Gênero Crotalus

Toyama MH, Toyama Dde O, Passero LF, Laurenti MD, Corbett CE, Tomokane TY, Fonseca FV, Antunes E, Joazeiro PP, Beriam LO, Martins MA, Monteiro HS, Fonteles MC.

A novel l-amino acid oxidase (LAO) (Casca LAO) from Crotalus durissus cascavella venom was purified to a high degree of molecular homogeneity using a combination of molecular exclusion and ion-exchange chromatography system. The purified monomer of LAO presented a molecular mass of 68 kDa and pI estimated in 5.43, which were determined by two-dimensional electrophoresis. The 71st N-terminal amino acid sequence of the LAO from Crotalus durissus cascavella presented a high amino acid sequence similarities with other LAOs from Colloselasma rhosostoma, Crotalus adamanteus, Agkistrodon h. blomhoffi, Agkistrodon h. halys and Trimeresurus stejnegeri. LAO displayed a Michaelis-Menten behavior with a kilometer of 46.7 microM and an optimum pH for enzymatic activity of 6.5. Casca LAO induced a dose-dependent platelet aggregation, which was abolished by catalase and inhibited by indomethacin and aspirin. These results suggest that the production of H2O2 is involved in subsequent activation of inflammatory enzymes, such as thromboxane. Casca LAO also inhibited the bacterial growth of Gram-negative (Xanthomonas axonopodis pv passiflorae) and Gram-positive (S. mutans) strains. Electron microscopy assessments of both bacterial strains suggest that the hydrogen peroxide produced by LAO induce bacterial membrane rupture and consequently loss of cytoplasmatic content. This LAO exhibited a high antileishmanic activity against the promastigote of Leishmania amazonensis in vitro, its activity was dependent on the production of hydrogen peroxide, and the 50% inhibitory concentration was estimated in 2.39 microg/ml.

(1,3)Sant`Ana, CD; 1Oliveira, DG; (1,2)Marcussi, S; (3)Ticli, FK; (3)Mazzi, MV; (3)DaSilva, JO; (4)Magalhães, MR; (1)Marins, M; (3)Sampaio, SV and (1)Soares, AM

Nucleotidic Activity on DNA induced by Crotalus snake Venom: Isolation and Biochemical Characterization of a Nucleotidase from C. durissus terrificus Venom.

Snake venoms are complex mixtures of proteins which include several enzymes displaying a large action range. Active nucleases, including phosphodiesterases, which hydrolyze DNA and/or RNA, have already been reported in venoms of snakes from several world regions. This work reported a screening of nucleotidic activity of different Crotalus species and the isolation of a nuclease from C. durissus terrificus. Venoms from C. atrox, C. durissus cunamensis, C. d. terrificus, C. d. collineatus and C. d. cascavella were assayed for nuclease activity in agarose gel plates containing 2.0mg DNA from sperm salmon, with different concentrations of venom (25-100µg). After incubation at 37°C, the plates were photographed under a UV light at different time intervals and the nucleotidic activity was expressed in cm of the resulting halos. Activity was also assayed by electrophoresis on agarose gel containing 400ng of DNA from E. coli and 1.0µg of each venom. Isolation of a nuclease from C. d. terrificus from venom was accomplished through an affinity chromatographic column, eluted with water followed by a salt concentration gradient (0 to 1M) in 0,01M Tris-HCl +0.001M EDTA, pH 7,4. The protein was assayed for purity by 12% SDS-PAGE and stained with Coomassie Brilliant Blue and Ag+. The nucleotidic activity was assayed by PAGE and radial diffusion in agarose gel. Venoms from species C. d. collineatus, C. d. cumanensis, C. d. terrificus, C. d. cascavella and C. atrox showed 0.8; 0.6; 0.8; 1.1; 0.65, respectively of nucleotidic activity. The highly purified enzyme, which was isolated in a single step, showed a single polypeptide chain and extremely active even at very low concentrations (0.5µg), snake venoms are promising sources of nucleotidic enzymes for biotechnological applications.

MARCUSSI, S.*; TICLI, F.K.**; SILVEIRA, L.B.*; GONÇALVES, M.A.*; NETO, M.M.*; PEREIRA, P.*; FRANÇA, S.C.*; SOARES, A.M.*

Introdução

As Fosfolipases A2 (PLA2s) de venenos de serpentes induzem diferentes efeitos tóxicos e farmacológicos como miotoxicidade, neurotoxicidade, edema, letalidade, hipotensão, inibição da agregação plaquetária, convulsão, anticoagulação e outros. Inibidores naturais de PLA2s (PLIs) foram encontrados em diferentes organismos como plantas, esponjas marinhas, plasma de serpentes e de gambá.

^aMaiorano VA , ^b,cMarcussi S, ^aDaher MA, ^a,cOliveira CZ, ^dCouto LB, ^dGomes OA, ^aFranca SC, ^cSoares AM, ^aPereira PS.

Antiophidian properties of the aqueous extract of Mikania glomerata

Aqueous extracts, prepared from dried or fresh roots, stems or leaves of Mikania glomerata, a plant found in Mata Atlantica in Southeastern Brazil, were able to efficiently neutralize different toxic, pharmacological, and enzymatic effects induced by venoms from Bothrops and Crotalus snakes. Phospholipase A₂ activity and the edema induced by Crotalus durissus terrificus venom were inhibited around 100 and approximately 40%, respectively, although this inhibition was only partial for Bothrops venoms. The hemorrhagic activity of Bothrops venoms (Bothrops altenatus, Bothrops moojeni, Bothrops neuwiedi, and Bothrops jararacussu) was significantly inhibited by this vegetal species, while the clotting activity of Crotalus durissus terrificus, Bothrops jararacussu, and Bothrops neuwiedi venoms was totally inhibited. Although, the mechanism of action of Mikania glomerata extract is still unknown, the finding that no visible change was detected in the electrophoretic pattern of snake venom after incubation with the extract excludes proteolytic degradation as a potential mechanism. Since the extract of Mikania glomerata significantly inhibited the studied snake venoms, it may be used as an alternative treatment to serumtherapy and, in addition, as a rich source of potential inhibitors of PLA₂s, metalloproteases and serineproteases, enzymes involved in several physiopathological human and animal diseases.