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200 1976 REPORT' of THE COUNCIL FOR TOBACCO RESEARCH-U.S.A., In THE COUNCIL FOR TOBACCO RESEARCH-U.S.A., Inc. 110 East 59th Street, New York, N.Y. 10022

Organization and Policy The Council for Tobacco Research-U.S.A., Inc. is the sponsoring agency of a program of research into questions of tobacco use and health. It is the out- growth of an organization formed early in 1954 by tobacco manufacturers, growers and warehousemen. Research support has been mainly through a pro. gram of grants-in-aid supplemented by contracts for research with institutions and~ laboratories. The Council does not operate any research faeility: The Scientific Advisory Board to The Council meets regularly to evaluate applications for grants-in-aid and for contracts, judging them solely on the basis of scientific merit and relevance. The Council awards research grants to independent scientists who are as- sured complete scientific freedom in conducting their studies. Grantees alone are responsible for reporting or publishing their findings in the accepted scien- tific manner - through medical and scientific journals and societies. Through December 1976;, The Council approved research projects for 358' investigators in~ 239 medical schools, hospitals and research institutions. These awards totaled more than $40,000,000. This Report includes a brief summary of The Council's present program in the cardiovascular area as well as lists of current and previous research projects supported' by The Council'. Also ineluded are abstracts of 80 researcK papers, acknowledging Council support, that were published in scientific journals dur- ing 1976. Project recipients have so far published 1,420 such papers. ADDISON YEAMAN Chairman and President

® 202 SCIENTIFIC ADVISORY BOARD to The Council for Tobacco Research-U.S.A., Inc. as of~ December 31, 1976 SHELDON C. SOMMERS, M.D., Chairman Director o f Laboratories, Lenox Hill Hospital Clinical'Professor of Pathology College of Physicians & Surgeons of Columbia University New York, New York .' 3. RICHARD M. BING, M.D. Director of Cardiology and Intramural Medicine Huntington Memorial Hospital~ Pasadena, California ~~. Professor of Medicine University of Southern California School of Medicine Los Angeles, California - JOSEPH D. FELDMAN', M.D. Head, Department of Immunopathology Scripps Clinic and Research Foundation La Jolla, California ~ WILLIAM U. GARDNER, PH.D:. Scientific Director, The Council for Tobacco Research-U.S.A, Inc E. K. Hunt'Professor of Anatomy (emeritus)' ~ l i h ool of~ Medicine Ya e Un versity Sc New Haven, Connecticut ROBERT J. HUEBNER, M.D. ~ Chief, Laboratory of RNA Tumor Viruses National Cancer Institute Y Bethesda, Maryland LEON O. JACOBSON, M.D. Director, The Franklin McLean Memorial Research Institute ~ Regenstein Professor of Biological Sciences ~ University of Chicago "` Chicago, Illinois ~ AVERILL A. LIEBOW„M.D. A Professor of Pathology (emeritus) ~: University of' California School of Medicine San Diego, California 4' HENRY T. LYNCH, M.D. °~ Professor and Chairman Department of Preventive Medicine and Public Health ,i Creighton University School of! Medicine Omaha, Nebraska =4

nc 203 FANS MEIER, D.V.M., Dr. Med. Vec, M.R.S;H. Senior Stafl Scientist The Jackson Laboratory Bar Harbor,,Maine LEE W. WATTENBERG, M:D. Professor of'Pathology Department of', Laboratory Medicine and Pathology University ofMinnesot'a Medical SchoolMinneapolis; Minnesota JOHN P. WYATT, M.D. Director Tobacco and Health Research Institute University of Kentucky Lexington, Kentucky Scientific Staff of The Council WILLIAM U. GARDNER, PH.D. Scientific Director ROBERT C. HOCKETT, Px.D. Research Director JOHN H. KREISHER, PH.D. VINCENT F. LISANTI, D.M.D. Associate Research Director Associate Research Director DAVID STONE,Px.D. Associate Research Director I H I

204 . CONTENTS Studies Related to Cardiovascular Diseases and Function Abstracts of Reports , Cancer-Related Studies The Respiratory System Heart and Circulation Neuropharmacology and Physiology Immunology and Adaptive Mechanisms Epidemiology Active Frojects Completed Projects Index of Principal Investigators Index of Senior Authors P,' Counci monar, relevar. Ir of card Cardi A: from p the Un stroke;, 6721 failure. to preci 7$f I Ati Epide) 0c•  Nu posstble eases, TII The "ris relations it might Am elevated personali i predispo!'. factor, bi the other scientfists to what operativestrong, a personal but has t( observatic coherents prove vali 30-498 O V I I C

Studies Related to Cardiovascular Diseases and Function Previous annual reports have presented the general plan and rationale of Council-sponsored' studies of carcinogenesis and the etiology of chronic pul- monary disorders. Their purpose was to provide a framework in whichi the relevance of individual contributions wouldi be more easily apparent. In the present issue, we describe similarly some of our approaches to study of cardiovascular diseases and function. Cardiovascular Diseases Among the many disorders of the cardiovascular system, those deriving from progressive atherosclerosis rank first as causes of disability and death in~ the UnitediStates. These include heart' attacks (myocardial infarction), angina,, stroke, arterial blockages irn the limbs, and some cases of congestive heart failure. Hypertension is believed not only to accelerate atherosclerosis, but also to precipitate acute events in damaged circulatory systems.. Atherosclerosis is, therefore, a principal focus of the present discussion. Epidemiology o f Atherosclerosis-Related Diseases Numerous epidemiological studies during the last 20 years have sought' possible "causes" (primary or contributory) of the atheroscleresis-related dis- eases. These studies often summarized their findings im terms of "risk factors." The "risk factor" is essentially a stati'sticali concept based upon mathematical relationships without necessarily any known or established mechanism, by which it might contribute to etiology. Among the many such "risk factors" frequently reported are hypertension, elevated serum cholesterol, diabetes or a prediabetic diathesis, cigarette smoking, personality type, inadeqpate physical' activity, and emotional stress. Genetic predisposition (e.g., hyperlipoproteinemia, homocysteinemia) is another such factor, both in its own right and as a contributor to most or possibly all of the others: Biochemists, physiologists, pharmacologists, psychologists, and other scientists have thus been challenged to fill the gaps and learni whether, how, to what extent and' in what kinds of persons each factor might actually be operative. The stimulus to conjecture, speculationi and hypothesis has been strong, and many scientists have apparently seized the occasion to test their personal hunches in the hope of a lucky strike. This was entirely legitimate„ but has tended to produce a welter of' fragmentary, confusing and inconclusivee observations subjected to speculative projections. Clarification may come as coherent, integrative theories emerge that can accommodate all the findings that prove valid. 5 30+498 0 ~- 78 - 14

Cigarette Smoking as a "Risk Factor" ~ The identification of a"risk factor" can be no more reliable than the statistics on which it is based. Smokers select themselves from among the .~; general population on a basis or bases that are little understood. Is it legitimate ~ to compare them with nonsmoker controls as animal experimenters eompare' ~ their test animals with genetically identical litter-mates? Tobacco use has many variations, both qualitative and' quantitative. To ~ what extent does voluntary adoption of any particular behavioral pattern select out a sub-population with innate characteristics or life-styles that' are'` linked to cardiovascular disease susceptibility? There are many evidences from studies of man that genetic predisposition'.~ plays an important part in determining which individ'uals are potentiali victims of atherosclerosis-related diseases. ln several types of rather extreme suscepti-,; bility, the patterns of inheritance have been well established. Review of the "risk factors" listed above indicates that most of them, are ; already known to be influencedi if not determined, by heredity. Studies of' spontaneous alcoholi consumption by inbred mice have shown ' substantiall strain differences in appetite, in behavioral responses and in, meta-` bolism. Analogous mouse studies of spontaneous nicotine and tobacco smoke ' intake have now been inaugurated by a Council grant to learn whether similarc genetic variations occur. a Twin Studies tary influences (even among twins reared apart), in determining initiation and ;: Identical twins provide the human~ counterpart of animals of inbred straini ~ Studies of all like-sexed twins in Sweden and! Finlan& are being assiste& by ~ The CounciP in an effort to determine broadly the relative influences of heredity ~ versus environment upon the incidence of atherosclerosis-based cardiovascular ~ diseases. A key strategy is to compare the incidence of such~ disDrders in smoking and nonsmoking identical twins, including symptomatology during life and, ~ eventually, age at death, the primary cause of decease and post-mortem evalua- tion of vascular pathology. Non-identical, like-sexed twin siblings serve as " controls. :. ;~ While the incidence of discordance in smoking practices is low among ' identical twins, an observation that in itself evidences a strong role of heredi- z~. maintenance of smoking; the numbers of discordants are great enough to accrue to increasingly significant levels in substantial twin populations followed over a sufficient period of time. These studies have now been extended beyond identical twins to other relatives of various defined degrees of consanguinity, such as half-sibs and the offspring of twins. These time-consuming, 'investigations will' require a considerable induction perio& before comprehensive new reports emerge. They may eventually provide more direct evidence whether cigarette smoking per se is a truly significant "risk factor" in these and perhaps some other diseases. They cannot, however, be expectedi to add greatly to elucidation of pathogenic processes or to ~ provide 6 V1 M M 0 e

207 strain. ed by nedity pscular Inoking e and, valua- rve as ,,. among heredi- n and accrue pd over beyond uinity, duction brovide pificant ~wever, `rovide I ~. methods usefuli in reducing risks due to biochemical aberrations imparted by the genes from ancestors. For this, other types of research are required. Cholesterol Metabolism Another prevalent "risk factor" in epidemiological studies has been "ele- vated serum cholesterol."' Contradictory findings over the years suggest that this concept; was an oversimplification, especially since many victims of heart attacks had no history of high cholesterol levels. That cholesterol is implicated in atherosclerosis has been assumed because mature arterial plaques contain the steroid both free and in combinations. Nevertheless, long-ter~m adminis- tration of drugs that lower serum cholesterol has not been as generally effective in arresting or reversing the process in man as had been hoped. Accumulating information about the states of combination of cholesteroli in blood is directing attention to the several cholesterol-containing combina- tions, classified as chylomicrons, very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL). The LDLs are the major carriers of' blood cholesterol and there are a number of experi- mental as well as epidemiological findings that an elevate& serum level of VLDL is correlated with progression of atherosclerosis, whereas elevated HDL is a favorable indication. Thus certain "lipoprotein profiles" are now considered by many to be better indicators of susceptibility to atherosclerosis than elevated totali ch& esterol. Certain~ of these profiles, associated; with very high susceptibility to disease, have been shown to be genetically determined. Barring some unforeseeable empirical discovery (which does occur im medical research and is a perennial hope), the rational route toward effective control is through systematic study of the pathogenesis of atherosclerosis, at biochemical' and physiological levels. This obviously must include a better understanding of the regulat'ion of synthesis, transport, function, and elimin• ation of cholesterol and of the aberrations in disease in the hope of altering these by targeted'treatment. The Council is presently reviewing the most promising concepts and leads that now exist in this complex field with the intent of increasing its support of basic study of athererosclerosis. The intent is not only to assist these im- portant developments, but also to seek more directly relevant assay systems for assessing the possible effects of cigarette smoke inhalation. The task is additionally difficult because the atherosclerotic disorders appear to be peculiarly human ones with few counterparts among animals under natural conditions. Highly contrived manipulations to produce animal "counterparts" tend to diminish probability of relevance to human experience. Human studies have been impeded by the lack of non-intervention tech- niques for assessing initiation and progression of the atherogenesis processes in the vasculature of man. New experimental techniques for visualizing the condition of the arterial walls, including scintillation photography, appear to show promise for the future. 7 1

208 In Vitro Studies of Arteries and Veins Meanwhile, in vitro techniques provide one method of studying synthests :~ and uptake of lipids an& cholesterol by human arteries an& veins, both _-` " " and atherosclerotie inct comparison with those of animals , dire. -, normal Though in vitro conditions cannot be made to duplicate the in vivo situation ~ perfectly, they may provide useful guides to in vivo research. Such studies have already been made with CTR support as.deseribed in the Council's Annual Reports for 1972-1975. It has been reported (and is reiterated in a current review) that human atherosclerotic and normal coronary arteries as welli as saphenous veins, perfuse& under pulsatile arterial pressures with human plasma containing labeled cholesterol an& acetate, do not synthe= size cholesterol from acetate and produce only small amounts of' cholesterol esters. Cholesterol is taken up in identical amounts by normal and diseased vessels and this uptake is increase& at higher pulse pressures, which seems consistant with the reputed effects of hypertension. Labeled acetate is incor- porate& into various lipids. Both normal and diseased coronary arteries and saphenous veins synthesize free fatty acids, triglycerides and phospholipids.' Addition of nicotine to the perfusion fluid did not influence cholesterol uptake. Analogous experiments with dog arteries appeared to shown an in- fluence of nicotine upon cholesterol uptake. If this is confirmed, it suggests caution in extrapolations from this species to man (1972' report). Carbon monoxide in the perfused plasma was reported' to enhance chol; esterol uptake by all arteries in this in virro system. Several' new studies report a dramatic inhibition of cholesterol uptake by `.' both~ human and animal arteries, under these in vitro conditions, by 7-keto-q~ cholesterol. : Living rabbits also showed am inhibition of cholesterol uptake, but to a much smaller extent. The low solubility of the 7-keto compound; imposed t technical problems. Improved techniques may increase the efficiency of the; ' effect. Meanwhile, the disparity, between the in vitro an& whole animal re-T sponses is another reminder that extrapolations must be cautious. ~A ° Other current studies deal with elucidation of possible mechanisms bf_` the inhibition and with the metabolic fate of the injected 7-keto compound iA; rabbits. Role o f Smooth Muscle Cells in Atherosclerotic Plaques The heretofore prevalent theory of atherosclerotic plaque formation posti-l-lated that the infiltration of fatty substances from the bloodstream into tbd ` arterial wall gives rise to cholesterol deposits that act as an irritant, causinr inflammation and proliferation of cells by processes akin to ordinary healiny This "insudation"' concept was consistent with the associations of atherosclet F, osis with elevated blood cholesterol, high blood pressure and high fat diets.. Animals fed large amounts of saturated fats and cholesteroli sometimes s* plemented with~ hormonal or other treatments, developed lesions superficiMresembling those observed in man at autopsy: These lesions often regressod in animals when the diets were altered, an observation, that stimulated hum22 dietary studies involving restriction of! cholesteroli and saturatedl fat consump'. 6 cc VE m ia ht is Ce sn to vc m m 01 tl rf 0 si st b, ai li1 ce th w cc in e% m E th hZ c r 0 s

ibed and' ron ressurt synthe~ leste ise see in ies xolipids: :)lesterd an ta~ ugge n postu- into the causing healing. TF, eroscler- '* ~t diets.~ es sup- _ ~~erficiallyregressed ` 4 human "` ~- ~onsump- _` 209 tion. Complicated by difficulties of control, the results in man have been equivocal. Recent electron microscopic studies of human arterial' plaques have re- vealed that the major component' of authentic plaques is proliferating smooth muscle cells like those normally composing the center (medial) layer of arter- ial walls rather than fibroblast cells such as proliferate to heal a wound. Early human arterial lesions (streaks) contain little lipid, suggesting that insudation is not the prime factor in typical atherosclerosis. Cholesterot deposits and cellular debris appear later. Present debate centers on what incites normali smooth muscle cells to migrate from the mediali layer and what causes them to proliferate. Presumably, damage to the inner surface cell' layer of the vessels is implicated and a number of theories as to how this endothelial layer may become damaged have been advancedi Many of the plaques generated in animal arteries by non-physiological manipulations are reported to be radically different in composition from those of genuine human atherosclerosis and their relevance to the human diseases is therefore questionable. Recognition of the difference has, however, led to reports that certain animals can develop human-type atherosclerosis under other more appropriate conditions. Smooth muscle cells from primate arteries are now being maintained successfully in culture media. Low-density lipoproteins added to the media stimulate them to multiply. Further, it has been reported that the ever-present blood platelets, normally involved in the clotting process in response to injury an& im other, functions, also secrete a substance that strongly stimulates pro- liferation of these smooth muscle cells. Damage to endothellal and' intimal cell layers of an artery, which ordinarily separate the blood from contact with the smooth muscle cells of the medial layer, can bring these cells into contact with~ platelets and presumably incite multiplication in the artery wall. Another concept holds that the smooth muscle cells multiply as the consequence of a mutatiom akin to that which transforms other normal cells into malignant ones. This "monoclonaV' theory„ which is supporte& by striking evidence but is nevertheless in some dispute, would suggest quite different mechanisms of action by external agents than those described heretofore. Endothelial Cells and Blood Platelets A single layer of endothelial cells composes the thin membrane lining the inner surfaces of arteries, performing, an important function in retaining the red corpuscles while allowing water and some other substances to pass through. As mentioned, damage to the endothelium is believed to contribute to all three of the mechanisms of atherosclerosis described. Many possible causes of endothelial damage have been suggested andl are being studied. It is possible that severali may be involved. The damaged endothelium can repair itself, rather slowly, but if damage is sustained or repeated too often, repair may not be achieved. Clots (thrombi) forming in an artery are thought to injure the endo- thelium by pressure and by impeding the access of oxygen. The blood platelets have long been• known to play a role in the complex events that produce

210 thrombosis in both arteries and veins, and may thus contribute indirectly endothelial injury. At the same time, it has been thought likely that the p lets may contribute in some way to maintaining integrity of the endotheli Recently developed techniques for growing human endothelial cells°` culture media, after harvest from umbilical cords, have made it possible~~- study factors that influence their replication. A Council:sponsore& investiga has reported that platelets added to the medium, as well as some individ~ substances secreted' by the platelets, will stimulate their growth. It is, theref ~ suggested that in vivo; platelets may expedite endothelial repair. He has , isolated from normal blood substances that inhibit platelet adhesion and impede the processes of thrombus formation. The implication is that a delicai balance among opposing functions maintains integrity under normal conditiotq Extension of such studies should' help delineate the mechanisms of thro bosis and atherosclerosis. ~ Another investigator has used in vitro techniques to culture rat' hea; muscle and epithelioid cells to determine the effects of cholesterol, cholesterol esters and 6-lipoprotein in producing cellular lipi& inclusions and' in labilizin lysosomes or mitochondria. Lecithin: Cholesterol Acyl T'ransferase (LCAT) Lecithin: cholesterol acyl transferase is an, enzyme thought, to be respon sible for the esterifi¢ation of lipoprotein cholesterol in plasma. It is postulate~i by several investigators to be important in the mechanisms that remove chol- esterotfrom the arterial i wal l. ~~ A number of previous papers, abstracted in these Annual Reports, have, reported clinical studies of LCAT activity in~ the plasma of animals and man under different circumstances to elucidate its clinical significance. Instability;` of the enzyme complicated these studies. Experimentation to delineate the enzyme's modes of action required' its isolation, purification and stabilization so that enzyme concentratiors could be controlled and substrate compositions better defined. . A recent publication reports that a concentrated effort has achieved the' isolation of LCAT in substantiali quantity ini a virtually pure state with greatly improved' stability. Development of a radioimmunoassay is reported to be under way. Two publications on applied studies of LCAT have also appeared recently. Availability of the purified enzyme and of its radioimmunoassay should stimulate and facilitate studies of cholesterol metabolism and the roles of the several lipoproteins and contribute to understanding of atherosclerosis. ~ MetabolicActicities of Pulmonary Endothelial Cells Fve yean of research asaisted by The Council have produced numerous reports on this subject, including several publishe& during 1976. It has been skwwtf that the vasoattive polypeptides bradykinin and angiotensin I are meta- boiiealty altered during a single passage through the vast pulmonary vascular bod. Bradykinin. a substance that tends to lower blood pressure, is completely 10 ; 71"-'.. , I .. ~~.it .-. ...}~. 3.:. a ,} rf 0 0 0

a l v to late,; lium. sin, le to gator du efore,~ also thus elicate itions. rom- e art ~ srol- h bilizing espoa 2ulated g chol l have d man stability ate the ilization 0 ositions ved the greatly itobe ppeared e V should s of the . '.'3 is. q <!. umerous has been re meta- vascular mpletely i 211 inactivated while angiotensin I is converted to angiotensin II, which is a potent' hypertensive agent. Bradykinin, under suitable conditions, can inhibit, this angiotensin conversion, These relationships suggest a role of the pulmonary circulation in blood pressure regulation. These metabolic changes have been traced to peptide hydroltise enzymes of the lung, which are not present in the bloodstream but are attached to the luminal surfaces of endothelial cells, especially those of capillaries and venules. The same enzyme inactivates bradykinin and converts angiotensin I. Steps in the hydrolytic process have been described'' in considerable detail; antibodies to the enzyme were prepared and labeled to provide tools for research. New functions of bradykinin are now being discovered; including an effect on prostaglandin synthetase which remains to be •explored in depth. It also remains for future studies to inquire how dysfunctions of these systems may be related to vascular or pulmonary diseases. Chronic Smoke Inhalation by Dogs '" A technique for chronic exposure of beagle dogs to cigarette smoke via tracheostomy was employed by a Council-supported scientist to look for possi- ble effects on clotting mechanisms and on cardiovascular function. Despite recognized limitations of this teehnique as a model of human practice, rather extensive experience with its use for other purposes suggested that extension of observations to the cardiovascular system might provide some preliminary data and guidelines for future studies. No clinically evident disease was found after 18! months' exposure under different dietary regimes. Some indications of possible enhancement of coagu- lation mechanisms and of relatively minor alterations in function were re- ported. The author described these minutely with cautions against transfer to man in view of experimentali limitations, species differences, and the greater complexity oT'human enviionmentF. Studies Involving Nicotine A number of past Council+sponsoredl studies, mainly using animals andd with a variety of objectives, have involved the administration of nicotine. Experience through the years suggests that' comments on some of the problems involved in selection of dosages are pertinent for evaluation, of past results and for designing'new and better experiments. Smokers receive nicotine by way of the oral cavity and the lung, in small successive doses over a period of several minutes and repeated at variable intervals. The entire dosage range they experience is very low in contrast to the levels often used by pharmacologists in exploratory studies of nicotine's pharmacological potentials. The view has been expressed that ordinary smok- ing rarely, if ever, produces high enough levels to act upon the sympathetic ganglia, but stimulates only the several special sensory receptors to' incite rapid but brief systemic responses of reflex origin. These responses may be quite Contradictory and paradoxicali in nature, depending on the conditions that predominate at any moment. 11 T 14 g 4 0 w ODA s'4 9#

212 - Yet, within the low range of dosages that can be achieved from sm 0 / there is still, very considerable variation. Rate of absorption via oral cavi lung is highly dependent upon the acid-base balance in the smoke. Maint of blood' level is strongly affected by the hydrogen-ion concentration of; bladder contents. The degree of acidity influences nicotine resorption altered by diet and' nervous state. Nicotine metabolism is rapid and me rates not only vary markedly among normal humans but are probably? fluenced by duration of smoking experience, a recognized but little undersG phenomenon. In addition are the variations due to choice of' tobacco product used individual differences in puff voltime, puff, frequency, depth of inhalatidM duratiom of smoke retention, etc. ' Design of animal experiments intended to mimic human smoking perience, long or short, musb include consideratios of' all the factors en ated and species differences as welL Experimental control or standardization of all the variables mentioned any large-scale or long-continued human study appears to be virtually possible. However, an expansion of informatiom om the actual ranges or dur tions of plasma nicotine levels attained by human smokers (and users of oth forms of tobacco)'• under actual conditions of life should be attainable. Recent studies by laborious methods have provided such data for sm: numbers of human subjects. These are a most valuable interim guide to the design of animal experiments. However, mass data on large representativoF populations are needed to define, on a sound statistical basis, the ranges, peaks and medians of plasma nicotine on a time scale, if important refinements are to~ be achieved in• epidemiological studies of smoking. ~ Radioimenunological Assay of Nicotine ~ Sensitive, specific and rapid assays for plasma nicotine and its major metabolites have long been needed. They should be able to be carried' out with very small samples of blood, with simple, inexpensive and mobile equipment, and by competent technicians without extraordinary special training, have long been needed for such purposes. Radioimmunoassays would' appear to have promise of meeting these re- quirements and The Council, is continuing to support studies in this area., Antibodies sensitive to nicotine have been obtained and reported (1975)., Continuing, efforts are now directed toward, improving sensitivity, testing for, specificity and simplifying procedures for broad application. , Nicotine Eff ects on Coronary Circulation in Conscious Dogs To avoid the effects of anesthetics on the pharmacological responses to nicotine, a method was perfected for intracarotid administratiorn of the alka- loid to conscious dogs in doses described by the investigator as "realistic." A striking increase in coronary blood flow was observed which was. traced to two separate factors. The major component was found' to be due indirectly to 12 a

N 1 13 9 ._,.. .• _,.,._ 213 an increase in the depth~ of respiration produced by nicotine, the minor one to a chemoreflex via a different pathway. EPIDEMIOLOGIC STUDY OF SMOKING' CESSATION A general difficulty in making an& interpreting studies of smoking cessa- tion is that those who discontinue smoking are not chosen at random but by their own decision, so that the influence of selection on comparability of the groups has been unknown. Nor has it-generally been possible to assess the influences of concomitant or "compensatory"' changes in life-style, such as alterations in diet, exercise, use of alcohol or drugs, etc,. A Council-sponsored comparison of continuing smokers, with smokers who have discontinued, and with, smokers who have stopped for a period and then resumed, sometimes repeatedly, is under way. In the population under study, a large body of data had been collected by uniform methods when alli were smokers, before any had stopped. Hence, some bases may be foun& for assessing selection biases that may have occurred in the subsequent groupings into the categories mentioned. 'X I- S A. 's

214 Following are abstracts, approved by the authors, of reports on search acknowledging support from The Council that have appeared in sci journals since publication of the 1975 Report'. The name of the recipi in italics. The abstracts are grouped under these headings: I. Cancer-Related'Stii II. The Respiratory System, III. Heart and Circulation, IV. Neuropharmaco an& Physiology, V. Immunology and Adaptive Mechanisms, VI. Epid'emio y. I. Cancer-Related Studies HYDROCARBON-NITROSAMINE SYNERGISM AS A POSSIBLE'.r AMPLIFYING FACTOR IN LUNG TUMORIGENESIS BY TOBACCO SMOKE A number of'studies have shown that inducers and'repressors of microsom mixed-function oxidases can powerfully influence the effects of chemical caZ cinogens. A significant incidence of pulmonary' zumors has been reported both rats and mice as a result of the simultaneous administration of 3-met'hylj compound is carcinogenic in the lung: Nitrosamines, including DMN', as well as 3,4-benzopyrene and~ 3,4-benzofiuoranthene whose carcinogenic properties are cholanthrene (MC) and dimethylnitrosamine (DMN) at~ levels at which neithe very similar to those of MC, are present in tobacco smoke. Therefore, it is pra; posed that a substantial proportion of the lung tumor incidence of smokersis~ ' due to synergism between the carcinogenic hydrocarbons and nitcosamines in the smoke, rather than to hydrocarbons alone. Two alternative mechanisms which may account for this synergism are considered. Preliminary results, how- ever;supportthe concept that DMN increases the hydrocarbon epoxide pool by lowering the activity of microsomal' epoxide hydrases in the lung while substran- tially increasing the arylhydrocarbon hydroxylase activity. The demonstration of a DMN,,hyd'rocarbon synergism in human lung carcinogenesis could en- courage unexplored approaches for the development' of partial means of pro- tection for smokers, such as the use of DMN-demethylase repressors and in- hibitors to block the DMN-hydrocarbon synergism. It is also suggested that dietary nitrosamines or prenitrosamine components, such~ as nitrites and secon- dary amines, may be foun& to have some role in the etiology of lung cancer. Argus, M. F: and Arcos, I. C. Journal of Theoretical'Biology 56:491-498, 1976,. Other support: National Cancer Institute. From the Seamen's Memorial Research Laboratory, U. S. Public Health Service Hospital, and' the Department of Medicine, Tulane University Medical Center, New Orleans. 1 miite comp logs. Othen rneth' difler and' repre sivel' on t' pres, tion crea of I and rate pret whe part dica diffe tiviti Zeit Otk Fro Hpc I*IeN DI11 IN( EF (D is } res ing cre ent cai chr pet

STRUCTURAL LIMITS OF SPECIFICITY OF METHYLCHOLANTHRENE-REPRESSIBLE NITROSAMINE N-DEALKYLASES. INHIBITION BY ANALOG SUBSTRATES In order to gain an insight into the structural specificity of dimethylnitrosa} mine (DMN)-demethylase, a systematic investigation was carried out on the comparative dealkylation of DMN, higher dialkylnitrosamines and DMNLana- logs; as well as on the inhibition of DMN-demethylation by the DMN-analogs. Other experiments in this framework explored the effect of pretreatment by 3- methyl~cholanthrene (MC), a potent repressor of DMN-demethylase, on these different; dealkylases. Results showed' that the dealkylation of dimethyl-, diethyl- and dipropylnitrosamine by hepatic microsomes of Sprague-Dawley rats was repressed by pretreatment of the animals with MC. This-repression progres- sively decreased with the increase of alkyli chain length. In contrast to its effect on the demethylation of DMN, in vivo phenobarbital induced rather than, re- pressed the deethylation of diethylnitrosamine (DEN). The rates of demethyla- tion of the DMNI analog substrates, although low as compared to DMN, in- creased with the acyl chain length. These analogs were potent in vitro inhibitors of DMN demethylation when used in combination with DMN as substrates, and the inhibition decreased withithe length of the acyl chain. Although the rate of demethylation, of methylphenylnitrosamine was not influenced, by MC- pretreatment„ the compound was, however, a potent inhibitor of demethylation when used, as substrate in combination with DMN. Moreover, beyond the ap- parent distinctness of DMN-demethy,lase and DEN-deethylase there is now in- dication that more than one enzyme, having the same substrate specificity but different kinetic and regulatory characteristics, underlie DMN-demethylase ac- tivity. Arcos, J. C et al. Zeitschrift fur Krebsforschung und Klinische Onkologie 86:171-183; 1976. Other support: National Cancer Institute. From the Seamen'S Memorial Research Laboratory, U. S. Public Health Service Hospitali and the Department of Medicine, Tulane University Medical Center, New Orleans. DIMETHYLNITROSAMINE-DEMETI-IYLASE: ABSENCE OF INCREASED ENZYME CATABOLISM AND MULTIPLICITY OF EFFECTOR SITES IN REPRESSION! HEMOPROTEIN INVOLVEMENT Evidence is presented here that the rate of decay of dimethylnitrosamine (DMN) demethylase following pretreatment with 3-methylcholanthrene (MC) is no greater than that to be expected from, normal enzyme eatabolismi These results also show that the observed decrease of DMN-demethy,lase VII1ex follow- ing MC administration is not due to increased rate of breakdown, but to de- crease& de novo synthesis. Other experiments in this study indicate that differ- ent receptor sites are involved in the repression of DMN-demethylase by hydro- carbons and by phenobarbital (PB), and that a P-450 type mcrosomal cyto- chrome is involved in the demethylation, of DMN.. The totality of these ex- perimental observations presents an apparent paradox which may be summar- I '-_«.-

ized as follows: (1) MC and PB s1 T ase 2 p are potent repressors of the DMN-de O MC and PB are otent inducers of a number of other mixed- Nticnts. oxidases as welll as of the s nthe i y s s of theil essenta components, cytoc „k att( P-448I and' P-450, and (3), the MC- and PB-repressible enzyme, D cw)mic methylase, is iiihibited', by carbon monoxide, just as are MC- and PB-ind ntr-incr mixed-function oxidases: The implications of these findings are consider ,tula ga " Argus, M. F., Arcos,, l. C., Pastor, K. M., Wu, B. C., and Venkatesan,' +~ausin Chemico-Biological lnteractions 13:127=140; 1976. Th~s disc ;cntal at ot embr Other support: National Cancer Institute and Hoffmann-La Roche Inc. From the Seamen's Memorial Research Laborato -. and the Hospital, and the Department of Medicine„ Tulane Uns erPsityliMedieal C• A 1erspe New Orleans. cnzy mes questior MALIGNANT DISEASE AND TRACHEOBRONCHIAL EPITHELIAL MULTINUCLEATION Tracheobronchial washin s of i g pat ents withid a we variety of extrathoi malignancies have been shown earlier to contain significantly more multinucle ciliated' cells than those nf a.. cancer. This study, while confirming the original Ifind ngs, also deterPm ned so factors which influence multinucle i ' at on One hlf fh .ao te total'group of 824 p~ tients had malignant disease and the other half', matched, by sex, age (decades and smoking habit but without redi p agnosed maliie gnancs, served as contro Multinucleation was found to be 203 times m f .orerequent in the cancer tients than in, the eontrolfi. Ih patients with invasive tumors without kno metastases, multinueleation was se f en our time f controls. Site ofs morerequently than in t origin sta e f , g o tumor adi ,n excessve smoking habit in control females influenced statistical significance, but smoking habit in males di& noL A prospective study is now being planned in which incidence an& degree of tracheobronchial epithellal multinucleation will' be used in conjunction witb biochemical tests for the diagnosis of occult eancer . Chalon, J. et al. Cancer 37(4):1874-1881,1976. Other support: U. S. Public Health Service. From the Departments of Anesthesiology and Pathology,, New York University School of Medicine, New York. ECTOPIC ISOENZYMES: EXPRESSION OF EMBRYONIC GENES IN NEOPLASIA ~ Awareness of the phenomenon of ectopic polypeptide hormone production by tumors has coincided with the recognition of ectopic isoenzymes in experi- mental rodent tumors and in the serum and tumor tissues of humam cancer neoplas' answer oncolog host ge cnzyme pred'om produci of f act, carcino regulati underst transfo appeara cells m viruses: Fishma In: Be Growt) pp. 57- Other From U nive7 A SIA ALKE SPEC E in the strong, bryon tremel maril) petitiv 0'

217 N-demethyl- ed-f'unction cytochromes e, DMN-de- PB-inducible sidered. xtrathoracic Itinucleated rediagnosed ined some of 824 pa= e (decades) as controls: cancer pa= out known than in the it in control es di& not: d degree of nction with patients. Many newly-recognized embryonic protein phenotypes are receivingg wide attention as "markers" of malignancy. A number of other properties both enzymic and nonenzymic are shared by embryonic and neoplastic cells. The ever-increasing accumulation of findings of embryonic gene products in~ neo- plasia gathered from research on isoenzymes, hormones, and protein antigenss is causing reevaluation of the current viewpoint regarding the nature of cancer. This discussion examines the authors' recent experiences with the carcinopla- cental antigen, Regan isoenzyme, as a model system for studying the regulation of embryonic gene expression in cancer cells,, the relevance of' the cell cycle,, and the nature of the gene product in membranes. Their hope was to construct a perspective on the nature of cancer from the point of view of ectopic iso- enzymes. The evidence is then examined from the standpoint of a single central qpestion: "Is the selective activation of embryonic genes a necessary step in neoplastic transformation?" Several areas are scrutinized in an attempt to answer this question. These include the relationship between embryology and oncology, the possible embryonic origin of viral transforming genes, normal host genes, and oncogenic mechanisms. It is concluded that: (1) ectopic iso- enzymes, present in tumor tissues but not in the tissue of tumor origin, are predominantly embryonic in type; (2) the Regan- and non-Regan-isoenzyme- producing HeLa cells provide a suitable modeli system for the study of a variety of factors which are invoUved in the expression,of ectopic isoenzymes of the carcinoembryonic category; and (3) information on cell cycle and hormonal regulation of embryonic gene expression in cancer cells may contribute to our understanding of the role of this phenomenon in the process of neoplastic transformation which may reflect a disorder of gene regulation with the re- appearance of trophoblastic properties. Such traits evidenced' by transforming cells may be a consequence of whatever oncogenic agent (chemicals; radiation, viruses) produces a specific loss of regulatory control of embryonic genes. Fishman, W. H. and Singer, R. M. In: Becker, F. F. (ed.): Cancer: Biology oJ' Tumors: Cellular Biology and Growth, New York, Plenum Publishing Corporation, 1975, voli 3{ chapt. 3, pp~ 57-80. Other support: National Cancer Institute. From Tufts Cancer Research Center an& the Department of Pathology, Tufts University School of' Medicine, Boston. A SIMPLE RADIOIMMUNOASSAY OF HUMAN PLACENTAL ALKALINE PHOSPHATASE (REGAN ISOENZYME) USING SPECIFIC ANTIBODY POLYMERS Ectopic placental alkaline phosphatase (Regan isoenzyme) has been found in the sera of cancer patients, reported in variant forms„ and considered to strongly reinforce the contemporary view of' the biological importance of em- bryonic gene activation during neoplastic transformation. However, the ex- tremely minute amount of Regan isoenzyme im sera has made its detection, pri- marily by enzymological quantitation, difficult. Now, this paper presents a com- petitive-protein-binding assay of Regan isoenzyme using a specific polymerized 17 r

218 antibody to facilitate the phase separation step. Only a minute quantity of, polymerized antibody particles is required for each assay in admixture with-l specially prepared labeled and unlabeled enzyme. By adding a small amount clinical specimens by this procedure. Chang; C-H., Raam, S., Angellis, D., Doellgast, G'., and Fishman, W. H. Regan isoenzyme and' Nagao isoenzyme, could thus be directly determined of the enzyme. Native variants of placental-type alkaline phosphatase, inclu phatases) and capable of detecting both~ cataly,tically, active and inactive fo being direct, specific (no interference by the nonplacental-type alkaline X says. However, radioimmunoassay is advantageous to the enzymic assay zyme protein per tube, is comparable to the sensitivity achieved by enzymic starch-gel particles before low-spee& centrifugation, complete phase separal can be achieved. This radioimmunoassay, which can detect 0.4 to 0.8' ng University School of Medicine;, Boston. From Tufts Cancer Research Center and the Department of Pathology, T Cancer Research 35 c 1706-1'7'1'2; 1975. Other support: National Cancer Institute. only nanogram levels of the sample and that offers advantages not provided by, conveniently performed on cellulose acetate membrane (c.a.m.), that requir This paper reports an immunofixation electrophoresis technique that can CONVENIENT' IMMUNOFIXATION ELECTROPHORESIS ON CELLULOSE ACETATE MEMBRANE the use of agar gel as the supporting medium. Applying poiyspecmc antiserum directly upon the surface of c.a.m. after electrophoretic separation results in thk simultaneous fixation of multiple antigens as discrete immunoprecipitin~ bands; rather than the complex precipitin arcs observed in conventional immunoelectro= phoresis. Although the principle of this technique is similar to that performe~ on agar gel, using, c.a.m. for immunofixation electrophoresis has the followtng adNantaees: (Il Because of c.a.m.'s hiehlv norous nature. it nrovides an exceT~ action with a second-antibodyenzyme conjugate which ean be recovered and reused. These advantages thus have made the c.a.m. a convenient, acceptable, much less antiserum non-specifically than agar gel. (4) This technique is suited for effectively and conveniently amplifying the primary precipitin bands by re-a separation of proteins. (2) It greatly reduces the amount of' time neede& for antibody fixation~ and for subsequent washings: (3) The membrane absorbs lent molecular-exclusion effect and hence a highly satisfactory electrophoretic supporting medium for immunofixation~electrophoresis analysis. Chang; C-H. and Inglis; N'. R. (Fishman, W. H.) Clinica Chimica Acia 65t91-97, 1975. " Other support: National Cancer Institute. From Tufts Cancer Research Center and the Department of Pathology, Tufts University School of Medicine, Boston. 18 R Pl 1 T r I

REACTION OF CONCANAVALIN A WITH ALKALINE PHOSPHATASES EXTRACTED FROM VARIOUS HUMAN TISSUE SOURCES Abundant evidence indicates that most' vertebrate alkaline phosphatase, whether originating in normal or malignant cells, is intimately associate& with the plasma membrane of the cell in which the enzyme is synthesized and that the enzyme invariably contains carbohydrates. In order to shed some light on the nature of the carbohydrate moeity associated with the enzyme, it would be of interest to know whether there is a specific interaction between alkaline phosphatase and concanavalin A (con A) of jackbean, a wellcharacterized lectin which has the property of reacting with cell-surface sugar residues. A simple and sensitive electrophoretic technique on cellulose acetate membrane was selected to differentiate the mobilities of the free enzyme and the enzyme- con A complex, based' on the fact that the complexed form is retarded at the origin of application due to its state of aggregation, while the free enzyme has normal mobility. The specific binding of alkaline phosphatase was then studied by con A affinity chromatography. The results demonstate that there is a spe- cific reaction between con A and a fraction of alkaline phosphatases extracted from various human tissue sources. This phenomenon was also observed with highly purified placental alkaline phosphatase with a specific activity of 200 µmole phenol/mg proteinL'min• In~light of the well-establishe& specificity of con A reactivity toward the carbohydrate moeity, the authors conclude that the re- acting human alkaline phosphatases may contain branched polysaccharides with a terminal a-D-mannopyranose, a-D-glucopyranose, D-fructofuranose, their glu- cosides, or sterically related structures. Chang, C-H., Angellis, D. and Fishman, W. H. _ Molpcular & Cellular Biochemistry 9(1i):55-57; 1975. ` Other support: National Cancer Institute. From Tufts Cancer Research Center and the Department of Pathology, Tufts University School of Medicine, Boston. THE IMMUNE RESPONSE AT THE TUMOR SITE IN LUNG CARCINOMA In this study, histologic and immunologic means were used to investigate the local immune response to lung cancer. To begin with, sections from 50 cases of different types and grades of' lung carcinomas were examined com- paratively in order to evaluate the morphology of! the local reactions. Distinc- tive patterns of stromal cellular reaction, characteristic for different histologic types of lung carcinoma, were recognized. The amount of cellular infiltration was highest in squamous cell carcinomas and lbwest' or nonexistent in oat cell carcinomas, Within the various histologic categories, the well-differentiated tumors appeared' to be accompanied by more reactive cells than the poorly dif- ferentiated ones; there was no relation between tumor necrosis and cellular infiltration. The plasma cells were distinctly associated with squamous cell car- cinomas; their number in the stroma was proportionate to the degree of dif- ferentiation and the presence of keratin produced by the tumors. In the im, munologic tests, eluates - with a high content of immunoglobulins were re- 19

220 covered from pleural' effusions and from solid lung carcinomas by dissociati of antigen,antibody compiexes. In indirect immunofluorescence tests, th preparations reacted, positively with tissue cultures and with fresh suspensio of! lung carcinoma cells, but not with cultured cells of most' nonpulmon ~~ tumors or with cell' suspensions of normal adult and, fetal lung. Althoughs~ far only a limited number of cases has been examined, the results consistenti indicate that tumor-reactive antibodies are present at the tumor site in amoun~ significantly larger than in the circulating bloodi It also appears that these im'i munoglobulins are largely the product of the plasma cells and lymphocyt ~ accumulated in the tumor stroma. - ~ loachim, H: L., Dorsett, B. H. and Paluch, E. . Cancer 38'(6) :2296-2309; 1976. ` . °.F From the Departments of Pathology; Lenox Hill Hospital and the College o Physicians & Surgeons of Columbia University, New York. A ACTIVATION OF DEVELOPMENTAL GENES IN NEOPLASTIC' TRANSFORMATION Activation of embryonic genes, as evidenced by the detection of their pro-', tein products, is being recognized as a genuine manifestation of neoplasia.'' Since such proteins are being identified on the basis of germ-layer origin and of extra-embryonic tissues, it seems important to fix the stage of development( corresponding to the pattern of expression of such proteins in cancer tissue. ' From a map of development focused on events beginning with the zygote and ending with the establishment of fetus and placenta, it' is possible to explain why certain developmental gene products are restricted, to fetus or placenta or` distributed' in both. It also suggests which products can be expected to occur concordantly in neoplastic transformation and in neoplasia. In, the paper pre- sented here, the expression of term placental, chorionic and amnionic alkaline phosphatase isoenzymes in preneoplastic and neoplastic lung and other cancers is anticipated from a preliminary study of epithelial cell sonic extracts from the tracheobronchial tree of a patient with bronchogenic cancer. This study ex-' plores, first the onco-developmental~ relationship between chorionic alkaline phosphatase and chorionic gonadotropin, both of which are localize& in syn• citiotrophoblast and expressed in choriocarcinoma. Then, the tracheobronchial tree as a human model of neoplastic tt•ansformatiom is presented in relation to the known tumor and' developmentat alkaline phosphatases, and the widely recognized oncofetal proteins are identified with their counterparts and position in early development. Such a developmental perspective may, whem it has been fully explored,, lead to a rational~ interpretation of the significance of' expression of any product or combination of products in neoplastic transformation and in neoplasia: ' Fishman, W. H. Cancer Research 36(9 pt. II):3423-342&, 1976. Other support: National Cancer Institute. From Tufts Cancer Research Center and the Department of Pathology, Tufts University School of Medicine, Boston. VIR I"ih NOF been mun, muM virus was mun tivit} tion. the i agai cont seru derr prel and' for of t ge n, Lev, Pr©4 A m~ Oth tute Fro of t Car Hat FE LE dec the chrr ane cer inv lesi inc: Thi sim 20 A G.~ ~ ~ C!t 4 0

4 221 . ciation s; these ensions monary ugh so istently mounts 'ese im- hocytes :fi_-7 eir pro- plasia. n and pment tissue. te and xplain nta or occur r pre- lkaline ancers mthe dy ex- lkaline n syn- nchial ion to widely'_ osition s beed ression and in ;! VIRUS-SPECIFIC NEUTRALIZATION BY A SOLUBLE NON- IMMUNOGLOBULIN FACTOR FOUND NATURALLY IN NORMAL MOUSE SERA Two types of immunologic responses to endogenous C-type viruses have been detected in normali mouse sera: virus binding as detected by radioim- mune precipitation assays and virus neutralization, However, while mouse im- munoglobulins in the sera bind to both ecotropic and xenot'ropic (X-tropie)) viruses, these same sera neutralize only the X-tropic virus. This inconsistency was at first considered to be related to differences in the affinity of the im- munoglobulins. The data presented here now indicate that the neutralizing ac- tivity is not due to the antiviral antibodies detected by radioimmunoprecipita- tion. Rather, virus neutralization is associated with a factor whch has none of the characteristics of, any known class of mouse immunoglobulin. It is effective against several X-tropic virus isolates, but not ecotropic viruses, and requires contact of the serum with X-tropic virus. The decrease of this factor in mouse serum only after absorption with X-tropic, but not ecotropic, virus further demonstrates its specificity for, this class of endogenous C-type virus. Although preliminary, these data suggest that binding of the factor to the virus occurs and is presumably involved in its neutralization. The specificity of this factor for X-tropic virus suggests that it represents a newly recognized type of responsee of the host to an endogenous virus. Its possible role in the regulation of endo- genous C-type viruses is considered. Levy; I. A. et a1: Proceedings of the National Academy of Sciences of the United' States of America 72(12) :5071-5075, 1975. Other support: National' Institutes of Health and the National Cancer Insti- tute. From the Department of Medicine and Cancer Research Institute, University of California; San Francisco, the Basic Cancer Research Program, Frederick Cancer Research Center, Frederick, Md., and the Clinical Research Centre, Harrow,, Middlesex, England! FEUIrGEN MICROSPECTROPHOTOMETRY OF ORAL CANCER AND LEUKOPLAKIA Early in the development of cancer, a celli line emerges whose nuclearr deoxyribonuclt;ic acid (DNA) content remains constant throu-a,hout the life of the tumor. The amount of DNA appears to be largely dependent on the actual chromosome number„ or karyotype, and can thus be diagnostic of diploidy, aneuploidy or polyploidy. The demonstration of abnormal cell lines in in situ cervical carcinoma has permitted the inference that this change occurs before invasion, while the fact that this characteristic change is found in cervical lesions diagnosed as mild, moderate or severe dysplasia would indicate that an increase in nuclear DNA might occur much earlier than heretofore suspected. This study attempts to discover whether oral leukoplakias have DNA contents similar to oral carcinomas, and to determine the valtle of Feulgen microspectro- 21. ,a

l 222 photometry in predicting the transformation of leukoplakia into carcinoma. The technique of using two wavelengths was applied to 10 µ sections of 33 lesions and showed that five out of ten carcinomas and 12 out of 16 Ieukaa plakias had diploid cell lines. Instead of demonstrating a correlation between the degree of atypia and abnormal Feulgen-DNA stem~ lines; however, the re- sults suggest that DNA content increases at a much earlier stage and that seem- ingly innocuous hyperkeratotic lesions, as well as oral leukoplakias„ contain amounts of DNA similar to oral carcinomas. This technique, therefore, seems to have minimal value as a predictor of malignant transformation because the sigtrificance of a diploid stem, line would', always be uncertain. .. ;, ,., fFt Doyle, J. L. and Manhold; J. H., Jr. ~zI . . ,x? Journal of Dental Research 54(6) :1196-1199, 1975. Other support: American Cancer Society. From the Department of General and Oral Pathology, School, College of Medicine and Dentistry, JerseyCity: IDENTICAL GENETIC BASIS FOR LYMPHOSARCOMA AND HEMOLYTIC ANEMIA IN THE RABBIT -- In rabbits; both lymphosareoma and hemolytic anemia whichi occur in two genetically relrited strains, WH/7i and X/J respectively, are inherited in auto- somal recessive fashion. Tests for identity of the hemolytic anemia (ha), and lymphosarcoma (Is) genes show that they are both, allelic an& identical by descent and that the two different conditions result-from interaction of these genes with the host genotype. Hemolytic anemia is the primary cause of death in compound heterozygotes, ha/ds, with increasing lympho-proliferative disease with age. Ages at death of the animals with histologically confirmed disease ranged from five days to 22.5 months, the average being about 10!5 months.' The symbol ha will henceforth be used to represent the gene for either disord'er. Fox, R. R. and Meier, H. , The Journal of Heredity 67:99-102, 1976. Other support: National Institutes of Health's Division of' Research Re-; sources, National Institute of' Child Health and Human Development and the Nationali Eye Institute. From The Jackson Laboratory, Bar Harbor, Me. PRESENCE OF A HIGH-MOLECULAR-WEIGHT RNA AND RNA-DIRECTED DNA POLYMERASE IN RABBIT HEREDITARY in several animal species, it seemed possible that rabbits with lymphosarcoma might harbor type C virus(es) or viral markers: To test this, the assay for LYMPHOSARCOMA [ In light of the association of' type C RNA virus with~ lymphosarcoma' 22 R C' b; st ti, er ti( fr, pr PI pr nc Bt C, 0 St E' P( be in otl tio Po (R C thE gr, na US( en: vir an pn W1 in tur Be Ca Ot

/- 223 kia into carcinoma. 10 µ sections of 33, 2 out of 16 leuko= correlation between es,, howevery the re-! stage and that seem- ukoplakias, contain ue, therefore, seems rmation because the 1rn .sit which occur in two e inherited in auto- ic anemia (ha) and lic and identical by interaction of! these ary cause of death' proliferative disease ly confirmed disease about 10.5 months 3 Le for either disorder. I _:~ . ~+t4Y .:•xcSa A AND EREDITARY Ir with lymphosarcoma ' with lymphosarcoma t ~~ this, the assay for s RNA-directed DNA polymerase (RDDP) was used as an indicator of a type C virus in lymphosarcomatous rabbits. An RDDP associated' with particles that band in the density range of type C RNA viruses and viral cores was demon- strated in 85% of' the tissues taken from these rabbits. Endogenous RDDP ac- tivity that was sensitive to treatment with RNase was detected in, the crude enzyme obtained from, tumorous tissues. The RDDP associated with the par- ticles could be distinguished from cellular DNA polymerases by salt elution from phosphocellulose. The partially purified enzyme preferred the template primers poly(rA)•(dT)1Q-1;8 and poly(rC)•(dG)12_18 over other synthetic tem- plate primers and also utilized viral 70 S RNA as template. While this and, previous studies indicate that type C viral markers are present in rabbit tissues, no virus from tumor cells or normal cells in culture has been isolated yet. Bedigian, H. G., Fox, R. R. and Meier, H.. Service. From The Jackson Laboratory, Bar Harbor, Me. Other support: National Institutes of Health and the U. S. Public Health Cancer Research 36:4693-4698, 1976. EVIDENCE FOR A PARTICLE-ASSOCIATED RNA-DIRECTED DNA POLYMERASE IN, RABBIT PLACENTAL AND UTERINE TISSUES Because of evidence that type C RNA viruses exist in rabbit tissues, and because type C particles have been found in rabbit blastocysts and uterine cells, in primordial germ cells an& in the embryonic or placental tissue of severali other species, it seemed possible that rabbit placental tissue, as welli as progesta- tional and estrus uterus, could express type C virus(es) or virali markers. Re- ported here is the isolation of a distinct rabbit RNA-directed DNA polymerase (RDDP) associated with, particles that band at a density characteristic of type C RNA viruses. That this enzyme is biochemically an& biophysically similar to the RDDP of mammalian type C RNA viruses is shown by column chromato• graphic characteristics, templt<te primer preferences, molecular weight determi- nation, and an absolute requirement for the divalent cations. The methodology used provides the sensitivity require& to detect low intracellular levels of the enzyme and to distinguish the cellular DNA polymerases (a, je, and y) from viral RDDP. The accumulated evidence indicates that the rabbit may harbor an endogenous type C RNA viral genome and thus contain viral markers. The presence or absence of viral markers in tissues of rabbits, particularly strain WH/J which has a high incidence of lymphosarcoma; is also being investigated in hopes of elucidating the role of' pardially expressed' type C virus(es) in tumorigenesis. Bedigian, H. G.,,Fox, R. R. and Meier„H: Cancer Research 36(,12) :4687-4692, 1976. Other support: National Institutes of Health and U. S; Public Health Service., From The Jackson Laboratory, Bar Harbor, Me. 23

224 'rv errcaT ACFwTAT rARI'TNTI(;FNTC`. FFFFCT.4 ClF DIETHYLNITROSAMINE IN' MICE ~ This study investigates the prenatal carcinogenic effects of a single dose of diethylnitrosamine (DEN) administered to mice at different times during,preg- nancy. Lung tumors and leukemias were' observed in the offspring, of DEN'-.,- treated SWR/J females and AKR/J males when the carcinogen was adminis-.t tered any time between the fifteenth and eighteenth day of gestation. The inci-; dence of lung tumors, histologically multiple pulmonary adenomas, was high-1 est (87% ) when the mice were treated on day 18. Since the DEN did not7 induce any tumors in these animals when administere& before day 15; it appeais, that' susceptibility of the offspring to this carcinogen begins at that~ time or later and that the target organ is the lung;,' although leukemia was common as well. Previous reports suggest~ that, in rats, the fetal enzyme system neces- sary' for the activation of "indirect"' alkylating carcinogens becomes operative, only in later prenatall stages. A study of the synthesis of dealkylating enzymes in fetal tissues at different times during gestation could be important for es-1 tablishing a positive correlation between transplacental carcinogenesis by these'; substances and the development in fetal tissues of enzymes capable of metabo- lizing them, Diwan, B. A. and Meier, H. Die Nnturwissenschaften 63(10):487-488, 1976. Other support: Leukemia Society of America, Inc. From The Jackson Laboratory, Bar Harbor, Me. THE BIOSYNTHESIS AND BIOLOGICAL PROPERTIES OF 6-HYDROXYMETHYLBENZO[a]PYRENE As part of' a series of studies on the properties of aryl hydrocarbon hy-; droxymethyl synthetase, this paper shows that the enzyme is present in lung and liver both as a microsomal membrane-bound form and as a soluble form;, and that each form of the enzyme is activated by a-naphthoflavone. In addition,, `, the biologic properties of benzo(x)pyrene (BP) derivatives as carcinogenic, agents in mice are studied, and the effects of cytochrome P-450' inhibitors on: 7 the side-chain methyl oxidation and the hydroxymethyl synthetase reaction are., ~ compared. Results show that the aryli side-chain methyl oxidation, is inhibited, by the classic inhibitors of cytochrome P-450; whereas the aryl' hydroxymethyl synthetase reactioni functions independently of the cytochrome P-450 pathway.: - Also included in this paper are observations on: (1) the mechanism of the synthetase, reaction; (2) tumorigenicity of derivatives of BP;, (3) induction of the synthetase by BP; (4) transformation of cells im culture, and (5) trans-' formation,of mouse embryo' cells by BP and BP in the presence of 1-benzyllmi- dazole: Moreover, data are presented to demonstrate that WI-38 human lung diploid fibroblasts treated with BP and 1-benzylimidazole clone more effectively than with, either treatment alone. The sum, of these data indicates that 1-ben-, zylimidazole, an inhibitor of' cytochrome P-450, in combination with BP allows 24 trans also tivity the i arom Sloan In: F nucle New Otht Frorr Heall tion, STU ACT tive i aryl trach prefe MCA cMai aftFr press. DBA termi (1) ' to in cytoc quani AHI mice of m was sults majc indu Kou: Chen Fron Beth,

dose ot' ng preg f DEN- adminis= he inci= as high did nox t appears time oi: common m neces- operative; enzymes t for es= by these' f inetabo- O carbon hy-: nt in lung, luble f'orm,, In addition, ; arcinogenic, hibitors on reaction are , is inhibited roxymethyl 50 pathway.. nism of the !induction of 4 (5) trans- ~ ~ 1-benzylimi- I human lung re effectively 6 that 1-ben- i[th BP allows i 225 transformation of mouse embryo cells at~ passage 8 and that this combination also affects WI-38 cells' cloning efficiency. The relationship between, this ac- tiuity, and! transformation~ is currently under study in a total program evaluating the role of the nonhydroxylative pathways in carcinogenesis by polycyclic aromatic hydrocarbons. Sloane, N: H., Chen, H., Diwan, B., Bedigian, R., and Meier, H. In: Freudenthal, R. I. and Jones, P. W: ('eds:): Carcinogenesis, Yol. 1. Poly- niiclpar Aromatic Hydrocarbons: C/remistry; Metabolism, and Carcinogenesis, New York: Raven Press, 1976, pp, 171,-180. Other support: American Cancer Society. From the Department of Biochemistry,, University of Tennessee Center for Health, Sciences, Memphis, and The Jackson Laboratory, Virus Leukemia Sec- tion, Bar Harbor, Me. STUDIES ON PULMONARY ARYL HYDROCAP,)3QN HYDROXYLASE ACTIVITY IN INBRED STRAINS OF MICE This report describes some of the major parameters regulating the constitu+ tive and 3-methylcholanthrene (MCA) induced levels of, pulmonary and hepatic aryli hydrocarbon hydroxylase (AHH) activity in inbred strains of mice. Intra- traeheal instillation of 188 pg MCA in sterile 0.2% gelatin in saline resulted in preferential induction of pulmonary AHH. After treatment with this dosee of MCA, the pulmonary AHH levels of strains C57BL/6Cum, C57BL/6J, BALB/ cMai, C3H/fMai and C57L/J were observed to be induced within 24 hours after treatment. Strains DBAI2Cum,, AKR/J, SJL/J, DBA/2J and RF/J ex- presse& no such increase. At a dose of 500 µg MCA, the pulmonary tissue of DBA/2 mice did express a fourfold increase. This increase in AHH was de- termined to be quite different from the increase observed in C57BL/6 mice by: (1) specific activity of the enzymes; (2) genetic regulation; (3) susceptibility to inhibition by 7;8-benzoflavone, and! (4) spectral properties of the associated cytochromes. Thus, this paper shows that there was both a qualitative and quantitative differencebetnveen the increased levels ofAHH'observe6 in the AHH' "non-inducible" mice and the increase observed in the AHH "inducible" mice. Moreover, in crosses involving the C57BL/6Cum and DBA/2Cum,strains of mice, pulmonary AHH responsiveness to intratracheally administered MCA was regulated by a single autosomal dominant gene. On the basis of these re- sults, it seems that this genetically controlled enzyme response may play a majpr role in the ultimate susceptibility of pulmonary tissue to chemically induced'cancer. Kouri, R. E. et af. (Microbiological Associates) Chemical-Biological Interactions 13:317-331, 1976, From~ the Department of Biochemical Oncology, Microbiological Associates, Bethesda, Md. 25

226 MACROMOLECULAR DETERMINANTS' OF PLASMINOGENI ACTIVATOR SYNTHESIS -;t Neoplastic transformation of primary or early passaged fibroblasts is a6 companied by enhanced production of plasminogeni activator (PA) which n~' probably directly related to the transformation process, since increases in PA~ levels do not occur after infeetionlof fibroblasts with cytocidal viruses or leuk ~ emia viruses. The experiments initiated here aim to define some of' the factoitT- which may be involved in the controli of plasminogen activator synthesis. Chic1C l embryo fibroblasts infected with a temperature-sensitive mutanu of Rous sai,- ~ ~ coma virus, Ts 68, were selected as a model for study because such cells are, normal by morphologic and biochemical criteria when grown at 41 °C, but ait, transformed at 36°C. Since these phenotypic properties are fully reversibl ; within a few hours after appropriate shifts in temperature, this system is,' very favorable one for exploring variables that might governn activator produCs: tion. Earlier work showed that the properties of plasminogen activator are; determined by the cell, rather thaniby the transforming agentt The effects proa duced by the addition, of! several metabolic inhibitors to cultures at the time of' the shift from the permissive (36°C) to the nonpermissive (411°C) temper- ature suggest that intracellular enzyme can be maintained at a high level if RNA synthesis is inhibited. The data also show that: (1i) marke& fluctuations in, plasminogen activator production occur very rapidly following temperature shifts; (2) the formation,of plasminogen activator in this system is closely cor- ' . related with the expression of the viral transforming function; and (3) RNA ` synthesis must, precede termination, of activator formation when cultures arE shifted upward to temperatures which are nonpermissive for transformation, al- though though the nature of the relevant RNA species is unknown. The ability of ~ actinomycin to limit the disappearance of inducible enzymes following transfer4 to noninducing conditions has been~ reported for several systems and various A interpretations have been offered as explanations for these phenomena. These " include possible decreases in the catabolism of specific proteins and inhtbtthon 4; , of the synthesis of an inferred RNA species with a rapid turnover, which might ~` function as an inhibitor of translation. The data presented'~ here are as yet in-~ sufficient to permit either discrimination between these hypotheses or the ,~ elimination of other plausible explanations. RiJkin, D. B., Beal, L. P. an&Reich,, E. In: Reich, E., Rifkin, D. B. and Shaw,, E. (eds.) : Proteases and Biological'Con- trol: Cold' Spring Harbor Conferences on Cell Proliferation, Cold Spring Har- bor, N. Y.:,Co1d Spring Harbor Laboratory, 1975, vol. 2, pp. 841-847. Other support: National Institutes of Health. From the Department of Chemical Biology, The Rockefeller University, New York. PROTEASES PRODUCED BY NORMAL AND MALIGNANT CELLS IN'CULTURE This paper describes the control of plasminogen activators (PA) synthesis in two cell types, chick embryo fibroblasts infected with a temperature-sensitive mutant of Rous sarcoma virus and human embryonic lung cells. Induction of 26 PAne% RIs tioi cat, resi tral . . bN acti~ e.tr pea The pea Thi' con diN Rif In: lati 11, Ckv Nrc Yol Sto: beei pol} to I cret mic hal bee cult epit secn . prel epit big neoo in v Son. In: Am Fro; I

whic~: es ia ; ~or% e fa is. otisr; cells > but reve temi prod ator ects p the ti tempoc level tuatio , peratu, sely coQ~ ) RNA res tion, iliry o transfe various ' . Thesg hibitiq h might yet in;; or th9 227 ~-~..~.. ~ PA reqµires both RNA and protein synthesis. Deinduction of PA requires a n¢µ• RNA and, perhaps, protein to be made. If the synthesis of this deinduetive RNA is inhibited, PA production can continue for many hours under eondi- tions that are normally nonpermissive. Many different experiments have indi- cated that the observed increase in productiom of PA by neoplastic cells is the result of transformation, A number of the phenotypic changes associated with . transformation are dependent's at' least in part, upon the generation of plasmin by the PA secreted by these cells. One of these changes, the loss of intracellular actin-containing cables in neoplastic cells, can be correlated with~ the level of extracellular proteases. Moreover, these same-proteases can cause the disap- pearance of actin-containing cables in normal cells when supplied exogenously.. Therefore, plasmin acting from the external side of the plasma membrane ap- pears to be capable of causing the dissolution of these intracellular structures. ` This paper also attempts to demonstrate how, the production of PA, a particular ` component of the fibrinolytic system{ may be implicated in the etiolbgy of diverse patnological conditions. Ri/kin; D. B. and Pollack, R. . . - Y In: Ribbons, D. W. and Brew,, K. (eds.) : Proteolysis and Physiological Regu- lation. Miami Winter Symposium, New York: Academic Press,, Inc., 1976, vol. . ll1i, pp. 263-285.. Other support: NationaliInstitutes of Health, From the Department of Chemicali Biology, The Rockefeller University,, New York, and the Department of Microbiology, State University of New York at Stony Brook. UNUSUAL MANIFESTATIONS OF CANCER Among the many unusual properties of neoplasms, great attention has been focused recently on ectopic hormone secretion. Macromolecular forms of polypeptide hormones, such as big ACTH and big gastrin, have been shown to be produce& by pulmonary oat cell carcinomas, certain thymomas, pan- creatic islet carcinomas, and other related' cells. In this investigation of electron mierographs of dysplastic and normal human bronchial neuroendocrine epithe- liall cells, two statistically significant differences in organelle ultrastructure have beeni uncovered whichi appear to be relevant to the size of the hormone mole- cules produced.. Compared to the normal human bronchial neuroendocrine epithelial cells, dysplastic epithelial cells had smaller Golgi vesicles and fewer secretory granules., These ultrastructural differences, may correlate with the preferential secretion of big ACTH by dysplastic and neoplastic pulmonary epithelium. It seems, therefore, that macromolecular peptide hormones such as big ACTH or big gastrin are secreted ectopically from some dysplastic and neoplastic neuroendocrine cells partly because of underdeveloped Golgi vesicles in which the trypsinizatiomof prohormones may normally occur. Sommers, S. C. In: Finckhi E. S. (ed.): The Eflects of Environment on Cells and Tissues, - Amsterdam: Excerpta Medica, 1976, pp. 175-178. From the Department of Pathology, Lenox Hill Hospital, New York. 27

228 benzo(x)pyrene (BaP)' could enhance the clearance rate of BaP, duplicate " Pentobarbital, an anesthetic used in this laboratory for the study of pul- monary tumor induction in mice by aromatic hydrocarbons, is a very weak inducer of aryl hydrocarbon hydroxylase (AHH). Because of the possibility, however, that pentobarbital combined with the intratracheal instillation of COMPARISON OF' TWO ANESTHETICS FOR THEIR EFFECTS ON THE CLEARANCE RATE OF BENZO(a)PYRENE FROM MOUSE LUNGS experiments were conducted to compare the BaP clearance rates from mouse lungs with two different types of anesthetics, pentobarbital an&methoxyflurane. Pentobarbital was given intraperitoneally, the methoxyflurane by inhalation. Methoxyfiurane had a very slight effect on the AHH system, There were no significant differences, however, in the clearance rates of' BaP from the mouse lungs in parallel experiments employing these two anesthetics. These observa-I Francisco. From the Ihstitute of Chemicall Biology, University of San Francisco, S because mortality is minimal at the proper dosage level. Furst, A. and Wilcox, K. (University of San Francisco) Proceedings of the Western Pharmacology Society 19:32-35s 1976. tions support the preferential use of pentobarbital anesthesia in these animal studies on the basis of its ease of administ'ratiom and long-lasting effects and II. The Respiratory System CONSTITUENTS. III. SERUM ANTITRYPSIN AND BRONCHOMOTOR RESPONSES IN RATS ts,. FOLLOWING INHALATION OF CIGARETTE SMOKE AND ITS FUNCTIONAL AND BIOCHEMICAL EFFECTS ON THE LUNG _ i , . controls an& bronehodilatiom in the emphysematous animals, the nature of the *; : -~A While smoke inhalation caused bronchoconstriction in the normal activity s aggerate the functional changes in the lung nor further reduce the enzyme ditional' exposure of these animals to cigarette smoke, however, did not ex- Ad ~ ' ~ perimentally, induced emphysema showed decreased serum antitrypsin activity. ' As ia some forms of' human pulmonary emphysema; rats with evidence of ex-„AX. lationship between bronchomotor responses in rats and! lung biogenic amines. way resistance, Unexpectedly, the data eonsidere& here indicate a possible re' smoke exaggerates emphysematous lesions in animals and further increases air- effects of! cigarette smoke, this report examines whether exposure to cigarette As part of a continuing series ot investigations on the tfroncnoputmonary 28 It T F Iv

of pul- weak ibility; ion of , plicate mouse ' urane. lation.' Fre no mouse serva- nimal t, and San 229 bronchomotor response was altered by pretreatment of the rat with sympa- thomimetic amines. Because norepinephrine and dopamine evoke a broncho- constrictor response to hypoxia whereas epinephrine and synephrine elicit bronchodilation„the authors postulate that preloading catecholamine stores with an N-methylated amine (epinephrine or synephrine) causes bronchodilation, while similar preloading withi nonmethylhted amines (norepinephrine or dopa- mine) results in bronchoconstriction. This hypothesis suggests that epinephrine is the neurohumoral transmitter in the bronchial muscles of the rat and that it can also be released by such inhalants as cigarette smoke, 100% nitrogen, halothane„ and aerosoli propellants. Support for this new theory is being ob- tained by analysis of epinephrine in the blood draining the airways.. Ito;,H., Watanabe, T., Shore, S.R. and A viado, D. Toxicology and Applied Pharmacology 35(3) :403-412, 1976. From the Department of Pharmacology, Uhiversity of Pennsylvania School' of Medicine, Philadelphia. TRACHEOBRONCHIAL AND PULMONARY CYTOLOGIC CHANGES IN SHOCK Tracheobronchial smears from 1&patients who hadld'eveloped hemorrhagic shock while undergoing general endotracheal anesthesia for surgery contained morphologically abnormali histiocytes. These cells were overloaded with, Papani- colaou stain whichi compressed the nucleus against the cell membrane. No such cells were seen in smears from the 10 patients used as controls. Cytochemical staining methods were undertaken to discover the composition of the substance in the histiocytes. There was no excess ingestion of any substance detectable by PAS, Sudan 3 or alkaline phosphatase stains. All cases in shock yielded smears with histiocytes which had phagocytized material which was Prussian blue positive, indicating that' the substance contained inorganic iron. The maxi- mum proportion of histiocytes containing Prussian blue granules was 40% ini shock patients and, only 2% in normal controls. Histologic studies conducted oni rats submitted to hemorrhagic shock were carried! out to investigate this phenomenon. Ironfladen histiocytes were found in the kidneys, spleen and lungs of both shocked and control animals., However, substantially more histio- cytes containing Prussian blue positive granules were discovered in the lungs of rats in shock than in controls. It may be, therefore, that iron is deposited in the lungs in low flow states. Friedman-Mor, Z., Chalon, J., Katz, J. S., Gorstein, F., Turndorf, H., and Orkin, L. R. The JournaloJTrauma16(1)i:58-62„ 1976. Other support: U. S. Public Health Service. From the Department of Anesthesiology, Albert Einstein College of Medicine, The Btonx, N.Y.,, and'the Departments of Anesthesiology and Pathology, New York University Medical Center, New York. 29

VIRAL INCLUSION BODIES IN TRACHEOBRONCHIAL EPITHELIUM OF ASYMPTOMATIC SUBJECTS t Cytoplasmic inclusion bodies have been found in the urinary tract epij thelia of both symptomatic and asymptomatic patients, but such cells have only, been reported thus far in the respiratory tract of subjects with viral disease and' bronchogenic carcinoma. During a survey conducted for the cytodiagnosis of+ early bronchogenic carcinoma, however, cytoplasmic viral inclusions werC, . found sporadically in tracheobronchial' smears of asymptomatic patients oj~ both sexes, 18 to 80 years of age, undergoing general endotracheali anesthesiaA' for various surgical procedures. Of the 3,049'smears screened'; 1.1% contained5 ciliated' epithelial cells withi eosinophilic cytoplasmic inclusion bodies. Statistical~ analysis did not disclose any relationship between the presence of viral inclusion,i bodies and other abnormalities noted in the smears, nor was there any cor- relation between their presence and' the age, sex or smoking habits of the pa= tients. When the frequency of occurrence was assessed in relation to the sea-V son of the year, however, 60% of all these smears were found to have been', collected during January through March: It is thus probable that the majorttyl, of these patients actually had asymptomatic coronavirus, respiratory syncytial~ virus and rhinovirus infections. According to the authors, the low postoperative~ li i d n is d f cat ons rate note series seems to in comp i th icate that the presence o virall inclusion bodies is not a precursor of respiratory disease, even under stres ~ conditions. Katz, J. S., Chalon, J. and Turndorf, H. . Chest 69(4),:556•558,,1976. Other support: UL S. Public Health Service. From the Department of Anesthesiology, New York University Medical Center, New York. TRACHEOBRONCHIAL EPITHELIAL MULTINUCLEATION IN PREECLAMPSIA Multinucleated ciliated epithelial cells have been seen in the past in the sputum of patients who had undergone a bronchoscopy 48 hours previously and, in a different study, in the tracheobronchial washings of' patients under- going surgery for a wide variety of extrathoracic tumors. Now, smears were made from the tracheobronchiat washings of 40 nonsmoking parturient women who were undergoing cesarean section or forceps delivery under general anes-'~ thesia administered via an endotracheali tube. Results showed that the smears "' from 12 patients with preeclampsia of more than 24 hours' duration contained'.,, significantly more multinucleated ciliate& cells than, those of 13' healthy con='' trols, eight women with preeclampsia of less than 24 hours' duration, and seven ': women with associated diseases other than preeclampsia. While the explanation .; of this phenomenoni is still in doubt, it may be that it is related to immune , mechanisms that accompany preeclampsia. Chalon;,J., Marx, G. F. and Katz, J. S. Archives of Pathology and Laboratory Medicine 100:427-428, 1976. 30

231 tract epi- ]ls have only disease and diagnosis of ions were patients of anesthesia' o contained . Statistical al inclusion re any cor- of the pa- to the sea- have been he majority: syncytial , stoperative nresence of nder stress i5 •.'t'f T7 ast in the,?':; previously~ nts under- ears wele ;S ~ nt womefl eral anesf4~ he smears - contained~ althy con-k~ and sevelt - , ~xpianation ir p immune II -~ Other support: U. S: Public Health Service. From the Department of Anesthesiology, New York University Medical' Center, New York, and the Department of Anesthesiology, Albert Einstein, College of Medicine, The Bronx, N. Y. ay-ANTITRYPSIN AS A SYSTEMIC DETERMINANT OF LUNG STRUCTURE AND FUNCTION The first section of this review demonstrates that slow, progressive loss of lung parenchyma begins in mid to later life in persons with a homozygous de- ficiency of xl-antitrypsin and the ZZ phenotype for this protein, and, perhaps also im smokers with a heterozygous deficiency of al-antitrypsin and the MZ phenotype for this protein. The al-antitrypsin is made andlor destroyed in the liver and' its d'eficiency' has been corrected by liver transplantation. If' this de- ficiency causes the lung destruction, then al, antitrypsin is indeed a systemic determinant of lung structure and function. Evidence that the lung is a meta: bolic orgaw and that in this role it influences organ function elsewhere in the organism is also presented here. Most research on the etiology of this genetical- ly-determined type of emphysema now focuses on the hypothesis that this condition~ is caused by a deficiency of a1-antitrypsin, which results in the unre- strained action of one or more enzymes on pulmonary tissues. However, it is equally passible that al-antitrypsin acts primarily at' a remote site and sends toxic products to the lung. Indeed; it is even possible that a1-antitrypsin has other as yet undiscovered functions, or that a1-antitrypsin deficient patients have othei7 defects that could cause emphysema. However, this genetic disease remains the only solid clue to the cause of some forms of human emphysema and may eventually enable investigators to understand' the origins of other forms as well. Should some common biochemical mechanism be proved,, it is possible that' the same drugs would be effective in the prevention of all types of emphysema. Cohen, A. B. Inz Crystal, R. (ed.) : The Biochemical Basis of Pulmonary Function, New York: Marcel Dekker, Inc., 1976, pp. 313-360. Other support: National Heart and Lung Institute. From Temple University Medical School, Philadelphia. HYDROXYPROLINE CONTENTS AND PROLYL HYDROXYLASE ACTPVITIES' IN LUNGS' OF RATS EXPOSED TO LOW LEVELS OF OZONE Collagem synthesis in the lungs of adult rats exposed to 0.8 ppm ozone was studied by estimating hydroxyproline (Hyp) content and, by following the activity of prolyl hydroxylase (PH)i, a crucial enzyme in the pathway of' col- lagen biosynthesis. In• the early phases (1-2 days) of ozone-induced injury,, PH' activity was increased twofold over eontrol' values and the amount of coll~gerr synthesize& (as estimated by Hyp formation) was double that of the noncol- 31

232 lagenous protein synthesized. This resulted, by the third day, im a significant increase (291°!0 ) in total lung collagen. However, with continued exposure (up to seven days), noncollagenous protein synthesis increased to the point where the ratio of collagen to noncollagenous protein synthesized was comparable to that of controls. When the exposed (0.8' ppm 03/7 days) animals were placed in filtered ambient air, PH activity returned to normal in 13 days whereas Hyp content remaine& elevated for up to 28 days. These results indicate that in- creased lung PH activity is temporally related to ozone exposure and suggest that this modeli may be useful inithe study of lung injury and repair processes. Hussain; M. Z., Cross, C. E.,,Must'afa, M. G., and Bhatnagar, R. S. Life Sciences 18(9):897-904, 1976. ' Other support: U. S. Public Health Service. From, the Department of Internal Medicine and California Primate Research Center, University of Cali)ornia, Davis, and the Connective Tissue Biochemistry,_'. Laboratory, Uhiversity of California School of Dentistry, San Francisco. ' EXPOSED TO CIG,ARETTE SMOKE BIOCHEMICAL EFFECT OF OZONE EXPOSURE IN RATS Pulmonary glucose-6-phosphate dehydrogenase (G-6-PD)~ activities in the cytosol-rich fraction from control, ozone-exposed, cigarette-exposed, an& ciga- rette-plus-ozone-exposed rats are reported in this paper. Whereas ozone-exposed rats show an 11% decrease from the activity level of untreated controls, rats exposed! to cigarette smoke for five weeks show a significant ( 27 %) increase: Subsequent exposure to acute, high-dose ozone (3 ppm for 4 hr) decreasesl the augmented G-6-PD activities to near normal levels, indicating that rats ex posed to cigarette smoke-induced augmentation of G-6-PD are still susceptible to G-6-PD inhibition following ozone exposure. However, G-6-PD activities in the rats exposed to both cigarette smoke and ozone remain slightly elevated above control levels: The importance of this enzymatic effect with respect to the susceptibility of smokers to oxidant' exposures is unknown. +- .,,'_r ; I f . Peirce,,T. H., York, G. K.,,Franti, C. E., and Cross, C. E. ; rlrchives of Environmental Health 31(6) :290-292, 1976. '~ Other support: U. S. Public Health Service, From the Department of Internal Medicine;, University of California School Medicine, Davis. OZONE INTERACTIONS WiTH LUNG TISSU!E. BIOCHEMICAL APPROACHES .~ ~ a Summarizing the biochemical effects of ozone (03) on, the lung; the . thors review the molecular mechanisms of 03-induced damage, the acute hi dose effects, the chronic low-dose effects, and the decreased and increased s ceptibility to lung 03 dpmage., It is most likely that the molecular mechanis of 03; cytotoxicity operate through oxidation~ and/or oxidation products, inte!- 32

233 action with cellular membranes and resulting depressions of many cellular enzymatic activities. Some experiments suggest that, in animals, a wide variety of antioxidant substances provide relative protection against 03 exposure. That high-d'ose exposures severely depress several' enzymatic activities almost cer- tainly indicates acute 03-induced lung cytotoxicity. However, if the animal5 are allowed to recover from nonlethal acute exposures, some of the lung metabolism parameters are greatly augmented, apparently reflecting the cellular reparative and proliferative response to injury;. Low-leveli 03 exposure has a similar effect within 24-28 hours. According to some morphologic observations, prolonged exposure of experimental animals to 03 causes mild fibrosis of lung parenchyma. The concurrent increase in lysosomal enzyme activity also suggests the accumu+ lation of inflammatory cells, mainly macrophages, in the lung. This perhaps ac- counts for the reported increase in collagen synthesis during the early injury and repair phases. Current evidence also suggests that a significant' aspect of susceptibility to prolonged exposure is "adaptive" tolerance, a concentration- dependent phenomenon whereby the metabolic augmentations induced by low- level' exposures reach their maximum two to four days after the initial exposure then diminish„disappearing completely if exposure is continued. Thus far, how- ever,, there is only a suggestioni that helpfuli adaptations to chronic low-level 03 exposures occur in man. Since numerous variables can affect' an organism's response to toxic substances, the authors recommend caution in interpreting the overall effects of environmental pollutants such as 03 on parameters of lung biochemical activities, Cross, C. E. et al. The Arnerican Journal'of'Medicine 60(7,) :929-935, 1976. Other support: National Institutes: of'Health: From the Section of' Pulmonary Medicine, Department of Internal Medicine, University of' California Schools of Medicine, Davis and Los Angeles. STIMULATION BY CIGARETTE SMOKE OF GLUTATHIONE PEROXIDASE SYSTEM ENZYME ACTIVITIES IN RAT LUNG Glutathione peroxidase is part of the enzyme systemi involved in generating reduced nicotinamide adenine dinucleotide phosphate (NADPH ), and as such constitutes part of a potentiallyimportantint'errelated reductive antioxidant defense mechanism in erythrocytes an& lung tissue. This study investigates the short-term, in vivo effects of experimental cigarette smoke exposure on gluta- thione peroxidase-related enzyme systems in pulmonary tissue, and attempts to correlate any changes with histopathologic observations within the range of light microscopy. Male Sprague-Dawlfry rats, 30-days-old, Caesarean d'erived, and specific pathogen-free; were exposed to 13 cigarettes daily for 21 consecutive days withi a Walton reverse-smoking exposure apparatus, At the end of that time, spectrophotometric assay of, homogenized lung tissue showed increased activity of gllrtathione peroxidase (34% ), glutathione reductase (24%), and glucose-6,phosphate dehydrogenase (38%) compared to control values. This particular level of smoke exposure, however„did not cause detectable histolbgic lesions. It is suggested that short-term, low-level cigarette smoke exposure cam

initiate metabolic alterations in lung cells without evoking histologic changes sensitive method for assessing the effects of cigarette smoke or some of its posures. Certainly, the presence of these biochemical abnormalities suggest 2 lead to more overt structural changes following more intense or prolbnged ex-' li~ alterations represent but' one stage in~ a multistage process that could' eventually detectable by light microscopy. It is possible, however, that such biochemical constituents on the mammalian lung, York, G. K., Peirce, T. H., Schwartz, L. W., and Cross, C. E. Archives of Environmental Health 31(6) :286-290, 1976, Other support: U. S: PublirHealth Service. From~ the Department' of Internal Medicine, Medicine, Davis. tst University of California School of ASPIRIN AND EXERCISE-INDUCED ASTHMA Since exercise causes release of prostaglandin and;sometimes also induces Y~~g asthma, it' seems possible that exercise-induced bronchoconstriction may be due, to the release of prostaglgndin. Aiso, because acetylsalicylic aeid' inhibits pros-j ~ taglandin synthetase, thus diminishing prostaglandin release, it may be that as-; ' pirin decreases or inhibits the bronchoconstriction of exercise-induced asthma. In order to test this hypothesis, four asymptomatic subjects with a history of f~ exercise-induced' asthma and, a 10% or more drop in Forced Expiratory Voltime in one second (FEVL) were selected to study the effect of'aspirin~on pulmonary` "~ function following exercise. The double blind method was used to administer, ;t aspirin (500 mg) an& placebo prior to exercise. Pulmonary function tests did f: not show' any difference in response between aspirin or placebo. It is concluded ~ thab in these four subjects aspirin did not prevent the bronchovascular response,' ~` perhaps suggesting that prostaglandins have no significant role in exercise-in- ~' duced asthma. The authors suggest, however, that a larger series of patients must be studied before this hypothesis can be completely rejected: '~ Taveira da Silva, A. M. and Hamosh, P. Prostaglandins 111(1),:71-75; 1976. From the Department of Physiology„ Biophysics and Medicine, Georgetown Universit Schools of' Medicine and Dentist Washi n D t C '""a y ry; , . ng o LIPOPROTEIN LIPASE IN RAT LUNG: EFFECT OF DEXAM.ETHASONE Lipoprotein lipase, an enzyme active in the capillary endothelium, regulates ' the uptake of blood triglyceride-fatty acids. Lipoprotein lipase activity was de- termined in the lungs of rats after administratiom of four hormones - dexa- methasone, 1-thyroxin, estradiol-17,B and progesterone - which have a known effect on lipid metabolism lipoprotein lipase activity, or lung surfactant synthe- sis. In addition, lung lipoprotein lipase activity was studied in diabetic and lac- ; tating rats, Dexamethasone administration caused, a rise of 70% in the level of 34 H B G F ]V cy~ int' pre the pet the wh dis;, trec ene cell inc bec mu wei the] filai syst regl, per~ Lau Cell Froi giun

235 activity of lipoprotein lipase in acetone powders of lung and a 100% increase in the amount of enzyme released during heparin infusion into isolated, perfused lungs. Enzyme activity was higher in lungs of females than of male rats; how- ever, the level of activity was unaffected by estrogen or progesterone administra- tion to either male or ovariectomized rats. Diabetes, hyperthyroidism or lacta- tion di& not change lipoprotein lipase activity in the lung, The constant presence of lipoproteinilipase activity in the lung suggests that this organ is able tolmain- tain a steady supply of triaeylglycerol4fatty acids under circumstances that markedly impair the ability of other tissues to utilize this substance. Stimulation of enzyme activity by dexamethasone could lead to increased' uptake of triacyl- glycerol-f'atty acids by the lung and may thus be a contributing factor to corti- costeroid-induced enhanced surfactant synthesis. Hamosh, M., Yeager, H., Jr., Shechter, Y., and Hamosh, P. Biochimica et Biophysica Acta 431:519-525, 1976., Other support: .Washington Heart Association. From the Department of Physiology and Biophysics, and the Department of Medicine, Georgetown University School of Medicine, Washington, D. C. INTRACYTOPLASMIC FILAMENTS IN PULMONARY LYMPHATIC ENDOTHELIAL CELLS Earlier observations by various investigators have suggested that intra- cytoplasmic filaments may be actinoid parts of a contractile system. Since fine intracytoplasmic filaments occur in pulmonary lymphatic endothelial cells, the present investigators used tissue blocks from 18' neonatal rabbits to compare the cytochemistry and ultrastructure of these filaments with myofilaments of thee peribronchial and pulmonary vascular smooth~ muscle cells. Two types of endo- theliali filaments were observed in the pulmonary lymphatic cells: thin filaments which lie close to the abluminal cell membrane and thick filaments which are dispersed throughout the cell eytoplasmi Following heavy meromyosin (HMM) treatment„ characteristic arrowhead complexes formed in the thin lymphaticc end'othelial filaments as well as in the actin filaments of the smooth muscle cells. There was no detectable reaction of HMM with the thick filaments. After incubation with EDTA„ the thim filaments were labile, andl the thick filaments became the major filamentous component in the endothelial cells. In smooth muscle cells, the actin myofilaments were also labile while the larger filamentss were stable. These observations support the hypothesis that the actin-like endb- theliali lymphatic filaments form part of a contractile system, while the thick filaments constitute a plastic cell skeleton. The significance of the contractile system in lymphatic endothelial cells might lie in a mechanism for the active regulation of' the endothelial intercellular junctions and gaps, and hence the permeability of the lymphatic endothelial cell lining. Lauweryns, J. M., Baert, J. an&DeLoecker, W. Cell & Tissue Research 163 (2) :111-124, 1975. From the Katholieke Uhiversiteit te Leuven School of Medicine, Leuven, Bel- gium. 35 I

v 236 FINE FILAMENTS IN LYMPHATIC ENDOTHELIAL CELLS Severall types of cells contain fine cytoplasmic filamentswhichcloselyri ~1.: resemble the myofilaments of muscle cellfi. The staining characteristics of these,* various cells suggest that they may be actinoid and form part of a contractile ~ system. Since fine intracytoplasmic filaments occur ini lymphatic endothelial '.4 cells, the authors postulated that they belong to a contractile filamentous sys--'. ' tem present in these cells and investigated their fine structUre after incubation with heavy meromyosin (HMM) and EDTA. Results of subseqµent electron F microscopy showed that pulmonary endotheliali cells of neonatal rabbits contain a large number of fine cytoplasmic filaments measuring either, 5-6 nm (thin) or'' 9-10 nm (thick). The thin, filaments react with HMM, forming arrowhead structures, and appear labile when treated with EDTA. The thick filaments d'o' notI react with HMM and are stable in EDTA. Comparison ofl the thin filaments ~:+ with myofilaments of smooth muscle cells suggests that the thin filaments are actin-like and form part ofl a contractile system, while the thick filaments con-' ' stitute an, elastic celll skeleton. The chemicali structure of these filaments remains I , to be explored. It is suggested that a contractile system in, lymphatic endothelial `~ cells might be involved in the active regulation of the endothelial intercellular ?',~ gaps or junctions and, hence, the permeability of the lymphatic wall. Such a system may be involved as well in, other cellular processes such as endocytosis, transport and secretion. Lauweryns, I. M., Baert, J. and de Loecker, W. The Journal of Cell Biology 68 (1) :163-167; 1976. From the Laboratory of Histopathology and the Laboratory of' Pathological Biochemistry, Katholieke Uhiirersiteit te Leuven School of Medicine, Leuveni ri Belgium~ ALVEOLAR TYPE II CELLS: STUDIES ON THE MODE OF RELEASE OF LAMELLAR BODIES '} A Although the evidence increasingly suggests that type II alveolar cells are responsible for the production of pulmonary surfactant, whether they actually ,, synthesize it is still inconclusive. One of the problems remaining to be solved before this can be stated unequivocally is that of secretion, Previous studies suggest that lamellar bodies are released by merocrine secretion. Here, mor- phologic evidence is presented! which supports the view that lamellar bodies are . in fact releasedI by exocytosis. Freeze-fracturing and electron microscopy of rat lung tissue show that there are sites at the apical! surface of type IIi cells where,,. the limiting membrane of the lamellar body is clearly fused with the cell plasma , membrane and that an open channel exists between the contents of the lamellar body and the alveolar space. At these sites, the lamellar contents extrude into ' the airspace with consequent loss of the highly compact organization of intra- cellUlar lamellar bodies. The intactness and continuity of the membranes eam' be traced for the full extent of the exocytosis site. Freeze-etch replicas of type II cell membranes show, depressions which may represent the sites of dis- charged lamellae. Also seen are tongue-like folds which may be cytoplasmic extensions surrounding the releasing lamellar body and which may flap over 36

237 hich closely tics of these ~a contractile endothelial entous sys: r incubation ent electroii ~~ bits contatn : m (thin) or arrowhead A laments do'3 in filaments Raments are ments con-` ts remains ~ endothelial tercellular~I 11. Such a { ; ndocytosis, 1 cells are , ~ actually, e solved : k's studies ~re, mor- : odies are ; (py of rat' r ; s where, plasma . I ]amellar lude into bf intra- lnes can I of type ~ of dis- ~lasmic 4p over the exocytosis pit after discharge. Electron-photomicrographs of the alveolar space show disorganized lamellar whorl5 which appear to be unraveling to produce tubular myelin, In view of the unusually large size and lipid compo• sition of larnellar bodies, a mechanism involving hydration of mucopolysac- charide contents as an aid to expulsion of lamellar contents is suggested. Ryan, U. S., Ryan, J. W: and Smith, D. S. Tissue&Ce1l, 7(3) :587-599, 1975. Otlier support: U. S. Public Health Service, Hartford Foundation and' the Heart Association of Palm Beach County, Fl13. From the Papanicolaou Cancer Research Institute and the Department of Medi* cine, University of Miami School'of Medicine, Miami„Fla. RAPID PURIFICATION OF HUMAN TRYPSIN AND CHYMOTRYPSIN I Highly purified, maximally active enzymes are needed in order to evaluate the properties of humani pancreatic proteases, particularly the stoichiometric relationship between the proteinases and their inhibitors. Earlier reports have shown that the Kunitz bovine pancreatic trypsin inhibitor, commercially avail- able as Trasylol, reversibly complexes with cationic human trypsin an& chy- motrypsin I but does not inhibit any of the other humani pancreas proteinases. A one-step method for the purification of bo'h human cationic trypsin an& chymotrypsin I is described whichiis basedion these observations. The technique involves the adsorption of the enzymes onto a Sepharose-Tnasylol affinity col- umn; Subsequent elution and'separation of the two enzymes is accomplished by a gradient of decreasing pH. Thus treated, some samples of bovine trypsin with aslittle as 50% titratable active sites have beem converted to chymotrypsin- free trypsin preparations with 87 90% available active sites. Johnson, D. and Travis, J. Analytical Biochemistry 72:573-576; 1976. Other support: National Institutes of Health. From the Department, of Biochemistry,, University of Georgia„ Athens. HUMAN LEUKOCYTE GRANULE ELASTASE: RAPID ISOLATION AND CHARACTERIQATION It has been proposed that, the chronic obstructive lung, disease which de- velops in, individuals deficient in a-l-proteinase inhibitor (x-1-antitrypsin) re- sults from the elastolytic degradation of lung alveoli' by enzymes in alveolar macrophages and the granules of polymorphonuclear leukocytes. At least four such enzymes with different electrophoretic mobilities have been observed in the leukocytic granules, but none has been successfully isolated. Here, the au, 37

238 thors describe a simple two-step procedure for isolating granule elastases, based om the weak inhibitory activity of' bovine Kunitz basic trypsin inhibitor (Tra-. ~__ sylol) toward elastase. The technique involves affinity chromatography of crude. leukocytic granules extracts on Sepharose-Trasyl'ol„ followed by ion-exchange ~ chromatography on CM-cellulose to resolve the isoelastases. All were found to be glycoproteins. The major form had a carbohydrate content essentially com- posed of only neutral sugars and a molecular weight near 30;000 daltons, with'` i" that of other forms being slightly higher. Preliminary structural analyses indicate ~ that all the elastase isoenzymes have identical NH2-terminal sequences, sug- ~ gesting, that the observed differences in mobility are due to minor changes in carbohydrate content rather than to different degrees of activation of a com-''; mon zymogen. Human granulocytic elastases are less active on ligament elastin than porcine pancreatic elastase but both are inhibited by synthetic elastase „ active site-directe& low moleeultir weight compounds, as well as by plasma a-1- proteinase inhibitor. Wi'th the latter, a stable complex having a molecular weight " ' of 78,000 daltons is formed, indicating combination in a 1: I ratio. `' Baugh, R. J. and Travis, !. From the Department of Biochemistry,,Uni'versity of Georgia, Athens. Biochemistry 1i5(4) :836-841, 1976. Other support: National Institutes of Health. HUMAN ALPHA-I-PROTEINASE' INHIBITOR MECHANISM OF ACTION: EVIDENCE FOR ACTIVATION BY LIMITED PROTEOLYSIS Although the role of human al-proteinase inhibitor (al-PI) in controlling tissue destruction by endogenous serine proteinases is well-known, the mech-, anism~ by which this inactivation occurs is not yet understood. To gain insight, into this process, an attempt was made to, clarify the steps involved during _ ' inactivation of serine proteinases by re-isolating the a1-PI (formerly called x,-anti'trypsin) from complexes formed:, with, porcine trypsin. The re-isolated protein ('xl-PI`) was found to be noninhibitory and to have a lower molecular. i° weight thanthe native inhibitor (45,000 vs 53.000 for ai-PI). Sequence analy- ` sis of al-PI * showed that an amino terminal peptide had been lost, apparently' the result of cleavage aU a Lys-Thr bond. These results indicate that al-PI exists as a pro-i'nhibitor which is activated by limited proteolysis. As a conse- quence of this activation; the inhibitor traps the activating proteinase by form* . ing a stable complex, presumably by acyJationi of the serine hydroxyl of the en- zyme to a carboxyl group of the inhibitor. It may be concltided, therefore, that zyme limited! proteolysis is the first step in this inhibitory mechanism. ". Johnson„ D. A. and Travis, I. Biochemical and Biophysical Research Communications 72(1) :33-39; 1976. Other Pupport: National Institutes of Health. From the Department of! Biochemistry, University of Georgia, Athens. :~ b f p. . u fc o, p tv g S' 0 c r t ,

based ( Tra- ; crude hange ~nd to com- , with ° dicate sug- ges in' com-` elastin lastase .: a a-1- eight 239 ISOLATION OF ALBUMIN FROM VWHOLE' HUMAN PLASMA AND FRACTIONATION OF ALBUMIN-DEPLETED PLASMA The isolation of proteins from blood plasma or serum is often complicated by the presence of albumin as a contaminant. Particularly significant in plasma fractionation is the fact that~ many proteins of current biologic interest have physical properties similar to those of albumin{ especially with regard to molec- ular weight and electrophoretic mobility. A previously described proceduree for the separation of albumin from those other plasma proteins by adsorption on Sepharose-Blue Dextran columns had major disadvantages. In the present paper, the authors recommend a modification of the affinity gel, which substi- tutes the dye Cibacron Blue F-3-GA for Blue Dextran, resulting in~ much greater capacity for albumin (40 mg albumin bound/ml'of gel) and in a stabler Sepharose-ligand conjugate. Passage of whole human plasma through this type of column removes about 98% of the albumin. This can be quantitatively re- covered by desorption with NaSCNL The albumin-deplete& plasma is readily resolved into discrete fractions by conventional biochemical! techniques. In par- ticular, plasma proteins with properties similar to those of native human plasma albumin are readily resolved by ion-exchange chromatography of the Sepharose- dye-treated plasma on DEAE-cellulose. Even thou& it has been shown that the dye has an affinity for enzymes utilizing AMP for reactions and even though it also has been suggested that, it resembles a nucleotide in shape thus binding to any protein with a "nucleotide fold4° the mechanism by which Cibaeron Blue- Sepharose interacts with human plasma albumin has not yet been determinedi Nevertheless, the technique described here is simple, inexpensive, easily re- peatedj and sufficiently mild that denaturation is minimized, which makes it most useful for analyzing fluids with~a high albumin content. Travis, J. et al: Biochemical Journal 157:301-306, 1976. Other support: National Institutes of Health. From the Department of Biochemistry, University of Georgia, Athens, and Marshfield Clinic Foundation, Marshfield, Wis. LUNG ANTIPROTEINASE: A POTENTIAL DEFENSE AGAINST EMPHYSEMA DEVELOPMENT Extracts of certain polymorphonuclear leukocytes andi alveolar macro- phages are capable of inducing experimental emphysema. This paper reports the presence of an antiproteinase in canine lung lavage which is different from the known serum antiproteinases and might function as a potential regulator of lung proteolysis, hence aiding in~ the defense against those proteinases thaY may cause pultnonary emphysema. In this study, lung antiproteinase was ex- tracted by bronchial' saline (0.95% NaCI) lavage of freshly exeised. saline- perfuse& dog lungs. The crude antiproteinase concentrates prepared from the 20 dog samples were tested against various proteinases. As could be seen, the 39

240 ,t inhibition index (amount of inhibitor necessary to cause 50% inhibition of p ) ;:. specific enzyme) of the lung antiproteinase diffcred from the indexes of serum,*~ soy bean trypsin inhibitor andl ovomucoid. This high molecular weight anti-,,- proteinase was heat labile and a strong inhibitor of pancreatic elastase esteroly tw sis. Although elastase alone or elastase mixed with a sham protein~ instilled' into ; a bronchus gave emphysematous lesions, no lesions were induced when the ;' elastase was first mixed~with lung antiproteinase. Weinbaum, G. et al. American Review of Respiratory Disease 113(2)1:245-248,,1'976. Other support: National Heart and Lung Institute. From the Pulmonary Disease Section and Research Laboratories, Albert Eiir ~ stein Medical Center, Philadelphia. ROLE OF LEUCOPROTEASES IN THE GENESIS OF EMPHYSEMA ` Papain an& other exogenous enzymes with strong elastolytic properties7 have been shown to produce experimental emphysema in animals. In the ,- J~ search~ for endogenous proteases which could! damage alveolar walls in a way similar to papain, dogs were exposed to aerosolized homogenates of dog poly- morphonuclear Ipucocytes (PMN). The ability of other dog cells and PMN from, different species to produce experimental emphysema in dogs was also "`' investigated. The contents of human and dog PMN, as well as dog alveolar 4~ macrophages; produced emphysema-like lesions which were quite similar to ~ those induced by papain, while dog monocytes and rabbit PMN did not. Fur- r. ther work showed that only cells which have significant proteolytic activity at ` neutral or alkaline pH were capable of producing emphysema. Partial purifica- ~ tion of the active agent was obtained from an acetone-precipitated and desic- ;~' cated preparation of the crude homogenate. The fraction soluble in 0.15M NaCI was most active. This impure fraction did not have esterolytic or elastoly- tic properties when measured by the usual procedure with synthetic substances. ~; The production of experimental emphysema took place at the air-lung interface ~ and not by the circulatory route in, this model. Finally, an inhibitor fbr this , enzyme fraction and pancreatic elastase has been found in lung lavage material. The inhibitor is not alphal-antitrypsin but has some immunologic and' biochemi- ,' Kimbel, P. and Weinbaum, G. ' cal similarities to alpha2-macroglobulin. The significance of these findings in ~ relationship to human emphysema remains to be determined. ~ In: Junod, A. and DeHaller, R. (eds.): Lung Metabolism, New York: Aca- From the PultnonaryDisease Section, Albert Einstein Medical Center, Phila- delphia~ demic Press, Inc., 1975, pp. 25-41. Other support: National Heart and Lung Institute. 40 IN AC 7-k, tak( Stil. vasc rab. bini h ov mei inh aor cen inti twc sim nar ' Co, by hib: upt.. ketc inhi coni Sari Pro( 197 Ott Me( Fro Sou~ ME BY

241 III. Heart and Circulation INVIV'O INHIBITION OF CHOLESTEROL UPTAKE IN RABBIT AORTAS' BY 7-KETOCHOLESTEROL Previous reports indicate that in man and some animals, the addition of 7-ketocholestcrol to the perfusion fluid' inhibits in vitro arterial cholesterol up- take, possibly as a result of' the competitive inhibition of HMG-CoA reductase. Still lacking, however, are published data concerning such inhibition in the vascular wall in vivo. The experiments described here demonstrate that in rabbits, the intravenous injection ofl 7-ketocholesterol, rendered soluble by com- bining it with~ bile salts, inhibits aortic cholesterol uptake. The effect was not, however, as markedl or as uniform as previously note& infthe in vitro experi- ments, probably because in the injected animals plasma levelk did not reach the inhibitory threshold. Administration by gastric intubation also failed to inhibit aortic cholesterol uptake, probably for the same reason. Plasma cholesterol con- centrations plotted against plasma 7-ketocholesterol concentrations after multiple intravenous injections of the latter show that there is a direct reltitionship be- tween the two steroids, suggesting that they bind to plasma lipoproteins in a similar manner. Contrary to the previously demonstrated fact that human~ coro- nary arteries do not synthesize cholesterol in vitro, indicating a lack of HMG- CoA reductase activity, there was significant inhibition of cholesterol uptake by these vessels in the presence ofl 7-ketocholesterol. Thus, it is unlikely that in- hibition of this enzyme plays a significant role in the inhibition of cholesterol uptake by aortas or coronary arteries. It is concluded that the effect of 7- ketocholesterol on aortic cholesterol uptake is mediated through competitive inhibition, especially in view of the direct relationship between the plasma concentrations of the two steroids. _ Sarma, J. S. M., Fischer, R.,,Ikeda, S., and Bing; R. J. Proceedings of the Society for Experimental Biology and Medicine 151:303-306, 1976. Other support: Hoover Foundation, Norris Foundation, and Ross and Merle McCollum. From the Huntington Memorial', Hospital, Pasadena, Call, and the University of Southern California, Los Angeles. MECHANISM OF INHIBITION OF CHOLESTEROL UPTAKE BY THE ARTERIAL WALL The studies described here deal with lipid synthesis and cholesterol uptake in isolated perfused coronary arteries and aortas of mani rabbit and'! pig, and, most specifically, with the inhibitory effect of 7-ketocholesterol in both in vitro and in vivo animal preparations. In vitro; cholesterol uptake was significantly inhibited im human coronary arteries and aortas of pigs an& rabbits by 7-keto- cholesteroli concentrations of approximately 700 nmoles/ml plasma in the per- fusion fluid. The inhibition was reduced when 7=ketocholesterol concentrations dropped below 50 nmoles/ml. Lipid synthesis was never affected by this steroid. In vivo, the inhibitory effects of 7-ketocholesterol in rabbits were more difficult 41

242 to demonstrate. They could only be shown after repeated intravenous injectioti~ of 7-ketocholesterol solubilized with bile salt (Na•glycocholate). The metabo fate of 7-ketocholesterol and the nature of its effect on cholesterol' are dis• cusse& at length. There is the possibili'ty, as yet unproven, that both cholester6~ and 7-ketocholesterol actively compete for identical and specific binding sites ~t`bi the cell surface. It also might be possible that an increase in 7-ketocholestero~ in plasma leads to an increase in i intracellular, concentrations of this steroid thu~- i hibitin ite l t l t f th ll b fi h H d o es er across rane. n g c o ero rans e ce mem owever, e n c II nh Bing,,R.1. et al: clusions on the nature of inhibition must await further experimentation. I In: Day, C: E. (ed:) : Atherosclerosis Drug Discovery, New York: Plenttt>; Publishing Corporation, 1976, pp. 419-435. ", "•' Other support: The Hoover and Norris Foundation. From the Huntington Memorial Hospital, Pasadena, Cal., and the University o th lifo l ' ~ S C i L A ou ern~ rn os nge es. a a, LIPID METABOLISM IN PERFUSED HUMAN AND DOG CORONARY ARTERIES' Using materials perfused in vitro, these investigators studied lipid metabo; lism, in human and dog coronary arteries and in human saphenous veins. The formation and uptake of lipids in human arteries were studied under a variety of experimental' conditions, including exposure to carbon monoxide. The effecU of eollagenase on lipi&synthesis and transport in carotid arteries of dogs was" also studied. Cholesterol uptake in human blood vessels was inhibite& by the addition of 7-ketocholesterol to the basic test perfusate (human 'plasma with; [3H]-cholesterol and [h4C]+acetate. Both atherosclerotic and' normal human eoro- nary arteries incorporated [14C]-acetate into lipids but failed to synthesize either ` cholesterol or cholesteroll esters. Similar results were noted in human saphenous N ' veins perfused at arterial pressure. Cholesteroll uptake from the perfusion fluid' F` was demonstrated in atherosclerotic and normal human coronary arteries as: well as in human saphenous veins. Carbon monoxide increased permeability ` of the arterial wall to cholesterol uptake. In dog arteries exposed to collagenase, marke& increases in cholesteroli uptake were found, but total lipid synthesis I was reduced; the relative synthesis of individual lipids:remained unchanged. The. J results presented in this summary paper illustrate that human coronary arteries, as well as human saphenous veins, synthesize lipids but not cholesterol. Cho1~ esterol flux into the artery is augmented by carbon monoxide an& colltigenase '' through competitive inhibition. Sarma, J. S. M. et al. (Bing, R. J.) The American Journal of Cardiology 35(4) :579-587; 1975. while 7-ketocholesterol activity inhibits cholesterol uptake in the arterial wall S Other support: Norris Foundation, Hoover Foundation~ an& Wright Founda- '' "j, -, tion. From the Huntington Memorial Hospital, Pasadena, Cal., and the University of r Southern California, Los Angeles: 42

243' etabo= The'' . variety I: effect t igs was )f" ~ by the,t , a with'', 3:= n coro-%? I# either 0henous lon fluid ~ eries as ` eability ~ agenase, ynthesis ~ged. The.,,? arteries, 7", 'L Chol- agenase,ff ~ rial wall ;;- nisms: METABOLIC FATE OF INGESTED AND INJECTED 7-KETOCHOLESTEROL IN THE INTACT RABBIT Having previously, shown that 7-ketocholesterol inhibits cholesterol uptake into coronary, arteries in vitro and in vivo, the authors now describe the fatee of injected and ingested 7-keto (4-14C) cholesterol in the intact rabbit. The animals were sacrificed 24 hours after administration of the radioactive steroid; and different specimens were collecte& from the aorta, stomach, and iatestinal contents for lipid analysis by thin-layer chromatography. All samples showed measurable amounts of radioactivity in the 7-ketocholt:sterol fraction as well as in the other lipid fractions on the thin-layer chromatography plate. The aortic uptake of 7-ketocholesterol was relatively low. These results demonstrate that rabbits are capable of absorbing as well as partially metabolizing 7-ketocholes- terol and indicate that they excrete it in conjugated form, mainly through~ the bile. In contrast to what occurs with 7-ketocholesterol, the amount of cholesterol already present in the vascular wall probably determines its uptake. It is sug- gested, therefore, that these two steroids may trigger different uptake mecha- Sarma, J. S. M.,,Bing, R. J., Ikeda, S:, and Fischer, R. Artery 2(2) :153-160, 1976. Other support: The Hoover Foundation„ Wright Foundation and Norris Foundation, From the Huntington Memorial Hospital and the Huntington Institute for Ap. plied Medical Research, Pasadena, Cal., and the University of Southern Cali- fornia School of Medicine, Los Angeles. IN VITRO INHIBITION' OF CHOLESTEROL, UPTAKE IN HUMAN AND ANIMAL ARTERIES BY 7 KETOCHOLESTEROL This report demonstrates that 7-ketocholesterol, an analog of cholesterol, can inihibit the uptake of cholesterol, by the arterial wali of several species. Pulsatile pressure was used' to perfuse human and pig coronary arteries and rabbit aortas in~ a modified Lindbergh apparatus with blood plasma obtained from the same species. Uptake of cholesterol by the arterial wall was measured using [3H]-cholesterol as tracer. Percent distribution of synthesized; lipid frac- tions was determined by thin-layer chromatography and liquid scintillation counting: Inhibition of cholesterol uptake by the arterial wall was studied by the inclusion of 7-ketocholesterol (0.05 to 1 µmoles/ml) in the perfusate. The addition of 7-ketocholesterol to the perfusate reduced cholesterol'uptake by the vessel by an average of 90%. At concentrations between 0.1 and 1 µmoles/ml of perfusate, 7- ketocholesterol inhibition appeared to be the same. However, this inhibition was reduced at concentrations of 0.05 µmoles/m1. In all three species tested, inhibi- tion was presenti and was not due to the oxidation of cholesterol to 7-keto- cholesterol intheperfhtsate. Since these results suggest competition between~ cholesterol and, 7-ketocholesterol for binding sites within the vascular wall, the data also may indicate a new, and different approach to the prevention of eholesterol~ buildup in the arterial wa1L 43

244 Bing, R. J. and Sarma, J. S. M, Biochemical and Biophysical Research Communications 62(3):711-716, 1975. " Other support: Hoover Foundation and the Norris Foundation From the Huntington Memorial Hospital, Pasadena, Cal., and the University of Southern California, Los Angeles. FACTORS AFFECTING THE ESTERIFICATION OF LIPOPROTEIN CHOLESTEROL BY LECITHIN: CHOLESTEROL ACYL . TRANSFERASE Lecithin:cholesteroliacyltransferase (LCAT) 1 is the enzyme responsible for the esterification of lipoprotein cholesterol in plasma. In order to gain more insight into the lipoprotein specificity of LCAT, studies were carried out using highly purified enzyme and pure lipoproteins. Results showed! that LCAT' esterified relatively small amounts of cholt sterol fromivery low density lipo- proteins (VLDL), low density lipoproteins (LDL) or high density lipoproteins (HDL) in the presence of 5% human serum albumin (HSA). However, when the very high density plasma fractioni (F-4) substrate base was substitute& for the HSA, VLDL cholesteroli was esterified at rates up to tenfold higher than LDL or HDL cholesterol. Thus, it appears that VLDL together with some com- ponent present in F-4 may provide a highly efficient complex resulting in a favorable configuration of' substrate lipids for the enzyme. David, J. S. K., Soloff L. A. and Lacko, A. G. Life Seienees18(;7)!:701-7,06, 1976. Other support: U. S. Public Health Service. , From~ the Department of Biochemistry, Texas College of Osteopathic Medicine, North Texas State University, Denton; and the Lipid Research Laboratory, Temple University Health Sciences Center, Philadelphia. SERUM CHOLESTEROL ESTERIFICATION IN FAMILIAL HYPERBETALIPOPROTEINEMIA There is relatively little information concerning the quantitative aspects of lipoprotein cholesterol utilization by the enzyme lecithin: cholesterol acyltrans- ferase (LCAT), since previous studies have been done with purified lipoproteins and sonicated' lecithin/cholesterol emulsions„ The LCAT uses intact lipoproteinss as substrates in vivo and'; it has been postulated, helps maintain the normal composition and shape of plasma lipoproteins and plasma membrane integrity. This report deals with the influence of abnormal lipoprotein concentrations on LCAT activity as observed im eight patients with familial hyperbetalipopro- teinemia (Type II). Serum cholesterol esterification was studied in these sub- jects an& in a matched control group by measuring the initiat and fractional rates of LCAT activity. The Type II patients had a slightly higher mean rate 44

245 of cholesterol esterification (expressed as mµmoles./min/mil), but a significant- ly lower fractional rate (expressed as c1o /min) thani the presumably healthy 1975~ ~ controls. In a group of normal children of Type II parents,, however, both the ;; mean and! the fract'donal rates of esterification were significantly lower than those `V of healthy controls, indicating that the decreased~ fractional rate of esterification niversity may be a yet unrecognized early feature of Type II hyperlipoproteinemia and that! LCAT assays may eventually aid' its diagnosis im phenotypic yet unaffectedi kindred. Additional experiments using heated serum as substrate indicated that A- the decreased fractional esterification rates of' Type II patients were most likely due to substrate rather than enzyme deficiency. t ~EIN Sofoff, L. A., Rutenberg, H. L. and Lacko;,A. G. A „ ~ Artery 2(3) :238-249, 1976: sible for 0ther support: U. S. Public Health Service. in more ~ From Temple University Health Sciences Center, Philadelphia, and Texas Col- ut using s~ le e of' Osteopathic Medicine, Nbrth Texas State University, Denton, t LCAT iity lipo- ~, protetns ~ r, when ~ A METHOD FOR THE PURIFICATION OF MILLIGRAM utedJor' ., QUANTITIES OF STABLEHUMANI PHOSPHATIDYLCHOLINE- er tha4 0 , t" CHOLESTEROL ACYLTRANSFERASEE e eom 4 Phosphatidylcholine-cholesterol acyltransferase seems to play an important ing in a role im lipid metabolism, but it has been difficult to assay in human plasma ~~ and no previously attempted method has been found satisfactory. In view ofl the various problems encountered with other techniques, however, radioim- ~- munoassay seemed promi'sing. This reports therefore, describes a procedure for ' processing large quantities of human plasma that' yields milligram amounts of highlypur~ified and stable enzyme suitable for~ theproductioniof ant'ibodies. This involves, successively, (NH.r) _SO:r fractionation~ citric acid treatment, and' edicinet. ~ DEAE-cetlulose and hydroxyapatite chromatography,, eachi step increasing the oratory, ,, enzyme concentration so that the specific activity of the finall preparation is ap- prorimately8,0©0i times greater than that of the original plasma. This purifiedl Product appears to be freeo6 lipoproteins, as determined by immunoelectro- phoresis phoresis against antihuman serum, and is minimally contaminated! with albumin, j (l0ssthan 30 µ.g/mg of enzyme protein) as determined byimmunodiffusion: At 4°C„ the enzyme remains stable for four days but most of its activity (80%)) disappears by the twentieth day. Electrophoresis on 5% polyacrylamide gel yields an enzymatically active fast-moving band a nonactfive slow-movin band g 0 spects and a faint band of albumin. Extracts of enzymatically active bands cut from e cyltranr-'... I I . 10 gels which are pooled and extracted with 0.15M NaCI/4mM NaeH'PO4, pH oproteitis,~, 7•0; produce a single band' on re-electcophoresis on 5% polyacrylbmide gel. proteitts,,t, V 0 arma, K. G. and Solo , L. A. e normpl;iintegrity 8iocheniical Journal 155:583-588, 1976. ations o0 , alipopr4' . Other support: U. S. Public Health Service. ese snb- Frorn the Lipid Research Laboratory;, Department of Medicine, Temple Uni- rean actional rittg Ytrsity, Health Sciences Center, Philadelphia. '` - 45 0 0 I IB ® .,~, - _

246 CHOLESTEROL AND P-LIPOPROTEIN' ON LIPID INCLUSIONS AND `1 LYSOSOMAL AND MITOCHONDRIAL PERMEABILITY OF CULTURED HEART MUSCLE AND ENDOTHELIOID CELLS a~ = While the most universally-accepted explanations of atherogenesis involv'Y- one or more lipid abnormalities, experimental evidence is still sketchy, some- times even contradictory. In the present experiments cultures of rat heart muscle~ (M) and'endothelioid (E) cells were treated with /4-lipoprotein, cholesterol andl M cells were then evaluated for evidence of' lysosomal or mitochondrial perme, . a production of lipidosis was determined with E-cells after 24, 48, 72 and 96X hours. Lipid inclusions were measure& by staining with oil red O. Both E an~ The effect of, cholesterol and (or) P-lipoprotein on the~ cholesterol ester (or) ability after 72-hour treatment with O-lipoprotein with and without added cho- lesterol or cholesterol esters. Results showed that P-lipoprotein enhanced the production and maintenance of' lipid inclusions. In the concentration empl'oyec (20mg9'b) it alone produced no effect after 72 hours on lysosomal or mitochori° Research Communications in Chemical' Pathology and' Pharmacology 12(4):,, Wenzel, D. G., Acosta, D. and Kretsinger, W. B. e e aonstp ~ , genesis. { 1~ en cellWar cholesterol or cholesterol ester untake and athero- : tw 1 ti - b genesis,, to altered function of lysosomes or mitochondria. In view of' this, the't observe& changes in endothelioid cells support the possibility of a causali re-1 vestigators have reported a relationship of! cellular injury, particularly athero- cholesterol linoleate (5mg%) there was a trend toward labilization. Other in drial lability, but with cholesterol, cholesterol stearate, cholesterol oleate, o Other support: U. S. Public Health Service and the University of Kansas .:. 789-792, 1975. General Research Awards. From, the Department of Pharmacology and Toxicology, University of Kansas School of Pharmacy, Lawrence. AND BINDING A TI T OF THE MOLECULE This report details the participation of the three distinct structural regions~' of - of' the Hageman factor molecule in the two known biological activities C' VI IES W1TH SEPARATE REGIONS THE RELATIONSHIP OF STRUCTURE AND FUNCTION IN P~~ HUMAN HAGEMAN FACTOR. THE ASSOCIA.TION, OF ENZYMATIC ~~ Hageman factor, namely enzymatic activity and ability to bin& to negative sur ,: f'aces. Radiolabeled Hageman factor, functional assays of' activity, and immuno logic techniques were used to probe the structure of the native molecule. Based. : on the analysis of fragments obtained by enzymatic cleavage during fluid-phase ` I activation, there are three distinct molecular segments with different properties: (1) cregiomwi'th a molecular weight of 40;000 has binding capacity with nega-;' tively charged surfaces but no detectable enzymatic activity; (2) d region, lo-, _ C, 7 ( a

egions ties of - ve sur-, Z muno- . Based ; -phase krties: ' ' nega-, In, lo-,, 247 cated between c and e fragments, has a molecular weight' of 12;000; can~ bind firmly to negatively charged surfaces, but could not be detected as a freely existing polypeptide; (3) e region with a molecular weighv of 28,000 has en- zymatic activity and does not bind to negatively eharged surfaces. The prepa- ration of specific c and e antibodies is described as well as their use in defining the electrophoretic characteristics of the five (cde, cd, de, c and e) polypeptide fragments of Hageman factor which can be isolated after selective proteolytic cleavage. Evidence indicates that the e region„but not the c or d, is released from a negatively charged surface when bound Hageman factor is exposed to pro- teolytic enzymes or whole plasma, and that when this occurs in the presence of normal' plasma the e fragment becomes bound to C1 esterase inhibitor. Cochrane, C. G. and Revak, S. D. The Journal of Clinical]nvestigation 57:852-860, 1976. Other support: U. S. Public Health Service. From~ the Department of Experimental Pathology, Scripps Clinic and Research Foundationj La Jolla, Cal. METABOLISM OF PROSTAGLANDIN Fl IN THE PULMONARY CIRCULATION Because of its stable ring, structure, 3H'S.B-prostaglandin Fl,(3H-PGFl,)) was selected to test the hypothesis that enzymes of intact rat lungs metabolize circulating prostaglandin Flm(PGFla): As determined by perfusion followed by thin layer chromatography, 3H-PGFIa is removed from the circulation during a single passage through the pulmonary vascular bed probably by cellular up- take. According to the data, the volume of distribution and& mean transit time of the radioactivity are greater than those of an intravascular marker, blue dextran. The lungs retain 25-30% of the radioactivity, mostly in the form of a metabolite less polar than PGFlQ itself, while the pulmonary venous effluent contains the same metabolite along with some unmetabolized! PGFia. The metabolite can be distinguishe& from prostaglandins of the A, B, D, E and F series. It is reduced by borohydride, but this reaction does not yield PGFla. Sodium hydroxide has no effect on the metabolite, ruling out the presence of a j8-hydroxy-ketone group in the ring structure. The chromatographic behavior of the metabolite and its reaction with borohydride are,those expected of 9,1L- hydroxy-l5-ketoprostanoic acid. Ryan, J. W., Niemeyer, R. S. and Ryan, U. S. Prostaglandins 10(1):101-106, 1975. Other support: U. S. Public Health Service, Hartford Foundatiom and the Heart, Association of Palm Beach County, Fla. From the Papanicolaou Cancer Research, Institute and the Department of Medi- cine, University of Miami, Miami~ Fla. 47

248 METABOLIC'ACTIVITIES OF PLASMA MEMBRANE AND CAVEOLAE OF PULMONARY ENDOTHELIAL CELLS, WITH A NOTE ON PULMONARY PROSTAGLANDIN SYNTHETASE While studying the mechanisms by whichi the vasoactive polypeptides,''; bradykinin, angiotensin, I and angiotensin IL are formed and then d'egraded;'~' the authors came to consider an active metabolic rolc for pulmonary endothelial'' cells. A summary of' the results of the experiments that lcd to this consideration` shows that bradykinin and angiotensin I are metabolized during a single pas=4 , sage through the pultnonary vascular bed. While metabolism of bradykinin~~ yields biologically-inactive products; metabolism of angiotensin I yields, among; other products, angiotensin 11. Angiotensin II is not metabolized by the lungs:` ~ Other data show that metabolism of the vasoactive polypeptides is accomplished~ by peptide hydrolase enzymes of the lungs. These enzymes are not seereted, but appear to be attached to the luminal surface of endothelial' cells, especially :: those of capillaries an&venules. The ability of a single lung enzyme, angiotensin- ; converting enzyme, to inactivate the hypotensi've agent, bradykinin, and to`', form the hypertensive agent, angiotensin II~ suggests a role of the enzyme in blood pressure homeostasis. Globular particles, approximately the size of' angio-i tensin•converting enzyme, are associated with caveolae andl undifferentiated _ plasma membrane of' endothelial cells, Methods now are being developed for localizing prostaglandin synthetase at the level of electron microscopy, because ~ of' the pharmacological data implicating bradykinin as an activator of'this en-- zyme in the lung. ~ Ryan, J. W. and Ryan, U: S: In: Junod, A. F. and Dehaller, R. (eds.): Lung Metabolism. Proteolysis and Antiproteolysis: Biochemical'Pharmacology. Handling of' Bioactive Sub'stances,4 N Yo k A d i P 1 76 399 4 ew r : ca em c cess, „pp. -42 . 9 Other support: U. S. Public Healthi Service an& the Hartford Foundation. Fromithe Papanicolaou Cancer Research Institute, Miamii Fla. KININASEID (ANGIOTEN'SIN! CONVERTING ENZYME) AND ENDOTHELIAL CELLS IN CULTURE This report represents a fine focus on the localization of kininase II using a specific cell type in culture, namely pulmonary arteryy and aortic endothelial cellk. Electron microscopy; immunocytochemistry„ immunofluorescent micro- scopy and kininase IL assay were the several' techniques used to further test the hypothesis that the enzyme is localized on the pltisma membrane of endo- thelial cells. Results confirm not only the presence of kininase II in endotheliai cell cultures but also its locus on endothelial plasma membranes and associated caveolae as well as on, endothelial projections. The results obtaine& with aortic cells indicate that kininaseII is not unique to pulmonary endothelium. If! the lhng enzyme is exclusively endotheliall or more prevalent in this type of cell, angiotensini converting enzyme could provide a useful marker for endothelial cells. R}. Ac O1 Hc Fr- Scl A C( zy br a~ to qt pl cc an ar tu lut pn mi of m: tu sih, PC an tic PC cc ca PE C B 0 V' F M L. el

f--~-- --° 249 .,xZ,.,.. ides, ~ _ ided, „ ieiial . ition Pas4~ : in111: .i ong nngs: ~ shed ted, ially ~: lstn- :t d to` e iq"" Igio-' i" eted for s ause ;~ en-,, L . ~ es, a ;ing fliai ,ro= test do- aial ted rtic the Lell, ial Ryan, U. S., Ryan, J. W: and' Chiu, A. Advances in Experimenral Medicine and Biology 70t2ii7-227, 1976. Other support: U'. S. Public Health Service; Hartford Foundation and the Heart Association of Palm Beach County, Fla. From the Papanicolaou Cancer Research Institute and the University of Miamii School of Medicine,, Miami, Fla. . A SENSITIVE RADIOCHEMICAL ASSAY FOR ANIGIOTENSIN~ CONVERTING ENZYME (KINASE II) This study examines the reactivity of pig lung angiotensin-converting en- zyme with an analogue of bradykinin of high specific radioactivity, [[i1-sI]TyrF)- bradykinin, in order to develop a highly sensitive assay using a commercially availhble substrate. This analogue is degraded by angiotensin-converting enzyme to [i25I]Tyr-Arg, which can be separated' completely and recovered nearly quantitatively from intact substrate by cation-exchange chromatography. Em- phasis was placed on sensitivity so as to enable the identification of angiotensin- converting enzyme in isolated cell lines and cultures. Iniorder to test this assay and its actual sensitivity. endothelial cells from the main-stem rabbit pulmonary artery were isolttted oni strips of! cellulose acetate paper and maintained in cul1 ture for 1$'days. The cellfi were washed and incubated in Hanks basic salt so- lution; pH 7.4, at 37°C for 60 minutes with [[1=51]11yr8]-brad'y'kinin (01125 pmol) in a final volume of 0.5 mll Samples were assayedi after 15, 30 and 60 minutes of incubation. Hydrolysis of the substrate proceeded linearly at a rate of 0~84 fmol/min„and the only reaction product was [''''I9Tyr-Arg: Incubation mixtures devoid ofl cells di& not degrade the substrate. Since the reaction mix- tures used in these experiments contained about 1 x 10; cells, it may be pos- sible to use this assay to measure the amount of' angiotensin-converting enzyme per cell. The assay is highly sensitive, reasonably precise, apparently specific, and allows the use of [[t"-'I]Tyre]-bradykinin at the same or lower concentra- tions of bradykinin as are thought to occur iit vivo. However, while it is thus possible to measure the small quantities of angiotensin-converting, enzyme en- countered in small-scale cultures ofl pulmonary endothelial cells, certain modifi- cations of the present assay may be required to eliminate the influence of other peptidases for testing biological'fllrids or tissue homogenates. Chiu„A. T:, Ryan, J. W.,,Ryan;, U. S., and Dorer, F. E., - Biochemical Journal 149:297 300, 1975. Other support: U. S. Public Health Service, Hartford Foundation and the Veterans Administration. From the Papanicolaou Cancer Research Institute, and the Depart'ment of Medicine, University of Miami; Miami. Florida; an&the Hypertension Research Laboratory, Veterans Administration Hospital and the Department of Bio- chemistry, Case Western Reserve University, Cleveland. 49 .. _ _ . . . . . . .. ,.:a,:

250 FURTHER EVIDENCE ON THE' SUBCELLULAR SITES OF KININASE II (ANGIOTENSIN CONVERTING ENZYME) Indirect evidence suggests that circulating bradykinin and angiotensin I'; are metabolized by enzymes located along the luminal surface of the pulmonar}~~~ endothelfal cells. A recently isolated enzyme from~ pig lung (angiotensin-con=~ verting enzyme or kininase II) has the same bradykinin and angiotensin I me- tabolizing capacity as the intact lung and shows the same selectivity. Antibodies!z- to pure preparations of the pig lung enzyme have been prepared andl used to ~ localize the cellular andl subeellular sites of the enzyme in intact lungs and in,`' pulmonary endothelial cells in culture. Subsequent studies having shown that antibody bound to 8-microperoxidase (18-MP, a heme-octapeptide of cytoo-,le chrome c) is more suitable for electron microscopy, the authors report on the f use of 8-MP conjugated to antikininase II using bis-succinyl succinate. The im-' ~ munocytochemical results provide further support for the hypothesis that cir-~ culating bradykinin and angiotensin~ I are metabolized by enzymes on the,~ luminal surface of pulmonary endothelial cells. Other data indicate that aorticts`',; endothelial cells also have kininase II on their surfaces. That this may be true of~' ' endothelial cells from~ other vascular beds as well would not lessen the tm;4"1 portance of the pulmonary vascular bed as the primary site for inactivation of*circulating bradykinin and for angiotensin II formation, as the lungs are unique;;}~ in that their venous effluent enters the systemic arterial circulation. Ryan„J. W., Ryan, U. S., Schultz, D. R., Day, A. R., and Dorer, F. E. School of Medicine, Miami, Florida, and the Cleveland Veterans Hospital and t',,, Department of' Biochemistry, Case Western Reserve University, Cleveland. ~ r ' From the Papanicolaou Cancer Research Institute and University of Miamit Administration, anA the Heart Association of Palm County, Fla. Advances in Experimental Medicine and Biologyy 70:235-243, 1976. n~ Other support: U. S. Public Health Service, Hartford Foundation,, Veterai% FORMATION OF ANGIOTENSIN III BY ANGIOTENSIN- ~; CONVERTING ENZYME , , zatii~t In this biochemical study; rates of hydrolysis of either angiotensin I oY'4' des-Asp1-angiotensin I by pig lung angiotensin-converting enzyme were mea- e'~. sured in terms of the formation of the dipeptide His(histidine)-Leu(leucine) by`?`;- an automated fluorescence technique. Except for studies for estimating KM and'I,~ _ Vo, fl.„ each substrate was used in a final concentration of 25 or 50 µM and r~~~ actions were begun by adding angiotensin-converting enzyme at a final concen- ,: tration of 0.286 ng of protein/mi. It has been known that angiotensin II is formed by metabolic conversion of angiotensim I, an& this paper shows that s` angiotensin III, a.possible primary secretogogue of aldosterone, is formed frotn.: des-Aspt-angiotensin I by angiotensin-converting, enzyme. The K~, (11 µM) of. {. ; this reaction is one-third of that for the conversion of angiotensin I into angio-;~;~ tensin II. In view of the different pharmacological effects of angiotensin II and q" III, whether converting enzyme has a greater affinity for des-Asp?-angiotensin I I than for angiotensin I could! well be a question of physiological significance. ;1 50 Sh tht mi ter F1ee the Cl-. Bi, On I' I U of C ! I I 6

251 tensin I '' ~rtonaO` in-con-'.,ia, iImeS~ , ~ibodies • _ used tp~~ and in ~ Hn that . `.; f cyto-2 ~ on the' X. ~ e im.' ~ t i ~ a c r- on the ~ ' t' aortic ~` I true ofl~ ; e im-•s~" tion of ,- r unique, a~ . ~ htss. ' in I o!'rt ; e mea= `" ' ine) by',a M and 'I and re~ ,, oncen- nllis'3 ~ s that froin~~ M)of~ angio-„ l II and t, ensin I 1 cance. , Should des:Aspi-angiotensin I and angiotensin I occur in equimolar quantities, the formation of an aldosterone secretogogue would be favored over the for- mation of a pressor agent. A higher affinity of' the enzyme for des-Asp1-angio- tensin I is favored by data showing that angiotensin I and II, bradykinin an& peptide BPPea are better inhibitors of the hydrolysis of angiotensin I than of the hydrolysis of des-Aspl-angiotensin I. Chiu, A. T: et'al: (Ryan, U: S.) Biochemical Journal 155:189-192, 1976. Other support: L,T. S. Public Health Service and the Veterans Admini'stration, From the Papanicolaou Cancer Research Institute and Department of Medicine,. University of Miami, Miami, Florida, Department of Biochemistry, University of Colorado Medicali Center, Denver, and .Veterans Adtninistration Hospital, Cleveland. , . EFFECTS OF PLATELETS AND CERTAIN PLATELET COMPONENTS ONI GROWTH OF CULTURED HUMAN' ENDOTHELIAL CELLS Human and nonhuman endothelial cells have recently been grown success- fully in tissue culture, opening the way for a study of factors that may influence their replication rate. One report indicates that the presence of platelets in the nutrient medium~ enhances endothelial cellJ growth. This has been confirmed' here and these observations have been extended to include the effects of certain platelet components on endothelial replication in the belief that platelets and' certain ofl their components might influence this growth rate in vivo as well as in vitro: Various agents were tested for their possible effects on endothelial cell growth. Results show that isolated platelets and two platelet components,, ADP and serotonin, each~ enhance such growth in tissue culture, while epinephrine has no discernible effect. Isolated platelets had the greatesj effect, and ADP had a concentration-dependent one. At equimolar concentrations, serotonin was less stimulating than ADP and its effect did not seem to be produced through direct enhancement of cell' metabolism: Platelets are known to participate in the formation of thrombii and indirectly damage endothelium, but they may welli play a role in the healing of vascular lesions by enhancing the growth, of endothelial cells that will cover the wound area. Knowledge of such a function may permit reinterpretation of' the influence of' infused platelets and platelet components on the hemostatic potential of thrombocytopenic patients and ani- mals. Thus,, this newly-discovered growthi enhancing effect of platelets on endo- thelial cells may have far-reaching implications in our understanding of the mechanisms of hemostasis, thrombosis and atherosclerosis. Saba, S:, R. andl Mason, R. G. Thrombosis Research 7:807-812, 1975. I Other support: National Institute for Arthritis, Metabolism and Digestive Diseases. From the Department of Pathology, University of NorthiCarolina, ChapeliHi11. 51,

252 INTERACTION OF THE CHEMOREFLEX AND THE PULMONARYi INFLATION REFLEX IN THE REGULATION OF CORONARY CIRCULATION IN CONSCIOUS DOGS The goal of this study was to examine the relative roles of pulmonary iDt~ flationi and' chemoreflex control of the coronary vascular bed in, conscious dogs by comparing ( I) the responses to intracarotidl administrationi of nicotine wheu,, the dogs could', increase ventilation and when ventilation was controlled, and(2 ) the effects of forced mechanical hyperinflation{ hyperinflation induced b"` chemoreflex stimulation and, finally; spontaneous hyperinflation. When th~~ heart rate was constant, intracarotidly administered nicotine induced an it~ crease in the depth of respiration follbwed closely by an increase inilate dias=~ tolic coronary flow and a reduction in late diastolic coronary resistance. Aftet~ beta-receptor and cholinergic blockade, a similar coronary dilation iniresponse~ nicotine occurred only when ventilation was allowedi to increase. Howev when, ventilation was controlledj intracarotidly administered nicotine increa4: coronary resistance after combined beta-receptor and cholinergic blockades,; The reflex coronary dilation was not observed after carotid sinus nerve section:, or after alpha-receptor blockade. Thus, the results of this study propose a nev/- aspect of reflex coronary control in conscious animals. Nicotine stimulation o the carotid' chemoreflex results im a striking coronary dilationi that has tw components. The minor component involves a chemoreflex with its efferent- pathway in the vagi. The major component of coronary dilation follows an in='_ crease inithe depth of respiration, and its efferent component appears to involve';,. withdrawal of'alpha,adrenerg4c constrictor tone.. Ani almost identical perio& of reflex coronary dilation follov.ed either forced mechanical or spontaneou3„ hyperinflation inithe conscious dog. Vatner, S. F. and McRitchie, R. J. Circulation Research' 37:664-673, 1975., Other support: Ui. S. Public Health Service and the American Medical Aj_~ sociation. From the Department of Medicine, Harvard Medical School and Peter Bent"' Brighami Hospital, the Department of, Cardiology, Children's Hospital, Medicai _rCenter, Boston. and the New England Regional Primate Research Centei4"~ Southborough, Mass. CHRONIC SMOKING INI AN ANIMAL MODEL: EFFECTS ON CLOTTING AND FIBRINOLYSIS The effects of chronic smoking oni the cardiovascular system were studied"r." prospectively in beagles maintained on a chronic smoking program for 18 = months. Clotting and fibrinolytic tests were carried! out before the initiation of smoking and were repeated at 6, 12 and 18 months in the unanesthetized test ` animals and their corresponding controlS. At the end of, the 18-month smoking period, fibrinogen, turnover rates and platelet function were determined. An ad- ` ditional group of four dogs on high hpid diet and chronic smoking program 52

dogs ~; pvhen ` ; and sd by ; i~ the ` n iq- dias- ' After ise to `' ,ever„ ease& ades. ` ction new '` )n of ' two erent n in- `= volve ; )d of teous 253 for nine months was included in the fibrinogen decay studies only. Results showed that at the end' of 18 months the smokers appeared to have significant- ly shorter clotting time than the control group: The same pattern of significance at 18 months between the two groups was found for partial thromboplastin time, with a borderline difference at' six months, Also at the end of the smok- ing, period, the turnover rate of fibrinogen was found significantly increased in the chronic smoker, particularly when combined with high lipid diet'. It would seem, therefore, that the data obtained from this small group of animals sug- gest significant enhancement of the coagulation mechanism in the smoking group. Moschos, C. B.,,Ahmedy S. S., Lahiii; K., and Regan, T. J. Atherosclerosis 23:437-442, 1976. Other support: The American Medical Association Edueatiom and Research Foundation. From the Department of Medicine, College of Medicine and Dentistry of New Jersey, New Jersey Medical Schoolti Newark. CARDIOVASCULAR EFFECTS OF LONG-TERM CIGARETTE SMOKING AND NICOTINE ADMINISTRATION The relative rarity of angina pectoris among smokers with cardiac disease suggests that myocardiall alterations may contribute to clinicalf abnormalities independently of coronary vascular disease. Beagles, selected for their character- istic relative lack of coronary atherosclerosis - which could obscure a direct myocardial response - were studied to determine if left ventricle function, mor- phologic features and composition were altered by prolonged cigarette smoking: Observations on young beagles which inhaled cigarette smoke over long periods of time (up to 22 months) were compared with data fromi another group re- ceiving ani equivalent daily amount of intramuscularly administered nicotine to determine if the cardiac responses of the first dogs were at~ least partially at- tributable to the alkaloid, All the animals were also matched against a control group. Heart rate, stroke volume, left ventricular end' diastolic pressure and volume, as well' as intraventricular conduction time, did! not differ significantly in the three groups. Left ventriculhr ejection fraetion, was highest in the con- trols, lower in the dogs that smoked and lowest among those given nicotine, despite similar values for end-diastolic variables in the three groups. The first derivative of left ventricular pressure (dP/dt) normalized for pre- and! afterload followed the same pattern. Although~ mean aortic pressure was significantly elevated in both experimental groups, there was no significant correlation with the contractility indices. Reduction of afterload to normal levels did not affect the abnormal ventricular performance. Hypertrophy„ inflammation and~ cellular ultrastructural abnormalities were absent, and myocardial lipid and cation com, position were normal. Since interstitiali fibrosis was evident in all' experimental animals, however, an alterationi of elastic elements may be operative. These cardiovascular abnormalities appear to be predominantly dependent on the nico- tine content of cigarettes. 53~.

254 From the Departments of Medicine and Pathology, College of Medicine an Dentistry of New Jersey, New Jersey Medical School, Newark. Foundation. Other support: American Medical Association Education and Research J., and Regan, T. l. - The American Journal of Cardiology 37(1i):33-40, 1976. Ahmed, S. S., Moschos, C. B., Lyons, M. M.,, Oldewurtel, H. A., Coumbis, R. IV. Neurophar.nacology and Physiology termine whether the difference between the two enantiomers could, be used to ~ BEHAVIORAL EFFECTS' AND BINDING AFFINITIES' OF TWO STEREOISOMERIC PSYCHOTOMIMETIC GLYCOLATES As part of a long-range study aimed at investigating the structure-activity relationships of the psychotomimetic glycolate esters, interest has focused on the stereoconfigurational aspects of the drugs and the physieochemical nature of the pharmacophore. It has been shown that the availability of the non- bonded electron pair on the heterocyclic N is a crit'icat factor in determining psychotomimetic potency, and that' the conformation of the heterocyclic ring and other steric factors can affect accessibility of the electron pair to a pharma- cophore. Because of the recent observation that dextro- and levo-isomers have differing central nervous system potencies„ the authors attempted here to de- characterize the pharmacophore. A special computer-controiled program was ~ used to compare the behavioral effects in~ cats of d- and l-3-quinuclidinyl benz:il- ~ ate (QB). Psychophys:cal parameters such as the rate of, response and tendency `:; to use the left or right paw for lever pressing were used primarily to evaluate ~ errors in lever pressing. The 1.isomer was at least 100 times more potent than ,'_.~'~~, . the d form, both having a similar qualitative effecL After a single dose of 1-QB i drug efficacy. The drug did not alter auditory threshol& or the percentage of (5µg/kg), the cats' performance did not return to normal until 5-7dayslater. ~ Pretreatment of' the animals with atropine (,2mg/kg), an antimuscarinic agent, ,. did not prevent the behavioral effects of 1-QB. The binding affinity of the two isomers for synaptic membranes and phosphatidyl serine was identical. It is '~ ~ suggested that the interaction of 1-QB with the active membrane site may indUce' a configurational change that is either not reversible or requires metabolic turn- ;; over or some protein or omer moiecuie comprising tne pnarmacopnore. Lowy, K., Abood, L. G. and Raines, H. ' lournal'of Neuroscience Research 2:157-165, 1976. Other support: Ui. S. Public Health,Service. From the Center for Brain Research and, Department of Biochemistry, Univer- ' sity of, Rochester Medical Center, Rochester, N. Y. Fr vei PR BIP SY:~ anit The func man resis pone forP as fo voted

~bis, esearcfi ane an ctivity sed on, ~.; nature e non- ' ~, mining -, ,. ic ring ,,'~: arma- s have Ito de- .a sed to'~ mwas = ` benzil- . dency aluate age of t than f 1=QB later. agent, e two It is niver- >m 255 ENHANCEMENT OF STEREOSPECIFIC OPIATE BINDING TO NEURAL MEMBRANES BY PHOSPHATIDYL SERINE Previous studies based on evidence indicating that a proteolipit fraetionn from brain tissue may be the opiate pharmacophore have shown that phos- phatidyl serine, a major acidic lipid of mammalian biomembrane, binds mor- phine. This binding is stereospecific only and appears to be related to the phar. macologic potency of the opiate, though it is recognizably different from the high affinity binding of opiates to brain tissue demonstrated by other investi- gators. Since it seemed possible that phosphatidyl serine, in the form of a com- plex with some membranous protein, might be a component of the opiate pharmacophore, the authors tested its exogenous effect and that of' other lipids on the high affinity stereospecific binding of' 3H-dihydromorphine to various membranous preparations of rat brain. The addition of phosphatidyl serine to suspensions of' either synaptic membranes or a microsomal fraction significantly enhanced both high and lower affinity binding. Phosphatidyl ethanolamine and ly,sophosphatidyli ethanolamine inhibited opiate binding, Sulfatides enhanced it slightly but other acidic lipids and lecithin had no effect. Incubation of 3H', dansyl phosphatidyl serine with the microsomal fraction established that enough exogenous lipid associates with the neural membrane to account for the en- hancement of opiate binding. The results, which are discussed from the stand- point of the modification of membrane lipids and their possible significance for .he binding of' opiates and other ligands, suggest that phosphatidyl serine may be an important component of the opiate pharmacophore. That the high af- finity opiate binding is primarily associated with the synaptic membrane is still inconclusive, however, in spite of the fact that the microsomal fraction con- tained synaptic components. A6ood, L. G. and Takeda, F. European Journal of'Pharmacology 39:71-77, 1976. Other support: U. S. Public Health Service. From the Center for Brain Research and the Department of Biochemistry, Uni- versity of Rochester Medical Center, Rochester, N. Y. PREPARATION AND CHARACTERIZATION OF CALCIUM- BINDING AND OTHER HYDROPHOBIC PROTEINS FROM SYNAPTIC MEMBRANES Several types of calcium-binding proteins have been isolated from various animal tissues im an effort to elucidate the role of Ca++ in cellular function. These authors have focused their interest on the characteristics and possible function ofl hydrophobic proteins derived from isolated synaptic membranes of mammalian brains. In the present study, preparative acrylamide geli electropho- resis in dodecyl sulfate was used to separate and' purify a number of com- ponents of the hydrophobic complex. The individlial bands were then, anallyze& for N-terminal amino acids, amino acid composition and peptide profile, as welli as for their ability to bind~ Ca++' and' ATP. The majpr effort, however, was de- voted to the purification and characterization of the Ca' +-binding protein which , 55 I ! 1

256 ping, there appears to be a similar acidic component in a number of proteins ex- hibiting Ca+'+'-binding. Binding of ATP was associatedl mainly with the high molecular weight proteins, particularly those which consisted of' numerous seems responsible for its Ca+*-binding activity. Judging from the peptide ml studied. It is an acidic tryptic peptide derived fromi this particular protein which * and a Km of 1.5 X 10-", but represents only 6% of the4ot'al protein in the fractiom *~ has a molecular weight of about 16,000, a binding capacity of 4 Ca++/molecule, ` From, the Center for Brain Research, and the Department of Biochemistry, Uni- basic tryptic peptides. Abood, L. G. et al. Biochimica et Biophysica Acta443:414-427, 1976. Other support: U. S. Public Health Service: versity of Rochester Medical Center, Rochester, N. Y. EFFECT OF CHRONIC ADMINISTRATION OF NICOTINE' ON METABOLIC RESPONSES AND INTESTINAL GLUCOSE TRANSPORT Nicotine induces several adaptive changes in the physiologic system, in- cluding, alterations in the turnover rate of catecholamines. Since norepinephrine and other amines are implicated in, the central regulation of body temperature, part' of this study attempts to determine the influence of prolonged nicotine ad- ministration upon, the rat's thermoregulatory responses. Several other physio- logic parameters were examinedi as well. Animals injected with five subcutaneous nicotine doses (1 mg/kg)' daily for severali weeks displayed a number of adap- tive reactions, including increased resistance to hypothermia. After four weeks of treatment„ while oxygen consumption remained maximal, cooling time in- creased significantly as did the blood pressure. However, the continuous decline in heart rate (which ar the end of five v eeks was unquestionably lower than in the saline-treated controls) may have been a compensatory response to the ele- vated systolic blood pressure. Initially reduced„ food intake was notably greater in the sixth week without any effect on the growth rate, suggesting a possible enhancement, of the glucose absorptive ability of the intestine. Actually, ileal glucose absorption rose significantly but independently of any changes in glucose metabolism by intestinal mucosal tissue. That this may indicate increased glu- cose transport is supported by the fact that a nonmetabolized glucose analogue was also absorbed more rapidly following chronic nicotine treatment. This is consistent with the observation that, after an initial decline; oxygen consump- tion did not differ from control values: Except for the adrenals, which were heavier, there were no significant changes in organ weights or morphology. Bhagat, B., van Beaumont, W. and Ellert, M. S: Drug Addiction 4:127-136, 1974., Other support: U. S. Public Health Service. From the Departmen& of Physiology, Saint Louis University School of Medi- cine, St. Louis. 56 B T O' H F T oll Sc D A re Tl 5- an tw pr arw 4-

lecule, action which map- as ex- ; high ierous Utti- ORT ~I. ~ in- rine ture, ' ad- c sio- ~ eous d'ap- eeks in- line nI in iele- ater siblt ileal cose glu- `~ ogue _, is is mp- vere . EFFECT OF NICOTINE' ON SERUM SECRETIN ANDEXOCRINEPANCREATIC' SECRETION Nicotine appears to markedly depress exocrine pancreatic responses to exogenous secretin and„ it has been suggested, also may inhibit the release of secretin in response to intestinal acidification. However, one classic study deal- ing with smoking and the alimentary tract failed to reveal any effect of nicotine on pancreatic secretions. Because of this discrepancy and the clinicali impor- tance of a possible inhibitory effect of nicotine, these authors have examined the influence of nicotine alkaloid on HCl~stimulated secretin release and on secretin*induced pancreatic secretions. In dogs with pancreatic fistulas, intra duodenal infusion of HC1 (9.6 mEq/30' min) induced the release of immuno- reactive secretin (IRS). Pancreatic flow rate, as well as bicarbonate and protein secretions, were stimulated either by intestinall acidification or infusion of exo- genous secretim (1.0 IU/kg/hr). Nicotine (100µg/kg/hr) infused together with HC1i delayed the appearance of peak IRS concentrations by about 20 minutes. Nicotine had no effect, however, on the total amount of IRS released nor was the observed delay accompanied by a similar delay in the appearance of peak bicarbonate output. Furthermore, nicotine did not affect either the pancreatic secretory function stimulated by HCII and exogenous secretin or the metabolic clearance of the latter. Since most of the dogsstudied'initially showed an increase in pancreatic secretions rather than a decrease;, following a pattern produced' by parasympathetic stimulation, it may be that the effects of nicotine observed here are largely due to a similarmechanism: In view of the inconsistent results obtained in various laboratories because of the slightly different experimentall protocols used, it is the authors' opinion that far-reaching conclusions as to the effecti of nicotine on duodenal ulcer formation are not warranted ati this time. Boden,:G. et al. The American Journal of Digestive Diseases 21(11),:974-977„ 1976. Other support: UL S. Public Health Service and the National Institutes of Health. From the Department of Medicine and the General Clinical Research Center, Temple University Health Sciences Center, Philadelphia, and the Department of Surgery, College of Medicine and Dentistry of New Jersey, Rutgers Medical School, Piscataway. DIURNAL DIFFERENCES' IN ALTERED BRAIN 5-HYDROXYTRYPT- AMINE-RELATED REGIONAL PROTEIN SYNTHESIS The rate of cerebral protein synthesis in the rodent brain varies with the regional and subcellular contents of' endogenous 5-hydroxytryptamine (5-HT). This study examines the relationship between diurnal variations in endogenous 5-HT levels and microsomal protein synthesis in the male CF-IS mouse brain, and' attempts to determine how changes in this interaction can be influenced at two opposite points in the diurnal cycle (8 A.M. and 8 P.M.). Microsomal protein synthesi§ which was evident in the cortex and regions of the limbic area in the evening when endogenous 5-HT levels were low, diminished in the 57 A -::•. ~" r:. I" ., t!t -1 N MA g 257 t-

258' morning when the 5-HT levels were high. Electroconvulsive shock, which ele- vates 5-HT, induced! significantly higher amounts, especially in the forebrain, when applied in the evening: This response was accompanied by a correspond- ing decrease improtein synthesis. Forebrain 5-HT increments resulting from the administration of 6-methyl-tetrahydro-,8-carboline were regionally specific for different phases of' the diurnal cycle, but the negative correlation between microsomali protein synthesis and tissue 5-HT still prevailed. Reduced 5-HT contents following treatment with p-chlorophenylalanine were associated with increased microsomal prot'ein synthesis in the morning, which was consistent with the greater magnitude of 5-HT depletion at this time. The accumulated data support the hypothesis that microsomal protein synthesis in various regions of the mouse braini depends upom 5-HT lpvels. This relationship, moreover, is dependent on diurnal variations in endogenous 5-HT content, or oni diurnal dif- ferences in the magnitude of 5-HT alterations effected~ through their physiologic or pharmacologic variables: The author also suggests that some of the proteins, whose synthesis is diurnally, as well as 5-HT, dependent, could well comprise enzymes with a significant role in the process within the central nervous system~ and in the regulation of the metabolism of amines and other molecules of sig- nificance for, brain function. Essman, W. B. Journal de Pharmacologie (Paris) 6(3) :313-322; 1975. From Queens College of the City University of New York, Flushing. PROTECTION BY NICOTINE FROM BEHAVIORAL DISRUPTION~ CAUSED BY RETICULAR FORMATION' STIMULATION IN' THE RAT ~ Chronic nicotine treatment has been shown to improve performance of a~` visual attention task by rats. Short trains of appropriate levels of! electrical cur- '~ rent delivered to the reticular formation (RF), disrupu ongoing,conditioned be- .~ havior, presumably because the induced state is one of general overarousal. If the RF and limbic systems are mutually inhibitory, then the hyperstimultited state resulting from increased RF activationi might be counteracted by increased limbic activation. This study represents a preliminary investigation of nicotine's efficacy (through its supposed effects on limbic structures), as ani antagonist of the behavioral disruption resulting from RF stimulation. Male Sprague-Dawley rats with electrodes permanently implanted in their mesencephalic reticular for- mation, were trained to perform a visual attention task. Short' trains of eleetric current delivered to the RF effectively blocked' performance in a reversible and reproducible fashioni Subcutaneous administration of nicotine (100 µg/kg, the base dissolved in saline) served to attenuate the behavioral disruption caused by reticular stimulationi The suggestion that it is a nicotine-induced limbic system activation which antagonizes the behavioral disruption caused by eleetricalLy- induce& reticular overactivation is discussedl Extension of these results to hu- mam smoking behavior, furthermore, suggests a physiologic mechanism to ac- count for nicotine self-administrations namely that the smoker may be attempt- ing to manipulate his relative arousal state. Thus, it is conceivable that one underlying motivation in smoking behavior is the desire to reduce an RF activa- tion level as manifested ini a; hyperstimulated or anxious state inappropriate for

ele- rain, ond- i the ; for ween i-HT with stent iated gions :r, is I dif- logic teins, prise rstem f sig- i RAT ; ; I ofa : cur- .~ d be- ` al. If s ilated eased tine's . ist of awley : r for- ect'ric ' e and g, the - e&by :: ystem cally- ohu- o ac- ° empt- - t one {~tiva- te for 259 effective behavior, and to engender what might~ be considered a state of useful behavioral arousal. Nelsen, J. M., Pelley, K. and Goldstein, L. Pharmacology Biochemistry & Behavior 3:749-754, 1975. From the Department' of Psychiatry, College of Medicine and Dentistry of New Jersey, Rutgers Medical School, Piscataway. NICOTINE-LIKE ACTIONS OF cis-METANICOTINE AND trans-METANICOTINE , Isolated rabbit aortic strips and ileal segments were employed in a com- parative study of the biological activities of' cis- and trans-metanicotine. Accord- ing to the data, both isomers have a nicotine-like effect on these preparations. This interpretation is supported in part by the fact that hexamethonium, cocaine, phenotolamine, reserpine, and atropine blocked or reduced the contractile re- sponse produced by the metanicotine isomers in a manner similar to that, pre- viously observed in nicotine studies. Dose-response studies on, the aortic strip showed that trans-metanicotine is significantly less active than, nicotine, while cis-metanicotine is the least active of all. When four pyridino compounds (3- pyridyljicetic acid, N-(3-pyridylacetyl)glycine, nicotinuric acid and trans-4(3- pyridyl)-3-butenoic acid) were also tested for both agonist and antagonist ac- tivity, they had no stimulatory effect' on either physiologic preparationi Pre- treatment with either pyridylacetic acid or N-(3-pyridylacctyl)glycine reduced the contractile response of aortic strips to trans-metanicotine moderately to markedly, while pretreatment with trans-4-(3-pyridyl)-3-butenoic acid did so only slightly. Thus, the present data clearly indicate that the metabolism of nicotine and metaAicotine to 3-pyridylacetic aeid' and its glycine conjugate is accompaniedlbygreat loss of adrenergic and cholinergic activity. I Wilson~ K. L., Jr., Chang; R. S. L., Bowman, E. R., and McKennis, H., Jr. The Journal of Pharmacology and Experimental Th'erapeutics 1i96(13),:685-696, 1976. Other support: American Tobacco Company, American Medical Association- Education Research Foundation and National Institutes of Health. From the Department of Pharmacology, Medical College of Virginia, Richmond. PROPOSED ROLE OF THE PLACENTAL CHOLINERGIC SYSTEM IN THE REGULATION OF FETAL GROWTH AND DEVELOPMENT All three components of a aholinergic system, acetylcholine (ACh)-cho- line acetyltransferase (ChA)-acetylcholinesterase (AChE), are present in the human placenta. They are localized in the syncytiotrophoblast and their activitiess vary with, gestational development. ACh levels are triphasic, being low at early gestation periods (6 to 13 weeks) and at the time of parturition, while exhibir~ ing a peak at mid-pregnancy (16 to 20 weeks) . ChA and AChE activities cor- 59 0 a H h tJ 1 0 ^N.~~;

1. 260 Aspects of Perinatal Pharmacology, New York: Raven Press, 1975, pp. 107- In: Morselli. P. L., Garattini, S. and Sereni, F. (eds: ): Basic and Therapeutic nance of placental function and„subsequently, fetallgrowth and'development. Harbison, R. D., Olubadewo, J., Dwi'vedi, C., and Sastry, B. V. R. system, as well as the placental uptake and transfer of various substances, indi- cate a possible role of the eholinergic system in the modulation and mainte- linergic system. The gestation-dependent variation of the placental cholinergic and their uptakes fluctuate inversely to the development of the placental cho= uptake of substances which are dependent on the gestational age. Diphenyl- hydantoin uptake is high during early and late gestation and low during the midgestation period. The same pattern holds for the uptake of a-aminoiso- butyric acid. Both of these compounds are concentrate& by the placental tissue, relate well with ACh levels. In addition, there are differences im the placental Other support: U. S. Public Health Service. of' Biochemistry, Vanderbilt University School of Medicine, Nashville„Tenn. From the Department of' Pharmacology and Center im Toxicology, Departmenf ACETYLCHOLINE IN HUMAN PLACENTA DISTRIBUTION AND VARIATION WITH GESTATIONAL AGE OF HUMAN PLACENTAL CHOLINERGIC SYSTEM OCCURRENCE, tion of the transport of nutrients and chemicals across the syncytiotrophoblast that the placental cholinergic system may play a significant role in the regula- tiogenesis and functional maturation. The sum of these observations indicates ChA had a similar pattern of! variation wi'thi gestational age, it would seemi that the placental' cholinergic system is fully formed at the early fetal period of his, gestational age;, the placental ACh levels being higher during the second tri- mester of pregnancy than during the first and third trimesters. These observa- tions indicate that ACh~ is possibly localized in the syncytiotrophoblast. Since segments„ the very segments where villi are localized. ACh content varied with ripheral segment had lower concentrations of ACh than the central concentric the placenta, the concentric segments next to the umbilical cord and the pe- in membranes. While there were high concentrations of ACh in all segments of ponents of the ACh-like activity in the human placenta have not. In this study, gas chromatography was used to ascertain~ that the major component of the ACh-like activity of the term placenta was ACh (112 nm/g of wet tissue). This result was confirmed by the separation of ACh from!other quarternary am- monium compounds by eolumn chromatography. Althoughi the placenta could be stored at 4?C for a number of days without significant' loss of ACh, freezing and thawing,of the placenta destroyed ACh, indicating that ACh is bound with- While human, placental ChA and, AChE have been characterized, 'the com- (ChA) andl acetylcholinesterase ('AChE)' are present in the human placenta. Various published observations indicate that all three components of the cholinergic system, acetylcholine (ACh) hke activity, choline acetyltransferase an&thereby regulates the fetal growth. Sastry, B. V. R. et al,

*i 0 1 M1 0 261 Biochemical Pharmacology 25 (4) :425-431, 1976. Other support: Ui. S. Public Health Service. From the Departments of Pharmacology and Biochemistry, Vanderbilt Uni- versity School of Medicine,,Nashville, Tenn. NORMAL FUNCTIONS OF THE THYROID GLAND OF THE PYGMY GOAT The purpose of this study was to gather hitherto unavailable information about the normal physiologic and biochemical values for thyroid function in the pygmy goat which is extensively used as a laboratory animal model. Normal values were obtainedJor blood serum PBI, T3 index, T4„ and! cholesterol in a colony of 55 pygmy goats of mixed sex and age. Serum PBI values averaged 8.1 ± 1.2 µg/ 100 ml with, no significant sex differences. The mean T3 index and T4 values were 1.1 ±0:1 and 7.2+ l.l µg/100 ml respectively. No sex differ- ences were noted in these values. The mean serum cholesterol value was 90!0 mg/100 ml, an&sex differences were apparent. In females, the average level was significantly lower (84.9 mg/ 100 ml; n=44) tham in intact males (97.4 mg/100 ml; n=8) and it was stilli lower in castrated males (69.2' mg/ 100 ml; n=6). Serum cholesterol values, however, consistently and significantly increased with age in the females, an unexplaine& phenomenon also observed in humans. The animals studied showed no evidence of either thyroid malfunc- tion or pituitary thyrotropic deficiency. Castro, A. et al. Laboratory Aniinal Science 25(3) :327-330, 1975. Other support: Florida Juvenile Diabetic Research Foundation. From the Department of Medicine, University of' Miami School of Medicine, Miami, Fla., and the Heart' Research Laboratory, University of Oregon Medical School, Portland. V. Immunology and Adaptive Mechanisms LOCALIZATION OF ANGIOTENSDN CONVERTING ENZYME (KININASE II). I. PREPARATION OF ANTIBODY-HEME- OCTAPEPTIDE CONJUGATES Antibodies to pure preparations of pig lung angiotensin-converting enzyme. (kininase II) have been prepared and are now being used' to help localize the cellular and subcellhlar sites of! kininase II in intact lung an& in~ lung cells in~ culture. This paper describes a method of conjugating these antibodies to a peroxidase marker suitable for electron microscopy. In the work presented!here,: a heme-octapeptide (8-microperoxidase, 8-MP) derived from cytochrome c 61 H 0

was coupie& to antibody in a two-step procedure using a bifunctional active, ester, bis-succinyli succinate. In the first, step, 8-MP, which has only one reac! tive amine, was reacte& with an excess of bis-succinyl succinate to yield' 8-Mp: succinyl succinate a stable compound which can be stored: In a second ste ~ , p the remaining active ester was used for coupling to reactive amines of the anti.~ body. The conjugate consists of' 1•6-2•3 MP moieties per antibody. Using these; procedures, the formation, of complex polymers is avoided. Eachi molecule oG conjugate possesses both immunoreactivity and peroxidatic activity. The coii~`, jugate has been used to localize angiotensin-converting enzyme (kininase II)' along the plasma membrane and associated caveolae of' pig aortic endothelia~1, cells in culture. Ryan, J. W., Day„A. R., SChultz, D. R., Ryan, U. S:, Chung, A., Marlborouj D. L, and Dorer, F. E. ^ Tissue & Cell 8(1),:111-124, 1976. Other support: U. S. Public Health Service, Hartford Foundation, Veterans Administration, and the Heart Association of Palm Beach County,,Fla: .11 From the Papanicolaou Cancer Research Institute and the Department of Medi;~ eine, Uhiversity of Miami School of Medicine, Miami, Florida; Hypertenston,,: Research Laboratory, Veterans Administration Hospital and the Department of,- Biochemistry, Case Western Reserve University, Cleveland. ('KININASEIIq. 11. IMMUNOCYTOCHEMISTRY AND IMMUNOFLUORESCENCE LOCALIZATION OF ANGIOTENSIN CONVERTING ENZYME In this part of the localization study of angiotensin-converting enzyme r, (kininase II),, the cellular and subcellular sites of the enzyme in~ lung tissue and endotheliali cells in culture were examine& by immunocytochemical and im-Is munofluorescence techniques. The immunocytochemical studies at' the electron F microscope level used, goat anti-(pig lung angiotensin-converting enzyme) ' coupled to 11-MP (11-microperoxidase) via glutaraldehyde or to 8-MP (8-~ mieroperoxidase) via a bifunctional active ester, bis-succinyl succinate. The' latter conjugate, which does not contain complex polymers, has been eharac- terized in detail in terms of immunoreactivity and peroxidatic activity. BothX conjugates yield similar results: angiotensin-converting enzyme appears to be-.= ~ localized along the luminal surface of pulmonary endotheliall cells. Endothelial cells of large and small vessels react with the antibody conjugates, but reactions ` are most prominent' at the level of the capillaries and venules. The conjugates ; are also reactive with, pig lung and aortic endothelial cells ini monolayer culture. . These cell lines were shown to possess converting enzyme which is capable of ; inactivating bradykinin and of, converting angiotensin I to its potent homolog, , angiotensin II. Converting enzyme was also shown to be associate& with pul- monary endothelial cells in culture by direct and indirect' immunofluorescence. ; These results support the conclusion that circulating bradykinin can be inacti- . vated and angiotensin can be activated by an enzyme situated on the luminal

surface of pulmonary endothelial cells: The ability of' pulmonary converting en- zyme to inactivate a hypotensive substance. bradykinin, and to form a hyper- tensive substance, angiotensin II, may bespeak a role of the enzyme in blood pressure homeostasis. Ryan, U. S. et al. Tissue & Cell 8(li) :125-145, 1976. Other support: Ui. S. Public Health Service, Hartford Foundation and the Heart Association of Palm Beach County, Fla. From the Papanicolaou Cancer Research Institute and the Department of Medi- cine, University of Miami Schooliof Medicine, Miami, Fla., ACCESSORY SPLEENS IN DOMESTIC RABBITS (ORYCTOLAGUS cuniculus): II. INCREASED FREQUENCY IN HEMATOLOGICAL DISEASES AND EXPERIMENTAL INDUCTION WITH PHENYLHYDRAZINE This report documents the association of increased frequency of accessory spleens with ('1) certain inherited hematological diseases in rabbits and (2) thee productiomof accessory spleens byphenylhydrazine. In this study, data fromiin- bred rabbit strains of commoni ancestry with a high frequency of hereditary autoimmune hemolytic anemia or lymphosarcoma showed that all of these strains also had a high frequency of accessory spleens. A total, of 21% of! rabbits in these strains, though phenotypically normali had globulin-coated (Coombs'-positive) erythrocytes. These findings supported' observations of in- creased frequency of accessory spleens in human beings with similar diseases and~ suggested that accessory spleens, rather than being passive developmental anomalies, represent physiological responses to demand for phagocytic capacity provide& by the reticuloendothelial system im the spleen. This hypothesis pre- dicted that simulation, of the basic defect might yield a laboratory model for the induction and study of accessory spleens in rabbits. Phenylhydrazine was used to alter hematopoiesis and it was shown that the frequency of accessoryy spleens in 80 rabbits so tteated was 45%, as compared to a naturally occurring frequency of! about 7.5%. The rabbit phenylhydrazine model offers the first direct proof that a biologicall basis exists for the induction of accessory, spleens in, response to hematolbgical stress. No evidence was found to relate anemia per se or cardiovascular anomalies to the increased frequency of accessory spleens in the rabbits. Weisbroth, S. H. et a!: (Meier, H.) , Teratology 13(3):253-262, 1976, Other support: NationaliInstitutes of Health, From the Division of Laboratory Animal Resources, Health Sciences Center, State University of New York, Stony Brook, and The Jackson Laboratory, Bar Harbor, Me.

264 VI. Epidemiology THE RELATIONSHIP OF SCIENTIFIC OBJECTIVES TO POPULATION SEL.ECTIONAND ATTRITION IN LONGITUDINAL STUDIES: THE CASE OF THE NORMATIVE AGING' STUDY The Normative Aging Study (NAS), a lifelong study of 2,280 initially healthy male subjects, has been active for 12 years now, and the issue of at- trition has become important because of the nature of' the original population. Since the NAS subjects were selected on the basis of scientific objectives, not representativeness;, it seemed necessary to assess attrition rates and to determin0 whether attrition had changed the population's characteristics. Over its first ~ dozen years, results showed the, NAS' had a remarkable capacity to retain itsA population with a 1% annual attrition for all causes and a 0.5% annual attri- tion due to loss of interest. Nb remarkable health, age or social differentials were found between those who remained in the NAS and, the small group that left, and it seemed likely that' the initial highly selective nature of the popula- tion, as dictated by' scientific objectives, reduced both attrition and any differ- ence between, the smalli number of dropouts and those who remained, in the study. Thus, the original population characteristics have been maintained since the inception of the Study in 1963 and it appears that the problem of attrition in longitudinal studies should be viewed in, the broader context of scientific objectives. Rose, C. L., Bosse, R. andlSzretter, W. T. The Gerontologist 16(,6)':508 516, 1976. Other support: Medical Research Service of the Veterans Administration. From the Normative Aging Study, Veterans Administration Outpatient Clinic, Boston, and Hellenic College, Brookline, Mass. RELATIONS OF AGE AND PERSONALITY DIMENSIONS TO COGNITIVE ABILITY FACTORS _ This study examined the relation between three cognitive ability factors -A Informationi Processing Ability (IPA), Manual Dexterity (MD) and' Pattern' Analysis Capability (PAC) - and three personality dimensions - Anxiety, ; Extraversion and, Openess to Experience - in 969 male volunteers ranging in ;: age from 25 to 82. Also, since most measures of these cognitive ability factors ~ have shown cross-sectional declines in performance with increasing age, this study tried to determine whether these declines might be mediated by person- ality ality factors. On the whole, results showed small but significant relations be- tween ~ tween personality and cognitive ability factors; most of which could not be ac- ; counted for solely by education or social class. Anxious subjects scored lower y than adjusted subjects in all three forms of intelligence, while subjects more ; open to experience scored higher than closed-minded subjects on IPA and PAC. ' Introverts showed some superiority over extraverts,, but only in PAC. Just as in other studies, younger subjects generally did better than older ones, but sew : erali hypothesized interactions of age an& personality were not foundi This sug- 64 C Ic - 0 th F B S

ain its attri'- ntials 'p that ~ _y pula- j~ ~ iffer- n the ~' since '` rition ntific '_. 265 gests that while personality and intelligence are subtly related, the decline in cognitive functioning with age cannot be attributed to the influence of per- sonality variables. Costa, P. T., Jr. et al. lournalof Gerontology 31(6):663-669,1976. Other support: Medical Research Service of the Veterans Administration and the National Institutes of Health. From~ the Normative Aging Study, Veterans Administration Outpatient Clinic, Boston. SMOKING' CESSATION AND SEX ROLE CONVERGENCE As noted in previous reports, smoking rates are becoming more similar among males an& females. This study now examines whether blurring or de- crease in sex role differentials as a result of social change also affects male and female smoking cessationi rates. As shown by the literature, there already is convergence in proportion of smokers among males and females from older to younger age cohorts, suggesting, an equalitariani shift over time. That a similar effect would be evident, in giving-up smoking seemed a likely possibility. Fur- thermore, convergence in responsiveness to interpersonal' influences due to the equalitarian shift forms the basis for another hypothesis: across age, from older to younger, there is a diminished sex differential when smoking cessation is related to interpersonal factors. Age cohort analyses on a national probability sample of current and former smokers (N=3,364) strongly supported these hypotheses. In addition, the data presented here may have significant implica- tions for research on aging. Bosse, R., and Rose, C. L. Journal of Hea1t4 and Social Behavior 17(1) :53-61, 1976. Other support: National Clearinghouse for Smoking and Health and the Veterans Administration Nbrmative Aging Study. From the Veterans Administration Outpatient Clinic. Bostonj and! Hellenic Col- lege, Brookline, Mass. IS THE INCREASED RISK OF MYOCARDIAL INFARCTION IN CIIGARETTESMOKER& DUE TO PSYCHOLOGICAL TRAITS?AI*T ATTEMPTED EXPLORATION USING PSYCHOLOGICAL QUESTIONNAIRE RESPONSES This paper explores the question of whether the relationship between ciga- rette smoking and myocardiali infarction might be based on underlying psycho- logicall differences rather than on smoking, itself. In this epidemiological study,, cigarette smoking assessed in a multiphasic health checkup was almost twice as frequent in 222 men who subsequently develope& a first myocardial infarction tham it was in 228 controls. Also given at this health checkup were 155 psycho- logical questionnaire items that had been derived mostly from the Minnesota 65

266 Multiphasic Personality Inventory. For most (94/ 155, or 61% ) of the items, the case-control differences were parallel to the smoker-nonsmoker differences. Subdividing the study group according to their responses to suchi items failed to eliminate the apparent relation of smoking to subseqpent infarction in most subjects. However, as many as one-fourth of the subjgcts could be identified by the questionnaire as subjects in whom smoking was not predictive of inf'arction.. The present data raise the possibility that the importance of smoking as a coro- nary risk factor varies with psycholbgical status. Although cigarette smoking remained a risk factor in most subjects, the investigators did identify a subgroup in whom it apparently was not' a predictor of myocardial infarction. Friedman, G. D. et al. Preventive Medicine 4(4) :526-532, 1975. Other support: National Institutes of Health and the Kaiser Foundation Re- search Institute. From the Department of Medical Methods Research and Department of Medi- cine, Kaiser-Permanente Medical Care Program, Oakland, Cal. 66

267 S. d t P. Active Projects Following is a list of' the principal investigators, or institutions, of projects under way or activated in the period since the previous Report, together with the respective project titles. Completed projects are listed in a later section. PRINCIPAL INVESTIGATOR OR INSTITUTION LEO G: ABOOD, Ph.D., Professor of Biochemistry and Brain Research, Cen- ter for Brain Research, The University of Rochester Medical Center, Roches- ter, N. Y. JOSEPH C. ARCOS, D.Sc:, Professor of. Medicine, Tulane University School of Medicine, New Orleans. DOMINGO M. AVIADO, M.D., Profes- sor of Pharmacology, University of Pennsylvania School of Medicine, Phil- adelphia: CARL G. BECKER, M.D., Associate Professor of Pathology, Cornell Uni~ versity Medical College, New York. WILLIAM E. BENEDICT,, M.D„ Assist- ant Professor of Pediatrics, University of Southern California School of Medi- cine, Division of Hema*.ology and Med- ical Genetics, Childrens Hospital of Los Angeles, Los Angeles., RICHARD.J: BING, MLD., Professor of Medicine, University of Southern, Cali- fornia School of Medicine, Los An- geles; Visiting, Associate in Biomedical Engineering;, California Institute of Technology;, Director of Cardiology and Intramural Medicine, Huntington Memorial Hospital, Pasadena; Cal. GUENTHER BODEN, M,D., Associate Professor of, Medicine; Assistant Direc- tor, General' Clinical Research Center, Temple University Health Sciences Cen- ters Philadelphia. RAYMOND BOSSE, Ph.D.,, Associate Director, Normative Aging Study, Vet- erans Administration ~ Outpatient Clinic,, Boston. A. SONIA BUIST, M.D., Assistant Pro- fessor of Medicine and Physiology, University of Oregon Health Sciences Center, Portland. PROJECT TITLE Cholinergic correlates of behavior Synergistic effects of polycyclic hydrocar- bons and nitrosamines in pulmonary carcinogenesis. Potentiali repressors of metabolic aetivation of nitrosamines. Influence of cigarette smoke on pulmo- nary emphysema and bronchospasm Investigatiom of the role of allergy to tobacco constituents in the pathogenesis of arteriosclerosis and myocardial in- farction . Malignant transformationa mutagenesis an& fibrinolysin production of cigarette smoke condensate fractions Inhibition of cholesterol uptake by ar- teries in r.itro and in vivo Effect of nicotine and cigarette smoke on secretin ~ secretion A smoking research project in the Nor- mative Aging Study The role of alpha-l-antitrypsin deficiency as a risk factor in the development of chronic airways obstruction 67 ® 11 a 3

268'. PRINCIPAL INVESTIGATOR OR INSTITUTION ALBERT CASTRO„Px.D., Director, Hor- mone Laboratory;, Associate Professor of Medicine, University of Miami School of Medicine, Miami, Fla. JACK CHALON;, M.D.,,Associate Profes- sor of Anesthesiology, New York Uni- versity Medical Center, New York., CHARLES G: COCHRANE, M.D.,,Mem- ber, Scripps Clinic and•Research Foun- dation, La Jolla, Cal. ALLEN B! COHEN, M.D., PH.D.,, As- sociate Professor of' Medicine, Temple University Health Sciences Center, Philadelphia. PAUL T. COSTA, Jr. Px.D., Associate Professor of Psychology, University of Massachusetts at Boston. CARROLL E. CROSS, M.D., Associate Professor of Medicine and Human Physiology; Director, Section of Pul- rnonary Medicine, University of Cali+ fornia School of Medicine, Davis. DAVID W. CRUMPACKER„Ptt.D:, Pro- fessor and Chairman;, Department of Environmental, Population and Orga.- nismic Biology, University of Colorado, Boulder. H. FRED DOWNEY, PH.D., Assistant Professor of Physiology, Uhiversity of' Texas Healthi Science Center at Dallas; Director, Cardiovascular Research, Car- diopulmonary Institute, Methodist Hos- pital of Dallas;,Dallas. WALTER B. ESSMAN, M.D., PH,D., Professor of Psychology and Biochem- istry, Queens College of the City Uni- versity of New York, Flushing. HUGH E. EVANS, M.D.,, Director, De- partment of Pediatrics, Jewish Hospi- tal and Medical Center of Brooklyn, Brooklyn, N.,Y. HANS J. EYSENCK, PH.D.,, D.Sc., Pro- fessor of Psychology, Institute of Psy- chiatry, University of London, Lon- don. GAD FEINSTEIN, PH.D., Senior Lec- turer in Biochemistry, The George S. Wise Center of Life Sciences, Tel Aviv University, Tel Aviv; 'Tsrael. Epidemiology of tracheobronehial multi-" nucleation The genetic defect in alpha-l-antitrypsin deficient patients The relations between smoking motives; personality and feelings Cigarette smoke effects on certain aspeets_ of7atilung metabolism Genetic and environmental factors af- fecting fecting smoking behavior f Effects of tobacco smoke and nicotine on coronary collateral blood flow Metabolic response to stress-tobacco: smoke interactions Relationship of non-MM phenotypes afld lung disease among infants + The inheritance of the smoking habit Studies on peptide bond specificities, aar~4' tive site and inhibition of human letr cocyte proteases which are implicated;; in the pathogenesis of pulmonary eo?;,- physema. ' r 68 LE c f ., JO1 b' si' %fA A' a, ~ si tr PAL fe ar Sc W' NOF. Fa Da HER] A ss Gec icin

PROJECT TITLE WILLIAM H. FISHMAN, PH.D., Pro- fessor of Pathology; Director, Tufts Cancer Research Center, Tufts Uni- versity School of Medicine, Boston. LARS FRIBERG, M.D., Professor and Chairman, Department of Environ- mental Hygiene, The Karolinska Insti- tute, Stockhoimi GARY D. FRIEDMAN, M.D., Senior Epidemiologist, Department of Medical Methods Research, Kaiser Foundation Research Institute, Oakland, Cal. JACQUES E. GIELEN, PH.D., Assistant Professor, Laboratory of' Medical Chemistry, Toxicology and Hygiene, Institute of Pathology;, University of Liege, Liege, Belgium. GERALD J. GLEICH, M.D., Consultant in Medicine, Research Laboratory for Allergic Diseases, Mayo Clinic, Roches- ter, Minn. LEONIDE GOLDSTEIN, D.Sc., Asao- ciate Professor of Psychiatry, Institute for Mental Health Sciences, College of Medicine & Dentistry of New Jersey, Rutgers Medical School, Piscataway. JOHNI W. GORROD, D.C.C., Lecturer in Biopharmacy;, Chelsea College, Univer- sity of London„ Londbn. MARGIT HAMOSH, PH.D., Research Associate, Departrnenr of Physiology and' Biophysics, Georgetown Uhiver- sity Schools of Medicine and Dentis- try, Washington, D.C. PAUL HAMOSH, M.D., Associate Pro- fessor of Physiology and Biophysics,, and Medicine, Georgetowm University Schools of Medicine and Dentistry, Washington, D.C. NORMAN W. HEIMSTRA, PH.D., Pro- fessor of Psychology; Director, Human Factors Laboratory, University of South Dakota, Vermillion. HERBERT B. HERSCOWITZ, Px.D., Assistant Professor of Microbiology, Georgetown University Schools of Med- icine and Dentistry, Washington4 D.C. Cancer phenotype profile which may pre- sage bronchogenic cancer Epidemiological studies on the new Swed- ish twin registry Causes of death in relation to smoking habits and other behavioral and' en- vironmental factors. A study on the Swedish Twin~ Registry Characteristics of smokers and non- smokers Twin registry feasibility study. Cigarette smoke and polycyclic hydro- carbon metabolism in rat and mouse lung and kidney Hypersensitivity to antigens from tobacco as P: factor in the pathogenesis of chronic bronchitis The "chronic nicotine state" and anxiety: a behavioral and electroencephalb- graphic analysis of induced an& spon- taneous hyperactivation in rats The metabolism of "pyridines" in relation to the induction of xteoplastic disease The effect of cigarette smoke on lung metabolism (Initiated under Vidic, B.) The effect of smoking on the "small air- ways" Effects, of smoking deprivation on risk- taking behavior Behavioral effects on nonsmokers of ex- posure to smoking Effects. of cigarette smoke exposure om developmental, cellular and molecular aspects of the immune response 69 30-498O- 7B : - 18

270 PRINCIPAL INVESTIGATOR OR INSTITUTION ALPHONSE J. INGENITO, PH.D., Asso- ciate Profe.ssor of' Pharmacology, The Albany Medical College of Union Uni- versity, Albany, N. Y. (Now at East Carolina University School of Medi- cine, Greenville„ N. C.) HARRY L. IOACHIM, M.D., Attending Pathologist, Lenox Hill Hospital;,Clini- cal Prof,essor of Pathology, Columbia University College of Physicians & Surgeons, New York. WILLIAM J'. JUSKO;, PH.D:,, Associate Professor of Pharmacetuics; Director, Clinical Pliarmacokinetics, Laboratory, Millhrd Fillmore Hospital, Buffalo. EDWARD L. KLAIBER, M.D:, Senior Scientist, The Worcester Foundation for Experimental Biology, Inc., Shrews- bury, Mass, ST'iG KULLANDER, M.D., Professor and Chairman, Departntenl of Obstet- rics arrd Gynecology, Uhiversity of Lund, Lundj Sweden., MICHAEL E. LAMM, M.D., Prof,essor of Pathology, Ntw York University Medi- cal Center, New York. PAUL S, LARSON, PH.D., Haag, Prof es- sor of Pharmacology, Medical College of' Virginia, Richmond. JOSEPH M., LAUWERYNS, M.D., Pt[.D., Professor Ordinarius and Chair- man, Department of Pathology and Microscopic A'natomy;, Experimental Laboratory of Pulmonary Hlstopathol, ogy, Katholieke Uhiversiteit te Leuven, Leuven, Belgium. RICHARD~A. LERNER, M.D., Associate Member, Scripps Clinic and Research~ Foundation, La Jolla, Cal; JAY A. LEVY, M.D., Assi.stant, Clinical Professor of Medicine; Research Asso, ciate, Cancer Research Institute, Uni- versity of California School of Medi- cine, San, Francisco. CLAYTON G. LOOSLI. PH.D., M,D... Hastings Professor of Medicine and Pathology, University of' Southern Cali- fornia School of Medicine„Los Angeles. PROJECT TITLE Actions of carbon monoxide and tobacco smoke on retinal metabolism and func- tion The immune response at the tumor site in lung carcinoma , Effect of smoking and its cessation on drug disposition Centrali nervous system adrenergic func- i t ` gare te smoking tioning and~c Studies of a gonadal and central nervous system syndrome that~ differentiates smokers from nonsmokers Influence of smoking on human foetal growth and' postnatal development, and on, fibrinolysin in the blood of pregnant women. Accumulation of nicotine andfor damage to human pla- cental andf'oetal lUng, tissues Immune mechanisms of mucous mem- branes Publication of SupplemenC III to Tobacco, 1961 The neuroepitheliali bodies: their role and structure as intrapulmonary neuro (chemo)receptors in normal and vari- ous physiological, pharmacological and pathological conditions Studies on persistent viral infection Oncornaviral gene expressioni in normal = and malignant tissues ~ Possible genetic determinants of chemical carcmogenests Effects of freshicigarette smoke inhal8tio0.. on the respiratory tracUof'imice ~` 70 7 1' ( 1 I L IN G r i

271 PRINCIPAL INVESTIGATOR OR iNSTITUTION'. HENRY T. LYNCH, M.D., Professor and Chairman, Department of, Preven- tive Medicine and' Public Health, Creightom University School of! Medi+ cine, Omaha; Neb. REGINALD G. MASON, M.D., Ptt;D., Pathologist-in-Chief, Memorial Hospi- tal, Pawtucket; R. I. GERALD E. McCLEARN, PH.D:,, Di- rector, Institute for Behavioral Ge- netics, Department'of Psychology, Uhi- versity of Colorado School of Phar- macy, Boulder. HERBERT McKENNIS, Jr.,,PH.D., Pro- fessor of' Pharmacology, Medical Col- lege of Virginia, Virginia Common- wealth University, Richmondi . HANS MEIER, D.V.M., Senior StafJ'Sci- entist, The Jackson Laboratory, Bar Harbor, Me. DOV MICHAELl, PH.D., Assistant Pro- fessor of Biochemistry and Surgery, University of California School of Medicine, San Francisco: MICROBIOLOGICAL ASSOCIATES, INC.,, Bethesda, Md. PROJECT TITLE Smoking history in families with low and high~cancer incidences Aryl hydrocarbon hydroxylase (AHH): cancer genetics Effects of' nicotine on interactions of platelets an& endotheliali cells Heredity and tobacco-related behavior in, the mouse Cooperative studies aimed at the devel- opment of' immunoassays for nicotine and nicotine metabolites Oncogenesis, in the rabbit: genetic sus- ceptibility, vertical transmission oflvirus and environmental influences Transplacental effects of nitrosocom. pounds in inbred strains of mice and rabbits Effects of cigarette smoke on pulmonaryy fibroblasts an& collagen and its rela- tiom to emphysema Development of a mouse model system for genetic susceptibility and its relation- ship to in vivo lung carcinogenesis Development of in vitro and in vivo model sy,stemsfor thestudyofl lung chemical susceptibility and carcino- genesis Smoke inhalation carcinogenesis studies in mice - Human AHH studies. Research services in support' of other projects (see Levy, Jay A. and Wang, I.), J. ANDREW MITCHELL, PH.D., Assist- ant Professor of Anatomy, Wayne State. University School of Medicine, Detroit. GEORG B. NEURATH, PH.D.,, Micro- analytical Laboratory, Hamburg, West Germany. A study of the effects of nicotine on gesta- tion in the rat'with particular reference to implantation and the time of onset of parturition Nitrosamines in, tobacco and its smoke:, occurrence, formation and transfer 71 /

272 PRINCIPAL INVESTIGATOR OR INSTITUTION KENNETH PAIGEN„ Px:D., Director, Department of Molecular Biology, Health Research, Inc., Roswell' Park Division, Buffalo. CARL W. PIERCE, M.D., Px.D.,, As- sociate Professor of Pathology, Har- vard Medical Schoolj Boston. ILARI RANTASALO, M.D., Professor and' Chairrnan, Department of Public KeatthScience, University of Helsinki, Helsinki. RONALD E'. R'ASMUSSEN,, PH.D., As- sociate sociate Research Physiologist, Depart- ment of Community and Envi'ronment- al Medicine, University of' California College of Medicine, Irvine. TIMOTHY J. REGAN, M.D., Professor of Medi'cine;, Director, Division of, Cardiovascular Diseases,, College of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark. LYNNE M. REID, M.D., Wolbach Pro- fessor of Pathology, Harvard Medicall School, Boston; Chairman, Department of Pathotogy; Children's Hospital, Medical' Center, Bostoni DANIEL B. RIFKIN„ PH.D., Assistant Professor of Chemical Biology, The Rockefeller Uhiversity, New York. CHARLES L. ROSE, PH.D., Clinic Direc- tor; Director, Normative Aging Study, Veterans Administration Outpatient Clinic, Boston.. JOHN A. ROSECRANS, PH.D., Asso- ciate Professor of Pharmacology, Medi- cal College of Virginia, Richmond. UNA S. RYAN, Px.D:,, Senior Scientist; Papanicoldou Cancer Research Insti- tute, Miami; Assistant Professor of Medicine, University of' Miami School of Medicine, Miami, Fla. B. V. RAMA SASTRY, D.Sc., Pk.D.,, Professor of Pharmacology; Vand'er- bilt' University School of Medicine, Nashville, Tenn. JAKOB SCHMIDT, M.D., Prt.D:, As} sistant Professor of Biochemistry, Di- vision of Biological Sciences, State University of New York at Stony Brook, Stony Brook. PROJECT TITLE A genetic test of glucoronidase in blad- der cancer Biolbgy of suppressor T cells The Finnish Twim Registry Effect of cocarcinogens and tumor pro- moters on DNA repair in mammalian cells susceptible to chemical transfor- mation Variables affecting the cardiovascular re- sponses to chronic smoking The effects of inritation and drugs on airway epithelium - An experimental study of mechanisms Proteases produced by mammaliam lung tissue A smoking research program in the Nor- mative Aging Study State dependent' properties of nicotine- related compounds Endocrine functions of the lungs Influence of nicotine on the release of acetylcholine in the human placenta and its implications on ~ the fetal growth Central nicotinic receptors 72 I I

273 PRINCIPAL INVESTIGATOR OR INSTITUTION SCRIPPS CLINIC AND RESEARCH FOUNDATION, La Jo11a, Cal. CARL C. SELTZER, Pa.D., Honorary Research Associate, Peabody Museum, Harvard University, Cambridge, Mass. CHARLES R. SHAW, M,D.,,Chief, Sec- tion of Medical Genetics, M. D. An- derson Hospital and Tumor Institute; Professor of Biology, The University of Texas at Houston, Houston. NATHAN H. SLOANE, PH.D., Profes- sor of Biochemistry;, The University of Tennessee Center for the Health Sciences, Memphis. LOUIS A. SOLOFF, M.D.,, Blanche P. Levy Distinguished Service Professor; Professor of Medicine; Director, Re- search Lipid Laboratory, Temple Uni- versity Health Sciences Center, Phila- delphia: DAVID W. TALMAGE, M.D., Director, Webb-Waring, Institute, University' of Colorado Medical Center, Denver. MARC D. THAMES, M.D., Senior Re- search Fellow, Mayo Clinic and' Foun- dation, Rochester, Minn. JAMES TRAVIS, PH.D., Associate Pro- fessor of Biochemistry, University of Georgia, Athens. GERALD M. TURINO, M,D.,,Professor of Medicine, Columbia University Col- lege of Physicians & Surgeons, New York. D. M. TURNER, Ptt:D., Head, Depart- ment of Drug Metabolism and Bio- chemistry;, Hazleton Laboratories, Eu- rope, Ltd.,, Harrogate, Yorkshire, Eng- land. 4 EMIL R. UNANUE, M.D., Mallinckrodt Professor of Immunopathology, Har- vard Medicali Sehool„ Boston. UNION CARBIDE CORPORATION, Nuclear Division, Oak Ridge, Tenn. 73 Immunological competence and chemical carcinogenesis Constitutional studies relative to smoking and coronary heart disease Hydrocarbon metabolizing enzymes and lung cancer a Effect of benzo[o]pyrene and derivatives on mammalian lung cells Role of lecithin: cholesterol acyl trans- ferase (LCAT) in cholesterol metabo- lism in health, disease and' during smoking Purification of an antibody production to lecithin: cholesterol' acyltransferase The role of macrophage-induced factors in cancer immunity Cardiac mechanoreceptors and renin re- lease Biochemistry of chronic obstructive lung disease Chemical basis of tissue destruction in obstructive lung disease Buccal absorption of nicotine in the anesthetized cat Physiopatbology of normali and activated macrophages Characterization of animal exposure sys- tems Characterization of animal inhalation ex- posure devices Dosimetty studies

274 PRINCIPAL INVESTIGATOR OR INSTITUTION PROJECT TITLE UNIVERSITY OF SAN FRANCISCO, San Francisco. A study of squamous cell carcinoma ini mouse lungs Exposure of mice to asbestos and ciga- rette smoke - STEPHEN F. VATNER, M.D., Associate Nicotine-induced reflex coronary vasodi. Professor of Medicine, Harvard Medi- lation cal School; Associate in Medicine, Peter Bent Brigham Hospital, Boston. Nicotine-induced reflex activation BRANISLAV VIDIC; D.S., Professor The effect of cigarette smoke on lung of Anatomy, Georgetown University metabolism Schools of Medicine and Dentistry, Washington, D.C. (See Hamosh, M.) IRENE Y. WANG, PH.D., Assistant Pho- fessor of Basic and Clinical Immunol- ogy and Microbiology, Medicali Uni- versity of South Carolina, Charleston. LEE W. WATTENBERG, M.D., Profes- sor of Pathology, University of Min- nesota Medicali Sehoolj Minneapolis. GEORGE WEINBAUMs PH,D., Biosci- entist, Pulmonary Disease Section, Al- bert Einstein Medicali Center, Phila- delphia. THOMASC. WESTFALL, PH.D., Profes- sor of Pharmacology, University of' Vir- ginia School of Medicine, Charlottes- ville. JAMES A. WILL,, D.V.M., PH.D., Pro- fessor and Chairman, Department of Veterinary Science, University of Wis- consin, Madison. GEORGE WOLF, D. PHIL., Professor of Physiological Chemistry, Department of Nutrition and Food Science, Massa- chusetts Institute of Technology, Cam- bridge, Mass. KOJI YOSHINAGA, PH.D., Associate Professor of Anatomy, Laboratory of Human Reproduction and Reproduc- tive Biology, Harvard Medicali Sehoolj Boston. Genetic differences in the in vitro metabo- lism of ehemicali carcinogens by human and mouse tissues Inhibition of carcinogenesis by benzyliso- thiocyanate and related compounds Lung proteinase:antiprotinase balance and the effect of cigarette smoke on this interaction Action of nicotine on peripheral an&cen- tral neurons in animals chronically ex- posed to nicotine _ Morphologic and functional correlations of the APUD cells of the lung The effect of vitamin A on glycoprotein synthesis in normal and' precancerous respiratory epithelium Effect of carcinogens on glycoprotein synthesis Effects of nicotine on pregnancy 741 1 1

r 275 Completed Projects Following is a list of the principal investigators, or institutions, of projects that have beem completed prior to the period covered in this Report: Several of the individuals named are deceased. The titlt s and affilia- tions listed are those in effect at the time the work was completed. CLARENCE M. AGRESS, M.D., Asso- ciate Clinical' Professor of, Medicine, University of California Medical Cen- ter, Los Angeles. ANTHONY A. ALBANESE;, PH.D., Di- rector of Laboratories, The Burke Re- habilitation ~ Center, White Plains, N.Y. ANTHONY P. AMAROSE, Px.D:, In- structor in Obstetrics and Gynecology, The Albany Medical College of Union University, Albany, N.Y. E. T. ANGELAKOS, M.D., Ptt;D., Pro- fessor of Physiology, Boston University School of Medicine, Boston. D. MURRAY ANGEVINE, M.D., Uhi- versity of Wisconsin School of Medi- cine, Madison. STEPHEN M. AYRES, M.D., Director, Cardiopulmonary Laboratory, Saint Vincent's Hospital, New York. OSCAR J. BALCHUIvI; Px.D., Hastings Professor of Medicine, University of Southern California School of Medi- cine, Los Angeles. FREDERIK B. BANG; M.D., Professor and Chainnan; Department of Path'o- biology, The Johns Hopkins University School of Hygiene and Public Health, Baltimore. A. CLIFFORD BARGER, M.D., Robert Henry Pfeifler Professor of Physiol- ogy, Harvard Medical' School, Boston. BRODA A. BARNES, MLD., PH.D., Pro- fessor (ABiliate) of Physiology; Colo- rado State University, Fort Collins. FREDERICK W. BARNES, JR., M.D., Associate Professor of Medicine, The Johns Hopkins University School of Medicine, Baltimore. T. C. BARNES,, D.Sc., Research Scien- tist, Philadelphia State Hospital, Phila- delphia. R, FREDERICK BECKER, PH.D., Asso- ciate Professor of Anatomy and Direc- tor, Laboratory of Perinatal' Science, Duke University Medical Center, Dur- ham, N. C. RALPH S. BECKER, PH.D., Professor of Chemistry, University of Houston, Houston. BENJAMIN', BELL, M.D.,, Director Em- eritus, Normative Aging Study, Veter- ans Administration Outpatient CGnic,, Boston. SAMUEL BELLET, M.D., Director, Di- vision of Cardiology, Philadelphia General Hospital, Philadelphia. BARUJ BENACERRAF, M.D., Fabyan Professor and Chairman; Department of Pathology, Harvard Medical School, Boston. JOHN A. BEVAN, M.D.,, Professor of Pharmacology, University of Califor- nia School of Medicine, Los Angeles. --BUDHDEV BHAGAT, PH.D., Professor of Physiology, Saint Louis University School of Medicine, St. Louis. CESARE BIANCIFIORI, M.D.,, Division of Cancer Research, University of Perugia, Perugia, Italy. HYLAN A. BICKERMAN, M.D., Assist- ant Professor of Medicine, and AL- VAN L. BARACH, M.D., Consultant in Medicine, Columbia University Col- lege of Physicians & Surgeons; Gold- water Memorial Hospital, New York. BIO-RESEARCH CONSULTANTS, INC;, Cambridge, Mass. BIO-RESEARCH INSTITUTE, INC., Cambridge, Mass. FRED G. BOCK, PH.D:,, Associate Can- cer Research Scientist, Biological Sta- tion, Roswell Park Memorial Institute, Springville,,N. Y. HERMAN' V. BOENIG, PH.D., Head; Department of Chemistry and Biochem- istry, Spindletop Research Center, Lex- ington, Ky. 75 ® a ® ® a 0 0 I a tj

11 JAMES F. BONNER, PH.D., Professor of Biology, California Institute of Technology; Pasadena. WALTER M. BOOKER, PH.D.,, Profes- sor and Head; Department of Pharma- cology, Howar& University, Washing- ton, D.C. TOM G. BOWERY, PH.D., Research Professor, Pesticide Residue Labora- tory, Nbrth Carolina State College, Raleigh. GEOFFREY L. BRINKMAN; M.D., As- sociate Professor of Medicine, Wayne State University Sehooli of Medicine, Detroit.. ROBERT E. BROOKS;, PH.D., Associate Professor of Pathology, University of Oregon Medical School, Portland. BARBARA B. BROWN, PH.D., Chief; Experimental Psychiatry; Veterans Ad- ministration Hospital, Sepulveda, Cal. RAYMOND R. BROWN; PH;D., Profes- sor of Clinical Oncology, University of Wisconsin Medical School, Madison. JOSEF BROZEK, PH;D., Professor Cnd Chairman, Department of Psychology, Lehigh University, Bethlehem, Pa. SUE BUCKINGHAM, M.D., Assistant Professor of Pediatrics, Columbia Uni: versity College of Physicians & Sur- geons, New York. BENJAMIN BURROWS;, M.D., Asso- ciate Professor of Medicine;,University of Chicago;,Chicago. E. M. BUTT, M.D., Chief Pathologist, Los Angeles County Generali Hospital, Los Angeles. RICHARD U. BYERRUM, PH.D., Pro- fessor of Chemistry, Michigan State University;, East' Lansing: SISTER M. EMILY CAHILL, PH.D., Chairman, Department of' Chemistry, Regis College, Westonj Mass. BRUCE F. CAMERON, M,D., PH.D., Howard Hughes Institute, University of Miami School of Medicine, Miami, Fla. WILLIAM H. CARNES, M.D., Univer- sity of Utah College of Medicine, Salt Lake City. 76 276 MARCUS N. CARROLL, JR.,, PH.D., Chief, Division of' Pharmacology, The Brookdale Hospital' Center, Brooklyn, N. Y. WILLIAM ALVIN CARTER, M.D., Assistant Professor of Medicine and Microbiology, The Johns Hopkins Uni- versity School of Medicine, Baltimore. LEOPOLD R. CERECEDO;, PH;D., Pro- f'essor of Biochemistry and' Nutrition, University of Puerto Rico School of Medicine, San Juan. CHILDREN'S HOSPITAL OF LOS AN- GELES, Los Angeles. SANFORD CHODOSH, M.D., Assistant Professor of Medicine, Tufts Univer- sity School of Medicine, Boston. NAITER M. CHOPRA, Pii:D., Profes- sor of Chemistry, North Carolina Ag- ricultural and Technicali State Univer- sity, Greensboro. WILLIAM G. CLARK, PH.D., Director, Psychopharmacology Research Labora- tory; Veterans Administration Hospital, Sepulveda;, Cal. HANS T. CLARKE, D:Sc., Professor of Biochemistry, Columbia University College of Physicians & Surgeons, New York. JAY D. COFFMAN;, M.D.,, Section Head, Peripheral Vascular Department, University Hospital, Boston. DANIEL COHEN, D.V.M., M.P.H., As- sistant Professor of Veterinary Epi- demiology and Public Health, Univer- sity of Pennsylvania School of Ve.ter- inary Medicine, Philadelphia. JULIUS H. COMROE, JR., M.D., Direc- tor, Cardiovascular Research Institute, University of California Medical Cen- ter, San Francisco. DEAN M. CONNORS, M.D., Associate Director, Department of, Laboratory Medicine, StL Mary's Hospital, Madison, Wis. PHILIP COOPER, M.D., Clinical Pro- fessor of Surgery and Director, Surgi- cal Laboratory of Cellular Physiology, Albert Einstein College of Medicine of 7 . Yeshiva University; Chief, Surgical 15'kl Service, Veterans Administration Hos- ~~ pital, The Bronx, N. Y. JOHN! E. CRAIGHEAD, M.D:,, Profes- '!~ sor of' Pathology, University of Ver- mont College of Medicine, Burlington. RA] 0) fc N JA11 fe L, RIC Pt si: JOH Pr U1 cil BER In Sc to• HYh ing pit i t

277 ROBERT L. CRAIN, PH.D., Assistant Professor of, Sociology, University of Chicago, Chicago. T. TIMOTHY CROCKER, M.D., Profes- sor of Medicine, University of Cali- fornia College of Medicine, Irvine. -CECIL E. CROSS, Research Department~ Sti Joseph Hospital, Burbank, Cal. ALBERT DAMON, M.D., PtnD., Lec- turer on Anthropology; Research Asso- ciate in Medical' Anthropology;, Pea- body Museum;, Harvard University, Cambridge, Mass. THOMAS R. DAWBER, M.D., Associate Professor of Medicine, Boston Univer- sity School of Medicine, Bbston.. R. F. DAWSON, PH.D., Professor of Bot- any, Columbia University, New, York. JOHN P. DELANEY, M.D., PH.D., As- sociate Professor of'Surgery, University of Minnesota;,Minneapolis. ANDREW S. DIBNER, PH.D., Exec- utive, Psycho-Research, The Age Cen- ter of New England, Inc., Boston. EDWARD F. DOMINO, M,D., Profes- sor of Pharmacology, University of Michigan, Ann Arbor. RALPH L. DORFMAN, Px.D., Director of Laboratbries, Worcester Foundation for Experimental Biology, Shrewsbury,. Mass. JAMES J. DYAR; PH.D., Assistant Pro- fessor of Biology, Bellarmine Coll'ege,, Louisville, Ky. RICHARD H. EARLE, M.D., Chief„ Pulmonary Function Laboratory; As- sistant' Professor of Medicine, Univer- sity of Chicago, Chicago. JOHN W. ECKSTEIN, M.D., Assistant Professor of Internal' Medicine, State University of Iowa College of Medi- cine, Iowa City. BERTRAM EICHEL, D.D.S., Director, Institute of' Stomatological Research, Science Resources Foundationj , Water- town, Mass. HYMAN ENGELBERG, M.D., Attend- ing Physician, Cedars of Lebanon Hos- pital) Los Angeles. 77 CARLTON K. ERICKSON, PH.D., As- sociate Professor of Ph'armacoiogy and Toxicologyl, The University of Kansas Schooliof Pharmacy, Lawrence. HENRY J. ESBER, PH.D., Research Im- munologist, Mason Research Institute, Worcester, Mass. JOHN R. ESTERLY, M.D., Associate Professor of Pathology, University of Chicago Pritzker School of Medicine, Chicago. : HANS L. FALK, PH.D., Adjunct Associ- ate Professor of Pathology, University of Southern California School of Medicine, Los Angeles. DANA L. FARNSWORTH, M.D., Henry K. Oliver Professor of Hygiene and Director, University Health Services, Harvard University, Cambridge,,Mass: FRANK C. FERGUSON, JR.,, M.D., Chairman; Department of Pharmacol- ogy, The Albany Medical College of Union University, Albany,, N.Y. THEODORE N. FINLEY, M,D., Direc- tor, Pulmonary Research Laboratory, Mount Zion Hospital, San Francisco. WILLIAM I. FISHBEIN, M.D., Chief of Epidemiology, Chicago Board of Health, Chicago. EDWIN R. FISHER, M.D., Director of Laboratories, Shadyside Hospital; Pro- fessor of Pathology, University of Pittsburgh School of' Medicine, Pitts- burgh. RUSSELL S. FISHER, M.D., Universityy of Maryland School of Medicine, Bal- timore. B. L. FREEDLANDER, M.D., Director of' Cancer Research„ Mount Zion Hos- pitali and Medical Center, San Fran- cisco. FREDERIC A. FRENCH, A.B., Direc- tor of Cancer Chemotherapy Research, Mount Zion Hospital and Medical Center, San Francisco. JACK FREUND, M.D., Assistant Pro- fessor of Pharmacology, Medical Col- lege of Virginia,, Richmond. GILBERT H. FRIEDELL, M.D., Chief of Pathology, St. Vincent HbspitalJ Worcester;, Mass. ;~,

278 H. HUGH FUDENBERG; M.D., Profes- sor of Medicine, University of Cali- fornia Medicali Center, San Francisco; Professor of' Bacteriology and Immu- nology, University of California, Berke- ley. ARTHUR FURST, Px.D., Director, In- stitute of Chemical Biology, University of San Francisco, San Francisco. MURRAY B. GARDNER, M.D., Asso- ciate Professor of Pathology, Univer- sity of Southern California School of Medicine, Los Angeles. GEORGE O. GEY, M.D., Director, Fin- ney-Howell Cancer Research Labora- tory; Associate Professor of Surgery, The Johns Hopkins University School of Medicine„Baltimore. THOMAS M. GOCKE, M.D., Associate Professor of Preventive Medicine and Community Health, Seton Halli Col- lege of Medicine and Dentistry, Jersey City, N. J. DAVID M. GOLDENBERG, Sc.D., M.D., Associate Professor of Pathol- ogy, Temple University Health Sci- ences Center, Philadelphia. PAUL GOLDHABER, D.D.S., Associate Professor of Periodontology, Harvard' School of Dental Medicine, Boston. IRA GORE, M.D., Professor of Pathol- ogy, Boston University School of Medi- cine;, Chief of Laboratory Service, Veterans Administration Hospital, West Roxbury;, Mass: GERTRUDE Y. GOTTSCHALL, PH.D., Assistant Professor of Biochemistry, Columbia University College of Physi- cians & Surgeons, New York. A. CLARK GRIFFIN, PH.D., Head, Department of' Biochemistry, M. D. Anderson Hospital and Tumor Insti- tute, University of Texas Medical Center, Houston. ARTHUR L. GROSS, M.S:, Senior Bio- chemist, Southwest Research Institute, San Antonio, Tex. MORTON I. GROSSMAN, M.D., Pa.D., Associate Clinical Professor of Medi- cine, University of California Medical Center, Los Angeles. CARL C. GRUHZIT, M.D., PH.D., As- sociate in Physiology and Pharmacol- ogy, University of Pennsylvania Grad- uate School of Medicine, Philadelphia. JOSEPH J. GUARNERI, PH.D., 'Ane ing Microbiologist; Director, Microb& ology Laboratories, Long Island Jew= ish-Hillside Medical Center, Queens Hospital' Center Affiliation,, Jamaica, N:Y. vs:, FRANK E. GUTHRIE, Pa.D., Profes- sor of Entomology, North CarolinA State College, Raleigh. H. B~ HAAG, M.D., Professor of Ph macology, Medical College of Virgini~ Richmond. ' F. J. HADDY, M.D., PH.D., Professar and Chairman, Department of Physi- ology, University of Oklahoma Medical' Center, Oklahoma City. ,a .., . - ~; JOSEPH H. HAFKENSCHIEL, M.D., Director, Cardiopulmonary Unit,, The Lankenau Hospital; Associate in Medi- cine, University of Pennsylvania School' of Medicine, Philadelphia: BERNARD HANES, PxD., Professor of Health Science, California State Univer- sity, Northridge. RICHARD J.. HAVEL„ M.D., Assistant Professor of Medicine, University of California Medical Center, San Fran- cisco. :~ HERBERT R. HAWTHORNE, M.D., ;, Chairman, Department of' Surgery, University of Pennsylvania Graduate School of Medicine, Philadelphia. JOHN A. HAYES, M.D., Associate _.~ Pathologist, Mallory Institute of Pa- thology, Boston City Hospitalj Boston. CLARK W. HEATH,,M.D.,,Professor of ; Medicine and Director of Health Serv- ices, Tufts University, Medford, Mass. PAULINE HEIZER, PH.D., Research Associate in Cytology and Cytochemis- try, San Francisco Institute of Medical Sciences, San Francisco. LAWRENCE L. HESTER, JR., MD., Professor and' Chairman, Department of'Obstetrics and Gynecology, Medical College of' South Carolina, Charleston. EBBE CURTIS HOFF, MD., PH.D.,. Professor and Chairman, Division of Psychiatric Research, Medica[ College of Virginia, Richmond. RU a c FR c t RO c 1 III GI JE 1L M O: A', A A H F

279 Ws SHIRLEY L. KAUFFMAN; M.D., Pro- fessor of Pathology, State University of New York Downstate Medical Cen- ter, Brooklyn. ANCEL KEYS, Px.D., Director, Labora- tory of Physiologicat Hygiene„ Univer- sity of Minnesota School, of Public Health, Minneapolis. JOSEPH' B. KIRSNER, M6D., Professor of Medicine, University of Chicago School of Medicine, Chicago. JEROME KLEINERMAN, M,D., Head, Division of Pathology Research and' Cinical Pathology, St. Luke's Hospital, Cleveland; Professor of Pathology,. Case Western Reserve University School of Medicine, Cleveland. RUSSELL L. HOLMAN, M.D.,, Louisi- ana State University Sehool' of Medi- cine, New Orleans. OLE A. HOLTERMANN, M.D., Re- search Scientist, Lobund Laboratory, University of Notre Dame, Notre Dame, Ind. FREDDY HOMBURGER, M.D., Presi- dent and Director, Bio-Researeh Insti~ tute„Inc., Cambridge, Mass. ROBERT W. HULL, Ptt,D., Professor of Biological Sciences, Florida State University, Tallahassee. IIT RESEARCHINSTITUTE„ Chicago. GEORGE JACOBSON, M.D., Professor and Head„ Department of, Radiology, University of Southern California e School of Medicine, Los Angeles. PETER H. KNAPP, M.D., Research i- Professor of Psychiatry, Boston Uni- bl JERRY HART JACOBSON, M.D., Di- versity School of Medicine, Boston. rector, Division of Electrophysiology, New York Eye and Ear Infirmary, KENNETH P. KNUDTSON, M.D., Uni- New York. versit ton School' of Medi- of Washin bf y g cine Seattle. JULIUS' H. JACOBSON II, M.D., Asso- , ciate Professor of Surgery and Director ALVIN I. KOSAK, PH;D., Associate of Surgical Research. University of Professor of Chemistry, New York Vermont College of Medicine, Bur- University, New York. lington. MURRAY E. JARVIK, PH.D., Associate Professor of Pharmacology, Albert Ein- stein College of Medicine of Yeshiva University, The Bronx, N. Y. 0 OSWALD R. JONES, M.D., St. Luke's Hospitalj New York., ANDREW A. KANDUTSCH, Ptr.D., StaB Scientist, The Jackson Labora- tory, Bar Harbor, Me. I ARNOLD R. KAPLAN; PH.D., Direc- tor, Laboratory of, Medical Genetics,. Cleveland Psychiatric Institute and Hospital, Cleveland. ATTALLAH KAPPAS, M.D., Professor and Senior Physician, The Rockefeller University, New York. HRATCH KASPARIAN, M.D., Assist- ant Director, Cardiovascular Labora- toryr Instructor in Medicine,, Hahne- mann Medical College and Hospital, Philadelphia.. ELIHU KATZ, PH.D., Associate Profes- sor of, Sociology, University of Chi- cago;, Chicago. ROBERT A. KUHN, M.D., Associate Professor, Division of' Neurosurgery, New Jersey State College of Medicine, Jersey City. MARVINI KUSCHNER, M.D., .4ew York University Medicali Center, New York. CHARLES W. LABELLE, PH.D., Assist- ant Professor of Environmental Hy- giene, Jefferson Medicali College, Phila- delphia. AARON J. LADMAN„ Pa.D., Professor and Chairman, Department of Anat- omy, The University of New Mexico School of Medicine, Albuquerque. THOMAS C. LAIPPLY, M.D.,, Profes- sor of Pathology; Northwestern Uni- versity Medical' School, Chicago. ROGER K. LARSON, M.D., Chief, of Medicine, Fresno County Hospital, Fresno, Cal. GUSTAVE A. LAURENZI, M.D., Chief of Medicine, St. Vincent Hospital, Worcester, Mass. 79 t 0 0 0 R

280 EDWARD LEETE, PH.D., D.Sc., Profes- sor of Chemistry, University of Minne- sota, Minneapolis. CECILE LEUCHTENBERGER, Px.D., Head, Department of Cytochemist'ry, Swiss Institute for Experimental Can- cer Research, Lausanne, Switzerland. AVERILL A. LIEBOW,, M.D.,, Chair- ntan,, Department of Pathology, Yale University School of Medicine, New. Havena Conn. ESTEN O. LINDSETH, M.D., PH.D., St. Joseph's Hospital Research Laboratory, St. Paul, Minn. ROBERT H. LINNELL, Pt-C.D.,, Associ. ate Professor of' Chemistry, University of Vermonts Burlington. HERBERT L. LOMBARD, M.D., M.P.H., AJJiliate, Cancer Research In- stitute, New England Deaconess Hos- pital„ Bostom J. P. LONG, PH.D., Professor of' Phar- macology, State University of Iowa College of Medicine, Iowa City. DONALD B. LOURIA, M.D., Associate Professor of Medicine, Cornell Univer- sity Medical College, New York. KENNETH MERRILL LYNCH, M.D., Sc.D., LL.D., Professor Emeritus of Pathology and Chancellor, Medicall College of South, Carolina, Charleston: INES MANDL, PH.D., Assistant Profes- sor of Biochemistry;, Columbia Univer- sity College of' Physicians & Surgeons;, New York. MASON RESEARCH INSTITUTE, Worcester, Mass. DONALD J. MASSARO, M.D., Associ•ate Professor of' Medicine, George Washington University School of Medicine, Washington, D. C. i CHARLES McARTHUR, PH.D., Psy- chologisty University Health Services; Harvard University, Cambridge, Mass. CHARLES B. McCANTS, PH.D., Asso 1 ciate Professor of Soils, Nbrth Caro- lina State College School of Agricul-, , ture, Raleigh. HENRY C. McGILL, JR., M.D., ActiniJ Head; Department of Pathology,, Loui- siana State University School of Medi- cine, New Orleans. HENRY D. McINTOSHi M.D., Profes-' sor of Medicine and Director, Cardio- vascular Laboratory, Duke University Medical Center,,Durham, N. C. FORDE A. McIVER, M.D., Associate Professor of Pathology, Medical' Col- lege of South Carolina, Charleston. '" EDWARD McKEE, M.D., Professor and Acting Chairman, Department of Pathology;, Medical College of South Carolina, Charleston KELLY T. McKEE, M.D., Associate Professor of Medicine, Medical Col- lege of South Carolina, Cbarleston. VICTOR A. McKUSICK, M.D., Profes- sor of Medicine, The Johns Hopkins University School of Medicine, Balti- more, JOHN H. MANHOLD, JR., D.M.D.,. Professor and Director, Department of Pathology and Oral Dfagnosis, New Jersey College of Medicine and Den- tistry, Jersey City. DAVID E. MANN, PH.D.,, Associate Professor of Pharmacology, Temple University School' of Pharmacy, Phila- delphia. JOHN P. MANOS, M.D., instructor in Virology and Bacteriology, Medical College ofl South Carolina, Charleston. CHRISTOPHER M. MARTIN, M.D., Assistant Professor of Medicine and Director, Division of Infectious Dis- eases, Seton Hall College of Medicine, Jersey City. ROSS L. McLEAN, M.D:,, Associate Professor of Medicine,, Emory Univer- sity School of Medicine, Atlanta. WILLIAM F. McNARY, Jtt., PH.D., As- sociate Professor of' Anatomy,, Boston University School of Medicine, Boston NEAL L. McNIVEN„ Pa.D., The Wor- cester Foundation for Experimental Biology, Shrewsbury, Mass. JULIA MEYER, PH.D., Associate Pro- fessor of Oral'PatJiology, University of Illinois College of Dentistry, Chicago. BERNARD J. MILLER, M.D., Assistant Professor of Anatomy; Jefferson Medi- cal College, Philadelphia. 80 CH. . D C t( HUU c 0 I P. ] G1 K H E' Vi R E t ]

JAMES G. MILLER, M.D., PH.D., Pro- fessor of Psychiatry and' Psy,chology;, Director, Mental Health Research In- stitute, University of Michigan, Ann Arbor. CHARLES MITTMAN; M.D.,, Director, Department of Respiratory Diseases, City of Hope National Medical Cen- ter, Duarte, Call HUGH MONTGOMERY, M.D., Asso- ciate Professor of Medicine, University of Pennsylvania School of' Medicine, Philadelphia. P. O'B. MONTGOMERY, JR., M.D., Professor of Pathology, University of Texas Southwestern Medical Schoolt Dallas. GEORGE E. MOORE, M.D., Ptt:D., Di, rector, Roswell Park Memorial Insti- tute, Buffalo, N'. Y. KENNETH M. MOSER, M.D., Assistant Professor of Medicine, Georgetown University Medical School, Washing- ton„D. C. HURLEY LEE MOTLEY, M.D.,,Profes- sor of Medicine and Director, Cardlo- Respiratory Laboratory, University of Southern California School of Medi- cine, Los Angeles. EDMOND ANTHONY MURPHY, M.D., Sc.D., Associate Professor of Biostatistics and Medicine, The Johns Hopkins Uhiversity School of Medi- cine, Baltimore. WILLIAM S. MURRAY, Sc.D., Senior Staff' Scientist, The Jackson Labora- tory, Bar Harbor, Me. RICHARD L. NAEYE, M.D.,, Professor and Chairman, Department of Pathol- ogy, Pennsylvania State University Col- lege of Medicine, Hersbey: ALBERT H. NIDEN, M.D., Professor of Medicine, Drew Postgraduate Medical School and University of Southern California; Chief, Pulmonary Disease Section, Martin Luther King Hospital, Los Angeles. OAK RIDGE NATIONAL LABORA- TORY, Oak Ridge, Tenn. DONALD M. PACE,, PH.D., Professor of Physiology and Director, Institute for Cellular Research, University of Nebraska, Lincoln. 81 281 ALBERT B. PALMER, PH-D., Assistant Professor of Pathology, University of Toledo, Toledo, O. ROSE MARIE PANGBORN; M.S., As- sistant Food Technologist and Lecturer, Department of Food Science and Tech- nology, University of California, Davis. JOHN W. PARKER, M.D:,, Associate Professor of Pathology, University of Southern California School of Medi- cine, Los Angeles. MARY STEARNS PARSHLEY, PH;D., Assistant Professor of Anatomy in Ob- stetrics and Gynecology, Columbia University College of' Physicians & Sur- geons, New York. EDWARD W. PELIKAN; M.D., Chair- man, Department of Pharmacology and Ezperilnental Th'erapeutics, Boston University Sehooli of Medicine, Boston, MALCOLM C. PIKE, PH;D., Professor of Community Medicine and Pediat- rics. University of Southern California School of Medicine, Los Angeles. OTAKAR J. POLLAK, M.D., PH.D., Executive Director, Dover Medical Re- search Center, Inc., Dover, Del. MORRIS POLLARD, PH.D.,, Director, Lobund Laboratory, University of Notre Dame, Notre Dame, Ind. C: M. POMERAT, PH.D., Director of Biological Research, Pasadena Founda- tion for Medical Research, Pasadena, Cal., S. N. PRADHAN, M.D., PH.D., Profes- sor of Pharmacotogy, Howard Univer- sity College of Medicine, Washington, D.C. H., R. PRATT-THOMAS, M.D., Profes- sor of Pathology, and Dean, Medical College of South Carolina, Charleston. PROCESS & INSTRUMENT CORPO- RATION, Brooklyn, N. Y. MARTIN S. PROTZEL, B.S., D.D.S., Chief, Department of Oral Pathology, Newark City Hospital, Newark, N. J. WALTER REDISCH, M.D., Associate Professor of Clinical Medicine, New York University Sebooli of Medicine, an&NYU Research Service, Goldwater Memorial Hospital, New York.

WILLIAM REGELSON, M.D., Professor and Chairman, Department of Medical Oncology, Medical College of Virginia, Richmond. HOBART A. REIMANN, M.D., Profes- sor of Medicine, Hahnemann Medical College and Hospital, Philadelphia. ROLLAND C. REYNOLDS, M.D., As- sistant Professor of Pathology, Univer- sity of Texas Southwestern Medical School;, Dallas. VICTOR RICHARDS, M.D., Chief of Surgery, Presbyterian Medical Center, San Francisco. WILLIS H. RIESEN, PEt.D., Senior Bio- chemist, Life Sciences Division, IIT Research Institute, Chicago. R. H., RIGDON„ M.D., Professor of Pa- thology, University of Texas Medical Branch, Galveston. SYDNEY C. RITTENBERG, PH.D., Professor of Bacteriology, University of Southern California, Los Angeles. BENSON' B. ROE„ M.D., Associate Pro- fessor of Surgery; Chief, Cardiac Sur- gery, University of California School of Medicine, San Francisco. JOSEPH H. ROGERS, M.D., Holy Name of Jesus Hospitall Gadsden, Ala. ROBERT C. ROSAN, M.D., Associate Professor of Pathology and Pediatrics, St'. Louis University School of Medi- cine; Associate Pathologist, Cardinal Glennon Memorial Hospital for Chil- dren, St. Louis. JOHN R. ROWLANDS, PH.D., Stafj Scientist, Southwest Research Institute, San Antonio, Tex. BENJAMIN A. RUBIN, PH:D., Assistant Professor of Public Health, Baylor University College of Medicine, Hou• ston. RONALD P. RUBIN, PH;D., Professor of Pharmacology, Medical College of', Virginia, Richmond. HENRY I. RUSSEK, M.D., President, The Russek Foundation, Inc., Staten Island. N. Y. W. C. RUSSELL, M,D., University of Texas Medical Center, Houston. 282 WAYNE L. RYAN, Px.D., Professor oP" Biochemistry, University of Nebraska~ College of Medicine, Omaha. PETER F. SALISBURY, M.D., PH.D., , Head, Intensive Treatment Cettter, `.. Saint Joseph Hospital„Burbank, Cal. a~ PAUL SALTMAN, PH:D., Assistant Pro-'', fessor, Department of Biochemistry and Nutrition, University of' Southern ' California School of Medicine Los" Angeles. ULRICH H. SCHAEPPI, M.D., Direc- tor of Neuropharmacology, Mason Re- search~Institute, Worcester, Mass., JORGEN U. SCHLEGEL, M.D., PH,D., Professor and Chairman, Department of, Surgery, Tulane University Schoo ' of Medicine, New Orleans. ALVIN R. SCHMIDT, PH.D., Director ': ; of Counseling, Tufts University, Med-'; ford, Mass. ISAAC SCHOUR, D.D.S., PH.D.. D.SC., Dean, University of Illinois College of ' Dentistry, Chicago. MAURICE S. SEGAL, M.D., Clinicafl-Professor of Medicine, Tufts Univer-., - sity School of Medicine; Director, De-' partmem of Inhalation Therapy Bos- ton City Hospital, Boston. J"' ' LUCIO SEVERi, M.D., Director and , Dean, Institute of Anatomy and'Pathob ~ ogy, Division of Cancer Research, ~ University of Perugia, Perugia; Italy. ~~ CHARLES E. SHERWOOD, M.D., As- '~ sistant Professor of, Radiology, Univer- ~ sity of Rochester School of Medicine `~ and Dentistry, Rochester, N. Y. SHOJI SHIBATA, M.D., PH.D., Profes- .sor of Pharmacology, University of ~ Hawaii School of Medicine, Honolulu. DAVID L. SIMON, M.D., Instructor in Internal Medicine, Cincinnati General Hospital,, Cincinnati. , ERIK SKINHOJ, M,D., Chief, Depart- ment of Neurology, Bispebjerg Hospi- tal, Copenhagen. THEODORE A. SLOTKIN, Pe.D., As- ;- sistant Professor of Pharmacology, Duke University Medical Center, Dur- ham, N.C: G C, f. L S I I 1 82 4 W ~ »a C11 -1 W 0) nN> . „ ."ktm....

.,.d. ,.. .D., ter. -fi ro-.,, stry ern '. Loa `. Vi'ec- Re- _ V t~ ,D., ent 001 ctor ~ ed- e of 7" nical iver- De- Bos- .Y' "A s- iver- icine pfes- !yart- ;ospi= As- logy,. IDur- M GEORGE W: SME9'TERS, M.D.,, Asso- ciate in Pathology, Northwestern Uni- versity Medical School, Chicago: GENE M. SMITH, PH.D.,, Assistant Pro- fessor of Psychology, Harvard Medical School, Massachusetts General Hospi- tal, Boston. LUCILE SMITH, PH.D., Professor of Biochemistry, Dartmouth Medical School, Hanover, N. H. SHELDON C. SOMMERS;, M.D., Direc- tor of Laboratories, Lenox Hill Hos- pital; Clinical Professor of Pathology, Columbia University College of Physi- cians & Surgeons, New York. _ ERNEST SONDHEIMER, PH.D., Asso- ciate Professor of Biochemistry, Col- lege of' Forestry, State University of New York, Syracuse. T. M. SONNEBORN, PH.D., Distin- guished Service Professor of Zoology, Indiana University, Bloomington. SAM SOROF, PH.D., Head, Department of Macromolecular Chemistry;, The Institute for Cancer Research, Phila- delphia. SOUTHWEST RESEARCH INSTI- TUTE, San Antonio, Tex. DAVID M. SPAIN, M,D., Director, De- partment of Patholbgy, The Brookdale Hospital Center, Brooklyn, N. Y. ALEXANDER SPOCK, M.D., Assistant Professor of Pediatrics„ Duke Univer- sity Medical Center~ Durham, N. C. FREDERICK J. STARE, M.D., Profes- sor of Nutrition, Harvard University School of Public Health, Boston. C. HAROLD STEFFEE,, M.D., Director of Laboratories, Methodist Hospital, Memphis. JACK P. STRONG, M.D., Associate Professor of Pathology, Louisiana State University School of Medicine, New Orleans. MARION B. SULZBERGER, M.D., Pro- fessor and Chairman of Dermatology and Syphilology, New York Univer- sityBellevue Medical Center, New York. RENATO TAGIURI, PH.D., Associate Professor of Psychology, Graduate School of Business Administration, Harvard University;, Boston, !7 283 CAROLINE BEDELL THOMAS, M.D.,. Professor Emeritus of Medicine, The Johns Hopkins University School of Medicine, Baltimore. JEROME F. THOMAS, PH.D., Professor of Sanitary Engineering:, University of California, Berkeley. JAMES E. P TOMAN, PH.D., Profes- sor and'Chairman; Department of Ph'ar- macology, Chicago Medical School, In- stitute for Medical Research, Chicago: JANET TRAVELL, M.D., Associate Professor bf Clinical' Pharmacology, Cornell University Medical College, New York. LIE SHA TSAI, Ptt.D., Research Asso- ciate in Pathology, Y'ale University School of Medicine, New Haven{ Conn, UNIVERSITY OF SOUTHERN CALI- FORNIA, Los Angeles. ELLIOT S. VESELL, M.D., Pro fessor and Chairman, Department of Pharmacoll ogy, Pennsylvania State University C& lege of Medicine, Milton S. Hershey Medical Center, Hershey: ROMEO A. VIDONE, M.D., Associate Professor of Pathotogy;, Yale Univer- sity School of Medicine, New Haven, Conn, PETER K. VOGT, Ptt.D:, Professor of Microb'iology; University of Washing- ton School of Medicine, Seattle. E. D. WARNER, M.D., Professor of Pa- thology, State University of Iowa Col- lege of Medisine, Iowa City: SHIELDS WARREN; M.D.,, Director of Laboratories, Cancer Research Insti- tute, New England Deaconess Hospi- tal, Boston. YASUSHI WATANABE, Px.D., Asso- ciate Member, The Wistar Institute of Anatomy and Biology, Philadelphia. BARBARA K. WATSON, PH.D., Assist- ant Bacteriologist, Massachusetts Gen- eral Hospitall Research Associate in Bacteriology and Immunology, Har- vard Medical School, Boston. JOHN S. WAUGH, PH;D., Professor of Chemistry, Massachusetts Institute of Technology, Cambridge. 83 A Cj  

284 RICHARD L. WECHSLER, M.D., Clin- FREDERICK E. WHISKIN, M.D. C.M., ical Physiologist,, Montefiore Hospital Director, Division of Heaith and Per- Institute of Researcha Pittsburgh. sonality Equilibrium, The Age Center Inc., , Boston. JOHN V. WEIL M.D., Assistant Pro- of New England, fes.sor of Medicine, University of Colo- ROGER J. WILLIAMS, M.D., Professor rado Medical Center, Denver. of Chernistry;, Director, Clayton Foun- - dation Biochemical Institute, The Uni- WEINSTOCK P D R h Bi A , B. ., eseare . o chemist, Life Sciences Division, IIT versity of Texas, Austin. Research Institute, Chicago. DANIEL H. WISEMAN, M.D., Assist- RUSSELL W. WELLER, M.D., Pathol- ant Professor of Pediatrics, University ogist; Memonial' Hospital of Chester of Southern California; Children's Di- County„ West Chester, Pa. vision, Los Angeles County General A. STANLEY WELTMAN; PH.D., Asso-~ Hospital, Los Angeles. ciate Professor of Pharmacology and J Instructor in EDWIN WOOD M D , .., , Research, Brooklyn College of Phar- Medicine, Boston University School of Br kl N Y macy, oo yn, . . Medicine, Boston. SIMON H. WENDER Ptt Research D , . ., Professor of' Biochemistry, University SUMNER WOOD, JR., M.D., Assistant of Oklahoma, Norman. Professor - of Pathology;, The Johns Hopkins University School of MedI- DUANE G. WENZEL, PH.D., Professor cine, Baltimore.. and Chairman, Department of Pharma- cology and Toxicology, The Uhiversity JOHN P. WYATT, M.D., Professor o of Kansas School of Pharmaey; Law- Pathology, Saint Louis University rence. School of Medicine, St. Louis. 84

ofessor Foun- e Uni- cror in hool of ssislqnJ Johns Medi- sor q versity Abood„L. G., 54, 55 Arcos, J. C:, 14, 15 Aviado, D. M.,28 Bhagat, B., 56 Bing, R. J., 41„42, 43 Boden, G., 57 Bosse„ R., 64, 65 Castro; A., 61 Chalon, J., 16,29, 30, Cochrane, C. G., 46 Cohen, A. B., 31 Costa, P. T., Jr., 64 Cross, C. E., 31, 32„ 33 Essman, W. B., 57 Fishman, W. H., 16, 17; 18, 19, 20 Friedman, G. D., 65 Furst, A., 28 Goldstein, L., 58 Hamosh, P., 34 loachim, H. L., 19 Kouri{ R. E., 25 Lauweryns, J. M., 35, 36 Levy, J. A., 21 Manhold„ J. H., Jr., 21 Mason, R. G., 511 McKennis, H., Jr:, 59 Meier, H., 22, 23, 24, 63 Regan, T. J., 52, 53 Rifkin, D. B., 26 Rose, C. L., 65 ( Ryan+ U. S., 36, 47, 48, 49, 50, 61, 62 Sastry, B. V. R., 59, 60 Sloane, Nl H., 24 Soloff, L. A., 44, 45 Sommers, S. C., 27 Travis, J., 37, 38, 39 Vatner, S. F., 52 Weinbaum, G., 39, 40 Wenzel, D. G., 46 ~.~.,: ~:,•~.~, ,

286 INDEX OF SENIOR AUTHORS'. Abood, L. G., 55 Ahmed, S. S., 53 Arcos, J. C., 15 Argus, M. F., 14, 15 Baugh, R. J., 37 Bedigian~ H. G., 22, 23 Bhagat, B., 56 Bing,R.J.,41,43 Boden, G.,, 57 Bosse, R.,, 65 Castro, A., 6I Chalon, J., 16, 30 Chang, C-H., 117, 18,,19 Chiu, A. T., 49, 50 Cochrane, C. G.,, 46 Cohen, A. B., 31 Costa, P. T., Jr., 64 Cross, C. E., 32 David, J. S. K., 44, Diwan, B. A., 24 Doyle, J. L., 21 Essman, W. B., 57 Fishman, W. H., 16, 20 Fox, R. R., 22 Friedman, G. D., 65 Friedman-Mor, Z., 29 Furst, A., 28 Hamosh, M.,, 34 Harbison, R. D., 59 Hussain, M. Z., 31 loachim, H. L., 19 Ito, H., 28 Johnson, D. A., 37, 38 Katz, J. S., 30 Kimbel, P., 40 Kouri, R. E., 25 Lauweryns, J. M., 35, 36 Levy, J. A., 21 Lowy, K., 54 Moschos, C: B., 52 Nelsen, J. M.,, 58 Peirce, T. H., 32 Ritkin~ D. B., 26 Rose, C. L,, 64 Ryan„J. W.,,47, 48,,50, 61 Ryan, U. S., 36, 48, 62. Saba, S: R., 51 Sarma„J. S. M.,,41, 42, 43 Sastry, B. V. R., 60 Sloane, N. H., 24 Soloff, L. A., 44 Sommers, S. C., 27 Taveira da Silva, A. M., 34 Travis, J., 39 Varma, K. G'., 45 Vatner, S. F., 52 Weinbaum, G.,, 39 Weisbroth, S. H.,,63. Wenzel, D. G., 46" Wilson, K. L., Jr., 59' York, G. K.,, 33 I 1: J K

287 RESEARCH GRANTS Since the publication of the 1976 Annual Report, the following research projects have been funded by The Council for Tobacco Research - U1.S.A., Inc.: MARIO D. ACETO, Ph.D., Associate Professor, Depart- ment of Pharmacology, Virginia Commonwealth University, Richmond. "Stereospecific binding of nicotine." _ LESLIE BAER, M.D., Associate Professor of Medicine, Columbia University College of Physicians & Surgeons, New York, "Cigarette smoking in normotensive and hyper- tensive subjects: blood pressure, renin, aldosterone and catecholamine response." _ _,., FRANCOIS M. BOOYSE, Ph.D., Associate Professor of Bio- chemi~stry, Rush-Presbyterian-St. Luke's Medical Center, Chicago. "In vivo and in vitro responses of endothelial celLs,and pTatelets to nicotine and extracts from stan- dardized cigarette smoke condensates." . ELROY T. CANTRELL, Ph.D., Chairman, Department of Pharma- cology, Texas College of Osteopathic Medicine, Denton. "Aryl hydrocarbon hydroxylase in single cells and subpop~- ulations of human lymphocytes and'other cells." :RANCI,S C. CHAO, M,D., Ph.D., Senior Investigator, Center for B1ood Researchy Boston. "Platelet activstion and blood hypercoagulability." , RONALD W. GILLETTE, Ph.D., Senior Researcher and Associate Director, Basic Science Unit, University of Hawaii, Cancer Center of Hawaii, Honolulu. "Effect of tobacco byproducts and other environmental contaminants on lymphoid cell homing and function." I r I

CAROLINE HALL, M.D., Assistant Professor of Pediatrics and Medicine, University of Rochester School of Medicine, Rochester, N.Y. "The relationship of lower respiratory tract disease in infancy to the development of chronic lung disease in adults: development and time course of physiologic abnormalities indicative of early obstructive airways disease." LINDA M. HALL, Ph.D., Assistant Professor of Biology, Massachusetts Institute of Technology, Cambridge, Mass. "Hereditary alterations of nicotine sensitivity." ABEL LAJTHA, Ph.D., Research Foundation for Mental Hygiene, Inc., Albany, N'.Y. "The effect of nicotine and carbon monoxide on the transport of amino acids into brain and on protein metabolism."' FRANK A. MANNING, M.D., Women's Hospital, Los Angeles County-University of Southern California Medical Center, Los Angeles. "Fetal and maternal effects of cigarette smoking, nicotine injection and carbon monoxide inhalation in the pregnant rhesus monkey model." HERBERT Y. REYNOLDS,, M.D., Associate Professor of Internal Medicine and Head, Pulmonary Section, Yale University School' of Medicine, New Haven, Conn. "Bronchoalveoliar lavage fluids in pulmonary carcinoma: secretory compo- nent of immunoglobulin A as a marker of neoplastic growth." THOMAS P. STOSSEL, M.D., Chief, Medical Oncology Unit, Massachusetts General Hospital, Boston. "Functional anatomy of the pulmonary macrophage." A. M. TOMETSKO, Ph.D., President and Director of Research, Litron Laboratories Ltd., Rochester, N.Y. "'Probing nicotine receptor sites." This brings to $46,000,000 the amount The Council has provided for smoking and health research since its formation in 1954. t t f m. .

r ( 289 Im addition, in the period 1964 through 1972, the industry gave $15,005,000 to the Education and Research Foundation of the American Medical Association. These funds were used to support approximately 195 research grants made by the Foundation. The tobacco industry has also funded the following re- search projects at various universities (all of which are still in progress): - 1. . Harvard University. In 1972, ,the industry awarded s $2,792,750 to Harvard University for investigation into any effect ` *_obacco smoke may have on pulmonary and cardiovascular diseases. Since that time, an additional $,2,376,122.301has been committed to this project for a totali amount to date of $5,1168,872.30. 2. Washington University at St. Louis. In 1971, the industry awarded $2,000,000 to Washington University for basic research in cancer immunology. Since that time, additionali sums totaling $1i,600,000 have been committed to this project, for a total of $3,600,000. 3. UCLA. In 1974, the industry awarded $1,700,000 to UCLA for research involving possible effects of tobacco smoke on defense mechanisms in the lung. Q p 9 tr