ARTICLE |
Correspondence to: Timo J. Nevalainen, Dept. of Pathology, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. E-mail: timo.nevalainen@utu.fi
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Summary |
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Group II phospholipase A2 (PLA2) is an acute-phase protein and an important component of the host defense against bacteria. In this study we investigated the distribution of PLA2 protein by immunohistochemistry and the distribution of mRNA of PLA2 by Northern blotting and in situ hybridization in rat tissues. PLA2 protein was localized in the Paneth cells of the intestinal mucosa, chondrocytes and the matrix of cartilage, and megakaryocytes in the spleen. By Northern blotting, mRNA of PLA2 was found in the gastrointestinal tract, lung, heart, and spleen. By in situ hybridization, PLA2 mRNA was localized in the Paneth cells of the small intestinal mucosa but in no other cell types. Our results show specific distribution of PLA2 in a limited number of cell types in rat tissues. The reagents developed in this study (the anti-rat PLA2 antibody and probes for Northern blotting and in situ hybridization of mRNA of rat PLA2) will provide useful tools for future studies concerning the role of PLA2 in various experimental disease models. (J Histochem Cytochem 48:14691477, 2000)
Key Words: immunohistochemisty, in situ hybridization, Northern blotting, phospholipase A2, polyclonal antibody, rat, recombinant protein
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Introduction |
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PHOSPHOLIPASE A2 plays an important role in inflammation as a rate-limiting enzyme in the production of proinflammatory mediators derived from arachidonic acid (
The gene coding for rat Group II phospholipase A2 was cloned from the spleen (
The purpose of this study was to investigate the distribution of PLA2 protein and mRNA in rat tissues. We produced recombinant rat PLA2 and polyclonal antibodies against it, and localized the PLA2 protein by immunohistochemistry and the mRNA of PLA2 by Northern and in situ hybridizations. Our results show that PLA2 is expressed in few specific cell types. The strongest expression of PLA2 is found in the Paneth cells of the intestinal mucosa.
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Materials and Methods |
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Reagents
Rat Group II phospholipase A2 cDNA sequence cloned in the EcoRI and XbaI sites of pGEM4 (Promega; Madison, WI) was obtained from Shionogi Research Laboratories (Osaka, Japan). The bacterial expression vector pQE-60 was purchased from QIAGEN (Chatsworth, CA). Restriction enzymes were purchased from Boehringer Mannheim (Mannheim, Germany) except for BspHI, which was from New England Biolabs (Beverly, MA). DNA ligase was from Life Technologies, Gibco BRL (Paisley, UK). Primers were synthesized at the University of Kuopio (Kuopio, Finland). E. coli JM109 cells were obtained from Department of Microbiology (University of Turku, Turku, Finland) and M15 and SG13009 cells from QIAGEN. Unless otherwise noted, all other reagents were from Sigma (St Louis, MO).
PCR Amplification
Three oligonucleotide primers of sense and antisense orientations based on the sequence of rat PLA2 cDNA were synthesized. The forward primer sequences were (412) 5'-ATA TCC ATG GAT GAA GGT CCT CCT GTT GC-'3 and (551) 5'-ATA TTC ATG AGC CTT CTG GAG TTT GGG-'3 and the reverse sequence (413) 5'-ATA AGA TCT GCA ACT GGG CGT CTT CCC-'3. The region of rat PLA2 cDNA that encodes the signal peptide and the mature protein was amplified by PCR. Primer 412 has a unique NcoI site. Biotinylated primer 551 contained the sequence for BspHI and the initiating methionine. Primer 413 has a unique BglII site. Rat PLA2 contains the BspHI site in the protein coding sequence starting at nucleotide 258. PCR was carried out in 100-µl reaction volume containing 100 mM Tris-HCl (pH 8.3), 0.2 mM dNTP, 1 µM antisense primer, 1 µM sense primer, and 2.5 U Taq DNA polymerase (Finnzymes; Espoo, Finland). Conditions for 35 cycles of PCR were 95C/30 sec, 58C/60 sec, and 72C/45 sec.
Cloning and Expression of Rat Group II Phospholipase A2
Cloning of rat PLA2 was carried out in two steps (Fig 1). The first PCR was done with primers 412 and 413. The product was extracted from the gel and digested with NcoI and BglII. The digested product was ligated to pQE-60 vector previously digested with NcoI and BglII. This construct (pQE-60-PLA2-sham), which is not in frame, was used as the first step in cloning and later as a negative control. The second step was PCR with primers 551 and 413. The PCR product was attached to streptavidin-coated magnetic beads (Boehringer Mannheim). The product was digested with BamHI, washed, digested with BspHI, and recovered. The product was ligated and cloned to the pQE-60-PLA2-sham vector previously digested with NcoI and BamHI, resulting in the plasmid pQE-60-PLA2. Finally, successful construction was confirmed by sequencing.
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The plasmid pQE-60-PLA2 was transformed into E. coli strain JM 109. Transformants were selected on LBagar plates supplemented with 100 µg/ml ampicillin. For induction of gene expression, E. coli JM109 cells containing pQE-60-PLA2 were grown at 30C in LB medium containing 100 µg/ml ampicillin. After reaching an OD550 = 1.0, isopropyl-ß-D-thio-galactoside (IPTG) was added to a final concentration of 1 mM. The cells were harvested by centrifugation and stored as pellets at -70C.
Preparation of Recombinant Rat Group II Phospholipase A2
Recombinant PLA2 was found exclusively in the inclusion bodies of E. coli. A cell pellet from 300 ml of bacterial culture (1.5 g wet weight) was thoroughly washed with buffer containing 100 mM Tris-HCl (pH 7.3) and 150 mM NaCl. The cells were lysed with an ultrasound sonicator (Labsonic L; Braun Diessel Biotech, Melsungen, Germany) at maximal power twice for 30 sec. Lysozyme was added to a final concentration of 1 mg/ml, incubated for 30 min at room temperature (RT) and sonicated. The pellet was harvested by centrifugation at 10,000 x g for 10 min and resuspended in 20 ml buffer containing 6 M guanidine hydrochloride and 100 mM Tris-HCl (pH 7.3). After incubation overnight at RT, silica was added to a final concentration of 1% and the cell lysate was pelleted by centrifugation. The supernatant was recovered and ß-mercaptoethanol was added to a final concentration of 0.2%. The recombinant PLA2 was further purified by using nickel affinity chromatography. Briefly, a 20-ml sample was loaded onto an affinity column charged with Ni2+ and washed with 6 M guanidine hydrochloride, pH 7.6 and pH 5.3, and PLA2 was eluted with 6 M guanidine hydrochloride, pH 4.1. The eluate was dialyzed against water overnight at 4C. Amino-acid sequence analysis was performed with an Applied Biosystems (Foster City, CA) model 477A protein sequencer equipped with an on-line Applied Biosystems model 120A phenylthiohydantoin amino acid analyzer. The sample for mass spectrometry was mixed with sinapinic acid matrix and analyzed by a matrix-assisted laser desorption/ionization mass spectrometer (MALDI-MS; Lasermat, Finnigan Mat, Bremen, Germany).
Measurement of the Catalytic Activity of Phospholipase A2
The catalytic activity of phospholipase A2 was measured by a modified radiometric method (-1-palmitoyl-2-arachidonyl-containing phosphatidyl ethanolamine (NEN Life Science Products; Boston, MA) as a substrate.
Preparation of Antiserum
Three New Zealand White rabbits were immunized SC with 250 µg of purified recombinant rat PLA2 in Freund's complete adjuvant. Four booster injections of 200 µg of PLA2 in Freund's incomplete adjuvant were administered after 2, 4, 6, and 8 weeks. Blood was collected from each rabbit before each booster injection.
Western Blotting
Purified recombinant rat PLA2 and ileal lysates from SpragueDawley rats were separated in 415% SDS-PAGE gels (Phastsystem; Pharmacia, Uppsala, Sweden) and transferred to nitrocellulose filters (Schleicher & Schuell; Dassel, Germany). Anti-rat PLA2 antiserum diluted 1:5000 was used as the primary antibody. The immunoreaction was detected with a Vectastain avidinbiotinperoxidase complex (ABC) kit (Vector Laboratories; Burlingame, CA) according to the manufacturer's instructions.
Immunohistochemistry
Tissues from various rat organs were fixed in 10% phosphate-buffered formalin and embedded in paraffin. Sections were incubated with several different dilutions ranging from 1:5000 to 1:20,000 of anti-rat PLA2 antiserum with 0.15 M NaCl in 0.05 M Tris buffer, pH 8.6, containing 1% bovine serum albumin. Rabbit anti-human von Willebrand factor (DAKO; Glostrup, Denmark) was diluted 1:1000. The immunoreaction was localized with a Vectastain ABC kit (Vector Laboratories) according to the manufacturer's instructions. For control staining, the primary antibody was replaced by preimmune rabbit serum. The sections were counterstained with hematoxylin.
Northern Hybridization
RNA was isolated by the guanidine isothiocyanate/acid phenol method (
In Situ Hybridization (ISH)
ISH was performed on sections of formalin-fixed, paraffin-embedded tissues as described previously (
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Results |
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Cloning and Expression of Rat Group II Phospholipase A2
PCR was used to amplify the protein coding sequence from rat PLA2 cDNA. The cDNA contains a BspHI site in the protein coding sequence at nucleotide 258. To avoid addition of extra amino acids to the N-terminus of the recombinant protein, PCR was done in two steps as described in Materials and Methods. A 378-bp DNA product was obtained (Fig 2). The DNA product was inserted into NcoI/BspHI and BglII sites of the pQE-60 expression vector to produce pQE-60-PLA2. This expression system produces a recombinant protein with six additional adjacent histidine residues at the C-terminus of the inserted gene. Plasmids were transformed into JM109 E. coli and selected bacteria were submitted to induction protocols. After the induction of plasmid pQE-60-PLA2 with IPTG, significant production of rat Group II phospholipase A2 recombinant protein could be achieved in JM109 E. coli, as visualized by the appearance of a protein band of approximately 18 kD in SDS-PAGE (Fig 3). We also found duplicates of recombinant PLA2 when more protein was loaded in SDS-PAGE (Fig 3). Recombinant PLA2 was found exclusively in the inclusion bodies of JM109 E. coli, whereas no recombinant protein was found in the soluble fraction of JM109 E. coli. The maximal rate of production of the recombinant protein was reached at 12 hr after the induction. For practical purposes, production was allowed to continue overnight. The induction of plasmid pQE-60-PLA2 was also tested in strains M15 and SG13009 of E. coli, but no production of the recombinant protein was seen in these bacteria.
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Large-scale Expression and Purification of Recombinant Rat Group II Phospholipase A2
JM109 E. coli harboring the plasmid pQE-60-PLA2 were grown in 1 liter of LB medium and induced with isopropyl-ß-D-thio-galactoside (IPTG). When bacterial cells were ruptured by sonication under nondenaturing conditions, all recombinant rat PLA2 protein was recovered in the insoluble pellet. The recombinant protein was purified by nickel affinity chromatography under denaturing conditions. Typically, yields of approximately 40 mg of recombinant protein per 1 liter of culture, with a purity of >97%, were achieved. Sequencing the first six amino acids of the product revealed that the initiator methionine was uncleaved in 17% of the recombinant proteins. In 83% of the protein, the first amino acid was serine as in the native protein (
Production of Antibody
Once a sufficient amount of recombinant protein was available, antiserum was raised in rabbits against rat PLA2. To verify that the antibody produced to the recombinant protein would recognize native rat Group II phospholipase A2, an extract of rat ileum was analyzed by Western blotting. The antibody recognized a 14-kD protein (Fig 4). The antibody was used to detect the distribution of PLA2 protein in rat tissues by immunohistochemistry.
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Crossreactivity of Anti-rat Group II Phospholipase A2 Antibody
Crossreactivity of the antibody was studied with sections of formalin-fixed, paraffin-embedded samples of human small intestine (
Distribution of Group II Phospholipase A2 in Rat Tissues
Tissues from SpragueDawley rats of both sexes were studied by immunohistochemistry for the presence of PLA2 protein and by Northern and in situ hybridizations for the mRNA of PLA2. The results are summarized in Table 1.
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Gastrointestinal Tract. The Paneth cells were strongly immunoreactive in the duodenum, jejunum, ileum, and cecum. The immunoreaction was localized in the secretory granules of Paneth cells (Fig 5). Many goblet cells showed moderate immunoreaction in the small intestine and colon. No immunoreaction was found in the walls of vessels in the lamina propria or in the smooth muscle cell layer. There was no immunoreaction in the esophageal mucosa, mucosa of the glandular stomach, or pancreas. Periportal and centrilobular hepatocytes were lightly stained, whereas other cell types in the liver were unstained.
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Spleen. There were large multinuclear cells with strong immunoreaction in the red pulp area of the spleen (Fig 6). The same cells were immunoreactive for von Willebrand factor (Fig 7), suggesting that these cells were megakaryocytes.
Northern hybridization showed approximately an 800-base transcript in the esophagus, glandular stomach, duodenum, jejunum, ileum, cecum, colon, and spleen (Fig 8). The largest amount of PLA2 transcript was found in the ileum. By in situ hybridization, mRNA of PLA2 was found in the Paneth cells only (Fig 9). In situ hybridization with the control riboprobe gave negative results in the intestinal mucosa (Fig 10) and all other tissues studied.
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Respiratory Organs and Cartilage. There was moderate immunoreaction in the chondrocytes and the matrix of the cartilage of the trachea and in the main bronchus. Costal cartilage was also immunostained. There was no immunoreaction in the other cell types of the lung. Northern blotting detected mRNA of PLA2 in the lung, but in situ hybridization gave negative results.
Heart and Circulatory System. There was no immunoreaction in the myocytes of the heart or the aortic wall. Northern blotting detected mRNA of PLA2 in the heart, whereas in situ hybridization gave negative results.
Urinary and Reproductive Organs. Neither immunoreaction or Northern blotting signal was observed in the kidney, prostate, seminal vesicle, testis, epididymis, ovary, Fallopian tube, and uterus. Urinary bladder gave a signal in Northern blotting but no immunoreactive material was observed.
Neural, Muscle, and Lymphoid tissues. Neural tissue and skeletal muscle were devoid of immunoreaction and Northern blotting signal. Neither lymph nodes nor adipose tissue contained immunoreactive material. Northern blotting gave negative results in the latter tissues as well.
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Discussion |
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In this study we expressed rat Group II phospholipase A2 in E. coli and produced antibodies against the recombinant protein. Furthermore, we investigated the distribution of PLA2 protein and its mRNA in various rat organs and cell types.
The recombinant protein was generated by expressing the protein-coding sequence of rat PLA2 with an attached C-terminal histidine tag for rapid purification. Rat PLA2 cDNA was cloned and sequenced earlier (
In this study we investigated the distribution of PLA2 and its mRNA in rat tissues. We used immunohistochemistry to detect the PLA2 protein and Northern and in situ hybridizations to detect the mRNA. As far as we are aware, the present study contains the first data on in situ hybridization of mRNA of Group II phospholipase A2 in the rat. We found the mRNA in the Paneth cells of the intestinal mucosa. Despite positive results in Northern blotting, no in situ hybridization signals were detected in other cell types. This finding indicates that the level of the gene expression of PLA2 in rat tissues may be below the detection limit of the current methods of tissue preservation and in situ hybridization.
Despite the high homology between human and rat Group II phospholipase A2 proteins, we have not previously found any crossreactivity between human anti-Group II phospholipase A2 antibody and rat PLA2 (unpublished data). In this study, we show crossreactivity between the anti-rat PLA2 antiserum and human Group II phospholipase A2 in immunohistochemistry. The Paneth cells of the human intestinal mucosa were immunostained with the anti-rat PLA2 antisera, and the latter crossreacted with the tissues containing human PLA2 in human Group II phospholipase A2-transgenic mice (
Earlier, the distribution of PLA2 was studied in the gastrointestinal tract of humans (
It has been proposed that Group II phospholipase A2 has antibacterial effects in the gastrointestinal tract. PLA2 appears to be involved, together with other enzymes (e.g., lysozyme) and cryptidins in the protection of the small intestinal crypts against microbial invasion (
Group II phospholipase A2 has been purified from rat spleen (
Recently, PLA2 was cloned from rat heart (
Synovial fluid, chondrocytes, and the matrix of cartilage contain large amounts of PLA2 in the human (
In summary, we describe the production of large amounts of recombinant rat Group II phospholipase A2 and polyclonal rabbit antibodies against PLA2. Immunohistochemical, Northern blotting, and in situ hybridization analyses allowed a detailed characterization of the distribution of PLA2 in rat tissues. These methods provide useful tools for studies on the role of PLA2 in various experimental disease models.
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Acknowledgments |
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Supported by the Turku University Hospital and the University of Turku Foundation.
We thank Dr Jun Ishizaki for providing the cDNA of rat Group II phospholipase A2, Dr Jukka Hellman for sequencing the recombinant PLA2, and Ms Sinikka Kollanus, Ms Anne JokilammiSiltanen, and Mr Jaakko Liippo for technical assistance.
Received for publication December 29, 1999; accepted May 24, 2000.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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Chomzynski P, Sacchi N (1987) Single-step method of isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156-159[Medline]
Crowl RM, Stoller TJ, Conroy RR, Stoner CR (1991) Induction of phospholipase A2 gene expression in human hepatoma cells by mediators of the acute phase response. J Biol Chem 266:2647-2651
Cupillard L, Koumanov K, Mattei M-G, Lazdunski M, Lambeau G (1997) Cloning, chromosomal mapping, and expression of a novel human secretory phospholipase A2. J Biol Chem 272:15745-15752
Dennis EA (1997) The growing phospholipase A2 superfamily of signal transduction enzymes. Trends Biochem Sci 22:1-2[Medline]
De Windt LJ, Willemsen PHM, Pöpping S, Van der Vusse G, Reneman RS, Van Bilsen M (1997) Cloning and cellular distribution of a group II phospholipase A2 expressed in the heart. J Mol Cell Cardiol 29:2095-2106[Medline]
Emadi S, Mirshahi M, Elalamy I, Nicolas C, Vargaftig BB, Hatmi M (1998) Cellular source of human platelet secretory phospholipase A2. Br J Haematol 100:365-373[Medline]
ForemanWykert A, Weinrauch Y, Elsbach P, Weiss J (1999) Cell-wall determinants of the bactericidal action of group IIA phospholipase A2 against Gram-positive bacteria. J Clin Invest 103:715-721
Harwig SSL, Tan L, Qu X-D, Cho Y, Eisenhauer PB, Lehrer RI (1995) Bactericidal properties of murine intestinal phospholipase A2. J Clin Invest 95:603-610[Medline]
Inada M, Tojo H, Kawata S, Tarui S, Okamoto M (1991) Preferential distribution of group-II-like phospholipase A2 in mononuclear phagocytic cells in rat spleen and liver. Eur J Biochem 197:323-329[Abstract]
Ishizaki J, Ohara O, Nakamura E, Tamaki M, Ono T, Kanda A, Yoshida N, Teraoka H, Tojo H, Okamoto M (1989) cDNA cloning and sequence determination of rat membrane-associated phospholipase A2. Biochem Biophys Res Commun 162:1030-1036[Medline]
Kallajoki M, Nevalainen TJ (1997) Expression of group II phospholipase A2 in human tissues. In Uhl W, Nevalainen TJ, Buchler MW, eds. Progress in Surgery. Vol 24. Phospholipase A2. Basic and Clinical Aspects in Inflammatory Diseases. Basel, Karger, 8-16
Kiyohara H, Egami H, Shibata Y, Murata K, Ohshima S, Ogawa M (1992) Light microscopic immunohistochemical analysis of the distribution of group II phospholipase A2 in human digestive organs. J Histochem Cytochem 40:1659-1664
Komada M, Kudo I, Inoue K (1990) Structure of gene coding for rat group II phospholipase A2. Biochem Biophys Res Commun 168:1059-1065[Medline]
Kramer RM, Hession C, Johansen B, Hayes G, McGray P, Chow EP, Tizard R, Pepinsky RB (1989) Structure and properties of a human non-pancreatic phospholipase A2. J Biol Chem 264:5768-5775
Kriegsmann J, Müller W-D, Richter W, Wunderlich J, Wallukat G (1993) Demonstration of membrane-associated phospholipase A2 in cultivated heart muscle cells by immunogold-technique in surface replicas. Acta Histochem 95:61-66[Medline]
Laine VJO, Grass DS, Nevalainen TJ (1999) Protection by group II phospholipase A2 against Staphylococcus aureus. J Immunol 162:7402-7408
Nevalainen TJ, Grönroos JM (1997) Serum phospholipase A2 in inflammatory diseases. In Uhl W, Nevalainen TJ, Buchler MW, eds. Progress in Surgery. Vol 24. Phospholipase A2. Basic and Clinical Aspects in Inflammatory Diseases. Basel, Karger, 104-109
Nevalainen TJ, Grönroos JM, Kallajoki M (1995) Expression of group II phospholipase A2 in the human gastrointestinal tract. Lab Invest 72:201-208[Medline]
Nevalainen TJ, Haapanen TJ (1993) Distribution of pancreatic (group I) and synovial-type (group II) phospholipases A2 in human tissues. Inflammation 17:453-464[Medline]
Nevalainen TJ, Haapanen TJ, Rajala P, Ekfors T (1998) Expression of group II phospholipase A2 in Paneth cells of an adenoma of the rectum. APMIS 106:780-784[Medline]
Nevalainen TJ, Kallajoki M, Pesonen E, Andersson S, Kärkkäinen P, Höckerstedt K (1996) Origin of circulating group II phospholipase A2 in hepatocytes in a patient with epithelioid hemangioendothelioma of the liver. Lab Invest 74:585-591[Medline]
Nevalainen TJ, Laine VJO, Grass DS (1997) Expression of human group II phospholipase A2 in transgenic mice. J Histochem Cytochem 45:1109-1119
Nevalainen TJ, Märki F, Kortesuo PT, Grütter MG, Di Marco S, Schmitz A (1993) Synovial type (group II) phospholipase A2 in cartilage. J Rheumatol. 20:325-330[Medline]
Ono T, Tojo H, Kuramitsu S, Kagamiyama H, Okamoto M (1988) Purification and characterization of a membrane-associated phospholipase A2 from rat spleen. J Biol Chem 263:5732-5738
Schädlich HR, Büchler M, Beger HG (1987) Improved method for the determination of phospholipase A2 catalytic activity concentration in human serum and ascites. J Clin Chem Clin Biochem 25:505-509[Medline]
Seilhamer JJ, Pruzanski W, Vadas P, Plant S, Miller JA, Kloss J, Johnson LK (1989) Cloning and recombinant expression of phospholipase A2 present in rheumatoid arthritic synovial fluid. J Biol Chem 264:5335-5338
SenegasBalas F, Balas D, Verger R, de Caro A, Figarella C, Ferrato F, Lechene P, Bertrand C, Ribet A (1984) Immunohistochemical localization of intestinal phospholipase A2 in rat Paneth cells. Histochemistry 81:581-584[Medline]
Tischfield JA (1997) A reassessment of the low molecular weight phospholipase A2 gene family in mammals. J Biol Chem 272:17247-17250
Vadas P, Browning J, Edelson J, Pruzanski W (1993) Extracellular phospholipase A2 expression and inflammation: the relationship with associated disease states. J Lipid Mediat 8:1-30[Medline]
Van Schaik RHN, Verhoeven NM, Neijs FW, Aarsman AJ, Van den Bosch H (1993) Cloning of the cDNA coding for 14 kDa group II phospholipase A2 from rat liver. Biochim Biophys Acta 1169:1-11[Medline]
Weinrauch Y, Abad C, Liang N-S, Lowry SF, Weiss J (1998) Mobilization of potent plasma bactericidal activity during systemic bacterial challenge. J Clin Invest 102:633-638
Weinrauch Y, Elsbach P, Madsen LM, Foreman A, Weiss J (1996) The potent anti-Staphylococcus aureus activity of a sterile rabbit inflammatory fluid is due to a 14-kD phospholipase A2. J Clin Invest 97:250-257