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We investigated the antiophidic properties of isohemigossypolone (ISO), a naphthoquinone isolated from the outer bark of the Pachira aquatic Aubl. The inhibition of phospholipase A2, coagulant, fibrinogenolytic, hemorrhagic and myotoxic activities induced by Bothrops pauloensis venom (Pb) was investigated. For this, we use samples resulting from the incubation of Pb with ISO in different concentrations (1:1, 1:5 and 1:10 w/w), we also evaluated the condition of treatment using ISO after 15 min of venom inoculation. The activities of phospholipase A2, coagulant, fibrinogenolytic, hemorrhagic and myotoxic induced by the B. pauloensis venom were significantly inhibited when the ISO was pre-incubated with the crude venom. For in vivo neutralization tests, the results were observed even when the ISO was applied after 15 min of inoculation of the venom or metalloprotease (BthMP). Also, to identify the inhibition mechanism, we performed in silico assays, across simulations of molecular coupling and molecular dynamics, it was possible to identify the modes of interaction between ISO and bothropic toxins BmooMPα-I, Jararacussin-I and BNSP-7. The present study shows that naphthoquinone isohemigossypolone isolated from the P. aquatica plant inhibited part of the local and systemic damage caused by venom proteins, demonstrating the pharmacological potential of this compound in neutralizing the harmful effects caused by snakebites.

Grains, brans, and vegetable meals may contain non-starch polysaccharides (NSP), which increases viscosity in the gastrointestinal tract (GIT) and interfere with the digestion and absorption of nutrients. This study aimed to evaluate the performance and determine the metabolizable energy of a sorghum-based broiler diet with and without the supplementation of an enzymatic complex. The experiments were conducted in a completely randomized design with 1200 chickens, using sorghum-based feed with and without the addition of 50 g of enzyme-CCE complex (?-glucanase and ?-xylanase), and with two levels of metabolizable energy (ME kg-1): ME; ME + CCE; reduced ME (-50 kcal kg-1); and reduced ME + CCE. The data were subjected to an analysis of variance and the means were compared using a Tukey’s test at the 5% significance level. At 42 and 47 days of age, the living weight of the birds fed with the reduced ME was low, while birds fed with reduced ME + CCE had the same weight as those fed with other energy diets (ME and ME + CCE). Feed conversion was poorest at 47 days of age for the birds on reduced ME diet. In the metabolic test (with fattening diets) to determine AME and AMEn, the reduced ME diet had the lowest result, confirming the effect of the addition of enzymes. The addition of CCE to sorghum-based diets provides enough enzymatic activity to increase the metabolizable energy of the diet (50 kcal of AME) and influence the growth performance of broilers at the slaughtering age.

In this work, we describe the molecular cloning and pharmacological properties of an acidic phospholipase A(2) (PLA(2)) isolated from Bothrops pauloensis snake venom. This enzyme, denominated BpPLA(2)-TXI, was purified by four chromatographic steps and represents 2.4% of the total snake venom protein content. BpPLA(2)-TXI is a monomeric protein with a molecular mass of 13.6 kDa, as demonstrated by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis and its theoretical isoelectric point was 4.98. BpPLA(2)-TXI was catalytically active and showed some pharmacological effects such as inhibition of platelet aggregation induced by collagen or ADP and also induced edema and myotoxicity. BpPLA(2)-TXI displayed low cytotoxicity on TG-180 (CCRF S 180 II) and Ovarian Carcinoma (OVCAR-3), whereas no cytotoxicity was found in regard to MEF (Mouse Embryonic Fibroblast) and Sarcoma 180 (TIB-66). The N-terminal sequence of forty-eight amino acid residues was determined by Edman degradation. In addition, the complete primary structure of 122 amino acids was deduced by cDNA from the total RNA of the venom gland using specific primers, and it was significantly similar to other acidic D49 PLA(2)s. The phylogenetic analyses showed that BpPLA(2)-TXI forms a group with other acidic D49 PLA(2)s from the gender Bothrops, which are characterized by a catalytic activity associated with anti-platelet effects.

Snake venom metalloproteinases (SVMPs) participate in a number of important biological, physiological and pathophysiological processes and are primarily responsible for the local tissue damage characteristic of viperid snake envenomations. The use of medicinal plant extracts as antidotes against animal venoms is an old practice, especially against snake envenomations. Such plants are sources of many pharmacologically active compounds and have been shown to antagonize the effects of some venoms and toxins. The present study explores the activity of triacontyl p -coumarate (PCT), an active compound isolated from root bark of Bombacopsis glabra vegetal extract ( Bg ), against harmful effects of Bothropoides pauloensis snake venom and isolated toxins (SVMPs or phospholipase A 2 ). Before inhibition assays, Bg or PCT was incubated with venom or toxins at ratios of 1:1 and 1:5 (w/w; venom or isolated toxins/PCT) for 30 min at 37 °C. Treatment conditions were also assayed to simulate snakebite with PCT inoculated at either the same venom or toxin site. PCT neutralized fibrinogenolytic activity and plasmatic fibrinogen depletion induced by B. pauloensis venom or isolated toxin. PCT also efficiently inhibited the hemorrhagic (3MDH – minimum hemorrhagic dose injected i.d into mice) and myotoxic activities induced by Jararhagin, a metalloproteinase from B. jararaca at 1:5 ratio (toxin: inhibitor, w/w) when it was previously incubated with PCT and injected into mice or when PCT was administered after toxin injection. Docking simulations using data on a metalloproteinase (Neuwiedase) structure suggest that the binding between the protein and the inhibitor occurs mainly in the active site region causing blockade of the enzymatic reaction by displacement of catalytic water. Steric hindrance may also play a role in the mechanism since the PCT hydrophobic tail was found to interact with the loop associated with substrate anchorage. Thus, PCT may provide a alternative to complement ophidian envenomation treatments.

Four compounds (isoquercitrin, myricetin-3-O-glucoside, catechin and gallocatechin) were isolated from lyophilized aqueous extract of Schizolobium parahyba leaves by chromatography on Sephadex LH-20, followed by semipreparative HPLC using a C-18 column, and identified by 1H and 13C NMR. The compounds were then, tested against hemorrhagic and fibrinogenolytic activities of Bothrops crude venoms and isolated metalloproteinases. The inhibitors neutralized the biological and enzymatic activities of Bothrops venoms and toxins isolated from B. jararacussu and B. neuwiedi venoms. The results showed that gallocatechin and myricetin-3-O-glucoside are good inhibitors of hemorrhagic and fibrinogenolytic activities of metalloproteinases, respectively. Gallocatechin also inhibited the myotoxic activity of both B. alternatus venom and BnSP-6 (Lys49 PhospholipaseA2 from B. neuwiedi). Circular dichroism and docking simulation studies were performed in order to investigate the possible interaction between BnSP-6 and gallocatechin. This is the first time these compounds and their anti-ophidian properties are reported for S. parahyba species. Forthcoming studies involving X-ray co-crystallization, will be of great importance for the development of new therapeutic agents for the treatment of ophidian accidents and for the better understanding of the structure/function relationship of venom toxins.

A fibrino(geno)lytic nonhemorrhagic metalloproteinase ( BleucMP ) was purified from Bothrops leucurus snake venom by two chromatographic steps procedure on DEAE-Sephadex A-25 followed by CM-Sepharose Fast Flow column. BleucMP represented 1.75% (w/w) of the crude venom and was homogeneous on SDS-PAGE. BleucMP analyzed by MALDI TOF/TOF, showed a molecular mass of 23,057.54 Da and when alkylated and reduced, the mass is 23,830.40 Da. Their peptides analyzed in MS (MALDI TOF\TOF) showed significant score when compared with those of other proteins by NCBI-BLAST2 alignment display. As regards their proteolytic activities, BleucMP efficiently acted on fibrinogen, fibrin, and was inhibited by EDTA and 1.10-phenanthroline. This enzyme was also able to decrease significantly the plasma fibrinogen level provoking blood incoagulability, however was devoid of hemorrhagic activity when tested in the mice skin and did not induce relevant biochemical, hematological and histopathological alterations in mice. The aspects addressed in this paper provide data on the effect of BleucMP in envenomation from B. leucurus snakes in order to better understand the effects caused by snake venom metalloproteinase.

The damaging effects of neuwiedase, a non-hemorrhagic snake venom metalloproteinase from P-I class, on gastrocnemius muscle are studied herein. Following neuwiedase injection, ultrastructural alterations were detected early showing disarrangement of skeletal muscle fibers (characterized by discontinuity of Z lines), mitochondrial swelling, and disruption of plasma membrane and basal lamina. Degradation of skeletal muscle and the appearance of an amorphous substance, primarily composed of cellular debris, were noted after 24 hours. The presence of neuwiedase at the injection site (detected by immunocytochemistry) revealed highly specific labeling of myofibril components of damaged myocytes. In addition, proteolysis of muscle proteins assayed through myofibrils extracted from gastrocnemius muscle indicated that neuwiedase provoked degradation of myofibrils, especially myosin. These results suggest that skeletal muscle damage, induced by neuwiedase, is probably due to its proteolytic action on myofibrils, which are responsible for the maintenance of the cellular architecture.

The herbal extract of Schizolobium parahyba leaves is used commonly in the Brazil central region to treat snakebites. This study evaluates the acute toxicological effects of Schizolobium parahyba aqueous extract in mice 24 h after intraperitoneal administration. Acute toxicity was evaluated using biochemical, hematological and histopathological assays. Alterations in the levels of transaminases, bilirubin, albumin and prothrombrin time were observed, and these are likely to occur due to hepatic injury, which was confirmed by light microscopy. Liver histopathological analysis revealed the presence of lymph plasmocitary inflammatory infiltrate, but no other histopathological alterations were observed in any of the other organs analysed. The data confirm the low toxicity of the extract of Schizolobium parahyba and provide a model for the selection of a dose that does not cause injuries in the organism.

The present work evaluates both in vitro and in vivo antitumor activity of BPB-modified BthTX-I and its cationic synthetic peptide derived from the 115-129 C-terminal region. BPB-BthTX-I presented cytotoxicity of 10-40% on different tumor cell lines, which were also susceptible to the lytic action of the synthetic peptide. Injection of the modified protein or the peptide in mice, 5 days after transplantation of S180 tumor cells, reduced 30 and 36% of the tumor size on day 14th and 76 and 79% on day 60th, respectively, when compared to the untreated control group. Thus, these antitumor properties might be of interest in the development of therapeutic strategies against cancer.

The Snake Venom Metalloproteinases (SVMPs) play a relevant role in the multifactorial inflammatory response induced by Bothrops envenomations. Neuwiedase, an SVMP isolated from Bothrops neuwiedi venom, is devoid of hemorrhagic activity on skin tests, but is able to induce myonecrosis and degrade fibrinogen, fibrin, type I collagen, fibronectin and laminin. In this study, we analyzed the inflammatory reaction induced by neuwiedase in gastrocnemius muscle, with special focus on cytokines release. Our results showed clear evidence of inflammatory infiltrate in the gastrocnemius muscle and an increase of MMP-9, and the cytokines KC, IL-1 beta and IL-6 in the early periods after toxin injection. The cytokine release was also evaluated in inflammatory and muscular cell culture. Both murine peritoneal adherent cells (MPACs) and muscle cells (C2C12) released pro-inflammatory cytokines after stimulus with neuwiedase. MPACs showed increased production of KC, IL-1 beta and IL-6 in the cell culture supernatant while in C2C12, the predominant chemokine expressed was KC. These data reinforce the importance of SVMPs in the inflammatory response caused by envenomation and point out the role of muscle cells in this event by releasing pro-inflammatory mediators able to attract leukocytes to the muscle, thus starting and amplifying the setting of the inflammatory reaction.

Crotalus durissus rattlesnakes are responsible for the most lethal cases of snakebites in Brazil. Crotalus durissus collilineatus subspecies is related to a great number of accidents in Southeast and Central West regions, but few studies on its venom composition have been carried out to date. In an attempt to describe the transcriptional profile of the C. durissus collilineatus venom gland, we generated a cDNA library and the sequences obtained could be identified by similarity searches on existing databases. Out of 673 expressed sequence tags (ESTs) 489 produced readable sequences comprising 201 singletons and 47 clusters of two or more ESTs. One hundred and fifty reads (60.5%) produced significant hits to known sequences. The results showed a predominance of toxin-coding ESTs instead of transcripts coding for proteins involved in all cellular functions. The most frequent toxin was crotoxin, comprising 88% of toxin-coding sequences. Crotoxin B, a basic phospholipase A2 (PLA2) subunit of crotoxin, was represented in more variable forms comparing to the non-enzymatic subunit (crotoxin A), and most sequences coding this molecule were identified as CB1 isoform from Crotalus durissus terrificus venom. Four percent of toxin-related sequences in this study were identified as growth factors, comprising five sequences for vascular endothelial growth factor (VEGF) and one for nerve growth factor (NGF) that showed 100% of identity with C. durissus terrificus NGF. We also identified two clusters for metalloprotease from PII class comprising 3% of the toxins, and two for serine proteases, including gyroxin (2.5%). The remaining 2.5% of toxin-coding ESTs represent singletons identified as homologue sequences to cardiotoxin, convulxin, angiotensin-converting enzyme inhibitor and C-type natriuretic peptide, Ohanin, crotamin and PLA2 inhibitor. These results allowed the identification of the most common classes of toxins in C. durissus collilineatus snake venom, also showing some unknown classes for this subspecies and even for C. durissus species, such as cardiotoxins and VEGF.

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.

Many medicinal plants have been recommended for the treatment of snakebites. The aqueous extracts prepared from the leaves of Schizolobium parahyba (a plant found in Mata Atlantica in Southeastern Brazil) were assayed for their ability to inhibit some enzymatic and biological activities induced by Bothrops pauloensis and Crotalus durissus terrificus venoms as well as by their isolated toxins neuwiedase (metalloproteinase), BnSP-7 (basic Lys49 PLA2) and CB (PLA2 from crotoxin complex). Phospholipase A2, coagulant, fibrinogenolytic, hemorrhagic and myotoxic activities induced by B. pauloensis and C. d. terrificus venoms, as well as by their isolated toxins were significantly inhibited when different amounts of S. parahyba were incubated previously with these venoms and toxins before assays. However, when S. parahyba was administered at the same route as the venoms or toxins injections, the tissue local damage, such as hemorrhage and myotoxicity was only partially inhibited. The study also evaluated the inhibitory effect of S. parahyba upon the spreading of venom proteins from the injected area into the systemic circulation. The neutralization of systemic alterations induced by i.m. injection of B. pauloensis venom was evaluated by measuring platelet and plasma fibrinogen levels which were significantly maintained when S. parahyba extract inoculation occurred at the same route after B. pauloensis venom injection. In conclusion, the observations confirmed that the aqueous extract of S. parahyba possesses potent snake venom neutralizing properties. It may be used as an alternative treatment to serum therapy and as a rich source of potential inhibitors of toxins involved in several physiopathological human and animal diseases. Copyright © 2008 John Wiley & Sons, Ltd.

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 15.8 kDa and p I 4.3. Its N-terminal sequence revealed a high homology with other Asp49 acidic PLA 2 s from snake venoms. Its specific activity was 585.3 U/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 24 h 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.

Several metallic compounds recognized as potent antitumor agents, have been developed and tested in vivo and in vitro . In this work, we evaluated the toxic, therapeutic, and cytotoxic properties of the cis -dichlorotetraammineruthenium(III) chloride. Transplanted animals with Sarcoma 180 cells were treated with ruthenium(III) complex and injected i.p., at different time intervals. After the 15th day, tumoral postimplant, the animals were sacrificed and their lungs, kidneys, liver, and tumors were removed and processed for histopathological analysis. Blood samples were also taken for haematological and biochemical analyses. Interaction between the ruthenium complex and the DNA was also investigated. Besides being cytotoxic for the S180 cells, the metallic compound induced tumoral volume reduction and increased survival time of the animals treated. Serum levels of LDH, creatinin, and bilirubin increased, but no serious irreversible histopathological alterations were observed in the analyzed tissues. The compound did not cause anemia, but reduced the number of leukocytes in the treated animals. The absence of viable S180 cells, necrotic cells, and the presence of granulation tissue were observed in tumor tissue of treated animals. The Ru(III) complex, in the presence of the reduction agent, caused plasmid DNA to fragment. These results suggest that cis -RuCl 2 (NH 3 ) 4 Cl compound is a potent antitumoral drug in vitro and in vivo , which seems to involve binding to DNA molecule.

Two basic myotoxic PLA(2)s, namely BnpTX-I and II, were isolated from Bothrops neuwiedi pauloensis snake venom through three chromatographic steps: ion-exchange chromatography on CM-Sepharose, gel filtration on Sephadex G-50 and reverse phase HPLC on a C18 column. Both PLA(2)s showed a M(r) around 14,000 for the monomer and 28,000 for the dimer (as estimated by SDS-PAGE), pI approximately 7.8 and approximately 121 amino acid residues cross-linked by seven disulfide bonds. The N-terminal sequences revealed significant homology with Asp49 basic myotoxic PLA(2)s from other snake venoms. The catalytic and anticoagulant activities of BnpTX-I were higher than those of BnpTX-II. Both were able to induce cytotoxicity in vitro, as well as, myotoxicity, edema and lethality in mice. BnpTX-I also induced neurotoxic effect on mouse neuromuscular preparations and bactericidal activity on Eschericia coli and Staphylococcus aureus. After chemical modification of BnpTX-I with BPB or incubation with EDTA or Mn(2+) ions, the catalytic activity was completely abolished, while the toxic and pharmacological activities were partially reduced. Interaction with heparin inhibited the cytotoxic and bactericidal effects. Anti-BthTX-I, anti-BthTX-II and anti-115-129-C terminal antibodies strongly recognize both BnpTX-I and II. It is shown that the neurotoxic effect induced by B. neuwiedi pauloensis venom is due to the presence of myotoxic PLA(2)s. The data also corroborate the hypothesis of a partial dissociation between toxic and enzymatic domains. In addition, BnpTX-I displays a heparin binding C-terminal region, which is probably responsible for the cytotoxic and bactericidal effects.

In the present work, we demonstrate some biochemical and functional properties of a new PI snake venom metalloproteinase (SVMP) isolated from Bothrops pauloensis snake venom (BpMP-II), in addition we evaluated its capacity to inhibit endothelial cell adhesion and in vitro angiogenesis. BpMP-II was purified after a combination of three chromatography steps and showed molecular mass of 23,000 Da determined by MALDI-TOF, an isoelectric point of 6.1 and the sequence of some fragments obtained by MS/MS (MALDI TOF\TOF) presented high structural similarity with other PI-SVMPs. BpMP-II showed proteolytic activity against azocasein, was able to degrade bovine fibrinogen and was inhibited by EDTA, 1.10 phenantroline and β-mercaptoethanol. BpMP-II did not induce local hemorrhage in the dorsal region of mice even at high doses and did not affect plasma creatine kinase (CK) levels when administered intramuscularly into the gastrocnemius muscle of mice. Moreover, this metalloproteinase decreased tEnd cells viability at concentrations higher than 20 μg/mL. With sub-toxic doses this metalloproteinase affected tEnd cell adhesion and was also able to inhibit in vitro angiogenesis. BpMP-II showed very important functional properties suggesting considerable therapeutic potential for this class of protein.

Aqueous extract from Casearia sylvestris leaves, a typical plant from Brazilian open pastures, was able to neutralize the hemorrhagic activity caused by Bothrops asper, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi and Bothrops pirajai venoms. It also neutralized two hemorrhagic metalloproteinases from Bothrops asper venom. Proteolytic activity on casein induced by bothropic venoms and by isolated proteases, including Bn2 metalloproteinase from B. neuwiedi venom, was also inhibited by the C. sylvestris extract in different levels. The α-fibrinogen chain was partially protected against degradation caused by B. jararacussu venom, when this venom was incubated with C. sylvestris extract. We also observed that this extract partially increased the time of plasma coagulation caused by B. jararacussu, B. moojeni and B. neuwiedi venoms. C. sylvestris extract did not induce proteolysis in any substrate assayed.

The crude aqueous extract from the leaves of Casearia sylvestris, a plant found in Brazilian open pastures, was assayed for its ability to inhibit phospholipase A2 (PLA2) activity and some biological activities of bee and several snake venoms, and of a number of isolated PLA2s. The extract induced partial inhibition of the PLA2 activity of venoms containing class I, II and III PLA2s. When tested against the purified toxins, it showed the highest efficacy against class II PLA2s from viperid venoms, being relatively ineffective against the class I PLA2 pseudexin. In addition, C. sylvestris extract significantly inhibited the myotoxic activity of four Bothrops crude venoms and nine purified myotoxic PLA2s, including Lys-49 and Asp-49 variants. The extract was able to inhibit the anticoagulant activity of several isolated PLA2s, with the exception of pseudexin. Moreover, it partially reduced the edema-inducing activity of B. moojeni and B. jararacussu venoms, as well as of myotoxins MjTX-II and BthTX-I. The extract also prolonged the survival time of mice injected with lethal doses of several snake venoms and neutralized the lethal effect induced by several purified PLA2 myotoxins. It is concluded that C. sylvestris constitutes a rich source of PLA2 inhibitors.

A basic serine protease which is active on casein and fibrinogen was purified from Bothrops moojeni venom using a single step chromatography on a CM-Sepharose fast flow column. The enzyme, MOO3, was not hemorrhagic and presented only a trace of blood-clotting activity. Synthetic chromogenic substrates (azoacasein and azoalbumin) where not hydrolyzed by MOO3. Using polyacrylamide gel electrophoresis at pH 4.3, MOO3 showed as a single protein band. Using sodium dodecyl sulfate-polyacrylamide electrophoresis, MOO3 behaved as a single-chain protein with an approximate mol. weight of 27,000, both in the presence and absence of beta-mercaptoethanol. Its pI was 7.8 by electrofocusing. The enzyme did not contain neutral carbohydrates and its N-terminal amino acid was alanine. The amino acid composition showed 249 residues/mole, a high content of hydrophilic amino acids and 14 half-cystine residues, which should account for 7 disulfide bonds. The protease cleaved the A-alpha chain faster than the B-beta of bovine fibrinogen and showed no effect on the delta-chain. Specific esterolytic activity of MOO3 on alpha-N-tosyl-l-arginine methyl ester was 29.64 mumol min-1 x mg-1. MOO3 represented 1.42% (w/w) of the initial desiccated venom. Its proteolytic activity was inhibited by beta-mercaptoethanol, leupeptin, phenylmethylsulphonyl fluoride and ethylenediamine tetraacetate.

(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 approximately equal to 8.6 and 8.8, respectively) which were isolated by ion-exchange chromatography on CM-Sepharose. (3) From BnMG, three myotoxic isoforms (pI approximately equal to 8.2 and M(r) = 13,600) 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.

Bothrops moojeni snake venom was fractionated on a CM-Sepharose column which was previously equilibrated with 0.05 M ammonium bicarbonate buffer at pH 8.0 and subsequently eluted with an ammonium bicarbonate concentration gradient from 0.05 to 0.5 M at constant pH (8.0) and temperature (25°C). The fraction which eluted last (M-VI) showed, after direct lyophilization, a single band by polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE, indicating an approximate M r of 14 000 and 27 000, in the presence and absence of dithiothreitol, respectively. Its amino acid composition revealed a high level of hydrophobic and basic amino acids as well as 14 half-cystine residues. Its isoelectric point and extinction coefficient ( E 1.0 cm 1.0 mg/ml at 278 nm and pH 7.0) were 8.2 and 1.170, respectively. M-VI was devoid of phospholipase A 2 (PLA 2 ) activity on egg yolk, as well as of hemorrhagic, anticoagulant and coagulant activities, but could induce drastic necrosis on skeletal muscle fibres as well as rapid and transient edema on the rat paw. Its N-terminal sequence: SLFELGKMILQETGKNPAKSYGVYGCNCGVGGRGKPKDATDRCCYVHKCCYK... revealed high homology with other Lys 49 PLA 2 -like myotoxins from other bothropic venoms. Orthorhombic crystals of M-VI, which diffracted to a maximal resolution of 1.6 A, were obtained and indicated the presence of a dimer in the asymmetrical unit.

The antibothropic complex (ABC) from opossum (species Didelphis albiventris) serum was purified by chromatography on DEAE-Sephacel. It showed an acidic character and two polypeptide chains of ca. 45 kDa and 48 kDa, respectively. Lyophilized opossum serum or the ABC (100 micrograms), as well as ethylenediamine tetraacetate (0.25 mumoles) were able to completely neutralise the hemorrhagic effect of 50 micrograms of the desiccated venoms of Bothrops moojeni, Bothrops pirajai and Bothrops jararacussu. The myotoxic (100 micrograms venom in mice) and edematogenic (90 micrograms venom in rats) activities of Bothrops moojeni and Bothrops jararacussu venoms, as well as of the major myonecrotic protein (myotoxin-I) isolated from Bothrops moojeni venom, were also totally inhibited by the ABC (200 micrograms and 270 micrograms, respectively). The lyophilized opossum serum (30 micrograms) and the ABC (30 micrograms) reduced to 50% the phospholipase A2 activity of Bothrops moojeni venom (10 micrograms). The clotting activity of Bothrops alternatus and Bothrops moojeni (20 micrograms) on bovine plasma was also significantly inhibited by the ABC (60 micrograms).

The herbal extract of Schizolobium parahyba leaves is used commonly in the Brazil central region to treat snakebites. This study evaluates the acute toxicological effects of Schizolobium parahyba aqueous extract in mice 24 h after intraperitoneal administration. Acute toxicity was evaluated using biochemical, hematological and histopathological assays. Alterations in the levels of transaminases, bilirubin, albumin and prothrombrin time were observed, and these are likely to occur due to hepatic injury, which was confirmed by light microscopy. Liver histopathological analysis revealed the presence of lymph plasmocitary inflammatory infiltrate, but no other histopathological alterations were observed in any of the other organs analysed. The data confirm the low toxicity of the extract of Schizolobium parahyba and provide a model for the selection of a dose that does not cause injuries in the organism.

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-1 beta showed the highest level. Modification of BnSP-7 with BPB decreased the release of IL-8, IL-6 and IL-1 beta 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.

In the present study, a thrombin-like enzyme named BpSP-I was isolated from Bothrops pauloensis snake venom and its biochemical, enzymatic and pharmacological characteristics were determined. BpSP-I is a glycoprotein that contains both N-linked carbohydrates and sialic acid in its structure, with M r = 34,000 under reducing conditions and p I ∼ 6.4. The N-terminal sequence of the enzyme (VIGGDECDINEHPFL) showed high similarity with other thrombin-like enzymes from snake venoms. BpSP-I showed high clotting activity upon bovine and human plasma and was inhibited by PMSF, benzamidine and leupeptin. Moreover, this enzyme showed stability when examined at different temperatures (−70 to 37 °C), pH values (3–9) or in the presence of divalent metal ions (Ca 2+ , Mg 2+ , Zn 2+ and Mn 2+ ). BpSP-I showed high catalytic activity upon substrates, such as fibrinogen, TAME, S-2238 and S-2288. It also showed kallikrein-like activity, but was unable to act upon factor Xa and plasmin substrates. Indeed, the enzyme did not induce hemorrhage, myotoxicity or edema. Taken together, our data showed that BpSP-I is in fact a thrombin-like enzyme isoform isolated from Bothrops pauloensis snake venom.

An L-amino acid oxidase (Bp-LAAO) from Bothrops pauloensis snake venom was highly purified using sequential chromatography steps on CM-Sepharose, Phenyl-Sepharose CL-4B, Benzamidine Sepharose and C18 reverse-phase HPLC. Purified Bp-LAAO showed to be a homodimeric acidic glycoprotein with molecular weight around 65kDa under reducing conditions in SDS-PAGE. The best substrates for Bp-LAAO were L-Met, L-Leu, L-Phe and L-Ile and the enzyme showed a strong reduction of its catalytic activity upon L-Met and L-Phe substrates at extreme temperatures. Bp-LAAO showed leishmanicidal, antitumoral and bactericidal activities dose dependently. Bp-LAAO induced platelet aggregation in platelet-rich plasma and this activity was inhibited by catalase. Bp-LAAO-cDNA of 1548bp codified a mature protein with 516 amino acid residues corresponding to a theoretical isoelectric point and molecular weight of 6.3 and 58kDa, respectively. Additionally, structural and phylogenetic studies identified residues under positive selection and their probable location in Bp-LAAO and other snake venom LAAOs (svLAAOs). Structural and functional investigations of these enzymes can contribute to the advancement of toxinology and to the elaboration of novel therapeutic agents.

The aqueous extract prepared from Schizolobium parahyba (Sp) leaves, a native plant from Atlantic Forest (Brazil), was tested to analyse its ability to inhibit some biological and enzymatic activities induced by Bothrops alternatus (BaltCV) and Bothrops moojeni (BmooCV) snake venoms. Sp inhibited 100% of lethality, blood incoagulability, haemorrhagic and indirect haemolytic activities at a 1:10 ratio (venom/extract, w/w), as well as coagulant activity at a 1:5 ratio (venom/extract, w/w) induced by both venoms. BaltCV fibrinogenolytic activity was also neutralized by Sp at a 1:10 ratio, resulting in total protection of fibrinogen Bbeta chain and partial protection of Aalpha chain. Interaction tests have demonstrated that, at certain extract/proteins ratios, Sp precipitates proteins non-specifically suggesting the presence of tannins, which are very likely responsible for the excellent inhibiting effects of the analysed ophidian activities. Sp aqueous extract chromatography on Sephadex LH-20 was carried out aiming at the separation of these compounds that mask the obtained results. Thus, the fractionation of Sp resulted in three fractions: F1 (methanolic fraction); F2 (methanol:water fraction, 1:1 v/v); and F3 (aqueous fraction). These fractions were analysed for their ability to inhibit the BaltCV fibrinogenolytic activity. F1 inhibited 100% the venom fibrinogenolytic activity without presenting protein precipitation effect; F2 showed only partial inhibition of this venom activity. Finally, F3 did not inhibit fibrinogen proteolysis, but presented strong protein precipitating action. We conclude that Sp aqueous extract, together with tannins, also contains other compounds that can display specific inhibitory activity against snake venom toxins.

Many medicinal plants have been recommended for the treatment of snakebites. The aqueous extracts prepared from the leaves of Schizolobium parahyba (a plant found in Mata Atlantica in Southeastern Brazil) were assayed for their ability to inhibit some enzymatic and biological activities induced by Bothrops pauloensis and Crotalus durissus terrificus venoms as well as by their isolated toxins neuwiedase (metalloproteinase), BnSP-7 (basic Lys49 PLA(2)) and CB (PLA(2) from crotoxin complex). Phospholipase A(2), coagulant, fibrinogenolytic, hemorrhagic and myotoxic activities induced by B. pauloensis and C. d. terrificus venoms, as well as by their isolated toxins were significantly inhibited when different amounts of S. parahyba were incubated previously with these venoms and toxins before assays. However, when S. parahyba was administered at the same route as the venoms or toxins injections, the tissue local damage, such as hemorrhage and myotoxicity was only partially inhibited. The study also evaluated the inhibitory effect of S. parahyba upon the spreading of venom proteins from the injected area into the systemic circulation. The neutralization of systemic alterations induced by i.m. injection of B. pauloensis venom was evaluated by measuring platelet and plasma fibrinogen levels which were significantly maintained when S. parahyba extract inoculation occurred at the same route after B. pauloensis venom injection. In conclusion, the observations confirmed that the aqueous extract of S. parahyba possesses potent snake venom neutralizing properties. It may be used as an alternative treatment to serum therapy and as a rich source of potential inhibitors of toxins involved in several physiopathological human and animal diseases.

The major aim to the present study was to determine the effects of neuwiedase, a metalloproteinase isolated from Bothrops neuwiedi snake venom, on invasion and replication of Toxoplasma gondii in human fibroblasts in vitro. Neuwiedase treatment was done on host cells previously infected with T. gondii or on parasite before fibroblast infection. When treatments were done after or before infection, infection rates were inhibited in 71% and 61%, respectively. Considering that therapy protocols currently used in T. gondii infection cause considerable side effects, particularly in immunocompromised individuals and pregnant women, the results of neuwiedase treatment described herein could be taken into account for the development of new synthetic therapeutic agents, mainly due to the capacity of this enzyme to degrade extracellular matrix components, such as laminin, fibronectin and type I collagen, which is important to interfere in T. gondii host cell invasion.

In this work, a new weakly hemorrhagic metalloproteinase (BthMP) was purified from Bothrops moojeni snake venom. This enzyme was homogeneous by native and SDS-PAGE. It showed a polypeptide chain of 23.5kDa, pI=7.1, and N-terminal blocked. BthMP is comprised of high proteolytic activity on casein, fibrin and bovine fibrinogen, with no coagulating, esterase or phospholipase A(2) activities; it was inhibited by EDTA, EGTA and 1,10-phenanthroline and maintained its activity on pH from 7.0 to 9.0 and temperature from 5-40 degrees C. Assays with metal ions showed that Ca(2+) is an activator, whereas Zn(2+) and Hg(2+) inhibited about 50 and 80% of its activity, respectively. The edema evidenced the important role of the toxin in the inflammatory activity of the venom. BthMP also caused unclotting, and provoked histological alterations in the gastrocnemius muscle of mice inducing hemorrhage, necrosis and leukocytic infiltrate. The molecular mass and the inhibition assays suggest that the metalloproteinase BthMP belongs to class P-I of SVMPs.

A proteinase, named BmooMPalpha-I, from the venom of Bothrops moojeni, was purified by DEAE-Sephacel, Sephadex G-75 and heparin-agarose column chromatography. The enzyme was purified to homogeneity as judged by its migration profile in SDS-PAGE stained with coomassie blue, and showed a molecular mass of about 24.5 kDa. 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 metalloproteinases showed a high structural similarly, mainly among class P-IB proteases. The enzyme cleaves the Aalpha-chain of fibrinogen first, followed by the Bbeta-chain, and shows no effects on the gamma-chain. On fibrin, the enzyme hydrolyzed only the beta-chain, leaving the gamma-dimer apparently untouched. It was devoid of phospholipase A(2), hemorrhagic and thrombin-like activities. Like many venom enzymes, it is stable at pH values between 4 and 10 and stable at 70 degrees C for 15 min. The inhibitory effects of EDTA on the fibrinogenolytic activity suggest that BmooMPalpha-I is a metalloproteinase and inhibition by beta-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Aprotinin and benzamidine, specific serine proteinase inhibitors, had no effect on BmooMPalpha-I activity. Since the BmooMPalpha-I enzyme was found to cause defibrinogenation when administered i.p. on mice, it is expected that it may be of medical interest as a therapeutic agent in the treatment and prevention of arterial thrombosis.

Two fibrinogenolytic enzymes, Bothrops alternatus metalloprotease isoform (BaltMP)-I and II, were purified from Bothrops alternatus venom using Diethylaminoethyl (DEAE) Sephacel, Sephadex G-75 and Heparin-Agarose column chromatography. Purified BaltMP-I and II ran as single protein bands on analytical polyacrylamide gel electrophoresis and showed molecular weights of 29000 and 36000, respectively, under reducing conditions in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). BaltMP-II, but not BaltMP-I, displayed blood-clotting activity in bovine plasma, which was about 10-fold higher than that of the crude venom. Both enzymes were proteolytically active against bovine fibrinogen as substrate. When fibrinogen and each enzyme were incubated at 37°C, at a ratio of 1:100 (w/w), BaltMP-II cleaved preferentially the Aalpha -chain and more slowly the Bbeta -chain. The action of BaltMP-I was similar, but lower. None of the proteases degraded the gamma-chain of fibrinogen. The fibrinogenolytic activity of the enzymes was inhibited by 1,10-phenanthroline, suggesting they are metalloproteases. Since both enzymes were found to cause defibrinogenation when intraperitoneally (i.p.) administered to mice, they can be of medical interest as a therapeutic agent in the treatment and prevention of arterial thrombosis.

In this work we describe the isolation of a new l-amino acid oxidase (LAAO) referred to as BpirLAAO-I from Bothrops pirajai snake venom, which was highly purified using a combination of molecular exclusion, affinity, and hydrophobic chromatography steps. BpirLAAO-I homodimeric acid glycoprotein (approximate Mr and pI of 130,000 and 4.9, respectively) displays high specificity toward hydrophobic/aromatic amino acids, while deglycosylation does not alter its enzymatic activity. The N-terminal LAAO sequence of its first 49 amino acids presented a high similarity between a amino acid sequence with other LAAOs from: Bothrops spp., Crotalus spp., Calloselasma rhodostoma, Agkistrodon spp., Trimeresurus spp., Pseudechis australis, Oxyuranus scutellatus, and Notechis scutatus. BpirLAAO-I induces time-dependent platelet aggregation, mouse paw edema, cytotoxic activity against Escherichia coli, Pseudomonas aeruginosa, Leishmania sp., and tumor cells, and also a typical fago (M13mp18) DNA fragmentation. Platelet aggregation, leishmanicidal and antitumoral activities were reduced by catalase. Thus, BpirLAAO-I is a multifunctional protein with promising biotechnological and medical applications.

The cDNA encoding BthaTL, a serine peptidase from the venom of the snake Bothrops alternatus, was cloned and sequenced. The deduced primary structure shows over 62% of identity with snake venom thrombin-like enzymes (SVTLEs), molecules with high substrate specificity toward different natural substrates. Indeed, a phylogenetic reconstruction by two different methods clustered this enzyme close to other SVTLEs. These enzymes generally affect the hemostatic system in several ways, and therefore are used as tools in pharmacology and clinical diagnosis. A three-dimensional model of BthaTL was built by homology modeling using TSV-PA (Trimeresurus stejnegeri venom plasminogen activator) crystal structure as template. BthaTL model showed that the typical catalytic triad conformation of serine peptidases was preserved. The calcium coordination ligands were absent or adopt an unfavorable conformation, preventing interactions with metals. On the other hand, the Asp97–Arg174 saline bridge of TSV-PA was not found and its specificity determinant Phe193 is replaced by a Gly in BthaTL. The substitution of essential residues in the neighborhoods of the catalytic site cleft of BthaTL indicates that these two proteins do not share the same enzymatic specificity, what means that BthaTL will probably not activate plasminogen. Such observations may be helpful in the understanding of the molecular mechanism for substrate specificity of these enzymes.

The use of plants as medicine has been referred to since ancient peoples, perhaps as early as Neanderthal man. Plants are a source of many biologically active products and nowadays they are of great interest to the pharmaceutical industry. The study of how people of different culture use plants in particular ways has led to the discovery of important new medicines. In this work, we verify the possible activity of Musa paradisiaca L. (Musaceae) against the toxicity of snake venoms. Musa paradisiaca, an important source of food in the world, has also been reported to be popularly used as an anti-venom. Interaction of Musa paradisiaca extract (MsE) with snake venom proteins has been examined in this study. Phospholipase A2 (PLA2), myotoxic and hemorrhagic activities, including lethality in mice, induced by crotalidae venoms were significantly inhibited when different amounts of MsE were mixed with these venoms before assays. On the other hand, mice that received MsE and venoms without previous mixture or by separated routes were not protected against venom toxicity. Partial chemical characterization of MsE showed the presence of polyphenols and tannins and they are known to non-specifically inactivate proteins. We suggest that these compounds can be responsible for the in vitro inhibition of the toxic effects of snake venoms. In conclusion, according to our results, using mice as experimental model, MsE does not show protection against the toxic effects of snake venoms in vivo, but if was very effective when the experiments were done in vitro.

The aqueous extract from the leaves of Casearia mariquitensis (C. m.), a plant found in Brazilian open pastures, was assayed for its ability to inhibit some hematological and hemostatic effects induced by neuwiedase, a 22 kDa class P-I metalloproteinase from the venom of the South American pit viper Bothrops neuwiedi pauloensis. The aqueous extract from C. m. was able to neutralize the hematological alterations induced by the crude venom (C.V.) upon erythrocytes when the venom was incubated at a ratio of 1:10 (w/w, venom/extract), but it did not neutralize the platelet decreasing ability of C.V. The plasma fibrinogen concentration decreased approximately 36% and 83% when 0.6 LD(50) of the C.V. or neuwiedase, respectively, were injected by i.p. route in mice, and the aqueous extract from C. m. was able to inhibit this effect. The Bbeta fibrinogen chain was protected against degradation caused by crude venom and neuwiedase when the venom or toxin were incubated with C. m. extract. We also observed that this extract exerted a very slight effect on the clotting time, prolonging it only to a little extent. The pulmonary hemorrhage induced by neuwiedase when injected intravenously with 0.6 LD(50) was completely inhibited when this toxin was incubated with the extract at a ratio of 1:10 (w/w, toxin/extract). It is concluded that C. m. displays components able to inhibit some hematological and systemic alterations induced by C.V.

Background Hyaluronate is one of the major components of extracellular matrix from vertebrates whose breakdown is catalyzed by the enzyme hyaluronidase. These enzymes are widely described in snake venoms, in which they facilitate the spreading of the main toxins in the victim’s body during the envenoming. Snake venoms also present some variants (hyaluronidases-like substances) that are probably originated by alternative splicing, even though their relevance in envenomation is still under investigation. Hyaluronidases-like proteins have not yet been purified from any snake venom, but the cDNA that encodes these toxins was already identified in snake venom glands by transcriptomic analysis. Herein, we report the cloning and in silico analysis of the first hyaluronidase-like proteins from a Brazilian snake venom. Methods The cDNA sequence of hyaluronidase was cloned from the transcriptome of Bothrops pauloensis venom glands. This sequence was submitted to multiple alignment with other related sequences by ClustalW. A phylogenetic analysis was performed using MEGA 4 software by the neighbor joining (NJ) method. Results The cDNA from Bothrops pauloensis venom gland that corresponds to hyaluronidase comprises 1175 bp and codifies a protein containing 194 amino acid residues. The sequence, denominated BpHyase, was identified as hyaluronidase-like since it shows high sequence identities (above 83%) with other described snake venom hyaluronidase-like sequences. Hyaluronidases-like proteins are thought to be products of alternative splicing implicated in deletions of central amino acids, including the catalytic residues. Structure-based sequence alignment of BpHyase to human hyaluronidase hHyal-1 demonstrates a loss of some key secondary structures. The phylogenetic analysis indicates an independent evolution of BpHyal when compared to other hyaluronidases. However, these toxins might share a common ancestor, thus suggesting a broad hyaluronidase-like distribution among venomous snakes. Conclusions This work is the first report of a cDNA sequence of hyaluronidase from Brazilian snake venoms. Moreover, the in silico analysis of its deduced amino acid sequence opens new perspectives about the biological function of hyaluronidases-like proteins and may direct further studies comprising their isolation and/or recombinant production, as well as their structural and functional characterization.

In the present study, an acidic PLA(2), designated Bl-PLA(2), was isolated from Bothrops leucurus snake venom through two chromatographic steps: ion-exchange on CM-Sepharose and hydrophobic chromatography on Phenyl-Sepharose. Bl-PLA(2) was homogeneous on SDS-PAGE and when submitted to 2D electrophoresis the molecular mass was 15,000Da and pI was 5.4. Its N-terminal sequence revealed a high homology with other Asp49 acidic PLA(2)s from snake venoms. Its specific activity was 159.9U/mg and the indirect hemolytic activity was also higher than that of the crude venom. Bl-PLA(2) induced low myotoxic and edema activities as compared to those of the crude venom. Moreover, the enzyme was able to induce increments in IL-12p40, TNF-α, IL-1β and IL-6 levels and no variation of IL-8 and IL-10 in human PBMC stimulated in vitro, suggesting that Bl-PLA(2) induces proinflammatory cytokine production by human mononuclear cells. Bothrops leucurus venom is still not extensively explored and knowledge of its components will contribute for a better understanding of its action mechanism.