Purification, characterization, crystallization and theoretical molecular modeling of gyroxin fraction from Crotalus durissus terrificus venom (Laurenti, 1768)

The venom of Crotalus durissus terrificus snakes presents various substances, including a serine protease with thrombin-like activity, called gyroxin, that clots plasmatic fibrinogen and promote the fibrin formation. The aim of this study was to purify and structurally characterize the gyroxin enzyme from Crotalus durissus terrificus venom. For isolation and purification, the following methods were employed: gel filtration on Sephadex G75 column and affinity chromatography on benzamidine Sepharose 6B; 12% SDS-PAGE under reducing conditions; N-terminal sequence analysis; cDNA cloning and expression through RT-PCR and crystallization tests. Theoretical molecular modeling was performed using bioinformatics tools based on comparative analysis of other serine proteases deposited in the NCBI (National Center for Biotechnology Information) database. Protein N-terminal sequencing produced a single chain with a molecular mass of similar to 30 kDa while its full-length cDNA had 714 bp which encoded a mature protein containing 238 amino acids. Crystals were obtained from the solutions 2 and 5 of the Crystal Screen Kit (R), two and one respectively, that reveal the protein constitution of the sample. For multiple sequence alignments of gyroxin-like B2.1 with six other serine proteases obtained from snake venoms (SVSPs), the preservation of cysteine residues and their main structural elements (alpha-helices, beta-barrel and loops) was indicated. The localization of the catalytic triad in His57, Asp102 and Ser198 as well as S1 and S2 specific activity sites in Thr193 and Gli215 amino acids was pointed. The area of recognition and cleavage of fibrinogen in SVSPs for modeling gyroxin B2.1 sequence was located at Arg60, Arg72, Gln75, Arg81, Arg82, Lis85, Glu86 and Lis87 residues. Theoretical modeling of gyroxin fraction generated a classical structure consisting of two alpha-helices, two beta-barrel structures, five disulfide bridges and loops in positions 37, 60, 70, 99, 148, 174 and 218. These results provided information about the functional structure of gyroxin allowing its application in the design of new drugs.

Ovine leptospirosis in Brazil

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

The extract of the jellyfish Phyllorhiza punctata promotes neurotoxic effects

Phyllorhiza punctata (P. punctata) is a jellyfish native to the southwestern Pacific. Herewith we present the biochemical and pharmacological characterization of an extract of the tentacles of P. punctata. The tentacles were subjected to three freezethaw cycles, homogenized, ultrafiltered, precipitated, centrifuged and lyophilized to obtain a crude extract (PHY-N). Paralytic shellfish poisoning compounds such as saxitoxin, gonyautoxin-4, tetrodotoxin and brevetoxin-2, as well as several secretory phospholipase A2 were identified. PHY-N was tested on autonomic and somatic neuromuscular preparations. In mouse vas deferens, PHY-N induced phasic contractions that reached a peak of 234 +/- 34.7% of control twitch height, which were blocked with either 100 mu m of phentolamine or 1m m of lidocaine. In mouse corpora cavernosa, PHY-N evoked a relaxation response, which was blocked with either L-NG-Nitroarginine methyl ester (0.5 m m) or 1m m of lidocaine. PHY-N (1, 3 and 10 mu g ml(-1)) induced an increase in tonus of the biventercervicis neuromuscular preparation that was blocked with pre-treatment of galamine (10 mu m). Administration of 6 mg kg(-1) PHY-N intramuscularly produced death in broilers by spastic paralysis. In conclusion, PHY-N induces nerve depolarization and nonspecifically increases neurotransmitter release. Copyright (C) 2011 John Wiley & Sons, Ltd.

Methodology for fast evaluation of Bacillus thuringiensis crystal protein content

O desenvolvimento da produção e uso do Bacillus thuringiensis no Brasil em escala comercial enfrenta certas dificuldades, entre elas o estabelecimento de metodologias para a quantificação de produtos tóxicos a serem comercializados. Atualmente, a quantidade de toxinas é expressa como porcentagem do total de proteínas presentes em amostras em consideração. Tal metodologia, entretanto, não mede a quantidade real de uma determinada proteína presente em um produto qualquer, além do fato de diferentes linhagens bacterianas possuírem diferentes genes codificadores para endotoxinas e mesmo para b-toxina. Desde que os diferentes tipos de toxinas apresentam diferentes características antigências, este trabalho tem como objetivo a utilização de técnicas imunológicas para quantificar específicamente o conteúdo de proteína cristal presente em diferentes amostras. A proteína cristal produzida pela subespécie B. thuringiensis var. israelensis foi purificada por ultracentrifugação e utilizada para imunizar coelhas e produzir soros hiperimunes. Tais soros foram posteriormente usados para avaliar o nível de proteína cristal em bioinseticidas comerciais e em culturas de laboratório desta bactéria utilizando-se a técnica do imunodot. Os resultados foram obtidos por comparação de reações com concentrações conhecidas de proteína cristal permitindo assim avaliar com segurança os níveis desta proteína em várias preparações.

Atividade tóxica de isolados de Bacillus Thuringiensis a larvas de Aedes Aegypti (l.) (diptera: culicidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Cardiopulmonary and acid-base effects of desflurane and sevoflurane in spontaneously breathing cats

The cardiopulmonary effects of desflurane and sevoflurane anesthesia were compared in cats breathing spontaneously. Heart (HR) and respiratory (RR) rates; systolic (SAP), diastolic (DAP) and mean arterial (MAP) pressures; partial pressure of end tidal carbon dioxide (PETCO(2)), arterial blood pH (pH), arterial partial pressure of oxygen (PaO(2)) and carbon dioxide (PaCO(2)); base deficit (BD), arterial oxygen saturation (SaO(2)) and bicarbonate ion concentration (HCO(3)) were measured. Anesthesia was induced with propofol (8 +/- 2.3 mg/kg IV) and maintained with desflurane (GD) or sevoflurane (GS), both at 1.3 MAC. Data were analyzed by analysis of variance (ANOVA), followed by the Tukey test (P < 0.05). Both anesthetics showed similar effects. HR and RR decreased when compared to the basal values, but remained constant during inhalant anesthesia and PETCO(2) increased with time. Both anesthetics caused acidemia and hypercapnia, but BD stayed within normal limits. Therefore, despite reducing HR and SAP (GD) when compared to the basal values, desflurane and sevoflurane provide good stability of the cardiovascular parameters during a short period of inhalant anesthesia (T20-T60). However, both volatile anesthetics cause acute respiratory acidosis in cats breathing spontaneously. (c) 2004 ESFM and AAFP. Published by Elsevier Ltd. All rights reserved.

Effects of levomepromazine and different desflurane concentrations upon electrocardiographic variables in dogs

Objectives To investigate the effects of levomepromazine and different desflurane concentrations upon electrocardiographic variables.Animals Twenty adult mongrel dogs of both sexes weighing 6-28 kg.Methods Dogs were divided into two groups of 10 animals. Group I received 1 mg kg(-1) lV of levomepromazine and 15 minutes later anesthesia was induced with propofol (3 mg kg(-1) IV). Desflurane end-tidal concentration was set at 1.6 MAC. After 30 minutes at this concentration, measurements were taken and the end-tidal concentration was reduced to 1.4 MAC. Thereafter, it was reduced to 1.2 and then 1.0 MAC at 1.5-minute intervals. The same procedure was followed for group 2, except that levomepromazine was replaced with 0.2 mL kg(-1) of 0.9% saline solution and more propofol was needed for induction (7 mg kg(-1)). The animals' body temperature was maintained between 38.3 and 39 degreesC using a heating pad. The electrocardiographic tracing was obtained from lead II throughout the experimental period. The measurements were taken immediately before the administration of levomepromazine or placebo (T-1), 15 minutes after pre-medication (T-2) and 30 minutes after the establishment of 1.6 MAC (T-3)The other measurements were made at the concentrations of 1.4, 1.2, and 1.0 MAC, respectively (T4-6). The numerical data were submitted to analysis of variance plus F-test (p < 0.05).Results the dogs that received levomepromazine had a decrease in heart rate. However, in both groups it increased with desflurane administration. Levomepromazine, in association with desflurane, did not induce significant electrocardiographic changes, and all mean values (except P-wave duration) were within the reference range for this species.Conclusions and clinical relevance This study documented that levomepromazine, in association with desflurane, does not induce significant changes in electrocardiographic variables, suggesting that this drug combination has minimal effect on myocardial conduction.

Inhibition of the myotoxic activity of Bothrops jararacussu venom and its two major myotoxins, BthTX-I and BthTX-II, by the aqueous extract of Tabernaemontana catharinensis A. DC. (Apocynaceae)

Partial neutralization of the myotoxic effect of Bothrops jararacussu venom (BV) and two of its myotoxins [bothropstoxin-I (BthTX-I), catalytically inactive, and II (BthTX-II), showing low PLA(2) activity], by the lyophilized aqueous extract of Tabernaemontana catharinensis (AE), was studied in rat isolated soleus muscle preparations (in vitro) and through i.m. injection in the gastrocnemius muscle (in vivo) by determination of creatine kinase (CK) activity and histopathological analysis. Incubation of soleus muscle for 1 h with BV or toxins (20 mug/ml) plus AE (400 mug/ml) added immediately after BV, BthTX-I or BthTX-II reduced CK levels by 53%, 37% and 56%, respectively. The myonecrotic effects of BV (20 mug/ml) upon soleus muscle was reduced 24%, 35% and 36% when AE (400 mug/ml) was added 1 h after BV and CK was evaluated 30 min, 1 and 2 h later, respectively. For BthTX-I these values were 46%, 48% and 47%, while for BthTX-II no inhibitory effect was detected. Histological analysis of soleus muscle after incubation with AE (400 mug/ml, I h) did not reveal any change in muscle fibers, but severe necrosis induced by -BV or toxins (20 mug/ml) was clearly in evidence, and decreased significantly when soleus muscle was protected by AE. This protection was also observed when AE was administered 1 h after BV or BthTX-I, but not after BthTX-II. AE did not inhibit the catalytic PLA(2), activity of BthTX-II or BV and did not change the PAGE pattern of BV, BthTX-I or BthTX-II. In vivo assays were performed in 100-g rats and maximal CK release was attained at a dose of 100 mug of BV, 3 h after injection. AE was not effective when injected 20 s after BV or toxins. However, injecting BV or toxins (100 mug), which were pre-incubated with AE (2 mg) caused an inhibition of 57%, 59% and 51%, respectively, with zero time pre-incubation, but was less effective with I h pre-incubation. This plant represents a potential source of promising myotoxin inhibitors. (C) 2004 Elsevier GmbH. All rights reserved.

Cardiovascular effects of desflurane following acute hemorrhage in dogs

Objective: To determine the cardiovascular effects of desflurane in dogs following acute hemorrhage.Design: Experimental study.Animals: Eight mix breed dogs.Interventions: Hemorrhage was induced by withdrawal of blood until mean arterial pressure (MAP) dropped to 60 mmHg in conscious dogs. Blood pressure was maintained at 60 mmHg for 1 hour by further removal or replacement of blood. Desflurane was delivered by facemask until endotracheal intubation could be performed and a desflurane expiratory end-tidal concentration of 10.5 V% was maintained.Measurements and main results: Systolic, diastolic, and mean arterial blood pressure (SAP, DAP and MAP), central venous pressure (CVP), cardiac output (CO), stroke volume (SV), cardiac index (0), systemic vascular resistance (SVR), heart rate (HR), respiratory rate (RR), partial pressure of carbon dioxide in arterial blood (PaCO2), and arterial pH were recorded before and 60 minutes after hemorrhage, and 5, 15, 30, 45 and 60 minutes after intubation. Sixty minutes after hemorrhage, SAP, DAP, MAP, CVP, CO, Cl, SV, PaCO2, and arterial pH decreased, and HR and RR increased when compared with baselines values. Immediately after intubation, MAP and arterial pH decreased, and PaCO2 increased. Fifteen minutes after intubation SAP, DAP, MAP, arterial pH, and SVR decreased. At 30 and 45 minutes, MAP and DAP remained decreased and PaCO2 increased, compared with values measured after hemorrhage. Arterial pH increased after 30 minutes of desflurane administration compared with values measured 5 minutes after intubation.Conclusions: Desflurane induced significant changes in blood pressure and arterial pH when administered to dogs following acute hemorrhage.

Pré-compostagem de cadáveres de bovinos acometidos pelo botulismo

Foram pré-compostados, em células individuais e isoladas, cinco cadáveres de bovinos acometidos pelo botulismo com a finalidade de monitorar a presença de esporos de Clostridium botulinum e de toxina botulínica antes e após o processo de decomposição em leira estática com fonte de carbono. O diagnóstico da intoxicação nos animais foi baseado nas características clínico-patológicas, epidemiológicas ou laboratoriais. Dos cinco bovinos com evolução clínica crônica de botulismo, cujos cadáveres foram pré-compostados, três foram acometidos pela toxina tipo D, um pelo complexo CD e um dos animais foi negativo na tentativa de detecção da toxina e de esporos da bactéria nas vísceras pelo bioensaio e neutralização em camundongo. O processo de pré-compostagem foi realizado em leira estática, com o uso de material carbonáceo umidificado como substrato e esquartejamento do animal, vedado individualmente com lona plástica e sem aeração por um período de 50 dias. A temperatura das leiras foi monitorada durante o período e oscilou de 40,5-52,4ºC. Após a abertura das leiras, pôde-se constatar a completa decomposição de todo material mole, com redução significativa do seu peso (de 26,5-44,5%), restando apenas os ossos. Não foi detectado esporo ou toxina botulínica no interior dos ossos (n=5 para cada cadáver). Nas 200 amostras examinadas do homogeneizado restante (n=40 para cada cadáver), em apenas duas amostras de uma leira foram detectados esporos de C. botulinum tipo C, enquanto que todas foram negativas para a tentativa de detecção da toxina botulínica pelo bioensaio em camundongo. da forma como foi avaliado o processo de pré-compostagem de bovinos mortos pela intoxicação botulínica não contribuiu para a proliferação de C. botulinum.