Bp-13 Pla2: Purification And Neuromuscular Activity Of A New Asp49 Toxin Isolated From Bothrops Pauloensis Snake Venom.

A new PLA2 (Bp-13) was purified from Bothrops pauloensis snake venom after a single chromatographic step of RP-HPLC on μ-Bondapak C-18. Amino acid analysis showed a high content of hydrophobic and basic amino acids and 14 half-cysteine residues. The N-terminal sequence showed a high degree of homology with basic Asp49 PLA2 myotoxins from other Bothrops venoms. Bp-13 showed allosteric enzymatic behavior and maximal activity at pH 8.1, 36°-45°C. Full Bp-13 PLA2 activity required Ca(2+); its PLA2 activity was inhibited by Mg(2+), Mn(2+), Sr(2+), and Cd(2+) in the presence and absence of 1 mM Ca(2+). In the mouse phrenic nerve-diaphragm (PND) preparation, the time for 50% paralysis was concentration-dependent (P 0.05). The main effect of this new Asp49 PLA2 of Bothrops pauloensis venom is on muscle fiber sarcolemma, with avian preparation being less responsive than rodent preparation. The study enhances biochemical and pharmacological characterization of B. pauloensis venom.201582605

Characterization of proteolityc, hemorrhagic and edematogenic activities of BmHF-1 metallproteinase, isolet from Bothrops marajoensis crude venom

Orientadores: Sergio Marangoni, Luis Alberto Ponce SotoDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Neste trabalho, descrevemos o isolamento e as caracterizações funcional e estrutural de uma nova metaloprotease, denominada BmHF-1. A proteína foi isolada da peçonha de Bothrops marajoensis, com um alto grau de pureza e homogeneidade molecular, pela combinação de dois passos, cromatográficos: exclusão molecular em coluna Superdex G-75 acoplada ao sistema HPLC-biocompatível; e seguida de uma cromatografia em HPLC de fase reversa numa coluna C-18 -Bondapack. A nova metaloprotease BmHF-1 apresenta massa molecular de 27162,36 Da obtida por espectrometria de massas (MALDI-Tof) ...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digitalAbstract: In this present work we described the isolation, structural and functional characterization of a new metalloproteinase, named BmHF-1. This protein was isolated with a high degree of purity from the Bothrops marajoensis snake venom by the combination of two chromatographic steps, size exclusion in a Superdex G-75 column coupled to HPLC biocompatible system, followed by a HPLC-RP chromatographic procedure in a C-18 -Bondapack column. This new metalloproteinase had a molecular mass of 27162.36 Da determinate by MALDI-Tof mass spectrometry.....Note: The complete abstract is available with the full electronic digital thesis or dissertationsMestradoBioquimicaMestre em Biologia Funcional e Molecula

Characterization of local and systemic effects of the metaloproteinase BtaHF purified from Bothriopsis taeniata crude venom

Orientador: Sergio MarangoniTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Neste trabalho descrevemos a purificação, caracterização estrutural e os efeitos locais e sistêmicos de uma nova metaloprotease de baixa massa molecular, denominada BtaHF (Bothriopsis taeniata Hemorrhagic Factor). A proteína foi purificada a partir do veneno total da serpente Bothriopsis taeniata usando cromatografía exclusão molecular convencional (Sephadex G-75), seguida de uma cromatografia de alta eficiência de troca iônica (coluna DEAE 8HR APMinicolumn). A nova metaloprotease BtaHF apresenta um alto grau de pureza e homogeneidade molecular e possui uma massa molecular de 25968,16 Da. A BtaHF apresentou atividade caseinolítica com uma temperatura e pH ótimos de 37-40 oC e 8, respectivamente. A atividade caseinolítica da BtaHF foi inibida por EGTA, EDTA e DTT, mas os inibidores PMSF e SBTI não apresentaram efeito. O íon Ca+2 é importante para a estabilidade da metaloprotease BaHF, aumentando atividade caseinolítica da enzima, entretanto, os íons Zn+2 e Mn+2 inibem a atividade enzimática desta enzima. A BtaHF é uma enzima ?-fibrinogênolítica por degradar rapidamente à cadeia A do fibrinogênio após 15 minutos de incubação, enquanto a cadeia B? é completamente degradada após 6 horas. Por outro lado, a BtaHF não possui atividade fibrinolítica ou arginine amidase. A análise de composição de aminoácidos mostrou que a BtaHF possui caráter ácido e apresenta 6 resíduos de cisteína. No estudo de homologia a BtaHF apresentou maior identidade sequencial com metaloproteases com atividade hemorrágica da classe P-I como a BaP1 (Bothrops asper). A metaloprotease BtaHF possui atividade hemorrágica fraca com uma dose hemorrágica mínima de 20,14 ?g/animal. Esta metaloprotease mostrou atividade edematogênica que mostrou ser dose-dependente e teve um efeito permaneceu após 6 horas de injeção. Ambos os efeitos estão relacionados à atividade proteolítica e ao correto enovelamento da BtaHF, una vez que foram inibidas por agentes quelantes (EDTA e EGTA) e redutor (DTT). A metaloprotease BtaHF não possui atividade miotóxica, embora tenha atividade citotóxica em fibroblastos C2C12. A análise histológica do músculo gastrocnêmio de camundongo confirmou a atividade hemorrágica e edematogênica da metaloprotease, assim como, a ausência de atividade miotoxica. A metaloprotease BtaHF produz efeitos sistêmicos específicos quando injetado via endovenosa em camundongos. A nível do tecido pulmonar, a toxina produz alteração da estrutura alveolar com hemorragia significativa, engrossamento dos septos alveolares e inflamação. A BtaHF não produz hemorragia no tecido renal, mas alterou a estrutura glomerular, além de alterações no citoplasma das células dos túbulos proximal e distal. Contrariamente, esta toxina não produz efeito nenhum a nível hepático. A BtaHF consome o fibrinogênio plasmático de modo dose e tempo-dependente, produzindo incoagulabilidade sanguínea após uma hora. Estudos in vitro revelaram atividade pro-coagulante dose-dependente, ao reduzir os índices PT e APPT. A utilização de metodologias de purificação de alta eficiência permitram a purificação da metaloprotease BtaHF. Assim, esta abordagem pode ser aplicada nos estudos bioquímicos, estrutura-função, fisiológicos e farmacológicos, podendo deduzir o papel desenvolvido pela metaloprotease purificada nos efeitos biológicos produzidos pelo veneno total de Botriopsis taeniata. As informações produzidas no presente trabalho permitiram sugerir que as principais funções das metaloproteases de veneno de serpente da classe P-I são manter a hemorragia característica dos envenenamentos botrópicos e participar da imobilização da presa promovendo rápidos efeitos próinflamatóriosAbstract: In this work we described the purification, structural characterization and the local and systemic effects of a new low molecular weight snake venom metalloproteinase, named BtaHF. This protein was purified from the Bothriopsis taeniata crude venom combining conventional molecular exclusion (Sephadex G-75 column) followed by an ion exchange (DEAE 8HR APMinicolumn) chromatography on a HPLC system. The new metalloproteinase BtaHF showed a high degree of purity and molecular homogeneity with a molecular mass of 25968.16 Da. BtaHF showed caseinolytic activity with an optimum temperature and pH of 37-40 °C and 8, respectively. The caseinolytic activity was inhibited by EGTA, EDTA and DTT, but PMSF and SBT-I did not show inhibitory effect. The Ca+2 ion shown to be important for protein stability enhancing its caseinolytic activity, on the contrary, Z+2 and Mn+2 showed inhibitory effects upon the enzymatic activity of this protein. The metalloproteinase BtaHF is an ?-fibrigenolytic enzyme, rapidly degraded fibrinogen A? chain within 15 minutes, while fibrinogen B? chain is degraded after 6 hours. This enzyme did not show fibrinolytic or arginine amidase activities BtaHF is an acidic protein due the mayor proportion of acidic amino acid in its amino acid composition with 6 cysteinyl residues. By the homology study BtaHF share a high sequence identity other weakly hemorrhagic P-I class SVMP such BaP1 isolated from Bothrops asper snake venom This new metalloproteinase, BtaHF, have a weak hemorrhagic with a minimum hemorrhagic dose of 20.14 ?g. Also, this metalloproteinase showed dose-dependent paw edemaforming activity, this effect remain six hours after inoculation. Both hemorrhage and edemaforming activities were related to the enzymatic activity and correct fold of BtaHF, since chelating (EDTA and EGTA) and reducing (DTT) agents inhibited these activities. On the other hand, this enzyme did not show miotoxic activity, although, showed citotoxic activity on C2C12 fibroblast. Histological analysis of mice gastrocnemius muscle confirmed that the metalloproteinase produce local hemorrhage and pro-inflammatory effects, as well as, the absence of miotoxic activity. This metalloproteinase, BtaHF, produce specific systemic effects when injected endovenously in mice. On lung tissue level these toxin produce alteration of alveolus structure with significant hemorrhage, thickness of alveolus septum and inflammation. On renal tissue BtaHF did not produce hemorrhage, but, was observe alteration of glomerulus structure, along with cytoplasmatic alterations of distal and proximal tubulus cells. On the other hand, were not observed any alterations on hepatic tissue. BtaHF deplete completely plasma fibrinogen levels in a time and dose-depended manner, reaching blood incoagulability after one hour. BtaHF shown dose-dependent pro-coagulant activity "ex vivo" reducing PT and APPT index. The metalloproteinase BtaHF was purified using a high efficient system. Thus, this approach can be applied to biochemical, structure-function, physiologic and pharmacologyc studies which allow infer the role of this enzyme in the biological effects produce by Bothriopsis taeniata crude venom. The information here presented suggest that the mainly functions of P-I class snake venom metalloproteinases are the maintenance of the characteristic hemorrhagic effect of the botropic venoms and participate of prey immobilization by promoting rapid inflammatory effectsDoutoradoBioquimicaDoutor em Biologia Funcional e Molecula

Chromium (VI) bioremediation potential of filamentous fungi isolated from Peruvian tannery industry effluents

The tannery is an important trade in various Peruvian regions; however, tannery effluents are a serious local environmental threat due to its highly toxics components and lack of efficient treatment. The untreated effluents produced by tannery factories in Arequipa Rio Seco Industrial Park (PIRS) have formed a lake in the region nearby. In this work, we study the capability of filamentous fungi species found in this effluents lake with potential for chromium (VI) bioremediation. Fourteen species of filamentous fungi were isolated; only two species were identified Penicillium citrinum and Trichoderma viride, and third strain identified as Penicillium sp. The filamentous fungi showed that are fully tolerant to chromium (VI) concentrations up to 100 mg/L. These fungal strains showed significant growth in chromium (VI) concentrations up to 250 mg/L. Tolerant index (TI) analysis revealed that P. citrinum and T. viride began adaptation to chromium (IV) concentrations of 250 and 500 mg/L, after 6 and 12 days, respectively. When exposed to higher Cr (VI) concentrations (1000 mg/L), only T. viride was able to show growth (enhance phase). Interestingly, one of the significant responses from these fungal strains to increasing chromium (VI) concentrations was an increment in secreted laccase enzymes. Our results show tolerance and adaptation to elevated concentrations of chromium (VI) of these fungal strains suggesting their potential as effective agents for bioremediation of tannery effluents

Local and systemic effects of btamp-1, a new weakly hemorrhagic snake venom metalloproteinase purified from bothriopsis taeniata snake venom

A new weak hemorrhagic metalloproteinase named BtaMP-1 was purified from Bothriopsis taeniata snake venom by molecular exclusion followed by anion exchange chromatographies. This protein showed a molecular mass of 25,968.16 Da and is composed of 218 amino acid residues. The multiple alignments of its partial amino acid sequence showed high structural identity with other P-I class SVMP. BtaMP-1 showed caseinolytic activity that was enhanced by Ca2+ ion, completely inhibited by chelating and reducing agents and can be classified as an α-fibrinogenolytic enzyme. Locally, BtaMP-1 induces hemorrhage and edema, but not myotoxicity. These findings were confirmed by histological analysis of mouse gastrocnemius muscle. “In vitro” studies suggest that BtaMP-1 induce cytotoxicity in myoblast C2C12 but not in the myotubes cell line. BtaMP-1 induced systemic alterations in mice with one MHD and two hours exposure; histological analysis of lungs showed hemorrhagic areas, congestion, and increase the thickness of alveolar septum. Also, this protein induced mild effects on kidney and disruption of coagulation by depletion of fibrinogen plasma levels. This work provides insights into the importance of BtaMP-1 biological effects in envenomation by Bothropsis taeniata snake venom and providing further evidence to understand the role of P-I class SVMP in ophidian envenomation14110441054The author's thanks to Mr. Paulo A. Baldasso for technical assistance, to Prof. Dr. Stephen Hyslop for reviewing and correcting the English language, and the Mass Spectrometry Laboratory at Brazilian Biosciences National Laboratory, CNPEM/ABTLUS, Campinas, Brazil, for its support with the MS analyse

Local and systemic effects of BtaMP-1, a new weakly hemorrhagic Snake Venom Metalloproteinase purified from Bothriopsis taeniata Snake Venom

A new weak hemorrhagic metalloproteinase named BtaMP-1 was purified from Bothriopsis taeniata snake venom by molecular exclusion followed by anion exchange chromatographies. This protein showed a molecular mass of 25,968.16 Da and is composed of 218 amino acid residues. The multiple alignments of its partial amino acid sequence showed high structural identity with other P-I class SVMP. BtaMP-1 showed caseinolytic activity that was enhanced by Ca2+ ion, completely inhibited by chelating and reducing agents and can be classified as an α-fibrinogenolytic enzyme. Locally, BtaMP-1 induces hemorrhage and edema, but not myotoxicity. These findings were confirmed by histological analysis of mouse gastrocnemius muscle. “In vitro” studies suggest that BtaMP-1 induce cytotoxicity in myoblast C2C12 but not in the myotubes cell line. BtaMP-1 induced systemic alterations in mice with one MHD and two hours exposure; histological analysis of lungs showed hemorrhagic areas, congestion, and increase the thickness of alveolar septum. Also, this protein induced mild effects on kidney and disruption of coagulation by depletion of fibrinogen plasma levels. This work provides insights into the importance of BtaMP-1 biological effects in envenomation by Bothropsis taeniata snake venom and providing further evidence to understand the role of P-I class SVMP in ophidian envenomation

Danmark som international aktør 705-2005

Four proteins with phospholipase A2 (PLA2) activity, designated P9a(Cdt-PLA2), P9b(Cdt-PLA2), P10a(Cdt-PLA2) and P10b(Cdt-PLA2) were purified from the venom of Crotalus durissus terrificus by two chromatographic steps: a gel filtration and reversed phase HPLC. The profile obtained clearly shows that three of them have a similar abundance. The molecular mass, 14193.8340Da for P9a(Cdt-PLA2), 14134.9102Da for P9b(Cdt-PLA2), 14242.6289Da for P10a(Cdt-PLA2) and 14183.8730Da for P10b(Cdt-PLA2), were initially evaluated by SDS-PAGE and confirmed by ESI-Q-TOF spectrometry, and all of them displayed a monomeric conformation. Also, partial amino acid sequence of each protein was obtained and their alignments with other crotalic PLA2 revealed a high degree of identity among them. Additionally, we studied some pharmacological activities like neurotoxicity, myotoxicity and lethality, which prompted us to pick two of them, P9a(Cdt-PLA2) and P10a(Cdt-PLA2) that resulted to be less toxic that the others, and further characterize them to be used as immunogen. We next injected these last proteins in mice to produce antitoxins against them and ELISA and dot blots reveled that both toxins do not show immunogenic differences, unlike those other pharmacologic activities tested. Furthermore, the antibodies produced cross-reacted with all the isoforms purified demonstrating the feasibility of using only one of them and ensuring the cross-reaction of all. The results obtained show that P9a(Cdt-PLA2) isoform has the lowest toxicity and also a good purification performance; thus this protein may be a promising candidate to be employed in the production of crotalic antitoxins

P9a(Cdt-PLA2) from Crotalus durissus terrificus as good immunogen to be employed in the production of crotalic anti-PLA2 IgG

Four proteins with phospholipase A2 (PLA2) activity, designated P9a(Cdt-PLA2), P9b(Cdt-PLA2), P10a(Cdt-PLA2) and P10b(Cdt-PLA2) were purified from the venom of Crotalus durissus terrificus by two chromatographic steps: a gel filtration and reversed phase HPLC. The profile obtained clearly shows that three of them have a similar abundance. The molecular mass, 14193.8340Da for P9a(Cdt-PLA2), 14134.9102Da for P9b(Cdt-PLA2), 14242.6289Da for P10a(Cdt-PLA2) and 14183.8730Da for P10b(Cdt-PLA2), were initially evaluated by SDS-PAGE and confirmed by ESI-Q-TOF spectrometry, and all of them displayed a monomeric conformation. Also, partial amino acid sequence of each protein was obtained and their alignments with other crotalic PLA2 revealed a high degree of identity among them. Additionally, we studied some pharmacological activities like neurotoxicity, myotoxicity and lethality, which prompted us to pick two of them, P9a(Cdt-PLA2) and P10a(Cdt-PLA2) that resulted to be less toxic that the others, and further characterize them to be used as immunogen. We next injected these last proteins in mice to produce antitoxins against them and ELISA and dot blots reveled that both toxins do not show immunogenic differences, unlike those other pharmacologic activities tested. Furthermore, the antibodies produced cross-reacted with all the isoforms purified demonstrating the feasibility of using only one of them and ensuring the cross-reaction of all.The results obtained show that P9a(Cdt-PLA2) isoform has the lowest toxicity and also a good purification performance; thus this protein may be a promising candidate to be employed in the production of crotalic antitoxins.Fil: Fusco, Luciano Sebastian. Universidad Nacional del Nordeste. Facultad de Cs.exactas Naturales y Agrimensura. Departamento de Bioquímica. Laboratorio de Investigación En Proteinas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rodríguez, Juan Pablo. Universidad Nacional del Nordeste. Facultad de Cs.exactas Naturales y Agrimensura. Departamento de Bioquímica. Laboratorio de Investigación En Proteinas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Torres Huaco, Frank. Universidade Estadual Do Campinas. Instituto de Biología; BrasilFil: Huancahuire Vega, Salomón. Universidade Estadual Do Campinas. Instituto de Biología; BrasilFil: Teibler, Gladys Pamela. Universidad Nacional del Nordeste. Facultad de Cs.veterinarias. Departamento de Clinica. Cátedra de Farmacología; ArgentinaFil: Acosta, Ofelia Cristina. Universidad Nacional del Nordeste. Facultad de Cs.veterinarias. Departamento de Clinica. Cátedra de Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Marangoni, Sergio. Universidade Estadual Do Campinas. Instituto de Biología; BrasilFil: Ponce Soto, Luis. Universidade Estadual Do Campinas. Instituto de Biología; BrasilFil: Leiva, Laura Cristina Ana. Universidad Nacional del Nordeste. Facultad de Cs.exactas Naturales y Agrimensura. Departamento de Bioquímica. Laboratorio de Investigación En Proteinas; Argentin

Pharmacological analysis of hemodynamic responses to lachesis muta (south American bushmaster) snake venom in anesthetized rats

In this work, we examined some mechanisms involved in the hypotension caused by Lachesis muta (South American bushmaster) venom in anesthetized rats. Venom (1.5 mg/kg, i.v.) caused immediate hypotension that was maximal after 5 min and gradually returned to baseline over 60 min. Pretreatment of rats with the non-selective nitric oxide synthase (NOS) inhibitor N-omega-nitro-L-arginine methyl ester (L-NAME) did not attenuate the early phase of venom-induced hypotension, but abolished the recovery phase and resulted in rapid death; a similar effect was observed with the soluble guanylate cyclase (sGC) inhibitor ODQ. In contrast, the hemodynamic responses to venom were not attenuated by the nonselective NOS inhibitor N-G-monomethyl-L-arginine, the inducible NOS inhibitor aminoguanidine, the phosphodiesterase 5 inhibitor sildenafil, the adenylate cyclase (AC) inhibitor SQ-22.536, the nonselective muscarinic receptor antagonist atropine, the bradykinin B2 receptor antagonist HOE-140 and the non-selective cyclooxygenase inhibitor indomethacin. Preincubation of venom with the PLA(2) inhibitor pBPB had no effect on the immediate hypotension but tended to improve the recovery phase. Neither AEBSF (a serine proteinase inhibitor) nor EDTA (a metalloproteinase inhibitor) prevented the venom-induced hypotension, but AEBSF and not EDTA protected against the lethality of a high dose (3.0 mg/kg, i.v.). There were no marked changes in the ECG parameters with the various treatments, except with L-NAME and ODQ that increased the RR interval. Pulmonary thrombus formation was markedly enhanced by L-NAME and ODQ, and to a lesser extent by pBPB, especially in small vessels, whereas AEBSF and EDTA inhibited thrombus formation. Venom relaxed phenylephrine-precontracted thoracic aorta and pulmonary artery in vitro, with the latter being more sensitive. The relaxation was endothelium-dependent and was inhibited by ODQ but not by H-89, a protein kinase A (PKA) inhibitor. Together, these findings indicate involvement of the NO/sGC/cGMP, but not the AC/cAMP/PKA signaling pathway, in the hemodynamic responses to L muta venom in rats. Muscarinic mechanisms, kinins and arachidonic acid metabolites are apparently not involved. (C) 2016 Elsevier Ltd. All rights reserved.1232544FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL E NÍVEL SUPERIORCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO2010/51034-402-P-6889/2014; 01-P-3488/2014; 01-P-1748/2016134397/2013-4; 141691/2013-1; 307105/2010-5In this work, we examined some mechanisms involved in the hypotension caused by Lachesis muta (South American bushmaster) venom in anesthetized rats. Venom (1.5 mg/kg, i.v.) caused immediate hypotension that was maximal after 5 min and gradually returned to baseline over 60 min. Pretreatment of rats with the non-selective nitric oxide synthase (NOS) inhibitor N-omega-nitro-L-arginine methyl ester (L-NAME) did not attenuate the early phase of venom-induced hypotension, but abolished the recovery phase and resulted in rapid death; a similar effect was observed with the soluble guanylate cyclase (sGC) inhibitor ODQ. In contrast, the hemodynamic responses to venom were not attenuated by the nonselective NOS inhibitor N-G-monomethyl-L-arginine, the inducible NOS inhibitor aminoguanidine, the phosphodiesterase 5 inhibitor sildenafil, the adenylate cyclase (AC) inhibitor SQ-22.536, the nonselective muscarinic receptor antagonist atropine, the bradykinin B2 receptor antagonist HOE-140 and the non-selective cyclooxygenase inhibitor indomethacin. Preincubation of venom with the PLA(2) inhibitor pBPB had no effect on the immediate hypotension but tended to improve the recovery phase. Neither AEBSF (a serine proteinase inhibitor) nor EDTA (a metalloproteinase inhibitor) prevented the venom-induced hypotension, but AEBSF and not EDTA protected against the lethality of a high dose (3.0 mg/kg, i.v.). There were no marked changes in the ECG parameters with the various treatments, except with L-NAME and ODQ that increased the RR interval. Pulmonary thrombus formation was markedly enhanced by L-NAME and ODQ, and to a lesser extent by pBPB, especially in small vessels, whereas AEBSF and EDTA inhibited thrombus formation. Venom relaxed phenylephrine-precontracted thoracic aorta and pulmonary artery in vitro, with the latter being more sensitive. The relaxation was endothelium-dependent and was inhibited by ODQ but not by H-89, a protein kinase A (PKA) inhibitor. Together, these findings indicate involvement of the NO/sGC/cGMP, but not the AC/cAMP/PKA signaling pathway, in the hemodynamic responses to L muta venom in rats. Muscarinic mechanisms, kinins and arachidonic acid metabolites are apparently not involve