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Sample records for neuwiedi pauloensis jararaca

  1. Phylogenetic relationships within Bothrops neuwiedi group (Serpentes, Squamata): geographically highly-structured lineages, evidence of introgressive hybridization and Neogene/Quaternary diversification.

    PubMed

    Machado, Taís; Silva, Vinícius X; Silva, Maria José de J

    2014-02-01

    Eight current species of snakes of the Bothrops neuwiedi group are widespread in South American open biomes from northeastern Brazil to southeastern Argentina. In this paper, 140 samples from 93 different localities were used to investigate species boundaries and to provide a hypothesis of phylogenetic relationships among the members of this group based on 1122bp of cyt b and ND4 from mitochondrial DNA and also investigate the patterns and processes occurring in the evolutionary history of the group. Combined data recovered the B. neuwiedi group as a highly supported monophyletic group in maximum parsimony, maximum likelihood and Bayesian analyses, as well as four major clades (Northeast I, Northeast II, East-West, West-South) highly-structured geographically. Monophyly was recovered only for B. pubescens. By contrast, B. diporus, B. lutzi, B. erythromelas, B. mattogrossensis, B. neuwiedi, B. marmoratus, and B. pauloensis, as currently defined on the basis of morphology, were polyphyletic. Sympatry, phenotypic intergrades and shared mtDNA haplotypes, mainly between B. marmoratus and B. pauloensis suggest recent introgressive hybridization and the possible occurrence of a narrow hybrid zone in Central Brazil. Our data suggest at least three candidate species: B. neuwiedi from Espinhaço Range, B. mattogrossensis (TM173) from Serra da Borda (MT) and B. diporus (PT3404) from Castro Barros, Argentina. Divergence estimates highlight the importance of Neogene events in the origin of B. neuwiedi group, and the origin of species and diversification of populations of the Neotropical fauna from open biomes during the Quaternary climate fluctuations. Data reported here represent a remarkable increase of the B. neuwiedi group sampling size, since representatives of all the current recognized species from a wide geographic range are included in this study, providing basic information for understanding the evolution and conservation of Neotropical biodiversity. PMID:24140980

  2. Molecular cloning of a hyaluronidase from Bothrops pauloensis venom gland

    PubMed Central

    2014-01-01

    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

  3. Bothrops pauloensis snake venom toxins: the search for new therapeutic models.

    PubMed

    Rodrigues, Veridiana M; Lopes, Daiana S; Castanheira, Leticia E; Gimenes, Sarah N C; Naves de Souza, Dayane L; Ache, David C; Borges, Isabela P; Yoneyama, Kelly A G; Rodrigues, Renata S

    2015-01-01

    Snake venoms constitute a mixture of bioactive components that are involved not only in envenomation pathophysiology but also in the development of new drugs to treat many diseases. Different enzymatic and non-enzymatic proteins, such as phospholipases A2, hyaluronidases, L-amino acid oxidases, metalloproteinases, serine proteinases, lectins and disintegrins have been isolated and their functional and structural properties described in the literature. Many of these studies have also explored their medicinal potential focusing mainly on anticancer, antithrombotic and microbicide therapies. Bothrops pauloensis is a species found in Brazil, whose venom has been the focus of our studies in order to explore the biochemical and functional characteristics of their components. In this review, we have presented the main results of years of research on different toxins from B. pauloensis emphasizing their therapeutic potential. Studies concerning snake venom toxins to search for new therapeutic models open perspectives for new drug discovery.

  4. Coagulopathy after snake bite by Bothrops neuwiedi: case report and results of in vitro experiments.

    PubMed

    Dempfle, C E; Kohl, R; Harenberg, J; Kirschstein, W; Schlauch, D; Heene, D L

    1990-12-01

    Coagulation studies were performed in a patient who had been bitten by a snake of the species Bothrops neuwiedi. The patient presented with hemorrhagic necrosis at the envenomization site and considerable bleeding from venous puncture sites. He developed a severe defibrination syndrome with a clottable fibrinogen level of approximately 0.1 g/l. Fibrinogen was not measurable by clotting time assay. Fibrin degradation products were greatly elevated. Treatment with antivenom caused an anaphylactic reaction within ten minutes and serum sickness after three days. In vitro experiments revealed that B. neuwiedi venom directly activates Factors II and X, but does not activate Factor XIII. In vivo consumption of Factor XIII after B. neuwiedi envenomization is ascribed to the action of Factor IIa. At low venom concentrations clotting is initiated by activation of prothrombin by the venom either directly or via Factor X activation. Treatment with heparin might be beneficial in coagulopathy secondary to snake bite by reducing circulating active thrombin. The venom contains thrombin-like proteases which cause slow clotting of fibrinogen, and plasmin-like components causing further proteolysis of fibrinogen and fibrin. Antivenom has no effect on the proteolytic action of the snake venom. The in vivo effects of antivenom are presumably caused by acceleration of the elimination of venom components from the circulation. Intravenous administration of antivenom caused normalization of blood coagulation parameters within 48 h. PMID:2291986

  5. Bradykinin is involved in hyperalgesia induced by Bothrops jararaca venom.

    PubMed

    Chacur, M; Picolo, G; Teixeira, C F P; Cury, Y

    2002-07-01

    Bradykinin is involved in hyperalgesia (pain hypersensitivity) induced by Bothrops jararaca venom-intraplantar injection of B. jararaca venom (5microg/paw) in rats caused hyperalgesia, which peaked 1h after venom injection. This phenomenon was not modified by promethazine (H(1) receptor antagonist), methysergide (5-HT receptor antagonist), guanethidine (sympathetic function inhibitor), anti-TNF-alpha or anti-interleukin-1 antibodies or by the chelating agent CaNa(2)EDTA. Venom-induced hyperalgesia was blocked by the bradykinin B(2) receptor antagonist HOE 140. On the other hand, des-Arg(9), [Leu(8)]-bradykinin, a bradykinin B(1) receptor antagonist, did not modify the hyperalgesic response. These results suggest that bradykinin, acting on B(2) receptor, is a mediator of hyperalgesia induced by B. jararaca venom.

  6. Bothrops jararaca envenomation: Pathogenesis of hemostatic disturbances and intravascular hemolysis

    PubMed Central

    Senise, Luana V; Yamashita, Karine M

    2015-01-01

    To attain fully functional biological activity, vitamin-K dependent coagulation factors (VKDCF) are γ-carboxylated prior to secretion from liver. Warfarin impairs the γ-carboxylation, and consequently their physiological function. Bothrops jararaca snake venom (BjV) contains several activators of blood coagulation, especially procoagulant enzymes (prothrombin and factor X activators) and thrombin-like enzymes. In order to clarify the relative contribution of prothrombin and factor X activators to the hemostatic disturbances occurring during experimental B. jararaca envenomation, warfarin was used to deplete VKDCF, prior to BjV administration. Male Wistar rats were pretreated with saline (Sal) or warfarin (War) and inoculated subsequently with BjV or saline, thus forming four groups: Sal + Sal (negative control), Sal + BjV (positive control), War + Sal (warfarinization control), and War + BjV. Three hours after inoculation, prothrombin and factor X levels fell 40% and 50%, respectively; levels of both factors decreased more than 97% in the War + Sal and War + BjV groups. Platelet counts dropped 93% and 76% in Sal + BjV and War + BjV, respectively, and plasma fibrinogen levels decreased 86% exclusively in Sal + BjV. After 6 and 24 h, platelet counts and fibrinogen levels increased progressively. A dramatic augmentation in plasma hemoglobin levels and the presence of schizocytes and microcytes in the Sal + BjV group indicated the development of intravascular hemolysis, which was prevented by warfarin pretreatment. Our findings show that intravascular thrombin generation has the foremost role in the pathogenesis of coagulopathy and intravascular hemolysis, but not in the development of thrombocytopenia, in B. jararaca envenomation in rats; in addition, fibrinogenases (metalloproteinases) may contribute to coagulopathy more than thrombin-like enzymes. PMID:26080462

  7. Bp-13 PLA2: Purification and Neuromuscular Activity of a New Asp49 Toxin Isolated from Bothrops pauloensis Snake Venom

    PubMed Central

    Sucasaca-Monzón, Georgina; Randazzo-Moura, Priscila; Rocha, Thalita; Vilca-Quispe, Augusto; Ponce-Soto, Luis Alberto; Marangoni, Sérgio; da Cruz-Höfling, Maria Alice; Rodrigues-Simioni, Léa

    2015-01-01

    A new PLA2 (Bp-13) was purified from Bothrops pauloensis snake venom after a single chromatographic step of RP-HPLC on μ-Bondapak C-18. Amino acid analysis showed a high content of hydrophobic and basic amino acids and 14 half-cysteine residues. The N-terminal sequence showed a high degree of homology with basic Asp49 PLA2 myotoxins from other Bothrops venoms. Bp-13 showed allosteric enzymatic behavior and maximal activity at pH 8.1, 36°–45°C. Full Bp-13 PLA2 activity required Ca2+; its PLA2 activity was inhibited by Mg2+, Mn2+, Sr2+, and Cd2+ in the presence and absence of 1 mM Ca2+. In the mouse phrenic nerve-diaphragm (PND) preparation, the time for 50% paralysis was concentration-dependent (P < 0.05). Both the replacement of Ca2+ by Sr2+ and temperature lowering (24°C) inhibited the Bp-13 PLA2-induced twitch-tension blockade. Bp-13 PLA2 inhibited the contractile response to direct electrical stimulation in curarized mouse PND preparation corroborating its contracture effect. In biventer cervicis preparations, Bp-13 induced irreversible twitch-tension blockade and the KCl evoked contracture was partially, but significantly, inhibited (P > 0.05). The main effect of this new Asp49 PLA2 of Bothrops pauloensis venom is on muscle fiber sarcolemma, with avian preparation being less responsive than rodent preparation. The study enhances biochemical and pharmacological characterization of B. pauloensis venom. PMID:25789175

  8. Hypericum brasiliense plant extract neutralizes some biological effects of Bothrops jararaca snake venom.

    PubMed

    Assafim, Mariane; de Coriolano, Eduardo Coriolano; Benedito, Sérgio Eufrázio; Fernandes, Caio Pinho; Lobo, Jonathas Felipe Revoredo; Sanchez, Eladio Florez; Rocha, Leandro Machado; Fuly, André Lopes

    2011-01-01

    Alternative treatments for snake bite are currently being extensively studied, and plant metabolites are considered good candidates for such purpose. Here, the ability of a crude ethanolic extract of Hypericum brasiliense plant in neutralizing Bothrops jararaca snake venom was investigated by in vitro (coagulation, hemolysis or proteolysis) and in vivo (hemorrhage, lethality and edema) biological assays. We describe for the first time the ability of H. brasiliense extracts to inhibit some pharmacological effects of a Brazilian snake venom. Inhibitory assays were performed by incubating B. jararaca venom with H. brasiliense extracts for 30min at room temperature before the assays were performed. The results showed that H. brasiliense extracts impaired lethality, edema, hemorrhage, hemolysis, proteolysis as well as fibrinogen or plasma clotting induced by B. jararaca venom. This indicates that H. brasiliense extracts can provide promising agents to treat B. jararaca envenomation.

  9. Ontogenetic Variation in Biological Activities of Venoms from Hybrids between Bothrops erythromelas and Bothrops neuwiedi Snakes.

    PubMed

    Santoro, Marcelo Larami; do Carmo, Thaís; Cunha, Bruna Heloísa Lopes; Alves, André Fonseca; Zelanis, André; Serrano, Solange Maria de Toledo; Grego, Kathleen Fernandes; Sant'Anna, Savio Stefanini; Barbaro, Katia Cristina; Fernandes, Wilson

    2015-01-01

    Lance-headed snakes are found in Central and South America, and they account for most snakebites in Brazil. The phylogeny of South American pitvipers has been reviewed, and the presence of natural and non-natural hybrids between different species of Bothrops snakes demonstrates that reproductive isolation of several species is still incomplete. The present study aimed to analyze the biological features, particularly the thrombin-like activity, of venoms from hybrids born in captivity, from the mating of a female Bothrops erythromelas and a male Bothrops neuwiedi, two species whose venoms are known to display ontogenetic variation. Proteolytic activity on azocoll and amidolytic activity on N-benzoyl-DL-arginine-p-nitroanilide hydrochloride (BAPNA) were lowest when hybrids were 3 months old, and increased over body growth, reaching values similar to those of the father when hybrids were 12 months old. The clotting activity on plasma diminished as hybrids grew; venoms from 3- and 6-months old hybrids showed low clotting activity on fibrinogen (i.e., thrombin-like activity), like the mother venom, and such activity was detected only when hybrids were older than 1 year of age. Altogether, these results point out that venom features in hybrid snakes are genetically controlled during the ontogenetic development. Despite the presence of the thrombin-like enzyme gene(s) in hybrid snakes, they are silenced during the first six months of life. PMID:26714190

  10. Ontogenetic Variation in Biological Activities of Venoms from Hybrids between Bothrops erythromelas and Bothrops neuwiedi Snakes

    PubMed Central

    Santoro, Marcelo Larami; do Carmo, Thaís; Cunha, Bruna Heloísa Lopes; Alves, André Fonseca; Zelanis, André; Serrano, Solange Maria de Toledo; Grego, Kathleen Fernandes; Sant’Anna, Savio Stefanini; Barbaro, Katia Cristina; Fernandes, Wilson

    2015-01-01

    Lance-headed snakes are found in Central and South America, and they account for most snakebites in Brazil. The phylogeny of South American pitvipers has been reviewed, and the presence of natural and non-natural hybrids between different species of Bothrops snakes demonstrates that reproductive isolation of several species is still incomplete. The present study aimed to analyze the biological features, particularly the thrombin-like activity, of venoms from hybrids born in captivity, from the mating of a female Bothrops erythromelas and a male Bothrops neuwiedi, two species whose venoms are known to display ontogenetic variation. Proteolytic activity on azocoll and amidolytic activity on N-benzoyl-DL-arginine-p-nitroanilide hydrochloride (BAPNA) were lowest when hybrids were 3 months old, and increased over body growth, reaching values similar to those of the father when hybrids were 12 months old. The clotting activity on plasma diminished as hybrids grew; venoms from 3- and 6-months old hybrids showed low clotting activity on fibrinogen (i.e., thrombin-like activity), like the mother venom, and such activity was detected only when hybrids were older than 1 year of age. Altogether, these results point out that venom features in hybrid snakes are genetically controlled during the ontogenetic development. Despite the presence of the thrombin-like enzyme gene(s) in hybrid snakes, they are silenced during the first six months of life. PMID:26714190

  11. Cloning, Characterization and Anti-Inflammatory Properties of Bothrops jararaca Snake Antithrombin.

    PubMed

    Morais-Zani, Karen de; Grego, Kathleen F; Torquato, Ricardo J S; Silva, Caroline S; Tanaka, Aparecida S; Tanaka-Azevedo, Anita M

    2015-01-01

    Antithrombin inhibits blood coagulation through the interaction with serine proteases in both intrinsic and extrinsic pathways. In addition, antithrombin also shows anti-inflammatory properties, which are independent of its effects on coagulation. This work shows for the first time the cloning and sequencing of antithrombin from a snake species. This predicted protein is composed by 430 amino acids and presents about 64.5% sequence identity to human antithrombin. Biacore experiments revealed that the binding affinity of Bothrops jararaca snake antithrombin to heparin was ~30 times higher than that of human antithrombin. Furthermore, Bothrops jararaca antithrombin is more effective in preventing acute inflammation induced by carrageenan when compared to human antithrombin. Hence, the results showed herein suggest that Bothrops jararaca antithrombin can play a key role in the control of acute inflammation and that this molecule might be used as a pharmacological tool and as a prototype for drug development. PMID:25687119

  12. Inhibitory effect of the plant Clusia fluminensis against biological activities of Bothrops jararaca snake venom.

    PubMed

    de Oliveira, Eduardo Coriolano; Anholeti, Maria Carolina; Domingos, Thaisa Francielle; Faioli, Camila Nunes; Sanchez, Eladio Flores; de Paiva, Selma Ribeiro; Fuly, André Lopes

    2014-01-01

    The ability of extracts of the plant Clusia fluminensis Planch & Triana (Clusiaceae Lindl.) to neutralize proteolysis, clotting, hemolysis, hemorrhagic and lethality activities of Bothrops jararaca snake venom was studied. Clusianone and lanosterol from the flower and fruit extracts, respectively, were also tested. The extracts of different organs of C. fluminensis inhibited proteolysis and hemolysis induced by B. jararaca venom, but with different potencies. Only the stems prevented blood clotting. Only the acetone extract of the fruit protected mice from hemorrhage while the acetone or methanol extracts prevented mice from death. Clusianone and lanosterol did not inhibit clotting or hemorrhage, but the former inhibited proteolysis and the latter hemolysis.

  13. Appraisal of Antiophidic Potential of Marine Sponges against Bothrops jararaca and Lachesis muta Venom

    PubMed Central

    Faioli, Camila Nunes; Domingos, Thaisa Francielle Souza; de Oliveira, Eduardo Coriolano; Sanchez, Eládio Flores; Ribeiro, Suzi; Muricy, Guilherme; Fuly, Andre Lopes

    2013-01-01

    Snakebites are a health problem in many countries due to the high incidence of such accidents. Antivenom treatment has regularly been used for more than a century, however, this does not neutralize tissue damage and may even increase the severity and morbidity of accidents. Thus, it has been relevant to search for new strategies to improve antiserum therapy, and a variety of molecules from natural sources with antiophidian properties have been reported. In this paper, we analyzed the ability of ten extracts from marine sponges (Amphimedon viridis, Aplysina fulva, Chondrosia collectrix, Desmapsamma anchorata, Dysidea etheria, Hymeniacidon heliophila, Mycale angulosa, Petromica citrina, Polymastia janeirensis, and Tedania ignis) to inhibit the effects caused by Bothrops jararaca and Lachesis muta venom. All sponge extracts inhibited proteolysis and hemolysis induced by both snake venoms, except H. heliophila, which failed to inhibit any biological activity. P. citrina inhibited lethality, hemorrhage, plasma clotting, and hemolysis induced by B. jararaca or L. muta. Moreover, other sponges inhibited hemorrhage induced only by B. jararaca. We conclude that Brazilian sponges may be a useful aid in the treatment of snakebites caused by L. muta and B. jararaca and therefore have potential for the discovery of molecules with antiophidian properties. PMID:24141284

  14. Biochemical and functional characterization of a C-type lectin (BpLec) from Bothrops pauloensis snake venom.

    PubMed

    Castanheira, Letícia Eulálio; Nunes, Débora Cristina de Oliveira; Cardoso, Thomaz Monteiro; Santos, Paula de Souza; Goulart, Luiz Ricardo; Rodrigues, Renata Santos; Richardson, Michael; Borges, Márcia Helena; Yoneyama, Kelly Aparecida Geraldo; Rodrigues, Veridiana M

    2013-03-01

    In the present work, we report the isolation and partial biochemical characterization of BpLec, a C-type lectin purified from Bothrops pauloensis venom by one chromatographic step on an affinity agarose column immobilized with d-galactose. This protein was homogeneous by SDS-PAGE under reducing and nonreducing conditions, and was shown to be a 33.6 kDa homodimer by MALDI TOF analysis. BpLec presented an isoeletric point of 5.36. Its partial sequence of 132 amino acids for each subunit, determined by Edman degradation, revealed high identity (between 86% and 95%) when aligned with sequences of other related proteins. BpLec was capable of agglutinating native dog and cat erythrocytes and this activity was inhibited by β-galactosides and EDTA. Its hemagglutinating activity was abolished at high temperatures and stable in any pH range. BpLec was effective in inhibiting Gram-positive but not Gram-negative bacteria. In addition, BpLec agglutinated promastigote forms of Leishmania (Leishmania) amazonensis. PMID:23178369

  15. Insights into anti-parasitism induced by a C-type lectin from Bothrops pauloensis venom on Toxoplasma gondii.

    PubMed

    Castanheira, Letícia; Naves de Souza, Dayane Lorena; Silva, Rafaela José; Barbosa, Bellisa; Mineo, José Roberto; Tudini, Kelly Aparecida; Rodrigues, Renata; Ferro, Eloísa Vieira; de Melo Rodrigues, Veridiana

    2015-03-01

    Here we evaluate the effects of BpLec, a C-type lectin isolated from Bothrops pauloensis snake venom, on Toxoplasma gondii parasitism. BpLec (0.195-12.5 μg/mL) did not interfere with HeLa (host cell) viability by MTT assay, whereas higher doses decreased viability and changed HeLa morphology. In addition, the host cell treatment before infection did not influence adhesion and proliferation indexes. BpLec did not alter T. gondii tachyzoite viability, as carried out by trypan blue exclusion, but decreased both adhesion and parasite replication, when tachyzoites were treated before infection. Galactose (0.4 M) inhibited the BpLec effect on adhesion assays, suggesting that BpLec probably recognize some glycoconjugate from T. gondii membrane. Additionally, we performed cytokine measurements from supernatants collected from HeLa cells infected with T. gondii tachyzoites previously treated with RPMI or BpLec. MIF and IL-6 productions by HeLa cells were increased by BpLec treatment. Also, TGF-β1 secretion was diminished post-infection, although this effect was not dependent on BpLec treatment. Taken together, our results show that BpLec is capable of reducing T. gondii parasitism after tachyzoite treatment and may represent an interesting tool in the search for parasite antigens involved in these processes. PMID:25541358

  16. Proteomic Analysis of the Ontogenetic Variability in Plasma Composition of Juvenile and Adult Bothrops jararaca Snakes

    PubMed Central

    de Morais-Zani, Karen; Grego, Kathleen Fernandes; Tanaka, Aparecida Sadae; Tanaka-Azevedo, Anita Mitico

    2013-01-01

    The ontogenetic variability in venom composition of some snake genera, including Bothrops, as well as the biological implications of such variability and the search of new molecules that can neutralize the toxic components of these venoms have been the subject of many studies. Thus, considering the resistance of Bothrops jararaca to the toxic action of its own venom and the ontogenetic variability in venom composition described in this species, a comparative study of the plasma composition of juvenile and adult B. jararaca snakes was performed through a proteomic approach based on 2D electrophoresis and mass spectrometry, which allowed the identification of proteins that might be present at different levels during ontogenetic development. Among the proteins identified by mass spectrometry, antihemorrhagic factor Bj46a was found only in adult plasma. Moreover, two spots identified as phospholipase A2 inhibitors were significantly increased in juvenile plasma, which can be related to the higher catalytic PLA2 activity shown by juvenile venom in comparison to that of adult snakes. This work shows the ontogenetic variability of B. jararaca plasma, and that these changes can be related to the ontogenetic variability described in its venom. PMID:24062950

  17. Anti-hemorrhagic activity of four Brazilian vegetable species against Bothrops jararaca venom.

    PubMed

    Nishijima, Catarine Massucato Nishijima; Rodrigues, Clenilson Martins; Silva, Marcelo Aparecido; Lopes-Ferreira, Mônica; Vilegas, Wagner; Hiruma-Lima, Clélia Akiko

    2009-03-09

    Around 20,000 snakebites are reported annually in Brazil and 90% of them are inflicted by species of the genus Bothrops. Intravenous administration of antibothropic antivenom neutralizes the systemic actions, but it is of little effect on the reversal of local symptoms and often induces adverse reactions, a context that drives the search for complementary treatments for snakebite accidents. Vegetable extracts with a range of antiophidian activities constitute an excellent alternative. In this study, we investigated the anti-hemorrhagic effects of Mouriri pusa Gardn. (Melastomataceae), Byrsonima crassa Niedenzu (Malpighiaceae), Davilla elliptica St. Hill. (Dilleniaceae) and Strychnos pseudoquina St. Hil. (Loganiaceae) against Bothrops jararaca venom. The methanolic extracts from M. pusa (leaves), B. crassa (leaves) and D. elliptica (leaves) showed total neutralization capacity against local hemorrhages. The amenthoflavone and quercetin fractions from B. crassa and the flavonoids fractions (quercetin and myricetin) from M. pusa and D. elliptica also showed total neutralization capacity. We conclude that flavonoids derived from myricetin, quercetin and amenthoflavone play an important role in the anti-hemorrhagic potential of these Brazilian vegetables species against B. jararaca venom.

  18. Phylogeography of the Bothrops jararaca complex (Serpentes: Viperidae): past fragmentation and island colonization in the Brazilian Atlantic Forest.

    PubMed

    Grazziotin, Felipe G; Monzel, Markus; Echeverrigaray, Sergio; Bonatto, Sandro L

    2006-11-01

    The Brazilian Atlantic Forest is one of the world's major biodiversity hotspots and is threatened by a severe habitat loss. Yet little is known about the processes that originated its remarkable richness of endemic species. Here we present results of a large-scale survey of the genetic variation at the mitochondrial cytochrome b gene of the pitviper, jararaca lancehead (Bothrops jararaca), and two closely related insular species (Bothrops insularis and Bothrops alcatraz), endemic of this region. Phylogenetic and network analyses revealed the existence of two well-supported clades, exhibiting a southern and a northern distribution. The divergence time of these two phylogroups was estimated at 3.8 million years ago, in the Pliocene, a period of intense climatic changes and frequent fragmentation of the tropical rainforest. Our data also suggest that the two groups underwent a large size expansion between 50,000 and 100,000 years ago. However, the southern group showed a more marked signal of population size fluctuation than the northern group, corroborating evidences that southern forests may have suffered a more pronounced reduction in area in the late Pleistocene. The insular species B. alcatraz and B. insularis presented very low diversity, each one sharing haplotypes with mainland individuals placed in different subclades. Despite their marked morphological and behavioural uniqueness, these two insular species seem to have originated very recently and most likely from distinct costal B. jararaca populations, possibly associated with late Pleistocene or Holocene sea level fluctuations. PMID:17054497

  19. Structural and functional properties of Bp-LAAO, a new L-amino acid oxidase isolated from Bothrops pauloensis snake venom.

    PubMed

    Rodrigues, Renata S; da Silva, Juliana F; Boldrini França, Johara; Fonseca, Fernando P P; Otaviano, Antônio R; Henrique Silva, Flávio; Hamaguchi, Amélia; Magro, Angelo J; Braz, Antônio Sérgio K; dos Santos, Juliana I; Homsi-Brandeburgo, Maria Inês; Fontes, Marcos R M; Fuly, André L; Soares, Andreimar M; Rodrigues, Veridiana M

    2009-04-01

    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. PMID:19135502

  20. BnSP-7 toxin, a basic phospholipase A2 from Bothrops pauloensis snake venom, interferes with proliferation, ultrastructure and infectivity of Leishmania (Leishmania) amazonensis.

    PubMed

    Nunes, Débora C O; Figueira, Márcia M N R; Lopes, Daiana S; De Souza, Dayane L Naves; Izidoro, Luiz Fernando M; Ferro, Eloísa A V; Souza, Maria A; Rodrigues, Renata S; Rodrigues, Veridiana M; Yoneyama, Kelly A G

    2013-06-01

    This paper reports the effects of BnSP-7 toxin, a catalytically inactive phospholipase A2 from Bothrops pauloensis snake venom, on Leishmania (Leishmania) amazonensis. BnSP-7 presented activity against promastigote parasite forms both in the MTT assay, with IC50 of 58.7 μg mL(-1) of toxin, and a growth curve, inhibiting parasite proliferation 60-70% at concentrations of 50-200 μg mL(-1) of toxin 96 h after treatment. Also, the toxin presented effects on amastigotes, reducing parasite viability by 50% at 28.1 μg mL(-1) and delaying the amastigote-promastigote differentiation process. Ultrastructural studies showed that BnSP-7 caused severe morphological changes in promastigotes such as mitochondrial swelling, nuclear alteration, vacuolization, acidocalcisomes, multiflagellar aspects and a blebbing effect in the plasma membrane. Finally, BnSP-7 interfered with the infective capacity of promastigotes in murine peritoneal macrophages, causing statistically significant infectivity-index reductions (P < 0.05) of 20-35%. These data suggest that the BnSP-7 toxin is an important tool for the discovery of new parasite targets that can be exploited to develop new drugs for treating leishmaniasis. PMID:23442579

  1. Combined venomics, venom gland transcriptomics, bioactivities, and antivenomics of two Bothrops jararaca populations from geographic isolated regions within the Brazilian Atlantic rainforest.

    PubMed

    Gonçalves-Machado, Larissa; Pla, Davinia; Sanz, Libia; Jorge, Roberta Jeane B; Leitão-De-Araújo, Moema; Alves, Maria Lúcia M; Alvares, Diego Janisch; De Miranda, Joari; Nowatzki, Jenifer; de Morais-Zani, Karen; Fernandes, Wilson; Tanaka-Azevedo, Anita Mitico; Fernández, Julián; Zingali, Russolina B; Gutiérrez, José María; Corrêa-Netto, Carlos; Calvete, Juan J

    2016-03-01

    Bothrops jararaca is a slender and semi-arboreal medically relevant pit viper species endemic to tropical and subtropical forests in southern Brazil, Paraguay, and northern Argentina (Misiones). Within its geographic range, it is often abundant and is an important cause of snakebite. Although no subspecies are currently recognized, geographic analyses have revealed the existence of two well-supported B. jararaca clades that diverged during the Pliocene ~3.8Mya and currently display a southeastern (SE) and a southern (S) Atlantic rainforest (Mata Atlântica) distribution. The spectrum, geographic variability, and ontogenetic changes of the venom proteomes of snakes from these two B. jararaca phylogroups were investigated applying a combined venom gland transcriptomic and venomic analysis. Comparisons of the venom proteomes and transcriptomes of B. jararaca from the SE and S geographic regions revealed notable interpopulational variability that may be due to the different levels of population-specific transcriptional regulation, including, in the case of the southern population, a marked ontogenetic venom compositional change involving the upregulation of the myotoxic PLA2 homolog, bothropstoxin-I. This population-specific marker can be used to estimate the proportion of venom from the southern population present in the B. jararaca venom pool used for the Brazilian soro antibotrópico (SAB) antivenom production. On the other hand, the southeastern population-specific D49-PLA2 molecules, BinTX-I and BinTX-II, lend support to the notion that the mainland ancestor of Bothrops insularis was originated within the same population that gave rise to the current SE B. jararaca phylogroup, and that this insular species endemic to Queimada Grande Island (Brazil) expresses a pedomorphic venom phenotype. Mirroring their compositional divergence, the two geographic B. jararaca venom pools showed distinct bioactivity profiles. However, the SAB antivenom manufactured in Vital Brazil

  2. Effect of diterpenes isolated of the marine alga Canistrocarpus cervicornis against some toxic effects of the venom of the bothrops jararaca snake.

    PubMed

    Domingos, Thaisa Francielle Souza; Vallim, Magui Aparecida; Cavalcanti, Diana Negrão; Sanchez, Eládio Flores; Teixeira, Valéria Laneuville; Fuly, André Lopes

    2015-01-01

    Snake venoms are composed of a complex mixture of active proteins and peptides which induce a wide range of toxic effects. Envenomation by Bothrops jararaca venom results in hemorrhage, edema, pain, tissue necrosis and hemolysis. In this work, the effect of a mixture of two secodolastane diterpenes (linearol/isolinearol), previously isolated from the Brazilian marine brown alga, Canistrocarpus cervicornis, was evaluated against some of the toxic effects induced by B. jararaca venom. The mixture of diterpenes was dissolved in dimethylsulfoxide and incubated with venom for 30 min at room temperature, and then several in vivo (hemorrhage, edema and lethality) and in vitro (hemolysis, plasma clotting and proteolysis) assays were performed. The diterpenes inhibited hemolysis, proteolysis and hemorrhage, but failed to inhibit clotting and edema induced by B. jararaca venom. Moreover, diterpenes partially protected mice from lethality caused by B. jararaca venom. The search for natural inhibitors of B. jararaca venom in C. cervicornis algae is a relevant subject, since seaweeds are a rich and powerful source of active molecules which are as yet but poorly explored. Our results suggest that these diterpenes have the potential to be used against Bothropic envenomation accidents or to improve traditional treatments for snake bites.

  3. Effect of photobiomodulation on endothelial cell exposed to Bothrops jararaca venom.

    PubMed

    Franco, Ana Tereza Barufi; Silva, Luciana Miato Gonçalves; Costa, Marcília Silva; Zamuner, Silvia Fernanda; Vieira, Rodolfo Paula; de Fatima Pereira Teixeira, Catarina; Zamuner, Stella Regina

    2016-07-01

    Bleeding is a common feature in envenoming caused by Bothrops snake venom due to extensive damage to capillaries and venules, producing alterations in capillary endothelial cell morphology. It has been demonstrated, in vivo, that photobiomodulation (PBM) decreases hemorrhage after venom inoculation; however, the mechanism is unknown. Thus, the objective was to investigate the effects of PBM on a murine endothelial cell line (tEnd) exposed to Bothrops jararaca venom (BjV). Cells were exposed to BjV and irradiated once with either 660- or 780-nm wavelength laser light at energy densities of 4 and 5 J/cm(2), respectively, and irradiation time of 10 s. Cell integrity was analyzed by crystal violet and cell viability/mitochondrial metabolism by MTT assay. The release of lactic dehydrogenase (LDH) was quantified as a measure of cell damage. In addition, cytokine IL1-β levels were measured in the supernatant. PBM at 660 and 780 nm wavelength was able to increase cellular viability and decrease the release of LDH and the loss of cellular integrity. In addition, the concentration of pro-inflammatory cytokine IL1-β was reduced after PBM by both wavelengths. The data reported herein indicates that irradiation with red or near-infrared laser resulted in protection on endothelial cells after exposure to Bothrops venom and could be, at least in part, a reasonable explanation by the beneficial effects of PBM inhibiting the local effects induced by Bothrops venoms, in vivo. PMID:27147074

  4. Anti-parasitic effect on Toxoplasma gondii induced by BnSP-7, a Lys49-phospholipase A2 homologue from Bothrops pauloensis venom.

    PubMed

    Borges, Isabela Pacheco; Castanheira, Letícia Eulalio; Barbosa, Bellisa Freitas; de Souza, Dayane Lorena Naves; da Silva, Rafaela José; Mineo, José Roberto; Tudini, Kelly Aparecida Yoneyama; Rodrigues, Renata Santos; Ferro, Eloísa Amália Vieira; de Melo Rodrigues, Veridiana

    2016-09-01

    Toxoplasmosis affects a third of the global population and presents high incidence in tropical areas. Its great relevance in public health has led to a search for new therapeutic approaches. Herein, we report the antiparasitic effects of BnSP-7 toxin, a Lys49 phospholipase A2 (PLA2) homologue from Bothrops pauloensis snake venom, on Toxoplasma gondii. In an MTT assay, BnSP-7 presented significant cytotoxicity against host HeLa cells at higher doses (200 μg/mL to 50 μg/mL), whereas lower doses (25 μg/mL to 1.56 μg/mL) produced low cytotoxicity. Furthermore, the toxin showed no effect on T. gondii tachyzoite viability when evaluated by trypan blue exclusion, but decreased both adhesion and parasite proliferation when tachyzoites were treated before infection. We also measured cytokines in supernatants collected from HeLa cells infected with T. gondii tachyzoites previously treated with RPMI or BnSP-7, which revealed enhancement of only MIF and IL-6 cytokines levels in supernatants of HeLa cells after BnSP-7 treatment. Our results showed that the BnSP-7 PLA2 exerts an anti-Toxoplasma effect at a lower dose than that required to induce cytotoxicity in HeLa cells, and also modulates the immune response of host cells. In this sense, the anti-parasitic effect of BnSP-7 PLA2 demonstrated in the present study opens perspectives for use of this toxin as a tool for future studies on toxoplasmosis.

  5. Anti-parasitic effect on Toxoplasma gondii induced by BnSP-7, a Lys49-phospholipase A2 homologue from Bothrops pauloensis venom.

    PubMed

    Borges, Isabela Pacheco; Castanheira, Letícia Eulalio; Barbosa, Bellisa Freitas; de Souza, Dayane Lorena Naves; da Silva, Rafaela José; Mineo, José Roberto; Tudini, Kelly Aparecida Yoneyama; Rodrigues, Renata Santos; Ferro, Eloísa Amália Vieira; de Melo Rodrigues, Veridiana

    2016-09-01

    Toxoplasmosis affects a third of the global population and presents high incidence in tropical areas. Its great relevance in public health has led to a search for new therapeutic approaches. Herein, we report the antiparasitic effects of BnSP-7 toxin, a Lys49 phospholipase A2 (PLA2) homologue from Bothrops pauloensis snake venom, on Toxoplasma gondii. In an MTT assay, BnSP-7 presented significant cytotoxicity against host HeLa cells at higher doses (200 μg/mL to 50 μg/mL), whereas lower doses (25 μg/mL to 1.56 μg/mL) produced low cytotoxicity. Furthermore, the toxin showed no effect on T. gondii tachyzoite viability when evaluated by trypan blue exclusion, but decreased both adhesion and parasite proliferation when tachyzoites were treated before infection. We also measured cytokines in supernatants collected from HeLa cells infected with T. gondii tachyzoites previously treated with RPMI or BnSP-7, which revealed enhancement of only MIF and IL-6 cytokines levels in supernatants of HeLa cells after BnSP-7 treatment. Our results showed that the BnSP-7 PLA2 exerts an anti-Toxoplasma effect at a lower dose than that required to induce cytotoxicity in HeLa cells, and also modulates the immune response of host cells. In this sense, the anti-parasitic effect of BnSP-7 PLA2 demonstrated in the present study opens perspectives for use of this toxin as a tool for future studies on toxoplasmosis. PMID:27212627

  6. N-terminal amino acid sequences and some characteristics of fibrinolytic/hemorrhagic metalloproteinases purified from Bothrops jararaca venom.

    PubMed

    Maruyama, Masugi; Sugiki, Masahiko; Anai, Keita; Yoshida, Etsuo

    2002-08-01

    We determined the N-terminal amino acid sequences of the fibrinolytic/hemorrhagic metalloproteinases (jararafibrases I, III and IV) purified from Bothrops jararaca venom. The N-terminal amino acid sequences of jararafibrase I and its degradation products were identical to those of jararhagin, another hemorrhagic metalloproteinase purified from the same snake venom. Together with enzymatic and immunological properties, we concluded that those two enzymes are identical. The N-terminal amino acid sequence of jararafibrase III was quite similar to C-type lectin isolated from Crotalus atrox, and the protein had a hemagglutinating activity on intact rat red blood cells. PMID:12165326

  7. Bothrops jararaca Venom Metalloproteinases Are Essential for Coagulopathy and Increase Plasma Tissue Factor Levels during Envenomation

    PubMed Central

    Yamashita, Karine M.; Alves, André F.; Barbaro, Katia C.; Santoro, Marcelo L.

    2014-01-01

    Background/Aims Bleeding tendency, coagulopathy and platelet disorders are recurrent manifestations in snakebites occurring worldwide. We reasoned that by damaging tissues and/or activating cells at the site of the bite and systemically, snake venom toxins might release or decrypt tissue factor (TF), resulting in activation of blood coagulation and aggravation of the bleeding tendency. Thus, we addressed (a) whether TF and protein disulfide isomerase (PDI), an oxireductase involved in TF encryption/decryption, were altered in experimental snake envenomation; (b) the involvement and significance of snake venom metalloproteinases (SVMP) and serine proteinases (SVSP) to hemostatic disturbances. Methods/Principal Findings Crude Bothrops jararaca venom (BjV) was preincubated with Na2-EDTA or AEBSF, which are inhibitors of SVMP and SVSP, respectively, and injected subcutaneously or intravenously into rats to analyze the contribution of local lesion to the development of hemostatic disturbances. Samples of blood, lung and skin were collected and analyzed at 3 and 6 h. Platelet counts were markedly diminished in rats, and neither Na2-EDTA nor AEBSF could effectively abrogate this fall. However, Na2-EDTA markedly reduced plasma fibrinogen consumption and hemorrhage at the site of BjV inoculation. Na2-EDTA also abolished the marked elevation in TF levels in plasma at 3 and 6 h, by both administration routes. Moreover, increased TF activity was also noticed in lung and skin tissue samples at 6 h. However, factor VII levels did not decrease over time. PDI expression in skin was normal at 3 h, and downregulated at 6 h in all groups treated with BjV. Conclusions SVMP induce coagulopathy, hemorrhage and increased TF levels in plasma, but neither SVMP nor SVSP are directly involved in thrombocytopenia. High levels of TF in plasma and TF decryption occur during snake envenomation, like true disseminated intravascular coagulation syndrome, and might be implicated in engendering

  8. Bothrops jararaca Peptide with Anti-Hypertensive Action Normalizes Endothelium Dysfunction Involved in Physiopathology of Preeclampsia

    PubMed Central

    Benedetti, Gabriel; Morais, Katia L. P.; Guerreiro, Juliano R.; de Oliveira, Eduardo Fontana; Hoshida, Mara Sandra; Oliveira, Leandro; Sass, Nelson; Lebrun, Ivo; Ulrich, Henning; Lameu, Claudiana; de Camargo, Antonio Carlos Martins

    2011-01-01

    Preeclampsia, a pregnancy-specific syndrome characterized by hypertension, proteinuria and edema, is a major cause of fetal and maternal morbidity and mortality especially in developing countries. Bj-PRO-10c, a proline-rich peptide isolated from Bothrops jararaca venom, has been attributed with potent anti-hypertensive effects. Recently, we have shown that Bj-PRO-10c-induced anti-hypertensive actions involved NO production in spontaneous hypertensive rats. Using in vitro studies we now show that Bj-PRO-10c was able to increase NO production in human umbilical vein endothelial cells from hypertensive pregnant women (HUVEC-PE) to levels observed in HUVEC of normotensive women. Moreover, in the presence of the peptide, eNOS expression as well as argininosuccinate synthase activity, the key rate-limiting enzyme of the citrulline-NO cycle, were enhanced. In addition, excessive superoxide production due to NO deficiency, one of the major deleterious effects of the disease, was inhibited by Bj-PRO-10c. Bj-PRO-10c induced intracellular calcium fluxes in both, HUVEC-PE and HUVEC, which, however, led to activation of eNOS expression only in HUVEC-PE. Since Bj-PRO-10c promoted biological effects in HUVEC from patients suffering from the disorder and not in normotensive pregnant women, we hypothesize that Bj-PRO-10c induces its anti-hypertensive effect in mothers with preeclampsia. Such properties may initiate the development of novel therapeutics for treating preeclampsia. PMID:21858206

  9. Late embryos and bony skull development in Bothropoides jararaca (Serpentes, Viperidae).

    PubMed

    Polachowski, Katja M; Werneburg, Ingmar

    2013-02-01

    In recent years, developmental anatomy received increasing interest as a potential new source for phylogenetic research. For skeletal development, studies mainly rely on the first appearance of ossification centers. However, informative events occur during the whole course of skeletogenesis; interactions between external and internal development occur and morphometric changes take place - all of which present potential sources for phylogenetic analyses. Therefore, the Standard Event System (SES) was used to traceably describe the external development of the snake species Bothropoides jararaca and external measurements were analyzed. We then applied micro-computed tomography (μCT), clearing and double-staining, and 2D and 3D morphometric methods to describe, illustrate, and analyze the development of the head in great detail. We found a 3D flattening of the skull during ontogeny, a pattern that is not reflected in external development. This may be explained by a different relationship of skeletogenesis and external characters to the developing jaw musculature or simply by the different type of data. Clearing and double-staining and μCT-scanning revealed a broadly similar sequence in the onset of ossification. Minute differences may be due to the treatment of embryos. Bones of the dermatocranium are among the first to ossify and the development of the calcified endolymph may reflect its function as a calcium source during development. The value of phylogenetic observations using the sequence of first ossifications is critically discussed. The related heterochronic changes are interpreted to contribute at least to the very first phase of divagating skull formation among taxa. PMID:23348050

  10. Aqueous Leaf Extract of Jatropha gossypiifolia L. (Euphorbiaceae) Inhibits Enzymatic and Biological Actions of Bothrops jararaca Snake Venom

    PubMed Central

    Félix-Silva, Juliana; Souza, Thiago; Menezes, Yamara A. S.; Cabral, Bárbara; Câmara, Rafael B. G.; Silva-Junior, Arnóbio A.; Rocha, Hugo A. O.; Rebecchi, Ivanise M. M.; Zucolotto, Silvana M.; Fernandes-Pedrosa, Matheus F.

    2014-01-01

    Snakebites are a serious public health problem due their high morbi-mortality. The main available specific treatment is the antivenom serum therapy, which has some disadvantages, such as poor neutralization of local effects, risk of immunological reactions, high cost and difficult access in some regions. In this context, the search for alternative therapies is relevant. Therefore, the aim of this study was to evaluate the antiophidic properties of Jatropha gossypiifolia, a medicinal plant used in folk medicine to treat snakebites. The aqueous leaf extract of the plant was prepared by decoction and phytochemical analysis revealed the presence of sugars, alkaloids, flavonoids, tannins, terpenes and/or steroids and proteins. The extract was able to inhibit enzymatic and biologic activities induced by Bothrops jararaca snake venom in vitro and in vivo. The blood incoagulability was efficiently inhibited by the extract by oral route. The hemorrhagic and edematogenic local effects were also inhibited, the former by up to 56% and the latter by 100%, in animals treated with extract by oral and intraperitoneal routes, respectively. The inhibition of myotoxic action of B. jararaca reached almost 100%. According to enzymatic tests performed, it is possible to suggest that the antiophidic activity may be due an inhibitory action upon snake venom metalloproteinases (SVMPs) and/or serine proteinases (SVSPs), including fibrinogenolytic enzymes, clotting factors activators and thrombin like enzymes (SVTLEs), as well upon catalytically inactive phospholipases A2 (Lys49 PLA2). Anti-inflammatory activity, at least partially, could also be related to the inhibition of local effects. Additionally, protein precipitating and antioxidant activities may also be important features contributing to the activity presented. In conclusion, the results demonstrate the potential antiophidic activity of J. gossypiifolia extract, including its significant action upon local effects, suggesting that

  11. Contribution of mast cells and snake venom metalloproteinases to the hyperalgesia induced by Bothrops jararaca venom in rats.

    PubMed

    Bonavita, André Gustavo C; da Costa, Aline S; Pires, Ana Lucia A; Neves-Ferreira, Ana G C; Perales, Jonas; Cordeiro, Renato S B; Martins, Marco A; e Silva, Patrícia M R

    2006-06-15

    Bothrops jararaca venom (Bjv) is known to induce local inflammation and severe pain. Since, mast cells are able to secrete mediators involved in algesic processes, in this study we examined the putative role of these cells in the hyperalgesia triggered by Bjv in the rat paw. We noted that treatment with mast cell stabilizer sodium cromoglicate as well as with histamine and 5-hydroxytriptamine receptor antagonists meclizine and methysergide, respectively, inhibited the Bjv-induced hyperalgesia. In addition, we showed that stimulation of isolated rat peritoneal mast cells with Bjv in vitro resulted in the release of stored and neo-generated inflammatory mediators such as histamine and leukotriene C(4), respectively. Bjv-induced histamine secretion was clearly sensitive to treatment with sodium cromoglicate and sodium nedocromil. We further observed that metalloproteinase inhibitors 1,10-phenantroline and DM43 inhibited mast cell degranulation in vitro, under conditions where inhibitors of phospholipase A(2) as well as of serine- and cysteine-proteinases were inactive. Altogether, our findings indicate that mast cells seem to contribute to the hyperalgesia caused by Bjv in the rat paw, and also provide evidence that this response might be dependent on the ability of the Bjv to activate directly mast cells. PMID:16730041

  12. [Bacterial flora of the oral cavity, fangs and venom of Bothrops jararaca: possible source of infection at the site of bite].

    PubMed

    Jorge, M T; de Mendonça, J S; Ribeiro, L A; da Silva, M L; Kusano, E J; Cordeiro, C L

    1990-01-01

    Culture of fang, fang sheath and venom of fifteen healthy freshly captured Bothrops jararaca were analyzed. The bacteria most frequently encountered were group D streptococci (12 snakes), Enterobacter sp. (6), Providencia rettgeri (6), Providencia sp. (4), Escherichia coli (4), Morganella morganii (3) and Clostridium sp. (5). The bacteria observed are similar to those found in the abscesses from Bothrops bitten patients. Since these snake mouth bacteria may be inoculated during the snake bite, bacterial multiplication and infection may occur under favorable conditions. PMID:2259834

  13. Venom-Related Transcripts from Bothrops jararaca Tissues Provide Novel Molecular Insights into the Production and Evolution of Snake Venom

    PubMed Central

    Junqueira-de-Azevedo, Inácio L.M.; Bastos, Carolina Mancini Val; Ho, Paulo Lee; Luna, Milene Schmidt; Yamanouye, Norma; Casewell, Nicholas R.

    2015-01-01

    Attempts to reconstruct the evolutionary history of snake toxins in the context of their co-option to the venom gland rarely account for nonvenom snake genes that are paralogous to toxins, and which therefore represent important connectors to ancestral genes. In order to reevaluate this process, we conducted a comparative transcriptomic survey on body tissues from a venomous snake. A nonredundant set of 33,000 unigenes (assembled transcripts of reference genes) was independently assembled from six organs of the medically important viperid snake Bothrops jararaca, providing a reference list of 82 full-length toxins from the venom gland and specific products from other tissues, such as pancreatic digestive enzymes. Unigenes were then screened for nontoxin transcripts paralogous to toxins revealing 1) low level coexpression of approximately 20% of toxin genes (e.g., bradykinin-potentiating peptide, C-type lectin, snake venom metalloproteinase, snake venom nerve growth factor) in body tissues, 2) the identity of the closest paralogs to toxin genes in eight classes of toxins, 3) the location and level of paralog expression, indicating that, in general, co-expression occurs in a higher number of tissues and at lower levels than observed for toxin genes, and 4) strong evidence of a toxin gene reverting back to selective expression in a body tissue. In addition, our differential gene expression analyses identify specific cellular processes that make the venom gland a highly specialized secretory tissue. Our results demonstrate that the evolution and production of venom in snakes is a complex process that can only be understood in the context of comparative data from other snake tissues, including the identification of genes paralogous to venom toxins. PMID:25502939

  14. Contribution of metalloproteases, serine proteases and phospholipases A2 to the inflammatory reaction induced by Bothrops jararaca crude venom in mice.

    PubMed

    Zychar, Bianca Cestari; Dale, Camila Squazoni; Demarchi, Denise Soares; Gonçalves, Luis Roberto C

    2010-01-01

    Various toxins isolated from Bothrops snake venoms induce inflammatory reactions and have been claimed to contribute to the severity of local symptoms present in this envenomation. Notwithstanding, the relative participation of serine proteases, metalloproteases and phospholipases A(2) in the inflammatory reaction produced by crude Bothrops venoms is poorly understood. Herein, crude Bothrops jararaca venom was treated with phenylmethanesulfonyl fluoride (PMSF), 1,10-phenanthroline (oPhe), or p-bromophenacyl-bromide (p-BPB) to inhibit those classes of enzymes, respectively, and inflammatory parameters were evaluated and compared to those induced by the control crude venom. The intensity of edema and hyperalgesia/allodynia was remarkably reduced in animals administered with oPhe-treated venom. Leukocyte-endothelium interactions (LEI), such as adhesion and migration of leukocytes, were also modified at 2h and 24h. Edema and LEI parameters induced by p-BPB-treated venom were similar to those observed with the control venom, but hyperalgesia/allodynia was significantly lower. Inflammatory parameters induced by PMSF-treated venom were similar to those induced by the crude venom, except for a mild reduction in edema intensity. Our results indicate that metalloproteases have a pivotal role in the inflammatory reactions induced by B. jararaca venom, and phospholipases A(2) and serine proteases have a minor role.

  15. The Anti-Inflammatory Effects of the Methanolic Extract and Fractions from Davilla elliptica St. Hil. (Dilleniaceae) on Bothrops jararaca Envenomation

    PubMed Central

    Nishijima, Catarine Massucato; Delella, Flavia Karina; Rodrigues, Clenilson Martins; Rinaldo, Daniel; Lopes-Ferreira, Monica Valdyrce dos Anjos; da Rocha, Lucia Regina Machado; Vilegas, Wagner; Felisbino, Sergio Luis; Hiruma-Lima, Clélia Akiko

    2015-01-01

    Inflammation and haemorrhage are the main characteristics of tissue injury in botropic envenomation. Although some studies have shown that anti-venom prevents systemic reactions, it is not efficient in preventing tissue injury at the site of the bite. Therefore, this work was undertaken to investigate the anti-inflammatory effects of the methanolic extract and fractions from D. elliptica and to evaluate the role of matrix metalloproteinases (MMPs) in this process. Effects of the extract and fractions from D. elliptica were evaluated using a carrageenan-induced paw oedema model in rats, and leukocyte rolling was visualized by intravital. The quantification of MMPs activities (MMP-2 and MMP-9) extracted from the dermis of mice treated with extract and fractions alone or incubated with venom was determined by zymographic analyses. Our results show that intraperitoneal (i.p.) injection of fractions significantly reduced paw oedema after the carrageenan challenge. Treatment with the tannins fraction also resulted in considerable inhibition of the rolling of leukocytes and this fraction was able to decrease the activation of MMP-9. These results confirmed the anti-inflammatory activity of the methanolic extract and tannins fraction of D. elliptica and showed that the dermonecrosis properties of B. jararaca venom might be mediated through the inhibition of MMP-9 activity. PMID:26042466

  16. Neutralization of the oedematogenic activity of Bothrops jararaca venom on the mouse paw by an antibothropic fraction isolated from opossum (Didelphis marsupialis) serum.

    PubMed

    Perales, J; Amorim, C Z; Rocha, S L; Domont, G B; Moussatché, H

    1992-11-01

    The pharmacological modulation of mice paw oedema produced by Bothrops jararaca venom (BJV) has been studied. Intraplantar injection of BJV (1-30 micrograms/paw) produced a dose- and time-related oedema, which was maximal 30 min after injection, reduced gradually thereafter and disappeared over 48 h. BJV heated at 100 degrees C for 5 or 15 min blocked local hemorrhage and caused partial inhibition of its oedematogenic activity. The BJV oedema was not inhibited by the anti-histamine meclizine, the inhibitor of histamine and serotonin, cyproheptadine, PAF-acether antagonist WEB 2170 or by the anti-leukotrienes C4/D4, LY 171883. Dexamethasone, aspirin, indomethacin, and the dual cyclooxygenase and lipoxygenase inhibitor BW 755C inhibited BJV-induced oedema indicating that arachidonic acid metabolism products via the cyclooxygenase pathway participate in its genesis and/or maintenance. The antibothropic fraction (ABF) (25-200 micrograms/paw) isolated from Didelphis marsupialis serum neutralized the oedema induced by the venom with and without heating, the hemorrhage induced by BJV and partially blocked the oedema induced by bradykinin and by cellulose sulphate. The oedema produced by histamine, serotonin, PAF-acether or leukotriene C4 was not inhibited. PMID:1295374

  17. The defensive strike of five species of lanceheads of the genus Bothrops (Viperidae).

    PubMed

    Araújo, M S; Martins, M

    2007-05-01

    We studied the defensive strike of one species of each of five recognized lineages within the genus Bothrops, namely, B. alternatus, B. jararaca, B. jararacussu, B. moojeni and B. pauloensis. The defensive strike of the studied species was in general similar to that of Crotalus viridis and C. atrox, but some important differences were observed. Bothrops alternatus and B. pauloensis struck preferentially from a tight body posture, whereas B. jararaca and B. moojeni from a loose body posture. Defensive strikes were either true or false (during the latter, the mouth remains closed or partially open). Almost all strikes were successful; only on a few occasions snakes missed their target (flawed strikes). Strike variables were very conservative among the five species, especially strike distance and height, and one possible explanation may be related to constraints imposed on strike variables as a way of increasing strike accuracy. PMID:17876444

  18. Evaluation of the effect of aqueous extract of Croton urucurana Baillon (Euphorbiaceae) on the hemorrhagic activity induced by the venom of Bothrops jararaca, using new techniques to quantify hemorrhagic activity in rat skin.

    PubMed

    Esmeraldino, L E; Souza, A M; Sampaio, S V

    2005-08-01

    Aqueous extracts of Croton urucurana (Sangra D'agua), a plant popularly considered a cicatrizant, were analyzed for anti-Bothrops jararaca venom activity. The plant extracts antagonized the hemorrhagic activity of the venom and proanthocyanidins were involved in this activity. Two new methods for the quantification of hemorrhagic activity evoked by bothropic venoms were employed. The first consists of graphic computer analysis of the hemorrhagic halo evoked in rats by dorsal intradermic administration of venom. The second method involves quantification of the hemoglobin present in the hemorrhagic halo. Based on the results, we suggest that these methods, easily implemented in the laboratory routine, allow for quantification of venom-induced hemorrhagic activity. In addition, this study demonstrates that the rich extracts of proanthocyanidins are powerful inhibitors of bothropic venom metalloproteinases.

  19. Preclinical assessment of the neutralizing capacity of antivenoms produced in six Latin American countries against medically-relevant Bothrops snake venoms.

    PubMed

    Segura, A; Castillo, M C; Núñez, V; Yarlequé, A; Gonçalves, L R C; Villalta, M; Bonilla, C; Herrera, M; Vargas, M; Fernández, M; Yano, M Y; Araújo, H P; Boller, M A A; León, P; Tintaya, B; Sano-Martins, I S; Gómez, A; Fernández, G P; Geoghegan, P; Higashi, H G; León, G; Gutiérrez, J M

    2010-11-01

    Species of the genus Bothrops induce the vast majority of snakebite envenomings in Latin America. A preclinical study was performed in the context of a regional network of public laboratories involved in the production, quality control and development of antivenoms in Latin America. The ability of seven polyspecific antivenoms, produced in Argentina, Brazil, Peru, Bolivia, Colombia and Costa Rica, to neutralize lethal, hemorrhagic, coagulant, defibrinogenating and myotoxic activities of the venoms of Bothrops neuwiedi (diporus) (Argentina), Bothrops jararaca (Brazil), B. neuwiedi (mattogrossensis) (Bolivia), Bothrops atrox (Peru and Colombia) and Bothrops asper (Costa Rica) was assessed using standard laboratory tests. Despite differences in the venom mixtures used in the immunization of animals for the production of these antivenoms, a pattern of extensive cross-neutralization was observed between these antivenoms and all the venoms tested, with quantitative differences in the values of effective doses. This study reveals the capacity of these antivenoms to neutralize, in preclinical tests, homologous and heterologous Bothrops venoms in Central and South America, and also highlight quantitative differences in the values of Median Effective Doses (ED50s) between the various antivenoms. PMID:20621114

  20. Preclinical assessment of the neutralizing capacity of antivenoms produced in six Latin American countries against medically-relevant Bothrops snake venoms.

    PubMed

    Segura, A; Castillo, M C; Núñez, V; Yarlequé, A; Gonçalves, L R C; Villalta, M; Bonilla, C; Herrera, M; Vargas, M; Fernández, M; Yano, M Y; Araújo, H P; Boller, M A A; León, P; Tintaya, B; Sano-Martins, I S; Gómez, A; Fernández, G P; Geoghegan, P; Higashi, H G; León, G; Gutiérrez, J M

    2010-11-01

    Species of the genus Bothrops induce the vast majority of snakebite envenomings in Latin America. A preclinical study was performed in the context of a regional network of public laboratories involved in the production, quality control and development of antivenoms in Latin America. The ability of seven polyspecific antivenoms, produced in Argentina, Brazil, Peru, Bolivia, Colombia and Costa Rica, to neutralize lethal, hemorrhagic, coagulant, defibrinogenating and myotoxic activities of the venoms of Bothrops neuwiedi (diporus) (Argentina), Bothrops jararaca (Brazil), B. neuwiedi (mattogrossensis) (Bolivia), Bothrops atrox (Peru and Colombia) and Bothrops asper (Costa Rica) was assessed using standard laboratory tests. Despite differences in the venom mixtures used in the immunization of animals for the production of these antivenoms, a pattern of extensive cross-neutralization was observed between these antivenoms and all the venoms tested, with quantitative differences in the values of effective doses. This study reveals the capacity of these antivenoms to neutralize, in preclinical tests, homologous and heterologous Bothrops venoms in Central and South America, and also highlight quantitative differences in the values of Median Effective Doses (ED50s) between the various antivenoms.

  1. [Bites by snakes in the genus Bothrops: a series of 3,139 cases].

    PubMed

    Ribeiro, L A; Jorge, M T

    1997-01-01

    Medical records of 3,139 patients bitten by Bothrops snakes and attended at Vital Brazil Hospital (HVB) from 1981 to 1990 were reviewed. They were more frequent in males (75.7%). In 1,412 cases (45.0%) the snake was classified by species, and 1,376 were B. jararaca, 20 B. jararacussu, 11 B. neuwiedi, 2 B. moojeni, 2 B. alternatus e 1 B. pradoi. The most frequent bitten anatomic regions were: foot (47.5%) and hand (21.3%). Tourniquet was used in 38.2% of the cases and its frequency fell down during the study period (p < 0.05). The clinical features at the bite site were: pain (95.6%), swelling (95.4%), ecchymosis (56.1%), blisters (13.8%), necrosis (16.5%), and abscess (11.0%). Systemic manifestations were: bleeding (12.3%), acute renal failure (1.6%), and shock (0.7%). There were blood coagulation disorders in 1,730 (57.9%) of the 2,990 cases. There were 21 amputations (0.7%) and 9 deaths (0.3%). The average serum dose that was used in treatment fell down during the study period (p < 0.001). PMID:9463193

  2. Determination of Toxic Activities in Bothrops spp. Snake Venoms Using Animal-Free Approaches: Correlation Between In Vitro Versus In Vivo Assays.

    PubMed

    de Souza, Letícia Lopes; Stransky, Stephanie; Guerra-Duarte, Clara; Flor-Sá, Ana; Schneider, Francisco Santos; Kalapothakis, Evanguedes; Chávez-Olórtegui, Carlos

    2015-10-01

    The main purpose of this study is to investigate the in vitro toxic effects of 5 Bothrops spp. snake venoms, which are part of the antigenic mixture used for the production of Brazilian antivenom, and evaluate their correlation with the in vivo toxic activities of Bothrops spp. venoms. The correlation analysis could be helpful for the replacement of living animals experimentation for in vitro bioassay. Cytotoxicity, L-amino acid oxidase (LAAO), proteolitic (serine and metalloproteinase), hyaluronidase (Hyal), and phospholipase A2 (PLA2) activities were estimated and the correlation coefficient was determined for each activity in relation to lethality, edema, hemorrhage and necrosis induced in live animals by B. jararaca, B. alternatus, B. jararacussu, B. neuwiedi, and B. moojeni venoms. The lethal activity in mice was highly related to Hyal activity (r = 0.94, p < .05), edema related to PLA2 activity (r = 0.94, p < .05), whereas the necrotizing activity showed high correlation with LAAO activity (r = 0.83, p < .05). A very significant correlation between in vitro cytotoxicity and LAAO activities was also observed (r = 0.97, p < .05).

  3. Determination of Toxic Activities in Bothrops spp. Snake Venoms Using Animal-Free Approaches: Correlation Between In Vitro Versus In Vivo Assays.

    PubMed

    de Souza, Letícia Lopes; Stransky, Stephanie; Guerra-Duarte, Clara; Flor-Sá, Ana; Schneider, Francisco Santos; Kalapothakis, Evanguedes; Chávez-Olórtegui, Carlos

    2015-10-01

    The main purpose of this study is to investigate the in vitro toxic effects of 5 Bothrops spp. snake venoms, which are part of the antigenic mixture used for the production of Brazilian antivenom, and evaluate their correlation with the in vivo toxic activities of Bothrops spp. venoms. The correlation analysis could be helpful for the replacement of living animals experimentation for in vitro bioassay. Cytotoxicity, L-amino acid oxidase (LAAO), proteolitic (serine and metalloproteinase), hyaluronidase (Hyal), and phospholipase A2 (PLA2) activities were estimated and the correlation coefficient was determined for each activity in relation to lethality, edema, hemorrhage and necrosis induced in live animals by B. jararaca, B. alternatus, B. jararacussu, B. neuwiedi, and B. moojeni venoms. The lethal activity in mice was highly related to Hyal activity (r = 0.94, p < .05), edema related to PLA2 activity (r = 0.94, p < .05), whereas the necrotizing activity showed high correlation with LAAO activity (r = 0.83, p < .05). A very significant correlation between in vitro cytotoxicity and LAAO activities was also observed (r = 0.97, p < .05). PMID:26160116

  4. Microbiological evaluation of different strategies for management of snakes in captivity.

    PubMed

    Campagner, M V; Bosco, S M G; Bagagli, E; Cunha, M L R S; Jeronimo, B C; Saad, E; Biscola, N P; Ferreira, R S; Barraviera, B

    2012-01-01

    Keeping snakes in captivity to produce venom for scientific research and production of inputs is now a worldwide practice. Maintaining snakes in captivity involves capture, infrastructure investments, management techniques, and appropriate qualified personnel. Further, the success of the project requires knowledge of habitat, nutrition, and reproduction, and control of opportunistic infections. This study evaluated the management of snakes in three types of captivity (quarantine, intensive, and semiextensive) and diagnosed bacterial and fungal contaminants. A bacteriological profile was obtained by swabbing the oral and cloacal cavities, scales, and venoms of healthy adult snakes from Bothrops jararaca (Bj) and Crotalus durissus terrificus (Cdt). There was predominance of Enterobacteriaceae, especially non-fermenting Gram-negative bacilli excluding Pseudomonas spp and Gram- positive bacteria. Statistically, intensive captivity resulted in the highest number of bacterial isolates, followed by recent capture (quarantine) and by semiextensive captivity. No statistical difference was found between Bj and Cdt bacterial frequency. In vitro bacterial susceptibility testing found the highest resistance against the semisynthetic penicillins (amoxicillin and ampicillin) and highest sensitivity to amicacin and tobramycin aminoglycosides. To evaluate mycological profile of snakes from intensive captivity, samples were obtained from two healthy Bj and one B. moojeni, one B. pauloensis, and one Cdt showing whitish lesions on the scales suggestive of ringworm. Using conventional methods and DNA-based molecular procedures, five samples of Trichosporon asahii were identified. Despite the traditional role of intense captivity in ophidian venom production, semiextensive captivity was more effective in the present study by virtue of presenting superior control of bacterial and fungal transmission, easier management, lowest cost, and decreased rate of mortality; therefore, it should be

  5. Biological and immunological properties of the venom of Bothrops alcatraz, an endemic species of pitviper from Brazil.

    PubMed

    Furtado, M F D

    2005-06-01

    Bothrops alcatraz is a new pitviper species derived from the Bothrops jararaca group, whose natural habitat is situated in Alcatrazes Archipelago, a group of marine islands near São Paulo State coast in Brazil. Herein, the biological and biochemical properties of venoms of four adult specimens of B. alcatraz were examined comparatively to a reference pool of Bothrops jararaca venom. Both venoms showed similar activities and electrophoretic patterns, but B. alcatraz venom showed three protein bands of molecular masses of 97, 80 and 38 kDa that were not present in B. jararaca reference venom. The i.p. median lethal dose of B. alcatraz venom ranged from 5.1 to 6.6 mg/kg, while it was 1.5 mg/kg for B. jararaca venom. The minimum hemorrhagic dose of B. jararaca venom was 0.63, whereas 2.28 mug/mouse for B. alcatraz venom. In contrast, B. alcatraz venom was more potent in regard to procoagulant and proteolytic activities. These differences were supported by western blotting and neutralization tests, employing commercial bothropic antivenom, which showed that hemorrhagic and lethal activities of B. alcatraz venom were less effectively inhibited than B. jararaca venom. Such results evidence that B. alcatraz shows quantitative and qualitative differences in venom composition in comparison with its B. jararaca relatives, which might represent an optimization of venom towards a specialized diet. PMID:16002343

  6. Anti-snake venom properties of Schizolobium parahyba (Caesalpinoideae) aqueous leaves extract.

    PubMed

    Mendes, Mirian M; Oliveira, Carolina F; Lopes, Daiana S; Vale, Luís Henrique F; Alcântara, Tânia M; Izidoro, Luiz Fernando M; Hamaguchi, Amélia; Homsi-Brandeburgo, Maria Inês; Soares, Andreimar M; Rodrigues, Veridiana M

    2008-07-01

    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.

  7. Protective effect of schizolobium parahyba flavonoids against snake venoms and isolated toxins.

    PubMed

    F Vale, Luis Henrique; Mendes, Mirian M; Fernandes, Renata S; Costa, Tassia R; S Hage-Melim, Lorane I; A Sousa, Maicon; Hamaguchi, Amelia; Homsi-Brandeburgo, Maria I; Franca, Suzelei C; Silva, Carlos H T P; Pereira, Paulo S; Soares, Andreimar M; Rodrigues, Veridiana M

    2011-01-01

    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.

  8. Haematopoiesis in snakes (Ophidia) in early postnatal development.

    PubMed

    Dabrowski, Z; Sano Martins, I S; Tabarowski, Z; Witkowska-Pelc, E; Spadacci Morena, D D; Spodaryk, K; Podkowa, D

    2007-05-01

    The occurrence of haematopoiesis has been studied in various parts of the spine and in the ribs in four species of snakes (Boa constrictor L., Elaphe guttata L., Lamprophis fulaginosus Boie., Bothrops jararaca Wied.) from hatching until 150 days of postnatal development. Marrow spaces are formed by chondrolysis with various time frames depending on the studied species. Marrow cells egress to the general circulation in two ways: via migration through the endothelial cells lining the venous sinuses or by the rupture of protrusions. Erythroblasts are present in the lumen of marrow sinuses suggesting their final maturation there. Various relationships of the spleen to the pancreas have been found. No myelopoietic foci occur in the spleen, liver or kidney of any of the studied species. However, erythropoiesis (sparse islets) has been observed in Bothrops jararaca spleen.

  9. [Effect of reduction in the Bothrops antivenin dose administrated in patients bitten by the Bothrops snake].

    PubMed

    Jorge, M T; Ribeiro, L A

    1994-01-01

    Seven hundred and thirty accidents by Bothrops snake bite attended at Hospital Vital Brazil-Instituto Butantan (HVB-IB) were studied: 411 of in 1983/84 (group A) and 319 in 1986 (group B). All the patients were seen in the Hospital 6 hours after the bite. As an average, all the 411 patients of the group A and the 319 patients of the group B received antivenom capable of neutralizing 215 mg and 117 mg of Bothrops jararaca, respectively. Both groups were similar concerning to: sex and age of the patients; anatomic region bitten; frequency of incision and tourniquet use at the site bite; time in between of the accident and the admission at HVB-IB; classification of B. jararaca either young or adult; frequency of the coagulation impairment. There was no difference in the evolution of the local envenoming manifestations and there were no cases of death. PMID:8061698

  10. Plant natural products active against snake bite--the molecular approach.

    PubMed

    Mors, W B; Nascimento, M C; Pereira, B M; Pereira, N A

    2000-11-01

    The article surveys the substances identified in plants reputed to neutralize the effects of snake venoms. Protective activity of many of them against the lethal action of the venom of the jararaca (Bothrops jararaca) snake was confirmed by biological assays. It was shown that all belong to chemical classes capable of interacting with macromolecular targets--receptors and enzymes. In a few cases it has been shown that exogenous natural micromolecules can mimic the biological activity of endogenous macromolecules. From the evidence presented, it can be inferred that micromolecules which neutralize the action of snake venoms mechanistically replace endogenous antitoxic serum proteins with venom neutralizing capacity such as produced by some animals. PMID:11130675

  11. Comparative analysis of viperidae venoms antibacterial profile: a short communication for proteomics.

    PubMed

    Ferreira, Bruno L; Santos, Dilvani O; Dos Santos, André Luis; Rodrigues, Carlos R; de Freitas, Cícero C; Cabral, Lúcio M; Castro, Helena C

    2011-01-01

    Bacterial infections involving multidrug-resistant strains are one of the ten leading causes of death and an important health problem in need for new antibacterial sources and agents. Herein, we tested and compared four snake venoms (Agkistrodon rhodostoma, Bothrops jararaca, B. atrox and Lachesis muta) against 10 Gram-positive and Gram-negative drug-resistant clinical bacteria strains to identify them as new sources of potential antibacterial molecules. Our data revealed that, as efficient as some antibiotics currently on the market (minimal inhibitory concentration (MIC) = 1-32 μg mL(-1)), A. rhodostoma and B. atrox venoms were active against Staphylococcus epidermidis and Enterococcus faecalis (MIC = 4.5 μg mL(-1)), while B. jararaca inhibited S. aureus growth (MIC = 13 μg ml(-1)). As genomic and proteomic technologies are improving and developing rapidly, our results suggested that A. rhodostoma, B. atrox and B. jararaca venoms and glands are feasible sources for searching antimicrobial prototypes for future design new antibiotics against drug-resistant clinical bacteria. They also point to an additional perspective to fully identify the pharmacological potential of these venoms by using different techniques. PMID:18955360

  12. Partial in vitro analysis of toxic and antigenic activities of eleven Peruvian pitviper snake venoms.

    PubMed

    Guerra-Duarte, C; Lopes-Peixoto, J; Fonseca-de-Souza, B R; Stransky, S; Oliveira, D; Schneider, F S; Lopes-de-Souza, L; Bonilla, C; Silva, W; Tintaya, B; Yarleque, A; Chávez-Olórtegui, C

    2015-12-15

    This work used eleven Peruvian snake venoms (Bothrops andianus, Bothrops atrox, Bothrops barnetti, Bothrops castelnaudi, Bothriopsis chloromelas, Bothrocophias microphthalmus, Bothrops neuwiedi, Bothriopsis oligolepis, Bothriopsis peruviana, Bothrops pictus and Bothriopsis taeniata) to perform in vitro experimentation and determine its main characteristics. Hyaluronidase (HYAL), phospholipase A2 (PLA2), snake venom metalloproteinase (SVMP), snake venom serine protease (SVSP) and L-amino acid oxidase (LAAO) activities; toxicity by cell viability assays using MGSO3, VERO and HeLa cell lineages; and crossed immunoreactivity with Peruvian (PAV) and Brazilian (BAV) antibothropic polyvalent antivenoms, through ELISA and Western Blotting assays, were determined. Results show that the activities tested in this study were not similar amongst the venoms and each species present their own peculiarities, highlighting the diversity within Bothrops complex. All venoms were capable of reducing cell viability of all tested lineages. It was also demonstrated the crossed recognition of all tested venoms by both antivenoms. PMID:26365916

  13. Use of microarrays for investigating the subtoxic effects of snake venoms: insights into venom-induced apoptosis in human umbilical vein endothelial cells.

    PubMed

    Gallagher, Paul G; Bao, Yongde; Serrano, Solange M T; Kamiguti, Aura S; Theakston, R David G; Fox, Jay W

    2003-03-01

    The pathological effects of only a small percentage of the total number of protein components of snake venoms are well documented, yet this knowledge has led to a general understanding of the physiological consequences of snake venom poisoning. The aim of this study was to assess the effect of subpathological levels of Crotalus atrox (Western diamondback rattlesnake) and Bothrops jararaca (Jararaca) snake venoms on the gene expression profile of human umbilical vein endothelial cells (HUVEC) in culture. Analysis of the data demonstrated that HUVECs treated with C. atrox venom had 33 genes up-regulated with significant fold changes of 1.5 or greater compared to untreated control cells. Ten genes were down-regulated with 1.5 or greater fold changes. In cells treated with B. jararaca venom, 33 genes were observed to be up-regulated and 11 genes were down-regulated with a fold change of 1.5 or more. More than half of the up-regulated genes and approximately half of the down-regulated genes detected in cells treated with the venoms were found in both data sets underscoring both the similarities and differences between the two venoms. Ontological categorization of the up-regulated genes from endothelial cells treated with either C. atrox or B. jararaca venom gave the cell growth/maintenance and signal transducer groups as having the most members. The ontology of the down-regulated genes from both venom-treated cell samples was more varied but interestingly, the predominant ontology class was also cell growth/maintenance. Many of the up-regulated genes are involved in the Fas ligand/TNF-alpha receptor apoptotic pathway. In summary, these experiments demonstrate the power of gene expression profiling to explore the subtoxic effects of venoms on gene expression and highlight its potential for the discovery of novel insights into a variety of biological processes and signal transduction pathways. Furthermore, these studies illustrate the subtle functional differences between

  14. The conserved structure of snake venom toxins confers extensive immunological cross-reactivity to toxin-specific antibody.

    PubMed

    Harrison, R A; Wüster, W; Theakston, R D G

    2003-03-01

    We have demonstrated previously that antisera from mice immunised with DNA encoding the carboxy-terminal domain (JD9) of a potent haemorrhagic metalloproteinase, jararhagin, neutralised over 70% of the haemorrhagic activity of the whole Bothrops jararaca venom. Here, we demonstrate that the JD9-specific antibody possesses extensive immunological reactivity to venom components in snakes of distinct species and genera. The polyspecific immunological reactivity of the antibody showed a correlation with amino acid sequence identity and with predicted antigenic domains of JD9-analogues in venoms of snakes with closest phylogenetic links to B. jararaca. This study further promotes the potential of DNA immunisation to generate toxin-specific antibodies with polyspecific cover. An analysis of the reactivity of the JD9-specific antisera to B. atrox complex venoms that exhibited intraspecific variation in the venom proteome revealed, however, that the toxin-specific approach to antivenom development requires a more in-depth knowledge of the target molecules than is required for conventional antivenoms. PMID:12657313

  15. Serological analysis of venoms and antivenins*

    PubMed Central

    Schöttler, W. H. A.

    1955-01-01

    The immunological relationship between the venoms of six species of the snake genus Bothrops (alternata, atrox, cotiara, jararaca, jararacussu, neuwiedii) was investigated by assay against the corresponding species-specific antivenins in more than 11,000 intravenous and subcutaneous mouse tests. The observations were statistically analysed after the probit method. Both ways of antivenin assay gave numerically identical results, within the limits of error, in the majority of the tests. The width of the neutralization spectra of the monovalent sera anti-atrox, anti-jararaca, and anti-neuwiedii against the heterologous venoms is similar to that of a polyvalent antivenin obtained by immunization with all the six species of venom. The titre of a polyvalent antibothropic serum may vary with the venom species used in the assay. The following are discussed: reasons for occasional difficulties in reproducing results, variations in the toxicity of different samples of the same venom species, divergences in the susceptibility of various species of laboratory animals to venom, and inter-relations between venoms and antivenins. PMID:13240449

  16. Evaluation of an Antimicrobial L-Amino Acid Oxidase and Peptide Derivatives from Bothropoides mattogrosensis Pitviper Venom

    PubMed Central

    Gomes, Diego G.; Porto, William F.; Batista, Carla L.; Ramos, Carmel S.; Holanda, Hortência H. S.; Dias, Simoni C.; Franco, Octavio L.; Moreno, Susana E.

    2012-01-01

    Healthcare-associated infections (HAIs) are causes of mortality and morbidity worldwide. The prevalence of bacterial resistance to common antibiotics has increased in recent years, highlighting the need to develop novel alternatives for controlling these pathogens. Pitviper venoms are composed of a multifaceted mixture of peptides, proteins and inorganic components. L-amino oxidase (LAO) is a multifunctional enzyme that is able to develop different activities including antibacterial activity. In this study a novel LAO from Bothrops mattogrosensis (BmLAO) was isolated and biochemically characterized. Partial enzyme sequence showed full identity to Bothrops pauloensis LAO. Moreover, LAO here isolated showed remarkable antibacterial activity against Gram-positive and -negative bacteria, clearly suggesting a secondary protective function. Otherwise, no cytotoxic activities against macrophages and erythrocytes were observed. Finally, some LAO fragments (BmLAO-f1, BmLAO-f2 and BmLAO-f3) were synthesized and further evaluated, also showing enhanced antimicrobial activity. Peptide fragments, which are the key residues involved in antimicrobial activity, were also structurally studied by using theoretical models. The fragments reported here may be promising candidates in the rational design of new antibiotics that could be used to control resistant microorganisms. PMID:22438972

  17. Another new and threatened species of lancehead genus Bothrops (Serpentes, Viperidae) from Ilha dos Franceses, Southeastern Brazil.

    PubMed

    Barbo, Fausto E; Gasparini, João Luiz; Almeida, Antonio P; Zaher, Hussam; Grazziotin, Felipe G; Gusmão, Rodrigo B; Ferrarini, José Mário G; Sawaya, Ricardo J

    2016-01-01

    A new insular species of the genus Bothrops is described from Ilha dos Franceses, a small island off the coast of Espírito Santo State, in southeastern Brazil. The new species differs from mainland populations of B. jararaca mainly by its small size, relative longer tail, relative smaller head length, and relative larger eyes. The new species is distinguished from B. alcatraz, B. insularis and B. otavioi by the higher number of ventral and subcaudal scales, relative longer tail and smaller head. The new species is highly abundant on the island, being nocturnal, semiarboreal, and feeding on small lizards and centipeds. Due its unique and restricted area of occurrence, declining quality of habitat, and constant use of the island for tourism, the new species may be considered as critically endangered. PMID:27394563

  18. Hematopoiesis in snakes (Ophidia).

    PubMed

    Dabrowski, Z; Tabarowski, Z; Sano-Martins, I S; Spadacci-Morena, D D; Witkowska-Pelc, E; Krzysztofowicz, E; Spodaryk, K

    2002-01-01

    Locations of the hematopoietic tissue have been described in the following ophidian species: Bothrops jararaca, Bothrops jararacusu, Waglerophis merremii, Elaphe teniura teniura, Boa constrictor, and Python reticulatus. Studies were carried out on perfusion fixed vertebrae, ribs, spleen, liver, thymus, and kidney. Routine histological technique was applied using both light and electron microscopy. Hematopoietic tissue was found in the following locations of the vertebrae: neural spine, neural arch, postzygophysis processes, hypapophysis, vertebral centre. Moreover, intense hematopoiesis was found inside the ribs. In the spleen and thymus, only lymphopoiesis was found. Hematopoietic islets in the spleen were sporadically found only in young specimens. No hematopoiesis was observed in the liver and kidney. In the studied species, there were no differences in the location of hematopoietic tissue. A new model of mature and immature blood cell release to the lumen of marrow sinuses different from that known to operate in higher vertebrates is proposed.

  19. High-level expression, purification, characterization and structural prediction of a snake venom metalloproteinase inhibitor in Pichia pastoris.

    PubMed

    Shi, Yi; Ji, Ming-Kai; Xu, Jian-Wen; Lin, Xu; Lin, Jian-Yin

    2012-03-01

    Snake venom metalloproteinase inhibitor BJ46a is from the serum of the venomous snake Bothrops jararaca. It has been proven to possess the capacity to inhibit matrix metalloproteinases (MMPs), likely based on its structural similarity to MMPs. This report describes the successful expression, purification, and characterization of the recombinant protein BJ46a in Pichia pastoris. Purified recombinant protein BJ46a was found to inhibit MMPs. Structural modeling was completed and should provide the foundation for further functional research. To our knowledge, this is the first report on the large scale expression of BJ46a, and it provides promise as a method for generation of BJ46a and investigation of its potential use as a new drug for treatment of antitumor invasion and metastasis. PMID:22307654

  20. Inhibition of hemorrhagic and edematogenic activities of snake venoms by a broad-spectrum protease inhibitor, murinoglobulin; the effect on venoms from five different genera in Viperidae family.

    PubMed

    Ribeiro Filho, Wilker; Sugiki, Masahiko; Yoshida, Etsuo; Maruyama, Masugi

    2003-08-01

    In order to obtain basic data on the effect of broad-spectrum protease inhibitor against local symptoms of Viperidae snake envenomation, inhibitory capacity of rat murinoglobulin on local hemorrhagic and edematogenic activities of venoms from Crotalus atrox, Bothrops jararaca, Lachesis muta muta, Trimeresurus flavoviridis and Echis carinatus sochureki were examined. Murinoglobulin, pre-incubated with the crude venoms at 37 degrees C for 15 min, inhibited hemorrhagic activity of all five venoms to various extents. The activity of C. atrox was almost completely inhibited at the murinoglobulin/venom ratio (w/w) of 20. The activity of B. jararaca, Lachesis muta muta and T. flavoviridis venoms was considerably inhibited at the ratio of 20 (77.2, 80.0 and 86.2% inhibition, respectively), however some of the activity still remained even at the ratio of 40 (84.2, 79.8 and 86.2% inhibition, respectively). Among the five venoms, E. c. sochureki venom is quite resistant to murinoglobulin treatment and statistically significant inhibition was only found at the ratio of 40 (64.1% inhibition). Fibrinolytic and gelatinase activities were more susceptible to murinoglobulin inhibition. The treatment at the ratios of 10 and 20 almost completely inhibited respectively the fibrinolytic and the gelatinase activities of all the venoms. Murinoglobulin treatment also significantly inhibited the edematogenic activity of L. muta muta, T. flavoviridis and Echis carinatus sochureki. The treatment of murinoglobulin at the ratio of 40 considerably suppressed the swelling up to 60 min after subcutaneous injection of L. muta muta and E. c. sochureki venoms, and up to 30 min after T. flavoviridis venom injection. Murinoglobulin is a potent inhibitor against local effects of multiple snake venoms in Viperidae family. PMID:12906888

  1. Functional variability of snake venom metalloproteinases: adaptive advantages in targeting different prey and implications for human envenomation.

    PubMed

    Bernardoni, Juliana L; Sousa, Leijiane F; Wermelinger, Luciana S; Lopes, Aline S; Prezoto, Benedito C; Serrano, Solange M T; Zingali, Russolina B; Moura-da-Silva, Ana M

    2014-01-01

    Snake venom metalloproteinases (SVMPs) are major components in most viperid venoms that induce disturbances in the hemostatic system and tissues of animals envenomated by snakes. These disturbances are involved in human pathology of snake bites and appear to be essential for the capture and digestion of snake's prey and avoidance of predators. SVMPs are a versatile family of venom toxins acting on different hemostatic targets which are present in venoms in distinct structural forms. However, the reason why a large number of different SVMPs are expressed in some venoms is still unclear. In this study, we evaluated the interference of five isolated SVMPs in blood coagulation of humans, birds and small rodents. P-III class SVMPs (fractions Ic, IIb and IIc) possess gelatinolytic and hemorrhagic activities, and, of these, two also show fibrinolytic activity. P-I class SVMPs (fractions IVa and IVb) are only fibrinolytic. P-III class SVMPs reduced clotting time of human plasma. Fraction IIc was characterized as prothrombin activator and fraction Ic as factor X activator. In the absence of Ca2+, a firm clot was observed in chicken blood samples with fractions Ic, IIb and partially with fraction IIc. In contrast, without Ca2+, only fraction IIc was able to induce a firm clot in rat blood. In conclusion, functionally distinct forms of SVMPs were found in B. neuwiedi venom that affect distinct mechanisms in the coagulation system of humans, birds and small rodents. Distinct SVMPs appear to be more specialized to rat or chicken blood, strengthening the current hypothesis that toxin diversity enhances the possibilities of the snakes for hunting different prey or evading different predators. This functional diversity also impacts the complexity of human envenoming since different hemostatic mechanisms will be targeted by SVMPs accounting for the complexity of the response of humans to venoms. PMID:25313513

  2. Functional Variability of Snake Venom Metalloproteinases: Adaptive Advantages in Targeting Different Prey and Implications for Human Envenomation

    PubMed Central

    Bernardoni, Juliana L.; Sousa, Leijiane F.; Wermelinger, Luciana S.; Lopes, Aline S.; Prezoto, Benedito C.; Serrano, Solange M. T.; Zingali, Russolina B.; Moura-da-Silva, Ana M.

    2014-01-01

    Snake venom metalloproteinases (SVMPs) are major components in most viperid venoms that induce disturbances in the hemostatic system and tissues of animals envenomated by snakes. These disturbances are involved in human pathology of snake bites and appear to be essential for the capture and digestion of snake's prey and avoidance of predators. SVMPs are a versatile family of venom toxins acting on different hemostatic targets which are present in venoms in distinct structural forms. However, the reason why a large number of different SVMPs are expressed in some venoms is still unclear. In this study, we evaluated the interference of five isolated SVMPs in blood coagulation of humans, birds and small rodents. P-III class SVMPs (fractions Ic, IIb and IIc) possess gelatinolytic and hemorrhagic activities, and, of these, two also show fibrinolytic activity. P-I class SVMPs (fractions IVa and IVb) are only fibrinolytic. P-III class SVMPs reduced clotting time of human plasma. Fraction IIc was characterized as prothrombin activator and fraction Ic as factor X activator. In the absence of Ca2+, a firm clot was observed in chicken blood samples with fractions Ic, IIb and partially with fraction IIc. In contrast, without Ca2+, only fraction IIc was able to induce a firm clot in rat blood. In conclusion, functionally distinct forms of SVMPs were found in B. neuwiedi venom that affect distinct mechanisms in the coagulation system of humans, birds and small rodents. Distinct SVMPs appear to be more specialized to rat or chicken blood, strengthening the current hypothesis that toxin diversity enhances the possibilities of the snakes for hunting different prey or evading different predators. This functional diversity also impacts the complexity of human envenoming since different hemostatic mechanisms will be targeted by SVMPs accounting for the complexity of the response of humans to venoms. PMID:25313513

  3. Proteomics application exercise of the Swiss Proteomics Society: report of the SPS'02 session.

    PubMed

    Binz, Pierre-Alain; Abdi, Fadi; Affolter, Michael; Allard, Laure; Barblan, Jachen; Bhardwaj, Sanjeev; Bienvenut, Willy V; Bulet, Philippe; Burgess, Jennifer; Carrette, Odile; Corthals, Garry; Delalande, François; Diemer, Hélène; Favreau, Philippe; Giuliano, Elia; Gueguen, Yannick; Guillaume, Elisabeth; Hahner, Stephanie; Man, Petr; Michalet, Sophie; Neri, Dario; Noukakis, Dimitrios; Palagi, Patricia; Paroutaud, Pierre; Pimenta, Daniel Carvalho; Quadroni, Manfredo; Resemann, Anja; Richert, Sophie; Rybak, Jascha; Sanchez, Jean-Charles; Scherl, Alexander; Scheurer, Simone; Schweiger Hufnagel, Ulrike; Siethoff, Christoph; Suckau, Detlev; van Dorsselaer, Alain; Wagner Redeker, Winfried; Walter, Nadia; Stöcklin, Reto

    2003-08-01

    After the success of the mass spectrometry (MS) round table that was held at the first Swiss Proteomics Society congress (SPS'01) in Geneva, the SPS has organized a proteomics application exercise and allocated a full session at the SPS'02 congress. The main objective was to encourage the exchange of expertise in protein identification, with a focus on the use of mass spectrometry, and to create a bridge between the users' questions and the instrument providers' solutions. Two samples were sent to fifteen interested labs, including academic groups and MS hardware providers. Participants were asked to identify and partially characterize the samples. They consisted of a complex mixture of peptide/proteins (sample A) and an almost pure recombinant peptide carrying post-translational modifications (sample B). Sample A was an extract of snake venom from the species Bothrops jararaca. Sample B was a recombinant and modified peptide derived from the shrimp Penaeus vannamei penaeidin 3a. The eight labs that returned results reported the use of a wide range of MS instrumentation and techniques. They mentioned a variety of time and manpower allocations. The origin of sample A was generally identified together with a number of database protein entries. The difficulty of the sample identification lay in the incomplete knowledge of the Bothrops species genome sequence and is discussed. Sample B was generally and correctly identified as penaeidin. However, only one group reported the full primary structure. Interestingly, the approaches were again varied and are discussed in the text.

  4. Recombinant expression of the precursor of the hemorrhagic metalloproteinase HF3 and its non-catalytic domains using a cell-free synthesis system.

    PubMed

    Menezes, Milene C; Imbert, Lionel; Kitano, Eduardo S; Vernet, Thierry; Serrano, Solange M T

    2016-09-01

    Snake venom metalloproteinases (SVMPs) participate in snakebite pathology such as hemorrhage, inflammation, and necrosis. They are synthesized as latent multi-domain precursors whose processing generates either catalytically active enzymes or free non-enzymatic domains. Recombinant expression of the precursor of P-III class SVMPs has failed due to the instability of the multi-domain polypeptide structure. Conversely, functional recombinant non-catalytic domains were obtained by prokaryotic expression systems. Here, we show for the first time the recombinant expression of the precursor of HF3, a highly hemorrhagic SVMP from Bothrops jararaca, and its non-catalytic domains, using an E. coli-based cell-free synthesis system. The precursor of HF3, composed of pro-, metalloproteinase-, disintegrin-like-, and cysteine-rich domains, and containing 38 Cys residues, was successfully expressed and purified. A protein composed of the disintegrin-like and cysteine-rich domains (DC protein) and the cysteine-rich domain alone (C protein) were expressed in vitro individually and purified. Both proteins were shown to be functional in assays monitoring the interaction with matrix proteins and in modulating the cleavage of fibrinogen by HF3. These data indicate that recombinant expression using prokaryotic-based cell-free synthesis emerges as an attractive alternative for the study of the structure and function of multi-domain proteins with a high content of Cys residues. PMID:27209197

  5. Effects of Schizolobium parahyba Extract on Experimental Bothrops Venom-Induced Acute Kidney Injury

    PubMed Central

    Martines, Monique Silva; Mendes, Mirian M.; Shimizu, Maria H. M.; Melo Rodrigues, Veridiana; de Castro, Isac; Filho, Sebastião R. Ferreira; Malheiros, Denise M. A. C.; Yu, Luis; Burdmann, Emmanuel A.

    2014-01-01

    Background Venom-induced acute kidney injury (AKI) is a frequent complication of Bothrops snakebite with relevant morbidity and mortality. The aim of this study was to assess the effects of Schizolobium parahyba (SP) extract, a natural medicine with presumed anti-Bothrops venom effects, in an experimental model of Bothrops jararaca venom (BV)-induced AKI. Methodology Groups of 8 to 10 rats received infusions of 0.9% saline (control, C), SP 2 mg/kg, BV 0.25 mg/kg and BV immediately followed by SP (treatment, T) in the doses already described. After the respective infusions, animals were assessed for their glomerular filtration rate (GFR, inulin clearance), renal blood flow (RBF, Doppler), blood pressure (BP, intra-arterial transducer), renal vascular resistance (RVR), urinary osmolality (UO, freezing point), urinary neutrophil gelatinase-associated lipocalin (NGAL, enzyme-linked immunosorbent assay [ELISA]), lactate dehydrogenase (LDH, kinetic method), hematocrit (Hct, microhematocrit), fibrinogen (Fi, Klauss modified) and blinded renal histology (acute tubular necrosis score). Principal Findings BV caused significant decreases in GFR, RBF, UO, HcT and Fi; significant increases in RVR, NGAL and LDH; and acute tubular necrosis. SP did not prevent these changes; instead, it caused a significant decrease in GFR when used alone. Conclusion SP administered simultaneously with BV, in an approximate 10∶1 concentration, did not prevent BV-induced AKI, hemolysis and fibrinogen consumption. SP used alone caused a decrease in GFR. PMID:24551041

  6. Characterization and cDNA cloning of aminopeptidase A from the venom of Gloydius blomhoffi brevicaudus.

    PubMed

    Ogawa, Yuko; Murayama, Nobuhiro; Fujita, Yoshiaki; Yanoshita, Ryohei

    2007-06-15

    The aminopeptidase activities of snake venoms from Gloydius blomhoffi brevicaudus, Gloydius halys blomhoffii, Trimeresurus flavoviridis, Bothrops jararaca and Crotalus atrox were investigated. Aminopeptidase A (APA), aminopeptidase B and aminopeptidase N activities were present in all snake venoms. The strongest APA activity was found in venom from G. blomhoffi brevicaudus. The susceptibility to metallopeptidase inhibitors and the pH optimum of the partially purified enzyme from G. blomhoffi brevicaudus venom were similar to those of known APAs from mammals. A G. blomhoffi brevicaudus venom gland cDNA library was screened to isolate cDNA clones using probes based on highly conserved amino acid sequences in known APAs. Molecular cloning of APA from G. blomhoffi brevicaudus venom predicted that it was a type II integral membrane protein containing 958 amino acid residues with 17 potential N-linked glycosylation sites. It possessed a His-Glu-Xaa-Xaa-His-(Xaa)(18)-Glu zinc binding motif that allowed the classification of this protein as a member of the M1 family of zinc-metallopeptidases, or gluzincins. The deduced amino acid sequence shows approximately 60% sequence identity to mammalian APA sequences. This is the first study to report the primary structure of APA from a reptile. PMID:17383704

  7. Transcriptome analysis of the Amazonian viper Bothrops atrox venom gland using expressed sequence tags (ESTs).

    PubMed

    Neiva, Márcia; Arraes, Fabricio B M; de Souza, Jonso Vieira; Rádis-Baptista, Gandhi; Prieto da Silva, Alvaro R B; Walter, Maria Emilia M T; Brigido, Marcelo de Macedo; Yamane, Tetsuo; López-Lozano, Jorge Luiz; Astolfi-Filho, Spartaco

    2009-03-15

    Bothrops atrox is a highly dangerous pit viper in the Brazilian Amazon region. We produced a global catalogue of gene transcripts to identify the main toxin and other protein families present in the B. atrox venom gland. We prepared a directional cDNA library, from which a set of 610 high quality expressed sequence tags (ESTs) were generated by bioinformatics processing. Our data indicated a predominance of transcripts encoding mainly metalloproteinases (59% of the toxins). The expression pattern of the B. atrox venom was similar to Bothrops insularis, Bothrops jararaca and Bothrops jararacussu in terms of toxin type, although some differences were observed. B. atrox showed a higher amount of the PIII class of metalloproteinases which correlates well with the observed intense hemorrhagic action of its toxin. Also, the PLA2 content was the second highest in this sample compared to the other three Bothrops transcriptomes. To our knowledge, this work is the first transcriptome analysis of an Amazonian rain forest pit viper and it will contribute to the body of knowledge regarding the gene diversity of the venom gland of members of the Bothrops genus. Moreover, our results can be used for future studies with other snake species from the Amazon region to investigate differences in gene patterns or phylogenetic relationships. PMID:19708221

  8. Proteomic and Glycoproteomic Profilings Reveal That Post-translational Modifications of Toxins Contribute to Venom Phenotype in Snakes.

    PubMed

    Andrade-Silva, Débora; Zelanis, André; Kitano, Eduardo S; Junqueira-de-Azevedo, Inácio L M; Reis, Marcelo S; Lopes, Aline S; Serrano, Solange M T

    2016-08-01

    Snake venoms are biological weapon systems composed of secreted proteins and peptides that are used for immobilizing or killing prey. Although post-translational modifications are widely investigated because of their importance in many biological phenomena, we currently still have little understanding of how protein glycosylation impacts the variation and stability of venom proteomes. To address these issues, here we characterized the venom proteomes of seven Bothrops snakes using a shotgun proteomics strategy. Moreover, we compared the electrophoretic profiles of native and deglycosylated venoms and, in order to assess their subproteomes of glycoproteins, we identified the proteins with affinity for three lectins with different saccharide specificities and their putative glycosylation sites. As proteinases are abundant glycosylated toxins, we examined the effect of N-deglycosylation on their catalytic activities and show that the proteinases of the seven venoms were similarly affected by removal of N-glycans. Moreover, we prospected putative glycosylation sites of transcripts of a B. jararaca venom gland data set and detected toxin family related patterns of glycosylation. Based on our global analysis, we report that Bothrops venom proteomes and glycoproteomes contain a core of components that markedly define their composition, which is conserved upon evolution in parallel to other molecular markers that determine their phylogenetic classification. PMID:27297130

  9. Parasitological and immunological diagnoses from feces of captive-bred snakes at Vital Brazil Institute.

    PubMed

    Souza, Janaína Lima de; Barbosa, Alynne da Silva; Vazon, Adriana Prado; Uchôa, Claudia Maria Antunes; Nunes, Beatriz Coronato; Cortez, Myrian Bandeira Vianna; Silva, Valmir Laurentino da; Más, Leonora Brazil; Melgarejo, Aníbal Rafael; Bastos, Otilio Machado Pereira

    2014-01-01

    Fecal samples from 56 snakes at the Vital Brazil Institute, in the city of Niterói, Rio de Janeiro, were tested using the sedimentation and flotation techniques to investigate the evolutionary forms of parasites such as helminths and protozoa, and using enzyme immunoassay techniques to detect antigens of Cryptosporidium sp. and Giardia sp. Among the animals tested, 80.3% were positive for parasites. Out of these, there were 16 Bothrops jararaca, 16 B. jararacussu and 13 Crotalus durissus. The prevalence of parasitic nematodes was 41.1%, and nematodes were found in all three snake species. Among these, the most frequent finding was eggs of Kalicephalus sp., which were diagnosed in 25% of the snakes. The positivity for protozoa detected using parasite concentration techniques was 75%, including oocysts of Caryospora sp. in 75%, cysts with morphology similar to Giardia sp. 3.6%, amoeboid cysts in 41.1% and unsporulated coccidia oocysts in 8.9%. Immunoassays for Cryptosporidium sp. antigens produced positive findings in 60.7%. Pseudoparasites were detected in 64.3%. These results show that there is a need to improve the sanitary handling of captive-bred snakes, and also for the animal house that supplies rodents to feed them. The results also highlight that diagnostic tests should be performed periodically on stool specimens from captive-bred snakes.

  10. Alpha1-adrenoceptors trigger the snake venom production cycle in secretory cells by activating phosphatidylinositol 4,5-bisphosphate hydrolysis and ERK signaling pathway.

    PubMed

    Kerchove, Celine M; Luna, Milene S A; Zablith, Mariana B; Lazari, Maria F M; Smaili, Soraya S; Yamanouye, Norma

    2008-08-01

    Loss of venom from the venom gland after biting or manual extraction leads to morphological changes in venom secreting cells and the start of a cycle of production of new venom. We have previously shown that stimulation of both alpha- and beta-adrenoceptors in the secretory cells of the venom gland is essential for the onset of the venom production cycle in Bothrops jararaca. We investigated the signaling pathway by which the alpha-adrenoceptor initiates the venom production cycle. Our results show that the alpha(1)-adrenoceptor subtype is present in venom gland of the snake. In quiescent cells, stimulation of alpha(1)-adrenoceptor with phenylephrine increased the total inositol phosphate concentration, and this effect was blocked by the phospholipase C inhibitor U73122. Phenylephrine mobilized Ca(2+) from thapsigargin-sensitive stores and increased protein kinase C activity. In addition, alpha(1)-adrenoceptor stimulation increased the activity of ERK 1/2, partially via protein kinase C. Using RT-PCR approach we obtained a partial sequence of a snake alpha(1)-adrenoceptor (260 bp) with higher identity with alpha(1D) and alpha(1B)-adrenoceptors from different species. These results suggest that alpha(1)-adrenoceptors in the venom secreting cells are probably coupled to a G(q) protein and trigger the venom production cycle by activating the phosphatidylinositol 4,5-bisphosphate and ERK signaling pathway.

  11. Cloning and characterization of a basic phospholipase A2 homologue from Micrurus corallinus (coral snake) venom gland.

    PubMed

    de Oliveira, Ursula Castro; Assui, Alessandra; da Silva, Alvaro Rossan de Brandão Prieto; de Oliveira, Jane Silveira; Ho, Paulo Lee

    2003-09-01

    During the cloning of abundant cDNAs expressed in the Micrurus corallinus coral snake venom gland, several putative toxins, including a phospholipase A2 homologue cDNA (clone V2), were identified. The V2 cDNA clone codes for a potential coral snake toxin with a signal peptide of 27 amino acid residues plus a predicted mature protein with 119 amino acid residues. The deduced protein is highly similar to known phospholipases A2, with seven deduced S-S bridges at the same conserved positions. This protein was expressed in Escherichia coli as a His-tagged protein that allowed the rapid purification of the recombinant protein. This protein was used to generate antibodies, which recognized the recombinant protein in Western blot. This antiserum was used to screen a large number of venoms, showing a ubiquitous distribution of immunorelated proteins in all elapidic venoms but not in the viperidic Bothrops jararaca venom. This is the first description of a complete primary structure of a phospholipase A2 homologue deduced by cDNA cloning from a coral snake.

  12. Interaction of toxic venoms with the complement system

    PubMed Central

    Birdsey, Vanessa; Lindorfer, Jean; Gewurz, H.

    1971-01-01

    Thirty-nine venoms from various vertebrate and invertebrate species were tested for their ability to consume haemolytic complement (C) activity upon incubation in fresh guinea-pig serum. Nineteen had `anti-complementary' activity, and these were provisionally sorted into the following groups: Pattern I—exemplified by the Naja haje (Egyptian cobra) and six other Elapidae species (all cobras), which induced selective consumption of C3—C9, and led to formation of a stable C3—C9-consuming intermediate; Pattern II—exemplified by the Agkistrodon rhodostoma (Malayan pit viper), Bitis arietans (puff adder), Bothrops jararaca (South American pit viper), Bothrops atrox (Fer de Lance) and three other species, which induced marked consumption of C4 and C2, as well as C3—C9, but did not form a stable C3—C9-consuming intermediate; and individual animals, e.g. the Lachesis muta (bushmaster), which induced other patterns (III—VI) of complement component consumption. Active fractions of representative venoms were partially purified by ion exchange and gel filtration chromatography and their interactions with the complement system characterized further. It is anticipated that these enzymes, with a capacity to activate the complement system in unique ways, will prove to be of further experimental usefulness. PMID:4398349

  13. Seasonal, daily activity, and habitat use by three sympatric pit vipers (Serpentes, Viperidae) from southern Brazil.

    PubMed

    Rocha, Marcelo C; Hartmann, Paulo A; Winck, Gisele R; Cechin, Sonia Z

    2014-04-25

    Viperid snakes are widely distributed in the South America and the greater distribution range of the family is found at the Crotalinae subfamily. Despite the abundance of this snakes along their geographic distribution, some ecological aspects remain unknown, principally at subtropical areas. In the present study, we evaluated the activity (daily and seasonal) and the use of the habitat by Bothrops diporus, B. jararaca and B. jararacussu, in an Atlantic Forest area at southern Brazil. We observed higher incidence of viperid snakes during the months with higher temperatures, while no snakes were found during the months with lower temperatures. The data suggest the minimum temperature as environmental variable with the greatest influence on the seasonal activity of this species. Considering the daily activity, we observed a tendency of snakes to avoid the warmest hours. Bothrops jararacussu tend to avoid open areas, being registered only inside and at the edges of the forest. We compared our results with previous studies realized at tropical areas and we suggest the observed seasonal activity as an evolutive response, despite the influence of the different environmental variables, according to the occurence region.

  14. Diagnostic uses of snake venom.

    PubMed

    Marsh, N A

    2001-01-01

    Snake venom toxins are invaluable for the assay of coagulation factors and for the study of haemostasis generally. Thrombin-like enzymes (SVTLE) are used for fibrinogen and fibrinogen breakdown product assays as well as detecting dysfibrinogenaemias. Since SVTLE are not inhibited by heparin, they can be used for assaying antithrombin III in samples containing heparin. Snake venom prothrombin activators are utilised in prothrombin assays, whilst Russell's viper venom (RVV) can be used to assay clotting factors V, VII, X and lupus anticoagulants (LA). Activators from the taipan, Australian brown snake and saw-scaled viper have also been used to assay LA. Protein C (PC) and activated PC (APC) resistance can be measured by means of RVV, Protac (from Southern copperhead snake venom) and STA-Staclot (from Crotalus viridis helleri) whilst von Willebrand factor can be studied with Botrocetin (Bothrops jararaca). Finally, snake venom C-type lectins and metalloproteinase disintegrins are being used to study platelet glycoprotein receptors and show great potential for use in the routine coagulation laboratory. PMID:11910187

  15. Use of snake venom fractions in the coagulation laboratory.

    PubMed

    Marsh, N A

    1998-07-01

    Snake venom toxins are now regularly used in the coagulation laboratory for assaying haemostatic parameters and as coagulation reagents. Snake venom thrombin-like enzymes (SVTLE) are used for fibrinogen and fibrinogen breakdown product assay as well as detecting dysfibrinogenaemias. Significantly, because SVTLE are not inhibited by heparin, they can be used for defibrinating samples that contain the anticoagulant before assay of haemostatic variables. Prothrombin activators are found in many snake venoms and are used in prothrombin assays, for studying dysprothrombinaemias and preparing meizothrombin and non-enzymic prothrombin. Russell's viper (Daboia russelli) venom (RVV) contains a number of compounds useful in the assay of factors V, VII, X, platelet factor 3 and lupus anticoagulants. Activators from the taipan, Australian brown snake and saw-scaled viper have been used to assay lupus anticoagulants. Protein C and activated protein C resistance can be measured by means of RVV and Protac, a fast acting inhibitor from Southern copperhead snake venom and von Willebrand factor can be studied with Botrocetin from Bothrops jararaca venom. Finally, phospholipase A2 enzymes and the disintegrins, a family of Arg-Gly-Asp (RGD)-containing proteins found in snake venoms, show great potential for the study of haemostasis including, notably, platelet glycoprotein receptors GPIIb/IIIa and Ib. PMID:9712287

  16. Parasitological and immunological diagnoses from feces of captive-bred snakes at Vital Brazil Institute.

    PubMed

    Souza, Janaína Lima de; Barbosa, Alynne da Silva; Vazon, Adriana Prado; Uchôa, Claudia Maria Antunes; Nunes, Beatriz Coronato; Cortez, Myrian Bandeira Vianna; Silva, Valmir Laurentino da; Más, Leonora Brazil; Melgarejo, Aníbal Rafael; Bastos, Otilio Machado Pereira

    2014-01-01

    Fecal samples from 56 snakes at the Vital Brazil Institute, in the city of Niterói, Rio de Janeiro, were tested using the sedimentation and flotation techniques to investigate the evolutionary forms of parasites such as helminths and protozoa, and using enzyme immunoassay techniques to detect antigens of Cryptosporidium sp. and Giardia sp. Among the animals tested, 80.3% were positive for parasites. Out of these, there were 16 Bothrops jararaca, 16 B. jararacussu and 13 Crotalus durissus. The prevalence of parasitic nematodes was 41.1%, and nematodes were found in all three snake species. Among these, the most frequent finding was eggs of Kalicephalus sp., which were diagnosed in 25% of the snakes. The positivity for protozoa detected using parasite concentration techniques was 75%, including oocysts of Caryospora sp. in 75%, cysts with morphology similar to Giardia sp. 3.6%, amoeboid cysts in 41.1% and unsporulated coccidia oocysts in 8.9%. Immunoassays for Cryptosporidium sp. antigens produced positive findings in 60.7%. Pseudoparasites were detected in 64.3%. These results show that there is a need to improve the sanitary handling of captive-bred snakes, and also for the animal house that supplies rodents to feed them. The results also highlight that diagnostic tests should be performed periodically on stool specimens from captive-bred snakes. PMID:25054488

  17. Crotalid snake venom subproteomes unraveled by the antiophidic protein DM43.

    PubMed

    Rocha, Surza L G; Neves-Ferreira, Ana G C; Trugilho, Monique R O; Chapeaurouge, Alex; León, Ileana R; Valente, Richard H; Domont, Gilberto B; Perales, Jonas

    2009-05-01

    Snake venoms are mixtures of proteins and peptides with different biological activities, many of which are very toxic. Several animals, including the opossum Didelphis aurita, are resistant to snake venoms due to the presence of neutralizing factors in their blood. An antihemorrhagic protein named DM43 was isolated from opossum serum. It inhibits snake venom metalloproteinases through noncovalent complex formation with these enzymes. In this study, we have used DM43 and proteomic techniques to explore snake venom subproteomes. Four crotalid venoms were chromatographed through an affinity column containing immobilized DM43. Bound fractions were analyzed by one- and two-dimensional gel electrophoresis, followed by identification by MALDI-TOF/TOF mass spectrometry. With this approach, we could easily visualize and compare the metalloproteinase compositions of Bothrops atrox, Bothrops jararaca, Bothrops insularis, and Crotalus atrox snake venoms. The important contribution of proteolytic processing to the complexity of this particular subproteome was demonstrated. Fractions not bound to DM43 column were similarly analyzed and were composed mainly of serine proteinases, C-type lectins, C-type lectin-like proteins, l-amino acid oxidases, nerve growth factor, cysteine-rich secretory protein, a few metalloproteinases (and their fragments), and some unidentified spots. Although very few toxin families were represented in the crotalid venoms analyzed, the number of protein spots detected was in the hundreds, indicating an important protein variability in these natural secretions. DM43 affinity chromatography and associated proteomic techniques proved to be useful tools to separate and identify proteins from snake venoms, contributing to a better comprehension of venom heterogeneity. PMID:19267469

  18. Biochemical and biological properties of phospholipases A(2) from Bothrops atrox snake venom.

    PubMed

    Kanashiro, Milton M; de Cássia M Escocard, Rita; Petretski, Jorge H; Prates, Maura V; Alves, Elias W; Machado, Olga L T; da Silva, Wilmar Dias; Kipnis, Thereza L

    2002-10-01

    Phospholipases A(2) (PLA(2)s), of molecular mass 13-15kDa, are commonly isolated from snake venom. Two myotoxins with PLA(2) activity, BaPLA(2)I and BaPLA(2)III, with estimated molecular masses of 15kDa were isolated from the venom of Bothrops atrox using Sephacryl S-100-HR and reverse-phase chromatography. BaPLA(2)I was basic, with a pI of 9.1, while BaPLA(2)III was neutral with a pI of 6.9. On a molecular basis, BaPLA(2)III exhibited higher catalytic activity on synthetic substrates than BaPLA(2)I. Comparison of the N-terminal residues of BaPLA(2)I with other PLA(2) proteins from snake venoms showed that it has the highest homology (94%) with B. asper myotoxin II and homology with a PLA(2) Lys(49) from B. atrox (89%). In contrast, BaPLA(2)III demonstrated 75, 72, and 71% homology with PLA(2) from Vipera ammodytes meridionalis, B. jararacussu, and B. jararaca, respectively. BaPLA(2)I and BaPLA(2)III were capable, in vitro, of inducing mast cell degranulation and, in vivo, of causing creatine kinase release, edema, and myonecrosis typical of PLA(2)s from snake venoms, characterized by rapid disruption of the plasma membrane as indicated by clumping of myofilaments and necrosis of affected skeletal muscle cells. BaPLA(2)I- and BaPLA(2)III-specific monoclonal and polyclonal antibodies, although incapable of neutralizing PLA(2) edematogenic activity, blocked myonecrosis efficiently in an in vivo neutralization assay. The results presented herein suggest that the biological active site responsible for edema induction by these two PLA(2) enzymes is distinct from the myonecrosis active site and is not dependent upon the catalytic activity of the PLA(2) enzyme. PMID:12234622

  19. A multifaceted analysis of viperid snake venoms by two-dimensional gel electrophoresis: an approach to understanding venom proteomics.

    PubMed

    Serrano, Solange M T; Shannon, John D; Wang, Deyu; Camargo, Antonio C M; Fox, Jay W

    2005-02-01

    The complexity of Viperid venoms has long been appreciated by investigators in the fields of toxinology and medicine. However, it is only recently that the depth of that complexity has become somewhat quantitatively and qualitatively appreciated. With the resurgence of two-dimensional gel electrophoresis (2-DE) and the advances in mass spectrometry virtually all venom components can be visualized and identified given sufficient effort and resources. Here we present the use of 2-DE for examining venom complexity as well as demonstrating interesting approaches to selectively delineate subpopulations of venom proteins based on particular characteristics of the proteins such as antibody cross-reactivity or enzymatic activities. 2-DE comparisons between venoms from different species of the same genus (Bothrops) of snake clearly demonstrated both the similarity as well as the apparent diversity among these venoms. Using liquid chromatography/tandem mass spectrometry we were able to identify regions of the two-dimensional gels from each venom in which certain classes of proteins were found. 2-DE was also used to compare venoms from Crotalus atrox and Bothrops jararaca. For these venoms a variety of staining/detection protocols was utilized to compare and contrast the venoms. Specifically, we used various stains to visualize subpopulations of the venom proteomes of these snakes, including Coomassie, Silver, Sypro Ruby and Pro-Q-Emerald. Using specific antibodies in Western blot analyses of 2-DE of the venoms we have examined subpopulations of proteins in these venoms including the serine proteinase proteome, the metalloproteinase proteome, and the phospholipases A2 proteome. A functional assessment of the gelatinolytic activity of these venoms was also performed by zymography. These approaches have given rise to a more thorough understanding of venom complexity and the toxins comprising these venoms and provide insights to investigators who wish to focus on these venom

  20. Comparison of indirect and direct approaches using ion-trap and Fourier transform ion cyclotron resonance mass spectrometry for exploring viperid venom proteomes.

    PubMed

    Fox, Jay W; Ma, Li; Nelson, Kristina; Sherman, Nicholas E; Serrano, Solange M T

    2006-05-01

    In a sense, the field of snake venom proteomics has been under investigation since the very earliest biochemical studies where it was soon recognized that venoms are comprised of complex mixtures of bioactive molecules, most of which are proteins. Only with the re-emergence of 2D polyacrylamide gel electrophoresis (2D PAGE) and the recent developments in mass spectrometry for the identification/characterization of proteins coupled with venom gland transcriptomes has the field of snake venom proteomics began to flourish and provide exciting insights into the protein composition of venoms and subsequently their pathological activities. In this manuscript we will briefly discuss the state of snake venom proteomics followed by the presentation of several straightforward experiments designed to explore approaches to investigating venom proteomics. The first set of experiments used 1D gel electrophoresis (1D PAGE) of Crotalus atrox venom followed by slice-by-slice analysis of the proteins using liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). In the second set of experiments, C. atrox and Bothrops jararaca venoms were subjected to in-solution digestion followed by Fourier transform ion cyclotron resonance (FTICR) LC/MS/MS. The peptide ion-maps of these venoms were compared along with the proteins identified. In addition, the results were compared to the results observed from the 1D PAGE approach. From these studies it is clear that sample de-complexation/fractionation before mass spectrometry is still the best approach for maximum proteome coverage. Furthermore, comparison of venom proteomes based on tryptic peptide identities between the proteomes is not particularly effective since there does not appear to be a sufficient number of such identical peptides, derived from related proteins, present in venoms. Finally, as has previously been recognized without either better databases of venom protein sequences or facile and rapid de novo sequencing

  1. Fluorometric assay using naphthylamide substrates for assessing novel venom peptidase activities.

    PubMed

    Gasparello-Clemente, Elaine; Silveira, Paulo Flávio

    2002-11-01

    In the present study we examined the feasibility of using the fluorometry of naphthylamine derivatives for revealing peptidase activities in venoms of the snakes Bothrops jararaca, Bothrops alternatus, Bothrops atrox, Bothrops moojeni, Bothrops insularis, Crotalus durissus terrificus and Bitis arietans, of the scorpions Tityus serrulatus and Tityus bahiensis, and of the spiders Phoneutria nigriventer and Loxosceles intermedia. Neutral aminopeptidase (APN) and prolyl-dipeptidyl aminopeptidase IV (DPP IV) activities were presented in all snake venoms, with the highest levels in B. alternatus. Although all examined peptidase activities showed relatively low levels in arthropod venoms, basic aminopeptidase (APB) activity from P. nigriventer venom was the exception. Compared to the other peptidase activities, relatively high levels of acid aminopeptidase (APA) activity were restricted to B. arietans venom. B. arietans also exhibited a prominent content of APB activity which was lower in other venoms. Relatively low prolyl endopeptidase and proline iminopeptidase activities were, respectively, detectable only in T. bahiensis and B. insularis. Pyroglutamate aminopeptidase activity was undetectable in all venoms. All examined peptidase activities were undetectable in T. serrulatus venom. In this study, the specificities of a diverse array of peptidase activities from representative venoms were demonstrated for the first time, with a description of their distribution which may contribute to guiding further investigations. The expressive difference between snake and arthropod venoms was indicated by APN and DPP IV activities while APA and APB activities distinguished the venom of B. arietans from those of Brazilian snakes. The data reflected the relatively uniform qualitative distribution of the peptidase activities investigated, together with their unequal quantitative distribution, indicating the evolutionary divergence in the processing of peptides in these different

  2. Isolation and characterization of cotiaractivase, a novel low molecular weight prothrombin activator from the venom of Bothrops cotiara.

    PubMed

    Senis, Yotis A; Kim, Paul Y; Fuller, Gemma L J; García, Angel; Prabhakar, Sripadi; Wilkinson, Mark C; Brittan, Helen; Zitzmann, Nicole; Wait, Robin; Warrell, David A; Watson, Steve P; Kamiguti, Aura S; Theakston, R David G; Nesheim, Michael E; Laing, Gavin D

    2006-05-01

    In this study, we isolated a novel prothrombin activator from the venom of Bothrops cotiara, a Brazilian lance-headed pit viper (Cotiara, Jararaca preta, Biocotiara), which we have designated "cotiaractivase" (prefix: cotiar- from B. cotiara; suffix: -activase, from prothrombin activating activity). Cotiaractivase was purified using a phenyl-Superose hydrophobic interaction column followed by a Mono-Q anion exchange column. It is a single-chain polypeptide with a molecular weight of 22,931 Da as measured by mass spectroscopy. Cotiaractivase generated active alpha-thrombin from purified human prothrombin in a Ca2+-dependent manner as assessed by S2238 chromogenic substrate assay and SDS-PAGE. Cotiaractivase cleaved prothrombin at positions Arg271-Thr272 and Arg320-Ile321, which are also cleaved by factor Xa. However, the rate of thrombin generation by cotiaractivase was approximately 60-fold less than factor Xa alone and 17 x 10(6)-fold less than the prothrombinase complex. The enzymatic activity of cotiaractivase was inhibited by the chelating agent EDTA, whereas the serine protease inhibitor PMSF had no effect on its activity, suggesting that it is a metalloproteinase. Interestingly, S2238 inhibited cotiaractivase activity non-competitively, suggesting that this toxin contains an exosite that allows it to bind prothrombin independently of its active site. Tandem mass spectrometry and N-terminal sequencing of purified cotiaractivase identified peptides that were identical to regions of the cysteine-rich and disintegrin-like domains of known snake venom metalloproteinases. Cotiaractivase is a unique low molecular weight snake venom prothrombin activator that likely belongs to the metalloproteinase family of proteins. PMID:16647309

  3. Argininosuccinate Synthetase Is a Functional Target for a Snake Venom Anti-hypertensive Peptide

    PubMed Central

    Guerreiro, Juliano R.; Lameu, Claudiana; Oliveira, Eduardo F.; Klitzke, Clécio F.; Melo, Robson L.; Linares, Edlaine; Augusto, Ohara; Fox, Jay W.; Lebrun, Ivo; Serrano, Solange M. T.; Camargo, Antonio C. M.

    2009-01-01

    Bj-BPP-10c is a bioactive proline-rich decapeptide, part of the C-type natriuretic peptide precursor, expressed in the brain and in the venom gland of Bothrops jararaca. We recently showed that Bj-BPP-10c displays a strong, sustained anti-hypertensive effect in spontaneous hypertensive rats (SHR), without causing any effect in normotensive rats, by a pharmacological effect independent of angiotensin-converting enzyme inhibition. Therefore, we hypothesized that another mechanism should be involved in the peptide activity. Here we used affinity chromatography to search for kidney cytosolic proteins with affinity for Bj-BPP-10c and demonstrate that argininosuccinate synthetase (AsS) is the major protein binding to the peptide. More importantly, this interaction activates the catalytic activity of AsS in a dose-de pend ent manner. AsS is recognized as an important player of the citrulline-NO cycle that represents a potential limiting step in NO synthesis. Accordingly, the functional interaction of Bj-BPP-10c and AsS was evidenced by the following effects promoted by the peptide: (i) increase of NO metabolite production in human umbilical vein endothelial cell culture and of arginine in human embryonic kidney cells and (ii) increase of arginine plasma concentration in SHR. Moreover, α-methyl-dl-aspartic acid, a specific AsS inhibitor, significantly reduced the anti-hypertensive activity of Bj-BPP-10c in SHR. Taken together, these results suggest that AsS plays a role in the anti-hypertensive action of Bj-BPP-10c. Therefore, we propose the activation of AsS as a new mechanism for the anti-hypertensive effect of Bj-BPP-10c in SHR and AsS as a novel target for the therapy of hypertension-related diseases. PMID:19491403

  4. Haematopoiesis and a new mechanism for the release of mature blood cells from the bone marrow into the circulation in snakes (Ophidia).

    PubMed

    Sano-Martins, I Sigueko; Dabrowski, Z; Tabarowski, Z; Witkowska-Pelc, E; Spadacci Morena, D Denelle; Spodaryk, K

    2002-10-01

    This is the first description of haematopoiesis in snakes. Studies were carried out on the following species belonging to Ophidia: Bothrops jararaca, Bothrops jararacusu, Waglerophis merremii, Elaphe taeniura taeniura, Boa constrictor,and Python reticulatus. Smears of the peripheral blood and histological preparations from the vertebrae, ribs, liver, and spleen were studied under a light and electron microscope. Myeloid cells were present in the following locations in the vertebrae: the neural spine, zygoapophysial processes, floor of the neural canal, lacunae in the bodies of vertebrae and also inside the ribs. Although the vascular system was well developed, especially around the ribs, vessels inside the marrow cavities were scarce, both in the ribs and elsewhere where haematopoiesis was found. Venous sinuses were well developed in the vertebrae and in the rib regions from their costal head towards the middle area. They consisted of one layer of fine endothelial cells. Mature cells in the process of migration into the general circulation were only sporadically encountered when venous sinuses were studied on perfusion-fixed specimens. In contrast, almost every sinus venosus contained protrusions directed towards the lumen, filled mostly with mature and immature blood cells. Various stages of their formation were seen in the cross sections of venous sinuses ranging from small, newly formed to large, elongated ones, filled with many fully developed and some maturing blood cells. In many cases the apices of the protrusions were ruptured, and mature blood cells, as well as a few immature ones, were seen in their vicinity. This observation led us to a new hypothesis that blood cells are released from the extravascular space into the lumen of venous sinuses. In snakes, these cells are released into the systemic circulation mainly via the rupture of protrusions filled with mature blood cells and, to a lesser degree, by transcytosis as known in mammals. In the spleens

  5. Practical applications of snake venom toxins in haemostasis.

    PubMed

    Marsh, N A; Fyffe, T L

    1996-01-01

    Snake venom toxins have an established role in the coagulation laboratory for the assay of haemostatic parameters and a potential role for therapeutic treatment of thrombotic disorders. In the laboratory, snake venom thrombin-like enzymes (SVTLEs) are used for the assay of fibrinogen and detection of fibrinogen breakdown products and dysfibrinogenaemias. Importantly, because SVTLEs are not inhibited by heparin, they can be used for assaying antithrombin III and other parameters in samples which contain heparin. Prothrombin activators occur in many snake venoms and these have become established in the assay of prothrombin, in the study of dysprothrombinaemias and in the preparation of meizothrombin and non enzymic forms of prothrombin. Russell's viper (Daboia russelli) venom contains a number of useful compounds including toxins which can be used to assay blood clotting factors V, VII, X, platelet factor 3 and lupus anticoagulants (LA). More recently, activators from the taipan, Australian brown snake and saw-scaled viper have been used to assay LA. Proteins C and S can be measured by means of protac, a fast acting inhibitor from Southern copperhead snake venom and von Willebrand factor can be studied with botrocetin from Bothrops jararaca venom. The disintegrins, a large family of Arg-Gly-Asp (RGD)-containing proteins found in snake venoms, show great potential for the study of platelet glycoprotein receptors, notably, GPIIb/IIIa and Ib, and in the treatment of arterial thrombotic disease. Established SVTLEs used in clinical practice include ancrod and defibrase although success with these agents has been limited. A further group of enzymes under consideration as thrombolytic agents are the fibrinogenases. PMID:9425723

  6. Practical applications of snake venom toxins in haemostasis.

    PubMed

    Marsh, Neville; Williams, Vaughan

    2005-06-15

    Snake venom toxins affecting haemostasis have facilitated extensively the routine assays of haemostatic parameters in the coagulation laboratory. Snake venom thrombin-like enzymes (SVTLE) are used for fibrinogen/fibrinogen breakdown product assay and for the detection of fibrinogen dysfunction. SVTLE are not inhibited by heparin and can thus can be used for assaying antithrombin III and other haemostatic variables in heparin-containing samples. Snake venoms are a rich source of prothrombin activators and these are utilised in prothrombin assays, for studying dysprothrombinaemias and for preparing meizothrombin and non-enzymic forms of prothrombin. Russell's viper (Daboia russelli) venom (RVV) contains toxins which have been used to assay blood clotting factors V, VII, X, platelet factor 3 and, importantly, lupus anticoagulants (LA). Other prothrombin activators (from the taipan, Australian brown snake and saw-scaled viper) have now been used to assay LA. Protein C and activated protein C resistance can be measured by means of RVV and Protac, a fast acting inhibitor from Southern copperhead snake venom and von Willebrand factor can be studied with botrocetin from Bothrops jararaca venom. The disintegrins, a large family of Arg-Gly-Asp (RGD)-containing snake venom proteins, show potential for studying platelet glycoprotein receptors, notably, GPIIb/IIIa and Ib. Snake venom toxins affecting haemostasis are also used in the therapeutic setting: Ancrod (from the Malayan pit viper, Calloselasma rhodostoma), in particular, has been used as an anticoagulant to achieve 'therapeutic defibrination'. Other snake venom proteins show promise in the treatment of a range of haemostatic disorders. PMID:15922782

  7. Insights into cardiovascular effects of proline-rich oligopeptide (Bj-PRO-10c) revealed by structure-activity analyses: dissociation of antihypertensive and bradycardic effects.

    PubMed

    Paschoal, Juliana F B; Yamaguchi, Juliana; Miranda, José R R; Carretero, Gustavo; Melo, Robson L; Santos, Robson A S; Xavier, Carlos H; Schreier, Shirley; Camargo, Antonio C M; Ianzer, Danielle

    2014-02-01

    We have previously reported that the proline-rich decapeptide from Bothrops jararaca (Bj-PRO-10c) causes potent and sustained antihypertensive and bradycardic effects in SHR. These activities are independent of ACE inhibition. In the present study, we used the Ala-scan approach to evaluate the importance of each amino acid within the sequence of Bj-PRO-10c (Pyr(1)-Asn(2)-Trp(3)-Pro(4)-His(5)-Pro(6)-Gln(7)-Ile(8)-Pro(9)-Pro(10)). The antihypertensive and bradycardic effects of the analogues Bj-PRO-10c Ala(3), Bj-PRO-10c Ala(7), Bj-PRO-10c Ala(8) were similar to those of Bj-PRO-10c, whereas the analogues Bj-PRO-10c Ala(2), Bj-PRO-10c Ala(4), Bj-PRO-10c Ala(5), Bj-PRO-10c Ala(9), and Bj-PRO-10c Ala(10) kept the antihypertensive activity and lost bradycardic activity considerably. In contrast, Bj-PRO-10c Ala(1) and Bj-PRO-10c Ala(6) were unable to provoke any cardiovascular activity. In summary, we demonstrated that (1) the Pyr(1) and Pro(6) residues are essential for both, the antihypertensive and bradycardic effects of Bj-PRO-10c; (2) Ala-scan approach allowed dissociating blood pressure reduction and bradycardic effects. Conformational properties of the peptides were examined by means of circular dichroism (CD) spectroscopy. The different Ala-scan analogues caused either an increase or decrease in the type II polyproline helix content compared to Bj-PRO-10c. The complete loss of activity of the Pro(6) → Ala(6) mutant is probably due to the fact that in the parent peptide the His(5)-Pro(6) bond can exist in the cis configuration, which could correspond to the conformation of this bond in the bound state. Current data support the Bj-PRO-10c as a promising leader prototype to develop new agents to treat cardiovascular diseases and its co-morbidities.

  8. Dexamethasone antagonizes the in vivo myotoxic and inflammatory effects of Bothrops venoms.

    PubMed

    Patrão-Neto, Fernando Chagas; Tomaz, Marcelo Amorim; Strauch, Marcelo Abrahão; Monteiro-Machado, Marcos; Rocha, José Roberto Da Silva; Borges, Paula Alvarenga; Calil-Elias, Sabrina; Melo, Paulo A

    2013-07-01

    In the present work we investigated the toxic activities of two Bothrops snake venoms using in vivo and in vitro experimental protocols in mice and tested the protective effect of dexamethasone (DEXA) in different conditions, comparing it with the polyvalent antivenom. We also expanded the investigations on the antiophidic effect of the Eclipta prostrata (EP) crude extract. The administration of Bothrops jararaca and Bothrops jararacussu snake venoms induced muscle damage demonstrated in vivo by the elevation on plasma creatine kinase (CK) activity in mice and by the decrease in CK content in the extensor digitorum longus (EDL) muscle of these animals, and in vitro by the increase in the rate of CK release from the isolated EDL muscle. We also observed inflammatory response following perimuscular injection of B. jararacussu venom (1.0 mg/kg). Treatment with DEXA (1.0 mg/kg) preserved over 50% of the EDL muscle CK content in vivo when evaluated 24 and 72 h after the injection of B. jararacussu venom in mice, and likewise reduced about 20% of the edema induced by this venom. DEXA reduced in 50% the presence of inflammatory cells and their activity in EDL muscle. The EP extract (50 mg/kg) showed similar ability in preventing the induction of edema and the decrease in muscle CK content, and its association with DEXA showed additive effect. EP reduced over 77% of the plasma CK activity induced by the B. jararacussu venom. In the in vitro experiments, DEXA was not able to change the rate of CK release from EDL muscles exposed to 25 μg/mL of B. jararacussu venom, neither to prevent the fall in the amplitude of the indirectly evoked twitch at the phrenic-diaphragm preparation. EP extract showed otherwise a protective effect on these protocols, reaching up to 100% of protection when concentrations of 50.0 and 100.0 μg/mL were used. Altogether our results show that inflammation is at least in part responsible for the tissue damage induced by Bothrops snake

  9. [Epidemiology of accidents due to bites of poisonous snakes: a study of cases attended in 1988].

    PubMed

    Ribeiro, L A; Jorge, M T; Iversson, L B

    1995-10-01

    The attendance given to patients by ta specialized Hospital, in S. Paulo, Brazil, during 1988 is studied. The study is based on the medical records of 322 patients and on questionnaires filled out by author during interviews with 209 patients or their companions. The 322 snake-bites occurred mainly between October and April, in the diurnal period, mainly in the afternoon. Most of patients were adult males, mainly between 10 and 20 years of age. The parts of the body most frequently affected were the feet, hands and legs. The snakes of the genera Bothrops, Crotalus and Micrurus were responsible, respectively, for 306 (95.0%) 14 (4.4%) and 2 (0.6%) of the accidents under study. Among the 160 snakes that were classified at the Herpetological Section of the IB, 152 were Bothrops; 142 B. Jararaca, mostly young reptiles, and 8 were of the genus Crotalus. Of the patients, 90.4% recovered completely, 2.2% presented sequelae, 7.5% were transferred and thus it was impossible to follow them up. Of the 209 persons interviewed, the occupational group most prone to snake bites was agricultural workers, followed by studentes; nearly 60% of the accidents ocurred during work; most of the patients had their inferior extremities unprotected at the moment of the bite. On hundred and sixty patients (76.6%) submitted to some from of treatment before coming to the HVB-IB, the more common being the use of a tourniquet (50.2%), local squeezing in an attempt to remove part of the venom (33.5), application of substances on the site of the snake bite (36.8%) and the ingestion of others (12.9%). Slightly over a quarter of the patients underwent some kind of medical treatment before coming to the HVB-IB, the most common being antissepsis (8.2%), administration of antivenom (6.2%), antihistamines (5.7%) and analgesics (5.3%). The snake was seen before it struch by 187 (89.5%) of the 209 persons interviewed and in most cases it adopted the strike posture just before the first bite. PMID:8731278

  10. Occurrence of sulfated fucose branches in fucosylated chondroitin sulfate are essential for the polysaccharide effect preventing muscle damage induced by toxins and crude venom from Bothrops jararacussu snake.

    PubMed

    Monteiro-Machado, Marcos; Tomaz, Marcelo A; Fonseca, Roberto J C; Strauch, Marcelo A; Cons, Bruno L; Borges, Paula A; Patrão-Neto, Fernando C; Tavares-Henriques, Matheus S; Teixeira-Cruz, Jhonatha M; Calil-Elias, Sabrina; Cintra, Adélia C O; Martinez, Ana Maria B; Mourão, Paulo A S; Melo, Paulo A

    2015-05-01

    Snake envenoming is an important public health problem around the world, particularly in tropics. Beyond deaths, morbidity induced by snake venoms, such as myotoxicity, is of pivotal consequence to population. Bothrops jararacussu is the main venomous snake in southeast region of Brazil, and particularly presents strong myotoxic effect. The only available therapy, antibothropic antivenom, poorly affects venom-induced myotoxicity. The aim of this study is to assess the ability of fucosylated chondroitin sulfate (fucCS), a glycosaminoglycan with anticoagulant and antithrombotic properties, and its derivatives to inhibit toxic activities of B. jararacussu crude venom and its isolated toxins, named bothropstoxins (BthTX-I and BthTX-II). The in vitro myotoxic activities induced by crude venom, by BthTX-I alone and by toxins together were abolished by fucCS. Carboxyl reduction (fucCS-CR) kept this ability whereas defucosilation (defucCS) abrogates myoprotection. We observed the same pattern in the response of these polysaccharides in antagonizing the increase in plasma creatine kinase (CK) levels, the reduction of skeletal muscle CK content and the rise of myeloperoxidase (MPO) activity induced by crude venom and isolated toxins. FucCS inhibited edematogenic activity and partially prevented the reduction of total leukocytes in blood when pre-incubated with crude venom. Furthermore, the venom procoagulant effect was completely antagonized by increasing concentrations of fucCS, although this polyanion could stop neither the tail bleeding nor the skin hemorrhage induced by Bothrops jararaca venom. The B. jararacussu phospholipase, hyaluronidase, proteolytic and collagenase activities were inhibited in vitro. The results suggest that fucCS could be able to interact with both toxins, and it is able to inhibit BthTX-II phospholipase activity. Light microscopy of extensor digitorum longus muscle (EDL) muscle showed myoprotection by fucCS, once necrotic areas, edema and