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NYSA CTR 1

Sincerely

Date: 04 Aug 1992
Length: 28 pages

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nysa_ctr1 50610061-50610088

Abstract

Enclosed please find a brief narrative summary of our grant proposal, curriculum vitae of the co-investigators, and relevant publications.

Fields

Named Organization
Council for Tobacco Research - USA (CTR) (Formerly Tobacco Industry Research Committee (TIRC))
Originally organized as the Tobacco Industry Research Committe(TIRC) in 1954, and renamed Council for Tobacco Research - USA, Inc. (CTR) in 1964.
Harvard Medical School
Illinois College
Massachusetts General Hospital
Memorial Hospital
National Institutes of Health (NIH)
Northwestern University
Plenum Press
Red Cross
University of Chicago
University of Chicago Hospitals and Clinics
University of Cincinnati
University of Illinois (at Champaign-Urbana)
University of Minnesota
Wayne State University
Western Reserve (Medical School)
Named Person
Cordon, Leo I.
Eisenberg, Arthur D., Ph.D. (CTR Assoc. Research Director 1991, Asst. Secretary 1997)
Defense
Gordon, David L. (BW RD&E Project Planning Manager 1985)
Defense
Gordon, Leo I.
Li, Gordon
Reese, Michael
Res, Michael Reese
Robbins, Kenneth C.
Roe, Von
Schmeichel, Cynthia
Sharp, Elwood A.
Weiss, David
Williams, R. W. J. (Rothmans Research Director)
Type
Letter
Date Loaded
11 Jan 2006
Box
0240

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Page 1: 50610061
Nt~rlh,¢~c.,,tcrn U.nivcr.Mt.v Mcdic;d ~cht.~i August 4, 1992 Arthur D. Eisenberg, Ph.D. The Council For Tobacco Research-U.S.A., Inc. 900 Third Avenue New York, NY 1022 Dear Eisenberg: Enclosed please find a brief narrative summary of our grant proposal, curriculum vitae of the co-investigators, and relevant publications. We look forward to hearing whether this proposal meets your qualifications for a full application. Sincerely, lilt
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Title. Purification and cloning of soluble phosphatidylcholine-preferring phospholipase C Introduction, The phospholipase C OaLC) isoenzymes have been found to be present in many ma._mmalian cells and in other organisms (1). The phosphatidylinositol-specific isoenzymes of PI_.C (PI-PLC) are critical components of the signal transduction system in which extracellular signals are transmitted across the cell membrane by mechanisms that utilize second messenger molecules (2). Among these molecules axe diacylglycerol (DAG) and inositol phosphates, derived from inositol lipids by the action of membrane PI-PLC (3). This enzyme h.as been well characterized, purified to homogeneity and recently cloned from mammalian species. Recent data have suggested, however, that at least some of the DAG involved in signal transduction comes from the hydrolysis of phosphatidylcholine (PC) rather than phosphatidylinositol (PI), and therefore from the action of another PLC with specificity for PC (PC-PLC) (4-7). We have shown that this enzyme is present at extracellular sites (8), can be isolated from sonicates of human smooth muscle cells, and may influence events associated with the inflammatory response (9). Thus, we believe that the isolation, characterization and cloning of PC-PLC is an important objective, and it will be the major aim of this proposal. We believe that the purification and cloning of an endogenous enzyme which may have anti- inflammatory properties is important, since recent data suggest that tobacco related carcinogenesis may be influenced by inflammatory mediators. Aims. We propose to 1) purify and clone soluble PC-PLC from human smooth muscle cells using affinity chromatography techniques and oligonucleotide probes, and 2) to characterize soluble PC-PLC with respect to its protein characteristics and enzymatic activity. Background and Preliminary Data. There is now considerable interest in the mechanisms involved in signal transduction, and central to this question is the role played by membrane or cytosolic PLC. Many agonists exert their effects through G-protein mediated stimulation of PLC activity, leading to the production of DAG and inositol phosphates (1,2). This has led to attempts to purify PLC, and recently the purification and cloning of several forms of PI-PLC has been described (10-15). More recently, there are data which suggest that at least a portion of the DAG mass derives from phosphatidylcholine, and hence from the action of a PC-PLC. These data come from Extort (16) in studies examining the fatty acid composition of the mass of DAG, from Boeekino (17) in hepatoeytes stimulated with various agonists, and from Besterman (4) in the pre-adipocytic cell line 3T3-L1 stimulated with phorbol esters and platelet derived growth factor (PDGF). All of these investigators have found that at least some of the DAG mass is derived from hydrolysis of phosphatidylcholine. Perhaps more intriguing is the observation by Lacal (18) that the p21 proteins encoded by ras genes result in an increase in DAG mass in transformed 3T3 cells, and that this derives from choline lipids rather than inositol iipids. All of the~ data argue for the importanoe of a phosphatidyleholine cycle in addition to an inositol cycle in signal transdw~dion events, and thus implicate PC-PLC as an important mediator in biological systems. An example of this has been our own observation that bact~ially derived PC-PLC can inhibit the respiratory burst in human neutrophils (9), and that ~ effect is accompanied by aite~ti~o~s i~ calcium flux in the cells. Our data suggest that 5061(X)62
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effects of PC-PLC are on the membrane components of the isolated NADPH oxidase. We found, in a cell free syst~a, that incubation of the purified membrane component of the oxidase (but not its cytosolic component) with PC-PLC inhibited arachidonate mediated activation of the oxidase. This was accompanied by a time dependent depletion of phosphatidylcholine as measured by thin layer chromatography. Indeed, the inhibitory effect of PLC on the oxidase could be reverr, ed by the addition of exogenous phosphatidylcholine. The inhibitory effect could also be reversed by addition of exogenotts phosphatidic acid (a product of phospholipase D hydrolysis of PC) directly to the inactivated membrane component. In order to determine if this enzyme is present at inflammatory sites, we assayed supematants of a variety of cells and cell lines that might be present at inflammatory sites. We found that PC-PLC activity could be found in the supernatants of maerophages, fibroblasts and human smooth muscle cells (8), and more recently we found that it could be found in larger quantities in sonicates of smooth muscle cells. Our preliminary data show that this enzyme activity can be separated by ion exchange and affinity columns, and that lysates can be pooled and stored frozen in order to obtain enough enzyme to carry out purification studies. Methods. In all experiments, PC-PLC activity will be screened using a colorimetric assay (alkaline phosphatase assay) or turbidity assay. Final activity will be measured using radiolabeled substrate (PC, PI, phosphatidylserine [PS], phosphatidylethanolamine [PE]) to measure hydrolysis by the enzyme. Purification of smooth muscle cell lysates will be carried out by first separating proteins on an ion exchange column. Our preliminary data suggest that most of the PC-PLC activity can be eluted with 1 M NaCI. After several purification steps, affinity columns using PC, P1, PE or PS linked to Sepharose beads will be constructed and the lysate eluted with the appropriate salt concentration. From our preliminary data, we know that the affinity purification is possible because under the conditions of our assay the Sepharose bound ligands are not hydrolyzed by the enzyme. We will select that activity(ies) which selectively bind to phosphatidylcholine. When sufficient amounts of enzyme are sequentially purified, a partial sequence will be obtained at the Northwestern University Biotechnology facility. We anticipate, that since we are dealing with a secretory protein, the amino terminus is not blocked. If it is blocked, an alternate method utilizing in situ trypsinization to generate tryptic fragments will be utilized. These fragments, separated by HPLC, will then be sequenced. Once the partial sequence is determined, oligonucleotides will be prepared. These will then be used to screen a poly A selected eDNA library obtained from the RNA of human fibroblasts. Hybrids that are generated will be detected, plaque purified and expanded by standard techniques utilizing lambda gt 11 phage infecting E Coli. These expanded cDNA's will then be used to express PC-PLC. In order to characterize the purified enzyme, we will measure effects of pH, substrate- concentration-velocity relationships, PLC stoichiometry, and the effects of cations and detergents on PC-PLC activity. We anticipate that these studies as proposed will take three years to complete, and that the yearly direct cost e~tiraal~ will be approximately $.55,000.
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References 1. Rhoe SG et al. Science 244:546-550, 1989. 2. Majerus PW et al. Science 234:1519-1526, 1986. 3. Berridge lvIJ. Bioehem J 220:345-360, 1984. 4. Besterman JM et al. PNAS (USA) 83:6785-6789, 1986. 5. Grillone LR et al. J Biol Chem 2632658-2663, 1988. 6. Irving HR and Exton JFI. J Biol Chem 262:3440-3443, 1987. 7. Daniel LW et al. J Biol Chem 261:9128-9132, 1986. 8. Gutstein D et al Blood Suppl 76:182a, 1990. 9. Gordon LI et al. Cellular Immunology 128:503-515, 1990. 10. Ryu SH et al Biochem Biophys Res Comm 141:137-144, 1986. 11. Ryu SH et al. J Biol Chem 262:12511-12518, 1987. 12. Ryu SH et al. PNAS (USA) 84:6649-6653, 13. Suh PG et al. Cell 54: 161-169, 1988. 14. Homma Y et al. J Biol Chem 263:6592-6598, 1988. 15. Banno Yet al. Bioehem and Biophys Res Comm 167:396-401, 1990. 16. Exton JH. Cell Membranes: Methods and Reviews Vol. 3 New York, Plenum Press p 113- 181, 1987. 17. Bocckino SB et al J Biol Chem 260:14201-14207, 1985. 18. Laeal JC et al. Nature 330:269-272, 1987.
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. FF P~o~ ~mz/~jmm D~o~ (~. ~. m~): Dr. Leo I. Gordon SKETCH NAME POSITION TITLE Leo I. Gordon, H.D. Associate Pro£essor INSTITUTION ANO LO~TtON DEGREE CONFERRED FIELD OF STUDY University of Chicago, Chicago, IL B.A. 1969 Biology University of Cincinnati, Cincinnati, OH M.D. 1973 Medicine RESEARCH AN~ PROFES.SWJNAL EXPERIENCE: Corclu<lic~ with present position, list, ~ chronological or, tier, pc~ious employment, exl~rience, and honors. Key pe~,.~:)nnel include tim principal investigator and any other individuals who participate in the scientific development or exe,'-..ution of the project. Key I>ersonnel typically ~ include all i¢~vid~els wi~ doctoral or other Wofe~onal degrees, but in some projects will include individuals at the masters or I:~ce.aJaureate ler~el pcovided they contdtluta in a substar~ way to the scientific developmec~t o¢ ex, eoaliOn of the project. Irclude present membership on any Fecleral Government public aclviso~y committee. List, in chronological order, the titles, all author, and comp4ete references to all pubhcations during the past three years arid to representative e~rlier pubi~:ations pedinent to Ihis application. DO NOT EXCEED TWO PAGES. profe~.sional Expe~'ience 1990- 1985- 1984-87 1983- 1986- 1982-1986 1979-82 1979-85 Assistant Dean for Graduate and Continuing Medical Education Associate Professor of Medicine, Northwestern University Medical School Board of Trustees, Northwestern Medical Faculty Foundation Director, Joint Fellowship Training Program in Hematology/Ontology Attending Physician, Northwestern Memorial Hospital Associate Attending Physician, Northwestern Memorial Hospital Adjunct Attending Physician, Northwestern Memorial Hospital Assistant Professor of Medicine, Northwestern University Medical School postcloctor~l Training 1973-1974 1974-1975 1975-1976 1976-1978 1978-1979 Intern in Internal Medicine, University of Chicago Hospitals and Clinics Junior Assistant Resident in Medicine, University of Chicago Hospitals and Clinics Senior Assistant Resident in Medicine, University of Chicago Hospitals and Clinics Fellow in Hematology, University of Minnesota Fellow in Hematology/Ontology, University of Chicago Hospitals and Clinics Selected P~tblications Gordon LI, Douglas SD, Kay NE, Yamada O, Osserman EF and HS Jacob: Modulation of neutrophil function by lysozyme: A potential negative feedback system of inflammation. ~ 64:226, 1979. Yaehnin S, Streuli R, Gordon LI, R Hsu: Alteration of peripheral blood cell membrane function and mothology by oxygenated sterols: A membrane insertion hypothesis. Current Top.ies in He~aatol 2:245-27 t, 1979. Gordon LI, Miller WJ, Branda RF, Zanjani ES and HS Jacob: Regulation of erythroid colony formation by bone marrow maerophages. ~ 55:1047-1050, 1980. Gordon L[, Bass J, and S Yachain: Modulation of polymorphonuclear leukocyte (PMN) function by oxygenated sterol compounds (OSCs). ~oc Had Acad Sci (USA) 77:4313-4316, 1980. Gordon LI, Douglas SD, Kay lqE, lacob HS, and $iltzbach LE: Inhibition of ueutrophil (I>MN) migration by sa~-~d sera with partial rever*,al by the tri~ccharide of N-acetyl glucof, amine (NA, GI,~b, a lycot3tme iallibitor, in: Williams W.B. and Davies BJ. (edit) 1~¢~-__~41 _a~t of the 8th lm~.aatimml C.onfefeace on FF 5061O065
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Gordon 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. Gordon Li, Douglass SD, Kay NE, Yamada O, Osserman EF, HS Jacob: Modulation of neutrophil (PMN) 6ruction by iysozyme, a macrophage secretory product. Williams WJ and Davies BH (eds) ]~g~gIJ~gL~" the 8th International Conference on Sarcoidosis and other Granulomatous Diseases. London: Alpha Omega Publishing, Ltd., 19g0:76-83. Rosen ST, Wittlin Flq, Epstein AL, Gordon LI, Kies MS, Kucuk O, Kwaan HC, Winter JN, Vriesendrop HM, A Molteni: Estrogen receptor analysis in chronic lymphocytic leukemia. Blood 62:996-999, 1983. Larson R., Gordon LI, and S Yachnin: Neutrophil assisted DNA synthesis by human lymphocytes in response to mevalonic acid: Enhancement by eytochalasin B. Cell lmm..unol 81:357-372, 1983. Kucuk O, Gordon, Kies MS, Spi~ S, Spies W, and HM Vrieseadrop: Estimation of hemopoietic potential by CFUc and bone marrow scan in cancer patients. EXp Hem.atol0gy 12:101-106, 1984. Ueshima Y, Rowley JD, Variakojis D, Winter J and Gordon LI: Cytogenetic studies in patients with chronic T cell leukemia/lymphoma. Blood 63:1028-1038, 1984. Gordon LI, Rosen ST, Vriesendrop HM, Kies MS, O Kucuk: Separation of clonogenic tumor cells from small cell lung cancer bone marrow and small cell lung cancer cell lines: Implications for autologous bone marrow transplantation. Cancer R~earch 44:5404-5408, 1984. Rosen ST, Zimmer AM, Goldman-Leikin R, Gordon LI, Kazikeiwicz JM, Variakojis D, Marder RS, Dykiewicz M, Grammer L, Blei AI, Piergies A, Silverstein A, goenigk H, and SM Spies: Radioimmunodetex.tion and radioimmunotherapy of cutaneous T-cell lymphoma utilizing ~3tl labeled monoclonal antibody. J CliaOnc 5(4):562-573, 1987. Kies MS, Gordon LI, Kucuk O, and HM Vriesendorp: Autologous bone marrow transplantation in breast cancer: Separation of clonogenic tumor cell colonie-s by gradient fractionation. Exp Hematology 16:190-194, 1988. Gordon LI, and SA Weitzman: The respiratory burst and carcinogenesis. In The Respiratory Burst, AJ Sbarra, (ed), Plenum Press, NY, 1988, pp. 277-298. Vasconcelles M, Weitzman SA, Lee SN, Prachand S, and Gordon LI: Inhibition of human polymorphonuclear leukocyte respiratory burst activity and aggregation by 6-ketocholestanoi. Free Red Res Comm 8(3): 185-193, 1990. Weitzman SA, and Gordon LI: Inflammation and Cancer: Role of phagocyte generated oxidants in carcinogenesis. Blood 76:655-663, 1990. Gordon LI, Schmeichel C, Prachand S, and Weitzman SA: Inhibition of polymorphonuclear leukocyte oxidative metabolism by exogenous phospholipase C. Cellular Immunology 128:503-515, 1990. Gordon LI, Weiss D, Prachand S, and Weitzman SA: Scavenging of superoxide anion by phosphorylethanolamine: Studies in human neutrophiis and in a cell free system. Free Red Comm 15(1):65- 71. 1991. McDunn S, Winter JN, Variakojis D, Rademaker A, Von Roenn J, Tallman M, Gordon LI, and Bauer K. AIDS-related lymphomas: Measurement of DNA-content and p105, a novel proliferation associated nuclear antigen, by dual parameter flow cytometric analysis. J Clin Oncol 9:1334-1340, 1991. Kucuk O, Stoner-Picking J, Yachnin S, Gordon LI, Williams RM, Lis L and Westerman MP: Inhibition of natural killer cell cytotoxicity by oxysterols. Cell lmmunol 139, 541-549, 1992. Gordon LI, Gutstein D, Prachand S, Weitzman SA. Constitutive extracellular release of phosphatidyl choline preferring phospholipase C (CPC-PLC) by human fibroblasts and smooth muscle cells, in "The Molecular Basis of Oxidative Damage by Leuckoyte, s," CRC Press, 1992, pp. 325-328. Gordon LI, Harrington D, Andersen J, Colgan J, Gliek J, Neiman R, Mann R, Resniek GD, Barcos M, Gotlieb A and O'Connell M. Randomized Phase 1II trial of CHOP vs m-BACOD in patients with advanced diffuse large cell and diffuse mixed non-Hodgkins lymphoma: An Eastern Cooperative Oneology Group (ECOG) and Cancer and Leukemia Group B (CALGB) study. New Ea_~land Journal of Medicine (in press 1992).
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BIOGRAPHICAL SKETCH Sigmund A. ;ei:zman, M.D. Principal Investigacor ,NSr:TUTION AND LCCAT,CN DEGRE~ CONFERRED FIELD OF STUOv Temple t'niversi~, Philadelphia, PA B.A. 1966 Biology Temple Universi=[~, Philadelphia, PA M.D. 1970 Medicine UniversiE~: of l-[ichigan, Arbor, Michigan Intern/ ~ Residen= 1970-1973 Internal >[edicine RESEARCH AND PRO;ESSIONAL EXPE!qlENCE. Conc~uaing '.~m oresent posdion, list. ~n c.~ronolo(J,Cal oKler. ~nors. Key ~r~ ,~:u~ the ~nc:Oal ~nvest~a~o~ and any otb~ ,~a~ ~o ~e Key ~onnei :y~lly w,d ,~lu~ all ~nO~v~als w,th aoc:o~l or Diner o~ies~l d~r~s, but oa~alauream ~evel ~ ~ conm~le ~n a suostant~ve way to :~e S~ ~e~men~ of ex~on of ~e p~. IncluGe Ofesent memDetsn:c any Federal Gouernmen~ ou~< a~so~ ~mm~ee L~SL m ChrOnOtOg~Ca~ o~er. ~e titles, all ~ul~. ~St th(ee years ann Io reore~nCatwe ear~,er publica¢~ons pe~men¢ :o th~s aO~l~cat~on. DO N~T ~XCEED ~O PAGES. Fellowships: 1975-1977 Clinical and Research Fellow in Medicine (Oncology), Massachusetts General Hospital. Research Fellow in Medicine, Harvard Medical School. Academic Aopointrne~ts: t990 Professor of Medicine, Northwestern University Medical School. 1986 Associate Professor of Medicine, Northwestern University Medical School. 1986 Associate Professor of Medicine, Harvard Medical School (for Iuly, 1986). [980 Assistant Professor of Medicine, Harvard Medical School. 1977 Instructor in Medicine, Harvard Medical School. Principal C!inicat and Hospital Service Responsibilities: 1986 Chief, Section of Hematology/Oncology, Northwestern Memorial Hospital and Northwestern University Medical School. 1978-1986 Director, Medical Oncology Clinic. Massachusetts General Hospital. Director of Massachusetts Generai Hospital Oncology Fellowship Training. Publications (represen.~ative from a total of 82) 1. Weitzman SA, Aisenberg AC. Siber GR, Smith DH. Impaired humoral immunit3, in treated Hodgkin's disease New ~ J_ Me__~d, 1977;297:245-248. 2. Siber G, Weitzman SA, Aisenberg AC, Weinstein H. Schif'f'man G. Impaired antibody response to pneumococcal vaccine in treated patients with Hodgkin's disease. New ~ J ~ 1978;299:442-448. 3. Weitzman SA, Stossel TP. Drug-induced immunological neutropenia, l~cet i, 1978;8073:1068-1072. 4. Weitzman SA, Stossel TP. Mutation caused by human phagocytes. ~, 198l;212:54,6-547. 5. Weitzman SA. Stosset TP. Effects of oxygen radical scavengers and antioxidants on phagoe~e-induced mutagenesis. J_ ~, 1982:128:2770-2772. 6. Weitberg AB, Weitzman SA, Destrempes M, Lart SA, Stossel TP. Stimulated human phagocytes produce eytogenetic changes in cultured mammalian cells. N En~/_ Med, 1983;308:26-30, 7. Weitzman SA, Graceffa P. Asbestos catalyzes hydroxyl and superoxide radical generation from hydrogen peroxide. Arch Biochem ~, 1984;228:373-376. 8. Weitzman SA, Stossel TP. Phagocyte-induced mutation in Chinese Hamster ovary cells. 1984; 22: 337-341. 9. Weitzman SA, Weitberg AB. Asbestos-catalyzed lipid peroxidation and its inhibitionby desfer~xamine. [, 1985;225:259-262. 10. Weitzman SA, Weitberg AB, Clark E, Stossel TP. Phagocytes as carcinogem: maligtwm transformation produced by bum,,an neutmphils. ~, 1985;227:1231-1233. 11. g/e|tberi All, Weilzllwn SA, Clark E. Stossel TP. ERects of antioxidants on oxidant-induced siste¢ chroraatid .._ ~x~ge tbrmatlon. ~[ ~li, In~e~t. 198J:75:1835-1841; ._ . . F.= 50610067
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Z I2. ~'eitzma~n SA, Stossel TP, H~mon DC, D~Lniels G, Malcof F, Ridgway EC. An~neu~ophil ~to~ti~ies in Grav~" dis~e: implications of ~yrotropin binding to ncu~ophils. [985:75: I I9-I23. t3, Ch~ter JF, R~ss JS. MaR RA, We[~m~ SA. Acute colitis pr~uc~ by che~mmic ~ . :-8 ~90. ra~ ~d mi~s. Am I P~thol, 19~5;I2~ ~ ~ t4. Weitberg AB, Wei~ SA. ~e ~ffe=~ of virgin C on oxygen radic~-induc~ sister ~hromatid exchange. Muta~ ~, ~985; 15. Von Roe~ 3H, Mushy R, Weber K. Willia~ L, Weimm~ SA. Meg=s~ol ac=t~o: Truant for ~e eacSexia ~soeiate~ wi~ hum~ immun~eficiency vies infection. Ann lnt M~, 19~8: 16. Weimm~ SA, Weitberg AB, Stossel ~, S~hw~ I, Sh~m G. Effecm of hydrogen p~roxi~= on o~ cmc~nog~is in ~ers. ~ P~:~ont01 t9~:57:685~88. 17. Von Roe~ JH, Mushy R, Weber K, Williams L, Wei~m~ SA. A curriculum in p~liativ~ for intern~ medicine housestaff: A pilot project. ~. Canc=~ Edu~, 1988, 3:259-263. 18. Jac~n JH, Schraufsta~er IV, Hyslop PA, Vosbeck K, Sau=rh=ber R, Weimm~ SA, Co,brae CG. Role of oxidan~ in DNA ~amaging ~ffec= of ~bestos ~d ~igm=~e smok~. 1987;80: 1090-1095. 19. Graceffa P, Wei~man SA. Asbestos eat~yzes ~= formation of ~ 6-Oxobe~o [ai pyren= ra~ic~ ~om 6-hydroxybe~o [al pyrene. ~ ~ ~, 1987:257:481~84. 20. Wei=man SA, Chester IF. Graceffa P. Bin~ing of def~rox~in~ to ~b~stos ~ ~ ~ in vivo. Carcinogenesis, 1988.9: 16~3-1645. 2l. W=i=man SA. Schmeichel C, Turk P. Stevens C, Tolsma S, Bouck N. Phago¢~-m~iate~ c~cinogenesis: DNA ~om p~ago~yte-~ransforme~ C3H 10T 1i2 c~lls e~ tr~sfo~ NIHi3~ ¢=tls. Ann NY Acad Sc~, t988:551:103-1t0. 22. Wei~man SA, Lee RM, Ouellete AI. Alterations in e-abl gene me~ylation in cells ~sfo~ by phagocyte-generated oxidants. Bioch~m ~ ~ ~, 23. Ch~st=~ IF. G~isse~ HA, Matt RA, Weimman SA. Coloni~ e~c~r in~uCe~ by ~ime~ylhydr~ine: Promotion by experiment~ colitis. ~ri~ £ Cancer, t989:59:70~-705. 24. Kamp DW. Dunne M, Wei~man SA, Dunn M. ~ interaction of ~stos ~ neu~rophils injures cultured human pulmonary ~pithelial c~lls: role of hydrogen peroxide. ~ ~ C1i~ ~e~.. 1989:114:604-612. 25. Ra~osevich CA, Wei~man SA. Hydrogea peroxide ~n~ue~s squ~ous metapl~ia in a h~ter trache~ organ explant cul~r= model. Care~0.~gen~sis 1989: i0:~943-19~. 26. Gor~on LI, Sc~meichel C, Prachand S. Weimman SA: I~ibition of polymo~honucl~ oxidative metabolism by exogenous phospholipm= C. ~ [mmunol 1990;128:503-515. 27. Kamp DW, Dunne M, Anderson IA, Weimm~ SA, Dunn MM: Se~m promot~ mO~tos-in~uc~ injury to human pulmonary epi~elial c~lls. ~ ~ ~ ~ t990:116:289-29~. 28. Wei~man SA, Gordon LI. Inflammation ~d e~cer: rot~ of phagocyte g~n=rat~ oxid~m c~cinog~nesis. Blood 1990;76:656~63. 29. Schm~ichel C~, Wei~man SA. Oxidant in~ue~ restriction polymo~hism maps to ~in~ region of e-~ Oncog~n~. Fr~ Rad]ca~ ~ Com~ 1991" 12-13:761-769. 30, Gordon LL W~iss D. Prachan~ S, Wei~m~ SA: $¢avenging of supemxi~ ~ion ~y phosphow/lethanolaminm Fr~ ~ Res ~ 199 t ; 15:65-7 i. 31. K~mp DW, Grac,ffa P, Pryor W, Weimm~ SA: Rol~ of ~e= radi~s in mb~tos-indue~ dise~es. Fre~ Radical Bioi M~ Vol. 12,pp.29S-315,1992. 32. Wei~man SA. Tur~ PW, Rund=lI K. Hy~rog,n p~roxid= causes ~pli~cation of vir~ ~ c~l~ gen~s, in "Oxidativ~ Damags ~ R~air: Ch=mie~, BiologieS, ~ M~J¢~ AspS," P~r~n Press. 1992.pp.~9-783. 33. Gor~on LI. Gust=in D, P~hand S, Wei=m~ SA. Co~timtiv= ~1~ rsl~ of phosphat~dyi choline preferring phosFholipme C (PC-PLC) by human fibrobi~ ~n~ ~o~ m~ele cells, in "~e Moi~ular. B~is of Oxidative Damage by L~k~." CRC l~.pp. 325-32~. , 1810088
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Dr. Leo I. Cordon BIOGRAPH~AL SKETCH Kenneth C. Robbins. Ph.D. Research Pro~essor of Hed£c[ne INSTITUTION AND LOCATION University of Illinois, Urbana, IL University of I11inois, Urbana, IL University of Illinois, Chicago, IL DEGREE M.S. Ph.D. YEAR CONFERRED 1939 1940 1944 FIELD OF STUDY Chemistry Biological Chemis Biological Chemistri RESEARCH AND PROFESSIONAL EXPERIENCE: Concluding ~ present position, list, in chronological order, previous employment, experience, and honors. Key personnel irckJde the pm'mipal i,'west~gator and any other incividu=~ who perl~t_.e in the scientific development or execution of the pro~e<~t Key pee~ortl'tel typic~gy will include ell in@vicbJals with doctoral or o4her profese3onel degrees, f0ul in some projects will in~ude individuals at I~e masters or baccalaureate level provide~ t~ey contribute in a substantive way to the =cienti~ development or execution of the project. Include present membership on any Federel Government public advisory committee. List, in chronological order, the titles, all authors, and complete references to all publications during the past three years and to representati,~e eadier publicaUor~ pertinent to thins appliceti0~. DO NOT EXCEED TWO PAGES. PROFESSIONAL EXPERIENCE 1944-47 1947-51 1951-58 1958-73 1970-88 1973-84 1984-86 1986-88 1987- 1989- Instructor, Dept. of Biol. Chem. Univ. of Illinois College of Med., Chicago, IL. Assistant Professor, Dept. of Path., Western Reserve Univ. School of Med., Cleveland, OH. Head, Protein Sect., Bioehem. Res. Dept. Armour Laboratories, Chicago, IL. Director, Bioehem. Res. Dept., Michael Reese Res. Foundation, Chicago, IL. Professor, Depts. of Meal. and Path., Pritzker School of Med., University of Chicago, Chicago, IL. Scientific Director, Michael Reese Re.s. Found, Chicago, IL. Director, Div. Experim. Path., Dept. of Pathology, Michael Reese Hospital and Med. Center, Chicago, IL. Professor, Joint, Section of Hematol.lOncol., Dept of Med., Michael Reese Hospital and Med. Center, Pritzker School of Med., Univ of Chicago, Chicago, IL. Professor Emeritus, Pritzker School of Med., University of Chicago, Chicago, IL. Research Professor, Div. of Hemat./Oncol., Department of Med., Northwestern Univ. Me(]. School, Chicago, IL. AWARDS 1971 1980 Fourth Elwood A. Sharp Lecturer and Prize. Nineteenth Annual Symposium on Blood, Wayne State Univ. School of Med,, Detroit, MI. The Prix Servier Medal and Prize, Fifth International Conference on Fibrinolysis, Malmo, Sweden. CAREER~RELATED ACTIVITIES 1970-72 1971-75 1973-76 1974-75 1975-80 19"/6-~0 Chairman, Medical Research Institute, Michael Reese Hospital and Medical Center. Member, Hematology Study See., Div. of Res. Grants, NIH. Member, Advisory Committee, Amer. National Red Cross-National Fractionation Center. Gordon Conference on Flemostasis, Vice Chairman 1974, Chairman 1975. Melttber, E, ditorhtl Board, Journal of Biological Chemistry. Member, Blood Diseases and Resources Advisory Committee, National Heart, Lung and Blood ltastitute, Memb~, Hematology Study $e¢., Div. of Re~. Grants, NIH. FF 5061O(O9
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1984-88 1987-90 Member, Committee on Appointments and Promotions, Division of Biol. Sciences. University of Chicago. Associate Editor, Fibrinolysis. PROFESSIONAL MEMBERSHIPS American Assoc. of Immunol. (1950); American Soc. of Biochem. and Mol. Biol. (1967); American Soc. of Hematol. (1974); International Soc. of Thrombos. and Haemostas. (1970). RELEVANT PUBLICATIONS (1979-1992) (over 100 publications since 1944) 12. 13. 14. 15. 16. Wohl RC, Summaria L, Robbins KC: Physiological activation of the human fibrinolytic system. Isolation and characterization of human plasminogen variants, Chicago I and Chicago II. J BIOL CHEM 254:9063-9069, 1979. Wohl RC, Summaria J, Chediak J, Rosenfeld S, Robbins KC: Human plasminogen variant Chicago Ill. THROMB HAEMOSTAS 48:146-152, 1982. Gonzalez-Gronow M, Robbins KC: In vitro biosynthesis of plasminogen in a cell-free system directed by mRNA fractions isolated from monkey liver. BIOCHEMISTRY 23:190-196, 1984. Robbins KC, Tanaka Y: Covalent molecular weight 92,000 hybrid plasminogen activator derived tom human plasmin amino-terminal and urokinase carboxyl-terminal domains. BIOCHEMISTRY 25:3603- 361 l, 1986. Scharrer IM, Wohl RC, Hath V, Sinio L, Boreisha I, Robbins KC: Investigation of a congenital abnormal plasminogen, Frankfurt I, and its relationship to thrombosis. THROMB HAEMOSTAS 55:396- 401, 1986. Robbins KC, Barlow GH, Nguyen G, Samama MM: Comparison of plasminogen activators. SEM THROMB HEMOSTAS 13:131-138, 1987. Robbins KC: The pl&sminogen-plasmin enzyme system. IN HEMOSTASIS AND THROMBOSIS: Basic Principles and Clinical Practice. (Eds) R Colman, J Hirsh, VJ Marder, EW Salzman. 2nd Ed. Lippincott, Philadelphia, Chapter 21, 1987, pp. 340-357. Robbins KC, Boreisha I: A covalent molecular weight 92,000 hybrid plasminogen activator derived from human plasmin fibrin-binding and tisue plasminogen activator catalytic domains. BIOCHEMISTRY 26:4661-4667, 1987. Summaria L, Boreisha I, Barlow GH, Robbins KC: The isolation and characterization of ternary human plasmin light (B) chain-streptokinase-plasminogen complex. THROM HAEMOSTAS 58:772-777, t987. Robbins KC: Dysplasminogenemias. ENZYME 40:70-78, 1988. Le~ PP, Wohl RC, Boreisha I, Robbins KC: Kinetic analysis of covatem hybrid plasminogen activators: Effect of CNBr-degraded fibrinogen on kinetic parameters of Glu-l-plasminogen activation. BIOCHEMISTRY 27:7506-7513, 1988. Robbins KC: Classification of abnormal plasminogens: Dysplasminogenemias, SEM THROMB HEMOSTAS 16:217-220, 1990. Robbins KC: Fibrinolytic therapy: Biochemical mechanisms. SEM THROMB HEMOSTAS 17:1-6, I991. Robbins KC, Boreisha I, Godwin JE: Abnormal Plasminogen Maywood I. THROMB HAEMOSTAS 66:575-580, 1991. Robbins KC, Boreisha IG, Hach-Wund~'le V, Sch~rrex I. Congenital plasminogen deficiency with an abaormal plasmiaogett: Fr~mkfurt ll, Dy~asminogenemia-Hypoplasminogenemia. FIBRINOLYSIS 5:145.-153, 1991. Robbins KC: Dytpla.smiaogeaemias. P~OG CARDIOV#._S DIS 34:295-308, 1992. 50610070
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F..'e .~d ~'¢~ Cc~u . Vol. I~. ~No I. pp 65-'1 Repnnts a~a~Labl~ d~recc|y from tl~ pub~,~t P~olocul:)~.~! ~-rm,tlcd b~ ]~'ecn¢ onl~ Printed ,~ the Umt~d Km~;Iom SCAVENGING OF SUPEROXIDE ANION BY PHOSPHORYLETHANOLAMINE: STUDIES IN HUMAN NEUTROPHILS AND IN A CELL FREE SYSTEM LEO 1. GORDON~. DAVID WEISS. SHEILA PRACHAND and SIGMUND A. WEITZMAN Northwestern ~,~iversit) Medical School. Department of Medicine. Section Of Hematology,:Oncology. 303 E. Chwago ..1 re. Chicago. I11 60611 USA i Received Jul.l 31. l~9tt- in re~t~ed form Ot't~,t~t'r On the basis of previous obsetn..atton~. ~.e attempted to characterize the effects of various products of pho~phohpid hydrol.',sis on neu~rophtl {PXINI re~pm~tor.~ bur~t act~t.~ We ~;tudied the effect~ o1" pho~- phorylchohn¢ IPCI and phosphorylethanoline IPE~ on supero~de anion pr~duction m PMN and in a tell free system. We found that PE but not PC inhibited measured ~upcroxidc anion, but that thi~ v.a~ not due to inhibition of cellular ~uperoxide generation but to :;cavenging of generated ~,uperoxide amon. Further. utilizing a system ba~ed upon the photo-oxidation of O-diam~idlnc ~cn~ttized by riboflavmo we w.ere abte to determine that the sca,,enging effect of PE ~va~ not ~uperoxide d~,,muta,,e (SOD~-Iike but rather a general scavenging or glutath~one (GSH)-hke eff~t. The~e data under~cor~: the importance of identifying the mechanism o1" inhibition of supero~ctdc generation b) ptltat:~e mhtbitor~ a~ being due to a direct cellular effect or to a scavenging property. KEY WORDS: Phosphor~,lethanolamine. phosphor.~lchohne ~,.'a~cngmg. supere\~dc anion. INTRODUCTION We have recently shown that phospholipase C (PLC). a membrane bound enzyme responsible for the hydrolysis of membrane phospholipids to diacylglycerol (DAG) and phosphoryl derivatives phosphorylinositol (PI), phosphorylcholine (PC), phos- phorylethanotamine (PE), or phosphorylserine (PS) (depending on the substrate preference of the phospholipase) can inhibit certain aspects of neutrophil {PMN) oxidative function while stimulating other PMN functions? In our experiments we used an enzyme which perferentially hydrotyzed phosphatidylcholine, not phos- phatidylinositol. In an attempt to better characterize the effects of various products of phospholipid hydrolysis on PMN respiratory burst activity, we studied the effects of PC and PE on superoxide anion production in PMN and in a cell free system. We found that PE, but not PC, inhibited measured superoxide generation in a dose dependent manner. However, we found that this lowering of measured superoxide was not due to inhibition of cellular superoxide generation but rather to scavenging of generated superoxide anion by this phospholipid hydrolysis product, and that the PE acted as a general free radical scavenger rather than as a specific superoxide dismutase type scavenger. • To whom reprint requests should b¢ addrcss¢d 65 50610071
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~ L I GORDON ET .-~! MATERIALS AND ,METHODS Reagent~ Phorbol myristate acetate (PMA) was purchased from Consolidated Midland Corp, Forrester, NY, and stored at - 4* C at a stock concentrat,on of 10 mg/ml in dimeth~l- sufoxide (DMSO), and used in a final concentration of 10-100ng/ml. N-formyl- methionyl-phen),lalanine (fMLP) was purchased from Sigma Chemicals, St. Louis, Mo., stored at a stock concentration of" 10-6M in DMSO and used at a final concentration of 10-7 M. Dianisidine and superoxid¢ dismutase (SOD) were obtained from Sigma Chemicals and riboflavin from Eastman Organic Chemicals. PE and PC ~'ere all obtained from Sigma and stored dessicated at 0* C. Preparations at the appropriate concentrations were made fresh each day in Hanks Balanced Salt Solu- tion (HBSS}. Xanthine and xanthine oxidase were purchased from Sigma. Xanthine (purity 99-100 %) was stored at room temperature. Xanthine oxidase was stored at 4°C and had a specific activity of 1.2U/mg protein. lsoladon of Pol.~'morphonuclear Leukocytes PMN ~ere isolated as previously describedz. Briefly, 30-50cc of blood were drawn into a syringe containing I ulml of heparin and were allo~ed to sediment with one half volnme of 6.5 % dextran and 0.98 % sodium chloride. The supernatant was collected. the remaining red cells lysed by hypotonic lysis and the pellet was resuspended and layered onto a Ficoll-Hypaque gradient and centrifuged at 450 × g for 30min. at 20° C. The pellet contained approximately 95 % PMN and was suspended in HBSS with 0.2 % human serum albumin (HBSS-HSA~ or HBSS alone. Superoxkh' Get~eration Superoxidc anion generation was determined by two methods. Both relied on the superoxide dismutase (SOD) inhibitablc reduction of cytochrome C. In PMN the assay ~'as performed as de~zribed by Qoldstein et al.~ PMN were incubated with varying concentrations of PE and PC, then stimulated with agonists PMA (10 ng/ml) or fMLP (10 7 IV[). Reduction of cytochrome C at 550nm (using a molar extinction coet/icient for this change in absorption of 21.000) was determined at 15 rain, and in some experiments was measured in a continuous assay using a Varians spectro- photometer. Superoxide generation v, as also measured in a cell free system with xanthine-xanthine oxidase (X:XO) as the source of free radical I.I U XO) with and without added phosphoryl derivative. Biochemi~al Augmentation Assay The photo-oxidation of O-dianisidine sensitized by ribofla~'in was measured as des- cribed by Misra and Fridovicha. Illumination for the photochemical reactions was provided by two parallel 20-W Sylvania Gro-Lux fluorescent bulbs mounted in the center of an aluminum foil lined box. Reaction mixtures containing riboflavin (stock solution 1.3 × 10 ~M in 0.01 M potassium phosphate buffer at pH7.5) and o-diani- sidine (10-" M) in ethanol were added to quartz cuvettes with and without SOD. Absorbance was measured at 460ram on a Varians spectrophotometer before and after 4 minutes illumination and the difference in absorption was calculated. This 50610072
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~C-~VENGING OF SUPEROXIDE ANION BY PHO~PHORYLETHANOLAMINE ~7 assay can be used to classify scavenging compounds as SOD-like or giutathione (GSH)-Iike. since SOD-like compounds produce an augmentauon and GSH-like compounds an inhibition of absorption,s RESULTS Effects of Phosphoryl Derivati,.es on Superoxide Generation We found that phosphorylethanolamine (PE) but not phosphorylcholine (PC) (data not shown) inhibited PMA and fMLP induced production of O.,- when pre-incubated with PMN (Table 1). Thix effect was dose dependent and was seen at concentrations as low as 2 mM PE when fMLP was the agonist and 4mM PE when PMA was the agonist. The inhibition occurred whether PMN were pre-incubated with PE (Table I) or when agonist and PE were added simultaneously (Figure la, b) and superoxide generation measured in a continuous assay system. The high concentrations required suggested that the effects were mediated by scavenging or toxicity. Effects of Pho.Tphor.vl Derivatires in a Cell Free Srstem In order to help determine whether the inhibitory effect of PE on measured generation was due to scavenging or inhibition of production of free radical, v,e repeated the experiments with varying concentrations of PE in a cell free system, measuring the production of O_, by a mixture of xanthine and xanthine oxidase. We found that there ~vas maximum inhibition of O_: generation after approximately 1 min of incubation, but that over time the SOD inhibitable reduction of cvtochrome C in presence of PE returned to close to normal levels (Figure 2), suggestit~g consumption or inactivation of the PE. Photo-oxidt~tion of Dianisidine To determine whether the observed scavenging of O., by PE was due to a specific SOD-like effect or was due to a general free radical scavenging activity, we measured TABLE l' Addition to PM,V A,¢oni~t 0,-: 0 c.~t B ~ FMLP' 248 ± 25 PE 20raM* 2 b + ' t~ PE 15ram 28 +__ 0 PE 10ram 74 ± 2~, PE J, mM 12~¢ _.+ 26 PE 2ram 14.5 -- 0.9 0 PNIA' 48 8 --- 0.95 PE 20mM 0 PE 15ram .L7 -.- 028 PE 10ram 8 5 ~ 057 PE .J, mM 21.9 ± 2.0 * Resul[,~ are expressed as mean __. S.E. of a repre~entat,ve experiment m duphcate Similar data found in tv, o additional experiments for each agoni~;t. :O.," *~ expressed as nanomole$ of cytochtom¢ C reducedll(P PM N ~Cytochala~;in B 5ug/ml and FMLP t0-* M. PMA 10 ng/ml v, ithout c],tochalasin B. *PMN are pr¢-incubated with PE for 10ram. prior to activation. 50610073
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L I. GORDON ET 25 5 I . I ~ ~ - ~ :2t t , lt6 2 4 6 8 10 1 14 Minutes FIGURE l Human pol>morphonuclear leukoc.~tes (PMN) ~,ere lneabated u, tth varying ¢oncentration~ of pho~phor~lethanolamine t PE~ (control ~, 20 mM PE ~, ~5 mM PE ~. 10ram PE ~, 5 mM ~ PE~ then s~multaneou~l~ ~timulated ~tth ¢~tochalas~n B 5tt~'ml and FNILP IO-~M Su~roxtde generation, measured as SOD inhibitable r~uction ofc~ tochrome C ~as as~a~ed at l-minute time inter~ls on a Varians spectropholometer. There ~ai dose de~ndcnt inhibition ~hen PE incuba~ PMN were compared to COlll~t)[~ the effects of PE on the photo-oxidation of dianisidine. We found that when PE was added to the reaction mixture (described in Materials and Methods). there was inhibition of the photo-oxidation ofdianisidine (Table II). This effect is similar to that produced by glutathionc (GSH) and leakotrienes.~ and is opposite of the augmenta- tion that is seen with SOD alone (Table Ill.~s TABLE 1I : .4ddrtion .4bJorptton aider 8 nun ?i. inhtb t} 00877 +_ ooo[ PE 5mM 0.0107 ~- 00011 85.4 PE 2 5m,',,| 0.0,155 + 0.0032 52 SOD 10/.*g.ml~ 0.3453 +_ 0.0275 ~ The effects of PE on the photo-oxidation of O-dianisidin¢ v,'ere measured as described in Materials and Methods :Data v, ere e,tpre~;~ed a~, mean - S.E. ab~;orption of a repr~entati~ • e~pertment in duplicate. Similar data were found in 2 additional experiment~. ~ When supcroxid¢ dismuta~e ISODI wa,~ added, there w~$ expected" -~ augmentation of photo-oxiflation of O-diantsidine. 50610074
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SCAVENGING OF SUPEROXIDE ANION BY PHOSPHOR'~LETilANOL~MI.NE (~9 35 "o 2~ o 10 5 M~nutes FIGURL 2 Control PMN ('~--~1') treated as abo'.e, but the stimulus ts PMA ~ 10 raglml~. 20 mM PE o--o. 15ram PE a--t,. 10MM PE CF--G. 5ram I!--II PE I)ISCUSSION We have demonstrated that phosphorylethanolamine but not phosphorylcholine diminishes measured O,, generation in human PMN. but that this effect is due to scavenging of generated O.,-. Moreover, using a unique assay which relies upon the photo-nxidation of dianisidine to a colored compound th,".t can be spectrophotome- trically measured at 450 ram. we found that the effect of PE ~as due to a scavenging effect and was not the result of a specific SOD-like catalytic effect. The study of superoxide production by human PMN and the subsequent charac- terization of the biochemical pathways which regulate this production have been the focus of intense interest in biology over the past several years.* Several recent reviews highlight the importance of the respiratory burst system in human PMN,' ~ and weft 10 II and others • have shown that regulation of PMN respiratory burst activity in PMN has important consequences in a variety of biological systems. A number of com- pounds which have been found to inhibit various components of the respiratory burst system have now been identified,*-" and clarification of the site of action and mechan- isms of these putative inhibitors is warranted. We v-ere interested in further characterizing the effects of hydrolysis products of 50610075
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70 25 -~ 20 0 10 L I GORDON EF 60 2O 30 60 90 120 150 180 210 240 Secor~ds FIGURE 3 Supcroxide generation is measured in a celt free n~stem using a mixture of xanthine and xanthme o'~lda~e (specttic activity 1.2 U/rag protein~ as described i'n material, and methods, in the ab~nce (~---~) and presence fc,~-o} of20mM PE, Percent tnhlbition with t~me I ,~--,~) i~ sho~,n on the ordinate on the r~g.ht. phospholipases, since we have recentl> shown that phosphatidylcholine-preferring phospholipase C can inhibit PMN respiratory burst activity while stimulating other PMN functions.~ This apparently paradoxical effect led us to investigate the effects o[ various phospholipid hydrolysis products on PMN function. We found that phos- phorylethanolamine, but not phosphorylcholinc, inhibits measured O; generation. and that this effect is due to scavenging rather than to a direct cellular'effect on the oxida~e. These experiments did not, however, elucidate the mechanism of the effect of phosphatidylcholine preferring phospholipase C on PMN O, generation but instead appeared to identify unexpected scavenging properties c~f phosphoryleth- anolamine. Finally, we sought to investigate whether the scavenging effect of PE was SOD-like, that is, specific for the dismutation of superoxide anion, or was a more general scavenger of free radicals. To answer thi~ question we utilized a system which is based upon the observations by Misra and Fridovich that the photo-oxidation of O-diani- sidine involves a complex series of free radical chain reactions which utilizes the superoxide anion as a chain propagating species.~ Thus: a) Riboflavin (Rf) + light (hv) --, R£ b) Rf + DH.,-*RfH° + DFI'~ 50610076
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SCA~.ENGING OF SUPFRO'KIDE ~.NION BY F~iOSPHORYLETHANOL.-~MINE c) RfH~ + O,----Rf- Of ~ H" DH° + Oz + H" ~DtI: - O: DH° ~ D~I°--,D + O~ + Of + 2H--,H:O: + O, In a~ the riboflavin is excited by light, and ~n b) *t oxidizes dianisidine, giving a flavin semiquinone and a dianisidine radical which would spontaneously dismute as in e) to yield the colored product D, which absorbs at ~-60 ram. However, the r]a~,'in semiqui- none produced in b) can reduce O: to 02 (reaction c) ~,hich will reduce the dianisidine radical (reaction d). SOD, by scavenging O., ~reaction f~ prevents the reduction of the dianisidine radical, allowing more of the colored D to accumulate. On the other hand. any radical scavenging species acting on reaction b would diminish the formation of D, and a decrease in absorbtion x,,ould be observed. In our system, PE t.-aused an inhibition of absorbance, thus indicating that it was acting on reaction b as a general free radical ~cavenger. This is exactly, as has been described for GSH, and different from what would be expected~ and what we observed lTable IlL for SOD. Thus, we have shown that PE scavenges generated free radicals, and that this effect is not SOD-like. Further description of putative inhibitors of PMN respiratory burst acti~.it.v should include evaluation of their ability to scavenge generated free radicals. as this information may have pharmacologic implications as .systems to modulate free radical production are developed, in addition, an understanding of the scavenging properties of physiologic compounds may provide insight into important biological phenomena. Re.l'ert.~tct,~• L I Gordon, C Sehmeichel. S. Prachand and S A Wettzman t 1'-3'901 tnh~b~t~on of pol,smorphonuclear Ieukoc~,te oxidative metabohsm by exogenou~ phosphohpa~¢ C t~c'lh~k~e It~t~nt,noh~e.~. 128. 503-515 2 LI Gordon. S.D. Douglas. N.E Ka), O iomad,i. E F O~,rm:m and H S. Jacob 11979) Modula- tion ofneutrophd function b) I)soz~nw Potential ncgal~e f~'dback ~,tem of in~ammat~on Journal ol Cloth'd [nve.~tt.¢atu~n, ~. 266-281 I M Goldstem. D. Roo~. HB Kaplan. ,tnd D. ~Ve~ssman 11'/75~ Complement and imn~unt~glt~bulin ~tin~ulatc ~u~roxide production b5 hhman leukt,cyte~ J~,ur~n~l ,I Clinical 4 H g. M~sra. and I. Frido~ich (19771 Supcro~de thsmuta,e ~ photochcnucal uugmcntatio~ asia) .4rcht*cs tn Btochemt.strt" and Biophysics. 181. 308-312 M Chopr,t. JJ.F, Belch andW.E Sm~thl1988~Acoa)pan,onofthe freerad~cal of Icukotriene~ and prostaglandms Free Ra~hc:~l R~'¢t'~;et11 ~oe~t~tl~ttt~,l~totls'. M W Verghese. CD. Smilh and R Snyde~an I 19~gl Role of guanine nu¢le~t~de regulator~ m pol~pho~hatidc degradation and actisat~on of phagt~'~t:c leukoQ:es b~ chcmoattrdctants Journal ~,t C~4t Btochemia'tr.l'. 32. 59. 7 A J Sbarra and RR. Straussledsl. lhe Respiralor~ Burst and ~t~ Ph)siological York. NY Plenum Publishing. 11988~, B Babior { 1988) The respiratory burst oxidase, blt'm~ltob,g)and Onc,,lo.et ('ltt~ ,V,~rth dttl~'rt~t. ~. 201 b A We~tzman and L I. Gordon 1199~11 Infl:~mmation and Cancer Role of phagocyte generated IO J [ Marx 11987} Ox)gen free rad~¢aI i~nked to many d~sca~e~ Stt¢tl~e. 235. II R I Lehrer (19881 Neutrophd~ and ho,t defen~ ..It~t~al, ol Infertility,real .~[¢thtl¢l¢. 109. 120-142 12 A R. Cross (1990) Inhibitorg of the leukocyte ~uperoxide generatin~ ox~dasc: Mechanisms of and method~ for their elucidation Frt', Rad~c~tl~ Bmb~g3 .~/cdwin¢. 8. 71-93. Accepted by Prof. J.V. Bannister 50610077
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Inhibition of Polymorphonuclear Leukocyte Oxidative Metabolism by Exogenous Phospholipase C LEO I. GORDON,I CYNTHIA SCHMEICHEL, SHEILA PRACHAND, AND SIGMUND A. WEI'I-ZMAN (,'na~erstt*" De'~me~t ol'Med~c~ne. Sec~toa qf Hemmo~og)./Oncotogy. 303 East Chicag,~ .4 venue. Chicago. lll, nots ,50,511 Re~'¢tved Janua£v 19. 1990" accepted.Warch .~. 1990 We studied the eft'~ts of e,~ogenous, punfied phosphohpas¢ C (PLCI on neutrophd o~idative metabolism, lysosomal enzyme release and aggregation. We found that PI.C inhtbited 0.~ and ! lzO: generation and oxygen consumption, but did not alter glucose oxidation via the hexos¢ monophosphat~ shunt. In contrast. ~,e found a striking stimulation of aggregation and release of the I.vsosomal enzymes lysozyme and ~-~ucuronidase. In experiments desiSned to further characterize the mechanism of the PLC effect on membrane activation we studied the erect of PLC on intraeellular calcium concentration [Ca:'l, and found that PLC did not interfere with the fMLP-mediated ri~ in [Ca:'],, suggesting that its inhibitory, effect on the respiratory burst does not m~ol,.e inhtbiuon of early signal transduction ¢ven~. In addition, we found that PLC alone results in mobilization oF intracellular Ca:" stores, consistent with its stimulatoo effect on aggregation and lysosomal enzyme release ¢. ~.),,'o ~"~m,c ~ ~nc INTR.ODUCT[ON Human polymorphonuclear leukocytes (PMN), when triggered by any one of a number of agonists, can metabolize oxygen to reactive oxidants (l). These cells can take up oxygen at a rapid rate, and utilize a membrane-bound NADPH oxidase sys- tem which generates superoxide anion radical (O,), a one-electron reduction product. The NADPH oxidase requires flavin adenine dinucleotide (FAD) (2.3L and the reac- tion may be characterized as Follows: NADPH + H* ~- 20: --,- NADP" + 2H- + Concomitamly. there is oxidation of glucose via the hexose monophosphate shunt. a process which provides NADPH as a source of electrons for the production or" O,~. We (4) and others (5) have previously shown ~hat certain cellular en~'mes, pro- duced by inflammatory phagoc~es at sites of inflammation may regulate oxidative metabolism in PMN. Recemly. Styrt et al. (6) have shown that exogenous bacterial phospholipase C (PLC) alters oxidative metabolism in bovine PMN, and suggest that this enzyme could play a regulalory role in phagocyte-bacteria interactions. In our current studies, we examined ~he effects of r~o forms of'purified bacterial phospholi- ' To whom reprint requests should be addressed. 503 0008-$74')/90 $3.00 50610078
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! • ,)(ll,47tl~ .il.,[flilaa [I;It)l jo • lid I II llliitldit till HINd ItlelmU!li pue ltU!ll~l jo 1 tlt.,|~uJ3dn~ '~,OiJ I aq ol • ill lllU~i:i~[ illlllll~iO llilo.l. °81i~JItl(tlli 1! .~ ,'l|HllOJII3111~ [(iu.~i|dO.ll!U-~/,"lil!~;ll tio!ll;l.I iliql ll~ lllltlllOdl~i.' "(If I OI ) crl I,'~t .io (lUl[~u 01 ) VINd lll!~ P.~ll;InUl!l~; uaql "<)" hi .,I )li'tdlllllbl.~i')l I,t .%11 Nt 111.V(IIXO Ill I,/OH I I I-IN ,1( i N( II I IIIII I *IV z OI Jtl UU!ll:.lI 'Slr'3!tu°t D etuih$ LIIOjj pa~l,,tl3Jnd sl;#~ (d'IV#J) 0UlUl,'llq~u.~qd-l~,)llal.l~uollllOLU.l,~lli. . ~oJ ItUJU 001-01 jo UO!li~lU;)auo:) letli.l I~ u.l l:~In ptm "(O~l~(lJ SUOII.L'._IIN CINV S']VI~I,H.LVIAI IV .I I NI, XlllOl)
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.(~ ~ ~l~p) ~UOIl~ d' i~ io auof .Yhl ol p~xkuoa uaq,~ ( ;.,t)~ 09) .~ ~ ...... -s.~JonLiolnV uul~u,~lsns Ila3.1o liU ~ ol ',q~!l,~l~J "(ill IId) V.I (I.I 11' i'0 'S!ll If ~'OJO I~ 0~ pu~ I~1-~ m~liJ.l. (~7~) % I jil in 08 ,I° UOil!pp~ ,~lll .4q ll~il!mi~l.III "°3tl!t~V ~e ~! '(ll~!JDtuoJ°ll~ p~i~eal ~i~ ~d ll~ll~tll ~'~ln:l .~lll ill iD~til~llI '[ ,:~Jl ~il~ "~n I!lii~ ~! till Itl~ pin) (li!lll~qll~ ililll~ lillniniI ';,71)0 I) ill!~l ~D£)Vd PlOD'~al ti! Itti/illaD ~01 x 7 lU ll~p~iill~ ~J~ ~l~d ll~ptrOl 2"~Jil:l DIll IId '[IO~N 1~' tO'O ~u~ 'qD@~ I~m I 'z[D~D 1~m 9(} "[./~ t~' ~00'0 "l.)Z~N 1~' I [ 0 "(sadld) (P!3~ a!Uqlln~auuttla'E)',N'N'aUlZe~od!d IV ~0'0 :ttl!~Ullqt ~lll jo tl3~tl~ 'j~llq ~30"~ i1~ pal~qn~u! IJa~ N~d ~lll~l~u,ldlO af~ill uI "(L I ) /t~ ~,J ihlll I • IILUI.~I",~ ,hoih/olldtoq,I 'I'7 l:l N(Rllltitl
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NOIS~JCI.)SICI .) .~ll~¥dl'lOlldl~l)lld .~.ll NOII V(I1Xt) "lllhl()H It I~IN I() N( )I I lillllNl o~ ! o~ .! i
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GI)RI×)N ET AI.. "llme (rain) lmalt'd as in Fig 2o hill fells arc p¢,eineubaled wilh cytofhalasin 13 5/~g,/ml then l;hmu la|cd There i.¢ af.am dose-dependcnl inhihilion of supcroxidc ttencration. lha[ the Sllc of PI.C aclion is at or near the common pathway for respiratory bu~t aclivily, the NADPll oxidase. We als<~ r=,u.d thai PI.C caused release of the lysosomal c=v.ymc.s ly~sosyn',.c and fl- Iducuronida.~,. and slimulated aggregation of PMN to a levcl [hat was comparable to lhal sccn with known PMN a~onis[s. These s[imulalory cff~(s on PMN by PLC ~'ere conlra~, to what might hav~ been expired from the respirato~ burst data. and st~c~l perhaps that Ihcrc a~ (li~ercnt mechanisms of action o~exogenous bacterial PI.C on diffcmnl PMN flmclions. Fo ddermine the point in the signal tmn~udion pathway at which exogenous I'I.C might ~ acling, we examinM the integrity of the ~LP-stimulated calcium response, an curly signal tran~luction cvcnl. In PMN preincubat~ wilh PLC at con- 7 ^II[.E I .__ __ I!l'l'¢cls ¢~f PI (" ~n ()~ f'nn~unlptu~n° (nil-~/l 0? PMN/Mirt) Nnnt, lg.9 ~ 1.1 P[ ('~" 10.9 _~ {1.07~ hl~ ~ PI ( "r, 1 .~ml ~s I~rm~ ul~,~l~l s~ills PMN fl~r In nun INIIIItl IION Of' NEll I R()I'III[. ()XII)A'I'I(IN IIY |'11('/~11|)1.11~/~,~1:. (" I AIII.I' 2 PI (" Does Nill Inhibll Pt~4A Slimtllal~l ()~kkillom ccnlr;ltmns which inhil'filed respiratory burst activity. PMLP in [Ca~' I~ (Fig. 5). "rhe kinetics of the fM LP-stimulaled in¢l~ll~ cal 1o that observed in PMN stimulated without PLC. Wllml ILL(7. to PMN. there wasan almchst immediate rise in [Ca~l,. Within there was nn increase iu JCaZ'], from hti:al le~ls. There was effect by EGTA. su~.esting tlmt this rise was du~ to n~ili=lliml cellular stores rather than from influx ofextmcdlular Ca=" dl~ PMN membrane. These data make it unlikely that PLC on the respiratory burs1 by inhibiling early signal the observation that PI.(" induces a rapid ri~ in l('az' I, with its observed stimulatory c~.~(s on PMN d~ranu~al~n surest Iha[ exogL'nous bacterial P[.C may ~ ~ratins by nism. All of these cffccls were ob~rvcd for difl~n[ ~l~¢al~ that contaminants were likely nol responsible ~o~ the ~rio~ I l<, 4 I'hw,pholipasc (', ~hcn added Io hum=m PMN, r¢~ull¢d *11 II drilt'.slfl~ lil'll~ e~l a I{I.Iiiill IIIiW tx'rh~d
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G{)RI)ON I.t • ^ II1~ 1 I'ltt'ct~nfpl C~n~ I'MN I.y~+ulm Ifntyme Rclea~-" 27•.1 ÷ 3"/02 ~ ,4.6 (tt = 5) 11.17 ± I.I in Illc~" eXperimcnt,~ x,,'~s derived From ('. wt,lt'hii and preferred Pho.sphalidylcholine ~ts su h.~trale, Ft~rthcrtnorc, there was no Io.~ of cellular viability by trypan blue exclu- sion t~r by I.I)]1 rcle:lsc al doses t~p Io II'~)#g/ml forat least 30-40 rain ofincubation. In an allempt Ii~ elucidate [he mechanism of Ibe observed inhibition of Oj and I I~(~: hy PI ,(', we sought to examine the effects of PLC on glucose oxidation via Ihc hc~o~e monophoxPhate shunt, the syslem which, in PMN, is responsible for genera- ~-- FMIP A~Mm~, 3O0 gecond~ I .. ~ I'MN prcmtuh.Ht~l s~llh t'~hm'halal I ~ 15 #~ml) I++r 5 tmtl are Ihen (rt'~lt%l w~(h P(.(* (7() ~ I ~; I 6 {ll AI ~ ram. I'MN ~te Illen e~l Io ~1 P and [('a~']. measur~l "lhc~ is a roll,old gre~tcr 1( ',1I' ], ~rc ick'nlwal I,"41111~111()N 01 NI!i I I'R()PI III. O~II)A I I(~bl IW PI ll~Plll|l.llqdil{ (" ~ I,l lilm cff NAI)PI I clcctrmas. We f~mnd that preinc,hntion conditim~s identical ~o tlmse (le~ earlier r~ul~ in oxiclatim~ (mcasur~ as '~CO~) c~mpar~J to t~ntml~ with alone c~m~cd no oxidalion of gluco~. Th~qe ~ult~ a~e activity during the respira~o~ burxl is fell Io ~ ~n~ tO cellul~r 1 t~O~ and depletion of NADP! 1, b(ah ~.~¢u~in~ ~ a oxida~ activity. We cannot e~sity ~ncilc th~ ~t~ in tion~, unless the timcpoint of me~+uremcnl o£ '~+ ( 15 mint an inhibitory We ob~crvt, d di~rcnces in th~ de$ree of inSibiti(m of l in the degree of inhibition of Lotal oxygen consumption a~ We cannt>t readily a~unt for the qu~nLiLati~ diffc~n¢~ but may ~ulale that this diffexn~ ~uld ~ due Io ~l~li~ tion oFone ~cies over the other. I t is unclear by what m~baoi~m VLC is ca~si~ the ~ our dala poln{ to a site in the memb~ne signal tran~uct~ receptor complex (for ~LP) and the NADFII oxids~. branex ~r in cytosol, is known to hydroly~ in~itol inosilol triphosphate and dizcyl~rol ( 19~ F~ teinx (20) and tia~ events alimony ~$nal atcd with tl~e ~pi~to~ bunt (21). lnd~d, th~ s~ pr~lucls of PI ,(" aclivati(m in "pfimin$'" of the ~u~ the FLC u~d in Ihe~ studi~ ~e~ phosphatidylinositol (PIt) as sul~tmte we cannot ~termlne pathways were involved, in fact, the m~haoi~m of the ments may not be related to the ~nd m~n~ ~)t~ alteration of the configuration or orien~atlon (~ie electron tranxpoo ~y~tem. ~rbaF~ indu~ b~ 5~i~is plaolipids. Thus we can,o~ I~ ceaain f~)m our data w~tSe~ the ~ invt)lvt,x ~ccond messenger. If not, then the stehe eff~ts PC preferring PLC could ~ impo~anl, lf~. ~nl accumed~,tc~ by the action of PC p~fe~ng PLC a~ ~11 as PI an unde~tanding oftt~e cffect~ ofexo~non~ PLC ~le~e~l. shown that I)AG and phosphocholine can ~ ~nemt~ in following slimulmtion with phorl~l dieslem, serum and P~P. thut at lemst ~mc of the DAG is de~ From ~ti~tion Similarly. Irving (24) ham shown that a PC ~a'~mnl PI+P liver cells a,d co~lribulc Io hepmlie DAG le~ls ~ ity. More recently, Griihmc (25) has pawkled e+~len~ G+ as well a~ DA(; nnd phoxpl~h~line from ~m~h rccept~>r, nctivt~tion ofwhich in Itl~ activutes t~ah PCand ~ ~~. ducidation of the potential a~le of P(' as su~trate for the xcngcrx in PMN api~ars warrnnt~t. l:~r some agonists, NAI)PI I t>xida~e activation is fflal¢~ Iofatim~ from the cytosol Io the membrane (26), a pn~ I~1 by diacylglyceml, a product ~f Pl.Gm~ial~l h~ ~~ For Ihix reason, our observations that PI.C inhi~ (~
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gener:tlcd dincylgiyccrol would be expecled Io activate protein kina~ C and iniliate cellular ws~nses (including ()~ generation). It is po~iNe, however that a Ohosphati- dylOmlmc prcl~rring Pi,(" acl~ in ~ome other way'on PMN membran~ to inhibit I'K(" activatmn, and thu~ ~r~e as a termination or negative feedback ~ignal. The recent experiments by Jone~ el al. (2g) sup~)~ this concept, sin~ they show Ihal c~ogcnoux PI.C. incubat~ for long ~ri~s with mouse epidermal (HEL-37) ceils. inhibit PK( aclmty hy inhibiting cylosol-mcmhranc Iranxiocation. A similar system may I~" tq~ralive in PMN. clear. ~ lhe results as they wlate to chang~ in intracellular me~enge~ or changes in inlracdlular compartmcnl~ mnst he inlcrprclcd with caution. rhc=c have I~'cn prcvitms xtudies examining the effects of exogenous PLC on PMN function Rotti and his group dem~nstralcd stimulation of ~xpiralo~ burst activity hy a PI .C prcparalion in guinea pig ncutmphils and poslulat~ involvement of prod- ucl~ of Ihc phoxphoinositide pathway ax m~iato~ nflhix effect (29.30). However. Styli r/td (fi) rt~enlly Ihtmd no eliot of (7o.ariditml pe([?ingen.~ or Bacillus" cereus Pl,('on bovine PMN (b production, bul did find a small priming effect when the PM N were subsequently stimulated with NaF. Inlerestingly they found inhibition of ~tygen mnsumotion alice particulate stimulation of PMN. a finding similar to the oh~rvalionx de~ribed herein. We found no di~ct effect of two preparations (purified h~ homogeneity by Iwo mcth~x and p~ferring phospfiatidylcholine as su~trate) of ¢' weMtfi PLC on hu man O~ or tt~O~ ~ne~tion. but found striking dose-dependent inhihitiot= Ihat ~as most pronounced during the first 8-10 rain following stimulation I~y PMA or ~1,1~. Some of these diffe~nccs may be due to the purity of the prepa~- tinn~ utiliteM, or may reflect differen~ in human ve~u~ bovine or guinea pig PMN. In summary then, we de~ri~ inhibition of Oi and l t~Oz generation in human PMN hy ext~genous PI,C following ~imulation with soluble stimuli, yet find no alter- alton m other membrane~lel~ndent functions. We also dc~fibe a striking and ina- mvdiale increase in intracelhdar Ca~' in P~xpo~d PMN. but no inhibition of ~1 P stimulated Ca~' mobilitation. These data surest thai exogenous bacterial I'1 (' (ptvl?==mg pht~sl~fi;tlidylcholi/te) effects PMN respirato~ burst activity by a medmnism thai is likely to invoh,¢ a site in the membrane between the ~LP r~ep- lot coloplex and the NADPt[ oxida~. REFERENCES 1 Ilabmt, n . Ilh~l~.q~q. liaR4 4 (it~,ltm. I I.Ihmglat S D.K~.N ~.Yamadn. O..Ot~rman. U.l'..andJ=eoh. ll.$.J Chn In*r~t 64. 266. IqTq O Slvtl, II, Walker. R I). a~tl While. J. ('. J tub {'hn 3hxl 114.5 I. ~ Maicrut. P. W. f'~mm~l% 1. M.. l~¢kmyn. II.. Ro~. T. Ifftm. "1. E.. Ishu. IL. Ban~d. V S. and ~ ( iold~lem. I. M. Rom. D.. Kaplan. ~ I. B.. aml Wei~man. G.. J ( 7m hnc~t 56. I 155. 1975. Keuwh. G l , Inh'~ Immtm g. 414. lqT~ I I I tmld~k. P ~ . I lammcm.hmldl. D, Wh~l¢. J. (~. l)alm~x~. A P., and J~b. II. S...I ('Ira bin'31 I~ l'wk, I . iml K¢~m. ~ . I h~ututm~ M~tlt~h 38, Ibl. INIIIIIrlION OF NI~IFIROI'IIII, OXIIIAIION |l'~' I k McPhatl, I ('.. I len~on. P. M.. and Johnston. R. B.. J ¢'lm In~.q ~7, ~ I~. I~1. 14. Takah~d~. I.. S~t~hara. T. anti Phyla. A.. ht "~¢t~ m I~Tmh~" II. v¢~l 71. pp. 720-725. I~1. 16 Mar'kin. W. M aml Steve ~. T. M. J l,cttk Bml ~, ~. 17. I~ien. R Y. Pnzzan. "r.. and Rink. T. J., J. ('all Bid ~ 325. I~. I ~. New~r~er. P. E.. Cho~ni~, M. E., and Cohen, I I. J.. Ilbe~ ~. I~. I ~ Smith. ('. I ).. ~n¢, B. C.. K~ I,, V¢~¢~. M. W., I~ ~n~¢tm~n, 2it. Smith. C. D., Co~, C. C.. an~ 5a~erman. R, &wan" ZIL 9L 21. Smith. C. I).. Uhning. R, J.. and Sny~an. R.. J. B~ (~*n ~61]1. I~. 22. Baxs. D. A., Gera~, C., OIh~nlz, P.. Wil~n, J., MCtE G. E., I~ ~¢~ L. ¢., 1, ~. ('~ 262. 66~3. Iqg7. 23 Besterman. J. M. Duronio. V .and ('aut~% P.. ~tw. Nag .4¢~ 24. Irving I I. R.. and Extort. J. t I.. d. ~id. ('hem. ~ ~. 25. Grdlone. I R. ('l;trk. M. A.. ~fr~. R. W.. ~. F.. =~d ~1. I 26 ('nx, J. &. leng. A. Y.. Shatk¢?, M. A., Rlum~ P. M., and Ta~t, A. I., Z (~ ~. ~ I$~. 27 ('ox. R. I.. [)auO~e~y. R. W.. (;aro~. II. R.. J. hnmtmal 136. 461 ~. 19~. 2g h)nt't. M,. and Murm% A. W.. gUa'kem, gi¢~h)'x. ~tt ek~m~. 2q Pairings. P.. ~ui. M.. Cromer. R.. and R~i. F., IJk.~L 9.~t.
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3~ CONSTr['UTIVE [~XTRAC~ULAR RELEASE OF PHOSPHATIDYLCHOLINE ~NG PHOSPHOLIPASE C (PC-PLC) BY HUMAN FIBROBLASTS AND SMOOTH MUSCLE C~ LEO 1 GORI:K)N, M.D., DAVID GUTSTEIN, SHEILA PRACHAND. AND SIGMUND A.WEITZMAN, M.D. NORTHWF_.S'~RN UNIVERSITY MEDICAL SCHOOL, DEPAR~ OF MEDICINE. DIVISION OF HEMATOLOGY/ONCOLO~Y, CHICAGO, ILL. I. INTRODUCTION The phospholipase C ('PLC) isoenzymes have been fo~u~d to be present in man), mammaliaa cells ~ well ~ in a variety of other organism.s.~ The phosphatidylinositol-specific isocnzy~es (PI-PLC) have well ~haracterized, purified to homoleneity and recently cloned.:~' These enzymes are critical components of the si[naI t.~_nsduction system in which e:~racellular signals are transmi~ed from the cell membrane by a number of mechanisms that u~ilize second me~enger molecules.' Among th~ molecule~ axe diacylglycerol (DAG) and inosi~oi phosphates, both derived from inosito| pbospholipids by the action of PI-PLC.s Recent data have suggested that some of the DAG molecules derive from the action of a different PLC. with specificity for phosphatidy|choline (PC-PLC).''~ We have shown th~,t bacterially derived PC-PLC, when incubated with intact human neu~rophits (*PMN), may influence e',,eu~ (including transmission or" signals) associated with the infi~amatory re~po~.se.~ In this repor~ we demonstrate that PC-PLC activity is measurable in supernatants from human fibroblasts and smooth muscle cells. This obse~,ation may have implications ['or the inflammatory response. II. MATERIALS AND METHODS Human smooth muscle ceils were a gift from Dr. Gerald Soft. Cells were grown in DME with 10% fetal calf serum and incubated at 37° C and 5% CO:. Cells were harvested for experiments when confluent. Human fibroblasts were a gift from Dr. J~.mcs Koztowski and were obtained from b~ign prostatic hypet'ra'ophy (BPH) dssue. C~lls were grown in RPMI at 2,7" C and 6% fetal calf serum and 5% CO.,. and most experimen~ were carried out with cells in passage 8-13 (BPH p8-13). Cells (> 90% viable at all ~ime points measured) were incubated in m~dia (Earles MEM or Hams F-12I for varying period~ of time. In some experimeu~, the super'natant that was collected after incubation w~ lay-red onto a C~nt.ricon l0 (10 kD cutoff) filter yielding retentate and t'iltra~e. Both were assayed ['or PC-PLC activity. Phosuholioase C A¢~i~city Phospbolipa~e C activiu/wa~ m~asured by a modification of ~ t~hniqu~ p~pos~ by Mac~in R~ioI~ ~ph~idyi~olia, ~C} w~ pr~ by mixing 1~1~ wi~ d~ing u~ ni~gen ~¢ 37° C. ~¢ subs~ate w~ ~en r~uspend~ ~d ~nica~ ~r t5 minute. Aliquo~ ~ uM, 0.IuCi ~H) wer~ ~d~ ~ 250 uL of0.~ M Tris acute buffer ~maining 5 uM CaCI ~ 0.5 ~ of d~xycholate. Five uL of protein ~ b~ ~ay~ w~ ~n add~ ~d ~ mixture in~ a~ 37" C ~r 30 minute. ~ r~ctiou w~ t~in~ by ~di~on of I.S ~ of bu~l/c~o~12 N HCI (1~: I~:0.6 v/v/v) ~ ~n 0.45 ~ of 1.0M HCI. vo~ ~ ~ at [~ x g. Aliquo~ of ~e ~u~us ph~e (250 ~) r~a~ ~ ~h~p~l~oli~ ~r PC-PLC) we~ ~mov~ ~ ~un~ by liqu~ i
Page 26: 50610086
326 Molecular Basis of Oxidative Damage by Leukocytes III. RESULTS W~ fotmd lh~ i~ hum~s smooth ~ ~ ~ w~ ~i~ r~ of ~-PLC, ~ ~ ~ ~e PL-P~ ~ity (~ ~ ~). TABLE 1 i~.~c~ of PC-PLC by Smoo~h M~.~le Cells~ 595+_:6 0 757+__3 6 11124-36 24 ~PC-PLC activity was measurM as d~ctibcd in Materials and Methods at the time intervals shown, ;Lad expressed as The I~-PLC activity was demo|'.graed in the rc, cmtate of a Centricoa filter with a molecular weight cutoff of I0 kD, as shown in Table 2. Tabl~ 2 Demonstration of PC-PLC activity at M.W > lOkD < I0 kD 781+.._2 > I0 kD 1322+$3 IV. DISCUSSION
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327 Z. 9. I0. V. REFERENC~ Rh~e, S.G. ct al, Studi~s of inositol phospholipid-specific phospholipase C. ~ 2~, 5~, 1989. R~. S.H. et ~, Two fo~ of ph~phafidylinosi~l-sp~ific phospholip~ C from bovine brain. Biochem Biophys R~ Comm 14t, 137, 1986. R~, S.H. ~ ~, ~rification ~d ch~acter~tion of ~o i~u~logic~ly distin~ phosphoinositide-sp~iftc phospholip~e C from ~vin~ brain. J Biol ~em 262, 12511, 1997. R~, S.H. et N, ~vine brain c~osol ~n~ ~r~ i~logi~ly disfin~ fo~ of i~sitolphospholipid-sp~ific phmpholip~e C. ~ ~SA) 84, ~, S~, P.G. et ~, Cloning ~ s~e of ~lfiple ~ of p~p~Hp~e C. ~ 54, 161, 1988. Hom~a, Y. ~ ~, l~lation ~d ch~act~ion of ~O differ ~ of inosi~l phospholipid- sp~ific p~p~lip~e C ~m ra~ b~in. ~ 263, ~, l~S. Maje~s, P.W. ~ ~, ~e m~l~m of phosphoi~si~id~eriv~ ~er m01~ul~. ~ 234, 15~9, 19~6. Be~id~, MJ., Inosiwl uiph~phate ~d dia~ylgly~rol ~ s~ m~enge~. ~ 220, 345, I9~4. ~, J.M., ~ ~, Rapid fo~a~ion o~ dia~ylglyc~ol ~m ph~h~idyi~oiine: A pa~way for gentian of s~ m~seng~. PNAS ~SA~ ~3, 67~, ~. 8 0087 I IIIIIII I | II IIII I 1~ IIII
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328 Molec,.dar Basis of Oxidative Damage by Leu]cocytes 11. 13. 14. 15. 16. 1"7. 18. I~, H.IL and Extmt. I.H., I~ylcfimlime bt~Mo~own in r~ liv~ pl~ ~. ~ 2~, ~, 19~. ~, ~W., A ~ ~ M ~ ~n: 12~r~l-13-~e ~ ~ ~ ~ ~ ~ ~ p~~li~. ~ 26t, 9128. ~n, ~I. ~ ~, ~ ~ P~ ~ m~l~m by e~ p~p~li~ C. ~ ~ A.S., ~: T~ ~ ~mr~ si~. ~ 56, 615, p~li~e D ~m. ~ ~, I~, 1987. ~, $.B. ~ ~, S~oa of l~~eml ~umulation in hepamc~ by ~pr~, ~hr~ ~ ~ ~ ~ 2~, 1420t. 1985. ~wn, LH. s .... , Pleura P~, Hew Yotk, 1987, I13.

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