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• W2013076235 abstract "An 85-kDa Group VI phospholipase A2 enzyme (iPLA2) that does not require Ca2+ for catalysis has recently been cloned from three rodent species. A homologous 88-kDa enzyme has been cloned from human B-lymphocyte lines that contains a 54-amino acid insert not present in the rodent enzymes, but human cells have not previously been observed to express catalytically active iPLA2 isoforms other than the 88-kDa protein. We have cloned cDNA species that encode two distinct iPLA2 isoforms from human pancreatic islet RNA and a human insulinoma cDNA library. One isoform is an 85-kDa protein (short isoform of human iPLA2 (SH-iPLA2)) and the other an 88-kDa protein (long isoform of human iPLA2(LH-iPLA2)). Transcripts encoding both isoforms are also observed in human promonocytic U937 cells. Recombinant SH-iPLA2 and LH-iPLA2 are both catalytically active in the absence of Ca2+ and inhibited by a bromoenol lactone suicide substrate, but LH-iPLA2 is activated by ATP, whereas SH-iPLA2 is not. The human iPLA2gene has been found to reside on chromosome 22 in region q13.1 and to contain 16 exons represented in the LH-iPLA2 transcript. Exon 8 is not represented in the SH-iPLA2 transcript, indicating that it arises by an exon-skipping mechanism of alternative splicing. The amino acid sequence encoded by exon 8 of the human iPLA2 gene is proline-rich and shares a consensus motif of PX 5PX 8HHPX 12NX 4Q with the proline-rich middle linker domains of the Smad proteins DAF-3 and Smad4. Expression of mRNA species encoding two active iPLA2 isoforms with distinguishable catalytic properties in two different types of human cells demonstrated here may have regulatory or functional implications about the roles of products of the iPLA2 gene in cell biologic processes. An 85-kDa Group VI phospholipase A2 enzyme (iPLA2) that does not require Ca2+ for catalysis has recently been cloned from three rodent species. A homologous 88-kDa enzyme has been cloned from human B-lymphocyte lines that contains a 54-amino acid insert not present in the rodent enzymes, but human cells have not previously been observed to express catalytically active iPLA2 isoforms other than the 88-kDa protein. We have cloned cDNA species that encode two distinct iPLA2 isoforms from human pancreatic islet RNA and a human insulinoma cDNA library. One isoform is an 85-kDa protein (short isoform of human iPLA2 (SH-iPLA2)) and the other an 88-kDa protein (long isoform of human iPLA2(LH-iPLA2)). Transcripts encoding both isoforms are also observed in human promonocytic U937 cells. Recombinant SH-iPLA2 and LH-iPLA2 are both catalytically active in the absence of Ca2+ and inhibited by a bromoenol lactone suicide substrate, but LH-iPLA2 is activated by ATP, whereas SH-iPLA2 is not. The human iPLA2gene has been found to reside on chromosome 22 in region q13.1 and to contain 16 exons represented in the LH-iPLA2 transcript. Exon 8 is not represented in the SH-iPLA2 transcript, indicating that it arises by an exon-skipping mechanism of alternative splicing. The amino acid sequence encoded by exon 8 of the human iPLA2 gene is proline-rich and shares a consensus motif of PX 5PX 8HHPX 12NX 4Q with the proline-rich middle linker domains of the Smad proteins DAF-3 and Smad4. Expression of mRNA species encoding two active iPLA2 isoforms with distinguishable catalytic properties in two different types of human cells demonstrated here may have regulatory or functional implications about the roles of products of the iPLA2 gene in cell biologic processes. bromoenol lactone base pair(s) 4′, 6-diamidino-2-phenylindole fluorescence in situ hybridization isopropyl-1-thio-β-d-galactopyranoside kilobase pair(s) polymerase chain reaction reverse transcription Spodoptera frugiperda, type 9 phospholipase A2 Group IV PLA2 Group VI PLA2 secretory PLA2 long isoform of human iPLA2 short isoform of human iPLA2 Phospholipases A2(PLA2)1 catalyze hydrolysis of sn-2 fatty acid substituents from glycerophospholipid substrates to yield a free fatty acid and a 2-lysophospholipid (1Dennis E.A. J. Biol. Chem. 1994; 269: 13057-13060Abstract Full Text PDF PubMed Google Scholar, 2Gijon M.A. Leslie C.C. Cell Dev. Biol. 1997; 8: 297-303Crossref PubMed Scopus (60) Google Scholar, 3Dennis E.A. Trends Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (758) Google Scholar, 4Balsinde J. Dennis E.A. J. Biol. Chem. 1997; 272: 16069-16072Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 5Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar, 6Tischfield J.A. J. Biol. Chem. 1997; 272: 17247-17250Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 7Stafforini D.M. McIntyre T.M. Zimmerman G.A. Prescott S.M. J. Biol. Chem. 1997; 272: 17895-17898Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). PLA2 is a diverse group of enzymes, and the first well characterized members have low molecular masses (approximately 14 kDa), require millimolar [Ca2+] for catalytic activity, and function as extracellular secreted enzymes (sPLA2) (3Dennis E.A. Trends Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (758) Google Scholar, 6Tischfield J.A. J. Biol. Chem. 1997; 272: 17247-17250Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). The first cloned PLA2 that is active at [Ca2+] achieved in the cytosol of living cells is an 85-kDa protein classified as a Group IV PLA2 and designated cPLA2 (3Dennis E.A. Trends Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (758) Google Scholar, 5Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar). This enzyme is induced to associate with its substrates in membranes by rises in cytosolic [Ca2+] within the range achieved in cells stimulated by extracellular signals that induce Ca2+ release from intracellular sites or Ca2+ entry from the extracellular space, is also regulated by phosphorylation, and prefers substrates with sn-2 arachidonoyl residues (5Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar). Recently, a second PLA2 that is active at [Ca2+] that can be achieved in cytosol has been cloned (8Tang J. Kriz R.W. Wolfman N. Shaffer M. Seehra J. Jones S. J. Biol. Chem. 1997; 272: 8567-8575Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 9Balboa M.A. Balsinde J. Jones S.S. Dennis E.A. J. Biol. Chem. 1997; 272: 8576-8580Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 10Ma Z. Ramanadham S. Kempe K. Chi X.S. Ladenson J. Turk J. J. Biol. Chem. 1997; 272: 11118-11127Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). This enzyme does not require Ca2+ for catalysis, is classified as a Group VI PLA2, and is designated iPLA2 (3Dennis E.A. Trends Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (758) Google Scholar, 4Balsinde J. Dennis E.A. J. Biol. Chem. 1997; 272: 16069-16072Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). The iPLA2 enzymes cloned from hamster (8Tang J. Kriz R.W. Wolfman N. Shaffer M. Seehra J. Jones S. J. Biol. Chem. 1997; 272: 8567-8575Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar), mouse (9Balboa M.A. Balsinde J. Jones S.S. Dennis E.A. J. Biol. Chem. 1997; 272: 8576-8580Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), and rat (10Ma Z. Ramanadham S. Kempe K. Chi X.S. Ladenson J. Turk J. J. Biol. Chem. 1997; 272: 11118-11127Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) cells represent species homologs and all are 85-kDa proteins containing 752 amino acid residues with highly homologous (approximately 95% identity) sequences. Each contains a GXSXG lipase consensus motif and eight stretches of a repeating motif homologous to a repetitive motif in the integral membrane protein-binding domain of ankyrin (8Tang J. Kriz R.W. Wolfman N. Shaffer M. Seehra J. Jones S. J. Biol. Chem. 1997; 272: 8567-8575Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 9Balboa M.A. Balsinde J. Jones S.S. Dennis E.A. J. Biol. Chem. 1997; 272: 8576-8580Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 10Ma Z. Ramanadham S. Kempe K. Chi X.S. Ladenson J. Turk J. J. Biol. Chem. 1997; 272: 11118-11127Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The substrate preference of these iPLA2 enzymes varies with the mode of presentation (8Tang J. Kriz R.W. Wolfman N. Shaffer M. Seehra J. Jones S. J. Biol. Chem. 1997; 272: 8567-8575Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar), but each is inhibited (8Tang J. Kriz R.W. Wolfman N. Shaffer M. Seehra J. Jones S. J. Biol. Chem. 1997; 272: 8567-8575Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 9Balboa M.A. Balsinde J. Jones S.S. Dennis E.A. J. Biol. Chem. 1997; 272: 8576-8580Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 10Ma Z. Ramanadham S. Kempe K. Chi X.S. Ladenson J. Turk J. J. Biol. Chem. 1997; 272: 11118-11127Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) by a bromoenol lactone (BEL) suicide substrate (11Hazen S.L. Zupan L.A. Weiss R.H. Getman D.P. Gross R.W. J. Biol. Chem. 1991; 266: 7227-7232Abstract Full Text PDF PubMed Google Scholar, 12Zupan L.A. Weiss R.H. Hazen S. Parnas B.L. Aston K.W. Lennon P.J. Getman D.P. Gross R.W. J. Med. Chem. 1993; 36: 95-100Crossref PubMed Scopus (55) Google Scholar) that is not an effective inhibitor of sPLA2 or cPLA2 enzymes at comparable concentrations (4Balsinde J. Dennis E.A. J. Biol. Chem. 1997; 272: 16069-16072Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 11Hazen S.L. Zupan L.A. Weiss R.H. Getman D.P. Gross R.W. J. Biol. Chem. 1991; 266: 7227-7232Abstract Full Text PDF PubMed Google Scholar, 12Zupan L.A. Weiss R.H. Hazen S. Parnas B.L. Aston K.W. Lennon P.J. Getman D.P. Gross R.W. J. Med. Chem. 1993; 36: 95-100Crossref PubMed Scopus (55) Google Scholar, 13Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 6758-6765Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar, 14Ma Z. Ramanadham S. Hu Z. Turk J. Biochim. Biophys. Acta. 1998; 1391: 384-400Crossref PubMed Scopus (44) Google Scholar). Proposed functions for iPLA2 include a housekeeping role in phospholipid remodeling that involves generation of lysophospholipid acceptors for arachidonic acid incorporation into P388D1 macrophage-like cell phospholipids (4Balsinde J. Dennis E.A. J. Biol. Chem. 1997; 272: 16069-16072Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 15Balsinde J. Bianco I.D. Ackerman E.J. Conde-Friebos K. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8527-8531Crossref PubMed Scopus (257) Google Scholar, 16Balsinde J. Balboa M.A. Dennis E.A. J. Biol. Chem. 1997; 272: 29317-29321Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Signaling roles for iPLA2 in generating substrate for leukotriene biosynthesis (17Larsson Forsell P.K.A. Runarsson G. Ibrahim M. Bjorkholm M. Claesson H.-E. FEBS Lett. 1998; 434: 295-299Crossref PubMed Scopus (35) Google Scholar) and lipid messengers that regulate ion channel activity (10Ma Z. Ramanadham S. Kempe K. Chi X.S. Ladenson J. Turk J. J. Biol. Chem. 1997; 272: 11118-11127Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 18Gubitosi-Klug R.A. Yu S.P. Choi D.W. Gross R.W. J. Biol. Chem. 1995; 270: 2885-2888Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar,19Eddlestone G.T. Am. J. Physiol. 1995; 268: C181-C190Crossref PubMed Google Scholar) and apoptosis (20Atsumi G. Tajima M. Hadano A. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 13870-13877Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar) have also been suggested. Recent observations with human iPLA2 suggest that the enzyme might serve distinct functions in different cells that involve regulatory interactions among splice variants (17Larsson Forsell P.K.A. Runarsson G. Ibrahim M. Bjorkholm M. Claesson H.-E. FEBS Lett. 1998; 434: 295-299Crossref PubMed Scopus (35) Google Scholar, 21Larsson P.K.A. Claesson H.-E. Kennedy B.P. J. Biol. Chem. 1998; 273: 207-214Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Human iPLA2cloned from B-lymphocyte lines and testis differs from iPLA2 cloned from cells of rodent species in that it is an 88-kDa rather than an 85-kDa protein and contains a 54-amino acid insert interrupting the eighth ankyrin repeat (21Larsson P.K.A. Claesson H.-E. Kennedy B.P. J. Biol. Chem. 1998; 273: 207-214Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). The human B-lymphocyte iPLA2 sequence is otherwise highly homologous to hamster, mouse, and rat sequences and includes the seven other ankyrin-like repeats and a GXSXG lipase sequence (21Larsson P.K.A. Claesson H.-E. Kennedy B.P. J. Biol. Chem. 1998; 273: 207-214Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Catalytically active iPLA2 other than the 88-kDa isoform have not yet been observed in human cells (21Larsson P.K.A. Claesson H.-E. Kennedy B.P. J. Biol. Chem. 1998; 273: 207-214Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Human B-lymphocyte lines do express truncated, inactive iPLA2 sequences that contain the ankyrin repeat domain but lack the catalytic domain and are thought to arise from alternative splicing of the transcript (21Larsson P.K.A. Claesson H.-E. Kennedy B.P. J. Biol. Chem. 1998; 273: 207-214Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Co-expression of the truncated sequences with full-length human iPLA2 attenuates catalytic activity (21Larsson P.K.A. Claesson H.-E. Kennedy B.P. J. Biol. Chem. 1998; 273: 207-214Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Because the active form of iPLA2 is an oligomeric complex (8Tang J. Kriz R.W. Wolfman N. Shaffer M. Seehra J. Jones S. J. Biol. Chem. 1997; 272: 8567-8575Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 22Ackerman E.J. Kempner E.S. Dennis E.A. J. Biol. Chem. 1994; 269: 9227-9233Abstract Full Text PDF PubMed Google Scholar) that may result from subunit associations through ankyrin repeat domains (8Tang J. Kriz R.W. Wolfman N. Shaffer M. Seehra J. Jones S. J. Biol. Chem. 1997; 272: 8567-8575Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar), this suggests that formation of hetero-oligomeric complexes represents a means to regulate iPLA2 activity (21Larsson P.K.A. Claesson H.-E. Kennedy B.P. J. Biol. Chem. 1998; 273: 207-214Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). That mechanisms of iPLA2regulation differ among human cell types is suggested by the fact that stimuli that induce iPLA2-catalyzed arachidonate release and leukotriene production in human granulocytes fail to induce these events in human lymphocyte lines, even though both classes of cells express iPLA2 and leukotriene biosynthetic enzymes (17Larsson Forsell P.K.A. Runarsson G. Ibrahim M. Bjorkholm M. Claesson H.-E. FEBS Lett. 1998; 434: 295-299Crossref PubMed Scopus (35) Google Scholar,21Larsson P.K.A. Claesson H.-E. Kennedy B.P. J. Biol. Chem. 1998; 273: 207-214Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). One human cell type in which iPLA2 may be biomedically important is the pancreatic islet beta cell. Impaired beta cell survival and signaling functions underlie development of types I and II diabetes mellitus, respectively; these are the most prevalent human endocrine diseases. In rodent islets, iPLA2 has been proposed to play a signaling role in glucose-induced insulin secretion (8Tang J. Kriz R.W. Wolfman N. Shaffer M. Seehra J. Jones S. J. Biol. Chem. 1997; 272: 8567-8575Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 23Ramanadham S. Gross R.W. Han X. Turk J. Biochemistry. 1993; 32: 337-346Crossref PubMed Scopus (123) Google Scholar, 24Ramanadham S. Bohrer A. Mueller M. Jett P. Gross R. Turk J. Biochemistry. 1993; 32: 5339-5351Crossref PubMed Scopus (73) Google Scholar, 25Ramanadham S. Wolf M.J. Jett P.A. Gross R.W. Turk J. Biochemistry. 1994; 33: 7442-7452Crossref PubMed Scopus (64) Google Scholar) and in experimentally induced beta cell apoptosis (26Zhou Y.-P. Teng D. Drayluk F. Ostrega D. Roe M.W. Philipson L. Polonsky K.S. J. Clin. Invest. 1998; 101: 1623-1632Crossref PubMed Scopus (119) Google Scholar). Human islets express a BEL-sensitive PLA2 activity that does not require Ca2+ (27Gross R.W. Ramanadham S. Kruszka K. Han X. Turk J. Biochemistry. 1993; 32: 327-336Crossref PubMed Scopus (113) Google Scholar, 28Ramanadham S. Bohrer A. Gross R.W. Turk J. Biochemistry. 1993; 32: 13499-13509Crossref PubMed Scopus (57) Google Scholar), but iPLA2mRNA has not been demonstrated in human islets. We have cloned human beta cell iPLA2 cDNA here and find that human islets express mRNA species encoding two iPLA2 isoforms with different sizes (85 and 88 kDa) and catalytic properties. We have also determined the human iPLA2 gene structure and its chromosomal location and find that the transcript encoding the short isoform arises from an exon-skipping mechanism of alternative splicing. The compounds [32P]dCTP (3000 Ci/mmol), [35S]dATPS (1000 Ci/mmol), andl-α-1-palmitoyl-2-[14C]arachidonoyl-phosphatidylethanolamine (50 mCi/mmol) and ECL detection reagents were obtained from Amersham Pharmacia Biotech, and the BEL ((E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one) iPLA2 suicide substrate was obtained from BIOMOL (Plymouth Meeting, PA). A human placental genomic DNA library in lambda FIX II was obtained from Stratagene (La Jolla, CA). Human promonocytic U937 cells (30Sundstrom C. Nilsson K. Int. J. Cancer. 1976; 17: 565-577Crossref PubMed Scopus (1954) Google Scholar) were obtained from American Type Culture Collection (Manassas, VA) and cultured as described (20Atsumi G. Tajima M. Hadano A. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 13870-13877Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 31Rzigalinsky B.A. Blackmore P.R. Rosenthal M.D. Biochim. Biophys. Acta. 1996; 1299: 342-352Crossref PubMed Scopus (41) Google Scholar). Sources of other common materials are identified elsewhere (10Ma Z. Ramanadham S. Kempe K. Chi X.S. Ladenson J. Turk J. J. Biol. Chem. 1997; 272: 11118-11127Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 14Ma Z. Ramanadham S. Hu Z. Turk J. Biochim. Biophys. Acta. 1998; 1391: 384-400Crossref PubMed Scopus (44) Google Scholar, 23Ramanadham S. Gross R.W. Han X. Turk J. Biochemistry. 1993; 32: 337-346Crossref PubMed Scopus (123) Google Scholar, 24Ramanadham S. Bohrer A. Mueller M. Jett P. Gross R. Turk J. Biochemistry. 1993; 32: 5339-5351Crossref PubMed Scopus (73) Google Scholar, 25Ramanadham S. Wolf M.J. Jett P.A. Gross R.W. Turk J. Biochemistry. 1994; 33: 7442-7452Crossref PubMed Scopus (64) Google Scholar, 28Ramanadham S. Bohrer A. Gross R.W. Turk J. Biochemistry. 1993; 32: 13499-13509Crossref PubMed Scopus (57) Google Scholar). Rat islet iPLA2 cDNA was isolated (10Ma Z. Ramanadham S. Kempe K. Chi X.S. Ladenson J. Turk J. J. Biol. Chem. 1997; 272: 11118-11127Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), labeled with32P, and used to screen a human insulinoma cDNA library (32Ferrer J. Wasson J. Permutt A. Diabetologia. 1996; 38: 891-898Crossref Scopus (43) Google Scholar) provided by Dr. Alan Permutt of Washington University. Insert sizes in clones that hybridized with the probe were determined by digestion with restriction endonucleases, and their sequences were determined from the double strand (33Sanger F. Nickeln S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52769) Google Scholar). Two cDNA species were obtained that contained about 1.80 and 1.59 kb, respectively, of the 3′-sequence of human iPLA2 cDNA, including the poly(A) tail. Neither contained the 5′-end of the full coding sequence, and RT-PCR was therefore performed with human islet RNA. Islets were isolated from human pancreata in the Washington University Diabetes Research and Training Center (34Ricordi C. Lacy P.E. Finke E.H. Olack B.J. Scharp D.W. Diabetes. 1988; 37: 413-420Crossref PubMed Google Scholar) and cultured as described (35Ramanadham S. Hsu F.-F. Bohrer A. Nowatzke W. Ma Z. Turk J. Biochemistry. 1998; 37: 4553-4567Crossref PubMed Scopus (68) Google Scholar). Total RNA was isolated from human islets and promonocytic U937 cells and first strand cDNA prepared by reverse transcription (RT) using standard procedures (36Davis L.G. Kuehl W.M. Battery J.F. Wonsiewicz M. Greenfield S. Basic Methods in Molecular Biology. 2nd Ed. Appleton and Lange, East Norwalk, CT1994: 335-338Google Scholar). PCRs were performed under described conditions (10Ma Z. Ramanadham S. Kempe K. Chi X.S. Ladenson J. Turk J. J. Biol. Chem. 1997; 272: 11118-11127Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), and products were analyzed by agarose gel electrophoresis (36Davis L.G. Kuehl W.M. Battery J.F. Wonsiewicz M. Greenfield S. Basic Methods in Molecular Biology. 2nd Ed. Appleton and Lange, East Norwalk, CT1994: 335-338Google Scholar). Primers used to generate the 5′ portion of human iPLA2 cDNA were sense (5′-GATGCAGTTCTTTGGACGCCTGG-3′), antisense (5′-T-CAGCATCACCTTGGGT-TTCC-3′), and nested antisense (5-AATGGCCAGGGCCAGGATG-C-3′). Two distinct cDNA fragments were obtained, subcloned, sequenced, and found to extend from the 5′-initiator codon through about 1.59 and 1.79 kb of DNA, respectively. The cDNA fragments obtained from screening the human insulinoma cell cDNA library overlapped at their 5′-ends with 3′-ends of cDNA fragments from RT-PCR of human islet RNA, and the overlapping region contained anNcoI site. The fragments were subcloned into pBluescript SK. Fragments from RT-PCR of human islet RNA contained the iPLA2 5′-coding sequence and were released from plasmids with EcoRI and NcoI. Products were isolated by agarose gel electrophoresis and ligated with a plasmid containing the 3′-end of human iPLA2 cDNA that had been treated withNcoI. Ligation product plasmids were used to transform bacterial host cells and sequenced. The resultant cDNA species contained complete coding sequences of human iPLA2 isoforms and were inserted into appropriate vectors for expression and used to prepare 32P-labeled human iPLA2 cDNA for genomic screening. The cDNA species encoding full-length human islet iPLA2 isoforms were subcloned in-frame into theEcoRI and XhoI sites of pET-28c (Novagen). The constructs were analyzed by restriction endonuclease digestion, sequenced, and transformed into bacterial expression host BL21(DE3) (Novagen). Cells transformed with pET28c without insert were negative controls. Protein expression was induced by treating cells with 0.5 mm isopropyl-1-thio-β-d-galactopyranoside (IPTG) and assessed by SDS-polyacrylamide gel electrophoresis analyses with Coomassie Blue staining and by immunoblotting under described conditions (10Ma Z. Ramanadham S. Kempe K. Chi X.S. Ladenson J. Turk J. J. Biol. Chem. 1997; 272: 11118-11127Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) with a rabbit polyclonal antibody against recombinant rat islet iPLA2. The Spodoptera frugiperda(Sf9) insect cell-baculovirus system used to express other PLA2 enzymes in catalytically active forms (37Becker G.W. Miller J.R. Kovacevic S. Ellis R. Louis A.I. Small J.S. Stark D.H. Roberts E.F. Wyrick T.K. Hoskins J. Chious G. Sharp J.D. McClure D.B. Rioggin R.M. Kramer R.M. Bio/Technology. 1994; 12: 69-74PubMed Google Scholar, 38de Carvalho M.S. McCormack A.L. Olsen E. Ghomaschchi F. Gelb M.H. Yates III, J.R. Leslie C.C. J. Biol. Chem. 1996; 271: 6987-6997Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) was used to express human iPLA2 isoforms. The iPLA2cDNA inserts were released from pBluescript SK plasmids by digestion with EcoRI and XhoI and subcloned intoEco RI and Xho I sites of pBAC-1 baculovirus transfer plasmid (Novagen). Sf9 cells were co-transfected with this transfer plasmid and linearized baculovirus DNA (BacVector-2000, Novagen) to construct recombinant baculovirus with human islet iPLA2 isoform cDNA inserts. Infection and culture were performed under described conditions (47Nowatzke W. Ramanadham S. Ma Z. Hsu F.-F. Bohrer A. Turk J. Endocrinology. 1998; 139: 4073-4085Crossref PubMed Scopus (55) Google Scholar). At 48 h after infection, Sf9 cells were collected by centrifugation, washed, resuspended in buffer (250 mm sucrose, 25 mmimidazole, pH 8.0), and disrupted by sonication. Cytosolic and membranous fractions were prepared by sequential centrifugations (10,000 × g for 10 min and 100,000 ×g for 60 min) and used for PLA2 activity assays. The protein content of Sf9 cell cytosolic and membranous fractions was determined by Bio-Rad assay, and iPLA2 activity was measured in aliquots (approximately 20 μg of protein) added to assay buffer (200 mm Tris-HCl, pH 7.0; total assay volume, 200 μl) containing 5 mm EGTA with or without 1 mmATP. Some aliquots were pretreated (2 min) with BEL (10 μm) before the assay. Reactions were initiated by injecting substrate (l-α-1-palmitoyl-2-[14C]arachidonoyl-phosphatidylethanolamine; specific activity, 50 Ci/mol; final concentration, 5 μm) in ethanol (5 μl). Assay mixtures were incubated (3 min at 37 °C), and reactions were terminated by adding butanol (0.1 ml) and vortexing. After centrifugation (2000 × g for 4 min), products in the butanol layer were analyzed by silica gel G TLC in hexane/ethyl ether/acetic acid (80:20:1). The TLC region containing free arachidonic acid (R F, 0.58) was scraped into vials, and its14C content was determined. A 32P-labeled human islet iPLA2cDNA was used to screen a human placental Lambda FIX II genomic DNA library (Stratagene). Clones that hybridized with the probe were isolated and plaque-purified, and the lambda DNA fragments containing genomic DNA inserts were purified by standard procedures (36Davis L.G. Kuehl W.M. Battery J.F. Wonsiewicz M. Greenfield S. Basic Methods in Molecular Biology. 2nd Ed. Appleton and Lange, East Norwalk, CT1994: 335-338Google Scholar). Inserts were excised with NotI and subcloned into a pBluescript SK plasmid for restriction site mapping. Sequences of intron-exon boundaries were determined by comparing sequences of genomic DNA and cDNA. Intron sizes were estimated from lengths of PCR products from reactions using genomic DNA as template and primers that hybridize to sequences in adjacent exons. A human iPLA2genomic DNA clone was biotinylated with dATP (40Heng H.H.Q. Squire J. Tsui L.-C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9509-9513Crossref PubMed Scopus (521) Google Scholar, 41Heng H.H.Q. Tsui L.-C. Chromosoma. 1993; 102: 325-332Crossref PubMed Scopus (431) Google Scholar) and used as a probe to map the chromosomal location of the human iPLA2gene. Fluorescence in situ hybridization (FISH) detection of the locus of hybridization of the fluorescent probe with chromosomal DNA was performed by See DNA Biotech Inc. (Downsview, Ontario, Canada) using described methods (40Heng H.H.Q. Squire J. Tsui L.-C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9509-9513Crossref PubMed Scopus (521) Google Scholar, 41Heng H.H.Q. Tsui L.-C. Chromosoma. 1993; 102: 325-332Crossref PubMed Scopus (431) Google Scholar). Human blood lymphocytes were cultured in α-minimal essential medium supplemented with 10% fatal calf serum and phytohemagglutinin at 37 °C for 68–72 h. Bromodeoxyuridine (0.18 mg/ml, Sigma) was used to synchronize the cell populations, which were then washed three times with serum-free medium to release the block and recultured (37 °C, 6 h) in α-minimal essential medium with thymine (2.5 μg/ml, Sigma). Cells were harvested and slides were prepared by standard procedures, including hypotonic treatment, fixation, and air-drying (40Heng H.H.Q. Squire J. Tsui L.-C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9509-9513Crossref PubMed Scopus (521) Google Scholar, 41Heng H.H.Q. Tsui L.-C. Chromosoma. 1993; 102: 325-332Crossref PubMed Scopus (431) Google Scholar), and the slides were baked (55 °C, 1 h). After RNase treatment, slides were denatured in 70% formamide and dehydrated with ethanol. Probes were denatured (75 °C, 5 min) in a hybridization mixture (50% formamide, 10% dextran sulfate, and human cot I DNA). After incubation (15 min, 37 °C) to suppress repetitive sequences, probes were loaded on denatured chromosomal slides, which were incubated overnight, washed, and subjected to detection and amplification procedures. FISH signals and DAPI banding patterns were recorded in separate photographs. Assignment of FISH mapping data with chromosomal bands was achieved by superimposing FISH signals with DAPI banding pattern on the chromosomes (40Heng H.H.Q. Squire J. Tsui L.-C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9509-9513Crossref PubMed Scopus (521) Google Scholar, 41Heng H.H.Q. Tsui L.-C. Chromosoma. 1993; 102: 325-332Crossref PubMed Scopus (431) Google Scholar). To determine whether human pancreatic islet beta cells express mRNA species encoding iPLA2, a human insulinoma cell cDNA library (32Ferrer J. Wasson J. Permutt A. Diabetologia. 1996; 38: 891-898Crossref Scopus (43) Google Scholar) was screened with a 32P-labeled rat iPLA2 cDNA (10Ma Z. Ramanadham S. Kempe K. Chi X.S. Ladenson J. Turk J. J. Biol. Chem. 1997; 272: 11118-11127Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) probe. Two clones (INS-C1 and INS-C2) of about 1.59 and 1.80 kb in length, respectively, hybridized to the probe and were sequenced. Both clones contained identical 3′-sequences that in" @default.
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• W2013076235 title "Human Pancreatic Islets Express mRNA Species Encoding Two Distinct Catalytically Active Isoforms of Group VI Phospholipase A2 (iPLA2) That Arise from an Exon-skipping Mechanism of Alternative Splicing of the Transcript from the iPLA2 Gene on Chromosome 22q13.1" @default.
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