Bothrops moojeni

Stabeli RG, Amui SF, Sant'Ana CD, Pires MG, Nomizo A, Monteiro MC, Romao PR, Guerra-Sa R, Vieira CA, Giglio JR, Fontes MR, Soares AM.

MjTX-II, a myotoxic phospholipase A(2) (PLA(2)) homologue from Bothrops moojeni venom, was functionally and structurally characterized. The MjTX-II characterization included: (i) functional characterization (antitumoral, antimicrobial and antiparasitic effects); (ii) effects of structural modifications by 4-bromophenacyl bromide (BPB), cyanogen bromide (CNBr), acetic anhydride and 2-nitrobenzenesulphonyl fluoride (NBSF); (iii) enzymatic characterization: inhibition by low molecular weight heparin and EDTA; and (iv) molecular characterization: cDNA sequence and molecular structure prediction. The results demonstrated that MjTX-II displayed antimicrobial activity by growth inhibition against Escherichia coli and Candida albicans, antitumoral activity against Erlich ascitic tumor (EAT), human breast adenocarcinoma (SK-BR-3) and human T leukemia cells (JURKAT) and antiparasitic effects against Schistosoma mansoni and Leishmania spp., which makes MjTX-II a promising molecular model for future therapeutic applications, as well as other multifunctional homologous Lys49-PLA(2)s or even derived peptides. This work provides useful insights into the structural determinants of the action of Lys49-PLA(2) homologues and, together with additional strategies, supports the concept of the presence of others "bioactive sites" distinct from the catalytic site in snake venom myotoxic PLA(2)s.

Fernandes, VC(1); Marcussi, S(2); Amui, SF(3); França, SC(1); Pereira, AMS(1); Soares, AM(3)

Efeito inibitório do extrato aquoso de folhas de Scleria pterota sobre diferentes atividades tóxicas e farmacológicas de venenos de serpentes

Estudos de mecanismos de ação de proteínas de venenos estão sendo desenvolvidos, através de ensaios com diferentes extratos de plantas e inibidores naturais isolados destas. O extrato e os venenos/ou toxinas foram incubados por 30 min a 37°C em diferentes proporções e posteriormente submetidos aos diferentes ensaios enzimáticos e biológicos. O extrato mostrou-se eficiente em inibir a atividade fosfolipásica dos venenos de Bothrops jararacussu e B. alternatus, não tendo efeito sobre os venenos de B. moojeni, B. neuwiedi e Crotalus durissus terríficus, nas proporções de 1:100 e 1:200. A coagulação induzida pelos venenos foi totalmente inibida na presença do extrato em proporção de 1:10. A hemorragia induzida por B. jararacussu, B. moojeni, B. alternatus e B. neuwiedi foi inibida pelo extrato na proporção de 1:50, mostrando maior inibição para os dois primeiros venenos. A eletroforese demonstrou que o extrato não degrada as proteínas dos venenos de serpentes, sugerindo a possibilidade de interações específicas entre os inibidores vegetais e as toxinas alvo. Assim, extrato de S. pterota inibiu as atividades enzimáticas e tóxicas dos venenos de serpentes interagindo de forma diferente para cada um deles, sem alterar a integridade das proteínas que os compõe. Os aspectos abordados neste trabalho poderão trazer informações complementares sobre mecanismos de ação destes venenos.

Marcussi, S.*; Stábeli, R.G.**; Monteiro, M.C.***; Romão, P.R.T.***; Oliveira, E.B.**; França, S.C.*; Soares, A. M.*

A L-aminoácido oxidase (LAAO) é uma enzima glicoprotéica que catalisa a deaminação oxidativa de L-aminoácidos, produzindo ?-cetoácidos, peróxido de hidrogênio e amônia. Estas enzimas encontram-se presentes nos venenos de serpentes e participam do envenenamento atuando sobre plaquetas, induzindo citotoxicidade e apoptose celular.

Malta-Neto, N.R.1; Stabeli, R.G.5; Marcussi, S.1,2; Nomizo, A.4; Sant’Ana, C.D.4; Giglio, J. R.2; Sampaio, S. V.4; Oliveira, E.B.2; Soares, A.M.1,4.
(smarcussi@rbi.fmrp.usp.br)

L-Amino acid oxidases (LAAO, EC 1.4.3.2) are flavoenzymes which catalyze the stereospecific oxidative deamination of an L-amino acid substrate to a corresponding α-ketoacid with the production of hydrogen peroxide and ammonia, via an imino acid intermediate. These enzymes are widely distributed in many different organisms such as bacteria, fungi, green algae and venomous snakes and are involved in the utilization of nitrogen sources. The present investigation reports isolation and biochemical characterization of L-amino acid oxidase (BmooLAAO-I) from Bothrops moojeni venom,
anti-Tumor effect upon murine melanoma, human breast adenocarcinoma, human leukemia T as well as upon peripheral human mononuclear cells. The citotoxic assay was done by using the MTT method described by Mosmann. It still shows the capacity of BmooLAAO-I to induce apoptosis in human leukemia cells compared to paclitaxel. It was tested in flux citometer after incubation with anexin V and propidium iodide. BmooLAAO-I is an acidic glycoprotein with about 130kDa arranged in two 66kDa monomers. It displays a high specificity toward hydrophobic and basic amino acids, while deglycosylation does not alter its enzymatic activity. BmooLAAO-I also displays dose dependent and anti-Tumor activities. The mechanism by its acts seems to be oxidative effects of hydrogen peroxide. Other mechanism involved in citotoxicity is apoptosis induction. SV-LAAOs are therefore interesting multifunctional enzymes, not only for a better understanding of the ophidian envenomation mechanism, but also due to their biotechnological potential as model for therapeutic agents.