Crystallization and preliminary X-ray diffraction analysis of suramin, a highly charged polysulfonated napthylurea, complexed with a myotoxic PLA(2) from Bothrops asper venom

Suramin is a highly charged polysulfonated napthylurea that interferes in a number of physiologically relevant processes such as myotoxicity, blood coagulation and several kinds of cancers. This synthetic compound was complexed with a myotoxic Lys49 PLA(2) from Bothrops asper venom and crystallized by the hanging-drop vapor diffusion method at 18 degreesC. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell parameters a=49.05, b=63.84 and c=85.67 Angstrom, Diffraction data was collected to 1.78 Angstrom. (C) 2004 Elsevier B.V. All rights reserved.

Antitumor effect of Bothrops jararaca venom

MANY experimental studies have been carried out using snake venoms for the treatment of animal tumors, with controversial results. While some authors have reported an antitumor effect of treatment with specific snake venom fractions, others have reported no effects after this treatment. The aim of this study was to evaluate the effect of Bothrops jararaca venom (BjV) on Ehrlich ascites tumor (EAT) cells in vivo and in vitro. In the in vivo study, Swiss mice were inoculated with EAT cells by the intraperitoneal (i.p.) route and treated with BjV venom (0.4 mg/kg, i.p.), on the 1st, 4th, 7th, 10th, and 13th days. Mice were evaluated for total and differential cells number on the 2nd, 5th, 8th, 11th and 14th days. The survival time was also evaluated after 60 days of tumor growth. In the in vitro study, EAT and normal peritoneal cells were cultivated in the presence of different BjV concentrations (2.5, 5.0, 10.0, 20.0, 40.0, and 80 mug) and viability was verified after 3, 6, 12 and 24 h of cultivation. Results were analyzed statistically by the Kruskal-Wallis and Tukey tests at the 5% level of significance. It was observed that in vivo treatment with BjV induced tumor growth inhibition, increased animal survival time, decreased mortality, increased the influx of polymorphonuclear leukocytes on the early stages of tumor growth, and did not affect the mononuclear cells number. In vitro treatment with BjV produced a dose-dependent toxic effect on EAT and peritoneal cells, with higher effects against peritoneal cells. Taken together, our results demonstrate that BjV has an important antitumor effect. This is the first report showing this in vivo effect for this venom.

Crytallization and high-resolution X-ray diffraction data collection of an Asp49 PLA(2) from Bothrops jararacussu venom both in the presence and absence of Ca2+ ions

Snake venom PLA(2)s have been extensively studied due to their role in mediating and disrupting physiological processes such as coagulation, platelet aggregation and myotoxicity. The Ca2+ ion bound to the putative calcium-binding loop is essential for hydrolytic activity. We report the crystallization in the presence and absence of Ca2+ and X-ray diffraction data collection at 1.60 Angstrom (with Ca2+) and 1.36 Angstrom (without Ca2+) of an Asp49 PLA(2) from Bothrops jararacussu venom. The crystals belong to orthorhombic space group C222(1). Initial refinement and electron density analysis indicate significant conformational. changes upon Ca2+ binding. (C) 2004 Elsevier B.V. All fights reserved.

Calcium-Independent Membrane Damage by Venom Phospholipases A(2)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Vascular effects and electrolyte homeostasis of the natriuretic peptide isolated from Crotalus oreganus abyssus (North American Grand Canyon rattlesnake) venom

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Antitumor effect of snake venoms

The search for biological antitumor agents has been pursued for over half a century. Snake venom has been shown to possess a wide spectrum of biological activities. The objectives of the present review are to evaluate the existing controversies on this subject published in a number of papers and to propose probable explanations for the phenomena observed. We reported our results obtained in a study, in which we evaluated the action of the venoms of Crotalus durissus terrificus and Bothrops jararaca on Ehrlich ascites tumor cells. We noticed an important antitumor effect, mainly with Bothrops jararaca venom, as well as an increase in the functional activity of macrophages. We also observed an increase in the number of mononuclear and polymorphonuclear cells with Bothrops jararaca venom. Considering these findings, we postulate that both Borhrops jararaca and Crotalus durissus terrificus venoms can act directly on tumor cells. In addition, we propose an indirect mechanism, based on the stimulation of the inflammatory response, to inhibit tumor growth and to promote its rejection.

Cytokine profile of Ehrlich ascites tumor treated with Bothrops jararaca venom

WE previously demonstrated that Bothrops jararaca venom (BjV) has an antitumor effect on Ehrlich ascites tumor (EAT) cells and induces an increase of polymorphonuclear leukocytes in early stages of tumor growth. It has been reported that this venom presents an important inflammatory effect when inoculated in animal models and in human snake-bites, and that cytokine levels have been detected in these cases. To evaluate whether the cytokines can be involved with the suppression of the tumoral growth, we evaluate the cytokine profile in the peritoneal cavity of mice inoculated with EAT cells and treated with BjV. Swiss mice were inoculated with EAT cells by the intraperitoneal route and treated with BjV venom (0.4 mg/kg, intraperitoneally), on the 1st, 4th, 7th, 10th, and 13th day. Mice were evaluated for cytokine levels on the 2nd, 5th, 8th, 11th and 14th day. Analysis was performed using an enzyme-linked immunosorbent assay for interleukin (IL)-1α, IL-2, IL-4, IL-6, IL-10, IL-13, and tumor necrosis factor-α (TNF-α) levels in the peritoneal washing supernatant. Results were analyzed statistically by the Kruskal-Wallis and Dunn's tests at the 5% level of significance. We observed that EAT implantation induces IL-6 production on the 11th and 14th days of tumor growth, IL-10 on the 11th day and TNF-α on the 14th day. The treatment with BjV suppresses production of these cytokines. In addition, IL-13 was produced by animals that were inoculated only with venom on the 11th and 14th days, and by the group inoculated with EAT cells and treated with venom on the 2nd and 14th days. Furthermore, we suggest that the IL-6 detected in the present study is produced by the EAT cells and the suppression of its production could be associated with the antitumor effect of BjV.

Action of anti-bothropic factor isolated from Didelphis marsupialis on renal effects of Bothrops erythromelas venom

Acute renal failure is the most common complication in the lethal cases caused by snakebites in Brazil. Among the Brazilian venom snakes, Bothrops erythromelas is responsible for the majority of accidents in Northeastern Brazil. Didelphis marsupialis serum could inhibit myonecrotic, hemorrhagic, edematogenic hyperalgesic and lethal effects of envenomation determined by ophidian bites. In the present study, we evaluated the action of the anti-bothropic factor isolated from D. marsupialis on the renal effects promoted by B. erythromelas venom without systemic interference. Isolated kidneys from Wistar rats were perfused with Krebs-Henseleit solution containing 6% bovine serum albumin. We analyzed renal perfusion pressure (PP), renal vascular resistance (RVR), glomerular filtration rate (GFR), urinary flow (UF), and the percentages of sodium and potassium tubular transport (%TNa +, %TK +). The B. erythromelas venom (10 μg mL -1) decreased the PP (ct=108.71±5.09 mmHg; BE=65.21±5.6 mmHg*) and RVR (ct=5.76±0.65 mmHg mL -1 g -1 min -1; BE=3.10±0.45 mmHg mL -1 g -1 min -1*) . On the other hand, the GFR decreased at 60 min (ct 60=0.76±0. 07 mL g -1 min -1; BE 60=0.42±0.12 mL g -1 min -1*) and increased at 120 min (ct 120=0.72±0.01 mL g -1 min -1; BE 120=1.24±0.26 mL g -1 min -1*). The UF increased significantly when compared with the control group (ct=0.14±0.01 mL g -1 min -1; BE=0.47±0.08 mL g -1 min -1*). The venom reduced the %TNa + (ct 90=79.18±0.88%; BE 90=58.35±4.86%*) and %TK + (ct 90=67.20±4.04%; BE 90=57. 32±5.26%*) The anti-bothropic factor from D. marsupialis (10 μg mL -1) incubated with B. erythromelas venom (10 μg mL -1) blocked the effects on PP, RVR, %TNa +, and %TK +, but was not able to reverse the effects in UF and GFR promoted by venom alone. However, the highest concentration of D. marsupialis serum (30 μg mL -1) reversed all the renal effects induced by the venom. In conclusion, B. erythromelas venom altered all the renal functional parameters evaluated and the anti-bothropic factor from D. marsupialis was able to inhibit the effects induced by the venom in isolated kidney. © 2005 Elsevier Ltd. All rights reserved.

Structure of myotoxin II, a catalytically inactive Lys49 phospholipase A2 homologue from Atropoides nummifer venom

Lys49 snake-venom phospholipase A2 (PLA2) homologues are highly myotoxic proteins which, although lacking catalytic activity, possess the ability to disrupt biological membranes, inducing significant muscle-tissue loss and permanent disability in severely envenomed patients. Since the structural basis for their toxic activity is still only partially understood, the structure of myotoxin II, a monomeric Lys49 PLA2 homologue from Atropoides nummifer, has been determined at 2.08 Å resolution and the anion-binding site has been characterized. © 2006 International Union of Crystallography. All rights reserved.

Bothrops leucurus venom induces nephrotoxicity in the isolated perfused kidney and cultured renal tubular epithelia

Bites from snake (Bothrops genus) cause local tissue damage and systemic complications, which include alterations such as hemostatic system and acute renal failure (ARF). Recent studies suggest that ARF pathogenesis in snakebite envenomation is multifactorial and involves hemodynamic disturbances, immunologic reactions and direct nephrotoxicity. The aim of the work was to investigate the effects of the Bothrops leucurus venom (BlV) in the renal perfusion system and in cultured renal tubular cells of the type MDCK (Madin-Darby Canine kidney). BlV (10 μg/mL) reduced the perfusion pressure at 90 and 120 min. The renal vascular resistance (RVR) decreased at 120 min of perfusion. The effect on urinary flow (UF) and glomerular filtration rate (GFR) started 30 min after BlV infusion, was transient and returned to normal at 120 min of perfusion. It was also observed a decrease on percentual tubular transport of sodium (%TNa+) at 120 min and of chloride (%TCl-) at 60 and 90 min. The treatment with BlV caused decrease in cell viability to the lowest concentration tested with an IC50 of 1.25 μg/mL. Flow cytometry with annexin V and propidium iodide showed that cell death occurred predominantly by necrosis. However, a cell death process may involve apoptosis in lower concentrations. BlV treatment (1.25 μg/mL) led to significant depolarization of the mitochondrial membrane potential and, indeed, we found an increase in the expression of cell death genes in the lower concentrations tested. The venom also evoked an increase in the cytosolic Ca2+ in a concentration dependent manner, indicating that Ca2+ may participate in the venom of B. leucurus effect. The characterization of the effects in the isolated kidney and renal tubular cells gives strong evidences that the acute renal failure induced by this venom is a result of the direct nephrotoxicity which may involve the cell death mechanism. © 2012.

Caracterização biofísica e estrutural da metaloproteinase não-hemorrágica do veneno de Bothrops moojeni e da endo-β-glicanase do Bacillus subtilis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Vellozia flavicans Mart. ex Schult. hydroalcoholic extract inhibits the neuromuscular blockade induced by Bothrops jararacussu venom

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structural bases for a complete myotoxic mechanism: Crystal structures of two non-catalytic phospholipases A(2)-like from Bothrops brazili venom

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A structure-based proposal for a comprehensive myotoxic mechanism of phospholipase A(2)-like proteins from viperid snake venoms

Envenomation via snakebites is an important public health problem in many tropical and subtropical countries that, in addition to mortality, can result in permanent sequelae as a consequence of local tissue damage, which represents a major challenge to antivenom therapy. Venom phospholipases A(2) (PLA(2)s) and PLA(2)-like proteins play a leading role in the complex pathogenesis of skeletal muscle necrosis, nevertheless their precise mechanism of action is only partially understood. Recently, detailed structural information has been obtained for more than twenty different members of the PLA(2)-like myotoxin subfamily. In this review, we integrate the available structural, biochemical and functional data on these toxins and present a comprehensive hypothesis for their myotoxic mechanism. This process involves an allosteric transition and the participation of two independent interaction sites for docking and disruption of the target membrane, respectively, leading to a five-step mechanism of action. Furthermore, recent functional and structural studies of these toxins complexed with ligands reveal diverse neutralization mechanisms that can be classified into at least three different groups. Therefore, the data summarized here for the PLA(2)-like myotoxins could provide a useful molecular basis for the search for novel neutralizing strategies to improve the treatment of envenomation by viperid snakes. (C) 2014 Elsevier B.V. All rights reserved.