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.

Molecular determinants of binding of a wasp toxin (PMTXs) and its analogs in the Na+ channels proteins

The structural specificity of alpha-PMTX, a novel peptide toxin derived from wasp venom has been studied on the neuromuscular synapse in the walking leg of the lobster. alpha-PMTX is known to induce repetitive action potentials in the presynaptic axon due to sodium channel inactivation. We synthesized 29 analogs of alpha-PMTX by substituting one or two amino acids and compared threshold concentrations of these mutant toxins for inducing repetitive action potentials. In 13 amino acid residues of alpha-PMTX, Arg-1, Lys-3 and Lys-12 regulate the toxic activity because substitution of these basic amino acid residues with other amino acid residues greatly changed the potency. Determining the structure-activity relationships of PMTXs will help clarifying the molecular mechanism of sodium channel inactivation. (C) 2000 Elsevier B.V. Ireland Ltd. All rights reserved.

Comparative studies of the anti-leishmanial activity of three Crotalus durissus ssp venoms

In this study, we compared the anti-leishmanial activity of three crotalic venoms (Crotalus durissus terrificus-Cdt, Crotalus durissus cascavella-Cdca, and Crotalus durissus collilineatus-Cdcol). Different concentrations of each venom incubated with Leishmania (Leishmania) amazonensis promastigotes were used. Cdt venom exhibited a higher anti-leishmanial activity (Inhibitory concentration-IC50-value of 4.70 +/- 1.72 mu g/ml) in comparison with that of Cdca venom (IC50 value of 9.41 +/- 1.21 mu g/ml), while Cdcol venom increased parasite numbers in 50% at a concentration of 44.30 +/- 2.18 mu g/ml. In addition, this venom showed a low anti-leishmanial activity in higher concentrations (IC50 value of 281.00 +/- 9.50 mu g/ml). The main fractions of Cdca venom were isolated and assayed under similar conditions used for assessing crude venom. The most active fractions were gyroxin and crotamine that had IC50 values of 3.80 +/- 0.52 mu g/ml and 19.95 +/- 4.21 mu g/ml, respectively. Convulxin also inhibited parasite growth rate, although this effect was not dose-dependent. Crotoxin was the least effective fraction with an IC50 value of 99.80 +/- 2.21 mu g/ml. None of the protein fractions presented cytotoxic effects against J774 cells in culture. In vivo assays using BALB/c mice revealed that crotoxin and crotamine were the main toxic fractions. In conclusion, C. durissus cascavella venom has three main fractions with anti-leishmanial activity. These results open new possibilities to find proteins that might be used as possible agents against cutaneous leishmaniasis.

Phospholipase A(2) - A structural review

Phospholipases A(2) (PLA(2)) are widely distributed in nature and are well characterized proteins with respect to their catalytic and pharmacological activities, A wealth of structural information has recently become available both from X-ray diffraction and NMR studies, and although a detailed model of the catalytic mechanism of PLA(2) has been proposed, the structural bases of other aspects of PLA(2) function, such as interfacial activation and venom PLA(2) pharmacological activities, are still under debate. An appreciation of the PLA(2) protein structure will yield new insights with regard to these activities, the salient structural features of the class I, II and III PLA(2) are discussed with respect to their functional roles. Copyright (C) 1996 Published by Elsevier B.V. Ltd

A molecular mechanism for Lys(49)-phospholipase A(2) activity based on ligand-induced conformational change

Agkistrodon contortrix laticinctus myotoxin is a Lys(49)- phospholipase A(2) (EC 3.1.1.4) isolated from the venom of the serpent A contortrix laticinctus (broad-banded copperhead). We present here three monomeric crystal structures of the myotoxin, obtained under different crystallization conditions. The three forms present notable structural differences and reveal that the presence of a ligand in the active site (naturally presumed to be a fatty acid) induces the exposure of a hydrophobic surface (the hydrophobic knuckle) toward the C terminus. The knuckle in A contortrix laticinctus myotoxin involves the side chains of Phe(121) and Phe(124) and is a consequence of the formation of a canonical structure for the main chain within the region of residues 118-125. Comparison with other Lys(49)-phospholipase A(2) myotoxins shows that although the knuckle is a generic structural motif common to all members of the family, it is not readily recognizable by simple sequence analyses. An activation mechanism is proposed that relates fatty acid retention at the active site to conformational changes within the C-terminal region, a part of the molecule that has long been associated with Ca2+-independent membrane damaging activity and myotoxicity. This provides, for the first time, a direct structural connection between the phospholipase active site and the C-terminal myotoxic site, justifying the otherwise enigmatic conservation of the residues of the former in supposedly catalytically inactive molecules.

Biological and biochemical properties of the Brazilian Potamotrygon stingrays : Potamotrygon cf. scobina and Potamotrygon gr. orbignyi

Stingrays of the family Potamotrygonidae are widespread throughout river systems of South America that drain into the Atlantic Ocean. Some species are endemic to the most extreme freshwater environment of the Brazil and cause frequent accidents to humans. The envenomation causes immediate, local, and intense pain, soft tissue edema, and a variable extent of bleeding. The present study was carried out in order to describe the principal biological and some biochemical properties of the Brazilian Potamotrygon fish venoms (Potamotrygon cf. scobina and P. gr. orbignyi). Both stingray venoms induced significant edematogenic and nociceptive responses in mice. Edematogenic and nociceptive responses were reduced when the venom was incubated at 37 or 56 degrees C. The results showed striking augments of leukocytes rolling and adherent cells to the endothelium of cremaster mice induced by both venoms. The data also presented that injection of both venoms induced necrosis, low level of proteolytic activity, without inducing haemorrhage. But when the venoms of both stingray species were injected together with their mucus secretion, the necrotizing activity was more vigorous. The present study provided in vivo evidence of toxic effects for P. cf. scobina and P. gr. orbignyi venoms. (c) 2006 Elsevier Ltd. All fights reserved.

Gabaergic-benzodiazepine system is involved in the crotoxin-induced anxiogenic effect

The behavioral effects of crotoxin (CTX), the major component of Crotalus durissus terrificus venom, were studied in rats submitted to the open field, holeboard, and social interaction tests. CTX (100, 250, and 500 mu g/kg, IP) was administered 2 h before the tests. In the open field, CTX reduced ambulation (250 mu g/kg) and rearing (250 and 500 mu g/kg) and increased grooming (100 and 250 mu g/kg) and freezing (250 mu g/kg). In the holeboard and social interaction, all the CTX doses evaluated decreased, respectively, head dip and head dipping, and social interaction time. The CTX-induced behavioral alterations could be attributed to its neuromuscular transmission blockade, but this possibility was ruled out because CTX (250 and 500 mu g/kg, IP, 2 h before the rotarod test) was unable to modify the rotarod performance of rats. The involvement of the benzodiazepine receptor in the CTX-induced behavioral alterations was investigated through the pretreatment (30 min before the tests, IP) of the animals with diazepam (1.2 mg/kg), or flumazenil (4 and 10 mg/kg). Both diazepam and flumazenil antagonized the CTX induced behavioral alterations in the open field, holeboard, and social interaction tests. This study demonstrated that: (1) CTX is an anxiogenic compound; and (2) the gabaergic-benzodiazepine system may play a role in the CTX-induced anxiogenic effect. (C) 1999 Elsevier B.V.