Insularis Venom Research Articles

Venom Composition in a Phenotypically Variable Pit Viper ( Trimeresurus insularis) across the Lesser Sunda Archipelago.

The genus Trimeresurus comprises a group of venomous pitvipers endemic to Southeast Asia and the Pacific Islands. Of these, Trimeresurus insularis, the White-lipped Island Pitviper, is a nocturnal, arboreal species that occurs on nearly every major island of the Lesser Sunda archipelago. In the current study, venom phenotypic characteristics of T. insularis sampled from eight Lesser Sunda Islands (Flores, Lembata, Lombok, Pantar, Sumba, Sumbawa, Timor, and Wetar) were evaluated via SDS-PAGE, enzymatic activity assays, fibrinogenolytic assays, gelatin zymography, and RP-HPLC, and the Sumbawa sample was characterized by venomic analysis. For additional comparative analyses, venoms were also examined from several species in the Trimeresurus complex, including T. borneensis, T. gramineus, T. puniceus, T. purpureomaculatus, T. stejnegeri, and Protobothrops flavoviridis. Despite the geographical isolation, T. insularis venoms from all eight islands demonstrated remarkable similarities in gel electrophoretic profiles and RP-HPLC patterns, and all populations had protein bands in the mass ranges of phosphodiesterases (PDE), l-amino acid oxidases (LAAO), P-III snake venom metalloproteinases (SVMP), serine proteases, cysteine-rich secretory proteins (CRISP), phospholipases A2 (PLA2), and C-type lectins. An exception was observed in the Lombok sample, which lacked protein bands in the mass range of serine protease and CRISP. Venomic analysis of the Sumbawa venom also identified these protein families, in addition to several proteins of lesser abundance (<1%), including glutaminyl cyclase, aminopeptidase, PLA2 inhibitor, phospholipase B, cobra venom factor, 5'-nucleotidase, vascular endothelial growth factor, and hyaluronidase. All T. insularis venoms exhibited similarities in thrombin-like and PDE activities, while significant differences were observed for LAAO, SVMP, and kallikrein-like activities, though these differences were only observed for a few islands. Slight but noticeable differences were also observed with fibrinogen and gelatin digestion activities. Trimeresurus insularis venoms exhibited overall similarity to the other Trimeresurus complex species examined, with the exception of P. flavoviridis venom, which showed the greatest overall differentiation. Western blot analysis revealed that all major T. insularis venom proteins were recognized by Green Pitviper ( T. albolabris) antivenom, and reactivity was also seen with most venom proteins of the other Trimeresurus species, but incomplete antivenom-venom recognition was observed against P. flavoviridis venom proteins. These results demonstrate significant conservation in the venom composition of T. insularis across the Lesser Sunda archipelago relative to the other Trimeresurus species examined.

Involvement of Nitric Oxide on Bothropoides insularis Venom Biological Effects on Murine Macrophages In Vitro.

Viperidae venom has several local and systemic effects, such as pain, edema, inflammation, kidney failure and coagulopathy. Additionally, bothropic venom and its isolated components directly interfere on cellular metabolism, causing alterations such as cell death and proliferation. Inflammatory cells are particularly involved in pathological envenomation mechanisms due to their capacity of releasing many mediators, such as nitric oxide (NO). NO has many effects on cell viability and it is associated to the development of inflammation and tissue damage caused by Bothrops and Bothropoides venom. Bothropoides insularis is a snake found only in Queimada Grande Island, which has markedly toxic venom. Thus, the aim of this work was to evaluate the biological effects of Bothropoides insularis venom (BiV) on RAW 264.7 cells and assess NO involvement. The venom was submitted to colorimetric assays to identify the presence of some enzymatic components. We observed that BiV induced H2O2 production and showed proteolytic and phospholipasic activities. RAW 264.7 murine macrophages were incubated with different concentrations of BiV and then cell viability was assessed by MTT reduction assay after 2, 6, 12 and 24 hours of incubation. A time- and concentration-dependent effect was observed, with a tendency to cell proliferation at lower BiV concentrations and cell death at higher concentrations. The cytotoxic effect was confirmed after lactate dehydrogenase (LDH) measurement in the supernatant from the experimental groups. Flow cytometry analyses revealed that necrosis is the main cell death pathway caused by BiV. Also, BiV induced NO release. The inhibition of both proliferative and cytotoxic effects with L-NAME were demonstrated, indicating that NO is important for these effects. Finally, BiV induced an increase in iNOS expression. Altogether, these results demonstrate that B. insularis venom have proliferative and cytotoxic effects on macrophages, with necrosis participation. We also suggest that BiV acts by inducing iNOS expression and causing NO release.

Bothropoides insularis venom cytotoxicity in renal tubular epithelia cells

Bothropoides insularis (jararaca-ilhoa) is a native endemic snake limited to the specific region of Queimada Island, on São Paulo coast. Several local and systemic effects have been described due to envenomation caused by it, such as edema, tissue necrosis, hemorrhage and acute renal failure. Our previous studies have shown that Bothropoides insularis venom (BinsV) demonstrated important functional and morphologic alterations in rat isolated kidney, especially decrease in tubular electrolyte transport, osmotic clearance and tubular necrosis. In order to elucidate the direct nephrotoxicity mechanism, the aim of the present study was to investigate BinsV cytotoxicity effect on renal epithelial cells. The treatment with BinsV over MDCK culture decreased cell viability in all concentrations tested with IC50 of 9 μg/mL. BinsV was able to induce membrane rupture and cell death with phosphatidilserine externalization. Furthermore, BinsV induced ROS overproduction and mitochondrial membrane potential collapse, as well as Bax translocation and caspases 3 and 7 expression. Therefore, these events might be responsible by BinsV-induced cell death caused by mitochondrial dysfunction and ROS overproduction in the direct cytotoxicity process.

“Insularin, a disintegrin from Bothrops insularis venom: Inhibition of platelet aggregation and endothelial cell adhesion by the native and recombinant GST-insularin proteins”

Insularin (INS) was obtained from Bothrops insularis venom by reversed-phase highperformance liquid chromatography using a C18 column and characterized as a disintegrin by peptide mass fingerprint and inhibition of ADP-induced platelet aggregation. A cDNA coding for P-II a metalloproteinase/disintegrin was cloned from a cDNA library from B. insularis venom glands. The deduced protein sequence possesses 73 amino acid residues, including the N-terminal, internal peptides of native insularin, the ARGDNP-sequence and 12 cysteines in a conserved alignment. This cDNA fragment was subcloned in the pGEX-4T-1 vector and expressed in a prokaryotic expression system as a fusion protein with glutathione S-transferase (GST-INS). Both native and recombinant insularin inhibited ADP-induced platelet aggregation and endothelial cells (HUVEC) adhesion with similar activities indicating that GST-INS folded correctly and preserved the integrin-binding loop. Insularin may be a tool in studies that involve platelets and endothelial cell adhesion dependent on alphaIIbeta3 and alphavbeta3 integrins.

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 α and β. 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.

Purification and biological effects of l-amino acid oxidase isolated from Bothrops insularis venom

Bothrops insularis is a snake from Ilha da Queimada Grande, an island located about 20 miles away from the Southeastern coast of Brazil. Compared with other Brazilian species of Bothrops, the toxinology of B. insularis is still poorly understood, and so far, no fraction from this venom with amino acid oxidase activity had been isolated or its biological activity tested. We investigated the biochemical and biological effects of one l-amino acid oxidase enzyme isolated from B. insularis snake venom (BiLAO), which was purified using HPLC and sequence grade. We also evaluated the renal effects induced by BiLAO. Chromatographic profile of B. insularis whole venom disclosed seven main fractions (I, II, III, IV, V, VI and VII) and the main LAO enzymatic activity was detected in fraction II. The group treated with BiLAO showed a decrease in perfusion pressure ( C 120=110.28±3.69; BiLAO 120=82.2±5.6 mmHg*); renal vascular resistance ( C 120=5.48±0.53; BiLAO 120=4.12±0.42 mmHg/mL/g/min*), urinary flow ( C 120=0.160±0.020; BiLAO 120=0.064±0.012 mL/g/min*), glomerular filtration rate ( C 120=0.697±0.084; BiLAO 120=0.176±0.017 mL/g/min*), sodium ( C 120=79.76±0.56; BiLAO 120=65.39±6.19%*), potassium ( C 120=69.94±6.86; BiLAO 120=60.26±2.24%*) and chloride tubular reabsortion ( C 120=78.53±2.33; BiLAO 120=64.58±6.68%*). Acute tubular necrosis foci were observed in the group treated with the LAO fraction of the B. insularis snake venom. Some findings have the same morphological aspect of apoptosis, more evident cortically; otherwise, reversible degenerative phenomena represented by hydropic ballooning with extensive cytoplasmic vacuolization and discontinuity of the cell brush borders in the proximal tubular epithelium were observed; furthermore, necrotic detachment of these cells into the tubular lumina, and increased amount of protein deposits in the distal and proximal tubules were observed. In conclusion, the slowness of blood flow and of glomerular filtration resulted in more time for filtration and tubular reabsorption, with elevation of the total percentage of sodium and chlorine reabsorption. The maintenance of the decrease in glomerular filtration rate would determine the subsequent decreases, which were noticed in these parameters. The necrosis observed was the result of damage cell induced by l-amino acid oxidase isolated from B. insularis venom.

Purification and renal effects of phospholipase A 2 isolated from Bothrops insularis venom

Bothrops insularis venom contains a variety of substances presumably responsible for several pharmacological effects. We investigated the biochemical and biological effects of phospholipase A 2 protein isolated from B. insularis venom and the chromatographic profile showed 7 main fractions and the main phospholipase A 2 (PLA 2) enzymatic activity was detected in fractions IV and V. Fraction IV was submitted to a new chromatographic procedure on ion exchange chromatography, which allowed the elution of 5 main fractions designated as IV-1 to IV-5, from which IV-4 constituted the main fraction. The molecular homogeneity of this fraction was characterized by high-performance liquid chromatography (HPLC) and demonstrated by mass spectrometry (MS), which showed a molecular mass of 13984.20 Da; its N-terminal sequence presented a high amino acid identity (up to 95%) with the PLA 2 of Bothrops jararaca and Bothrops asper. Phospholipase A 2 isolated from B. insularis (Bi PLA 2 ) venom (10 μg/mL) was also studied as to its effect on the renal function of isolated perfused kidneys of Wistar rats ( n=6). Bi PLA 2 increased perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF) and glomerular filtration rate (GFR). Sodium (%TNa +) and chloride tubular reabsorption (%TCl −) decreased at 120 min, without alteration in potassium transport. In conclusion, PLA 2 isolated from B. insularis venom promoted renal alterations in the isolated perfused rat kidney.

Enzymatic and immunochemical characterization of Bothrops insularis venom and its neutralization by polyspecific Bothrops antivenom

Herein we compared the biological activities of Bothrops insularis and Bothrops jararaca venoms as well as their neutralization by polyspecific Bothrops antivenom (PBA). On account of that, we investigated their antigenic cross-reactivity and the neutralization of lethal, myotoxic and defibrinating activities by polyspecific and species-specific antivenoms. Silver-stained SDS-PAGE gels evidenced many common bands particularly above 47 kDa between B. jararaca and B. insularis venoms. However, some protein bands between 46 and 28 kDa were observed exclusively in B. jararaca venom. Both venoms presented gelatinolytic, caseinolytic, fibrinogenolytic and phospholipase A 2 activities. No hyaluronidase activity was detected in both venoms by zymography. Polyspecific and species-specific antivenoms showed similar titers to B. jararaca and B. insularis venoms by ELISA, and recognized similar components by immunoblotting. The PBA was effective in neutralizing the lethal, myotoxic and defibrinating activities of both venoms as well as to abrogate microcirculatory disturbances induced by B. insularis venom. No statistically significant difference was observed for minimal hemorrhagic doses between both venoms. Antigenic cross-reactivity was evident between both venoms. Since toxic and enzymatic activities were similar, we speculate that B. insularis venoms can induce a local damage in humans comparable to that observed in other Bothrops venoms. Besides, the PBA was effective in neutralizing the toxic activities of B. insularis venom.

Purification, sequencing and structural analysis of two acidic phospholipases A 2 from the venom of Bothrops insularis (jararaca ilhoa)

Bothrops snake venoms contain a variety of phospholipases (PLA 2), some of which are myotoxic. In this work, we used reverse-phase HPLC and mass spectrometry to purify and sequence two PLA 2 from the venom of Bothrops insularis. The two enzymes, designated here as BinTX-I and BinTx-II, were acidic (p I 5.05 and 4.49) Asp49 PLA 2, with molecular masses of 13,975 and 13,788, respectively. The amino acid sequence and molecular mass of BinTX-I were identical to those of a PLA 2 previously isolated from this venom (PA2_BOTIN, SwissProt accession number Q8QG87) while those of BinTX-II indicated that this was a new enzyme. Multiple sequence alignments with other Bothrops PLA 2 showed that the amino acids His48, Asp49, Tyr52 and Asp99, which are important for enzymatic activity, were fully conserved, as were the 14 cysteine residues involved in disulfide bond formation, in addition to various other residues. A phylogenetic analysis showed that BinTX-I and BinTX-II grouped with other acidic Asp49 PLA 2 from Bothrops venoms, and computer modeling indicated that these enzymes had the characteristic structure of bothropic PLA 2 that consisted of three α-helices, a β-wing, a short helix and a calcium-binding loop. BinTX-I (30 μg/paw) produced mouse hind paw edema that was maximal after 1 h compared to after 3 h with venom (10 and 100 μg/paw); in both cases, the edema decreased after 6 h. BinTX-1 and venom (40 μg/ml each) produced time-dependent neuromuscular blockade in chick biventer cervicis preparations that reached 40% and 95%, respectively, after 120 min. BinTX-I also produced muscle fiber damage and an elevation in CK, as also seen with venom. These results indicate that BinTX-I contributes to the neuromuscular activity and tissue damage caused by B. insularis venom in vitro and in vivo.

Purification and biological activity of the thrombin-like substance isolated from Bothrops insularis venom

The venom of Bothrops insularis snake, known in Brazil as jararaca ilhoa, contains a variety of proteolytic enzymes such as a thrombin-like substance that is responsible for various pharmacological effects. B. insularis venom chromatography profile showed an elution of seven main fractions. The thrombin-like activity was detected in fractions I and III, the latter being subjected to two other chromatographic procedures, so to say DEAE and Hi Trap Benzamidine. The purity degree of this fraction was confirmed by analytical reverse phase HPLC, which displayed only one main fraction confirmed by SDS-PAGE constituting fraction III. About 5 μg of fraction III protein potentiated the secretion of insulin induced by 2.8 mM of glucose in rats isolated pancreatic β-cells treated; the increase being around 3-fold higher than its respective control. B. insularis lectin (BiLec; 10 μg/mL) was also studied as to its effect on the renal function of isolated perfused rat kidneys with the use of six Wistar rats. BiLec increased perfusion pressure (PP), renal vascular resistence (RVR), urinary flow (UF) and glomerular filtration rate (GFR). Sodium (%TNa +) and chloride tubular reabsorption (%TCl −) decreased at 120 min, without alteration in potassium transport. In conclusion, the thrombin-like substance isolated from B. insularis venom induced an increase in insulin secretion, in vitro, and transiently altered vascular, glomerular and tubular parameters in the isolated rat kidney.

Bothrops jararaca venom gland transcriptome: Analysis of the gene expression pattern

Bothrops jararaca is a pit viper responsible for the majority of snake envenoming accidents in Brazil. As an attempt to describe the transcriptional activity of the venom gland, ESTs of a cDNA library constructed from B. jararaca venom gland were generated and submitted to bioinformatics analysis. The results showed a clear predominance of transcripts coding for toxins instead of transcripts coding for proteins involved in cellular functions. Among toxins, the most frequent transcripts were from metalloproteinases (52.6%), followed by serine-proteinases (28.5%), C-type lectins (8.3%) and bradykinin-potentiating peptides (BPPs) (6.2%). Results were similar to that obtained from the transcriptome analysis of B. insularis, a phylogenetically close sister of B. jararaca, though some differences were observed and are pointed out, such as a higher amount of the hypotensive BPPs in B. insularis transcriptome (19.7%). Another striking difference observed is that PIII and PII-classes of metalloproteinases are similarly represented in B. jararaca in contrast to B. insularis, in which a predominance of PIII-class metalloproteinase, which present a more intense hemorrhagic action, is observed. These features may, in part, explain the higher potency of B. insularis venom. The results obtained can help in proteome studies, and the clones can be used to directly probe the genetic material from other snake species or to investigate differences in gene expression pattern in response to factors such as diet, aging and geographic localization.

Cloning, characterization, and structural analysis of a C-type lectin from Bothrops insularis (BiL) venom

Lectins are carbohydrate-binding molecules that mediate a variety of biological processes. In this work, we identify and characterize a lectin from Bothrops insularis venom, with respect to its biochemical properties and theoretical structure. Initially, from a venom gland cDNA library, we cloned and sequenced a cDNA encoding a protein with high identity to snake venom lectins. A lectin molecule was purified to homogeneity from the venom by affinity column and gel filtration. This protein named BiL displayed hemagglutinating activity that was inhibited by galactose, lactose, and EDTA. Mass spectrometry analysis and sodium dodecyl sulfate–polyacrylamide gel electrophoresis showed that BiL is a disulfide-linked dimeric protein consisting of monomers with 16,206 m/ z. The amino acid sequence, deduced from its cDNA sequence, was confirmed by Edman sequencing and by peptide mass fingerprint analysis. BiL shows similarity to other C-type lectin family members. Modeling studies provide insights into BiL dimeric structure and its structural determinants for carbohydrate and calcium binding.

Pharmacological characterization of mouse hind paw oedema induced by Bothrops insularis (jararaca ilhoa) snake venom

Bothrops snake venoms produce marked local effects, including oedema, haemorrhage and necrosis. The ability of Bothrops insularis venom to induce oedema in mice was investigated. Venom was injected into hind paws and the change in volume over time was measured by plethysmometry. B. insularis venom (0.01–2.5 μg/paw) induced paw oedema which, at high doses (≥0.5 μg/paw), was accompanied by haemorrhage. The peak oedematogenic response occurred 3 h after venom injection with all doses and decreased gradually thereafter, but was still elevated with high doses after 24 h. Pretreating the mice with cyproheptadine (histamine H1 and serotonin 5-HT2 receptor antagonist), mepyramine (histamine H1 receptor antagonist), l-NAME (inhibitor of nitric oxide synthase), indomethacin and rofecoxib (inhibitors of cyclooxygenases), and dexamethasone (indirect inhibitor of PLA2) significantly attenuated venom-induced oedema, whereas methysergide, a serotonin 5-HT1/5-HT2 receptor antagonist, had no effect. The administration of antivenom 30 min before or immediately after venom injection also significantly inhibited venom-induced oedema. These results show that B. insularis venom causes oedema in the mouse hind paw and that this response is mediated by histamine, nitric oxide, and arachidonic acid metabolites formed by cyclooxygenases 1 and 2. The neutralization by commercial antivenom indicates that the venom components responsible for oedema are recognized by the antivenom and share immunological identity with their counterparts in the venoms of mainland Bothrops species.

Molecular Cloning and Expression of a Functional Snake Venom Vascular Endothelium Growth Factor (VEGF) from theBothrops insularis Pit Viper: A NEW MEMBER OF THE VEGF FAMILY OF PROTEINS

During the generation of abundant expressed sequence tags from the Viperidae snake Bothrops insularis venom glands, we identified for the first time a cDNA coding for a putative vascular endothelial growth factor-like (VEGF-like) protein. The deduced primary sequence, after complete sequencing of the longest snake venom VEGF (svVEGF) cDNA, displayed similarity with vertebrate VEGFs and with the hypotensive factor from Vipera aspis venom. Its cDNA was subcloned, expressed in Escherichia coli with a His(6) tag as an insoluble monomer, and purified by Ni(2+)-affinity chromatography after 8 m urea extraction. Antiserum against svVEGF was generated and tested in Western blot against proteins from snake venoms and cellular extracts. The mature svVEGF appears to be ubiquitously distributed throughout snake venoms and was also confirmed by Northern blot studies of other related Viperidae species and by cDNA cloning of svVEGF from Bothrops jararaca pit viper. The produced recombinant protein dimerizes after refolding processes and was biologically characterized, showing ability to increase vascular permeability. These results established that svVEGF is a novel and important active toxin during the early stages of bothropic snake bite envenoming and represents a new member of the VEGF family of proteins.

Histopathological changes in avian kidney caused by Bothrops insularis (jararaca ilh a) venom and a phospholipase A2-containing fraction.

The histopathological changes induced in avian kidney by the intramuscular injection of Bothrops insularis (jararaca ilh a) venom and its phospholipase A2 (PLA2)-containing fraction were examined. Acute experiments (3 h and 24 h) with B. insularis crude venom (20 microg and 80 microg) or its PLA2-contaning fraction (10 microg and 40 microg) resulted in significant structural damage to the kidneys of 5-12-day-old chicks. Histopathological analysis indicated that the venom and its fraction acted on the renal tubules and glomeruli. The morphological changes, although widespread, varied in intensity from cell to cell, and from tubule to tubule in venom-injected chicks. The tubular and glomerular changes produced by the venom and its PLA2-containing fraction may be the result of a direct cytotoxic effect potentiated by ischemia-related disturbances in the regional hemodynamics. The venom and its fraction affected more segments along reptilian-type nephrons than along mammalian ones. This divergent sensitivity to the venom and its fraction may reflect the species-specific characteristics of B. insularis snake, an example of geographical isolation influencing its diet which is almost exclusively avian.

Effect of Bothrops insularis venom on the mouse and chick nerve-muscle preparation

J. C. Cogo, J. Prado-Franceschi, M. A. Cruz-Hofling, A. P. Corrado and L. Rodrigues-Simioni. Effect of Bothrops insularis venom on the mouse and chick nerve-muscle preparation. Toxicon 31, 1237–1247, 1993.—The effects of Bothrops insularis venom were examined in vivo in mice and chicks and in vitro using the mouse phrenic nerve diaphragm and chick biventer cervicis muscle preparations. Incubation of the indirectly or directly stimulated mouse preparation with B. insularis venom (20–80 μg/ml) produced an initial increase in twitch tension followed by irreversible blockade. With direct stimulation in the presence of d-tubocurarine, no increase in twitch tension was observed prior to the onset of blockade. A venom-induced effect on presynaptic activity was suggested by the marked increase in the frequency of the mepps recorded in vitro 5–15 min after venom addition. A direct muscular effect was shown by the dose- and time-dependent reduction in the resting membrane potential of the diaphragm. Chick preparations were more sensitive than those of the mouse. In the isolated chick biventer cervicis muscle preparation, B. insularis venom induced a contracture and a dose-dependent block of responses to indirect stimulation. At low venom concentrations (1–5 μg/ml), no significant release of creatine kinase (CK) was observed from this preparation. However, a dose-dependent release of CK was detected at higher doses (10–80 μg/ml). For morphological studies, B. insularis venom was injected into the chick left pectoralis muscle. At low doses (0.4 μg), only an inflammatory reaction was present, while at high doses (20–80 μg) increasing numbers of necrotic fibers were observed as well as occlusive thrombosis and hemorrhage. The muscular effect, also observed on the incubated muscle, points to a direct myolytic action of the whole venom.

Morphological effects of B. insularis venom on chick pectoral muscle: Maria Alice Cruz-Hofling, 1José C. Cogo, 2Léa Rodrigues-Simioni2 and Júlia Prado-Franceschi2 ( 1Dept Microscopia Eletrônica, I.B./UNICAMP and 2Dept Farmacologia, FCM/UNICAMP, Campinas-SP., Brazil)

Morphological effects of B. insularis venom on chick pectoral muscle: Maria Alice Cruz-Hofling, 1José C. Cogo, 2Léa Rodrigues-Simioni2 and Júlia Prado-Franceschi2 ( 1Dept Microscopia Eletrônica, I.B./UNICAMP and 2Dept Farmacologia, FCM/UNICAMP, Campinas-SP., Brazil)

The effects of B. insularis venom on isolated nerve muscle preparations: José Carlos Cogo, Léa Rodrigues-Simioni, Júlia Prado-Franceschi (Departmento de Farmacologia, FCM/UNICAMP, P.O. Box 6111, 13081-Campinas-SP, Brazil)

The effects of B. insularis venom on isolated nerve muscle preparations: José Carlos Cogo, Léa Rodrigues-Simioni, Júlia Prado-Franceschi (Departmento de Farmacologia, FCM/UNICAMP, P.O. Box 6111, 13081-Campinas-SP, Brazil)