Rapid purification of serine proteinases from Bothrops alternatus and Bothrops moojeni venoms

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

Heterologous expression and biochemical and functional characterization of a recombinant alpha-type myotoxin inhibitor from Bothrops alternatus snake

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

Is rainfall seasonality important for reproductive strategies in viviparous Neotropical pit vipers? A case study with Bothrops leucurus from the Brazilian Atlantic Forest

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

Biogeografia histórica das jararacas do grupo Bothrops alternatus (Serpentes, Viperidae)

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

Skeletal muscle degeneration and regeneration after injection of bothropstoxin-II, a phospholipase A2 isolated from the venom of the snake Bothrops jararacussu

A myotoxic phospholipase A2, named bothropstoxin II (BthTX-II), was isolated from the venom of the South American snake Bothrops jararacussu and the pathogenesis of myonecrosis induced by this toxin was studied in mice. BthTX-II induced a rapid increase in plasma creatine kinase levels. Histological and ultrastructural observations demonstrate that this toxin affects muscle fibers by first disrupting the integrity of plasma membrane, as delta lesions were the earliest morphological alteration and since the plasma membrane was interrupted or absent in many portions. In agreement with this hypothesis, BthTX-II released peroxidase entrapped in negatively charged multilamellar liposomes and behaved as an amphiphilic protein in charge shift electrophoresis, an indication that its mechanism of action might be based on the interaction and disorganization of plasma membrane phospholipids. Membrane damage was followed by a complex series of morphological alterations in intracellular structures, most of which are probably related to an increase in cytosolic calcium levels. Myofilaments became hypercontracted into dense clumps which alternated with cellular spaces devoid of myofibrillar material. Later on, myofilaments changed to a hyaline appearance with a more uniform distribution. Mitochondria were drastically affected, showing high amplitude swelling, vesiculation of cristae, formation of flocculent densities, and membrane disruption. By 24 hr, abundant polymorphonuclear leucocytes and macrophages were observed in the interstitial space as well as inside necrotic fibers. Muscle regeneration proceeded normally, as abundant myotubes and regenerating myofibers were observed 7 days after BthTX-II injection. By 28 days regenerating fibers had a diameter similar to that of adult muscle fibers, although they presented two distinctive features: central location of nuclei and some fiber splitting. This good regenerative response may be explained by the observation that BthTX-II does not affect blood vessels, nerves, or basal laminae. © 1991.

Preliminary X-ray crystallographic studies of a Lys49-phospholipase A(2) homologue from Bothrops pirajai venom complexed with p-bromophenacyl bromide and alpha-tocopherol inhibitors

PrTX-I, a non-catalytic and myotoxic Lys49-PLA(2) from Bothrops pirajai venom has been crystallized alone and in complex with bromophenacyl bromide (BPB), alpha-tocopherol and alpha-tocopherol acetate inhibitors. These crystals have shown to diffract X-rays between 2.34 and 1.65 angstrom resolution. All complexes crystals are isomorphous and belong to the space group P2(1) whereas native PrTX-I crystals belong to the P3(1)21.

Ac2-26 mimetic peptide of annexin A1 inhibits local and systemic inflammatory processes induced by bothrops moojeni venom and the lys-49 phospholipase A2 in a rat model

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

L-Aminoacid oxidase from bothrops leucurus venom Induces nephrotoxicity via apoptosis and necrosis

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

l-amino acid oxidase from Bothrops marajoensis causes nephrotoxicity in isolated perfused kidney and cytotoxicity in MDCK renal cells

Renal alterations caused by Bothrops venom and its compounds are studied to understand these effects and provide the best treatment. Previously, we studied the renal effect of the whole venom of Bothrops marajoensis and its phospholipase A2 (PLA2), but these effects could not to be attributed to PLA2. To continue the study, we report in this short communication the effects of l-amino acid oxidase from B. marajoensis venom (LAAOBm) on renal function parameter alterations observed in the same model of isolated perfused kidney, as well as the cytotoxic effect on renal cells. LAAOBm caused a decrease in PP, RVR, UF, GFR, %TNa(+) and %TCl(-), very similar to the effects of whole venom using the same model. We also demonstrated its cytotoxicity in MDCK cells with IC50 of 2.5 μg/mL and late apoptotic involvement demonstrated by flow cytometry assays. In conclusion, we suggested that LAAOBm is a nephrotoxic compound of B. marajoensis venom.

Thrombocytopenia and platelet hypoaggregation induced by Bothrops asper snake venom Toxins involved and their contribution to metalloproteinase-induced pulmonary hemorrhage

Thrombocytopenia and platelet dysfunction occur in patients bitten by Bothrops sp snakes in Latin America. An experimental model was developed in mice to study the effects of B. asper venom in platelet numbers and function. Intravenous administration of this venom induces rapid and prominent thrombocytopenia and ex vivo platelet hypoaggregation. The drop in platelet numbers was primarily due to aspercetin, a protein of the C-type lectin family which induces von Willebrand factor-mediated platelet aggregation/agglutination. In addition, the effect of class P-III hemorrhagic metalloproteinases on the microvessel wall also contributes to thrombocytopenia since jararhagin, a P-III metalloproteinase, reduced platelet counts. Hypoaggregation was associated with the action of procoagulant and defibrin(ogen)ating proteinases jararacussin-1 (a thrombin-like serine proteinase) and basparin A (a prothrombin activating metalloproteinase). At the doses which induced hypoaggregation, these enzymes caused defibrin(ogen)ation, increments in fibrin(ogen) degradation products and D-dimer and prolongation of the bleeding time. Incubation of B. asper venom with batimastat and α 2-macroglobulin abrogated the hypoaggregating activity, confirming the role of venom proteinases in this effect. Neither aspercetin nor the defibrin(ogen)ating and hypoaggregating components induced hemorrhage upon intravenous injection. However, aspercetin, but not the thrombin-like or the prothrombin-activating proteinases, potentiated the hemorrhagic activity of two hemorrhagic metalloproteinases in the lungs. © 2005 Schattauer GmbH, Stuttgart.

Estudos estruturais de complexos entre fosfolipases A2 homólogas isoladas do veneno de Bothrops moojeni e inibidores da atividade miotóxica

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

The golden lancehead Bothrops insularis (Serpentes: Viperidae) relies on two seasonally plentiful bird species visiting its island habitat

Adult individuals of the island pitviper Bothrops insularis have a diet based on birds. We analysed bird species recorded in the gut of this snake and found that it relies on two out of 41 bird species recorded on the island. When present, these two prey species were among the most abundant passerine birds on the island. A few other migrant birds were very occasionally recorded as prey. A resident bird species (Troglodytes musculus) is the most abundant passerine on the island, but seems able to avoid predation by the viper. Bothrops insularis is most commonly found on the ground. However, during the abundance peak of the tyrannid passerine Elaenia chilensis on the island, more snakes were found on vegetation than on the ground. We suggest that one cause may be that these birds forage mostly on vegetation, and thus cause the snakes to search for prey on this arboreal substratum.

Isolation and characterization of moojenin, an acid-active, anticoagulant metalloproteinase from Bothrops moojeni venom

A fibrinogenolytic metalloproteinase from Bothrops moojeni venom, named moojenin, was purified by a combination of ion-exchange chromatography on DEAE-Sephacel and gel filtration on Sephacryl S-300. SDS-PAGE analysis indicated that moojenin consists of a single polypeptide chain and has a molecular mass about 45 kDa. Sequencing of moojenin by Edman degradation revealed the amino acid sequence LGPDIVSPPVCGNELLEV-GEECDCGTPENCQNE, which showed strong identity with many other snake venom metalloproteinases (SVMPs). The enzyme cleaves the A alpha-chain of fibrinogen first, followed by the E beta-chain, and shows no effects on the gamma-chain. Moojenin showed a coagulant activity on bovine plasma about 3.1 fold lower than crude venom. The fibrinogenolytic and coagulant activities of the moojenin were abolished by preincubation with EDTA, 1,10-phenanthroline and beta-mercaptoethanol. Moojenin showed maximum activity at temperatures ranging from 30 to 40 degrees C and its optimal pH was 4.0. Its activity was completely lost at temperatures above 50 degrees C. Moojenin induced necrosis in liver and muscle, evidenced by morphological alterations, but did not cause histological alterations in mouse lungs, kidney or heart. Moojenin rendered the blood uncoagulatable when it was intraperitoneally administered into mice. This metalloproteinase may be of medical interest because of its anticoagulant activity. (C) 2012 Elsevier Ltd. All rights reserved.

Local inflammatory events induced by Bothrops atrox snake venom and the release of distinct classes of inflammatory mediators

Bothrops atrox is responsible for most accidents involving snakes in the Brazilian Amazon and its venom induces serious systemic and local effects. The local effects are not neutralized effectively by commercial antivenoms, resulting in serious sequelae in individuals bitten by this species. This study investigates the local inflammatory events induced in mice by B. atrox venom (Bay), such as vascular permeability, leukocyte influx and the release of important inflammatory mediators such as cytokines, eicosanoids and the chemokine CCL-2, at the injection site. The effect of Bay on cyclooxygenase (COX-1 and COX-2) expression was also investigated. The results showed that intraperitoneal (i.p.) injection of BaV promoted a rapid and significant increase in vascular permeability, which reached a peak 1 h after venom administration. Furthermore, BaV caused leukocyte infiltration into the peritoneal cavity between 1 and 8 h after i.p. injection, with mononuclear leukocytes (MNs) predominating in the first 4 h, and polymorphonuclear leukocytes (PMNs) in the last 4 h. Increased protein expression of COX-2, but not of COX-1, was detected in leukocytes recruited in the first and fourth hours after injection of BaV. The venom caused the release of eicosanoids PGD(2), PGE(2), TXA(2) and LTB4, cytokines TNF-alpha, IL-6, IL-10 and IL-12p70, but not IFN-gamma, and chemokine CCL-2 at different times. The results show that Bay is able to induce an early increase in vascular permeability and a leukocyte influx to the injection site consisting mainly of MNs initially and PMNs during the later stages. These phenomena are associated with the production of cytokines, the chemokine CCL-2 and eicosanoids derived from COX-1 and COX-2. (c) 2012 Elsevier Ltd. All rights reserved.