Avifauna da Ilha Queimada Grande, SP: diversidade, estrutura trófica e sazonalidade

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Estudos estruturais com fosfolipases A2 e fosfolipases A2 homólogas isoladas no veneno de Bothrops moojeni

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Caracterização do efeito anti-inflamatório e anti-hemorrágico dos extratos e frações enriquecidas de espécies do projeto temático BIOTA/FAPESP

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

Estudos estruturais do precursor dos peptídeos potenciadores de Bradicinina e da proteína nudel: nuclear distribution element-like

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Relações filogenéticas entre as espécies de Rhinocerophis Garman, 1881 (Serpentes; Viperidae; Crotalinae)

Pós-graduação em Ciências Biológicas (Zoologia) - IBRC

Critérios técnicos e de participação social para a recuperação florestal: quais as diferenças na definição de áreas prioritárias?

The Jararaca River Basin suffers significative environmental impacts caused by inaccurate land use. In Brazil, the areas for permanent preservation are defined and protected by the 2012 Federal Law 12.651. These areas are located in the bank side and other specific places. The objective of this paper was to analyze the results of possible alternatives in function of different procedures used in the proposal elaboration. The methodology used was the elaboration of a priority map for the recovery of these areas using the Geographic Information System with multicriterial analyses and comparing it with the guidelines from the Jararaca River Basin Management Plan. As a result, there were identified differences in the priorities defined by technical issues from the priorities defined by public consultation process.

Peptidomics of Three Bothrops Snake Venoms: Insights Into the Molecular Diversification of Proteomes and Peptidomes

Snake venom proteomes/peptidomes are highly complex and maintenance of their integrity within the gland lumen is crucial for the expression of toxin activities. There has been considerable progress in the field of venom proteomics, however, peptidomics does not progress as fast, because of the lack of comprehensive venom sequence databases for analysis of MS data. Therefore, in many cases venom peptides have to be sequenced manually by MS/MS analysis or Edman degradation. This is critical for rare snake species, as is the case of Bothrops cotiara (BC) and B. fonsecai (BF), which are regarded as near threatened with extinction. In this study we conducted a comprehensive analysis of the venom peptidomes of BC, BF, and B. jararaca (BJ) using a combination of solid-phase extraction and reversed-phase HPLC to fractionate the peptides, followed by nano-liquid chromatography-tandem MS (LC-MS/MS) or direct infusion electrospray ionization-(ESI)-MS/MS or MALDI-MS/MS analyses. We detected marked differences in the venom peptidomes and identified peptides ranging from 7 to 39 residues in length by de novo sequencing. Forty-four unique sequences were manually identified, out of which 30 are new peptides, including 17 bradykinin-potentiating peptides, three poly-histidine-poly-glycine peptides and interestingly, 10 L-amino acid oxidase fragments. Some of the new bradykinin-potentiating peptides display significant bradykinin potentiating activity. Automated database search revealed fragments from several toxins in the peptidomes, mainly from L-amino acid oxidase, and allowed the determination of the peptide bond specificity of proteinases and amino acid occurrences for the P4-P4' sites. We also demonstrate that the venom lyophilization/resolubilization process greatly increases the complexity of the peptidome because of the imbalance caused to the venom proteome and the consequent activity of proteinases on venom components. The use of proteinase inhibitors clearly showed different outcomes in the peptidome characterization and suggested that degradomic-peptidomic analysis of snake venoms is highly sensitive to the conditions of sampling procedures. Molecular & Cellular Proteomics 11: 10.1074/mcp.M112.019331, 1245-1262, 2012.

Interaction between bradykinin potentiating nonapeptide (BPP9a) and beta-cyclodextrin: A structural and thermodynamic study

Herein, we demonstrate the physical and chemical characterizations of the supramolecular complex formed between beta-cyclodextrin (beta CD) and bradykinin potentiating nonapeptide (BPP9a), an endogenous toxin found in Bothrops jararaca. Circular dichroism results indicate a conformational change in the BPP9a secondary structure upon its complexation with beta CD. Nuclear magnetic resonance results, mainly from NOESY experiments, and theoretical calculations showed a favorable interaction between the tryptophan residue of BPP9a and the beta CD cavity. Thermodynamic inclusion parameters were investigated by isothermal titration calorimetry, demonstrating that beta CD/BPP9a complex formation is an exothermic process that results in a reduction in entropy. Additionally, in vitro degradation study of BPP9a against trypsin (37 degrees C, pH 7.2) showed higher stability of peptide in presence of beta CD. This beta CD/BPP9a complex, which presents new chemical properties arising from the peptide inclusion process, may be useful as an antihypertensive drug in oral pharmaceutical formulations. (C) 2011 Elsevier B.V. All rights reserved.

A NEW AND THREATENED INSULAR SPECIES OF LANCEHEAD FROM SOUTHEASTERN BRAZIL

We describe a new species of Bothrops from Vitoria Island, off the coast of Sao Paulo, southeastern Brazil. The new species differs from the mainland coastal populations of B. jararaca mostly in its smaller and stouter body, number and form of scales, and hemipenial morphology. From B. insularis and B. alcatraz, both related species endemic to islands in southeastern Brazil, B. otavioi sp. nov. differs mainly in its body form and number of scales. The new species has the twist common mitochondrial haplotype for mainland populations of B. jararaca, which is also found in B. alcatraz. A mitochondrial genealogy (gene tree) shows the new species nested within the northern clade of B. jararaca. This genealogical pattern can be explained by a recent speciation event for B. otavioi sp. nov. The isolation of insular species of Bothrops from continental ancestor populations are probably related to the same vicariant process, the oscillations of sea level during the Pleistocene. The new species feeds on small hylid frogs, and attains sexual maturity at 388 mm snout-vent length (SVL; males) and 692 mm SVL (females). Bothrops facial sp. nov. is endemic to Vitoria Island, and should be listed as critically endangered because it is known from only a single area (an island), its geographic range covers less than 100 km(2), and there is a projected continuing decline in the quality of its habitat because of increasing human settlement.

Batroxase, a new metalloproteinase from B. atrox snake venom with strong fibrinolytic activity

The structures and functional activities of metalloproteinases from snake venoms have been widely studied because of the importance of these molecules in envenomation. Batroxase, which is a metalloproteinase isolated from Bothrops atrox (Para) snake venom, was obtained by gel filtration and anion exchange chromatography. The enzyme is a single protein chain composed of 202 amino acid residues with a molecular mass of 22.9 kDa, as determined by mass spectrometry analysis, showing an isoelectric point of 7.5. The primary sequence analysis indicates that the proteinase contains a zinc ligand motif (HELGHNLGISH) and a sequence C164I165M166 motif that is associated with a "Met-turn" structure. The protein lacks N-glycosylation sites and contains seven half cystine residues, six of which are conserved as pairs to form disulfide bridges. The three-dimensional structure of Batroxase was modeled based on the crystal structure of BmooMP alpha-I from Bothrops moojeni. The model revealed that the zinc binding site has a high structural similarity to the binding site of other metalloproteinases. Batroxase presented weak hemorrhagic activity, with a MHD of 10 mu g, and was able to hydrolyze extracellular matrix components, such as type IV collagen and fibronectin. The toxin cleaves both a and beta-chains of the fibrinogen molecule, and it can be inhibited by EDTA. EGTA and beta-mercaptoethanol. Batroxase was able to dissolve fibrin clots independently of plasminogen activation. These results demonstrate that Batroxase is a zinc-dependent hemorrhagic metalloproteinase with fibrin(ogen)olytic and thrombolytic activity. Published by Elsevier Ltd.

Biochemical characterization and comparative analysis of two distinct serine proteases from Bothrops pirajai snake venom

This study reports the isolation and biochemical characterization of two different serine proteases from Bothrops pirajai snake venom, thus providing a comparative analysis of the enzymes. The isolation process consisted of three consecutive chromatographic steps (Sephacryl S-200, Benzamidine Sepharose and C2/C18), resulting in two serine proteases, named BpirSP27 and BpirSP41 after their molecular masses by mass spectrometry (27,121 and 40,639 Da, respectively). Estimation by SDS-PAGE under denaturing conditions showed that, when deglycosylated with PNGase F, BpirSP27 and BpirSP41 had their molecular masses reduced by approximately 15 and 42%, respectively. Both are acidic enzymes, with pI of approximately 4.7 for BpirSP27 and 3.7 for BpirSP41, and their N-terminal amino acid sequences showed 57% identity to each other, with high similarity to the sequences of other snake venom serine proteases (SVSPs). The enzymes showed different actions on bovine fibrinogen, with BpirSP27 acting preferentially on the B beta chain and BpirSP41 on both A alpha and B beta chains. The two serine proteases were also able to degrade fibrin and blood clots in vitro depending on the doses and incubation periods, with higher results for BpirSP41. Both enzymes coagulated the human plasma in a dose-dependent manner, and BpirSP41 showed a higher coagulant potential, with minimum coagulant dose (MCD) of similar to 3.5 mu g versus 20 mu g for BpirSP27. The enzymes were capable of hydrolyzing different chromogenic substrates, including S-2238 for thrombin-like enzymes, but only BpirSP27 acted on the substrate S-2251 for plasmin. They also showed high stability against variations of temperature and pH, but their activities were significantly reduced after preincubation with Cu2+ ion and specific serine protease inhibitors. In addition. BpirSP27 induced aggregation of washed platelets to a greater extent than BpirSP41. The results showed significant structural and functional differences between B. pirajai serine proteases, providing interesting insights into the structure-function relationship of SVSPs. (C) 2012 Elsevier Masson SAS. All rights reserved.

Análise fitoquímica e avaliação das atividades anti-inflamatória, antipeçonhenta e citotóxica de extratos aquosos de Aspidosperma pyrifolium e Ipomoea asarifolia

Envenomation caused by venomous animals, mainly scorpions and snakes, are a serious matter of public health. Tityus serrulatus is considered the most venomous scorpion in South America because of the high level of toxicity of its venom. It is responsible for causing serious accidents, mainly with kids. The species Bothrops jararaca is a serpent that has in its venom a complex mixture of enzyme, peptides and other molecules. The toxins of the venom of B. jararaca induce local and systemic inflammatory responses. The treatment chosen to serious cases of envenomation is the intravenous administration of the specific antivenom. However, the treatment is not always accessible to those residents in rural areas, so that they use medicinal plant extracts as the treatment. In this context, aqueous extracts, fractions and isolated compounds of Aspidosperma pyrifolium (pereiro) and Ipomoea asarifolia (salsa, salsa-brava), used in popular medicine, were studied in this research to evaluate the anti-inflammatory activity in the peritonitis models induced by carrageenan and peritonitis induced by the venom of the T. serrulatus (VTs), and in the local oedema model and inflammatory infiltrate induced by the venom of the B. jararaca, administrated intravenously. The results of the assays of cytotoxicity, using the MTT, showed that the aqueous extracts from the plant species presented low toxicity to the cells that came from the fibroblast of the mouse embryo (3T3).The chemical analysis of the extracts by High Performance Liquid Chromatography revealed the presence of the rutin flavonoid, in A. pyrifoliu, and rutin, clorogenic acid and caffeic acid, in I. asarifolia. Concerning the pharmacological evaluation, the results showed that the pre-treatment using aqueous extracts and fractions reduced the total leukocyte migration to the abdominal cavity in the peritonitis model caused by the carrageenan and in the peritonitis model induced by the T. serulatus venom. Yet, these groups presented anti-oedematous activity, in the local oedema model caused by the venom of the B. jararaca, and reduced the inflammatory infiltrate to the muscle. The serum (anti-arachnid and anti-bothropic) specific to each venom acted inhibiting the inflammatory action of the venoms and were used as control. The compounds identified in the extracts were also tested and, similar to the plant extracts, showed meaningful anti-inflammatory effects, in the tested doses. Thus, these results are indicating the potential anti-inflammatory activity of the plants studied. This is the first research that evaluated the possible biological effects of the A. pyrifolium and I. asarifolia, showing the biological potential that these species have.

Purification and renal effects of phospholipase A(2) isolated from Bothrops insularis venom

Bothrops insularis venom contains a variety of substances presumably responsible for several pharmacological effects. We investigated the biochemical and biological effects of phospholipase A(2) protein isolated from B. insularis venom and the chromatographic profile showed 7 main fractions and the main phospholipase A(2) (PLA(2)) enzymatic activity was detected in fractions IV and V. Fraction IV was submitted to a new chromatographic procedure on ion exchange chromatography, which allowed the elution of 5 main fractions designated as lV-1 to IV-5, from which lV-4 constituted the main fraction. The molecular homogeneity of this fraction was characterized by high-performance liquid chromatography (HPLC) and demonstrated by mass spectrometry (MS), which showed a molecular mass of 13984.20 Da; its N-terminal sequence presented a high amino acid identity (up to 95%) with the PLA(2) of Bothrops jararaca and Bothrops asper. Phospholipase A(2) isolated from B. insularis (Bi PLA(2)) venom (10 mu g/mL) was also studied as to its effect on the renal function of isolated perfused kidneys of Wistar rats (n = 6). Bi PLA(2) increased perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF) and glomerular filtration rate (GFR). Sodium (%TNa+) and chloride tubular reabsorption (%TCl-) decreased at 120 min, without alteration in potassium transport. In conclusion, PLA(2) isolated from B. insularis venom promoted renal alterations in the isolated perfused rat kidney. (c) 2007 Elsevier Ltd. All rights reserved.