Histological and functional renal alterations caused by Bothrops alternatus snake venom: Expression and activity of Na(+)/K(+)-ATPase

Background: Acute renal failure is a serious complication of human envenoming by Bothrops snakes. The ion pump Na(+)/K(+)-ATPase has an important role in renal tubule function, where it modulates sodium reabsorption and homeostasis of the extracellular compartment. Here, we investigated the morphological and functional renal alterations and changes in Na(+)/K(+)-ATPase expression and activity in rats injected with Bothrops alternatus snake venom. Methods: Male Wistar rats were injected with venom (0.8 mg/kg, iv.) and renal function was assessed 6.24, 48 and 72 h and 7 days post-venom. The rats were then killed and renal Na(+)/K(+)-ATPase activity was assayed based on phosphate release from ATP; gene and protein expressions were assessed by real time PCR and immunofluorescence microscopy, respectively. Results: Venom caused lobulation of the capillary tufts, dilation of Bowman`s capsular space. F-actin disruption in Bowman`s capsule and renal tubule brush border, and deposition of collagen around glomeruli and proximal tubules that persisted seven days after envenoming. Enhanced sodium and potassium excretion, reduced proximal sodium reabsorption, and proteinuria were observed 6 h post-venom, followed by a transient decrease in the glomerular filtration rate. Gene and protein expressions of the Na(+)/K(+)-ATPase alpha(1) subunit were increased 6 h post-venom, whereas Na(+)/K(+)-ATPase activity increased 6 h and 24 h post-venom. Conclusions: Bothrops alternatus venom caused marked morphological and functional renal alterations with enhanced Na(+)/K(+)-ATPase expression and activity in the early phase of renal damage. General significance: Enhanced Na(+)/K(+)-ATPase activity in the early hours after envenoming may attenuate the renal dysfunction associated with venom-induced damage. (C) 2011 Elsevier B.V. All rights reserved.

The Snake Venom Peptide Bj-PRO-7a Is a M1 Muscarinic Acetylcholine Receptor Agonist

Proline-rich peptides from Bothrops jararaca venom (Bj-PRO) were characterized based on the capability to inhibit the somatic angiotensin-converting enzyme. The pharmacological action of these peptides resulted in the development of Captopril, one of the best examples of a target-driven drug discovery for treatment of hypertension. However, biochemical and biological properties of Bj-PROs were not completely elucidated yet, and many recent studies have suggested that their activity relies on angiotensin-converting enzyme-independent mechanisms. Here, we show that Bj-PRO-7a (

Jararhagin, a snake venom metalloprotease-disintegrin, activates the Rac1 GTPase and stimulates neurite outgrowth in neuroblastoma cells

It has been shown previously that the snake venom metalloprotease-disintegrin jararhagin stimulates cell migration and cytoskeletal rearrangement, independently of its effects on cellular adhesion but possibly associated with the activation of small GTP-binding proteins from the Rho family [Costa, E.P., Santos, M.F., 2004. Toxicon 44(8), 861-870.] Here we show that jararhagin stimulates spreading, actin dynamics and neurite outgrowth in neuroblastoma cells, and that this effect is accompanied by the translocation of the Rac1 small GTPase to the membrane fraction, suggesting its activation. Stimulation of neurite outgrowth was observed within minutes and was dependent on the proteolytic activity of the toxin. These results suggest that jararhagin may stimulate neuronal differentiation, being potential tool for neuronal regeneration studies. (C) 2008 Elsevier Ltd. All rights reserved.

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.

Biochemical and functional properties of a thrombin-like enzyme isolated from Bothrops pauloensis snake venom

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

Cell cycle arrest evidence, parasiticidal and bactericidal properties induced by L-amino acid oxidase from Bothrops atrox snake venom

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

Platelet aggregation and antibacterial effects of an L-amino acid oxidase purified from Bothrops alternatus snake venom

The isolation and biochemical/enzymatic characterization of an L-amino acid oxidase, Balt-LAAO-I, from Bothrops alternates snake venom, is described. Balt-LAAO-I is an acidic glycoprotein, pI similar to 5.37, homodimeric, M-r similar to 123, 000, whose Nterminal sequence is ADVRNPLE EFRETDYEVL. It displays a high specificity toward hydrophobic and basic amino acids, while deglycosylation does not alter its enzymatic activity. Bait-LAAO-I induces platelet aggregation and shows bactericidal activity against Escherichia coli and Staphylococcus aureus. In addition, this enzyme is slightly hemorrhagic and induces edema in the mouse paw. Bait-LAAO-I is a multifunctional enzyme with promising relevant biotechnological and medical applications. (C) 2004 Elsevier Ltd. All rights reserved.

Treatment of venous ulcers with fibrin sealant derived from snake venom

Venous ulcers of the lower limbs complicated by infection or chronicity represent a serious public health problem. The elevated number of those afflicted burdens the health services, interferes in quality of life and causes absenteeism. Although there are 2,500 items on the market, ranging from the simplest dressing up to the most complex types of dressing, treatment remains a challenge. Among the substances used, fibrin sealant is the one that promotes diminution of bacterial colonization and of edema, controls hemorrhaging, alters the pain threshold by protecting the nerve endings, hydrates the wound bed and forms granulation tissue that favors healing. Its disadvantages include higher cost and utilization of human fibrinogen that can transmit infectious diseases. The Center for the Study of Venoms and Venomous Animals (CEVAP) at São Paulo State University (UNESP) developed a new sealant made up of fibrinogen extracted from large animals and from an enzyme obtained from snake venom. The present study, developed in the Health Education Clinic (CEPS) of Sacred Heart University (USC) aimed to evaluate the effect of the new sealant on the healing process of venous ulcers in 24 adult patients, seven of whom were male and 17 female. Two study groups were formed as follows: Group 1 (G1) - control group of 11 patients treated with essential fatty acid (EFA) and Unna's boot, and Group 2 (G2) - 13 patients treated with essential fatty acid (EFA), fibrin sealant and Unna's boot. The follow-up lasted eight weeks and the sealant was applied at only the first and fourth weeks. The results showed that Group 2 presented worse lesion conditions as to healing, but, when comparing the two groups, it was noteworthy that the the sealant was effective in healing venous ulcers. There is evidence that the new sealant is recommended for leg ulcers with the following advantages: ease of application, preparation of the wound bed, diminution of pain and a higher number of discharges in the eighth week. More important, other positive characteristics are non-transmission of infectious diseases, absence of adverse reactions, and economic advantage of being produced by Brazilian technology. Finally, it is suggested that the weekly application of sealant, for at least eight weeks, could improve the healing process and consequently life quality.

Treatment of venous ulcers with surgical adhesives derived from snake venom

A Brazilian dictionary states that "ulcer", from the Latin word ulcuserise, is a superficial lesion on skin or mucous membrane characterized by an inflammatory process and loss of tissue. Ulcers are a relatively frequent problem that may be worsen by infection or long-time duration. In these cases, they can represent a serious public health issue, since the increased case number contributes to the burden on the public health service. In recent decades, wound treatment has advanced in technological and scientific aspects regarding both development of new products and techniques. Ongoing research in this area is providing new products for wound treatment. Among newly tested substances that have presented successful results is fibrin glue, a snake venom derivative that promotes reduction of infection and edema as well as bleeding control and pain decrease. Thus, the present study aimed to evaluate fibrin glue effects on ulcers of venous origin in 24 patients that were divided into two groups. The first one (G1) consisted of 11 patients treated without the adhesive while the second group (G2) was composed of 13 persons who were under therapy with the adhesive. The results of this study revealed that patients treated with fibrin glue evolved to a satisfactory healing when compared to the other group. Therefore, fibrin glue is an appropriate alternative for treating leg ulcers with several advantages: easy application, less pain, early hospital discharge, not transmiting diseases, lower cost than the adhesive currently available in Brazil and, finally, it is a Brazilian technology.

Antitumoural effect of an L-amino acid oxidase isolated from Bothrops jararaca snake venom

An L-amino acid oxidase (BjarLAAO-I) from Bothrops jararaca snake venom was highly purified using a stepwise sequential chromatography on Sephadex G-75, Benzamidine Sepharose and Phenyl Sepharose. Purified BjarLAAO-I showed a molecular weight around 60,000 under reducing conditions and about 125,000 in the native form, when analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. BjarLAAO-I is a homodimeric acidic glycoprotein, pI similar to 5.0, and N-terminal sequence showing close structural homology with other snake venom LAAOs. The purified enzyme catalysed the oxidative deamination of L-amino acids, the most specific substrate being L-Phe. Five amino acids, L-Ser, L-Pro, L-Gly, L-Thr and L-Cys were not oxidized, clearly indicating a significant specificity. BjarLAAO-I significantly inhibited Ehrlich ascites tumour growth and induced an influx of polymorphonuclear cells, as well as spontaneous liberation of H(2)O(2) from peritoneal macrophages. Later, BjarLAAO-I induced mononuclear influx and peritoneal macrophage spreading. Animals treated with BjarLAAO-I showed higher survival time.

Bothrops moojeni myotoxin-II, a Lys49-phospholipase A(2) homologue: An example of function versatility of snake venom proteins

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-PLA2 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. (c) 2005 Elsevier B.V. All rights reserved.