Biological characterization of Bothrops marajoensis snake venom

This study describes the effects of Bothrops marajoensis venom (Marajó lancehead) on isolated neuromuscular preparations of chick biventer cervicis (CBC) and mouse phrenic nerve-diaphragm (PND). At low concentrations (1µg/ml for CBC and 5µg/ml for PND), the venom exhibited a neuromuscular blocking without any damaging effect on the muscle integrity. At higher concentration (20μg/ml for PND), together with the neuromuscular blockade, there was a moderate myonecrosis. The results show differences between mammalian and avian preparations in response to venom concentration; the avian preparation was more sensitive to venom neurotoxic effect than the mammalian preparation. The possible presynaptic mechanism underlying the neuromuscular blocking effect was reinforced by the observed increase in MEPPs at the same time (at 15min) when the facilitation of twitch tension occurred. These results indicate that the B. marajoensis venom produced neuromuscular blockade, which appeared to be presynaptic at low concentrations with a postsynaptic component at high concentrations, leading to muscle oedema. These observations demand the fractionation of the crude venom and characterization of its active components for a better understanding of its biological dynamics

Estudo comparativo : bioquimico e imunofarmacologico do veneno total, da crotoxina e suas isoformas de crotapotina e PLA2 de crotalus durissus terrificus. C. d. ruruima C.d. cascavella e C. d. collilineatus

Orientador: Lea Rodrigues SimioniTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias MedicasResumo: Este trabalho está dividido em três momentos, o primeiro trata-se de um estudo comparativo do efeito dos venenos brutos de Crotalus durissus terrificus; C. d. ruruima; C. d. cascavella e C. d. collilineatus, bem como da crotoxina e da crotamina sobre a junção neuromuscular. O veneno C. d. ruruima foi purificado pela primeira vez através de HPLC em uma coluna Protein Pack SW 300, onde foi obtida a crotoxina e a crotamina (os demais venenos já foram purificados e os dados publicados). O estudo neurotóxico foi realizado em duas preparações nervo-frênico diafragma de camundongo e biventer cervicis de pintainho. Dos experimentos realizados em mamíferos e aves obteve-se o efeito neurotóxico esperado nas concentrações de 10 mg/ml. Os venenos brutos e as crotoxinas das serpentes C. d. terrificus e C. d. ruruima em preparações de mamífero produziram aumento da amplitude seguido de bloqueio total da resposta contrátil, diferentemente dos venenos e das crotoxinas das serpentes C. d. cascaavella e C. d. collilineatus, que causaram apenas bloqueio total neuromuscular. Em preparações de aves os quatro venenos e crotoxinas estudados induziram bloqueio total, porém sem causar facilitação. As crotaminas-positivas, estudadas em nervo-frenico diafragma de camundongo das serpentes C. d. terrificus e C. d. ruruima, na concentração de 10 mg/ml, causaram efeito facilitatório retornando à amplitude controle após 120 min de incubação. Ao recombinar a crotoxina 10 mg/ml e crotamina 5 mg/ml houve o aumento da amplitude seguido de bloqueio total da resposta contrátil. Nas preparações ensaiadas em aves, apresentou um efeito facilitatório fugaz seguido de bloqueio neuromuscular total. No segundo momento, as isoformas de crotapotina e PLA2 isoladas a partir da crotoxina dos venenos das serpentes de C. d. terrificus, C. d. cascavella e C. d. collilineatus, foram ensaiadas em mamíferos e aves nas concentrações de 10 mg/ml. As crotapotinas e PLA2 isoladas não causaram diferenças significativas em relação ao controle em preparações de mamíferos. O mesmo não ocorrendo com as PLA2 testadas em aves que causaram bloqueio neuromuscular, efeito este não esperado. Após a recombinação (5 mg/ml crotapotina) + (5 mg/ml PLA2), o complexo crotoxina causou bloqueio neuromuscular total em ambas preparações. Na terceita etapa deste trabalho, foram produzidos antivenenos específicos contra o veneno bruto e crotoxina do veneno C. d ruruima e PLA2 do veneno de C. d. collilineatus. O título de anticorpos e a especificidade dos antivenenos produzidos foram avaliados por ELISA. A neutralização da atividade neurotóxica foi avaliada em preparações biventer cervicis de pintainho. A capacidade neutralizante dos antivenenos produzidos em coelhos foi comparável ao soro anticrotálico comercial contra o veneno e crotoxina na proporção 1:1. O veneno, a crotoxina e os antivenenos estudados induziram liberação de CK significativamente diferente do controleAbstract: This work is divided at three moments, the first one if terrificus deals with a comparative study of the effect of the crude venoms of Crotalus durissus; C. d. ruruima; C. d. cascavella and C. d. collilineatus, as well as of the crotoxin and the crotamine on the junction neuromuscular. Venoms C. d. ruruima was purified for the first time through HPLC in a column Protein Pack sw 300, where it was gotten the crotoxin and the crotamine (the too much venoms already had been purified and the published data). The neurotoxic study nerve-frênico was carried through in two preparations diaphragm of mouse and to chick biventer cervicis. The experiments carried through in mammals and birds the waited neurotoxic effect in the was gotten concentrations of 10 mg/ml. The crude venoms and the crotoxin of rastnaks C. d. terrificus and C. d. ruruima in preparations of mammal had produced increase of the followed amplitude of complete blockade of the contractil reply, differently of the venoms and the crotoxin of rastnakes C. d. cascavella and C. d. collilineatus, that they had caused only complete blockade to neuromuscular. In preparations of birds the four studied venoms and crotoxin had induced complete blockade, however without causing facilitation. The crotamine-positive, studied in nerve-frenico diaphragm of mouse of snakes C. d. terrificus and C. d. ruruima, in the concentration of 10 mg/ml, had caused facilitatori effect returning to the 120 after amplitude have controlled min of incubation. When crotoxin being recombined 10 mg/ml and 5 crotamine mg/ml had the increase of the followed amplitude of complete blockade of the contractil reply. In the preparations assayed in birds, fast followed of blockade presented a facilitatori effect to neuromuscular complete. At as the moment, isoforms of isolated crotapotin and PLA2 from the crotoxin of the venoms of the rastnakes of C. d. terrificus, C. d. cascavella and C. d. collilineatus, had been assayed in mammals and birds in the concentrations of 10 mg/ml. Isolated crotapotin and PLA2 had not caused significant differences in relation to the control in preparations of mammals. The same not occurring with the PLA2 tested in birds that blockade caused to neuromuscular, effect this not waited. After the recombination (5 mg/ml crotapotin) + (5 mg/ml PLA2), the crotoxin complex caused blockade to neuromuscular complete in both preparations. In the third stage of this work, specific antivenoms against crotoxin the crude venoms and of C. d. ruruima had been produced and PLA2 of the poison of C. d. collilineatus. The heading of antibodies and the especific of the produced antivenoms had been evaluated by ELISA. The neutralization of the neurotoxic activity was evaluated in preparations to chick biventer cervicis. The neutralizing capacity of the antivenoms produced in rabbits was comparable to commercial the anticrotalic serum against the venoms and crotoxin in ratio 1:1. The venoms, the crotoxin and the antivenoms, studied had induced significantly different release of CK of the controlDoutoradoDoutor em Farmacologi

Efeito farmacologico de novos componentes do complexo Crotoxina (Crotalus durissus terricus) e suas isoforma sobre a junção neuromuscular

Orientador: Lea Rodrigues SimioniDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: As espécies Crotalus durissus, popularmente conhecidas como cascaveis, são serpentesde grande importância médica na Américado Sul, principalmente no Brasil.A peçonhadessa serpentetem sido objetode estudos por maisde um século,como mostrado pelos estudos realizadospor BRAZIL,1903, SLOTTA & FRAENKEL CONRAT, 1938, VITAL BRAZIL, 1966 e muitos outros. A peçonha crotalica isolada através de gel filtração do veneno bruto em coluna de SephadexG 75, mostraa presença de quatropicos, (pico I) convulxina, (pico II)giroxina, (pico III) crotoxina e (pico IV) crotamina. O pico III foi testado, mostrou seu característico bloqueio neuromuscular em preparação nervo-frênico diafragma de camundongo. Na purificação do veneno bruto crotálico em HPLC de exclusão molecular, usando uma coluna Protein Pack SW 300, obteve-se além dos picos principais três novos componentes do complexo crotoxina picos: cdty III; cdty V e cdty VI, sendo a crotoxina identificada como pico cdty IV. O uso desta cromatografia determinou a dissociação dos efeitos facilitadores e bloqueadores neuromusculares característicos da crotoxina .Assim, o pico cdty III e cdty V exibiram praticamente somente o efeito facilitador da crotoxina (pico cdty IV) nos 120 min de observação. A repurificação da crotoxina em HPLC de fase reversa em coluna µ- Bondapack c 18, resultou em duas isoformas de crotapotina (F 5 e F 7) e três de fosfolipase A2 (F15, F16 e F17). Foram escolhidas as isoformas F7 (crotapotina) e as F16 e F17 (PLA2), as quais foram recombinadas em três diferentes proporções: 1: 1; 1:2 e 2: 1. Ao associarmosa crotapotina(F7) com a PLA2(F16), obtivemos o efeito "chaperone' descrito na literatura ou seja, a inibição da resposta contrátil, o efeito bloqueador irreversível. O mesmo não ocorreu ao recombinarmos crotapotina (F7) com PLA2(F17), o qual não determinou qualquer alteração na resposta contrátil, isto é, mostrou-se destituída de atividade biológica. Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digitalAbstract: The species Crotalus durissus, popularly known as rattle snakes,includis serpents of great medical importance in South America. The poison of that serpent has been object of studies for more than a century, as shown by the studies accomplished by BRAZIL, 1903, SLOTTA & FRAENKELCONRAT, 1938 and VITAL BRAZIL, 1966 and so many others. Through gel filtration of the crude poison in a column SephadexG 75, shows the presence of four picks, (pick I) convulxin, (pick 11)gyroxin, (pick III) crotoxin and (pick IV) crotamine were obtained. Whem the (pick III)crotoxin was tested, in the phrenic nerve-diaphragm preparation it showed the characteristic block neuromuscular effect. Whem Crotalus durissus terrificus (cdty) was subjected to the molecular exclusion chomatograpy (protein pack SW 300) at isocratic conditions, besides the main picks three new components were obtained pick: III V; and VI. Crotoxin was there identified, as pick IV. Able of inducing initial facilitation followed by an irreversible block. Peak cdty III showed a facilitation effect that dures at least 120 mino The Pick V presented a light facilitation and a decrease of the contradile response during the course time of experiments. The use of this chomatograpy determined the dissociation of the facilitatory effects and blocking characteristic neuromuscular of the crotoxin. Note: The complete abstract is available with the full electronic digital thesis or dissertationsMestradoFarmacologiaMestre em Farmacologi

Beneficial Effect Of Crotamine In The Treatment Of Myasthenic Rats.

Crotamine is a basic, low-molecular-weight peptide that, at low concentrations, improves neurotransmission in isolated neuromuscular preparations by modulating sodium channels. In this study, we compared the effects of crotamine and neostigmine on neuromuscular transmission in myasthenic rats. We used a conventional electromyographic technique in in-situ neuromuscular preparations and a 4-week treadmill program. During the in-situ electromyographic recording, neostigmine (17 μg/kg) caused short-term facilitation, whereas crotamine induced progressive and sustained twitch-tension enhancement during 140 min of recording (50 ± 5%, P < 0.05). On the treadmill evaluation, rats showed significant improvement in exercise tolerance, characterized by a decrease in the number of fatigue episodes after 2 weeks of a single-dose treatment with crotamine. These results indicate that crotamine is more efficient than neostigmine for enhancing muscular performance in myasthenic rats, possibly by improving the safety factor of neuromuscular transmission.47591-