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Council for Tobacco Research

Report of the Council for Tobacco Research - U.S.A., Inc. 1987 [St]

Date: 1987
Length: 97 pages
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1987 REPORT uf 'fHE COUNCIL I+OK't'UIIACCO KESL:AItCI•1-U.S.A., INC. TI16: CUUN('I1. FOlt'i'UIiA('('U RI:S1:,1R('il-U.ti.:1., INC. 9UU'I'hird Avenue, New 1'ork, N.Y. IIN)22
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°> c St'Ih.N'1'Ih'lC AI)VISURY I3UAR1) lu'fhr Clrunril I'nrTuharcu Rcuarrh--l1,S-A., Inr, ,u ul Ikrrudx:r 31, 1987 110N ( ). JA('( )IiS( )N, M.I)., ('huirrnun Jrrmplt Rrmrndr'in l'ru/i-.esrrr u/ Medircrl uncl Rinlo,qirrtl.Srirarrs (r•rnrriln.,) I'rr,/rasur t,/ tlir• I)rpdrntcen( ufMerliricte (e»trrilux) Univcrtiity ul ('Itiral:u ('hiragu, Illinois RI('I IAkI) J. I31N(i, M.I)r l)irer•)ur u/ l::tprriuu'ntul ('urdiulul;y cutrl Seietui/it' l)rrrlulnnetti I luntingtun Medical Research Inailulcs, I'asadrna, Calilivntia Vi.siiing i1s.suriulr' ('aliliirnia Institute uf Tcrhnulugy, Pasadena I't rt/r.c.%ur u/'Mrrlir•inr (euterincs) University cti Suuthrrn ('alifurnia School uf MLYiirinc l.us Angeles, C'alilirrnia RUSWIit.t. K. 13Oll'fWl?I.1., I'H.D. l'rr./V ssur n% Onc'ulug v McAidle LaMrratury litr Cancer Research University ul Wisconsin MuJiscm, Wisrunsin I)kl1MMUNl) li. 13OWI)IiN, M.I). Pru%r.s.cur cucrl llc'ud Ihhcurturrnt crf I'alhuluuy lMivcrsi(y ol Manitoba I Ir:dth Srirnrc. ('rnter Winnilx:g, ('anada MI('I IAI?I. J, I3RI?NNAN, M.I). l'residen( und Merlic•ul 1)irer(ur Mirhig:m ('.utrrr Foundation l7o/i•.s'.vur rr/ Ahvlirinr Waync Sta1c lMivcrsi(y School ul' Mcdirinc Iktruit, Michig:ut 1USF.I'I I I). Flil .I)MAN, M.I). Mrud,r•r (r-tttrriucs), Research Institute uf Scripps Clinic Srripp. ('linic and Research Foundation I.a Jolla. ('aliktruia JIi17RIiY R. IDI.I's, I'u.U. Kr•urlrr in Phurntrcru,t;ene(ic•s and Ib'ellrrunr I'rtc.sr Srttiur l.ernrrer I)rpartment ul I"harmareslugy St- Mary's Hospital Medical School Lundon, IinglanJ MANI-ktsl) I.. KAIWUVSKY, I'u.U. lluro/d'I'. lt"hi(r• I'ru/i•s.snr u/'lliulu,>;irrt/ ('ltrrtustry wtd Mrrlrrtulur I'lwrnturrtlo,rv Ilarvard Medical Srhu+d Hu,tcm, Maa,achu,clh ALFICIiI) (J. KNUIriON, JR., M,I)., PtLU, Sr•nir u- hl rnchr,t. Imlitutc lur ('anccr Research fux ('ha,r Cancer ('enter I'hiL•alrlphia, I'rnn.ylvania IIIiNItI' "I'. I.YN('II, M.I). l'ru/rs u» • und ('huirrnrut Ihpartinrnt ul I'rrvcntivc Medicine and I'uhlir Hcalth l'ru/rssur u/ Mr•rlirine l'rr•.sidrnt, Ilcreditury Cancer ImtitWc ('rrightnn llnivcr.ity School ul Medicine Omaha, Nebraska G. 13ARKY 1'Ilat('li, M.1). Antrrirrut ('unr•r•r ,Snric•tv ('c'ntc•tutiul Reseru-clt I'rufi.rsur l Inivrrsity ol ('uluradrr lieallh Sciences ('enter Iknver, Colorado GOItllON II. SATO, I'tt.p. I )irrrtur W. Ahun Juttcs ('cll Science C'cnlcr I.akr Placid, New York SI11?I.I)UN C. SOMMERS, M.I), Srirnti/ic• l)ic•rc7ur The C'uuttril liOr 7bbaccu Itescarrh- II.S.A., Inc. (Yinir •ul /'ru/r.c.sur u/Tu(kulcrgv ('ullegr uf Physirians & Surgeons uf Culwnbiu University New York. New York PliTlik K. VO(;1', ('huirnturn I)cparuucnt uf Micnrhiulogy University ul Suulhrrn Calilornia School ul Mcdirinc I.os Angeles, ('alilitrnia Scientilic Staff of'1'he Council SI II?LI)UN C. SOMMI:KS, M.I). Srienli/ir !)in•c(ur JAMtiS F. (;I.IiNN, M.I). A.uut-iu(e Sc•ientiJic• 1)irerlur HAKMUN C. Mc'ALLIS'f1iR, JN., PtLU. Reseurrh /)irer(ur I)ONAI.I) II. FORI), Pu.D. VINC'ENT F. LISAN'I'I, I).M.I). Asxuc•iure Reuurrlt l)irrrlur Assvriutc• Re.eeurrlt /)irerlur OAVII) S'I'ONIi, PU.I). nssur-iute Resrarrh I)irer7ur
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coNTF.NTs Introduction ................................................... S Abstracts ol RMrrts .... : ........ , ....... . . . . . . . . . ........ 7 I. Canrcr-kclut4til Stuclic, ........................... . . . . 7 I I. The Respiratory Syztcun ............... ........ . . , , . 31 ~ III. Neart and ('irrul.niian . . .. ....................... 47 IV. Ncuruphurmarulugy and 111hyr~iolugy ................... 7-1 V. I'h,u-niaculugy. Biochemistry anil Cell liiarlugy ........... 'JI VI. Imtnunulugy and Adaptive Mechunisnts ................ 1-1'- VII. I?Pidemiulugy .......................... . . . . . . . . 159 Act i ve I'ruJrrt. ................................................. I twl ('umpl2tecl Projects . . . . ....... ' ....... ' . . . . . . . . . . . . : . . . .... 177 Index ut I'rinripal Invctitigaturs ..... . ....... . . . . . . . . . . . . . . ........ 1')5 Introduction This annu1tl rcport-insludtis 25(1 abstracts of pubdishcJ se irntilis reports wckncrwl- cclgiug Council su(+port.'I'he nurotxrul'.uch cIM1}rumcnta that have stppearcd'sinre "I'hc C'uuncil began Cunding studies into smokinl; and health in 1954 nnow is at least 3,348; Ihis rcprc!,ctus a substantial contribution to thc scientific literature. Peter K, Vugt, I'h.l)., Chairman ut the I)epurttncnt of Microbiology at the University uf tiuuthern C'ali4irrnia School of McJirinc, l.ox Angelcs,,luutcJ the SCiCn- titic Adviuory Board tu'I'hc Council during the year. James F. Ulrnn, M.I)., formerly President of Mownt Sinai Medical C'rntrr, New York. was uplxointrd Associate Srirntilir I)irtrtur ol 'I'he Council during 1987. I)r. (ilenu also had served as Ihwt uf limury University School of Medicine in Atlanta. The Council has provided more Ih:m $12O,(Xlt),(MN) for research by independent investigators .inrr its establishment 33 years ago. It has supported studics by G?9 ,c•ienti,ts lior I,tM)N original projects in nearly 31N) medical xchiwl., hu.pitals unJ research inaitutiom.. 5
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Abstracts of Reports Nulluwiug mrc :rbstrcrtts, upprovrd by the aut)wr.r, ul' reports on new rcsr.rrrh urhrnowlydgrinY .uplxtrl Irurn '1'hr Council Iha1 h:rvc.arlxarrd in ,ti•iuntilir _juurnalx siure puhlicatiun ut the I'1ti6 kcputl. The nanir ul thr grant recipient i% in italics. The ab%tract% are gruulxd unJer these hraJingti: I. Cancer-keluted Studies, II. '1'he Respiratory Syarni. I11. Ilean and Circulation, IV. Nrunopharmaculogy and I'hysrulugy. V. Ph:umaculugy, 13ionhemi:.lry and Cell l3iulugy, VI. lmntunulugy and Adaptive Mcchaniam, VII. GpiJcnuohogy. 1. Cancer-Related Studies IiIT1:C"fS Oh INJURY ANt) kEl'AIk ON 7111i I'IILMUNARY IiNIM)'1'ItI:LIUM ON Ll1N(i Mli'I'AS"I'ASIS AF'1'F:k RI.LOMYC'IN Acute rutlulhelial injury induced by blicunrycin has been shown to enhance Ihe h>Laliiatiou anJ mrtaxlasis ul'rirculating tumwur cells. In the present study we wished to tlocrminc whether increased nretaslases lu the lung is related to the degree uf enduthrli:d damage a% indicated by morphology and protein leakage tu alveoli and whether the progression tu repair with pulmun:try lihrusix also rflccls metastatic lu- nwur gruwth. C'57hl/h mice were iqjcrlcJ with a single inlravcnuus dose ofblcomycin (120 mY./F,g). A(tcr 5 days. severe cndulhrlial iqjury was Jciuunstrated by morphology and by incrca.cd Icvcls uf prutcin in lung lavage tluiJ. When 1`11-iixhxkoxyuriJinc labeled tiynl;enric lihrouircuma cells were injected inlravcnously ut this time, a N-/iilJ increase in their luraliialiun was Jrlcrtrd 24 h later in hlcumycin-trratcJ lungs cunt- pared with saline controls. By electron microscopy tumuur cells were observed at sitcs irf dcnudrd vascular basement mctubranr. Tltrrc was also a signjlicant increase in Ihc numlxr uF gru.+ nutasta.cs which JcvclujvJ subsequently and in thu Ixrccntagc ul' lung occupied by lunruur in the hleumycin group. Animals examined IIl Jays altur hirnnrycin showed less endolhclial damage and a smaller increase in tuinuur cell kKaliialiun and mrtaaascs. AI 21 Jay., when cnJuthelial xtructure :unJ alveolar rru- trin levels had returned to normal, and at 6 weeks, when Ihcre wax a lixal lihru,is, no increase in tumuur cell hx:aliiatinn or metatitauti was IirunJ. It is concluded that damage tu Ihc pulnrunary cnJulhclium is a key factur in enhancing the trapping uF circulating unuuur cells and increasing nirtastatic lumour growth atier hlcunrycin. Adamsan, l. Y. R., Orr, F. W. and Young, L. Journal ol' Pathology 151):279-?K7, 191i6. 7
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Other >,uplrurt: Naliunal C'ancer Institute uf CanaJu and Medical Research Council of C'anuda. Frum the I)epartment itf I'enhuhigy, University ut' Munitohu, Winnipeg, C'anndu, and I)eparUUent uf Pathulugy anJ Oncology Research (.GrOup, McMaater University MrJr cal C'enter, Hamitlun, Ontario. HUMAN I'LA"I'liLl l'-Ola{IVEf) GROWTH FACTOR AND THIi S/S/PIXiF-2 GIiNI: Significant prugre!,s has been made in the understanding of (he .sis/I'I)GF-2 gene and its transfurming prurluct. 'I'here are rnany imponunt yueslionr yet to he answereJ. For example, dte mechanisms involved in Ihe activation of the c-six gene :md the roles of Iransfurming viruses, other oncogenes, prumoters, and "carcinogens" in these processes rcmain tu be explained. The controlling elements of si.v gene expression are yet to be eluciJ:ued. The mechanisms af actiun of the.sia/I'1)(;F-2 gene product i..till debated. Understanding of these ntechanisntti is necessary in order to develop rational approaches for the control uf the sis gene and its transforming pnxlucts. The fate of the elusive I'UGF-I chain and its whereabouts are still unclear. The pu,sihility exists that other oncogenc(x) code for the homuJimer of the PI)GF-I chain. On the bright side is the f'act that the recent adv:mces in this area have attracteJ new diverse and vigorous talent as indicated by the increasing volume of publications on PIXiF and thc si.s gene. Aruuniu(k% N. N.. Pantacis, I'. and Owen, A. J. In: Guruff, (;. (editor): Oncogenes, Genes, and Growth Factors, John Wiley & Suns, pp. 1-40, 19M7. Other support: National Institutes ut' I lealth and American Cancer Society. Frum the C'enler for Blood Research and Ihpantncnt of Nutrilion, HarvarJ School of Public Health, Boston. SYNTIII',SIS ANl) Sli(.'ftiCl'IUN UF 1'LATELI:T-DI:RIVED GROW'1'U I'AC"1'OR BY I IUMAN 13R1?AS'I' CANCER C'Lil.l. LINES We report that human breast cancer cells secrete a growth factur Ih:rt is biologi- cally and immunologically similar to platelet-derived growth factor (1'I)(lF). Scnun- free medium conditioned by etitrugen-independent MI)A-MB-231 or etitrugen-dcpen- dent M('1'-7 cells contains a mitugcnic or "competence" aclivity that is capable of inducing incorporation uf I'Uithymidine intu yuiexcent Swiss 3T3 cells in the presence of platelet-poor plasma. In uddition, the conJitiut,_d meJium contains an :rclivity that competes with "I labeled I'I)GF for binding tii PD(;F receptors on normal human hhrohlasts. The secretion of I'IX,F-Iike activity by the hornwne-respun.ivc cell line MC'I--7 is stimulated by 17(3-etitradiol. Like authentic PU(iF, the Pl)(iF-like activity produced by breast cancer cells is stable after acid and heat treatment (95"(') and inhibited by reducing agents. The mitogenic activity comigrate% with a material of - lO kl)a on NaDmISO,/pulyacrylamide gels. Innnunoprecipitation with PIx;F antisc- rum of proteins fram metakxilically labeled cell lysates and conditioned medium lirl- luwed by analysis on nunreducing NaUttJ•S(),/Iwlyarrylumidr gels iJs:ntiti,ed proteins of 30 and 3-1 kl)a. Upon reduction, the il)- aruL;-l-kl)a bunJs were canvened to 15. and 16-k1)u h;mJ, sul!gcatin)! Ihat the imnnunoprecipitatul pruteini were nuiJc up u/'twu Jisultidc-linked Ixrlylxptides similar tu PI)(iF. liyhridiiatiisn sluilic+ with cUNA prulx:s fur the A chain of I'I)GF and the B chain af I'Ixl'/S/S idcutilied tran.cript.lor both 1'1)GF chain. in thc M('F-7 and MI)A-M13-211 cells. l'he J:n7l.ulnmarized above provide conclusive evidence tor the aynthexis unrl hurnwnally regulated secretion uf a PIXiF-like mituge'n by breast carcinoma cells. I'nxluctiun uf a PI)GF-Ilke growth Iacnor by breast cancer cell lines may Ix: impnnant in nuJiating paracrine .tiruulatiun of tumur growth. lironicrt, I). A., Pantaiis, N., rlnruniuuh•s, il. N„ Kasid. A., t)aviJtiun, N.. Uick:,on, R. li., and Lippman, M. E. Proceedings of the National Academy of Sciences, USA R4:57Ga-5767, August 19ti7. Other suplxm: Natiunal Institutcs of Health. Fram dre Medicine Branch. National C'ancer In.litute, Bethesda, MI); C'cnter fur Nluusl Research anJ Ihpartment of Medicine. Boston University School uf Medicine, Bu.tun; anil Ikpanmcm of Nutrition, Harvard Sclurol uf Public Hcalth, Boston. ' S'IR(IC"fl IRAI. AND FUNCI'IONAL 11)1?NTIFI('A`I'IUN OF I'LA'1'lil.li'f-!)IiRlVlsl) (71tOW'1'li FACTOR-I.IKE k'RO'1'I:INS 1'ROI)UC'lil) BY MAMMAI.IAN ('IiLI.S 'I'hiti curnnwnicatiun describes pnxcJurex applicable for the identiticatiun and charucterii.ation uf thc structural and f unctiunal propinies uf thc .sm unr gene products derived from SSV-transfircmed cells, or frum human malignant cells uf ms:senchymal origin expressing the c-sis/1'IXiF-2 gene. Recent irdbrmatiun has shown thad several leukemic cell lines also produce 1'I)QF-Iikc proteins. Huwever, these proteins du not appear to Ix- cixleJ by the c-sis/l'DCiF-2 hxus since no sis t»RNA was tlelectahle in tlmsc cells (I II: (rl). MI: ;, K562. IIIiL). This underlines the inywrtance uf tht diverse proreJure, descritx;d here which can yield tiignificant information on structural anJ functional pruperties of the I'1)GF-like proteins produced by certain cells in culture. Since these PI)CiF-Iike products are present only in trace amounts in the cell cultures, extreme care ntu,t be exercised in the design of each study, including all appropriate cuntrul~, in order tu obtain valid and meaningful data. , AiuuniuJrx, /l. N. anil Pantazis, 1'. Methods in Enzynwlogy 147:22-4O, 1987. Other.uppon: National Institutes of Health and American Cancer Society. From'lhe C'enter lior Blood Research, IlarvarJ Medical Schrwl, Buxtun. 8 9
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'I'III: I:1•Fli("I:ti OF NIC(YI'INh, ('O'I1NINlt ANU ANARASINI: uN kA'P AUk1iNAl. Il4i•111'I)kOX1'l.Ay;l{ ANI) 11-IIYI)kUXI'LASIi I:ruh0dnstrloil cancrl ; arr e.wnogcn delkndenl ncuplatian, iti Iht fwurlb mu,t ruutuKun tualil!ia:wiy of wunirn. Women whu xnwkr have only hall Ihc risk u1 developing cnulunu•u ial canrrr when rumparcd to wunten who do nut unoke. The nrrrhani,uw hy which ,nuokille pnrtrcls against (he d¢vcluptucnl ol cndunnctrial ranrrr iti unknown. I hiwrvrr. vunkurg may allrr r,trugen prrxluction, mrlalxrlism cmJ action. Our lahura- tury has uh.urvril th:u nicuUnr, eulininti unJ anaha.inr inhibit estrogen pnnlurtton rn r•irro by iuhtbiting thc c•ytur:hrumc P-45O drl>LnJcnt cnrymr, arumatau. In adnluiun, cuinp+mrnls of cigarrttr ,mukc increase the adrenal prixlurtiun rrt prugracrunr iit rivu I'rugcr,lcnmr is known to protect against the devch>pment uf enJumrtnad cancer. Therefore, rumpomrut, of cigarette xntokc may protect against the clrveluptntinl ul' rudumrtrial ranrcr lxith by decreasing estrogen production and increaxing prugcstcr- ane hrixlucliun: Research is in prugrCsx to examine llte eftccls ul stnukinE un estrogen mctahul i+m artd arl iun. Itrr6ieri, R. L., York, C. M., C'hcrry, M. 1.., :mJ Ryan, K. J. Journal uf Slrruid I3iut:hemi.try 28(1):25-2ti, 1987. From the Lahuratury uf Ilutnan Reproduction and Reproductive Biolugy, IlarvarJ Medical Schuul, liuaun. Illil'A'l'O('AFt('IN(>(~IiN•INUUCED AI:1'IiRA'IIONS IN NUC LI?AR RNA ('( )M I'A ItTM I :N'1'A'I'I ( )N l'rrlatxlcd rat liver nuclei were purilicd, drtcrgrnl-rin.rJ to remove rytuhlasmir RNA cuntanuuanl. autd incubated in two in vitro RNA transport assays. Appropriate a0utpartntcnl:uiun nf nuclear KNA sequences was maintained in an assay cuntaining pulyvinylpyrrulidonc (I'VI'), which prevents nurlear.welling in ayuruus mcdia. l In- drr these rundilion., Iw1y/A)' RNA released in the presence uf PVP (and A7'P) was tiil;nilirantly murr active in directing in vitro prutcin synthesis than Ixdy(A)' RNA released in Ilte absence of rithrr additivr. Further, Ihe excised lilih intrun uf (r,-arid glyruprutcm rcmaincd nuclrus-rc.triclcd, while release uf Iioly(A)' prairssing calah- ulitcti uccunrd in Iltr ah.rnrc uf 1'VI'. 'I'hr apprupriatc contpartrocnl:dion of nuclear sequences was lost IitllowinR Ire;dmrnl of rats with the hcpalurarrinugrn thiuacc- lamidc, parallrling Ilu iit riru xiluatiun. This assay should prove usctitl liOr.uhuyuvul sturlics un nuclruryluplauuir kNA IranslNrrt in vitro, in particular lior invr.tigatiuns rclaling lu the aherrd I(NA Ir:uulxirl a.axiatcd with the initiation uf carcinugrnrsi.. ('luu,sun, (i. A., 13ullun, J. I)., and Wiw, C. II. (':trcmogcncsts H(9):1L 15-1232i, 19R7. Other support: U.S. I'ublir I lcalth Service and Research Career Ihhvrlupmcnt AwarJ. Front thr I)cpartntcnt uf I'athulugy, School of MeJicinc, University of C'alifornia, San Vranci.ro. Itlil'k(1VIklJSI'-.S AS ('Ak('INU(;IiNS ANI) I'ATIIUGIiNS: IiXI'I:CI'A'1'IUNti ANI ) RI?A1.I"I'Y htrtmvirusrx (wialurut Iranxfcurmiu): genes) arc thought ut cause lcukrmias and utirrr ranc•cr. in :minuil.:md hwuan. because they were originally uuL•dcil Irunt Ihu4e Jlsc•ax, :mJ tkcuusc rxlxrimcnlal infcrtiunx uf nrwtaorn, ntay induce Icukcmiati with prohahilitir, uf t) to'NV4, AcrurJinE to this hypothesis viral canrcrr .huuld hr runla- gious, lxdyclunal, and preventable by imnumiialiun. Iluwever, r4truviruu:x arc rather widespread in hcalthy auinnah and huntans where they typically cause latent infcrtinn. anJ anliviral imntunily.'1'hc Icukcmia risk u1'surh infcrtiuns is less than Q.I':4. and thus about as low as that ul' viru.-Ircc controls. Inderd rrtroviruus are nul .uflicicnl lu initicnc Uanaitrnuuiun (a) because ul thr low percentage uf tiymptumatic virus rarrier+ and Ihr complete lack of tranaitrntinl; funrtiun in rin•r,; (G) because of Ihr striking discrepancies Iklwrrn the long latent (xrirxi, uf 0.51u 10 years fur carcinugcncsiti :md Iltr short eclipse of days tu weeks fur virus replication and direct Iralhogcnic and inununugrnir cllcct.; (r) because there is no gcnc with a latc Inm+timning funrtiun, sincr aU guncs arc essential l<rr rrplicatiun; (d) because host gene,, whirlt do nut inhibil viru., inhibil Iunwrigcnesis ur tu I(NNk if inlart and determine the nature otlhc luntur il dcfrrlive: und above all (e) because ul the nwnmklunal origin ol viral Icukemia., Jctincd by viral inlcgration sites that auc difl'crcnl in carh Iuntur. On these bases Ihc pmhability Ihet a viru.•inlartrrl cell will become IransfurntcJ is estimated to Ix: about IU ".'fhr viruks are alw nut necessary to maintain IranaCorntatiun, since many animal ancl :dl Iwvinc and hutn:m tumurx do nut cxprcxs viral antigens or RNA or contain unly inrumplrtr pruviru,cti. 'I'hux as rarrinul;rns retroviruses du nul nrces.arily litl(ill KoK•h', lir.t postulate vrd do not or only very rarely (10 ") fullill thc Ihird.'('hcrctorr it has Inrn prupo.rd that rctrrtviruus tramlirrnt inrl7icicntly by activating latent ccllular unrugrnrs by fur examplc provirus intrgr:uion.'I'his prcJicts dipluiJ Iwuorx with great rhvrrrily, hticau.r intcgraliun sites are dilicreni in each twnur. I lawcvcr, thc unitirnn- ity uf ditirrrnl viral and even nonvir.d tumor+ of Ihc samr lineagr, their rummun susw•rltlihility to Ihe sautr luntur rc.islanrc gcncs, anJ Iranslitnualirrn-slxcilic rhruiuw- surnc ahnuunalitirs shared with non-viral twnurti each argue fur cellular Iranstiirnting grnr.. Iudrril clunal rhruntu.onte ahnormalilies are the unly known translimnatiun- ,I*crilir dclrrminanlti of viral tumarrti. Since lumurs originate with thcsr ahnormalitirs, these or associated rvcnt., ralhcr than preexisting viruses, ntuxt iniliatc Irantifurnta- tiun.'I'hcrcliirr it is proposed that tr:utslurmaliun is a virus-indepcndvnt event and that rlun:d viral tntegr:uiun ,ilr, arc cunuyurncrs ul'rlonnI prulilrratiuu itl Iranstircmcd rrllti.'I'hr role of dte viius in carrinugenc.ia is limitrd Iu the inductiun ul'hylxrplasia whtrh i, nerratiary htn nut srdticirnl lirr rarcinuEcnti.is. Ilylxxrphrsia Jcpends un rhrunir vitcnria or high virus expression which are very rare in anintalx outside Ihe laboratory and have nrvrr Ixxn observed in humnns. Since lalcnt viruscs, which are tyhiral of nrarly all nalural infccliun., arr ncilhrr direct nur iudirrrl rarrinugrns, they ate uu1 targets liOr cancer prrvcntiun. Vuuus arc also not lar)!c1s lur cancrr therapy. since tuntui:s arr nul mainlaincJ and not directly initialcd by viral gencx and occur naurally despitc activc untiviral iiuuumuy. I)ue.%hrr.4, 1'. Il. ('anrer Research -17:1 NH)-I?'_'(1, March I. 19M7. Othrr,up)wrt: National C'unrer Institute and the Fogarty Inicrnational ('rntrr. Frunt thr F)eparlntrnt uf Molecular Biology, llnivrr.ity of C'alilorwa, Berkeley. 10 11
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C'AN('IiR (;I;NIiS: RARIi Rli(Y)M13INANTS INS'1'I:AU OF A("I'IVATIa) (1N('U(il:Nl•;ti (A 1{IiV11:W) 'I'he 20 knitwn n•anslotmimg (woc-) I;encs itd'retruviruscs are ilrainck) by sequences that are trvt.tluccd Iruw cellular gcnex tenued prtwtormcugenes or ccllular unrugenes. • liascd un these sequences, viral unr genes have been postulated lu lx tran.Ju,reJ cellular cancer gene%, and prutu-urtr genrs have been postulated to tk latent cancer grnr. Ilrat cau Ix activated lium within the cell tu cause virus-negalive lunuir.. 'I'hc hypothesis is popular because it prantiu:. direct access tu cellular canccr genes. I low- ever, Ihr cxislence of tat¢nt cancer genes presents a p•rradox, since such gcne. ;uc clearly undesirable. 'I'hr hypothesis predicts (i) that viral one gene% and prutu-ruu• genes are isugenic: (ii) that expression uf pruto-unc genes induces tunrurs; (iii).Ihau activated pruuruiu, genes Iramliirnt diploid cells ulxm lramlcctiun, like viral.aiir grncti: and Giv) that diploid tumors exist. As yet, none of these prcdicliuns is cun- /irmcJ. Instead: (tf Structural cumrarisom between viral unr genes, exsential rc- truvtral genes, anrl prctlu-unr gencs show that all viral one genes are indeed new genes, rather than tranYJuced cellular cancer genes. They are recuntbinants hul together Irunt nvncated viral amtl truncated prodo-unr genes. (ii) Prutu-otr genes are lrcyruenlly expressed in nurmal cells. (iii) •I'u dale, not one activated prulu-wir gene has been isolated that tr;m.lbrnts diploid cells. (iv) Above all, no diploid tumors with activatcd prutu unr gcnes have been firunrl. Moreover, Ihe probability uf slxrntanraux Ir,mstor- mauon in riru is at least 10" timc, luwer than predicted from the mechani.ms thought to activate prutu-u,tc l;encx. 7'herelitre. the hylxuhe,is that prolorurtr genes are latcnt cellular uncugencti aplxars to he an overinterpretatiun uf seyuence homology to struc- tural and luncliunal homology with viral one genes. Here it is prulwscd that only rare truncations and illegitimate rccuntbinatiuns that alter the germ-linc cun/iguratiun uf cellular genes generate viral and possibly cellular cancer genes. The clonal chrumo- wme ahnunttalities Ihat are consistently lound in tutuurcells are micruxupic evidence tur rcarrangemcnt% that may generate cancer genes. The clunality indicates th:u Ihc tunuors arc initiated with, and possibly by. these ahnunualitieti, a., predicted by liuveri in 1914. lhrr.vbrr.q, P. ll. PrerceeJings ol dtc National Academy ul'Sciences, USA It4(M):2117-2124, April 1987. (hhcr support: National ('anccr Institute and National Institutes uf llealth. Prum the Department ul Modecular Biology, Univeristy ul Calilornia, Berkeley. 'lliti'lil'll)IiMIOLOUIC NI?CROI'SY' UNE?(Pl:("1'EU t)li'I'l:("I IUN:ti, I)I:MOGRAI'l1IC SI?LGC"PIONS, ANI) ('I1AN(;IN(; RAl'IiS OFLl)NG CANCER When rising rcdes ul occurrenct are reported fur a particular Jisraw. clinicians oIten c:mnut determine whcthcr the disease has increased in actual occurrence or in dte unrnrvrd detectiun pru% iJrJ by better diagnostic technology untl exparnbeJ access to ntrdtcal carc. The cpidemiolugtc use uf necrupsy data, which might help to answer these questions, has been inhibited by fears of bias in demugraphic anJ clinical ulcc- tuon of paticnts lirc necrup.y. The denwgraphic problem can be managed by suitable adjustment and standardization ol the disease rates found at necropsy, and the clinical I pruhletn can he reduced or avoided by studying the rates with which the disease is liiunrl unexpectedly in nerrupsiea perfirrmeJ frtr uther, unrelated clinical rcuwns. 'fhe,: rc,uhs, obtained in (opulatian gruups "urcenrd" via necrup.y, can!,uggest the magnitude ul' the "undetccted reservuir" that coexists an(1 \u1111ICn1entS the rates t,ll relxortcd uccurrence fur a di,eau . ht a atuJy of necrupsies at Yale-New 1lavcn I lu.pital Irunt 1972 to 191{I, the necropsy Jetectiun ratcs lor lung cancer were slightly higher Grr wutuen than Iior mcn, and were substantially higher ti~r Mrth gcnJct:~ lhan the cu.tumar- ily repurtuJ rates in due gcueral lxtpulatiun. The results suggest that dtc reported rates may cuntinuc to rise in both genders until they become estienttally equal at a xi/c appruximating that of the currently undetected reservuir. 'I'he "epiJemiolugir ne- crupsy" offers a potentially valuable method to help distinguish the true occurrence rates iif Jixease lrunt the changes attributable tu improved diagnostic detection with modern tcchnulugy. McFarlane, M. J., Fainsrein, A. R., Wells, C. K., and Chan, C. K. Journal ul the American Medical Association 251i(3031-338, July 17, 1987. Other support: Andrew W. Mellon Foundation. Frum Ihc 1)epatlment uf MrJicinc and Epidemiology and the Robert Wood Johnson ('linical Scholars 1'rogr.un, Yale University School ul Medicine, New liaven, Cf. INIIIBI•IlON UI-' IIUMAN MAMMARY CAR('INOMA C'ELI- PROLINLiRA•I'ION BY R(ifINpIb)S ANI) INTRAC'GLLULAR cAMI'-ii1.EVA'fING COMPOUNDS Retinoids and cAMP-clevating agents markedly inhibited the proliferation of' human mammary tumnr cells. '1'heir response has been previously correlated with the prc.ence uf cstrugen receptor (IiR) positivity. MDA-M13-231 cells were 6R negative antl insensitive to thc :mtipruli(crative effectx uf rctinuids. However, their growth was markedly inhibited by agents that elevated intracellular cAMP levels, i.r., li-bruntu- cAMP, cholera toxin (l°f), /iorxkulin, and the phu+phrxliesterase inhibitor Rapaverine. The C"f anJ fur.kolin inhibition uf the I:R-Ixtsitive cells (MCF-7) was associated with an elevaliun uf adenylate cyclau activity und intracellular cAMP levels; however, similar clev:dions in inlracellular cAMP levels were nut observed fullowing (I' or t'ur,kulin inhibition of MI)A-M13-2:i1 cells but only lolluwing the addition of the photiphurlicsterax inhibitor 3-isobulyl-t-nuthylxanthinc. l imranu, J. A., Miksis, G., Miranda, I). M., and Durham, J. P. Juurnal of the National C'ancer Institute 78(6):I107-1112, June 1987. Other support: U.S. 1'uhlic Ilealth Service and National Cancer Institute. From the Section uf Ilematology/Uncohogy, Department of Medicine, West Virginia Univer.ity Medical Center, Murgantown. 12 1 13
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INTIikA("1'IUN OF klil'INOII)S ANI)'1'AMOXIFEN ON TIII: INIIIIi1TIUN OI~ HUMAN MAMMARY CARCINOMA C'I?t.l_ L'kOLIhGkA7'ION The growth ttf c7hc:rtiically induced tuumm;try tumors is inhihitWd4by buth hcrrlnttn4 manipulation as well a!, hy rctinuich. Numerous mamnwry carcinnrnaccll Itnc%arr also inhibited by rctinuida. C'u-lrcauncnt of estrogen receptor (lik)-positivc breast cancer cells resulted in an additive cffeet in terms of inhihititrn of cellular proliferation. l'he addition of varying conccntratiuns of rctinoic acid (RA) to varying c•uncentratium ul' tamoxifcn (TMX) resulted in an additive effcct on the inhibition urprulifcralion of Ihc Ek-Ix,.itivc human carcinoma cell lines (MC'N-7). C'u-trcauncnt of M('F-7 cclls uvcr timr with RA and'I'MX resulted in enhanceJ inhihitiun ofgruwth. A similar phcnumc- nun was observed when other synthetic retinuid:+ were combined with TMX. This enhanc•cd inhibition by the combination of rctinoiJs ancl TMX was also observed with uthcr Gk-Iw,itive cell lines ("!.k-75, T47-L)), while no elTecl was noteJ on the ER- negative cell lines (Mt)A-MB-231, Iis578T). Funrunu, J. A. lixperimental Cell Biology 55:136-144, 1987. OIher.uplxtn: National Cancer Institute. Pront the Ikpartntcnt% of Medicine and Biochemistry. West Virginia University, Murganlown. AI)IINUAN'1' SYN'I'IIIiSIS OF FIIN("1'IONAI. IIUMAN T-('I:LL I.I:UKIiMIA VIRl1S'I'YI'li I 1A1Y I'kO'I'IiIN IN IiU('AKYO'I'I(' C'I:LI-S BY USING A l3A('ULOVIkIIS I'.XI'kIiSSION Vli(°1'Ok The hunutn T-crll Icuhcnua virus type I(1 fl'LV-U Ir111• prulcin is a-l0-kilud:dtun pulyhrpliJr encoded in (hr 3'-ICnuinal region nf thr virus. This protein is responsible lur Ih,.itivc lrmtsrriptitrnal rrun.t-artivatiun of promoter clcmcnlv located within the II'17.V-I long Icrurinal reIkat. We introduced the prutcin-cuding region uf II'I1.V-I Ir111• tntu the grnomr ul Ihc haruluviru. Aurugrrrphn ruli(urniru nurlear polyhedrosis vtru.. Altrr inlecliun uf thr imrct Spt,rluprrruJrugipr rdu ( SI Y)) cell lutr, lhis rcrumhi- nanl .tram of haruluvirun produced approximately 2(X) ntg of intac•I (xl(P prutein per 2.5 x I(P crlls. 'I'hr pnncin was biologically active in rruns-activauun of an 1Ill.V-I long Irrnunal rrlxat-luunan (3-ghrbin ronstrurt. 13itn:hcmical analyus of Ithc larutcin, suggest Ihat Ihc It-Itl• pulylxntitlc undrrwcnt lau.ttranslatiunal ntudihruiun in these curaryuur tiFt) rell.. Jcang, K. I' .(;ium• ('.-L., Nrrcnlxrg, M., and Khuury, (i. Journal uf Vitulu);y blLl):7118-71 i, March 1987. Frum tltr Laharatury of Molecular Virology, National Cancer Institutr, lirthc,da, MI). ISC)I.A•1'lill C'ANINIi MASTOCYTOMA C'I:I.I.S: PROPAGATION ANl) C'IIAkAC"1'LikIZA'1'ION OF'I'WO C'lild. LINIiS t^ivc dillcrcnt Jnl; nt,u,tttcytuma tumors were successfully Irauisplantccl atedprup-_ agatcJ in liAl.lilr nuJr mire. Cells front two uf thtisc tumurs were passaged serially through at least fnur generations uf micc without morphological or funrlittnul change. 'I'he average yield I'nmt a 2-cm tumor harvested Irwn a muutic waa 1?!=2.8 x I/l" mast cells with -''MI'! viability. ('rlls ufunc line were larger and more heavily granulated than the uthrr, and contained 1.29 !(1,74 p)g histantinclccll (mcan %Sl)). Calcium ionuphurr A23187 and rtmtlwund 4til!{l1 caused dusr dependent histamine release with no .iguiliranl diflcrrncc in release I'runt generation to generation. The smaller cells runtainrcl (1.(1(i ! U-(F6 pg histamine/ccll. tlistantinc release aftlr culrium itrnuphurc ur cuntpuund 4R/80 was dose dependent and unchanged thruugh,crial passages. Falluw- ing passive ticntiitir:dion.antigrn caused dose-dependent histamine release cunlirming thr presence of IgE receptors un these crll,. In both cell lines histamine release was inhibited by Icrhut:dinc. Jtbulyryl adenosine 3',5'-ryclic: tntmc,pho.phatr, or izubtuyl- mcthylxanthinc. '1'hc,r ntctlntJs provide a nturpht,lugirally and functionally stable Ix,pulaliun of nearly pure caninu ntast rclls for biochemical and physiological studies. l.;u:uus, S. C'., IkkVinncy, R., McCabe, L. J., hinkla:inc:r, W. E., Glius, t). J., and Gulrl, W. M. Antcriatn Journal ul Physiulogy 251 (C'cll Physiolugy 20):C935-C94•i, 1986. Othcr.uppon: National Ilcart, L.ung and Blood Institute, National ln,titut¢% ul'I lrallh, and the Shuwaltcr'I'ru,t. Front the ('arJiuva.rular Research Institute ancl Ihpanntcnts of Mcdicinc and 1'athttl- ugy, University uf C'alilitrnia, San Francisco. M111:1'IPl.li ('/S- ANI) 7'RANS-AC"I'IN(i IiLIsMEN'1'S MI?t)IA'I'li 'I'Iiti TkANS('kll''1'IUNAI. k1iSl'ONSIi'1'O I'I IOkl)Ul. I:S'I'1?kS Protein Kinasc (' is iutpt,rtant in the tranxduction of signals grnrratcd at the plasma ntrtnbranc. •I'hr physiological artiv:uurs of protein kinau C are Jiacylgly- ccrul., anJ tltr tuntuur-prumnting plturhtd cacrx, such a.12-(l Ictradreanoyl-phurhul- 1-I ac'rlulc (•I'I'A), coustitutr annthcr group uf axrilic activators. Many cellular suh- titrates li,r phu.phurylatiun by protein L.ina.r (' havu hccn dr.rrilxd, but proteins that directly control Iranarriptiun in rrspunuu to protein 6ina+c C activation are yet to hr ulcntilird. TI'A uraunrnt Irado ta induction uf various protu-unrugrnrs, growth fartur gcncs, and gcnrs rncuiltttg acrrctrd prutrascs. In ad.lition, TI'A increases the uctivity of viral enhancer clcmcnts. Tu iJentify trrurs-artinl; factors that mediate the tranx•rnp- linual rr%lhmu tu'I'1'A we chutic Ihe sintian virus -ll) /SV4O) cnhanrrr as a nunlcl, because it is known to be composed uf scvcra discrete rir-acting cl¢mtints which atr rrragnv.cd by wultiplr n-uru-arting fartors. We report here that thc SVa(1 rnhanrcr runtainl al least fuurdillcrcnt'fl'A reslHtnsivc clrmcnt. whuac artivity ic drlxntbctd nn rrll-type. The inductiun rcaxrnu is likely tu involve at least two distinct Iwtit-tran.la- tinnal steps which modulate thc artivity uf (hc proteins that rccugniie these rlrmcntx, 14 1 15
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Chiu, R., Imagawa, M., hnhra,, R. J., Bockoven, J. R. and Kurin, M. Nature 3'_9>(tiaH-h51, (k:tuher 15, 1997. National In,titute of linvironmcntal Health Sciences, Environmental Prutection Agency and Ikpartment uf I:nergy. Other tiuplwrt. Frum Ihc Ikpartment uf' Pltamtacology, School of' Medicine, and ('enter tior Molecular Genetics, University uf Califurnia, San I)iegu, l.a Jolla. ANALYSIS OF kAS GLiNL'S AND LINKED VIRAL SEQUENCES IN RA1' HEPATUCAICC'INOGLiNESIS Afier long-term Ieeding ol'a choline-devoid diet to rats, the authurs analyzed raa'', rus", and rus` transcripts and gene structure in livers and liver tumurs. They conlrulled their analysis by studying cell lines derived from chemically induced hepatutnas. Transcripts from all three genes were elevated in all tunxxs, but not in the livers frum which they arose. The transcript elevations may represent an ellect of active cell pruliferaliun in (lie tunrurx. C'lune Hilli-3, derived frum the Kirsten murine sarcuma virus, detected a large number of hybridization hands, most of which were not part of the ras'-p21 gene. Most tumurti had an altered hand at 2.6 kh; some had other altered bands. No alteratiuns were u:en in liver ONA, and nunc uf lhe cell lines showed the 2.6 kh hand. At low stringency, a rus" prulx, which contains a short segment of a similar viral sequence, al.o detectcd altered hand% in tumors amd a single treated liver. These changes in cndugcnuu, viral .Lyuenceti uf the rat genomc appear to he characteristic ul' carcinogencsis by a choline-devoid diet. ('handar, N., I.unihnrdi, 13., Schulz, W., and Lurkrr, J. American Journal uf 1'athulugy 129(2):?32-241, Nuvcnther 1987. Other support: National Cancer Institute and American Cancer Society. From Ihr 1)cpartmcot uf Pathology, University ol' 1'ittsburgh, School of Medicine, Pitt.hurgh, PA. "I'-POS'I'I.ABIiI.ING ANALYSIS OF LIVER I)NA AI)I)L1C1'S IN RATS ('IIRONI('At.LY 17:1) A ('IIUI.INI: 1)IiVOIU Uluf Livci ONA, obtained at variuue time inlcrval% from rat+chrunically Ied a chulinc- devuid diet, was analyied (ur the prc,cncc of aramatic or alkyl adducts by dir `1'- pu.tlahehng assay. Alkyl adducts were nut detected. Aromatic I)NA adduct Irsiuns were revealed, but only at levels (I adduct per 0.5 - 3 x 10" nuclcutides) which are at the limilx uf the extremely high sensitivity uf the method used, levels which rem~tined cuatitatn throughout the period uf feeding, Thus. contamination of the total envi,run- nttint of thc animals with chemical carcinogens does not appear to he respunsihle fur Ihe genesis uf the hcpatocellular carcinomas (hat dkvelup in rats chrunically fed a chulinc-devuid diet. The diet, thercliire, either acts as a complete carcinugen, or prumutes the cvulutinn to cancer of eadugenaus, "spcmtaneously" initiated liver cells. Gupta. R. C., Earlry, K., /on•Arr, J., and Lontbardi, B. C'arcinogencsiti R( I ):187-189, 1987. Othcr,uplxort: National Institutes of Hcalth and American Cancer Society. Frum the 1)kpartment of 1'harmaculogy, Baylor College of Medicine, Houstun, TX, and (kpartnient uf Pathology, School of Medicine, University of Pittsburgh, Pitts- burgh. PA. a-Ft:COPROTtiIN GEN1: METHYLATION ANI) HEPATOCARCINOGENESIS IN RATS FED A CHOLINE-DEVOID DIET We studied the ar-feluprutein (AFP) gene methylation of DNA from livers of rats fcd choline-devoid or control chulinu-supplemented diets, and from hepatucellular carcinumas induced by the choline-devoid diel. Chronic choline deficiency caused a reduction in the level but not the pattern uf inethylation in hepatckytes. The tumors, however, had an altered methylatiun pattern with a marked increase in methylatiun at the 3' end, even those turnun that had low average DNA methylation. At the same tinic, thcre was a decrease in 1)NA mcthylation at the 5' end. The tumor methylatiam pattcrn resembled that of the active AFI' gene of f'etal liver but there was no increase in the steady-state level uf AFP mRNA in the tumurs. The 3' demethylated region is characteristic utthe inactive adult liver AFP gene, but it disappears in the final stages of ncuplastic transformation uf hepalexytes. The methylation changes are not sufficient lu activate the gene. Lurk<-r, J., Ilutt, S., and [A)mbardi, B. Carcinogcncsis 8(2):241-246, 1987. Other support: National Cancer Institute and American Cancer Society. Frum (he 1)epartment of 1'athulogy. University of Pittsburgh, School of Medicine, Pittsburgh, PA. a-L-FL)('OSII)ASIi VARIANT ANI) LIPID-ASSOCIATED SIALIC ACID IN HERI:DI'fARY OVARIAN CANCER In a previous study of a-l: fucosidast (ALF) activities in plasma samples from three uvarian cancer-prone kindreds, we observed a significant inverse correlation of cancer su.ceptibilily :md enzyme actlvlty. Because a low level of plasma ALF:Ictlvlty is an inherited characteristic that has been termed the "fucosidase variant," the above demonstration has provoked great interest in the fucusidase variant relative to the 16 1 17
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grurtic tran*,mis.riun ul' uvuriwt cancer tiu:,ccpaibilily. We have now observed in these tiantc pla.mu x;utlplcti thut tltc cuncentratiuns uf lipid aswn i:ucil tiialic acid (LAS) are al,u invcrscly cuircl:ard with the ALF aclivitira attd. Ihcrcl'urc, directly currrlatcd wbth owun.m cancer tiusccptihtlity. In the fui;usidase variaut indiviiluul, the Al.h is nw.hhcd intraccllularly. but the nuxliltcd enzyme only exhibits decreased activity in the pla.ma. For this rca+un, ALF r.mnut txc involved in tumurigcnc.ix. R;uhcr. the lactur that nuxhlics AI.I:tti probably involvcJ anJ ie thr inhcriicd characlrr.'I'hcrrlorc, we hypothesize that thts ntoxlifier is responsible not only tior the "Iucu+idasr v:uianl" hu1 also fur the clcv:ued cuncentrutiun of LAS and plays a ratc in hcreditary uvarian cancer. Wrll,, L('., l.Ynrli, ll. 7'. and Lynch, J. F. (';inccr Cicnclics and ('ytogcnctics 25:247-251. 197i7. From the 1)cpartntent ut' Bioxhrntiary, Ikparttucnt uf I'rcvcnlivc Mcdicinc/l'ublic I Ucalth, ('rcighlun lJnivcr.ity School uf Medicine, and The Hcrcditary ('ancer In}ti- tule, Ontulta. Nl:. FAMILIAL OVARIAN C'AR('INOMA --C'I.INICAI- Nl1ANC'GS Famili:d ovarian carcinoma ha.lxen recognized with increased freyuency during thc past decade cununrn.uratc with phy.ician attention to l:nnily history. Putative autusoro;d duntinant inheritance uf this heterogeneous problem mandates attentiun to both paternal and ni:ncrnal lineages. A family with probable p:uernal transntis.iun uf bream/uvarian carcinuma is prerented. One family mcmtx:r had finJings cun.onartt with papillary unws ailenncarcinunta uf ovarian origin, which may have arisen di- rcclly trom extru-uvarian pelvic nlCtiUitIlCllunl. IhCSC antl other clinical nuanccs uf t:uuilial ovarian carcinunta are discussed in urJcr to aid physicians iu rmJrr.tcntJing the natural hi+tory, survcillancc, and managcnunt ut' I:wtilial ovarian rarcinunnt. l.rnrh. N. 7'.. 13cwtra. C, and Lynch. J. F. 'Chc American Journal ul' Medicine SI:1071-IU76, 1)ecemtx:r 1986. Frum the Ikpartmrnt ul' Preventive Mcdicinc/Public Health and the Ihpanmcnt ul' Pathulugy, ('reightun University School of Medicine, and The Ilcrcditary C'anccr Institute, Umaha, Nls. GGNIi'rIC' PRlil)I("1'At31LfI'Y ANI) MINIMALCANCI?R CLl11S IN LYNC'tI SYNI)RUMG II Increamng attcntiun has been given tn hereditary nunpolylwsiti culoreclal cancer (HNP('C), a disorder which a:curs liour or live timtis more frcyucntly than its hcredi- I tary counterpart, I'atnilial multiple ;alcnamatnus polyposis culi (17'('). liccam: of the lack uf prcnurnitnry physical signs in I INPC'(', its diagnur•is ntttst cncuntpa..r pertinent family cancer history. 'I'his report dcscrihcx u{,inctrcJ with a tiuhtypc of'tINI'('(', the cancer I':nnily syntlruuiti also rcferre~l to as Lynch syndronte 11. liturhaas has tkcn given to the temporal evolution uf' this disorder and the manner in which ntinmwl clinical-genetic clues nu)!ht Iwm Ix rntpluycJ (ur its diagnosis. L vnrh. ll. 7'. anJ I.ynch. 1. Diseases of the Colon & Rectum .i(l(4):?43-2a6, April 1987. Othcr sultlHUt: Natiunad ('ancer Institutc and'1'hc Hcalth Futures Fuundation, Inc. Frum Ihr 1)cpartntrnt uf Preventive Medicine and Public Ilcaldt, and IicrcJilary Cancer Institute, an41 the HcrcJitary Cancer ('un.ullatian C'cuter, Omaha, NE. FAMILIAI. IiLAI)1)1?R CANCER IN AN UNCOLUGY ('LINI(' We have evaluated t;mtily history and cigarette smokin)g history un 41) conucu- tively ascertained patients with urinary bladder carcinoma and compared them with 95(t cun.ecutivcly asrcrtaincd paticnts with histologically vcrificJ canccr (all zitcs), all ut' whunt were being treated in thc same oncology clinics. 1)ata were cullrcted through interviews, questionnaires, and primary nueJical and pathology doxunocnts. ('anccr risks were comparcJ oil the basis ul"anaumtic sites involved and with cuntulativc risk cstimatcd I'ront d:da in thc Third National Cancer Survcy. A)xrnmtatiun tcxt was also empluycd. Significant heterogeneity uf'risk (z = 4.85, p-1~ 0.02) titr bladilcrcanccr was founil iu relatives ul' prohands with bladder cancer. Significant heterogeneity or increase in risk of bladder cancer was nut observed when familics with lung, other smoking related canccr, und n+mstnul.ing related cancer wCrC;lnaly'LCII. 'I'hOul;h based un a Iimited numhcr uf vcrilieJ bladder cancer patients, we have provided statistical cvidcnce in support of uur hylwlhesis that bladder cancer ntay have a!,Irunger familial etiulugic contlxntcnt than Itcrctulorc rccugnizcJ. l.vnrh. Il. 7'.. Kimtxrling, W. J., Lynch, J. F., and Brennan, K. C anccr Genetics and ('ytugcnctics 27:161-165, 1987. Other support: Health Futures Foundation. Inc. Frum the 1)cparlmcut uf I'rcvcntivc Medicine/Public 1lealth, and Ikpanmcnt of Otu- laryn);ulugy, ('reightun lJniver.ity School ol' Medicine, and Heredity C ancer Institute, Oniaha. NI;. ('IIARA("fl?RI?.A'llON OF A M1C ROTtlllNl.f: ASSI?MB1.Y I'RUMOTING FAC"f()R IN I)IITERENTIA'ftiU NEUROBLASTOMA CEl_I S ('lonal ccll. ( NIR) itf thc mousc ncuatbla,tuma ('-l l(M) can hr induced to undergo nturphulugical diffcrcntiauun characterized by the outgrowth uf very long nnuritc. I8 1 19
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cuntaining many micrutuhules. 'fhi. differentiation event ikres nut require a uta,jur cbutnge in the anununt uf tuhufin or its synthesi,, I luwever, high wlxctl sulx:rnatr+ frum dil'ferentiateJ cell extrsuts can promote the in rirro Ixilymeriicuiun uif puritied brain tuhulin; in contra.t yulx:rnates from undil'ferentiated neuruhla.tuwa cells I:aiIrJ tu promote assenibly. 'I'his neurublaxtoura microtuhule assentbly promoting factur Inh- MAI'F) i, heat and trypsin labile and has a native Mr = I(rl),INM). NbMAPF activity is seen at I hr. after the induction ufJilferentiation and reaches maximal levels by 4-8 hr., while neurite outgrowth is still mininral; long neurites are nut seen until I day ou murc. The appearance of' NhMAPF is insensitive to cycluheximide, suggesting that the activity results frum a Iwsttranslatiunal m4xliticatiun of a pru-factur molecule. tnduc- tiun uf' NbMAI'F requires that the cells be attached to a suitable substratum where ncurite growth can occur. Furtherniure, cyhxhalasin B blocks the induction ul' the factor. Interestingly, the NbMI'AF Jixss not cycle with micrutubules during in vitro as.embly, and additional cycles of assembly require the readdition uf the supernatant fraction frum the previous assembly step, suggesting the NbMAPF inay play a cata- lytic role or be inactivated during the assembly process. A rabbit antilxxly tu native NbMAPF has been prepared Ihat blocks NbMAPF activity in a concentration depen- dent fashion, but Jots not affect mouse MAP-2 or tau promoted micrutubule assembly. These findings suggest that NbMAPF is a unique protein involved in mirrutuhule assembly associated with neurite outgrowth. Seeds, N. W., HinJs, K. R., and Mucriani, R. B. In: Maccioni. R. 13. and Arechaga, J. (eJs.): The ('ytoskelctun in Cell Differentianon and Development. First International Symlosium, Granada, Spain, 1987. pp. lii7-192, IRL Press, 1987. Other suplxon: U.S. Public I leahh Services and Milheint Fuunrlatiun fur Cancer Re- search. From the Dcrartments uf 13iochemislry, Biophysics and Genetics, University of C'ulo- rado Medical School, I)enver. 13UMAN GAS3RIN-RI?LEASING I EP'fIDE GENE IS LC)CATt:D ON CHKUMOSOMt: IR' Gustrin-relca%ing peptide (GRP), a Ixonihestn-hke Ixptide, increases Irlatima lev- els of gastrin, pancreatic IwlYlxptiJe, glucagon, gastric inhibitory Ixptide, and m,u- lin. GRP is produced in large quantities by small-cell lung cancer and acts as a growth factor for these cells. To determine il' chrumusuntal changes in small-cell lung cancer are related to the expression of (iRP, we chruntusomally mapped the gene using human-muuse somatic cell hybrids. Twenty hybrids, characterized for human chro- mosomes, were analyted by Southern filter hybriJizatiun of DNA digested with FcoRl. Human I)NA cut with EcoRl yields a major band of 6.8 kb and a minor hand of 11.3 kh. The 6.8-kb hanJ segregated concordantly with chromosome I}f and the marker peptidase A. The chromosome 3 abnormalities seen in small-cell lung cancer do not correlate with the rhrununrrnutl location ul'(IRI', suggesting that the elevated expre.- Sinn uf Ihis gruc tuay be due tu ntechanisms uthcr than chruntu.unwd rearrangement. Nuvla•, 5, /.., Sakaguchi, A. Y., SpinJcl, li. and Chin, W. W. Somatic Cell and Molecular Genetics 13(1):K7-91, 1987. Other tiuhpurt, National ('nncer Inxtitute. Frum the 1)epartment o1' ('rllular and Structural Biology, Universiry ul"1'exas Health Science ('enter at San Antonio, and 1uslin I)iaf>Lte. Center, LaMmitury of Mulecular Genetics, Urparuntent uf MeJicine, I3righam and Wumcn's Hiispital, 6ostun. QUAN,TII7C'A'I'ION OF MISI'ASTATIC TUMOR GROW'I'II IN 131.EOMY('IN-INJIIRI?D LUNGS 'flnc lung is a camnaun target organ in experimental nuxlels uf tumur mctastatiis in which yuantificatiun usually involves cnunting labeled tumur cells shortly after iqjec- tiun, or enumeratiun uf grussly visible pleural tumurs. In this study, these approaches were used in addition to autoradiographic and nturphometric methods to analyse the cll'cct uf hlcumycm-meJiateJ injury on the develupment, distribution andyuantifira- tiun uf pulnronary metasta,es. One day afier intravenous injectiun uf 2 x 10' librusar- cuma ccll,, thr lungs uf ('57 bl/6 mice, pretreateJ with hleomycin (120 mg/kg i.v., 5 Jay. Ixfure) containeJ aFwut nine times as many 1"ll iixludeoxyuridine-lalxlcd cells as the lung% uf control anintals given saline injections. At this tinu, auluraJiographte counts uf, 1Ithymidine-IahelcJ tunwr cells in lung suxtiuns tihowed a similar incrrase in turnur cell hxaliiation afier bleomycin, with labeled cells distributed equally be- tween parenchymal and pleural areas. However, subsequent tumur growth was Jem- un.trateJ microscopically to be preduminantly in pleural and peribronchial areas, especially a1 sites ul' lung injury induced by bleomycin. Counts uf grossly visible pleural tunwr. I'aileJ to Jemunstratc a dill4rence between bleumycin groups auJ cuntrul. at 7 days whereas counts of mxiules in hmg sections, and yuantilicatiun of lung area uccupieJ by tumnr both showed significantly greater tunwr involvement in hleomycin-trcated animals. As tunwrs became cunfluent, murphumetric meaaure- mcnts ilemunstrated tumor gruwth in the lung more accurately than did nodule counts. We conclude that hlcumycin-induceJ injury greatly enhances met:utadic tumor growth and that murphumeu•ic methods are more sensitive than lung colony counts in their ability to yuantify pulmnnary mrtastascs. Murphometry and auturadiugraphy have also demunstraued that while there is a unif4m distribution ul' arrested twnor cells in the lung initicdly, there is preferential development of inetastatic tunwrs at sites uf pulmunary damage, in particular at the pleura. Orr, F. W., AJamson, I. Y. R. and Young, L. Clinical and lixlurintental Metastasis d(2):1135-116, 1986. Othcr suppcrrt: National Cancer Institute uf CanaJa and the Medical Research Council uf C'anada. Frum the I)epannunt uf Pathology, University of Manitoba, Winnipeg, Canada. 20 1 21
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(;I.Y('OI'kOTI; .INS I)IS'1'1NGU15IIING NUN-SMALL Clil.l. FltOM SMALL ('I:I.I. IIUMAN I.t IN,(; CARCINOMA kI:CU(;NI'LIiI) BY MUNO('LONAI. AN'1'lli( )l)Y -l3-yl: C'rll Ime, dcrivccl Irunt huiuan,yuatncrua lungrarcinuma release latgc utuntcnth td a,ulublr glyruprutein intu the culture mcdia, having very high ntulrrular weight (•? x I(Y) ;md rourin-lil.c• prolxxrlirs. A nxtntclunal antitxxly rallrcl -13-`)Fha, been f,rnrt atrJ thal rcrugnvcti a c:nbahyclratr rpttolxx un thu f.lyrtxuqju f,atc.'fhr ehuul>v is al,ru Ilrcx nt un a divrr,e set ul ,ntallcr glycuprutcins (+ti1, 50,(NN)-?llO,(N)l)) di,lrihulcd Itrintarily on the,url'arr ul Ihr syuantuus lung carcinoma cells. A urnsitivr a„ay using the -1 3-')I'antihcxly in a dot bhN prtx:eJurc has been devised that is able tu detect an: atununt of antt f,cn less th:m that possessed by a single squamous lung carrinuma cell. This a,,:ry, and also conventional immunulluurcucncc and imntunuhisdlxlrocnlical a„ay prucrdures, have been used to screen diffcrent normal cells, nonnal tis,ucx, cancer rcll,, anJ tunuu• htup,y,lxrinlen, for Ihc antigen. In the nurmal lung the-1;-')1' antigcn is litund only on cells of ,otnc uf tho u:romucuus glanJs. In thc nurntai Jigc,tivr ;y,lrtu it i, a,uxiatrd in certain organs only with u IimitcJ population ul'- muru,al rpithclial cells. Other organ,y,tern, lack any reactive rell,. The crll, ul nlu,t hunlan ntm-,mall cell lung carcinomas and their released gIyriKUqjugate, have large anwunt. (if th¢ -l1 -)Frpitunl, while small cell lung carcinomas and thc glyrtx:unju- g:nc, released by uuall cell lung cancer cells lack the cpitulte. The uligo,archaridc rerustniied by the 4i-91::nuilxxly may thcrcfurc prtrvitlr a u,cful marker to di,tinfui,h Ihe clillercnl lung rarrinuma, anJ fur investigating the Jiltcrenl cells uf urigin uf thr,c tuutur,. Prnijulut. 1). E., Str:ntahan, 1'. L., I)uc, C.. Ronne, E., Sorcnscn, 11. R., and Uls,un. L. ('ancrr Research -17(4):1161-11b9, February 15, 1987. Otlrer .uplxut: 1)ani,h Medical Research Council and the Danish Cancer Society. Frum the ('anrer 13iulu fy Lahuratury, Ikparttncnt ul'"1'hurac•ir Surgcry. St:u. lJnivrr- sity Ilu,pital, ('ul>LnhaE¢n, I)cnmau•k, and IkparUnent nl liitx:hrnli,try, Biophysics and Grnctir,, University ul ('oluraJu Ilcallh Sciences Center, Iknver. IINIIS(IAL GLY('OI'ItOl'I:INS 01: IIIIMAN SQUAMOUS ('I:I.I. LUN(; ('Alt('INOMA In our laboratories. mcmullonal antilwdic, (Mahs) have been produced that rrr- ugntic tuut,ual uligu,acrhandc sequences un thr Elycoprutcin, ul cells ul hunsm ,yu;nnuu, cell lung rarcinunta, ( SI.('). One uCthe,e MaM, callyd-(1 -)P, is,lxcilir lior an uligu.arrhariclr that is associated with many Jillcrrnt glyrohrutcin, (at least 50) found on thr,url:uc ul cloned SI.('rcllx. It is also present on nlurin, ,rcrctrd by SI.(' cell line,. 111e uligu,arrharitlc rccugniicd by the 43-')h Mab wa, fuund un 7ll'k uf Ille dillerenl SI_(' l I6utn ul 2'_), 5 out ul• 5 uf (he adentx:arrinumas, and none (0 uut ul Ib) , I ul lhe .ntall-c•cll lung rarrinuuta, that were wrvryecl. This antigrn may thcrrlirrr proviilc a u,ri'uI marlst Itx• di,tinguitihing nun-anall cell I'mm ,mull cell human lung l'anx,'l'M. Frum farrntal Chx1C,, ,ulx•IIInL`, Call be selected that are mctJe up exclusively ul' cells either having in nut having thr 43-9F carlxohydratr (callrJ -ll-t)I' pos and -i3-')h nrg clones, rrax•rluvrly). In nude micc, thc tunwrigcnicity ul'thc,,: variant rluncs ha, hrcn invr,tigatrcl. Thc-I;-')F liwn rlunc, arr,Irungly tumurigcnic, whilr 1hr 4i-91' nrg clones were )tt tuml only weakly tumnrigcnir. 'I'he results suggest that huna:m ,clua- utuus cell lung cancer ccll, have acyuirrtl cell xurlarc glycuprutcina with nxxlilicd uligu,arrharitlr, that ruhanrc thuir nsdignanl putcntial.'I'his cl'Icct utay c>lrur because thr nligu,aa.•hariJc+ ullcr protection against the host ,y,ICm litr rcll-meJiatcJ intmunity- l'rtup,/rn, /). /s.. OI„un, L., Siran:daan, 1'. L., and I'I'cnningrr. O. ('hca VIS:20S-21S, March 1N87. Othur,uplairt: I)ani,h Medical Research C'uunril. Ftunt 1he 1)rpartntrnt af Iliclchrnli,try, 13iuphy,ic, anJ Qcnctic,, University ul ('ulo- radu Ilcalth Sciences ('enter, l)rnvcr, anJ C'ancer Biology Laharatury, State Univrr- ,ity Ilutipital. ('ulx•nhagrn, I)cmnark. IN"I'I:(;FtA"I'ION ANI)'I'ILANS('RII'"I'ION Oh IIUMAN 1'AI'II-t.OMAVIRUS 'I•YI'li 6 l(li('OMI3INAN'I' 1)NA IN MOUSE ('IiLI.S Iluroan papilluncrviru, (III'V) I)NA is fuunJ in nature nw,(ly in an rpixumal It )rnt. Iluwccrr, in prrmanrnt cell line, caabli,hcJ frunl cervical rarrinuntas in which I(PV sequences are nrrsunt, thcy are tt,ually intrgratcd in the host genumc. In ritru ,tudie, ul I II'V have been hanllxrcd because ol• thc dilliculty al ,tahly ntaintaining IIPV scyucnrr, in tran,IcrteJ cclls. We have cluned the entire Iil'V6 genunu: into Ihrrr vectors: pMl?, a drrivativc ul plik 322 lacking thc "Ixli.on,rclurnccx": pId?-6, a pla,mid runtaininN the romplctc bovine papilluntavirus type I(131'V-I) gcnunre which u,ually rrrnain, cxtracllrunw,unwl in tran,IrrlcJ mouse relts, and pll)2-3, a plu,mid cunt;uning thr hactcrial gene cunlcrring resistance to nconrycin. Each ul'thu Ihrer rumtrurt, ha, hcrn Iran,frctrd unlo ('127 nluuu rrll,. In thu ra,r ul thr rrcurtlhi- naut ntulrrulc, rluned into pMl2 and inta p-l2-b, Ihe tran,lcrtiun had been clune lugrthrr with Iltti p.lO?-3 pla,micL (;-IIK rrsi,tatn rulunirs wcrr,clrrtrcl autl au:dyiccl lur thrit I)NA runtrnt. In alI cases (5/5) tltr Iran,lrrtrd molecules were present anJ intcgr:nrcl in thr ccllular grnumc. No lite rpi,umal I)NA was tlrtcrtrcl, even in the cells Iran,lcrtrd with thr 131'V-1 containing plasntiJ. Onc junrtiun with the Ilanl.utg crllular ,ryurnce, is located in the late open reading frames region uf the IIPV(t frouiuc and there is a disruption uf tttc early region, as has been described lilr the III'V18 );rnumti intrgratr~l into thc cervical carcintnna cell line Hrl.a. 'I'ran,rriptinn analysis revealed transcripts of hctcrogcnrous,iccs, spanning the distal early region 11:31:41?5) uf lhc III'V6 gcnumc, a, happens in riru in C'unth•luntutu acmxiuutu, 22 1 23
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al.tharurh in the.re gcnit;tl lesions the III'V6 gettnntc is maintained as ;t nnn-intcgrateJ episuinc. blrrruni•Ru.~rnh<nrm. S. and Kilrun. N. Virus Research K:);5-1.47, 1987. NFrom the I)cpv1mcnt uf Vtrulu~y,'fhc Ilebrcw University-IluJassala Medical Schuul, Jcru.ulcm, Israel. USE UI: I;YMP'II IN C'GI.I, ('UI:I•URI?'1'O MOI)I:L, HURMONAI. ANt) NlJ'1'RI'I'ION,At. CONSTRAINTS ON TUMOR GROWTIi IN VIVO "1•ranaitrnoed BAI.B/3T3 cclls, which prolil'erate without restraint in culture, cunsistcntly pnnluce rapidly growing seurcumas when IO' or nture cell% are inixulatcJ into nude mice hut produce sarcumus, ul wirlcly varying Idtent periods and growth rates, or neHativcs when II)' or Icwrr cell. are inoculated. In un attempt to simulatc the in riru constraints on Iuntur ilevclupntcnt, Ihc.e cells were cultured un plastic surl:accs in coucentratinns ul lymph up to ItN)'h .('ulf Iyutph was less eflcctivc in suppauling multiplicatiun th:tn calf .erunt at all concentrations up to about 50'/r•. "fMte rate ul' ccll multiplicutiun progressively decreased with increasing concentratiuns ol hutlt fluids above 5U'! . Nunethele.s, rapid multiplication could he achieved even in IlN)'/ urunt or lytuph by aupltlctnenting thcm with the high concentratiuns u( nutrients used in Ihe syntltctic mrdium M('UI3 402. Supplrmentatiun oCcy,tine and glutamine was e.scn- tial liar the growth-enhancing el'Iccts ul' the uther nutrients. When the cell, were suspended in agar, lymph was much less cClcctive than serum in pronruttng cxduny lircm;ttiun even wheat Ixath were supplemented with cyslinc and glutantine. or with all (he cunstrlucnt% ol tlte synthetic medium. We conclude that part ul the low cllicicncy ol' twuur pnxluctiun antl reduced growth rate uf the Iransfi,rmed cells in mice resultrd Irunt a cuntbinatiun u( (a) the paucity atl l:ruwth factun in interstitial IluiJ, (b) thc marked reduction in cunccntratiun u(e.sentinl amino acids encountered by the cells in passing Iront culture into mice, and (c) the Gtct thit cell. multiplying in s.c. space du so without benefit ul attachntcnt to a solid substratum. Other factors, such as the growth inhihiting ell'cct, of direct contact with quiescent nluscle and cunneclive tissue cells, remain to he evaluated. I{uhin, ll, and Numura, T. ('ancer Rcscarch 47:492,t-49i1, Septetulxr 15, 19R7. Uthrr support: U.S. Public Uealth Scrvice, National C'ancer Institute anrl Anurican ('anccr Soxiety. I'rutu the I)epartntrnt of Molecular Biology and Virus Laboratory, University ol ('ahlirrniu, tierkeley. f 4 ARE MI°I'()('II()NI)RIAL DNA MU'I'ATI()N!i INVOLVEU IN Tltli ('AR('IN()( H:N.IC' 1'R( )('IiSS? In this review we have pretwntcil evidence liar the involvement ul'nwluliunx in the minx•hundrtal gcrn,mc in tlre carcinogenic process, As slated by Fcinhcrg anal (.'ul'fcy 119tS21, when an area uf, rc.carch is faceJ with npparently paradoxical p)sitions, the cunflict is nftcn resolved by the develuptuent ol unother hypothesis th;u cnruntpa+ses lhe essence ul Ix,lh arpuments. We Ixlieve the evidence fur structural and lunctinn:d JJfcrrncc+ in tumur mifuchundrin is yuitc striking, just as lhc evidence litr mutations in nuclear I!cnc% in tumur cells is striking. ('rrtainly, at present, no convincing J:na have Ixrn rcpurteJ that would deny u role tu ntittxhondria in the carcinogenic process. In reality a cuntbinatiun u( huth rnita>chundrial and nuclear mutations may be critical events. .ShrY, ./. 1i'., anr.l Wcrhin, FI. Mutation Research It{6:139-16(1, 1987. Othrr support: National Cancer In.titute and Anterican Cancer Society. Frunt llte 1)epartment uf ('cll Biology anJ Anatumy, The University uf "1'exas tlealth Science Ccntcr at I)ullas, anJ Program in liiulol;y, The University uf'I'CXJs at I)allus, RidtarJsun. IN•I'tiRLli(JKIN 2 IIIGIi-AFFIN1TY R1iC'IiPTY)R EXPRESSION RIiQUIRL•S TWO I)IS'I'INC"f BINI)IN(; PRO'I'IiINS A cell line established frutn a patient with acutc tymphuhlastic leukemia was li,und tu express IL-2 binding sites with a novel, intermcdiate al'linify compared with the chcuacteristic high-allinity II: 2-rcccptors and luw-affinity IL-2 hituling sites de- sc•rilx•d previou.ly. ('lunes were isolated from this cell line that displayed solely this new 11.-2 binding prot4in, aud were littutd to lx unreactive with unti-7 ac, the mAh that compctes with Il: 2 lur binding. Moreover, these samc cluneJ cells did not express mRNA dctectuhle by hybridization with radiolabeled cDNA encrxling Ihc Tac prutcin. In contrast, the original cell line and sintilar clnncs expressed low levels of Tac mRNA ,md cell surlacc Tac antigen, both ul' which could be augmented by explnure to nterlium conditioned by adult T' Icukentia cell lines. Particularly nutewurthy, induction ul 'I'ac antil:en expression was paralleled hy an increase in the number uf high-affinity II: 2-R rlrtrctable. Since the expression of the Tac antigen protein by itu If ntakcs only li,r luw-affinity II: 2 binding, these data prompted a reevaluation ol' the structural cumpositiun uf high-:affinity 11: 2-R. Analysis of the IL-2-hinding proteins expressed by Icukernic cell lines lat:king high-affiuity receptors revealed only a single prutein, larger than the Tac antigen protein (df, = 75,01X) vs. 55,(1(X)). In contrast, clones induced to express high-aftinity receptors had clearly both uf Ihcse II: 2-hinJing pro- teins. Mnreuver, when II. 2 binding to nurntal T cells was perlormcJ under conditions that 1'avored the prulxartiun of high-affinity receptors occupied, Iwu distinct proteins identical to those already identified on the leukemic cells could he crosslinked cova- lently tu radiulaheleJ It.-2. The interpretations derived from these varied, assemhleJ data, point tu Iwu 11: 2-binJing proteins, both of which are required for high•atiinity II: 2 binding. 24 1 25
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'I'c.higawara, K., Wang, II.-M., Kato, K., and Smith, K. A. Juua•naI of li.xpvrilucnta.l Medicine Ih5:"_'_1-21ii, January ISlR7. Udtcr tiuhpurt: Natiancd ('anccr Institute and lili Lilly ('orlwratiun. 1'rum Ihr I)epartunrnt uf MeJirine, I)artinouth Medical Schcutl, Ilannver, Nil. UIRIiC"t' ( iliNl; TttANSFI:k INTO IIUMAN ('llt;rllRla) (.'IiLI,S FA('II.I'I'A'11iU IiY I.AStiR MI('RUPUNCI'URIi OFTHIi CIa.L MGM13;kANIi '1'he.elertive altes:niim urthe ecllulargenumeby lakr uiicroheam irradiation has been rxten.ively applied in cell biology. We report here the use ot'thc third hariuunic (155 nm) ut an yttriunralwninum garnet laser tu facilitate the direct lranxlcr uf the nru gene into cultured human II'110R0-(if(i cells. The resultant trnnslurmantti were se- Iec1rJ In mcdium containing an aminoglycu.ide antibiulic, G411t. Integration ot lhe nru gene into Individual human chromosomes and expression ul"the gene were Jeniun• slr:ued by Southern blot analyses, micnxell-mediadeJ chromosome transfer, and chru- mu.onu analyses. 'I'hc stability ul' the integrated rter, gene in the tr:m.furmants was shuwn by a comparative growth assay in selective and nonselective media. Tran,lirc- watiun anJ incurporatiun ul the nru gene intu thr host genonre oxcurcJ at a Ireyuenry ut ft x Ill '-.i x I/) '. '1'his mcthoxl appears lu be 1(K)-fuld more el licient than Ihe aanelatd calcium phu,phate-mcJiateJ method ul DNA transfer. I'au. W., Will.insun, J., StunbrUgr•, h.'. J., and lierns, M. W. 1'ronrcdingti uf the National Academy ul' Sciences, USA 1i4:418(t-41R4, June 1987. Uthcr support: National Institutes ul' Ile:dth and Oflire ul Naval Research. Front the liecl.mcw I.aser ht..tiunc :unl Medical ('Iinie, and I)cpartnvcnt of Micrahiol- ugy aud Molecular (icnetic., University ul ('aliturnia, Irvine. INl'ROUll("I'1ON OF IIUMAN ('IIROMUSOMli II VIA MI('RO('IiI.1. "1'I{ANS1=I'.1( C'ON'I'IZUIS'I'l1M(H{IGIiNIC tiXI't2tiSSION C)P tlrt.a ('Ii1.t.S Ituth tumurigenic ,egregant I IrLa x human IihruMasl hyhriJs and tunturigenic I Icl.a (U')ISIA11-2) cells can Ix• converted to a nun-twnurigenic state lidluwin): intru- Juctiun ut a single cupy uf a Iihrohlast t( X:I l) chromosome. 'fhe Iranshx:ated chrumu- ,umc ronuain, -y5'/ ul Ihe I I chromsume and the y26-yter lxmiim ul lhe X chrunw .ume which contains the hylwxanthinc )!uanine phu.phuribosyl Iran.ler:ue (I11'RT) eene. Intruthtctiun ut a human X chruma.ume haa nu eflect un tuuorigenic expres- sion. Supprc+sian ul tumurigenirily is relieved by selecting rrlls which have lu.t the n X;I1) chruntu.umc by t'ruwth io mcdium runlaining b-IhiuEuanine (()-'1'(i). Furthrr, reinaruLIuruan ut Ihe I(X;I I) chruntuwme mIU tunwrigenic (i I'( i" cell, again .uppre„c. tunuorigrnicity. 'I'hus. Ihe introduction ot a.ingle copy ut a human chrunw.unie II is .uttirirnt 14) ronupletely suppress the tumoriEcnir phenotype ut I lel ;i cells and is .uEee,uve of Ihe presence ut lwnor-xuppre,aor gene(s) on this chrumusume. ti,ixun. I'. J., Srivat%an. li. S. and S(unhrirl,tir, E. J. 'I'he IsM13U Journal 5(1 i):,i.161-taGb, 19}ib. Other support; Natiunzil Institutes , uf I Icalth, I- - rum the I)cparUnrnl irf Micruhinlogy and Mnlecular(icnetirs, llniver.ilyul'C'alifiur- nia, Irvine. GI:NI:II(' L(L•GULA7'ION ON't'UMUI(IC7tiNIC t:Xl'RIiSSION IN SUMA'I'IC C'IiLI. IIYIikII)S "I'hr lrhenumenun ul' Iunuur aupprestiiun tncaaaireJ in xetuuutic cell hyhriJ;a huz moved Irom a highly controversial area to a generally ac4eptecl t'act. It would be naive tu expect that the lumurigenic phenotype uf all cancer cells will behave in a recessive nr.inner and be xuppressed by the intnxluctian uf genetic in!lormatiun liunt normal cells, I luwever, this can occur with many cancer cells, including those Ihnt express apparently Juminantly acting oncogenes. Methiids are now available to proxeeJ with Ihe mulecularrharaclerizationul'tunrur-suppressorgenex. Whcnthi,itiaccomplished, it should Icad to a better understanding uf Ihe molecular basis u('cancer and Iws.ihly lead tu lhe identitication ul recestiive oncogenes that may play an intlwrtant role in the neupla.rtic process. SturiLrid,4c'. !i. J. In: Kc•lin, G. (editor): Advances in Viral Onculugy, Raven I'rexs, New York, pp. 113- I/ll, I9tl7. (hher support: Natiunal (':meer Institute and Philip and Clarisse Fay Fund. Fromn Ihe I)erartment ul Microbiology and MulecularGenetics, College uf MeJicine, tJnivrrsity uf C'alitiirnia, Irvine. IMt'LI('A'I'ION O1' ('I IKOMOSOMli I I IN '1'lIl? SlJPP121iSSION OF NliOPLASI'IC EXPRESSION IN I IUMAN C'ta.l. HYBKII)S' ('ytugenetir analysca ul'in(rastxries human llel.a x lihrubla.,t hybrid cell ~)pula- tiunx have pr<wided tentative evidence for the correlation uf luss of a single copy nf chrunwsome. II anJ 14 with reexpression of' lumurigenicity. In this study paired cumhinatium ut nuntumurigenic and tunwri);utic segregant UcLa x lihruhlaxl hybrid cells lroni two indetx nJent futiion events were examineJ lirr the presence or abscnce ul nomnal rhrumusomes II and 14. In human hybrid cell lines the parental origin uf chrumusumes can tx- di.tinguisheJ on the basis uf restrictiun fragnrent Icn~th Ixolymur- phisnts. (irnes for c-Il:rrrrs, insulin, anJ alxdilwprolein A-I on chrunw,ume II and a Iwlynwrphir Iocus AWIUI un chrunrutiutne 14 were used as Sewthcrn hybridiiatiun pnohe%. Analy.is of I)NA front the parriUal libruhl:ut and Ilrl.;i cell line, anJ their nuntunwrigenir and tumurigenir hyhriJs showed the loss uf a lihrubla.t chrumu.ume I I in Iour uf lhc tumurigenic ugrc):ants ancl a I lrl.a chromosome I I in a tilth hybrid cell line. "fhis latter xegre);ant has, interestingly, also lost a copy of chromutiomc 14 of libniblatit urigin.'1'here was nu obvious correlation of h>s.ti af a copy ul normal chronro- umee 1-1 anJ reexpressiun ul tunwrigenirity in any ul'the other hybrid cell Ixorulatiuns. 26 1 27
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Our conclusion frutu these observations is that gene(s) that map to normal chrn- muNstntc I I miEht Ix: involved in control of tunwrigcnic expression in these human hybrid cells. Srivutaan, E. S., licncdtct, W. F. and Srunbridge•, E. J. ('ant:cr Research 46:b174-617y, December 19H6. Othcr support: Nuttunal In,titutes of Ilealth. Fruro the l)cpartntrnt of Microbiology and MulecularGenetics, llniversity of C'alifur- nta, Irvine, attd I)ivisiun of Hematolugy-Onculogy, Children's Iluspitad of t-os Angeles. INTItOI)l1("I'ION UF A NORMAL I IUMAN CIIROMOSOMI', II IN"1'O A WILMS' TUMOR C't:Ll. LINE CONTROLS ITS"fl1MO12IGl'sNI(' tiXP12ESSI6N 'I'he rlevelupnunt uf Wilms' tumur, a pediatric nephrublastuma, has been associ- ated with a Jeletiun in Ihe p13 region uf chruuwsume II. The structure anJ limctiun or functions of thi, deleted genetic material are unl.nuwn. "I'hc role uf thiti deletion in the prucexs uf maliknant tran,limuatiun was investigated by intrtalucing a normal human chrunwsunie II into a Wilnts' tunwr ccll Iinc by means of the mtcrucell transfer techniyue. The,c variant cells, derived by micrtKell hybridiration, expressed similar tranaormcd traits in culture as the parental cell line. Furthermore, cxpreaiun of several ptotu uncugenc. by the parental cell% was unaffcctetl by the introduction of tltis chrutmuwtue. Iluwcver, Ihe ability uf the.e cells to lirrnt tunwn in nude micc was completely suppressed. 'I'ransfcr of other chrrnnutiontex, namely X and 13, had no rflcct un the tunrurigenicity of the Wilms' tumur cells. These stuJies provide support litr the exixtrnce of genetic informatiun on chrotnosumc II which can control the mcdtgnatu expression of Wilms' tumur cells. Weiaman, li. I?., Sitxun• 1). J., Pa.yuale, S. R., Jones, (i. R., Gci.cr. A. (l.. and .St.tnllrid,qr• li. J. Science 236:175-IHO, April 10, 1987. Othcr support: National Institutes uf Iicalth and U.S. Public Flcalth Service. I-rum thc I)ivi,inn of I Icmatulugy attJ Onculugy, Children's Ilospital ul' Lun Angclcs, and I)cpartniciu uf Malecular (lenetics and Micrubiulugy, lJniversity uf C:dilirrnia, Irvine. A('1•.LLl11.AK I:NI IANC'I:R OF RETROVIRUS GI'sNl: EXPRESSION IN I:MIIRYONAI. C'Abt('INOMA ('t:LLS Murinc crnbryunal carcinuma ( F:C) cclls are refractory to infectiun by retruvirusec because rctruvrral lungtennincd repeat (I:IR)cnhancers have littlc activity in IiCcclls. A prcviuus report descrilxJ the isolation uf clonal EC cell lines that eKprc» the integrated ncumycin-re.mance gene (neo) linked to the Moluney nturinr Icukcmia virus I:I'It. The cxpreawn of the neo gene was explained by a ci.-acting mechauti.nt. Front une tiuch t:C cell line, we i&ulatcJ the Ilanking cellular sequence 5' ta the proviral gcnatne. ligated it lu various test con+truCts, and transfected it into the parental EC cells. 'I'he cellular scy(ccnce increased expression trf the LTR-linked neo gene signili- catnly, in a manner independent uf its orientation and position. The neu mFtNA was initiated ad the btma litlc promoter ul' the LTR. By deletion analyses, we defined a region of I)NA essential liir the enhancer activity and determined its sequence. This region contains distinctly characterixtic stretches as well as some similarity to various viral anJ cellular cnhancrrs. '1'hus, the I: fR-IinkeJ neo gene is expressed because the pruvirus is integratrd in the vicinity of this enhancer that is active in undifferentiated li(' cells. "I'uRrm, hl. and'1'anaka. M. Prucecding+af thc Nauunal Academy uf Scicnces, USA 84(II):374H-3752, June 1987. Other support: National Institutes of Health. Frum Thc Jackson Labnratory, Bar Harhor, ME. IIIIMAN PROTO-ONCO0iNE C-JUN ENCODES A!)NA BINDING PROTEIN Wfl'lI SI'Itll("I'IIRAL ANI) FUNCI'IONAL PItUPL"RTIF•S OF 'I'ItANSC'RIPTION I:ACI'OR AP-I Nuclcar oncogene products have the potential to induce alteratiuns in gene regula- liun IcaJinti tu the genesis of cancer. The biochemical mechanisms by which nuclear uncuprutrins act rcmain unknown. Recently, an oncogene, v jwr, was founJ tu share humolugy with ttre DNA binding domain of a yeast transcription Gtctur, GCN4. Furthennure, ( 7C'N-I and the phorbol ester-inducible enhancer binding protein. AP-I, recogniic very situdar DNA sequences. The human proto-uncugene c jun has now been isolated, and the deducerl amino acid seyuence, indicates more than Ht) percent identity with v-jun. Expression of cloned r jun in bacteria produced a protein with scyuencc-tipecilic I)NA binJing propenies identical to AP-I. Antibodies raised against two distinct pcplides derived from v jun reacted specifically with human AP-I. In adJdion, partial aminu acid sequence uf purifieJ AI'-1 revealed Iryptic IxptiJes in cununun with the c-jrur protein. The stmctural and functional aimilarities between the rjun prutluct ,mJ the enhanccr binJing protein suggest that AP-1 may be encoded by c- jmt. 'I'ht,e findings demonstrate that the proto-oncogene product of c jun interacts directly with xpecilic target 1)NA tieyuences to regulate gene expression, anJ therefore it may now Ix: possible tu idcntify genes under the control of c-jun that affect cell gruwth :rnd neuplasia. Buhmann, I)., Bus, T. J., AJnwn, A., Ni.himura, T., Vugl, P. K., anJ Tjian, R. Science 23H:13H6-92, 1)ecember 4, 1987, UOther support: National Institutes of I lealth and a Deutsche Furschungsgeancinschaft Felluwship. Frum Ihe lhpanment of 13ituhcmistry. IlowarJ tiughes Medical Institute, University uf ('ahlitrnia, Berkeley, and Ihpannrent of Micrubiology, University of Suuthern California, School ul' Medicine, l.tts Angeles. 2H 1 29
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I'AI'ILLOMAVIRIJS-SI'l:C'II7C INUUC1'IONS OF C'LiI.LLJLAR 1'RO'1'LiINS IN MURINIs ('127 ('liI.l.ti ll,ing tdtru-hil;h-n:wnlutiun giant two-dimensional );elx; we have anwJyied the protein phenotype of ('127 and NIH-3'I'3 cell lines tranaorntcd by intact and sub- gcnumic .egmcnt% ul'huvinc papilluntaviruy type 1(13PV-1). Analysis of Ihe etitah- li.hcd ItPV-I-tranaormcJ C'127 cell line, IDI4, revealed a set uf six prutein.,, that werc either ab.cnt or present at extremely low levels in the parental cell line. One uf the,c proteins is also present in v-rus-transli,rmed C127 cells, but none of (he ulhcrs are Iirund in cells transformed by a variety uf l)NA sequences, including the oncogenes, v- rnus, or v-/ia; these proteins may therelirre be papillumavirus-ulx:ci/ic inductiun,. The genomc uf IiPV-I contains twu separate open reading frames (URPs), E5 and 1:(i, that can act independently to tranali,rm ('127 cells. We examined cell lines transfurmed by tiuhl;enumic clones uf BI'V-I containing ES and/ur E6 in combination with variuus other IIPV-I uyuences. Only cell lines translixnteJ by the E2-1:5 region exhibit the Papillumavirus-specitic induction,.. Levenson. R. M., lirinckmann, U. C;., Androphy, E. J., Schiller, J. T., Turck, L., ('htn, M., 13roker,'I'. R., ('how, L. T., and Young, U. A. Cancer Cells 5/Papillumaviruses 137-144, 1987. Other support: James P. Wilmot Cancer Research Foundation, German Research Society and National Inatitutes uf Health. From the 1)cpartmcnts of Medicine, Biophysics and Biochemistry, University af Rochester School uf MeJicine and L)enli,try, Rochester, NY; Laboratory uf Cellular Oncology, National Cancer Institute, Bethesda, MI); and Lhpartment uf Pathohrgy, Veterans Administration Medical Center anJ University of Iowa College of Medicine, Iowa City. SI'li('I17(' PROTIiIN SECRBTION BY Md)USG CELI-S TRANSNORMlil) BY BOVINE PAI'ILLOMAVIRUS TYPE I AND BY V-hYiS, V-MOS ANl) V-I I A-RA S In many systems, cellular tranzfurmatiun results in the increased secretion uf xnLcitic prerteinti, including transforming growth factors and prateases (e.g-, pla.mina Yen activator and cathepsin. l) and L.); these ntay play a role in tumurigenicity and invasiveness. Using giant two-dimensional gels, we examined the proteins.ccreted by C'127 cells transfurnuJ by the bovine papillonwvirus type-I (LJI'V-I ) virion and various plasmiJs containing viral sequences, and we found that the protein profile of.ecrrtiem depends un the tramfonning 1)NA. Of appruximately 5(M) detectable secreted proteins, about 70 translirrmatiun-resPonsive proteins were found, many ol' which are phus- phorylated. C'uinplex, partially overlapping, secretory phenotypes exist in ('127 cells tramfurnud by a variety of vird agents, including the E2-E5 and E6-li7 open reading frames (()R1=ti) ul 1JI'V-L. These indicate the existence ul' alternative pathway% to tran.formatiun and sugges( avenues for investigative and possibly theralxutic manipu- lation. 13rinckmann, U. G., Levenson, R. M.,13roker, T. R., Chow, L.. T., anJ Young, 1). A. C'ancer Cells 5/Papillumaviruses 151-157, 1987. Other support: J. I'. Wilnurt hirundatiun, (.ieruuan Research Society, United Cancer C'uuncil, and Natiunal In.tituteti of Health. Frum Ihe Ihptrrtmenl, uf liicxhemistry. Medicine and l3iuPhy.icx. University ul' Rochester School uf Mcilicine and Dentistry, Rochester, NY, Il. The Respiratory System lil7'/i("I'ti OI~ ('IS-IIYUROXYI'ROLING ON'I'YPLi II CELL 1)I;VIiLC)PMI:NT lN Fli'fAL RA'f LItN(i "I'he role uf cullagen in pulmonary cpithclial cell division and dil7crentiatiun during 11,119 development was investigated by injectin)g the proline analogue cis-hy- Jnixy-l.-pruline (cis-IIYP) to tinteJ pregnant rats. Lung weight, DNA anil 'H thymi- dine incorlx,ratiun were unchanged when compared to controls; epithelial cell divi- xiun, determined by auturaJiugraphy, was also unchanged. Huwever, air sac development and phospholipid sytuhesis by epithelial cells were retarded in cis-NYP treated animals. '17te percentage uf epithelial cells containing lamellar hrKlies was reduced at days 20-22 of gestatiun and the level oI dlSaluraled phOtil'IhatldylchOltne (I)SI'(') was also significantly lower in treated rats. Asex-relateJ Jifference was not elimincUed by cis-HYI'treatntent, lung maturation was still more rapal in females. Non-Jialyxahlc hydroxyproline levels were decreased, and morphologically, Iess lihrill:rrcullagen was scen at the epithelial-interstitial cell interfuce, though direct cell- cell cuntacts were m,t reduced. 'I'he results indicate that the injection of cis-I IYP diJ not ;dter lung growth, hut caused a reduction in Iibrillarcullagen which was associated with reduced surfaclant synthesis by Type 11 epithclial cells. Kinl!, G. M. and Aclurnsr,n, 1. Y. R. Nxlxrinunlal Lung Research 12:.i47-362, 1987. UOther support: Medical Research Council of CanaJa. Frum thc Ikparunent of I'athology, University uf Manituba, Winnipeg, Canada. TUMOR M1:'1'AS'I'ASIS ANI'1?R UYPGROXIC INJURY ANI) REPAIR pF'I'lll: I'lJ1.MONARY NNIXYI'Hta.IUM Pulnwnary meta.ta.i, is a ciimmun event. The studies described herc werc dunc tu te,t the hyruthe.is that there is a relationship Ix~tween enJrrthelial injury anJ the hkahiatirm and mctatita.iv uf circulating tumor cells. Mice were exposed liir lxeriiKlx uf up to 4 day% tu an atmosphere of 90% oxygen; sonic were allowed to recover in 1100111 air lirr 3 or 14 days. Air-expwseJ animals served as controls. f:'nJuthelial injury, Jenum.trateJ norphuh,gically and quantitated by measuring protein levels in brun- chualveolar lavage Iluid, increased front 2 tu 4 days exposure when injury was maxi- 30 1 31
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mal. MrtaMatic events increased in frequency cancurrcndly. Wlw:nradiulate4cd synYe- nctc hhrusarcnma cells were tnjected, a 5•fitld increase in hcalitatiun at 24 hhuura was s,:en in the 3-day exposed group and a 36-fuld increase wa:k found i~n the 4-day );noup. Tumor cell, were fuunrl by electron microscopy at sites uf denuded entluthelial basc-- ment memhranc• often associated with small platclet-tibrin thronthi and sometimes, aftcr-l-day exlwsure, with sequestered neutruphtls. The subsequent develupment u/' ntrtastatic nodules in the lung and the percemage uf lung occupied by tumur were also maximal in the 4-day group. Increased ntetastasis and endothelial injury were also uen in mice returned tu air fiir 3 days, but by 14 days the enduthclium was nurmal and ntetasta,es were at control levels. The results demonstrate a relationship Ixtween the degree of pulmonary enduthelial damage produced by hyperoxia and the extent of' metastatic tunwr growth. Adumswt, l. Y. R.. Young, L. and Orr, F. W. Laboratory Investigation 57(1):71-71, 1987. Other support: National Cancer Institute of Canadu and Medical Research C'uuncil of Canada. From the Department of Patholugy, University of Manitoba. Winnipeg. and I)cpan- ment of' Pathology, Oncology Research Group, McMaster University, Ilantiltun, Ontario. t.-A'L(:TIDINE-2-C'ARBOXYLIC ACII) RETARDS LUNG GROW'I'H ANI) SURFACTANT SYNTIIESIS IN FETAI. RATS Maturation of the pulmonary epithelium during late fetal development is cun-- trolled at least in part by the underlying fibrublasts. To further investigate this cellular interdependence and the role of collagen in type 2 cell differentiation, we studied the effects of inhibiting fibroblast function in vivo by injecting the proline analog t.- azetidine-2-carhoxylic acid (LACA) tu timed pregnant rats, and examining changes in cell pruliferatiun and surfactant synthesis in fetal lungs. LACA (2lXl mg/kg) was injected twice daily for 2 days and rats were killed 2 days later at days 19, 20, 2I, amd 22 of gestation. Fetal lung weight and DNA content were about 50°k, of controls, hydroxy- proline per dry weight was reduced and by electron microscopy, there appeared to be less librillar collagen in the lung. Autoradiography after 1'H1thymidine pulse-labeling showed reduced cell proliferation on days 19 anrl 20 niainly due to lower fibroblast growth with a smaller reduction in epithelial labeling. Lung development in LACA- treated rats was retarded; air sacs were slow to open, epithelial cells retained glycogen longer and fewer cells developed laniellar bodies compared with age-ntalchcd cun- truls. There was a reduction in the incidence of epithelial-interstitial cell contacts at day 20 only. Measurements of disaturated phosphatidylcholine showed a 5tl'%, reduction per dry weight and a lower disaturated ph<isphatidylcholine/lipid ratio after LACA. The results indicate that LACA administration in vrvo slows fibroblast growth and greatly reduces librillarcollagen deposition with an accompanying reduction in puhuu- nary surfactant. This suggests that secreted matrix influences growth and differentia- tion of the alveolar epithelium. Adamson, 1. Y. R. and King, G. M. I.aboratury Investigation 57(d):43N--1d5, 19tt7. Olher tiuplxnl: Medical Research Council uf C;utada, Frum thc Ikpartmcnt uf I'athulug,y, Univcr,ity of Manitoba, Winnipeg, Canada, I'1.A'I'IiLli'I' AC"1'IVA'I'IN(; FACI'OR STIMULATES SI:C'RtiTlON OF MUC'IN BY IiXI'I.AN"I"S OF. ROI)IiN'I' AIRWAYS IN ORGAN CULTURIi I'huclet activating factur (PAF: I-O-alkyl-2-(t-acetyl•sn-glyccrn)-.i-phu.phtKltr)- line) a pulcntial meeliutur uf anaphylaxis, stimulatcs secretiun of mucin by explants r~f trachca fram liiur separate rixlent species (guinea pig, rat, rabbit, ferret) in organ culture. Enhanced secretion is not a result uf cell damage or release uf hislantine by cells within thc explants (r.4.. platelets). 11 is inhibited by equiniolar cunccntratiuns of' thc putcnt PAF-rcccptur antagonist, Ru 19-37Q-F. PAF provokes production of im- numurcactivc peptidyl Icul.otrienes (ir-I:1'(,„ L't I)„ 1:1'G,) within thc explants. The ,tinmlauury cl Icct uf I'AF`un mucin secretion is blocked by eyuinwlar concentrations uf nurdihydruguiarctic acid (NIX;A), a"nuxed" inhibitor uf binh cyclo- and lipimy- genase pathways uf ara.•hidunic acid metalwlism. Lcukutriencs are localized within tracheobrunchial cpiUtefiwn by imntunohistuchemical staining, and physical removal uf epithelium Irom explants inhibits prtiductiun uf leukatrienes in virru under non- stimulated conditions and after exposure to PAF. In addition, the stimulatury eflect of I'AF un mucin secretion is not altered by FPI: 55712, a receptor antagonist of L'I'I),. These results arc consistent with the hypothesis that PAF stimulates secrctiun of mucin by activating biusynthcsis of lipoxygenase products (r.g., peptidyl leukutrienes) within epithclial cells uf the respiratory nwcosa. Adlrr. K. ll., Schwarz, 1. li., Anderson, W, H., and Wclton, A. F. lixlxrimcntal Lung Research 13:25-43, 1987. Other suppurt: National ('ystic Fibrosis Founrlation, National Heart, l.ung and Blood htstilute, and Ilultlnann La-Roche, Inc. Frum thc Ikkpartmcnt of' Pathulugy, College of' Medicine, University of' Vermont, Iturlmgtun, and 1)cpannunt of Pharmacology, IlufTntann La-Roche, Inc., Nutley, NJ. MOIJ.('tILAR BIOLO(;Y ANl) ('ARI)IAC FUNC"1'ION, PAST, I'RI:SI.NT ANI) Fll'I'l1Rt! Suiue uf thc contributions of nwlccular biology to cardiac functiun. wcrc dis- cusscJ. Partirular cmphasis was placcd un thc rrlatiunship ~if ccllular calcium trans- purt tu niyixanhal failurc• un molecular pathways detcrmining cardiac contractility and on ntulccrdar mechanisnu controlling vascular reactivity. 'I'lue relationship of' events ut the ntulccular level to cardiac function in general was stressed. Bing, R. J., Saecd, M., tlartntann, A., Sutsch, (;., Mett, M. Z., and Navas. R. 32 33
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In: I)halla, N. S., Pierce, G. N. and Bcami.h, R. 6. (eds.): Heart Function and Metabolism. l:ighth Annual Meeting of the American Section of Ihe International Society I'ur Heart Researdh, Winnipeg, INBti, pp. 1-21 (Martinus Nijherf/', Boston, 19R7). Other support: '1'hc Margaret W. and Herbert Hrwver, Jr. Foundation. Frorn the IluntinEtim Medical Research In.titutes, 1'asadena, CA. A NEW MIiTHO1) FOR PULMONARY Hl)EMA RESf'sARCH: SCANNING 13LliC"fRUN MICROSCOPY OF FROZEN-HYDRATGD EI)L:MA'1-OL1S LUNG Wc present an integrative method litt studying alveolar fluid accumulation during pulmonary cdcma using scanning electron microscopy (SEM) of frozen-hydrated Iung. We induced cdcma in dogs by fluid over-loading the vascular system while uwnituring vascular arrd airway pressures. Terminally, we freez.e-fixed the lungs in the open thorax while controlling airway pressure. Using a cryosample preparation chain- her attached tu a scanning electron microscope equipped with a low temperature stage, we in ruruu freeze-fractured the lung, gold coated the fractured surface and uvicd secondary electron emission scanning electron microscopy to examine the froicn- hydr;dcd lung with scanning electron micrographs of freeze-dried and airway fixcd, critical puint dried lung. We discuss thc advantages of frozen-hydrated xcannin(g lung prcurvauiun for puhuonary edema reuarch, especially in regard to quantifying alveo- lar fluid distribution by stereo phutugrammetry, and suggest areas uf /'uture research tu improve the mcthrxl. Huuk, G. R., liastacky, J., ('unlraim, R. l.., Staub, N. C., and Hayes, T. L.. Scanning 9( 2):71-79, 1987. Other support: Ll. S. Department of Energy and National Institutes of Health. Frum the Surgical Neurology Branch, National Institutes uf Health, Bethesda, MI); Ihmner I.aMrratory, University of Califirrnia, Berkeley; and Cardiovascular Research Institute, University of California, San Francisco. PI?RMF.AI3ILI'1'Y OF ISOI-ATGD RAT LUNGS PIiRFUSED WI"I't( I'LJRINIi ANI) XANTIIINIi OXIDASIi UNI)ER CONSTANT PIikFUSING PRESSURIi Oxidant-induced Iung injury can result from O, radicals produced by I) environ- mental sources, such as hyperoxia, air Iwllulants, such as ozone and nitrogen oxides, and cigarette smoke; 2) prrxlucts of inflammatory cells and inflammatory reactions; and 3) intracellular metatwlites, especially those generated under conditions uf ische- mia. Acute uxidant lung injury, as with most models of acute lung injury, is manifested primarily by edema, which results from an excess uf (luid filtration over fluid resorp- ttun. We have found that perfusion of isolated rat lungs with purine and xanihine uxida%e under constant pufnwnary artery pressure conditions caused a significant decrease in p+:rfu.ate flow (41%) 45 min aftcr xanthine uxidaw additiun as conrpared 1 , with the base-line flow rate. lifl'ective pulmonary capillary pressure did not change sig- nili~cantly. The reduction in 19ow in the face of unchanging pressures at all level:ti resulted frvrm a(Mi% increase in precapilfary vascular resistance and a 141^k, increase in Ixrstc:yiillary resistance. (n c•ur nr,rsI to u r ' , significant but modest rl'fccts on puhtrunary vascular rcxistanrc, pcrfutiiun with purinc and xanthinc oxidase caused a very large ( 32-fuld ) increase in the capillary liltratinn coefficient (K,) as compared with the basc-line permeability. This change occurred in the absence of any significant mechanical stresses such as increased pressures in either the vascular or airway compartments. 7'huti, thr present studies shown that perl usion of iuilated rat lungs under constant pressure conditions with an enzynwtic generatur of Q. radicals, the purine and xanthine uxid:r.x systeru, causes moderate increases in (xnh pre- and pustcapillary resistances and a morc marked increase iu K,. f'u.t, K. K., Parad, R. B., Demling, R. H., and Merrigan, M. J. In: Physiology of Oxygcn Radicals. American Physiology Society, pp. 163-168, 1986. National I lcart, Lung and Blood Institute. Other support: From the 1)ivisions of Pulmonary and Cell Biology, Children's Hospi- tal; Longwood Area Trauma Center, Brigham and Women's Hospital, and lkpart- ments of Pediatrics and Surgery, Harvard Medical School, Boston. ALVI;OLAR MACRUPHAGI:/PGRIPHtiRAL 13L(?OL) MONOCYTE-DGkIVEU FA("fOkS MOI)ULA'I'G PROLIFIiRATION OF PRIMARY LINI?S OF HUMAN LUNG FIBROBI-ASl'S Pulmonary fibrosis is characterized by an alteration in lung collagen synthesis and deposition, as well as by increased fihroblast proliferation. It is also characterized by an intcrmittcnt influx uf immune and inllammatory cells in the lung. Tu investigate the nalurc of the target cell in this disorder, we established a series of primary lines of' human adult lung librublatits and studied the effu t uf mcdiatun released from acti- vated normal human alveolar macrophagcs (AM) and peripheral blood monrxytes (1'13M) on fhc proliferation ol'buth normal lung fihroblasts and librubla.ts established from lung tissue uf paticnts with active librosis. Ourdata show that nwnc>4yte supema- tanls cuntaining a 15-18 kD monokine frunt either AM or PBM inhibits growth of logarithmic phase prulifcrating lung fibroblasts in a dose-dependent manner. This effect can be entirely abrogated by treating the fibroblasts with indumethacin and is recunatitutrd by adding exogenous PC;B,. A study uf the kinetics of this interaction shows that exposure to monocyte supernatant for l(l min to I hr is sufficient to cause significant inhibition of fibroblast proliferation and that this effect can be halted, but not reveraed, at any stage by incubation with indumethacin. We also show that /ibroblasts derived from patients with pulmonary fibrosis are affected nture quickly by exposure to the mediators, although the final extent of inhibitiun uen at each cuncen- Ir.uiun ul' mediators is similar in normal and "fibrotic" fibrobhrsts. These studies indicate that activated AM or PBM release cytokines (including Il: 1) which inhibit the growth uf proliferning normal and fibrotic fibrublasts through activation of the intrin- sic arrchidunic acid pathway of this cell and also that this effect requires a continuous activation of this pathway to IxL fully expressed. 34 1 35
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Jordana, M., Newhouse, M. "I'. and (;uuldir, J. Juurnzd uf IAttk,oc,yte Liin:hr)!y -11:51-(rU, 1987. (rihrr.uppurt: Metlical Rescarch C'uuncil ul'Cunrtla, OnlarioThnraric Society and Ihe St. Juseph'. I lurpilal I;uundaliun. Frutn the Ikparlinent nf I'athulu(;y, MuMa.ter (Jnivcnity, and th+: I)rlwrtmenl uf Mcdicinc, St. Ju.eph'x I lu.pit:d, Ilatntiltun, Onturiu, Canada. NIiU"I'RUYIiII. INJUkY'fO AN AC'I:IJLULAR AMNION MGM63LtANh. Alteration uf Ihc connective tissue ntalrix nt' the lung occurs in a variety uf puhuunary tli:eases including emrhy.cma, idiopathic pulmonary lihru.i4 (IPF), adult respiratory distress syndrome (ARI)S) and tutn+tr invusiun nf the lung, In lihrulic disorders uf pulmonary itucrstitium, IMdymUrphUnuclear ICukl><yteti (L'MN) pnxhntn- nate tn the alveoli in early and late stages od'the disease. At prescnt, it is diflicult to obtain isulated accllular lung mcmhrane,. Huwever, human amniunic mcntbr:utcs have been u.cJ to study PMN nugraliun.'1'his cummunicution describes Ihe injury ul' a huntan umniun nrenthrane malrix by human peripheral blood PMN. "I'hcu: titudics show that thi% membrane model is uuful for studying PMN injury mcclr.tni,nu. The cclls rrspond appropriately to activating agents and. with PMA, adhere tu and invudr the ntembranr. Comparative stuJics of Ihe cffects uf I'MN .ulxrnat:uua, iutacl adhering PMN and uf various antipruteau:x and untiuxidants can hc rxantincd. We show here that claxtaxc, rather than ctxiJanls, is impon:mt in tiulubi= liimg I;N. We .how that a low molecular weight clastau inhibitor Jintinishes FN xuluhiliiation frum Ihr .mouth ba.rcntcnl ntcmbrane surface but acents ineffcclive om (hc suumal .urfacc. Whether higher mulccular weight inhibitors, such as ulpha-I- prutcau inhibilur, behave .intilurly is currently hcing examined. 'I'hix memhrane .yartu ruay contplrwenl uther approaches which employ either artificial surl:urs, (r.S., librunrclin rualrd plates or lihruncctin cuatetl rlaain) or utih/c cell sccrclyd ntairtcr, (r.,q.. frunt .muulh musclc ccll.). The accllular antniutic mcnthr:,ne is cun- venicut, readily availahtr and easy lo prepare. It ullcra somc advantage in that thr nratrix cumpunrnls exist as an urganicrtl memhrane. Sihillc, Y., I.wchu);a-Mul.a.a, J. S.. I'ulomski, I.., Merrill, W. W.. In);bar, I). II., and (;rr. J. B. !.. In: I'ulntunary lintphyuma and Prulculy.is. Academic 1'res., pp. -125-d3-1, 1986. Ulhrr.uhpurt: ('linical Invrtitigatitm Award frum the National Ilrart, I.ung and IihwJ In,Uttue, Anurtc:m I.ung Association Research Training Fclluwshtp and National In,titulrs of I lealth I'hy.ician Scientist Award. Frunt Ihr I'uumanary Section, Ikpartnunt uf MrJicine, Yale University School uf Medicine, New I laven, CI'. AC'()'I7: lil 17:C"I' UI NI"I'k2U(;I:N I)IUXIDIi L:XI"(riUl~;I? ()N "1'Llli Id)Nt'I'IUNAL A("1'IVI'I'Y Uh AI-.I'IIA-I-IyLLU'I'):ASf: INF111J1'1'OLt IN l)IdONC'l (d)ALVL„OLALt LAVAGE FLUIyI) t)N NOkMAL SUIiJGCTS Nrtrngcn dioxide is uuc liorm uf an uxiJiiing frrc radical that is suflicicntly xtuble to exist in relatively high cancrntr;mmuns in ambient air ajttl cigarette stnole. We ¢t:nuiucd thc cflcct ul' NO, exposure on the funcliunal uclivity again.l pancreatic cl:ulasc uf tdpha-I-Itrutrasc inhihitur (ir,l'I) in hrunchuulveuhu• lavage (L3AL) fluid ul' nun.nuol.in); xuhjects. Ten nonanukers (tnean aFe, 25 ! 2 SF•' yr) were expt.ed to NO. 0 trr4 ppm) for 3 h with intermittcnt exercise. Seven nonsmokers (ntcan age, 24 _c 2 Sli yr) underwent a similar protocol but were exposed to NU; frce air and served as cuntrol subjects. Hrnnchctalveular lavage was IxrlirrnuJ 3.5 tu 4 h after the en+l of exposure. Exposure to NU, caused a 45% decrease in functiunal activity ol' a,Pl in 13AL. Thcrc was no signilicant dillcrence in intntunoreactivc a,Pl txaween the groups whether expressed as nticrugrums per 1(X) tnl ul' rccovcrt:J fluid or per milligram of alhumin. '1'his inactivation uf a,PI was not associated with any neutruphil ntigratiun intu thr air spaces of the lung. The "chtstuulike" activity uf' L3AL using synthetic cla.tinlikc cltrumuphurr substrate succiuyl-trialaninc-nitruaniliJc showed no signili- cant dilTerence between Ihc NU; exrw.etl group (221 * 39 SE ng/dl BAL) and the control group (1'/6 ! 61 SI'. ng/JI IiAL). Assay for human leukix:ytc clastase (NLI:) in concrntratetl BAIL using the synthetic substrate Mclhnxy.uc-Ala,-Pro-Val-aminu- mcthylcuuntarin did nut detect any I ILIi activity in thc 6AL.. 7'hcse results showed that nunanuking ,ubjccls exposed to relatively low concentrations of NO. for a short timc havc a agnilic.mt inactivation of a,Pj in lhe lower respiratory tract fluid compared with nunsnwl.ing control subjects. Muhscnin, V. and Grr, J. K. /.. Amcrican Review uf Respiratory I)iseastu 136:646-650, 19R7. Otlter.uplxort: Natiunal Institutes of )lcallh and I1ulTmann-L.aRuche, Inc. Frunt Ihe Juhn li. Pierce Fuundatiun Lalwralory and Pulntonary Section, Lkpartmcnt uf Medicine, Yale University School uf MeJicine, New Haven, C"I'. IMM(1NU1.O(il(' AND Nt)NIMMUNOLt)(;I(' RESPONSIVENESS IN h'A( 7W GIiU-SI:NSI'I I Vli I)(x;S 'I'hr relationship hctwcen airway reslxmsivenrrs tu inhaled antigen and hi.1a- nunr, Inmtunulugic rrlra,c of' lung histantine, intnutnulugic responsiveness of .l.tn, and spciilic tntnwnugluhulin Ii (Igli) antihaxlies were examined in II inbred allergic dogs inuuuniird with cxtract, uf ragwced antl gra.rx anJ 5 nuninnnuniirrl control dogs hum Ihr.ante colony. Airway rc.pmtiivenrti,,tu antigcn antl hia.miinc was character- i/ed by thc doses that incrcaseJ thr airflow rexi.ta,ncc ol'the total rcxpiratury xyxlrm to twirr thc cuntrul values (I:I),.,). Ilighly .igniticant corrclatiuns were found txtwccn airway responsiveness and cutaneuus rcaxmstvcnc.rs lu antigen :uul other inununu- luEic ch:arartrri,tic. (r.,q.. Igli;mJ hi,laminc released linat lung by inhaled antigen) in all rluEs. In ragwrcJ-,rmitind dogs, there was an inverse correlation Lxtwccn immu- nulugic reslxnn,ivcnes. (rclkcted by the cutaneous response to antigen and hisiwttmc released Irunt Iung by inhaled antigen) and n+roimtnunulugic reslxrn.ivcness ol'airways (histamtne Et),,,: r=t).73, P•:0.05 and r=0.75, i'<0.01, respectively). Antigen 36 i 37
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la),,, was al4u correlated with hixtamine relcasc front lung al'tcr antigen inhalation (r=O.74; P•.1).01). We conclude that airway rcactiuns tu inhaled antigen in allergic dugs are JcwnJent not only on immunologic factors but also on the degree of nnnim- munulugic airway rcslxrn.iveness lo hi.tamine and that these facturs are correlated inverxly. Mapp, C., Ilarliala, J., Frick, O. I-„ ShiclJs, R. L. and Guld. W. M. Journal uf Applied Physiology 61(4):1467-1474, 1986. Other suprurt: National Heart, Lung and Blood Institute, National Institute uf Allergy and Infcctiuus Uiu a.c, and Northern Cali/irrnia Allergy Society. I'rum the Cardiovascular Research Institute and I)epartments ul' MCdicine aud I'cdiat- rics, Univcrsity of Calilirrnia, San Franciscu, and Animal Resources Services Facility, School uf Vcterinary Mcdicine, University of C'alifurnia, Davis. INIIIRITION OF AN"1'1(;IiN-INDUC'IiI) AIRWAY HYPI:RRGSPONSIVFiNI:SS BY A'l'lIROM13UXAN1: SYNTIiiiTASE INHIBITOR (OKY-046) IN AI.L.I-'RGIC 1)(1GS Tu dcterminc the role of thronnhuxane A, in the airway hypcrresp+m.ivcne,z induced by antigen cltallenge, we studied the effect of a thrumhuxane symhctase inhibitor, OKY-(W6, i.e., sodium (I?)-3-14-(I-imiclazolylmethyl)-phenyll-2-pru- panoate, in 6 ragweed-sensitized dogs. Airway reslxmsiveness was assessed with Juse-re+lxmtie curves uf acetylcholine aerosol versus total pulmonary resistance Ix lirre and 6 and 24 h alicr inhalation with ragweed antigen. This procedure was repeated in each dog Jurin)g intravenous infusion of OKY-046 (1IX) µg/kg/min). OKY-046 did not alter the acute increa.c in total pulmonary resistance altcr antigen. At 6 h, there was a 7-tirlJ increase in airway resMmsiveness, an effect that was prevented by OKY-046 (p- 11.(H)1). At 24 h, I>'f h aftcr OKY-046 was stopped, hylxxrreslxrnsivcncss was still significantly inhibited. OKY-O46 did not alter the influx of neutruphils recovered by brunrhualvcular lavage perliormeJ at 6 h al'tcr antigen challenge. Antigen-inJuceJ airway hylx:rrespumrvcncss in dogs may depend upon the thrunrtwxanc A, generation frrmi tnflanunatory cells (r.,ti., neutrophils). ('hunl;, K. F., Aiiawa, 1I., Becker, A. B., Frick, 0., Gr,(d, W. M., and Nadel,l. A. Amcriran Review of Respiratory 1)iseaus 134:258-261, 19R6. Other suplwrt: National Institutes of Health and the A.G. Spanos Allergy Research Fund. From Ihc ('ardiuvascular Research Institute and fkpartments of McJicine. I'hysiulugy and I'ediatrics, University uf Califirrnia, San Francisco. k MIiC'l1ANISMS U); ANTIGI:N-INI)UCLiD RI:ACI'IUNS IN SKIN AND LUNG Invealigaturs have tried to dcvelur various experimental prcparaticIns suitaMle to study the cflccts uf antigen un skin ancl airway. in urJer to understand the mechanisnrs involved in a•rthma and to develop new methods uf diagncrsis, Irreventiun and trcat- Inent. We have gained considerable experience in studies of specilic anmigens in inbred dogs with incrcascd levrls of serum IgE antibodies to aeroallergcns. By using a new mtthud of sensitization, combining an attenuated live virus with a specific and 1xHCnt anugcn, we have J1a+eloped an experimental preparation that has many I'catures rese. m bling htunan asthma: (I) High scrum levels uf Igl? antilxxlies were specifically directed against ragweed antigen. (2) Skin and airway respornses to antigen were specific and reprialuciblc. (3) Airway reaxmsivencss tuhistamine and methachuline acrusuls were significantly increased in immuniied dogs. (4) Similar to human asthma, ragweed- induced brunclux:unstrictiun in ragweed-sensitized dogs was severe and asssxiated with rrulixtnJ ahnunnalities in cardiopulm+mary function and appeared to be ofgreater magnituJc than usually seen in Auaris-inJuccJ reactions. (5) Ragweed-sensitized dogs had atupic dermatitis, particularly durinlg grass Ixrllen season, confirmed by biopsy and described previously in atopic dogs. (6) Ragweed-sensitized dogs did not have slwntancuus wheezing, but usually had diffuse, severe wheezing during induc- tiom of anc.thesia; cardiac arrhythmias were also common during anesthesia. (7) RagwceJ-scn.itiicd dogs appcared to clil'fcr frum Ascaris-sensitive dogs with respect to skin respomxivene.s. Thcre was a surprisingly narrow range of skin responsiveness tu antigcn, whereas Snapper el al. in 1980 relwrteJ a wide range of skin responsiveness to Ascaris suum antigen in a group uf mongrcl dogs. Skin responsiveness to histaminc was greater in ragweeJ-scnsitizeJ dogs than in nunimnwnizeJ dogs. (8) Similar to human akrpic paticntx, we found frequent late-pha:u reactions to ragweed antigen. As dcscrilxd below, we showed that antigen-induced rttast cell degranulatiun in skin and airways was associated with inflammation which, in turn, was associated with in- cruruJ airway reslxmsivcness to pharmaculugic agents. (9) The generation uf thrunr Iwxane by inflammatory cells induced by antigcn challenge in ragweeJ-uunsitized dogs appears to play a critical role in altering the rezpcnt.w: of hcrih airways and skin to pharmaculogic agonists. (:uhl, W. M. Respiration 50(Suppl. 2):42-56, 1986. Other suppurt: National Ilcarl, Lung and I;hxx) Institute, National Institutes of Heal(h, University uf ('alitirrnia'x Acarlemic Senate C'ommiuec on Research and the Research livaluatiun anJ Allocation ('ummitlec. Frum the Cardiovascular Research Institute and lhpartmcnt of Medicine, University of Califurnia, San Frattciscu. cAMI' IMMt)N(K'YI'(M'IIIiMIS'I'RY 1'ROVIl)ES IiVII)IiNC)? FOR FUNC"1'IONAI. V)I' LtIX'Lil''1'URS IN '1'RAC'HIiA Va.uactivc intcstinal pcptidc (VIP), first isolated from pCrrcinc intestine has been idrntiticJ in pustganglionic aulunumic axons uf many tissues. VII' has lxricnt regula- tory rllects un the function crf various cell tylxs within these tissues, ranging frum relaxation ol srourrth musclc to ion transport. Reccntly, VIP has been implicated in the 39 38 4
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+14CP rc):ulalicrn ul' mucus,ccrction in the respiratory trart, a 1?ruccsti involving release nf macruniulcculcr I+utu exucrinc ccll, uncl tr:uttilwrt uf inn:, and watcr acru,x the airway mucu,a. lhtwcvcr, because airway glands and mucu±;a buth, consist ut mixed cell Itupulatinn,, it was unclear which,)xcific cells cuntuincd VIP receptors anJ cuntnh- utcJ tu V II'-cvokc+J reslwn,c,. We irlcntificJ thc,e,)xcilic cells by using iuunumx•ytu- chcmical teclmtyuc, tu monitor concentration changes in adenosine 3',5'-cyclic munuphu,phutc (CAMI'), thc intracellular compound known to mediate VII' rc- ,pun,c,. Scntu, and nwruus cells ul Icrrct tracheal submucosal g1anJ,;md ciliated and ba, +l cells ul dog tracheal iuucu,a alI incre;.wd cAMP in re,lwnse tu VII' atinudatiun. We conclude that thc,c cell types twsxs+ VIP receptors and thus participate iu VII'- ,unwlatrJ rc+lxmu,. In cuntraxt, Icrru tracheal epithclium and dog cpithrlial goblet ccll„huweJ little nr no reartivity al'ter VII', and thus we believe that these cells lac•k V 11' rcccpturti. laiurus, S. C'.. 13a,haum. C. 13., liarnca, 1'. J. and (iuld, W. M. American Journal ul I'hy,iulogy 251 (('cll Physiology 2():C'II5-C11+1, 1986. Othcr,uptx+rc National Iicart, Lun)g and Blood Institute, Francis S. North, Sr. houn- Jatiun and the Strobel Medical Research Fund ul thc American Lung As,+u:iatiun ul' San Francisco. Frum the ('arJiavaxular Research Institute and Ikrpardncnt, of MrJicine anJ'Anat- unty, University ul (:diturnia, San Francisco. lil•I+("l7i OF f41AS'C C'Iil.l: I)IiRIVIiI) MI:I)IA'f ORS ON 1:I'I'I'IIIiI.IAL ('I•:I.I.S IN ('ANINI: "fkA('IIIiA We cxantine+l the interaction hrtwcrn ma.t cell-derived mcJiatur, and d+r clcrui- cal and iun tran,purl prupcrlir, ul' c:minc trachcal cpilhcliun+. We cun+parcd the el Icct ul rncJi;+tur, released by inimunulugic challenge +d sensitized lung parcnc•hyma with that ul mcJi:nur, released Irunt canine mu,tucyloma cells challenged w•ilh calcium iunupho+c A3.iI>37. Shurl-c+rcuit cunenl (/,) increased by 19.2 +- 3.0 µA/cn+' in response to ntcdiatun rrlc;+.ud Iront stin,itizcd lung f'ragmcnl, challenged with r;+g- wcrJ antigen. 'I'hi, cll¢ct was nut due to hi,taminc. When thc epithclial ti„uc, were p+cnraled with indorurthacin. the,ame mcd+atur xupernatant increased /_ by only i.8 !-1.1 µA/rm'. The mcJiatur, released Crum 10' tna,t+>Lywnta cells challcnl;rJ w+lh c:dc+un+ ionophore incrcax•d /, by 25.1 !. 13.6 µAlc+u'. In the prc,cncc ul indumrlha- cin, tltc /„ incrca4cd by 2.(t U-1 µA/cm'. Mu,t+x ytu+u:rJc+ivrd nrcdi:dur, prcxlucrtl am inrrca.4 in net chloride xxcrcti+m without a,ignificant cffrct un net ,uJiuin ah,urp- uun. l'hi„tudy pruviJr, direct evidence th tt mast cell-derived mtdialnr, can sliuwlate rp+lhrl+al iun tran,lwrt in canine trachra and suggests that the cffecl is indirect anJ dependent un +ntact rychruxytirnau pathways in the tracheal cpithchum. Laiarm, S. C'., Mc('alx:, l.. J., Nadel. J. A.. Gnld, W. M., and Lcik;wf, (;. I). American Journal uf 1'hy,iulugy 251 (C'ell Physiology 20):('387-C'39-1, 1986. 40 ()thcr supp+trf: Nalianrl Ibcurt, L.ung and Blood Inxtitutc, Nali,unal C'ytitir I-ihrusis Puundaliun, Univcr,.iry +rf C'alif+trnia, Acudrmic Senate Cumntntlrc un krsrurch, and Ihr Calilitrnua Research and Medical Education Fund uf Ihc Antcrican Lung Aa,t>4ia- tiun uf C'aldornia. I'rum the ('arJiuva,cular Research Institutu and Ih:µartrocnt u/' Mcdicinc, University oI'('aliJurnia, San fr+nci,cu. SI'li('IALV.I:U T6iti (' AR'1 ICl.li AI)MINIS'TRA'1'ION: NOSE ONLY I:XI'OSUFtI: ANI) IN"IRATkACI11iAL INUCULA'l lON The rc,pir:nury trac•t serves as the twrtal of cn+ry into the body litr u great variety ul ai+h+m+c substances, h,tth gaseous and particulate. 'lloc lung has over 40 dil7crcnt cell tylws, including cell, ufepitheli:d origin, connective lissuc cells, cartilage, bi+xxl vc,scl,, nurxcle, nrrvou, tissue, and gl:mJulnr tissue. Rcspirablc acrosuda, thu,, are in cunl:rcl with this cntirc complex ul cclls anJ tissues and,uch acru,ol, may play a,ig- nificanl role in lnitiating or exacerbating a variety of' pulmonary, cardiovascular, or infccliuu, diseases. 'I'hc tncch:+ni,m by which cnvirontncntal :lgcn+s cxcrt such wide-ranging tuxic+tlugi- cal and/ur carcinogenic cl7cct, is not known. (;iven the complexity uf thc biological rc,pun,c and in order tu,tudy mcchanum,, it is imtx+rtant to lintit the route uf cxlxaurc to only the lung.'I'hi, can I>< accontplished in model anim.d,ystcml by either intrtxluc- ing malcrial +)ircctly intu the lungs or by providing matcrial to hc inhaled only through thc rc.ltirutury tract, avoiding dcrmal or oral exposure. In thi, article, we discuss ancl evaluate Iwu cumplcnerntary tcchniyucs which ach+cvc thctic rnJ, au+l which have been u,cd extensively in our laboratories: nose-only inhalatinn exposure a+xl intratrachcal in+x:ulaliun. Ik•nry, C. J. and Kuuri• K. E. (Mi+•rubiulu,qi+•ul A.ta+N•iur+•s) In: S:+Iru+, 11. (rd.l: hthalatiun Tuxic+tlutiy Research Mclhud,, Applications, ;u+J livalu:+tiun. Marcel I)rkkcr. Inc., New York, PP. 121-134, 1987. Frum Microbiological Associates, Bethesda, MI). I3ltON('I1O('ON:ti'IRI("I'OR ANI) IIYI'(YI'IiNSIVIi f:Nfl?CTS IN RI'sI_A'1'ION 'I'O I'l1ARMA('UKINI:I'IC :ti OI''I'AC'HYKININS IN 7'llli (;tIINIiA-I'I(l - IiVII)I:N('Ii I•Ok IiX"IIZANI:UItONAL (.'UiAVAGG O1: NlitlkO1'1:1'l1Ul'. K TO NI•.llltOKININ A I The hiulugiral cflicts of thc lachykinins substance 1' (SP), ncurukinin A (NIiA) an+l nc+n+tpcptn6c K(NI'K) wcrc,tudic•d in relation to their pharmarukincuc Ix+tlx•n+c, +n the gu+nra-piE in rn•u. 2. NKA and N1'K exerted a considerably larger brunch++cun,tncturrllcct than SI'. Thr cliccl ul NI'K wa„low in umct :u+d had a long dwatinn. 'fhr three l;+chykinin„huwcd similurhylwtrn,ivc cflrct, allhuugh NI'K had a longer +luraliun ul action than SP and NKA. 3. 'I'he Ji,aplx•arance ul' NI'K-likc inrmunurcactivity (-1.1) lrunl pla,ma after i. v. infusiun ol"synthrtic NI'K was biph;rtiic with apparent hall-livcs o10.9 min and 6 min. The plasnra half-lileof NKA-U was less 41
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6P C'hc+l')Itl): 21-?5, January 1987. ()ther support: Nutitxtal I Icu:rl, Lung and Iihxx.l Inwti(utc. Fruro the I)cpartntcnt of Inlcrna Medicine artJ Physiology, lJnivcrsity uf ('incimtali Srh4xrl of Medicinc, ('incinnati, 011. lu„-Pl1ttINO('IiP'IY)I{ RI:UULA'I'ION Oi: SUIthA("1'AN'r Sl:('ktla'1UN 1'kt)M KA`' ISULA'I'Iil) AI.VU:Uf-AR TYI'I's II C'I:LLS IS ASSOCIA77il) Wl'I'll . MO1311-I'LATION UI; INIRACGt.LUI-AR CALCIUM 1.'I'hc cl Iect uf nnethylcne, thin, ttntl imidusutMtituteJ analuguex t)I'atlCnarnine 5'- tnrho,phatc (A'I'1') un surl'actant phu!,hhulipiJ +ecretiun and calcium nrohili.itttittn in rat isolated alvrnlar Tylx: II cells was studied. 2. A'!'P was (he most potent secrctagugue ul' adcninc nuclcutidcx sluJied. "I"he rank order of aguniat (ronency Ibr I'Hj-phosphatiJylchulinc uxrctiun was A'I'P -> adcnusinc 5'-U-0-thiotriphusphale) (yS-ATP) > (3, y-imidu adenosine 5'-triphu.- phate (AMPPNI') •P, y-mcthylene adcnttsinc 5'-triphurphate ((3, y-('11; A'I"P) :-. u, (3-methylene adenusins: 5'-triphoxphate (a, (i-CH; ATP). The respective IiC,,,s were 10 "M, 2 x 10 "M, 2 x IO 'M, 5 x I(1 'M, and > 2.5 x 10 'M. 3. lixugcnuu. A'I'P also induced a rapid mobilizatiun of intracellular calcium nxmilurcd by changcs in Fura 2 Iluurcuencc. The rank order of agonist Ptdcncy litr ralcium nxthilii.atiun was similar to the rank order of' agunist IxNency fur tiurGirtant secretion: A'll' - yS-ATI' > AMPPNP > tr. (3-CH.-ATP. 4. "I'herc was no cffcct uf 1:(; fA on A'fl'-induced calcium muhiliiatiun, cun.i,t- ent with the hypothesis that exugenuus ATP induces release uf calcium from iruracellu- lar stores. 5. -I'heu d:da are cun,iaent with a 1'.,-purintxxptor regulating surlitctant ucrc- tiun Irum i,trlated'I'ylx• II cells via mubihiation ul intraccllular calcium, since: (a) nun- hydruly.cd an:dogues ul A'I'1' arc Iwient secrelagugues, (b) (i, y-('I I.-A'I'I' waa a murc puteul sccrctaguguc Ihan tr, 13-('l I,-A'I'P and (c) the rank orders ofagunist potency lur calctum nwhiliianiun and phusphulipid secretion were the tianu. Kirr. W. R. and Singlelou, F. M. British Journal uf Pharnmaculugy 91:1(33-H3R, 1987. Other,uppurl: ('hildren'.Iluspital Ne.earch FuunJatiun. 1'rom the Univenity ol Cincinnati College of' Medicine, Ihpanutent ol I'edicdric,, ('tncinnati, ( )I I. S(IRFACI'AN"f AStiO('IA"I'Ii1) I'M(Y1'EIN IN1IIt3ITS I'I(()SI'llOl,lI'll) Sh•.('RI:fION I~KuM 1'YI'I:II C'LLl ti Sccrrtiun u( 1'ItIpho.phatidylchnline (I'FlI l'C) I'rotu isolated ra1 pulnwnary lype II epithelial ccll% wa, inhibited by the .urfactant-as.rx:iateJ protein of M, =.15,(M(1O 44 (SAP-35) purilied Irunt canine lung xurfacta.nl. SAI'-z5 inhibited I'I I 11'(-' 4ecreliun in a duu-Jelxtulcnt Inanncrand,ignilicantly inhibited husal, phorhirlrster, P-atlirencrttic, and I'.-puriner(:ic agunisl-intluceJ 1'Ii1PC' secretion. SAP-35 signtficantly inhibited ,ccretiuu Irum I to 3 h aftcr trealmcnt. The IC, lirr inhibition of 1'II1I'(' .ccreliun by c;winc SAP-.1S was I- 5 x 10 ^g/ml and was xiuutar fur inhibitiun of Ix)lh basal aarJ ucrclagugue-stimulaled release. Ileat clcnaturatiun uf SAP-35, addition ul ntonochonal:urlU-SAI'-.15 anlitxxly. rcJuctiun anJ alkylation uf SAP-35, uras.okialiun of SAP-35 with phu.phulipiJ vcxiclcx reversed the inhibitory ellcct on wcrelagugue- induccd ,ecrctiun. hthibitory effectti uf' SAI'-35 were observed 3 h alter cells were washed wilh bul Icr that did not contain SAI'-35. Although SAP-35 enhanceJ rcasux•i- ation uf surlact:un phospholipid with isolated type II cella, its inhihitury elTecl on ucretiqm of 1'Ii11'(' did nul result Irum stintulation uf reuptake ul'secreted 1'll1l'C' by 9ypc II'cclls. 7'hc inhibniun of phospholipid secretion by'SAP-35 was adsu not due to inhibition ut' I'C urdisaturatcd I'C synthesis by SAP-35. SAI'-35, the tnajorphu.pholi- pid-asuocialcd prutcm in pulrtmunarry surfactant, is a Iwlent inhibitor of sur/actanl ucretiun Ironi tylx II cells in rirrrr and iuay play an imlxrrtant role in humeosta.is uf' surlactant in the alveolar space. Kir•e, W. R., Ross, G. F., Singleton, F. M., 1)ingle, S., and Whitutt, J. A. Journal of Applied Physiology 6.1(2):692-698, 19H7. Othcr support: National Institutes af Health, American Lung Association and ('hi)- Jrcn's Iluspi(al Research FounJatiun. Frr>m the Pcrliatrics/Ncunatnlogy 1)ivision, University of Cincinnati College uf Medi- cinc, C'incinnati, 011. NGIfIROPIIII. kl{('Itl/l'I'MI:N'I' ANI) 1)li(;RANIII-ATION 1)IIkIN(; INUII("fION OVIiMI'IIYSIiMA IN TNG RA'I' BY NI7R(x;tiN DIUXIUIi Ratx exposed to 30 pPm NO, continuously lirr 3 to 6 wk developed miW cen- triacinar air-space enlargcmenl and mild interstitial lihrusts. -I'he crnphysematuus changcs did not becuntc niure severe afier 6 wk with continued NO, exposure litr as long as I-I additional weeks. tilaslau inhibitory capacity (IiIC) and the total protein cunccnlranuu increased acutely in lung lavage IluiJ, consistent with the histologic evidence of puhmmary cdcnW, then subsided hut renwineJ above conrrul levels tirrthe enl irc exlw.urc Ix ruxl.'I'he ratiu of 1:1(' Iu total protein remaineJ unchangeJ relative to that in control animals. Neutruphils accumulated in the lung and were recoverable by lavage with the satuc rclativc concentration prufile as observed lirr total protein. 'I'hc avcragc clast:r,e activity per neuu•ophil in cells recovered frum the air space by lung lavage with 6(I1k less than in cells recovered frum peripheral bhxKL'I'his was true Ior control or NO.-exponcd rats. Thus, emphyunratous changes in the NO; exlwscd rat were accumpanicd by a marked neutrophil recruitment concomitant with an increased ncutruphil elastase burden in the lung, which may have directly contributed to lesion devclupment. 45
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70 ~ 0 (ila.guw, J. li., Pictra, G. U., Ahranis, W. R.. Blank, J., Oplxxnhcim, L). M., aud tYwrthuuon, (;. Anieriran Review of Respiratory 171istiase 135(5):I129-II:Z6, May I9M7. Other yurlwrt: National Heart, I.ung, and L31uuJ Institute and National Intititutcs uf Ilcalth. 1•rum'fhr (;raduate Iluspital. 1)epanment uf Medicine, Research I)ivisiun, and thii- vcr,ity ul 1'cnnxylvania School ul' MeJicinc, Ihpartment of Pathology. Philadelphia. IILIMAN ALVIcOI.AR MA(RUPIIAGIiS tiEC'I2NTLi AN 1NH113ITOFZ Oh MI 1'ALLUPR(YI7'.INAS131:LASTASG litasta,r activity directed against lung exiracellular matrix is currently hel'icved to he itulxalant in the pathugene+is ol'pulmonary cntphyxtna. Although human alveolar nwcropha);ex degrade clastin when in direct contact with this substrate in viu-u, studies of free ela,tux uctivitv in meilium conditioned by hunwn alveular macrophages have yielded variable result.. A+ human alveolar ntacruphages secrete the tiasue inhibitor uf mrtalhoprutrinaus (11M1'), an inhibiturof colla(!ena.e and of otlurconnective-tiaue- derived mammalian mctallupndcinases, we speculated that this inhibitor's effects mil;ht extend to nwcrurhage clastase. Using metallupruteinase clastau liuro the tnu- nnr rnacnohhagclikc cell line P3881)I, we observed that human alveolar ntacrophage runJitiuned medium inhibits metalloproteinase clustatie and that this inhibitory activity could he blocked by .Ixcilic antibody tu "fIMP. Alpha: macruglubulin, another pro- icina.e inhibitor secreted by alveolar macruphages, also inhibited nuaalluprutcinau cla.ta:,c, hut its inhibitory capacity was nut blocked by antilxxly tu TIMP. Because detergents are often included in clastatie assays, we cxantined the effcct% uf sodium dudreyl sulfate (SUS). l3ullcrs containing SDS and Sl)S-lrcateJ clastin were IiiunJ Iu cxert diverse eIICCIS on InetallUprUiL'InaSe elatitaSP., TIMI', and alpha: ntacrug luhulin activities, including a marked inhibition of mctalloproteinase elasta.u activity by SI)S- cuntaining huf Icrti. These findings suggest that detection of secreted metalluproteinau eL•ttitase activity by hwnan alveolar nracruphages is complicated by the concomitant release by these cells of inhibitur% of nietalloproteinaua, and that assay conditions can maukrJly tnlluence the results. Albin. R. J., Seniur, R. M., Wel);us, li. C., Connolly, N. L., and ('ampbell, E. J. Amertc:m Review uf Krtipuatury 1)isease 135(6):1281-1285, June 1987. Qther suplxirt: U. S. Public I lealth Service, National Institutes uf Health and American Lung Association of Eastern Missouri. Frum the Respiratory and ('riticul Care and Ihrniatology 1)ivisiuns, 1)epartment of MeJicine, Jewish Hospital at Washington University Medical Center, St. Louis. 46 III. Heart and Circulation VON WII.I.IiIikANI)1'A("I'OR IS PRESI:NT UN lllls SURFACE OF PLA'IYiLlil'S STIMULA'1'EI) IN PLASMA BY ADP vun Wilfchrand factor (vWf) can bind to glycoprutcin ((;P) 11b/Illa ont activated platrlets. '1'he significance nf thiti interaction is unclear, however, because it has not IxrenlxatlbletudetcctvWfhln(hngtU(;PIIb/IllaunplateletsStlmulated /npla.Snla, We have developed an indirect, fluw cyturnetry assay that uses /luoresccin-IaheleJ anti- bodies to detect v W f and fibrinogcn on platelets. Using this assay, we found vWf an the awlace of platelets stimulated in plasma by AI)I'. The number of phatelcts that hounJ vWf increased in proportion to ADP concentration and incubation time. Washed platclc(s in a prutcin-free btu7eractivateJ by I µmul/I-calciunt ionophore A23187 or IQ: µmul /I, ApP alsu hyrund vW f, suggesting that we were detecting surface binding uf (x- )!ranule-iberived vWf. Monoclonal antiMxliex against the vWf binding site un GI'Ih (61)I) and thc vWf and fibrinogen binding sites on UPIIb/Illa (LJPS and LJ-C'PR, respectively) were used to characterice the mechanism uf vWf binrcling to stimulated platelets. Riatucetin-induccd binding uf vWf was inhibited by 6U1, and AI)1'-induccJ binJinE uf fibrinugen was inhibited by U-('P8. None of these antibodies inhibited AI)1'-induccd vWI binding. Aspirin and prustaglandin L, also inhibited AUP-induccd hinrling uf vWf in platelet-rich plaxnta. 1)uring platelet activation in plasma, vWf derive front cx-granules becomes bound au the platelet surface possibly Ix ing iransler- rcJ already associated with a binding.ite. Adrbnwi, lf., C'arlwn, P_, and Piiwerti, P. Blood 7(1(5):1362-130h, November 1987. Other suppcm: Virginia AI'filiate of the American Heart Ass4xiatiun and the Veterans Administration Research Program. Frum Ihc Ihpartmcnts uf Medicine and Laboratory Services and the 1)ivi.ion of Hrmatulugy/Oucolugy, Medical College uf Virginia, and Hunter Holmes McGuire Vetcr:ms AJministratiun lluspital, Richmond. I IIIMAN II,IAC ARTERY IiNDOTHI'sIJAL C'Fil-LS EXPRESS l30'I'H (7ENIS IiNiC'OUIN(; Tllt'i CHAINS OF I'LATfiLFT-UF'ItIVEU(;KOW'rH FAC"fOR (I'IX;F) ANI) SYNTIIIiSf/.G PIX;1 -I.IKIi M1T(X.;GN In huntan umbilical vein and bovine aurtic endothelial cells in culture c-si.r gene expression and secretion uf platclet-derived growth factor (1'UGF) have been previ- ously demonstrated. We now relxin the presence of PI)(;F-I and PIX;I'-21sis mkNA transcripts in primary cultures of human iliac artery endothelial cells (HIA-I:C'). ('un- cnmitantly, these cells synthesiie and secrete PIX;H-Iikc proteins identified by direct nnnumoprccipitatiun with specific PIX;F antiserum. The PIX;F protcins sccreteil by I IIA-IiC have molecular weights of 31 and 35 kd under nonreducing conditions. Ulxm reduction these proteins are converted to the monomeric 15- and 16-kd lirrmti. Condi- tioned iuedia derived from HIA-tsC stimulated the incorporation of '11-thymidine by 47
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iTt Crll" ;InJ rulnplrlr(I wlth'"I-I'U(il: lur i1, hmJinp. to i'I'; rr:ll memhr:lnr rrreIt- lurti. The; hpoluklC UCIIvII) Na, sI:IhlC to hCUtillg nl I(N)"(' I*ur II/ ntitl and sensitive (u I rJuctn); aprnt,, I'rnlprrur, ,InuLa' to Ihit,o of :ruthrntir I'U( il-• I'urdurlum nl I'I )( iF like muuzru by Ihr hunrnn :nlrricd rndulhcliul rrlls nury play :tn inlp+ulant role in Ihr Itara.•rlnr InuduLurun of arlrnal w:dl rr):rucrattun Iitlluwiug vascular iujury. Slturu,, N. M., S,uih;lu• 1{., I'ant;vis, 1'., %xttcr, Ii., and Arunniudrx, ll. N. hwrnul ul ('rlluLlr I'hy,iohogy I 3_':37G-3tc(1, 1987. OIhrr,ultpurt: N:uiomal Instilulr, ul Ilcal(It. Frrnn Ihe I)rlrnvurnl ull Nutriliun, I larvard Srhtwl u( Public Ilralth` L:uhur:dury ul ('Iiniral I'hannarulugy. I):(aa-F:rrlxr ('anecr In,lituac: Ihpartntrut ul Physiology aud Surgrry, ('hiJclrru'. I lu.pit:tl Medical C'cnlrr, I Inrvard Medical Srholll: and'I'hr ('en- trr lirr IiluuJ ke,r:rrrh, Boston. 'I'I I1: IN VI7R[) tiYNTl II:SIS OI., I'i)LYI'Ii1'l'II)IiS FUR'I'Illi 1'I.A'I'lil.El' MIiMIikANE (iI,Y('UI'KU'fI:INS Ilh ANI) Illa 'I'hc pl:llclrt lurmhranr glycuprutrin, IIh ((lpllh) and (ipllla lurm calriurn- dr)xmlcnl hrtcruclinlrr, runl:lininlg binding ,itrs litr lihrinugcn, vun Willrhraud I:Ic- (ur, :md lihrunrrtin. AhhnuLh ( ipI lh and ( iltllla are dislinct pruteins, lwtlt are dclic•icnt in plalrlrt, lrum inJividual, with Ihr rcre„ive disurJcr Glancnuurn's Ihrunrha,thcni:l. 'fu gaiu a hrltcr unrlrr.t:tndinE ul thc genetic ba.is litr Gpllh and (ipllla synthesis, we studied their ,ynth¢,i, by two human Irul.rmia cell lines. I IEI. and K562. I II:L rrlls cuntuinrd cumplrxc, of ( ipllh and UpIIIa, and K562 cells expressed Gpllla, hut noM GpIlb, when,tinurl:nrd with phurhul-l2-myritilatc-13-acctatc (1'MA), RNA I'ront I11:1, cclls Jircrlrtl the in vitru,ynllresi, ul'a I I(I,INNI-M, precursor lar (ipl lb and a')2,ll(MI-M, jw4cur.or fur (ipllla, which indicates that thc synthesis ul'Gpllh and (iplllu by I IIiI. cells is directed by separate mRNA,. In rontra,l, RNA lrunt PMA-stirnulatcd K563 rcll, only dhcctrJ thr ,ymhc.i, uf a r1?,(KN)-M, precursor I'ur Gpllla. The dissociation of ( ipl Ib and ( ipl I la ,yn(hr,i, in K5b? rcll„uggr,ts that Gpllb anJ ( ipllla may be thc products ul,ehar:ar genes. Silver, S. M11., Mcl)unuueh. M. M., Vilairr, (i., and Krnnrtt, J. S. 131oixl b9141:I11.i1-I(Li7, April 1987. (11hcr ,uphia-t: N:uiunal In,titutcs ul' I icalth. I-rulu thr I IrntaaAugy-( )nrolugy Srrtiun and (':ntrrr('cnlcr, llnivcrsity of Nrnn,ylva- nla School of Mudiclnc•. I'luladrlphet. INI111i1'11ON O1• VASUA("I'IVN A(iE.Nl'S BY I'I:RFI.UOR(H'III{MI('Al. IiMUI.SION 1'rrllworuchrntir;d, are widely used in clinical and ex)nrinmrnt:d ,tuJir. a, vol- uluc cxpanJer, with hlgh oxygen carrying capacity. We nutircd that cunlrartiun, ul rabbit aurlir ,trlp,, inclucrd by nurrrincplrrinc, were inhibited in FC'-4 i rlnul,iun as rranlt:lrrd tu Krrh,-Ilul,rlrn solution (KIIt. 'I'u study thi, uthihitlun, a cumparaUivc cv:duatiun wati matlr of Ihr romtrarlilc rr,lwnx, of norcpilttiphrinr, scrtrtunln anJ hi.lununr in F('-4.1 rmuhiom aud KIL 'fhe rllrc•t ul"Ihrk agents wzt, sl):niliruutly dimml,hcJ in F('--{ i rmul,iun: cuncrntratiuu-rr.punsc curve, were ,hilicd lu the right :uul thc tuaxinuun rra>onse was Jiminl,IkcL A search wo, made lilr the indlviJtud run,tuucnl, ul I-('-43 rc,)>,msihlr lirc dmtinuliun uf cunlr;rrliun tit vascular snnKqh nm,clr. Altrr rrntrilugatiun of F('-4 t rmul,iun. Ihc sulx.rnatanl caused a rcductiun ul runtrnctility and reduced I:C',; v:duc, uf nurrpinrphrmc to the s:uuc degree as the lully run.titutcil Cmuhlun. 'I'hi, cxcludrd Ixxrlluurutrihutylamine, the oxygen carrying par- nclr., as Iking responsible for this inhihititm. When the detergent I'luronir F-6K was added Iu KI I in conrrntratium equal to that in FC'-43 ewulsiun, inhibition olcuntrac- liuu urrurrrcl :mJ 1{('.; v:ducs inrrra,cJ. ('untr:rctiun% induced by nurcpincphrint were cyually inhihited by Ihr adcliliun tu KI I ul a volume cxpauJtir, hydtuxyrthylaarrh, in a cunccnlratwn equal tu that in F('-43 rtuulsiun. It is conrluclyd that FC43 rmulsion inhibits va,uaclivr a~rnt, and uurlluatr, its pharnlacuhrgir cllert. un vascular smooth muulc. 'I'hr Irartiuns of hC'--13 rmul,iun responsible for inhibiting Iho ullcrt of vaurarlivr agents ure I'lurunic F-fi8 :InJ hydruxycthyl,larch. Sarrcl, M., I Iartnt:lnn, A. anJ Bitt);, R. J. I.ifc Sciences -III(21)1:IN71-IN79, I')K7. OtJrrr.uppurt: Mar);arct W. and Ilrrl)crt Ilrwvrr, Jr. Foundation. hruru Iltc Iluntingtou Medical Research In,tilutcs, I'u.adena, C'A. AI)VAN-I'AWiS OI~ I'IiRI:LIIUR(M'III:MIC'AL PERFUSION IN'I11E ISUI.A'I'lil) WOI{KINU RAI31317' HI:AR'I' PREPARATION USING "I'-NMK Quantitative 'RNMR and cniynratic analysis uf high-energy phosphates were used to ch:lrtctrriic ;In isolated I>LrfusrJ working rahhit heart prcparatiom. In this mtxlel, Ilte Icll side ul tltc heart wurs c against a physiological altcr-luad• Two pcrlus- :ur+, Krrbti-I Irmrlrlt,alinc anJ thr IxrlluurucarMm cmulsiun FC-43 ((xrlluurutrihu- tylantinr), were evaluated in thrir ability tu maint:tin cardiac limrtiun and high-energy phutiphatc nlctabulitr, ovvr a(xriud ul 2-.1 h. Adrninc nuclruticlrs A"1'P. AI)P, pltus- phuc•rcalinr antl inurganic phu,ph:ur (P.) were measured hy'T-NMR while ntunituring cardiac uutlnd and rurun:uy Iluw. Intraccllular plt was Jrtrrnrincd u,ing Ihe chemical .hiIi ul I'.. At Ihc end of each rxlxrimcnt, hr:rts were frcric clamped and cniym;ui- rally a„xyrrl lor :Idrninr nurleutidrs, phusphtrrrratinc and I'. In every rxlxxrinlenl, hc:u•Is Ixrrluucl wuh FC'--J.i cmultiinn utaintainrd dtc same rate ul cardiac output as hrarh I>Lrlu,cd witll Krrh,-Ilcnxlril ,alinc, hut with hell'thr carunary Iluw rate: I-('- 4{ i, 22 1 2.5 (it = S). Krrh,-Ilruticlrh s:tlinr-12' 2.7 (rr=-6) ml/min. I''- I).(W)I. Ilcarls prrln,ril wllll P('--13 rntnl,itm,huwed highrr IrM„phtFrrcatincl :mJ IATI'I nrca,urcrd hv "I'-NMIl. Fon• Ipho,phucreatincl: F('--{ i 3.2 t 0.7 (rt=51. Kreb,-Ilrn,rlcil saline 1.7 ' U-? Ut - bl µnwl/g wcl wl., I'• (1.01. I'or IA-PPI: FC-43 I.8! (1.7(n -- 5), Krrb,- Ilrn,rlril saline ILV 1 1).2 (rt 6) µnrol/g wrl wl., /'- 0•/13. IhIIU.phoKrr:nincl anrl IA'I'I'I drlcrlnincJ hy `I'-NMK valtlcl were iJentiral withinrxl>Lrinuut:rl errurtu Ihu,r values uhtainrd by cniymatir :m,•dy,is. ('onlparinl; I I' I drtermiucJ by both ntrthonh, 36'/4 ul P in I•('--1; IxrliluJ hcan,, and unly 24% (411 . in Krrh,-I Irnsclrn ,alinr- Ix'rluud hrart, were vi,lblc by NMR, indir:ning that a large proportion uf P is hrntnJ in the Intarl lunctiuning hran. Similar results were obtained lirr IAI)PI. Using thc 421 49
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'm connhtWd tcrhniyucs uf "I'-NMIt and cnxymatic asaay, we have shown in this m+xlcl of the isolated working rabbit heart preparuion, that FC-43 emulsion maintains signiti- cantly better lianctiim and high-energy phosphate levels than Kreh,-Henscleit sahne. Freeman, I)., Mayr, H., SchmiJl, P., Rolxrts, J. D., and Bing, R. J. l3iix•Itimica et I3iuphysica Acta 927:3511-35t3, 1987. Other support: Jame. A. Itoswell Foundation, Henry A. l3raun Foundation antl Max Kade Foundation. From the I)ivi.iun uf ('hcmi,try and ('hemical lingineering, California Institute ol' "I'echnulugy, and I luntingtun Medical Research Institutes, Pasadena, CA. 7'H1: GITG("I' O1' )3-13KOMO-('YCLIC GMP ON RABBIT VASCULAR SM(x)'rH MUSCI.L: The cffect uf a substituted cyclophosphate derivative, 8-bromo-cGMP (l; bromo cyclic guanusine-3',5'-munuphosphute) on rabbit aortic strips with and without endu- Ihelium was investigated. In arterial suips without endotheliwu precontracled with nurepincphrinc (3 x IO " tu 1(1 ' rooll, M-brotnu-cGMI' caused more relaxation than in intact preparations. Increased relaxation also ox cwred with cumulative cuncentratiunx of }t-hrumu-cGMI' (IO " to 10 ' ntnl). The relaxing effecl of 8-bromo-cGMI' was diminished at high cunccntralions of norepinephrine in intact and, to a Iesurextent, in denuded strips. In the absence ul' 8-brumo-cGMP, low concentrations of nore- pinephrine (a X 10 ', 10 ', and 3 x h) ' nwl) signilicantly increased the contraction af the denuded artery. At concentrations of norepinephrine greater Ihan IO " mol, no change in tension between the intact and denuded artery preparations was noted. The response tu cumulative concenlralions of S-brontu-cGMP was not influenced by low (Il) ' ur 3 x 10 ' mul ) concenlratiuns of norepinephrine. C umpurcd with intact ptepara-, liun denuded arteries ilenwnslrated significantly higher degree of rclaxatiun. SaecJ, M., Schmidli, J., Mclz, M. and lting, R. J. Current Theralxutic Research 41(?):199-20A), February 1987. Other suplxm: "1'he Margaret W. and Herbert Hrwver, Jr. Foundation and the Charles A. Lindbergh Fund. From (hr Ituntinl;tun Medical Research Institutes, Huntinl;lun Memorial Iluspital, I'asadena, CA. IiNFL•("I' OFA NI?W 1'HOSI'llOI)IIS'I'IiRAtili'I'YI'li 111 INHII31'I'OR AS A C'ORONAI(Y VASOUILA'I'(llt ANI) POSITIVE INO7'NUI'ICAGI:N'I' Isolated rabbit curunary artcrie., aurtic strips, and a working rabbit heart prcpara- nutt were used to assess llu positive inotropic and vasixlilaktr effet:ls of a newly developed positive mulrupir contlwund, C'1914. The substance had a relaxing el7ccl on histamine precunstricled coronary artcries and aunic strips. At concentratiuns uf 10 ' utul•litrc ' the relaxing effect on the coronary arteries was significant only at a hi.ta- 50 tuinr concentration ol'.3 x 10 ' mul•lilre ' (pcll.(11) whereas at a concentration uf Ill ' mul•lilrc ' thc relaxin)g eflcct was slgnilicant at histamine concentrations uf I'rom 3 x I(1 ' urol•Iitrc ' to 3 X I(1 ' rnul'lilre '. Annic strips were more sensitive to the relaxing cl7ecl uf ('I 914, which was significanl with IO ' mol•litre ' at histamine cunccntrations of 3 x Il) " lu i x I(1 '(p- l/.l)5 tu p<(L0I). In the isolated working heart preparation topically sprayed histamine significantly increased total and large corunary artery resistances (r:0.(N)I and p<.01). This cftcct was,il:niticantly reduced (p•.11.01 and p•.0.O2) when CI 914 I(1 ' nwl'litre ' was present in the Ix:rfusiun IluiJ. These lintlings slwiw that the phusphuJicstcrasc inhibitor CI 914, a positive inutrupic agcnt, induces vasixlilatiun in isolated coronary arteries and aurtir strips uf the rabbit and reduces the constriction uf curunary arteries induced by histamine in the isolated working rabbit heart prcparatiun. Hartmann, A., SaccJ, M., Sutsch, G., and Bing, X, J. C'.uJiovauular Research 2I(8):593-G(M), August 1987. Other support: Margaret W. and Herfxsrt Hoxwer, Jr. Foundation, Charles A. Linrl- Iergh Fund, and Saoo S. Stein and Rose Stein Foundation. From the tluntingtun Medical Research Institutes, Huntington Memorial Hospital, I'asadena, ('A. NtiLIiASE OF I?NI)(YI'HI:LIUM-DGItIVIiU RELAXING FACI'OR FROM h'lt1iSIILY HAl2V1iSTl:1) PORCINE LiNfXyl'IIN.I,IAL-CELL S Vascular relaxalion in rabbit aurtic preparations induced by acetylcholine is enJo- thcliutn-Jclxndcnl.'fhe naturcof thecuJolhchum-derivcJ relaxing factor(FDIZF) has not been ascertained because it is very labile (reprrteJ half-life b-5(1 seconJs). 'I'o obtain a stable source of IiURF, a system was developed in which the relaxing factor was continuously produced by freshly harvested porcine cnJiNhelial cells. lindothclial cells were collected frunm aortas by exposing the enduthelial lining to cullagrnase (1.1'/.. Cells were washed :mJ concentrated by repeated cenlrifugatiun to obtain a high cell count (7.2 x 1(1" cells/mI). t;ndatheliunrdcprived aunic strips frum rabbits were incubated in these cells suspended in tissue culture medium and felal calf sertuu. 'I'he strips were prccuntrac(ed with histamine. Acelylchuline was added to induce I:URF relrau. Si)!nificant relaxation of enJothelium-dcprivcd aurtic strips was ubservcd. Superoxide dismuta.e, am entymr known lu protect IiDRFagainsl inaclivatiun, caused firthcr rclaxation, which was inhibited by the.rdditiun uf hcmoglubin, an agent known lu inhihil the relaxing aclion uf GI)RF. I:ven without the adJition of acetylchuhne, hemoglobin caused contraction of the denuded aortic strips in suspension uf Ixorcinc cnJuthelial cclla. demonstrating slwnlancuus lil)RF release. I lemoghihin haJ no ef- frcl in ccll-frre mcdium. Endothelial ccll-dcpendent relaxation occurred withuut :it- lacluncnl uf, cnd+Mhcli.d cells lu the cnduthelirun-deprivcJ aurtic :.Irips: when thr cell suspension was replaced by ccll-I'rec ntcdium, relaxation did not occur aftcr acctyl- chulinc. Scanning electron microscopy showed no attachment uf enJiuhrlial cells lu the sulxnJulhelial layer. It can he concluded that freshly harvested endothelial cells produce cnduthelium-dcrived relaxing factor with and without stimulation by arcrlylchulinc. Hanmann, A., Saced, M- and Bing, R. J. 51
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('ircul:niun Research 01(4):54K-554, October 1987. OIhct .uppirt:'fhc Margarct W. and Herbert Htxwcr, Jr„ Fuundatitm, and Sam and Rose Stein ('h;umahbc Trusis. From the fluntingtun MLYlical Research Institutes, Huntingdun Memorial Hutipital, Pa,adena, CA. THIi VASUI)II,ATOK lil9Fli("I' OF('OIiONARY VASCULAR ENI)O'I'UIiLIIJM /NS/7'll: ITS INA("I'IVATION BY IIYI)KO(1UINONli Studiics were carried out to demonstrate the presence of cndothcliunt derived rclaxing Iaclur (L1)I(F) in coronary arteries in .vim and the cf(ect u( hyJroyuinonc on this Iacuir. The studies revealed the presence uf I:URF in coronary artcrics of rabbit hean., remaining in sinr. I:I )kF:was inactivatcJ by Icft atrial injection of hydnxluinunr. In situ cuustriction ul curunary arteries by vauiprcaain was markedly increased litlluw- ing exposure to hyJruiluinunc, and the dilatory rcxl>trnu: to acetylchuline waz ahunt. The cxlxrieutcntx confirm the presence uf'the cnJothcfiunt derived relaxing factur in coronary arteries iut siru and its inactivation by hydroquinone. /)ing, K. J., Saecd, M. and liantnann, A. The Journal ul C'cllular C'arJiulugy 19:343-348, 1987. Othcr suppurl:'I'hc Margaret W. and Hcrlx:rt Iluuvcr, Jr., FHunJ:diun. From the Iluntiugton Medical Research Inxtitutrs, Iluntingtun Memorial Huspital, Pa,adctw, CA. I:FFIi(°f OI' NISOI.I)II'INIi ON I)I: I'.NI)(YI'llliUAI.IZIiD LAK(ili ('URONAkY Ak'I'I:kY IN ISUI.A"fGt) WORKING KABIiI'1' IIGAR'I'S, The effect of uiu,ldipinc on coronary vascular rctiistanccs, cardiac Ierfurmance and ntyurardtal oxygrn cumumptiuu was dctermincJ. Rabbit hcans wrtc (mrliurJ with a Prrlluaruchcntical (F('•43) cmulsiun. I:nduthclium was removed from the (ibtu4c m:uginal and the uhlxr Purtton of the antcriur descending corunary anrry. ('haul:cti in the iutcrnal di:unctcr of these arteries were vitiualiicJ using iqjrctiun of Patent I31uti I)yc (cuhtr artrtitgrahhy). Mran internal Jiantrlcruf curun;try artcrirsand tiurltce arra/unn length wrrr cuminucr ralctd:nctl. Niwldipinr rcduccd the cun,tric. uun of cutunary m•Irrnrti and the drercax on coronary flow induced by histatmnc. NttiulJipinr ahulitihctl or reduced Ihc dccrcasc in cardiac uutput. I.VIiSP, dP/dt and myurardial uxygcn con,untptinu. "I'h+r rrsuit, drmuntitratr that in the supportrd rabbit heart prrparauun, ni>oIdipinc is an cllrrtivr dilatur of large xtthepicardird cnrun:uy artcnr,, drpmcd Of cnduthrlial lining, aud cuunlcrarts cunstrictiun ul curonary rcsi.t- anrc vessels As a cunxyuencc, nisolJtpinr improvcs Ixrlirrnt:urcc of the itichcwic myur:udium. SaccJ M.. IL•tnmann. A..:mJ Bin.q, R. J. I'harutaculugy 35:256-201, 1987. Othcrxuplxtrt: Margaret W. and Flrrlxrn Iloover, Jr. Naunrlaliun, Miles Lalwraturiex, Inc., and Sam and Rose Stein Charitablc Trusls. From the Ilunling(un Mcdic:d Research Irntilulca, Huntington Mentorial Iluspital, I'aaadwna, ('A. A kI:LAXING FA("I'OR RGI.IiASlil) BY PHOSI'HULIPASI: A. IN THI: AIfSIiN('li 01: IiNU(YfIIIiLIUM Phoxphulipatir A. IPLA.) produced slow dou: dependent relaxation in intact and enilulltclium-dcprivcd prccunlractcJ rabbit aorlic strips. In cnJothcliunt-Jcprived preparations, relaxation induced by PI.A, is inhibited by hemoglobin, tncthylcne blue and parabrunwnhrnarylhruntiJc (P131'13), and is Ixitcntiatcd by sulxruxidc Jismutasc (SOI)). Induntcthacin has no cllcct. Relaxation is accompanied by a rise in c-GMP. Phtisphulipasc (' causcs a significant increase in tension, while 11ho,phulipasc Dhas no effects. In intact aortic titrips I'LA. causes a hiphasic response with no elevation in c- GMI'. The rc%ultr indicatc several cuntnwn Icatures ol' the PLA: released I:rctur with cnduthrlium-dcrivcd relaxing factor (IiI)RF). Howcvcr, I'LA, induced relaxatiun is not dcpendcnt on enJuthclial cells. Apparently in addition to nitric oxide which may be the cndothcliunrdcrivcJ relaxing factor, a,ccund snuwth ntuulc relaxing larturcxist+ which is initiatcd by I11.A. and is independent ul'cnJuthclium. The production ul'thc PLA. produced relaxatiun is dependent un its specific hydrolytic activity. We call this relaxing factur the pho,pholipid-dcrivcJ relaxing 1'actor (PURF). Bing, K. J. and Saccd, M. Mulcrul,tr and ('cllular Iiiuchcmititry 78:81-}iti, 1987. Other support: Margaret W. and Flcrl-wrl Ihruvrr, Jr. Foundation, Sam S. Stein and Rose Stein ('haritablc 'I'rust, and the I'anrun Saint FounJatiun. From the I)rpanmrnt of Experimental C'arJiulogy, Iluntingam Medical Research Imtitutc., Pasadrna, C'A. AI:I'I:kA'I'IONti IN (iL.ll'I'A'I'IIIONI? I)IIRINCi S'I'UItACiti OI' Hl1MAN I'I.A'I'IiL.l:'f C'( )N('I:N'I'RATiiS Ghttathiunc and Elutathiunc tli%ullidc dccline rapidly and progressively in huutan platelet cuncrntr:ucti sturcd lirc uP lu 7 days at 22"('.'I'utal Hlutathtuuc declines progres- tiivcly thruughout the sturag-c Ix:riuJ, with in cstimatcd half-ilisaplx:anmcc tituc ul' 2 days. (Hut:uhiunc Jixullidr remaim cunstant during the first 2 days ul',tur,tgc but drelincs progressively thcrcal'Icr. with an c,timalcJ Itall-disaplxarancc tinue uf 3.2 J:rys. Based oil mcatiurenocnts ul gIruuthionc ditiullide in freshly cullectrd platclrtti, the authurs pusttdatc Ihat glutalhionc disulhJr is clcvatcd during the pnnlucuun of platclct conccntratcti or during the first 2 days uf titur,rgr, accounting fur thc lag in its dccay curve. Since total glututhionc decreases more rapidly Ihan glutathionc dtsullidc, the I'ractiun of glutathiune present in the oxiJiicd lurm increases throughout the storage period. Glutathiunc-dclxxndcnt mechanisms maintain esu:ntial cell proteins in the appropriate redox tilatcs and provide avenues for the clctoxilicatiun of Putentially 52 53
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unxiuu. rumpuund. pencrated by lhr cell or within its environment. C'hanges in platclet Flutathiune and glut;uhiune tlt.rullitle during storage may hc rc~rpunablc fur stura)!e- deprnt)cnt altertniuns in in vitro Iunctiunx and ntay also atfect suhycy,ucnt ur riru recovery and survival ulxm reinlusion. Ilurch, 1'. T. and Burr/r, J. W. 1'tan,lusiun 271-!): 3-13-3d6, July-August, IN2{7. Other support: Antcriran Red C'russ. Frum the Research 1)cpartment, American Red Cross l3hxxl Services, Rochester Region, Rochester, NY. I:FFE("I'S OF IiPINGPHRINIi ON CORONARY MICROVASCULAR DIAMGTI:RS '1'his study was designed to examine the hypothesis that epinephrine has nanuni- lircm ellictu un coronary naicrnva.scular diameters. Measurements uf curunary mi- cruva.cuL•rr dianteter were completed in anesthetized, open-chest cat preparations in which the epicardial microcirculatiirn was viewed through an intravital microscope using stroboscopic cpi-illuminatiun. Images uf corunary microvessels were digitized and analyzcJ un a video monitor. With arterial pressure controlled, mcasurentents in the absence and presence uf (3-adrencrgic blockade (propranolol I mg/kg) were irb- tatned during epinephrine infusion (1-2 µg/kg/rnin). In the absence uf /i-aJrenergic blockade, erinephrine produced a 2519, increase in myocardial perfusion. Under these conditions, coronary vasox)ilatiun was observed in all classes of coronary arterial anJ artrriular vessels. In the preunce of p-aJrenergic blockade, epinephrine produced a tiignificant decrease in myix•arJial perfusion ( - 20%). Nonuniform eflects on Ji:une- tcr were observed in arterial and venous segments of the coronary circulation. "I'hetic Jata are consistent with the view that in the absence of (3-adrenergic blockade, the functiunal coronary hyperemia associated with epinepluine administration is produced by unitorm coronary arterial and arteriolar dilation, In the presence of (3-adrenergic blockade, with mctahulic effects controlled, epinephrine produced a decrease in ntyucurdial rrrfusiuu, which is related tu a nonuniform decrease in coronary micruva.- cular Jiameters. Such heterogeneous effects on micruvascular diameters result in a redistribution uf carunary microva.cular resistance. ('luliun. W. 61., IKryne, S. M., Gaslhant, ('. L., and Marcus, M, L. Circulation keuarch 61(Suppl 11):II-47-11-53, 1987. Other support: U.S. 1'ublic Health Service, Ischemic Specialized ('enter of Research, and the Iowa Aftiliate o( tlte American Ileart Asscxiation. From the Ikpartment uf Internal Medicine and the Cardiovascular Center, University uf luwa College uf McJicine, Iowa City. INTRAVASCULAR KliI.IiASI: c)F IN'I'A(I' ('I:I.LUI.Att FIIiRONtiCl'IN 1)lll{IN(i OXIUAN'1'-INI)l1C'EI) INJURY OF'I'III:/N VITRO I'I:I2NUSGU RAltliI'I' I.UNG Fihruncctin (fn) is produced by cell% in blood vessels at mflammalury sites ut riro. Fn release into the circulation thus may be a marker litt vascular injury. In support of this, we liounil that uxiJant-induced vascular injury of isulatcd perfuuJ rabbit lungs caused elevated circulating Fn levels. Western blot analysis indicated that Fn released fruru the injured blood vessels was intact, Jinteric, and possessed electrupharetic mobility identical with Fn produced by tibrohla.ts. Unlike F'n isulatea fntm rabbit rla.ma, Fn derived frum lung Ixrfusate or produced by librublasts reacted with anti- IxtJics raised tu a synthetic peptide containing sequences frotn the extra type III Pn domain that i.lranscritxd in fibrubfa.tx but not hepattx:ytes. Vascular injury by prute- are was also associated with intravascular release ol' Fn, but with cleavage. Oxidant- induced vascular injury causes release ofti+sue-JerivcJ hn, which can be distinguished frum plasma Fn by its size and content of antigenic determinants of the extra type Ill Juntain. Peters, J. H., Ginsberg, M. H., 13uhl, B. P., Sklar, L. A., and C'orhranr, C. (;. Journal uf C'linical Investigation 78:1596-16O3, L)ecember 1986. Other zuppun: U.S. Public I lealth Service. Frum Ihe 1)epartment of Immunology, Scripps Clinic and Research houndatiun, L.a Jolla, ('A, and Pulmonary Division, Repartment (if Medicine, University of Calitiu- nia, San Diego. IIIiPARIN-ItINI)ING hRAGMI:NTS OP FIBRONGC"1'IN Altli P(YI'IiN'1' INI II13I'1'ORS OI' F,NUOTHIa.IAL CELL GROWTH: S'IRUCTl1RG-1'UNCI'IUN CORRELATIONS Ilcparin-binJing fragnunts derived from the amino- and carboxyl-terminal re- gions ul human plasma fibronectin appear to txx at least relatively specific potent iuhihiturs uf thr growth of Iwvine aortic endothelial cells in culttrre by as yet unknown mech:utism.. In order to understand better the sites which subserve this activity, we have compared the relative potency uf uther majur fragments of fibnmutin, most of which have dissimihu• properties and do nut bind heparin. We have alw proteolytically digested and chemic;dly mrxlifieJ the most potent of these Iragntcnts, the aminu- terminal 211-k1)a fragntent, in order to test whether structural alterations that affect hrparin-hindingal+o affect the inhibitory property. Not all chemic:rl mnJitir:uiuns Ihal abolished heparin-hinding alx+t abolished cnduthelial cell growth. Neither an ammu- terminal 20-L1)a nur a carhoxyl-terminal 13-kUa subfragment of the 2r)-kDa fragment huund heparin; huwever, huth were as inhibitory as native 29-kDa fragment. kcJuc- liun of the JisultiJes uf the 20-k1)a and 8-kl)a fragments did not abolish inhibitory activity. We thrretitre conclude that the activity is not strictly cunfirrmation-depenJent and that although the inhibitory activity i+JixtributeJ throughout the 29-k1)a,egment, it can he expressed by an R-kDa cartwxyl-terminal segment containing residues ot' liue last 'I'ylx I loop structure. 54 55
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I Iumarnlhcrg, (i. A., Kr:uncr-I3jcrke, J., (iranl. I)., ('hristiansun. (i., :,nel liiu•u.,trin, R. 13iex^,hionic;i cl Iiiuphy.ica Acla H74:61,b7, 1986, Othcr tiupperrl: National In.titutea etI' I Icalth. Prutu thc I)cparuncnts ul' Medicine anJ I'athuh,gy. The University ul' Wisconsin McLhcal School, Mtlwaukre Clinical C:unpus, and Mount Sinai Medical ('enter, M,il• waukcc. PAC"I'OK XIIIa F°OKMA'I'ION PkOMUTIiI) 13Y COMFLGXING OF a=I IIKOMBIN, FIBRIN, ANl) PLASMA 1'ACTUK XIII Fihrtn Ix,lymrrs (des A,li fihrinugen) reduced tite concentration uf ec-thrwnhin rcyuired lur 5(NX activatiuno(plasma 1'arutr XIII (a,h. teuanrer) by uppruxi,ntately I(H)- li,hl. In Ihc pre,cncc uf fihrin, the amount ufy-Ihrutnbin required li,r activaliun was tio( aflcrtcJ. ('atalytirally inactive i-Pr.l'- anJ u-Phc•1'ru-Arg-CFI_ a Ihruntbin wcte luunel to inhihit over 95'h• uf Ihe activatiun uf a-thrumhin in the presence uf Iihriu. Unlikr pla.ma facuu XI I I, the cuncentatiun uf ex-thrumhin required li,r 51)'!.activatiun uF hlatelet Iactur XIII (:,. Jimer) was luwer, and the activation was not enhanced by li'hrin. I luwcver. when the a. platelet factor XIII was incubated with punhcJ b-chain., Ihe «- anrl y-thrunthin cuncenlraticros reyuireJ li,r activation increased tenlidd and reached Icvels simil:u-lu those required li,r activation ufthc plasnta I'actur XIII. When fibrin was present, the er-thrumhin cetnccntratiuns necJed li,r activatiun ul' Ihe a.h. cumplexes were reduced, and Ih1 presence ul' fibrin had no cllcct un -y-thrunthin cleavage e,l' Ihe a.h, cemtplrxes. -I'hcrefure, Ihe b-chains must inhibit a-chain cleavage by er-thrumbin in lltc ahxncc uf lihrin. 'I'hcse results imply that the furnwtion of a cucuiultlcx invulving a-thrunthin, fihrin, anil plasnta factur XIII causes sumc cunlnr- matiunal change in plasnr.t factur XIII such that the h-rhains no longer inhibit cleavage of thc a-ch:uns. Grevnlu'r,q, ('. S., Achyulhan, K. li and Fenton II, J. W. Bluud, 69(1):867•871, March 1987. Other.uppun: National Institutesof Itcahh. Front the I)epartmcnts of Mcdicine anti Pathology. I)uke University Medical Center, 1)urham, N(', and Wadsworth ('enter fur Laboratories and Research, New York State Department uf Ilcalth, Alh:my. GLY-1'KU-AK(i-I'KU MUI)IFIIiS "1'Hli (;LUTAMINI? KIiSIUl11S IN Tlili er- ANI) -t-('IIAINS OF 17BKIN(X;I:N: tNlI1B1'flON OF TKANSGI.UI'AMINASIi ('kOSS-I-INKING During blrxxl clotting Factor XIIIa, a transglutaminase, catalyies the fornt:rtiun uf covalent hemds betwcen the r-amino group uf lyune anJ Ihe y-cartxrxamide gruup uf pcptide-huunJ glutamine resiJucs between librin molecules. We report that glycyl-I: prolyl-t -arginyl-t -prulmc (GI'KI'), a tetrapeptiJe that binds to the fibrin polynteriza- lion.itcs (1)-duntain) in fihnn(ugen), inhihits tctmElutaminase cruss-linkin f, by nuxli- I)ing the glutaminc re.iJues in Ihe er- and y-ch:tins uf fihnnugcn. I'urilied platelet Factur XlIla, and tt,tiuc tran.glulaminase Irunt adult bovine attnic cntlulltclial cells were used Iur Ihc cross-linking studit:%. (Hy-Pro (UP) and Gly-Pro-Gly-Gly ((it'(i(i), Ix:ptiiles whirh do not bind to fihrinugen, had no cftcc( un tran+glulammasc cross- linking. (il'KP inhibited platelet Factor XIIla-calalyied cruss-linking hr:Iween the y- chains uf Ihe li,lluwing lihrin(e,gen) derivatives: fibrin munonterti, librinogen and pulymcriicd fibrin lilhrs. GI'KI' limetierocd as a reversible, nuncumlxtitive inhihitor uf I-iactur XIIla•cat:dyicJ incerrpe,ratiun uf I'Iilputreuinc and I"Clmethylamine ,nto fihrinugrn and I'ragment 1)I. GI'K1'did not inhthit '-"I-FacturXIIIa hinding u,pulymcr- ind lihrin, dentun.Uading that Ihe Factor XIIIa binding sites un fibrin were not nukl- ilicrL GI'KI' also had no clfect on Factur Xllla cruss-linking etl' I'lliputreuinc to casciu.'I'his demun,trates that GI'Kp slxcifically nuxtified the glutauti,ne cross-linking sites in lihrinugen, and hael no cffccl un either Factor XIIIa or the lysinc residues in librinugcn. GI'KI' alsu inhibited I "Clputrescine incorporation into the a-:mJ ry-chains utfihrinufcn without inhibiting /3-rhain incorporation, suggesting that the interntulec- ular crass-linking >hes were selectively aficcted. Furthermore, GI'KP inhibited tissue tr:msglut:uninasc-catalyccel incurpa,ratiun ol' I'lF1putrescine into both librinugen anJ Fraf.nu:nt 1)I, withuut tuudil"yin)! 1'll1putrescinc incorporation intucasein. GI'kF':dsu inhihitcd intcrnu,Iecular er-cr-chain cross-linking catalyzed by tisxue transglutaminase. 'I'hts elemonsu:ues that thr glutantine rc%iducs in the a-chains involved in intermolccu- larcrua-linking arc modifieJ by GI'KI'. 'I'his is tlte lirst dcmunslratiun Ihat a molecule binding to lhc fibrin pi,lyntcriration sites on the 1)-Jnnwin of fihrinugen nrudifies the glutamine cruss-linking sites un Ihti: ce- and y-chains of lihrinogen. Achyuthan, K. I:., I)uhsun, J. V. and Grrcvtherg. ('. S. Biex:himica ct Bluphysica Acla ti72:261-2GR, 1986. Other support: National Institutes of Ilealth and American Hear1 Association. Prom the 1)cpartntcnts of McJiciinc and I'athulugy, I)uke University Medical Center, I)urham, NC. 'I'Illi "IRANSGLU'I'AMINASIi IN VASCULAR C'NLLS AND TISSIIIiS ('OUL1) PROVIDE AN AI:I'I:KNATF: PATHWAY FUK FIBRIN STABILIZATION A thromhiu-indcpendcru transglutaminase (TG) has been iJentified in vascular cells and Iistiues frunt human, rabbit, rat, porcine, and Iwvine s+,urces. 'fhe vascular '1'G had several prulkrties th:u were siniilar hut not identical to guinea pig liver'I'G. Both enrymes haJ similar chruntutugraphic and electruphuretic properties, preleren- tially cross-linked the eY-chains of librinuf,~en, and reacted with polyclonal and muntr clun:d anti-guinea-pig liver TG antibodies. Iluwever, Ihe TG frum adult bovine aortic enduthelial (ABAIi) cells exhibited a novel C'a" /Mg" dependence litr enzymatic activity lhat was distinct frunr that of purilicJ guinea pig liver TG. The mul wt of the vascular TG (79 !:( kd) determined by sodium dexiecyl xulfatr-pulyacrylamide gel elcclrophuresix (SDS-PA(il:) was slightly htwer than the purifieJ guinea pig liver TG (R5 ± 9 kd). The TG antigen was detected by immunuhiskxhemical techniques in association with the cndolhelial and smooth ntuscle cells of arteries, veins, venules, and capillaries. 'I'he TG antigen also corlistrihuted with the lihronectin antigen along the hepatic sinusuiJs. '1'he ABAE cell TG cross-linked a; plasmin inhibitor tu librino- 56 57
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gcn andcauu:d Ihr mudilirJ lihrinogrn to hc 4(t-[uld nrure rr.ititant to plasminulytii+. A thtundrnn-inJclx•ndcnt'I'(; in vuticualr cells u[ hhnod vessels cuuhl pruvtde an alternate pathwuy tu inhthit Idmuuolysiti and promote librin xtabiliiatiun. (.irrnhrrg, C. .4., Achyuthan, K. li., lioruwiti, M. J., and Shum:m, M. A. 131uoJ 7(I(31:7(12-7(K), September 19ti7. OIhcr ruPport: National Institutes ul Ilcalth, Amrrican HCart AstilKGdiun, W. I'. Innrut I'vnd, and BhMrd li:mk Training. Frum the I)epartrncnt, ul' Medicine and Pathulugy, [)ukc University Medical Crntrr, I)urh;un, NC. and Univcr,ity uf Culiliirnia Medical Center. San I'rancisca. POI°I?N'I' I:Fhli("l:ti ON IVIiUKOPIiP'1'll)I: Y AN[) CALCI"I'ONIN (;IiNli-KI:LA"1'L•I) 1'lil''I'II)liON HUMANC'UKUNAKY VASCULAK'I'UNE IN VI7R(! The present finding..how that two peptides which are present in coronary Ixri- va.cul:u nerves exert potent rf[ccts on human curunary artcrics. 'I'hc relaxation ul Iltc NPY-induced cuntractions ohurveJ allcr nifcJipine, an agent u[ten used in trc:Umcnt ul vasospastic angina, is likely to Ix related tu interference with C'a" uuchaniam.. In the pig, the relaxation ol' curonary arteries induced by CGKI' mimics the cl7ccts ul' arlivatiun u[.en,ory nerves by cap,aicin. Since CGKP was a IxMtrnt dilator ol'human rurunary ancric,, it will lie u[ interest tucstabliah whethcrCUKP is released from local ('-lihcr allcrcnt, during cardiac ischactnia and contributes tu thc reactive hylxractnia. Francu-Ccreccda. A. anil Lundbrrg, J. M. Acla I'hy,iulupica ScanJinuvica 131:159-I6O, 1987. Othrr suppurl: Swedish Medical Research Council, Swedish Tobacco ('umrany. I'ctrus and Augusta I lcdlunds Foundation, anil the Karolinska Institute. Froni thc Dcpartntrnt of Pharmacology, Karulin.ka Institute, Stuckhulm, Sweden. IS('I II:MIA ANI) ('I IANGI:S IN ('ONTKAC"I'Il.ff Y INDUCE KIi1.GAS1{ OF ('AL('I'1'UNIN (;IiNI: KI?LA'I'EI) PGI'11I)I: 13[1'I' N(Yf NIiUKUI'lil''f1Ul's Y FKOM'I'Illi ISOLA'IIiI) I'I:KFl1SNl) GUINI:A-1'IU HGAK'1' Thr ptc.cnt dala show that lulal iachcmia as well us agents which increase cun- Irarltlily rnhanrr uvrrlluw ul ('(iKP-L1 I'rum the isolated per[uticd guinra-pig hcart, suggrsting Iha1('(;KI'relcau huth upon increased metabolic drmand anJ myucardial t,chrnua may increase curunary blood tluw as well as induce changes in cuntractihty. Thr rnhancrd ('GKI' uverfluw by capsaicin is likely to ix due to a direct action nn ,cnwry nrtvr. whily drr site of action lor nicotine, NA and uuahain remains to hc established. Nicutinr is well known to release NA from cardiac sympathctic ucrvr, and the Prisout data, in addition, show an increased overflow uf Ihe co-slurcd Ixptidc NPY. 'I'he chmnutrupic action u[rahsaicin is probably related to the release o[C'GKI' and thr initial increase in heart rate seen during the rclxr[uviun aficr i,chemia may al,u he due to release ul' this Ixpti+Jc. Anulher reason liir the observed tachycardia after i,chcmia would Ik Ixta-aJrcnoxcptur aclivaliun duc to release u[ NA. No clear-cut uvcrllnw of NA .cent., however, tu occur during the pretietu relatively short i.chrmic period. Furthrrntorr, in contrast to the passive leakage of NA upon prulungcd i,chc- mia, (he CC;KI'-uvcrlluw was calcium-dependent, suggesting active relcatir. hr:urcu-C'crurJa, A., Saria, A.. anJ LunJGc-r.r, J. M. Acla Phy%iodugica Scandinavica 131:319-.i21), 1987. OOther support: Swedish Medical Research Council, Swedish "I'ubaccu Company, Petrus and Augusta I icJltmJs Foundation, the Karolinska Institute, Austrian Scicutilic Research Funds, and thc'fhysun Foundation fiir Cardiovascular Research. From Ihr 1)rparuucnt u[ I'harmaculugy, Karolinska Institute, Shxkhalnt, Sweden, and Dcpurlntrnt of Experimental and Clinical Pharmacology, University ul' (;raz, (;rac, Austria. INU'tROI'IC lihh'liC 1'S OF CALC'ITONIN GENE-KIiLA'I'I:I) PIiP"I'II)E, VASOAC"1'IVG INTI:S7'INAI. POLYPIiP'I II)E ANI) SOMA"1'C)S'1'ATIN ON'I'HI: I IUMAN KIUH'1' A"1'KIl1M IN VI'fKO Iuuu•opir responses to calcitonin gcnurclatcJ pepliJe w-CGKI'), substance It, nrurokiniu A, capsarcin, ncurolx:ptidc Y, vasoactive intestinal [xilYlxptiJc ( VIP) smd som:duslatin (Sann) 14 :md 28 were analyzcd using the isolated, electrically driven auricle uf the huntan right atrium. a-('GKP and VIP stimulated atrial contractility cunccnlratiun Jclxndrntly. (x-C'GKP was about IU-/i0ld murc Ixncnt than nuradrenalinc (NA) as an inotrupic agrnl. I'Itcntulaminc plu% mctuprulol decreased Ihe aqrial rc- apouuu tu NA.il;ni6cantly while thc (r-('GKI'clI'cct remained unchanged. Som did not influencr the basal contractility uf thc atria, which, however, was inhibited hy acrtyl- rltulinr (A('h). A('h, Sum 14 and Sum ZR inhibited the NA-induced stimulation of atrial cunlractilily, whereby Sum 28 was more potent than Som 14. The inhibitory clfrct, of A('h were completcly blocked by atropine which did not inllucncc the response to Sum. ('ap.aicin, substance 1', neurokinin A, ncurulxplidc Y(NPY) and the NPY fra)!ments 1-19 and 26-3G did not induce any changes in contractility of thr clccartcally driven human atrium. "I'hr present results suggest that some o[ thc recently discovered ncuropcptidc, I(r-CGKP , VIP and Som) could be of impurtancc in lhe regulation uf cardiac cunlractility in num. Fr:mtu-Cerrct^tla, A., Henglswn. I.. and l.nndhr•rg, J. M. Iituulkaut Juurnal uf Pharmacology 134:(i9-76, 1987. Other .upliort: Swedish Medical Research Council, Swedish 'I'uhacco C'untpany, Prtrm and Augusta IlydlunJs Foundation, and Ihc Karolinska lnailulc. Prum the I)eparttnrnt uf 1'harntacolugy, Karolin.ka Institute, and Ikrpartmcnt u1"Thu- racic Surgery, Karolimka Huspital, Stockholm, Swcdcn. 58 59
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NIiKVOtIS ('ON'I'KOI.UI''I'KACIIIiAL 13L(X)I) FLOW IN'I'llli ('AT MI:ASLIKIiI) l3Y'llIl: LASIik I)OI'I1LGk'1'kiC'IINI(lllli 'fhe trachral hluud Iluw aa detemtined by the laxer I)uppler lechniyue wati ciintin- uuutily mirnitured in ane.thetiieJ cuti. Iaerlriral stimulation uf the right superior Jaryn);cal nerve caused an :rlrupinc-re+istant increase in bhKxl Ilow itl' the upp+:r tra- ehri. l luil;ueral vagal nerve stimulatiun at the rervic)d level in the presence of alrupmc tndurrJ a Ireyurnry-drpendenl increase in blood Ilow ol the lower trachea. Intermit- tenl `tumuLwrm with hunts of impulurs it a high frequency resulted in a considerably larger blood Iluw incrrau than it continuous low Ireyuc:ncy stimulation giving the same total number of itupulus. The gangliunic blocking agent chhrrisond;uuinr aholnhcJ most rrl the vagally induced increase in tracheal blood flow when using low thretihuhl paranirtrrti (2 V, 0.2 mti) presumably activating preganglitrnic nerves. I ligh threshold sUmulatiun (IO V, 5 nn) huwrver, still rctiulteJ in an increased blood Iluw suggesting antidranrir activation (il sensory (' fihres. Lrkal mechanical irritatiun or ncemic;d irritation by capsaicin also increased tracheal blood Ilow. Furthermore, local applica- lion ul calcituntn gene-related lxptiJe on to the mucrna caused a slowly developing, lung-laaing incrut%e in hhKUl Iluw. I;lectrical stimulation of the cervical tiyntpathetir trunk and local applicatiun of adrenaline reduced tracheal blood Ilow. In conclusion, vagal nerve .tintul:uiun induces an alrupine-retiistant increase in tracheal blood Iluw probably mainly by activating preg;ut);lirmic paraxyntrathetic nerve, and possibly also by anlidrumir stimulation ul' C-lilx:r allcrents. Martling, C.-k., (iai.eliu,, B., anti l.um(brrg, J. M. Acta Physiulugira Scandinavica 130:419-417, 1987. Other support: Swedish Medical Research Council, Swedish Tobacco Cumpnny, Swedish Society of Medicine, and the Karolinska Institute. From the Ihpartment nl Anesthetii;r, Karulina.a I lutipit;d and Departments ul' Pharma- rulogy and lincludontics, Karulinxku Institute, Sux:khulm, Sweden. ('AKI)IOVASC'III.AK IiFFli("t'S OF CAL('ITONIN GliNli-KI:LATIiU I'IiI'"I'II)IS I AND II IN MAN ('alrilonin gene-rehuetl Ixplide (C(iKI') is present in sensory nerve lihrrs in the hrart and :uuund peripheral arteries. On interaction with slxcific C'(iKl hinJing sites and activ.uiun of adcnyl;ote cyrlase, C'GKI'rauuus vawJilatiun;rnd has positive inutro- ptr and chrunutrorir rlf'ect.% on the heart. In the present study, human ('OKI' I anrl II exerted positive inutrupic ellcrts on isolated huntam right auricles ;rnrl relaxed ,unall ar7rnes Irom human skeletal muscle precontracted with nurepinephrinc Iur CGKP 10.59 nM and for C'(iK1' 110.37 nM). C'Clkl' I and If (3.2 nnwl) administercJ t, v. tu 6 normal subject:, exerted positive inutrupic actions un the hunrm heart ruucum- itant with positive rhronutrupte ellcrts, hyputen,iun, und vau,dil:uion. ('(]KI' may, thrreture, be uf imlaurtanre for cardiovascular control in man. I'r.utru-('ereceJ;i, A.. (iennari. C'., Nami, R., Agnuxdci, I).. I'ernuw, 1.. Lundbrr,ti, J. Ai.. and Fischer, J A. ('irrulauun Research (dt: l9 i- iy7, 14)37. Other support: Swedish Medical Research Council, Swedish Tobacco Company, Swiss Naliunul Science Faunclatian, and the ('anton uf "Lunch, From the Ikpartment ul Pharmacology. Karulinmka Institute, Slokkhnhu, Sweden. Imtitute of Medical Senteiulirs, University uf Sien;r, Siena, Italy, and ketiearch Lahyr- ratury for Calcium Mrtalr+di+nt, IkF;trtmentx ul Onhulx;Jie Surgery and McJicine, University ui 'Lurirh, Zurich, Swilcerland. VASO('UNS'1'kICI'OK IiFFI:Cl'S IN VIVO AND 1'LASMA 1)ISAPI'I:AKANCIi kA'1'li OF NIiIIKOI'I:NTII)E Y IN MAN Vascular ellccts uf neurulnxrliJe Y(NPY) and nuradrenaline (NA) were studied in six human vulunteers. Systemic infusion uf hwnan NPY litr 40 min (5 pinol x kg ' x min') increased arterial plasma NPY-like immunureartivity (NPY-I-1) from 12 i 2 to 356 !: 10 pM. 'I7iir concentration caused no systemic cardiovascular effect,. The disappearance curve lirr NI'Y-1.1 was hiphasic: the slopes uf the two phases curres- pundinE to half lilcti ul -{.I t 0.4 and 20 !? min respectively. C'lose i.a. infusiun uf human NI'Y in the lirrrarm rautied u,lowly developing and duu depcndent decrease in littrann blood flow ( FBF) and inrreaxe in venous tcrne with maximal values ril 44 ± 6 and 235 1 81 'k• uC rtrntrul respectively at 5 nnwl x min '. The curreslxmcling values li,r NA (5 nntol x min') were 21 ± 9 and 489 ± 78 'h, of ronlroL A threshold ronrentratiun fur a Jrcreau in F131-'wax ubtaineJ at a plasma NPY-LI of 3.7 -t 0.6 nM. The elecreast: in 1,13F raused by NI'Y was maintained for a ntuch longer period curn- p:rreJ to that uf NA. Pcrnuw, J., Lwrdhrr);, J. At., and Kaijser, L.. Lile Sciences 40:47-54, 1987. Other suplxnl: Swedish Medical Research Council. Swedish 'I'ohaccu C'umpany, Swedish Work I?nvirunment Fund, Petrus and Augusta Hedlunds Foundation, SweJ- ish Society of, Medicine, and the Karolinska Institute. From the I)rpartment uf ('harmacolugy, Kamlimka Institute, and lkpartment uf Clini- ral Physiology, Kantlinsl.a I-luspital, Stockholm Sweden. IIIGII AND LOW AFFINITY I3IN1)IN(7 SITIiS FOR L3ASIC FI13KO131.AS"I' (ik( )W'I'll FA(''Ok ON ('I'IIKI?I) ('I:LI.S: A13SGN('li OF A kOLI; FOR 1.OW A1FINIl'Y 13IN1)IN(i IN'1'111i S'I'IMULA'I'1ON OF PLASMIN(HiliN A("1'IVA'1'OK I'KOl)U("PIUN BY 130VING CAPII.LAKY IiNI)U'I'IIIiLIAI. ('GI.I S Sc;urharJ ;m;tly.is of binding ul"l-ha,ir IibruhL•rst growth factur 1 F(iF) to hahy hatmter LtJncy (131IK) cells revealed the presence of two hinJtng ,ites a high allinity site witlt K,, uf 2U pM and >;t),(XX) sites Ixrcell and a luw allinity site with K,, ul ahout 3 nM and 6(M),(NX) sitrs per rell. The binding to the two sites could be separated by lint washing the cells with 2 M NaCl at p11 7.5 which released the low ;dlinity hindiog anJ then extracting the rells with 0.51%,Tritun X-I(K) to recover the "`I-ba,ir F(lF huunJ to high alfinity ,ites. '1'he binding to the high a(linity sue was acid sensitive, suggesting (A) 61
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that it represented binding to the receptor. Binding to the low affinity site could be comlxaeJ titrongly by heparin and less strongly by heparan +ulfate but not by chondroi- tin wlfate, i3crntat,m sulfate, or keratan sulfate. Treatment of 1311K cells with hcparina,e aholi,hed 621Y, uf the low affinity binding, suggesting that the low aflinuy binding represented binding to cell-asuxiated, heparin-like molecules. A variety ol' uther ceil types, including bovine capillary enJothelial (1)CE) cell., alw Jrmunstnded' both low and high al linity binding sites. Tu te.t whether the low affinity btnJing might play a role in the huaic FGF.timulation ol'plasminogen activator (I'A) productiun by 13('I: celh, heparin was adJed tu 13CIi cultures at concentratiuns which totally blocked binding of "'1-ha.ic FGF tu low affinity sites. Addition uf the heparin Jitl not diminish the increased PA PnaJuctian induced by basic FGF. This suggests that the low aflinity binding has no direct role in the stimulation uf PA production in BCE cells. Muscutell i. 1). Juurnul of Cellular Physiology 131:123-130, 1987. Other support: National Inatitute% of Health, American Cancer Society and New York Ilcart Association. Frnm the I)cparnnent of Cell Biology and Kaplan Cancer Center, New York Univer- sity Medical ('enter, New York. PRESLNCE OF BASIC I'II3RO13LAST (;ROW'f FI FACTOR IN A VARIETY 01: CGLI-S ANI) I'1'S I31N1)IN(; TO C'IiI.LS I)uring tumur angiogenesis, angiogenic factors released by the twnors stimulate endothelial cells of the neighboring capillaries to penetrate into the surrounding tissue, to migrate toward the wurce uf thc angiogenic factor, and to multiply, thereby funuing a new network uf capillarics supplying nutrients to the tissue. It is thought that these re.txrnses are currelated to three in ritru re,lxmu:x of capillary enJuthelial cells to angiogenic factors:.tinmlatiun of the production ol'the prutcascs plasminogen activa- (ur and cullagenase, increascd clrenwtaxis and elevated rate of proliferation. We have used one of these responses, the increased prixlurtion ol' plasntinogen activator tI'A) and collaf!cnax by cultured bovine capillary endutheli:d (l3Cli) cells, as an a.tisay lur the purificattion nf an angiogenic factor fi•unt tenn human placenta. The purilied ntule- culc was shown to have all three of the above prulxnies proposed for an angiugcnic factor, 'I'he purified molecule was identified as basic fibroblast growth factor (bF(;F) on the basis of it..imilar chemical prupenies and a 98% amino aciJ ,cyuence homul- ogy with bovine pituitary bFGF. We have demonstrated that a variety of cells, from both normal and tumor tissues, contain bF(;F like ntolecules, suggcstingthat angiogenesis can be induced by injury to many tylxs uf cells. In nonnal cultures, most of the bFGF remains cell-assoxiwed. 'I'his may represent intracellular, nunxecreteJ bFGF. However, since a wide variety of cell types have luw-aflinity binding sites fur hFGF, part of the cell-associated MF(iF may he secreted bF(;F that has Mrund to heparin-like molecules on the ccll .urface or in the extracellular matrix. Several of the cultured cell types investigated (e.g.. lihroblasts and B('Ii cells) both produced hF(iF and had high-affinity receptors for the molecule. This suggests that for umie cells hFGF inay act as an autocrine hormone. Mr,xcutrlli, b., Prestx, M., Jirxeph-Silveruein, J., and Rifkin, 1). H, In: Rifkin, I), F3., Klagshru.n, M. (eJs. ): Angiugenex.is Mechanitints and Pathnhiulugy, Current ('urnmunic:.uiun, in Molecular Biology. Cold Spring Hurbur Laboratory, pp. 47-5I, 1987. Otlter tiuplwn: National intititutes of He;dth, American Cancer Society and New York Ilean Assix:iaticm. Frunt thc 1)epartment uf C'eIl 13iulugy and Kaplan Cancer ('enter, New York Univer- sity Mcdical ('enter, New York. 'I'lll: OPPOSING I:FFI:C"I'S OF BASIC FIBROBLAST GROWTH I•~'AC"fOR ANI) TRANSFORMI!NG (iROWTH FACTOR BETA ON TIII: REGUI.A"1'ION OF I'LASMINOGFN ACTIVATOR ACTIVITY IN ('AI'ILLARY ENUOTHELIAL ('EI-LS Itasic fibrciblast growth fa sti ctur (hFGF), a~)tent inducer of angiogenesis in viru, mul:nec the prnJuctiun of brnh urokinase- and tixsue-typr: plasminogen actlvaturs (PAs) in cultured bovine capillary cnrluthelial cells. The observed increase in protcu- lytic activity induced by hFGF was effectively JiminitiheJ by picogram amounts of transfirrming growth factor beta (TGF(3), but could not he abolished by increasing the amount ufTGFP. I luwever, the inhibition hy TGFP was greatly enhanced if .1he cells were rrctrcated with T(;h'P before addition of hl-(;F. Afier prolonged incubation of' cultures treated .intultanwu,ly with hFGF and TGF/3, the inhibitory cf7ect of'I'GFf3 diiuinishcd and the stimulatury cf tect of thc added hFGF duminated as assayed by PA Icvcls. T(;F/3 did not aher the receptor hindin)g uf IabeleJ hFGF, nor did a 6-h pretrcat- ment with'I'(;F(3 reduce fhe amount of bFGF hounJ. The majordif/crcnce between the , cllccts uf hF(7F and "f GI•/3 was that while bFGF el'fectivrly enhanced PA activity , expressed by t7te cclls, 'IGF(3 decreased the amounts of htrth cell-associated atul ucreted PA activity by decreasing enzyme production. l3uth bFGF and T(;F(3 in- creased the secrction uf the enJothelial-IyPe plaantinogen activator inhibitor. Sakscla. 0., Moscutelli. l). and Rifkin, I). B. 'l'he Journal uf C'cII Biology 105:957 ')6a, August 1987. Other suppurt: National Institutes of Hcahh and Amcrican Cancer Society. From Ihe I)epartnunt of ('ell Biology, New York University Medical ('enter, New , York. Rli(;(ILA'1'1ON ANI) ROl.li OF GUANYLATE CY('I.ASE-CYCLIC GMP IN VASCULAR RELAXATION "fhrce classes of vauxlilaturx ntediate their effccts through Ihe activation of guanyl:de cyclau and the increased synthesis ol'cyclic GMP. Nitrova.uxlilators.uch ax nitroglycerin, nitruprussiJe, hydroxylamine, aziJe, etc., result in the generatinn of the nitric oxide free radical that activates the cytusulie (soluble) itioenzynu firrm of guany- late cyclase. These agents have been useful in increasing cyclic GMP synthesis in 63 62
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numerous ntudrl xy.tenu and these cl7ects arc independent uf cxlraccllular calcium. 'fhc increased xynthe,is of cyclic (iMP and the activation of cyclic CiMI'-rlelxndcnt pratein kinasc retiult in the altered Phu+phurylraitrn of roany smouth muncle pruteinx including the dephusl7huryiatiun utnryutim light chain, which is a.sociated with vascu- lar and tracheal tiuuxoth nruule relaxation. Another class uf vaxoxlilatars, de.ignatcil L'nllUthCllunl-llelx'ntlent vasodilators, includes a long list of agents such acetylchuline, histamine. A23187, A"I'P, thrurnbin, etc., that relax vcxselsunly when the en>Juthelirun ix intact. These agrnta result in the increa.ed endothelial synthesis andlur release uf a fati•tur(s) deaign:ned enduthelial-JeriveJ relaxant factor ( lil)12F), the structure uf which is unknown. This labile factur also activates the soluble iurentyme forw uf )!uanylate cyclasc tn the .muonh uru.rle, resulting in cyclic GMP accurnulalrun and thc same cascade uf evcnts as above. Therc is evidence that even under hasal, nunstinrulatcJ cwrJitiomv there is lil)RF release that influences vascular tonc Juc to the increased xynthc.is of cyclic (iMP. A third clas+of vasodilators, utrial n;t(riuretic Cactor (ANt ) or atriulx:ptim, includes a family of Pcptidcs that are produced in cardiac atria and other tissues and influence carJiuvast:ular volume and Jynamics by causing natriure- sis, diureni., va.oKhlatiun anJ decreased renin, alduxtenyne and vasopressin ucretiun. 'I'hese Ix•ptidc hurrnuncs also increase cyclic GMP synthesis in vascular, renal, aJrenal anJ uther tissues. 'f'hese effects are mediated through slx:citic ANF receptors that cuuplc to anJ activate thc membrane (particulate) iuteniymc: litrnr of guanylate cyclase antl incrcare cyclic GMI'-Jependent protein kinase activity. 'I'hesc studies have permitted us to develop a useful model uf the regulation and role ol'cyclic (iMP in vascular snxxtth muscle and other tissues that are xummarizeJ m this review. ILlurnd. F., Waldtuan, S., Molina. C., 13ennett, 13., and Leiunan,•1). In: Mechani.mti uf Signal Tr:uisJuctian by Hurnrones and Gruwlh Facturs. Alan R. t.iss. Inc., pp. 65-76, 1y87. Othcr tiuplwrt: National Institutes ut' I lealth and the Veterans Administration. Frorn thc I)ePartnunt, of Medicine and Phannacolugy, Stanlurtl University School uf MrJicine, Slanfurd, ('A, and Veterans Administration Medical Center. Palo Alto, ('A. lil l li("f (11 IN VIVO NI'1'KOGI,Y('I:RIN'Illl:fdAPY ON I•.NI)U'1'I11:1.II1M-I)1?1'IiNt)I:N'I' ANI) INI)lil'lsNl)IiNT VASC(11.AR RIiI.AXAI'ION ANI) ('YC'I.IC GMP AC('IIMULA'I'ION IN RAT AORTA Va.cular relaxation by the organic (nitroglycerin) and inorganic (sodium nitru- prusaide) nilruva.udil:dun and Ihe endutheliuut-dcpendent va,oJtlaturs (acetylchulute and htxtanrincl has been associated with cyclic UMP accunrulatioxt. Tulerance tu vauudilatiun by nitroglycerin commonly occurs following pruiunged exlxo+urc lu niuu- glyccrin. "fhi. xtudy investigatc. the clTects uf in rivu nilruglyccrin therapy un vascular rclaxatiun and cyclic GMP accumulation induced by the nitruvasudilalur, and the cmlutheliwu depenilent vasualilalurs. Rats were injected with nitroglycerin ur the pru- pylene glycol Jilucnt cuntrul for 4-7 days. 'I'horacic aortas from the nrtrul;lyccrin- ucatcd rat% were 750-lidd less sensitive to the relaxant effcct+ uf nitroglycerin. In cuutraa, the.e aorta% were only threefuld less sensitive to thc relaxant cftccts uf ,udium nitropru..ide, while the maximum relaxation tu acetylcholine and hiaumine w•u, depressed by 5,0 and 41`k., respectively. IkMn,it'valiun tu relaxation was anux:i- ated with reduced cyclic GMP clcvatiunx loall the vasodilators kelaxatiun to R-hrumu cyclic GMI', dihalyryl cyclic AMI', or diltiai.em war unaffectcd by nitroglycerin therahy. 'I'uleranre was alsu atixtk iated with an incrc:r.ud unsitivity tu the cuntractile cllects uf low cuncentratiuns of nurcpincphrine. 'I'his increauJ scn.itivtry to nuru pinePhrinr w:rs aswciatcd with a decrease in cyclic GMI' levels. The present results suggest Ihat: (I) Jesemitizatiun to nitruglycerin, sixliutn nitrnrprusxide, acetylchuline, anJ histanrine by nitroglycerin therapy may txx at the level of cyclic GMP accumula- tiun; (2) cyclic (iMl' is thc conunlrn nreJiator of relaxation induced by the nitro- and cnduthcliwu-Jclkndcnt va.oJilaturx; (3) the mechanismx involved in the activation of guanylate cyclarc and relaxation by sodium nitroprusside, acetylcholine, aml hi,ta- tnine trre probably diltcrent than those of nitroglycerin; and (4) cyclic GMP may be acting a% a physiological negative feedback signal in agonist-inJuccJ contractiun. Mulina, C. R., Andresen. J. W., Rapoport, R. M., Waldman, S., and Mlurud, F. Journal of CarJiuvascular Pharmacology 10:371-37t{, 1987. Othcr support: National Institutes of I lealth and the Veterans Administration. From the Department of MeJicine, Stanlitrd University School ol' Medicine, StanfurJ, CA, and thc Veterans Administratiun Medical C'enter, Palo Alto, CA. OUAN'I•I'1'A7'ION AND ('HARAC"I'ERIZA'rlON OF HUMAN PLA'I'GUiT (;LYC'UPROTGIN Illa BY RAI)IC)IMMUNOASSAY (llycoprutcin (lia was quantitated in human platelets by raJiointmuniratisay using antisera specific to platelet numbranes and purified glycuprutein Illa. Glycuprutcin IIla and glycoPrutein llb were isulated, frwm washed platelets by Triton X-114 extrac- twn followed by preparative sudiutu dodecyl sull'ate-Iwlyacrylamide gcl electrophore- sis. KaJiuialinaterl glycupmtcin Illa was funher puriticd by affinity chrunratugraphy on Lentil Icctin-Sepharuxe 413. PuritieJ glycoprotein Ilb.howed little crossreactivity with '"I-IahelcJ glycuprotcin Illa using the anti-platelet membrane or anti-glycopru- tcin Illa antiura on a competition inhibition radioimmunoas,ay.'I'hc expression uf glycoprotein I lia epitupcs was the same titr the purified glycoprutein Illa and glycopro- tein I Ila in Triton X-I(lO sulubiliteJ platelets. A 66 kDa protein derived Irum glycopru- tcin 11I<t by limited pru(culysis uf platclct membranes also expressed the same eritupcs as intact glycuprutein llla. SolubilixeJ platelets contained approximately 16 µg of tul:rl glycuprutein Illa antigen per 10' cells. The level of glycuprotein Illa dctennineJ by rarliuimnwnuaxsay in one patient with Glanzmann's thrombaxthcnia amounteJ lu 6.71k, uf normal and it was close to the values obtained by uther methods. C'ierniewski, C. S., Niewiurowski, S., Ilershock, I)., Rucinski, B., and Schntaier, A. I1. Biuchimica et 13iuPhysica Acta 924:216-224, 1987. UOther support: National Institutes of Hcalth. Nrom the Dcparnnems of Physiology and Medicine, Thrombosis Research C'enter, Temple University I lealth Science Center, Philadelphia, and Department of Biophys- ics, Medical School of l_ixlx, Lrxlz, Poland. ('4 65
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MYOC'AkI)IAI. I3IOENIiRGGTIC'S STUI)IEl) WI'I'H "P NUCU?AR MAGNI:TIC RCsSONANCIi The rcccnt development of oP nuclear magnetic resonance (NMR) spectru,copy techniyues has provided the impetus fur new basic and clinical research. liven though the use ul' in vivo spectroscopy techniques to evaluate problems uf clinical cardiology is in its inl:mcy, a fair number of "1' NMR studies of perfused heart prcparatiun.% amd of antmul mudefs uf hearl disease have been cunducted. The goal uf these techniques is to study in vivo myrrcardi:d bii>Lnergetics in relation to mechanical function during vari- uus loading conditions and disease processes. Examples uf in vivo studies perfirmted in open- and closed-chest anintal uuxlels are presented, and the fx>tential for future development is addressed. l)sGukken, M., Young, M., Cluster, J., Iluddell, J., and Chance, B. lieart Failure: 149-155, Augusl/Scptcmbcr 1987. Other support: American Heart Association, Southeastern Pennsylvania Afiiliate, and '1'hc Mary Smith Lead Trust. From the I)epanmcntx of Anesthesia and Bioa:hemistry/Biophysics, Universiry of Penntiylvania, I'hiladelphia. CLINIC'AI. l1SliS OF NUCLEAR MAGNETIC RESONANCE SPIiCTROSCQPY IN CARDIOVASC'ULAR DISEASE Extensive research efforts are being expended to develop nuclear niagnetic reso- nance ( NMR ) spectroscopy into a mndality that can be used for in vivo characterization of bicx:hemical limctiun of the heart in health and disease. Information concerning changes of ntetalwlites under different physiologic and pathuphysinlogic conditions, relative change in mobility and interaction of molecules with metal (paramagnetic) ions, change in intracellular pti, and enzyme-mediated reaction rates can be rrbtained using NMR spectruscapy techniques. These techniques may be of unparalleled impor- tance in delineating in vivo myocardial biuchcmistry and physiology. O.qulche,,. M. American Journal uf Cardiac Imaging 1(3):242-253, July 1987. Other supprm: American Heart Asuxiation, Pennsylvania Affiliate, and The Mary Smith LA`alf'I'rutit. Frunr the lepartntents of Anesthesia and Biochemistry/Biuphysics, University of 1'ennsylvania, Philadelphia. I I i ('OMI'AF(ISON Of~ ATHEKOSCLIiFtO'fIC LIiSIONS ANI) I11)L: LIPID LtiV1iIS IN MALE, FEMALE, ANI)'I'ESTOSTIiRONL•=fI2EATla) I°BMAI.Ii MICE FROM STRAINS C57L3L/6, 13AL13/c, ANl) C3H Iu order to determine whether male and female mice dif lered in I IDL-fipid levels or in atherosclerotic response to a high fat diet, we examined 3 inbred strains which dilTerecl in susceptibility to atherosclerusis; C57BL./6, BALB/c, and C 3H. Mice were fecl normal chow ur an atherogenic diet containing 1.25% cholesterol, 15'%, fat, and (1.5°!r, cholic acid. l.esiun number and.iie were determined after 14 weeks on the diet; plasnui I IUt: lipid levels were determined at O and 4 weeks on the diet. For C311, the most athrrnsclerusis-resistant strain, HDI: lipid levels were very high and nut affected by scx or diet, For BALB/c,1iDL-lipid levels were intemtediate between 1he other two strains. Male levels were significantly higher than the females, and the atherugenic diet causcd a drop in HDL-lipid levels of 14-27°k+ depending on x:x. Fur('57131./6, the most atherusclerusis-susceptible strain, HDI- lipid levels were low compared to the other two strains. Males and females on normal chow did not diflcr in HDL-lipid, but Icmales showed a 59% decrease in HDL when fed the athrro- g,enic diet. For both BALB/c and C57BL/6, Icstosterane-treated females resembled Ihe males. 'I'he HDI: lipid levels in mice on atherogenic diet dif'fered over a 3-Rild range among the nine groups. When (-11)I: fipid levels were compared to the number of atherosclerotic lesions or the total lesion area, a high degree of correlation was ob- served (r --(1.95 for lesion numher and -(1.93 for total lesion area). This suggests HDL-lipid levels arc important in determining atherosclcrosis susceptibility in mice. /',ugen, B., Holmes, 1'. A., Mitchell, D., and Albee, D. Atherosclerosis 64:215-221,19ti7. Other support: California Affiliate of American Heart Association, National Heart, I.ung and Blood Institute and National Institutes of Health. Frum tht Bruce Lyon Memoriall Research Laboratory, Children's Hospital Medical Center, Oakland, CA. GliNli'fIC ANALYSIS OF MURINE STRAINS C57f3L/6J AND C3H/HeJ TO C'ON17RM'I'Hti MAI' POSI'I'ION QFAth-l, A G6NE DGTIiRMINING ATIIfiROS('LI'sROSIS SLISCEPTII3ILITY Previous results suggested that strains C57BL/6J and C3H/IIeJ dif7ered in a sinEle gene li,r atherosclerosis susceptibility, called Arii-l. Ba.u:d on data from rcconr hinant inbred strainx Ath-I was tentatively assigned to chromtxu,me I linked tu Alp-2. In this report, a cross between C'57111./6 and C3H/HeJ was carried uut in order to test whether the tentative map position was correct. Parental strains and F, and K progeny were examined. Susceptible alleles of Arh-I , found in C5761./6, are associated with relatively low levels of high-density lipuprntein (IIUL)-cholesterol in animals led an athcrogenic diet; resistant alleles of Ath-I are associated with relatively high levels u/' I11)L-chuleacrul. F, progeny have HI)L levels that are intennediate between these of the two parental strains. Among the F, progeny, Alp-2 and Arh-1 cosu:grcgatcd, provid- ing confirmatury evidence that Adt-I is linked to Alp-2 on chromosome I. Three mice recombinant forAlp-2 and Ath-I were found among the 60chrumosomes tested, giving 66 - 1 67
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an c.nmatccl nrap clitilance Ix:twcen these two gcncs uf 5.111 2.11 (Sli) cM. "1'he pltcnutypir charactcrititic. ol'Ar/r-! resemble a genetic trait in humans, hyperalphalilw- prulctncruia, which is clr,rractcrv.ed by elevated levels of Ht)I.-chu6crtcrol• reduced risk of heart disease. and increased longevity. I'ai•qrrr, B., Allxx, I)., Ilulmes, P. A., and Mitchell, I). Biurhemical Genetics 25(7/8):501-511, 1987. OIher.uppurt: National I leart, Lung and Blood Institute and American I leart A,+ucia- liun. From the Bruce I.ynn Memorial Research l.atxrratory, C hildren'. I hnpil;tl Medical ('enter, Oakland, ('A. nrh-l, A(iliNli I)I~ftiRMINING ATHEROSCLEROSIS SUSCGf'"I'IBILITY ANI) IIIGH L)ENSI'I'Y I.II')PRUTI:IN LEVIiI S IN MICE I ligh elensity lipoprotein (111)I.) is the majur plasntu lipoprotein fuund in mice fed normal laboratory chow containing 4'k. fat. When fcmale nrice from some inbred titrains, such as ('571i1./(r, are fetl a high fal diel ( I?5%choleaterul, 15% ful, and f1.5'7v chulic acid). the levels uf HUI.-chuletitcrul decrease by about 50%, and lipid staining le.iuns furm in the aorta within 14 weeks. In other strains of mice, such as C.i11 and IiALB/c, IIUI.-IipiJ levels decrease only slightly, and few or no aortic lesions are ubticrvcJ at 14 weckti. 'Phr genctir basis uf these phenotypic differences was analyied by using recombinant inbred strains derived I'rum C'5713L/h and BALB/c and also from C'571iL/6 and ('311/He. The Iwu phenotypes segregated as simple Mendeli:rn traits, ancl no rccomhinatiun was observed between thcm. Thus, III)L-clwlesterol levels and susceptibility lu atherosclerosis appear tu be determineJ by the ,ame genc (or by two closely linked genetic facturs that are a maxinutm of 1.7 centimorganx apart).'I'hiti gene was named Adrl, fur atherti,clerosis susceptibility, with allcles r for resiaance and.c lirr susceptibility. Adrl maps on chromosome I nearAlp-?, a gene thal determine+ the structure uf aprdiprrprutein A-11, one of the two majur proteins forurd in I11)L. Arh-I is clearly separable from Alp-J, anJ the distance between these genes is 6 0 crntinrurgans with a standard error of d.2 centinuirgans. In humans, levels of 1)I)L are inheritcd anJ are inversely correlated with atherosclerosis; familial hyperalphalilw- pruleinrnria is associated with high levels of HUL-cholesterul and decreased risk of heart disease. The human trait phenotypically resembles Adr-t in the mouse. Prn,4en• B., Mitchell, I)., Reue, K., Morruw, A., Lusis, A. J., and Lelicxuf, R. ('. 1'ruceedings uf the National Academy uf Sciences, USA 84:3743-3767, June 19H7. Othrr.uppurt: National I Ican, Lung and Blood Institute and American ilcan Aszokia- tiun. Prunr the Bruce I.yun Memorial Research Latwratory, Children's Huspilal Medical C'cnter, OakLmd, ('A; I)ep;rrtnienls of Medicine and Micrubiulugy, lJniversity ul' California, Los Angeles; and Veteran's Administration tluspitnl, LArs Angeles. I'LA'1'lil.l l'-A("I'IVA'1'IN(i FACI'OIZ IilTli("I:ti ON BOVINE PULMONARY ARTERY IiNI)(YI'I IL•LIAL ('G.LS I?ndrhlheluil cell. (Ii('s) were isolated frutn bovine puln.uurary artery and m;rin- laincd in Iun);-trnn culture. On reaching cunlluenry, 9:C. formed a characteristic '.cuhhlr,tunr" munulayer. One hour ,dtcr addition uf I nM platclcl-activating I;rctur 11'AH tu Ihe growth mcdium, li('s underwent dramatic changcs in shape I'rum their normal polygonal morphology to nrUre elongated spindle-shaped lirrm.. More pro- nounced rffcrlti were evident in the presence uf 0.1 nM phorhyrl-13-nryristale-Li- acctatc t I'MA). u putrnt artivaturufC kinase. It was lirund thal ;rt cuncentraliuns front I(1 "- II) M, I'A1= ainwlate.lhc phusphcrinusilidc lurnuvcr in I:C'.'I'hc half-maxim;d activatiun in Ihe release of inusitul phu.phates was at 10 " M. "fhiti finding suggested Ihat an increase in inlracrllular ('a" concentration and aclivatiun of pttilein kinase (' were invulveJ in the mechani,m ol' ac•tion uf 1'AF rrn EC. "1'he nutalxdic responses uf li(' wcrr rvaluated hy measuring the activily of ji-aJrcncrgic receptor-coupleJ adeny- late eycla.e (A(') in a crudc membrane fraction and by assay of prtr+tucyclin and Ihrumh+rx;ute released by cullured IiC. AC from control membranes was activated by isuprolrrriwl in a Ju,e-rlelknJent nranrrer (l?C',,= 30 nM) I'rom 0.R-5.5 pmol cAMP/ nrin/nrg protein. If thc mcmhranes were isolated after preincubation of ECs with I nM I'Af: or 0.1 nM 1'MA, Ihe A(' uctivity wati decreased by 70 ancl 9O'!•, reslxxtivcly; in both cures, adlinity lur i.uprntercnol was lowered threefold (EC„= I(NI nM). Our data suggest that PAF interaction with I:C leads tu an apparent /3-aJrenergic receptor Je.rtn,itiiatiun that probably acts via a phosphorylation mechanism involving (' ki- nase. Incubatiun of 1:(' lirr 30 minutes with 0.1-1.(t nM PAFcuu.cd inhibition of buth prostaryclin and Ihrundxrxanc production (55 and 75%, respectively) inclicating that I'AF acls at a level cuurmun to both pathways uf arachidonate metabolism. Similar results were obtained using I'MA (0.1 nM) hul not with phurlwl-12,13-Jtderanoate, an inactive analogue ul' PMA. Taken together, these data indicate that C kinase is in- volved in PAF-induced alteratiun in receptur.en.ilivily at Ihc plasnra ntemhrane level a% well as in intracellular eniymcs responsible for prostacyclin anJ thrumhux;ure .ynlhe.is by E('. "I'hi% down-regulation of ntetahulic aclivity ul' li(' is arcunrpanieJ by cuncumrlanl shape ch;mgcx. Grigorian, (i. Y. and HY(tir, (/. S. ('irculaliun Research 61(3):32i9--i95, Septemlxr 1987. Othcr support: National Ileart, l.ung and Blood Institute anel National In.titute,, ul' I leahlt. Frum the I)eparlnunt uf MeJirine, University uf Miami School ol'Medicine, Miami, 1•1., and lhc Cardiology Rr.carrh•Cruter of lhr IISSk, Moscow. AC"I'IVI'I'II:S O1' 5-Mli'I'IIYI.I-'IIKI-'III{YI:1'RIMIi'l'IIYLAMMONIIIM IUI)11)I; ANI) KI•.LATI•.1) ('UMI'UIINI)S AS VAS('l1LAR IiNDOllI1:LIAI. MIIS('ARINI(' RIiC'IiPTORS O1='I'H): RA'I' AORTA 5 Mrlh)•lfurfitryllrinrethylamnumitun iodide (5-MI'f) is a hrran analug uf nru.- cartnr and wa. studied for its rhulinrrgic urtivity at va.cular cnduthtiltal receptors ul' the rat aurta. Other related compounds have diflcrent tiubstitutiun, :n pusitiun 5 o/ tlre furan ring and include the following compounds: 5-hyJruxy-methyl-(5-HMPI'), 5- (rg 69
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chluromethyl-lS-C MFI'), 5-hromumcthyl- ( 5-13Mrf), 5-icrdhrmcthyl- ( 5-IMFI'), and 5-inethoxy- (5-MUI'f) lurluryltrimethylaumumium salts. '1'he furan analogs relaxed helical strips ul-rat aurta which contracted with norepinephrine (1(/" M). 'I'he.e relaxa- tiunx were endothelial cell-depr:nJent.'1'he (iD„'s lix muscarinic activiltes incrr:rtied in the li0lluwint; urder: 5-MFI' = ACh < d/-musc:arine < 5-IIMI'f = 5-('Ml'f '- 5- MOt'I' •: 5-13MhT •-_ 5-IMh`I'. Amung the luran analugs, 5-MNI' was found to he a liull agonist at the cnduthelial cell.; other luran analogs were only partial af.uni,ts.'I'he afttnnir, and relative intrimic eflicacies uf the most potent analogs decreased in Ihc liolluwuig order: A('h = 5-MI'f > d!-nwuarine > 5-IIMFI' = 5-('MFI'. Atropine and scopolamine antagonized relaxations by furan analogs. K„ values for atropine and scopolamine against ACh, 5-MhT or 5-HMI-T as agonist were nut rhffcrent, indicating that these agonists and antagonists were acting at the same mus- carinic recclrtur.. Thc K,, uf atropine and of uulxilannine increased when 5-CMt-Twas used as an agonist, indicating that 5-CMIT may cause relaxation by acting at other sites besides endothelial muuarinic recepturs. The endothelial nw.carinic receptor might he classified tentatively as of M, or M, type. These studies did not exclude the possible heterogeneity uf the endothelial muscarinic receptors. Ilorst, M. A., Susrrv, B. V. R. and Landon. E. J. Archives internalionales de Pharmacudynamie et de Therapie 282i(1):R7-99, July 1987. Other support: Natiunal Institutes uf I lealth. From the I)epartment uf Phannacolugy, School uf Medicine, Vanderbilt University, Na.hville, 'I'N. SYN'fl)1;1'I(' I11(ilt I)I'-NSI'1'Y I-IPUPftU'I'1?IN PARTIC'LIiS- AI'I'LI('A'I'IUN'I'O S'I'(11)IIiS OF'I'Uli AI'OPROTI:IN SPEC'IPICITY FOR SI:LECI'IVIi UI''I'AKIi OF ('I IULI?S'I'IiRUI. GS'I'NRS* Particles clauly resembling rat high density lilx}protcim (11DI.) in terms of eyuilihrnim den.ity profile and particle size were pacpared by sonicatiun of apuA-I with a micnxxntul.iun made with egg lecithin anJ cholesterol olcate. These p:aticlcti, like authentic 111)L, allowed srlcctive uptake uf their cholesterol ester nruirties by cultured cells without parallel uptake uf the particle itsLlf. That uptake was saturable and competed by I IUI.. In rats, the plasma decay kinetics and sites uf uptake of a- chulesteryl ether tracer were similar whether that tracer was incorporated into synthetic or authentic 111)L. Synthetic particlcs cuntaining other alxrproteins were made by generally tiu tiame method, but using in place uf a1wA-I either a mixture ul' rat alw('ti or apoE that was either competent or reductively methylated to prevent interaction with the Ii/li rrcep- tur. 'I-heu panicles, of lower density and larger Stukes radius than those made with apuA-I, also alluwed selective uptake of cholesterol esters, albeit with a lower degtce of selectivity than in the case uf apiA-L Thus a specific alwprutcin cunqwnent in Ihe subject lipoprotein particle is not required fur selective uptake. Iluwevcr, selective uptake was ,huwn tu tx a func(ion uf particle density or siie, and part uf the Jil Ierence in rates uf selective uptake from the particles made with various alwproteins was explained by their differences in density or sit.c. I'tttnum, R. C'., Glass, C. K.. Atkinurn, 1)., and Small, 1). M, (Srrlnhr),q. l).) The Journal uf Itiulugical C'iumiary 2G2(6):24.i5-2d-12, February 25, 1987. Othrr suppurt: National Ile:u-t, I.ung and Blood Intitittue. From thr I)ep:utmrnt of Medicine. University ufC'ahlornia, San 1)ieEu, Lo Jolla, and the Biophysics Institute, Boston University School of Medicine, Boston. A NONtiNl1O('Y'fO'I'I(' MI:C'IIANISM FOIt'I'IiG SI:LIiC'1'IVIi l)I'TAKI? OF 111(i11 UIiNSI'I'Y L11't)1'kO'1'IiIN-ASSU('IAT1il) C'IIOLFSI'liltOL 1:S'IliKS We have previously dcurilkJ in rats the selective uptake of HI)l, a,siwiateJ chobraerul esters (traced by 1'If1chulestcryl oleyl ether) in excess uf the uptake ul' 111)1: asuk ia1cJ apuA-I. In the present studies we show that the mechanism also exists in cultured cells uf human and ntouse origin as well.'fhis selective uptake represents a net uptake of chulesterul esters and not an isotope exchange, as shown by mass flux Studies in adrenal cells. Inhibitors uf receptor recycling, chlunryuine, uroncnsin, and colchicine, inhibited uplake uf alanA-I from I IUL by I lcp G-2 human hepatoma cells to about the tiamr extent as a reference protein, asialu-fetuin, hut inhibited uptake uf the chuletiteryl ether tracer much less. Levels uf NaN, which effectively inhibited sucrose pmucytn.is inhibited uptake uf alxrA-I tu about the same extent but diJ not inhibit uptake of the chulestcryl ether at all. Thus, rnat only receptur recycling, but cndocytusis a. well, appears not tu be involved in selective uptake. 'fhis conclusion was xuppttteJ by studies in which synthetic I II)1. particles were made tu contain twu nrutral lipid core Iracers; une of them, the I')lIrhulesteryl ether previously used, was selectively taken up, whereas the uthrr, I 'K'lsucrose oktaulcate, was excluded I'rum selective uptake. 'fhu., selective uptake cannot involve enJix:ytumti uf the entire lipid core, hut may involve other specific u-an.fer mechanixm.. I'iuni:m, R. (,'., Knrcht,'I'. P., Rusenhaum, M. S., and'1'aylur, C. A. Jr. (S7rinHerg. I).) "1'he Journal of t)iulugical Chemistry 262(b):2443-2451), February 25, 1987. Othcr suplwrt: National Ileart, Lung and Blood Institute. Frum the Department uf Mcdicine, University ol'Califittnia, San I)iego, La Jolla, ('A. Mli('IIANISM OF ACI'IVA'1'IUN QF COAGULATION FACI'UR XI BY FACTOI2 XIIA S"1'UI)IG) WITII M(JN(X'LONAL ANTIBOI)IES The interaction nf h'actur XIIa with Factor XI was investigated using Iwo nrunu- clunal antihixliea, one (1C1) directed against the heavy chain of Factor XIa and the 70 71
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uthrr (5I-4) a(;aiml its light chain. 3('I either as intact Ig(i or as fcdh' fra);nicnt, enhanced Ihc rate of I actur Xla gencratiun in the fluid phase bul inhibited it in Ihc Irrcscnce of kaolin and high tnulecul:ur weight (IIM W) kininogen. In cuntraa, thr Fah' Ira(auents of 51`d iuluhilcJ only the Iluid phase activation and had no eflcct on the .urlacc-mrJiatcd acnvatiun. 3C'I wa.r IiounJ tu Muck thc binJing of Factur XI tu I(M W kinmuEcn, whereas 5F-3 did not. We cuncluJc: (a) a domain on the heavy chain region uf Factor XI ts essential for binding to hIMW kininogen and for uplimal xurl'acc- mcJiaucd activahun by Factor Xlla; and (h) binding of 3C'I to Iactur Xl changes ils conformation rendering it a more favorable substrate fur Factor Xlla in the Iluid phase. Akiyama, II., Sinha, I)., Seaman, F. S., Kirby, E. P., and Walsh. 1'. N. Journal of Clinical Investigation 78:1631-Ib37, 1)ecemtx:r 1986. Other tiupport: Natiunal Institutes uf Health, the W. W. Smith C haritahlc Trust and the American Ilcarl As,uciatiun, Pennsylvania Af7iliatc. From Ihe 1)epartmrnts uf MeJicine and liioxhcmistry,'fhrumMosis Research Centcr, Tcmple Univer.ity School uf Medicine, Philadelphia. ROLI: OFC'ALC ItJM IONS ANI) THl3 til?AVY CHAIN OF FACTOR XIA IN TIII: AC"fIVATION OF IiUMAN COAGULATION FACTOR IX Since optimal rates of I'actur IX activation by factor XIa require the presence of calcium ions anil the heavy chain of the cniyme as well as the active-sile-containing light chain, we have studied the effccts uf calciunt ions and the heavy chain utt the reaction kinetics. Whereas the amidulytic activitics ol'1'actor Xla and of its activc-sitc- cuntaininR light chain were alnto.t indistinguishable, the two cnzymc+ behaved yuite differently when factor IX was the substrate. Factor Xla was I(K)-fulJ more potent in lhc presence of C'a" than in ils absence. On the contrary, the presence or abuncc of C'a" ntaik very little dil7crcnce in the case of the isolated light chain uf factor Xhi. Moreover, the rnrymatic activity of the light chain was almost identical with that of uuact factor Xla when ('a" was ahsant. Using an optimal cuncenlratiun uf ('a" , we %tudicJ lhe activation in the pretience of various cuncentrations ul' (wu nwnuclunal auuihudic+, one (5F4) directcd against tlte light chain of factor XIa and the other (3('I) agaimt its heavy chain. Analysisuf I/V I•s. I/Srluis.huwed that whereas inhibition by SFd was noncoml>Ltitive, 3CI neutr.dizeJ the cnzyme in a classical competitive lash- ion. We conclude that in Ihe calcium-dclaendent activation of factor IX by factor XIa the heavy chain uf the cnzyme is involved in the binding o1' thc substrate anJ this is essential for uptimal reaction rates. Sinha. I)., Scanian, F. S. and Wulsh, P. N. Biochemistry 26:3762i-3775, 1987. Other suplwre U.S. I)cpanmcnt of Ilcalth and Human Scrvices; thc W. W. Smith ('haritable Trusr American Heart Association, Pennsylvania Aftiliatc; and National In.titutes of Ilcalth. From thc Ikparlments of MeJicine and Biochemistry, Thromlw,is Research Center, '1'emple Univertiity School uf MeJicine, Philadelphia. IV. Neuropharmacology and Physiology Tllli ROLE OF MAS I' CEI.LS IN INFLAMMATORY PRCK'EiSSES: EVIDENCE FOR NIiRVI?/MAS'1-('IiLL INTI:RACIIQNS In the rat, therc is considerable evidence of mast cell/nerve interactiun twth in the nunnal and infcctcd intcainc. Between 67 and 87% of all ntas-t cells in the intestinal lamina propria of rat. infected 22-35 days earlier with Nippustrun,4vlus brasiliensis were touching ncrves.'fhetie mcmbrane cuntacts wcre between subepithelial mast cells and nanmyclinatcd nerves cuntaining substance P, calcitonin gene-related Ixptidc and ncurone sIx:cific cnolase. 2.5S nerve growth factur (NGF) has a significant rnhance- ment cllcct on antigen-induced histamine release without addhiun of phosphati- dyl,erinc, and the iit viru administration of NGF to rat% causes Mnh connective tissuc :mJ mucu4al mast cells tuJramatically increase in numlx:r. All of thesc effects are both dose Jclxndent and NGF specific, as evidenced by inhibition with anti-NGF. 2.55 N(iF alw causes in vitro increase uf colunics in methylcellutux: cultureti of human Pcriphcral blood. The eflccts of NGF in this system arc synergistic with other T ccll- Jerived growth 1'acturs and relatively specific firr mctachrumatic cell growth. l'heu observations ,uphurt Ihe conclusions that nerves and mast cells may cun- stantly cunrmunicate and provide a structural and conceptual framework whereby the ccntral nervous SyxtCm may communicate with inllammatury events. Liirncnaurk, J., Tumioka, M„ Matsuda, I1., Stead, R. Ii., Quinoncz, G., Simon, G. '1'., ('oughlin, M. I)., and Ihnburg, J. A. (GrrrdJir•, J.). International Archives of Allergy and Applied Intmunulogy 82:2311-243, 1987. UOther suppurt: McJical Research Council of Canada. Fruiu thc Ilu,t Resislancc Program and 1)epartmcnts uf Pathology and Medicine, McMastcr University, Hamil7un, Ontario, Canada. MASf ('IiLL INVOLVEMENT IN VARIOUS INFI.AMMATOkY PkU('IiSSI'sS Mast cells frum dif fcrcnt ti.suc sites may have dil7crent histr+chcmicd, chemical, and functiunal propenics. Whatever the basis for these differences, they mutit be impunant in trrnis of their biologic significance. Through their mediators, ma:,t cells arc involved in utany diffcrent actde and chronic inflwumatury processes. They act in delayed hypersensitivity, inuuediatc hyperunsitivity, :md in granulumatuus reactions. 'I'hey can influence phagircyluai.. chenwkinesix, and many aspects of immune activity in several dilTercnt "1' anJ 13 cell pathways, to mention only a fcw el'fects. Matit cells are invulved in repair pnkc.scs including fibroblast tirnctiun and fibrosis. 'I'heir growth may he influenccd by T cell-dcrived factors as well as factors derived f'rum lhc epithe- lirun.'fhey appear tu he inlimately involvcYd with nrrves and can form apparent cunt- municaliuns with ncurunes, especially thou containing Substance P, which causes all types uf nw.t cell% to degranulate. Mast cells may therefore act as central switchboards between the central nervous .ystent and migrating and sessile cell types in inflannna- tury processes. 72 73
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a Bienenstock, J., Tomioka, M., Stead, R., Ernst, P., Jordana, M., Gn«ldie, J., Dolovich, J., and Denburg, J. , Ameri~an Review of Respiratory Disease 135:S5-S8, 1987. Other s~pport: Medical Research Council of Canada and Fison's Pharmaceuticals, England. From If{e Departments of Pathology and Medicine and the Molecular Virology and Immunology Program, McMaster University, Hamilton, Ontario, Canada. DEVELOP1v1ENT OF NEURAL. RECEPTORS FOR SEROTONIN IN T}lli MUR'IN13 BOWEL During the ontogeny of the crtterie nervous system, the varicosit.ies of' maturc neurons contact dividing neural precursors, which persist in the developing niurine gut for several weeks postnatally. This phenomenon has led to the hypothesis that the release of transmitter from mature neurons may influence the subsequent development of uncommitted neuroblasts. In order to test this hypothesis, it is necessary to know the timing of the expression of postsynaptic receptors for enteric neurotransmitters. Since serotoninergic neurons are among the earliest of enteric neurons to develop (appearing on day EI2 of development in the mouse), the ontogeny of enteric neuraV serotonin receptors (5-HTR) was studied. These receptors have previously been characterized in adult guinea pigs and rabbits. In the current experiments, 5-I-ITR were identified in the adult murine howel; their properties were compared with the 5-HTR of guinea pig and rabbits; their ontogeny was followed throughout the length of the developing mouse gut; arld'the properties,of 5-HTR in the developing murine bowel werecoimpared with those Nf the mature gutt. The 5-HTR were assayed by measuring the binding of 3H- serotonin ('H-S-HT) to isolated enteric membranes by rapid filtration, and to frozen sections of bowel by radioautography. A single saturable, high at'finity'H-5-14T bind- ing site was found in membranes from the adult mouse gut (K„ = 3.9 ± 0•5 nM; B,„„ = 1.6 ± 0.3 pmoles/mg protein), Binding of'H-5-HT at this site was not antagonized by compounds known to be antagonists at receptors for other neurotransmitters or at the 5- ~ HT, or 5-HT, class of CNS 5-HTR• Hydroxylation of the indole ring ot' anak>gues of ._I serotortin was required f'or affinity at the enteric''H-S-HT binding site. B inding of `H-5- HT wa; inhibited by N-acetyl-5-hydroxytryptophyl-5-hyroxytryptophan dipeptide, a compound that has been dcmonstrated to antagonize those responses of myenteric neurons to serotonin that resemble slow excitatory postsynaptic potentials, but not by ~ ICS 205-930 (Sandoz), a serotonin antagonist that does not block these responses. All o('these properties ofadult murine'1•I-S-HT bindirtl: sites are virttnrily identicai o1'tlto:;e offm of guinea pigs and rabbits, which have previously been shown to by 54l'fR; therefore, I41+ nturine enteric 'H-5-1dT binding sites are probably 5-I-1TR ux wcll. Thc 54ITR were found radioatnugraphically in both enteric plexuses and in the mucosa ol'all regionv of' 0 the adult nwrine bowel with the exception of' Ihe fundus (rumen) of' th'e stomach, ~~,, During ontogeny the binding of''hl-S-HT, eva,4uated radioaut<~~grapltic'ally, appeared +~+' for the first time on dav E14, whcn it wns found in the stomach and small intestine i'rom j,a the pylorus to the level ot' the mid-jejunum. 'fhe 'H-S-I l'!' binding sites Ihen sprcad proximodistally, reaching the ilcocolic sphincter on day EI5, the proxinwl colon on r~ days Ei6-18, and the distal colon on day P2• The acquisition of '1-1-5-1-IT hinding sites W by the rectum occurred entirely postnatally and was not comple-te until duy P22. The 74 t l I , properties of the 'H-5-HT binding sites of developing animals were not distinguishable from those of adults, and thus were considered to be 5-HTR. Additional'H-5-HT binding sites were found in the perianal skin of adult mice. The characteristics of'H-5- HT binding sites to these cutaneous sites were similar to those of the gut, which suggests that there are 5-HTR of the enteric type in the skin. These observations indicate that enteric neural 5-HTR make their appearance later in development than do serotoninergic neurons; however, 5-HTR are present during the period when prolifer- ating neural precursors are present in the enteric nervous system. It seems unlikely, therefore, that 5-HTR on precursors are important in determin- ing the scrotoninergic phenotype. Yet, the observations are consistent with the hypoth- esis that serotoninergic neurons iniluence the development of other types of neuron that sequentially follow the appearance of sercitoninergic neurons in development. Branchek, T, A, and-Ger,r/ton, M. D. The Journal of Comparrative Neurology 285:597-610, 1987. Other support: Pharmaceutical Manufacturers Association Foundation. From the Department of Anatomy and Cell Biology, Col lege of Physicians & Surgeons of Coiumbia University, New York• ANALYSIS OF PROLACTIN AND GROWTH HORMONE PRODUCTION IN HYPERPLASTIC AND NEOPLASTIC RAT PITUITARY TISSUES BY THE . HEMOLYTIC PLAQUE ASSAY ' " The reverse hemoPytic plaque assay (RHPA) was used to detect hormone release from cultured normal, hyperplastic, and neoplastie rat pituitary cells. Hyperplastic pituitary cells were produced by s.c. diethylstilbestro) (DES) treatment (10 mg in Silastic tubes) for3, 6, and 9 weeks. Neoplastic pituitary cells from rats with MtT/W15 transplantable tumors treated with DES for3 weeks were also analyzed. Aliquots of the same cells were also analyzed by immunocytochemical staining. DES treatment re- sulted in an increase in prolactin (PRL)-producing cells in hyperplastic pituitaries compared to untreated pituitaries after 9 weeks of treatment by the RHPA 161.2± 5.2 (SE) vcrsus 32±3.0i and by immunocytochemical staining i70.9+2.4 versus 36 ± 1.4J,. The percentage of mamrnosomatotropic cells decreased from 11.3 ± 3.8 to 4.2 ± 2.6% in pituitary cells from these same groups of animals, After 3 weeks of DES treatment in rats with MtT/ W 15 tumor, there was an increase in growth hormone (GH)- producing cells and a decrease in PRL-producing cells when analyzed by the RHPA (control: percentage of G1-1, 36.3 ± 6.2; percentage of PRL, 39.0 ± 1.6 versus DES- treated tumors: percentage of GH of 68.2 ± 1.9; and percentage of PRL, 3.2 ±1.8%). 1'he percentage of mammosomutotropic cells declined from 12.4 ± 2.3 to 0.77 ± 2.4~'la. A combined procedure of RHPA followed by immunocytochemicaPstaining on the same slides also revealed a decline in mammosomatotropic cells afterchronic DES treatment in hyperplastic and neoplastic MtT/W 15 tumor cells. These results show that DES has dilTerent effects on PRL and GH secretion and storage in hyperplastic pitui- tary and in the MtT/W15 pituitary tumor cells. Llo.vd, R. V., Coleman, K., Fields, K., and Nath, V. Cancer Research 47(4):1087-1092, February 15, 1987. 75 4
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~ ~ Other %uppurt: National Institutes uf I lealth. I•rotn the Ihpartnrcnt of Pathology, University of Michigan Medical ('enter, Ann Arbor. SIiNSORY NIiRVI?S C'ONTAINING TACIiYKININS AND CGRP IN '1'IIG LOWER AIRWAYS-FUNCTIONAI. IMPLICATIONS FOR IiRUN('l IUC'ONS'I'RIC"flON, VASODILA'CATION AND I'R(yf GIN IiX'1'RAVASATIUN. An inve-,tigatiun ul' the distribution and origin of tachykinin- and C'GRP-L.I re- vc:ded thut SI', NKA and NPK are colocalized with C'CiRP-LI in nerve fibers supplying bronchial smoxHh muscle, close to and within the lining epithelium, around blood vesselti unJ aruunJ local trucheobrunchial ganglion cells in animals and man. SP-IR and C'(7RP-1R were also present in the same cells of jugular, norlose and thoracic spinal ganglia, hut only in nerve libers in local parasympathetic ganglia and the stellate ganglia. suggesting a sensory origin. CGRI'-IR, hut not SP-LI, was also present in cpithelial endocrine cells. A C'u" -dependent release uf tachykinin-LI and C'GRP-LI was demonstrateJ upon Ihrlusiun of the guinea-pig lung with K' as well as capsaicin. Othersuhstances such as hradykinin, hislaminc and the nicotinic receptor agonist DMPP were also able to induce uvertluw, suggesting the release uf Ixoth SP-Ll and NKA-LI, The material released up+m stimulatiun with capsaicin co-cluteJ with HLI: on HI'LC, anJ a unaller peak eluteJ in the position uf NKA, but no cleareviJence for the release of material cu- cluting with NI'K was observed. This suggests that SP and NKA are the main tachy- kinins released front sensory nerves, while the nature of the GLG-LI remains tu be further clarified. Significant differences in pharmucukinctical properties of the various tachykinins make it Jifficull tu draw major conclusions about tachykinin receptor subtypes based un hiulogicallwtenliesin riru. SI', NKA anil NPK were fuunJ to exert bronchoconstrictor effects irr vivu as well as in vitro. NKA was the most potent and SP the least potent uf the three tachykinins.' 'fhe ilt rirrn data support thc presence of SP-E/NK-A receptors in bronchial snumth muulr ul both thc guinea pig and man. C'GRP had no effect un bronchial smooth nwscle, neqher in rirn nor in wrro. Substances known to aclivale sensury C'-libers, such as cupsairin and rther, caused brunchue:unstriction in normal guinea pigs hut nul in guinca higs prelrc:ned with capsaicin. Funhermore, thr histamine-induced hrunchu- cunstrictiun was rcduccJ alier capsaicin treatment, suggesting a rclatiunship between tachykinins released frum sensory nerves and btunchoKonstriction induced by these agcnts. ll,ing laser I)uppler Iluwmetry, it was shown that vagal nerve stinudatiun in atrupiuve.l c:us causrJ :m atrnpine-resistant tracheal vasudilatation, pan of which was atill present :dtrr tre;dmcnt with the ganglinnic blocking agent, chlurisundaminr. ('apsaicin aeru.ul and topically applied C(iRP also increased thc tracheal blood flow. hi ruro studies on isulaled pulmonary arteries suggest that tachykinins and CCiR1' were eyuiputent to relax vascular smuoth muscle, indicating a tachykinin action via SI'-Pl NK-I' rrcchlur.. 'Ihe protein exuavasauun in the rat airways seen upon exposure to cigarette smuke was dependent un vapor phase cumlwnenls uf the smoke but not on the nicotine. 76 'I'hts efl'rct was atwlisheil in animcds pretrcated with capsaiein or by i.v. administratiim of SI' an;tlul!ues with tachykinin-anlat;onistic Irrulx:nies, suggesting thal Iachykinin release from scmory nerves is u prerequisite fur thc extravaxatiun uf marrunwlecules in (lie airway mucasa. Acid and gastric juice also caused protein extravasu6un in the lower airways. "I'he resulls suggested that acid-induced airway edema involved hinh sensory nerves and other mechanisntx dePcnJing om the pll. 11 is concluded that population oI sensory nerves in the lower airways can he visualveil due tu their content uf slxcific peptides (tachykinins and C(;RP) and cla.- sified as helunging tu the C-lilxrr group due to thcir sensitivity to capsaicin. "I'hexe Ixptide-cuntamin); cupsaicin-unsitive sensory nerves are activated by irritant stimtdi such as cigarettc smoke, ether anJ aciJ as well as by histantine and bradykinin iniliat- ing huth central reflexes and local release of SP, NKA and CGRP.'1'hese lx:ptides then inleract with specific receptors on target tissues leading tu vasoclilatatirnt, protein extravasation and hrunchorunstriction. Martling, C. R. U.wi(lhrrg, J. ) Acta Physiologica ScanJinavica 130(563):I-57,19R7. Other support: Swedish Medical Research Council, Swedish Tobacco ('umrany, I'etrus and Augusta LIeJIunJs huunJatiun, Swedish Society of MeJicine, Swedish Work I?nvirunment Fund, and the Karolinska Institute. Frum the Department uf Pharmacology, Karolinska Institute, Stockholm, Sweden. NEUROKININ A IN ('APSAI('IN-SIiNSiTIVG NEURONS OF Tllli GUINEA-PIG INFERIOR MESL?NTLiRIC GANGI.IA: AN AL)I)ITIONAL I'L1TA'I'IVIi ML:UTAI'OR FOR TLLG NUN-C:IIULINIiRGIC EXCITATORY POSTSYNAP1'IC POTLiN'fIAI. Tlu presence of ncurukinin-A-like inmtunurcaclivity in guincr-pig inferior roe- unlcric ganglia was detected by radiuimmunoassay procedures. 1'retreating Ihe ani- mals with capsaicin 7 dayx prior to cxpcrimentutiuns reduced the mcan content of neurokinin-A-like immunurcactivity by 85%, frum its control value of 15Qt 31.3 fnwl per ganglion. Iligh-Lxrliirmance liquid chromalography revealed that neurukinin-A- like immunureactivity was heterogenous as in addition to neurukinin A, peaks correx- punding to the amphibian tachykinin eleJuisin and to neuropeptide K were dctected, .md they tuu were depleted by capsaicin. Iaectrophysiulugical studies showed that ncurukinin A applied either by superfusiun or by pressure ejection evoked a slow depodariratiun in the majority uf inferior mesenterie ganglia neurons in <vrro. Ncuru- kinin-A-evukcd dcpularvations in the majority of celis tested were assix:iated with a small increasc in membrane input resistance. Ilowever, Ihc responses were increased by numhrane hyperpuLarizatiun; the extrapolatcd mcan eyuilibrium potential of ncuru- kinin-A-induced Jelwlari•r.atiun was-36 mV. Removal of extracellular suJiwn hut nut chluride iuns suppressed the ncurukinin-A-inJuceJ delxdarizatiun. 'I'he sluw dclwlar- iiatiun elicited either by exogenously applied substance P or by repetitive stinmlatiun of hypogastric nerves was reversibly eliminated in the presence of neurukinin A. (bllectively, our studies suggest that ncurukinin-A-likc immruwrcactivity may coexist with substance-P like immunureaclivity in capsaicin :uxnsitivc filer% in the guinea-pig prevertebral ganglia and that the similarity uf ihe actions uf ncurokinin A on the one hand and substance 1' on the other raises the Ixnsibility that nun-chulincrgir 77
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excitatory potentials elicited in the inferiur meseuteric ganglia may he generated by not irne hut a numlxr of closely related tachykinins. Saria, A., Ma, R. C., Dun, N. J.,'1'heoJorsson-Norheim, 1:. andl.unulGerfi, J. M. Ncuruscience 21(3):951-95t3, 1987. Oltur tiuppurt: Ikparhuent uf Hcalth and Fluman Services and USAMItU(', Austrian Scicntilic Research Funds, Swedish Medical Research Council, and SwcJiah'fuhaccu Company. From the I)Lpartment of Phannacolugy, Loynla University Medical ('enter, May- wood, IL; I)epartment uf Clinical Chemistry, Karulinxka Huspital, Stonkhulm, $we- dcn; and the Ihpartmcnt of Pharmaculogy, Karulinska Institute. NIiUROPIiP't'll)F. Y ANI) NON-AUKIiNERGIC SYMPATHETIC VASCULAR CONTROL OF'[ I Ili ('AT NASAI. MUCOSA Neuropeptide Y(NI'Y) coexists with nuradrenaline (NA) in a population o1' perivascular nerves in Ihe cat nasal nwcosa. In the present study, NI'Y was fiwnd to exert non-adrenergic nasal vasux:omtrictor actions. Pustganglionic sympathetic nerve stimulation induced a release of NPY-Iike immunoreactivity (LI) concomitant with vasox:unstrictiun in the nasal mucusa. AExwt 6O and 70%, of the va.OCUnstrlLlur re- sponses upon sympathetic stimulation at 2 and 10 Hz, respectively, remained alter pretreatment with phcnoxylxnzamine and propranolol which abolished the efl'ects of exogenous NA. Preganglionic denervation one week prior 1t) the experiments did not change the vasoconstrictor response to sympathetic nerve stimulation or the NA or NPY contents of the nasal nmcusa. "I'he levels of NPY-LI in the superior cervical gangliun were however reduced. After reserpinc treatment, which depleted the nasal NA content by almost 1N)'Yn and the NPY content by 50%, a vasocunstrictur response to nerve.timulatiun was still present. After reserpine treatment cuntbineJ with prcgangli- unic denervatian, nerve stimulation simultaneously induced an increased output of NPY-LI and a marked long-lasting vasoconstriction which was not influenced by pheuoxyhcnzamine and prupranulol. The reserpine-inJuced depletion of NA was not itdluenced by pregangliunic denervation while the reduction in the nasal content of NI'Y-LI was prevented. In conclusion, NPY could he a non-adrenergic mediator of sympathetic vascular effects in cat nasal nwcusa. Lundblad, L.., Anggard, A., Saria, A., and l.undhrr,G, J. M. Journal of the Autonomic Nervous System 20:189-197, 1987. Other support: Swedish Medical Research Council, Swedish Work F.uvironmcntal FunJ, Swedish'IbhaccoCumpany, Wibergs Faundation, Petrus and Augusta I1eJlund Foundation, and the Karulinska Institute. From the lk:partments of Oto-Rhino-Laryngology and Pharmacolugy, Karulinska Institute, Stockhulm, Sweden, and Ihpartmem of Experimental and Clinical Phamta- colugy, University of Graz, Graz, Austria. 78 I)li'I'li("I'ION 01: NLiL1ROl'I:I'"I'IUIi Y ANI) ITS mItNA IN MI:GAKAKYOC'Y'1'IiS: IiN11ANCliI) LNVI'.LS IN Clsl{TAIN AU"I'OIMMUNIi MICE Neurulxptide tyrosine (neurupeptidc Y, NPY) is a Iwtent vasoconstrictor with a wide ditiutbuuon in the central anJ peripheral nervous systerm. I Iere we shuw that high levels nf rat NI'Y ntRNA are also fiiunl in peripheral blood cells, Iwne nrarruw, lung, and splecn. hurlhcrnwrc, radiuitumunuas,uy revealed high levels ul' NPY-likc !xp- tidcs in these tissues. In mice, the levels of splenic NPY mItNA and immunareacuve Ikptiilc diflcreJ extensively between strains and were greatly elevated in several strains (N%li. N'LI3xW, and BXSB) that develop a disease resembling human systemic lupus crythcmatu.us. Like the rat, the N%B mouse showed a high cuntent of NI'Y niRNA in the peripheral blood cells and bonc marruw. Immurnthi.toxhemical staining revealed NPY-like inuuunurcactivity in large cells morphuh)gically irlentiliaMe as megakaryucytes in rat bune marrow and in the spleen of the M1.6 mouse strain. Expression of NI'Y mKNA in mcgakaryix:ytcs in rat bnne marrow and N'Ll3 muuse spleen was cunfirmed by in situ hybridiialion. Thr;se results indicate that NI'Y is symhesiied in megakaryucytes, implying that NPY can 6c released from platelets and function as a vasoconstrictor during hhwd-vcssel damage. In addition, the increase in splenic NI'Y in certain aukoimmune mouse strains adds to the list uf ahnurmalitics associated with these strains. Gricsson, A., Schalling, M., Mclntyre, K. R., l.undherS, J. M., Larhammar, 1)., Senxigy, K., Ilokfch,'I'., and PersHm, IE. I'roceedings of the National Academy uf Sciences, USA ii-1:55135-5589, August 1987. Other support: Kabi Gen A13, the Swedish Natural Science Research Council, Swedish Medical Research Council, Magnus Bergvalls Foundation, Petrus and Augusta Iledlunds Foundation, Swedish Tobacco Company, and Fredrik anJ Ingrid Thurings Foundation. From the Ihspartmcnts o/' Medical Genetics and Immunology, the Biomedical Center, Uppsala University, Uppsala, Sweden, and Depanmr;nts of Histology and 1'harmacul- ogy, the Karulinska Institute, Stockholm, Sweden. A("fIONS OF CAI.CIUM ANTAGONISTS ON PRE- AND POS'I'JUNC'1'IONAL E:FFIiC'I'S OF NIiL1ROPE1'TIUG Y ON IIUMAN PERIPHERAL BLOOD VFSSGLS IN V!"!'kO The niechanisms underlying the contractile effects of neurupepliJe Y(NPY) in relation tu those ul' noraJrcnaline (NA) on small human blood vessels were studied in vitro. NPY caused contractions uf mcsenteric veins, renal and skeletal muscle arteries but nut uf mcuntcric artcries. NPY was about 5- to 10-fu1J more porient than NA. The maximal contractile responses to NPY (5 X 10' M) were 3R ! 4, 37 -* !i and 95 ± 16% ol'the response evoked by NA l0' M in the mesenteric vein, renal and skeletal muscle arteries respectively. The NPY ef'fects were resistant to aJrenoxeptor antagonists. The calcium antagonist nifedipine reduced the effect of NA but not the contractile response to NPY on mesenteric veins. Nidetipine and felodipine reduced the contractile re- sponse to both NA and NPY on renal and skeletal muscle arteries. In contrast to the contractile e/Tects of K' , the responses to NPY and NA were largely uninfluenced by 79
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chanFc. in extracclhdar Ca" cunccntratiurr.. Nifctlilrinc still inhibited thc NPY cun- fractton, in a('a" dfrrr urcdium whilc high rxtraccllularCu" ((7.5 mM) partly reduced the ndedihuic cllcct. NPl' reduced the nerve stimulation-evoked 1'II1NA overflow frunr thc mrticntcric vcin% via a niledipine resistant mechanism. 'I'hc stable analogue (i.li-mcthylcnr adrnu.inc triphmphatc (mATP) was nmre putrnt than A'I'I' and harl ntl'ctlrpinc-.cn.itivc contractile rllrcts similar to those ul' NA on dre human blood vc.scl, without ualucncing the nerve-evoked 1'IIjNA cl'llux. In cunclu,tun, NI'Y cxcns a potent tutcJipmr-,cn.itivc vaatxun.trictur activity, especially on htuuan .Lrl - etal mu.cle arteries in vitru, although the inllux uf cxlraccllulur calciunr nray not hc a crucial ntcchanunr. 'I'hr NI'Y-inJucctl contractions of mc.rntcric vrind anJ the inhihi- tieru of nerve-evoked 1'II1NA clllux seem to be- mediated via nifedipinc resistant inc,.cnl;cr ,y,tents. I'crnuw, J., Svcntarrg,'I'., and Lundberg, J. M. Gurulxan Journal uf I'h«rmaculugy 136:2Q7-218, 1987. Other support: Swedish Mcttical Research Council, Swedish Tahacca ('uwpany, Swedish Work lawirunmctu Fund, Pctnr. anJ Augusta Ilcdlundl Foundation, SwrJ- i.h Sucicty uf Mcdicine, and thc Karolinskn Institute. frunt the I)rpartnrrnt uf Pharmaculugy, Karolinska Institute, and 1)cpartnrcnt uf Sur- l:cry. 6:arultml,a Ilu.pital, Stuckholnt, Sweden. C'AL('fI'ONIN (iliNk-RI:LATI?O PI:P'('IDIi BUT N(YI' SUI3STAN('li P MIMIC:S ('AI'SAI('IN-INI)ll('EU CORONARY VASO1)It.A'I'IUN IN'1'Illi PIG 'I'hc v;itiudilatwr cffect, ul' the Itunuut calcitunin gCnc-rclatcJ IxptiJcs (r (h('( iNI'a) and 11 (hC(il(JI'(i) were studied in rdrru and in viru in relation to thccffcct, uf substancc I' (SP) and capsaicin on coronary vascular tunc in the pig. Both hC'Gkl'o: and -ji induccd a concrntratitm-dc~nJrnt, long-lasting relaxation uf prccuntracictl snuJl (tlinnutcr0-5 nuu) pig currrnary artrrics in vitro. SI' was slightly murc pulcut hut caustrJ a tran.icnt rclax«niun with a stnaller maximal response than ('(iRP.'I'hr rclax;r lion induced by h('(iRl'a and -(i as well as SP was resistant to propranolol and atrupinc. ('apsaicin also inducctl a long-lasting relaxation uf Ixnaysiunr antl I'OF.,,- prrcuntractcd curunary artcrirs. After tachyphylaxis tu SI' had developed thc rrlaxant cffccty of ('GI{I' and cap.aicin were unchanged. Rubbing the vessels tu remuvc thc cnduthcliuni completely abolished !hc relaxant cffccts ul' SI' while the vasotlilation induced by hC(ikl'u ;rti wcll ax capsaicin remained unchanged. Injections uf h('GRI'a, SI' or calrs;ucin into thc constantly IxrfusrJ Iclt antcriorJcsccndint; coronary artery ul' thc pig in vivo caused a dose-dependent decrease in Ix:rfusiun pressure, suggesting coronary vatiuJilatiun. In cunclusiun, the va.uJilator cfl'ccts of SP in virru dil7er fruni the response tu ('GRI' both with regard to their transient nature, the develupmcnt uf tachyphyLtxiti and rnJuthrliwn dependence. The capsaicin-induccd curunary vasrrdi- hniun i, lhcrrlirre murc likely to depend on release of CGRP rather than tachykinin, fronr sensory nerves since neither endotht:lium remuval nor SP-tachyphylaxis in- Iluencctl the cap.aicin and CGRP responses. CGRP and tachykinins are present in cap.aicin-scnsitivr sensory nerves around coronary vessels of many species and these results therclirrr indicate an important ('unction for ('(7RP rather than SP in regulation of curunary blood flow. I~rancu-('crccctla, A., Rudehill, A., antl l.undGer,q, J. M1. I:urulx«m Juurnal uf I'harmaculul:y Id2:235•2-i•1, 1987. Othcr support: Swedish Medical Research ('uuncil, Swedish Tobacco ('ump;my, I'cu•us and Augusta I ledlunds 1«uundatiun, lacrtlalx Foundation, and the Karulin,l.a Institute. . Frum thc I)rlr,irtnunt uf Pharnraculul;y. Karulmska Institute, and Ikpartmcnt uf An- ctitlrc,ia, Karultna,a I lu.pilal, SlrxhhuPrn, Swrtlctt. F(I{SI{RI'INIi-INU(I('I:U I)GPl.li'I'IUN OF NIiIIROPIiP'I'll)li Y IN '1'tlt: (iUIN1iA-I'I(i: 'I'ISSll.li-SI'IiC'II~IC' IiFhli("'S ANI) MI:C'I IANISMS OI' ACIIUN fhc cl Icct~ ul lreauncnt with rescrpinc (5 mg • bg ', ti.c., 2-1 Ir prior to sacrilicr) or 6•hydruxy,lup;uuine (6-OIII)A; '5O rog •{.g'- x.c., lirr 4 days. one week helirrc .ucrilicr) un thc content uf ncurnlxlrlidc Y(NPY),Iikc imnwuurcactivity (L) and nuralrcn;dinc (NA) were cunqiarrd in a variety of titixuc. I'rum the guinca-pig. Rcticr- pine antl b-OI II)A trratnrrnt mar{.cJly reduced the NA content ul'all Ixripher;d organs invc,tigatcd. Itracrpinc trcatmcnt also cau.cd depletion of thr cuntent uf NI'Y-I.I in laiger blood vraclx and in orEans containing mainly Ixrivascular ncrvcs. Furthrr- murr, rcxupinc trcaunent depleted NI'Y-LI in the hran, spleen antl atlrcnal gland. In uthrr urgaus duminatcd by parcnchymal adrenergic inncrvatiun xuch as gcnital urg;ars (vas tlcfrrcna, utrruti) or iris, trcatnrcnt with 6-OI II)A but nut resrrpinc caused signili- cant rlrhlctiuns uf NI'Y-1.1. 'I'hc urinary bladder and gastrointestinal tract tiecm to be wainly innrrvatcd by nun-aJrcnrrEic NPY-contuining ncumns resistant to rcxrpinc and 6d)1II)A treatmrnt-'I'ltr content ul' NPY-LI was clrvutcd in .ympathrtic ganglia altrr rc.crpinr trcauucnt whilc nu increase in axonal tran.pun uccunrd in thr .ciatic nrrvr. ('crchellunr was the only audicd «u•ca in the central nrtvuus sy,trtu where the NI'1' conlcnl was depleted by rescrpinc or (t-OI II)A treatment tiuggc.ting prt:urncc of NI'1'-I.1 in pcrrvascular ncrvrs, 7'hc rcurrpinc-induccJ dcplctiun of NI'Y-LI iu the .plrcn. kidncy anJ.Lclctal muscle was prcvcuted by prcganglianic dcncrvatiun.'I'hi. suggc.Iti that rnhttncrd ncrvr iurpul,t: diuh;rrgc in synrpathctit• ncrvcs curutl a situa- tinn whcic NI'1' rclrau exceeded thc annuunt which could tx replaced by axonal tran.purt. In runclu.iun, IrrclrratmcrN with a high dose of rrscrpinc is associated with a Jrplctirm of NI'Y-LI in cardiovascular adrrnrrgic ncuruns antl the adrenal ghind. "I'he NI'Y cuntrnt of parcnchymal adrenergic ncrvc% except fur in the hcan ;mtl ,plccn, however, does nut sccm to he inllucnccd by rescrpinc trcatment. In cunrbinatiun with thr inhihitury ellerl on the rcscrhinr-inJuccd NI'Y depletion by prrgangliunic dcnrr- v«uiun, thi..u(;LC.t. that ccnain Ixqrulatiuns o/ syrupathctic nrtiruns are ,clcctivrly activ;ncd ;dtcr rrxrpinr trcabncnt and changes in NI'Y-Icvcls nwy thuti serve as an indicator ol sympathclic ncurunal activity. Nagata, M.. Francu-Crrcccda, A., Saria, A., Amann, R., antl Lunrlhrr,4, J• M1l. Juurnal uf thc Autunumic Nervous System ?0:257-26.i, 1987. 80 81
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.r OIher .uplxrrl: Swcdhtih Medical Research ('uuncil. tiwedi,h 'I'uhacco ('utupany. I'rqu,:ual AuEusta I lrillund. Fuundatiun, Auariam Scicntitic Ite.rarch Funds, anl Ihe K:uulm.ka (n%lilulc. From the I)epanmrnt of I'harmaculo);y, K.uolin>ka Instttute, Sukkhulm, tiweden, and I)epartment of I?xlxrimental and Clinical 1'hannaculugy, University uf, (;rai, (;rat, Austria. I'Ol-1'I'lil'1'll)li-l'ON'I'AINING NI:URONS IN AIRWAY SM(HYI'II MIISC'LIs Acctylchuline and noradrenaline have long been the only identified neuronal meJiaturs in the bronchial smooth muscle of thr: airways. Huwever, increasing evi- dence suggesls that several biologically active rxdypcptides are also present in nerves ul the respiratory Iract. 'fhe present review concentrates on peptides in parasynrpa- thetic and sensory nerves that have been most thoroughly characteriredl with regard to functiunal aspects. The roles ut' VII' and Pill in parasympathetic nerves are discussed as are the actiunti uf CGRI' and tachykinins on sensory neurons. The tachykinins involved are structurally related and include substance P, neurokinin A, neuropeptiJe K and an eleduisin-related Ixptide. 'I'his family of peptiJcs, as well as a CGRP-like immuno- reactivity, is detected in the lung alter exposure to eapsaicin; these reactions are discussed. Also detailed are the capsaicin- and tachykinin-inJuced reawnses uf vaui- dilaUan iu nasal mucosa and trachea, protein extravasation in airway mucusa, and contraction uf tr.rcheuhronchial sm{xrih muscle. Airway C-/iher afferrnts seem to he signilirant sources Iiir the release of local bioactive IxptiJes and are important in local and rellrxol;cnic respunses to irritant chemicals and as medialur, of the allergic reaction. l.mrdbr•)w, J. A1. and Saria, A. Annual Review ul I'hysialuRy 49:557-572, 19H7. Other support: Swedish Medical Research Council, Swedish Tobacco Company, 1'etrus and Augusta IieJlunJs Fuundation, Swedish Work linvirunmental Fund, Karolinska In,titutc, and the Austrian Scientilic Research Funds. 1=rum the 1)rrarnucnt uf Pharmucolugy, Karulinska Institute, Stockholm. Sweden, and the I)elr,trtntent iif lixlxrimental anJ C'linical I'harmacolugy, University ulGr:v, Urai, Austria. NI'.IIROPlil''I'II)li Y ANI) NORADRIiNALINF. MG('IIANISMS IN RFLATION TO KI:SIiRI'IN1: INI)ll('1'.I) IMPAIRMI:NT 01: SYMPAI'Illi7'IC NIiIJROTRANSMISSION IN TIIG C'A'I' SPLEEN '1'he ntech,:uti.ms underlying the reserpine-induced impairment uf the functional resprmx, tu sympathetic nerve stimulation and output uf nuradraline (NA) and neuru- Ix.ptide Y(NI'Y)-like inmmnorcactivity (-LI) were studied using the isolated bkmd- IxrtuseJ cat.pleen. Splenic nerve stimulation (101ii lior 2 min) during control condi- ltuns caused per(usion-pressure increase, volume reduction and an increased output uf H2 NA and NI'Y-LI. After alhninistratiun uf phenoxyMrni;rminc, the nerve stinudatiun- induceJ Ixrfusiun-pres.ure increne was almost abolished, the volume reduction in- lubited and the output uf NI'Y-LI enhanced. After subsequent addition uf prapranulul, a clear-cut increa.c in I)crl'usian pressure upon nerve stimulation reappeared. Local intiuiun ol NI'Y caused a potent, lung-Iaaing, arlrenx:eptor-resistant increase in Ixrfu,iun pressure and a relatively smaller volume reduction ol'the spleen. Twenty- tour hours aitcr rexerpme pretreatinent (I mg kg' i.v.), which depleted the splenic cuntent of NA -r15'G and NI'Y-L) by about 50°/r., the functional responses upon nerve stimulation were markedly reduced. I'repanglinnic denervation or pretreatment with the gangliunic-hioxking agent chlorisondamine did not inltuence the NA deplctiun alter rcserpine trealmcnl. A considerable, aJremoceptur antagonist-resistant, lung- la.ung functiunal response as well as a markedly enhanced output of NPY-LI then occurred upon nerve stimulatiun. In conclusion, reserpine treatment combined with interruption of pregangliunic impulse flow reveals non-adrenergic, nerve stinwlation evoked splenic functiunal responses which could be rrtediated by release of a cotrans- nritter peptide like NPY. LwtdGer,G. J. M., Pernow, J., Fried, G., and Anggard, A. Acta Physiologica Scandinavica 131:1-10, 1987. Other support: Swedish Medical Research Council, Swedish Tobacco Company, Petrus and Augusta IieJlunJs Huundation, Swedish Work Environment Fund, and the Karolinska Institute. hrum the I)epartntenls of PhannaculuNy, Karolinska Institute, and Dcpartmmnt uf Oto- khinu-laryngulugy, Karulinska Ilo.pital, Stockholm, Sweden. I'lIliN"I'OI.AMINIi- ANI) CLONII)INI:-INI)UCED C'NANGtiS IN PLASMA NIi(JRUI'IiP'I'II)li Y-LIK1: IMMIJNUIt1iACfIVfI'Y I)URIN(.; SYMPA"I'It6TIC A('"I'IVATIUN IN MAN '1'hr aim of the present study was to investigate whether the increase in plasma NI'Y-Ll during >ympathctic activation in man, such as physical exercise, could be altered by pretreatment with the alpha,- and alpha.-adrenoceptor antagonist phento- lanune or the allrha.-adrcnuceptor agunist chmidine. The present results .uggcxt that •alpha-adrenrx:eptor blockade by phentulamine increases and alpha.-aJrernKeptor stitnulation by cluniJme reduces the exercise-in- duced release of NI'Y-1.1 from sympathetic nerves in man. Since phenhilamine is a nun-selective alpha-aelreno:eptor antagunisl, the enhanced release uf NPY-l.1 atier administration of this drug may be related to blockade uf pre-synaptic alpha; receptors and/ur tu a harurccrplur mediated increase in sympathetic nerve aclivity to cumlx n.ate Iur peripheral vasuahhua/iun due tu post-synaptic alpha,-receplor blockade. Since the ulcctive ulpha,aJrrnrKeptur antagonist praiuxin causes an increase in plasnw NA, it is likely that the I:rller mechanism also contributes to the elevated levels of NPY-LI. 'fhe reduction in pla.ma NI'Y-LI seen al'ter clunidinr cumrared tu the control exercise is in parallel with luwer.ystemic plasma levels of the co-existing classical tr:msmittcr NA during exercitic :dter claniJine treatment. This effect is most likely due to a central actiun uf clunidine an alpha,-recepturs resulting in reduced sympathetic nerve activity, although peripheral inhibition uf NI'Y release due to stimulation uf pre-synaptic al- pha: rcceptun cannot he ruled uut. ln conclusion, the alpha-aJren><eptor antagunist 83
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phrntulanunc and Ihc alpha.-aJrcntcrptur agunist cloniJine enhances and rctlucc%, rr.lkctivcly. Ihc inclease in plasma NPY-LI during physical cxcrciuc, ruFticsting that thc release ul NPY-LI in man is regulated hiuh via peripheral and central alpha- adtc noxchtur.. Pcrnuw, J,/.miJber,q, J. M., and Kaij.er, L. ('lintcal I'hy.iulugy 7:4.i1--1 34, 1987. Other support: Swedish Medical Research Council. Pctrus and Augusta Ilcdlundti Fuund:uiun, antl Swedish Tobacco Company. Frum thc t)rp:trtiurnt uf Pharmnculugy, Karulinxka Institute, and t)cpartment uf Clini- cal I'hy.iulugy, Karulutaka Ilutipilal, Shxkhulm, Sweden. ('Al.('I'fONIN (iliNli-RI:I.A'I'lil) 1'I:1'I'll)li ICGRP) IN C'APtiA1C'IN-SI:NS1TIVli SUI3S"I'AN('li I'-IMMUNURIiA("1'IVB St:NSORY NIiURONS IN ANIMALS ANI) MAN: 1)ISI'RI13lJTIUN ANI) RGI.t:ASE BY CAPSAICIN The Prc,cncc of calcitunin-grnc related IxrptiJc (C'GRI')-likc immunurcactivity (-I.I) in sensory neurun+ was established by inuuunuhi.ux:hcmistry and radiuimmu- mtauay (RIA) in cumbinatiun with high (xrtitrmancc liyuid chruntahigraphy ll II'L.C'). ('(JRI'-imtuunurcactive (-IR) nerve fihres were present in many peripheral organs includtnE heurt, urctrr, utcrua and gall bladdrr uf guinra pig anJ man. 'I'hc dititrihutiun of ('(iRP-IR nerves in the dorsal hurn of the spinal cord, of' positive cell hudirs in thoracic tipinal and nudu,e ganl!lia anJ nerves in Ixriphcral urgans was closely rehncd to that of suhstamcr I)uublc staining cxperintcnts revealed that in nw.t cases peripheral ('GRI'-IR nerve tcrminals also contained SI'-LI. I luwever, difli•rcnt lurali- /ation uCSI'• anJ ('GRI'-IR neuruns was observed in the nucleus uf tht solitary tract its well a+ in the ventral horn uf the spinal cord. In the heart, CGRI'-IR nerves wur associated witlt myocardi:d cells (mainly atria), curunary vessels, local para.yntp:t- tltctic ganglia as well as willt Ihc epi- and endocardia. Three to 4-liiltl higher levels ul native ('GRI'-I.1 were observed in the atria than in thc vrntriclcs u( thc hcart. III'I.(' :tncdysis revealed that the ntajur peak ul` C'GRI'-LI in Ihn heart uf rat antl man had the tiatuc retention Iimcs ati the synthetic equivalents. Systemic captiaicin prrtrcattncnt of adult gtunra-pig, c,tu,cd a loss uf ('GRI'-IR Icnninais in thc dorsal hurn uf thc .Irinal cord as well as in hcripheral organs including the he:trt. Allercap,aicin uratmcnt, the run,rm of ('GRP-IR was reduced by 7lt'7k in the heart and by (rlNf in thc Jur.al P:u•t ul dtr .hwal cunl. In ,uprrlu.iun cxnlrimcnts with slices frum thc rat }pin:d cord, a rclca,r of ('(;RP-1.1 was induced byYrO mM K' and 3 µM capsaicin in u calciuur dclxnJcnt manncr. Francu-('crcccila, A., Ilcnkc, Ii., l.undher);, J. hf., Pctcrntann, J. 13., Ilukfclt, 'I'., :md Fischer. J A. Pcpudc, 8: 3t)9-a1U, Nm7. Other.ruplwn: Swedish Medical Research Council, Knul and Alice Wallentkrgs Foun- dation, Swedish Tobacco Cumpany, Pcttu% and Augusta Iledlunds Foundation, the Karuhn.rka Inxtitutc, Swedish Work 1'nvirontncnt Fund, Swiss National Science Fuun- datiun, Canton ul 7.urich, and the Schweizerische Verein Balgrist. Frum thc~lkpartnrcntti uf Pharmacology and Histology. Karolinska Inatitute, Stcxk- hohn. Sweden, and Reuarch Laboratory lur Calcium MetaMrlism, 1)epartments of Orthopedic Surgery and Medicinc, University uf'Lurich, Zurich, SwitLcrland. C'I IRQNI(' NI('O'I'INE ADMINISTRATION INCRI:ASIiS BINDING OF 1'li1l)OMPIiRII)ONIi IN RAT NUCLEUS ACCUMBENS An apparent inverse relationship between smoking und Parkinson's disease pruntptcd an investigation of the ctlccl ut' chronic nicutinc aJntinistration on Jo- paminerl;ic and x:rutuncrgic receptors in ral brain. Nicotine, 0.8 ntg/kg, was iqjectcd oncc Jaily, live timcs per week, Iitr 6 wecks. In nucleus accumbens the K, tiir j'N jtlum- Ixrridunc was increased 2-4liild, and the 13,,,, was increased I.S-2-titld. Nu changcs were observed in the binding uf j'H1dumperidunc in cauJatcpulamcn or in that ul 1'IlIkrtan.crin in frontal cortex. It is concluded that chmnic nicotine aJministratiun may havc asuppressanl cl Icct on central nervous system releak ut' Jopaminc that in prc-p:trkin.wmian persons causr, all aversion to thc ct7ccts of snwking. Rrilly, M. A., Lapin, li P., Maker. Ii. S., and l.ujrhrr, A. Juurnal ul-Ncuroscicncc Research Iti:621-625, 1987. Uther.uplxxt: New York State Basic Research Support Grant. Frum thc Ihpartnoent ul' Ncurulugy, Mount Sinai School ul' McJicinc, New Yurk: Ihpartmrnt ul Netn•ulugy, 13cth Israel Medical C'cntrr, New York; and ('enter fitr Nrwik•hcmititry, Nathan S. Klinc In.titute titr Psychiatric Research, Ward's Island, NY and Nathan S. Klinr Intititutc fur Paychiatric Research, Orangeburg, NY. R1:7'Rt )(iRAI)li 'I'RA('ING Sl IOWS 'I'IiAT CGRI'-IMMUN(')RIiACTI Vli NI?RVI:S OFRA'I"IRAC'IINA ANI) LUNG ORIGINA'1'G FROM VAGAL AND I)ORSAI. RUOT GANGLIA 't'hc urigins ul scnsury innervation of thc lower respiratory tract arc thought lu Ik prmripally the nudosc anil jugular Eanglia ol"thc vagus ncrvc. It ha.lxxn suggctitrd and parti:tlly tlcnwn+trared that there ix also a cumlwncnt arising 1'rum Jursal root ganglia. hut (hr xgmental levelti invulvcd are not known prcciticly. We have Ihcrclitrc invcati- gatcd thc origms uf scmury ncrvcs within the rat respiratory Iract, p:ulicularly thn.c cuntaining calcttunin gcnc•rrlatcd IxhtiJc /C'GRI'), using the trchnitlur uf rctrugradr axunal iraring camhinrJ with immumuhititochcmi.try. Injrcliun% uf 7'rttr blue were marlr intu cxtra-thuracic trachea (rt =-1 rats) and Ixrcutancuu.ly into the right and Irti IunL (n =-l c:tch). Rctrugradcly labelled ncurunal perikarya were drtrctcd in vagal anJ Jnr.al ruut ganglia, anJ sympathetic chain ganglia. C'GRP-imntunurractivc cells were srcn unly in vagal and dorsal rout ganglia. Trachcal inncrvation arose bilatcrally in the vagul sensory ganglia but those on tha right side represented the principal .uurcc; thc 84 85
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~ M majority uf CGRP-cuntaininti neurons occurred in the jugular ganglion. A very small cumlwnent uf labelling occurred in spinal ganglia at levels C,-Ca.'I'he sensory inner- vation of the lungs was seen to arise predominantly from the ipsilateral dorsal root ganglia (45% of cells C'GRP-immunoreactive) at levels 7',-T,. In contrast to the trachea, the contribution of vagal sensory neurones to the lungs appeared tu be l,ess than that uf the spinal ganglia.'I'hese results show that the sensory innervation of the rat lung has a nmjur origin in the dorsal ra)t ganglia, in which almost half of the involved neurons contain C(;RP, and cunlirm that most CGRP-immunoreactive nerves in the trachea arise in the right jugular ganglion. Springull, D. R., Cadieux, A., Oliveira, H., Su, H., Ruyston, !)., and l'uluk, J. Mf. Journal of the Autonontic Nervous System 20:1:55-166, 19t37. Other support: Asthma Research Council. Frum the 1)epartment of Hititrx:hemistry, Royal Postgraduate Medical School, and I)epartment uf Ancsthetics, Hammersmith Hospital, lAmdon, l:ngland. ACTIVATION OF 1'RQ•fI:IN KINASE C AUGMENTS GVQKEI) •f RANSMI'ITI?R RI:LIiASIi In view uf the cmerging role of the phusphoinusitiJe system in cclkdar cummuni- catiun we ex;uoincd' its involvement in yuantaltransmitter release, which is a key element in synaptic trantimissiun. 'I'ransmitter release is nurmally activated by an increase in in,racelluhir calcium, achieved either by entry uf calcium ions through the presynaptic memhrane or by in(racellular calcium liberation. One of Ihc targets of the phusphumusitide signalling system is the enzyme protein kinase C ll'KC'), which can he activated experimentally by tumur promoting phorhol esters, including 12-O-tctra- dccanuylpharhyil Li-arelalc ('I'I'A). Such activatiun of PKC may be implicatcd in tran.mitter relcau in two ways. First, phorhul esters were fuund to incrcase sccretiun and enhunce calcium currents; it might thereliOre Ix: expected that they would increase synaptic transmitter release. fiut phorhMd esters also inhibit the calcium current in Jorwl root gangliun ncurunes. We report that the phurfwl e,ter'fI'A auf!mcnts synaptic tiansmisviun;u the nrurumuscular junction by increasing transmitter liberation. Acti- vauun uf PI:(' alw deepcns synaptir depression. Shahira, R., Silhencerg, S. 1)., Ginsburg, S., and RuhurninurfJ; R. Nature 325(6099):5H-bU, January I, 1987. Other wppom: Muscular I)ystrophy Association, Israeli Academy of Sciences and Huntanitie., and thc fiay Fuundatiun. Frum the I)cpar(ment uf I'hysiulogy, Hebrew University-Hamlassah Medical School, Jeru.alem, and Gveryman'.llniversity,'I'eI Aviv, Israel. 86 Rli(IIILA'I'ION OF SURFACTAN'I' SECRETION FROM ISQLATIiD TYPE il I'NEUMOC Y•ITS BY SUt3S't'ANC'E P Subst;mce P, an eleven amino aciJ neurirpeptide, significantly inhibited release ol' j'II Iphusphatidylchulinc ftom pulmunary 7'ype 11 epithelial cells in vitro. Basal release ;md release in re.punsc to the 1i-adrenergbc agonist, terbutaline and 12-O-tetradeca- nuylphorlwl 1.1-acclate (TPA) were signilicantly decreased in the presence of sub- stancc 1'. Inhibitory cl7ccts of substance P were noted lirlhrwing a I h exposure ul' primary cultures uf'I'ype 11 cells in vitro and persisted up to 3 h in the presence uf the ucrrlagogurs, 'I'PA and terbutaline. The IC„ values for substance P inhibition of I'I I IPC release were Itl µlVl lor basal release, 40 µM Iiir'fPA-inJuceJ release and 50 µM fiir terbutaliuc-induced release. The related neuropeptiJe, physalaemin and the stable active analog uf substance P, jpGlu', MePhe', MeGIylsubstance P, had no sig- nilicant inhibitory cl7ccts un surl'actant release whether in the presence or absence of TI'A or tcrhutaline. 'I'hesc data support the hypothesis that NH2-tcrminal basic groups uf .uhstance P are necessary for inhibition uf surfactant secretion from isolated Type II cells and support thc concept that an inhibitory system contributes to mediation of surfactant secretion from Type 11 epithelial cells. Kire, W. R. and Singleton, F. M. fliurhimica et 13iuphysica Acta 889:123-127, 1986. Other support: American Lung Association, Children's Hospital Research Foundation and National Institutes of Health. Frum the University uf Cincinnati Medical Center, PeJiatrics/Newborn Division, Cincinnati, OII. RI?LEASIi UF S1113S"1'ANC'G P, ACIiTYLCHOLINI? ANI) MIiTIIIONINIi I:NKEI'IIAI.IN FROM MOUSE C6RGIiRAL SLICIiS: EFFGC"1'S OF NICOTINE Substance 1' (SP) enhanced acetylcholine (ACh) release while methionine cnkephalin ( MIiK ) depressed it. Tu cvaluate the Icedback mechanism liir AC'h release, the cl7ects uf nicirtine un the s.imultaneaus release of AC'h, MNK, and SP from mouse cerebral slices were investigated using micrusupcrfuxiun. ACh release was IiilluweJ by thc cfllux uf 'U-A('h. The peptides released were measured by raJiuimmunuassays. A low concentratiun ul' nicotine (61.6 x I(1" M) increased the rate of spontaneuus release uf AC'h and SI' anJ decreased the release of MEK. A high concentratiun of nicutine (bl.6 x I(1' M) decreased the spimtaneous release of A('h and MEK and incrcaseJ the spuntancuus release of SP. The high cuncentratiun of nicutinc depressed the evoked release uf all three, ACh, SP and MGK. Thc.se irbservations support the hypothesis that MEK and SP may participate in Ihe regulation of A('h release in the brain. 'f aycb, O. S. and Su.clr)•, lt. V. R. In: Henry, J. L., ('uiuure, R., Cuellu, A. C., Pelletier, G., Quirinn, R., and Retiuli, 1). (eJ..): Substance P and Neurukinins. Springer-Verlag, pp. 350-352, 1986. From the Deparuncnt of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN. 87
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SL113STANCI: 1' IN IIUMAN SPI:FtMATOZOA ANI) MODULATION OF SPERM MOTI1.1'fY BY SUI3STANC'I: P ANI) ITS AN'fACiONIS'I'S I I>jacwlatcJ huutan .Ienuatur.ua anJ se:minal plasma were extracted for SP-Iike Ix:pUdcs. SP tn the extracts was identified by selective radiuimmunuassay.utd hiuati..ry un electrically stimulated rat vas Jclcrcns. "1'o evaluate the role uf SP, the influcnce uf SI' and two SP:uuagunia., D-pru',l)-Irp'"-SP and D-arg', D-trp'"-Icu"-SP were aud- icd on human .Ikrm mutibty index (MI). At 4 hours, the MI uf SI'-trcateJ ( i_.5 x 10 ' x M).perm was higher than that ot' the untreated controls by about 201G'. I lowever, the MI was depressed with higher cuncentrations of SP (:> Il/'• M/. liuth SI'- antaguni.h (10 '• to 10 ` M) increased the M) by about 30-50%. Higher concentratium ol' these antagonists (1l)' to 10' M) did not exibit a significant effect on the M1. SP is known to incrrau ('a' ' uptake by nerve cells. It is possible that SP-antagonists are antagunii.ing endogenous SP and thereby down-regulating the ('a' ' channels. I?nkrphalins may serve as endrigcncous physiological antagonists ol' SP. Sustrv, B. V. R. and Janson, V. E. In: Henry, J. L., C'outure, R., Cucllu, A. C., Pelletier, G., Quirion, R., and Regoli, I). (eds.): Substance P and Neurokinins, Springer-Verlag, pp. 179-181, 1986. Other suplxrrt: ll, S. Public Ileallh Service. Frum the I)epatrtment of' Pharmacology, Vanderbilt University School of MeJicine, Nashville, TN. I)AMAGG'IY)'1'Hli I11OI(-AFFINfI'Y y-AMINOI3UTYRIC ACII) (GA13A) UP'I'AKH SYSTI;M IN MO(1S1: BRAIN BY HORSERADISH PIiROXIDASIi (IIItP) Presynaptic nerve terminals when delwlarized are sensitive to nxirphuhigical and functianal alteratiun by horseradish (xruxidase. Mouse brain slices, 0.1 mm, dcpolar- itcd by a K' -I Ilil'IsS huffer and exposed to horseradish peruxiJase exhibited altera- tions in both synaptic vesicle membrane structure anJ in high-at7inity I"C ly-antinubu- tyric acid uptake. The post stintul:nary retrieval nl' synaptic vesicles from the nerve terminal plasma membrane in the presence of horseradish peroxidase resulted in a decrease in lhc synaptic vesicle population with a concurrent increase in non-synaptic vesicle membrane structures. High-affinity I"C'Iy-aminuhutyric acid uptake into l).I- mm slices ol' muuse cerebral cortex and Iwns-medulla-spinal cord was inhibited by 11'G• and 2-I'k , respectively, aftcr incubation for (rl) ntin in K'-HtiPHS hul7er comtain- ing horseradish pcruxirlase. Superoxide dixmutatie protected hnth the synaptic vesicle memhranr anJ the high-af liniry uptake system frum the deleterious elfects uf hurscrad- ish pcruxiJa,e, porinting to the pussihle involvement of supenrxide aniun radicals in the hururadi.h pcruxidatie-related effccts. These horseradish peroxidase induceJ altcra- tiuns appear tu hi directed towards the exposed synaptic vesicle menthrane, since uun- stmwlcncd brain sliccs exposed to horseradish peroxidase do not exhibit a reduction in cnhcr high- or low-aflinity I"CIy-antinubulyric acid uptake. Low•aflinity uptake of I"Cly-aminuhutyric acid and I "Clu-aminuisohutyrie acid into cortical slices was not alfcctcrl altrr incubation in K'-HIiPIS with horseradish peroxidau. Luw-aflinity uptake, however, is reduced by the high-K' /Na'-free stinmlatury incubation Pnor tu uptake. It appears, thus, that high- and luw-affinily uptake are diainct and dilfcrcnt systems with the high-aflinity transport systcnt structurally associated with synaptic vesicle memhrane. Ikhler, E. A., .tirr.%Arn, ll., Lajtha, A. and Gcnnaru, J. F., Jr. Neiuokhemiaty International 10(2):I-{3-15i, 1987. Frum the, ('enter lur Ncurrn:hcmistry, 'fhe Nathan S. Kline Institute 1'ur Psychiatric Research. Ward's Isl;uul, New York, and New York University, Ikpartment uf Biul- ugy. NIC'O'1'INI: INI)UC hU CHANGGS IN THG Mli-l'AI30LISM OF SPECIFIC BRAIN PKO'1'IiINS The eflcct uf acute and chronic nicotine on the metabolisnt ol' specific brain pnrteins was examined by nteasuring incorporation of Iatx:Ied valine into protcin, with Jensitomctric scanning ol'protcins resolved by gel electrophoresis. Acute and chronic adhninislration ofnicutine (0.4 mg/kg per 3(1 ntin /iir 2 huurs, s.c., or0.5 ntg/kg per 30 tnin for 5 (lays (Alzel ntini-puntp implanteJ sulx:utaneuusly)) reduced incorpuratinn of I"Clvaline administered by approximately 6-7'1v. The results with chronic nicotine administration indicated a lack uf tolerance for this eflect uf nicotinc. Mecantylamine, a nicutinic ganglinnic antagonist, does not seem to block the inhibition uf protein synthesis. Small increases in protein content were observed in a high- and a low- molecular-weight region of SUS-Iwlyacrylantide gel, used to separate proteins from newborn brain. In adult brain after chronic nicotine administration, selective increases and a decrease were seen in selective banJs. Results are consonant with selective effects uf nicutine on the synthesis or degradation of specific brain proteins. Srra•hrn, Il., 13anay-Schwartz, M., Dunlop, D. S., I?ebler, E. A., and Reith, M. E. A. Neurachemical Research 12(2):I97-202, 1987. Frum the C'cnter far Neurochemistry, The Nathan S. Kline Institute tiir Psychiatric Research, Ward's Island, New York. LFFIiCI' 01: AS('OR13IC ACID ON THE SYNAPTOSOMAL UPTAKE OF I'1lIMPP', I'HIUOPAMING, AND I"CIGAf3A The effects iit'ascurbic acid on the synaptosumal uptake of I'HIMPP', I'NIJo- pamine, and I"C'IUA13A were examined in attempts to understand the mechanisnt of' ascorbic acid attenuation of MPTP neurotoxicity. I'H II)opamine uptake was increased at lower levels (0.01 and 0.1 mM) and decreased at higher levels (10 mM) of-ascarhic acid. Ascorbic acid inhibited I'IIIMPP' uptake (IC,,,=0.1 mM) and I"CIGAI3A uptake (IC",=10 mM). Washout uf ascorbic acid restored uptake ul I'HlJupantine and I'1I IMPP', suggesting that ascorbic-aciJ-inJuced lipid peroxidation was not involved in the effect on uptake. In addition tu the possible involvement of antiuxiJant mecha- nisms in the in vivo attenuation of the neurotoxicity of MPTP by ascorbic acid, the present results indicate a direct el'fect of ascorbic acid in inhibiting the uptake of MPP' into dupamincrgic nerve terminals. xx 89
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Srrslrrn, ll., l)ebler, l•. A. and Lajtha, A. Journal uf Ncurowcience Research 17298-3QI, 1987. Frum the ('enter fur Neuruchemistry, The Nathan S. Kline Institute for Psychiatric Research, Ward's Island, New York. BLiIiAVIOFtAL ANI) 131OCHEMICAL I:FFECI'S OF NICO"flNli IN AN MIP'1'P-INI)UC'lil) MOUSIi MODF:L OF PARKINSON'S I)1SIiASG This study examined the effects of nicotine on locomotor activity and on the level ul'dupamine (DA) and its metabolites 3,4-dihydroxyphenylacetic acid (t)OPAC) and hornovanillic acid (IIVA) in the striatum and ollactory tubercle of mice that had been treated with the ncurutoxin 1-methyl-4-phcnyl-1,2,3,6-tetrahydropyrkline (MP'I'P). MI'TP significantly lowered the spontaneous locomotor activity 1-2 weeks and 2 months after 2 injections of MPTP (30 mg/kg SC, 24 hr apart) in young adult (.l nwnths) and old mice (22-24 months old). The effect of nicotine on locomotion was biphasic; an initial stimulation uf hu:umotnr (0-5 ntin after nicotine) followed by a depressant period lasting I'rom 5 to 20 min after injection. Tolerance to the depressant clTect uf nicutine developed after the 51h day of daily injections uf nicotine (0.4 mg/kg SC, twice daily). Tolerance did not occur by day 8 to the initial stimulatory ef7cct of nicotine. A similar eflect of nicotine on locomotor activity was seen in mice treated with MPTP. The levels ol IX)PAC and HVA in the striatum were reduced by amount 21111- in (lte chronic nicotine-Ircated animals. The levels of IX)PAC, 1)A, and I IVA were reduced in the MPTP-treatcd mice; however, acute and chronic nicotine did not cause an additional change in the amine levels. The results suggests that nicotine has an influence on hxomotor activity in MI'TI'-treated mice and that this ellcct is not due lu changes in I)A receptor activity in the striaturn caused by chronic nicutine. Srrshrii, ll., Hashinr, A. and I.:rjtha, A. Pharmacology Biochemistry & Behavior 28(2):299-303, 1987. From the ('enter li,r Neurochemistry, The Nathan S. Kline Institute lior Psychiatric Research, Ward's Island, New York. cantly faster twitch half-relaxation time than pups receiving nicotine postnatally only. Pups treated with nicotine during prenatal and postnatal life as compared to control animals showed an increase in tetanic contraction duration indicating an intproved ability lo sustain tetanus. Offspring receiving nicotine pre- and pustnatally had signili- cantly longer tetanic contraction times than those pups receiving nicotine Iostnatally only. Adult EI)l. muscle, made up ul' predominantly fast-type fibers, initially has, during nurmal devehrpment, contractile parameters similar to those of slow-rype fihers. Nicotine may accelerate this maturation process, while AC"I'H/MSH 4-II) may have little ef Iect as seen two weeks afier birth. Rose, K. J. and Strand, F. L. Anna~ls of the New York Academy of Sciences 494:319-322, May 27, 1987. From the Biology Ikpartment, New York University, New York. V. Pharmacology, Biochemistry and Cell Biology Rl?VIiRS113L1?, SLOW,'I'I(;LLT-LiINbIN(i INHIBITION OIAIUMAN LEl1KUCY'I'G ELASTASE ('lic-Ala-Ala-Pro-amhu-Val-CF, was synthesized. The compound inhibits hu- man Icucoxyte clastase with K, = LQ x 10 ° M. "I'his inhibitor is a reversible, slow, tight- binding inhibitor with k„„= 2 x 10' M' s' k,,, = 1.9 x 10 ` s'. For the soluhilization ef elastin by HI.IE by I I.C'.,,, = 110 nM. This inhibitor is the most effective aldehyde or ketone inhibitor of a serine proteinase yet described. I)unlap, R. P., Stone, P. J. and Abeh•s, R. ll. Biochemical and 13iophysical Research Communications 145(I):5(tr)-513, May 29, 190. From thc Department of Bioxhcmistry, Boston University School ol'Medicine, and Graduate 1)epartntcnt of Iliixhemistry, Brandeis University, Waltham, MA. RI:SPONSIi OF TI Ili 1)NVIil.OPING NhUROMUSCl1LAR SYS'f1;M OF'I71E M RA"1"f0 NIC'O"1'INI? ANU'I'llli NEUROTROPIC PLiPT1UE FRAGMENT ACI'H/MSH 4-10 Results show no change in twitch or tetanic contraction amplitude between or within the groups tested. No nicotine- or ACT11/MSH 4-10-induced changes occurred in either post-rest or post-tetanic potentiation. No differences were seen in muscle wet weights. Pups treated with nicotine during prenatal and postnatal life as compared to control animals (saline-saline) showed shorter twitch contraction durations indicating a more rapid twitch. Prenatal ACfH/MSH 4-10-treated pups had somewhat longer twitch durations than pups treated with nicotine postnatally. In pups treated with nicotine prenatally and poslnatally as compared to controls, twitch hall'-relaxation durathons shortened. Offspring receiving nicotine pre- and postnatally had a signili- 90 EXTENDED BINDING INHIBITORS OF CHYMOTRYPSIN'fHAT INTERACI' WI'I'll I.EAVING GROUI' SUBSITIiS S,'-S,' We have synthesized inhibitors of chymutrypsin, based on Iluoronrethyl ketones, that bind at S and S' subsites. "Small" inhibitors of serine proteases, which have previously been synthesized, only interact with S subsites. The parent compound is Ac-Leu-amho-Phe-CF,H (1) (K, = 25 x 1d)• M). This inhibitor was miu)i/ied by successively replacing H of the -CF,H group by -CH,CH,CONHCH, (4), -CH; CH,CONH-Leu-NHMe (5), -CH,CH,CONH-Leu-VaI-Ofit (6), and -CH,CH; 91
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('ONI I-Lcu-Argd)Mc (7). Curccvltunding h', valucs arr 7.8 (4), 0.23 (5).0.21(6), anJ ll.l)I4 (7) µM. lixtcnding 5 to 6 by addition of Val-Olit at P,' tk>Ls not decrease K,. In cnntrast, cxtcn.inn uf 5 to 7 by incorporating Arg-OMe at P,' decrcascti K, ttppruxi- matcly I5-IuIJ, suggesting interactiun between Arg and the S,' subsite hut nu currc- srunding interaction at that tiubsite with Val. These results are in accordance with re.ults uhtaincd with Ihc homuluguu. family ul' avian uvomucuiJ third Jumaut pru- tcin.. I'rutctns with Arg at the P.' position show highly lavorable interactium wtth the prutcatic at Ihe S,' .uh.itc. 'I'hese « tiults establish that incorporation ul re.iJues which interact with S' sul.t.ite. .ignilicanUy increases the eFlicacy uf inhibitors and that valuable intircmatiun concerning the most effective amino acid cunt)w.itiun uF small inhihiturs can bc obtained Frunt the .mtinu acid xyuence of protein inhibiturs. Irnpcriali. B. and Ahclrs, R. ll. Biochemistry 2G;4474-4-177, 1987. OOther support: National Institutes of Hcalth. from the Graduate Department nf liiochemistry, Brandeis University, Walthum, MA. FLUORIDI:-MGUTATED ACTIVATION OF (iUINGA PIG NEU'1'RUPIIILS In guinea pig Ixrituncal neutraphilx NaFat a concentration ol'aFarve 5 rnM elicited a Juu-dclxndcnt. delayed and sustaincd activation of NAUI'H uxidau. Unlike in human neutrophils, in guinea pig cells, this response was inJclxndrnt ul cxtraccllular calcium. Fura211uuresccnce measurements indicated also a IluuriJe-mcdiatrJ iuudcr- ate elevation in the level ul'cytu.olic calcium concentration. Prctreatmcnl of neutru- phils with penutisis toxin, blocked fluoridc-pronwteJ activation cif NAI)I'lI oxidasc, indicating that NaF titinmlatiun was mediated by a G protein which ix a Ixxrtussiti toxin sub>tratc. NaF-clicited calcium elevation was insen.itivc lo the toxin. Upon translcr uf NaI'-stiniulnteJ cclls to a Iluoride-(rce ntcdium, superoxide release declined and cal- ciunt levels dimini.hod. '1'hc response uf the deactivated, Iluuride-prestimulatcd guinea pig neutruphils tu a.rcunJary atintulation with phurhul ntyriaate acctatc (I'MA) or IMet-Leu-Phc, was either un:d7ccted by the previous challenge with NaF (PMA) or augmented by it (the chcnwtactic peptide). In parallel to the activation of NAI)PII uxidase, Nah also induced Iran.hKalion of protein huuise (' to cell mcm- hrancs. This cll'cct was also abolished by a pretreatment with pertussis tuxin. 'fupcr, K., Avir:un, A., and Avirwm. l. liiochimica ct Biophysica Acta 931:262 ?66, 1987. Frum the I)rpartnunt ul' 13itkhetnistry, "I'cl Aviv Univcrsity,'1'cl Aviv, I,rarl. A SIMI'I.I? I'RO('lil)IIRE I;Ok NMR MIiASIIREM1iN'fS OF IN'fkA- ANI) I:X'I'KA('IiI.LIJLAK SOI)IUM IN INTACI' TISSUES The total Na' wtd both the intra- and extraccllular Na' content uf cxcisxd rat and frog tissues wa, yuantitated by ='Na N M It at 95.51 Mi Ir.. An external capillary cun- taming 33 mM Na.jI)y(I',O,,,1j, resonating ahout 30 ppm upfielJ relative to lhe 00) hptn ul'the intraccllular Na', was it~urted intu the Iis.ucs.'I'hc capillary was calibrated against a cuncctuatioro range uF rurc Na('I Suluttun. for nteatiurcmrut od' intrati'cllul:u Na', and agaimt thc Samc concentrations ol' NwCI xolutiunv containing -1-6 ntM K,jhy(I',O,,,) I in 50 mM histiJinc.('I and IIYq mM cholioe.C'I, lur mrasuretucnl (iI extracellular Na'. Spectra were recorded un tis,ucti lint in the abscncc ul Ihc .hift reagent Iur Jrlcnuinaliun ul lhc lotal Na' . AI'tcr additiun uf a K,j1)y(P,O,,,)] Sulutiun to the sample. lhe "Na ,pectra were recorded inrotediatcly s,i that data accumuladion was coinplrtcJ within 15 min. llnJcrlhcseconJitiuns, thecxlraccllular Na' resonated Irunt 10 tu 20 ppnt upliclJ relative Iu intracellular Na', and no loss in the intensity uC the cxu•accllular Na' resonance occurred due to the lability of dyspro.iunt(III )triixrlyphus- phate. 'I'hc intra- and extrucellular Na' content of thc tissue was calculated from the integrated areas uC the rcspcctive Na' resonances and that oF the calibrated capillary, frum the known weight of the lissue, and I'rum the known volumes oF the .ulutiuns added. Thti .unt uF tlic inlra- and exlracrllular Na' content ol' the lissue was in agrecment with its total Na' cuntent, detcrmined by N M R at the level ul' intact tistiue, or with Ihe total Na' content Jetermined by N M R ulicr liberation ol all the Na' Fronu the tis.ue with trichluruacelic acid or Tritun X-I(KI. Nurutiv. M. and Venkatasuhrantanian, 1'. N. Physiological C'hcmistry and Physics and Medical NMR 18233-241, 1986. Other support: Muscular Dystrophy Association. From the I)cltartnunl uf Biological Chemisu•y, College ul' Medicinc, University uf Illinois. Chicago. SIIKFA('li ('O11, ttO1.1)IiR FOR SIGNA A surlacr coil huldcr is described litt thc Gcneral L:Icctric Signa srcctromrtcr. Thi, alluw. lur,iccuratr positioning uf thc "P cuil relative to that u('thc'I I coil used lior ti,tiuc slice lucaliiattun. Itmurn•. M., Mcnc, (i.'I'., Ivicic, J., Mok. E.. Sltigu., I). G., anJ Kleps. R. A. Magnetic Resonance in Mrdiciuc 5:175-176, 1987. OIhrr.uppurt: Mu.cular I)ystruphy As.ucialinn. Frunt the I)cp:uuncnt od tiiulugic:d C'henti.try, Research Resuurccti ('entcr, and Mag- nctic Re.unance ('enter. lJniver.ity of Illinois. C'hicagu. Iiti'I'IMA'I'ION Of "1'ISSUI? I'IIOSPIIO1.11'II)S BY NA'I'IIRAI. ABIINI)AN('li '.('-NMR 'I'hc tutal phu.lthulipid cunt¢tn ul exciscd r.n inu.clc, liver, brain anJ kiJucy and ut' hwnan muscle hiopsic% was eaimatcJ by natural ahunJancc "C'-NMR alier cum- plrte aoluhiliiation ol thc tissue membranes with excc.s halothanr. An external diux- anc capillary, calibrated against pure palutitic acid and phu.phulipid ve,icles with known phosphate cuncrntration, was inserted into the tissues, and Ihc repeating methy- 92 93
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Icne carbon peak area in the spectra uf the haluthane-treuteJ tissues was integratcd versus the dioxane relerence pcak area. 'rhe amount of tissue used lirr NMR analysis was quantitated by dty weight dcterminalion aftcr "C spectroscopy was cumplclcJ. The phu.phulipid cuntenl estimated by the indirect NMR method wus in good agrec- mcnl with that measuied by direct phosphate analysis and with literature data. Fur human ntuaclc biopsies, the NMR mcthrK) can also estimate the fractiun of the total hhu,phulipids which arc mobile without treating the muscles with haluth:me. In this rcaxct human muscles could be separated into three different groups: (I) nornial and nunaxcitic n,u.clc di.entie,, (2) myotonia and myopathy, (3) huchcnne dystrophy; with uicrcasing fractiun of the mobile phospholipids in this order. Burum•. M. and Venhatayubramanian, P. N. 13iiuhimica et Biophysica Acta 923:339-346, 1987. Olher suplwn: Muscular I)ystruphy Association. Frum the 1)rpartnrent ut' Biological Chemistry, College of Medicine, University uf Illinois, C'hicagu. 111iPARIN SliOUIiNC'I:S IN'I'llli III:PAkAN SULFA'I'li CHAINS OF AN IiNUU"1'I II:IJAL CI'sLl. 1'kO'I'liOGLYCAN The structure of the glycusanrinuglycan chain uf a heparan sulfate pruteuglycan isolated fruni the cunrlitinned merliunt of an endothelial cell line has been analyied by using various degradative rnzynres (heparitinase 1, heparitinaxx 11, hcparina.e, Fly- curqniJase, sulfatascs) from l•7uvuburrerium hrpurinum. This proteoglycan inhibils the thromhoplastin-activated pathway of cuagulatiun; as a consequence, the catalytic conversion of pruthrumbin tu thrombin is arrested. Ileparitinas4: I(GC 4,2.2.8), an eniyme with slncilicity restricted to the heparan sulfate portion of the polysaccharide, releases fragmcnls with the electruphurelic mobility and the structure ol' heparin. Conversely, an atises+ment uf the size and Jistribution ol'the heparin sulfate regions has been provided by the use uf heparinase (NC 4.2.2.7), which, by degrading the hcparin uctium uf thc chain, rclcases two segments that exhihit the structure uf heparw sulf:Ue. One uf thesc segments is attached lu lhe prolein core. On the basis of these lindings, thc hrlraran sulfate chain can he defined as a cu)rulynrer containing heparin tcgwns in its structure. The cumbineJ use ot' these eniyrocs has made it possible lu establish the disaccharirle uyuence uf parts of the glycosaminuglyc:ut nruicty of this prrucoglyc:ur. Nader, 11. B., 1)ictrich, C. P., Buunussia•i, V.. and Colhurn, 1'. Proceedings of the National Academy of Scicnces, IISA ti4:15b5-1569, 1987. OOther support: hund:tcau dc Amparu a I'esyuisa do listado de S3u Paulo, ('un,rlhu Naciunal de Ihsenvulvinuntu Cicnlilicu c Technulrigico, Brazil, .md k. J. keynulJs, Inc. From the W. Ahnn Jones Cell Science Center, Lake Placid, NY. ('IIAkA("I'I:kI7.A'fION ANI) N-TIikMINAI. SI?Olll?NC'k: OF A I IG('ARAN Slll.l'IIATIi PRUI'liOCiLYC'AN SYN'1'lI1:SI"LtiD BY ENIH)'I'lIE1.IAL C'EI.LS IN C'UI:I'tlkE We have i~ulatcd frum the conditioned nudium nt' an establi.heJ enJutheli:tl cell linc a hrparcm tiulphate protcuglycan whose involvement in dre inhibition of Ihe extrin- ,ic coagulation pathway was'relanncd in previous studies. The proteoglycan was punlirJ by gel hhtatiun and iun-excltange chrunratugraphy, and appears to tx Irce of comt:uninating pruteins a. determined by polyacrylanridc-)!cl electrophure.is of the radiuiuthnated pratcin cure hcfure and after removal nf thc glyco.atninul!lycan chains hy trcaunrnt with heparitina,e. By this prixedure the M, of the protein core was c.tim:derl tu be 22/NN1. "I'he N-lernrinal end was uyuenceJ up to amino acid 25. 'I'he 21.t residue is likcly to Ix glycosylated. Analysis of the purilied krutcuglyrm by gel- lihratiun chromatography yielded K,, values uf 0.2 fur the whole molecule and 0.35 lirr the Elycusaminuglycan chains. The structure that emcrges from these data is that nf a heparan sulphate proteoglycan characterized by a relatively xnrall protein core and lew glycosaminnglycan ch:tins. ('astillu, C. J., Colhurn, P. anJ Buunussisi, V. Biochemistry Journal 247:687-693, 1987. UOther support: National Institules of Health. From the W. Altun Jones Cell Science Center, lake Placid, NY. N-GI.Y('ANSIJLFA'1'I:D 17BRON1?C"1'IN: ONB OF THG SEVIikAL SULHA'I'GI) (iLY('O1'kOTIi1NS SYN'I'IiIiSI"LED BY ENDOTIII'sLIAL CELLS IN CUI:fUkf: N-glycana.u, an endoglycosidase that cleaves Ihe bond t><tween asparal;ine and glucusanrine, releases oligosaccharides with various degree of sulfation frunr enJothe- lial cell librunectin. As shown by analysis by Iwlyacrylamide gel electrophuresis ul' culture medium cunditioncJ by cells exposed to I "SIsulfate, endutheliul cell librunec- tin is one uf a number ul' glycopruteins hearing sulfatcd oligosaccharides synthesized by this ccll type. ('ulburn, 1'., Uuunussisi, V., 1)ietrich, C. P., and Nader, H. B. Biochemical antl 13iuphysical Research Communications 147(3):920-926, Seplemlkr 30, hJR7. Othrr support: RJR Nabisco and National Institutes of Health. from the W. Allon Jones Cell Science Center, Lake PlaciJ, NY, and IAparlment uf Birn:hemistry, I:scola I'aulista de Medicina, S5o Paulo, Brazil. 94 95
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RI•;1'AIk Of~ I31iN'LOIn[l'YI(I'Nli-INI'1'lATlil) 1)NA 1)AMAGB IN IIUMAN ('IiLLS Rli(7UIItl;S A("I'IVA'l'ION Oh DNA 1'OLYMI;KASI: ALI'UA Normal human lihrubla,ts treated with r-7,r-S-dihydruxy-r-9,1/)-epuxy-7,R,'),Itt- Ictrahydrulkuiulc.lpyrcne t 131''I)1:) yielded DNA prlymera.e alpha with clevated lev- el% ul activity• incorporated I'll lthymitline as a lunctiun ul un.chcdulcJ 1)NA synthe- .i., :utJ exhibited rc,torauun ul' nurmcd I)NA-.tran11 length as a lunctiun ul' unscheduled I)NA synlhe.i,. Lipoprutein-delicient tibrublaats treated with 131'I)l: did not show elevated levels ul' I)NA Ixdyntera.c alpha activity, exhibited minimal ('IIphymiJiur incurh+uatiun, and had fragmented DNA atter 24 h of repair in t)te absence ul lipuprutem or hhu+phatiJyliuusihtl supplementation. When I)NA poly- merase bcta activity was inhihiteJ, cells witlt normal lipuprutcin uptake exhibited I'I I Il6tymidine incorporation into 131'UI:-damal!eJ UNA but Jid nut.huw an increase in UNA-titranJ lenl;lh. I)NA Iwlymeratie alpha activity and I'llIthyntidine incorporation in lipt~prutein-dclicicnl lihruhla.t+ increased tu nurnnal levels when the cells were permcahilized and luw-den.ity Iipoproteins or phuxphatiJylinu,ilul were introduced into the cells. ONA pulyntrrase alphu isolated Lrum normal human lihruhlaat+, but not Lrum lip+iprutrin-delicient hhruhla+t., showed increased specific activity alier the cells were treated with lil I)li. When 13PD1:-lreated lilxiprutein-delicient fibruUla.ts were Ikrmeahiliied and "I'-ATI' was introduced into the cells along with IilHopruteins, `P- IabcleJ I)NA polymerase alpha with signilicantly increased specific activity was i,u• I:ncd Rum the cells. '1'hcse data suggest that treatment of human libruhlast, with 13PI)G initicues unscheduled I)NA xynthesis, as a function ul I)NA excision repair, which is correlated with increased activity ol hNA pilymerau alpha, and that increa.ed I)NA Iwlymcratie alpha activity may be currclated with phusphorylatiun uf the eniynte in a rcaction that is.timulatcJ by luw-dcmity lipoprutein or by the lilwprutein cumpunent, phnyphatidylinusitul. Joe, C. O., Sylvia, V. I.., Nurman. J. O., and BmGre. 1). l.. Mutation Research lit-1:129-1.37, 1N87. Othcr.uppurt: National Institutes of Ilcallh, Texas Agricultural lix)xriment Statiun and U.S. I)epartntent ul Agriculture. From the I)cpartmentti uf Anatumy and Ccll Biology, and Physiology and 1'harrnacul- ogy, ('ullege ul Veteruiary Medicine, Tcxas A and M University. College Statiom, and Veterinary "foxiculugy anil lintumulugy Research Laboratory, U.S. I)cpartuncnt (iI Agriculturr, ('ul lege Station. I'YRII)INIi Nll('LI:U'I'II)li ('YCI.IN(i ANI) I'OL.Y(AI)I'-1tI130S1.i) SYNTIIIiSIS IN NGS'1'INCi IIIIMAN LYMI'IIO('YI'IiS NAI) is a critical culactur for the oxidation uf fuel molecturs. '1'he exposure ul human peripheral blood lymphucytes (('131.) tu agents that cause I)NA ,trand hrcal.% tu arcumul:ue can deplete NAI) puuls by increasing NAD consumption fur Iwly (AI)I')- nho»e) lurm;uiun. I Inwcvcr, the pathways oL NAl) synthexix, and degrad.uiun in viable 1'I3L have not Ixrn c;urlully do<umented.'fhe present exlxritnrnts have used radioac- live labeling tcchniyuc, tu trace the routes ol'NAI) metaMilisnt in resting 1'131.. '1'he cells could generate NAI) from either nicotinamide or nicotinic acid. P13I. incubated with I"Clnicutinic acid excreted I "C'Inic«tinamiJe into the medium. Approximulcly 5O'!• of a nrclahcled I"('INAI) pool was mclalwlitcd during 6 to R hr in tissue culture. Basal NAI) turnover was prolonged thretitidJ to liourtirld by 3-ununuhenzatniJe (1- AI3A), an inhihitur of poIy(ADI'-ritwne) xyntheta.e. Supplementation ul the nuedium with 3-ABA also prevented the accelerated NAll deEradatiun that emucd alier expo- sure ul 1'131. to deuxyadrnu.ine plus Jemycufitrmycin at cuncentratiuns previously shown ttt cau.e I)NA strand break accunmlation. These results demonstrate that yuies- crnt huatan 1'131. cuntinually produce NAD and utihze the nucleuliJe litr puly(AI)I'- rihutic) syntltesi.. C'ursuu. 1). A., Seta, S. and Wasson. D. B. '1'hr Journal ut' lumwnolugy 138(6):I()(kl-INO7, March 15, 1910. ( )thcr .upputl: National Institutes uf liealth. hrum thc I)cpartntent ul' 13.uic and Clinical Rexardi, Scripps Clinic and Research Foundation, Ia Jolla, CA. VI'I'AMINS li ANI) K INI)ll('G ARYL HY/)ROCARHUN HYI)ROXYLASL AC"fIVI'I'Y IN HUMAN C'IiLL CUL7'UKES "1'wo fat soluble vitamins, Vitamins E and K, when added into culture nteJium, were liwnd to increa.e aryl hydrocarbon hydroxylase activity in human cultured cells. 'I'he extent ul inductiun in a hcpatoma-th;riveJ cell line (I Iep G2) by these vitamin. is ol similar magnitude to those cells receiving Ixnzjajanthracene; whereas in a mammary tunwr-deriveJ cell line (MCF-7), henzla/anthracene is the best inducer 1'or Ihe hy- druxylau activity. 7'he increase of the hydroxylase activity is associated wilh in- crea.rJ levels ol a slxcilic ml{NA coding liir polynuclear aromatic hydrocarbons- induced lornt ul'cyttx:hrunm 1'-4511 with Vilamins li and K trcatmenl.'I'he.iie ol the induced mItNA i.3-3 biluhauu which is the same as that of' hy:niIalantihracene treat- nlcnt. ('hrrt. Y. 7'. and I)ing, J. II. Biochemical and Biophysical Research ('unuuunications 14.3(3):863-871, March 1(l, 1987. Frum Ihe I)epartnunt ul Pediatrics, I)ivitiiun of Genetics and Metalxtlism, l)uke lJniver,iry Medical C'enter, Durham. NC. 1'11KIFI('A"IIUN 01: HUMAN I:RYTIIROII) C'OU)NY-fORMING UNITS ANI) I)IiMONS'1'ILA'I'IUN 0FSPIiCIFIC 13IN1)IN(i Ui' I:ILY'I'lIROI'OII fIN Morphological and biochemical studies ulhuman colony-forming rmits-erythruid (('flI-li) have heen hindered by their extreme rarity. Since hurst-lunning units-cry- thnnd t IiFI l-li) develop into ('FU-li, we used normal htunan blood I3Pll-L• to generate large nuutber% uf highly purilieJ CFU-G in rirro. Using density ccntrititgation, sheep erythnxyte rosetling, surface immunngluhulin-Iwsitive cell Jepletiun, adherence to pla.tic, and negativc panning with monuclonal antilxxlic., human blood BPU-1: wcrc purified Irutu 0.017 to 0.36R%, a 22-fitld purificatiun with a 43'k, yield. The panned 96 97
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cells were cultured in rnethylcelluluse with n.•c:unihinant crythrulwictin (rlip) anJ ennditiuned medlunt lirr 9 d. These cells were then collected and ('fU-G were lurther Purilied using adherence and density centrifugation. This yielded almost 10' erythroiJ colony lurwtng cells with a purity ol' 7U+ 18%. Analysis uf these cells by light antl elcrtrun microscopy showed 9414- crythroid cells. The prominent cell was a primilive blast with high nuclear/cytuplasmic ratio, disper.asJ nuclear chroinatin and a distinct large nucleuluti.'I'he relation between the numlkruferythruidculoniesand the numtkr of day 9 cells plated in pla.ma clots was a str.u(!ht line through tite origin wilh a niaxinwnt number uf crythruiJ colonies at I U/ml uf rEp and no erylhruiJ colonies withuin rlip. Slxcific binding with'"I-rl:p showed that 601h, of the binding was inhib- ited by excess pure crythrolwietin (Ep), hut not by albwnin, fetal call +erum, and a variety of growth factur% or glycoproteins. By days 12-13 uf cell culture, when the prugenitur cells matured tu late erythruhlasts, specific binding markedly declined. In this .tudy, hum:m ('I;ll-li have been isolated in xul'ficient purity to charactcrii.e the morphology uf these rare cells anrl in aufficient numbers to measure slx:citic binding ut' l.r. Sawada, K., Krantz, S. i3., Kans, J. S., Dessypris, E. N., Sawyer, S., Glick, A. I)., and C'irin, C. l. The Journal ul' Clinical Investigation 80:357-366, August 1987. Other support: Veterans Administration Medical Research Funds and National Inxti- tutes of Health. Frunt the I)epartnunts uf Medicine and Pathology, Vanderbilt University School uf Medicine, and Veterans Administration Medical Center, Nashville, TN, and The John. Ilupkins Oncuhigy C'enter, Baltimure, MD. . ('l.ONING OFRA'l''I'ItlON-KIi1.A'I'GI) PRO'I'I?IN' cI)NA Rat prion-related protein (I'rP) cUNA has been cloned and.eyuenced. ('umpari- ,on of this cDNA with cuNA from human, hanmtitcr, and mouse indicates extremely high cunservatiun (about 95%). 'I'hc deduced partial rat PrP possesses: (a) a highly cumervcd region cumpuseJ of repetitive uyuenre, in what is luesuruably an extracel- Iular domain. (h) a hydrophobic tr:msmcmhrane domain, /cl a highly charged region which should stup membr:me translcr, ldl asuh.tantial cyn,rla,tni< domain (which cunlains all of the nonconservative substitutions and a high proportion of cunservative .uh.Iitutions), and (e) a hydrophobic C-terminus. I)ut and Northern blut analyses .uEge,t a limited expression of Prl' in raI tissues and indicate that I'rP expression is decreased in the brain during the acute phase response systemir.illy. Our results Icnd support tu the notion that PrP is a highly conserved, nornial cellular niemhrane protein of eti.cntial (althuugh unknown) biologic functiun, which may bc deposited in librillar amyluid turm as a result uf abnormal processing. Liao. Y.-C., "I'ukes.'L., Lim, li., Lackey, A., Woo, C., Button, J.D., and (Yawsrm, G. A. Laboratory Investigation 57(4): 370-374, 1987. Other support: National In.titutes of Health, I~roin the I)epartment ul'1'athulugy, School ufMedicine, University irfCalifurnia, San l r mcixcu. Tllli MAJC)K'NUC'LI:OSII)G'I'RIPHOSPHA'fASI: OF NUCLI?AR SCAhFOl.l) IS I)IS'I'INC"1' 1'ROM AC"fIN The nuyur nucleuside triph+ixnlranise uf rat liver nuclear k:afloIJ, a 46 kD protein thuught to Irarticipate in nucleikytuplasmic RNA tramlrn:adion, is rliuinct from immu- nulol;ically-iJentilieJ u:aftiilJ actin on Western bhHs, has a substantially difterent amino acid composition, and its eniyntatic activity is not affecteJ by anti-actin anti- hudie.. Thus, although lhe contractile protein actin is limnd in nuclear,caftiold and appears tu interact with RNA, it is not associated with the nucleosiJe triphusphatase activity in such preparations. C7unacun, G. A., Lackey, A., Button, J., and Smuckler, E. A. Experimental Cell Research 167:559-562, 1986. Other.uprxirt: L).S. Public Health Service, Research Career Development Award, and Academic Senate Award, University uf Califumia, San Francisco. Frunt the I)epartment uf I'atholugy, School of Medicine, University ol'Calilornia, San Franciuu. IiARLY IIYPOMH'I'FIYLA"1'IUN Uh 2'-O-RIL;OSL•: MOIETIES IN I Ilil'ATO('Y'L'l; ('Y'I'OPL.ASMIC RIBOSOMAL RNA UNDERLIES THL: PROTI:IN SYN'I'Ii1:fI(' Dlil'GC"f L'ROI)UCNI) BY CCI, ('arhun lrtrachluride (('CI,) treaunent of rats prouluceti an carly Jel'ect in methyla- lion of hepatucyte rihu.umal RNA, which occurs concurrently with a Jefect in the protein synthetic capacity cif isulated riMosumes. The CC'I; induceJ tnethylatiun defect is sIkcitic fur thc 2'-O-riMi.e position, and a corresponding proportional increase in ta'(; base methylatiom occurs in viru. Underntethylated rihonomal subunits (rRNA) f rum ('C'I,-treateJ preparations can ia: methylateJ in vitru to a much greater extent than those ti•um control preparations, and in vilru tnethylatiun restores their functiunal rap;icity. In virru methylatiun of treated rihusumal subunits (which restores functiunal capacity) occurs ;u 2'-O-ribose positions (largely G residues). In contrast, in rilru methylatiun uf cuntrul ribu.omal subunits (which does not affect functional activity) represents base methylation as rn'G, sites which are apparently methyhucJ in treated prcparatiun, in riru. Mcthylatiun/demethylation u/'2'-U-ritxru sites in rRNA exposed un the aurlace ul'cytuplatimic rihowni:d subunits may represent an importunt cellular mechani,m liir controlling protein synthesis in quiescent hepatixytes, and it appears that C'C'I, disrupts protein synthesis by inhibiting the 2'-O-rihtnc methylrtiun. ('luirsun, (:. A., MacDonald, J. R., and Woo, C. 11. 'fhe Journal of Cell Biology 105:705-711, August 1987. 98 99
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Othcr,uppurt: U.S. Public Ileallh Service and Research Career Ikvchipment Award. Frum the I)cp;lrtnlcnt uf I'athulul;y. School uf MeJicinc, University ufC'alifurniu, San Fr;mciscu. IN'fltA('IiI.I,IILAk ('ALt:'IUM IiOMGOSTASIS OULtING IIYUROGl'sN I'IiKO)(IUIi INJURY TOC'UL'I'UKlil) 1'3881), CIiLIS 'I'he effect of exposure of cultured P3881), cells to H.O; un intracellular frec calcium ( IC'a' ' I,) wa. invc,tigcdcd utiliting the intracellular fluorescent calcium chcla- tor "Quin 2." [('a' ' 1, rose frum approximately 151) nM to >2 µM ovcr a limc course that wati arungly dependent un the concentration uf H.O. used (5 x 10 'to 5 x Ill 'M). Alter cxlw,ure uf 1'388U, cells lu 5 x 10 ' M H,O., Quin 2 was fully saturatcd hctwccn 15 antl ill min exposure. I)uring this time, nu apparent change in the rate uf eyuilibru- (iun of "('a" from the extracellular medium could Ix detected, whereas in cells preloaded with "('a, nct "('a was lost from the cells at a greater rate than controls. Mca,urcmcnts uf tulal cellular calcium by atomic absorption spectroscopy cuntirnud that there was a net Iusa uf calcium frum the cells (luring the first 30 min. At time points :~-!5 min after exposure tu 11.O, the influx uf extracellular "Ca and net intracellular ('a" , Na' and K' rapidly increased. Ilalf Iimes furH.O,catatxilitimby thecelltivarieJ Irum about 8 min at 5.1) x I/l ' M 11.0. to 14.0 min at 5,0 x IO ' M. When the total (('a" I,-hulfering capaciry of lhe Quin 2 pintl was varied by increasing the loading of intracellular (luin 2 by 6K-fuld ( I.I x 10' - 7.6 x IQ' amul per cell), lhe rate uf rise of j('a' ' 1, was depressed by only 1.6-fold fulluwing exposure to 5 mM 11,0,. Ihtrinl; the rise uf intracellular jC'a " 1cell murphldu f,y was uhKrvcd by both light and scanning election microscopy and revealed that ".urface blchs" appeared during this phase of injury. Iluth the ri.c in j('a' ' 1, and "blcbhing" were observable lkliorc any loss in cell vwhility was drtccted by either loss u('I'rypan blue exclusion or luss ul' prcluaded "('r Irum the ccllti. Frum Ihe.e re.ult% we conclude the fulluwing, 1) 11.0. exposure induces a Ju,c-depcndrnl disturbvtce of intracellular calcium humcusla.iv; 2) the rise in j('a.' 1i, mediated by exposure to 11.O, in the early phase uf the iqjury, and is not Jcprndcnl un the continuing presence ul' the oxidant; 3) the rate of ritie of (('a" 1, is lar f,ely independent uf the yuantity irf calcium nwhiliied to the (?uin 2 pool; 4) during the early phase (•'_3O ntin) of rise ul' [C'a" I„ only intracellular calcium is involved in Ihc re,lxtnxe: 5) these event% occur cuncumitanlly with gross morphological ch;utge, to the plasma membranc; and lin;dly, 6) these events precede luss uf integrity uf lhc plasma mcmbrane as a permcabilily harricr. I Iy%lup,1'. A., I limhaw, l). It., Schraufaattcr, I. U., Sklar, t.. A., Sprigg, R. G., anJ ('urhrunr, ('. (;. luurn;d ol Cellular Physiology 129:356-366, 1980. Other support: U.S. I'uhlic Ilealth Service. Frum the I)cpartrncnt of Inmmnulogy, Scripps Clinic and Research PuunJ;uiun, la Julla, ('A, and Ikpartmcnt of MeJicine, University of Califitrnia, San 1)iegu. IN l'I'IRl) S'I'UI)IIiS ul• IIUMAN MUNOC'Y'I'li MIGRA'I'ION A('ROSti IiNI)(7flllil.IUM IN I{IiSPONSI?'I'l) l.I:UKU'I'ItIliNli l3, ANI) Ii•MI I'•LGU-I'IIII: Relatively little is known about muntx•ytc emigration frum the vasculature or about the fartur. that rc);ulate this process. In this study, a human in ritru nrodel of a bhwd vessel wall was used litr cxaminatiun uf munucytc transcml,nhcllal utigraliun. llmbilic;d vein endothelial cells were grown lu conllucncy trn amniou connective tis.ue, and hunlan munocytes were stinudatcd to cross the munnL•tyer in response to the chcinuatlractant. Icukotricnc 13, or 1=Mct-Lcu-Phc. The pattern and time course uf monucyte migration were similar for the two chcmotaclic facturs. In both cau:s, approximately d0-5p7 of the adherent momtx:ytcs extended single or multiple pxeudu- pixls inlo the apical cnJuthelial surlacc. This indenting behavior was also observed in thr absrncc uf chcmulactic factors. It was not ;dfcctcd by the medium (M 199 or Gey's) or mclhtxl uf mantk:yte isolation. Ncutruphils also displayed this behavior, hul only about half as many ncutruphils as monuryteti inJcntcd the endothelial surface. The integrity of thc cnJuthclium remained intact as the muntxyles traversed thc monolayer. When the nwnocytcs reached the basal .urface of the endulhelium, they frcyuenlly wedged thenisclves Ixxtwecn the basal surface uf'Ihe cnJoUtelium and its basal lalnina. 'I'he monucytcti then invaded the basal lamina and accumulated in thc connective tissue. In response lo both f-Mct-l.cu-Plte and Icukotrienc B„ munucytc migration across the cnJuthelium began as early as IQ minutes. 'fhc average rate uf accumulation in the cunnective tissue peaked at .l(1 minutcs; and by 60 minutes, 25-35% of Iltc numucytcs had traversed the monolayer. Approximately two to three times as many montr:ytcs Irtrvcrsed the endonhclium under conditions ul' chenwlaxis as under conJi- Iions of chemukincsi. or randam mi);ratiun. 'fhe.e studies pruvide the basis for under- standing the process uf monucyte migration out uf the bhxxlstream and lay Ihe IounJa- tiun lur the study uf their JilTercnti;uion into nwcruphagcs in the connective tissue. Migliuritii, G., Fulhcs, I:., Pawluwski, N., and Crunu•r, E. !J. American Journal ul' Pathology 127:157-167, 1987. Other,uppurl: National Institutes of Ile;tlth and New York Ilcart Aswciatiun. frum Ihe I)epartntent uf Anatomy and ('eII Biology, State University uf New York, (ktwn.udc Medical ('enter. Bruoklyn, and Lahuratury of C'cllular Physiology and Immunulogy,'fhc Rockefeller University, New York. '1'1(li Ii17FliC"f OF NLU'fROP1iIL MI(;RATION ON LPITHGLIAL PI:ItMIiAl3Il.I'I'Y Tu reach an inflammatory Ie.iuu, neutruphilz must frequently traverse the epithc- lium of an inl'cctcd organ. Whether Ihe actual migration of neutrophils alters the cpithelial permeability is unknown. Thruugh the use ofan in vitro model system it was po..iblc lu directly Jllermine the cfl'ect of neutrophil emigration on the transepithelial electrical resistance of the monolayer. Iluman ncutrophils (5 x IO" cells/ml) were placed in the upper compartment of a cumbined chemolaxis/resistance chamber and sunmlated litr40 min by a gradient uf 10' Mie-liirmyl-mcthiunyl-lcucyl-phenylalanine to traverse a confluent mtmolayer of canine kidney cpithelial cells grown un micropure filters. Neither the chenwattractant alone (10'-10' M) nor the accumulation of' an I(N) 101
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average of eight nruuruphils pcr millimcter trf cpithclium lowered the transcpithclinl clrctric;il resitit:mcc. Ilnwcvcr, under ccnuin conditions Ihc mi);ratiun of ncutruphils tcmporurily iurrcascd tltc Ix:rmcahility of Ihc munolaycr. The resistance tell •• 4M% wuhin 5 min it thr migratory cells were stinwlatrJ to reverse their migr:niun acruss Ihr samc monulaycr. As rc-mi):ruUun continued, the resistance returned Iu its tnitial levels within (>!) min. I)uubling the initial nculruphil concentration to II) x I(P ccll.lnil rc,ultcd tn lhc accunmLttitm nf an average uf 6(i neutrophils per milliuu(cr uf cpithc- lium and an :rvcravc fall in rr.islancc uf 46'k. ir = 0.98; /' < Q.IM)I ) in 40 min. If thc resistance had fallcn •45%, removal uf thc neulruphils remaining in the upper cuin- p:ultucnt resulted in n return uf lhc transepithelial electrical resistance to its initial level within 65 min. Ituwrvcr, when the fall was -451',(r., the resistance only recovered tu 23.5% uf its initial levels within lhe samc lime framc. Thus, these results suggest that the integrity uf an cpilhcliutn can, unJer ccrtain conditions, be affcctcJ by Ihc cmitira- liun of ncutrophils, hut that this cffcct is cithcr cumplctely or partially reversible within 65 min. Milks, L. C., Conyers, (i. P., and L'rumer, E. /l. The Journal uf C'cll Biology 10.1(Nu.6, Pt. 2):2729-2738, Decemter 1986. Other support: Natiun:d Institutes ol' Heallh and New York Ileart Association. hrum the t)cparuncnt uf An:uonry and ('ell liiolugy, Stale University of New Yurk, Hcallh Science Ccntcr at Brooklyn. II)ENTIFICA'I'1ON OF('HROMAFF`IN (iRANUI-Ii-BINI)ING PRU'I'EINS- RI:LATIONS111P OF'1'I Ili C'HItOMOBINI)INS TO CALHLIiC"1'RIN, SYNIIIIiIN, ANU'I'III? TYItOSINI? KINASE SUBSTRATES p35 ANI) p36 At least 23 soluhlr proteins (chrumohindins) bind to chronr,diin granulc mem hr:rnc, in the presence of (':t" . ht order to further Ihc identification of thc chrumabin- Jins and to determine the roles they may play in exocytosis or other aspects uf chra maffin cell biology, several of these proteins were compared to other known mcmbranc-hinding proteins. Chrumuhindin 4 was identilieJ as a 32-kDa protein called calelectrin or cntluncxin. immunolugically related proteins were JctcclcJ in bovine brain and human plalclcts. C'hrunwbindin 20 was identified as a 67-k1)a variant of . calclcctrin and was litund to have the activities of lhe synexin inhibitory protein, synhibin. C'hronwhindin 8 was identified as p36, a substrate fur Ihc tyrusinr-spccific kinase. pp(,0-. ('hrumobindin tt was also dentonstrateJ to unJergo phusphurylatiun preJomin:utUy un alkali-scnsilivc sites during stimulation uf thc chrum:tffin cell with 20 µM nicutinc. ('hrunwhinJin 6 was iJentifieJ as p35, a suhstratc lior thc lyrusinc kin:nc actrvuy a,ux iatcd with the cpidermal growth factur receptor. ChrumobinJin 9, which is knuwn tu he a substrate tor protein kinase C(Ca" lphusphulipid-drpcntlcnt cncymr), was litund to he immunolugically related to p35 and may hc a prccursur of chronwbinJin b. 'I'he identification uf thesc proteins from the chromaflin system may be uscful m the charactcrizatiun uf similar, complex groups of mcmbranc-binding proteins that have been observed in other systems. C'reutz, ('. 1:., Zaks, W. J., Hatmnan, H. C., Cranc, S., Martin, W. H., Gould, K. L., Oddic, K. M., and Parsons, S. J. i The Journal of 13iulugical ('hemistry 262(4):1)ih0-1868, I chruary 5. 1987. Other suppure National Instllutes of Hcalth, Nutionttl Science FuunJetiun and Alncri- can 1lcart Association. Prom thr I)cparlmcnts uf 1'harmuculul;y and Micrtubiolugy. Univcrsity uf Virginia, Charluttcsvillc, and llte Molecular Biology and Virology LaMrr:uury, the Salk htsti- lutc, San I)icgu, ('A. M1r.CHANISMS UFTHE ItGA(."PIUNS OI' SOMIi COI'PIiR CUMI'LI;XIiS IN TIIIi PRliS1:NC'l: Of DNA WITU O. , ILO;, ANI) MOLECULAR OXYGEN 'I'he kinetics anJ the reaction mechanisnt uf sonic copper complexes of 1,10- phcmmthrulinc, 5-nin•u-I,f0-phcnanthrulinc, and 2,2'-bipyriJinc with O, , H,O„ and O. in the presence tif calf Ihyntus I)NA have been investigated with use of the pulse radiolysis tcchniyuc. We have founJ that both copper (II) anti copper (I) complexes bind lo I)NA. The tcrnary complexes react very slowly with O, relative tu lhe free complexcs, while lhe rates uf thc oxidation of frcc and hnund cuprous complexes by I LOt arc :dmost Ihe same. Thcrcfitrc, these ternary copper complexes turned out tu be good caUalysts uf thc reaction between U; and H.O;. GUI(ISICnI. S. aINI ('-z(ll)Skf, G. Journal uf thc American Chcntical Society IQ8(t)):2244-225O, 19R6. Other support: Gcscll.chal't lur Strahlen Forschung Ncuhcrk-rg, Federal Republic of Gcrnrany. Frunt the department of Physical C hcmistry, The Hcbrew Univertiity uf Jerusatem, Israel. t)ISTIN("I' AN'I'I(iliNI(' CIIARACI'GRISTICS OF MURING PAItIhTAL YOLK SA(' LAMININ Two monoxlunal antibodies (LAM-A and LAM-li) specific for luminin from uormal and ncoplastic parictal yolk sac (PYS) cells were produced in rats imntuniicd with a muusc yolk suc carcinoma cell line. Both antibodies inuntmuprccipitatcd the 4(N),(MM)- and 2(M),(MM)-I)a chains uf laminin and reacted with purified PYS laminin and 1{LISA. LAM-A reacted with mouse anJ rat I'YS lamintn, whereas LAM-B reacted only with mouse PYS laminin. Formaldehyde- anJ mcthanul-lixcd aJult and fctal sumatuc tissues were immtmuhialuchemic:dly unreactive with either uf the two anti- bodies. In arctunc-fixcJ tissue sections, both antibodies reacted weakly with sumc mcdullary tuhulrs of thr kiJney, the fitllicular baumcnl membranc of thc ovaries, anJ Qic.cminifcruus tubules. The antibodies appear tu react with lhr pulyprptidc Jelcrmi- nant, residing un lhc Icrminal portion uf thc lunfg arm uf laminin. Il is concluded that huninin derived frum normal or malignant PYS cells has distinct antigenic sites that are inuuuntxhemically not apparent in most other basement membranes. Wewcr, U. M., Tichy, D., Damjanov, A., Paulssun, M., and Drunjanuv, l. 102 1 103
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x« ~ ~ I)evrlulrorcnlal liwlupy 1? 1:.1')7--1117, 1987. Othrr ,uhlu,rt: Ucani,h ('ancer tiuc•iery und National httilituteI of I le:dth. I•rum Ihe Lahulatury 411 1'athuhoEy, Naliunal ('anrer Institute, Ilethexla, MI); I)cparl- ment of 1'allwlugy,'I'hutna, Jcllersun (Inivet.uy, I'hdaclelphtu; and Max I'l:mcl. Iutiti: tute lut 14uorhetwe, M:ultnaied, Federal Republic of (icnuany. I.li("fIiNS AS MAktKIiltS fOR IsQ)SINOI'MII.IC I-1i11KOC"Y'I'IiS Lectiu Irum Gtillimia .implicitiilia ((iSA-I) and soybean (SIIA) are rcliahle markers furhum:m eutiinuhhilx. In thiti.tudy we have shown that IluuroKhrome Lthti led (iSA-I antl Sltn ran Ix• used t'or a{x:cific labeling iafeu.inophtlx in parallin emheclded ti..ue sections, in peripheral blood sntears and in cell suslxxn.iunti prepared lior Iluw rytumetry. fhe,e two Irctins are u.rlul diagnostic reagents which could he applird lur Iurther rharaclcrii:uiun of rytuplaxmir runt(xtnents selectively Ii,unJ in human ru.imophils. Lei. M. ('.,'furrtnuv, I), anJ.l)unijunur, l. I It.tuchrnustry Hb:209-37 3, 1987. OIher tiuphurl:'fhe Lalur Foundatiun. Prum the IkparUuent ul I'atftolugy, Jetfer.un Medical C'ullege, Thomas Jcllcrsun University, Philadelphia. IiVll)I:N('Ii FOR 171Ii I:XIS'17'.NC'Ii OF'1'llRliI: C'lII('K LUNG "IR(H't)I:LAti l'INS Three trapoelaain pulyprplides are identifieJ anwng the cell-Iree translation products ol rhicl. embryo lung mRNAs and organ rtdtures extract.. "I'he trolwoela,tins are JititinguixhtiJ by one and two dinten.ional gel eleclruphoretic systems and :u•e all irnmunurearlive wilh nwno.pecitic chirk trupticla,tin antixrum. '1'he ratiu uf the three Irulxx•la.tin, doks not vary .ignific•antly between 11) and 16 days nf lung embryugene- ati. The third truhoela,tin (c) is fiwnJ lu co-migrate wilh trulxielasun h on SUS- pulyarrylantide gel clutruphorrestti but is visible al'ter ryanugen hrutuide cleavage of reticuluryte lysate pruteins. Immunuprecipitates (rum lung organ culture also cunt:tin trupuel,•itiWts a, h and c. Rich. C. 13. and I•isl<•r, J. A. Riurhemir:d and Biophysical Research Cnnmtunirations 146( t):1291-1295, August 14, 1987. OIher.upport: National Institutes of Health. I•rum the 1)epanment of 13iuloE,y, Syracuse University, Syracuse, NY. 104 t I I I INS(II.IN-LIKIi UILOWl'I1 fA('"I'UR I, SOMA'fOMlil)IN C, INI)(J('IiS'I'III'. SYN"fI11iSIS OF'fltUl'OIiI.AS'fIN IN AOR'fIC 7'ISS(Ili 'I'he arliun uf I( i{~-1 ( in.ulin-hl.e growth factor I) on the .yulhea% of Iruptn•laain in chick emhryontr aurtae was examined. Maxintal and selective .tit,nulatiun of Ihe relative (-IO'f 1 and absolute (145'/ ) rates ul uulxkla.lin synthe.is over control ic- rurred al an I(iF-I concentration uf I(X) ng/nil ut meJtunt. 1'arallel tu the increase in ,~ynthe,i, was a9?1/r increase iu Ihe auwunt uf trulxklaain artivuy Ixr UN) ng uf puly (A)' RNA IransLneJ in a rell-lire system. 'fhe relative rate ul trulxicla,tm synthesis achieved :u nutxtntal stimul:uiun is greater Ihan that observed during normal aurlir eiuhryuErne.i.. 'I'he aimulatury artiun uf the hurmune un ela.tut ayuthesis ap(xars to he at a prelramiuunal level perhaps invul'vinE increauJ transcription ur stahili/anun ul' the Irupiklaslin mRNAs. These result, suggest that ICiF'-I may play a key role in the reculatiun uf ela,tugene.is in arterial (ixaue. I•i,•ctrr• J.• Rich, C. I3. and Flurini, J. R. Collagen ancl Related Research 7:161-16'), I92i7. U(her suplwrt: National In.tilute, ul IIcalth. hrum tlle l3iulugy I)ep:ulmenl, Syracuse University. Syracuse, NY. MI:'1'IIUI)S IN l.At3ORATUKY INVI:S'I1(;A'fION. I'ULMONAI2Y FIIiKOIiI.AS'I:ti: A MOI)lil. SYSl1iM POk SI'l)I)YINCi IiLAS'fIN SYN'1•IIIiSIS I'uImunary intertilitial tihruhla\Iti were I.UIateJ frum 4-day neuncdal ra1 lung :md rulttnrd lior v;uytnt; periuds of lime al'ter lir.t passage. "fhe ability of Ihe rell, tu .ynthe,iir huth suluhle anJ insoluble cla,lin was examined by hiurhemical, immmnu- lul:ir:d, :md uhraxtrurtural techntyue.. The rellx .ynthexiie a trulxwl:tain of 75,1NN) J.dtun% which is rrus,-linked in the rxtrarellular nrnrix to fiorm .ul lirient anwunl.r of in.uluhle ela,tin tu he characleriied directly hy antinu acid analy.is. AlthuuEh Ihe cells prnilure a.uluhle lirnt of ela,lin immediately atler cuulluency :utJ llte depusitiun in the ntau•ix begins ,hurlly thcreafter, the artual rhetnical Jefinition of alMnli-retii.I:mt clasttn does nut occur unlil 4 weeks alier cuntluency. The ahhaurnl mulecular weighl of trupuela,tin .yndte.iied by the cells agree. wuh Ihe tuulerul:u %in of trupoxlastin fuund in urgan culture of neun:ual IuuLs. 'I•he hulrnuu:uy Iihruhla,l cultures provide :m imlortant nwdel ,y.t4nt lor exantining Ihe retuLuiun ancl re.pun,e ul ela,lin synthe.is in lung interstitium. ('ampagnome. R., Ite);an. J., Rich. ('. lt., Miller, M., Keene, 1). k., Sal.ai. I.., auJ /•ieiwr, J. rl. l.ahuratury Inveatigatiun 50(2):324-230, 1987. Other .uplxn1: National Itt,litutes of I lcalth. From the Biology 1)eparlment, Syracuse lJnivertiity, Syracu.x, NY, and Shriners Ifuspit:d Iur C'rippled ('htWren, I'urtland, UR. 105
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Mti('IIANISM OFT1It: ('YANII)I:-('A'I'ALY'Gt:u OXIL)A'flON c)1' u-Kli'I'OAI_I)I'slll'UHS ANI) a-KI:`I•OALCUIIO1 S ('yanidc catalyic. thc reduction ul'Jioxygcn uruf Icrricytochrumr v by dihytlrux- yacctunc phu,phatc.'I'hc rapid initial phase of these reactions, but not Ih+: subscyucnt slow pha.e, wa, aul!mmntcrl hy incuhatmg thc Iriu.c phusrhatc ar:rubically or auacmhi• cally ;n Itll 9.0 priur to aitdmg Ihc cyanidc. 1'hr acrubic incubation, which wasmust cl Icctivc, waa as+ucialcd with u dcchnc in cncJiul, whcrca.lhc Ics cl lcrtivc anucrubtc incubation Was accump;uued by an increase in enediol conlcnt.'I'his suggested that the rc-LcluaklchyJc product of autuxiJatiutt of Ihe cncJiul, rather than the cnediul it.cll, was responsible lor tltc rapid phase rcacitun which fulluwcJ addition uf cyanidc. 'I'ht. wa+% conlirmcJ by exploring the cyanidc-catalyzcJ oxidation uf thc cx-kctualdchydc, phcnylglyuxal. 1'hc inhibitory clTcct of the manganesc-cuntaining su(x:ruxidc disnut- la,r mdic;rtcJ Ihal O0 was a Linctically inqxtrt•ant inlcnncJiatc rtf thc rapid phase reaction. A reactirm nuchani.m is proposed which is consistent with Ihc results prc- }cntcJ. Matihinu, 'I'. and I• i-idurirlt, 1. Archive, nf Biochcmislry and Biophy.icti 252(11:143-170, January 1987. Othrr suplrort: Natiunal Science Foundation and the U.S. Anny Research O17ice. Frunt the I)cpartmcnt of Itirxhemititry, I)ul.c University Medical ('enter, I)urhant, NC. STA1311.1'LA'f1ON ANI) 1'AR'I'IAI. ('t1ARA("1'I:RI'LA'I'IUN 01: 1'.NI)O'I'IIIiI.IIIM-I)I:kIVI?I) ILI:LAXING FAC I'UR FROM ('l1l:fllRlil) lil)VINIi AOk'fl(' l:NU(YI'l IIiLIAL ('IiLIS 'fhc cxtrcntc lahility uf thc chcntirally unJrlincJ vasurc(;ulatory ntcdi;uur. cndo thcliunrJrrivcd relaxing Iarlur (IiI)I21.), ha% been ovcrccmtc. 'I'he activity ul I:UItP. Irunt cullurcll Ixivtnc aurtir cmh)lltclial ccllti .%tintul:ucrl with A231H7, wati tilahiliicd by aciJiGraliun. An adJiiiunal action uf lil)RF, platelet Jitia(;Errgatiun was uu•d a+ a un.itivc and convenient hiuassay to monitor purilicatiun. EURI appears tu tx a hydruphilic molecule, rapidly inactivatcd unJeralkalinccomJitiuns. I Iuwcvcr, activity is rcanr«I upant rcaci:lilicatiun suggesting that this instability results Irum a readily reversible chemical rrocr,,, 'I'hc aahiliiatiun and partial purilication uf lil)KI:.ct, thc s1agc lin its further biochemical and nceuucal characlcriruiun. Murr;ry, 1. 1., l•ridurirh, l.• Makhuul, R. (iG and Ilagrn, 1'. O. Iiioxhcmical;mJ Biophy.ical Rcscarch('untmmnicatiuns 141(2):689-')6.1)cccnthcr 15, N)Kb. Other.uppurl: II.S. I'uhlic Hcalth Service, National Science Fuundatiun, U.S. Army Research OI'licc, auul 121R Nabisco, Inc. Frum the 1)uhe University Medical Center, Ikparnncnts o( Mcdicinc, Biuchenti,try, and Surgery, I)urham, N('. I:Nl+("I'OF IIYI)ROCiliN I'IiROXII)tiON'1'lIN IItON-C'ONTAININ(i Sl1I'likOX11)li I)ItiMlll'ASf; OF l:S('lll•:Kl('11101 C'Ol.l '1'hr irun-cuntainint, superoxide di.muta.c frum I::sr1u•rirhiu rnli is inactivated by IL( ). tu a liniit ul -9014. Whcn currertcd hrr the I I.( ).-rcsi.tant portion, this inactiva- liun was Itr.t order with respect tu residual artivity and exhihiteJ a p.ruJu-lirst-urikr rutr cunstwu uf 11.1N><i ntin ' at 25 "C tn /t.?-i mM IIA, at ptl 7.R. The superoxide Jiwiutasc activity rcm;rintng attcr trcaunrnt with 110. diffcrcd Irunt lltr activity uf thc native cniyrnc with respect to heat .tabihty• inhibitiitn by ui.idc, and inactivatiom by Iighl in Ihc prcknrc uf rose hcngal and by N•hromosuccinitnirlc. 'fhc native and Ihe ILU.-ro:nlihcJ eniyutes were indistinguishable by clcctrupharcxis un ptlyacrylautirlc prl.. Inartrvatiun uf Ihc cniynoc by 11.0. was accontpwticrl by lu,s of tryptuphan and xtnic lu.tis uf iron, but there was no dctcctablc luss uf hisudinc ur uf uthcr aminu aciJ.. 11.0. trcautunt caused changes in the uptical .Ixclrum uf thc enzyme. Inactivation ul' thc cnEymc by 1 L( ). depends upon thc rrun at tho active site. 'I'hu., thc aprx:ncymc and 1hc m;rngancsc-suh..litutcd cniyntc were uncdlccteJ by ILO,• We conclude Ihat rcac- tiom itf tI O with the iron at Ihc activc site generates a potent oxidant capable of altar!`ing trylttaphan residues. A ntcchani.nr is proposed. lieycr, W. F., Jr. :md 1•'ridurirh, l. 13iuchcmistry 26:1251-1257• 1997. Othrr,upp+rrl: National Science FtiunJatiun and U.S. Army Research Oflicc. Fram Ihe I)cparhncnt uf liiuchcmistry, I)ul.c CJnivcr.ity Medical Center, vurham, NC. ASSAYIN(i FOR Slll'I:ROXIUfi I)ISMIffASH A("fIVITY: SUMIi LAR(i1: ('ONSIiOtIHNC'IS OP MINOR ('IIANGI:S IN ('UNI)I'I'IUNS Most assays lor superoxide di.ntutasc depend ulwn competition between the cniymc and suutr indicating scavenger lor O, . We have invc.tigatcd the cffects ul cxlkrintcntat variablc, on a,.ay% based upon the use uf cithcr fcrricytuchruntc r or nitro blue tr(raiulium. Our results should help invctitigalor. Io avoid the nwncruus putcntial pitl:diti which nrcczaatily surround these assay methuyh. licycr, W. F.. Jr. anJ 1•'riduriclt. 1. Analylical Bi:xhcmitilry Ib1:55N-5bb, 1987. OOther support: U.S. Arnty Research OIlicc and Naunnal Science Fuundatiun. I'ront the I)cparuntint uf Biochemistry. Duke University Medical ('enter, I)urhant, NC. 106 107
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4. d' (e,43 I)11IYI)ItOXYISOVAl.liRA'I'li 1)I•.IIYI)RA"I'ASL•- A S111'L•ItOXII)Ii-SIiNSI'I'IVIi I:NZYMI: Inrrraring the inuurcllular Ilux el O, by incuhaling aerobic lim-lrrrirhtu culi wult harayual ur plumh11Ln1 nr:nkCtll)' lowered the lx,(3-dihydruxyisuvalrralr Jrhydiala.c nrti% ily clctectablc in rxtrarl. Rum these cells. This clfcct was not u:cn iu thr ub+rnre ul Jiuxy):cn :md was rxarrt ha1cJ by inhibiting protein hio.ynlhctiix with chlurauythrn- i.•oL l'hru cltcct, of I,.ir,iyual and ul pluntbagin were Mtth time- anll ranrrntr,nulrn- clcprnllcnl. Tramvlrr of li. ruli froin aerobic to anaerobic conditions caused a rchlwncl of dtr Jrhyllr:da.c activity, in thr cuntinucd presence nl parayuat and of rhluramphrnr rul, imhcatmg the ptc.cnrr of a mtich:mism fur reactivating this cniyinr. "1'he in+tahil- ity of thc JchyJtatau activity in cell extracts was exacerbated by selective removal ld' .uperux iJr Jisrnulasc:. but nut of catalau, by intnrunuprccipilation. Addition nf cxugr- nc,uv .ulx:mxicle di.mutaux reversed Ihc: cflcct ol' irnanunuprecipitatiuu; w•hereas cala- lase ur inactivc superoxide Ji.nrutase were inel7eclive. We conclude that the JchyJra- ta,c is inactivated by O, .'I'his could account litr the bactcriuxtatic c(Icrts uf diuxygcn and ul' paraclu:n. Kuu, C. F., Ma,hiuu, '1'- and hridurirh, l. '1'hc Journal of Biological Chemistry 2G2(I(I):4724-4727, April 5, Il)tS7. Other support: Natiunal Science FuunJatiun and U.S. Army Research OI'licc. Frum thr Department ul liiurhemistry. Duke University Medical C'entrr. I)urham, NC. '1'lili UXII)A'I'ION UFNAI)tI BY "11:7RAVALI:N"1' VANAI)IIIM VanaJyl (V,,,,) .:ills autuxidiie in ncutral ayueuus wlutiun yielding O. plu. v:mad:nc ( V,,,) anll thc,c, in turn, cau.e: lha oxidation ul NAUI I• hy a free radical ch:un rcarliun. "1'hiti nxidatiun ul NAI)I I was inhibited by auperuxidc di,tuuta.c• hul nut hv a sti•avrngrr of I1t )'. Whrn 11.0. wa. prrunl V,,,, caused rapid uxidatiun of NAI)I I by a nrurr,. which was un:rlTcrtid by .ulxroxide di.nuuatic btn was inhihitrd by a u•:rvru- grr ol I I( Y. "I'his :qppr:urcl tu Iie drpendrnt ulwn reduction of I 1.( ), tu O1I" plu. I 1( )'• by V,,,,• lirlluwed by uxidatiun ul NAI)II by I1U'. Since there are rcductanls within crlls capable ul reclucing V,,, lu V,,,,, these reaclitms are likely tu contribute to Ihe toxicity of vanad:ur. I-iac•hrv, S. anll I•') idul•u•h. l. Archives ul liiunceini,try :md Biophysics 255(2):274-278, 1987 Otlirr.up)x,rt: National Science huundatiun, ll. S. Arnty Research OIlicc, Interna- tional Atumic Iinrrt_y Agenry• and Bulgatrian AcaJrnty ol' Scirnrr.. Fmni thr Intililulr ul I'hy.iulugy, Bulgarian Academy ul' Scicnrr., Suha, Btdg:tria, and I)rpanmcnt ul liiuchrmi+lry. I)uke llnivenity Medical ('enter, 1)urham, N('. lux Tllli OXIUA'llON 01; NAI)II BY VANAI)ATI: PLUS Sll(;ARS Sugars and sugar phosphates enable vanadate lu catalytc the uxidatiun of NAI)LI. Superoxide dt.muta,r inhibit.lhis oxidation. Incuhalilm uf sugar. with vanadate. prior to addhiun of NAl)I1, accclerates this oxidation of suhsryucntly added NAUIi and climinatrv the lag phase otherwise noted. Incubation uf sugars with vanaJatc also results in the reduction ul vanaJale to vanaJyl• with appearance of a blue-green color probably associated with a vanadyl-vanadatc complex. It appears that sugars reduce vanadatc to vanadyl which• in turn reduces O, to O; and that vanaJalc plus O; thrn cat:dyics dte uxiJatiuu ul NAI)(I')l I by a Iicc radical chain reaction. Such oxidation ot' NAI)(1')II may account litt several of thc biological ellcctx ol vanadatc. l.icnhcv, S. and Fridorirh, l. Biochimica ct lliaphyvica Acta 924:119-122, 19H7. Other support: National Science Foundation. U. S. Army Research Oliicc, Inlcrna- liunal Atomic Energy Agency, and Bulgarian Academy of Sciences. Frum thc Institute u( Physiology, Bulgarian Academy uf Scicnccti, Sulia, Bulgaria, and lkpannunl ul Liilxhcmistry, 1)uke University Medical Center, 1)urhant, NC. A I)I?MONSTRA"I'It)N'I'IiAT U' IS A CRUC'IAL INTI?ItMIiUTATE IN TUG HI(iIl QUANTUM YIGLI) LUMINI?SCENC'E OF LUMINOL. '1'hc chcmiluminc.ccncc ol' luminol, due to its reaction with alkaline h1.O„ is inhibited by superoxide dismutasc uf by hydroxyl radical scavengers. I Icnr.r(in ntark- rclly enhanrrs this 11.1),-inclucecl luminescence ol luminul and Irsun., but does not rliminatr, the scnsitivity towards these inhihiturs. Reaction ntechanisnts are proposed to accuunt lia• these rr!crd1.. Since luminul luminescence dalxnds upon a reaction txlwren the luntinul radical and (Y„ and since the Iuntinol radical can reduce diuxygcn tu O: , superoxide di.nmtau-inhibitahlc Iuminul luminescence cannot k- reliably used as a Jclrclur lif U,' pruducliun. Miller, I:. K. and !•ridryvirH, l. Journal uf Frrc Radicals in Biology and Medicine 2:1(17-110, 1986. Udter suppurt: U. S. Army Research Ollirc and Natiunal Science Foundation. Frlnu the I)rparUnrid uf Biochemistry, 1)uke University Medical ('entrr, t)urham, NC. INI)tl("IlON OFStIPIiROXII)N 1)ISMIPI'AS6 IN I::VC'Nl:lU(YIIA ('O!.l BY I11iATSI IOCK lixpuan•r ul midfluFai ithmic-pltasc culturrs ul lsmrhernrAiu ruli Ii lu 4t{"(' lur I hr elicited :m induction ltl the mang:mcu-runtaining superoxide Ji.mula.cu (MnSUI)), which became more pronounced during I hr of recovery at .17"C'. 'I'hu inductum required protein biu.ynthti.i., ,incr it was suppressed by chluramphcnicul. Indurtlun
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aa of 11InSOU aplx•arrcl tci hc a rr.rxmst: to a hcat-nurdiatcd increase in O: pruJucuun because it was Jiuxygen dependent and because heating to 4t;"C' duuhlyd the ryaruclc- resistant fraction of thc total respiration. Privallr, C. 'I'., and hriclurich, 1. PrurrrJings uf Ihr National Academy uf Sciences. USA 84:272.1-2726. May 1987. OIhcr support: II.S- Arnry Research Ullice, National Science Foundation, and Na- ticrnal hrsututc, of I Icalth. Fruan the Department ul' 13iurhemislry, I)uke University Medical Center, Durham, NC. SUI'I:ROXII)li I{AITI('AL INITIATI:S'1'llli AUTOXIDATION OF UIIIYUROXYA('I-I'ONli' The acruhic xanthinc oxidase reaction causes the cooxidation of dihydroxyacc- tone in a Irnxrss which is strongly inhibited by superoxide Jisnwtasc but not by catala.c, I IO• uavenEers, or iron-inactivating chelating agents. Several molecules ul the sugars can hc uxiclii.ed per O: introduced. A free radical chain nuchani>m, in whrrh U; acts both an an initiauar anJ ax a clwin propagator, iti prulwuud. Simplc sugars capable ul tautuntcrinng to enediols may now be added to the list uf biologically relevant targets lur O:. Ma,hino,'1'. and 1•ridurirh, 1. Archives ol' 13ica:hrmistry and Biophysics 254(2):547-551. 19R7. Other support: U.S. Army Research Ollire and thc: National Science Fuundatiern: Front the I)cpartmcnt of l3icxhemiatry, I)ukc University Medical ('enter, Uurham, N('. 'f11I: I:Ftli("I'S OF PARAQl1AT ON h.'S('111cY11('lllA COI-l: DISTIN("fIUN 13t:I'WliliN I3A("I'I:RIOS'fASIS AND I.IiTtIALI'I'Y Parayual exerted a progressively murc pronounced bactcriastatic cl7cct un l:.c- clu•richiu ruli as its concentration wati raised in the range 0 L0 µM. In ccinlrast, cunccntratiunl of I(H) µM or greater were required hclirrc.ignilicant Iethalily could he observed. 'fhis bactrriustatic rl'fcct of paraquat could tx: climin:drd by .upplcmrnta- ticrn of the glucose plus-salts medium with either yeast extract or a casein hyJruly,:uc. This protection was seen whether the supplcnrrnt was added a few minulrs prior to or lolluwing the addition uf parayuat and was thus not duc to the inhibition of active uptake ol' paraquat by the cells. The lethal cffect uf high levels ol' parayuat was not influenced by supplementation of the medium with yeast extract. It fulluws that thc hactrrio.tatic anJ lethal cffccts of paraquat involve attack upon distinct targets within the cell. 110 Kitilrr, J. and h'riduric•h, 1. Journal of Free Radicals in Biology & Medicine 2:245-248, 1986. Other suplxirt: U. S. Army Research Qllicc and the Natiunal Science Foundation. Fruhr the I)rparturent uf Biochemistry, 1)uke University Medical ('entcr, Durham, NC. I'sFTl:(`I :5 OFTAkA(111A't' ON Tl lt; GRliliN ALGA I)UNA11l:7.1-A SALINA: I'RO'1'G("1'ION 13Y 7'IIE MIMIC OF Sl1PliROXIUtr I)ISMU'fASI:, I)IlS17iRA1: Mn11V) I'arayuat caused a timc-, dose-, and light-dependent bleaching of thu halophilic green alga 1)ucnrlicdlu salincr. Sublethal lcvrl% of paraquat elicited increases in ccll content of both .ulx:roxide ditimutasc and CalalaxL, with changes in the pattern of elcctrunwrphs ofthc.r encymex. Iksfrral-Mn(IV), which catalyies the disnwtatiun of O,-- in rhru, protected against the toxic cltcrl of paraquat. Desfcral (Jcsferoxaminc mcsylalc) alone was toxic tol). ,culinu, and Ihe salts uf Mn(II), Mn(III), and Mn(IV), in (he absence of t)c.feral, did not protect. 1)c:sferal-MnIIV) is grecn, hnt its absorb- ancc was 15% or less than Ihc peak absorbances due tu the chlorophyll in 1). srrlirw under the conditions ol'exlxaure; hence, masking of incident light could not have been Ihe basis of thc protective cflcct ut'Ihe complex. Incubation of the cells with Dcsferal- Mn(IV), for up to 8 h prior to thc addition uf parayuat, did not increase its protective action, and hrief watihing, fulluwing 30 tnin incubation with the romplcx, clinunatcJ ils protective rlJccl. Nrithrrcalalatic nor superuxidc Jisntutau, added tu Ihe mudium, provided protection against parayuat. 71re.r results xuplwrt the view that I)esferal- MrNIV) gains entry into 1). sulinrr and protects against Ihe lethal cffcct uf parayuat by lhrrr ratalyiing the Jismutation ol' O. -- into H,U, + p,. kabiuowitrh, 11. 1)., Privallc, C. T. and h'riduvirh, 1. Free Radical Biology & Medicine 3:125-131. 1987. Othcr support: National htxtitutes of llralth, National Science Foundation and U.S. Army. From the I)rpartntrnt of Biochemistry, 1)uAe University Medical C entcr, Durham, N('. AN A("I'IVI'I'Y S'I'AIN FOR t)IIIYI)RUXY-A('ID I)IillYl)KA'1'ASIi An activity .tain has been dcviuJ for thc dihydroxy-acid dchydratasc. When applied to IwlyarrylanuJc gel clrrtrulnceruErams uf cruJc soluble extracts uf litichcri- chia ruli, it dtileclyd a single elcctrontorph.'I'he intensity of.taining increased with the anwunt of extract protein applied tu the gel. Activity staining demonstrated than (a) :rnarruhically grown cells contain tnorc extractable dehydratau activity than do acru- brcally grown cells; (b) exposure ofF.. ('uli lu 4.2 atm 0,causcd virtually complete loss of activity; (r) exposure uf rulls to paraqual or pluntbagin in the presence ufdiuxygcn, hut not in its absence, rauslJ a massive loss of activity. Thusc data illustrate the utility III
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of this activity tit.un anJ dcmun,trate that the JehyJratau is inactivated by O. gcncr- atcd within cells. Kuu, C. F., Mashina,'I'. anJ Fridrn•ic•h, l. An:dytical I{icxhrtnt.try I(A:52G-53(1, 1987. Othrt support: U.S. Army Research uflicc, National til'IenCe I'UUnilatlUn an(I National In>tuutrs of I Iralth. Frum the I)ep:rtment uf liuwhemistry, I)uke University Medical Center, L)urhum, NC. IiXAMINA'I'ION OF'fI4F ROI.I: OF AR(iININIi-143 IN Tlll? IIUMAN CO1'1'IiR ANI)'LIN(' SUPI:ROXII)1: I)ItiMU'fASli BY SITI: SPfC'IFIC MLPI'AC;IiNIsSIS Thr active site argininc-14i uf human ('u,'Ln sulxruxide Jisnwtasc has hrcn replaced by lysine or by i.ulcucine. The mutant proteins were expressed at high Irvrl+ in yeast, purilicJ, and Ilte amino acid substitution explored through the use of gruup specilir reagents. The .perilic activitirs of theu: cnzynres, measured by the xanthine uxida.elcytckhrumc r methcal and by using dry weight determination to establish protein cunrentratiun, wcre: native cnzyme, 65711 units/mg: Lys-substituted enryntr, 'K40 unitamg, Ile-tiubatituted enaymc, 708 unilx/mg. The active site arginine thus plays an impurtant, hul not an essential, role in the catalytic process. licyrr, W. F., Jr., !''riduric•h, l., Mullcnhach. G.'I'., and llallcwell, R. The Journal uf Itiulugical C'hemistry 26?(?3):III82-IIIK7, August 15, 1987. Othrr support: ('htrun ('urpur:uiun, National Science Foundation and U,S. Army. Research OIlice. Frum the I)rpartmcnt of Biurhemi>try, I)ukr University Medical ('enter, 1)urhatu, N(', anJ tltr ('hitun ('urpuratiun, limrryvillr, ('A. ('ON'IkASfIN(; FA'I'IS OF"I'III: I'ARAOtJA"I' MUNOCATION ItAI)ICAL IN liJ'(7NtXl('ll1A ('Ol.l Ii ANI) IN I)l/NAl.ll:Zl.A SALINII Anaerobic cultwrs of l:rrltrrirltia ruli exlx)ud tu parayu:u 11'O" ) arrtunulatrcl Ihr corrrtipccndin f, munuc:uiun radical 1'(1, both within the cell.:md tn thr %u.IxnJing mrdnime l hr grrcn algaa l)uncrlie llu .~ulinu, which i»utircptihlr tu a Itfht- and O,clr- prnJrnt toxicity of I'Q', was nevertheless unahle to rausc accumttlatiun of Ptl'whrn illuwinatrcl anaicruhti all,v and cuuld, murcuvcr, discharge Ihe L:SR +ign:d anJ tltc hluc culor of Pt1 arrunwlatrcl by 6. ruli. Spin trapping allowed dcmunwatiun of Ihc phutuprucluctiun u(O - within /). sulinu and ui'Ihr augmintatiun ol'th;d U: prudurtruu by 1'(1'' . 1). molicuc ahlxat s to contain an elertron.ink aud a heat-lahtle mechani,m Iur Iran.lcrring elcrtrun, frum P(1'tu that sink. This mechanism wa.Jcnronarahlr:marru- htrally hut did not prevent I'(1=mcJtatcJ O, production tinder aerobic conditions. I Il:dbinuwilrh, 11. I)., Rcr,en, (;. M. and l'ridurirh, l. Arrhives ul' I;nxhrmi,try and liictphy.irs ?57(3):,152-356, 1987. Other tiuhpurt: National Science Foundation. U. S: Army Research C)1'licc and Na• ttunal Institutes uf I le:dth. frum Ih¢ Ikp:rtmcnls of liickhcruistry :mJ I'hannacology, t)uke Univcrsity Medical ('rntrr, Ourhaut, N(', and Ikpartnrrnt of Field and Vegetable ('rup., Faculty of Agriculture. '1'he I lebrew University uf Jeru.alrm, Israel. A MIMI(' OF SLII'IiROXIDE UISMU'I'AS'J: AC"I'IVI'I'Y L3ASG1) lIPON' I)I3SI7skRlOXAMINIi 13 ANU MANGANISE (IV) MnO, reacted with dcxl'crrioxamine B yielding agrecn, water-soluble complex, wilh absorption ntaxinra at 315 and 635 nnt whose extinction rueflicfcnts were'135 and (>!) M' cm', respectively. Increasing the proportion uf ligand tu metal increased Ixuh color yield and ability to scavenge U„ with ntaximal color yield and activity hcinF achieved at a 1:1 ratio. 'I'he complex catalyzed the Jisnwtation uf U, and I µM was equivalent to I unit ol' tiulxroxide dismuttt.xc activity in the xanthirre oxidase-ryto- rhrume r assay. 'I'he complex thus exhibited appruxintately O.Ic/ as much activity as did the manganrse-containing,upcruxide Jisntutaur, on the hasis uf man);anese ron- trnt.'I'he artivity uI'thecumplex was not.upprcti.eJ by txwine urum alhumin orby the u,lublc proteins extracled from Lur(ubuc•ilhcs plcutrurunt. In contrast, (he activities ul' ('ulll) complexes id's:rlicylate or Gly-His-Lys were suppressed by these proteins. I)arr, I)., Zarilla, K. A. and Fri(lur•irlr, l. Archives uf Bickhemistry and Biophysics ?5t{(2):351-355, November I, 1987. Other xuplwn: United States Army Research Ofliuc, National Science Foundation and Natiunal Instilutcs of I lealth. Frum the 1)epartment of Mcdicine, I)ivisiun ol' I)ennatulogy, and Department ol' Bicn:hemislry, Duke University Medical ('enter, 1)urham, NC. IiI Fli("fS OF (IRIiA ANI)'17(IMIiT11YLAMINI?-N-UXIUI: ON l:N"LYMI: A("I'IVITY ANI) STABILITY The interartiuns ul' urca, trimclhylaminr-N-uxidr (I'MAU), and related sulutrs un a nwuher of enzymrs were examined. Urea inhibited enzymatic activity and acrel- cratrd the therural inartivatiun of catalaa, whereas 7'MAO activated suntc rniyturs but inhihited uthcrs. 'I'he cffccts ul' urea anJ of 'I'MAO, whcthrr parallel or in uplxi.i- tiun, were exerted independently. Thus, in those caus wherc'I'MAO increases rniy- m:dir :trtivity, it did so to the same relative degree, whether or not urea was preu nt. 'fMAU markedly Jerreaud lhe rate of thcrmal inactivation uf catala.c, indicating that it dcies lavor compact protein structures. The assumption Ihat'1'MAO factun romp:rc- tiun uf prcuein structure, whereas urea has the contrary et/ert. Jcks not lead to the expectation that'I'MAO must always oppose the eflcct of urea on enzymatic activity, since the most cuntpart form uf an enzyme may not always he the most active /isrm. 112 t 113
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1 Mashinu, T. and Friduvirh, l. Archives ul' 13iuchemistry and Biophysics 25M(2)::156-16(1, November I, 192i7. Oher.uppnn: llniteJ States Army Research Oflice, National Science 1=ownJatii~n and Nauunal Institutes ul'I lealth. From the I)epanment uf Biochemistry, Duke University Medical Center, Derh.un, NC. ANTIOXII)AN'I' ACfIVITY OF SERUM CERULOPLASMIN ANI) '1'RANSFI:RRIN AVAILABLE IRON-BINDING CAPACCfY IN SMOKERS ANt) NONSMOKERS We investigated in 39 healthy smokers (20 li:male, 19 male) and 18 healthy nonsmokers (I8 fcmale, 20 male): (1) the impact of smoking and smoking-related facturs un the concentration of serum ceruloplasmin, transferrin, transferrin available iron-binding capacity (AII)C'), and serum antioxidant activity (AOA); (2) the relative contribution ul'scrum ceruluplasmin and All3C to serum AOA; (3) the association and possible interactive effccts ul'serum ceruluplasmin, AIBC, and smoking with serum AOA; and (4) the relation ul' pulmonary functian in healthy smokers to.erum AOA, cernlOplatimin, and AI13C. We fuund that: (I) as compared with healthy nunsmukers, healthy smokers had higher +erum ccruhtipla.min concentration, similar serum trans- ferrin and AI13Ccunvntration, and lower serum AOA; (2) ceruloplasmin, A113C, and smuking acted additively, accounting liv about 60% of'the variability of serum AOA; (3) the impact uf A I I3Cun scrum AOA was significantly greater than that uf ceruluplas- min; anJ (4) pulmonary functiun in the smokers was not significantly related to serum AOA, ceruluplatimin, or AII3C. Our finJings suggest that: serrun AIBC has a greater role in serum AOA than has previously been attributed lo it; suppression of the AOA uf uerutn cunstituent(s) other Ihan those we measured may account lior the significantly lowor.crum AOA in healthy smokers than in healthy nonsmokers; and normal air fluw in healthy cigarette .mukers is unrelated to serum AOA, ceruloplasmin, or A113(' concentration. Guldsvun, h9., FclJman, J. G., Lcvytska, V. and Magnusson. It. American Review uf Respiratory I)isease la5(4):78a-787, April 1987. Other uippurt: Thc Louis anJ Roxx Klu.k Fund. From the I)epannu•nt of Medicine and Ihe Chest Scrvice, liellrvue lkupital, New York (Inivcr.ity Medical ('enter, and Ihparunent of I'reventrve MeJicine, I)own,tate Medical Center, New York. AN'I'IOXIDANT ACTIVITY OF SI:RUM CERULOPLASMIN ANI) TRANSFERRIN AVAILABLE IRON-BINDING CAPACI'f Y IN SMOKERS ANI) NONSMOKERS The antic>xiJant activity (AOA) uf setum has been ascribed chiefly to ceruloplas- min, with ,erum transferrin available iron-binding capacity (AIBC) having a minor I I i I rule. The ferruxtJa.e activity uf cerulopla+min (catalysis of' Fe" to Pe" ) and the murked accelerating effect uf ccruloplasmin on tttc rate uf binJing Fe" to i,run-frce transl'crrin suggested that these two proteins act cowrdinately in extracelludar defense against irun-catalyied cellular and tissue iniury. Significant concentrations of cerulu- platimin and tr:m.ferrin exist in broncualvculcr lavage fluiJ ul' smokers and non- smnkers. We speculated that these twu extracellularly-JistributeJ antiuxiJants prevent ox- iJative inactivation ul"alpha-I-pruteina.uu inhihitur (aIPI) by cigarette smoke and other air pollutants. 'I'his would help maintain a normal protease-antiprotease balance in the lower respiratory tract. We recently reported that serum AOA is lower anJ scrum ceruhiplasmin cimcLn- tratiun higher in smokers than in nonsmokers, anJ that ceruloplasmin can prevent oxidative inactivation of all') by cigarette smoke and by chluramine T. We have shown that mean ceruluplasmin concentration was significantly higher in smokers than in nonsmokers and in I'emales than in males, whether or nut either smoked. However, mean serum AI13C concentration was not significanlly JilTerent in smokers and nun.ntukrrs ol'buth sexes, but mean serum AOA was significantly higher (I I'%•) in nunsnwkers than in smokers and not Jil7crent in I'emale and male smokers and nonsmokers. None of' the fulluwing smoke-related facturs had an impact on serum ceruloplasmin, AI13C and AOA: pack years smoked; current daily packs of cigarettes smoked, their tar and nicotine content; and car(xoxyhemoglubin concentration Ixxfure and aftcr smoking. Despite the considerably higher ceruloplasmin-specitic AOA, Ihe 15-liild greater scntm AIBC concentration than that uf ceruluplasmin offsel.r• the 10-IiiIJ greater ceru- luplasmin-specilic AOA. Since none ul'the smoking-relateJ factors tested had an impact on serum AOA, cerulupla.min and AI13C concentratiun, the mechanism ul'the smoking effect was not clarilied by this study. Guldswn. M.. Levytska, V., Feldman, J. G., and Magnusson, B. In: 1'ulmunary liniphysema and Pmteolysis. Academic Press, pp. 459-46b, 1987. Other support: Louis and Rose Klosk Fund. Frum New York University Medical C'enter, New York; Department of I'reventive Medicine, I)ownstate Medical C'enter, New York; and Reykjalundur Rehabilitation ('enter, Iceland. 1)ISPI:RSIiI) RNPIi"I'I'I'IVN- SIiQUENChS (lh THE MOUSE GENOME ARE I)IFI'LRIiN'fIAI-LY RIiPRGSt:N'1'GD IN EXTRACHROMOSOMAL CIRCULAR I)NAs IN VIVO Eukaryotic cells contain extrachromu.onral circular (ecc) DNAs which aue hu- modugous to chrinnosumal seyucnces. We have isolated eccDNAs frurn whole tissues uf C57liL/6 mice. Using hybridization techniques, we show that R-, MIF-, BI-, and 132-Jispersed repetitive sequences of the mouse genume are differentially represented in heart, brain, and liver tissues. Moreover, we show that the relative abundance of 132 u:yuences in heart and IivereccDNAs is higher than the relative abundance of the other repetitive sequence families studied. 114 1 115
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1 ,,,M1...n Flurrn, S. C., Muurc,'f. K. and Guuhutc, J. W. I'latimid I7:357 Nd), I')ti7. (hhcr.uppurt: Ilc:ua I•ounrlaliun and American Federation for Aging ltr.e:nch. . Fruin thr I)rpartntrnl uf Iiioxhcmistry. Univrrsily of Suuth Alahama, College of MrJlcinr, Muhilr. MAS'1' C'lil.l, I II:'I'EROGIiNIiITY IN 1)(Xi SKIN 'I°hc dcgr¢r ur inrtachrnma:,ia of mast cell granules is known to vary ivith tile type uf li;,sur lixatiun and amomg diffcrent tissues and species. The present study sought to dclcnuinr whether nta,t cells in dog skin are heterogeneous with respect to lixatiun and ~(aining propertirs, We rwrt'urmcJ skin biopsies in six anesthetized, atupic dogs and one mongrel dug.'Unc biopsy was lixed in furmalin and a second, from a parallel abdominal site, was fixeJ in basic lead acetate (Mola's solution). Adjacent scctious from each biopsy were stained with alcian blue (1'k-, pH 0.5) or liir chluroacetate cacra.c activity. In :dcian trtue-slainlJ sr:cliuns, one-third fewer mast cells were dcicclcJ in skin fixed in fonnalin (I,R36 ± 454 mast cellx/mm', mcan ! SF:M) than in ,Lin fixed in basic lead acetate (2,684 -_+. 527 maa cells/mm') (I'<: 0.05). The chlu- ruacctatc rstcra.c reaction detected thc larger nunther ul'tttasl cells regardless ul the lixattvc used. We conclude that nta.t cell heterogeneity, as demumirated by metachru- tnalic .taining tidluwing dillrrent lylxs of tissue lixatiun, rxi.tx in dog skin. ""fypi- cul" mast cells stain with alcian blue regardless ol lixatiun: huwever, "atypical" mast cells exhibit mctachrutuasia only after lixatiun in basic lead acetate. 13u1h thc typical and atypical types uf ma,t cells have chluruacetatr estera.e activity. Becker, A. 13., ('hung, K. F., Mcl)rmald, I). M., I-azarus, S. C., Frick O. L., and lluld. W. M. 'fhr Anatunucal Record 213:-177-4111), 1985. Other support: National Institutes uf I lcalth, National Institute ul Allcrgy and Inlec- tiuus I)i.casc., University ul ('alifimtia'ti Research Evalualiun and Allocation C'um- nuttec, and thc A. (;. Spanos Allergy Research Fund. Frum Ihe Cardiovascular Research In.titutc and I)eparttucnts uf Medicine, PrJi:drirs and Au:dumy, University of ('alifurnia, San Francisco. I)I:MONS"LItA"I'ION c)F"I'WO I)1S fINC"f IIIGII MOI.E('ULAR WGI(;IfI' I'KOl'IiASIS IN RABBIT NI•:1'IC'ULO('Y"1'I?S, UNiiUF WIIICH I)I:GRAUIiS l1BIOUll'IN ('UNJII(iA'fliS Ileltculucyles cuntain a nunlysosumal pruieulytic pathway that requires A"I7' and ubiquitin. By I)IiAli chrom:uugraphy and gel tiltration, we were able to fracllOltalC thC A'fl'-JeIxndent system into a 3t1-3(K)-kUa fraction that catalyzes the ATP-Jelx:ndcnt coqlugatiun of ubiyuitun tu substrates ("Ccmjugatiun Fractiun") and a high mass fractiun (-•-{50 kl)a) necessary liir hydrolysis of the conjugated proteins. The latter cuntains two distinct prulca+es. 116 i One proHca.r is unusually large, approximalrly 15(K) LI)a, and degrades proteins uuly when A'1'I' and the conjugating fractions arr addcd. "I'hix activity precipitates at 0- iH1r (NI1,) SO,,aturation and is c.racnlial for A'fl'-dclsenJcnt Irrutruly.i.. l.d.e crude exuracl,, it i% labile in the ahsence uf nuclrotidr. anJ is inhibited by hrpann, I>,dy((;Iu- Ala= I:Yr), S-.{-dichluruir-ocaumarin, hcmin, dreavanadatc, N-ethyhn:Jcimidr, and various Ikptidc chlurumrthyl krluneti. It lac6.s anlinulxplidasc and in.ulin-Jcgradmg activitic% and dtk, nul require tFiNA linr activity. '1'hr uhiyuitin-cunyugatc degraJin)g rniyoie, which we suggest he namtJ ll('I)h-N, is inactive al;allltil substrates that cannut undergo ubiyuilin cuqjuFaliun. I'hc auallrr prutcau (6701 Ua1, which prccipilalcs at 4O- HO'/• (NI I,).SU, tiaUura- Iiun, do•s not require Al'I' or ubiyuitin and is therrfiire not required lur A'I'1'-depen- Jrnt hronruty.i.. It is stimulated by N-cthyhnalcimide and i,.1-dirhluruisixoutnarin and is stable at 37"('. 11 hydrolyics Iluurumctric 4etra)xplides and proteins, including pnrtcuts which cannut be coniugatcd to uhiyuilin.'fhusu rcticulrkyteti conlain two large cylo.ulic pruteascs: une is essential for the degradation uf uhiyuitin conjugates, while the funclinn ol the other is unconain. Waxinan, L., Fagan, J. M., and Guldbrr,S, A. 1.. 'f he Journal ul 13iulugic:d Chcmis(ry 262(6):'-451-2457, February 25, 1987. Other support: National Institute of Neurological and Cunmutnicative 1)i,urders and Strukc. Frum Ihc t)cparlntcnl of Physiology and liiuphy.ics, I-IarvarJ Medical tichuul, Bu.- tun. SKIiI.I:I'AI. Ml1S('LI: ANI) t.IVNR C'ON'I'AIN A SOLUBI.1? ATI'+ 11131(1111'I'IN-INiI'IsNUIiN'I' I'RO7'liOI-Y'I'I(' Sl'S'1'I:M Although prutcin breakdown in nwsl cells uents lu require mrtabulic energy, it ha% unly been possible to establish a soluble A"I'I'-Jclx'ndcnl prolculytic system in rxtraclx of rcttculurytes anJ erylhrulcukaemia cells. We have now succeeded in Jent- uiutratinl; in suluhlc extracts anrl nwre purified preparations Irum rabhit .hclctal mus- CIC a 12-IOI/l 111111ulaUUn by A I I' l1I hrCahlJUwll UI C1111UgenCOIIS 11rUICln\ atlll aO-IUh) stimulaliun of "I ly.ur.yme degradation. Iluwevcr, it has still nul been possible to rlrnwntilratc such large cl lccl. ul A'(7' in +imilar preparatiun, fruiu liver. Nevertheless, after Iracliunanun by UI:AIi-chrum:uugraphy anJ gel fillratiun, we firunJ that extracts Lrunt liver ati well as musclc cuntatn both ihc rnr.ymrs which cunjugatr ubiquitin tu'"1- lysuiymr and an rnrymc which slxcitically degrades Ihe uhuluitin-prulrin conjugatrx. When Ihis prulcolytic activity was recombined with the conjugating cotytncti, A"I'P + uhiyuitin-drlknJcnt degradation ul many prutcins was observed. This proleinase is unusually large, approx. 15(M ll)a, requires A"17' hydroly.i. liir aclivily anJ resembles (lie uhiyuitin-protcin-cunlupatc degrading activity isolated 1'rum rrlicuhKytes. '1'hus the A'I'1' t uhiyuitin--delxnJent pathway i.likely tu hr: present in all mammalian cells, allltough certain tis.ucs may contain inhibitory Lacturti. Fagan, J. M., Waxman, L-., and Gn1dLr-r);, A. 1.. Biochemical Journal 243:335-i43, 1987. 117
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Othcr .upl,urt: N:wunal Institute ul C'untntunii:nn•c and Neurological I)i.cirse :rntl 1tul.c. Mu,iul:u Uy.nuphy Association nl Arnrrtca, (ltuk C'ullrge-RulEcrs (lnivcr. tiqy, autl Ihe New Jersey Agricultural I:xlarrimenl Station. Frum the hcpartntrnl ul Physiology anJ Biophysics, Ilarvartl Medical Schuul, Iiu.tun. II)I?N-1'I17CA'I'IUN 01: A (iUANOSINI? TRIPIiOSPIIATIi-BINI)IN(i Sfl'li ON CIUINIiA PIG L.IVI:R TRANSGL.UTAMINA5E Guanusine 5'-Iriphu,phatc (GTP) was found to inhibit guinea pig liver transl;lu- taminase activity as ntea.ured by I'li1putreuine incorporation inlo catiein. GI)P and GTI'-y-S also inhibited enxynte activity (GTP-y,S > GTP > GDP). Kinetic studies showed that GTP acted as a reversible, noncompetitive inhibitor and that C:aCI, par- tially reversed (;1't' inhibition. GTP also inhibited rat liver and adult bovine aurtic endothelial cell tran.glutarninau:, but did not inhibit Factor XIII, activity. Guanusinc nwnnphusphatc (GMI'), cyclic GMP, and polyguanylic acid did not inhibit enryme activity. Guinea pig liver Iranxglutantinase adsorbed well to UI'P-agaruu aflinity columns, but not tu ("I'I'-agaroxe columns, and the binding was inhibited by the presence of calcium ions. Specilic binding of GTP tu Iranxglutaminuse was demum stratcd by pholuallinity labeling with 8-azidoguanosine 5'-ly-'°L'j triphnsphate, which was inhibited by the presence uf GTP or CaCI., GTP inhibited trypsin prutculysis ut guinea pig liver trantiglutaminase without aflecting the trypsin pruteuly~sis af chrunro- gcnic substrates. 1'roteulytic protection was reversed by the addition of calcium. This study dcnwnarates Ihal (PI'P hinds to transglutarninase anJ that both GTP and calcium ions Lunctiun in concert to regulate transglutaminase .truclure and function. Achyutltan, K. G. and Gra'<•rtberS, C. S. 'L'he Journal ol Biulugical Chemistry 262(4):hH)I-1906, 1987. Other support: Natiunal htstitutes ol' I lcalth. From the I)cpartntentti ul McJicinc and P.rthology, I)ukc University Medical Center, I)urham, NC. FA("fUR XIII I3INUS-I'O Tllti Arr- ANI) lip-C'HAINS IN TIIN U-UOMAIN OF FIILRINU(;IiN: AN IMMLJNOBLU7"fING STUI)Y The binding sites in fihrinogen for Factor XIII were localized using an int- ntunuhluttrng techniyue. Platelet Factur XI II bound to Iibrinugen and tu plasmin Jegr:r J.dion product, uf lihrin(ogcn) including Fragments: X, I),-U„ and 1)-dinrcr, hut did nut hind to Fragment 1?. l3inJing ol Platelet Factor XI11 was independent u( calciurn iuns but could he inhibited by the presence uf 0.5 M NaCl. Btndiul; could also he inhibited by preincubating Factor XIII with a I(K)-IitIJ molar'excess ul librinogen hu1 not by I(X)-tu1J mul:tr exccss ot Fragment li. Binding of Factur XI II tu hhrtnugcn was specilic, .uue several uthcr proteins texlerl (avalhwnin, bovine xcrurn albumin, (r; tnacruglubulin, P-galactu`rJa.e, fructose kinase, lactic dehyJrogen:rae, Iriuse phus- i , I I I 1 phalc i,unucraw:, lunsrra.x and pyruvatr kmatie) did not hind Factor XIII. Furthcr- ntarc, himinrlg was mH observed either when Factur XIII was left out or when :tnul :.tctur XIII antiticrunt was substituted with n+rnint:mune xerum. When librinttgen w:ts reduced priur to clcclrophorc.i., Factur Xlil bound lu the Aa arnl li/i chains uf' lihrinugcn and Je, A,li hbrinugen, dte Ilp-chain uf Fragment X, hut nut Ihe y-ch:rins. Lucalii.:diun u( thc Factor XI11 binJing .ile% lu the cartwxy terminal segments of Ihe Arr and Iip chain% in the Fragment I)-Jomain u( librinogen could have intpurtant physiological consequences. Mary. A., Achyuthan, K. F. and Grrrnhc•r,q, ('. S. I)itxhcmical and 13iuphy.ical Reuarch Communications 147(2):6O5-614, September 15, 1987. Other support: American Meart Association, Walter P. Innian Fund, Blood Bank 7-raining, and National Institutes uf liealth, Frunt (he I)cpartrnents of Medicine and L'athology, L)uke University Medical Center, 1)urham, NC. 'I'llli MNCIIANISM OF OSMU"1'IC TRANSFLsCT1ON OF AVIAN EMBRYONIC I:RY'I'LIROCY'1'ES: ANALYSIS OF A SYSTEM FOR STUDYING I)I:Vl1.OPMENTAI. GGNB I:XI'RIiSSIUN We have undertaken a study of the mechanism of 1)NA transler into primary chicken erythrttcytcs by a nteth4xl named usnwtic tramfection. The cells are,uhjecteJ lu controlled usnwtic swelling in NI I,C'I anJ then ruptured in a lower osnwtic strength udutiun containing 1)NA and DGAIi-dextran. The usntutic rupture results in transient lirrmatiun uf a single hule in the cell membrane, which is lulluwed within huurs by recovery ul near nrxmal levels uf RNA and protein synthesis. 7-he association of DNA with the cclls is much greater Grr ruptured than fur unruplured cells or (orcells that have Ixen lysed and resealed before I)NA is added. '1'ransienl litnnatiun of pure. in the cell membrane is apparently essential lirc high rates of ntacromulecular Iranafer into the cell, l)IiAI: dextran increases the antuunt of DNA associated with the cells, especially alier cell rupture. Our understanding nf the mechanism has allowed uti to extend the applicaliun uf u.nrutic transtectiun to essentially all Jevelupmental stages of avian crythruid Jifferentiation. Osmotic Iranslcctiuns were done with plasmiJs cuntaining tite chlorantphenrcul acctyl tran,lerase (CAT) gene placed Ixtween the chicken (3-Eluhin pruntuter and Ihe 3' P-globin enhancer. The pattern uf ('AT expression at uyucntial dcvelupmenlal stages parallels that ot the endugenuus gene, showing that u.mutically tranaected cells appear to retain developmental IiJelity. The approach pruvides a cunvenicnt, sensitive, anJ Ilexible system litr the study ut transient gene expression as a functiun ol Jevclupmcnt. l.incer, M. R., Ilctix, J. li., Nickol, J. M., and Fclscnteld, G. The Journal uf CrII Biology 105:1055-I(H)5, September 1987. Othcr support: National ('ancer Imtitule. From the Lahrn•atury of Molecular Biology, National Institute ul I)iahetes and Uiges- tive and Kidney l)i.cascs, Betltesda, MI). 118 119
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1)1'NAMI( :ti UF'I'IiRMINA'I'IUN I)l1RING IN VI'I'R(J RI:I'I.I('A'I'ION OI~ UI:1'FtAVIOLlil'-lKKAI)IA'I'I'.I) I)NA WI'I'11 I)NA I'ULYMI'.kASli III IIOLUIiN%YMIi OI,• lt.S('llliRl('llU C'(1IJ I)urtn)g in rhro tcplicauun ul l1V-irrarhatcJ single-stranded DNA with l::%rhrri rhiu ruli 1)NA pulynicra.c III huhxncyn,c termination I*rcyucntly occur, at pynmiJinc IrhuluJinrcr. 'I'hr tcriuinatiun stage is Jynamic and charactcrii.rd by at least thtcc .Iillcrrnt events: (a) rclxatrd di,aKi:niun-rcinitiatiun cyclcs ul thc pulynurasc at the blocked tcrmini; ( h) cxten.ivc hydrolysis ul A'I'P ItI AI)P and inurEamic phu.phatr; (c) ntrnuver u1 JN'I'I', into dNMI'. 'I'hc reinitiatiun events are nonproductive and arc not lulluwed by /Lrthcr clungatiun. '1'hc tunrover ul JNTP. into dNMI'~ is likely to rctiull Irum rclkatctl ryclcs ul in.cniun ol dNMP residues opposite the hhk•kint 'Ic.uuns IitlilowcJ by Ihctr excision by the 3'-+5' exunuclculytic activity ul the polynterasr. Although all JN'I'I'ti are turned over, there is a preli:rcnce lirr JA'1'I', indicating that I)NA pulymcrax I11 huhkniymr has a prclerencc fur inserting a dAMP residue opposite hhrcking pytimidine phrituJintcn. We suggest that the inability ul'the lauly- rnera.e tu hypa+s rhuuKlimen during terminatiun is due to the Cunnatiun of dclcctivc initiatiun-likc cumplcxcs with reduced %tahility al the blocked Icrnrini. Shwarti. Ii. and l.ir•nrh, Z. Thr Journal ol liiulugical C'hcmi,Iry 362(?2):I(151H-10523, August 5. 1987. Other support: National In.titute% of Ileahlt. Frum Ihc !)cparuuenl uf Iiirxhemislry,'I'he Wcinnann Institute ol' Science, Rchuvut, Israel. kliGIII.A'I'ORY ASI'I:("1:1 OFTllli C'OLC'IIICINE INTI:ItAC"fIONS Wfl'll 'I'IIIlU1.IN I.imitcd prutculy,iti rrl tubulin with .uhtilisin results in the cleavage ul' huth tuhulin subunits yielJing S-tuhultn heterudhncr and 4 kl)a peptiJc fragmculs cuiuatn- int; the carhoxyl-terminal Juntains uf a- and P-pulylxxpaide chains. S-tuhulin hinJs culchicinc anl thc charactervatiun ul the hinJing uf culchicine to S-tuhulin nwlrcule, ,huweJ a decreased ratr uf dccay ul colchicine hinding uctivity as compared to that ul undigested tuhulin. I luwever, S-luhulin exhibited a lower culchicine binding cun.uun than tuhulin. I'eptidr li-aginent% resulting lrom the controlled tryrtic prutrulytiis uf buth pwc tuhulin anJ S lrihulin were pru•ilicJ by lilu•atiun duumatrigraphy and presented a strong rulchicinc hinding activity with a.,rki.uiun constants ul 4.5 x Il/' and 2.7 x 10' M', respectively. Furthcrnuure, these studies suplwrrt our initial IinJingx un the luc:di- caniun ul the tubufin site I'ur culchicinc and deline the colchicine binding dumain in a Juiuain ul rr-uihunn Itum the point ot' IimiteJ tryptic cleavage to the site uf.ubtifi.in cnntnrlled prutcody,u ul that tuhulin ,uhunu. Un the basis uf these :dtrratiun. tn thc itucracuun oI culchirine uhun icmuval ul Ihc C-tenninal moiety ul Iuhuliu and since nu changr in the number ul hinrling xitcs was lirund after xubtili.in digc.tiun, we sug):c,t Qtat the carhuxyl-tcrininal region al tubulin .%ubunits mudulatex the binding of cuirhi- cinc. Avila. J., Scrrana, L. and ,blucriuni, R. B. Mulecular and Cellular I3io<hemistry 73:29-36, 1987. OOther support: hurulxan Molecular Biology Organization, Antericun Cancer SrKiely, Nauoltal Institutes uf' IJealth, and C'umision Asesora, Spain. Frrmr the ('cntru de {;iulugia Molecular, llniversirlaJ Autrnioma, Madrid, Spain, and Ikpartrncnt uf 13iurhemistry, Biophysics and (ienetics. University oI'C'ulurado Health Sciences Center, lhhnver. BIO('lll?MICAI. ASPI:C"IS OF7'IIIi It1iGULA'I'ION 01, MICKOTUIiUI.Ii ASSIiM13LY 1'he molecular basis underlying the regulatory role ul' the car(wxyl-terminal do- main u( tubulin have been studied using controlled pruteuly,i., interaction with micru- luhule associated protcins and structurc-activity analyses. Controlled pruteulysis of tubulin with subtilisin results in Ihe cleavage ul buth a anrl (3 subunits yielding S-tuhulin and two 41,1)a fragmentti containing the cartwxyl- terntinal domain ol tuhulin subunits. S-luhulin exhibits an increaurW propensity tu.elf- assrxiate ax compared with tuhulin. Treatment with S. uureus V-H prutca.e resulted in removal u( a4kl)a C-terminal fragnrent ( V-tuhulin) with a concumitant ,tintulation of' thc atiticmhly. The propagation cunstant lix V-H treated tuhulin, where unly (3 -subunit is cleaved, was one-third ol'that tirr S-tubulin assembly. Polymers of S-tubulin do nut incorporate MAI's, but we observed a partial incorporation ol' MAPs into V-tubulin Iwlymers relative to roicrutubule controls. On the other hand cleavage with tryp+in, which removes a 16 kl)a C-terminal 1'ragnxnt from a-xubunit or limited digestion uf tuhulin with carhuxYlx:ptiJase Y, which removcti only 6-9 anrinoacid residues 1'rum both subunits, have nu regulatory eflect while removal uf the 4kUa fragmem does. In both caus, treatment with the endo- and exolxptiJaus produces inhibition of the assembly assrniate.l with significant changes in tubulin cunfixmation. 'I'he substructure ol' the tubulin's regulatory domain was further examined by hinding iff 'I 1-acetylateJ peptiJes from the variable region of the ('-ternunal dumain, a (430-441) and 4i (422-433) to MAf's. The binding data showed a prelerential interac- tiun ul j3 -pcptide with MAP-2 and tau as analyzed by Airluge ultracentrifugatiun and ronc liltratiun chruntatugraphy. The a(430-441) peptide interacted with tau with a higher allinity than MAP-2. These studies support our hylxrthe,is that the C-terminul dumain hinders interactions responsible firr mhulin assembly and that cleavage at or near the 4kl)a end is critical to relieve this hindering ef7ect. hfurriuni, K. K., ICivas, C. and Vera, J. C. In: Macciuni, R. It. and Arechaga, J. (eJs):'I'he C'ytotikelcton in ('c1I I)iflcrentiatiun and 1)evelopntent. First International Syntpu.imn, Granada, Spain, 1')H7, pp. 39--IH, IRI. Pres., 1987. Othcr support: Milheim Foundation. hrum the University ril Colorado Health Sciences Center, Ihrnver. 120 I 121
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NEW MI:'I'HODS TOWARD I'URIFICATION AND STRUC'TURAL STUDIES 01: MICROTUBUL-N ASS(X'IATED PROTEINS Because of limitations inherent in the methaloingy utilized tu purit'y MAPs, we do not know practically anything alwut the biochemical and functiunal properties of MAPs present in most non-neuronal cells and tissues. In an attempt tu overcome these limitatiuns, we have searched for new experimental approaches toward the purification and characteriratiun of MAPs. As a result of this, we have developed an af7inity purilication method that takes advantage of two well known properties of MAPs: heat stability and binding to calmixiulin. This method has been successfully applied to obtain MAI's from tissue of neuronal and non-neuronal origins. The results obtained cunlirm the wide distribution of MAPs in Jifferent systems and provide reliable inli,r- matiun on the in r•ivu presence of different isoforms of MAPs. Vera, J. C., Rivas, C. I., and Maccioni, R. B. In: Maccioni, R. B. and Arechaga, J. (eds): The Cytoskeleton in Cell Differentiatian and Ihwelupment. First International Symposium, Granada, Spain, 1987, pp. 119-130, IRI. Press, 1987. From the 1)epanments of Bio<hcmistry, Biophysics and Genetics, University of C'cilo- raJu I lealth Sciences Center, Denver. RIiURGANIZATION OF TUBULIN P(X)LS DURING EARLY MOUSE EMBRY(X;IiNIiSIS Microtubules aplxxar to be involved in the morphological changes of blastomers during prcimplantatiun cmbryogencsis, namely in cellular polarization. We have ex- amincd the intracellular free and assembled tuhulin prxils as an attempt to understand the molecular aspects of micrutubule involvement in early mouse emhryogene.is. After embryo's humogeni/ation at 4`C, nrbulin levels in the soluble fraction did not change signilicantly throughout the precompacteJ stages but decreaud in cumpacteJ nwrulac anil blastocyst tu 36-40% uf that uf preliminary stages. On the other hanJ, tubulin in NI'-40 suluhilized cellular fractions was significantly higher in compacted nmruL•rc. Sucrose gradients centrifugation at 32°C of supernatants and soluhiliicd pelletti obtained alter homot!eniiation ol' embryo, in warm bul'fcrs also indicated a preferential distribution of astiembled tuhulin in the membrane domain of blatitomrrs ot compacted embryos. Indirect inununulluorescence studies using arttitubtdin anti- hotlirti.howed topographic diflcrencex in the distribution uf tubulin :U blasuxy,t stage with a prcl'crcntial urganiratiun uf assembled tubulin in the inner cell mass. Arechaga, J., Rivas. C., Vera, J. C., and Mrrrciorri, l{. B. In: Maccioni, R. B. .md Arechaga, J. (eds.): The Cytuskelelon in Cell l)rfferentiatiun and Devclupntcnt. First International Symposium, Granada, Spain, 1987. pp. la 3-151, IRL I'ress, 1987. Other support: Milhcim Foundation and CAICYT. From the 1)epartments uf Bioc:hemistry, Biophysics and Genetics, University of Culo- radu I lealth Sciences ('enter, Ih:nver. TIiE STUI)Y ANI) DESIGN OF SPECIFIC INHIBITORS TC) ELASTASE At the beginning o( this project, insuflicient knowledge alxrut the binding of benzoxazinone inhibitors to eluxtr.e was available. We therefnre undertook the study uf sevcral crystal structures, using Lwrcine pancreatic elastase (PI'1:) as the receptor protein. As the structure of human leucocyte clastase has just become available, we tlreretirre are able to report initial binding studies with both elazttr.un. This method promixs tu place QSAR stuJies on a lirm liwting by providing a geometric description of active site interactiuns. Me~w'r, Iz. F. and Bode, W. In: liaJzi, I). and Jernian-Blazic, B. (eds. ): QSAR in Drug Design and Toxicology, Elsevicr Science Publishers B. V., pp. 247-254, 1987. Other support: I('I Americas, Robert A. Welch Foundation, the Office of Naval Research, the '1'exas Agricultural Experiment Station, and Deutsche Forschungsge- mcindschaft. Frum (he Biographies Laboratory, Biixhentistry 1)epartment, Texas A&M Univer- sity, College Station, and StrukturRirschung 11, Max Planck Institut fur Biochemie, Martmsried, Federal Republic of Germany. M, 25,(MX) IIEPARIN-BINUING PROTEIN FROM GUINEA PIG BRAIN IS A HIGH MOLECULAR WGIGHT FORM OF BASIC FIBROBLAST GROWTII FA("IY)R A M, 25,(X10 furm of banic fibroblast growth factor (bFGF) has been isolated f rurn guinea pig brain along with the typical M, 18,(XX) form. Both forms were purified to homogencity by a combination of heparin-aftinity chromatography and ion-exchange chromatography on an FPLC Mono S culunm. Thc M, 25,(XX) form, like the M, IR,(XM) lirrm, was nut clutcJ liom the heparin-aflinity column with 0.95 M NaC'I, but was eluted with 2 M NaCI. The M, 25,(XX) guinea pig protein stiniulatcd plasminogen activator production by cultured bovine capillary enJuthelial cells in a dose-JepenJent manner at cuncentrations of (1.1-IO nglml, the sanu: range that was eltective for guinea pig and human M, IR,(M)) hFGFs. 'I'he binding of human "`I-lalxled bF(iF to baby ham.ter kidney cells is inhibited equally by thc M, 25,(XX) guinea pig protein and the M, 18,(NX) guinea pig and human hFGFs. Polyclonal antibodies raised against human hF(;f rccol;nitc both the M, 25,(XX) and I8,(1lX) guinea pig protcins in an imnrunublut analysis. In a radiuinununuas.ay, both the M, 25,(RXl and M, Iti,(MN) guinea pig proteins cumlkte equally well with ioxlinateJ human hF(;F for binding to the anti-human hFGF antitxa)ic.. When treated with luw concentratiuns of trypsin, the M, 25,(XX) guinea pig hF(iF was converted to a M, 18,(HX) protein. These results show that the two molecules are closcly related and suggest that the M, 25,1K1O protein shares substantial hunrulugy with the M, Iti,(MN) bFGF. Mumrarrlli, U., Joseph-Silverstein, J., Manejias, R., and Rilkin, D. B. Proceedings of the National Academy o/' Sciences, USA 84(16):5778-5782, August 1987. 122 1 123
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Other .uppurt: Natiunal Institutes uf Ilealth, Atuerican Cancer Suciety and Juvenile l)ialkles Fuundatiun. frum the l)epartnrent uf C'el) Biology and Kaplan Cancer Center, New York llniver- tiity Mcdical Center, New York. A'I'RIAI. NA'I'RIURI:I'1(' FA('I'OR ANl) SODIUM NITROI'RUSSII)N IN(.'IiliASl? ('Y(.'t.IC' (;MI' IN CULTUREI) RAT LUNG FIl3RUBLAS'I'S BY A(°I'IVATIN(l UPI'FIiRIiN'I' FURMS OF GUANYLATE CYCLASG We used cultured rat Im,g tibrnblaay tuevcduate the role of particulate and soluble gu:utyhrtc cyclase in the atrial natriuretic factor (ANF)-induccd stimu6ation ol'cyclic (;Mf. ANFrecepturs were identified by binding of "'I-ANF to confluent cells at 37"('. Specific ANF binJin(; was rapid and saturable with increasing concentratiuns of' ANF. 'I'he eyuilibriuni Jixxuciatiun constant (K„) was 0.6fi*_O.077 ntyt and the 8,,,, was 216 t,3.3 1'mul tnwnd/1(1" cells, which correslwnJs to 13(1(M)Qt2(NXK) sites/cell. 'I'he mulecularcharacteristics ufANFhinJing sites were examineJ by affinity cross-linking uf'"I-ANF to intact cells with Jisuccinirnidyl suberate. ANF slxcifically labelled two sites with molecu lar sizes uf 66 anJ 130 kDa, which we have identified in other cultureJ cells. ANF and sodium nitruprusside pnxluceJ a time- and concentratiun-Jelx:ndent increase in intracellularcyclic GMP. An increase in cyclic GMI' by ANF was detected at I nM, and at I(X) nM an approx. I(JU-liild increase in cyclic GMI' was observed. Nitroprusside stintulated cyclic GMP at 10 ntM and at I mtN a 5(X)-MX)-tblJ increase in cyclic GMP tx•curreJ.'I'he sirnultancuus additiun of 1(M) nM-ANF and 10 µM-nitroprus- side to cells resulted in cyclic GMI' levels that were additive. ANF increased the uctivity ut particulate guanylate cycfa+c by alwul IO-fofd, but had nu effect on suluble guanylate cyclase. In contrast, nitropru.side did nut alter the activity of particulate t!uanylale cyclase, but increased the activity uf soluble guanylate cyclase by 17-IitIJ. '1'hcse results demunstrate that rat lung lihruhla.ts cuntain ANF recepturs and suggest that the ANF-inJuceJ stimulatiun of cyclic GMI' is mediated entirely by particulatc Nuauylatc cyclase. Lciunan, I). C., Agnmt, V. L., Tuan, J. J., Andresen, J. W., and Murcul, l". liiuchcmical Juurnal 2-3-1:bt)-7-F, 19R7. Other suplwrt: Natiunal Institutes 01' Ilcalth and the Veterans Adntiui,tration. Frunt the I)epartments uf MrJicine and I'harmaculugy, SumfitrJ University Schuul ttf Medicine, Stanlurd, ('A, anJ lhe Veterans Administration Medical ('enter, Palo Alto, ('A. ('OMI'AKISON OF I.AN'I'IIANIDI? (111) ANI) ('AI)MIl1M (11) AS C'AI.('Il)M (l1) MUI)I:IS: MIiTA1: IUN INI)ll('IiI)('ONFOI2MA'I'IONALCIIANGNS IN S)'IN-LAtiG.I:U ('ALMODUt.IN ('ahnudulin (('afbl), unc ufa groupuf high uftinity cafcium (11)-binding proteins, has been tihuwn to have a number of' imlwrtant regulatory functions. 'I'he natural calcium (II) cu-factur, huwrver, does not have physical pruperues which can be expduited tu study C'aM and .ru lanthanides have often been used us calciunt (11) analugs in the audy uf c:dciwn (l1)-binding prateins. Mure recently a number uf CaM studies have etupluyed the trant<uiun metal cadmium (II). Recent sludie, by the authurs and uthers have indicated that, in lhe ca.re ul' C'aM, the lanthanides may nut be gtxKl nuxlels, while cadtnium ( I I) aprxar, to be quite g(xx). In order tu explore this further, spin-lalx•IeJ C'aM has heeu prepared and metal iun titratians with calcium (11). cad- mirun 11I), terbium (III) and lutctium (111) have been perfurmcJ. The preliminary electron paramagnetic rexunance (EPR) spectra are compared in terms uf protein confurmatiunal changes induced by thc variuus nmetal inns. (C'alcium (IU-induced cunliOrmatiunal changes have already been demuustrated by uthers.)'1'he significance uf these preliminary results, and thusc already repurted, tu the suitability of Ln(111)- C'aM and C'd(II)-C'aM as mtxlela for ('a(II)-C'aM is discussed. liuccigro,s, J. M. and Nc•lsun, !). J. Journal uf the Less-Cunmwn Metals 126:343-350, 1986. From the I)cpanmcnt of Chemistry, Sackler Sciences Center. Clark University, Worcester, MA. III'.Mli-LINKNI) ION17.A'I'IONS OF MYELOI'EROXII)ASIi UIiT1:C'TEU BY RAMAN I)IFFIiR1iN('G SPI:CTF2OSC'UPY-A COMPARISON WITH PLANT ANI) YEAST I'IiROXIDASES Thc pH-Jependlnce of the oxidation state marker line v, of human leucocyte myelulxruxidasc is determined in dte absence uf chloride using Raman Jil7erence spectruscopy (RI)S). A transition in the freyuency uf v, with pK uf 4.2 *_ 0.3 is limntl. 'I'he pK compares favorably with that previously determined by spectruphotumetric titratiun and kinetic stuJieti. The shil't in r, acrus. the transitiun is -1.3 cm'.'fhe shift in v, and uther Raman marker lines indicates enhanced ir charge in the chlurin ring below the transition. '1'he low frequencies ol'the uxidatiun state ntarker lines indicate that a structural change tKcurs near the chromophure, which results in the liormatiun of a nwre 7r-charge donating protein envirunment litr the chlurin ring at low pH. The Raman results are discussed in terms taf a profxt.eJ catalytic ciintrol mechanism based on charge stahiliruiun uf the energy of ring charge-depleteJ fcrryl intermediates uf Ihe reactiun with IxroxiJc. 7'he myclulxruxiJase IinJings are cumpareJ with similar RUS results lor ferruus horseradish peroxiJase and ferric cytuchrome r Ix:ruxiJase. Stump, R. F., 1)eanin, (;. G., Olit•er, J. M., and Shelnutt, J. A. 13itiphysica) Journal 51:(rO5-610, April 1987. Other support: U. S. I)epartntent of I:ncrgy, Gas Research Institute and National Institutes uf I lealth. Front the 1)cpanment of 1'atholugy, University ol' New Mexico, and the Process Research Division, Sandia National Lalwraturies, Albuquerque. 124 1 125
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O('C'URRIiN('I:, U)S'rRllilJ'I'ION nNU UN'IY)(lL•'NY Of C'GRP IMMl1NOR1;A("I'IVI'I'Y LN'I'Itli RA'I' LOWI:R RIiSI'IRA'1'ORY'('RAC'T: lil l I:("f OI ('nPSnl('IN "I'IdtiA'1'MI:N'I' nNU SURGICAL I)IiNIiRVA"I'IONS '1'he accurrcncc and distribution uf calcitonin gene-relateil peptide (CYiRP) im- munurcacuvuy in the rat respiratory tract were investigated by means uf immimucytO-. chctw.try and radiunnmruwassay using antilxxhcs raised in rabbits to synthetic rat C'(IRP. Suhstantial amuunta of C'(iRP immunnreactivity (range 5-37 pniol/g) were drtrcted in all pans uf the respiratory tract, the highest bcing in the stem bronchus. Gel liltratiun chromatography uf extractahle CGRP immunorcactivity revealed one single peak, eluting a( the Ix)sition of synthetic rat CGRP. CGRP immumrrcactivity was hx:alticd both in mucu.al endocrine cells and nerve fibres from the larynx down to the peripheral lung. ('ORI'-inmwnurcactive endocrine cells were IounJ singly in trachea and stein bronchi and in groups in intrapuhnonary airways. They appeared at a late stage of gestation (17 days), reached a maximum number near lenn and decreased alter hinh to maintain a population similar to that uf the adult animals by postnatal day 21. Similarly, ('GRP-immunureactive nerve libres were first identified by day 1K of the gestation period and reached the adult distribution by postnatal day 21. ('GRP-immunureactrvc nerve fibres were hxaliieJ among smooth muscle, seru- mucuus glands, beneath and within the cpithelium of the airways and around blood vessels. ('( 7RP was also foimd in sensory ganglia and in motor end plates of the larynx musculature. Neonatal pretreatment with capsaicin caused a marked reduction in ('GRl' immurnoreactivity ul' nerve fibres in the respiratory tracts as well as a less marked decrease in the population uf C(iRP-containing endocrine cells uf the lung. No change was tiecn in mutur end plates immunostaining. Vagal ligation experiments revealed that ('GRI'-immunureactive nerve fibres travelling in the vagus originate mainly from neurons located in the jugular ganglion. Infranixlusal right vagal ligation induced a marked loss in CGRP-immmmureactive nerves of the trachea, and uf the ipsilatcral stein bronchus, but no changes were observed in peripheral lung. By con- trast infranodusal Icft side vagal ligation caused a decrease in CGRP-immunorcactive nerves of the ipxilateral lung and bronchus without allccting the peptiJe content in thc trachea. Left vagal ligation also induced a marked increase in both the intensity of staining and number uf C'GRP-immunureactivc endocrine cells in the lung. We conclude that CGRP immunoreactivity is localized in both nerve fibres and endocrine cells and is associated principally with the afferent (sensory) innervation uf' the respiratory tract. The apparent changes in the CGRP-immunorcactive endocrine cell pupulatiun following capsaicin treatment and vagal ligation suggest that these cells may be under neurunal influence. ('aJieux, A., Springall, I). R., Mu)derry, P. K., Rodrigo. J., Ghatel, M. A., Tercnghi, G., Bhxmi, S. R., and Polak, J. M. Ncuru,ciencc )9(2):6U5-627, 1986. Other support: Cancer Research Campaign. Frenn the I)Lpartments of Ilisttxhemistry and Medicine, Royal Postgraduate Medical School, London, England. Tllli FRlits RAI)I('A1. CI II?MIS'I'RY OF('IL)AR)3Tr): SMOKG ANI) TIIL' INACI'IVA'I'ION OF At,l'IIA-I-PROTIiINASN tNtlltil'I'OR Clgarcttc smoke can he separated into gas-phase .muke anl) tar, and fw(h phuses contain very high cuncrntratiun% of I'ree rudicals. Tar contains stable radicals that can be observed directly by electrun spin resonance (USR). (ias-phase smoke cuntains react ive carbon- and uxygen-centereJ radica)s that can not he observed directly by ESR but can be observed using ate HSR .pin-trap method. 'I'he tar radical is identified as a yuinonc-hyJnxluinunc-scmiyuinane interacting sytitem in a low mulccular weight, tarry matrix. The tar radical can Ix extracted into water, and this aqueous tar radical .ystem reduces oxygen to superoxide and hydrogen peroxide and also catalyzes titc conversion uf hydrogen peroxide to the hydroxyl radical. Aqueous solutions uf the tar radical• therefiore, nick DNA. (ias-pltau smoke consists uf small, reactive radicals that should have lifetimes of much less than one second. Paradoxically, however, the observed lifetime uf radicals in gas-phase unuke is about five min. To rationalize this, we have suggested that radicals are continuously produced in gas-phasc smoke by reactions that are similar to those that occur in .rnwg. 'I'his view of the radical chemistry of gas-phase smoke suggests that cigarette smoke is an NO,-rich .mug. Both tu demonstrate that a sntug- like mixture of ga.es can duplicate lhe radical chemistry of gas-phase smoke, and also to provide a utieful and tar-free model of cigarettc smoke, we have developed a simple model system. Our model consists uf mixtures ul' nitric oxide (NO) and isoprene at levels that mimic those in cigarette smoke. The reactions of NO and isoprene in air produce mixtures of raJicals that are virtually identical to those in cigarette smoke. Gas-phase cigarette smoke inactivates alpha-l-proteinase inltibitor (alPl) in a hiphasic process consisting uf a 1'ast reaction folluwcd by a slow rcactiun that occurs over several days. The fast reaction is observed only when fresh smoke is bubbled through a buffer containing aIPI. The slow reaction is observed lxrth using fresh gas- phase smoke and using previously prepared aqueous extracts of smoke. The NO/air/- isuprenc model system also inactivates alpl, and also by a biphasic process very similar to that observed for gas-phase cigarette smluke. A number of subxtances, most notably amines• ascurbic acid, and thiols such as glutathione, protect aIPI against inactivation by cigarette smoke. f'reur, W. A. Pulmunary Emphysema and PruwYo)ysis 369-392, 19K6. Other support: National Institutes of I lealth and National Foundation for Cancer Rc- scarch. Fnmt Ihe l3iodynamics Institute, Louisiana State University, Batim Rouge. P.-PURINO('IiP'1'ORS RIiGULATI: SURFACfANT SECRf3TION FROM RAT ISOI.A"I'GI) ALVIiOI-AR TYPE II CGLLS I. Rat isolated alveular Type 11 cells were utilized to examine the cf fect of purine and pyrimiJine analogues on secretion of pulmonary surfactant. 2. ATI' potent)y stimulated /'l l l-phosphatidylcholine (1'H 1-PC) secretion in a time- and dose-Jependent manner. The ef fect uf ATP was noted by one hour of exposure and persisted t'or three huurs. The EC,(concentration producing 1/2 the maximal response) for ATP-induced 126 1 127
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I'11I -I'(',ccrctk in wa. I(MII uM. 3. AI)I' was also a potent wecrctagu);ue fur surfactatn aecrrtiun, hut AMI' anJ :nleno:,ine had nu signilic;mt cl7ert uot ,urlactant rerrettun at iomrrntratiun. •251) µ.M. 1 he fur AUP-incfuceJ 1'II1-I'C'tiecretiun was 250 uh1. -J. Uthrr purine and pynnucline nuclentiJe.11'1'I', GfP, ("Il',were exanunrcl It n• their cllrct on 1'I11-1'(' .ccrcucm. All purinc and pyrimidine triphu.phateti examinecl signilic:mtly auy.meturJ u:cretiun, hut were much less poncnt that A"1'P. 'I'he li(',,,s wric 1'I'P- Ill µM: IINI µM: C"1'P= 250 µM:'fTl'-= 1(K/ µM. 5. Neither 8-phenyltheuphylline (IO µM, a 1', purin~xc~lur antaguniat), propranolol (I/N) µM, a 4i-aJrcnoKCptur :mtagunist), nur indoniethacin (10 µM, a prostaglandin synthelase inhibitor) inhibited A1l'-incluccJ 1'll1-PC ucretiun frrnn isolated '1'ype II cells. b. 'I'hc,e datu provide evidence lior regulation of surfactant secretion from alvcolar'I'ypc If cells by a 1',-purinuceptor. Rirr, W. R. and Singleton. F. M. British Journal uf Pharmaculugy 8y:485-491, 1986. UOther support: American I.ung Association. C'hildren's Iluspital Research Foundation and Natiun;d Institutes of Ilcallh. From the t)epartnicut uf I'cJiatrics, lhtiver,ity uf Cincimiati Medical ('enter, C'incinm nati, OH. MHASllltl?MIiN'I' OI• INTI:RSI'RANI) C'RUSS-t.INK FREOUI?N('Y ANI) I)IS'I'AN('F R1' 1'Wt:1:N IN'I'I:RRUP"I'IONS IN DNA F,XPUSI:D'fc) 4,5',n=1'ItIMI l'IIYI,PSOKALIiN ANI) NIiAR-lll:l'RAVIUI.ti'I' LIGHT Ilifunrtiunal pwral'ens react Phutochemically with DNA to form single-strand adductti and interstrand, chcnrical cru..-links. ('ross-link formation is first order with JI'l, ihe cuncentratiun of aJJeJ psoralen. when 11'1 •4- K„ the p,uralen - I)NA dis.urr atiun constant. I)NA molecules containing intersiranJ cruss-linke are reversibly bihrli- cal and su :ue readily detected. It was not heretofore possible to detemuine cross-link frcyurncy in polydisperse I)NA fruni the mass F uf I)NA spared cru,s-linkage. We have derived a.tati.tiral relation lo calculate cross-link frequency at fixed light expa .ure and variable JPl. We show here that S, the initial slope of the curve described by - In F as a liinction of l I'/, is proportional to M, the weight-average molecular weight of nick-free I)NA. "Ilte cross-link frcyuency at any JPl can he determined Irunt k, a constant measured for I)NA of knowin Mf at low cross-linkage. This relation is v:Jicl lior DNA uf :my molecular weight distribution. In experiments with unilurtn length I)NA, - In )•' (cro,s-hnk frequency) increased in simple proportion to [I'l. Intact and restriction rnclnnurlcaue llindlll digested nhage Jl I)NA nwlecules have discrete lengthti. S for each w:t, prupurtiunal tu bf of the twin helix even though the molecular weight ili,tnhuuun ol the restriction fragnrents was skeweJ. S was prulwrtiunal to M1/ and to thc meJian uwlerular weight uf.heared cellular DNA over a wide range. Also, we fuunil the IIS was linear with exposure of cellular 1)NA tci y radiation. S ran therefore tk used tu calculate L, the average distance tx;tween interruption% in the double helix. Matsuo, N. and Ross, l'. M. Biu<•hemistrv 2A(7):2(X)l-2(X19, /987. Other support: '1'he Skin Cancer Foundation and National Institutes of Health. hruin the Laboratory for Investigative Dermatology, The Rockefeller University, New York. t)NA REPAIR IN VITRO I)NA repair genes from Escltrrichiu c•oli, yeast and man have recently been cluneJ. The enzymes responsible for phcttoreactivatiun, nuclecxicle excision repair and recumbinatinn-prstreplication repair in E. ccrll have been purified tu homogeneity from genetically engineered strains carrying the corresfwnding genes. This paper describes the overproduction and purification of UNA photolyase (phutureactivating enzyme) which mediates phntureversal uf pyrimidine Jimers and of ABC excinuclease which mediates the removal ul' pyrimiJinc dimers as well as base adducts caused by the ma,jority ofchcmical carcinogens. 1)NA photolyase was purified to homogeneity fram a strain that overPnxluces the enzyme by a factor of 15000. The purified enzyme has a blue color due tu one uf its chrumaphores, the FAD neutral radical. In addition, it contains a.econJ chronuiphure of unknuwn structure. Photulyau binJs with high af linity to eitherdouble-ur single-stranded DNA cuntaining pyrimidine Jimers hut not to undamaged DNA. By investigating the activities and absolute action spectra of eniyme preparations containing FAU,,,, FADH', and FADiIL we have obtained strong evidence that upon excitation with 3Q0-5(X) nm the enzyme donates an electron to pyrimicfine dimers to generate a dimer anion which collapses to two pyrimidines with cuncomitant return of the electron to the chromuphure which enables the enzyme to turn over. The pure enzyme repairs 2.4 dimers min', in agreement with the in vivu values uf 4-5 climerr min'. ABC excinuclease was reconstituted t7um individually purilieJ suhunits, the UvrA, UvrB and lJvrC proteins. The three subunits do not associated in solution hut assemhle on the substrate. The enzyme binds to the 1)NA faice opposite the one containing thc damaged nucleoliJe(s) and removes the nuxlifieJ bases by hydrolyi.ing the Kth pho.phcxJietiter bond 5' and the 41h or 5th phosphodiester bond 3' to the damaged base. The excinuclease does not tum over in the absence of adJitiunal lacturs. In the presence of DNA Iwlymcrase I, helicase 11 and I)NA ligase, A'I'I' and 4 dNTI's, the eniyme excises damaged nucleotides near the in civu rate (0.12- O.2d dimers min') and complete repairuf the damaged DNA is achieved. Scutrur, A. I'hutubiochemi,try and Phutohiophysics Suppl.: 301-315, 19K7. Other auplwn: National Institutes uf Health. Frutu the lJnvertiity uf Nurth Carolina, School of Medicine, Department uf 1)ioxhemis- try, Chapel Ilill. REPAIR UI N Mli'1'l1Yt: N'-NITRO-N-NI'1'ROSCK;UANIDINI: INDUCI:D DNA I)AMA(7li BY A13C IiX('INLJCLEAS6 Esrheric•hiu culi has several overlapping DNA repair pathways which act in concert tu eliminate the DNA damage caused by a diverse array of physical and 128 129
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chemical agents. The AI)C excinuclease which is encoded by the urrA, uvrB, and uvr(' gcnes mcdiatcs both the incision and exci.ion.teps of nucleotide excision repair. Traditionally, this repair pathway has been assumed to be active against I)NA adducts that cause major helical distortions. To detennine the level of helical deformity re- quired for recognition and repair by ABC excinuclease, we have evaluated the sub- strate zpeciticity uf this encyme by using DNA damaged by N-methyt-N'-nitru-N- nitrutioguanidme. ABC cxcinucleaxe incised methylated [)NA in vitro in a dose-dependent manner in a reaction that was ATPdcpendent and specitic for the fully reconstituted enzyme. At vivo studies with various alkylation repair-deficicnt mutants indicated that the excinuclease participated in the repair of DNA damage induced by N- methy I-N' -nitru-N-nit ro.oguanidirn. Van Houtun, B. and Suru•ur, A. Journal of Bacteriology 169(2):540-545, February 1987. OOther support: National Institutes of Health. From the 1)epartment of [3ioLhemistry, University of North Carolina School of Medi- cine, Chapel klill. INVES'1-IGATION OF E3ENZOjajPYRENIi-GLOE3IN ADDUCPS The nature of the adducts lixnud between benzola1pyrene (BP) and glubin was investigated in animals treated with I'H113P. Modification levels on globin were deter- mined by radioactivity measurements. Since BP tetraols can be released from Ixn- «tl(r IPyrene diol epoxide modified protein and DNA by acid treatment, globin samples were treated with acid, released tetraols separated by HPLC and quantitated by uintil- latian counting. In addition, acid released material was measured in a competitive enzyme linked immunosorfx:nt assay (ELISA) using antibodies which recognize I)P tctrauls. 13oth measurements indicate that only 2% of hound radioactivity could he released as free 13P tctraols. These studies indicate that benrol«lpyrene diol epoxide may not be the major meta[wlite of BP involved in blobin binding. Wallin, 11., Jcffre, A. M. and Santellu, R. M. Cancer Letters 35:139-146, 1987. From the Cancer Center/Institute of Cancer Research, Columbia University, New York. S-AD[iNOSYI: L-MIiTII1nNINE MEDIATED ENZYMATIC Mlil'lIY1.A'fIONS IN THl? PLASMA MEMBRANES OF'fHF? HUMAN TROPHOBLAST In recent years, several enzymatic reactions mediated by S-adenusyl-I-- methiuninc (SAM) have been described. Of these, (a) stepwise conversion of inem- brane phusphatidylethanolamine (PE) to phu.phatdiyl-N-methylethanulamine (1'Mli) and phosphatidylcholine (PC ) by phosphilipid N-nrethyltranferaus (PMT I and 11), (h) litrmation of protein carlxtxymelhylesters (PCME) by protein carboxymcthylase (PCM), and (c) mcthyla(iun of endogenous fatty acids by fatty ac id carlxmymrthylascs (FACM) have received considerable attention because they have been implicated in several cellular functions and in biosignal transfer across membranes. Inhibition of SAM-mediated enzyme reactions in intact human placental villus has depressed the uctive uptake of alpha-aminoisobutyric acid (AIB) by this tissue. Amino acid uptake systems are known to be localized in the pla.nta membrane. Therefore, the plasnw membrane of the human Irophoblast was prepared and SAM-mediated methylapuns were studied to evaluate as to which one of the above enzymes ntight play a role in depressing the Alit uptake. The plasma membrane of the human full term placental villus was prepared by exfbliation of microvilli front syncytiotrophoblast and differential centrifugation. Al- kaline phosphatase and 5'-nucleotidase are marker enzymes for placental surface membranes. '1'heir activities were enriched by about 25-foh) in the plasma membrane as compared to the villus homwgenate. The plasma membrane contained enzyme systems which formed PME (PMT 1: K„ 4 < µM, pH optimum 8.6) and PC (PMT 11: K„ > 20 µM, pH optimum 10.5). PMT I was enriched in the plasma membrane. Part ol' the PMT 11(abimt 40%) was solubilized and possibly lost during the preparation of the plasma membrane. l:ven then, PMT 11 was about 4 limes that of PMT I. The activities of PCM and FACM were considerably lower in the plasma membrane than those in the villus homogenate. These investigations suggest that plasma membrane phospholipid N-methylation plays a significant role in the uptake of amino acids by the human trophoblast. Transient accumulation of PME in the membrane lipid bilayer during phoxpholipid N-methylation may alter membrane fluidity which, in turn, influences the lateral microdisplacement and alignment of amino acid carrier systems in the mem brane and their interactions with amino acids. Barnwell, S. L. and Sactry. B. V. R. Trophoblast Research 2:95-120, 1987. Other support: U.S. Public Health Service and Pharmaceutical Manufacturers Associ- atiun Foundation. Front the 1)epartment ol' Pharmacology, Vanderbilt University School nl' Medicine, Nashville, TN. I RIiGIONAI. ANI) DIFF7iRE?NTIAL SLNSITIVITY OF UMRILICQ-PLACGNTAL VASCULATURE TO 5-HYDROXYTRYPTAMINE, NICOTINE, ANI) E:'1'HYL ALCOHUL Perfusiun of human placenta in vitro does not permit distinguishing its sites ol' vascular resistance. Therefore, helically cut strips of (a) umbilical, (b) churiunie, and (c) villuti stem arteries were examined for their sensitivity to 5-H'[', nicotine, and ethyl alcohol (l:l'OFI) in Krehs bicarbonate buff'er (pH 7.4, 37°C). In these preparations, increased vascular resistance is indicated by increased tension or contraction uf the vascular strips. These experiments gave the following results: (a) 5-HT was very potent for causing contractions of umbilical, chorionic, and villus stein ([iC,,,, 84, 47, and 8t) nM, respectively) arterial strips; (h) nicotine induced contractions in umbilical, chorionic and villus stein arterial strips (EC., 2.4, 3.3, and 2.4 mM, respectively); (c) 1:'fO11 (0.5 to 2.0%, v/v) had differential effects on umbilical and chorionic arterial segments. Its eftcct on umbilical arterial strips was minimal contractions or flaccidity. 5-FIT was less effective in the umbilical arterial strips pretreated with ETOH. About 130 ~ 131
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511'd uf tlte contraction height induced by 5-HT was reduced when the umbilical strips were pretreated with lil'UIi. However, l:I'UH IxotentiateJ the contractile reslxtuses of umhtlical strips tu nicutine. (J) IiTOI I, by ilulf, caused cuntracuun4 uf churirnnic anrl villus ,tem arterial ,tripd anJ potentiated the effects uf 5-I fl' and nicotine by about 115- I(dl'ti, depending upon the cuncentratiun uf ETUII and the nature uf thc strip. 11 also enhanced the clfectti of KCI on these strips. The.c observations suggest that ('a' ' nwventenls required for nicotine induced contractions may Ix facilitated in all vessels wherea. ('a' ' ntuvements frunt its sources litr 5-HT induced contractions are partially hindered in umbilical arteries. Susrrv, B. V. R., :md Owens, L. K. Trophoblast Research 2:289-304, 1987. Other support: U.S. Public Heal'dh Service and National Institutes of Ifealth. Frunt the Department uf Pharmaculugy, Vanderbilt University Scluxd of Medicine, Nashville, "I'N. MITOCIIONDRIAL (.]GNOML?S IN INTRASPECIES MAMMALIAN ('L:LL I IYBR11)S 1)ISI'LAY ('Ul)UMINANT OR tH)MINANT/RL:CBSIVE BIiI1AVIOR A unique type uf nunaochaalic mitochondrial I)NA (nttl)NA) segregation wati tuund in m:mmudi:ut cells. In human cell hybrids isolated from the futiiun uf I Icl.a cell. with 23, ( iM639, A5-19, or 293 cells, I IcLa nul)NA was always lost frunt the hybrids, whcreas both parental mtI)NAs were maintained in hybrids of IIcl.ax 1d31iTK . Similar phenomena were observed in nruuu cell hybrids isolated by thc fusion of ccllx wnh different nul)NA types. 'I'ypcs I, 2, and 3, can be distinguisheJ frum each other by reslnctiun IYagment-length polyntorphisms. The ntuuu cell hybrids between cells with type I and type 2 uul)NA :dwary` lost type 2 mtt)NA, whereas the hybrids between cells with type 2:uul type 3 tntl)NA retained both types stably. These observations Suggest Ihat cither a ctxluminanl or a Jumin:uu/reces.ive relationship may he present in inlratilxries ntiuKfiuuJriul gcnumcs ttf huntsm and ntause cells. When the ntihochun- drial genumes in cell hybrids are citduntinant, snx:hastic segregation occurs while nonaochastic segregation occurs when they are in the dontinant/recessive relatiun- .hip. These concepts may help elucidate organelle heredity in animals. H:ryashi, J.-l., Yanehawu, II„ Murak:tmi, J., Tagashira, Y., I'ereira-Smith, O. M., and S/ruv, J. W. L:xlxcrmtlntal ('elI Research 172:218-227, 1987. Other.uppurt. Ministry of Education, Science and Culture uf Japnn, National Science I-uuttJatiun, National Cancer Institute, National Inslitutes of Ileallh, and American ('anrer Society. 1)epanrrrent of Biochemistry, Saitama Cancer Center Research Inctitute, Saitama, Japan; l)eltanmetn uf Virolugy and Epidemiology, BaylorCullege cd'Medicine, I luu.- tun; and I)epanment uf Cell Biology and Anatomy, The University uf Texas Health Science ('enter, Dallas. Mli('IIANISMS 01: CELL FUSION AND SI:LLC"I'ION IN'1'Hli (;6Nlil(ATION 01: IIYBI(IIH)MAS A ntajirr problem associated with the hybriduma, technology is chirontoutntc xta- bility and the reduction or loss of nwnix:lonal antibody production. To ntinitniie the unpredictability of nunpnxlucing variantx, ntost investigators plate out, at clonal Jen- .ity, 5(A)-1lM)t/ fusion pruJuct., screen them I'ur positive untibor)y production, and tinally, reclone llte Itu.itivc wellti two to three times to select fur the chrunrosomally stable hyhridomas. Thix is one ttf the nnnt tinte-cumuming anJ labur-intensive aspects uf the hyhridunt;i prucedures, and methods that would ntinintize the problem ol"nunim- nwnugluhulin synthesiiing cells would be intlwnant. In order lu uuiler.tand why certain positive hybridomas cease producing im- munughrhulinti, we would probably have to know why there is chromosumal instability in cell hybrids. lhtfurtunately, this question remains unanswered. There have been several hylxHheses fur chrunwwmal instability (e.g., elimination or inactivation) in hybrids which lm.ttdate effects of the mitutic spindle, centrioles, miux:hun.lrial in- cuntp:tCtbilitics, and even variatiotu in cell cycle. Ilowever, there is little direct evi- dence to support these hypotheses. We have recently considered the possibility that the st:de of gene activity may he important in regulating chrumo.ome segregation in hybrids. In preliminary experiments when fusing ntouse to human cells, we observed the typical luss of huntan chromosomes. II' variations in the stale of gene activity are inryturt,utl in regulating chromosome segregation, (lten one would predict that exl>Lri- nrental changes in genc activity might intluence the direction uf segregatiun ufhyhrids. To test this, we prctreateJ the human cell line with the hylx)methylating drug 5- azacytidine (unpublished observations). 5-Aiacytidine induces various cell types tu differentiate by an epil;enetic nxxlilicatiun uf the methylation patterns of nuclear 1)NA. Aftcr treatment af lhe human cell line (I ITI080) with 50 aiacyliJute (5 µg/ml. lor 24 h), we fused nrousc (LM"fK-) cclls to the Ircated human cells anJ observed that Ihe mean numher of human chromosontes )termanently retained in the hybrids zignili- cantly increased. In xupprn of this observation, Pravtcheva and Ruddle reported that the nwuu X chromosome has the ability to switch the direction uf chruntusunre segregation in muusc/('hinctie ham.ter somatic cell hybrids, :mJ they suggested that there may be xxgregviun reversal gcnes on the X chromosome. It could be prupused that in ntust hybrnlumaa the loss uf spleen cell chromo.ontes is an important cause of unstable antihudy production and Ihal, by activating certain genes via hylxmoethylatiun or by sonic uthrr ntech:mi,m, one may improve the stability of tltc hybriduntas. It has previously been demonstrated in hybriJumas that heavy-chain synthesis is lost before light-chain syothesis, and that these losses are correlated with the elimination ttfspleett cell chrunrusumes encoding Ihe immunugluhulin genes. Thus, ntethol. /itr selecting hyhriduma, Ihat do not u.e I IAT selection may prumote the growth of those hybridu- ntas retaining spleen cell immunughthulin genes. In adJitiuu, di/ferentiateJ cells are usually Ixtstmitotic urunly slowly dividing, anJ using the I IAT system the investigator usually selects Ihe fusion )troJucts that most rapidly Jivide, Since it is believed that less than I"/r, uf the initial heterokaryons ultimately give rise to dividing hybrid clones, il is Iw.,ible Ihal by using I IAT seleclion une ntay inaJvertently bias the results against the exprc.siun uf dilfcrentiated function and toward a highly selected suhtiet. Further- nx,re, inmtonality of fusiun prcxiucts (r./;., nryeluma cell x spleen cells) is not neces- sarily Juntinant. It has been observed that mouse cells are easier to transfurrn to immortality than hum:m cells. This could he an explanation for why mouse myeloma cells fuuud to spleen cells grow well, whereas human myelomr cells fused to spleen cells do not. 132 1 133
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Finally, recombinant DNA technologies may uffer pramise in the hybriduma lield. Ochi e•t ul. reported functional immunoglubulin pnxluction after transfectiun of' cloncd wnuunugluhulin genes in lymphuid cells. If translation uf imntunugluhulin genes can tk accumpltshed on bacterial host cells, then large-scale proJuctiun,of human nwnnclunal antihox)ies may hecume economically more feasible. Shav, J. W. In: liartal, A. H. and I linhaut, Y. (editor): Methods of Hybridoma Formation. The liuntana Press, 1987, pp. 63-75. Other .rupport: National Science Foundation and National Cancer htstitute. Front the l)eparUment of Cell Biology and Anatomy, The University of Texas Health Science Center at Dallas, n ALLOSTERIC AND NON-ALLOSTERIC FORMS OF RAT LIVER 3-IIYDROXY-3-METHYLGLUTARYL COENZYME A REDUCTASE: 1)IFFERENTIAL INIIIBITION OF ACTIVITY BY ADENOSINE 2'-MONOPHOSPHO-5'-D I PHOSPHOR I BOS E Adenosine 2'-mont)phospho-5'-diphnsphoribose (P-ADP-Rib) is a structural an- alog of NADPH which was reported to competitively inhibit (Ki,,,=21.7 µM) solubi- lized rat liver 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductau:. Huw- ever, microsomal HMG-CoA reductase, which at low thiol concentrations exhibits allosteric properties, is only poorly inhibited by P-ADP-Rib Ki,r,=55O µM at 4.5 mM (iSH). Gradual shill of the microsomal reductase towards a non-allosteric form by increasing glutathionc (GSH) concentrations resulted in a higher inhibition by P-AhP- Rih. Under these conditions, Ki values for P-ADP-Rib were 165 µM and 53 µM at 9 mM and 27 mM GSII, respectively. The largest change in the degree of inhibition by P-ADI'-Rib was observed within the IO mM range of GSH. By contrast, freeze-thaw solubilized HMG-CuA reductase, which does not display allosteric propenies, is readily iniftbited by P-ADP-Rib, even when assayed at a low concentration uf GSH (Ki„g,= 50 µM at 4.5 mM GSH). Assaying the solubilized reductase in the presence of increased thiol concentration results in a minor decrease in the apparent Ki for P-ADP- Rih (22 µM at 27 mM GSH). Microsomal HMG-CuA reductase is allosterically .t"" activated by various nucleuudes. When acttvated hy NADH, thc enzymc ts el fecttvely inhibited by P-ADP-Rih even at a 4.5-mM GSH concentration (Ki,w= 175 µM in the presence of 3(N) µM NADII). These results support a proposal that thiul-depcndent reduction uf enxyme disullide(s) or binding of an allosteric activator to it cause confur- ~~~ titatiunal change. in the micrusumal reductase protein that alter the properties uf its catalytic NAI)PII-hinding site(s). ~.., Ruitelman, J. and Sherhler, 1. ~~,,.,A Journal of Lipid Research 27:828-835, 1986. ~,,,,,;,," Frum the Department uf Biochemistry, George S. Wise Faculty of Life Scicnces,'1'cl ~ F+^~~ Aviv Untverstty, Israel. 134 SI/.li OF'I'tlli DIREC"1'ING MOIIiTY AT CARBON 5 OF CYTOSINE AND THE AC"1'IVI'I'Y OF HUMAN 1)NA (CY'I'OSINE-5)METHYI:I'RANSFERASE M13 UNAx in which carbon 5 of cach deoxycytidine residue in one strand is replaced with a bulky group are very good substrates I'or hunian DNA (cytusine-5) methyltransferase. Rate cnhancements uf up to 35 liold arc obtained depending on the size ol'the moiety :n C-5. The enzyme appears optimally suited to sense a methyl group in one strand a1 this position. Alkaline density gradient analyses iif'the distribution of methyl groups applied to 5-13rdCyd or 5-IdCyd substituted DNA reveal that these groups serve to direct the enzyme to methylate (he unsubstituted strand. Hardy, T. A., Baker, 1). J., Newman, E. M., Sowers, L. C., Gocximan, M. F., and .S)sith. S. S. Biochemical and 13iophysical Research Communications 145(1):146-152, May 29, 1987. Other suppttrC National Institutes of Health. From the L)ivisiuns uf Surgery and Pediatrics, City of Hope National Medical Center, Duarte, CA, and Department ul' Biological Sciences, Molecular Biology Section, University of Southern California, Los Angeles. THE INFLUENCE OF THE dT•dG MISPAIR ON THE ACTIVITY OF THE I IUMAN 1)NA (CYTOSINE-5)METHYLTRANSFERASE Synthetic oligixlectxynuelcutides containing a dT•dG mispair at a centrally Io- catcd d(pCG) dimcr are methylated at a moderate rate by highly purified human 1)NA (cytosine-5)methyltransferase (E.C. 2.1.1.37). The presence of a mispaired dT in one strand induced the enzyme to preferentially methylate (he opposite strand. Baker, 1). J., Hardy, T. A. and Smith, S. S. Biochemical and 1)iophysical Research Cummunications 146(2):596-6O2, July 31, 1987. Other support: National Institutes uf Health. From the 1)ivisiun of Surgery, City of Hope National Medical Center, Duane, CA. S'1'RLICI'l1RAI. ORGANIZATION AND DNA METHYLATION PATTERNING WITIIIN "I'HE MOUSE 1.1 FAMILY We have studied stable differences in patterns of I)NA methylaticro seen in the repeated sequences uf mnuse cells. A cloned 1330-base pair fragmenl of mouse repeti- live 1)NA (pFS-13) was used as a probe in Southern blotting experiments. Mouse spleen anJ 1-121O lymphoma DNA appeared to be normally methylated at lll.ull sites probed by this sequence. Friend erythroleukemia cell, and Sp2 cell 1)NA both showed an abnurntal banding pattern in Ilpull digests. Hybridization nt sini to nutaphase chromosomes showed that probed sequences were broadly interspersed along the anns 135
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of each mouse chromosome. The DNA sequence of the 1330-base pair insert in the clone was detertuined; a copy of the R sequence of 1.1 was found at its 5' end. Walking experimentti using M13.ublcunes from pFS-13 permitted the construction uf a map for d(pCC'(;G) sites at the 3' end uf the mouse LI family. The unmethylated d(pCCG(;) sites in Sp2 and Friend cells could then he assigned to polymorphic-repeated sequencc groups within LI, honwlugous to the region spanned by I3AM5 and R. Since there are uveral thousand copies uf cach of the fragments seen in autoradiographs, these sc- yuences mutit ponxess a comon methylation state at many genomic locations. C'on- cened (noarandom) hypomethylation of certain suhfamilies of LI appears to hc a stable characteristic uf uveral cell lineages. These findings suggest that certain LI families ponsess cummunalitiex that permit and perhaps require differential DNA methyfation in established cell lineages. Tulberg, M. E., FunJerhurk, S. J., Klisak, I. and Smith, S. S. The Journal uf Biological Chemistry 262(23):11167-11175, August 15, 1987. Other support: National Institutes of Health. From the I)ivisions of Surgery and Biology, City of Hope National Medical Center, f)uartc, CA, and 1)epartment of Pediatrics, University of California at Los Angeles, School of Mcdicine. HUMAN I)NA (('Y`I'OSINL: 5)MF."1'HYLTRANSFIiRAS13 SGLECTIVNLY Mf:'fIIYLA'I'hS DUPLEX 1)NA CONTAINING MISPAIRS The presence of the C-C mispair in a defined duplex oligodeoxynucleotide cn- hanccd its capacity to serve as a substrate for highly purified human I)NA methyltrans- ferase. Analysis uf tritiated reaction pnxfucts showed that the C•C mispair acted as a "methylatiun acceptor" in that it was itself rapidly methylated. The m`C'•G base pair also enhanced the capacity of the oligodeoxynucleotide to serve as a substrate for the enrynu. However, this complementary base pair was found to act as a"ntethylation dtrector."'I'hat is, the presence uf (he m'c in one strand induced the enzyme to rapidly methylate at the cylosine residue on the opposite strand in an adjacent C•G base pair. Suiiih, S. S., llardy, 1'. A. and Baker, I). J. Nucleic Acids Research 15(17):6899-6916, 1987. Other support: National Institutes of Health. From the t)ivision of Surgery, C ity of Hopc National Medical Center, Duarte, CA. TAC ANTIGI:N FORMS DISULFIDE-LINKED HOMODIMERS Intcrlcukin 2(Il: 2) is responsible for stimulating T-cell proliferation via interao- tion with spccific, high-affinity membrane receptors. Although an IL-2 binding protein has been identified by virture of its reactivity with a monoclonal antibody that competes with Il: 2 for binding (anti-Tac), the complete and precise structure of functional It: 2 receptors is still unknown. To define further the composition of IL.-2 receptors, both IL-2 itself and anti-Tac were used aa ligands to adsorb membrane proteins for analysis by sodium dodecyl tiull'atelpolyacrylamide gel electrophoresis (NaDix1SO,/I'AGI?). A variety of experimental approaches yield results indicating that the Tuc antigen, which migrates as a single protein on NaIAxISO,/1'AGE under reducing conditions, is also expressed as disulfide-linked homodiniers and oligomera. Examined under nonreduc- ing conditions, Wth activated norntal human'I' cells and cell lines from patients with adult T cell leukentia (HUT-f02; MT-I) express Tac antigen howudimers (M, 105 (XX)) in addition to tnononucrs (M, 54 (kMl). Formation of the disulfide bond is not a conse- yucnce of Ihe cxperimental procedures used to isolate the proteins fior analysis, inas- much as identical results are obtained when the receptor proteins are iodinated and extracted in the presence of N-ethylmaleimide or prepared for electrophoresis in the absence of heat denaturation. Accordingly, these findings point to Tac antigen a.csoci- atin); with itseff prcferentially. The physiologic significance of homodimer and oligo- mer liirmatiun, especially as it relates to the formation of htgh-affinity II: 2 receptors, is presently unknown. Kato, K. and Smith, K. A. Biochemistry 26:5359-5364, 1987. Other support: National Cancer Institute and Eli Lilly Corporation. From the Department of Medicine, Dartmouth Medical School, Hanover, Nil. TIIE IN'fl:RLlil1KIN 2 RBCfiPTOR-FUNCTIONAL CONSE(lUENCGS OF ITS BIMOLECULAR STRUCI'URG High-affinity Il: 2-R binding results from an exceptional type of cooperative interaction between two Il: 2-binding proteins termed a and (3. When expressed to- gether on the cell surface, these two distinct chains form a noncovalent kinetic hybrid receptor complex that exploits a rapid association rate contributed by the p55 fi chain and a slow dissociation rate characteristic fitt the p75 « chain. The p75 a chains signal cell growth, whereas the p55 0 chains only facilitate IL-2 binding by serving as helper binding sites, haveing no discernible signaling role themselves. The unique functional implications of this structural organization indicate that this cooperative bimolecular arrangement reflects a general mechanism by which the efficiency of surface receptors cantw enhanced markedly. Wang, H.-M. and Smith, K. A. Journal of Experimental Medicine 166:1055-1069, Cktober 1987. Other support: National Cancer Institute and Eli Lilly Corporation. From the Department of Medicine, t)artmouth Medical School, Hanover, NH. i 136 137
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IN'I'GRLIiUKIN 2 IMMUNOASSAY USING MONOCLONAL ANTII3ODIIiS 'fo design a sentiitive and reproducible itnnwnitassay for interlcukin 2(IL: 2), two II: 2 reactive ntom`c3onal antibodies (UMS-I and DMS-5) were characterized exten- sively. The unique IL-2 binding characteristics of DMS I and DMS 5 were exploited tii cuntitruct an IL-2 inuuunoassay that is sensitive to 10 pM murinc and human IL-2 (15O pg/ ml ), antcnable to both radioisotopic and cnzymatic detection, and readilyavailabk to otherlaboraturie.. 13udd, R. C. and Snuth, K. A. 13iu/'hcchnolugy 4:983-9R6, November 1986. Other suppwt: National Cancer Institute and Eli Lilly Corporation. From Ihe L)epartment of Medicine, I)artmouth Medical School, Hanover, NH. INTERLEUKIN-2 MON(K LONAI. ANTIBODY AFFINITY ADSORPTION- TH13 C RITIC'AI. ROLIi OF BINDING KINETICS FOR OPTIMAL IMMLJNOADSORPTION With the ready availability of monexlonal antibodies reactive with an extensive spectrum uf antigens, immunoaffinity adsorption has become more widely applicable for protein purification. Huwever, given several monoclonal antilxxiies reactive with the same antigen, most investigators have found that only a few antibodies are useful liir solid-phase immunuaffinity antigen purification. Accordingly, in order to deter- tninc the paramctcrs of monoclonal antifxxly-antigen binding most important forcfl'cc- tivc inununoaflinity adsorption, equilibrium and kinetic binding experiments were performed using radiolafLled interleukin-2 (II: 2) as antigen and four different 11: 2- rcactive momxlonal antibodies. The antibodies were found to diffcr primarily with respect to their kinetic binding characteristics; at 37°C IL-2 Ixound to two of these antibodies very rapidly, while it bound to the other two more slowly. When binding was Ixrfonned at 4°C, thc equilibrium dissociation constants for all of the antibodies decreased due to a more marked prolongation uf thc dissociation rate than the assucia- tion rate. Ilowevcr, at 4°C the association rates of the two slow-reactive antibodies became retarded so markedly that efficient affinity adsorption did not occur. By cuur parison, for both of these antibodies, efficient removal of IE: 2 could be obtained if adsorption was performed at 37°C, provided the column Ilow rate was adjusted accord- ing tu the II: 2 association rate. Kinetic considerations also dictated 11: 2 adsorption to mixtures of two or murc nionoclonal antitxxiies: IL-2 immunoadsotption correlated with the association rates ot' the individual antibodies, rather th:m Ihe equilibrium binding constants. These results indicate that the most important parameter fur cf- ficicnt affinity adsorption is the association rate constant. In addition, Ihe results obtained indicate that monoclonal antibodies may differ markedly as regards Ihcir kinetic binding characteristics, and that all antibodies can serve as effective inuuu- noadsorlxnts, provided their antigen binding characteristics are known. l3udd, R. C. and Smith, K. A. Journal of Immunological Methods 95:237-248, 1986. Other support: National Cancer Institute and Eli Lilly Corpittation. From the Department of Medicine, Dartmouth Medical School, Hanover, NH. REVERSIf31LITY OF GELSOLIN/ACTIN INTERACTION IN MACROPHAGES-EVIDENCE OF Ca"-DEPENL)ENT AND Ca"-INDEPENDENT PATHWAYS Rapid and reversible assembly of an actin matrix in the peripheral cytoplasm is associated with the motile functions of mammalian phagix:ytes (f), Although certain agonists produce a transient rise in the intracellular Ca" concentration which precedes Ihe onset of leukocyte actin assembly and motility, it is also apparent that Icukocytc actin assembly and asslxiated movements can also take place in some cases without a detectable increase in the cytosolic Ca" level. Therefore, besides Ca", an additional signal is implicated firr the regulation of actin assembly associated with leukocyte motility. Gclsolin, a Ca"-binding protein of'mammalian leukocytes and othercclls, is a potent moxlulatur of actin filament length and gelation. In the presence of micromolar Ca" concentration, purified gelsolin severs actin filaments and remains bound to two actin monomers at the fast-growing end of the severed filaments where it blocks further exchange of monomers at the end. If these eff'ects were reversible, gelsolin could, in concert with other actin-binding proteins, regulate the state of polymeriration of actin in response to changes in the cytusolic Ca" concentration. However, whereas the binding of purified gelsolin to actin is highly Ca"-dependent, it is only partially reversed in vitro by a lowering of the calcium concentration. The addition of the calcium chelator EGTA to the 2:1 actin/gelsolin complex permits only one of the two hound actins to dissociate, resulting in a 1:1 actin/gelsolin complex which is incapable of severing actin filaments even though the complex can block the fast-growing end of actin ftlanlents. If another mechanism, not directly involving changes in C:a" concen- tration, was able to dissociate the EGTA-resistant actin/gelsolin complex, it would suggest that gelsolin has a central role as a regulator of the actin matrix in vivu. Chaponnier, C., Yin, H. L. and Stossel, T. P. Journal of Experimental Medicine 165:97-106, January 1987. Other support: National Institutes of Health. From the I Icmatology-Oncology Unit, Massachusetts General Hospital, and Ehpart- ment of Mcdicine, Harvard Medical School, Boston. POLYPIiOSPIIOINOSITIDIi M/C'ELLES AND POLYPHOSPHOINOSITIDE- CONTAINING VESICLES DISSOCIATE ENDOGENOUS GI?LSOI.IN-ACI-IN COMPLF.XF.S ANI) PROMO'I'F. ACTIN ASSEMBLY FROM THE FAST- GROWING ENE) OF ACTIN FILAMENTS BLOCKED BY GELSOLIN The Ca"-activated actin-binding protein gelsolin regulates actin filament length by severing preformed filaments and by binding actin monomers, stabilizing nuclei for their assembly into filaments. Gelsolin binds to phosphatidylinositol, 4,5-bisphos- phate (PIP,), with consequent inhibition of its filament severing activity and dissocia- 138 139
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lion of E(i I'A-resistant complexes made with rabbit macrophage or human plasuta gelsulin-and rabbit muscle actin. This study provides evidence for an interaction trf' gelsolin with phusphatidylinusitol momtphosphate (1'IP) as well as PIf', and further descriMa their effects on gelsolin's function. Both phosphoinusitides completely dis- suciate E(; ( A-inscnsitive rabbit macroplwge cytoplasmic gelsolin-actin complexes and inhibit gelsolin's severing activity. The magnitude of inhibition depends strongly on the physical state uf Ihe phusphuinusitides, ixing maximal in preparations that contain small micelles ol' either purified PIP or I'IP_. Aggregation uf PII' or PII', micelles by divalent cations or insufficient sonication or their incorporation into vcii- clcs cuntaining other phospholipids decreases burt does not eliminate the inhibitory properties uf thc pulyphusphuinusitides. The presence of gelsolin partly intiihits the divalent cation-induced aggregation of PIP, micclles. PIP; in combination with EGTA inactivates gelsolin molecules that block the fast-growing end ol' actin filaments, thereby accelerating actin polymerization. Regulation of gelsolin by the intracellular ntcsscngers C'a" and pulyphosphoinusitides allows for the formation of scveral diffcr- cnt gelsolin-actin intermediates with distinct functional properties that may be in- volved in changes in the state uf ctyoplasmic actin following cell stimulation. Janmcy, 1'. A., lida, K., Yin, H. L. and Sius.rel, 7'. P. The Journal of Biological Chemistry 262(25):12228-12236, September 5, 1987. Other support: U.S. Public Health Service, Whitaker Health Sciences Fund, and American IJcart Association. From the Ilcmatulugy-Oncolugy lJnit, Massachusetts General Hospital, and Uepart- mcnt ul' Medicine, I larvard Medical Schcxtl, Boston. MUI)llI'.ATION 01: GELSULIN F(1NCfION BY PIIUSPHATIL)YLINUSITUt. 4,5-BISI'l IUSP1 fATE The actin-hinding protein gelsolin requires micromolar concentratiuns uf calcimu iuns to sever actin filamcnts,ln potcntiate ils binding to the end of Ihc lilameiu and to prumotc the po~lymeriiation of monomeric actin into filaments. Because transient increases in huth intracellular jC a" I and actin polymerization accompany the ccllular response to certain stimuli, it has beeu suggested that gelsolin rcguhUes the reversible assembly of achn filaments that accompanies such cellular activations. But other evidence suggests that these activities do not need increased cylUplatimlC lC'a" I and that once actin-gclxulin complexes form in the presence of Ca" in rirru, removal uf' free C'a" causcs dissociation of only one of twu hound actin monomers from gelsolin aud the resultant binary cumplexes cannot sever actin filantents. 'I'he finding that cellular gclutlin-actin complexes can be dissociated suggests that aC'a" -independent regulation of gelsolin alsu occurs. I lere we show that, like the dissociation ul' prutifin- actin complexes, phusphatidylinositul 4,5-bisphosphate, which undergoes rapid turn- over during cell stimulation, strongly inhibits the actin filament-severing properties of gclsalin, inhibits less strongly the nucleating ability of this protein and restores the potential for lilament-uvering activity to gclsulin-actin complexes. Jiumy, P. A., and .Suossid, 7'. P. Nature 325(64(12):362-364, January 22, 1987. Other support: U.S. Public Hcalth Service and Whitaker Health Sciences Fund, From the Ilentatolugy-Onciilugy Unit, Massachusetts General Hospital, and I)epart- ntent of Mcdicine, Ifarvard Medical SchoHd, 13uston. FLUORES('I?NCI's RA"1'IO IMAGINC; MICROSCOPY: TEMPORAL AND SPA'fIAI. MEASUREMENTS OF CY"I'QPLASMIC pH Fluorescence ratio imaging microscopy has been used to ntcasurc thc spatial variations in cytuplasmic pH of individual quiescent and nunyuiescent Swiss 3'I'3 cclls. Frmdamcntal issues ul ratio imaging that permit precise and accurate temporal and spatial mcasuremcnts have been addressed including: excitation light levels, lamp operation, intracellular probe cuncentratiitns, methods of threshold selection, photob- leaching, and spatial signal-to-noise rutiu measurements. Sulx:ellular measurements can be measured accurately (<3%. cokflicient of variatiun) in an area of 3.65 µm' with the present imaging system. Quiescent Swiss 3'1'3 cells have a measured cytoplasmic p11 of 7.0+) (0.01 SEM ), whereas nonquiescent cells have a pH of 7.35 (0.01 SEM) in the presence ol' hicar(wnate hut7cr. A unimoxlal distribution oflncan cytoplasmic pH in both quiescent and nonyuiescent cells was identified from populations of cclls ntca- surcd on a cell by cell basis. Therclitre, unlike earlier studies based on cell population averages, it can be stated that cells in each population exhibit a narrow range of' cytoplasntic p11. I lowever, the mean cytoplasmic pli can change bas4d on the physio- logical state of the cells. In addition, there appears to be little, if any, spatial variatiun in cytuplasmic pl l in either quiescent or nonyuicuem Swiss 3T3 cells. Thc p11 within the nucleus was always the same as the surrounding cytoplasm. These values will serve as a reference puint for investigating the role ol' temporal and spatial variations in cytoplasmic pll in a variety of cellular processes including growth control and cell rrtuvcmcnt. Bright, G. R., Fisher, G. W., Rogowska, J., and'1'aylur, 1). L. The Journal uf CcII Biology 1(k3:1(/I9-1O33, April 1987. Other support: National Institutes uf Health and National Science Foundatiun. From the 1)cpartment of Biological Sciences, Center for Fluorescence Research in Biomedical Sciences, Carnegie-Mellon University, Pittsburgh. t1INl)IiRIiU I)117F(1S1ON OF INERT TRACER PARI'ICLES IN TIIE C'Y'1'OPLASM OF MOUSE 37'3 ('I:LLS Using fluorescence recovery afler phuto-bleaching, we have studied the dif7usion ol IluUresCL'In-IabCh:d, sitc-1'racti~onated Ficoll in (he cytoplasmic space of living Swiss 3T3 cells as a probe of' the physical chemical properties of cytoplasm. The results reported here corroborate and extend the results of earlier cxperiments with Iluures- ccin-L•Q>ibed, size-fractiunated dextran: diffusion of nonbinding particles in cytoplasm is hindered in a size-dclendent manner. Extrapolation of the data suggests that parti- cles larger than 260 Ain radius may be completely nondiffusable in the cytuplasmic space. In contrast, diffusion of Ficoll in protein utlutions of concentration comparable 140 141
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to the range reported for cytoplasm is not hindered in a size-dependent manner. Although we cannot at present distinguish amung several physical chemical mixlels for thc organiration of cytoplasm, these results make it clear that cytoplasm Ixrsticsses some sort of higher-order intermolecular interactions (structure) not fimnd in simple aqueous protein solutions, even at high concentration. These results also suggest that, tor native cytuplasmic particles whosc smallest radial Jimcnstion approaches 260 i~ size may Ixx as nnlwrtant a determinant of cytoplasmic Jiffusibility as binding specific- ity. -1'his would include most enJosonoes, Polyribosomes, and the larger multienzynm complexes. Luby-I'helps, K., Castle, 1'. G., 'I'uvlor, D. L., and Lanni, F. Proceedings of the National Academy of Sciences, USA 84(14):491O-4913, July Io)87. Other support: Hamamatsu Photonics, K. K., and National Institutes of Health. From the Ikpartmcnt uf Chcmistry and Department of Biological Sciences, ('enter for Fluorescence Research in Biomedical Scicnces, Carnegie-Mellon University, Pitts- burgh. VI. Immunology AN'I'IGI:NIC ANALYSIS OF IIGMATOPOII'sSIS. V1. FLOW CYTOM(:TRIC C'HARAC"f13RI'LATION OF MY-IO-PUSITIVE PROGENITOR CELLS IN NORMAI. HUMAN BONE MARROW We have previously shown that the anti-My-10 murine monoclonal antibcxly detected an epitolx of a 115-kUa glycoprotein expressed specifically on KB-la Icuke- mia cells and a small subset of normal human twne marrow cells. This My-I0+ marrow cell subset was shown to contain a highly enriched Ixopulation of morphologic blast cells and hematopiictic coluny-fiirming cells. In this relwn. My-10+ cells were characterii.cd, by flow cytumctry, as an approximately 1°k, sublwpulation of normal human hone marrow cells. My-I0+ cells were slightly larger than lymphocytes and al!ranular, as determined by their tluoreseence-activating cell sorting (-er) light-scat- tering properties. In two-color immunotluoresecnce experiments, My-10 t• cells cikx- presscd the I ILA-I)R antigen. However, there was no detectable cellular coexpression of My-10 with either the Lcu I--5, 7, 9, 11, 15, M3, or My-18 antigens. 'I'here was an average of approxima(ely 50,(NN) My-IO molecules per My-10 + marrow cell. 'I"his PruviJcs further evidence that the My-IO molecule is expressed, at relatively low levels, selectively on early human marrow cells hut not on mature lymphohemato- Iwictic cells. ('ivdrr. C. l., Banquerigo, M. L., Strauss, L. C., and L.oken, M. R. Exlxrintental Hematology 15:10-17, 1987. Other support: National Institutes of Health and the 13ecton-Dickinson Monoclunal Ccntcr, From the l)ivision of Pediatric Oncology, Johns Hopkins Oncology Cemer, The Johns Hopkins University School of Medicine, Baltimore, Ml), and 1)ecton-L)ickinson MonoKlunal Center, Mountain View, CA. ANTIGENIC ANALYSIS OF HEMATOPOII:SIS. IV. THE"s MY-11 IIEh1ATOPOI1 1'IC CGLL SURFACE ANTIGEN IS 1:XPRL^:SSI:D BY MYI:LOMON(1CYTIC AND LYMPHOID, BUT NOT GRYTHROIt), PROGENITOR CELLS The anti-My-II murine monoxlunal antibody reported on in this work identifies a KG-la cell surface glycoProtein with apparent molecular mass of 210,tHf0 daltons. Peripheral blood B-Iymphocyles, and a novel subset of'T-lymphoxytes (not coinciding with helper or cytutoxic subsets) express My-11 antigen; granulocytes, red cells, and platelets are antigen negative. In normal Iwne marrow, lymphoid progenitors (TdT positive) anJ most granulocyte-monor:yte progenitors express My-I I, but erythroid and nmltilineage progenitors are My-II negative. Approximately half of acute leukemia blast cell specimens are My-II fx)sitive. The My-11 antigen distinguishes between lyntphohernatopuietic cells on the basis ol' lineage, and assists in the purification of hematopoietic progenitor cells and the suhclassification of leukemias and normal lym- phocytes. Strauss, L. C., Brovall, C., Fackler, M. J., Schwartz, J. F., Shaper, J. H., Loken, M. R., and (.'ivin. C. l. Experimental Hematolugy 14:935-945, 1986. Other suplx>rt: National Institutes of Health. From tioc Ikpartments of Pediatrics and Pharmacology and Experimental Thcralx:u- tics, I)tvisioms of 1'ediatric Oncology and Cell Structure and Function, Johns Hopkins Oncology ('enter, The Johns I lopkins University Scluwl of McJicinc, Baltimure, MD, and t3ccton-Dickinsun Monoclonal Center, Mountain View, CA. IiXPRI:SSION OF TIIG Fcy RECEPTOR ON Ly-I' B LYMPHOCYTBS The expression of IgG Fc receptor (FcR) molecules was examined on Ly-l' B cells anJ 13 cells tuntor.. FcR molecules were f'ound on a representative Ly-l' B cell lymphoma of the pre-B and as well as of the mature cell B phenotypes. The expression of the FcR did not change on these cells during their differentiation to B cells or to antiMhJy-sccreting cells, respectively. Ly-I' 1) cells were tiwnd at low freyuency (--- 2%.) in the spleens of normal mice, and in the Peritoncal cavity where their repre- sentation was greater. The frequency of Ly-1' 13 cells declined after birth, although their numbers increased in both organs. These L.y-I • B cells expressed the FcR njole- cule throughout ontogeny. Furthermore, the amount of FcR expressed on Ly-I ' B cells was similar to the levels expressed on their "conventional" (Ly-I ) B cell counter- 143 142
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parts. '1'he Pclt was alw fuunJ un Ly-1' It cells from auluimmune mice. The signiti- cancc of the expression uf FcR molecules on Ly-I' B cells is discussed. lhxtcr, C. M. and C'urlrv, R. B. Furopean Journal of hnmunolugy 17:R67-871, 1987. Other ,uppun. National Institutes of Ilealth and National Science Foundation. Frum the 1)epartuuent of Microbiology and Immwaulogy, Duke University Medical (.'enter, I)urham, NC. TFlI? kOLli OF ADHI:RENT ACCESSORY CELLS IN THE GENERATION OF tiFFEC'TOR SUPPRESSOR T CELLS The role uf accessory cell populations in the generation of effector suppressor ("I's,) cells was studied. By using an in vitro culture system, it was previously deter- mined that the induction of NP-specific effcctor suppressor activity requires T cells, antigen, and an anli-iJiutypic B cell population. We now demonstrate that the genera- tiun ofTs, cells in this system also requires accessory cells. The accessory population appears to play a role in the processing and presentation of antigen. These antigen- presenting accessory cells are required early in the induction phase of'fs, generation. '1'hcse accessory cells can present NP coupled to immunogenic or nun-imntunugenic polypeptide carriers, including polymen of L-antino acids. However, NP coupled to polymers of Fxwrly metatwlited D-amino acids fail to induce suppressor T cell genera- tion. Furthermore, Ihc data demonstrate that an H-2 homology must exist between lhe Ts, precursors and the antigen-pretienting cell population if suppressor activity is to Ix: gencrated. We also characterite the differential genetic restrictions that govern the induction of Ts, cells that control suppression of either T cell or B cell responses. The data suggest tltat although I-J region encoded gene products control the induction and. et f ec tor phases of suppressur cel I act i,vity as measured on T eell respunses, the xuppres- siun of li cell respun.es appear to he controlled by I-A gene proolucts. Possible cellular mechanisms that might explain these findings are disussed. Kawasaki, H., 7.upku, K., Diamond, B., Minami, M., and Dorf, M. E. The Journal of Immunology 13H(7):2(M)3-2OG8, April I, 1987. Other support: Natiunal Institutes rrf Hcalth. Frunt the 1)epartment of I'athulugy, liarvarJ Medical Schoo)l, Bostun, anJ I)epartrurnl of Micruhiuhrgy and Immunolugy, Albert Einslein College of MeJicine, 7'he Brunx, NY. AN'I'IBOI)Y INt1IBl'I'ION OF SUPRI:SSOR Cla.l. INDUCTION The IJ genetic restrictiuns of suppressor T(Ts) cells are controlled by 11-2-relatcd determinanl% that are expresud on antigen-presenting cells This has led to the hypnthe- si% that '1'ti cells carry receptors for a self' H-2-related ligand that is expressed on specialized antigen-presenting cells. We refer to this H-2-rclated ligand as the IJ intcr.tctin)g muleculc. This report evaluates the ability of rabbit antibodies directed against idiotypes utl nuinuclunal anti-IJ antibodies (the latter are presumably reactive wtth Ihe "fr cell receptor) fu hind IJ interacting molecule and to inhtbit antigen preu nta- uun tu 'I's celis. Such anti-iJiotypic reagents were prepared against 7' cell-reactive monoclonal anti-1' and anti-IJ° antihcxlies. The F(ab'), fragments of thc.e anti-idiotypic reagents hhx•keJ'fs cell mduction. 'fhc inhibition was haplotype specific and mapped to the IJ rcl!ion. The anti-iJiotypic antibodies blocked the generation uf Ts„ '1's., and 'I'ti, cells. The cellular target of the blocking activity mediated by Iheu: anti-idiotypic antihyxlies is a m:arnophage. 'fhi. was shown by using a cloned ntacrophage hybriJuma line for IxMh 'I's induction and absorption uf antitxrJy activity. The combined data support the concept that ntacrophages express IJ interacting determinants that are respunsible fur Ts cell inducticzn. Kawasaki, H.. Zupko, K., Diamond, B., Minami, M., and Dorf, M. F.'. The Journal af Intntunolugy I:i8(7):211b3-2(1b8, April I, 1987. Other.upprtn: National Institutes of Health. From the Departntent of Pathulogy, Harvard Medical School, Boston, and Department of Microbiolugy and Inununolugy, Albert Einstein College of Medicine, The Bronx, NY. F11NCfIONAI. ANALYSIS OFCLONED MACROPHAGE HYHRII)OMAS V. INl)11("17ON Oh SUPPRESSOR T C'EI.L RESPONSGS It has been suggested that macrophage-like accessory cells are involved in tiup- pressar'I' cell (-fs) inductian. '1'o further analyzA! this issue, we obtained several cloned utacruphaHe hyhrirhoma cell lines by somatic cell fusion uf the macrophage tumor P31tliI), uf UIIA/2 (I1-2°) origin with splenic adherent cells ot' C'Kli mice (11-2'). Several cloned linrti displayed Ihe serolugical and functional characteristics of macro- phages. We evaluated the ability of these hyhridumas to induce third order or eflcctur 'I's ('fs,) to suppress the contact sensitivity resprmsc: against the hapten 4-hydruxy-3- nrlruphenyl acityl ( NI'). In contrast to the parental P3881), and two other inacrophage hybriJomas, onc macruphage hyhridonta clone, ternteJ 63, when conjugated with NP, induceJ Ts, which suppressed contact sensitivity responses against NP but nnt I)NFB, showing that the'I'ti, were antigen specific. Macrophage hyhridoma 63 could specili- cally inducc'I's, activity in either H-2', H-2", or H-2'/11-2' heterozygous hosts. Thus, macruphage hyhrirfonta 63 functionally expressed major histucuntpatibility complex- related restricting Jeterminanti, and Ihe fusion with cells from a H-2` macruphage donor caused a functional cumplementatiun of F1-2'-relateJ, 'I'ti-inducing elements. 'I'he genetic restrict5on governing induction ofTs, was controlled by genes Ihat mapped to I-1 region. F'unhermure, NI'-coo,jugated inacrophage hybriduma 63 could serve as a target for elicitation of suppressor responses after administration of I-1', bul nut I-1", restricted suppreswr Iactor. 'I'he data suggest that inacrophage hybriJumas represent a mcans to disuct heterogeneity within the macrophage polwlation. The data also imply that the I-J determinants expressed on ntacrophages represent a ligand litr the antigen receptor ol'Ts. Kawasaki, !(., Martin, C. A., Uchida, T., lJsui, M., Noma, T., Minanti, M., and I)urf; M. E. 144 . ~ 145
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The Journal ul' Immunulutiy 137(7):2145-2151, October I, 1986. OOther suppnrl: National Inslitutes of' Iicalth. From the I)epanment uf Pathology. liarvard Medical Schowl, Boston. NI;W C'ONCI:P'fS IN ANTIBODY STRUCTURE In this chapter we have outlined a new statistical analysis of the antibody structure a~ it rclates to its antigen binding and interaction in the immune network. With this approach we recognize certain aurface accessible areas uf the Ig molecule which express idiutypic determinants. These Idiutopc Determi.ning Regions (IDR) overlap to a large extent with previously defined complementary determining regions (C'1)Rs) implicated in providing contact residues l'or the antigen. Experimental data un idioty- pic sequence regions are in excellent agreement with the predicted location of II)Rs. The estahlishment uf IDRs leads to the development of a new concept of the antibody structure-functiun relationship and provides a better rationale fur immuno-moelulatory approaches based on the use of antitxxlies. Another advantage of thc proposed multiple variable surface model relates to a reconciliation of previous views on the antilxxly structure with models ol an immunc network. According to these views the interaction of twoconcave or "f'emale" binding sites would have tu take place during network interactions. Removing the restriciton fur a binding site to be concave and for an idiotupe to be protrusive allows to build a better molecular ntudcl for the immune network. As a consequence of'this, we propose to abandon the idiotype terminology of Ab2 alpha, txaa, gamma and epsilon. Kohlrr, tl. and Kielx:r-Nmmons, T. In: Szentivanyi, A., Maurer, P. Ii. and Janicki, t3. W. (eds.): AntihlAdies, Plenum Publishing C'urroration, 1987, pp. 11-17. Other support: American Cancer Society and Center for Applied Molecular Biology and Inmwnulugy. Frant the t)epartment nf Molecular Immunology, Roswell Park Memorial Institute, I1ulTalo, NY. "f(JMUR-SI'GCIFIC II)IUTYPLi VAC'CINIS II. ANAI.YSIS UI 'fllli "I'UMOR KIa.A'I'GU NETWORK RESPONSIi INL)(JC'IiI) 13Y'TIIIiTUMUR ANl) BY INTIiRNAI.IMAGG AN'I'I(;I:NS (Ab24i) In this study the tunmor-specitic immune respim.e induced by nradiated tumor cells (1.1210/(YLI.) and by anti-idiutype antitxadies was analyzed. 'fhc anti-idiotype antibodies (Ah2) were made a)tainst Ihe paratope of a nionoclonal antitwuur antihody (IIC'l/ that recognizes a tumor-associatcd antigen which cross-reacts with the muux mammary tumor virus-encoded envelope glycoprotein 52. Two Ab2, 2110 and 3A4, induced idiurypes expressed by the monoclonal antitumor antibodies IICI and 2132. Cytotoxic T cells, generated by immunization with irradiated tumor cells, lyse 2F70 and 3A4 hyhridoma cells. Furthermore, immunization with Ab2 induces tumor-slk- citil cytotoxic'f lymphocytes, The frequency of tumor-reactive cytiuuxic T lympho- cyte was found to Ixx sint,ilatr in tnicc immunized with Ab2 or irradiated tumor cells when cxarninrd at the pt, -, in,ur level. Huwever, only 2FI(1 induces protective inmw- nity against thc growth ut 1.121O/C'LL tumor cells. The deplctiun uf a L.iT4' T cell pupulation frnm 2PIQ immune mice was found to increase the effectiveness of transfer- red'I' cell. to induce inhibition of tumor growth. The inability of 3A4 to induce anti- tunwr immuniry could be correlated with the presence of a population of Lyt2' regulatury T cclls. Collectively, these results demonstrate the existence of a regulatory network controlling the expression of eflcetive tumor immunity. Our results demon- strate that aelection of binding site-related Ab2 may not Ix a significant criteria fbr the development iif an idiutype vaccine. A better understanding uf the regulatory interac- tions induced by anti-idiotypes is needed fiir the design of effective antitumor inununothcrapy. Raychaudhuri, S., Saeki, Y., Chen, J.-1., Irihe, H., Fuji, fi., and Xohler, H. The Journal of Inununulogy 139(I):271-278, July I, 1987. Other support: American Cancer Society and the National Cancer Institute. From the Department of Molecular Immunology, Roswell Park Memorial Institute, [3uffalo, NY. FUNC"1'IC)NAI. ANALYSIS OF T CELL SUBSETS AND CLONES IN HUMAN RENAL ALLOGRAIT REJECTION We previously refwrted that is was possible to propagate activated donor-specitic T I'ymphoxytes I'rum biopsy tissue of human renal allografts undergoing episodes of rejection. The ability of interleukin 2 to stimulate division of such in vivo-activated T lymphocytes has been conlirntcd by others frunt heart, lung, and liver grafts. While it is clear that there is enrichment for relevant donor-specific proliferative and cytotoxic T lymphocytes in rejecting grafts, it is important to investigate the mechanisms by which damage is ultimately mediated. While there is evidence that cytotoxic cells can mediate damage, other groups have emphasized the contributions ut' helper/inducer subsets in causing rejection via adclayed type hypersensitivity-like reaction. Toevalu- ate the contribution of individual cell subsets, we have used a cell sorter to enrich CD4', CI)8', and a populatiun ol' IL 2-respimsive CD4'/8' cells found within the graflx. These subsets were assayed lor donor recognition to further our understanding uf thc process uf dunor recognition by lymphocytes present at the sites of tissue injury dut ing rc~jcction. There was readily deleclable donor specificity with respect to prolil'- eratirFn in secondary MLR for both CD4' and CD8' soned subsets, hut only the sorted CDti' cells were specifically cytotuxic. The CD4'/CD8' cells lacked donor specific functiun. In addition, we have prepared clones from limiting dilutions of fymphocyles obtained frum biopsy tissue. The clonal analyses indicated a pronounced enrichment lor dunur-sIxcific lymphocytes within the grafl buth befitre significant nr virru selec- tion (cloning was pcrfiormed within 48 hours atter culture initiation) and after two weeks of gruwth in IL 2. Stegagno, M., Boyle, L. A., Preffer, F. I., Leary, C. P., Colvin, R. B., Cosimi, A. B., and Kuntick, J. 7'. 146 147
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'I'ransptantatiun Pnx:eedings 19(I):394-397, February 1987. Other support: National Institutes of' I Icalth and March uf Dimes• From the Dcpanrucnts uf Pathology and Surgery, Massachusetts General Hospital, . Butium. TWO-C`OLOR FLOW CYTOML:TRY AND FUNCTIONAL ANALYSIS UF LYMNI IOcY'I'IiS C'ULTURIiD FROM HUMAN RENAL ALLO(iRAI•TS: II)L•N'I'IFICA7'ION OF A LGLJ-2' 3' SUBPOPULATION The phenutypc uf T lymphox:yte subsets present in renal biopsies showing acute cellular allogralt rejectiun in six patients on cyclosporine have been charactcrired iri sim by hnnwnoperuxidase staining, and atierexpansion in vitro in in(crleukin 2(I1= 2) by two-culur Iluw cytonutry, soning, and functional analysis. After 8 tu 42 days in organ culture, Iwth Lcu-3' (CD4) and lxu-2' (CD8) subsets were fiwnd in each culture, in a ratio that varied from 0.21u 5.(1, which was not significantly dif ferent than the results uf in situ intntunoperoxidase staining of the uncultured biupsy. 77te cultured cells were almost all Leu-4' (('1)3) T cells (89% :t 4), which expressed the activation markers 1)R (2i2'l. ± 6) and the IL 2(CD25) receptur(15% ± 4). The L.eu-3' cells were largely Leu-R (9O%- ± 6), whereas a minority uf the Leu-2' cells were Lcu-I5' (('1)I I) (26^k• ± 4). Only a small fractiun of the Lcu-2' cells stained fitt Leu-7 (811, !- 6). Functiunal analysis of FAC'S-purilied Leu-2 3' and Leu-2'3 populations indi- cated that both subsets prolil'erated in response to graft donor antigens in a mixed lyntphucyte reaction (MLR) and produced 11.2. Only the L.eu-2' 3 population denwn- strated donor-specilic cytutoxic activity. A minor subpopulation in each culture were both Lcu-3' and L.eu-2' (2.0%). l.cu-2' 3' cells front one biopsy were purified to homogeneity (99.8%), and were f'uund to express the T cell antigen receptor complex Ti/CD3 ( W'I'-3I ', Lcu-4' ), but nut the common thymucyte antigen CUI (OKTb). 'I'he l.cu-2' 3' cells neither responded in the MLR, nor showed any cytotuxic capacity. The I.eu-2' 3' cells were capable ot' Il. 2 but not interferon-y production. None of the purilied cultures demonstrated NK activity. A subset of the purilied l.eu-2' 3' cells lost L.eu-2' during I to 3 wk in culture, and became Leu-2 3'. '1'hese studies pruvide' evidence that the cells that inliltrate renal allografts during rejection include allupru- liferative lymphokine-producing cells of Ixnh Lcu-2' and Lxu-3' subsets. The Lcu- 2' 3 cells are also highly cytotuxic against donor lymphocytes, indicating the pres- ence ul hellxr independent cytotuxic T Cells. A minor population of l.eu-2' 3' T cells that do not express donor specilic function was also identilied. Preffcr, F. I., Colvin, R. B., Leary, C. P., Boyle, L. A., Tuazon, T. V., Lacarovits, A. L, C'osinti, A. 13., and Kurnick, J. T. The Journal uf lmmunology 137(9):2)123-2830, November I, 1986. Other support: National Institutes of llealth, March of Dimes, Shrintr's Burns Insti- tute, Bcctun l)ickinson, U.S. Public Health Service, Medical Rmarch Cc>uncil-C'an- ada, and ('hina Medical Board. From the Imntunopathulugy Unit of the Department of Pathology, and Transplantation Unit of the General Surgical Service, Massachusetts General Hospital, and Harvard Medical School, Boston. LiXTRAC'lIROMOSOMAL. DNA SUBSTRATES IN PRI:-B CELLS UNDGRGO INVERSION OR I)I:LIiTION AT IMMUN(xiLOBULIN V-(D)-11OINING SIGNALS Seyuences encrx.ling inmrunuglubulin variable domains are known to he assrm- bled frum variable ( V), diversity (I)), and joining (J) segments by sile-specific recum- hination. We present a.entiitive and rapid assay fur V-(D)-J rccumbinatiun that uses plasmid 1)NA transiently introduced into transfurnred pre-B cells, and demonstrates that the recombinatiun is independent irf any unique chrumusomal context. Sequences suf ficient to constitute recumbination sites are contained within the 8-1 and 42 hp Ilank- ing, respectively, (he murine J,I and V,L.8 segments, which include the known hepta- nur-nonamer V-(D)-J juining signals. I)cletiun and inversion occur at comparable freyuencies. Thus, V-(D)-1 recombinatiun may be relatively insensitive to the tupolug- ical arrangement uf sites, and events at the two novel junctions produced by the reaction ntay be coupled. Hesse, J. E., Lieber, M. R., Gellert, M., and Mizuuchi, K. C'elI 49:775-7H3, June 19, 1987. Other support: National Cancer Institute. Frum the Laboratory of Molecular Biology, National Institute uf Diatxaes and Diges- tive and Kidney I)isea•ues, National Institutes of Health, Bethesda, MD. DIiVELOPML3NTAL STAGE SPECIFICITY OF THE LYMPHOID V(D)l RECOMILINATIUN ACTIVITY We have examined the level uf immunoglohulin gene V(D)J recombination activ- ity in a number uf cell lines derived front lymphuid or nonlymphoid lineages. The assay we employed uses extrachrumosomal DNA as substrate and thereby avoids difticultics associated with the use of chrumosotnally integrated substrates. The re- conthinatiun activity decreases during B-Iymphsrid development. It is highest at the earliest stages of comntitted B-cell differentiation and then falls progressively, reach- ing undetectable levels at the mature B-cell stage. The activity is also present in muiltipritenlial progenitors of myehrid cells and in pre-T cells but not mature T cells. No activity was lirund in several nunhentatupoietic cell lines. Recombination was seen only among substrate molecules which had replicated in the eukaryotic cells. Several possible interpretations of this result are discussed. l.irhrr, M. R., Hesse, J. E., Mizuuchi, K., and Gcllert, M. Genes & 1)evelupment 1:751-761, 1987. Other support: National Cancer Institute and National Institutes of Health. Frum the Laboratory of Molecular Biology, National Institute of Diahetes and Diges- tive and Kidney Diseases, Bethesda, MD. 148 149
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r M ('APSAIC'IN-SENSI"I'IVIi NIiRVIiS ANU'fIIE CUI'ANEOUS ALI.I:R(;Y RI:A('T1( )N IN MAN -- I'OS`11n1.I; rNV(M.vI:M91:NT OI- StiNtiURY NGUROPta'rn)tiX IN 'I tlh. M.ARI: RI•.A(-I IUN The elfrcts uf local capsaicin pretreatment on the cutaneous triple response reac- lion induced by allergen exlxosure or anti-Igfi were studied in man. Acute exlxnure uf (he human,kin to capsaicin caused a buming sensation and a clearcut flare reaction hut no wheal response. Upon repeated administration these local reactions to capsaicin ditiappeared. '1'he flare component and the subjectivc itching sensation of the cutaneous allergy reaction tu rat antigen in sensitized persons or anti-IgE in non-allergic persons were then markedly reduced. Two weeks after capsaicin pretreatment the flare rc- sponsc to allergen was not significantly changed compared to the control reaction, suggesting a reversible cffcct of capsaicin treatment. The wheal component ol' the allergy or anti-IgE reaction was, however, not influenced by capsaicin pretrcatntent, indicating that the wheal and flare components are caused by different mechanisnts. It is concluded Ihat capsaicin sensitive sensory nerves are of importance for the human cutaneous triple response reaction induced by allergen exposure. Thus, secondary release uf inediaturs, such as CGRP or tachykinins from sensory nerve branches, may contribute to the flare component of this reaction. Furthermore the itching sensation seems to be dependent to a large extent on capsaicin-sensitive nerves. However, sensory nerves seem to have less importance for the wheal reaction, i.e., the protein extravasatiun response. Lundblad, L., l.undGerg, J. M., Anggard, A., and Zetterstrom, O. Allergy 42:20-25, 1987. Other support: Swedish Medical Research Council, Swedish Work Environment Fund, Swedish "1'obacco Company, Petrus and Augusta Hedlunds Fund, and the Karo- Iinska Institute. From the Departments of Oto-rhino-laryngology and Thoracic Medicine, Karolinska Hospital, and Department of Pharmacology, Karolinska Institute, Stockholm, Swe- den. EXPRESSION OF IIUMAN IgG SUBCLASSES Iluman mlmunUglobunn l, (tgl,) can DC (nvlueu tnto IUur surn:tasses mat are selectively expressed. For instance, carbohydrate antigens preferentially elicit IgG2 antibodies, whereas protein antigens usually elicit IgGI and IgG3. Elucidating Ihe biological basis of the selective expression of these (gG subclasses is important to our understanding immunodcficiencies and li lymphocyte development. To investigate clinical importance of lgG subclass deficiencies, a sensitive and specific assay has been developed for IgG subclasses using particle concentration Iluorescence immunoassay. Preliminary clinical studies have already shown that infection-prone individuals ultcn have selective IgG2 subclass deficiency. Normal levels of IgG2, however, do not rule out an imnmmx)eliciency in the infection-prone individuals because some individuals have normal levels of IgG subclasses and are poorly responsive to antigens of bacteria. Based on animal studies, two contrasting models of B cell development have been advanced. One nuxlel of B cell development proposes a single lineage and proposes that a B cell can successively switch and produce any IgG subclass. The other model 150 proposes multiple lineages and proposes that a Li cell can express only some IgG subclasses. It has been found by us that anti-PC antibodies are mostly IgC;2 with some IgGI, and that the V region of IgGl and auti-PC antibody is dilfercnt I'rum that uf (gG2 antibody. Our finding, therefixc, suggests that B cells producing anti-PC antiMxlies are progeny of not one ancestral II cell that has successively swttched, but two indcpcn- dcnt ancestral B cells. Cellular studies using polyclonal activators also suggest that regulatory mechanisms litr IgGI and IgG3 are dil'ferent from those of IgG2 ;md IgG4. When the two models are taken together, we favor the multilincage one better than the single lineage onc. Nalan, M. ll., Scott, M. G. and Shackclli>rd, P. G. Annals ol' C'linical and Laboratory Science 17(3):183-196, 1987. Other support: Public Health Service. From the I)ivision ol'Laboratory Medicine, Department of Pathology, and Division of Infectinus Diseases, Department of Pcdiatrics, Washington University School of Med- icine, St. Louis. CONCENTRATIONS OF ANTIBODIES IN PAIRED MATERNAL AND INFANT SERA: RELATIONSHIPTO IgG SUBCLASS Previous studies comparing IgG subclass concentrations in cord and maternal sera have indicated that IgG, is transported across the placenta to a greater extent than is IgG,. The purpose of our study was tu examine tlte relationship between the transport o6IgG, and IgC;, and the transport of specific antibodies that are relatively restricted to a particular sutxlass, either IgG, or IgG,. The concentrations of total serum IgG, and IgG; and those of IgG-anti-tetanus toxoid (7T) and anti-group A streptoc(x:cal carfw- hydrate (GAC) were measured in 30 paired maternal and cord sera. Previous studies have shown that anti=fT in adults in predominantly IgG,, whereas anti-GAC is pre- dominantly Ig(i,. The mcan cord/maternal concentration ratios of IgG, and anti-Tf were similar (1.77i0.56 and 1.93i0.67, respectively), but differed significantly (P=(l.(Nx)I) from those of IgG, and anti-GAC (0.99±0.39 and 1.01*_0.45, respcc- tively). We condirmed the difference in curd/maternal concentration ratios ol' (gG, and IgG, antib<xlics by mcasuring IgG, and IgG, antibodies specific for flaentuphilus in/lurnzue typc b capsular polysaccharide; the mean cord/maternal concentration ratio of I(;G,-anti-Hib I'S was significantly higher than that of IgG,-anti-Hib PS (2,23-±-0.83 compared with 0.94! 0.49, P=0.01). These results indicate that placental transp(m of IgG antibodies is related to their subclass composition. Einhorn, M. S., Granoff, 1). M., Nahnt, M. H., Quinn, A., and Shackelliord, P. G. The Journal of Pediatrics I II(5):783-788, November 1987. Other support: U. S. Public Health Service. From the Edward Mallinckrodt Department ol' Pediatrics and the f)ivision of Labora- tory Medicine, Department of Pathology, Washington University School of Medicine, and the Division of Infectious Diseases, Children's Hospital, St. Louis, MO. 151
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IIUMAN ANTIBOUIES'f0 PHUSI'HOCHOLINE. IgG ANTI-PC ANTIBOI)IIiS EXPRESS RES"I'RI(."17iI) NUMBERS OF V ANI) C REGIONS We examined the IgG subclass composition and isoelectric focusing (ItiF) slxo- trutype pattern uf naturally occurring human IgG antibodies that bind phnsphochuline (I'C) and found direct evidence for restricted expression of both V and C regions among these antibodies. In most individuals, the isurype of these IgG anti-PC anti(xxl- ics was primarily Ip(J2. However, serum from some individuals contained significant amiiurur of IgG I and IgG3 anti-PC antibodies. We also found that in individual .era, anti-P(' antilxxlies are pauciclonal, as demonstrated by restricted spectrotypic patterns of the anti-PC antibodies. The IFF pattern of these antibodies were for the most part unique firr each individual. In some sera, certain anti-PC antibodies with isoelectric points of basic pH Iwund PC conjugated to bovine serum albumin (PC-BSA) but did not bind pneumoxoxcal C-carbohydrate bearing PC determinants. In two individuals, we found that the spectrotypes that bound only PC-BSA were of the IgG I subclass. Taken together, these findings demonstrate that within individual sera, human antibud- ies to PC are quite restricted in both V and C region expression, and furthermore, these V and C regions of human Ig may not randomly associate. Scott, M. G., Brilcs, D. G., Shackelford, P. G., Smith, D. S., and Nuhm, M. il. The Journal of Immunology 138(Ip):3325-3331, May 15, 1987. Other support: U. S. Public Health Service. From the Mallinckrodt, Inc., Diagnostic Products Division, St. Louis, MO; University of Alabama, Birntinl;ham; Department of Pediatrics and Division of Laboratory Medi- cine, Departntent uf Pathology, Washington University School of Medicine, St. Louis, MO. S(1BPCIPULATIONS UF 13 LYMPIIOC'YTES IN GERMINAL CIiNTIiRS With two new monoclonal antibodics and flow cytometry, we defined duee' suhprrpulatious among B cells expressing binding sites for peanut agglutinin (i.r•., B cell% of the germrnal center). One mornx:lonal antibody (5B5) hinds glotwtriausyl ceramide. The B lymphocytes binding 5135 have binding sites for Ixxanut agglutinin on the tiurlace and express only small amounts of slgD and slgM. When tested against a panel of B cell lines, only Burkitt's lymphoma cells were 5135 r. Moreover, the 5135' cells have Iarger average sizes and a large fraction of prolifcrating cells. The other monoclonal antibody ( I IK2:1) binds a 9(1,(liK) protein. Lymphocytes binding EIK2 3 are 5135 and wclude'I' cells and a.ubpopulatiun uf B cells. In contrast to 5135' cells, the IIK23' and peamn agglutinin positive B cells express a large amount i('slgM.'I'hese two subpopulatiuns uf germinal centers are distinct from the germinal center B cell subpopulation expressing the CI)23 (131ast-2) antigen. The CD23' B cells are 5115 and express an intermediate level uf HK23 antigen. In addition, C'D23' B cells are highly variable in numlxr, whereas the proportions of HK23' and 5135' cells are relatively stable. Fyfe, G., Cebra-Thomas, J. A., Mu+tain, E., Davie, J. M., Alley, C. D. and Nrdun, M. H. The Journal of Immunology 139(7):21f37-2194, Qctober I, 1987. Other support: U. S, Public Health Service and the Procter and Garnble Company, Frtmr the Ikpartment uf Pathology, 1)ivision of I,atxoratury Medicine, and the lhpart- ment of Microbiology and Immunology, Washingtun, University School of Medicine, St. Luuis, MQ, and Department of Cell Biology and Anatomy, University of Ala- bama, Birmingham. AN ANALOGY BETWEEN FETAI. HAPTOGLOBIN AND A POTENT IMMUNOSUPPRESSANT IN CANCER In spite uf the numerous reports indicating the presence of humoral immuttosup- pressive factors in cancer patients, only a few of these factors have been biochemically identified. Furthermore, their role as effective immunosuppressors in vivo remains to be established. Our laboratory has attenrpted to isolate and identify the major immuno- suppressive factor in the malignant effusions derived from ovarian and lung cancer patients. We have previously demonstrated that the M,52,000 immunosuppressive factur isolated frum the ascites Iluid of an ovarian cancer patient inhibited T-dependent immune responses in viru and in virru including the inhibition of G-roseiting. '1'hus, this immunosuppressive factor was named "suppressive E-receptor" (SLiR). Our current study demunstra(es that this SIiR factur purified from malignant efi'usiuns derived from ovarian, lung, or head and neck cancer patients had a common compo- nent which dissociated equally intoM,38,(NX)-42,(KK) and 17,000-19,(RX) muietieson sodium dodecyl sulfate-Iwlyacrylamide gel electruphoresis under vigorous reducing conditions. Electnxlutiun of these two compiments followed by a limited amino acid sequence determination revealed these two components to have N-terminal amino acid sequences identical to the (3 and a, subunits of normal adult haploglobin. Immunix•lec- Irophuresis of SGR using a pulyclonal antiserum to neonatal cord bloaxf demonstrated that SER, unlike nonnal haptuglobin, has slower clectrophoretic mobility than the normal adult haptuglobin. Western blotting analysis of SER separated on sodium dixlecyl sulfate-polyacrylamide gel electrophuresis under denaturing conditions failed to recugnize a monoclonal antihixly directed.pecifically to SER. However, this mono- clonal antibody exclusively reacted with the SER separated by an analytical polyacry- lamide gel electrophoresis gel under nondenaturing conditions while normal adult haptuglobins or purified but denatured haptoglubin obtained frunr the same malignant fluid as SIiR all failed tu react with this antibody. Thus, SER appears tu bear an additional epitolx:(s) that is absent in normal adult haptoglobin. Since the SER as well as the neonatal haptughThin has at least I(X) to 1(XX)-fiold more potent immunosuppres- sive activity than the normal adult haptoglobin, (his additional epitope(s) present in SER may be responsible for the potent immunosuppressive property uf S6R. Oh, S.-K., Very, 1). L., Walker, J., Raam, S., and Ju, S.-T. Cancer Research 47:5120-5126, Octoher I, 1987. Other support: National Institutes of Health. From the Depanment of Microbiology and Arthritis Center, Boston University School of Medicine, Natick Army Research Center, Natick, MA, and Depanment of Medi- cine, "I'ufts University School uf Medicine, Boston. 152 ' 153
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t)NI'-I'lIY('OHILIPRO'1'IiINS, 1'LUORF.S('IiN`t' ANTIGIiNS TO STOI)Y I)YNAMI(' 1'ROI'IiRTIIiS OF ANTIGhN-Igti-RL?C'EI'TUR COMPLIiXES ON RBL-2113 RA'I' MAS`I' ('ELLS' In RBI. 2113 rat nwcusal mast cells, the crosslinking of cell-surface Igli-receptor complexez by multivalent andigens initiates a seyuence of responses leading to dc- granulatiun. We have developed a family of dinitrophenol (DNP)-conjugated Iluorres- cent antigens to sludy dynamic membrane events associated with these responses. Ly.yl groups on the phycohiliproteins, B-phycocrythrin and C-phycocyanin, were labelled with DNP, yielding fluore.x:ent conjugates that cause the release of 1'll1 serotonin from anti-DNP-IgE-primed RBL-2H3 cells. The binding of these antigens to IgF.-rcceptor ccimp(exes was observed by fluorescence microscopy and yuantified by flow cytometry. Incubation with I µg/ml DNP,r B-phycoerythrin stimulates maximum degranulation from IgE-saturated cells. Under these conditions, approximately redi (0' molecules of DNP,. B-phyc<xrythrin are bound per cell at equilibrium. The rate and extent of antigen binding and of antigcn-stimulated mcdiator release decrease in paral- lel as the concentration and DNP:protein ratio of the fluorescent conjugates is reduced. Secretion stops immcdiately when the nonfluore.uent monovalent antigen, DNP-ly- sine, is added to degranulating cell suspensions. DNP-lysine also displaces surface- Mound anti(;en when added during the first minutes after multivalent antigen. However, lhe ahility ul' DNP-(ysinc to displace surface-twund DNP,; B-phycoc:rythrin from I)±1: reccptar complexes decreases progressively with time. Treatment with dihydrocyto- chal:uin B and several analogs that prevent antigen-stimulated F-aclin assembly en- hances secretion and delays the transition of antigen to its DNP-lysinc-resistant furtn. Cytochalasin treatment also permits the long-range movement of antigen into surface caps. Based on these data, we propose that secretion is triggered by the act uf IgE- rrccptur cros.linking or by a short-lived excited atate of the crosslinked antigcn-Igl: receptor complex. We prupusc further that amtigen-stimulated F-actin assembly con- tributcs to the transition of-antigcn-Igf-3-reccptor complexes to a DNP-lysine-resistant 1'unn that does not trigger secretion. Two pinsibie mechanisms liar the transition to I)NI'-lysine resistance are discussed. Scagrave, J C., Ihanin, G. G., Martin, J. C., 1)avis, B. H., and lNiver, J. M. ('ytumetry ti:2K7-295, 19K7. Other suppun: Anicrican Cancer Six:icty, National Institutes of' Health and U. S. I)epartment of L:nergy. Fnom the Department uf Pa(hnhogy, University nf New Mexico, School of Medicine, Albuquerque, and Life Sciences Division, Los Alamos National Laboratory, t.os Al:unos, NM, and Ikkpartment of Pathology, Dartmouth-Hllchclkk Medical Center, I(anuver, NU. A Rt)l.li Ft)It IIIS'1'AMINI's TYPIi 1I (11-2) RECEPTY)R BINDING IN 1'kt)I)tlCl'It)N OF'1'Ilti LYMI'HOKINE, SOLUBLE IMMUNE RESPONSE SLIPPRIiSSOR (SIRS) Soluble imnnme response suppressor (SIRS) is an immunosuppressive protein produced by human and murine suppressor cells activated by a variety of agents. Because histamine has been repittted tu activate suppressor cells, the possibility that it also,induced SIRS production was investigated. Human lymphocytes treated with 1() • M fiistaminc for less than I hr released a suppressive substance into culture super- natants that was physically, functionally and antigenieally similar to human SIRS. Cimetidine and ranitidine, structurally distinct histamine type 11(H-2) receptor antago- nists, prevented histaminc-induced SIRS production. In further experiments, suppres- siun of human polyclunal IgM PFC responses by Con A and interfcruns, substances that activate the SIRS pathway, was inhibited by H-2 receptor antagonists. Activation of lymphocytes to prikluce SIRS by Con A or interferons was blocked by cimetidine or ranitidinc. These data demonstrate that pnxiuctiun of SIRS is induced by histamine, and raise the possibility that H-2 receptor binding may play a role in the SIRS pathway. Sclnuper, fl. W., Aune, T. M. and Roby, R. K. The Journal of Immunolugy 139(4):1185-119O, August 15, 1987. Other supp<m: National Cancer Institute. From the Department of Pathology, Jewish Hospital of St. Louis, and Departments of Pediatrics and Pathology, Washington University School of Medicine, St. Louis. • LYMPHOMA MOI)ELS FOR B CL:LI. ACTIVATION AND TOLERANCE. Ill. CELL CYCLE DEPENDLiNCE FOR NEGATIVE SIGNALLING OF WEIII-231 B LYMPHOMA CELLS BY ANTI-µ It has become increasingly apparent that B lymphomas provide clonal models for antigen-specific lymphocytes and for the analysis of Iwth positive and negative signal- ling in B Cells. In addition, anti-Ig reagents have (x:en used to mimic antigen/tolerogen in their interaction with B cell receptors at a polyclonal level. We have been studying the effects of anti-Ig on a group of unique B cell lymphomas as models for either tolerogenic or immunogenic signalling. One such line, WEHI-231, has been shown to be readily inhibited in its growth by anti-Ig reagents. We cunlirnted the sensitivity of this line to growth inhibition by anti-µ and anti-K reagents, and determined its kinetics, spec ilicity, and site of the block in the cell cycle at the G,/S interface. In this report, we have enriched W I?I11-231 lymphoma cells at various points in G, to S and analyced the effects uf anti-µ on progression through S phase. Our data suggest that critical events ix:curcarly in U, and are uniquely sensitive lu nuxlulation by anti-µ. These studies now will allow sensitive molecular approaches towards an understanding of B cell signal- ling, as well as methods to regulate lymphuma growth per se. S'rua, 1). W., Livnat, 1)., Pennell, C. A., and Keng, P. Journal of Experimental Medicine 164:156-164, July 1986. Other support: U.S. Public Hcahh Service and the Leukemia Society of America. Front the Immunology and Experimental Therapeutics Units, University of Rochester Cancer Center, Rochester, NY. 154 155
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Rt:GULA'1'IQN Uh I3 1-YMI'HOMA GRUW'I"H I; ('ell lymphomati have been us,erl ns models fur negative signalling in growth rcgulatirrn ul rnormal It cells and as a mudcl sytitem for unrespunsivencsti. C'nrsxlinl.ing nl .urfacc UgM receptors ix a critical event which can he mrululated by phorbol etiter,. LI'ti or T cell Carlurs. Studies un calcium mobilitation indicate that while such a process occurs in une uf thc,e cell lines, it is not a universal observation and it I'ails tti currrl:ne with the neEative signalling process. Indeed, the role uf calcrum in positive srl:n.dhng is un much lirrucr ground. We believe (hat receptor crosslinking imtiatcs a process which IeaJ, tu changes in certain IgM associated proteins causing physiologi- cal run.cyuences that arc at least temporarily reversible with agents xuch as I'MA. 'I'he rule ul pralein kina.se C activation in this process in under investigatiun. Srurl.l). W. Molecular Mechanisms in the Regulation of'Cell Behavior 39-44, 1987. Other suplwrl: U.S. Public Health Service. Prutu the Immunology Unit, Cancer Center, and the IDiepartmcnt of Microbiology and Imrouttolugy, University uf Rochester School of Medicine and Fkntistry, Rochester, NY. I.YMI'I IOMA MUI)IiLS FOR B C'F'sl.l. AC."('IVATION ANU TOLERANC'E. V. AMI'I-Ig MEUTA'1'ED (iRUWTH INHIIiI'1'ION IS RI:VF.RSEU BY PHORBOI. MYRIS'fA'17: ACIi'1'Al'I: IiUT UOES NO'I' INVOLVE CHANGES IN ('Y'f USULIC F'RIi6 ('ALC IUM I3 cell lymphomas which can be growth inhibited by erosslinking their surface 11rM receptors by anti-Ifg reagents provide models for nunnal B cell regulation and tulerance. WtiHI-231 and CH31 are two independently derived lines that are exyui- sitcly umsitivc tu negative signalling by antibodies specific for nm or kappa chains, hul arc unaffcctcJ by antrhixliesa);ainst MHC class I or 2 antigens. In order to determine (he mechanism of (his growth inhibition as a model lirr tolerance, we have examined the roles played by protein kinase C activation and calcium mobiliiation/inllux during negative signalling in (hcu cells. We found that growth inhibition caused by anti-mu cru+tilinking was reversed in the presence ofeither phorhul myristate acetate (I'MA) or by lipopolysaccharide (I,I'S) frum E. coli. The effect of PMA on negative signalling was a true reversal since phorbol esters could be added after anti-mu treatment, thus allowing nearly nurm:d cellular progression into the S phase uf the cell cycle. In contrast, pretreatment with PMA did non provide protection against the growth inhibi- tiun frum anti-mu. Indeed, a"desensitizatiun" protocol demonstrated that I'MA prctrcatment actually decreased reversal by MHh PMA and LPS of the eflectx of anti- mu un Il lymphuma growth. '1'hetic studies suggest that both LI'S and PMA act via .n least one cnmmun intermediate, which is assumed to involve activation and trarnluca- tiun uf protein kinatic C. Analysis uf changes in calcium ion concentration after treatment with anti-Ig reagents showed both mahilitatiun from internal stores and influx via calcium channels in WliI11-231, as has been reported for normal 13 cells. However, these changes did not conclate with ncgative.ignalling furthe several reasons. Firslly, anti-mu inhibition of the growth uf WEI II-2 1I could be induced in the relative absence of extraccllularCa' ' or in yuin-2 loaded (hulTcred) celk Secundly, pretrcatrneni with high concentrations ul PMA ahlateJ calrrum mubiliiation, yet faileJ to muJulale growth inhibition in WL111-211 cells, Moreuver, l.l'S provided prutectiun frum thc effects uf anti-mu yet diil nut alter cellular ('a,' ' 1. In adJiliun, I'MA Ixosttrcutment (under conditions caus- mg a reversal ul the eftcct+ uf anti-mu) can Ix: applied as long as I'our hours after the ininal exposure tu anti-mu anJ the rapid measurable changes in calcium flux. Indeed, such changes in intracellular frce calcium occurred in clutriatcJ WEIII-231 lymphuma cells at all phases uf tlte cell cycle, although we have previously iilentitieJ early G, as the only critical period in which negative signalhng can txx Jelivercd. Finally, using hinh yuin-2 and inJu-I, no calcium signal can Ix? observed upon anti-Ig crosslinking in CI131. which is anuther auti-Ig sensitive li cell lyrnphuma. 'fltus, negative signalling by anti-Ig Joes not u:em to require changes in intracel- lular calcium levels rclxorteJ to occur during activation of mature B lymphucytes. 'fhc mmnncr by which activation of protcin kinase C reverses B cell growth inhibition and alternative negative signalling pathways is currently under investigation. Srurr,l). W., Livnat, l)., Whitin, J., I)illon, S. B., SnyJcrman, R., and Pennell, C. A. Journal of Molecular and ('ellular Intntunulugy 3:1O<)-120, 1987. Other support: National Institutes of Flealth and University ol' Rcx:hcster Cancer Center C'ure. hrum the Immunolugy and Pediatric Hcmatulogy Units of the Cancer Center and Ikpartments uf Microbiology and Immunology and I'ediatrics, University ol Rokhes- ter School uf McJicine and Iknti.try, Rochester, NY, and Howard flughes Medical Instilute and I)ivi.iun uf Rheumatulugy and Immunulogy, F)epanntcnt uf Medicine, I)uke (lniversity Medical Center, Durham. NC. NI('O'I'INI(' BINnING.SI'IES IN THE BRAIN: PROt'EFtTIFiS, REGULATION, ANI) I'(1TA7'IVI'- ENDOGENOUS LIGANDS Several birKhentical, clectrophysiological, and behavioral changes have been :nsuciated with nicotine action in brain. Huwevcr, Ihe exact mechanisms and .pecilic rcccptur.itc(s) invulved arc not known. We liiund alterations in brain protein mctalw- lisrn in rit•u and in rirru tidluwing nicatine achninistration, which may be meJialed by intracellular sites of •rctiun. Of special interest is the eflcct of nicutine on ncurutrans- mitters and receptors and whether.pecific synaptic receptor(hinding) sites for nicotine exist that mclhate its action on the nervous system. The possible existence uf enJuge- nous ligand.lirr.uch sitcs anJ the interaction of niccutine with neurotransntittersystems in various brain cell+:ue under investigation. C'entral catccholaminergic, GA13Acr):ic, cnkephalinergic, and prnsibly noncholinergic nicotinic sites have been inrplicated. As many as 5 hinJing sites have hcen reported tirr I'lliniculine. "1'he nanonwlar high- attutity site rcxemhles lhe binding site associated with 1'IljacetylchUllnc. I hese two IiganJ., however, differ in regional distributiun, auturadiugraphic patterns ol'Jititribu- ti<m, and responses to receptor changes atter exposure to chronic nicotine. The low- aflinity (rnicrumular) binding site apparently exists. Its pharmacologic role is not known, bul if aticr smoking, nicotine levels in specitic brain regions reach low micro- nwlar level., these sites may be relevant to somre of the central actions af nicotine that cannot be explained by the nicotinic-cholincrgic site. 156 157
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W-4 ^j1 33 70 Se•rshe-n, H. and Lajtha, A. In: Martin, W. R., Van Lexin, (;. R., Iwamoto, li.'1'., and 1)avis, L. (cds.):'fobaccu Smoking anJ Nicotine, Plenum Publishing Corporation, pp. 481-491, 1987. From the C'entcr for Neurochemistry, The Nathan S. Kline Institute for Psychiatric Research, Ward's Island, New York. T ct:LL GROWTII Wff11UUT SERUM Most in vitru T cell prulif'eralion experiments are performed by using tierwit- supplementcd medium, yct the actual contributions of scrum cumponcnts tu cell cycle progression remain ifl-deiined, thuscumplicating attempts to fully define requirements for cell division. I)y utilii.tnK a functional separation between T cell receptor-triggered "com(xntence" and IL 2-promoted "progression" to independently as:;rss serum re- yuirements during each cell cycle state, it was shown that serum serves an essential, active role only during the early events of the competence phase (G,; G, transition) uf'l' cell activation. Serum is required for optimal IL 2 production and the cell surface expression uf IL 2 receptors af'ter the stimulation of the T,/T, antigen receptor cotn- plcx. In contrast, serum does not function actively during IL 2-mediated progression through the (l, phase of the cycle. Serum proteins serve only a passive role at this stage, preventing the adsorption of IL 2. This same effcct can be provided by any number of proteins including Il. 2 itself, or even a high cell concentration. Supplementation of scrum-frec T cell cultures solely with IL 2 and transferrin is sufficient for maximal'1' cell proliferation, although the time uf thc peak response is delayed owing to a suhopti- mal rate of Il. 2 receptor expression. Accordingly, the realiration that serum is unly necessary for the earliest stage uf T cell activation will now enable studies designed to identify the critical individual serum components and to define their mechanism of action. tlevtx:rg, V. L. and Smirh, K. A. The Journal 01' Immunulogy 119(4):99H-I(X)4, August 15, 1987. Other support: Charles If. Houd Foundation, National Cancer Institute and Eli Lilly Corporation. From the Department of Medicine, I)artnwuth Medical School, Hanover, Nil. IN'I'I:RLGUKIN-2 INI)U("I'ION OF T-('hLL (i, 1'ROGRfiSSION ANI) c-nvh IiXPRESSION In titudies to ilctermine the biochemical mcchanitims rc.pnn+iblc forcell prolilera- tiun, synchrunized 'I' cells were used a-, a model for cellular growth cuntrul. Ry mctabulic and nwtphulugic criteria, it was firund that activation of thc'1'-ccll antigen receptor rendered the cells reslwnsivc to interieukin-2 (IL: 2), but did not move thcm through the cell cyclc. Instead, IL-2 stimulated G, progression to S phase, or lymphu- cyte "blatiuc transfiirmation." During IL-2-prumoted G, progression, expression of the cellular proto-oncogene c-myh was induced transiently at six to seven times basal levels, maximal levels occurring at the midpoint of G,. Stern, J. B. and Smidt, K. A. 158 Science 2;41:203-2(16, July It, 1986. Other support: National Cancer Institute and lili LillyCorpuration. Front the 1)epartment of Medicine, Dartmouth Medical School, Flarntver, NH. I)Ih1=I:RliNTIAL RL:QUIREMIiNT FOR ACCESSORY CELLS IN POLYCLONAL'I'-CI:LL ACTIVA'f1UN Highly purificd rat la-nepative (UX-6 ) and la-positive (QX-6' ) T cells were cmployed lu examine the requirement for accessory cells (AC) and/ur soluble factors in (he activation uf resting T cells with Con A, PI IA, sodium periodate, ur antigen. A variety uf cells were cntployed as AC, including la-positive and la-negative macro- phages ( M4)), -y-irradiated (2O(X) rud) or nonirradiated OX-6' T cells, and several la- negativc adcnovirus-transformed rat embryo ebrobla,t cell lines. Our results sug- gested that for the expression of Il: 2 receptors (II. 2R) and prolif'eration of OX-6 T cells in response to ('on A. PHA, or antigen, there was an obligatory requirement for the presence uf ACwhich could not he overcome by the addition of IL. I and/or IL: 2. Activation uf UX-6 T cells with antigen required the presence of la' AC, while activation with mitof,ens could be initiated with la AC. M+ were efiicient in AC functiun in all responses tested, while the AC function of OX-6' T cells (TAI'C) proved discriminatory under different conditions. The optimal response to PHA re- quired much higher concentrations of'I'APC as AC than titt the Con A response. TAPC failed to stinmlate sodium Ixxriodae-treated T cells under any conditions tested. Fur- thcrnwre, when TAPC were employed as AC, their antigen-presenting ability was radiosensitive, while their AC function for Con A and PIiA was radioresistant. Theu results suggest that molecules involved in T cell - AC interactions may differ, depend- ing un the source of AC and/ur type of the pruliferative stimulus provided to T cells. These data have heen discussed in the context of T-cell activation. Supuri, M. 1.., Llun, Y. (.. Cherian, S., Kaplan, A. M., and Diamantstein, T. C'ellular Immunology 105:174-Iti6, 1987. Other support: Tobacco and Hcalth Research Institute. Frum the l)epartment of Medical Microbiology and Immunology, L)niversity ut' Kcn- tucl.y Medical Ccnter, lxxington, and the Immmnology Research Unit, Steiglitr. Clinic, Free University of West Berlin, West Germany. VI l. F pidemiology SELP-RGPORTED ALC'ULIOL USE: A LONGITUDINAL STUI)Y OF 12 ,'}94 ADULTS "I'he internal validity of the questions on alcohol use in the questionnaires was assessed by calculating the proportion of inconsistent reports of yuantity and frequency of alcohol use. These figures were in 1975 for txxer 0.6%, wine 0.8%. spirits 1.1% and 159
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in 1981 for beer 0.7'! , wine 0.8% and spirits 0.7%. To assess the changes of alcohol use fuur groups were formed: (I) teetotalers (2) occasional drinkers, (3) moderate drinkers and (4) heav,y drinkers. The cut-points for groups were I, 4()0 and 9110 grams of alcohol per month. The stability of alcuhol consumption by sex is displayed in the Tables I and 2. With follow-up of xix years it was found that there were not any remarkable changes in the proportiun, of diffcrent groups of drinking. Their distribution were nearly similar in both yuestionnaires. The stability of alcohol consumption was assessed by comput- ing the proportion of probands staying in the same consumption category during the I'oI Iow-up. Results were that 65% of males stayed in their consumplion category while slightly more than 17't6 moved to a highercategory and 17% moved to a lowercategory. Similarly, 78% of females remained in the same category while approximately 9% moved to a higher category and 12% moved to a lower category. Among the teetotalers, a change to start drinking alcohol can be observed: one third of male teetotalers in 1975 began using variables amounts of alcohol during the follow-up. Nearly the same numberof individuals using alcohol at Stage I did not drink alcohol at Stage II. As a consequence the percentage of male teetotalers remained nearly the same, 9.7"k, in 1975 and 10.7% in 1981. Remarkable changes are also fuund among heavy male drinkers; half of them cut down their alcohol consumption by moving to the lower categories during the follow up. However, simultaneously almost the same number of males moved to the heavy drinking group from the lower consump- lion categories. Among the females the same trends can be observed; over 20% of teetotalers in 1975 were drinking alcohol in 1981 and over 30% of teetotalers in 1981 were drinking alcohol in 1975. The proportion ot' teetotalism increased during the follow-up from 23.7% to 28.8%. Among fentale heavy drinkers, roughly 80% reduced their drinking by 1981. 1981. Of thosc who were heavy drinkers in 1981 over 70% were not in the category in 1975. 1)cspite these changes between difl'erent consumption groups among both sexes, there were only minor changes in the distribution of alcohol consumption between Stages I and I1. Romanov, K., Rose, R. J. , Kaprio, J., Koskenvuo, M., Langinvainio, H., and Sarna, S. Alcohol & Alcoholism Suppl. 1:619-623, 1987. Other support: Finnish Foundation of Alcohol Studies and U.S. National Institute on Alcohol Abuse and Alcoholism. From the Department of Public Health, University of Helsinki, Helsinki, Finland, and Indiana University, Bloomington. SOCIAL ANI) GENI 1'IC INFLUf:NCES ON DRINKING PATTERNS OF ADUL'I' MEN: A STIJI)Y OF 5638 FINNISH TWIN BROTHERS I(ierarchical multiple regression analyses of questionnaire data reported by male twins in the Finnish Twin Cohort were used toevaluate genetic and social influences on use and abuse of alcohol. The similarity in drinking patterns of twin brothers is related to the frequency of their scxial interaction, and monoiygotic co-twins are in more frequent social contact. But the stepwise multiple regressions show that the greater surtal interaction uf idcntiud twins cannot account fur their greater similarity in drink- ing padterm. We cunclude that genetic variance significantly con.trihule,, to population variance in the frequency, quantity, and den.ity uf so<ial drinking. Kapriu, J., Koskenvuu, M., Langinvainio, 11. Romanov, K., Sarna, S., and Rose, R. J. Alcohol & Alcoholism Suppl. 1:373-377, 1987. Other suplwrt: Finnish Foundation for Alcohol Studies, John E. Fogarty International Center of Ihe U. S., National Institutes of Health, and the U. S. National Institute on Alcohol Abuse and Alcoholism. Frum the I)cpartment of Public I Icalth, University of Helsinki, Helsinki, Finland, and Depanment of Psychology, Indiana University, Bloomington. OCCURRENCE OF RHEUMATOID ARTIiRITIS IN A NATIONWIDE SERIES OF TWINS The nationwide Finnish Twin Cohort was linked with the Sickness Insurance Register on the basis of the unique idenlilication number assigned to each Finnish citizen. The study u:ries consisted of 4137 monorygotic (MZ) and 9162 dizygotic (D"L) same-sexed twin pairs Mirn Ixxliire 1958 and alive in 1975. Altogether. 261 subjects in the series had the right to receive free medication tiir rheumatoid arthritis (RA) under the Sickness Insurance Act that covers the entire Finnish population. The pairwise concordance percentage liir RA was 12.3 in MZ twins and 3.5 in D7.lwins. The age and sex adjusted ratio of ubserved per expected numbers of concordant pairs (relative risk ) was 8.6 for MZ pairs and 3.4 for DZ pairs. These /igures are lower than those previously reported on twins but compatible with results frotn family studies on the genetic cotnponenl ol' RA. Aho, K., Koskenvuu, M., Tuominen, J., and Kaprio, J. Journal of Rheumatology 13(5):1i99-902, 1986. Other suppnrt: Sigrid Juselius Foundation of Finland. From the National Public Ifealth Institute, Helsinki, Finland, and 1)epartment of 1'uhlic Health Science, University of Helsinki. SNORING AS A RISK FACTOR FOR ISCHAEMIC HEART DISEASE ANI) STROKE IN MI:N The association of snoring with ischaemic heart disease and stroke was studied prospectively in 4388 men aged 40-69. The men were asked, in a yuexlionnaire scnt to theni, whether they snored habitually, frequently, occasionally, or never. Ilospital records and death certificates were checked for the next three years to establish how many of the men developed ischaemic heart disease or stroke: the numbers were 149 160 1 161
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and 42, respectively. Three categories uf snoring were used I'ur analysis: habitual and frerluent snorers (n= 1294), occasional snorers (n=2614), and non-snorers (n=42tO). The age adjuaed relativc risk ul'ischacntic heart disease between habitual plus freyucnt snurerti and nun-snrrrers was L()I (p-0.01) and liir ischaemic heart discase ur.tn,kc, or txnh, 2.3K (p< .0,Ot)l ). There were no cases of' stroke among the nun-snurcrs. Adjust- mcnt fur age, body mass index, history uf' hypertension, smoking, and alcohol use did not significantly decrease the relative risks, which were 1.71 (p=-0.O5) fur iu:hacmic heart disease and 2.08 (p•:•Q.(tl) for ischaemic heart disease and stroke combined. At the beginning uf liilluw up in 1981, 462 men reported a history of augina pecturis or myacardial infarctiun. For them thc relative risk of ischaemic heart disease between habitual plus freyuent snurers and non-snuren was 1.30 (NS); Ibr men without pre- viirur i,chaemic heart disease 2.72 (p<(1.05). Snoring seems to be a potenlial detertninanl ol'risk of ischaemic heart Jiscase anJ stroke. Koskenvuo. M., Kaprio, J., Telakivi, T., Partinen, M., Heikkila, K., and Sarna, S. British Medical Journal 294:16-19, January 3, 1987. Other support: Paavo Nurmi Foundation. Frum the Department of Public Health, University of Helsinki. Helsinki, Finland. M)OR SLGIiPQUAI.I'fY, EMOTIONAL S'E'RIiSS ANI) MORB11)ITY-A SIX YEAR FOLLOW-UP STUI)Y UF 1(1778 PI:RSUNS AGEI) 35-59 YEARS The quality ot sleep of 10778 Finnish aJults aged 35 to 59 years was asked in a postal questionnaire in 1975. Sleep quality was grouped in three categuries; good (38%•), quite good (51)%,) and bad (12%). The association ol' poor sleep quality with neuruticism, stress of Jaily activities and life Jixsatisl'actiun were very clear, showing, liolluwing corrclatiun crxf ficients: 0.31,0.22 and 0.28. Poorquality of sleepcurrelated also to short sleep length (r = 0.23) and to unemployment (r = 0.11). Individuals were 'IiillowcJ up using the computeriseJ nationwide registries to collect death certificates and hospital records. Person-ycars of fidlow-up was cuni- pu1cJ until the first event or end of folluw-up in end of 1981. Age-adjusted risk ratios (RR) rur had sleep quality compared tu good quality were calculated litt men and womcn in natural deaths (RR for men 2.60** *, and for women 0.92 ns), violent deaths (4.59***, 2.63 us), mental disorder huspitaliration or suicide (2.86***, 2.20 ns), hospitalization due to neurosis (I1.7O***, 4.6)***) and ischcmic heart disease inci- dence (death or hospitalization) (2.(W**, 2.21**). When variables indicating emo- tianal stress (neurulicism, stress of daily activities, lifc satisfaclion) were adjusted in nrultiple logistic models, risk ratios for had sleep quality remained essentially un- chant!ed. When only healthy men in 1975 were analyzed (exclusion uf all men with any ol' the fullowinp, events: hospital care due a disease in 1972-1974, reported angina Iwc- Iori., hypertension or chronic bronchitis in 1975 questionnaire; totally 2212 men (41%) were excluded including 73% of deaths during follow-up), the numtx:rof cases became quite small fur analyses. It seems that hail sleep quality is a strong indicator ol'mental and somatic disease. It remains unclear whether had sleep quality is a consequence or cause of disease. t I i Auskrnruo, M.. Kaprio, J., Partincn, M., Langinvainio, H., Sarna, S., Rita, H., anJ Hcikkila, K. Psychialria Fennica Supplementum: 115-120, 1986.' Frum lhe Ik:parunlnl uf Public Health Science, Univetsity of Helsinki, Helsinki, Finland. I>ISIGN ANl) SAMPLING CONSIDERATIONS, RLSPUNSL's RATES, AND RI:PRISLiNTATIVGNESS IN A FINNISH TWIN FAMILY STUDY Kinships cot r rpo,mlor twin parents, thcir sfxiuus and ehilJren, uffcr a robust anJ IlexiMe sainpling design liir research in genetic cpidemialogy. Families-uf-Iwins de- signs circumvent some of thc sampling problems that arise when independent data sets are combined, and these designs provide unique evaluations of maternal influences, axsurtativc mating and X-linkage. Unfortunately, empirical studies uf ('amilies of twin parents have been limited by relatively small samples and by self-selcction biases intrinsic in asccrtainment of families frum volunteer twin registries. A large and representative cuhurt uf' monuiygotic and dizygotic twin parents, drawn rrom a pupulatiun-based twin registry, provides the optinwl sampling frame Rrr Iwin-fantily research. This paper reviews the sampling considerations underlying the initial family study based on the Finnish Twin Cohort anJ evaluates the representative- ness ul'the sampled twins. Spouses and aJull children (over 18 years) uf 236 pairs ol' twins, about equally divided by gender and zygusity, were evaluated by a postal questionnaire. Individual response rates exceeded 86% and in 464 uf the 472 nuclear families (98.3%), at least one member of the twin's family completed the question- naire. The sampled twins, selecteJ fi,r IccunJity to maximize statistical power of the obtained data, were broadly representative of non-selected twins drawn from the Cohort, with whum they were matched on age, gender, and cygusity. Such results suggest that the Finnish Cohort has excellent potential li,r extended twin-family re- search designs. Kaprio, J., Rose, R. J., Sarna, S., Langinvainio, H., Ka.ckenvuu, M., Rita, H., and Heikkila, K. Acta Genelicae MeJicae et Gemellologiae 36:79-93, 1987. From the Lh:partment uf Public Heallh, University of Hclsinki, Helsinki. hinland, and Ihhpartmcnt of Psycholugy, Indiana University, Bhwmington. 162 ' 163
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Active Projects l.'ulluwinlt i% u list trl'lhc principal investigators, or institutions, whusi nrujt:cts are urnict way or were activated in the pr:ritxJ since the nrevious Rrlxtrt, together with thc respective prttjcct lil4es. Completed projccts are listed in a later uxlitm. 1'ItIN('ll'AI. INVb:.ti'1'Il'A'1'()N ON INS'1'I'1'll'I'Il)N ~ ROPiliRl' I1. AItI:I.1:S, Ptl.l). I'rulc..ur td ItitKhcntistry, Br,tnJais Uni- versity, Wallhtun, MA. I.G(1( i. Ali( )(11), I'll.l)Y t'rod6\Ll/r UI t)ralr) I(m-n1'ch antl Iiln6'hCnD istry, ('cntcr for Itratn Rcacur.h, llnivcr+ny nf Rtkhcstcr Mcdical C'cntrr, Rochester. NY. NI(oJrx°I"1'tTI.H: 1)rvcluptuent of clastau tnhthnurn Nicotine tran.fcr-disnusitiun in livrr cells IM)1.1'll O. AI)AMS, M.I)., Pn.l). I'hufcs.ur ul 1'atltulugy, I)uke University Medical ('etucr, 1)urhunt, NC. IAN Y. K AI)AMS(1N, I'n-I). 1'rute.sur ut I'athoingy, University of Man• ituha, Winoqx:g, Mamtuha, ('anaJu. ItIIH'I' AUI:LMAN. M.1). Assistant Prule%utr ol MeJicine, MeJicul ('ollcgc uf Virginia, ltichntund. JOI IN J. ALIIIiRS. 1'u.l), Rcuarch A.stxiatc I'rulcs+ur uf Mcdicinc, llnivcrsity ul Washtngtnn Schrhrl ul' McJi- cmc. Scaulc. 1( )R( il: li. AI.I.I:NI)li. I't1.1). 1'rulcswtr ut Itittchcnuauy. Univcr.ity of ('lulo, Santnrgo. IIARItY N. AN'1'ONIAI)IiS, I'n I). I'tulcssur of Iiukhcnuatry. Il:vvarJ lJni- vcrslly Schnul ul I'uhttc Ilcahlt, &r.tun. ALAN 1). A'ffll-., Asstsnml I'rulcs.nr n( Ititchemistry. Uni- vcrsny ul Wt+cunsin, Madtuut. IRPI' AVIRAM. 1'n.U. I)c ,mutcnt of Bnx'hcnustry,'lltc Pacrdty ul Ltlc ticlctrlts,'fcl Avtv llutvctany,'fcl Avlv, IsriK't. Iil?RNA6U) M. ISAIiIUK. M I)., Pu.l). IIeuJ, 1)tvisrotr ol Nirxhcnu+try, Scri~ft+ ('hmc anA kcuatdt t•oundauun, l.a Jn la, ('A. RUIC anJ rcgulathut ul Irrntcin'rylrutiplritry4u- tron during rn.n:rnphagc rtcuvntutn C'cll interactions at the air hhMtd harricr IifCccl ul' lihrinulyttc acttvahun on Irl.nrlct funcuon Iligh density lilxtprutcin quantituhun Ititx hcmical signal transdacuun in tnwytcs ltiutiynlhcsis runl prrK-rssin(t ol I'1)(;h-14kc Ixrlylwptid x in hutuan muhgnanl cclls tu culturc Mctahulism nf law density Iqxttuutrm Irwu pigs with alxthpuprutcrn-li nunaunns Isolation, pntpcnics and (rhysnoingtcal hm(•, ttnn uf nculruphtl t'ylrK''trumc b Studies on the mechanism of au nvauun oI thr . respiratory burst in ncutrophtls PF(IN('IWAI. INVH:STI(;A'1'Ok OR INti 1'ITtI'f1UN ROI11iR'I' 1.. IlAl(IiIGHt, M.1). Assistant I'rulc.sar of Obstetrics and Gyrur- culugy, Ilarvard Medical Schtxd, lttmton. LAURIIi IiAR('LAY, M.U. C'hmcal I)irecnrr,'I hc Iturke Rchahililruon C'cntcr, Whuc I'lains, NY. MI('/IAI?I. IIAKANY, M.D.. Pn.U. YrufcaMUr of Ilrolo~rcal ('henai.try, lJmvcr- sny of Ilhnttis, ('htcagu. I)AVII) W. ItARNIi,';, I'n.D. Associate I'rnfcst,nr af IJitx:hrmiury/ Itinphysic., Oregon State llnivcratty, Cnr- val I t s. S'flil'I II'tN lt. ISAYI,IN, M.1). Ass~x'iatc 1'rulcssorul'(lncnlogy anrl Mcdi- ctnc,'fhc Johm I lopkins llnlvcrsity Schurrl nl' McJrcinc, liultinturc, Ml). ( J) PRQJI?(.'T'1'1'1'1.8 li(Trcle uf cnmqxnknls uf cigurrttc Mtnrrkc (in steroid Prrxlucunn, rnctutxrtism and action Tabacca use in Alihcunur's disease Idfl"cYl uf drugs on rnemhrancx uf livr tis.uex Irlrntiticutiun of uncugrncx involved in human lung carcinuma (;tanc hyn,:mxthylation in humun lumora I:I. S. HI:NNI:1 I, M.I). CharactCriiatinn of the platclct lihrinogcn Associate I rolc}zor ol MaJicinc. Iluxpital receptor nf tltc Univcr.iuy ul' I'cnnsylvania, Philudcl- phia. Mh'l1Al'sI. ItNNNkTI', M.l). Imntunnrcgulation ul' NK cclla 1 rulcs:,tuuf 1'athulugy, University ol'Trxa. ' licaldt Sctcnccx ( entcr, I)allay. I2IC'HAkI)1. HIN(i, M.1). Prulcsaer of McJicinc (cmcritus), l)nivcr- sity ul' Suuthcrn ('.ddurnia Scluwrl of Merli- cinc, l.ui Angclcx; Visiun g Associate, ('ali- Guntia Institute of Tcchnalrrgy; 1)trcclnr of lix(jerutn•nlal ('ardtulul;y unJ Scicntilic Ik- vcl)pmcnt. I tuntingl~tn Medical Rc`earch In+lnutcs, I'asarlcnu. ('A. ('ONti'I'AN'I'IN A. liONA, M.D. Ihuilcs%ur of Micruhinlugy Mrrunt Sinui Schtwl u/ Mcncctnc, Ncw York. 11. I.I;t)N ItRAt)LOW, Ad)unct I'rulcsaur, 'fhc Rtk•kcfellcr Uni- vcr.ny, New Yutk. 'I'tIUMAS R. ItKOKlik, Iht.l). Asmx'r,De Prulcsu,r uf Iinwherrri.try, I lni- vwsity ul krkhcstrr Schtxr) nl MrJtcmc, I(IKIICsICr, NY. tx)RUTIIY l.L lill('1111A(ll:N, Ptt.b. Asstntant Profexsnr, State llntvcrstty of New Yurk, IAtwm,lalc Medical C'cnlcr, Rnxtklyn, NY. , I6S Coronary sp•rxm; cerebral micnx:irculation I'vnction anil rnnlccular studies of V gcnc+cn- crxling autrrantihrxlicx Snwking untl cetrul;cn mclaMdistrt in rclauun to cancer rtsk ('¢Ilular tnntsfnrmatinn by rapillunra vtrus rc- cutubtnants Oncogene expression in Iclal mousc lung
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I'RIN('II'AL INVh:S'1'I(:A'fOR 1'ROJECT TI'I'l.P; PRIN('II'AL INVF.S'1'ICATOR P'ROJF:L f TITI,I? OR INS'1'ITII'I'lON OR INSTI'1'U'1'1ON VINCIiNLO 1311UNASSI3I. M.I). Hcraran sulfate rrudcuglycanti unil hhitKl Bl[RNI('li 11, CUIII:N, 1'n.U. Airways obstruction and smoking in black and Srmur Sncntra and Ikpuly I)ircclur, W. huiucn.tuttc mcchanums Professor ul' GpiJcmiulugy, The Johns whttc adults Alluo Junc. ('ell Science ('cnter, L.ake Hupklns Umveni(y. Ilalumare, MD. Placid, NY. S'I'ANLEY CUIItiN. 1'u I). The role of lipoconin in the cellular re.punsc ' ' Prufesuoruf I3iixfiemtMry Vanderbilt Lhti- toEGF JOHN W. BUIt( I1, M.I). i*nlrul uf arachtJontc acid oxygenation in hu ( , vcraity School ul Mcdlcinc Nashvdle 'I'N A+tioK'iatc McJtcal 1)irector, Amrrican Red man platclets , , . ('ru+. Rorlw,tcr I)iviaun Rochester NY , , , . ROBERT l.. CONIIAIM, I'n.U. Routes of alveolar Ihxxling and clearance Associate Scientist, Univcr;,ity irf Wis+:on- lil)WARI)1. ('AMI'Iil?LL, M.I). Moxlulators of inllanlmatriry crll pru7culylic sin, Madiurn. A!.sut:ml I'nolc+.or of Medicine, Wushing- activity lutt llnivcnny Srhad ul MrJirinc, St. RONALI) I3. C'ORI.IiY, I'It.l), Gene expression in I-Y - 1+ B lymphocytes Luuiti. MO. Associate Prulcssur uf Inununuilogy, I)uke University Medical C'cnter, Durham, NC. DENNIS A. ('ARtiUN, M.I). Mechanism uf immune Jysfunctian after uxi- A.}+Kiatc Mc"uhcr, Scrilap. C'linic and Rc- Jant exposure (iIiRALD R. C'RAI3TRIili, M.I). Retrnviral insertion and aclivution of the 11: 2 karch Fuun.laliun, La Julla, C'A. Assikiate I'rufcssorof Path+dogy; Stanford gene University, StanGrrrl, ('A. DONNA ('IIAI'RONII•:RE, 13.Sr., 1'rt.U. ('nntrol of pruliferatinn nf ccll+ frum the adult Sirangeways Rcuarch Luhuratory, C'am- human pnntate I:VA BROWN CRAMER. Pn.D. Studies of inllammalion using an in virro bridgc, IinglnnJ. Associate I'rol'essur of Anatomy and C'cll uuKlcl 13iulut;y, Ihiwn.tale Medical Center, Y(IAN'fSONG ('IIIiN, M.D., Pu.l). Recombinant DNA approaches to assess ri.k Bnwklyn, NY. A+tiiatant I'ru/cssor (if 1'eJiatrics, I)uke I Iniversity Medical Center I)urham NC litr lung cancer CARI. H. CRllli'I"/., Pn.l). ' ' Role of protein phosphorylation in nicotine- , , . Assistant Prul essor ul 1'hnrmacology, Uni- induced catccholaminc release vcr.ity of Virginia School of Medicine MORI)I:C'IiAI ('HL:VION, I'n.D. The effects of vitatnin C anJ Iransitiun mctals , Charlottesville. ('hairntan, Insutute of liioa:hemistry , 'I'hc on cnagulaliun processes Ilchrew University of Jeru+aletn, lerusa- SUSAN t?. C'111.1.GN, I'n.D. Synthesis and function of invariant chain prn- Icm, Israel. I'rufcssnr of Microbiology, Imttunnlopy Icuglycan and (ienclics, Wa+hington l)niverstty WILI.IAM M. ('IIILIAN, Pathophysiology of the coronary micnxircu- School of McJicine, St. Luuis, MU. Assi.tanl 1'rulcs.ur, 'I'cxus A&M (Iniver- latinn .ily Slation. GII)ON ("/.APSKI, M 1r., I'u.U. Role of tmtal ions un superoxide and vitamin 1'rulc..or of Physical ('hcmistry, The Ile- C toxicity in biological systenis CURT I. ('IVIN, M.U. Hir><hcanistry and function uf huuwn granulu- hrcw University of Jerusalem, Jcrosalem, Assistcml I'rufcswr id' Oncology and I'cJi- prielic antigens Israel. utrics, The Johns Ilapkins OnruUugy ('en- tcr, Baltimurc, MI). IVAN DAMJANOV, M.I)., Pu.l). Developmentally plunlxdcnt human lung can- I'rulcssor of I':nhohogy, Jcflcrsun Medical cer stetn cells ' ' ' ('entcr '1'honras Jetlcr+on Universit Phil- RUIiIiR I A. C I.ARK, M I). 13iosynlhe.is of human ncutruphil clastase , y. adclphia Professor of McJrr:mc, llnivcrsily of Iowa, . Iowa ('ity . MARTIN 1?. 1)ORI', I'u.U. Macrophage-like cells involved in inmrune Professor of Palholugy liarvard Medical suppression GARY A. ('I.AWSON, M.I)., Pn.U. Nuclear NTPasu: and selective RNA splicing/- , SchoKrl Boston. AsuK•lalc Prole..ur ul' Pathology. Gcor~e Iransfkirt , Washington lhtivcrmuy, Washington, IX . 1'I:TI'sR 11. DLIGSILLiR(;, Ptt.l). '1'ranslirnning genes of two acute leukemia Molecular mechanisms of response to in,lury I'rutcswor ol' Mulcrular Biology, University viniscs of ('ahliornia, Berkeley. BRIAN L. C'LE:VIN(;IiR, Pu.D. Role of J segment in V segment expression Assistant Pwfc.%ur ul' BiomcJical Science, REUBEN I:ISENSTIiIN, M 1). Ilepurin-hinJing proteins and enJulhcliul cells Wa+hmgtun lhrivcrsity School of Lk:ntal Prufesti,rr of Pathology, Mount Sinai Medi- Medicine, St. Luuis, MO. cal ('entcr, Milwaukee, WI. ('IIARLIiS G. ('O('IIRANIi, M.1). Mediation systems in inllammatury lung Jis- GDWARI) A. 17SH1?R. M.I)., Pn.l). Nutritional, hormonal and cellular /'ar:tors a1= Memlxr, Ihpartuknt ut Immunopathol- ea.u: Asustant Professor of 13itxhetnistry, The frxaing alwprutein gene cxpression ugy, Scri pti ('linic and Research Founda- Medical ('ullege ol'I'enmylvania, Philarbel- tion, La Julla, CA. phia. 166 167
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1'RIN('IPA1. INVESTIGATOR OR INS'1'I'1'll'17ON llll)I'fll ANN mSTlil(, Ptt.l). I'ndc,uir uud ('hairpersun, Ikpurmrcnt of Itwlogy, Syracuse University. Syracuse. NY. RI('IIAKI) It. FUX, M I). I IraJ. I'ulmunury I)ivt,ion, Minneapolis ('htldren's Medical ('enter. PROJECT TITLF. Involvement of claxtin hhcrs in lung Jt4eaw ole of glycosaminoglycans in lung cJcma 1'ItIN('IPAL INV1+:8'1'ICA'I'OR OR INS'1'I'1'U'1'ION NOltlll'OSIII IIA(iINO, M.U., Plt.l). I'rufcsu,r of Anatomy. Univcrsity of Tcxus I Ical(h Science ('cntcr, San Amnniu. LINI)A M. IIALL. Pu.l). A%. mwtr Prulr%,ur of (icnelicti and Nruroscicncc, Albert lantitrin College of Medicine of Yeshiva University, The ISrunx, NY. PRaJI^:cT Trri.b: Nicu(me on prolactin u;creliun in Jevelop- utenl (icnctic Jillercnceti in tucutine unsilivily in Uru.wpHilu rnelunugimrrr strains II(WIN hRII)OVI('II, Iht.D. Control of the biusynthesis ul'supcroxidc dis- I'rulcti,+u of Bioxhcmistry, t)uko Univer- ' nnutases III.I.A MAN(iklA'li IIANSI?N, I'u.t). ('Irrutnatin eff c~:ta on RNA prrl mcra»e II Iran- cntcr, 1)urhant, NC. tiny Medical ( ('htcl, I.ntkrrrnury of liukaryotic frujmrip• y scnptiau ttun. I )ana-Farlx:r ('anccr Inslitutr 8utitun I:RROL ('. FRII:I)IIfKU, M.D. Complementing liuman cells with cloned , , Atitiuc'talc I'rufrswr of 1'alholugy Stanford yeast DNA repair genes , Stanlirrd, CA. llnivcrsity IIFNRY 1). IIOIII?RMAN, M.I)., Pu.l). Reacliun ill aldehydrs cuntaincd in cigarette , Profcmur. Albert liinslcm ('ollc ~r of Mcdi- sutukc with hcrnuglnhin KJIi1 t-l1XI: M I) 1 Nicotinc, calechulamincx, and neuraknclu- c+nc of Yc,htva University, We 13r1nx, . , . . . I'rule»ur of I litilulo y, The Karolinska In- crmc functiuns NY. ~ +titutr, Shwkhulm, Sweden. Srrutkin ~, dupo-rminc, neurnpcptidrs and RO131i1Y'I' M. 11OhFMAN, Pu.U. Methianine JepenJcncc, mcthylanon and ur- mtnle~s of Parkinscrn's disease Assistant Prulcssur of I'cdtatrics in Kcsi- ganic translrrrmation dcncc, llnivcrsiry of ('cdilirrnia School of JACK 6Al11.UIti, Pu.l). The mast cell in interstitial pulmonary lihru- Medicine. San 1)icgu, La Jolla. Prolcssur of I'alhulogY, McMaster Univcr- tiis. sny, I lantiltun, Ontario, ('.uraJa. RI('IIARU L. IltlGANIK, Pu.U. The nicotinc acetylcholinc receptor: rcgula- Assistant Proles,ur, 'fhe Rockefeller IJm- tiun by protein phospharylabun J. 1(IiRNARI) L. (ilils, M.U. Tissue matrix and phal,axytc injury: relative vrrsny- New York. Pruftswr of MeJicine, Yale Univerzity contributions of proaeaxs and uxtJ.mtti School ul' MrJtcine, New I laven, ('T. J.(i JOSIII- Alumtnum and Icrritins in Alnceinrer's Jis- Prulimot ill Birkhcnrislry University of ca u MI('IIALI- I). GI:ItSIION, MAY Nicotine effecls un neural development: a , fcnucurr. Knoxvdlc . I'tulrstiur of Anatomy and ('ell Biulugy, study of the acccssihlc nervous %ystrm ill . ('ulunahta llnrvctsity ('ullcge ol Physicians the gut ' & Surgeons New York. MIC I IAlil. KARIN, 11n.l). tudaliun and characlerireniun of a heritable , As+,x'ialr 1'rulr+tior of Mcdicinc. llmvcr- Iragde site on human chturousume IiN GIAM I'n 1). ('110(1 Y Immunoghohulin enhanrer elemcnts in tisnur +ny of California Schrnd of Medicine, San . , I'usIJok'tur:d Fclluw, National Institutes of ,pecilic gene cxprestiion I)irgu, Lu Jolla. I lralth. Hcthcula, MI ). M(NdRISJ KAkNOVSK1'. M.li., B.('u. The nrolrcular basis ul' pulm+mary surliu-t:uu GORDON Nlil tiON (ill.l., M.1). liptJerntal growth laclar receptor gcnc in cpi Slculuck I'rulr„ur of I'alldngtc;d Anat- sccrcuun by ly(x. 11 pncum,xytcs: studies in I'rulcswr of Mcdirtnc, llnrvcrsity ill ('ali- JcrmrnJ carcinuma uluy, Ihnv:nll MClhcal tiihoN7I, I3Ul1Un. int;trt ccllti anJ a rcll - frcr iystcm lurnia, Stm I)irl;u, La Jolla. R()Itl•.bt'I' W. KARft, M.I). Ikvclnpnttut and JilfcrcnlialUon of nurm;d GAItKIkI. C. (70I)MAN, M.D. ('ytuskclctal organi/ation of the cnduahrhal A+sulmtl l'tolisu~rufMrdicinr, lhtivcrsily and Icukcnnc mutnw'ylcs Prulissnr of I'alhulugy. ('ulumbia Univcr- cell in regulation of shape cunlrarttlity and ul Iuwa, luwa ('uy. vny College ul I'hyucians & Surl:cuns, surface nuwenrcnt New Yurk. IIHIN% KOIII.1ik- M.I) , I'n.U. Multilarl:cling with hybridnmas on tutuar I)ireclor Ihp;trrmcnt ill Mulcrular Inrnm- rell, AI.PRIin L. ciul.l)lil{Ftci. Pu.l). Selective degradation of damaged ccllul:n . nulupv. Kuswcll Park Mcnturial In+tnutc I'rulestior ill I'hysiulrrgy, UarvarJ Medical proteinx , 13ul1aFu, NY. School, Bu,wu. WII.I.IAM 1? (1OI.I)MAN, Pn.D. A»iutmu I'nrfe+anr ill Microhiulul;y and Immunulu ,y, Wir+hm ~ton University School uf Mtdic'ine, St. ~.ouis, MO. MARK I. (IRP.hNft, M.I)., 1'u l). I)ucctur ill lumwnuhiolupy, University of I'cntnylvama, Philadelphia. IlordrrrlLt prrnr.rsi.+ tracheal cytutoxm Suppressarcells in syngcncir ttmrur imtnuuuy MARKKII KOSKI?NVIIO, M.D. Prulrsurr and ('hntrnran. Ikparlmrnt uf I'uhlic Ilcalth Science, Univcrsny uf Ilcl- stnkt, I lrltiinki- Finland. The I-'iuni,h'I'win Cnhnrt Folluw-up Study ROBI'.N'f 11. KRk'I'SIN(iliR, 1'tr.U. I'rulcsWIr of Biulucy, Univcrsity ul' Vir- giuct, ('harlottesville. C'rytitallographic stuJy ill Jrug-calmodhdrrr cumplexcs 168 169
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1'RINCII'AI. INVES'1'IGA'1'OR OR IN.ti'I'I'I'U'I'ION AR(IN I' KIILKARNI. Iht.U. AS1uCl:ne Prulessur, Umvcrsity of South hlunda, Tampa. JAMI:S'1'. KL1kNI('K. M.1). AsuKtate Pathologist, MassachusettsGcn- cral I luspnal, lioston. PA(11. I.EHOWITZ, M.I). Senior Resrarch Scientist, Yule University, New Haven, Cf. JOSIiI'lI LGI(iH'1'ON, M.D, Prol'csutr of /'athohrgy, Medical College ot' 1'ennsylvania, Philadelphia. LAWRENCIi LIl'-KIN(i I.NUNG, M.I). Asststunt Prufcsxor of Mcdicine, Stanford Univcrsny, Stanford, CA. MIC'HAlil. R. LII:BIiR, M.D., Pt1.D. Lah+iratory of Molecular Riolugy, National Institute of Arthritis, Uiatxaes and Utges- lcvc and Kidney I)isca.xs, Bethesda, INI). JON I_INI)S'I'ROM, Pu.l). Associate 1'rufcssor and Mcmlxxr, 71tc Salk Irntitutc for Biological StuJies, San I)icgu, CA. JOSGPH S. I.II'SI('K. M.D., Pn.D. Assistant Prufessur of Pathology in Resi- dcnce, lfiiversily of ('aldirrnia School of MeJicine, San I)icgu, l.a Jolla. PROJM:("P TI'1"LE Bcnio(a)pyrcne activation in human placcnta Lung cancer study uf the iu situ inllenunatury respunsc c-Fus cell proliferation and Jiff'erentiution Atypia and neoplasia of stratificd epithelium in gradient culture Molecular biulngy of moncxtye and platelet interactions with enJothelium Site specific recombination of antigcn receptor gertcs Ncuronal nicotinic receptor structure Interactions of nuclear oncogenc pnnlucts ZVI LIVNIiII- Pu.l). Mechanism of S.O.S. error-prone repair Scientist, The Weirmann Institute of Sci- ence, Rchovot, Israel. RI('ARDO V. LLOYI), M.I)., Pu.D. Assistant Professor of Pathology, Univer- sity of Michig•an, Ann Arbor. Analysis uf pnuitury nctrplasnrs whh mono- c6unal anuMKlies J(1Siil'II 1). L(X'KIiR, M-I)., Ptl.l). Assistant Prufes.or of Pathology and IJio- chcmistry, llnivcrsity uf Pittsburgh School (it Medicine. RONALI)1 I.11KAS, I'u I). I)ircctur- Laboratory of Neurochemistry, St. louuph's Hospital and Medical Center, 1'hoenix, AL. JAN M. LUNDHIiRG, M.h. As.i+lant 1'rofcssor uf Phannacology, The Karolinska Institute, Snx:kholm, Sweden. DNA nnthylation in neoplasta Influences of nicotine on neuronal expression of acctylcholinc receptors Scnsory neuropeptides and smuke-anduceJ ir- rilauon in the respiratory tract 170 1'RIN('IPAI. INVF:S'I'ICATOR OR INS'fl'1'UTION HI:NRY'f. LYN('I1, M.U. I'rulcti.or and ('hainuan. I)cpannu•nt of' Preventive Medicine ,mJ I'ubhc Ilealth, ('rciEhton llnivctsny School uf Medicine, Umalaa, Nli. RI('ARIX) 13. MAC'('IONI. I).Sr. A,sistatu Prul'cssor- Univenity of('uluraJo I Icatth Sciences ('cntcr, I)cnvcr. 13ILLY R. MARTIN, Pn.l). Associate Profeasur, Medical ('ullcgc of Virginia, Virginia Commonwealth lJniver- sity. Richmond. ROI)l.R'I' W. MASON, Pn.l). I'ust-Ihk•tcual Research Scientist. Strange- ways Research laboratory, Cambridge. lingland. WALLAC'li I.. Mc•K@f:IIAN, PtI.D. Senior Scientist, W. Ahun Jones Cell Sci- cnce Center. Lake 1'IaciJ, NY. IaH;AR F. MEYER, JR., PH.U. Associate Professor. 'lexas A&M Univcr- sily, College Station. 1)AVII) A. MOS('A'I'IiLLI, 1'u.l). Research Assistanl Profcssctt, New York University Medical Center, New York. FI:RID MLIRAD, M.D., Pu.D. Prufeasor of Medicine and Phamwcudogy, Stanford lJniversiry, Stanfivd, CA, and ('hicf of McJicine, Veterans AJministra- uun Medical ('enter, Palo Alto, CA. CIIRISTOI'111:R Ml1RLAS, M.D. Assistant Profcssorof Medicinc, University of Cinctnnati, Cincinnati, OH. M(M1N 11, IIAHM, M.l). Assistant Prufess)ruf 1'athalo~~,y, Washing- Inn Univcrsity, St. Louis, MO. SUSAN NAYLOR, PIt.D. Associate Professor of' Human (ienetics, The University of Texas Hcalth Science ('entcr, San Antonio. IM)NALD J. NELSON, Pw.D. Associate Professor of Chemistry, Clark lJnivcrsity, Worcester, MA. S'f1iV1?N K. NORDEEN, Pit.l). Assistant Professor, Univenity of Culoraclo Ileallh Sciences Center, Uenver. 171 PMOJ1':("1' '1'1'1'LF: (icnctic and hiunrarkcr .tuihes of cancers of the respiratory tracl, panrrcas and urinary bladder Rcgulatiunaf microtuhule avunrbdy in nomtal and transfurtncJ cells C'haractcrixation of ccntral niculine receptors Role of carhepsin L in cntphytema Endocrine control of human endathelial cell regeneration Structural studies of clastase AnViogenic factor-enduthclial cell interac- ttons Mechanism of nitric oxide activation of guauylatc cyclase Role of cyclic GMP in stnooth muscle relaxa- tion Atrial nalriurelic factor receptors l:lectromechanical properties of airway muscle Ikvelopment of human B Cells Molecular and genetic analysis of small cell lung cancer Calm+xlulin interactions with target proteins and synaptic vesicles Regulation of expression of the c-mos proto- oncogene
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1'KINCII'AI. INVh'.S'1'IGA'1'OR ' ' ' PKOJb:Cr TITLE P!t I N('I I'A 1. I N V P;5'1'I G A'I'( )R PROJr:C'T TrrLE OR INS I fl 11T10N OR INS7'1'1'll'fl(1N SI: KYl1N(i OI I, I'tt I). Role rrf the uumumr,~upprev.usr SI:Fi in (ruhnrt- LOLA M. RIiII) I'u.l). lu vilru assay prediction of nuelu.tmtie ptnen- A.suctatc 1'rnleti.ut of Microbiology, BarY- nary inflammation and malignancy , Associate Prulcssor, Allxrt F.inytetn ('ol- ual ' ton University Sch00l of Medirine. Icgc of Menccine (rl' Ye.hrva University, The Itrunx, NY. JANIi'f M OLIVI:R, I'u.U. Kcgulatiunuf themcmhntneuxiJaxufhuman I'tofc.xor of I'alhulu)!y. Ilnivcrxlty ul' NeW Ixrlymorphonuclear leuktxytc. WARI) RI('IIAKI) KI('li. M.p Pu l) NcurnpepuJe hormnnc regulation ol' surfac- Mextat School of Medictne, Albuquerque. .. . . Assistant I'nrfesxor, University (it C'mcin- tant secretion naU, Cincinnati, OH. YOSIIIO (ISAWA, 1'n.l). Arumatase inhibitors in cigarette snwke and HcaJ, lkpartmem ol knJ+krine 13icxhem- lobaccu JUIIN N1. ROBINSON I'n l) Ncutrophil »timulation: hiixhemical and ccll ititry. Medical FoamJattun of Bulfalo. Iluf- . . . Associate Pmlc%sor ol' Pathulogy Harvard biological studies luln, N Y. , Medical Sclxxd, liuston. MARY 1). USI3AKKEN, M-I). A>,.ititant Prul'essur uf Anesthesia and Itiuchemititry(fdiuphy.ucs, University of' Penn.ylvania, Philadelphia. I3NV@RLY PAIGEN. Ptt.l). ('hildren's Ilo.pilal Medical Center of Nunhern ('alifurnta, Oakland. ISRAI:I. PEC'IIT, Ptt.l). Prul'exsur ot Chemical Immunology, The Wciimann Institute of Science, Rehovu7, Israel. SC(YI"I' W. PI:TERSON, Pu.l). Associate Professor of 6iology, Arkansas ('ollege, 13ate.rville. DANII'sl-1?. Pli 1-1'IJOHN, 1'u.D. Prulessor of Iliochcmntry/13iuphysics, llniveraty of ('ulurada, 1)enver. EI)GAR PI('K. M.l)., Pu.l). I'rofesxir of Inununolugy, Tel Aviv Uni- verstty, Tel Aviv, Israel. "P N MR uuxly trf cardiac nmetahnlism in hcalth and disease Mapping genetic detenninants o/' athcmxclc- ro.is susceptibility The role of CA" ions in hasnphil and most- cell degranulatiun Insulin receptor down regulation in crythro- eytes NAI)IA KOSI:N-I'l1AL, 1'tt.D. Children's Ilaspital, Boston, MA. IIARRY N1113IN, Pu.l)., I).V.M. I'rul'esaurnl Molecular Biology, University ot ('alilirrnia. Berkeley. UNA S. KYAN, Pn.D. Research Profc+.urnf MeJicine, University uf' Mtami Scharl of Medicine, Miami, FL. JGFFNNY 1). SAFFER. 1'tt.l). Atiuxiatc Stalf Scientiat. The Jackson Lab- uratury, Bar I1arMfr, ME. AZIZ SANCAIt, M.1)., Pn.D. A.uxiate Professor of l3iix:hemixt llni- versity ul' Nonh Carnlina, Chapel Iill. REGINA M. SANTGI.LA, Pu.I). Associate Prolessor of Medicine and Envi- ronmental Sciences, Columbia University, New Yurk. Regulatory factors in muscle gene expression Adaptive vs. selective effeclx of alkylatjng agents Interactions of hurntunes with cells of the pul- monary vascular wall HMG proteins in chromatin NuclcotiJe excision repair Ikvelopntent ul' umtnxlunal antitxxlies tu carcinogen-DNA adducts Role of specific cell +urface carbohydrates in the development of hunwn u{uamous lung carcinoma The biochemical basis of enhanced nxygen radical production of lyntphokine-acti- vated macruphuges 13.V. RAMA SASTRY. 1).Sc., Pu.l). Profcs.ur of I'Itann:rcnlugy Vanderbilt JAMES M. PIPAS, Pu.D. Uncugenex active in colon cancer , University School ol' Medicine Nashville A.ustant Professor, l/niver+ity of Pitts- , , TN. hurgh. (iOI(3)ON 11- SATO 1'tt 1) SALVA'fORE V, 1'1YJO, M.I)., Pu.U. ' Protease regulation and cellular ntetaMolistn . . I)trertor W. Alton Jones Cell Science C'en- I'athulogy, I)uke Atiux:iate Prolessor ol , ter Lake Placid NY University Medical Center, 1)urhanr, NC. , , . WILLIAM S('IiNAI'I?f( I) 11 M JULIA M. POLAK. I).St•., M.I). Investigation of the « de of regulatory Ixplides , . , . Ad1uuct in Pathulogy '1'he Jewish Ho.pital Senior Iarturer in Ihmtopathuingy, Royal in human lung disease , uf til Louis MO Po.t ~~,raJuate Medical SchrNrl, liammer- . , . xmitl1 1loxpital, Lrmdnrt, England. ('IIARLl3S li. S('(Xi(iIN, M.1). IlcttJ I)tvtuun of ('linical Applications; LOUIS PON(!. I'tl.l). Birx:hemiary and inmwmrlogy uf Psrudu- . A.xociale Prul'essor of Medicine Univer- Senior Research Asuwiate, ('ax Western rnonas proteinases . .ny of ('olorado Ilealth Sciences Center Rcxervc Umvcraty, Cleveland, UH. , Iknver. RAMI RAIIAMIMUFF, M.I). I lum+rral effects uf smull cell carcinonw of the I)AVIU W S('OTI' Pn b Prulc..or o/ Phystulogy. Hebrew llniver- lung on neurumux:ular transmission . , . . I'ruleawr of Inrmunulu~y University of aty-HadaUah Mcdual School, Jerusalem, , Rochester Rochester NY Israel. , , . Maternal smoking and blood concentratiuns of amino acirls in umbilical arteries and veins I%olation of growth factors frorn blue-green algae Biulapy ol'the lymphukirte, soluble immtme response suppressor (SIRS) The somatic cell genetics of lung cancer Inmmne respimut to nuxlilied sclf-regulauon ol murint B lymph+nn.r growth Regulation of murine B lymphoma growth 172 173
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I'ItIN('ll'AI. INVF.S"I'I(:A'1'Qlt PROJW:t"1' TI'1'LN: PRINCIPAL INVI?S'fICA'1'Ult PR(UEC'1' TI'1'LE OR INSI'I'1'll'1'1ON OR INS'1-1TU'1'ION RUItLR'I F SCl)'I-I• M.I)- Cunuuitmeut control and carctnogcnctiia tn THOMAS P. STOSSI:L, M.h. Functional analumy uf the lung maurorvhage . I'ndcuor ul I',uhulugy, Mayo C'linmc and norntal prrn.xtplasttc and malignant hu C'hiel. Medical Onroluy.y llnit, Massachu- I•uundanun. It+Khc.lcr, MN. , man cplthelial cclls sclts ( icncral I luspnal, Boston. GINI:I-I I: SI• RKlil(() I'n I) Chatarlrriiatiun ufadilxtx Jdlcrcnttaliun in- I•l.fllR L. S'I'KANI), I'n.l). Prenatal and P+ntnaud cflects of nic+ttinc anJ , • . . Scnwr Scientist. W Altun Jones ('el'1 Sci- hihuur Prule.snr of I4+ulogy, New York l.)niver- AC"fH peptiJes un neuruntuscular develop- cncc ('enter. I.ake Placid. NY. sity, New Yurk. mcnt and motor behavior in rats A I))NNY S'IROSBI:RG I'tt l) Structure and function of the muscarinic re- III?NRY SF.KSIII:N, I'u.I). Ihhvelupmrnt of an anintul m+xlol of I:atktn- . , . . ('hatr Prul'css+tr uf 14inchetnislry and Immu- ceptors of a huntan embryonic lung Kc,r:uch Scicnti,t IV, Ncur+xhemislry I)i- >tm's disease Pasteur In+utute France nology Paris lihrublast visiun. Nathan S. Kline Rcsu:•arch Institute, . , , . Ward's I+IanJ New York , . MAK(yl'O'fAKIiTO, M.U., Pn.l). Gene regulation in terantcarcinoma stcm cells ~ ' Associate Slaff Scicnlisl 'fhe Jackson I ab- JI?RRY W. SI IAY. 1'u.h. cytoPlasntic elcmonts in Ihc induction Role ol ' . , otatury 13ar Ilartxrr ML•: A,++u+•iale Ihulcssor, University of 'I'cxus and suppression ul Iunwi tgenicily , , , Ilcalth Science ('rntrr, 1)ullus. JANGI TANNkNBAIIM, Pn.l). Cytoskclrtal m+xlulatiun of gene expression Assistant Pnde+surut PJIhuhllLy C'olunrhia anJ surfacc functiuns in endnthelial cells ISAIAIIII SIif•,( 'Ifl'I:K, Pn 1). Gflcct ul'lhiolx and disulhdes on cholesterol , Ilniversity College of Physicians & Sur- Scniur Lcrturrr tn lin><hemistry, The nuaulMtlism feuns New York. (ieurl:c S. Wise Farulty lur Lilc Sciences, , Tel Aviv Universily,'1'eI Aviv, Isracl. I). LANSIN(i'fAY1.QR, PILD. The structural and metabolic chemistry ol' I'rufc...ux of Ihuloyical Science, Carnegie- cytuPlasm KI•.NI)Al.l- A. SMfl71, M.I). Uissrction of the eukaryotic I)NA rcpliratiun Mclluu University, 1'itlshurgh. I'rnli.sur of Medtcinc, 1)arunuuth Medical pathway School, Ilamrver, NI1. JOSlil'll ('HARI.ES'I'AYLUR, 1'n.l). Ceruloplasmin abnormality in chronic uh- AsuKiute Research Scientist, ('ity of Ilope structive pulmonary disease S'1'I?VF.N S. SMI'I'I1, I'n.b. Selectivity uf UNA methylation in nnrnml:mJ Research Institute, t)uane, CA. Assistant Resrarch Sctcntist. 13eckman Re- oncogenically trunslinmed cells search In.ulute ul lhe ('ny uf tlupc, Uuane, VI('fOR 1'.'I'IiRItANUVA, I'n.U. 1)irected migration of ntctastatir Iunn+r cella CA. Associate Prulcssor, State University of New York, Bullalu. ASIIWANI K SOl)1) I'n I) Mutant MIIC class I anti cns in ntitumnr inr I . , . Cancer Research Scientist III, Ituswell Park g a mmnlty JOIIN A. 'I'IIOMPSON, Pti.l). ChromatoEraphic uparuliun and comparative Menwrial Institute, Buffalo, NY. Ass+x-iatc I'rufessur of Phanuaceutlcal metabolism of d- and I-niculine ('hctniytry, University of Colorado School MUI IAN SOI'( )N1. I'n.l). Cigarette smoke-induced alteration of inr (if Pharmacy, Boulder. Scicntist 11, !_ovelace Medical FuunJatiun, mmnc resP+mse Albuyucryue, NM. WAYNE M. TRGBIiIN, M.I). The cffccts ul' renal function on nicotine mc- Associate Program I)irertor ol' Intemal lalxtlism TIM(y1'lIY A. SI'KIN(ilik, Iht.l). Sludies uf mucruphage suhlwltulatiuns and Medicine Kesi+bcncy, Salem Huspital, Sa- ~ A.+tstamt 1'rolessur of I'athulogy; ('Itief, Jitlcrcntiatiun using munorlanal antibodies lent, MA, . ~ L;itx r,uury ul Memhrane hnmunnchctnis- Iry. I )ana-Farher (':mcer Institute, tius(un. IiMll. R. UNANUI:, M.1). Physiolunhulogy of nurnul and activated mac- ('h:urnwn and I'rulcssor, Ikpartntent uf I'a- rophages IiRK' J. STANIiRIIXiIi, I'u.l). Tnrns/'er of specilic individual human chr+nnu- tholugy, Washinl;ton University School ol' Aeukuuc I'rul'essur of Micruhiology, Uni- sanecti to recipient cells Mcdtctnc, St. Louia, MO. virsity uf ('ahlinnia, Irvine. LYUTA VILLA-KOMAROFF Control of ncumlt+:ptiJe synthesis in rodents UANIIiI. S'17iIN1iGR(i, M.D., 1'r1.D. \ Anerial degradation of low density lilwpu+tcin , Ass+kiale Professor, Children's I-lusPilal, 1'rulrsu+rul Mediciuc, I Inivcnily ofC alilirc- in vivu If+rton. nw School of McJicine, San Ot+:go, l.a J+ulla. I'I:f{iN K. V(Xif, Pn.l). New ON(' gencs frum acute « truviral Icuke- NIiWMAN 1.. STGI'lIF.NS M.1). F.R.('.I'. Cellular mechunisms of airway xnuxah nnixle I'rulctisor and ('hainnan, 1)cpartment of mias 'r ..+"• , , I'tutc,HtrufP9rysialugy llmvct~ity afMani- cuntral liun MicruMolugy, University of Southcrn ('uli- ; ~ , tuha, W innyxg, Man+toha, C'anaJa. furnia, t.us Angeles- 1)AVII) M. S'17?RN, M I). Modulation ol' cnJuthelial cell anticuagulan HGLIiN VAN VUNAKIS. Pn.l). ' Purificaliun and propenies nf a uduhle Associate Mcmlxr, t)kl,+huma Medical Re- nuxhatusms etiutr of Bi+x:hcmislry, Brandeis Uni- Prol NAI)(P) glycohyJrolasc isolated frunt Ihc search Foundatiun, Oklahoma C'tty. versity, Wuhhanl, MA, spange, M. pruliJrru 175 174
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PRINCIPAL.INVFai77(:ATOR PROJti(:TTITLE Grantees of Completed Projects OR INSTITUTION PL:TLiR N. WALSU, Pn.l). I'rofrsux (il Medicutr. 9 enrp6e University School of Medicine, Philadephia. HOWARI) G. WELGLIS, M.1). A.aulant Prulc+wr of Medicine, Jewish Ho,pual at Washm~,~tun University Medical ('cntcr. St. Low., MO. MICIIAI;I.J. WI's1.SH, M.I). Aaststant Prufesx+truf Mcdicine, University of Iowa C'ollege af Medicine, Iowa City. AKIi WENNMALM, M.1). Profesux and ('hairman, Department of ('Iinical Physiology, University ol Gothen- burg. Guthenhurg, Sweden. ALIiXANUtiR S. WHI'f1:111iAD, D.1'hi1. A..iilunl Professur of Ikdiatrics, Chil- dren's Hotipital, Boston. PAUL V. W(X)LEY, 11I, M.b. Professor of Medicine and Pharmacology, Georgetown University Medical Center, Washington, [X'. STANLF.Y YACIININ, M.t). I'rofe,sor of Medicine and Chiel'. Seclion trf HemaloingylOnculugy, 1'he University (if Chicago Medical ('enter. IX)NALI) A. YOUNG, M.I). Prufe.,ox nf Medicme, University of Roch- ester, Rochester. NY. MAl1KI'I.IO 'I.ANIi I'f1, M.I). Asuxtale Prol'c4sor of Medicine, llniver- aily of ('ahfornia School of Medicine, San I)icgo, la Jolla. 176 Interaction uf pIr,rtelets with coagulation fuc- tar. IX and X lluntan macrophage cullagcnau: and colla- genase inhibitor Mechanisms controlling ion transport in air- way epithelia Nicotine as inhibitor of prusta glandin furnw- tion: localization of the inhibitory step and characterization of the cardiovascular inr plicatiuns Muux serum amyloid P component: a model acute phase reactant for the study of inllam- mation at the molecular level. Effects of chemical carcinogens upon gene hxi in the pancreas Models for the pathugenesis of atherosclcro- sis: A) biological effects of uxygen:rted sterol comptwnds; B) mevatunic acid and cholesterol biosynlhesn and the hio.ymhe- .is and regulation of cell growth Papilloma virus proteins and cell transtircma- tion Idiotype mapping by site-directed nrutagcne- ais Fulluwing is a list uf the principal investigators, or institutions, whose projccts lurve been completed prior to the period covered in this Report. Several of the indivtduals nannnd are deceased. The titles and affiliations listed were those in el7ect at Ihe time the work was in progress. MARIO 1). A('t:'1'U, I'u.I). Associate Professlir uf I'harmaculugy, Medical College ot' Virginia. Virginia C'onununwealth University, Richnrond. KIiNNE'I'll B. ADLER, Pn.D. Assistant Prufessor of 1'utholugy. Univer- sity of Vermont College of Medicine, Burlingtnn. ('I.ARN.N('E M. AGRESS. M.I). A,sinialc Clinical Profcsuir of Medicine, University of ('alifurnia Medical Center, Los Angeles. ANI'/iONY A. ALBANESE, Pu.D. I)ircctur of lahoratories, The Burke Rcha- hilnation Centcr, White Plains, NY. AN'I'HUNY 1'. AMAROSIi, Pn.D. Instructor in Obatctrica and Gyncculoay, The Albany Medical College of Union Univcrsity, Albany, NY. li. T. ANGIit.AKOS, M.1)., I'u.[). Professor (il' I'hysiolugy, Boston Univer- sity School of Medicine, Boston. THUMAS M. AUNG, Pn.l). Assistant Professor of Pathology, The Jewish Hospital uf St. I.ouis, Mo. UOMINGO M. AVIAIX), M.I). Professor of Pharmacology, University of Pennsylvania School of Medicine, Phila- delphia. STEPIIEN M. AYRIiS, M.D. l)trcctor, Cardiopulmunary Laboratory, Saint Vincent's Hospilal, New York. LESI-IE BAI:R, M.l). Associate Professor of Medicine, Colum- bia University College of Physicians & Surgeons, New York. OSCAR J. BALCHUM, Pn.t). Hastin gs ProfCssor of Medicine. lJniver- sity of Southern California School of Med- icine, Lus Angeles. SAMUEL BALK. M.D., Pn.D. Patholo gist, New England Deaconess Hospita, Boston. FKEI)ERIK li. BANG. M.I). Professor and Chairman, I)epartment of Pathr>[riology,'1'hc Johns Hopkins Univcr- sity School of I lygiene and 1'ublic Health, Baltimore, MD. I); MURRAY AN(;EVINE, M.I). tltlivcr+oty of Wisconsin Schoul of Medi- cine, Mathson. JOSNI"H ('. AR('OS, U.Sc. Professor of Mcdicinc, Tulane University Schuol of Medicine, New Orleans, L.A. AI.AN K. ARMI'I'AGE, Pu.D. Rcuurch 1)ircclur, Iluzlctun Lahoratorics I?urupc, Harrogate, North Yorkshire, F.ng- land. MARILYN S. ARNO'fT (RASCO). I'n,U. Assistant Biologist and Assistant Profes- sor of Biulugy, The Univcrtiity of Texas Sy+tem Cancer Centcr, M.D. Anderson Ilo+pitul and'furnur Institute, Huustan. A. ('LIFFORI) l3ARG1:R, M.I). Robert Henry Pfeiffcr I'rofe.%ur of Physi- ology, Harvard Medical School, Boston. BROI)A O. BARNES, M.I)., Pn.l). Professor (Al/di:uc) of Phy%iolugy, Colu- rado State University, Fort Collins. FREI)F.RI('K W. BARNES, Ja., M.U. Associate Prufesaor of Mcdicine, The Johns Hupkiny lJniversity School uf Mcdi- cine, Baltimore, MI). T. C. BARNES. D.SL•. Research Scientisl, Philadelphia State Huapital. CARt-G. BE('KER, M.I). Associate Prolessor of I'athuhrgy, Cornell University Medical College, New York. 177
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t i R. fRltl)IiRI('K HI:('KIiR, Pn.l). Aswciatc 1'rulcssnr of Anatomy and I)i- rcctur. I.ahoransry ul 1'erinala9 Science, I)ukc (Jnivcrsity MrJical Center, 1)ur- ham, NC. RAI.I'H S. BG('KI'sR, Pu.D. Protc.sur of ('henuslry, University of Iluuston. HI:NJAMIN Blit.l., M.1). l)ircctur limcritus, Nurniative Aging Study, Veterans Administration Oulpa- ticnt Clinic, Hrruuu. SAMIJIiL BELLET. M.D. Ihrcrtor. l)ivt.ion uf Cardiology, Phila- Jelphia General Ilnspltal, Philarlclphia. HARU1 BIsNACk:RRAN, M.I). Fahyan Prol'essur and Chairman, 1)epart- ment uf Pathulogy, Ilarvard Medical Schuol, Boston. WILLIAM P. Ht:NLI)IC"1', M.D. Assistant Prufcssor ul' Pediatrics, llnivcr- shy uf Suuthcrn ('alrfurnia School uf Mcrl- icinc, I)ivtstun of Ilcmatulugy and Medi- cal (;cnetics, C'hilJrcn's Huspital of Los Angclc., Lus Angeles. BARBARA J, van den BERG. M.l). Rcscatch I'cJiatrician, Adjunct I'rufcssor in Hinaatistics, llnivcrsity uf ('alilixnia Srhuul of I'ubhc Ilralth, Oakland. 1OIIN A. BEVAN, M.I). 1'rufcssor of I'hannaculogy, lJnivcrsity uf ('aliliornia School uf Medicine, l.os Angclc.. B(11)III)IiV BIIA(;A'I', Prt.h. 1'ralessnrul Physiology. Saint Louis Uni- vcrsity School ol' Medicine, St. Louis, M( ). ('IiSARI? IiIAN('It7OR1, M I). bivisirnr af ('anccr Rcxcarch, University uf Pcrugia, I'crugia, Italy. IIYLAN A. Itl('KIiRMAN, M.I). A..i.tant Prulc,sui ul Mcrlictnc, and AI: VAN L. IIARA('II, M.l).. ('unsultant in Medicine. ('ulumbia lJntvcrsny ('ullcgc ol Physicians & Surgeons; (Joldwatcr Me- nwnul Iluspnal, New Yurk, NY. HIO-RISIiAR('I1 CONSUI:1'ANTS, IN(', ('atnhriage. MA. HIO-RIiS1iARC'H INSTITII-1'E, INC. C'amhnJgc, MA. 178 UfiHAJIT K 13ISWAS, PIr.U., D 4r. Assuciate Prufesxx of Oral Hiulngy, larh- oralury uf Pharnracoloy.y, Ilarvard School of Ikntal Medicine, Boston. IRA B. BLACK. M.I). Prufcssrtt anil Chtel', Division of Ikvclup- mental Neurology ('ornell Univcrsity Medical College, Ncw York. PHYLLIS B. BLAIR, Pu.D. Prufessor of Inununolugy, University of California. Berkeley. FRED G. HOCK, Pu.l). Associate Cancer Research Scicnti.t, Biu- logical Station, Roswell Park Memorial Institute, Springvillc, NY. G(JENTH6R BODIiN, M.I). Associate Profeselrc uf Medicine, Assisb ant I)irector, (lenerul Clinical Research Center, Tcrnplc University Ilcalth Sci- enccs Center. Philadelphia. HI:RMAN V. BOIiNIG, I'tt.U. IIcaJ, 1)epartment of Chcmistry anJ Hio- chcmistry, SpinJlctup Research ('entcr, Lexington. KY. JAMES I'. BONNER. Pn.t). 1'rulcssur of Biulugy, C'alifurnia Institute of Technolugy, I'a}arlcna. WAI:I'liR M. BOOKER, Pu.l), Professor and IIcaJ, I)cpartmcnt uf Phar- mnculofy, IiowarJ lJnivcrsity, Washiuti- tun, I)C. I-RANt;'OIS M. BOUYSIi, Ihl.l). Senior Investigator, Michael Rccsr Re- search Fuunrlatiun. ('htrago. RAYMOND HOSSE, I'u.l). Associate I)nrcctur, Nonn:uive Agmg Sturly, Veterans Admini+tr:uiun Outpa- ticnt Chnic, Buston. TOM (i. HOWERY, Ih1.U. Rrscarch Profcsux, I'esticidr kcsiJue Laboratory, North ('arulina State ('olicgc, Raleigh. J. MARK BRAUGIILIiR, I'u.D. Assistant Profe+snr (rl I'harntacolugy, Northeastern Ohio Univcrsitic. College of Medicine, Rrwlsluwn. EI)WARI) BRESNICK, Pu.l). Prul'cskrr and Chairman, (hpartntent ol' Hirkhcmistry,'fhc University ul Vcnnunt College of Mcrlicinc, Burlington. (;I:(*I1t1iY L. I3RINKMAN, M.I). AssuKi,ne Professor of Mcdtctnc, Wuync Stu4c University School ul' Med.lcine, Ih- truit, MI. R(11iliR"1' I:. IiR(H)KS. 1'u.D. Associate Prulc.<ut of Palholugy, Univer- sity ul Urcgun Medical School, Portland. HARBARA B. BROWN, Pu.IJ. ('hicl', Gxperinuntal 1'sychlatry, Veterans Adnuniatrauun Iluspital, ScpulveJa, CA. RAYMONI) R. HROWN, Pu.U. I'rofcasor of Clinical Oncolo~•,y, Univcr sity ul' Wisconsin Medical Scl'rwl, Marli- snn. JOSLI^ IIRULI:K, PILI). I'rulcssur and C'hairman, I)crarlmctn ol' I'+ychulogy, l,chigh Universtry, Hethlc- henr, PA. REBECCA BRYSON, PH.I). Associate Prolcssor uf I'sychulol;y, San 1)icgu Statc University, San Diego, ('A. SUE liU('KINGHAM, M.t). Assistant I'rafcssor of PcJiatrics, Culurn- bia University College of Physicians & Surgeons, New York. A. SONIA B111S'I', M.U. Associate Pruli ssur uf Medicine and 1'hys- iulugy, University uf Orcgun Hcalth Sci- ences ('cnter, Portland. BENJAMIN BURROWS, M.1). Associate I'rolcssor of Mcdicinc, lJnivcr srly of ('hicago. 1)AVII) L. HIJSHHIi, I'n.D. Prulcssur uf Tuxicolu •y, Texas A&M Univcrsity ('ollege uf etcrinary MeJi- cinc, C'ullcgc Slation. 14. M. BUTT, M.1). ('hicf I'athnlugist, Los An~•.eles ('oun(y (kneral Hu.pital, Los Angeles. RI('IIARU U. HYI:RRIJM, Pu.l). I'rulctiurr uf C'henristry, Michigan Slate lhtivcrsity, tiast Lansurg. SISTER M. IiMII-Y CAI111.1., Prr.l). ('hairman, IkPartmenl uf C'hrmistry, Re- gis C'ollege, Wcstun, MA, IiRI!('L' F. ('AMIiRON, M.I)., Prr.l). Ihiward Hughcs Imtitute, lJnivcrsitY of Miami School of Medicine, Miami, FL. 179 IA.RUY'I'. C'AN'I'Rlild-, I'it.l). ('hairinan, I)ep:ulnrcnt of I'hsuutaculol;y, liexa. Cullcge ol (htcupatluc Medicine, North Texas State llnivcrsity, I)rntnn. WIl.L1AM H. ('ARNIiS, M.I). llntvcrsity u/' Utah College ol Medicine, Salt Lakc (lty. MARCUS N. CARROLL, Ju„ Ih1.U. ('hlef, Division of Pharmacirlogy, The Broukdalc Hospital Ccnter, Brooklyn, NY, WII,LIAM A. CARTER, M.D. Prufes.wrof Hematology anJ Medical On- colnl X, Hahncmann Medical College, L'IrrIaUClphla. WILLIAM AI.VIN CARTI?R, M.D. Assistant I'rufessur uf Merlicine and Mi- cruhiolugy, The John% l lurkins University School ol' Medicine, Baltimore, MI). AI.ItIiRT CAS'rRO, PH.D. 1)trcclor, Hnrmane Research Lahur:uury; l'ru/i•s.vur u( Nruirulugv and A4rJirinr, University of Mianri School ol Medicine, Miami, FL. LI:OI'OI D R. ('I:REC'GI)O, Pu.D. Professor of' BirK•lur:nrislry and Nutrilion, lJnrversity uf Pucrtu Rico School uf Medi- cine, San Juan. lA('K CHALON, M.I). Associate Profcswr uf AnesthcsioluEy, New York University Medical ('enter, New Yurk. 1'RANC'IS C. CHAO, M.I)., 1'u.l). Senior Investigator. CeMer krr Blood Re- x::uch, Boston. LAN HO CHLiN, 1'u.U. Associate Professor of Pathulogy, I)ana- Farlxxr Cancer Institute. Bustan CHIL1)RIiN'S HOSI'1'1'AI. OI-' I.OS AN- GI'-LIiS JAN F. CIILEHOWSKI, I'u.t). Assistant Prufesuttuf IlirKhctuistry, Med- ical College of Virginia. Richmunrl. SANFORD CHOf)OSH, M.I). Assistant Professnr ul' Medicine, Tults University School of Medicine, Buston.
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t I WII.I.IAM I I•ISIIIN:IN, M 1). ('hu 1 ul I:pnfennulutiy'• ('hliagu Iiturrd ul Ilridllt. I:I)WIN R I•ISIILI(, M I) I)nrclut ul I.ahuralutt.•., Shad}.idc Ilua- pual. l'rulc.sur of t'.athulupy, l)nrverxily ul I'ttl%blugh School ul Mrdtrlne. I'Al II. It. I-151II.K. Ihl 1) Srtuur Kr.r.trrh AN.nrcue. Ihparlurrnl of Mtcrohtnlugy, ('ulutnhia lhnverxuy ('ul- Icpe ul I'Irytiirtanti & Surgcuna. New York. KUSSI:LI. S. 14tilll:K, M.U. lJntvetvtp of Met yland Srhthal uf Merh- eunr. 1lalutnure• tv11L WPLI.IAM 11. fIS11MAN. I'II.I). Uht•,tdcnl. I.a Julla C'uneet Research Puun- damun. I.u lulla. ('A. IIIK(;I1'I'A I•I.OI)I;KI)S M1'KRIIIiU, I'nl1 A.utanl I'rnleaur nl litivtrunnn:ntul 11 y- ~.trnr, 'Ihr Katultn4,t Imululc. Shnk- halnl, Swcdcn. 1OSlil'II A. 1•ON'fANA• M I)., I'n.l). Asvtlt;wt Ihnlc,xvt of Medlclnc nntil Riro rltctnt+ny• Wcot Vugtutu Ilntveraty Med- ical ('entcr. Murgcunnwn. JA('K W. I•RANK1:1., I'n I). ('trn.ultunt w Medical Kc,c:rtch. Vetcrans AtltttlltlitraltUn Medical ('cntcr, Ilay I''ancn• Pl.. It. I. FI(Iail)LANI)IiK. M.1). I)rrcrtur nl ('unrer Kcxr:rrrh, Muunl'Litrn Ilu+pnal and Medical C'cnter, San Prun- l•rhl'tl. ALLAN I' 1•KI:1?pMAN. M I). Aa+istunl Prufcs.nr u/ Medicine, liahne- m:mn Medical e'ullel:r, 1'hdudclphiu. AARON I'.. PKI?I:MAN, Pn.l). Staff Scicnhtit, ('aldtn•nut Iliontetlicrl Kc- uarrh Fuund:nron, Lu Jolla, ('A. I'Klil)IiKt(' A. fKI:N('ll, A Ii. Uucrtur uf ('anra•r ('hcmothcrupy Ke- +ratch• Mount %tuu Ilu+pual anrl Medical ('enlrr. S:m hrunrt.ru. )A('K I-RfaINI), Ivt l). Av,r,l:rnt Ihulen.nr nl 1'hartrtacolul:y, Medical (`ollcgc of Vtrgtnta, KichtnonJ. LAKS I KII1I:Kli, M 1) I'tUll•\Snr :nul ('ItAttn6nt, Ue1TnrIt1M11t ol' I:nvtrnnmrnlul I lyptrnc. '1'he Kurolmaka Inauule, Shkkhulm. Sweden, 182 (iI1.111:K1' II. ('Kllil)I:I.1., NI I). ('htcl ul' 1'uthnlnpy SI. Vtnrrnt'a Iluspa- uil, Wurcetiter, MA. UARY I). f'KII'.UMAN, NI I). A%.Ltan1 I Arrrlnr, I h•parUrw nt ul h1edtr.rl Me4htKl+ Research, Katt,r/ htnutdatnun Kr srarrh In.mute, OuklanJ, ('A. li, IIIJ(id1 IPUI)I:NIII:K(i, M.U Prulc.xnrul Medicure, l)ntvrr+rly of l'ah- lurnia Medical ('rntrr, Sun I•ranctu°u; Prulcv.ur of Bu.•trtiololty und Inununul- ogy• llntvcrsity uf ('ulilutnw, lirrkeloy. AK'1'Ii(1K FIII;S'I'. Pu I) Uitcctur, In.lilute nl ('ltruriral Ntnlugy, t)ntversity o~ Sun 1•rrnrtaro. MORTON (;Ald)S'I'UN, NI 1). A%sut•iate I'rul'rsu,r nl' Medteine, New Ytnk University Mrdlrul ('ctuer, Nuw York. MURRAY It. (;AKUNGK• M.I). A%ti1rCIJ(e ('rulesSUr t/I I'alhulugy, l intver • titty of Suuthern C'altiiunia Srllnul ull Mctl ictne, le,a Angeles, JAMIiS W. (;AIIIIA'1"L, I'ILt) A.aalnnt 1'rules.urnl Nurr•hrrui%try, lJm- vcr+tty ul'South Alahantu, Mubilr. MI('IfAlil. C. (i13OKAS. M.I).. Ihl.l). Professor and Vice•('hairmun. I)epart.. hr, ment r~ , tl Mu fun~•• , lImvrntly rIC,t•h , nta Schrxd of Mcdicunc, 1)avti,. 'll'.KIiSA (i1tSSNIiK. Ir'll'.1). As.rorialc C'anccr Kctic:rrrh SrienutiC, Ilculth Ke+curch, Inc., Ku%wcll l'ark Ihvi- .iun, Buflalu, NY. (;liOK(;1: Q. GI:Y• M.I). I)trcctor• hinttcy-Ilnwell ('anrcr Kcscurch Luboratory; Associate I'rufcs.or of Sur- K y, '1'he Julun Ilu+ktua llntveraty rhuul of Mcthcinc, lia~titnure, ttifl). JA('(111NS F. (IIGt.IiN. I'n 1). Asaiatunt Prolc.sor, I.ahoratury of Mcdt- r:al ('hemi.try, 'l'uxicolugy and Ilygtcne. In.tilutc u( 1'alhuingy, University ul' l.tcgc, Ltcge, lielgium. RONAI.I) W. (;ILI.I I'1'1;, Iht.l). Oircrtur. Iiaatc Science Kc.eurch llnn• ('anrcr ('enter ul Ilawau. l/nivcraly ul' Ilawaii at Manuu, Ilunululu. (i1sKA1.I)1• (;Llil('11, M.I). (bmullurnl ut Medreutc, Kexcurrh Lafrtura- tury lur Allrrhtr 1)i+cau+, Mayu Cltnir anQ I•uunrLmorr I'rolc..ur uI' lulernul Methrtue antl Imtuunott,l y, Mayo Medt- cal Sch,ti,l, Ktk-hc,ter, MN. 'I'llOMAS M. (iO('Kli, M.1). Asurct,uc I'rulc.wv of 1'IevcNtve M.di- cmc :md ('urmuunuy Ileallh. Sclun Ilall ('ullrgc ul Medtcine and Ihntistry, ler.ey ('Iry, NJ. WAK'RI:N M. (iOI.1). M.l). Prnlr.at,r of Medicine, ('anlinvusrular Research In.litute• llntvcr.uy of C'ahli,r- niu, S:ut Frunrncu. hAVII) M. (i(Dl.t)IiNI4I;K(i• U.Sr., M.I). Ax.+n'latr Prulcvtiar of 1':nllulugy•'I'ettiplc Univcr+ny Ilredth Sciences ('enter. I'lula- tlclphtu. SII)NI:Y (tO1 I)PlS('l IIiK, M.I). Prulcau,r of I;nhuluky. Allx:rt Isinstcin C'u11cFe ul Mt•dicinc• fhc Ilrunx. NY. 1'AUI. (701.1)I IAIIIiK. D.U.S. A+..nri:uc I'rulc.u,r uf 1'cntKlontulul!y, Ilarvutd Schuul of Ueutul MeJictne, Ru.- h,n. Lf:ONII)li GO1.1)S'I'1?IN• I).Sr•. A.auriale I'ro/'c+.or ol 1'.ychiatry, Imti- lute lur Mental Itc.rlth Sciences, Cbllege ul' Medicine & I)enlixlry u/ New Jersey, Rutgers Medical Srhuol, I'tssalaway. IRA (;t)Kli, M.I). I'rule.anrtni 1':ah4rlugy, 6u,tou Univcr>rily Schottl ul Medicine; (.'htc( of Luhoralury Servtcc• Veterans Admini.trulion Hmpi- lal• West Kuxhury, MA. Jl)IIN W. GOKKOI), 1).('.C. t.cclurer in Itiophurntacy, ('hel+ea ('ol- Irgc, Univcrsity ul I.onrlon, l.undnn, 1inl;land. lilil('IRllllli Y. (;U'I-I'S('IIAI.I,. Pll.l). Aatit.lanl Prnles.ur of Kirrr•hcnuay, ('o- Iutuhta l)niver.ity ('ullcgc of Physicians & Surpcuua, New Yurk. MAlIR1C'G(;KHIiN, M.I). Ihtctlur• In+lnule lur Mulcrular Virui- uFy, St. l.uui, Ihtivcrsity Mcdtral C'cnt.r• SI I.ourz, MO. ('l1AKLHS S. UKI>IiNIl1iK(i. M.1). A%aaant 1'rule+.ur of Metlirine, lluke llniver.ity Medical ('enter, Durh•rm, NC. . 183 A. ('LAI(K (iKIF17N, Nt(.1). Ibcad, Ihrsutntenl uf IiuK•hemiary, M.I). Anrlerwn Ihr.lulal unrl Turnur In.litule, lhriver+ity of '1'cxn. Medical ('enter, IlnuaVun. AK'I'H(IK L: (iK(riS, M.S. Senior linrr•hcnu•d. Struthwcsl Ke.earcb In.ttlutc, San Autuniu, '1'X. htl)K ION L(,Kl)SSMAN, M,D., I,n.U. Asstkiate C'hntcul I'rulesaor ul' Metlirine, University of C'alrluruta Medical ('entert, I.o. Anti~ . .„le. ('AKI. ('. (;KUI1%1'1'. M I)., I'tt.l). {, Associate in I'hynurlutiy and Nhurntucml- ogy• Unrvcrsily ul I'cnmyIvania (;ruJuale Sclwul u1 Mcdirinc, Phil•.trltilphia. :, , 1OSLII11. (iIIAKN1iKt, I ILI). Attrndtng Micrubtulul;tsl; I)ircrlnr, Mt- cruhiulul:y LuhunaHtrnc., Long IsI:mtl Jrwr.11-IIt11siJc Methr;d ('enler, Ou.etw Ilu.pilul ('cnter Alhhutiun, Jantatcu, N` . l1ODA A. GI1IK(;IS. I'I(1). Axxtktalc hn11L'Js1H nl' ('ommunily antl linvirunmcnlal Medicine, lJntverstty of Culilirrniu C'ullrl:e of Medicine. Irvine. IIIRA L. (;11K'IYK)• I).V,M., M.V.S('., I'n.D. A!,xariutc ('uncer Research Seientist, Ik- p.trtmcnt of ExPt:rinrcnlal Tlacrn~:ulica, Kuswcll Park Mcnurrial Institute, 13uffulu. NY. FRANK li. GlJ'fI1KI1:, I'rna'exStrr uf I:mutnuingy,, North C'arolinn Slale College. Raleigh. li. li. HAAG, M.I). I'rufe++ur of I'hurmaculogy, Menccul C'ul• lege ul Virginiu• Ktrhurond. F. J. IIAI)I)Y, M.I) , Iht.U. Prole.aur and ('huirntan, Ikpartntcnl 01' I'hystulug7• Univer.ity of Oklal'rutnu Medical ( enter, Uklahuma C'ity• J()SI:I'II II. I IAI'KIiNS('I Illil., M 1). 1)ircrtur, Cardtupulntomuy llnq, '1'Itc I.ankcnau Ilu.pilul; A.sortate in McJi- emr, llniv.r.ity u/ l'rnn+ylvantu Srlauul u( Menccine. I'hdudt'Iphia. C'AK(11.INIi It. I IAI.L, M.I). A+uK•tute PrulC..nr of I'cdiatrir% and Medtetne, l/niver%ily of Rnchc+terSrhtxd u( Medicinc, RrKhe.ter, NY.
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NAI'I li6t M. ('IIUI''RA, Ihl.l) I'rnlcsurr of ('hcnuslry, Nurth Carolina Apnavtlural and l'echntcul State llnivrr- suy, Urrecn.huro. I 0 r IH)l1(iI.AS L3K(x'K CINIiS, M.I). 1'rnl'eseor Ir1 Medicine. Iluapdal of the t Imvrr.uy ul' I'rnnsylvama, Philadelphia. WII.LIAM (i. ('I.AKK, I'u.l). Ihrcclnr, Psychupltarmacolopy Research I.aMtralury, Vrteran. Administration Hnx- ptlal, Sepulveda, ('A. IIANS'I'. ('LAICKIi, I).Sr'. Iholrssor of Iluk•hrmiary, Culumbia llni- vrr.tly College ul I'hysictan. & Surgeuns, Ncw Yurk. JAY I). l'OFFMAN, M.I). Scctiun I Icnd, I'ertpheral Vascular 1)cpart- aicnl, Ilmvcrsuy Iluspital, 13oslun. AI.I.I:N It. t.'OIIF.N• M.1)., Pu.l). Associate I'rufeSyur uf Medicine, Chief, I'ulnttmary Section, 'I'emple University Ilcalth Sciences C'entcr, Philadelphia. I)ANII?I. COIIIiN, I).V.M., M.1'.H. A.sititanl I'ruli'sunr nf Veterinary IspiJe- nnnlopy and Public llculth, University ul' PennsyIvania Scharl ul' Veterinary Medi- cine. 1'hdntlelphia. ALLAN C. C'ULLINS, Pn.U. Associate Prolcsnr of Phurmacolopy, In- .Illute Iru ILehavlnral (icnetlcs, University ol,C'oluradrr, ISuulder. KOI31iK'r W. ('OI.MAN. M.I). 1'rules.ur ol Medtctnr,'rrmple University Schuul of Medicine. Philadelphia. J111.1US 11. (Y)MltOli, Jn., M.D. I)ireclur, (:rrdtovasculnr Research Insti- truc, University ul C'olilurnia Medical Crnter. San Francisco. DEAN M. CONNORS, M.U. Associate 1)ircctor, Ikpartment of (.aho- ratnry Mcnccine, St_ Mary'. Hospttal, Mudt.un. WI. I'I IILII' C'OOI'IiR. M I). ('linlcal Ihulessnr ut Surgery and Ihrcc- tnr. Surgical l.ahur;rtury, u('r Ilular I'hysi- ulagy, Allxrt tiinstetu ('ullege ot Mrdi- cine ul Yrshiva University; Chief, Smgir;d Servicr, Veterans AJmtnistruriun Iluspual, The Brunx, NY. PAUI.'r. co5•rA, ht . P)I.l). Asvcurtale I'rutes+ur ut' I'S ychnlqy, llni- versrty uI' Massachusctts at ISostun, I)ut- che+ler. JUIIN di. C'LtAIGi111iA1), M.). PrulrsNor nf Parhulugy, I Inivrrvity ol' Ver- nrunt College of Medicine, lturhngtun. F(C)I11iRT I.. CRAIN, PII.D. Assistant Profc.utrul Stxiolugy, lJnivrr- sity of Chicago. IRVING 1'. CRAWFORD. M.I). 1'rul'esxtr anJ Chainnan, 1)epurtmenl u1' Micruhiolnl;y, (ltriveraityu(IuwaCulle/;c ut Medicine. Iowa City. T. TIMOTIIY CF(t7CKliR, M.1). Prnfcssorof McJicine, llniver.ity of Cali- fornia College of Merlicine, Irvine. CARROLI. li. C'RUSS, M.I). Asuxiate I'rolc+uu of Mcdicine and I Iu- man Physiology; I)ireclur, Sccliun ul I'ul- nwnury McJlctne, llniversilY ut Calilnr- niu Schonl of Medicine, I)avts. CEC IL G. CROSS, Research 1)epartment, St. Joseph Ilunpi- tat, Burbank. C'A. 1)AVII) W. C'KUMPA('KGR. 1'n l). Professor and C'hutrman, DepartmeN o/' Gnvironmental. I'upulution and Orpani.- mic Biology, University of ('oluraJo, 13oulder. ALBERT DAMON. M.D., Pn.n. l.ecturer on Amhrolwlugy; Research As- sociate in Medical Anthropulugy, Pear hrdy Museum, Harvard University, ("am- hrtJge, MA. TI1OMAS R. DAWI3HR, M.1). Associate I'rofessur ot' Medicine, liuaun lJniver.ity School of Medicine, Hushvn. R. F. DAWSQN, 1'n.h. I'rolessor ol' liotuny. C'olunrhin llniver- suly, New York. ALBERT It. f)Ii1SS1iNCy/'li, M.U., I'tu I). I'rnlessoruf Menccinc, Vetrrans Attminia- tratiun Medical ('enter, San Franci:.ccr. JUIIN I'. I)I:LANI:Y, M-I)., Ihr I). Associate Prulessur ul' SruKcry, Unlver- sity of Mtnnestirta• Minneopults. ANI)RNW S. 1)IISNIiR• Pu.l). lixrcutive, I'sycho kcaearch, The Age C'enter of New Gngland, Inc., Ifnstnn. I:UWAktU F. I)UMINO, M.1). I'ralcsvnr o(' I'harmaculnpy, lInivcrsity ud' Mrchigun, Ann Arbor. RALI'II L. DOkFMAN, I'u.l). I)itrcturuflaMuatoncs, Wurec.IMFwun• d;urun lor 1?xlxrirnentul litohrgy, Shrewx- hury• MA. II. FRED IH)WNIiY, I'n.1). A:,aul.rnl Prole+sur uf I'hysiula~ry., llni- versity u1"1'exaa I(ecdth Sctcnee ('enler al I)rdlaa; I)uector uf CurJiovascular Re- u nrch- ('ardrupuhnunary Inailule, Meth- txhsl I luspil.tl u/ 1)allaa. I IANULI) F. I)V(ARAK, M.U. ('hirl, 1)epdrtmcnt ul Patlpulugy, lteth Is- rtcl I losputal, 8oaon. MA. 1AMPS 1. I)YAK, 1'Ir t). Assistant 1'rulca+or uf 13iuluHy• Itellar- nunc C'ollege, Luutt,vdle, KY. kl('I IAKI) 11. IiARUi, M.I). ('hicl, I'ulnnmary Funclian l.uhuratory; AKai%lant Prulessur ut Medicine, Univer- Sily o( C'hicagu. ROIIF.R'1' IiC'11'I'. Iht.l). I'tulc.+nr ul Anatomy, Michigam State llntvcrstly, East lansing. JOIIN W. GC'KS1'Ii1N, M.U. Assistant Prulcsutr uf Internal Medicine, State University n/' Iowa C'o Ilcl,u t r I McJi- cinr, Iowa ('ity. IIF:R'rRAM I:I('l lla., t).I).S, 1)trectur. Inslilulr ul Slarnatulitpical kr- urarch• Science Rrsource:. FitunJuuon, Walertuwn, MA. 111'h1AN IiNGIiI.BIiR(;, M.1). Attcmhng 1'hy+ictun, ('rdars nf Lebanon I lu,pital. Lna Angclc., CA. ('Ahtla'ON K I:KI('KSON, Pn I). I'rutcawrr ul Pharmarulogy, 'I'hr llniver- vty of'rcxas College ul'''hrrmacy, Aus- tin. V. (iliNli IiRWIN, I'tl I). 14nicsur, ul I'hurmacoloyy; (kan, llni- vcrstty nf ('ulorudu Schrxrl ol I'harmacy, (tuulr6cr. IIFNKY J. ISISIiR, I'n l). Rcacarch Inrmunulogt.l, Mawn Research In+tttute• Wnrcestcr, MA. 180 181 ~~ . WAl:3til2 B. IiSSMAN, M.1)., Pn.U. prnfrasor of 1'sythnlugy and liuklrcnris• W Queens ('ullrge ul tlrc ('tty llmversity New York. Ilu.hing, NY. JUIIN R. IiST1:R1,Y, M 1). Assoclale I'rulc+tinr nl I'alhnlagy, (lniver- .ity of ('htcago Prutkrr SchtKd ul Mcdi• cine. Illl(ilI E. EVANS. M.l). I)trectnr, I)epartment of Pediutrics, Jew- inh Hoapnal and Medical ('enterul linxtk- lyn, lirrwklyn, NY. IIANS J, EYSENCK. I't/.l)., p.Sr. I'rulcaur nf I'aychulu~',y, In~lilutc ul' I'sy- chtatry, lJniversrry uf I.unr3un, l.onrlon, l:nllanJ. IIANS t.. FALK, Adjunci A..ocutte I'rofessnr ul 1'adtulugy, llntversity ul Southcrn ('ahfurnia Schtatl nf Medicine, Loa Anleles. 1)ANA L. FARNSWOR'I'I1, M.I). I Icnry K. Oliver I'rufc+sor ol' Hygienc and I)irectnr, llnrver.ity Ilealth Servtc", tlar- vard Universuy, Camhridge, MA. ALVAN R. FLiINS'I'IiIN, M.1). 1'rofe!.aur oI' Mrrlicinc :mJ lipidemiulul;y, Yale University Schuol of MeJtcine, New I laven, ("1'. GAD FL:INSTI:IN, Senior 1.ccturr r in 13irKhenu,tr 'ncc Y.• George S. Wise ('enter uf Lifc Sciences, Tel Aviv lJmverzity,'I'cl Aviv. Israel. JOSldl'FI I). Fla-l)MAN, M.I). Inununupathulol;ia, Scripps Clinic and Research Fuundation, l.a Julla, CA. RIC'HAKI) FtiN'rON, Pn.D. In+tructar in 1'hysinlo~, y, lJnivrrsity u1' Maxsachuuutts ScltoNtl of' Mcdiclne, W urc ea,lcr. FRANK ('. FI:RQ(ISON, Jn., M.D. ('hatrman, I)epartmetu of PharmaculoFy, rhe Alhany Medical ('ollege of lJnrun University, Alhany, NY. 1'IiOMAS H. FINLAY, I'n h. Associate Prufcssur of Uhsletrics and Gyneculugy, New York Univer.uy McJt cul Center, New York. TIIGOIX)R1; N. FINLEY. M.I) Directnr, Puhnonary Research I.ulwra- tnry, Mount-Ltnn Ilo.pital, San I;ranctsco.
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MARGrr HAMOSH, Pu.D. EBBF: CURTIS HOFF. M.L)., Pu.D. AARON JANOFF, Pn.D. INGEGERD M. KEITH Pti D Research As+uciate, 1)epartment ol' Phyt,i- Professor and Chairman, I)tvisiun of Psy- Prulessur ul' Pathology. Ilcalth Sriences , . . Assistant Professor of Anatomy Univer- ulogy and Btophystcs, Gcorgetuwn Uni- chiatric Research, Medical Collegc of Vir- ('enter, State University of New York at , sity uJ' Wisconsin School of Veterinary vcrsny Schuulaot Medicincund Lk:ntiary, ginia. Richmond. Stony Brortk, Stony Brook. Medit;inc Madison Washinl;ton, DC , . JEROME L. HOJNACKI, Pii.D. MURRAY E JARVIK Pt/ D ANCEL KEYS Ptt D PAlll. HAMOSH, M.L). Assistant Professor of Biological Sci- . , . . Asuxiate Professor o1 Pharmacolo AI- , . . 1)irector, Laboratory uf Physiolo~~ ical Ity- Associate Professor of Physiology and Biophysics and Medicine Georgetown ence+, Urnversity o(L.owell, Lowell, MA. gy, hert liinstcin Cullc ge uf Medicine of Ye- , ~.iene, University of Minnesota School of , , University Schools ol' Medtcine and DLn- RUSSELL L. I IOLMAN, M.1). shnr linivcrsity, Tluc Bronx, NY. 1'uhlic Health, Minncapnli.. ti,,try, Washington, IX'. Louisiana State University School of Med- New Orleans. icine DAVII) A. JOHNSON, Pn.D. JOSEPH B. KIRSNI:R, M.D. BERNARI) HANES Pu D , Assistant Professor nf Biochemistry East Profesxx of Medicinc. University of Chi- , . . Professor uf Health Science, California OLE A. HOLTERMANN, M.D. , Tennessee State University College of cago School of Medicine. Slate llniversily, Nonhrtdge. Research Scientist, Lohund l-aburatory, Medicine, Johnson ('ity. University of Notre Dumc, Nu1rc 1)vue, EDWARI) I-. KI-AIBER, M.1). RONALD G. HARVEY, 1'u.D. IN. IIAROLI) I'. JONES Pt1.I). Senior Scientist, The Worcester Founda- ' I'rolc.sor of Organic Chemistry, The Uni- , Assistant Profcssorof Biochemistry Uni- tion 1 or Expcrimental Biology, Inc., ver.ily of Chicago. FREDDY HOMBURGER, M.D. , versity of South Alabama, Mobile. Shrewsbury, MA. President and Director, Bio-Research In- RICHARD J. HAVEL M I) stitutc Cambridge Inc MA JEROME KLEINERMAN M I) , . . Assistant Professor of Medicine, Univer- ' , , . .. OSWALI) R. JONES. M.D. St Luke's Hospital New York , . . Professor and Chairman, Department of sity ol California Medical Center, San WAYNE HOSS, PH.D. . , . Pathology, Mount Sinai School of Medi- Francisco. Aswciate Professor, Center for Brain Re- cine, New York. search, University of Rochester Medical WILLIAM J. 1USKO, Ptt.D. tIERBL:R'f R. HAW'rHORNIi, M.D. Center, Rochester NY. Asukiatc Professorof Pharmaccutic.; Di- PETER H. KNAPP, M.1). Chairman. Departmcnt uf Surgery, Uni- rector, Clmical Phannacokinetics Labora- Research Professor of Psychiatry. Boston versity of Pennsylvania Graduate School ROBERT W. HUL(-, Pn.D. tory, Millard Fillnrore Hospital, Buffalo, University School of Medicine Boston. of Medicine, Philadelphia. Professor of Biological Sciences, Florida YN . Slate University, Tallahassee. KENNfifH P. KNUDTSON M I) JOHN A. HAYES, M.1). ANI)REW A. KANDUTSCII, PH.D. , . . University of Washington School of Medi- Associate Pathodol;ist, Mallory Institute of RONALD R. HUTCHINSON, Ptt.D. Staff Scientist, The Jackson Laboratory, r:inc, Seattle. Pathology, Boston City Hospital, Boston. Research Director, Foundation for Behav- Bar Harbor, ME. iural Research, Augusta, MI. ALVIN I KOSAK Pu t) C LARK W. 111?ATI1, M.D. ARNOI I) R KAPI AN Pu D . , . . Associate Professor of Chemistry New Prufessor of Medicine and Director, ' ' HT RESEARCH INSTITUTE . . - , . . I)irectur Laboratory of Medical Genetics , York University New York Health Services, rut ts University, Med- ford MA Chicago. , , Cleveland Psychiatric Institute and Hospi- , . , . tal, Cleveland. ROBERT W KREILICK PH D NORMAN W HEIMSTRA P l) ALPHONSE J. INGENITO, Pu.D. ' . , . . Professur of Chemisl~ University ol' . , n. . Professor of Psycholo gy;1)ircctor, Human Associate Professor ol Pharmacology, East Carolina University School of Medi- ATTAI-I-AH KAPPAS, M.D. Rochester, Rochester, IVY. Factors Laboratory, University of South cinc, Greenville, NC. Prolcssor and Senior Physician, The I)akota, Vennillion. Rockefeller University, New York. KLAUS E. KUETTNIiR, Pu.D. HARRY L. IOACHIM, M.D. Professor and Chairman. Department of PAl1LINl: HEI"LER, Pn.l). Attending Patholugist, Lenox Hill Hus i- SIMON KARPATKIN M.D. Biochemistry, Rush ('ollep,e of Hcallh Sci- Research Associate in Cytolof y and Cyto- tal; Clinical Professor ol' Pathology. C o- , Professor of Medicine. New York Univer- ences and Rush Medical College, Ruah- chemi+try, San Franriu:u Institute of t41ed- lumbia University College of Physictans fk sity Medical Center New York Presbyterian-St. Luke's Medical Center, ical Sciences, San Francisco. Surgeons, New York. , . Chicago. R I H C G IIRATCH KASPARIAN, M.D. ROBERT A KUHN M D A O J. ENRY, Prt.D. 1)i Lk f E i l O EORGE JACOBSON, M.I). P I Assistant I)irectur, ('ardiovascular Laho- . , . . As o t P i f I)i i i f rcctur, partmcnt u xper mcnta n- l Mi bi l i l A i I rofessor and Head, )epartmcnt of Radi- l U i i f S h l f ratory; Instruclor in Medicine, Hahne- s c a e ro essor, v s on o Ncuro- sur cr Ncw J St C ll f M co ogy. cro o og ca ssoc ates, nc., Bethesda, MI). o ogy, n vers ty o out ern Ca i ornia School of Medicine, Los Angeles. mann Medical College and Hospital, Phtl- adelphia. g y, cr.uy ate o ege o erl- icine, Jersey City. HERIiERT 1t. HERSC'OWfI"L, Pti.D. JERRY HART JACOBSON, M.I). STIG KULLANDIiR, M-1). Associate Profcssor ol' Microbiology, Director, Division ol'I:Icctrophysiulogy, Fl.lIlll KAT"L, Pn.D. Professur and C'hairntan, Department of (ieorgctown University Schools of Medi- New York Eye and I:ar Inlirrnary, NY. AsaK iate Prolcssur of Srx iology, Univer- Obstetrics and (;ynecology, University of cine and Dentistry, Washington, DC. sity of ('hicago. Lund, Lund, Sweden. JULIUS H. JACOBSON II, M.D. LAWREN('E L. HESTER, Jtt., M.D. Associate Professor of Surgery and Direc- SHIRLEY L. KAUFFMAN, M.D. LAWRENCE L. KUPPER, Pn.D. Professor and Chairman, Department of tor of Surgical Research. University of Proles.or of Pathology, State University Associate Professur of Biustatistics. Uni- Obstetrics and Gynecolol,y, Medical Col- Vermont College of Medicine, uf New York Downstate Medical Center, versity of North Carolina School of Public lege of South Carolina, Charleston. Burlington. Brooklyn, NY. Health, Chapel Hill. 184 185
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MARVIN KL1SC'FINI?K, M.I). New York llntvcr.ny MeJical ('enler, NCw York ('HARLIS W. LABlil.l.li, I'n.U. Assrslaut 1'rutcs.nr ol I:nvirunntental I ly- taenr, lcllcnnn Medical C'ollcge, Philn- drlphia. AARON J. LAUMAN, Pn.t). Prulrssnr and ('hairntan, Department crf Anatumy- T1tc llnivcrsity ul' New Mexico School uf Medicine, Albuquerque. THOMAS C. LAIPPLY, M.I). I'rolcsMrr ot' I'atholugy, Northwestern llnrvcrsity Medical SchrKrl, Chicago. AtiE1. LAJTHA. Pu.D. Uircclor, New York State Research Insti- une for Ncunxhemistry and Drug Atldic- Iion, New York. MI('IIALI. Li. LAMM. M.I). Professor of Pathulugy, New York Uni- vcrsily Mcdieal C'etner, New York. Ix')N LAPIiNAS- M.I). Assitilant I'rufcssur of Pathology, llnivcr- sity uf Vcrntont ('ollcge ot' Medicine, Burlington. PAUL S. LARSUN, 1'n.l). I laag t'rnlcssurof Pharntacology, Medical C'ollcgc ul' Virl;inia, RichmunJ. RUGHK K. LAKSON, M.1). ('hicf ut Medicine, Fresno County I lospi- lal, Fresno, C'A. (il1S'I'AVIi A. LAIIRIiN%I, M.I). ('hie(u( McJicinc, St. Vincent's Hospilal, Worrrarr, MA. JOSIiI'H M. LALIWI:KYNS, M.I)., I'u.l). 1'rolcssurr Ordinariu. anJ ('h:urman, Ih:- partment ul 1'atholuy.y and Microscopic Analumy, I'xltrrimcntal Lahuralury ol 1'ulrnunary I lrslultathulul;y, Kalhulickc linivcrsitcn tc Leuven, Lcuven, BclKiunt. li. ('LIN'fON LAWMI:NC'li, M.I). Asaiuanl Prolrssut of Medrrinc, Baylor College (il MrJicinc, Ilouaun. JAMI:S ('- LF.N, Pn.I). Assistant Prufcssur uI liakhcnnistry, Saint Luuis School of Medicine, St. Louis, MO. NI)WARD LFt:'fli, I'n I)., D.Sc. I'rules.nr ut ('henuslry, University of Minnesota, Minncalxrlk. 1'EIILIP M. LI•. (1111iSNF., 1'u I)., U.Sc•. I'ruleaMrr nf Chcmislry, Nut thcastcnt l hu- eerwny, liostun. IiICIIAKD A. L1?RNI•:R, M I). Assckiale Member, Scrrpps('Imlr and Re- scarch FuunJaliun, La Jolla, C'A. C'li('ll.f I.I:U('Ifl'I:NHIiKGIiK, 1'u 1). llcacl, Lkpartmcnt ul ('yluchcmistry, Swiss Institute litr I:xlwriutental ('anrcr Research, Lausannc, SwitterlanJ. JAY A. LEVY, M.I). Associate Prolcssor uf MeJirinr; Re- search Associate, Cancer Research Inst/- lute, University al' ('alifarnia Sncotd uf Medirine, San Francisco. PALII. I). LE:WIS, M.D. Seniur L.erturer in Histopathulut;y, Royal Postgraduate Medical School. Ilammcr- snrith Ilu.pital, LonJun, England. AVIi12IL1. A. LIEBOW, M.1). Chairmmn, lkpartntent af Patholugy, Yale University School af Merlicinc, New Ila- ven, Cf. IRVIN E. LIENER, 1'tl.l). Professor of Bioehcmistry, University al' Mtnnesuta, St. Paul. VALIiRIE K. LINIXJRIiN, 1'u l). (3uest Rescarthcr. National Cancer Inat- lute, BethcMJa, MI). GSTIiN O. LINUSIiTH, M.I)., I'n.l). St. Joseph's Ilospital Research Lahur:r lory, St. Paul, MN. ROBI?R'1' H. LINNELL, 1'u I). Associate Professurul ('hcnti+try, Univcr- sity of Verutont, Burlinglan. FABIAN J. 1.I()NI"1"1'I, I'n.l). 1'rofcasctr ol' liicx•hcmizlry, Ituston Uni- vcrsily School of Medicine, Boston. Illil{BliliT L. LOMIiARI), M.I)., M.I'.I1. Aftiliale, Cancer Research Institute, New England Deaconess Hospital, liuauu. J. P. LONG, Pu.D. I'rul'e+sur of I'harnutcnlogy. Stalr I htivcr- sity of Iowa C'ullcy,c of Mcdirrne, luwa City. GISINA L.. LONGE:NEC Kl{H Associate Prafcaxor (il' P'harntacoingy, University of South Alabama ('ullege uf McJicrne, Mobile. ('LAY'I'ON (i. LOUSI.I, M.1)., I'u.l). Ilasnnks 1'rulcs.ur ul htedictnc und Pu- tholul y, llnrvcrstty nt'Suudtcrn ('alilitrnta S.hunl ul' hh'Jicinl, Lu. Anl!clr.. IMtNALI) li. LOl1RIA- M I). Assuc'ialc Prulctivrr of Mcdicine, ('urncll University Medical ('ullegc, New York. Itl)NALI) Ii. L.111-TI(i, Prt.l). I'ruti'ssuranrl Ilcctd, I)cpartntcnt qf Mirru- hiolut;y and Inuntuoolugy, t.SU Medical ('cnlcr, New Orleans, I.A. KIiNNI•°I'II MIiRR11.L LYNCH, M.1)., Sc•.)., L.L..1). Prulcssitr lancritu% of Palholnv and C'hanrcllnr, Medical College ul South Carolinn, ('harlratun. 13KU('G A. MA('HIiR, Pu.l). Assistant 1'rulcs>,x of Phannacculical ('hcntistry, University of California, San Franciu:o. IiUWAR1) S. MAKIiR, M.I). Associate I'rofessor al Ncurolo~y, Muunt Sinai School of McJicinc, New Yurk. INI:S MANI)I., Pn.l). Assislant Professor of Iiicx•hcmistry, Cu- Iuntbia University Cullcgc uf Phyairians & Surtieuus. New York. JO11N II. MANHOLD, Jn., U.M.I). I'rulissor aud Uircclor. I k Itartmcnl uf Pa- thulugy and Oral I)iagnosrs, New Jcrxcy ('ullrge o( Medicine ancl Iknli.lry, Jersey City. I)AVII) li. MANN. L'n.U. Associate 1'rolcssur of Pharmacaluy,y, 'I'ernple llniversity Schuol of 1'harmacy, Philadelphia. FRANK AR'1'1I1/R MANNING, M.1). Assrstanl Professor ot' Obstetrics and Oynecuto gy, Wonten's Hnsltital, Los An(clcs ('ounty/Univcrsity of Southern ('ahlirrnia Mcdreal C'entcr, Los Angeles. JOIIN 1'. MANOS, M.I). Instructor in Virology and Bacteriology, Medical C'ollcge uf Suuth C'aruhna, ('hac Icslnn. RI('l1ARl) A. MARKHAM, M.1). Assistant Prudbssurr ol' Mcdicrnc and of Mi- crubiology and Immunolu gy, The Jewish Hospital ul' St. Louis, St. Louis, MO. C'IiRIS"I'OI'lI1iH M. MAI{'I'IN, M.D. Assistant PruGess+tr ul' Medicine and I)t- rcclur, 1)ivisiun uf (ctli•rtnnu. i)IwrusCS, Srlan Ilall C'ullegc ol Medicine. Jersey (.'ity, NJ. Lt. RLISSI:LI. MARTIN. M.I). I'ntfcssur of MeJtttne, and Mirruhrulu ~y anJ Iutnwnolugy, Iiuylur('olleEeof Mc~i- rotc. Iluuslun. RIiGINAI.I) (i. MASON. M.1)., PIt.U. 1'rulrsuor anJ ('hairntan. I)cPartnront of Padruluy,y, Univcni(y ol' Utah College ol MeJictne, Salt Lake ('ity. MASON RESFAR('II INS'I'I'I'U'fls Wurresler, MA. IX)NAL.D J. MASSARO, M.1). Associate Prufessur al' Meclicinc, Genr~.~e Washinl;tun Univcrsity School of Medt- cinc, Washtngton. I).C. CLIARLEiS McARTL1UR. I'n.l). P+yclwlugisl, University Ilcalth Services. Harvard University, ('ambrirlge, MA. CHAI(LIiS WCANTS, Pn. U. Associate 1'rufesutr uf Suils, North ('aro- lina State College School of Agriculture, Raleigh. (;EKALI) L?. M(Y'LL:ARN, Ptt.U. hirertor, Instilulc for Behavioral Gene1- ic., Ihpartnrenl ul' I'sychulogy, Univer- aity ol' C'olorado School of Pharruary, Buuldcr. ALAN ( '. Mc•LAUGIII.IN, I'n-D. Lrrlurer in BicK:hrntistry and liiuphyaies, llnivcraity ul- Pennsylvania Schc>yd of Medicine, Philadelphia. HENRY ('. Mc'(iIl.l., Jlr., M.1). Acunl: I IeaJ. I)cparlnrent of 1'atholugy, Louisiana State University School of Med- icine, New Orlcans. HI:NRY 1). McIN'fOSII, M.1). Professor of McJicinc and I)irectur, C'ar- Jiavascular LaLruralury, I)uke University Medical C'cnter, Uurham, N('. FORI)L: A. McIVER, M.I). AsuK iate Prufeasor uf Pathlrlol;y, Mcrl iral College of South Carohna, C'harleslon. EI)WAKI) Mc•KEIi, M.I). Profcsurr anrl Acting ('hairman, Ihpart- ment of Palholo y, McJiral Cnllcgc of South Cantlina, ~harlcalun. 186 187
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KELLY T. MCKEE, M.D. Associate Professor of Medici:ne, Medical C'ollege of South Carolina, Charleston. HNRBERT McKENNIS, Jk., PN.D. Rescarch Professor of PatholoPy, Univer- sity of Miami School of Medicine, Miami, kL. VI("1'OR A. McKUSI('K, M.D. Professor of IMedicine, The Johns Hopkins llnrversity School of Medicine, Balti- more, MD. ROSS L. McI.EAN, M.D. Associate Professor ol' Medicine, Emory University School of Medicine, Atlanta, GA. WIL.LIAM F. Mc•NARY, itt., Ptt.D. Associate Professor of Anatomy. Boston University School ol' Medicine, Boston. NI:AI. McNIVEN, Ptt.D. The Worcester Foundation lirr lixperimen- tal Biology, Shewsbury, MA. HANS M1:11iR, I).V.M. Senior Staff Scienust, 'I'hc Jackson Labo- ratory. Bar Harbor, ME. I. WISTIiR MIiIGS, M.D. ('linical Professor of 1?pideminingy; I)i- rector, ('onnecticut Cancer Epidemiology Unit, Yale University School of Medicine, New Havcn, ('1'. JI.11.IA MEYER, Pn.D. Associate Professor of Oral Pathology, University of Illinois ('oI lege ol' 1)erttistry, Chicago. DOV MICHAELI, Ptt.l). Assistant Profesurc of Biochemistry and Surgery, University of Cahlornia School of Medicine, San Hrancisco. MIC'ROBIOL(IGICAI-ASSOCIATES, INC., Bethcsda, MD. BERNARI) J. MII.LIiR, M.D. Assistant 1'rofetisnr uf Anmomy, Jefferson Medical College, Philadelphia. JAMES (l. MILI.ER, M.1)., Pn.L). Profc.ux of Psychiatry and I'sychology; Director. Menial Hcallh Research htstitute, University ul' Michigan, Ann Arbor. 1P.RALD. A. MI'I'CHELL, I'tt.D. Associate Professor ol Anatomy. Wayne State (Jniver.uy Schood of Medicine, De- tran, MI. STELLA MITRANI-ROSENBAUM, Pu.D. Pruf'essor of Virolugy, Hcbrcw Universi(y-Hadassah Medical Schoul, Je- ruxalem, Israel. CHARLES MITTMAN, M.D. . Director, Department of Respiratory Dis- eases, City of Hope National Medical Cen- ter, Duane, CA. HUGH MONTGOMERY, M.D. Associate Professor of Medicinc, Univer- sity of Pennsylvania School of Medicine, Philadelphia. P. O'B, MONTGOMERY, Ju., M.D. Professor of Pathology, University of Texas Southwest Medical School, Dallas. GEORGE E. MOORE, M.I)., Ptt.D. Director, Roswell Park Memorial In,ti- tute, Buffalo, NY. KENNETH M. MOSER, M.D. Assistant Professor of Medicine. George- town University Medical School, Wash- ington, DC. IILIRLEY LEE MOTI.I:Y, M.D. Professor of Medicine and Director, Card io- Respiratory Laboratory, lhtiver- sity of Southern California School of Med- icine, Los Angeles. EDMOND ANTHONY MURPHY, M.D., Sc.D. Associate Professor of Biustatistics and Medicine, The Johns Hupkins Univcrsity School of Medicine, lialumore, Mf). WILLIAM S. MURRAY, Sr.D. Senior Staff Scientist, The Jackson lahu- ratory, Bar Harbor, ME. JAY A. NADEL, M.D. Professor of Medicine, Physiology and Radiolugy, Cardiovascular Research Insti- tute, University of Calil'ornia. San hrun- cisco. RICHARD I.. NAEYE, M.D. Professor and Chairman, Department of Pathology, Pennsylvania State University College of Medicine, I lcrshcy. GEORG B. NEURATH. Pn.l). Micruanalytical Laboratory, Hamtburg, West Germany. HAROLI) H. NIiWBALL, M.D. Associate Professor of Medicinc, The Johns Hopkins University Schowfof Mcdr cine, Baltimore, MD. AI.HIsRT H. NIDEN, M.D. Professor uf Medicine. l)rc vv Poslgraduate Medical School and University al"South- ern California; ('hief, Pulmonary l)iscase Scctiun, Martin Luther King Hospital, Ln Angeles. STIJ:AN NIIiW1AROWSKI, M.l)., Ptt.D. Prodeasur of 1'hyswlugy, Thrombosis Re- search Center. Temple University School of Medicine, I'hdadclphia. OAK RIDGIi NATIONAL LABORATORY Oak Ridge, TN. FRANZ OI:S('H, Pu.1). Proteswrof Pharmacology; Head, Section on Itirxhctnical Pharmacol%y, Llniver- sity of Mainz, Mainz, West Germany. F. WILL.IAM ORR, M.D. Associate Prafcssor of Palhology Univer- sity of Manitoba, Winnipeg, Manitoba, Canada. Ix)NAL1) M. PACE, Ptt.D. I'rofessor of Physiology and I)irector, In- stitute IorCellul•.rr Research, University of Nebraska, l.incoln. KNNN1:1'I I PAIGEN. Ptt.D. I)trector, I)cpanment of Molecular Biol- o}ty, Health Research, Inc., Roswell Park 1)ivision, Buffalo, NY. ALBI:R'I' l3. PALMER, Pu.D., Assistant Professur of Pathology, Univcr- sity uf Toledo, Tuledo, OH.ROSE MARIE PANGBORN, M.S. Assistant Ftxxl Technologist and Lecturer, Ik'Irannant of Food Science and'1'echnology, University of C'alifornia, Ilavis. JOIiN W. PARKIiR, M.D. Associate Profcssor of Pathoingy, University of Southern California School of Medicine, Lus Angeles. MARY STEARNS PARSHLI:Y, Pu.D. Assistant Professor of Anatomy in Obstetrics and Gynecolol;y, ('olumbia Univer+ny Col- legc of Physictans & Surgeons, New York. BIiNDIC'H'1' U. 1'AUI.I, D.V.M. Associate Professor of Pathology, Rush- Presbyterian-St. Luke's Medical Center, Chi- cago. EDWARD W, PELIKAN, M.D. Chairman, Department of Pharmacology and Experimental Therapeutics, Boston University School of Merlicine, Boston. BORIS M. PETERLIN, M.D. Assistant Professor of Medicine, Section ol' Rhcumatolo y-Clinical Immunology, University of California School of Medi- cine, San hrancisco. DENNIS R. Pk:"PERSEN, Ptt.D. Professor of Pharmacology, University of Colorado School of Pharmacy, Boulder. CARL W. PIERCE, M.D., Pu.D. PrnletiMlr, Departments of Pathology and Microbiology-Immunology, Washington University School aI Medicine, Director, Patholo and Laboratory Medicine, '1'he lewish Hospital of St. Louis, St. Louis, MO. MALCOLM C. PIKE, Prt.D. Professor of Commumty Medicine and Pe- diatrics, University of Southern Calif'ornia School of Medicine, Los Angeles. OTAKAR J. POLLAK, M.D., Ptt.D. Executive Director, I)over Medical Re- search Center, Inc., Dover, DE. MORRIS POLI.ARI), Pu.D. Director, Lobund LaMoratory, University of Notre Dame, Notre Dame, IN. C. M. POMERAT, Pu.D. Director of Biological Research, Pasadena Foundation for Medical Research, Pasa- dena, CA. S. N. PRADHAN, M.I)., 1'tt.D. Professor af Pharmacoluby, Howard Uni- versity College of Medictne, Washington, DC. H. R. PRATT-THOMAS, M.D. Professor of Pathology and Dean, Medical College of South Carohna, Charleston. I'ROCESS & INSTRUMIiNI'S CORPORA- TION Brooklyn, NY. MARTIN S. PRO'F7.El., D.U.S. Chief, Department of Oral Pathology, Newark City Hospital, Newark, NJ, WILI.IAM A. PRYOR, Pu.D. Boyd Professor of ('hemistry, Louisiana State University, Baton Rouge. 188 189
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MI('IIAfa. S RAIISON, M I). Rcuarch Scicnttsl, Lwisluutury of Palhnl- l I I) Nutirnral ('anccr InrlMutc, Itethesda, RONAI,1> 1!. RASMUSSEN, Pn.D. Aas,KtaleAdjunct Prrnlcsor, lkpantncntx of ('umamunity anJ I:nwronmcntul MuJi- cine. University ol' C'alilirrnia ColJcgc of Medicine, Irvrnuc, CA. WALTER RIiDIS('ll, M.D. Associate Pnnlessur uf ('linical Medicine, New York University SchlKrl of McJicine, und NYU Kescarch Service, Coldwater Memuriul Hospihtl, New York. TIMQ'I'lIY J RliOAN, M.D. I'iulessor ot Medtcnre; I)irector, Division ul ('ardtova4cuLu Utacaacti. ('o16cEe of Mcdictnc und I)enliitry of New Jer%cy, New Jersey Mclhcal School, Newark. WII.LIAM RI;(;liI.SQN, M.1). I'rulesurr ,ind ('hairntun, Ikpannrcnt of Medical (/nculogy, Medical College of Virginia. Rirhnrund. L.YNNI'i M. RGI)- M.I). W(Ilba.h I'rofcseoruf Puthology, Harvard Medical Selwud, Bustnn; ('hairman, Ik- Ir:uluu-nt of 1'atludu.y, ('Itddran's Ilospi- tat Medical ('clncr, luslrm. IIIANAR'I' A. REIMANN. M,D. 1'rol'csurr ul' Medicinc, I lahucntann Mcdi- Cal College atnl IIUspI1al, Philadelphia. IiII.1:IdN RIdMOI,ILO'IX)NNlil.l., Ptl.b. Pnnttpnl Rcscarch Asslwiute, Ilarvard h1cdtral Schlwrl; Invcstigatur, Center li>r INond kesearch, Ituston. JOlIN N. RP.PINI?, M.I). Asstatant I)ireclor, Wchh-Wurina Lung In+utute; AxslKiale 1'rulessor ul Medr cine, l)nivcrsity uf Colurado Health Sci- cnces C'cntcr, Ihnvcr. ROIiIiRT Rl?:SNI('K, M.D. AsuKtate Pru(esoruf Kepnxluctive Mcd- icmc, Univeraity uf ('alifurnia Medical Center, San 1)icgo, I II?RIIIiRT Y. RLYNOI.f)S, M.I). 1'rulcstitr of Mcdicine; Hcad, Pulmonary Srctiun, Yale llniversuy School ut Medi- crue, New Ilaven, (`I'. ROLLANn ('. RIsYNQI-I)S, M.D. Assistant I'rolessut of 1'athnlogy, (lniver- any ol '1'exas Suuthwestern Medical Sclilwt, 1)allas. VI("I'OR RI('IIARI)S, M.l). ' J()IIN R RUW'LANI)S I'1t 11 MAl1R1CI:S, SEGAL, M.1). hiel of Surkery, Ptcshyteriun Medical C ' . , . . Staff Scientisl Southwest Research I'nztl- Clinical Professor ltI' Mellicine, TufIS lJnr C eulcr, San hranrtsco. , hne, San Antonio, TX. veraity Scluail of Medicinc; I)irector, lk:- panntent of Inhalatiun 1'herapy, Ronrm VIRGINIA L. RI('IIMOND, Pn.U. BENJAMIN A. RURIN, Ptt.D. City Huspital, 13onton. kesearch Ass.>Liute, 1'acilic Nurthwest Assistant Professuruf Puhlic Health,l3•ry- Research fuundution, Seattle, WA. lur University College of Medicine, Houx- CAR!- C. SELTZIiR, Pt1. D. tun. Hunorary Research Associate. Peabody ARNIS RICHTIiRS, I'u.U. I Museum, Harvard Univeniry, C'ant- Asuxiate Professor ut' I'athningy, Univcr- RUNAI-I) P. RUI31N, Pn.D. hridgc, MA. sityofSouthcrn Cahfurnia School uI Med- Professor of Pharmacology, Medical Cnl- icinc, Llrs Angeles, Icge of Virginia, Richntond- LUCIO SIiVHRI, M.D. WILI IS H Pu D RIESEN D Ht:NRY 1 RUSSfiK M D'ircctor and Dean, Institule of Anatnmy . . . . , Senior liu><hemist ifc Scien I ;,es I)ivi . . , . The Russck Nundation Inc Presidenl und Pathology. 1)ivisiun of Cancer Re- , , , - rion II'1' Research Institute Chica o , ., , NY Staten Island search, University of Perugia, Perugia, It- , , g . , , aly. DANIEL ft RIPKIN Pu h W. C. R(JSSl31,1., M.D. . , . . Assislant Prntessor of ('hcuoirad I!liullul;y tlntversuy of '1'exas Medical C'enter, CIIARI-IiS R. SIIAW, M.I). ' , '/)oe Rlkketeller llntversit New Yulk I luustun. hoel, Section uf Medicul Genetics, M.1). ( ' ' y, . untur Insbtule; Andersun Ilnspital amd 1 R ll RICIM)N M I) WAYNIi I-. RYAN, Pu.l). 1'ru/essor ol' Kiuingy, 'I he University of . , . . I'rufcaaur of 1'athldugy University of I'rulesux of IJia:henus,ry, University of TCxas at Hnustnn. , Texas Medical Branch (ialvestun Nebraska College of Medicine. Urttaha. , . CHARLLS E. S1IIiKWOOD, M.D. SYI)NNY (' RI7TFNIIIiRG Iht D I'li1'IJ( F. SAI-ISISIIRY, M.D.. Pn.D. Assistanl Prufessoruf Radiulul;y, Univer- . , . . Professor of l'3acleriology, University of Head, Intensive Trcutntcnt Center, Saint h Hos ital Hurhank CA Jose sity of Rochcstcr Schad of Medicine and NY. Ihnlistry Rochester Southern C•rlilitrnia, Los Angeles, p , , . p , , ' 1'Altt- SALTMAN, Pu.l). Pu l) SIIOJI SHIIIATA M D faJQ1:NH ROBIiRI S, I'tLD. ' Assistant Prufcssur of Biochemistry and . , , . ., lJniversity of 1'rafcsurr of Pharmaculotiy Research Scictnrst, ( uy of Iiolx Rcscarch ' Nutritiun, lJnivenity uI Southern C'alifur- , Ilunululu Iiawaii Schaol uf Medicine In.,litute, 1>uartc, C A. nia School of Medicine, I.us Angeles, . , ISt:NSAN B. RQC, M.1). 1SRAHMI P Ptt D. SANI GHRAI-1) SHKLAR, D.D.S. Associate 1'ratessur of Surgery; Chief . . . Southern Re- Protein lihx:hemistry Ilead Charles A. firackctt Professor of Oral Pa- , Cardiac Surgcry, tJniversity nf C'alifornia , , AL, Himtingham sl:arch Institute thology; Head, Deparunent of Oral Medi- Schoul of Medtcine, San Pranciscu, , . cine and Oral Pathulugy. Ilarvard School Ul-RICH If. SCHAF:PI'I, M.1). of Dental Medicine, lSoston. JOSHPII H. RO(;I?RS, M D. Uirector of Neurupharmacolu~;y, M•rson I l y Narne of Jesus Iluspital, (;ade.dcn Reuarch Institute, Worcester, MA. DAVID L. SIMON. M.I). A ~l , Inaructur in Internal Medicine, Cincinnati JORGIiN U. SCHI-G(iI31., M.I)., Pn.D. General Hospital. Cincinnati, 011, RUI31iRT C. ROSAN, M.1>. Profes.+rr and C'huirman, Ikepartntent of Associate Prulessor of Patltolnl;y and 1'c- Surgery, Tulanc University School of ERIK SKINHNJ, M.l). dtatrics, St. Louis Ilmvcr.nY Schuol of Medlcine, New Orleans, LA. Chief. Ikpartmenl of Neurology. Hispeb- Medicine; Associate I'athologrst, l'tudinal jerg Hoapnal, Copenhagen, Denmurk. Glcnnlro Memunul Hospital fur ('hildren. ALVIN R. SCHMII)T, Ptt.D. St. t.rruis, MU. Director of ('uunseling, Tufts University, ' ROISGRT J. SKLARHW Pn.D. Medl ord, MA. , Kesearch Associate PruIL~sWrr of I'athol- CHARLES L. ROSE. New York University Reseurch Ser- o ty Clinic I)irector; I)irector, Nurmativc Ag- JAKOIS SCIIMID'f, M.I)., Ptt.D. l , vtce, Goldwater Memurtal I luspital, inl; Study. Veterans Admirtutrutinn Out- Assistant Prolessuruf liirs:heuristry , Divi- Roosevelt Island, NY. patrent C linic, liustun. tilnll nt Iln1ln ~ICaI SctelrceS, Stute Univer- ~ urk at Stony Urook, Stony sity u/' New JOIIN A. ROSGCRANS, I'u.l). Itnatk. NATHAN 11. SLOANE, I'tt.l). I'rufcssnrol'Ituxhctmslry,'1'hc (Iniveraity Associate I'rulessur of Pharnulcolugy, D.Sc. 1).S. Pn.D. 1) ISAAC SC'IIOUK of l'cnnesr.ce ('enter (ur thc 1lcaltlr Sci- Medical College of Vtrginia, Vugmta ' , , , . University of lllinois College of Ikan ences, Memphis. C omnurnwealth University, R ichmond. , Ikntistry, Chicago. Ptt.l). HAN(>CH SLOR PIdT1iR M. RoSS, Pn.D. S('KII'PS C'LINIC AND R4S8ARCI1 , Asslxiatc Professor of Iluman Genetics, Research Assuctatc, The Rot'kcfellcr tJnr FOt1N1)ATION Sackler School of Medicine, Tel Aviv versity, New Yurk. I-a Julla, CA. University, Tel Aviv, Israel, 190 191
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Tllli()I)()Ntli A. SI.O•rKIN, Pt1.1). AsiittanJ 1'ruleaanr u4' I'hnnnactrlogy, I)ukc University Medical C'entcr, Uur- hatu, NC. (iIiURGI: W. SMI'sl-I'IiRS• M.1). AsuKtatc in Pathology, Northwestern ltmveraily Medical Schtxrl, C'lnicago. l)IiNNIS M. SMI'1'll, Pu.D. A%hislant Prufca+ar nf 1liulugIcal Sct- em•es, wefle,ley CnUege. WCIIeaICy, MA. FRI?I)I:RIC'K J. STARIs, M.U. 1'rutesauruf Nutriuun, Ilarvartl llnivertiity School of Public Ilaalllt, tiumun. NORMAN C. S'I'AUB, M.I). 1'rulc+aor of Phyaiolqy, C'arJinvavrul•rr Research Institute, University of ('altlur- nia, San Franciacu. G6iO'RC;H W. SMfa'TIiRS, M.I). Associate in Pathology, Nurthwe.,,tern University Medical School, Chicagu. JANIiT'I'RAVI:I.I., M.D. Asn+kialc Prufessnr ot Clinical Pharma- culugy ('nroell Universny Medical CoL Icy.e. Ncw York. JAMIiS'I'RAVIS, Pn.D. I+rulcxurt ot Itnx•hemtstry, The University ol Georgia. Alh.tn.. Llli SIIA'I:SAI, Pn.l). Re.earch Associate in Pathnln gy, Yale Uuivcr.ny School af Medicine, New Ha- ven, C I'. lI1KiNll'sW WAI,AS%IiK, P'tLD. Research Scientist. Ohru State University. C'olumhus. 1dV14l.YN WAL.DS'I'EIN, I'o.D. Senior Lccturer, 1)cpartment of Itiochcnt- i+try, Tel Aviv University. '1'el Aviv, Ia- ntc'. IRENG Y. WANG, Ptt.U. Anniatant Prufc>,urr ul Basic and Clinical Imnrunology and Microhinlugy Medical University (il South Caralina, Charleston. GlsNli M. SMI'rll, Pu.U. 1ACK P. STRONG, M.D. PITGR A. WARI)• M.I). Aa+istant I'rtrtasyur of Psychology, liar- Associate 1'rufessur a/ I'athnluHy Iertrisi- (iIiRALI) M 'I'tIRINO M D Profcshor and C'harrman, lkpartnrcnt of vaid Medical St•h+Ntl Maa sacltuullx(i ana State llnrvcratty School of Medicine, . , . . ' ' Pathnlugy The University of Michigan , , en- rnd I Iuspital, I luslun New Orlcana. I rules+ur uf Mcrltcune, ( ulumhia llnivcr- uly ('nllelre of I'hyatiinna & SarEcunn„ , , Ann Arhur. New Yoik. Ll l('I l.li SM f l'I I, MARION H. S1/1.7.1dlik(iliR, M.1), E. I). WARNER, M.I). I'rulc.%na of Ititachcmislr 1)arttnauth Prulct.srrr and ('hatrman, Ikpartmcm ud ' Prulcxanr of Pathulu~,y. State University y Medical Schtxrl Hanover NH Iknnatology and Syphdulugy, Ncw•Yurk rl IRNI:R, Pu. U. I). M. of Iowa College of Mctlicine, Iuwa City. , , . University-Uellcvue Mledical ('enter NY. IIeaJ, 1)eparunent of I)rug Metabolism , anJ ISita:henristry, Huiletnn Lahuratnries DS WARREN M D SHII?I LOUIS A. SOLOI7F, M.I). ' linrote , Ltd., Ilarrol:ate, Yorkshire. Hn- , . . . Directarof LaMrratnriea Cancer Research 8lanche P. Lcvy 1)tstinEuiaheJ Service ' ' RI:NATOTA( rIURI, Prt.D. glanil. , Institute New En land Deaconess Hrupi- 1'rol e,aur; 1'rofes,ur ul Medicine; Drrcc- Associate Pmfcasnr of Psychology, (irad- g , tal Boston lor, Research Lipid Lahoratory Temple uute School of Business AJrninratruttun . , , University I Icalth Sciences ('enter Phila- , llarvurd University Hoaon UNION ('ARtiIUG C'ORPORATION , dclPhia. , . Nuclear Division. Oak RiJge,'fN. YASUSIII WATANABE, Pn.D. Associate Memher, The Wistar Institute of DAVID W. TALMAGI:, M.D- Anatomy and Biology Philadelphia. SIIIiLIX)N C. SOMMERS, M.D. Direcmr, Webb-Waring l.ung Institute. UNIVERSITY OF SAN FRANCISCO . Uirectur nl' Lahuraturicx, Lennx 11i11 Hux- University of Colorado Medical Centar, WATSON Pu D BARBARA K pitaL' Clinicul 1'rufcsaor uf 1'atholol;y, Co- Denver. UNIVERSITY OF SUl1TLIGRN C'ALIFOR- , . . . Assistant Bactcriolul;ist Massachusetts lumhla l)mvcrsity ('ollcge of Phy+rctanx & NIA , General Hospitul; Research Associate in SurPeona, New York, LYNN M. TAUSSIG, M.D. Los Angeles. Ractcriolugy and Inuuunology, I larvard Associate Professor and Axstxiate Chair- Medical School, Boston. l4RNliS1' SONbHI:IMLiR, Plt.l). ' ' mun, DaePanment of Pediatrics, Arieuna IIAROI,D H. VARMUS, M.D. Prolcs or ol' Microhiolo and Im,nunul- Associate Prolessnr ul Ilitn•hemiptry, C nl- ' I leahh Sciences Center 'I'ucsun a gy ' LEE W. WAT'rHNBIiRG, M.1). lel;e itl Fure+try, State Univcraity of New , . alif`wnia, San Fran- olty, University of ( I'rol'csurr at Pathoingy University of Yurk, Syracuse. clun. , Minnesotu Merlicul School Minneapolia MARC D. THAMIiS, M.D. . , Senior Research Felluw Ma o ('hnic and VA'fN1iR M S'rIsPI11SN F I) T M. SONNIiIIORN, Pn.l). , y FounJatiun Rrx•hestcr MN , . . . I larvarrl uk•iate I'rufcsMU of MeJicine A. JOIIN S. WAUGII, 1'u.l). IA.ImguishcJ Service Professor of?.twl- , , . . . Mediccd Srhekd New England Regional Prufessoruf ('hcntialry, Massachusetts In- ' ugy, Indiana LJniveraity, lihnnnington. ' ' , Snuthtroro Prunate Research ('cnter +titutc ol Tcchnulugy, Canrhrirlge. SAM SOROF, Pn.D. CAROLINE t11:DIiLI. I IIOMAS, M.D. Professor Bmerilus (it' Medicine, '1'he , , MA.; Associate in MeJirine, Peter Bent Huatun (Srigham I (o+Pital TLIOMAS E. WRIHi, Pn.f). I lead. I)wPurtntent of Mucramnleculur Jnhns Hopki.ns University School ot McJi- , . Prufcst,or of I'hysiulo ~~icnl Chemistry, ('henri+try, 1'lu: Institute liv Cancer Re- cinc, Haltimore, MI). lil.l .1( )'1' S. V IiSEI.I. M.I). Ohiu State Llnivcrstty (:ullege of MeJi- uarch, 1'liilatlclphia. , I'rutc,.a+rr und ('hairntan, Ihfwrtntent ol' cine, C'olutnhun. JEROME F. 'rl IOMAS, Pn.l). I'harmaculnlty, Pennsylvania Slutc Uni- SOl1Tl 1 W IiST R IiS I:A RC'I I I N STITUTF. 1'rofesatrr of Sanitary I:nl;meering, Uni- vcraity C'ulleFc ol' Medicine, Mthon S. RICIIARU L. WL4C'IfSLIiR, M.I). San Antnnru,'I'X. versity ol' C'alilurntu, Berkeley. I lcrshcy Medical ('cntcr, I lernhcy. C'Itnical 1'hyaiulugi.t, Montchnrc Hospital ' Inatitutc ul Reu:arch, Pittshurgh. I)AVII) M SI'AIN M I) JAMES G. P. TOMAN Pu l) RRANISLAV VII)IC`, D.S. . , . - I)irectur, I)cpartmcnt of Pathulogy The , . . Prufeakrc unJ C'hairman 1)+parunent ol' I'rnletis,u of Analuury, Georgetown tlnl- J()IIN V. Wlill.. M 1). ' ' , Ilnmkdale lirapital ('entcr Brooklyn , Pharmacology, C'hicago Medical School vcroi(y Schtnrl+ol'MeJicinc and Lkhntintry, Assistant Prol es+or ol McJicine, l)nivcr- , , NY. , Institute for Medical Research, Chicago. Washington. I).C. sfty of Coluradu Medical Center. I)cnver. ROMGO A VII)QNIi I) M GEORGE WIiINBALIM• Pu.D. AL1?XANI)IiR SPOCK- M.1). ANI)RBW M. TOMETSKO. Pn.l). , . . . A+ukiatc I'rufcaurr of Patholo~y Yale Assistant Chatrman fur Research, Ikpan- Atisistant I'rulc.urr ut 14:rliatrics, /)uke President and Directnr ul' Reu:arch, l.nron , New Ha- School of Mcdit:ine Universit 'flu Graduate Iluspn•rl ment of Medicine llniversny Medical ('enter, Uurham, NC. 1.ahorahuics, LIJ., Rochester, NY. • y vcn, Cf. , , I'hilaJelphia. 192 193
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1. IiI:RNARD W I:INSTtiIN, M.D. Prutessor of Medicine and linvironmental Sciences, Columhia University, College of Physicians & Surgeons, New York. A. WI:INST(K'K, Pu.D Research Buxhemist, Life Scicncex Divi- sion, IIT Research Institute, C'hicago, IL. SAMUIa. B. WEISS, Pu.p. 1'rolessnr of Biochemistry and Micrubiol- ogy, The University of C hicago. SIGMUND A. WEITZMAN, M.D. A.aHanl Professor of Medicine, HematnlogY-Oncolugy Unit, Massachu- sctts Gencral Hospital. lioston. RUSSELL W. WELLER, M.D. Pathologist, Memorial Hospital ol'Chester ('uunty, West Chester, PA. A. STANLEY WELTMAN, Pu.D. Associate Professor of Pharmacology and Research, Brooklyn ('ullcge of Pharmacy, Brooklyn, NY. SIMON H. WENDER, Pu.l). Research Ptutcssur ul' Uiochemistry, Uni- versity ot' Oklahoma, Norman. DUANEG. WI?NZE1., Pn.D. I'sulcssur and Chatrtnan, Ihpanntenl ot' I'hartnacology and 7'oxiculugy, The Uni- versity of Kansas School of Pharmacy, La- wrcnce. THOMAS ('. WtiSTFAI.L., Pti.D. Prufcssor of Pharmacology, University of Virginia School of Medicine, Charlottes- ville. FREDERICK E. WIIISKIN, M.D.C'.M. I)ircctor, Division uf Hcrlth and Pcrsonal- ity 1?yuilibrium, The Age Center ul' New I?ngland, Inc., Boston. JAMES A. WILL, D.V.M., Pti.D. Prufessor and C'hairman, Department of Veterinary Science, Universityuf Wiscon- sin, Madison. ROGER J. WILLIAMS, M.D. Prolessur of ('hemixlry; 1)ireclur, Clayton foun+' ,:, ,i Biochemical Institule, The . t,ny of Texas, Austin. JOIIN'1'. WILSON, Pu.l). Assistant Prol"c.sor ol Cell and Molecular 13iulogy, Medical College ul'Georgia, Au- gusta. ALVIN L. WINTERS, Pu.D. Assistant Professor of Micrubiolugy and Biochemistry, The University of Ala- hama, University. 194 I)ANIL•a. H. WISEMAN, M.D. Assistant Professor (if Pediatric:,, Univer- sily of Southern Cabfomia; Children's Di- vision, LA)s Angeles Cuunty General Hos- pital, Los Angeles. BRUCE A. WODA, M.I). Associate Professor of Pathology, Univer- sity of Massachusetts, Worcester. GEORGG WOLF, D.Puu.. Professor o(Physiological Chemistry. Dc- U mcnl of Nutrition and Food Science, assaehuselts Institute of Technology, Cambridge. J. EDWIN WOOD, M.D. Instructor in Medicine, Boston University School of Medicine, Boston. SUMNER WOOD, JR., M.D. Assistant Professor of Pathoingy, The Johns Hopkins University School uf Medi- cine, Baltimore, MI). JOHN P. WYATf, M.D. Professor of Pathology, St. Louis Univer- sity School of Medicme, St. Louis, MO. KOJI YOSHINAGA, Pit.D. ('enter for Population Research, NICIII), National Institutes of Health, liethesda, M 1). INDEX OF PRINCIPAL INVESTIGATORS Abeles, R. H., 91 Adamson, 1. Y. R., 7, 31, 32 Adelntan, B., 47 Adler, K. B., 33 Antoniadcs, H. N., 8, 9, 47 Aviram, I., 92 Barany, M.; 92, 93 Barbieri, R. L., 10 Bennett, J. S., 4t{ Bing, R. J., 33, 48-53 Buonassisi, V., 94, 95 Burch, J. W., 53 Busbee. D. L., 96 Carson, D. A., 96 Chcn, Y. T., 97 Chilian, W. M., 54 Civin, C. 1., 97, 142, 143 Clawson, G. A., 1(t, 98, 99 Cochrane, C. G., 55, 100 Conhaim, R. L., 34 Corley, R. B., 143 Cramer, E. B., 1(11 Creutz, C. E., 102 Ct.apski, G., 103 Damjanov, 1., 103, 104 Dorf, M. L^:., 144, 145 Duesberg, P. H., 11, 12 Eisenstein, R., 55 Feinstein, A. R., 12 Fontana, J. A., 13, 14 Foster, J. A., 104, 105 Fox, R. B., 34 Fridovich, I., 1O6-113 Galdston, M., 114 Gauhatz, J. W., 115 Gauldie, J., 35, 73 Gee, J. B. L., 36, 37 Gershon, M. D., 74 Giam, C. Z., 14 Gold, W. M., 15, 37-40, 116 Goldberg, A. L., 116, 117 Greenberg, C. S., 56-58, 118 Karin, M., 15 Kohler, H., 146 Koskenvuo, M., 159-163 Kurnick, J. T., 147-148 L.ajtha, A., 85 Lieber, M. R., 119, 149 Livneh, Z., 120 Lloyd, R. V., 75 Lxker, J., 16, 17 Lundberg, J. M., 41, 42, 58-61, 76-84, 15t1 L.ynch, H. T., 17-19 Macr:ioni, R. B., 19, 12'0-122 Meyer, E. F., 123 Microbiological Associates, 41 Mitrani-Ro.u:nbaum, S., 23 Moscatelli, D., 61-63, 123 Murttd, F., 63-64, 124 Murlas, C. G., 43 Nahm, M. H., 150-152 Naylor, S. L., 20 Nelson, I). J., 124 Niewiarowski, S., 65 Oh, S. K., 153 Oliver, J. M., 125, 154 Orr, F. W., 21 Osbakken, M., 66 Paigen, B., 67-68 Pettijuhn, D. E., 22 Pnlak, J. M., 85, 126 Pryor, W. A., 127 Rahamimoff, R., 86 Rice, W. R., 44, 87, 127 Ross, P. M.. 128 Rubin, H., 24 Ryan, U. S., 69 Sancar, A., 129 Santella, R. M., 130 Sastry, B. V. R., 69, 87, 88, 130, 131 Schnaper, H. W., 154 Scott, D. W., 155, 156 Sershen, II., 88-90, 157 Shay, J. W., 25, 132, 133 Shechter, 1., 134 Smith, K. A., 25, 136-138, 158 Smith, S. S., 135, 136 Sopori, M. L., 159 Stanbridge, E. J., 26-28 Steinberg, D.; 70, 71 Stossel, T. P., 139, 140 Strand, F. L., 90 Taketo, M., 28 Taylor, D. L., 141, 142 Vogt, P. K., 29 Walsh, P. N., 72 Weinbaum, G., 45 Welgus, H. G., 46 Young, D. A., 30 195

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