Characterization of a basic phospholipase A2-homologue myotoxin isolated from the venom of the snake Bothrops neuwiedii (yarará chica) from Argentina

A basic protein was isolated by CM-Sephadex C-25 chromatography from the venom of Bothrops neuwiedii from Argentina, and named B. neuwiedii myotoxin I. This protein exerted local myotoxic and edema-forming effects in mice, with potencies comparable to other myotoxins isolated from Bothrops spp. venoms. When injected by i.v. route at doses up to 4.7 mg/kg of body weight, the toxin was not lethal. In vitro, the toxin had no detectable phospholipase A₂ activity on egg yolk phospholipids. B. neuwiedii myotoxin I appeared as a homodimer in sodium dodecylsulphate–polyacrylamide gel electrophoresis, with a subunit molecular weight of 15 kD. Gel immunodiffusion revealed a pattern of partial antigenic identity between the newly isolated myotoxin and myotoxin II from Bothrops asper venom. The sequence of B. neuwiedii myotoxin I was determined for the first 40 amino acid residues, showing high homology to several class II phospholipase A₂ myotoxins of the Lys-49 family from crotalids. Altogether, results suggest that this toxin is a new member of the Lys-49 phospholipase A₂-homologues with myotoxic, cytolytic, and edema-inducing activities.     

Neurotoxicological effects of a thrombin-like enzyme isolated from Crotalus durissus terrificus venom (preliminary study).

A thrombin-like enzyme, purified from the venom of Crotalus durissus terrificus by gel filtration and affinity chromatography, showed a single protein band in Sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) with a molecular weight of about 33kDa. Clear cellular morphological changes, deep ganglioside level modifications in some brain areas and behavioral alterations in pup rats injected with this protein were detected. Ganglioside composition, one of the chemical markers of brain maturation, was altered specially in the hypothalamus, hippocampus and prefrontal cortex. The most reliable behavioral effects were a delayed, maturation of the righting reflex, posture and motor response after treatment. These effects were consistent with the histological changes revealed in the cerebellum and prefrontal cortex of treated neonate rats, areas related to motor activities.     

Bhalternin: Functional and structural characterization of a new thrombin-like enzyme from Bothrops alternatus snake venom

A serine protease from Bothrops alternatus snake venom was isolated using DEAE-Sephacel, Sephadex G-75 and Benzamidine-Sepharose column chromatography. The purified enzyme, named Bhalternin, ran as a single protein band on analytical polyacrylamide gel electrophoresis (SDS-PAGE) and showed molecular weights of 31,500 and 27,000 under reducing and non-reducing conditions, respectively. Its complete cDNA was obtained by RT-PCR and the 708 bp codified for a mature protein of 236 amino acid residues. The multiple alignment of its deduced amino acid sequence showed a structural similarly with other serine proteases from snake venoms. Bhalternin was proteolytically active against bovine fibrinogen and albumin as substrates. When Bhalternin and bovine fibrinogen were incubated at 37 °C, at a ratio of 1:100 (w/w), the enzyme cleaved preferentially the Aα-chain, apparently not degrading the Bβ and γ-chains. Stability tests showed that the intervals of optimum temperature and pH for the fibrinogenolytic activity were 30-40 °C and 7.0-8.0, respectively. Also, the inhibitory effects of benzamidine on the fibrinogenolytic activity of Bhalternin indicate that it is a serine protease. This enzyme caused morphological alterations in heart, liver, lung and muscle of mice and it was found to cause blood clotting in vitro and defibrinogenation when intraperitoneally administered to mice, suggesting it to be a thrombin-like enzyme. Therefore, Bhaltenin may be of interest as a therapeutic agent in the treatment and prevention of thrombotic disorders.     

Pharmacokinetics of thrombin-like enzyme from venom of Agkistrodon halys ussuriensis Emelianov determined by ELISA in the rat

A thrombin-like enzyme (TLE) was separated and purified from the venom of a northeast Chinese snake Agkistrodon halys ussuriensis Emelianov. Experiments were performed in rats to determine the pharmacokinetic parameters following an intravenous (i.v.) or a subcutaneous (s.c.) injection of the thrombin-like enzyme. The plasma levels of TLE were estimated by enzyme-linked immunosorbent assay. The method exhibited high reproducibility and accuracy in correlating optical densities with TLE concentrations (0.2–30 ng ml⁻¹, r=0.99). The plasma concentration-time course after i.v. administration of 50 μg kg⁻¹ TLE was well fitted by a two-compartment open model. The half-life of the α-phase was 18.0±3.2 min, and that of the β-phase 3.9±0.7 h. The apparent volume of distribution was 1.8±0.5 l kg⁻¹, and clearance was 5.4±0.5 ml min⁻¹ kg⁻¹. When the TLE was injected s.c. at a dose of 0.75 mg kg⁻¹, the changes in plasma concentration were best described by a two-compartment model with a first-order absorption. The maximal plasma level of 51±2.7 ng ml⁻¹ was reached at 5.2±0.5 h. The absorption rate constant was 0.3±0.03 h⁻¹. The area under the plasma concentration-time curve (AUC) was 2.8±0.8 μg h⁻¹ ml⁻¹.     

Biochemistry and toxicology of toxins purified from the venom of the snake Bothrops asper

The isolation and study of individual snake venom components paves the way for a deeper understanding of the pathophysiology of envenomings – thus potentially contributing to improved therapeutic modalities in the clinical setting – and also opens possibilities for the discovery of novel toxins that might be useful as tools for dissecting cellular and molecular processes of biomedical importance. This review provides a summary of the different toxins that have been isolated and characterized from the venom of Bothrops asper, the snake species causing the majority of human envenomings in Central America. This venom contains proteins belonging to at least eight families: metalloproteinase, serine proteinase, C-type lectin-like, l-amino acid oxidase, disintegrin, DC-fragment, cystein-rich secretory protein, and phospholipase A₂. Some 25 venom proteins within these families have been isolated and characterized. Their main biochemical properties and toxic actions are described, including, in some cases, their possible relationships to the pathologic effects induced by B. asper venom.     

Functional and structural characterization of a new serine protease with thrombin-like activity TLBan from Bothrops andianus (Andean Lancehead) snake venom

A new serine protease with thrombin-like activity (TLBan) from Bothrops andianus (Andean Lancehead) was isolated in two chromatographic steps in LC molecular exclusion and reverse phase-HPLC. TLBan is a glycoprotein that contains both N-linked carbohydrates and sialic acid in its structure, with Mr ∼29 kDa under reducing conditions and non-reducing ∼25 kDa conditions and confirmed by MALDI-TOF mass spectrometry (25,835.65 Da) and exhibited high specificity for BAρNA, Michaelis-Menten behavior with Km 5.4 × 10⁻¹ M and the V_max 7.9 × 10⁻¹ nmoles ρ-NA/L/min for this substrate and high stability when was analyzed at different temperatures (25 to 60 °C), pHs (4.0 to 8.0), was inhibited by soybean trypsin inhibitor, EDTA and phenylmethylsulfonyl fluoride (PMSF).The total amino acid sequence was obtained through sequencing of selected tryptic peptides and by inference obtained using SwissProt database http://br.expasy.org/ with the search restricted to serine proteases from Crotalinae snakes and show high amino acid sequence identity with other serine proteases from snake venom.TLBan showed the presence of His(44), Asp(91) residues and Ser was deduced (187) position, in the corresponding positions to the catalytic triad established in the serine proteases and Ser(187) are inhibited by phenylmethylsulfonyl fluoride (PMSF).In this work, we investigated the ability of TLBan to degrade fibrinogen and we observed that it is able to cause α- and β-chain cleavage. Enzymatic activities as well as the platelet aggregation were strongly inhibited when were incubated with PMSF, a specific inhibitor of serine protease. TLBan showed a potential medical-scientific interest to understand the pathophysiological mechanism of the snake venom action and identification of new blood coagulation cascade acting enzymes of natural sources.     

Insights of local tissue damage and regeneration induced by BnSP-7, a myotoxin isolated from Bothrops (neuwiedi) pauloensis snake venom

Envenomations caused by Bothrops snake venoms are characterized by prominent local tissue damage due to myonecrosis, hemorrhage, edema and acute muscle damage which is widely correlated with phospholipases A₂ (PLA₂). In the present study, the progression of local tissue damage and inflammation induced by BnSP-7, a myotoxin isolated from Bothrops (neuwiedi) pauloensis snake venom, was evaluated. Local tissue damages characterized by edema, necrosis and inflammation were evaluated until 24 h after inoculation of BnSP-7. The regeneration of myofibers, analyzed by light microscopy, was observed from 72 h to 2 weeks post-inoculation of toxin. MMP-2 was expressed in gastrocnemius muscle at all time points tested, while the expression of MMP-9 increased expressively at the same time interval of regenerating muscle, suggesting the involvement of MMP-9 in the regeneration process. The production of pro-inflammatory cytokines was also increased, whereas IL-1β showed the highest level. Modification of BnSP-7 with BPB decreased the release of IL-8, IL-6 and IL-1β when compared to native BnSP-7. These data suggest that BnSP-7 acts as pro-inflammatory incentives (mediators), inducing MMP and cytokine production from the inflammatory and satellite cells, and thus it may play an important role in inflammatory process and, consequently, in the evolution of local tissue damage and regeneration.     

Local and systemic pathophysiological alterations induced by a serine proteinase from the venom of the snake Bothrops jararacussu.

The local and systemic pathophysiological alterations induced by BjussuSP-I, a thrombin-like serine proteinase from the venom of the snake Bothrops jararacussu, were assessed in mice. BjussuSP-I induced a mild edema but no local myonecrosis or hemorrhage. It did not induce any microvascular alteration in the cremaster muscle. Intramuscular injection of BjussuSP-I promoted an increase in the expression of proMMP-9, but it did not induce the activation of proMMP-2 or proMMP-9 synthesized in muscle tissue injected with a myotoxic phospholipase A(2) homolog. BjussuSP-I induced defibrin(ogen)ation upon intravenous and intramuscular injections, with reduction in plasma fibrinogen concentration and increments in the levels of fibrin degradation products and D-dimer. When compared with animals having normal coagulation, mice defibrin(ogen)ated by BjussuSP-I developed a slightly larger hemorrhagic lesion in the skin when injected with metalloproteinase BaP1. Intravenous injection of sublethal doses of BjussuSP-I promoted a series of behavioral and motor changes similar to those previously described for 'gyroxin', i.e. opisthotonus and a circular body movement along the longitudinal axis.     

A study on the venom of Bothrops alternatus Duméril, Bibron and Duméril, from different regions of Argentina

We report a comparison of toxic and enzymatic activities of Bothrops alternatus venoms collected from specimens of different regions of Argentina with a pool of these same venoms. We determined lethal, hemorrhagic and pro-coagulant (plasma and fibrinogen) doses, as well as indirect hemolysis, hydrolysis of gelatin and fibrinogen. The electrophoretic pattern of the venoms and that of fibrinogen incubated with each of the venoms was studied by SDS-PAGE. The toxic activities were consistent with those reported for these venoms but with noticeable differences between samples which do not seem related to geographical or climatic characteristics. In some cases the pooled venom showed values close to the individual samples, but it was not representative since at least the half of the venoms (4/9) exhibited very different patterns of toxicity. The potency of some activities in the individual venoms ranged from much higher to much lower than those determined in the pooled venom (0.1 to 3 fold). Not all venoms exhibited thrombin-like activity but all hydrolyzed α and β chains of fibrinogen; however, degradation patterns were not always similar. Variation in activities and toxicities do not seem to be related to geographical or climatic characteristics.     

Isolation and characterization of a new serine protease with thrombin-like activity (TLBm) from the venom of the snake Bothrops marajoensis

The thrombin-like serine protease TLBm from Bothrops marajoensis was isolated in one chromatographic step in reverse phase HPLC. Its molecular mass was 33239.95 Da, as based on the determined primary structure and confirmed experimentally by MALDI-TOF mass spectrometry (33332.5 Da) and it contains 12 half-cysteine residues. This TLBm exhibited high specificity for BAρNA, Michaelis-Menten behavior with K_m 2.3 × 10⁻¹ M and the V_max 0.52 × 10⁻¹ nmoles ρ-NA/lt/min for this substrate. TLBm also showed ability to coagulate bovine fibrinogen and was inhibited by soybean trypsin inhibitor, EDTA and S(Dm) from the serum of the species Didelphis marsupialis. The primary structure of TLBm showed the presence of His(45), Asp(103) and Ser(228) residues in the corresponding positions of the catalytic triad established in the serine proteases and Ser(228) are inhibited by phenylmethylsulfonyl fluoride (PMSF). Amino acid analysis showed a high content of Asp, Glu, Gly, Ser, Ala and Pro as well as 12 half-cysteine residues and calculated pI of 6.47; TLBm presented 285 amino acid residues. In this work, we investigated the ability of TLBm to degrade fibrinogen and we observed that it is able to cause α- and β-chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with PMSF, a specific inhibitor of serine protease. Also, TLBm induced platelet aggregation in washed and platelet-rich plasma, and in both cases, PMSF inhibited its activity.     

Experimental pathology of local tissue damage induced by Bothrops asper snake venom

Envenomations by Bothrops asper are often associated with complex and severe local pathological manifestations, including edema, blistering, dermonecrosis, myonecrosis and hemorrhage. The pathogenesis of these alterations has been investigated at the experimental level. These effects are mostly the consequence of the direct action of zinc-dependent metalloproteinases (SVMPs) and myotoxic phospholipases A₂ (PLA₂s). SVMPs induce hemorrhage, blistering, dermonecrosis and general extracellular matrix degradation, whereas PLA₂s induce myonecrosis and also affect lymphatic vessels. In addition, the prominent vascular alterations leading to hemorrhage and edema may contribute to ischemia and further tissue necrosis. The mechanisms of action of SVMPs and PLA₂s are discussed in detail in this review. Venom-induced tissue damage plays also a role in promoting bacterial infection. A prominent inflammatory reaction develops as a consequence of these local pathological alterations, with the synthesis and release of abundant mediators, resulting in edema and pain. However, whether inflammatory cells and mediators contribute to further tissue damage is not clear at present. Muscle tissue regeneration after venom-induced pathological effects is often impaired, thus resulting in permanent tissue loss and dysfunction. SVMP-induced microvessel damage is likely to be responsible of this poor regenerative outcome. Antivenoms are only partially effective in the neutralization of B. asper-induced local effects, and the search for novel toxin inhibitors represents a potential avenue for improving the treatment of this serious aspect of snakebite envenomation.     

Experimental pathophysiology of systemic alterations induced by Bothrops asper snake venom

Moderate and severe envenomations by the snake Bothrops asper provoke systemic alterations, such as systemic bleeding, coagulopathy, hypovolemia, hemodynamic instability and shock, and acute renal failure. Systemic hemorrhage is a typical finding of these envenomations, and is primarily caused by the action of P-III snake venom metalloproteinases (SVMPs). This venom also contains a thrombin-like serine proteinase and a prothrombin-activating P-III SVMP, both of which cause defibrin(ogen)ation. Thrombocytopenia, predominantly induced by a C-type lectin-like protein, and platelet hypoaggregation, caused by the two defibrin(ogen)ating enzymes, also contribute to hemostatic disturbances, which potentiate the systemic bleeding induced by hemorrhagic SVMPs. Cardiovascular disturbances leading to shock are due to the combined effects of hemorrhagic toxins, other venom components that increase vascular permeability, the action of hypotensive agents in the venom and of endogenous mediators, and the potential cardiotoxic effect of some toxins. Renal alterations are likely to be caused by direct cytotoxicity of venom components in the kidney, and by renal ischemia resultant from hypovolemia and hypoperfusion. Lethality induced by B. asper venom is the consequence of several combined effects among which the action of P-III SVMPs is especially relevant.     

Inflammation induced by Bothrops asper venom

Inflammation is a major characteristic of envenomation by snakes from viperine and crotaline species. Bothrops asper snake venom elicits, among other alterations, a pronounced inflammatory response at the site of injection both in humans and experimental animals. This review describes the current status of our understanding of the inflammatory reaction, including pain, triggered by B. asper venom. The experimental studies on the action of this venom as well as the complex network of chemical mediators involved are summarized. Moreover, aspects of the molecular mechanisms orchestrating this important response to envenomation by B. asper are presented. Considering that isolated toxins are relevant tools for understanding the actions of the whole venom, studies dealing with the mechanisms of inflammatory and nociceptive properties of phospholipases A₂, a metalloproteinase and serine proteinases isolated from B. asper venom are also described.     

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 microg of fraction III protein potentiated the secretion of insulin induced by 2.8 mM of glucose in rats isolated pancreatic beta-cells treated; the increase being around 3-fold higher than its respective control. B. insularis lectin (BiLec; 10 microg/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 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, 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.     

Structural and functional characterization of brazilitoxins II and III (BbTX-II and -III), two myotoxins from the venom of Bothrops brazili snake

We report the purification and biochemical/pharmacological characterization of two myotoxic PLA₂ (BbTX-II K49 PLA₂ homologue and BbTX-III PLA₂) from Bothrops brazili venom. Both were purified by a single chromatographic step on reverse phase HPLC, showing M_r 14 kDa for both myotoxins, showing high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The BbTX-II K49 PLA₂ homologue and BbTX-III PLA₂, had a sequence of 121 amino acid residues. BbTX-II: SLFELGKMILQETGKNPAKSYGAYGCYCGVLGRGKPKDATDRCCYVHKCCYKLTGCDNKKKDRYSYSWKDKTIVCGENNPCLKELCECDKAVAICLRENLNTYNKKYRYHLKPLCKKADAC with pI value 8.73. BbTX-III: SLWEWGQMILKETGKNPFPYYGAYGCYCGWGGRRKPKDATDRCCFVHDCCRYKKLTGCPKTNDRYSYSRLDYTIVCGEDDPCKEICECDKAAAVCFRENLRTYNKKYMAHLRVLCKKDKPC with a pI value of 8.46. BbTX-III presented PLA₂ activity in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 35–45 °C. Maximum PLA₂ activity required Ca²⁺. In vitro, BbTX-II K49 PLA₂ homologue and BbTX-III PLA₂ caused a blockade of the neuromuscular transmission in young chick biventer cervicis preparations in a similar way to other Bothrops species. In mice, BbTX-II K49 PLA₂ homologue and BbTX-III PLA₂ induces myonecrosis and edema-forming activity. All these biological effects induced by the BbTX-II K49 PLA₂ homologue, occur in the absence of a measurable PLA₂ activity in vitro, further supporting the concept of catalytic independent mechanisms exerted by Lys49 proteins.     

A new acidic myotoxic, anti-platelet and prostaglandin I2 inductor phospholipase A2 isolated from Bothrops moojeni snake venom

Phospholipase A₂ (PLA₂, EC 3.1.1.4), a major component of snake venoms, specifically catalyzes the hydrolysis of fatty acid ester bonds at position 2 of 1,2-diacyl-sn-3-phosphoglycerides in the presence of calcium. This article reports the purification and biochemical/functional characterization of BmooTX-I, a new myotoxic acidic phospholipase A₂ from Bothrops moojeni snake venom. The purification of the enzyme was carried out through three chromatographic steps (ion-exchange on DEAE–Sepharose, molecular exclusion on Sephadex G-75 and hydrophobic chromatography on Phenyl–Sepharose). BmooTX-I was found to be a single-chain protein of 15,000 Da and pI 4.2. The N-terminal sequence revealed a high homology with other acidic Asp49 PLA₂s from Bothrops snake venoms. It displayed a high phospholipase activity and platelet aggregation inhibition induced by collagen or ADP. Edema and myotoxicity in vivo were also induced by BmooTX-I. Analysis of myotoxic activity was carried out by optical and ultrastructural microscopy, demonstrating high levels of leukocytary infiltrate. Previous treatment of BmooTX-I with BPB reduced its enzymatic and myotoxic activities, as well as the effect on platelet aggregation. Acidic myotoxic PLA₂s from Bothrops snake venoms have been little explored and the knowledge of its structural and functional features will be able to contribute for a better understanding of their action mechanism regarding enzymatic and toxic activities.     

Peptide mimicking antigenic and immunogenic epitope of neuwiedase from Bothrops neuwiedi snake venom

Peptides derived from a phage display library may mimic essential features of epitopes (mimotopes), including their immunogenicity. A recombinant peptide library of 12 amino acids displayed on the phage capsid was used to obtain peptides that mimic epitopes of antigens that are reactive to specific polyclonal antibodies anti-neuwiedase (NEU), a toxin from Bothrops neuwiedi snake venom. These polyclonal antibodies are protective against NEU activity and were used as target for the peptide library biopannings, resulting in the selection of 80 peptides. Antibody-binding epitopes were obtained by sequence alignment with the primary and tertiary structures of the NEU protein. Antigenicity and specificity of the mimotopes mixture were confirmed by dot blot, immuno dot blot, plaque reduction and Western blot assays. Their immunogenicity was demonstrated by immunization of BALB/c mice and ELISA tests. The NEU toxin is an important antigen that has many common structural regions to several toxic venom metalloproteinases, in which two epitope regions have been detected. The two mapped epitopes were found in primary sequences of several snake venom toxins, thus demonstrating the potential application of these NEU mimotopes as possible antigen components that are toxicity free.     

Primary structure of a thrombin-like serine protease, kangshuanmei, from the venom of Agkistrodon halys brevicaudus stejneger.

The complete amino acid sequence of the thrombin-like serine protease, named kangshuanmei, isolated from the venom of a Chinese snake Agkistrodon halys brevicaudus stejneger, was determined by Edman degradation. The serine protease was composed of 236 amino acid residues and conserved the catalytic triad as His43, Asp88 and Ser182. The protease had four sites of asparagine-linked glycosylation at 81, 99, 148 and 229, and contained fucose, N-acetylglucosamin, galactose, mannose and N-acetylneuraminic acid. The amino acid sequence exhibited considerable similarities with other thrombin-like proteases isolated from the snake venoms of the Viperidae family. However, the enzymatic characteristics of kangshuanmei distinct from that of thrombin and the other protease from the venom of Viperidae family may be derived from the structural difference of the sequence in the functional regions, especially corresponding to thrombin exosite 1, 2 and hydrophobic pocket.     

Natural history of the terciopelo Bothrops asper (Serpentes: Viperidae) in Costa Rica

The terciopelo Bothrops asper is the only lancehead species widely distributed in the humid lowlands of Middle America and northwestern South America. Its large body size, relative abundance and cryptic habits contribute to the high incidence of snakebites induced by this species throughout its distribution. The terciopelo plays an important role in ecosystems, both as prey and as a generalist predator. Diet comprises a great variety of prey items, including some species that are considered nuisances. B. asper, as other lancehead species, exhibits a notable ontogenetic shift in diet, consuming ectotherms (mainly frogs and lizards) when young, and increasingly incorporating birds, rodents, and other small mammals with maturity. Adult terciopelos also consume large anurans, especially when endothermic prey availability is low.Using radiotelemetry we determined home range and movement patterns from 28 individual B. asper at La Selva Biological Station, Costa Rica. Overall home range estimates are relatively small compared with other pitvipers, averaging between 3.71 ha and 5.95 ha; home range size did not differ between males and females. Movement patterns are largely aseasonal and consist of short (<10 m) movements between daytime shelter and nocturnal ambush sites within a given area, interspersed with longer distance (>50 m) movements to new foraging areas. Habitat use is related to prey availability and therefore to foraging strategy. Our data support a strong preference for areas near swamps by both sexes.Reproduction in B. asper is highly seasonal, and – apparently – biannual. Reproductive cycles in Costa Rica are tightly related to rainfall patterns. Therefore, the timing of breeding differs between populations in the Caribbean and Pacific lowlands. Bothrops asper is adapted to areas with low levels of disturbance along the agricultural frontier, and consequently it is not rare to find it in or near human dwellings. However, despite popular belief, no evidence supports a purported increase in population density of this species in Costa Rica. Despite human persecution and substantial modification of habitat, B. asper is a species with a conservation status of least concern, and probably will likely persist well into the future. Thus, it is important to learn how to coexist with this species, and to improve mechanisms for the prevention and treatment of accidental snakebite and its consequences.     

Isolation and functional characterization of a new myotoxic acidic phospholipase A(2) from Bothrops pauloensis snake venom.

This article reports the purification procedure and the biochemical/functional characterization of Bp-PLA(2), a new myotoxic acidic phospholipase A(2) from Bothrops pauloensis snake venom. It was highly purified through three chromatographic steps (ion-exchange on CM-Sepharose, hydrophobic chromatography on Phenyl-Sepharose and RP-HPLC on a C8 column). Bp-PLA(2) is a single-chain protein of 15.8kDa and pI 4.3. Its N-terminal sequence revealed a high homology with other Asp49 acidic PLA(2)s from snake venoms. Its specific activity was 585.3U/mg. It displayed a high indirect hemolytic activity and inhibited platelet aggregation induced by collagen or ADP. It also induced in vivo edema and myotoxicity. Pretreatment of Bp-PLA(2) with BPB reduced the enzymatic activity, the inhibitory action on platelet aggregation and myotoxicity in vitro. Morphological analyses indicated that Bp-PLA(2) induced an intense edema, with visible leukocyte infiltrate and damaged muscle cells 24h after injection. Acidic myotoxic PLA(2)s from Bothrops snake venoms are still not extensively explored and knowledge of their structural and functional features will contribute for a better understanding of their action mechanism regarding enzymatic and toxic activities.