Unmasking Snake Venom of Bothrops leucurus: Purification and Pharmacological and Structural Characterization of New PLA(2) Bleu TX-III

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Bleu TX-III was isolated from Bothrops leucurus snake venom on one-step analytical chromatography reverse phase HPLC, was homogeneous on SDS-PAGE, and was confirmed by Q-Tof Ultima API ESI/MS (TOF MS mode) mass spectrometry in 14243.8 Da. Multiple alignments of Bleu TX-III show high degree of homology with basic PLA(2) myotoxins from other Bothrops venoms. Our studies on local and systemic myotoxicity "in vivo" reveal that Bleu TX-III is myotoxin with local but not systemic action due to the decrease in the plasmatic CK levels when Bleu TX-III is administrated by intravenous route in mice (dose 1 and 5 mu g). And at a dose of 20 mu g myotoxin behaves like a local and systemic action. Bleu TX-III induced moderate marked paw edema, evidencing the local increase in vascular permeability. The inflammatory events induced in the mice (I. M.) were investigated. The increase in the levels of IL-1, IL-6, and TNF-alpha was observed in the plasma. It is concluded that Bleu TX-III induces inflammatory events in this model. The enzymatic phospholipid hydrolysis may be relevant to these phenomena. Bothrops leucurus venom is still not extensively explored, and the knowledge of its toxins separately through the study of structure/function will contribute for a better understanding of its action mechanism.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Signalling pathways regulating human neutrophil migration induced by secretory phospholipases A(2)

This study was designed to elucidate the signalling pathways by which secretory phospholipases A(2) (sPLA(2)s) induce in vitro neutrophil migration. The cell migration assays were performed with Naja mocambique venom PLA(2) (sPLA(2) with high catalytic activity), bothropstoxin-I (sPLA2 devoid of catalytic activity) and platelet-activating factor (PAF), using a 48-well microchemotaxis chamber. Both the non-selective protein kinase inhibitor staurosporine (30-300 nM) and the selective protein kinase C (PKC) inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpyperazine (H7; 50-200 muM) as well as the Gi inactivator pertussis toxin (30-300 nM) caused a concentration-dependent inhibition of the neutrophil migration induced by either N. mocambique venom PLA(2) (100 mug/ml) or bothropstoxin-I (100 mug/ml). Pertussis toxin nearly abolished PAF-induced migration, while staurosporine and H7 partly (but significantly) inhibited the chemotactic responses to PAR The dual inhibitor of cytosolic PLA(2) and Ca2+-independent PLA(2) (iPLA(2)), arachidonil-trifluoromethyl-ketone (ATK; 0.2-20 muM), or the specific iPLA(2) inhibitor bromoenol lactone (1-30 muM) caused a concentration-dependent inhibition of the migration induced by either sPLA(2)s. At the maximal concentration used for each compound, the migration was almost suppressed. In contrast, both of these compounds caused only slight inhibitions of PAF-induced migration. No rise in intracellular Ca2+ Was observed in neutrophil-stimulated sPLA(2), as determined in cells preloaded with fura 2-AM. In the experimental condition used, pertussis toxin, staurosporine, H7, ATK or bromoenol lactone did not induce cytotoxic effects, according to MTT assay. Our results suggest that activation of an endogenous PLA(2) through activation of GTP-binding protein and PKC is the main mechanism by which exogenous sPLA(2)s cause neutrophil migration. (C) 2004 Elsevier Ltd. All rights reserved.44547348

Human neutrophil migration in vitro induced by secretory phospholipases A(2): a role for cell surface glycosaminoglycans

The purpose of this study was to examine the ability of type I- (porcine pancreas and Naja mocambique mocambique venom), type II- (bothropstoxin-I, bothropstoxin-II, and piratoxin-I). and type III- (Apis mellifera venom) secretory phospholipases A(2) (sPLA(2)s) to induce human neutrophil chemotaxis, and the role of the cell surface proteoglycans, leukotriene B-4 (LTB4), and platelet-activating factor (PAF). in mediating this migration. the neutrophil chemotaxis assays were performed by using a 48-well microchemotaxis chamber. Piratoxin-I, bothropstoxin-I. N. m. mocambique venom PLA(2) (10-1000 mug/mL each), bothropstoxin-II (30-1000 mug/mL), porcine pancreas PLA(2) (0.3-30 mug/mL), and A. mellifera venom PLA(2) (30-300 mug/mL) caused concentration-dependent neutrophil chemotaxis. Heparin (10-300 U/mL) concentration-dependently inhibited the neutrophil migration induced by piratoxin-I, bothropstoxin-II, and N. in. mocambique and A. mellifera venom PLA(2)s (100 mug/mL each), but failed to affect the migration induced by porcine pancreas PLA(2). Heparan sulfate (300 and 1000 mug/mL) inhibited neutrophil migration induced by piratoxin-I, whereas dermatan sulfate and chondroitin sulfate (30-1000 mug/mL each) had no effect. Heparitinase I and heparinase (300 mU/mL each) inhibited by 41.5 and 47%. respectively, piratoxin-l-induced chemotaxis, whereas heparitinase 11 and chondroitinase AC failed to affect the chemotaxis. the PAF receptor antagonist WEB 2086 (3-[4-(2-chlorophenyl)-9-methyl-6H-thienol-[3,2-f] [1,2,4]-triazolo-[4.3-a] [1,4]-diazepine-2-yl]-1-(4-morpholynil)-1-propionate) (0.1-10 muM) and the LTB4 synthesis inhibitor AA-861 [2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-1,4-benzoquinone] (0.1-10 muM) significantly inhibited the piratoxin-I-induced chemotaxis. Piratoxin-I (30-300 mug/mL) caused a concentration-dependent release of LTB4 Our results suggest that neutrophil migration in response to sPLA(2)s is independent of PLA activity, and involves an interaction of sPLA(2)s with cell surface heparin/heparan binding sites triggering the release of LTB4 and PAF. (C) 2002 Elsevier Science Inc. All rights reserved.UNICAMP, Dept Pharmacol, Fac Med Sci, BR-13081970 Campinas, SP, BrazilUNICAMP, Dept Biochem, BR-13081970 Campinas, SP, BrazilUSP, Dept Biochem, Ribeirao Preto, BrazilUNIFESP, Dept Biochem, São Paulo, BrazilUNIFESP, Dept Biochem, São Paulo, BrazilWeb of Scienc

Inhibition of inducible nitric oxide synthase-derived nitric oxide as a therapeutical target for acute pancreatitis induced by secretory phospholipase A(2)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Background Nitric oxide is a key signalling molecule in the pathogenesis of inflammation, but its role in acute pancreatitis and related abdominal pain induced by secretory phospholipase A(2) (sPLA(2)) from Crotalus durissus terrificus (Cdt) venom has not been investigated. Methods Male Wistar rats were i.v. injected with L-NAME (20 mg/kg), aminoguanidine (AG, 50 mg/kg), 7-nitroindazole (7-NI, 10 mg/kg) or vehicle 10 min before or 60 min after the injection of sPLA(2) (300 mu g/kg) into the common bile duct. After 4 h of sPLA(2) injection, abdominal hyperalgesia and inflammation were assessed in addition to serum amylase, nitrite/nitrate (NOx), pancreas lipoperoxidation and 3-nitrotyrosine (3-NT) contents. Results sPLA(2)-induced acute pancreatitis, related abdominal hyperalgesia, hyperamylasemia and increased concentration of NOx were not correlated with lipoperoxidation or increased 3-NT in the pancreas. Pretreatment with all the nitric oxide synthase (NOS) inhibitors significantly reduced abdominal mechanical hyperalgesia, but only iNOS blockade by AG suppressed pancreas oedema and serum NOx increase. The therapeutic approach with all the NOS inhibitors produced a similar reduction pattern of the abdominal hyperalgesia, but AG treatment also inhibited serum hyperamylasemia and NOx concentrations and pancreatic myeloperoxidase. The nNOS blockade by 7-NI treatment also inhibited myeloperoxidase activity in both pancreas and lung. Conclusions Therapeutic blockade of iNOS or nNOS provides benefits in terms of inhibition of the acute pancreatitis-related abdominal hyperalgesia, while iNOS inhibition also ameliorates the inflammatory cell influx to the pancreas and reduces the resultant hyperamylasemia and NOx levels, thus representing alternative pharmacological strategies for treatment of clinical pancreatitis associated with increased PLA(2).185691700Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundacao de Apoio a Pesquisa e Inovacao Tecnologica do Estado de Sergipe (FAPITEC-SE) [019.203.00941/2009-6]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP [06/61591-2, 07/00529-0]Fundacao de Apoio a Pesquisa e Inovacao Tecnologica do Estado de Sergipe (FAPITEC-SE) [019.203.00941/2009-6

Purification and inflammatory edema induced by two PLA(2) (Anch TX-I and Anch TX-II) from sea anemone Anthothoe chilensis (Actiniaria: Sagartiidae)

The Anch TX-I and II PLA(2) were purified from Anthothoe chilensis (Lesson, 1830) from the extract of the anemone after only two chromatographic step using molecular exclusion chromatography (Sephadex G-75) and reverse phase HPLC on mu-Bondapak C18 column. Both PLA(2) showed a molecular mass of similar to 14 kDa determined by MALDI-TOF mass spectrometry and showed a high catalytic activity (data not showed). Although homologous with mammalian or snake venom group I PLA(2)s, Anch TX-I and II is sufficiently structurally different for the question of its placement into the existing PLA(2) classification scheme to arise. In addition, Anch TX-I and despite possessing many common structural features, also differ in some important structural properties. The amino acid sequence of both PLA(2) (Anch TX-I and III) showed high amino acid sequence identity with PLA(2) Rhopilema nomadica and Bunodosoma caissarum Cnidaria and PLA(2) of group III protein isolated from the Mexican lizard Heloderma horridum horridum and Heloderma suspectum. In addition, Anch TX-I and Anch TX-II showed high amino acid sequence identity with PLA(2) from group III also showed significant overall homology to bee Apis dorsata, Bombus terrestris and Bombus pennsylvanicus and PlA(2). We also investigated the in vivo edematogenic activity of Anch TX-I and Anch TX-II in a model of paw and skin edema in rats and observed that both are able to induce dose-dependent edema. (C) 2011 Elsevier Inc. All rights reserved.161217017

Effects of Low Molecular Weight Sulfated Galactan Fragments From Botryocladia Occidentalis on the Pharmacological and Enzymatic Activity of Spla2 From Crotalus Durissus Cascavella

Low molecular weight fragments of sulfated galactans (Boc-5 and Boc-10) from the red algae Botryocladia occidentalis significantly inhibited Crotalus durissus cascavella sPLA2 enzymatic activity. Equimolar ratios of sPLA2 to Boc-5 or Boc-10 resulted in allosteric inhibition of sPLA2. Under the conditions tested, we observed that both Boc-5 and Boc-10 strongly decreased edema, myonecrosis, and neurotoxicity induced by native sPLA2.FAPEPSConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)UNESP, Sao Vicente, BrazilUniv Presbiteriana Mackenzie, São Paulo, BrazilUniversidade Federal do Ceará (UFC), Dept Engn Pesca & BioMol BioMar, Fortaleza, Ceara, BrazilHosp Univ Clementino Fraga Filho HUCFF UFRJ, Rio de Janeiro, BrazilUniv Estadual Campinas, Dept Bioquim, Inst Biol, São Paulo, BrazilUFC, Dept Bioquim, São Paulo, BrazilUNESP, Sao Vicente, BrazilFAPEPS: 54714-3CNPq: 558193/2009-