Outras

Sant`Ana, CD^(1,3); Curtarelli, MB⁽¹⁾; Pereira, ALA⁽¹⁾; Ticli, FK⁽³⁾; Mazzi, MV⁽³⁾; Marcussi, S^(1,2); Magalhães, MR⁽⁴⁾; Giglio, JR⁽²⁾; Sampaio, SV⁽³⁾; Pereira, PS⁽¹⁾; Marins, MA⁽¹⁾; Soares, AM⁽³⁾

Inibição da atividade nucleotídica de venenos de serpentes por extratos de plantas

Venenos de serpentes são compostos principalmente por enzimas das classes PLA2s, proteases, LAAOs, entre outras. Essas proteínas têm sido intensamente estudadas e utilizadas como ferramentas em laboratórios para fins diagnósticos, sendo até mesmo empregadas para o entendimento de patologias e com aplicação na clínica médica. Neste trabalho foi avaliada a inibição da atividade nucleotídica de venenos de serpentes dos gêneros Bothrops e Crotalus, por extratos aquosos de diferentes plantas (Pentacletra macroloba, Mikania glomerata, Casearia sylvestris e Mandevilla velutina). As partes das plantas utilizadas para a elaboração dos extratos foram escolhidas baseando-se em conhecimentos populares de seu uso anti-ofídico, sendo utilizados o caule de P. macroloba, as raízes de M. glomerata, folhas de C. sylvestris e xilopódio de M. velutina. As amostras de venenos e extratos foram previamente incubadas em volume final de 5µL durante 60min a 37ºC (n=3) e posteriormente avaliadas através da técnica de enzimodifusão, sendo utilizado um gel composto por agarose 2%, tampão Tris-HCl 0,05M com MgSO4 0,01M pH 7.4 acrescido de DNA de esperma de salmão como substrato e brometo de etídio para possibilitar a visualização da atividade em UV. Os extratos de P. macroloba e M. glomerata foram eficientes em inibir 100% da atividade nucleotídica induzida pelos venenos de Bothrops jararacussu (50µg), B. neuwiedi (50 e 100µg), C. d. terríficus (50 e 100µg) e C. d. collineatus (50µg), nas proporções de 1:15 e 1:30 (m/m). Os extratos de C. sylvestris e M. velutina inibiram 100% da atividade nucleotídica do veneno de B. jararacussu apenas nas primeiras 6 horas, mostrando o mesmo efeito para C. d. collineatus em 24 horas de experimento, na proporção de 1:12 (m/m). Os resultados apresentados geram dados para a identificação de novos inibidores naturais dos efeitos tóxicos do envenenamento ofídico, além de servirem como uma ferramenta na compreensão do mecanismo de ação das nucleotidases presentes em venenos de serpentes.

Silveira, LB(1); Marcussi, S(2); Ticli, FK(3); Urzeda, MA(2); Biondo, R(2); França, SC(2); Pereira, PS(2); Soares, AM(2)

Natural And Artificial Inhibitors Of Snake Venom Phospholipases A₂

Phospholipases A₂ (PLA₂) from snake venoms induce different toxic and pharmacological effects including myotoxicity, edema, hypotension, platelet aggregation inhibition, convulsion, anticoagulation and others. Natural PLA₂ inhibitors were found in different organisms as plants, marine animals, snake and opossum plasmas. Functional characterization of natural (MMV from Taberna montana extract; manoalid A, from marine animal; heparin, from human blood; BmjMIP, from B. moojeni plasma; CAB from opossum plasma) and synthetic inhibitors (manoalid B, p-bromophenacil bromide BPB, EDTA), upon the enzymatic and pharmacological activity of PLA2 from Bothrops and Crotalus snake venome. PLA₂ was assayed in gels, while the myotoxic and edema inducing were evaluated in vivo. PrTX-I and III (from B. pirajai), BthTX-I and II (from B. jararacussu), crotoxin and CB (from C. durissus terrificus), in the presence or absence of different inhibitors, following an incubation of 30 minutes at 37ºC were used. Among the natural inhibitors, the following decreasing range of activity was observed: BmjMIP > manoalid A > CAB > heparin > MMV; for the synthetic ones, the sequence was: BPB > manoalid B > EDTA. Different levels of inhibition among PLA_2s from Bothrops and Crotalus venoms were also detected. Natural inhibitors were more efficient than synthetic ones for neutralization of the toxic and enzymatic effects induced by PLA_2s. Structural and interaction studies are needed to a better understanding of inhibitory effect in order to design new models of more potent synthetic inhibitors.

Tan NH, Lim KK, Jaafar MI.

The antigenic cross-reactivity of four Ophiophagus hannah (king cobra) venom components, the neurotoxin (OH-NTX), phospholipase A2 (OH-PLA2), hemorrhagin (OH-HMG) and L-amino acid oxidase (OH-LAAO) were examined by indirect and double sandwich ELISAs. The indirect ELISAs for OH-NTX, OH-PLA2 and OH-HMG were very specific when assayed against the various heterologous snake venoms and O. hannah venom components, at 25 ng/ml antigen level. At higher antigen concentrations (100-400 ng/ml), there were moderate to strong indirect ELISA cross-reactions between anti-O. hannah neurotoxin and venoms from various species of cobra as well as two short neurotoxins. However, anti-O. hannah hemorrhagin did not cross-react with any of the venoms tested, even at these high antigen concentrations, indicating that O. hannah hemorrhagin is antigenically very different from other venom hemorrhagins. Examination of the indirect ELISA cross-reactions between anti-O. hannah PLA2 and several elapid PLA2 enzymes suggests that the elapid PLA2 antigenic class has more than two subgroups. The antibodies to O. hannah L-amino acid oxidase, however, yielded indirect ELISA cross-reactions with many venoms as well as with OH-NTX, OH-PLA2 and OH-HMG, indicating that OH-LAAO shares common epitopes even with unrelated proteins. The double sandwich ELISAs for the four anti-O. hannah venom components, on the other hand, generally exhibited a higher degree of selectivity than the indirect ELISA procedure.