Synergism between basic Asp49 and Lys49 phospholipase A2 myotoxins of viperid snake venom in vitro and in vivo

artículo (arbitrado) -- Universidad de Costa Rica, Instituto de investigaciones Clodomiro Picado. 2014Two subtypes of phospholipases A2 (PLA2s) with the ability to induce myonecrosis, ‘Asp49’ and ‘Lys49’ myotoxins, often coexist in viperid snake venoms. Since the latter lack catalytic activity, two different mechanisms are involved in their myotoxicity. A synergism between Asp49 and Lys49 myotoxins from Bothrops asper was previously observed in vitro, enhancing Ca2+ entry and cell death when acting together upon C2C12 myotubes. These observations are extended for the first time in vivo, by demonstrating a clear enhancement of myonecrosis by the combined action of these two toxins in mice. In addition, novel aspects of their synergism were revealed using myotubes. Proportions of Asp49 myotoxin as low as 0.1% of the Lys49 myotoxin are sufficient to enhance cytotoxicity of the latter, but not the opposite. Sublytic amounts of Asp49 myotoxin also enhanced cytotoxicity of a synthetic peptide encompassing the toxic region of Lys49 myotoxin. Asp49 myotoxin rendered myotubes more susceptible to osmotic lysis, whereas Lys49 myotoxin did not. In contrast to myotoxic Asp49 PLA2, an acidic non-toxic PLA2 from the same venom did not markedly synergize with Lys49 myotoxin, revealing a functional difference between basic and acidic PLA2 enzymes. It is suggested that Asp49 myotoxins synergize with Lys49 myotoxins by virtue of their PLA2 activity. In addition to the membrane-destabilizing effect of this activity, Asp49 myotoxins may generate anionic patches of hydrolytic reaction products, facilitating electrostatic interactions with Lys49 myotoxins. These data provide new evidence for the evolutionary adaptive value of the two subtypes of PLA2 myotoxins acting synergistically in viperid venoms.Funding support by the Graduate Studies Program, Universidad de Costa Rica; International Centre for Genetic Engineering and Biotechnology, Italy (CRP/COS13-01); and Vicerrectoria de Investigacion, Universidad de Costa Rica (741-B4-100).UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Structural bases for a complete myotoxic mechanism: Crystal structures of two non-catalytic phospholipases A(2)-like from Bothrops brazili venom

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Bothrops brazili is a snake found in the forests of the Amazonian region whose commercial therapeutic antibothropic serum has low efficacy for local myotoxic effects, resulting in an important public health problem in this area. Catalytically inactive phospholipases A(2)-like (Lys49-PLA(2)s) are among the main components from Bothrops genus venoms and are capable of causing drastic myonecrosis. Several studies have shown that the C-terminal region of these toxins, which includes a variable combination of positively charged and hydrophobic residues, is responsible for their activity. In this work we describe the crystal structures of two Lys49-PLA(2)s (BbTX-II and MIX-II) from B. brazili venom and a comprehensive structural comparison with several Lys49-PLA(2)s. Based on these results, two independent sites of interaction were identified between protein and membrane which leads to the proposition of a new myotoxic mechanism for bothropic Lys49-PLA(2)s composed of five different steps. This proposition is able to fully explain the action of these toxins and may be useful to develop efficient inhibitors to complement the conventional antivenom administration. (C) 2013 Elsevier B.V. All rights reserved.18341227722781Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

O estudo da prova calórica em pacientes idoso The study of caloric testing in senior patients

OBJETIVO: realizar o estudo da prova calórica em pacientes idosos com queixa de tontura, acompanhada ou não de zumbido com finalidade de traçar um perfil dos idosos quanto os sintomas vestibulares. MÉTODOS: fizeram parte da amostra 143 pacientes, com faixa etária variando de 60 a 90 anos de idade; todos realizaram o exame otoneurológico. Foram excluídos pacientes idosos com queixa de desequilíbrio e aqueles que apresentaram a queixa de zumbido desacompanhado da tontura. RESULTADOS: o estudo demonstrou que a normorreflexia, em valores absolutos, encontra-se entre 82,3% a 91,2% e os valores relativos são de 93%. Referente aos dados colhidos na anamnese, o que chamou a atenção, foi que do total de pacientes analisados, encontrou-se a tontura acompanhada de zumbido em 69% dos casos e o elevado uso de medicação em pacientes nesta faixa etária. CONCLUSÃO: na população estudada houve um predomínio da tontura acompanhada de zumbido. Em relação à prova calórica, o trabalho demonstrou a alta prevalência de pacientes com valores absolutos e relativos normais.<br>PURPOSE: to accomplish the study of caloric testing in senior patients with dizziness, accompanied or not of with buzzing. METHODS: 143 patients were analyzed with age varying from 60 to 90 years; all of them did an otoneurologic evaluation. Senior patients with unbalance complaints and with buzzing unaccompanied with dizziness were excluded from this study. RESULTS: 82.3% to 91.2% of the patients obtained normoreflexia in absolute values and over 93% obtained normal results in relative values. Referring to the data obtained in the anamnesis, what got attention was 69% of dizziness accompanied with buzzing. And the high medication of the patients in this age group. CONCLUSION: there was a prevalence of dizziness accompanied with buzzing in the studied population. Concerning the caloric testing, the study demonstrated the high prevalence of patients with normal absolute and relative values