a variety of protein and 
peptide inhibitors of the enzyme. In addition, a new labeled polymeric 
substrate hs^s been devised and studied. 

Major Findings : I. Using our double isotope assay for angiotensin I we 
have continued our comparison of plasma renin levels with samples simultan- 
eously assayed by the method of Dr. Anne Gould in her laboratory at the Mt. 
Sinai Hospital, Cleveland, Ohio. Two new series of samples, in addition 
to the ones we described in last year's report, were studied. In the two 
series the correlation coefficients for the two assays were 0.8 and 0.9 
However, some individual samples were widely discrepant with the two methods. 
In the Gould technique, incubation conditions are nearly identical to our 
own, but the angiotensin I generated is quantitated by bioassay. It is 
difficult to reconcile these findings and extremely unlikely that the dis- 
crepant results are caused by technical errors. The possibility must now 
be seriously entertained that the angiotensin I generated by certain indi- 
viduals is chemically and biologically different from the majority of in- 
dividuals in the population. It would be possible with modification of 
presently available techniques to determine the amino acid composition of 
angiotensin I generated by individual human subjects. If significant vari- 
ations exist, these might have great importance physiologically and in 
certain hypertensive states. 

II. Further work with various immunoassay procedures for angiotensin 
using a variety of commercial kits as well as antibody and other reagents 
prepared in our own laboratory have convinced us that all of the methods 
in current use are unpredictable and, in our hands at least, lacking in 
precision. As such they are poorly suited to the type of physiologic 
studies we have previously proposed, namely, correlations of plasma renin 
activity and concentration with the secretion of aldosterone in normal 
aging man. Our collaborative efforts with Lnvestigatois at the Johns 
Hopkins University have therefore been abandoned for the present. 

III. In addition to the previously described synthetic substrate for renin, 
N-acetyl-poly-L-glutamyl- (M.W. 
Leu-Val-iyr- Ser labeled with-" 



;.5p,000)-Arg-Val-Tyr-Ile-Hs-Pro-Phe-His-Leu- 

1 I, we have now preparec a new synthetic j 



FJ-42 



substrate. This material was prepared primirily in the hope that it would 
not be attacked by pseudorenin of plasma. The structure of the new poly- 
meric substrate more closely resembles that of the natural substrate for 
renin in that its cleavage yields the N- terminal rather than the C-terminal 
peptide fragment of the synthetic tridecapeptide (or tetradecapeptide) 
renin substrate. The new material is Arg-Val-Tyr-Ile-His-Leu-Leu-Val-Tyr - 
Ser -poly- L- Lysine, M.W. 50,000. The poly-L-Lysine is partially acvlated 
by trifluoroacetyl residues. The substrate has been labeled with ^^^l. 
Renin cleaves this substrate to yield Arg-Val-Tyr-(-'-^^I)-Ile-His-Pro-Phe- 
His-Leu. This nonapeptide was prepared separately by Edman degradation 
of angiotensin I and then labeled with ■'^^^I. 

The cleavage of this new polymeric substrate has been only partially studied. 
The Kj^for the reaction is approximately that of the poly-L-glutamyl substrate 
(about 0.5xlO"°M) but the 'V^^^ appears to be considerably less. In addition 
the cleavage reaction is strongly inhibited by human plasma. Cleavage of 
the new substrate by plasma has not yet been studied. Although the rather 
low Vw^ is not encouraging, the results do already indicate a strong influ- 
ence ot sites remote from the Leu- Leu sequence and suggest that it may well 
be possible to inpart a greater degree of specificity to other synthetic 
substrates. Several other syntheses of soluble substrates are now under 
consideration as are further studies on the lysine polymer. 

IV. Studies of the inliibition of renin by hemoglobin and other Leu-Leu con- 
taining proteins was reported last year. This study has now been greatly 
extended. The working hypotJiese.s that Leu-Leu sequences account for the 
inhibition of the enzyme and that renin may attack sequences other than the 
Leu- Leu sequence of natural or synthetic renin substrate have now been tested. 

Hemoglobin chains (horse)were prepared by ion exchange chromatography, 
cleaved with cyanogen bromide, and two particular peptide fragments iso- 
lated, the al-32 sequence which contains no Leu-Leu sequences, and the 
61.55 which contains two. The Leu-Leu sequence was clearly shown not to 
be an absolute requirement for inhibition of renin. This unexpected find- 
ing led as to explore a number of other small synthetic peptides as potential 
inhibitors of renin. A number of these proved to be quite potent. All con- 
tained at least one leucine, but the Leu-Leu sequence was not required. 
Surprisingly sudi a single peptide as Leu-Leu-Leu was effective while Leu- 
Giy-Leu was not. Leu-Tryp -Leu was also quite potent as was Leu-Tryp-Met- 
Arg-Phe-Ala. Thus a large number of peptides have now been shown to be 
inhibitors of renin, and a Leu-Leu sequence is not necessary. 

While we were still entertaining the possibility that renin might interact 
v>/ith and cleave Leu-Leu sequences in hemoglobin, i.e., that hemoglobin 
inhibition was due to Leu- Leu sequences which were acting as alternate 
substrates, we prepared hemoglobin labeled with 14C-glycine, This material 
has been shown by others to be a sensitive acid protease substrate. We then 
showed tliat renin preparations cleave this labeled hemoglobin. Such prepa- 
ations l\ave been previously believed to be free of protease activity primarily 
because tlioy arc free of peptidase activity as tested with angiotensin I or 
11. Thus, we now provide evidence tliat renin may have a lower degree of 



c.1-43 



specificity than heretofore suspected. Such a conclusion is in keeping with 
the relative specificity characteristics of other acid proteases of wide 
biological distribution. Recognition of renin as an acid protease, similar 
to other acid proteases in that it is inhibited by pepstatin, a peptide of 
microbial origin, has been very recently reported, but the specificity of 
renin has not been previously challenged. Further confirmation of this view 
will require purification of the enzyme. 

Our recent kinetic studies of the inhibition of renin by proteins and peptides 
have shown tliat the inliibition is strictly competitive for some inhibitors 
(e.g. Leu-Leu-Val-Tyr-Methyl ester and lactoglobulin B) but that some contri- 
bution by non -competitive factors is involved in the inhibition by others 
(e.g. hemoglobin and Leu-Trypt-Met-Arg-Phe-Ala) . Control experiments estab- 
lished that the inhibition seen with the proteins and peptides was not due 
to interaction of these materials with the syntlietic labeled polymeric sub- 
strate. For example, hemoglobin was found to inhibit the action of renin 
on the natural protein substrate (from hog plasma) with the decreased angio- 
tensin I formation being measured by both radioimmunoassay and by double 
isotope derivative assay. In addition, it has previously been shown that 
the peptide Leu- Leu-Val-T/r -Methyl ester inliibited the action of rabbit renin 
on rabbit plasma substrate, lliese observations rule out the possibility that 
the inhibition seen by the peptides studied here were in some way related to 
use of the labeled polymeric substrate. 

Significance to Bio-Medical Research and the Program of the Institute : 
Hypertension is an age-related disease of major importance. Tlie enzyme 
renin is somehow involved in the pathogenesis of this disease. Our own 
efforts are related to the development of unproved diagnostic techniques 
for measurement of the enzyme, assays for which have assumed a significant 
role in the differential diagnosis of hypertension^ its treatment, and in 
further research in the area. In addition, our work is relevant to under- 
standing basic aspects of the chemistry of the enz)'me renin and of angio- 
tensin related peptides. 

Proposed Course of Project : Studies will continue of the peptide inhibitors 
of human renin. Specifically, new inhibitor studies with pepstatin, leu- 
peptin and diazo compounds will serve to relate renin to the general class 
of enzymes known as acid proteases. Tlie inliibitor studies will allow 
choice of suitable compounds for the purification of renin by affinity 
chromatography. Additional synthetic labeled polymeric substrates will 
be prepared in an effort to obtain the specificity required for lise of 
these substrates with human plasma. An effort may be made to identify the 
amino acids of angiotensin I in individual human subjects. 

Honors and Awards: Irr. Robert Gregerman was elected to Membership in The 
American Society for Clinical Investigation at the Annual Meeting of The 
Society in Atlantic City, N. J. , April 30, 1973. 

Publications : Bath, N.M. and Gregerman, R.I. Labeled polymeric substrate 
for renin. Synthesis of N-acetyl-poly (L glutamyl)- [^25] tridecapeptide and 
usr for cnryine assay. lUochemistiy 11:2845, 1972. 



FJ-44 



Gregerman, R.I. and Kowatch, M.A.: Isotope derivative assay of nanogram 
quantities of angiotensin I: Use for renin measurement in plasma. 
J. Meienhofer (editor), Chemistry and Biology of Peptides , Proceedings of 
the Third American Peptide S>Tnposium, p. 533, Ann Arbor Science Publishers, 
Inc., 1972. 



Serial No. HD-AG22(c) 

1. Gerontology Research Center 

2. Clinical Physiology Branch 

3. Endocrinology Section 

4. Baltimore, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Relationship of age to tlyroid function and thyroid hormone 
metabolism. 

Previous Serial Number: HD-AG22(c) 

Principal Investigators: Robert I. Gregerman 

Barry S. Handwerger 

Other Investigators: Paul J. Davis* 

Cooperating Units: Baltimore City Hospitals 

Man Years: 
Total : 

Professional: 
Other : 

Project Description: 

Objectives: This study was a long term project aimed at elucidating the 
control mechanisms for the metabolism of tliyroid hor.Tiones. In the recent 
past the work centered around the role and isolation of the intracellular 
binding proteins for thyroxine and tri iedothyronine . 

Methods Employed : A varity of teclmiques including protein electrophoresis^ 
equilibrium dialysis, etc., have been previously described. 

Major Findings : With the resigjiation of one of the major investigators 
(Dr. Handwerger) The project was discontinued in this Section, but the 
work has progressed in the cooperating unit (Dr. Davis) and has been de- 
scribed with their annual report. 

Significance t o Bio-Medical Research and the Program of the Instit ute : 
The project area remains of great interest to Gerontology and to Endocrin- 
ology. The role of intracellular binding proteins in the action of thy- 
roid hormones remains to be defined. The mechanism of the age-related 
slowing of thyroid hormone metabolism is still unknown, and its elucida- 
tion would be an important clue to other age-related alterations of hor- 
mone and drug metabolism. 



1 
FJ-46 I 



Proposed Course of Project : Active work in this area closed within the 
Endocrinology Section soon after July 1, 1972. Although an informal colla- 
boration with the Endocrinology Division of Baltimore City Hospitals (Dr. 
Davis) will continue, no further active efforts are planned with the Endo- 
crinolgy Section for the immediate future. 

Honors and Awards : None 

Publications : None 



Serial No. HD-CP 18 

1. GerontQlQgy Research Center 

2. Clinical Physiology Branch 

3. Endocrinology Section 

4. Baltimore, Maryland 

PHS-NIH 
Individual Project Report 
July 1,1972 through June 30, 1973 

Project Title: Hormone receptors and aging: Effects of age on hormone med- 
iated alterations of the adenyl cyclase, tissue cyclic AMP 
and factors controlling these metabolic regulators. 

Previous Serial Number: New Project. 

Principal Investigators: Robert I. Gregerman 

Martin J. Kalish 

Other Investigators: None 

Cooperating Units: Baltimore City Hospitals 

Man Years: 
Total: 

Professional: 
Other: 

Project Description: 

Objectives : Adenyl cyclase is a membrane -bound enzyme which catalyses the 
conversion of adenosine triphosphate (ATP) to adenosine-3' -5' cyclic 
monophosphate (cyclic AMP). The enzyme is very widely distributed in nature. 
It is well established that in their initiation of effects various hormones 
activate adenyl cyclase and thus increase the cellular concentration of 
cyclic AMP. Cyclic AMP then in turn initiates a variety of metabolic effects, 
depending on the tissue involved. This process is known as the "second 
messenger" concept of hormone action. 

Despite a large and growing literature on the cyclase-cyclic AMP system, 
no studies are reported which relate to the effect of aging in this area, 
despite the fact that a number of hormone responses are known to be greatly 
altered at advanced age. Recently reported studies indicate decreased 
cyclase activation by several hormones in adipose tissue during maturation, 
increased phosphodiesterase (an enzyme responsible for cyclic AMP degrada- 
tion and thus partially for the control of tissue levels of cyclic AMP) 
and a differential rate of loss of responsiveness depending on the hormone 
involved. In addition, basal levels of cyclase activity decrease sharply 
durinj; the Tirst few months (maturation) in the lifespan of the rat. 



FJ-48 



Our present study aims to investigate effects of aging, and especially of 
senescence, on hormone mediated activation of adenyl cyclase and the produc- 
tion of intracellular cyclic AMP. The information developed will be useful 
in determining whether the already loiown alteration of tissue sensitivity to 
certain hormones is the result of an age-related alteration of the initial 
step in hormone action. The study will also allow an examination of the effect 
of senescence on one aspect of membrane function. 

Methods Employed : Adenyl cyclase is being assayed by an established labeled 
substrate method which allows the determination of P-labeled cyclic AMP 
produced from a-labeled P-ATP. Tissue cyclic AMP will be determined by an 
established competitive protein binding method which is now being standardized 
in our laboratory. Phosphodiesterase will be assayed by one of several publish- 
ed methods. Rats of both sexes and various ages are being obtained from the 
animal colony at the Gerontology Research Center. Basal enzyme levels, tissue 
concentration of cyclic AMP, and phosphodiesterase will be examined in rats of 
various ages. 

Major Findings : Initial results indicate that basal levels of hepatic adenyl 
cyclase in crude homgenates are not different m one year (mature) and two 
year old (senescent) rats. However, the response to activation by epinephrine 
is greatly increased ( approximately doubled) in the senescent animals (p^.05; 
n=6) for both males and females . Dose response data are available for epiner - 
phrine and show that the age difference exists at all concentrations studied 
to date (10 ■■'■^M to 10 "°M) In contrast the cyclase response to glucagon does 
not appear to be affected over a wide range of concentrations nor does the 
activation by the non-specific effect of fluoride ion. 

Significance to Bio -Medical Research and the Program of tlie Institute : 
These studies should provide an ijnportant insight into the mechanisms by 
v^ich age affects the organism's response to hormones at advanced age. The 
results already indicate a selective alteration of cyclase responses during 
senescence. In addition, our studies offer an opportunity to study a membrane 
bound enzyme and will thus give an insight into age effects on membrane function. 
The work is directly related to the mission of the Gerontology Research Center 
and the Institure to provide new information in the area of research on Aging. 

Proposed Course of Project: We plan to pursue and to expand our present work. 
A variety of hormone effects will be studied in a number of tissues. The 
selection of tissues and hormones is dictated by information already available 
on altered tissue responses with aging. 

Honors and Awards: Dr. Gregerman was an invited Speaker at the 9th Intemation- 
al congress of Gerontology, Kiev, USSR, July, 1972. He spoke at an Interdiscip- 
linary Symposium on Honnones and Aging. 

Dr. Gregerman was also Co-Chairman of a Section meeting on Honnones at the 
same Congress. 



Dr. Gregerman was invited to contribute a Chapter to the new (5th) Edition 
o£ Williams' Textbook of Endocrinology. His Chapter "Hoimones and Aging" 
is the first of its type in a leading standard text on Endocrinolog-v- . 
The Chapter has been submitted and accepted. 

Publications : Gregerman R. I . , Mechanisms of age related alteration of 
hormone action and metabolism: Adrenal and thyroid hormones. Proceedings 
9th International Congress of Gerontology, 2: 392,1972. 



1 
FJ-50 li 



Serial No. HD-CP7 (c) 

1. Gerontology Research Center 

2. Clinical Physiology Branch 

3. Metabolism Section 

4. Baltimore, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: The kinetics of glucose and insulin metabolism in man 

Previous Serial Number: Same 



Principal Investigators; 



Other Investigators: 



Cooperating Units: 



Jordan Tobin 
Reubin Andres 
Karl Kramer 
Paul Insel 

Ralph DeFronzo 
John Rowe 
*Mones German 

*National Cancer Institute 
Baltimore City Hospitals 



Man Years: 




Total: 


4.1 


Professional: 


2.2 


Other: 


1.9 



Project Description: 

Objectives : These studies are aimed at delineating various parameters of 
insulin release, distribution, and metabolism in man and the changes in 
such parameters that occur in altered physiologic states, including aging, 
diabetes mellitus, and obesity. Because of insulin's role as a prime de- 
terminant of glucose utilization, a definition of insulin kinetics will 
provide a more precise understanding of how the metabolism of glucose is 
controlled. Current studies are being performed to develop an insulin 
kinetic model, a glucose kinetic model, and to describe the homeostatic 
relationship between those two models in normal, diabetic, and obese man 
of differing ages. From such an analysis, important physiologic controls 
will be described and clues to pathogenesis of altered metabolic states 
may be forthcoming. 

Methods Employed : The glucose clamp technique was utilized to maintain 
human subjects at basal arterial blood glucose concentrations after exper- 
imental perturbations of the arterial plasma insulin level. The clamp 
technique employs a controlled variable-rate IV infusion of glucose solu- 
tion with rapid measurement of blood glucose concentration by an automated 
method. A negative feed-back formula is applied at 5 minute intervals in 



FJ-51 



order to servo-correct the glucose infusion rate; thus, the blood glucose 
concentration is maintained at euglycemic levels. The amount of infused 
glucose, an index of glucose utilization, provides an estimate of sensiti- 
vity to exogenous insulin. 

Intravenous infusions of insulin were given by 2 techniques: (1) single 
shot intravenous injection of .025 crystalline porcine insulin per kg body 
weight or (2) prime plus continuous infusion at 1 or 2 mU per kg body 
weight per minute. The prime was given over a 10 minute period such that 
the total insulin infused in that time was twice the rate infused continu- 
ously from 10 to 80 minutes. The infusion during the 10 minute prime was 
at a logarithmically falling rate which approached the continuous infusion 
rate asymptotically. The goal of this technique was to create a nearly 
instantaneous square wave of insulin concentration at a new steady state 
level . 

In some studies glucose-l-^'*C was injected as a primed continuous infusion 
during the pre-clamp period to achieve a steady state specific activity 
and specific activity of glucose (corrected for metabolites and recycled 
glucose by established procedures) was followed after the administration 
of insulin. Such radioactive glucose data allows determination of endo- 
genous glucose production and of glucose utilization as a means of analyzing 
insulin action. 

Plasma immunoreactive insulin concentrations, plasma glucose radioactivity, 
and plasma glucose concentrations were utilized in mathematical modeling 
of insulin kinetics and glucose kinetics by the SAAM program of Berman and 
Weiss to determine compartmental dimensions, transfer rates, and to inves- 
tigate the mechanisms by which insulin regulates glucose utilization. 

M ejor Findings : Work has continued on the computer modeling of glucose and 
insulin metabolism. The 3 compartments and their exchange rates for glucose 
-and insulin are now defined, and these parameters are being used in a 
comprehensive»analysis of our servo-control glucose-clamp studies: 

(l)The development of the glucose model has made it possible to compute 
moment-to-moment glucose content in the three glucose compartments. Under 
experimental conditions in which blood glucose concentration is changing, 
as, for example, in the early part of the hyperglycemic servo-control studies, 
the infused glucose needed to maintain the desired blood glucose concentration 
can now be accurately split into two components: (1) the glucose which has 
simply been added to the (extracellular) glucose spaces and (2) the glucose 
which has been metabolized. Previous techniques for carrying out these 
computations had to be based upon the assumption that glucose was distributed 
in a single compartment. The error involved in the single compartment assump- 
tion has now been shown to be very large. The availability of the 3 compart- 
ment parameters has added greatly to the accuracy of the estimates of glucose 
metabolism under these controlled conditions. 

(2) These parameters have also enabled us to derive the insulin con- 
centration pattern in interstitial compartments from the complex biphasic 
plasma insulin curves induced by programmed steady-state hyperglycemia. 
Thus, we now have better estimates of changes in insulin concentration at the 



FJ-52 



^ 



the site of its action. The metabolic response to the induced insulin 
responses (i.e. uptake of glucose) is much better related temporally to 
insulin concentration in compartment 3 (interstitial fluid of peripheral 
tissues) than to concentration in plasma per se. These results thus further 
validate the importance of the compartment 3 insulin concentrations; we 
previously described the controlling effects of this compartment in our 
euglycemia "insulin - clamp" studies. 

(3) The computer modeling of the glucose and insulin compartnants uf 
the body has been supplemented by the development of a model of the pancreatic 
beta cell. The model consists of rapidly and slowly releasing insulin 
compartments, both fed from a common source. Control of the system can be 
exerted either at the source or between the source and the releasing pools. 
The model is able to simulate the complex pattern of pancreatic output of 
insulin, and by coupling the beta cell model to the 3 compartment insulin 
model (extrapancreatic) we can simulate plasma insulin responses. These 
models were able to fit data from both young and old subjects by adjusting 
the controlling parameters. The ability to define these parameters will 
provide better understanding of factors which result in altered beta cell 
function, for example, age, obesity, diabetes, and uremia. 

Significance to Bio-Medica1_Research and the Program of the Institute : 
The remarkable prevalence~(50%) of abnormal glucose tolerance tests in the 
older population of the United States coupled with the increased morbidity 
and mortality of patients with diabetes mellitus demands a delineation of 
the effects of aging on the pathophysiology of carbohydrate metabolism. 
These studies in man define kinetic parameters in the glucose-insulin 
homeostatic system and will apply such analysis to patients with disorders 
in that system. 

Proposed Course of Project : Further refinement of the pancreatic beta cell 

model will be done. The interaction of obesity and age with insulin and 

glucose kinetics will be explored. Further I'+C-glucose studies will be done 
to investigate basal glucose turnover as a function of age. 

Honors and Awards : 
Dr. Andres was an invited speaker at a symposium in Glasgow, Scotland in 
September 1972 on Recent Advances in Geriatric Medicine. 

Dr. Tobin was an invited speaker at an American Geriatric Society Symposium 
in April 1973 on Geriatric Medicine for the Practicing Physician. 

Dr. Rowe was an invited speaker at a Diabetes Seminar at the New York Medical 
College Department of Medicine in November 1972. 

Dr. Andres was an invited member of the faculty at the 4th Summer Biology of 
Aging Course at the University of Santa Cruz in August 1972. 

Dr. Andres was elected as Vice-Chairman of the Clinical Medicine Section of 
the Gerontological Society. 

Dr. Andres was appointed as Chairman-Elect of the Research Committee of the 



Gerontological Society. 

Dr. Tobin was Visiting Professor of Medicine (Gerontology) at the University 
of Nebraska, February 1973. 

Publications: None 



FJ-54 



Serial No. HD-AG 10 (c) 

1. Gerontology Research Center 

2. Clinical Physiology Branch 

3. Metabolism Section- 

4. Baltimore, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Effect of age on carbohydrate metabolism 

Previous Serial Number: HD-AG 10 (c) 

Principal Investigators: Reubin Andres 

Jordan Tobin 

Other Investigators: Karl Kramer 

John Rowe 

Cooperating Units: Baltimore City Hospitals 

Man Years : 

Total: 3.0 

Professional : 0.9 

Other: 2.1 

Project Description: 

Objectives : This project is part of the long-term prospective study of human 
9-ging, the Longitudinal Study of the Gerontology Research Center. The 
complex interplay of aging, carbohydrate metabolism and diabetes mellitus 
offers a prototype of one of the serious challenges to clinical gerontology, 
the differentiation of normal aging changes from specific disease states. 
The objectives of this project are: (1) to describe cross -sectional changes 
during the entire adult span of life in performance on the commonly-used 
clinical tests for the diagnosis of diabetes, (2) to provide age-adjusted 
standards of normality for these tests, (3) to define the relative sensi- 
tivity and specificity of these tests, (4) to establish the role of certain 
experimental variables which influence performance on these tests, (5) to 
investigate the mechanisms underlying the age changes, and (6) to evaluate 
the significance of the decline in tolerance [a] by noting tJe influence 
of poor performance on mortality and [b] by testing for correlations be- 
tween performance and certain other variables known to be clearly associated 
with the overt diabetic state. 

Methods Employed : Subjects are primarily those in the Longitudinal Study 
supplemented oy college students and laboratory and hospital employees to 
increase the sample size of the 3rd and 4th decades. Subjects are given 



FJ-55 



one of the following tests per visit: oral glucose tolerance (OGTT), intra- 
venous glucose tolerance test (IVGTT), cortisone glucosettolerance (CGTT), or 
intravenous tolbutamide response (TRT). On the fifth visit the cycle is 
re-started. Diabetic patients are available primarily from the Baltimore 
City Hospitals Out-Patient Department; "chemical" diabetes and a small 
number of overt diabetics are available from the Longitudinal Study Group. 

M ajor Findings : We described previously the development of a "hot box" 

technique for arterial izing venous blood draining the hand. The goal of this 
technique was to enable us to study participants in the Longitudinal Study 
with the glucose clamp method but without the necessity of brachial arterial 
cannulation. The clamp technique provides much more information about 
pancreatic beta cell responsivity and insulin sensitivity than can be 
obtained from the routine clinical tests for diabetes. We have studied 
28 of the participants in the Longitudinal Study who had characteristics 
which were needed for the completion of these studies. Our goal has been 
to have at least 5 subjects in each of 9 categories: 3 age groups (20-39, 
40-59, 60-79 years) each with 3 degrees of obesity (obesity index 0.85-1.14, 
1.15 - 1.29, and 1.30-1.44). With this grid we should have an adequate 
description of age differences and of the effect of both mild and moderate 
obesity. This grid also provides the necessary control data for judging 
responses of subjects with diseases of interest, especially patients with 
diabetes mellitus. 

This 9 category grid is needed Both for the hyperglycemic studies and 
for the euglycemic insulin clamp studies. The minimum number of total 
control subjects required is therefore very large (90) and the ability to 
carry out this complex test on the Longitudinal Participants is an important 
achievement. 

In the past year we have studied 28 of these volunteers: 10 were normal 
weight controls, 8 v;ere obese but non-diabetic, and 10 were diabetics. The 
procedure -.was very well -tolerated, the only complication being an unusual 
hand-edema which was diagnosed by dermatologic consultants as thermal allergy; 
it passed off without sequel lae in several hours. 

A series of 5 studies were done in order to evaluate neuro-endocrine 
responses to hypoglycemia. It has been proposed that a rapid rate of fall 
in glucose concentration could stimulate counterregulatory mechanisms even 
in the absence of hypoglycemia. We therefore created steady-state hyper- 
glycemia (300 mg%) with the glucose-clamp technique and then stopped the 
glucose infusion. As blood glucose levels fell arterial blood samples 
were collected for assay of counterregulatory hormones (catecholamines, 
glucagon, adrenal corticosteroids, and growth hormone). The studies have 
been completed and endocrine assays are in progress. 

Statistical analysis of the routine diagnostic tests for diabetes will 
be resumed next year. These variables had a lower priority than others 
in the Longitudinal Study this past year. 

Proposed Course of Project : (1) Analyze the routine diagnostic tests for diabetes 



FJ-56 



through the end of Cycle G (June 30, 1973). These computations in association 
with the other variables measured in the Longitudinal Study will provide 
the evidence to meet goals (1) to (4) and goal (6) as outlined in the 
Objectives section. The interrelated variables are the following: (a) 
glucose and insulin responses on the clinical tests, (b) serum lipids, (c) 
body composition, especially lean body mass and obesity, (d) physical 
activity, (e) dietary factors, and (f) presence of atherosclerotic disease. 

Honors and Awards : None 

Publications: None 



FJ-57 



Serial No. HD-CP12(c) 

1. Gerontology Research Center 

2. Clinical Physiology Branch 

3. Metabolism Section 

4. Baltimore, Maryland 



i 



PHS-NIH 
Individual Project Report 
July 1, 1972 through July 30, 1973 

Project Title: Cytoxan-induced water intolerance 

Previous Serial Number: Same 



4 



Principal Investigator: 
Other Investigators: 

Cooperating Units: 



Ralph A. DeFronzo 

Paul J. Davis** 
Michael Colvin* 
Hayden Braine* 
Gary Robertson*** 

Baltimore City Hospitals 

* Oncology Service 

**Endocrine Service 

***Indianapolis VA Hospital, Indianapolis, Indiana 



Man Years: 

Total: 0.20 

Professional: 0.20 

Other: 0.00 

Project Description: 

Objectives ; High dose Cytoxan therapy (30-120'mg/kg/day) has been employed 
with increasing frequency in organ transplantation and in the treatment of 
neoplasia. With these high doses a syndrome of weight gain, hyponatremia, 
and decreased urine output with high urine osmolality has been observed. 
The goals of this study are to determine the frequency with which the 
syndrome occurs and to define the underlying mechanism. 

Methods Employed : Twenty- two studies were performed in 17 patients. All 
had normal renal function and excreted a water load normally prior to 
Cytoxan administration. All subjects received 3 liters of normal saline IV 
for one day preceeding and throughout the period of Cytoxan administration 
(15-100 mg/kg/day). Fluids were permitted ad lib. Urine flow and osmolality 
and serum sodium and osmolality were followed every four hours; weights 
were obtained every 6 hours. Because of the possibility of a cytoxan-induced 
vascular leak, combined ^^GRBC volume and i^ij albumin space were determined 
before and during Cytoxan administration. 



FJ-58 



i 



Major Findings : Are summarized in the following table 



Increase 


Decrease in 


Wt. Gain 


Decrease in 


Decrease in 


in Uosm 


Urine Flow 


(kg) 


Serum Na 


Serum Osm 


(mOsm/L) 


(ml/hr) 




(niEq/L) 


(mOsm/L) 


CY Responders 










(N=17) 










Average 514 


118 


2 


9 


15 


Range 305-798 


28-203 


0.4-5.6 


3-20 


6-4.1 



A close temporal and quantitative relationship was found between the 
excretion of Of active alkylating metabolites XC{-M) and changes in urine 
osmolarity. Creatinine clearance and urine sodium excretion remained 
unchanged (12 studies) , AVP levels were normal prior to CY and suppressed 
physiologically following the decrease in serum osmolality. 

Significance to Bio-Medical Research : With low circulating levels of AVP and 
without change in glomerular filtration or urine sodium excretion we have con- 
cluded that CY-AM exerts a vasopressin- like effect on the distal renal tubule. 
Studies of the effect of O'-AM on the urinary toad bladder are presently in 
progress. With the development of an in vitro assay system which simulates 
the effect of CY-AM on the renal tubule hopefully specific therapy can be 
developed which would prevent the water retention that at times has resulted 
in congestive heart failure, especially in elderly patients with comiDromised 
cardiac function. 

Proposed Course of Project : Study has been completed. 

Honors and Awards : None 

Publications : Water Intoxication in Man Following Cyclophosphamide. Time 
Course and Relationship to Drug Activation. Ralph A. DeFronzo, H. Braine,O.M. 
Colvin, P.J. Davis. Accepted for Publication, Annals of Internal Medicine, June 
1973. 



FJ-59 



Serial No. hd-cp 19 

1. Gerontology Research Center 

2. Clinical Physiology Branch 

3. Metabolism Section 

4. Baltimore, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Multiple Myeloma and the Kidney 

Previous Serial No: New Project 

Principal Investigator: Ralph A. DeFronzo 

Other Investigators: Richard L. Humphrey* 

John Wright** 
J. Robert Cooke*** 



Cooperating Units: 



Baltimore City Hospitals 

*Oncology Service 
**Department of Pathology 
Johns Hopkins Hospital 

***Renal Division 



Man Years: 




Total : 


0.2 


Professional : 


0.2 


Other: 


0.0 



Project Description: 

Objectives : Renal failure is present in 20-50% of individuals with multiple 
myeloma on initial presentation and develops in greater than 50% of such 
individuals at some time during their course. To date the pathogenesis of 
this renal failure has not been delineated. The classical teaching suggests 
that obstruction of tubules by myeloma protein is primarily responsible for 
the deterioration in renal function. However, in 
studies myeloma casts were not found in up to 15- 
severe renal dysfunction. No prospective studies 
have been reported and consequently the pathogenesis of the renal failure 
remains largely speculative. This prospective study was undertaken to cor- 
relate changes in glomerular and tubular function with renal histopathology 
and myeloma protein abnormalities. 

Methods Employed ; 35 patients with multiple myeloma consecutively admitted to 
the Baltimore City Hospitals Oncology Service were studied. Renal acidifica- 
tion was evaluated with the standard NH4C1 test (0.1 gm/kg); concentrating 
ability was examined after a 12 hour overnight dehydration followed by a one 
hour intravenous infusion of 300 mU of aqueous vasopressin. PAH clearance 



several large retrospective 

of individuals with 
on renal function in rnyeloma 



FJ-60 



were performed to determine renal blood flow. Twenty four hour creatinine 
clearance was used as an index of GFR. Urine and serum protein was examined 
both quantitatively and qualitatively (immunoelectrophoresis) . After comple- 
tion of the renal functional study, a renal biopsy was performed. All tissue 
was examined by routine light microscopy (with amyloid and calcium stains also) 
electron microscopy, and immunoflourescence. 

Major Findings ; 13/35 (37%) patients had definitely impaired renal function as 
defined by a creatinine clearance less than 40 ml/min. Renal tubular function 
was impaired disproportionately to GFR. Severe acidifying and concentrating 
defects were noted in many patients with normal creatinine clearance. PAH was 
also diminished disproportionately to GFR. There was a strong correlation 
between impaired glomerular and tubular function and the presence of Bence- 
Jones proteinuria. No patients with balanced immunoglobulin synthesis '(i .e. 
absence of Bence-Jones protein) had a creatinine clearance less than 40 ml/min; 
12/23 (52%) patients with demonstrable Bence-Jones protein had Ccr less than 
40. Lambda Bence-Jones protein (60%) was more conmonly associated with renal 
function than Kappa (43%). 

Renal histologic examination revealed that tubular atrophy and degener- 
ation and not tubular casts correlated best with impaired renal function. 
Furthermore, only patients with demonstrable Be ce-Jones protein manifested 
renal structural abnormalities. 

Significance to Bio-Medical Resear ch: (1) This prospective study clearly demo- 
strates a close relationship between the presence of Bence-Jones protein and 
impaired renal function. Furthermore, the marked tubular dysfunction with 
spared glomerular function suggests that the B-J protein is a direct tubular 
toxin. Recent studies which show that 98-99% of B-J protein is reabsorbed 
and catabolized by the renal tubule support our observations. The knowledge 
that (a) patients with the B-J protein are at risk to develop renal disease 
and (b) that the presence of impailred renal tubular function is the earliest 
manifestation of renal dysfunction, allows us to select high risk patients 
for agtiressive chemotherapy early in the course of their disease before 
severe renal disease has occurred. 

(2) Little has been done to support the patient with myeloma who develops 
renal failure since the renal disease has been felt to be irreversible. How- 
ever knowledge that such renal disease results from a direct toxic effect of 
the B-J protein affords new hope for future therapy. If the patient's myeloma 
can be arrested with appropriate chemotherapy, yet renal failure persists, 
such patients would be candidates for chronic dialysis and renal transplanta- 
tion. If the abnormal plasma cells can be eliminated from the marrow and as 
a consequence the B-J protein is eradicated, then the transplanted kidney 
would not be at risk to recurrent "myeloma renal disease". 

We have recently transplanted one such myeloma patient. This 62 year 
old white female presented with plasmacytosis B-J proteinemia and 
proteinuria, lytic bone lesions, and endstage renal failure. She was treated 
with intermittant high dose cyclophosphamide with healing of her skeletal 
lesions, disappearance of marrow plasmacytosis, and loss of B-J protein. 
However, there was no return of renal function. She was supported with twice 

FJ-61 



weekly hemodialysis for ten months without evidence of recurrent myeloma. 
She subsequently underwent a cadaveitic renal transplant which continues to 
function well at present, some 4 months postransplantation. There was 
no evidence of recurrent myeloma kidney disease on renal biopsy performed 
2.5 months post-transplantation. 

Proposed Course of Project : The study has been completed; manuscripts are 
in preparation. 

Awards and Honors : None 

Publications: None 



.1 

I 



Serial No. hd-cp 20 

1. Gerontology Research Center 

2. Clinical Physiology Branch 

3. Metabolism Section 

4. Baltimore, Maryland 



PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Uremia and Carbohydrate Intolerance 

Previous Serial Number: New Project 

Principal Investigator: Ralph A. DeFronzo 

Other Investigators: Reubin Andres 

Jordan Tobin 
Karl Kramer 
Jack Rowe 
Gordon Walker* 
Dan Sapir* 
Paul Edgar* 



Cooperating Units: 

Man Years 

Total: 

Professional : 
Other: 

Project Description: 



Johns Hopkins Hospital 
* Renal Division 



1.0 
0.5 
0.5 



Objectives: Glucose intolerance occurs in greater than 50% of uremic 
individuals. However, despite numerous investigations the underlying 
mechanism (s) has not been agreed upon. Before the advent of dialysis 
and transplantation the finding of abnormal glucose tolerance in patients 
with chronic renal disease had little bearing on patient management. 
However, the availability of these newer therapeutic measures has created 
new significance for uremic carbohydrate intolerance. Where diabetics 
have been accepted for chronic dialysis or transplantation the incidence 
of serious complications and mortality has been 4-5 times higher than 
in non-diabetics. Thus it has become important to distinguish between 
true diabetes mellitus and uremia-induced glucose intolerance. 

Because of the diagnostic and therapeutic implications of carbohydrate 
intolerance to the patient with chronic renal disease, it was felt important 
to summarize the large body of literature that has accumulated on the 
subject of uremia-induced carbohydrate intolerance and to attempt to 
explain apparent discrepancies reported by different investigators. This 



FJ-63 



I 

was done and our review paper has been accepted for publication in Medicine. 
In this paper we propose the hypothesis that glucose intolerance occurs in 
those indi.viduals who are unable to augment insulin secretion sufficiently 
to overcome the peripheral antagonism to insulin that exists in uremic 
individuals. The studies to be reported here are designed to test this 
hypothesis. 

Methods Employed : Carbohydrate tolerance was evaluated in eight chronically 
uremic subjects accepted into our hemodialysis program employing the 
servo-control "glucose clamp" technique previously described by us and 
with standard intravenous and oral glucose tolerance tests. 

Major Findings : Beta cell sensitivity was examined by "clamping" the arterial | 
blood glucose concentration at 125 mg% above fasting levels and measuring 
the plasma immunoreactive insulin (IRI) response. Glucose metabolized, 
M, equals glucose infusion rate and is a measure of glucose tolerance. In 
all eight subjects diminished glucose tolerance was observed pre dialysis. 
To date four subjects have been restudied post-dialysis and all four have 
demonstrated significant improvement in glucose tolerance. In one of these 
four subjects the improvement was due to increased pancreatic insulin 
secretion. 

Insulin resistance was evaluated after a continuous 80 min. intravenous 
infusion of 1 mU insulin per kg body wt per min, hypoglycemia being pre- 
vented by a variable-rate infusion of IV glucose. Since arterial glucose . 
concentration is maintained at a constant level, all of the glucose infused I 
must be metabolized and the glucose infusion rate is a measure of sensitivity 
of tissues to insulin. In 7/8 subjects mild to severe degrees of insulin 
resistance were noted prior to dialysis. All four subjects studied post- 
dialysis have demonstrated increase in insulin sensitivity. 

In two of the four subjects studied post-dialysis, the improvement in 
glucose tolerance could not be entirely accounted for by the increased 
pancreatic beta cell response or by the enhanced insulin sensitivity. This 
suggests that a third factor may be important in the genesis of uremia- 
induced glucose intolerance - namely, an inability of the liver to shut 
off gluconeo-genesis in response to insulin and glucose. 

Compared to the glucose clamp technique the IVGTT and OGTT proved to 
be poor indicators of the degree of glucose intolerance pre-dialysis; i 
furthermore, in 1/4 patients studied post-dialysis was significant improve- 
ment noted despite marked improvement in the hyperglycemic clamp. 

Significance to Bio-Medical Research : These results substantiate our hypothesis 
that carbohydrate intolerance in uremia may result from either insulin 
resistance, diminished insulin secretion, or a combination of these. In 
addition they suggest that the liver may also play a central role in the 
genesis of this glucose intolerance. It is clear that with techniques 
currently available at most renal centers it is impossible to differentiate 
genetic diabetes mellitus from uremia-induced glucose intolerance. It 
therefore, seems unwise to recommend that mild elevation of the fasting 
blood sugar and mild to moderate glucose intolerance on the intravenous 
or oral glucose tolerance test to be used as criteria to exclude patients (I 
from transplant programs. [ 



FJ-64 



Proposed Course : The study will be completed by July 1, 1973. 
Honors a^nd Awards : None 

Publications : Carbohydrate Metabolism in Uremia: A Review. Ralph A DeFronzo, 
R. Andres, P. Edgar, and W.G. Walker, accepted for publication in Medicine, 
April 27, 1973. 



FJ-65 



Serial No. hd-cp 21 

1. Gerontology Research Center 

2. Clinical Physiology Branch 

3. Metabolism Section 

4. Baltimore, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: The response to solar-simulating radiation as a function of 
age 

Principal Investigator: K.J. Kramer 

Previous Serial Number: New Project 

Other Investigators: I. Willis 

Department of Dermatotology 

Johns Hopkins University School of Medicine 

Cooperating Units: None 

Man Years: 

Total: 0.2 
Professional: 0.2 
Other: 0.0 

Project Description: 

Objectives ; Actinic damage is a precursor to cutaneous malignancy. The 
normal sunburn response has therefore been a subject of great interest. 
This response may be influenced by a number of factors including age and 
skin pigmentation. The particular spectrum causing "sunburn" is from 
290-320 nm; longer wavelength ultraviolet radiation (320-400) causes 
"tanning" and had been felt to have no effect on sunburn or even to pro- 
tect the skin from damage caused by short UV radiation (290-320 nm) . It 
has however recently been shown (Willis) that the longer wavelength UV 
radiation actually enchances damage caused by the 290-320 radiation; this 
has been termed the photoaugmentative effect. In light of these new 
findings, coupled with the fact that older studies suffered from serious 
technical defects, the human sunburn response deserves re-evaluation. 
Specific objectives are: 

1) To determine susceptibility to sunburn as a function of age 

2) To determine the photoaugmentative effect of long UV wavelength light 
in relation to age 

3) To determine the time course of the sunburn response of individuals 
in relation to age 

4) To determine all of the above with respect to exposed and non-exposed 
skin 



FJ-66 



I 



Methods Em ployed; Longitudinal participants will be divided into decade 

age groups and into clinically determined skin pigmentation groups. A 

total of about 100 subjects will be tested. Individual areas of skin (6mm 

diameter each - total of 12 sites) on the arm and back will be exposed to 

increased levels of full spectrum radiation (290-700 nm) (4 sites); short 

UV irradiation (290-320) (4 sites) and short and long UV radiation (290-400), 

Exposures will be chosen above ind below the minimal amount of energy that 

will be anticipated to produce erythema over the entire test site (minimal 

erythema dose or MED). Responses will be read immediately and at 6,8,12, 

24,36, and 48 hours by either the investigators or nurses trained for the 
task . 

Results; Preliminary results are confirming the photoaugmentative effect. 
Somewhat unexpectedly, the immediate response does appear to anticipate 
the 12 and 24 hour responses even though the physiologic bases for these 
responses are different. This fact has potential clinical significance in 
that it could facilitate the time-consuming process of testing and would 
make screening for abnormal responses practical. 

Significance of Results : Confirmation of the photaugmentative effect will 

change the criteria for sunscreens designed to be prophylactic for the 

development of cutaneous carcinoma, since present preparations screen 
primarily short UV light. 

In addition, delineation of the evolution of the normal response to solar 
radiation with age will add new information concerning the aging process in 
an organ little studied, the skin. 

Proposed Course of Project : It is anticipated that the project will be 
completed by July. 

Honors and Awards: None 

Publications: None 



FJ-67 



NICHD ANNUAL REPORT 

July 1, 1972 through June 30, 1973 

Gerontology Research Center 

Laboratory of Cellular and Comparative Physiology 

FY '73 has been highlighted by a major change in the emphasis of program 
goals and in the accompanying research activities to achieve these goals. 

The current goals are three-fold: (a) to conduct studies on the nature 
of age-related deteriorating functional changes of cells and tissue systems 
whose growth and differentiation events are reasonably well understood, (b) 
to determine the underlying mechanisms of these changes, and (c) to develop 
methods for early detection of the deterioration and, hopefully, methods to 
control or reverse the deterioration. 

To implement these goals emphasis will be on the use of cells and 
tissues of mammalian species, including man, in in vitro and in vivo cul- 
tures. Moreover only a few closely related model systems will be analyzed, 
but they will be studied systematically. 

The major thrust in research activity has been in immunology and aging, 
with the creation of the Immunology Section, for the following reasons, (1) 
Associated with, or as a consequence of immunosenescence, the incidence of 
immunodeficiency diseases including autoimmunity, cancer and infection in- 
creases. (2) Cellular, molecular and genetic knowledge of the ontogeny, 
phylogeny and differentiation process involved with the immune system is 
well understood, probably more so than any other functional system. (3) The 
system is amenable to elegant cellular and molecular analyses, and, there- 
fore, offers great promise for successful manipulation of the system. 

During the past year, members of the Immunology Section expanded their 
effort in the creation of the laboratories and initiation of research 
projects. Hence fruition of their current research task should become 
apparent at the earliest during the next fiscal year. Nevertheless, basic 
findings were obtained in FY ' 73 , some of which were established just prior 
to their arrival at the GRC, and they are as follows. 

(1) Of the three cell types (thymus -derived T cells, bone marrow -derived 
B cells and accessory A cells) involved in the initiation of a humoral immune 
response, the T and B cells are altered in immunologically deficient old 
mice, but A cells appear normal. 

(2) The proliferative capacity of T and B cells is decreased as much as 
10-fold with age. This is a very critical function because the number of 
functional effector cells generated in an immune response is dependent upon 
these cells to divide efficiently. 

(3) Limiting dilution, reconstitution, and young cell-old cell mixture 
analyses bear out indirect evidence that either the relative number or the 
efficiency of regulator cells that suppress an immune response increases with 
age. For example, it was found that the humoral immune activity of a mixture 



FK-1 



i 



of spleen cells from young and old mice was lower than the sum of individual 
responses, indicating that there are cells in old mice that can inhibit young 
immunocompetent cells from responding to antigenic stimulation. 

(4) One of the deficiencies of radiation-induced allogeneic bone marrow 
chimeras was shown to reside in the thymus gland, the seat of immunodefi- 
ciency diseases in most cases. Previously it was shown that these chimeras 
are suitable model animals to study immunosenescence. 

(5) Limiting numbers of lymphoid cells undergo maximum antibody response 
in vitro only when they are cultured under optimum cell concentration, a 

variable which was not taken into consideration previously. This information A 
offers us the option of assessing quantitatively the activity of limiting 
numbers of lymphoid cells and, hopefully, one or two clones of competent 
cells . 

(6) Mice with an exceptionally long life span show a decline with age 
in cell-mediated immune activity and resistance to allogeneic tumor cells. 
Previously the decline in activity was observed in mice with short life 
spans. Therefore critics have argued that life-shortening diseases imposing 
on the immune system were responsible for the decline in cell-mediated 
Immunity. These results should minimize such criticisms. 

Based on these findings efforts are focused currently on the cellular M 
and molecular definitions of immunosenescence with emphasis on (a) isolation 
and characterization of promotor and suppressor cells, (b) ability of stem 
cells to generate thymus -derived T and bone marrow-derived B cells, (c) life 
spans of memory cells, (d) mitogen receptor sites and the cyclic AMP system 
of proliferating T and B precursor cells, and (e) characterization and repair 
of induced and naturally occurring immunodeficiency states. 

Research has also been initiated on a small scale in biomembrane 
physiology, with emphasis on the effect of age on hormone bindings, for two 
reasons: (a) it will complement ongoing molecular studies in immunology and 
(b) the time is "ripe" to initiate cellular and molecular studies focused on 
the nature of inefficient response to hormones and drugs by the elders. g 

Preliminary results showed that adipose tissue, skeletal muscle, brain and ^ 
prostate gland of aging rats exhibit a progressive decline in steroid hormone 
binding due, in part, to a reduction in the number of macromolecular binding 
sites per unit tissue mass. 

Another area of research that can contribute to the program goals of 
LCCP is mammalian genetics. An understanding of the genetic mechanisms of 
age-related functional deterioration at the molecular level is obligatory. 
To this end, research is being promoted to understand, at the cellular level, 
human and animal disorders manifesting altered development and aging. 
Currently a laboratory is being created to carry out two studies: (a) the 
role of chromosomal aneusomy in the impaired proliferation of fibroblasts 
and immunocompetent cells in relation to aging and (b) the influence of gene 
dosage and maternal environment on the life span of embryos and individuals 
carrying lethal genes. The long-term goal of these studies is to "sort out" 
and elucidate those genetic alterations which can impair the proliferative 

FK-2 



capacity of somatic cells. Information derived from this program should have 
a tremendous impact on cellular biology of aging, for impairment with age of 
the proliferative capacity is characteristic of certain somatic cells. 

Two sections in existence prior to FY '73 are the Nutrition and 
Morphology Sections. The former is concerned with the influence of nutrition 
and antimetabolites on the life span and various tissue enzyme activities in 
rats. The latter is concerned with structural and cytochemical changes asso- 
ciated with development and aging of myocardial and skeletogenic cells and 
with the growth patterns of coelenterates as influenced by temperature and 
endogenous rhythmic changes. 

During the past year, the Nutrition Section completed preliminary 
studies in two areas: (a) the effect of cycloheximide on the rate of devel- 
opment of chick embryos and (b) the effect of protein undernourishment on 
the life span of middle-aged rats. In the former study it was found that 
nontoxic levels of cycloheximide, an inhibitor of protein synthesis, when 
given to 1 day old chick embryos delay their rate of development at least 
until day 17, as judged by morphological parameters, heart beat and DNA 
content. However, the activities of lysosomal and mitochondrial enzymes were 
not affected. 

The latter study revealed that reduction in the dietary protein intake 
in 16 month old rats from the standard 247o to 8 or 47o had no influence on 
their subsequent mean life expectancy. However rats restricted to 127o casein 
diet lived on the average of 2 months longer. The life promoting effect of 
127o protein intake was manifested during the first 3-4 months following 
dietary restraint. In view of the profound implication this observation 
offers, a more systematic study is being initiated. 

Ultras true tural cytochemical studies by the Morphology Section relating 
lysosomal activities and aging processes in the left ventricular cells of 
&ging rats suggested that in mural myocardial cells there are (a) two differ- 
ent pathways by which primary lysosomes are formed and (b) two different 
mechanisms of lysosomal degradation of mitochondria. The two pathways of 
synthesis, transport and packaging of primary lysosomes are the nuclear pole 
zone route and the peripheral route. The latter, which bypasses the Golgi, 
provides the cells a more direct route to sites of action of the lysosomes. 
Lysosomal degradation of mitochondria occurs by the more common matrix 
densif ication process and the less common matrix clarification process. 
Studies on papillary myocardial cells of 24 month old rats showed that there 
are two types of changes: (a) cells that appear grossly normal in struc- 
tural appearance but showing marked intracellular changes in comparison to 
those of 6 and 12 month old rats, and (b) cells that appear grossly abnormal 
in structural appearance. Ultrastructural definition of age related deteri- 
oration of heart muscle cells may provide us a better insight how to 
resolve whether the changes observed are the cause or consequence of 
senescence of the organ. 

Studies on endogenous circannual rhythms in growth, development and life 
spans in marine coelenterates revealed that they can persist for over 3 years 
in the absence of periodic signals from the environment. The period for the 

FK-3 



cycles at three ambient temperatures, 10°, 17° and 24°C, was about one year. 
The life span of hydranths during prolific growth of the colony was consid- 
erably longer than during repressed growth. These observations suggest that 
there can be exceptions in certain invertebrates to the two widely held 
views on life span: (a) the span of life of a species is specific and (b) 
delay of growth can extend life expectancy. 



FK-4 



Serial No. : HD-CCP-12 

1. Gerontology Research Center 

2. Laboratory of Cellular and 

Comparative Physiology 

3. Immunology Section 

4. Baltimore, Maryland 

PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Characterization of Age-related Defect in the Proliferative 
Capacity of Lymphocytes 

Previous Serial Number: None 

Principal Investigator: Takashi Makinodan 

Margaret L. Heidrick 
Other Investigator: None 

Cooperating Units: Oak Ridge National Laboratory 

Oak Ridge, Tennessee 

Man Years : 

Total: 1.2 

Professional: 0.7 

Other: 0.5 

Project Description: 

Objectives : The proliferative capacity of stimulated lymphocytes is known 
to be defective in old mice. Both thymus -derived (T) and bone marrow- 
derived (B) cells are affected as determined by a decrease in the number of 
antibody-forming progeny generated per immunocompetent unit, a decrease in 
the mixed lymphocyte reaction and a depressed response to specific mitogens. 
The objective of this study is to determine the underlying cause (s) of this 
age-related defect. Three possible causes are being investigated: (1) an 
increase of regulator or repressor T-cells, (2) a decrease in the number of 
mitogen receptor sites and/or (3) a defect in the cyclic-AMP system. 

Methods Employed : Stimulation of lymphocyte proliferation with specific 
mitogens is being used as the model system. Spleen cells are separated 
into distinct populations by size and/or density. The component cells are 
then assessed for (1) their proliferative ability, (2) number of mitogen 
receptor sites and (3) changes in their cyclic-AMP system. 

Ma j or F ind ings : 

Presence of Repressor Cells . Recent reports in the literature indicate a 
sub-population of T-lymphocytes may suppress the response of immunocompe- 
tent cells and thereby regulate the magnitude of the response. It is thus 
possible that an increase of such repressor cells could be a causative 
factor in the depressed proliferative capacity of lymphocytes from old mice 

FK-5 



A search for such suppressor cells in the spleens of old mice has yielded 
the following information: (1) While the total number of lymphoid cells 
remain relatively constant with age, the proportion of lighter cells 
increases. (2) Light and heavy cells can be distinguished by their re- 
sponsiveness to mitogens, the lighter cells being responsive to pokeweed 
mitogen and the heavier cells to concanavalin A. (3) The responsiveness 
of the heavier T cells to concanavalin A can be decreased by mixing them 
with lighter cells, indicating that there are regulator cells in the light 
cell fraction that can suppress the heavier cells from responding to the 
mitogen. 

Mitogen Receptor Sites . It is known that specific mitogens bind to the 
surface of lymphocytes and stimulate the cells to divide by some unknown 
event occurring at the ceU membrane . A possible explanation for the de- 
creased response of aged lymphocytes would be a decrease in the number of 
mitogen binding sites. To investigate this possibility, the amount of 
■'•^-'l-labeled concanavalin A bound by young and old spleen cells was 
measured. Preliminary results indicate that both young and old cells bind 
the same amount of mitogen and suggests that on the average the number of 
mitogen binding sites on the lymphocyte surface does not decrease with age. 

Cyclic-AMP System . The earliest known biochemical event which occurs in 
lymphocytes after stimulation with mitogens is a sharp rise and then fall 
in the cyclic-AMP and cyclic-GMP levels of the cells. These changes occur 
within minutes after mitogen stimulation and there is evidence that changes 
in the intracellular levels of the cyclic nucleotides initiate and regulate 
cell proliferation. It is thus possible that a defect in the cyclic-AMP 
system of the old lymphocytes could be a contributing factor to their 
defective proliferative capacity. To investigate this possibility, the 
cyclic-AMP and cyclic-GMP changes in young and old lymphocytes after mito- 
gen stimulation are being measured. This study has just recently been 
started. To date, most of the preliminary experiments (i.e., determining 
optimum mitogen concentrations, cell nvraibers required for obtaining cyclic 
nucleotide levels, etc.) have been completed but comparative data of young 
and old cells is not yet available. 

Significance to Biomedical Research and the Program of the Institute : 
Precursor T and B cells generate effector progenies efficiently through 
proliferation. Therefore reduction in the proliferative capacity of aged 
precursor cells is an important factor in the reduced immune response of 
the aged. Correction of this deficiency should have a beneficial effect 
on the immune system of the aged which, in turn, may lower the incidence 
of immunodeficiency diseases . 

Proposed Course : If the cause or causes of the defective proliferative 
capacity of the old lymphocytes can be determined, then attempts will be 
made to correct the defect (i.e., removal of repressor cells, treatment of 
the cells with various agents to alter the cyclic nucleotide levels) . If 
a correction is achieved, it will then be determined if the ability of T 
and B precursor cells to function in an immune response is improved. 



FK-6 



Honors and Awards : 

Dr. Makinodan was invited to present a seminar at the Aging Research 
Center, Korman Research Pavillion, Albert Einstein Medical Center, 
Philadelphia, Pennsylvania on December 4, 1972. 

Dr. Makinodan was invited to chair a Session on Immunobiology and to 
present a paper at the 25th Annual Scientific Meeting of the Gerontological 
Society in San Juan, Puerto Rico, December 17-21, 1972. 

Dr. Makinodan was invited to give a seminar at the Dental Research Center*, 
University of North Carolina, Chapel Hill, North Carolina on February 19, 
1973. 

Dr. Makinodan was invited to present a seminar at the Department of Labor- 
atory Medicine, Medical School, University of Minnesota, Minneapolis, 
Minnesota on March 30, 1973. 

Dr. Makinodan was invited to present a seminar at the Department of Micro- 
biology, University of Vermont, Burlington, Vermont on April 2, 1973. 

Dr. Makinodan was invited to present a lecture series at the Department of 
Microbiology, School of Medicine, Johns Hopkins University, Baltimore, 
Maryland on May 2-3, 1973. 

Publications: Heidrick, M.L., and Makinodan, T.: Nature of cellular 

deficiencies in age-related decline of the immune system. 
Gerontologia . In press. 

Price, G.B., and Makinodan, T.: Aging: Alteration of 
DNA-protein information. Gerontologia . In press. 



FK-7 



Serial No.: HD-CCP-13 

1. Gerontology Research Center 

2. Laboratory of Cellular and 

Comparative Physiology 

3. Immunology Section 

4. Baltimore, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Characterization of Cellular Deficiencies in Age-related 
Decline of the Humoral Immune System 

Previous Serial Number: None 

Principal Investigators: Margaret L. Heidrick 

Katsuiku Hirokawa 

Other Investigator: T. Makinodan 

Cooperating Units: Oak Ridge National Laboratory 

Oak Ridge, Tennessee 



Man Years: 




Total: 


1.6 


Professional : 


1.1 


Other: 


.5 



Project Description: 

Objectives : The humoral immune activity of BC3F]^ mice declines with 
advancing age. The cause for the decline is due primarily to defects in 
the immunocompetent cells. Since three different cell types [accessory 
cells, thjmius -derived lymphocytes (T-cells) , and bone marrow-derived 
lymphocytes (B-cells)] are known to be involved in producing an immune 
response, the defect in the old mice could be due to a decrease in the 
number or function of one or more of these three essential cell types. 
The objectives of this project are to (1) separate the three cell types 
from diseased and nondiseased spleens of old mice, and (2) determine the 
functional capacity of each cell type in the old mice. 

Methods Employed : Several cell separation techniques are being used in 
this study in an attempt to separate the different cell types. These 
include: separation by differential adherence to plastic, differential 
centrifugation in a discontinuous Ficoll gradient, separation on glass 
bead columns and separation by size on a Stay-Put cell separator. The 
purity of the separated cell fractions is being assessed by use of spe- 
cific anti-sera, specific mitogens, size, and functional capacity. The 
ability of the old cell fractions to function is being evaluated by com- 
bining the old cell with young cell fractions and assessing the humoral 
response of the various combinations to sheep red blood cell antigen 
(SRBC) . 

FK-8 



Major Findings : 

Activity of the Accessory Cell . Spleen cell suspensions from young and old 
mice were separated into two populations on the basis of their ability to 
adhere to plastic Petri dishes and their activity was assessed in various 
combinations with SRBC. The accessory cell is present in the adherent 
population while the T- and B-cells are in the non-adherent population. 
Old adherent cells cultured with young non-adherent cells responded fully 
to the antigen, whereas old non-adherent cells combined with young adherent 
cells gave a response comparable to unseparated old spleen cells. Similar' 
results were obtained when adherent cells were exposed to SRBC, washed free 
of the antigen, then recombined with the nonadherent cells. The results 
demonstrate that the adherent cells of old spleens are indistinguishable 
from those of young spleens in their ability to initiate antibody response, 
but the activity of nonadherent cells of old spleens is impaired. To 
further evaluate the activity of the accessory cell, which is thought to be 
a macrophage, peritoneal macrophages from young and old mice were compared 
as to (1) number of SRBC engulfed, (2) time required to digest engulfed 
cells and (3) activity of three lysosomal enzymes --catheps in D, beta- 
glucuronidase and acid phosphatase. The results indicated the old macro- 
phages had slightly higher levels of all three enzymes and were equivalent 
to young macrophages in their ability to engulf and digest SRBC. Thus by 
various indices it appears that macrophages do not deteriorate with age. 

Activity of T and B Cells . In the Ficoll gradient, non-adherent cells 
separate into two fractions, a light fraction enriched in B-cells and a 
dense fraction enriched in T-cells. Combining these fractions in various 
combinations and proportions in vitro revealed that an optimal ratio of T 
and B cells was required to generate a maximum response to SRBC. For both 
young and old cells, the optimal ratio was about the same. When old spleen 
cells were separated in the Ficoll gradient, a different separation profile 
was observed. The old spleens contain an increased percentage of cells 
separating in the lighter fractions and a decrease in the percentage of 
cells in the dense fraction. To determine if the altered ratio of T to B 
cells in the old mice was a contributing factor to the defective response, 
old spleen cells were separated in the Ficoll gradient and then reconsti- 
tuted in the ratios observed in young mice. However, our preliminary 
attempts to improve the response of the old cells by changing the ratio of 
the Ficoll fractions have thus far been unsuccessful. Replacing individual 
fractions with young cells was not effective until both fractions were 
replaced with young cells. Our results indicate both T and B cells are 
defective in the old mice and that the defect is not due merely to a shift 
in the ratio of cell types present. 

Cell Interaction . To investigate possible deficiencies in the necessary 
cellular interactions required for an immune response, we have studied the 
ability of young spleen cells to supplement tlie defective old spleen cells. 
For these studies, dispersed pooled spleen cells from young (3 months) and 
old (24 and 30 months) mice were assessed individually in 1 : 1 mixtures for 
their antisheep RBC responsiveness. The response of old spleen cells is 
always lower than that of young spleen cells. However, the magnitude of 
response of the mixtures was dependent upon the disease status of the 



FK-9 



spleen of old mice and the type of culture used in the assay. In the case 
of mixtures of cells from old spleens with frank reticulum cell sarcoma, 
the response in vitro and in vivo was either equal to (additive) or less 
than (inhibitory) , but never greater than (synergistic) , the sum of the 
individual responses. This would suggest that old tumorous spleen cells 
can inhibit the response of young spleen cells. In the case of mixtures of 
cells from old spleens free of frank tumors, the response was influenced in 
part by the type of culture. Synergistic, additive, and inhibitory re- 
sponses were observed when the mixtures were cultured in vitro and in vivo 
in diffusion chambers. The synergistic response would suggest that the 
cells or factors which are limiting in the young spleen cell suspension are 
in excess in the old spleen cell suspension and vice versa. Interestingly, 
inhibitory responses were not observed when the mixtures were assessed in 
vivo in the spleen of irradiated syngeneic young recipients. This would 
suggest that irradiated young spleens may possess factors that can counter- 
act the inhibitory action of certain old spleen cells from donors free of 
frank lymphoreticular diseases. 

Significance to Biomedical Research and the Program of the Institute : The 
decline of the immune system with age has an obvious effect upon general 
senescence and longevity. If the cell type or types which are responsible 
for the decline can be separated and identified, this will be a significant 
step toward determining the cause for the decline at the molecular level. 

Proposed Course : Further studies will continue to separate and identify 
the cell types responsible for the decline of the immune system. When this 
is accomplished, studies will be initiated to determine the nature of the 
defect in the cells. 

Honors and Awards : 

Dr. Makinodan served as a member of the Workshop Panel of the Task Force 
on Immunology and Disease sponsored by NIAID. 

Dr. Makinodan was invited to present a lecture on Immunology and Aging by 
Dr. R. A. Good, Department of Pathology, University of Minnesota, Minne- 
apolis, Minnesota on November 14, 1972. 

Dr. Makinodan was invited to present a seminar at the Jefferson Medical 
College, Department of Biochemistry, Philadelphia, Pennsylvania on 
November 29, 1972. 

Dr. Makinodan was invited to attend and present a seminar on "Cellular 
Aspects of Age-Associated Impairment of the Immune System" at the Gordon 
Research Conference, Biochemistry of Aging, held 1/22-1/26/73 at Santa 
Barbara, California. 

Dr. Makinodan served as a member of the Public Policy Committee of the 
Gerontological Society. 

Dr. Makinodan was invited to present a seminar at the Department of Biology, 
University of Puerto Rico, Rio Piedras, Puerto Rico on December 22, 1972. 



FK-10 



Dr. Makinodan served as Associate Editor of the following journals: 
Journal of Cellular Physiology , Blood , Mechanisms in Aging and Development , 
and Journal of Immunology . 

Publications: Quinn, R.P., Price, G.B., Ellis, J.M., and Makinodan, T.: 
Catabolic half-lives of immunoglobulin and albumin as a 
function of age in mice. J. Gerontol. In press. 

Makinodan, T., Heidrick, M.L., and Nordin, A. A.: Immunode- 
ficiency and autoimmunity in aging. In Second International ' 
Workshop on Primary Immunodeficiencies in Diseases in Man . 
National Fdn. Press, Washington, D.C., 1973. In press. 



FK-11 



Serial No. HD-CCP-14 

1. Gerontology Research Center 

2. Laboratory of Cell-ular and 
Comparative Physiology 

3. Immunology Section 
U. Baltimore, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Celliilar Basis of Regulation of the Humoral Immune Response 

Previous Serial Nvmiber: None 

Principal Investigator: Albert A. Nordin 

Other Investigators: Barbara E. Lou^rmian 

John J. Parrar 

Cooperating Units: 

Man years: 

Total: 2.3 

Professional: 1.6 ^ 

Others : 0.7 

Project Description: 

Objectives : The goal of this project is to characterize the regulation of 
the immune response as expressed by cellular elements. Effoirts to deter- 
mine the origin and mechanism of action of these cells are of prime 
interest . 

Methods Employed : 

T) B-cell source-Young C$7Bl/6 md.ce (6 weeks of age) are thymectomized, 

iirradiated 2 weeks later and grafted with 10 x 10" syngeneic bone i 

marrow cells 21+ hours after irradiation. Pour to ei^t weeks later 
the spleens of these mice serve as a source of bone marrow derived 
lyoaphocytes (B-cells). 

2) T-cell source-six to ei^t week old C57B1/6 or B6D2P1 mice are used 
as donors for thymus glands. X- irradiated (850r) syngeneic mice are 
injected with 50-70 x 10" thymus cells with or with a simultaneous 
injection of antigen. Seven or ei^t days later the spleens of these 
mice are used as a source of thymiis derived lymphocytes (T-cells). 
Those cells from mice injected with thymocytes and antigen are referred 
to as activated T-cells and those from mice receiving only thymocytes 
are refeired to as normal T-Cells. 

3) The in vitro ciilture technique and the assay for plaque-forming cells "j 
are routine methods. ! 



I 



FK-12 



Mai or Findings : 

1) Activated T-cells partially reconstitute the in vitro response of 
B-cells. The ratio of T:B-cells is a critical parameter. 

2) A high ratio of activated T:B-cells results in significant suppression 
of the in vitro response. 

3) Significant numbers of B-cells contaminate adherent cell layers. 
Expression of these B-cells requires the addition of activated T-cells. 

4) Activated T-cells suppress the rn vitro response of normal spleen cells. 

Significance to Biomedical Research and the Program of the Institute : This 
proposal attempts to offer two main significant contributions. The basic 
cellular interactions involved in the total immune response extends into 
many areas of research. Of great interest in cell biology is the control 
of cellular functions. The regulation of the humoral immune response by 
direct cellular interactions and/or by products of "regulator" lymphoid 
cells is one of the main contributions to be derived from this research. 
Of equal significance is to demonstrate the role, if any, of this cellular 
regulation in aging. Any logical approaches to maintenance of a functional 
immune capacity in aging individuals can only be undertaken when the facts 
surrounding the problem are firmly documented. 

Proposed Course : 

1) Investigate the occurrence and distribution of suppressor T-cells. 

The preparation of normal T-cells as well as determination of naturally 
occurring suppressor cells in normal spleen cell populations is planned 

2) Identification and characterization of suppressor cells. Physical 
means of separation, i.e., discontinuously Ficoll gradients, gravity 
sedimentation, electrophoretic mobility as well as biological methods 
of preparation, i.e., cell transfer, antisera treatment will be used to 
obtain an enriched cell fraction for suppressor cell studies. 

3) Determine if suppressor cells also function in T-independent immune 
system. Some success has already been observed using detoxified 
endotoxin of E. coli as a T-independent antigen. 

4) Investigate the mechanism of suppression. It is necessary to establish 
if suppression is exerted by direct cellular interaction or by soluble 
cell products. 

5) The immunosenescence observed in aged mice will be examined by these 
established methods to determine what role suppressor cells play in the 
decline of the humoral immune response seen with increasing age. 

Honors and Awards: Dr. Albert A. NordLn participated as a visiting staff 

member in the Cell Culture Procedures for Aging Research 
course conducted at the University of Vermont on 
September 22, 1973. 



FK-13 



Dr. Albert A. Nordin is presently a member of the Council 
of the Regulatory Biology Section of the National Science 
Foundation. 

Publications: Nordin, A. A., and Makinodan, T.: Symposium on immunopathology 
of aging: Humoral immunity in aging. Fed . Proc . In press. 



FK-14 



Serial No. HD-CCP-15 

1. Gerontology Research Center 

2. Laboratory of Cellular and 
Comparative Physiology 

3. Imntunology Section 
h' Baltimore, Maryland 

PHS-MIH 
Individual Project Report 
July 1, 1972 throu^ June 30, 1973 

Project Title: Cellular Aspects of the Immune Response of Long-term 
Radiation Induced Allogeneic Chimeras 

Previous Serial Number: None 

Principal Investigators: Barbara E. Lou^iman 

Other Investigators: Albert A. Nordin 

Cooperating Units: None 

Man Years: 

Total: 0.3 

Professional: 0.2 

Other: 0.1 

Project Description: 

Objectives : The objective of the proposed research is to delineate the 
immune system of x- irradiated, allogeneic bone marrovf grafted mice in an 
attempt to use these mice as a model for investigating basic cellular 
aspects of the immune mechanisms and immunodeficiencies. 

Methods Employed : Previous work has establi.shed that allogeneic bone 
marrow chimeric (ABMC) mice can only survive in a controlled environment. 
This environment has previously been the germ free state but there is 
reasonable evidence to suggest that a clean environment would s\iffice. 
This clean environment will be created by using a low velocity laminar-flow 
tent. 

Major Findings : The findings listed below are relevant to this project 
even thou^ they were obtained prior to instituting this activity at the GRC 

1) AayK) mice show a normal life-span but the humoral immune response is 
drastically reduced. 

2) The lymphoid system of the ABMC mice is completely derived from donor 
bone marrow cells. 

3) The marked humoral immune deficiency has been associated with thymus 
gland malfunction. 



FK-15 



I|.) The thymic deficiency can be corrected completely in vitro hut as yet 
in vivo reconstitution has failed. 

Sigiificance to Biomedical Research and Program of the Institute ; The 
res^llts of these investigations will be significant in a practical sense. 
Clinical bone marrow grafting within a heterogeneous population has 
necessarily been restricted mainly as a resiilt of immunologic damage to the 
host. For obvious reasons, the parameters involved in these reactions are 
not as easily approached in human as they are in a representative animal 
model. Techniques are available to experimentally determine not only the 
favorable conditions, which permit the establishing of allogeneic bone 
marrow chimeric mice, but also to define the events resulting in immuno- 
regulation observed in such animals. Hopefiilly, these results will bear a 
semblance to those encountered in human bone marrow grafting. Information 
may then be available i/diich will reduce the risks in bone marrow grafting 
and result in the reconstitution of an intact immune mechanism. 

Basic information pertinent to the immune response is equally significant. 
Cellular immunology, during recent years, revolved aroimd cell cooperation 
as a basic requisite for the expression of antibody to many antigens. Al- 
thou^ the majority of such studies have considered the interactions 
between thymus dependent and bone marrow dependent cells, it is becoming 
increasingly obvious that other cell type cooperative efforts are involved 
in the complex immime systems of mammals. The deficiency of any one cell 
type most likely results in regulating the expression of immunity involving 
several lymphoid elements. The information which can be supplied in 
attempts to re-establish immunological competence to the allogeneic chimeric 
mice is expected to demonstrate and define the importance of cellular 
interactions in the both humoral and cell- mediated forms of the immune 
response. 

Studies concerned with immunodeficiency diseases will be of significance 
since many naturally occuring examples of such diseases have traced a 
causative factor(s) to the thymus gland. This model system with a well 
defined thymic defect shoiald provide meaningful data relevant to the 
immunodeficiency disease state. 

Proposed Coirrse ; 

The specific aims of the proposed research are: 

1) To establish long-term allogeneic radiation chimeras in a controlled 
laminar-air- flow environment . 

2) To characterize the humoral and cell-mediated immune responses of the 
allogeneic radiation chimeras prepared and maintained (at least l\. months) 
in a laminar flow environment. 

3) To locate and define, at the cellular level, the defects(s) involved 
in the severe regulation of the total immune response of allogeneic 
chimeric muce. 



TT-ir 1 /: 



k) To attempt to relieve this regulation by manipulating the intact 
animal. 

S) To investigate the possible reasons for the more normal life span 

afforded the allogeneic chimeras prepared in a controlled environment . 

Honors and Avrards: 

1) Dr. Barbara E. Loughman vra.s invited to present a seminar at the 
Trudeau Institute, Saranac Lsike, N. Y. , August 30? 1972. 

2) Dr. Albert A. Nordin presented a paper at the 25th Annual Scientific 
Meeting of the Gerontological Society held in Puerto Rico on 
December 18, 1972. 

3) Dr. Albert A. Nordin presented a seminar related to this project on 
January 10, 1973 to the Immunology Unit at Veterans Hospital, San 
Prancisco, California. 

ii) Drs. Bajrbara E. Lou^inan and Albert A. Nordin presented a seminar 
related to this project to the Johns Hopkins Immunology Group on 
Jan\iary 30, 1973. 

Publications: 

1) Loia^iman, B.E. , Nordin, A.A. , and Bealmear, P.: Studies of the 
immunological capacity of germfree mouse radiation chimeras. I. 
Chimerism and humoral immune response. Cell . Immunol . In press. 

2) Lou^iman, B.E. and Nordin, A.A. : Studies of the immunological 
capacity of germfree mouse radiation chimeras. II. Thymus cell 
function in a humoral immune response to sheep erythocytes. Cell . 
Trnrntmol. In press. 



FK-17 



Serial No.: HD-CCP-16 

1. Gerontology Research Center 

2. Laboratory of Cellular and 

Comparative Physiology 

3. Immunology Section 

4. Baltimore, Maryland 

PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: In Vitro Studies on the Capacity of Aged Mice to Develop 
Immunological Memory 

Previous Serial Number: None 

Principal Investigator: John J. Farrar 

Other Investigators: Barbara E. Loughman 

T . Makinodan 
Albert A. Nordin 

Cooperating Units: None 



Man Years: 




Total: 


1.5 


Professional: 


1.3 


Other: 


0.2 



Project Description: 

Objectives ; The objective of this research is to define the specific 
memory cell deficiencies in the aged mouse. 

Methods Employed : Modifications of the ±n vitro immunization procedure 
as described by Mishell and Button were used throughout the course of the 
research. The system involves culturing mouse spleen cells with antigen 
(sheep erythrocytes) in tissue culture for five days. The generation of 
antibody-forming cells (AFC) has been assessed by the Jerne and Nordin 
hemolytic plaque-forming cell assay. In several of the experiments, the 
spleen cells were separated into non-adherent (lymphoid) and adherent 
(non-lymphoid) cell populations prior to immunization in vitro . In 
several experiments, the capacity of bone marrow from aged mice to gener- 
ate bone marrow-derived (B) lymphocytes has been assessed. These experi- 
ments were performed using the in vivo cell transfer system of Claman, 
Chaperon and Triplett. 

Major Findings : Spleen cell dose-response experiments showed that the 
optimal cell density for development of AFC in vitro is the same for both 
the young and aged mouse cells. Using this cell density (10x10 cells per 
1 ml culture) , a series of experiments were performed to determine the 
ability of aged mice to develop immunological memory. Groups of young and 

FK-18 



aged mice were immunized intravenously with sub-optimal, optimal, and 
supra-optimal doses of antigen. At various time intervals thereafter the 
spleen cells were placed in tissue culture and rechallenged with the same 
antigen. In each case the secondary direct AFC response of the aged 
spleen cells was found to be enhanced above the level of the primary in 
vitro response. However, the secondary responses of the aged spleen cells 
were still severely deficient in comparison to the respective young spleen 
cell secondary responses. The number of indirect (IgG) AFC generated was 
not significant even in those cultures containing young primed spleen 
cells. 

Preliminary experiments on an assay system for assessing the degree of 
deficiency in the B-lymphocyte memory cell population have been completed. 
The system involved co-cultivating varying numbers of antigen-"activated" 
thymus -derived lymphocytes (ATC) with either unseparated spleen cells, 
non-adherent spleen cells, or anti-0 treated non-adherent spleen cells. 
In each case the ATC enhanced the response of the aged mouse B-cells but 
their response still remained deficient in comparison to the young spleen 
cell controls. The results suggest that aged mouse B-cells are deficient 
in their ability to generate AFC even when provided with a "purified" and 
"optimal" source of thymus -derived lymphocytes (T-cells) . 

The results of the first experiments using the In vivo transfer system 
suggested that aged bone marrow contained a significant thymus -derived 
lymphocyte (T-cell) contamination, which was not detected in young marrow. 
These observation.'', led to the us? of anti-mouse lymphocyte serum (ALS) and 
anti-9 to reduce the T-cell contamination. Subsequent experiments indi- 
cated that (1) bone marrow cells from ALS treated aged donors were defi- 
cient in their ability to generate AFC, and (2) anti-9 treatment of the 
bone marrow cells significantly reduced the T-cell contamination of the old 
marrow but did not depress its capacity to generate AFC. These results 
suggested that along with the T-cell contamination the aged marrow also 
contained an additional differentiated population. These cells apparently 
had an enhanced capacity to generate AFC, suggestive of a peripheral 
B-lymphocyte. In order to examine this possibility more directly, immuno- 
fluorescent studies using labelled antibody against mouse immunoglobulin 
were carried out. The results revealed an approximately 7-fold increase in 
the frequency of immunoglobulin-bearing cells in the bone marrow population 
of aged mice. B-lymphocytes are characterized by the presence of immuno- 
globulin on their surface. 

Significance to Biomedical Research and the Program of the Institute : The 
proposed research is significant in the field of gerontological research in 
that it is designed to specifically define the memory cell deficiencies of 
the aged mouse. Such a definition would be required before any attempt at 
antigen-specific immunological reconstitution of the aged animal could be 
undertaken. 

Proposed Course : In vitro experiments utilizing the T and B-cell co-culti- 
vation systems being developed will be used to assess the relative defi- 
ciencies (both immunological activity and turn-over) in the B and T-memory 
cell populations of the aged mice. 

FK-19 



Honors and Awards : 

Drs. Barbara E. Loughman, John J. Farrar and Albert A. Nordin presented a 
seminar related to this project to the Johns Hopkins Immunology Group on 
January 30, 1973. 

Dr. Makinodan was invited to present a paper entitled "Cellular Nature of 
Immunodeficiency in the Aging" at the 2nd International Workshop on Primary 
Immunodeficiencies in Man held in St. Petersburg Beach, Florida, February 
4-8, 1973. 

Dr. Makinodan was invited to present a seminar at the Wistar Institute, 
Philadelphia, Pennsylvania on March 14, 1973. 

Dr. Makinodan was invited to present a seminar at the Lobund Laboratory, 
University of Notre Dame, Notre Dame, Indiana on May 16, 1973. 

Dr. Makinodan was invited to present a seminar at the Department of Micro- 
biology and Immunology, Temple University, Philadelphia, Pennsylvania on 
May 23, 1973. 

Publications: None 



FK-20 



Serial No.: HD-CCP-17 

1. Gerontology Research Center 

2. Laboratory of Cellular and 

Comparative Physiology 

3. Immunology Section 

4. Baltimore, Maryland 

PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Pathology of Age-associated Kidney Disease of Wis tar Rats 
Reared at GRC 

Previous Serial Number: None 

Principal Investigators: Katsuiku Hirokawa 

Kenneth A. Rosenberg 

Other Investigators: None 

Cooperating Units: None 

Man Years : 

Total: 1.7 

Professional: 0.7 

Other: 1.0 

Project Description: 

Objectives : The objective of these studies is to establish the nature of 
the kidney disease of the rats reared at GRC. The severity of the disease 
was studied as a function of the age of the rats and to determine if the 
immune activity is associated with the kidney lesions. Prevention or 
amelioration of the disease is of major interest in this project. 

Methods Employed : Standard techniques suitable for light and electron 
microscopic observation were used. 

Major Findings : 

(1) Approximately 907„ of the male and 507o of the female rats died of or 
with kidney disease. As expected, the severity and frequency of kidney 
disease is always greatest in the male rats of any age group. 

(2) Kidney disease was detectable in some rats as young as three months of 
age whereas nearly every rat six months of age showed kidney disease 
manifested as focal glomerulitis . 

(3) A slight deposition of Ig-G was observed along the glomerular basement 
membrane. 



FK-21 



(4) Electron microscopic observation showed: (a) thickening of the glo- 
merular basement membrane correlated with age, (b) rare occurrence of 
lumpy lesions, (c) cytoplasmic fusion of podocytes of glomeruli and (d) 
occasional abrasion of podocytes. 

Significance to Biomedical Research and the Program of the Institute : 
Some diseases are thought to occur in aged animals, mostly or partly due to 
deficiency of immunological control. The kidney disease of rats reared at 
the GRC increases in its severity and frequency with age, and appears to 
have close relation with the age-associated decline of immune activity. An 
understanding of relationship between the decline of immune activity and 
increased occurrence of the disease in aged animals may enable us to pre- 
vent or ameliorate the disease. 

Proposed Course : (1) Elution and characterization of kidney-fixed immuno- 
globulin. (2) Detection of factor (s) causing glomerulonephritis in the 
serum of rats. (3) Induction of experimental autoimmune kidney disease in 
both young and old rats by passive immunization with anti-rat glomerular 
basement membrane. (4) Studies on the permeability of the thickened glo- 
merular basement membrane as compared to normal by using horseradish 
peroxidase, ferritin and immune complexes. 

Honors and Awards: None 

Publications: None 



FK-22 



Serial No.: HD-CCP-18 

1 . Gerontology Research Center 

2. Laboratory of Cellular and 

Comparative Physiology 

3. Office of the Chief 

4. Baltimore, Maryland 

PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Effects of Age on Steroid Hormone Binding and Responsiveness 

in Target Cells and Tissues 

Previous Serial Number: None 

Principal Investigator: George S. Roth 

Other Investigator: Gary R. Dunn 

Cooperating Units: None 



Man Years: 




Total: 


1.2 


Professional: 


0.95 


Others: 


0.25 



Project Description: 

Objectives : A general characteristic of aging organisms seems to be an 
altered ability to respond to hormonal stimuli. Evidence from several 
laboratories suggests that a long overlooked possible explanation may 
lie in age-related changes in hormone uptake and/or binding by target cells 
and tissues. Efforts are currently underway in an attempt to determine 
(1) whether specific binding of steroid hormones by target cells and 
tissues is altered as a function of post -developmental age, and (2) 
whether such possible alterations can be correlated with age-associated 
changes in cell and tissue biochemical responsiveness to these hormones. 

Methods Employed : A number of in vivo and in vitro assays will be 
employed to assess steroid binding, enzyme inducibility, viability, and 
various metabolic parameters in purified cell populations and tissues. 

Major Findings : Muscle, brain and adipose tissue cytosols of adrenalecto- 
mized, male Sprague-Dawley rats exhibit progressively decreased glucocor- 
ticoid hormone binding in vitro from 2 to at least 24 months. Such reduc- 
tions appear to be due to decreases in the number of specific hormone 
binding sites per unit mass (tissue weight and protein). Prostate glands 
yield similar reduced binding between 12 and 24 months. In contrast to 
other tissues, liver actually increases in binding sites at least until 
12 months of age. Changes in numbers of glucocorticoid binding sites in 
liver and muscle have been related to changes in the inducibility of 



FK-23 



tyrosine amino transferase by Cortisol in vivo . Age-related changes in 
steroid binding do not appear to be due to the effects of small organic 
molecules since cytosols preadsorbed with activated charcoal yield binding 
results similar to those of untreated cytosols. Thus, binding alterations 
would seem to reflect quantitative and/or qualitative changes in cytosol 
macromolecules . 

Brain cytosols of female mice aged 3 to 33 months exhibit progressively 
decreased numbers of estradiol and progesterone binding sites. Liver 
cytosols increase in these sites between 3 and about 18 months then de- 
crease until at least 33 months. Such data are probably comparable to 
those obtained for adrenal glucocorticoids in rats since both sex steroids 
and corticosteroids have been reported to bind to the same sites in 
non-sex tissues. Uterine cytosol data are still uncertain since fertility 
cycle phases influence the number of binding sites, while ovariectomy has 
been reported to reduce this parameter. 

Significance to Biomedical Research and the Program of the Institute : 
Binding of hormones to cell and tissue receptor sites is the initial step 
required for their physiological action. Present studies at this level 
offer an opportunity to elucidate the nature of the cellular and/or 
molecular phenomena which result in altered responsiveness in aged target 
tissues. Such information is essential to any attempts to alleviate or 
reverse age-related changes in biochemical vitality. 

Proposed Course : Studies will attempt to further correlate age-related 
changes in hormone responsiveness with alterations in hormone binding by 
target cells and tissues. Emphasis will be placed on in vitro cell culture 
systems that will support growth of defined homogeneous cell populations. 
This approach should allow assessment of age-related changes in these 
functions at the level of tissue parenchymal and stromal cells. The 
ultimate goal of this investigation will be the determination of the 
mechanism(s) of such age-related sensitivity changes in hormone target 
cells. 

Honors and Awards : None 

Publications: Roth, G.S., and Adelman, R.C.: Age-dependent changes in 

sensitivity of rat submandibular gland _in vivo prior to the 
initiation of isoproterenol-stimulated DNA synthesis. 
J. Gerontol . In press. 



FK-24 



Serial No.: HD-CCP-19 

1. Gerontology Research Center 

2. Laboratory of Cellular and 

Comparative Physiology 

3. Mammalian Genetics Section 

4. Baltimore, Maryland 

PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Effect of Chromosomal Aneusomy on Cellular Growth, Metabolism 
and Aging 

Previous Serial Number: None 

Principal Investigator: Edward L. Schneider (E.O.D. 5/23/73) 

Other Investigators: Stuart Shorr 

Gary Dunn 

Cooperating Units: Roger Chakley, University of Iowa, Iowa. City, Iowa 

R. Yanoshevsky, Livermore Radiation Laboratory, 
Livermore, California 

Man Years: 

Total: 1.75 

Professional: 1.25 

Others: 0.5 

Project Description: 

Objectives : Experimental evidence indicates that there is a direct 
relationship between chromosomal aneusomy and aging in humans. Moreover, 
patients with aneusomies are characterized by retarded growth, decreased 
life spans and altered immune responsiveness. At the cellular level the 
fibroblasts and lymphocytes are deficient in their proliferative capacity 
and immunocorapetence . The purpose of this project is to identify and 
elucidate cellularly and biochemically those alterations which impair 
the proliferative and immunological capacity of fibroblasts and lymphocytes 
in vitro . Another important goal of this project will be to investigate the 
relationship between advanced maternal age and chromosomal aneusomy. 

Methods Employed : Fibroblasts and lymphocyte stocks will be prepared from 
patients with trisomy-21, 13 and 18 and normal controls. Cellular repli- 
cations will be studied by measuring DNA synthesis, cell cycle time, and 
percent of replicating cells, and by examining cell cycle blockage, histone 
acetylation and phosphorylation, and DNA methylation. Ribosomal, messenger, 
transfer and mitochondrial RNA synthesis, turnover and degradation will be 
studied by utilizing polyacrylamide gel electrophoresis, pulse labeling 
and chromatographic methods. Protein synthesis will be examined by 
measuring incorporation of appropriate labeled precursors. Chromosomal 



FK-25 



analysis will be performed of preimplantation and postimplantation mouse 
embryos from young and old mothers . 

Major Findings : This project was initiated late in FY 1973 and therefore 
much of the effort was devoted to the creation of the laboratory and animal 
farm facilities and standardization of the various experimental procedures. 

Significance to Biomedical Research and Program of the Institute : 
Information derived from this program should have a tremendous impact on 
cellular biology of aging, for the proliferative capacity of certain somat- 
ic cells is one of the major functional properties that seem to deteriorate 
with age. In addition, these studies will provide insight into the de- 
ranged cellular metabolism in trisomy-21. Lastly, this work will provide 
an animal model for studying the chromosomal aneusomies which are maternal- 
ly age related in man (Down's syndrome. Turner's syndrome, Klinefelter 's 
syndrome, XXX, and XYY) . 

Proposed Course : Initially emphasis will be placed on cellular and bio- 
chemical characterization of the impairment in proliferative capacity 
observed in human trisomy-21 fibroblasts and lymphocytes. Subsequently 
other naturally occurring trisomic and aneusomic cells will be analyzed 
in conjunction with experimentally induced aneusomic cells. 

Honors and Awards : None 

Publications: None 



FK-26 



Serial No.: HD-CCP-20 

1. Gerontology Research Center 

2. Laboratory of Cellular and 

Comparative Physiology 

3. Mammalian Genetics Section 

4. Baltimore, Maryland 

PHS -NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Effects of the Agouti Locus on Development and Aging in Mice 

Previous Serial Number: None 

Principal Investigator: Gary Dunn 

Other Investigators: None 

Cooperating Units : None 

Man Years: 

Total: 0.60 

Professional: 0.50 

Other: 0.10 

Project Description: 

Objectives : An understanding of the genetic regulatory mechanisms operat- 
ing in cells is of basic importance for our understanding of age-related 
decline in cell function, especially of genes which are expressed through- 
out the life span of an organism. Our model system is the agouti locus of 
the mouse and specifically the yellow-agouti allele, A^. Homozygotes for 
this particular allele die as early embryos while heterozygotes lack black 
pigment in their hair, are obese, and have a shortened life span relative 
to their normally pigmented littermates. To date, our research has 
centered on obtaining a fuller understanding of the homozygous lethality 

of Ay. 

Methods Employed : The technique of experimental delay of the implantation 
process in gestation has been used. This involves ovariectomy once the 
female has become pregnant and maintenance of the preimplantation status of 
the uterus by progesterone injection. Implantation can then be induced at 
will by the administration of estrogen. 

Major Findings : These studies appear to indicate that A^/A^ embryos die 
during a fixed time period after fertilization irrespective of whether or 
not the process of implantation is occurring. This suggests that homozy- 
gosity for A^ may be inhibiting some function of all cells in the embryo 
rather than just those cells (trophoblast) responsible for implanting the 
embryo in the uterine epithelium as previously thought. The suggestion 
that Ay inhibits some function in all cells when homozygous is also 



FK-27 



consistent with its observed heterozygous effects which were not explained 
by the theory that it acts only on trophoblast. 

Significance to Biomedical Research and the Program of the Institute : The 
agouti locus appears to control some basic cellular process which is re- 
quired for normal development and proper physiological functioning through- 
out life. The agouti gene product appears to be involved in regulatory 
mechanisms which affect many or all organ systems. The shortened life span 
of yellow-agouti mice suggests that abnormalities in such a system may be a 
cause of the process of aging and that such abnormalities can be 
genetically determined. 

Proposed Course : Studies on the embryological effects of yellow-agouti 
will be continued since it is possible to examine the homozygous effects of 
this locus only during early development. However, the manifestations of 
this mutation in adult heterozygotes will also be studied. The adrenal 
glands of these animals are known to hypertrophy with age and this process 
is correlated with the development of obesity. Therefore, experiments will 
be done to determine whether the mechanism for this hypertrophy is due to a 
defect in the adrenal glands or in the hypothalamic -pituitary control of 
adrenal function. Since hypertrophy suggests elevated hormone production, 
these experiments will involve measurement of adrenal hormone levels in 
ACTH stimulated and unstimulated animals. Such experiments will better 
define the nature of the endocrine defect in yellow-agouti mice. Diet has 
also been shown to affect the development of obesity and the life span of 
yellow-agouti mice. The possibility that these effects are mediated by the 
adrenal gland will be examined by assaying adrenal hormone levels in the 
blood under different dietary conditions. 

Honors and Awards : None 

Publications: None 



FK-28 



Serial No. : HD-CCP2 

1. Gerontology Research Center 

2. Laboratory of Cellular and 

Comparative Physiology 

3. Environments and Genetics 

Section 

4. Baltimore, Maryland 

PHS -NIH 
Individual Project Report > 

July 1, 1972 through June 30, 1973 

Project Title: Dietary Proteins and Longevity 

Previous Serial Number: Same 

Principal Investigator: Charles H. Barrows, Jr. 

Other Investigators: None 

Cooperating Units: None 

Man Years : 

Total: 1,50 

Professional: ,75 

Other: ,75 

Project Description: 

Objectives : Restricted dietary protein intakes may increase life span by 
(1) retarding differentiation and growth and (2) delaying the onset of 
diseases associated with late life. Therefore, in this series of experi- 
ments, attempts were made to determine (1) the extent to which an inhibitor 
of protein synthesis, viz,, cycloheximide, would retard the development of 
chick embryos; (2) whether longevity can be increased by decreasing dietary 
protein in adult organisms, i.e., 16 month old rats and (3) whether a re- 
duced dietary protein intake would change the incidence and/or time of 
onset of leukemia in AKR mice. 

Methods Employed : (1) Attempts to regulate differentiation and growth in 
young growing animals were carried out by injecting 0.8 ^or l.OXof cyclo- 
heximide into one day old chick embryos. The levels employed here did not 
result in an increase in mortality or frequency of anomalies of the 
animals. When the embryos were 2, 3, and 4 days old, the variables meas- 
ured were size, heart rate, and the degree of differentiation as described 
by staging according to Hamburger and Hamilton. In older organisms (viz., 
8, 11, 14, and 17 day old embryos), DNA, protein, as well as various en- 
zymes (acetyl cholinesterase, cytochrome C oxidase, alkaline and acid 
phosphatase, and cathepsin) were determined by standard procedures. (2) 
In order to determine whether longevity could be increased by decreasing 
dietary protein in adult organisms, 16 month old rats were offered diets 
which contained either 24, 12, 8 or 47„ protein. The mortality statistics 



FK-29 



on these animals were determined. (3) AKR mice were fed diets which con- 
tained 26, 4, 3 or TL protein and efforts made to establish, by gross 
pathology and histological examination, whether the increase in life span 
associated with dietary protein restriction was associated with a change in 
the incidence and/or the onset of leukemia in these animals. 

Major Findings : (1) Injection of cycloheximide resulted in a reduction in 
the rates of differentiation and growth at 2, 3, and 4 days. In the older 
organisms a reduction in body weight was observed. However, the activities 
of alkaline phosphatase, acid phosphatase, cathepsin, cytochrome C oxidase 
and acetyl cholinesterase expressed either as per unit wet weight, protein, 
or DNA was not affected. (2) Reduction in the dietary proteins at 16 
months of age from 24 to 8 or 47o did not result in an increase in longevity. 
However, a reduction in dietary protein to 127„ resulted in a 247o increase in 
life span, (27.6 t 2.0 weeks to 34.2 1 1.7 weeks) (P = 0.01). (3) Mortality 
data in AKR mice indicate 50%, 207„, 87o and 407o in the animals fed 267„, 47o, 
37o and 27o dietary protein respectively. Thus far, on the basis of gross 
pathology, all mortality was associated with leukemia with the exception of 
the animals fed the 27. protein diet in which case malnutrition appears to 
be the cause of death. 

Significance to Biomedical Research and the Program of the Institute : 
These dietary manipulations may result in the retardation of the onset of 
various diseases and biological aging. 

Proposed Course : The dietary protein intake will be reduced in 16 and 3 
month old rats from 24 to 12, 8 or 47, and differences in life span will be 
established. The incidence of kidney pathology and age-associated changes 
in renal function will be assessed. 

Honors and Awards: None 

Publications: Barrows, C.H., Jr.: Nutrition, aging, and genetic program. 
Amer. J. Clin. Nutrit. 25: 829-833, 1972. 



FK-30 



Serial No. : HD-CCP-21 

1. Gerontology Research Center 

2. Laboratory of Cellular and 

Comparative Physiology 

3. Environments and Genetics 

Section 

4. Baltimore, Maryland 

PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Effect of Dietary Manipulations on Life Span, Incidence of 
Diseases and Immunologic and Related Functions 

Previous Serial Number: None 

Principal Investigators: C. H. Barrows, Jr. 

K. Hirokawa 



T . Makinodan 



0:her Investigators: 



G. 


S. 


Roth 


A. 


A. 


Nor din 


J. 


J. 


Farrar 


M. 


L. 


Heidrick 


G. 


R. 


Dunn 



Cooperating Units: None 

Man Years : 

Total: 1.20 

Professional: .95 

Other: .25 

Project Description: 

Objectives : Levels of intakes of various nutrients ingested at specific 
intervals of the life cycle which will optimize physiological performance 
are unknown. Perinatal undernutrition results in irreversible changes 
which markedly affect the organism throughout its life. Dietary restric- 
tion imposed immediately following weaning results in an increased life 
span of rodents with average life expectancies. However, no information 
is available if this latter effect can be brought about in rodents when 
the dietary restriction is imposed during adulthood or in animals with 
long life expectancies. More important is that the cellular mechanisms 
which regulate these observations are not known. Since the immune system 
plays a major role in the maintenance of health throughout the total life 
span and is well defined at both the cellular and molecular levels, the 
effect of various dietary manipulations imposed at specific ages will be 
assessed primarily on the immune system of mice with average and long life 
spans . 



FK-31 



Methods Employed : Various strains of mice will be fed, ad libitum , diets 
of nutritionally defined composition but varying in their protein content 
during either pregnancy, lactation, growth or adulthood. Actuarial and 
pathological studies will be carried out. These data will be correlated 
with assessments of the regulation of both cell mediated and humoral 
immune activities. In addition, the turnover of total proteins and 
specific enzymes in liver and muscle will be estimated enzymatically and 
immunologically. Therefore these data will provide information regarding 
the interrelationship among the enzymatic activity, immunocompetence, 
turnover of a number of specific proteins and the life span of the 
organism. The effect of dietary manipulations will also be related to 
age-associated changes in the specific binding of hormones in cells and 
in the cellular responses by measurement of enzymatic induction and 
glucose uptake. 

Significance to Biomedical Research and the Program of the Institute : 
Dietary manipulation could result in improvement of the immune activity 
and a decline in the incidence of immunodeficiency diseases of the aged. 

Proposed Course : Initially the effect of dietary protein intake on the 
regulatory factors in the immune system and on other physiological func- 
tions will be assessed. Other dietary manipulations, including the effects 
of saturated and unsaturated fats, calories and heavy metals will follow. 

Honors and Awards: None 

Publications: None 



FK-32 



Serial No. : HD-AG13 

1. Gerontology Research Center 

2. Laboratory of Cellular and 

Comparative Physiology 

3. Environments and Genetics 

Section 

4. Baltimore, Maryland 



PHS -NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Aging in the Rotifer 

Previous Serial Number: Same 

Principal Investigator: N. Meadow 

Other Investigator: C. H. Barrows, Jr. 

Cooperating Units: None 

Project Description: This project has been discontinued. 

Publications: None 



FK-33 



I 



Serial No.: HD-CCP-22 

1. Gerontology Research Center 

2. Laboratory of Cellular and 

Comparative Physiology 

3. Morphology Section 

4. Baltimore, Maryland 

PHS -NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Ageing and Genetically Defective Mammalian Skeletogenic Cells . 

and Connective Tissues ^ 

Previous Serial Number: None 

Principal Investigators: Dorothy F. Travis 

Other Investigators: None 

Cooperating Units: Laboratory of Biochemistry 

National Institute of Dental Research 
Bethesda, Maryland 

Johns Hopkins University % 

Baltimore, Maryland 



Man Years: 






Total: 





30 


Professional: 





15 


Other: 





15 



Project Description: 

Objectives : The broad objectives of this program are directed at the 
underlying mechanisms associated with changes in mitotic and post-mitotic 
skeletogenic cells and connective tissues in genetic models and with age 
in mammalian tissues including human and experimental mammalian model 
systems. 

Methods Employed : In these studies biochemical, ultrastructural, cy to- 
chemical, radioautographic and other procedures will be used to investigate 
these tissues in vivo and in vitro . 

Significance to Biomedical Research and the Program of the Institute : The 
significance of these studies will rest on their contribution to the 
general conceptual understanding of the poorly delineated cellular and sub- 
cellular mechanisms which regulate the changes that occur in both non-minei> 
alized and mineralized collagenous connective tissues of humans and other 
mammals with age and as a result of genetic defects. 



FK-34 



Proposed Course : New project. 
Honors and Awards : None 
Publications: None 



FK-35 



I 



Serial No. : HD-CCPl 1 

1. Gerontology Research Center 

2. Laboratory of Cellular and 

Comparative Physiology 

3. Morphology Section 

4. Baltimore, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Changes in Cells and Extracellular Matrices of the Crayfish 
Gastrolith Disc during Growth, Development and Aging 

Previous Serial Number: Same 

Principal Investigator: Dorothy F. Travis 

Other Investigators: None 

Cooperating Units: None 

Man Years: 

Total: 0.35 

Professional: 0.10 . 

Other: 0.25 f 

Project Description: 

Objectives : The purpose of this study has been to establish poorly under- 
stood structural and functional principles in mitotic and post-mitotic 
gastrolith epidermal skeletogenic cells during growth, development and 
ageing as these principles relate to: (a) synthesis, organization, and 
mineralization of the structural components of the matrix and (b) mineral 
metabolism including cellular storage and release mechanisms. 

Methods Employed : Ultras true tural and electron cytochemical , both modified 
and non-modified, procedures have been used in mineralized and EDTA demin- { 
eralized mitotic and post-mitotic skeletogenic tissues. Differential 
enzymatic digestion of the structural components of the gastrolith matrix 
(flamenous material and matrix vesicles ) and tissue components (precursors 
and/or structural) to characterize and identify the chemical nature of 
these components with age are being carried out by the method of Bodely 
and Wood (1972) . 

Major Findings : In growing mitotic skeletogenic cells, results indicate 
that (1) the function of lysosomes differs in epidermal cells: in feeding 
animals they function in normal maintenance activities, turnover of cell 
components, while in pre-molt, non-feeding animals, they function in 
degradation of basal regions of the cell that contain stored glycogen, 
lipid, and large protein crystals and in turnover of resorbed products 
from the old exoskeleton for resynthesis of gastrolith matrix structural 
components; (2) synthesis of sterols, which constitute 47o and phospholipids 

FK-36 



which constitute about 5% of the structural components of the gastrolith 
matrix, probably occur during pre-molt in the extensively and newly devel- 
oped apical smooth ER; (3) the carbohydrate moiety of filament material in 
the gastrolith matrix is synthesized in the Golgi where mineral particles 
also are bound to this structural moiety and are transported by Golgi 
vesicles to the cell surface for release; (4) mitochondria in pre-molt 
cells concentrate, store, and transport complexed inorganic calcium to the 
cell surface for release in the developing gastrolith; (5) pre-molt cells 
release alternately filamentous material or matrix vesicles from the cell 
surface into the developing gastrolith. The exocytotic matrix vesicles, • 
which arise from the plasma membrane and can account for the 5% phospho- 
lipid of the gastrolith matrix, contain amorphous mineral particles and 
very small crystallites and appear to serve as primary nucleating sites 
for amorphous calcium carbonate and calcite in the gastrolith matrix. 
These gastrolith matrix vesicles are distinctly analogous structurally 
and functionally to those that arise from mammalian chondrogenic , osteo- 
genic, and dentogenic cells, which also function as primary nucleating 
sites of hydroxyapatie in these tissues. Aged post-mitotic gastrolith 
epidermal cells differ markedly from young dividing cells in their exten- 
sive accumulation of lipid and degradation of mitochondria, swelling of 
the RER, and virtual disappearance of the cytoplasmic matrix; their 
adjacent basement membrane has thickened five-fold, mimicing reported age- 
related changes in the mammalian kidney. Furthermore, the aged animal 
shows marked changes in organization and infiltration of the structural 
components of the non-mineralized extracellular matrix with lipid and 
marked degradation and loss of cells in the sub-epidermal connective 
tissue. Several manuscripts relating to these studies are being prepared 
for publication. 

Significance to Bi omedical Research and the Program of the Institute : 
Both non-mineralized and mineralized connective tissues of mammals and the 
few invertebrate model systems studied show changes in organization, 
amount, composition, degree of cross linking, flexibility, permeability and 
rigidity of the structural components with age, which severely affects the 
functional capacity of the aged animal. To pinpoint the causes for these 
changes demands an understanding of poorly delineated cellular and sub- 
cellular mechanisms which regulate these changes. The establishment of 
cellular mechanisms in mitotic skeletogenic cells which synthesize and 
mineralize the extracellular connective tissues has clearly placed the 
primary role of regulation of mineral metabolism including concentration, 
storage, transport and release of the inorganic mineral phase on the call 
directly and not the extracellular matrix per se . These established prin- 
ciples make possible the marked contrast with post-mitotic, functionally 
impaired aged cells. 

Proposed Course : Further studies on this are being directed at under- 
standing the cellular mechanisms that underly the marked age-related 
changes in these post-mitotic skeletogenic cells and connective tissues. 
This project is being phased out. 

Honors and Awards: Dr. Travis serves as a member of the Council of the 

Washington Society of Electron Microscopy. 

Dr. Travis serves as an Advisory Editor to the journal, 
FK-37 



Calcified Tissue Research . 



Publications: None 



FK-38 



1 



Serial No. : 4D-CCP11 

1. Gerontology Research Center 

2. Laboratory of Cellular and 

Comparative Physiology 

3. Morphology Section 

4. Baltimore, Maryland 

PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Ultras true tural Studies of the Regenerating Sea Spine During 
Growth and Developmental Sequences 

Previous Serial Number: Same 

Principal Investigators: Barry M. Heatfield 

Dorothy F . Travis 

Other Investigators: None 

Cooperating Units: None 

Man Years : 

Total: 1.05 

Professional: 1.05 

Other: 0.0 

Project Description: 

Objectives : The purpose of this investigation was to delineate cell types 
in young regenerating spine tissues with the specific aim of identifying 
the skeletogenic cell type so that subsequent age-related changes could be 
studied in this cell of the non-growing spine stub. 

Methods Employed : Modern cytological and cytochemical techniques and pro- 
cedures of both light and electron microscopy are being utilized. 

Major Findings : The results indicate that in addition to numerous epiderm- 
al and dermal cell types, including those resembling coelomocytes which 
contain large, intracellular spherules, there are young, active skeleto- 
genic cells, the "calcoblasts ." Calcoblasts were characterized by numerous 
pseudopodia and a conspicuous nuclear protrusion encircled by the Golgi 
complex. These cells extend a thin, cytoplasmic skeletal sheath, which 
intimately surrounded the growing surfaces of each of the calcite micro- 
spines comprising the young skeleton. Distally, the sheath enclosed an 
extracellular channel containing an amorphous matrix in which skeletogene- 
sis occurs. 

This intimate morphological relationship between the young calcoblast and 
the growing mineral surface indicates that calcoblasts directly control 
skeleton morphogenesis and regulate the kinetics of skeletal growth, 

FK-39 



functioning in a manner similar to osteoblasts in vertebrate bone. 

The identification, for the first time, makes possible subsequent studies 
of age-related changes in this skeletogenic cell in old, non-growing 
calcified spine stubs. 

Significance to Biomedical Research and the Program of the Institute : 
The results of these studies have a direct relevance to our understanding 
of calcifying systems, particularly vertebrate bone, because the available 
evidence suggests that similar mechanisms are operative in a wide variety 
of calcified tissues. Moreover, the continuing study of concomitant 
changes in structure and function of the calcoblast cell-type throughout 
developmental sequences will contribute to our understanding of age- 
associated changes in calcified tissues generally. 

Proposed Course : Age-associated, ultrastructural changes in calcoblasts 
are being investigated by comparing this active skeletogenic cell in the 
regenerating spine tip, with those in the old, non-growing stub. This 
project is being phased out. 

Honors and Awards : None 

Publications: Heatfield, B.M., and Travis, D.F.: The fine structure of 
calcoblasts in the regenerating sea urchin spine. In 
Arceneaux, C. J. (Ed.): 30th Ann. Proc, Electron Microscopy 
Soc. America . The Proceedings, pp. 162-164, 1972. 

Cavies, T.T., Crenshaw, M.A., and Heatfield, B.M.: The 
effect of temperature on the chemistry and structure of 
echinoid spine regeneration. J. Paleontol. 46: 874-883, 1972. 



FK-40 



Serial No. : HD-CCP8 

1. Gerontology Research Center 

2. Laboratory of Cellular and 

Comparative Physiology 

3. Morphology Section 

' 4. Baltimore, Maryland 

PHS-NIH 

Individual Project Report 

July 1, 1972 through June 30, 1973 

Project Title: Age-related Changes in Mammalian Myocardial Cells 

Previous Serial Number: Same 

Principal Investigators: Dorothy F. Travis 

Trexler M. Topping 
Toshihide Sato 

Other Investigators: None 

Cooperating Units: None 

Man Years : 

Total: 2.35 

Professional: 1.75 

Other: 0.6 

Project Description: 

Objectives : The broad objectives of this program have been directed at 
understanding basic structural and functional principles underlying the 
activities of myocardial cells during developmental sequences and aging 
process. Encompassed in these considerations of highly differentiated 
post-mitotic cells, studies have been carried out on both left ventricular 
mural and papillary myocardial cells. 

Methods Employed : Ultrastructural and electron cytochemical, both modi- 
fied and non-modified procedures, have been used in these studies of 6, 
12, and 24 month old male rats. 

Major Findings : 

A. The left ventricular mural myocardial cells . The purpose of this in- 
vestigation was to elucidate mechanisms of lysosomal activity in the rat 
left ventricular mural myocardium, mechanisms not understood in any 
mammalian myocardial cells. Results indicated: (1) Two types of primary 
lysosomes, showing differences in acid hydrolase activity. (2) Two differ- 
ent pathways of synthesis, transport, and packaging of primary lysosomes. 
(a) Nuclear pole zone. Synthesis occurs in cisternae of the RER or granu- 
lar regions of the nuclear membrane or directly from other elements of the 
smooth ER to the Golgi cisternae for packaging and release from the 
swollen end sacs. (b) At distal cell sites from the Golgi, synthesis 
occurs in RER; transport, packaging, and release occurs directly from 

FK-41 



smooth ER, bypassing the Golgi, (3) Two lysosomal mechanisms of auto- 
phagic mitochondrial degradation. (a) By matrix densif ication to form 
residual bodies. (b) By matrix clarification to form residual bodies. 
(4) Lipid droplets were degraded by lysosomal and auto-oxidative activity 
to form residual bodies, all of which show acid hydrolase activity. This 
is the first investigation to establish these mechanisms in any mammalian 
myocardial cells. The establishment of these mechanisms bears directly on 
age-related changes in cellular structure and function. The latter encom- 
passes lysosomal synthesis and turnover rates, membrane phenomena in 
selection of components for breakdown, causal factors of residual body 
accumulation, and their different enzyme content with age. 

B. The left ventricular papillary myocardial cells . 

Age-related changes in mechanisms of lysosomal degradation in the rat left 
ventricular papillary myocardial cells . The purpose of this investigation 
was to elucidate mechanisms responsible for the marked age-related changes 
evident in 24 month old rat papillary myocardial cells in regions of the 
muscles in which the cells are grossly normal in structural appearance but 
show marked changes over the 6 and 12 month old cells. Changes evident in 
old cells were in: (1) the structural configuration of the nucleus; (2) 
mitochondria, with hypoxic or anoxic swelling, clarification of matrix, 
separation and disorganization of the cristae in some 12 and in most 24 
month old cells; (3) lysosome and residual body accumulation; (4) mechan- 
isms of autophagic degradation of mitochondria by redundant smooth ER 
segregating membranes; (5) lipid droplets with increased electron density 
and often degraded in autophagic vacuoles by lysosomal enzymes and perioxi- 
dation. These results indicate that slow subtle changes occur in the age- 
ing myocardial cell. Consequences of slow but evident hypoxic or anoxic 
changes in mitochondria could result in curtailed oxidative metabolic 
capacity, ion leakage and failure to membrane transport systems, inability 
of myofilaments to function normally, greater production of lysosomes with 
greater fragility or altered permeability of membranes, permitting slow 
leakage of acid hydrolases into the cytoplasmic matrix. Lipid droplet 
degradation in the old cells by perioxidation might similarly cause lyso- 
somal and other membrane damage by leakage of free peroxide radicles. 
Nuclear changes may also reflect acid hydrolase leakage leading to altered 
DNA and RNA at the genome or at the transcription levels and synthesis of 
defective proteins. Changes in the nature of lipid droplets with age sug- 
gests that altered membranes of the smooth ER could synthesize faulty 
lipids. Faulty macromolecules produced at these sites might well be re- 
sponsible for the production of seemingly redundant segregating membranes 
in the old cell. We feel that it is these slow but subtle changes, and not 
residual body accumulation per se , with time that alters the cell's capac- 
ity to function normally. This manuscript is prepared for review and will 
be submitted to the Journal of Ultras true ture Research for publication. 

Rat left ventricular papillary myocardial cell death . The purpose of this 

investigation was to elucidate the mechanisms responsible for myocardial 

cell death in regions of the muscles in which the old 24 month cells are 

grossly abnormal structurally, showing necrosis and fibrotic replacement. 

Results indicate that: (1) further, marked changes in the nucleus were 

evident; (2) most mitochondria showed extreme hypoxic or anoxic changes 

with reduction or loss of cytochrome oxidase activity; (3) increases in 

primary but decreases in secondary lysosomes and residual bodies were 

FK-42 



evident; (4) acid hydrolase activity was evident in the cytoplasmic 
matrix indicating leakage and loss from secondary lysosomes and residual 
bodies, thus accounting for their decreases in these severely affected 
cells; (5) myofilaments were degraded and Z-band thickening, disorganiza- 
tion and disruption were evident; (6) swelling of the T-tubules, cytoplas- 
mic vacuolation and breakage of the plasma membrane were evident; (7) 
Macrophages were trapped in the process of consuming both normal and ab- 
normal regions of the same cell; (8) invading fibroblast and collagen fiber 
replacement of dead cells were evident. These results clearly indicate 
that slow, subtle changes have occurred in the ageing papillary myocardial 
cell and that consequences of such changes as suggested previously have in 
fact occurred. Physiological mechanisms that start the chain of events 
may well be: (1) ventricu''ar hypertrophy causing mechanical stress and 
strain on the papillary muscles and (2) reduced coronary blood flow with 
age, the papillary muscles being the last to be supplied. This latter 
factor alone could cause slow hypoxia or anoxia, setting off the entire 
chain of events. The extensive cell death and fibrotic replacement with 
age in the papillary muscles would drastically alter the functional capac- 
ity of the heart. This manuscript is being prepared and will be submitted 
to the Journal of Ultras true ture Research for publication. 

Significance to Biomedical Research and the Program of the Institute : 
More than one-half of all human deaths in the U, S. occur secondary to 
cardiac disease. This demands an understanding of cellular mechanisms 
responsible for age-related decrements in valvular and cardiac function. 
The establishment of mechanisms of lysosomal activity in the rat left 
ventricular mural myocardium are the first to be established in any mam- 
malian myocardial cells. Similarly, the elucidation for the first time of 
cellular mechanisms responsible for the marked age-related changes that 
occur in old papillary myocardial cells and those responsible for papillary 
myocardial cell death have significant relevance to the understanding of 
decrements in valvular and cardiac function of older humans and other 
mammals , 

Proposed Course : Further studies require the biochemical quantitation of 
age related lysosomal and mitochondrial enzymatic activities in sub-cellu- 
lar fractions of the left ventricular papillary myocardial cells. 

Honors and Awards: Dr. Travis was invited to speak in a seminar at George- 
town University, School of Medicine, Department of 
Anatomy, Washington, D. C. 

Publications: Topping, T.M,, and Travis, D,F,: An electron cytochemical 
study of mechanisms of lysosomal activity in the rat left 
ventricular mural myocardium, J. Ultrastruc. Res, In press. 



FK-43 



Serial No . : HD-AG37 

1. Gerontology Research Center 

2. Laboratory of Cellulaj? and 
Comparative Physiology 

3- Morphology Section 
I|.. Baltimore, Maryland 

PHS-NIH 
Individual Project Report 

July 1, 1972 thro-u^ June 30, 1973 '^ IT^ 

, I « r r 9 

Project Title: Studies on the Comparative Physiology of Ageing and . ' 
Environmental Effects on the Ageing Process .■■svi ilaw Ysm 

- ■ ■ : ,-rr> -.- ' PL -< ■! J 

Previous Serial Number: Same 

a!fv.ij.& iojo&I 

Principal Investigator: Mary Anne Brock -i^-"o o n.zB o 

ax 9§B 

Other Investigators: None ^"'"^ 

Cooperating Units: None 

Man Years: • • 

Total: 1.0 JBibiao 

Professional: 1.0 anoqEsi 

Other: ■ -^^^ ^'^^ 

uiuojiansv 

Project Description: nsilsm 

Objectives : (l) To delineate both endogenous rhythmic changes aiid^"^^° 
temperature- induced changes in the longevity of poikilothermic and'^'^^°^'^ 
homoiothermic animals; (2) To define the age-related structtiral and^"'^^ 
functional characteristics of pre-and post-mitotic cell types as thes'e'^"' 
relate to cellular mechanisms associated with changes in the rate of 
ageing. ^.^^.^..fo^o^ 

Methods Employed : Colonies of the marine coelenterate Campanularia 
flexuosa were cultured in aerated, artificial sea water at k° t 10°, 17° 
and 2k° C. for ageing studies and other determinations at each of these 
temperatures. Golden hamsters, MesocricetuB auratua , were maintained 
either in a 2UP C. environment or periodically exposed to 5° C. to 
permit hibernation. Ultrastructural and biochemical techniques were used. 

Major Findings : 

A. Endogenous rhythms in the marine invertebrate , Campamilaria flexuosa . 
The delineation of endogenous rhythms in C_. flexuosa longevity challenges 
the generally accepted concept of specific age, an age at death which is 
characteristic of the species. It generally has been assxjmed that 
vertebrates and invertebrates grown under constant environmental conditions 
have an invariable life span. However, a contradiction to this concept is 
raised by the existence of rhythmic changes in C^. flexuosa hydranth lon- 
gevity. 

FK-44 



oH 



1. Anmial (cirarmual) rhythms in Ccunpan-ularia . EndogenoTis circaimual 
rhythms in growth, development and longevity of the marine coelenterate 
Campan\ilaria flexuosa have persisted for over three years in the absence 
of periodic signals from the environment. The free-running periods for 
the cycles at three constant ambient temperatures, 10°, 1?° and 2kP C, 
approximated one yeair, but were always more than 365 days. Temperat-ure 
switches at different times of the animal cycle have shown differential 
seasonal sensitivities to temperature in both hydranth development and 
longevity; these and related data have implicated temperature as a 
Zeitgeber. 

The endogenous circannual rhythms were marked by seasons of luxuriant 
colony growth that alternated with lulls in growth and development. During 
luxuriant growth, development of individual hydranths was initiated 
regularly in new positions and as replacements at regression sites on 
upri^ts. Nearly all hydranth development was normal and adult life spans 
were long. The profuse gq?owth was interrupted by seasons of repressed 
growth each summer and punctuated by recurrent, short periods of sharply 
curtailed growth in mid-winter. During repressed grovrth, the rate of 
hydranth initiation at all sites v/as depressed, and abnormal development 
aborted many individuals. Most strikingly, hydranth life spans fell to 
about half those observed during seasons of prolific growth. With the 
spontaneous return to the luxuriant growth habit, hydranth life spans rose, 
nearly doubling. 

To further define the endogenoiis control of the circannual cycle, new 
colonies of C_. flexuosa were obtained from their natural environment in 
late 1971 3XLd. cultured together with the older colonies in the same vessels. 
Despite this intimate association, the endogenous rhythms of the new 
colonies were cleaxly out of phase with the same rhythms observed in the 
long-term laboratory-maintained colonies, for they exhibited expansive 
growth when the growth of the older colonies was sharply curtailed. 

2 . Lunar rhythms in Campanizlaria . Hydranths of Campanularia not only 
exhibit rhythmic and predictable annual phases of increased longevity but 
also persistent lunar rhythms in hydranth development and longevity. 
Within a lunar cycle, three-to four- fold differences in life expectancy 
were observed > Each cycle began with the development of long-lived 
hydranths, and progressively shorter-lived individuals developed each day 
as the cycle vnfolded. The cycles overlapped and have continuoiisly 
repeated for over three years. Analysis of the lunar cycles as well as 
their relationship to the circannual rhythms is being completed. 

B. The effect of hibernation on the longevity of golden hamsters . 
Preliminary studies have shown a marked effect on the longevity of animals 
which have hibernated, their body temperature falling to near 6° C. during 
the hibernating season. These studies suggest that there is a marked 
extension of the life span associated with hibernation and that hibernation 
may have a more profoimd effect in increasing the longevity of male 
hamsters. Furthermore, the extension of the life span appears to bear no 
simple direct relationship to the length of time in hibernation. This has 
been clearly demonstrated with the use of a remote monitoring system that 

FK-45 



was developed for these studies and has successfully recorded the precise 
length of time each hamster spent in hibernation. The system depends on 
individual infra-red sensors positioned over each animal with leads to a 
multichannel scanner and recorder. 

Significance to Biomedical Research and the Program of the Institute : A 

primary objective of gerontologists is to extend the number of continuous 

years of productive, optimally functional and enjoyable life. To achieve 

this goal, an understanding of cellular functions in regulatory mechanisms 

associated with increased longevity such as that observed in vertebrates 

(mammals) and invertebrates either at reduced temperatures or during 

certain phases of endogenous rhythms in longevity is essential . The 

delineation of these regulatory mechanisms has significance in their -ni 

possible applicability in manipulating the rate of cellular ageing in 

other mammals, including man. Furthermore, this first delineation of 

endogenous rhythms in longevity is of significance not only to the field 

of ageing but in relation to similar yearly or seasonal rhythms that alter 

physiological and biochemical parameters such as ventilatory capacity and 

the immune response in mammals, including man. Circannual rhythms may well 

be as universal as circadian rhythms and of profound importance in 

biomecial research. 

Proposed Course : A series of papers on annual and lunar rhythms in 

C. flexuosa longevity are in preparation. This project is being phased 

out. Studies on age-related considerations of mammalian hemopoietic f 

connective tissue will be initiated. 

Honors and Awards: Dr. Brock was elected to the Board of Governors of the 

Society for Cryobiology. 

Dr. Brock served as Secretary of the Society for 
Cryobiology. 

Dr. Brock was an invited speaker at the 1972 AAAS 
Symposium, Developmental Biology of the Cnidaria. 

Dr. Brock was invited to speak at a AAAS Symposium on ,j 
Annual (Circannual) Biological Clocks. ' 

Publications: Brock, M. A.: Growth, developmental and longevity rhythms in 
Campanularia flexuosa . Amer . Zool . In press. 



FiM6 



NICHD ANNUAL REPORT 

July 1, 1972 through June 30, 1973 

Gerontology Research Center 

Laboratory of Molecular Aging 

The Laboratory of Molecular Aging has developed scientific data and theory 
fundamental to the understanding of molecular processes involved in aging. 
Research projects, utilizing a variety of model systems, have examined questions 
related to regulatory processes in the transmission of genetic information, 
mechanisms of control of enzymatic activity and cellular metabolism, and 
biochemical changes during aging that alter the responses and functions of 
subcellular organelles and cells. 

An extensive study of infection of human cells (WI-38) in culture by vesicular 
stomatitis virus and its prevention indicates: (1) Senescence of host cells 
is not detrimental to viral replication with the exception of a brief period 
before cellular death. (2) Cellular viral defense, _i. £• , the induction of 
interferon, is functional throughout the life span of the cells up to the 
moribund state, but its efficiency, i.e., the amount of poly IC required for 
induction, decreases with age. (3) In senescent cells the polynucleotide 
inducer of interferon produces the antiviral state faster than treatment 
with interferon itself, suggesting either another antiviral protection mechanism 
in senescent cells or a change in the relative rates of uptake of poly IC and 
interferon in the senescent cell. Related studies with this experimental 
model show that antiviral activity of the complex of poly IC is more critically 
influenced by substitutions in tho inosinate than in the cytidylate strand. 
Significantly, it is found that poly IC anchored to solid carriers suspended 
in the medium over the cell monolayer conveys antiviral protection. 

Synthetic analogs of nucleic acids, representing modifications of the structure 
of native genetic material, have been shown to interfere with specific biolog- 
ical processes and, therefore, can be used as probes to study biochemical 
mechanisms. Thus, polyvinyl uracil and polyvinyl adenine both inhibit murine 
leukemia virus infection, but they are rather ineffective against Sinbis and 
vesicular stomatitis viruses. The inhibition of the leukemia virus appears 
to be related to the inhibition of a RNA-dependent DNA polymerase. Polyvinyl 
uracil also inhibits protein synthesis in vitro and it is toxic to rapidly 
growing cells. On the other hand, polyvinyl adenine does not inhibit cell 
multiplication nor host cell synthesis of nucleic acids and proteins. 

Complexes of platinum have been used to probe the mechanism by which the 
replication of DNA and synthesis of RNA are frustrated by metal ions. It is 
known that the cis-form of a platinum complex inhibits DNA replication, cell 
division, and is an effective antitumor agent, whereas the trans-form of the 
complex is not active in these respects. It has now been found that the cis- 
isomer, but not the trans-isomer , forms complexes with DNA. The Pt-containing 
complex inhibits transcription in an in vitro system, and this inhibition is 
on the polymerization process, without affecting the initiation process. A 
technique has been developed to replace the zinc, the natural metal in RNA 
polymerase, with several metals including copper and to retain enzymatic 
activity. The substitution of copper, allowing electron spin resonance 
studies, may provide a valuable tool in elucidating the molecular action of 
the enzyme. 



The interactions of synthetic polypeptides with DMA have been used to examine 
the nature of his tone binding in chromatin. Physicochemical studies indicate 
that polylysine binding to DNA resembles that of histone I, and the similarity 
is augmented by introducing alanine residues into the polylysine (alanine- 
lysine copolymer). Polyarginine, on the other hand, resembles histone IV in 
its binding to DNA. Basic polypeptides with long side chains form weaker 
links to DNA than shorter chain polypeptides. DNA that is not bound to poly- 
peptide components appears to be required for the regulation of the conformation 
of polypeptide-bound DNA. This observation could explain the fact that only 
part of the DNA in chromatin is covered by protein, while all of the chromatin 
is repressed toward in vitro transcription. 

Studies on the mechanism of active transport of sugar in the kidney have 
continued to make significant progress and several underlying principles have 
been established. Glucose transport by isolated renal brush border membranes 
is comprised of at least two processes, a high affinity binding system and a 
carrier -mediated transport system. The presence of a stereospecific high 
affinity binding system has been unequivocally demonstrated by equilibrium 
dialysis. Kinetic studies of the binding have characterized its time course, 
reversibility, saturability, susceptibility to the presence of phlorizin, 
and have determined the effects of Na"*" as well as Ca2+ and Mg^"*". A particulate 
fraction derived from detergent-extracted brush border membranes has been 
prepared. This preparation has the same affinity and capacity as intact 
membranes but has lost about 90% of its protein content and shows no evidence 
of vesicular structure. Evidence for a glucose-bound protein has been obtained. 
The mechanism of uptake of high concentrations of glucose by the intact brush 
border membranes shows both influx and efflux components and is consistent 
with a hypothesis for a carrier-mediated transport into and out of vesicles. 
Prior loading experiments demonstrate the phenomenon of counter -trans port or 
facilitated exchange diffusion. Although Na"*" , per se, is not required for 
uptake of glucose by the brush borders, the rate of glucose transport by the 
membrane is greatly enhanced by a Na+ gradient from the outside to the inside 
of the membrane. 

Extensive studies on the control of pyruvate oxidation in mitochondria from 
blowfly flight muscle, and confirmed with mitochondria from rabbit heart 
muscle, have led to a novel mechanism for the regulation of Krebs cycle 
activity. Both ADP and inorganic phosphate are required for the uncoupled 
(FCCP) oxidation of pyruvate as well as for the coupled oxidation. The FCCP- 
stimulated oxidation is markedly inhibited by physiological levels of Ca2+. 
The apparent Kj^ = 10 )iM. The electron transport chain of the mitochondria is 
fully functional in the presence of Ca since the oxidation of a-glycerolphos- 
phate is not inhibited, indicating control at the dehydrogenase level. Kinetic 
studies show that the Ca inhibition is uncompetitive with respect to ADP and 
FCCP, but complex, and noncompetitive with respect to pyruvate. The results 
of these and other studies suggest that pyruvate oxidation and Krebs cycle 
activity is limited by NAD-dependent isocitrate dehydrogenase activity and Ca 
plays a role in its regulation. An additional mechanism for the control of 
pyruvate metabolism has been demonstrated in mitochondria for the first time. 
In analogy to glycogen phosphorylase, this involves the phosphorylation and 
dephosphorylation of the pyruvate dehydrogenase complex by a kinase and phos- 
phatase, respectively. In contrast, however, with pyruvate dehydrogenase the 
phosphorylated form is inactive whereas the dephosphorylated form is active. 



FIt2 



A correspondence between the decline in the ability of aged blowflies to fly 
and in the maximally stimulated respiratory rate (State 3) and respiratory 
control ratio of flight muscle mitochondria was reported previously. The 
biochemical mechanism and locus of this decrement with senescence has been 
investigated further by examining the bioenergetic properties of mitochondria 
from mature and aged flies for rates of oxidation during coupled oxidative 
phosphorylation, activities of the dehydrogenases, concentrations of components 
of the electron transport chain, phosphorylation of ADP, and the coupling 
mechanism, itself. Evidence, to date, indicates that the specific activities 
of a-glycerolphosphate and pyruvate dehydrogenases are unaltered by ag6. 
Similarly, the concentrations of cytochromes a, ao , b, and c+C]^ are the same 
for both age groups. ATP synthetase, measured as ATPase, is unchanged with 
senescence. In contrast, the uncoupled oxidation of substrates is signifi- 
cantly decreased with aging, showing the 30-407o decrement between mature 
(7-10 days) and aged (31-33 days) as seen previously for the maximally stimu- 
lated coupled rate of respiration. These observations narrow the possible 
loci of the defect in aging to the bioenergetic system between the primary 
dehydrogenase and the FCCP-sensitive uncoupling reaction. 

Hormonally (parathyroid, synthetic 1-34, and catacholamine) stimulated adenyl 
cyclase has been demonstrated in renal cortical membranes. Luminal brush 
border membranes bind c-AMP, as determined by equilibrium dialysis and Millipore 
filtration experiments. Kinetic parameters of the binding have been examined. 
It is anticipated that the binding of cAMP may initiate kinase activation and 
potentially exert a controlling role in renal tubular function. 



FIr3 



Serial No. HD-GMA.1 

1. Gerontology Research Center 

2. Laboratory of Molecular Aging 

3. Molecular Chemistry Section 

4. Baltimore, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Interaction of Metal Ions with Polynucleotides and 
Related Compounds 

Previous Serial Number: Same 

Principal Investigator: G. Eichhorn (357o time) 

J. Rifkind (407« time) 

Y. Shin (407o time) 

R. Srivastava (100% time) 

E. Tarien (707, time) 

Other Investigators: None 

Cooperating Units: Baltimore City Hospitals 

Man Years: 

Total: 3.75 
Professional: 2.85 
Others: .90 

Project Description: 

Objectives : (1) To understand the biological activity of metal complexes 
of nucleotides and polynucleotides on the basis of their structures and 
reactions. (2) To find specific reactions of the individual nucleotides. 
(3) To understand the biological significance of metal ions in nucleic 
acid function. 

Methods Employed : The interaction of metal ions with polynucleotides and 
their monomeric components is studied, using a variety of physical chemical 
techniques, e.g., optical rotatory dispersion, nuclear magnetic resonance, 
.ultraviolet and infrared spectrophotometry. From these data the site of 
interaction of the metal can be correlated with its effect on the reactions 
of the nucleic acids. 

Major Findings : A. Stereoselective reaction of platinum complexes with 
DNA . Complexes of platinum have been used to probe the mechanism by which 
metal ions bind to DNA and affect the mechanism of RNA synthesis (see also 
Report No. HD-AG 30). Platinum is extremely useful because it forms bonds 
which are much less labile than those of most other metal ions, so that 
intermediate stereoisomers are stable. Others have shown that 



FL-4 



cis[Pt(NH3)2Gl2-l is an effective antitumor agent, and that it inhibits cell 
division and DNA replication, while the trans complex is not effective. Im- 
portant progress has been made in this laboratory to explain these phenomena. 
The cis complex forms two types of complexes with DNA that are not produced 
with the trans complex. One contains approximately one platinum per ten DNA 
nucleotide residues; its properties suggest a structure in which platinum 
atoms are bound to the exposed DNA bases, which are the same distance apart 
(3.4 A) as the cis liganding positions of the platinum. The other contains 
one platinum atom per DNA residue; its properties suggest a structure (which 
is however not proved) in which platinum atoms, which form bonds with each 
other with 3.4 A distances, are costacked with the DNA bases. 

The 1:10 Pt/DNA nucleotide adduct is a manifestation of the most significant 
physical chemical differences exhibited by the cis and trans complexes in 
their interaction with DNA. The addition of small increments of the cis 
complex to DNA produces a marked enhancement of the ellipticity in the cir- 
cular dichroism spectrum; the enhancement is maximal at the 1:10 ratio, and 
further incremental addition of the complex produces decreases in ellipticity. 
In contrast, the ellipticity of the adducts of DNA and the trans isomer de- 
creases consistently with the addition of every increment of the platinum 
complex. The ellipticity does not increase when the cis complex is reached 
with denatured DNA, suggesting that the intact double helix is required for 
the reaction with platinum. The stoichiometry of the adduct of the cis com- 
plex with DNA can be explained if chelation of the platinum complex occurs 
to two adjacent bases only when a specific base sequence is present. We do 
not know at the moment which base sequence is involved. 

[Pt en Cl23j which contains the chelating ethylenediamine group instead of 
two coordinated ammonia molecules, and which must therefore have the cis 
configuration, reacts with DNA in a manner identical to that of 
cis [Pt(NH-^)?Cl7]. Trans[ pt(NH-^)9Cl?] reacts very differently, forming a 1:2 
Pt/DNA nucleotide adduct, which presumably involves platinum as a crosslink 
between the DNA strands. These differences in reactivity of the platinum 
isomers result in marked differences in template activity, as discussed in 
Report No. HD-AG 30. 

B. The higher stability of polydeoxynucleo tides vs. polyribonucleotides 
toward denaturation by metal ions . Metal ions are able to distinguish be- 
tween polyribonucleotides and polydeoxynucleo tides by destabilizing single 
or double helical conformations of ribopolynucleo tides much more readily than 
those of polydeoxyribonucleo tides. Moreover, the ease with which the confor- 
mations of the polydeoxynucleo tides are disorganized is sensitive to the 
nucleotide sequence. Thus Cu2+ ion, one of the most effective destabilizers 
of nucleic acid structure, has no effect on the poly dAT double helix, al- 
though it destabilizes DNA. Taken together with the previously determined 
greater susceptibility of RNA, compared with DNA, to cleavage by metal ions, 
it is apparent that DNA is much more able to withstand the various stresses 
to which metal ions subject the polynucleotide structure. Perhaps this is an 
advantage of DNA as a primary source of genetic information. 

Copper ions are capable of disordering the helical structure of poly dA, but 
all other metals that destroy the poly rA helix are ineffective toward poly dA, 



FL-5 



again demonstrating the greater stability of the polydeoxynucleotide struc- 
ture. 

C. Mechanism of destabilization of ordered structure of polynucleotides by 
metal ions . A combination of spectrophotometric, titration, and binding 
studies of the reaction of copper with oligonucleotides and polynucleotides 
has led to a mechanism of destabilization that involves crosslinks between 
phosphate and base groups. The high effectiveness of copper in disordering 
polynucleotide structure at neutral pH, where the free copper concentration 
is low, indicates that a hydroxylated species is responsible for the cross- 
links. Titration studies indicate that this species contain two copper atoms 
and two hydroxyl groups. At high polymer concentrations the crosslinks are 
predominantly intermolecular, and as the polymer concentration is decreased, 
intramolecular crosslinks are preferred. These results indicate different 
mechanisms for the denaturation of polynucleotides at different concentra- 
tions. 

D. The binding of copper(ll) to transfer RNA . Metal ions are necessary to 
maintain the active conformation of t-RNA. It has been previously shown by 
nuclear magnetic resonance studies that Cu^"*" ions are quite selective in 
binding to the bases of randomly coiled RNA. The binding of Cu(Il) to tRNA, 
on the other hand, does not proceed according to base preference, but occurs 
in response to the conformational requirements of the molecule. It appears 
reasonable that metal ions fit into crevices of the tertiary structure so as 
to maintain the active native conformation. 

Wnen the reaction of Cu(Il) with tRNA is studied in a DMSO-water mixture, in 
which the tRNA is denatured, the copper again exhibits preference for binding 
to the purines, and not to the pyrimidines. 

Significance to Bio-medical Research and the Program of the Institute ; The 
participation of metal ions in every aspect of genetic information transfer 
makes the study of the interaction of metal ions with the nucleic acids of 
major importance in understanding the perturbation of the genetic information 
that is presumably characteristic of the aging process. 

Proposed Course of Project ; It is intended to continue studies to determine 
the sites of binding of metal ions on nucleic acids, the conformational 
changes induced by such binding, and to correlate these effects with bio- 
logical function. We have begun to determine the concentration of metal ions 
in biological materials, and the changes in these concentrations with age. 
We intend to determine changes in the permeability of cells to metal ions or 
metals complexed to small or large molecules, as a function of age. 

Honors and Awards ; None 

Publications ; 

Rifkind, J. M. , and Eichhorn, G. L. ; Interaction of Metal Ions with Poly- 
nucleotides and Related Compounds. XXI. Metal Ions as Agents for the Stack- 
ing of Nucleotides. A Specific Interaction of Zinc(ll) and Adenosine Mono- 
phosphate. J. Amer. Chem. Soc. 94: 6526-6534, 1972. 



FL-6 



Berger, N. A., and Eichhorn, G. L. : Stereoselective Differentiation between 
Ribonucleosides and Deoxynucleosides by Reaction with the Copper(ll) Acetate 
Dimer. Nature New Biology 95; 237-240, 1972. / Ai,etace 



FL-7 



Serial No. HD-GMA2 

1. Gerontology Research Center 

2. Laboratory of Molecular Aging 

3. Molecular Chemistry Section 

4. Baltimore, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Degradation of Nucleic Acids by Metal Ions 

Previous Serial Number: Same 

Principal Investigator: J. Butzow (307= time) 

G. Eichhorn (5% time) 

Other Investigators: None 

Cooperating Units: Baltimore City Hospitals 

Man Years: 

Total: .55 
Professional: .35 
Others: .20 

Project Description: 

Objectives : (1) To render metal ion degradation of polynucleotides useful 
in the detection of nucleotide sequence and in selective degradation of 
ribonucleic acids. (2) To study the mechanism of the metal ion degradation 
reaction. (3) To utilize the ability of metal ions to degrade polynucleo- 
tides in the probe of the structure of biological macromolecules such as 
5S-RNA. • 

Methods Employed : After brief exposure of 5S-RNA to zinc ion, the RNA is 
subjected to conditions favoring unfolding of its ordered structure to 
release parts of the chain between the zinc cleavages. The fragments are 
separated by electrophoresis through polyacrylamide gel under these con- 
ditions, and are detected in the gel by ultraviolet absorption scanning. 

Major Findings ; We have previously analyzed the positions of zinc cleavage 
in 5S-RNA, at the level of 1 phosphodiester bond broken for each RNA chain 
(120 nucleotide residues long), using introduction of ■'^p.phosphate groups 
at the 5' hydroxyls into nonradioactive RNA. There were technical problems 
with this method, but the results were consistent with cleavage in sections 
of the RNA which are not base-paired. We have now directly invcsLigaLcd l.hc 
size distribution of the fragments produced by zinc cleavage under reaction 
conditions which are such that the initial RNA conformation should not be 
significantly altered from its native state (as determined by optical rota- 
tory dispersion). We find that reaction at 10 Zn/P and 40° will convert up 



PT.-R 



to 107« of the bulk of the RNA into a sharply moving fragment (or fragments) 
which is (are) of a chainlength between that of the undegraded 5S-RNA and 
"4S" or tRNA (about 80 nucleotide residues), as well as some material of the 
size range of "4S" RNA, before a heterogeneous gel profile results. We con- 
clude that brief exposure of 5S-RNA to high concentrations of zinc ion can 
produce coherent cleavage and assume that this cleavage reflects kinks in the 
structure of the native form of the 5S-RNA. 

Significance to Biomedical Research and the Program of the Institute : 5S-RNA 
is a component of the 505 subunit of the bacterial ribosome; it is the sim- 
plest ribosomal component and the one most readily capable of structural 
elucidation. In order to understand protein biosynthesis, the structure of 
the ribosome must be understood: the studies with zinc are designed to aid 
in this undertaking. The usefulness of zinc ion for selective degradation of 
RNA molecules is in itself of general technical biochemical interest. 

Proposed Course of Project ; On the basis of the currently reported results, 
we intend to probe the positions of zinc cleavage in the nucleotide se- 
quence of 5S-RNA using uniformly -^^P- labelled RNA, with the methods of analy- 
sis of 32p_ia5elled oligonucleotides already worked out by Sanger for this 
particular RNA. 

Honors and Awards : None 

Publications: None 



FL-9 



Serial No. HD-GMA3 

1. Gerontology Research Center 

2. Laboratory of Molecular Aging 

3. Molecular Chemistry Section 

4. Baltimore, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Structure of Nucleoprotein, Ribosome, and Interactions of 
Components Related to Protein Synthesis 

Previous Serial Number: Same 

Principal Investigator: J. Butzow (707„ time) 

G. Eichhorn (20% time) 
Y. Shin (607o time) 

Other Investigators: None 

Cooperating Units: Baltimore City Hospitals 

Man Years: 



Total: 


2.10 


Professional: 


1.50 


Others: 


.60 



Project Description: 

Objectives : (1) To determine the conditions for the binding of proteins 
' to nucleic acids, (2) to examine the mediation of this binding by metal 
ions, (3) to elucidate the structure of chromatin with the help of these 
model systems, (4) to understand how cellular differentiation and aging 
may be related to nucleoprotein structure, (5) to study the structure of 
the ribosome and its interaction with other substances engaged in protein 
synthesis. 

Methods Employed : (1) The interactions of proteins and nucleic acids, and 
the effect of metal ions on these interactions are studied by optical 
rotatory dispersion, circular dichroism, spectrophotometry, to determine 
conformational changes, and by infrared and nmr techniques, to determine 
binding sites. (2) Models containing selected characteristics of chromatin 
are examined to determine which characteristics are responsible for chro- 
matin function. (3) Conformational changes involved in the interaction of 
ribosomes, messenger RNA, transfer RNA, and other factors involved in pro- 
tein synthesis are studied by means of optical rotatory dispersion and 
fluorescence . 



Major Findings ; A. Interactions of synthetic polypeptides with DNA, and the 
structure of chromatin . The his tones that are bound to DNA in chromatin have 
been implicated in the regulation of gene expression. The various his tone 
fractions are rich in basic amino acids, and contain clusters of lysine and 
arginine. It is therefore very useful to compare the interaction of poly- 
lysine and polyarginine with DNA to the interaction of lysine-rich and argi- 
nine-rich his tones with DNA. Both synthetic polypeptides bind strongly to 
DNA and significantly stabilize it. They differ dramatically, however, in 
their effect on the confotrmation of DNA as demonstrated by optical rotatory 
dispersion (ORD). Polylysine produces an ORD curve with a large negative 
rotation at 290 nm, whereas polyarginine produces only a slight decrease in 
the magnitude of the ORD. Divalent metal ions influence the melting tempera- 
ture of both types of DNA complexes, but they do not influence the ORD of the 
polyarginine complex, in contrast to major effects on the polylysine complex. 
The effects of polylysine on DNA resemble those of the lysine-rich his tone I, 
and the similarity is augmented by introducing alanine residues into the poly- 
lysine(alanine - lysine copolymer). The effects of polyarginine resemble 
those of arginine-rich histone IV. 

A comparison of the stability of the DNA complexes of polylysine, polyargi- 
nine, and polyorni thine, using as criteria the melting temperature and the 
salt concentration required to break up the complex, reveals that basic poly- 
peptides with long side chains form weaker links to DNA than shorter chain 
polypeptides. L-polylysine and D-polylysine form complexes with DNA of simi- 
lar stability and properties but DL-polylysine foirms a much less stable com- 
plex with a lesser effect on the conformation of DNA. All of these phenomena 
can be readily explained by the use of molecular models, and they enable us 
to comprehend the forces that bind hi stones in DNA. 

DNA that is not bound to polypeptide components has a role in regulating the 
conformation of polypep tide-bound DNA. This observation could explain the 
fact that only part of the DNA in chromatin is covered by protein, while all 
of the chromatin is repressed toward in vitro transcription. 

B. Physical chemical studies of the interaction of ribosome, messenger RNA, 
and transfer RNA . The ORD of salt-washed E. coli ribosomes and of mixtures 
of such ribosomes with poly(rU) and tRNAP^e were determined at ~2 , pH 7.0, 
and .01 M Mg ion, at ribosome concentrations of the order of 10~%(P), and 
poly(rU) and tRNAP^^ concentrations of the order of lO'^M(P); no significant 
differences emerged between the ORD of the mixtures and the calculated com- 
binations. Direct-difference determinations, under the same conditions ex- 
cept at ambient temperature of 25-27°, unequivocally showed no significant 
differences, and so demonstrate that, under these particular conditions, no 
conformational differences measurable by ORD exist for these combinations. 
Experiments utilizing the fluorescence of the Y nucleotide of yeast tRNAP"^, 
which is located in the anticodon loop, were conducted with the same system 
and have also shown no net changes so far. Thus it appears that the compo- 
nents of the very simple system consisting of ribosome, messenger RNA, and 
transfer RNA do not influence each other's conformation, at least as de- 
tectable by these methods. If it can be shown that the addition of other 
factors does produce conformational changes, the inducers of these changes 
will be readily identified. 

FL-11 



Significance to Bio-medical Research and the Program of the Institute ; An 
understanding of the structure of chromatin is required to understand the 
mechanism of gene regulation, which is of direct consequence to the study of 
development and aging. The ribosome is the locus of protein biosynthesis, 
and the agency or locus of much of its control. Our physical studies are de- 
signed to provide direct information about structures and interactions which 
are intimately involved in the synthesis process and its ribosomal control. 
If we are able to understand these interactions, it will then be possible to 
determine how they may change with age. 

Proposed Course of Project : We expect to continue to analyze the interaction 
modes of DNA with the protein constituents of chromatin by comparison of the 
interaction with DNA of the chromatin proteins with synthetic polypeptides. 
We shall study the effect of translational cofactors on the conformation of 
the ribosome in the presence of message and both charged and uncharged trans- 
fer RNA. 

Honors and Awards ; None 

Publications; None 



T?T _i 9 



Serial No. HD-GMA.5 

1. Gerontology Research Center 

2. Laboratory of Molecular Aging 

3. Molecular Chemistry Section 

4. Baltimore, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Polymers as Biological Reagents 

Previous Serial Number: Same 

Principal Investigator: J. Pitha (90% time) 

H. Chou (1007. time) (E.O.D. 1/2/73) 
Other Investigators: D. Lowy 

N. Teich 

P. Pitha 

Cooperating Units: Laboratory of Viral Diseases, N.I.H. 

Johns Hopkins University 
Baltimore City Hospitals 

Man Years : 

Total: 2.40 
Professional: .40 
Others: 1.00 

Project Description: 

Objectives : The program focuses on the effects of synthetic polymers on 
human cells grown in tissue culture and on the interaction of polymers with 
viral systems (animal viruses). The main class of macromolecules studied 
consists of the vinyl analogs of nucleic acids of the general formula 
(-CH Base - CH2)n> furthermore a series of compounds with polynucleotide 
bound to protein or to polysaccharide has been investigated. The ultimate 
objectives are to determine which synthetic polymers can be used pharma- 
ceutically, to study viral infection in senescent cells and the possibility 
of its prevention by polymers, and finally, to determine whether the uptake 
of foreign macromolecules by cells changes with the age of the cells. 

Methods Employed : In the study a very broad range of chemical and bio- 
logical methods has to be used and several laboratories are involved in 
collaborating on this project. The majority of the chemical synthesis, 
some in vitro work with enzymatic systems (replicase) and the uptake of 
dextran by cells grown in tissue culture is done at the Gerontology Re- 
search Center. The study of the replication of murine leukemia virus is 
done in co-work with Drs. D. Lowy and N. Tcich from the Laboratory of Viral 
Diseases, N.I.H. , Bethesda. The rest of the work involving viruses is done 
in collaboration with Dr. P. Pitha, Department of Medicine, Johns Hopkins 

FL-13 



University. In vitro protein synthesis is studied with Drs. F. Reynolds and 
D. Gruenberger from Columbia University. 

Major Findings ; A. In vitro synthesis of proteins . The effects of poly(vU) 
on in vitro protein synthesis directed by mRNA has been investigated. The 
amount of protein formed was found to decrease in different degrees, depend- 
ing on the nature of the particular RNA. 

B. Polymers and living cells . Vinyl analogs of nucleic acid were found to 
be taken up by cells in tissue cultures. Polyvinyluracil at high concentra- 
tion is toxic to rapidly growing cells; polyvinyladenine (or the uracil ana- 
log at much lower concentration) does not interfere with cell multiplication 
and the synthesis of nucleic acids and proteins. 

C. Polymers and viral replication . Poljrvinyluracil and polyv^inyladenine as 
well as the corresponding polynucleotides polyuridylic and polyadenylic acid 
inhibit acute murine leukemia virus infection in mouse embryo cells, but do 
not significantly inhibit the replication of Sindbis and vesicular stomatitis 
viruses. These polymers were most effective when added during an early stage 
of murine leukemia virus replication. The inhibition of the leukemia virus 
appears to be related to an inhibition of an RNA-dependent DNA polymerase. 

D. Polymers and induction of interferon . The effects of different chemical 
modifications of the complex of polyinosinate and polycytidylate on its anti- 
viral activity have been studied. Using base substitutions which conserve 
the double helix of the complex, it was found that antiviral activity and 
interferon induction are more critically influenced by substitution in the 
inosinate than in the cytidylate strand. The rigorous steric requirements 

(as embodied by the poly I»poly C structure) for antiviral protection by poly- 
nucleotides are significantly decreased \^^en aggregated interferon inducers 
are used. Significant protection against virus infection was achieved by 
complexes of various polybases, such as polylysine or DEAE Dextran, with poly- 
inosinate; introduction of cytosine residues covalently bound to these poly- 
bases in the complex is without specific influence. Furthermore, the complex 
of polyinosinate with polycytidylate was attached to solid carriers, such as 
Sephadex or cellulose, v/hich may be suspended in the medium over the cell 
monolayer; it was also attached to surfaces which may be overgrown by the cell 
monolayer. Interferon production and antiviral protection achieved by these 
inducer systems were measured and it was found that such anchored poly IC con- 
veyed antiviral protection. However, in all systems studied, some release of 
polynucleotide from the carrier V7as observed and thus the results do not 
prove that mere contact of inducer with the external membrane is sufficient 
for induction of interferon. 

E. Polymers and senescent cells . A previously prepared series of radioactive 
dextran macromolecules of differing molecular weights has been used to study 
whether there is any change in the uptake of foreign macromolecules with 
aging of cells in tissue culture. No particular age differences have been 
detected but results are not yet final. 

F. Senescent cells and viral infection . An extensive study of abortive in- 
fection of senescent cells by vesicular stomatitis virus and its prevention 

FL-14 



has led to the following results: 1) Senescence of host cells is not de- 
trimental to viral replication with the exception of a brief period before 
cellular death. 2) Cellular viral defenses are functional throughout the 
life span of the cells up to the moribund state but the amount of antiviral 
agent required increases with age. 3) In senescent cells polynucleotide 
interferon inducer produces the antiviral state faster than treatments with 
interferon itself. 

Significance to Bio-medical Research and the Program of the Institute; 
Synthetic compounds of high molecular weight differ profoundly from low 
molecular weight compounds in their interaction with living cells. They are 
taken up by a different mechanism, the efficiency of which seems to depend 
more on the growing stage of the cell than on the age of the cell. Another 
difference is in their degradation in cells; synthetic macromolecules have a 
much longer life span since they are not susceptible to the usual cellular 
degradative processes. Both these differences have practical importance only 
when polymers with a selective biological action can be designed and synthe- 
tized. Vinyl analogs of nucleic acids do show selectivity thus pointing to 
their potential use as pharmaceuticals. 

Proposed Course of the Project ; The present results show that synthetic 
polymers can elicit a selective biological effect, in a manner similar to 
that of low molecular weight pharmaceuticals. By directed synthesis and 
study of the basic biological effects of polymers it is hoped to gain the 
knowledge necessary for the design of more potent preparations. 

Honors and Awards : None 

Publications : 

Reynolds, F. , Grunberger, D., Pitha, J., and Pitha, P. M. ; Inhibition of Cell- 
Free Protein Synthesis by Poly(9-vinyladenine) , Poly(l-vinyluracil) , and the 
Corresponding Vinyl Copolymer. Biochemistry 11: 3261-3266, 1972. 

Pitha, P. M. , and Pitha, J.: Nonenzymatic Synthesis of Oligoadenylates on 
Template lacking Steric Regularity. Nature New Biology 240: 78-80, 1972. 

Pitha, P. M. , Teich, N. M. , Lowy, D. R. , and Pitha, J.: Inhibition of MLV 
Replication by Polyvinyluracil and Polyvinyladenine. Proc . Natl . Acad. Sci . 
70: 1204-1208, 1973 . 

Pitha, P. M. , Teich, N. M. , Lowy, D. R. , and Pitha, J.: Inhibition of 
Leukemia Virus Replication by Vinyl Analogs of Polynucleotides. In Wells, 
R. D. , and Inman, R. B. (eds.): DNA Synthesis In Vitro . University Park 
Press, in press . 



PL- 15 



Serial No. HD-GMA.6(1) 

1. Gerontology Research Center 

2. Laboratory of Molecular Aging 

3. Molecular Chemistry Section 

4. Baltimore, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 197 3 

Project Title: Mispairing of Nucleotide Bases 

Previous Serial Number: Same 

Principal Investigator: G. Eichhom (107o time) 

J. Pitha (107o time) 
E. Tar i en (307„ time) 

Other Investigators: None 

Cooperating Units: Baltimore City Hospitals 

Man Years: 

Total: .70 
Professional: .50 
Others: .20 

Project Description: 

Objectives ; (1) To determine the conditions under which mispairing of the 
complementary bases occurs between polynucleotide strands in order (2) to 
understand the ways in which misreading of the genetic code can occur in 
order (3) to understand how errors that could lead to the aging of organ- 
isms can occur in the apparatus for the transmission of genetic information. 

Methods Employed : It is known that metal ions of the wrong type or in the 
wrong concentration can bring about the misreading of the genetic code in 
protein synthesis, resulting in the production of proteins containing in- 
correct amino acid sequences. Such errors can arise through mispairing of 
bases in codon-anticodon recognition. The following hypothesis is devised 
to account for mispairing. It is known that high concentrations of diva- 
lent ions stabilize the interaction of polynucleotide strands. In the 
absence of the divalent metal ions these interactions are therefore rela- 
tively unstable and require base complementarity; in the presence of these 
ions the interactions are stabilized to such an extent that sensitivity to 
complementarity is lost, and mispairing then occurs. 

This hypothesis can be tested by studying the binding of homopolynucleo- 
tides to heteropolynucleotides containing one base that is complementary 
and one base that is not complementary to the base in the homopolymcr. The 
extent of mispairing is measured by (a) stoichiometric studies of the 

FL-16 



reaction between the two polynucleotides and (b) by nuclear magnetic re- 
sonance studies that detect only non-hydrogen bonded bases. 

Major Findings : A. NMR studies on mispairing . It had been previously demon- 
strated that divalent ions can induce the mispairing of nucleotide bases. 
Nuclear magnetic resonance studies had shown that magnesium ions induce mis- 
pairing in the system poly(A,U) :poly(l) . It has now been shown in the same 
way that magnesium ions induce mispairing in the systems poly(U,C) :poly I and 
poly(A,C) :poly I. In every case NMR lines that are detectable in the absence 
of magnesium become broadened when magnesium is added, thus demonstrating the 
interaction of non- complementary base pairs in these systems as well. These 
studies show that the mispairing effect is general, and not restricted to any 
type of base pairing. 

B. NMR sequence studies . As a corollary to the mispairing studies described 
above, we have learned that the NMR peaks associated with a given nucleotide 
base may be shifted in different degrees by association with other nucleotide 
bases in the vicinity. Thus the adenine peaks in poly(A,C) appear to split 
into two groups that appear to be associated with adenines surrounded by 
other adenines and adenines surrounded by cytosines. This discovery may fur- 
nish a technique for the study of base sequence and will be pursued further. 

Significance to Bio-medical Research and the Program of the Institute : The 
accumulation of errors that would be produced during protein synthesis by 
mispairing of nucleotides of the kind being studied would lead to deleterious 
effects in an organism, according to one of the more popular aging theories. 
Thus the study constitutes a chemical model for a theory of aging. 

Proposed Course of Project ; We intend to determine whether metal ions in 
biological systems will produce the kind of errors that are induced in these 
model systems. We hope to investigate the usefulness of the NMR method in 
the determination of base sequence. 

Honors and Awards : 

G. Eichhom was elected Fellow of the Gerontological Society 

Publications: None 



FL-17 



Serial No. HD-AGl7(8)(c) 

1. Gerontology Research Center 

2. Laboratory of Molecular Aging 

3. Molecular Chemistry Section 

4. Baltimore, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Relation of Structure and Function in Hemoglobin 

Previous Serial Number: Same 

Principal Investigator: J. Rifkind (607o time) 

Other Investigators: None 

Cooperating Units: Baltimore City Hospitals 

Man Years: 

Total: .70 
Professional: .60 
Others: .10 

Project Description: 

Objectives : (1) To study the binding of ligands to hemoglobin, and the 
role of the protein in controlling this function. (2) To study the mecha- 
nisms for maintaining hemoglobin in its functional form. (3) To study the 
mechanisms involved in regulating the transport of oxygen to the tissues. 

Methods Employed ; Hemoglobin is purified by gel filtration. The purified 
hemoglobin is compared to hemolyzed cells with respect to the rate of 
autoxidation, the concentration of various metal ions and other red cell 
components, and the Cu(ll) activity. The oxidation is also investigated 
with and without the addition of various substances such as metal ions, 
complexing agents, and oxidizing agents. The relationship between the 
oxidation of hemoglobin and the ligands bound to hemoglobin are investi- 
gated by removing or changing the ligands. The direct interaction of 
copper with hemoglobin is studied by EPR spectroscopy. 

Major Findings : A. The involvement of copper in the autoxidation of 
hemoglobin . Oxygen only binds to reduced hemoglobin. Therefore, the au- 
toxidation of functional hemoglobin decreases its oxygen binding capacity 
and thereby its ability to transport oxygen to the tissues. Our previous 
studies on the stabilizing effect of metal complexing agents and the spe- 
cific catalytic oxidation of added copper suggested the involvement of 
copper in the autoxidation of hemoglobin. 

The validity of this hypothesis and the importance of this effect has been 
established by measurements of the total copper and Cu(ll) activity in 

PT-1R 



hemolyzed cells and purified hemoglobin preparations. Hemolyzed cells con- 
tain approximately one copper atom for every 1000 hemes. This concentration 
of copper is more than adequate to produce appreciable catalytic oxidation. 
When the hemoglobin is purified by gel filtration, all the copper chromato- 
graphs together with the proteins, and the ratio of copper to hemoglobin re- 
mains about the same. The autoxidation in both hemolyzed cells and purified 
hemoglobin can therefore be attributed to copper originating in the erythro- 
cyte. The dominant role of copper in the autoxidation of hemoglobin is sug- 
gested by the excellent correlation under various conditions of the rates of 
autoxidation with the Cu(Il) activity which must determine the extent to 
which copper is interacting with hemoglobin. About half of the copper can 
be separated from the proteins, lowering the rate of autoxidation, by adding 
a complexing agent to the hemolyzed cells prior to gel filtration. This re- 
sult suggests that only half of the erythrocyte copper is actually involved 
in catalyzing the autoxidation. The remaining copper is probably coordinat- 
ed to erythrocuprein (superoxide dismutase) from which copper cannot be re- 
moved by complexing agents at neutral pH. 

All investigators working with purified hemoglobin have found that almost 
anything you do with hemoglobin seems to increase the rate of autoxidation. 
We have also been plagued by this problem. However, the analysis of copper 
indicates that these problems can be related to the ubiquitous presence of 
trace amounts of copper on glassware, equipment and in solutions. The strong 
affinity of hemoglobin for copper tends to concentrate the copper in the 
hemoglobin solutions. Our studies have only been possible by extreme caution 
in minimizing copper contamination. When this is done the hemoglobin remains 
surprisingly stable with respect to autoxidation. 

B. The mechanism for the catalytic oxidation of hemoglobin by copper . Con- 
sidering the sig.nif icance of the oxidation of hemoglobin by copper we are 
attempting to elucidate the details of the mechanism. By studies involving 
deoxyhemoglobin, in the absence of oxygen, it has been possible to show that 
copper is able to directly oxidize the iron of hemoglobin. The role of oxy- 
gen for the catalytic effect is, therefore, probably to regenerate the Cu(Il) 
ion from the Cu(I) ion formed when copper oxidizes hemoglobin. We have also 
been able to show that the sixth ligand must be removed from hemoglobin prior 
to the oxidation. Therefore, the rate of oxidation by copper is greatest for 
deoxyhemoglobin where no sixth ligand is present. In the presence of a sixth 
ligand the rate is actually retarded by higher ligand concentrations or by 
replacing oxygen by carbon monoxide which dissociates from hemoglobin much 
more slowly. 

Finally the binding of copper to hemoglobin seems to be required for the 
oxidation. Thus the strong affinity of hemoglobin for copper is seen by very 
low copper activities in the presence of hemoglobin. EPR studies also in- 
dicate that there is very little free copper present when copper is added to 
hemoglobin. The involvement of the binding for the oxidation is consistent 
with the apparent difference in the EPR spectrum for copper bound to reduced 
and oxidized hemoglobin. Such a change in the nature of bound copper is ex- 
pected if the binding is to facilitate oxidation. The unique role of copper 
can possibly be explained by a combination of the right oxidation potential, 
to oxidize hemoglobin and be regenerated by oxygen, and the strong binding to 

FL-19 



a particular region of hemoglobin where electron transfer between the iron 
and the copper is facilitated. 

C. The regulation of oxygen utilization in the muscle . Oxygen storage and 
transport in the muscle involves myoglobin. This protein reversibly binds 
oxygen, and has a structure very similar to the individual hemoglobin sub- 
units. In a collaborative study with Dr. Lumry (Univ. of Minnesota) we are 
studying a possible mechanism for the regulation of oxygen release by myo- 
globin. In the preparation of myoglobin it has been found that a small ionic 
substance bound tightly to myoglobin, which can be oxidized and reduced, in- 
fluences the oxygen affinity of myoglobin. We have found that copper chro- 
matographs together with this regulatory substance suggesting that the sub- 
stance is perhaps copper or another substance which binds copper strongly. 

Significance to Bio-medical Research and to the Program of the Institute : 
The physiological role of hemoglobin is to transport oxygen from the lungs 
to the cells. The efficient uptake and release of oxygen requires coopera- 
tive oxygen binding and the proper regulation of the oxygen affinity. It is 
also necessary to limit the oxidation of hemoglobin in order to maintain an 
adequate concentration of functional hemoglobin. These studies thus help to 
elucidate a vital function of organisms. The aging process can involve 
changes in the ability of the organism to transport oxygen to certain tissues. 

Proposed Course of Project ; (1) We plan to continue our studies on the au- 
toxidation of hemoglobin in the erythrocyte and in purified systems. The 
possible role of superoxide dismutase will be investigated. (2) The mechan- 
ism for the oxidation of hemoglobin by copper will be further studied. (3) 
Numerous clinical conditions, requiring more effective oxygen transport, re- 
sult in increases in the level of 2,3-DPG which decreases the oxygen affinity 
of hemoglobin facilitating the release of oxygen to the tissues. An increase 
in the level of 2,3-DPG for human subjects above age 50 has also been report- 
ed. This finding suggests that a more effective release of oxygen from hemo- 
globin may be required to overcome certain degenerative changes occurring 
with age. We plan to test this hypothesis by measuring levels of 2,3-DPG in 
whole erythrocyte as a function of the age of the organism and correlating 
these levels with the oxygen affinity. (4) We also will attempt to change 
the level of 2,3-DPG in erythrocytes in vitro as well as in vivo in order to 
investigate the possible effect of these changes on particular aging pro- 
cesses. 

Honors and Awards : Dr. Rifkind was invited to present a seminar to the 
Division of Hematology, Johns Hopkins University School of Medicine, Balti- 
more, Md. 

Dr. Rifkind was invited to present a seminar to the Heme-Protein group at 
the National Institutes of Health, Bethesda, Md. 

Publications ; 

Rifkind, J. M. ; The Autoxidation of Horse Hemoglobin: The Effect of Gluta- 
thione. Biochim . Biophys . Acta 273: 30-39, 1972. 



FL-20 



Serial No. HD-AG30 

1. Gerontology Research Center 

2. Laboratory of Molecular Aging 

3. Molecular Chemistry Section 

4. Baltimore, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: The Function of Metal Ions in Enzymatic Reactions 

Previous Serial Number: Same 

Principal Investigator: P. Clark (100% time) 

G. Eichhorn (307., time) 

J. Froehlich (100% time) (E.O.D. 8/1/72) 

Other Investigators: None 

Cooperating Units: Baltimore City Hospitals 

Man Years: 

Total: 2.30 
Professional: 2.30 
Others: 

Project Description: 

Objectives : (1) To elucidate the mechanism by which metal ions activate or 
inhibit enzymatic reactions. (2) Specifically, to elucidate the role of 
bound metal ions in transcription by studying their mode of binding to RNA 
polymerase, and their role in substrate recognition, binding and message 
synthesis. 

Methods Employed : RNA polymerase obtained from E. coli contains approxi- 
mately 2 gram atoms of zinc/mole of core enzyme following prolonged dialysis 
with chelating agents. The weak, polarizability and diamagnetism of zinc 
make it an unsuitable probe of site conformation. The unpaired electrons 
of the metals of the first transition series provide a sensitive means with 
which to monitor (1) the local geometry of the binding site (2) the proxi- 
mity of neighboring paramagnets and (3) the accessibility of counterions 
(CI", Br") to the metal using the techniques of electron paramagnetic re- 
sonance spectroscopy and pulsed nuclear magnetic resonance spectroscopy. 
The effective use of these probes requires preservation of the native 
structure and partial retention of enzymatic activity. The kinetic pro- 
perties of the substituted enzyme can be used to assess the effects of metals 
on the initiation, polymerization, and termination of message. Preparation 
of the substituted enzyme will be attempted by (1) metal exchange in the 
presence of high concentrations of the desired probe (2) removal of zinc by 
chelation followed by stoichiometric addition of the paramagnetic probe in 

FL-21 



a zinc-free medium. Atomic absorption spectrophotometry and enzymatic 
activity will be used to follow the course and extent of substitution. 

A new method has been developed for determining whether a double helix is 
enzymatically cleaved by simultaneous attack on both strands of the helix, 
or by attack on only one strand at a time. The method consists of an analy- 
sis of the nucleotide content of the stabilized fraction. 

Major Findings ; A. RNA polymerase . Very tight binding of zinc is indicated 
by the failure to remove > 257<, of the bound metal after 8 days of dialysis 
vs. 5mM orthophenan thro line. It has however been demonstrated that the zinc 
can be displaced by Mg2+, Co2+ and Mn2+ with the retention of full activity, 
and by Cu2+ with 507<, activity. The copper enzyme contains 2-4 Cu ions per 
enzyme. Its electron spin resonance spectrum contains a dipole interaction 
signal which reveals that two copper atoms bound to the enzyme are 5-6 A 
apart. Cu ions the same distance apart are also present in an enzyme to 
which manganese had been bound and from which it was then removed. Thus 
Cu2+ ions readily attach themselves to a strong binding site which may be 
the intrinsic metal binding site of the enzyme. 

The cobalt enz5mie was subjected to kinetic studies which demonstrated kinetics 
similar to those of the native zinc enzyme. This behavior is in direct con- 
trast to the effect on the enzyme of free cobalt (which had not displaced 
zinc), which prolongs the initiation phase but enhances the rate and extent of 
polymerization. 

As reported in HD-GMA-1, cis [pt(NH-:i)?Cl?] forms a 1 Pt/10 DNA nucleotide 
adduct with DNA. Because of the biological activities of this complex it was 
decided to evaluate its effect on the ability of DNA to act as a template for 
RNA synthesis. The complex was found to confer little or no change in the 
initiation process of RNA polymerase, but a dramatic inhibition was observed 
in the polymerization phase of the enzymatic process. LPt en CI2I had an 
identical effect. In both cases polymerization was inhibited by approximately 
907o. These experiments were carried out by first incubating the DNA with the 
platinum complex for 24 hours to insure the formation of the adduct. Wlien 
other experiments were carried out without such pre-incubation, so that no 
binding of the platinum complex to the DNA occurred, a 507<, inhibition of the 
polymerization was nevertheless observed. The most likely interpretation of 
these experiments is that the platinum complex inhibits transcription in two 
ways, by binding to the DNA as well as by binding directly to the enzyme. 

B. Mechanism of DNA cleavage . Porcine spleen acid DNAse is known to cleave 
DNA by operating simultaneously the phosphodiester bonds of both nucleotides 
of a Watson-Crick base pair. The new method for determining whether cleavage 
occurs by simultaneous scission in both chains or by scission of individual 
strands consists of analyzing the base content of soluble oligonucleotides. 
If the analyses add up to complementarity, there must have been simultaneous 
scission. This is in fact what was found with the acid DNAse, indicating 
that the method does work. 

When applied to bovine pancreatic DNAse I, the base analyses did not display 
complementarity, when the enzyme had been activated with several metal ions. 



It is concluded that with this enzyme only one strand is cleaved at a time, 
thus reversing conclusions previously reached by more devious techniques. 

C. The binding of nickel ions in serum . In a collaboration with F. W. 
Sundetman of the University of Connecticut, it has been demonstrated that 
Ni'^ ions in serum are bound principally to aspartic acid in a 2:1 complex 
with apparently two carboxyl groups and an amino group coordinated to the 
nickel. This study is of some importance as a beginning in determining where 
metal ions introduced into a living organism are attached. 

Significance to Bio-medical Research and the Program of the Institute . The 
processes of RNA synthesis and DNA degradation are of fundamental importance 
to an understanding of cellular processes. Metal ions are required for these 
processes and variations in their effects may be of significance in the dele- 
terious changes accompanying aging. 

Proposed Course of Project : We shall continue to pursue the elucidation of 
the mechanism of transcription as well as the mechanism of enzymatic DNA de- 
gradation, as outlined in the Methods section. 

Honors and Award s: 

G. Eichhorn was invited to present an address at a Symposium on the "Role of 

Metal Ions in Biological Systems", at Argonne, Illinois, November, 1972. 

G. Eichhorn was invited to present a lecture at a Symposium on "Bioinorganic 
Chemistry" in Oneonta, New York, May, 1973. 

G. Eichhorn was invited to present a lecture at a Symposium of the American 
Chemical Society on "Bioinorganic Chemistry", Kalamazoo, Michigan, June, 1973. 

G. Eichhorn was invited to lecture at a Gordon Conference Symposium on Bio- 
inorganic Chemistry, New Hampton, N. H. , August, 1973. 

G. Eichhorn was asked to organize and to chair a Symposium on the "Regulation, 
Transport and Role of Inorganic Elements in Living Systems" at the American 
Chemical Society meeting in Chicago, Illinois, August, 1973. 

G. Eichhorn was invited to be a participant in the NATO Science Committee 
Conference on Catalysis, Cagliari, Sardinia, December, 1972. 

G. Eichhorn was invited to present seminars at various Universities. 

J. Froehlich was invited to present a seminar at Johns Hopkins University, 
Baltimore, Md. February, 1973. 

Publications ; 

Eichhorn, G. L. (Editor): Inorganic Biochemistry , Vol. I, 610 pp. Elsevier 

Publishing Co., in press . 

Eichhorn, G. L.: Binding of Nickel to Biological Substances. In Sunderman, 
F. W. (ed.): N ickel, Man and his Environment . National Academy of Science, 
in press . 

FL-2 3 



f 



Eichhorn, G. L.: Effects of Nickel on Enzymatic Activities. In Sunderman, 
F. W. (ed.): Nickel. Man and his Environment . National Academy of Science, 
in press . 



i 



FL-24 



Serial No. HD-GMA 7 

1. Gerontology Research Center 

2. Laboratory of Molecular Aging 

3. Section on Intermediary Metabolism 

4. Baltimore, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Cellular Regulation of Intermediary Metabolism 

Previous Serial Number: HD-AG19(8) and HD-AG20(8) 

Principal Investigators: Bertram Sacktor 

Bernard Bulos 
Walter Greenhouse 
Shri Shukla 
Peter Chiang 

Other Investigators: Stephen O'Brien (Guest Worker) 7/1/72 - 9/30/72 

Cooperating Units: None 

Man Years : 

Total: 7.2 
Professional: 4.5 
Others: 2.7 

Project Description: 

Objectives : The broad objective of this project is to describe the general 
mechanisms that regulate intermediary metabolism in animals, including man. 
These mechanisms range from the simplest types of control that involve the 
intrinsic properties of enzymes affecting the rates of reactions to those 
of increasing complexity that comprise the action of modulators on regulatory 
and allosteric enzymes; genetic repression and derepression; selective 
separation, sequestration and release of ions, metabolites and enzymes by 
intracellular membranous organelles; steady state and transient fluxes of 
intermediates; and the actions of endogenous and exogenous chemical and 
physical modifiers on entire metabolic pathways and coordinated systems. 
The development of this scientific data and theory is focused on those phys- 
iological functions that may show significant alterations with development 
and aging and, thus, are essential to the understanding of the molecular 
processes involved in development and aging. 

Methods Employed : Metabolic pathways are studied with purified enzymes, 
isolated organelles, cell free systems, cell cultures, slices of tissues, 
perfused organs and intact animals. Powerful tools of analysis of metabolites, 
measurements of enzyme activities, and isotope tracer techniques are employed, 
as appropriate. Computer analyses of kinetic models are used as required. 

FL--25 



Major Findings : A. Control of Krebs cycle activity in mitochondria . 
Extensive studies on the control of pyruvate oxidation in mitochondria from 
blowfly flight muscle, and confirmed with mitochondria from rabbit heart 
muscle, have led to a novel mechanism for the regulation of Krebs cycle 
activity. Both ADP and inorganic phosphate are required for the uncoupled 
(FCCP) oxidation of pyruvate as well as for the coupled oxidation. The FCCP- 
stimulated oxidation is markedly inhibited by physiological levels of Ca^"*". 
The apparent K^ = 10 yM. The electron transport chain of the mitochondria 
is fully functional in the presence of Ca^"*" since the oxidation of a-glycerol- 
phosphate is not inhibited, indicating control at the dehydrogenase level. 
Kinetics studies show that the Ca inhibition is uncompetitive with respect 
to ADP and FCCP, but complex, and noncompetitive with respect to pyruvate. 
The results of these and other studies suggest that pyruvate oxidation and 
Krebs cycle activity is limited by NAD-dependent isocitrate dehydrogenase 
activity and Ca^"^ plays a role in its regulation. 

B. Control of pyruvate dehydrogenase activity : Interconversion of active 
and inactive forms . In experiments to determine the mechanisms of regulation 
of the mitochondrial pyruvate dehydrogenase complex, it has beeii observed 
that the enzyme complex from blowfly flight muscle exhibits Michael is -Menton 
kinetics with respect to pyruvate and NAD, but shows sigmoidicity with CoASH. 
A Hill coefficient of 1.5 is obtained. The apparent Km values are: 0.21 iriM 
for pyruvate, 39 ]M for CoASH, and 0,28 niM for NAD. The Km value for pyruvate 
is similar to the Km value for State 3 oxidation of pyruvate in intact mito- 
chondria, and approximates the concentration of pyruvate found in the muscle, 
in vivo. The concentration of CoASH in blowfly flight muscle has been 
determined to be 35 nmoles/g wet wt. Acetyl CoASH is a potent competitive 
inhibitor with respect to CoASH. Both ATP and GTP are non-competitive in- 
hibitors with respect to pyruvate. Activity of the complex is influenced by 
the Mg and Ca^"^ concentrations and by the presence of chelating agents. 
CoASH forms a chelate with Mg2+. A stability constant of 5,000 M"-'- is ob- 
tained for the chelates of Mg-CoASH and Mg-thiamine pyrophosphate. 

It has been found that control of pyruvate dehydrogenase complex activity in 
mitochondria from blowfly flight muscle, and confirmed with mitochondria from 
rabbit heart, is mediated additionally by the interconversion of the complex 
from a phosphorylated (inactive) form to a dephosphorylated (active) form. 
In flight muscle, pyruvate dehydrogenase kinase, which converts the active 
form to the inactive form requires ATP and Mg^"*". By Hill plot analyses, the 
apparent Km for ATP is 0.3 mM and the coefficient of interaction is between 
2 and 3. Both pyruvate and inorganic phosphate are non-competitive inhibitors 
of the kinase whereas AMP is a competitive inhibitor. Changes in the con- 
centrations of these effectors in the muscle in situ during rest and exercise 
are in the range consistent with their modulation of enzyme activity iji vivo. 
Atractyloside, an inhibitor of adenine nucleotide translocation in mitochon- 
dria, does not block kinase activity. This may suggest that ATP is accessible 
to the kinase external to the atractyloside barrier. The phosphorylated pyr- 
uvate dehydrogenase is dephosphorylated and, thus, activated by a specific 
phosphatase. The phosphatase is inhibited by F" and chelating agents and is 
activated by physiological concentrations of Mg^"^ and Ca . 



FIj26 



C. Bloenergetics of mitochondria from flight muscle of aging blcx^7flies . 
A correspondence between the decline in the ability of aged blowflies to 
fly and in the maximally stimulated respiratory rate (State 3) and respira- 
tory control ratio of flight muscle mitochondria was reported previously. 
The biochemical mechanism and locus of this decrement with senescence has 
been investigated further by examining the bioenergetic properties of mito- 
chondria from mature and aged flies for rates of oxidation during coupled 
oxidative phosphorylation, activities of the dehydrogenases, concentrations 
of components of the electron transport chain, phosphorylation of ADP, and 
the coupling mechanism. Itself. Evidence, to date, indicates that the 
specific activities of a-glycerolphosphate and pyruvate dehydrogenases are 
unaltered by age. Similarly, the concentrations of cytochromes a, a-, b, 
and c+c^ are the same for both age groups. ATP synthetase, measured as 
ATPase, is unchanged with senescence. In contrast, the uncoupled oxidation 
of substrates is significantly decreased with aging, showing the 30-4070 
decrement between mature (7-10 days) and aged (31-33 days) as seen previously 
for the maximally stimulated coupled rate of respiration. These observations 
narrow the possible loci of the defect in aging to the bioenergetic system 
between the primary dehydrogenase and the FCCP-sensitive uncoupling reaction. 

D. Genetic regulation of enzyme activity . The malate dehydrogenases of 
Drosophlla melanogaster have been resolved into a cytoplasmic form (cMDH) , 
and a mitochondrial matrix form (mMDH) . Three isozymes of cMDH have been 
detected by starch gel electrophoresis and acrylamide gel isoelectric 
focusing. The probable structural gene for cMDH ( Mdh-1 ) has been mapped 
genetically by allozyme variants to II-35"t3, and cytologically by monitoring 
gene dosage in segmental aneuploids to between 28D and 29F on II-L of the 
Drosophlla salivary gland chromosome map. The structural gene for mMDH is 
neither Identical to nor in the near chromosomal proximity of Mdh - 1 . Never- 
theless, the two enzymes exhibit markedly similar properties with respect 
to: (1) catalytic activity, (2) pH optima, (3) pH optimum shift in response 
to different ionic environments, and (4) molecular weight as determined by 
sucrose density gradient sedimentation. 

A method for detecting possible structural genes in Drosophlla melanogaster 
based on gene dosage dependency has been developed. By making 30 crosses 
between Y-autosome translocations, and an attached 4 cross, it is possible 
to produce large duplications (approximately 150 salivary gland chromosome 
bands in length) for every autosomal region with the exception of 83DE. 
The usefulness of the technique has been demonstrated by dosage dependency 
of three known gene-enzyme systems: g-glycerophosphate dehydrogenase-l . 
alcohol dehydrogenase and malate dehydrogenase . A screen for genes affecting 
two enzymes localized on the inner membrane of the mitochondrion, a-glycero- 
phosphate oxidase (oGPO) and succinic dehydrogenase (SDH), produces a dosage 
sensitive region in each case. Region 50C-52E affects oGPO activity and 
region 28D-29F affects SDH activity. The latter region also includes the 
malic dehydrogenase-l gene. 

E. Molecular organization, biogenesis and turnover of mitochondrial 
membranes . The molecular organization of rat liver mitochondrial membranes 
is being studied by testing hypotheses on the mechanisms of mitochondrial 
turnover, e.g., by a dynamic process in which the membranes are assembled 



FL-27 



from multiple components, each turning over at an unique rate, or by a process 
of replication and degradation as a single unit. Previous studies have 
demonstrated the utility of altering mitochondrial function and perturbing 
the mitochondrial membranes by changing the thyroid state of the animal. 
We have now found that during the transition from the hypothyroid state 
(thiouracil fed for 4 weeks) to the thyroid repleted state (a single in- 
jection of 20 Jig T3/IOO g body wt) several enzymes, selected because they 
represent specific markers for intracellular membranes, behave in distinct 
fashion. For example, in confirmation of previous findings, the specific 
activity of a-glycerolphosphate dehydrogenase, an enzyme localized on the 
outer surface of the inner mitochondrial membrane, is decreased in tV\e hypo- 
thyroid state to about 20% that in the euthyroid animal. In the T2 repleted 
state, the specific activity is increased over 4007o within 40 hours. In 
contrast, succinic dehydrogenase, another marker enzyme for the inner membrane, 
but believed to be located on the inner surface, responds differently. The 
specific activity of succinic dehydrogenase is increased 150% by hypothy- 
roidism. To repletion does not cause a return to euthyroid levels by the 
first 40 hrs, however. Monoamine oxidase, a marker for the outer mitochon- 
drial membrane, shows a 2-fold increase in specific activity in the TU-fed 
rats and T3-repletion causes a fall in specific activity to euthyroid levels. 
However, when mitochondria are sub fractionated, the specific activities of 
the enzyme in the outer membrane from mitochondria of TU-fed and T3 -repleted 
animals are essentially the same. Therefore, these findings suggest that 
the changes in the specific activities of monoamine oxidase seen in whole 
mitochondria represent changes primarily in the protein content of the inner 
membrane, with the composition of the outer membrane remaining fairly con- 
stant. The enzymatic components of the microsomal membrane are also 
differently affected by the thyroid state. For example, the specific activity 
of glucose-6-phosphate dehydrogenase varies with the thyroid state but the 
specific activity of the rotenone-insensitive NADH-cytochrome c reductase 
does not. The phospholipid content of the different subcellular and sub- 
mitochondrial fractions have also been examined with respect to the thyroid 
state. No significant changes are found in total phospholipid nor phospha- 
tidyl ethanolamine, phosphatidyl choline and diphosphatidyl glycerol in 
the different membrane fractions. From these findings, the following 
tentative conclusions are indicated: (1) Protein is incorporated in the 
inner membrane of mitochondria independently of a change in the outer membrane; 

(2) A specific protein can be incorporated into the inner membrane without 
simultaneously incorporating other protein constituents of the inner membrane; 

(3) The depletion and repletion of a specific protein of the inner membrane 
is accompanied by corresponding changes in the phospholipid composition; and 

(4) Changes in membrane composition occur by additions and subtractions from 
a pre-existing mosaic structure which remains functional throughout the 
perturbation. 

Significance to Bio-medical Research and the Program of the Institute: The ac- 
quisition of basic knowledge on the biochemical mechanisms regulating the 
intermediary metabolism of animals is of crucial importance in understanding 
the cellular processes fundamental to the function of physiological systems. 
This information is essential for the better understanding of the changes 
that occur in the chemistry of the body during development and aging. 



FL-28 



Proposed Course : Mechanisms of the regulation of cellular metabolism will 
remain an area of intense investigation. Particular emphasis will be given 
to the control of bioenergetic pathways in mitochondria. Work in this area 
is immediately relevant to the determination of the basis for the decrement 
in excitation-contraction-bioenergetic coupling that is found during the 
aging process. 

Honors and Awards: 

Dr. Sacktor was invited to lecture at a Gordon Conference Symposium on The 
Biochemistry of Aging, Santa Barbara, California. 

Dr. Sacktor was invited to present a lecture and lead a discussion on 
Cellular and Molecular Aspects of Aging: Mitochondrial Processes at 
Temple University, Philadelphia, Pennsylvania. 

Dr. Sacktor was invited to present a seminar at Ohio State University, 
Columbus , Ohio. 

Dr. Greenhouse was invited to present a seminar at The Johns Hopkins Univer- 
sity, Baltimore, Maryland. 

Dr. Sacktor was elected Fellow of the Gerontology Society. 

Publications : 

Bulos, B. , Shukla, S. , and Sacktor, B. : Bioenergetic properties of 
mitochondria from flight muscle of aging blowflies. Arch . Biochem. Biophys . 
149: 461-469, 1972. 

Bulos, B. , Shukla, S. , and Sacktor, B. : Effect of thyroid hormone on 
respiratory control of liver mitochondria from adult and senescent rats. 
Arch . Biochem . Biophys . 151: 387-390, 1972. 

Bulos, B. , Shukla, S. , and Sacktor, B. : The rate of induction of the 
mitochondrial a-glycerolphosphate dehydrogenase by thyroid hormone in adult 
and senescent rats. Mech . Ageing Develop . 1: 227-231, 1972. 

Carafoli, E. , and Sacktor, B. : The effects of ruthenium red on reactions 
of blowfly flight muscle mitochondria with calcium. Biochem . Biophys . Res . 
Commun. 49: 1498-1503, 1972. 

O'Brien, S. J.: On estimating functional gene number in eukaryotes. 
Nature New Biol . 242: 52-54, 1973. 

Sacktor, B. : The bioenergetic properties and ultras true ture of mitochondria 
from flight muscle of aging blowflies. In Symposia Reports , 9th International 
Congress of Gerontology, Kiev, USSR. 1972, Vol. 2, pp. 84-86. 

Sacktor, B. : Biological oxidations and energetics in insect mitochondria. 
In Physiology of Insecta . In press. 



FL-29 



been unequivocally demonstrated by equilibrium dialysis experiments At 
equxUbrxum, when ^^C-L-glucose, which is used as a marker'for non!;petific 
interactions as well as a measure of the included volume in the brusEborder 
preparations is equally distributed in the inside and outside of the 
dialysis membrane 3H.D.gi^eose is concentrated many fold with the brush 
borders. The tubular cell luminal membrane has the capacity to bind "glucose 
100-times that obtained previously by short term Millipore filtration pro- 
cedures, and values of 5 nmoles per mg membrane protein are obtained 

ItTul" T I ?' '*:^ ''^''^^"^ '^^^^ characterized its time course, satur- 
ability (Km = 3-4 uM) , reversibility, inhibition by phlorizin, and have 
determined the effects of Na+ as well as Ca2+ and Mg2+. 

Brush border membranes extracted with U Triton X-100 (approximately 16 mg 

^^ Tn.^nnn"'''^ i\ ^° ""^^ "' ^°° ""^<^^^" ^''""^ 9°^° °' their protein, but 
the 105,000 X g pellet has the same affinity and capacity to bind glucose as 
has the intact membrane. Electron micrographs of the extracted membrane 
reveal complete disruption with no evidence of vesicular structure. This 
further distinguishes binding of glucose from uptake of the sugar into mem- 
brane vesicles. 

Further investigations of the low affinity interactions of D-glucose with 
the brush border membranes indicate that the system is characterized best as 
being consistent with the hypothesis for a carrier mediated transport. Both 
the rates of influx and efflux are temperature dependent and the time courses 
at the different temperatures have been established. At 20°, the initial 
rate of D-glucose uptake increases essentially linearly with 'increasing 
concentrations of the sugar from 0.8 pM to the tiM range, with progressive 
departure from linearity as the concentration exceeds 10 mM. However, the 
uptake as a function of concentration does not plateau at concentrations as 
high as 100 mM. Phlorizin significantly inhibits D-glucose efflux as well 
as influx, suggesting a mediated transport in and out of vesicles rather 
than simple binding where phlorizin would be expected to displace glucose 
and augment efflux. Prior loading of the brush border membranes with 50 mM 
D-glucose causes a 407o stimulation of the initial uptake of 25 iiiM D-glucose 
as compared to loading with 50 mM L-glucose or mannitol. Again, this effect, 
so-called countertransport or facilitated exchange diffusion, supports a 
transport phenomenon since in the case of binding the prior loading would be 
expected to inhibit subsequent uptake. 

There is no significant difference between the uptake of 1 itM D-glucose by 
brush borders prepared and incubated in Na+ or choline media. However, using 
membranes prepared in 130 niM choline, the uptake is 50% greater when the 
incubation medium contains 65 mM choline + 65 mM Na+, as compared to 130 mM 
choline or 65 mM choline + 65 mM Li"^. On the other hand, the uptake of 
D-glucose from a 65 mM choline + 65 mM Na"*" medium is 507,, greater if the 
preparation medium was 130 mM choline rather than 130 mM Na"*". These findings 
Indicate that although Na"*", per se , is not required for transport of D-glucose 
by the brush border membranes, the rate of glucose transport by the membranes 
is greatly enhanced by a Na+ gradient from the outside to the inside of the 
membrane. 



Fb31 



The bindings of D-galactose and several non-metabolizable sugars to isolated 
renal tubule luminal membranes have been estimated and the mutual interactions 
of these sugars and D-glucose have been examined. The order of binding of 
the various sugars is D-glucose X)!-methyl-D-glucose >D-galactose >2-deoxy-D- 
glucose >x>;-niethyl-6-deoxy-D-galactose. The binding of 2-deoxy-D-glucose is 
Na"*"- Independent, and sensitive to phbrizin and sulfhydryl reagents. Binding 
of a-methyl-D-glucose has a Na"''-dependent component. The apparent Km for 
D-galactose binding is similar to that for D-glucose but the capacity for 
the binding of D-galactose is only 257o that of D-glucose. D-galactose and 
D-glucose mutually inhibit, competitively, the binding of the other. These 
results suggest that D-glucose and D-galactose have common or closely 
associated binding sites on the renal brush border membrane. The inhibitions 
of the high affinity binding of D-glucose by the non-metabolizable sugars 
are competitive and the apparent Ki values have been estimated. These 
findings on the bindings of various sugars to the isolated luminal membrane 
and their interactions with D-glucose at the membrane relate closely with 
other results on the uptakes of these sugars by renal cortical slices and 
by the intact kidney, in vivo . This strongly supports the view that studies 
of the interactions of sugars with isolated membranes are valid models for 
studying the mechanisms of active transport in the renal tubule. 

Significance to Bio-medical Research and the Program of the Institute : 
This study has established a useful model to examine the molecular basis 
for the transport of substances across cell membranes. Physiological sys- 
tems, such as renal, cardiac, neural activity and p-cell secretion, are 
intimately dependent on transport of ions and metabolites across plasma and 
intracellular membranes. These systems show marked functional changes with 
age. The paucity of information on the biochemical mechanisms for the trans- 
port of vital cell constituents frequently precludes a full understanding 
of normal physiological activities as well as those showing age-related 
changes. These investigations are designed to provide this needed information. 

Proposed Course ; Studies will continue to define the molecular basis of the 
transport of substances across cell membranes. The interrelationship and 
physiological roles of two processes, the binding of glucose to the isolated 
membranes and the carrier mediated transport of the sugar across the membrane 
remains to be clarified. The mechanisms of the transport of ions, especially 
Na^, Ca and phosphate, and of amino acids, as well as the interactions of 
the transport of these substances with that of sugars is of immediate concern. 
The isolated renal tubule luminal membrane is also used as a prototype for 
other membrane systems, £•£.. , the plasma membrane of the pancreatic islet 
cell. A study of the mechanisms whereby transport of sugars induces the 
secretion of insulin is planned. As fundamental information becomes available, 
this knowledge will be applied to the understanding of the functional changes 
that occur during the aging process. 

Honors and Awards: 

Dr. Sacktor was invited to present a lecture on the Interaction of Sugars 
with the Renal Membrane" to the Philadelphia Physiological Society sponsored 
A. N. Richards Symposium on "Recent Advances in Renal Physiology and 
Pharmacology" at the Jefferson Medical College, Philadelphia, Pa. 



FIt32 



Publications; 



.i„^o '• ^^""^ ^^' function of trehalase in the active transport of 
glucose m the n,anm,alian kidney. In Piras, R. . and Pontis, H. G (Eds ) • 
Biochemistry of the Olycosidic Linkaee. PAABS Symposium, 'san c;rlos de' 
Bariloche, Argentina. 1972, Vol. 2, pp. 281-289. ^arios de 

Chesney, Russell, Sacktor, Bertram, and Rowen, Robert: The binding of D- 
glucose to the isolated luminal membrane of the renal proximal tubule. 
J. Biol . Chem . 248: 2182-2191, 1973. 

Chesney Russell, Sacktor, Bertram, and Kleinzeller, Arnost: The binding 
of phlorxdzin to the isolated luminal membrane of the renal proximal tubule 
lit K k"k^T''°"\°^ Phloridzin and D-glucose at the receptor sites on 
the brush border membrane. J. Biol. Chem. , in press. 

Chesney, Russell, and Sacktor, Bertram: Transport of sugar by isolated 
luminal membranes of the renal proximal tubule: binding of galactose and 
other sugars and their interactions with glucose. J. BioL Chem. in press 



Ffc33 



Serial No. HD-GMA4(2) 

1. Gerontology Research Center 

2. Laboratory of Molelular Aging 

3. Intermediary Metabolism Section 

4. Baltimore, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Control of RNA Synthesis in Senescent, Developing, and 
Neoplastic Systems 

Previous Serial No. : Same 

Principal Investigators: David Gillespie 

Project Description: 

This project has been discontinued. 

Publications : 

Gillespie, D. , Marshall, S. , and Gallo. , R. : RNA of RNA Tumour viruses 
contains Poly A. Nature New Biol . 236: 227-231, 1972. 

Marshall, S. , and Gillespie, D. : New rif ampin-resistant mutant of E. 
coll . J. Bacteriology 110: 782-783, 1972. 

Marshall, S. , and Gillespie, D. : On the absence of poly U tracts from RNA 
isolated from RNA -containing viruses. Nature New Biol . 240: 43-45, 1972. 

Patterson, D. , and Gillespie, D. : A temperature-sensitive mutant of E. 
coli defective in the chain elongation step of protein synthesis. Biochem . 
Genetics 8: 205-230, 1973. 

Patterson, D. , and Gillespie, D. : The effect of elevated temperatures on 
protein synthesis in E. coli. J, Bacteriology 12: 1177-1183, 1972. 

Patterson, D. , Gillespie, S. , and Gillespie, D. : Measurement of bacterial 
message RNA by RNA-DNA hybridization in formamide. Biochem . Genetics . 
In press. 



FIr34 



Serial No. HD-GMA 8 

1. Gerontology Research Center 

2. Laboratory of Molecular Aging 

3. Section on Intermediary Metabolism 

4. Baltimore, Maryland 

PHS-NIH 
Individual Project Report 
July 1, 1972 through June 30, 1973 

Project Title: Investigations on the Biochemical Mechanisms of Hormonal 
Regulation of Metabolism 

Previous Serial Number: None 

Principal Investigators: Bertram Sacktor 

Paul Insel 

Man Years: 

Total: 2.5 
Professional: 1.2 
Others: 1.3 

Project Description: 

Objectives - The broad objectives of this project are: (1) to understand the 
mechanisms by which metabolic processes are regulated by the action of hor- 
mones at the cellular and biochemical levels, and (2) to apply this fundamental 
knowledge to the understanding of the mechanisms whereby physiological control 
systems are altered during the aging process. Specifically, this new project 
has sought- (1) to localize in the renal cortex hormonally stimulated adenyl 
cyclase (2) to study the binding of cyclic AMP to cortical membranes, (3) 
to identify the endogenous membrane -bound cyclic AMP stimulated protein 
kinase (4) to identify and characterize the presumed phosphorylated enzyme, 
and (5) to determine the mechanisms by which the hormonally stimulated 
sequence of reactions regulate renal function. 

Metho ds Employed : The primary preparation used in these studies is the 
plasma membrane of the renal cortical proximal tubule. As appropriate, 
modern biochemical procedures required to answer the questions posed are 
applied, modified, or developed. 

Major Findings: An assay for adenyl cyclase, representing a modification 

rf^heKrishL technique, has been established for rabbit "-/ P"P-^^^°^=- 

Kinetic parameters, estimations of recovery, identification of product, 

stability of enzym;, and appropriate concentrations of activators and in- 

hibitors^re now known for this preparation Interestingly renal adenyl 

cyclase differs from the hepatic enzyme in its msensitivity to GTP. Para 

, , / ^u^no 1 ^i^ at- 10 ue/ml and catecholamines, most 
thyroid hormone (synthetic 1-34), at iu jig/mi, auu a , 

■, . ^ „^i «*- n 1 mM pffpct as much as 400/„ and 14U/o 

HTZTA'Z T^Zr^^X^O^- The sti™U.io„s ,.e no. ..di.lve 

Flr35 



with respect to F stinrulation. Luminal membrane preparations from proximal 
tubules contain measurable activity, although the specific activity of the 
enzyme in this membrane relative to established enzyme markers is not 
enhanced. 

Brush border membranes bind significant amounts of cyclic AMP, as determined 
by Millipore filtration and equilibrium dialysis procedures. The specific 
activity of the binding capacity is greatly enhanced in this isolated membrane 
preparation. Kinetic parameters for the binding have been partially 
established. 

Significance to Bio-medical Research and the Program of the Institute : 
Knowledge of the biochemical mechanisms by which cellular processes are 
controlled by hormone action is of fundamental importance in understanding 
how homeostatic physiological systems operate. This information is necessary 
to understand the mechanisms by which metabolic control systems may be 
deleteriously changed during aging. 

Proposed Course : It is planned to continue the elucidation of the mechanisms 
of hormonally-related processes in the renal tubule, as outlined in the 
Objective section. 

Honors and Awards: None 

Publications: None 



\^ 



2363 



1 



I 







DATE DUE 






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