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Report of the Council for Tobacco Research - U.S.A., Inc. 1992 [Report of the Council for Tobacco Research - U.S.A., Inc. 1992]

Date: 1992
Length: 146 pages
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Minnesota AG
Glenn, J.F. 1
Named Person
Feldman, J.D.
Research Institute Scripps Cli 2
Pierce, G.B.
University Colorado 3
Abood, L.G.
University Rochester 4
Arnason, B.G.
Bowden, D.H.
Brennan, M.J.
Erikson, R.L.
Gill, G.N.
Joklik, W.K.
Karnovsky, M.L.
Knudson, A.G.
Lynch, H.T.
Mcallister, H.C.
Sato, G.H.
Vogt, P.K.
University Manitoba 5
Michigan Cancer Foundation 6
Wayne State University 7
Harvard 8
University California 9
Duke University 10
Fox Chase Cancer Center 11
Creighton University 12
Council Tobacco Research 13
Walton Jones Cell Science Cent 14
University Southern California 15
UCSF Legacy ID


1. Glenn, J.F. Author
  • Affiliation:


2. Research Institute Scripps Cli Named Person
  • Affiliation:

    Research Institute Scripps Clinic

3. University Colorado Named Person
  • Affiliation:

    University Colorado

4. University Rochester Named Person
  • Affiliation:

    University Rochester

5. University Manitoba Named Person
  • Affiliation:

    University Manitoba

6. Michigan Cancer Foundation Named Person
  • Affiliation:

    Michigan Cancer Foundation

7. Wayne State University Named Person
  • Affiliation:

    Wayne State University

8. Harvard Named Person
  • Affiliation:


9. University California Named Person
  • Affiliation:

    University California

10. Duke University Named Person
  • Affiliation:

    Duke University

11. Fox Chase Cancer Center Named Person
  • Affiliation:

    Fox Chase Cancer Center

12. Creighton University Named Person
  • Affiliation:

    Creighton University

13. Council Tobacco Research Named Person
  • Affiliation:

    Council Tobacco Research

14. Walton Jones Cell Science Cent Named Person
  • Affiliation:

    Walton Jones Cell Science Center

15. University Southern California Named Person
  • Affiliation:

    University Southern California

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rn ~ REPORT `° 0 0 0 H H Of a 0 U THE COUNCIL FOR TOBACCO RESEARCH - U.S.A., Inc 1992 ~ ~
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Organization and Pol icy The Council for Tobacco Research --U.S.A., lnc. is the sponsoring agency of a program of research into questions of tobacco use and health. 11 is the outgrowth of an exganization formed early in 1954 by tobacco manufacturers, growers and ware- housemen. Research support has been mainly through a program of grants-in-aid supplemented by contracts for research with institutions and laboratories. The Council does not operate any research facility. The Scientific Advisory Board to The Council meets regularly to evaluate appli- cations for research sup(mrt, judging them solely on the basis of scientific merit and relevance. The Council awards research grants to independent scientists who are assured complete scientific freedom in conducting their studies. Grantees alone are responsi- ble for reporting or publishing their findings in the accepted scientific manner- through medical and scientific journals and socicties. .IAMr•.C F. Cii.t'•.NN, M.D. Chairman 1992 REPORT O,f THE COUNCIL FOR TOBACCO RN;SF,ARCH-U.S.A., INC. TIIF; ('OUNCII,FOR'IYIRA('(Y) RESFARCH-U.S.A., INC. 900 Third Avenue, New York, N.Y. 10022
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SCIF;NTIFI(' AI)VISORY ROARI) W. K..IOKI.IK, D. i'Im.. trr'171c Council for Tobacco Rcsearch-[ I S A Inc (7rairnrurr ~ . . ., . as of December 31 1992 Ikpartment of Micrnhiology ancl Immunology ~ H . I)uke tlniversity Medical Center d' O ' 0"'k I)urham, North Carolina O 0 .IOSIiPI 11). hI:LDMAN. M.D.. ( hairntan ~ Mrnncer (/Snu•rinc.c). Research Institute of Scripps Clinic MANIRI?D 1.. KARNOVSKY, PtI.D. N H Scripps ('linic and Research Founclation lfarnlcl7'. Whitc Prn%cs.cnrn/'Ricrln,qic•al Chr•mi.chyand H I.a Jolla. California Molecular !'lrarmarnln,ly (limerihrs) ~ a Harvarcl Medical School 0 G. BARRY P1F,RCF;, M.D.. Vice Chairman Bostcm, Massachusetts U 1)i.ctingni.chcd ('errtc•nnial Prn/'rssnr of Pctthnhr tiv , I Iniversity of Colorado I Icalth Sciences Center ALFRED G. KNIIDSON, JR., M.D., Pn.D. ~ [hmver, Colorado Senior Member Institute for Cancer Research I,liO G. ABOOD. PtI.D. Fox Chsrtie Cancer Center Prn%c,.c.cnr n/ !'lrar•mac•nlngv and Riorlremistr.v Philadelphia. Pennsylvania I)eparclment of Pharmacology HENRY T LYNCH M D University of Rochester Medical Center . , . . Rochester. New York Professor and Chairman Department of Preventive Medicine and Public Health BARRY G. ARNASON, M.I). Professor of Medicine Professor and ('hairntan. Department of Neurology President. Hereditary Cancer Institute I)irrc mr, Brain Research Institute Creighton University School of Medicine University of ('hicago Ornaha, Nebraska ('hicagn Illinois , HARMON C. McALLISTER, JH., PIt.D. DRl1MMONI) FI. BOWDEN. M.1). Scientifirc Director !'arnlh, rr/'Mc•dirinr. Department cif Pathology The Council for Tobacco Research -U.S. A., Inc. tlniversity of Manitoba I Icallh Sciences ('enter W innihcg. Canada MI('l1AFl, J. BRENNAN. M.D. I )ir~rtr» - Familial Cancer Prevention Clinic Il;irher I IcnhiUil Prr.cirh•nt ruul Mrdir•al Director (F,nrcritn,e) Michigan C'anccr Fnunclation l'rn%r.c%nr n/ Afrvlirnre (l;ntc•ritus) W:ryne Sl:rlc IInivcrtiitv School of Medicine hctrnil. Michir.,ni RAYMONI) I.. 1?RIKtiON, I't1.[). Amerir•rrn Crnrrc•r ,5'rrricll• l'rn(c•sanr of Cc'llrclm rm l !>rrclnlrnrenJcrl l3inhrg1' 13iolng'tc'aI I .:rhoralrrrics I Iarvarcl l Inivcrtiity ('amhridP-c. Mas,~achu~rlts GORI)ON N. G11,1,. M.I). ('Irir/: I)ivi.tiicrn of linrtocrincrhlky/Metahulism llniversity nl ('alifnrni:r. San I)iego School rif Medicine La .hllla, California GORDON H. SATO, Pn.D. Director W. Alton Jones Cell Science Center Lake Placid, New York PETER K. VOGT, Ptt.D. ('hairman, Department of Microbiology University of Southern California School of Medicine Los Angeles, California . Scientific fitaf'f of The Council HARMON C. McALLISTER, JR., Ptt.D. ,Sc•ic•rdi%tr Director ARTIIIIR D. I;ISENBI:R(7, Pn.D. GEORGE A. HASHIM. PII.D. Associate Research I)irrr •h,r Associate Research Director DONAL[) H. FORD, Pn.D. VINCENT F. LISANTI, D.M.D. Associate Research l)irertnr Associate Research Director DAVID STONE, Pn.D. Associate Research Director io I
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('ON'1'EIN'1;S hitroxhuctinn .................................................................................................... 5 Ahslract Titles and Authors ........................................................................... 7 ....................................................... Ahctracls of Rcporls ................................ 2 I. Canccr-Rclatcd Studies ...................................•...........•.......... 21 II. The Retipiratory Systcm......................................................... 47 Ill. Ilcart and Circulation ............................................................. 61 IV. Ncuropharmacnlcrgy and Physiology ..................................... 97 V. Pharmacnhogv. Ricrchemistry and ('cll f3iology ..................... 107 VI. Immunology and Adaptive Mechanisms ............................... 210 Active Projects ...............................................................:.................:............. 239 Completed Projccts ........................................................................................ 263 Indcx of Principat Invcslikalors ............................................... Inside Back Cover Introduction With its research funding continuing at a substantial level during 1992, the Council remains among the leading agencies in the country that are supporting scien- tists in various areas of basic biomedical investigation. Funding in 1992 was $19,5(Hl,(HN.I, bringing to more than $204,(HNI,(x10 the Councij's research program support since it began operations in 1954. NigMy-Ihree original projects were approved in 1992: many more continuing and renewal studies also were funded. To date, a total of 1.329 original investiga- tions havc been supported by the Council. Recipients for these arc 932 independent scientists at more than ?O0 medical schcols, hospitals and research centers. Council grantecs published 342 reports on their suppnrted research during the year. Abstracts of these are included in this report. The total for such publications now is at least 4,770. Two distinguished scientists joined the Scientific Advisory Board to the Council in 1992. They were Dr. Lco G. Ahond, Professor of Pharmacology and Biochemistry, Department of Pharmacology, University of Rochester Medical ('cntcr, Rochester. NY, and Dr. Raymond L. Erikson, American Cancer Society Professor of Cellular and Developmental Biology, Biological Laboratories. Harvard University, ('amhridge, MA. t)r. George A. Hashim, internationally recognized for his work on the structure and function of proteins and peptides,jnined the Council's scientific staff as an Associate Research Director. lie retired recently as a Senior Research Scicntist/Scholar at Columbia University's Departments of Microbiology and Surgery where he also had been an Associate Professor.
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ABSTRACT TITLES AND AUTHORS 1. Cancer-Related Studies MAI.IGNANT EPIT11ELIM, CFLI S IN PRIMARY HUMAN LUNG CARCINOMAS CGEXPRESS IN VNO PLATF.I-ET- DIiRI VFD GROWTIi FACTOR (PD(ih) AND PD(:F RECEPTOR mRNAS AND THF,IR PROTEIN PRODIf( TS. Anlnnindr.c Il N, Gdsmq»ulnc, T.. Nrville-GnlArn. J., am1 O'flarn. C.1 . ................................................................................. 21 EXPRESSION OF INSI/LIN-LIKr: GROWT11 FACTORS I AND II AND T11FIR RF.CEPTOR htRNAS IN PRIMARY HUMAN ASTR(X'YTOMAS ANI) MFNINGIOMAS: IN VIVO STUDIIiS USING IN SI'FU IJYRRIDIZATION AND IMMIIN(X'YT(X'IfEMISTRY. AnfnninAtc N. N.. Gelanrrywrkws, T., NcriIlc-('.nlAcn. J., and Maawcll, M ..............._.........._.. 22 EFTF•(T (N Tf1E FXPRF.SSION ()FTRANSFORMING GROWTII FACP(Nt-021N PRIMARY HUMAN GLIORLAST(?MAS ON IMMIINOS(IPPRFSSION AND 1.(1SS (1F IMMUNE SURVEII.I.ANCC Maawcll, M.. UalamyrnJox, T.. NrvilloOnkkn, l.. and An(nniru4t. N N. ......__._ ........................... .............. _................. ................ .................. 22 IiXPRI SSION (N' c-.v./PL.ATFI-FT-DERIVED (iROWTH FACTOR R, INSU9.IN-LIKF GROWTH FACTOR I. AND TRAN.SFORMING GROWTH FACTT)R a MESSENGER RNA.c AND TIIF,IR RFSPF(TIVE RF.CEPJY)R MF_SSI7)Gr:R RNAs IN PRIMARY HUMAN GASTRIC CARCINOMAS: IN VIVO STT)DIPS WITN fN SJTfI IIYRRIDIZATION AND IMMIINO('YTOCNEMISTRY. ChumR, C. K. ntd An(mrfn&s, A. N. ._....._...,_ ...._ ........__ ... _... 23 ~ EXPRFSSION OF MONOCYTF,CIIIiMOiA('T1C PROTF.IN-I IN HUMAN MFLANOMA INVIV(). Crravrs, D. T. Rarnhill. R_. (:alarnq.wMc. T., aard Anrnnindrs. N. N .............. ...... _... ................ ....... _.......... ..... _.................... ._.................. _.... 23 DISTINCT IIYPIiRMFT1IYLATlON PATTERNS O('CIIR AT ALTF-RFD CHROMOSOMF-LOC1 IN HUMAN 1.1fNG AND CDLON ('ANCIiR. Makns, M., Ndkin, fl. D.,1.crman. M. 1.1.a7iL F.,-l.Iwr, fl., nrnl Rndin. S- R _.. ,.. .._ .. 24 ARNORMAL MFTIIYLA7ION OF TIIF CAL('ITONIN OPNE MARKS PROGRFSSION OF CHRONIC MYF-I.fX1ENO11.S LIiUKEMIA. Ncikin. R. D, Pn.epiorka, D. flurkr. P. 1. TMune.c ii D., anrl Jlaylin, S. R . ...................._.... 25 ABNORMAI. PATITiRNS OF DNA MF7TIYLATION IN f 1I IMAN NF.OPI•ASIAr P(YF(NTIAL ('ONSFpIIEN(T•.S fXNi TIIMqR PR(XORIiSSK7N. RnvJin. S p, Maknc, M., Wu, l.. Ycn. R. W. (' , rk Rndroc. A., VMinn, P., anA Nrlkin. R. D. ....... 25 ISOI.ATION AND ('IIARACIYR17AT1ON OFTIIFcDNA RNCODING UIIMAN DNA MFTIIYI.TRANSI•TiRASF. Ycn, R-W. ('-, Vcrlinn, P. M., Nrlkin, R- D. Yu.l. J, lil-IXiry, W., ('umara.awamy, A., Lcnnnn, (i. G_, Tra.k, R. J, 26 DIFfT?RENTIAI-REGULATIONOFpmFAMILYGF-NF-EXPRE45IONRYT1fETI1MORPROMOT/iROKADAICACID. ~ Srhnrnhal. A.. AIhcnq A S.. Fnx61. A.. Fr.nmi.nn.l R 27 R10LIXiICM. AND RHXTfEMICAL ACTIVITY OF v-Crk CIIIMERAS ('ONTAQ3ING TIIF SI12/5111 RF.GIONS (N= MI(kSl'iIATIDYI.INDSITDI: SPE(1FIC P11O51'11OLIPA.SIi ('-y AND Sre. MalsmAa, M. Reechman. C. T., aml Hnmr/un. N ......................... ............. .......... ...... .. .. - . _.................... ........... ........................... ...................... .. 27 . .........,.. GFNFTIC AND MOI-ECIR.AR ANALYSIS OP TIIF All RECEPTOR AND OF CYPI AI GI:NF F,XPRf SSfON. Nnnlin.u+n. (1„ pmnks, R. A., Wcir-Rmwn. ll L, IMffman, F'_ C., Jnhnum. B. S., Nsn(hrer, J., Rcyex, ll.. and Wanrm. A. 1. ...... ........ __ ............. ...._...............__._................. ........................................ ............. ..... 28 DIf1XIN- AND AIl RECRPU)R-INiP1iNDF.NT PRO'fRIN BINDING TO XFNfHWOTIC RGSPONSIVIi ELIiMFNTS AND f) RI('ll DNA STIIDiED flY IN 1'IVO F(K)TT'RINTTNG. Wal.-. A.1. anrl Nnn4intnn, O. ................... ............ ......... ..... 29 INVIiS IT(;ATIONS OON TTlli AI I RF(1iPTt1R ANU ('YPI A I ORNIi EXPRILSSION. //nnkin.mn, O, Rrnnks, W. A.. I lnffman. F. (' . )uhnaw. R. S., NanIhnr. J, Rryrc 11 , W alnxt, A.1 . anA Wcir.Rmwn. K. I . ...................................................... 29 ABERRANT F.XI'RhS.SK1N (FTIIRc-rrirR~2/nru I*R(YfDONC(MPNF IN OVARIAN CANCER. Ihrng, M.-C., 7JianF.X_.Yan,D.11.,7hanR.11 7,IIr-O,7hanR.T.ammlShi.1) .................................._......._.......,.......,.._..................._ . u) SIi1.ECITVIi RIbI.STRIRiITION bF PRO11°.IN KINASI'. (' IS07YMIiS RY TTIAPS4GARGIN AND STAUR(15P(1RINF, Kilry, S(' . Parke.l' J..I:rhhro. D.. arxl.ln4rn .S . .... ................. ...... .... ........ ........,_.............. ...... .._ .- 11 P(H,Y('Y('1.1(' AR(1MMT(' IIYDR(X'ARRI)N MFTAROLISM IN RAT ADRENAL.OVARY, AND TIiSTIS MI('R(K()MIiS I•S ('A'1 AI.YZIiD RY T I II:.SAMI- Nf 1VI'.1. ('YTOCUROMF P450 (P45fIRAP). Oun, S., Rh~mn. hnrvya. K. K.. aixf lr( ~mrr. (" R .. . . , . _ . .. _ . . . . .. _.. - - . _ il ONt'(IUFNI(' AND TRANS(RUTIDNAI. ('(N)PHRATION WIT71 IIA-RAS REQIIIRFS PII(15PIIfNtYLAT1ON (1F r lun (1N SF.RINhS A 1 AND 71, Smral, T. Ilinclnry. R., Mrrcnla, D. A.. flirtt, M, and Knrin. M. ........._ 12 ARR(X;AITf1N RY r nnr (1P ( i 1 PIIASIi ARRF.S'f INDI I(•lil) RY NR PROTPIN RIIT N(Yf RY PS1. Gnnrlrich, D. W- andl.rr.W, II . . . . .. . . 11 MOI.I'.CI q.AR UI V IiRSITY 4I' NIi11RONAI. TYPIi ('AI.('II IM ('I IANNIi1.S II)fiN11f 11:D IN SMAI,I, (Ti1.1.1.1IN(; CAR('INOMA 4auru Ok-t M.(irir•mnnn, U li., Wirhrn. F. D.. Slaymnktt. S 1.. Snntch, T. P, andLrnnnn-1~A ....__ .. .. . .. .. . _ . __...... _... . .. _...._._.._.__ .. .... 11 ... _ 1111; MYR DNA.RINDING fN)MAIN IS IIIGIII.Y C(1NSIiRVFD IN PJ('7TOSTF.JJI JM pLS('OIPFUM SmMrf;ra«.rr,U.HryrhdLlt-Rin.X,f7ravcrr.I~,1'inrl,R A.andIrPnrA,1S 14 7
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A 111(;111 V 1'11NS1 RVI"I1('Y:S IITNI IN I IIh v A1\'ll NNA I11NI11N(; IxIMAIN I.S 1 SSI Nl'IAI ItiR IKANSI()RAtAII(1NANIIIRANS(KII'II()NAl.rnlvl A('IIVAIIIIN f~ra~.rr,h A InM,mray,n-,K. Whulakcr.L.Sinln.S,nmI/.i/,nrA J,1 - -. . , 15 ('ASI' RhM)R't (:YN!('(111K:1(' (-AN( h.R (T11V5 II)1.1'N( lI SVNIIRIIMh:II IIIA(iNr)SIS A I AMII Y RhP()RT~~~ ~ Ivwh,ll /.("avahrn,R ),Lyruh.l h,and('mr1,M J 16 IN'IRA('PLIJR,AR'1'ARGIiTIN(; (11 pP6)• L_\PRI SSI(1N I Ix'Ai I/A11/1N OP v ln 11) AhIIRSIt1N 17 AfJII1S IS Sllli I('llhNl" IOIRANSPnRM1'III( KI N 6MPKl-11I'IPROHI ASIS 1,rhl, 1~-(- amlMronn,G t l!. 11f151 RAN(RF MIIIANfS1)1: v:r, AI"IIiRA1K)NSINKINASI A('IIVITYANI)SIIIISTRA'IhINIl~RA(TIf)NS. 1,rhl F (',hnRlan.l,1. 1.I)r(lur,J.li,andMu,r. 1 17 'flll~ LI I('KI: RI'NAI. AbFN1K"AR('INf1MA ANh I'IS III~RPI-SVIRI IS AL k'rnurll, R G„Cnurrlxn. 14 Lud, l M., W dham., I W. Wilhann, t' S, Rnlhne Srnnh, I A. :nid ("arhnn, I). I. ... _ 1R MA1'RIX INI7 I IP.N('ICS (}Rf:AN SI (h SI'P('119f 11 Y ()1~ MhI A51 AShS IlY Rti(:I II,A I1N(f I"Rf q )I I( TI( )N ANI) R15IM1NSY 1'1) AII ITl(RINI' (;KOW'1111-N'1(1KS Kraf(. A, Lvrh9. 1, IArrr, R.. ('hnnrn, U, anl RnJ. I, 6( 1R PRf)IPINSYNl111:SIS,t'I'1.1 6R(1W1'l1ANb(1N('(x;l•NI-SIS Rhrwdl.R F. . . , 19 AHAI'7IVI'IiVfllil'lH1NOI~III'(iRIl3 ANIIKINbSOI~NhO1'IASI'1('TRANSPQRMKI'K/NINCIiiI t'1q.TlrRli . Ruhm.ll • ., .. . 411 INbIVII)I IAI 1RANSI'(1RMINf; I"VPN7:S IN IJ)N(; "IhNM ("1'11.('11I 1'11KK OI' Nlll 1'1-1('I:LI S AS PRI111IK'I:S OI' • ' ' H/ ItKFNI?II( INI)IR'lI(1N PIMnn.N I a,nlRuhm Il 41 111(;IIRKII:OhUIVhRSIIit~AIInNANhRI~VI:RSAI AMI)N(iSUPI-LONLS('NIiQP1AS'!I("AILY " l KANSFnNMhbNI1111'1('L(INI-S,Rnhm,A I .S.R•:xlrKrx•niF,A.andRuhrn,H .. _. ... 41 ,. . . . ._ .... _ .... .. . _.... SFNSI'IIVITY fll' TRANSFORMA N( IN l()SMA1,1. (111iliRliN('GS IN M)1'111,ATINN hPNSIIY P11RIN(: SRRIAI. 1`ASSAUh (>h NIII rl'1(ll I ti Ynn. A and Rubru /J 42 SI IPI'KLSSI(1N (11 'I IIM(IRK;I"NptiIS IIV 1'111i PRI'ASI ('AN('f;R ('I'I 1 LINF MI'I• 7 PI1I.IDW1Nf; 7'RANSITiR OI~ A NIIRMAI III rMAN ( IIKnMnS(1M1 I 1. Nrgrini, M./'a.lac~xdi. A,. S»Lh,nni. S, Rr(analini, h.. (;uwannim, (; , ` I o..aii. I. Hrmhndcr. F J, Nrm i, I, aud Rarl,nun Pndanu, l; . ., ...- 41 t'l1AN(;PS IN (:ROW 1'II ANI) TIIM(}RI(;I'Nl('I I Y POLI f)WING RECONSl ITI11'It1N (11' RtilINORLASTI}MA I:fNK ' I~I1N( IlON IN VARI(11IS III'MAN ('AN(TR ('PI 1. IYI'I:S ISY MII'R(('I:I1.1'RANSI7(R fDP(lIRf1M1)Sf)Mli 11. Ilanrrjrr. A, Xn, Il 1. IIn. S X, Ar»uln, 1) , I aknha•hr, H..lrnnhrrrJCr, I J, anrl Itrnttlial. W h.. .... ... .......... 41 IN71-RAI"1'K1NS oh Il -MI1$ (LI,IS WI I'II RASI:MfNIMPMRRANF.S.1rn.rnnr.r,1 P arnl Madnw, I) W, ,. _. 44 -" MM'IIANISM (1P pIRS "" PXPRI SSKIN IN IIIIMAN Nt}N SMALI ('I°I I ' ' ' ' I-I_I N(; ( ANII R ( I ! I INhS Krm 1 A I Ruh,mon K A r;:vd A I I d " .. . W~. . .. . . ar, nrmev. . . , nn P rm.v 1) R .. . . .. _ . , .. . , .. _ . ..... .. ._ _ ............... 45 h.XI'RhSSION(ld l'HF.N/f/t11'NIih.N('t1I)ILUI'RUI19N1PIx5•'^)INIIIIMANNON-SMALLI'19.h('AR('INOMAS t)I 1111 " I I IN(I l4'rrnn, /) 11. N,Rd7x•q•. 1. Kulnn.ml, R, Nnwr1l, I+ (' ,(;ardar. A., I;m•nr, M 1, W dham.. W V, fAM•n,J A.andKrrn.I A .. .- - - , 46 1'R(x;liS I P.R(1NI: AI I(:MI~N1S 1'KO11I7 RA1111N INhI l( liI) IIY P,I'IhFRMAL (:N(7W1) I hA(TOR IN A I941 1NP. MAMMARY AI)I-N(x'ARI'IN(1h1A ('PI 1 I INI~ M,.hann.l h. Kok:u, Y. R'rm.~r. II Il, I'vkru, M 1. Nnwcll P(' , . arnI I yNlr ( K 46 11. The Respiralory Syslem P1 A 1'I-1 hI IIbRIV1'h f iRf IW I71 I•A(TI1K IN IbIM'A111I(' I'l IIMf }NARY I-IRROSIS. ArunnrnAr,. If N" Rravo, M A, Mda. R li .(:xlannInmlm. I. N-dlr („dJrn, l, and Ma.wrll. M.. mrul Srirnan, M. .,,.... 1"\PRI:SSII)N (lh h1nN(x"YIf fllhM(IA 1'I'KAf'1"ANI-1'ROII'IN I mRNA IN It11MAN II)IfIPKI'I{I(' Pll1 MONARY 47 I~IItI(r1ti1S 4nf..nwd~..Jl N,N.•,dlo(:nl.lrn,lI.Kmdin.R I..Vakrnr,A J.and(;mvr~,1) T ............. 49 Rf111, CnR 111I:1'IRVI/'A1 SY-MPAIIIhIII'~IWINKINRI°(Hq.A'I'INRANAPIIYI.A(TI('ANI)I'.NIxITf)XIf' Sl lr x"h Waddrll. S ( Ila, iwn. I. S" Rrfu, 4/) , and Malluvm, R Il. .. . .. .._.._ , . _.. . ...-.. . .._......,... 49 I»4'I•NIRA1 I/AIIt1N (11 1111- SUI9 NIr1R (TRVI('AL(;AN(;I.IA ANb'fliP IMMFbIAIii IIYPIiRSt?NSII'IVITY RhSI'(1NSh. M:ahrwm. K ,1)AViwm 1 S , h, Srmre.. O , 6rcrn. F . and Rrjr., I) A 49 R(/1 I.IYIR I IIP SIrPMANU1H1Il nR IR ANh IN MnUI'll Al IN(; PI ILMf)NARI' INI I AMMATION W)LhOWIN(: INI)I I('1111N (R SYS'IlMI( ANAI'Ill I AXIS Mxlhnml, K.Iln4an, A, Ilrlnx•r, 1>-, I(aufr, I. W,x,lnrr, J., Ilnv;.nn.l. S-. S, hulv. ( i. and Fl IILXAMI'IIIASf/NHS141F("IIVHII'MIIIRIIAIhSIIASAI ANh111Y/P(11.YSAt'('IIARIUP1NbPCP.N MhI AI I f ll'Rf )1 HNASP A Nb I 15S11P IN I IIIIII (1R nF 4(1- I'A1 I J)PR( )1'Ii1NASI- PR( /bl l('11f 1N RY I II IMAN 511 AI VI,(M.ARMN'RUI`IIA(;1•S Sh:lpnn.S I).reml,hrll,F /,Kuhnya.hi,b K,an,IWrlFuc,ILO. ._._.... ,......._.. 5R Al(1W x'Y 1'li AIII/PRPN('h.'IYl FI1tKrlNI~("IIN" R(11 1-fK-('hl IA'hIR IN11i({KINS ANI) Rh:1 A'IlI1NS11IPll)OIIIP.R M(7N(1(~1"11-1-11N1-II(INS I)w,-n.( A,(-.rnqilwll J J,:uRISNxIIry,R A _ . ._ . ,. ... _.. .,- 91 IN('KIA9PhAI)IIhR'hN(li(1FM(IN(x)•Il'SI'111~IRRnNF('IININI1Rf)NCIIII'("I"ASIS »P1anAlrnNY1191r'IC,p nnr nwiv iuv/naY1ArrnnRUn ANNR-ai ru rnnAln. Owrn.l' A,Orrrrplvll,F /.IhILS.L..nndtiuKklry,R A. ,_. .. 52 PLASMA I,PVFIS 1)I F1 ASlASh~.SI7-('11-It" I IIIRININ'hP11f}BS('URRKLA'Ii? Wlrill'ROiIJNASI: , INIIIRITUR PIH.NI)IYI'I_ IVIInNI11.,aIN,R1A\In/1A\lA\1 Ar"(iVtll iN~l~nlll'I\WIr1111r1~H//Yr.fN~lAN1111111RN/YraI- nlln'IrNr"Y ra n. IW,IrI rN4t( INlllllrrrny Wt'tlr, l I. SII„'mian 1•- K_ Ihnnp. It . nnd (-nmphrll. F J 9 52 f h11/IJ~('I~LAK (T t1NING ANh l?XI'RISSSION OI' IIIIMAN AI VfOLAR Mh('R(1PIIA(71i('ATIIIiPSIN S, AN 1 I AS IlNI tl YlIC ('YSlI',INI: PRI ~IEIS('ARINI(' Rf ('1IT)R5 IN ANTIt1IiN ('11AL1.F.N(:Iil) ('i IINt'A I'1iS ... .. ............ pI~N( "1'II)N (/h PtILMONARY . A ../. ...... ....... _..... .. IS RI S 11)RI• U ItY I llil'ARIN ANT) IY/P.V ~( (:1.1 f fAMA Ili. Frw.. A/1 adI J»rnhy. P. B ..... . ... ..... CIIAl1 S! N(:lIMMI NtK!YIYKTII•MI.S"1'RVRRmkrnf ('1IL 11 ni..l r('RIrr1KR nrNl Hny¢„IY('OPCI( ~. SMtKlRS MINSESINTIIIiI. r~ .RY.,.•.RI.'..S. . NIIRII'OXIIN?MPbIAWc ARI'nIlle rlh I~RINI~(TbVASOUILATt1 AL•inp. K. Pnmhcm, C.. R• hR( MCAI'111•:PSINIL RcJly`lCl'S1AN.1•'~l.^ •'TSailnr'I.t7 W;knx.t)R.fMuenmR W.nd('hrTmnn.H AnJr.TIN(T 1'III--SI'I'('119CITY AND IiLASI'IN(7LY7'IC A(TIVlI1R.S IN' PqVINF. ('ATIII?PSINS S AND H Xin, X-Q.. - (;nM0rrn, R , aMI Mninn, R W . .. .. . ~` I{tx'II:%P11511RfiI1FAIIIIMANAIRWAYGPITTIIi1,lAL('fi1.1.1.INliPGCRI:ASr•.SITSI'LARMAMFiMRRANIs ~ V . NP.IITRALIINIx)PIiPlII)ASli.lanR.%.arxlMurlncC.(: AI'R(1501.1'All) NEII'lRAI, FNINIt'Iil'TIDASIi RCVRR51:5 O%ONP.IN1711CRb AIRWAY ..HYPF.RREACTIV............ .. ............. r/V ..... SPPSlAN("IiP Mwlna(' G.LanR."l...WilliamcG.l..»nd('hahmclla . ..............-..._......... ... . PNVIRONMI?NT'AI.AIRWAYINJIIRY M(K/KM (l/ANGPCANIIAtRWAYI1VMl11lEA('T'VRY,M//r/r/S,(' ('~ P,NINYIli1v1,IN IMMI INORIihtTIVITY (N' AIRWAY I?PlTlll(LI(IM IN A•STIIMATI(' PATIPNTS. SpAnpall. D. R.- " ~ Ilnwanh. P 11 .('nunihan, lt. Ujnkamwrr, R, IInIRa(r. S-'C. and Pnlnl.l. M._ ...........•......... ............... ". ® MOI)III.ATIONOPIlfP,LI)N(:f'OLON'V.AT1ON()1'RIh~I'1MGI.ANOMACCLI•SWY(9TRUSPF.(TIN•- ..... . ...............................•......... PI»IL 1). nnd Rn:. A...... -..._.._... __ ........ ......._....._-..........._....-... 52 RY 1'RIMARY CIR.TIfR!•S (}P TOR ' ~/ II PAC SI'ONTANP.f)lIS PRODII('ryON OI°TRANSPf}RMIN(: (:Rf1Wl RRbN(TIIAI.P.PITIIP.I,IA1.('I:I.I,S- 1r11.-1~oNIl'I1.nrunvN'rtrNen.nSnran,O.,Rnmbrrgcr . .D..Rir.inn,A.. ............ Pn~kmnnn, l. U., Rrnnnrd. S I, and SJxrrr rm. l.~-~" Hl. Hearl and (:ircolation TIIIi ROLG (}P TIIR I•lRST f;ROWTtI PAt'IT7R DOMAtN IH° IIIIMAN I'A(T()R IX» IN RINI)IN(i T(1 W 1T. r•NS. . ANT) IN hA( TQR X A("Il V Al I(1N- Ahnmd. S S. R»wnla~Shcikh. R.<lrcnnR, W: h. Stattrmml, b. W.. amI ('OMM)NI•7JTS ANI) ASSP.MPI.Y OP TI(li FA(`ff)R X A("11VA1'ING ('OMt'LRX Akmnd, S..S., R»w»Ia~Shcikh, R"• " ..................................... .................. . . .. ...... ..... .. andWalch,P N-......._. lry,Al1:LKT A("IIVATING FA(TOR ANI) SYSTEMIC' ANM`IIYLAXIS IN MPP(1.STROMGYLIIS RRASIIJFN.SLS- SIiNSfI'1%PU RATS_ DIt77{RITITIAI, Hllli(TS OP PAP ANTMN)NISTS. Mathiema R., t}av:enn. J S., ancl Rrf (e A I) . STIIDY Q!• T'IIR CNIN/l'RnTI'(M.YTII' (T,IiA V A(Jh.OI' M,ATRLF7l:V h~1 PR0T'P•~INrOR)lk~ 1. M"' OLIC'rONll('1.(SOTIDti-ML•UTATIiO M(ITAGI?NPSIS. KulnArirj. M. anrl RrnnrR.l 5. . _ . _ . . - _. , . . . - . 1947iCr UP Dti1.I•71ON OP CLYCOPRQTTIN IIR EXON 28 ON Tllli I?XPRli55ION OP Tllli PLATIiI.IiT " (;LY(Y)PRQ'ITi1N 1111RIIA ('I1MPI,GX. Kakd.irj, M. A_ Vilairc, G., Rifa7, S„ I'a+cz, M., anA Rrnnrft. J S.. A SIMM,IhItiD M1:77IOD 1q)R ~1'li!•, I)F:II`.RMINATK}N QP NITRIC OXIDR IN R/()j/XaCAL SOi,11T11)NS_ Trnnin, A., Iluffmann, M.. »nd RinX. R .I . . . .. . - . _. _ . - . .... ..... . . . .. . .__. 71 H°. IiPI•lttT OP IMMCINIi MRUTATORS (CYTOKINfiS) ()N T11R RP.hP,ASE OP RNDOTIICI•1'IIM-DF,RIVRD RCI,AXIN(7 I°A(T()R (GURPI AND (R;1'ROSTA('Y('I.IN RY FRCS111,Y HARVFSTIib IiNDOTHI(hIAL ('IiLIS. . .. _ . ,.. .,. _ . . , . Ihukk. R., Krhira, S-, Kahlrr, l., and Ring, R J TOR AND GNDOTIRi1.IN RI{LIiASIi (° 1'ROS'I'A('Y('1 /N I:NIK)T111i1.IlIM-UItRIV1iU RI'J,AXINC PA( R RY IRI°SIILV IIARVI:S'I191('hLI S A t'1 A( III'.D'I'O MI('R(K"ARRIISR RR•AIJS. Kibira, S-. Umkk. - _....._ Namyan, K S_ arxl Rmx. R .I ~ - " - " - " ~ - I,IK'AIiN(i A LOW PI1N511"Y I.IIY)PRI)ThIN l~ARGPTIN(11X)MAIN OPIIIIMAN AI''OLIP(WROTIiIN R-11N1 PY I°.XI'RIi.SSIN(: A MINI(:INP ('11NSIRU('I IN'IRANSGfNK' .. MIC!•,. XinnR, W.,7.aRmnnd-1'-•(:min. A M._ lc. • .._...... ............. ....... .... Lci. K Y. arNl f'Mm. L_ .. ~ PIRP.f 9" IiV Ib4°NCP Il)R 1111; . IN VUI VIiMI'.NT OP hRtiC RA6I('AIS IN I.SC I'Ih,MIC INSI ILT TO TI IE INTI:STINIi." - _,._...,.. ....._.. Itda..m. R-. Arirli, I. Ilackc/. Y., Knrr,.•kY, N.• ard (7+^ inn' ---"'•~ ~ 1•RI°.I' RAUI('AI- INDIK'I•.b I'lI1RINIMI N C(IA(iIR,ATI(1N: MODIR,ATION (11: NP.f}I°IRIi P(7RMATK)N BY ('UN('IiN'fR NfION. p11 AND'IT.MIM:RAII IRIi Ka+llrl, R.. Marx, G.. »nA ('hn•rnn. M- ... - ~ - - ~ Kitrrxs k y,i RAT INTI STIN!•.. SALI('YI •Al'li AS AN IN 1'll'O hRIiB RAI 'AI. TRAP: STI II71GS (1N IS('l IP.MI(' INS(ILT TOII ll Udawin. R . Arirl, I , I lavk,•I. Y . ky, N., and ('hrri..n, M . ~ ~ ~~ ~ ' ~ A POSSIRLP. RI l!K I11' PRKIi RAUK'AI S IN TI Ih.1~R ANSI'I.ANI'ATK)N Oh RH7"INAL , I'1(;MIiNT fsMT III+LIAI. ('I'.1 1 S f Ipl+;r, A :md ('hrrrnn. M ~ - ~~ ~" - I,(K'AI,'V.AlION O1' pls (ANNI'XIN h 1.IIYX'ORTIN Il IN Nt1RMA1. AD fl1.T RAT KIDNEY AND f)l1RIN(: RI:(Y1VI4RY IROM IS('IIIiMIA- Mr Kanna. J A.('hnrNharumro. A, Mun K. A. Rrcyrr. J. A.. Cnhrn. S." -" . ....... ......... . an111hlrrla,R.(- -.- -- .. - "-......... . 9
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IMI'AIRt 1)11FI'A I71-AIYII ll'()fY(()1 FIN 11 ANII F.7RANSI.A fll)N IN S'I'RI I'Tr)l.OIrx'IN I/1ARhI11' RA I'S Sl,a.k,. l 1,.7nRayhan H. SlwrAa.l' r..Cn,nh. n(-. nml fr,nn F A 1'IIF('t1NIRIRII"IINNIIhVA,S'('i11.ARINIR)'IiIF1.lAI XANIIIINI•IIFIIYDRIX'FNAC ~ ~ 1•N 70 . I)(IhASt• innXY(,I:N MFDIA i l•D t l I,1 INJI IRY Panu..l' (' . Wr;Rhr. S A.. Chumlrv. 1' 11 , R:nh, R, aml I•u.romn. R A .. 71/ 1 IIY1/'R()li•IN I 1PASF.IN('RF.AShs 1 f/W DI:NSI'1'Y 1 IP()I'R(III:IN RI:TI-N'fI(1N F1Y SIrR1:ND1)'1'i11;L1A1. ('1ii.l. MAIRIX (;,Vann,,I M an,lf:n/dhnl. /! I IIY)'R(y1l:IN LIPASG MRDIATi'D I IPIAKI. ANI) DC(;RADA IION Ili~ I OW IX!NSITY I.IIYII'RUI7:INS ItY I IRR0111.ASIN ANh MA( R(II'l1A(;hS Rum"rv, S('., Oln,nikr, J(', Arnd, Y., hrekrlhnum. 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I'I/VSi01 (XRC RtR.P UP I IMII'H/III~INIa1 /hufrrl, l' t' nnJ N;n~hman. R I. ..._., 76 IDFNTI19/'A IION (/1~ MG('IIANISMS i'l1AT MAY MI)1)1ILA7ii l'11Ii ROLL (7F I JI'OPRb77i1N1a1 IN TIIROMROSIS ANU AIIIFR(x)INICSIS lfnrPr~L I' !" arnllhnlh, W . 76 1 INKA(;F.11P A1'lllRrx;FN1('LIP(IPR(IIiiIN 19II:Nt71'Y1qi"1'(1Tlll! ibW I1F:NSIIY IJMR'HOTFIN RP(7iPfOR 1 rN'l I.S ON 1111- A11/)R'I ARM r)P CI IR/1MOS(IMI' 19 Nkhma,1' M, LJm,on..l r. NaRRCn, J K, nml Kraa... H M. 77 I XPRI'SSI(1N (11-'111F c,+n•I+PR(1'Th nN('Ix:1.N1- IN RnVINP VAS('1 U.AR SMtx)l'11 MII,IX'LL ('FLIS. Itrnun, K F. Km,h, M t, and S,mrn.hrin, (; 1; - ... .. . ........ ... . 7R M(NN q A11/1N (/1 ORNI7lIINI-D14'ARROXYI ASF mRN.A W)I I hWINO TRANSIPNI' IS('111.M1A IN i'lIF (;TRIIR RRAIN Ih•ml,ary.lt 1.('a,nrY.1 M.anriA'rn,/c.M.C . , . .. . . . .. . ...... ......... ........ 79 I I Y Iq-R(;I.YI'I- MIA SI IPPRI tiSFS cfn n,RNA F.XI'RFSSII )N t4)1.1 (1W ING TRANSIFNI' CRRFRRAI. IS('l IFMiA IN GF.RIII1 S('nnrhr, D 1. I/rny,.rv, R. ), ihmaid.rm, U, amrl Amdr. M S .. 74 MPVAI (1NAl1i KINASI' I.C 1(X'AL.ILIiD IN RAl I IVhR iq~ROXISOMFS Starnrllna, K. D., Shuckrlfmd, l. F. 1"anaka. R A' U. anJ rrl.uu , s K 1'lll"Rrll I'UhPI~HnX15OMhSINI'H(IItSIPRIDI MP7AROI,LSM Kmm..r,.S K........., I)1 t9(FSSOR I97fi('I nP RI IK'KIN(i ANI;IIYTI•NSIN Sl RiCI YMi I RhI'lil'T(7RS IN ANTFRIt)R IIYIY)TIIAI,AMIIS. Yan(;H II/mIiW J M ya. anJOInml S .,.... ('l/'I FI"ANINF RI 1IN1:C IIIF. Plll MONARY PRI :SSOR RhSP(1NSF 11) A('IrfI:IIYPqXIA IN RAI;S. J;n, 11- YanR.R Il.aminpen/.C SAI 1' SUPiq,l MPNI A I II1N Ixlh,S N07 Ai./l?R l1iF PRFSSOR FI•IT/TO6 RLfX'KINIJ ATRIAI. NA7RI(IRI•TI(' 1M"1'Iil)1•INNII('IF.IISIRAI`f11S5(R.II"ARII YanF.H,lin.ll,Wyc<,1 M.Chrn.Y F,aml()rnrrl..t . A'IRIAI.NArR111RPT1('PFI•IIUI:MODIIIAIT'SRAR()Rh(1?P111RRI'ilPXINSI't)N1"ANI'()US1 79 RR RI RI R2 IIYIq H 1'PNSIVI HAl Iin. 11 . Y;,nR, R IL CalMn;n. D A_ Wyt<, 1 M, nnd!)pnril,.5 . . .Y . RS ANbHIx;FN U(99:Nh1'N7 AN(;IOI1(NSIN(x;FN ANI) RIiNIN MICSSI'N(;IiR RNA L•'XPRI!SS/ON IN /I)'PhRIFNSIVPRAIS 1'hrn.l' F.NaRAan,A.l.anAOlmnl.S . I(M ~AI IlAI I(1N t R IIH AIN ANiI ATRIAI NA IRIIIHh.TI(' PhPTIDP IN IIIIMAN ANI) PORCIN1° 1 - R AR 84 : . T 11n r,. A, Wharn,n. I. Mhu.liui.l' , Grac.n, M. DirRoh. M, Nmdlrmnn. P. ViRam,, M, Mrncrnn. (i , anJ Pn/nJ, J M R4 FNINX ARhIAi Iflr'A1I/MIONANiI('i/ARA('ITRI7ATION07: NATRIIPRPTI('PF1'TIUF.RINUINf;SIT:S INIII,MANI1`1AI ANIIAIRq I IIhARC Rwhrrhnd.A 1),Whannml,i;nrdnn,i.'.Muvrnvy(;,,Yaawb.M 11. amllsdnJ,l M 85 IN'I1MAI. SM(x)1'll MII,SCLh.IYI.I PRULIPPRAIION AI--1TR RAL1.(X1N ('A'111F1TR INJIIRY - Nnnm I 1aensn~ Imamm-)r.wnWrnrAr'roa-(11um.NF.('hau-S Iindnrr,V„andRndw.MA . _ - - R6 NPhIN11MA1 PR/)I Ii:l'RAIIqN' ('1)N1R01 IN VAS('Iq ARSMIXPIi1MlIS('LP.('1l.LOROWI'll RnAv M A, hrkw+n, ('„ aml Lrminrr. V , .. . -. .. . . .__.- ........_... .. ....._ .... .. .. ..... ..._._ RR ~ ~ I'ROD11(TIhN Oh TRANSFnRMIN(; ORUWl111~A(lT)H I7 DIIRINO RIiPA1R lIP ARTP.RIAI.IN111RY MaP••AY. M W. Lindnrr. V-'1'wardiik, U H,.l'r hw.rv. S. M. and Rr,rA. M A R/ ll i,(11' BASIC hU1Rl7it1-AST (;RO WTI I 1'M'7 OR IN 1'R(11 IIRiR A'I'H )N (1F IiNiX)'I'I ILL111M AND.SMf K yrll Mlly('l lt AITPR 11T1I I I NIN(7INJURY IN C'll (1 1l V rn,nrr, aml Rrnh. M A 1'NIxYi'IIFI-IAL /'FL1 RF(7H17WI71 R.•nlv. Af A aml l.,minrr. V ,. RASI(' I OF ANI) (;R(1 Wi 1101 AR I PRIAI (F1.I S R. nlr. M A and 1 innrr. V~~ ~ R7 RR RR SPF('IFICINI9AMMA1()HYCYI(1KIN1_CRF(;tlLAll•I111'hXI'RFtiSh)NOFIIItMANM(1N ' ~ (N V R9 il: IsI1P()XYt;FNASh (,nna,l.i) -._....... ....__.-.. R4 10 A PRIMARY DiiT1IRMINAN T Fr)R I.II'OXY(;f?NASI'. POSITI(1NN, 51'Pf'It9('1'f Y- Slnano, D.1 •., LrunR, H., ('raik.(' S aml.Sicn).F INI'FRA('l1ONS IiITW19iN RAND ) ANDtttIif:R TRANSPORT"RIiLA1TD 1'R(111i1N.S SnMnnm. A K FFiI(CI' (1T P('MRS ON ANIUN TRANSIYX(T IN 1II IMAN RGD ('F,L1. MtiMRRANttS. Zhang. Z. aml.SnJrmann, A K A(`I'IVIi SITIC-RL(1CKIiD hA('tOR IXa PRFVFN'rS INIRAVASCIILAR 1TIROMRUS I~ORMATI(7N IN TI7F. CORONARY VAS('iII,AT(IRIi Wi 111OIlT INIIIRITIN(; IiXTRAVASCUi AR COA(iU1,ATION IN A CANINti TIIROMROSIS MOI/F,1- Rrnrdicr. C. R.. Ryan, /., Wnlihky. R, Ramrw. R.. Gorix'h, M.: TrjhmE, P.. and Srrrn, D...... ."ADV AN('liD PR(1TRIN UI.Y('OSYI.ATION INl)l/('FS TRANSFNIX)TI IF.LIAI• I II IMAN MON(%'YTF• CTIFMDTAXIS AND SP(RI:TION OF PI.Allil.la-DIiRIVP.U ORbW111 FA(TOR: ROLIi IN VAS('UT AR DIStiASC OF U(ARIiT1iS ............................~.-.....~.~.~.~~ ~-" ~--~ ANIt AGING. Kirakin, M., Rrcm. J.. Ra,InfC S, ORawa, S., 9rrn. D. and Vlacsara, 11 ........ ('ARDIA(' f)FNFRVATI()N IN T11F ('AIJ° USIN(i CRYOARI•ATION: FIINfTIONAI. FVIDIiNCF AND RFOIONAI. 97, 93 TISSI IIS('A'ill('11OLAMINF, ('qN'tliNT f;arr,1. A R., Whanrm, l.. (innMn.1.., Swift, R L. M1mah, C, InRtis, G. C. 94 1'n6k.1.M..amlTnvk.r,K M RFOI(7NAI. DISTRIRIITION AND RFODI,ATI(1N (W /'"'IICAL('ITONIN OFNr•. Rfil•A7E0 PF,PTIDF BINDIN(',SITIS IN CORONARY ARTIiRif:S. Knnr'k, I) A., Whamm, J,(;an, J. A R, Yacnuh, M I1., TmLv, K M., am1 Pnlak. J. M. ........... TORM'CO USF AND URINARY RXC'R(TI(NJ OPTIfROMBOXANR A, AND PR(1,CTA('YCT•IN MI•7ARfHJfFS IN WOMFN S'IRATII'IBf) BY AOF R9nRcma, C.. Rcnlhm, (7., Oranad:m.li. P.. Persana,1,.. Wincll, S., amI Wrn-fm. A RF.hIiASF Of• FNDOTHtiL1AL MEDIATORS ANI) SYMPAT11b71C TRANSMITTFR.S AT D11TF]IFNT C(7RONARY 19!)W RATfiS IN RABBIT IIFARTS Wrnnmalm. A , Rrmhm. G.. Karwa(nwda Pmkopnnk. F., I.mdlvrR.l M., amlPrlrr.cam.A..S .._....__ ........ ......._ .._ __....__...........-_._..........._._._....-....,....._..................__......_..._... IV. NeuropFlarmacolLfRy and Physifty PROTON MAGNETIC RFSONANCT: SPIiCTROSC(WY OF TIIF BRAIN IN SC11170PIIRFNIC AND AFfFC'iIVF PATIPNTS Sharma. R.. Vrnkaucuhramanian, P. N.. Rrvrfwv. M., aml Dav;a, I M. ....... _............. ............_................• .•....... ...- IiFITS-I' Ol: NICOTINF ON FXTRACFLLI R.AR LFVFI S OF NFIIROTRANSMITTIiRS ASSESSED BY MI('RUDIALYSIS IN VARI(1IIS BRAIN RPOIONS: R(N.F (1P GId(TAMICACID. TnIfi..F.,.Scrshrn, it..llasMim, A., Vvi,1i S.. am1 i.n11An. A .._...... ... N TYIMI CAL('111M CIIANNFI S ARE INVOI,VFD IN TIIF DOPAMINF RFLF.ASING FFFFCT OF NICOTINF. I lardnp. l.. (i., Jr.. Srrchrn. ll. Vizi. S. F., aad lnyhn. A . ........ ...... .........._..,......... ,.., ..,.• .,....... .,....,•.............. P1:RS(STV.NCT; OF CIfRONIC NIC(YflNli-INfH/(T>D ('(X;NiTiVf': F•A('IhITATION- 1.n•rn• E P, RdgRa. S. J,(Triclnphrr. N. C'., and Rnsc. J. F. ,.._..._.....•......_ ........... ....... ..._.........-,...................... ...._........._._... NI(YITINIf'SYS7EMSANDCOGNITIVP.FIINCTION.lirin,F, /) .............. ..... ........ ......... ............. ............... __............._.... It• IMPORTANCF OF D AND D INTPRA(Ti(}NS WITII NICOTINIC AND MU.SCARINIC SYSTEMS FOR n 94 qt 9(, 97 97 qR qR 99 . ................... WORKIN(: Mf':MORY Fll'M(TION. Lrwn. F. A. and Rrnr.l. l: .._................ ... ..............................__......_........ IfNt IMMIINOt1IST(XTIFMICAI. t,OCALI7ATION OF NICOTINIC AC'ETYLCNOLINF RFCFPTOR SIIRCINITS IN TUf': MTSENCEPNALON AND D1F,N('FPIIALON (1F TIIR (IRCK (GAIlIdS (IAlLIL9) Rrino. L R. G.. Kryscr, il'I(1 ......• ....................._.,.. ...............,..._.•......-.•.......•.............,........-........,.. .. K.1 • IJndr(rom.l M. aml Kancn.11. 1. . _. _.. (TfROMOSOMAI.I.CK'AIJ'l.ATION (1F SFVFN NFI)RbNAL NPCfTTINIC ACFTYLCN(N,INIi RECEPTOR SI IRI/NIT (;ISNPC IN III IMANS, Anaml. R. ami fAndrv.nn.l ......... •........... _..._.. ......•...•.......... .• .... ....... "•-. ..• .. .......... ........_. 101 FJ'ITOPR MAPPfN(J (1F P(N,YCI.ONAL AND MONOCI,ONAL ANTIRODIFS AGAINST TWO a-RUNOAROfOXIN- BINDING a SI(RIINII'S IROM NFIIRONAI, N1('OTINIC RFC'F,PI'(/RS, Mdanc, K.1:.. Wu, X..IJndstrnm.l M-M am1('m,li-Trnncm+;.R ............. ................ ..._ Itll .. ,_. _ ... .. _.._. -_ ............. _ ..._ ..__........... ._............. ._.. , ..,... .. . NKIIRONS (F T1IF ('lll('K BRAIN AND RIiTINA fiXPR1iSSING B(TI'N rt•RUNGAROTOXIN•SF•NSITTVF• AND rrRUNUAR(yTOXIN-INSIT1SIl I VF NtCO1TNI(' MRTYLCtIOLIN1i RF('EPPORS. AN IMM(INOIIISTOC11r•.MICAI. ANALYSIS Rrinn.l, R-(i .Itamacvaki Rriuu, 1) li„ Frrrn, li S., Keyar. K T., Kanrn. ll. l., amll.rnrl.rrmn, I M......._ L'P/1YR"F MAPPING OF MON(X'il)NAI, ANTIRODIFS TOTDRPEDO ACF•TYLCN(R,INF• RFC'F.PTOR Y SI IBI INITS WIII('li SPP('II9CAI 1 Y RP('(1GN17•ti TIIF r SUBIINIT OF MAMMALIAN M1ISCLF. 1R2 A(l•7Y1 c'lIt1LINli RF('P.ITOR Nrkrm. S_ Shrlu,n. G. Q. Isi.S., hf+rd.(mm.l M. aml Cnn)i•Trrarzv,ni, R. .....-..-....... .-.. 103 RfIK-L-DF.RIVIiD C'lil.l. hINFS 111AT PR(1D11(E BA,SIC F7BROBLAST GROWTJI FA(TOR. Bl1T NOT I'ARFN1'Al. RIIK.21 CIiLI S, tNl'IIATI' NPIIRONAh DIf77iRFNTIATION OF NFIIRAI, ('RFCT PR(X)1"NITORS. Rrill. (7.. Vaisman. N.. Nra/ Jd, (/. anA Knkhrim, ('. ........__.........._...._.•_............. ......... .. S119TYPIi 01° Ml1,S('ARINI(' RFCIiITOR C(X/%,FD TOTIIF ATTT•,NIIATION OF 11(N(MONF.STIMULATF,D cAMP A('('1IM111.AT1ON IN NGIOR.15 NFItRORLAST(1MA X GI•IOMA IIYBRID ('F,1.1 S. Stnphan, ('. C- and Smlrv. R (- R ..............._..................._.... ......... .....................................__..•. °SYNAPTIC MUS('ARINI(' RFC'FI'fORS AND T11F AUTORF(i(ILATION OF NF.IIRO7RANSMITIRR PR1 101 IM . RFI IiASP. Sn.qrv. R V R and (khi1M. R P __....... .......... ...... ..... ..................__.............._..........._ ._.. IRs I))PAMtNF EFILIIX FROM STRIATUM AI'IPR CIIRONI(' NI('(yfINli FVIDFNCF. FOR A1)TORF.CEPTOR Di31'NSfTIZAIlON. IlnrcinR. L. G.. Jc..4rrhrn.lf . and lajlha. A. -...__ ........._ ...................._........... ......_................-... IfIG I I 1-
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(IIR()NI('('r1N!"/NN(Rr,C NI(Y)'I'INF lNf~:A'IMI NT ('AI!CI V I1I f'RhAChU IIIrR51 I IHINI~ 11h NIt;RAI. Ixtl'AMINh N/i 'Rf)N.t IN HA I:S 1'ARIIAI f,Y 111 MIfRANCht'li-U A I 1111" Mhtil) Itll Nf "I 1•IIA1 It' 1UNI-I If1N Inrnh„rl I.Jn,n~•n. A M t„~nrvrn 7 JI nn.IFurr K V. PharmacolnKy, Iliochemisfry and Ce11 13iohJgy IIII rUNAs('t11)IN(;Ii)R1'111-,r ANU(1%1/1tlrNllS(ti•YI•N(IPIISIAFI'/.Sf'ASI°INKINASh(1 Irdli,ki,A, Ihnn, h., M V, AlIrndr, (' C, amI All.•nd.•, I G Illl" IIYUR(R Y.CLti OF PIfO51'7(A Ilf 1Yf IN(1SI ft/I. A.1'l lf 1ShIlA IT IN MhMRRANIS/lt RFN(7PN,S' 1AF125' tKx Y I"I:S IIIARA('ITRIS71(;S(/F A I'110SPtIbMONUP,S I'tRASh laanh, (; , Allcndr, ('. f' ,:md Allrnrlr, J F /)lli9 R(,NIIAt_ S11MIq A1111N (11f11h GIPa•r Af'I'IVII Y(7Ff; 1'HO114NS RY W)1 YI YSIN/i Ammwffi. M„ 01,111, 1,(;ra/, R, A/Irmk, (' t, anA.111rndr..l I 11)1 YI (Y11.Vf :1 l r I AMA I t? AN A1,t R;S ('AN INI IIRI1' ('A SI9N K 1NA.S1' 111•Rl1M Y('NnP(l,S LAFI'LC 'I'rllcr, R. Alkndr, f' (' . and Altrnrlr„1 I ANALYSIS (th 1•IIr15PI1111 YRRSINr: f'qN'/"AININC PRO'1f7NS 1'Rf+ShNi IN v u. INI'li('IIiU MVIiI OIU I`R(R:IINI l'(1R ('Iq 1 S I'.rmin, I I. and An,lrr,.,n.,l M !l 1.1 ('YI'Lf: UM77VNBN I 1(N'A1 fLA Cf(1N (tI~ ( ASIi1N KINASF 1 T(1 Mll'(11I('.l'I'INUI hS. Hrrrrkman, l 1-, tho.~, S U. Su..ncru. M R, urrl ,InJ• ~ o,n R 4 /'IR:F AMI1f i'f/1rN(T1(1NA1.(;R1tW'I'lll•A("tttH Arrnn,r,wlr,.Il N . AIIRAN("IIINt;1•R(RYtCSMI11714 tR (ihNi AMI'1II•1fAI1qNhOIIJnNN(:('(1R(IM(IS(/MIiRH6:AKA(;h KimmrLM,Arrhn,/,1) F.andWxhl.M (; . . 107 109 1 Ir) Iln .. . . .. . . _. - . . . .. . . ,. . ..._..... . I I I INIIhHI"1'AN('1: ANU RI~( I HISSIIIN T/)WAR!) I III .:A/l AN IN III:TPR(x;I'.NI'f )I I,S ('I:LI. IY7I'l I1.A"I I(/NS I:amrt 1 W nud A,rlrn,l 1) F 111hN'l l/9f'A I'R1N (R• A N)-K1U117A1.1(IN Hh RINIIIN(; NRUIP.IN. RhIYr11, "fl1AlAI,I UWS 1'IIF. Rh 1:21 ('tlMP1/iX It)ItINI)UNA Ray,S,K,Arrnyn.M RqC•hr (nnrtR h ' . :rvr mnlhnnl ., . . . . . . .. .. .... . . . ... . .tiFRIrM 1 H/•h Mr1lll'F ( M111tRY(1151 Mh) f'LI,t Y ANI)'1 kANSF R ORMIN(; t:RI/W I II hA(TOR CF(fl'>il, M:uay,nhi.I 112 11: .. ...- -. . .. ... ... .... . ... . .. .. fll PRf)IRk 'CIt1N(11 k' CIt1N(11RCIt1N(11 (;RItWTI1STIh11rLAIINGI'A('It1Rtil9(t)MANIMAI ( .. 1°I . IS . ('nR . .xii, . P.,am/ . Rnrnrr.N W . III 'llll((:MI`khltlt('IA5I f P ; NI UF I1II fh - NI MA In/:A.(("4RL(((/MC(R((Y71/7/SVAH SI/IrM (;nnd1.M..Hunrh.K., fihnnh.S,andltrwmvr,k AN hX IRI MPLY AItUNUAN7 OVAH/AN mHNA I°I(f1M 1III• PARASIl1C Nh.MATOUIiA.C('AHLC /.I7d1RR1('IIIO/{ti VAR C(qrA( IIAS Atl ll 1119 / R!-!`FA 1 A((/ flf S(; d ' nn l M( ar / Wil I ,,rr,..,mM1 , t:hanh, S , anJ Rrnnrrl. K L ALfNRNA 1'fVfir Y SPLI(lsU LIK MRNA IN NPUHbNS I•RI:UI('I:S A NI?( Nnrn.,(i A 't9'lY)R WI'l71 A 1,AH(:IiR PII'1'A'I7VF, /:YIHA('hI1.Ir1ARfx)MAfN Ilnu.cV,II.Cnrplrr.,A 1M.I'rnR.M N,KnuI,I),1.rRrau.N M, I',:rndRna.n.A.A ,,.. 114 114 SFQ(rPN/7 fll ArhNA('IttNl hNI'ItbINGARATRIi(:'I"Rfl'1751N Kamimrrrn,l,Woa((',unrlRrurlrr.F. .,. IIS CY If rKINh I'R(7U1 R'1'R IN I'Rf1M I•kIS/11J' 11ARVP.S 11-1) if( rMAN Mt iNnNl r('1 hAR ('19.1 S ATfAf'I II'11'Itl I`I AS Ill I(I•Al1S Iimq R I I7nd,4 H K hl 1 N . a n urayan KCA M -..,..., :m 'I YI'ISIMY'II7C AN'11Rn1)Il.S'1(11711i IY,A'IJ•LI•.T I)IiRIVI U t:Rf 1WI'II 4Af'I t)H Rh.('hPl'(iRS ROhh IN II 11( IUMINf; 11111 S'I'RI It'11IR1I. ANUPNF!('fl(/NAI,('l1ARA/"II'RI•Sl'I('SrR-RP('hI•IUR fYWsC Rnj S hanalxrni. 1•. Khan. S A anrl Nnlrvrm.~ {' , ._ . _-. 11'II11111AI S/'F('R9("(i1~Nf KI HA I )N/x "1' ~fXI'R1YSI(1NU11RIN(iUIPIPRI':NTIAI'If)N fNF SIHAI"Ihlll) hRIMARYIIt1MAN IPI'1911rR/S W~Lrnrf 1Il II ' ' .• anLS ( ( .,,nw..unrr. R hl IR(7 VIRAI I~I ANU(..' I'Rnll"INS SVIq'Y)H'I Rh1'I x"AI'lI7N 1tb II(1M(71 txRl(1S AND/Ri7FH(/1 (XN/ILC 1'A171/(1 MAVIRAf r 1HR,INtitl IIA I17 . n;mR,t M II 4tI'1'kAI'IqtP ,IAIac,M.StrnhnuhA..lln,I'I•,RmAr,-TR,and(~hrrw,7.'1 /IR tiLN ' KIIIVPtiI NIIIhS15l n AMhIAI/nl'Rn'/F/NA.SPIN~I;SII(rMSM-'IRANSfURMPU NHK t'hl I S flnrn. I f", Sr:,nhm M Jh V n I I / ,p : ,,..rrv.. and Mua III 1 1 Mt1I I('111 AR ('1 nNIN(: ANUI IIARA(")PRI7AI1(jNfR•v mn, A(`ffVATT-UI'RANSI4 I'Hr/1 )I(MAII(1N ASS(x' 1 IN5 11 IN5/ IN5 l A ~ " : : : ( urn I ( I l fU Sr.,~ ll / . nl n,M./h,mR. .1r:r,I R.YU,N,I(unR,M (".rrrmli.M,andl~.n.nnan.P M, ~ .-~~- RtNbt)1'/ NZYAfI"H!'(' ' /lv ; b! 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A. r MP('IIANISM OF SKIN M(1RPI((x:P.Nl'StS 1 ANALYSPS WIIFI AN'/IR1)INliti "1O AUlIIiS1UN MOLY('IILILS ITNAC('IN, N /'AM, ANtI (N'fF(:RIN IranR, 1 X aml I "bnnu.C. ( M PI'INl' I'VIU1'Nf'h Ff)R A1YP1 (11'PRt1X1VASF UF.PENU(iN1'IfYUROXY(. RARK'AL F(IRMATIbN RY RA ~ ~ 121 , . . . - SPIN I III IMAN NI'1 rl R(11M111 ti ANI1 Mt)NIx'YTFS Rmm~.. C L. h~w, S Rritigaa !( F.. ('nhrn, M S.. am) R.evn. G M. .. 124 /11?I'AT(x'Y Ili NII(1.1;AR hA(-IQR tp ('4N'IAINS TWftTRANCt'RII'll()NAL A(TIVATION txtMAINS.f1Nfi ()1' W III('I I IS NnV RI. ANI) ('(tNSPR VIiU W I I'l l l'I tt? IIR2C(1P1111 A hf )NK I ) PR(/11°.IN Panr, h_ f lvrrditt. U t ; , Prxcclla. A . V~an. X., l ai, h... a,xl ('rnrn. R ll P VA1.IN110N UI• TIII? ANNLXINS AS P/)Th.M'IAI. MIiD1A'ft}RS OP MIiMflRANIi PIISIUN IN li%O('Yl'OSIS. 7aks.W.1 and!'rrrrr..t' F _...... ,.,._..............-............ ........... .. .. ('AI,('1(IM UICPI:NURNl MI:MRRANR.RINUIN(7 PROTI°INS AS P)l IiN'nAl • MP.DIAT4RS Oh 51'IMI IL115- f 1ON ['411M IN[' ('r~u)- C F Uru+r 1) S.. Ilamrrwn.IL (' . Junkrr. M.. KumMmriv, N. <i.. Klcin,l, R.. CR a • ~ • ~ 125 125 . . • ~ --• SF ..............._........_........_.......-._.___........... . 126 Nrlvm. M. R. alx/ Snydrr. S 4 IMMI INOLfR'ALrLAT1UN (1F A(;1 YCOSYLPN(7,51'l1ATIDYLINOSIT(H: SPI?C'IP1(' h110SM1U1.IPASIi I) IN MASI'('1_'I IS (-(111ND IN N(RMAL'rISSl II! ANO NPI IRqI•IARUMA'fq•SIS I.IiSIONS MrV. ('. N., "tlwmac, P, and /hrrro-. M A . - . . . . . - . '- . . ._ -. - . t10RMON1[ RIiG1ILA1'IiU v-rrI I°STR(1C:IiN RPCfiI'T)R 1•l/S(ON P1N)1TIN' RI?VIiR.SIRI.ti INIYI/(T10N OP Cl:l,l, 'IRANSt(/RMA"f10N ANU C(iLLU/.AR CI'NI? I!XPRI :SSION. JJrrhmrh. [i., Walkrr, A.. Kahnm. N.. Mrllivtt. li.. ItrnR, l l.. Zrnkr. M., nml F'nnr(m. P l INI'fl(1N Of I)NA SYNTIIPSIS IN LIVIN(: ('Ii1.IS BY MI('RI)IN1K(Tf(1N OF (:; ANTROUI(CSS taMrwtc, V. J. (:rrkAmnh. l' K.. SpecFn•I, A. M.. Mrinkmh, J L . arxl Fr.nmirur, J. R , . . . . . .. - -~ ~ .~~- ~ f111i INUI R910N OP Rgr I EXPRIiSS/ON RY v F(n IS VIA A PR(1T1'.IN KINASti ('-1N'UGf9?NI)CNl' ' A('1•l I ltl AR it('NAt l'l1AT IS ChQIIfN'f1AI LY DP,PI9NDIiNT UM7N IlnRaa ANI7 Raf L ~ ~ R 1'h 127 128 . ... . . r . ~ INT 12R Alcsarnlrn(wxdrx, K.. () rrcsl+i, S A.. Rnukr.l.'I'., Rslqr, ll ~arut Fnqrr, P A .. . . . . •~. ~~ - ~ F.V1171iN('Ii 1"/IAT I/a~Ras MI?UTAll:S TWO UISTINGIIISIIARI.Ii INIRMT?LI.UTAR SI(iNA1S A(TIVATIiD RY v,Src. Qurcahi, S, A, Alrzandnqxm)nr, K. Rim. M.1ncrPlr, C. K, Rnxkr.l. T, Ra(q1.1/. R. enrl Frnrrr. h~A Al1NN(R•ATIIF.RM/)LARILPv.vr{;F.NIiPOR1ISI'.INRIiV1iRSIRI.Ii t N('ANbC11ARA(TPRU ( N/ 129 . . (l. TRANSFORMA7I/)N IR~ MAMMALtAN Cli1.I.S, Marrmry, A C, Uurcd+i. S, A, Fnrrr..0 A. and RrnRRc.l. S. .__ ..... ... I29 v 1'IK RR.SIYINSIVIiN1:S5IN lllR (iRr I PROMOT(iR IS MIiUTATRU RY S1?RUM RI:SP(MSI? EI-RMIiN7:S. Alczaexlnrl,rmkrs. K„ Vurc:hi. S. A„ Rim. M, Sukhwtmr, V- P. mnl F.r,rrr. t) A- ... ._ .. -. C IS"rAINI'U INUIIC)ION qh r r I RY v~vr ('ORRIiLATtiS WITII A IA('K (1P FOS MIiUTATh,U Rlil'RI:SS(ON 01: R l , 'I'llti rgr-1 {'Rf1Mf)TIiR. Qurcshd, S. A. Rim. M. AlraaruhrN,u)ns, K. Rng, K, Sukhatmr. V P, amI Fnrrrr, 7) A . I tl 1MMIJNO ISOI,ATION UI' Sli('7Pd'(lA'Il°.U fRANSPt)R"I' VI-'~SICLI:S hROMl'tl1? Yh•AST Sti('RI?TORY PATIIWAY- /rnn:unrlJ.A.lanvr.F.a,Mtlnw<ILKJ? .......... .... .... ...._........_......... ...... ..._............. "........ 1(I RI':AI ITY ANU TIIF, YIiAST: ('(M1PARl'MIiNTA1, OR6ANIZATIUN OP T111i SIiCRETORY PATI I W AY, - _ ..........................._ .._...__.,,._.._.... Fran:nrnA.A IIPfROX1UF C(RATFU RY QI U7AT1(iONI? RIiUU(TASI? 1N111ATT:.5 A VANADATI>URh/!NUf?NT PRI!li ~ S U2 . , RA(M('AI. ('l1AIN OXtUAT"I(1N /)1• NA1N1. Linchrv, 5.1. anr/ Frrdimrrlr, L...._ ... ................... 112 IiPl7i(TS OP QVIiRPRQUU(TIUN fN~ SIIMiR(7XIUI's UISMIITASIiS IN f..SCIlF.RI(.'I(U CM.! 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R, ' 1h 217 nINQi Rl /O CLASS SW IT( ..__.... .. ~ ... . . ..... . ........... ............ ......... .... ..... and CamMrr. l. ('. . -SINCI P R-IIYRRIf1nMA l7?LIS RY l'OTAI, IIiN'CIN(i REAVY- ANI) LIGNT-(l1AIN VARIABLh (•FNI:S OI• . r.. ~ 1fi SROI FTfLYMATI('AM19.If'•ICATI(7N•1iu,A.n..('rra,km.(:.,amlWvrrnki•l.J -.-- - ...... ... . . VIN'R NKl17ROMIII S- IN VIVO AND IN VITRO. OP SORfACI: EXI'Rf SS10N DF CDI R BY RO ILATI(1N 217 71N >19 . MOD( Gdhrn.R.(l.,Kim.C.A..anrlYrn,A .... . -~~ .__. 71v .'.'11 2?I ?71 227 221 1+1 19 19
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Abstracts of Reports Folkiwing arc abstracts of reports on new research acknowlcdging supporl from '1'hc Council that have appeared in scicntific journals sincc publication of the 1991 Rcpurt. Thc name of the grant recipient is in italics. The ahstracts arc grouped under these headings: 1. Canccr-Relalcd Studies, 11. The Respiratory System, III. Heart and Circulation, IV. Ncuropharmacology and Physinlogy, V. Pharmacotogy, Biochemistry and ('cll Biology, VI. Immunology and Adaptive Mechanisms. in ~ ~ ~ N ~ O ~ I. Cancer-Related Studies *0 N MALIGNANT EPITHELIAL CELLS IN PRIMARY HUMAN LUNG CARCINOMAS COEXPRE.SS IN VIVO I'LATELET-DERIVED GROWTH FACTOR (PDGF) ANI) PDGF RECfiPTOR mRNAS ANI) THEIR I'ROTEIN PRODUCTS Lung cancer represents one of the major human carcinomas with the highest degree of mortality. Epidemiologic studies have linked this disease to "chronic injury," largely induced by cigarette smoking. In the present studies, we demonstrate the in rivn expression of platclct-derived growth factor (PDGF) and PDGF receptor (PDGF-R) (3 mRNAs and their respective protein products in malignant cpithelial cells of primary human lung carcinomas. In contrast, nonmalignant epithelial cells in control, normal lung tissue specimen did not express PDGF and PDGF-R mRNAs and did not produce their respective protein products. Epithelial cells in lung speci- men from patients with idiopathic pulmonary fibrosis expressed only PDGF mRNA but not PDGF-R (3 mRNA. These Findings of the inappropriate coexpression of a potent mitogen, PD(;F. and its receptor in lung cancer epithelial cells suggest thee presence of a powerful in rirr, mechanism contrihuting to the self-stimulation and unregulated growth of lung cancer tumor cells. Anlaniudrs. H. N.. Gal.mopoulos, T., Neville-Golden. J., and O'Hara, C. J. Proceedings of the National Academy of Sciences USA R9:3942-3946. May 1992. Othcr.suPln7rt: National Institutes of 1lcalth. From the Dcpartnunts of Cancer Biology and Nutrition. Harvard School of Public Ilealth. Center for Blood Research, and Department of Pathology, New England Deaconess Hospital, Boston. 21
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F?XPRESSION OF 1NSUI.IN-I.IK1: GROWTH FACI'ORS I AND II AND THEIR Rls('NF''I'OR n.tRNAS IN PRIMARY HUMAN ASTRO('Yl'OMAS ANI) MI:NIN(iIOMAS: IN Vlt'l) S'I'FIDIF;S IISING IN.SlITI IIYIiRIDIlATION AND IMMIINOC'YTOCIIF,MISTRY These studies demnnstrate the expression of IGF-l. IGF-11, and their respective receptor mRNAs in primary human atitrocytomati and mcningiomas. In situ hyhridizatinn and imrnunocytnchcmistry have localited a strong expression of both IGF-I and IGF-11 mRNAs and of their protein products in the tumor cells of astroc:y- fomas and meningiomas. The expression of IGF-I and IGF-11 mRNAs in the tumor- cells was accompanied by the co-expression of their respective type-1 and type-11 IGF receptor mRNAs. Control, non-malignant human brain expressed IGF-I mRNA and IGF-I and IGF-II receptor mRNAs. lhere was no significant expression of IGF-11 mRNA in the control brain specimens. Control pachymeninges (dura mater) expressed low levels of IGF-1 mRNA and IGF-I receptor mRNA. There was no sig- nificant expression of IGF-11 and IGF-Il receptor mRNAs in pachymeninges. The co-expression of IGFs and their receptors in brain tumors may contribute in their development and maintenance. The strong inappropriate expression of IGF-11 mRNA and its protein product in the tumor cells of astrocytomas and meningiomas, hut not in normal brain spccimens, may serve as molecular markers for the early detection of these tumors. Anlnninrlr.c.11. N.. Galanopoulous, T.. Ncville-Goldcn, J., and Maxwell, M. International Journal of Cancer 50:215-222, 1992. Othcr support: National Institutes of Hcalth. From 'I'hc ('entcr for Rlood Research, Departments of Cancer Biology and Nutrition, I larvard School of Puhlic Ilcalth, and Neurosurgical Service. Massachusetts General Hoxpifal and Department of Surgery, Harvard Medical School, Boston. FsFT1;C°F' OF TI IF: I?XPRESSION OF l'RANSFORMING GROWTH FACTOR-(32 IN PRIMARY 11t IMAN (i1,1OF31.ASTnMAS ON IMMIiNOSFJPPRF,SSION AND LOSS OF IMMI INI? SlIRV1ilI,1.ANC'F. GliohlaNtnmas arc malignant brain tumors that arc attended by an immunosup- prc~ud,o:nc. I'hc authors have studicd the expression of transforming growth factor- (i2, which i" known to have pntent immunosuppressive and angiogenic properties. Transforming growth factor-/32 messenger ribonuctcic acid and its protein product are both found to he greatly nvercxpressed in these tumors and arc abscnt from nor- maI brain tismc. The overexpression of this growth factor may contribute to the escape of nenphi~tic astrocytes from immune surveillance and, furtherniore. to the immunosupprcsscd st:uc that is characteristic of many of fhctc patients. Maxwell. M.. Galanopoulos. l'., Ncvillc-Golden. J., and iln/nniodc.c, !!. N. Jourmd of Neurosurgcry 7fi:7c)9-RO4. May 1992. Other supporC National hictitincs of I lcahh. I:,i,m The Center for Blood Research and Neurosurgical Service. Massachusetts (;cneral Ihospital and Department of Surgery, Harvard Medical School: Department of ('anccr Biology and Nutrition. Harvard School of Public Health. Bnston: and 1)cpartmcnt of Neuropathology, Radcliffe Infirmary. and Department of C'linical Ni,lirohogy, Oxford University. England. EXPRESSION OF c-.si.s/PF.ATELET-DERiVED GROWTH FACTOR B. INSIJLIN-LIKE GROWTH FACTOR I, AND TRANSFORMING GROWTH FACTOR a ME.SSENGER RNAs AND THEIR RESPECTIVE RECEPTOR MF,SSENGER RNAs IN PRiMARY HUMAN GASTRIC CARCINOMAS: IN VIVO STUDIES WITH INSITU IIYBRII)I7.ATION AND IMMUNOCYTOCHEM1Sl'RY In silu hybridization and immunocytochemistry have been applied to investigate the expression of c-sis/platelet-derived growth factor (PDGF)-B, insulin-like growth factor (IGF)-l, and transforming growth factor a mRNAs and their respective recep- tor mRNAs in three primary human gastric carcinomas and in their adjacent nonma- lignant mucosas. Expression of c-sis/PDGF-B mRNA and PDGF-receptor (3 mRNA was seen in the tumor cells of the three gastric cancer specimens but not in their adja- cent nonmalignant mucosa. The mRNA expression was accompanied by the expres- sion of their respective protein products. ICiF-I, IGF-I receptor, and epidermal growth factor receptor mRNAs were seen in both the tumor cells of the gastric can- cer specimens and in nonmalignant mucosa. Transforming growth factor rx mRNA was expressed in gastric tumor cells hut not in nonmalignant mucosa. The coexpres- sion of a potent "competence° growth factor. PDGF, and "progression" growth fac- torti, IGF-I and transfonning growth factor a, in the tumor cells of gastric carcina- mas may contribute to their growth and maintenance. Chung, C. K. and Antnniadcs. ll. N. Cancer Research 52:3453-3459. June 15. 1992. Other support: National lnstitutes of Health. From The ('enter foi Blood Research and Departments of Cancer Biology and Nutrition. Ilarvard School of Public Ilcalth. Boston. EXPRFiSS1ON OF MONOCYTE CHEMOTACTIC PROTEIN-I IN HUMAN MELANOMA IN VIIY) A common feature of human melanoma is infiltration by monocytes at early stages of tumorigencsis. This infiltration may be highly significant since macrc,phag" have the capacity to alter the behavior of tumor cells. The authors pre- 23 22
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vinutily dcmomtratccl Ih:n Ihc hrcdnminanl mnnncytr chcm„anractant rr(,duccd by Immor cclls in rinv, wa,, mmtnc•ytc chcmnt:tcti( protein. 1( 1). 'I'hc authc,r. i(k•n- tify Ihc cvl,rc.einrt nl' M('I'-I in hatht,h,~,ic I,ccimcns nfi h,lh rrimat.v and mrlatit:dic human ntchuumia htd nnl in n„rmal %kin. 'Ihc f'in,fing Ihat Ml'1' I k produced by ntalignanl nx•lanrona snzgc.lN Ih:u ,)Tcc•ific zcncti are can'csscd in (untnr cells that cnn inducc thc recruitment oI'hxrnocvlcs in rirv,, ( iraves, 1). -I'., I):n'nhill. R.• (ial:tni,lnwhn. T., :tncl llnnuiin,le~e. 11. N. , Amcric•an Journal nf I'atht,h,gv 1-1(/(1 ):')-1 J, .I:muar_v 1992. OIhcr suppt,rt: N:tti<mal In,,tilutc% (,f Ilcallh. From thc I)chartmcnl nf Orad Ri,>h,ty ;md Ri,rchcmistry. Boston lhiivcr.eity Medical ('cntcr: I)ivisinn of I)(•rmal„Ir.uht,l)py, Maeea(`huscU~ (icncral Ihnpilal: ('cntcr for Ith,nd Rc.c:u'ch: and h'crartmrnl,~ „I ('anccr Biology :tnd Nulrilion. Il:rrvar(I School (nf 1'uhl ic I Icalth. Bn.tt,n. 1)I.S'I'1N("I' I IYI'I',RMI?TI IYI.A'I'ION I'A-I-T'TiRNS O('('l IRIR Al' Al:fl:RFl) ('IIROMOSOMI'. I,O('I IN IIIIMAN I.IIN(i ANI) ('OLON ('AN('HR Rcgional inc•rcascti in I)NA mcthylation cx•c•ur in normally unmcthylalcd c'ylo- sinc-rich arcw, in ncc,rlastic ccllti. 'I'hcsc changc; could hntcntialh• alter chrnnmatin "Iructurc to inactivatc gene tran.c•ripti,m or Lcncratc I)NA instahilitv. Wc nnw sh(~w Ih:u in hnman lum~ :tnd ct,lnn cancer I)NA. hyrcrmcthylalinn of such a region c(m- tiiilcnllv „cctnti nn chrmmotiuntc 171, in an area thal is frcqucntly rcciuc•cd to hom(vy- P-ity in hnth tumnr Ivpcs. Over thc progression stages ol' colon ncoplaxia• this nmlltyl:dion rhan,r.c incrr:t~c. ill extent and hrcccdcs thc allclie In..ws on 17p that arc ( h:nnCICfIStIC of c„hm c•nrcinoma". We also show on c'hronunnnmc 3p that regional hyl,crnr(•IhyI:rli„n mav nnnrandnmlv accomranv chromos(inic changcs in human nrnhla,i:t, hictratird mcth) laticm is c„nsictcnl in tiniall-ccll lung carcinoma DNA at Iw„ lh I„ri Ihal arr ct,n'ianlly rc<luccd to hnnx»ygosity in this Iumnr• hul it is not 'rcn in c„h*n riinccl' I)NA. in which Ihctic loci are infrcyucnlly %tructurally allcrctf. R1akc,ti. M.. Nclkin.It. I).. I.crm:tn, M. L. I,alif. F-,'l.har• 11., an(113rrrlin.,S. R. I'n,c(•cdini,; of ihc National Acatlcnry t,f' Sc'icncc~, IISA RO(5):Iq2,)-IO33, March 1902. Othcr support: Nati,mal ('an('cr Intitilulc• ('layltm Fund and Nalinnal hnlilulcs of Ilcalth. Frrnn Ihc Onct,lncy (`cntcr and hcparUncnl nf Mc(lic•inc, The .h,hn,, Ilopkins Mctlical Inqitutirn,ti• Raltimurc. MI). and National Cancer htstitutc, Frederick ('anccr Ri•ticarch Facilily. Vrc•drrirk, MI). ARNnRMAL METIIYI.ATION OF Tllli ('AI,CITONIN GENE MARKS pROGRI?SSION QF C'IIRONI(' MYIiL.OGliNnIIS LIiIJKF:MIA -fhc clinical acpccte nf' disease progression in chronic myclogcnous lcukcmia (('M1.) are well cstahlishcd, hut the nature of Ihc molecular cvcnts responsible is not known. We have previously reported a consistent pattern of novel sites of mcthyla- linn in the 5' region of thc calcitonin (CT) gene and other chromosome 1 Ip loci in acutc myclogenous and lymphcrid Icukcrnias. In the present study. CT gene mcthyla- tinn rattcrns were invcstigatc(1 in peripheral blood from 51 patients with CML. Abnormal patterns were found in only 2 of 31 patients in chronic phacc, hut in S(if R patients in accelerated phase, and in I I(if 12 patients in blast crisis (P <.(N)5). For one patient studied in blast cri.is. abnormal CT gene methylation was found in the peripheral blast cells hut not in the granulocytcs. In two of three patients sturlicd with ('ML and having normal peripheral cell patterns, abnormal patterns were found in marrow blast cells. In nne patient. only partial normalization of the C'T gene mcthy- Imion pattern was seen after chemotherapy induction of a second chronic phase and the patient relapsed 5 months later. Our findings indicate that abnormal mc(hylation nf Ih'c 5' rc€ion of the CT gcnc is regularly a marker of disease progression in ('ML which may prove clinically useful. This abnormal mcthylation site is part of an imbalance in DNA methylation that may play a rolc in the progressive gcnctic insta- hility which charactcri7cs advancing stages of CML. Neikin. B. D., Przcpi(,rka. D.. Burke. P. J.. Thomas, E. D., and 13uylin, S. B. RhNxl 77(1 1):241 I-2434, June I• 1991. Other support: National Cancer Institute and Clayton Fund of the Johns Ilorkins llnivcrsity School of Medicine. From the Oncology Center, Department of Mcdicinc, The Johns Ilnpkins Medical Instihttions, Baltimore. MD: Pittsburgh ('anccr Institute. University uf Pitttihurgh, Pittsburgh, PA; and Ihc Fred Ilutchinxon Cancer Research Center, Univcreity ol' Washingtnn, Seattle. ABNORMAL PA'I"I'ERNS OF 1)NA METHYLATION IN NUMAN NEOI'LASIA: 1'OTI;N'T'IAI,CONSF.O( INN('I;S FOR'I'IIMC)R PROGRESSION An imbalance nf I)NA mcthylaliun, involving widespread hypomethylatian, regional hyrcrmcthyl:di(m. and increased ccllular capacity for mcthylation, is cha ' r- actcrislic nf human ncohla%ia. This imbalance beginx in prcncoplastic cells and becomes more extensive thrt,ughnut subsequent stages of tumor progression. In nor- jnal ccllti• a pritnary I'unc•li,m nf DNA mcthylatian may hc to modulate c(timparlmcn- taliiation crf DNA to ensure that regional areas of transcriptionally active chr(,matin rcplicatc carlicr than the hulk transcriptiunally inactive chrornatin. We argue here that Ihc altered mcthylation patterns observed during tumnr progrescion, especially regional hy(?crmcthylatiom. may mark- or even help to cstahlish----ahnormaditics of chrontntin trrgani/alic,n. In turn. Ihc%c c'h:mgcs in chrnniatin %tructurc may, through direct transcriptirnnd inactivation of gencs, predisposition to mutations, an(1 allclic (lelctirnis. mc(Iiatc Ihc progressive Insseti of gene cxpression associated with tumar (Icvchormcnt. 24 25
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lirn-lirr, .4. R.. Makos, M., Wu, .I.. Yen, R.-W. ('., dc liustmc, A.. Vcrtinn, I'., :md Nclkin, li. I). Cancer Cells 1(IO) 3R3-39O, October 1991. Other tiupport: National ('anccr Inelilute and ('layton Rcscarch I'vnd. hrom the Oncology ('enter. Dcparlmcnt nf Mcdicinc. and Iluman Gcnctics (7raduatc Program, The Johns Ilnpkim. Medical Inslitution.',, Ballimorc, MI). ISOI.ATION ANI) C'IIARA('TGRI"I.AT'ION OFTII1;cDNA ENCODING 11(IMAN DNA MI;I'IIY1:I'RANSI,'I?RASI: We have chmcd ascrict of overlapping cDNA clones encoding a 5194 hp tran- ticripl fnr human I)NA mcthvllramlcracc (I)NA MT'a%c). This .cqucncc potentially rolc,~ Gor a protcin ol' 1495 amino acids with a predicted mnlccular weight of 169 kI)a. T hc huntan DNA MTatic cDNA has cighly percent hnmoingy at thc nuclcotidc level, and the prcdictcd protein has scventy-fiiur percent idcntity at the amino acid Icvcl, to the DNA MTasc cDNA cloned from moutic cells. Like the murinc DNA Ml'ncc, the amino terminal two-thirds cif the human protein containe a cystcinc-rich region .ug.gc%tivc oI' a mctal-hinding domain. The carhoxy terminal one-third of the prntcin .,.hows considerable similarity to prnkaryolic (cylnsinc-5)-mcthyltransfcr- asc.. Thc an;mgcmenl of multiple motifs conserved in the prokaryotic genes is prc- scrvcil in the human DNA M'I'asc, including the relative position of a prolinc-cys- Icinc dipcplidc thought to he an essential catalytic citc in all (cytosinc-5)-methyl- nan%fcratc%. A;inr'le 5.2 kh tnm.%cript was dctccted in all human lisxucs tested, with thc highc%l Icvcl. „f expression ohscrvcd in RNA from placcnla, brain, heart and Imig. DNA M'faw cl)NA clones were used to screen a chromosome 19 genomic cnmmid lihrary. '1hc DNA M-1'asc-pn.citivc cosmids which are ctitimatcd to span a grnnniic di"tancc (if 93 kh have Ixrn localizcd to I9pI3.2-p11.3 by fluoresccncc in mltu Irylnidiinlinn, KnLuuon nf the cDNA for human DNA MTa.cc wil9 allow further ,,tudy od the n•)wlatinn „I DNA M'Iasc cxprccsinn, and of the role of this cnzymc in c,.tahlishing I)NA mcthylatirni pallcrne in both normal and neoplastic cells. Ycn. R.-W. ('., Vcrtinn, 1'. M.. Nclkin. B. I).. Yu. J. .1. I?I-I)ciry. W.. Cumar.iswamy. A.. Lcnn„n. (;. ( i.. Trask. Il. .I.. ('clann. I'., and Ruldin. ,S. R. Nucleic Aciih Rc,,cnrch 2/)(9):22R7_Z291. 1992. Otltcr support: N;uitinal (':uiccr Institutc and ('laytcm Foundalinn. Fronn thc Oncokozy ('cnlcr ,ind I)cpnrtmcnt (1I' Mcdicinc, The ,lohns Hopkins Medical Inqilutiim., Baltimnrc. MU. and Iluman Gcminm ('ctHCr. /3inmcdical Scicnccti Divi.iom, I.awrcnc•c I.ivcrnu,rc National I.ahor;qorics, I,ivcrmnrc, C'A. Fronr the Oncology Center and Department of Medicine, The Johns Hopkine Medical Institutions, Baltimore. MD, and lluman Genome Center, Biomedical Scicnces Division, Lawrence Livermore National Laboratories, L.ivcrmore, CA. DIFFERENTIAL REGl1LAT1ON OF jun FAMILY GENE EXPRESSION BY THE TUMOR PROMOTER OKADAIC ACIL) Phosphorylation events are major regulatory mechanisms of signal transduction pathways that control cell growth and differentiation. The potential involvement of sertne/threonin-specific phosphoprotein phosphatases in pathways that regulate gene expression was analyzed. By use of okadaic acid, an inhibitor of protein phos- phatases I(PP-1) and 2A (PP-2A), we present evidence that expression of distinct members of the jun family of genes, c jun, jun8, and junD, are regulated differential- ly by serine/threonine-specific phosphoprotein phosphatases. Treatment of cells with okadaic acid induces the expression of jun13, and to a lesser extent c jun, but has only a marginal effect on junD expression. This induction involves transcriptional as well as post-transcriptional mechanisms. An analysis of defined elements in different promoters suggests that serine/thrconine phosphoprotcin phosphatases are involved in the regulation of the c-jun and the collagenase 12-O-tetradecanoyl phorbol- 13°acetate (TPA) response element (TRE) as well as the c fns serum response ele- ment (SRE). Since inhibition of PP° I and PP-2A leads to increased proto-oncogene expression, our results further support the view that certain protein phosphatases might act as negative regulators of growth. Schonthal, A., Alberts, A. S., Frost, J. A.. Fcramisco, J. R. The New Biologist 3(10):977-9R6, October 1991. Other support: National Cancer Institute. From the Cancer Center and Departments of Medicine and Pharmacology, University of California at San Diego. La Jolla. BIOLOGICAL AND BIOCHEMICAL ACTIVITY OF v-Crk CHIMERAS CONTAINING THE S112/SH3 REGIONS OF PHOSPHATIDYLINOSITOL- SPECIFIC PHOSPHOLIPASE C-y AND Src The chicken CT10 virus oncogenc product, p47'11"', contains SH2/SH; domains that have been identified as conserved domains among proteins involved in signal transduction. We studied the functional similarity of the SH2/SH3 domains by replacing those of v-Crk with those of phosphatidylinositol-speclfic phospholipase C--y. v-Src, or c-Src. The transforming activity of v-Crk was partially retained in a mutant with a v-Src S113 domain but not in the other mutants with heterologous SH2/SH3 domains. Mutant viruses with Crk-SH2/SH3 domains induced tyrosine phosphorylation of cellular proteins, hut mutants with phosphatidylinositol-specific phospholipase C--y or Src SH2/SH2' domains did not. However, the mutant proteins with heterologous SH2/SH2' regions were able to weakly associate with sonie phos- photyrosinc-containing proteins in virr•n. These results indicate that in the context of the P47^""' structure, the requirement of Crk-SH2/SH3 is more stringent for its activ- ity to induce cell transformation than to cause phosphorylation of cellular proteins. 26 27
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M:d1,uila, M.. Rcichman. ('. T.. an l!l nru%ucn, 1l. .Inarnal o/ Virology hh( I l:115 - 12 1..I:muarv I()(12. Othcr enhlxirl: National (':uiccr Inllilulc and Nalinnal Imtilulcs t,f I Icalth. Pri,m "I'hc Rackcfcllcr I Inivcr.ity. New Ytirk. GI',NIi"I•I(' ANI) MOLE('(II.AR ANAI.YSIS OPl'IIE: AII RP.('IiPTOR AND OP ('Y!'lA1 GIiNI? I?XI'RE.SSION The Ah receptor is a.u~luhlc protein complex that nudialcti carcinogcncsis by a wiclc range c,l cnvirnnmcnl;d (,t,llutants. inclucling Irnlycyc•lic ammatic hyclnxarlxins, hctcrtv yclic :unincti. and rolychhirinatacl aromatic c•t,mlrnunds. The best undcrsicind activity „f Ihc rcc•cpti,r cnnccrm il,~ role in the incluctinn of• cykx•hromc 1'4501A I• We undertook a umratic• c•cli genetic analv%ie of P45O1A I inductic,n using the mouse hcpatoma cell linc. Ilcpa-I. ('hmcs cif' Ilcpa-I were isolated that arc dcfcctivc in induction oI' P45O1AL. Eviclcnc•c was obtained that the chrncs are tmnational in ori- gin. C'cII fueinn crpcrimcnl.,, dcmometr:ttcd that a Icw ol Ihc nntlanls are d<mtinant, whilc thc majnrity arc recessive. '1'hc dominant mutantc were shown to xynthctii/c a rclrrcs,,tr ol I'45(l1A I Iranscriptirnt. The recessive mtn:mt.ti were a%signcd to 4 ccttn- plcnuntatiom groups (probably corresponding to 4 dif7crcnl gcne.,;). ('umrlcmcntation group A ctirrespnncls to Ihc I'45OIA I structural gene. Mutaliunti in the 13. C and D gcncN ntl affcc•I functiuning of thc Ah receptor. A"rcvcrxc sclcction proccclurc," w'hcrc•hy cclN that express I'45111A1 inducihility can Fx• solcctrd I'rom a majority pohulati„n of crlls Incking induc•ihility. was developed. The reverse sclcclion Prcxc- dnrc w;,t,. n.u•d In i«olafc Irantifcclants nl" rcrrc.cnt;uivc recessive mutants in which Ihr mulati„nal di•Ic•rl. ire c•„mhlcmrntcd by cxogcnc,usly applied gcnomic DNA. A htnnan DNA dviivcil han.fcctanf nl' a('- niutanl wa.e usccl to clone fhc human C gcnr. "1'hr (' gcnr i,; not the li} ;md-hincling ~nhunil (iI the All rccchtor hut isa rrnlcin thal i1 mynncd I'm Iraneh catiom of Ah rccchlott-Iigancl rutnplcxcs In,m c•ytoplasm Ici nnclcuti. In an:alnpnu~ t•rpcrimcnte Ihc clnminanl gene f"rnm one nf Ihc dontinanl mutanl~ .va. lian."fcctivl inUi wild lyhc .11cra-I ccllc. Suc•cces in Iransfccting the dnminant prnc "h„uhl ( ruvidc Ihc ineanti fnr cloning it. 1lundincrnr. O . lin,nk%, 13. A., Wcir-Rrnwn. II. I., Ihotlman1 li. C., .hihn,;on, 13. S., N;rnthur,.L, I I_, and Wals„n. A. I. Flinchimic 71:(,I 66, 1'/'/1. OIhcr.urh„rl: National (':inccr Insliluhv. Vnnn the I.ahor,itory (11 Iliomrdic•al and I:nvirommcnlal Sciences. I)cparlmcnt of PafhoIoLy. School (11 I)cnli~try, and School (if' I'nhlic Ilcalth, Ilnivcrsily nf ('aliforni:i, Loti Anlx•Irti. 1)InXIN- ANI) All RI:('1:1''I'OR-hl?PI;NI)I:NT 1'RO"I'I:IN I;INI)INO TO tliNOI31OTI(' RESI'ONSIVE EI.I:MIiN"I:S ANI) G-RICII I)NA STLII)II'sl) RY I.V 171Y) I;O(YI'PRIN'1"IN(i I)NA-prolcin inleractionti hcforc an(1 allcr Iranscriptional activalinn ol" thc rar- Cinngcn- :md ditrxin-inclucihlc cnh;mccr trl' thc murinc ("YP1A1 gene were (Iclcctcd ill ill rinl hy treatment with (limcthyl xulfatc liillowcd by ligalion-mcdiatcd, lolymcra.c c•h;iin rcaclion-aidcd genomic sequencing. Pollowing 2.3,7.R-letrachlorodihinzo-p- dit,xin (T('DD) treatment of mouse Ilcp;t-I hcpatcnna cclls, evidence crf' protein binding wax detected at thc sequence 5' C'ACC('NA(P 3' within two prcviouay clcfinccl xcnohiatic response elcmcnts (XRlis). The obscrvecl XRE footprint was sim- ilar to Ih;d previously idcntificcl by ilt ri(r•n mcthylalicm protection footprints and attributed to the binding protein for 2,1,7,R-tetrachlorcxlihenr.o-p-diuxin The Alt rcccptor. No XRE footprinling was observed in Ilcpa-I mutant cells possessing a dclcctivc illi receptor. Llncxpcctcdly, evidence ol' protcin binding was also dctccled at a G-rich DNA sequence inmicdiatcly adjaccnt to one nf the XRES. Footprinting nf the G-rich sequence element, like that of XREI and XRE,2, was dependent on the prcuncc of" a functional Alt rcccptor. The Ab receptor is therefore able to hind to its own DNA target sites ill rirn and is also required for the binding of a second factor to the G-rich clcmcnt. Watson. A. J. and Narrkinsnn, O. Thc Journal (if Biological Chemistry 2h(,( IO):fi874-6R7R, April 5, 1992. Other suplort: National Cancer lnstitutc and U.S. Department of Energy. From the Lat>trratory of Biomedical and Environmcntal Sciences. School of 1'uhlic Ilcalth and Department of` Pathology and Lahtttatory Mccficinc, University of" ('alifomia, Lns Angeles INVESTIGATIONS ON THE All RECEPTOR AND CYPIAI GE.NF. EXPRESSION Cytochromc P45O IAI and P45(1 lA2 arc the principal P450s involved in the activatinn of carcinogenic chcmicals. 1'45(1 IA1 is the principal P45O responsible for aryl hydrocarbon hydroxyla,,c (AlIll) activity, which is involved in the activation of polycyclic aromatic hyctmcnrhrni. (PAlls) such as hcnr.o(a)pyrcnc (RP). PAlls are iniportant carcinogenic Compunenl.% ol' cigarette smoke and smog. The mcchanism whcrchy the Arnl protcin transkxatcs the ligand-binding suh- unit of Ihc Ali receptor tn thc nucleus is being investigated. Efforts to clone Ihc B, I) and cloniinant genes arc Ix ing continued. The question as to whether allclic dilTcr- enccs in any nf the,;c genes explain The observed hetcrogeneity in degree crf inducihility ol' 1'45(1 IA I-dcpendcnt AIIH in The human is being pursucd. Thc hctcrci- gcncity in inducihiliry appears to lcacl to heterogeneity in susceptibility to cigarette- induced lung and nthcr cancers in thc huntan. '1'hc Ah receptor appears to he invnlvcd in thc diffcrcntiation and proliferation of cpithcli;tl Gssuc. The lissuc and developmental expression (if Ihc arnl gene in thc mouse will hc investigated. 'IYamgcnic mtwu strains hrnnorygous for an inactiva- ,(t 29
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linn mtttatinn in Ihe mrrl )tc•ne will he Pertcraleil in nt'rler to ~Iwlv Ihe role of Ihe encnded protein in devch,hntenl. 'I hctic qudir, will ;rl,r, he ;irl,li,•d In the other gence ;i. Ihey are ehtncd. `lhc ctact site on tlle upqrrmt region nf Ihe ('vPIaI Lcne, wherc the cletntinant rc•Lressnt' nclti i" hcing defincd. and Ihe mr dr of' actinn of this repressor, are being investiy;rrccl. !lundirrvm. (l.. Rrnok%, I;- A„ I Inl7man, li Jnhnenn, 13. S., Nanthur. J., Rcvcs, 11-. Wahr,n, A..1., and Weir-Rrrtwn. K. I. In: Rrx•k, K. W.. Gerr,k, W., Matern. S.. and Srhnticl, R. (eds.): Ileratic ntetaMtlisnt and hisltusitirttt r,f F,nrho- ancl Xcncthiotics. Kluwer Acaclentic• Puhlishers, Iktrdrecht/Rn~lrtn/Lnnd(1n. pP. 115- 12 1. I91N). Other ;uPrnrl: National ('ancer In.litule and 1)epartntent of Energy. Frnm I,ahr,r;rtrtry nl 13irn»eciical anrt F.nvirc,nrncntal Sciences, School of Public Ilealth. and I)eP;rrlntcnl nf 1';rlhuhtgy and Lahrtratrtry Meclic•itte, llniversity of ('alifnrnia. Lns Aqt'cles. A131iRRANT EXPRIiSSION Oh'I'I I1; c•-e,-hR-2/nerr I'ROTOONCO(;I;NI? IN OVARIAN ('AN(T.R Ovc•rexpres.inn rtf thc r-r•rhR-2/ncu prutonncrtgenc has recently been shown in ovarian tumrtrs collectc•cl from thc tlnited Statec It is known that envirrntmental and cuhurrl factnr% may cctntrihute to certain types of rmcer, therefore, we examined exPres.irm „f c-r•rhR-2/rrrn in ovarian luntnrs collected from China by immunnhisto- chemical aaining. Out of K I tumor xpecimenti, 57 (7O.4rYr) were found to he intntnmtPrttiitivc. wheresrs only one r,ul nf 17 (5.9'%.) nrtrntal ovarian tissuc samples was sli)thtlv hrnitivr, Our results indicate that nverexpressictn of c-erlr13-2/nr•u is a grneral Ithemomennn for ov;uian c'ancer regardlexs of ctilTerent popnlation. Tet search Ior a c-e•rhlthrrn nven•xlnessing cell line for future stuely nn ntobec•uL•rr mechanism, we also amnlvieci 1.3 cancer cell lines from the fentale genital tract for expression of c rrlrR 2/nen. The c-r•rhR 2/nrrr RNA was found to be overexpressed al least I(Hl- Iolel in r,ne rif Ihc four m,rrinn cancer cell lines exantinecl. An aberrant c-r•rhR-2/nru RNA ..a% uko Irtund to he overexpressed in this cell line. Southern blot analysis indir;ucd Ihal the c,•rhlt 2/rlcu was amPlificd 2-4-folcl in this linc, and some of these alleles have sunclurrl alteratittn which may ttccctunl for expression of the aberrant c-r•rhl3-2/nr•rr RNA. Since the 24-fold Eene amplification i.c nett profxtrtional to the ? IOtl-frrld rtvcrerprecsirtn in RNA, other ntechanisntc such as transcriptional or prtsllranticrilttir,n;rl c•rmtrrrl must he involvecl in overexpreti.cittn of this gene in ovarian cancer. /!rr»X. M.-C'.. 7.h;tng• X.. Ytm, I).-I I.. 7hang, I1.-7,., Ilc•, G., 7,hang, l'., and Shi, D. ( :utc'ct' LcUcrti (,I:r)5-I()i. 1942. Other 1 uPpnrt: Mnr(h of I)imc•ti Rirth Dcfccts I,'oumlatinn. I r,m the I)eparlment (if '1'Itmrtr Biology, Ilniverxity rtf `Fex;tx M.I). AnJcr.nn ('nnrcr Cenlcr. IHwqnn, and I)cpartment of I'athnlogy. C'ancer Ilo%pital/('anccr lil,litwc_ ShVrrtFhai Mcdical I Iniversity. Shanthai, Pcuplc's Republic of ('hina. SIiI,E('TIVE REDISTR113UTION OF PROTEIN KINASt; C 1SO"l,YMIS BY 1'l1Al'SIGARGIN ANL) STAUROSPORINE Protein kinase C(PK(') is the major cellular receptor tumor prnmoting phorhol e~tcrs. I'horhnl eslers activate rr-, S- and F-PKCs in GH CI rat pituitary cells and cmisc their redistribution frnm a sttluhlc to a particulate fraction. We have now rhar- ;rcteriicd the effect of several non-phorhol ester tumor promoterc on PKC isozyntc distrihution in (iF1 C, cells. The incomplete tumnr promoter ntezerein caused recliti- trihntinn of a-, (3-, Fi- and e-PKCs. Thus, it did not display partial agonist activity. 1-he rhnsphatase inhibitor okaclaic acid (licl not cause redistribution uf any ixozynte. The calcium ATPasc inhibitor thapsigargin and the scr/thr kinasc inhibitor stau- rnsporine caused redistribution of e-PKC and, to a lesser extent, fi-S-PKC. AlthnuFh the mechanism of the selective effect on Fi- and e-PKCs is not yet known. these clata clearly clemonstratc that their suhccllular distribution can be regulated by a pathway that does nnt influence a- and (3-PKCx. Phrtrhnl etiter activation of e-PKC was ;rssrt- ciatccl with appearance of a more slowly migrating immunnrcactive han(I in the par- tic•ulate fraction- 13nth e-PK(' forms accumulatecl phosphate during phorhol ester trcaunent. The phosphorylated forms of e-1'KC were preferentially recovered in the particulate fmction. Al(hnugh staurosporine cause(1 reclistrihution, it prevented the phnrhnl dihutyr;tle (PDBu)-mccliated appearance ol' the tipper hamd of the Qnuhtct ancl the increased phosphorylation of both hands. The PDliu-mediatecl redistrihntiim af (Y- and (3-PKCs was not inhibited by ltaurospnrinc, even though titauro%ptrinc effectively inhibited PKC catalytic activity. ThereAore, catalytic activity is not rcquired for redistrihutinn. Kilcy, S. C.. Parkc, 1'. .L, Fahhro, D., and Jrrkerr, S. Carcinogenesis 13(1 I): I rri)7-2(N) I, 1992. Other support: National ('anccr Institute. Frttnt W, Alton Jnne. C'ell Science ('entcr, Lakc Placid. NY: Imperial Cancer Rescarch Fund, Lonckm, England: and I''harmaccutical Division. CIRA-Geigy Ba~cl, Switierlancl. POI,Y('Y('LI(' AROMA'I'IC IIYI)ROC'ARRON METABOLISM IN RAT AI)RENAI,. OVARY, ANI)'IIiSTIS MI('ROSOMl?S IS CATAI,Y'l.1:1) BY TIIF, SAMV. NOVI?I. ('YTO('I IROMI:1'45O (I'45ORAP) A novel A('TII-imlucihlc P451), cvHtc•hrunte I'45ORAP, is responsible fnr INtly- cvclic arrnnatic hydrrx•m'hnn (I'AI1) metaholicnt in nude rat adrenal micrn~'antet . 10 11
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P45ORAI' is prc%cnl at similar Icvck in malc and k•malc adrenal microsomcs and is inmtunnchcntically distinct from P45t)IAI. Anti-P45ORAI' immunnhlolc a protein present in ovarian and tctiticular mit•rosomcti that is thc same siic as P450RAP and which coclutcs with the 1'45p fraction during chromatography on an inmiohili7cd artificial mcmhranc coluntn madc with rhosphatidylcholinc. Ral adrenal, ovarian• and Ic~licular micrononuc exhibit similar rcgio-sclcctivitics in thc mctaholism of two pnlycyclic aromatic hydrocarhon., dintcthylhcnz(a)anlhraccnc (DMRA) and hcn/o(a)Pyrcnc (R1'). llnlikc P45OIAI, these microsomes fnrm little or no 7-OH- I)M13A and RI'4,5-diol hut do catalyrc the formation of a high proportion of thc prc- sumrtivc hrocarcinngcn, UMF3A-3•4-diol. Thc rclativc ac•tiviticc of PAI I metaholisni by untreated adrenal, tetiticular, and ovarian rnicrosomcs arc approximately 60. 20, and 6 pmol/mg microsomal Prntein/min. respcctivcly. PMSG induced PAII metabolism 2- to 5-fold in ovarian microsumcs and also increased thc P45ORAP immunohlot. Hypophyxcctomy rcduct•d PAII mctahnlism 1-fold in Icsticular tnicrosomcs while also decreasing the P45ORAL' immunohlnt.'I'his close correlation between PAII metabolism and expres- sion of P45ORAP indicates the involvement of thc cytochromc in this activity. DMRA and 1311 metabolism by I'MSG-trcatcd rat ovarian microsomes and untreated tcsticular microxnmcs are each completely inhibited by anti-P45ORAP hut are not inhibited by anti-P45OIA I. Es%cntially all of the PA1I metabolism in rat adrenal, testis, and ovary is, therefore. c:nalyicd by P45pRAP, which is hormonally elevated in cach tissue hy;t variety of possible mcchanisms• including induction and sclcctive hmlifcralion rrf cclL. that cspresti this protein. (hto. S., 13hatlacharyya• K. K., and Jr•(rnnle. ('. R. I?ndncrinology I; I((i):3O(,7-1O7(i, 1992. (Other xuphort: National Inaitutcti nf Hcallh. From the Department of Pharmacnlot±y. University of Wisconsin Medical School. Madi."cm. ONC'OGL~.NI(' AND TRANS('RIP'I'IONAL COOPF?RATION W1T11 HA-RAS R1'sCJI IIRI:SI'IIY)SI'I IORYLA'1'ION Oh c-Jtm ON SI:RINfiS (,l AND 73 Rcccnt adwmrce indicate a link between tunmur Promotcrs, transforniation, and AI'-I activity. I'iatcin kinacc (' activation increases AP- I DNA-hinding activily inde- hcndcnlly ol new protein 1 ynthcsi.ti. AP-I is also stimulatccl by transforming onco- hrntcins and zrowrh faclors. Thc.c prntcins are Ihought to participate in a signalling cact•atlc affcctinp thc nuclear AI'- I t•trmr6cr comhoscd of Ihc Jun and Fos proteins. Rccausc c•-1un is thc mna Wcttl tt'amxactivator in thc AI'-I complex and is elevated in I la-rn.c-Ir:m.fnrmcd ccllti, in which c-Fos is dnwnrcgulatcd, we focuscd on it as a Pnlcntial targcl, c-.lun crnuld convert input from an oncogcnic signalling cascade into changcs in gene cxhressiun. Indccd• Iranstormatinn of rat embryo fihrohlasts by c- .lun rcyuires an intact transcrirlitmal activatitm domain and coopcration with onco- gcnit• Ila-rn.c. I?rprc"itm nf omcogcnic Il:t-rn.r augments transactivation by c-.lun and ctimulatc%. its Pho>phorylatinn. Hcrc we describe the mapping of thc Ha-r•u.c- resPrnnivc photphorylatiun ,itcti to urincs 63 and 73 of c-.Iun- Site-directed nnnagc- ,t,i.,i, indic:dcs that phosphorylaliim of Ihcsc scrincti is essential for stimulation nf c- Itttt ;Iclivity and for coopcratiom wilh I la-reu in ocogcnic tranxforniation. tintc;d.'I'.. Rinctruy, R., Mcrt'ola, D. A.. Rirrcr, M.• and Knrin, M. Nnturc 354:•J'14-49(r. December 12. 1991. Othcr muPPort: National Instittttc% of Ilcalth. I:rnm the Departments ol' Pharmacology. Biology and Pathology, Center for MoIccuhtr Genetics. Llnivcrsity of Catifornia at San Diego, La Jolla. and 1)ivision of Cancer Prcvcntion and C'ontrol, National Cancer Institutc, Bethesda. MD. A13ROGATION BY c-m,v(- OF GI PHASE ARREST INDUCED BY RB PROTEIN R111'N<)TB'Yp53 Inaclivating mtnations of the rctinnhlastoma (RB) arc found in a wide variety nf Iumour cells. Replacement of wild-type RR can suppress the tumorigcnicity of sonic nf these cclls, tiuggesting (hat the RB protein (Rb) may negatively regulate cell growh. As activation of c-nn•t• expression promotes cell proliferation and blocks dif- ferentiation, it may positively regulate cell growth. The c-mvc, protein is localizcd in the nucleus and can physically associate with RB protein in rilro, hence c-nivc may funclionally antagonize RB function. Microin,jection of Rh in GI phase reversibly arrests cell-cycle progression. Here we co-inject RB protein with c-mvc. PJ-ra.c, c-/im or c;jnn protein. Co-injcction of c-ntrc, hut not EJ-ras. c-(i).c or c-jmt, inhibits Ihc ability of Rh to arrest the cell cycle. The c-myr docs not inhibit the activity of an- othcr tumour suppretisor, p53. Thus, c-nrw• and RB specifically antagonize one another in Ihc cell. Gaodrich, D. W. and Lee, W.-H. Nature 36O:177-179, November 12, 1992. Other support: National Institutcs of Health. From the Center for Molecular Mcdicinc, Institute of Biotechnology. University of Texas Flcalth Science Ccntcr. San Antonio. MOLECI ILAR DIVF.RSITY OF NEURONAL-TYPE CALCIUM CHANNELS IDFNTIFIED IN SMALL CELL LUNG CARCINOMA Using the polymcrasc chain reaction (PCR), we identified RNA transcripts for Iwo dixtinct classes of neuronal-tylx voltage-galed Ca" channels (VGC'C) in a protn- typic small cell lung carcinoma (S('LCl cell line. SCC-9. Oligonucleotide primers were dcsignctl to encode amino acid sequences cornnron to a-subunitti of known ncuronal VGCC classcs. Scqucncing of complementary DNA (cDNA) cloncs 32 33
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dcrivcc) Ir(,nt two indchcntlt•nt I'('R Imoducl, rc•vc;!Ic'r) Ih:n mnc rnrr"llntnlccl to a hr:tin cl:ta A V(i('(' Itaimcnt prcdiclcrl In c•ncrulc :; I' Itlrr V(i('(' (imcnsilivc Icr <lihychc,hyriclinc~ and m-t'rmnlr \in) chnractcri"l!C nl c cn hcll:!r I'u!kinic ct•IIs hut not Irrcviou"lv i(Icntificcl in hum;ms. I-hc tict•rmcl 1'('R (irnrluct w•;ts identical (crt•cpl Inr onc crmscrvativc nuctcc,tidc dilli•rcnccl In a fr,tgmcnl of thc cla•. I) V(i('(' of ncu- trms and ncnmmndrrcrinc ccllti, which cncrtidcs an I.-lypc VG('(' (sensitive to clihy- clrnlrvriclincsl. fly Nrnlhcrn blot anatyst•1,, hnth cI)NAs hyhridiiccl to mcsccngcr RNA,, (tnRNAs) ohtainc(i hnm .S('(' <): cla« 1) hyhricliicd arhliticmally tn hunrnt cerebral cvortical mRNA, hul ncithcr hvhridiicd to mRNA from the ekclctal nutscic c•ell line 'I'I'.(r71. Alth!wzh no cl)NA t•rrrretiponcling lo e•laee R V(iC(' (N-type) was i<Icnlificd. S('I.('x arc known lo express VG('(' that arc• scn.ilivc to co-conotoxin and cnlnrcipitatc with '"I-lahclctl-rn-nmr~tctxin when complexed with scrum Ig(i from halicntc with Ihc L:mthcrl I::unn nt,y;tsthcnic cynclrontc. The ntulliplc classcs of ncu- t'nnal-lylm V(i('(' crrrc«cd in S('I.(' could c•rmccivahly have hnth unique and rc- I:ncd ;mtigcnic dctcrntin;tnts Ih:n mey rivi• ri.c tn antincurnnal auloirnrnunc respons- c%. 'lltiti w•rmhl acccwnt lirr ashcctnnn r,f pvancnpiaslic ncurvrlogic disorclcrs includ- ing Ihc I amhctl Iialon .ticnch-omc ;mcl 1 uh:rcutc c•crctx Ilnr dcgcncration. O}:um Oknnn• M.. (iricmt:mn. (i. I`... Wichc•n. I?. 1).. Slaymakcr. S..r., Sm,lch.'r'. P.. and Lr•lutnn. 1'. A. Mavo ('linic I'mcccdinw. (,7:1 I SO-1 15<), hcccmhcr 1992. Other euppnrl: N:ninu;tl (':mccr Instiltuc• Mcdical Research Council of Canada and Ih,warcl Ilughcc Mcclical In.tittnc Inlcrnalicmal Research Scholars Program. Frnnt Ihc 1)ivi.irnt (,f Intmunohatholugy and Ocpartntcnls of Imnnrnnlogy and Ncurr,h, cy. Rinchcntiary and Molct'ulnr Riolttgy, Neuroscience and 7,oology, and Biotechnology Iahor:unry, I Inivcrsity ol Rritish ('nlunihia, V;rncrn!vcr, Canada. 'IIII: . MYR I)Nr1 FiINI)IN(i DOMAIN IS I11(iIII,Y C'nNSI?RVI5I71N I)Il '7 ) t6I Fl ll rrL1 I)I,S7 Y)1l )li 1/A9 1'hc• r,rrt•h hrr,tn.nnc•oLonc encodes a prcrlcin Ihat is highly conserved among hink :tnrl mamnutl"- Ihc ontinntcrminal dr,m;tin of c-Myh contains three imlTCrfcc( tnnrlctn !rllr;nh r1f ahlnrrximatclv SO antinn aci(IN each. 'fhiti dnntain is rcquir'cd for I)N \ hinrhne ;!n<I ha~ ;ilc(, hocn crmsc•rvcrl ki varying degrees in invcrtchratcs• plants ;uul vr:lq. <;ivrn Ihal arr07-mhnccl kcnc,, arpcar to control ccllnl:;r dif7crcntialion in a v;tricl.- nl cnr:nvr,tic `.v"Icm.. Ihc prcscnc'c of a nrt•h gcnc in Ilrc cellular slime molcl 1)irh•frtrrlirnrt rlicrnirlr•mn ntlgh9 prnviclc ;t Ira!clahlt• sytilcm le~r tiltt<lying thc role of mrh in rlillr•!rntiatinn. I)r}tcncrsqc r,liinnvclcc~tirlc primcrti cncc,ding rcl!icmx Ihal are hiVhlv crm.r! X'ccl in the vcrtchr:uc am1 Orn.crqrhiln Myh l)NA-hinclitts clomainx .vcre uurt to nmhlifv a rcl;n(,d rlnmain Irnm I)irt , ~r,.c(r•lirrnr gcnnmic• I)NA, which was then uticd Uo itol:ur ot }tcnrrmit' rhmc. -1'hc hnl;nivc I)NA-hincling tlotnain of Uictwxtclitmt Mvh i~ ae rlnwIv tclatcd 14) crrtchr!Ic c-Myh m is I)rmnhlriln Myh l65(4, ictcntily), w•hcrc:t~ thc knmtn Myh-ti•I:rtccl lin!tcinti rrl ptank and yc:!~t :trc more ditit:tnllv rcla- crl Tltc crm.ct'vcd rk mnin c l I)irnn.tr<•lium Myh i,, capable r1f hintling to the same I)NA sctlu!•ncc n~ thc vcrtrhratc and I)r,acnhhilu Myh prolcin%- fhc remainder ol'Ihc dc<fitccd ,!minn :tcirl wqucncc of 1)ir rrncl<•lirmr Myh t how~ no hnntology to the div<'rgc'nl th,mains of Ihc knuwn anim:tl, plant ancl ycast Myh-rclatc<I hrndcine. I,r!lutic~n:<rv analysis imhlic" that thc dnrlic:dions thal generated thc rcpcalc (1l Ihc \I\h I)NA-hincling domatn hcg:m prirrr to the divertcncc cif anintals. Idant.. ccllular .litnc nmld,; and yeast. titnhcr-crasscr• ll., ltrytlulf, B.. Rin• X., Grmscr, F.. Firtel. R. A.. and hip.cit l..l. ,S. Oncrrgcnc 7:5)i9-59(i, 1992. OIhcr surrort: Antcrican Cancer Socit•ly and II.S. Public Hclath Service. 1:rom the I)cparimcnt of Microhiology. School of Mcdicinc, Statc University of New York. Slony Brook A I IIGHI,Y CONSERVED CYSTEINE IN THE v-MYB DNA-BINDING DOMAIN IS ESSI:N'1'lAL FOR TRANSFORMATION AND TRANSC'RIPTIONAL rrr.nNS-ACI'1VATION "1'he v-Myh protein is nuclear, binds to DNA in a sequence-specific fashion, regulates the transcription of various reporter genes and transfarms myelomnnoc:ytic cells. Cystcine is one ol' the most conserved residues during protein evolution and has been implicated in DNA hinding, protein-protein interaction and redox regulation ol' various proteins. Therefore, we have now individually substituted each of the seven cystcincs of v-Myb with a scrine. All seven mutant proteins bound to DNA when they were expressed in F.. t•uli. However, mutant C65S neither (ran.c-activated transcription in vivo nor transformed mycloi(1 cells, although it was transported into Ihc nucleus. This cystcinc is conserved in the Myb-related proteins of animals, plants, yeast and the cellular slime mold I)ir•tvn,clelium dist•oideurn. The C65S mutation and a nearby codon insertion mutation also abolished rr•arr.c-activation by fusion proteins. containing the v-Myh I)NA-hinding domain and the strong constitutivc activation domain of herpes simplex virus (IISV) VPI6. Because this domain of VPI(i appears to activate transcription whenever it is bound upstream of an appropriate promotcr, these resullti imply that C65 may he required for high- affinity DNA binding in rirn. In support of this hypothcsis, we have also shown that, in contrast to wildtypc v-Myh• mutant (Y,5S is unable to block transcription from a reporter gene in which Myb hincling siteti overlap the initiation site. Cirasscr, F. A., LaMontagnc. K.. Whittakcr, L.. Slohr, S., and Lip.cirk..l. S. Onc ngcnc 7: I(N)5- IOt)9• 1992. OIhcr snhporC U.S. Public I Icahh Scrvicc. From the Institut f(ir Mcditinischc Mikrohiologic und Hygicnc, Ahlcilung Virologic, t)nivcrsit:itskliniken des Saarlanclcc, Ilomhurg, Gcrmany, and l)cpartmcnt ol' Microhiology, Schonl of Medicine. State I Inivcrtiity nf Ncw York, Stony Brook. 3.1 15
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('A.tiI•. ItI'I'OI2T. I;YNIi('l)1 Ol;l(' ('AN('f;R l'I.I II?.ti 'I'O I YN('II SYNI)ROMli II I)IA(iN(/,CI.S: A FAMII 1' IZI'.I'ORI' I.ynch .yndrnmc II wa", di:,)1nrncd wficn twr, ~iqt•t. ntnnili•sk•d cat IV-rnttiel tiyn chrr,nnu" carrinr,m:t', nt' tile uvary and cndnmctritun and a third sister was firund In have I)uke'ti A c:,rcinr,ma nf' thc cecttm. A tlctaileel cancer I'amih, histury inelit'atecl paternal Ir:tn1 mis%ir,n r,l thc ck:Ictrrinu% gcnnlyl,c. The pattcrn nf carcinrana r,f tile cnlnrecltnn and crlrac•nkmir snce Ihmuzhnut tile crlenclcd Iamilv wa" then found tu Ix• ctrnmnanl wilh Ihi% hercclit:try ranccr prone disorder. I.ynch syndrome II may he exceetlingIv tlillicnlt to rliagnmc (luc to an ah%ence nl' premcmitory clinical sig.nti or hir,m:rrkers t,f cenntyl,ic %usceptihilily. Its recognition is therefnre dclmndenl on a detailed canrer f:unily hi%tr,ry (all an:deunic sites), coupled wilh knowledge of Ihe pancrn nl' Ihe cancer til,cctrum. ditittihutirm, ancl natural histc,ry, as manifctitccl in this hereditary clisr,reler. We descrilx tile decision logic that wax involvecl in tile diagnosis of I,ync•h synclromc II in Ihis family and indicate the importanl role of thc gynccolo- gia in this hrncc.«. I,rnr h, ll. 1'.. ('avaticri• R. .1., I.ync•h..l. F., and ('atc_v. M. J. (iynecc,lneic ('1ncolnzV 4-l:It)k-2(li, 1992. riOIher snppnrl: National ( ;mccr hiclilulc. Fnmt Ihe heparlmente rIf Preventive Meclicine/Puhtic• Health nnd Obstetrics and Qynccr,h,gy, ('rcighlon 1 Inivct,,ity School r,l' Mcclicinc. Omah:t. NIi. IN'I'RA('1,'.1.1.tII,AR TARGIiIINC; OF ppf.O^ l:Xl'RRSSION: LOCALIZATION OF v-.crc'IY1 ADIII:SION I'I,AQIIKS IS SIIFPIC'tl?NTTO'fRANSFORM C'I II('KFN I:MRRYO FIRI2Ol;l .AS'I :S Tr, rlrline the el'fecls rll p),h(T -° activity at different intracellular siles, we have cnn~thurtcd chimcric molecules which target tile pph(P ^ kinase to spcc•ific intraccl- lular Inc:dinn.. pp(,(P " was targeted to the nudcus by insertinn of the SV4(1 large T nntipen nnclcar k,calir,dion tiig.nal. Nuclear pphO• ^ was activc as a tyrosine kinase :nul hhr,.ldu,tyl:tlccl mtalcar proteins at tyrc,sine. Flowever. ccll.s cxpre.sxing the nuclc:u ),I,hl) • wcre hhenr,lypirally normal by a number of criteria. and nuclcar.crv kinm~r• rhd nr,t indtur tile expre%siun nf :tn mRNA (('F.F-4) whose induction is char- acn•rititir, i,l u~amfr~rmatinn hv wild-type v-a,-r~, ppf,()' .. was targeted to pcrinuclear mrmhrnnr~ by fu~i,m to rrrl growth hormone and vesicular strnnatis G protein wquenrc1 . ('cll,~ expressing Ihiti chimeric molecule were phennlypically normal by rnrr.t cr iterin. I low ever the rerinnclear .crr protein did inclttce elevaled levels of (1?F- 4 mRNA. indicaling that Ihe v-Arr kinaw expressed at this site inducc.% partial Irans- li,rmatir,n. "I'hc v,%rr ;md activated csrr• kinaties were targeted to adhesion plaques by fusion to tile Ialin-hindinL Seyuenc•e nf vinculin. Cells expressing these fusion prntcins were Irawf,n•mcrl hy nir,rphnh~Lical. phyeiriingical and hicxhcmical critcria, althnuf!h Ihc R,ci induced hy thc~c viru~es were distinct from those induced by wild- lype v-.crr. A c•hinieric protein which lar}eetecl c-,crc to a(Ihe.ion plaques was not IramG,rminL. Thu% targeting hph(1•• tr, adhesion playucs. although not sufficient to activate 1he Ir:m'lnrn,ing capacily r,f c.crr-, is wl7lcicnl Iu allow transformation hy v-.crr. Lirhl, l:. C• :rncl Mrrr(irt, G. S. Onroccnc 7:2417-2429. 1992. Othcr.%upport: National Institutes of Health. I:ram the Department of Molecular and Cc11 Biology. University of ('alifornia, I;crkcley. HOST RANGE MUTANTS OF v-.vc: ALTERATIONS IN KINASE ACTIVITY AND SUBSTRATE INTERACTIONS Host range mutants of Schmidt-Ruppin v-•crr that transform chicken embryo fihrohlasts (CEF) but not Rat-2 cells were generated previously by linker insertinn- (Iclction mutagenesis (J. E. DcCluc and 0. S. Martin, J. Virol. 63:542-554.19R9). nnc of these mutants, SRX5, in which Tyr-41fi is substituted by the sequence Ser Arg Asp, retained high levels of kinase activity in vitro and in r•ir•cr, Ixith in CEF and in Rat-2 cells. Phosphorylation of p36 (the calpactin I heavy chain) was drastically recluced in cells expressing SRX5 ,cre•, suggesting that the phenotype of SRX5 results from an alteration in substrate recognition by the srr kinase. Three mutants, SPX I, SIIX13, and XD6, containing linker insertions or sniall deletions within the .crr homology 2(SH2) region, induced reduced levels of kinase activity in both (TF aml Rat-2 cells. However, the resictual levels of kinase activity in Rat-2 cells were ahnve the threshold at which wild-type pp6(P "' transforms Rat-2 cclls, indicating that tile reduction in kinasc activity was not sufficient to accounl for the failure to transform. Cells infected by these mutants exhibited reduced levels of phosphorylation ol' 120- and 62-kDa proteins. We have reported elsewhere (M. F. Moran, C. A. Kcxh, I). Andcrson, C. Ellis, L. England. Ci. S. Marlin• and 1'. Pawson, Proc. Natl. Acad. Sci. I1SA 87:R622-8626, 1990) that r•ns GTPase-ac•tivating protein GAP and associated protein p62 are not tyrosine Phosphorylatcd in Rat-2 cells expressing SIIX 13 or XDfi. The transfonnation defect in Rat-2 cells may result from the failure to phos- phorylate those proteins. The fifih mutant, XD4. contains a deletion which rcmnves all of the srr• homology 3 (S113) and most of Ihc SH2 sequences of srr. The prntcin encoded by XD4 is active ati a kinase when expressed in CEF. indicating that in ('F'sF tile SI12 and S113 regions nf v-sr•c are not necessary for kinase activity and Iransfnr- mation. The XD4 .crr• product i-~ not tyroeine phosphorylated and is inactive as a kinase when expressed in R:u-2 cclle. 'f'hus, host cell factors can affect the Iyroeinc photphnrylation and activilv of thc r srr• kinase in the absence of the SI-12 and SH:( regions. These results indicate thal the hnst-clependcnt Iramformatitm phenotype results frrnn alleratinns in sr•r kinase activity and suh%trate specificity. Lichl, F's. ('., England. I,. J., 1)eCluc. .1. (i•_ and Mm•(in, (:. S. lournal of Virnlogy h(.(7):4315-4324..1u1y 1992. OIher suppnrt: National Intilitutes of Heallh. From the 1)cpartnicnl of Molccular and ('ell Bic,logy; llnivc"ity of ('alifornia. Berkclcv. .31. 37
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lllf: I.l I('KP; RFNAI. AI)HNO('AR('INOMA ANI) I'I:C III;RI'TiSVIIZIIS The m,rlhcrn leopard fnlg, Rruur is '4u-chtihlr tn fhc Luckc renal car- cimoma which mclasla.iic~ in a Icnlpcr,nurc dcllcn<Icnt nlanncr, 'fhc dilTcrcntiatinn pntc•nlial of Ihc tumnr }tcnnnlc can hc a«aycd hv nuclcar Iran.lllanlatinn (NT). N'T' of Ih1• I,ue•kc tuntor reeull. in .wimming Luvae lehich f•lil to feed and rlie. Rescue of N'I' titiwc. clcetincr) In llic, has been acc•nml11id1ccl by alingrafiing trilrloicl tumor NT cnihryn fragrncnl,~ to normal rlihhlid ho..tx. -I'hc alhlgralis pcr%ist ancl cliffc•rcntiatc a drvcr,,ity nf hisInhlgically normal tissues. The graliing prnccdurc, while permitting thc mr,st clcvclorrncnt yet reported from Iramlllantatirnl of a naclcus derived frc,m a frrig near or ;tl mctamorphnsis, is imrcrfccl hccauw uf Ihc nligralion of lymphocytes into the tissuc with suhsequcnl rejection. t'crnlutalirnl% ol' Ihc Lrafiing prnceclurc which avoid or abrogate immune rcjrctirm m;ly (xrmil furthcr ck'vclnpmcnl of Ihc alingraf)cc) tissuc. Thc ctinlnLical apcnt of thc• Luckc carcinorna is thc Luckc tumnn herpes virus I:I'V viri,me were harvc~tcd and rurificcl, and the viral DNA wa,~ cxtractccl, ~,iicd and cloned in a plasnlicl vector. Sludics arc in progress In investigate the Irans- Innning Ix,tcnlial rlf thcsc cloned scqucnccx. A tritium-IaIxIcJ viral DNA probe has been )lrcl.arcd fnr in sinr hyhriclirninn rlf Ihc Innlnr NT cmhryns and allc>,gralis. Strch t nidics c•an irlcntify whcthcr LTV pcrnisls in thc mitotic progeny (lf nunor cells which wclc induccd, to rliffcrcntinlc by N'T' amf alingrafling. h1rR'm„r11, R. (;., .Sr,nr'rhicr, W., Lust• J. M., Williams, J. W., Williams, C. S., Rnllins Smith, 1.. A. and ('arlcon, I). I.. In: Gahricch• K. (Mannhcim), Schlidgcr. R- (Frankfurt). 7,w;lrl, 1'. (Utrcc•ht), (F,ds.): 4. Intcrnalillnal ('r,llnqnium nn I'alhningy and Mcdicinc of Rcptilcs and Amphibians, Bad Nauhcim. Sclllcmhcr 27-:,4, 1')91. plt 2O4-21 R. OIhcr 4ullhnrt: Amcric•an ('anc•cr Sncic•ty- Nalinnal Inslilulcs nf I Icalth and National Science Fnund;llirnl. Frrml Ihc I)cp,lrlnunt rd (icnctics and ('cII Riology, l)niversity of Minnesota, Saint Paul: I)cll:rtnlenl nl Mic•rnhioingy. Ilniverxity nt' Minnesclla. Minneapolis; I)chartnrcnt nl ftiillogy. Tuxkcgcc University. "F'uskcgec• AT,: and F)epartment of Pcrluwics. Schnnl nf Mcdicinc, V;rnclcrhilt llniversity, Na,;hvillc. l'N. MA I RIX INI•I.I lf?N('I?S pRGAN-SIIT SI'1;('IFIC'ITY OF MIiTASTASES BY RF (;l II.A 1"IN(; I'RUI)1 t('I'ION ANI) RF'.SPONSI:I'O Atl'I'O('RINfi GROWTIT I A( "f(1Rti 'I'hc mr•t;ltilatic ellrcacl of Immor cells is not rl rrmdam prrx•c.s. ('harslclcrislic pal- tern"; rll rlieu'minatinn of vrccific• tttmor types have been known fnr more than I(W) vcarti and gac•c ri,,c U, tllc hvlrr,thrtiis <lf PaLCI in 1889, who su)*zcetcd Ihat varinhlcs in the lunu,r~ ('Ihc tiCC/I ) IIIClh wlth fllher,, Irl II1C IJSCIIeti ('Ihc wil') to diclalc nrgan- ~,ilc %Ix•rificilv of mcl;letatis- We havc identified variables thal ennstithnc il signifi- cant a"hcct of Ihi. '%rrcl-.r,il' ,IVnamic prncc%~: 'fhc inflncncc• of rrrgnn-tipccific ctlraccllul:u m;nrie chcmi.ry crn Ihc tumor cc•Ils' synthcsis of autucrinc growth fac- Inr, that in turn rctnl;nc ccll i1nw11t and dillcrcnti:nirm. Tissuc-srcc'ific extracts nrinc~cl in cxtraccllular matrix, hiomatric", were fnuncl to have diffcrcntial cf fccts gr„wlh in culture ol' nnrmal cellti versus tumors and minimally deviant versus lllct;l,latic tumor cells. Normal cells ouul minimally deviant tumor cells survived or _ri,c`, Only on hinmatricex from the titisuc frrnn which the cell IyPcs derived. By crnl- Inl~t. nlctaslatic (omnr cell lines were able to survive and grow at lower eccding den- %itics jrnrl c'll more types of hionutriccti. 'fhe ahilily to grow at low cell clensitics on li~~uc-,NPccific hi~rmatrices cnrrclatcd with thc organ-sitc-sfx'cificity of mctasta~is llf tl,c~c ccll lines in immunosuppressccl athymic nude mice. Studies In iclcntify thc rc•,rr,nxihlc component(s) in thc hiomatriccx indicated that only hcparins. the gly- c.is,mliru,glycan (C3ACi) chains of heparin protenglycans, nlatrix components present at the plasma membrane surfacc, restricted growth uf the normal cells and of the minimally deviant tumnrs, hut had no inllucnce on mctastatic tumor cells. Ilcparin's inhibition of growth nf a representative non-metastatic cell linc, the minimally clevihnl hepatocellular carcinoma. HerG2, correlated with its regulation of the xyn- thcsis and abundance of mRNAs encoding several atdcx:rinc growth factors, cspc- cially IGF II and TGF /3. Hcparin's regulation also strictly correlated with morphn- Ingical changes that included significent changes in nuclear size . Separate studies ongoing in the lab arc addressing the questions whether tissues that assume a quiescent statc, such as liver, contain precursor cclls slnwly producing a lincagc of ctaughtcr cells unclcrgoing a terminal cliffcrcntiation process. driven in part by matrix/hortnonc synergies. A concept derivative of the lineage hypothesis is that the precursor ccllc, not the mature cells in thc lineage, are thc targets for uncn- gcnic events. Thus, Ihc tumor cells arc predicted to hc transformed stem cells that are aberrant in their matrix/hormonal regulation: tharl tumor progression cnulci hc a reverse lineage proccss; and that increase in mctastatic potential should correlate with earlier anJ earlier stages nf thc lincagc, We discussed our data in the contest of these concepts anJ thc clinical implications if thesc hypotheses arc correct. Krafl, A., Zvihcl,l., Doerr, R., Chiutcn, f)., and Reirl. l.. M. In: Rahcs. H., Peters. P. I?., and Munk, K. (Eds.): Mctastasis: Basic Research and its Clinical Applications. Contrih Oncol., Ba.scl. Kargcr, Vol. 44, pp. 203-223. 1992. Other support: National Tnstitutcs of Health ancl American Cancer Society. From the Departments of Molecular I'harmacology and Microbiology and Immcmnlogy, Albert Einstein College nf Meclicine, The Bronx, NY. PROTEIN SYN'I'I IGSIS, ('I?I,1_ CROWTH ANI) ON(Y)Gf?NF;SIS Two lines nf investigation support a new hypothesis concerning thc role of pro- Icin synthesis in thc mitntcnic pathway. 'I'hc first is that a variety of mitngcns and rolcngcnc products incrca~c phoxphnrylation ancl thereby activate IF-4E?• which is involved in Ihc rdc-limiting Iranacr of mRNA to rihnsomcs. Thc second is that nvcrcxpression iir microinjcctinn ol cII -41: inclucce rapid cell proliferation ancl omcrl- gcnic transformation- ti {r)
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Rhnnrl.%, R. 1'. (-urrcnt C)hinicm in Cell Rinln)!v -1:I(llt)-1O24, I()')I. Other support: Nalinnal htstiltnc of (icnc'raI Meclical Sc•icn<•e.~. Frnm the I Inivercity nf Kcntucky. L.cxingtrni. ADAP-I IVI: EVOLUTION OF DEGREES ANI) KINDS OF NF,nPLASTIC 7RANSFORMAIION IN ('1'.1,1. ('ll1;l'URIi Iluman canc•ers undergo prntractcd complex development from benign to malig- nant states, a.ti most Ihnrrnighly dncmnentecl in the mnle-tn-melanoma eequcnce. The carly stage" of Ihe tiequrncc lend to redif7erentiate into normal tissues: the later stages prntiress to ever increasing multiplicatirni and malignancy. When placed trnder Ihe growth cnnsirainl nl' either crowding or low serum cnncentratinnsl the NIH 3T3 line uf mrnrse cells readily undergoes transformation, expressed in the development of fnci of ccll. Ihal c•nntinuc to mulliply :u confluence when the rest of the Pnpula- tinn has stnhrcd. If' Ihc nnntransfnrmcd cells arc maintained for 3 months by frc- qucnl Imv-dentiity ha,;eaRce in high concentrations of calf scrunt, they gradually lose Ihe capacity to undergo Irantfnrmalinn when the constraints are applied. The same crnxliticros of p:tesage have been uced to reverse Ihe transforniation, both processes re%emhling in principle the rever%al of Ihe early stagec of Ihe rnole-In-melanoma sequence. When fhe frequent low-density Pas%ages are made in high concentrations nl ft`tal hrn•ine scrunt• which curhorts a slightly lower growth ratc than an equal con- centralinn of call' serum, the degree of transfonnation is gradually increased, so that the I'oci hc•cnmc more numernus, hroader, and thicker, reaching a maximum in suc- ccs.ivc assa" :u about 3 mnnths of passaging. A cliversity of focal morphnlogics is %Pnradicalh e'ncr:uccl in fhc calf %crum passagc by exposing the cclls to various cnncantratinw, of calf .eruni for 14 days af growth and confluency before assaying Ihe•m. '1'hc elehondenc•c nf the ntnnher, density, and morphology of foci on the envi- rnnnicnt in which thc cells had been grown before assay reinfnrces the evidence that Ihe Irnmlorni;uinn is :m epigenetic Pnxess. The fact that these effects develop in cul- lurc gr,nltr,dlv c.ver an extendcd period of time suggetits parallels to the characteristi- calh' long lerm carly regression and later Progression• as well as the diversity of the molc-tn-rtulannma sequence, and may also he representative of other cancers. Rnhnr.11 Prnceedings of the Nalinnal Academy of Sciences USA R9:977-9R1, February 1992. Other support: U.S. Public Health Service. Front the I)cpartment of Molecular and Cell Biology, Division of Cell and DeveloPmcnt Biology, and thc Virus Laboratory. Stanlcy/Donner Administrative Scrvice% I Init, I Inivt•r~,ity ol Califurnia. Berkeley. d(1 INI)IVII)tIA1,TRANSFORMING 1iVl?N'I:ti IN LONGTHRM('1'.LL('Ill;l't+KI? f)I; N111 3T3 ('I?LI.S AS I'RO1)tt(°I:ti OI 1:1'IC~I.N1:11(' INU(I("1-ION "I'hc N11I 3T3 line nf mnut e fihrnhlasls undcrltncs "spnntancnus" Il:mtifnrmalinn in culture, exhibited in Ihe clevelnpmenl of foci of Iranefnrmecl cells overgrowing the ccmflucnt monolayer. I'.videnc'c is rrnvidcd here to support the hropncitinn that Ihe til„mtancnuti generation of indivichr.d Iransfnrmed variants is a prnclucl of epigenctie• inductiroi by various types nf growth inhibilion. Novel Irantiformed v:triants generally :rri•c aficr prolonged confluence ancl cesxatinn of net grnwth, with these new lylx•,, of foci appearing during a second round nf conf7uence. although not in the first round. I:ew or no Iransfonncd variants exist in Ihe cultures prior to growth constraint. 'I1hc susceptibility nf NIII 3'T:3 cells to incluctinn of transformation is itself shown tn Ix• >uhjcc'I to crigcnetic influence, with caPacily for u'ansformatirni rcllccting pas%age histnries. Cell suhlines niainlained in long-term suhconflucnt passage Ihat allows unimpeded growth hecrnnc more rel'raclory with time to spontaneous transformatirni. whilc suhlines passaged in more growth-retilricting conditions maintain their .cn~itivity. The ctiffcring capacitics of the suhlines are reffected in the degree of growth inhibition required to induce individual events of sprnttancnus Ir:msfnrmatinn, and in Ihc frequency at which such new variants arise. 1'hus growth inhihition not only induces individual transforming evcnts, hul even incrcascs cell susceptihilily to further induction of transfnnnatinn. These phcnomena :rrc consistent with the progressive xtate sclectinn model of heritable change. which pnaulates a sclf-regrdating selection of hetter ad:trted states Irom amnng those made available to a biological system during heterogeneous Iluc'luations in its total pattern nf chemic•al eyuilihria. EIlisnn, 13. J- and Rrrhin. ll. ('ancer Research 52:667-673. Fcbruary I. 1992. Other Support: U.S. Public I Ieallh Service. From the Departmenl of Molecnlar and Cell Biology and Virus Lahnrrtnry, (Iniversily of California. Berkeley. HIGII RATE OF DIVERSIFI('ATION AND REVERSAL AMONG SURCLnNI;S OF Nf:OPLAS'I'l('A1.I.1'"IRANSFORMfif) NIH 3T3 CLONIiS Nllt 3'1:3 cclls umlergn neoglaslic Irantifnrmation when exposed to cnnditirnis af mcxlerale physiological grnwth constrainl. One of several characteristics of Ihis trans- fnrmatinn that indicates its adaptational nature is its gradual reversibility under con- dilinns of unconstraincd growth. Wc explored the origins nf rcversibility by isolating cclls frnm each of three highly transfnmicd itx:i and comparing their focus- fomiing capacity with Ihal of derivative clones and suhclones. A high proportion nf' the parental cells made dense fea:i. Six of the nine clones nhtained frntn the three foci prcxluced faci, though the percentage varied widely. The other three clones procluced nn foci at all. The transformed clones were suhcloned and analyxed to evaluate the possibility that Ihe negative clones were genuine revertants, rather than heing derived from a.mall minnrity of nontrantifnnned cells surrounding or undcrlying the 41 1
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nriginal lirci. In c;tch e•a',e Ihr .ulx•Inne" v;nicd widelNl in Ihe Ix•rcentatte nf fncuti- fnrnrine cclle and tho ;rvera!•c wae much Inwcr Ih;m Ihr p,ircntal clone from which they were det'ived. Indecd. 15 nl tlrc 5.3 .uhcfnnal pnlntLnions prntluccrl no Gori. Thc high deLrce nf heteropencity, includinL complete revc•rs;il nl tircut-fimning capacity, prr,vides additirntal tiupprnt ferr Ihe hvpnthetii~ Ihat "ehnnlanaru.,~" Ir,rnsfirnnntinn is rbiven hy an adaptivc response to nrndcrate trnwth cnnsIraint raqier than by one or nxorc clleclively irrcver,,ihle nnnatirm,,. Ruhin. A. I.., Sncade-Kox nip., A., and Rubin. lL 1'r'ox•ecdinz~ of the National Academy nf Sciencec l ISA R9:a 1 R3-41 fi6, May 1992. (hhcr suhpnrt: National histilule nf I Icalth. Frcmi the Virus I,ahoratnry and 1)epartment of Molecutar and C elt Biology. l)nivcn-ity nf ('aliliirni;r, 13erkelcv. SF?NSITIVfF'Y OFTRANSFORMnTIC)N TO SMALL 1)IFFERf•,NCES IN POPl ILATION I)1?NSI-1'Y 1)11RING SI?RIAI, PASSAGF, OF NIH 3T3 CELF S Early pasal;es ol the NIII iT3 munsc cell line undergo spontaneous neoplastic transformation leading to Ihe development rrf transfonned frxi if gmwn to confluence in 2'4• (vol/vnl) rall scrunr (C ;5) ;mrl Ie/i Iherc for more than a week. Transfer of the pnNlcnnfluenl cuhureti resultc in Ihe appearance nf large numlxrs nl' transformed frtici: many nl them arc larger and denwr than those in the original culture. 11' the cells are continually kept at low population densities by I'requcnt passages in 10% (:S, they hr.c the capacity to undergo spontaneous transformation. If however the hrw-dcnsity pastia):es are made in 2ry„ CS or in 10% (vol/vul) fetal bovine serum, both of which support lower growth rates and saturatinn densities than does 10% CS, Ihev eaitt Ihe capacitics to grow to high saturatinrt densities and produce more foci when grciwn tn confluence in 2'7v CS. These incrcascs are proportional to the pnpula- linn den:,itics u%cd in the frequent passages, although the densities are all kept well Ix Inw comtluence. We cnnclude that Ihe combined constraints of suhmaximal serum pluti those r,f the linrited cell contacts of the low cell dentities used here elicit an artaptivc respunse that endcrws the entire population with incrr.tsed growth capacity. 'fhe increaud growth capacity nf the heterogeneous population in tum increases the r;tpacitv nl a fr;tclinn of Ihe pnpulatinn to initiate distinctive transformed foci. Sintilar ~In~lio, have indicated that Ihe capacity of cells to produce tumors and nreLi%t;i,c~ in micc and rns is enhanccd by prior maintenance at high density in cut- Ime. We propose the concept of pn,gre%sive state seleclinn to acenunt for the general increase in the growth capacity nf cells that is elicited by moderate constraints on thcir gr<iwih and nrelahnli%rn. Yan, A, and Rnhin. 1/ Pnocedinex nf Ihe National Academy ol'Sciences IISA R9:74R6-74(N), August 1992. Other suppnrl: U.S. I'uhlic I lealth Service. From the I)epartment of Molecular and Cell I3inlogy atxl Virus Laboralory, t hriver.ity of ('alifornia. 13crkclev. S111'1'RFSSInN OF'I'UMORI(:I:NI?SIS BY THE 13REAST CANCER CI:LI, LINE M('F-7 FOI,LOWING'I'RANSFI;R OF A NORMAL HUMAN ('IIROMOSOME I I Breast cancer development is a«aciatect with several genetic abnormalities. l,nss of hetercizygtrsily in the short arm nf chromosome I I has been observed in ;(N.1v of tumors. We found homozygosity at live chromosome I I polymnrphic loci in I.ennmic 1)NA of the MCF-7 breast carcinotna cell line, suggesting a pcissihle tocs ol' one chrumosomc 11. We have studicd the transformed and tumorigenic phcnotypes of MCF-7 cells following introduction of a normal human chromosome I I via nricrocefl fusion. MCF-7/III I cell hyhrids, containing chromosome 11. showed in r,ipn characteristics similar to the parental cell line. I-lowever, tumorigenicity in athymic mice was completely suppresseil. Since tumor formation by MCF-7 cells is "trogcn dependent, we have analysed the expression of the estrogen receptor and of Ihe estrogen-activatect gene'pS2. No difference was detected between the parental MC F-7 cells and the derived chromosome I I cell hybrids, indicating that the mecho- nism of MCF-7 tumor suppression by chromosome I I-associated functions does not directly involve the estrogen/cstrogen receptor molecular pathway. Negrini. M.. Castagnoli, A., Sabhioni, S.. Rcc:rnatini, E., Giovannini, G., Pnssati, L., Stnnhrid,¢c. E..l., Nenci, I., and Barhanti-Bmctano, G. Oncogcne 7:201:3-201 R, 1992. Other support: National Cancer Institute. From the Interdepartmcnt Center for Cancer Research and Institutes of Microbiology. Radiology, and Palhologic Anatomy and Histology, University of Ferrara, Ferrara. Italy; Institute of Biomedical Sciences, University of Ancona, Ancona, Italy; and Department of Microbiology and Molecular Genetics, University of California, Irvine. CFIANCrES IN GROWTH ANI)'F'UMORIGENICITY FOLF.OWING RF,CONSTITUTION OF RF,TINUF3I,ASTOMA GENE FUNCTION IN VARIOUS HUMAN CAN('ER CFLLTYI'F;S BY MIC'R(X'F,LL TRANSFER OF ('I IROMOSOMI: 1 3 Functional loss nf the retinnhlastnma (RR) gene has been implicated in the initi- ation or progression af several human tumor types including cancer nf the eye. harle, bladder, and prostate. To examine the consequence of adding one RB allele contain- ing its normal regulatory elements hack into representative examples of each of these cancer types, as well ax to compare the resulls, to those previously reported using. various RB complementary DNA constructs, a ncomycin resist<mt marked 1 ; chro- mctisnme was tran4erred by micrcxcll fusion. Several attempts to obtain RB po%itive ostcosarcoma cells failed. In addition, only one RB positive retinohlastoma clone was isolated. This clone contained many large cells, could not be maintained in long- term culture, and produced only RB negative tumors. Threc RB pnsitivc bladder can- cer cell cloncs were nhtained, all of which grew slower in culture than their RB ncg- alive parental cutmtcrpstrt and did not form colonies in sofl agar. Ttnnorigcnicity was 42 43
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markedly suppressed in these clones. One clone yiclded nrr tumors, and the other 2 clomes produced only one small tumor each, both of which were RB negative. In amtracf, file 2 RR positive prostate cancer cell clones itiolated had no differenceti in their cell culture growth propertics, including growth in sofl agar compared to the parental cells. One of the clnncs was nontumorigenic. while the other clone produced 4 cmall Itmiors, all of which were RB positive. These results indicate that file transfer of one Rli allele by micrncell Iransfer produces different levels of growth inhibition as well as tmnor suppression. dcpending on thc ccll type examined. In the case of prostate canccr, the function of the RB gene in Imnor suppression appears to be inde- pendent from its growth regulatory functinn, since no growth inhibition in cell cul- tore was noted in these cells, although tumor suppression was zignilicant. Banerjce. A., Xu. H.-,I„ Hu, S.-X., Araqjo, D.. Takahashi, R., Sianhridgc. E. J., and Benedicl, W. F. Cancer Research 52:6297-6304. November 15, 1992. Other cupport: National Institutes of Hcallh and Retina Research Foundation. From file ('enter for Biotechnology. Baylor College of Medicine, The Woodlands, TX, and I)epartment nf Microbiology ;md Molecular Genetics. College of Medicine, University of ('alifoniia, Irvine. INTERACTIONS OFT(IMOR ('I:LLS WITH BASEMENT Mf?MBRANE,S The ahility of inetastatic cells to modulate their behavior plays a crucial role in determining the overall success of the potential of a tumor cell to form distant tumorsc At each stage in file metastatic cascade, the interactinn of lumor cells with matrix componentti or other environmental factors can potentiate or perturb the abil- ity nf file cells to Inrm a mctastalic Icsirni. Tumor cells in the primary tumor interact with basement membrane and extracellular matrix, resulting in matrix degradation. ('hemnt;ictic ctimulation of motility in response to matrix degradation products, as well as collagennlytic eniymes, affect the intravasation step. The dissemination of file Unnnr cell is akn modulatcd by plasma fibronectin and prior cxfxnure to laminin. ('clltdar arrest dcrcndti. at leau in part, on adhesive interactions with capillary cell walls anrl mav crhihit specificity based on subendothelial basement membrane-spe- cific pnlyPepiidcc. 'fhe tiPecificity of this adhesion is indicated by the observation that many metrrstatic tumcir. ,,how a remarkable preference for certain target organs and specific crn•acellul:rr matrices. 13oth the adhesive properties ol' the tumor cells and file matrix cimstitucnts at the mel.rstatic tites are (if importance. Several compo- ncnts of the basement membrane interact uniquely with tumor cells by glycoprotein receptor molecules exprned on the tumnr cell surface. The presence of increased number of laminin receptors correlates with increased metastatic propensity. Fihremectin aplx,rrs to negatively nxodulate the expression of laminin receptors in some lumor ccllc. Thus, file high levels of fihronectin in serum may represent a nat- ural mechanism nf ciefense against the spread and Prolil'cration of certain metastatic nnnor ccll%. . While many aspects ol the interactions of tumor cells with basement membrane matrix and other environmcntal factors have been elucidated, considerably more 44 informatirni from in rirn spontaneous metatitases must be accumulated to crnifin file hypothcses based on the in rin•n model systems and to take into account file hci croeencity of lumor cells. Further exploitation of molecular, biological, and gen manipulativc techniques may permit definitive resolution of somc of the issues m vet .unenahle to analysis in rirn. Only then can we hope to transfcr this unclerslanc ing to practical clinical modalities. (r,rrunnra. l'. P. :md Maslow. D. W. In: Orr. Buchanan, and Weiss (f ds.): Microcirculation in Cancer Metastasis. CR Press Inc., pp. 23-44, 1991. Other support: Smokeless Tobacco Research Council. From the Laboratory of Tumor Biology and Connective Tissue Research, School Dental and Oral Surgery. Department of Pathology, Columbia University. Ne York. MECHANISM OF p185"'' EXPRE,SSION IN HUMAN NON-SMALL CELL LUNG CANCER CELL LINFS To identify mechanisms that allow pIR5"'" expression in lung cancer, we p formed Western. Southern, ancl Northern blot analyses of 14 cell lines derived fn human non-small cell lung carcinomas and one cell line derived from a hmr mesothelioma. Human hronchiole epithelial cells and rat type 11 pneumocytes w found to express pIRS"'"' at low to undetectable levels by Western blot technique, contrasl, 13 lung cancer cell lines expressed pIR51"". and eight of these 13 expres; p185"'"' at levels at least 2-fold higher Ihan that found in normal bronchiole epithe cells or type II pneumocytes. Genomic Southern analysis showed that amplifical of the HER2 gene was present in only one of the eight cell lines that expres pIR5"'"' at these higher levels. Increased levels of steady-state HER2 mRl occurred in the remaining seven cell lines. We conclude that in human non-smsdl, lung carcinoma cell lines the most common mechanism resulting in increa PIR5"'" expression is due to mechanisms that increase HER2 mRNA levels, v HER2 gene amplification occurring less commonly. Keni, J. A., Rcibinson, R. A., Gazdar, A., Torney, L.. and Weiner, D. B. American Journal ol' Respiratory Cell and Molecular Biology 6(4):359-363, A 1992. Other support: National Institutes of Health. American Lung Association University of Iowa Cancer Center. From the Departments of Internal Medicine and Pathology, University of L College of Medicine, Iowa City: Dcpartments of Medicine and Pathology Laboratory Medicinc, University of Pennsylvania School of Medicine, Philadclf anct National ('ancer Institute and Naval Hospital, Bethesda, MI). 45 1
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HXPRESSION OFTIIENL•'U GENF.-F.,NCODED PROTEIN (P1K5"') IN IIUMAN NON-SMALI. C'F,LL CARCINOMAS OF'1'IIE LUNG The nrrr protooncogene is a recently descrihed transforming gene originally iso- lated from ethylnitrosourea-induccd rat ncurohlastomas. We have cxamined the expression of the rrru gene in human non-small cell lung carcinomas using immuno- precipitation and immunohistuchcmistry. The rreu protein product (pIR5^^") was pres- ent in eight of 22 non-small cell carcinoma cell lines derived from human lung tumors. Expression o1' pIR5"^ was found in all histological subtypes of non-small cell carcinomas including large cell carcinomas, squamous cell carcinomas, and adeno-carcinomas. Extension of these data to biopsy specimens of human lung tumors demonstrated that normal ciliated bronchial epithelium of the peripheral air- ways expressed pIR5°^, at low levels. Neoplastic cells in four of 12 adenocarcinomas and three of five squamous cell carcinomas also expressed p185°•. at levels higher than the nomial ciliated bronchial epithelium. Together these studies indicate that p 1 R5^^" expression is a common feature of human lung tumors. Wriner, D. B., Nordberg. J.. Rohinson, R., Nowell, P. C., Gazdar, A.. Greene, M. 1., Williams, W. V., Cohen. J.A., and Kern, J. A. Cancer Research 50:421-425, January 15, 1990. From thc Departments of Pathology and Laboratory Medicine, Neurology and Internal Medicine. University of Pennsylvania School of Medicine, Philadelphia: Department of Pathology, Univorsity of Iowa College of Medicine, Iowa City; and National Cancer Institute, Bcthesda, MD. PROGESTERONE AUGMENTS PROLIFERATION INDUCED BY EPIDERMAL GROWTH FACTOR IN A FELINE MAMMARY ADENOCARCINOMA CELL LINF. Steroid honnoncs and peptide growth factdrs promote growth and development of nonnal nianunary tissues and some types of breast cancer. Ovarian steroids may influence mammary growth directly or indirectly. The epidermal growth factor (EGF), family of proteins may also regulate mammary growth. These two pathways may function independently of each other or they may act in concert, with steroids inducing transcription of genes that encode growth factors or growth factor receptors. We ueed a feline mammary adenocarcinoma cell line (K 12) to address whether there was an interrelation between progesterone (PGN) and EGF-associated growth pathways. K 12 cells responded to EGF by a dose-dependent increase in pro- liferation. PGN or promegestone (R5020, a synthetic progestagen) alone did not stimulate K 12 growth, hut when EGF and PGN, or EGF and R502O were combined, they were synergistic. This synergistic response was abrogated by the PGN receptor antagonist RU4Rh or by antibodies that blocked binding of EGF to its receptor. K12 cells expressed characteristic double-affinity EGF receptors, as well as p185 (a functionally and structurally related protein, product of the neu gene) on their surface. PGN receptors were also found on intact cells and in cleared cytosols. Stimulation of K 12 cells by PGN or by R5020 induced a two- to threefold increase in the number of high-affinity %urface EGF receptors after 24 h. Stimulation of these 46 cellc by P(7N also affected the relative levels of phosphorylation of the EGF recepl ~r~rl pl}t5 within minutcs, but not of other cellular phosphoproteins. Our results shc that PCiN enhances the EGF-induccd growth of K 12 cells and suggest that Ihis cffe rn,ry Ix medialed at least partly via an increase in the number or function of hig ;d7inity F.GF receptors. 1vlocliano, J. F., Kokai. Y., Weiner. D. 8.. Pykett, M. .I., Nowell, P. C.. and Lytt C. R. journal of Cellular Biochemistry 45:196-206, 1991. Othcr Support: National Institutes of Health. From the Departments of Pathology and Laboratory Medicine and of Obstetrics a Gynecology. University of Pennsylvania School of Medicine, Philadelphia. II. The Respiratory System PLATELET-DERIVED GROWTII FACTOR IN IDIOPATF-IIC PUL.MONARY FIBROSIS Fibrosis is a complex process involving an inflammatory reaction, fibrob proliferatinn, and abnormal accumulation of interstitial collagens. Mononuclear c are usually present in lung fibrosis. Activated monocytes and macrophages in cult have been shown to produce several growth factors including platelet-derived gro• factor (PDGF). PDGF is a potent mitogen and chemoattractant for fibrohlasts smooth muscle cells and a stimulator of collagen synthesis. We have studied expression of c-.ci,c/PDGF-2 mRNA in lung tissues derived from five patients i, idiopathic pulmonary fihrnsis (IPF) and from four control individuals without I Northern blot analysis of specimens obtained from four patients with IPF rever Ihe expression of the c-.ci.c/PF)GF-2 protooncogcne. A control lung tissue without (lid not express the c-sis protooncogenc. hr sila hyhridization extended these stur demnnstrating the expression of the c-sis mRNA in the five specimens with IPF not in the four control ;lxcimens without IPF. The expression of c-sis mRNA localimcl primarily in the epithelial cells. Invading alveolar macrophages : expressed c-sis mRNA. The expression of c-sis mRNA was accompanied by exprccsion of PDGF likc proteins in lung specimens with IPF hut not in control I spccimens. The.e I'indings demonstrate (he in viro expression of the c-.cis/ PDC protooncogene amd Ihe production of PDGF-Iikc proteins in the epithelial cells rnacrophagcs of thc fibrotic lissue. This localizcd and sustained productioi PDGF-like mitogen may constitute an important contributing factor in the ahnor fibro-hlast proliferation and collagen production. events assexiated with pulmo fibrosis. Anrnniorh•.c. H. N.. Rravo, M. A., Avila. R. E.. Gahmopoulos, T.. Neville-Goldel and Maxwcll, M., and Sciman. M. 47 l
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Jrnirnal of ('linical Invcstization Rh:l(K5-I(1(,4, October I()O(l. Other su),pi,rt: National Instilutcs nf I Icalth. Frnrn The ('enter for Rkmd Re•tiearch and Departmentx of Cancer Riolugy and Nutritinn, I larvard School of Puhlic Hcadth, Rnston, and I)ivixion clc Invectigacinn, Intitilntn Nac•iomd dc linfcrmc•dacic~ Rc%piratoriati, Mcxico C ity. Mexico. I:XPRESSION OF MONO('yIl; CHfiMOATTRACTANT PRO'I'EIN I mRNA IN IIFIMAN IDIOPA'1'IIICI'tILMONARY FIRROSIS Macrophagcs are thought to play an important role in the pathologic changes ati.oci;dcd with idiopathic Pulrntmary fibrosis (IPF). The mechanisms for increased mnnrx•ytc/macrophapc rci•ruitment in IPP are unknown, Monrx:yte c•hemoattraclant protein I(MCP-I ) is the prcdnminanl monnc•yte chemoattractant secreted by n variety c,f (lifl'crent cell types in culture. We examined Ihe expression of MCP-I mRNA and its protein product in rirn it) IPF and nnn-IPF Iung specimens by in silu hybridization and immunocylrx•hcmistry. The cell types expressing MCP- I in rirvr were identified by immunnstaining with spccilic antibodies. We demonstratcd Ihe expression of M('P-I mRNA in pulmcmary epithclial cells, in monocyteti/mac•rophages, and in vascular endcithclial and snx,nth mu%cle cclls. I.ung cpithclial cells in patients with II'I'xtnmgly expressed M('I'-I mRNA and its protein product. In cnntrast, epilhclial cells in nnn-11'I' .pccimcns did not express MCP-I mRNA. Macrophages and vaticular c•ndc,thclial and .nnx,th mwclc cells were shown to express M('1'-I in both II'I-. and non-11'I= lung specimenti. The•xe findings provide a hatiis for the under- standing ol the irr riro physiologic processes that mediate mrniocytc/mac•rnphage rccruitmcnt and infiltralinn in thc• Iung, intcrstitium and thc pathologic state c•ontrihutine to an incrcatic•d alveolar momicytc/macrnphagc pnpulation and inflanun;ilit,n in II'h. :IiUnitiude.c, !I N.. Neville (inldcn, .I.. Galanopnulne, T.. Kradin, R. I,., Valcnte, A. J.. and ( iravr.. 1). 1'. 1'nuc•ctlinz,, r,l thc Natitmal Academy of Scienccti USA R9:5371-5375, ,lunc 1992. 1=rnm The ('cntcr for Blood Research and Departments c,f Cancer Biology and Ninrition, Ilarvard tichonol c,f I'uhlic Ilcahh: I~rFxrratnry of 1'ulmnnary Inrmunningy and Mr,&•c,il:n- Ilinh,gy. Massachuccttc General Ilotipital, l;ostrnr Departmcnt of Palhnlniv. I[nivertiity r,f Texas Ilcalth Science Center, San Antonio: and I)cparnncnl of Or,il 13inh,ge, Boston I Inivcrsity Mcdical ('cntcr. 12OLI: POR T'HI'. ('F.RVICAh SYMI'A'I'11F,T1(' TRIINK IN REG( II.A-fING ANAI'l1YI.A(°fI(' ANI) I.NI)OIOXIC' SIIO('K This study tc.ls thc hyp+nhe%is that spinal nerves projecting down the cervical synrpathctic Irunk c~,nlrihutc to thc• regulation of systcmic imntunc responses. 4}{ I)crentraliiati()n or ablation (ganglionectnmy) ol' the superior cervical ganRlia Iti(•(;), which receive innervation from spinal segments ('tt-TR, was found to reduce thc pulmrnnny inll:unmatnry.re.pmrec consequent In inductinn nf anaphylaxis in rats .cmitilccl tn Ihe ncmatocfc Nippn.snrm,t;yln.s hrnsiliensis. Furthermore. Ihe hyputen- ,ivr re~Pt~rtti- to IV endotoxin were attenuated in sentiitiMl rats by these operations, ccherca% deccn(ralir.ation without ganglirmectomy protected against endoloxic shtxk in nnrmatl (unsentiitir.ed) rats. These results suggest that systemic inllammatc,ry c•vcnts are regulated by the cervical xympathetic nervous syztem at a level superior to Ihr quperior cervical ganglia. Further studies are warranted to investigate the role of the cervical and thoracic sympathetic nerves in the regulation of systemic immuno- h,gical function. Waddell, S. ('.. Davi.nn, J. S., Rrfin.s, A. D., :rnd Mathison. R. D. lournal of Manipulative and Physiological'Ilterapeutic 15(1):10-IS,.lanuary 1992. From the Departments of Medical Physiology and of Microbiology and Infectious Dfse:nes, Faculty of Medicine, University of Calgary, Alberta, Canada. DECENTRALI7.,ATION OF THE SUPERIOR CERVICAL GANGLIA AND THE IMMEDIATE HYPF..RSE,NSITIVITY RF,SPONSF, Bilateral decentralization of the superior cervical ganglia protects against pulmonary inflammation when measured 8 hr after induction of anaphylaxis in rats .cnsitized to the nematode. Nippnsn•arr,qi•lus hra.siliensi.s. Since anaphylactic shtxk produces immediate perturbations to tlie cardiovascular and respiratory systems, we examined whether bilateral decentralization of the superior cervical ganglia modified the responses of these two systems during the first 4 hr of the anaphylactic response. With the exception of the bronchioles, decentralization did not protect against ana- phylaxis-asscx:iated increases in extravasation of albumin, and the small changes in recpiratory function imluceel by anaphylaxis were unaffected by the denervatirni, Dcwentralizalion did not alter anaphylaxis-induced reductions in blood flow to the gastrointestinal tract: hmvever, blood flow to the kidneys and spleen of decentralil.ed rats was restored more rapidly lu normal valucs. These results suggest that the pro- tective efTect (if clecentralization on the late phase pulmonary inflammation of ana- phylaxis is unrelated In early c•hanges in respiratory mechanics, although the protec• tion may Ix facilitated by the mnre rapid re-establishment of normal cardiovasculai hnmcrntaxis. Mathison, R., Daviscm..l. S., I)e Sanctis, G., Green, F., and Be(us, D. A. Pnxrcdings of the Society for lixpcrimental Biology and Medicine 2(W):542-547 1492. From the Department of Medical Physiology. Respiratory Research Grnup, am Department c,t Microbiology and Infectious Diticases, Faculty of Medicine University of ('algary, Alberta. Canada. 49
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ROLIi FOR THr SFIRMANI)IRUI,AR GLANT) IN MOI)IILA'T'ING I'F)I.MONARY INFI.AMMA'I'ION 1'OI,LOWING INI)(t('TIt)N OPSYSTf:MIC ANAI'HYI,AXIS Previnus studies have shown that bilateral decentralization of Ihe superior cervi- cal ganglia (S(Y;: decentralizatinn) attcnuate,~allergen-indticc(I pulmonary inflam- matnry responses in malc rat,~ sensitired to the ncmatode Nippnsrrnn,4vlns hra.cilirn- .cis- The present report examines the neuronal and glandular niechanistns mediating lhe protectinn against pulmonary inflammation afforded by decentralization. Tissues and organs inncrvated by the SCG are responsible for this protection since, in a man- ncr similar to dccentralizatinn, bilateral removal of the SCG (ganglinnectomy) reduced anaphylaris-induced accnnudatinn nf inflamntatory cells in bronchoalveolar htvage fluid. Removal nf the suhmandibular gland (sialadenectomy) did not modify the severity ol' the pulninnar-y inflammation, but concurrent tiialadeneclomy and decentrali7ation ahnliched the protective effect of decentralization. Thus, we Postu- late that cervical sympathetic nerves tonically inhibit release of anti-inflammatory factnrs from submandibular glands. No relationship was found between noradrena- line and sernlonin content of .ubmandihular glands and the degree of protection against pulninnary inflammation offered by decentralization and ganglionectomy. 13oth decentralization and ganglionectbmy appeared to increase the level of tran- scripts that encode immunomcxlulatory growth factors (nerve growth factor and epi- dennal growth factor) in suhmandihular glands, hut these denervations evidently did not modify the transcripts for TGF(32. Systemic inflammatory events are regulated by the central nervious system al a level superior to the SCG probably through mod- ulation nf immunorcgulatory fac(nre in submandihular glands. Mathisnn, R.. Iingan, A., I Ielrner. D., Rauce, L., Wonlner..l., i)avison, J. S., Schultz, (i., and Re/is, 1). Brain. Rehaviur and Immunity 6:1 17-129, 1992. Other support: Medical Research Council of Canada. From the I)erartments nf Medical 1'hysiology, Microbiology and Infectious Disease, and Medical Riochemistry. Facnitv of Medicine. University of Calgary. Alberta, Canada. 1)FtAM1;'I1IASONTi SFa.E("('IVF,I.Y MODULATES BASAL AND I,IPOI'OI,YSA('CI IARIDh:-INDUC'ED MF.TALLOPROTFiINASf-; AND TISSUE INHIRIT(1R (11; MFTAI.1 OPROTHNASf? PRODU(°fION BY HI.IMAN ALVI?tN,AR MACRt)I'I IA(;I?S To define the capacity nf glucocorticnids to regulate tissue damage associated with inllammatirni more clearly, we have studied the effects of dexamethastme on human alveolar macrnPhagc sccretinn nf both a variety of inelalloproteinases and also the cnunter-regtdatnry tiseue inhibitor of inetallnprotcinaws (TIMP). We found Ihat dcxamethasonc wlcctivcly and coordinately inhibited expression of the follow- ing human mctallnpmtcinaNc>: interstitial cnllagenase, strotnelysin, and the 92-kDa type IV collagcnasc, a. well a% TIMP. Both basal and LPS-stimulated cells exhibited ,,imilar dt;crcc% nf inhihitinu, with >50'7, decrease in secretion of all cnr.ytnes and I'IMI' ohsau'vcd at dexamethasone concentrations of ?IO" M in scrum-cnntaining inrdium. The efTcclz of dcxamcthasnne were mediated at a pretranslalinnal level. In `tittittt,try. our results indicate that glucocorticoids suppress the malrix-degrading hlicnnlvP't' that is characteristic ot' mature human mononuclear phagocytes, and ),Int-l, Ihc elTects of the nmst potcnt known signal for upregulation of inetallopro- Icitt,iw Wcretirni. Similar actions in t•it•n would serve to limit tis-sue damage associat- cd with the inflammalnry response. Shapiro. S. 1)., ('nntlrhell, /i..L. Kohayashi, D. K., and Welgus, H. G. f hc Journal of Immunology 146(R):2724-2729, April 15, 1991. Other supporl: U.S. Public Health Service and I)epartment of Vcterans Affairs. From tltc I)ivisions of Dermatology and Respiratory and Critical Care, Department nf Mcdicine, Jewish Ilospilal at Washington University Medical Ccntcr, St. Louis, and I)ivision of Respiratory, Critical Care and Occupational Pulmonary Medicine, I)ep;trtment uf Me(licine, University of lhah Health Science Center, Salt Lake City. MONOCYTE ADHERENCE TO FIBRONF.CTIN: ROLE OF CDI I/CDIft INTf.(1RINS ANT) RELATIONSHIP TO OTIIF,R MONOCYTE FUNCTIONS Adherence of monocytes to extracellular matrix comPonents is critical for lheic accumulatinn at sites of infection. To gain insight into the factors Ihat regulate montx•yte recruitment, we have studied monocyte adherenee with regard to the regu- latory effects of bacterial IipnPolysaccharide (LPS) and the nrechanisms involved; nrnrcnver. we have contrasted the phenotypes of adherent and nonadhcrent cells. Our results show that only a minor suhpopulation of monocytes (20-25%) adhere sponta- neously to fihroncctin and Ihat LPS stimulated a threefold increase in the propnrtion of adherenl cells. Basal adherence and LPS-stimulated adherence of monocytes to fibronectin were substantially mediated by CDI I/C'DIR integrins. Further studies revealed that spontaneously adherent monocytes were 14-fold more actively phago- cytic, released 1.6-fold more superoxide anion, and contained 20-fold more Pcroxi- <lase activity than nonadherenl cells, whereas LPS-adhercnt cells had an intermediate phenotype. These results indicate that LPS may enhance the accumulation of mono- cytce with an antimicrobial phenotype and thereby promote resolution of tissue infectinn. Owcn, C. A.. C'antphrll, IS. .1., and Stockley, R. A. .lournal nf Lcukocyte Biology 51:4(H)-40R, April 1992. Other suPpnrC Chest, Heart and Stroke Association of London, U.S. Public Healtl Scrvicc and 1)cpartnunt of Vetcrans Affairs Medical Research Funds. From the 1)ivision nf Resriratnry, Critical Care and Occupational Pulmonar; Medicinc. Ucpartment of Medicine, University of Utah Health Sciences Cenler Veteran,~ Affairs Medical ('enter. Salt I,ake ('ity; and Lung Immunuhinchemica Resruch I,ahurvnry, thc General Hosritad, 13irminham, England. 50 5 I
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IN('121iAS1;l) AI)IIIiRUNC'li OF MONO('YTI?S TO FIIiRONI?("IlN IN RRON('1111?('.LASIS HI(;IqAlnltvCll'rrlS((I n~(nlilv Innl`ttlti"S:~<rllwin)I ANU K(il I111('1)I1/(•UIR " R(•Ltdsucd adhcrcncc nf n)nnncvtc% to crtraccllular n):Ih'ix is :I prcrcyui%ilc fnr accmmulrniOn (d m(,nnnu('Icar hh:tp('c'vlc% during huln)rn)arv infcc'Iinn an(I inl)amn)a- liom. Wc have nhtnincd n)(~nncvt(•> frOm ))alicnts wilh am inllanunalnry lun~ dierasc (hrOnchicclasi.l and Inm) cnnUnl tuhjccls and havc cotn))arcd their a(Ihcrcncc to Iihr(rncctin. Sl)nnlnncrnlti :I(Ihcrcncc of n)anncvlcs frOn) Ihc c•nntrol .ubjccts was 2(1 ): 2'/., whcrcw. that ol I)aticnl"' cclls was markedly higher and correlated witlrlhc severity of airway inflanm)alim): (i5 E51%. and 4O+X '%r in patients wilh purulent and n)ucOi(I ..pnlun). resl)cclivcly. I•:ndntOxin and cytokincs I'ron) arcati nf airway disease m•c Iikt'lv to hc rc,,hnntiihlc fo( thc nhscrvcd monncylc activatinn, since: ( 1) cn(Iotox- in woti dctccluhlc in all nI Ihc I):dic'nt% hul ill nrn)c' of tl)c control suhjccts: (2) 1.1'S I)nxluccd a(kxc-rclatc(I increase in adherence nf normal n)onOcytcs in 1•ifrn (maxi- n)al 65 + 2~; aclhcrcncc at I I(t./n)I nf 1.1'S): (3) recombinant cylnkincs and I,I?S pro- (lucccl ,Idditivc cllcc'Is nn mrn)ncytc adhcrcnc•c in 1'i1rn.1'hc adherence of Ihc paticnts' n)nnOc•ytc,, to fihrnncctin was substantially mc(liatcd by ('DI I/('DIR intcgrins, via hOIh RGI) (Icpcn(Icnl and R(il)-in(Icl)en(Icnt n)cc•hanistm. 'fhcsc clala indicate that sign:lls arising frrnn 166 i (11" infcctinn and inllan)nlation can influence the aclhcrcncc of m('nOcvtcs. an(1 thcy nre likely to be dclcrminants of the accun)ulatinn ol'rnonnnu- clcar ))hngc,cvlcs in the lune% nf paticnl% with hrnnchicclasis. Owen, ('. A.. ('mnphcll, I: •I., I fill. S. 1.., tm(I Stncklcy, R. A. American Rcvicw Of Rc%lriralnry I)iscasc 145:626-(i31. 1992. Other snhrnrl: ('hcsl, I lcarl and Stroke Association of I,ondon, l).S. I'uhlic Health Scrvicc, Nali(mal In~lilulcs nf' IlcalthI and hcrnrtn)cnt nf Vrtcr:rns Affairs Medical Rcwarch lilnd%. hrnn) the I.uni hnmunOhinchcn)icall Research Lahnralnry, Ihc (icncrat Hospital. Rirtningh:nn, I:.nplan(I: I)ivisiOn of Respiratory. ('ritical (':nr and Occupational I'ulmOn:uy Mcdic•inc. Ucparhncnl nf Mcdicinc, Univcr,eity ol l)tuh I lealth Sciences ('cntc(: and Vclcru)ti Adn)oli~lralion Medical ('cntcr, Salt Lakc ('ilY, (1T. 1'1•-\S.MA I.E.VITS OF I?I,AS'1'ASI; SI'I?C'IFI(' FIRRINOI'1:1'11D1iS ('ORRI'.I A fl'. WIII I PRO'1-ti1NASIi INIIIRfIY)R PIII;NnTYPI: --- rvtl)rN(r. FOR IN("ItI ACI I) 11 \CI,\CI- A( IIVII) IN SIUtItd'"I:ti WI111 II(1Mn7.Y(i(11IS ANI) III:;IPROYWGl1Rti I11'I I('II-N("N Of (r -I'INIIFINASI". INIII111II1R '1'hcrc it in(lirccl cvidcnc'c that unopposed human ncutro'phil claslasc (IINt?) is responsible Ii)I' emphysema in palicnlx wilh (r,-prOtcinasc inhibitor (Pi) deficiency. To dircc'llv explore Ihi~ lu)aihililv, we developed an assay for fihrinopcptidc A(r,-2I and its (Ictra(latinn hrnducl% and used it to mca%nrc IINfi activity in 128 suhjccts of knnwn I'i Incenntypc. •1'hc mean clatita.c-shccilic fihrinOpcplidc (ESh) level in 49 dcl"icicnl Pil• in(lividuak i" siznificanlly higher than that in 56 PiM7, hctcrnzygotcti (4.5 and 1.5 nM. rcsprclivcly I' <(1•01). while the n)can FSF value in hctcrOiygotcs is .CI,L`I1111C:mlly clcvatc(I nvc(- that in ?.1 normal I'iM subjects (1.5 and O.(i nM, I(•sp(•ctivcly: P<O.O1). consistent with increased HNE activity in those clcficicnt in th(, m;ljnr regulator nf Ihc enzyme. '1'hcse results arc not cluc to clit7crcnccs in sn)nk- ;IlV hislnry hccausc aftcr correction f(.r pack years of smnking, HSF values in Pi7• ~uhjrcls are fourfoI(I higher than Ihnsc in PiM7• individuals (P = 0.(N)5), while the 1;Sh levels in hclcroiygotcs are threefold higher than those in PiM suhjects (I' = 1).O2). In ad(lilion, this analysis suggests that cigarette smoking and (x,-protcinasc inhihitor deficiency have additive effects on ESF levcls, thereby explaining why I'i7, :nl(I N(m)c PiMZ in(livi(luals arc at especially high risk for the development of lung if they sn)oke. Finally, the observation that ESF levels, in nunsmoking Pi7. %uhjcc•Is are inverscly related to the percent ol" predicted forced expiratory volume in I s(FF.V,'%•) provides direct snpport for the concept that unregulated HNE activity (;;Irscs alvcohlr scplal destruction in paticnts with (x,-prntcinase inhibitor deficiency. WciV,.1. I.. Silvcrman, I's. K., Thong, li., and ('antphell, P" .I. Thc.lourm)I of Clinical Investigation R9:766-773. March 1992. Other support: Medical Research Council of Canada, Ontario Heart and Stroke Foundation. 1C1 I'harma Canada, U.S. Department of Veterans Affairs, and U.S. Puhlic Ilcalth Scrvicc. From the Deparlment of Medicine. McMaster University, Hamilton Civic Iiospitals Research Ccntre, Iiamilton, Ontarin, Canada; Respiratory and Critical ('nrc 1)ivisinn, 1)epartmcnt of Meclicine, Washington University School of Medicine, St. Louis: I)ivisinn of Rcspiralnry, Critical ('arc and (kcupational Pulmonary Meclic'inc, Department of Mc(licinc. University of Utah Ilcalth Scicnc•cs Centers: and Veterans Administration Medical Center, Salt Lakc (.'ity,11T. MOLECULAR CLONING ANI) EXPRESSION OF HUMAN ALVEOLAR MACROPHAGf: CATI IF,PSIN S, AN I:LASTINOLYTIC CYSTEINE PROTEASE Human alveolar macrophages (HAM) express an elastaee activity of acidic pH optimum inhihitahlc by cystcine protcase inhibitors. Recent studies indicate that the only known cukaryotic clastinnfytic cysteine protease, cathepsin L, cannot complctc- ly account for this activily. In order to scarch for additional cystcinc proteascs witF clastinnlytic activity. low degeneracy oligonuclcotidc primers based on rcgions of strong homology among Ihc known cystcine protcases were used to screen revcrsc" Iranwrihed HAM RNA for cy~teine prntestces by the Ixrlymerase chain reaction An)ong the cDNA %c(lucnccs generated was a 493-base pair product highly hon)olo gous to bovine cathcpsin S. Screening (if a HAM cDNA cukaryotic cxpressiol lihrary with this c'DNA vicklcd a I.7-kiloha.sc full-Icngth cDNA highly hmnologote to bovine cathcpsin S(-•R5"G identical). This cDNA predicts a 331-amino acid prc' pru-cathcpsin. F,xpression of Ihis cDNA in COS cells revealed the active enzyme tt hc a single chain 2R-kDa protcasc, as.juclgc(I by activc site laheling with a novel incl inalcd analogue of N-(1.-;-Irml.c-carhuxy(ixiranc-2-carhonyl)-t.-Icucylamiclo-(4 guani(I(1)hutanc (E-(i4). '1'hc recombinant cmyn)c was found to he clastinnlyti tuwar(I 'H-lahclc(I claslin (hnvinc ligamcntum nuchac) at pH 5.5 hut with 251%• o Ihiy activity rctaincd al pH 7.(l. Labeling of 11AM with the active site probe rcvcalc• thcsc c•clN express a 2R-kDa cv~lcinc prntcase, and Northern blot analysis rcvcalo 52 51
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the presence of a-1.7-kilohase cathepsin S mRNA. Thesc data cstablish that human macrnphages expre~,s at Icaist two cy,,teine hrntca%cs with ela,;tinolytic activily. The relatively broad p11 range of human cathepsin S activity uiggests this enzyme may contribute to Ihe contact-dependent elastase activity of live human alveolar rnacrophagcs. Shi. (;.-P.. Munger, J. S., Mcara, .I. P., Rich, D. I-L, and C'kupman, I/. A. The Journal of Biological Chemistry 2(i7(11):725R-7262, April 15, 1992. Other supporl: National Hcahh Service. Frmm the E)epartment of Mcdicine. Brigham and Women's Hospital, and Harvard Medical School. Boston, and School ol Pharmacy and Department of Chemistry, t Iniversity of Wisconsin, Macliton. FUNCTION OF PULMONARY M. MUSCARINIC RECEPTORS IN ANTIGEN-CI IALLI;NGED GUINEA PIGS IS RESTnRF.D BY HF,PARIN ANI) 1'ULY-t.-GLUTAMATE The ef7ect of heparin and poly-t.-ghrtamate on the function of inhibitory M, muscarinic autoreccptors on parasympathetic nerves in the lung was tested in anti- gen-challenged guinea pigs. After antigen challenge, M, receptor function is decreaseel. thus increasing release of acetylcholine from the vagus and potentiating vagally induced bronchoconstriction. Guinea pigs were anesthetiaed, tra- chcostrnnized, vagotomized, paralyzed, and ventilated. Electrical stimulation of the vagi caused hronchoconstrictinn and bradycardia. In controls, pilocarpine attenuated vagally induced hrrnichrxnnstrictinn by stimulating neuronal M; muscarinic recep- lors. ('onversely, blocking these autoreceptors with gallaminc potentiated vagally induced hronchoconstrictionl In challenged animals the effects of both drugs were markedly reduced, confirming M, receptor dysfunction. 20 min after heparin or poly- t_-glutamate, the elTects of both pilncarpine and gallamine on vagally induced hron- chncnnetriction were restored, demonstrating recovery of M, receptor function. Neither heparin nor poly-t -glutamate affected vagally induced responses in control aninialc. Thuti antigen-induced dysfunction of M,receptors can he reversed by pnlyanionic hnlysaccharides (heparin) or polyanionic peptides (poly-t.-glutamate). 'Ehis. sugg"tN thal a polycationic substance such as eosinnphil major basic protein, cationic protein, or peroxidase may he responsible for arrtigen-induced pulmonary M. receptor dy4umctiori: Fi.rer, A. 1). and Jacohy, D. B. Journal of Clinical Investigation 90:2292-229g, December 1992. Other support: National Heart, Lung and Blood Institute and American Lung Association. From the I)cpartment of Enviromnental Health Sciences, School of Hygiene and Public and ('ritical C'arc Medicine. Johns Hopkins Asthma and Allergy Center, Johns Hopkins I Inivcrsity, Baltimore, MD. 54 ('IIARACI'1?R17.ATION Of: TI II? INFI.AMMATORY REACTION IN Tltt; I'IiRII'lll'.RAL AIRWAYS OP ('I(;AR1;1"I'I.i SMOKERS USING I,,1Ml INO('Y"1'O('1II?MISTRY l.ung tissue from 20 patients undergoitig resectioti for a peripheral carcinoma was 1.tudicd using monoclonal antibodies to identify inflammatory cell typca in the Ix•riphcral airways and to determine the location of the hronchial-assoc:iated lym- hhnnl tissuc (BALT).'fhc paticnts were grouped according to their percent predicted f1:V, (IX,FF,V,) into obstructed (`7r1'I;V <HO^/n) and control (%rrX >RO'Yn).The reeccted lungs were filled with dilute cryocmhedding media (Tissue-Tek R), frozen nver liquid nitrogen. sliced into 2-cm sagittal slices using a band saw, and sampled ucing a cork hore. Ten serial histologic sections cut from these samples were stained with monoclnnal antibodies for specific inflammatory cell types. which were count- ed and expressed pc:r square millimetcr of airway wall area. The results showed that Ihe patients with airway obstruction had more B-lymphocytes in the airway aelvenli- lia than did the control suhjects (p<O.(H)I ) and that the number of submucosal poly- mnrphonucle:rr Icukixytes is related to Ihe amount smoked (p<0.02). They also show the BQL'I' has a diffcrent distribution in human than in rodent Iungs in that the lymphoid collections are found in the outer walls of the airway rather than in the .uhmucosa. Rosken. C. H., I lards, J., (;alter, K., and Ilo,¢,q..l. C. American Review of Respiratory Disease 145(4):91 I-)17, April 1992. Other support: Medical Research Council of Canada. From the Pulmonary Research Lahoratory. University of British Columbia. SI Paul'c I locpilat, Vancotrver, Canada. NITRIC OXIDE MEI)IATf;S C'1GARF;T1'E SMOKE-INDUCED VASODILATORY REiSPONSES IN THE LUNG The vasodilatory responses seen in the pig lung during challenge with the gas phas nf cigarette smoke is mainly mediated by NO. The particulate phase of cigarett smoke seems to counteract the NO-induced relaxatory effect in the puhnottary circt lation, but not in the bronchial circnlation. Alving, K., Pornhcm, C'.. WcitncL rg, E.. and Lundhrr•g,./. M. Acla Physiologica Scandinavica 146(.l):407-40R, November 1992. Other support: Swedish Medical Research Council, Swedish Society of Medicin AGA AB Medical Research Fund. Swedish Tobacco Company, and Swedis Environmcntal Protcction Board. Prom the Department of Pharmacology, Karolinzka Inslilute, Stockholm, Sweden. 55 I
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('IGARETI'E SMOKING INDl1('FS AN 1?I.ASTOI.Yl1(' ('Y'S"I'Li1NE PROTEINASF. IN MA('ROI'lIA(;I~S I)IS"I'INC'T FROM ('ATIII?PSIN L f)cgradation of the inlcraitiiun of Ihe lung by cla%tolytic• cn/ymcs is thought to he a critical component ol" Ihc pathogenesis of emphysema. Alveolar tnacrophages are incrcased in numhcrs in cigarette .mokcrs and contain Ihe clastolylic cystcine prolcinasc cathepsin L. We sought to determine if cigarette smoking induces a change in cathepsin I, Ievcl,~ in alveolar macrophages which woidcl, in turn, alter the expression of claslolytic activity. Lyssues of smokers' macrophages assayed at pH 5,5(1, degraded more than seven times as much I'Hlelastin as (lid Iysates from non- smokers' macrophages (44 ± 2f1"R vs. 6 ± 1.6 µg-I(>~ cells' • 24 h'). Little or no activity was demonstrable at neutral pll. Imrnunohlots of macrophage lysates dcmonstrated that smnkers' cells contain 3.7 ± 1.1 times as much 25-kDa cathepsin I, antigen as nonsmokers' cells. Howevcr, as judgcd by active site labeling, levcls of active cathepsin I, in smokers and nonsmokcrs are indistinguishable, suggesting that most uf thc 25-kI)a antigen found in smokers' macrophages is inactive. Inhibitors of cathepsin L had little effccl on lysate elastolytic activity, confirming that an enzyme other than cathepsin 1, is responsible for the increased elastolytic activity seen in smokers' mac'rophaget. Further experiments demonstrated that this second cnrymc(s) has a profile of inhibition indicating that it is a cysteine proteinase with optimal activity at pl15.50. It is this second elastolytic cyslcine proteinase(s) that is induced by exposure to cigarette smoke and is responsible for the sevenfold increase in clastnlytic activity found in snmkcrs' macrophage lysatcs. Reilly. J. J., .Ir.. ('hcn. P.. Sailor, L. 7,.• Wilcox, D.. Maa»n, R. W., and Chn/rnuur. 11. it...h American Journal of Physinlogy 261: (Lung Cell. Mol. Physiol. 5):L41-L4R, 1992. Other support: National Ilearl. I.ung and Blood Institute. From Ihe 1)cpartmenl of Mcdicine. Brigham and Wonien's Hospital, and Harvard Medical School, Boston: and 1)cpartmcnt of Binchcmistry and Nutrition, Virginia Poly9cchnicnl Institute and State IIniversity, Blackburg. l'lll? SI'F('Ihl('ITY ANI) EI,ASTINOLYTIC ACTIVITIES OF BOVINE ('A'I III"I'.CINS S ANI) II Cathepsim S and H were purified from bovine spleen and their catalytic proper- tics compared. "t'he en/ymes were shown to he similar by chrnmatographic propcr- lies and by the ability to hydrolyie Bz-Phc-Val-Arg-NHMec. They could however lie dittingui.hcd by Ihe facl that cathepsin S reacted with 'L-1"`I ITyr-Ala-CHN: and hyclrolyced '1.-Phe-Arg-NHMec whereas cathepsin li (lid not. The suhstrate and inhibitor specificities of cathepsin II suggest that unlike calhepsins B. L, and S, it cannot accommodate pcptides with aromatic sidc chains in P,. Cathepsins L and 5 can accommodate the aromatic side chain of tyrosine in P, readily, whereas cathep- sins H and B cannol. The specificities of cach enzyme for synthetic substrates and inhihitors have enahled the construction of models of the architecture of the active sitce of the mammalian cysteine prMCinases which clearly show the differences hrtwecn Ihe fonr enmymes. A significant characterislic of cathepsin S is that i1 cal hedrnlyic intioluh)c clastin al both acidic and ncutral pH: this clislinguishcs it fron njl of thc olher lyuw~nmal prnteinascx. `(in. X.-(1.. 0uncsckcra. B., uncl Mn.cnn. R. Nf. Archives of Biochcmistry and Biophysics 299(2): 334-139, 1992. Prom the Department uf Biochemistry. Virginia Polytechnic Institute and St:rt ( Inivcrsity. 131acktihurg. HOCI EXPOSURE OF A HUMAN AIRWAY F.PITHELIAL CELL LINE I)E('RI;ASES ITS PLASMA MEMBRANE NEUTRAL ENDOPEPTIDASE 11 has rccently been demonstrated that tuminal exposure of airway segments i rilrrr to HOCI produces airway muscle hyperresponsiveness to substance P and decrease in neutral cndopeptidasc (NEP) activity of tissue segment homogenatc suggesting that HOC'I may decrease airway cpithelial cell NEP activity. To confin that this effect occurs in humans and to investigate possible suhcclluhrc mechanixn for it. wc assessecl HOCI exposure of Ihc human airway epithclial cell line Calu- 'llicsc cells, grown to confluency in Dulhccco's modified Eaglc medium with 10, fetal bovine scnim and penicillin-slrcptomycin, were exposed in.cinr for 5 min lo I( µM FIOC'I in a phnsphatc-huffered saline solution (PBS: pH 7.0 at 37'C) or u, P13 ;done.'fhercafter, cells were rinsed and assayed li,r NI;P activity employing revers, phase high-pressure liquid chromatography. This activity was characterized by II generation of phosphoramidon-inhibitable product (ANA) cleaved from (lie synthcl substrate succinyl-(ala)3-p-nitroanilinc during a;O rnin incubation at 37°('. Cc viability was assessed by changes in LDH rcleasc, trypan blue exclusion, and cc volume. In some experiments, crude plasma membrane and soluble components - exposed cells were isolated and differential NEP activity was assayed. We found Ih a 5 min exposure to I'10C'I decreased whole cell NEP activity from 74.1 ± 4.4 (mr ±SE) to 54.3 ±(,.0 pmolcs of ANA/ min/I(' cells (p <0.05), while no parameter cell viability was affected. NEI' activity in the cntdc membrane fraction decreast 36.3 t 3.1% after exposure (p <0.(11), whereas NEP activity in the soluble fracti( increased 4.(1 ±(/.(,'Y~. Isolaled rnembrane NEP exposed by itself was not affccte Suhsequcnt experimcnts with reducing agents demonstrated that NF,P activity of c( cullurc% prctrcalcd with f(H) mM of either beta-mercaptoethanol or dithiothrict hefore IIO('I exposure wa~ not significantly different from control values. V conclude that whole cell 11O('I exposure decreases Calu-I plasma membrane NF. This loss appears to occur by intcrnali7.ation of ccll membrane NEP. Lang, '1.. and Mtn•lu.c, C. (;. Lung 169:311-324. 1991. Other support: National Institutcs of Ileahh and National Hcart, L.ung and Blo Institute. 1'roni Ihe Ikparlmcnt of Medicine (Pulmonary), Rush University. Chicago. 56 57
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AF,ROSOL17.F,D NE?E1TRA1. ENDI)I'P.1'TIDASI: RI;VF;RSfiS n%ONE INDUCED AIRWAY HYPEiRRI?AC'1-IVITY T() St tRSTAN('1? 1' We investigated the elTecls of ozone exposure (3.n ppm. 2 h) on airway neutral endopertidase (NEP) activity and bronchial reactivity kr suhstance P in guinea pigs. Reactivity after oione ott air exposure was determined by mcasuring specific airway resistance in intact unanesthelized spontaneously breathing animals in response to increacing doses of intravenous substance P holuses.'i'he effective (lose of substance P (in µg) that produced a doubling nf baseline specific airway resistance (ED,,,, SP) was determined by interpolation of ctlmulative substance P dose-response curves. NEP activity was measured in tracheal hrxnogenates made from each animal of other groups exposed to either o7crne or room air. By reverse-phase high-pressure liquid chromatography, this activity wati characterized by the phosphoramidon-inhibi(ahle cleavage of alanine-/)-nitroaniline from succinyl-(Ala) -I)-nitroaniline in the presence of I(X) µM amastatin. Mean values of the changes in log ED.,,,,SP were 0.27 ± 0.07 (SE) for the o7rnie-exPosed group and 0.09 ± 0.04 for the air-exposed group. We found that phosphnramidon significantly increased substance P reactivity in the air- exposed animals (P <O.01), hul it had no effect in the ozone-exposed group. This finding was associated with a significant reduction in tracheal homogenate NEP activity of oznne-exlxxed animals compared with controls: mean values were 18. 1 ± 1.9 mmol - min' - mg prntein' for the ozone-exposed group and 25.1 ± 2.4 nmol - min' • mg Protein' for air-exposed animals (P <(l.O5). Inhalation of an acrosolized NEP preparation, partially purified from guinea pig kidney, reversed the substance P hyperreactivity Produced by ozone exposure. Inhalation of phnsphoramidon post- NIiP inhibited this effect. Heat-inactivated NEP aerosol had no influence on ozone- induced hyperreactivity. Our data indicate that ozone exposure decreases airway NEP activity and increases substance P reactivity, which can he reversed by acrosriliced NEP. Murlu,c. ('- G.. Lang.7,., Williame, G. J., and Chodimella, V. Journal of Applied Physiology 72(3):I 133-1 141, 1992. Other cuphnrl: National Institutes of Health and National Heart, Lung and Blood Instittnc. From the Department of Medicine. Rush University. Chicago, and Department of Medicine. I hlivcr,~ity of Tennessee. Memphis. f?NVIRONMENTAI, AIRWAY INJURY --Mt1COSAt (•nANC;E:S ANn AIRWAY IIYPI~RREACIIVI lY Environmental or occupational airway disorders are common worldwide and are most prevalent in nverlxopulatcd or heavily industrialized urban areas. Hundreds of different agentc have been associated with these disorders which affect millions of people. Perhap. the most common type of these disorders is so-called occupatinnal, or environmentally related, asthma, which affects more Ihan a million people in the United States (Salvaggio. 19R2). The importance of this problem is further enhanced by the fact that it is a preventable and reversible disease. In certain situations, how- 58 cvcr, particularly those related to Ihe isocyanates or to western red cedar, the diq- nrder may persist for titonth,; or years after the last exposure. Agents that have been linked to this type of asthma can hc classified into thrct hrnad c•ategories (Moller e( :d., 19R(i): large molecular weight biological suhstancex smell molecular weight chemicals, and various fumes or gases. Biological agenL include dusts from animal danders and secretions, insects and cnlstaceans, vegetahh gunls, and plant bacterial enzymes. The palhogenesis of asthma associated witl these substances may or may not relate to their stimulating 1gE-medialed reaction when inhated. Small molecular weight chemicals linked with asthma include iso cyanates, acid anhydrides, platimmm, and resin. The pathogenesis of environmentall: related asthma caused by these agents and to various fumes and gases remains con Iroversial in most instances. Because of increasing air pollution and steadily risin industrial use of a wide variety of potcntially harmful agcnts, especially oxidants these envirunmental health hazards represent seri6us public health concerns. Murh.c, C. G. In: Len/ant, C., f•anner, and Hay, D. W. P.. (eds): The Airway Epithelium. pp. 21: 252. 1991. Other support: National Institutes of Hcalth and National Hcart. Lung and Blor Institute. From Rush University, Chicago. ENDOTIIELIN IMMUNOREACTIVITY OF AIRWAY EPITEIE.LIUM IN ASTFIMA'fIC PATIENTS There is extensive pharmacological and physiological evidence that endothelin influences airway calibre. In mammals, endothelin receptors occur on airway smex muscle, local storage and release of Ihe peptide have been demonstrated, and inha lion of endothelin- I induces hronchoconstriction. To investigate the relation hetwe enduthelins and asthma, the cxpression (if this peptide in endohronchial biopsy spi imens was cxamincd imnrunohistochcmcally with an antiscnlm against cndothclin Riopsy specimctrs from 17 asthmatic patients and I I atopic and non-atopic hcall controls revealed striking diflerences, with endothelin expression being evidenl airways epithelium and vascular endnthelium in I I of the 17 asthmatic patiente I in only I of I I contrulc Thesc resultc suggest (hat endothelins may play a part in exaggerated hmnchumntorr tonc uf asthma. Springall, I). R.. I Inwarth. P. 11., Counihan. H.,.D.jukanovic, R., Ilolgatc, S. T., : I'olnk..l. M. 'fhc I.ancet 337:697-701. March 23, 1991. Other %uPport: National Asthma Campaign and Medical Research Council. 1'rmn the t)epartnunt of Itistochemistry, Royal Postgraduate Medical Scht Lnndon, and I)epartmcnt nl Medicine. Elniversity of Southampton. Srnnhamf Cieneral I lrnpital, Solnhamplon. England. 59 I
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MODIII.A'fION OF"IIII: I,I IN(; ('OI.ONII,ATION (NFRIh F'I MELANOMA ('fi1.I.S BY ('I'1'RI IS 1'F('TIN ('nnlr•r(: Stucfies havc.hnwn thal the galac'totiiclc crrntaining simple ;ugars and anli Ralaclrrside-hinding Icctin anlihodies may affect experimental lunuor cell nn•I:INtaeiti. Ilowcver, Ihe limilcd numhcr of reagcntti used Ihu% far ncc•ecsitate further rrhwrvatitms. I'urpn.%r•: N:dural citrus pee•tin (('I') and PII-mndllled ('I' (MC'1'), rich in gafaclosc residucs, were uced to study Ihc invnlvemenl of r.lrhuhyclralcs crmlaining g,alac•Irlsidc rctiiducs in cellular interaction in ri(rn and in lung coluniialirm in rirn rrf 131h-FI nu•lanrnna cells. MeNrrld.c: RIf,-FI mclannma cells were inc•uhaled with various cr,ncentrntirms of ('P and MCP. Their ability to ftrnn homotypic aggregation in rilrn and lumrtr lung colonitati(in in rirr, in 8-week-old fcmalc ('5713L/(i mice was then analyicrl. Rrsnlls: The ('P hinds to the surface of I31h-11 mclanrmra c•clls: this hinding can Fx inhihitccf by laclcicc a( a concentration of 0. 15 M. Intravcnous injectirnt of the murine RIG-FI mcfanoma cells wilh the natural ('1' resultccl in a significant increase (uP to Ihrcefolcl) in the appearance of tunxlr cnlonics in thc lung and in increased homntypic aggregatirm properties of the cclls, while injec•lion of M('I' significantly decreased RIh-FI cxperinrental mctastasis (>9O%). (',ncln.%inn.c: -Ibmor galactuside-hincling proteins mediate cellular recr,gnitirrn by linking oligosaccharides with terminal I) galaclosidc residues rni acljaccnt cells. Succcssl'ul interference with such a process with MCP may lead to a reduced ability to fiwm Iumor cell emhali and melastasis. Implir•c,(ions: These findings imply that Ihc Ralactose-containing carbohydrate side chains ol' ('P might mimic or compete with thc natural figancl(s) of Ihc tumnr );alaclnsidc-hinding protein (gal-Icc•Iin ) and Ihno afTect cellular interacticros relevant fcrr metastasis. I'Ia1t. I). and Ruz-. ,4. .hiurnal nf thc National ('anc•cr Institute X4(f,):41X-442, 1992. OIher Nupprnl: I'aul Zuckerman Support Foundation for Cancer Research. Fnmi thc Mctasla.iti Research Program, Michigan Cancer Foundation, Detroit. SPON fANF , Ol IS I'ROI)1I('"T'ION Of-- l'RANSFORMING GROWTII FA('TOR-P2 BY I'RIM 11tY ('[ 11:1•I IRIiS OF RRON('IIIAL EI'I"I'llliLlAl, C'PI,I,S --la l tic. tx I1N ( I I I Itl II \\'If>R I1' I lllirl The ahifil~ of` airway c•Pilhcfial cells to produce trnnsfnnning growth factor-(3 ( I'( ;I•-P) may hr an imluortvll mechani~,m G,r the control of growth, dil7ercntiatinn. and repair of Ihr airway chilhelium. "I'ri dctermine whether airway cpitheli:d cclls are capahlc of I,IodncinL wc eramined primary c•ultures t1l' bovine bronchial eplthelial cclN. Using o hir/assay13 ac•tivity was deleclecl readily in media con- dititmcd by h+rvine bronchial crithclial cellti. Neutralizing anliscra to T(;F-/3I and IGF-G32 were used to dcnu,nstratc Iha1 Ihc nnw_j<irily nf Ihc activity was of the'I•GF- /i-2 itinf.rrm. Inlcretilingly, .omc of Ihe 'IYiF-/i activity was hresent in the cundi- linne•cl mcdia a.e "aclivc•" l'(;F (i, nol rcqttirink acid ae•tivatinn. The production <rf TGFP wa, variahle• cfehcndink rm cell density and the presence of retinnic acid. The Pre.ence of enehrgennusIv hrerduc•ed active T(;F-P in Ihe culture media was 'I,I/,N~lr to modulate Ihc behavior nf Ihc cell c•ullurec a; evidenc•ed by the effecl~ n1 I I(i (i-nclnraliiing anti,~era on cell sii.c :rnd fihruneclin production. Our retull,, tug I•r l th:tt active TGI'-1i produced by airway cpithclial cells may function in al ~ndt,crinc or haracrinc manncr to modulate cpilhclial cell behavior. S:Ircn. O.. Rnmhcrger. I)., Ri-_inn, n. ITcc•kmann. J.. I).. Rennard. S. I.. anc Spuri.cm..l. R. 1•hc.lnurnal crf Clinical Invcsligalicin 91):I;79-I 3X5, October 1992. OIhcr ,nprnrt: Department of Veterans Affairs. Fnmi the Research Service nf the I)epartment ol Veteran Affairs Medical C'enlel Omaha, NG. and I)cPartnient of Internal Medicine and F,pplcy Institute for Researcl in (;tnc•cr and Allied Sciences. l Iniversily of Nchraska Medical (.'enler. Omaha. III. Heart and Circulation TIIF? ROLE OF TNE FIRST GROWTH FACTOR DOMAIN OF HUMAN FACTOR IXa IN BINI)ING TO PI,ATF;LEI'S AND IN FACTOR X ACTIVATIO We have recently shown that thromhin-stimulated human platelets have slxci ic, saturable receptors for Factor IXa, occupancy of which promotes factor X activ tion (Ahmad, S.S., Rawala-Shcikh• R., anrl Walsh, P. N. (1989) J. Riol. Chem. 26 3244-3251, 20(112-2O016: Rawala-Shcikh, R., Ahmad, S. S., and Walsh, P. (I919Q) Biochemistry 29, 2606-2611). To study the structural requirements for fact IXa binding to platelets, we have carried out equilibrium binding studies with hum factor lXa after replacing the first epidermal growth factor (EGF) domain by the c( responding pnlypeptide region of faclor X(Lin, S.-W., Smith. K. J.. Wclsch, D.. a Slafford, I). W. (1990) •J. RioL ('hcm. 265. 144-I5(1). The chimeric prolein, fact as well a> the wild-lylx, fac•tcrr IX.,. produced in embryo kidney cells, a factor IX isol:rtcel from human plasma were radiolahcled with '"I and aclivaterl w factor Xht. F)ircct binding studies with Ihrombin-activatcd platclcts showed nom sloichiometry and alTinily of hinding of factor IXa„,,,, (566 si(es/platelet, K, = 0. nrwt) and factor IXa (590 sites/platclcl, K., = 0.61 nM) in the presence of faclor VI (5 unils/ml) and G•rctor X (1.5 µnt) compared to factor 1Xa (55X sites/pla(elel, K, 0.67 nM). The ctmcenlralion nf factor IXa, factor IXa , anit factor IXa,,,,,,,, requit for half-maximal ralcs of fac•tor Xa formalion were 0.63, 0.7, and 0.83 nM, indicat that Ihe K,app for binding (tl factor IXa,c,,.,,, to the factor X activating complex activatcd platelets is nnrmai. These studics suggest either that the IiGF-I domain factor IXa ic nrrl invtilvcd in factor IXa hinding to platelets or that (he fGF-I cloln Irom factnr X whcn inserted into factor IXa• sufficcs to promote normal factor I binding. 6I
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1/u,wr1..S..f.. Rawala Shciklt. R.. ('hcunR, W.-I•., .Slalli,rd, I). W., nncl Walsh, P. N. hhc p,urnal nf Rinln1"ical ('hcmisn-y 2(,7( 12):)i571-R57h. April ?5. 1992. Othcr m1,P„rl: National Intitilalcs rtf Ifcallh and W. W. Smith ('haritahlc Trust. Frrnn the Thrnmhosis Research ('cntcr, Dcpartnunts of Mcclicine :md Biochemistry. Tcmplc ltnivcr,;ily School of Medicine. I'hiladclhhia, and file 1)cl,artmcnt of Itiolngy, l Inivcrsity of Nc,rlh ('arc,lina, ('hapcl I lill. ('OMI'ONI?N'I:S AND ASSI:MI;I,Y OF TIIF FACTOR X ACTIVATING ('OMI'I,I?X The imlx,rtance of Factor V111 and Factor IX in hernnslatic reactions is evi- clcnccd by file fact Ih:rl hcmnPhilia A(Factor VIII deficiency) and hemophilia B (Factor IX deficiency) are Iwo serious congenital del'ecls, hc,lh producing severe, lifc-Ihrcatening, lilc-hmg hemorrhagic diseases. Ilotnorygous Factor X deficiency is extremely rarc and is al.n a scrious bleeding disordcr. Recent evidence from our lah- uratnry a% reviewed in Ihic article indicates that the interactions of Ihese three cnagu- I:rlirni hroteins normally cxcurs on the surlnce of activated platelels through receptor- mecfiated mechanisms, which are physiologically and hiocheniically important. T'hercfnrc, we believe that the extensive study and chicidation of the mechanisms by which the Factor X activating complex is assembled (in the platelet surface will pro- vide infc,rmaticrn essential fnr understanding file central role of platelets in promnling nr,rmal hcmrntasiti. Alrmod, S ,4., Rawala-Shciklt, R., and Walsh, P. N. Seminars in Thrnmhntiti and Ilcmcnlasis IR(;):3I 1-323, 1992. OIhcr suPhnrt: National Inslitutes c,f 1lcalth and W. W. Smith Charitable Trust. From the Thrcmthn.is Research Center. Deparlmenls of Medicine and Bicxhemistry, TcmpIc I Inivcr%ity School r,f Mcclicinc, Philadelphia. I'LA'IIFI.IiT A('TIVATIN(i FACTOR AND SYSTEMIC ANAPIIYLAXIS IN NI!'P(Lti7Fl)NI:Yl,I1,S BKA,SILII;N,S1S-SENSI'fI7.1?I) RATS: DIFFERENTIAL liFlli(°T:S 01: I'AI; ANTA(iONI3T;S I: The effccts of two hlatelct-activating factor (PAF) antagnnists, WEB 2086 and RN 52021, in reducing file changcs in extravasation (F,vans blue technique) and blood flow (radiofalxlcd micrnsphcrc method) to various organs and tissues follow- ing anaphylactic .hock in file Niplu,.clr ut,qrlu.c hrct.ci/irrccis-scnxitiiecl ral were invcs- tif:atcd. 2. RnIIt antagmtislti attenuated anarhylaxis-incluccd increases in plasma protein leak in file Irachca, ~tomach and small intcstinc. although they did not block cxtrava- ,;aticm in Ihc cc,lnn and kiclncysk t. Anaphylaxis-induced decreases in blood flow to the adrenals were effectivel rrtl;,Ynni,cd by WFR 2096. aalthough this antagonist did not reverse blood flm clccrcuscs to any othcr tissues. RN 52021. cm file other hand, did no1 aftcr anaphy laris-induced decreases in blood flnw to the adrenals, but effectively prevented dr, m;dic decreases in blood flow to file large and small bowel and spleen. .1. Anaphylactic shock procluced marked reduction in blood pressure tha( was pan Iv reversed by WEB 2096, wwhereas HN 52021 effectively blocked the decreases i carcliac output. 5. l'hus, PAF is responsible for sonic of the hacmnc(ynamic and cxtravasation c protein changes associated with systemic anaphylaxis in thc rat, although the diffe cntial inhibition observed with the two antagonists suggcsts that PAF altcrs vasculs responsiveness through different mcchanitims in selected tissues. Mathison, R.. Davison. J. S., and Bcftc.c. A. 1). Rritish.Inumal of Pharmacology IQ(i:2(ia-26(i. 1992. From the Gastrointestinal Research Group. I)epartments of Medical Physiology ar Microbiology and Infectious Discascs, Faculty of Medicine. lJnivcrsity of Calgar ('algary. Alhcrta, Canada. STUDY OF THF. ENDOPROTF,OLYTICC'LEA VAGE OF PLATELET GLYCOPROTEIN 1113 USING OLIGONUCLEOTIDE-MEDIATED MUTAGF,NESIS The precursor of platelet membrane glycoprotein Ilh (GPIIh) undergoes end pro(eolytic cleavage into heavy and light chains posl-translation. F..ndoproteolyr occurs within a 17-amino acid stretch of the precursor that contains 4 argini residucs, 3 in dihasic sequences (Lys-Arg (855-R56) and Arg-Arg (R5R-ft59)1 anc single argininc :u 971. Tn cletcnninc the site of GPIlh cleavage and its role in I ftrnctinn of file glycoprotcin Ilh/Illa helcrodimcr, we mtnatcd arginine 956. the c arginine sequence R5K-R59, and arginine 871 and coexpressed the mulants with gl coprutcin Illa (GPllla) in ('OS-I cells. Each GPIIh mutant formed recomhin: GPIIh-Illa hclcrncfinurs, hul mutants lacking arginine at 956 or R5R-859 failed undcrY.n clcavagc. Nevcrlhclcs. hctcrodimcrs containing the uncleaved CiPIIh wc cxprcc.cd cm thc ccll surfacc. Rccausc cndoprotcolysis most nftcn occurs af, arginincs in dihasic .cyucnccc, we ncxt expressed GPlib mutants containing lysinc 95(, or asparlic acid at 955 u.•ith CiPllla. Both mutants were cleaved and surfar cxpresscd, indicating that file dihatiic sequence at R5R-R59, hut not at R55-R56, required for 0Pllh cleavage. I.astly, we tcsled the function nf GPIlb-Illa containi unclcavcd GPIIh hy rncasuring adhcsinn of Iransfcctcd cells to immohiliMd fihrit gen. We found no dilTcrcncc in file adhesion uf cclls expressing either wild-type mutant GI'llh, indicating GI'Ilh-Iila hctcrudimcrti containing unclcaved GP11h ma Iain their ability to interact with fihrinoYcn. Knlocliicj, M, A.. Vilaire. (i., Grnntcr, I).. Ponci, M., and Rrtmell.J..S. 62 63
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The Journal of Rinlngical ('hcmislrv 2(,6(34):234r19-2 35fW, Ikccmhcr 5, 1991. (lther Nuhhnrt: N;itional In~litinCti (rf Ilt'alth. Frvom the Ilcmalolnw UncoloLy Scctir,n, Ilotihital rrf Ihc University of Pcnneylvani;t: 1)ivisinn of Ilcmatnlo)tv, ('hildren's Ilocl,ital ol I'hiladclphial and I)cparlmcnlti of Mcdirinc and Pcdi;nric~,. Univcrsity r1l 1'enn.ylvania School of Mcdicinc. 1'hi I;tdclphia. EFFIi('T OI' DI?I,I:TION OF GI.Y('OI'RO'fFIN IIR EXON 28 ON TI IF. fiXl'RGSSION OF 'I'HIi I'LAl'IiI.F:I'(~I,Y('OI'ROTI;IN IIR/IIIA ('OMPI,EX Wc have isolatcd from an I II?I. cell c•DNA lihrary an alternatively spliced Iran- script fnr the platelet membrane glycoprotein Ilh (0'IIh) that resulted from Ihe clelo- litm of the .14 amino acidk of cxon 2>{ of the (4lin gene. Confirming an carlicr rchnrt. we altin dctcc•tcd this Iranscripl in platelet mRNA. To dctcnninc the conso- cµicnces of exrni 29 deletion on Ihe errressiun of the GPllh/IIIa hcteroclimer, we cxnre%sc•d cl)NA lur (il'llh" in ('OS-I cclls, either individually or simultaneously with a cl)NA fior GPIIIa. When recombinant G11111) ' " was expressed alone. it did not acquirc resitilancc to thc cniymc cndn-(3-N.-acctylglucosaminiclasc H. was not c•Icnvccl inUn heavy and light chaint, and was nn1 transported lu Ihc c•cll surfacc. I lowcvcr, when rccomhinvil (iPllh " was c•ocxpresscd with recombinant GPIIIa. GI'llh/Ill;t hc(cmdimcr% were ;ts%cmhlcd. Ncvcrthclcss. these hetcrodimcrs failcd to cnrnplclc pn"lu;in0ational proc•cscing and were degraded iniraccllularly. F:xon 2R contain, one tiitc for Asnlinkcd glycrr.ylatirni, To dclcrntinc if Ioss uf this glycosyla- linn ,itc w;t% n•,,pomtiihlc• for thc cl7ccts of exon 29 dclctiun,wc rcmovcd the site from the exon 29 of intacl (iI'llh hy oligunuclcotide-meetiated mutagenesis. Ilnwever• ah.cncc of the c;trhrrhvdralc appended to exon 29 did not prevent normal (iPllh/Illa hclcroclimct gxprc.ti~ion. Our stnclicx indicatc that ahsence of Ilte amino acids encoded by ( il'Ith crnn 2S 1 nlTiricntly Imrlurhti thc quatcrnarv confignration nf Ihc GPllh/Illa hctcrrulimcr to imlrair its subsequent intraccllular transport and processing. They ;rlco inrlicatc th;n Ihi~'ahcrnativcly spliced forni oI Gl'IIh mRNA, although present in mcgakaryncytcti, i,~ unlikely to makc a significant contribution to the GPllh/llla c'runhlcrcti t•xhrc"1ed rni hLut•h•Is. KoIndiicj, M. A.. Vilairc. (;.• Rifal. S., Ponci. M.. and Rcrrnr•rr..l..S. Blood 7R(9):21.14-23(i3, November I, It191. Other support: Nalionnl Ihtitilulcs of I Icalth. From thc Hcmatnltrgv-On(-ningy Scction. Ilospilal of Ihc (Inivcrxity of I'cnnwlvania: 1)ivisitm ol Ilc•niatnhoLV, Children's Ilospilal of Philadclphia; and I)cpartmc•nt,~ nf Mcdiiinc aml I'cdiatrics. thc Univcrtiilv nf Pennsylvania School of Medicine. I'hiladclhhia. 64 A SIMPLIFIEI) METHOI) FOR TIIE DF,TERMINATION OF NITRIC OXIDE IN BIOL(H~ICAL SOLI ITIONS A new simplified procedure for determination of nitric oxide (NO) in biological ~olutions is clcscribed utilizing a new reducing sytilem of nitric oxide prior to chemi- luminescence. Advantages of the new method are that it makes heating of the recluc- ing snlution unnecessary and avoids cooling and condensation of generated vapors. Only traccs of acid with a high boiling point are used. The method permits analysis t,f small sample volumes (2(X) µ.L). Thc basal production of nitric oxide by freshly h:vvested endothelial cells ranged from I(X) to ARO picomoles. Tcrmin, A., Hoffmann, M., and Bing, R..l. I,ifc Scicnces 51:1621-1629, 1992. Olhcr support: The Margaret W. and Herbert Hoover, Jr., Foundation and Miles I,ahoratorics. From the Huntington Medical Research Institutes. Department of Experimental Cardiolcrgy, and California Institute of Technnlogy, Department of Environmental Engineering. Pasadena. THE EFFFCT OF IMMUNE MEDIATORS (CYTOKINES) ON THE RELEASE OF f;NDOT1iEL1UM-DERIVED RELAXING FACTOR (EDRF) AND OF PROSTACYCLIN BY FRESHLY HARVESTEI) ENDOTHELIAL CEL1 S The effect of recombinant tumor necrosis factor and other cytokines stimulated by LPS (lipnpolysaccharide), on the release of endothelial-derived relaxing factor and of prostacyclin was investigated using freshly harvested endothelial cells attached to plastic microcarrier beads. The results show that the cytokines failed to interfere with the release of EDRF and prostacyclin under the conditions of these experiments. Dudek, R., Kibira, S.. Kahler. J., and BinR, R. I. Life Sciences 50:RF3-R73, 1992. Other support: Margaret W. and Herherl Hoover, Jr., Foundation, Patron Saints Foundation and the Polish-American Congress. From the Huntington Medical Research Institutes, Department of Experimental Cardiology, Pasaclcna, CA. RELEASE OF PROSTACYCLIN ENDOTHF.,LIUM-DERIVED RELAXING FACTOR AND ENDOTI-IELIN BY FRESHLY HARVESTED CELLS AITACFIED TO MICROCARRIER BEAI)S Cultured endothelial cells have been used in the past as a source of cndothe- lium-derived relaxing factor (EDRF) and of prostacyclin (PGI,). Although cell cul- 65 ~
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lures arc cewnlial Gor nhscrv;llicm of prolonged exlxnurc uo mcdia or when Ihcrc i~ dehrycd re,;pun.c they are timr constnning and stcrile ctmditioms are es.ential. In fhe present %tudy, wc report Ihat cnthothclial cclls, freshly harvc%tcd from bovine aortas, readily allachcd Ihcrosclvcs to cylodcx-1 micmcarricn ceads and released an cndttthclitnn-dcrivcd relaxing factor (EDRF), rrostacyclin (1'(4h) and increased Ihc amount of cyclic GMP in vascular smooth muscle. Attachment lo micnrcarricn heads was es.scntial, sincc it incrcascd thc surfacc area and the nurnhcr nf altached cells and Ixvnnitcd collection ol' ccll frcc filtrates because of thc l"ormation of clcnsc networks ol' cells and heads. As a resull, superfusion of cells and heads on the filler did not dislodge hnund cells which remain on the filter. Conditioned filtrates frrm freshly harvested endothelial cells allachcd to micrncarricn ceads caused marked relaxation of cnchothclium-clehrivccl hnvinc Pnlmrniary artery strips. The degree of relaxation depended on Ihc number of cclls: maximal relaxation occurrcd with 50 million cells ;tt EDy of 14 million. Iligh values of cyclic GMP were found in vascular smooth muticle exlrnsed to cnnditioned fillratc. The calcium ionophore A211R7 further increased the amnunt ul'cyclic GMP. I,argc amounts of PGI, were released by fresh- ty harvestcd endothclial ccllti, particularly afier slimulation with the calcium inmophtuc. In contrasl, cndothclin production by freshly harvested cells attached to microcarricn ceads was barely dctcctahlc a(ler 30 min incubation and was beyond the limit of cletectirni by hioassay procedures. Freshly harvested endolhelial cells attached to micrncarricr Ixads appear to hc a useful adjunct to tissuc cultures undcr specific experimenlal conditions. Kibira, S.. Dudek. R., Narayan. K. S., and Ring, R..l. Molecular and ('cllular Biochemistry IOR:75-f14, 1991. Other sur(,nrt: Margaret and Hcrhert Hoover. Jr., Fnttndalion, Patron Saints Founclaticm, Tanahc Sciyaku Company (Japan). and Polish-American Congress. From Ihc I Iunlington Medical Research Institutcs, Pasadena, CA. I.O('ATING A I.OW DENSITY I,IPOPROTF,IN-TAR(iE'I'ING, DOMAIN OF III IMAN APOLIPOI'ROTEIN R-I(H) BY I;XPRI'sSSING A MINIGENE ('ONS'I'Rl I("I- IN TRANS(;f':NIC MI('E Through its interaction with thc low density lipoprotein (LDL) receptnr, apnlilmpro- Icin (apo) I3 I(H) iti a majtrr dc(crminanl of LE)L rnctabohxn, and plasma cholesterol. Ils rccclnon hinding ahility is confnrmalion•dePcndenl and requires its expression on the right lilx,protcin rarticlcs. '1'hc stniclural signal that largcts almR-I(N) to LDL is unknown. We have micminjecled a human apoR-I(10 minigene construct comprising <25''G• nf Ittc apoli-I(N) sequence driven by Ihc natural apoR promoter to produce Iransgenic mice. The tranxgene pmduct was expressed at a high level and was pre- sent exclusively in the 1,1)L of these anim;ds. Analysis of the reslxm.ihlc sequence (residues 2R7R-.i925 of apuR-I(W)) reveals unique structural fcatures that may be impnrtant in its role as :m I.UI,-targcting dumain. Xiong, W., 7sigmnnd, F.. Gotlo, A. M.. Jr., I,ci. K. Y., and (-han. L. The Journal of Biological ('hcmistry 26(i(31):20R93-20H9R, November 5. 199 1. Othcr suhprnrt: Specializcd ('enter of Research in Arteriosclerosis at Baylor College nf Nledicine. Natiunal Institutes of Ilcalth and March of Dimes Birth Defec(s I:t'und:Uion. I:rron the Departments of Cell Biology and Meclicinc, Baylor College of Mec(icine, and thc Mcthodi4 Hospital, I lous(nn, TX. pIRE("T EVIDENCE FOR THE INVOLVEMENT OF FREE RADICALS 1N 1S('lll?MI(' 1NSt (1; f TO THE INTESTINE Ischcmia of rat intestine was induced in rivn by occlusion of the superioi mcscntcric artery (SMA) for 15 min. Sodium salicylate. 100 mg/kg, given IP 30 mir prior to the ischemic event served as a specific trap fnr hydroxyl radicals and provid ed r1ire<•t cridcrrr e for the involvement of dircct radicals cluring the ischcmic insult I'ortions of the bowel were sequentially isolated and removed. 'i'he hydroxylatiot products, dihydroxyhenzoic acid (DHBA) derivatives were isolatcd, identified an( quantificd by IIPLC coupled with electrochemical clelection (ECD). The level o 2.5-DHBA. (Mean ± SE: ng/g tissue) in the preischemic bowel (N = 21) was 241.9 a 10.(l. It rose significantly tn 313.? ± 15.5 in the ischemic specimen (p = 0.0129) an( rcmainecl unchanged in the reperfusion pcriod (322.R ± 15.5). The hislologica examination correlated well with these levels: mild villi damage in the ischemic Ix rind with no further damage in the rcpcrfusion period. (idassin, R.. Arieli. I.. Haskel, Y.. Kitrossky, N., and Chrrinn. M. Free Radical Research Communications 12-1a:721-724, 1991. Other support: Joint Research Fund of the Hehrew University and Hadassah. From (he Department of Pediatric Surgery and Pathology, Hadassah Universit Hospital. Jerusalem, and Department of ('ellular Biochemistry. Hcbrcw Universit} Hadassah Medical School, Jerusalem. Israel. I12EI? RADI('AI: INDI K'f?I) FIBRINOGF,N COACrU1.ATION: MODULATION OF NEOFIRE FORMATION BY CONCENTRATION, pH AND TEMPERATUR The reaction uf fihrin-gcn wilh C'u(tl) (10--I(N) µM) and ascurhatc (0.1-2 mM) leads to thc fumiatiun of an insoluble clot-like material. "nenfibe," which (1cpcndent on cxperimenlal ctmditinnti. The reaction is observed with human ar bovine fihrinogcn, in the presence of tl.l-(1.3 N Na('I, is optimal at Ihe pH range 7.4 7.7, and has ch;vactcristics typical of a site-specific Fcnton reaction.'lhus, it inhibited by I?D'I'A and catalasc. Inhihitinn by mannitol is observed nnly at rclativ ly high crntccntratinns of this scavcngcr (>I(H) mM). Crnt(fomitantly, the rate of ux } cn utilication incrcasce lincarly with the cunccntration of reagents. The energy activsuirni of uxygcn utili"atiun by accorhatc and ('u(II) in thc ahsencc or presence 66 67
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fihrinogen is F.a = 6.1 and 7.9 Kcal/mol, respectively. These values suggest that Ihe ratc-limiting step is dictated by a reaction of oxygen with "frce° or protcin-hound copper cations. The lemperalure dcpendcncy of the extent of transformation of human and bovine fihrinogen into nenfihc is unusual in that il is biphasic-increasing from 5 to 25^C, and dccrcasing Ihercafter, to 43°C. This is not due to the tempera- ture stability of fibrinogen. The esscntial requirement for coppcr, ascorhate and oxy- gen or hydrogen peroxide, as well as the low cfficiency of mannitol as a scavenger, are in accord with the most likely interpretation of these data that fihrinogen under- gocs a site-specific Fenton reaction. This modifies Ihe protein and results in the fnr- mation of insoluble, pnlymeric, neofibe aggregates. Karpel, R., Marx. G., and Chcrinn, M. Israel Journal of Medical Sciences 27:61-66, 1991. From the Department of Cellular Biochemistry. Hebrew University-Hadassah Medical School, Jerusalern; C'oagulation Laboratory. Magen David Adorn Blood Services, Tcl-Hashomcr, Israel; and Oklahoma Medical Research Foundation, Oklahoma City. SA1,I('YI.ATF. AS AN IN VlVrl IREF, RADICAL TRAI': STUDIES ON IS('Hf;MIC INSI)LT TO THE RAT IN'1'ISTINE lschcmia of rat intestine was induced in riro by occlusion of the superior mesentcric artery (SMA) for 15 min. Sodium salicylate, 100 ntg/kg, given 1P, 30 min prior to the ischemic event scrvcd as a specific Irap for hydroxyl radicals. Portions of tfie bowel were sequentially isolated and remove(I- 2 min prior to ischemia, 2 min prior to declamping ol (he SMA, and IO min following reperfusion. The bowel seg- ments were homogenized in 3% TCA. The homogenate was centrifuged and filtrated Ihrnugh a n.2:.1 µ filter. The hydroxylation products of salicylate, dihydroxyhenzoic acid (I)I IHA) derivatives, were isolated, itlentifiecl, and quantified by HPLC coupled with etcctrnrhentical detcction (1;('D). The level of 2.5-DIIBA (M ± SE, ng/g tissue) in the prei.chemic bowel (N = 21) was 24 1.9 ± 10.0. In the ischemic specimen the level of 2.5-DHBA increased significantly to 313.3 ± 15.5 (p = q.O129), and reniaincd unchanged in the rcpcrfusion period (322.8 ± 15.5). The histological exam- ination cnrrelated well with these levels: mild villi damage in the ischemic pe-rind with no further exacerbation during the reperfusion period. This study in an in rivn animal model of intestinal ischcmia-repcrfusion provides clirect evidence for the involvement of free radicals during the ischemic insult. Udassin, R., Aricl, I., I laskcl, Y., Kitrossky, N., and C'hcrinn. M. Free Radical Biology & Medicine I0:1-Ci, 1991. From the Department of Pediatric Surgery and Pathology, Hadassah University Hospital, Jcrusalem, and Dcpartment of Cellular Biochemistry, Hebrew University- Hadassah Medical Schrol. Jerusalem, Israel. 68 A pOSSIRLI; ROLE OF FREE RAI)ICAI.S IN TI IE TRANSPLANTATION OF Rts fINAL PI(iMf;NT EPITIIF,LIAL ('fiLLS Oxygcn-dcrived free radicals have been implicated in tissue injury following i~chcntia and reperfusion (reoxygenation). It has been hypothesized that the radicals ;lre prnducetl during the early reperfusion stage. Recently, submacular implantation nl retinal pigment epithelium cells has been reported. It is probable that during the hrr,cedure, the transplant and the IR0-dcgree folded outer retina underwent a period n1 ischemia, followed by reoxygenation. We, therefore, infer that free radicals were proelucecl (luring the reoxygenation stage of the procedure, injuring both tissues. We urggesl that these hypotheses be investigated with the aim of improving the surgical rnitcome in eyes with age-related macular degeneration. Ophir, A. and Chcrh,n. M. nphthalmic Surgery 23:(4):284-2R7, April 1992. From Department of Ophthalmology. Hatlassah University Hospital. and Departmen of Cellular Biochemistry, Hebrew University-Hadassah School of Medicine Jcrusalem, Israel. LOC ALI7,ATION OF p35 (ANNEXIN I, LIPOCORTIN I) IN NORMAL ADULT RAT KII)NEY ANI) DURING RECOVERY FROM ISCHEMIA The 35-kDa protein (p35, lipncortin I, annexin 1), originally discovered as Ca-dependent substrate for the EGF receptor tyrosine kinase, binds CA" and phos pholipids. is developmentally regulated in embryos and has restricted expression i adults. Immunohistochemistry of normal rat kidney shows that p35 is enriched i, epithelia of Bowman's capsule, the macula densa, and medullary/papillary collectin ducts, suggesting that p35 is related to specialized renal functions. Light staining i nlnerved in the thick ascending limb; elsewhere, immunoreactivity is nil. Since ren, recovery from ischemia involves both hyperplasia and hypertrophy and reportedly i accclcrated by EGF, we examined p35 distribution during this process. After 4 hours of recovery, hnth the distribution and amount of renal p35 are alterec Immunoblots show p35 levels increased at least threefold in whole-kidne homogenates. The expression of p35 is still highly restricted in recovering kidney: however, the thick ascending limb now stains heavily. This is the first document; tion of altcrations in anncxin levels (luring a pathophysiologic response. Howcve our attempts to discern cl7ects of exogenous EGF on the recovery from ischem were negative for both mitotic index and renal function assays. McKanna, J. A., Chuncharunce, A., Munger, K. A., Breyer, J. A., Cnhcn. S., ar 1larris, R. C. Journal of ('ellular Physiology 151:4(i7-475, 1992. Other support: National Institutes of Health. From the I)epartments of Cell Biology. Medicine Biochemistry, Vandcrhi University School of Medicine, Nashville, TN. 69 I
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IMPAIRHI) III?PA'I'IC' APOI.IPOPR(YI'EIN R ANI) 1? TRANSI.ATION IN STRFP'f(Yl.t)TO('1N DIARI-"17(' RAI;S Studies of streptoiohx•in-induccd diabetes in rat% have demonstrated that hepat- ic apo R:md apo E prodnction are rcduccd. To determine if rcductions arc related to decreases in hepatic mRNAs, we Perfornred hlotling analysis of Iotal liver RNA with rat apo R, apo F., and alhumin cDNA probes. The cxpcctcd reduction in albumin mRNA levels to 4R"G, of control livers occurred in diabetic rat liver, while apo B and apo F mRNA levels were unchanged. The proportion of translational stop codon (BSTOP) mRNA avcragcd 43% of total in diabetic rats similar to control levels. Long-term labeling experiments using j"Sjmethionine in primary cuhures of rat hcp:nocytcs and specific immunoprccipitati<ms dcmonstratecl production of apo B and apo F;, and albumin by hcpakxytcs from diabetic rats was reduced to 37"/.-, 53%, and 23^ti. of controls. Pulse-chase studies, together with mRNA analyses, suggest that reduced hcpatic sccrction of apo 13 and apo E in diabetics is primarily a result of impnircd translation and not intracctlular degradation. Rihosomc transit studies directly conlinncd the prolonged clongation ratcs for api) B and apo E mRNAs in hepalorytes derived from diahetic rats. This effect was more pronounced on apo Btn (higher molecular weight) than on alxi RI. (lower molecular weight). Treatment of diahctic rats with insulin for 7 d Icd to normalization of hcpatic albumin mRNA lev- els with no substantial change in apo E mRNA Icvcls. In contrast, insulin treatment resulted in significanl increase in hepatic apo B mRNA over control levels. Results suggest hepatic alhumin and apo B mRNA Icvcls ;rrc respcrosive to insulin in the dia- hctic ctatc. Sparks, J. I).. "l,olfaghari, R., Sparks. C. E.. Smith, H. C.d and Fi.cner. E. A. Journal of ('linical Investigation R9:14IR-143O, May 1992. Other support: National Hcart, Lung and Blood Institutc, Arncrican Diabetes Am;cx•isuion and W.W. Smith ('haritahlc Trust. From the Dcpar(mcnt of Pathology and I,ahoratory Mcdicinc. University of Rochester School ol Medicine and 1)cntistry, Rochester. NY, and Departtncnt of I'hyeioh,gy and Biochemistry. Medical C'ollegc of Pennsylvania. Philadelphia. Tf11'. ('ONTRIRFITION OF VASCULAR ENDOTHELIAL XANTHINE DFaIYI)RO(iI:NASIi/OXIDASf? TO OXYGEN-MEDIATED C'ELL INJURY 'llu convcreion ol"xanthinc dchydrogcnasc (XDH) to xanthine oxidasc (XO) and the reaction of XO-derived partially reduced oxygen species (PROS) have been sug- gcslcd to Ix importanl in diverse mechanisms of tissuc pathophysiology, including oxygen toxicity. Bovine aortic cndothelial cells expressed variable amounts of XDH and XO aclivily in culture. Xanthine dehydrogenase plus xanthine oxidasc specific activity incrcased in dividing cclls, peaked after achieving cnnlluency, and decreased in postctmflucnl cells. Exposure of RAFi(' to hyperoxia (95'I. 0,: 5% CO=) for 0-48 h caused no.change in cell protein or 1)NA when compared to normoxic controls. C'ell XDII + XO activity decreased 9R^/o after 48 Ii of 95% 0, exposure and decreased 6A'%, after 49 h nonnoxia. 1)uring hypcroxia, the perccntagc of ccll XDH + XO in lhc 70 XO form increased to 100%, hu( was unchanged in air controls. Cell catalase activit) was unaffectcd by hyperoxia and lactate clchyclrogenase activity was minimally cle vated. Ilyperoxia resulted in enhanced cell detachment from monolaycrs, whicF increased 112% compared to controls. Release of DNA and preincorporatc( IK-"('ladenine was also used to assess hyperoxic cell injury and did not signrficantl) changc in exposed cells. Pretreatment of cells with allopurinol for I h inhibited XDL + XO activity I(N)"/o, which could be reversed after oxidation of cell lysalcs will lx,tassiurn ferricyanide (K Fe(CN),). After 49 h of culture in air with allopurinol, cel XDH + XO activity was enhanced when assayed after reversal of inhibition will K pe(C'N)n, and cell detachment was decreased. In contrast, allopurinol treatment o cells I h prior to and (luring 49 h of hyperoxic exposure did not reduce cell damage Ater K Fe(CN)« oxidation, XDH + XO activity was undetectable in hyperoxic ccl lysates. Thus, XO-derived PROS did not contribute to cell injury or inactivation o XDH + XO during hyperoxia. It is concluded that endogenous cell XO was not . significant source of reactive oxygen species during hyperoxia and contributes onl: minimally to net cell production of 0, and 14,0, during normoxia. Panu's, P. C., Wright, S. A., Chumlcy, P. H., Radi. R., and Freeman, R. A. Archives of Biochemistry and Biophysics 294(2):695-702, May I, 1992. Other support: American Heart Association and National Institutes of Health. From the Departments of Anesthesiology. Biochemistry. Pediatrics an. Pharmacology, University of Alabama at Birmingham. LIPOPROTEIN LIPASE INCREASES LOW DENSITY LIPOPROTEIN RETENTION BY SUBENDOTHELIAL CELL MATRIX Lipoprotein lipase (LPL), Ihe rate-limiting enzyme for hydrolysis of plasm lipnprotein triglycerides, is a normal constituent of the arterial wall. We explore whethcr LPL affects (a) Iipoprotein transport across bovine aortic enclothelial cells ( (h) lipoprotein binding to suhcndothclial cell matrix (retention). When bovine mil LPL was added to endothelial cell monolaycrs hefore addition of "9-laheled LDI LDL transport across the monolayers was unchanged; but, at all concentrations ( LDF, tested (1-1(N) µg), LDI, retention by the monolayers increased more than liw fold. "I-labeled LDL hinding to extracellular matrix increased when LPL was addc directly to the matrix or was added to the hasolateral side of the endothelial cc monolayers. Increased LDI, binding required the presence of LPL and was not asst ciated with LDL aggregation. LPI, also increased VLDL, but not HDL, rctentioi Monoclonal anti-LPL )gG decreased both VLDL and LDL, retention in the presenc of I,PL. l.,ipoprotein transport across the monolayers increased (luring hydrolysis 1 VLDL triglyceride (Tr). In the presence of LPL and VLDL, VLDI, (ransport acro the monolaycrs incrcased i f{"lo and LDL transport increased 37%. High molar co centrations of olcic acid to bovine serum alhumin (3:1) in the medium increas( VLDL transport 3(1^/v. LDL transport increased 42% when olcic acid was added the media. Thcrcfore, LPL primarily increased retention of LDL and VLDL. A Ic remarkable increase in lipx)protcin transport was found during hydrolysis of "I'G-co taining lipoprotcins. We hypothcsicc that LPL; mcdiatcd VLDL and LDL rctcntic 71 i
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wilhin the at'ICrial wstll pntcntiatcs crinvcrsiun of Ihc•ec IihnlxoIeitte to more atltcrtr ):cnic fnt'tns. Sarcna, IL. Klcin. M. G.. Vanni• T'. M. and f:„lrlhrr,~. l..l. hiurnal of ('linical Invcstizatirin Rt):37i-1R(1, Fchrttary 1992. Othcr suphr,rt: Natirmal Ilc•art, 1 11119 9 and Rlnod Instilutc and American 11car1 Association, New Ynrk ('ity Affili:uc. Frrmt Ihc I)charlmcnl Of Mcclicinc and Shccialiicd ('cnlcr of Research in Arteriosclerosis, C'tdumhia llnivcr,,ity ('nllcLC nl Physicians & Surgcrnis, New York. I.IPOI'RO'IIiIN LrI'ASI: ,MI',I)IATI: .U l IPTAKIi ANI) DI?CMAI)ATlON OF LOW I)I?NSI'fY LIPOPROTI31NS BY FII1RO131.AST;S ANI) MA('RUPIIAGFS I,ihnhmlcin lihit.c the rate limiting enzyme for hydrolysis of lipoprntcin Iri,elyrcridc, alsti mcdialcti nimcnzymatic interactions between firc,prtNCins and hclraran tiullatc hnncoglycan.. Trr clctcr•minc whcthcr ccll surl'ac•c I,PI, increases I-I)I. hin4ing to c•cllti, bovine milk 1,111, w;ts ;rddccl to uprcgulatcd and nonurrcgulat- cd hnman Iihrrrhla%tt along with media containing LDI,. 1.1)I. hincling to cells was incrriud ? I(Llold in a rhi.c-clchcndcnl manncr• by thc addition of (1.5-I(1 flf;/ml of I,I'I,.'I'hc amrrunl of L1)I, honrnd to Ihc cells in the hrcscncc of'1.1'L far cxcccclccl Ihc cahacity for LI)I, hincling via the I.I)I, rcccptor. 'frculnrcnl of fihrrrhlasts with hclutrinasc and hcParitinaW rctiullcci in a(,<1'/, clccrcasc in I,I'1.-mccliatcd I,UL hind- ing. Crrmintrccl In ttudic. Pcrfortnctl'withnul I,PI,• more I,I)1, w:rt inlcrnaliiccl and clc).traclcd in the In-cscncc nf I.PI.. hut the tintc c•nurnc was slower Ihan that cif' classi- cal IIlnohrUlrln rcccptor mediated pathw;rys. In 1,1)I, receptor negative fihrohlasts, I.I'I, incrcawd .urfacc hnund 1.1)1, > 14O.fnld. in(raccllular I,I)1.> 4O-fold, and 1.1)1, dcgradaurm > 6 hold.'I'hcsc clfccts were almml c•trmplctcly inhihitcd by heparin and mtti•I.I'I nu,noi•Innal antibody. 1.111, also increased the hincling and urt;tkc by fihruhLt.tti of aholilmhriitcin.lrcc Irizlyc'criclc cnuil.itms: hincling was increased > R- fr ld ancl r'llular uptake was incrc•uscd > 40-fold with I.PI,. I,1'1, increased I,U1. hin linp. Io'fI II' I m mucylcs, a/ttl inc'rcascd L11L uptake (d.5 frrlcl) and 1,1)1, dcgra- (I:uion (? S Inld) hy 'I I II'-I macrcrhha,ecs• In the absence of added I,I't., hchan•in and anti I.I'I nu0nncl0msil anlihcxlicti decreased I.I)L degradation hv > 4O'%, and IriFlyc•- cri lc• crmil1 u,n ul,takc by > 5()''S•. sngecstinz that cndngcnuu.ly pmduccd 1.1'1, mccli- ,nccl Ill id Ir,irliclc eyplakc ;mrl tlcgrathnirm. We conclude that I.I'I• increases lipid and lihulnrvlrin ul,lal.c h.v t•clk via a Ptultwav nnt invnlvingg tltc t,111. receptor. This rath- wa\msiv I,c impr rl+ml fr r lipid ,icc•umulatinn in 1,1'I, synthc.iiing cells. Runncy. S. ('., Ohunikc, .L ('.. Arad. Y.. I)cckclhanm. R. .I., and l:nlrlhrr.l, /. .1. .hornnal of ('linicstl Invc,,litsrliun 9/):15O.1 151?, (h-Inhcr 1992. OIhct ~ulih,rt: Nalirmal Ilcart, I.ung:ntd Blood htstitulc. From the I)chv-lmcnl% of I'cili;uric,, antl Mcciicinc, ('olumhi;t ( Inivcrsity C'ollcic of I'hv-%ician,, & Surpc mti. New 1'rnk. 1;11 ( ('"I;S OF IN'I'RAI,IPID-INI)I I('f;l) IIYPFRTRIG LY('I;RII)GMIA ON I'I.ASNIA IIIGH-I)I?NSI'I-Y I,II'OI'R(YI'1?IN MI:I'AROI.ISM IN TI II? ('YN/)MOI,GIIS MONKNY L,ow plasma Icvcls of high-dcnsity lipoprotcin (f 1D1,) and apnlipohratcin (aPr A I of)cn accompany human hypcrtriglyccriclcmia. In'an animal moclcl of hype Iriglyc•cridcmia. the IipoProtcin Iihasc (LPIJ-inhihilccl cynnmolgus mtmkcy. H rcpanicd that placma levels of apo A-I were decreased and the fractional c•atohnl rate (F('R) of' IIbL apo was incrcasccl. 'I'n explore whether hypcrtriglyccriclcm alone would allcr plasma apo A-I levclti and calaholism, hypertriglyccriclcntia w; produced by intravenous (IV) infusion of 20%, In(ralipid into female cynomolgt monkcys. Baseline plasma Iriglyccridc CTG) Icvcls avcragcd 106 mg/cIL. Wilh inf sion of 2(X) mg/kg/h Intralipid TG. plasma TG levels peaked at 967 mg/cll, (rang 413 to 1,Q(i9: n= 6). More prolonged or more severe hypcrlriglyccriclcmia caust serious cotnplicatirms in several monkeys. I)cspitc thc severe hypcrlriglyccridcmi IInL TG content, 111)1. apoprntcins, and plasma apo A-I levels did not marked change. suggesting that very little III)L remodeling had occurred. Kinetic studies I II)I, protein and apo A-I were performed in four pairs of monkcys. 'I'he two trace were rcnmved from the plasma at identical rates. In fivc pairs of animals, apo A turnover (luring control and Intralipiil-induccd hypcrtriglyccriclcmia was not tiigni cantly different. We hypothesize that apo A-I FCR is a function of HI)1, cxtmpn! linn. Because intralipid infusion did not altcr IIUI, composition to the samc clcgr as did I,PI, inhihitinn, its effects on I II)L apn catabolism were not apparcnt. (;nldhr•r,r. I..L, Vanni, T. M., and Ramakrishnan, R. Mclahnlism 41(1 1):1 17fi-1 1 R4, November 1992. Other support: American I leart Association and 13ristnl-Mycrs Squibb Co. From the I)cpartmcnt of Mcdicinc and Specialized Center of' Rcccorc•h Arlcritisclerosis, C'olumhia Univcrsity College of Physicians & Surgcons, New Yor fFFl?('T OF IIIJMAN rv-'I'IIRC)MRIN ON Tllfi TRANSFORMING GROW'1'II FA("I'OR-0 1-RINI)IN(i A('1'IVII'Y OI: IIIIMAN cv,-MA('ROGLORIILIN Thc rcac•tian of w-thrrmthin with human (r; macroglohulin (n2M), under con tinns specified here. resulte in Ihc f'trrniatinn of a,M-Ihromhin complexes in wh Ihc cr.M h;ts undcr;rtmc lmrtial or incomplete conforrnational change. 'lltc s1uJ pr"cnlcd in this invr,,tigatitm dcmnn~(ralc that rx,M-tltt'omhitt hinc)x TGF-I3I; level of hinding clctcctcrl hy nativc PAGE is increased compared with native re,M. cr.M-Ihrrtmhin. like other te.M fast liirms, is cleared from the plasma by (I clcnsity Iilurprrrlcin) rcccpttu-rchdccl protein (I,RP) located primarily in Ihc livcr.'1 tr•M-I'asI for7n-I.RI' intcractitm is not inhibited by T(it,'-fiI lltat is hottncl to tr,M. a r"u11, fast Grrms of (rr,M like rr,M-Ihmmhin may dclivcr T(iF-(31 to the surfacc ccll% that cxhres I.RP. Based tm Ihc irt virn experiments prcccntccl hcrc. Ihc fatc lY;F-G31 al'Icr delivery to t'hc cell as a c•omplcx with tr,M cannnt be clctcmiinccl. -1'hc factors determining affinity of TGF-13I fnr cr.M are c•crmplcx; however. crntfnrmalion of n•M iti ahharcntly imrtrrtant. We believe Ihat (luring tr.M conhon 72 73
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Iinnal c'hangcI a crilicat'r(I hirnlin"' "ilc• i" csl,n.c( l. I'rnlt•inascs Ihal hind Ic, o.M :md itn'm tcrnarv cnml,lcm.t inhihit T( iF-/;I hindin~,. 'I'hi% hninl is dcnn,mtrat- c l in thc lirescnl invt•~lipatinn hv icactini! hinary (r N9-Ihtomhin %.ith Irylxitt. Thc inhihitinn c,l T(il: IlI hintlinz cvnctl I+v hrt,tcina"c% m:rv hc ,tctic in nature (thc nrn- Icinatic c,helruclc Ihc T(iI'-f;I hlll(IInC ,itc). Allcrnalivclt., Pn,lcinascs may c•au.c %uhtlc chanccti in Ihc NIructurc (,f tht• (r•M ti-uhmlils. which dccrcaxc'I'(iP-(;I hinding affittihy. Futurc .luclic. xhnuAl inc•Indc a complete charac•ICriiatitm of Ihc 'rGF-GiI hincling "itr in rr.M and a dctrrminatinn of Ihc hioln):ic significancc n( (r.M-hrn- Icinaw cybikmc intcractit,n'. Wchh. I). .I.. IaPVlarrc..I.. and Grn,irrc, ,S'. l.. Scminart in Thrcnnhn.i,. and I Icnunlasit I R( Z):a(K-{ 1(L 1992. OIhc•r ,uhln rl: Schi htrs I'mptrani in Ihc Ricmicdical Sciences. Frc,m thc Ucpartntcnt of Patht,h~ty and Ricrchcmislry, lhtivcrsity of Virginia llcahh Scicnccti ('cnlcr. ('harb,ltctivillc. ('IIARA('-IliRl/.A'I'ION ANI) IMMI INOIIIS'rO('IIF.MI('AI.I.O('AI,r1,A"I'ION (/F cr -MA('RO(il.OI1III.IN RI{('I?I'TOR (I.OW-I)F.NSITY I.II'OI'R(Yr1;IN KF.('lil'lOR RIiI.A'fl'.I) 1'ROTIiIN) IN Ill IMAN BRAIN Pmtcinaw inhihitnrs have been implicatcd in hrain development and in clcgcncr- alivc hrnc•c,,scs ,nch a,~ Alihcimcr's diwaxc. Lt,w-dcneily lipnprtitcin rcccptnr-rclat- ctl hrtttcin (I RP) ic a mnllifunc•tirnml c•cll-surfacc receptor that hinck ac•tivatccl fnrms of Ihc htnl •inacc inhibitor, cr -macrnLlt,hulin (tr,Ml ancJ apnlipopmtcin F,. St,luhiliiccl plo.ma mcmhrancs of human cerebral c•c,nic-al gr,ly matter were sahjcct- rtl It, ;iflinit~ chromatography nn tr,M-mcthylamincscrharcxc. A single receptor was I,uritical: Ihi~ hmlrin was 1.10, as dctcrmincJ by molocular mass, pchtidc structurc. antl innnunorcactivity with numoc•lon:d and pnlvc•Innal antihndics. In adult human hrain, I RI' inmmn„rv;tctivity was abundant nn ncuronal cell hndics and proximal I,tocc.w.. /)titcr ccll" within Ihc ncurohil, includinR glia antl micrnvatlcular cells (cmFnlhcGnm antl hciicy(cs). were imnnmtmcgativc. Weak I.RI' imnmmorcactivity wa~ i lcnti)icJ in a I,riivaa•ular pattern ctirrespondinfg to Ihc location trf astmcytic Ir»d hrorctitt•1 . l'hc dieuihntitm nf I.RI' in Ihc ccntral nervous svztcm is consistent cvith Ihc Ixarnlial funrtinn of this receptor in the rcgulatic,n of pmlcinasc aetivity, cylt,kinc artivity, and chnlcstct'td mckthtilism. Wolf. R. Il., l.npcti. M. 13. S., VandcnRcre. S. R.. and Gnni(tr„S. /.. Amcrit'an .Inurnal t,l Pathnlc»•y 14I( I):37-42, .lulv 1992. OOther sul,rnrl: Natinnal htslitutc. of Ilralth and I'cw Sc•holar. PrnFram in the Ilitmtcdical Scicncc.. Frctin thc I)cpartmcnl til Ilinchcmititry and P:uhnh,gy, lhrivcrxity of Virginia llcallh Sciences ('cntcr. ('harhittc.villc. 74 AS,SI;MIILY 01: PI,ASMIN (iF.NERATING PROTEINS ON TF1E SURFA('E OI I Il IMAN I;NI)O'I'l Il?I,IAL ('EI.I.S Tratlitionally, plasmin gcncratitm has been cnnccptualiiccl as n process oricni nn Ihc curfacc of a fihrin-conlaining thrnmhus. Recent work, however, indicated t' plasrninngcn and its activators, tixsuc plasminogen activator (t-PA) ancl urokina can acticmhlc on the surface of cultured human tnnhilical vein cnclothclial cc (f1uvR('s). On binding to HIIVE('s, plasminogen is activated by t-PA apprtrxima ly 12-fnlcl more efficiently than fluid-phase plasminngcn, and is converted to a pI min-nuxlificd form, possibly unique to cell surfaces. In aclclition, I-PA interacts w IIt1VECs at two sites. Thc majnr binding site preserves its activity and represent truc (relative mnlccular weight 4(1,(NN)) mcmhranc-associaled exoreceplor. The h clcnsily lipoprotcin (I,DL)-tikc lipnprntcin, Fipnprrncin(a). is highly associated v, athcrosclcrnsis, hcars striking sequence homology to plasminogcn, and ccmipc with plasminogen fcir cell surface binding. In summary, functional assembly of p1 minogcn and I-PA tttay represent an important thromboregulatory system. Ilrrjjar, K. A. Annals ol' F;ridcmiology 2(4):419-42(i, •ruly 1992. Othcr support: National Institutes of Health and American Hcart Association. From the Division of Hcmatoingy-Oncology, F)cpartmcnts of Medicine : Pccliatrics, C'nrncll University Mcclical ('nllcgc, New York. LIPOPROTEIN (a) INHIBITS TI IE GENERATION OF TRANSFORMINC+ GROWTH FAC°fOR (3: AN F.NI)O(iENOIIS INHIRITOR OF SMOO'rFl MUSCLE C'ELI, MIGRATION ('onditionecl meclium (CM) derived from co-cuhures of bovine aortic endo lial cells (BAECs) and Ix)vinc smooth muscle cells (BSMCs) contains transforrr growth factor-(3 (T(iR(3) formed via a plasmin-depcnclcnt activation nf lalcnt TG (L:T0F1;), which occurs in hctcrotypic hut nnt in homotypic cultures (Sato, Y.. I). B. Rifkin. It)Rc). .l. ('r•ll Rit,l. 107: 1199-1205). The TGF-(,3 fornrccl is ahle block the migration ol' RSMCs or BAECs. We have found that Ihe simultane aclditiort tn hctcmtypic culturc medium of plasminogcn and the alhcrogcnic tipol Icin, lipoprt,lcin (a) (I.p(a)). which contains plasminogen-likc kringlcx, inhihits.- activatinn t,f I:rGF-(3 in a dose-dependent manner. Thc inclusion uf U)L in thc turc mcdium clicl not %hnw such an clfcct. C'ontrol experiments indicated that I, does not intcrfcrc with thc h;t.al level of cell migr:uic,n, the activity of cxogcr adclcd'I'(T-(3. thc rcicatc ttf I:r(iF-(i frrnn cells, the activation nf F:rcF-(3 eithe plasmin nr by trancicnt aciclific•ation, or the activity of plasminogen activator. adclititm nf Lp(a) to Ihc culturc mcclium clccrcasccl the almtwnt of Plasmin foun RAE('s/RSM('s cultures. Similar resulls were obtained using ('M derived f cocullures nf, human umhilical vein cndnthclial cells ancl human foreskin fihrnhl• Thcsc results wgtcst thal I,p(a) r.m inhihit thc activation of' LTGF-F3 by cnmpc with Ihc binding t,l' plasminugcn to cell or malrix surfaccs. Thcrcforc, high pla Icvc•I,; nl I.P(a) mighl cnhancc sntooth musclc cell migration by dccreasing thc Ic 75 I
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of Ihc mi}±r:uinn inhihitnr'f(iF /3. Ihus ccmtrihntin}: tn zcncratitm (if Ihc ;uhcrnma- Ir+m Ic"irm. Kojinur• S., I/ml~r•1, P. l'.. and Rilkin. 1). B. '1'hc.lnurnal of ('cll Rinhrgy I I.;((r):Id3'1-1445•.lunc 199 1. OOther support: Nalirmal Ilcarrt• I.unt and Blood InAitutc and Nalirnial Cancer Imtilulc. Frcrm the 1)crartmcnl of ('cll liialnl±y, New York Ilnivcrsily Mcdical School• and Raymond and Beverly Sacklcr Foundatiun lahoratory• Ncw Ynrk, and Division of Itcmalnhogy, 1)clr,trlmcnt rrf Mr•dic•inc• Mount Sinai School of Mcdicinc, New York. I'l1YSIOI.O(iI(' ROI.1? OF I.IPOPROTI:IN(a) I.ipnhrnlc•in(a) (I.h(a)) i% a Plasma IipnProlcin whose func•lion in humans has not as yct been dc•lincd (l ilcrmann 1989: Scanu and Flcss I')9(1). I?Pidcmiohogic stuclics have suggcstcd Ihal an increased hlnucl crmccntratiun of I.p(a) is an iniPortant and indcpcndcnt risk fac•Irrr for hnth ccrrnnary and c•crchrat vascular disease (Scanu Ir)XX). Thc :Ihility of L.h(a) in Ihc prescncc of ('a'' and (raneglulaminascs to incorfm- ralc primary amincs suggcsts that it may form poymers with suilahlc pro(cin part- ners. Thu% faclor XIII rr li.suc Iransglut:uninasc may calatlyic crosslinking of Lp(a) tu crll ~urLicc,, nr to vessel wall matrix prntcine. I,p(a) interacts wilh c•clts tn altcr surfacc rclatcd fihrinnfytic activiry or to stimulate I'AI-I production. Microvascular I.h(a) may orchc•slralc rcac•livity of fhc vessel wall lo inflammatory stinmli. In ad(li- lirm, I.P(a) may cnntrnl tietiuc cnnccntrations of "fY;F-(3 which may promote snxrnth muscle c•cll migration. 'fhc,,c studies thus define novel mcchanitims that allcr the LP(a) nrnlcculc and that could he invcrlvcd in nrodulalinf; its Ihrrmihntic and athcro- Pcnic• hnlcnli;d. Ilnrlrrl, 1' (' and Nachman. R. L. In: Bcarn. A. (i. (cd.): Gcnctics of ('rrrunary I)iscasc. Institute ril' Medical Gcnctics, I lnivcr~il) of Osln, pp. 53-(i2. 1992. riOOther -cuhlun•t: l I.S. Piihlic I lc•alth Service. Ficmt tI r 01r•i~irm nf Ilcmatrrhrgy, 1)cp,vtnunl of Medicine. Mount Sinai Mc(lical Crntcr. nnd I)ch;irtmcnt of Medicine and SPccinliicd ('enter of Rcscarch in llnrrmhmk. ('rnncll I lnivcrsiry Medical ('crllcgc. New York. II)FN'llFl('ATION OI: MI:('IIANISMS'hIIA'1' MAY MO1)IJI,ATIiTIIf? ROLfi OF I,11'OI'ROTI?IN(a) IN l'IIROMROSIS AND A'1'IIERO6F.NIiSIS In Ihi,, rclrnrt, we review recent findings concerning Ihc idcnlificatirm of mccha- nimm. that may nxodul;r(c the rnlc cil' IihoPrntc•in(a). or I.P(a). in Ihromhc,sis and ;uhcmgcnc,Js. I.r(a) hind,, to surfacc-immrrhili/cd plasmin-mndificd fihrin• Ihue Pr virling ar nu'c•hnnism ftrr incurpnratinf, LP(a) intu the vessel wall. We fnund II t Irrnnocystcinc and other sulfhydryl-c•nntaining amino acids markedly increase hindinz of I,P(a) to Platimin-modificd lihrin. Our resultc suggest thal homncystci nltcrs the structure of l.P(ar) to cxpct.c lysinc-hinding sites on Ihc apoliPoprolcin 1 pnrtirrn of Ihc molecule, and thu. Prr~vidc a potcntial biochemical link hctwc thrnmhnsis and athcrogcncsis. Wc also fuund that tranxglutaminases catalyic I incnrluoratinn nf primary amincs into Lp(a). Studies in cell culttrre systems ha found Ih;d LP(a) stimulates cndnthclial cells to synthesize and release plasritinng ;tc•tivatnr inhihiNrr-I. Further. Lp(a) inhibits thc activation of transforming grov factnr-Ixla in a coculture of bovine cndolhclial and smooth muscle cells. Plnrpel. P. C. an(1 Borth. W. Annals of lipiclctniology 2(4):41?-417, July 1992. Other support: U.S. Puhlic licalth Scrvicc. Frcmi Ihc I)ivisinn (if Ilcm:dnlogy, Mount Sinai School of Medicine. New York. LINKAGIi OF ATIif:ROGIiNK' LIPt7PRnTE1N PHF,NOTYPF. TO THE LOW 1)IiNSI'1'Y LIPOI'RO"I'EIN Rl's('I;P'IY)R LO('lIS ON "1'IIF; SI IORT ARM OF ('IIROMOSOMB 19 The athcrogcnic Iilrtiprolcin phenotype (ALP) is a common heritable Irait cl stctcriicd by a predominance of small, dense low density lipoprotcin (LI)l,) partii (sufxlass pattern R), increased levels nl' triglyccridc-rich lipoprotcins, rcduclinnm high density lipoprotcin, and a 3-fold increased risk of myocardial irrfarcti Significant two-point linkage was found between ALP and the I,DL receptor Ic on thc short arm of chromosome 19 in 51 rclativcs of ninc probands with AI.I' Icrn 13. The maximum logarithm of odds (LOD) score of 4.07 was observed rccomhination fraction of 0.04, assuming 100% Pcnc(rancc of AI,P pattern B. 4.27 at a rcconihination fractinn of zero. assuming 90% pcnetrancc of pallcrr IlaplotypinE data and nwltipnint IinkaFc analysis suggest that the gcn(. Inat AT7/,S fur:uhcrnsclcrnsis susccptihility (Iifxtprotcin-assnc:iated)l responsible for / is Icx•atcd dislal to 1)1r).S7h near rtr at the I,DL receptor Icx:us. This result sugg Ihc potisihility that gcnclic variation at thc LDL receptor locus or a closely Iin Incus nn chrmmo,,rnnc 19 may he respnntible for metabolic alterations in AI.1' tern 13 that account for a suhslamtial pmpnrtion of thc familial predisposition to c nary artery diseasc in Ihc general pnpulation. Nishina. ('. M.,.lnhrrcrm..1. 1'.. Nakgcr(,.f. K., and Krauss. R. M. Prnc•ccclings uf thc Nalirmal Academy of Sciences 1ISA Xc):70X-712, January Ir)S Other support: National 1)air,y Promotion and Research f3oard, National Institutt Ilcalth. National Ilcarl, I.ung and Blood Institute. U.S. I)cpartmcnt of Energy. Amcricsm Ilcart Associatirm. Frnm thc ('hildren's Ilospil;d Oakland Research Instittdc, Oakland, CA, and 1)o Lahnr:nory, Lawrence Berkeley Lahnralory. l)nivcrsily of ('alifornia, Rcrkclcy. 76 77
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Ii\I'RI•SSION OI IIIIi ( »,th I'ROfr) ON('()(;I•:NI: IN I(nVINt: VAS(-(tI,AR SM(N)I II Ml lt('1 li ('I'.I I S I'rc%inuS I)' wc h:tv,• .hi,%vn Ihal hnvinc l.s,S t ula, .nt„nlh ,nuu•Ic cclls (SM('N) cxln,•tiS c nnh tnltN.y (Ilvill\, ('. F., Kin,ly. M. S,. 13rnwn. K. I... R„Scnlx•ri, R. I)., ancl Scmctnht•in, ( i. I{. ( I')XV) ./ Rnr/ ('hr•m. '(r-1. (,')9O. R095). I lere we have c'h:trac- Icriicd changcs in Iltc I„cc level nl c rnn-h ntRNA crhrt•S.c,l in ytncticcnl scrurn deprived Suhrnnllncnl SN•L('ti ulr„n cnlrv int„ Ihc cell cycle. Aftcr scrunt ~Iintufalirm. Icvcl.,il c nivh mRN:1 incrcau l 3-.a li,id durinc Ltlc (i and remaincd aI Ihi. level durin!r S hh:tw. A 1.5 I,il„haw harti:tl c nrnc LI)NA t'hmc, ienlatcd it'rnn a Ix,vinc SM(' lihrary, wati Ir,rrtially vcyncnc'cd :md fnuttd I„ hc 89 and X5'G• h,tntohiinuti to Ihc Ituman and rmirinc t nn•h tcncs, rcShcctivclv. ltSinL hrn•inc and murinc c-nn•h ch,nr., nn changc in Ihc r,rlc nf c nrrh gene Iran%c'rihlirm or mRNA stability wns dclcrtcd dminr Ihc r•cIl cv, It•. 'fhu.. Ihc rcgulalirnt r,f chvtgcs in c nn•h mRNA Icv- clv in SM('e ahhc:n'" rliSlincl Inmt mceh:rnitinn .eccn in hcm:dr,hnictic or fihrc,hlastit' ccllti. Vcctr,rs cr,ntainin, nrt•h hin,linz sites linked Irt the thyntidinc kinase Irrumn1cr :mrl Ihc chlnrantlthrnic„I :rc'clyllrmfcrasc reporter Lcnc wcrc Irsmcicntly IransIcclcrl int,t SM(' cullur". liI IK-('A'1'-dAX. which contains ninc cnncalcnatctt nrv/, hinding sitc%, cxhihitcd 7-Ibhl more ac•tivitv than life parental tIAX-'I'K-('A'I'vcclr,r in cxrrn ncntially f:rr,wini SMO,. I'hc Icvcl, uf chlnramphcnic'nl ncclvltranslcrasc activity in cxlxmcmially grrrwing cclk, were approximately 2-fi,Icl higher than in cells Ihal had been scrunt deprived f'nr 24 It and were entering quiescence. '1'hnx, SM('x produce a limclinnal c-nn~h rrntcin Ihal cmt activ:Uc U:m.crirtion frnttt a hctcmlr,gnns rrrrntnt- cr. Ivrlhcrnmrc. intr,ufuctirtn of anti-scncc c-mt'h nligrmnclc<,lidcs to yuicsccnt ,wrum dchrivcd SM(' cuhmrs .cvcrclv inhihitcd entry of cells into S phase upon t crum additi,m. Thu.. cxlnctiei,nn r,l life c-mvh r,ncngcnc plays an intrrtrlanl rufc in ccll cvclc pn~arctisi,m nf SM('ti. lit'r,wn, K. I'... Kinrh•. M.S, anri Srntcn.hcirt. (7. F. ;Thc .luurn:rl uf 13ir,lngical ('hcntisliy 2h7(7):4(r25.4(,30, March 5. 1992. OIhcr ~nhr„rt: N:nirm:rl Imlitulcs nf I lcallh. Frnm thc I)cp;rrtmcnl of Rirrchcntivtty, R„tilr,n Ihtivcr:vily School of' Mcclicinc• 1t„,~I„n. aml I)cpartntcnl ul 13ir,chcntislry, I Inivcrtiity of Kcrnuckv, I.cxingl,m. MOI )I II.A'IION Oh ORNI'I'rIINI: I)I?C'ARROXYI.ASf; mRNA I~OLl,OWING 'I RANSII'N I IS('I II~:MLA IN'IIII'. GI:RI3II. I3RAIN (hnithinc dccarhmvlaSc (()I)(') iti Ihc r:nc-lintiting cniynrc Ihal cataly~cs the synthc.ie nl lu,h~antinc. hcnn r,rnithinc and iS thuught to hc invc,lvcd in life cellular rcslumsc to ,tn'r,wth. ,Iiffrrcntialir,n, :md SIrcss. I'rcvirrus studicS havc (Icntontitralcd that Irnnsicnt ccrchral iu•hcntia resullti in :m incrc:,tic in OIM' aclivily and pnlvaminc xvnthcsi.. We havc nScd Ihc Mrmg(rli:m gerbil ns a ntr,dcl system to I"t life hvpnlhc- sis Ihnl life ccllular tr.h,nt.c to iSchcntia in(loccv a distinct pattern nf OI)(' gene t•xl'rt'ssion• Onr rcutll" indicatc Ihal transicnl ischcmia, incluccd by bilateral carnlitl ncclusinn. clcvatcti OL)(' ntRNA wilhin I•I aflcr rchcrfusirm, which correlates wilh incrcascd OI)(' nt•Iivity and hnty:unin(. synlhcSiS. htcrc:ucJ OI)(' ntRNA can he IrV iIt'rI in life Inrchrain, slri:nunt, hipp-c'antpus. .ut(1 ntitlhrain hut not life ccrchc lui t. whiih iti not suhjccl U, itichcntic injnry. In cnnlrast. c-/i,.c ntRNA incrc:rScrl I 1:, mtn :r/)cr rclticrfuvinn :tnd octin ntRNA did not dcntrmsn':Uc ;tllcra(innti in Icv lt •, mcVnti:r. 1'cntnharhilal Itrcvcnlcd the inc'rc:tsc in OI)(' ntRNA, whcrca% II un:tic anta}:onist MK-X/lI had no cffcct rnt Ihc elevation of OUC gcnc exhrc „n :tllcr iuhcntia. We crmcludc that life ischcntia-inclucc(l increase in OI)(' cniyr ,)riivilv tn;ry hc altrihutcd in h:rrl U, Iranwriplimtal aclivalirm of thc nl)(' gcnc. I)r•nqxcx•. R. .I„ Carncy..l. M.. nnd Kinrlv. M. ,ti'. Ionrnal ol'('crchral 131nnd Flow and Metabolism 11 (6): t)7')-7X5, It)'r)I. Otlrcr Suhport: N:dional Institulcs of I It•allh. I,nnt life I)ivisir,n r,f Ncurosur}cry and Vclcrans Aclminislration Ilnspil Ikcp:rrttncntti of Pharmncr,logy amd 13it,chcntiary. Lahoratory nt ('cllular a Mnlccular Ncurahioingy, and ('enter rm L?xc'clIcncc on Stroke of Ihc S:mclcrs-l3ro• ('cntcr rm Aging. ('handlcr Medical ('enter. llnivcrsity of Kcntucky, I.cxineton. IIYI'FLYCF.MIA SIII' SSf:S r fn.c mRNA EXPRESSION FOI,LOWING 'IRANSII?NT CGRIi13RAL ISCI II:MIA IN GI?Rf3I1,S 'I'hc c fn.c prolo-oncogcnc is aclivatcd by transicnt cerebral ischcntia. This a vatirrn may xignify a specific genetic response to ischcmia affccting krlcranct ischcntia and ullintalc cell atrvival. I1yFcrglyccntia. which cnhanccx brain inj front transicnt itichctnia, was stuclictl ft,r its clTccls crn this gcnc system in gerbils mcasuring c-/r,.c mRNA 2 h allcr 20 min of hil,ttcral carotid artery occlutirnt. 13t cfi).r mRNA was increased hy 'ischctnia ( I 1.7 a 5.0. /, <_ 1).05, fold increase) ct parc•tI Ir, mtnischcrrtic crtntrols (1.0 ± 1.3). I'rctrcatmcnt with I g/kg of glucnsc I lially reduced pnstischcntic c-/i,.c niRNA (6.3 4: 1.6. p 5 0.05) while 4 g/kg (if gluc cnmplclcly suppressed pu.titichcntic c Jn.c cxltresxion (0.7 ± 1).3, p 5 0.O5), TI d:da indic:dc that hvperglvccntia suFpresScs normal postixe:hcmic gcnc cxpre's! and vu}gcsl thc hrntiihilitv that Surh suhrrcxxirtn is a prcdictnr or even a crntlrih to hyhcr}:lyc•cnti:r-cnhnncc(t ischcmic hrain damage. ('onths, l)..I., I)cmpmcy. R. .L, hnnaldsnn• I).. and Kinrlv. M. S. .Irntrnal (il ('crchr'aI Islosul How nnd Mclaholism 12(1):I69-172. 1992, OIhcr surlt„r1: Nati,mal InslilnicS nf I Icalth ancl Veterans Adntini%tralion. From Ihc I)c(i;n'tnunte nl Surgcry. I'hv%ioktgy and Biochemistry. Stroke ('cntc lixccl Icnc'c. lfnivcrtilv !d' Kcntucky. and I)crnrtmc•,nl of Snrgcry. Vc•ICi Adminititr:dinn Mcdicol ('cntcr, I c'cingjon, KY, Mf.VAl.ONA'1'I? KINASF IS LO('A1.I'/.11) IN RA'1' I.IVPR I'F.ROXISOMI:S Recent tlata SuggcSt Ihal ral liver Itcrnxisontcs Irlay a crilical mlc in chnics NymhcSis. Sl,ccifiarlh•. (icrnritir,nt" cnnl:rin a numhcr of cnryntcs rcquircd fcir 7t) 7X
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Ic.lt n,l svnlhc~i~ at ~tt 11 a. Icr„I ranit r Im,tc in '. I tn'Ihrrrn„rc. hcrmMntnt•" invnlcc•tl in Iht• m rrn,,.vnlht•"it t,f't•hnlr-"It•rnl Irnnt ntcc;rlnnalt• aml cnnl:tin `i)•nili canl Icvt h nf ;q,t,lil,t,hn,lc in I.:t maj,,, rnn~lhurnt t,f ~cvt•tal cl:tca•s of plaana Iila,hrtilcim. In tIhi..lu,lv wc havc invt•~lii.atrtl Ihc tiuhcclinlar h,caliialiam t,l' mt•val- nnalc kin:r.c (1;(' ?.7.1.3h: A`Il':mt•vnhmalt•-5-(iht,aihnlramfcra.c/. Mcvahtn:nc kin:t~c i. hclictrtf t„ I,t• a cytrnt,lic cniymc :md c'alalvir. Ihr hhn.rh„rylatit,n of mc•v:thm;nc to I,Itnt nut;tlnnal(• 5 hhnshhnlc. Mcvahtttalc kin:nc h:tt Ixt•n htnific•d Irt,m r:n livcr cvtn.nl and a cl)NA clone ct,dini fnr ral ntcvahm;nc kinast• ha.,; altin ht•cn itt,l;ncd :mtl chv;tctt•riird. In Ihiti titudy• uliliiing ntnn<,clonal :mtihntlice m:rth• :rcaiml Ihc I,urificd ral ntc.;tlnnalc kina.c. we dcntnnstratc Ihc prcticncc ol' ntcv:d- t,n:tlc kinaw in t'al livcr ht•noxientncd and in flit' c'vlt,~t,l. 1?ac•h t,f thcSt• t't,mrarlntcms ct,nl;tinctl a diflt•n•nl G,rnt t,l' Iht• I+rr,lcin. 'lhc rl mul Ihc• M t,f Ihc hcrnri.r,mal nro- It•in arc 6?,;nul •1?,INI(1, rt Ixclivcly. 'fhc hI and M of Ihc cvtnst,lic hrtrtcirt arc (~-9 :mtl '1/)•(I(1O• rt•"hct•livt•h•. 'I'hc hcrnriu,mal Im,lcin wati also .iLnificanlly induced hy :t numlx•r t,f diflcrcnl hyl,nfilridcmic thup%. In :rcldilitnt• wc present cvidcncr fm thc nncTx•t•It•d lintlin}t Ihal'thc hurilicd rncv:tlnnatc kinacc (ist,l:rtcd frnm Ihc c•yto%t,l :rntl ;t«nntctl In ht• a cylrnt,lic hrtutcint iti :tclu;tlh• a hcrrtxi•;nm:tl pmtcin. Sl:nnrllm. li. I).• Shackcllt,rtf, Tanaka• R. I).• mtd Arisant•,S. R'. 'I'hc It,urnal nf 13it,h,pic:tl ('ht•mi~n'y N,7(7{1:55(rO-55RIt. Mv'ch 15. Iqt)i. ()tht•r mhlu,rl: Nalit,nal Intililult•ti nl' Ilc:rllh. hrtnn Ihc I)chartnu•nl <,f I)inlncv. Snn I)icrn Slatc tlnivcrsitv• Sart I)icl:o. ('A, and 17claartntt•nt of Mct:tholit' Uivcn,~cs• Syuihh In,;tiiutc ft,r Mcdical Rcscarch, I'rit)ccttm. N.I. Illl RO!I.I? O1 I'I'.I2Oy(ISOMIS IN ('llOI•I;S'I'IiROI• Mlil'A13OI•ISM I hctr i~ ro,w cnmitlcrahlc• cvidcncc Ihat hcroxi11nntcs not only have a role in rht,lt•tilt•rnl t,tulalinn huI ah() in chnlc.tcrt,l hinsynihcxiti• Specifically, pcrttxisnmc~ c„main at Ir:tsl Iwo cniv'ntc. nrcc%.:try for thc initial stcrc in chnlcsfcrnl symhcsis. i r• . Ihiuln~c• antl ntcv;rlnnatt' kina,;c. Fhc ratc-IimitinL cnlymc in chnlcstcrol svnthc- tii~, t.htthrnr.l.n,rlhyl~Itn:ny'I cncniyntc A rcrluclasc. i~ altin Incali/ctl in hcrttsi- MInu, an,l u\hihil~ a t.clic v:trialit,n tliaincl frnm thal rl/ Ihc rctlucta.w Inuncl in Ihc rntl„I,f:mntrr rcticnhnn. The lar.eca c•onccntr:ninn of c•cIInL•tr stcrrtl carrier hrt~lcin-2 i• ha:,tiit•cI in Ix•rnvi",ntc" :Iti wcll ati a nuntlx•r nf cnivrncs rcquirccl ItirThc cnnvcr- .rnn „I I:rn,,,.trrnl 1n rhnlt•',(crnl. I:urthcrnu,rc. Ilcrnxistnnc1; arr• invnlvccl in Ihc in , rn ll ~t nth,'tiiti of cln,lc.lrrt,l :tnd dnlicht,l (rnnt ntcv:tlnnalc and h:vt• hct•n shown to rnnl:tin .rpnilrc;tnl Irw•l~ nl :qx,lihnhn,tt•in F. a majt,t-t•tmslitucnl of several clasNc•.,, of I,I:i.m;r Irl,nl,rt,tcim. Mt,rct,vt•r• t•ht,lcstcrul sytnhclic cnlr.tcity is imltaircd in cul- lurcrl.kin Iihrtrhl:t.lti „hlainccl fr„nt Ir,rlicnls wilh licrnxisnmal d(lit'icncy Jitic:rtics. A'lr.ttnn. .1. h' Anrcricsm .Innrnal of Kc.hir:ntn•y ('cll :mtl M„Iccul:n• ISiuloiy 7:15R-;(,a. Iqg'- Othc'r tinhl"nl: Natit,nal Imlilulc,, of I It•allh and Antcric:tn Ilcarl Astinciation. Frnm Ihc Ut•LMrlntrnt t,I Rrnlnev ;tntl Mt,lc•c•uLtr Rinlngy Inailtnc. San Diego State I Inivcr i11. S:tn I)tt•Lt,. ('A. I,I I'NI tiSl1K lil l l?("1'(NF RI.O('KIN(i AN(iIO-11:.NSIN SIIIiTYI'I? I I,I t l.l'IYIRti IN AN'll'.RIOK IIYI'(1TIIAI•AMIIS I,t•~ inan ~ludir. h:t;c d`t'wn hnmrt It t,rtcxttt cnna~~Il m~itricnsin al~ :u lhc cnrrcnl l fbr (cnu'alh' ntcdi:tlc•tl I tc. t t ttsr utJt• tta dc>i.ont•tl 14) Icst thc hyputhc~k Ihat cnclogcnnus antcrir hypothalamic i„I,•rt in II hlav'; a significanl role in blood hresavc c•tnttrtil.'I'ypc I angit,lcmin II n'tt'I'I"" in Ihc :mlcrinr hyl,nth:damic• arca were hlrn•kcd by loc:tt nticrctinjcctinn I I)ul' 751 ('_-n-hutyl-4-chlort,-5 (hydrnsymcthyl)-I-I(2'_(I11-tctraiul-5- lil iphrnyl-4- ~I)mclhjimidl ~''Ic~r P'h(/l/ nl tarl fic ticrl crrchrtypirrtl clluicl)t or l vehicle 1)ul 751 (;•I„I) or a I L. d„nr w;t` micrt,injcctcd into Ihc antcritrr hypnthnlantic arca ttl' conscirms NaC'I-ticn- dnr .hnntanctwslv hypcrtcnsivc ralti and Wia:tr-Kyolo c•nnlrols. 1)ul' 753 c:mscd ";nilic:mt dcrst•-rclatcd dccrcascti in mc•an arterial prc~surc Imaximnl dcc•rcn~c. „51 l.:( ntm 1Ig) w'ilh unchangcJ heart ratc in N:r('I-scnsitivc spontancotrsly hypcr- tt,tt ivc rtls hu1 cffcctccl ntt ch;mLC in Wislar Kynlo ralti. Injcctionti tif ctµral volumcs „f ;u'titicial ccrchro%rin:d Iluid into Ihc anlcrinr hvpothalamic arc:t hncl mt cf7ccl in c',Ihrr strain. furthcr. nticroinjcclitrn nf I)uP 753 into the pnclcrior hypothalamic arc: I,nxlncctl n() ; iRnilicanl cfl'cct t,n blood rressurc or heart rate in Nn('I-scnsitivc srttm Ittttr,ursly hypertensive rats. Microinjcctic,n into thc antcrittr hypttthalamic arc:t of Ihc sclcctivc type 2 angiotcnsin II receptor nntafamist I'I) 12;;It) did not affcc hlrrnd hresmurc or cearl ratc in Na('I ,~cnsitivc sronlatncously hypcrtcmivc rats.'I'hcm d;da prtrvidc the tirst dcmtm.Irntitm that cndngcnnns nngintcnsin Il in the antcritt hvpothnlamic area participates in Ihc trntic cnntrnl nf hlotrd pressurc in .alt-unsitive tipt,ntnnroutilv hyrcrtcnsivc r:us htd nol in nnrmntcneivc Wislar-Kyttlo r:tlc and Iha Ihis cf Icct is mcdialctl hy type I angitttcnsin II receptors. Y;uig. R.-I1,. Jin. I1.. Wyss. J. M..:trttl OI,uril. ,S'. Ilypcrtcnsion 19(5):475-4ftl, May 1992. OIhcr support: Natitmal Ilc.rct. 1-une and Rlnod Institute and National Institutce t Ilcnllh. Prrnn thc Vascular Biology ancl IIy(rCrlcnsian Program. 1)ivisictn ol ('ardinvascul: I)i.cascti, 1)cpartmcnt of Medicine and I)cpartmcnt of Cell Biology and Anattmr llnivcrsity ttf Alahamat at Rirmingham. ('I('LI;I•ANINI'. IlI.1IN'1'S'fltl;. I't11.MONARY PRF•SSnR RESPONSI? 9'O A('l l'I'1! IIY1'OXIA IN RA I'S ('iclctaninc (('I(') rccrntly has hccn shown to lower systemic arterial prcxtiurc hypcrtcnsivc animals and ntan by a mcchanism that may invtilvc potcntiation of f v;tcndil:rltrr cffcct uf alrial nntriurclic h(-plidc tANI'). We prcviomly h:rvc shown tI ANI' ptcvcnts aculc hvpt,Kia-incluccd pulmonary vastx•onslriction and mt,dul:rlcs I uvcrity of chrmtic hypoxic pulntnnary hypcrtcnsinn. Thc current stucly tested I hvhnlhc>i% thal ('I(' inhihil% thc pultntmary hresor resptmsc to acutc hypnxia M cyclic tuantuinc nu,nnphoshhalc Ic(M'1-tlcpcndcnl mcchanism. C'athctcrs wt I,lac•cd in thc pulmtmary arlcric,, ttf Shraguc I)awlcy rats through thc right jul:u 80 91
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,r•m n.ntr :t r In~crl chr•,I Ic•c hnt,lnc. :tnd in Iltr ,n,rl,t Iht„u h Ihr til4n Irnu,t;tl ;trl,•tc ~Ilcr ;t r,tn rr•rr„t•tt. ('I(' Ih(P(I tn,f il !) r,r „hic h,,a :nlntini.lcrcd r,t:tll, I t !•;tva'c Ir, cnnNrit,aN t:rlt 1 hryn'" I,rirn Ir, r\l,n rnc 10 I(1'; „\y,lrn ill :unhicnl I,tt.` "nrc r,r In rnnm air. Mr•an hulnn,n:uv :trtrti:tl I,rrti.urc (hIl'AI') ;tnrl nrc:rn ~y.tlcmi< arfc•ti:,l hrrti.urr rM.tiAI'1 an,l hr;ut t:uc /11121 wcrc mnnilr,rt•,I /r,r 1 hntnc• ('I(':qtcn u;ucd Ihc :rcutr I,tdnn,n.uc I,rr•ti.rn rry,nn~c In hvrnvi;t (MI'•11' I./1 mm Ilt• ill Ihr "h,ln,vic r(''l('_ I•rntrl, %". .'.').') t I.O mm IIL in Ihc "hvl,nxic + vclhiclc:• Crc,rqr I, •- 0.05, ;n 3 hntnti (,l hyl,nric c\l,nmuc•), hnl harl nr, xi}nil'icanl cllccl nn MI':\I'. MSAI', r,r I IR in air cr,nlrnI r;u~. At•uIC hvrnxia c:ntticd siRnilicanl incrca.c, in I,l:rtinta ANI' and r( iMl' :tnd rn hi<Inc•v c( iMI' cr,ntt•nl. hul ('I(' athninixlr;tlinn dirl 1101 allct Ihcu• I,,yrnnclrr% frirIhcr. •I hk iti Iht• lir%l dcmuntilr;tlinn Ihat acntc admilti` Ir';uinn nf, ('I(' :nlt•nualc•ti thr hulnxmarv I,rc.sr,r response k, aculc hyl,rtxi;t in cnn- t t'ir,uti rnlt. lin, IL. Y;tniL. anrl Thc Amcric;tn Irnnn:tl r,I Ihr• Mr•tlical Scicnc•cti t(l.l( l):1•a 19..Iniv Ir)')? OIhcr tinlthr,rL' Anrcrit an I,unj! A,;%r,cialinn. I)cpartrnctrf ,if' Vctcrans Affairti IZc,,carrh Scr. icc. N;uinnal In.linnc. r,f I lc;tllh• anJ Atncrican I lcau'I Astinc'alirm. I rnm Ihc Ih•I,orlcminn I'rrx'ram. I)ivi.inn of (;trclirn•a%ruLn I)iWa.cs, lhtivcrsitv of :11;th:tnm aI linminehmn. SAI :I'til lI'I'I+MIi .N'I'A'1'1ON IN)FS NO'I' AI l'FR 'fllli I'I21iSSOR EI+,1;("I' OI, RLO('KIN(; A I'RI:\I. Ni\'I RII IZfsI I(' I'I1'I'IUIi IN Nl1('I.IitIS "fRi1(°I'11S SOI.I f,\RII W4• ha,•i• I,ICVinuslv tihrn.n Ihn1 nticrninjcctinn nl, mant,ctnnal antibody to :rt)'ia1 rrtlritrartic la•laidr (ANI') inlr, Ihc t'atudal nuclcuv Iraclus tnfifnrii c•:mscs ;I pressor rc,,l,r,mr in .alt ~cn.ili, r• Imnt;mcnusly hyhcr1cn.ivc rols (SI IR ) led a haN:rl ( I'; l t:tll rlicl. mt •t•c.,Iin)• Ihal cndr,!'cnnus ANI' in this rczinm m~y Ix invnlvccl ill fhc cc n- h;tllv n,r,li:ur,l rrt•trl;,lirm of I,In,xl I,m-ntr in Ihk, mntlcl. 'Iltc I,rc~cnl vtucly tcslcd Ihr h,1,,,Ihr iti Ih:tl Iltr I,tcstit,r cffccl r,f hh,rkinL cndngcnrnrt ANI' in caud;tl nuclcus t•nhmnrrrl hy dirtarv N;tll surl)Icntcntatinn in "alt scnsifivc SIIR. cunih,,,h In ANI' 1(1,55 1~ 1 in 5(1 nl nrlificial rcrchrntil,inal Iluid r,r am- iu•I ut,n,„ir,hnhn (i w;t~ micrninjcrtcd inln Ihc cnudal nnclcuti Iraclus snlilarii of rnn., 1u1i•. ••: II tt•nvli,c tiI1R. ,nll rc"i.l;ml SIIR• anrl Wi%l;n' Kwrlr, r:tlti fctl I'ti nr :,Irrh,•I% li,r t csl•t•tit. Mic•rrtinjcrlit,n uf' Ihc mr,rn,clnn;tl nnl'ilx,dy into thc caudd nn, Ir m, u;trlui tir,lil:uit rvr,ticcl ,imilar increstw" in mcan ;trlcri:rl hrctisnrc• in .e:Ilt- >cntili,i .tiIIK r,n h„Ih I`•l ;tntl X'.*,, ~;tll tlie(" anrl rn ti;tll-rc~itil;un SIIR nn a I'; .e;tll rlirt h,tt h;ul Itrt t•IIrrI in \b'i~Iar Kvntc, ralti. In c'r,ntr:rel_ ntiCrninjcrtirm nl c'rmllr,l immunnylr,hnlin (i rrttr, Ihi, brain :ncat tfitl not :tftor tnrut ;trUCrial hrct>urc or heart ratc tn am rel,otimcntnl Vrnnl,. 'I'hu., cmh,!tcnnuN ANI' in (awlal nurlcus Irac'lus snlitarii tnr•rfialr. Ir,nir c„nlrnl nf hlnncl I,rc ~tiurc in hnlh s;rll ~cntitivc ;ntd tiall-rc.i,- lanl SI IIZ hul 11411 in \Vttl:u Kvr,tn ral,. and Ihi" cllcrl k indclx•ntlcnl of Ihc• s:tll 1 cn tili.ilN of It\I,ctlcn~ir,n aml ul (licl;nv tiall inl;rkc. Y;tnP. I2.- lin. II., Wv .. I_ M., ('hc•n. Y.-F.. antl U/,rnil..4 X2 II I''''Irn ic,n _'(1( 2):312--'-'1(,. Auy_utit 1992. tttl ,r ~,tl,l,,,rl: N;tlinnal Imlilutc. nf' Ilcallh ;tncl the Naticmal Ilcarl. I,un): and It)r,nd In,tilnl,•. Inan Ihr Vaarular Rinlrip y ancl Ilylxrlcminn Program, IJivisitm nf ('ardiova~cular I)cl,:uorncnl, of Medicine ;tnd ('cII Iiiolr,},y. IInivcrsity nf' Alabama at ISnntin"Iham. A fRIAI, NATRIURI;TI(' PIiPTIDI? MOI)lI1,A-1'ES BARnRL•CIiPT(7R RGr71.IiX IN SI'ONTAN1:OLISI.Y IIYPfRTPNSIVf: RAT Our prcviauti studies have suggcstcd that atrial natriurcti(• peptide in the can(h nuclcu, Iraclus sc,lilarii is involved in Ihc ccntrally mc(lialc(I rcgulatinn ol' hlnn pressurc in Ihc xalt-xcnsitivc sprmluncciusly hypertensive ral (SIIR). 'I'hc currcr qndv Ic%tcd the hypnthcsiz that cnckrgcnnus alrial natriurctic pcpticlc in the c•aud: nuclcm tr;tctussolitarii particil,cdcx in harnrcccplor reflex cnntrol nf hcarl ralc in thi hY1Icrlrnsivc mndcl. Salt-wn.qitivc SI IR and cunlnrl Wiaar-Kvr,ln (WKY) r;ttti mnir Iaincd rnn hasal ( I'Z ) sall intake were studiccl. Arlcrinl harc,rrc'cplor rcllcx. mcdiatc ch;tngcti in heart ratc were recorded in conscious unrcwaincd rats dut'inl; phcny c•phrinc (5-4O µg • kg-' • min ' infusinn: 31) rninutcs latcr, atri:tl natriurctic Rcptic (5O ng), monnckm;tl anlihcxly to elri;tl n:driurclic Rcpticlc (0.55 Ir.g), pnrificd mnua intmunoglohulin (i (0.55 µ•g), or nrlific:ial ccrchrotipinnl Iluid vchic'Ic (5O nl) w; micrninjcctcd into thc cau(lal nuclcu~ Iraclus solitarii. I'hcnylcphrinc infuzian w: then rcpcalcd and rnrrn arterial prcl.urc and heart rate were mnnitnrcd as Ix far 'fhc slope of Ihc hc;ut rdc/mc:m arlcrial prestiurc relation was significantly Ic (p < 0.05) in Ihc sall-scnxilivc SIIR thnn in the WKY control, indic•aling that harnr (cplur rcflcx ccintrrtl nl' heart ratc was blunted in Ihic hypertensive rnr,d( Micrainjcclinn of ntrinl nnlriurclic• pchtiJc inlo Ihc caudal nuclcuti Iracl(ts solil;t furthcr hltuilcd (/r <(l.(1.51 harnrc•ccl,lrnr rcllc•x control of hcnrl rate in sall-xcntiili tiIIR 11411 nnl ill WKY r;n". In ct,nlr;r,; , l, tnicrninjcctinn ttl' Ihc nrrnnx•hmal antiha cnhanc•crl Ihc scntiitivi)y of harnrcccptrn• rcf1cx contrnl uf' hcart rotc in ti:dl-xcneili tiI1R hnl n,rt in WK1' rat..'Ihr•-ic dnla ang}cit (hal cndogcnons alrial nalriurclic I,c lirlc in Ihc c:nrd;rl nnt Iru. uacius snlitarii nwdulalcs h;rrnrcccptt,r rcflcx cunlrnl hcarl ratt, in .sall-u•n.nivc SI IR hul ntd in WKY rats. .lin• I I.. Yang. I2. I I., ('athc,un. I). A.• Wy"..I. M.. and O/,nril, ,S. I lypcrtt•minn 2O(;): \7•1-j7c)• Scptcmhcr 1992 ' OIhcr suht,nrl: Naljnnal Ilc:ut. 1 .11,19 and RIurn1 Imlitulr :md Nalional Inslitulc- Ilcallh. I•rum thc Va,,cui;u• RinlnLy, anrl IIY1,crlcn~it,n Prugr:tm nI Ihc I)ivitiitro ('ardit,v:t~cular I)iscmc. Ik•(l:trtmcnt'. nf Mcdicinc ancl ('rlt Rioh,gy. llnivcr.ity Alah:nn;t al 13irminc•hant. K;
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ANI)R(1GI N I)1:1'I?NI)I•N I' iWGIO'll•.NSI'NO(;I;N •\NI) RI•NIN MI;SSI:N(;I Il RNA I'.XI'RE;C,till )N IN I Il'I'I'.IZ I l?NSIVI: RA I:` Our prcc inm .tu,lic•. rlcmr,n.lralr cl th;n fhc u•rualh' (limnrl,hic Ir.dlcrn nl hyl~•r Icminn in the tiluml:rncnn.It• hvI,crlcn"ivc r:u i, anrlnrJ cn rlclmndcnL (i,madcclnrn, rctarrlti Ihc dcvclr,lmtcnl „I hyrcrtcn"inn in ynung malcc• hut not in Icmalc~. an I :rdminiqr,tlir,n nf Icqr,tilt•rr,nr fnnhirm:ur• to fr,nadcctr,miicd "hc,nlancc,ntily Itvi",r Icmivc ralN uf hnlh .cu" cr,nfcr, a m:dc Imllcrn c,f blood Inctisurr dcvclnrnrcnl. Ilil• ctn•rcnt ~turly Ictilr•rl Ihc• Iryl,nthc.i,, Ih:tl rcnal and hchalic rcnin and angic,ICnxinnXcn zcnc cehrc%cir,n ;rrc al"n andrnLCn rlcl,cndcnt in Ihc spontaneously hyhcrtcnsivc• ral M:rlc and fcmalc til,nntancr,u.h' hyl,crtcn.ivc rats undcrwcnt }!unadcctnmy nr:t sham cnpcralinn tn •J ,ccrk. „f :r:lr•. SuhJ,rr,ul,s r,f Lrrnaclc•c•Ic,miic•cl rats crf hnlh Ncxce tiq.rr, imhl:nucd with a 15 t,ttn or tO-nmr SiL•rs-lic c•alnulc fillcd with Icslnacrc,nc ;n Iht Samc limc Ihc gnn:ulcclr,my w:t% I,crfnt'mcd: a third group rcccivccl an cmhly Sila%lic c:q,tiulc. Nrn•Ihcrn and shn hlul analysrs were uscd to characlcrirc and yuanlitalc rcnin and anuinlcminn)lrn mcsscngcr RNA (mRNA) in the kidncy and livcr Ik wcck,~ allcr Ihc ?u,n;nlcrl,rn»', ISh,nrl I,rc~tiurc, plasma rcnin aclivity. and hchalic :rngir,lcn,,inngcn nrRNA Icvcl% wcrc higher in intac-f m:tlct than in fcmalc%; Orchiticctnmy rclardcrl Ihc rlcvr•Inl,mcnt crf hyl,crtcnsinn and hrwcrccl phttima rcnin and renal and hclr,nic ancu,icnsinu};cn mRNA Icvcl%, and Ictilnslcrunc rcrl:rccmcnl rc.lnn•cl Ihr m:tlc Ir.ntcrn nf I,vlut'ICnsinn and I,latma rcnin and incrcacccl renal and hc•l,atic nngir,tcnsinngcn nrRNA. Ovaricctnmy did not allc•r hlnrnl rres%urc nr phtsnrt rcnin htd rlirl lnwcr rcnal rcnin and renal and hcratic angir,lcntiinngcn mRNA: Icsh,ti. Icrr,nc incrcwcd blood hrc%~,tnr• plasma rcnin, rcnal rc•nin and angintcnsinc,gcn mRNA and hclxnic anzintc•nsinogcn mRNA levels in crvaricctnmiiccl fcmalcs.'I•hcxc <lala .ugLC•N Ihal Ihc enclrngcn-dc•I,cndcnl ilcvclul,mcnt nf hypcrtcn-sion in shnnta- nrnu0v IYl,crtcntiivc r:n,, may hc rclalcd In andrt,gc•n-inducccl activatir,n of the rrnin an•;!iutcn"in .Y"tcm. ('hcn. Y. I.. Nal'Iil:rn. A. •I.. and l)Irmil..S'. I lyl,crtcn.ion Ir)(S):.JS(, •1(,3, 1992. OIhcr euhln,rt'. National Ilc:rrl. I.ung and Klc,ncl Inailutc• National Institutcs of I Ic:rllh and Ihr• Amrrfc•:tn I lc:trt A.~crcialirm. Frum Ihc V;rsc•nlar 13ir,lngy and Ilyncrlcnsinn Program nf Ihc 1)ivitiion of ( arrli,,,:rmular I)itic:t.cs. 1)c•Irtrlmcnl r,l Medicine, llnivcrsity c,f Alabama al Itnnrinsh:nn. I.O('A1.1/.ATION OF RRAIN AND A'1'RIAI. NA'1'RrLIRl~;1'I(' I'I?I'f'll)1; IN I ll IMAN ANI) I'OR('INI. I II:AR'T We havc cnml,nrccl the Incaliiatic,n „f• hr•ain and alri:rl nafriurclic rcrlirlc-likc inmrunnrr:rclivih' in human and Iu,rcinc hcarf.", u.ing inmtunnhistr,chcmical Icch- nirlurt al holh Ihc liJphl and ultr.rtilructural level and spccific :tntisc•ra It, nmuto-(c':v- (Iindil:uin) and c;vhnry-IC•rminal rc.pion~ c,f-Ihc :nrial natriurctic (,rccucarr nmlc•culc and In hr:rin tt:uriur,•lic I,chtidc. Alrr:rl merxarrli:rl ccllt in hum:ut fct:tl, nr,rmal adult and failing cxpl:utlc•d Ir I,I,wrJ immttnrncactivity fr,r hnth hr:rin and atrial nalriurclic luptidc' likc \I Ihr ~nhc'~ Ilul:rr level. hrain nalriurctic pchlidc-, cardic~dilatin- and nn,r` n.rl n,driurrtic hcptialr-likc immunc,rcac'livily were cu-Ic,caliicc lu >ccrc nr~ ole r rrr ;dri:tl mVncarclial cclls I nm!~r/ ) of 22rfaii ng cxplant~ Ichr.tris. hut not uJ ~•inrl t'irhl vcnlrirular firc wal .( h u r r:ud1°vcntricular frt`<r wallrsland tinmtunc,rcar:tiv~rty for both natrlailling ~iurctic I was :r n"• r:mulce in a suhpnpulation c,l' myc~c:tnlial l , I` •,ynrn~cti tc, sccrctc,t'y }~ c„ Ir,c•ali~.cd ,r,mccnlr:Ncd in suhcnclcrcarclial rcgir cwcrc alsotdcmc,n~t atccl inr,pctt and ll. cPtidc-likc immunc~rcacUvily o ',.il nah'irn'ctic r' rcinc nIV'x:rrdium and rells of Ihc vcnlricular cnc-likc hnnn norcacl virylsug}.c~ls I,trtr„rr „I carJiac hrain and atri:d natriurctic Ixplictc 11,101 rr)'ulatir,n and ca-storagc of Ihc natriurctic pcpticlcs in human and pnrctnc I'mt~ A.. Wharton..I.. Arhustini, f.?.. Cira,~so, M.. Dicgoli, M., Nccdlcman. P.. ~ i,ranr,, n4.. Mcricnsn. C.. and Po1uk..l. M. Inlrrnatinn:J .Iuurnal of C'atrdic,hogy 34:237-247. 1992. I rnm Ihc Ilcparlmcnl of Ili%tochcnii; ~y'c~K~ i'a and I)i tisic itc Medic Carcli(>chiru~ia. 1„rrrlnn. I:ngland: Institndn cli Anatom ! In liluln Ji Ricovcro c('ura a('ari oi.caSd I)c~ artmcntrof Mc I Malrnny.IKi ngs It:dy: Mnn~ant~ ('orpnratinn. St. L ('nllcec I hspital. Lc,nclon. 1?NI)O('ARDIAL LO('A1,I7.ATION ANI) ('P1ARA('TFRI7,AT1ON OF NAl•RI( IRE'11C P(:P'1'll)L;13INL)INU SITES IN II(1MAN I'T.'.TAI, ANI) Al)III,T 111:ART Spcc•ilic, high affinily binding sitcs for u.I-human-a-atrial nalriurctic pcptic c (1_2R)) ('"I-hANP-(1-2R)) were iclenti/iecl in humat) fetal and adult heart and thr binding charactcritccl ueing quantitativc in ritrn autoradiography. Bincling xites wcrr Incali7ccl to atrial and vcntric•nlar cnctocarctium. aorta. pulmonary arteries and epi c:uiclal mcsc,thctium. Kinclic ~urdic•s indicated a K,, value of 32 pM for vcntricuta cndrx•arclial '"I hANI' (I 29) hincling. •I'hc binding was completely inhibited by a excess ( I µM) ol' nnl:thclrct hANI'-( I 2R), human brain natriurctic pcptidc-( I-32 (hBNI'-( I•;2)) and by the "clcarnnce rc•c•cplc,r" spccific ring-dctctcd analc,guc ( ANP-(4 .2\). ('ompctilivc inhihilir,n studies indicatcct a relative inhibitory pntcnr li,r (hRNI'-(I 32) and ('-ANI' (4 23) uf 6`Y• and 3% rctipcclivcly. The data suRgc that a clistincl natriurctic• hcl,liclc rcccptor subtylx: is cxprctitic(I in Ihc cnck,cardiw and in additirni to a possible clcarmtcc fnnction. may rcprcticnt a%itc for fccdhac rcgutatic,n and pcpliclc interaction. Rulhcrfc,rcl. A. I).. Whartrnr. J.. Cnrdon• I... Moscc,~o, (i.. Yaccruh• M. IL• at Pnln6..1. M. - I?tut,pcan.hwrn:d ol I'h:trmacolc,gy 212:1-7. 1992. (lthcr tiupporl: Rrilitih Ilcarl Vc,undatinn. 84 KS
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i I-rnnt Ihr I)cl,arlmcnl t,l Ilitil„chrmitiIrV, IZ„t:rI I'n"Il'r:nlu:rlr Mrtlical \chn„ I.,,ndt,n. 1`.nl! l:rn(l: 1),•I,alInrcnl „f fl1hid •\n:rlnmy. n! ('~,IIrTt• Ilil.,l I,md,rnI anrl (':rnlintht,r;u u 1IniL I I:rrt lirl,l I h,,I,il:rl, Mitltll h ic rt. IN I IMAI. SMO(Yfl I Ml IS('LIi ('I"sl.l. I'ROI.IPIi1ZA'I'ION A11'1?R RAL1.OON ('A'f1IHI'I'.IZ INJI I)ZY ttn kr,l r nr n\titr l nnlnnt ,xS l r,RUw•III I At•trtR 'I'hc k,rrliinlinn :rntl " ynthc%iti nl hartiic fihrnhlatit gr•nwllt Ltcrt,r in Ih,• r:n cnn,lid arlcn «rrc invcslis;nccl al timc, nl chronic .mnr,th ntu,;clc c•(•II I,rnlifcra- Irnn. Imnmm~c)•It,chcnnral tilaining showed Ihc prcscncc nl hI~(71; in thc uninjtnrd ;n'ICrial wall, and :rllcr halhmn injurv. Ihis ccllnLu- slainin}t was decreased. Wcslcm and nnrtht•rn hh,l an:thut likc•wi.r ehnwc•d that Ihc anrnunl t,f hF(il' protein :md rnRNA decreased altt•r injmy. A nculraliiinR anliM,dy to hFGP was adminitilcrcd d :urd 5 d:ry~ :rltcr injury antl wa. found to have no cffccl on intimal smnnth mutic•Ic cell I,rnlili•ratit,n. "fhc~c tlnta suLZCsI that an inc•n•asc in the cxpressinn til hFGP is not nccc.s;rry f„r chn,nic .mnnlh mu,,c-Ic cell I,rnlifcralitm nh,,crvcd al'ICr hallnnn calhctcr injury ;rntl Ihal hFGF i~ not the major milt,pt•n rcti),onsihlc for inlimal timro,lh mu"clt• t•cII I,rnlifcralinn. OI.nn. N. I•:., (''hat,• S., I.inJncr. V., and llci(l,•, M. A Amcri(-;tn.lnurnal nf 1':uhnh,~y I4(1(5):IO17-IO?t. M;tv 1902, OIhcr wpl,nrl: N;rUnn;tl Inailulc% nf 1Ic•alth. Frnnt Ihc I)cl,arlnu•m n/' I'alhnhiay. I lnivcrtiity of Washinilun. Seattle. NI OIN I IMAI I''IZOI.II I'.RA'llON: ('ON'I ROL OI VAS(`I II.AR SMOOl'l l N11 Iti('I I ('1'.I I . ( iR( )W"11I \':,u'nl;n timnnlh mmclc ccllti (SM('s) havc rcccivcd rrruch allcnrinn over thc I1n•r 'Iri mlc in Ir,rrl hrrn.rtir Ihcy h;rv,c been finm<I In hc key cellular clcnx nls in alh- r„ h r, li, Ictiinn and Ihcir Irrc.cncc h;r% prnnrt,lctl a grc•al deal of allcntion and ,,,,rl, h, urnlrr.tarnd the idcnlity n/ Grc•Inr% invnlvcd in stimulating Ihcir', rcnlicalir,ti. \t', ala, kn,ru Ih;tl Iht nntlc~ircd rel,lic:uitm nf Ihcu• ccll,, is in I:u•gc I,au1 responsible Inr Ih,• c liniral rnml,lrc;rliunt which Ircyucnlly t'c'car following angiol,lasty and iml,Lrnl:ninn of ,au-nl:rr Ilccausc nl Ihc c,hvinu. eimil:u-ilics.in the palllolo)•y nl Ihr.c tn+n c„iuliti„n", ix . acc•unnrl:ni(m of SM('e, it is Icmpliatf; to suggctil Ihat sinrilar It,rccti aur invnlvctl in ctmtn,llinz Ihc replication of SM('s in cac•h c•asc.'fhcrc :nc. hnwc,rr, cliffcrcnccti hrtwccn :nhcrrnclcrnlic Ic.it,nti and a lesion fi,rrncd aftcr nnpit,hla.lv ;tn,l il iti nt,l intcndcd to rcvicw all I,+,ssihlc mcchanism,; thal have been I,rnl,mc,l to rrhlain SM(' I,rt,lil'cralinn. hntc•ad, Ihis hricf rcvicw will concentrate on Ihc rcNl,nnu of Iht• ;ulcrial w;lll to mechanical injrny which is I,crhap. Ihc hctil dc.crih,•cl m„dcl „I arlcrial injury and nnc where much prnpress ha% recently been n,adc in undcrt l:unlinL Iht• cvrnl. leading to the G,rmali<m of SM('-ric•h lesions. The xr, I i I pn„ I Ih,•sc 'ludic'; may have rclcv;utrc tn athcrogcncei,~ hul are I,crhapc more h, .ihlr In Ihc grnwlh t,i artcri;d Icsit,n. which dcvclnp aflcr surgic•al intrrvcnlit,n~ 11.11 ,m.•rnhl:r~ty. i lrr:thlc ad~ th nh•~mnnthtmu~clc!richrnrlc i il lcsitmti"I~n ctmtra~irtotin .,,I,nrlanl Irn' the F I,rl:r. I'I)GI doc% nnl ;tl,l,ca r,car to hc imlmrt:tnl fnr SMC rcplication in rirn in the rathut atttin tprcticnt t mc~ It~is I Ir'`r"It r~t~ Ilhrl~rttrµ,thtnf intirnal lcsfit,ns()t~ ..ud rcl "'I'r' "11 ~ II (;vll(itir~a kcytfac ory ittsl ilnulati tile r}.Itht.I rcplicatitmsof mcdialtSM( tilanul l n~' 11r ,rl", hl;ry a role in cell migralion. Neither c,f thcsc I'actnrs, howcvcr• appears ln „Ilu,.n(t• intinutl cell replication. More work is thc•rcforc required to determine lhc rrtit;rl lat-t,~rs, since inhibition t,f these ccll~ is Ihctught to hc c•rucial in preventing mlinrd Ic~it,n dcvclol,mcnt. M i1...lncku,n, ('.. and Lindncr, V. ~a cul;tr Medicine Rcvicw 3:156-167. 1992. Itihcr %nppt'rt: National Institulcs of Ilcallh. pr,mt Ihc I)cp:nlmcnl t,f I'ath<,h,gy, I Inivcrsity of Washington. Seattle. I'RO1)II('llON l)1''fRANSFORMING (;ROW'ftI PA('TOR 13, I)t112ING RHI'AIR 01: AR'I'I:RIAI. INJURY Repair r,f nrtcrinl injury prtxlurcd by h;tllorm angio),lasty Icaclx lo thc fnrmatirn nf a ncuintima and a narmwing of thc vascular lurncn. In Ihis study, we cxaminc( thc possihility Ihat smnoth mu%clc cc•Ils (SMC) in iqjurcJ rat curotid arteries an qinnd;tlcd to producc typc-I tratisftrrming growth f:tctor-,(3 (TGf,-(31) d'uring ncn intima furmatitm in rir,,. I,cvcls trf TIiI:-/3, tr:tnscripts (2.4 kb) were significatnll' mcrcawd wilhin 6 h aftcr ramticl injury autd reached a nutximum (five to scvcnfnld hy ?d h. Rcgcncrntinz Icf1 cnrtrlid'- had .u,tainrd increases in T(iP-13, rnRNA level (ahnuf I'ivcli,l(l) nvcr Ihc nc'ct. 2 wk. during which Iimc :t substantial nct,intinu Ihickcning wa,~ frnnud. N„ rhanec% in h;tvtl '1'GF-j3I tnRNA levels were fonnd i cnntr:tlalcral uninjrnrd c:rmlitl% ;tt ;my nl' Ihc timc.1, cxsnnincd. Immum,histnchcmic: . ~Indics .hnwcd that a large majority ttf nct,intimal SM(' wcre stained for 'I. GIA prrntcin in an intracclhrlar I,ancrnt crnn"itilcnl with active T(31' (3. synthctiis in Ihit li! muc. Ncnintima fnrmntinn and TGF-J3, immunnrcactivily wcrc cnrrclalcd wit incrca.r, in fihrtmcctin, ct,llagcn (v•(I). and collagen a1 (I11) gene cxpresSior hrl'usit,n nl Iwrifirtl. rccnmhinanl '1'GF-13, into rats with a prccxisting ncointima pn (luct•d a .ignitic•;ml stinnrlatinn c,f carolid ncttintirnal SM(' 1)NA synthc%i.. 'I'hc rr.nll" Sut}c%l Ih;n 'f( &-j3- I'Iny" :tn iny,nrtant mic as an cndngcntws growth rcgul tnrv fac,nr producc•tl by ncointimal SM(' themselves during prr,grasivc ncuintim thickcnine altcn citllnn aniinpl:t.ty. M;tjc~ky. M. W., I.indncr• V., I'w;udiik. I). R.. Schwarti• S. M.• and Reid,•. M A. .Inurnal t,f ('linical Invcstigatinn KS:t)fLl t)Il), Sc),ICmhcr 1991. x7
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OIhcr suhllnrL Amrric'an I Irud A.,.nciatlirm and N:,tiimal Inq itutrti rll Ilcallh. I•rcnn the I)c(lartmcnt of P:llhrrlniy. Ilniccr.ity of Wa.hinpttrm. Scaltlc. and ( )ncnr•cn. Inc.. Srnl lc- ROLF. O1' IiASI(' 1:113RO1tI.AST ( iROW'I'1I FA("IY)R IN I'ROI,IFERA"I'ION OI' F.NI)OI'IIlil.t(IM ANI)SMOO'lll MUS('I.I? API'IiR I)IiNlll)1NG INJ(tRY- /N I Yt'o Ralh,cm catheter injury of the rat c•r/nnnc,n carotid artcry causes complete clcnucl:uirm of, cnrhrlhclium. Iltiinf; this nmdcl, cncllht;lial regrowth from the aortic arch and the runticl hifurrltirnl is initially rapid hut cessation of cnclnlhclial nut- prrnvlh occurs :lllcr alllm)xinl:rtrly (, t~cck. This leaves a central portion of the crnn- mrnl c:u'nlid arlcry Ilcrmancntly clcnudcd nf cndclthcliuna. 1lnwcvcr, if'a Icss tr,tumatic Icchniquc (I'ihm)cnl hrnll denudation) is used to remove the cnclnthclintn. Ilrulifcra- tir/n of cndnthclium llnrcccdti at a continuously high rate unlil complete coverage of he vcsticl is achicvc<I witllin allllmeim:)Icly 12 weeks. The diffcrcncc• in cndofhclial rcgrnwth in these Iwn rtlr,dck allowed if,, In investigate w-hich faclnrs might be involved in c•nntmlling the growth of these cells. Smooth muec•Ic cell (SM(') proliferation with suhticqucnt intimal thickening is an imllcrrt:uH event in Iltc development of an athcrnsclcrntic Iceinn. Although growttl factrv, are Ihnughl lo he invnlvcd. there is currcnlly no direct evidence fnr a role of any milrrgr•n in this process. Following hallrnm catheter injury of the ral carotid arlcrv. %nhstantial dc;dh of medial SM('s nccurs which is followed by SM(' prolifcr- atiun and intinlal Ihicl.cning. One hyllnthc•sis to explain the rapid SMC prcllifcratinn in this )nnclcl is that mitr~ecne are released from these clcacl cclls. One such mitogcn rnnld be h;l'-ic• fihn~hl:)tit gnrwth fac•hor (h1"6F) sincc it was fnund to he Ilrc~cnl in tllc vessel wlll hy immunnhlnt analysis and we have prcviouxly shown that hF(il., is a Ittltlnll n11In,L'Cn for SM(\ in rirr,. Irnnlunclcvlochcmitilry showed hF(iF Irl-hc 1c)cal- iicd in Ihr nuclci r,l SM('N :)nd cndothcliol cells with no apparent staining in the cvtraccllul;)r matrie rll* the vessel wall. In this study we cxaminccl the role of cnclogc- ncrw, hl-( il and arterial in,jury and fmnld that the nrnlitcratiml of' SM('e aflcr haI- Inrrn cathclcr rlcnudatiom of the rat rtrnlid artery was tiil:nificantly reduced when a ncntrafiirn): antihr,dy rniticd :ly:lintil human rccomhin:nll hFGV (see ahnvc) was injrctr l Ilrir r Nr injutv. I indnrt. V. :mJ Nrh1v. Al :1. In: Slrinrr. R., Wci,,i. P, 13.. and I,angcr. R. (cds): Angiu;acncsic Key I'rinci,l/lcs - Science Tcchnoh~ev - Medicine. Rit'kh:wticr Vct'lag 13aticl/SwiVcrlattd, pp. 387- 3`{!{. 1992. I nml the I)cllar1mcnl of Pathology, Ilnivcrsily nf Wa%hintlcln, Seattle. I•.NI)O1'I II.I.IAI. ('11.1. RI:( iROW'I'I I I:.minthcli;ll Ucll", cilhct in ri-, rrr in riln,. grow as a mnnclLtycr and dctillitc thcir 6c•c I caLrnn in hhxul vc%Ncls and the Innctimn which they carrv onl, we know ,ca;qivclv little ahnul the nlcch:lixms which c•rlntrnl their growth in rirn.'I'his is tlc luql~ hrc;msc most cndnihclial cclls, at least in ri(rn, were fcltrnd In grow readily ar littlc altcntinn was givcn lil understanding what faclors might influence this prnccti 'I'hc wiclcsllrcacl use of vascular grafting and the clisc'ovcry that rccndudlcliali/atit it „ftcn severely Iimilcd, howcvcr, clearly dcnurostratc that cndrnhclial erowlh :u rnnintcnancc of an intacl cndnthclial monolayer are not simple autnmatic Ilrcx•cstic in Ihi> chapter we will diticuss those experiments which have focuscd an umlcrstan i„L, what factors are impnrtanl in contrnlling cndothclial cell replication and whcth these factorti can inllucnc•c cndnthclial c•cll growth in the denuded blood vcsscl. Rrirh•. At. A. and l.intlncr. V. In: Sinlirnlcscu, N, and Siminncxcu. M., (ccls): f:nclnthclial Cell Dysfunclinns. Plcnum Prctiti, New Ynrk, pp. 31-48, 1992. OIhcr suppnrt: National Inslitntcs of Ilcalth. From the 1)c)lartmcnt of I'atthningy, Univcrsity of Washinglnn, Seattle. RASI(' FGF AND GROWTH OF ARTIiRIA1, C'6LLS Our studics xhnw that hasic fihrnhlast growth factor (hFGF) appcars In pla' nuijor roilc in cnntrnling the growth of both cnclnthclial and smooth nlusclc cclls appears that cnclnthclial cells may require hF(;F for their prulifcralirnl and cxol nous addition of hFGF can rcinitiatc cndnthclial ccll replication in situations wl these cells appeared to have senesced. Basic f"•CiF is also a mitogcn for smonth in c'Ic cells (SMCs). and in our anirnal models hF( iF is capable of increasing rcplical to a much greater extent than other mitogcns. Of greater importance is the fact t the response cll' SM('x to vascular injury can be inhihitccl by action of an anti-hF antibody. An attractive hypothesis is that hFGF may play a role in athcrosclcn where fixal cell death followcd by release of hFGF from either cnclothclium. SM or nlacrnphagcs may be responsiblc for the dcvclopnlcnt of inlinlal lesions. Rridr. M. A. and I.inncr. V. Annals rlf Ncw Ynrk Academy of Sc•ictlccs 618:290-299, 1991. Othcr cuppnrt: National Imlilulcs of Hcallh. From the I)cllarlmcnl nf I'athnlncy. I lnivcrsity of Washingtcln. Seattle. SPI?('IFI(' INFLAMMATORY ('YTOKINfiS RFGULATG THE F.XPRI:SSION OFIII IMAN MONO('Y'll? 15-1,11'OXYGI'.NASF, Ar,tchidnnalc 15-lillrlxygcnasc (arachi(lrnlatc:nxygcn 15-oxiclorcductasc, 1.13.1 1.13) is a lillid-pcrnxicl:ninR cniynlc that is implicated in oxiclicinl± low (Icr 8 }{ 89
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lihr,hrnlcin Ir, itti :nhcrn)'cnic fnrm_ Mrmr,r.tr/nercn,l,ha,tc li-li(u,ry€cna.tic iti Lrcti rnl in hum:m ;uhcnnclrrnlic Ictiir,n." l n I,wwc :t h:t.i. Ir,r irnlnctinn of Iltc cniymc. cchich iN nt,l InrNcnl in hlomtl nu,nr,c\ tr%. Ihc ability (,l rrlc\ant cytc,l.inc" to rc-ulalc i1', cxl,rn wa~ invrtitil!atc,l. InIcrlcukin I f II-I), amnn), If, Iaclur.lctilcd. shccifi. c:rlh inthrccd I5-Iil,t,\v,rn:nc tnRNi1 :tnd I,rntcin in cullurctl hutnatt mt,nncytc.. Inlcrlc•nrn y:tntr Iredrrn nrtiti„nc inhihitccl this inductinn. I li)th-I,crlnt'nl:tnc'c liqui(I chrtnn:ntrir'al,hy an;th•~i~ of lipid crtracls Ir't,m II, J-trcatcd nwncx.vtcs dc•fcctccl IS- til,nr)grnasc Inruhrc'Iti c~tc•rilictl Ir, thc cclluLtr mcmhranc• Iihicl., imlic':nin€ cniym;tl- it' :tctinn nn cndc~ecm,u~ .uhtilr:rtc~. Slimulatir,n nf 11.-4-trrucd monnc•ylcs with cal- cium itnu,l,hnrc nr c,lnnniiccr iynxi.an A cnh:mccd thc furniatic,n cd' I5-lipc,xy€cnasc I,rnducL..Thctic dnln idcntif\ II-I ancl intcricmn y a~ I,hytinlc,€ic:tl rc€ul;Uor:ti of Iil,t,tyLCn:ttic ctl,rc,,.i,mt and ..u€LCtt an imlunt;mt link between 15-lihuxy€cnasc Ctntctinn ancl Ihc immunc/inll:unnlatnry rc,;hnn.(. in :rlhc•rcisclcrcniti as well as other tli,w;tw~. ('r,nr:rd. I)..1.. Kuhn. I1.. Mulkin.• M., Hi€hland, I?•, and,Si,Lrd. F. I'n,cccdint_', of Ihc N:ttitmal Academy c,l' Scicnccs (ISn Sc):217-221..lanuary 1992. t)Ihrr whlu,rl: (I,S. Nalicmal Intitilulcs nf Hcnlth and Ihc liunclcsntinititcritnn fiir 1 nr~chun> uncl'I'cchnt,ln€ic (Gcrmanv). Fn,m.thc ('ardinv;t%rular Rcsc;mch In~litutc and I)cpartmmnt r,l' Mccficinc, (htivcr5it_y of ('alifnrnia. San I:rancisrr,.:md Syntcz. Inc.. 1'aln nlln. ('A. A I'RIMARY hlsfl?RMINnNf FOR I•II'OXYG1:NASli1'OSI'1'IONAI, SI'I:('II:I('fI'1' . I'hc Ihrcc mammali:m lil,nry€cna.c•ti :trc namcd acc•nrdin€ to Ihc carhrnr pnsition 15. I' nr 151 :u whiclr Ihcy c:u;rly.c thc ory€cn;ttinn rif arachidnnic acid: thcy are iny,lic;nc,l ill inflamnuttr,ry clititrrcll•rt, ft,r cti;unplc 15-Iipr,Ky€cu;rse is induc•cd in :nhcrn,cdrr,ni` and can t,ridiic h,w-clcnsity lirnprotcin Iu its athcrn€cnic form. Tcr itlcnnfy „h:tl (lctranrincti Ihi% I,n.itir,n:tl shccilicity• wc have etc•h;m€cd conscrvcd rlil/crrra r. in Ihc i~r,hnnn r,f 1 2- and I5-Iipmy€cnasc•ti. Suhslitttlion uf ntctlticminc " rnc ~:rlmr ;rt I,n.iti„n 4 IK r,f hnman I S-lilu,xy€cn:t.c rccull~ in an cniymc (hat hcr- I t.' arnl IS Ii1,oN4,LCnalinn cqu;tlly. 'fhis cficct can hc mimickccl by incubating "rlil rkp• I S lihnxy,•en:tu• %cilrt a wttfhcticolly altcrcd suh.tr:rtc• which h:rc ile doubly :tlh•h,' rnt•Ihylcnc rarhnn..hificcl by unc carbon relative to arachidrmic ac•id. Other mul;tLrnm ;n Ihc ncilzhhrnn'in€ ;tmint, :tc•id. 4I(i ancl 417 €ivc an cniymc which rcr- Inrmti I' :rnd 15 lil,,,xvr,•n:rlinn in a ratio r,f 15:1. '1'hcsc rc•tults indicatc that Ihiti rc)tinn mi.0hf hrnitirm Ihc %nh.tralt• ill Ihc active sitc. Sloane. I) I... I cunt. R., ('rnik. ('. S. :md .Si,Vrr1. li. N:nurc 35.t:1-19 152. Nnvcmhcr- 1-1, Ic)ol• Othc•r %uhl,t,r1: Naticmal In.liltncti „I Ilcalth. Nntir,nal Science Foundaticm, and thc.l'arkc Uavi. 1'harm:tccutical Rcscarch I)ivisicm. Warncr-I,amhcrt Co. Frr,m Ihc (;rrclinva"ctrl:u' Rcwarch Intititulc :md Dcpartnuntti t,l I'harmacculical ('hcmi~tn• :mtl Mcdicinc. I Inivcrtiity nl ('alilrn'nia. San Francitico. I Ivll.RA('TIONS RI;TWfiI;N finNl) 3 ANI) OTl1I.R TRANSPOR'f-Rra.n'n;l) I,IZOII:INS I'wt, scts nf cxl,crimcntc were c:u'riccl c,ui to study interacticros anxm€ the Iran' I,rl rclatcd prutcim in thc human red cell. In Ihc carlicr series of cxpcriments• ca ricd r~trt hctwccn 1977 ancl I')RI. "1 nuclear magnetic resonance was utied lo auc bin lir~€ :rnd c,Ihcr inlcractinns between Ihc aninn ancl calion transport pmtcin, ar tl r, n, irrvetiti€;uc their rel:dicros with the €lycolytic enzymes in thc cytosnl. It w; litinrl Ih:r1 all Ihc €lyaolytic cniymcc between :dclc,lasc and pyruvatc kina.c wc- bnnnd lo€clhcr in a me€aulalNm complcx which was able to synthesize ATP frn Inrctou I,(,-dirhosphatc. Studies with insiclc-out vesicles showed that inclividu mcrnlxrs of thc ccrniplcx c•han€cd thcir conformation as they were hmmcl scqucnlii Iv n, thc cytcxolic membrane facc. ('cmfomiational information could be tr:msfcrrt ,tc.rtns thc mcmhranc, as shown by lhc ohscrvalion that either DII)S c,r ouahain, hnth. added to Ihe extracellular face, allosterically mcxlified the conformation of tl c.niymcs in the €lycolytic• complex hound to the cytnsolic facc. A sccrmcl series of cxpcrimcnts carried rnn between 1999 and 1991 uticd Ilu rcu'cnt probes to characterize thc kinetics of inhibitor binding to hand 3 and Ic, I €lucnse transport protein. '1'hese experimentti showed that perturbations inclucecl cither the Na',K'-ATPacc or the glucose transport protein produced allcrstcric cffct on thc confurmaUion of hand 3. These cxpcrimcnts show that these trantiRorl-rclatcd protcinx are hc,und lo€ctl wilh Ihc glycolytic enzymes in a mc€adalton complex linked to cytcnkclct;rl c mcnts to form a three-dimensional array cc,mpctent to carry out Ihc necessary me hranc traf fic. ('omrnunication can take place aniong the members of Ihc array tn rc ulntc thc energy supply ancl cnntrnl the internal rnilicu. .Cnlnrnnrt, n• K. In: Ranihcrg, E. and Pnssciw, H. (Eds.): Progress in ('cll Research. Vol 2. f{Iscv Science I'uhlishcr R.V., pp. 2(iy-283, 1992. Othcr support: National Instilutcs of Hcnlth, National Science hc,unclaticm, Amerk Ilcart Atisuciation, and Rristol-Mycrs Squibb Institutc for Medical Rcscarch. frnni the Biophysical Lahoratory, Ilarvarcl Medical School, Boston. i - p('MRS ON ANION TRANSPORT IN H11MAN RED CF,1.1, (iPPfi(°I' O1' M1:MRRnNFa •I'hc kinctic.,, (,f hindin€ of thc mercurial sulfhyclryl rca€cnt• pCMRS chlornmcrcurihcnicnc sulfnnatc), to the cxtracellular tiile(s) at which p('M inhihits walcr ancl urea Iranspnrt across the human red cell mcmbr:mc• have pr nusly been ch;tntcrcriicd. To cletcnninc whether pC'MRS hinclin€ ;dtcrs ('I Iransl- wc mc:rsurccl ('I /NO, exchange by Iluc,resccncc enhancement. using the (lye SPQ mcthc,ry-N /~ ~ulfupmpyllyuim,liniuml. An owntially instantancous cxlnrccll phaw c,t r('MRS inhibition is follnwcd by a muc•h slower intrnccllular phase. cc lalccl with ly('MRS pcrmcatirm. We attribute thc instantaneous phase to cumpcti inhibition r,f, ('I binding to band 3 by the (r('MRS anion. The II),,, of 2.0 -t I).I 90 91 ~
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agrc•c,~ with nthc•r nrg;mic wlfonatc•ti, hul is vct,v much grcatcr tltan that of p('N1kS inhihition ol urca an,l w;UCr transl,nrtf showing that P('MI3S reaction with walcr;in l urca Iranlpt,rt inhihition tiilcs ha" n„ cffect on anion cxchangc. Thc intraccllular hition by I mM h('MI1S ( I h) is apparcntly nnn-comhctitivc with K= 5.5 ±(,..i n~M prestnn;thly ;tn ;tllnslcric cffcct of h('MRS binding to an intr;tc•cllular han(1 i-rclalt•,1 %ullhyclryl group. Alter N-ethylmalc•imide (Nf•,M) trcatment to block these h;tntl ; sullhydryl groups, there is alqnrt•nt non-compclitive inhibition with K= 2,1 ~ I mM, which sul;gcsls thatt p('M13S rcacts with one of thc NIsM-inscnsilive sullitytlryl grottPs on a rtotcin that links h;tnel 3 to the cyloskcleton, perhaps ankyrin or hanth 4.1 antl 4.2. -/,hang. 7 ancl ,Snlmmmn. A. K. Riocltimica ct Riohhycica Acta 1 I(1(,:11-3c), I992. (lthcr support: Squibb Institute for Medical Research and American Hcart Assnciation. Frrrm Ihc Rinphytiic•al I.ahorotory, Ilarvarcl Mcclical School, Roslon. A("I'IVf: SI'I'1; RI,O('Kl;l) FACTOR IXa PRIiVf;N1:S INTRAVASCUI,AR 111ROMB11S FORMA-IlON IN'TIIIi('ORONARY VAS('(ILATURE WITHn(1T INIIII31llN(i IiX•IRAVAS('1)I.AR ('OA(;III,A'llON IN A ('ANINE '111ROMROSIS MUI)I?I. To aacss Ihc ccmtrihutinn of Faclor IX/IXa, to intravascttlar thromhosis, a canine coronary Ihrnmlxisis model was slucliecl. 7lvomhus formation was initiated by arhlying currc•nt to a needle in the circumllex coronary artcry. When 50% occlusion nl the v"ticl clevelopeci. Ihe current was stoPpecl and animals received an intra- vcmou,~ hnlu% of either salinc•, bovine f;lutamyl-glycyl-arginyl-Factor 1Xa (IXai), a c•ontpctitivc inhihitor nl' Iac•Inr IXa assembly into thc intrinsic Factor X activation e•„mhlex, bovine Factor IX, or heparin. Animals receiving saline or Factor IX devel- ohcd coronary t,cclu.iom (luc to a fihrin/platclet thrombus in 7(/+l I min. In contaast, mlueinn of IXai prevented thromhtts formatian completely (> IR(1 min) at doses of 4h/) ;trnl ;(IO fr,c/,y. and partially hlockcd throtnhus forn)ation at 150 Ft.g/kg• IXai ;utcmuatcd the acc•unntlatic,n of'"1-fihrinogcn/fihrin a1 the site of thc Ihomhus by h'1'.f (I' -t).(1O1) and rccultcd in 26'%, decrease in ticrotonin release from platelets in cor,m;nv tiinu~ (/' <(1.(/5), Ilcrnostalic variables in animals receiving IXai, remaincd within norm;tl lirnits. Animak- given heP;trin in a concentration sufficient to Prcvcnl occlusivc thrombosis had markedly increased hlccclin}t, whereas heparin Icvclti that mnintaincd cxtravaNculan cemostasis did not prevcnt intra-cc,ronary Ihnrmhntiis. -I'hiti suggests that Factor IX/IXa can contrihutc to Ilvomhus furmation, and thal inhihition nf IXa p;trticipation in Ihc clotting mechanism blocks intravascu- lar Ihnamhn.is without impairing ixtrav;tsc•ulan cemnstasis. Benedict. C. R.. Ryan. .I., Wt,li(iky. R., R;unos, R.. Gcriach. M.. Tijhurg. P.. and ,S7rrn• /). .I<,urn;d nf ('rinical Invt•stigation XR:17h(1-I7(,5, Novc•nilxr 1991. 1)` to 1-4 i N 0 on. I Icalth Scrvicc and SchultV Foundat i ' 0 c uhl upl ~ rc I I.S. I , I)i,P;trttncnt of Internal Medicine and l)ivision of ('ardiology, I Inivcrsity nf siology ancl Ccllutar l Ph 0 H thr y Iiral Sc•hool ;tt Ilouclon: I)epartment o York; .\ly( t l ~ i E1 t . Inc ~ Nutlcy. N•I , nyl aRoche It l`I" `"`' (, t~ll)Molct ular ( icnct c~' I h rt fm;m nd I)i hatlnnnl of 0 C) , I I .%I)VAN('fil) PROTEIN GLYCOSYLATIf)N INllUCES TRANSENDOTHGI.IAL IIItMAN MONOCYTfi ('HEMOTAXIS ANI) SECRfiTION OF PLATF.LE'I'- I)PRIVpp (;ROWTII FACTOR: ROLE IN VASCULAR DISEASE OF l)I>\RhTES ANI) AGING piafxlcs and aging are commonly accompanied by arterio- and athcrosclcrosis Infiltration of the arterial subendothelial intima by macrophages/monocytes is at iitiportanl early event prcceding the development of alhcromatous lesions: thes mncrophnges are known to produce mitogenic factors in early atherosclerotic Iesionr It has hccn previously shown that, over time, vascular matrix accumulates protein nonettzymatically modified by advanced glycosylation end products (AOES). I view of the fact that macrophages/montx•ytes have A(11:-specific receptors associa ed with the expression of several growth factars, we investigated the possibility th: A(71s mediate initial monocyte-vesscl wall interactions that occur hefore overt fo mation of vascular lesions. This study demonstratcs that (i) in vitro- and in rit's lirrmcd AGEs arc chcrnutactic for human blood monacytcs, (ii) subendolhcli AGEs can selectively induce monocyte migration across an intact endothelial c( monolayer, and (iii) subsequent montxyle interaction with AGE-containing matr results in the expression of platclet-derived growth factor. These results sttpPori t' cxisting hypothesis that irt rirn-forming glucosc-clcrivcd Protcin adducts can act signals for the normal turnover of senescent tissue protein by means of the A(3F s f cific receptor syslem. Tinte-dependent glucose-inctuced deposition of AGI?s matrix proteins may promote monocyte infiltration into the subcndothcliu Subsequent AG1; triggerett macrophage activation an(1 consequent elaboration prnliferalive factors may normally coordinate remodeling but may also lead to I divcrsc pathogenic changcs typical of arterio- and atherosclcrosis in diabetic aging Pnpulations. Kirstcin. M., Brcttt, J., Radoff. S.. Ogawa, S.. Stern. I)., and Vlassara, H. Proceedings of the National Academy of Sciences USA R7:901(1 -)O14, Novem 1990. Other sttpport: U.S. Public Health Service and New York Lung Association. From the Laboratory of Medical Biqchemistry, The Rockefeller Universily, f` York, and I)epartment of Physiology, Rover Physiology Lahoratories, Colutr llnivcrsity ('ollcgc of Physicians & Surgcons, New York. (Z
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('ARI)IA(' I)I•:NERVAIION IN -Il ll', ('AI.I-'I ISIN(; ('RYOAItLA/lON: H IN('TIONAI. I?VII)IiN('1: AND RI?(M)NAI. IISSI Il; ('Alli('llOI,AMINfi ('ON'I1FINT '1'wcnty ~ix calvc% wcrc "nhjcctcd 10 a Icc•hniyuc of c•ryrnhlatirm in order Icr rtitahfi%h an animal mndcl of comhlctc cardiac clcncrvatirm. All 26 survived thc prn- rt•durc. and 20 wcrc alivc to hc rc-cvaluatcd 2-4 wccks later. Mean hcarl ratc in Ihc clcncrvalcd animal, rutic from 77 4 7.9 hcats/min to 102 ± 16.4 (P <0.(ll). ('rr:thlatinn ahtrli,.hcd Ihc heart ratc retlrnnscs tu electrical titinnrlalion of thc va}!us ncrvc and thnracic .yrnrathclic n•unk-'I'hc rcductiim in mwocarclial iroradrenalinc crmccntr:uiims nvcragcd'1')'4, in Ihc right :drium, 9O'%• in Ihc Icl) atrium, ft5^/~ in the right ventricle and t)O'« in Ihc Iefl ventricle, when ctmiparccl with lissuc obtained Irrrm cnntrol anint:rk. ('ryn:thlation is a rclativcly sintplc means of acconrpli.hing ctmqplctc Iruncliomal c•artliac• clcncrvatitm in thc calf. On the batsis (if the crh,,crvcd chanrc in heart ralc, thc call ntndcl alrpcarN In hc more comparable with human heart tr:mplanl rcc'ihicnl. Ihan Ihc cloe. (Pacr,.I. A. R.. Wharkori,.I„ Gordtm. I.., Swill, R. 1., Munsch. ('.• higlix, G. C.. Polak, .1. M..:md lurbn, K. h1. liurnhcan.Inurnal of ('ardin-thnracic Surgery 6:2O1-2Oft, 1492. Fnmi thc I)cp:trlmcnt t1f (;rrdirrthoracic Surgery. Ilammcrsmitlt Ilospital, l.ondnn, 1?ngl:md: I)cr:rrlntcnl nf Ilietnchcntistry. Royal Postgraduate Medical Schcxil• I lannnc•r~mith 1 ho.pital: Medical Research ('ouncil Blood I'rc~.urc llnil. Western Infirmnry. (il:ntnw, Scotland. RI'.(ilONAI. I)ISl'RIRII'llON ANI) RIiGIII.AI'ION O1' I'"I1('ALCITONIN GI:NIi RIiI.AI'EI) I'I?I''l'II)1; I)INI)INC'i SI'll?S IN CORONARY ARTERIES (lwrntil:uivc in rirrn autnradioRraphic Icchniqucti were used to localize and char:rctcriic , 'I labeled htnnan c•alciUrnin f;cnc-rclatccl rcptidc 1"`'I1 binding sites in wctitrm of hovinc Icft antcritrr clc%ccniling cornnary arlc•ry (1.A1)). Spccific high alfinit\ (K, (1:1 nM) i"'Iih('(iRP hinding tiitc% were localized to tlrc media of both orir ardi:tl and myocartli:tl coronary artcricti. Binding site dcnsity was greater in dis- tpl chicanhal :mcl mvncardial arteries than in hrrrximal epicardial regions o(' the IAI>. Ilindmi ,ilcs cflhih{ICd a ~igni(ic•anlly higher tdfinity fur (r-hCGRI' (K 1.1 nM) Ihan Inr h('(;RI' (}{ ; I) (K 7./1 nM) and J('v%(A('M)"Ih('(;RI' (K 27.4 nM). (iuamo"inc 5O-(3 Ihinn•ipht;sphalc) inhibited f"`Ilh('(iRP binding in a concentra- lirm clclu•ndcnt m:tnncr. I?xIrimic clc•ncrvaticm of Ihc htrvinc heart resultcd in a dcplc- litm (il ('GRI'-likc irnmunoreaclivc Ixrivascular nerve Iihrcx and an increase in Ihc dcnsith nl' coronary artcrv 1'"lih('(IRP hindin)± sites (1) =(/.(H)c)2). l'he regional cliti- trihulitm of hinding SitcN in human c•orcmary arteries differed Irron that observed in hnvinc and lurrcinc vctisclt. II is crmcluJcd Ihat selective, (i pr„Icin-c•onplcd, CGRI' rcccplors are prc,,cnt in Ihc ntcdia nf bovine coronary arteries: thcrc arc both regional and shccics Jiflcrcncc% in Ihc cli.trihutinn rrf ('(iRl' hinding sites in ccironary arteries and cntlrrgc•nous ('( iRl' nr:ty crcrt a tr,nir inllucncc irn crnnnary vasrmurtcrr Irntc. Knock-, G. A.. Wharttm• .I., Gacr. .I. A. R., Yac•ouh, M. II., 7nelor. K. M., and Pr,lak. I. M. I nmpcsm ltwrn:(l of I'harmacrrkrgv'1'):4I5-4'5, 1992. Oqlrcr,ul'I"'rt: Briti,~h I Icarl Foundation. I rnm Iky':trlmcnts crl' Ilititcuhcmislry and ('arclicilhcrracic Surgcry, Royal I'oxl- Lradualc Medical School. Ilammcrsmith Ilo%pital. Lrnulon.,and ('arclinthoracic (htil• Ilarcficld Ilnspital. Midcllcscr, I;nl:lanJ. TOBACCO USL'• AND IIRINARY EXC'RETION OFTHROMBnXANF, A, AND I'ROSTA('YCLIN MI?TARnI,ITF..S IN WOMP.N STRATIFI6D BY AGE Bork,trnnnrl. Activated platelets have been implicated in both acute thronihus Gttm:ttion and athcrogcncsis. Rccausc smoking is a risk factor for cardiovascular (lis- cttsc in mcn and women cmJ male smokers have hiochemical evidence of increased platelet activation, we found it of- interest to stucly whether smoking augmcnls platelet activity in women as well. Mrvlu,d.c arrd Results. Data on smokinR hahits and a urinary samplc were obtained frtrm 125 hcallhy fernalc nonsmokcrs and an equal number of sntokcrs, etratifiecl by age in five gronps from IR to 59 years old. Urinary samples were ana- ly/etl with gas chromatography/mass spectrcrmctry for thc 2•3-clinormctaholitcx of Ihromfxtxane A, (Tx-M). reflecting platclcl activity, and proslacyclin (P(iI-M). icp- rcticnting platelet/vessel wal'I intcraction. Urinary Tx-M in smokers was higher Ihan in nonsmokers (p<0.(N)I ), increasing with the number of cigarettes smoked per day and with age. In mmsmnkcr., thcrc was no difference in Tx-M between the age groups. Urinary P( iI-M in smokers was higher than that in nonsmokers (p<(/.(N)I ) ancl clccrcascct with age in nommcrkcrs hul not in timokcrc. There was nn cliffcrcncc in l'x-M hctwecrt previnns smokers and Iifclnng nonsmokers. ('.nrrn.ciincc. The elevated Tx-M in women who smoke cigarettes indicates an increased platelet activity Ihal ie dcpcnclcnt on smoking intensity. hi parallcl, P(il-M is augmcntccl, tiuggcsting that hlatclct/vcssel wall interaction is stimulated. Quilting smnking is an cilcctivc mcam In restore platelet function. We propose that thc nhecrvcd inercasc in platelet activify in women who smoke cigarettes rnay hc related to suhxcquc•nt dcvchrrnrcnl nf cardiavascular disease and that quitting snioking ~hould hc considered a hith-prinrity medical target also in this sex. Ri)ngcma. ('., Rcnthin, G., Granelriim, Ii. F.. 1'crsson. L., Winell. S., and M-rrnrnrdnr. A. ('irculation M(5):14c)5-15(lt/, November 1992. OIhcr cnrptrrl: Swcdidh Medical Research Council and Swcdich Tobacco ('rnnp:my. Frnm the I)crartmcnt of ('linic:tl Physinhogy, (iothcnhurg University. (iothcnhurg• Sweden. 94 1 95
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RI: .I.EASIi OFI?NI)(YIIIPI.IAI. MF,DIA'IY)RS ANI) SYMI'A'1'III:I'I(' I'RANSMfI"1'I:RS n'I I)fI•I-I:.RI.NI'(Y)RONAI2Y H.OW RATFS IN ItAF1Rf1' I II?AMS I. Thc rclc:r,~c of thrcc endolltclial mcdiatrrrs• n;unclyl cndothclial-clcrivcd rt•I,yr ing laclrn (FI)R1). Irnnlacyclin (I'0.l anrl cndrrthc•lin. and (,f' Iw•o.ymp;dhctic nr•u. rntran.millcrs, nrrradrenalinc and ncumpeptidc Y (NI'Y). fnYm restini or symrathcli• cally uinrul:rlcrl cihhil I.anzcndnrff ht•arls was invcstigatcd aI nrmnal or clcv;ut•,I cnnrnary flnw. 'I•hc symhath(lic nerves Irl the hcarts were xtimulatcd at 5 I li for 30 ~ and tlrc cardiac clllucnl was analyscd firr nitrite (mctahr,litc of I;L)RI,*) wilh clcctrrnn paramapnrtic rct rmancc sIx•ctrrnnclry, fr,r (,-kclrr-I'C~I' Imctahnlitc of I'GIJ wilh g:rs chrnmatugraphy/nrrs .I,cctnnnctry. frrr cn(lrithcfin and NI'Y-Iikc immunorca livily with radioimmunoas-,ay. and fnr nuraclrcn:rlinc and purincs with liquid chro- mak,cral,hy. 2. 1)uring pcrfuaicrn r,f thc hearts ;u normal Ilr,w (35+ 1,4 ml min') the cfllucnt concentration of nitritc wa~ (1.151(1.02 ItM• Ihat nf (,-kc1o-PGF,„ 0.74+..(l.(1R m.t, and.lh:n ol cndnlhcfin-likc inmrunorcactivily (I.IR±(l.O1 pni. Nerve stimulation auE- mcnted thc rclc;tsc rrf h-kcki-I'(iP from 7(i4R lo rri):+_IO pmol (3 rnin)' (P <(1.115), hul rlid not aftccl fhc release uf nitritc or cndothcfin-tikc imnnmorcactivity. Nerve stimulation also facililatcd Ihc rtutflnw of nrrradrenalinc and ol' NFY-likc immtmnrc- activity by 521 I I pmol (I min)' and 1947 fnnrl (3 min) ', respectively. 11 . I?Icvaliun ril thc coronary Ilow to 7()+{.2 ml min ' did nrrl affcct Ihc cfflucnl cnnccntr:uirm, r,l nitritc. 6-kctn-I'(iFand cndothclin-likc immunnrcac•tivity, imply- ing that their rwtflrrwc were augmented. Symra(hctic slinmlatirm at elevated crrro- n;iry Ilow did not fru-lhcr :mgmcnl Ihc rnqflow of cndothclial mcdiatrrrs or of NPY- like inmrunorcactivity, hul increased Ihc oulflow rlf noradrenalinc by A2+12'%, in crrnil,;rrisnn tn stinrutalirm nt normal flow. Pcrfuv~irrn rif Ihc hcart with Ihc nnraclrcna- linc uhlakc blocker dcsiln;mrinc (5 unt) completely abolished Ihc promoting cffccling nf clcvatcrl currmary Ilrrw crn nrn•adrenalinc rmt/lrrw during sympathctic stimulation. 4. 'I'hc~c d:na indic:rlc that an incrcau in coronary Ilow in perfused rabbit hcar(e it p;rr;rllclcd by a corresponding facifitatiun rrf Ihc frmnalion of Ihc cndothclial mcdi- a1nr., FI)ILF• prrra;icyclin and cndnthclin. Such an elevation of mcdialor formalion docs nnt ;rllccl ncrvt' titimulatirm-incluccd release rif synrpathctic Iransmillcrs in the hcarl. If rnr,nurlm. A.. IScnlhin, G., Karwatow.ka-I'rokc,pciuk, I'.._ l,urrdhrr,~. .1.. nnd ..,m. A.-S. Ir„n i,il nf I'Iry~inlnzv •135:163-17;, 199 1. OIhci .o(1hnrl: Swcdi"h Mcdic:rl Research ('ouncil and Llpjc,hn Company. I-nnn thr 1)cl,arlmcm of ('linical I'hytiirrlnLy, (ir,thcnhur,g Ilnivcnily, Gothcnhurg• anrl the I)clrartmr m r,f 1'h;nrmacohrgy• Karnlimka Instiunc. Sluckholni• Swcdcn. I IV. Neuropharmacology and Physiology I Rr)TON 11RENIC AND AFFECTIVF. PAT FNTSCOPY OF THE BRAIN IN SPE <('I IILOP Watcr-srrppressed 'H magnetic resonance spectra were recorded from two brain r`ci n` nf psychratrrc patients and normal volunteers. The two regions studied were t;rl Ihc h:rsal gang~ta structures surrounding the anterior ho hosf ~~~Qatine creatine rnrl (h) Ilre occipital cortex. N-Acetylaspartate (NAA). p p t~( r( rl. choline intenr IS ostPCr-Cr peak integ al were calcutatedrforreach Rpe~ rum "'ctxhr,lr(e I'e artlal volume effects, comparisons between patients and controls I„ control for p «.crc n,ade only from identical regions i.e., basal ganglia vs basal ganglia, and r e «,itc for occipital cortex. Metabolite ratios from the occipital region of patients were tiirnilar to those from the occipital region of normal subjects. Bipolar patients being ircated with n~~ m h~eseepat entN also demonst at d elevated chol ne/PCr Cr and cnmpared innsilol/PCr-Cr ratios in the basal ganglia region. Sharma. R., Venkatasubramanian, P. N., 86runv. M.. and Davis, J. M. Schiiophrenia Research 8:43-39. 1992. F:rnm the Department of Psychiatry, Biological Chemistry and Magnetic Rcsonanc( (•enter, College of Medicine. University of Illinois at Chicago, and Illinois Statr p,ychiatric Institute, Chicago. F,fTECT OF NICOTINE ON EXTRACELLULAR LEVELS OF NEUROTRANS- MITTERS ASSESSED BY MICRODIALYSIS IN VARIOUS BRAIN RECiIONS: ROLE OF GLUTAMIC ACIL) We studied the effect of local administration of ~ipcnoctin pon the release c monoamines in striatum, substantia nigra, cerehellum, hi am us, cortex (fronta cingulatc), and pontinc nuclcusand on the release of glutamic acid in striatum of rat in rirn, using microdialysis for nirntine administration and for measuring extracellr lar amine and glutamic acid Icvcls. Following nicotine administration the extracellt lar concentration of dupaminc increased in all regions except cerebellum: scrotoni increaced in cingulate and frontal cortex; and norepinephrine increased in suhstant nigra, cingulate cortex, and pontinc nucleus. Cotinine, the major nicotine metabolit had no effect at similar concentrations. The cholinergic antagonists mecamylamir and adropine, the dopamincrgic antagonists haloperidol and sulpride, and the excit lory amino acid antagonist kynurenic acid all inhibited the nicotine-induced incrca of extraccllular dopamine in the striatum. The fact that kynurenic acid almost cor pletely prevented the effects of nicotine, and nicotine at this concentration producr a 6-fold increase of glutarnic acid release, suggests that the effect of nicotine mainly mcdiatc(1 via glutamic acid release. 96 97
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Tnth• li.• Scrshcn, I I., Hashim, A., Vizi, I:. S., and LrrjNui, A Ncurochcmic•al Rescarch 17(i):265-27I• 1992. From the Center for Neurcrchcmistry, The Nathan S. Kline Institute for Psych Research, C)rangchurg, NY. N-TYPE CALC'It IM CIIANNF,f S ARE INVOLVED IN THE f)nPAM1NB RELEASING' F,FhI?('T pP NICOTINE Mouse striaturn was incubated with I'HIdopamine (1'H1DA) and superfused with (Krehs bicarhonate) huffer, then the tritium efflux induced by nicotine, electri. cal stimulation, or simultaneous nicotine and electrical stimulation was measured. Ic, characterize the role ol different Ca" channels in the transmitter release. Nicotine stimulation ancl electrical stimulation exerted additive effects on tritium efflux. Separation of the released radioactivity on alumina columns indicated that nicotinc or electrical stimulation increases the release of f 711DA and that the outflow of'fl- lahelccl metaholites was similar with the two different stimulation procedures. Removal of ('a" from the supcrfusatc resulted in a marked reduction in the tritium release evoked by nicotine whereas the clectrical stimulation-evoked tritium release was completely dependent on external Ca". The L- and N-type calcium channel blockers omcga-conotoxin GVIA and Cd" inhibited the tritium release from the striatum evoked by either nicotine or electrical stimulation, whereas the I: type and 'f-tylx channel blockers dibiazem and Ni" ((lid not aher release of I'HIDA. We con- clude that N-type vollage-sensitive calcium channels participate in striatal dopamine release, and we speculate that nicotinic receptor-operated ion channels permeable to cations such as ('a•• and N-tyfx voltage-sensitive calcium channels may simul1ane- ouxlY open up, and thcy additivcly increase free intracellular Ca' concentration. I larsing, I,.. ( i., Jr., Sershen, I1., Vizi, S. F,., and Lajlho, A. Ncuroc•hcmical Research 17(7):729-734, July 1992. 1'rnnr the ('enter for Ncurae•hemisrry, the Nathan S. Kline Institute for Psychiatric Rc.ticarc•h, r)rarrgchurg, NY, and the Institute of Experimental Medicine, Hungarian Acsrdcmy of Scicnccs, Rudalxsl. I'f:RSISTI'N(T;O1'('HRONI(' NICOTIN1;-INDII(•Ef) C'O(;NITIVf? PA('ILITATI( )N Nicotine has been formd in a variety of species and behavioral paradigms to improve memory perGrmrancc•. The beneficial effect of nicotine has been seen after both acute and chronic• adminictratimr. Interestingly, improved performance has been seen 24 Ir after acute injection and for at least 2 weeks alier chronic administration. Ilowever, it is nnt c•Icar fromi previous studies whether the persistence of the t t,c,rf,rrrnancc represents a true canyovcr of the drug effect or is due to the r,rl c.xperrence while under nicotine's effect. The current study was p t`cx~(ormancercorrlc)ehe seen afterrwithct awal evenf if there was no behavior.l n I the riorl of chronic nicotine administration. Rats were administered ,mmeclurrng• ~fnr 3 weeks but werc not tested during that time. Starting I unr chronrcally t~ttc r withdrawal they were trained on a working memory paradigm in an cig t- r~~l~n~~'rh~~' h sowed s gnifcantly f aster learning astdetected by three levels i• rt' n ~ r,( choice accurac . B the final hase of testin the control sub'ects had Y Y p g J run ctit rrl, with the nicotine-treated rats. After the acquisition phase, acute cha enges „'tli thr rrrcotrd'fferential effects int henn cot neel eated and contro gr upseThe „t clicit any Ilrtccct audy demonstrated that nicotine-induced cognitive facilitation persists for at nndcr the influence oftthe drug~The m chan depend for~thiper i ting effec~is not clrtc,ntly understood. Alterations in nicotinic and muscarinic responsivity were not ,k•tcctcd in the current study. f ,rin. f.1)., Briggs• S• J.. Christopher, N. C., and Rose, J. E. Rchavinral and Neural Biology 58:152-158. 1992. Other support: Alzheimer's Association/Neit Bluhm Pilot Research Program and Natinnal Institute of Drug Abuse. Duke University, ctc aos Adm nistat onoMe ficalCeenlemDurham NC. ancl the V NIC'OTINIC SYSTEMS AND COGNITIVE FUNCTION Nicotinic acetylchotine receptors have been found to be important for maintain• ing optimal performance on a variety of cognitive tasks. In humans, nicotine-induce( improvement of rapid informatinn processing is particularly well documented. Ir experimental animals nicotine has been found to improve learning and memory on t variety of tasks, while the nicotinic antagonist mecamylamine has been found h impair memory performance. Nicotine has been found to be effective in attenuatinl memory deficits resulting from lesions of the septohippocampal pathway or aging ii experimental animals. Nicotinic receptors are decreased in the cortex of patients wit Alzheimer's cliscasc. Prcliminary studies have found that some aspects of the cogn livc deficit in Alzhcimer'S discase can be attenuated by nicotine. Nicotine may prov to be useful therapeutic treatment for this and other types of dementia. Lerin. F.. f). Psychopharrnacology 10R:4t7-431. 1992. Other support: Alzhcimer's Association/Ncil Bluhm Pilot Research program. From the Nicotine Research 1 -ibnratory, Department of Psychiatry, Duke Universi Medical Center, l)urharn, NC. 99 I 99
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THE IMPnRTAN(T O1' 1) ANI) 1), INTIiRA('7'IONS WITII NICOTINIC ANI) MI IS('ARINI(' SYSTf?MS IPOR WORKIN(; MEMORY FI INC'TION There arc potent interactions between cholincrgic and dnpamincrgic systems with regard to working memory performance. These interactions and their nature are specifically related to the different rcccptorsuhtypes of these systems: nicotinic and muscarinic cholincrgic rcccptors and L) and D dcrpamincrgic receptors. Sites of DA- A('h interactions have been identified that are likcly to be substrates for the observed interactions. Lesion studies and sitc-specific drug infusion s(udics will help deter- mine which of these systems are critical for the observed systemic drug interactions. l.crin, R. 1). and Rose, J. E. In: Levin, E. D., Decker. M. W., Butcher, L. L. (eds.): Neurotransmitter Interactions and Cognitive Function, Rcrkhauscr, Boston. MA, pp. 144-158, 1992. Other support: Alzheimer's Association/Neil Bluhm Pilot Research Program and National Institutc for Drug Abuse. From the Nicotine Research Laboratory, Department of Psychiatry, Duke University Medical ('cntcr, Durham, NC. IMMUNOHISTOCIII?MICAI, LOCALIZATION OF NICOTINIC A('ETYI,('1101,INE RE('1?PTOR SUBUNITS IN TIIE MESENCEPHALON ANI) DIEN('EPIIALON OF THE C'I IICK (GA1,LU,S GAIJ,(LS) Monoclonal antitxxlics against two a-bungarotoxin-binding subunits (a7 and (YR) nf the nicotinic acetylchuline receptors (nAChRs) were used as immunohisto- chemical prohes to map their distribution in the chick diencephalon and mesen- cephalon. 'fhe distribution of the a7 and aR nAChR subunits was compared to ihe dietrihution nf immunorcactivity produced by a monrxlnnal antibody against the 132 structural suhmrit of lite nA('hRs. Structurec that contained high numbers of a7-like immunoreactive (Li) somata inchtdcd lite intcrgcniculatc lca(let, nucleus intcrcalatus thalami, nucleus ovoidalis, organum pnravcntriculari., nucleus rotundus, isthmic nuclei, nucleus trochlcaris, ocu- homotnr complex, nucleus intcrslitio-prctecto-suhpretcctalis, stratum griseum ccntralc of lite optic tectum, and nucleus sctnilunaris. Neuropil staining for a7-LI was intcn.c in lite nucleus dorsomcdialis hypothalami, nucleus geniculatus latcralis ventralis, griscum tecti, isthrnic nuclei, nucleus Ientiformis mesencephali. nucleus of lite ba.al optic rcxot, and stratum griscum ct fibrosum superficiale of the tcctum. High numlx-rs of aR-LI somata were found in lite stratum griscum cl fibrosum superficiale of the Icctum and lite nucleus interstitio-pretecto-suhpretectalis, and intense neuropil staining for aR-LI was found in the dorsal thalamus, nucleus geniculatus lateralis ventralis, lateral hypothalamus, griscum tecti, nucleus Ientiformis mescncephali, nucleus inicrpcduncularis, and straturn griscum el fibrosuni superficiale of the tec- tum. Iligh numbers of (32-LI somata were found only in the nucleus spiriformis lat- cralis, whereas ncuropil etaining for (32-L1 was intense in the nucleus geniculatus lat- cralis ventralis, nucleul suprachiasmalicus, nucleus lateralis anterior, nucleus habe- nularis Iateralis, area prcicctalis, griscum tecti, nucleus Ientifnrmis mesencephali, t,uclcus extcrnus, and nucleus interpedoncularis, and in the stratum griseum ccntrale, tr.ttun1 griscum ct fihrosum supcrficralc, and stratum npticum of the tcctum. Trese results indicate that there are major disparities in the localization of the a- hrtnFamtoxin-hinding cx7 and aR nAChR subunits and the (32 structural nAChR sub- tmit in the chick diencephalon and mesencephalon. These nAChR subunits appear, I,(,wcvcr, to coexist in several regions of the chick brain. (3ritto, L. R. G•• Keyser, K. T., Lindctrnrn, .l. M., and Karten. H. J. Thc Journal of Comparative Neurology 317:125-340, 1992. Other suPport: Muscular Dystrophy Association, Smokeless Tobacco Research Council and Fogarty International Center (Latin American and Caribbean Initiative). 1 From the Department of Neurosciences, University of California at San Diego. La siology t of Ph t ~ y men Jolla; Neurosciences and Behavior Research Nucleus and Depar and Biophysics, Institute of Biomedical Sciences. Sao Paulo State University, Sac Pauln, Braail; and David Mahoney Institute of Neurological Sciences. University ol ~ 0 Pennsylvania, Philadelphia. NIC ~ ACET Y1CNnMNE RECF`PTORISUBOUN TGENES NRiOUMANSCOTI Frl ~ We have determined the chromosomal location of seven human neuronal nico tinic acetylcholine receptor subunit genes by genomic Southern analysis of ham stcdhuman somatic cell hybrid DNAs. The (32 subunit gene was localized to humat chromosome 1, the a2 and (33 subunit genes were localized to human chromosoim, R, the 0, a5, and (34 subunit genes were localized to human chromosome 15. an, the a4 subunit gene was localized to human chromosome 20. Mapping of the (3' subunit gene to chromosome I establishes a syntenic group with the amylase gen locus on human chromosome I and mouse chromosome 3, while mapping of the a subunit gene to chromosome 15 confirms the existence of a syntenic group with th mannose phosphate isomerase gene locus on human chromosome 15 and mous chromosome 9. Anand, R. and Lind.clrnm, J. Gcnomics 13:962-967, 1992. Other support: National Institutes of Health. Muscular Dystrophy Association an Smokeless Tobacco Research Council. From the David Mahoney Institute of Neurological Sciences, University r Pcnnsylvania, Philadelphia. F.PITOPE MAPPING OF POLYCLONAL AND MONOCLONAL ANTIBODIES AGAINST TWO a-BUNGAROTOXIN-BINDING a SUBUNITS FROM NEIIRONAL NICOTINIC RECEPTORS Recently, cDNAs for a subunits of two different neuronal a-hungarotoxin-bin ing proteins (aBgtBP) were isolated from chick brain designated aBgtBPal a- I(N) 1 101
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aRgIRPn2. These arc now also referred to as subunits rx7 and nR, respceliveh Expression studies in Xr•rrr,/rus cxocyles have indicated that rx7 snhunits are ahle form cation channels that are sensitive to nicotinic ligan(s, and thcrefore re t' hrma fide nicotinic• acetylcholine receptor subunits. Polyclonal and monoclon~lr~~n~, hndiex (mAhs) have been produced against: (i) affinit nrrt~ y-purificd chick brain rxBgq;h and (ii) fusion proteins containing the unique cytoplasmic sequences a7(i27-412i and (0293 -435). Hcre, synthetic overlapping peptides corresponding to the,r deduced amino acid sequenc•es are used to map the epilopes recognized by the differ, ent antibodies. The polyclonal response to affinity-purified rxRgtBPs and the fusinn proteins indicates that sequence segments 29O-42O of both subunits contain several major and minor epitopes. mAbs selected for their ability to bind both native an,(l denatured rxRglRl's isolated from chick brain also recognize subunit-specific sequential epitopes within the sequence segment 290-420. The epitnpes recognize,l by the mAbs correspond to the minor epilopes defined using antisera. The mAh, characterized in these studies will prnvide useful probes for further studies n( nBgIF3P structurc and histological localiz.uion. McLane• K. G., Wu, X.. Lindctrom, J. M.. and Cnnti-Tronconi, B. Journal of Ncuroimmunology iR:115-12R. 1992. Other support: National Science Foundation, National Institute of Drug Addiction, Muscular Dystrophy Association, Smokeless Tobacco Research Council, Myaslhcnia Gravis Pnundation, and National Institutes of Ilcalth. From The Department of Ricxhemistry, College of Biological Sciences, University of Minnesota. St. Paul, and Institu(e of Neurological Sciences, University of Pennsylvania Medical School, Philadeiphia. NF:I IRONS OF THF. ('lll('K BRAIN ANI) RETINA EXPRESSING BOTH rx-R1JN(;AROTt)XIN-S1iNSITIVf? ANl) rx-131)NGAROTOXIN-INSENSITIVE NI('O'I'INI(' A('I:'I'YLCHOLINF, RECEPTORS: AN IMMUNOHISTOCHEMI- ('AL ANALYSIS Immmnnhislochemic•al methods were used to study the possible co-localiza4ion ol tui, fx hungaroloxin-sensitive (rx7 and (YR) and two (x-hungarotoxin-insensitive ([iQ aml 1i3) subunils of• Ihe nicotinic acetylcholinc receptors in neurons of the chick brain and retina. Several structures contained neurons that were doubly-labeled with antibodies against the rx7 subunit and the /32 subunit. 'I7resc stnrctures included, for cxamplc, the intcrpcduncular nucleus, nucleus spiriformis latcralis, optic tcctum, prc•Iectal visual nuclei, and Ihe lateral hypothalamus. Douhle-lahcling with antihod- ies against the rx7 and rxR subunits was also seen in several regions, which included Ihe inlerlreduncnlar nucleus, visual pretectum, lateral hypothalamus, dorsal thalamus, and the halx•nular complex. In the retina, many cells in the inner nuclear layer were observed to contain rxR and r0 subunits, whereas neurons in The ganglion ccll layer were seen to contain rx7 and rxfi or, less frcquently, 07 and rx3 subunits. These results indicate that rx-hungarotr,xin-scnsitive and rx-hungarotoxin-inscnsitive subunits of the nic•otinic receptors are co-expressed by neurons of the chick brain and retina. IFamassaki-Britto, D. L;., Ferro, F.. S., Keyser, K. T., Karlen, H. 1.. I,r nn. 1,. M. pr in Rc~c:uch 59f1:193-2(1R, 1992. ~,p„r ~~~PFn~~ Muscular I)ystroPhY Association and Smokeless Tobacco Research t „Uniil. 1 r r~~ the Neurnsciences and Behavior Research Nucleus, Nucleus of Cellular an siology, Department of Physiology and Biophysics, Institute of ,ln)ccular PhY, It „jjedical cs1eUnivers ty of tCalifoma a at San Di goULa olla! a d Instritute of s;,•uroscicnc `,rrr„Ingical Sciences. University of Pennsylvania. Philadelphra. I,PITOPE MAPPING OF MONOCLONAL ANTIBODIES TO TORPF,DO (•1;1•YL('HOLINE RECEPTOR y SUBUNITS, WHICH SPECIFICALLY A A(,CHOUNE RECEPTOR OF MAMMALIAN MUSCLE Epitopcs for four monoclonal antibodies (mAbs) to the y subunit of Torpedo nicntinic acetylcholine receptor (AChR), and one mAb crossr5ant~etic with fdc ~ri h cubunits of Torpedo AChR were mapped using overlapping • y PeP rcgionding to the comptete amino acid sequence of Torpedo y subunit. The epitopes for all mAbs were within a 50 residue sequence region, on the cytoplasrnic surface nf the AChR. Three mAbs crossrcacted with mammalian muscle AChRs. Two of Ihem specifically recognized the e subunit of AChRs at adult neuromuscular junc• tion. Nclson, S., Shelton, G. D., Lei. S.. Lrndstrnm, .l. M.. and Cnnti-Tronconi, B. Joumal of Neuroimmunnlogy 36:13-27, 1992. Other support: National Science Foundation, National Institute of Dmhcn ~clGravi National Institutes of Health, Muscular Dy-strophy Assrrciation, My • Foundation, and Smnkeless'1'ubaccn Research Council. From the Department of Biochemistry. University of Minnesota, St. Paul, an Receptor Biology l,ahoratnry. The Salk lnstitute for Biological Sciences, Sa Dicgo, C'A. RIIK-2I-DERfV1iD CELI, LINES THAT PRODUCE BASIC FIBROBLAST GROWTH FA("TOR, BUT NOT 1'ARENTAL BHK-21 CELLS. INITIATF, NI;URONAL D11TER1•sN-I'IATION OF NF.IJRAL CREST PR(K;ENI r~uc n cc We present evidence that basic fihrohlast growth factor (hFGF~ p g stimulate primary dit7crcntiation of ncurons from neural crest progenitors. Ba 102 1 103
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hamster kidney (RHK-21) cells were stably cotransfected with plasmid pSV2/nery which contains the gene conferring vectors containing (he human hFGF cDNA. Various clones, which differed in their hFGF production levels, were isolated Homogeneous neural crest cells were cultured on monolayers of hFGF-producing, BHK-2I-derived cell lines. While the parental F1HK-21 cells, which do not prnduce detectable hF(iF, had poor neurogenic ability, the various hFGF-producing clone.% promoted a 1.5- to 4-fold increase in neuronal cell number compared to the parental cells. This increase was correlated with the levels of bFGF produced hy the different transfectcd clones, which ranged between 2.3 and 140 ng/mg protein. In contrast, no stimulation of neuronal differentiation was observed when neural crest cells were grown on monolayers of parental f3FIK cells transfected with plasmid pSV2/neo alone, or on a parental BHK-derived clone, which secrets high amounts of recomhi- nant vascular endothelial growth factor (VEGF). Furthermore, the ncuron-promoting ability of bFGF-producing cells could he niimicked by addition of exogenous bFGF to neural crest cells grown on the parental 131-1K line. A similar treatment of neural crest cells grown on laminin substrata, instead of BHK cells, resulted in increased survival of non-neurnnal cells, but not of neutrons (see also Kalcheim. C. 1989, Dev, F3:iol. 134, 1-1(1). 'I'akcn together, these results suggest that hFGF stimulates neuronal differcntiation of neural crest cells by a cell-mediated signalling mechanism. Rrill, G., Vaisman, N., Nru(rld, G., and Kalcheim, C. Development I 15:1059-1(K~9, 1991 Other support: National Council for Research and Development, Commission for European Communities. Familial Dysautonomia Foundation, Israel Academy of Sciences and Humanities, Israel Ministry of Health, and USA-Israel Binational Foundation. From the 1)cpartmenl of Anatomy and Embryology, Hehrew Univcrsity-Hadassah Medical School, Jerusalem, and Department of Biology, Technion-Fsrael Institute of Technology, Haifa, Israel. SI IRTYI'E OF MI IS('ARINICRF.CF,PTOR COUPLED TO TIIE ATTENUATION OVIICIRMONli-ST7MtJLATF,D cAMP ACCUMULATION IN NG 108-15 NI?tIRORLASTOMA X GLIOMA HYF3R11)CELLS The subtype of muscarinic receptor which mediates cAMP attenuation is not established. Thcreforc, several selective muscarinic antagonists were used to charac- terize thc subtypc of muscarinic receptor coupled to the inhibition of hormone-stimu- laled cAMP accumulation using NG 108-15 ncuroblastoma x glioma hybrid cells. These cells were prclahelcd with 12-'llI-adenine, washed, and resuspended in a cul- ture medium containing the phosphodieslerasc inhibitor 3-isobutyl-I-methylxanthine (0.5 mM). The labeled cells were preincubated with the different antagonists 12-15 min. before (hey were challenged with agonists. The formation of ('HI-cAMP was activated by PGfil (I µM) or forskolin (1 µM). In all cases, l'HI-cAMP formed was separated and measured. C'arbachol ( I(X) µM) and McN-A343 (10 mM) were used as standard muscarinic agonists. These studies gave the following results: a) McN- A343 (10 rnM), an M I receptor agonist, was only a partial agonist causing 40% inhi- f ,n of cAMP accumulation indicating Ihat this effect was not mediated by an A r ccPt"r: h) The MI-selective antagonist, pirenzepine, exhibited low affinity (p~ t,,2) further suggesting that an MI receptor was not coupled to the attenuation cAMP accumulation; c) Two selective M2 antagonists (AF-DX 116 and methc irarnine) and M3 antagonist (HIiSiD) were used to further characterize these mi carinic receptors. The order of all antagonists based on their affinities (pA2 valu, ,,idd he arranged in the following order: atropine (9.0) >methoctramine (7.6; IlIlSiD (6.9) > AF-DX 116 (6.6) > pirenzepine (6.2). HHSiD exhibits the sa ilcgrec of affinity to M2 receptors of other tissues as it does to those of NG cc ?hese observations suggest that an M2 muscarinic receptor may be coupled to ,1ttenuation of hormone-stimulated cAMP accumulation. They do not exclude pnssihility of the mediation of this response by M4 receptors. Stcphan, C. C. and Sastry, B. V. R. Cellular and Molecular Biology 3R(6):601-612, 1992. Other support: Smokeless Tobacco Research Council and National Institutes I(calth. From the Departments of Pharmacology and Anesthesiology, Vanderbilt Univers School of Medicine, Nashville, TN. PRFSYNAPTIC MUSCARINIC RECEPTORS AND THE AUTOREGULATIO OF NEUROTRANSMITTER RELEASE Two mechanisms have been proposed for the regulation of acetylcholine (A release in the nervous system: a negative feedback mechanism and an ACh-aml cation mechanism or a positive feedback mechanism. We have used 5-hydr methyl-furfuryltrimethylammonium (5-HMFf and 5-methoxyfurfuryllrimcl ammonium (5-MOI'1'), two furan analogs of muscarine, to study the above me nisms in Auerbach plexus of longitudinal ileal muscles of the guinea pig whicl rich in ACh (119-120 mmol/g). 5-HMFT (10" to 10' M) inhibited spontancoi well as nicotine-induced release of ACh. Atropine, enkephalins, and morphine : ished the effect of 5-HMIT. These observations indicate that 5-HMFT activa muscarinic receptor (Mi) and causes inhibition of ACh release. 5-MOFT caused traction of the guinea pig ileal muscle (EC50, 28 mm) which was potentiate physostigmine and blocked by atropinc. When the tissue was depleted of AC incuhation with hemicholinium-3, the response to 5-MOFT disappeared, indic that 5-MOFT activated a presynaptic muscarinic receptor (Ms) and released These results indicate that the Aucrbach plexus contains two muscarinic rcce (Mi and Ms), one excilatory and the other inhibitory, which maintain homeosta ACh release. Sa.rlrv, B. V. R. and (khilln, R. F. 104 1 105
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In: Wcgmann, R. J. and Wegmann, M. A. Recent Advances in Cellular and Molecular Biology. Pcctcrs Press, Letrven, Bclgium, pp. 273 279, 19t)2. Other support: U.S. Public Health Service, National Institutes of Health and The Study Center for Anesthesia Toxicology (Vanderbilt lJnivcrsity). From the Departments of Anesthesiology and Pharmacology, Vanderbilt University Medical Center, Nashvillc, TN. DOPAMINE EFTLI/X FROM STRIATUM AFTER CHRONIC NICOTINE: EVIDENCE FOR A1ITORECEP'F'OR DESENSITIZATION We examined the effect of chronic nicotine treatment on dopaminergic activity by measuring the effects of D, and D, dopamine (DA) receptor agonists and antago- nists on tritium release from mouse striatum prcloadcd with I'HIDA. The radioac- tivity released during supcrfusion was separated on alumina columns and the distribu- tion and efflux of 1'H1DA and its main 'H-laheled metabolites were quantified. After preloading by incubation with ['HIDA, the electrical slimulation-evoked tritium overflow was higher in striatum prepared from nicotine-treated mice, whereas in vin-n adclition of nicotine caused a similar increase in tritium release from stiiatum of untreated and chronic nicotine-treated mice. The overflow of 1'H1DA and its 'H- metatxolites exhibited similar clistribution patterns in I'HIDA-preloadcd striatum dis- sected from untreated and chronic nicotine-pretreated mice, indicating that repeated injections with nicotine did not alter the metabolism of I'HII)A taken up by Ihe tis- sue. (-)-Quinpirolc, a selective agonist for D' DA receptors, and apcrmorphine, a nonselective D,/D, agonist, inhibited Ihe electrical stimulation-induced tritium efffux from striatum of untrcated mice, whereas (±)-sulpiride. a D2 DA receptor antagonist, enhanced the evoked release of tritium. These changes in tritium efllux effected by (-)-quinpirolc and (±)-sulpiridc reflected changes in 1'H1DA release and not in DA mclabolism, as shown by separation of the released radioactivity on alumina columns. The D, receptor agonist (±)-SKF-3R393 did not affect the tritium overflow, whcrea% the 1). receptor antagonist (+)-SCH-23390 exerted a stimulatory action but only at a high oncentration. In contrast, neither the DA receptor agonists 1(-)-quin- pirnlc. alximorphinc, and (±) SKF-383931 nor thc antagonists 1(±)-sulpiride and (+)- S('H-23 ttN)1 altered the stirnulation-evoked tritium release in striatum obtained from mice repeatedly treaUed with nicotine. It is concluded that chronic administration of nicotine produces an increased release of I)A in the stria(um with a resulting eleva- tion of synaptic DA concentrations leading to development of D, DA autoreceptor subsensitivity. As a consequence, chronic nicotine may attenuate autoinhibition of dopaminergic neurotransmission in the strialum. Harsing, L, G., Jr., Senchen, H., and l ttjtha, A. Journal of Neurochemistry 59( I):4R-54, 1992. From the Center for Neurnchemistry, Nathan S. Kline Institute for Psychiatric Research. Orangcburg, NY. ('IIRONIC CONTINUOUS NICOTINE TREATMENT CAUSES DECREASF,D NfiMITRANS CTI',D AT THE MESO-DIENCEPA IC JUNCTIONRTIALLY Chronic continuous administration of nicotine (0.125 mg/kg/h 14 days) to n Sprague-Dawlcy rats with a partial hemitransection at the meso-diencephalic ji titm caused a significant reduction in burst firing of remaining dopamine (DA) t rnns in the zona compacta, substantia nigra, whereas neither the firing rate nor number of spontaneously active DA cells per track were altered in comparison ' ,aline-lreated, hemitransected controls. The reduced functional activity of rcmaining DA cells subjected to nicotine treatment provides a physiological c( Iatc to the previously observed, reduced DA utilization in these neurons. It may help to explain the increased nigral DA cell survival found after chronic nicc treatment in similar lesion experiments. Grenhoff, J., Janson. A. M., Sven,cson, T. H., and Fuxe, K. Brain Research 562(2): 347-351, October 25, 1991. Other support: Research Council Smoking and Health (Germany), Swedish Mei Research Council, Swedish Medical Society, Syskonen Perssons Donationsf Torsten och Ragnar Sfiderbergs Stiftelser, and Karolinska Institute. From the Departments of Pharmacology and Histology and Neurobiol- Karolinska Institute, Stockholm. Sweden. V. Pharmacology, Biochemistry and Cell Biology THE cDNAs CODING FOR THE a- AND f3-SUBUNITS OF XENOPUS LAF,6 CASEIN KINASE II Using a?cgI10 cDNA library obtained from Xenopus laevis oocytes and p derived from the known sequences of the human and Drosophila genes, a c coding for the a-subunit of the X. laevis casein kinase lI was isolated. The a sequence of this clone determines a polypeptide of 350 amino acids. The X. t sequence is 98% identical to the human and rat proteins in the first 323 amino ; Using the polymerase chain reaction to generate a 370-nucleotide-long probe, i- possible to clone and sequence a eDNA of 900 nucleotides that coded for t laevi.c R-subunit of casein kinase If. The derived protein sequence is 215 amino long and again shows an extraordinary degree of conservation with other specie Jedlicki, A., Hinrichs. M. V., Allende, C. C., and Allende, J. E. FEBS 297(3):2R0-2R4, February 1992. Other support: International Center for Genetic Engineering and Biotechnolog FONDECYT-Chile. From the Departamento de Bioquimica. Facultad de Medicina, and Depariame, Biolog(a, Facultad de Ciencias, Universidad de Chile, Santiago, Chile. 106 1 107
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TEIE HYDROLYSIS OF I'lIt7SPIIATIDYI.INOSITOL 4-PIIOSPHATE IN MEMBRANES OF XLNI)1'U,S LA/il'/.S DO('Y'f'f?S: CHARACTERISTICS OF A PI i()SPHOMONOESTf:R A SE I. Phosphatidylinositol 4-phosphate (Ptdlnt4P) is degraded by isolated mem- brancs from J(cnnlms lneris oocytcs. 2. Incubation of j4-"PjPtdIns4P with membranes yields only radioactive innr. ganic phosphate. indicating the presence of a phosphomonoesterase. 3. Membranes hydrolyzc Ptdj2-'Hjlns4l'to producc mainly Ptd[2-'HjIns in the lipid phase. In this incubation I'll1inositol and inositol monophosphate appear in the water phase. 4. Membrane incubations of Ptdl2-'H/Ins4P carried out in the presence of excess non-radioactive lns(1,4)P' allows the trapping of small amounts of 1'If jIns(1,4)P'. These results demonstrate the presence of a phospholipase C. 5. Testing several phosphorylated analogs, it is determined that fructose 1,6-bis- phosphadc and cr-glyccrophosphate are potent inhibitors of the oocyte PtdIns4P phos- phomonocsterase. Jacob, G., Allende. C. C., and Allende, J. F.. Comparative Biochemistry and Physiology IOOB(4):ft09-R16, 1991. Other support: National Fund for Science and Technology (FONDECYT) of Chile, and the Univcrsity of Chile. From the Departamento de Rioqufmia, Facultad de Medicina, and Dcpartamento de Rinlogia. Facultad de'Cicncias, Universidad de Chile. Santiago. DIFFF.RENTIAL STIMt ILATION OF THE GTPasc ACTIVITY OF G-PROTEINS BY 1'OI.YLYSINF. Pnlylytiine, pnlyornithinc and, to a lesser extent, polyarginine were found to aimulate the GTPase activity of the purified recombinant a subunit of the human Gi , tran.ducing Prntcn a, ,. Optimal stimulation of 4- to 5-fold was obtained with pnly%inc concentrations between I and 20 µm, higher concent.rations being inhibit- ory. Polylysine at similar concentrations stimulated by 50% Ihe GTPase of trans- ducin (G,), the visinn Iransducing protein, but had only a very slight effect on the (;TPace of the p21 product of the H-ra.s protooncogene. The stimulation of the a, GTI'ase caused by polylysine was due to a reduction of the apparent K~, for GTP from 3.9 to 1.3 µM. The stimulation by polylysine was observed at free Mg` con- centrations below I tiM. These results indicate that polylysine acts in a fashion simi- lar to mastoparan and substance P in tnimicking the action of an agonist-hound receptorrrn G-proteins. Antonclli, M., Olate, J., Graf, R., Allende, C. C., and Allende, .1. E. Biochemical Pharmacology 44(3):547-55I, 1991. tttlicr tirrPport. FONDECYT-Chile and University of Chile. I;r,~Tl the Departamento de Bioquim(ca, Facultad de Medicina, Departamento ~ Iiru,><nt c I Medicine Baylor College of Medic ne HotustonaTXtgo. Chile, at I~ip. I:OLYLPOLYGLUTAMATE ANALOGS CAN 1NH1B1T CASEIN KINASE 11 FROM XF,NOPUS LAEVIS polyglutamate analogs of folate and related compounds were tested as inhibit, Xeno nf cateroyl n4 amino ~1m thylt pteroyl (the methotrexate aromatictmoiety) ~and Ihc p ;rminobenzoil derivatives increased as the number of y glutamates attache w from 2 to 7. The nature of the aromatic head group was also important since hexa Flutamatic acid had no inhibitory activity while the folylhexaglutamate derivati were strong inhibitors with relative potency of inetho.tyre pxeate > pteroyl > p aminob zoic tive with ca ein and showed an apparent K of r90 uM. ntaglutamate was comp Tellez. R.. Allende, C. C.. and Allendr, J. F,. FEBS 30R(2):I 13-115, August 1992. Other support: International Centre for Genetic Engineering and Biotechnology FONDECYT-Chile. From the Departamento de Bioquimfca. Facultad de Medicina, Departamentr Biologfa, Facultad de Ciencias, llniversidad de Chile, Santiago, Chile. ANALYSIS OF PHOSPHOTYROSINE-CONTAINING PROTEINS PRESENI v-srr-INFF.CTED MYELOID 1'R(XiENITOR CELLS We examined the phosphotyrosine-containing proteins in v-src-infected my cells. Proteins with relative molecular weights (M,) of 180 ((N). 175 000. 135 125 O(Xl, 120 O(N), 9(11N1O, 75 (10), and 60 000 were present in v-src-infected bt in uninfected 32D cl3 cells. Stimulation of 32D c13 cells with interleukin 3( resulted in the tyrosinc phosphorylation of a protein of 1.50 000 (M), which wz phusphorylated in v-src-mfected 32D cl3 cells. A panel of rnonoclonal antib directed against pltosphotyrosme-containing proteins in v-src-transformet25h emhryo fibroblasts (3C4. 2A7. 2B12 and 4F11, directed against p210. p and pR5 respectively) was used to characterize these substrates. We did not ot tyrosine phosphorylation of proteins recognized by these four monoclonal antit in either I1.-3-stimulated or v-src-infected 32D cl3 cells. However, we did - tyrosine phosphorylation of proteins recognized by these tnonoclonal antihod v-srr-transformed NIH3T; cells. Tyrosine phosphorylation of GTPase-acti, 109 1 109
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protein (GAP) and the GAP-associated proteins p62 and pl()(l was observed in v,A,_, infected 32D cl.1 cells. Stimulation of 321) cIl cells with IL; 3 does not induce ph,,,phorylation of GAP or the GAP-associated proteins p62 or p19Q. These results gest that substrates for v-,vr vary between different cell types. Erwin, J. L. and Ander.cnn• S. M. Oncogene 7:1101-1107• 1992. Other support: New York Community Tnist. From the Department of Pathology, State University of New York at Stony Brook. CELL CYCLE-DEPENDENT LOCALIZATION OF CASEIN KINASE I TO MITOTIC SPINDLES Cascin kinase I (CKI) is a class of protein kinases ubiquitous to all eukaryotic cells. Recently, cDNA clones encoding several bovine CKI isoforms have been sequenced that show high sequence identity to this gene product of the budding yeast Sacr•ltarnntvres t'ererisiac; HRR25 is required for normal cellular growth• nuclear segregation. DNA repair, and meiosis. We have raised polyclonal antibodies to a human erythroid 34-kDa CKI and have sequenced a portion of this kinase. The amino acid sequence identifies the CKI as the a-CKI isofonn, which is 62% identi- cal to the HRR25 protein kinase. By use of immunofluorescence, the a-CKI has been kxalized to vesicular cylosolic structures and to the centrosome in interphase cells. As cells progress into mitosis, centrospheric staining increases and, in mitosis, a-CKI associates with kinetoihore fibers. This localization suggests that a-CKI, like HRR25, plays a role in the segregation of chromosomes during mitosis and may be cell cycle-regulated both in humans and in yeast. Rrockman• .t. L., Gross, S. D., Sussman, M. R., and Anderson. R. A. Proceedings of the National Academy of Sciences USA R9:9454-945R, October 1992. Other,;upport: National Institutes of Health. From the I)cpartment of Pharmacology, Cell and Molecular Biology Program, I Inivcr~ity of Wisconsin Medical School, Madison• and Department of Horticulture, College of Agriculturc; and F.ife Sciences, University of Wisconsin. PDGF: A MULTIFZINCTIONAL GROWTH FACTOR hPDGF is the major growth factor of human blood serum. In riro, it is apparently synthcsized by megakaryrticytes and is transported in blood stored in the a granules of platelets. hPI)GF is a hcterodimer of two homologous pnlypeptide chains (PDGF-I(A) and PD(;F-2(B)) linked togehter by disulphide bonds. The PDGF-I(A) 110 is c,rcrxled by a gene localized in chromosome 7 and the PDGF-2(B) chain ia het. h'"n the c-sis proo-oncogene localized in -chromosome 22. . The hPDGF homo tmers, are ncotlcd hy 4.,, dimcr and its two isoforms. the PDGF 1(A) and CrF ens and chemoattractants for tar~etbtc'nls '' ~hce11 at The PDGF II (A ~°~~ohlactsgarterial smooth muscle cells an 1~° ~~Fdi2(R~ homo-dimerobindstocboth recep or pa an tbe InPaDddFionto he remi e ID rl ~ idnand prr tagl ndio synthesi•SnIt cellular otbe an important factor in earl including tcm• hP ars to modulate tissue regeneration and remodellin Jc~•eloPment and in vivo appe eluning wound healing and osteogenesis. The inappropriate expression of PD Fenes and their mitogenic products has been linked to several proliferative disorde .uch as fibrosis, atherosclerosis and neoplasia. Antnniade.c, H. N. Fiailliere's Clinical Endocrinology and Metabolism 5(4):595-613. December 1991. nther support: Nationallnstitutes of Health. From the Department of Nutrition. Harvard School of Public Health, Boston. A BRANCHING PROCESS MODEL OF GENE AMPLIFICATION FOLLOWIt CHROMOSOME BREAKAGE We have devised a mathematical model of gene amplification utilizt experimental observations concerning dihydrofolate reductase (DHFR) g e amp cation in CHO cells. The mathematical model, based on a biological model wF proposes that acentric elements are the initial intermediates in gene amplificat includes the following features: (1) initiation of amplification by chromosomal bn age to produce an acentric structure; (2) replication of acentric DNA, once per cycle; (3) dissociation of replicated acentric DNA; (4) unequal segregation of a- tric DNA fragments to daughter cells at mitosis; (5) subsequent reintegratio acentric fragments into chromosomes. These processes are assumed of unequal setgregat on and i tegration cell at a given time. Thus. P occur in parallel, not necessarily in a unique sequence, and may be reiterated in or multiple cell cycles. These events are described mathematically as a Ga . Watson branching process with denumerable infinity of object types. This m matical model qualitatively and quantitatively reproduces the major elements c dynamical behavior of DHFR genes observed experimentally. The agree between the mathematical model and the experimental data lends credence t biological model proposed by Windle et al. (1991), including the importan chromosome breakage and subsequent gene deletion resulting from resection c broken chromosome ends as initial events in gene amplification. Kimmel, M., Arelrnd, D. E., and Wahl. M. G. Mutation Research 276:225-239. 1992. 111 In N N O O O H H a 0 U
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Other support: Charles and Joanna Busch Memorial Fund and Natiunal Institutes of Health. From the Department of Statistics, Rice University, Houston, TX; Waksman histitine, Rutgers-The State University, Piscataway, NJ; and Gene Expression Lahnratnry, The Salk Inctitmc for Biological Studies. La Jolla. ('A. INHERITANCE AND REGRESSION TOWARD THE MEAN IN HETF.ROrENF,OUS C'EL1. POPI ILATIONS Traits such as birth sim and lifetime can vary widely even among nonmutated progeny of the same cell proliferating in the same environment. On the other hand, population parameters of these traits may remain stable over many generations, and there may be a (listinct inheritance of these traits from mother to daughters. We have reconsidered the implication of mother-daughter correlations in light of linear regres- sion analysis. It is proposed that a non-mutant cell whose phenotype deviates from Ihe population mean produces progeny whose rate of regression toward the mean is prnportional to I -r, where r is the mother-daughter correlation coefficient of the trait under study. Theoretical support for this proposition is derived from linear regres- sion analysis. Empirical support is found in pedigree analysis of cell growth con- stants among N1113T3 mouse fibrohlast cells, where the presence of an activated human ra.c oncogene is associated with a decreased r and an increased rate at which the growth constantti of progeny regress toward the population mean. Gamel. J. W. and A.relrnd. 1). E. Cell Proliferation 24:2R 1-292. 1991. Other support: U.S. Public Health Service, Charles and Johanna Busch Memorial F'vnd, Veterans Administration, Kentucky Lions Eye Foundation, and Research to Prevent Blindness. From the Veterans Administration Hospital and Department of Ophthalmology and Visual Sciences. University of Louisville. KY, and Waksman Institute, Rutgers I tnivcreitv. Piuatawnv, NJ. II)f;NTIFI('ATlON OF A 6(1-KILODAI;fON Rh-BINDING PROTEIN. RbP6(1, TI IAT AI,I,OWS TFlIi Rb-E2F C'OMPLEX'1'O BIND DNA Several reports have indicated that the product of the retinohlastoma gene (Rh) cotnplexes with the transcription factor Fi2F. We present evidence that the DNA- binding of the Rh-E2F complex involveti another cellular factor. Addition of Rh to purified preparations of 1?2F does not generate an Rh-E2F complex that can bind 1)NA, and in fact, we see an inhibition of the DNA-hinding ability of E2F. On the other hand, addition of Rh to cruder preparations of 1's2F results in the formation of ;m Rh-F.2F complex (E2Fr) that can hind DNA and produces a distinct complex in Li.l retardation assays. We have identified and purified a 6(1-kDa protein that allows thc Rh-E2F complex to hind DNA, and we show that this 60-kDa protein exerts its ctfi•ct by directly interacting with Rb. Ray, S. K.. Arroyo, M., Ragrhi, S.. and Raychaudhuri. P. Molecular and Cellular Biology 12(10) 4327-4333, October 1992. Other support: American Cancer Society, Leukemia Research Foundation and U.S. puhlic Health Service. From the Department of Biochemistry. University of Illinois at Chicago. SERUM-FREE MOUSE EMBRYO (SFME) CELLS AND TRANSFORMING GROWTH FACTOR (TGF)(3, Serum-free mouse embryo (SFME) cells died with the addition of transforming growth factor (TGF)(3, in the absence of selenium. This cell death was reversed by vitamin E, indicating a lipid peroxidation mechanism. The cell death was also sup- presssed by either selenium or albumin or high density lipoprotein. The effect of TGF(i, was somewhat different with rac- or rreu-transfected SFME cells. TGF0, lowered the resistance of SFME cells to heat treatment. Masayoshi, l. and Barncs. D. W. In: Murakami, H. et al. (eds.): Animal Cell Technology: Basic & Applied Aspects. Kluwer Academic Publishers, The Netherlands, pp. 515-519, 1992. Other support: U.S. National Institutes of Health, U.S. National Cancer Institute. Ministry of Education, Science and Culture of Japan, and Kumamoto Prefecture. From the Nutrition Biochemistry Laboratory, Kumamoto Women's University. Kumamoto, Japan. and Department of Biochemistry and Biophysics, Oregon State University, Corvallis. PRODUCTION OF GROWTH STIMI ILATING FACTORS FROM ANIMAI. CELLS The work described in this chapter illustrates the progress made in producinf peptide growth factors from animal cells. Even though this work is in its infancy, th( knowledge gained will provide the foundation for further work aimed at developinl new expression systems and modifying existing ones to meet the specific needs fol the production and efficient processing of biologically active growth factors. Man} of the existing recombinant cell lines described in this chapter can produce and cor rectly process recombinant forms of several peptide growth factors thus makinl 112 1 113
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availahle a contiistenl and safe tiource of each growth factor for therapc++t++ research purposes. Further work is necessary to adapt these cell lines for L+rP+ culture as well as to modify Ihe purification sc•hemes presently in use trr cflicit•,.. pnx•ess large volumes of conditioned medirnn. CoIlodi. P., and Rarne.c.1). W. In: Animal Cell Biotechnology Vol. 5. Academic Press Ltd., pp. 247-277, 1992 From the Department of Biochemistry and Biophysics, Environmental Ilc,~l+t Sciences Center. Oregon State Ilniversity, Corvallis. THE GMP RF.DUCTASE GENE OF THE NEMATODE ASCARI,S LUMRRI- C•O1l)l::S VAR. ,S(I(IM Our laboratory has cloned the nematode guanosine monophosphate ((;Mpt reductase gene from an early embryonic cDNA library of the parasitic roundworm Asrari.s (umhrir.rides var. srnim. The GMP reductace gene codes for an enzyme directly involved in the purinc nucleotide salvage pathway, converting GMP to inn. sine monophosphate (IMP). The nematocfe gene has a 60% exact match, both at the nucleotide and the amino acid level, with the only other cloned GMP reductase, from the bacterium /:srherichio rnli. We have shown the RNA of this gene to be most abundant (luring early ernbryogenesis. We are pursuing in sitn techniques, to deter- mine if the GMP reductase RNA accumulates in the somatic cells of the 16-32-cell embryo and not in Ihe single gennline lineage cell. Gnridl, M.. Bunch, K., Gharib, S., and Rennett. K. L. Molecular and Biochemical Parasitology 52:271-274, 1992. Other support: National Institutes of Health, March of Dimes Birth Defects Foundati+,n and a National Science Foundation Career Advancement Award. From the Department of Molecular Microbiology and Immunology, University of Missouri, Columbia. AN EXTREMELY ABUNDANT OVARIAN mRNA FROM THE PARASITIC NF.MATOUE A,S(•ARLS l.(IMRRI('OIDF.,S VAR. SU(1M FIAS MULTIPLE RF.PFA'f MOTIFS The major focus of our laboratory is identification of genes required for gennline determination (luring embryogenesis in nematodcs. To search for germline- specific genes, our laboratory made a cDNA library in the Xgt22 vector using size- selected pnly A + RNA isolated from ovarian tissue. In probing this library with the 1>rn.cr+Phila rasa gene a germline-specific RNA helicase, we isolated a potential full- length cDNA clone that represents 10-15% of the cDNA library (as determined by ++t +t~ „f PnSitive plaques in filter hyhridi7ation experimentt) and therefore ~ t,, Ix nn a+hunJant mRNA in ovarian tiscue. Initial sequence analysis showed I~NA was not ;rn A.srcn•is lronhrirnides raso equivalent. However, Ihe +f ++++~I;+r~cc and the tissue source of the mRNA suggested to us that this gene In• !k r`lcvant to orrrclone the gene of which we have narrred oamr(ov~rian ahun- p ,n r „I Ihc cllNA ~fJ)d'rflcrcnt sels~ h n er repeats which makehup about 45% of thespre- ..~•n,c . rd prn!cin. I`I. Catcr, J., Wilcon, B., Gharib, S., and Bennett, K. L. ~t,d,cular and Biochemical Parasitology 56:177-180. 1992. ,+t+cr ~++Pp°rt: National Institutes of Health, March of Dimes Birth Defects ! „undation and National Science Foundation. Microbiology and Immunology, School of ~( Jirinc. ilnnrverrity of Missouri, Columbia. \( TERNATIVELY SPLICED LTK MRNA IN NEURONS PREDICTS A RECEP- ft)R WITH A LARGER PUTATIVE EXTRACELLULAR DOMAIN kinases Ltk is a new member of hl~ r1e/tnar or~ and fo ebrain neurronsCWe p eviously cxpresscd in murine B-lymp y P ec ref,K,rted that lymphoid !tk cDNAs predict a 69 kDa transmembrane glycopmtein. uhich uses a CUG translational start codon and has a 110 amino acid putative extra- ccllular domain. We now show that the predominant Irk mRNA in brain is alterna• tively spliced and predicts a protein with a substantially larger extracellular part. The human Irk gene maps to chromosome 15, bands q13-21, a region containing thc breakpoint of a recurring chromosomal abnormality in B-cell non-Hodgkin lym phomas. Haase, V. H., Snijders, A. J., Cooke, S. M., Teng, M. N., Kaul, D., Le Beau, N. M. Rnms, G. A. P., and Rer•nards, A. Oncogene 6:2319-2325, 1991. Other support: Whitaker Health Sciences Fund and U.S. Public Health Service. From the Molecular Genetic- Laboratory,.Cancer Center of Massachusetts Genen Hospital. Harvard Medical School. Boston; Section of Hematology/Oncolog: University of Chicago: and Genetics Division, Children's Hospital and Departme+ of Pediatrics, Harvard Medical School. SEQUF,NCF. OF A cDNA CLONE ENCODING A RAT REG-2 PROTEIN We report here the nucleotide sequ`~f en f~e encod d prote'n d cignatedrRc homologous to the Reg (regenerating) p 114 1 115
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2, shows 6(N%•, 7R''/,, and 61% similarities with Ihe reported amino acid sequcnce'~ „I the r,u, bovine and hmnan protein%, respectivelv. Karnimura,'1'., Wcsl. C., and Rrullrr, E. Gene I 1 R:299-1(N), 1992 . From the 1)epartment of Molecular and Experimental Medicine. Scripps Research Institute, La .lolla, CA. CYTt)KINF, PRODIiCTION FROM FRESHLY HARVESTED HUMAN MONONUCI.EAR C F;LLS ATT'ACHI:I) TO PLASTIC BEADS The release of tumor necrosis factor (TNF), interleukin-I/3 (IL-1) and granulo- cyte-macrnphage colony-stimulating factor (GM-CSF) from freshly harvested mono- cytes and lymphocytes attached to plastic heads was investigated. Previous studies had shown that freshly harvested endothelial cells attached to microcarrier beads release an endothelium-derived relaxing factor. Attachment of freshly harvested lymphocytes and nionocytes to plastic heads created a dense network, consisting of 25% monocytes and 75% lymphocytes as shown by flow cytometry. Viability of cells was 90%. Monocytes were characterized by phagocytosis and non-specific esterase stain. Freshly harvested cells stimulated with Iipoprotcin lipase (LPS) relcased TNF and ll.-I, Non-st'imulated cells also produced GM-CSF five hours after collection of hlood. Rin,r;. R-.1., Dudek, R., Kahler, J.. Narayan, K. S., and Ingram, M. Tissue and Cell 24(2):203-2(l9, 1992. Other suppnrt: Margaret W. and 1-lerbert Hoover. Jr.. Foundation, Miles Laboratories and the Polish-American Congress. From the Department of Experimental Cardiology. Huntington Medical Research lnstitute,~, Patadena, CA. TYPE-SPF,('IF1C ANTIBODIES TO THE PLATELET-DERIVED GROWTH FACTOR RECEPTORS: ROLE IN ELUCIDATING THE STRUCTURAL AND FUNC'TI(7NAL CHARACTERISTICS OF RECEPTOR TYPES Two types of platelet-derived growth factor receptors have been cloned and sequenced. Both are glycoprotcins with similar molecular weights. We have earlier established the ligand binding specificity, ligand-induced dimerization, and kinase activation of these Iwo receptor types jBishayee et al. (1989) J. Binl. Chem. 264, 1161)9-1J7f15: Kanakaraj et al. (1991) Rinrhemi.rtrv 30. 1761-17671. In the present studic%, we have investigated the biosynthesis, processing, and glycosylation of the n-receptor and compared its structural and functional characteristics to those of the I) reccPtnr. Unlike an anti-peptide antibody, AbP (amino acid residues 964-979). tc tl , hiin,;,n peceptor which detects a phosphoryiation-specific conformation of thc t',cPtor. an antibody. AbP j(amino acid residues 956-971), to the correspondinf ~cFi„n (,f the human a-receptor. failed to do so. However, our studies revealed tha tl1c ctahiHty of the a-receptor is comparable to that of the-(3-recept or. In addition, N- linked glyc°sylation of the a-receptor, like that of the (3-receptor, is not important it ki„a~e activation. We have exploited the lack of an effect of N-linked oligosaccha ridet on the functioning of the a-receptor to develop a simple and rapid method fo drcect demonstration of ligand-induced noncovalently linked a-(3 receptor heter ndimer formation. This method is based on the~nteraction between functionall! ;Ictive short and the long forms of two receptor t c which can be resolved by dena tnring gel electrophoresis. Raj• S.• Kanakaraj. P., Khan, S. A., and Bishavee, S. Rinchemistry 31:1774-1779, 1992. t7ther support: National Institutes of Health and W. W. Smith Charitable Trust. From the Coriell Institute for Medical Research, Camden, NJ, and the Wist'- Institute, Philadelphia. EPITHELIAL-SPECIFIC GENE EXPRESSION DURING DIFFERENTIATION OF STRATIFIED PRIMARY HUMAN KERATINOCYTE CULTURES Cultured epithelial cells are used to generate extensive patches of autologo skin equivalent for patients with burns or wounds and to investigate the growth at differentiation of epithelia in vitro. We have undertaken a comprehensive study the morphological and molecular events that occur during culturing of human fot skin keralinncytes at the liquid-air interface on a dermal equivalent consisting of collagen matrix containing fibroblasts. Using radioactively labeled RNA probes I mRNAs and monoclonal antibodies for proteins, we found that the expression ol comprehensive set of differentiation stage-specific genes was affected by the type fihroblasts included in the matrix as well as by the age of the culture. The expressi . of these genes was not always coordinated and could not he predicted from the his- logical appearance of the stratified epithelium. Surprisingly, the mouse fibrobla promoted epithclial differentiation much more closely resembling foreskin than < the homologous primary'foreskin fibroblasts. Wilson. J. L., Dollard. S. C., Chow, L. T., Rroker, T. R. Cell Growth & Differentiation 3:471-4R3, August 1992. Other eupport: U.S. Public Health Service. From the Department of Biochemistry, University of Rochester School of Medic and 1)cntistry. Roxhester, NY. 116 1 117
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VIRAL El ANI) E2 PROTEINS SUPPOR'1' REPLICATION OF HOMOLOGOUS AND HETEROLOGOUS PAPII,LOMAVIRAL ORIGINS We have shown Ihat F;I and E2 proteins of human papillomavirus type I I (IIPV-I I) were essential to support the replication of the homologous viral origin (nri) in a transient rcplication assay, similar to reports on bovine papillomavinis type 1(BPV-I). Unexpectedly, matched or even mixed combinations nf EI and E2 pro- teins from HPV- I I or BPV-I replicated either nri in human, monkey, and rodent cell lines of epithelial or fibroblastic lineage, albeit with varied efficiencies. Either set of viral proteins was also able to initiate replication of nri-containing plasmids from many other human and animal papillomaviruses. Thus, the interactions among the cis elements and trans factors of papillomaviruses are more conserved than expected from the other members of the papovavirus family, simian virus 40 and poly- ornavirus, for which large tumor antigen does not replicate a helerologous nri in either permissive or nnnpennissive cells. We infer that the stringent species and tis- sue specificities observed for papillomavinises in rirn are nnt entirely due to direct restrictirnie on viral DNA replication. Rather, transcriptional control of viral gene expression must play a dominant role. Chiang, C. M., Ustav, M., Stenlund, A.. Ho. T. F., Brnker, T. R., and Chow. L. T. Proweedings of the National Academy of Sciences USA R9:5799-5AO3, July 1992. Other support: 11.S. Public Health Service. From the Department of Biochemistry, University of Rochester School of Medicine and Dentistry. Rochestcr, NY, and Cold Spring Ilarhor Laboratory. Cold Spring Harbor. NY. TEMPI?RATI IRE-SF,NSITIVE SYNTHF-SIS OF A METALLOPROTEINASE IN "1'S I I0-MSV-M-"1'RANSFORMED NRK C'ELI,S Prcviously, we reported that transformation associated protein (TAP) was over- expresscd in the 6m2 line, hut not in their normal counterparts. 6m2 is a culture of NRK cclle Ir,insformcd by the ts-I 10 mutant of MSV-M. The synthesis of TAP and the cxhression nf trnnsfomiation properties in the 6m2 cells arc all temperature-sen- sitive. TAI' is secreted as two pnlypeptides of 64 kD and 68 kD (P64 and P6R). f:xperimgnt, were carried out to determine whether any metalloprotcinase (MP) activity w;ie a~snciated with TAP. Results of zymograms indicated that the two forms of piirified TAP (P64 and P6R) had MP activity, using gelatin or collagen type IV as suh,~trates. Scrum-free medium (SFM) of 61112 cells incubated at 33°C also showed two bands of MP activity, while the corresponding SFM from 6m2 cells at 39"C lacked such MP activity, indicating that the synthesis of MP was temperature- sensitive. The association of MP activity with the P(A and P6R hands of TAP (puri- fied or in SFM) was confirnud by simultancnux Western blot analysis, which showed the reactivity of the two MP hands with monnclonal or polyclonal antibodies tn TAP. Accnrdingly, what we previously designated as TAP is apparently one form of MI'. which arc known to hc involved in tumor cell metastasis. ('hun,.l. ('., Scanlnn, M.. Zhang. H. 7.., and Murray Ill. J. 1,. Biochemical and Biophysical Research Communications 17R(2):453-459, July 31. Iqql. OIhcr support: National Cancer Institule. From the University of Texas M.D. Anderson Cancer Center; Houston. MOLECULAR CLONING AND CHARACTERIZATION OF v-mns- ACTIVATi:D TRANSFORMATION-ASSOCIATED PROTEINS Using monoclonal antibodies, we previously detected two forms of transforma- tion-associated proteins, a 64-kDa protein and a 68-kDa protein, in temperature-sen- sitive I 10-Moloney murinc sarcoma vims-mutant-transformed rat kidney 6m2 cells. The identity and functions of the transformation-associated proteins were previously unknown. By molecular cloning techniques and immunoscreening, we have isolated two cDNA clones (34A and 79B3) that were found by Western blot analysis to code for a monoclonal anti-transformation-associated protein antibody-reactive polypep- tide of approximately 59 kDa. Limited restriction enzyme mapping indicated 34A and 79B3 arc two different cDNA clones. The nucleotide sequence of 34A cDNA was determined, and a search of GenBank revealed that it is identical to that of rat transin-2. The deduced amino acid sequence of 34A shares 71 % sequence identity with rat transin and 41-76% identity with six human metalloproteinases. The limited restriction enzyme mapping and partial nucleotide sequencing data indicated that 79B3 may be the rat transin gene. When either 34A cDNA or 79B3 cDNA was used as a probe in Northern blot analysis, one mRNA band of approximately 1.9 kilobases was detected in 6m2 cells grown at the permissive temperature of 33"C, at which the cells exhibited transformation properties, and a much lower level in 6m2 cells grown at the nonpermissive temperature of 39°C, atwhich the cells reverted to normal phe- notypes. These results suggest that at 39"C, these two genes were not. transcribed at the same level as at 33"C. "Lymogram and Western blot analysis of 6m2 cells further confirmed that the 64- and 6R-kDa proteins have metal-loproteinase activities and that the synthesis of mctal-Ioprnteinases was also temperature-sensitive. Apparently, the Iwn proteins we formerly designated transformation-associated proteins are members of the rat transin gene family. Therefore, within v-mo.c transformed 6m2 cells, the absence of transformatinn-associated protein (metalloproteinase) synthesis at the nonpermissive temperature was due to the absence of transcription of two rat transin gencs. C'han.,,L C'.. Scanlon, M., "1.hang, H.-Z., Jia, L.-B., Yu. D., Hung, M.-C., French. M., and Eastman. E. M. The Journal of Biological Chemistry 267(2):1Q99-1103, January 15, 1992. Other support: University Cancer Foundation Grant from University of Texas M.D. Anderson Cancer Center. From the Department of Tumor Biningy, University of Texas M.D. Anderson Cancer C'enter, Hnuston, and I,ark Sequencing Technoingies, Inc.. Houston. i
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R(1LE OF ENZYME RIX'F.PTORS ANI) INIIIRIT(lltS IN RE(;ULATINC PR(YTIiOLYT1C A('TIVITIES OF MA('ROPIIAGfS Expression of protcascs by neutrophils and rrther cells with a prominent reFr,_ latcd secretory pathway is determined largely hy stimulus-response secretion of ~rt' teins prepackaged in high concentration. The regulated secretory pathway is aprar. ently minor in macrophages, and instead proteases arc either channeled intn lysosomes or secreted constitutively. Posttranslational regulation of macrophagc protcases then depends on compartmentalizing enzymes to their sites of prirnary function. Available data suggest that cells,use both specific receptors and inhihitoN, to accomplish this. Viewed in this context protease inhibitors primarily function t(' inhibit enzyme not bound td their receptor. Consonant with this model of regulation connective tis,;ue turnover by macrophages is a contact-dependent process relatively resistant to exogenous macromolecular inhibitors. Although limited information iN available regarding determinants that modulate matrix metabolism by human macrophages, this model suggests that detemtinants of adhesion and cohocalization of croyntc and substrate would be as or more important than alterations of inhibitors in the micmmrivironrnent of thc cell. ('hapman. f/. A.. Jr. Annals of the New York Academy of Sciences 624:R7-96, May 22, 1991. Other support: American Lung Association. From the Respiratory Division. Brigham and Womcn's Hospital, and Harvard Medical School. 13oston. PARAQIIATTOXI('ITY IN PI,S(1M SA17V11M: EFFECTS ON SOLI/BLE AND MF.MItRANti-13l)t1ND PROTEINS Thc cffects of paraquat (PQ) on Pisum .ranirum L. proteins were investigated in rirn in a new experimental system utilizing IO-day-old plant cuts. A marked dccreaw in the specific activity of rnemhranc-hound Ca'•-dependent ATPase was rccnrded. while that of Mg',-dependent AT'Pasc remained unchanged. Concurrently wilh a(Imp in the total plant protein, the specific activities of the three cytoplasmic cnivmcs, malate dchydrogenase, hydroxypyntvate reductase and triose-phosphate isomcrasc, were also found to decrease. The effect on various enzymes involved in cellular clcfcnse mechanisms was also studied: glutathictne reductase and superoxide dismutatic at'tivitics incrcased, while ascorhate pcroxidasc was not affected. These findinge shed light on the selectivity of PQ-induccd iqjurious processes. focusing on protein homeostasis mechanisms in the membrane and cytoplasmic compartments at the cellular level as well as on the prominent role played by eroy- matic defense systcmti against PQ poisoning. Pclcg. I., l.cr, I1., attd ('lu•rinn, lVf. Physiologia Plantarum Rfi:131-1a5, 1992. ()tlrtr ;npport: (icscllschafl fiir Strahlen- und Umweltforschung, Neuhcrbe (icrn,:my. Lr„rr~ the Department of Cellular Biochemistry. Hebrew University-Hadas! ntcdical School, Jcrusalcm, Israel. pR(1TECTION AGAINST FREE RADICAL, INDUCED AND TRANSITION MPA-A AND "PUSH" L-MI:DIATED DAMAGE: TIiE USE OF "PULL" htFC1IANiSMS Free radicals have been incriminated in a variety of injurious processes incl ing the toxicity of the herbicide paraquat and the damage following ischemia rcperfusion of different organs. Based on the assumption that iron and copper could serve as mediators for transformation of relatively low reactive species (such as superoxide radicals, hyt gen peroxide, ascorhate, and others) to the highly reactive species,in the site-c cific metal-mediated mechanism, two new modes for intervention have been t out. The first is the introduction of specific chelators that "pull" out redox-active available metals, and by this reduce the apparent damage. Desferrioxamine shown to protect bacterial cells and mammals against the poisonous effect paraquat. Using the retrogradly perfused isolated rat heart, we have demonsir that the chelator neocuproine, which effectively binds both iron and copper prov a major protection against hydrogen peroxide-induced cardiac damage and ag: ischemia/reperfusion-induced arrhythrmas. Likewise. TPEN a heavy metal chel provides almost total (>90%) protection against ischemia/reperfusion-indt arrhythmias. The other mode of intervention is the use of redox-inactive metal ions that c compete for the binding sites of iron and copper, and by this "push" these metal out, lead to their displacement. and divert the site of free radical attack. Appl Zn(II) complexes provided a marked protection against metal mediated free rad induced damage in the copper-mediated paraquat toxicity to E. coli, and it arrhythmias induced by ischemia and reperfusion. It is proposed that the complex zinc-desferrioxamine would be the ultimate tector being effective by both the "pull" and "push" mechanisms. Cheriom, M. Free Radical Research Communications 12-13:691-696, 1991. Other support: Gescllschaft fur Strahlen- und Umweltforschung, Neuher' Gennany, and U.S. National Institutes of Health. From the Department of Cellular Biochemistry. Hebrew University-Had: Medical School, Jerusalem. Israel. RI.E()MYCIN-DETF,C'TABLE IRON IN BRAIN TISSUE The normal brain contains regions with high concentrations of iron, p which appears to he in a low molecular mass chelatable form. Iron complexes - 120 1 121
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1 niolecular masti of helow I(l,O(1f), were measured in ultrafiltrates of homogcili;,e,l gerbil hrains using the hlcomycin atisay, and were found to average 20.5 4 1.5 f,.nl (n = R). As cxpcctcd, no hlcnmycin dctcctahlc iron was found in thc plasma of thcsc animalt. No ohvious difference in the fissue levels of hleomycin-detectahle iron was recorded following ischacmia and reperfusion. This is probably due to the already abundant presence ol iron in the brain and the likely release of iron from protected sites due to structural damagc inherent in the preparative procedures used. C7uttcridgc, J. M. C., Cao, W„ and (7icrian, M. Free Radical Research Commtmications I I(6):317-.120, 1991, Other support: National Institutes of Health. From Molecular Toxicology Research Group, Oklahoma Medical Research Foundation, Oklahoma City, and Department of Cellular Biochemistry. Hebrew l Iniversity-lladassah Medical Schnol, Jerusalem, Israel. ('RYSTAI.I,I7.ATION ACTIVITY ASSAY AND PRFLIMINARY X-RAY DIITRA('TION ANALYSIS OF THE IINC'LI;AVFD FORM OFTHE SERPIN ANTI('I IYMOTRYI'SIN Crystals nf recombinant wild-Iype antichymotrypsin have been prepared by the methnd of vapor diffusion with polyethylene glycol 4(N)(1 as a precipitant at pH 5.7. Two crystal forms are ohccrved. One form belongs to tetragonal space group P 4 2 2 (or P4 2 2) and has unit cell dimensions a = h= 126 R, e~ = 243 /~, with two moie- cules in the asymmetric unit. The other crystal form belongs to orthorhombic space group P2,2,2, and has unit cell parameters of a= 73 A, h= 78 A and r= 80 A, with one molecule in the asymmetric unit. Diffraction intensity measurements have been made nn the Ietragonal crystal form to a limiting resolution of 4-1 A, and reflections have hcen oh%erved on X-ray sfill photographs to a limiting resolution of 2•5 A for the orthorhomhic form. An activity assay of redissolved letragonal form crystals indicates that fhe uncleaved, functional serpin has been crystallized. Wei, A., Ruhin, I I., Coopemian, B. S.. Schechter. N., and ('lu•i,cria,r.cnn, /), W. I"mmil nf Mnlecular Biology 226:273-276, 1992. Other ~uppnrl: National Science Foundation. H and Q l.ife Sciences and National Intil itute~ of Hcalth. From the I)cpartments of Chemistry. Medicine and Dermatology. University of Pcnmylvania, Philadclphia. GRADIF.NTS OF HOM1?OI'ROTEINS IN DEVF,LOPING FEATHER BUDS Homcoprntcins arc functionally involved in pattern formation. Recently, homeo- proteins have been shown to hc distributed in a graded fashion in developing limb buds. Hcrc we examine the expression of homeoprotcins in chicken feather develop- 122 „icnt by immunocytochemical localization. We find that XlHbox I antigen is pres, in cell nuclei and is distributed in a gradient in the mesoderm of developing featl hn(ls with strongest expression in the anterior-proximal region. The gradient is m „hvious in feather buds from the mid-trunk level. Feather buds from the scapc level express very high levels of XlHbox I and feather buds from the cau region express no XlHbox 1, suggesting that a broad gradient along the body axi: .uperimposed on a smaller gradient within each individual feather bud. Feat cr.tndcrm also expresses XlHbox I antigen but without an obvious graded pattc Another hnmeoprotcin, Hox 5.2, is also expressed in developing feather buds i graded way, and its distribution pattern is partially complementary to that of XIHI i. These observations suggest that homeoproteins may be involved in setting up antcrnpnstcrior polarity of cell fields at different levels, first for the body axis, t! (nr the limb axis and finally for the feather axis. ('hpnn,q, C.-M.. Oliver. G., Ting. S. A., Jegalian. B. G., Chen, H. M., and Rnhertis, E. M. Development 110:1021-1030, 1990. Other support: National Institutes of Health and American Cancer Society. From the Department of Pathology. University of Southern California Schoo Medicine, Los Angeles, and Department of Biological Chemistry, Universit, California School of Medicine, Los Angeles. MECHANISM OF SKIN MORPHOGENESIS. I. ANALYSES WITH ANTIBODIES TO ADHESION MOLECULES TENASCIN, N-CAM, AND INTEGRIN To understand cell interactions during induction of skin appendages, we stu the roles of adhesion molecules N-CAM, tenascin, integrin, and fibronectin du feather development. Tcnascin appeared in a periodic pattern on.epithelia and ws far the earliest molecule detected in placodes. Three placode domains were id fied: the anterior was positive for tenascin, the distal positive for N-CAM, an< posterior lacking both. Integrin appeared in dermalepidermal junctions of plac( In feather huds, sagittal sections revealed a transient anterior-posterior asymn with tenascirt and N-CAM enriched in the anterior mesoderm. Tangential sec . revealed a lateral-medial asymmetry with tenascin distributed in a ring shape an CAM in an "X" shape. Integrin was diffusely distributed within buds. Later ten: and N-CAM were enriched in dermal papilla, the inducer of skin append: Perturbation of embryonic skin explant cultures with antibodies showed that integrin (il and anti-fibronectin blocked epithelial-mesenchymal interaction, an CAM caused uneven segregation of inesenchymal condensation, and anti-ten inhihited feather bud elongation. Dose-response curves showed gradual effec these antibodies. The results indicated that these adhesion molecules are ind( dently regulated and each contributes in different phases during morphogene5 skin appendages. .liang. T.-X and Clwon,t. ('. M. 123
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1)cvelopmcntal Biology 150:R2-9R, 1992. Other support: National Institutec of Hcalth. From the Department of Patholrtgy, University of Southern California School ~,f Medicine, Los Angcl". SPIN TRAPPING EVI[)ENCE FOR MYELOPEROXIDASE-DEPENDENT HYDROXYL RADICAL FORMATION BY HUMAN NEUTROPHILS AND MONOCYTES llsing the electron spin resonance/spin trapping system. 4-pyridyl I-oxide N-rerr-hutylnilrone (4-PBOBN)/ethanol, hydroxyl radical was detected as the rx-hydroxycthyl spin trapped adduct of 4-POBN, 4-POBN-CH(CH)OH, from phorhol 12-myristate 13-acclate-stimulatcd human neutrophils and monocytes without Ihe addition of supplemental iron. 4-POBN-CH(CH,)OH was stable in the presence of a neutrophil-derived superoxide flux. Hydroxyl radical formation was inhibited by trcatmcnt with superoxide dismutase, catalase, and azide. Treatment with a series of transition metal chelators (lid not appreciably alter 4-POBN- ('ll(('H)OH• which suggested that hydroxyl radical generation was mediated by a mechanism independent of the transition metal-cataly7ed Haber-Weiss reaction. Kinctic differences between transition metal-dependent and -independent mechanisms of hydroxyl radical generation by stimulated neutrophils were demonstrated by a greater rate of 4-POBN-CH(CH ,)-O1-t accumulation in the presence of supplemental iron. Detection of hydroxyl radical from stimulated monocyto-derived macrophages, which lack mycloperoxidase, required the addition of supplemental iron. The addition of purified mycloperoxidase to an enzymatic tiulxroxide generating system resulted in the detection of hydroxyl radical thal was dependent upon the presence of chloride and was inhibited by superoxide dismutase, calalase• and a7ide. These findings implicated the reaction of hypochlorous acid and superoxide In produce hydroxyl radical. 4-PORN-C'H(CH)OH was not observed npon %timnlatir,n of mycloperoxidase-deficient neutrophils, whereas addition of myclnhcrnridasc to thc reaclion mixture resulted in Ihe detection of hydroxyl radiril. 'I'hew resultc cupport the ability of human neutrnphils and tnonocyles to gcnriatc hvdrnryl radical through a mycloperoxidasc-dependcnt ntechanism- Ranint, ('. I.., Pou, S.,, Britigan, B. E., C'nlrcrt, M. S., and Ruserr, G. A4. The Journal of Biological ('hemistry 2(,7(12):R307-R312, April 25,1992. Olhcr support: National Institutes of Health. Veterane Administration Research Service and National Science Foundation. From the Department of Pharmacology and Toxicology, University of Maryland School of f'harniacy, Baltimore: Research Service and [)cpartnient of Internal Medicine• Veterans Administration Medical ('enter, Iowa City. IA; Department of Internal Medicine, University of Iriwa College of Medicine, Iowa City: Departments of Medicine, Microbiology and Immunology. Flniversity of North ('arolina, Chapel Hill; and Vcterams Administration Medical ('en(cr, Baltimore. III I, ~ 1O{'YTE NUCLEAR FACTOR 3(3 CONTAINS TWO IK~N~( RlPTIONAL ACTIVATION DOMAINS, ONF,OF WH1CH IS NOV[,l. `~I) (.ONSf;RVI;D WITH THE I)RO,Sl)PHILA FORK HEAD PROTEIN ) hc hepatcxyte nuclear factor 3(HNF-3) gene family is composed of three prc c~cral liver genes. AII three proteins share strong hom logy in thei eDNAehinc ilnmains (region I) and are able to recognize the same DNA sequence. They als ail 1"" c~nd anfnurthrsegment of1homology atttthe am no te minus(( ctg onllV III) / I-nrthermoce tf rk h ad in regions 1, 11,~ nd illc oggesting that HNF-3 mayrheri hnncotic g „i;immalian homolog. In order to define HNF-3/3 protein domains involved in tra ,criptinnal activation, we have used a reporter gene, whose transcription is depe dcnt on 1{f•)F-3 hinding, for hepatoma cell cotransfection assays with expression ve p,rs that produced different truncated HNF-3(3 proteins. A position-independent ac vation domain which contained conserved regions Il and lll was identified at the c: Ix,xyl terminus of the HNF-3(3 protein (amino acids 361 to 458). Moreover, sit directed mutations that altered the sequences within regions Il and IIl demonstrat their importance to transactivation. The region II-111 domain does not possess ami acid sequences in common with other transcription factors and may define a no, activation motif. HNF-3(3 amino-terminal sequences defined by conserved region also contributed to transactivation, but region IV activity required the participati nf the region 11-Ilt domain. Region IV is abundant in serine amino acids and c( tains two putative casein kinase I phosphorylation sites, a feature similar to protl motifs described for the transcription factors Ptt-I/GHF-I and HNF-I. Pani, L., Overdier, D. G., Porcella, A., Qian, X., Lai, E•, and Costa. R. H. Molecular and Cellular Biology 12(9):3723-3732, September 1992. Other support: National Institute of General Medical Sciences and American Can Society. From the Department of Biochemistry, University of Illinois College of Medic• ('hicago, and Division of Endocrinology and Program of Cell Biology and Genet Memorial Sloan-Kettering Cancer Center, New York- EVALIIATION OF'1'FIFi ANNEXINS AS POTENTIAL MEDIATORS OF MI;MBRANE FFISION IN F,XOC'YTOSIS Membrane fusion is a central event in the process of exocytosis. It oc between secretory vcsicle membranes and the plasma membrane and also air secretory vesicle membranes themselves during compound exocytosis. In many the fusion event is regulated by calcium. Since the relevant membranes do not ur go fusion in vitro when highly purified, much attention has been paid to lxrs protein mediators of these calcium-dependent fusion events. The annexins com a group of calcium-dcpcndent memhrane-aggregating proteins, of which synex the prototype, which can initiate contacts between secretory vesicle tnemhr 124 1 125
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which will then fu%c if' the memhranee are /Lrther perWrbed hy the ;uhlitini, exogenous free fatty acids. This review discusses the ucrctnry pathway an4 Ihc,, dcncc obtained front in rinn titudics thal suggcstti Ihc anncxins may he mcdiatr,r, rckulators ofmcmhranc ' futiion in cxocytcrcis. 7.aks, W. J. and ('rrrrr.. ('. /:. Journal of Rinencrgc•tics and Riomcmbrancs 22(2):97-12(l, 1990. Other support: National Institutcs of Hcalth, National Science Foundation anrl American Hcart Association. From the Department of Pharmacology and Molecular Biology Institute, Univenit, of VirFinia• ('harlrittesvillc. CALCI(fM-DF,PF:NDF,NT MEMRRANE-BINDING PROTEINS AS POTENTIAL MEDIATORS OF STIMULUS-SE('RETION COUPLING In this chapter we will outline the development of model systems that have been particularly succcesful at recreating calcium-dependent membrane contact and fusion steps in ria•n_ In addition we will dcscribc a number of proteins that interact with c•hrom;dfin granulcs in Ihe presencc of calcium. Sontc of these proteins have been used in the modcl systcros we will describe and others are likely to become elements of new model systems. Hopcfully, some or all of these proteins underlie events occurring in exocytosis. Our discussion will extend to recent observations we have made in thc yeast system. C'reur., C. F. , Drust. 1). S., Hamman, H. C.. Junker, M., Kambnuris, N. G., Klein, J. R.. Ncl.eon, M. R., and Snydcr. S. L. In: Snrith, V, L. and Dcdman, J. R., (cds): Stimulus Response Coupling: The Role of lntraccllular ('afcium-binding Proteins. CRC Press, Boca Raton. FL, pp. 279-310, 1990, Othcr su(,pnrt: National Inttitutes of Health, National Science Foundation, Office of Naval Rctir,irc•h, and American Hcart Assoriation. Fmm Ihc Dcpartmcnt of Pharmacology and Molecular Biology Institutc, University r,f Virpinia, (•harhoitcsville. IMM(INOLOCALI7,ATfON OF A GLYCOSYLPHOSPHATIDYLINOSITOL- SPI?CIFICPHOSPf10LIPASB D IN MAST CELLS FOUND IN NORMAL TISSUE AND NEUROFIRROMATOSIS LESIONS A large number of eukaryotic proteins have been shown to be anchored to the cell membrane hy glycosylphnsphatidylinositol (GPI). This glycolipid anchor can ~„bstrate for anchor-specfic phospholipases that convert the GPI-anchored I,roteins into soluble fotTrts. Soluhle forms of many GPI anchored pro- hr,rnc h,r~c been icfentified in rirn in connective tissue, plasma, and urine. ' e l r It;rve discovered that mammalian plasma contains a GPI-specific phospholi- I~ (t.pl_pLD). Because it recognizes a portion of the conserved glycan core turc. ,rll GPl-anchorcd proteins are potential substrates. The authors report the `I I rclcnt of a murine monoclonal antibody specific for one form of the human I,I hI 1) ;md the immunohistochemical localization of this enzyme to mast cells. `I Ir C. N., Thomas, P., and Darifz, M. A. `,,,crican journal of Pathology 140(6):1275-1281, June 1992. t: Lucille P. Markey Charitable Trust and a Whitehead Presidential Qrhpor /tihcr I rll(,Wship. Division of Immunology, Denartment of Pathology, New York Universit) W(lical Center, New York. lIORMONE-REGCILATED v-re! ESTROGEN RECEPTOR FUSION PROTEIN: RF,VERSIBLE INDUCTION OF CELL TRANSFORMATION AND CELLULAR GF.NE EXPRESSION We describe the construction of a v-re/ estrogen receptor fusion protein (' rcfER) which allows the regulation of v-re! oncoprotein activity by hormone. In tI presence of estrogen, v-relER readily transformed primary chicken fibroblasts at hone marrow cells in vitro. In both cell types, v-rel-specific transformation was cri cally dependent on the presence of estrogen or the estrogen agonist 4-hydroxytamo ifen (OHT). Withdrawal of estrogen or application of an estrogen antagoni IC'1164,3R4 (ICI) caused a reversal of the transformed phenotype. We also demc strate that the r-relER protein binds to NF-KB sites in an estrogen-dependent m: ner, thereby showing that sequence-specific DNA binding of v-re/ER is critical I the activation of its transforming capacity. In transient transfection experiments, failed to demonstrate a clear repressor or activator function of the v-re! moiety in reIF,R. However, in v-relER-transforrned hone marrow cells, estrogen and 01 induced elevated mRNA levels of two cellular genes whose expression is consti tive and high in v-re/-transformed cells. These results suggest that v-r•el might e) part of its activity as an activator of v-rel-responsive genes. Boehmelt, G., Walkcr, A., Kabrun, N., Mellitzer. G., Beug, H., Zenke, M., ; F.rtriclln, P..l. The EMBO Journal I I(12):4641-4652, 1992. Other support: National Cancer Institute. From the Institute of Molecular Pathology. Vienna. Austria, and Departmen Microhiology, State University of New York, Stony Brook. 126 127
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INIIIIIITION OF DNA SYNTFIESIS IN LIVIN(7 ('FI.IS BY MICROIN.IF,C`T1ON OF G,, ANTIRODIIiS Hctcrotrimcric guanine nuclcotidc binding prrncins function in the coupling trf;t divcrse span of ccll surface receptors to a variety of intraccllular signaling path ways, some of which stimulate cellular proliferation. With the recent cliscovery Ihal mutated forms of G proteins are present in specific tumors, there has been ap increased interest in the determination of The role of specific suMypes of G Proteim in the regulation ot ccllular growth. We have attempted to determine which subtypex of G proteins arc directly involved in scrum-stimulatcd DNA synthesis through nricrniqjcctiun of inhibitory antibodies into living cells. Inhibitory rabbit polyclonal antiFxldics directed against specific G n subunits were introduced into living Balh/c 3T3 fibrohlasts by microinjection, and the effect upon scrum-stimulatc(1 DNA syn- thcsis was examined. Results of these experiments indicate that G, plays a direct role in scrum-stinmlatccl DNA synthesis in living cells and suggest that Ci proteins may function in a variety of mitogenic signaling pathways initiated hy serum growth factors. LaMorte, V. J, GoIdsrnith, 1'. K„ Spiegcl, A. M., Mcinkoth, J. L., and Fer(lmi.ccn, .LR. The .loumal of Biological Chemistry 267(2):691-694, 1992. Other support: National Cancer Institute. From the Oepartment of Pharmacology. F)epartment of Medicine, Cancer Center, llniversity of ('alifornia at San Diego. La Jolla, and the Molecular Pathophysiology Branch, National Institute of Diahetes and Digestive and Kidney Diseases, Bethesda, Ml). THI; . 1ND11('TION OF Egr-I EXPRESSION BY v-Fps IS VIA A PROTEIN KINASI? C'-INI)fil'EN1)ENT INTRACELLULAR SIGNAL THAT IS SF.QUI?NI'IAI.I,Y DEPFNI)F.NT UPON HaRas AND Raf-I Activating the protcin-tyrosinc kinase activity of v-Fps leads to The rapid Irans- criptiOmal activation of the l:;;r-I gene, which encodes a mitogen-responsive trans- criptinn fackor. Activalinn of Fgr-t by v-Fps was insensitive Io protein kinase C dchlctiom. muggcsting that a protein kinase ('-independent signal activatccl by v-Fps Ic,uL• n) tlhc induction of F.gr-I. Expression of vFps in transient expression assays in(htccd Iigr-I hrmmolcr activation. v-HaRax and v-Raf alsu activatcd The Egr-I pro- motcr. To charactcriic IFaRas and Raf-I involvement in v-Fps-inctuccd F,gr-I expres.eiom, we used recently characterized dominant negative mutants of IlaRas and Raf-I, v-Fhs-induccd Fgr-I promoter activation was inhibited by the dominant ncga- livc mutants of Ixoth IlaRas and Raf-I. v-IlaRas-induccd Fgr-I promoter activation was hlnckcd by the negative Raf- I nwtnt: howcvcr, v-Raf-I-induced F:gr-I pro- molcr activatinn was unaffcctcd by The inhibitory IIaRas mutant. Thcsc data suggest that v-Fpti activates a protein kinase C-independent intracellular signaling pathway that is dcpcndcnt un both HaRas and Raf-I, where Raf- I functions downstream of I taRa%. Alex;mdroprndos, K., Qureshi, S. A.. Rrudcr, J. T., Rapp, tl., and Fnsrrr, U. A. ; II (imwth & Diffcrcntiation 3:731-737, October 1992, il,,,r •tthlmrt: National Institutes of Health and City Univcrsity of Ncw York. I t,tt the Institutc I'or Riomolccular Structure and Function, Department of Itmvi,~'tI ~~~ ~n~ ~~t~~s'ntcFrcdcr~ k Ctanc clr tRc seI rch and Development Ccntcr. I I «•jcrick. MI). I;~I(7ENCE THAT Ha-Ras MEDIATES TWO DISTINGUISHABLE IUTRACF,LLULAR SI(iNALS ACTIVATED BY v-Src v-Src activates promoters under the control of 12-O-tetradecanoylphorhol-I3- acctatc (TPA) response clements (TRF,s) and serum response elemcnts (SREs) vil Iwn distinguishaMc intracellular signaling mechanisms. The induction of TRF,- anc SKE_mcdi;ded gene expression by v-Src could be distinguished by a differential sen oivity to depleting cells of protein kinase C (PKC) and to a dominant negative Raf I mutant. Thus. PKC depletion and the dominant negative Raf-I mutant were able tt (listinguish Iwo intracellular signaling mechanisms activated by v-Src. Both of thcs, v-Src-induced intraccllular signals were sensitive to a dominant negative mutant o Ila-Ras. These data suggest that Ha-Ras functions to coordinately regulate multipl intracellular signaling mechanisms activatecf by v-Src. Qureshi, S. A., Alcxandropoulns, K.. Rim. M., Joseph. C. K.. Bruder, J. T., Rapl (I. R-, and Fo.cler,1). A. The Journal of Biological Chemistry 267(25):17635-17619, September 5,1992. Other support: National Institutes of Health and City University of New York. From The Institute for Riomolecular Structure and Function, Department ~ Biological Sciences, Hunter C'ollege, City tlniversity of New York, and Laboratoi of Viral C'arcinogenesis, Frederick Cancer Research and Development Cente Frederick. MD. C'LONING ANI) ('IIARA("1'ERI7.ATION OF A THERMOLABILE v-srr GF,NE FOR USE IN RF:VI?RSIRLI? TRANSFORMATION OF MAMMALIAN CELLS The usc of tcnipcraturc-scnsitivc (ts) ,crc• mutants for studics of cell transforn Iion and differentiation ha. been limited by the availability of cloned ts,err get that are inactivated at tempcratures compatible with growth of mammalian cclls. this report, we describe the cloning and characterization of the tsLA90.crr gc which displays tight thennal sensitivity at 39.5^C. Nucleotide sequence comparv of tsl.A9t1 nd'wild-typc .crr genes from the Schmidt-Ruppin subgroup A n( straim of Rous sarcoma virus (RSV) revealed four amino acid differences tsLAc)O.err. Substitution of one of these residues (Lys-2R0) from tsLA9(l.crr with 129 1 129
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wild-t,ylx• homolog ((ilu 2R(1) caueccl a revertiinn lo a wild-type .crr phenotype. 17ir c-hmcd Is1,Ao)I) gcnc, clctignatcd IsI11 I, was inlrrrdutt•d inln avian and mamm:rli:rn rctroviral vectors. ('hic•kcn cmhryo fihrcrhla~ls and inunortalicccl ninusc 3'1'; c•clk infected with Ihcsc viral vcc•tors displaycd a tcmlxrnturc-dcpcndent transfonncd phenotype as assc.sccl by cell mnrlrhoingy, secretion of plasminogen activator, Iran, - criptional activatinn of the primary reslxmsc gcncs, ligr-I and TIS 10, and slimula- lion of lyrosinr phnsphorylatinn. In adclilinn, chickcn mynhlaslx (infcctcd wilh RSVIs(II'I) showcd a Icmpcraturc-dcpcndcnt diffcrentiation into myMUhcs. Thu,;, this cloned .crr gene should hc ideally suitcd for inducing reversible transformalinn and cliffcrcntialinn of mammalian cells in culture. Maroncy. A. C.. Qureshi. S. A.. Fnarrr. !L A.. and Rruggc, J. S. Onc•ogcnc 7:12(17-1214, 1992. OIhcr surporl: National Institutes of I lcallh and National Cancer Institute. From the Hocuard Ilughcs Medical Institute. Department of Microhiology, University of Pennsylvania School of Medicine, Philadelphia: Institute for Binmolccular Structure and Function. I)cparlmcnl of Biological Sciences. Fluntcr ('rrllcgc, City I Inivcrsity of New York. v Fhs-RIiSI'ONSIVIiN1:SS IN Tlff? f:gr-I 1'ROMOTER IS MF;DIATED BY SI;RI IM RI;SPONSIi IiLf?MI?NT;S Egr-I, a milogcn-respCrnsivc Iranscription factor, is rapidly induced by v-Fps in Ihc ahscncc of protein synthesis. Thus, 1?gr-I is a primary response to Ihe protcin- tyroeinc kinase activity of v-Fps. To determine the v-fps-responsivc elements in the I'.gr-I promotcr, deletion mutants ot' the I'sgr-I promoter were used in transient cxprc,ttinn assayc. A v-Fps expression vector was cotransfcctcd into NIFI 3Ti cells with chlnramphcnic•ol ac•ctyl transfcrasc (('A'f) gene expression vectors undcr the cnntrol of Ihc Hgr-I prnmolcr or the Egr-I promoter containing various deletions. Rv~ronNivcnca to v-Fps was restricted to a region that contained repeated ('('(A/I1,(;G sequences, known as ('Ar(i boxes. C'ArG boxes form the core of acrum rca~nnsc clctncnt (SRI;s). v-Fps-induccd F?gr-I promoter aclivation was lost hy scrlucntial removal of four tandcmly repeated SRI:s. This region, containing four SRI; e, wati liwncl Ior Ix sufficic•nt for tnarirnal I:gr-I induction by v-Fps when placed upslrcam frnm a hctcrologous promoter. Individual SRI?s from lhis region were able lo rc%rrmcl to v-Flrs, howcvcr, thc activatinn of the individual SREs was lower than that observed for Ihc clusrcrcd SREs. These data suggest that v-Fps-resfxmsivcncss in the ligr-I promoter is mediated by SREs. Alcxandrnpnulns, K.. Qurcshi, S. A., Rim, M.. Sukhatme, V. P., and /•'n.clcr,1). A. Nucleic Acids Research 20(c)):2355-2359, 1992. Other support: National Institulcs nf Ilcallh, City Univcr.ity of New York, Howard 11ughc•s Medical Instilutc, and Itcllcnic Flnivcrsity Club. I rr,r~r Ihc Institutc for Riomolecular Structure and Function. 1)epartmcnt o' Itinlr'F'~a~ Scicnccs, Iluntcr College. ('ity Ilnivcrsity of New York, and Dcparlmcn ~ Mrdre•mc, Molecular Cicnctics and C'elI Biology, and Howard Hughes Mcdica In tilulc, Fhiivcrsity of Chicago. 51ISTAINGD INDUCTION OF cRr-I BY v-srr CORRELATES WITH A LACK t)F 1.OS-MF,DIATIiD REI'RFSSION OF l'HE rgr-I PROMOTER Semm stimulation of quiescent fibroblasts leads to a transient induction of th Iranscription factor e,r,>r-L However, the induction of egr-I by v-src• was found to F ostaincd rather than transient. The proto-oncogene fns has been reported to hc ca regulated with cgr-I and to repress senrm-induced rlGr-I expression. We found th c• fn.c prevents v-,cr•c-induced gene expression regulated by the rgr-I promoter. Thu Ihe sustained induction of c,rr-I by v-src• could be explained by a lack of cfcr,c indu~ tirrrr by v-srr. Consistent with this hypothesis, egr-I and C fns were co-induced I scruni, hut not by v-src, in 13alb/c 3T3 cells; v-sr•c• did not induce c fns expression Ihese cells. We propose that sustained expression of eRr-I induced by v-.crr• in Balh 311 cells is due to a lack of c fn.c down-regulation of rgr-I. (}ureshi, S. A., Rim, M., Alcxandropulos, K., Berg, K., Sukhatme, V. P.. and rn.cre 1). A. Oncogcnc 7:121-125, 1992. Other support: National Institutes of Health, City University of New York a Huward Hughes Medical Institute. From the Institute for Biomolecular Structure and Function, Department Biological Sciences. Hunter ('ollege, City University of New York, and Deparlmr of Medicine, Molecular Genetics and Cell Biology, and Howard Hughes Medi, Institutc, University of C'hicago. IMMUNO-ISOLA-I'IUN 01: SEC'7P-CC)ATF,D TRANSPORT VF.SICLF,S FROIV THE YEAST S8('RF:I'ORY I'A'fIIWAY The transfxrrt of proteins destined for post-endoplasmic reticulum locatiow Ihe secretory pathway is mediated by small vesicular carriers. Transport vesi( have been generated in cell-free assays from the yeast Sac•rharomyce.c cercri.c. and marnmalian systems. Yeast genes enccxJing cylosolic components that part pate in vesicular traffic were first identified from the collection of conditional Ie sir (secretory) mutants. Mutations in the yeast ,SF.C7 gene disrupt protein transl in the secretory pathway at the nonpermissive temperature. The .SF.'C'7 gene prcx is a phnsphoprntcin of relative molecular mass 2l(l,(N)0 that functions from the c plasmic aspect of intracellular membranes. We report that in a yeast cell-free tr. FCrrl assay, the introduction of antibodies to Scc7 protein (Sec7p) results in the al 130 1 131
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mulalinn of tramPnrl vcsiclce. '1'hc,,c vosit'Ic% are rclricvccl with Scc71i-sfx•cific anti- hndics by intmuncr i.nlaticrn for hicKficmical :md clcctrnn micnrticnpic charactcriia- tinn. Scc7p nn the surfacc of thc act'utnalatcd Ir:mxlxirl vcsiclcs, in comhination with previous genetic and hinchcmical studics, inthlicatc Sec7p as part nf a(non-clathrin) vcsiclc ccrat. 'I'Ititi Scc7p-cc,ntaining ccrat %fructurc is propnsccl to hc essential for viceiclc huddinR at multipic stages in the yeast sccrctory pathway. Grrrq: nsn%/; A., I.autc, N., and I lowell. K. F. Nalurc 355:173-r75, January 1992. Ulhcr support: National Institulce of I Icalth. Front the 1)cpartmcnt of ('cllular and Slruclural Biology. llrtivcrsity of Colorado Medical Schnnl. I)cnvcr. RI:AIrfY AND'Illl's YIiAS'I': (Y)MPARTMENTALORGANI7.ATION OF'Tllf? Sli('R1;1'ORY PA'fI1WAY Our Ixrccptinn of immccllular organcllcs and cellular architccturc was initially based nn xtrikin}; Iight and clcctrnn rnicrogralths of animal and plant cells. The high degree of compartmcnlal crrganiraticm within slx•calized mammalian sccrctnry cells aidcd carly clTorts to track the movcmcnt uf proteins through thc organcllcs of the sccrctcrry pathway. In cnnlrast, the morphological detail of thc yeast ,Carrlrarnmvres rrrericiui• appeared superficially sitnhlc, even primitive, by comparison with Ihc higher cukaryutic cells. I lowcvcr, the combination of gc-nctic Irtols and the dcvcinp- mcnt nf atisayti rcccrostituting vesicular traffic in yeast have facilitated the ictentfica- tirnt and charactcri,,atimt of indiviclual proteins that function in the sccretnry path- way. Analogies Ixtwccn the funclinn of yeast and mantmalian proteins in vesicular traffic arc being drawn with increasing frcyucncy. In this rcview, thc combination of Vcnctic,, hinchcmical, molecular and cell biological approaches used to study cotn- partntcnl;d organization in the yeast sccrctnry pathway will he discussed. The rapid Pmgress in our understanding of ycast membrane traffic has revealed the beauty of working with this orgarti.cnt Fra,r. rccn(/. A ('cll Binloyy i:,lf)c)-324, 1992. OIhcr whhc,rt: Amc•rit'an Cancer Society. Frrnn the I)cpartmcnt ctf ('cllular and Structural Riology, llnivcrsity of C'oloraclo I Icalth Sciences ('cntcr. 1)cnvcr. St)1'1?ROXII)1: G1?NIsRATED BY GLUTATHIONE REDIJCTASE INITIATFS A VANAhAI'1:-Ulil'1?NDliN'1' FRIiF, RADICAL ('IIAIN OXII)A'I'rON OF NADH Vanaclatc V_ markedly stimulalcd the oxidation of NADPII by GSSG rcduc- tasc and this oxidation was accompanied by thc consumption of O, and the accumu- lation of 11,0,. Snhcroxidc dismutascs completely eliminated this effect of V,,,, whcrcas ratalasc was without cffcct, as was exogenous H O, added to 0, 1 mne. lltcs effects could he seen equally well in phosphate- or in 4-(2-hydroxyethyl)-I-pi)Kraz nccthancsulfonic acid-huffered solutions. Under anaerobic conditions there was n V„ stintulatcct oxidation of NADI'H. Approximately 4% of the electrons flowin frunt f•IADPH to O,, through GSSG rcductase. resulted in release of O,. The averag length of the free radical chains causing Ihe oxidation of NADPH, initiated by ( l,lrr,s V~ was calculated to he in (hc range 140-2(X) NADPH oxidized per O, intr( duced. Wc conclude that GSSG reductase, and by extension other O; producin 11;rvnprotcin dchydrogenases such as lipoyl dchydrogenase and ferredoxin reductas cataly/.c Vv; stimulatcd oxidation uf NAD(P)H because they release 0, and hccau! O, plus V, initiatc a free radical chain cixidation of NAD(1')H. Thcrc is no rcason, suplxnc that these cnr.ymcs can act as NAD(P)H:Vv, oxidorcductascs. I,imhcv, S. I. an(I Fridovir{t. 1. Archives of Biochemistry and Biophysics 294(2):403-406, May I, 1992. Other support: National Science Foundation, Johnson & Johnson Focused Givir Program and National Institutes of Hcalth. Frnm the Department of 13inchcmi.etry. Duke University Medical Center. Durhat NC. EFFECTS OF OVERPRODUCTION OF SUPEROXIDE DISMUTASES IN ESC'HPRIC'lllA C'Ol.l ON INHIBITION OF GROWTH AND ON INDUCTION (. GI.UC'OSE-6-PHOSPHATE DEIIYDROGENASF: BY PARAQUAT Stationary phase inocula were more susceptible to the growth inhibitory cffi of paraquat than were log phase inocula and this difference was exacerbated strains overproducing superoxide dismutnees (SOD). Glucose-6-phosphate dehydr genase (G-6-PD), a memlxr of the soxR regulon, was induced by paraqual promp in the case of log phase cells; hut only alter a lag in stationary phase cells and II difference was also exaggerated in strains overproducing SOD. The negative con, qucnces of overproduction of SOI) on the adaptation of stationary phase cells paraquat may be attrihutccl to competition for cellular resources with an attenda delay in hiosyn- thesis <rf nthcr cnmPrmcnls of soxR. Sincc overproduction of S( did not prevent log phase cells frottt inducing G-6-PD in response to paraqual. appears likely that soxR can respond to aspects of rcdox status other than 0,. '1' conclusion is in accord with data which is already in the literature. I,iochcv. S. and Fridcmich, l. Archives of Binchcmixtry and Biophysics 294( I):13R-143, April 1)92. Other support: National Science Foundation, National Institutes of Ilcalth z I;ulg:rcian Academy of Sciences. Front the I')cpartmcnt of Biochemistry. Duke University Medical Centcr, Durh: NC. 132 1 133
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Tllf? ROI.F.S OF 0,. 11O", ANI) Sf:(Y)NDARII,Y DI?RIV1?I) RADI('ALS IN OXII)A'F'ION RGA("I'IONS ('Al'AI,Y'l.HD 13Y VANAhI1IM SALTS V(IV) decornPoscd Fl O,,wilh evolution nf O„in a free radical chain process involving O,:rnd IlO'. When V(IV) was limitinl;. the presence of V(V) augmented 0, evolutinn because il allowed production of additional V(IV) from the recluction of V(V) by O,.Gradual additicm of V(IV) increascd the yield of O, evolved, per V(IV) acleled, to greater than I a clear inclication of a free raclicat chain reactinn. Rcductants such as eth:mol, Ilcpes, and NAI)1I imfxiscd a phasc of O' consumption because of 110'-initiated oxiclalirnl reactions. The radical produced from the reaction of 110' with cthanol was unable to directly oxidize NADH, whereas that produced from Flepes was able to do so. F;Ihanol consequently inhibited the oxidation of NADI I hy anaernhic V(IV) + 11,O,, whereas Hepes clid not. These results, and others repnrted herein, are explained on the basis of a coherent set of reactions. Data already in the literature are also clarified on the basis of these reactions. Licx•hcv, S. I. and Ffidnrirh, 1. Archives nf Ricx•hemistry, and Biophysics 291(2):379-3R2, 1)ecember 1991. Other support: National Science Foundation and Bulgarian Academy of Sciences. From the I)cpartment of Riochemistry, Duke University Medical Center, Durham, NC. Sl)PI'RIiSSION OF OXIDA'I'1VE f•.NVEI,nI'F; DAMAGE BY 1',SIiIIDOREVERSION OF A SFIPfiROXIDE DISMI)TASIi-DEFICIENT Mt ffAN'1-OF /i,S('ILIiRI('llln ('OI,1 Mutantx of L•:crhrric-h coli that are dcvoid of superoxide dismutasc (SOD) fail to grow in acrohic minimal medium. This is largely because of the Olsensitivities of several sunino acicl hiosynthetic pathways, since amino acid supplements can restore }rowlh, alhril at a show rate. We now report that growth in amino acid-supplemenled medium can he further stinlulatecl by the presence of cxtracellular osmnlytes. O.mnlytcti alco hartially strppress the amino acid requirements nf thc SOF) mutant. hhc•w data %uggest that the curnhinatiem of oxidative injury and turgor pressure Per- mcahiliics the cell envelope and that critical metaholites. including the limiting Prnduct% of danrafted hiosynthetic pathways, escape from the cell. External otmnlytes may nffcr protection by countervailing the usual turgor pressure and thus stahilii.ing Ihc damaged envelope. This model is consistent with the previous obser- vatinn that deficicncy of cell wall components is lethal to SO1) mutants. A pseudorc- verl:mt that can grow al a moderate rate in ncirmosmntic medium without amino acid supplcmcntatinn has been nhtainecl (J. A. Inilay and 1. Fridovich, Mol. Gcn. Gcnct. 22R:4tO-41(i, 1991). Analysis suggests that the suppressor mutation allows the enve- lope cithcr to resist or to tolcratc oxidative Iesicins. Study of the pscudorevcrtant may illuminale thc nx,lecuhtr hasis of this cixidativic envelope injury. Imlay,.I. A. and I•i-idnrirh, L Journal of Ractcrinlogy f74( 3):953-9f,1, Fchruary 1992. Other support: National Science Foundation, National Institutes of Flcalth and Ja (,uffin ('hilcls Memorial Fund for Medical Research. F:pmi the Department of Riochemistry, Duke University Medical C'enter, Durha N('• 1NACTIVATION-REA("TIVATION OF ACONITASE IN F.,SCIIF.RICIIIA C'OLI A SIiNSIIIVIi MBASURB Ot' SIII'riROXIDG RADICAL The rapid inactivation of aconitase by O7, previously seen to occur in vilrn, ~ cxplore(1 in vivo. A fraction of the aconitase in growing, aerobic, E,rrherirhia rn1 inactive at any instant but can he activated by imposition of anaerobic conditic This reactivation occurred in the absence of protein synthesis and was inhibited the ferrous chelator a,at'-dipyridyl. This fraction of inactive, but activatahlc, acr tase was increased by augmenting 0, production with paraquat, decreased by ele tion of superoxide dismutase, and increased by inhibiting reactivation with n, dipyridyl. The balance between inactive and active aconitase thus represented a pseu equilibrium between inactivation by 0, and reactivation by restoration of Fe(II), it provided, for the first time, a measure of the steady-state concentration of 0, w in E. rnli. On this basis. 10 ' / was estimated to be v2O-40 pM in aerobic log pfiasi coli containing wild type levels of supcrnxiclc dismutase and -300 pM in a rnu strain lacking superoxide dismutasc. Gardncr, P. R. and Fridnrirh, l- Thc Journal of Biological Chemistry 267(13):R757-R763, May 5. 1992. Othcr support: Johnson & Johnson Focused Giving Program and National Sci( Foundation. From the Department of Biochemistry. Duke University Medical Center, DurF NC TRANS('RIPTIONAI. ANl) MATURATIONAL EFFECTS OF MANGANESF ANI) IRON ON Ttll: RIOSYNT'IIESIS OF MANGANESE-SUPEROXIDE I)ISMUTASF, IN l:,ti(71I:K1(711~ ('OL! Anacrohically grown F.srhrrichia coli contain an enzymatically active superoxide dismutasc (Fe; FcSOD) and an inactive iron-substituted manga superoxide dismutasc (Fc; MnSOD). The anaerobic electron sink, nitrate p,vacluat, cnhanccd biosynthcsis of the MnSOD lxrlypcptide, with accumulatic inactive Fc: MnSOD. '1'hc oxidant, diamide, in contrast, allowed anaer production of the active forms of MnSOD, i.e., Mn MnSOD and Mn/Fe-Mn` Nutritional supplementation with Mn(1I) favored occupancy of the MnSOD a sitc with mangancse and allowed anaerobic accumulation of Mn. MnSOD i 134 1 135
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ahscncc of diamidc. I:nrichmcnt of Ihc anacrnhicc growth mcdium wilh hc(II) hoth suppresscd hiosynthcsis of thc MnSOI) fa,lypcrtidc and inhibited fomtalinn r,f the active mangancsc-containing fnrms. A rur,crnlA operon Ibsinn wa% used to examine Ihc effects of chelating agents and mctals on maturalion of nascent MnSnD, independent from thc transcriptional cf7ct•Is thcsc agents impose. Isnhrnpyl-I-thio-(3-t)-gatactopyranrxidc (IP'F'G) elicited anacrohic• hiosynthcsiti ul MnSOI), which accumulated as the inactivc Pc -MnSOD. I)iarnidc, with IPT(i, alhowcd formation of active Mn/Fc-MnSOD whilc I,10- hhcnanlhrnlinc with IP'1'G resulted in accumulation nf Mn -MnSOD. These results suggest that iron participates in the rcdrixscnxitivc control (if the formation of active MnSOD al Iwr, Icvcls, i.r., that nf, transcriPtion as well as thal ol• maturation. During maturation rtl the nascent MnSOD pulypt•Ptidc, iron and manganese compctc for the mctal-hindin}± site: anacrnhic contliticm,, favor iron binding, whereas oxidanls such ati dicixyf;cn or diamidc Iavor hindinR of numgancsc. I'rivaltc, C. T. and Frirlnrir•h, l. The Journal of Biological ('hcmistry 2(,7(1 t):9140-9145, May 5. 1992. Othcr Support: National Science Foundation. National Instilutcs of Ilcalth and Iohnson & Johnson Fricuscd (iiving Program. Prnm the Dcharhncnt nl 13iochcrnislry, 1)ukc linivcrsity Medical Ccntcr, Durham, NC. 1'l IMARASIs C,'I-I IF. STA131.N 1't IMARASE OF I::SCIII:RI(711A C011.1113 ('ON'I R( )I .LED BY TI IF?.cntR,S RF;Gl ILON 1'umarasc C was stron};ly induced by paraquat in a parental strain of l:srhr•rir hiu rnli hul was not induc•cd in a strain lacking the .cnrR.S reponse. Murcrwr•r, a tilriin that conslitutivcly expresses the ,cr,rRS regulon contained more Inmarasc (' Ihan did Ihc parental strain. '1'hc Mn-containing supcroxidc dismulasc and plucotic (, Phoerhatc dchydrogcnasc, mcmtx.rs of the sntRS regulon, were simi- larly induccd by haraquat. Mutational dcfccts in glucosc-6-phnsphatc dchydrngcnasc incrca.crl thc inductiom of fumarasc (' by parayual. For Mn-containing supcroxidc rlismul;isc, rcslwn~ivcncss to parayuat was also enhanced in the giucosc-6-phosphatc dchydnrgcn;isc-dcfcclivc strains. Overproduction of the Mn-containing superoxide dismutasc, clicilcd by ixnPmpl (1-u-thiogalactnsidc in a tur,enrU fusion strain, did nol diminish induction r,l Iumarasc C or of glucrnc-6-phoxphatc dchydrogcnasc by haraqu;u, and induction of these cnzymcs was more scnsitivc to parayuat when the t•cliti were growing c,n soccinatc ratltcr than on 1.11 medium. These results indicate Ihal fumarasc (' is a ntcmlxr uf the sr!rR,S rcgulon and that this regulon does not respond to changcti in O; concentration hul perhaps does respond to sonic consc- qncncc of a decrease in the ratiri of NADPI I to NADI'+. I.itx'hcv, S. I: and 1•ridnrirh. l. Prrx•ccdings of Ihc National Acadcmy cif Scicnccs USA R9:5}i92-5R96, July 1992. 136  OIhcr suplort: Johnson &.Inhnsr,n Focused Giving Program and National Science 1;nundatirm. prom the I)cpartmcnt of Biochemistry. 1)ukc University Medical Center. Durham. N('• GXO(;E,NOUS QUINONES DIRECTLY INHIBIT THE RESPIRATORY NADH DI?IIYUROGENASE, IN L;SC11ER1(7IIA ('OLI The ability of naphthnyuinones to generate reactive oxygen species has been widely exploited in studies of oxidative stress. Hnwever, excess superoxide distnu- Iasc and catalasc failed to protect E.ar•herir•hia roli in rich medium against growth inhibition by plumhagin; indicating thad its toxic effect was not due to the productinn of partially rcduccd oxygen species. Respiration failed immediately upon the addi- lion of growth-inhihitory levels of plumbagin. Sludics in t•ilrn showed that plumba- gin and other rcdox-activc yuinoncs intercept electrons from NADH dchydrogcnasc, the primary respiratory dehydrogenase in gluctnc-containing media. An excess ol oxidative substratc, such as plumhagin inactivatcs this cnzymc, which appears to be redox-regulated. The resultant respiratory arrest is a cautionary example of ineta- hnlic dysfunction from rcdox-cycling drugs that cannot bc attributed to supcroxidc or hydrogen peroxide. Itnhry, J. and Fridnvirh, l. Archives of Biochemistry and Biophysics 29fi(1):337-346, July 1992, Other support: Johnson & Johnson Focused Giving Program and National Scicna Foundation. From the Department of Biochemistry, Duke University Medical Center, Durham NC. ALUMINUM(1II) f ACII,ITA-I-ES TIIE OXIDATION OF NADH BY TIIE, SUPEROXII)E ANION AI(III) augments the oxidation of reduced nicotinamidc adeninc dinuclcotid (NADII) by cnzymatic or pholochcmical sources of O,. Superoxide dismutasc, ht not catadasc, inhihitcd this at•tion of AI(III). It thus appears that AI(III) forms a con picx with O, which is a stronger oxidant than is 0, itself and which may contrihui to Ihc advcrsc biological effects (if AI(111). Kong. S., Liochcv, S., and Fr•idnrirh, l. Free Radical Biology & Medicine 13:79-8I, 1992. Other support: Johnson & Johnson Focused Giving Program and National Scicn( Foundation. From the Oukc University Medical Center, Durham, NC. 117
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HIIMAN'1'-('IiLL LYMI'HOTROI'I(' VIRIIS'T'YPF. I (II'f1.V-I) 'fRANS('RIP"IlONA1, A('TIVA'I'OR. TAX, FiNI IAN('IiS ('RER BINDING TO f1Tl.V-I 21-RASE-PAIR RI;PF.Al;ti BY I'R(yT'FIN-I'RO"17?IN INTERA(TION fITLV-I Tax protein activates transcription from Ihrce 21-hase-pair (hp) repeat sequences in file viral enhancer. The Ifl'LV-1 21-hp repeal contains a TGA('GT molif that is hnmologou, to file cAMP-responsive element (C'RE) ancl crucial for Iax transactivation. Tax exhibits marginal affinity for 1)NA hut rather interacts with cel- lular ('RE-hincling proteins to enhance their affinity for the IITLV-1 21-hp repeals. llsing file H"I1,V-I 21-hp repcat and Jurkat T-Iymphocyte nuclear extract in a gel eleclrophorclic mohilily-shifl assay. we previously detected three protein-DNA com- plexes that are specific for the CRE in file 21-bp repeal (complexes I, 11, and IV). Complexes I:md II hut not IV interacted with Tax. We now show that complexes 1, 11, and IV are composed of ('REB (CRE binding protein) homodimer, CRF,B/ATF-I (activating transcription factor I) hctcrodimcr, and ATF-I homcxiimcr, respectively. Tax stahili/es complexes I and II via a direct interaction with file CRER moiety. In Ihe absence ol 1)NA, ('RER an(i Tax continue to form a complex that can he immunoprccipitatccl by a Tax-specific antibody. These results suggest that one mechanism by which Tax activates transcription may be mediated through the direct interaction with ('RER homndimer and/or C RF.B/ATF-I hcdcrodimer tn stabilize their assembly on Ihc Tax-responsive CRE molifs in the IITLV-I enhancer. 7hao. L.-.I. and Giam. ('.-7,. Proceedings of the National Academy of Sciences USA ft9:7(17O-7O74, August 1992. Other suppnrts: National Institutes of Heallh. Frnm the Division of' Infeclious I)iscases, Departments of Medicine and Molecular Biology and Microbiology. Case Western Reserve llniversity School of Medicine, ('Icveland, 011. RINf)ING (ll:TRANSFORMING GROWTH FA(TOR-RI TO METHYLAMINE= MO1)IHF:I) rr2-MA(ROGE.f)RIILIN AND TO BINARY ANI) TERNARY a2- MA('ROGI,rYREII,IN I'RO'T'F:INASE('OMI'LI:XIiS '1'hc binding of "I-lahelcd transforming grnwlh factor-/3I (TGF-(31) to human a, marrrrghrhulin (rr,M) was studied by native PA(iE and autoradiography. T(iF- /il.hcnmd prclerentially to a,M mclhylarnine and minimally, if at all, to native a,M. Prcparatinns rrf rv,M protcinasc complex were generated by incubating a standard conccnlraliun of rv;M (l).4 µm) with differcnl concentrations of trypsin, chy- molrypsin or ncturnphil clastasc (0.04 -2-O µre). The "I-TGF-(3I-hinding activity clepende<I tm the initial ratio of active prntcinasc to a,M, or r value, uscd to fonn the (x,M -protcinase complex. Wilh all three proteinases. r values of 2 or greater yielded preparations with unchanged or decreased TGF-(3I-hinding activity relative to native a,M. Ry contrast, r values near I yielded preparations with significantly increased TGF-(31-hinding activity. The results nf I'H)Ihymidinc-incorporation studies per- formed in mouse kerafimrcytcs were consistent with Ihe "IT(iF-p-hinding expcri- ments. rv.M trypsin and a.M chymotrypsin prepared at an r value of 1.0 counteracl- ccl the activity of TGF-(3I, whereas the equivalent complexes prepared at an r v of 3.0 had no effect..As determined by SDS/PAGE, "`I-TGF-(31 hindin n,M methylamine was at least R(NZ, non-covalent. Reaction of a,M -methylar with icxioacetamide or 5,5'-dithiohis-(2-nitrohenznic acid) decreased the percen of covalent binding hut had no effect on total binding, Neuraminidase treatment no effect on the binding of "9-TGF-/3I to a,M-methylamine. Cleavage of the ' regions" in a,M-methylamine by prolonged treatment with trypsin also har effect. These studies suggest that TGF-(3I binding to alM is enhanced by confo tional change in the prolcinase inhibitor resulting from reaction with proteinas amine. If both protcinase-hinding sites in a single a,M molecule are oc:cupied.'1 (3I-binding activity is decreased or perhaps eliminated. Hall, S. W., LaMarre, J., Marshall, L. B., Hayes, M. A., and Gonia.c, S. L. Biochemical Journal 281:569-575, 1992. Other support: Pew Scholars Program, Medical Research Council of Canada 11.S.National Hearl, l.,ung and Blood Institute. From the Departments of Pathology and Biochemistry, University of Virginia H Sciences Center, Charlottesville, and Department of Pathology, Universi Guclph, Guelph, Ontario, Canada. BINDING OF TRANSFORMING GROWTH FACTOR-(31 TO IMMOBILIZE HUMAN a2-MACROGLOBULIN Native ar macroglohulin ((v M) and a.M-methylamine were immohlilized i well microtiter plates. "`I-lahelcd transforming growth factor-(3I (TGF-(3I ) hou both a,M variants; however, greater binding was observed with a,M-methyla: Binding of "`I-TGF-(il (().2nM) to immoholized a,M-methylamine was inhihit, nonradiolabeled TGF-(il (up to 74% with 0.4 µM TGF-0I). Approximately 1( Ihe TGF-(31-a2M-methylamine complex was covalent. Treatment of azM-mr amine with iodoacetamide prior to immohilization completely eliminated coi TGF-(3I hinding: the total amount of "I-TGF-(3I-a?M-methylaminc complex d ec1 was unchanged. The binding of "9-TGF-f31 to immobilized a M-methyls was not significantly inhibited by increasing the ionic strength to (.6 M. Bindin complex dissociation were also unaffected by changes in pFl within the 6.9 -R.9. Acidic pH dramatically decreased binding and promoted complex dis tion; no binding of "111JF-(31 to immobilized a,M-methylamine was detec plt i.5, The interaction (if TGF-(3I with immohilized a,M-methylamine was m nificantly changed by 1.0 mM EDTA or 1.0 mM CaCl1. 7,nCl (1.0 mm) com f eliminated binding. 'I'his result was not due to TGF-(3I, precipitation or aggrcg Inhibition of '-9-TGF-(3I binding to a,M-melhylamine was 50% complete with 30 µM 7,nC'I,. Native a M, thromhnspondin, and a,M-methylamine (in sol decreased binding of 'A-TGF-RI to immobilized a,M-methylamine. The I(',,,' for these three proteins were 520, 100, 79 nM, respectively. The TGF-(31-hi activity of native a,M may have reflected, at least in part, trace-contaminatioi a.M-protcinasc complex. 139 1 139
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Wchh, 1). .I., ('onkston. K. I'.. 1IaII, S. I 1., and (inniu.c..S 1._ Archives of Biochemistry and I;irtphy.icc 292(2):4H7-4r)2. 1902, Other supprtrl: Pew Schnlar. 1'n)gmm and Re~rlrch ('arecr I)cvch)pnx•m Awar(I. I'rrnn Ihe 1)epartmenls of 1'alhoh)iy and Rilx•hcmixtry. tlniversity of Virginia Hcallh Scienc" ('enter. ('harlottetivillc. rr,-MACROOLORI IL1N: A I'RO'T1iIN A'I' TI IH INTIiRFACTi OF FIRRINOLYSIS AND ('F.I.1,I ILAR (iROWl'II RIiG1I1,A'I'ION During the middle decades Irf Ihe twentieth century, an extraordinary number of eniymes were purified, characterired, and calegoriied. I;niyme category was gener- ally determined hy the hinchcmistry r,f the cnrytnc active site, the nature of the suh- strale. and Ihe hiologic;d system in which the enzyme functioned. Most enzymes were considered biological calaly.ts for one reaclion within one pathway or biologi- c;t1 pmcesx. 'Ihere arc approximately 3.3 X IO" base pairs in Ihe human gennmc; however, only a.cmall fraction of this DNA actually encodes protein. Therefore, the numher of eniymes available lo funcliun in diverse biological processes is not limitless. Recent studies have demonstrated that enzymes in the blood and cxtraccllular spaces frc- quently have numerou.s physiological substrates relevant to many different biological processes. '11u functional (liver';ily of individual cniyt»cs limits the burden placed on cells to synthesiie dil-ferent proteins :md provides an opportunity for coordinated regulation nf different enzWnte systems. Perhap:e there is no hctter example of this concept than the eniymes of the fihrinolylic system. Gnnirtc. ,S'. L. 1?xperimcntal I lctnalolngy 2O: 3O2-i 1 I, 1992. Other support: National Heart. I.ung and Blood Institute and Pew Scholars Program in the I3iontedical Scir•ncee. From lhe /)epnrlmcnts ol I'athology and RiOchcmistry. lfniversity of Virginia Health Science,, ('enter, ('harlntlesvillc. II)IiNTIFI('A"IlON OF a,-MAC'ROGLOBULIN ('ONFORMATIONAI, IN'fF.RM1:DIA'fF.S BY l?l.l?('TRON MI('ROS('OPY AND IMAGF ANALYSIS-- ('nMt'ARISON 01: (r,-MA('R(N;If)It111.IN-t7IRnMn1N ANh a -MA('R(X:U)nt!I.IN RI'A(`ItiU WI111 (7.f-hl('III (tKf)UI~MA11Nt'.1'LKIINIIM(II) ANI) TRYI'.SIN Human at-macrr)glohulin ((YrM) exists in two well defined, highly distinct con- ti)rmations and in Icsx well descnbed intermediate conformations. In this study, pre- viously characterized reactions were used to partially or completely transform lhe conformation of (r,M. lileclrrnt micrographs of each preparation were suhjected to image analysis. Ternary a,M-tr-ypsin (2 mol of trypsin/mnl of a M) was analyzed ati a control lor The fully transformed state. (.'orrespondence anafysis (CORAN) and hierarchical ascendant classification (IIA(') generated five image clusters from ;;l/ aligned a,M-trypsin complexes. Average images of each cluster resembled the letter "H" with four nearly equivalent lateral arms. Abnormally shaped lateral arms were not demonslrated by IIAC, using a variety of factor sets. In a native polyacrylamide gel clectrophoresis syslcm, a,M-thromhin migrated in a diffuse band partially be- hind a,M-lrypsin, suggesting c(mformational heterogeneity. CORAN and HAC of 733 a,M-thromhin complexes identified two neighhnring clusters, The average images of which showed an H-like structure in which one arm was replaced by a glohular stain-excludinF body. The two a,M-thromhin clusters included 125 images (17.1% of image population). The complete absence of atypical lateral arm structure in the a,M- trypsin clusters suggests that this variation is not the result of orientation or staining artifact. Native a,M was reacted with ris-dichlorodiammineplalinum(II) and then with trypsin to form a,M-Pt-trypsin, a preparation that includes partially transformed a,M structures. COkAN and IIAC of 590 arM-Pt-trypsin complexes generated five clusters, the average images of which showed atypical lateral arm structure equivalent to that demonstrated with arM-thromhin. The five a)M-Pt- trypsin clusters accounted for 15.2"G. of the image population. These studies suggest that a,M conformational change intermediates demonstrate common structural char- acteristics, permitting an elucidation of the steps involved in this complex transfor- mation. Marshall. L. B.. Figler, N.1,., and Gonias, S. L. The Journal of Biological Chemistry 267(9):6347-6352. March 25. 1992. Other support: Pew Scholars Program. From the I)eparlment of Pathology and Biochemistry, University of Virginia llealth Sciences Center, Charlottesville. STRIIC"TURAL ANALYSIS AND GXI'RESSION OF HUMAN DESMOGLEIN: A C'ADI II;RIN-LIKE C'OMPONhNT OF THF, DESMOSOME Desmosomes are adhesive cell junctions found in great abundance in tissues that experience mechanical slrc.s. The transmembrane desmosomal glycoprotcins have been proposed lo play a role in cell adhesion; desmoglcin 1(D(I) is a major member of this class of desmoenrnal molecules. However, evidence supporting a role for I)GI in cell adhesion or in the plaque is lacking. In order to begin to understand DGI function, we have identified human cDNA clones encoding the entire maturc polypeptide of I(1(X) amino acids. Our data suggest that like Ihc bovine D(;I molecule, ttuman DGI is highly related to the calcium-depen(lent class of cell adhesion molecules known as cadherins. Four related extracellular domains loc:ated in the atnino-terminal domain of the molecule contain putative calcium binding sites originally identified in the cadherins. The highest degree of similarity between human N-cadherin and human 1)GI, and likewise between bovine DGI and human DGI, is greatest in the most amino-terminal extracellular domain. This suggests a 140 1 141
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c•nnticrvcd frmctirniarl role for Ihc crlracrllular clomains, pcrh:rp% in calcium-mcdiated cell acfh"ion. 'fhc cytohlasmic portion nf Ihc mnlc(-ulc rc~nlain~ a cadhcrin-like region and, like bovine I)GIL a carhnxy terminal tail that i% not present in Ihe cacfhcrins, cumpricin}t three addilicmal domains. One nf thc~c contains a nnvcl rclx•ating motif of 2')+ I resitlucs, first identified in bovine D( iL I?:tch of thc highly homnlognu.s rclxating units iti likcly to cnnsist of two (;-xtramN and two turns with ~;pccial charactc•ristice. Five amim0 acids that are iclcntical in bovine and human D(il lie in the second ol* thc two predicted G3-strands, and intriguingly contain putative t:nRct sites for pmtein kinase (•. On the hasi; nf' structural analysis, a model prcdicting the disposition of human DGI clomains in the desmosomc is proposed. - Northern analysis sugf:cas that unlike bovine epidermis, which expresses a singlc mRNA of reported sim 7.6 kh, human Goreskin and cultured keratinocytes display a complex panern with hands of 7.2, 4.0 ancl 3.Ukb. Each (if these cross- hyhriclizing mRNAs is coordinately expressed in nonn:d human kcratinocytcs in response to hmg-dcrm cullurc and incrcatiecl calcium. Nilles, l,. A., Parry. I). A. I)., Pnwcrs, E. F.. Angst. B. F)., Wagncr, R. M., and Grrr'n, K. .l Journal nf ('cll Science 91):R(M)-}{2I. 1991. Othcr snppnrt: American Cancer Society and N:uional Institutes of Hcalth. Frnm Ihc 1)cpsvtment of I'athnhogy and ('ancer ('cnter, Northwestern l)niversity Medical School, (•hicagn, and Massey t Inivcrsily, 1'almcrston North, New Zealand. MOI,li('III.AR S"T-RlI('TIIRF,OFTI11; HIIMAN DI'SMOPI.AKIN I ANI) II AMINO'1-1?RMINIIS Dcnmuplakins (Dl's) 1 ancl 11 are clnscly related proteins louncl in the innermost region of the desmcr.,omal plaque. which serves as a cell surface attachment site for c•ytc,plasniic inlcrmecliate filamems. Overlapping cDNA clones comprising 9.2 kilo- hascs nl 1)1'-I- hrcclictccl to encode a full-Iength 3I0-kDa Pnlylxptidc (2677 amino acid reci(lncs), have mow been identified. Ilere we report the predictecl protein w(ncncc and %tmctural analysis of the N terminus of I)I', extending our previous snidv nl the rnd emd carhnxyl domains. The N terminus contains groups of heptad rcpcnt% th:it nrc hrrclickYl to forni :il least two major n-hclical-rich bundles. Unlike the rod and c:uhoxyl dnmains, the N termittus did not display a periodic distribution of ch:rrf!ccl rc.iducsi Northern blot mapping and gcnomic sequence analysis were also undcrtakcn tn examine thc organization of thc DP mRNAs. A I-kilohasc intron was Incateel at the 3' boundary eif a I)P-1-xpec•ific region; however, instead of an intron at thc S' junctirni, a pn.xihle splice donor site was observed within a potential codinF scqucncc- suggctting ahcrn:divc RNA splicing from an internal drmor sitc. Virala, M. 1.. A.. Wagncr- R. M.. Parry. I). A. D., and Green, K..1. Pnxeeclingc nf Ihe Naticm:d Academy of Sciences IISA R9:544-54R,.lanuary 1992. Other mupport: National In%titutcti c,f Ilcalth, March of Dimcs Birth I)cfccts Fnundatiom and Amcrican ('anccr Socit•ty. t Fmm the Department of Pathology and Cancer Ccnter, Northwestern llnivcrsity Medical School. Chicago. and I)cpartmcnt of Physics and Rinphysics• Ma~~cy lhiivcrsity. l'almerston North, New Zealand. THE DESMOPLAKIN CARROXYL TERMINUS COALIGNS W1T11 AND SPECIFICALLY DISRUPTS INTERMEDIATE FILAMENT NETWORKS WHEN EXPRESSED IN CFILTUREI) CELLS Specific interactions between desmoplakins I and 11 (DP I and 11) and other desmosomal or cyto-skeletal molecules have been difficult to determine in part because of the complexity and insolubility of the desmosome and its constituents. We have used a molecular genetic approach to investigate the role that DP I and Il may play in the association of the dexmosomal plaque with cytoplasmic intermediate filaments (IF). A series of mammalian expression vectors encoding specific predict- ed domains of DP I were transiently expressed in cultured cells that form (COS-7) and do not form (N1H-?T3) desmosomes. Sequence encoding a small antigenic pep- tide was added to the 3' end of each mutant DP cDNA to facilitate immuno-localiza- tinn of mutant DP protein. Light and electron microscopical observations revealed that DP Polypeptides including the 9O-kD carhoxy-terminal globular domain of DP I specifically colocalized with and ultimately resulted in the complete disruption of IF in both cell lines. This effect was specific for IF as microtuhule and microfilament networks were unaltered. This effect was also specific for the carboxyl terminus of DP, as the expression of the 95-kD rocl domain of Dl' I did not vicihly altcr IF net- works. lmmunogold localization of COS-7 cells transfected with constructs includ- ing the carlxoxyl terminus of DP demonstrated an accumulation of mutant protein in, perinuclear aggregates within which IF subunits were sequestered. These result,; sug- gest a role for the DP carboxyl terminus in the attachment of IF to the desmricome in either a direct or indirect manner. Stappenbeck, T. S. and Green. K. J. The Journal of Cell Biology 116(5):1 197-12(19, March 1992. Other support: National Institutes of Health, March of E)imes Birth 1)cfects Founclatinn and American ('anccr Society. From the 1)cparlnient of Pathology and Cancer Center. Northwestern University Medical School. ('hicago. ISOLATION OF INTERCELLULAR .IUN('TIONS Early clecton microscopic studies revealed the existence of a number of distinct types of intercellular junctions. Specific physiological properties of a cell or tissue could often he correlated with the presence of a particular type of junction, tiuggest- inF very different functions for each type. Some types of cell contacts, the tlesmo- 142 1 143
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.nrncs and adhcrcns junctirnrs, apPcarcd to Iclhcr diffcrcnt clcmcnts of thc ccll's cytn4clelnn to the plasma membrane while fnrming adhesion Pnintti between cells. Othcrs were proposed In provide a harricr to paraccllular difTusirni of solutes (light junctinne) or facilitatc intercellular communcatirnr (gap junctions). Roles were hypntheci7ed for these junctinn,; in ti%sue modeling, and in regulatirni of cell func- lirni and ccll proliferation. The ahility to isolate intcrccllular junctinns has led to the development of more dctailcd molecular models of thccc junctinns- -and to many surprixes. For gap junc- tinnti, a family of structural prntcinx, the connexins, has now been idcntificd, prc- sumably with individual properties which influence Ihe activity of intercellular chan- nel.. 'I'ight junctions are now known tn not only form a diffusion ban-ier for solutes, hut for plasma membrane proteins and lipids, thereby creating di.ecrete domains within the cell's limiting membrane. Adhesive junctions arc now being found to share somc protcim and overlap in thcir interactions with cell adhesion molecules, a li•ature in common with contact pnint% Ixtween cells and substrata and the extracel- lular nratrix. While complexity has incrcascd, common principles are being found. And the ahility to Icst Ihc hypnthctical rolcc (if thctic interccllular junctions in cellular and devcfopmental Pracetice% is now. with the isolation and characteri/atinn of intercellu- lar junctirnrt, yielding to experimental analysis. I lcrtzlx•rp_, f;. I.., Stevenson, 11. R., Green, K-.I.. and Tsukita. S. In: Slcvcnsun, B. R., ('nllin., W. J., and Paul, I.). L. (eds.): Cell-Cell Interactions, a Practical Aprrn:rch.Oxl'nrd (Inivcrsity I'rc,;s, London. pp. I I I-142, 1992. OOther surprrrt: National hrstilutes of Ilealth, Irma T. Ilirschl Career Scientist Award. Anrcrican ('anccr Society. March of I)imcs Birth 1)efccts Foundatinn. Ministry (if Educatirnr. Science and ('ulturc of .Iapan, Medical Research C'ormcil of C'anada, and Allxrta Hcrilagc Fnundatirni fnr Medical Research. From the 1)chartment of Pathology and the ('anccr Centcr, Northwestern University Medical Scho+rl, ('hicagn. I)I:VI?LOI'MI;N'f AND IISI: OF Rli('I;P'fOR 13IN1)ING PFTrI1DIS DERIVED I•ROM AN'1 IR('.(TI'TOR ANTIRODIIS Many lig:mds have hccn described that interact with specific receptors on cclls. witlr suh,,cqucnl cffccls nn ccllular physiology. Recent advanccs, in inrnrunningy and mnlccular hiningy have greatly enhanced our understanding of how ligands and reccptnrti interacl. Thc development of hnth pnlyclrnral and mnmi- clnn,rl amtirccchtnr antihndics has allowed the isolation and subsequent characlcri- iatinn of a wide variety of receptor structures. Mnlccular cloning techniques have allnwed the cfucid;ilinn of the primary amino acid sequences of many receptors and Ihcir cognate Iip:mds. Hcre we dcticritx a mcthud for the development of receptor binding Ixptid" based un the primary structure nf antircccptor antihodics. Williams. W. V., if'eincr, I). B., Cohen, J. ('., and t':rrrirr. M- I. 13intcchnnlngv 7:47-475, M.rv 1999. Other Support: National Institutcs nf I Icalth, National ('anccr Institutc and American C anccr Society. From the t)ivisinn of Rhcumatology, Department (if Medicine. 1)ivisirnr of Immunningy, I)cpartmcnt of Pathology and Laboratory Medicine, and 1)cpartnrcnt of Ncurolugy, University of Pennsylvania School of Mcdicine, Philadelphia. CYTOSINE METHYLATION CAN INDIICE LOCAL DISTORTIONS IN TI1E STRUC'T(IRE OF DUPLEX DNA Methyl groups at the C5 position of pyrimidines located within oligopurinc- nlignpyrimidine tracts in DNA have been shown previously to modulate curvature generated hy those tracts. However, it was not known whether the influence of such methyl groups is consequent to the altered helical structure within the tracts them- selves. In the current study, it is demonstrated that methylatinn of cytosincs tip to three base pairs away from a(dA),(dT), tract (A-tract) can still result in alterations of the nct curvature of the A-tract-containing DNA, as measured by aherations in clcc- trophoretic mobility. This latter effect depends strongly on both the sequence of the non-A-tract DNA and the positions of the methylated C residues. The current results lend further support to the notion that the biological consequences of cytosine methy- lation may be effected through local alterations in I)NA structure as well as through direct protein-DNA interactions. Hodges-Garcia, Y. and Hqkkerman, P. J. Bioc:hcmistry 31(:13):7595-75<)9, 1992. Other support: National Cancer Institute. From the Department of Biochemistry, Biophysics and Genetics, University of Colorado Health Sciences Center, Denver. THE PRODt IC'f OF THE CELLULAR crk GENE CONSISTS PRIMARILY OF SH2 ANI) S113 RE(ilONS We have cloned and sequenced a complementary 1)NA encoding the cellular homnloguc of (hc Iran%forming cmcogcnc v-rrk of avian sarcoma virus CTIO. l'his complementary 1)NA contains an open reading frame of 915 base Pairs that encodes a lxrlypcptidc of 305 amino acids. The first 20.5 amino acids of this c-Crk protein were identical to those nf thc CTIO encoded v-Crk protein, with the exception of 5 amino acids. Like v-C'rk, this portion of c-Crk contained one cach nf thc Src homolo- gy domains SIf2. SH2', and S113. The I(X) carhnxy-terminal amino acids of c-Crk protein. which are not cnded for in the CTIO viral genomc, contain another S113 region. We found limited sequence homology hetwcen c-rrk and Ihe avian rctrovirns genome, which explains rccombinatinn events in the transduction of this protoonco- gene. llsing a pnlyclnncrl anliscrum made against bacterially expressed v-rrk, we 144 1 145
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idcntificd a 35-kihodallnn protein in normal chickcn cmhrvrr fihrnhlatite and in all cmhrynnic chicken tissucs cxamincd. This 35-kilndaltnn protein was indistinguish- ahlc from a Pcilypcptidc made by in vitro hantilatinn of c-rr k cnmplcmcntnry I)NA. Rcichman, C. 'I'.. Mayer. R. .L, Kcshav, S., and llunq/'ir.ca. /l. ('cll Growth & Diffcrcntiation 3:451-46O, .luly 1992. Othcr support: National ('anccr Institulc. From The Rockefeller Univcrsily, New York. l'YROSINIi-PI1OSPIIORYI,ATEO EPIf)FiRMAI, GROWTH FACTOR RE('I:P'T'OR ANI) ('F?LLIII,AR P130 PROVIDE FIIGH AFFINITY BFNI)ING S1113STRA'I-T:S'I'O ANAI,Y7,1; ('rk-I'llO.SI'IIOTYROSINF.-hEl'F,NI)F.NT INTERA("IIONS IN L7TRl) The genome nf ('TIO avian sarcoma virus encodes a 47-k1)a fusion protein that consists nf viral gqg sequences fused to a ccll-dcrived sequence containing S112 and S113 domains (v-(r&). Genetic and biochemical evidence suggests that v-Crk can induce Irancformatinn uf chicken embryo fibroblasts by influencing the activity of cellular proteins involved in growth regulatirm. In this rcpnrt, we have developed an in rilrn micrcrtitcr aseay to study the binding of bacterially expressed glulathionc S- tr:msfcrasc-fusiun proteins rrl'v-Crk and its cellular hmmoing, c-('rk, to the phospho- rylated cpidcnnal growllt factnr « ccptor (1?(;F12). ('ompetitive binding data are pre- scntcd that compare the ahilitics of hclcrologuus glutalhionc ,S-tramfcrasc-fusinn pro- tcim containing GAI'S112jNj, AhIS112, Src:S112, and Pf.('-ySll2jNj sequences to in- hihil ('rk hinding. Results indicate Iha1 both full-Ienglh Crk and GAPSI121NI bind the phrrerhorylaterl 1;(il•12 with high affinity and can quantitatively compete the hind- ing nf cach other by competitive cniymc-linkcd immunntiorlxent assay. Binding of Ibll-Icngth ('rk or the isolated S112 domains crf (iAP or AhI resulted in a significant prntcc•tir,n ol phnsphnrylatcd li(;FR against dcphriephorylation by ccllular phoz- phatase activil,v. but did not appear to stimulate the intrinsic tyrosine kinase activity of the NGFR. 'fn extend these findings to pl.iO, the major phnsphrityrosinc-cnntain- ing prnrcin in ("fIt) Iransfnnncd cclls• we utiliicd a nitmcclluhisc filler binding a«ay. Rrtiult~ +Icmnmtratc high alfinity binding nf ('rk Ioward dcnaturccl p110 ancf, w, i,~ thc cumc f++r phrnph„rylalcd Ii(;FR. C'rk binding can partially protect pl l0 from ph+nhh;d:vw arlivity. Ilnwcvcr, no apparent compctitinn nf ('rk binding was noted wilh hclcn+h+t~rnis 5112 containing proteins including GAI?SI12(NI, suggesting a pns- %ihlc lpccifrcil, y of ('rk pil ZO hinding.'lltese data are consistcnt with a direct role of S112 in the nrn+lulatinn of c•cllular phrnphntynKinc stanrs irr rirn. Birgc. R. B.. F,rjardn, J. F.. Maycr• I3. J._ and llunuM.ca. 11. The Journal of Ririhrgical ('hcmi+;try 2h7(15):105f{R-10595, May 25, 1992. Othcr supprirl: Natinnal Institutcs of Ilcalth, American Cancer Sncicly• Damon Runynn-Wallr•r Winchcll ('ancrr Research Fund. U.S. Public Itcallh Service, and Nalirmal ('anccr Institutc. Frnm the F)cmrtmcnl of Molecular Oncnhr}±y, Thc Rockefeller Onivcrsily, New York. MOLECULAR CLONING ANI) I:XPRESSION OF CIiICKEN C-TERMINAL Src KINASE: LACK OF STARLE ASSOCIATION WITFI c-Src PROTEIN Cloning and sequencing of chicken C-tcrminal Src kinatc (CSK), a tryosinc kinasc that phosphorylates the regulatory C-lerminal tryosine residue present on cyloplaxmic tyrosine kinases of thc Src family, demonstrated a high degree of inler- species conservation as well as src homology 2 and 3 domains N-terminal to the kinase domain. The lack of ardophnsphorylation sites distinguishes CSK from other tyrosinc kinaces. C'SK is unique and does not belong to a gene family, suggesting that il may phosphorylate other members of the Src family of tryosine kinases in addition to c-Src. Since complex formation between c-Src and CSK seemed a likely regula-Inry step in the ccmtrol of c-Src kinase activity, such an association was inve5- tigaled by immunoprecipilation and Western blotting as well as intracellular local- ization studies. Although some pnrtions of CSK were found in a membrane fraction, no complex formation between CSK and c-Src was observed, suggesting that the src homology 2 domain of CSK does not play a role in the direct interaction of c-Src. Salx, IL, Knud.scn, B., Okarla, M., Nada, S., Nakagawa. H., and I-lanqfilsa, N. Proceedings of Ihc National Academy of Sciences l ISA 89:2190-2194, March 1992. Other support: National Cancer Institute. From the Laboratory of Molecular Oncology, The Rockefeller University, New York, and Division of Protein Metabolism, Institute for Protein Research, Osaka 1)nivcrsily. Osaka. Japan. MECIIANISM OF ACTION OF A REPRESSOR OF DIOXIN-DEPF.NDF.NT INDUC"I'ION OFC'YPIAI GENE TRANSC'RIP'1'ION A dominant mutant of Hepa-1 cells, cat, expresses a repressor that prevents 2,3,7,R-tctrachlorcxlihenzo-p-dioxin (TCDb)-dependent stimulation ol'('vplal trans- cription. The repressor acts via the xennhiolic-retiponsive elements (XREs), which are the DNA-hinding sites for the aryl hydrocarbon (Ah) receptnr-T('DF) complex cluring transcriptional activation of the gene. Nigh-salt nuclear extracts prepared from c3I cells grown with T('I)I) contained normal levels of the Ah receptor which Ixxmd the XRE with normal affirtity, as jurlged by in r'irrn gel mobility shift assays. Furthermore, extracts prepared from thesc cclls, grown either with or withrnd'1'('I)1), contained no novel XRE-binding proteins compared with extracts from wild-type Ilepa-I cells. Ilnwcvcr, in envr g,cnomic Gxrlprinting demonstrated thal TCDD trcal, ment Icads to binding of the AIi receptor to the XREs in I lepa- I hut not mutant cells. llris finding tiuggesl.ti that the repressor associates with the Ah receptor to prevent its binding to thc XRI?s and that high-salt treatment either causes clissrxiation of the receplor/repressor complex nr fails to extract Ihc repressor from nuclei. 9'hc results underscnrc the importance of using both in rirn and in virrr, assays for analyAng I)NA-protcin interactions. Watson, A, J., Weir-Rmwn, K. L, Bannister. R. M., ('hu, F.-F.. Rcisz Prxs/asi. R., Fujii-Kuriyama, Y.. Sngawa• K.• and llnnkin.cnrr, O. Molcc•ular and ('cllul:rc Biology 12(5):21 15-2121, May 1992. 146 1 147
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OIher suppnrt: II.S. I)cpartmcnt nf I?nergv, ('alifiirnia lwlitidc for ('ancer Rescarclt and (';mcci Research ('aordinating ('nmmiltec of file I lnivcrsitN- ilf (':difiornia. Prnm the I.aboratory of Biomedical and I:nvirunmental Se•iences, School of Puhlic I lealth, I)cpartmmnt cif I':Ithohogy and I~ahnratory Meclicinc. l inivcrsity nf ('alifornin• Los Ang,eles, :ind I)erartntenl oit ('hemiary, Facuhy (if Science. Tnkoku l Inivercity, Scndai, .lahan. TuWS A('TIVATION OP TI IE SIMIAN V1Rl IS 40 LATE PROMOTER BY I.AR(;t: T ANTIGP,N RI; .(?LIIRI?S I;INI)ING SITES FOR THE ('F1,LIILAR 'I'RANS('RII'TIUN FA('l'OR "1'IsF-I Simian virus 40 (SV40) Tatttigen stiniulates the level of Iranscription from sev- cral RNA pcrlymcrase II promoters, including the SV4(1 late promutcr. The nmcha- nism ~~f hun.c aclivati~m aplx nrs to be indirect since hincling of 'T antigen ln spccific 1)NA sequences is not reyuirect. Ilowever• specific promoter elements that respond to 'f antigcn havc not previously been defined. We identified L)NA scyuenccs from the SV4fI late prrnnnleo whnsc ahility to stimulate transcription is induced by the expres- sinn of '1' antigen. In particuLir, file Sph I + Il motifs uf the SV4(1 enhancer can confer 'f-antigen inducihility to Ihc nnrmally uninclucihte herpes simplex virus thymidine kinase gene promoter when multiple copics of file sequence are inscrted 5' of the trana•riptinn initiatinn sile and '1'A'1'A seyuence. Binding sites for the cellular tran- scriptiom factor'll?1"I and oxtamcr hinding pmlcins are cclntainccl within file Sph I + If mutifs, as wcll ati at other pcnitioms in the SV411 promoter. To study the role of indiviclual prutcin-hinding sitcs in nnns activaUinn hy'1' antigcn. mut:dions were con- strttcted in various'IIiF-I and ocl:mter protein-binding sites of the SV4(1 late promo- ler. These mulatioms did not significantly affect hasal promoter activity. However, mutalion nf all Ihree'fl?p-I sites prevented detectable activation hy T antigen, DNasc I footprinting of file mutatcd pinmotcrs with purifiecl proteins demonstrated that induc•ihility hv'I' antigen cnrrclateel with hintling aflinily of'1'I?F-I for the I)NA and not with hindingallinity (rf :ni oclamer binding prntcin, ('ns:v• I'., Snmcth. R.. and !lu/r«•n. ll. Inurnal of Vitoh t•v (,5( I.')-65 t5 6543, Decemlxr 1091. OIhcr %iihhort: March (i/ I)imcs Birth I)cfcc•ts Prnmdalion, American Cancer Society ,uul N;uion;il lmlilutc,; of I leallh. I,rnm thc (omnnittce (m Virnhigy, I.ahoratory of F?ukaryotic Trmiscription, I)ana I•arhe•i (':mc •r Imlinne• and 1)cp:lrtmenl n( Micrnhinhogy and Molecular Genetic•s, I Inrvard Mcdhc al Schoud. Itndun. AI.TI?RNA'I'IVI: . RNA SI'I,I('IN(i IN'I'ltli ('ONTROI. OI; GI?NI; I?XPRNSSION IN MtIS('LF AND NONMIIS('I.I•.('1'.I,I,S Ilaving idi•nlificcl ccHular factors Iltat intcract with specific scyucnccs in file Inc niRNA. .+r c:un now ctircct onn clforl, In a greater understanding of how these factnrs act to regulate alternative splicing, Whether or not the facaxs identifieel in Ihe present .etudics are expressed in a tissue specific-manner remains to he determined. What is clear is that the binding of these proteins to the pre-mRNA is involved in regulated alleniative splic•ing and that this interaction is required for blocking the usc of the skeletal muscle exon in mtinmusclc cells. C'Iearly,,adclitional factors cnul(I be involved in tissue-specific alternative splice site selection. Although the use of thee skclet:d muscle exon in nonniuscle cells is suh.ject to negative regulation via block- ing faclors, it remains to he cleterntincd how regulation is achieved in skeletal muscle cells. Our results do nnt rule out the possibility that positive-acting factors will be requirecl in skeletal muscle cells to promote the use of exon 7. In the future, we hope to determine the nature of the factors iclentificd in the present studies. Work is in progress to purify the factor(s) that interact with the RNA, to precisely identify the sequences to which they bind, and to develop in rin•o assays for their function. Guo, W., Mulligan, (i. J., Wormsley, S., antllhlfinrmt, n. M. In: Kclly, A. M. and Blau. H. M.(Eds.): Ncuromuscular Development and Discase. Raven Press, Ltct, New York, pp. 157-172, 1992. Other support: National Institutes of Health, National Cancer Institute and Muscular Dystrophy Association. From the Cold Spring Harbor Laboratory. Cold Spring Harhor, NY. DIFFERENTIAL REGULATION OF PROTEIN KINASE C ISOZYMES BY TIIYROTROPIN-RE,LEASING HORMONE IN GH~C, CELLS GH c, cclls, which express Ca"-ctepcndcnt a- and (3- as well as Ca"-indepcn- dent y-, e- and t-protcin kinase C (PKC) isozymes, provide a cell culture model for studying isozyme-specific properties and functions. Hormonal activation of PK('s regulates the differentiated functions of these cells, namely secretion and synthesis of prolactin (PRL). We previously reported that lhyrotropin-rcleasing hormone (TRI1) selectively clnwn-moclulates E-PKC with no effect on rx- or (3-PKCs (Kiley. S. C.. Schaap. I)., Parker. P.. I Isich. I..-L., and Jaken• S. (1990) J. Biol. C'hem. 265, 1 5704-1 57 1 2). We now crtend those studies to explore the relationship between TRH-stimulated diacylglycerol (DAG) levels and E-PKC down-moclulation. TRII stimidates three distincl I)AG phnscs in (7II cells. Phase I DAG peaks at 15 s, is accompanied by a 6-fnld increase in intracellular Ca", and causes the redistribution of o-, (3-, fi. and F 1'K(' i,~orymc,; from a soluble to a dctcrgenl-insoduhlc particulate compartment. Phase 2 I)A( ; peaks at I(1 min, is not associated with a('a" signal, and does not activate PKC by any criteria tested. Phase 3 DA(i peaks at 6 h and is sus- taincd through 12 h. -Ihis novel 1)AG phase is not associated with incrcasc(I intracel- lular Ca''. The time course of phase 3 DA(i fonnation corresponds to the time coune of TRII-stimulatecl e-PKC down-regtdatiun: niaximal effects are ohservect at 6-12 h for both events. Unlike n-, /3-, and Fi-PKCs which arc preferentially distributed in the soluble fraction of resting GH cells. e-PKC is also distributed in the detcrgcnt-insol- uhle particulate frac•tion. The selective compnrtmentilirdiem of e-PK(' in the particu- late fraction may render this pool uniquely susceptihle to proteolytic degradation. The linu c•ourse of phase 3 1)A(i fnrmatiim and e-I'K(' clown-modulaticm corre- 14R 1 149
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sponds Io the time course of dccrca';ing I'RI. mc«agc eynlhcsi~, in (iFl ccllg. The d;da suggest that loss of e-1'K(' may hc assncialcd will) Ihc down rcgulation of pro- lactin synlhcsiz and that rcgulauion of PRI. gene tramcriplirm may hc an e-PKC'-spc- cific function in GII cells. Kiley. S. C., Parkcr, T'. .T., Fahhro, D., and.lukrn„S. 'Fhc.lonrnal of Bioingical ('hcmistry 2h6(l5):2;761-2376R, December 15. 1992. Othcr tiurprnt: Natinnal ('anccr Institulc. froni W. Allon Jones C'cll Science ('enler, Lake Placid, NY: Imperial Cancer Research Ptmd, I,ondrn), I:ngland; and the Pharmaceutical I)ivision, ('IBA-(iE.ICiY, Bascl, Switicrland. IIORMONE- ANI) PIIORROL F;STER-A(TIVATED PROTEIN KINASE C ISO7.YM1?S MI?I)IA'1'T; A RliOROANI%A'T'ION OFTIIE ACTIN ('Y'1'OSKE.I.1:'I-ON ASSO('IATFiI) WfIII I'ROI.A("1'IN SECRETION IN (iII,(', ('EI,t,S 'fRIT regulates PRI. secretion and synthesis in OI1,(', rat pituitary cells. TRH respm)scz are asscxiatcd with aclivatinn nf protcin kinase C(I'K(') itiozymcs and clc- vatinn of cytnsnlic calcium. To determine which 1'K(' isozymes are involved in TRI I-dircctcd reelrnnscti, we cvaluatccl the effect of TRFi on GFI cell Ix-, (3-. Fi-, and e-PK(' isnzymcs. Inm)unohlot analysis demonstrated that TRI I caused rapid redistri- hutirm of all isnrym~cs to st I'riton X-10O-insoluhlc (i.r., cytockclctal) fraction. ('nrollary innnnnncytofluoresccncc studics clcmonstratcd that redistributed PKCs accumul:uc in c(•II peripheries. I?xncytosis involves rcorganiiation of thc cytoskclc- Irnt, thcrcforc. each of Ihc (ilI cell PK('x is aprropriately located to phosphorylatc proteins imprnlamt for cytoskclcton nrganirttion. To determine the relative contrihu- lionc nf'calcium .md I'K(' signal Iransduclirn) pathways in rncdiating'1RF1 responscs, Ihc cffccl% of hnlasximn dcrolaritalion (which increases cylosulic calcium) and hhorhol dihutryalc (which activalcs all PK(' itiozymcs without increasing calcium) were crnnharcd. "Ihc data indicalc Ihat TRII-tnediatcd reorganiration nf vincttlin pro- cccdti via a talciun)-n)cdialcd pathway, whereas fragmcntation of actin filaments pnxrcd% via a I'K(' dependent pathwary. Selective down-mcxlulation of E-F'KC with prolonged TRIl-trrnmcnl was uscd to demonstrate Ihat e-I'K(' is not necessary for ccrtain'1'RI I-stimulalcd biological resEonsct. Kilcy, S. ('., I'arkcr, P..I., Fahbro, I)., ancl.ln6•r)r„S. Molecular FAidocrinnlogy A( I):12O-131, 1992. Oihcr suphorl National Cancer In,~Iitulc. Frorn W. Alton Jones Cell Science Center, Lakc Placid, NY: Imperial Cancer Research Fund. I.ondon, F;ngland: and 1)cpartmcnt of Virology and Oncology. Pharrnaccutical I)ivsiun. liascl. ('IRA-Gcigy SwitVCrland. IiTI,V-I TAX IS A 7.INC-BINDING PROTEIN: ROLE OF ZINC IN TAX S'TRLI("f11RE ANI) FUNCTION We have examined the functional significance of the cysteine- and histidinc-rich region (amino acids 22-53) of IiTLV-I Tax. A modeling of this region suggests two possible overlapping zinc-fingcr-likc motifs. Using a zinc blotting tcchniyue, we show that Tax hinds zinc. An N-terminal deletion in Tax that removed this zinc- finger region abolished the ability to bind zinc. Site-directed mutagencsis was used to gcneratc 10 separate mutations so as to discriminate between the two alternative zinc-binding structttres. Each Tax mutant was studied for its ability to trans-activate the I ITI,V-1 LTR. Five of Ihe ten mutations inactivated trans-activation. Our results support that one of (he two putative zinc fingers is an integral element of Tax stntc- lurc. Semmcs, O. J. and.Jenng, K.-T. Virology IRR:754-7(,4, 1992. From Ihe Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD. KINE'F'IC'S OF IIIV-I LONG TERMINAI, REPEAT iknvs-ACTIVATION-Ilsr: rn: IN1RAGfiNt(' RIRUlYMI~. "f0 ASSESS RAl'f-LIMI'I'IN(G S"17iPS We have examined, using self-cleaving rihozymcs, the intracellular rrnrr.c- aclivation kinetics of the human immunrxlcficicncy virus type I(111-1) long tcrntinal repeat (l;!'R) by viral protein Tat. Experiments were designed to effect a comlxlition (during RNA chain elongation) between cleavage of a nascent RNA containing Ihe Tat-responsive target sequence (TAR) and Tat interaction with the same TAR in the process of LTR-trans-aclivation. We found that fast self-cleavage of nascent TAR- containing RNA abolished Tat trnn.c-activation, Slowing the cleavage reaction kinetically rescued trans-activation. Based on our results, we conclude that the rate- limiting step in IIIV-I LTR truns-activatinn is the initial contact made between Tat/TAR/LTR rather than the promoter proximal pausing of RNA Fx)lymerascs that are tethered to functional l'AR. Jenng. K.-T. and Bcrkhout, B. 'I'hc.lottrnal of Biological ('hcn)istry 267(25):17R91-17R99, Scptembcr 5, 1992. From the I.alxrratory of Molecular Microbiology, National Institute ol' Allergy and Infcctious I)iscascs, National In%titutcs c)f Ilcallh, Rcthcscla, MD. 1?LEC'lRON MICRnSC'O1'I(' IMMUNOCYTOCIiF•:MI('AI. I,OCALI7,ATION 011' 1.II'II) I'1?ROXIDATION PROD(I("I:S IN 1'TIArO('Y'fOSING I1(IMAN NI'.l1TR( )I'I I II S lhunan neutrophils produce superoxide anion (O, ) when stimulatcd by a vari- cty of %nluhlc and particulatc stimuli, including phorhol myrist:dc acctatc (1'MA) and 151 151) ~_
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hcat-killcd opsrmiicd .Sraplrvlnrorw.c aurru.c. Supcrnridc anion gcncratcd by acti- v:ucd ncutrrrphitN can react witlr a mmnhcr of lixsuc targctc, including pnlyunxatu- rated fally acids- of neighboring ccll mcmhrancs. I.ipid pcroxidatirnr resulling from frcc radical chain rcactirms initiated hy O, attack results initially in Ixroxidizcd frcc L•uty acids and phospholipids which subsequently break down into short chain clcav- agc products such as hydroxyalkcnals and hydruxyaldchydcs. One such product is 4-hydmxynunclal (4-IINE). 4-IINE has been shown to have a number (if toxic hio- logical cffccts, and it has been suggestcd that 4-I INE protein adducts might be gcn- cratcd at sites of free radical-mcdialed inflamnratirm and, thcrcforc, could serve as markers uf Iipid pcroxidation and possibly memhranc damage. I,inncr, J. G., Rucschcr, G. S., Siemscn, 1).. Dram E. A., Quinn, M. T., and .lr.cailis. A. .1. In: Molecular Basis of Oxidative 1)amagc by Lcukocyles. Proceeding of the Montana State I Iniversity. ('R(' Press, Boca Ratnn, 1'l., pp. 277-2R1, 1992. Other support: Anicrican Lung Association. From the I)epnrlment c,f Chemistry and Riochcmistry, Montana State llniversity, 13o7eman, and I)cparlnicnt of I'cdiatrics and Infectirws Disease. tlnivcrsity of Tcxas lieallh Science ('enler, I louston. TIaR1iSI1OI.I) I'1IFNOMF:NA ANI) I.ONG-1)ISTAN('E ACTIVATION OF TRANS('RII''1'ION BY RNA I'OLYMERASE II 't'n explore the underlying mechanisms by which genes are regulated in cukary- nlcs. Inng-ditil:uicc transcriptional activation and threshold effects were reconstituted in rin». Lnng rmgc activation of transcriptinn by GAL4-VPI6 prolciti located 13(X) base pair uh~trram of Ihe RNA start site was dependent on packaging of thc template into hitilomc III conlaininft chmmatin. A Iranscriptirmal thrcyhold effect by GAL4- VI'It, wa.-, „h.crved with repressed chromatin templates but not naked DNA tem- hlalcs. 't'hc experimental data with the chromatin templates were similar to the thco- n•lic:rl activation profile that is predicted if thc .rctirm of each DNA bound promoter (if GAIA-VPIh were indclx•ndcnt and additive in Icnnx of free energy. Layhuurn. 1'. J. and KarL,nr{la, .l. '/'. Science 257:IhR2-I(iR5, September IR. 1992. OOther support: Naliomal Institutcs of Ilcallh. National Science Foundation and I.ucillc P. Markey Charitable Trust. From the Depariment of Biology and ('enter of Molecular Geneticti, llniversity of California at San 1)icgn, I.a Jolla. ISOLATION, CI IARACTERI"/.A'I'ION. ANI) REGIONAL MAPI'ING OF MI('RO('LONES FROM A 11(.1MAN ('I IROMOSnMIi 21 MICRODISSI's(TION 1.I13RARY Thirty-four unique-scqucnce microcluncs were isolated from a previously deticrihed microdissection library of human chromosome 21 and were regionally mapped using a cell hybrid mapping panel which consists of six cell hybrids and divides chromosome 21 into eight regions. The mapping results showed that Ihc micnx:lnncs were unevenly distributed along chromosome 21, with the majority of microclones located in the distal half portion of the long arm, between 2lq2l.3 and 2lqter. The number of unique-sequence clones began to decrease significantly from 21q21.2 to centromere and extending to the short arm. This finding is consistent with those reported in other chromosome 21 libraries. Thus, it may be inferred that the proximal portion of the long arm of chromosome 21 contains higher proportions of repetitive sequences, rather than unique sequences or genes. The micrcxlones were also characterized for insert size and were used to identify the corresponding genom- ic fragments generated by lfindlil. In addition, we demonstrated that the microclones with short inserts can be efficiently used to identify YAC (yeast artificial chromo- some) clones with large inserts, for increased genomic coverage for high-resolution physical mapping. We also u.ced 2(X) uniquc :eequence microclones to screen a human liver cDNA library and identified two cDNA clones which were regionally assigned to the 21q21.3-q22.1 region. Thus, generation of unique-sequence microclones from chromosome 21 appears to he useful to isolate and regionally niap many cDNA clones, among which will he candidate genes for important diseases on chromosome 21, including Down syndrome. Alzheimer's disease, amyotrophic lateral sclernsis, and one form of epilepsy. Yin, J., I lartz, J., Ku, Y., Gemmill, R. M., Korenberg. J. R., Patterson. I)., and Kan, F. T. American Journal of Human Genetics 51:263-272, 1992. Other support: National Institutes of Nealth, Department of Energy. American Health Assistance Foundation, Alzheimcr's Association. March of 1)inies Birth Defects Foundation, and Lucille P. Markey C haritaMe Trust. From the Eleanor Roosevelt Institute for Cancer Research. Department of Biochemistry, Biophysics and Genetics, and Cancer Center, University of Colorado Ilcalth Science ('cnter, I)cnver, and Ahmanson Department of Pediatrics, Cedars- Sinai Medical Center. l lniversity of California, Los Angeles. A SIMI'LE AND HIGIILY EFFICIENT PROCEDURE FOR RESCUING AtJTONOMOI /S PLASMIDS FROM YEAST In order to take advantage of the extremely powerful techniques of molecular genetics which are available in the yeast Sarcharamvres rereri.cirre, it is often neces- sary to recover shuttle vectors replicating in yeast back into F,. e•ali. Nowever, many workers have found this step troublesome due to what has been described as a"pcr- sistent inhibitor of F. rali transfnnnation" which accompanies yeast DNA prepara- 152 1 153
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i tion%. None nl Ihc previously puhli.hcd protnrolx complctcly eliminalcc this pmh- Icnr. Here we clcccrilx a mcthrxl thal rnutincly, yiclds greater than II)' bacterial trans- lirnnants using DNA prepared from a 1.5 ml yeast culture. This represents an cffi- cicncy which is I 2 ordcrs of magnitudc highcr than that of previously reported results. In addition to being very reliable, our method is simple and easily applied to many samples. Furthermore, this rnr•thnd reyuires neither expensive matcrials such as iymolyasc or purilication kit, nor thc organic solvents phenol and chlnrofomi. In fnct, those who use the boiling mcthrxl for preparing plasrnid DNA from E. cnli, probably have all of the ncrc«ary snlutinns preparccl. Rnhzyk, K. and Kns.cir, Y Nucleic Acids Research 2O(14):1790, 1992. Other tiupport: Israel Academy nl Science an(1 Humanitics. Frnm Ihc Faculty ol Biology. Tcchnion-Isracl Inxtitutc of Tcchnology, I laifa, Israel. SWITCII RE('OM13INATION RRFAKPOINTS ARE STRICTLY C'ORRELATF,D WTl'H I)NA RI;(YCNI'I-ION MOTIFS FOR 1MMUNOGLORtII,IN Sy3 I)NA BINDING I'R(Yft:INS The ctclction looping out model nf switch (S) rccomhination predicts that the intcrvcning I)NA between switch regions will hc excised as a circle. Circular exci- sion products of irnmtmoglohulin switch recombination have been recently isolated Ironr lipupnlysacchariclc (1,RS)-stimulated spleen c•clls. The rccomhination break- points in these large circles were found to fall within switch regions. Since switch rccomhinatirnr is clearly focusccl on switch rcgionti, we hypothesized that sonic DNA-hincling protein Grctnr might hc involved in specifically recognizing and facili- tating thc alignment nf switch regions before recombination. Two DNA-hinding pro- tcins that tilnc'ifically intcract with two cliscrctce regions of Ihc Syl tandem repeat have been identificcl in crude and partially purified nuclear extracts derived f'rom I.1'S- and elextrm .ulfate (I)xS)-activated splenic B cells. The first factor has been I'nuncl indistinguishahlc Irnm NF-KB mohility shift assays, mcthylation interference, compctitirm binding studics, and supcrshiff analysis using an antiscnnn spccific for Ihc I'SO c<,mlrnncnl. Thc Wcond appears to be composed of two closely traveling mohiliti" Iha1 dn not separate upon partial purification. This second coniplcx is uniquc and >prcific fnr S-y3 by nuthylation interference assays and cornpctition- hinding analv~i~. '11ic eitcs at which recombination occurs in the S-y3 switch region have been analyicd and frnmd to strictly correlate with the binding sitcs of the Syi cwitch binding pnrlcins. Wucrffcl, R.,.lamic%rni. ('. li., Mnrgan, I,., Mcrkulov, G. V., Scn, R., and Kentr•r, A. L. Jcrunial of F,xfxrimcntal Medicine I76:3:(c)-:449, August 1992. Othcr suppnrt: National Intititutcs nf Hcalth and American ('anccrScx•icty. From Dc.pnrlmcnt nf Microbiology and Immunology, Elnivcrsity of Illinois College nf Medicine at ('hicagn, and Rmcnsticl Research ('cntcr, Brandeis llnivcrtiity, Walth;rm. MA. IN VITRO CIIROMATIN ASSEMBLY PROMOTF?E) BY THF XF,NOPFIS I,AIa'LS S-lSp CELI, FRI'sE EXTRAC'T IS F;NHAN('ED BY TREATMFNT WITH RNasc A Ccll-free extracts employed as chromatin assembly systems contain a myriad of proteins, polyanions and nucleic acids. The roles of ATP, MgCIZ and other cofactors in the catalysis of nucleosome formation by the Xennpns laevis nocyte S-ISb have yet to be established unequivocally. In Ihis study we examine the influence of RNA in the assembly process. Under reaction conditions that inhibit nucleosome forma- tion (+EDTA), pretreatment of the extract with RNasc A revives the chromatin assembly machinery while the rate of DNA supercoiling is stimulated significantly. Addition of purified RNA blocks DNA supercoiling. Taken together, thcsc data sug- gest that the parameters surrounding in vitro chromatin assembly are variable and subject to modulation by endogenous factors. Sckiguchi, J. M. and Kmier, E. R. Nucleic Acids Research 20(4):RR9-R95, 1992. From the Department of Molecular Pharmacology, Jefferson Cancer Institute, Thomas .Fefferson University School of Medicine. Philadelphia. IN VITRO ANALYSIS OF A TYPE I DNA TOPOISOMERASE ACTIVITY FROM ('ULTURED TOBACCO C'ELLS The role of DNA topoisomcrates in plant cell metabolism is currently under investigation in our laboratory. Using a purified type I topoisomerasc from cultured tobacco, we have carried out a biochemical characterization of enzymatic behavior. The enzyme relaxes negatively supercoiled DNA in the presence of MgCI,, and to a lesser extent in the presence of KCI. Phosphorylation of the tnpoisomerase does not influence its activity and it is not stimulated by the presence of histones HI or H5. The enzyme may act in either a processive or distributive manner depending on reac- tion conditions. The anti-tumor elrug, camptothecin, induces significant breakage by the enzyme on purified I)NA molecules unless destabilized by the addition of KCI. The tobacco tnpoisomerase I can catalyze the formation of stable nucleosomes oil circular I)NA temphHes, suggesting a role for the enzyme in chromatin assembly. Cole. A. D., Hcath-Pagliuso, S., Raich, A., ;md Kmicr, E. R. Plant Molecular Biology 19:2(i5-276, 1992. From thc Department nl' Pharmacoln€y, Jefferson Cancer Institute. Thomas Jefferson University, Philadelphia, and Department of Biology, Southern Illinois I Inivcrsity, Edward.villc. OXYRADICAIS AND MEIF.TIVITAMIN TABLETS Ingestion nf a single multivitamin tablet Icads to hydroxyl radical production equivalent to a racliatinn dnw rate of 51 Gy/h. 154 ' 1 155
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Mnskos, "!.. amd Koppenol. W. I/. Free Radical Biology & Medicine 1 1:6(i9-610, 1991. Frorn the Biodynamics Inslitute ancl Departments of Chemistry and Biochemistry, Louisiana State University. Baton Rouge. 'I'HF: HYDROXYI,ATION QF PHENYF,ALANINE AND TYROSINE: A('OMPARISON WI-fll SALICYLATE AND TRYPTOPHAN The hyclroxylation of phenylalanine by the Fenton reaction and -y-radiolysis yields 2-hydroxy-, {-hydroxy-, and 4-hyclroxyphenylalanine (tyrosine), while the hydroxylation of tyrosine results in 2.3- and 3.4-dihydroxyphenylalanine (dopa). Yields are determined as a function of pH and lhe presence or absence of oxidants. I)uring -y-radiolysis and the Fenton reaction the same hyclroxylated products are formed. The final product distribution depends on the rate of the oxidation of the hyclroxyl radical adducts (hydroxycyclohexadiene radicals) relative to the competing dimeri/.ation reactions. The pH profiles for the hydroxylations of phenylalanine and tyrosine shov, a maximum at pH 5.5 and a minimum around pH 8. The lack of hydroxylated products around near pF1 R is due to the rapid oxidation of dopa to mclanin. The relative abilities of iron chelates (HLFe(11) and a HLFe(III) to promote hydroxyl radical f.rrrrrdion from hydrogen peroxide are nitrilMriacetate (nta) > ethyl- enediaminediacetate (edda) » hydroxycthylethylenediarninctriacetate > citrate > eth- 'ylencdiaminctctraacetatc > diethylenctriaminepcntaacctatc > adenosine 5'-triphos- phate > pyrcrphosphalc > adenosinc 5'-cliphosphate > aclcnosine 5'-monophosphate. The high activity of ironnta and -edda chelatcs is explained by postulating the fonna- linn of a ternary Fe(III)-t.-dnpa complex in which dopa reduces Fc(III). The hyclroxy- latiorn of phenylalaninc and tyrosine are similar to that of salicylate (Z. Maskos, J. I). Rush. and W. 11. Koppenol, 1990, Free Radical Biol. Med. R, 153-162) and tryp- tnphan (preceding paper) in that oxiclants augment the formation of hydroxylated pnxlucts hy catalyzin}; the disnmtation of hydroxyl radical adducts to the parent com- prwncl and a stable hydroxylatecl product. A comparison of salicylate and the amino acids tryptophan, phenylalanine, and tyrosine clearly shows that salicylate is the best indicator of hydroxyl radical production. Maskos, 7... Rush, J. D. and Koppenol. W, 11. Archives of Biochemistry and Biophysics 29(ti(2):521-529, August I, 1992. From the Riodynamics Instilutc and F)cpartments of Chemistry and Birxhemistry, Iewisiana State Universily. Raton Rouge. INIIIBITION OF DNA PRIMAS(; ANI) POI,YMERASE a BY ARARINOF( IRANOSYI.N( I('LEOSIDE TRIPI IOSPHATES AND RF.LATF.D ('OMI'O(INDS Inhibition of 1)NA primase and polymcrase a from calf thymus was examincd. I)NA priniase requires a 3'-hydroxyl on the incoming NTP in order to polymerizc it, i i while the 2'-hydroxyl is advantageous, but not essential. Amazingly, primase prefers to polymerize araATP rather than ATP by 4-fold (k,,,/KM). However, after incorpora- Iion of an araNMP into the growing primer, further synthesis is abolished. The 2'- and 3'-hydroxyls of the incoming nucleotide appear relatively unimportant for nuclcotide binding to primase. Polymerization of nucleoside triphosphates by DNA polymerase a onto a DNA primer was similarly analyzed. Removing the 3'-hydroxyl of the incoming triphosphate decreases the polymerization rate >IOOtI-fold (kjK„), while a 2'-hydroxyl in the ribo configuration abolishes polymerization. If the 2'- hydroxyl is in the ara configuration, there is almost no effect on polymerization. An araCMP or ddCMP at the 3'-terminus of a DNA primer slightly decreased DNA binding as well as binding of the next correct 2'-dNTP. Changing the primer from DNA to RNA dramatically and unpredictably altered the interactions of pol a with araNTPs and ddNTPs. Compared to the identical DNA primer, pol a discriminated 4-fold better against araCTP polymerization when the primer was RNA, but R5-fold worse against ddCTP polymerization. Additionally, pol a elongated RNA primers containing 3'-lerminal araNMPs more eff iciently than the identical DNA substrate. Kurhta, R. D., Ilsley, D., Kravig, K. D., Schubert, S., and Harris, B. Biochemistry 31:4720-4728, 1992. Other support: National Science Foundation. From Department of Chemistry and Biochemistry, University of Colorado, Boulder. INHIBITION OF DNA PRIMASE BY 9-(3-u-ARAI3INOFURANOSYLADENOSINE TRIPHOSPHATE 9-/3-D-Arabinofuralnosyladenosine triphosphate (araATP) is a potent inhibitor of DNA primase. Primase readily incorporates araATP into primers, and primers containing araAMP are then elongated by DNA polymerase a(po1 a) upon addition of dNTPs. AraATP did not inhibit utilization of primers under conditions where the ability of pol a to elongate primers was independent of the dATP concentration. The fraction of primers elongated by pol a was reduced by araATP only when elongation was dependent upon the dATP concentration. When the K for prima.se was meas- ured in terms of the inhibition of the synthesis of primers tfiat can he utilized by pn1 a, we obtained K = 2.7 µM (37°C) and 2.0 µM (25°C). Inhibition was competitive with ATP. Inhibilion of pol a activity by araATP was measured under conditions where primase-catalyzed primer synthesis was required for the pol a activity. The decreased pol a activity was due to primase inhibition, and at constant dATP, araATP inhibition was competitive with ATP and gave K= 1.2 µM, similar to the K for primase alone. Increasing the dATP concentration hail no effect on inhibition. Iri combination with previously reported in vivo data, we conclude that DNA primase is Ihe primary in vivo targe( of'the arabinofuranosyl nucleotictes, not pol a. Kurhta. R. l). and Willhelm, L Biochemistry 30:797-R03, 1991. Other Suppcrrt: Univcrsity of ('olorado. From the Department of Chemistry and Biochemistry, University of Colorado, Boulder. 156 157
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I INVOI,VEMENTOFcDNA IN HOMOLO(iOUS REC'OMBINATION BETWEEN 'I'v I:I,EMIiN7:S IN SA('(YIAROMY('Is,S (YiRF,6ZS/AE Strains carrying a marked Ty element (TyUra) in the LY,S2 locus were trans- formed with plasrnids hearing a differently marked Ty I element (Ty I Neo) under the control of the GAL promoter. When these strains were grown in glucose, a low level of gene conversion events involving TyUra was detected. Upon growth on galactose an increase in the rate of gene conversion was seen. This homologous recombination is not the consequence of increased levels of transposition. When an intron-contain- ing fragment was inserted into Ty I Nco, some of the convertants had the intron removed, implying an RNA intermediate. Mutations that affect reverse transcriptase or reverse transcription of TylNeo greatly reduce the induction of recombination in galactose. Thus. Ty cDNA is involved in homologous gene conversion with chromo- somal copies of Ty elements. Our results have implications about the way families of repeated sequences retain homogeneity throughout evolution. Mclamed, ('., Nevo, Y., and Kupier, M. Molecular and Cellular Biology 12(4):1613-1620, April 1992. Other support: Israeli Cancer Research Foundation. From the 1)cpartment of Molecular Microbiology and Biotechnology, Tel Aviv University, Rainat Aviv, Israel. E('TOPICRE('OMBINATION BETWEEN TY ELEMENTS IN ,SA('(71AROMY('P.,S (YiRF.VLtiIAE IS NOT INDUCED BY DNA DAMAGE Mitotic recombination is increased when cells are treated with a variety of phys- ical and chemical agents that cause damage to their DNA. We show here, using Surrlmrnmwr•s c•rrrrisae strains that carry Ty elements, that recombination between members of this family of retrotransposons is not increased by irradiation or by treat- menl with the radiomimetic drug methyl methanesulfonate. Both ectopic recombina- tion and mutation events were elevated by these agents for non-Ty sequences in the same strain. We discuss possible mechanisms that can prevent the induction of recombinalion between Ty elements. Parket, A. and Kuph•r. M. Molecular and Cellular Biology 12( I(1):4441-444R, October 1992. Other support: 11.S.-Israel Binational Science Fund. From the Dcpartment of Molecular Microbiology and Biotechnology, Tel Aviv Oniversity. Tel Aviv, Israel. IILTRAVIOI.ET NIC'KING OF LARGE DNA MOLECULES FROM PULSED- FIELD GELS FOR SOFffIIERN TRANSFER ANI) I-IYBRIDIZATION Large 1)NA molecules separated in pulsed-field gels are not efficiently trans- ferred from the gel for Southern hybridization. Various procedures for fragmenting the DNA prior to transfer are in use, but quantitative details that permit reproducible application have not been reported. We have determined the optimum level of energy for uv nicking of large DNA needed to promote efficient Southern transfer and detec- tion by hybridization. To ensure consistent results we have used a uv oven equipped with a detector that measures only 200-400 nm wavelengths, and we report the total energy delivered. Using uv nicking and the transfer techniques described, we can obtain hybridization signals overnight with single-copy DNA probes on Southern blots of large DNA fragments separated by pulsed-field gel electrophoresis. Lee, H., Birren, B., and Lai, E. Analytical Biochemistry 199:29-34, 1991. Other support: North Carolina Biotechnology Center and Elsa U. Pardee Foundation. From the Department of Pharmacology, University of North Carolina at Chapel Hill, and Division of Biology, California Institute of Technology, Pasadena. FACTORS DETERMINING THE SPECIFICITY OF SIGNAL TRANSDUCTION BY GUANINE NUCLEOTIDE-BINDING PROTEIN-COUPLED RECEPTORS- I. COIIPt.1NG OF a=-ADRENGERIC RECF.PTOR SUBTYPES TO DISTINCT G-PROTF.INS a1 Adrenergic receptor (a -AR) subtypes couple to pertussis toxin (PT)-sensitive G-proleins to elicit both stimulatory and inhibitory cell responses. Signal specificity may be generated by the ability of the receptor subtypes to "recognize" distinct G- proteins with different affinity. To address this issue we stably expressed three a-AR subtypes, RNGaI (a,w- AR), RGIO (ax-AR), and RG20 (a?~~ AR), in NIH-3T3 fibroblasts, which express two PT-sensitive G-proteins (G~Z, Gp~), and analyzed receptor/G-protein interactions by determining: I) functional couplmg to adenylylcyclase and 2) the ability of the receptors to exist in a high affinity state for agonist. In az-AR transfectants express- ing 200 or 2,200 fmol of receptor/mg of protein, epinephrine (10 µM) inhibited forskolin-induced elevation of cellular cAMP by 26 ± 4.8% and 72 ± 6.2%, respec- tively. Similar results were obtained in a w AR transfectants. However, in a-AR transfectants (200 fmol/mg) the forskolininduced elevation of cellular cAMP was not altered by agonist treatment. In a1 -AR transfectants expressing higher receptor densities (650-1,200 fmol/ mg), epineplrrine inhibited the effect of forskolin by 30 ± 3.2%. This difference in functional coupling among the a. AR subtypes is reflected at the receptor/ G-protein interface. In membrane preparations of azp and a7D -AR hut not a7,.-AR transfectants, agonist competition curves were biphasic, indicating high and low affinity states of the receptor for agonist. The high affinity state was guanyl- 5'-yl imidodiphosphate- and PT-sensitive, indicative of receptor/G-protein coupling. These data suggest that the aN-AR differs from the alp and a -AR subtypes in its ability to recognize PT-sensitive G-proteins expressed m NIHJT3 fibro- blasts. The an-AR may couple preferentially to PT-sensitive G-proteins (G", G„"I) not expressed in NIH- 3T3 fibroblasts and thereby elicit different cellular responses. Duzic, E., Coupry, I., Downing, S., and Lanier, S. M. The Journal of Biological Chemistry 267(14):9844-9R51, May 15, 1992. 159 1 159
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Other support: National hutitutes of Health. From the Cellular and Molecular Research Laboratory, Massachusetts General Ilocpital. tlarvard Medical Sch(x)I, Boston. FACTORS DETERMINING THE SPECIFICITY OFSIGNALTRANSDUCTION BY GUANINE NU(1.EOTIDE-BINDING PROTEIN-COUPLED RECEPTORS- 11. PRI'.I7'RYNTIAI. ('Olit'I tNG (lr: 1111i (Y,-ADRIiNERGn' RE('lil•fOR TO lNE GIJANINE NI I('PLO1'IDti-RINDING PROIFIN, (7„ Cell to ccll communication by many hormones and neurotransmitters involves three major entities: receptor (R), G-protein (G), and effector molecule (E). Plasticity in this system is conferred by the existence of each entity as isoforms or closely related subtypes that arc expressed in a tissue-specific and developmentally regulat- ed manner. Factors that determine signal specificity in this system are poorly under- stood. Such factors include the relative affinity and stoichiometry of R-G or G-E and the possible colocalization of R-G-E in cellular microdomains. Utilizing the a2 adrenerglc receptor (a,-AR) system as a representative subfamily of this class of sig- nal transducerti. we dctemlined the relative importance of these factors. By analysis of R- (; coupling in manlmalian cells cotransfected with a,-AR genes and G cDNA. we demonstrate preferential coupling between an a,-AR subtype and G, tJur data implicate R-G affinity as an important determinant of signal transduction specificity smd indicate that a critical level of G cr is required for coupling. This report indicates the utility of R-G cotransfection in mammalian cells as a"natural environment mOdel" to characteriic events occurring at the R-G and G-F, interface. ('oupry. 1., Duzic. E., and Lanier. S. M: The Journal ul' Biological Chemistry 267(14):9852-9857, May 15. 1992. Other support: National Institutes of I le:dlh. From Ihe Cellular and Molecular Research Laboratory, Massachusetts General Ilospital, Ilarvard Medical School. Boston. PtIRIFICATION AND ('HARAC'TERI7,ATION OF MITOCHONDRIAL IMIDA7,OI.IN1;-(;tIANII)INIUM REC'EPTIVE SITE FROM RABBIT KIDNEY The imida/oline-guani(linium receptive site (IGRS) is a memhrane-tx)und pro- tein that may medialc some of the pharmacological effects of imidazoline and guani- diniunt cumpounds. The structure and functionality of this protein are unknown but, in addition to its location at the plasma membrane, it is found in high density in the outer membrane of mitochondria (Tesson. F., 1'rip-Buus, ('., Lemoine, A.. Pegorier, J.-P., and Parini. A. (1(J91).I. BioL ('hcm. 266, 155-I60). Using a two-step procedure, we report the purification of mitochondrial IGRS irom rabhit kidney to the apparent homogeneity. After solubilization of mitochondrial membranes with digitonin, an apparently homogeneous IGRS preparation was obtained by two sequential purification steps, chromatofocusing and hydroxylapatite-agarose chromatography. One- and two- dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified preparation after silver staining or radioiodination indicated that IGRS binding subunit was purified at the apparent homogeneity since a single band (M, - 60,000) was observed. IGRS behaves as an acidic protein (p1 5.5) whose binding activity is regulated by H' concentration near a physiological pH of 7.4. The ability to achieve rapid purification of IGRS should facilitate efforts to define molecular properties and functionality of this protein. Limon, I., Coupry, I., Lanier, S. M., and Parini, A. The Journal of Biological Chemistry 267(30):21645-21649, October 25, 1992. Other support: Contrat de Recherche Exteme, Institut National de la Sant6 et de Ia Recherche M6dicale; North Atlantic Treaty Organization Collaborative Research Grant; and U.S. National Institutes of Health. From the Centre National de Ia Recherche Scientifique, Facult6 de M6decine Necker-Enfants Malades, Paris, France, and the Department of Pharmacology, Medical University of South Carolina, Charleston. FACTORS DETERMINING THE SPECIFICITY OF SIGNAL TRANSDUCTION BY GUANINE NUCLEOTIDE-BINDING PROTEIN-COUPLED RECEPTORS. ill. COUPLING OF aj ADRENERGIC RECEPTOR SUBTYPES IN A CELL TYPE-SPECIFIC MANNER A number of diverse signaling pathways can be activated by G-protein coupled receptors. However, the factors involved in selection of a particular transduction pathway by a single receptor are not well understood. We are attempting to address this issue utilizing the a= adrenergic receptor (a= AR) subfamily as a representative model system. In this report, we demonstrate that the cellular response mediated by an a -AR subtype is cell-specific and thus depends on its environment. Receptor coupiing to adenylylcyclase was determined following stable expression of the rat aZ - and a= -subtypes in three functionally distinct cell types (NIH-3T3 fibroblasts, DaT. MF-5 smooth muscle cells, and the pheochromocytoma cell line PC-12). When the receptor subtype gene is expressed in NIH-3T3 and DDT. MF-2 cells, receptor activation inhibits basal and forskolin-induced increases in cellular cAMP. However, in PC- 12 transfectants the same receptor subtype actually increases basal cAMP and augments the effect of forskolin. Potentiation of the forskolin effect in PC-l2 cells is insensitive to pertussis toxin but is blocked by loading the cells with BAPTA (bis-(n-aminophenoxy)-ethane-N,N,N,1V'-tetraaeetic acid) which minimizes changes in Ca", by calcium chelation. These data and the functional demonstration of a Ca''/calmodulin-sensitive adenylylcyclase in PC-12, but not NIH-3T3 and DDT~MF-2 cells, suggest that the cell-specific effects of epinephrine are due to receptor coupling to both different G-proteins and types of adenylylcyclase. Duzic, E. and L.anier, S. M. The Journal of Biological Chemistry 267(33):24045-24052, November 25, 1992. 160 1 161
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i Other supporl: National Institutes of I lcalth. From the Department of Pharmacology, Medical University of South Carolina, Charleston. MOLECULAR CLONING OF CELLULAR GENES ENCODING RETINOBI.ASTOMA-ASSO('IATED PROTEINS: IDENTIFICATION OF A GENE WITH PROPIiR'fIES OF TI-fE TRANSCRIPTION FACTOR E2F The retinohlastoma protein interacts with a number of cellular proteins to form com- plexes which are probably crucial for its normal physiological function. To identify these proteins, we isolated nine distinct clones by direct screening of cDNA expres- sion libraries using purified RB protein as a probe. One of these clones. Ap12, is expressed predominantly at the G,-S houndary and in the S phase of the cell cycle. The nucleotide sequence of Apt2 has features characteristic of transcription factors. The C'-tcrminal region binds to unphosphorylated RB in regions similar to those to which T antigen binds and contains a transactivation domain. A region containing a potential Ieucine zipper flanked by basic residues is able to bind an E2F recognition sequence specifically. Expression of Ap12 in mammalian cells significantly enhances E2F-dependenl transcriptional acivity. These results suggest that Ap12 encodes a protein with properties known to he characteristic of transcription factor E21;. Shan, B.. 7.hu, X.. ('hen. P.-1,., Durfee, T., Yang, Y., Sharp, D., and Lee. W.-Ll. Molecular and C'ellular Biology 12(12):562(1-56i I, December 1992. Other support: National Institutes of Health. From the ('enter for Molecular Medicine/Institute of Biotechnology, University of Texas Iiealth Science Center, San Antonio. EPITHELIALTISSUE FORMATION IN MATRIX CULTURE AND IN HISTOPI-IYSIOLOGI(' GRADIENT CULTURE Formation of epithelial tissues in culture so that they become facsimiles in their structure of such tissues in nature requires procedures (hat comply with several spa- tial imperatives: a) three-dimensional growth; b) histophysiologic conditions that provide, concurrently, gradients of maturation and of diffusion of inetabolites: and c) growth as layers of cells without free edges. Many steps have been required in the evolution of these methods. Two systems are descrihe(1 here in sufficient detail to serve as a manual. Three-dimensional growth of masses of epithelial tissue is accom- plished in matrix cuhure using Gclfoarn sponge and collagen-coated cellulose sponge. Radial gradient culture, a recent development, provides conditions that com- ply with the requirements of histophysiologic gradients and of epithelial tissue growth in layers without interruption in their continuity. i I I L,eiRhlon. J. Journal of Tissue Culture Methods 14:201-208, 1992. Other support: National Institutes of Health and American Fund for Alternatives to Animal Research. From the Peralta Cancer Research Institute, San Leandro, CA. MONITORING CHOLESTEROL AUTOXIDATION PROCESSES USING MUI; fIDEUTERIATED CHOLESTEROL Deuterium-labeled cholesterol is used to monitor for artifactually produced cho- lesterol oxidation products during analysis. 1'H9)Cholesterol, labeled on the side chain, is added to the sample immediately upon isolation, and the ratios of labeled to unlabeled oxides and of labeled to unlabeled cholesterol are monitored by capillary gas chromatography-mass spectrometry. The analytical methodology involves an ini- tial solvent extraction followed by silica gel LC and reversed-phase HPLC to isolate and concentrate the oxide fraction. The feasibility of the technique for analysis of cholesterol oxides in foods and biological samples at the part per mIllion level with an accuracy of better than ± 5% is demonstrated. Wasilchuk, B. A., Le Quecne, P. W., and Vouros, P. Analytical Chemistry 64(10):1077-1087, May 15, 1992. Other support: Biomedical Research Support Grant. From the Department of Chemistry and Barnett Institute of Chemical Analysis, Northeastern University, Boston. PROTEIN TRUNCATION IS REQUIRED FOR THE ACTIVATION OF THE c-myh PROTO-ONCOGIiNE The protein product of the v-myb oncogene of avian myeloblastosis virus, v-Myh, differs from its normal cellular counterpart, c-Myb, by (i) expression under Ihe control of a strong viral long terminal repeat, (ii) truncation of both its amino and carboxyl temiini, (iii) replacement of these termini by virally encoded residues, and (iv) substitution of I I amino acid residues. We had previously shown that neither the virally encoded termini nor the amino acid substitutions are required for transfomia- tiun by v-Myh. We have now constructed avian retroviruses that express full-length or singly truncated forms of c-Myb and have lested them for the transformation of chicken hnne marrow cells. We conclude that truncation of either the amino or car- boxyl terminus of c-Myb is sufficient lor transformation. In contrast, the overexpres- 162 1 163
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zSZOOO IZ ?IOD sion of full-length c-Myh does not result in transformation. We have also shown that the amino acid substitutions ol v-Myb by themselves are not sufficient for the activa- tion of c-Myb. Rather, the presence of either the normal amino or carboxyl terminus of c-Myb can suppress transformation when fused to v-Myb. Cells transformed by c-Myb proteins truncated at either their amino or carboxyl terminus appear to he granulated promyclocytcs th:u express the Mim-1 protein. Cells transformed by a doubly truncated c-Myb protein are not granulated but do express the Mim- I protein, in contrast to monohlasts transformed by v-Myb that neither contain granules nor express Mim-I. These results suggest that various alterations of c-Myb itself may determine the lineage of differentiating hematopoietic cells. (;rasscr, F. A., (;raf, T., and Lipsirk,.1. ,S. Molecular and Cellular Biology 11 (R):39R7-1996, August 1991. Other support: American Cancer Society and U.S. Public Health Service. From the Institut fiir Medizinische Mikrobiologie und Hygiene, Abteilung Virologie, Universitatskliniken des Saarlandes, Homburg. Germany; 1)ifferentiation Program, European Molecular Biology LaMxatory, Heidelberg, Germany; and Department of Microbiology. School of Medicine. State University of New York at Stony Brook. FSTRAMUSTINE-PH(>SPIIATE BINDS TO A TUBULIN BINDING DOMAIN ON MI('RO'L'UBtILI:-ASSO('IATED PROTEINS MAP-2 AND TAU I;stramustine-phosphate (EMP), a phosphorylated conjugate of estradiol and nor-nitrogen mustard hinds to microtubule-associated proteins MAP-2 and tau. It was shown that this cstramustine derivative inhibits the binding of (he C-terminal tubulin peptide (3-(422-434) to both MAP-2 and tau. This tubulin segment consti- tutes a main binding clomain for these microtuhule-associated proteins. Interestingly, estramustine-phosphate interacted with the synthetic tau peptides V'"'-G"" and V""- G"`, representing two major repeats within the conserved microtubule-binding domain on t:w and also on MAP-2. This observation is corroborated by the inhibi- tory effects of estramusline-phosphate on the tau peptidc-induced tubulin assembly into microtuhules. On the other hand, the nonphosphorylated drug estramustine failecl to block the MAP peptide-induced assembly, indicating that the negatively charged phosphatc moiety of estramustine-phosphate is of importance for its inhibitory effect. These findings suggest that the molecular sites for the action of estramustinc-phosphate,arc located within the microtubule binding domains on tau and MAP-2. Moraga, D., Rivas-Bcrrios, A„ Farias, G., Wallin, M., and Marcinni, R. B. Biochimica ct 'Biophysica Acta 1 121:97-1O3, 1992. Other support: Directorate-Gcneral Xll of (he European Community, CONICYT- Chile, Swedish Research Council, and Phamiacia Leo. From the International Center for Cancer and Developmental Biology, Santiago, Chile: Faculty of' Sciences, Universidad de C'hile. Santiago; and Department of 7.oophysiology, llniversity of GoteMorg, Goteborg, Sweden. , I , t SPECIFIC MACROMOLECULAR INTERACTIONS BETWEEN TAU AND THE MICROTUBULE SYSTEM The microtuhule-associated protein Tau, a major component of brain micro- tubules, shares common repeated C-terminal sequences with the high molecular- weight protein MAP-2. It has been shown that tau peptides V'`-G"'° and V21"-G"`, representing two main repeats, induced brain tubulin assembly in a concentration- dependent fashion. The specific roles of these repeats in the interaction of tau with microtubules, and its antigenic nature were investigated using synthetic tau peptides ~ and site-directed monoclonal antibodies. Tau peptides appeared to compete with C-4 MAP-2 incorporation into assembled microtubules. The interactions of the tau frag- 0 ments with /i-tubulin peptides bearing the tau binding domain on tubulin were ana- ~ lyzed by fluorescence spectroscopy. The specificity of the binding was further N demonstrated by the reactivity of tau and the tau peptides with a monoclonal anti- p.( idiotypic antibody produced after immunization with the (3-11(422-434) tuhulin pep- o tide, as assessed by enzyme-linked immunoassay. Western blots confirmed the inter- action of tau with the monoclonal antibody. In addition, immunoassays revealed a ~ competition between the MAP-r ti l l eac ng monoc ona antibody and the tubulin peptide (3-11(422-434) for their interaction with the tau molecule. Farias, G. A., Vial C., and Macrioni, R. B. Molecular and Cellular Biochemistry I 12:81-R3, 1992. Other support: CONICYT-Chilc and Directorate-General XII of the European Community. From the Departmento de Biologia, Facultad de Ciencias. Universidad de Chile, and International Center for Cancer and Developmental Biology, Santiago. Chile. SURFACE ACTIVATION OF PRO-CATHEPSIN L Procathepsin L is an inactive zymogen that has been shown previously to under- go autolysis at pH 3.0 to give mature forms of the enzyme. We have now been able to demonstrate that this enzyme can undergo activation at pH 5.5 in the presence of negatively charged surfaces. Activation could also be measured at pH 6.0, but no activation occurred at pH 6.5 or higher. The initiation of activation depends upon (he presence of a small percentage of active pro-enzyme, and this is then followed by a more rapid activation to give mature forms of the enzyme. No significant intermedi- ate molecular forms of the enzyme were seen. The time taken for processing of the pro-enzyme to single-chain mature enzyme is comparable to that seen in biosyn- Ihetic pulse-chase experiments. Masnn, R. W. and Massey, S. D. Biochemical and Biophysical Research Communications IR9(3):1659-1666, December 30, 1992. Other support: American Lung Association of Virginia. From the Department of Biochemistry, Virginia Polytechnic Institute and State University, Blackshurg. F~°I ~ 164 165
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INIIIBITION OF CYSTEINE PROTF,INASES IN I.YSOSOMES AND WHOLE ('E[.LS Inhibitors of cysteinc protcinascs have been used extensively to dissect the roles of these proteinases in cells. Surprisingly though, little work has been performed to demonstrate unequivocally that the inhihitors reach and inactivate their target pro- teinases in cell culture or in rirn. In the present study, the permeability of lysosomes and whole cells has been studied. Benzyloxycarhonyl (Z)-l"`Iliodo-Tyr-Ala-dia- zomethane (CHN,), an inhibitor of cathepsins L and B, has been shown to label active forms of these enzymes in lysosomes and whole cells. The ability of other cysteine proteinase inhibitors to block this labeling has been used to indicate (he per- meation of these compounds. All thc inhibitors were able to block labeling by Z- I"`I liodo-Tyr-Ala-CHN in lysosomal extracts. In intact lysosomes or cells, however, only N-IN-t.-3-rrans-ethoxycarbonyloxirane-2-carbonyl)-L.-leucyll-3-methylbutyl- arnine ('F:-64d') "7,=fyr-Ala-('HN, Z-Phe-Ala-CHNI and Z-Phe-Phe-CHN, were able to block labeling by Z-I"`llicxlo-Tyr-Ala-CFIN,, N-(t.-3-Ir(in.c-Carboxyoxirane-2-car- bonyl)-t.-Icucyllamino-4-guanidinobutane (E-64) and leupeptin were unable to block labeling by 7.-I"`Iliodc-'1'yr-Ala-CHN,in lysosomes or in cells. The ability to block labeling in lysosomes is an indication of the ability of (he inhibitor to diffuse across membranes. Thus, E-64 and Icupeptin do not readily permeate membranes and, therefore, their uptake into cells probably only occurs via pinocytosis. Wilcox, 1). and Masnn. R. W. Biochemistry Journal 285:495-502, 1992. From the Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State t)niversity. Blacksburg. ROLE OF ANTIOXIDANTS IN PROTECTING CELLULAR DNA FROM [)AMAGE BY OXIDATIVE STRESS We have previously derived 2 V79 clones resistant to menadione (MDlcells) and cadmium (Cdl cells), respectively. They both were shown to be cross-resistant to hydrogen peroxidc.'ihere was a modification in the antioxidant repertoire in these cells as compared to the parental cells. Mdl presented an increase in catalase and glutathione peroxidase activities whereas Cdl cells exhibited an increase in metal- hothionein and glutathione content. The susceptibility of the DNA of these cells to the damaging effect of HOt was tested using the DNA precipitation assay. Both Md I and CdI DNAs were more resistant to the peroxide action. In the case of MdI cells it seems clear that the extra resistance is provided by the increase in the two HZO scav- enger enzymes. catalase and glutathione peroxidase. In the case of Cdl ceils the activities of these enzymes as well as of superoxide dismutases (Cu/7.n and Mn) are unahered as compared to the parental cells. The facts that parental cells exposed to I(X) µM 7.n" in the medium exhibit an increase in metallothioncin but not in glu- tathione and that these cells become more resistant to the DNA-damaging effect of H Ol suggest that this protein might play a protective role in vivo against the OH rad- ical attack on DNA. Martins, E. A. L., Chubatsu, L. S., and Meneghini, R. Mutation Research 250:95-101, 1991. Other support: FAPESP (Brazil). From lhe Department of Biochemistry, University of Sao Paulo, Sao Paulo, Brazil. GLUTATHIONE IS THE ANTIOXIDANT RESPONSIBLE FOR RESISTANCE TO OXIDATIVE STRESS IN V79 CHINESE HAMSTER FIBROBLASTS RENDERED RESISTANT TO CADMIUM By manipulation of Cd and Zn concentrations in the medium, several pheno- types, differing in the contents of glutathione (GSH) and metallothionein (Mt), were derived from a parental clone of V79 Chinese hamster fibroblast. In some of these phenotypes, resistsnee to Cd and cross-resistance to oxidative stress was developed. The highest levels of GSH and Mt were found in cells which were rendered resistant to Cd by stepwise increases of Cd and Zn in the cell medium for over 50 passages. Upon removal of Cd/7.n from the medium of these cells or addition of Cd/7,n to the parental cell medium, changes of cellular GSH and Mt levels occurred to different extents. At the same time, changes in the resistance to Cd and HIOz were observed. Good linear correlations were observed for Mt levels X resistance to Cd and for GSH levels X resistance to H70 . Poor linear correlations were found for Mt levels X resistance to HZO2 or for GSIi levels X resistance to Cd. Moreover, addition of Zn to the medium produced an increase in Mt content without affecting the GSH con- tent. In this case no cross-resistance to oxidative stress was developed. Therefore, Mt which has been shown to be an excellent antioxidant in in vitro experiments, does not seem to play any major role against oxidative stress in Zn and Cd challenged cells. Most of the cross-resistance to oxidative stress in Cd challenged cells seems to be accounted for by the parallel increase in GSH. Chubatsu, L. S., Gennari, M., and MeneRhini, R. Chemico-Biological Interactions 82:99-1 10, 1992. Other support: FAPESP (Brazilian research support agency). From the Department of Biochemistry, Institute of Chemistry, University of Sao Paulo, and Instituto Biologico, Secfio de Imunologia, Sao Paulo, Brazil. HUMAN 1L-3 INDUCTION OF C-JUN IN NORMAL MONOCYTES IS INDEPENDENT OF TYROSINE KINASE AND INVOLVES PROTEIN KINASE C We have used normal human monocytes as a model system to begin elucidating the signal transduction mechanism associated with the IL-3R. Normal human mono- cytes deprived of human serum and CSF become quiescent in vilro. Stimulation of these cells with rll. 3 induces expression of the c-jun protooncogene, as detected by 166 1 167
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Northern blotting of total monocytc RNA. Thix protooncogene is also induced in Ihesc cells by phorhol csler through direct stimulation of protein kinase C. Concentrations of the protein kinasc C inhibitor I-(5-isoyuindinyl-sulfonyl)-2 methylpiperazine (11-7) between 30 and I(H) uM (5-20xK ) inhibit this induction by phorhul ester. The same conccntration range of H-7 completely inhibited the induc- tion of c-jun by hwnan II: 3. A structural analog of H-7 designated HA-I(X)4 prefer- entially inhibits cyclic nucleotidc-dependent protein kinase rather than protein kinase C. IIA-I(N)4 at 5 to 20xK did not inhibit II; 3-induced c-jun mRNA accumulation. Further 3(1 µM genistcin tliat is an effective inhibitor of cellular tyrosine kinases did not inhibit IL-Z-induced c-jun expression. Immunoprecipitation of lysates from I"l'horthophosphatc labeled cells with antiphosphotyrosine polyclonal antibody showed that II: 3-stimulated phosphorylation of a 70-kDa protein and a I10-kDa protein on tyrosine, and that these protein phosphorylations were completely inhibit- ed by 30 µM genistcin. As further confirmation that IL-3 is stimulating protein kinase (' in human rnonocytes we have found that IF: 3 stimulates phosphorylation of the unique protein kinase C substrate myristoylated alanine-rich C kinase substrate in these cells. It is therefore likely that the interaction of IL-3 with its receptor gener- atcs diacylglycerol and stimul:ucs the C'a''/phospholipid-dcpendent protein kinase C. Muf,%nrt. R. A., Szaho, J., and Eckert, 1). The Journal of Immunology 148(4): 1129-1 135, February 15. 1992. Other support: American Red C'rnss, Oliver S. and Jennic R. Donaldson Charitable Trust and l I.S. I'ublic health Service. From the American Red ('rotis. Holland Laboratory for Biomedical Scienees, Rockvillc. MI). I?NI IAN('I?MIiNT ANI) Qt IIiN('HING OF FL.(/ORESC'EN('E OF QUIN-2 BY MI:"I'AI.1ONS Addition of ayueous solutions of mctal ions (I.a", Dy" Nd''. Eu", Sc", AI", and I3e'') to the fluoreuence prrrlx Quin-2 induced changes in the 490 run emission hand (excitation at 334 nm). l'he resulting spectra were yualitatively different and provi- (led a"signalure" for Ihc individual metal ions. The lanthanide metal ions showed decreasing Iluorescence emission al 490 nm. Lu" and Sc" caused a decrease in the cmisxion intcnsity at 490 nm and a substantial increase in emission intensity at 4(X) nm or 412 nm, reslkctively. In all instances, stoichiometry indicated an apparent I:I binding nuio. except for AI" and 13e", which showed no significant effect on the flu- orescence. Joncs. E. 13., Jr., Nelson. I). J.., Turnhull, M. M. Journal of Inorganic Biochemistry 45:85 -)2, 1992. From the I)cpartmcnt of ('hemistry, ('lark University, Worcester, MA. 1"CD NUCLEAR MAGNETIC RESONANCE STUDY OF THE BINDING OF CD'' AND LU" TO SILVER HAKE PARVALBUMIN The binding of Cd" and Lu" to a parvalbumin isotype isolated from silver hake (Merlurcius hilinearis), SHPV-B, has been studied by `Cd nuclear magnetic reso- nance (NMR). Titration at pH 6.7, with "'CdCI=, of either apo SHPV-B or native SHPV-B (i.e. Ca" loaded) resulted in the emergence of two "'Cd resonances in the "'Cd NMR spectrum, one at about -94 ppm and another at about -97 ppm (relative to a"'Cd'' signal at 0 ppm arising from (unenriched) 2 M CdSO,), consistent with the presence of two distinct binding site environments on the protein for cadmium ion. Addition of Lu" to "'Cd"-substitutcd SHPV-B resulted in the preferential loss of the low field (i.e. -94 ppm) "'Cd NMR signal, consistent with the existence of one high- ly selective site on the silver hake parvalburnin (for the binding of ions that are very close to Ca"in their ionic radii and charge density, such as Cd") and a second, less selective site that can accommodate ions of slightly differing size (and charge densi- ty). Comparison of our Lu" addition results (on the silver hake parvalbumin) with the results of similar studies in the literature on pike and carp parvalbumin indicates sig- nificant similarities with the former and significant differences from the latter. These studies may bear on the evolutionary relatedness of parvalbumins from these three species. 7,hang, C. and Nelson, l). ,1. Journal of Alloys and Compounds 180:349-356, 1992. From the Department of Chemistry. Sackler Sciences Center, Clark (Iniversity, Worcester, MA. RELEASE OF CELL SURFACE-ASSOCIATED BASIC FIBROBLAST GROWTH FACTOR BY GLYCOSYLPHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE C Heparan sulfate proteoglycans (HSPG) are ubiquitous constituents of mam- malian cell surfaces and most extracellular matrices. A portion of the cell surface HSPG is anchored via a covalently linked glycosyl-phosphatidylinositnl (Pl) residue. which can he released by treatment with a glycosyl-PI specific phospholipase C(PI- PLC). We report that exposure of bovine aortic endothelial and smooth muscle cells to PI-PLC resulted in release of cell surface-associated, growth-promoting activity that was neutralized by aniihasic fibroblast growth factor (bFGF) antibodies. Active hFGF was also released by treating the cells with bacterial heparitinase. Under the same conditions there was no release of mitogenic activity from cells (BHK-21, NIFI/3T3, PF-HR9) that expressed little or no bFGF, as opposed to PI-PLC-mediated release of active bf-GF from the same cells transfected with the bFGF gene. The released bFGF competed with recombinant hFGF in a radioreceptor assay. Addition of PI-PL(' to sparsely seeded vascular endothelial cells resulted in a marked stimula- tion of cell proliferation, but there was no mitogenic effect of PI-PLC on 3T3 fibro- blasts. Studies with exogenously added "`I-bFGF revealed that about 6.5% and 20% of the cell surface-bcrond bFGF were released by treatment with Pl-PLC and hepar- itinasc, respectively. Both enzymes also released sulfate-labeled heparan sulfate 168 1 169
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from metabolically labeled 3T3 fibroblasts. PI-PLC failed to release "`I-bFGF from the %utx:ndothelial extracellular matrix (E('M). as compared to release of 60% of the 1:('M-txiund hF(;P by heparitinase Our results indicate that l--R% of the total cellu- lar content of bFGF is associated with glycosyl-PI anchored cell surface HSPG. This I`G& may exert hoth autocrine and paracrinc effects, provided that it is released by PI-1'L.(' and adequately presented to high affinity hFGF ccll surface receptor sites. Bashkin, P., Ncujrld. G.. Gitay-(;orcn. H., and Vlodavsky. I. Journal of Cellular Physiology 15:126-1 i7, 1992. Other support: National Cancer Institute, USA-Isracl Binational Science Foundation. Gerrnan-Israel Foundation for Scientific Research and Development, and Israel Ministry uf Ilealth. From the 1)cpartment of Oncology, Hadassah-Hebrew University Hospital, Jerusalem, and Department of Cell Biology. Technion-Israel Institute of Technology, Ilaifa.Isracl. A 43 KD c-mo.c PROTEIN IS ONLY EXPRFSSED BEFORE MEIOSIS DURING RAT SPERMATOGENESIS We have investigated Ihe RNA and protein expression pattern of the rat c-mos proto-nncogene during spermatogcnesis. In mouse testis a 43kD c-mos protein is expressed throughout spennatogenesis, which is in agreement with one report detect- ing a 1.7 kh c-mns RNA in pachytene spermatocytes and in early spermatids. However, sevcral other reports show that the mouse 1.7 kb c-mos RNA is exclusive- ly exprested in post-meiolic male germ cells. We report that in rat male germ cells three c-mns RNA species of 5. 3.6 and 1.7kb arc detectable by Northern blotting analysis both before and after rneiosis, with highest levels in early spermatids. However, western immuno-blot analysis reveals the presence of a 41kD c-mns pro- tein in total testis and pachytcnc spermatocytes, but not in post-meiotic cells. These finding combined with those made in the mouse system strongly suggest that c-mos protein may be a regulator of mciosis during spermatogenesis. Van der Hoorn, F. A.. Spicgel, J. E.. Maylic-Pfenningcr, M.-F., and Nnrdeen. S. K. Oncogene 6:929-912. 1991, Other support: Yamagiwa-Yoshida Memorial International Cancer Study Award (Intcrnational Union Against Cancer). From the 1)cpartnicnt of Medical Biochemistry, University of Calgary, Alberta, Canada, and Department of Pathology and Department of Cellular and Structural Biology, Flnivcrsity of C'olorado. Denver. SSZOOO is H0D i REGULATORY MECHANISMS FOR SKELETAL MUSCLE DIFFERENTIATION AND THEIR RELEVANCE TO GENE EXPRESSION IN THE HEART ~ The discovery of the MyoD family of skeletal muscle-specific regulatory fac- tors, which bind DNA and activate muscle-specific transcription in collaboration with widely expressed factors, has led to dramatic progress toward understanding the mechanisms responsible for activation of muscle-specific gene expression during differentiation of skeletal muscle. In contrast, relatively little is known of the mecha- nisms responsible for activation and maintenance of cardiac muscle transcription. Many muscle-specific genes that are directly regulated by the MyoD family in skele- tal muscle are also expressed in the heart, which does not express known members of the MyoD family. The different embryonic origins of skeletal and cardiac muscle and the differences in responsiveness of skeletal and cardiac muscle-specific genes to growth factor signals suggest that, if MyoD-like proteins participate in cardiac muscle development, they are likely to be expressed much earlier in development than the MyoD family and may have diverged substantially from these skeletal muscle regulatory proteins. Regulatory pathways independent of or in addition to those controlled by MyoD-like proteins appear more likely to be involved in specifi- cation of the cardiac muscle developmental program. Olson, E. N. Trends in Cardiovascular Medicine 2(5):163-170, 1992. Other support: National Institutes of Health, Muscular Dystrophy Association and Robert A. Welch Foundation. From the Department of Biochemistry and Molecular Biology. University of Texas M. D. Anderson Cancer Center, Houston. INTERPLAY BETWEEN PROLIFERATION AND DIFFERENTIATION WITHIN THE MYOGENIC LINEAGE In muscle cells, as in a variety of cell types, proliferation and differentiation are mutually exclusive events controlled by a balance of opposing cellular signals. Members of the MyoD family of muscle-specific helix-loop-helix proteins which, in collaboration with ubiquitous factors, activate muscle differentiation and inhibit cell proliferation function at the nexus of the cellular circuits that control proliferation and differentiation of muscle cells. The activities of these myogenic regulators are negatively regulated by peptide growth factors and activated oncogenes whose prod- ucts transmit growth signals from the membrane to the nucleus. Recent studies have revealed multiple mechanisms through which intracellular growth factor signals may interfere with the functions of the myogenic regulators. When expressed at high lev- cls, members of the MyoD family can override mitogenic signals and can cause growth arrest independent of their effects on differentiation. The ability of these myogenic regulators to inhibit proliferation of normal as well as transformed cells from multiple lineages suggests that they interact with conserved components of the 170 I 171
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cellular machinery involved in cell cycle progression and that similar types of regu- huory factors participate in differentiation and cell cycle control in diverse cell types. OIcnrr, E. N. Developmental Biology 154:261-272, 1992. Other support: National Institutes of Health, Muscular Dystrophy Association and Robert A. Welch Foundation. From Ihe Department of Biochemistry and Molecular Biology, University of Texas M. I). Anderson Cancer Center, Houston. CYCLIC AMP-DF,PENDENT PROTEIN KINASE INHIBITS THE ACTIVITY OF MYOGENIC HELIX-LOOP-HELIX PROTEINS 1)ifferentiation of skeletal muscle cells is inhibited by the cyclic AMP (cAMP) signal transduction pathway. Here we report that the catalytic subunit of cAMP- dependent protein kinase (PKA) can substitute for cAMP and suppress muscle-spe- cific transcription by silencing the activity of the MyoD family of regulatory factors, which includes MyoD, myogenin. myf5, and MRF4. Repression by the PKA cata- lytic (C) subunit is directed at the consensus sequence CANNTG, the target for DNA binding and transcriptional activation by these myogenic regulators. Phosphopeptide mapping of myogenin in rilro and in vinn revealed Iwo PKA phosphorylation sites, both within the basic region. However, repression of myogenin function by PKA does not require direct phosphorylation of these sites but instead involves an indirect mechanism with one or more intermediate steps. Regulation of the transcriptional activity of the Myol) family by modulation of the cAMP signaling pathway may account for the inhibitory effects of certain peptidc growth factors on muscle-spe- cific gene expression and may also determine the responsiveness of different cell types to rnyogenic conversion by these myogenic regulators. Li, L., Hcllcr-Harrison, R., Czech. M., and Olson, E. N. Molecular and Cellular Biology 12(10):447R-44R5, October 1992. Other support: National Institutes of Health and Robert A. Welch Foundation. From the Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson C'<mcer ('enter, Houston, and Program in Molecular Medicine, I)epartment of Biochemistry and Molecular Biology, llniversity of Massachusetts Medical Schcxil, Worcester. ISOLATION OF A XENOBIOTIC METABOLIZING CYTOCHROME (P-45(l~,) WITH NO AROMATASE ACTIVITY FROM PLACENTAL MICROSOMI;S OF ROTH CIGARETTE-SMOKING AND NONSMOKING MOTI IERS The presence of different monooxygenase activities has been documented in human placental microsomes. The predominating cytochrome P-450, that of andro- 9GZOO0 Is 'doo I l gen aromatase, catalyzes the biosynthesis of estrogens. Aromatase P-450 has been the object of numerous studies to determine its mechanism of action and to purify it. Gough ef al reported the presence of another enzyme system which catalyzes the hydroxylation of' xenobiotic substances. It has also been reported that a placental cytochrome P-450 is responsible for the hydroxylation of benzolalpyrene via aryl hydrocarbon hydroxylase. The low content of the responding cytochrome P-450 in human placenta and instability during purification have made it difficult to isolate any other active P-450 enzyme. As a side benefit gained from our procedure to puri- fy and characterize catalytically active aromatase P-450, we found a fraction from the immunoaffinity.chromatography which showed a high level of xenobiotic activi- ty with little contaminating aromatase activity. Through the use of conventional purification procedures, we obtained a xenobiotically active P-450 with no aro- matase activity. Previous studies on aryl hydrocarbon hydroxylase activity have demonstrated higher levels of this activity in smokers but not in nonsmokers. We compared these metabolic activities of (he purified human placental non- aromatase P-450. Osawa. Y., Higashiyama, T., Shimizu, K., and Yoshida, N. In: Soma, H. (ed): Placenta: Basic Research for Clinical Application. International Conference on Placenta. Tokyo, 1990..Basel, Karger, pp. 105-114, 1991. Other support: U.S. Public Health Service. From the Endocrine Biochemistry Department, Medical Foundation of Buffalo Research Institute, Buffalo, NY. CHARACTERIZATION OF THE HEPATITIS B VIRUS ENnI ENHANCER AND X PROMOTER COMPLEX The hepatitis B virus Enhl enhancer element overlaps the promoter of the X gene. By performing methylation interference experiments, four protein factor bind- ing sites clustered in a I20-bp region were found to control the EnhI enhancer and X promoter activities. Deletion mapping experiments indicated that the two upstream protein factor binding sites constituted a basal enhancer module. This module, likely bound by a liver-specific factor and a ubiquitous factor, could activate the herpes simplex virus thymidine kinase gene promoter by 5- or 10-fold, depending on the orientation, in Huh7 cells, a liver-derived cell line, but not in other cell types tested. The two downstream protein factor binding sites interact with the upstream basal enhancer module in an orientation- and distance-dependent manner to increase the enhancer activity by another 10-fold. In addition, at least one of the two downstream protein factor binding sites is also essential for the X promoter activity. Guo. W., Bell, K. D., and Ou, J.-hl. Journal of Virology 65(12):6686-6692, December 1991. Other support: National Institutes of Health. From the Department of Microbiology, University of Southern California, Los Angeles. 172 173
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LSZOOO LL HOD ('OLCEMID AND THE MITOTIC CYCLE We have reviewcd the evidence that, for many cells, disruption of the mitotic spinclle with Colcemid. colchicine and similar drugs delays hut does not inhibit pro- gression through the mitotic cycle. Whether a particular cell type can exit C-mitosis depends on its ability to overcome the spindle-formation surveillance checkpoint in the absence of a spindle, an ability that may depend on whether the cell can ultimate- ly degrade cyclin B while in C-milosis. C-mitotics capable of passing through this checkpoint normally advance to interphase by way of a C-anaphase. C-anaphase is indicate(1 by the separation of sister chromatids and this event does not depend on forces generated by the spindle. Ricdcr, C. L. and Palazzn. R. E. Journal of Cell Science 102:3R7-392, 1992. Other support: National Institutes of Health and American Cancer Society. From Wadsworth Center for Laboratories and Research, Albany, NY; Department of Biomedical Scicnces. State University of New York, Albany; and Marine Biological Lahoratory, Woods Hole. MA. O1.IGOMI3RIC Sl'RUC"I'URE ANI) AUTOPHOSPHORYLATION OF N(I('LEOSIDE DII'HOSPHATE KINASE FROM RAT MUCOSAL MASTCELL.S Nucleoside diphosphate (NDP) kinases have been found to he involved in a widc range of fundamental biological processes ranging from developmental control to signal transduction and metastasis. We have recently cloned and sequenced a cDNA encoding an NDP-kinase of the rat mucosal mast cell line RBL-2H3 Illemmench, S., Yarden. Y., & Pecht, I. (1992) Binrhemisny (preceding paper in this issue)j. The cnzymc itself has been isolated by means of its affinity to the his- chromone cromoglycate. Here we report several of its biochemical characteristics: A structural model for the native protein is proposed in which two disulfide-Iinked pairs of similar ISkDa subunits (p18) associate to form a 72-kDa tetramer (p72). This is based on the migration properties of the purified enzyme on gel filtration columns, sodium dodecylsulfate gel electrophoresis. and two-dimensional elec- trophoresis, together with peptide mapping data. In the absence of NDP, both intact p72 and the dissociated I8-kDa subunits (p18) were shown to undergo Mg" depen- (lent stoichiometric autophosphorylation utilizing adenosine and guanosine triphos- phate or y-thiotriphosphate as phosphate donor. This autophosphorylation activity was found to he retained by the I8-kDa subunits even following fractionation by SDS-PAGE and electrophoretic transfer to nitrocellulose. The Michaelis constant of this ainophosphorylation reaction with either ATP. ATPyS, GTP, or GTPyS was determined to be 6.5 + I µM. and maximally 2 mol of phosphate were found incor- porated per p72 molecule, thus indicating that phosphorylation occurs at a single site on only two of the four I8-kDa subunits of the holoenzyme. This covalently bound phosphate is labile to hyctroxylarnine and alkaline treatment hut is acid stable, sug- gesting the fomiation of an activated aspartyl or glutarnyl phosphate as the reaction intermediate that is characteristic for the ping-pong reaction mechanism of NDP- kinases. At a much lower stoichiometry (<0.01 mol of phosphate per mole of p72), serine residues were also found phosphorylated. Moreover, at micromolar enzyme concentrations, p72 was found to phosphorylate serine recidues in casein and histone 2b. Using antibodies raised specifically to this NDP kinase, p72 was shown in the rat to be present in most organs. In mice and humans, immunologically cross-reactive auto-phosphorylating proteins were also identified. Hemmerich, S. and Pechl,l. Biochemistry 31:4580-4587, 1992, Other support: The Yeda Fund and Government of Lower Saxony, Germany. N From the Department of Chemical Immunology, The Weizmann Institute of Science, O Rehovot, Israel. ~ N ~ 0 ~ DIMERIZATION KINETICS OF THE IgE-CLASS ANTIBODIES BY DIVALENT HAPTENS. 1. IIIE FAB-11APTEN INTERACI'IONS The binding of divalent haptens to IgE-class antibodies leads predominantly to their oligomerization into open and closed dimers. Kinetics of the open dimer formation was investigated by fluorescence titrations of Fab fragments of mono- clonal DNP-specifc IgE antibodies with divalent haptens having different spacer length (I' = 14-130 A). Binding was monitored by quenching of intrinsic trytophan emission of the Fab. Addition of divalent haptens with short spacers (I' 14-21 A) to the Fabs at rates larger than a distinct threshold value caused a significant decrease of Fab-binding site occupation in the initial phase of the titration. This finding was interpreted to reflect a nonequilibrium state of hapten-Fab-binding. Such nonequilib- rium titrations were analyzed by inserting a kinetic model into a theory of antibody aggregation as presented by Dembo and Golstein (Histamine release due to bivalent penicilloyl haptens. 1978. J. Immunol. 121, 345). Fitting of this model to (he fluores- cence titrations yielded dissociation rate constants of 7.8 • 10' s' and 6' 10' s' for the Fab dimers formed by the flexible divalent haptens N",N'-di(dinitrophenyl)-t.- lysine (1' = 16A) and bis (N®-2,4-dinitrophenyl•ananyl)-meso-diamino-succina(e (I' = 21 A). Making the simplifying assumption that a single step binding equilibri- um prevails, the corresponding dimer formation rate constants were calculated to be 1.9 • 10` M' s' and 1. 1 • 10' M's', respectively. In contrast, all haptens with spacers longer than 40A (i.e., bis(N"-2,4-dinitrophenyl-tri-o-alanyl)-1,7-diamino-heptane, and di(N•-2,4-dinitrophenyl)-6-aminohexanoate-aspartyl-(prolyl)p t; lysyl (n = 24, 27, 33) exhibit a relative fast dimerization rate of the Fab fragments (>7 • 10"M' s'). These observations were interpreted as being caused by orientational constraints set by the limited solid angle of the reaction between the macromolecular reactants. Thus, ligands having better access to the binding site would react faster. Schweitzer-Stenncr, R., Licht, A., and Pech1, 1. Biophysical Journal 63:551-562, August 1992. Other support: Government of Lower Saxony, Germany. ~ ~ 174 175
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8SZOOO IS 2iOD From the Institute of Experimental Physics, University of Bremen, Bremen, Germany, and Department of Chemical Immunology. The Weizmann Institute of Scicncc, Rchovot, Israel. line), a dosc-dependent and an InsP, indcpendent increase in (from resting Icvcls ol R3-I5U nM to 6(N)-6R0 nM), and a secretory response amounting to 30-50% of that observed upon FceRI clustering. The TG induced rise of (Ca" I., is most prob- ably provided by both arrest of its uptake by the endoplasmic reticulum and influx from the medium. Thus, Ca" influx in mast cells may be modulated by the (Ca"I,, level. A CROMOGLYCATE - BINDING PROTEIN FROM RAT MAST CELLS OF A LEUKEMIA LINE IS A NUCLEOSIDE DIPHOSPHATE KINASE Recently, we have shown that a membrane-permeant derivative of the antiasth- matic drug cromoglycate (CG) effectively inhibits the Fc -receptor-mediated secre- tory response of rat mucosal mast cells (line RBL-2H3) at a stage preceding the tran- sient rise in the cytoplasmic free calcium concentration lHemmench, S.. Sijpkins, D., & Pecht, 1. (1991) Biochemistry 30, 1523-1532]. In contrast to cromoglycate itself, which is membrane impermeant and ineffective in these cells, its his-ace- toxymethyl ester derivative (CG/AM) can diffuse across the plasma membrane into the cytosol. where it is hydrolyzed into the impermeant CG dianion, which presum- ably may interact with intracellular components involved in the Fc R signal trans- duction pathway. In order to identify cytosolic components involved in the stimulus- secretion coupling that interact with this drug, we coupled CG to an insoluble matrix. This matrix was indeed effective in the affinity isolation from RBL cells of a cytosolic protein that exhibits an apparent molecular mass of 18 kDa on reducing SDS gels. This protein was purified to homogeneity and then fragmented, and the amino acid sequence of three resultant peptides was determined. Using the corre- sponding synthetic oligonucleotides, we cloned and sequenced a cDNA that encodes the full-length 18-kDa polypeptide (pIR). The protein sequence deduced from this cDNA is identical to that of rat nucleoside diphosphate kinase (Kimura N., Shimada, N., Nomura. K., & Watanahc, K. (1990) J. Biol. Chem. 265, 1 5744-1 5749j and high- ly homologous (SR"h) to the human NM23 gene product whose expression is associ- ated with reduced metastatic potential, as well as with the Drosophila awd gene product (77% sequence identity). The plR isolated from RBL cells was indeed shown to catalyze phosphate transfer from nucleoside triphosphates to nucleoside diphosphates. This activity was inhibited by cromoglycate (/v~ 2 mM). The amino acid sequence of pIR and its enzymatic activity indicate that this cromoglycate bind- ing protein is a nuc leoside 5'-diphosphate kinase, which, through control of the cyto- plasmic pool of nucleoside triphosphates (e.g.. GTP), may affect the transduction of the Fc,-receptor-mediated signal. Hemmerich, S., Yarden. Y., and Perlrt, l. Biochemistry 31:4574-4579, 1992. From the Department of ('hemical Immunology, The Weizmann Institute of Science. Rchovot, Israel. Fce RECEPTOR MEDIATED Ca` INFLUX INTO MAST CELLS IS MODULATTD BY THE CONCENTRATION OF CYTOSOLIC FREE Ca" IONS The relationship between the Fce receptor mediated stimulation of mast cells and the Ca" signal it induces were studied using thapsigargin (TG), a blocker of the endoplasmic reticulum Ca" pump. TG induced, in mucosal mast cells (RBL-2H3 Dar, O. and Pec•ht, l. FEBS 310(2):123-128, September 1992. Other support: Fritz Thyssen Foundation. From the Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot, Israel. 1 AUTORADIOGRAPHIC LOCALIZATION AND ANALYSIS OF ENDOTHELIN-I BINDING SITES IN HUMAN SYNOVIAL TISSUE Ohjertive. To determine the localization of endothelin binding sites and immunoreactivity in human synovial tissues. Methods. Quantitative in vitro autoradiographic and immunohiskochemical tech- niques were used to hrcalize and characterize "9-labeled endothelin-I ("`I--ET-I) binding sites and endothelin-like immunoreactivity in sections of rheumatoid, ostcoarthritic, and normal synovium. Results. Specific "`I-E'('-I-binding sites, characteristic of the ET, receptor, were localizcd to the media of synovial blood vessels in all groups. No difference was found in vascular binding site density in rheumatoid and osteoarthritic syno- vium. Endothelin-like immimoreactivity was localized to endothclial cells in blood vessels clisplaying "9-ET- I binding sites. C'onrlusion. We conclude that endothelin may act locally, modulating synovial perfusion and exacerbating hypoxia in chronic arthritis. Wharton, J., Rutherford. R. A. D., Walsh, D. A., Mapp. P. I., Knock. G. A.. Blake, D. R., and Pnlak,l. M. Arthritis and Rheumatism 35(8):894-R99, August 1992. Other support: Arthritis & Rheumatism Council (England) and British Heart Foundation. From Ihc Department of Histochemistry, Royal Postgraduate Medical School; The Inflammation Group. Arthritis & Rheumatism Council Building: and the London Hospital, Whitechapcl, London, England. DI('HOTOMY IN THE LAMININ-BINDING PROPERTIES OF SOLUBLE AND MEMBRANE-BOUND HUMAN GALACTOSIDF,-BINDING PROTEIN Recent studies indicate that galactoside-bincling proteins may bind Ihe poly-N- acetyllactosamine sequences of laminin. We questioned whether human galactoside- ~ I 176 1 177
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s5zooo IS 2IOD binding protein (hL-31) binds to laminin and whether cells that express hL-31 on their surface use it as a laminin receptor to promote cellular attachment. The data show that both lectin and cells bind to immobilized laminin. The binding of soluble lectin to laminin is inhibited by lactose, while cell adhesion to it is not. The results indicate that laminin may be a ligand for soluble galactoside-binding proteins. Ochieng. J., Gerold, M., and Raz, A. Biochemical and Biophysical Research Communications IR6(3):1674-1680, August 14. 1992. Other support: National Institutes of Health. From the Metastasis Program, Michigan Cancer Foundation, Detroit. DEVELOPMENTAI. PATTERN OF GENE-SPECIFIC DNA METHYLATION IN THE MOUSE EMBRYO AND GERM LINE Methylation patterns of specific genes have been studied by polymerase chain reaction and found to undergo dynamic changes in the germ line and early embryo. Some C'pG sifes are methylaled in sperm DNA and unmodified in mature oocytes, indicating that the parental genomes have differential methylation profiles. These differences, however, are erased by a series of early embryonic demethylation and postblastula remodification events, which serve to reestablish the basic adult methyl- ation pattern prior to organogenesis. During gametogenesis, all of these sites are unmethylated in primordial germ cells but eventually become remodified by 18.5 days postcoitum in both males and females. The final methylation profile of the mature genn cells is then formed by a multistep process of site-specific demethyla- tion events. These results form a basis for the understanding of the biochemical mechanisms and role of DNA rnethylation in embryonic development. Kafri, T., Ariel, M., Brandeis, M., Shemer, R., Urven, L., McCarrey, J., Cedar, H., and Razin. A. Genes & Development 6:705-714, 1992. Other support: National Institutes of Health and U.S.-Israel Binational Science Foundation. From the Department of C'ellular Biochemistry, Hebrew University Medical School, Jerusalem, Israel, and I)epartment of Genetics, Southwest Foundation for Biomedical Research, San Antonio, TX. MOLECULAR CLONING AND DNA SEQUENCE ANALYSIS OF cDNA ENCODING CHICKEN HOMOLOGUE OF THE Bcl-2 ONCOPROTEIN We have isolated a 2228 bp cDNA clone encoding a chicken homologue of the human Bcl-2 oncoprotein by low-stringency hybridization screening of a Xgt I O cDNA library derived from a chicken B-cell lymphoma. DNA sequence analysis of this cDNA revealed an open reading frame predicting a polypeptide of 232 amino acids and a M of 25839. The predicted protein is highly homologous to the human (73%) and mouse (70%) Bcl-2 proteins, and contains a hydrophobic stretch of amino acids within its carhoxyl-end (213-229) consistent with an integral membrane pro- tein. Areas of very high sequence homology shared by all three Bcl-2 proteins at the NHE terminus (amino acids 1-33) and within the last 150 amino acids of these pro- teins suggest the presence of at least two evolutionarily conserved domains within the family of Bcl-2 proteins that may be important either for their targeting to mito- chondria or their ability to block programmed cell death. Cazals-Hatem, D. L., Louie, D. C., Tanaka, S., and Reed, J. C. Biochimica et Biophysica Acta 1132:109-113, 1992. From the Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia. THE LIVER AS A STEM CELL AND LINEAGE SYSTEM We propose that the liver is a stem cell and lineage system with many parallels to lineages in the bone marrow, gut, and epidermis, varying from them only in kinet- ics. All are organized with three compartments: a slow cycling stem cell.compart- ment with cells expressing a fetal phenotype and responding slowly to injury; an amplification compartment with cells of intermediate phenotype rapidly proliferating in response to regenerative stimuli or acute injuries; and a terminal differentiation compartment in which cells increasingly differentiate and gradually lose their ability to divide. In all systems, both those with slow or rapid kinetics, the various compart- ments are positioned in a polarized organization, are associated with a gradient in the chemistry of the extracellular matrix, and show lineage-position-dependent growth responses, gene expression, pharmacological and toxicological responses, and reac- tion to viruses and radiation. In general, known oncogens selectively kill cells in the differentiation compartment inducing chronic regenerative responses of the cells in stem cell and/or amplification compartment. Tumors arise by subsequent transforma- tion of the activated stem cells or early precursor cells. The evidence for a lineage model consists of the data implicating gradients in cell size, ploidy, growth potential, and antigenic and gene expression in the liver parenchyma along the sinusoidal plates. The traditional explanation for this heterogeneity is that it represents adaption of cells to a changing sinusoidal microenvironment dictated by the direction of blood flow. However, we review the extant data and suggest that it more readily supports a lineage model involving a maturation process beginning with stem cells and precur- sors in the periporlal zone and ending with sensescing parenchyma near the central vein. Support for this theory is provided by the studies on phenotypic heterogeneity in liver, investigations into the embryology of the liver, and analyses of the re- sponses of liver to chemical and viral oncogens that induce rapid proliferation of small cells with oval-shaped nuclei, "oval cells;" now thought to be closely related to liver stem cells. The lineage model provides clarity and insights into many aspects of liver biology and disease including the limited proliferative ability of in vitro 179 179
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09Z000 IZ HOO parenchymal cultures. liver regeneration, gene expression, viral infection, hepatocel- Iuhu carcinogcnesis, liver ccll transplantation, and aging. Sigal. S. Ii., Brill, S., Fiorino. A. S., and Reid, L. M. American Journal of Physiology 263(26):G I39-G 148. 1992. Other suporl: National Institutes of Health, American Cancer Society and Genetic Therapy. From the Departments of Molecular Pharmacology and Microbiology and Immunology, and Marion Bessin Liver Research Centcr, Albert Einstein College of Mcdicine, The Bronx, NY. TRANSCRIP7IONAL AND POSTTRANSCRIPTIONAL CONTROL OF ('ONNEXIN mRNAS IN PERIPORTAL AND PERICENTRAL RAT HF,PATO('YTES Distinct patterns of expression of gap.junction, or connexin, mRNAs were observed in periportal vs. pericentral hepatocytes. The Iwo cellular fractions (iso- latcd from rat livers by perfusion) were more than 90% parenchymal, as determined by flow cytometry for a hepatocyte-specific marker. The periportal and pericentral fractions were identifiable due to enrichment in enzymatic activities previously shown to he differentially expressed in the respective regions of liver. Northern blot analyses revealed that mRNA encoding connexin 26 was 2.8 times more abundant in the periportal than in the pericentral cells, while connexin 32 mRNA was equally distributed. Messenger RNA from each fraction was radiolabeled in order to com- pare the relative ahundancc of the connexin mRNAs in each fraction. The ratio of connexin 26 to connexin 32 mRNA in the portal fraction was about 0.085, and in the central fraction about 0.038. Connexin 26 mRNA was transcribed, however, at a faster rate Ihan connexin 32 mRNA by nuclei isolated from both cellular fractions. Connexin 26 mRNA was transcribed a( 3.9 times the rate in nuclei from the peripor- tal than from the pericentral cells. These data suggest that while the zonation of con- nexin 26 rnRNA synthesis in liver appears to he controlled transcriptionally, post- transcriptional regulatory mechanisms determine the relative abundance of the con- nexin mRNAs. Rosenberg. E.. Spray, 1). ('., Reid, L. M. European Journal of Cell Biology 59:21-26, 1992. Other support: National Institutes of Health and American Cancer Society. From the 1)cpartmentti of Molecular Pharmacology, Neuroscience, and Microbiology and Immunology. Albert Einstein College of Medicine, The Bronx, NY. EXTRA('F,LLUI,AR MATRIX GRAI)IF,NTS IN THE SPACE OF DISSE: RELEVANCE TO LIVER BIOLOGY The observation of' McGuire et al. on the importance of matrix to sinusoidal biology can he understood and further highlighted if one considers the liver as a lineage system extending between the portal triads and the central vein. We suggest that the liver has many parallels to other epithelial lineage systems such as epider- mis, intestine and hemopoiesis, in which matrix chemistry is altered in composition and quantity in a pattern that is lineage-position specific. New ultrastructural studies reveal a matrix gradient in the liver in the space of Disse. In a recent review we dis- cussed in some detail the evidence implicating the liver as a stem cell and lineage system, including the newly discovered matrix gradient. Critical aspects of this model will he given here to facilitate the discussion. Reid, L. M., Fiorino, A. S., Sigal, S. H., Brill, S., and Holst, P. A. Hepatology 15(6):I19A-1203, 1992. Other support: American Cancer Society, National Institutes of Health and Genetic Therapy, Inc. From the Departments of Molecular Pharmacology and Microbiology and Immunology and Marion Bessin Liver Research Center, Albert Einstein College of Medicine, The Bronx, NY. ras TRANSFORMATION OF CLONED RAT EMBRYO FIBROBLASTS RESULTS IN INCREASED RATES OF PROTEIN SYNTHESIS ANI) PHOSPHORYLATION OF EUKARYOTIC INITIATION FACTOR 4E Eukaryotic initiation factor 4E (e1F-4E) is a 25-kDa phosphoprotein that binds to the 7-methylguanosine cap of mRNA and acts, along with other eIF-4 polypep- tides, to unwind mRNA secondary structure at the 5' terminus. Recent studies have indicated that e1F-4E acts as a protooncogene, but only in its phosphorylated state. In order to determine the role of e1F-4E in oncogenesis, we examined its regulation and expression in cloned rat embryo fibroblasts transformed with the Harvey ras (Ha- ras) oncogene. The expression of Ha-ras increased the rate of protein synthesis but did not increase the levels of eIF-4E mRNA or protein. However, a dramatic increase (7-fold) in phosphate incorporation into eIF-4E was observed. The percent- age of eIF-4E in the phosphorylated state was the same in transfected and control cells, indicating that both phosphorylation and dephosphorylation of e1F-4E were increased. Phosphopeptide mapping of e1F-4E from transformed cells indicated a single site of phosphorylation at Ser-53, which is the same as that identified previ- ously in eIF-4E from reticulocytes and HeLa cells. These results indicate that p21'" is part of the signal transduction pathway leading to phosphorylation of eIF-4E. These findings also provide a potential mechanism for cell transformation by p21'" which involves the preferential stimulation of translation of certain mRNAs. Rinker-Schaeffer, C. W., Austin, V., Zimmer, S., and Rhnadr, R. F,. The Journal of Biological Chemistry 267(15):10659-10644, May 25, 1992. Other support: National Institute of General Medical Sciences. From the Departments of Biochemistry and Microbiology and Immunology, College of Merlicine. University of Kentucky, Lexington. 180 1R1
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T9Z000 IS 2iOa 1'RFiFERENTIAL TRANSLATION OF HEA"I' SHOCK mRNAs IN HeLa CELI S I)I?FI('IEN"1' IN PROTEIN SYNTHESIS INITIATION FACTORS cIF-4E \O M ~ AND eIF-4y ~ Expression of antisense RNA against eukaryotic translation initiation factor 4E (eIF-4E) in HcLa cells causes a reduction in the levels of both e1F-4E and eIF-4y (p220) and a concomitant decrease in the rates of both cell growth and protein syn- thesis (Dc Benedetti, A., Joshi-Barve, S., Rinker-Schaffer, C., and Rhoads, R. E. (1991) Mol. ('ell Biol. 11, 5435-5445). The synthesis of most proteins in the anti- sense RNA-expressing cells (AS cells) is decreased, but certain proteins continue to he synthcsized. In the present study, we identified many of these as stress-inducible or ccat shock proteins (IISPs). By mohilities on sodium dodecyl sulfate-polyacryl- amide gel electrophoresis and by reactivity with monoclonal antibodies generated against human HSPs, four of these were shown to he HSP 90, HSP 70. HSP 65, and IISI' 27. The steady-state levels of HSP 90. 7(l, and 27 were elevated in relation to total protein in AS cells. Pulse labeling and immunoprccipitation indicated that HSP 90 and 11SP 7(/ were synthesized more rapidly in AS cells than in control cells. The accelerated synthesis of' HSPs in the AS cells was not due, however, to increased mRNA levels: the levels of 11SP 90 and 70 mRNAs either remained the same or decreased afier induction of antisense RNA expression. Actin mRNA, a typical cel- lular mRNA, was found on high polysomes in control cells hut shifted to smaller pnlysomes in AS cells, as expected from (he general decrease in translational initia- tion caused by cIF-4E and eIF-4y depletion. HSP 90 and 70 mRNAs showed the opposite behavior: they were associated with small polysomes in control cells hut shifted to higher polysomes in AS cells. These results demonstrate that HSP mRNAs have little or no requirement in riro for the cap-recognition machinery and suggest that these mRNAs may utilize an alternative, cap-independent mechanism of transla- tional initiation. Joshi-Barve, S., I)e Bencdetti, A., and Rhucfd.c, R. E. The Journal of Biological C'hemistry 267(29):210aR-21(14 i, October 15, 1992. Other supporL• National Institute of General Medical Sciences. From the Department of Biochemistry, University of Kentucky, Lexington. A RAPID ANI) RELIABLE METHOI) FOR THE PURIFICATION OF HIGH- QUALI'I'Y PLASMID 1)NA FOR DOUBLE-STRANDED SEQUENCING Double-stranded dideoxy DNA sequencing is becoming an increasingly popular method by which to sequence DNA templates. However. the success of this method is largely depcndent upon the purity of Ihe I)NA template to be sequenced. To resolve these difficulties. we devised a rapid and reliable method for isolating and purifying plasmid DNA as a template for dideoxy sequencing. This procedure requires less than two hours to perform and consistently provides 10-20 µg of seyuencing-yuality pl,asmid DNA without phenol: chloroform:isoamy) alcohol extractions or precipitations in polyethylene glycol and ethanol. Tiesman, J. and Rizzino, A. Biotechniyucs 1O:119-i2(l, 1991. Other support: National Institute of Child Hcalth and Human Development, National Cancer Institute and American Cancer Society. From the Eppley Institute for Research in Cancer and Allied Sciences, University of Nebraska Medical Center, Omaha. EFFECTS OF CELL DENSITY AND PHORBOL ESTERS ON THE EXPRESSION OF EPIDERMAL GROWTH FACTOR RECEPTORS Previously, it has been shown that the binding of epidermal growth factor (EGF) by a wide range of cells decreases as cell density increases. In this report, we demon- strate that KB cells treated chronically with phorbol esters continue to exhibit decreases in EGF receptor binding as cell density increases. This finding suggests that protein kinase-C may not be essential for density-induced down regulation of EGF receptors, since phorbol esters are known to down regulate protein kinase-C. We also report that short-term and long-term effects of phorbol esters on the binding of EGF are affected by density. As shown previously for several cell lines, the phor- bol ester 12-O-tetradecanoylphorbol-l3-acetate transiently reduces EGF binding. We now show that the magnitude of this reduction diminishes as cell density increases. In addition, we determined that long-term treatment of KB cells with phorbol ester increases EGF binding. Again, this effect is diminished at high cell densities. Finally, we report that the increases in EGF binding induced by long-term treatment with phorbol esters are due to increases in the number of EGF receptors. Rizzino, A., Huebert, C., Kuszynski, C., and Wilder, P. J. Cytotechnology 7:85-92, 1991. Other support: Nebraska Department of Health. National Cancer Institute and American Cancer Society. From the Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha. I DIFFERENTIAL REGULATION OF THE TRANSFORMING GROWTH FACTOR TYPE-(32 GENE PROMOTER IN EMBRYONAL CARCINOMA CELLS AND THEIR DIFFERENTIATED CELLS Previous studies have shown that EC cells do not express detectable levels of TGF(32 or its mRNA until they differentiate. Thts suggested that differentiation influences the transcription of the TGF-02 gene in this model system. To address this possibility, we have examined the activity of the TGF-/32 promoter in EC cells and their differentiated cells using gene constnrcts containing various portions of the TGF-(32 promoter inserted upstream of' the reporter gene, chloramphenicol acetyl- 192 183 r `
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transferau (('A'll. We determined that the level of ('AT increases approximately ninefolcl when F,(' cells were induced to clifferentiate. Our studies also indicate that thc TGI'-/32 promoter contains ad least two positive regulatory elements thal are sep- araled by a negativc regulatory element. Finally, we have identified a CRE/ATF-like tiite 111.11 appears to be responsible for a positive regulatory clcmenl located between 77 aml 40. Kelly. D., O'Reilly. M. A., and Ri..:ino, A. Devclopmcntal Biology 153:172-175, 1992. Olher suplwrt: National Cancer Institute and American Cancer Society. From Ihc Eppley Institute for Research in Cancer and Allied Diseases. Department of Pathology ancl Microbiology, University of Nebraska Medical Center, Omaha, ancl Laboratory of ('hcmoprevention, National Cancer Institute, Bethesda. MD. ('O-LO('ALI7.ATION OF AN ENDO('YTIC MARKER AND ACID I'I IOSPHATASE IN A TUBl1LAR/RETIC'l1LAR COMPARTMENT IN MA(ROPHAGt;S Cuhured resident murine macrophages are incubated in the continuous presence of the Iluorescenl endocytic market Lucifer Yellow and a phorhol ester that activates protein kinasc ('. lJnder these steady-state labeling conditions the fluorescent tracer was, for the most part, in a tubular/relicular compartment. Enzyme cylochernical localization of acid phnsphatase in Ihe same cells showed essentially a one-to-one correhdion Ixtwecn the Lucifer Yellow- and acid phosphatase-containing compart- rnents. Procedures for epifluorescence observation anel subsequent enzyme cylo- chemical examination of the same whole mount cells are described. In addition, chemical fixation methods for the preservation of this labile tubular/relicular com- partment are presented. Luo, "1.. and Rnhinsnn, J. M. The Journal of I listnchemistry and Cytochemistry, 40(1):93-103, 1992. Other support: American Ileart Association and National Institutes of Health. From the Department ol' Cell Biology, Neurobiology and Anatomy, Ohio State University. Columbus. STIMULUS-DEPF,NDENT RELOCATION OF THE MICROTUBULE ORGANIZING ('fiNTER IN HUMAN POLYMORPHONUCLEAR LFUKO('YTI:S Polymorphonuclear Ieukocytes (PMNs) exhibit extensive directional migration (chemotaxis) and phagocylic activities. We have developed an in vitro model to eval- uate the organization of the microtubule organizing center (MTOC) in PMNs as the latter interact with various substrata, including immobilized antigen-antibody com- plexes. PMNs were layered on poly-L-lysine substrata containing ferritin (PL+F) or ferritin-antiferritin complex (PL+F+AF) and the location of MTOCs was determined by indirect immunofluorescence of tubulin using conventional epifluorescence microscopy and confocal laser scanning microscopy. The MTOCs in the majority of the PMNs attached to PL+F occupied an apical location (81.29% ± 3.34%), while in the majority of PMNs layered onto PL+F+AF, a basal location (79.37% ± 5.26%) was observed. Following disruption of microtubules (MTs) by nocodazole before layering the cells on the substrata, the proportions of PMNs with apical MTOCs were 65.2% ± 6.27% for PL+F and 47.2% ± 4.1% for PL+F+AF substrata, while the proportions of PMNs with basal MTOCs were 26.11% ± 8.89% for PL+F and 39.6% ± 4.4 for PL+F+AF substrata. The results indicate that MTOCs in human PMNs in ritro (i) occupied a°pre-defined" apical location; (ii) translocated to a "newly defined" basal location upon stimulation with immobilized antigen-antibody com- plex; (iii) and depended on intact MTs for placement of MTOCS in both situations. Chiplonkar, J. M., Vandre, D. D., and Rnbinson, J. M. Journal of Cell Science 102:723-728, 1992. Other support: National Institutes of Health and National Science Foundation. From the Department of Cell Biology, Neurobiology and Anatomy, Ohio State University, Columbus. DESIGN OF MODIFIED PYRROLINE N-OXIDE DERIVATIVES AS SPIN TRAPS SPECIFIC FOR HYDROXYL RADICAL Nitrones, 4-(2-(ethoxycarbc)nyl)elhyll-3,3,5,5-tetramethyl-I-pyrroline N-oxide (7a) and 4-12-(ethoxycarbonyl)ethyl l-5,5-di(JIH,Jmethyl)-3,3-dimethyl-I-pyrrolidine N-oxide (7b) have been synthesized. The ability of the nitrone (7a) to spin trap hydroxyl and superoxide radicals has been compared with nitrones lla and 5,5- dimethyl-I-pyrroline N-oxide (DMPO, 8). Nitrone l la bears a carbelhoxy group at C-4, whereas nitrone 7a has a spacer of two methylene units between the carbethoxy and the basic nitrone ring skeleton. Nitrone Ila trapped both hydroxyl and superox- ide radicals, while nitrone 7a trapped only hydroxyl radical. The hydroxyl radical adducts of 7a and I la were more resistant toward superoxide-mediated reduction than the hydroxyl radical adduct of DMPO (8). Arya, P., Stephens. J C., Griller, D.. Pou, S., Ramos, C. L., Pou, W. S., and Rosen, G, M. The Journal of Organic Chemistry 57:2297-2301, 1992. Other support: National Science Foundation and Chemistry of Life Processes Program. From the Department of Pharmacology and Toxicology, University of Maryland School of Pharmacy, Baltimore, and Veterans Administration Medical Center, Baltimore. 184 1 185
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THE MYOSIN LIGHT CHAIN ENHANCER AND THE SKELETAL ACTIN PROMOTER SHARE A BINDING SITE FOR FACTORS INVOLVED IN MUSCLE-SPECIFIC GENE EXPRESSION The myosin light chain (MLC)1/3 enhancer (MLC enhancer), identified at the 3' end of the skeletal MLC'1/3 locus, contains a sequence motif that is homologous to a protein-binding site of the skeletal muscle a-actin promoter. Gel shift, competition, and footprint assays demonstrated that a CArG motif in the MLC enhancer binds Ihe proteins MAPFI and MAPF2, previously identified as factors interacting with the muscle regulatory element of the skeletal a-actin promoter. Transient transfection assays with constructs containing the chloramphenicol acetyltransferase reporter gene demonstrated that a I I5-bp suhfragment of the MLC enhancer is able to exert promoter activity when provided with a silent nonmuscle TATA box. A point muta- tion at the MAPFI/2-binding site interferes with factor binding and abolishes the promoter activity of the 115-bp fragment. The observation that an oligonucleotide encompassing the MAPFI/2 site of the MLC enhancer alone cannot serve as a pro- moter element suggests that additional factor-binding sites are necessary for this function. The finding that MAPFI and MAPF2 recognize similar sequence motifs in Iwo muscle genes, simultaneously activated during muscle differentiation, implies that these factors may have a role in coordinating the activation of contractile protein gene expression during myogenesis. Ernst, H., Walsh, K., Harrison, C. A., and Rosenthal, N. Molecular and Cellular Biology I I(7):3735-3744, July 1991. Other support: Mathers Foundation, Life and Health Insurance Medical Research Fund and National Institutes of Health. From Ihe Department of Medicine, Division of Rheumatology and Immunology, Medical University of South Carolina, Charleston; Department of Physiology and Biophysics. School of Medicine, Case Western Reserve University, Cleveland, OH; and Department of Biochemistry, School of Medicine. Boston University, Boston. EFFECT OF INTF.RCLONAL HETEROGENEITY ON THE PROGRESSIVE, CONFLUENCE-MI:DIATED ACQUISITION OF THE FOCUS-FORMING PHENOTYPE IN NIH-3T3 POPULATIONS Confluence is an agent that promotes the progressive acquisition of the focus forming phenotype in clones of cells within NIH-3T3 populations. This conclusion is based on ('our results. (a) Even in cultures which have been confluent for more than 2 weeks without making foci, some cells in those cultures have been affected by the confluent state and will make foci if replated and allowed to grow into new saturated cultures. Because replicate dishes are very similar in the number and type of foci formed after replating, we conclude that the progression toward focus formation is substantially completed before replating, while the cells are in their original contlu- ent cultures. (h) Diffcrent NIH-3T3 populations were produced by expansion of small or large numbers of starting cells. When plated without exposure to confluence there was little difference in focus production among cells from these different sized starter P populations. However, confluence caused foci to arise more frequently in platings ol 10' cells derived from large starting numbers than from platings of 10' cells derived from small starting numbers of cells. This implies that the confluent state successful- ly promotes the acquisition of the focus forming phenotype in a limited percentage of cells in an NIH-3T3 culture and that those cells are absent from many small starting populations. (c) There is a progressive temporal effect of the confluent state on focus formation; the number and density of foci that emerge from replated confluent cul- tures increase with the length of time the cells spend in the confluent state. (d) There is heterogeneity among different batches of NIH-3T3 cells in the ability of the con- fluent state to induce acquisition of the focus forming phenotype. Also, the morphol- ogy of the foci that do arise after confluent treatment differs substantially among cell populations. Nonetheless, the foci formed from a single batch of cells are typically similar in morphology, indicating that those foci arose from one clone, or very few clones, of cells. Gmndel, R. and Rubin, H. Cancer Research 51:1003-1013, February I, 1991. Other support: U.S. Public Health Service. From the Department of Molecular and Cell Biology and Virus Laboratory, University of California, Berkeley. HUMAN NUCLEOTIDE EXCISION NUCLEASE REMOVES THYMINE DIMERS FROM DNA BY INCISING THE 22ND PHOSPHODIESTER BOND 5' AND THE 6TH PHOSPHODIESTER BOND 3' TO THE PHOTODIMER By using a human cell-free svstem capable of nucleotide excision repair, a syn- thetic substrate consisting of a plasmid containing four thymine dimers at unique locations, and deoxyribonucleoside 5'-[a-thioJtriphosphates for repair synthesis, we obtained DNA fragments containing repair patches with phosphorothioate linkages. Based on the resistance of these linkages to digestion by exonuclease III and their sensitivity to cleavage by Ia, we were able to delineate the borders of the repair patch to single-nucleotide resolution and found an asymmetric patch with sharp bound- aries. That the repair patch was produced by filling in a gap generated by an excision nuclease and not by nick-translation was confirmed by the f-inding that the thymidine dimer was released in a 27- to 29-nucleotide oligomer. Huang, J.-C., Svoboda, D. L., Reardon, J. T., and Sanc•ar, A. Proceedings of the National Academy of Sciences USA 89:3664-3668, April 1992. Other support: National Institutes of Health. From the Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill. 196 187
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THE REPAIR OF UV-DAMAGED DNA In Ihis review we have summarized our current understanding of UV damage to DNA and the three pathways for the repair of UV-induced DNA damage: direct reversal mediated by photolyase, base excision repair, and nucleotide excision repair. All three pathways arc well characterized in F.. coli. We have devoted consid- erable space to nucleoticle excision rcpair, as this pathway is implicated in the repair of all types of l1V-incluccd damage as well as the repair of lesions induced by other physical and chemical agents. We rely heavily on review articles for many of the results published prior to 1989 and, with apologies to Ihe scientists responsible for Ihis work, we direct our readers to the cited reviews for the complete references to Ihe appropriate research articles. Some of the more exciting advances in the past few years have involved the elucidation of the reaction mechanisms of photolyase and pyrimidine dimer glycosylase-endonuclease at Ihe mechanistic level and the realiza- tion of Ihe stepwisc recognition mechanism of (A)BC excinuclease. Equally exciting has been the discovery of iranscription-stimulated strand selective repair. The field of nucleotide excision repair in eukaryotic organisms remains an area of great chal- lenge and promise. We expect major breakthroughs in this area in the near future. Reardon, J . T. and Sanem•. A. In: Eckstein, F. and Lilley, D. M. J. (cds.): Nucleic Acids and Molecular Biology, Springcr-Verlag, Berlin, Heidelberg, pp. 54-71. 1991. Other support: National Institutes of Health. From Ihe I)epartment of Biochemistry and Biophysics, School of Medicine, I Inivcrsity of North ('arolina, Chapel flill. I IOMF.OTI(' GENE ANTh.'NNAPF.DIA mRNA CONTAINS 5'-NONCODING SE(1lIEN('ES "1'IIAT CONFER TRANSLATIONAL INITIATION BY INTERNAL RIBOSOMF, BINI)ING The Anrennnahedia /Antp] homeotic gene of Urnsnplrila melanoRaster, has two promoters. PI and 1'2. The resulting Antp mRNAs contain 1512-nucleotidc (PI ) and I727-nuclcotidc (1'2) 5'-noncoding rcgions, composed of cxons A, B. D, and E(PI ) or exons C, I), and F: (P2). respectively. Multiple AUG codons are present in cxons A, B, and ('. We have found that 252-nuclcotidc exon D, common to mRNAs from both transcription units and clevoid of AUG codons, can mediate initiation of transla- tion by internal rihosomc binding in cultured cells. Many mRNAs in Drn.ccrphila contain long 5'-noncoding regions with apparently unused AUG ccxlons, suggesting that internal rihosomc binding may be a common mechanism of translational initia- tion, :mcl possibly its regulation, in l)ro.cnphila. Oh, S.-K., Scott, M. P., and Sarnow. P. Cicncs & I)cvclopment G:1643 1651, 1992. Othcr.upport: Lucillc P. Markey Charitable Trust and National Institutes of Health. From the Departments of Biochemistry, Biophysics and Genetics, and Microbiology and Immunology, University of Colorado Health Sciences Center. Denver, and Departments of Developmental Biology and Genetics, Stanford University School of Medicine, Stanford, CA. TRANSLATIONAL ENHANCEMENT OF THE POLIOVIRUS 5' NONCODING REGION MEDIATED BY VIRUS-ENCODED POLYPEPTIDE 2A Genetic and biochemical studies have revealed that the 5' noncoding region of poliovirus mediates translation of the viral mRNA by an unusual mechanism involv- ing entry of ribosomes in internal sequences of mRNA molecules. We have found that mRNAs bearing the 5' non-coding region of poliovirus were translated at an enhanced rate in poliovirus-infected mammalian celis at a time when translation of cellular mRNAs was not yet inhibited. This translational enhancement of the poliovi- ral 5' noncoding region was mediated by the expression of encoded polypeptide 2A. This indicates that 2A is a multifunctional protein involved directly or indirectly in the activation of viral mRNA translation, in addition to its known roles in viral polyprotein processing and in inhibition of cellular protein synthesis. Thus, 2A rep- resents an activator of translation of a viral mRNA that is translated by an internal ribosome binding mechanism. A likely consequence of this role of 2A is the efficient translation of viral mRNAs early in the infectious cycle, when host cell mRNAs can still compete with viral mRNAs for the host cell translation apparatus. Hambridge, S. J. and Sarnow, P. Proceedings of the National Academy of Sciences USA 89:10272-10276, November 1992. Other support: U.S. Public Health Service, National Institutes of Health and Lucille P. Markey Charitable Trust. From ihe Departments of Microbiology and Immunology, and Biochemistry, Biophysics and Genetics, University of Colorado Health Sciences Center, Denver. MOLECULAR ASPECTS OF REGENERATION IN DEVELOPING VERTEBRATE LIMBS We review embryological as well as molecular evidence that emphasizes the idea that both the regenerate and the developing vertebrate limb bud utilize a similar set of signals that regulate pattern formation. Evidence is presented to implicate the Hox-7.I gene in the developmental regulation of growth, differentiation, and posi- tional assignment during progress zonc during limb outgrowth. Finally, we review the limited information known about the regenerative capabilities of limb buds in organisms that cannot regenerate as adults. We contend that a solution to the prob- Icm of regenerative failure among higher vertebrates will come progressively 188 189
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through a stepwise analysis of impaired regeneration associated with increasing developmental age. Muncoka, K. and ,Sassoon.l). Developmental Biology 152:37-49, 1992. Other support: Monsanto Company. From the Department of CcII and Molecular Biology and the Molecular and Cellular Biology Program, Tulane University, New Orleans. LA, and Department of Birxhemistry, Boton University School of Medicine, Boston. Mtiox: A MESODERMALLY RESTRICTED HOMEODOMAIN PROTEIN THAT BINDS AN ESSENTIAL SITE IN THE MUSCLE CREATINE KINASE ENHANCFR Myogenic helix-loop-helix (HLH) proteins, such as myogenin and MyoD, can activate muscle-specific transcription when introduced into a variety of nonmuscle cell types. Whereas cells of mesodermal origin are expecially permissive to the actions of these myogenic regulators, many other cell types are refractory to myo- genic conversion by them. Here we describe a novel homeodomain protein. MHox, that hinds an A-T-rich element in Ihe muscle creatine kinase (MCK) enhancer that is essential for muscle-specific transcription and trans-activation by myogenic HLH proteins. MHox is completely restricted to mesodermally derived cell types during emhryogenesis and to established cell lines of mesodermal origin. In contrast to most other homeolxix genes, MHox expression is excluded from the nervous system, with the highest levels observed in limb bud and visceral arches. In adult mice, MHox is expressed at high levels in skeletal muscle, heart and uterus. The DNA-binding prop- erties and pattern of MHox expression are unique among homeolmx genes and sug- gest a role for MIlox as a transcriptional regulator that participates in the establish- ment of diverse mesodermal cell types. Cserjcsi, P., Lilly, B.. Bryson, L., Wang, Y., ,Sas.conn, D. A., and Olson, E. N. Development I 15:1087- I 101, 1992. Other support: National Institutes of Health and Muscular Dystrophy Association. From the Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson ('ancer Center. Houston and Department of Biochemistry, Boston University School of Medicine, Boston. A TRANSGENE TARGET FOR POSITIONAL REGULATORS MARKS EARLY ROSTROC'AUDAL SPECIFICATION OF MYOGENIC LINEAGES In transgenic mice, muscle-specific regulatory elements from the myosin light chain (ML(') 1/3 locus drive graded expression of a linked CAT reporter gene in 190 selected fast muscles along the anteroposterior axis of the adult animal. The gradient of MLC-CAT transcripts is established early in development, during the generation of somites from the paraxial mesoderm and the activation of myogenic factor gene expression, and is not reflected in the expression of the endogenous MLCI gene. At later embryonic stages, the gradient of MLC-CAT transcripts persists in intercostal and intervertebral muscles, but is not maintained in other axial muscles. Profiles of CAT transgene activity reveal that the gradient is generated during the maturation of increasingly caudal somites, opposite to the direction of somite development, and is retained in dissociated somite cultures. We propose that coexpression of myogenic factors is necessary but not sufficient to regulate expression of the MLC-CAT trans- gene, which is responsive to additional positional cues in the embryo. Grieshammer, U., Sassoon, D., and Rosenthal, N. Cell 69:79-93, April 3, 1992. Other support: National Institutes of Health. From the Department of Biochemistry, Boston University School of Medicine, Boston. THE PRIMARY CULTURE OF MOUSE ADIPOCYTE PRECURSOR CELLS IN DEFINED MEDIUM 1. A defined medium supporting the proliferation and differentiation of adi- pocyte precursors isolated from inguinal fat pads of 8-12-day-old mice was devel- oped. 2. It consists of a I:1 mixture of DME and WAJC404A media supplemented with insulin (10 µg/mI), transferrin (10 µg/ml), fibroblast growth factor (10 ng/ml) and high density lipoproteins (HDL) (90 µg protein/mI). 3. DME-F12 medium (1:1 mixture) used as a nutrient mixture in the defined medium of rat and human adipocyte precursors was inadequate for cultivating mouse adipocyte precursors. 4. HDL had a definite beneficial effect on both preadipocyte growth and differ- entiation. 5. Differentiation was enhanced by addition of dexamethasone (10 "M) but could be almost completely inhibited by transforming growth factor (31 (TGF-(31). 6. TGF-/31 was shown to he effective only when present in the early stages of differentiation. Litthauer, D. and Serrero, G. Comparative Biochemistry and Phyiology 101 A( I):59-64, 1992. Other support: National Institutes of Health. From the W. Alton Jones Cell Science Center, Lake Placid, NY. 191 -mono
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PROSTAGLANDIN F2« INHIBITS TfIF, DIFFERENTIATION OF ADIPOCYTE PRECURSORS IN PRIMARY CULTURE Influence of arachidonate metabolite pathway on adipose differentiation was investigated using primary culture of adipocyte precursors in defined medium. Treatment of the cells with cyclooxygenase inhibitors stimulates adipose dfferentia- lion by at least 2-fold. Anong the various arachidonate metabolites tested, only prostaglandin F,, (PGF,.,) was found to inhibit Ihe differentiation of adipocyte precur- sors in a (lose dependent fashion. Other eicosanoids tested did not have any effect. A 50% inhibition of adipose differentiation was observed with a dose of PGF,,, of 3x 10"M to 7x 10"M according to the strain of rats used. Maximal inhibition occurred at PGF,, concenlrations equal or higher than 10"M. PGF,,, inhibited not only the expression of late markers of adipose differentiation such as G3PDH and triglycerides accumula- tion but also Ihe mRNA expression of early markers of adipose differentiation such as clone 154, lipoprotein lipase and ap2 gene. These results indicate that PGF, repre- sents a physiological negative modulator of adipose differentiation. Serrero. G., L.epak, N. M., and Goodrich, S. P. Biochemical and Biophysical Research Communications 183(2): 43R-442, March 16, 1992. Other support: National Institules of Health. From the W. Alton Jones Cell Science Center, Lake Placid. NY. MOLE('ULAR ('LONING OF A DIFFI;REN'I'IATION-REE,ATED mRNA IN THE ADIPOGENICC'ELI, LINE 1246 The 1246 cell line is a C3H mouse Icratoma-derivcd adipngenic cell line that can proliferate and di/7erentiate in defincd medium. We have constructed a recombinant phage library containing complementary DNAs (cl)NAs) prepared from mRNA of diflcrentiated 1246 cclls. This lihrary was screened using a differential hybridization technique. We have isolated live different cDNA clones corresponding to mRNAs that are induced during adipogenesis of 1246 cells and one cDNA clone correspimd- ing lo mRNA that is decreased chiring adipogenesis. Among the mRNAs expressed during adiposc differentiation, some are not expressed in undifferentialed cells, whereas sonic are expressed at very low levels under these conditions. Moreover, the level of' induction during differentiation and the temporal expression of the mRNAs corresponding to Ihetie cDNAs varied. Our results indicate lhat one af the cDNA clones isolated, called 154, which sclecls a 2.2-kilohase mRHA, was induced I(K)- fnld at a very early tinu during the onset nf Ihe differentiation program in 1246 cells and also in adipix.yte precursors in primary culture. t)irect sequencing of 154 cDNA insert revealed no homology with sequences in Genl3ank and PIR protein databases. The expression of 154 mRNA was stimulated by accelerators of differcntiation such a% dexamethasnne and isobutylmethylxanthinc and inhibited by tumor necrosis factor (r. Iran.fnrming growth factor f3. and cpidernial growth factor, which arc known inhibitors of 1246 cell differentiation. In addition, 154 mRNA level in adipoxytcs was down-regulated by tumor necrosis factor ot. hut not by transforming growth factor (3 or epidermal growth factor. These results suggest that the increase in 154 mRNA expression is related to the onset of adipose differentiation. Further analysis of this clone should allow characterization of a novel protein induced early during the process of'differentiation. Jiang, H.-P., Harris, S. E., and Serrero. G. Z Cell Growth & Differentiation 3:21-30, January 1992. ~ Other support: National Institutes of Health. F~{ From the W. Alton Jones Cell Science Center, Lake Placid, NY. ADIPOGENIC ACTIVITY PRODUCED BY HEPATOCYTE-DERIVED CELL LINES AND BY NORMAL HEPATOCYTES IN PRIMARY CULTURE Culture media conditioned by several hepatocyte derived cell lines were ana- lyzed for their ability to stimulate adipose differentiation of the adipogenic cell line 1246. The results presented here show that culture media from HcpG2 and Hcp3B cell lines contain a high level of Ihe activity, whereas media from hepatoma and hepato adencxarcinoma cell lines Huh-7. PLC/PRF/5, and SK-Hep-I do not contain adipogenic aclivity. Conditioned medium from HepG2 cells also stimulated differen- liation of iT3-L, cells and of rat epididymal adipoc:ytc precursors in primary culture. Partial biochemical characterization of the adipogenic activity carried out using HepG2 conditioned medium indicates that the hepaloc:yte derived adipogenic faiclor has an apparent molecular weight between 445 and 232 kDa, is destroyed by treat- menl at I(NP"C, with protease, with 2-mercapUmthanol and in acidic conditions. The activity is stable at alkaline pH. Culture media conditioned by normal rat hcpato- cytes in primary culture also conlained adipogenic activity. In contrast, medium con- ditioned by primary culture of nonhepatocyte cells also isolated from liver was deprived of this activity. The data presented in this paper suggest that hcpalocytes could be a physiological site of production of adipogenic activity. Zaitsu, H. and Serrern, G. Journal of Ccl1idar Physiology 149:339-346, 1991. Other support: National Institutes of Heallh. Frnrn the W. Alton Jones C'cll Science Center, Lake Placid. NY. DnaJ IIOMOLOGS AND PRO"1'EIN TRANSPORT • A variety of intracelhdar protein Iransporl processes require or are stimulated by members of the evolutionarily conserved 70 kDa heat shock protein (HSP7(q family and other unidentil'ied I'actors. The ahility of HSP70s to control protein fnltling and 192 193
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assembly is thought to Ix thc basis for their role in protein transport. In Esc•herirhia (nli. genetic and biochemical evidence suggests that Ihe unique bacterial 1-ISP70. DrraK, does not act alone hul functions in concert with Iwo other heat shock pro- teins. Dna.l and Grpli, to mediate such protein folding and assembly. Complete and partial DnaJ homologs have now been identified in other bacterial species as well as in the yeast, .Sucrlruromrres rererisine. Genetic evidence suggests a role in protein transport for three of fhe four yeast homologs. 13ased on the functional complex of UnaJ and f)naK in E. rnli, we propose that DnaJ homologs will interact with HSP7(/s via an evolutionarily conserved J-region to mediate protein folding and assembly rcactions important for protein transport processes. It will he of great inter- est to determine if the unidentified factors (hat are required along with HSP70s for nraximum stimulation of some transport processes correspond to DnaJ homologs. Kurihara. T. and Silver. l'. A. In: Neupert. W. and I,ill, R. (Eds.): Membrane Biogenesis and Protein Targeting. El.civer Science Puhlishers B.V., pp. 309-327, 1992. Other support: National Institutes of Hcalth. From the Department of Molecular Biology, Princeton University, Princeton. NJ '1'IIYROII) IIORMONIi AND APOTRANSFERRIN REGULATION OF (;ROW"I'H HORMONE SECRETION BY GH, RAT PITUITARY TUMOR CELLS IN IRON RIiSTRI('TI:1) SERUM-FREE DEFINEI) MEDIUM Growth hormone (GH) production by GH, rat pituitary tumor cclls in iron restricted .crunrfree clcfined medium requires apotransfcrrin (apoTf) and triiodothy- rmiinc ('T ). As measured hy radioimmunoassay, apoTf plus T, induced Gil levels 2 to 4-fold above controls. Deletion of either apoTf or T, arrested GH secretion. ApoTI'/l', defined medium regulated GH production as effectively as whole serum. Because gfucocnrticoids enhance GH secretion in serum containing cultures, the effects of dexanuthasonc were evaluated in apoTpT, defined medium. The steroid hormone showed no enhancing effects unless the cells were exposed to serum prior to incubation in apoi 17/I', defined medium. Even under these conditions, the response to dcxamethasonc remained T, dependent. These observations indicate that a yet to he charactcrircd serum factor(s), other than apoTf, regulates (he reponse to the steroid honnone. This is (he first report of Ihyroid hormone regulation of GH secre- lion by rat pituitary tumor cells under completely scrum-free chemically defined con- ditions. Sirhar6rr.1). i1.. Pakala, R., Sato, fl.. anci Eby, J. E. In Vitro Cellular & I)evclopmcntal Biology 28A: 67-71, January 1992. Other support: Anrerican Cancer Society and National Cancer Institute. From the Department of Biochemistry and Molecular Biology. University of Texas Medical Schonl, Houston. APOTRANSFERRINS FROM SEVERAL SPECIES PROMOTE THYROID HORMONE-DEPENDENT RAT PITUITARY TUMOR CELL GROWTH IN IRON-RESTRICTED SERUM-FREE DEFINED CULTURE Previously, we have studied thyroid hormone-dependent growth of GH, rat pitu- itary tumor cells in iron-restricted serum-free defined medium (Sirbasku, D. A., et al. (1991) Biochemistry 30, 295-304, 7466-7477). Proliferation was promoted by tri- iodothyronine (T) and any of seven forms of horse serum-derived apotransfen•in (apoTf). In this report, we have asked if apoTfs from other species also acted as thy- romedins and if other metal ion chelalors served this role. To address these issues, three thyromedins were isolated from human serum and identified as apoTf. Fe" depletion, and assay in low-Fe medium, gave ED,~s of 1.4-1.7 nM.Fe" saturation abolished their activities in high-Fe medium. To ask if apoTf was the major thy- romedin in human serum, hormone-depleted preparations were iron saturated and shown to no longer support T dependent GH, cell growth. Next, commercially pre- pared human, rat, horse, dog, rabbit, guinea pig and mouse apoTfs were shown to be as active under iron-restricted conditions as those isolated from human serum. Bovine apoTf and colostrum lactoferrin were > 100-fold less active: human milk apo-lactoferrin and apo-ovotransferrins were inactive. Transferrins which displayed thyromedin activity blocked the binding of "'1-rat 2Fe • Tf to GH, cell receptors while those without thyromedin activity were ineffective. Finally, the metal ion chelators EDTA, cilrate and deferoxamine did not show thyromedin activity indical- ing that apoTfs uniquely were able to promote T,-dependent cell growth in defined culture. Sato, H., Eby, J. E., Pakala, R., and Sirhasku, n. A. Molecular and Cellular Endocrinology 83:239-251, 1992. Other support: American Cancer Society and National Cancer Institute. From (he Department of Biochemistry and Molecular Biology, University of Texas Medical School. Houston. PREPARATION OF IRON-DEFICIENT TISSUE CULTURE MEDIUM BY DEFEROXAMINE-SEPHAROSE TREATMENT AND APPLICATION TO THE DIFFERENTIAL ACTIONS OF APOTRANSFERRIN AND DIFERRIC TRANSFERRIN We have shown that Iriirxiothyronine-dependent GH rat pituitary cell growth in serum-free defined culture required apotransferrin (apcif1) (D. A. Sirhasku, el al., Biochemistry 30, 295-aO4, 7466-7477, 1991). These studies were done in "low-Fe" medium without Fe(III)/Fe(II) salts. Nonetheless, significant concentrations of iron may have been contributed by other components, making this medium unsuitable for study of (he differential effects of apoTf and diferric transferrin (2Fe•Tn. Measuring residual iron in culture medium has been troublesome because (he most sensitive method (i.e.. atomic absorption) detccted levels only in excess of 10 ng/ml and did no( distinguish between the forms of iron present. To estimate the Fe(III) available to hind to apoTf, we developed a more sensitive and specific method. Urea-polyacry- 194 1 195
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lamidc gel clcctrophorcxis (PAGIi) %cparatcs apoil'I. thc Iwo monofcrric transferrins. and ?Frfl'. 1 '"IJapiil'I was incubated with mcditun, or components, and the forma- tirm of I'"I1-2J~c-f1 was monitored by urca-I'AGIi/autoradiography. By this method, thc conccntration of Fc(III) in low-Fe mcdium was estimated at 9.4 to 20 ng/rnl and Ihc sources were idcntilicd. We next sought to remove the Fc(III). Standard chelators wcrc incffcctivc or cytotoxic. In cuntrast. an affinity method with defcroxaminc- Scpharrnc depleted `tNN%, of Ihc Fc(III). In this mcdium, apoTf and 2Fc•Tf showed dif fcrcnlial cffccts. I:hy, .I. li., Sato. H., :md .Sirhas6ri, I). A. Analytical Biochemistry 2O3:317-325, 1992. Othcr.upport: American Cancer Society and National Cancer Institute. From thc I)cp:utmcnt of Biochcmistry and Molecular Biology. University of Texas Medical School. I louslott. I,A('K OF 5-MI?111Y1.('YTOSINE IN I)1('TYl),S77iL/(IM!)LSCOIDF,UM DNA We find no evidence for thc presence of 5-mcthylcytosinc in (he DNA of I)irtrnalelinm disrnidermr. Mcthylation was absent from CCGG sites in repetitive I)NA and in 1)NA Ironi Ihc aclin multigcnc family. Nor was 5-rncthylcytosinc dctcct- cd in total I)NA when hasc comlxnition was determined by means of h.p.Lc. ,Smith. ,S. .S, and Ralncr, 1). I. 13irrchcmical .Inurnal 277:273-275, 1991. OIhcr,ouhhorl: National Instilutcs ol Ilcalth, National Science Foundation and March of I)imcs I;irlh Ucfccts Foundation. From thc I)cpartmcnl of ('clt and Tutnor Biology, ('ity uf Hope National Medical ('cntcr and Bcckman Research Institute, 1)uartc, CA. and Department of Biology, Amherst ('ollcgc, Anthcrst, MA. Rli('OGNI'flON 01: FOLDBACK I)NA BY THI? Hl1MAN 1)NA (('Y"1'(1SIN1: 5-h M(:"I"I IYLTRANSFI;RASE In order to specify thc recognition requirements of Ihc human I)NA (cy(osinc- 5) mcihyltransfcrasc. Iwu isomcric 4Rmcrs were synthesized so as to link a long block nI 1)NA with a tihortcr complementary block of )NA through a tether consist- ing of f'ivc Ihymidinc residucs. Thcsc isomcric foldhack molcculcs, diffcring only in thc lucatiom of Ihc 5-mcthyldcoxycytosinc. were shown to hc unimolccular, to con- tain a rcginn of duplcx I)NA, and to contain a region of singlc-slrandcd DNA. When used ati tuhwatcs lor thc I)NA mcthyltransfcrasc, only onc of the isomers was tncthyl:ucd. A cunr),arisnn of thc structures of the two isomcrs allows us to begin to I define thc potential sites of interaction between the enzyme and the three nuclcotides forming a structural motif consisting of 5-methyldcoxycytosinc, its base-paired dcoxyguanosinc, and a deoxycytosine 5' to the paired dcoxyguanosine. Smith. S. .S., Lingcman, R. G., and Kaplan, B. E. Biochemistry 31:850-R54, 1992. - Other support: National Institutes of Health and City of Hope Cancer Center. From the City of Hope National Medical Center and Beckman, Research Institute, Duarte, CA. ME('HANISM OF HUMAN METHYL-DIRECTED DNA METHYLTRANSFERASE AND'1'HE FIDELITY OF CYTOSINE (v1ETHYLATION The properties of the methyl-dirccted DNA (cytosine-5-)-methyltransferase (EC suggest that it is the enzyme that maintains patterns of inethylation in thc human genome. Proposals for the enzyme's mechanism of action suggest that 5- methyldeoxycylidine is produced from deoxycytidine via a dihydmcytosine intenne- diate. We have used an oligcxleoxynuclcotide containing 5-tluonxlcoxycytidinc as a suicide substrate to capture the enzyme and the dihydrocytosine intermediate. Gcl retardation experiments demonstrate the formation of the expected covalent complex between duplex DNA containing 5-Iluorodeoxycytidine and the human enzyme. Formation of the complex was dependent upon the presence of the methyl donor S- adenosylmethionine, suggesting that it comprises an enzyme-linked 5-substitutcd dihydrocytosine moiety in DNA. Dihydrocytosine derivatives are extremely labile toward hydrolytic deamination in aqueous solution. Because C-to-T transition muta- tions are especially prevalent at CG sites in human DNA, we have used high-perfor- mance liquid chromatography to search for thymidine that might he generated by hydrolysis during the methyl transfer reaction. Despite the potential for deamination inherent in the formation of the intermediate, the methyltransferase did not produce detectable amounts of thymidine. The data suggest that the ability of the human methyltransferase to preserve genetic information when copying a methylation pat- tern (i.e., its fidelity) is comparable to the ability of a mammalian DNA polymerase to preserve genetic information when copying a DNA sequence. Thus, the high fre- quency of ('-to-T transitions at CG sites in human DNA does not appear to hc due to the normal enzymatic maintenance of methylation patterns. ,Saritlr, S. S.. Kaplan, B. E.. Sowers. L.C., and Newman. E. M. Proceedings of the National Academy of Sciences USA R9:4744-4748, May 1992. Othcr supporl: National Institutcs of Health. From the City of Hopc National Medical Center and Beckman Research Institute. 1)uartc, CA. 196 ~ 197 fi
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MOI.1'.('lI1.AR I)IAGNOS'1'I( :ti FOR MYELIN I'R(YI'EOLIPID PROTEIN (;ENE Mll'I-AllONS IN I'I;LI%AIiUS-MIiR'l.RA('III;R DISIiASIi I'cliiacus-Mcrihacher disease (I'MD) is a clinically heterogeneous, slowly pro- gressive Icukrrdyxlrophy. 'I'he recent dctcction of mutations in Ihc myelin protcolipid prntcin (I'I.P) gene in several PMI) paticnts offcrs Ihc opportunity both to design DNA-hascd tesls Ihsu would be u.cful in diagnosing a proportion of I'MD cases and, in particular, to evaluate the diagncistic utility rrf singlc-strand confonnation poly- nunhhi%m (SS('1') analysis tiir this disease. A combination of SS('P analysis and direct sequencing of 1'('R-amPlified 1)NA was used to screen for PLP mutations in 24 Patientc alTcctcd with Icukodyslrophics rrf unknown etiology. Two heretofore undescrihcd mutation; in Ihe 1'1,1' gene were identifietl. Asp202His in exon 4 and (ily73Arg in exon 3. The ease and efficiency of SSCP analysis in dctecting new ntutalicm..tiuppnrl Ihe ulilizairnm rrf this techniyue in screening for PLP mutations in paticnts with uncxplaincd Icukodystrophics. I)oll. R., Natowici, M. R., Schiffmann, R., and,Srnilh, F. 1. Amcrican.Inurnal of Iluman (7cnetics 51:Ifi1-I(i9. 1992. Olhcr suppurl: Naticmal Institutes of Hcalth. Fremi the 1)ivisinn uf Riochemistry and Molecular Riology, Division of Medical Genelics. liunice K. Shriver ('enter for Mental Retardation, Waltham, MA; 1)cpartmcnts ul' Pathology, Harvard Medical School and Massachusetts General Ilnspital, Roslrm: and Pedialric Neurology (Init, Iladassah University Medical ('cnter, Jertisa1cm, Israel. SIMI'I.E 1.1 IMINOM1;I'RI(' ASSAY TO DETE('T Pl1OS1'IIOINOSITOI, I.INKI:D Rf:('1;1''1'OR F.XI'RIiSS1ON IN X1iNl)P11S OO('YTES A tiimplc and sensitive method to measure the expression of phosphoinositol- linked reccptnre in Xrvm/ru.c Gerls oncytes is described. O<xytes are co-injected with Ihe calcium phr,toprotcin acyuorin and RNA. encoding the receptor of interesl. 'I'he binding of ligand to thc expressed receptor increases intracellular calcium that induccs Ihe acquorin to lunrinesce. With an autosampler-equipped luminorneter, Ihis Irrovidcs a Iully anlrnu;ucd astiay of receptcrr expression of oorytcs. This method was applied to cloning thc homhcsin/GRI' receptor expressed in Swiss 3T3 fihrohlasts. (kicytes expressing Ihe chmcd BR tihuwed up to a IO,(MX)-Rild increase in light emis- sion in response to hrmthetiin. The sensitivity of this procedure allowed detcction of positive lunrincmreler signals in single'oocytes injected with RNA transcribed from cl)NA pools uti largc as 25.0(X) chmes. These findings show Ihc potential value of this procedure for rapid screening of expression lihraries, structure/function analysis of rcccpt<rrs and analysis of receptor antagonists or agonists. 0ladi, f:. and .Shindel. F. R. RioTechniyucs IO(f,):744-747, 1991. Other support: National Institutes of Health. Medical Research Foundation of Oregon and American Lung Association of Oregon. From the Division of Neuroscicnce, Oregon Regional Primate Research Center, Beavcrton. HYPOXIA-INDUCF.D INCREASED PERMEABILITY OF ENDOTHELIAL MONOLAYERS OCCURS 7'HROIJGH LOWERING OF CELLULAR cAMP LEVELS Prolonged exposure to hypoxia, as at high altitude, results in increased vascular permeability that may be ameliorated by administration of glucocorticoids. To under- stand mechanisms underlying these observations, cultured bovine aortic and pul- monary artery endothelial cells (ECs) were subjected to hypoxia, and changes in monolayer permeability and adenosine 3',5' -cyclic monophosphate (cAMP) levels were assessed. Exposure of fmth types of cultured ECs to hypoxia (PO --14 Torr) Icd to a time- and dose-depcndent increase in monolayer permeability, as measured by diffusion of radiolabeled sotutcs, which was associated with a progressive decrease in F,C cAMP levels from (9) to 15 pmol/mg protein, and a decrease in EC atlenylate cyclase activity. The change in endothclial barcier function was prevented by addi- lion of cAMP analogues. Pertussis toxin protected EC monolayers from hypoxia- mediated increase in permeability while maintaining cAMP levels and adcnylate cyclase activity. Addition of dcxamcthasone to E(' monolayers before or simultanc- ously with their incubation under hypoxic conditions blocked the hypoxia-mediated increase in monolayer permeability. Dexamethasone pretreatment also prevented (he decline in cAMP and adenylate cyclasc levels in oxygen-deprived cultures. These data indicate that hypoxia decreases F..C harrier function by lowering adenylatc cyclase activity and cellular cAMP levels. They suggest that dexamethasone may exert its protective effect, in part, by preventing the hypoxia-induced decline in adenylate cyclase activity, leading to an increase in cellular cAMP and maintenance of EC harrier function. Ogawa, S.. Kuwahara, K., Brett. J.. Murrow, B., Morris. S. A.. Bilezikian. J. P.. Silverstein. S. C., and Stc•r•rr, l). American Journal of Physiology 262 (Cell Physiol 31):C54fi-C554, 1992. Other support: National Heart, Lung and Blood Institute. New York Heart Association, Schultz Foundation, and Juvenile Diabetes Foundation. From Ihe Departments of Physiology and Cellular Biophysics, Medicine and Pharrnaculogy, ('olumhia llnivcrsity College of Physicians & Surgeons. New York: IIYPOXIA INDU('ES I;NDOTH'F,LIA1. C'f:LL SYNTHESIS OF MFiMRRANE- ASSOCIA'fF;D PROTI;INS Hyprmemia is associated with a prothrombntic tendency. In this study we report Ihc purification and partial characteriratiom of an activator of a central cnagulation cnmpunent, factor X, induced in endnthclium by exposure to hypoxia (hypnxia- 19fi 1 199
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OLZ000 IZ 2IOa induced factor X activator or X,,,). I:xpression of X,,, occurred in a reversible manner when endothelial cell cultures were exposed to hypoxia or sodium azide but not in response to a variety of other alterations in the cellular milieu, such as heat shock or glucose deprivation. The activity of X,,,, which was not detected in normoxic endothelial cells, was maximal under acidic conditions, pH 6.0-6.8, which often coexist with hypoxia in an ischemic milieu. By sequential isoelectric focusing and preparative SDS/PAGE of endothelial membrane-rich fractions, X,,, was purified 19,000-fold and found to he a single-chain, 100-kDa polypeptide with pl 5.0. Activation of factor X by purified Xy, was not affected by blocking antibodies to other coagulation proteins or by phenylmethylsulfonyl fluoride or leupeptin but was prevented by mercury chloride or iodoacetamide. In addition to the induction of X,,,, Iwo-dimensional gel analysis of membrane fractions from metabolically labeled hypoxic endothelial cultures revealed two groups of 10 additional spots: (i) a group for which expression was maximal after 24 hr and (ii) a group for which expression continued to increase tip to 49 hr. The pattern of hypoxia-mediated modulation of protein expression was distinct from that seen with other cellular stimuli bu( could be duplicated, in pan. by sodium azide. These results indicate that hypoxia elicits a spe- cific biosynthetic response, including the expression of endothelial cell-surface mole- cules that can alter cellular function and may potentially serve as markers of hypox- emic ves.wl-wall injury. - Ogawa, S., Causs, M.. Kuwahara, K., Shreeniwas, R.. Butura, C.. Koga, S., and .Slr'rn. I). Pnx•ecdings of the National Academy of Sciences USA 88:9897-9901, November I()91. Other support: U.S. Public Hcalth Service, Schultz Foundation. Stony-Wold Fuundsninn, and New York Lung Association. From the Department of Physiology and Cellular Biophysics. Columbia University, ('nllege of I'hysicians & Surgeons, New York. prenatally treated %aline males. In additiom, an increase in plasma ACTII in adult- hoixl was observed in animals injected postnatally with sdine. 'This study indicates that the dccrease in sexual fxhavior observed in males treated prenatally with AC'71-1 is associated with increased scrotonin levels in the prcoptic area, which suggests that ACTH may act as a neuromodulator during sexual differentiation of the brain. 1t also demonstrates that the effect of perinatal manipulations on the development of male sexual behavior may vary depending on the ontogenetic period of the brain. Segarra, A. C.. Luine, V. N., and Sr'rand, F. L. Physiology & Behavior 50:6R9-697, 1991 From The Rockefeller University, Laboratory of Neuroendocrinology, New York; Hunter College, City University of New York: and Department of Biology and C'cnter for Neural Science, New York University. SPECTRAL CHARACTERIZATION OF BRAIN AND MACROPHAGE NITRIC OXIDE SYNTI{ASES -('YT(l('tIROMti P-450-t.IKt: HIiM1iPROTEINS TlIAT('ONTAIN A I1.AVIN StiMIQU1NONF. RADIC'AI. Nitric oxide (NO) is synthesized in mammals where it acts as a signal molecule for neurotransmission, vasorelaxation, and cytotoxicity. The NO synthases isolated from brain and cylokinc-activatcd macrophages are FAD- and FMN-containing Ilavoproteins that display considerable sequence homology to NADPH-cytoc:hromc 1'-450 reductasc. However, the nature of their catalytic centers is unknown. We have Rotmd that both isucnzymcs contain 2 mol of iran-protoporphyrin IX/mol of enzyme homodimer. The optical and EPR spectroscopic properties of the hcme groups were found to be remarkably similar to those of high-spin cyt(xhrome P-450. The heme iron in the resting NO synthase is ferric and five-coordina(e with a cystcine Ihiolate as the proximal axial ligand. In addition, the EPR spectra of the resting NO syn- thases contained a free radical signal attributable to a bound flavin semiyuinone that appeared to interact magnetically with the ferric hetne iron. NO production was inhibited by carbon monoxide, implying a role for the heme groups in catalysis. Snrehr, n. a. and Ikeda-Sailo, M, SEXIIAI, BEHAVIOR OF MALE RATS IS DIFFERENTIALLY AFFECTED BY TIMING OF PI;RINATAL ACTH AI)MINISTRATInN The lalxrratory ra1 was used as a model to investigate the effect of pre- and/or postnatal AC'TH administration on sexual differentiation of the brain. Pregnant Spraguc-I)awlcy rats were injected with ACTH 1-24 (10 µg/kg/2X/day or 5(N) µg/kg/2x/day); postnatally treated neonates were injected with the above dosages once a day. Perinatal treatment with ACTIi (1O µg/kg/2 X/day) altered several sexu- al behavior measurements, but did no( have an overall effect an the number of males that exhibited sexual behavior. At a higher dose (5(K) µg/kg/2 x/day) prenatal ACTH administration decreased sexual behavior in male rats, as measured by an increase in the percent of males that did not mount or intromit. In contrast, all males treated post- mtlally with ACTII (5(H) µg/kg/2x/day) completed 2 ejaculatory series and initiated a third-scri". No significant differences were observed in adult plasma testosterone or prolactin levels: howevcr, scrotonin levels in (he preoptic area of adult male rats treated prcnatally with AC'TH (5(l) µg/kg/2x/day) were significantly higher than in The Journal of Biological Chemistry 267(29):20547-2055(l, October 15, 1992. Other support: National Institutes of Health. From the Immunology Section, Cleveland Clinic, Cleveland. 011, and Department of Physiology and Biophysics, Case Western Reserve University School of Medicine. Cleveland. IN('REASEI) TYROSINE KINASE ACTIVITY OFc-SRC DURING ('ALCIIIM-INDFICED KERATINO('YTFi I)IFFERENTIATION In cultured human epidcnnal keratimocytes, induction of differentiation by Ca" and ionophore treatment was found to result in rapid elevation ot' c-Src tyrosine kinase activity and inactivation of the c-Ycs tyrosinc kivasc. Activation of c-Src 2(Wl 1 201
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kinatic was accompanicd by tyrotiinc dcphnsphrirylatirm, which might he explained hy a rapid increase in intraccllular protein-tyrosine phosphatase activity. ('a"- inducerl <lil7erentiatiun was also associ:ued with ahered tyrosine phosphorylation of .everal ccllular proteins and correlated with a marked redistribution of intracellular phrnphotyrosine from membrane and adhesion sites to the nucleus. Some of the c- Sic protcin was also found in the nucleus after ('a" Ircatmenl, and ('a''-activatetl c- Sic bound to three ccllular protcins ( 120 kDa, 65 kDa, and 34 kUa). In agreement wittr Ihc.c restdts, inmmnohistochemistry on human epidermis revealed ;tn increase in c-Src expression and tyrosine phosphorylation in cells undergoing differentiation, which ctrongly suggests a po%siblc role of non-receptor-type tyrosine kinases in cpithclial cell maturatiun. 'l.h:ro, Y., SuJr,l. M., I Ianafusa, I I., and Krueger, J. I'rrxredings of the National Academy of Scicnces USA ft9:R29R-RiO2, September 1992. Other support: Skin I)iscase Society, Thomas Fitzpatrick Research Award and National InNitutcs ol' I Icalth. From Ihe I)epartments (if Molecular Oncology and Investigative Dermatology, Rnckcfclfcr I Inivcrtiily, New York.' MAI'I'IN(i OF RI?('OMBINANT RI;'I'ROVIRtIS INTI'sGRA'I'ION SITES THAT ('Al ISI? F.XI'RI•:SSION OFTIIF. VIRAL GI:NOMI's IN MURINE EMBRYONAL ('AR('INOMA ('I;I.LS Murine embryonal carcinoma (E(') cells do not normally express Moloney murine leukemia virus genes. F;arlier, rare IiCcell lines were isolated thad expretised prrwiral neomycin resistance (ner,) gene. This expression was dependent on cellular cnhancer or promoter sequences that flank the proviral integration site. Four such integration tiitcs, dceign:nccl as Mint (I'or Moloney murine leukemia virus integration and expression siles in 1'.(' cells), have tnen mapped on mouse chroniusonies. Minla. Mirnh, Mirrrr and Mirued are unlinked :tn(I mapped on dif'feretd chromosomes (Chr), ('hr I(1. ('hr I, ('hr 5 and the X('fir, respectively. None ol' these loc:i appear to be linked to :uiy known Mo-MuI.V proviral integration sites previously rnapped. These enhancer and pnnnoter hoci may represent a new set of genes active in unelit7erenti- :ncd enrhryimic cells. 7'u4eln, M.. I lowanf,'f: A., and Seldin, M. I'. Mamntalian ( ;cnomc 2:2411-245. 1992. Other supporl: National Institute% of Ilcalth. From the I)epartntent uf Microhiology and Immunology, and I)epartment of Mcdicinc, I)ukc Ilnivcr,,ity Medical ('cntcr. I)urham. NC. SINGLE-STEP PURIFICATION OF BACTF.RIALLY EXPRESSED POLYPEPTIDES CONTAINING AN OLIGO-I1ISTIDINE DOMAIN Plasmid expression vectors have been constructed that direct the synthesis in F'sc•heric•hia roli of fusion proteins containing a stretch of six histidine residues at either the N or C terminus. This oligo-histidine domain allows the single-step purifi- cation of the fusion proteins, tinder nondenaturing conditions, by immobilized metal affinity chromatography on Ni" bound to iminodiacetic acid-agarose. Several eukaryotic transcription factors (e.g.. the upstream stimulatory factor for the adeno- virus major late promoter) have been successfully purified, in an active state, by this method. Van Dvke, M. W., Sirito, M., and Sawadogo, M. Gene I I 1:99-1(kt, 1992. Other support: Robert A. Welch Foundation. From the Department of Tumor Biology and Molecular Genetics, The University of Texas M.D. Anderson Cancer Center, Houston. CORRUPTION OF GENOMIC DATABASES WITH ANOMALOUS SEQUP,N('F. We describe evidence that DNA sequences from vectors used for cloning and sequencing have been incorporated accidentally into eukaryotic entries in the GenBank database. These incorporations were not restricted to one type of vector or to a single mechanism. Many minor instances may have been the result of simple editing errors, but some entries contained large blocks of vector sequence that had been incorporated by contamination or other accidents during cloning. Some cases involved unusual rearrangements and areas of vector distant from the normal inser- tion sites. Matches to vector were found in 0.23% of 20,000 sequences analyzed in GenBank Release 63. Although (he possibility of anomalous sequence incorporation has been recognized since the inception of GenBank and should be easy to avoid, recent evidence suggests that this problem is increasing more quickly (han the data- base ilsclf. The presence of anomalous sequence may have serious consequences for the interpretation and use of database entries, and will have an impact on issues of database management. The incorporated vector fragments described here may also bc useful for a crude estimate of the fidelity of sequence information in Ihe database. In alignments with well-defined ends, the matching sequences showed 96.R"l, iden- Iity to vector: when poorer matches with arbitrary limits were included, the aggre- gate itlentity to vector ticyuence was 94.ft"/,, Lampcrti, E. D., Kit(cllxrgcr, J. M., Smith. T. F., and Villu-KomarnJ'f L. Nucleic Acids Research 2(1(1 I):2741-2747. 1992. Other support: National Institutes of Ilealth. From the Department of Neurology, ('hildren's Ilospital, Ilarvard Medical School, and Molecular Biology ('omputer Research Resource. Dana-Fartxr ('ancer Institttte. Iiarvard School of Puhlic I lcalth, Boston. 202 1 203
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O1'T'IMI7INGTIIF NORTIIIiRN RLOT I'RO('EDIIRF We describe methods for preparing fonnaldehycle-agarose gels for use in Northern hlotling which yield conxislcnt high quality results. Using these methods, we tcs'lcd seven different commcrcially available nrcmbrancs in Northern blots. Each ntcmhrane was handled as specified by the manufacturcr in a course of hyhridiza- lirm, sIripping and rehyhridi/:uirm. Fillcr hackground was low in all cascs. but the intcnlity of lite signal gcncraucd by specific hybridization varied markedly hclwccn Iilcrs alicr INrth lite first and second hybridization. Rosen. K. M., I vnpcrli. I?. I)..:m([ l'illu-K.nnurnJ( l.. Itioi Fcchniquc. R(4): t9H 4(11, 1990. Othcr wpporl: National Inslitulcx of Ilcallh. Front lite ('hildren'ti Iluspital, Ilarvard Medical Schaol, and University of Mnv.acltuscltt Medical School, Boston. hIti1I1.FII)I?-13ONI)IiI) 1)IS('ONTINIIO(IS fipIT'Op1iS ON T111? GI.Y('OI'ROTI:IN 01' VI?SICI II.AR STOMATITIS VIRUS (NFW .IFiRSFY SI;R(YI'YI'I?) Intrachain disulfidc hunds between paired cytitcincs in lite glycopmtcin (G) of vcxicular qrrmaliti. virus (VSV) arc required for Ihc recognition nl' disctmlinuouti cpikopes by specific ntunochmal antihodiee (MAhs) (W. Kcil and R. R. Wagner, Virology I7(l:t92 407. 1')R9). ('Icavagc by ,S'luphvlncncru.c uurrac VR pmlcasc ol' Ihc 517-;tmino-acid VSV-New Jersey G pmtcin, limited Io Ihc glutamic acid at residuc 110, rcsnflcd in a protcin (dcxignsucd G,w) with greatly retarded migration by poly,tcrylnmidc gel e1ccUophoresis (1'A(;F) under ncmrcducing conditions. By Wctilcrn hhot (imnnmuhhH) analytis. protcin (i was found to lose discontinuous cpitopc IV, which maps within lite firtil 193 NII -Ienninal amino acids. These clala, cowplcd wilh Ihcnc ohlaincd by I'A(il; migralicin of a vcclor-cxpressccl, truncated pnolcin ((i, ") tutdcr reducing and nonrcducing conditions, lead us to postulate the c+cititcncc o l a majrr loop structure within lite first 19.3 NII,-ICrminal amino acids of thc ( i prntcin, pn.ihly anchored by a disulfidc bond between cysicinc IOH and cys- Icinc I(,9, cncomtpa..ing cpitopc IV. Sitc-clircclcd tnulants in which 10 ol'tltc 12 cys- Icines were individually convcrted to scrincx in vaccinia virus-based vectnrs express- ing tJtc%c .inglc-tiitc tnulanl G protcins were also constructcd, each of which was then te.led by immurnoprecipil:uitm for its capacity to recognize epilnpe-specil'ic MAhs. T'hcvc resulls showed that mutations in NI I,-Icrminal cyxlcincti 13(l, 174, ancl, to a Icucr extent. 193 all resulted in lite loss of nculrafization epitope Vlil. A muta- tiim al NI I.-ICrmin.d cy%lcinc 130 also resulted in lite loss of neutralization epitope VII. as did a mulation sU cyslcinc 108 to a lesser extent. Both cpik,pes VII and VI11 disappeared w•hcn mutalioms were made in ('OO11-dislal cysteinc 215, 240. or 271, lite general m;rp lncatiim', uf cpitopcs VII ;tnd VIII. These studies also reveal tltat distal, av wcll a,, hrurimal, cyacinc residues markcdfy influence lite disulfide-hond sccnndarv titrurturc, which n.lcn.ihly dctcrmincti the crrnfurmationaf structure of the VSV New Jcr~cy (; protein rcyuircd fur prescntation ol the major discontinunus epi- krpcs rccirgnvcd hv ncutraliiing MAbs. Grigera, P. R., Kcil, W., sutd Wagner. R. R. Journal ol' Virology (i6(6):3749-1757, June 1992. Other support: National Inxlitule uf Allergy and Infcclious Diseases and Fogarly Inlernal ional Fcl lowship. F'rom lite Department of Microbiology and Cancer C'cnlcr, University ol' Virginia School of Mcdicinc, Charlottesville. FNDOTHFLIN-I SECRETION BY HUMAN FIBROBLASTS IN CULTURE: rr•F-FC'T:S OF C'FLL DENSI'1'Y AND IFN-(3 Evidence is presented of FT-I release by cultured human fihrohlasts. Such a conclusion is supported by (hc parallelism in displacement curves obtained using dilutions of extracts prepared from fihrohlast conditioned media and the synthetic Fl'-I stanclards, and the significant time dependent increase in FT-I content in media from IFN-P trealcd human fihrohlastx. An increase in cell density significantly cle- valccl the total amount of FT-I in the conditioned media, although a linear rclaliun- ship between these two variables was not observed. 7.challos, (i. A., An S., and Wn..l. M. Biochcmislry Intcrnaliotial 25(5):R45-R52, I)cccmhcr 1991. e)Iher support: Philip Morris ('ompanies. Smokeless Tobacco Research ('ouncil and National Institutes of I Icalth. Proni lite 1)eparhncnts of Physiology and Riuchcmistry and Molecular Biology, New York Medical C'ollcge, Valhalla. TIIIORFDOXIN GENF FXPRFSSION IS TRANSCRIPTIONAL.LY F/I'- REGULATEI) BY RIiTINOL IN MONKEY CONDF/CTING AIRWAY F,PIT'IIGLIAI, ('FLI S Using lite diffcrential hyhridir,uion tcchniquc, a cDNA clunc, MT7R, was iso- latcd frum lite cDNA library of' rctinol-Ircalcd monkcy trachcohronchial (TBF) epithclial cells. MT7ff has a high sequence hotnohogy to human thiurecluxin. "I'hc cDNA insert containt 506 nuclcotides which encodes a peptide ol- 105 arnino acids. The deduced peplide contains lite highly conserved sequence Cyx-Gly-I'ro-('ys, found al lite active site ol' all Ihiuredoxins. The expression of the (hiorccloxin gene is stimulated R-1O fold by vitamin A(retinol) in monkey TBF cells. The expression is significantly cnhanccd within 4 h allcr lite vit:unin A treatment and concurrent pro- Icin tiynthc:ix is not rcyuircd for this cnhanccmcnt. T'hcsc rcxults, in cunjuncticm with lite nuclcar run-an transcriptional assay, support the conclusion that lhiorccloxin gene is transcriptionally up-regulatccl by rctinol and/or its ntctahnlilcs. An, ( i. attd Wit. R. 204 1 205
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Iii(x'hcn)ical ,md lii(rlrhy%ical Rcscarch ('nnmronic:nirn)ti IRi(I):170-175, February 'K. 1992 OIhcr.upp(rrl; National Imlilutcs of Ilcallh. Fr(rm thc ('alifornia l'rimauc Research ('enter, I Inivcrsity of Califortlia, I)avix. M11('IN-I,IKI: GLY(Y)I'ROTI?IN SE('RF?TEI) BY C'U1;1'IJRI:D HAMSTER TRA('HI:AI. I1PfH I171.IAt. ('F'.I,I.S nIU('IIPMI('AL AN11 IMMIINOI (Hn(•Al. ('I I ARA(' 11-ItI/A I I( )N We icolatc(I mucin-likc glycnprntcins from Illc conditioned medium of primary han)stcr Irachcal c11i1hcli,(I (11'1'I?) cell cullurc and charac•tcrizcd them biochemically and imn)unol(1g.icsllly. 'Il)ese glycopruteins were purified on Sepharose CI,4B after Snepn,nr)•('es hyaluronidase treatment and then by ('s('I-dens i(y-gradicnt centrifuga- li(1n in thc presence of 4 M-guanidinium chloride. The purificcf gfycoprotcins were rctiistanl 14) iligcslinn hy ch(lnelrnitin AC lyase, heparinase, heparilinase and endo-N- arctylglucnsan)inidasc~ A, 1) and II. bul susceptible to cndo-(3-galactosidasc and kcratanw,e. SI)S/PAGIi demonstrated no crn)lamination by low-molccular-mass pro- tcins. The 1lurificcl }.lycnprrncins showed a peak buoyant density of I.5(i g/ml in ('1 ('I dcntiity-gradient ccntrifugati(m. and contained IO'%, pcplidc and 9O"/., carhohy- dratc by wcight. ('arhnhyclratc% in these glycnprnlcins contained N-acctylglu- c(naminc. N-acelylf.,llaclotiaminc. galactose, fuc•nsc, sialic acid and a Iracc amount of mannusc, hut no uronic acid. Scrine and Ihrcnninc together accounted for 27`Y, of Ihe Inlal ;m)inn acid residues, In acldili(m. Ihc mucin-likc glyc•oprntcins exhibited hln(rd-}.roup A and 13 activilicti, and very tin'(mg inhibitory activity for influenza A viruti hacmagglutinati(1n. Wilh the use n/ Ihc purificcl glycuprntcin as an antigen, six nulnoclunal antibodies that stained mucus granulcs in han)stcr tracheal epithelium were (lhtainc•d. We ch:u'actcriicd the antibody produced by one nf the clones. HM D4(1. We conclude Ihat Iffl: cells cultured in Ihe serum-frce n)ecliurn secrete a gly- cnpr(11cin with plrysic(lchcmical properties similar to those known in various airways nwcins. Wn. R.. Ph,pPcr. ('. ( i.. and Cheng, P.-W. Rinc•hcn)ical Journal 277:71 3-71R, IOOI. Othcr urplnrrt: N,niunal Institutes nf I Icallh and ('ystic Fibrosis Foundation. Frrnn Ihe ('alifinnia 1'rin)atc Research ('enter. IInivcrsity of ('alifornia, I)avis, and 1)cparlment (11 I'r(liatricti an(I I)epartntcnt (1f 13ioe•hcmislry, Flnivcrsily of North ('arulina. ('hapcl I lill. AN I INI IStIAL 1?XI'RIiSSION OF A SQIIAMOIIS CELL MARKER, SMALL 1'ROI.INH-RI('lI I'RO'I•EIN GI?NI;, IN "IRA('lll:ORRON('HIAL til'TI'HEI.fUM: DIFI I'.RIiN'fIAI . RI?( ;l II.ATION ANI) GENE MAI'PIN( ; An unusual expression (1f a putative syuamrnrs cell markcr. small prcllinc-ric•h protein (tiprl), in muc•(rriliary cpithclial cells uf concluc•ling airways was demonstrated in a serum-free culture system. A cDNA clone wa% isolated from Ihe cl)NA library of monkcy trachcobrnnchial cpilhelial (TBE) cells by dil7crcnlial hyhridic;dion This cDNA clonc, MTS, exhibited 98% homology to a DNA sequence obtained from human keratinocytes Ireatecl with either UV light or phorlx)I esters (T. Kartatiova er (il., 1998, Mol. Cell. RioL 8:2195-2230). The predicted peptide of MT5 is unusual for its high content of proline (29°/r,), glulamine (18%), and cysteine (9%.,) and its repeated PKVPEPC units The level of sprl mRNA in cultured cells was inhibited more than 90% by vitamin A. In contrast, phorbol 12-myristate 13-acctate (PMA) stimulated the level of sprl mRNA by 3- to R-fold. This differential regulation coin- cided with the effects of these chemicals on the cornification of cultured TBE cells. Using MTS as a probe, we have Iclcalized the tracheal .prl gene on the human chro- mosome I by a Southern blot analysis using a panel of human-rodent somatic cell hybrid DNAs. The gene was further sublocalized to bands q22-23 by in sim hybridization., An, G., Huang, T. H.-M.. Tcsfaigzi, J., Garcia-Heras, J., Ledbetter, D. H., Carlson, D. M., and Wit, R. American Journal of Respiratory Cell and Molecular Biology 7:1(l4-I 1 I, 1992. Other support: National Institutes of Health, Cystic Fibrosis Foundation and Baylor Mental Retardation Research ('enter. From the California Primate Research ('enter and Department of Biochemistry and Biophysics. University (1f C'alifornia, 1)avis, and Institute for Molecular (icnctics, Baylor ('odlcgc of Mcclicine, I Icluston,'I'X. 1,25-DIHYDROXY VITAMIN D2 INDUCES LEUKEMIA ('EI,I. F)IFFEREN"1'IA'TION 1,25-dihydroxy vitamin D2, like I,25-dihydroxy vitamin D3, can modulate the prOlifcrati(1n and cliffcrentiaticm of human pro-myclncytic Icukenlia cclts. HI,-GO human promyelncytic leukemia eells, which are known to undergo terminal difTeren- tiatinn along the n)nnncytic lineage in response to 1,25-dihydroxy vitamin D1, were c•ultured in lilc presence nl• varying concentrations ol' I,25-clihydroxy vitatnin D2. -1'hr cells underwent (;1/O spccific• growth arrezl and mumxytic clif•fercntialirn).'I'hc kinclic:s uf growth arrest and 1lhcnotypic diffcrcnliation were similar to that nbscrvecl previously f•or 1,25-dihyclruxy vil:unin D3, 1,25-clihyclroxy vitamin D2 was effective at ccrncenlrations of 10'•M a( c•ffecling growth arrest and phenotypic dil7crentiation. Rcducing Ihc conccnlration lo 10 ° M resulted in no apparent growth arrest or clilTcr- entiatinn. Dntie respnnse studies indicate that (il/o specific grnwfh arrest and phcno- lypic diffcrcntiatinn duc to I•25-(lihydrnxy vitamin 1)2 arc coupled. Yen. A., 131uc• J., ancl Forbes, M. In Vitro Cellular & 1)cvclnnntcnlal Biology 27A:5IR-52t), July 199 1. 206 1 207
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Other uippnrt: NationaI Institutes ol' I lcalth. Anterican Institute fnr C'ancer Research, ('nurcll ('rmsolidatcd and Biutcchnnlogy I'nogramti, II.S. Army Research Office, and Naliomal Science Foundatinn. From ('ancer 13irringy I.ahorati~ry, Deparlment of Pathoingy, Veterinary College, ('nrnell l Inivcrlity. Ithaca. NY. INI)11('fiRS OF LI?UKF,MI(' ('I?I,I, I)IFFERENTIATION (.'AUSE 1)OWN-RI'.GItI.ATION Of RB GLNf:I?XPRESSION lixpreti%irm of tlu retinohlastoma (RB) tumor suppressor gene (luring ccll differ- cntiatinn induced by dimcthyl sulloxidc or sodium hutyratc was studicd in HL, fiU human prrnnyelocytic Ieukemia cells. As cells progressed through the cell cycle, the anxwnt of R13 protein per cell increased with homcnstasis maintained, so that the amcwnt or RB prntcin rclative to the total ccll mass remained almost constant. I)imetliyl .ullnxide was used to induce these promyclocytic leukemia cells to under- go terminal diffcrcntiation into mattrrc mycloid cclls. There was an early reduction in Ihe RB protcin expressed per cell. The reduction in expression was similar for cellti in all cell cycle phace1 . '1'here was also prtrgrestiively reduced expression at later timce as cellti terminally dillerentiated. 'I'hix was compared to the case in which tindium hutviatc wa% uscd to induce the dil7crentiatirm of I11.-bO cells into mature mtnux•ytic ccllti. An ruiy reduction in 1213 protein expression per cell also occurred. It occurred fiu cells in all cell cycle phases as well. Thus. (he induced differentiation oI 1I1.-hl) ccll, along either the mycloid or the mrmncytic dilfcrcntiation lineage involvc,; an earfv rrduction in RB expressiom. which is common ko both pathways. Thc rcductirm antcccdcd prtrlitcrativc arrest or diflcrentiation. hr both cascs. Ihc linal, 1"ulting Q. dilfercntialcd ccll% had less RB protein per cell Ihan Ihc prtiliferat- ing. immaturc, Icukcmic prccursor cells. Ycn. A. and ("han<Ilcr. S. Proceeding~, of Ihe Sncicly Ror Expcrimental Bink,gy and Medicine 199:291-297, 1992. riOther suppnrt: 1I.S. I'uhlic Ilculth Service. National Institutes of Ilcalth. American Institute fnr ('anccr Rcticarch. and ('urncll ('onsolidatcd and Biotechnology I'mgr:uns. Froni the (':mcer ('cll Bioingy 1:rhoratory. Department ol' Pathohogy, Veterinary ('oIlegc. ('orncll I hrivcrtiity. Ithaca, NY. ('OIIPI,ta) 1)OWN R1:(iIII.ATION OFllll? RB RE, IINOBLASTOMA AND c MY(' GF:NI?S ANTI;,('I;I)I:S ('lil.l. DlFFl?RI?N'11A'llON: POSSIBI,E ROLE OF RB AS A"S'I'ATI IS (1l tO" GIiNli '1'hc ahilitv (if Ihc well knowm morrhogcn, rctinoic acid (RA). as well as 1,25- dihvdrnrv-vitamin I)3 (VI)), whose receptor complex hinds a I)NA consensus scqucncc related to that of Ihe rctinoic acid receptor, to regulate expression of thc rctinnhlastoma (RB) ttunor suppressor gene in a context or induced cell differentia- tion was characterized. IIL-60 human promyclocylic Icukemia cells were induced to undergo myclnid or monncytic terminal cell differentiation by these agents. To investigate the potential coupling between down-regulation of RB and c-myc onco- gene expression with cell differentiation, dose response relationships for the induced down-regulation of' RB and c-myc expression were compared with each other and with induced cell differentiation. The total amount of RB protein per cell increased as cells advanced through the cell cycle, but the amount of RB protein relative to the total cell mass remained approximately, constant. Treated with RA or VD, an early progressive decrease in cellular content of the RB protein occurred in all cell cycle phases well before any cell cycle modulation or phenotypic dilTerentiation. For a dif- fcrentiation-defective variant I-ll. CiO cell line, failure to differentiate was preceded by a failure to down-regulate cellular levels of the RB protein. In dose response experiments, progressively increasing RA or VD concentrations caused progressive- ly greater reductions in RB as well as c-myc expression with an increasing fraction of cells terminally differentiating. For both RA and VL), the dose response relation- ships for reductions in RB and c-myc expression were similar, suggesting that their down-regulation may be coupled. These observations are consistent with a model whereby RB expression acts as a cellular brake to sustain a developmentally ordained statc of differentiation (i.e., preserve the "status quo"): and the down-regu- lation of heterogeneously distributed RB protein per cell below a threshold is part of' the metabolic cascade cuhninating in terminal cell diffcrentiaticw. Thus. RB may have a role in this developmental context. Yen. A., Chandler, S.. Forhes, M. E?., Fung, Y.-K., T'Ang, A.. and Pearson. R. Burapcan Journal of Cell Biology 57:210-221, 1992. Olhcr support: National Institutes of Health. American Institute for C'ancer Rescarch, and C'orncll Consolidated and Biotechnology Programs. From the Cancer Cell Biology LaExirataries, Department of Pathology. Veterinary College. Cornell University, Lthaca, NY, and Department of Hematology/- Oncology and Ophthalmology, Children's Hospital of Los Angeles. REGULATION OF ('ELL PROLIFERATION: LATE DOWN-REGULATION OFc-MYB PREi('F.DING MY6LO-MONOCYTIC CELL DIFFERENTIATION Expression of thc c-myh nuclear oncogene during the cell proliferation and dif- ferentialion of H1: 60 human promyclacytic leukemia cells was characterized and compared to the expression uf c-fos, another nuclear oncogene with transcriptional regulatory activity. During progression through the cell cycle, the amount of c-myh protein increased. lhe increase was commensurate with total cell size, thus preserv- ing the relative abundance of c-myb protein present at (ht: rtntiet of the cell cycle. In lIL-(rll cells, the induced metabolic casca<le Ieading to terminal myeloid or mono- cytic differemiation segregates into two steps occurring over two division cycles. Expression of c-myb did not diverge frorn the control until latc in this metabolic cas- cade when it decline(1 prior to onset ol' terminal differentiation. This course of 2nK 1 209
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expression was similar for both the retinoic acid induced myeloid or the 1,25-dihy- droxy vitamin D2 induced monocytic terminal differentiation of the cells. Bromodeoxyuridine. which induces proliferative arrest but not phenotypic differenti- ation of these cells, induced the same course of c-myb expression as the inducers of terminal differentiation. The same course of c-myb expression with growth arrest induced by these three different means is consistent with a potential proliferation regulatory role for c-myb in late but not early events leading to terminal differentia- tion. The dynamics of c-myb expression during this process were qualitatively, but not quantitatively, similar to the course of c-fos expression. Thus, taken with previ- ous results, then amongst the nuclear oncogenes or tumor suppressor genes, c-myc, RB, c-fos, and c-myb, only c-myc and RB expression exhibit early regulation during induced HL-60 cell differentiation. Yen, A., Samuel, V., and Forbes. M. Journal of Cellular Physiology 153:147-156, 1992. Other support: U.S. Public Health Service, National Institutes of Health, American Institute for Cancer Research, and Cornell Consolidated and Biotechnology Program. From the Cancer Biology Laboratory, Department of Pathology, College of Veterinary Medicine, Cornell University, Ithaca, NY. VI. Immunology and Adaptive Mechanisms INHIBITION OF NEUTROPHIL CHEMOTAXIS AND ACTIVATION FOLLOWING DEC'ENTRALIZATION OFTHE SUPERIOR ('ERVIC'A1. GANGLIA Recent studies have shown that bilateral decentralization (sympathectomy) of the superior cervical ganglia (SCG) of rats sensitized to the parasite NippnstrnnRylus hrasiliensis attenuated the development of pulmonary inflammation following aller- gen challenge. Syrnpathectomy inhibited total leukocyte infiltration into lung lavage fluids, particularly neutrophil infiltration. To define the effects of decentralization of the SCG on neutrophil responses, peripheral blood neutrophils of rats were isolated and lested in in vitro chemotaxis and phagocytosis assays. Neulrophils from rats that were sympathectomized 7 (lays previously displayed a marked reduction in chemo- taxis to N-formyl-methionyl-leucylphenylalanine and leukotriene B, compared to neutrophils from sham-operated or unoperated groups. Although the degree of chemotaxis was greater in blood neutrophils from parasile-infected rats than from uninfecled rats, sympathectomy markedly reduced the chemotactic responses of both groups. In addition, nculrophils of sympalhectomized rats were unresponsive to lipopolysaccharide-induced mctabolic activation as assessed by in vitro phagocytosis and oxidative reduction of nitroblue tetrazolium. Thus, decentralization of the SCG of rats affects the chemotactic responses and functions of neutrophils. Understanding the role of the sympathetic nervous system in modulating the behavior of neutrophils will shed light on the interactions between the nervous and immune systems. Carter, L., Ferrari, J. K., Davison, J. S., and Befus, D: Journal of Leukocyte Biology 51:597-602, June 1992. From the Department of Microbiology and Infectious Diseases and Department of Medical Physiology, University of Calgary, Alberta, Canada. PHENTOLAMINE BUT NOT PROPRANOLOL BLOCKS THE IMMUNOPOTENTIATING EFFECT OF COLD STRESS ON ANTIGEN- SPECIFIC IgM PRODUCTION IN MICE ORALLY IMMUNIZED WITH SHEEP RED BLOOD CELLS The effect of cold stress on immunocompetence was investigated in mice intra- gastrically intubated with sheep red blood cells. Cold stress was found to consistently augment total IgG and IgM production by splenic lymphocytes. In addition, antigen- specific IgM production by cultured splenic lymphocytes obtained from cold stressed animals was enhanced compared to unstressed mice. However, serum levels of total and antigen-specific immunoglobulins were suppressed or unaltered following cold stress. The a-adrenoceptor antagonist, phentolamine, could block the effects mediat- ed by cold stress while the (3-adrenoceptor antagonist, propranolol, potentiated the action of cold stress. Taken together, the data indicate cold stress-mediated enhance- ment in immunoglobulin production by orally immunized animals takes place through the activation of a-adrenergic pathways. The results also suggest a- and a- adrenergic pathways independently regulate antibody production following oral administration of antigen. These observations illustrate the integrative nature of the immune and neuroendocrine systems. Carr, D. J. J., Woolley, T. W., and Blalock, J. E. Brain Behavior and Immunity 6:50-63, 1992. From (he Department of Physiology and Biophysics and Biostatistics, University of Alabama at Birmingham. SELF-REACTIVE REPERTOIRE OF TIGHT SKIN (TSK/+) MOUSE: IMMUNOCHEMICAL AND MOLECULAR CHARACTERIZATION OF ANTI-CELLULAR AUTOANTIBODIES The tight skin (TSK/+) mouse has been proposed as an experimental model for progressive systemic sclerosis because of the biochemical alterations in collagen synthesis and pathological similarities to the human disease. Here, we report the analysis of tight skin mice sera for the presence of anti-cytoplasmic and anti-nuclear 210 1 211
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autoantihodies and determination of the frequency of hybridomas producing anti-cel- lular autoantibodies. The binding specificity of TSK mAbs to nuclear and cytoplas- mic antigens such as keratin, actin, vimentin, and mitochondria was determined. Of 71 monoclonal antibodies that we have studied, only 3 appear to bind to foreign as well as sel('-antigens, indicating that the majority of these antibodies do not belong to the class of natural autoantibodics. Our results also showed that the frequency of hybridomas producing anti-nuclear and anti-cytoplasmic antibodies was higher in TSK mice than in C57BL/6 pa/pa, the control mouse strain, used in these studies. The results of the analysis of V gene usage showed that the majority of anti-cyto- plasmic and anti-nuclear antibodies are encoded by genes from a restricted number of V,, and VK genes families. In the sera of TSK mice we have detected the presence of autoantibodies specific for cytoplasmic antigens in addition to anti-nuclear and antitopoisomerase I antibodies which are characteristic of scleroderma. Since the presence of anti-cytoplasmic antibodies has not been described in scleroderma, the significance of their production in tight skin mice is not clear. However, the presence of such autoantitodies in the animal model provides a basis for investigation of this type of antibodies in human disease. Muryoi, T., Andre-Schwartz, J., Saitoh, Y., Daian, C., Hall, B., Dimitriu-Bona, A., Schwartz, R. S., Bnna. C. A., and Kasturi, K. N. Cellular Immunology 144:43-54, 1992. Other support: National Institute of Allergy and Infectious Diseases. From the Department of Microbiology, Mount Sinai School of Medicine, New York, and F)ivision of Hematology-Oncology, New England Medical Center, Tufts Flniversity, Boston., ANALYSIS OFTHF, EXPRESSION IMMUNOGLOBULIN GENE REPERTOIRE BY S('RI:ENING LIBRARIES DERIVED FROM PCR-AMPLIF7ED cDNA The set of variable region (V) genes expressed by the population of B lympho- cytes at a particular time represents the antibody repertoire of the individual. The V genes from 3' end V,gene families and certain V. gene families are relatively over- expressed during necinatal development but usage of VI and V gene families nor- malizcs to random pattern in adults. At present it is not certain whether or not autoantibrxlies are encoded by a few restricted V genes. Analysis of the expression of V an V or V gene repertoires in autoimmune animals is essential to determine whetber or not certain V genes are overexpressed in autoimmune diseases. Hence, a rapid, reliable and convenient method is essential. The currently available methods for such analysis: Northern blotting and sequence analysis of V genes expressed in hyhridomas or F;BV transformed B cell lines, in situ hybridization of LPS stimulated B cell colonies, and in silu RNA hybridization of differentiated B cells have limita- tions. First of all, isolating, maintaining and analyzing a large number B cell clones is time consuming. Secondly, EBV transformed B cell lines are unstable. Thirdly, in vitro culture methods introduce selection. We report here a novel method that is effi- cient. reliable and provides data reflecting the in rivn levels of individual V gene mRNAs expressed. Kasturi, K. and Bona, C. A. Nucleic Acids Research 19(22):6339-6340, 1991. Other support: New York Chapter of Arthritis Foundation. From the Department of Microbiology, Mount Sinai School of Medicine, New York. ANTITOPOISOMERASE I MONOCLONAL AUTOANTIBODIES FROM SCLERODERMA PATIENTS AND TIGHT SKIN MOUSE INTERACT WITH SIMILAR EPITOPES We have generated for the first time monoclonal antibodies (mAbs) specific for topoisomerase I (topo I) from scleroderma patients, and tight skin mice which devel- op a scleroderma-Iike syndrome. The epitope specificity of these antibodies has been determined using a series of fusion proteins containing contiguous portions of topo I polypeptide. Western blot analysis demonstrated that both human and mouse mAbs bound strongly to fusion protein C encompassing the NHZ terminal portion of the enzyme, and weakly to fusion proteins F and G containing regions close to the COOH-terminal end of the molecule. This crossreactivity is related to a tripeptide sequence homology in F, G, and C fusion proteins. It is interesting that a pentapep- tide sequence homologous to that in fusion protein C was identified in the UL70 pro- tein of cytomegalovirus, suggesting that activation of autoreactive B cell clones by molecular mimicry is possible. Both human and mouse mAbs exhibiting the same antigen specificity, also share an interspecies cross-reactive idiotope. These data sug- gest that B cell clones producing antitopo autoantihodies present in human and mouse repertoire are conserved during phylogeny, and are activated during the development of scleroderma disease. Muryoi, T., Katuri, K. N., Kafina, M. J., Cram, D. S., Harrison, L. C., Sasaki, T., and Bona, C. A. Journal of Experimental Medicine 175:1103-1109, April 1992. Other support: National Institute of Allergy and Infectious Diseases. From the Department of Microbiology, Mount Sinai School of Medicine, New York; Second Department of Internal Medicine, Tohoku University School of Medicine, Sendai, Japan: and Burnet Clinical Research Unit, Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia. PRODUCTION OF HUMAN PAPILLOMAVIRUS AND MODULATION OF THE INFECTIOUS PROGRAM IN EPITHELIAL RAFT CULTURES Human papiltomavinises trophic for anogenital epithelia cause benign warts, and certain genotypes are closely associated with cervical neoplasia. By using our 212 I 213
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modifications of the epithelial raft culture system, we were able to recapitulate and modulate the infectious program of a papillomavirus in vitro for the first time. Small pieces of a condyloma containing human papillomavirus type I I were explanted onto a dern)al equivalent consisting of a collagen matrix with fibroblasts and were cultured at the medium-air interface. The infected stem cells proliferated rapidly across the matrix, stratified, and differentiated, as judged by histology. The results correlated with the state of epithelial differentiation, which, in turn, was dependent on the type of fibroblast in the matrix. Under conditions where the epithelial out- growth underwent terminal differentiation, the entire productive program took place, leading to virion assembly. In contrast, using an alternative condition where the out- growth failed to achieve tenninal differentiation, only the E-region RNAs from the El promoter accumulated to any appreciable extent. The proliferating cell nuclear antigen was induced in the differentiated suprabasal cells in the productive cyst growth, which also exhibited high copy viral DNA and abundant E6-E7 RNAs. Comparable cells in the nonproductive cyst outgrowth were negative for all three. These results suggest that the E6 and E7 proteins may play a role in establishing a cellular environment conducive to vegetative viral replication. The culture condi- lions described should be useful for genetic analysis of this family of important human pathogens and for testing potential pharmacological agents. Dollard, S. C., Wilson, J. L., Demeter, L. M., Bonnez, W., Reichman, R. C., Broker, 7'.R..andChow,L.T. Genes & Dcvelopment 6:1 131-1142, 1992. Other support: U.S. Public Health Service. From the Departmentti of Biochemistry and Medicine, Infectious Diseases Unit, llnivernity of Rochester School of Medicine and 1)entistry. Rochester. NY. HUMAN MONO('I.ONAL STRIATIONAL AUTOANTIBODIES ISOLATED FROM THYMI(' B LYMPHOCYTES OF PATIENTS WITH MYASTHENIA GRAVIS USE V,, ANI) V. GENE SEGMENTS ASSOCIATED WITH TIIE Alfi'OIMMUNE REPERTOIRE The prmluction of autoantihixlies reactive with components of skeletal muscle is characteristic of myasthenia gravis (MG) and is strongly associated with the presence of thymic epithclial tumors in patients with MG. The nucleotide sequences of the heavy and light chain variable regions (V„ and Vd of three human monoclonal striat- ed muscle antihodies (SIrAb) isolated from thymic B lymphocyte lines from two patients with MG and thymoma were analyzed. The V,) and V, gene segments used by these anti-striated muscle antibodies appear to be derrved from the same repertoire of gene segments as have been found in other autoantibodics isolated from patients with a number of different autoimmune diseases. While the IgM StrAb SA-IA is a direct copy of germ-line V,. and V, gene segments, an analysis of the IgG StrAb SA- 4A and SA-4B, which may be more representative of antib(xlies found in the serum of MG patients, suggests that the processes of antigenic selection and somatic muta- tion may contribute to the generation of autoantibodies in MG. 214 ~ ~ Victor, K. D., Pascual, V.. Williams, C. L., Lennon, V. A., and Capra, J. D. European Journal of Immunology 22:2231-2236, 1992. Other support: National institutes of Health. From the Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, and Neuroimmunology Laboratory, Department of Immunology, Neurology, and Laboratory Medicine and Pathology, Mayo Clinic and Mayo Foundation, Rochester, MN. ~ V,, 4-21, A HUMAN V GENE SEGMENT OVERREPRESENTED IN THE , AUTOIMMUNE REP&TO1RE The molecular study of human autoantibodies, under normal as well as under autoimmune conditions, shows the recurrence of particular V„ genes which, in asso- ciation with a diverse array of D and J,, segments and light chains, give rise to differ- ent specificities and represent a vast portion of the repertoire. Although we have not addressed the real forces which drive the preferential expression and/or selection of these V„ segments, the example of the V,, 4-21 gene segment makes us envision a hypothesis in which the repertoire of self-renewing B cells in the bone marrow is expanded before encountering the fine specific antigen that its "conventional" bind- ing sites recognizes, based on a broader, and very likely lower, affinity toward its own milieu, in this case represented by the contiguous pool of renewing red blood cells. It might well be that cells displaying high affinities are lolerized upon encounter with an overwhelming amount of antigen in the bone marrow. It might also happen that T cells control the ability of V„ 4-21-bearing B cells to proliferate and switch under normal circumstances, avoiding the generation of highly specific self antibodies. The preferential finding of a particular V„ gene expressed in the repertoire of human antibodies encoding very different specificities could simply be the reflection of how the potential repertoire is set: less diverse than we think, but undoubtedly extremely plastic. The absence of the same V„ gene in antibodies to exogenous anti- gens might indicate that tolerance is indeed an important factor in shaping the normal repertoire. Pascual, V. and Capra, J. 0. Arthritis and Rheumatism 35(I):I 1-18, January 1992. Other support: National Institutes of Health and Robert A. Welch Foundation. From the Department of Microbiology. University of Texas Southwestern Medical ('enter, Dallas. V,, RESTRICTION AMONG HUMAN COLD AGGLUTININS-Tnr: v„ 4-21 Gr•.NI: SIHiMtiNT IS RI':QUIRIiD TO IiN('OUF. ANTI-1 AND nNTI-i SPF.('lI7CITn:S We previously reported that human autoantihcxlies with cold agglutinin activity contained a single human V„ gene segment ( V„ 4-21) which was also responsible for the cross-idiotypic specificity characteristic of the cold agglutinin response. To con- 215 ,,
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firm and extend this observation we have analyzed at the nucleotide level the H and I, chains of six new cold agglutinin molecules derived from different patients. We f2wnd that regardless of whether the antibody recognizes the i or the I red cell Ag, restriction at the V,, gene segment level is absolute. We also found that even in the absence of somatic mutation, the V11 4-21 gene segment can encode both anti-i and anti-I specificities. Finally, although the V,, 4-21 gene segment is essential for cold agglutinin activity, the other genetic elements that contribute to the V region of the antiMidy molecules can be extremely diverse. The structural information provided in this report sharply restricts Ihe requirement for encoding pathogenic cold agglutinin activity to one of the components of the H chain V region, specifically (he VH gene segment. The implications of this apparently absolute requirement for a single V,, gene segment, unprecedented in the human autoimmune response, are discussed. Pascual, V., Victor, K., Spellerberg, M., Hamblin, T. J., Stevenson, F. K., and Capra, J. I). The Journal of Immunology 149(7)2337-2344,October 1, 1992. Froni the Department of Microbiology, University of Texas Southwestern Medical ('enter, Dallas, and Lymphoma Research Unit, Tenovus Research Laboratory, General Hospital, Southampton. England. IgM RIIEUMA'fOID FACTORS IN PATIENTS WITH RHF,UMATOID ARTHRITIS DERIVE FROM A 1)IVERSE ARRAY OF GERMLINE IMM(JNOGLOBIII.IN CHiN1:S ANI) 1)ISPLAY LITTLE EVIDENCE OF SOMAT'I(' VARIATION Rheumatoid factors (RF) are present in the plasma of patients with rheumatoid arthritis (RA) although the site of synthesis of most of these antibodies is within the synovium. This report primary concerns RF of the IgM isotype. While a few of the RF derive from patients with systemic lupus erythematosus or from normal individu- als, the remaining derive from the inflamed synovial tissue of patients with RA. Two RF are encoded by members of the V„I gene family, 8 from the V„3 family and 2 from the V„4 family. Two polyreaclive antibodies derive from the V„3 family and 2 come from thc V,,4 family. This distribution is not fundamentally different from the distributions seen in a large array of autoantibixties and antibodies to external anti- gens. Similarly, the light chains derive from most of the known kappa and lambda V, families. It is hard to escape the preliminary conclusion that gene segments from vir- tually any light chain variable region can contribute to RF or polyreactive antibody structures. Most IgM RF and polyreactive antibodies are direct copies of germline genes in one of their pnlypeptide chains or at most are 2 nucleotides away in one of their chain~ from a known gemiline gene. Paseual, V.. Victor, K., Randen, I., Thompson, K., Natvig, J. B., and C'apra.J. D. Journal of Rheumatology 19 (Supplement 12):5t1-53, 1992. Other support: National Institutes of Health and Robert A. Welch Foundation. From the Department of Microbiology, University of Texas Southwestern Medical Center, Dallas; Institute for Immunology and Rheumatology, University Hospital, Oslo, Norway; and the MRC, MIP Unit, Department of Immunology, the Institute of Animal Physiology and Genetics, Cambridge, England. NUCLEOTIDE SEQUENCE ANALYSIS OF RHEUMATOID FACTORS AND POLYREACTIVE ANTIBODIES DERIVED FROM PATIENTS WITH RHEUMATOID ARTHRITIS REVEALS DIVERSE USE OF V„ AND V, GENE SEGMENTS AND EXTENSIVE VARIABILITY IN CDR-3 The heavy and light chain nucleotide sequences of 17 monoreactive and polyre- active rheumatoid factors largely derived from the inflamed synovial tissue of two patients with rheumatoid arthritis are described. Some of these sequences have been the subject of a previous report from our laboratories. Additionally, a few rheumatoid factors from the peripheral blood of patients with systemic lupus erythematosus and Sjogren's syndrome as well as a normal individual are included. A review of our pre- vious results as well as the new data provided within this paper lead to the following major conclusions: (I) Rheumatoid factors and polyreactive antibodies derive from a diverse array of V,, and V, gene segments; (2) While many rheumatoid factors and polyreactive antibodies are direct or nearly direct copies of germline genes, sonie show clear evidence of somatic mutation; (3) The CDR3 of all of these antibodies is extraordinarily diverse in length and composition. Certain "restrictions" do appear in this very large sample: (a) the polyreactive antibodies are exclusively lambda, and (b) there seems to be a preponderance of a particular subset of V,,3 genes beyond that one would expect based on random utilization. Pascual, V., Victor, K., Randen, 1., Thompson, K., Steinitz, M., Fon•e, O., Fu, S: M., Natvig, J. B., and Capra, J. D. Scandinavian Journal of Immunology 36:349-362, 1992. Other support: National Institutes of Health and Robert A. Welch Foundation. From the Department of Microbiology, University of Texas Southwestern Medical Center, Dallas; Institute for Immunology and Rheumatology, University Hospital, Oslo. Norway; Department of Pathology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel; Oslo Sanitetsforenings Rheumatism Hospital, Oslo; and Department of Medicine, University of Virginia School of Medicine, Charlottesville. SOME CAUTIONARY NOTES ON INTERPRETING IMMUNOGLOBULIN VARIABLE GENE SEGMENT RESTRICTION IN AUTOIMMUNE DISEASE Since the development of molecular biological techniques and, in particular, the introduction and routine use Epstein-Ban- virus (EBV) transformation of human B lymphocytes, there has been an explosion of data concerning the utilization of vari- ous human V„ and V, gene segments in autoantibodies of diverse specificities. 216 1 217
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Although these data have provided critical information about the extent of the human immune receptor repertoire, as well as certain insights about mechanisms of antibody diversity, Ihere are some potential problems with interpreting them that need to be understocxl as additional information accumulates. Molecular biological techniques, particularly the synthesis of cDNAs and the use of the polymerase chain reaction to generate V„ and V, structures, have resulted in nearly 1(K) human autoantibcxly structures being sequenced at both the V„ and V, level. An area of considerable interest has been the interpretation of these data as they apply to re.etrictions in the V-gene usage in immune responses in certain dis- eases. The implications of this are obvious therapeutically, as should a single V,, or V, gene be utilized in Ihe vast majority of antibodies of a particular immune response, an avenue of therapy directed against such immune receptors would be opened. This chapter is intended to provide some areas for which caution should be exercised in these interprctations. It is quite possible that we know so little of why immunoglobulin genes rearrange in the first place and why certain gene segments are ulilized in particular responses Ihat much of Ihe available data and the insights that they provided may be misleading. Victor. K., Andris, l., Pascual, V_, and C'apra, J. D. In: Bigazzi. P. F. and Reichlin, M. (eds.): Systemic Autoimmunily. Marcel Dekker. Inc.. New York. Basel, Hong Kong, pp. 1-12, 1991. Other support: National Institutes of Health and Robert A. Welch Foundation. From the University of Texas Southwestern Medical Center. Dallas. SMALI. IIUMAN V GENE FAMILIES SHOW REMARKABLY LITTLE POLYMORPHISM After only a few human myeloma proteins were sequenced in the early 1960s, was it obvious that the human B cell repertoire displayed remarkable heterogeneity in that no two sequences were more than 85% identical. As murine proteins were sequenced in the late 1960s and early 1970s, more homogeneous structures were seen and, as an understanding of the V, D, and J gene segments became apparent, it was obvious that, although rare, occasionally two independently derived murine mycloma proteins would he found with identical V,, regions. At the present time, with well over 3(X) human and murine myelomas and hybridomas sequenced, it is still very unusual to find two V„ regions that are identical. Viewed in this light, the human V,, genes appear no different than the human genes for any gene family, be they pancreatic clastases, albumin, globin, or the low density lipoprotcin (LDL) receptor. In these instances, exonic sequences are known to vary at most at the level of l).I%, which is approximately the level we are seeing here. The notion that human V„ genes are quite polymorphic is, therefore, incorrect and it may well be Ihal all human antibody genes are as similar to each other as are all other genes in the human germ line. Thus, the remarkable variation that is seen in the human population among expressed antibodies would have to be explained by somatic mutation. It may well be that the reason this level of germ line variation has not been seen in thc murine system is because most murine antibodies that have been studied are directed toward small haptens and the techniques used for the isolation of hybridomas differ from the human work. Nonetheless, the findings reported above suggest a remarkable homogeneity of human VH sequences (particularly among the smaller human V" families), and these similar structures may explain the structural basis of cross-reacting idiotypes among human antibodies, particularly autoanti- bodies that typically are IgM and much closer to germ line. Williams, C., Weigel, L., Sanz, I., and Capra, J. D. In: Cazenave, P.-A. (ed.): Anti-Idiotypic Vaccines. Springer-Verlag New York, Inc., pp. 22-30, 1991. Other support: National Institutes of Health. From the Department of Microbiology, University of Texas Southwestern Medical Center, Dallas. INTERACTION OF THE HUMAN T-CELL LYMPHOTROPHIC VIRUS TYPE I (HTLV-1) TRANSCRIPTIONAL ACTIVATOR TAX WITH CELLULAR FACTORS THAT BIND SPECIFICALLY TO THE 2 I-BASE-PAIR REPEATS IN THE HTLV-1 ENHANCER The human T-cell lymphotrophic virus type I(HTLV-1) Tax protein activates transcription from three 21-base-pair (bp) repeat sequences in the viral enhancer. Using gel electrophoretic mobility-shift assays, we now show that Tax interacts directly with the nuclear proteins, Tax activation factors (TAFs), that bind the 21 -bp repeats. This interaction is demonstrated by decreased electrophoretic mobilities of the TAFs-2I-bp-repeats complexes upon supply of Tax exogenously. Formation of the TAFs-2I-bp-repeats and Tax-TAFs-2I-bp-repeats complexes correlates with in vivo transactivation by Tax. Furthermore, interaction of Tax with TAFs enhances their binding to the 21-bp repeats. These data indicate that trans-activation by Tax is most likely mediated by interaction of Tax with TAFs. Zhao, L.-1. and Ciam, C.-Z. Proceedings of the National Academy of Sciences USA 88:11445-I 1449, December 1991. Other support: National Institutes of Health. From the Division of Infectious Diseases, Departments of Medicine and Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, OH. SEQUENCES OF THE CELL-ATTACHMENT SITES OF REOVIRUS TYPE 3 AND ITS ANTI-IDIOTYPIC/ANTIRECEPTOR ANTIBODY: MODELING OF THEIR THREE-DIMENSIONAL STRUCTURES Previous studies have identified an area of amino acid sequence similarity shared by Ihe reovirus type 3 cell-attachment protein rr I and an anti-idiotypic/antire- 21R 1 219
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ceptor monoclonal antibody (mAb) 87.92.6 that mimics reovirus type 3 by attaching to the same cell-surface receptor. We found that synthetic peptides corresponding to this area of primary sequence similarity bind a neutralizing mAb 9BG5 against which the mAb 87.92.6 is directed. The synthetic peptides compete with mAb 87.92.6 and reovirus type 3 for binding by mAb 9BG5 and displace mAb 87.92.6 and reovirus type 3 from binding to the cell-surface reovirus type 3 receptor. Such observations show that the shared primary structure between reovirus type 3 rr I polypeptide and antireceptor mAb 87.92.6 defines the oligopeptide neutralizing/cell-attachment epi- tope of rcovirus type 3. Computer modeling of this epitope, by use of sequence simi- larities of known immunoglobulin hypervariable loop conformations, permits an examination of the rudimentary three-dimensional structure of this epitope. Williams. W. V., Guy, H. R., Rubin, D. H., Robey, F., Myers, J. N., Kieber- Emmons, T., Weiner, D. B., and Greene, M. /. Proceedings of the National Academy of Sciences USA 85:6488-6492, September 1988. Other support: National Institutes of Health, American Cancer Society and National Cancer Institute. From the Departments of Pathology and Microbiology, University of Pennsylvania; Research Medicine, Veterans Administration Medical Center, Philadelphia; Laboratory of Mathematical Biology, National Cancer Inslitute, Bethesda, MD; Lalxrratory of Oral Biology and Physiology, National Institute of Dental Re,cearch, Bethesda: and IDEC Pharmaceutical Corporation, La Jolla. CA. MUTATIONAL ANALYSIS OF HUMAN T-CELL LEUKEMIA VIRUS TYPE I TAX: RF.GIONS NECESSARY FOR FUNCTION DETERMINED WITH 47 MUTANT PROTF,INS We have made 47 mutations that span the length of the human T-cell leukemia virus type I(HTLV-1)) Tax open reading framc. Of the 47 mutations. 38 were substi- tutions of single amino acids, 5 were missense changes in two or more amino acids, and 4 were deletions. A subset of these mutations includes individual changes of all 26 naturally occurring scrines to alanines. By assaying each mutant protein separate- ly on the XVI long tenninal repeat (LTR) and the human immunodeficiency virus type I(HIV-I ) LTR in parallel, we were able to identify regions of Tax selectively necessary for each promoter. A small region in the carboxyl terminus, amino acids 315 to 325, was found to be selectively important for activation of the HTLV-I LTR. Three changes at scrine 113, scrine 160, and serine 258 were found to (specifically affect function on the HIV-I I,TR. Surprisingly, we found that the great preponder- ance of missense changes (32 of 42) in Tax did not affect function. Semmes. O.J. and JconR, K.-T. Journal of Virology 66(12)7183-7192, December 1992. From the Labcxatory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Bethesda, MD: THE BASIC RNA-BINDING DOMAIN OF HIV-2 TAT CONTRIBUTES TO PREFERENTIAL TRANS-ACTIVATION OF A TAR2-CONTAINING LTR Human immunodeficiency viruses HIV-I and HIV-2 encode a Tat protein that trans-activates the respective viral genome through RNA targets (TARI and TAR2). Tat-I and Tat-2 have considerable homology. However, an interesting biological observation has been that Tat-I activates the HIV-I and HIV-2 LTRs equally while Tat-2 activates the former, in comparison to the latter, poorly. Here, we present evi- dence that it is the TAR2 RNA target together with the basic RNA-binding protein domain of Tat-2 that dictate this non-reciprocity in trans-activation. Chang, Y. and Jeang, K.-T. Nucleic Acids Research 20(20):5465-5472, 1992. Other support: National Institutes of Health. From Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Bethesda, MD. ADENOVIRUS INFECTION INHIBITS THE PHOSPHORYLATION OF MAJOR HISTOCOMPATIBILITY COMPLEX CLASS I PROTEINS Major histocompatibility complex (MHC) class I molecules act as peptide receptors to direct the recognition of foreign antigens by cytolytic T cells. The cell surface expression and trafficking of these peptide receptors is thought to be con- trolled by the conformation of the MHC molecule and pussibly by the phosphoryla- tion of the cytoplasmic portion of the heavy chain protein. It is of some interest that adenoviruses (Ads) have evolved proteins that interfere with the expression of MHC molecules. One of these proteins, called E3/19k, binds to newly synthesized MHC molecules in the rough endoplasmic reticulum (RER) and inhibits their trafficking to the cell surface. Here we show that during the infection of a human cell line with Ad2, the phosphorylation of the endogenous MHC molecules is inhibited. We also observe that the phosphorylation of the endogenous HLA molecules is grossly impaired in a human cell line transfected with the Ad2 EcoRID fragment containing the E3/19k gene. We conclude that the E3/19k protein inhibits the phosphorylatiim of the MHC heavy chains and that this may be one of the important functions of this protein in infected cells. In addition, we show that a mutant of the E3/19k protein, which lacks an RER retention signal but which retains its ability to bind to HLA molecules, does not inhibit the phosphorylation of HLA molecules and that phospho- rylated molecules are not Endo H sensitive. This suggests that HLA molecules are phosphorylated after leaving the medial-Golgi compartment, thus providing the most compelling evidence yet that HLA molecules are phosphorylaled at or near the cell surface. Finally, to our knowledge, this is Ihe first study under which the phosphory- lation of MHC molecules is shown to be altered and may have some relevance for other pathogenic conditions. Lippe, R., Luke, E., Kuah, Y. T., Lomas, C., and JPfferie.r, W. A. Journal of Experimental Medicine 174:1159-1 166, November 1991. 220 1 221
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~ ~ Other support: National Cancer Institute of Canada Natural Science and ® , Engineering Council of Canada and British Columbia Health Care Research ~ Foundation. ~ From the Biotechnology Laboratory, Departments of Microbiology, Zoology and ® Medical Genetics. University of British Columbia. Vancouver. Canada. 1~-t SEROTONERGIC ASPECTS OF THE RESPONSE OF HUMAN PLATELETS TO IMMUNf; ADJ(IVANT MURAMYL DIPEPTIDE The human platelet scrotonergic responses of aggregation and uptake were shown to be modulated by muramyl peptides. Muramyl dipeptide inhibited serotonin uptake in a temperature dependent and stereospecific manner. It also blocked the binding to platelets of I'Hlirnipraminc, a well-known inhibitor of serotonin uptake. Muramyl dipeptide decreased scrotoninl (5-HT) mediated platelet aggregation, and blocked thc binding of a 5-HT:-specific ligand: lysergic acid diethylamide. Since rnuramyl peptides are released upon degradation of bacteria, the findings offer a pos- sible mechanism for neuro-immune modulation by emphasizing the interaction between 5-FIT (a ncurotransmi(ter) and muramyl peptides (immuno-adjuvants). Polanski, M. and Karnrrrskv. M. L. JoumaLof Neuroimmunology 37:149-160, 1992. Other support: National Institutes of Health. From the I)epartmcnt of Biological Chemistry and Molecular Pharmacology. Harvard Medical Schuol, Boston. SITE-SPECIFIC RECOMBINATION IN THE IMMUNE SYSTEM Site-specific DNA recombination has been identified in a wide variety of bio- logical systems. In vertebrates, however, the only identified use of this genetic device is in the immune system. Here it plays a critical role in generating a diverse repertoire of surface receptors to intercept invading microbes and parasites. The mechanism and orchestration of this reaction are intriguing and are relevant to a broad array of rel;ucd biological and biomedical issues. l.ieher, M. R. FASEB Joumal 5(14):2934-2944, November 1991. Other support: National Institutes of Health. From the Laboratory of Experimental Oncology, Department of Pathology, Stanford University School of Mcdicinc, Stanford, CA. I)EVELOPMENTALLY RECit1LATED ASSOCIATION OF A 56-kD MEMBER OF THE SURFACE IMMUNOGLOBULIN M RECEPTOR COMPLEX Immature and mature B cells differ in the signals generated and transduced through their antigen receptor, surface immunoglobulin M (sIgM). Whereas signals generated through sIgM on mature B cells initiate a program leading to the positive activation of these cells, signaling through this receptor at the immature stage of development leads to a state of induced unresponsiveness or tolerance. Our previous studies have described developmental differences in sIgM transmembrane signaling that are independent of ligand-receptor affinity. In an attempt to understand the mol- ecular basis for signaling differences between immature and mature B cells, we have analyzed the s1gM receptor complex in neonatal and adult mouse splenic B cells. While previously described components of this complex do not exhibit marked developmentally regulated differences in their association with slgM, we have iden- tified a 56-kD protein that associates with sIgM in mature (antigen-responsive), but not immature (tolerance-sensitive) B cells. This protein (p56) associates with slgM as a homodimer, is constitutively phosphorylated on tyrosine, and is coimmunopre- cipitated with 1gM but not IgD. The observed inability to iodinate p56 suggests it is an intracellular component of the receptor complex. Based upon its migration in one- and two-dimensional gel electrophoresis we show, however, that p56 is distinct from the blk, lyn, or fyn src family kinases that have been shown to be associated with slgM in mature B cells. The developmentally regulated participation of p56 in the B cell antigen receptor complex suggests a role in the differential signaling mediated via slgM on immature and mature B cells. Yellen-Shaw, A. J. and Monroe, .l. G. Journal of Experimental Medicine 176:129-137, July 1992. Other support: American Cancer Society, National Institutes of Health and Lucille P. Markey Charitable Trust. From the Department of Pathology and Laboratory Medicine. University of Pennsylvania School of Medicine, Philadelphia. SIGNALING THROUGH SURFACE IgM IN TOLERANCE-SUSCEPTIBLE IMMATURE MURINE B LYMPHOCYTFS-Dt:vet.oPMt:NTALLV RECtA.ATED DIF17iRENCGS IN 7RANSMF.MBRANE SIGNALING IN SPLENI(' B CELt S FROM ADtA.T AND NEONATAt. MIC'E During the course of B lymphocyte development, newly emerging surface Ig' B cells pass through a stage when Ag-Ag receptor interactions lead not to immune responsiveness but to a state of functional tolerance. We have explored the molecu- lar basis of antigenic nonresponsiveness and tolerance susceptibility using tolerance- susceptible surface Ig' splenic B lymphocytes from neonatal mice and anti-µ chain antibodies as a polyclonal ligand. In this population of cells, surface IgM is uncou- pled from the inositol phospholipid (PI)-hydrolysis pathway at a point proximal to the receptor: anti-µ antibodies did not stimulate inositol phosphate generation de- spite the fact that PI-hydrolysis was observed after treatment with AIF,, implicating the existence of a functional G protein and phospholipase C. Further evidence for a dif- 222 1 223
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ference early in the signal transduction pathway stems from the finding that anti-µ stimulation does not induce the expression of two immediate/early PKC-linked genes egr-I and r.(as. This appears to be the primary signaling difference between the mature and immature B cells from the neonatal mouse splenic population, as these cells undergo a G,G, cell cycle phase transition when surface IgM is bypassed using phorhol diester and calcium ionophore. Interestingly, despite undetectable levels of PI-hydrolysis, we observed equivalent receptor-mediated changes in intracellular cal- cium when comparing the immature and mature populations. These results indicate incomplete coupling of surface IgM to the signal transduction machinery operative in mature, immunocompetent B cells and suggests a molecular mechanism accounting for the differential processing of surface 1gM signals into activation vs tolerogenic responses observed in these two stages of B cell development. Yellen. A. 1., Glenn. W., Sukhatme, V. P., Cao, X., and Monroe..l. G. The Journal of Immunology 146(5):1446-1454, March I, 1991. Other support: American Cancer Society, National Institutes of Health and Lucille P. Markey Charitable Trust. From the Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, and Department of Medicine, Howard Hughes Medical Institute. University of Chicago. PHORBOL F,STER REDUCES CONSTITUTIVE NUCLEAR NF B AND INIIIBI"1'S 1lIV-I PRODUCTION IN MATURE HUMAN MONOCYTIC CELLS NF B is a potent mediator of specific gene expression in human monocytes and has been shown to play a role in transcription of the HIV-1 genome in promonocytic Icukemias. There is little information available on the response of NF B to cytokines in normal human monocytcs. We have used a"P-labeled oligonucfeotide derived from human immunodeficicncy virus (HIV-1) long terminal repeat, which contains a tandem repeat of the NF B binding sequence, as a probe in a gel retardation assay to study this transcription factor. Using this assay, we have detected NF B in extracts of nuclei from nomial human monocytes. Treatment of normal monoeytes with 12-0- tetradecanoyl phorMol-1 i-acetate (TPA) for 4-24 h caused the complete disappear- ance of NF 13 from nuclear extracts of monocytes. A similar result was obtained with the mature monocytic leukemia cell line THP-I. The constitutive transcription factor SPI was unaffected by addition of TPA. The disappearance of NF B from the nu- cleus was concentration dependent between 10 and 50 ng/ml of Aorhol ester. In TIIP-I cells, TPA also induced a new, faster-migrating NF.B species not induced in monocyles. Protein kinase C inhibitor staurosporine. hut not cyclic nucleotide- dependent protein kinase inhibitor HA-I(X)4, also dramatically reduced constitutive levels of nuclear NF B. Finally. TPA addition to monocytes infected with HIV-1 inhibited IIIV-I replication, as determined by reverse transcriptase assays, in a con- centration-dependent manner. Thcse results are in striking contrast to the increase in nuclear NF B and HIV-1 replication induced by phorhol esters in promonocytic leukemia cells U937 and I ll. 60, and emphasize the importance of studying cytokine regulation of HIV-I in normal monocytcs. Mufsnn, R. A., Myers, C., Turpin, J. A., and Meltzer, M. Journal of Leukocyte Biology 52:637-644, December 1992. Other support: American Red Cross and National Institutes of Health. From the Cell Biology Department, Jerome H. Holland Laboratory, American Red Cross. Rockville, MD, and Cellular Immunology Department, Walter Reed Army Medical Center, Rockville. THE EVOLUTION OF IMMUNE MEMORY AND GERMINAL CENTERS Antibody responses in homoiothermic and poikilothermic vertebrates are signif- icantly different in their heterogeneity and affinity range, and in the speed of the sec- ondary response following repeated antigenic stimulation. This article presents the hypothesis that the evolutionary development of unique lymphoid structures, the ger- minal centers, in combination with the development of a distinct B-cell lineage, is a determining feature of these diferences. Nahm, M. H., Kroese, F. G. M., and Hoffman, J. W. Immunology Today 13(1 I):43ft-442, 1992. From the Department of Pathology, Washington University School of Medicine, St. Louis, Department of Histology and Cell Biology, University of Groningen, Groningen, The Netherlands, and Department of Pathology, St. Louis University School of Medicine. FUNCTIONAL PROPERTIES OF HUMAN GERMINAL CENTER B CELLS Germinal centers (GCs) are histologically defined areas where B cells undergo extensive proliferation and maturation, or die of apoptosis. GC B cells isolated from human tonsils can be phenotypically identified by expression of peanut agglutinin (PNA)-hinding sites and can be further divided into suhpopulations based on their expression of CD77. To assess the functional potential of GC B cells, we studied CD77' PNA' B cells isolated from tonsils by examining their differentiation status and their ability to proliferate in vitro to various cytokines and costimulants. We found that CD77' GC B cells are less differentiated than CD77- GC B cells; CD77 GC 13 cells less frequently express cytoplasmic IgG and IgM, and spontaneously secrete less Ig compared to CD77 GC B cells. To identify conditions capable of inducing GC B cell proliferation, we examined IL-4. IL-2, 1FN-y, low molecular weight B(YAF (LMW-BCGF), and an MLR supernatant along with costimulants such as anti-IgM antibody, ,Staphvlncnr•cus aureus Cowan I(SAC), PMA, and pokeweed mitogen (PWM). While non-GC B cells proliferate strongly in response to these stimuli, GC B cells did not proliferate. However, CD77' as well as CD77 GC B cells mounted a rapid and strong proliferative response upon stimulation with IL-4, but 224 1 225
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only in the presence of anti-CD40 antibody. Moreover, although nine additional cylokines were examined, only IL-4 was capable of supporting CD77' GC B cell pro- liferation in the presence of anti-CD4O antibody. When cells were stimulated with IL-4 and anti-CD40 antibody, we also found that IFN-y consistently decreased the proliferative response of CD77' GC B cells without affecting the response of non-GC B cells. Taken together, these data indicate that GC B cells have characteristic growth requirements and that IL-4 may he important for GC cell growth in riva. Butch, A. W. and Nahm, M. H. Cellular Immunology 140:331-344, 1992. From the Division of Laboratory Medcine, Department of Pathology, Washington University School of Medicine, St. Louis. CLONAL CHARACTERIZATION OF THE HUMAN IgG ANTIBODY REPERTOIRE TO 1-lAEMOPNILUS INFLUFN7.AL TYPE b POLYSACCHARIDE. IV. 1111; . LIiSS FRtiQUENTI.Y IiXF'RFSSI?I) VI. ARE Fili7T•,R(XiF,NOUS We previously demonstrated that the human anti-Haemophilu.c influenzae type b polysaccharide (Hib-PS) VL repertoire is dominated by a product of the Vrdl-gene, A2, and that VKII-A2 anti-Ilib-PS antibodies have little or no somatic mutation in VL. To further study this VI, repertoire, we studied non-A2 anti-Hib-PS antibodies that were identified either scrologically or by amino-terminal amino acid sequence analysis. Of 15 non-A2 anti-Hib-PS antibodies from 12 vaccinated adults, we found four V.1IV, five VKI, one non-A2 VKI1. four VKIII, and one VKIV antibodies. As expected, all but two of these subjects also produced VKII-A2 antibodies. Interestingly, one of these subjects lacks the A2 gene in the germ line. However, both subjects who did not produce detectable VKH antibody did produce normal amounts of total anti-Hib-PS antibody after vaccination. Candidate Vrc genes for the non-A2 antibodies were identified by comparison of up to 60 VL amino acid residues, including CDRt and CDR2, with all sequenced VK genes. VKI antibodies appear to be products of three newly sequenced Vul genes, 08, 018, and LI I, that arc reported here. The 08 and 019 genes encode identical amino acid sequences. The non-A2 VKH antibody is a likely product of the Al or A17 genes, the VK III antibodies are likely products of the A27 gene, and the VKIV antibody is a products of the single VKIV gene. 133. Unlike VKII-A2 antibodies, the Val, VKIII, and VKIV antitxxlies differed by one to five CDR residues from the germ line product of the candidate genes, suggesting the presence of somatic mutations. Thus, anti-Hib-PS antibodies can be divided into Iwo types, the most frequently observed A2 antibodies with little or no somatic mutation and non-A2 antibodies that likely contain somatic mutations. Scott. M. G.. Crimmins, D. 1.., McCourt, D. W., Chung, G., Schable, K. F., Thicbe, R., Qucnzcl. I:.-M., Zachau, H. G.. and Nahm. M. H. The Journal of Immunology 147(1 1):4(X)7-4013, December I, 1991. Other support: Bundesministerium fur Forschung und Technologic and Fonds der Chemischcn Industric. From the Department of Pathology, Division of Laboratory Medicine, Howard Hughes Medical Institute, Core Protein/Peptide Facility, Washington University School of Medicine, St. Louis, and the Institut fur Physiologische Chemie, Physikalische Biochemie und Zellbiologie der Universitat Munchen, Munich, Germany. CHARACTERIZATION OF THE HUMAN IgG ANTIBODY V REPERTOIRE TO HAF.MOPHILUS INFLUENZAE TYPE b POLYSACCHARIbE The human antibody response to the capsular polysaccharide of Haemophilus influenzae type b(Hib) is a good model for examining human V region repertoires. While the VL repertoire of human antibodies to Hib polysaccharide is relatively simple and dominated by the product of a germline V.II gene named A2, at least five other V, genes can be expressed by some individuals. These include at least two V I products and at least one V 1I1, one V~IV, and one V~ product. The epitope recog- nized by a monoclonal anti-idiotype antlbody is on the A2 V.11 product. Scott, M. D. and Nahm, M. H. The Journal of Infectious Diseases I65(suppl I):S53-S56, 1992. From the Department of Pathology. Division of Laboratory Medicine, Washington University School of Medicine, St. Louis. AN IDIOTYPIC MARKER ASSOCIATED WITH A GERM-LINE ENCODED K LIGHT CHAIN VARIABLE REGION THAT PREDOMINATES THE VACCINE- INDUCED HUMAN ANTIBODY RESPONSE TO THE HAEMOPHILUS INFLUENZAE b POLYSACCHARIDE Human antibodies specific for the Haemophilus influenzae b polysaccharide (Nib PS) frequently express a cross-reactive idiotype (CRI), and commonly utilize a V, region that is the product of the VFII gene A2. To examine further anti-Hib PS V region expression and to determine whether CRI expression is correlated with the. V IIA2 chain, we isolated a monoclonal antibody (MAb) reactive with an idiotypic determinant of anti-Hib PS antibodies. This MAb inhibited Nib PS binding but did not react with Ig isotypic determinants. The CR1 recognized by this MAb, designated Hibld-I, was associated with the Nib PS-combining site since the reactivity of the MAb with anti-Hib PS antibodies could be inhibited by Hib PS. Hibld-I was expressed by 17 of 17 clonally purified and sequence-defined anti-Hib PS antibodies having V IIA2 L chains. In contrast, 0 of 10 anti-Hib PS antibodies having either Vy, V.1, or V III chains expressed Hibld-I. Western blot analysis showed that Ihe MAb anti-CRI 'reacted with isolated anti-Hib PS V IIA2 L chains but not with H chains or other L chains, indicating that the Hibld-l determinant is localized to the V IIA2 chain, and does not require pairing with H chain for expression. . Anti-Hib PS antibodies bearing Hibld-I were present in at least 85% of subjects immuniz,ed with either free Nib PS or Nib PS coupled to diphtheria toxoid (Hib PS- 226 1 227
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DT), and comprised on the average 60% of the total vaccine-induced serum anti-Hib PS. Hihld-I expression was not related to age at vaccination inasmuch as infants, children, and adults had similar distributions of Hibld-I ,positive anti-Hib PS after vaccination with Hib PS-DT. Hibld-1 was expressed at a lower frequency and com- prised a smaller fraction of the total anti-Hib PS antibody in adult preimmunization sera as compared to post-Hib PS immunization sera, suggesting that immunization preferentially stimulates Hibld-I-positive B cells. These data demonstrate that anti- bodies hearing Hibld-I/V IIA2 comprise a predominant component of the anti-Hib PS response induced by immunization, and that this pattern of V, expression is estab- lished early in ontogeny. Lucas, A. H., tangley, R. J., Granoff. D. M., Nahm, M. 1-1., Kitamura, M. Y., and Scott, M. G. Journal of Clinical Investigation R8:IA1 1-181R, December 1991. Other support: National Institute of Allergy and Infectious Diseases. From the Children's Hospital Oakland Research Institute, Oakland, CA, and Division of Infectious Diseases, the Edward Mallinckrodt Department of Pediatrics, and I)ivision of Laboratory Medicine, Department of Pathology, Washington University School of Medicine, St. Louis. TYROSINE PHOSPHORYLATION OF PHOSPHOLIPASE C COUPLES THE FCe RI;CEPTOR MEDIATED SIGNAL TO MAS'I' CELLS SE'CRETION Mast cells respond to clustering of the type I Fce receptor (FceRl) on their membranes by mediator secretion. Recently, a marked enhancement of tyrosine phosphorylation on several proteins has been observed as a result of antigen-induced FceRt aggregation on thcse cells. We report here that the phosphatidyl inositide spe- cific phospholipase C,, (PLC,,) is one of Ihe prime proteins that undergoes tyrosine phosphorylation as a result of this stimulus. This was determined by immunoprecipi- tation of phosphotyrosine containing proteins from detergent lysates of rat mucosal mast cells (rat basophilic leukemia cells, sublinc 2H3; RBI, 2H3) and Western blot- ting analysis of the separated components. A fast appearance of phosphorylated tyro- sine residues on PL.C,, was observed, reaching its maximal intensity at -1-3 min after stimulation and declined afterwards to basal levels. Moreover, the phosphoryla- tion depended on maintaining the aggregated FceRl as did other cellular responses (e.R., phosphatidyl inositides hydrolysis and secretion). The time course of both FceRI induced phospholipase C,, activation, as monitored by the formation of inosi- tol phosphates, and of the secretory response of the cells followed that of the PLC,, phosphorylation. Furthermore, Ihe tyrphostin AG490, a protein tyrosine kinase inhibitor, caused similar inhibition of the FceRI-induced PLC,, phosphorylation, inositol phosphates formation, and mediator secretion. Significantly, no tyrosine phosphorylation of PLCy was induced by the Ca" ionophore, ionomycin, even at doses that cause optimal secctory response. Taken together, these results clearly sug- gest that PI.C,, phosphorylation on tyrosine residues constitutes a key element in the cascade coupling the FcERI clustering stimulus to the secretory response. Moreover, FceRl clustering is dynamically coupled through tyrosine phosphorylation/dephos- phorylation to the PLC,, activity and to the ensuing secretory response. 228 Schneider, H., Cohen-Dayag, A., and Perhl,l. International Immunology 4(4):447-453, 1992. Other support: Thyssen Foundation. From the Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel. VARIANTS OF THE MUCOSAL MAST CELL LINE (RBL-2H3) DEFICIENT IN A FUNCTIONAL MEMBRANE GLYCOPROTEIN We have isolated and characterized subpopulations of the rat mucosal mast cell line, RBL-2H3, carrying either high or low density of a glycoprotein, recently estab- lished as mast cell function-associated antigen (MAFA, Ortega et a1., 1991), on their surface. These populations were investigated in order to better define the involve- ment of the MAFA in coupling the immunological stimulation of mast cells to medi- ator release. The MAFA density on the cell surface of the deficient subpopulation was 510-20% that of the parental population and this phenotype was found to he sta- bly maintained for several months. In contrast, the MAFA-enriched cells had maxi- mally twice the number of copies per cell surface than that of the parental population and this phenotype was less stable. Significantly, low copy number of MAFA on the cell's surface was accompanied by a markedly different secretory response, i.e., (i) a considerable decrease in the secretory response to the FceRI-mediated stimulus (ii) a marked enhancement of the ionomycin induced secretion. In order to gain insight into the causes for this decrease in cellular response to the FceRI- mediated stimulus, we measured the amplitudes of several biochemical processes which are assigned to the stimulus-secretion coupling cascade. The FceRl- mediated uptake of "C.a" by the MAFA-deficient cells was considerably lower than that of the parental and MAFA-enriched cells. Similarly, these cell's FceRl-induced rise in ICa2'j, (both the initial transient as well as the sustained elevation) was markedly lower than that of the parental line and the MAFA-enriched cells. Moreover, the low initial transient rise in ICa"j was found to be correlated with the decrease in FceRl-mediated IP, levels. We therefore examined the cell's content of the phosphatidyl-inositides hydrolyzing enzyme, phospholipase Cy~. This was found to be similar in the parental line and in derived subpopulations. However, PLC ~ acti- vation, as measured by the time course of phosphorylation of its (yrosines, showed a marked difference; while PLCy~ tyrosine phosphorylation, in the parental cells, was, only transient (detected already I min after antigen addition and declined afterwards to basal levels at ca. 10 min) in the MAFA-deGcient cells, tyrosine phosphorylaled PLCy) was also observed I min after antigen addition, yet showed no decrease with time in its phosphorylation intensity for up to 30 min. Thus, in analogy to the current mechanisms proposed for the coupling between other antigen-receptors, such as that of T cell or surface Ig on B cells, with their associated membrane components and cylosolic protein tyrosine kinases (PTKs) or phosphatases (PTPs), it is suggested that the MAFA may serve as a regulatory element of the coupling between the FcERI and the PTKs and/or PTPs which in turn carry on the cascade to the cell's secretory response. 229
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Cohen-Dayag, A., Schneider, H., and Perhl, l. Immunobiology 185:124-149, 1992. Other support: Government of Lower Saxony, Germany. From the Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot. Israel. SURFACE Ig RECEPTOR-INDUCED NUCLEAR AP-I DEPENDENT GENE EXPRESSION IN B LYMPHOCYTES The relationship between signals generated via the sIgR complex of B lympho- cytes and subsequent changes in gene expression is poorly understood at the molecu- lar level. To illuminate mechanisms that may couple these events, we examined the expression and function of tetradecanoyl phorbol acetate-response element (TRE)- binding proteins (i.e., activator protein I, (AP-l)) in the murine B lymphoma cell line BAL-17.7.1 (BAL-17), which models primary B lymphocyte responses in a number of respects. Cross-linking of sIgR led to substantial induction of nuclear AP- I, in BAL-17 B cells, that bound the TRE, as detected by electrophoretic mobility shift assay. The slgR-induced TRE-binding activity consisted of both Jun and Fos proteins, on the basis of immunoreactivity of nucleoprotein complexes with specific antisera. In addition, immuno-precipitation with specific antisera showed that de novo synthesis of Jun-B and c-Jun proteins, accompanied by c-Fos, was stimulated after cross-linking of slgR on BAL-17 B cells. Transient transfection of BAL-17 B cclls with reporter gene constructs showed that B cell AP-l failed trans-activate the TRE-containing human collagenase gene promoter, for which activity is dependent upon functional expression of cellular c-Jun. In contrast, slg-induced AP-1 tran.c- activated a HSV-tk promoter that contained three TRE; this pattern of gene expres- sion is consistent with the presence of functional Jun-B-containing AP-I in B lym- phocytes. These results are the first to attribute a functional role to slgR-mediated AP-I in B lymphoid cells and suggest that AP-I functions to couple the slgR com- plex to changes in nuclear gene expression. Chiles. T. C. and RodcctPin, T. L. The Journal of Immunology I49(3):R25-83I, August I, 1992. Other support: U.S. Public Health Service and National Institutes of Health. From the Departments of Medicine and Microbiology, and Evans Memorial Department of Clinical Research, Boston University Medical Center, Boston. ASSOCIATION OF HEAT SHOCK PROTEIN 70 WITH ENTEROVIRUS CAPSID PRECURSOR PI IN INFECTED HUMAN CELLS Members of the human heat shock (HSP) family of related proteins are involved in the intraccllular folding. transport, and assembly of proteins and protein com- plexcs. We have observed that human heat shock protein 70 (HSP70) is associated with the capsid precursor PI of poliovirus and coxsackievirus BI in infected HeLa cells. Antiserum generated against HSP70 coimmunoprecipitated the poliovirus pro- tein PI, an intermediate in capsid assembly. Similarly, a-virion serum coimmuno- precipitated HSP70 from virus-infected cell extracts, but not from mock-infected cell extracts. The HSP70-PI complex was stable in high-salt medium but was sensitive to incubation with 2 mM ATP, which is a characteristic of other known functional complexes between HSP70 and cellular proteins. The P1 in the complex was pre- dominantly newly synthesized, and the half-life of complexed Pl was nearly twice as long as that of total PI. The HSP70-PI complex was found to sediment at 3S to 6S, suggesting that it may be part of, or a precursor to, the "5S promoter particles" thought to be an assembly intermediate of picomaviruses. The finding that HSP70 was associated with the capsid precursors of at least two enteroviruses may suggest a functional role of these complexes in the viral life cycles. Macejak, D. G. and Sarnow, P. Journal of Virology 66(3):1520-1527, March 1992. Other support: U.S. Public Health Service, National Institutes of Health and Lucille P. Markey Charitable Trust. From the Molecular Biology Program, Department of Biochemistry, Biophysics and Genetics, and Department of Microbiology and Immunology, University of Colorado Health Sciences Center, Denver. CAN PERITONEAL B CELLS BE RENDERED UNRESPONSIVE? Ly-I' B cells have been reported to produce a number of autoantibodies, and to be involved in the selection and regulation of the conventional B cell repertoire. It is not known if these B cells, which are found in high numbers in the peritoneum of normal adult mice, themselves can be regulated. In this study, we evaluated the sen- sitivity of peritoneal B cells (PBCs) versus conventional splenic B cells to regulation in a model system for tolerance. Normal splenic (conventional) or PBCs (containing both CD5' and CD5 'sister' cells) were cultured overnight with either F(ab')T or intact IgG anti-mouse Ig, washed, and then challenged with fluorescein(FL) coupled to Brue•ella ahorlu.c (BA), trimethylammonium (TMA)-BA or lipopolysaccharide (LPS), and the IgM rises to the FL and TMA haptens measured. In contrast to spleen cells, which exhibited up to a 90% reduction in anti-FL responsiveness, pretreated PBCs were mostly resistant to this form of tolerance regardless of challenge. The anti-TMA response of PBCs, which reflects the skewed V"I I usage by peritoneal CD5 B cells, was also resistant to tolerance. However, splenic• TMA-specific B cells appeared to be sensitive to unresponsiveness induced by anti-Ig. Signaling studies show that PBCs have a blunted initial Ca" response, suggesting that the consequence of anti-Ig crosslinking may he defective in these cells. Furthermore, phorbal myris- tate acetate and/or ionomycin treatment of both PB and splenic B cells led to hypore- sponsiveness to LPS challenge. This suggests that PBCs may he defective in a sig- naling pathway, perhaps involving protein kinase C activation. Our studies reveal that antibody production by PBCs is not subject to conventional tolerance pathways, a control which may be important in terms of their ability to regulate other B cells. 230 1 231
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l.iou, L.-B., Warner, G. L., and Scorl. 1). W. International Immunology 4(1):I 5-21, 1992. Other support: U.S. Public Health Service. From the Immunology and Immunotherapy Division, University of Rochester Cancer Center and Department of Microbiology and Immunology, University of Rochester School of Medicine, Rochester, NY. TIIMORI(;ENIC'ITY OF INTERLF,UKIN-2 (11. 2)-cDNA-TRANSFECTED 1,1210 I,YMPHOMA ANI) ITS IN VIVf) VARIANTS IS MODULATED BY CHANGES IN ll: 2 1?XPRF.SSION; I'OTENTIAL THERAPEUTIC IMPLICATIONS Tci study parameters that affect the tumorigenicity of L1210 lymphoma, we have analyzed the structure of MHCclass I antigens of this tumor. In addition, this tumor was transfected with interleukin-2 (IL-2) cDNA in order to determine the effects of high concentrations of I1,-2 within the tumor environment. The nucleotide sequence of the class I K', 1T and L`' mRNAs from this tumor showed that the encod- ed amino acid sequence of the corresponding antigens is normal, thus suggesting Ihat the tumorigenicity of 1,1210 lymphoma is not due to defective antigen presentation to tumor-specific cytotoxic T cells. In contrast, induction of IL-2 expression by cDNA transfeclion led to loss of tumorigenicity of the IL-2-secreting tumor cells. Ilowever, a fraction of long-term surviving mice developed progressively growing variant tumors that showed substantial decrease or loss of IL-2 expression. These results suggest that II,-2 secretion by tumors is suicidal but, because of tumor hetero- geneity, II,-2-Ioss-variant tumors may arise that are able to escape the immune defenses of the host. The observed consistent loss of Il: 2 expression in variant tumors implies that specific targeting of large quantities of IL-2 to tumor cells may be a valuable appmach to immmnotherapy of cancer. In addition, we find that under specific gamma ray irradiation IL-2-secreting tumor cells lose their ability to multi- ply yet continue to secrete Il; 2 at levels equivalent to those secreted by unirradiated cells. Such I1: 2-sccrcting irradiated tumor cells were found to he superior immuno- gens in comparison to Ihe irradiated parental tumor cells, suggesting their use as tumor vaccines. ('hakravarty, P. K..1'vji, H., Abu-hadid, M. M., Hsu, S. C., and Sond, A. K. Cancer Imrnunology Immunotherapy 35:347-354. 1992. Other support: Elsa U. I'arclec Foundation and National Institutes of Health. From the 1)epartment of Molecular Immunology, Roswell Park Cancer Institute. Buffalo. NY. COORDINATE INTERACTIONS OF PROTEIN TYROSINE KINASES AND PROTEIN TYROSINE PHOSPHATASES IN T-CELL RECEPTOR-MEDIATED SIGNALING T-cell receptor stimulation leads to a rapid increase in tyrosine phosphorylation which is regulated by both the CD45 transmembrane protein tryosine phosphatase and by intracellular protein tyrosine kinases. The Src-family members, Fyn and Lck, have been implicated in T-cell receptor signaling and may be regulated by CD45. Shaw, A. and Thomas, M. L. Current Opinion in Cell Biology 3:862-868, 1991. Other support: American Heart Association and U.S. Public Health Service. From the Washington University School of Medicine, St. Louis. CD45: A TRANSMEMBRANE PROTEIN TYROSINE PHOSPHATASE INVOLVED IN THE TRANSDUCTION OF ANTIGENIC SIGNALS Antigen recognition by T lymphocytes is mediated through the T-cell receptor (TcR) complex, an assembly of three distinct transmembrane components: an a(3 or -y8 heterodimer, which can physically interact with antigenic peptides; CD3, a group of three distinct polypeptides; and aC chain homodimer. How this complex mediates signal transduction upon binding to foreign peptides has not yet been elucidated. Nonetheless, changes in a variety of second messengers have been described upon signalling through the TcR complex including changes in tyrosine phosphorylation, an increase in intracellular calcium, phosphoinositol turnover, and activation of p21,° Weaver, C. T., Pingel, J. T.. Nelson, J. O., and Thoma.s, M. L. Biochemical Society Transactions 20:169-174. 1992. Other support: Lucille P. Markey Charitable Trust, U.S. Public Health Service and American Heart Association. From the Department of Pathology. Washington University Medical School, St. l,ouis. B-CELL SUBSETS DEFINED BY THE FceR The data summarized herein demonstrate the utility nf the low-affinity FceR in delineating murine B-cell subsets. In the peritoneal cavity, the FcER appears to be a reliable marker in distinguishing between conventional (FceR') and Ly-I/sister (FceR ) B cells. In the spleens of normal animals, flow cytometric and histologic studies established that a distinct population of FceR B cells is also present and 232 1 233
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comprises the marginal zones. Thus, in the spleen, the FceR may be the first murine marker to allow for selective purification and analysis of marginal zone B cells. Although it is unlikely that splenic FCER B cells are directly related to peritoneal FceR Ly-1/sistcr B cells, further studies will be required to address this question. Analysis of autoimmunc mice revealed that (he splenic FceR subset is greatly expanded in these animals and indicates that the FCER may be a sensitive indicator of abnormalities within the B-cell compartment. Additional studies compared the func- tional capacity of FceR' and FCER B cells and tested the ability of these populations to isotype-switch and respond to polyclonal stimuli. The results showed that FceR' and FCER B cells from both the peritoneum and spleen can switch to produce IgG, and all but the peritoneal FCER B cells can switch to the IgE class. This latter result is certainly interesting and demonstrates an important functional difference between peritoneal and splenic FcER B cells. Finally, experiments with B-cell mitogens showed further differences between the FceR' and FceR subsets. Whereas FCER B cells appeared to be more sensitive to LPS stimulation, FceR' B cells were clearly more responsive to an anti-IgM signal. Taken together, the results show that the FceR is likely to be useful in separating B cells with different phenotypic, histologic, and functional characteristics. Wald.cchmidr, 7'.. Snapp, K., Foy, T., Tygrett. L., and Carpenter, C. Annals of New York Academy of Sciences 651:84-98, 1992. Other support: Biomedical Research Support and University of Iowa Spelman Rockefeller Research (;rant. From the F)epartmcnt of Pathplogy, University of Iowa College of Medicine, Iowa ('ity. THE 1,OW AFFINITY IgE Fc RECEPTOR (CD23) PARTICIPATES IN B ('ELI, ACTIVATION No clear function for the surface form of the B cell FceRll has been identified. Most investigators have focused on the role of the cleaved receptor, and have shown that the soluble FceRII can serve as a growth co-factor for immature and mature T cells, myeloid precursors, and B cells, prevent macrophage migration, and potentiate IgE secretion. Although no dominant role for the surface FceRII has been found, several groups have demonstratecl that crosslinking this receptor with IgE or anti- ('D2i antibodies downrcgulates IgE secretion, while other investigators have reported that IgE immune complexes can be effectively taken up by B cells resulting in pro- cessing and presentation of the complexed antigen. Our laboratory has taken another approach in searching for the function of the FceRII. A large body of evidence has demonstrated that the FcyRII delivers a strong inhibitory signal to the B cell when crosslinked with surface Ig; and can effect downregulation of B cell activation, pro- liferation, and differentiation. The inhibition only occurs if the FcyR1I is crosslinked to surface Ig, since crosslinking the FcyRll with itself has no demonstrable effect on B cells. This slg-FcyRII crosslinking can be perfomed experimentally with intact rabbit IgG anti-mouse IgM or IgD, where the Fc portion of the rabbit antibody binds to the FcyR1I and the antigen binding sites of lhe molecule bind to surface 1g. In vivo, this crosslinking is thought to be mediated by IgG immune complexes or anti- idiotypic antibodies, and has been proposed as a means of antibody feedback regula- tion. Based upon the ability of the FcyRII to exert its function when crosslinked with surface lg, we sought to examine whether the FceRlt might also deliver a negative signal to the B cell when similarly crosslinked. Accordingly, we developed a unique reagent which would allow one to test the effect of slg-FceRII crosslinking, and thereby lend information as to the function of this receptor on B cells. Waldschmidr, T. J. and Tygrett, L. T. In Gupta, S. and Waldmann, T. A. (Eds.): Mechanisms of Lymphocyte Activation and Immune Regulation IV: Cellular Communications, Plenum Press, NY, pp. 149- 156, 1992. Other support: National Institutes of Health. From the Department of Pathology, University of Iowa College of Medicine, Iowa City. MOLECULAR ANALYSIS OF PRIMARY T CELL INVOLVEMENT IN THE IDIOTYPIC NETWORK UTILIZING IMMUNOGLOBULIN-DERIVED PEPTIDES The development of anti-idiotypic antibodies during immune responses has been implicated in a wide variety of immunologic phenomena including immunoreg- ulation, development of autoantibodies and modulation of immune response. These studies indicate that primary helper T cells can be stimulated by immunoglobulin variable region products. This finding suggests that if the T cell repertoire is determined by self peptides associated with MHC molecules during development, then IgH linked products must be considered as elements that can influence the receptor selection of at least some T cell subsets. We have previously suggested that apparent IgH linked restrictions of certain T cells mighl be deter- mined by receptor driven processes. It is apparent that this sort of mechanism may play a variety of roles in the development of many specific immune responses, with immunoregulatory effects on Fxoth B cells and T cells. Williams, W. V., Weiner, D. R., and Greene, M. 1. In: Cruse. J. M., Lewis, R. E., Jr. (eds.): The Year in Immunology 1989-90. Molecules and Cells of Immunity. Year Immunol. Basel, Karger, vol. 6, pp. 152- I(i1, 1990. Other support: National Institutes of Health, American Cancer Society, National Cancer Institute, National Eye Institute, and Lucille P. Markey Charitable Trust. From the Departments of Medicine and Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine. Philadelphia. 234 1 235
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B-CELL PROLIFERATION INITIATED BY la CROSS-LINKING AND SUSTAINED BY INTERLEUKINS LEADS TO CLASS SWITCHING BUT NOT SOMATIC MUTATION IN VITRO Somatic mutations that are acquired by antibody V genes of antigen-stimulated B cells ultimately provide the clonal diversity from which memory B cells are select- ed during immune responses to T-cell-dependent antigens. Somatic mutations appar- ently are not acquired when B cells are stimulated by mitogens nor when they partici- pate in immune responses to T-cell-independent antigens. Since the basis of T-cell- dependent humoral immunity is T-cell recognition of processed antigen in the con- text of class 11 major histocompatibility glycoproteins (la) on the B-cell surface, we sought to determine whether the ligation of la on B cells induces somatic mutation. B cells were stimulated in vitrn by a procedure in which their proliferation was depen- dent upon ligation of surface la with antibody. Sequences of hybridoma V genes derived from these B cells revealed no somatic mutations despite prolonged stimula- lion in vilra and the induction of immunoglobulin secretion and switching to isotypes characteristic of T-cell-dependent humoral immunity. We infer that la-mediated sig- naling and isotype switching are not causally related to somatic mutation. The avenue of differentiation that leads to somatic mutation in memory B cells is apparently sep- arable from that leading to proliferation, immunoglobulin secretion and switching. Wv.aorki, L. .I.. Creadon, G., Lehmann. K. R., and Cambier, J. C. Immunology 75:115-121, 1992. Other support: National Institutes of Health. From the Division of Basic Sciences, Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO. SEQUENCING HEAVY- AND LIGHT-CHAIN VARIABLE GENES OF SINGLE B-FIYBRIDOMA CELLS BY TOTAL ENZYMATIC AMPLIFICATION We have devised a protocol to obtain accurate and complete sequences of the immunoglobulin heavy- and light-chain variable-region (V,, and Vd genes of single B-hybridoma cells Ihat express defined V genes. The amplification achieved ranges from 2 X 10"- to I X l0'°-fold. Only one potential Taq DNA polymerase error was observed in 7590 nucleotides of sequence, thus permitting the identification of natu- rally occurring somatic mutations. The two-step nature of the amplification protocol provides sufiicient 1)NA for a minimum of 160 sets of sequencing reactions of both the V,, and V, genes from one cell without cloning. The amplification of relatively long segments of DNA in the first step of the protocol permits second-step amplifi- cation and sequencing of regions that flank V,. and V,codons. Fractionating cellular lysates prior to the first step of amplification permits the separate amplification of V genes on opposite sister chromatids and possibly on opposite strands of the same 1)NA duplex. Accurate sequencing of V,, and V, genes of defined germ-line origin that are expressed by single B cells taken directly from the animal is thus made fea- sible by this approach. Liu, A. H., Creadon, (;., and Wv.cnrki, L..l. Proceedings of the National Academy of Sciences USA 89:7610-7614, August 1992. Other support: American Cancer Society, University of Colorado Health Sciences Center, U.S. Public Health Service, and Arthritis Foundation. From the Division of Basic Sciences, Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, and Department of Microbiology and Immunology, University of Colorado Health Sciences Center, Denver. MODULATION, IN VIVO AND IN VITRO, OF SURFACE EXPRESSION OFCDI8 BY BOVINE NEUTROPHILS A series of experiments was designed to elucidate some of the factors that may influence surface ression of CDI8 by bovine neutrophils. Expression of CDI8 was determined by immunofluorescence flow cytometry. Neutrophils recovered from the uterus of cows (n = 9) after intrauterine administration of sterile oyster glycogen so- lution expressed (mean ± SD) 123 ± 21% more CD18 than did circulating neu- trophils recovered simultaneously from the same cows (P = 0.003). In 8 cows given 20 mg of dexamethasone 1M daily for 3 days, expression of CD18 on blood neu- trophils was 29.6 ± 8% less after treatment than before treatment (P = 0.0078). Neutrophils from 12 cows or bulls exposed to phorbol myristate acetate in vilrn increased expression of CDI8 by 137 ± 37% (P = 0.0035). Likewise, exposure of neutrophils from 8 cattle to zymosan-activated bovine plasma increased CDI8 expo- sure by 10.6 ± 3.8% (P = 0.029). These findings indicate that expression of CD18 by bovine neutrophils is a dynamic system, capable of responding to inflammatory stimuli. Inadvertent activa- tion of neutrophils may be responsible for some of the variance in expression observed when examining large groups of cattle for CD 18 expression by neutrophils. The ability of bovine neutrophils to respond rapidly to various stimuli by increasing surface expression of CDI8 indicates that a pool of intracellular CD18 may be avail- able for inclusion in the plasmalemma, as has been reported for human neutrophils. Gilbert, R. 0., Kim, C. A., and Yen, A. American Journal of Veterinary Research 53(9):1675-1678, September 1992. Other support: National Association of Animal Breeders, Eastern Artificial Insemination Cooperative, U.S. Department of Agriculture, Animal Health and Disease Program, National Institutes of Health, and American Institute for Cancer Research. From the Department of Clinical Sciences and Pathology, College of Veterinary Medicine, Cornell University, Ithaca, NY. 236 1 237
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Active Projects Following is a list (if Ihe principal investigators, or institutions, whose projects are under way or were activated in the period since the previous Report, together with the respective project tilles. Completed projects are listed in a later section. PRINCIPAL INVF,STI(:ATOR ()R INST1'I'ItTI()N IAN Y. R. ADAMSON. Pn.D. Profccsor of Pathology, University of Man- itoha, Winnipcg. Manitoba. Canada. PROJECT TITLE Epithelial-fihroblast interactions in lung iojury and repair SYED S. AIIMAD, M.I).. Ptt.D. Research Associate Professor. Temple tini- vcrsity, I'hiladelphia. KA'(HRYN M. ALBERS, Pn.D. Assistant Professor of Pathnlogy, Universi- ty of Kenmcky, Lucille P. Markey Cancer Center. I.exington. JORGE F.. AI,LENI)E, Pn.D. Prnfessor of Biochemistry, llniveraity of Chile. Santiago. RI('HARI) A. ANDF.RSON, Ptt.D. Assistant Prol'eanr ol' Phamracology. Ilni- vcrcity of Witicon~in Medical School. Madisnn. STIiVF.N M. ANDERSON. Pn.D. Assistant Profcstior of Pathology. State IIniversity ol New York, Stony Brook. HARRY N. ANTONIADfiS. Pn.D. Professor of Bicxhcmistry, Harvard Uni- versity School of Public Heallh, Boston. SUSAN A. ASTRIN, Pn.D. Senior Member, Inslitute for Cancer Research. Philadelphia. ALAN D. ATl'IE. Pn.t). Assistant Profcssor of Biochemictry, llni- vertity of Wi~consin, Madison. DAVID E. AXELRt7D. Pn.D. Associate Professor of Microbiology. Waks- man Institutc, Rutgers University, Pi.cataway, NJ. Interaction of platelets with coagulation factor Vlil Keratin protein function in epithelial cell growth and differentiation Nuclear casein kinase Il: its regulation and pcnsible role in signal Iransduction Regulation of PIP kinases and protein 4.1 by growth factor receptor tyroxine phosphory- lation Substrates for the v-.crr oncogenes Platelet-derived growth factor in human pro- liferative diseases Deregulation of c-mvc in colon carcinoma and AIDS lymphoma Functional domains of apolipoprotein-B Ra.c oncogene: suppression/modification of cell transformation PRINCIPAL INVE.STI(:AT()R PROJECT TITLE OR INSTITUTION STEVEN BALK, M.D., Pti.D. Regulation of CDI gene expression during Instructor in Medicine, Beth Israel Hospi- human T lymphocyte development tal, Boston. DAVID W. BARNES, Pn.D. Oncogenic transformation in serum-free cul- Associate Professor of Biochemistry/- ture BioPhysics, Oregon State University, Cor- vallis. STEPHEN B. BAYLIN, M.D. Regulation of the DNA methylase gene in Associate Professor of Oncology and Med- normal vs. cancer cells icine, The Johns Hopkins University School of Medicine, Baltimore, MD. ELAINE L. BEARER, M.D., PH.D. Xeno us rel/dorsal homologues and the con- As.cistant Professor of Pathology and Laho- tmfof cell migration during gastrulation ratory Medicine, Brown University, Provi- dence, RI. A. DEAN BEFUS, Ptt.D. Neuromodulation of pulmonary inflammation Professor, University of Calgary, Catgary, Alberta, Canada. TIMOTHY P. BENDER, PH.D. Expression of c-myh mRNA expression dur- Assistant Professor of Microbiology, Uni- ing B-lymphopoiesis versity of Virginia, Charlottesville. WILLIAM F. BENEDI(T, M.D. The role of the retinoblastoma gene in lung Professor of Biotechnology, Baylor College cancer of Medicine, The Woodlands, TX. JOEL S. BENNETT, M.D. Associate Professor of Medicine, Hospital of the University of Pennsylvania, Philadel- phia. Characterization of the platelet fibrinogen receptor KAREN L. BENNETT, Pn.D. Assistant Professor. University of Missouri, Columbia. Germ line-specific gene expression in early nematode development ANDRE BERNARDS. PH.D. Growth factor receptor gene mutagenesis Assistant Professor of Medicine (Genet- ics), '1'he Cancer ('enter, Massachusetts ( ieneral Hospital, ('harlestown. BRIJ('F, BF.IITI,F.R, M.D. The role of cytoplasmic rihonuclease inhibitor Associate Professor of Internal Medicine, in regulation of gene expression University of Texas Southwestern Medical Center, 1)allas. SRILATA BA(7CI1I, I'tt,D. Papillomavirus E7 proteins and transcription Assistant Professor. University of Illinois, regulation Chicago. F.RNFST BF,UTLER, M.D. Cloning the hemochromatosis gene ('hairman, F)epartment of Molecular and I:xperimental Medicine. Scripps Clinic and Research Foundation, la Jolla. ('A. 239 1 239
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PRINCIPAL INVESTI(:ATOR OR INSTITUTION RICHARD 1. BING, M.D. Professor of Medicine (emeritus), Universi- ty of Southem California School of Medi- cine, l,os Angeles; Visiting Associate, Cali- fornia Institute of Technology: Director of Experimental Cardiology and Scientific Development. Huntington Medical Research Institutes. Pasadena. CA. GAIL A. BISHOP. Ptt.D. Assistant Professor of Microbiology and Internal Medicine, University of Iowa, Iowa ('ity. J. EDWIN BLAL(x'K, Ptt.D. Professor, llniversity of Alabama. Birm- ingham. MARIANN BLtIM, Ptt.D. Assistant I'rofessor, Fishherg Research ('entcr for Neurobiology. Mount Sinai Schrxrl of Medicine, New York. CONSTANTIN A. BONA. M.D. Professor of Microbiology. Mount Sinai School of Medicine, New York. S('OT1'T. BRADY, Pn.[). Associate Professor of C'elI Biology and Neuroscience, University of Texas South- westem Medical ('entcr. I)allas. GARY R. BRIGII'I'. Pu.l). Assistant 1'rofessor, Case Western Reserve llniversity, Cleveland. OH. CONSTANCE li. BRIN('KI'sRHOFF, Pn.D. Associate Professor of Medicine and Bio- chemistry. I)artmouth Medical Schnol, Ilanover, NI1. TI IOMAS R. BROKF:R 1'n.D. Associate Professor of Bimhemislry. Uni- versity of Rrxhetler, Roxhetter, NY. MEL.ISSA A. BROWN. IOt.D. Research Assistant Professor of Medicine. Microbiology and Immunology. Oregon I lcal,h Sciences I Inivertity, Portland. KATIB,EFN M. BU('KI.EY, F'n.l). Assistant Profetisor nl Neurnhinlogy, Har- vard Mcdical Schoxd, Rostnn. DAV 11) F.. lit iRS'1'I?IN, M.I). Ascixtant Professor of Pnahology, Mount Sinai School of Medicinc, New York. PROJECT TITLF, Biochemical and biophysical mechanisms of vascular relaxation B cell subset activation in normal and autoimmune mice Molecular cloning of the delta-class opioid receptor EGF and TGF-alpha gene expression in the Purkinje cell degenerate mutant mouse Autoantihodies in experimental and human sclerodcrma Recombinant antibody analysis of kinesin- related polypeptide function Role of second messengers in chemotaxis Receptors for serum anyloid A (SAA) in col- lagenase induction Human papillomavirus replication and gene expression in primary human keratinocyles Mechanisms of interleukin 4 action: identifi- cation of 11.A inducible genes Function of synaptic vesicle proteins Suppression of neoplasia by N-ra.c oncogene 240 PRINCIPAL INVFSTIGATOR OR INSTITUTION YURI BUSHKIN, Ptt.D. Assistant Member. Public Health Re- search Institute, New York. EDWARD 1. CAMPBELL, M.D. Associate Professor of Medicine. Universi- ty of Utah, Salt Lake City. J. DONALD CAPRA, M.D. Professor of Microbiology and lnternal Medicine, University of Texas South- westerrt Medical Center, Dallas. ANN M. CARROLL, Pn.D. Assistant Professor of Microbiology/- Immunology, Albany Medical College, Albany. NY. DENNIS A. CARSON, M.D. University of California. San Diego, La Jolla. JOHN J. CASTELLOT, JR., Ptt.D. Associate Professor, Tufts University. Boston. ANDREW 1. CATON. Pti.D. Associate Professor, The Wistar Institute of Anatomy and Biology. Philadelphia. DONALD A. CHAMBERS, Ptt.D. Professor and Head. De artment of Bio- chemistry, University of Plinois, Chicago. JAMES C. CHAN, Ptt.D. Associate Professor, University of Texas M. D. Anderson Cancer Center, Houston. LAWRENCE CHAN, M.D. Professor of Cell Biology and Medicine, Baylor College of Medicine, Houston, TX. HAROLD A. CHAPMAN, M.D. Associate Professor of Medicine, Brigham and Women's Hospital, Boston. MORDECHAI CHEVION. Ptt.D. Chairman, Institute of Biochemistry. Hebrew University of Jemsalem-Hadassah Medical Schcxil, Jemsalem, Israel. ROBERT H. CHIU, Pn.D. Assistant Professor in Residence, llniver- sity of California. Los Angeles. KATHLEEN R. CHO, M.1). Instructor, Department of Pathology. The Johns Ilnpkins Flospital, Baltimorc, MD. PROJF,CT TITLE Expression and function of (i2m-free MHC class I Human lung elastin turnover in emphysema Human VHDn genes VDl recombination and T cell receptor genes in developmentally arrested snd thymo- cytes Mechanism of immune dysfunction after oxi- dant exposure Growth regulation in pulmonary artery smooth muscle cells Tolerance and au(oimmunity to influenza IIA as a transgenic self antigen Neuroimmune interactions: mechanisms of catecholamine effects on lymphocyte func- tirm. Molecular studies of a new transforming growth factor, "TAP" Over-expression of high density lipoproteins and apoproteins in atherosclerosis research Role of macrophages in lung injury Zinc protects against free radical damage The effects of E I A on TGF-/3 inducible JunB expression Genetic alterations in cervical and vulvar tumorigenesis 241
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~ ~ PRIN(7PAL INVESTIGATOR PR()JM:('T TITI,F, O PRINCIPAL INVFSTIGATOR PROJF,CT TITLF. OR INSTITUTION ln OR INSTITUTION N 1)AVII) W. CHRISTIANSON. Pn.D. X-ray studies of novel elastase-inhihitor com- ~ FREDERICK C. DE BEER, M.D. Function of serum amyloid A protein (SAA) Assistant Professor of ('hemixtry. I lniversi- plexes O Professor of Medicine, University of Ken- ty of 1'ennsylvania, Philadelphia. tueky College of Medicine, Lexington. ('I IF.NG-MING ('EIUONG PII M D. D Adhesion molecules in wound healing: model ~ EVANS S. DENERIS, Pn.D. Regulation of neuronal nicotinic receptor . • . , . Assistant Professor of Pathology, Universi- for tumor growth control and potential (her- Assistant Professor, Case Western Reserve gene expression ty of Southern California• l.os Angeles. apeutic value University, Cleveland, OH. H ~ MICHAEL S DuBOW Ph D Molecular mechanisms of aluminum arsenic AARON J. CIF:CIIANOVF.R• M.D., PIt.D. Mechanisms of degradation of oncoproteins . . , . Professor of Microbiolog and Immunol- , and nickel toxicity Professor of Biochemistry• Technion- Isnml Institute of Technology Ilaifa Israel and the ubiquitin proteolytic system ogy, McGill University, Montreal. Quebec, . • . Cananda. BERNICE H Pn ('OHEN D Airways obstruction and smoking in black . , . . Professor of F pidemiology The Johns and white adults PETER H. DUESBERG, Ptt.D. Retroviral onc and cellular proto onr genes . , Hopkins Elniversity, Baltimore, MD. Professor of Molecular and Cell Biology, University of Califomia, Berkeley. MYRON S ('OHEN M D Effect of lactoferrin on the free radical chem- . , . . Assistant Professor of Medicine Microbi- istry of monocytic phagocytes WILLIAM R. ECKBERG, PH.D. Activation of M phase by phorbol'esters , ology and Immunology, University of Professor of Zoology, Howard University, Washington, D.C. North Carolina, ('hapel I lill. VICTOR H. ENGELHARD PH.D. T cell res onses to human class 11 MHC mol- STANLEY ('OHEN• 1't+.D. Functions of p35 (lipocortin I) and F.GF dur- , Professor of Microbiology University of p ecules in human CD4 transgenic mice Professor of Bicx•hemistry, Vanderbilt Uni- ing embryogenesis , Virginia. Charlottesville. versity School of Medicine, Nashville• TN. ELLIS ENGLESBERG, PH.D. Genetic studies of the insulin receptor's mito- MARY SIIE COL.EMAN, Pn.D. The role of TdT in the developing immune Professor of Microbiology. University of genic signal Professor of Biochemisty, Associate Pro- system: construction of a transgenic TdT- Califotnia, Santa Barbara. vost and Dcan o/' Rescarch• I lnivcrsity of deficient mouse model North ('arolina• ('hapcl Hill. PAULA J. ENRIETTO, Pn.D. Identification of rel-regulated genes Assistant Professor of Microbiology, Stale ('I.AI IDIO J. ('ONTI• D.V.M.• PIt D. Tumor suppressor genes in squamous carci- University of New York, Stony Brook. Asscx iatc I'rofessor and Assoc iatc Director. nomas Science Park-Re.seardh Division, llniversi- JAMES E. ENSTROM, PH.D. Mortality trends among smokers and non- ty nf Texas M.1). Anderson Cancer ('cnter, Associate Researcher, University of Cali- smokers Ilouston. fomia, Los Angeles. BIANCA M. CONTI TRON('ONI, M.I). Molecular recognition of the nicotinic recep- ABRAHAM FAINSOD Ptt.D. Gene networks during embryogenesis in ver- Professor of Biochemistry and Phannacolo- tor in murine myasihenia gravis , Lecturer, Hebrew University-Hadassah tebrates gy. Ilniversity of Minnesota. St. 1'aul. Medical School, Jerusalem, Israel. RONALt) B. CORLEY PII.D. Transgenic mouse models for Ihe study of B DOUGLAS V. FALLER. M.D., PH.D. Molecular mechanisms controling blood flow • Associate Professor of Immunology, Duke cell repertoire development Professor of Medicine, Biochemistry: Pedi- in the lung - t Iniversity Medical Center• I)urham, NC. atrics and Pathology, Boston University. ROBERT H. COSTA, PIt.). Transcriptional regulation of the transthyretin JAME,S R. FERAMISCO, Plt.l). Protein dephosphorylation in growth control Assislant Professor of BioloFical Chem- gene in (he choroid plexus of Ihc brain Professor of Medicine, University of Cali- istry. l lniversity of Illinois. ('hicago. fomia, San E)iego. G. STANLEY ('OX• PtI.D. Cloning and characterization of novel nega- THOMAS H. FINLAY, Ptt.D. Anticarcinogenesis and protein pmtease in- Associate Professor of Bioxfiemisly, Uni- tive rrnn.c-acting factor Professor of Obstetrics and Gynecology. hibitors versity of Nebraska Medical ('enter, New York University Medical Center. ( hnaha. DANIEL FINLEY• Ptt.D. Apparent involvement of a novel multiuhiqui- MI('EIAEI. A. DAVf1Z• M.I). Development of a murine model of the GPI- Assistant Professor of Cellular and Molecu- tin chain in DNA repair Assistant Profcssor of Pathology, New specific phospholipase D lar Physiology, Harvard Medical Schox>I, York Elniversity School of Medicine. Boston. 242 243
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PRIN('IPAL INVESTIGATOR OR INSTITUTION I)AVII) A. FOSTER, Pn.D. Asauciate Professor, Hunter College of the City University of New York. ALEX J. FRANZUSOPF, Ptt.D. Assistant Professor. Llniversity of Colorado Health Sciences Center. Denver. BRUCE A. FRF,EMAN, Ptt.D. Professor of Ancsthesiology. Biochem- istry and Pediatrics. University of Alaba- ma. Bimiingham. BA1.7. FREI. Ptt.D. Assistant Professor of Nutrition. Harvard School ol' Puhlic Health, Boston. IRWIN FRIDOVICH. Pn.D. Professor of Qicxhemistry, Duke Universi- tV Medical ('enter, I)urham. NC. STEPHEN H. FRIEND. M.D.. Ptt.D. Associate Memher, Massachusetts General HospitaL Charlestown. ALLISON 1). FRYER. Ptt.D. Instructor, The Johns Hopkins University School of Ilygiene and Public Health. Bal- timore, MI). PROJECT TITLF; V-src-induced F,gr-I expression Genetic and biochemical analysis of cellular organization Vascular endothelial binding of xanthine oxi- dase LDL oxidation and antioxidant protection Tumor necrosis factor (TNF): effect on produc- tion of superoxide radical by mitochondria Tumor suppressor genes, tumor formation and development Airways disease and neuronal muscarinic receptors in the lung JOF;1. P. GALLAGHER, Ptt,D. Nicotine and its direct inhibitory action in the Profcssor. llniversity of'hexas Medical rat septal nuclei Branch. Galveston. ROBERT E. GALLAGHER, M.D. Professor of Oncology and Medicine. Mon- tefiore Medical Cenler, The Bronx. NY. Retinoic acid receptor proteins in leukemia MARTIN F. GF.LLP.RT, Ptt.D. ('hief, Section on Metabolic Enzymes, Laboratory of Molecular Biology, National Institutes of Health, Qethesda, MD. CECILIA M. GIACI1f?LLI, Pn.D. Research Aesociatc, University of Wash- ington School of Medicine. Seattle. ('HOU ZEN GIAM, P t.D. Associate Professor of Medicine, Molecu- lar Biology and Mirrohiology, ('ase West- em Reticrve Lhiiverity. ('levehmd, 011. GORDON NF.I,SON GILL, M.1). ('hief, Division of lindix•rinology/Metalxo- Iism, Universitv of California School of Medicine at San I)icgo. La Jolla. Mechanism of immunoglobulin gene rearrangement CYPIAL expression in vascular SMC ('ellular factors involved in HTLV-I trans- cription and ra.i mediated rrans-activation Receptor mechanisms in carcinogenesis 244 PRINCIPAL INVE,STIGATOR OR INSTITUTION IRA J. GOLDBERG, M.D. Assistant Professor of Medicine, Columbia University College of Physicians & Sur- geons, New York. STEVEN L. GONIAS, M.D.. PH.D. Assistant Professor of Pathology and Bio- chemistry, University of Virgmta, Health Sciences Center, Charlottesville. DIXIE GOSS, Pn.D. Professor of Chemistry, Hunter College, New York. SANNA M. GOYERT, PH.D. North Shore University Hospital. Manhas- set, NY. JEFFREY A. GRAY, PH.D. Professor and Head, Department of Psy- chology. Institute of Psychiatry, London, England. ANN M. GRAYBIEL, PH.D. Professor of Neuroanalomy, Massachusetts Institute of Technology, Cambridge. CAROL W. GREIDER, PH.D. Staff Scientist, Cold Spring Harbor Labora- tory, Cold Spring Harbor, NY. KATHLEEN J. GREEN, PH.D. Assistant Professor of Pathology, North- western University Medical School, Chicago. MARK I. GREENE, PH.D. John Eckman Professor and Head of Immu- nobiology, for Receptor Biology, University of Pennsylvania School of Medicine, Philadelphia. ALLAN D. GRINNELL, Pn.D. Pmfes.sor of Physiology. University of Cal- ifornia, Los Angeles. SIDNEY E. GROSSBERG, M.D. Walter Schroeder Professor, Medical Col- lege of Wisconsin, Milwaukee. JUN-LIN GUAN, Ptt.D. Assistant Professor of Pathology, Cornell University, Ilhaca, NY. PAULJ. HAGERMAN, M.D. Associate Professor of Biochemistry, Bio- physics and Genetics, University of Col- orado, [)enver. 245 PROJECT TITLE Li oprotein li p.ase-mediated increases in Ppopmtein atherogenicity Electron microscopy studies of alpha2- macrogtobulin Regulation of mRNA translation Myeloid differentiation genes: CDI4 The role of aminergic systems in the cogni- tive effects of nicotine in the rat Molecular effects of nicotine in the meso- striatal reward system Telomerase in cellular imortalization Epilhelial differentiation and neoplasia: desmosome biosynthesis and assembly Receptor interactions in cancer Calcium influx and regulation of lung carci- noma growth Characterization of the human JHK virus Role of protein tyrosine Phosphorylation in integrin-mediated cell adhesion The mle of DNA methylation in gene expres- sion
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PRINCIPAL INVFSTIGATOR OR INSTITUTION KATIIERINF. A. f1AJ1AR, M.D. Assistant Professor of Pediatrics and Medi- cine, Cornell University Medical ('ollcge, New York. HIDFSABURO HANAF(ISA. Pri.D. Professor, The Rockefeller University. New York. MARIE H. I IANIGAN, Pu.D. Research Fellow. University of Virginia, Charlottesvil le. OLIVER HANKINSON, Pn.D. Associate Professor of Pathology, Universi- ty of California, Los Angeles. t1LLA M. HANSEN, Pn.D. ('hief, Laboratory of Eukaryotic Trans- cription, Dana-Farber Cancer Institute. Boston. PRQ/N;CT TITLE Membrane receptor function in cell surface fibrinolysis Regulation of protein phosphorylation and cell transformation The role of gamma-glutamyl transpeptida.ce in hepatocarcinogenesis Characterization of repressor of cytochrome P4501 A I transcription SV40 T antigen stimulation of transcription PETER C. IfARPEL, M.D. Lipopmtein (a): structure and function Professor of Medicine, Mount Sinai School of Medicine, New York. STEVEN E. HARRIS, Pn.D. Biology of lipocorlin I in thymic epithelial Asscxiate Professor. IJniversity of Texas cells I lealth Science ('enter, San Antonio. SUSAN P. HAWKES, Pn.D. Associate Adjunct Professor of Pharmacy and Pharmaceutical ('hemistry, University of California, San Francisco. ISUMI HAYASHI, Pn.D. Assistant Research Scientist, Beckman Research Institute, City of Hope. Duarte, ('A. Purification and characteriaation of the 21K protein Molecular and biological aspects of a neu- rotrophic factor in Drosophrla DAVID M. HELFYv1AN, Pti.D. Senior Staff Investigator, ('old Spring I lar- Ixir Laboratory, ('old Spring Hartxx, NY. AVRAM HERSHKO. M.D. Ptt.D. Professor of Biochemistry. Technion - Israel Institute nf Technology, Haifa, Israel. ROBERT M. HOFFMAN, Pn.D. Associate Profesax of Pedialrics, Universi- ty of California at San Diego. La Jolla. JONATHAN M. HOROWIT7., PnD. Assistant Professor of Microbiolog y and Immunology. Duke llniversity Medical Center, Durham, NC. Molecular basis for tissue-specific alternative RNA splicing Mechanisms of regulation of cyclin degrada- tion Genes controling methionine dependence in human cancer Molecular genetic analysis of an Rb-like path- way in yeast 246 00 ~ O kn OR INSTITUTION N ~ 0 PRIN(:IPAL INVFCTI(:ATOR DEBORAH K. HOSHIZAKI, PHD. Assistant Professor of Biological Chem- istry. University of Illinois. Chicago. RICHARD L. HUGANIR, PH.D. ~ Associate Professor, The Johns Hopkins University School of Medicine, Baltimore, M MD. _ MIEN-CHIE HUNG. PH.D. Assistant Professor, University of Texas M. D. Anderson Cancer Center, Houston. JEFFREY R. IDLE, PH.D. Professor of Pharmacogenetics. University of Newcastle upon Tyne, Newcastle upon Tyne, England. ANIL K. JAISWAL, Pti.D. Associate Member, Fox Chase Cancer Cen- ter, Philadelphia. SUSAN JAKEN, Pn.D. Senior Scientist, W. Alton Jones Cell Sci- ence Center, Lake Placid, NY. GRAHAM A. JAMIESON, Pn.D. Astociate Professor of Microbiology, Uni- versity of Minnesota, Minneapolis. KUAN-TEH JEANG. M.D., PH.D. Laboratory of Molecular Biology, National Institutes of Health, Bethesda, MD. COLIN R. JEFCOATE, PH.D. Professor of Pharmacology and Director, Environmental Toxicology Center, Uni- versity of Wisconsin Medtcal School, Madison. WILFRED JEFFF,RIES, D.PHU.. Assistant Professor, University of British Columbia, Vancouver, Canada. RONALD JEMMERSON, Pti.D. Associate Professor of Microbiology, Uni- versity of Minnesota, Minneapolis. ALGIRDAS J. JESAITIS, Pn.D. Research Professor of Chemistry, Montana Slate l)niversity, Bozeman. WOLFGANG K. JOKIJK, D.Ptrn.. Chairman. Department of Microbiology and Immunology, Duke University Medical Center, Durham. NC. PROJF,(,T TITLE Isolation and expression of a Drnsophila lipid gene Mutagenesis of phosphorylation sites in the nicotinic acetylcholine receptor Ligand isolation and its cDNA cloning for the neu-encoded receptor Pharmacognetic epidemiology of lung cancer Role of NAD(P)H: quinone oxidoreductase I (NQOI ) in carcinogen and drug metabo- lism Transformation sensitive properties of protein kinase C PCP - A new probe of platelet function Role of Tat and cellular factors in HIV ex- pression Control of two novel rodent p450 cyto- chromes The role of the E3/19K protein of adenovirus- 2 in lowering cell surface expression of HLA molecules and in viral persistence The molecular basis for antigenicity Molecular targeting of oxidants by reorgani- zation of the neutrophil superoxtde generat- ing system The structure and function of reovirus pro- teins. 247
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PRINCIPAL INVESTIGATOR OR INSTITIITION J.(i. JOSI11, Pn.D. Professor of Bitxhemistry, University of Tennessee, Knoxville. MARSIIALI. E. KADIN, M,I). Associate Professor of Pathology. Harvard Medical School. Beth Israel Hospital, Boston. JAMES T. KADONAGA, Ptt.D. Assistant Professor of Biology. University of California at San Diego, La Jolla. LEE M. KAPLAN, M.D., Ptt.D. Assistant Professor of Medicine, Harvard Medical School. Massachusetts General Ilospital, &nlon. MI('HAI?I. KARIN, Ptt.D. Asscxiate Professor of Medicine. Universi- ty of California School of Medicine at San I)icgn, I,a Jolla. MORRIS J. KARNOVSKY. M.B.. B.Cn. Shattuck I'rofessor of I'athological Anat- onty. Ilarvard Medical Schixd, Boston. YONA KASSIR, Pit.D. Senior I.ccturer, Technion Isracl Institute uf"I"echnnlogy, I laifa, Israel. MIC'IIAF.I. B. KASTAN. M.I)., Ptt.D. Assistant Professor of Oncology and Pedi- atrics, Johns Hopkins llnivcrsity, Balli- morc. MI). 1•I,ORA KA'1"L. Ptt.D. Assistant Professor nf Bioxhemistry, lJni- versity of Texas Southwestern Medical ('enler, Dallas. AMY KENTI?R, Pn,l). Acsistant Professor of Microbiolo&y and Immunology, University of Illinois. (-hicago. MARK S. KINDY, PtrD. Assistant Professor of Biochemistry. Ilni- versity of Kentucky, Lexington. DONNA KIN(;. Ptt.l). Assistant I'rofessor of Pharmacology and Molecular Biology, University of Health St:iences, Chicago Medical Schcxol, North Chicago. PROJF,('T TITLF. Aluminum and iron metabolism in Alzheimer's disease l,ymphomatoid papulosis: a cutaneous model of lymphomagenesis Mechanisms of gene activation in eukaryotes Estrogen regulation of pituitary gene expres- sion Regulation of AP/I activity by tumor promot- cn:and oncogenes Characterization and therapy of rat vein graft lesions Signal pathway leading to iniliation of ineio- sis in ,S. crrct•i.riae. P53 and the response to DNA damage in Li- f•'raumeni and A.T. Neural carbohydrate function in Drosophila Molecular assays for Ig switch recombination Analysis of gene expression in ischemia tnediatctl stroke Regulation of expression of a reinducible juvenile heta globin gene in transgenic mice 248 PRINCIPAL INVFSTIGATOR OR INSTITUTION ERIC B. KMIEC, Ptt.D. Associate Professor, Thomas Jefferson University, Philadelphia. VICTORIA P. KNUTSON, PH.D. Assistant Professor of Pharmacology, Uni- versity of Texas Medical School, Houston. TAD H. KOCH, PH.D. Professor of Chemistry, University of Col- orado, Boulder. WILLEM H. KOPPENOL, DR. PttYS. St't. Associate Professor, Louisiana State Uni- versity, Baton Rouge. ROBERT H. KRETSINGER, Pn.D, Professor of Biology, University of Vir- ginia, Charlottesville. SKAIDRITE KRISANS, PH.D. Professor of Biology, San Diego State Uni- versity, San Diego, CA. ROBERT D. KUCIITA, Pn.D. Assistant Professor, University of Col- orado. Boulder. ARUN P. Kl1LKARNI, Ptt.D. Associate Professor, University of South Florida. Tampa. STEVEN L. KUNKEL, PH.D. Associate Professor of Pathology, Universi- ty of Michigan Medical School. Ann Arbor. MARTIN KUPIEC, Pn.D. Assistant Professor, Tel Aviv University, Tel Aviv, Israel. RYOKO Kl1R1YAMA, Ptt.D. Associate Professor of Cell Biology and Ncuroanatomy, University of Minnesota, Minneapolis. ERIC LAI, Ptt.D. Assistant Professor of Pharmacology, Uni- versity of North Carolina, Chapel Hilt. ABEI. LAITHA, Ptt.D. Director, ('enter for Neurochemistry. The Nathan S. Kline Institute for Psychiatric Research. Orangeburg, NY 249 PROJECT TITLE Transcriptional regulation by ci,r-acring ele- ments The transport of insulin across the vascular endothelium Nucleic acid photocleavage and photocross- linking to associated proteins The interaction of peroxynitrate with metal complexes Polyproline (i-turn helices: syntheses and stmctures The role of peroxisomes in cholesterol metab- olism DNA primase as a target for chemotherapeu- tOcs Benzo(cn)pyrene activation in human placenta Cytokine interactions between cells of the alveolar wall during inflammation Yeast mutants that affect recombination between repeated sequences Molecular analysis of an end-specific mitotic MAP Structure and organization of human T-cell receptor genes Glutamatergic effects of nicotine
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PRINCIPAL INVE,STIGATOR OR INSTITUTION THOMAS A. LANGAN. Pn.D. Professor of Phannacology, University of ('olorado School of Medicine, Denver. STF.PHEN LANIER. Pn.D. Assistant Professor of Pharmacology, Med- ical University of South Carolina. Charleston. GORI)nN W. LAURIE. Pn.D. Assistant Professor of Anatomy and Cell Biology, University of Virginia. Char- lottesvdle MURIEL I,EDERMAN, Pn,D. Assistant Professor of Biology, Virginia Polytechnic Institute and State University, Blackshurg. [.vn LEE. Ptt.D. AssOxiatc Professor, Institute of Biotech- nohogy, University of Texas Health Science Center, San Antonio. WEN-HWA LEE., Ptt.D. Professor and Director, Institute of Bio- chemistry. University of Texas Health Sci- ence ('enter, San Amonio. STUART E. LEf•T, Pn.D. Assistant Professor of Pharmacology, Stan- ford University, Stanford, CA. J1/1.lAN L. LEIBOWIl7.. M.I).,14t.D. Associate Professor of Pathology and Lab- oratory Medicine, University of Texas Health Science ('enter, Houston. VANDA A. I.IiNNON, M.1)., Pn.D. Professor of Immmrology and Neurology, Mayo ('linic, Rochester, MN. EI)WARI) B. LE(1P, Ptt.D. ('onsultant and Associate Professor, Mayo Foundation. Rochctitcr, MN. BI:N-7.ION LFiVI, I'it.D. Senior Lecturer, Technion-Isracl Institute of Technology, I laifa, Israel. EDWARD 1). LEVIN, Pn.D. Assistant Medicad Research Professor of 1'sychiatry. Duke University Medical ('en- ter, Durham, N('. El1GENE ( i. LEV IN, I1t.D. Assistant Member. Scripps Clinic and Research Potmdation, l.a Jolla, ('A. PROJECT TITLE Regulation of mitosis by CDC2R/cdc2' pro- tein kinase substrates Alpha; adrener$ic receptor subtypes and the plasticity of signal transduction Molecular control of alveolarization Interaction of viral and cellular proteins with origin of replication of bovine parovirus Biological function of a retinoblastoma-asso- ciated cellular protein Role of tumor suppressor genes in the genesis of human prostate carcinoma Purification and cloning of RNA binding pro- teins that regulate alternative RNA splicing The molecular genetics of prothrombinase induction by mouse hepatitis virus Novel ligands for small cell lung cancer TGF (31 regulation of lung epithelial cell pro- liferation The cloning and characterization of human I)NA binding protein which interacts with interferon consensus sequence DA-ACh interactions and chronic nicotine- induced cognitive facilitation Regulation of endothelial cell tissue plas- minogen activator 250 PRINCIPAL INVESTIGATOR OR INSTITUTION STUART B. LEVY, M.D. Professor of Medicine, New England Med- ical Center, Tufts University School of Medicine, Boston. MICHAEL R. LIEBER, M.D., Ptr.D. Assistant Professor, Stanford University, Stanford, CA. HOWARD B. LIEBERMAN, Ptt.D. Assistant Professor of Radiation Oncology, Columbia University College of Physicians & Surgeons, New York. MICHAEL W. LIEBERMAN, M.D., PH.D. W. L. Moody, Jr., Professor and Chairman of Pathology, Baylor College of Medicine, Houston, TX. JON LINDSTROM, Pn.D. Trustee Professor, University of Pennsylva- nia School of Medicine, Philadelphia. JOSEPH S. LIPSICK, M.D., PH.D. Associate Professor of Microbiology, State University of New York, Stony Brook. ZVI LIVNEH, PH.D. Scientist, The Weizmann Institute of Sci- ence, Rehovot, Israel. REUBEN LOTAN, PH.D. Professor and Deputy Chairman, Univer- sity of Texas M.D. Anderson Cancer Cen- ter, Houston. JAN M. LUNDBERG, M.D. Associate Professor of Pharmacology, Karolinska Institute, Stockholm, Sweden. HENRY T. LYNCH. M.D. Professor and Chairman, Department of Preventive Medicine and Public Health, Creighton University School of Medicine. (hnaha, NE. RICARDO B. MACCIONI, D.Sc. Director General, International Center for Cancer & Developmental Biology, Santi- ago, Chile. F.(1GENE E. MARCANTONIO. M.D., Pn.D. Assistant Professor of Pathology, Columbia University Health Sciences Center. New York. 251 PROJECT TITLE Transport changes in early adriamycin resis- tant leukemia cells LySmphDoid VDJ recombination in human Molecular aspects of DNA repair in S. /xrmbe Mechanisms of neoplasia in transgenic mice Neuronal nicotinic receptor structure Retroviral transduction of oncogenes in vitro Mechanism of S.O.S. error-prone repair Regulation of endogenous lectin expression in tumor cells Neuronal and endothelial mechanisms upon airway irritation Tumor variation in hereditary breast cancer and hereditary nonpolyposis colorectal cancer Regulation of microtubule assembly in nor- mal and transformed cells The role of integrin receptors in targeting of lymphocytes in human disease J
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~ PRINCIPAL INVESTIGATOR PROJECT TITLE ~ ~ PRINCIPAL INVESTIGATOR PROJECT TITLE OR INSTITUTION OR INSTITUTION ~ G. STEVE MARTIN, Pu.D. Berkeley. Pmfes t Iniversity of ('alifomia ux Proto-oncogenes, integrins and signal trans- duction N ~ WILLIAM J. MORRISON, Pu.D. Instructor, Department of Pharmacology, CD4 down-regulated lymphocyte activation . . , ~ Oregon Health Sciences University, Port- ROBERT W. MASON, Mi.D. Identification of novel cysteine proteinases F--1 land. D W roEGF in cell-to- re into the role of t d A Virginia Polytechnic Institute, Blackshurg. TIMOTHY E. M(fRAW, Mi.D. involved in elastin degradation Random mutagenesis of the transferrin recep• ~ SKI, Pn. . BARBARA MROCZKO Staff Scientist, California Institute of Bio- logical Research, La Jolla. p p u y s cell communication Assistant Professor of Pathology; Columbia Health Sciences Center Universit tor ~ R. ALLAN MUFSON, PH.D. The human IL-3 receptor , y New York. FRANK D. Mc-KEON. Mi.D. Assistant Professor of Cellular and Molec- ular Physiology, Harvard Medical School. Boston. ROBERT G. Mc•KINNF.LL, Mi.D. Professor of Genetics and ('ell Biology, University of Minnesota, St. Paul. PAIIL J. MF,E('HAN. Pn.D. Assistant Professor of Biological Sciences, Northern Illinois F)niversity, F)eKalb. ROrER1O MENEGHINI, Pn.D. Professor, University of Sao Paulo, Sao Paulo. Brazil. JOHN P. MERLIE. PII.D. Professor of Molecular Biology and I'hannacology, W ashinglon Flniverxity, SI. Louis. EDWIN M. MEYER, Pn.D. Associate Professor, University of Florida ('ollege of Medicine, Gainesville. ODED MEYFIHAS, Mi.D. Senior Lccturer, Institute of Biochemistry, Ilebrew Univcrsity-Hadassah Medical School, Jerusalem, Israel. JEFl7tEY B. MILLER. Pn.D. Assistant Professor of Neuroscience, Day Neuromuscular I,aMoratory. Massachusetts Gcneral I lospital. Boston. ROBERT L. MILLF,TTE. Pn.b. Professor of Biology, Portland State Uni- versity, Portland. OR. JOHN G. MONROI:. Ptr[). Assistant Profcssor of Pathulogy. Universi- ty of Pennsylvania School of Medicine. Philadelphia. Cytoplasmic retention signals ocyte nuclear transfer to abrogate a malig- nant phenotype Isolation and injection of chromosome(s) complementing XP mutations Cellular iron and DNA damage argeted mutations of the mouse neuromus- cular synapse icotine in a long-term model for Alzheimer's disease Translational repression of ribosomal protein mRNAs (luring tumor growth arrest Molecular and cellular regulation of neuro- muscular development A cellular transcription factor that regulates viral and cellular genes Developmental biology of B lymphocyte acti- vation signaling Senior Scientist. American Red Cross HoI- land Faboratory, Rockville, MD. CORNELIS MURRE, Pn.D. Assistant Professor of Biology, University of California at San Diego, La Jolla. MOON H. NAHM. M.D. Assistant Professor of Pathology, Washing- Um University, St. Louis. JAVIER NAVARRO. Pn.D. Profeasor of Physiology and Biop hysics, University of Texas Medical Branch, Galveston. DONALD 1. NELSON, Ptt.D. Associate Professor of Chemistry, Clark University, Worcester, MA. GERA NEUFELD, PH.D. Senior Lecturer, Technion-Israel Institute of Technology, Haifa, Israel. JOHN E. NIEDERHUBER. M.D. Professor of Surgery/Oncology, Stanford University, Stanford, CA. GAJANAN NILAVER, M.D. Associate Professor of Neurology Cell Biology and Anatomy, Oregon Health Sci- ences University. Portland. STEVEN K. NORDEEN, Pn.D. Assistant Pmfessor, University of Colorado Health Sciences Center, Denver. ERIC N. OLSON, Pti.D. Associate Professor and Associate Bio- chemist in Biochemistry and Molecular Biology, University of Texas M. D. Ander- son Cancer Center, Houston. SUZANNE OPARIL, M.D. Professor of Medicine. University of Alabama, Birmingham. he role of E2A in pediatric pre-B acute lym- phoblastoid leukemia Selective isotype expression in germinal cen- ters Expression of the fMet-Leu-Phe receptor from neutrophils almodulin interactions with neurotoxic met- als and the functional role of lysine-75 olecular basis for nuclear accumulation of bF(iF olecular analysis of abnormal tyrosine kinase expression Dopamine receptors in Alzheimer's hypothal- amus Structure and function of c-mos Regulation of muscle differentiation by growth factors and oncogenes Role of endothelin in the pathogenesis of hypoxic pulmonary hypertension 253 252
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' N ~ t- rn CV O O PRIN(:IPAL INVFSTI(:ATOR T TITLN; PROJF( PRINCIPAL INVFSTI(:ATOR PROJF,CT TITLF. 0 OR INSTITUTION 0 ~ OR INSTITUTION H YOSIIIOOSAWA, Ptt.D. I lead, Department of Endncrine Biochem- Medical Founrlation of Buffalo, Buf- istry Aromatace inhibitors in cigarette smoke and tobacco N !~"I 0 DAVID M. PRESCOTT, Ptt.D. Distinguished Professor, University of Col- Boulder. orado Reordering of DNA segments to produce functional genes in ciliates H a , falo. N. Y. MICHAEL C. OSTROWSKI. Pn.D. Assistant Professor of Microbiology and Duke University Medical Immunology enetic analysis of ras-sipal transduction pathways in transgenic mice -t , CATHERINE PRODY, Ptt.D. Scientist, The Hos ital for Sick Children, Toronto, Ontario. Canada. Factors controling vascular smooth muscle development 0 U , ('enter. 1)urham, N. C. H H LINDA C. QUATTROCHI, PH.D. Assistant Research Scientist, University of Tissue-specific regulation of the human CYPI A2 gene 1. JAMES 011. Pn.D. Assistant Professor. University of Southern ('alifomia, I .os Angeles. LfiSTER PA('KER, Ptt.D. Professor of Molecular and ('ellular Biolo- gy, University of ('alifomia, Berkeley. BEVERLY PAIGF,N, Pn.D. The Jackson Lahoratory, Bar Harbcx, ME. ROBERT f;. PALA7.7,O, Ptt.D. Assistant Professnr of Physiology and Cell Bioingy, Ilniversity of Kansas, I awrcnce. BIN= fAO PAN, PI I.D. Assistant Professor, Lucille 1'. Markey ('ancer ('enter. llnivercily of Kentucky. Lexington. SANKHAVARAM R. PANINI, Ptt.D. Institule Fellow. Eleanor Roosevelt Insti- lute for ('ancer Research. Iknver, ('O. ARTHUR B. PARDEE. Ptt.f). Professor of Biological Chemistry and Molecular Pharmacology, Dana-Farher ('anccr Institute, Boston I IENRY PAlI1.l1S, PtrD. 1)cpartnient 1)irector. Boston Biomedical Rcscach Imtitute, Boslon ISRAEI. Pli('HT. Ptt.D. Professor, The Weizmann Institute of Sci- ence. Rehnvol, Israel. ARTHl1R PENN. Ptt.D. Research Professor of Environmental Med- icine. New York University. MORTIMER PON('Z„ M.I). Associate Professor of Pediatrics, Joseph Stokes, Jr., Research Institule, ('hildren s Hospital of Philadclphia_ Molecular analysis of hepatitis B virus X gene Low-density lipoprotein protection by antiox- idant recycling apping genetic determinants of atheroscle- rosis susceptibility Reactivation of isolated mitotic apparatus Oncngenic ras protein and cell cycle regula- lion iological actions of 24(S), 25-oxidolamxsteml cgulation of the TK gene promoter during the cell cycle Adaption of yeast to osmotic stress Mast cell membrane function-associated com- ponents Molecular characteri7ation of the transform- ing gene(s) in cockerel arteriosclerotic plaque DNA Regulalion of platelet protein expression California at San Diego, La Jolla. RAMI RAHAMIMOFF, M.D. Professor of Physiology, Hebrew Univer- sity-Hadassah Medical School, Jemsalem, Israel. AVRAHAM RAZ, Ptt.D. Director, Metastasis Research Program. Michigan Cancer Foundation, Detroit. AHARON RAZIN, PH.D. Professor of Medical Sciences, Hebrew University-Hadassah Medical School, Jerusalem. Israel. JOIIN C. REED, M.D., Pn.D. La Jolla ('ancer Research Foundation, La Jolla, CA STEVEN 1. REED. Pn.D. Associate Member, Scripps Research Institute, La Jolla, CA TIMMOTHY J. REGAN, M.D. Professor and Director, Division of Cardi- ology, University of Medicine and Den- listry of New Jersey, New Jersey Medical School, Newark. RONALD R. REICHEL, Pti.D. Assistant Professor, University of Health Sciences, ('hicago Medical School, North Chicago LOLA M. REID, Pn.D. Associate Professor, Albert Einstein Col- lege of Medicine nf Yeshiva llniversity, The Bronx, NY. MI('IIAEL A. RF.IDY, Pn.D. Research Associate Professor. Universily of Washington, Seattle. ROBERT F. RH(SADS, Pn.D. Head, I)epartnrent of Biochemistry and Molecular Biolory, Lousiana Slale llni- versity, Shreveport Humoral effects of small cell carcinoma of the lung on neuromuscular transmission arbohydrate-binding protein and metastasis ynamic changes in DNA methylation 'loning of novel genes that regulate pro- grammed cell death Cell cycle control in animal cells ardiac neurohormones and the chronic interaction of nicotine and ethanol ctivation of liver gene expression upon F9 teratocarcinoma cell differentation Liver stem cells and Ihe relationship of hepatopoicsis to hcmopoiesis Regrowth of arterial endothelium Protein synthesis initiation factor 4E and oncogcncsis 254 255 I .
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OD rn tV 0 ~ O O PRINCIPAL INVFSTI(:ATOR PROJECT TITLE ~ PRINCIPAL INVESTIGATOR PROJECT TITLE OR INSTITUTION ® OR INSTITUTION H ~ H I IFIMO RIEDEL, Ptt.D. Investigator. Joslin Diabetes ('enter. A genetic model to dissect protein kinase C signaling N J. DF.NRY SATO, Pn.D. Research Scientist, W. Alton Jones Cell Tumor-derived growth factors a 0 Boston. ® Science Center, Lake Placid, NY. U A. ANGIE RIZZINO, Pn.D. Associate Member, University of Nebras- ka Medical ('enter. F.ppley Institute, Omaha. Transforming growth factor beta and mam- malian embryogenesis =-1 WALTER SAUERBIER, PH.D. Professor of Microbiology, University of Minnesota. St. Paul. Genetic studies of tumor suppressor genes in a haploid cell/animal system ~ LUCIA SCHUGER, M.D. Role of laminin in mouse lung development JOHN M. ROBINSON. Pn.D. Neutrophil stimulation: biochemical and cell ~ Mallory Pathology Associates, Boston. Associate Professor, Ohio State University Col lege of Medicinc, C olumbus. 1.ORNA W. ROLE, Pn.D. Assistant Professor of Anotomy and Ccll Biology, ('olumhia University College of Physicians & Surgeons. New York. 1)IANA RON. Pn.D. Assistant Professor. Technion-Israel Insti- tute of Tcchnology, flifa, Israel. GFRALI) M. ROSEN. PrI.D. Professor of P'harrnacology and Toxicolo- gy, l/niversity of Maryland School of Phanmcy, Baltimore. NAI)IA ROSF.NTI IAL. Pn.1). Assistant Prulcssor (if Biochemietry. Bncton liniversity. THOMAS L. ROTIISTEIN, M.1). Associate Professor of Medicine and Microbioingy, Bostun Ilnivcrsity Medical ('entcr, Boston. IIARRY RUBIN, Pn.l).. D.V_M. Professor of Molecutar Biology, IInivcrsi- ty of ('alifornia, Berkeley. A/.1"L SAN('AR, M.1)., Pttl). Associate Professor of Birxhcrnistry. Uni- vcrsity of Nanh (':mrlina, ('hapcl Hill. PF.TER SARNOW. Pn.D. Aasistanl Professor (if Molecular Biology, llniverxity of ('olorado Ilcallh Sciences ('cnter, 1)cnvcr. f)AVID A. SASSOON. Pn I). Assislanl Professor nl' Binchemistry, Boston I Inivcrsity. B.V. RAMA SASTRY, I'n.D. Professor of Pharmacology, Vanderbilt llniversity, Nashville. TN. biological studies Modulation of neuronal nicotinic acetylcho- line receptors tructure/function analysis of bFGF ree radical mediated cytotoxicity egulation of myosin light chain expressinn in tranxgcnic muscle cells TRE/AP- I I)NA binding proteins in B lym- phocytcs uantitative studies of spontaneous transfor- mation in cell culture Nucleotide excision repair Fukaryotic initiation factor of elF-4F-inde- pendent translation of mRNAs Molecular basis of limb development Mechanisms of smoking-induced compen- sauory responses in human placenta DAVID W. SCOTT, Pn.D. Professor of Immunology, University of Rochester, Rochester. NY. SIDNEY A. SCUDDER, M.D. Assistant Professor of Hematology/Oncol- ogy, University of Califomia, Davis. STEWART SELL. M.D. Professor, University of Texas Health Sci- ence Center, Houston GINETTE SF.RRERO, Pn.D. Senior Scientist, W. Alton Jones Cell Sci- ence Center, Lake Placid. NY. JACQUELINE SHARON, Ptt.D. Assistant Professor. Boston University 6chool of Medicine ISAIAHU SHECHTER, PH.D. Senior Fellow, Eleanor Roosevelt Institute for Cancer Re.cearch, Denver, Co. ELLIOTT SIGAL, M.D., PH.D. Assistant Professor of Medicine, Univer- sity of Califomia, San Francisco PAMELA A. SILVER. Pu.D. Assistant Professor of Molecular Biology. Princeton University, Princeton, N.J. MICHAGI. SINENSKY, Ptt.D. Ilead, Lipid and Lipoprotcin Metabolism Division, Eleanor Roosevelt Institute for Cancer Research. Denver. CO. DAVID A. SIRBASKU, PH.D. Professor of Biochemistry and Molecular Biology. University of Texas Ilealth Sci- ence ('enter, I louston. FRANCES 1. SMITfI, Pn.D. Shriver ('entcr for Mental Retardation, Waltham, MA. B lymphomas in aging mice: role of idiotype and tolerance Role of multidrug resistance in transitional cell carcinoma of the bladder Rabbit AIDS urification of serum-derived adipogenic factor Antibodies as DNA-binding proteins he role of regulatory protein in the coordi- nated regulation of hepatic cholesterol synthesis Transcriptional control of 15-lipoxygenase by cytokines Function of a new class of eukaryotic heat shock proteins Translational control of HMG-CoA reduc- tase Thyroid hormone mechanism of action in growth and development Detection of mutations in genes involved in human disease 256 257
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PRIN('IPAL INVESTI(:ATOR OR INS'1'ITUTION PRO,IE("1' TITLE 111 ~ PRINCIPAL INVFSTI(:ATOR ® in OR INSTITUTION PROJECT TITLE STIiVF:N S. SMITII, Ptt.D. Selectivity of DNA methylation in normal ~ FLEUR L. STRAND, PH.D. Prenatal and postnatal effects of nicotine and Assistanl Research Scientist. City of I lope and oncogenically transformed cells ~ Professor of Biology, New York University. ACTH pepttdes on neuromuscular devel- Duane ('A National Medical Center opment and motor behavior in rats . , ® AR'17111R K. SOLOMON. Pn.D. Prolein:protcin and prolein:cytoskeleton DAVID S. STRAYER, M.D., Ptt.D. The role of EGF-like growth factors in onco- 1'rulessor of Biophysics, I:mcritus, Ilar- interactions in the red cell and kidney Z Professor of Pathology, Thomas Jefferson genesis vard Medical Sch~xd, Boston. membrane 1`al University, Philadelphia. LAI IRIiN SOMPAYRAC. 1'n.D. (;enes involved in SV40 transformation NANCY E. STREET. Ptt.D. Determination of the role of CD4+ murine T Assoe'iate 1'rnlescor, University of Col- Assistant Professor of Microbiolog , Uni- helper cells in B cell tumor dormancy orado, 13oulder. versity of Texas Southwestern Medical School, Dallas JANI:f 1). SPARKS,1'n.D. Apolipoprolein B phosphorylation in rat Scientist. University nf Rochester, hepatocyles DENNIS 1. STUEHR, Pn.D. Molecular analysis of inducible and constitu- R(xhester. NY. Section of Immunology, The Cleveland tive nitric oxide synthesis Clinic Foundation, Cleveland, OH. 1)AVII)1-. SPE("fOR, Pn.D. Spatial and temporal organization of DNA Senior Staff Investigator, ('nld Spring replication SURESH SUBRAMANI, PH.D. Recombination genes in S. pombre and Ilarlxtt Lahoralory, ('old Spring Harbor, Associate Professor of Biology, Universi- mammalian cells NY- ty of California at San Diego, La Jolla. SARAI I SPIECYL, Pn.D. Molecular mechanisms of the regulation of MARIUS SUDOL, Pn.D. Functional analysis of yrs and .crc protoonc- Asxistant Professor of Biochemistry anrl differentiation and growth of tumor cells Assistant Professor, The Rockefeller Uni- genes Molecular Biology. Gcor~ctown Univer- by ganglioside GM I versity, New York, sity Medical ('enter, Washington. I).('. KATHY A. SUPRENANT. Pn.D. Endogenous inhibitors of microlubule lil.lO'f R. SI'INDF:I,. M.D., Pu.). Structure and function of receptors for the Assistant Professor, University of Kansas, assembly Scienlixt, Oregon Regional Primate bnmbesin like peptides Lawrence Research ('enter, F3eavenon. LORRAINE S. SYMINGTON, Pn.D. Molecular and biochemical analysis of DNA F.RI(' 1. STANNRIIH;Ii. Pn.l). Role of activated oncogenes in the tumori- Assistant Professor, Columbia University. repair Atiseciate 1'rofescor of Microbiology, I Ini- genic expression of human cancer cells New York, NY. versity of ('ali/omia. Irvinc. KENNETH D. TARTOF, PH.D. Cloning the renal carcinoma gene 11?AN R. S'I'ARKL'Y, I'n.D. Adhesive receptoraigand interactions in Senior Member, Institute for Cancer Assncialc Professor of Microbiology. tumor melastasis Research, Philadelphia. Monlana State l)niversity, Bozeman. D. LANSING TAYLOR, Pn.D. Regulatory peptides in the innervated and KARI STEFANSSON. M.F). Hexabrachion (Tenascin): an adhesion/anti- Professor of Biological Science, Carnegie- denervated heart Professor of Neurology, University of adhesion molecule Mellon University. Pittsburgh. ('hic-ago. R P STEPIII:NS M D F C' NEWMAN 1 Cellular mechanics of contraction in airway KENNETH M. TAYLOR, M.D. Regulatory peptides in the innervated and . . , . ., . . ,. Professor of Physiology, Ilniversity of smooth muscle; gene expression of con- Professor and Chief of Cardiac Surgery. Royal Postgraduate Medical School L on- denervated heart Manitoha. Winnipeg. Manitoba, Canada. tractile proteins , . don, England 1)AVID M. STERN, M.D. Modulation of endothelial cell anticoagulant 1)ANIEL G TF NEN M D Identification of retinoic acid responsive Asscrciate Prufessor, ('ulumhia University mechanisms . . , . . Assistant Professor of Medicine Harvard genes ('ollege of Physicians & Surgeons, New . Medical School Beth Israel Hospital, York. . Boston. S'I'OE('KLE M D MARK Y Cytokine gene regulation: control of mRNA , . . . Cornell Assistant Professor of Medicine stability CORNELIUS P. TF.RHORST, PII.D. Intracellular transport and assembly of the T . New York. Ilniversity Medical College Associate Professor of Pathology, Beth cell receptor . Israel Hospital, Boston. THOMAS P. STOSSEL. M.I). Functional anatomy of the lung macrophage Divition nl F.xpcrimenlal Medicine. IFead VICTOR P. TERRANOVA. F'n.D. Directed migration of tumor cells , Brigham and Women's 1lospital, Boston. Associate Professor, Columbia University. New York. 259 258
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0 0 th 0 0 0 PRINCIPAL INVESTIGATOR OR INSTITUTION MATI'HEW L. THOMAS, Pn.t). Assistant Professor of Pathology, Microbi- ology and Immunology, Washington Uni- versny School of Medicine, St. Louis. PIIILIP N. TSICH'LIS, M.D. Senior member, Fox Chase C'ancer Center, Philadelphia. PHILIP W. TUCKF:R, PH.D. Professor of Microbiology. University of Texas Southwestern Medical C'enter, Dallas. ANGELA L. TYNER, Pn.D. Assistant Professor of Genetics, University of Illinois. College ol Medicine. Chicago. JONATHAN W. lII IR, M.D. Professor and ('hairman of Microbiology, Professor of Internal Medicine, University of Texas Southwestern Medical Center, I)allas. MI('llAla. W. VAN DYKF„ Pn.D. Assistant Professor of Biology, University of Texax M.1). Anderson ('ancer C'enter, Ilou%ton. ~ 1NDIiR M. VERMA, Pu.D. 1'rotcssor, 'I'hc Salk Institute. San Diego, ('A. TIIOMAS W. VI('KROY, PIt.D. Assistant 1'rofessor, llniver.ity of florida, (7ainesville. LYI)IA VILLA-KOMAROfI', Pn.D. Associate Professor. ('hildren's Hospital, Boston. PF.TI:R K. V(XiT, Pn.D. Professor and ('hairman, Department of Microhiningy, IInivcrsity ol Soulhcnt ('al- ifumia, I.ox Angeles. ROBERT R. WAGNER. M.1). Professor of Microbiology. llniversity of Virginia, Charlottesville. TIIOMAS J. WAI.US('IIMID"r, Pn.D. Assistant I'rofessor ol' Pathology. Univer- sity of Iowa, Iowa ('ity. Lll HAI WANG, Pn.l). Associate Professor, Mount Sinai School of Medicine. New York. PRQIF,('T TITLE Leukocyte-common antigen. CD45: molecu- lar interactions Progressipn of retrovirus-induced lym- phomas Role of 2p13 translocations in childhood leukemia Negative regulation of AFP transcription in fetal gut Mechanisms underlying tumor dormancy Isolation and characterization of TFIID- ascociated proteins Regulation of rel/NF-KB by IKB: mediators of signal transduction Neuropharmacological investigations of nicotine interactions with CNS cholinergic and mesolimbic doparninergic neurons C'ontrol of neuropeptide synthesis in rodents New ONC genes from acute retroviral leuke- mias Molecular immunology of vesicular stomati- tis virus The Fc R distinguishes Ly I from conven- lional B cells F.xpression and function of proto-oncogene c-srr 260 PRINCIPAL INVESTIGATOR OR INSTITUTION LAWRENCE 1. WANGH, PH.D. Associate Professor of Biology, Brandeis University, Waltham, MA. DAVID B. WEINER, PH.D. Director of Biotechnology, Wislar Insti- tute, Philadelphia. MICHAEL W. WEISS, M.D., Pn.D. Assistant Professor of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston. SAMUEL B. WEISS, Pn.D. Professor of Biochemistry and Molecular Biology, University of Chicago. NANCY K. WELLER. Prt.D. Assistant Professor of Medicine, Case Western Reserve University, Cleveland, OH. SAMUEL A. WELLS, JR., M.D. Bixby Professor of Surgery, Washington University, St. Louis. MARIANNE WFSSLING-RF.SNICK, PH.D. Assistant Professor of Nutritional Bio- chemistry, Harvard School of Public Health, Boston. JEFFREY WILUSZ,Pn.D. Assistant Professor of Microbiology and Molecular Genetics, University of Medi- cine and Dentistry of New Jersey, New Jersey Medical School, Newark RICHARD M. WRIGHT, Pt(.D. Senior Instructor, Webb-Waring Lung Institute, l)niversity of Colorado. Denver. JOSEPH M. WU, Prt.D. Professor of Biochemistry. New York Medical College, Valhalla. REF.N WU, Pn.D. Associate Adjunct Professor, California Primate Research Center, University of ('alifomia, Davis. ' LAWRF,NCE 1. WYSOCKI. Pu.D. Staff Researcher, National Jewish Center for Immunology and Respiratory Medicine, Denver, C'O. GARY YEI,LEN, Pn.D. Associate Pmfessor of Neumbiology, Mass- chusetts General Hospital, C'harlestown. H H PROJECT TITLE Effects of glycation on DNA replication in tZ Xenopuseggs O Structural basis for receptor activation in cel- lular transformation Cancer-related transcription factors: struc-* ture and mechanisms DNA recombinant events induced by acti- vated hydrocarbon Molecular characterization of pulmonary alveolar type I cells and consideration of their relationship to alveolar type 11 cells Gene mapping of multiple endocrine neopla- sia type I The role of rab stmcture in vesicle biology Characterization of pre-mRNP structure Regulation of yeast xanthine oxidase gene expression Interferons and 2-5A expression in Alzheimer's disease fibroblasts Genetic analysis of mucous cell differentia- tion Immunogenicity of peptide fragments Mulagenesis studies of the activation bind- ing site of the nicotinic acetylcholine receptor 261
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PRIN('IPAL INVFSTI(:ATOR OR INSTITUTION ANDREW YF.N, Pn.D. Associate Professor, New York Slate ('ol- Iegc of Veterinary Medicine. ('omcll Flni- vcreity. Ithaca. TIF:N-S7.E B. YF.N, M.I)., Ptt.D. Assistant Professor of Pathilogy, tlniver- sity of California. San Francisco. ('110-YAl I YEUNG. Ptt.D. Assistant 1'rofetsor, University of Illinois, Chicago. 7.AI IRA 7.AKERI, Pn.D. Assistant Professor of Biulo~y, Queens College of the ('ity University of New York. Flushing. MAF /Rfl.IO'7.ANF.TTI, M.1). Associate Profescor af Medicine. Univer- sity of ('alifomia School of Medicine, San 1)icgo, La Jolla. MAFIRI('I; 7.At1DERER. Pn.D. Associate Profcstnr of Oncology, t)nivcr- sity of Rnrhetiter. Rixhesler, NY- PROJN:('T TITLE 1lalting progression of neoplastic disease by inducing terminal differentiation Mechanisms of trans-activation by the hep- atitis B vintx X protein Trans-regulatinn by family members of a serum tnducible gene Mechanisms of programmed cell death Idiotype engineering, a structure/function study Tumor immune responses to melanoma 262 Grantees of Completed Projects Following is a list of the principal investigators, or institutions, whose projecte have been completed prior to the period covered in this Report. A number of the individuals named are deceased. The titles and affiliations listed were those in effect at the time the work was in progress. ROBERT H. ABELES. Pn.D. Professor of Biochemistry, Brandeis Uni- versity, Waltham, MA. LEO G. ABOOD, PH.D. Professor of Brain Research and Biochem- istry, Center for Brain Research. Univer- sity of Rochester Medical Center, Roches- ter, NY. ANTHONY P. AMAROSE, PH.D. Instructor in Obstetrics and Gynecology, The Albany Medical College of Union University, Albany, NY. E. T. ANGELAKOS, M.D., Ptt.D. Professor of Physiology, Boston Uni- versity, School of Medicine. MARIO D. ACETO, Pit.D. Associate Professor of Pharmacology, Medical College of Virginia, Virginia Commonwealth University. Richmond. DOLPH O. ADAMS, M.D., Pn.D. Professor of Pathology, Duke F)niversity Medical Center, Durham, NC. BURT AI)ELMAN, M.D. Assistant Professor of Medicine, Medical College of Virginia, Richmond. KENNETH B. ADLER, PH.D. Assistant Professor of Pathology, University of Vermont College of Medicine, Burlington. D. MURRAY ANGEVINE, M.D. University of Wisconsin School of Medicine, Madison. JOSEPH C. ARCOS, D.Sc. Professor of Medicine, Tulane University School of Medicine, New Orleans, LA. ALAN K. ARMITAGE, Pn.D. Research Director, Hazleton Laboratories Europe, Harrogate. North Yorkshire. England. MARILYN S. ARNOTT (RASCO), PH.D. As,sistant Biologist and Assistant Professor of Biology, The University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston. CLARENCE M. AGRESS, M.D. Associate Clinical Professor of Medicine, KAREN J. AUBORN, Pt+.D. University of California Medical Center, Research Scientist. Long Island Jewish l.os Angeles. Medical Center, New Hyde Park, NY. ANTI IONY A. ALBANE.SE, Ptt.D. THOMAS M. AUNE. PH.D. Director of Laboratories. The Burke Assistant Professor of Pathology, The Rehahilitalicm Center, White Plains, NY. Jewish Hospital of St. Louis. JOIIN J. ALBERS, Pn.D. DOMINGO M. AVIADO, M.D. Associate 1)irector, Northwest Lipid Professor of Pharmacology, University of Research C'enter, Ilarborview Medical Pennsylvania School of Medicine, ('enter, Seattle, WA. Philadelphia. 263 ~
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~ VAN nt:N BERG M D BARBARA 1 IRff AVIRAM. 1'n.l). The Faculty I)epartment of Biochemistry FREDERICK W. BARNES. JR., M.D. Associate Professor of Medicine, The ~ . . . , Research Pediatrician, Adjunct Professor , Tel Aviv University Tel of Life Sciences Johns Ilopkins University School of ~ in Bioslatistics, University of California , , Aviv, Israel. Medicine, Baltimore, MD. ~ School of Public Health, Oakland. ~ ® BEVAN M.D. JOHN A STEPIIEN M D AYRES M D Sc. T BARNES C , . . . , . Oirector, Cardiopulmonary Lahoratory, . . . Research Scientist. Philadelphia State Professor of Pharmacology, University of Los California School of Medicine New York Saint Vincent's Hos ital Hospital. , . p . Angeles. BERNARD M. BARIOR, M.I)., Pn.D. F--I CARL G. BECKER. M.D. F®1 BUDHDEV BHAGAT, PH.D. Ilead, 1)ivision of Biochemistry, Scripps Associate Professor of Pathology, Cornell ~ St. Louis Professor of Physiology Clinic and Research Foundation, I,a Jolla, University Medical College, New York. , St. Louis. University School of Medicine ('A. , FREDERICK F. BECKER, M.D. CESARE BIANCIFIORI. M.D. L.FiSI,IE BAER. M.D. Professor of Pathology, University of Division of Cancer Research, University of Associate Professor of Medicine, Texas M. D. Anderson Cancer Center, Perugia. Perugia, Italy. Columbia University ('ollege of Physicians I louston. & Surgeons. New York. HYLAN A. BICKERMAN, M.D. R. FREDERICK BECKER, Pn.D. Assistant Professor of Medicine, and OSCAR J. RAL('HI.IM, Pn.D. Associate Professor of Anatomy and ALVAN L. BARACH, M.D.. Consultant Ilastings Professor of Medicine, University lalxttatory of Perinatal Science, Director in Medicine, Columbia University College of Southern California School of Medicine, , 1)uke University Medical Center, Durham. of Physicians & Surgeons; Goldwater Me- I,ris Arigeles. NC. morial Hospital, New York, NY. SAMIIEI. BALK. M.D., Pn.D. RALPH S. BECKER. Pia.D. BIO-RESEARCH CONSULTANTS, INC. Pathologist. New England Deaconess Professor of Chemistry, University of Cambridge, MA. I lospital, Boston. I Ioustnn, Houston, TX. BIO-RFSEARCH INSTITUTE, INC. FRF•,DI;RIK B. BANG, M.1). BENJAMIN BEI,1., M.1). Cambridge, MA. Professor and ('hairman, Department of Director Emcritus, Normative Aging Pathohiolugy, The Johns Ilopkins fJniver- Veterans Administration Outpatient Study sity School of Hygiene and Public Health. . Boston. ('linic SUBAL BISHAYEE, Pn.D. Ballimore, MI). , Associate Professor, Coriell Institute for Medical Research, Camden, NJ. MI('l1AEL BARANY, M.D.. Pn.D. SAMUEL BELLET, M.D. Professor of Biological (,'hemistry. 1)irector, Division of Cardiology, University nf Illinois, ('hicago. Philadelphia General Hospital. ROBF.RT L. BARBIERI, M.I). Assistant Professor of Obstetrics and Gynecology. Ilarvard Medical School, Boston. BARUJ BENACERRAF. M.D. Fahyan Professor and Chairman, Department of Pathnlogy, Harvard Medical School, Boston. I,AIIRIE BAR('LAY, M.1). tlniversity of South Florida,'I'ampa. A. CLIFFORO BARGER, M.D. Robert Henry 1'feiffer Professor of Physi- ology, Iiarvard Medical School. Boston. WILLIAM F. BENEDICT. M.D. Assistant Professor of Pediatrics, llniversity of Southcrn California School of Medicine. Division of Hematology and Medical Genetics, Children's Hospital of I,os Angeles, Los Angeles. DALE E. BOCKMAN. PH.D. Professor and Chairman, Department of Anatomy, Medical College of Georgia, Augusta. GUENTHER BODEN. M.D. Associate Professor of Medicine, Assistant Director, General Clinical Research Center. Temple University Health Sciences Center, Philadelphia. HERMAN V. BOENIG, PH.D. Head, Department of Chemistry and Biochemistry, Spindtetop Research Center, Lexington, KY. JAMES F. BONNER. PH.D. Professor of Biology. California Institute of Technology, Pasadena. WALTER M. BOOKER, PH.D. Professor and Head. Department of Pharmacology, Howard University, Washington. DC. FltANt:O1S M. BOOYSE. PH.D. Senior Investigator, Michael Reese Research Foundation, Chicago. RAYMOND BOSSf, Pn.D. Associate Director, Normative Aging Study, Veterans Administration Outpatient Clinic, Boston. TOM G. BOWERY, PH.D. Research Professor. Pesticide Residue Laboratory, North Carolina State College. Raleigh. DEBAJIT K. BISWAS, PH.D., D.Sc. Associate Professor of Oral Biology, H. LEON BRADLOW. PH.D. Laboratory of Pharmacology, Harvard Institute for Hormone Research. New York. School of Dental Medicine, Boston. IRA B. BLACK, M.D. Professor and ('hief, Division of I)evelopmental Neurology, C ornell University Medical ('ollege, New York. J. MARK BRAUGHLER, Pn.D. Assistant Professor of Pharmacology. Northeastern Ohio Universities College of Medicine, Rootstown, EDWARD BRESNICK, Pn.D. PIiYL.LIS B. BLAIR, Pn.D. Professor and Chairman, Department of Professor of Immunology, University of Biochemistry, The University of Vermont C'alifornia, Berkeley. College of Medicine, Burlington. FRED G. BO('K, Pn.D. GEOFFREY L. BRINKMAN, M.D. Associate Cancer Research Scientist. Associate Professor of Medicine. Wayne Biological Station. Roswell Park Memorial S1ate University School of Medicine, De- Institute, Buffalo, NY. troit. MI. MI('HAEL BENNETT. M.D. BRODA O. BARNf:S, M.D., Pit.D. Professor of Pathology, University of Professor (Affiliate) of Physiology, Texas Southwestern Medical Center. Colorado State University. Fort Collins. Dallas. 264 1 265
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~ D PIt ROBFRT E BR(X)KS M.D. ~ M. BIJTT F. . . , . Associate Professor of Pathology. . , ('hief Pathologisl. Los Angeles County ~ FRANCO CF.LADA, M.D., Pu.D. Senior Immunologist and Professor of CURT 1. ('IVIN, M.D. Assistant Professor of Oncolol nd University, of Oregon Medical School, General Hospital, Los Angeles. Immunology, Hospital for Joint Diseases, y a Pediatrics. The Johns Hopkins Oncology Port land. Orthopedic Institute, New York. Center, Baltimore, MD. RICHARD U. BYERRUM, Ptt.D. BARBARA B. BROWN, Ptt.D. Professor of Chemistry. Michigan State ® LEOPOLD R. CERECEDO, PH.D. ROBERT A CLARK M D C'hief, Experimental Psychiatry, Veterans ' llniversity, F.asl Lansing. Professor of Biochemistry and Nutrition, . , . Professor of Medicine University of Iowa A. Administration Hospital. Sepulveda, ( University of Puerto Rico School of , , Iowa City. MYLES CABOT, Pu.D. Medicine, San Juan. RAYMONI) R. BROWN, Ptt.D. Senior Scientist, W. Alton Jones Cell ~ WILLIAM G. CLARK PH D I'rofessor of Clinical Oncology, University Science Center, Lake Placid, NY. ~ JACK CHALON M.D. , . . Direclor Psychopharmacology Research of Wisconsin Medical School. Madison. ~ , Associate Professor of Anesthesiology, , Laboratory, Veterans Administration 1OSEF BRO7,EK, PIt.D. SISTER M. EMILY C'AHILI,. Pu.D. New York University Medical Center. Hospital. Sepulveda, CA. Department of Professor and ('hairman Chairman, Department of Chemistry, . chigh University. I Ps chology Regis College. Weston, MA. FRANCIS C. CHAO, M.D., PH.D. HANS T. CLARKE, D.Sc. , y , PA. Bethlehem Senior Investigalor, Center for Blood Professor of Biochemistry, Columbia . EDWARD J. CAMPBELL. M.D. Research, Boston. University College of Physicians & Surgeons, New York. RI:BE('CA BRYSON, PII.D. Assistant Professor of Medicine, Associatc Professor of Psychology, San Washington University School of DONNA CHAPRONIERE, B.SC., PH.D. SUSAN CLASTER M D I)iegn Stale University, San I)iego, CA. Medicine, St. Louis. Strangeways Research Laboratory, , . . Assistant Research Hematologist Cambridge, England. . Children's Hospital Oakland Research NAN('Y 1.. R. BlJ('l1ER, M.D. BRUCE F. CAMFRON, M.D., Ptt.D. , Institute Oakland CA Rctcarch Professor of Pathology, Boston Boslon. Schrxil of Medicine Universit Howard Hughes Instilute, University of LAN BO CHEN, PH.D. , . . . y Miami Schail of Medicine, Miami, Fl,. Associate Professor of Pathology, GARY A. CLAWSON, M.D., Pn.D. IH)ROTIIY I.. B(I('IIHAGI:N, 1'n.D. DanaFarber Cancer Institute. Boston Associate Professor of Pathology, George Assioain Professor, Stale University of ELROY T. CANTRELL, Pn.D. Washington University. Washington, D('. New York. Downstale Medical ('enter, Chairman, Department of Pharmacology. YUAN-TSONG CHEN M Prt D D B nxAlyn. Texas ('ollege of Osteopathic Medicine. , . ., . . Assistant Professor of Pediatrics Duke BRIAN L. CLEVINGER. Prt.D. North Texas State University. Denton. . Assistant Professor of Biomedical Science S(IE t3t I('KIN(i1IAM, M.D. University Medical Center, Durham, NC. . Washington University School of Dental Columbia Assistant Profcssor of Pcdiatrics Medicine, St. Louis. . University ('ollegc of I'hysicians & WILLIAM 11. CARNIiS, M.D. University of Ihah College of Medicine, CHILDREN'S HOSPITAL OF LOS AN- New York Sur eons GELES CHARLES G COCHRANE M D . . g Salt lake City. . , . . Member, Department of Immunopalhol- M SONIA BI IISf I) A ogy, Scripps Clinic and Research . . . . Associate I'rofessor of Medicine and MARCUS N. CARROLL., JR., Pu.D. WILLIAM M. CHILIAN, Pn.D. Foundation, La Jolla, CA. l)nivcrsity of Oregon Ilcalth siolugy Ph Chief, 1)ivision of Pharmacology, The Assistant Professor, Texas A&M , y Sciences Center. Portland. Brookdale Hospital Cenler, Brooklyn. NY. University Station. JAY D. COFFMAN, M.D. Section Head. Peripheral Vascular VIN('F.N7.O BIIONASSISI, M.D. WILLIAM A. CARTER, M.D. JAN F. CHLEBOWSKI, Ptt.D. Department, University Hospital, Boston. Senior Scientist and Deputy 1)irector, W. Professor of Ilematology and Medical Assistant Professor of Biochemistry, Med- Altnrr Jones ('cll Science ('enter. Lakc Oncology. Ifahnemann Medical College, ical College of Virginia, Richmond. ALLEN B. COHEN, M.D., Pu.D. Placid. NY. Philadelphia. Associate Professor of Medicine, Chief, Pulmonary Section Temple University JOIIN W. BUR('II, M.D. SANFORD CHODOSH. M.D. Assistant Professor of Medi in T ft , Health Sciences Center,,Philadelphia. American Red Asuwiatc Medical 1)ircctor WILLIAM ALVIN CARTFR, MD. c e, u s , NY. R(xfiester Rrxfiester 1)ivisinn ('rusx Assistant Professor of Medicine and llniversity School of Medicine. Boston. . , , Micrnhiology, The Johns Hopkins DANIEL COHEN, D.V.M.. M.P.11. BIiNJAMIN BURROWS, M-U. University Schcml of Medicine, Ballimore, NAITER M. CHOPRA, Pn.D- Assistant Professor of Veterinary lipidemiology and Public Health Assuciate I'rnfessor of Medicine. MD. Professor of ('hemistry, North Carolina . l/niversity of Pennsylvania School of IInivcrsily of ('hicagn. Agricultural and Technical State Veterinary Medicine. Philadelphia. ALBI?RT C'ASTRO, Pn.D. llnivcrsity, Grecnslxrro. Pn.D. BI ISBP.F nnvID I 1)irector. I lormone Research LaMoratory: . .. Texas A&cM Professor ol Toxicology 1'rofessor of Pathology and Medicine. ALI,AN C. COLLINS, Ptt.D. . ('ollcgc of Veterinary llnivertiit University of Miami Schnul of Medicine, DOl1G1,AS BROCK CINES, M.D. Associate I'rofessnr of Pharmacology. y ('nllegc Statinn Medicinc Miami. FI,. Profcesor of Medicine, Hospital of the Institute for Behavioral (ienetics, . , University of Pennsylvania. Philadelphia. I lniversity of ('olorado, Boulder. 266 267
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ROBERT W. COLMAN. M.D. Profescor of Medicine, Temple University School of Medicine, Philadelphia. JULIUS H. COMROE. JR., M.D. Director, Cardiovascular Research Institute, University of California Medical Center. San Francisco. ROBERT L. CONHAIM, Pn.D. Associate Scientist. University of Wisconsin, Madison. DEAN M. CONNORS. M.D. Associate Director, Department of Laboratory Medicine, St. Mary's Hospital, Madison, W1. PHILIP COOPER, M.D. Clinical Professor of Surgery and Director, Surgical Laboratory of Cellular Physiology. Albert Einstein College of Medicine of Yeshiva University; Chief, Surgical Service. Veterans Administration Hospital. The Bronx, NY. PAUL T. COSTA, Jn., Pn.D. Associate Professor of Psychology, University of Massachusetts at Boston, Dorchester. GF,RALD R. CRABTREE, M.1). Associate Professor (if Pathology. Stanford l)niversity, Stanford, CA. JOIIN E. CRAIGHEAD. M.D. Professor of Pathology, University of Vermont College of Medicine. Burlington. ROBERT L. CRAIN, Pn.!). Assistant Professor (if Sociology, University of Chicago. EVA BROWN CRAMFR, Pn.1). Associate Professor of Anatomy and Cell Biology, Downstate Medical ('enter. Brooklyn. NY. CARROLI, E. CROSS, M.D. Associate Professor of Medicine and Human Physiology; Director, Section of Pulmonary Medicine, University of California School of Medicine, Davis. CECIL E. CROSS Research Department. St. Joseph Hospital. Burbank, CA. CARL E. CRUETZ, Pn.D. Assistant Professor of Pharmacology, University of Virginia School of Medicine, Charlottesville. DAVID W. CRUMPACKER, Ptt.D. Professor and Chairman, Department of Environmental. Population and Organismic Biology, University of Cotorado, Boulder. SUSAN E. CULLEN. Pn.D. Professor of Microbiology, Immunology and Genetics, Washington University School of Medicine, St. Louis. GIDON CZAPSKI, M.Sc., Pn.D. Professor of Physical Chemistry, The Hebrew University of Jerusalem, Jemsalem, Israel. IVAN DAMJANOV, M.D., Pn.D. Professor of Pathology, Jefferson Medical Center, Thomas Jefferson University, Philadelphia. ALBERT DAMON. M.D., Pn.D. Lecturer on Anthropology; Research Associate in Medical Anthropology, Peabody Museum, Iiarvard University, Camhridge, MA. LARRY W. DANIEL, PH.D. Associate Professor. Bowman Gray School of Medicine, Wake Forest llniversity, Winston-Salem, NC. TIIOMAS R. DAWBER, M.D. Associate Professor of Medicine, Boston University School of Medicine. JOHN P. DELANEY, M.D.. Pn.D. Associate Professor of Surgery, University of Minnesota, Minneapolis. JEROME A. DEMPSEY, PN.D. Professor of Preventive Medicine, University of Wisconsin, Madison. ANDREW S. DIBNER, PH.D. Executive, Psycho-Research, The Age Center of New England, lnc., Boston. EDWARD F. DOMINO. M.D. Professor of Pharmacology, University of Michigan, Ann Arbor. MARTIN E. DORF, Pn.D. Professor of Pathology, Harvard Medical School, Boston. RALPH L. DORFMAN, Pn.D. Director of Laboratories, Worcester Foundation for Experimental Biology, Shrewsbury, MA. H. FRED DOWNEY, Ptt.D. Assistant Professor of Physiology, University of Texas Health Science Center at Dallas; Director of Cardiovascular Research, Cardiopulmonary Institute, Methodist Hospital of Dallas. LAURENCE A. DOYLE, M.D. Assistant Professor, University of Maryland Cancer Center, Baltimore. HAROLD F. DVORAK, M.D. Chief, Department of Pathology, Beth Israel Hospital. Boston. JAMES J. DYAR, PH.D. Assistant Professor of Biology, Bellarmine College, Louisville, KY. RICHARD H. EARLE, M.D. Chief, Pulmonary Function Laboratory; Assistant Professor of Medicine. University of Chicago. ROBERT ECHT, Ptt.D. Professor of Anatomy, Michigan State University. Fast Lansing. IRVING P. CRAWFORD. M.1). Professor and Chairman, I)epartment of R. F. DAWSON. Pn.D. Microbiology, University of Iowa ('ollege Professor of Botany. Columbia l)niversity, of Medicine, Iowa City. New York. "L TIMOTHY ('ROCKER, M.I). ALBERT B. DEISSEROTH, M.D., Pn.D. Professor of Medicine. University of Professor of Medicine, Veterans Admin- ('alifornia ('ollege ol Medicine, Irvine. istration Medical Center, San Francisco. JOHN W. ECKSTEIN. M.D. Assistant Professor of Internal Medicine, State University of Iowa College of Medicine, Iowa City. BERTRAM EICIfiEL, D.D.S. Director, Institute of Stomatological Research, Science Resources Foundation, Watertown, MA. REUBEN EISENSTEIN. M.D. Professor of Pathology, Mount Sinai Medical Center, Milwaukee, WI. HYMAN ENGELBERG, M.D. Attending Physician, Cedars of Lebanon Hospital. Los Angeles. CARLTON K. ERICKSON, Ptt.D. Professor of Pharmacology, The University of Texas College of Pharmacy, Austin. V. GENE ERWIN. Ptt.D. Professor of Pharmacology; Dean, University of Colorado School of Pharmacy, Boulder. HENRY J. ESBER. PH.D. Research Immunologist, Mason Research Institute, Worcester, MA. WALTER B. ESSMAN. M.D., PH.D. Professor of Pathology and Biochemis- try Queens College of the City University of New York, Flushing. JOHN R. FSTERLY, M.D. Associate Professor of Pathology, Univer- sily of Chicago Pritzker School of Medi- cine. HUGH E. EVANS, M.D. Director, Department of Pediatrics, Jew- ish Hospital and Medical Center of Bnxrk- lyn, Brooklyn, NY. HANS J. EYSENCK, Pn.D., D.Sc. Professor of Psychology, Institute of Psy- chiatry. University of London. London. England. HANS L. FALK. Pii.D. Adjunct Associate Professor of Pathology, University of Southern California School of Medicine, Los Angeles. 1)ANA L. FARNSWORTH, M.D. Henry K. Oliver Professor of Hygiene and Director, University Health Services. Har- vard University. Cambridge, MA. 268 1 269
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~ ALVAN R. FEINSTEIN, M.D. WILLIAM It. FISIIMAN, N.D. ~ ERROL C. FRIEDBERG. M.D. TERESA GESSNER. Pn.D. 1'rofessor of Medicine and Epidemiology, President. La Jolla ('anccr Research Foun- ~ Associate Professor of Pathology, Stan- Associate Cancer Research Scientist, Yale University Schrx)l of Medicinc, New dation. La Jolla, CA. ~ ford University, Stanford, CA. Health Research. Inc., Roswell Park Divi- Ilaven, ('l'. Buffalo sion NY H , , . BIRGITTA FLODERUS-MYRRf1ED, N.D. ~ GILBERT H. FRIEDELL, M.D. GAD FEINSTF,IN Pn D. Assistant Professor of Environmental Hy- Chief of Pathology Vincent's Hospi- St GEORGE O. GEY M.D. . , Senior Lecturer in Biochemistry. The gicne, The Karolinska Institute, Stock- d , . tal, Worcester, MA. , Director, Finney-Howell Cancer Research George S. Wise Center of Life Scicnces, holm. Sweden. Laboratory; Associate Professor of Sur- Tcl Aviv University, Tel Aviv, Israel. GARY D FRIEDMAN M D. gery, The Johns Ho kins University P JOSEPH A. FONTANA, M.D., N.D. ~ . , . Assistant Director. Department of Medical imore, MD. School of Medicine. Ba M JOSEPH D FELDMAN D Assistant Professor of Medicine and Bio- Methods Research, Kaiser Foundation Re- . , . . C i i Med- hnl West Vi inia Universit hemistr search Institute, Oakland, CA. JACQUES E. GIELEN. PH.D. Immunopathologist, Scripps c and l n La Jnlla ('A Research Foundation y c y. rg ical Center. Morgantown. F_~ Assistant Professor, Laboratory of Medi- , . , H. HUGH FUDENBERG, M.D. cal Chemistry, Toxicology and Hygiene. Professor of Medicine, University of Cali- Institute of Pathology, University of LiPge, RICHARD FENTON, N.D. JUDITH ANN FOSTER, N.D. fornia Medical Center, San Francisco; Lfte, Belgium. Instructor in Physiology, University of Professrrr and Chairperson, Department of Professor of Bacteriology and Immunol- Massachusetts School of Medicine, Biology, Syracuse University, Syracuse, ogy, University of California, Berkeley. RONALD W. GILLETTE. PH.D. Worcester. NY. Director, Basic Science Research Unit, ARTHUR FURST, PH.D. Cancer Center of Hawaii, University of FRANK C. FERGUSON, JR., M.D. RICI IARD B. FOX, M.D. Director, Institute of Chemical Biology, Hawaii at Manoa, Honolulu. C'hairman, 1)cpartment of Pharmacology, Head, Pulmonary Division, Minneapolis University of San Franciso. The Albany Medical ('ollege of Uninn Children's Medical Center, Minneapolis, GERALD J. GLEICH, M.D. University, Albany. NY. MN. KJELL FUXE M.D. Consultant in Medicine, Research Labora- , The Karolinska In- Professor of Histology tory for Allergic Diseases, Mayo Clinic J. STIiP11EN FINK. M.D., N.D. JACK W. FRANKEL, N.D. , stitute, Stockholm, Sweden. and Foundation; Professor of Internal Medicine and Immunology. Mayo Medi- Assistant Professor of Neurology. Massa- Consultant in Medical Research, Veterans cal School. Rochester, MN. ('harlestown. chuscas General Hospital Administration Medical Ccnter, Bay Pines, MORTON GALDSTON, M.D. , FL. Associate Professor of Medicine New , York University Medical Center. THOMAS M. GOCKF„ M.D. Tlif•.Ofx)RF. N FINLEY M D. Associate Professor of Preventive Medi- . , . Pulmonary Research I,ahnra- Director B. L. FRF,F.DLANDF.R, M.D. cine and Community Health, Seton Hall , San Francisco tory Mount Zion Hospital Director of Cancer Research, Mount Zion MURRAY B. GARDNER, M.D. College of Medicine and Dentistry, Jersey , , . Hospital and Medical Center, San Fran- Associate Professor of Pathology, Univer- City, NJ. cisco. sity of Southern California School of FISHBEIN M WILLIAM I D Medicine, Los Angeles. . . , . y ('hicago Board of ('hief of I: idemiolo GABRIEL C. GODMAN, M.D. g , p Health ALLAN P. FREEDMAN, M.D. Professor of Pathology Columbia . Assistant Professor of Medicine, Hahne- JAMES W. GAUBATZ, Ptt.D. University College of Physicians & mann Medical ('ollege, Philadelphia. Assistant Professor of Biochemistry, Uni- Surgeons, New York. EDWARD A. FISHER, M.D., N.D. versity of South Alabama, Mobile. Assistant Professor of Biochemistry, The BARRY GOLD, PH.D. e of Penns lvania Phila- Medical Colle AARON E. FREEMAN. N.D. University of Associate Professor , y g rnia Biom Staff S ientist Calif dical Re- JACK GAULDIE, N.D. , delphia. , c o e McMaster Professor of Pathology Nebraska Medical Center, Eppley lnstitute, search Foundation, La Jolla. , Omaha University, Hamilton, Ontario, Canada. . EDWIN R FISHER M 1) . , . . b t i d id H Di t f L Sh FREDERIC A. FRENCH, A.B. WARREN M. GOLD, M.D. or o a ora or es, a ys os- rec e f l it f i P f P th Il i l Director of Cancer Chemotherapy Re- J. BERNARD L. GEE, M.D. Professor of Medicine. Cardiovascular essor o vers y o p ta ro a o ogy, n ; Pi h S h l f M di b i search, Mount Zion Hospital and Medical Professor of Medicine, Yale University Research Institute. University of Califor- oo tts o e nc. urg c c Center. San Francisco. School of Medicine, New Haven, CT. nia, San Francisco. FISt1ER PAUI B N D MICHAEL C GEOKAS M D N D , . . . . M JACK FREUND D . , . .. . . ALFRED L. GOLDBERG. N.D. Senior Research Associate. I)epartment of bi l i C' U i ' l i bi l . . . Assistant Professor of Pharmacology, Professor and Vice-Chairman, Depart- University of Califor- ment of Medicine Profecsor of Physiology, Harvard Medical o um vers a n ty ( o - M cro o ogy, lege of Physicians & Surgeons. New York. Medical College of Virginia, Richmond. , nia Schortl of Medicine, Davis. School, Boston. LARS FRIBERG, M.D. MI('HAEL. D M (iERSf1ON D. DAVID M. GOLDENBERG, D.St'., M.D. RIISSELL S. FISHER, M.D. Professor and Chairman, Department of . . , Professor of Anatomy and Cell Biology, Associate Professor of Pathology. Temple University of Maryland School of Medi- Environmental Hygiene, The Karolinska Columbia University College of Physi- University Health Sciences Center. Phila- cine, Baltimore. Institute. Stockholm, Sweden. cians & Surgeons, New York. delphia. 270 1 271
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SII)NEY GOLDFISCHER, M.D. Professor of Pathology. Albert Einstein College of Medicine of Yeshiva Univer- sity, The Bronx, NY. PAE/l. GOLDHABER. D.D.S. Associate Professor of Periodontology, Harvard School of Denial Medicine, Bos- lon. WILLIAM E. GOLDMAN, Pti.D. Assistant Professor of Microbiology and Immunology, Washington University School of Medicine, St. Louis. LEONIDF. GOI,DSTEIN. D.Sc. Associate Professor of Psychiatry, Insti- tule for Mental Health Sciences, College of Medicine & Dentistry of New Jersey, Rutgers Medical School. Piscataway. IRA GORE. M.D. Professor of Pathology, Boston University School of Medicine; Chief of I,aMttatory Service, Veterans Administration Ilospi- tal, West Roxhury, MA. JOHN W. (iORROD, I).('.('. Leclurer in Biopharmacy, Chelsea Col- Icgc. University of London. London, 1?ngland. GERlRl1DE Y. GO'1'I:S('HALL. PIt.D. Assistant Professor of 13inchetnistry. Co- lumbia llnivereily College of I'hysicians & Surgeons. New York. MAI IRI('F. GREIiN, M.I). Direc'lor. Intitilutc for Molecular Virol- ogy, St. Louie I Inivereitv Medical ('enter. ('l1ARI.ES S. GRF?I?NBF.RG, M.D. Assistant Professor of Medicine, Duke Itniversily Medical ('enter, Durham, NC. A. ('LARK GRIFFIN, Pn.l). Head, 1)epartment of Biochemistry. M.I). Anderson Iluspilal and Tumor Institute. University of Texas Medical ('cnter, I loustun. ARTHUR I.. GROSS, M.S. Senior Biochemist, Southwest Research Institute, San Antonio. TX. MORTON I. GROSSMAN. M.D., Ptt.D. Associate Clinical Professor of Medicine, University of California Medical Center, Los Angeles. CARL C. GRUHZIT, M.D., PH.D. Associate in Physiology and Pharmacol- ogy, University of Pennsylvania Graduate School of Medicine. Philadelphia. JOSEPH J. GUARNERI, Pn.D. Attending Microhiologist: Director. Mi- crobiology Laboratories, Long Island Jewish-Hillside Medical Center, Queens Hospital Center Affiliation, Jamaica, NY. HODA A. GUIRGIS, Pn.D. Associate Professor of Community and Environmental Medicine. University of California College of Medicine. Irvine. HIRA L. Gl1RTOO, D.V.M., M.V.S('., Pn.D. Associate C'ancer Research Scientist, De- partment of Experimental Therapeutics, Roswcll Park Memorial Institute, Buffalo, NY. FRANK E. GUTHRIE, Ptt.D. Professor of Entomology, North Carolina State ('ollege, Raleigh. FI. B. HAAG. M.D. Professor of Pharmacology. Medical Col- lege of Virginia, Richmond. F. J. HADDY, M.D., Pn.D. Professor and C'hairman, Department of Physiology, l)niversily of Oklahoma Medical Center, Oklahoma ('ity. JOSEPI I H. I IAFKENSCHIEL, M.D. Director, ('ardiopulmonary l)nit, The Lankenau Ilospital; Associate in Medi- cine. University of Pennsylvania School of Medicine, Philadelphia. NOBUYOSHI HAGINO, M.D., PtI.D. I'rofessor of Anatomy, University of Texas Hcalth Science ('enter, San Anto- nio. CAROLINE B. IIALL, M.D. Associate Professor of Pediatrics and Medicine. University of Rochester School of Medicine, Rochester, NY. 272 A LINDA M. HALL. Pn.D. Associate Professor of Genetics and Neuroscience, Albert Einstein College of Medicine of Yeshiva University, The Bronx, NY. MARGIT HAMOSH, PH.D. Research Associate, Department of Physi- ology and Biophysics, Georgetown Uni- versity Schools of Medicine and Dentistry, Washington, DC. PAUL HAMOSH. M.D. Associate Professor of Physiology and Biophysics, and Medicine, Georgetown University Schools of Medicine and Den- tistry, Washington, DC. BERNARD HANES, PH.D. Professor of Health Science, California State University, Northridge. RONALD G. HARVEY, PH.D. Professor of Organic Chemistry. The Uni- vereity of Chicago. RICHARD J. HAVEL. M.D. Assistant Professor of Medicine, Univer- sity of California Medical Center, San Francisco. FIERBERT R. HAWTHORNE, M.D. Chairman. Department of Surgery, Uni- versily of Pennsylvania Graduate School of Medicine, Philadelphia. ('AROI. J. HENRY, Pn.D. Direcotr, Department of Experimental On- cology. Microbiological Associates, Inc., Bethesda, MD. HERBERT B. HERSCOWITZ, PH.D. Associate Professor of Microbiology, Georgetown University Schools of Medi- cine and Dentistry, Washington, DC. LAWRENCE L. HESTER, JR., M.D. Professor and Chairman, Department of Obstetrics and Gynecology, Medical Col- lege of South Carolina, Charleston. HENRY D. HOBERMAN, M.D., Pt(.D. Professor. Albert Einstein College of Medicine of Yeshiva Universtiy, The Bronx. NY. EBBE CURTIS HOFF. M.D., PH.D. Professor and Chairman. Division of Psy- chiatric Research, Medical College of Vir- ginia. Richmond. JAMES C. HOGG. M.D., Pn.D. Professor of Pathology. University of British Columbia. Vancouver, British Columbia. Canada. JEROME L. HOJNACKI. PH.D. Assistant Professor of Biological Sci- ences, University of l.owell, Lowell, MA. RUSSELL L. HOLMAN, M.D. Louisiana State University School of Medicine, New Orleans JOHN A. HAYES, M.D. OLE A. HOLTERMANN, M.D. Associate Pathologist, Mallory Institute of Research Scientist, Lobund Laboratory. Pathology, Boston City Hospital. University of Notre Dame, Notre Dame, IN. CLARK W. LIEATH, M.D. Professor of Medicine and Director, Health FREDDY HOMBURGER. M.D. Services, Tufts University. Medford, MA. President and Director, Bio-Research In- stitute, lnc., Cambridge, MA. NORMAN W. HEIMSTRA, Pti.D. Professor of Psychology; Director, Human WAYNE HOSS, PIt.D. Factors Laboratory, University of South Associate Professor, Center for Brain Re- Daknta, Vennillion. search, University of Rochester Medical ('enter, Rochester, NY. PAULINE HE17.ER, Pn.D. Research Associate in Cytology and Cyto- ROBERT W. HULL. PH.D. chemistry. San Francisco Institute of Med- Professor of Biological Sciences, Florida ical Sciences. State University. Tallahassee. 273 to 0 cY) 0 0 0 H H a O U I
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RONALD R. HUTCHINSON, Pn.D. JOLYON JESTY. Ptt.[). ROBERT W. KARR, M.D. WILLEM KOPPENOL. Ptt.D. Research 1)irector, Foundation for Behav- Associate Professor of Medicine. State Assistant Professor of Medicine, Univer- Associate Professor, Louisiana State Uni- ioral Research, Augusta. MI. University of New York at Stony Brook. sity of Iowa, Iowa City. versity, Baton Rouge. IIT RESF.AR('H INSTITUTE DAVID A. JOHNSON, Ptt.D. HRATCH KASPARIAN, M.D. ALVIN 1. KOSAK, Pn.D. Chicago. Assistant Professor of Biochemistry. East Assistant Director, Cardiovascular Labo- Associate Professor of Chemistry. New Tennessee State University College of ratory; Instructor in Medicine, Hahne- York University. ALPHONSE J. INGF;NITO, Pti.D. Medicine,lohnson City. mann Medical College and Hospital, Phil- Associate Professor of Pharmacolo Fast adelphia. gy. MARKKU KOSKENVUO M D. Carolina University School of Medi- M.D. JOHN P. JOHNSON . , Professor and Chairman Department of cine, Greenville. NC. , Associate Director and Clinical Geneticist, ELIHU KATZ, Ptt.D. , Public Health Science, University of Hel- Children's Hospital Medical Center, Associate Professor of Sociology, Univer- sinki, Helsinki, Finland. HARRY L. IOACHIM, M.D. Oakland, CA. sity of Chicago. Attending Pathologist, Lenox Hill Hospi- tal; Clinical Professor of Pathology. Co- ROBERT W. KREILICK, PIt.D lumbia University College of Physicians & HAROLD P. JONES. Pn.D. SHIRLEY L. KAUFFMAN, M.D. Professor of Chemistry, University of Surgeons New York. Assistant Professor of Biochemistry. Uni- Professor of Pathology, State University of Rochester, Rochester, NY. , versity of South Alabama, Mobile. New York Downstate Medical Center, Brooklyn. PETER C. ISAKSON. Pn.D. KLAUS E. KUETTNER, Ptt.D. Assistant Professor of Pharmacology, OSWALD R. JONES. M.D. Professor and Chairman, Department of University of Virginia School Medi- St. Luke's Hospital. New York. INGEGERD M. KEITH, Ptt.D. Biochemistry, Rush College of Health Sci- c ine, ('harlottesville. Assistant Professor of Anatomy, Univer- ences and Rush Medical College, Rush- sity of Wisconsin School of Veterinary Presbyterian-St. Luke's Medical Center, WILLIAM J.IUSKO, Pn.D. Medicine, Madison. Chicago. KY(X;O ITOIi, M.D. Associate Professor of Pharmaceutics; Di- Assistant Profcssor of Surgery. University ' rector Clinical Pharmacokinetics Labora- fexas M. I). Anderson Cancer Center, of , ANCEL KEYS PH D ROBERT A KUHN M D I louston tory, Millard Fillmore Hospital, Buffalo, . , . . , . . . NY. Director, Laboratory of Physiological Hy- Associate Professor, Division of Neuro- giene, University of Minnesota School of surgery, New Jersey State College of Med- BARBARA J. JACKSON. Pn.D. Public Health, Minneapolis. icine, Jersey City. Staff Scientist. Boston Biomedical Re- ANDREW A. KANDUTSCH, Pn.D. search Institute. Staff Scientist, The Jackson Lahoratory, JOSEPH B. KIRSNER M.D. STIG KULLANDER M D Bar Harbor. ME. , Professor of Medicine, University of Chi- . . . Professor and Chairman, Department of GEORGE JA('OBSON, M.D. cago School of Medicine. Obstetrics and Gynecology, University of Professor and Head. Department of Radi- FA-TEN KAO, Pn.D. Lund, Lund, Sweden. ology, University of Southern California Professor, Eleanor Roosevelt Institute for EDWARD L. KLAIBER, M.D. School nf Medicine, Los Angeles. Cancer Research, Denver, CO. Senior Scientist, The Worcester Founda- LAWRENCE L. KUPPER, PH.D. tion for Experimental Biology, Inc., Associate Professor of Biostatistics, Uni- M.D. JERRY f1ART JACOBSON Shrewsbury, MA. versity of North Carolina School of Public , Director, Division of Electrophysiology, ARNOLD R. KAPLAN, Pn.D. Health, Chapel Hill. New York E e and Ear Infirmar Director, Laboratory of Medical Genetics, y y. Cleveland Psychiatric Institute and Hospi- JEROME KLEINERMAN, M.D. Cleveland OH. tal Professor and Chairman, Department of JAMES T. KURNICK, M.D. JULIUS H. JACOBSON 11, M.D. , , Pathology, Mount Sinai School of Medi- Associate Pathologist, Massachusetts Associate Professor of Surgery and Di- cine, New York. General Hospital, Boston. rector of Surgical Research, University of ATTALLAH KAPPAS. M.D. Bur- Vermont College of Medicine Professor and Senior Physician, The MARVIN KUSCHNER D M , lington Rockefeller University, New York. PETER H. KNAPP, M.D. . , . New York University Medical Center . Research Professor of Psychiatry, Boston . University School of Medicine. AARON JANOFF, PttD. MANFRED L. KARNOVSKY PH.D. CHARLES W. LABELLE, PH.D. Professor of Pathology, Health Sciences , Harold T White Professor of Biological Assistant Professor of Environmental Hy- State University of New York at Center . KENNETH P. KNUDTSON, M.D. giene, Jefferson Medical College, Phila- . Stony Brook Chemistry and Molecular Pharrnacology, University of Washington School of Medi- delphia. . Harvard Medical School, Boston. cine. Seattle. MURRAY E. JARVIK, Pn.D. AARON J. LADMAN, PH.D. Associate Professor of Pharmacology, Al- SIMON KARPATKIN, M.D. HEINZ KOHLER, M.D., Pfl.D. Professor and Chairman, Department of bert Einstein College of Medicine of Ye- Professor of Medicine, New York Univer- Adjunct Professor of Pathology, Univer- Anatomy, University of New Mexico shiva University. The Bronx, NY. sity Medical Center. sity of Califomia, San Diego, La Jolla. School of Medicine. Albuquerque. 274 1 275
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THOMAS C. LAIPPLY, M.D. RICHARD A. LERNF,R, M.D. ~ JOSEPH D JR., D.M.D. MANHOLD JOHN H D Pn D M LOCKER Professor of Pathology, Northwestern , . . ., . . . . Associate Member, Scripps Clinic and Re- ® Assistant Professor of Patholo and Bio- Professor and Director. Department of Pa- University Medical School, Chicago. gy search Foundation. La Jolla, CA. thology and Oral Diagnosis. New Jersey 40 chemistry, University of Pittsburgh School ' N of Medicine. College of Medicine and Dentistry, Jersey MI( HAEL E. LAMM, M.D. CECILE LEl1CHTENBERGER, PH.D. ~ City. Professor of Palhology, New York LJni- Head, Department of Cytochemistry, Swiss HERBERT L H D M P M LOMBARD ® versily Medical Center . ., . . . . . Institute for Ex rim ntal Cancer . pe e Affiliate. Cancer Research Institute, New DAVID E Pn.D. MANN , . Research. Lausanne, Switzerland. England Deaconess Hospital, Boston. Associate Professor of Pharmacology, ~ DON I,APF.NAS, M.D. Temple University School of Pharmacy, Assistant Professor of Pathology. llniver- LAWRENCE LIT-KING LEUNG, M.D. 1. P. LONG, PH.D. Philadelphia. si(y of Vermont College of Medicine, Assistant Professor of Medicine. Stanford Professor of Pharmacology, State lJniver- Burlington. University Stanford CA. sily of Iowa College of Medicine, Iowa , , FRANK ARTHUR MANNING, M.D. City . Assistant Professor of Obstetrics and PAUL S. LARSON, Pn.l). JAY A. LEVY, M.D. Gynecology, Women's Hospital, Los Haag Professor of Pharmacology, Medical Associate Professor of Medicine; Re- GESINA L. LONGENECKER Angeles County/University of Southern College of Virginia, Richmond. search Associate, Cancer Research Insti- Associate Professor of Pharmacology, California Medical Center, Los Angeles. tute, University of California School of University of South Alabama College of ROGER K LARSON M D Medicine. San Francisco. Medicine, Mobile. . . , . Chief of Medicine. Fresno County Ilospi- HAIM MANOR, Pn.D. Technion-Israel Associate Professor tal, Fresno. CA. PAUL D. LEWIS, M.D. Senior Lecturer in Histopathology, Royal , CLAYTON G. LOOSLI, M.D., PH.D. Hastings Professor of Medicine and Pa- Institute of Technology, Haifa. Israel. Postgraduate Medical School Hammer- thology. University of Southern California GL~STAVE A. LAl1REN7,I, M.D. ' , smith Hospital London England School of Medicine Los Angeles. JOHN P MANOS M D Chief of Medicine St. Vincent s Hospital , , . . . . . , , , Worcester MA Instructor in Virology and Bacteriology. , . RANDOLPH V. LEWIS. Ptt.D. DONALD B. LOURfA, M.D. Medical College of South Carolina, Associate Professor and Head, Depart- Associate Professor of Medicine, Cornell Charleston. 1OSEPH M. LAIIWERYNS. M.1)., Pn.f). ment of Molecular Biology, University of University Medical College. New York. Professor Ordinarius and Chairman, Dc- Wyoming, Laramie. M D MARKHAM RICHARD A partmcm of Pathology and Microscopic Anatomy. Experimental Laboratory of . , , . RONALD B. LUFTIG, Pn.D. Assistant Professor of Medicine and of Mi- Pulmonary Ilistnpathology. Katholicke AVERILL A. LEBOW. M.D. Professor and Head. Department of Micro- crobiology and Immunology, The Jewish tlnivcrsilcit Ic l,cuvcn, I,euvcn. Belgium. Chairman, Department of Pathology, Yale biology and Immunology. Louisiana State Hospital of St. Louis. University School of Medicine, New Ha- University Medical Center, New Orleans. ven. CT. 1?. ('I,INTON LAWREN('E 1) M BILLY R. MARTIN. Prt.D. . , . Assistant 1'rofessor of Mcdicinc Ba lor RONALD 1. LUKAS, Ptt.D. Associate Professor, Medical College of , y IRVIN E. LIENER. Ptt.D. Laboratory of Neurochemistry, Vir Director inia Commonwealth Uni- Vir inia College of Medicine. Ilouskm, TX. Professor of Biochemistry, University of , g , g St. Joseph's Hospital and Medical Center, versity, Richmond. Minnesota. St. Paul. Phoenix. AZ. PAl)l. LIiBOWITZ, M.D. Senior Research Scientist, Yale University. VALERIE K. LINDGREN, Pn.D. KENNETH MERRILL LYNCH. M.D.. CHRISTOPHER M. MARTIN, M.D. New Haven, ('f. Guest Researcher, National Cancer Insti- LL.D. Assistant Professor of Medicine and Di- Sc.D. Iute, Belhesda, MD. , Professor Emeritus of Pathology and rector, Division of Infectious Diseases, JAMI:S C Ptt LEE D Chancellor, Medical College of South Seton Hall College of Medicine, Jersey . . . , Assistant Professor of Biochernistry, St. ESTEN O. LINDSETH, M.D., Pn.D. Carolina, Charleston. City. NJ. Luui% School of Medicinc. St. loseph's Hospital Research Labora- tory, St. Paul, MN. BRUCE A. MACHER. Ptt.D. R. RUSSELL MARTIN, M.D. F EDWARD I I;rli Ptt D D Sc• Assistant Professor of Pharmaceutical Professor of Medicine, and Microbiology , , , . ., . ' ROBERT H. LINNF.LI„ Ptt.D. University of California Chemistr e of Medi- San and Immunolo Ba lor Colle Professor of ( hcniistry, University of Associate Professor of ('hemistry, Univer- . y, g y gy, Franciso cine Ho ston TX Minnesota. Minncapulis. sity of Vcrmont, Burlington. . , , . u ' HOWARD S MAKER M.D. JOSEPLI LF:IGII IY)N, M.D. FABIAN J. LIONFTTI, Pn.D. . , Associate Professor of Neurology, Mount REGINALD G. MASON, M.D., Pn.D. 1'rofessnr of Pathology, Medical Coldcge of Professor of Biochemistry, Boston Uni- New York. Professor and Chairman. Department of Sinai School of Medicine 1'cnnsylvania, Philadelphia. versity School of Medicine. , Palhology. University of Utah College of Medicine, Salt Lake City. Pn.D. INES MANDL PHILIP M. Lt: (1t11 :SNf?, D.S<•. RIC'ARfx) V. LLOYD, M.D., Ptt.D. , Assistant Professor of Biochemistry. Co- Professor of ('hemistry, Northeastern llni- Assistant Professor of Pathology, Univer- lumbia University College of Physicians & MASON RESEARCH INSTITUTE vcrsity, Boston. sity of Michigan. Ann Arbor. Surgeons. New York. Worcesler, MA. 276 i 277
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DONALD J MASSARO M D VICTOR A McKUSICK M D JERALD. A. MITCHELL, PH.D. EDMOND ANTHONY MURPHY, M.D., . . , . . , . . ~ Wayne Associate Professor of Anatomy D Sc Associate Professor of Medicine, George Washington University School of Medi- Professor of Medicine. The Johns Hopkins University School of Medicine Balti- , State University School of Medicine. De- . . Associate Professor of Biostatistics and cine, Washington. D.C. , ~ more MD troit, Mt. Medicine, The Johns Hopkins University . . ~ School of Medicine, Baltimore. MD. CHARLES McARTHUR, Pn.D. P°'I ROSS L. McLEAN M.D. STELLA MITRANI-ROSENBAUM, PH.D. Psychologist. University Health Services, , Emory ® Associate Professor of Medicine Professor of Virology, Hebrew University- WILLIAM S. MURRAY, Sc.D. Harvard University, Cambridge, MA. , University School of Medicine, Atlanta, Hadassah Medical School, Jerusalem, Senior Staff Scientisl, The Jackson Labo- GA. 0~-a Israel. ratory, Bar Harbor. ME. CHARLES McCANTS, Pn.D. ~ Associate Professor of Soils, North Caro- WILLIAM F McNARY JR Pn D CHARLES MITTMAN, M.D. JAY A. NADEL, M.D. lina Slate College School of Agriculture, . , . . ., H Associate Professor of Analomy, Boston Director, Department of Respiratory Dis- Professor of Medicine, Physiology and Raleigh. University School of Medicine. ~ eases, City of Hope National Medical Cen- Radiology, Cardiovascular Research Insti- ter, Duarte, CA. tute. University of California, San Fran- cisco. GERALD F,. McLF.ARN, Pn.D. NEAL McNIVF,N, Pn.D. I)irector, Institute for Behavioral Genet- The Worcester Foundation for Experimen- M.D. HUGH MONTGOMERY ics, Department of Psychology, llniver- tal Biology, Shewshury, MA. , Associate Professor of Medicine, Univer- RICHARD L. NAEYE, M.D. sity of Colorado School of Pharmacy, sity of Pennsylvania School of Medicine, Professor and Chairman, Department of Boulder. Philadelphia Pathology, Pennsylvania State University HANS MEIER. D.V.M. . College of Medicine, Hershey. Senior Staff Scientist, The Jackson Labo- ALAN C. Mc9.AUGHLIN, PH.D. Lecturer in Biochemistry and Biophysics, ratory, Bar Harbor, ME. P.O'B. MONTGOMERY. JR., M.D. SUSAN NAYLOR, PH.D. University of Pennsylvania School of Professor of Pathology, University of Associate Professor of Human Genetics Medicine, Philadelphia. J. WISTER MEIGS. M.D. Texas Southwest Medical School, Dallas. , The University of Texas Health Science Clinical Professor of Epidemiology; Di- Center. San Antonio. rector, Connecticut Cancer Epidemiology PH D MOORE M D GEORGE E HENRY C. McGILL, JR.. M.D. A i H 1) d f P h l Unit, Yale University School of Medicine, . . , . ., . Roswell Park Memorial Institute Di t r ct ng ea . epartment (i at o ogy, Louisiana State Unrversity School of Med- New Haven. CT. . rec o . Buffalo, NY GEORG B. NEURATH, PH.D. New Orleans. icine Microanalytical Laboratory, Hamburg, . West Germany. EDGAR F MEYER Ptt JR D HENRY I) McINTOSH M D . , . ., . Associate Professor Texas A&M Univer- DAVID A. MOSCATELLI, PH.D. . , . . Professor of Medicine and Director, Car- , sity College Station. Research Assistant Professor, New York D. NEWBALL M HAROLD H diovascular Laboratory, Duke University , University Medical Center. , . . Associate Professor of Medicine, The Medical (-enter, Durham. NC. Johns Hopkins University School of Medi- JULIA MEYER, Ptt.D. KENNETH M. MOSER, M.D. cine, Baltimore, MD. FORDE A. McIVER, M.D. Associate Professor of Oral Pathology, Assistant Professor of Medicine, George- Associate Professor of Pathology Medical University of Illinois College of Dentistry, town University Medical School, Washing- . College of South Carolina Charleston Chicago. ton DC ALBERT H. NIDEN, M.D. . . , . Professor of Medicine, Drew Postgraduate Medical School and University of South- EDWARD McKEF., M.D. DOV MICHAELI, Pn.D. Pulmonary Disease ern Catifornia; Chief Professor and Acting Chairman, Depart- Assistant Professor of Biochemistry and HURLEY LEE MOTLEY. M.D. , Martin Luther King Hospital Los Section ment of Pathology, Medical College of Surgery University of Califomia School of Professor of Medicine and Direclor, , , Angeles South Carolina, Charleston. , Medicine San Francisco Cardio-Respiratory Laboratory, Univer- . , . sity of Southem California School of Med- icine, Los Angeles. STEFAN NIEWIAROWSKI, M.D.. Pn.D. KELLY T. McKF.E, M.D. MICROBIOLOGICAL ASSOCIATES. INC. Professor of Physiology, Thrombosis Re- Associate Professor of Medicine. Medical MD Bethesda search Center, Temple University School College of South Carolina, Charleston. . . FF,RID MURAD, M.D., Ptt.D. of Medicine, Philadelphia. Professor of Medicine and Pharmacology, WALLACE L McKF .F.fIAN Pn D BERNARD J. MILLER, M.D. Stanford University, Stanford, CA, and . . , . , Senior Scientist W Alton Jones Cell Sci- Assistant Professor of Anatomy, Jefferson Chief of Medicine, Veterans Administra- OAK RIDGE NATIONAL LABORATORY , . ence Center, lake Placid, NY. Medical College, Philadelphia. tion Medical Center, Palo Alto, CA. Oak Ridge. TN. HERBERT McKENNIS JR Pti D JAMES G. MILLER, M.D.. Pn.D. CHRISTOPHER MURLAS, M.D. FRANZ OESCH, Pn.D. ., , . . R h P f f P h l - i Professor of Ps chi tr d P h l Associate Professor and Assistant Chair- Professor of Pharrnacobgy: Head. Section esearc ro essor o at o ogy, Un ver sity of Miami School of Medicine Miami y y an a syc o ogy; Director. Mental Health Research Institute, man, Department of Medicine, Rush Uni- on Biochemical Pharmacology. Univer- , , FL. University of Michigan, Ann Arbor. versity. Chicago. sity of Mainz, Mainz, West Germany. 279 278
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SE-KYUNG OH, Pn.D. A ssociate Professor of Microbiolo BORIS M. PETERLIN, M.D. kn JULIA M. POLAK, D.Sc., M.D. RONALD E. RASMUSSEN, Pta.D. Section of Assistant Professor of Medicine gy. Boston University School of Medicine. , 00 Senior Lecturer in Histopathology, Royal Associate Adjunct Professor, Departments Rheumatology Clinical Immunology, University of California School of Medi- 0 Postgraduate Medical School, Hammer- of Community and Environmental Medi- JANET M OLIVER Pn D cine, San Francisco. ~ smith Hospital, London, England. cine, University of California College of . . , . Professor of Pathology University of New Medicine. Irvine, CA. N , Mexico School of Medicine Albu uer ue F1 , q q . DENNIS R. PETERSEN, PH.D. OTAKAR J. POLLAK, M.D., PH.D. WALTER REDISCH M D ® , . . Professor of Pharmacology. University of Executive Director, Dover Medical Re- Associate Professor of Clinical Medicine F. WILLIAM ORR, M.D. , Colorado School of Pharmacy. Boulder. search Center, lnc., Dover, DE. New York Universily School of Medicine Associate Professor of Pathology, Univer- , ~ and NYU Research Service, Goldwater sity of Manitoba, Winnipeg, Manitoba, Memorial Hospital New York Canada . . SCOTT W. PETERSON, Pn.D. MORRIS POLLARD, PH.D. . Associate Professor of Biology, Arkansas ~-i Director, Lobund Laboratory University , College, Batesville. H of Noire Dame, Notre Dame WILLIAM REGELSON IN D M MARY D. OSBAKKEN, M.D. , . . , . Professor and Chairman Department of ~ Assistant Professor of Anesthesia and , Medical Oncology Medical College of Biochemistry/Biophysics, University of DAVID E. PETTIJOHN, PH.D. , C. M. POMERAT, PH.D. Virginia Richmond 1'ennsylvania. Philadelphia. Professor of Biochemistry/Biophysics, , . Director of Biological Research. Pasadena University of Colorado, Denver. Foundation for Medical Research Pasa- 1)ONALD M. PACE, Pii.D. , dena, CA. LYNNE M. REID, M.D. Professor of Ph and Director In- siolo Wolbach Professor of Pathology, Harvard y gy . EDGAR PICK. M.D., PH.D. Medical School; Chairman De artment of stitute for Cellular Research, University of Professor of Immunology, Tel Aviv Uni- , p Palholo Children's Hos ital Medi l Nehraska, Lincoln. versity, Tel Aviv, Israel. gy, p ca LOUIS PONCZ, PH.D. Center Boston , . Senior Research Associate, Case Western KENNETH PAIGFN Pn D Reserve University, Cleveland, OH. , . . Director. Department of Molecular Biol- CARL W. PIERCE. M.D., Pii.D. Departments of Pathology and Professor HOBART A. REIMANN, M.D. Roswell Park ogy Health Research Inc. . Professor of Medicine, Hahnemann Medi- , , , Microbiology-Immunology, Washington S. N. PRADHAN, M.D., PH.D. cal Coll d H it l Phil d l hi Division, Buffaln, NY. University School of Medicine. Director. ege an osp a , a e p a. Professor of Pharmacology, Howard Uni- Pathology and Laboratory Medicine, The versity College of Medicine, Washington, ALBERT B. PALMER. Pn.D. Jewish Hospital of St. Louis. DC. EI'LEEN REMOLD-O'DONNELL, Ptt.D. Assistant Professor of Pathology, Univer- Principal Research Associate, Harvard sity of'Tolcdo, Tolcdo, 011. Medical School; Investigator Center for G. BARRY PIERCE, JR.. M.D. , H. R. PRATT-THOMAS Blood Research Boston M D Distinguished Centennial Professor of , , . . . Professor of Pathol d D M di l ROSE MARIE PANGBORN, M.S. Pathology, University of Colorado Health ogy an ean, e ca Colle e of South C li Ch l t Assistant Food Technologist and Lecturer, Sciences Center Denver. g aro na, es ar on. JOHN E. REPINE M.D. 1)epartment of Food Science and Technol- , , Assistant Director, Webb-Waring Lung ogy. l Iniversity of Califomia, Davis. Institute; Associate Professor of Medi- MALCOLM C. PIKE. Pii.D. PROCESS & INSTRUMENTS CORPORA- cine University of Colorado Health Sci- JOHN W PARKt;R I) M Professor of Community Medicine and Pe- , TION ences Center, Denver. . , . . Associate Professor of Pathology, Univer- diatrics, University of Southern California Brooklyn, NY. siry of Southcm California School of Med- School of Medicine, Los Angeles. icine. Los Angeles. ROBERT RESNICK, M.D. EDWARD V. PROCHOWNIK. M.D. Associate Professor of Reproductive Med- JAMFS M. PIPAS, Pii.D. Associate Professor of Pediatrics, Univer- icine, University of Cahfornia Medical MARY STEARNS PARSHLEY Pn D. Assistant Professor, University of Pitts- sity of Michigan, Ann Arbor. Center, San Diego. , . Assistant Professor of Anatomy in Ohstct- burgh. rics and Gynecology Columbia llnivcr- . sity College of Physicians & Surgeons, RAY C. PITTMAN PH.D. MARTIN S. PROT7.E1., D.D.S. HERBERT Y. REYNOLDS, M.D. New York, , Research Biochemist, University of Cali- Professor of Medicine; Head. Pulmonary Chief. Department of Oral Pathology. Section Yale Universit School f M di- San Diego la Jolla fornia , y o e Newark City Hospital, Newark, NJ. , , . cine. New Haven, CT. BENDI('HT U. PAUI,1. D.V.M. Associate Professor of Pathology, Rush- ' ' SALVATORE V. PIZZO, PH.D. WILLIAM A. PRYOR, PH.D. ROLLAND C REYNOLDS M D Presbyterian-St. l.uke s Medical C enter, ' Associate Professor of Pathology, Duke . , . . Boyd Professor of Chemistry. Louisiana Assistant Professor of Path l Univ ( hicago. University Medical Center, Durham, N.C. o ogy, er- State University, Baton Rouge. sity of Texas Southwestern Medical School, Dallas. F.I)WARD W. PELIKAN, M.I). CHR1S D. PLATSOUCAS, PH.D. MICHAEL S. RABSON D M Chairman. Departmem of Pharmacology Professor of Immunology and Deputy , . . Research Scientist Laboratory of Pathol- SOLON L RHODE Ptt III M D D and Experimental Therapcutics, Boston University School of Medicine Chairman, University of Texas M.D. Houston Anderson Cancer Center , . , , . ., . . ogy National Cancer Institute, Bethesda, Professor. University of Nebraska Medical . , . MD. Center, Omaha. ztto 281 1
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rh 0 O O WARD RICHARD RICE, M.D.. Pn.D. Assictant Professor, University of Cincin- nati, Cincinnati, OH. VICTOR RICHARDS, M.D. Chief of Surgery, Presbyterian Medical ('enter, San Francisco. VIRGINIA L. RICHMOND. Pn.D. Research Associate, Pacific Northwest Research Foundation, Seattle. WA. ARNIS RICHTERS. Pn.D. Associate Professor of Pathology, Univer- sity of Southern California School of Med- ieine. Los Angeles. WILLIS H. RIESEN, Ptt.D. Senior Biochemist, Life Sciences Divi- sion, IIT Research Institule, Chicago. DANIEL B. RIFKIN. Pn.D. Assistant Professor of Chemical Biology, The Rockefeller University, New York. R. H. RIGIxJN, M.D. Professor of Pathology, Univcrsity of Texas Medical Branch. Galveston. SYDNEY C. RITTENBF.RG. Pn.D. Professor of Bactcriology, University of Southern California, Los Angeles. EUGENE ROBERTS, Pn.D. Research Scientist, City of Hope Research Institute, Duarte, CA. BENSON B. ROE, M.D. Associate Professor of Surgery ; Chief, Cardiac Surgery. University of C'alifomia School of Medicine. San F'vancisco. JOSEPH li. ROGERS. M.D. Holy Name of Jesus Hospital. Gadsden, AL. JOHN A. ROSE('RANS, PH.D. Associate Professor of Pharmacology. Medical College of Virginia, Virginia Commonwealth University, Richmond. PETER M. ROSS, Pn.D. Research Associate, TheRockefeller Uni- versity. New York. JOHN R. ROWLANDS, Ptt.D. Staff Scientist, Southwest Research Insti- tute, San Antonio, TX. BENJAMIN A. RUBIN, PH.D. Assistant Professor of Public Health. Bay- lor University College of Medicine, Hous- ton, TX. RONALD P. RUBIN, Pn.D. Professor of Pharmacology, Medical Col- lege of Virginia, Richmond. HENRY I. RUSSEK, M.D. President, The Russek Foundation, Inc.. Staten Island. NY. W. C. RUSSELL. M.D. University of Texas Medical Center, Houston, T'X. UNA S. RYAN, Pu.D. Research Professor of Medicine, Univer- sity of Miami School of Medicine. Miami. FL. WAYNE L. RYAN, Pn.D. Professor of Biochemistry, University of Nebraska College of Medicine, Omaha. JEFFREY D. SAFFER, Pn.D. Associate Staff Scientist, The Jackson Laboratory, Bar Harbor, ME. REGINA M. SANTELLA, PH.D. Associate Professor of Medicine and Envi- ronmental Sciences, Columbia Univer- sity, New York. BRAHMI P. SANI, PH.D. Head, Protein Biochemistry, Southern Re- search Institute, Birmingham, AL. GORDON H. SATO. Pn.D. Director, W. Alton Jones Cell Science Center, Lake Placid, NY. JEROME SCHAACK, PH.D. Assistant Professor, University of Colorado Health Sciences Center, Denver. ULRICH H. SCHAEPPI, M.D. Director of Neuropharmacology, Mason Research Institute, Worcester, MA. CHARLES D. SCHER, M.D. Professor of Pediatrics and Human Genetics, Joseph Stokes Research Institute, Children's Hospital of Philadelphia. JORGEN U. SCHLEGEL. M.D., Pn.D. Professor and Chairman, Department of Surgery, Tulane University School of Medicine, New Orleans, LA. ALVIN R. SCHMIDT, PH.D. Director of Counseling, Tufts University, Medford, MA. JAKOB SCHMIDT, M.D., PH.D. Assistant Professor of Biochemistry, Divi- sion of Biological Sciences, State Univer- sity of New York at Stony Brook. H. WILLIAM SCHNAPER, M.D. Assistant Professor of Pediatrics, Wash- ington University School of Medicine, St. Louis. ROBERT E. SCOTT, M.D. H Professor of Pathology, University of H Tennessee, Memphis. ~ SCRIPPS CLINIC AND RESEARCH U FOUNDATION La Jolla. CA. MAURICE S. SEGAL, M.D. Clin'tcal Professor of Medicine, Tufts Uni- versity School of Medicine; Director, De- partment of Inhalation Therapy, Boston City Hospital. CARL C. SELTZER, Prr.D. Honorary Research Associate, Peabody Museum, Harvard University, Cam- bridge, MA. HENRY SERSHEN, PH.D. Research Scientist IV, Neurochemistry Division, Nathan S. Kline Research Insti- tute, Ward's Island, New York. LUCIO SEVERI, M.D. Director and Dean, Institute of Anatomy and Pathology, Division of Cancer Re- search, University of Perugia, Perugia, Italy. CHARLES R. SHAW, M.D. Chief, Section of Medical Genetics, M.D. Anderson Hospital and Tumor Institute; Professor of Biology, The University of Texas at Houston. JERRY W. SHAY, PH.D. Associate Professor, University of Texas Health Science Center, Dallas. ROBERT C. ROSAN. M.D. Associate Professor of Pathology and Pe- diatrics, St. Louis University School of Medicine; Associate Pathologist, Cardinal Glennon Memorial Hospital for Children, St. Louis. PETER F. SALISBURY, M.D., Ptt.D. Ifead, Intensive Treatment Center, Saint Joseph Hospital, Burbank, CA. PAUL SALTMAN, Pn.D. Assistant Professor of Biochemistry and Nutrition, University of Southern Califor- nia School of Medicine, Los Angeles. CHARLES E. SHERWOOD, M.D. Assistant Professor of Radiology, Univer- sity of Rochester School of Medicine and Dentistry, Rochester, NY. ISAAC SCHOUR, D.D.S., Pn.D., D.Sc. SHOJI SHIBATA, M.D., PH.D. Dean, University of Illinois College of Professor of Pharmacology, University of Dentistry, Chicago. Hawaii School of Medicine. Honolulu. CHARLES H. SCOGGIN, M.D. GERALD SHKLAR, D.D.S. Head, Division of Clinical Applications; Charles A. Brackett Professor of Oral Pa- Associate Professor of Medicine, Univer- thology; Head Department of Oral Medi- sity of Colorado Health Sciences Center, cine and Oral Pathology. Harvard School Denver. of Dental Medicine, Boston. CHARLES L. ROSE, Pn.D. Clinic Director; Director, Normative Ag- BARBARA M. SANBORN, Pn.D. ing Study, Veterans Administration Out- Professor University of Texas Medical patient Clinic, Boston. School, Houston. 282 1 293
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- N r-t rr) O 0 DAVID L. SIMON. M.D. SHELDON C. SOMMERS, M.D. 00 DANIEL STEINBERG, M.D. P".D. LYNN M. TAUSSIG, M.D. O Cincinnati Instructor in Internal Medicine Director of Laboratories, Leonx Hill Hos- 0 Professor of Medicine, University of Cali- Associate Professor and Associate Chair- , (:cneral Hospital, Cincinnati. OH. pital; Clinical Professor of Pathology, Co- lumbia University College of Physicians & ~ ~ fornia School of Medicine. San Diego. La Jolla. man. Department of Pediatrics, Arizona Health Sciences Center. Tucson. H H ERIK SKINHI6J, M.D. Chief. Department of Neurology, Bispeb- Surgeons, New York. 1p1~ ® JACK P. STRONG, M.D JOSEPH CHARLES TAYLOR, PH.D. a O jerg Hospital, Copenhagen, Denmark. ROBERT J. SKLAREW, P".D. ERNEST SONDHEIMER, PH.D. Distinguished Service Professor of Zool- ogy, Indiana University, Bloomington. ~ Associate Professor of Pathology, Louisi- ana State University School of Medicine, New Orleans. Associate Research Scientist. City of Hope Research Institute, Duarte, CA. U Research Associate Professor of Pathol- New York University Research Ser- ogy ~ A. DONNY STROSBERG, PH.D. MARC D. THAMES M.D. Senior Research Fellow Ma o Clinic and , vice, Goldwater Memorial Hospital. Roosevelt Island. NY. NATHAN H. SLOANE, Pti.D. Professor of Biochemistry, The University of Tennessee Center for the Health Sci- ences, Memphis. HANOCH SLOR, Ptt.D. Associate Professor of Human Genetics, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel. THEODORF, A. SLOTKIN. Pn.D. Assistant Professor of Pharmacology, Duke University Medical Center. Dur- ham. NC. GEORGE W. SMF.TTERS. M.D. Associate in Pathotogy, Northwestern University Medical School, Chicago. DENNIS M. SMITH, Pn.D. Assistant Professor of Biological Sci- ences. Wellesley College, Wcllesley, MA. GENF. M. SMITH, Pn.D. Assistant Professor of Psychology, Har- vard Medical School. Massachusetts Gen- cral Hospital, Boston. KENDALL A. SMITH. M.D. Professor of Medicine, Dartmouth Medical School, Hanover, NH. LUCILE SMITH, Pn.D. Professor of Biochemistry. Dartmouth Medical School, Hanover, NH. I.OUIS A. SOLOFF, M.D. Blanche 1'. Levy Distinguished Service Prnfessor; Professor of Medicine; Direc- tor, Research Lipid I,atxttatory, Temple University Hcalth Sciences ('enter, Phila- delphia. T. M. SONNEBORN, PH.D. Distinguished Service Professor of Zoology, Indiana University, Bloomington. ASHWANI K. SOOD, P".D. Cancer Research Scientist 111, Roswell Park Memorial Institute, Buffalo, NY. MOHAN SOPORI, Ptt.D. Scientist 11, Lovelance Medical Founda- tion. Albuquerque, NM. GEORGE D. SORENSON, M.D. Professor of Pathology, Dartmouth Medical School, Hanover, NH. SAM SOROF. P".D. Head, Department of Macromolecular Chemistry, The Institute for Cancer Re- search, Philadelphia. SOUTHWEST RESEARCH INSTITUTE San Antonio,TX. DAVID M. SPAIN, M.D. Director, Department of Pathology, The Brookdale Hospital Center, Brooklyn, NY. ALEXANDER SPOCK, M.D. Assistant Professor of Pediatrics. Duke University Medical Center, Durham, NC. TIMOTHY A. SPRINGER, Pn.D. Senior Investigator and Vice President, The Center for Blood Research. Boston. FREDERICK J. STARE, M.D. Professor of Nutrition, Harvard University School of Public Health, Boston. NORMAN C. STAUB, M.D. Professor of Physiology Cardiovascular Research Institute, University of Califor- nia, San Francisco. ~ ChairProfessor of Biochemistry and Immu- nology, Pasteur Institute, Paris, France. ARION B. SULZBERGER, M.D. Professor and Chatrman, Department of Dermatology and Syphilology, New York University-Bellevue Medical Center. MARY E. SUNDAY, M.D.. PH.D. Assistant Professor of Pathology, Brigham and Women's Hospital, Boston. JOHM P. SUNDBERG, D.V.M., PH.D. Staff Scientist, The Jackson Laboratory, Bar Harbor, ME. RAQUEL SUSSMAN, PH.D. Associate Scientist, Marine Biological Laboratory, Woods Hole, MA. TORGNY H. SVENSSON. M.D. Professor. Karolinska Institute, Stock- holm. Sweden. RENATO TAGIURI, PH.D. Associate Professor of Psychology. Grad- uate School of Business Administration, Harvard University, Boston. MAKOTO TAKETO, M.D.. PH.D. Associate Investigator, The McLaughlin Research Institute, Great Falls, MT. DAVID W. TALMAGE, M.D. Director, Webb-Waring Lung Institute, University of Colorado Medical Center, Denver. JANET TANNENBAUM, Pn.D. Assistant Professor of Pathology, Colum- bia University College of Physicians & Surgeons, New York., , y Foundation, Rochester, MN. CAROLINE BEDELL THOMAS. M.D. Professor Emeritus of Medicine. The Johns Hopkins University School of M®di- cine, Baltimon:, MD. JEROME F. THOMAS, Ptt.D. Professor of Sanitary Engineering. Uni- versity of Califomia, Berkeley. JOHN A. THOMPSON, PH.D. Associate Professor of Pharmaceutical Chemistry, University of Colorado School of Pharmacy, Boulder. JAMES E. P. TOMAN, Pn.D. Professor and Chairman, Department of Pharmacology, Chicago Medical School, Institute for Medical Research. ANDREW M. TOMETSKO, PH.D. President and Director of Research, Litron Laboratories, Ltd.. Rochester, NY. ANNAMARIA TORRIANI-GORINI, Ptt.D. Professor of Biology, Massachusetts Institute of Technology, Cambridge. JANET TRAVELL, M.D. Associate Professor of Clinical Pharma- cology. Cornell University Medical Col- lege, New York. JAMES TRAVIS, P".D. Professor of Biochemistry, The University of Georgia, Athens. WAYNE M. TREBBIN, M.D. Associate Program Director of Internal Medicine Residency, Salem Hospital. Sa- tem. MA. 284 295
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LIE SHA TSAI. PH.D. ELLIOT S. VESELL, M.D. LEE W. WATTENBERG M.D RUSSELL W. WELLER, M.D. Research Associate in Pathology, Yale Professor and Chairman, Department of , . Professor of Pathology University of Pathologist, Memorial Hospital of Chester tlniversily School of Medicine, New Lla- Pharmacology. Pennsylvania State , Minnesota Medical School. Minneapolis. County, West Chester, PA. ven. CT. University College of Medicine, Milton S. Hershey Medical Center Hershey , . JOHN S. WAUGH, PH.D. MICHAEL l: WELSH, M.D. BEN Y. TSENG. Pn.D. Professor of Chemistry, Massachusetts In- Univer- Assistant Professor of Medicine Associate Adjunct Professor. University of BRANISLAV VIDIC, D.S. stitute of Technology, Cambridge. , Iowa sity of Iowa College of Medicine California, San Diego, La Jolla. Professor of Anatomy, Georgetown Uni- , City. versity Schools of Medicine and Dentistry, Washington, D.C. THOMAS E. WEBB, Ptr.D. GERALD M. TURINO, M.D. Professor of Physiological Chemistry, A. STANLEY WELTMAN, PH.D. Professor of Medicine Columbia Univer- Ohio State University College of Medi- ciat Prof ssor of Pharmacolo and A , ROMEO A. VIDONE, M.D. cine Columbus gy sso e e sity College of Physicians & Surgeons, Associate Professor of Pathology, Yale , . Research, Brooklyn College of Pharmacy, New York. University School of Medicine, New Ha- Brooklyn, NY. ven, CT. MICHAEL 1. WEBER, PH.D. JOHN TURK, M.D. Professor of Microbiology. University of PH.D. SIMON H WENDER Assistant Professor of Medicine and Pathol- ZBIGNIEW WALASZEK, PH.D. Virginia Medical Center, Charlottesville. , . Research Professor of Biochemistry, Uni- ogy, Washington Univer;ity, St. Louis. Research Scientist, Ohio State University. vetsity of Oklahoma, Norman. Columbus . RICHARD L. WECHSLER, M.D. D M TURNER Pn D Clinical Physiologist, Montefiore Hospital X . . , . . EVELYN WALDSTEIN, PH.D. Institute of Research Pittsburgh KE WENNMALM, M.D. Head. Department of Drug Metabolism Senior Lecturer, Department of Biochem- . . Professor and Chairrnan, Department of and Biochemistry, Hazleton Laboratories Tel Aviv University. Tel Aviv, Is- istry Clinical Physiology, University of Europe. Ltd., Harrogate, Yorkshire, En- , rael. JOHN V. WEIL, M.D. Gothenburg, Gothenburg, Sweden. gland. Assistant Professor of Medicine. Univer- PETER N. WALSH, Pn.D. sity of Colorado Medical Center, Denver. DUANE G. WENZEL, PH.D. EMIL R. UNANUE. M.D. Professor of Medicine, Temple University Professor and Chairman, Department of Chairman and Professor. Department of School of Medicine, Philadelphia. GEORGE WEINBAUM, Ptt.D. Pharmacology and Toxicology, The Uni- Pathology, Washington University School Assistant Chairman for Rresearch, Depart- versity of Kansas School of Pharmacy. of Medicine, St. Louis. IRENE Y. WANG, Pn.D. ment of Medicine, The Graduate Hospital, Lawrence. Assistant Professor of Basic and Clinical Philadelphia. Medical Immunology and Microbiology UNION CARBIDE CORPORATION , University of South Carolina Charleston THOMAS C. WESTFALL, PH.D Nuclear Division, Oak Ridge. TN. . , 1. BERNARD WEINSTEIN, M.D. Professor of Pharmacology, University of Professor of Medicine and Environmental Charlottes- Virginia School of Medicine PETER A. WARD. M.D. Sciences, Columbia University, College of , ville UNIVERSITY OF SAN FRANCISCO Professor and Chairman, Department of New York. Physicians & Surgeons . Pathology, The University of Michigan, , Ann Arbor. UNIVERSITY OF SOUTHERN CALIFOR- A. WEINSTOCK PH.D FREDERICK E. WHISKIN, M.D.C.M. d P l l h f NIA , Life Sciences Divi- mist R h Bi ch ereona - Hea t an Director, Division o Los Angeles. E. D. WARNER, M.D. Professor of Pathology, State University of esearc o e , sion, IIT Research Institute, Chicago. ity Equilibrium. The Age Center of New England, lrrc., Boston. Iowa College of Medicine, Iowa City. HELEN VAN VUNAKIS D Pn , . . Professor of Biochemistry, Brandeis Uni- SHIELDS WARREN M D JUDITH WEISZ, M.D. ALEXANDER S. WHITEHEAD, D.Pntt. versity, Waltham. MA. , . . Director of Laboratories, Cancer Research Professor of Obstetrics and Gynecology, Milton S. Hershey Medical Center, Assistant Professor of Pediatrics, Chil- Institute, New England Deaconess Hospi- Pennsylvania State University, Hershey. dren's Hospital, Boston. HAROLD E. VARMUS, M.D. tal, Boston. Professor of Microbiology and Immunol- V PH JAMES A WILL D M D San Fran- ogy University of California YASUSHI WATANABE, PH.D. SIGMUND A. WEITZMAN, M.D. . . . , . ., . , , cisco Associate Member The Wislar Institute of I Assistant Professor of Medicine, Professor and Chairman, Department of . , Anatomy and Biology Philadelphia Hematology-Oncology Unit, Massachu- Veterinary Science, University of Wiecon- . . setts General Hospital, Boston. sin, Madison. STEPHEN F. VATNER, M.D. Associate Professor of Medicine, Harvard BARBARA K. WATSON, Pn.D. Medical School, New England Regional Assistant Bacteriologist, Massachusetts HOWARD G. WELGUS, M.D. ROGER J. WILLIAMS, M.D. Primate Research Center. Southhoro, MA.: General Hospital; Research Associate in Assistant Professor of Medicine, Jewish Professor of Chemistry; Director, Clayton Associate in Medicine, Peter Bent Brigham Bacteriology and Immunology, Harvard Hospital at Washington University Medi- Foundation Biochemical Institute, The Hospital, Boston. Medical School, Boston. cal Center, St. Louis. University of Texas. Austin. 287 286 j
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JOHN T. WII SON, Pn.D. Assistant Professor of Cell and Molecular Biology, Medical College of Georgia, Au- gusta. ALVIN L. WINTERS, Pn.D. Assistant Professor of Microbiology and Biocemistry, The University of Ala- bama, University. DANIEL H. WISEMAN, M.D. Assistant Professor of Pediatrics. Univer- sity of Southern California; Children's Di- vision, Los Angeles County General Hos- pital, Los Angeles. BRUCE A. WODA, M.D. Associate Professor of Pathology. Univer- sity of Massachu.setts, Worcester. GEORGE WOLF, D.Pnn.. Professor of Physiological Chemistry. De- partment of Nutrition and Food Science. Massachusetts Institute of Technology, ('anihridge. DEAN F. WONG, M.D. Assistant Professor. Johns HoPkins Hospi- tal, Baltimore, MD. PAUL V. WOOLEY. 111, M.D. Professor of Medicine and Pharmacology. Georgetown University Medical Center. Washington, DC. THOMAS C. WRIGHT,IR., M.D. Assistant Professor of Pathology. Columbia University College of Physicians & Surgeons, New York. JOHN P. WYATT, M.D. Professor of Pathology, St. Louis Univer- sity School of Medicine. ROBERT L. WYKLE, PH.D. Professor of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winstnn-Salem, NC. STANLEY YACHNIN, M.D. Professor of Medicine and Chief. Section of Hemato4ogy/Oncology, The University of Chicago Medical Center. KOJI YOSHINAGA, Pn.D L F.DWIN WOOD, M.I). Center for Population Research, NICHD. Instructor in Medicine. Boston University National Institutes (if Health, Bethesda, School of Medicine. MD_ SUMNER WOOD.1R., M.f). Assistant Prufcssor of Pathology, The IX)NALD A. YOUNG, M.D. Johns Hopkins University School of Medi- Professor of Medicine. University of cinc, Baltimnre, MI). Rochester, Rorchester, NY. INDEX OF PRINCIPAL INVESTIGATORS AMrud, S. S., 61, 62 Aik;nde, l. E., 107-109 Anderson, R. A., 110 Atxkrson, S. M., 109 Anlotmadea,H. N.,21-23,47,48,110 Axe}rod, D. E.,111,112 BaBehi, S., 112 B1nlny, M., 97 Batrtea, D. W.,113 Baylin, S. B., 24-26 Befus, A. D., 48-50, 62, 210 Btamatt, J. S., 63.64 BemK.u, K. L.,114 Btxrqmda, A.,11S Beutter, E.,115 Bina, R. J., 65,116 Biahq+ee, S., 116 Bla{odc, J. E., 211 Bone, C. A., 211-213 Broku, T.1t.,117,118, 213 CamPbeB, E. J.. 50-52 Ca4xa, l. D., 214-218 Chmt, J. C., 118, 119 Chan, L, 66 Chqmat, H. A., h., 53, 56,120 Chevion, M., 67-69,120,121 Christianson, D. W., 122 Chuong, C: M., 122, 123 Cohen, M. S., 124 Cohen. S., 69 Costa, R. H.,123 Creutz, C. E.,125,126 Davitz, M. A.,126 Enrietoo, P. J.,127 Feraanisco, J. R., 27,128 F'rsher, E. A., 70 Foster, D. A., 128-131 Frrmzuaoff, A., 131,132 Freeman, B. A., 70 Fridovich,1.,132-137 Fryer, A. D., 54 Giam, C: Z., 138,219 Goldberg,l. J., 71-73 Heapel, P. C., 75.76 Helfman, D. M., 148 Hogg,1. L., 55 Hung, M.-C., 30 Jaken, S., 31,149,150 Jeali& K T.,151, 220, 221 lefoottle, C. R., 31 )efJaiaa, W, A,, 221 JestZtit, A. J.,151 Jolwat, l. P., 77 Kadotr,8a, J. T., 152 Kao, F: T:,153 Karimt, M„ 32 ICarmordry, M, L, 222 Kmair, Y.,133 Kenlbr, A. L.,154 Klndy, M, S., 78,79 Kmiec, 8,13.,155 Koppettal, W. H.,135,156 Kriaftm S.1L, 79, 80 Kuc1Ma, R, D.,156,157 Kuplec, M,,158 La9, E.,158 LajrttH, A., 97, 98,106 Lanier, S. M., 159-161 Lee, W: H., 33,162 Leigfitar, J.,162 Lennon, V. A., 33 Le Queene, P. W., 163 Leviat, E. D., 98-100 Lieber, M. R., 222 Lindatrom, J., 100-103 Lipsict,l. S., 34, 35,163 Landbers, J. M., 55.96 Lynch, H. T., 36 Maecioni, R. B.,164,165 Marfn, 0. S., 36, 37 Maron, R. W., 56,165,166 R G 38 11ysKimell . ., , Il k ~dtoe8hini, R.,166,167 Gonias, S. L., 73, 74,138-140 Great, K. l., 141-143 Greene, M.1., 144, 219, 235 Hagerman, P. J.,145 Hajjar, K. A., 75 Hanafusa, H., 27,145-147 Hankinson, 0., 28, 29, 147 Hansen, U.. 148 Mouoioe, J. G., 223 Mufson, R. A.,167, 224 Murlas, C. G., 57, 58 Naltm, M. H., 225-227 Nelson, D. J., 168,169 Neufeld, G.,103, 169 Nordeen, S. K., 170 Olson, E. N., 171, 172 Oparil, S., 81-84 Osawa, Y., 172 Ou, J.-H., 173 Palam, R. E.174, PecM, L, 174-176,228,229 Polak, J. M., 59, 84, 85,177 Raz, A., 60, 84, 85,177 Raziat, A., 178 Reed, J. C.,178 Reid, L. M„ 38,179, 180 Reldy, M. A„ 86•89 Rhuadt, R, E„ 39,181,182 Ritii®o, A., 60,182,183 Robimroo, J. M.,184 Raaem, 0. M,,124,185 Raeatdml, N.,186 Rotltpe6t, T. L., 230 Rb61n, H„ 40-42,186 Satt:ar, A„ 187,188 9unoM, P.,188,189, 230 Sarloon, D. A.,189,190 S1rpp, B. V. R.,104, 105 Sluhbitx, W., 38 Sooa, D. W., 231 Stxehen, H.,106 Sara+o, G„ 191-193 Sipl, E., 89, 90 Silver, P. A., 193 Sit6uku, D. A.,194,195 Smith, F.1.,198 Smidt, S. S., 196,197 Sulatton, A. K. 91 Sood, A. K., 232 Spkdel, E. R.,198 Stanlxidae, E. l., 43 Sfan, D., 92, 93,199 Strard, F. L, 200 Stuetr, D.1., 201 Sndal, M., 201 Svatsaon, T. H., 107 Taketo, M., 202 Taylor, K. M., 94 Ttxrartova, V. P., 44 Thornaa, M. L., 233 Van Dyke, M. W., 203 ViIlrKomwoN, L., 203,204 WaBtter, R. R., 204 Waldaclnnidt, T.1., 233, 234 Weiner, D. B., 45, 46,144, 219, 235 Wemtmalm, A., 95, 96 Wu, J. M., 205 Wu, R., 205,206 Wysocki, L l., 236 Yen, A., 207-209, 237 288

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