Triple bothropic envenomation in horses caused by a single snake

In the present study, snakebites caused by a single Bothrops pauloensis simultaneously affecting three male adult horses are described. Whereas the first two affected horses were six years old, the third was 16 years old; they weighed respectively 555, 550 and 500 kg. All horses presented swollen muzzles. The first animal received an initial antivenom dose of 19 vials and adjuvant medication, it was also subjected to tracheostomy due to the progression of acute respiratory failure. The second and third horses respectively received 15 vials of antivenom each, in addition to adjuvant medication. Due to continuous changes in blood coagulation observed during hospitalization, the first and the second horses received five more vials of antivenom, respectively, in the second and third days of treatment. The first animal was discharged on 15(th) day and the other on the 8(th) day of hospitalization. Several factors including the main actions of Bothrops venom (coagulation, proteolytic and vasculotoxic activities), the importance of early diagnosis based on clinical history and clinical examination suggesting the diagnosis of Bothrops snakebite, the adequate doses of antivenom, and finally the immediate tracheostomy are herein discussed. The tracheostomy, required to save the life of the first horse, should only occur after the administration of antivenom and control measures for changes in blood coagulation.

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.

Determination of snake-necked turtle Hydromedusa tectifera (Cope, 1870) (Testudines: Chelidae) plasma protein concentrations by refractometry and the biuret method.

The purpose of this study was to evaluate the accuracy of hand-held refractometer in determining plasma protein concentrations in South American snake-necked turtle (Hydromedusa tectifera) as compared with the standard biuret method. The results indicated that plasma protein values may be accurately determined in snake- necked turtle with a hand-held refractometer.

Neutralization of snake venom phospholipase A(2) toxins by aqueous extract of Casearia sylvestris (Flacourtiaceae) in mouse neuromuscular preparation

Aqueous extract of Casearia sylvestris (Flacourtiaceae) has been shown to inhibit enzymatic and biological properties of some Bothrops and Crotalus venoms and their purified phospholipase A(2) (PLA(2)) toxins. In this work we evaluated the influence of C sylvestris aqueous extract upon neuromuscular blocking and muscle damaging activities of some PLA(2)S (crotoxin from C. durissus terrificus, bothropstoxin-I from B.jararacussu, piratoxin-I from B. pirajai and myotoxin-II from B. moojeni) in mouse phrenic-diaphragm preparations. Crotoxin (0.5 mu M) and all other PLA2 toxins (1.0 mu M) induced irreversible and time-dependent blockade of twitches. Except for crotoxin, all PLA2 toxins induced significant muscle damage indices, assessed by microscopic analysis. Preincubation of bothropstoxin-I, piratoxin-I or myotoxin-II with C. sylvestris extract (1:5 (w/w), 30 min, 37 degrees C significantly prevented the neuromuscular blockade of preparations exposed to the mixtures for 90 min; the extent of protection ranged from 93% to 97%. The vegetal extract also neutralized the muscle damage (protection of 80-95%). Higher concentration of the C. sylvestris extract (1: 10, w/w) was necessary to neutralize by 90% the neuromuscular blockade induced by crotoxin. These findings expanded the spectrum of C. sylvestris antivenom activities, evidencing that it may be a good source of potentially useful PLA2 inhibitors. (c) 2007 Elsevier B.V.. All rights reserved.

Bothrops moojeni myotoxin-II, a Lys49-phospholipase A(2) homologue: An example of function versatility of snake venom proteins

MjTX-II, a myotoxic phospholipase A(2) (PLA(2)) homologue from Bothrops moojeni venom, was functionally and structurally characterized. The MjTX-II characterization included: (i) functional characterization (antitumoral, antimicrobial and antiparasitic effects); (ii) effects of structural modifications by 4-bromophenacyl bromide (BPB), cyanogen bromide (CNBr), acetic anhydride and 2-nitrobenzenesulphonyl fluoride (NBSF); (iii) enzymatic characterization: inhibition by low molecular weight heparin and EDTA; and (iv) molecular characterization: cDNA sequence and molecular structure prediction. The results demonstrated that MjTX-II displayed antimicrobial activity by growth inhibition against Escherichia coli and Candida albicans, antitumoral activity against Erlich ascitic tumor (EAT), human breast adenocarcinoma (SK-BR-3) and human T leukemia cells (JURKAT) and antiparasitic effects against Schistosoma mansoni and Leishmania spp., which makes MjTX-II a promising molecular model for future therapeutic applications, as well as other multifunctional homologous Lys49-PLA(2)S or even derived peptides. This work provides useful insights into the structural determinants of the action of Lys49-PLA2 homologues and, together with additional strategies, supports the concept of the presence of others bioactive sites distinct from the catalytic site in snake venom myotoxic PLA(2)s. (c) 2005 Elsevier B.V. All rights reserved.

Molecular characterization and phylogenetic analysis of JussuMP-I: A RGD-P-III class hemorrhagic metalloprotease from Bothrops jararacussu snake venom

Snake venom metalloproteases (SVMPs) embody zinc-dependent multidomain enzymes responsible for a relevant pathophysiology in envenomation. including local and systemic hemorrhage. The molecular features responsible for hemorrhagic potency of SVMPs have been associated with their multidomains structures which can target these proteins them to several receptors of different tissues and cellular types. BjussuMP-I. a SVMP isolated from the Bothrops jararacussu venom, has been characterized as a P-III hemorrhagic metalloprotease. The complete cDNA sequence of BjussuMP-I with 1641bp encodes open reading frames of 547 amino acid residues, which conserve the common domains of P-III high molecular weight hemorrhagic metalloproteases: (i) pre-pro-peptide, (ii) metalloprotease, (iii) disintegrin-like and (iv) rich cysteine domain. BjussuMP-I induced lyses in fibrin clots and inhibited collagen- and ADP-induced platelet aggregation. We are reporting, for the first time, the primary structure of an RGD-P-III class snake venom metalloprotease. A phylogenetic analysis of the BjussuMP-1 metalloprotease/catalytic domain was performed to get new insights into the molecular evolution of the metalloproteases. A theoretical molecular model of this domain was built through folding recognition (threading) techniques and refined by molecular dynamics simulation. Then, the final BjussuMP-I catalytic domain model was compared to other SVMPs and Reprolysin family proteins in order to identify eventual structural differences, which could help to understand the biochemical activities of these enzymes. The presence of large hydrophobic areas and some conserved surface charge-positive residues were identified as important features of the SVMPs and other metalloproteases. (C) 2006 Elsevier B.V. All rights reserved.

Phospholipase A(2) myotoxins from Bothrops snake venoms: Structure-function relationship

Phospholipases A(2) constitute the major components from Bothrops snake venoms and have been extensively investigated not only because they are relatively very abundant in these venoms but mainly because they display a range of many relevant biological effects, including: myotoxic, cytotoxic, edema-inducing, artificial membrane disrupting, anticoagulant, neuromuscular, platelet aggregation inhibiting, hypotensive, bactericidal, anti-HIV, anti-tumoural, anti-malarial and anti-parasitic. The primary structures of several PLA(2)s have been elucidated through direct amino acid sequencing or, inderectly, through the corresponding nucleotide sequencing. Two main subgroups were thus described: (i) Asp49 PLA(2)s, showing low (basic, highly myotoxic) to relatively high (acidic, less or non myotoxic) Ca++-dependent hydrolytic activity upon artificial substrates; (ii) Lys49 PLA(2)s (basic, highly myotoxic) , showing no detectable hydrolytic activity on artificial substrates. Several crystal structures of Lys49 PLAs from genus Bothrops have already been solved, revealing very similar fold patterns. Lack of catalytic activity of myotoxic Lys49-PLA(2)s, first related solely with the fact that Lys49 occupies the position of the calcium ion in the catalyticly active site of Asp49 PLA(2)s, is now also attributed to Lys122 which interacts with the carbonyl of Cys29 hyperpolarising the peptide bond between Cys29 and Gly30 and trapping the fatty acid product in the active site, thus interrupting the catalytic cycle. This hypothesis, supported for three recent structures, is also discussed here. All Asp49 myotoxins showed to be pharmacologically more potent when compared with the Lys49 variants, but phospholipid hydrolysis is not an indispensable condition for the myotoxic, cytotoxic, bactericidal, anti-HIV, anti-parasitic, liposome disrupting or edema-inducing activities. Recent studies on site directed mutagenesis of the recombinant Lys49 myotoxin from Bothrops jararacussu revealed the participation of important amino acid residues in the membrane damaging and myotoxic activities.

Rosmarinic acid, a new snake venom phospholipase A(2) inhibitor from Cordia verbenacea (Boraginaceae): antiserum action potentiation and molecular interaction

Many plants are used in traditional medicine as active agents against various effects induced by snakebite. The methanolic extract from Cordia verbenacea (Cv) significantly inhibited paw edema induced by Bothrops jararacussu snake venom and by its main basic phospholipase A(2) homologs, namely bothropstoxins I and II (BthTXs). The active component was isolated by chromatography on Sephadex LH-20 and by RP-HPLC on a C18 column and identified as rosmarinic acid (Cv-RA). Rosmarinic acid is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid [2-O-cafeoil-3-(3,4-di-hydroxy-phenyl)-R-lactic acid]. This is the first report of RA in the species C. verbenacea ('baleeira', 'whaler') and of its anti-inflammatory and antimyotoxic properties against snake venoms and isolated toxins. RA inhibited the edema and myotoxic activity induced by the basic PLA(2)s BthTX-I and BthTX-II. It was, however, less efficient to inhibit the PLA(2) activity of BthTX-II and, still less, the PLA(2) and edema-inducing activities of the acidic isoform BthA-1-PLA(2), from the same venom, showing therefore a higher inhibitory activity upon basic PLA(2)s. RA also inhibited most of the myotoxic and partially the edema-inducing effects of both basic PLA(2)s, thus reinforcing the idea of dissociation between the catalytic and pharmacological domains. The pure compound potentiated the ability of the commercial equine polyvalent antivenom in neutralizing lethal and myotoxic effects of the crude venom and of isolated PLA(2)s in experimental models. CD data presented here suggest that, after binding, no significant conformation changes occur either in the Cv-RA or in the target PLA(2). A possible model for the interaction of rosmarinic acid with Lys49-PLA(2) BthTX-I is proposed. (c) 2005 Elsevier Ltd. All rights reserved.

Molecular and functional characterization of a new non-hemorrhagic metalloprotease from Bothrops jararacussu snake venom with antiplatelet activity

BjussuMP-II is an acidic low molecular weight metalloprotease (Mr similar to 24,000 and pI similar to 6.5), isolated from Bothrops jararacussu snake venom. The chromatographic profile in RP-HPLC and its N-terminal sequence confirmed its high purity level. Its complete cDNA was obtained by RT-PCR and the 615 bp codified for a mature protein of 205 amino acid residues. The multiple alignment of its deduced amino acid sequence and those of other snake venom metalloproteases showed a high structural similarity, mainly among class P-I proteases. The molecular modeling analysis of BjussuMP-II showed also conserved structural features with other SVMPs. BjussuMP-II did not induce hemorrhage, myotoxicity and lethality, but displayed dose-dependent proteolytic activity on fibrinogen, collagen, fibrin, casein and gelatin, keeping stable at different pHs, temperatures and presence of several divalent ions. BjussuMP-II did not show any clotting or anticoagulant activity on human citrated plasma, in contrast to its inhibitory effects on platelet aggregation. The aspects broached, in this work, provide data on the relationship between structure and function, in order to better understand the effects elicited by snake venom metalloproteases. (c) 2007 Elsevier B.V. All rights reserved.

Geographic variations in the composition of myotoxins from Bothrops neuwiedi snake venoms: biochemical characterization and biological activity

(1) Venom pools from Bothrops neuwiedi (Bn) and from two subspecies, namely Bothrops neuwiedi pauloensis (Bnp) and Bothrops neuwiedi urutu (Bnu), collected in the States of São Paulo (SP) and Minas Gerais (MG), Brazil, were electrophoretically examined. Basic toxins with different isoelectric points were identified in the venom collected in São Paulo (BnSP). These toxins were absent in the corresponding pools from Minas Gerais (BnMG, BnpMG and BnuMG). (2) BnSP, but not BnMG, BnpMG or BnuMG, showed two myotoxins (pI congruent to 8.6 and 8.8, respectively) which were isolated by ion-exchange chromatography on CM-Sepharose. (3) From BnMG, three myotoxic isoforms (pI congruent to 8.2 and M-r = 13600) were isolated by chromatography on CM-Sepharose followed by reversed-phase high-performance liquid chromatography. (4) the chemical and biological characterization of these toxins showed a high similarity with the Lys-49 myotoxins from other bothropic venoms. (5) Doses up to 5 LD50 (i.p.) of p-bromophenacyl bromide alkylated BnSP-7 caused a total loss of lethality in 18-22-g mice, thus indicating that the LD50 was increased by greater than 5-fold. At this dose myotoxicity was also not detectable, but the edematogenic activity on the rat paw apparently did not change. (C) 1998 Elsevier B.V. All rights reserved.

Structural and functional characterization of an acidic platelet aggregation inhibitor and hypotensive phospholipase A(2) from Bothrops jararacussu snake venom

An acidic (pI similar to 4.5) phospholipase A(2) (BthA-I-PLA(2)) was isolated from Bothrops jararacussu snake venom by ion-exchange chromatography on a CM-Sepharose column followed by reverse phase chromatography on an RP-HPLC C-18 column. It is an similar to13.7 kDa single chain Asp49 PLA(2) with approximately 122 amino acid residues, 7 disulfide bridges, and the following N-terminal sequence: 'SLWQFGKMINYVMJGESGVLQYLSYGCYCGLGGQGQPTDATDRCCFVHDCC(51). Crystals of this acidic protein diffracted beyond 2.0 Angstrom resolution. These crystals are monoclinic and have unit cell dimensions of a = 33.9, b = 63.8, c = 49.1 Angstrom, and beta = 104.0degrees. Although not myotoxic, cytotoxic, or lethal, the protein was catalytically 3-4 tithes more active than BthTX-II, a basic D49 myotoxic PLA(2) from the same venom and other Bothrops venoms. Although it showed no toxic activity, it was able to induce time-independent edema, this activity being inhibited by EDTA. In addition, BthA-I-PLA(2) caused a hypotensive response in the rat and inhibited platelet aggregation, Catalytic, antiplatelet and other activities were abolished by chemical modification with 4-bromophenacyl bromide, which is known to covalently bind to His48 of the catalytic site. Antibodies raised against crude B. jararacussu venom recognized this acidic PLA(2), while anti-Asp49-BthTX-II recognized it weakly and anti-Lys49-BthTX-I showed the least cross-reaction. These data confirm that myotoxicity does not necessarily correlate with catalytic activity in native PLA(2) homologues and that either of these two activities may exist alone. BthA-I-PLA(2), in addition to representing a relevant molecular model of catalytic activity, is also a promising hypotensive agent and platelet aggregation inhibitor for further studies. (C) 2002 Elsevier B.V. All rights reserved.

Snake venom phospholipase A(2) inhibitors: Medicinal chemistry and therapeutic potential

Phospholipases A(2) (PLA(2)s) are commonly found in snake venoms from Viperidae, Hydrophidae and Elaphidae families and have been extensively studied due to their pharmacological and physiopathological effects in living organisms. This article reports a review on natural and artificial inhibitors of enzymatic, toxic and pharmacological effects induced by snake venom PLA(2)s. These inhibitors act on PLA(2)S through different mechanisms, most of them still not completely understood, including binding to specific domains, denaturation, modification of specific amino acid residues and others. Several substances have been evaluated regarding their effects against snake venoms and isolated toxins, including plant extracts and compounds from marine animals, mammals and snakes serum plasma, in addition to poly or monoclonal antibodies and several synthetic molecules. Research involving these inhibitors may be useful to understand the mechanism of action of PLA(2)s and their role in envenomations caused by snake bite. Furthermore, the biotechnological potential of PLA(2) inhibitors may provide therapeutic molecular models with antiophidian activity to supplement the conventional serum therapy against these multifunctional enzymes.

Molecular characterization of BjussuSP-I, a new thrombin-like enzyme with procoagulant and kallikrein-like activity isolated from Bothrops jararacussu snake venom

A thrombin-like enzyme, named BjussuSP-I, isolated from Bothrops jararacussu snake venom, is an acidic single-chain glycoprotein with M-r = 61,000, pI similar to 3.8 and 6% sugar. BjussuSP-I shows high proteolytic activity upon synthetic substrates, such as S-2238 and S-2288. It also shows procoagulant and kallikrein-like activity, but is unable to act on platelets and plasmin. These activities are inhibited by specific inhibitors of this class of enzymes. The complete cDNA sequence of BjussuSP-I with 696 bp encodes open reading frames of 232 amino acid residues, which conserve the common domains of thrombin-like serine proteases. BjussuSP-I shows a high structural homology with other thrombin-like enzymes from snake venoms where common amino acid residues are identified as those corresponding to the catalytic site and subsites S1, S2 and S3 already reported. In this study, we also demonstrated the importance of N-linked glycans, to improve thrombin-like activity of BjussuSP-I toxin. (c) 2007 Elsevier Masson SAS. All rights reserved.

Influence of Quaternary Conformation on the Biological Activities of the Asp49-phospholipases A(2)s from Snake Venoms

One of the main components of snake venoms are the Asp49-phospholipases A(2), also known as svPLA(2)s. The study of these toxins is a matter of great scientific interest due to their wide variety of biological effects. In this work we present strong evidences found in literature and other aspects which strengthen the importance of quaternary assembly for understanding the activities and molecular evolution of svPLA(2)s.