An ultraviolet photoacoustic spectroscopy study of the interaction between Lys49-phospholipase A(2) and amphiphilic molecules

We have used near ultraviolet photoacoustic spectroscopy (PAS) over the wavelength range 240-320 nm to investigate the complex formed between the homodimeric bothropstoxin-I, a lysine-49-phospholipase A(2) from the venom of Bothrops jararacussu (BthTx-I), with the anionic amphiphile sodium dodecyl sulfate (SDS). At molar ratios > 10, the complex developed a significant light scatter, accompanied by a decrease in the intrinsic tryptophan fluorescence intensity emission (ITFE) of the protein, and an increase in the near UV-PAS signal. Difference PAS spectroscopy at SDS/BthTx-I ratios < 8 were limited to the region 280-290 nm, suggesting initial SDS binding to the tryptophan 77 located at the dimer interface. At SDS/BthTx-I ratios > 10, the intensity between 260 and 320 nm increases demonstrating that the more widespread tyrosine and phenylalanine residues contribute to the SDS/BthTx-I interaction. PAS signal phase changes at wavelengths specific for each aromatic residue suggest that the Trp77 becomes more buried on SDS binding, and that protein structural changes and dehydration may alter the microenvironments of Tyr and Phe residues. These results demonstrate the potential of near UV-PAS for the investigation of membrane proteins/detergent complexes in which light scatter is significant. (c) 2006 Elsevier B.V. All rights reserved.

Crystal structure of a dimeric Ser49 PLA(2)-like myotoxic component of the Vipera ammodytes meridionalis venomics reveals determinants of myotoxicity and membrane damaging activity

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Pseudechis australis Venomics: Adaptation for a Defense against Microbial Pathogens and Recruitment of Body Transferrin

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

Influence of the Bilayer Composition on the Binding and Membrane Disrupting Effect of Polybia-MP1, an Antimicrobial Mastoparan Peptide with Leukemic T-Lymphocyte Cell Selectivity

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The Venomics of Bothrops alternatus is a Pool of Acidic Proteins with Predominant Hemorrhagic and Coagulopathic Activities

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

Snake Venomic of Crotalus durissus terrificus-Correlation with Pharmacological Activities

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

Selectivity in the mechanism of action of antimicrobial mastoparan peptide Polybia-MP1

Many potent antimicrobial peptides also present hemolytic activity, an undesired collateral effect for the therapeutic application. Unlike other mastoparan peptides, Polybia-MP1 (IDWKKLLDAAKQIL), obtained from the venom of the social wasp Polybia paulista, is highly selective of bacterial cells. The study of its mechanism of action demonstrated that it permeates vesicles at a greater rate of leakage on the anionic over the zwitterionic, impaired by the presence of cholesterol or cardiolipin; its lytic activity is characterized by a threshold peptide to lipid molar ratio that depends on the phospholipid composition of the vesicles. At these particular threshold concentrations, the apparent average pore number is distinctive between anionic and zwitterionic vesicles, suggesting that pores are similarly formed depending on the ionic character of the bilayer. To prospect the molecular reasons for the strengthened selectivity in Polybia-MP1 and its absence in Mastoparan-X, MD simulations were carried out. Both peptides presented amphipathic alpha-helical structures, as previously observed in Circular Dichroism spectra, with important differences in the extension and stability of the helix; their backbone solvation analysis also indicate a different profile, suggesting that the selectivity of Polybia-MP1 is a consequence of the distribution of the charged and polar residues along the peptide helix, and on how the solvent molecules orient themselves according to these electrostatic interactions. We suggest that the lack of hemolytic activity of Polybia-MP1 is due to the presence and position of Asp residues that enable the equilibrium of electrostatic interactions and favor the preference for the more hydrophilic environment.

Snake venomics of the Siamese Russell's viper (Daboia russelli siamensis) - Relation to pharmacological activities

The venom proteome of Daboia russelli siamensis, a snake of medical importance in several Asian countries, was analysed by 2-D electrophoresis, subsequent MS/MS and enzymatic assays. The proteome comprises toxins from six protein families: serine proteinases, metalloproteinases, phospholipases A(2), L-amino acid oxidases, vascular endothelial growth factors and C-type lectin-like proteins. The venom toxin composition correlates with the clinical manifestation of the Russell's viper bite and explains pathological effects of the venom such as coagulopathy, oedema, hypotensive, necrotic and tissue damaging effects. The vast majority of toxins are potentially involved in coagulopathy and neurotoxic effects. The predominant venom components are proteinases capable of activating blood coagulation factors and promoting a rapid clotting of the blood, and neurotoxic phospholipase A(2)s. The analysis of the venom protein composition provides a catalogue of secreted toxins. The proteome of D. r. siamensis exhibits a lower level of toxin diversity than the proteomes of other viperid snakes. In comparison to the venoms of Vipera ammodytes ammodytes and Vipera ammodytes meridionalis, the venom from D. r. siamensis showed quantitative differences in the proteolytic, phospholipase A2, L-amino acid oxidase and alkaline phosphatase activities. (c) 2009 Published by Elsevier B.V.

Study of the mechanism of action of anoplin, a helical antimicrobial decapeptide with ion channel-like activity, and the role of the amidated C-terminus

Anoplin, an antimicrobial, helical decapeptide from wasp venom, looses its biological activities by mere deamidation of its C-terminus. Secondary structure determination, by circular dichroism spectroscopy in amphipathic environments, and lytic activity in zwitterionic and anionic vesicles showed quite similar results for the amidated and the carboxylated forms of the peptide. The deamidation of the C-terminus introduced a negative charge at an all-positive charged peptide, causing a loss of amphipathicity, as indicated by molecular dynamics simulations in TFE/water mixtures and this subtle modification in a peptide's primary structure disturbed the interaction with bilayers and biological membranes. Although being poorly lytic, the amidated form, but not the carboxylated, presented ion channel-like activity on anionic bilayers with a well-defined conductance step; at approximately the same concentration it showed antimicrobial activity. The pores remain open at trans-negative potentials, preferentially conducting cations, and this situation is equivalent to the interaction of the peptide with bacterial membranes that also maintain a high negative potential inside. Copyright (C) 2007 European Peptide Society and John Wiley & Sons, Ltd.

Purification, crystallization and preliminary X-ray diffraction analysis of crotamine, a myotoxic polypeptide from the Brazilian snake Crotalus durissus terrificus

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

Avaliação do desempenho dos reagentes do tempo de tromboplastina parcial ativada utilizados para detectar o anticoagulante lúpico

INTRODUÇÃO: O anticoagulante lúpico é uma imunoglobulina pertencente à família dos anticorpos antifosfolípides. A sua ação in vitro é interferir nos testes de coagulação dependentes de fosfolípides. O tempo de tromboplastina parcial ativada (TTPA) é um teste utilizado como screening na pesquisa do anticoagulante lúpico. Os reagentes utilizados neste teste apresentam grandes variações quanto à sensibilidade. OBJETIVO: Avaliar o desempenho dos reagentes do TTPA e detectar a presença do anticoagulante lúpico através de diferentes testes da coagulação. MATERIAL E MÉTODO: A pesquisa do anticoagulante lúpico foi realizada em 50 amostras plasmáticas de pacientes do sexo feminino através dos testes do TTPA, do tempo de coagulação do caulim (TCC), do tempo de tromboplastina parcial ativada diluída (TTPAd) e do tempo do veneno da víbora de Russel diluído (TVVRd). Três cefalinas comerciais foram avaliadas pelos testes do TTPA e do TTPAd. Na comparação entre os reagentes estudados foi aplicado o cálculo do intervalo de confiança (95%). RESULTADOS: Os três reagentes avaliados apresentaram boa concordância e os métodos utilizados responderam bem à pesquisa do anticoagulante lúpico. DISCUSSÃO E CONCLUSÃO: As três cefalinas comerciais avaliadas podem ser utilizadas na rotina laboratorial para a pesquisa do anticoagulante lúpico.

Effects of 60Co gamma radiation on crotamine

Ionizing radiation can change the molecular structure and affect the biological properties of biomolecules. This has been employed to attenuate animal toxins. Crotamine is a strongly basic polypeptide (pI 10.3) from Crotalus durissus terrificus venom composed of 42 amino acid residues. It induces skeletal muscle spasms leading to a spastic paralysis of hind limbs in mice. The objective of the present study was to carry out a biochemical study and a toxic activity assay on native and irradiated crotamine. Crotamine was purified from C.d. terrificus venom by Sephadex G-100 gel filtration followed by ion-exchange chromatography, and irradiated at 2 mg/ml in 0.15 M NaCl with 2.0 kGy gamma radiation emitted by a 60Co source. The native and irradiated toxins were evaluated in terms of structure and toxic activity (LD50). Irradiation did not change the protein concentration, the electrophoretic profile or the primary structure of the protein although differences were shown by spectroscopic techniques. Gamma radiation reduced crotamine toxicity by 48.3%, but did not eliminate it.

Intoxicação por veneno de sapo em um canino

O sapo do gênero Bufo possui nas suas glândulas paratóides uma secreção mucóide contendo toxinas como bufaginas e Bufotoxinas, que são esteróides cardiogênicos. Os cães podem atacar os sapos, entrando em contato com o veneno por meio das mucosas. Um canino, da raça Bulldog Francês, foi encaminhado ao Setor de Patologia Veterinária da Universidade Federal do Rio Grande do Sul (UFRGS) para a necropsia com histórico de provável intoxicação por veneno de sapo. Na necropsia o canino apresentava pulmões aumentados de volume, avermelhados e com edema, e rins de coloração vermelho-escura. As alterações microscópicas indicaram congestão, hemorragia e edema pulmonar. Nos rins, no baço e nos linfonodos foi observada congestão. As análises toxicológicas para os venenos de rotina foram negativas. Porém, a investigação do veneno de sapo a partir de cromatografia por camada delgada e gasosa demonstrou resultado positivo, revelando ser esta a causa da morte do canino.

Liver dysfunction in patients bitten by Crotalus durissus terrificus (Laurenti, 1768) snakes in Botucatu (State of São Paulo, Brazil)

Os autores estudaram 32 doentes picados por serpentes venenosas, sendo 16 picados por Bothrops spp. e 16 por Crotalus durissus terrificus. Trinta doentes eram do sexo masculino e dois do feminino com idades variando entre 8 e 63 anos (méda 33±15). A prova da retenção da bromosulfaleína apresentou-se aumentada na maioria dos doentes picados por serpentes Crotalus durissus terrificus. Houve correlação positiva entre a retenção da bromosulfaleína e os níveis séricos de alanina aminotransferase e entre alanina e aspartato aminotransferase apenas nos doentes do grupo Crotalus. Um dos doentes evoluiu para o óbito e apresentou no exame anatomopatológico do fígado degeneração hidrópica e lesões mitocondriais. Os autores concluem que as alterações hepáticas são causadas por pelo menos dois mecanismos a saber: lesão mitocondrial por efeito do veneno crotálico; efeito das citoquinas, especialmente a interleucina-6.

The venomous toadfish Thalassophryne nattereri (niquim or miquim): report of 43 injuries provoked in fishermen of Salinópolis (Pará State) and Aracaju (Sergipe State), Brazil

Os peixes da família Batrachoididae causam um grande número de acidentes em pescadores das regiões Norte e Nordeste do Brasil. O gênero Thalassophryne apresenta várias espécies no Brasil e a espécie Thalassophryne nattereri é a mais comum, todas apresentando veneno. O veneno é inoculado por duas espículas ocas na nadadeira dorsal e duas nas regiões pré-operculares, ligadas a uma glândula de veneno na base. Os envenenamentos causaram dor intensa, edema e eritema iniciais em 43 pescadores observados em Salinópolis (Pará) e Aracaju (Sergipe). em todos os casos, não ocorreram fenômenos sistêmicos dignos de nota, mas ocorreu necrose local em oito pacientes e infecção bacteriana em dez. As circunstâncias dos acidentes são comentadas, assim como aspectos terapêuticos. Nossa conclusão foi que o envenenamento por Thalassophryne é importante e freqüente e deve ser considerado de média gravidade, em função de não haverem fenômenos sistêmicos, como observado nos acidentes por peixes-escorpião (Scorpaena) ou arraias marinhas e fluviais.