Structural and Functional Studies of a Bothropic Myotoxin Complexed to Rosmarinic Acid: New Insights into Lys49-PLA2 Inhibition

Snakebite envenoming is an important public health problem in many tropical and subtropical countries, and is considered a neglected tropical disease by the World Health Organization. Most severe cases are inflicted by species of the families Elapidae and Viperidae, and lead to a number of systemic and local effects in the victim. One of the main problems regarding viperidic accidents is prominent local tissue damage whose pathogenesis is complex and involves the combined actions of a variety of venom components. Phospholipases A2 (PLA2s) are the most abundant muscle-damaging components of these venoms. Herein, we report functional and structural studies of PrTX-I, a Lys49-PLA2 from Bothops pirajai snake venom, and the influence of rosmarinic acid (RA) upon this toxin's activities. RA is a known active component of some plant extracts and has been reported as presenting anti-myotoxic properties related to bothopic envenomation. The myotoxic activity of Lys49-PLA2s is well established in the literature and although no in vivo neurotoxicity has been observed among these toxins, in vitro neuromuscular blockade has been reported for some of these proteins. Our in vitro studies show that RA drastically reduces both the muscle damage and the neuromuscular blockade exerted by PrTX-I on mice neuromuscular preparations (by ∼80% and ∼90%, respectively). These results support the hypothesis that the two effects are closely related and lead us to suggest that they are consequences of the muscle membrane-destabilizing activity of the Lys49-PLA2. Although the C-terminal region of these proteins has been reported to comprise the myotoxic site, we demonstrate by X-ray crystallographic studies that RA interacts with PrTX-I in a different region. Consequently, a new mode of Lys49-PLA2 inhibition is proposed. Comparison of our results with others in the literature suggests possible new ways to inhibit bothropic snake venom myotoxins and improve serum therapy

Biological characterization of Bothrops marajoensis snake venom

This study describes the effects of Bothrops marajoensis venom (Marajó lancehead) on isolated neuromuscular preparations of chick biventer cervicis (CBC) and mouse phrenic nerve-diaphragm (PND). At low concentrations (1µg/ml for CBC and 5µg/ml for PND), the venom exhibited a neuromuscular blocking without any damaging effect on the muscle integrity. At higher concentration (20μg/ml for PND), together with the neuromuscular blockade, there was a moderate myonecrosis. The results show differences between mammalian and avian preparations in response to venom concentration; the avian preparation was more sensitive to venom neurotoxic effect than the mammalian preparation. The possible presynaptic mechanism underlying the neuromuscular blocking effect was reinforced by the observed increase in MEPPs at the same time (at 15min) when the facilitation of twitch tension occurred. These results indicate that the B. marajoensis venom produced neuromuscular blockade, which appeared to be presynaptic at low concentrations with a postsynaptic component at high concentrations, leading to muscle oedema. These observations demand the fractionation of the crude venom and characterization of its active components for a better understanding of its biological dynamics

Recuperação funcional do nervo ciático reparado pela cola de fibrina.

The molecules of cellular adhesion are been used for the coaptation of injured nerves as an alternative of the well know neurorrhaphy, however, there are still missing experimental studies of these two techniques. The aim of this study was to evaluate the functional recovery of the sciatic nerve repaired by the fibrin glue, by the means of isolated indirect contractions applied to the myoneural complex, sciatic nerve – extensor muscle of the long fingers (EMLF). Sixteen Wistar rats were divided into two groups: control group (CG), that didn’t have any lesion and the glue group (GG) that had their sciatic nerve injured and repaired with the glue. After 10 weeks, the myoneural complex was collected for the stimulus. The EMLF of the animals of the GG responded with contractions after the stimulus of the motor nerve. Even with greater amplitude than the CG, there wasn’t any significant statistical result. Nerves repaired with glue are capable to conduct nerve impulses to the skeletal muscle.As moléculas de adesão celular vem sendo utilizadas na coaptação de nervos lesados como alternativa a consagrada neurorrafia, entretanto, faltam estudos experimentais definitivos em relação às duas técnicas. O objetivo deste estudo foi avaliar a recuperação funcional do nervo ciático reparado pela cola de fibrina, por meio de contrações indiretas isoladas aplicadas ao complexo mioneural, nervo ciático - músculo extensor longo dos dedos (EDL). Foram utilizados 16 ratos Wistar divididos em dois grupos: grupo controle (GC), que não sofreu nenhuma lesão e grupo cola (GCL) que teve o nervo ciático lesado e reparado pela cola. Após 10 semanas o complexo mioneural foi coletado para estímulo. O músculo EDL nos animais do GCL respondeu com contrações frente ao estímulo de seu nervo motor. Mesmo com amplitude maior do que o GC, não houve diferença estatisticamente significante. Nervos reparados pela cola são capazes de conduzir impulsos nervosos até o músculo esquelético

Structural and functional studies with mytoxin II from Bothrops moojeni reveal remarkable similarities and differences compared to other catalytically inactive phospholipases A2-like

Lys49-phospholipases A2 (Lys49-PLA2s) are proteins found in bothropic snake venoms (Viperidae family) and belong to a class of proteins which presents a phospholipase A2 scaffold but are catalytically inactive. These proteins (also known as PLA2s-like toxins) exert a pronounced local myotoxic effect and are not neutralized by antivenom, being their study relevant in terms of medical and scientific interest. Despite of the several studies reported in the literature for this class of proteins only a partial consensus has been achieved concerning their functional-structural relationships. In this work, we present a comprehensive structural and functional study with the MjTX-II, a dimeric Lys49-PLA2 from Bothrops moojeni venom which includes: (i) high-resolution crystal structure; (ii) dynamic light scattering and bioinformatics studies in order to confirm its biological assembly; (iii) myographic and electrophysiological studies and, (iv) comparative studies with other Lys49-PLA2s. These comparative analyses let us to get important insights into the role of Lys122 amino acid, previously indicated as responsible for Lys49-PLA2s catalytic inactivity and added important elements to establish the correct biological assembly for this class of proteins. Furthermore, we show two unique sequential features of MjTX-II (an amino acid insertion and a mutation) in comparison to all bothropic Lys49-PLA2s that lead to a distinct way of ligand binding at the toxin's hydrophobic channel and also, allowed the presence of an additional ligand molecule in this region. These facts suggest a possible particular mode of binding for long-chain ligands that interacts with MjTX-II hydrophobic channel, a feature that may directly affect the design of structure-based ligands for Lys49-PLA2s. © 2013 Elsevier Ltd

Que bicho te mordeu? Prevenção de acidentes por animais peçonhentos no município de Botucatu

Accidents caused by venomous animals are a public health problem in Brazil because of the high incidence and the high risk of morbidity and mortality they present. Frequently, these accidents can lead to consequences that incapacitate the victim to work, causing a major economic and social impact on the lives of the injured and their families. Given this situation, we have developed a project to involve undergraduates and graduate students of the Institute of Biosciences of the University of São Paulo State, campus of Botucatu, in a program to prevent accidents caused by venomous animals in Botucatu city, state of São Paulo, Brazil. Firstly an epidemiological survey was carried out to characterize the incidence of these accidents in the region and strategies were subsequently developed to reach the target audience, including the preparation of informative and entertaining materials which were distributed during lectures. Data provided by the Epidemiological Surveillance of Botucatu in the period 1998-2014, through databases SINAN NET and SINAN WINDOWS of the Ministry of Health were analised. In the period were reported in 1338 envenomations, mostly caused by scorpions (35%), followed by bees (26%), spiders (19%) and snake (16%), than those generated by an unidentified animal (4%). Accidents predominated in the hot and rainy seasons (between the months of September to April), in the suburban area, reaching individuals of both sexes, especially in the age group of 20-49 years. The main sites were chopped hands and feet, and most of the attendances occurred within 3 hours after accidents and 20% of victims were treated with serum therapy. From these results it has been prepared information material to the guidelines on prevention, first aid and correct treatment for this type of accident, as well as biological information allowing the identification of venomous animal. This material is distributed during lectures for parents and students from elementary schools and secondary public network, located on the outskirts of Botucatu. During the presentations are also exposed preserved examples of animals causing accidents. At the end of the lectures, students are invited to participate in recreational activities, through interactive games in order to consolidate learning on the subject. The project drew a picture of envenomations in the city and made possible the dissemination of basic information that can significantly reduce the incidence and severity. To the students, the project provided the improvement of training and the ability to act with transforming agents of society.Os acidentes causados por animais peçonhentos são um problema de saúde pública no Brasil devido à elevada incidência e ao alto risco de morbidade e de letalidade que apresentam. Muitos destes acidentes podem gerar sequelas que incapacitam o indivíduo para o trabalho, causando um grande impacto econômico e social na vida do acidentado e de sua família. Diante deste quadro, desenvolvemos um projeto de extensão para envolver alunos de graduação e de pós-graduação do Intituto de Biociências da Universidade Estadual Paulista, campus de Botucatu, em um programa de prevenção de acidentes por animais peçonhentos no município de Botucatu, estado de São Paulo, Brasil. Inicialmente foi realizado um levantamento epidemiológico para caracterizar a incidência destes acidentes na região e, subsequentemente, foram elaboradas estratégias de interferência que consistiram no desenvolvimento de material informativo e na divulgação da informação à população. Foram analisados dados disponibilizados pela Vigilância Epidemiológica do Município de Botucatu, no período entre 1998 a 2014, por meio dos bancos de dados SINAN WINDOWS e SINAN NET do Ministério da Saúde. No período foram notificados 1554 acidentes por animais peçonhentos, sendo a maioria causado por escorpiões (35%), seguido por abelhas (26%), aranhas (19%) e serpentes (16%), além daqueles infligidos por animais não identificados (4%). Os acidentes predominaram nas épocas quentes e chuvosas do ano (entre os meses de setembro a abril), na zona periurbana, atingindo indivíduos de ambos os sexos, especialmente na faixa etária de 20 a 49 anos. Os principais locais da picada foram mãos e pés, sendo que a maioria dos atendimentos ocorreu em até 3 horas após os acidentes e 20% das vítimas receberam soroterapia. A partir desses resultados foi elaborado material informativo com as orientações sobre prevenção, primeiros socorros e tratamento correto para esse tipo de acidente, além de informações biológicas que permitem a identificação do animal peçonhento. Esse material é distribuído durante palestras ministradas para pais e alunos de escolas de ensino fundamental e médio da rede pública, localizadas na periferia do município de Botucatu. Durante as apresentações também são expostos exemplares conservados dos animais causadores de acidentes. Ao final das palestras, os alunos são convidados a participarem de atividades lúdicas, através de jogos interativos, visando consolidar o aprendizado sobre o tema. O projeto traçou um panorama dos acidentes por animais peçonhentos no município e possibilitou a difusão de informações básicas que podem reduzir consideravelmente a incidência e a gravidade dos mesmos. Aos acadêmicos, o projeto proporcionou o aprimoramento da formação e a possibilidade de atuarem com agentes transformadores da sociedade

Interactions between RA and PrTX-I atoms in the PrTX-I/RA complex.

<p>The surface charge distribution for the PrTX-I/RA crystallographic model and specific interactions between RA with some PrTX-I atoms are shown. Only the interactions with interatomic distances shorten than 3.7 Å are represented between chain A and the RA molecule (black dashes). To represent the interactions between RA and the residue Pro123 of chain B a larger distance cut-off was considered (blue dashes). Residues whose contacts with RA are established through water molecules are not shown. The electron density map for RA molecule was calculated with coefficients 2|F_obs|-|F_calc| contoured at 1.0 standard deviation.</p