ANALYSIS OF PR0(;RAM AaiVITlES NATIONAI. INSTIlirrES OF HEALTH

1956

NATIONAL INSTITUTE OE DEiNTAL RESL\RCH - CUNICAL CENTER

NATIONAL INSTITUTES OF HEALTH PUBLIC HEALTH SERVICE U. S. DEPARTMEiNT OF HEALTH, EDUUTION, AND WELFARE

ionai institui^-

U,S NATION,'iL INSTITUTES OF DENTAL RESL'.RCH SUI'M'VRY OF RESR'.RCH ACTIVITY

January - December 1956

In the are?, of cral bacteriology, the lu-.jor program goals arc (a) to conriuct systematic studies on individual types of oral micro- organisms, (b) to study host-parasite relationships of tlie oral nicrobic flor? in humans and experimental animals, (c) to further ex- plore the relationships of systemic and local oral conditions to dental disease, and (d) to evaluate the effectiveness of various preventive and theraneutic agentj and procedures for control of dental disease. Although steady progress has been made in these fields of study, as evidenced by such accomplishments as the determination of gror-jth re- quirements and unzymatic activities of many heretofore poorly described oral microorganisms, it should be emphasized tlxat the experimental production of dental caries and periodontal disease continues to pose new factors of etiologic significance. Studios planned for the future which should contribute much to our kna^jlcdge of these diseases include latent virus infections of the mouth, and observations on "germ-free" animals using nutritionally defined diets and knc-jn typos of organisms.

In the area of oral and biological chemistry, a number cf signi- ficant contributions may be cited. For -xample, the role of dietrjry protein in experimental caries was fijrther substantiated by evidence that procussed wheat cereal diets are highly cariogenic and that- L-lysine has anticaries properties in a purified lysine deficient zein- base diet, ^'inother important achievement was the isolation of a new pancreatic enzyme named basic carboxypeptidase, which miiy be re- sponsible for the rapid availability of lysine and arginine from digested nrotein. i.^. further observation vjas the role of lysine in pro- moting the d-,position of calcium in the growing ends of long bones. This 'is of significance in that it supports the evidence of a relation of protein to calcium meta.bolism.

Improved analytical technics have also benefited the study of ion- exchange behavior of C-'-^ labeled amino acids as well as the effect of C ^ on a cation-exchange column. By further study of single cell lines grovm in tissue culture, it has be^^n shoi-.m that tjTosinc, phenylalanine and £lut"mine arc all used directly for .protein synthesis.

In the category of periodont,al disease, oral soft tissues of v:hite rats have been subjectf,d to a newly devised technic for creating a con- trolled and reproducible inflammation reaction. This procedure has permitted a detailed study of chemical and cnzymological changes as -i^ll as a more definitive histopathological picture of inflammation.

Recognizing the role that saliva might play in the etiology of oral diseases, pure parotid secretions have been under analysis for thoir amino acid and lactic acid content, as well as for electro- phoretic patterns.

Continuing to present evidence of the safety of water fluorida- tion, analysis of skeletal tissues of humans exposed to 6 ppm fluorine for prolonged periods, revealed a maximum content of 0.653% fluoride. Although this is ap-nroximately ten times above noraial, no deleterious effects vjcre noted. Another study concerned with urinary elimination of fluorine showed ttiat similarly efficient excretion, rates exist in artificially and naturally fluoridated communities..

In the field of histology and pathology, long-range objectives include the development of fundamental informatj.on concerning the morphological, physical, and histocheraical characteristics of the normal oral hard and soft tissues, and the application of this ac- cumulated data in studies of the causative factors and pathological processes involved in oral disease. Morphological studies of the ultra-structure of dental tissues as viewed by the electron microscope are continuing to contribute new knoij ledge fundamental to our under- st^inding of oral disease. Other work, in which both electron micro- scopy and electron diffraction are employed, concerns the effects of various chemical agents, such as fluoride compounds, on the structure and properties of the outer enamel surfaces of teeth. Particularly illustrative of file important gains made by fhe collaborative work with the Section of Molecular Biophysics of the 'National Institute of Arthritis and Metabolic Diseases is the exploration of applications of the newly developed projection X-ray microscope to the examination of hard and soft tissues.

In the field of histochemistry, studies are being extended in which protein constituents and enzymatic activities in various tissues are characterized chemically and observed iijith the optical microscope. Emphasis in "these studies is placed on determining and comparing the localization of proteolytic activities in normal and diseased tissues inclu.ding salivary glands, mucous membranes and tongue. In the use of this approach, it is necessary to develop new and advanced chemical procedures for revealing hitherto unexplored cellular components. Other continuing investigations concern the histochemical properties of connective tissue in general, with special attention focused upon normal and diseased supporting tissues of the teeth.

As a part of continuing studies of dental caries at both the microscopic and gross levels, animal experimentation is being pursued in an effort to standardize a regime for the initiation of uniform and reproducible lesions in hamsters. It is believed that this study will facilitate future restjarch on the etiological and pathlogical factors in dental caries.

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In the- field of epidcmiologj^ and biometry, the broad goals in- cludu an a mnlifi cation of the descriptive and detcr:ninativ(.i epidemi- ology of oral disease . It is significant that a study of dental caries by this approach developed the fluoride-caries relationship, as well as established the safety and practicability of fluoridation of domestic vjater supplies. Further work is in progress to describe more fully the sequence of caries inhibition and, thereby, establish hypoth- eses on the mechanics of the fluoride effect.

Recognizing .that a majority of adults arc victims of periodontal disease and that. this condition is a most important cause of tooth loss in middle and later life, emphasis is currently being placed on this category of oral 'lisease. Following the development and testing of a field mc;thod for mensuration of the periodontal diseases in pop- ulations, the broad foum.lations of a descriptive epidemiology have been laid. Patterns of prevalence relr, ted to age, sex, race, and social status have been und^r study, as well as an analysis of the ctiologic role of v rious chrond.c systemic illnesses.

In the course of establishing the prevaL.nce of certain hereditary defects of the enamel and. dentine, a group of "racial isoliite" people in Southern Maryland were selected for study. This group of inter- related people, consisting of some 5,000 persons, has been under study for the past year. Preliminary surveys to datj have shown that not only as the prevalence of hereditary dental defects quite high, but also an unusual prevalence of other genetically-determined diseases exists. These include albinism, deaf -mutism, and mental deficiency. It is hoped th''t .information will be gained not orly regarding the cellular level in tooth development, where these genetic defects are initiated, but also of the interrelationship of the v;irious types of defects.

During 1958 the program of MIDR will continue to include a wide range of research activities, "

Work in electron microscopy will be extended to include more complete examination of tissues involved by caries and periodontal disease (pyorrhea). Also, nev; histochemical methods for studies of protein and enzyme components of soft tissues, tooth and bone will be explored and developed, and applications made of these histochemical methods in the fiuld of diagnostic surgical pathology. It is further planned to emphasize the investigations of periodontal disease. In addition to the currently omoloyed pathological approach, using principally diseased human material collected at iDlopsy or post-mortem, studios on experimentally- induced disease in ajiimals will be empha- sized.

Work on experimental caries will also be continued to the end of exploring and evaluating various potential agents for control' of this disease. In the area of epidemiology, emphasis will be placed on the

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relationship of systemic illness, aging and socio-eoonorrac factors to periodontal disease. These studios will be expanded to utilize patient material from other public health service hospitals as well as the Clinical Center, NIH, Another area of increased activity will be the genetic study in a population group of racially isolated indivi- duals for whom exhaustive and accurate pedigrees and histories are being obtained. '• "

Expanding studies in oral biochemistry, namely, the chemical com- position of saliva and the metabolism and function of salivary glands as secreting cells, should provide more basic knowledge of those diseases affecting both the calcified and the soft tissues of the mouth. Also to be continued will be studies on the relation of dietary protein to dental diseases, major emphasis being placed on the relation of such proteins to calcification of bones and teeth. Other projected investigations, which may shed additional light on the mechanism of dental decay, will include an evaluation of the chelating agents which possess the ^property of decalcification in a non-acid Fedium. Studies on the relation of protein chemistry of the cells involved in the tooth formation process will be continued. In the field of oral bacteriology, the studies to develop more effective mi ans for minimizing the danger of bacteremia and endocarditis, subsequent to oral surgical procedures in rheumatic fever patients, will be emphasized,-

CLINICAL INVESTIG.\TIONS

The Clinical Inv^jstigations Branch of NIDR has a major ro- "Sponsibility for encoura' ing the clinical apnlication of basic lab- oratory findings as they might relate to cause, diagnosis, treatment and prevention of oral disease. By such coordination of clinical and basic research, an ever-increasing benefit may be contributed to the dental health needs of tlie nation. With this realization of a broadened scope of responsibility for the future, a variety of major fields of clinical study will be errphasizcd. For examjile, studies of periodontal disease (pyorrhea) will include an evaluation of the efficacy of standard methods of treatment. Coupled with continued studies clarifying the relationship of systemic disease to pyorrhea, much progress is promised for -combating this difficult public -health -problem. In a similar manner,' studies of dental caries will continue to be directed toward a better understanding of the types of microorganisms involved, their enzymatic activities, the bioehemical role of saliva in the disease process, and the relationship of caries to the organ system as a whole. Other studies will continue- to be related to such fields as the physio- logic response of ambulatory patients to gerieral anesthesia; histo- cheraioal reactions of oral tissues in health and disease with particular attention to the salivary glandsj and prosthetic reconstruction for maxillo-facial defects with formulation of principles of design that might contribute to improved function of speech as well as the teeth and jaws.

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In the important area of growth and development, studies of normal and abnormal patterns of cranial and facial 'provrth will con- tinue to be made by special cephalometric and laminographic tochnics. Particular emphasis in these studies will be placed on young children with congenital and acquired deformities.

Relative to some current problems in practical dentistry, the biologic effects of certain ins trtimentation procedures (high speed drilling and ultrasonic cutting) are being evaluated on both the dental pulps and si:irroundi.ng tissues.

EXTRA MUR.;L mOGRAM

The increased Conf rcssional appropriations (FY-1957) d-Liririg 1956 allovred for the first time in the history of NIDR the development of a well-rounded and integrated Extramural Progrc?m consisting of Research Grants, Fellowships, and Training Grants,

Beginning in ?lay, 1956, steps were taken to develop "Research Training Centers" using a block of funds allocated from research grants. However, in July when specific training grant funds became available, the "Centers" idea was modified into a full-scale graduate training program. Project site visits were m^^de to all dental schools that evoked an interest in having a training rrogrnm. As a result, applications have been received totalling in tneir requests far in ex- cess of the funds available. Additional schools have indicated they plan to send in applications early in 1957. In order to develop the overall training program and to initiate training in as many institu- tions as possible where such programs might well be carried on, the NADRC found it advisable to reduce drastically the budgets requested. It is hoped that future appropriations will allow for supplemental re- quests so that funds will be adequate in terms of the training program which a given institution is competent to handle. Almost all schools submitting a graduate training program proposal requested a yearly increment for future years of support. The K..DRC, faced with the problem- of over -commitment for future support, decided at its October meeting to mainta.in future year moral commitments to tj-aining grants at the first year level. This procedure has not been favorably received by grantee institutions, inasmuch as a well-devised training program should bo allowed to increase gradually to its maximum potential over a period of years. It is felt that the potentialities in the graduate training area in dental medicine have been barely touched and that additional support for development of this program is imperative. Aost grants have been made to institutions where some training has been already underway and nersonnel, research facilities, and trainees arc readily available. On tiie other hand, efforts also have been made to initiate training programs on a small scale in those institutions where the need is obvious for more rese.arch- trained personnel. Op-

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portunities for future development along these Irenes are limited only by the availability of funds.

The availability of funds for fellowships allowed for an increase of the part-time dental students fellowships per institution from h to 6. This program has been highly praised by numerous dental schools as one of the most worthvjhile programs- available in terms of initiating undergraduate students to research. It is hoped to raise the number per institution to 8 in the near future.

A broader usage of the Special Research Fellowship was introduced during the last half of 1956 in order to supply adequate stipends for holders of a dental doctoral degree who desire to obtain specialized research training, and who will use such training because of tlrioir association vrith a dental school. It is already indicated that by this means more individuals are being encouraged toward development of an interest in research than ordinarily would be the case. Finally, all dental schools have been individually informed as to the details of each type of fellowship available, and where appropriate, they have been encouraged to integrate- and supplement "their training program by means of fellowships.

The response of dental schools to the Senior Research Fellowship program was somewhat disappointing] however, steps have been taken to have dental schools use this special type of fellowship to build up their basic science disciplines. The next deadline date of September 195? is certain to bring forth more worthwhile applications from dental schools for tiie Senior Rcsea.rch Fellowships.

The impetus given to dental research by the seven-fold, increase in Congressional appropriations for Research Grants is adequately attested to by the influx of applications between July 1 and Docaaber 1, 1956, for review by the March 1957 Council, These applications now total over 3 million dollars with only 1.3 million available as of December 31, 1956, to pay these requests. Based on the iiO - 50 percent approval rate expected, the FY 1957 appropriation is expected to fall short of the needs.

Consideration was :,iven late in 1956 to the development of program grants in the field of oral pathology in order to supply the needs in this special area. . Several dental schools have evinced an interest in such a orogram. Applications for these grants should be submitted in time for review by the i^hrch 1957 Council under special study section assignment.

Future plans call' for program type grants to schools now conducting little or no research. By means of such grants it is hoped that research will be stimulated and developed in those institutions where only a sma.ll amount of research is now being done.

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At the October nc,jting of the ADA a program sponsored by the ADA Council on Dcnt-^.l Research effectively presented a panel discussion of the NIDR Extraiaural Programs.

Form Mo. ORP-1 - 1 - Calendar Year 1956

October 1956

PUBLIC HEALTH SERVICE - - NATIONAL H-JSTITUTES OF HEALTH INDIVIDUAL PROJECT REPO^^T Part A, Project Description Sheet 1. NIDW - 1

SERIAL NW'ffiER

2. Dental Research 3. Oral and BiolOjg;ica].' Chemistry

TNSTlfUTfi Or DIVISION LABORATORY, BPJINCH, OR DEPARTMENT

U. -- 5.

SECTION OR SERVICE [ LOCATION: (IF OTHER THAN BETHESDA)

6. Nutrition and Dental Caries ^ith Enrphas-is on Protein and Heat

^ II I - " ■■ fc ^ I H II ■■■■! ■■- I I -. - ^ ■■■!■ M I ■■ I i M

Processed Foods

PROJECT TITLE

7. F. J. McClure '

PRINCIPAL INVESTIGATOR .

8, J. E. Folk:"" and A.Bavetjta (Visiting Scientist)

OTiMTN '.^S T lOATOP^

9. IF THIS PR.OJECT PESEIiBLES, COWLEMENTS, OR PARALLELS RESEARCH DONE ELSEIV!#RE IN. THE PUBLIC, rHEALTH SERVICE (VflTHOTJT INTERCHANGE OF PER- SON?-!EL, ^ACI-LTTIES.ORFTJNDS), IDENTIFY' SUCH RESEARCH:

M6ri#'- '■■■■■ ' ■; ■'■ '■ ■' ■' '■■' ■, -■ '-■I 10. PROJECT DESCRIPTION:

■■s - ' ./i? t. ; ti

. ' ' This ■''prof?ram is. directed toward obtaining information on the ■■■■■''■"'•■•' relfition of experimental caries to' protein losses of milk

powders and cereal foo.^s caused by excessive heat processing. Results of the past year confirm the role of lysine in ■'• preventing caries- induced by special heat processed skim milk ."■::''■'■■ powder, diets . L^ysine given b;jr water, stomach tube and food, ' ' '_ ^.' si'f^bifioantly reduced caries. ""It appears, however, that •'■-■' •■ L-lyBine ■Jivan -by intraperitoneal injection is probably not ''■■'^'••' effective,. .which permits some speculation as to how L-lysine '■'■■;"' "■ ' ^cts to '-reduce caries, in this particular diet. Special

">*■ Research Associate, American Dental .Association, at the National Institute of Dental Research.' •■ ■•

Form No. ORP-1 - 2 - Calendar Year 1956

October 1956

10. PROJECT DESCRIPTION:

attention has been paid to diets containing a large percent- age of wheat cereal protein, i.e. diets containing (a) native raw wheat flour, (b) conmercial whole wheat flour, (c) whole wheat flour autoclaved with lactose and (d) shredded wheat biscuit. Severe smooth surface caries results from these inadequate diets. Supplements ' of calcium, phosphorus and L-lysine have had pronounced inhibitory effects on the cariogenic property of these diets. A role of post- eruptive minerals in the etiology of caries is suggested and . ,. bears investigating. Further studies are attempting to relate heat processing of cereal foods to dental caries.

In cooperation with Dr. L. AvBavetta (Visiting Scientist), i it was shown that irradiation of a spray process skim milk

powder, did not alter its cariogenic potential. Experiments supervised by Dr. Bavetta demonstrated that synthetic diets which contained 1J% casein, particularly after autoclaving , , . the casein with lactose. Were highly cariogenic. V/ith 2k% -■.v.r", •■7 casein very little caries was produced, emphasizing the fact that the quantity of protein poses an important considera- tion in these diets. Addition of 11^ casein to a skim milk pov;der diet also i*educed caries , again pointing to the - ;■• . significance of the quantity of protein.

In cooperation with Dr-. Bavetta, it was shown that the B- . vitamins added in above optimum quantities, did not affect caries produced by Diet 636 which contained autoclaved

skim milk powder.

Two significant experiments (completed in cooperation with Dr. Bavetta) gave further evidence of a ^possible role of L-

lysine in caries etiology. Synthetic diets containing Zein

(lysine free) as a source of protein were distinctly cario- genic. Addition of 1,00 L-lysii;ie,; however y had a significant anti-caries effect, 'T^iese are our first successful studies pertaining to L-lysine as a supplement in "synthetic" lysine- def icient diets .

The accumulated evidence' of 'the past year in retrospect has advanced the possible role of quality of. protein (L-lysine), as well as quantity of pi'otein (13% vs. 2[i^ casein), as cariogenic factors. It has injected the possible inter- relation of minerals and protein, especially through informa- tion concernin-"': the cariogenic effects of inadequate cereal protein diets, particularly in' the presence of an inadequacy or an imbalance of calcium and phosphorus ,

V.ri-MT

Form No. OPP-1 October 1956

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Calendar Year 1956

PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH INDIVIDUAL PROJECT REPORT

Part B: Budget Data

11. NIDR - 1

SERIAL NUIfflER

12. BUDGET DATA:

ESTIi'lATED OBLIGATIONS

MN YEARS

DIP.ECT REIMBUR.SEMENT TOTAL

PROF

OTHER

TOTAL

31,205 3,6lii 3U,819

2

2 2/3

h 2/3

FY '57

BUDGETED POSITIONS

PATIENT DAYS

PP.OF OTHER 5 2

TOTAL 7

■"

FY'57

13. BUDGET ACTIVITY;

RESEARCH /TJ

REVIEVT & APPROVAL / 7

BIOLOGIC STAND/iRDS / 7

ADMINISTRATION

r~/

PROFESSIONAL &

TECHNICAL ASSIST- ANCE / 7

lit. IDENTIFY ANY COOPERATING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSONTffiL FOR THIS PROJECT IN FY 1957. IF COOPERATING UNIT IS WITHIN NIH INDICATE SERIAL NO(S):

Dr. Howard Andrews, Radiation Branch, National Cancer Institute.

Form No. ORP-1 - h - Calendar Year 1956

October 1956

PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH INDIVIDUAL PROJECT REPORT

Part C: Honors, Awards & Publications 1$ . NIDR - 1

SERIAL NUlviBER

16. LIST PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS PROJECT DURING CALENDAR YEAR 1956:

McClure, F. J., Folk, J. E. and Rust, J. D. Smooth Surface Caries in V/hite Rats. Effect of Fluoride, lodoacetate, Penicillin, Crisco, Butterfat and a Salt Mixture. J. Am. Dental Assoc. 53:1-5, 1956.

17. LIST HONORS AND AWARDS TO PERSONNEL RELATING TO THIS PROJECT DURING CALENDAR YEAR 1956:

None

Form Mo. ORP-1 - 1 - " Calendar Year 1956

October 1956

PUBLIC HEALTH SER'TICE - - NATIONAL INSTITUTES OF HEALTH INDIVIDUAL PROJECT REP07^T Part A. Project Description Sheet 1, NIDR - 2

SERIAL NUMBER

2. Dental Research 3. Oral and Biological Chemistry

INSTITUTE OR DIVISION LABORATORY, BRANCH, OR DEPARTIvIENT

U. - 5. - . -

SECTION OR SERVICE LOCATION (IF OTHER THAN BETHSSDA)

6. Biochemistry of Cra" Soft Tissue

PROJECT TITLE

"^ * Bernard Kt Forscher

PRINCIPAL IN^/ESflGM OT

8. H. R. Stanley,' Jr.

OTHER INVESTIGATORS

9. IF THIS PiiOJECT RESHvIBLES, COJ£PLEIffiNTS , OR PARALLELS RESEARCH DONE ELSEIWEPE IN THE PU8LIC HEALTH SERVICE (WIT'IOUT IPTTERCHANGE OF PER- SON NEL, FACILITIES Or^, FUNDS), IDENTIFY SUCH RESEARCH:

None

10. PROJECT DESCRIPTION:

Objectives;

The objectives of this project are to elucidate the pattern of biochemical changes which characterize a classic inflammatory response in oral soft tissue and to determine how this pattern is nodified by various systemic conditions.

Kethods Sn'ployed;

An inflammatory reaction is generated in the palate of the white rat by stimulation with a controlled pulse of radio- frequency current. Tissue samples are taken before and at

Form No . ORP-1 - 2 - Calendar 'Year I956

October 19^6

10. Methods Employed (Continued):

standard intervals after such treatment. These samples are subjected to histopatholoaical study and chemical analysis. This procedure is carried out on normal rats and on rats previously subjected to some specific systemic condition..

Major Findings; ^ ■;;

Curves describing the concentration changes of a number of '■'■ tissue components during an acute inflammatory response have

been developed. These components are: total tissue water, glycogen, hexosamine, tyrosine, proline, hydroxj^roline, inorganic phcspliate, acid-soluble phosphorus, residue phos- phorus, total nitrogen, non-protein nitrogen, ribonucleic acid and deoxyribonucleic acid.

The method of inducing inflammatory change has been found to be consistently reproducible.

The effect of prolonged oral administration of Dilantin on the acute inflammatory response was a marked change in the concen- tration of the acid-soluble phosphorus fraction.

Effects of alloxan diabetes in rats were studied anci it was found that acutely inflamed palatal tissue showed a signifi- cantly altered glycogen curve.

Significance to Denbal Research;

■Periodontal diseas.es involving the supporting structures of the teeth cause almost half the tooth loss in this . country. This project represents a fundamental approach to etiology and is based on long standing clinical observations that certain systemic conditions predispose subjects to periodontal disease, Compiarisons between the biochemical pattern of inflammatory response in normal tissue and in s"ystemically predisposed tissue may lead to elucidation of the factors that control resistance and susceptibility to inflammation, and, by extension, to periodontal disease. The animal tissue used in these studies is analagous, to' human gingival tissue in many respects, thus allowing application of these findings to later studies on human material.

Proposed Course of Project ;

Studies on the effects of diabetes will continue. Arrangements

Form Mo. ORP-1 - 3 - Calendar Year 1956

October 19^6

10. Proposed Course of Project (Continued):

have been completed for initiation of studies on the effects of age on the inflammatory response. Other systemic factors to be studied in the immediate future include the effects of splenectomy as well as pretreatment v;ith ACTH and with cortisone. Further studies will be made on samples of human tissue from patients on Dilantin therapy in order to verify findings on animal tissues. Studies also will continue in an effort to produce a standard experimental chronic inflammation.

Form No. ORP-1 October 1956

- h -

Calendar Year 1956

i; '•;

PUBLIC HEALTH SERVICE - - NATIONAL H^ISTITUTES OF HEALTH BTDIVIDUAL PROJECT REPORT

Part P; Budget Data

11. NIDR - ?

SERIAL NUMBER

12. BUDGET DATA:

ESTII.iATED OBLIGATIONS

MN YEARS

FY'57

DIRECT REBifBURSE'ffiNT TOTAL

PROF

OTHER

TOTAL

1«,036 3,513 21,6i}9

1

2 l/U

3 1/U

FY'57

BUDGETED POSITIONS

PATIENT DAYS

PROF OTHER TOTAL 1 h 5

13. BUDGET ACTIVITY;

RESEARCH /TJ

REV im & APPROVAL / 7

BIOLOGIC STANDARDS /~7

ALMNISTRATION

n

PROFESSIONAL &

TECHNICAL ASSIST- ANCE / 7

li;. IDENTIFY ANY COOPEKJ^TING UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER ORGANIZATIONS, PROVIDING FUNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN FY 1957. IF COOPEPJLTING UNIT IS WITHIN NIH INDICATE SERIAL NO(S):

None

Form No. ORP-1 - 5 - Calendar Year 1956

October 1956

PUBLIC HE.1LTH SERVICE - - NATIONAL INSTITUTES OF HEALTH INDIVIDUAL PROJECT REPORT

Part C: Honcrs, Avrards & Publications 15. NIDR -2

SERIAL NUIIBER

16. LIST PUBLICATIONS OTHER THAN ABSTRACTS PTfOM THIS PROJECT DURING CALENDAR YEAR 1956:

Forscher, P. K, and Stanley, H. R., Jr. A New Experimental Method to Produce Acute Inflammation, Arch. Pathology, In Press.

17. LIST HONORS AND A'TARDS TO PERSONNEL RELATING TO THIS PROJECT DURING CALEmAR YEAR 1956:

None

Form Mo. ORP-1 - 1 - Calendar Year 1956

October 1956

PUBLIC HEALTH SER\n:CE - - NATIONAL INSTITUTES CF HEALTH INDIVIDUAL PROJECT REPORT Part A. Project Description Sheet NIDR - 3

SERIAL NUIvIBER

2. Dental Research 3. Oral and Biological Chemistry

INSTITUTE"OR DIVISION . LABORATORY, BRANCH, OR DEPARTi>fflNT

ii. - 5.

SECTION OR SERVICE LOCATION (IF OTHER TH/lN BETHESDA)

6 . Proteolytic Enzymes; Organic Chemistry and Kinetics PROJECT TITLE ~" ~

7. J. E. Folk "

PRINCIPAL INVESTIGATOR

8. J. A. Gladner, K. Lakl (NIAtAD Subject A)

OTHhR INVE.STTGaTOP.S

9. IF THIS PROJECT RESEIfflLES, COMTLEMENTS , OH PARALLELS RESEARCH DONE ELSElfflERE IN THE PUBLIC HEALTH SERVICE C'/ITHOUT INTERCHANGE OF PER- SONNEL, FACILITIES OR FUNDS), IDENTIFY SUCH RESEARCH:

None

10, PROJECT DESCRIPTION:

Objectives:

A. The isolation in pure form and the thorough characterization of a new carboxypeptidase ( carboxypeptidase B) first observed in this laboratory,

B. The preparation of synthetic low molecular weight peptide substrates for various proteases and peptidases and determina- tion of enzyme specificity by means of these compounds.

C. The comprehensive study of the effect of the glucose-araino acid reaclion on enzymatic digestion.

# Research Associ- te, American Dental Association, at the National Institute of Dental Research.

Form No. ORP-1 - 2 - Calendar Year 19^6

October 193'6

Methods Employed;

■■'• A. A good deal of time is being devoted to isolation of ■carboxypeptidase B. Standard methods of protein separation are being employed as well as uncommon procedures such as digestion of contaminating protein and fractional crystalliza- tion of certain interfering enzymes. One difficulty encoun- tered during separation is an apparently strong protein- protein interaction between classical carboxypeptidase and the new enzyme, which complicates satisfactory separation. How- ever electrophoretic patterns show that enormous amounts of impurities have been removed during the purification proce- dures .

Activity and percent-recovery experiments are being conducted employing both arginine and lysine substrates. Low molecular weight peptide derivatives are being prepared and tested to learn (1) if basic amino acids, other than lysine and arginine, are cleaved from the carboxyl terminal position, (2) what effect the adjacent amino acid has on the release of the basic ainino acids, (3) if the enzyme has esterase activity and (k) if the enzyme will split carbon-carbon bonds.

Kinetic studies, inhibitor studies and complete electro- phoretic and ultracentrifical pictures are being obtained,

B. Peptide derivatives of proline and hydroxyproline have been prepared by the carbobenzoxy masking method in conjunc- tion with the azide, chloride, mixed anhydride and carbodi- mide coupling methods and these are being tested as possible substrates for bacterial collogenase and as a searching tool for a possible mammalian collagenase or gelatinase.

C, Many studies employing paper chromatographic and paper electrophoretic methods for identification of products, have been conducted on peptide substrates for the major pancreatic enzymes. Methods of identifying products of sugar-peptide combinations before and after enzymatic hydrolysis have been devised.

Major Findings;

A. Carboxypeptidase-B has been identified as an individual hitherto unreported major proteolytic enzyme of bovine pancreas,- It has been shown to be an enzyme specific for the release of carboxyl terminal basic amino acids. It has been partially purified and preliminary studies have been made on its specificity, kinetics and inhibition.

Form No. ORP-1 - 3 - Calendar Year 19^6

October 19'^6

Major Findings (Continued):

B. A few of the proline and hydroxyproline peptide deriva- tives which have been prepared are: prolyl- 3-naphthylamide, hydroxyprolyl-(3-naphthylamide , CBZO-glycylprolylhydroxy- prolylglycine ethyl ester and CBZO-glycylprolylhydroxyprolyl- glycine amide. Testing of these compounds as substrates is under/fay at present,

C. Data have been accumulated which indicate that the decrease in nutritive value of proteins which have been heated, cooked, or stored with reducing sugars is primarily a result of their incomplete digestion by pancreatic enzyme.

Significa nee to Dental Research :

A and C. An understanding of the alterations in the release of lysine from protein before and after its reaction v.'ith sugar, is important in evaluating factors affecting the production and prevention of experimental smooth surface dental caries. Even more fundamental is an understanding of the mechanism of release of the basic amino acids from ingested protein. The specificity of action of carboxy- peptidase B at least partially explains this phenomena. Moreover, a practical tool for the determination of protein structure is foreseen in the isolation of carboxypeptidase B.

B. Thus far practically nothing is known of the specificity of bacterial or mammalian collagenases. Keeping in mind the possible Lnportance of collagenase in dental disease, it is essential to learn as much as possible concerning their mechanism of action.

Proposed Course of Project;

A. During the coming year a good deal of time will be devoted to comprehensive studies of carboxypeptidase B, Studies on its specificity must be extended to protein and high molecular weight polypeptides and its value as a tool for protein structural determination must be demonstrated,

B. Further attempts to classify the mode of action and specificity of collagenases are underway.

C. The original aims o^ this project have been satisfied, i.e. to demonstrate the fact that pancreatic digestion was signifi- cantly influenced by the sugar-lysine reaction. However an additional study of the effect of the sugar reaction of argi- nine and possibly histidine on pancreatic protein digestion may be made.

Form No. ORP-1 October 1956

- h-

Calendar Year 1956

PUBLIC HEALTH SERVICE NATIONAL INSTITUTES CF HEALTH

riDIVIDUAL PROJECT REPORT

Part B; Budget Data

11 . NIPR

SERIAL NUMBER

12. BUDGET DATA:

ESTH'IATED OBI.TGATIONS

MN YEARS

DIP.ECT PEDfBURSES^ENT TOTAL

PROF

OTHER

TOTAL

7,009 3,131 10,220

2/3

1

1 2/3

FY' 57

BUDGETED POSITIONS

PATIENT DAIS

PROF OTHER TOTAL

1 1

FY'57

RESEARCH

/T-/

ADMiraSTRATION

REVIEW I- APPROVAL

n

PROFESSIONAL &

TECHNICAL ASSIST-

BIOLOGIC STANDARDS

ri

ANCE

r~i

n

Ih. IDENTIFY ANY COOPEHJVTTNG UNITS OF THE PUBLIC HEALTH SERVICE, OR OTHER Qf^GANI^ATTONS, PROVIDING FITNDS, FACILITIES, OR PERSONNEL FOR THIS PROJECT IN FY 1957. IF COOPERATING UNIT IS WITHII^' NIH IPIDICATE SERIAL NO(S):

American Dental Association

Form No, ORP-1 - ^- Calendar Year 1956

October 1956

PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH INDIVIDUAL PROJECT REPORT Part C: Honors, Awards & Publications 15. NIDR - 3

SERIAL NU?.ffiER

16. LIST PUBLICATIONS OTHER THAN ABSTRACTS FROM THIS mOJECT DURING CALENDAR YEAR 1956:

Folk, J. E. Reactions of Glucose with Lj-^ine. Arch. Biochem, & Biophys. 6l:150 (19?6).

Folk, J. E. The Influence of the Lysine -Glucose Reaction on Enz^^matic Digestion. Arch. Biochem, & Biophys. 6h:6 (1956).

Folk, J, E. and Burst one, U, S. Enzymatic Hydrolysis of

Prolyl- and Hydroxyprolylnaphthylamides . Arch, Biochem, & Biophys. 6l:257 (1956).

Folk, J. E. A New Pancreatic Carboxypeptidase. J. Amer. Chem. Soc. 78:35Ul (1956).

McClure, F. J,, Folk, J. E. and Rust, J. D. Smooth Surface Caries in lATiite Rats: Effects of Fluoride, lodoacetate, Penicillin, Crisco, Butterfat and a Salt Mixture. J, Amer. Dental Assoc. 53:1 (1956).

Burstone, M. S. and Folk, J. E. Histochemical Deronstration of Aminopeptidase. J. of Histochem, & Cytochera. 1^:217

(1956).

17. LIST HONORS AND AWARDS TO PERSONNEL RELyiTING TO THIS PROJECT DURING CALENDAR YEAR 1956:

None

Form No. ORP-1 - 1 - Calendar Year 19^6

October 19^6

PUBLIC HEALTH SERVICE ~ NATIONAL INSTITUTES OF HEALTH INDIVIDUAL PROJECT REPORT Part A. Project Description Sheet 1, NIDR - h

SERIAL NTOffiER

2. Dental Research 3, Oral and Biological Chemistry

INSTITUTE OR DIVISION . LABORATORY, BRANCH, OR DEPARTMENT

h. 5.

SECTION OR SERVICE ' LOCATION (IF OTHER THAN BETHESDA)

6^ Amino Acid and Protein Biochemistry PROJECT TITLE

7. K. A. Piez

PRINCIPAL INVESTIGATOR

H. Eagle (NIAID, subprojects 2 and k) , F. Irreverre and B. Witkop

8 . (NIAt.lD, subproject 3), R. C. Likins (NIDR, subproject 7) '

OTHER IrJVESTIGATOrtS ~~~~"

9. IF THIS PROJECT RESEI^IBLES , COMPLEMENTS, OR PARALLELS RESEARCH DONE ELSEi-^fHERE IN THE PUBLIC HEALTH SERVICE (WITHOUT INTERCHANGE OF PER- SONlJfEL, FACILITIES OR FUNDS), IDENTIFY SUCH RESEARCH:

None

10. PROJECT DESCRIPTION:

Objectives : ' ' ■■•

It is the broad purpose of this project to provide both basic information relating to amino acid and protein chemistry of ■general interest and specific information of possible appli- cation to dental problems. This bread objective may be sub- divided as follows :

1. To' establish a general theory of pH gradient elution to aid in the selection of conditions for the analysis of amino acids.

.2, To examine a C-^'^ isotope effect on the ion exchange chromatography of amino acids.

Form No. ORP-1 - 2 - Calendar Year 19^6

October 19^6

Objectives (Continued):

3. , To provide methods for the quantitative analysis of hydroxyproline, allohydroxyproline , and other cyclic imino acids and to use these methods to examine collagens from viur-ious sources, normal and pathological, for possible structural variations related to disease or metabolic state.

ii. To study the amino acid and protein metabolism of human cells grown in tissue culture.

5. To isolate and analyze the proteins of the teeth and to compare them with sim.ilar proteins from other sources,

6, To measure th.e metabolic turnover of tooth collagen in relation to a possible role in the caries process.

Methods Employed; , .

The major laboratory tools employed are ion exchange methods of amino acid analysis and radioactive tracer techniques (C^^). In addition, paper chromatography, spectrophotometry, and other analytical methods are used. Tissue' culture techniques are used by NIAID in cooperative work.

Major Findings;

1. Suitable equations have been derived which allow the prediction of the form of a pH gradient given the experimen- tal conditions. Various types of gradients have been consi- dered and methods for obtaining them described,

2. The ion exchjange behaviour of a large number of C ^- labeled amino acids has been studied. It has been shown that labeling of an amino acid in the 1 or 2 position results in slower movement, relative to the unlabeled amino acid, on a cation exchange column. Labeling in positions not adjacent

to an ionized group has no effect on the behaviour of the amino acid.

3. Methods for -the quantitative analysis of cyclic imino acids have been developed and applied to a preliminary analysis of dates. i

ii. Employing several different cell lines grown in tissue culture, the composition of the free amino acid pool has been examined quantitatively. Using improved techniques, it appears that though the cells concentrate some of the essentiaLj

Form No. ORP-1 - 3 - Calendar Year I956

October 19^6

Major Findings (Continued):

amino acids, they are lost when removed from the medium. The utilization of tyrosine, phenylalanine, and glutamine has been extensively studied. All are used directly for protein synthesis; glutamine amide N is not exchanged; phenylalanine and tyrosine are not interconverted except by one variant line which can synthesize a small amount of tyrosine from phenylalanine. A rapid intercellular turnover of protein has been shov/n to exist.

5. Metliods are being devised to obtain enamel protein, and possibly enamel cuticle protein, free from other materials.

6 . Preliminary experiments have been planned to measure collagen turpover in dentin and other collagens in the rat, employing G-^^-labeled lysine.

Significance to Dental Research:

In general, this basic information concerning proteins and amino acids is of equal importance to dental research as well as various divisions of the medical sciences. The proteins of teeth, saliva, oral soft tissues, and bone have, in some respects, received less attention than proteins from other sources.

1, 2, and .The three subprojects related to pH gradient elution, a C '^ isotope effect, and the analysis of cyclic iraino acids have contributed to the methods available for the analysis of amino acids and should have wide utility,

U. The tissue culture experiments are providing basic information on metabolism in cells derived from human tissues. The simplified system allows studies to be done which could not be done on a whole animal because of the difficulty of interpretation in a com.plex system. At the same time the results are mere readily transferable to the whole animal than in the case of bacteria. Of considerable importance is the fact that malignant tissues can be grown and studied by these techniques,

Similarly, very little is known about enamel protein even though many consider that it olays an important part in the caries process.

6. The turnover of dentin collagen is being studied in an attempt to evaluate the metabolic activity of dentin and to determine whether the protein can or can not be affected by systemic or dietary conditions. It is of interest to know

Foi'm No, ORP-1 - h - Calendar Year 19^6

October 1956

Significance to Dental Research (Continued):

whether the collagen, once formed, plays only a passive role in the caries process or whether it can contribute by -/irtue of systenically induced modifications.

Proposed Course of Project;

1, 2 and 3- These problems, concerned with methods, are complete and either published or in press.

k. Studies on the free amino acid pool, glutamine utilization, and tyrosine and phenylalanine utilization in tissue culture are well advanced. It is planned to study the u:ii±lization of other amino acids, intercellular protein turnover, and amino acid transfer across the cell membrane.

5. It is planned to isolate pure enamel protein and characterize it chemically, and to initiate animal studies in which dietary protein and amino acid variations may be related to enamel protein chemistry,

6. It is proposed to determine turnover rates of collagen from various tissues of the rat and to examine the effect of age and perhaps other variables.

Form No. ORP-1 October 1956

- 5 -

Calendar Year 1956

PUBLIC HEALTH SERVICE - - NATIONAL INSTITUTES OF HEALTH INDIVIDUAL PROJECT REPORT

Part B: Budget Data

11. NIDR - h

SERIAL MUI'ilBER

12. BUDGET DATA:

ES TUTTED OBLIGATIONS

I/iAN YEARS

<

DIRECT REDffiURSEI-ffiNT TOTAL

PROF

OTHER

TOTAL

20,060 3;613 23^673