Structure of myotoxin II, a catalytically inactive Lys49 phospholipase A2 homologue from Atropoides nummifer venom

Lys49 snake-venom phospholipase A2 (PLA2) homologues are highly myotoxic proteins which, although lacking catalytic activity, possess the ability to disrupt biological membranes, inducing significant muscle-tissue loss and permanent disability in severely envenomed patients. Since the structural basis for their toxic activity is still only partially understood, the structure of myotoxin II, a monomeric Lys49 PLA2 homologue from Atropoides nummifer, has been determined at 2.08 Å resolution and the anion-binding site has been characterized. © 2006 International Union of Crystallography. All rights reserved.

Laboratory evaluation of young ovines inoculated with natural or 60Co-irradiated Crotalus durissus terrificus venom during hyperimmunization process

Laboratory profile of young ovines was studied in order to evaluate and compare their antiserum production from natural and Cobalt-60 irradiated Crotalus durissus terrificus (C.d.t.) venoms. The parameters analyzed included complete blood count, and urea, creatinine, aspartate aminotransferase, total proteins, albumin and globulin serum measurements. Three groups of six animals each were used. Group 1 (G1) received natural C.d.t. venom; Group 2 (G2) received irradiated C.d.t. venom; and Group 3 (G3) was used as control and did not receive venom, only adjuvants, using seven venom inoculations. During the experimental period, animals were fortnightly weighed. According to clinical and weight evaluation, sheep in post-weaning phase showed no changes in their physiological profiles but had excellent weight gain. The parameters analyzed were not statistically different (p<5%) among the groups tested. The hyperimmunization process was successfully accomplished with the production of specific antibodies against Crotalus durissus terrificus venom. Results bring a new possibility of utilizing ovines in the commercial production of anticrotalic serum, which may be used to treat human and animal envenomation. Its production cost may be reduced by subsequent use of hyperimmunized sheep for human consumption.

Biochemical and hematological study of goats envenomed with natural and 60Co-irradiated bothropic venom

Venoms from snakes of the Bothrops genus are proteolytic, coagulant, hemorrhagic and nephrotoxic, causing edema, necrosis, hemorrhage and intense pain at the bite site, besides systemic alterations. Many adjuvants have been added to the venom used in the sensitization of antiserum-producer animals to increase antigenic induction and reduce the envenomation pathological effects. Gamma radiation from 60Co has been used as an attenuating agent of the venoms toxic properties. The main objective was to study, comparatively, clinical and laboratory aspects of goats inoculated with bothropic (Bothrops jararaca) venom, natural and irradiated from a 60Co source. Twelve goats were divided into two groups of six animals: GINV, inoculated with 0.5mg/kg of natural venom; and GIIV, inoculated with 0.5mg/kg of irradiated venom. Blood samples were collected immediately before and one, two, seven, and thirty days after venom injection. Local lesions were daily evaluated. The following exams were carried out: blood tests; biochemical tests of urea, creatinine, creatine kinase (CK), aspartate amino-transferase (AST) and alanine amino-transferase (ALT); clotting time; platelets count; and total serum immunoglobulin measurement. In the conditions of the present experiment, irradiated venom was less aggressive and more immunogenic than natural venom.

Modulation of the pharmacological effects of enzymatically-active PLA 2 by BTL-2, an isolectin isolated from the Bryothamnion triquetrum red alga

Background. An interaction between lectins from marine algae and PLA 2 from rattlesnake was suggested some years ago. We, herein, studied the effects elicited by a small isolectin (BTL-2), isolated from Bryothamnion triquetrum, on the pharmacological and biological activities of a PLA 2 isolated from rattlesnake venom (Crotalus durissus cascavella), to better understand the enzymatic and pharmacological mechanisms of the PLA 2 and its complex. Results. This PLA2 consisted of 122 amino acids (approximate molecular mass of 14 kDa), its pI was estimated to be 8.3, and its amino acid sequence shared a high degree of similarity with that of other neurotoxic and enzymatically-active PLA2s. BTL-2 had a molecular mass estimated in approximately 9 kDa and was characterized as a basic protein. In addition, BTL-2 did not exhibit any enzymatic activity. The PLA2 and BTL-2 formed a stable heterodimer with a molecular mass of approximately 24-26 kDa, estimated by molecular exclusion HPLC. In the presence of BTL-2, we observed a significant increase in PLA2 activity, 23% higher than that of PLA2 alone. BTL-2 demonstrated an inhibition of 98% in the growth of the Gram-positive bacterial strain, Clavibacter michiganensis michiganensis (Cmm), but only 9.8% inhibition of the Gram-negative bacterial strain, Xanthomonas axonopodis pv passiflorae (Xap). PLA2 decreased bacterial growth by 27.3% and 98.5% for Xap and Cmm, respectively, while incubating these two proteins with PLA2-BTL-2 inhibited their growths by 36.2% for Xap and 98.5% for Cmm. PLA2 significantly induced platelet aggregation in washed platelets, whereas BTL-2 did not induce significant platelet aggregation in any assay. However, BTL-2 significantly inhibited platelet aggregation induced by PLA2. In addition, PLA 2 exhibited strong oedematogenic activity, which was decreased in the presence of BTL-2. BTL-2 alone did not induce oedema and did not decrease or abolish the oedema induced by the 48/80 compound. Conclusion. The unexpected results observed for the PLA2-BTL-2 complex strongly suggest that the pharmacological activity of this PLA2 is not solely dependent on the presence of enzymatic activity, and that other pharmacological regions may also be involved. In addition, we describe for the first time an interaction between two different molecules, which form a stable complex with significant changes in their original biological action. This opens new possibilities for understanding the function and action of crude venom, an extremely complex mixture of different molecules. © 2008 Oliveira et al; licensee BioMed Central Ltd.

Cortisol response to acute stress in jundiá Rhamdia quelen acutely exposed to sub-lethal concentrations of agrichemicals

Exposure to agrichemicals can have deleterious effects on fish, such as disruption of the hypothalamus-pituitary-inter-renal axis (HPI) that could impair the ability of fish to respond to stressors. In this study, fingerlings of the teleost jundiá (Rhamdia quelen) were used to investigate the effects of the commonly used agrichemicals on the fish response to stress. Five common agrichemicals were tested: the fungicide - tebuconazole, the insecticide - methyl-parathion, and the herbicides - atrazine, atrazine + simazine, and glyphosate. Control fishes were not exposed to agrichemicals and standard stressors. In treatments 2-4, the fishes were exposed to sub-lethal concentrations (16.6%, 33.3%, and 50% of the LC50) of each agrichemical for 96 h, and at the end of this period, were subjected to an acute stress-handling stimulus by chasing them with a pen net. In treatments 5-7 (16.6%, 33.3%, and 50% of the LC50), the fishes were exposed to the same concentrations of the agrichemicals without stress stimulus. Treatment 8 consisted of jundiás not exposed to agrichemicals, but was subjected to an acute stress-handling stimulus. Jundiás exposed to methyl-parathion, atrazine + simazine, and glyphosate presented a decreased capacity in exhibiting an adequate response to cope with stress and in maintaining the homeostasis, with cortisol level lower than that in the control fish (P < 0.01). In conclusion, the results of this study clearly demonstrate that the acute exposure to sub-lethal concentrations of methyl-parathion, atrazine + simazine, and glyphosate exert a deleterious effect on the cortisol response to an additional acute stressor in the jundiá fingerlings. © 2008 Elsevier Inc. All rights reserved.

Myotoxic phospholipases A2 isolated from Bothrops brazili snake venom and synthetic peptides derived from their C-terminal region: Cytotoxic effect on microorganism and tumor cells

This paper reports the purification and biochemical/pharmacological characterization of two myotoxic phospholipases A2 (PLA2s) from Bothrops brazili venom, a native snake from Brazil. Both myotoxins (MTX-I and II) were purified by a single chromatographic step on a CM-Sepharose ion-exchange column up to a high purity level, showing Mr ∼ 14,000 for the monomer and 28,000 Da for the dimer. The N-terminal and internal peptide amino acid sequences showed similarity with other myotoxic PLA2s from snake venoms, MTX-I belonging to Asp49 PLA2 class, enzymatically active, and MTX-II to Lys49 PLA2s, catalytically inactive. Treatment of MTX-I with BPB and EDTA reduced drastically its PLA2 and anticoagulant activities, corroborating the importance of residue His48 and Ca2+ ions for the enzymatic catalysis. Both PLA2s induced myotoxic activity and dose-time dependent edema similar to other isolated snake venom toxins from Bothrops and Crotalus genus. The results also demonstrated that MTXs and cationic synthetic peptides derived from their 115-129 C-terminal region displayed cytotoxic activity on human T-cell leukemia (JURKAT) lines and microbicidal effects against Escherichia coli, Candida albicans and Leishmania sp. Thus, these PLA2 proteins and C-terminal synthetic peptides present multifunctional properties that might be of interest in the development of therapeutic strategies against parasites, bacteria and cancer. © 2008 Elsevier Inc. All rights reserved.

Dietary fish oil replacement with lard and soybean oil affects triacylglycerol and phospholipid muscle and liver docosahexaenoic acid content but not in the brain and eyes of surubim juveniles Pseudoplatystoma sp

Triplicate groups of juvenile suribim were fed for 183 days one of four different isonitrogenous (47.6% crude protein) and isolipidic (18.7% lipid) diets formulated using three different lipid sources: 100% fish oil (FO, diet 1); 100% pig lard (L, diet 2); 100% soybean oil (SO, diet 3), and FO/L/SO (1:1:1, w/w/w; diet 4). The tissue levels of fatty acids 18:2n - 6 and 18:3n - 3 decreased relative to corresponding dietary fatty acid values. The 20:5n - 3 and 22:6n - 3 composition of muscle and liver neutral lipids were linearly correlated with corresponding dietary fatty acid composition. In contrast, the 22:6n - 3 composition of the brain and eye were similar among treatments. The 22:6n - 3 level was enriched in all tissues, particularly in the neural tissues. Similar results were observed for tissue polar lipids: fatty acids content reflected dietary composition, with the exception of the 22:6n - 3 level, which showed enrichment and no differences between groups. Given these results, the importance of the biochemical functions (transport and/or metabolism) of 22:6n - 3 in the development of the neural system of surubim warrants further investigation. © Springer Science+Business Media B.V. 2008.

Resposta imune e capacidade de neutralização de anticorpos produzidos em ovinos jovens imunizados com veneno de Crotalus durissus terrificus nativo e irradiado com Cobalto 60

The ELISA technique was used to evaluate and compare young ovine humoral immune response during crotalic antiserum production. Animals were clinically evaluated throughout this process, and the neutralizing capacity of antisera raised against natural (NV) and Cobalt-60 irradiated (IrV) venoms of Crotalus durissus terrificus (C.d.t.) was verified by means of in vitro challenges. Three groups of six animals each were used: G1 received NV; G2 was inoculated with IrV; and G3 was used as control. Animals received six immunizations during 84 days at 14-day intervals. ELISA of antibody profile showed significant difference (p<5%) between experimental groups (G1

Structure of a novel class II phospholipase D: Catalytic cleft is modified by a disulphide bridge

Phospholipases D (PLDs) are principally responsible for the local and systemic effects of Loxosceles envenomation including dermonecrosis and hemolysis. Despite their clinical relevance in loxoscelism, to date, only the SMase I from Loxosceles laeta, a class I member, has been structurally characterized. The crystal structure of a class II member from Loxosceles intermedia venom has been determined at 1.7. Å resolution. Structural comparison to the class I member showed that the presence of an additional disulphide bridge which links the catalytic loop to the flexible loop significantly changes the volume and shape of the catalytic cleft. An examination of the crystal structures of PLD homologues in the presence of low molecular weight compounds at their active sites suggests the existence of a ligand-dependent rotamer conformation of the highly conserved residue Trp230 (equivalent to Trp192 in the glycerophosphodiester phosphodiesterase from Thermus thermophofilus, PDB code: 1VD6) indicating its role in substrate binding in both enzymes. Sequence and structural analyses suggest that the reduced sphingomyelinase activity observed in some class IIb PLDs is probably due to point mutations which lead to a different substrate preference. © 2011 Elsevier Inc.

Processamento da ração no desempenho de juvenis de jundiá (Rhamdia voulezi) cultivados em tanques-rede

The aim of this study was to evaluate the zootechnical performance and chemical composition of jundia juveniles Rhandia voulezi fed with mash, pelletized and extruded diets and cultivated in cages. One hundred and fifty fish were distributed in fifteen cages (0.20 m3), arranged in cages (5m3), in the hydroelectric plant of Governador José Richa, in Iguaçu River (Paraná-Brazil). The fish were fed five times a day, with mash, pelletized and extruded diets, containing 30% of protein and 3250 Kcal/kg of digestible energy/kg. At the end of 60 days, significant differences (p<0.05) were observed on fish performance. The fish that were fed with pelletized diet, presented higher average of final weight (44.08g), final length (16.51cm) and weight gain (36.65g). The different processes did not influence fish survival. The averages of protein and mineral matter of carcass chemical composition did not differ statistically (p<0.05), while the values of humidity and lipideos presented differences among treatments. The highest average of lipideos (8.59%) was observed in peletized diet. The mash diet provided fish with lower lipideo (5.45%) and higher humidity (75.98%) rates. A pelleted diet is most suitable for juvenile catfish R. voulezi cultivation in cages.

Artemia sp. na alimentação de larvas de jundiá (Rhamdia quelen)

A considerable problem in the catfish larvae rearing is related to the first feeding. This study aimed to determine the larvae growth and the survival of silver catfish submitted to feeding with Artemia sp. in different time periods. The study was conducted in the Aquaculture Laboratory of the Universidade Estadual do Oeste do Paraná, with four treatments and five replications, constituted by feeding with Artemia sp. in 5, 10, 15 and 20 days, and after each period of time, by its replacement by ration. We used 200 larvae distributed into 20 aquariums, soon after the eggs hatched. The larvae were fed 4 times a day until apparent satiation for 30 days. The Artemia sp. feeding for 15 days provided the best results on weight gain, final length and survival.

Structural insights into selectivity and cofactor binding in snake venom l-amino acid oxidases

l-Amino acid oxidases (LAAOs) are flavoenzymes that catalytically deaminate l-amino acids to corresponding α-keto acids with the concomitant production of ammonia (NH 3) and hydrogen peroxide (H 2O 2). Particularly, snake venom LAAOs have been attracted much attention due to their diverse clinical and biological effects, interfering on human coagulation factors and being cytotoxic against some pathogenic bacteria and Leishmania ssp. In this work, a new LAAO from Bothrops jararacussu venom (BjsuLAAO) was purified, functionally characterized and its structure determined by X-ray crystallography at 3.1å resolution. BjsuLAAO showed high catalytic specificity for aromatic and aliphatic large side-chain amino acids. Comparative structural analysis with prokaryotic LAAOs, which exhibit low specificity, indicates the importance of the active-site volume in modulating enzyme selectivity. Surprisingly, the flavin adenine dinucleotide (FAD) cofactor was found in a different orientation canonically described for both prokaryotic and eukaryotic LAAOs. In this new conformational state, the adenosyl group is flipped towards the 62-71 loop, being stabilized by several hydrogen-bond interactions, which is equally stable to the classical binding mode. © 2012 Elsevier Inc.

Envenenamento por caravela (Physalia physalis) manifestando-se com erupção papulopurpurica

We report the case of a 42-year old woman who was envenomed by a Portuguese man-o'-war (Physalia physalis). She presented an anomalous reaction manifested by purpuric papules that appeared after the initial phase of envenoming (around 24 hours later), when linear erythematous and edematous papules were observed. Late-onset reactions in accidents involving cnidarians commonly include chronic eruptions and local pigmentation. © 2012 by Anais Brasileiros de Dermatologia.

Agelaia MP-I: A peptide isolated from the venom of the social wasp, Agelaia pallipes pallipes, enhances insulin secretion in mice pancreatic islets

Peptides isolated from animal venoms have shown the ability to regulate pancreatic beta cell function. Characterization of wasp venoms is important, since some components of these venoms present large molecular variability, and potential interactions with different signal transduction pathways. For example, the well studied mastoparan peptides interact with a diversity of cell types and cellular components and stimulate insulin secretion via the inhibition of ATP dependent K + (K ATP) channels, increasing intracellular Ca 2+ concentration. In this study, the insulin secretion of isolated pancreatic islets from adult Swiss mice was evaluated in the presence of synthetic Agelaia MP-I (AMP-I) peptide, and some mechanisms of action of this peptide on endocrine pancreatic function were characterized. AMP-I was manually synthesized using the Fmoc strategy, purified by RP-HPLC and analyzed using ESI-IT-TOF mass spectrometry. Isolated islets were incubated at increasing glucose concentrations (2.8, 11.1 and 22.2 mM) without (Control group: CTL) or with 10 μM AMP-I (AMP-I group). AMP-I increased insulin release at all tested glucose concentrations, when compared with CTL (P < 0.05). Since molecular analysis showed a potential role of the peptide interaction with ionic channels, insulin secretion was also analyzed in the presence of 250 μM diazoxide, a K ATP channel opener and 10 μM nifedipine, a Ca 2+ channel blocker. These drugs abolished insulin secretion in the CTL group in the presence of 2.8 and 11.1 mM glucose, whereas AMP-I also enhanced insulin secretory capacity, under these glucose conditions, when incubated with diazoxide and nifedipine. In conclusion, AMP-I increased beta cell secretion without interfering in K ATP and L-type Ca 2+ channel function, suggesting a different mechanism for this peptide, possibly by G protein interaction, due to the structural similarity of this peptide with Mastoparan-X, as obtained by modeling. © 2012 Elsevier Ltd.

Bothrops leucurus venom induces nephrotoxicity in the isolated perfused kidney and cultured renal tubular epithelia

Bites from snake (Bothrops genus) cause local tissue damage and systemic complications, which include alterations such as hemostatic system and acute renal failure (ARF). Recent studies suggest that ARF pathogenesis in snakebite envenomation is multifactorial and involves hemodynamic disturbances, immunologic reactions and direct nephrotoxicity. The aim of the work was to investigate the effects of the Bothrops leucurus venom (BlV) in the renal perfusion system and in cultured renal tubular cells of the type MDCK (Madin-Darby Canine kidney). BlV (10 μg/mL) reduced the perfusion pressure at 90 and 120 min. The renal vascular resistance (RVR) decreased at 120 min of perfusion. The effect on urinary flow (UF) and glomerular filtration rate (GFR) started 30 min after BlV infusion, was transient and returned to normal at 120 min of perfusion. It was also observed a decrease on percentual tubular transport of sodium (%TNa+) at 120 min and of chloride (%TCl-) at 60 and 90 min. The treatment with BlV caused decrease in cell viability to the lowest concentration tested with an IC50 of 1.25 μg/mL. Flow cytometry with annexin V and propidium iodide showed that cell death occurred predominantly by necrosis. However, a cell death process may involve apoptosis in lower concentrations. BlV treatment (1.25 μg/mL) led to significant depolarization of the mitochondrial membrane potential and, indeed, we found an increase in the expression of cell death genes in the lower concentrations tested. The venom also evoked an increase in the cytosolic Ca2+ in a concentration dependent manner, indicating that Ca2+ may participate in the venom of B. leucurus effect. The characterization of the effects in the isolated kidney and renal tubular cells gives strong evidences that the acute renal failure induced by this venom is a result of the direct nephrotoxicity which may involve the cell death mechanism. © 2012.