Efeito da interação entre Bothropstoxina-I e Crotapotina na junção neuromuscular de camundongos “in vitro”: estágio em iniciação científica

A bothropstoxina-I (BthTX-I) é uma fosfolipase A2 (PLA2) Lys49 miotóxica isolada do veneno da Bothrops jararacussu. Embora seja desprovida de atividade neurotóxica in vivo, esta toxina bloqueia a transmissão neuromuscular in vitro. A relação entre as atividades miotóxica e paralisante da BthTX-I ainda não está esclarecida. A crotapotina corresponde à subunidade não-enzimática da crotoxina, principal fração tóxica do veneno da Crotalus durissus terrificus. Isoladamente a crotapotina é atóxica, porém atua como carreadora da PLA2 Asp49 da crotoxina, potencializando sua ação neurotóxica. Esta proteína também é capaz de se complexar com outras PLA2s (Asp49 ou Lys49) de venenos ofídicos, alterando suas toxicidades. Neste trabalho avaliamos a influência da crotapotina sobre o bloqueio neuromuscular e a atividade miotóxica da BthTX-I in vitro. Preparações do nervo frênico-músculo diafragma de camundongos machos foram montadas em cubas para o registro das contrações musculares evocadas direta e indiretamente. Cortes transversais do músculo foram submetidos à coloração por hematoxilina e eosina para a avaliação do padrão morfológico. A BthTX-I (1 μM) isoladamente, ou pré-incubada com crotapotina (2 M) à 35 ºC por 30 minutos, foram adicionadas às preparações. A análise dos dados foi realizada por testes não paramétricos (p<0.05). A BthTX-I induziu bloqueio irreversível e tempo-dependente das contrações musculares diretas e indiretas. O tempo para o bloqueio de 50% das contrações indiretas (18,98 ± 1,94 min, n=4) foi significativamente menor que o das diretas (45,97 ± 5,61 min, n=5). A pré-incubação com a crotapotina não alterou de forma significativa o bloqueio das contrações diretas ou indiretas induzidos pela BthTX-I. Isoladamente, a crotapotina não afetou as contrações... (Resumo completo, clicar acesso eletrônico abaixo)

Caracterização por espectrometria de massas e investigação das atividades anti-inflamatória e gastroprotetora do extrato etanólico e diterpenos clerodânicos de folhas de Casearia sylvestris Swartz

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Caracterização por espectrometria de massas e investigação das atividades anti-inflamatória e gastroprotetora do extrato etanólico e diterpenos clerodânicos de folhas de Casearia sylvestris Swartz

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The bactericidal effect of human secreted group IID phospholipase A(2) results from both hydrolytic and non-hydrolytic activities

The Human Secreted Group IID Phospholipase A(2) (hsPLA2GIID) may be involved in the human acute immune response. Here we have demonstrated that the hsPLA2GIID presents bactericidal and Ca2+-independent liposome membrane-damaging activities and we have compared these effects with the catalytic activity of active-site mutants of the protein. All mutants showed reduced hydrolytic activity against DOPC:DOPG liposome membranes, however bactericidal effects against Escherichia coli and Micrococcus luteus were less affected, with the D49K mutant retaining 30% killing of the Gram-negative bacteria at a concentration of 10 mu g/mL despite the absence of catalytic activity. The H48Q mutant maintained Ca2+-independent membrane-damaging activity whereas the G30S and D49K mutants were approximately 50% of the wild-type protein, demonstrating that phospholipid bilayer permeabilization by the hsPLA2GIID is independent of catalytic activity. We suggest that this Ca2+-independent damaging activity may play a role in the bactericidal function of the protein. (C) 2012 Elsevier Masson SAS. All rights reserved.

Antiviral and antiparasite properties of an L-amino acid oxidase from the Snake Bothrops jararaca: Cloning and identification of a complete cDNA sequence (Retracted article. See vol. 80, pg. 288, 2010)

L-Amino acid oxidases (LAAOs, EC 1.4.3.2) are flavoenzymes that catalyze the stereospecific oxidative deamination of an L-amino acid substrate to the corresponding a-ketoacid with hydrogen peroxide and ammonia production. The present work describes the first report on the antiviral (Dengue virus) and antiprotozoal (trypanocidal and leishmanicide) activities of a Bothrops jararaca L-amino acid oxidase (BjarLAAO-I) and identify its cDNA sequence. Antiparasite effects were inhibited by catalase, suggesting that they are mediated by H(2)O(2) production. Cells infected with DENV-3 virus previously treated with BjarLAAO-I, showed a decrease in viral titer (13-83-fold) when compared with cells infected with untreated viruses. Untreated and treated promastigotes (T. cruzi and L. amazonensis) were observed by transmission electron microscopy with different degrees of damage. Its complete cDNA sequence, with 1452 bp, encoded an open reading frame of 484 amino acid residues with a theoretical molecular weight and pl of 54,771.8 and 5.7, respectively. The cDNA-deduced amino acid sequence of BjarLAAO shows high identity to LAAOs from other snake venoms. Further investigations will be focused on the related molecular and functional correlation of these enzymes. Such a study should provide valuable information for the therapeutic development of new generations of microbicidal drugs. (C) 2008 Elsevier Inc. All rights reserved.

Neutralization of pharmacological and toxic activities of Bothrops snake venoms by Schizolobium parahyba (Fabaceae) aqueous extract and its fractions

The aqueous extract prepared from Schizolobium parahyba (Sp) leaves, a native plant from Atlantic Forest (Brazil), was tested to analyse its ability to inhibit some biological and enzymatic activities induced by Bothrops alternatus (BaltCV) and Bothrops moojeni (BmooCV) snake venoms. Sp inhibited 100% of lethality, blood incoagulability, haemorrhagic and indirect haemolytic activities at a 1:10 ratio (venom/extract, w/w), as well as coagulant activity at a 1:5 ratio (venom/extract, w/w) induced by both venoms. BaltCV fibrinogenolytic activity was also neutralized by Sp at a 1:10 ratio, resulting in total protection of fibrinogen B beta chain and partial protection of A alpha chain. Interaction tests have demonstrated that, at certain extract/proteins ratios, Sp precipitates proteins non-specifically suggesting the presence of tannins, which are very likely responsible for the excellent inhibiting effects of the analysed ophidian activities. Sp aqueous extract chromatography on Sephadex LH-20 was carried out aiming at the separation of these compounds that mask the obtained results. Thus, the fractionation of Sp resulted in three fractions: F1 (methanolic fraction); F2 (methanol:water fraction, 1:1 v/v); and F3 (aqueous fraction). These fractions were analysed for their ability to inhibit the BaltCV fibrinogenolytic activity. F1 inhibited 100% the venom fibrinogenolytic activity without presenting protein precipitation effect; F2 showed only partial inhibition of this venom activity. Finally, F3 did not inhibit fibrinogen proteolysis, but presented strong protein precipitating action. We conclude that Sp aqueous extract, together with tannins, also contains other compounds that can display specific inhibitory activity against snake venom toxins.

Identification and characterization of a new member of snake venom thrombin inhibitors from Bothrops insularis using a proteomic approach

Snake venom C-type lectin-like proteins (CLPs) are ubiquitously found in Viperidae snake venoms and differ from the C-type lectins as they display different biological activities but no carbohydrate-binding activity. Previous analysis of the transcriptome obtained from the Bothrops insularis venom gland showed the presence of two clusters homologous to bothrojaracin (BJC) chains a and P. In an effort to identify a new BJC-like molecule, we used an approach associated with proteomic technologies to identify the presence of the expressed protein and then to purify and characterize a new thrombin inhibitor from B. insularis venom. We also constructed homology models of this protein and BJC, which were compared with other C-type lectin-like family members and revealed several conserved features of this intriguing snake venom toxin family. (C)0 2007 Elsevier Ltd. All rights reserved.

ACUTE KIDNEY INJURY CAUSED BY Crotalus AND Bothrops SNAKE VENOM: A REVIEW OF EPIDEMIOLOGY, CLINICAL MANIFESTATIONS AND TREATMENT

SUMMARY Ophidic accidents are an important public health problem due to their incidence, morbidity and mortality. An increasing number of cases have been registered in Brazil in the last few years. Several studies point to the importance of knowing the clinical complications and adequate approach in these accidents. However, knowledge about the risk factors is not enough and there are an increasing number of deaths due to these accidents in Brazil. In this context, acute kidney injury (AKI) appears as one of the main causes of death and consequences for these victims, which are mainly young males working in rural areas. Snakes of the Bothrops and Crotalus genera are the main responsible for renal involvement in ophidic accidents in South America. The present study is a literature review of AKI caused by Bothrops and Crotalus snake venom regarding diverse characteristics, emphasizing the most appropriate therapeutic approach for these cases. Recent studies have been carried out searching for complementary therapies for the treatment of ophidic accidents, including the use of lipoic acid, simvastatin and allopurinol. Some plants, such as Apocynaceae, Lamiaceae and Rubiaceae seem to have a beneficial role in the treatment of this type of envenomation. Future studies will certainly find new therapeutic measures for ophidic accidents.

Comparative evaluation of adverse effects in the use of powder trivalent antivenom and liquid antivenoms in Bothrops snake bites

INTRODUCTION: Snake bite, a problem in public health, generally occurs where there is no electric power. METHODS: A comparative clinical study was conducted with 102 victims of Bothrops snake bite, from the state of Amazonas, Brazil; 58 victims were treated with liofilizated trivalent antivenom serum (SATL) and 44 victims treated with liquid bivalent and monovalent antivenom serum (SAMBL). RESULTS: 17% (10/58) of patients presented adverse effects with the SATL and 25% (11/44) with the SAMBL. CONCLUSIONS: There was no statistic difference in number of adverse effects between the two types of snake bite antivenom.

The pharmacological effect of Bothrops neuwiedii pauloensis (jararaca-pintada) snake venom on avian neuromuscular transmission

The neuromuscular effects of Bothrops neuwiedii pauloensis (jararaca-pintada) venom were studied on isolated chick biventer cervicis nerve-muscle preparations. Venom concentrations of 5-50 µg/ml produced an initial inhibition and a secondary increase of indirectly evoked twitches followed by a progressive concentration-dependent and irreversible neuromuscular blockade. At venom concentrations of 1-20 µg/ml, the responses to 13.4 mM KCl were inhibited whereas those to 110 µM acetylcholine alone and cumulative concentrations of 1 µM to 10 mM were unaffected. At venom concentrations higher than 50 µg/ml, there was pronounced muscle contracture with inhibition of the responses to acetylcholine, KCl and direct stimulation. At 20-24ºC, the venom (50 µg/ml) produced only partial neuromuscular blockade (30.7 ± 8.0%, N = 3) after 120 min and the initial inhibition and the secondary increase of the twitch responses caused by the venom were prolonged and pronounced and the response to KCl was unchanged. These results indicate that B.n. pauloensis venom is neurotoxic, acting primarily at presynaptic sites, and that enzyme activity may be involved in this pharmacological action.

Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper

A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13% of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 µg) and fibrinogen (minimum coagulant dose = 4.2 µg) in vitro, and promotes defibrin(ogen)ation in vivo (minimum defibrin(ogen)ating dose = 1.0 µg). In addition, when injected intravenously in mice at doses of 5 and 10 µg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the `gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.

Sympathetic outflow activates the venom gland of the snake Bothrops jararaca by regulating the activation of transcription factors and the synthesis of venom gland proteins

The venom gland of viperid snakes has a central lumen where the venom produced by secretory cells is stored. When the venom is lost from the gland, the secretory cells are activated and new venom is produced. The production of new venom is triggered by the action of noradrenaline on both alpha(1)- and beta-adrenoceptors in the venom gland. In this study, we show that venom removal leads to the activation of transcription factors NF kappa B and AP-1 in the venom gland. In dispersed secretory cells, noradrenaline activated both NF kappa B and AP-1. Activation of NF kappa B and AP-1 depended on phospholipase C and protein kinase A. Activation of NF kappa B also depended on protein kinase C. Isoprenaline activated both NF kappa B and AP-1, and phenylephrine activated NF kappa B and later AP-1. We also show that the protein composition of the venom gland changes during the venom production cycle. Striking changes occurred 4 and 7 days after venom removal in female and male snakes, respectively. Reserpine blocks this change, and the administration of alpha(1)- and beta-adrenoceptor agonists to reserpine-treated snakes largely restores the protein composition of the venom gland. However, the protein composition of the venom from reserpinized snakes treated with alpha(1)- or beta-adrenoceptor agonists appears normal, judging from SDS-PAGE electrophoresis. A sexual dimorphism in activating transcription factors and activating venom gland was observed. Our data suggest that the release of noradrenaline after biting is necessary to activate the venom gland by regulating the activation of transcription factors and consequently regulating the synthesis of proteins in the venom gland for venom production.

The three-dimensional structure of bothropasin, the main hemorrhagic factor from Bothrops jararaca venom: Insights for a new classification of snake venom metalloprotease subgroups

Bothropasin is a 48 kDa hemorrhagic PIII snake venom metalloprotease (SVMP) isolated from Bothrops jararaca, containing disintegrin/cysteine-rich adhesive domains. Here we present the crystal structure of bothropasin complexed with the inhibitor POL647. The catalytic domain consists of a scaffold of two subdomains organized similarly to those described for other SVMPs, including the zinc and calcium-binding sites. The free cysteine residue Cys(189) is located within a hydrophobic core and it is not available for disulfide bonding or other interactions. There is no identifiable secondary structure for the disintegrin domain, but instead it is composed mostly of loops stabilized by seven disulfide bonds and by two calcium ions. The ECD region is in a loop and is structurally related to the RGD region of RGD disintegrins, which are derived from I`ll SVMPs. The ECD motif is stabilized by the Cys(117)_Cys(310) disulfide bond (between the disintegrin and cysteine-rich domains) and by one calcium ion. The side chain of Glu(276) of the ECD motif is exposed to solvent and free to make interactions. In bothropasin, the HVR (hyper-variable region) described for other Pill SVMPs in the cysteine-rich domain, presents a well-conserved sequence with respect to several other Pill members from different species. We propose that this subset be referred to as PIII-HCR (highly conserved region) SVMPs. The differences in the disintegrin-like, cysteine-rich or disintegrin-like cysteine-rich domains may be involved in selecting target binding, which in turn could generate substrate diversity or specificity for the catalytic domain. (C) 2008 Elsevier Ltd. All rights reserved.

Umbelliferone induces changes in the structure and pharmacological activities of Bn IV, a phospholipase A(2) isoform isolated from Bothrops neuwiedi

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Neutralization of snake venom phospholipase A(2) toxins by aqueous extract of Casearia sylvestris (Flacourtiaceae) in mouse neuromuscular preparation

Aqueous extract of Casearia sylvestris (Flacourtiaceae) has been shown to inhibit enzymatic and biological properties of some Bothrops and Crotalus venoms and their purified phospholipase A(2) (PLA(2)) toxins. In this work we evaluated the influence of C sylvestris aqueous extract upon neuromuscular blocking and muscle damaging activities of some PLA(2)S (crotoxin from C. durissus terrificus, bothropstoxin-I from B.jararacussu, piratoxin-I from B. pirajai and myotoxin-II from B. moojeni) in mouse phrenic-diaphragm preparations. Crotoxin (0.5 mu M) and all other PLA2 toxins (1.0 mu M) induced irreversible and time-dependent blockade of twitches. Except for crotoxin, all PLA2 toxins induced significant muscle damage indices, assessed by microscopic analysis. Preincubation of bothropstoxin-I, piratoxin-I or myotoxin-II with C. sylvestris extract (1:5 (w/w), 30 min, 37 degrees C significantly prevented the neuromuscular blockade of preparations exposed to the mixtures for 90 min; the extent of protection ranged from 93% to 97%. The vegetal extract also neutralized the muscle damage (protection of 80-95%). Higher concentration of the C. sylvestris extract (1: 10, w/w) was necessary to neutralize by 90% the neuromuscular blockade induced by crotoxin. These findings expanded the spectrum of C. sylvestris antivenom activities, evidencing that it may be a good source of potentially useful PLA2 inhibitors. (c) 2007 Elsevier B.V.. All rights reserved.