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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. Antigenic, microbicidal and antiparasitic properties of an l-amino acid oxidase isolated from Bothrops jararaca snake venom.

    PubMed

    Ciscotto, P; Machado de Avila, R A; Coelho, E A F; Oliveira, J; Diniz, C G; Farías, L M; de Carvalho, M A R; Maria, W S; Sanchez, E F; Borges, A; Chávez-Olórtegui, C

    2009-03-01

    Venoms from the bee Apis mellifera, the caterpillar Lonomia achelous, the spiders Lycosa sp. and Phoneutria nigriventer, the scorpions Tityus bahiensis and Tityus serrulatus, and the snakes Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi, Crotalus durissus terrificus, and Lachesis muta were assayed (800mug/mL) for activity against Staphylococcus aureus. Venoms from B. jararaca and B. jararacussu showed the highest S. aureus growth inhibition and also against other Gram-positive and Gram-negative bacteria. To characterize the microbicidal component(s) produced by B. jararaca, venom was fractionated through gel exclusion chromatography. The high molecular weight, anti-S. aureus P1 fraction was further resolved by anion exchange chromatography through Mono Q columns using a 0-0.5M NaCl gradient. Bactericidal Mono Q fractions P5 and P6 showed significant LAAO activity using l-leucine as substrate. These fractions were pooled and subjected to Heparin affinity chromatography, which rendered a single LAAO activity peak. The anti-S. aureus activity was abolished by catalase, suggesting that the effect is dependent on H(2)O(2) production. SDS-PAGE of isolated LAAO indicated the presence of three isoforms since deglycosylation with a recombinant N-glycanase rendered a single 38.2 kDa component. B. jararaca LAAO specific activity was 142.7 U/mg, based on the oxidation of l-leucine. The correlation between in vivo neutralization of lethal toxicity (ED(50)) and levels of horse therapeutic antibodies anti-LAAO measured by ELISA was investigated to predict the potency of Brazilian antibothropic antivenoms. Six horses were hyperimmunized with Bothrops venoms (50% from B. jararaca and 12.5% each from B. alternatus, B. jararacussu, B. neuwiedii and B. moojeni). To set up an indirect ELISA, B. jararaca LAAO and crude venom were used as antigens. Correlation coefficients (r) between ED(50) and ELISA antibody titers against B. jararaca

  3. 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

  4. Antitumor effect of Bothrops jararaca venom.

    PubMed Central

    da Silva, Reinaldo J; da Silva, Márcia G; Vilela, Lízia C; Fecchio, Denise

    2002-01-01

    Many experimental studies have been carried out using snake venoms for the treatment of animal tumors, with controversial results. While some authors have reported an antitumor effect of treatment with specific snake venom fractions, others have reported no effects after this treatment. The aim of this study was to evaluate the effect of Bothrops jararaca venom (BjV) on Ehrlich ascites tumor (EAT) cells in vivo and in vitro. In the in vivo study, Swiss mice were inoculated with EAT cells by the intraperitoneal (i.p.) route and treated with BjV venom (0.4 mg/kg, i.p.), on the 1st, 4th, 7th, 10th, and 13th days. Mice were evaluated for total and differential cells number on the 2nd, 5th, 8th, 11th and 14th days. The survival time was also evaluated after 60 days of tumor growth. In the in vitro study, EAT and normal peritoneal cells were cultivated in the presence of different BjV concentrations (2.5, 5.0, 10.0, 20.0, 40.0, and 80 microg) and viability was verified after 3, 6, 12 and 24 h of cultivation. Results were analyzed statistically by the Kruskal-Wallis and Tukey tests at the 5% level of significance. It was observed that in vivo treatment with BjV induced tumor growth inhibition, increased animal survival time, decreased mortality, increased the influx of polymorphonuclear leukocytes on the early stages of tumor growth, and did not affect the mononuclear cells number. In vitro treatment with BjV produced a dose-dependent toxic effect on EAT and peritoneal cells, with higher effects against peritoneal cells. Taken together, our results demonstrate that BjV has an important antitumor effect. This is the first report showing this in vivo effect for this venom. PMID:12061431

  5. New methodology for the obtainment of antibothropic factors from the South American opossum (Didelphis marsupialis) and jararaca snake (Bothrops jararaca).

    PubMed

    Neves-Ferreira, A G; Valente, R H; Sá, P G; Rocha, S L; Moussatché, H; Domont, G B; Perales, J

    1999-10-01

    The antibothropic factor (ABF) from D. marsupialis was collected from perforated hollow plastic golf balls which were surgically implanted subcutaneously in anesthetized opossums, a technique originally described for the production of polyclonal antibodies. Two months after the implantation of the balls, approximately 15 ml of seromatous fluid from D. marsupialis (SFDm-50 mg total protein/ml) could be recovered monthly. Opossum serum as well as SFDm showed similar SDS-PAGE profiles and antihemorrhagic potencies against Bothrops jararaca snake venom (Bjv). The presence of ABF in SFDm was confirmed by immunoblotting, using rabbit polyclonal antibodies raised against ABF isolated from opossum serum. ABF isolated from SFDm or from serum by ion-exchange chromatography showed identical chromatographic and electrophoretic profiles. ABF fromboth sources displayed very similar antihemorrhagic and anticaseinolytic activities against Bjv. In the case of B. jararaca, polyethylene perforated tubes were inserted in the abdominal cavity and two months after implantation, approximately 4 ml of seromatous fluid from B. jararaca (SFBj-23 mg total protein/ml) were recovered. B.jararaca serum and SFBj showed the same native and SDS-PAGE band pattern. Both serum and SFBj inhibited Bjv hemorrhagic activity. We conclude that this new methodology is very suitable for continuously obtaining opossum ABF and SFBj, in large scale and in an easier way, avoiding animal suffering and eventual sacrifice. PMID:10414866

  6. Biochemical and functional characterization of Bothropoidin: the first haemorrhagic metalloproteinase from Bothrops pauloensis snake venom.

    PubMed

    Gomes, Mário Sérgio R; Naves de Souza, Dayane L; Guimarães, Denise O; Lopes, Daiana S; Mamede, Carla C N; Gimenes, Sarah Natalie C; Achê, David C; Rodrigues, Renata S; Yoneyama, Kelly A G; Borges, Márcia H; de Oliveira, Fábio; Rodrigues, Veridiana M

    2015-03-01

    We present the biochemical and functional characterization of Bothropoidin, the first haemorrhagic metalloproteinase isolated from Bothrops pauloensis snake venom. This protein was purified after three chromatographic steps on cation exchange CM-Sepharose fast flow, size-exclusion column Sephacryl S-300 and anion exchange Capto Q. Bothropoidin was homogeneous by SDS-PAGE under reducing and non-reducing conditions, and comprised a single chain of 49,558 Da according to MALDI TOF analysis. The protein presented an isoelectric point of 3.76, and the sequence of six fragments obtained by MS (MALDI TOF\\TOF) showed a significant score when compared with other PIII Snake venom metalloproteinases (SVMPs). Bothropoidin showed proteolytic activity on azocasein, Aα-chain of fibrinogen, fibrin, collagen and fibronectin. The enzyme was stable at pH 6-9 and at lower temperatures when assayed on azocasein. Moreover, its activity was inhibited by EDTA, 1.10-phenanthroline and β-mercaptoethanol. Bothropoidin induced haemorrhage [minimum haemorrhagic dose (MHD) = 0.75 µg], inhibited platelet aggregation induced by collagen and ADP, and interfered with viability and cell adhesion when incubated with endothelial cells in a dose and time-dependent manner. Our results showed that Bothropoidin is a haemorrhagic metalloproteinase that can play an important role in the toxicity of B. pauloensis envenomation and might be used as a tool for studying the effects of SVMPs on haemostatic disorders and tumour metastasis. PMID:25261583

  7. Interaction of Bothrops jararaca venom metalloproteinases with protein inhibitors.

    PubMed

    Asega, Amanda F; Oliveira, Ana K; Menezes, Milene C; Neves-Ferreira, Ana Gisele C; Serrano, Solange M T

    2014-03-01

    Snake venom metalloproteinases (SVMPs) play important roles in the local and systemic hemorrhage observed upon envenomation. In a previous study on the structural elements important for the activities of HF3 (highly hemorrhagic, P-III-SVMP), bothropasin (hemorrhagic, P-III-SVMP) and BJ-PI (non-hemorrhagic, P-I-SVMP), from Bothrops jararaca, it was demonstrated that they differ in their proteolysis profile of plasma and extracellular matrix proteins. In this study, we evaluated the ability of proteins DM43 and α2-macroglobulin to interfere with the proteolytic activity of these SVMPs on fibrinogen and collagen VI and with their ability to induce hemorrhage. DM43 inhibited the proteolytic activity of bothropasin and BJ-PI but not that of HF3, and was not cleaved the three proteinases. On the other hand, α2-macroglobulin did not inhibit any of the proteinases and was rather cleaved by them. In agreement with these findings, binding analysis showed interaction of bothropasin and BJ-PI but not HF3 to DM43 while none of the proteinases bound to α2-macroglobulin. Moreover, DM43 promoted partial inhibition of the hemorrhagic activity of bothropasin but not that of HF3. Our results demonstrate that metalloproteinases of B. jararaca venom showing different domain composition, glycosylation level and hemorrhagic potency show variable susceptibilities to protein inhibitors. PMID:24433992

  8. 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

  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. 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

  11. Expression and partial biochemical characterization of a recombinant serine protease from Bothrops pauloensis snake venom.

    PubMed

    Isabel, Thais F; Costa, Guilherme Nunes Moreira; Pacheco, Isabela B; Barbosa, Luana G; Santos-Junior, Célio D; Fonseca, Fernando P P; Boldrini França, Johara; Henrique-Silva, Flávio; Yoneyama, Kelly A G; Rodrigues, Renata S; Rodrigues, Veridiana de Melo

    2016-06-01

    Snake venom serine proteases (SVSPs) are enzymes capable of interfering at several points of hemostasis. Some serine proteases present thrombin-like activity, which makes them targets for the development of therapeutics agents in the treatment of many hemostatic disorders. In this study, a recombinant thrombin-like serine protease, denominated rBpSP-II, was obtained from cDNA of the Bothrops pauloensis venom gland and was characterized enzymatically and biochemically. The enzyme rBpSP-II showed clotting activity on bovine plasma and proteolytic activity on fibrinogen, cleaving exclusively the Aα chain. The evaluation of rBpSP-II activity on chromogenic substrates demonstrated thrombin-like activity of the enzyme due to its capacity to hydrolyze the thrombin substrate. These characteristics make rBpSP-II an attractive molecule for additional studies. Further research is needed to verify whether rBpSP-II can serve as a template for the synthesis of therapeutic agents to treat hemostatic disorders. PMID:26965926

  12. Appraisal of antiophidic potential of marine sponges against Bothrops jararaca and Lachesis muta venom.

    PubMed

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

    2013-10-01

    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

  13. Diversity of metalloproteinases in Bothrops neuwiedi snake venom transcripts: evidences for recombination between different classes of SVMPs

    PubMed Central

    2011-01-01

    Background Snake venom metalloproteinases (SVMPs) are widely distributed in snake venoms and are versatile toxins, targeting many important elements involved in hemostasis, such as basement membrane proteins, clotting proteins, platelets, endothelial and inflammatory cells. The functional diversity of SVMPs is in part due to the structural organization of different combinations of catalytic, disintegrin, disintegrin-like and cysteine-rich domains, which categorizes SVMPs in 3 classes of precursor molecules (PI, PII and PIII) further divided in 11 subclasses, 6 of them belonging to PII group. This heterogeneity is currently correlated to genetic accelerated evolution and post-translational modifications. Results Thirty-one SVMP cDNAs were full length cloned from a single specimen of Bothrops neuwiedi snake, sequenced and grouped in eleven distinct sequences and further analyzed by cladistic analysis. Class P-I and class P-III sequences presented the expected tree topology for fibrinolytic and hemorrhagic SVMPs, respectively. In opposition, three distinct segregations were observed for class P-II sequences. P-IIb showed the typical segregation of class P-II SVMPs. However, P-IIa grouped with class P-I cDNAs presenting a 100% identity in the 365 bp at their 5' ends, suggesting post-transcription events for interclass recombination. In addition, catalytic domain of P-IIx sequences segregated with non-hemorrhagic class P-III SVMPs while their disintegrin domain grouped with other class P-II disintegrin domains suggesting independent evolution of catalytic and disintegrin domains. Complementary regions within cDNA sequences were noted and may participate in recombination either at DNA or RNA levels. Proteins predicted by these cDNAs show the main features of the correspondent classes of SVMP, but P-IIb and P-IIx included two additional cysteines cysteines at the C-termini of the disintegrin domains in positions not yet described. Conclusions In B. neuwiedi venom gland

  14. 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

  15. Cystyl aminopeptidase activity in the plasma, viscera and brain of the snake Bothrops jararaca.

    PubMed

    Alponti, Rafaela Fadoni; Zambotti-Villela, Leonardo; Murena-Nunes, Cristiane; Marinho, Camila Eduardo; do Amaral Olivo, Renata; Silveira, Paulo Flavio

    2005-07-01

    The relationship between plasma osmolality and cystyl aminopeptidase was characterized in the snake Bothrops jararaca and comparisons were made with the emerging picture of this relationship in rats. The profile of cystyl aminopeptidase activity under basal conditions was determined in the soluble and membrane-bound forms in visceral organs and in the central nervous system in comparison with that of alanyl aminopeptidase. The regional localization of cystyl and alanyl aminopeptidase activities was studied in the central nervous system. The basal level of plasma cystyl aminopeptidase, four- to six-fold higher than in rats, suggests its importance to help regulate circulating levels of neurohypophysial peptides in B. jararaca snake. The osmotic sensitivity of this plasma enzyme, undetectable in male, but about three-fold higher in female snakes than in rats, reveals a sexual dimorphism. In marked contrast to those observed in rats, low levels of soluble and particulate forms in the kidney indicate that cystyl aminopeptidase plays a minor metabolizing role at this anatomical location in B. jararaca. Despite of the regional-specific divergence between the levels of rat and snake enzymes, the bilaterally symmetric pattern of the diencephalic distribution of alanyl aminopeptidase reflects functional homologies between these two distantly related species. PMID:16006161

  16. Intraspecific variation of biological activities in venoms from wild and captive Bothrops jararaca.

    PubMed

    Saad, Eduardo; Curtolo Barros, Luciana; Biscola, Natalia; Pimenta, Daniel C; Barraviera, Silvia R C S; Barraviera, Benedito; Seabra Ferreira, Rui

    2012-01-01

    The venom of Bothrops jararaca is composed of complex mixture of molecules, mainly lectins, metalloproteinases, serinoproteinases, desintegrins, phospholipases, and peptides. This composition may vary according to the snake's age, gender, and region of origin. The aim of the was to determine individual variation in Bothrops jararaca venom in the Botucatu region, Sao Paulo State, Brazil, by means of enzymatic, biochemical, and pharmacological characterization, utilizing in vitro tests and biological assays. The activities were compared with those of Brazilian Reference Venom (BRV). Protein concentration varied between adult and juvenile groups. The electrophoretic profiles were similar, with molecular masses ranging between 25 and 50 kD, but with intraspecific variations. Reverse-phase high-performance liquid chromatography (RP-HPLC) revealed protein concentration differences. Coagulant activity did not differ significantly among adult groups, but there was a large variation between juvenile venom and BRV, which coagulated more extensively. Venoms from adults displayed greater hemorrhagic activity, especially in males recently obtained from the wild. In contrast, juveniles kept in captivity and adult males showed higher values. Edematogenic activity displayed an increase in edema in all groups. At the mean lethal dose (LD₅₀), toxicity varied significantly between groups, with venom from captive females being threefold more toxic than juvenile venom. Data illustrate the intra- and interspecific complexity that occurs in snake venoms, which may be attributed to ontogenetic, sexual, and environmental factors that affect variability in Bothrops jararaca venom. Further, it is proposed that Brazilian public health authorities document the constitution of pooled venom employed in the immunization of serum-producing animals due to this variability in venom properties. Given the large Brazilian territory, this variability requires regional monitoring and evaluation of

  17. Absence of oxytocin in the central nervous system of the snake Bothrops jararaca.

    PubMed

    Lazari, Maria Fatima Magalhaes; Alponti, Rafaela Fadoni; Freitas, Thalma Ariani; Breno, Maria Cristina; da Conceicao, Isaltino Marcelo; Silveira, Paulo Flavio

    2006-11-01

    We used four complementary techniques to investigate the presence of oxytocin peptide in the hypophysis and brain of the snake Bothrops jararaca. A high-pressure liquid chromatographic analysis failed to show oxytocin in extracts of hypophysial and brain tissues but provided estimative values of the amounts of vasotocin (12 ng/mg hypophysis and 0.5 ng/mg brain) and mesotocin (500 pg/mg hypophysis and 8 pg/mg brain). Western blots with a polyclonal anti-oxytocin antibody failed to detect oxytocin in both tissues but detected compounds with higher molecular weight than oxytocin, as well as a relatively weak cross-reactivity with mesotocin. The reverse transcription-polymerase chain reaction analysis failed to detect the expression of oxytocin gene transcript, but detected a transcript related to the mesotocin-neurophysin precursor in both tissues. Immunohistochemistry with the same anti-oxytocin antibody detected strong staining in the neurohypophysis and in few fibers in the inner zone of the median eminence, which was not abolished by pre-adsorption of this antibody with oxytocin, vasopressin, vasotocin or mesotocin and might not be attributed to oxytocin. In conclusion, our data demonstrate the absence of oxytocin in the central nervous system of the snake B. jararaca and underline the pitfalls that can result from the use of a single technique to investigate the presence of peptides in tissues. PMID:16838134

  18. Antivenom Effects of 1,2,3-Triazoles against Bothrops jararaca and Lachesis muta Snakes

    PubMed Central

    Domingos, Thaisa F. S.; Moura, Laura de A.; Carvalho, Carla; Campos, Vinícius R.; Jordão, Alessandro K.; Cunha, Anna C.; Ferreira, Vitor F.; de Souza, Maria Cecília B. V.; Sanchez, Eladio F.; Fuly, André L.

    2013-01-01

    Snake venoms are complex mixtures of proteins of both enzymes and nonenzymes, which are responsible for producing several biological effects. Human envenomation by snake bites particularly those of the viperid family induces a complex pathophysiological picture characterized by spectacular changes in hemostasis and frequently hemorrhage is also seen. The present work reports the ability of six of a series of 1,2,3-triazole derivatives to inhibit some pharmacological effects caused by the venoms of Bothrops jararaca and Lachesis muta. In vitro assays showed that these compounds were impaired in a concentration-dependent manner, the fibrinogen or plasma clotting, hemolysis, and proteolysis produced by both venoms. Moreover, these compounds inhibited biological effects in vivo as well. Mice treated with these compounds were fully protected from hemorrhagic lesions caused by such venoms. But, only the B. jararaca edema-inducing activity was neutralized by the triazoles. So the inhibitory effect of triazoles derivatives against some in vitro and in vivo biological assays of snake venoms points to promising aspects that may indicate them as molecular models to improve the production of effective antivenom or to complement antivenom neutralization, especially the local pathological effects, which are partially neutralized by antivenoms. PMID:23710441

  19. Isolation, structural and functional characterization of a new Lys49 phospholipase A2 homologue from Bothrops neuwiedi urutu with bactericidal potential.

    PubMed

    Corrêa, Edailson A; Kayano, Anderson M; Diniz-Sousa, Rafaela; Setúbal, Sulamita S; Zanchi, Fernando B; Zuliani, Juliana P; Matos, Najla B; Almeida, José R; Resende, Letícia M; Marangoni, Sérgio; da Silva, Saulo L; Soares, Andreimar M; Calderon, Leonardo A

    2016-06-01

    Snake venom is a complex mixture of active compounds consisting of 80-90% proteins and peptides that exhibit a variety of biological actions that are not completely clarified or identified. Of these, phospholipase A2 is one of the molecules that has shown great biotechnological potential. The objectives of this study were to isolate, biochemically and biologically characterize a Lys49 phospholipase A2 homologue from the venom of Bothrops neuwiedi urutu. The protein was purified after two chromatographic steps, anion exchange and reverse phase. The purity and relative molecular mass were assessed by SDS-PAGE, observing a molecular weight typical of PLA2s, subsequently confirmed by mass spectrometry obtaining a mass of 13,733 Da. As for phospholipase activity, the PLA2 proved to be enzymatically inactive. The analyses by Edman degradation and sequencing of the peptide fragments allowed for the identification of 108 amino acid residues; this sequence showed high identity with other phospholipases A2 from Bothrops snake venoms, and identified this molecule as a novel PLA2 isoform from B. neuwiedi urutu venom, called BnuTX-I. In murine models, both BnuTX-I as well as the venom induced edema and myotoxic responses. The cytotoxic effect of BnuTX-I in murine macrophages was observed at concentrations above 12 μg/mL. BnuTX-I also presented antimicrobial activity against gram-positive and negative bacterial strains, having the greatest inhibitory effect on Pseudomonas aeruginosa. The results allowed for the identification of a new myotoxin isoform with PLA2 structure with promising biotechnological applications. PMID:26927324

  20. Detection of an antibothropic fraction in opossum (Didelphis marsupialis) milk that neutralizes Bothrops jararaca venom.

    PubMed

    Jurgilas, P B; Neves-Ferreira, A G; Domont, G B; Moussatché, H; Perales, J

    1999-01-01

    An antibothropic fraction (ABF) from Didelphis marsupialis (opossum) serum, which is responsible for the neutralization of Bothrops jararaca venom was isolated by Perales et al. [Perales, J., Moussatché, H., Marangoni, S., Oliveira, B. and Domont, G. B. (1994). Isolation and partial characterization of an antibothropic complex from the serum of South American Didelphidae. Toxicon 32, 1237-1249]. The aim of this work was to verify the presence of this factor in opossum's milk, which could represent an additional protection for the neonatal opossum against bothropic venoms. An active milk fraction was isolated and showed similar physicochemical, structural, antigenic and biological properties when compared to ABF, indicating that they are probably the same protein. PMID:9920488

  1. 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

  2. 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

  3. 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. PMID:25699595

  4. Structural characterization and neuromuscular activity of a new Lys49 phospholipase A(2) homologous (Bp-12) isolated from Bothrops pauloensis snake venom.

    PubMed

    Randazzo-Moura, Priscila; Ponce-Soto, L A; Rodrigues-Simioni, Léa; Marangoni, Sérgio

    2008-09-01

    Bp-12 was isolated from Bothrops pauloensis snake venom in only one chromatographic step in reverse phase HPLC on micro-Bondapack C-18. The molecular mass of 13,789.56 Da was determined by mass spectrometry. The amino acids composition showed that Bp-12 presented high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA(2). The sequence of Bp-12 contains 122 amino acid residues: SLFELGKMIL QETGKNPAKS LGAFYCYCGW GSQGQPKDAV DRCCYVHKCC YKKITGCNPK KDRYSYSWKD KTLVCGEDNS CLKELCECDK AVAICLRENL NTYNKKYRYF LKPLCKKADA AC, with a pI value of 8.55 and with a high homology with Lys49 PLA(2) from other snake venoms. In mouse phrenic nerve-diaphragm, the time needed for 50% paralysis was: 45 +/- 6 min (1.4 microM) and 16 +/- 6 min (3.6 microM). Bp-12 can induce indirect and directly blocked evoked twitches, even in the preparations in which Ca(2+) is replaced by Sr(2+), being the addition of d-tubocurarine required for direct blocking. These results identify Bp-12 as a new member of the Lys49 PLA(2) family and shows that this toxin might contribute to the effects of the crude venom on the neuromuscular junction. PMID:18769889

  5. 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

  6. 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

  7. Inhibition of the hyperalgesic activity of Bothrops jararaca venom by an antibothropic fraction isolated from opossum (Didelphis marsupialis) serum.

    PubMed

    Rocha, S L; Frutuoso, V S; Domont, G B; Martins, M A; Moussatché, H; Perales, J

    2000-06-01

    The antibothropic fraction (ABF) already isolated from Didelphis marsupialis serum, inhibits the haemorrhagic, oedematogenic, myonecrotic and lethal activities of Bothrops jararaca venom (Bjv). The aim of this work was to verify the capability of ABF to inhibit the hyperalgesic activity of Bjv. Intraplantar injection of Bjv induced hyperalgesia in a time- and dose-dependent manner and ABF administered in situ concomitantly with Bjv or i.v. 30 min before venom injection reduced the induced hyperalgesia. This same effect was observed when ABF was intravenously injected at 5 and 15 min after Bjv. Our results show that ABF inhibits also the hyperalgesia induced by Bjv. PMID:10695972

  8. 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

  9. 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

  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. Subpopulations of mononuclear leukocytes associated with inhibition of Ehrlich ascites tumor growth by treatment with Bothrops jararaca venom.

    PubMed Central

    de Vieira Santos, Mariana Morena; da Silva, Reinaldo José; da Silva, Márcia Guimarães; Fecchio, Denise

    2004-01-01

    Snake venoms have been used as antineoplastic substances in several experimental models. We demonstrated in previous studies that Bothrops jararaca venom (BjV) induces inhibition of Ehrlich ascites tumor (EAT) growth accompanied by an increase of mononuclear (MN) leukocytes in all groups inoculated with EAT and/or venom. The objective of the present study was to characterize the subpopulations of MN leukocytes involved in the inhibition of EAT growth by treatment with BjV. Swiss mice were inoculated with 1.0x10(3) EAT cells by the intraperitoneal route and treated with 0.4 mg/kg of BjV by the same route (Group TV). Treatment was started 24 h after tumor cell inoculation and consisted of five intraperitoneal injections performed at 72 h intervals. After 2, 8 and 14 days, groups of animals were sacrificed and the number of B, TCD4 and TCD8 lymphocytes, macrophages and natural killer cells present in the peritoneal cavity was determined by flow cytometry. The control group consisted of animals inoculated with EAT and treated with 0.1 ml of saline under the same conditions as the experimental group (Group T). Two additional control groups consisted of animals not inoculated with EAT and treated with saline or venom. Data were analyzed statistically by the Kruskal-Wallis non-parametric test for independent samples. On the 2nd and 8th day we observed a difference between groups T and TV (group T > group TV) for all cell types, except natural killer cells, that only differed on the 2nd day. However, on the 14th day there was no difference in MN cells among groups. These data suggest that the inhibition of EAT is related to the toxic action of BjV on tumor cells and/or to the proteolytic effect of the venom on the mediators produced by the cells for growth modulation. PMID:15203562

  13. The central nervous system as target for antihypertensive actions of a proline-rich peptide from Bothrops jararaca venom.

    PubMed

    Lameu, Claudiana; Hayashi, Mirian A F; Guerreiro, Juliano R; Oliveira, Eduardo F; Lebrun, Ivo; Pontieri, Vera; Morais, Kátia L P; Camargo, Antonio C M; Ulrich, Henning

    2010-03-01

    Pyroglutamyl proline-rich oligopeptides, present in the venom of the pit viper Bothrops jararaca (Bj-PROs), are the first described naturally occurring inhibitors of the angiotensin I-converting enzyme (ACE). The inhibition of ACE by the decapeptide Bj-PRO-10c (

  14. Venom-related transcripts from Bothrops jararaca tissues provide novel molecular insights into the production and evolution of snake venom.

    PubMed

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

    2015-03-01

    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

  15. 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

  16. An alternative micromethod to access the procoagulant activity of Bothrops jararaca venom and the efficacy of antivenom.

    PubMed

    Oguiura, N; Kapronezai, J; Ribeiro, T; Rocha, M M T; Medeiros, C R; Marcelino, J R; Prezoto, B C

    2014-11-01

    The assessment of the capacity of antivenoms to neutralize the lethal activity of snake venoms still relies largely on traditional rodent lethality assay (LD50). However, adequately validated in vitro tests should be introduced for assessing antivenom neutralizing capacity in plasma of immunized horses as well as for in-process quality control. The dynamic of fibrin formation in recalcified avian plasma samples is extremely slow, when compared to that presented by mammalian plasmas. In this study, we present one new coagulant assay, by performing dose-response curve after plotting the clotting time (CT) parameter of the ROTEM profile of recalcified chicken plasma samples (target) against semi-logarithmic doses of Bothrops jararaca venom (agonist), either in absence or in presence of the semi-logarithmic doses of anti-bothropic serum (ABS) (antagonist). The mean coagulant dose 50% (CD50) was defined as the quantity of venom (in μg) which reduces CT to 900 s, between minimum and maximum responses. The CT induced by 5CD50 of the venom was used as the control for calculating the effective dose (ED) of each batch of ABS. ED was defined as the ABS dose (nanoliters, nL) at which CT induced by one amount of venom corresponding to 5CD50 is displaced to the maximum threshold (1800 s). Five batches of the ABS, previously assayed for their lethality neutralizing activity (ED50) were assayed. The correlation coefficient (r) between both in vitro (ED) and in vivo (ED50) values was 0.87 (p value < 0.05). We propose this micro method as highly sensitive for characterization and quantification of possible procoagulant activity of small doses of snake venoms (nanograms) and for detecting small doses (nanoliters) of specific antibodies against this effect in little volume samples of biological fluids. PMID:25128708

  17. 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

  18. 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

  19. Brain nitric oxide production by a proline-rich decapeptide from Bothrops jararaca venom improves baroreflex sensitivity of spontaneously hypertensive rats.

    PubMed

    Lameu, Claudiana; Pontieri, Vera; Guerreiro, Juliano R; Oliveira, Eduardo F; da Silva, Carlos Alberto; Giglio, Joyce M; Melo, Robson L; Campos, Ruy R; de Camargo, Antonio Carlos Martins; Ulrich, Henning

    2010-12-01

    Baroreflex sensitivity is disturbed in many people with cardiovascular diseases such as hypertension. Brain deficiency of nitric oxide (NO), which is synthesized by NO synthase (NOS) in the citrulline-NO cycle (with argininosuccinate synthase (ASS) activity being the rate-limiting step), contributes to impaired baroreflex. We recently showed that a decapeptide isolated from Bothrops jararaca snake venom, denoted Bj-PRO-10c, exerts powerful and sustained antihypertensive activity. Bj-PRO-10c promoted vasodilatation dependent on the positive modulation of ASS activity and NO production in the endothelium, and also acted on the central nervous system, inducing the release of GABA and glutamate, two important neurotransmitters in the regulation of autonomic systems. We evaluated baroreflex function using the regression line obtained by the best-fit points of measured heart rate (HR) and mean arterial pressure (MAP) data from spontaneously hypertensive rats (SHRs) treated with Bj-PRO-10c. We also investigated molecular mechanisms involved in this effect, both in vitro and in vivo. Bj-PRO-10c mediated an increase in baroreflex sensitivity and a decrease in MAP and HR. The effects exerted by the peptide include an increase in the gene expression of endothelial NOS and ASS. Bj-PRO-10c-induced NO production depended on intracellular calcium fluxes and the activation of a G(i/o)-protein-coupled metabotropic receptor. Bj-PRO-10c induced NO production and the gene expression of ASS and endothelial NOS in the brains of SHRs, thereby improving baroreflex sensitivity. Bj-PRO-10c may reveal novel approaches for treating diseases with impaired baroreflex function. PMID:21132021

  20. Bothrops jararaca venom (BjV) induces differential leukocyte accumulation in mice genetically selected for acute inflammatory reaction: the role of host genetic background on expression of adhesion molecules and release of endogenous mediators.

    PubMed

    Carneiro, Adriana S; Ribeiro, Orlando G; Cabrera, Wafa H K; Vorraro, Francisca; De Franco, Marcelo; Ibañez, Olga M; Starobinas, Nancy

    2008-10-01

    The dynamics of the local inflammatory events induced by Bothrops jararaca venom (BjV) inoculation in footpad of mice genetically selected for maximal (AIRmax) and minimal (AIRmin) acute inflammatory reactivity (AIR) was investigated. The BjV injection induced a marked inflammatory cell infiltrate with predominance of neutrophils, with increased blood cell numbers before its accumulation, suggesting a stimulatory action of BjV on mechanisms of cell mobilization from bone marrow. The process of cell migration is regulated by different cell-adhesion molecules (CAM). Our results showed that neutrophil cells from both lines had the same pattern of response concerning CAMs expression, presenting the involvement of l-selectin, Mac-1 and PECAM-1 adhesion molecules in BjV-induced neutrophil accumulation. The effect of BjV on the release of pro-inflammatory cytokines and chemokines related with cellular migration was also studied and IL-1beta, IL-6, TNF-alpha and MIP-2 levels could be detected after venom injection. The AIRmax mice were shown to be more responsive than AIRmin with respect to leukocyte influx, expression of MIP-2 and release of IL-1beta and IL-6. These results demonstrate the importance of host genetic background in the local response and the involvement of alleles accumulated in AIRmax mice in the inflammatory events induced by BjV. PMID:18723041

  1. 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

  2. A bradykinin-potentiating peptide (BPP-10c) from Bothrops jararaca induces changes in seminiferous tubules

    PubMed Central

    2013-01-01

    Background The testis-specific isoform of angiotensin-converting enzyme (tACE) is exclusively expressed in germ cells during spermatogenesis. Although the exact role of tACE in male fertility is unknown, it clearly plays a critical function in spermatogenesis. The dipeptidase domain of tACE is identical to the C-terminal catalytic domain of somatic ACE (sACE). Bradykinin potentiating peptides (BPPs) from snake venoms are the first natural sACE inhibitors described and their structure–activity relationship studies were the basis for the development of antihypertensive drugs such as captopril. In recent years, it has been showed that a number of BPPs – including BPP-10c – are able to distinguish between the N- and C-active sites of sACE, what is not applicable to captopril. Considering the similarity between tACE and sACE (and since BPPs are able to distinguish between the two active sites of sACE), the effects of the BPP-10c and captopril on the structure and function of the seminiferous epithelium were characterized in the present study. BPP-10c and captopril were administered in male Swiss mice by intraperitoneal injection (4.7 μmol/kg for 15 days) and histological sections of testes were analyzed. Classification of seminiferous tubules and stage analysis were carried out for quantitative evaluation of germ cells of the seminiferous epithelium. The blood-testis barrier (BTB) permeability and distribution of claudin-1 in the seminiferous epithelium were analyzed by hypertonic fixative method and immunohistochemical analyses of testes, respectively. Results The morphology of seminiferous tubules from animals treated with BPP-10c showed an intense disruption of the epithelium, presence of atypical multinucleated cells in the lumen and degenerated germ cells in the adluminal compartment. BPP-10c led to an increase in the number of round spermatids and total support capacity of Sertoli cell in stages I, V, VII/VIII of the seminiferous epithelium cycle, without affecting BTB permeability and the distribution of claudin-1 in the seminiferous epithelium. Interestingly, no morphological or morphometric alterations were observed in animals treated with captopril. Conclusions The major finding of the present study was that BPP-10c, and not captopril, modifies spermatogenesis by causing hyperplasia of round spermatids in stages I, V, and VII/VIII of the spermatogenic cycle. PMID:24195771

  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. Sympathetic outflow activates the venom gland of the snake Bothrops jararaca by regulating the activation of transcription factors and the synthesis of venom gland proteins.

    PubMed

    Luna, Milene S A; Hortencio, Thiago M A; Ferreira, Zulma S; Yamanouye, Norma

    2009-05-01

    The venom gland of viperid snakes has a central lumen where the venom produced by secretory cells is stored. When the venom is lost from the gland, the secretory cells are activated and new venom is produced. The production of new venom is triggered by the action of noradrenaline on both alpha(1)- and beta-adrenoceptors in the venom gland. In this study, we show that venom removal leads to the activation of transcription factors NFkappaB and AP-1 in the venom gland. In dispersed secretory cells, noradrenaline activated both NFkappaB and AP-1. Activation of NFkappaB and AP-1 depended on phospholipase C and protein kinase A. Activation of NFkappaB also depended on protein kinase C. Isoprenaline activated both NFkappaB and AP-1, and phenylephrine activated NFkappaB and later AP-1. We also show that the protein composition of the venom gland changes during the venom production cycle. Striking changes occurred 4 and 7 days after venom removal in female and male snakes, respectively. Reserpine blocks this change, and the administration of alpha(1)- and beta-adrenoceptor agonists to reserpine-treated snakes largely restores the protein composition of the venom gland. However, the protein composition of the venom from reserpinized snakes treated with alpha(1)- or beta-adrenoceptor agonists appears normal, judging from SDS-PAGE electrophoresis. A sexual dimorphism in activating transcription factors and activating venom gland was observed. Our data suggest that the release of noradrenaline after biting is necessary to activate the venom gland by regulating the activation of transcription factors and consequently regulating the synthesis of proteins in the venom gland for venom production. PMID:19411547

  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. 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. PMID:17225172

  7. Permanent genetic resources added to Molecular Ecology Resources Database 1 February 2012 - 31 March 2012.

    PubMed

    Andris, Malvina; Arias, M C; Barthel, Brandon L; Bluhm, Burton H; Bried, Joël; Canal, D; Chen, X M; Cheng, P; Chiappero, Marina B; Coelho, Manuela M; Collins, Angela B; Dash, M; Davis, Michelle C; Duarte, Margarida; Dubois, Marie-Pierre; Françoso, E; Galmes, M A; Gopal, Keshni; Jarne, Philippe; Kalbe, Martin; Karczmarski, Leszek; Kim, Hun; Martella, Mónica B; McBride, Richard S; Negri, Valeria; Negro, J J; Newell, Annakay D; Piedade, Ana F; Puchulutegui, Cecilia; Raggi, Lorenzo; Samonte, Irene E; Sarasola, J H; See, D R; Seyoum, Seifu; Silva, Mónica C; Solaro, C; Tolley, Krystal A; Tringali, Michael D; Vasemägi, A; Xu, L S; Zanón-Martínez, J I

    2012-07-01

    This article documents the addition of 171 microsatellite marker loci and 27 pairs of single nucleotide polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Bombus pauloensis, Cephalorhynchus heavisidii, Cercospora sojina, Harpyhaliaetus coronatus, Hordeum vulgare, Lachnolaimus maximus, Oceanodroma monteiroi, Puccinia striiformis f. sp. tritici, Rhea americana, Salmo salar, Salmo trutta, Schistocephalus solidus, Sousa plumbea and Tursiops aduncus. These loci were cross-tested on the following species: Aquila heliaca, Bulweria bulwerii, Buteo buteo, Buteo swainsoni, Falco rusticolus, Haliaeetus albicilla, Halobaena caerulea, Hieraaetus fasciatus, Oceanodroma castro, Puccinia graminis f. sp. Tritici, Puccinia triticina, Rhea pennata and Schistocephalus pungitii. This article also documents the addition of 27 sequencing primer pairs for Puffinus baroli and Bulweria bulwerii and cross-testing of these loci in Oceanodroma castro, Pelagodroma marina, Pelecanoides georgicus, Pelecanoides urinatrix, Thalassarche chrysostoma and Thalassarche melanophrys. PMID:22642264

  8. 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

  9. [Snakes from the urban area of Cuiabá, Mato Grosso: ecological aspects and associated snakebites].

    PubMed

    de Carvalho, M A; Nogueira, F N

    1998-01-01

    This study presents data on snakes recorded in the urban area of Cuiabá, Mato Grosso, Brazil. Sources of information included specimens captured by local residents (1986-1993) and turned over to the Mato Grosso Regional Ophiological Center (Normat), and data from the Anti-Venom Information Center (Ciave), regarding urban snake bites (1988-1993). Thirty-seven species of snakes from 25 genera and three families were recorded. Diurnal and terrestrial habits predominated, as well as a diet based on amphibians and/or lizards. From a total of 307 snake bites recorded, some 56% were of no clinical importance, caused by non-venomous snakes, whereas 44% were clinically relevant. Approximately 99% of the latter were attributed to vipers of the genus Bothrops, and especially the Bothrops moojeni and Bothrops neuwiedi species The colubrids Philodryas olfersii and Waglerophis merremii were probably responsible for most of the non-venomous snake bites. PMID:9878908

  10. Peptide T, a novel bradykinin potentiator isolated from Tityus serrulatus scorpion venom.

    PubMed

    Ferreira, L A; Alves, E W; Henriques, O B

    1993-08-01

    A bradykinin-potentiating peptide was isolated and characterized from venom of the scorpion Tityus serrulatus by chromatographic techniques followed by biological assays. The complete amino acid sequence (13 residues) of peptide is presented. The peptide potentiated the contractile activity of bradykinin on the isolated guinea-pig ileum, and inhibited the hydrolysis of bradykinin by angiotensin-converting enzyme from B. jararaca plasma and the conversion of angiotensin I to angiotensin II by kininase II from guinea-pig ileum tissue. The peptide also increased the depressor effect of bradykinin on arterial blood pressure in the anaesthetized rat. PMID:8212046

  11. Detection of calcium-binding proteins in venom and Duvernoy's glands of South American snakes and their secretions.

    PubMed

    Gonçalves, L R; Yamanouye, N; Nuñez-Burgos, G B; Furtado, M F; Britto, L R; Nicolau, J

    1997-10-01

    Calcium-binding proteins (CaBPs) have been described as involved in the stimulus-secretion coupling mechanisms in secretory glands. CaBPs were revealed with 45Ca, after electrophoresis in SDS-PAGE and transference to Zeta probe membranes, in Duvernoy's or venom gland homogenates from three families of South American snakes: Viperidae (Bothrops jararaca and Crotalus durissus terrificus); Elapidae (Micrurus corallinus), and Colubridae (Phylodrias patagoniensis and Oxyrhopus trigeminus). A band with an estimated molecular weight of 12 KDa was found in all glands studied. Bands with 17, 28, and 67 KDa were found in all glands, except in O. trigeminus Duvernoy's gland. A 18 KDa band was found in Viperidae and Elapidae venom glands, and a 88 KDa band was observed only in Viperidae venom gland homogenates. Some of these CaBPs were identified by Western blotting or by immunohistochemistry, as parvalbumin (12 KDa) and calbindin (28 KDa). When the secretion of these glands were analyzed, CaBPs were detected only in B. jararaca venom, with bands of 14, 35, 42, and 72 KDa. The profile of CaBPs was not modified at different phases of the secretory cycle of the glands, as well as after isoproterenol treatment. PMID:9440247

  12. 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

  13. The proteolytic action of Arvin on human fibrinogen

    PubMed Central

    Ewart, M. R.; Hatton, M. W. C.; Basford, J. M.; Dodgson, K. S.

    1970-01-01

    1. Human fibrinogen was subjected to proteolysis by enzyme preparations (clinical Arvin and IRC-50 Arvin) from the venom of Agkistrodon rhodostoma. 2. IRC-50 Arvin releases three peptides from fibrinogen, and these were identified as fibrinopeptides AP, AY and A. 3. The less purified `clinical' Arvin releases, in addition to fibrinopeptides AP, AY and A, small amounts of two heptapeptides derived from fibrinopeptides AP and A, probably because it contains another enzyme as well as Arvin. 4. No fibrinopeptide B is released by either Arvin preparation. 5. Thus, although Arvin is known to differ from `reptilase' from Bothrops jararaca in that it does not activate the enzyme that cross-links fibrin (fibrin-stabilizing factor), it is identical with reptilase with respect to the peptides that it liberates from fibrinogen. PMID:5529716

  14. Novel pharmaceutical composition of bradykinin potentiating penta peptide with beta-cyclodextrin: physical-chemical characterization and anti-hypertensive evaluation.

    PubMed

    Denadai, Angelo M L; Ianzer, Danielle; Alcântara, Antônio Flávio de C; Santoro, Marcelo M; Santos, Cynthia F F; Lula, Ivana Silva; de Camargo, Antônio C M; Faljoni-Alario, Adelaide; dos Santos, Robson A S; Sinisterra, Rubén D

    2007-05-01

    This work describes chemical properties and anti-hypertensive activity of an oral pharmaceutical formulation obtained from the complexation of beta-cyclodextrin (beta-CD) with bradykinin potentiating penta peptide (BPP-5a) founded in the Bothrops jararaca poison. Physical chemistry characterizations were recorded in order to investigate the intermolecular interactions between species in complex. Circular dichroism data indicated conformational changes of BPP-5a upon complexation with beta-CD. ROESY and theoretical calculations showed a selective approximation of triptophan moiety into cavity of beta-CD. Isothermal titration calorimetry data indicated an exothermic formation of the complex, which is accomplished by reduction of entropy. The anti-hypertensive activity of the BPP-5a/beta-CD complex has been evaluated in spontaneous hypertensive rats, showing better results than pure BPP-5a. PMID:17196774

  15. Software system for three-dimensional volumetric reconstruction of histological sections: a case study for the snake chondrocranium.

    PubMed

    Hofstadler-Deiques, Clarice; Walter, Marcelo; Mierlo, Fábio; Ruduit, Rodrigo

    2005-10-01

    Volumetric digital computer-assisted reconstruction of histological sections is an attractive possibility for developmental studies. Commercial solutions are very expensive for many educational institutions. Therefore, we developed a software system for three-dimensional reconstruction of anatomical virtual models. The input data for the system are the digitized images from the histological samples of the chondrocranium of two crotalines, Bothrops jararaca and Crotalus durissus terrificus, and one colubrid, Philodryas olfersii, using a stereomicroscope connected to a digital camera. These images are then manually registered and segmented. We use computer graphics visualization algorithms such as marching cubes and ray casting to generate three-dimensional visualizations of the volumes. The results show that the digital reconstruction is as good as the manual reconstruction with the advantages of speed of reconstruction, accuracy, and flexibility to handle and study the volume. Compared with commercial options, our system has approximately the same features, and it is available free for the scientific community. PMID:16114067

  16. 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. PMID:12056654

  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. 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

  19. Isolation and characterization of DM40 and DM43, two snake venom metalloproteinase inhibitors from Didelphis marsupialis serum.

    PubMed

    Neves-Ferreira, A G; Cardinale, N; Rocha, S L; Perales, J; Domont, G B

    2000-05-01

    From Didelphis marsupialis serum, two antihemorrhagic proteins were isolated by DEAE-Sephacel, Phenyl-Sepharose and Superdex 200 and characterized. Their masses by mass spectrometry were 40318 AMU for DM40 and 42373 and 43010 AMU for DM43, indicating the presence of isoforms for the last. Molecular masses of 44.8 and 47.3 were obtained by SDS-PAGE, respectively for DM40 and DM43. Both inhibitors showed isoelectric points lower than 3.5 and glycosylation percentages varying from 20.5 to 29.0%, as estimated by chemical deglycosylation and amino acid analysis. N-terminal sequences of the first 17 residues of DM40 and DM43 were identical except for the exchange of R9 for P9. Both were homologous to oprin, a similar inhibitor from Didelphis virginiana serum. No evidence of complex formation between DM40 and DM43 was observed either by native PAGE or gel filtration chromatography. In addition to the antihemorrhagic activity, DM40 and DM43 inhibited the hydrolysis of casein, fibrinogen and fibronectin by Bothrops jararaca venom. DM43 also showed antilethal, antiedematogenic and antihyperalgesic activities. None of the inhibitors showed enzymatic activity on casein. Both proteins formed stable complexes with jararhagin and inhibited its hemorrhagic effect as well as the enzymatic activity of this toxin on fluorogenic substrate. PMID:10779682

  20. PO41, a snake venom metalloproteinase inhibitor isolated from Philander opossum serum.

    PubMed

    Jurgilas, Patrícia B; Neves-Ferreira, Ana G C; Domont, Gilberto B; Perales, Jonas

    2003-11-01

    PO41 was isolated from Philander opossum serum by DEAE-Sephacel, Phenyl Superose and Superdex 200 chromatographies and showed a molecular mass of 41,330 Da by MALDI-TOF MS. Molecular masses of 81.5 and 84.5 kDa were obtained by size exclusion chromatography and dynamic laser light scattering, respectively, suggesting that PO41 is dimeric. Its isoelectric point was estimated to be lower than 3.5. PO41 presented similar amino terminal sequence to those of DM40 and DM43, two antihaemorrhagins previously isolated from Didelphis marsupialis serum and was recognized by polyclonal antibodies raised against D. marsupialis antibothropic fraction. To study the inhibitory properties of this protein, the metalloproteinases bothrolysin and jararhagin were isolated from Bothrops jararaca venom by chromatographies on Superdex 200 and Phenyl Superose. Jararhagin was further submitted to a Mono Q column. The proteolytic and haemorrhagic effects of these haemorrhagins were neutralized by PO41. Both snake venom metalloproteinases formed stable complexes with PO41. The stoichiometry of the complex PO41-jararhagin was one inhibitor subunit to one molecule of the enzyme. These results show that PO41 has physicochemical, structural, immunoreactive and biological properties similar to other metalloproteinase inhibitors belonging to the supergene family of immunoglobulins. PMID:14602117

  1. Purification and renal effects of phospholipase A(2) isolated from Bothrops insularis venom.

    PubMed

    Machado Braga, Marcus Davis; Costa Martins, Alice Maria; Alves, Claudênio Diógenes; de Menezes, Dalgimar Beserra; Martins, René Duarte; Ferreira Barbosa, Paulo Sérgio; de Sousa Oliveira, Isadora Maria; Toyama, Marcos Hikari; Toyama, Daniela Oliveira; Dos Santos Diz Filho, Eduardo Brito; Ramos Fagundes, Fabio Henrique; Fonteles, Manassés Claudino; Azul Monteiro, Helena Serra

    2008-02-01

    Bothrops insularis venom contains a variety of substances presumably responsible for several pharmacological effects. We investigated the biochemical and biological effects of phospholipase A(2) protein isolated from B. insularis venom and the chromatographic profile showed 7 main fractions and the main phospholipase A(2) (PLA(2)) enzymatic activity was detected in fractions IV and V. Fraction IV was submitted to a new chromatographic procedure on ion exchange chromatography, which allowed the elution of 5 main fractions designated as IV-1 to IV-5, from which IV-4 constituted the main fraction. The molecular homogeneity of this fraction was characterized by high-performance liquid chromatography (HPLC) and demonstrated by mass spectrometry (MS), which showed a molecular mass of 13984.20 Da; its N-terminal sequence presented a high amino acid identity (up to 95%) with the PLA(2) of Bothrops jararaca and Bothrops asper. Phospholipase A(2) isolated from B. insularis (Bi PLA(2) ) venom (10 microg/mL) was also studied as to its effect on the renal function of isolated perfused kidneys of Wistar rats (n=6). Bi PLA(2) increased perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF) and glomerular filtration rate (GFR). Sodium (%TNa(+)) and chloride tubular reabsorption (%TCl(-)) decreased at 120 min, without alteration in potassium transport. In conclusion, PLA(2) isolated from B. insularis venom promoted renal alterations in the isolated perfused rat kidney. PMID:17953979

  2. 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

  3. Exploiting the antithrombotic effect of the (pro)thrombin inhibitor bothrojaracin.

    PubMed

    Assafim, Mariane; Frattani, Flávia S; Ferreira, Marcos S; Silva, Dione M; Monteiro, Robson Q; Zingali, Russolina B

    2016-09-01

    Bothrojaracin is a 27 kDa C-type lectin-like protein from Bothrops jararaca snake venom. It behaves as a potent thrombin inhibitor upon high-affinity binding to thrombin exosites. Bothrojaracin also forms a stable complex with prothrombin that can be detected in human plasma. Formation of the zymogen-inhibitor complex severely decreases prothrombin activation and contributes to the anticoagulant activity of bothrojaracin. In the present study, we employed two rodent models to evaluate the antithrombotic effect of bothrojaracin in vivo: stasis-induced thrombosis and thrombin-induced pulmonary thromboembolism. It was observed that bothrojaracin interacts with rat prothrombin in plasma. Ex-vivo assays showed stable complex formation even after 24 h of a single bothrojaracin dose. As a result, bothrojaracin showed significant antithrombotic activity in a rat venous thrombosis model elicited by thromboplastin combined with stasis. The antithrombotic activity of bothrojaracin (1 mg/kg) persisted for up to 24 h and it was associated with moderate bleeding as assessed by a tail transection method. Formation of bothrojaracin-prothrombin complex has been also observed following intravenous administration of the inhibitor into mice. As a result, bothrojaracin effectively protected mice from thrombin-induced fatal thromboembolism. We conclude that bothrojaracin is a potent antithrombotic agent in vivo and may serve as a prototype for the development of new zymogen-directed drugs that could result in prolonged half-life and possible decreased hemorrhagic risk. PMID:27179421

  4. 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

  5. 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

  6. Duvernoy's gland secretion of Philodryas olfersii and Philodryas patagoniensis (Colubridae): neutralization of local and systemic effects by commercial bothropic antivenom (Bothrops genus).

    PubMed

    Rocha, Marisa M Teixeira; Paixão-Cavalcante, Danielle; Tambourgi, Denise V; Furtado, Maria de Fátima D

    2006-01-01

    Colubrids involved in human envenomation in Brazil are mainly from the genera Helicops, Oxyrhopus, Thamnodynastes and Philodryas. There is a relatively large number of clinical descriptions involving the Xenodontinae snakes, Philodryas olfersii and Philodryas patagoniensis, in human accidents. The most common manifestations of envenomation are local pain, swelling, erythema and ecchymosis and regional lymphadenopathy with normal coagulation. The aims of this study were to characterize the biochemical and biological properties of P. olfersii and P. patagoniensis venoms, and to investigate their immunological cross-reactivities by using both specific antisera and anti-Bothrops sp serum used for human serum therapy in Brazil, in neutralizing the lethal and hemorrhagic effects of these venoms. We show here that P. olfersii e P. patagoniensis venoms present proteolytic and haemorrhagic activities but are devoid of phospholipase A2 activity. Haemorrhage and lethality induced by P. olfersii and P. patagoniensis are associated with metal-dependent proteinases, since EDTA could block these toxic activities. P. olfersii and P. patagoniensis venoms were immunogenic and the antisera produced were able to recognize several bands in P. olfersii, P. patagoniensis venoms in Bothrops jararaca venom. PMID:16360723

  7. 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

  8. 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

  9. Kallikrein-kinin system in the plasma of snakes.

    PubMed

    Picarelli, Z P; Prezoto, B C; Hiraichi, E; Abdalla, F M

    1992-01-01

    Using pharmacological preparations suitable for assay of mammalian kinins, it was shown that Bothrops jararaca (Bj) venom and other kininogenases were unable to release kinins from snake plasma. The kallikrein-kinin system presents species-specificity in birds. In order to detect such a specificity in snakes, the effects of Bj venom on snake blood pressure and the effect of incubates of snake plasma with trypsin, on snake blood pressure and snake uterus, were studied. The possibility of activating snake plasma kallikrein with ellagic acid, glass beads or kaolin was also investigated. Whereas plasma of the snakes Waglerophis merremii (Wm) and Crotalus durissus (Cd), were shown to contain factor XII, prekallikrein, kininogen, kininases and to present a low but definite activation rate of the kinin system, the plasmas of Bj, Bothrops mojeni (Bm) and Oxyrophus trigeminus (Ot), yielded only kininogen and kininases. Activation of the system was not even detected by the sensitive substrate Ac-Phe-Arg-Nan (acetyl-phenylalanyl-arginyl-4nitro-anilide), indicating that the plasma of these species does not possess either factor XII and/or prekallikrein. Snake plasma may constitute an interesting model for the study of blood clotting, fibrinolytic and complement systems. PMID:1609651

  10. Inhibition of melanoma cells tumorigenicity by the snake venom toxin jararhagin.

    PubMed

    Corrêa, Mário César; Maria, Durvanei A; Moura-da-Silva, Ana M; Pizzocaro, Kazumi F; Ruiz, Itamar R G

    2002-06-01

    Skmel-28 human melanoma cells were treated with jararhagin (Jara), a metalloproteinase disintegrin isolated from Bothrops jararaca snake venom, and Jari (Jara with the catalytic domain inactivated). Following treatments, monolayer cells lost cytoplasmic expansions acquiring round shapes, detached and formed cell clusters in suspension. Cytotoxicity effect of Jari was dramatically increased at concentrations higher than 0.4 microM, whereas cell adhesion responses did not differ significantly between similar concentrations of Jara and Jari. Treated cells were significantly inhibited to adhere to non-coated wells, as to ECM proteins-coated plates. Migration and invasion were also significantly inhibited in vitro. A decreased proliferation rate was observed in toxin-treated cells. Immunofluorescence staining showed a wide distribution of Jari across the cells. Jara treated cells (67.5%) steady bound anti-jara antibodies after 90 min, while Jari treated cells steady bound only after 6h (57.3%), as determined by FACS. Skmel-28 melanoma cells tumorigenicity was evaluated 180 days after s.c. injections in AIRmin mice. A statistically significant decrease in the ability of Jara and Jari treated cells to promote lung metastasis was observed. These results point to the potential use of this toxin as a tool for applied researches in the clinical field. PMID:12175610

  11. 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

  12. Pioneers of anti-venomous serotherapy: Dr Vital Brazil (1865-1950).

    PubMed

    Hawgood, B J

    1992-01-01

    Dr Vital Brazil was a great humanitarian and pioneer of medical science. His main work arose from his concern with poisonous snakebite accidents to labourers working the land. Vital Brazil estimated that, at the beginning of this century, deaths due to crotaline snakebites in the State of São Paulo, Brazil, were nearly 3000 per year, representing a mortality rate of about 25%, the majority being due to bothropic envenomation. After reading a report of Calmette's anti-Naja serum, Vital Brazil raised monovalent serum against the venom of Bothrops jararaca and the venom of Crotalus durissus terrificus. In 1989 this led to the first demonstration of the specificity of anti-venomous serum and later, the first production of polyvalent serum for therapeutic use. As Director of the newly founded Institute Butantan in São Paulo, Vital Brazil was actively engaged in every aspect of serotherapeutic treatment. This included organizing a unique system of exchanging anti-ophidic serum for snakes as well as a wide-ranging teaching programme. His many outstanding contributions to the fields of immunology, public health, toxinology and herpetology required not only a very high level of observational, deductive and practical ability but also an unswerving vision and sense of duty; this was allied to great administrative skill and exceptional energy. PMID:1519249

  13. Functional analysis of DM64, an antimyotoxic protein with immunoglobulin-like structure from Didelphis marsupialis serum.

    PubMed

    Rocha, Surza L G; Lomonte, Bruno; Neves-Ferreira, Ana G C; Trugilho, Monique R O; Junqueira-de-Azevedo, Inácio de L M; Ho, Paulo L; Domont, Gilberto B; Gutiérrez, José M; Perales, Jonas

    2002-12-01

    Bothrops snake venoms are known to induce local tissue damage such as hemorrhage and myonecrosis. The opossum Didelphis marsupialis is resistant to these snake venoms and has natural venom inhibitors in its plasma. The aim of this work was to clone and study the chemical, physicochemical and biological properties of DM64, an antimyotoxic protein from opossum serum. DM64 is an acidic protein showing 15% glycosylation and with a molecular mass of 63 659 Da when analysed by MALDI-TOF MS. It was cloned and the amino acid sequence was found to be homologous to DM43, a metalloproteinase inhibitor from D. marsupialis serum, and to human alpha1B-glycoprotein, indicating the presence of five immunoglobulin-like domains. DM64 neutralized both the in vivo myotoxicity and the in vitro cytotoxicity of myotoxins I (mt-I/Asp49) and II (mt-II/Lys49) from Bothrops asper venom. The inhibitor formed noncovalent complexes with both toxins, but did not inhibit the PLA2 activity of mt-I. Accordingly, DM64 did not neutralize the anticoagulant effect of mt-I nor its intracerebroventricular lethality, effects that depend on its enzymatic activity, and which demonstrate the dissociation between the catalytic and toxic activities of this Asp49 myotoxic PLA2. Furthermore, despite its similarity with metalloproteinase inhibitors, DM64 presented no antihemorrhagic activity against Bothrops jararaca or Bothrops asper crude venoms, and did not inhibit the fibrinogenolytic activity of jararhagin or bothrolysin. This is the first report of a myotoxin inhibitor with an immunoglobulin-like structure isolated and characterized from animal blood. PMID:12473101

  14. 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

  15. The interaction of the antitoxin DM43 with a snake venom metalloproteinase analyzed by mass spectrometry and surface plasmon resonance.

    PubMed

    Brand, Guilherme D; Salbo, Rune; Jørgensen, Thomas J D; Bloch, Carlos; Boeri Erba, Elisabetta; Robinson, Carol V; Tanjoni, Isabelle; Moura-da-Silva, Ana M; Roepstorff, Peter; Domont, Gilberto B; Perales, Jonas; Valente, Richard H; Neves-Ferreira, Ana G C

    2012-05-01

    DM43 is a circulating dimeric antitoxin isolated from Didelphis aurita, a South American marsupial naturally immune to snake envenomation. This endogenous inhibitor binds non-covalently to jararhagin, the main hemorrhagic metalloproteinase from Bothrops jararaca snake venom, and efficiently neutralizes its toxicity. The aim of this study was to apply mass spectrometry (MS) and surface plasmon resonance (SPR) to improve the molecular characterization of this heterocomplex. The stoichiometry of the interaction was confirmed by nanoelectrospray ionization-quadrupole-time-of-flight MS; from native solution conditions, the complex showed a molecular mass of ~94 kDa, indicating that one molecule of jararhagin (50 kDa) interacts with one monomer of DM43 (43 kDa). Although readily observed in solution, the dimeric structure of the inhibitor was barely preserved in the gas phase. This result suggests that, in contrast to the toxin-antitoxin complex, hydrophobic interactions are the primary driving force for the inhibitor dimerization. For the real-time interaction analysis, the toxin was captured on a sensor chip derivatized with the anti-jararhagin monoclonal antibody MAJar 2. The sensorgrams obtained after successive injections of DM43 in a concentration series were globally fitted to a simple bimolecular interaction, yielding the following kinetic rates for the DM43/jararhagin interaction: k(a) = 3.54 ± 0.03 × 10(4) M(-1) s(-1) and k(d) = 1.16 ± 0.07 × 10(-5) s(-1), resulting in an equilibrium dissociation constant (K(D) ) of 0.33 ± 0.06 nM. Taken together, MS and SPR results show that DM43 binds to its target toxin with high affinity and constitute the first accurate quantitative study on the extent of the interaction between a natural inhibitor and a metalloproteinase toxin, with unequivocal implications for the use of this kind of molecule as template for the rational development of novel antivenom therapies. PMID:22549991

  16. 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

  17. Proline rich-oligopeptides: diverse mechanisms for antihypertensive action.

    PubMed

    Morais, Katia L P; Ianzer, Danielle; Miranda, José Rodolfo R; Melo, Robson L; Guerreiro, Juliano R; Santos, Robson A S; Ulrich, Henning; Lameu, Claudiana

    2013-10-01

    Bradykinin-potentiating peptides from Bothrops jararaca (Bj) discovered in the early 1960s, were the first natural inhibitors of the angiotensin-converting enzyme (ACE). These peptides belong to a large family of snake venom proline-rich oligopeptides (PROs). One of these peptides, Bj-PRO-9a, was essential for defining ACE as effective drug target and development of captopril, an active site-directed inhibitor of ACE used worldwide for the treatment of human arterial hypertension. Recent experimental evidences demonstrated that cardiovascular effects exerted by different Bj-PROs are due to distinct mechanisms besides of ACE inhibition. In the present work, we have investigated the cardiovascular actions of four Bj-PROs, namely Bj-PRO-9a, -11e, -12b and -13a. Bj-PRO-9a acts upon ACE and BK activities to promote blood pressure reduction. Although the others Bj-PROs are also able to inhibit the ACE activity and to potentiate the BK effects, our results indicate that antihypertensive effect evoked by them involve new mechanisms. Bj-PRO-11e and Bj-PRO-12b involves induction of [Ca(2+)]i transients by so far unknown receptor proteins. Moreover, we have suggested argininosuccinate synthetase and M3 muscarinic receptor as targets for cardiovascular effects elicited by Bj-PRO-13a. In summary, the herein reported results provide evidence that Bj-PRO-mediated effects are not restricted to ACE inhibition or potentiation of BK-induced effects and suggest different actions for each peptide for promoting arterial pressure reduction. The present study reveals the complexity of the effects exerted by Bj-PROs for cardiovascular control, opening avenues for the better understanding of blood pressure regulation and for the development of novel therapeutic approaches. PMID:23933300

  18. Neuromuscular action of venom from the South American colubrid snake Philodryas patagoniensis.

    PubMed

    Carreiro da Costa, Roberta S; Prudêncio, Luiz; Ferrari, Erika Fonseca; Souza, Gustavo H M F; de Mello, Sueli Moreira; Prianti Júnior, Antonio Carlos Guimarães; Ribeiro, Wellington; Zamunér, Stella Regina; Hyslop, Stephen; Cogo, José Carlos

    2008-07-01

    Snakes of the opisthoglyphous genus Philodryas are widespread in South America and cause most bites by colubrids in this region. In this study, we examined the neurotoxic and myotoxic effects of venom from Philodryas patagoniensis in biventer cervicis and phrenic nerve-diaphragm preparations and we compared the biochemical activities of venoms from P. patagoniensis and Philodryas olfersii. Philodryas patagoniensis venom (40 microg/mL) had no effect on mouse phrenic nerve-diaphragm preparations but caused time-dependent neuromuscular blockade of chick biventer cervicis preparations. This blockade was not reversed by washing. The highest concentration of venom tested (40 microg/mL) significantly reduced (p<0.05) the contractures to exogenous acetylcholine (55 microM and 110 microM) and K(+) (13.4 mM) after 120 min; lower concentrations of venom had no consistent or significant effect on these responses. Venom caused a concentration- and time-dependent release of creatine kinase (CK) from biventer cervicis preparations. Histological analysis showed contracted muscle fibers at low venom concentrations and myonecrosis at high concentrations. Philodryas venoms had low esterase and phospholipase A(2) but high proteolytic activities compared to the pitviper Bothrops jararaca. SDS-PAGE showed that the Philodryas venoms had similar electrophoretic profiles, with most proteins having a molecular mass of 25-80 kDa. Both of the Philodryas venoms cross-reacted with bothropic antivenom in ELISA, indicating the presence of proteins immunologically related to Bothrops venoms. RP-HPLC of P. patagoniensis venom yielded four major peaks, each of which contained several proteins, as shown by SDS-PAGE. These results indicate that P. patagoniensis venom has neurotoxic and myotoxic components that may contribute to the effects of envenoming by this species. PMID:18455482

  19. 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

  20. 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

  1. 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

  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. Peptidomics of Three Bothrops Snake Venoms: Insights Into the Molecular Diversification of Proteomes and Peptidomes*

    PubMed Central

    Tashima, Alexandre K.; Zelanis, André; Kitano, Eduardo S.; Ianzer, Danielle; Melo, Robson L.; Rioli, Vanessa; Sant'anna, Sávio S.; Schenberg, Ana C. G.; Camargo, Antônio C. M.; Serrano, Solange M. T.

    2012-01-01

    Snake venom proteomes/peptidomes are highly complex and maintenance of their integrity within the gland lumen is crucial for the expression of toxin activities. There has been considerable progress in the field of venom proteomics, however, peptidomics does not progress as fast, because of the lack of comprehensive venom sequence databases for analysis of MS data. Therefore, in many cases venom peptides have to be sequenced manually by MS/MS analysis or Edman degradation. This is critical for rare snake species, as is the case of Bothrops cotiara (BC) and B. fonsecai (BF), which are regarded as near threatened with extinction. In this study we conducted a comprehensive analysis of the venom peptidomes of BC, BF, and B. jararaca (BJ) using a combination of solid-phase extraction and reversed-phase HPLC to fractionate the peptides, followed by nano-liquid chromatography-tandem MS (LC-MS/MS) or direct infusion electrospray ionization-(ESI)-MS/MS or MALDI-MS/MS analyses. We detected marked differences in the venom peptidomes and identified peptides ranging from 7 to 39 residues in length by de novo sequencing. Forty-four unique sequences were manually identified, out of which 30 are new peptides, including 17 bradykinin-potentiating peptides, three poly-histidine-poly-glycine peptides and interestingly, 10 l-amino acid oxidase fragments. Some of the new bradykinin-potentiating peptides display significant bradykinin potentiating activity. Automated database search revealed fragments from several toxins in the peptidomes, mainly from l-amino acid oxidase, and allowed the determination of the peptide bond specificity of proteinases and amino acid occurrences for the P4-P4′ sites. We also demonstrate that the venom lyophilization/resolubilization process greatly increases the complexity of the peptidome because of the imbalance caused to the venom proteome and the consequent activity of proteinases on venom components. The use of proteinase inhibitors clearly showed

  4. 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

  5. A new structurally atypical bradykinin-potentiating peptide isolated from Crotalus durissus cascavella venom (South American rattlesnake).

    PubMed

    Lopes, Denise M; Junior, Norberto E G; Costa, Paula P C; Martins, Patrícia L; Santos, Cláudia F; Carvalho, Ellaine D F; Carvalho, Maria D F; Pimenta, Daniel C; Cardi, Bruno A; Fonteles, Manassés C; Nascimento, Nilberto R F; Carvalho, Krishnamurti M

    2014-11-01

    Venom glands of some snakes synthesize bradykinin-potentiating peptides (BPP's) which increase bradykinin-induced hypotensive effect and decrease angiotensin I vasopressor effect by angiotensin-converting enzyme (ACE) inhibition. The present study shows a new BPP (BPP-Cdc) isolated from Crotalus durissus cascavella venom: Pro-Asn-Leu-Pro-Asn-Tyr-Leu-Gly-Ile-Pro-Pro. Although BPP-Cdc presents the classical sequence IPP in the C-terminus, it has a completely atypical N-terminal sequence, which shows very low homology with all other BPPs isolated to date. The pharmacological effects of BPP-Cdc were compared to BBP9a from Bothrops jararaca and captopril. BPP-Cdc (1 μM) significantly increased BK-induced contractions (BK; 1 μM) on the guinea pig ileum by 267.8% and decreased angiotensin I-induced contractions (AngI; 10 nM) by 62.4% and these effects were not significantly different from those of BPP9a (1 μM) or captopril (200 nM). Experiments with 4-week hypertensive 2K-1C rats show that the vasopressor effect of AngI (10 ng) was decreased by 50 μg BPP-Cdc (69.7%), and this result was similar to that obtained with 50 μg BPP9a (69.8%). However, the action duration of BPP-Cdc (60 min) was 2 times greater than that of BPP-9a (30 min). On the other hand, the hypotensive effect of BK (250 ng) was significantly increased by 176.6% after BPP-Cdc (50 μg) administration, value 2.5 times greater than that obtained with BPP9a administered at the same doses (71.4%). In addition, the duration of the action of BPP-Cdc (120 min) was also at least 4 times greater than that of BPP-9a (30 min). Taken together, these results suggest that BPP-Cdc presents more selective action on arterial blood system than BPP9a. Besides the inhibition of ACE, it may present other mechanisms of action yet to be elucidated. PMID:25091347