Actions of Crotalus durissus terrificus venom and crotoxin on the isolated rat kidney

Many studies have reported the occurrence of lethal acute renal failure after snakebites. The aim of the present investigation was to determine alterations in renal function produced by Crotalus durissus terrificus venom and crotoxin as well as the histological alterations induced by these venoms. Isolated kidneys from Wistar rats weighing 240 to 280 g were perfused with Krebs-Henseleit solution containing 6 g% of previously dialyzed bovine serum albumin. The effects of Crotalus durissus terrificus venom and crotoxin were studied on glomerular filtration rate (GFR), urinary flow (UF), perfusion pressure (PP) and percentage sodium tubular transport (%TNa+). The infusion of Crotalus durissus terrificus venom (10 µg/ml) and crotoxin (10 µg/ml) increased GFR (control80 = 0.78 ± 0.07, venom80 = 1.1 ± 0.07, crotoxin80 = 2.0 ± 0.05 ml g-1 min-1, P<0.05) and UF (control80 = 0.20 ± 0.02, venom80 = 0.32 ± 0.03, crotoxin80 = 0.70 ± 0.05 ml g-1 min-1, P<0.05), and decreased %TNa+ (control100 = 75.0 ± 2.3, venom100 = 62.9 ± 1.0, crotoxin80 = 69.0 ± 1.0 ml g-1 min-1, P<0.05). The infusion of crude venom tended to reduce PP, although the effect was not significant, whereas with crotoxin PP remained stable during the 100 min of perfusion. The kidneys perfused with crude venom and crotoxin showed abundant protein material in the urinary space and tubules. We conclude that Crotalus durissus terrificus venom and crotoxin, its major component, cause acute nephrotoxicity in the isolated rat kidney. The current experiments demonstrate a direct effect of venom and crotoxin on the perfused isolated kidney

Renal Effects Of Supernatant From Macrophages Activated By Crotalus Durissus Cascavella Venom: The Role Of Phospholipase A2 And Cyclooxygenase

In Brazil, the genus Crotalus is responsible for approximately 1500 cases of snakebite annually. The most common complication in the lethal cases is acute renal failure, although the mechanisms of the damaging effects are not totally understood. In this work, we have examined the renal effects caused by a supernatant of macrophages stimulated by Crotalus durissus cascavella venom as well as the potential role of phospholipase A2 and cyclo-oxygenase. Rat peritoneal macrophages were collected and placed in a RPMI medium and stimulated by crude Crotalus durissus cascavella venom (1, 3 or 10 μg/ml) for 1 hr. They were then washed and kept in a culture for 2 hr. The supernatant (1 ml) was tested in an isolated perfused rat kidney. The first 30 min. of each experiment were used as an internal control, and the supernatant was added to the system after this period. All experiments lasted 120 min. A study of toxic effect on perfusion pressure, glomerular filtration rate, urinary flow, percent of sodium tubular transport and percent of proximal tubular sodium transport was made. The lowest concentration of venom (1 μg/ml) was not statistically different from the control values. The most intense effects were seen at 10 μg/ml for all renal parameters. The infusion of the supernatant of macrophages stimulated with crude venom (3 or 10 μg/ml) increased the perfusion pressure, glomerular filtration rate and urinary flow, decreased the percent of sodium tubular transport and percent of proximal tubular sodium transport. Dexamethasone (10 μM) and quinacrine (10 μM) provided protection against the effect of the venom on glomerular filtration rate, urinary flow, percent of sodium tubular transport, percent of proximal tubular sodium transport and perfusion pressure. Indomethacin (10 μM) and nordiidroguaretic acid (1 μM) reversed almost all functional changes, except those of the perfusion pressure. These results suggest that macrophages stimulated with Crotalus durissus cascavella venom release mediators capable of promoting nephrotoxicity in vitro. Moreover, phospholipase A2 and cyclooxygenase products are involved in these biologic effects.9211420Azar, S., Tobian, T., Ishii, M., Prolonged water diuresis affecting solutes and intersticial cells of renal papilla (1971) Amer. J. Physiol., 221, pp. 75-79Balhlmann, J., Giebisch, G., Ochwadt, B., Micropuncture study of isolated perfused rat kidney (1967) Amer. J. Physiol., 212, pp. 77-82Barnes, P.J., Adcock, I., Anti-inflammatory actions of steroids: Molecular mechanisms (1993) Trends Pharmacol. Sci., 14, pp. 436-441Barraviera, B., Curso sobre acidentes pot animais peçonhentos: Acidentes por serpente do gênero crotalus (1989) Arq. Bras. Med., 64, pp. 14-20Bowman, R.H., Gluconeogenesis in the isolated perfused rat kidney (1970) J. Biol. Chem., 245, pp. 1604-1612Ferreira, S.H., Are macrophages the body's alarme cells? (1980) Agents Actions, 10, pp. 229-230Ferreira, M.L., Moura-da-Silva, A.M., França, F.O.S., Cardoso, J.L., Mota, I., Toxic activities of venoms from nine Bothrops species and their correlation with lethality and necrosis (1992) Toxicon, 30, pp. 1603-1608Fonteles, M.C., Forti, C.A., The effect of indomethacin and reserpine on renal vascular escape (1993) Chem. Pathol. Pharmacol., 81, pp. 103-110Fonteles, M.C., Cohen, J.J., Black, A.J., Wertheim, S.J., Support of renal kidney function by long-chain fatty acids derived from renal tissue (1983) Amer. J. Physiol., 244, pp. 235-246Fonteles, M.C., Greenberg, R.N., Monteiro, H.S.A., Currie, M.G., Forte, L.R., Natriuretic and Kaliuretic activies of guanylin and uroguanylin in the isolated perfused rat kidney (1998) Amer. J. Physiol., 44, pp. 191-197Greg, G.M., Cohen, J.J., Black, A.Y., Espeland, M.A., Feldstain, M.C., Effects of glucose and insulin metabolism and function of perfused rat kidney (1978) Amer. J. Physiol., 235, pp. 52-61Gutiérrez, J.M., Lomonte, B., Local tissue damage induced by Bothrops snake venoms (1989) Mem. Inst. Butantan, 51, pp. 211-233Hamilton, R.L., Benny, N.M., Williams, M.C., Severin-Ghaus, E.M.A., Simple and inexpensive membrane "lung" for small or-gan perfusion (1974) J. Lipid. Res., 15, pp. 182-186Hanson, R.W., Ballard, F.S., Citrate, pyruvate and lactate contaminants of commercial serum albumin (1968) J. Lipid. Res., 9, pp. 667-668Korzeniewski, C., Callewaert, D.M., An enzyme-release assay for natural cytotoxicity (1983) J. Immunol. Meth., 64, pp. 313-320Laskin, D.L., Pendino, K.J., Macrophages and anti-inflammatory mediators in tissue injury (1995) Annu. Rev Pharmacol. Toxicol., 35, pp. 655-677Lima, A.A.M., Monteiro, H.S.A., Fonteles, M.C., The effects of Escherichia coli heat-stable enterotoxin in renal sodium tubular transport (1992) Pharmacology & Toxicology, 70, pp. 163-167Mancin, A.C., Soares, A.M., Andriao-Escarso, S.H., Faca, V.M., Greene, L.J., Zuccolotto, S., Pela, I.R., Giglio, J., The analgesic activity of crotamine, a neurotoxin from Crotalus durissus terrificus (South American rattlesnake) venom: A biochemical and pharmacological study (1998) Toxicon, 36, pp. 1927-1937Marshall, J.S., Leal Berumen, I., Nielsen, L., Glibetic, M., Jordana, M., Interleukin (IL)-10 inhibits long-term IL-6 production but not performed mediator release from rat peritoneal mast cell (1996) J. Clin. Invest., 97, pp. 1122-1128Martins, A.M.C., Monteiro, H.S.A., Júnior, E.O.G., Menezes, D.B., Fonteles, M.C., Effects of Crotalus durissus cascavella venom in the isolated rat kidney (1998) Toxicon, 36, pp. 1441-1450Monteiro, H.S.A., Da Silva, I.M.S.C., Martins, A.M.C., Fonteles, M.C., Effects of Crotalus durissus terrificus venom and crotoxin on the isolated rat kidney (2001) Braz. J. Med. Biol. Res., 34, pp. 1347-1352Nancy, G., Ahlstrom, M.D., Luginbuhl, M.D.W., Tisher, M.D.C., Acute anuric renal failure after pygmy rattlesnake bite (1991) South. Med. J., 84, pp. 783-785Nathan, C.F., Secretory products of macrophages (1987) J. Clin. Invest., 79, pp. 319-326Nishiitsutji-Uwo, G.M., Ross, B.D., Krebs, H.A., Metabolic activities of the isolated perfused rat kidney (1967) Biochem. J., 103, pp. 852-862Pegg, D.E., Vascular resistance of the isolated rabbit kidney (1971) Cryobiology, 8, pp. 431-440Ribeiro, L.A., Albuquerque, M.J., De Campos, V.A., Katz, G., Takaoka, N.Y., Lebrao, M.L., Jorge, M.T., Deaths caused by venomous snakes in the State of São Paulo: Evaluation of 43 cases from 1988 to 1993 (1998) Rev Assoc. Med. 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Thalidomide and pentoxifylline block the renal effects of supernatants of macrophages activated with Crotalus durissus cascavella venom

Because thalidomide and pentoxifylline inhibit the synthesis and release of tumor necrosis factor-alpha (TNF-alpha), we determined the effect of these drugs on the renal damage induced by supernatants of macrophages activated with Crotalus durissus cascavella venom in order to identify the role of TNF-alpha in the process. Rat peritoneal macrophages were collected with RPMI medium and stimulated in vitro with C.d. cascavella venom (10 µg/ml) in the absence and presence of thalidomide (15 µM) or pentoxifylline (500 µM) for 1 h and washed and kept in culture for 2 h. Supernatant (1 ml) was tested on an isolated perfused rat kidney (N = 6 for each group). The first 30 min of each experiment were used as control. The supernatant was added to the perfusion system. All experiments lasted 120 min. The toxic effect of the preparation of venom-stimulated macrophages on renal parameters was determined. At 120 min, thalidomide (Thalid) and pentoxifylline (Ptx) inhibited (P < 0.05) the increase in perfusion pressure caused by the venom (control = 114.0 ± 1.3; venom = 137.1 ± 1.5; Thalid = 121.0 ± 2.5; Ptx = 121.4 ± 4.0 mmHg), renal vascular resistance (control = 4.5 ± 0.2; venom = 7.3 ± 0.6; Thalid = 4.5 ± 0.9; Ptx = 4.8 ± 0.6 mmHg/ml g-1 min-1), urinary flow (control = 0.23 ± 0.001; venom = 0.44 ± 0.01; Thalid = 0.22 ± 0.007; Ptx = 0.21 ± 0.009 ml g-1 min-1), glomerular filtration rate (control = 0.72 ± 0.06; venom = 1.91 ± 0.11; Thalid = 0.75 ± 0.04; Ptx = 0.77 ± 0.05 ml g-1 min-1) and the decrease in percent tubular sodium transport (control = 77.0 ± 0.9; venom = 73.9 ± 0.66; Thalid = 76.6 ± 1.1; Ptx = 81.8 ± 2.0%), percent tubular chloride transport (control = 77.1 ± 1.2; venom = 71.4 ± 1.1; Thalid = 77.6 ± 1.7; Ptx = 76.8 ± 1.2%), and percent tubular potassium transport (control = 72.7 ± 1.1; venom = 63.0 ± 1.1; Thalid = 72.6 ± 1.0; Ptx = 74.8 ± 1.0%), 30 min before and during the stimulation of macrophages with C.d. cascavella venom. These data suggest the participation of TNF-alpha in the renal effects induced by supernatant of macrophages activated with C.d. cascavella venom

Characterization of a new platelet aggregating factor from crotoxin Crotalus durissus cascavella venom

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOIn this article we investigated the platelet aggregating activity of whole crotoxin and its subunits isolated from Crotalus durissus cascavella venom. During the purification protocols of the venom, using HPLC molecular exclusion, we detected the presence of two different serine protease activities in the gyroxin fraction, and another in the crotoxin fraction, which induced strong and irreversible platelet aggregation, in addition to blood coagulation. From crotoxin, we isolated PLA(2), crotapotin (both fractions corresponding approximately 85% of whole crotoxin) and another minor fraction (F20) that exhibited serine protease activity. After a new fractionation on reverse phase HPLC chromatography, we obtained three other fractions named as F201, F202 and F203. F202 was obtained with high degree of molecular homogeneity with molecular mass of approximately 28 kDa and a high content of acidic amino residues, such as aspartic acid and glutamic acid. Other important amino acids were histidine, cysteine and lysine. This protein exhibited a high specificity for BApNA, a Michaelis-Menten behavior with Vmax estimated in 5.64 mu M/min and a Km value of 0.58 mM for this substrate. In this work, we investigated the ability of F202 to degrade fibrinogen and observed alpha and beta chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with TLCK and PMSF, specific inhibitors of serine protease. Also, F202 induced platelet aggregation in washed and platelet-rich plasma, and in both cases, TLCK inhibited its activity. The N-terminal amino acid sequence of F202 presented a high amino acid sequence homology with other thrombin-like proteins, but it was significantly different from gyroxin. These results showed that crotoxin is a highly heterogeneous protein composed of PLA(2), thrombin-like and other fractions that might explain the diversity of physiological and pharmacological activities of this proteinSpringer253183192FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO2004/00040-3300211.2004New York, N

Effects Of Morin On Snake Venom Phospholipase A2 (pla 2)

Flavonoids are potent anti-inflammatory compounds isolated from several plant extracts, and have been used experimentally against inflammatory processes. In this work, a PLA2 isolated from the Crotalus durissus cascavella venom and rat paw oedema were used as a model to study the effect of flavonoids on PLA2. We observed that a treatment of PLA2 with morin induces several modifications in the aromatic amino acids, with accompanying changes in its amino acid composition. In addition, results from circular dichroism spectroscopy and UV scanning revealed important structural modifications. Concomitantly, a considerable decrease in the enzymatic and antibacterial activities was observed, even though anti-inflammatory and neurotoxic activities were not affected. These apparent controversial results may be an indication that PLA2 possess a second pharmacological site which does not affect or depend on the enzymatic activity. © 2005 Elsevier Ltd. 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Chemother., 15, pp. 410-421Harborne, J.B., (1988) The Flavonoids: Advances in Research, , Chapman & Hall LondonHodnick, W.F., Duval, D.L., Pardini, R.S., Inhibition of mithocondrial respiration and cyaned stimulated generation of reactive oxygen species by selected flavonoids (1994) Biochem. Pharmacol., 47 (3), pp. 573-580Holzer, M., MacKessy, S.P., An aqueous endpoint assay of snake venom phospholipase A2 (1996) Toxicon, 34 (10), pp. 1149-1155Kitagawa, S., Sakamoto, H., Tano, H., Inhibitory effect of flavonoids on free radical-induced hemolysis and ther oxidative effect on hemoglobin (2004) Chem. Pharm. Bull., 52 (8), pp. 999-1001Koduri, R.S., Grönrooss, J.O., Laine, V.J.O., Calvez, C.L., Lambeau, G., Nevelainen, T.J., Gelb, M.H., Bactericidal properties of human and murine groups I, II, V, X and XII secreted phospholipases A2 (2002) J. Biol. 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Effects of morin on snake venom phospholipase A(2) (PLA(2))

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFlavonoids are potent anti-inflammatory compounds isolated from several plant extracts, and have been used experimentally against inflammatory processes. In this work, a PLA(2) isolated from the Crotalus durissus cascavella venom and rat paw oedema were used as a model to. study the effect of flavonoids on PLA(2). We observed that a treatment of PLA(2) with morin induces several modifications in the aromatic amino acids, with accompanying changes in its amino acid composition. In addition, results from circular dichroism spectroscopy and UV scanning revealed important structural modifications. Concomitantly, a considerable decrease in the enzymatic and antibacterial activities was observed, even though anti-inflammatory and neurotoxic activities were not affected. These apparent controversial results may be an indication that PLA(2) possess a second pharmacological site which does not affect or depend on the enzymatic activityPergamon Press467751758FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO04/00040-3Oxfor

Antiophidic Solanidane Steroidal Alkaloids From Solanum Campaniforme

Three new solanidane alkaloids bearing a 22,23-epoxy ring (1-3) and four known compounds were isolated from leaves of Solanum campaniforme. The structures were determined using spectroscopic techniques, including 1Dand 2D NMR, and HRESIMS experiments. The antiophidic activity of the alkaloids was tested against Bothrops pauloensis venom. Compounds 1-3 completely inhibited myotoxicity without inhibiting phospholipase A 2 activity of the venom, while hemorrhage and skin necrosis were significantly reduced in the presence of alkaloids 1 and 2. © 2011 The American Chemical Society and American Society of Pharmacognosy.741021682173Wink, M., (2003) Phytochemistry, 64, pp. 3-19Nino, J., Correa, Y.M., Mosquera, O.M., (2009) Pharm. Biol., 47, pp. 255-259Ikeda, T., Tsumagari, H., Honbu, T., Nohara, T., (2003) Biol. Pharm. Bull., 26, pp. 1198-1201Kodoru, S., Grierson, D.S., Van De Venter, M., Afolayan, A.J., (2007) Pharm. Biol., 45, pp. 613-618Sun, L., Zhao, Y., Li, X., Yuan, H., Cheng, A., Lou, H., (2010) Toxicol. in Vitro, 24, pp. 1504-151Emmanuel, S., Ignacimuthu, S., Perumalsamy, R., Amalraj, T., (2006) Fitoterapia, 77, pp. 611-612Pandurangan, A., Khosa, R.L., Hemalatha, S., (2010) J. Asian Nat. Prod. Res., 12, pp. 691-695Fenner, R., Betti, A.H., Mentz, L.A., Rates, S.M.K., (2006) Rev. Bras. Cienc. Farm., 42, pp. 369-394Harrison, R.A., Hargreaves, A., Wagstaff, S.C., Faragher, B., Lalloo, D.G., (2009) PLoS Negl. Trop. Dis., 3, p. 569Mors, W.B., Nascimento, M.C., Pereira, B.M.R., Pereira, N.A., (2000) Phytochemistry, 55, pp. 627-642Correa-Netto, C., Teixeira-Araujo, R., Aguiar, A.S., Melgarejo, A.R., De-Simone, S.G., Soares, M.R., Foguel, D., Zingali, R.B., (2010) Toxicon, 55, pp. 1222-1235Gutiérrez, J.M., Rucavado, A., Chaves, F., Díaz, C., Escalante, T., (2009) Toxicon, 54, pp. 958-975Caron, E.J., Manock, S.R., Maudlin, J., Koleski, J., Theakston, R.D., Warrell, D.A., Smalligan, R.D., (2009) Toxicon, 54, pp. 779-83Cotrim, C.A., De Oliveira, S.C., Diz Filho, E.B., Fonseca, F.V., Baldissera, Jr.L., Antunes, E., Ximenes, R.M., Toyama, M.H., (2011) Chem. Biol. Interact., 15, pp. 9-16Lomonte, B., León, G., Angulo, Y., Rucavado, A., Núñez, V., (2009) Toxicon, 54, pp. 1012-1028Coelho, R.M., Souza, M.C., Giotto, M.H.S., (1998) Phytochemistry, 49, pp. 893-897Lawson, D.R., Green, T.P., Haynes, L.W., Miller, A.R., (1997) J. Agric. Food Chem., 45, pp. 4122-4126Chiesa, F.A.F., Moyna, P., Alcaloides Esteroidales (2004) Farmacognosia: Da Planta Ao Medicamento, pp. 869-883. , In, 5 th ed. Simões, C. M. O. Schenkel, E. P. Gosmann, G. Mello, J. C. P. Mentz, L. A. Petrovick, P. R. Ed.a da UFRGS: Porto AlegreDraper, R.W., Puar, M.S., Vater, E.J., McPhail, A.T., (1998) Steroids, 63, pp. 135-140Jiang, Z.H., Wang, J.R., Li, M., Liu, Z.Q., Chau, K.Y., Zhao, C., Liu, L., (2005) J. Nat. Prod., 68, pp. 397-399Daneluttea, A.P., Costantina, M.B., Delgadob, G.E., Braz-Filho, R., Kato, M.J., (2005) J. Braz. Chem. Soc., 16, pp. 1425-1430Slimestad, R., Fossen, T., Verheul, M.J., (2008) J. Agric. Food Chem., 56, pp. 2436-2441Friedman, M., Lee, K.-R., Kim, H.-J., Lee, I.-S., Kozukue, N., (2005) J. Agric. Food Chem., 53, pp. 6162-6169Milner, S.E., Brunton, N.P., Jones, P.W., Brien, N.M.O., Collins, S.G., Maguire, A.R., (2011) J. Agric. Food Chem., 59, pp. 3454-3484Corrêa, L.C., Marchi-Salvador, D.P., Cintra, A.C.O., Sampaio, S.V., Soares, A.M., Fontes, M.R.M., (2008) Biochim. Biophys. Acta, 1784, pp. 591-599Cintra-Francischinelli, M., Pizzo, P., Angulo, Y., Gutiérrez, J.M., Montecucco, C., Lomonte, B., (2010) Toxicon, 55, pp. 590-596Montecucco, C., Ornella, R., Caccin, P., Rigoni, M., Carli, L., Morbiato, L., Muraro, L., Paoli, M., (2009) Toxicon, 54, pp. 561-564Fernandes, C.A., Marchi-Salvador, D.P., Salvador, G.M., Silva, M.C., Costa, T.R., Soares, A.M., Fontes, M.R., (2010) J. Struct. Biol., 171, pp. 31-43Silva, J.O., Fernandes, R.S., Ticli, F.K., Oliveira, C.Z., Mazzi, M.V., Franco, J.J., Giuliatti, S., Sampaio, S.V., (2007) Toxicon, 50, pp. 283-291Esmeraldino, L.E., Souza, A.M., Sampaio, S.V., (2005) Phytomedicine, 12, pp. 570-57

Quercetin As An Inhibitor Of Snake Venom Secretory Phospholipase A2

As polyphenolic compounds isolated from plants extracts, flavonoids have been applied to various pharmaceutical uses in recent decades due to their anti-inflammatory, cancer preventive, and cardiovascular protective activities. In this study, we evaluated the effects of the flavonoid quercetin on Crotalus durissus terrificus secretory phospholipase A2 (sPLA2), an important protein involved in the release of arachidonic acid from phospholipid membranes. The protein was chemically modified by treatment with quercetin, which resulted in modifications in the secondary structure as evidenced through circular dichroism. In addition, quercetin was able to inhibit the enzymatic activity and some pharmacological activities of sPLA2, including its antibacterial activity, its ability to induce platelet aggregation, and its myotoxicity by approximately 40%, but was not able to reduce the inflammatory and neurotoxic activities of sPLA2. These results suggest the existence of two pharmacological sites in the protein, one that is correlated with the enzymatic site and another that is distinct from it. We also performed molecular docking to better understand the possible interactions between quercetin and sPLA2. Our docking data showed the existence of hydrogen-bonded, polar interactions and hydrophobic interactions, suggesting that other flavonoids with similar structures could bind to sPLA2. Further research is warranted to investigate the potential use of flavonoids as sPLA2 inhibitors. © 2010 Elsevier Ireland Ltd. All rights reserved.18901/02/15916Kini, R., Excitement ahead: Structure, function and mechanism of snake venom phospholipase A2 enzymes (2003) Toxicon, 42 (8), pp. 827-840Nevalainen, T., Graham, G., Scott, K., Antibacterial actions of secreted phospholipases A2. Review (2008) Biochim. Biophys. Acta, 1781 (12), pp. 1-9Burke, J., Dennis, E., Phospholipase A2 biochemistry (2009) Cardiovasc. 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