Antimicrobial activity of an L-amino acid oxidase isolated from Bothrops leucurus snake venom

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Purification, crystallization and preliminary X-ray diffraction analysis of a class P-III metalloproteinase (BmMP-III) from the venom of Bothrops moojeni

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Clinical and hematological alterations in dogs during experimental envenomation with Crotalus durissus terrificus venom and treated with antiophidic serum

The present work aimed to evaluate the clinical and hematological aspects during experimental envenomation by Crotalus durissus terrificus in dogs treated with different antiophidic serum doses. Sixteen dogs were divided into two groups of eight animals each. Group I received 1mg/kg venom subcutaneously and 30mg antiophidic serum intravenously; Group II received 1mg/kg venom subcutaneously and 60mg antiophidic serum intravenously. In the clinical evaluation, we observed ataxia, moderate sedation, dilated pupils, sialorrhea, flaccid paralysis of mandibular muscles, and discreet edema at the site of venom inoculation. Evaluating red and white blood cells, we observed a decrease of hemoglobins, globular volume and erythrocytes, and an increase of plasmatic proteins, leukocytes, neutrophils, monocytes and lymphocytes. Clotting time increased and there was blood incoagulability with return to normal clotting time six hours after antiophidic serum administration. Animals treated with six antiophidic serum flasks had a faster recovery than the animals that received three serum flasks.

A new class of neurotoxin from wasp venom slows inactivation of sodium current

The effects of alpha-pompilidotoxin (alpha-PMTX), a new neurotoxin isolated from the venom of a solitary wasp, were studied on the neuromuscular synapses in lobster walking leg and the rat trigeminal ganglion (TG) neurons. Paired intracellular recordings from the presynaptic axon terminals and the innervating lobster leg muscles revealed that alpha-PMTX induced long bursts of action potentials in the presynaptic axon, which resulted in facilitated excitatory and inhibitory synaptic transmission. The action or alpha-PMTX was distinct from that of other known facilitatory presynaptic toxins, including sea anemone toxins and alpha-scorpion toxins, which modify the fast inactivation of Na+ current. We further characterized the action of alpha-PMTX on Na+ channels by whole-cell recordings from rat trigeminal neurons. We found that alpha-PMTX stowed the Na+ channels inactivation process without changing the peak current-voltage relationship or the activation time course of tetrodotoxin (TTX)-sensitive Na+ currents, and that alpha-PMTX had voltage-dependent effects on the rate of recovery from Na+ current inactivation and deactivating tail currents. The results suggest that alpha-PMTX slows or blocks conformational changes required for fast inactivation of the Na+ channels on the extracellular surface. The simple structure of alpha-PMTX, consisting of 13 amino acids, would be advantageous for understanding the functional architecture of Na+ channel protein.

Dissociation of enzymatic and pharmacological properties of piratoxins-I and -III, two myotoxic phospholipases A(2) from Bothrops pirajai snake venom

Piratoxins (PrTX) I and III are phospholipases A(2) (PLA(2)s) or PLA(2) homologue myotoxins isolated from Bothrops pirajai snake venom, which also induce myonecrosis, bactericidal activity against Escherichia coli, disruption of artificial membranes, and edema. PrTX-III is a catalytically active hemolytic and anticoagulant Asp49 PLA(2), while PrTX-I is a Lys49 PLA, homologue, which is catalytically inactive on artificial substrates, but promotes blockade of neuromuscular transmission. Chemical modifications of His, Lys, Tyr, and Trp residues of PrTX-I and PrTX-III were performed, together with cleavage of the N-terminal octapeptide by CNBr and inhibition by heparin and EDTA. The lethality, bactericidal activity, myotoxicity, neuromuscular effect, edema inducing effect, catalytic and anticoagulant activities, and the liposome-disruptive activity of the modified toxins were evaluated. A complex pattern of functional differences between the modified and native toxins was observed. However, in general, chemical modifications that significantly affected the diverse pharmacological effects of the toxins did not influence catalytic or membrane disrupting activities. Analysis of structural changes by circular dichroism spectroscopy demonstrated significant changes in the secondary structure only in the case of N-terminal octapeptide cleavage. These data indicate that PrTX-I and PrTX-III possess regions other than the catalytic site, which determine their toxic and pharmacological activities. (C) 2001 Academic Press.

AMINO-ACID-SEQUENCE OF TSTX-V, AN ALPHA-TOXIN FROM TITYUS-SERRULATUS SCORPION-VENOM, AND ITS EFFECT ON K+ PERMEABILITY OF BETA-CELLS FROM ISOLATED RAT ISLETS OF LANGERHANS

Highly purified Tityustoxin V (TsTX-V), an alpha-toxin isolated from the venom of the Brazilian scorpion Tityus serrulatus, was obtained by ion exchange chromatography on carboxymethylcellulose-52. It was shown to be homogeneous by reverse phase high performance liquid chromatography, N-terminal sequencing (first 39 residues) of the reduced and alkylated protein and by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and tricine. Following enzymatic digestion, the complete amino acid sequence (64 residues) was determined. The sequence showed higher homology with the toxins from the venoms of the North African than with those of the North and South American scorpions. Using the rate of Rb-86(+) release from depolarized rat pancreatic beta-cells as a measure of K+ permeability changes, TsTX-V (5.6 mu g/ml) was found to increase by 2.0-2.4-fold the rate of marker outflow in the presence of 8.3 mM glucose. This effect was persistent and slowly reversible, showing similarity to that induced by 100 mu-M veratridine, an agent that increases the open period of Na+ channels, delaying their inactivation. It is suggested that, by extending the depolarized period, TsTX-V indirectly affects beta-cell voltage-dependent K+ channels, thus increasing K+ permeability.

Structural and functional characterization of myotoxin I, a Lys49 phospholipase A(2) homologue from Bothrops moojeni (Caissaca) snake venom

Myotoxin-I (MjTX-I) was purified to homogeneity from the venom of Bothrops moojeni by ion-exchange chromatography on CM-Sepharose. Its molecular weight, estimated by SDS-PAGE, was 13,400 (reduced) or 26,000 (unreduced). The extinction coefficient (E-1.0 cm(1.0 mg/ml)) of MjTX-I was 1.145 at lambda = 278 nm, pH 7.0, and its isoelectric point was 8.2 at ionic strength mu = 0.1. When lyophilized and stored at 4 degrees C, dimeric, trimeric, and pentameric forms of the protein were identified by SDS-PAGE. This heterogeneous sample could be separated into three fractions by gel filtration on Sephadex 6-50. The fractions were analyzed by isoelectric focusing, immunoelectrophoresis, and amino acid composition, which indicated that heterogeneity was the result of different levels of self-association. Protein sequencing indicated that MjTX-I is a Lys49 myotoxin and consists of 121 amino acids (M-r = 13,669), containing a high proportion of basic and hydrophobic residues. It shares a high degree of sequence identity with other Lys49 PLA(2)-like myotoxins, but shows a significantly lower identity with catalytically active Asp49 PLA(2)s. The three-dimensional structure of MjTX-I was modeled based on the crystal structures of three highly homologous Lys49 PLA(2)-like myotoxins. This model showed that the amino acid substitutions are conservative, and mainly the beta-wing region, and the C-terminal extended random coil. MjTX-I displays local myotoxic and edema-inducing activities in mice, and is lethal by intraperitoneal injection, with an LD50 value of 8.5 +/- 0.8 mg/kg, In addition, it is cytotoxic to myoblasts/ myotubes in culture, and disrupts negatively charged liposomes. In comparison with the freshly prepared dimeric sample, the more aggregated forms showed significantly reduced myotoxic activity. However, the edema-inducing activity of MjTX-I was independent of molecular association. Phospholipase A(2) activity on egg yolk, as well as anticoagulant activity, were undetectable both in the native and in the more associated forms. His, Tyr, and Trp residues of the toxin were chemically modified by specific reagents. Although the myotoxic and lethal activities of the modified toxins were reduced by these treatments, neither its edema-inducing or Liposome-disrupting activities were significantly altered. Rabbit antibodies to native MjTX-I cross-reacted with the chemically modified forms, and both the native and modified MjTX-I preparations were recognized by antibodies against the C-terminal region 115-129 of myotoxin II from B. asper, a highly Lys49 PLA(2)-homologue with high sequencial similarity. (C) 2000 Academic Press.

Understanding the in vitro neuromuscular activity of snake venom Lys49 phospholipase A(2) homologues

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

Histopathological evaluation in experimental envenomation of dogs with Crotalus durissus terrificus Venom

The present work evaluated histopathological aspects in experimental envenomation of dogs with Crotalus durissus terrificus venom. Twenty-eight mixed breed adult dogs were divided into three groups of seven animals each: Group I only venom; Group II - venom + 50ml antiophidic serum + fluid therapy; Group III venom + 50ml antiophidic serum + fluid therapy + urine alkalization. Lyophilized venom of Crotalus durissus terrificus was reconstituted in saline solution and inoculated subcutaneously at the dose of 1mg/kg body weight. Three animals of each group were subjected to euthanasia, and their muscular tissue, brain, spleen, kidneys, heart, lungs, stomach, small and large intestines, and popliteal lymph node fragments were collected for histopathological evaluation. There was myonecrosis in the inoculated limb, renal tubular degeneration, lymphoid hyperplasia of spleen, and unspecific reactive hepatitis. These results show the antigenicity and action of the venom on the immune system.

Bioinsecticide action of the venom of Africanized bees Apis mellifera Linnaeus 1758 (Hymenoptera: Apidae): I - The most susceptible age of Diatraea saccharalis Fabricius 1794 (Lepeletier: Pyralidae) eggs to the venom action

The present paper aimed at testing the action of non-lyophilized venom of Africanized bees Apis mellifera through topical applications on Diatraea saccharalis egg masses. The CL50, DL50 and the most susceptible age of eggs to the venom topic application were also determined. Three-day-old eggs were the most susceptible to the venom action with CL50 equal to 8.6 mg/ml and DL50 equal to 0.173 mg/mass. The venom loses its action after being stored for 15 days.

Polybitoxins: A group of phospholipases A2 from the venom of the neotropical social wasp paulistinha (Polybia paulista)

The neotropical wasp Polybia paulista is very aggressive and endemic in south-east Brazil, where it frequently causes stinging accidents. By using gel filtration on Sephadex G-200, followed by ion-exchange chromatography on DEAE-Cellulose under a pH gradient, a group of four toxins (designated as polybitoxins-I, II, lII and IV) presenting phospholipase A2 (PLA2) activities was purified. These toxins are dimeric with mol. wts ranging from 115,000 to 132,000 and formed by different subunits. The four toxins contain very high sugar contents attached to their molecules (22-43% w/w) and presented different values of pH optimum from 7.8 to 9.0; when dissociated, only residual catalytic activities were maintained. The catalytic activities of polybitoxins (from 18 to 771 μmoles/mg per minute) are lower than that of PLA2 from Apis mellifera venom and hornetin from Vespa basalis. The polybitoxins presented a non-linear steady-state kinetic behavior for the hydrolysis of phosphatidylcholine at pH 7.9, compatible with the negative co- operativity phenomena. All of the polybitoxins were very potent direct hemolysins, especially the polybitoxins-III and IV, which are as potent as the lethal toxin from V. basalis and hornetin from Vespa flavitarsus, respectively; polybitoxin-IV presented hemolytic action 20 times higher than that of PLA2 from A. mellifera, 17 times higher than that of neutral PLA2 from Naja nigricolis and about 37 times higher than that of cardiotoxin from Naja naja atra venom.

Use of fibrin glue derived from snake venom in testicular biopsy of rams

Sequelae due to testicular biopsy such as hemorrhage, adhesion and fibrosis may be limiting factors to the use of this surgical procedure. Fibrin glue (FG) derived from snake venom was used to minimize these sequelae, as well as to evaluate its healing property in tunica vaginalis and scrotal skin of rams. Applicability of fibrin glue derived from snake venom was tested in different tissues of other animals such as in sciatic nerve and colon of rats and skin of rabbits. In the present study, 30 healthy adult rams were used. They were divided into 3 groups of 10 animals each as follows: G1: fibrin glue group (application of fibrin glue on puncture sites and skin incisions after bilateral testicular biopsy with a Tru-Cut needle); G2: swab/nylon group (hemostasis by compression with a swab on puncture sites and skin suturing with nylon after biopsy) and G3: control group (the animals were not subjected either to biopsy or to surgery). On the 20th day after biopsy, the presence of adhesion strands between the sites of skin incision and testicle was evaluated by palpation Adhesion strands were found in three testicles (15%) in G1 and in two testicles (10%) in G2. One hundred days after biopsy, orchiectomy was carried out and the material collected was assessed for subcutaneous (SC) and/or tunica vaginalis adhesions. G3 did not present any abnormality. Groups G1 and G2 presented four testicles each (20%) with adhesion between the tunics at biopsy site. On the other hand, subcutaneous adhesions were found once (5%) in G1 and three times (15%) in G2. Fibrin glue showed to be of easy application, required short postoperative monitoring, presented fast and good-quality healing property and tended to reduce formation of subcutaneous adhesion.

Detection and neutralization of venom by ovine antiserum in experimental envenoming by Bothrops jararaca

In this study we optimized an enzyme-linked immunosorbent assay (ELISA) to evaluate bothropic venom levels in biological samples. These samples were obtained by two distinct protocols. In the first one, Swiss mice were injected with 1 LD 50 of Bothrops jararaca (B. jararaca) venom and 15 minutes later, animals were treated with ovine antibothropic serum. Blood and spleen homogenate samples were obtained 6 hours after antiserum therapy. Ovine antibothropic serum significantly neutralized venom levels in serum and spleen. In the second protocol, BALB/c mice were injected with 1 LD 50 of bothropic venom by either intraperitoneal (IP) or intradermal (ID) route and venom levels were evaluated 1, 3 and 6 hours after, in blood, spleen homogenates and urine. Serum and splenic venom levels were significantly higher in animals envenomed by IP route comparing with animals envenomed by ID route. Higher venom levels were also detected in urine samples from animals envenomed by IP route. However, these differences were not statistically significant. These results demonstrated that the optimized ELISA was adequate to quantify venom levels in different biological samples. This assay could, therefore, substitute the in vivo neutralizing assay and also be useful to evaluate the severity of human and experimental envenomations.

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.