Permeabilization of E-coli K12 inner and outer membranes by bothropstoxin-I, A LYS49 phospholipase A2 from Bothrops jararacussu

Although lacking catalytic activity, the Lys49-PLA(2)s damage artificial membranes by a Ca2+-independent mechanism, and demonstrate a potent bactericidal effect. The relationship between the membrane-damaging activity and bactericidal effect of bothropstoxin-I (BthTx-1), a Lys49-PLA(2) from the venom of Bothrops jararacussu, was evaluated for the wildtype protein and a series of site-directed mutants in the active site and C-terminal regions of the protein. The membrane permeabilization effect against the inner and outer membranes of Escherichia coli K12 was evaluated by fluorescence changes of Sytox Green and N-phenyl-N-naphthylamine, respectively. With the exception of H48Q, all mutants reduced the bactericidal activity, which correlated with a reduction of the permeabilization effect both against the inner bacterial membrane. No significant differences in the permeabilization of the bacterial outer membrane were observed between the native, wild-type recombinant and mutant proteins. These results suggest different permeabilization mechanisms against the inner and outer bacterial membranes. Furthermore, the structural determinants of bacterial inner membrane damage identified in this study correlate with those previously observed for artificial membrane permeabilization, suggesting that a common mechanism of membrane damage underlies the two effects. (C) 2007 Elsevier Ltd. All rights reserved.

Shared structural determinants for the calcium-independent liposome membrane permeabilization and sarcolemma depolarization in Bothropstoxin-I, a LYS49-PLA(2) from the venom of Bothrops jararacussu

The structural determinants of myotoxicity of bothropstoxin-I (BthTX-I), a Lys49 phospholipase A(2) from Bothrops jararacussu venom, were studied by measuring the resting membrane potential in the mouse phrenic nerve-diaphragm preparation. This method proved to be around 100-fold more sensitive than the creatine kinase release assay, and was used to evaluate a total of 31 site-directed BthTX-I alanine scanning mutants. Mutants that reduced the resting membrane potential were located in a surface patch defined by residues in the C-terminal loop (residues 115-129), positions 37-39 in the membrane interfacial recognition surface (Y46 and K54), and residue K93. These results expand the known structural determinants of the biological activity as evaluated by previous creatine kinase release experiments. Furthermore, a strong correlation is observed between the structural determinants of sarcolemma depolarization and calcium-independent disruption of liposome membranes, suggesting that a common mechanism of action underlies the permeabilization of the biological and model membranes. (C) 2009 Elsevier Ltd. All rights reserved.

Equine laminitis: Bites by Bothrops spp cause hoof lamellar pathology in the contralateral as well as in the bitten limb

The envenoming caused by Bothrops snakebite includes local symptoms, such as pronounced edema, hemorrhage, intense pain, vesicles, blisters and myonecrosis. The principal systemic symptom consists in the alteration of blood clotting, due to fibrinogen consumption and platelet abnormalities. The horses involved in this study had this symptomatology and one of them exhibited symptoms consistent with laminitis in the bitten and in the contralateral limbs. Laminitis lesions were characterized by separation of the hoof lamellar basement membrane (BM) from basal cells of the epidermis. These results demonstrated that Bothrops snake venom can induce acute laminitis. We conclude that components of the venom, probably metalloproteinases, cause severe lesions in the hoof early in the envenoming process. Antivenom therapy must be initiated as soon as possible in order to prevent complications, not only to save the life of an envenomed horse, but also to avoid the dysfunctional sequels of laminitis. (c) 2006 Elsevier Ltd. All rights reserved.

Predictors of Bothrops jararaca venom allergy in snake handlers and snake venom handlers

Since allergic sensitization to snake venom has been reported, anaphylactic reactions to snake venom might be an underestimated factor contributing to fatal snakebites, independently from the toxicity of the venom itself. However, little information is available on the determinants of such reaction. Hence, we studied a group of workers exposed to Bothrops jararaca venom (BJV), in order to clarify the factors related with snake venom allergy. The aim of this work was to investigate the prevalence and predictors of venom allergy among workers exposed to BJV and to confirm the involvement of IgE-mediated mechanisms in this condition. Workers exposed to BJV were assessed for venom allergy using questionnaires and immunological tests. The presence of BJV sensitization was determined through quantification of specific IgE. Allergens were studied using the Western blots and inhibition assays. Of the 67 workers evaluated, 7 (10.4%) presented specific IgE antibodies to BJV. Of those, 6 presented typical symptoms of an IgE-mediated allergic reaction when exposed to BJV. Venom sensitization was associated with length of employment (P = 0.042), high levels of total IgE (P = 0.034), atopy (P = 0.051), and specific tasks, primarily the handling of dried venom (P = 0.014). Our observations suggest that exposure to BJV can result in allergic sensitization in snake handlers through IgE-mediated mechanisms. The prevalence rate of this condition appears to be high among these workers, and the handling of dried venom, total IgE level above 100 kU/L, length of employment, and probably history of atopy were predictors of its occurrence. (C) 2008 Elsevier Ltd. All rights reserved.

Involvement of circulating platelets on the hyperalgesic response evoked by carrageenan and Bothrops jararaca snake venom

Background: The role of platelets in hemostasis is well known, but few papers have reported their role in pain and edema induced by inflammatory agents. Objective: To evaluate the role of circulating platelets in the local injury induced by two diverse inflammatory agents, Bothrops jararaca venom (Bjv) and carrageenan. Methods: Rats were (i) rendered thrombocytopenic by administration of polyclonal anti-rat platelet IgG (ARPI) or busulfan, or (ii) treated with platelet inhibitors (aspirin or clopidogrel). Edema formation, local hemorrhage and the pain threshold were assessed after intraplantar injection of Bjv or carrageenan in rat hind paws. Additionally, whole platelets or platelet releasate were tested whether they directly induced hyperalgesia. Results: Platelet counts were markedly diminished in rats administered with either ARPI (+/- 88%) or busulfan (+/- 96%). Previous treatment with ARPI or busulfan slightly reduced edema induced by Bjv or carrageenan. Injection of Bjv, but not of carrageenan, induced a statistically significance increase in hemorrhage in the hind paws of thrombocytopenic rats. Remarkably, hyperalgesia evoked by Bjv or carrageenan was completely blocked in animals treated with ARPI or busulfan, or pre-treated with aspirin or clopidogrel. On the other hand, intraplantar administration of whole platelets or platelet releasate evoked hyperalgesia, which was inhibited by pre-incubation with alkaline phosphatase. Conclusions: Thrombocytopenia or inhibition of platelet function drastically reduced hyperalgesia induced by injection of carrageenan or Bjv; moreover, platelets per se secrete phosphorylated compounds involved in pain mediation. Thus, blood platelets are crucial cells involved in the pain genesis, and their role therein has been underestimated.

In Vivo Effects of Bothrops jararaca Venom on Metabolic Profile and on Muscle Protein Metabolism in Rats

This study investigated the in vivo effects of the Bothrops Jararaca venom (BjV) on general metabolic profile and, specifically. oil muscle protein metabolism in rats. The crude venom (0.4 mg/kg body weight, IV) was infused in awake rats, and plasma activity of enzymes and metabolites levels were determined after 1, 2, 3, and 4 hours. BjV increased urea, lactate, and activities of creatine kinase. lactate dehydrogenase. and aspartate aminotransferase after 4 hours. The content of liver glycogen was reduced by BjV. Protein metabolism was evaluated by means of microdialysis technique and in isolated muscles. BjV induced increase in the muscle interstitial-arterial tyrosine concentration difference. indicating a high protein catabolism. The myotoxicity induced by this venom is associated with reduction of protein synthesis and increase in rates of overall proteolysis, which was accompanied by activation of lysosomal and ubiquitin-proteasome systems without changes in protein levels of cathepsins and ubiquitin-protein conjugates.

Neuromuscular activity of BaTX, a presynaptic basic PLA(2) isolated from Bothrops alternatus snake venom

We have previously isolated a Lys49 phospholipase A(2) homolog (BaTX) from Bothrops alternatus snake venom using a combination of molecular exclusion chromatography and reverse phase HPLC and shown its ability to cause neuromuscular blockade. In this work, we describe a one-step procedure for the purification of this toxin and provide further details of its neuromuscular activity. The toxin was purified by reverse phase HPLC and its purity and molecular mass were confirmed by SIDS-PAGE, MALDI-TOF mass spectrometry, amino acid analysis and N-terminal sequencing. BaTX (0.007-1.4 mu M) produced time-dependent, irreversible neuromuscular blockade in isolated mouse phrenic nerve-diaphragm and chick biventer cervicis preparations (time to 50% blockade with 0.35 mu M toxin: 58 +/- 4 and 24 +/- 1 min, respectively; n = 3-8; mean +/- S.E.) without significantly affecting the response to direct muscle stimulation. In chick preparations, contractures to exogenous acetylcholine (55 and 110 mu M) or KCl (13.4 mM) were unaltered after complete blockade by all toxin concentrations. These results, which strongly suggested a presynaptic mechanism of action for this toxin, were reinforced by (1) the inability of BaTX to interfere with the carbachol-induced depolarization of the resting membrane, (2) a significant decrease in the frequency and amplitude of miniature end-plate potentials, and (3) a significant reduction (59 +/- 4%, n=12) in the quantal content of the end-plate potentials after a 60 min incubation with the toxin (1.4 mu M). In addition, a decrease in the organ bath temperature from 37 degrees C to 24 degrees C and/or the replacement of calcium with strontium prevented the neuromuscular blockade, indicating a temperature-dependent effect possibly mediated by enzymatic activity. (C) 2009 Elsevier Inc. All rights reserved.

Insights on calcium-independent phospholipid membrane damage by Lys49-PLA(2) using tryptophan scanning mutagenesis of bothropstoxin-I from Bothrops jararacussu

Bothropstoxin-I (BthTx-I) is a homodimerie Lys49-PLA(2) from the venom of the snake Bothrops jararacussu, which lacks hydrolytic activity against phospholipid substrates, yet permeabilizes membranes by a Ca2+- independent mechanism. The interaction of the BthTx-I with model membranes has been studied by intrinsic tryptophan fluorescence emission (ITFE) spectroscopy. Nine separate mutants have been created each with a unique tryptophan residue located at a different position in the interfacial recognition site (IRS) of the protein. The rapid and efficient Ca2+-independent membrane damage against unilamellar liposomes composed of DPPC/DMPA in a 9:1 molar ratio was unaffected by these substitutions. Binding studies revealed low protein affinity for these liposomes and no changes were observed in the ITFE properties. In contrast, the binding of all mutants to DPPC/DMPA liposomes in a 1:1 molar ratio was stronger, and was correlated with altered ITFE properties. The blue-shifted emission spectra and increased emission intensity of mutants at positions 31, 67 and 115-117 in the interface recognition surface of the protein suggest these regions are partially inserted into the membrane. These results are consistent with a model for the Ca2+-independent membrane damaging mechanism that involves a transient interaction of the protein with the outer phospholipid leaflet of the target membrane. (C) 2007 Elsevier Masson SAS. All rights reserved.

Identification and characterization of a new member of snake venom thrombin inhibitors from Bothrops insularis using a proteomic approach

Snake venom C-type lectin-like proteins (CLPs) are ubiquitously found in Viperidae snake venoms and differ from the C-type lectins as they display different biological activities but no carbohydrate-binding activity. Previous analysis of the transcriptome obtained from the Bothrops insularis venom gland showed the presence of two clusters homologous to bothrojaracin (BJC) chains a and P. In an effort to identify a new BJC-like molecule, we used an approach associated with proteomic technologies to identify the presence of the expressed protein and then to purify and characterize a new thrombin inhibitor from B. insularis venom. We also constructed homology models of this protein and BJC, which were compared with other C-type lectin-like family members and revealed several conserved features of this intriguing snake venom toxin family. (C)0 2007 Elsevier Ltd. All rights reserved.

Analysis of the Ontogenetic Variation in the Venom Proteome/Peptidome of Bothrops jararaca Reveals Different Strategies to Deal with Prey

Previous studies have demonstrated that the pharmacological activities displayed by Bothrops jararaca venom undergo a significant ontogenetic shift. Variation in the venom proteome is a well-documented phenomenon; however, variation in the venom peptidome is poorly understood. We report a comparative proteomic and peptidomic analysis of venoms from newborn and adult specimens of B. jararaca and correlate it with the evaluation of important venom features. We demonstrate that newborn and adult venoms have similar hemorrhagic activities, while the adult venom has a slightly higher lethal activity in mice; however, the newborn venom is extremely more potent to kill chicks. The coagulant activity of newborn venom upon human plasma is 10 times higher than that of adult venom. These differences were clearly reflected in their different profiles of SDS-PAGE, gelatin zimography, immunostaining using specific antibodies, glycosylation pattern, and concanavalin A-binding proteins. Furthermore, we report for the first time the analysis of the peptide fraction of newborn and adult venoms by MALDI-TOF mass spectrometry and LC-MS/MS, which revealed different contents of peptides, while the bradykinin potentiating peptides (BPPs) showed rather similar profiles and were detected in the venoms showing their canonical sequences and also novel sequences corresponding to BPPs processed from their precursor protein at sites so far not described. As a result of these studies, we demonstrated that the ontogenetic shift in diet, from ectothermic prey in early life to endothermic prey in adulthood, and in animal size are associated with changes in the venom proteome in B. jararaca species.

Antitumoural effect of an L-amino acid oxidase isolated from Bothrops jararaca snake venom

An L-amino acid oxidase (BjarLAAO-I) from Bothrops jararaca snake venom was highly purified using a stepwise sequential chromatography on Sephadex G-75, Benzamidine Sepharose and Phenyl Sepharose. Purified BjarLAAO-I showed a molecular weight around 60,000 under reducing conditions and about 125,000 in the native form, when analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. BjarLAAO-I is a homodimeric acidic glycoprotein, pI similar to 5.0, and N-terminal sequence showing close structural homology with other snake venom LAAOs. The purified enzyme catalysed the oxidative deamination of L-amino acids, the most specific substrate being L-Phe. Five amino acids, L-Ser, L-Pro, L-Gly, L-Thr and L-Cys were not oxidized, clearly indicating a significant specificity. BjarLAAO-I significantly inhibited Ehrlich ascites tumour growth and induced an influx of polymorphonuclear cells, as well as spontaneous liberation of H(2)O(2) from peritoneal macrophages. Later, BjarLAAO-I induced mononuclear influx and peritoneal macrophage spreading. Animals treated with BjarLAAO-I showed higher survival time.

Carios fonsecai sp nov (Acari, Argasidae), a bat tick from the central-western region of Brazil

Males, females, and larvae of Carios fonsecai sp. nov. are described from free-living ticks collected in a cave at Bonito, state of Mato Grosso do Sul, Brazil. The presence of cheeks and legs with micromammillate cuticle makes adults of C. fonsecai morphologically related to a group of argasid species (mostly bat-associated) formerly classified into the subgenus Alectorobius, genus Ornithodoros. Examination of larvae indicates that C. fonsecai is clearly distinct from most of the previously described Carios species formerly classified into the subgenus Alectorobius, based primarily on its larger body size, dorsal setae number, dorsal plate shape, and hypostomal morphology. On the other hand, the larva of C. fonsecai is most similar to Carios peropteryx, and Carios peruvianus, from which differences in dorsal plate length and width, tarsal setae, and hypostome characteristics are useful for morphological differentiation. The mitochondrial 16S rDNA sequence of C. fonsecai showed to be closest (85-88% identity) to several corresponding sequences of different Carios species available in GenBank. Bats identified as Peropteryx macrotis and Desmodus rotundus were found infested by C. fonsecai larvae in the same cave where the type series was collected. C. fonsecai showed to be aggressive to humans in the laboratory.

Estudo histopatológico das lesões causadas pelo veneno de urutu (Bothrops alternatus) em músculo esquelético de camundongos

Veneno bruto de urutu (Bothrops alternatus) dissolvido em solução salina fisiológica foi injetado no músculo tibial anterior direito de camundongos adultos na dose de 80 μg. Os músculos foram examinados em cortes de parafina, corados por Hematoxilina e Eosina. Aos 10 minutos já havia intensa hemorragia difusa no M. tibial anterior, mas apenas raras fibras musculares estavam necróticas. Nas horas seguintes, contudo, observou-se rápido aumento do número de fibras afetadas, sendo que às 24 hs o músculo apresentava-se totalmente necrótico. Vasos sangüíneos intramusculares e nas proximidades do M. tibial anterior mostravam necrose hialina da camada média e por vezes trombose. A fagocitose dos restos celulares ocorreu da periferia para o centro e acompanhou-se de regeneração muscular. Após 1 a 2 meses, em vários animais houve recuperação considerável do músculo, embora com persistência de cicatriz. As fibras regeneradas possuiam núcleos centrais e variavam em diâmetro, estando muitas atróficas. Em outros camundongos a regeneração do M. tibial anterior foi muito precária, tendo este sido substituído por tecido fibroadiposo com apenas raras fibras musculares. Os resultados mostram que, apesar da gravidade das lesões iniciais devidas ao veneno, ocorre regeneração muscular em grau variável de animal para animal. Sugere-se que a má regeneração observada em alguns casos poderia ser devida, ao menos em parte, a dano vascular permanente.

Acidentes ofídicos causados por bothrops moojeni: relato de 37 casos

Foram estudados 37 casos de acidentes ofídicos causados por Bothrops moojeni na região de São José do Rio Preto, São Paulo, durante um período de 5 anos (outubro 1982 a setembro 1987). Desse total, 34 apresentaram sintomatologia clínica, sendo indicado administração de antiveneno. São apresentados aspectos epidemiológicos relacionados à idade, sexo, horário do acidente, atividade do paciente no acidente, segmento corpóreo atingido e atitude inicial do paciente frente ao acidente: os resultados são semelhantes aos padrões já conhecidos no Brasil. O quadro clínico revelou dor e edema (usado como critério para avaliação da gravidade) em 100% tempo de coagulação prolongado/incoagulável em 72,7% e hemorragia sistêmica em 5,8%. As complicações descritas em 29,4% dos casos, surgiram apenas nos grupos em que a avaliação inicial era moderado ou grave (óbito, síndrome compartimental, necrose, infecção e retrações musculares) e não puderam ser evitadas mesmo com administração maior e mais precoce de antiveneno. Os acidentes por Bothrops moojeni produzem maior gravidade local (edema necrose infecção) e maior percentagem de tempo de coagulação prolongado incoagulável quando comparado aos causados por Bothrops jararaca.

Acidentes ofidicos causados por Bothrops moojeni: correlação do quadro clínico com o tamanho da serpente

Durante um período de 19 meses (março 1986 a setembro 1987) foram estudados 22 casos de acidentes ofídicos causados por Bothrops moojeni na região de São José do Rio Preto, São Paulo, nos quais o tamanho da serpente foi sistematicamente medido. Foram constituídos dois grupos de pacientes de acordo com o tamanho da serpente: grupo I - 9 casos de serpentes pequenas (30 a 53 cm) e grupo II - 13 casos de serpentes grandes (80 a 147 cm). Os resultados mostraram: 1. efeitos locais iniciais - dor e edema - mais leves no grupo I; 2. tempo de coagulação prolongado/incoagulável levemente mais freqüente no grupo I; 3. complicações locais - necrose, infecção e síndrome compartimentai - exclusivamente, e em mais da metade dos casos do grupo II, apesar da terapia com antiveneno ter sido mais rápida e em doses maiores nesse grupo. Conclui-se que as serpentes Bothrops moojeni maiores apresentam grande incremento nas suas ações locais - edema, necrose e infecção secundária - e leve perda em sua ação coagulante.