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Sample records for inducida por bothrops

  1. Detección y estudio mediante Fluorescencia Inducida por Láser de radicales libres formados por Disociación Multifotónica Infrarroja

    NASA Astrophysics Data System (ADS)

    Santos, M.; Díaz, L.; Torresano, J. A.; Rubio, L.; Samoudi, B.

    Una de las principales aplicaciones actuales de los procesos de disociación multifotónica inducidos por radiación láser infrarroja (DMI) es la producción de radiales libres, con el fin de estudiar sus propiedades cinéticas y espectroscópicas. La disociación de moléculas poliatómicas en el IR con láseres de CO2 tiene lugar desde la superficie de energía molecular mas baja y conduce generalmente a la formación de fragmentos en el estado electrónico fundamental, con diversos grados de excitación vibracional. En el Grupo de Procesos Multifotónicos del Instituto de Estructura de la Materia del C.S.I.C. hemos puesto a punto la técnica de Fluorescencia Inducida por Láser (LIF) para la detección y análisis en tiempo real de los fragmentos producidos en la DMI inducida mediante uno o dos campos láseres de diferentes longitudes de onda. Objetivos de nuestro trabajo han sido el estudio de los canales de disociación mayoritarios y de las especies transitoria producidas, así como de la distribución de energía interna con que éstas son generadas. En particular hemos detectado mediante LIF las especies: C2, CF, CH, SiH2, CF2, CH2, SiHCl, y CF3 a partir de la disociación de, entre otras, las siguientes moléculas: C2H3Br, C3F6, C4H8Si, C2H5ClSi y CH5ClSi. En este trabajo presentamos algunos de los resultados obtenidos mediante el estudio por LIF de estos radicales: estudio temporal de la señal LIF obtenida con determinación de tiempos de vida, espectros de excitación y fluorescencia, temperaturas vibracionales de formación, variación de la intensidad LIF con el tiempo de retraso entre los láseres de disociación y prueba, etc.

  2. Análisis clínico y epidemiológico de los accidentes por mordeduras de serpientes del género Bothrops en Venezuela [A clinical and epidemiological analysis of accidental bites by snakes of the genus Bothrops in Venezuela].

    PubMed

    Rodríguez Acosta, A; Uzcategui, W; Azuaje, R; Aguilar, I; Girón, M E

    2000-01-01

    Clinical register of 60 patients bitten by Bothrops snake who assisted at Leopoldo Manrique Hospital and the Institute of Tropical Medicine (HLM-IMT) in Caracas during 1996-1997 were analysed. The accident was more frequent in males (45/75%). In 32 cases (53.3%) the snake was classified and 26 were Bothrops lanceolatus, 4 Bothrops venezuelensis and 2 Bothrops atrox. Anatomic regions more frequent bitten were superior members (40/66.6%): hands (36/60%), forearm (2/3.3%), elbow (1/1.6%) and arm (1/1.6%). On inferior members (20/33.3%): legs (6/10%), feet (10/16.7%), ankle (2/3.3%), and the hip (2:3.3%). The most frequent clinical manifestations in moderate and severe cases (33 patient) were pain (100%), oedema (98%), ecchymosis (76%), blisters (20%), necrosis (12%), abscess (6%) bleeding (19%), heart failure (1/1.6%), renal failure (1/1.6%). The blood clotting was evaluated in 60 (100%) cases and it was altered in 33 (55%) patients. No deaths were recorded.

  3. Experimental Bothrops atrox envenomation: Efficacy of antivenom therapy and the combination of Bothrops antivenom with dexamethasone.

    PubMed

    Santos Barreto, Gabriella Neves Leal; de Oliveira, Sâmella Silva; Dos Anjos, Isabelle Valle; Chalkidis, Hipocrates de Menezes; Mourão, Rosa Helena Veras; da Silva, Ana Maria Moura; Sano-Martins, Ida Sigueko; Gonçalves, Luis Roberto de Camargo

    2017-03-17

    Bothrops atrox snakes are the leading cause of snake bites in Northern Brazil. The venom of this snake is not included in the antigen pool used to obtain the Bothrops antivenom. There are discrepancies in reports on the effectiveness of this antivenom to treat victims bitten by B. atrox snakes. However, these studies were performed using a pre-incubation of the venom with the antivenom and, thus, did not simulate a true case of envenomation treatment. In addition, the local lesions induced by Bothrops venoms are not well resolved by antivenom therapy. Here, we investigated the efficacy of the Bothrops antivenom in treating the signs and symptoms caused by B. atrox venom in mice and evaluated whether the combination of dexamethasone and antivenom therapy enhanced the healing of local lesions induced by this envenomation. In animals that were administered the antivenom 10 minutes after the envenomation, we observed an important reduction of edema, dermonecrosis, and myonecrosis. When the antivenom was given 45 minutes after the envenomation, the edema and myonecrosis were reduced, and the fibrinogen levels and platelet counts were restored. The groups treated with the combination of antivenom and dexamethasone had an enhanced decrease in edema and a faster recovery of the damaged skeletal muscle. Our results show that Bothrops antivenom effectively treats the envenomation caused by Bothrops atrox and that the use of dexamethasone as an adjunct to the antivenom therapy could be useful to improve the treatment of local symptoms observed in envenomation caused by Bothrops snakes.

  4. Experimental Bothrops atrox envenomation: Efficacy of antivenom therapy and the combination of Bothrops antivenom with dexamethasone

    PubMed Central

    dos Anjos, Isabelle Valle; Chalkidis, Hipocrates de Menezes; Mourão, Rosa Helena Veras; Moura-da-Silva, Ana Maria; Sano-Martins, Ida Sigueko; Gonçalves, Luis Roberto de Camargo

    2017-01-01

    Bothrops atrox snakes are the leading cause of snake bites in Northern Brazil. The venom of this snake is not included in the antigen pool used to obtain the Bothrops antivenom. There are discrepancies in reports on the effectiveness of this antivenom to treat victims bitten by B. atrox snakes. However, these studies were performed using a pre-incubation of the venom with the antivenom and, thus, did not simulate a true case of envenomation treatment. In addition, the local lesions induced by Bothrops venoms are not well resolved by antivenom therapy. Here, we investigated the efficacy of the Bothrops antivenom in treating the signs and symptoms caused by B. atrox venom in mice and evaluated whether the combination of dexamethasone and antivenom therapy enhanced the healing of local lesions induced by this envenomation. In animals that were administered the antivenom 10 minutes after the envenomation, we observed an important reduction of edema, dermonecrosis, and myonecrosis. When the antivenom was given 45 minutes after the envenomation, the edema and myonecrosis were reduced, and the fibrinogen levels and platelet counts were restored. The groups treated with the combination of antivenom and dexamethasone had an enhanced decrease in edema and a faster recovery of the damaged skeletal muscle. Our results show that Bothrops antivenom effectively treats the envenomation caused by Bothrops atrox and that the use of dexamethasone as an adjunct to the antivenom therapy could be useful to improve the treatment of local symptoms observed in envenomation caused by Bothrops snakes. PMID:28306718

  5. Preclinical testing of Peruvian anti-bothropic anti-venom against Bothrops andianus snake venom.

    PubMed

    Schneider, Francisco S; Starling, Maria C; Duarte, Clara G; Machado de Avila, Ricardo; Kalapothakis, Evanguedes; Silva Suarez, Walter; Tintaya, Benigno; Flores Garrido, Karin; Seraylan Ormachea, Silvia; Yarleque, Armando; Bonilla, César; Chávez-Olórtegui, Carlos

    2012-11-01

    Bothrops andianus is a venomous snake found in the area of Machu Picchu (Peru). Its venom is not included in the antigenic pool used for production of the Peruvian anti-bothropic anti-venom. B. andianus venom can elicit many biological effects such as hemorrhage, hemolysis, proteolytic activity and lethality. The Peruvian anti-bothropic anti-venom displays consistent cross-reactivity with B. andianus venom, by ELISA and Western Blotting and is also effective in neutralizing the venom's toxic activities. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Human antibody fragments specific for Bothrops jararacussu venom reduce the toxicity of other Bothrops sp. venoms.

    PubMed

    Roncolato, Eduardo Crosara; Pucca, Manuela Berto; Funayama, Jaqueline Carlos; Bertolini, Thaís Barboza; Campos, Lucas Benício; Barbosa, José Elpidio

    2013-01-01

    Approximately 20,000 snakebites are registered each year in Brazil. The classical treatment for venomous snakebite involves the administration of sera obtained from immunized horses. Moreover, the production and care of horses is costly, and the use of heterologous sera can cause hypersensitivity reactions. The production of human antibody fragments by phage display technology is seen as a means of overcoming some of these disadvantages. The studies here attempted to test human monoclonal antibodies specific to Bothrops jararacussu against other Bothrops sp. venoms, using the Griffin.1 library of human single-chain fragment-variable (scFv) phage antibodies. Using the Griffin.1 phage antibody library, this laboratory previously produced scFvs capable of inhibiting the phospholipase and myotoxic activities of Bothrops jararacussu venom. The structural and functional similarities of the various forms of phospholipase A2 (PLA₂) in Bothrops venom served as the basis for the present study wherein the effectiveness of those same scFvs were evaluated against B. jararaca, B. neuwiedi, and B. moojeni venoms. Each clone was found to recognize all three Bothrops venoms, and purified scFvs partially inhibited their in vitro phospholipase activity. In vivo assays demonstrated that the scFv clone P2B7 reduced myotoxicity and increased the survival of animals that received the test venoms. The results here indicate that the scFv P2B7 is a candidate for inclusion in a mixture of specific antibodies to produce a human anti-bothropic sera. This data demonstrates that the human scFv P2B7 represents an alternative therapeutic approach to heterologous anti-bothropic sera available today.

  7. 21 CFR 864.8100 - Bothrops atrox reagent.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Bothrops atrox reagent. 864.8100 Section 864.8100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8100 Bothrops atrox reagent. (a...

  8. 21 CFR 864.8100 - Bothrops atrox reagent.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Bothrops atrox reagent. 864.8100 Section 864.8100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8100 Bothrops atrox reagent. (a...

  9. 21 CFR 864.8100 - Bothrops atrox reagent.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Bothrops atrox reagent. 864.8100 Section 864.8100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8100 Bothrops atrox reagent. (a...

  10. 21 CFR 864.8100 - Bothrops atrox reagent.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Bothrops atrox reagent. 864.8100 Section 864.8100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8100 Bothrops atrox reagent. (a...

  11. Envenomation by Bothrops punctatus in southwestern Colombia.

    PubMed

    Cañas, Carlos A; Vallejo, Alexandra

    2016-12-15

    Bothrops punctatus (Chocoan forest lancehead) is a semi-arboreal pitviper species distributed in Panamá, Colombia, and Ecuador, whose human envenomation is poorly characterized. We describe two patients bitten by B. punctatus, whose most relevant clinical feature was the development of a severe coagulopathy with few local manifestations (mild edema without signs of necrosis) at the site of the bite. Patients quickly improved their clinical and laboratory abnormalities after polyvalent antivenom application. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Hemostatic properties of Venezuelan Bothrops snake venoms with special reference to Bothrops isabelae venom.

    PubMed

    Rodríguez-Acosta, Alexis; Sánchez, Elda E; Márquez, Adriana; Carvajal, Zoila; Salazar, Ana M; Girón, María E; Estrella, Amalid; Gil, Amparo; Guerrero, Belsy

    2010-11-01

    In Venezuela, Bothrops snakes are responsible for more than 80% of all recorded snakebites. This study focuses on the biological and hemostatic characteristics of Bothrops isabelae venom along with its comparative characteristics with two other closely related Bothrops venoms, Bothrops atrox and Bothrops colombiensis. Electrophoretic profiles of crude B. isabelae venom showed protein bands between 14 and 100 kDa with the majority in the range of 14-31 kDa. The molecular exclusion chromatographic profile of this venom contains five fractions (F1-F5). Amidolytic activity evaluation evidenced strong thrombin-like followed by kallikrein-like activities in crude venom and in fractions F1 and F2. The fibrinogenolytic activity of B. isabelae venom at a ratio of 100:1 (fibrinogen/venom) induced a degradation of A alpha and B beta chains at 15 min and 2 h, respectively. At a ratio of 100:10, a total degradation of A alpha and B beta chains at 5 min and of gamma chains at 24 h was apparent. This current study evidences one of rarely reported for Bothrops venoms, which resembles the physiologic effect of plasmin. B. isabelae venom as well as F2 and F3 fractions, contain fibrinolytic activity on fibrin plate of 36, 23.5 and 9.45 mm(2)/microg, respectively using 25 microg of protein. Crude venom and F1 fraction showed gelatinolytic activity. Comparative analysis amongst Venezuelan bothropoid venoms, evidenced that the LD(50) of B. isabelae (5.9 mg/kg) was similar to B. atrox-Puerto Ayacucho 1 (6.1 mg/kg) and B. colombiensis-Caucagua (5.8 mg/kg). B. isabelae venom showed minor hemorrhagic activity, whereas B. atrox-Parguasa (Bolivar state) was the most hemorrhagic. In this study, a relative high thrombin-like activity was observed in B. colombiensis venoms (502-568 mUA/min/mg), and a relative high factor Xa-like activity was found in B. atrox venoms (126-294 mUA/min/mg). Fibrinolytic activity evaluated with 10 microg protein, showed that B. isabelae venom contained higher

  13. 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.

  14. 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

  15. Comparative analysis of local effects caused by Bothrops alternatus and Bothrops moojeni snake venoms: enzymatic contributions and inflammatory modulations.

    PubMed

    Mamede, Carla Cristine Neves; de Sousa, Bruna Barbosa; Pereira, Déborah Fernanda da Cunha; Matias, Mariana Santos; de Queiroz, Mayara Ribeiro; de Morais, Nadia Cristina Gomes; Vieira, Sâmela Alves Pereira Batista; Stanziola, Leonilda; de Oliveira, Fábio

    2016-07-01

    Bothropic envenomation is characterised by severe local damage caused by the toxic action of venom components and aggravated by induced inflammation. In this comparative study, the local inflammatory effects caused by the venoms of Bothrops alternatus and Bothrops moojeni, two snakes of epidemiological importance in Brazil, were investigated. The toxic action of venom components induced by bothropic venom was also characterised. Herein, the oedema, hyperalgesia and myotoxicity induced by bothropic venom were monitored for various lengths of time after venom injection in experimental animals. The intensity of the local effects caused by B. moojeni venom is considerably more potent than B. alternatus venom. Our results also indicate that metalloproteases and phospholipases A2 have a central role in the local damage induced by bothropic venoms, but serine proteases also contribute to the effects of these venoms. Furthermore, we observed that specific anti-inflammatory drugs were able to considerably reduce the oedema, the pain and the muscle damage caused by both venoms. The inflammatory reaction induced by B. moojeni venom is mediated by eicosanoid action, histamine and nitric oxide, with significant participation of bradykinin on the hyperalgesic and myotoxic effects of this venom. These mediators also participate to inflammation caused by B. alternatus venom. However, the inefficient anti-inflammatory effects of some local modulation suggest that histamine, leukotrienes and nitric oxide have little role in the oedema or myotoxicity caused by B. alternatus venom. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Bothrops fonsecai snake venom activities and cross-reactivity with commercial bothropic venom.

    PubMed

    Collaço, Rita de Cássia O; Randazzo-Moura, Priscila; Tamascia, Mariana L; da Silva, Igor Rapp F; Rocha, Thalita; Cogo, José C; Hyslop, Stephen; Sanny, Charles G; Rodrigues-Simioni, Léa

    2017-01-01

    In this work, we examined some biochemical and biological activities of Bothrops fonsecai venom, a pitviper endemic to southeastern Brazil, and assessed their neutralization by commercial bothropic antivenom (CAv). Cross-reactivity of venom with CAv was also assessed by immunoblotting and size-exclusion high performance chromatography (SE-HPLC). Bothrops fonsecai venom had PLA2, proteolytic and esterase activities that were neutralized to varying extents by venom:antivenom ratios of 5:1 and 5:2 (PLA2 and esterase activities) or not significantly by either venom:antivenom ratio (proteolytic activity). The minimum hemorrhagic dose (69.2μg) was totally neutralized by both ratios. Clotting time in rat citrated plasma was 33±10.5s (mean±SD; n=5) and was completely neutralized by a 5:2 ratio. Edema formation was dose-dependent (1-30μg/site) and significantly inhibited by both ratios. Venom (10-300μg/mL) caused neuromuscular blockade in extensor digitorum longus preparations; this blockade was inhibited best by a 5:2 ratio. Venom caused myonecrosis and creatine kinase release in vivo (gastrocnemius muscle) and in vitro (extensor digitorum longus) that was effectively neutralized by both venom:antivenom ratios. Immunoblotting showed that venom components of ~25-100kDa interacted with CAv. SE-HPLC profiles for venom incubated with CAv or specific anti-B. fonsecai antivenom raised in rabbits (SAv) indicated that CAv had a higher binding capacity than SAv, whereas SAv had higher affinity than CAv. These findings indicate that B. fonsecai venom contains various activities that are neutralized to different extents by CAv and suggest that CAv could be used to treat envenoming by B. fonsecai.

  17. Interlaboratory study for the establishment of Brazilian Bothrops Reference Venom and Antivenom for potency evaluation of Bothrops antivenom.

    PubMed

    Araújo, Humberto Pinheiro de; Lucas, Elizabeth Porto Reis; Moura, Wlamir Corrêa de; Fátima Barbosa, Celia de; Rodrigues, Raquel Joane; Morais, Josefina Farina; Almada, Joseana Zaja; Guimarães, Barbara Carino; Almeida, Antonio Eugenio Castro Cardoso de; Boller, Maria Aparecida Affonso

    2017-09-01

    A collaborative study was performed for the establishment of the 5th lot of Brazilian Bothrops Reference Venom and the 1st lot of Brazilian Bothrops Reference Antivenom. All Brazilian manufacturers of Antibothrops Immunoglobulins and the National Control Laboratory participated of the study. The declared potency of the 5th lot of the Bothrops Reference Venom is 40.29 μg/0.5 ml, and the potency of the 1st lot of Bothrops Reference Antivenom is 6.51 mg/ml. For the potency evaluation of Bothrops Reference Venom the inter assay precision (gCV) was 3.25% in lab 01; 3.51% in INCQS; 4.71% in lab 03 and 25.11% in lab 02, and the inter laboratory precision was 13.76%. The intra assay precision of Bothrops Reference Antivenom determinations was 4.38% in INCQS; 8.47% in lab 02; 10.51% in lab 03 and 20.05% in lab 01. The inter assay precision was 3.51% in INCQS; 9.65% in lab 02; 18.03% in lab 01 and 20.23% in lab 03. The inter laboratory precision was 15.85%. Despite the high number of invalid results (55.6% for the pharmacopoeial method and 69.4% for the proposed method) the parallel line assay, have better inter laboratorial precision (gCV = 16.62%) than the pharmacopoeial potency assay (gCV = 38.28%). Copyright © 2017. Published by Elsevier Ltd.

  18. Biological characterization of Bothrops marajoensis snake venom

    PubMed Central

    Cavalcante, Walter LG; Hernandez-Oliveira, Saraguaci; Galbiatti, Charlene; Randazzo-Moura, Priscila; Rocha, Thalita; Ponce-Soto, Luis; Marangoni, Sérgio; Pai-Silva, Maeli Dal; Gallacci, Márcia; da Cruz-Höfling, Maria A; Rodrigues-Simioni, Léa

    2011-01-01

    This study describes the effects of Bothrops marajoensis venom (Marajó lancehead) on isolated neuromuscular preparations of chick biventer cervicis (CBC) and mouse phrenic nerve-diaphragm (PND). At low concentrations (1µg/ml for CBC and 5µg/ml for PND), the venom exhibited a neuromuscular blocking without any damaging effect on the muscle integrity. At higher concentration (20μg/ml for PND), together with the neuromuscular blockade, there was a moderate myonecrosis. The results show differences between mammalian and avian preparations in response to venom concentration; the avian preparation was more sensitive to venom neurotoxic effect than the mammalian preparation. The possible presynaptic mechanism underlying the neuromuscular blocking effect was reinforced by the observed increase in MEPPs at the same time (at 15min) when the facilitation of twitch tension occurred. These results indicate that the B. marajoensis venom produced neuromuscular blockade, which appeared to be presynaptic at low concentrations with a postsynaptic component at high concentrations, leading to muscle oedema. These observations demand the fractionation of the crude venom and characterization of its active components for a better understanding of its biological dynamics. PMID:22091348

  19. 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

  20. Isotopic analysis of Bothrops atrox in Amazonian forest

    NASA Astrophysics Data System (ADS)

    Martinez, M. G.; Silva, A. M.; Chalkidis, H.; de Oliveira Júnior, R. C.; Camargo, P. B.

    2012-12-01

    The poisoning of snakes is considered a public health problem, especially in populations from rural areas of tropical and subtropical countries. In Brazil, the 26,000 snakebites, 90% are of the genus Bothrops, and Bothrops atrox species predominant in the Amazon region including all the Brazilian Amazon. Research shows that using stable isotopes, we can verify the isotopic composition of tissues of animals that depend mainly on food, water ingested and inhaled gases. For this study, samples taken from Bothrops atrox (B. atrox), in forest using pitfall traps and fall ("Pitt-fall traps with drift fence"). The analyzes were performed by mass spectrometry, where the analytical error is 0.3‰ for carbon and 0.5‰ to nitrogen. The results of the forest animals are significantly different from results of animal vivarium. The average values of the tissues and venoms of snakes of the forest for carbon-13 and nitrogen-15 are: δ13C = -24.68‰ and δ15N = 14.22‰ and mean values of tissue and poisons snakes vivarium (Instituto Butantan) to carbon-13 and nitrogen-15 are δ13C = -20.47‰ and δ15N = 8.36‰, with a significantly different due to different sources of food animals. Based on all results isotopic δ13C and δ15N, we can suggest that changes as the power of the serpent, (nature and captivity), changes occur in relation to diet and environment as the means of the isotopic data are quite distinct, showing that these changes can also cause metabolic changes in the body of the animal itself and the different periods of turnover of each tissue analyzed.

  1. Action of anti-bothropic factor isolated from Didelphis marsupialis on renal effects of Bothrops erythromelas venom.

    PubMed

    Martins, Alice M C; Sousa, Fabiola C M; Barbosa, Paulo S F; Toyama, Marcos H; Toyama, Daniela O; Aprígio, Cleidiana C; Queiroz, Maria G R; Guarnieri, Mirian C; Havt, Alexandre; de Menezes, Dalgimar B; Fonteles, Manassés C; Monteiro, Helena S A

    2005-11-01

    Acute renal failure is the most common complication in the lethal cases caused by snakebites in Brazil. Among the Brazilian venom snakes, Bothrops erythromelas is responsible for the majority of accidents in Northeastern Brazil. Didelphis marsupialis serum could inhibit myonecrotic, hemorrhagic, edematogenic hyperalgesic and lethal effects of envenomation determined by ophidian bites. In the present study, we evaluated the action of the anti-bothropic factor isolated from D. marsupialis on the renal effects promoted by B. erythromelas venom without systemic interference. Isolated kidneys from Wistar rats were perfused with Krebs-Henseleit solution containing 6% bovine serum albumin. We analyzed renal perfusion pressure (PP), renal vascular resistance (RVR), glomerular filtration rate (GFR), urinary flow (UF), and the percentages of sodium and potassium tubular transport (%TNa+, %TK+). The B. erythromelas venom (10 microg mL(-1)) decreased the PP (ct = 108.71+/-5.09 mmHg; BE = 65.21+/-5.6 mmHg*) and RVR (ct = 5.76+/-0.65 mmHg mL(-1) g(-1) min(-1); BE = 3.10+/-0.45 mmHg mL(-1) g(-1) min(-1)*). On the other hand, the GFR decreased at 60 min (ct60 = 0.76+/-0.07 mL g(-1) min(-1); BE60 = 0.42+/-0.12 mL g(-1) min(-1)*) and increased at 120 min (ct120 = 0.72+/-0.01 mL g(-1) min(-1); BE120 = 1.24+/-0.26 mL g(-1) min(-1)*). The UF increased significantly when compared with the control group (ct = 0.14+/-0.01 mL g(-1) min(-1); BE = 0.47+/-0.08 mL g(-1) min(-1)*). The venom reduced the %TNa(+) (ct90 = 79.18+/-0.88%; BE90 = 58.35+/-4.86%*) and %TK+ (ct90 = 67.20+/-4.04%; BE90 = 57.32+/-5.26%*) The anti-bothropic factor from D. marsupialis (10 microg mL(-1)) incubated with B. erythromelas venom (10 microg mL(-1)) blocked the effects on PP, RVR, %TNa+, and %TK+, but was not able to reverse the effects in UF and GFR promoted by venom alone. However, the highest concentration of D. marsupialis serum (30 microg mL(-1)) reversed all the renal effects induced by the venom. In

  2. Counteraction of Bothrops snake venoms by Combretum leprosum root extract and arjunolic acid.

    PubMed

    Fernandes, Fabrício F A; Tomaz, Marcelo A; El-Kik, Camila Z; Monteiro-Machado, Marcos; Strauch, Marcelo A; Cons, Bruno L; Tavares-Henriques, Matheus S; Cintra, Adélia C O; Facundo, Valdir A; Melo, Paulo A

    2014-08-08

    Serotherapy against snakebite is often unavailable in some regions over Brazil, where people make use of plants from folk medicine to deal with ophidic accidents. About 10% of Combretum species have some ethnopharmacological use, including treatment of snakebites. We evaluated the ability of the extract of Combretum leprosum and its component arjunolic acid to reduce some in vivo and in vitro effects of Bothrops jararacussu and Bothrops jararaca venoms. The protocols investigated include phospholipase, proteolytic, collagenase, hyaluronidase, procoagulant, hemorrhagic, edematogenic, myotoxic and lethal activities induced by these venoms in Swiss mice. Oral pre-treatment with arjunolic acid reduced the Bothrops jararacussu lethality in up to 75%, while preincubation prevented the death of all the animals. Hemoconcentration effect of Bothrops jararacussu venom was confirmed two hours after i.p. injection, while preincubation with arjunolic acid preserved the hematocrit levels. Both Combretum leprosum extract and arjunolic acid abolished the myotoxic action of Bothrops jararacussu venom. Preincubation of Bothrops jararacussu venom with the extract or arjunolic acid prevented the increase of plasma creatine kinase activity in mice. The hemorrhagic activity of Bothrops jararaca crude venom was reduced down to about 90% and completely inhibited by preincubation with 10 mg/kg or 100 mg/kg Combretum leprosum extract, respectively, while the preincubation and the pretreatment with 30 mg/kg of arjunolic acid reduced the venom hemorrhagic activity down to about 12% and 58%, respectively. The preincubation of the venom with both extract and 30 mg/kg arjunolic acid significantly reduced the bleeding amount induced by Bothrops jararacussu venom. The extract of Combretum leprosum decreased the edema formation induced by Bothrops jararacussu venom both in preincubation and pretreatment, but not in posttreatment. Similarly, arjunolic acid preincubated with the venom abolished edema

  3. Comparison of phylogeny, venom composition and neutralization by antivenom in diverse species of bothrops complex.

    PubMed

    Sousa, Leijiane F; Nicolau, Carolina A; Peixoto, Pedro S; Bernardoni, Juliana L; Oliveira, Sâmella S; Portes-Junior, José Antonio; Mourão, Rosa Helena V; Lima-dos-Santos, Isa; Sano-Martins, Ida S; Chalkidis, Hipócrates M; Valente, Richard H; Moura-da-Silva, Ana M

    2013-01-01

    In Latin America, Bothrops snakes account for most snake bites in humans, and the recommended treatment is administration of multispecific Bothrops antivenom (SAB--soro antibotrópico). However, Bothrops snakes are very diverse with regard to their venom composition, which raises the issue of which venoms should be used as immunizing antigens for the production of pan-specific Bothrops antivenoms. In this study, we simultaneously compared the composition and reactivity with SAB of venoms collected from six species of snakes, distributed in pairs from three distinct phylogenetic clades: Bothrops, Bothropoides and Rhinocerophis. We also evaluated the neutralization of Bothrops atrox venom, which is the species responsible for most snake bites in the Amazon region, but not included in the immunization antigen mixture used to produce SAB. Using mass spectrometric and chromatographic approaches, we observed a lack of similarity in protein composition between the venoms from closely related snakes and a high similarity between the venoms of phylogenetically more distant snakes, suggesting little connection between taxonomic position and venom composition. P-III snake venom metalloproteinases (SVMPs) are the most antigenic toxins in the venoms of snakes from the Bothrops complex, whereas class P-I SVMPs, snake venom serine proteinases and phospholipases A2 reacted with antibodies in lower levels. Low molecular size toxins, such as disintegrins and bradykinin-potentiating peptides, were poorly antigenic. Toxins from the same protein family showed antigenic cross-reactivity among venoms from different species; SAB was efficient in neutralizing the B. atrox venom major toxins. Thus, we suggest that it is possible to obtain pan-specific effective antivenoms for Bothrops envenomations through immunization with venoms from only a few species of snakes, if these venoms contain protein classes that are representative of all species to which the antivenom is targeted.

  4. Comparison of Phylogeny, Venom Composition and Neutralization by Antivenom in Diverse Species of Bothrops Complex

    PubMed Central

    Peixoto, Pedro S.; Bernardoni, Juliana L.; Oliveira, Sâmella S.; Portes-Junior, José Antonio; Mourão, Rosa Helena V.; Lima-dos-Santos, Isa; Sano-Martins, Ida S.; Chalkidis, Hipócrates M.; Valente, Richard H.; Moura-da-Silva, Ana M.

    2013-01-01

    In Latin America, Bothrops snakes account for most snake bites in humans, and the recommended treatment is administration of multispecific Bothrops antivenom (SAB – soro antibotrópico). However, Bothrops snakes are very diverse with regard to their venom composition, which raises the issue of which venoms should be used as immunizing antigens for the production of pan-specific Bothrops antivenoms. In this study, we simultaneously compared the composition and reactivity with SAB of venoms collected from six species of snakes, distributed in pairs from three distinct phylogenetic clades: Bothrops, Bothropoides and Rhinocerophis. We also evaluated the neutralization of Bothrops atrox venom, which is the species responsible for most snake bites in the Amazon region, but not included in the immunization antigen mixture used to produce SAB. Using mass spectrometric and chromatographic approaches, we observed a lack of similarity in protein composition between the venoms from closely related snakes and a high similarity between the venoms of phylogenetically more distant snakes, suggesting little connection between taxonomic position and venom composition. P-III snake venom metalloproteinases (SVMPs) are the most antigenic toxins in the venoms of snakes from the Bothrops complex, whereas class P-I SVMPs, snake venom serine proteinases and phospholipases A2 reacted with antibodies in lower levels. Low molecular size toxins, such as disintegrins and bradykinin-potentiating peptides, were poorly antigenic. Toxins from the same protein family showed antigenic cross-reactivity among venoms from different species; SAB was efficient in neutralizing the B. atrox venom major toxins. Thus, we suggest that it is possible to obtain pan-specific effective antivenoms for Bothrops envenomations through immunization with venoms from only a few species of snakes, if these venoms contain protein classes that are representative of all species to which the antivenom is targeted. PMID

  5. Bothropic antivenom based on monoclonal antibodies, is it possible?

    PubMed

    Frauches, Thiago S; Petretski, Jorge H; Arnholdt, Andrea C V; Lasunskaia, Elena B; de Carvalho, Eulógio C Q; Kipnis, Thereza L; da Silva, Wilmar D; Kanashiro, Milton M

    2013-09-01

    Neutralizing monoclonal antibodies against three major toxic components of Bothrops atrox venom were produced and tested. The mAbs against phospholipase A2, hemorrhagic metalloprotease, and thrombin-like enzymes were produced in large amounts and purified with caprylic acid followed by ammonium sulfate precipitation. Purified mAbs were analyzed by SDS-PAGE and their ability to neutralize the respective toxins was tested. Five Swiss mice were injected i.p. with 13.5 mg of pooled mAbs and challenged via s.c. route with venom. Survival rate was recorded for the next 48 h. All mice treated and challenged with venom survived, whereas only one mouse in the control group survived. Bleeding time in mice treated with mAbs was similar to that observed in control mice. Our results show that monoclonal antibodies neutralized the lethal toxicity of Bothrops venom and indicate that there is a reasonable possibility of developing antivenoms based on humanized mAbs to treat victims of venomous animals in the future.

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

    PubMed

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

    2009-03-15

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

  7. Proteomic identification of gender molecular markers in Bothrops jararaca venom.

    PubMed

    Zelanis, André; Menezes, Milene C; Kitano, Eduardo S; Liberato, Tarcísio; Tashima, Alexandre K; Pinto, Antonio F M; Sherman, Nicholas E; Ho, Paulo L; Fox, Jay W; Serrano, Solange M T

    2016-04-29

    Variation in the snake venom proteome is a well-documented phenomenon; however, sex-based variation in the venom proteome/peptidome is poorly understood. Bothrops jararaca shows significant sexual size dimorphism and here we report a comparative proteomic/peptidomic analysis of venoms from male and female specimens and correlate it with the evaluation of important venom features. We demonstrate that adult male and female venoms have distinct profiles of proteolytic activity upon fibrinogen and gelatin. These differences were clearly reflected in their different profiles of SDS-PAGE, two-dimensional electrophoresis and glycosylated proteins. Identification of differential protein bands and spots between male or female venoms revealed gender-specific molecular markers. However, the proteome comparison by in-solution trypsin digestion and label-free quantification analysis showed that the overall profiles of male and female venoms are similar at the polypeptide chain level but show striking variation regarding their attached carbohydrate moieties. The analysis of the peptidomes of male and female venoms revealed different contents of peptides, while the bradykinin potentiating peptides (BPPs) showed rather similar profiles. Furthermore we confirmed the ubiquitous presence of four BPPs that lack the C-terminal Q-I-P-P sequence only in the female venom as gender molecular markers. As a result of these studies we demonstrate that the sexual size dimorphism is associated with differences in the venom proteome/peptidome in B. jararaca species. Moreover, gender-based variations contributed by different glycosylation levels in toxins impact venom complexity. Bothrops jararaca is primarily a nocturnal and generalist snake species, however, it exhibits a notable ontogenetic shift in diet and in venom proteome upon neonate to adult transition. As is common in the Bothrops genus, B. jararaca shows significant sexual dimorphism in snout-vent length and weight, with females being

  8. Effect of Bothrops bilineata snake venom on neutrophil function.

    PubMed

    Setubal, Sulamita da Silva; Pontes, Adriana Silva; Nery, Neriane Monteiro; Bastos, Jéssica Silva Félix; Castro, Onassis Boeri; Pires, Weverson Luciano; Zaqueo, Kayena Delaix; Calderon, Leonardo de Azevedo; Stábeli, Rodrigo Guerino; Soares, Andreimar Martins; Zuliani, Juliana Pavan

    2013-12-15

    The aim of the study was to evaluate the in vitro effects of Bothrops bilineata crude venom (BbV) on isolated human neutrophil function. We proved that BbV isn't toxic towards human neutrophils. During an incubation of human neutrophils with BbV hydrogen peroxide was produced. Moreover, BbV was able to stimulate neutrophil release of proinflammatory mediators such as IL-8 and IL-6 as well as PGE2 and NETs liberation. There is no data in the literature showing the effect of BbV on the production of IL-6 and IL-8 or NETs liberation by isolated human neutrophils. Taken together our results testify that BbV triggers relevant proinflammatory events in human neutrophils. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Neuromuscular activity of Bothrops fonsecai snake venom in vertebrate preparations

    PubMed Central

    Fernandes, Carla T; Giaretta, Vânia MA; Prudêncio, Luiz S; Toledo, Edvana O; da Silva, Igor RF; Collaço, Rita CO; Barbosa, Ana M; Hyslop, Stephen; Rodrigues-Simioni, Léa; Cogo, José C

    2014-01-01

    The neuromuscular activity of venom from Bothrops fonsecai, a lancehead endemic to southeastern Brazil, was investigated. Chick biventer cervicis (CBC) and mouse phrenic nerve-diaphragm (PND) preparations were used for myographic recordings and mouse diaphragm muscle was used for membrane resting potential (RP) and miniature end-plate potential (MEPP) recordings. Creatine kinase release and muscle damage were also assessed. In CBC, venom (40, 80 and 160μg/ml) produced concentration- and time-dependent neuromuscular blockade (50% blockade in 85±9 min and 73±8 min with 80 and 160μg/ml, respectively) and attenuated the contractures to 110μM ACh (78–100% inhibition) and 40mM KCl (45–90% inhibition). The venom-induced decrease in twitch-tension in curarized, directly-stimulated preparations was similar to that in indirectly stimulated preparations. Venom (100 and 200μg/ml) also caused blockade in PND preparations (50% blockade in 94±13 min and 49±8 min with 100 and 200μg/ml, respectively) but did not alter the RP or MEPP amplitude. In CBC, venom caused creatine kinase release and myonecrosis. The venom-induced decrease in twitch-tension and in the contractures to ACh and K+ were abolished by preincubating venom with commercial antivenom. These findings indicate that Bothrops fonsecai venom interferes with neuromuscular transmission essentially through postsynaptic muscle damage that affects responses to ACh and KCl. These actions are effectively prevented by commercial antivenom. PMID:25028603

  10. Compartment syndrome after Bothrops jararaca snakebite: monitoring, treatment, and outcome.

    PubMed

    Bucaretchi, Fábio; de Capitani, Eduardo Mello; Hyslop, Stephen; Mello, Sueli Moreira; Madureira, Paulo Roberto; Zanardi, Veronica; Ferreira, Daniel M; Meirelles, Guilerme V; Fernandes, Luciane C R

    2010-01-01

    To report the outcome of a patient who developed compartment syndrome after Bothrops jararaca snakebite. A 39-year-old male was admitted 5 h after being bitten on the lower right leg. Physical examination revealed tense swelling, ecchymosis, hypoesthesia, and intense local pain that worsened after passive stretching, limited right foot dorsiflexion, and gingival bleeding. The case was classified as moderate/severe and eight vials of bothropic antivenom (AV) were infused 1 h postadmission. The main laboratory findings upon admission were incoagulable blood (incoagulable PT, aPTT, and INR), thrombocytopenia, serum creatine kinase (CK) of 580 U/L (reference value < 170 U/L), and a serum venom level of 33.7 ng/mL (ELISA; cutoff = 2.3 ng/mL). High anterior compartment pressure (60 mmHg) was identified 8 h post bite, with progressively lower pressures after AV administration and limb elevation (36 mmHg; 19 h post bite). However, moderate pain and limited foot dorsiflexion persisted. In addition, there was a progressive increase in serum CK (6,729 U/L; 45 h post bite), as well as marked edema and hemorrhage of the anterior compartment detected by magnetic resonance imaging (MRI) at 48 h post bite. A fasciotomy done after a further increase in intracompartmental pressure (66 mmHg, 57 h post bite) revealed hemorrhage/necrosis of the anterior tibial muscle that subsequently required partial resection. The patient developed a local infection (day 15 post bite) and a permanent fibular palsy. Compartment syndrome is an unusual but severe complication of snakebites. MRI, in conjunction with subfascial pressure measurements, may be useful in the diagnosis of compartment syndrome after snakebites.

  11. Aqueous Leaf Extract of Jatropha mollissima (Pohl) Bail Decreases Local Effects Induced by Bothropic Venom

    PubMed Central

    Gomes, Jacyra Antunes dos Santos; Geraldo Amaral, Juliano; Lopes, Norberto Peporine; Tabosa do Egito, Eryvaldo Sócrates; da Silva-Júnior, Arnóbio Antônio; Maria Zucolotto, Silvana

    2016-01-01

    Snakebites are a serious worldwide public health problem. In Brazil, about 90% of accidents are attributed to snakes from the Bothrops genus. The specific treatment consists of antivenom serum therapy, which has some limitations such as inability to neutralize local effects, difficult access in some regions, risk of immunological reactions, and high cost. Thus, the search for alternative therapies to treat snakebites is relevant. Jatropha mollissima (Euphorbiaceae) is a medicinal plant popularly used in folk medicine as an antiophidic remedy. Therefore, this study aims to evaluate the effect of the aqueous leaf extract from J. mollissima on local effects induced by Bothrops venoms. High Performance Liquid Chromatography with Diode Array Detection analysis and Mass Spectrometry analysis of aqueous leaf extract confirmed the presence of the flavonoids isoschaftoside, schaftoside, isoorientin, orientin, vitexin, and isovitexin. This extract, at 50–200 mg/kg doses administered by intraperitoneal route, showed significant inhibitory potential against local effects induced by Bothrops erythromelas and Bothrops jararaca snake venoms. Local skin hemorrhage, local edema, leukocyte migration, and myotoxicity were significantly inhibited by the extract. These results demonstrate that J. mollissima extract possesses inhibitory potential, especially against bothropic venoms, suggesting its potential as an adjuvant in treatment of snakebites. PMID:27847818

  12. 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

  13. Determination of inorganic elements in blood of mice immunized with Bothrops Snake venom using XRF and NAA

    NASA Astrophysics Data System (ADS)

    Lopes da Silva, L. F. F.; Zamboni, C. B.; Bahovschi, V.; Metairon, S.; Suzuki, M. F.; Sant'Anna, O. A.; Rizzutto, M. A.

    2015-07-01

    In this work, mice genetically modified [HIII line] were immunized against different Bothrops snake venoms to produce anti-Bothrops serum (antivenom). The Neutron Activation Analysis (NAA) and Energy Dispersive X-Ray Fluorescence (EDXRF) techniques were used to evaluate Ca and Fe concentrations in blood of these immunized mice in order to establish a potential correlation between both phenotypes: antibody response and blood constituents after Bothrops venom administration. The results were compared with the control group (mice not immunized) and with human being estimative. These data are important for clinical screening of patients submitted to immunological therapy as well as the understanding of the envenoming mechanisms.

  14. 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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Molecular characterization of metalloproteases from Bothrops alternatus snake venom.

    PubMed

    de Paula, Fernando Fonseca Pereira; Ribeiro, Juliana Uema; Santos, Livia Mara; de Souza, Dulce Helena Ferreira; Leonardecz, Eduardo; Henrique-Silva, Flávio; Selistre-de-Araújo, Heloisa Sobreiro

    2014-12-01

    We have previously demonstrated that alternagin-C (ALT-C), a disintegrin-like, Cys-rich protein isolated from Bothrops alternatus snake venom, induces human vascular endothelial cell (HUVEC) proliferation and angiogenesis in in vitro and in vivo assays. Therefore this protein could be interesting as a new approach for tissue regeneration studies. However, its primary sequence was not completely determined since the protein isolated from crude venom is usually a mixture of isoforms. Here we describe the transcriptome analysis of B. alternatus from the venom glands of a single male specimen. About 800 good-quality contigs were screened for snake venom metalloproteases/disintegrins, resulting in the following expression profile for these enzymes: 4% for P-I, 7% for P-II and 89% for P-III SVMPs. The PII-SVMP sequence code for RGD-disintegrins and all the expressed PIII-sequences have the ECD adhesive motif. A cDNA sequence coding for an ALT-C homolog was completely sequenced and characterized. Comparative sequence and structural analyses suggested new features that distinguish SVMP classes such as two prolyl endopetidase cleavage sites. All these data add new information on the expression pattern of metalloproteases of B. alternatus venom and may have practical applications for the production of recombinant disintegrins for cell adhesion studies. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Toxicity of Bothrops sp snake venoms from Ecuador and preclinical assessment of the neutralizing efficacy of a polyspecific antivenom from Costa Rica.

    PubMed

    Laines, Johana; Segura, Álvaro; Villalta, Mauren; Herrera, María; Vargas, Mariángela; Alvarez, Gladys; Gutiérrez, José María; León, Guillermo

    2014-09-01

    The toxicological profile of the venoms of the snakes Bothrops asper and Bothrops atrox from Ecuador was investigated, together with the venom of a population of B. asper formerly classified as 'Bothrops xanthogrammus'. The three venoms exerted lethal, hemorrhagic, myotoxic, coagulant and defibrinogenating effects, in agreement with the characteristic toxicological profile of Bothrops sp venoms. A polyspecific antivenom (bothropic-crotalic-lachesic) manufactured in Costa Rica was assessed for its preclinical efficacy against the toxic activities of these Ecuadorian venoms. Antivenom was effective in the neutralization of the five activities tested in the three venoms. These observations are in agreement with previous reports on the extensive cross-reactivity and paraspecific neutralization of antivenoms manufactured in Latin America against the venoms of Bothrops sp snakes.

  17. Antigenic cross-reactivity and immunogenicity of Bothrops venoms from snakes of the Amazon region.

    PubMed

    Furtado, Maria de Fátima D; Cardoso, Silvia Travaglia; Soares, Oscar Espellet; Pereira, Aparecida Pietro; Fernandes, Daniel Silva; Tambourgi, Denise Vilarinho; Sant'Anna, Osvaldo Augusto

    2010-04-01

    Snakebites are still a critical public health problem in developing countries or isolated areas. In Brazil, the North Region has a high distribution coefficient worsened by the significant number of eventually unreported cases, due to difficulties in access to health services, to the natural geographic barriers and the vast territory. In the Rio Negro area, the species Bothrops atrox, Bothrops brazili, Lachesis muta muta and Bothriopsis taeniata are thought to be the major species responsible for snakebites. The aim of this study was to qualitatively and quantitatively determine the antigenic cross-reactivity and expression of toxins and the immunogenicity of Bothrops venom species of the Amazon and to evaluate the general efficacy of the therapeutic sera. The in vivo assays demonstrated that the defibrinating activity of B. taeniata venom was absent but that the lethal and hemorrhagic properties were more intense than in the B. atrox venom. The results evidence venom variability among the two B. atrox populations from two distinct Amazonian regions, which may reveal a subjacent speciation process. The results point to new aspects that may guide the improvement of anti-Bothropic therapeutic serum. Copyright 2010 Elsevier Ltd. All rights reserved.

  18. Naa Technique for Clinical Investigation of Mice Immunized with BOTHROP Venom

    NASA Astrophysics Data System (ADS)

    Zamboni, C. B.; Aguiar, R. O.; Kovacs, L.; Suzuki, M.; Sant'Anna, O. A.

    2009-06-01

    In the present study Neutron Activation Analysis (NAA) technique was used to determine sodium concentration in whole blood of mice immunized with Bothrops venom. With this value it was possible to perform clinical investigation in this animal model using whole blood.

  19. Enzymatic and Pro-Inflammatory Activities of Bothrops lanceolatus Venom: Relevance for Envenomation

    PubMed Central

    Delafontaine, Marie; Villas-Boas, Isadora Maria; Mathieu, Laurence; Josset, Patrice; Blomet, Joël

    2017-01-01

    Bothrops lanceolatus, commonly named ‘Fer-de-Lance’, is an endemic snake of the French Caribbean Island of Martinique. Envenomations by B. lanceolatus present clinical aspects characterized by systemic thrombotic syndrome and important local inflammation, involving edema and pain but limited hemorrhage. To investigate mechanisms of venom-induced inflammation, B. lanceolatus venom was characterized, its cross-reactivity with bothropic antivenom explored, its cytotoxicity on human keratinocytes and vascular cells, and the production of cytokines and chemokines were analyzed. We used electrophoretic separation, zymography, colorimetric or fluorimetric enzymatic assays, and immunochemical assays. Therapeutic South American bothropic antivenom cross-reacted with B. lanceolatus venom and completely or partially abolished its PLA2, hyaluronidase, and proteolytic activities, as well as its cytotoxicity for keratinocytes. The substrate specificity of B. lanceolatus venom proteases was emphasized. B. lanceolatus venom cytotoxicity was compared to the B. jararaca venom. Both venoms were highly cytotoxic for keratinocytes (HaCaT), whereas B. lanceolatus venom showed particularly low toxicity for endothelial cells (EAhy926). Patterns of cytokine and chemokine production by cells exposed to the venoms were highly pro-inflammatory. Thus, the results presented here show that B. lanceolatus venom toxins share important antigenic similarities with South American Bothrops species toxins, although their proteases have acquired particular substrate specificity. Moreover, the venom displays important cytotoxic and pro-inflammatory action on human cell types such as keratinocytes and endothelial cells, which are important players in the local and systemic compartments affected by the envenomation. PMID:28783135

  20. [Biological and immunological characteristics of the poison of Bothrops cotiara (Serpentes: Viperidae)].

    PubMed

    de Roodt, Adolfo Rafael; Estévez, Judith; Dolab, Jorge Adrián; Manzanelli, Marcelo Víctor; Piñeiro, Nicolás; Paniagua, Jorge Francisco; Vogt, Alejandro Urs

    2006-09-01

    Bothrops cotiara is a venomous snake sporadically found in the province of Misiones in Argentina, South of Brazil and Paraguay. Data on the clinics of the envenomation produced by its bite and on its venom are scarce. There is no information on the neutralizing capacity of the antivenoms available. In this study, the lethal potency, hemorrhagic, necrotizing, coagulant and thrombin-like, defibrinogenating, indirect hemolytic and fibrinolytic activities of the venom of B. cotiara specimens from the province of Misiones were determined. The toxic activities were within the range of those described for the other Bothrops species from Argentina, and the electrophoretic and chromatographic studies showed similarities with those described for the other bothropic venoms. The immunochemical reactivity of six South American anti Viper antivenoms (ELISA) have a strong reactivity with all the antivenoms studied. The neutralizing capacity of three of these therapeutic antivenoms against the lethal potency and hemorrhagic, necrotizing, coagulant, thrombin-like and hemolytic activities showed a very close neutralizing capacity. Our data strongly suggest that the antivenoms for therapeutic use available in this area of South America are useful to neutralize the toxic and enzymatic activities of the venom of this uncommon specie of Bothrops.

  1. NAA TECHNIQUE FOR CLINICAL INVESTIGATION OF MICE IMMUNIZED WITH BOTHROP VENOM

    SciTech Connect

    Zamboni, C. B.; Aguiar, R. O.; Kovacs, L.; Suzuki, M.; Sant'Anna, O. A.

    2009-06-03

    In the present study Neutron Activation Analysis (NAA) technique was used to determine sodium concentration in whole blood of mice immunized with Bothrops venom. With this value it was possible to perform clinical investigation in this animal model using whole blood.

  2. Bothrops jararaca and Bothrops erythromelas Snake Venoms Promote Cell Cycle Arrest and Induce Apoptosis via the Mitochondrial Depolarization of Cervical Cancer Cells

    PubMed Central

    Gomes, Dayanne Lopes; Martelli Palomino, Gustavo; da Silva, Wilmar Dias; Gonçalves, Ana Katherine; Fernandes-Pedrosa, Matheus de Freitas; Crispim, Janaina Cristiana de Oliveira

    2016-01-01

    Bothrops jararaca (BJ) and Bothrops erythromelas (BE) are viper snakes found in South-Southeast and Northeast regions of Brazil, respectively. Snake venoms are bioactive neurotoxic substances synthesized and stored by venom glands, with different physiological and pharmacological effects, recently suggesting a possible preference for targets in cancer cells; however, mechanisms of snakes have been little studied. Here, we investigated the mechanism responsible for snake crude venoms toxicity in cultured cervical cancer cells SiHa and HeLa. We show that BJ and BE snake crude venoms exert cytotoxic effects to these cells. The percentage of apoptotic cells and cell cycle analysis and cell proliferation were assessed by flow cytometry and MTT assay. Detection of mitochondrial membrane potential (Rhodamine-123), nuclei morphological change, and DNA fragmentation were examined by staining with DAPI. The results showed that both the BJ and BE venoms were capable of inhibiting tumor cell proliferation, promoting cytotoxicity and death by apoptosis of target SiHa and HeLa cells when treated with BJ and BE venoms. Furthermore, data revealed that both BJ venoms in SiHa cell promoted nuclear condensation, fragmentation, and formation of apoptotic bodies by DAPI assay, mitochondrial damage by Rhodamine-123, and cell cycle block in the G1-G0 phase. BJ and BE venoms present anticancer potential, suggesting that both Bothrops venoms could be used as prototypes for the development of new therapies. PMID:28050190

  3. [Neutralizing capacity of ++antiophidic sera against the venom of Bothrops moojeni (lance-headed viper)].

    PubMed

    de Roodt, A R; Dolab, J A; Hajos, S E; Fernández, T; Segré, L

    1997-01-01

    In the production of current therapeutic antisera used in Argentina for Bothrops snakebite, the Bothrops moojeni venom is not used as immunogen, since this snake is not considered a serious public health problem. Accidents caused by this species have not been reported in this country even though Bothrops moojeni is not unfrequent in some regions of Misiones. Despite the high degree of immunological cross reactivity found among the Crotalinae venoms and, in this particular case, among the venoms from the Bothrops Genus, there exists a significant intraspecific variation in venom composition, particularly in specimens arising from different geographic regions. In this study, the antivenoms prepared at the Instituto Nacional de Producción de Biológicos A.N.L.I.S. Dr. Carlos G. Malbrán have been tested for their immunochemical cross reactivity and neutralizing ability of enzymatic and toxic activities of venom from Argentinian specimens of Bothrops moojeni from Misiones. Immunological cross-reactivity was tested by double-immunoprecipitation, immunoelectrophoresis, Western-blot and ELISA. Neutralizing ability of antivenoms against proteolytic, indirect hemolytic activity, procoagulant activity, he-morrhagic activity and necrotizing activity. The Lethal Dose 50 was 1.5 mg/kg body weight; this value is located in the range to those obtained with the venom from Brazilian specimens. It was observed that all the antivenoms exhibited a strong immunochemical cross reactivity and that they were able to neutralize in different degree both, enzymatic and toxic activities of B. moojeni venom. From these results, it can be assumed that the antivenom tested could be employed successfully in cases of B. moojeni snakebites.

  4. [Inefficacy of bothropic antivenin in the neutralization of the coagulation activity of Lachesis muta muta venom. Report of a case and experimental confirmation].

    PubMed

    Bard, R; de Lima, J C; de Sa Neto, R P; de Oliveira, S G; dos Santos, M C

    1994-01-01

    In the State of Amazonas and in areas nearby Manaus, the snakes responsible for the majority of accidents are Bothrops atrox and Lachesis muta muta, with a percentage of confirmed species of 76% and 17%, respectively. Frequently, in the absence of the laquetic and bothropic-laquetic antivenoms, the Instituto de Medicina Tropical de Manaus (IMTM) has been using bothropic antivenom in the treatment of laquetic accident. In this paper is related a case of accident caused by L. muta muta; the patient was treated with bothropic antivenom, and after received twenty ampoules of this antivenom, maintained blood incoagulability until the 13th day after the accident. Experiments to obtain the potency of the bothropic antivenom for the coagulant and hemorrhagic activities has been done, using Bothrops atrox venom as control. The results showed that the potency of the antivenom for the hemorrhagic activity was similar, and the potency for the coagulant activity for the L. m. muta venom was 9.2 times minor than that for B. atrox. The antibodies titles from three different lots of bothropic antivenom varied for the L. m. muta venom, and were constant for the B. atrox venom. Due to the inefficiency of the bothropic antivenom on the neutralization of the coagulant activity for the L. m. muta venom, the use of bothropic antivenom is not recommended in the treatment of Lachesis muta muta accidents.

  5. Efficient muscle regeneration after highly haemorrhagic Bothrops alternatus venom injection.

    PubMed

    Garcia Denegri, María Emilia; Teibler, Gladys P; Maruñak, Silvana L; Hernández, David R; Acosta, Ofelia C; Leiva, Laura C

    2016-11-01

    Bothrops alternatus snake venom is particularly characterized for inducing a prominent haemorrhage and affecting hemostasis as a consequence of 43.1% of metallo-proteinases and less than 10% of PLA2 (almost all non-myotoxic phospholipases) in its venomics. In addition, myonecrosis is the major local effect in viper envenoming which might lead to permanent sequela. Then, the rebuilding of the microvasculature at the local injured site acquires significance since represents one of the pivotal stages for subsequent skeletal muscle regeneration either at morphological or functional aspects. Due to the significance played by vasculature in this process, it is important to study by histology and immunohistochemical techniques, the muscular damage and the sequence of skeletal muscle reconstruction (degree of damage, reconstitution of muscle fibres and capillaries). In this work, we injected intramuscularly 50 or 100 μg per mouse of B. alternatus venom in gastrocnemius muscles. We provided a complete description and characterization of the different stages of myogenesis after mild (50 µg) and severe (100 µg) local injury induced by B. alternatus venom toxins. The regeneration was evaluated 24 h, 3, 7, 14 and 28 days after receiving venom injection. Finally, both doses induced an extended necrosis at the site of injection where, when critical steps in the regenerative process are taking place, an efficient tissue rebuilding is achieved. B. alternatus venom is characterized by the high percentage of exclusively class P-III metalloproteinases, and by the lack of class P-I metalloproteinases in its venom composition. This could explain the effectiveness of muscle regeneration after venom injection despite the severity of the initial phase of envenoming.

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

    PubMed Central

    Senise, Luana V; Yamashita, Karine M

    2015-01-01

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

  7. Neutralization of toxicological activities of medically-relevant Bothrops snake venoms and relevant toxins by two polyvalent bothropic antivenoms produced in Peru and Brazil.

    PubMed

    Estevao-Costa, Maria I; Gontijo, Silea S; Correia, Barbara L; Yarleque, Armando; Vivas-Ruiz, Dan; Rodrigues, Edith; Chávez-Olortegui, Carlos; Oliveira, Luciana S; Sanchez, Eladio F

    2016-11-01

    Snakebite envenoming is a neglected public pathology, affecting especially rural communities or isolated areas of tropical and subtropical Latin American countries. The parenteral administration of antivenom is the mainstay and the only validated treatment of snake bite envenoming. Here, we assess the efficacy of polyspecific anti-Bothrops serum (α-BS) produced in the Instituto Nacional de Salud (INS, Peru) and at the Fundação Ezequiel Dias (FUNED, Brazil), to neutralize the main toxic activities induced by five medically-relevant venoms of: Bothrops atrox, B. barnetti, and B. pictus from Peru, and the Brazilian B. jararaca and B. leucurus, all of them inhabiting different geographical locations. Protein electrophoretic patterns of these venoms showed significant differences in composition, number and intensity of bands. Another goal was to evaluate the efficacy and safety of lyophilized α-BS developed at INS to neutralize the detrimental effects of these venoms using in vivo and in vitro assays. The availability of lyophilized α-BS has relevant significance in its distribution to distant rural communities where the access to antivenom in health facilities is more difficult. Despite the fact that different antigen mixtures were used for immunization during antivenom production, our data showed high toxin-neutralizing activity of α-BS raised against Bothrops venoms. Moreover, the antivenom cross-reacted even against venoms not included in the immunization mixture. Furthermore, we have evaluated the efficacy of both α-BS to neutralize key toxic compounds belonging to the predominant protein families of Bothrops snakes. Most significantly, both α-BS cross-specifically neutralized the main toxicological activities e.g. lethality and hemorrhage induced by these venoms. Thus, our data indicate that both α-BS are equally effective to treat snake bite victims inflicted by Bothrops snakes particularly B. atrox, responsible for the largest numbers of human

  8. Treatment of snake bites by Bothrops species and Lachesis muta in Ecuador: laboratory screening of candidate antivenoms.

    PubMed

    Theakston, R D; Laing, G D; Fielding, C M; Lascano, A F; Touzet, J M; Vallejo, F; Guderian, R H; Nelson, S J; Wüster, W; Richards, A M

    1995-01-01

    Bothrops xanthogrammus/asper, B. atrox and Lachesis muta are probably responsible for most cases of severe envenoming in Ecuador. In recent years, the most widely used antivenom ('Myn' Ronti, imported from Mexico) has proved clinically ineffective. There is an urgent need to identify an effective alternative for clinical testing. Five antivenoms with activity against Bothrops venoms were compared using standard World Health Organization rodent and in vitro assays: (i) 'Myn', Ronti Mexico SA ('B. atrox', 'Crotalus terrificus'), (ii) Instituto Butantan (Bothrops polyvalent, Brazil), (iii) Instituto Nacional de Hygiene y Medicina Tropical (Bothrops polyvalent, Ecuador), (iv) Instituto Nacional de Salud (B. asper, C. durissus and Lachesis muta, Colombia), and (v) Laboratorios Probiol (Bothrops, Lachesis and Crotalus, Colombia). The venoms against which these antivenoms were tested were Ecuadorian B. atrox, B. asper and B. xanthogrammus. Brazilian antivenom proved to be the most effective, followed by the Ecudorian and Colombian antivenoms. Mexican antivenom was completely ineffective in neutralizing the lethal effects of Ecuadorian Bothrops venoms. Monospecific Brazilian L. muta antivenom (Instituto Butantan) proved effective against Ecuadorian L. muta venom, but the Colombian polyspecific antivenoms did not. Clinical trials of Brazilian and Ecuadorian antivenoms are planned in the Amazon region of Ecuador in the near future.

  9. Snake Venomics and Antivenomics of Bothrops diporus, a Medically Important Pitviper in Northeastern Argentina.

    PubMed

    Gay, Carolina; Sanz, Libia; Calvete, Juan J; Pla, Davinia

    2015-12-25

    Snake species within genus Bothrops are responsible for more than 80% of the snakebites occurring in South America. The species that cause most envenomings in Argentina, B. diporus, is widely distributed throughout the country, but principally found in the Northeast, the region with the highest rates of snakebites. The venom proteome of this medically relevant snake was unveiled using a venomic approach. It comprises toxins belonging to fourteen protein families, being dominated by PI- and PIII-SVMPs, PLA₂ molecules, BPP-like peptides, L-amino acid oxidase and serine proteinases. This toxin profile largely explains the characteristic pathophysiological effects of bothropic snakebites observed in patients envenomed by B. diporus. Antivenomic analysis of the SAB antivenom (Instituto Vital Brazil) against the venom of B. diporus showed that this pentabothropic antivenom efficiently recognized all the venom proteins and exhibited poor affinity towards the small peptide (BPPs and tripeptide inhibitors of PIII-SVMPs) components of the venom.

  10. [Snake bites by Bothrops moojeni: correlation of the clinical picture with the snake size].

    PubMed

    Kouyoumdjian, J A; Polizelli, C

    1989-01-01

    During a period of 19 months (March 1986-September 1987), 22 cases of snake bites caused by Bothrops moojeni in São José do Rio Preto, São Paulo (South-east Brazil) were studied. The animals have their length measured and two groups were constituted: group I - 9 cases with length between 30 and 53 cm and group II - 13 cases with length between 80 and 147 cm. Our results revealed: 1. the local effects--pain and swelling--were frequently mild in group I; 2 - non-clotting blood (bed-side test) occurred in two groups, a little more frequently in group I; 3. local complications--necrosis, compartment syndrome and infection--occurred in more than a half and exclusively in group II, in spite of larger and faster antivenom therapy. We have concluded that larger Bothrops moojeni produced great increment in swelling, necrosis and infection and mild decrement in coagulation action.

  11. Snake Venomics and Antivenomics of Bothrops diporus, a Medically Important Pitviper in Northeastern Argentina

    PubMed Central

    Gay, Carolina; Sanz, Libia; Calvete, Juan J.; Pla, Davinia

    2015-01-01

    Snake species within genus Bothrops are responsible for more than 80% of the snakebites occurring in South America. The species that cause most envenomings in Argentina, B. diporus, is widely distributed throughout the country, but principally found in the Northeast, the region with the highest rates of snakebites. The venom proteome of this medically relevant snake was unveiled using a venomic approach. It comprises toxins belonging to fourteen protein families, being dominated by PI- and PIII-SVMPs, PLA2 molecules, BPP-like peptides, L-amino acid oxidase and serine proteinases. This toxin profile largely explains the characteristic pathophysiological effects of bothropic snakebites observed in patients envenomed by B. diporus. Antivenomic analysis of the SAB antivenom (Instituto Vital Brazil) against the venom of B. diporus showed that this pentabothropic antivenom efficiently recognized all the venom proteins and exhibited poor affinity towards the small peptide (BPPs and tripeptide inhibitors of PIII-SVMPs) components of the venom. PMID:26712790

  12. 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

  13. Kallikrein-kinin system in the plasma of the snake Bothrops jararaca.

    PubMed Central

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

    1989-01-01

    1. Bothrops jararaca venom (BJV) caused a fall in the carotid artery blood pressure of the anaesthetized snake. This effect was tachyphylactic and was potentiated by captopril, a kininase II inhibitor; it was partially antagonized by promethazine plus cimetidine and was not affected by atropine. 2. Similar hypotensive effects were obtained by administration of trypsin or a partially purified BJV kininogenase to the snake. 3. Incubation of Bothrops jararaca plasma (BJP) with trypsin released a substance (or substances) that produced hypotension in the snake but not in the rat; this hypotensive effect was also potentiated by captopril. 4. The trypsinised plasma contracted Bothrops jararaca isolated uterus, a pharmacological preparation weakly sensitive to bradykinin. Trypsinised plasma was inactive on pigeon oviduct and rat uterus and displayed a weak action on the guinea-pig ileum. Similar effects were observed with incubates of a fraction of BJP, containing globulins, with a partially purified BJV kininogenase. 5. Like mammalian kinins, the substance(s) was(were) dialysable, thermostable in acid but not in alkaline pH, and inactivated by chymotrypsin but not by trypsin. Its(their) inactivation by BJP or BJP kininase II was inhibited by captopril. 6. These findings strongly suggest that, besides releasing histamine, BJV or trypsin release a kininlike substance (or substances) from the snake plasma. 7. Since BJV and other kininogenases active on mammalian plasma were shown to be unable to release kinins from BJP, in experiments conducted on pharmacological preparations suitable for the assay of mammalian kinins, these data also suggest that the snake Bothrops jararaca, like birds, may have developed its own kallikrein-kinin system. PMID:2804549

  14. Reversible Posterior Leukoencephalopathy in a Venomous Snake (Bothrops asper) Bite Victim

    PubMed Central

    Delgado, Miguel E.; Del Brutto, Oscar H.

    2012-01-01

    An 18-year-old man developed posterior reversible leukoencephalopaty after being bitten by a venomous snake (Bothrops asper). It is possible that this previously unrecognized neurological complication of snake bite envenoming occurred as the result of endothelial dysfunction induced by the venom of the offending snake. This pathogenetic mechanism has also been implicated as the cause of cerebral infarctions in snake bite victims. Alternatively, the leukoencephalopathy might have been a complication of antivenom therapy. PMID:22403325

  15. Ability of a synthetic coumestan to antagonize Bothrops snake venom activities.

    PubMed

    Melo, Paulo A; Pinheiro, Diogo A; Ricardo, Hilmar Dias; Fernandes, Fabrício F A; Tomaz, Marcelo A; El-Kik, Camila Z; Strauch, Marcelo A; da Fonseca, Tatiane F; Sifuentes, Daniel N; Calil-Elias, Sabrina; Buarque, Camilla D; Brito, Flávia V; Costa, Paulo R R; Da Silva, Alcides J M

    2010-01-01

    We investigated a synthetic coumestan named LQB93 and similar compounds abilities to antagonize activities of Bothrops jararacussu and Bothrops jararaca crude venoms in different protocols. The antimyotoxic activity was evaluated in vitro by the rate of release of creatine kinase (CK) from isolated mouse extensor digitorum longus muscle (EDL) induced by B. jararacussu (25 g/ml). For in vivo studies, B. jararacussu venom (1.0 mg/kg) was preincubated with LQB93 (0.1-30 mg/kg), during 30 min, for later injection in mouse tight and evaluation of the antimyotoxic and anti-edematogenic effects. LQB93 antagonized in vitro, the increase of CK release from the EDL muscle (IC(50)=0.0291 M). It also showed in vivo, antimyotoxic and anti-edematogenic effects that were dose-dependent with ID50 of 0.17 mg/kg and 0.14 mg/kg, respectively. The hemorrhage induced by B. jararaca (1.0 mg/kg) venom in the mouse skin, was abolished by LQB93 (10.0 mg/kg) preincubated with venom. Like wedelolactone, LQB93 protected rat isolated heart on a Langendorff preparation, from the cardiotoxicity of B. jararacussu venom. LQB93 inhibit the effects of Bothrops venoms like wedelolactone, a natural compound isolated from the plant Eclipta prostrata. Copyright 2009 Elsevier Ltd. All rights reserved.

  16. 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

  17. Nomenclatural instability in the venomous snakes of the Bothrops complex: Implications in toxinology and public health.

    PubMed

    Carrasco, Paola Andrea; Venegas, Pablo Javier; Chaparro, Juan Carlos; Scrocchi, Gustavo José

    2016-09-01

    Since nomenclature is intended to reflect the evolutionary history of organisms, advances in our understanding of historical relationships may lead to changes in classification, and thus potentially in taxonomic instability. An unstable nomenclature for medically important animals like venomous snakes is of concern, and its implications in venom/antivenom research and snakebite treatment have been extensively discussed since the 90´s. The taxonomy of the pitvipers of the Bothrops complex has been historically problematic and different genus-level rearrangements were proposed to rectify the long-standing paraphyly of the group. Here we review the toxinological literature on the Bothrops complex to estimate the impact of recent proposals of classification in non-systematic research. This assessment revealed moderate levels of nomenclatural instability in the last five years, and the recurrence of some practices discussed in previous studies regarding the use of classifications and the information provided about the origin of venom samples. We briefly comment on a few examples and the implications of different proposals of classifications for the Bothrops complex. The aim of this review is to contribute to the reduction of adverse effects of current taxonomic instability in a group of medical importance in the Americas.

  18. [The toxicity of venom of Bothrops (Rhinocerophris) alternatus in different areas of Cordoba State in Argentina].

    PubMed

    Rocco, Daniela M; Reati, Gustavo; Costa de Oliveira, Vanessa; Lanari, Laura C; Laskowicz, Rodrigo D; de Roodt, Adolfo R

    2013-01-01

    Snake venoms can show biochemical and toxicological variability even in specimens from the same specie. The geographical localization of the snakes is one of the factors that can influence those variations. By these reasons the venom from specimens of Bothrops (Rhinocerophis) alternatus ("crucera", "yararágrande"), one of the snakes of highest medical importance in Argentina, from three different regions of Córdoba was studied. Lehtal potency, hemorrhagic, coagulant on plasma and thrombin like activities as well as the electrophoretic patterns of venom from snakes of Calamuchita, Traslasierras and the East of the province were determined. The venom from the snakes of the three regions showed the characteristic activities of the venom of the majority of Bothrops, causing hemorrhage, hemostatic disturbances acting on plasma or directly on fibrinogen with a "thrombin like activity". The different samples were very similar regarding their biochemical characteristics and toxic potencies at difference of previous observations on venoms from the same specie in different regions of other provinces fro Argentina. Bivalent antivenom, the one used by the Provincial Ministry of Health to treat the bothropic accidents, neutralized in all the cases the toxic activities of the venom in very similar range of neutralizing potency.

  19. Some toxic and enzymatic activities of Bothrops ammodytoides (yarará ñata) venom.

    PubMed

    de Roodt, A R; Dolab, J A; Hajos, S E; Gould, E; Dinápoli, H; Troiano, J C; Gould, J; Dokmetjian, J C; Carfagnini, J C; Fernández, T; Amoroso, M; Segre, L; Vidal, J C

    2000-01-01

    Bothrops ammodytoides, the smallest representative of this genus, is found only in Argentina. Venom was extracted from thirty adult specimens (35-70 cm in length, 90-300 g in weight) captured in the Province of Buenos Aires and kept in captivity. Venom yield was 3-30 mg. SDS-PAGE showed strong bands at 14.0; 23-25; 45; 54 and 63 kDa and weak bands at 17.0; 30.0; 40.0 and 85.0 kDa. Toxic activities were: LD50 (intravenous, mice) 0.5+/-0.2 microg/g; minimal procoagulant dose on human plasma (MPD-P) 35+/-2 mg/l; and minimal defibrinogenating dose (MDD, mice) 6-12 microg. Hemorrhagic and/or necrotic activities appear to play a major role in lethality; minimal hemorrhagic dose (MHD, mice) is 10+/-2 microg/g and minimal necrotizing dose (MND, mice) is 38+/-5 microg. The LD50, MPD-P and MND are among the lowest in venoms from Bothrops species found in Argentina. B. ammodytoides venom exhibited high proteolytic and phospholipase A2 activities. Most of the B. ammodytoides venom components cross-react with Bivalent Bothropic antivenom (Instituto Nacional de Producción de Biológicos ANLIS Dr. G. Malbrin, against B. alternatus and B. neuwiedii venoms). One ml of antivenom neutralizes 1.2 mg of B. ammodytoides venom.

  20. A transcriptomic analysis of gene expression in the venom gland of the snake Bothrops alternatus (urutu)

    PubMed Central

    2010-01-01

    Background The genus Bothrops is widespread throughout Central and South America and is the principal cause of snakebite in these regions. Transcriptomic and proteomic studies have examined the venom composition of several species in this genus, but many others remain to be studied. In this work, we used a transcriptomic approach to examine the venom gland genes of Bothrops alternatus, a clinically important species found in southeastern and southern Brazil, Uruguay, northern Argentina and eastern Paraguay. Results A cDNA library of 5,350 expressed sequence tags (ESTs) was produced and assembled into 838 contigs and 4512 singletons. BLAST searches of relevant databases showed 30% hits and 70% no-hits, with toxin-related transcripts accounting for 23% and 78% of the total transcripts and hits, respectively. Gene ontology analysis identified non-toxin genes related to general metabolism, transcription and translation, processing and sorting, (polypeptide) degradation, structural functions and cell regulation. The major groups of toxin transcripts identified were metalloproteinases (81%), bradykinin-potentiating peptides/C-type natriuretic peptides (8.8%), phospholipases A2 (5.6%), serine proteinases (1.9%) and C-type lectins (1.5%). Metalloproteinases were almost exclusively type PIII proteins, with few type PII and no type PI proteins. Phospholipases A2 were essentially acidic; no basic PLA2 were detected. Minor toxin transcripts were related to L-amino acid oxidase, cysteine-rich secretory proteins, dipeptidylpeptidase IV, hyaluronidase, three-finger toxins and ohanin. Two non-toxic proteins, thioredoxin and double-specificity phosphatase Dusp6, showed high sequence identity to similar proteins from other snakes. In addition to the above features, single-nucleotide polymorphisms, microsatellites, transposable elements and inverted repeats that could contribute to toxin diversity were observed. Conclusions Bothrops alternatus venom gland contains the major toxin

  1. Active immunization of cattle with a bothropic toxoid does not abrogate envenomation by Bothrops asper venom, but increases the likelihood of survival.

    PubMed

    Herrera, María; González, Katherine; Rodríguez, Carlos; Gómez, Aarón; Segura, Álvaro; Vargas, Mariángela; Villalta, Mauren; Estrada, Ricardo; León, Guillermo

    2017-03-01

    This study assessed the protective effect of active immunization of cattle to prevent the envenomation induced by B. asper venom. Two groups of oxen were immunized with a bothropic toxoid and challenged by an intramuscular injection of either 10 or 50 mg B. asper venom, to induce moderate or severe envenomations, respectively. Non-immunized oxen were used as controls. It was found that immunized oxen developed local edema similar to those observed in non-immunized animals. However, systemic effects were totally prevented in immunized oxen challenged with 10 mg venom, and therefore antivenom treatment was not required. When immunized oxen were challenged with 50 mg venom, coagulopathy was manifested 3-16 h later than in non-immunized oxen, demonstrating a delay in the onset of systemic envenomation. In these animals, active immunization did not eliminate the need for antivenom treatment, but increased the time lapse in which antivenom administration is still effective. All experimentally envenomed oxen completely recovered after a week following venom injection. Our results suggest that immunization of cattle with a bothropic toxoid prevents the development of systemic effects in moderate envenomations by B. asper, but does not abrogate these effects in severe envenomation.

  2. Venomics and antivenomics of Bothrops erythromelas from five geographic populations within the Caatinga ecoregion of northeastern Brazil.

    PubMed

    Jorge, Roberta Jeane B; Monteiro, Helena S A; Gonçalves-Machado, Larissa; Guarnieri, Míriam C; Ximenes, Rafael M; Borges-Nojosa, Diva M; Luna, Karla P de O; Zingali, Russolina B; Corrêa-Netto, Carlos; Gutiérrez, José María; Sanz, Libia; Calvete, Juan J; Pla, Davinia

    2015-01-30

    The Caatinga lancehead, Bothrops erythromelas, is a medically relevant species, responsible for most of the snakebite accidents in most parts of its distribution range in northeastern Brazil. The spectrum and geographic variability of its venom toxins were investigated applying a venomics approach to venom pools from five geographic areas within the Caatinga ecoregion. Despite its wide habitat, populations of B. erythromelas from Ceará, Pernambuco, Juazeiro, Paraiba, and Ilha de Itaparica exhibit highly conserved venom proteomes. Mirroring their compositional conservation, the five geographic venom pools also showed qualitatively and quantitatively overlapping antivenomic profiles against antivenoms generated in Vital Brazil (BR) and Clodomiro Picado (CR) Institutes, using different venoms in the immunization mixtures. The paraspecificity exhibited by the Brazilian SAB and the Costa Rican BCL antivenoms against venom toxins from B. erythromelas indicates large immunoreactive epitope conservation across genus Bothrops during the last ~14 million years, thus offering promise for the possibility of generating a broad-spectrum bothropic antivenom. Biological Significance Accidental snakebite envenomings represent an important public health hazard in Brazil. Ninety per cent of the yearly estimated 20-30,000 snakebite accidents are caused by species of the Bothrops genus. Bothrops erythromelas, a small, moderately stocky terrestrial venomous snake, is responsible for most of the snakebite accidents in its broad distribution range in the Caatinga, a large ecoregion in northeastern Brazil. To gain a deeper insight into the spectrum of medically important toxins present in the venom of the Caatinga lancehead, we applied a venomics approach to define the proteome and geographic variability of adult B. erythromelas venoms from five geographic regions. Although intraspecific compositional variation between venoms among specimens from different geographic regions has long been

  3. Proteolytic, edematogenic and myotoxic activities of a hemorrhagic metalloproteinase isolated from Bothrops alternatus venom.

    PubMed

    Gay, C C; Leiva, L C; Maruñak, S; Teibler, P; Acosta de Pérez, O

    2005-10-01

    A hemorrhagic metalloproteinase has been isolated from Bothrops alternatus venom from specimens that inhabit the north-east region of Argentina. The present study aimed at evaluating the proteolytic, hemorrhagic, edematogenic and myotoxic activities of the purified metalloproteinase, in order to consider its participation on the phatophysiology of the intoxication by Bothrops alternatus venom. The hemorrhagic metalloproteinase was isolated by a combination of DEAE-Cellulose chromatography and gel filtration on Sephadex G-75. The enzyme showed a molecular mass around 55k Da, it exhibited a hemorrhagic activity with a minimal hemorrhagic dose of 1.9 microg, almost two fold minor than the whole venom (3.6 microg). The enzyme showed a weak proteolytic activity on casein (18.72 U/mg enzyme), similar to the one exhibited by the whole venom (20 U/mg venom). Besides, the ability to degrade casein could be detected by SDS-PAGE; beta-casein was the fraction that showed the higher degradation, followed by alphas(1)-casein and kappa-casein degradation. The hemorrhagic metalloproteinase rapidly hydrolysed the A alpha-chain of fibrinogen, followed by B beta-chain degradation and leaving the gamma-chain unaffected. Proteolytic activities were inhibited by EDTA whereas they were not inhibited by benzamidine and PMSF. The metalloproteinase showed several polypeptides chains after autocatalytic processing, including a chain of 28k Da, it could be the processed disintegrin-like and cysteine-rich domains. The isolated enzyme exhibited myotoxic activity with high CK levels at 6h, due to local ischemia resulting of its hemorrhagic activity, and a significant edema-inducing effect (MED=1.3 microg), corroborated both results by the histological observations of samples of gastrocnemius muscle. These findings showed that this hemorrhagic metalloproteinase, possesses high edematogenic and myotoxic activities and, in despite of exhibiting a weak proteolytic activity, it is able to degrade

  4. Antagonism of the myotoxic effects of Bothrops jararacussu venom and bothropstoxin by polyanions.

    PubMed

    Melo, P A; Homsi-Brandeburgo, M I; Giglio, J R; Suarez-Kurtz, G

    1993-03-01

    The effects of heparin and other polyanions on the myotoxicity of Bothrops jararacussu venom and purified bothropstoxin (BthTX) were investigated. The release rate of creatine kinase (CK) from isolated extensor digitorum longus muscle and the plasma CK activity of mice were used to quantify the results. The myotoxic effects of B. jararacussu venom or BthTX were inhibited by preincubation of these agents with one of the following: a heterogeneous heparin preparation (designated 'heparin'), low mol. wt heparin (H-4500) or dextran sulfates (DS-8000 and DS-500,000). Non-sulfated dextran (D-40,000) and two chondroitin sulfates were ineffective. The antimyotoxic effects of the polyanions are ascribed to their forming inactive acid-base complexes with the basic myotoxins of Bothrops venoms. Gel-filtration experiments in Sephadex provided direct evidence for complex formation between heparin and BthTX. Intravenous (i.v.) administration of H-4500 or DS-8000 opposed the increase in plasma CK activity induced by a subsequent i.m. injection of venom or BthTX. In contrast, pretreatment with i.v. heparin or DS-500,000 enhanced the venom-induced increase in plasma CK activity. This effect was not observed (1) when the animals were treated with a polyvalent antivenom, which inhibits the coagulation and local stasis induced by Bothrops venoms, and (2) when BthTX, which has no thrombotic or hemorrhagic properties, was the myotoxic agent. The potentiation of the venom-induced increase in plasma CK activity by heparin and DS-500,000 is ascribed to improved washout of the CK released from damaged fibers, because of the anticoagulant properties of the drugs.

  5. Vellozia flavicans Mart. ex Schult. hydroalcoholic extract inhibits the neuromuscular blockade induced by Bothrops jararacussu venom

    PubMed Central

    2014-01-01

    Background Snakebite is a significant public health issue in tropical countries. In Brazil, some of the most common snake envenomations are from Bothrops. Bothrops bites trigger local and systemic effects including edema, pain, erythema, cyanosis, infections, and necrosis. Vellozia flavicans is a plant from the Brazilian “cerrado” (savanna) that is popularly used as an anti-inflammatory medicine. Since inflammation develops quickly after Bothrops bites, which can lead to infection, the aim of the present study was to observe possible anti-snake venom and antimicrobial activities of V. flavicans (Vf). Methods The chromatographic profile of the main constituents from the Vf leaf hydroalcoholic extract was obtained by thin-layer chromatography (TLC). The anti-snake venom activity was measured by Vf’s ability to neutralize the in vitro neuromuscular blockade caused by Bothrops jararacussu venom (Bjssu) in a mouse phrenic nerve-diaphragm model (PND). After a 20 min incubation, preparations of PND were added to Tyrode’s solution (control); Vf (0.2, 0.5, 1, and 2 mg/mL); 40 μg/mL Bjssu; pre-incubation for 30 min with Bjssu and 1 mg/mL Vf; and a Bjssu pretreated preparation (for 10 min) followed by 1 mg/mL Vf. Myographic recording was performed, and the contractile responses were recorded. The antimicrobial activity (minimum inhibitory concentration [MIC] and minimum bactericidal concentration [MBC]) was obtained for Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Enterococcus faecalis, using gentamicin and vancomycin as positive controls. Results TLC analysis yielded several compounds from Vf, such as flavonoids (quercetin) and phenolic acids (chlorogenic acid). Bjssu completely blocked the contractile responses of PND preparations, while Vf preserved 97% (±10%) of the contractile responses when incubated with Bjssu. In the PND pretreated with Bjssu, Vf was able to inhibit the neuromuscular blockade progress. MIC and MBC of Vf ranged

  6. Inactivation and fragmentation of lectin from Bothrops leucurus snake venom by gamma irradiation

    NASA Astrophysics Data System (ADS)

    Nunes, E. S.; Souza, M. A. A.; Vaz, A. F. M.; Coelho, L. C. B. B.; Aguiar, J. S.; Silva, T. G.; Guarnieri, M. C.; Melo, A. M. M. A.; Oliva, M. L. V.; Correia, M. T. S.

    2012-04-01

    Gamma radiation alters the molecular structure of biomolecules and is able to mitigate the action of snake venoms and their isolated toxins. The effect of γ-radiation on the folding of Bothrops lecurus venom lectin was measured by a hemagglutinating assay, intrinsic and bis-ANS fluorescence. Intrinsic and bis-ANS fluorescence analyses indicated that irradiation caused unfolding followed by aggregation of the lectin. Our results suggest that irradiation can lead to significant changes in the protein structure, which may promote the loss of its binding property and toxic action.

  7. Neutralizing effects of Nectandra angustifolia extracts against Bothrops neuwiedi snake venom.

    PubMed

    Torres, Ana M; Camargo, Francisco J; Ricciardi, Gabriela A L; Ricciardi, Armando I A; Dellacassa, Eduardo

    2011-09-01

    Leaves extracts and essential oil of Nectandra angustifolia were explored for the first time for neutralization of Bothrops neuwiedi diporus snake venom. The ethanol extract was the most active and inhibited both venom activities (hemolytic and coagulant), while the oil was only active on the coagulant activity. These observations confirmed that certain medicinal plants from Corrientes and Chaco Provinces possess significant snake venom neutralizing capacity and need further examination for their active constituents. Analysis by GC and GC-MS of the essential oil and the enantiomeric excess found for alpha-pinene, beta-pinene and limonene allowed a better characterization of this species.

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

    PubMed

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

    2013-07-01

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

  9. Neutralization of Bothrops mattogrossensis snake venom from Bolivia: experimental evaluation of llama and donkey antivenoms produced by caprylic acid precipitation.

    PubMed

    Fernández, Gil Patrick; Segura, Alvaro; Herrera, María; Velasco, Williams; Solano, Gabriela; Gutiérrez, José María; León, Guillermo

    2010-01-01

    Polyspecific bothropic/crotalic and bothropic/lachesic antivenoms were produced in Bolivia by immunizing two donkeys with the venoms of Bothrops mattogrossensis and Crotalus durissus terrificus and one llama with the venoms of B. mattogrossensis and Lachesis muta. These antivenoms are currently being used for snakebite envenomation in Bolivia. The rationale for using these animals is that donkeys and llamas are better adapted than horses to the high altitudes in South America and constitute good alternatives for antivenom production in these regions. Plasma was fractionated by caprylic acid precipitation of non-immunoglobulin plasma proteins, to obtain whole IgG preparations. Donkey-derived antivenom showed one band of 150 kDa when analyzed by SDS-PAGE, whereas llama antivenom presented two immunoglobulin bands, of 170 kDa and 120 kDa, the latter corresponding to the heavy-chain antibodies present in camelid sera. The effectiveness of these antivenoms to neutralize lethal, hemorrhagic, myotoxic, edema-forming, and defibrinogenating activities of the venom of B. mattogrossensis from Bolivia, a species formerly known as Bothrops neuwiedii, was assessed at the experimental level. Although llama antivenom has a total protein concentration four times lower than donkey antivenom, both preparations have similar neutralizing capacity against all toxic activities assessed. Llama and donkey IgG-based antivenoms are effective in the neutralization of B. mattogrossensis venom and represent valuable alternatives for antivenom manufacture in highland regions of South America. Copyright 2009 Elsevier Ltd. All rights reserved.

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

    PubMed

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

    1990-01-01

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

  11. Fatal stroke after Bothrops snakebite in the Amazonas state, Brazil: A case report.

    PubMed

    Silva de Oliveira, Sâmella; Freitas-de-Sousa, Luciana Aparecida; Alves, Eliane Campos; de Lima Ferreira, Luiz Carlos; da Silva, Iran Mendonça; de Lacerda, Marcus Vinícius Guimarães; Fan, Hui Wen; Moura-da-Silva, Ana Maria; Monteiro, Wuelton Marcelo

    2017-11-01

    Bothrops atrox is the snake responsible for the majority of snakebites in the Brazilian Amazon. Patients generally evolve to local manifestations such as edema, pain and ecchymoses. Systemic effects of B. atrox venom are usually restricted to blood incoagulability and spontaneous bleeding. However, in a few cases, bleeding in the central nervous system may occur, which can lead to sequels and deaths. Here, we report a case of a 59 year-old woman who presented edema, pain and ecchymoses on the right foot, headache, nausea, diarrhea, hypertension and blood incoagulability after the bite by Bothrops snake in the Brazilian Amazon. This case evolved with stroke resulting in death despite the antivenom and conservative therapy employed. In addition, we were able to identify the presence of venom in the patient's brain tissue after death. Direct action of toxins present in the snake's venom in the induction of systemic hemorrhage allied to blood incoagulability and hypertension presented by the patient could be involved in the mechanism of stroke in this case. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. The Triterpenoid Betulin Protects against the Neuromuscular Effects of Bothrops jararacussu Snake Venom In Vivo

    PubMed Central

    Ferraz, Miriéle Cristina; de Oliveira, Jhones Luiz; de Oliveira Junior, Joel Reis; Cogo, José Carlos; dos Santos, Márcio Galdino; Franco, Luiz Madaleno; Puebla, Pilar; Ferraz, Helena Onishi; Ferraz, Humberto Gomes; da Rocha, Marisa Maria Teixeira; Hyslop, Stephen

    2015-01-01

    We confirmed the ability of the triterpenoid betulin to protect against neurotoxicity caused by Bothrops jararacussu snake venom in vitro in mouse isolated phrenic nerve-diaphragm (PND) preparations and examined its capability of in vivo protection using the rat external popliteal/sciatic nerve-tibialis anterior (EPSTA) preparation. Venom caused complete, irreversible blockade in PND (40 μg/mL), but only partial blockade (~30%) in EPSTA (3.6 mg/kg, i.m.) after 120 min. In PND, preincubation of venom with commercial bothropic antivenom (CBA) attenuated the venom-induced blockade, and, in EPSTA, CBA given i.v. 15 min after venom also attenuated the blockade (by ~70% in both preparations). Preincubation of venom with betulin (200 μg/mL) markedly attenuated the venom-induced blockade in PND; similarly, a single dose of betulin (20 mg, i.p., 15 min after venom) virtually abolished the venom-induced decrease in contractility. Plasma creatine kinase activity was significantly elevated 120 min after venom injection in the EPSTA but was attenuated by CBA and betulin. These results indicate that betulin given i.p. has a similar efficacy as CBA given i.v. in attenuating the neuromuscular effects of B. jararacussu venom in vivo and could be a useful complementary measure to antivenom therapy for treating snakebite. PMID:26633987

  13. Local inflammation, lethality and cytokine release in mice injected with Bothrops atrox venom.

    PubMed Central

    Barros, S F; Friedlanskaia, I; Petricevich, V L; Kipnis, T L

    1998-01-01

    We have provided evidence that: (a) lethality of mice to crude Bothrops venom varies according the isogenic strain (A/J > C57Bl/6 > A/Sn > BALB/c > C3H/HePas > DBA/2 > C3H/He); (b)BALB/c mice (LD50=100.0 microg) were injected i.p. with 50 microg of venom produced IL-6, IL-10, INF-gamma, TNF-alpha and NO in the serum. In vitro the cells from the mice injected and challenged with the venom only released IL-10 while peritoneal macrophages released IL-10, INF-gamma and less amounts of IL-6; (c) establishment of local inflammation and necrosis induced by the venom, coincides with the peaks of TNF-alpha, IFN-gamma and NO and the damage was neutralized when the venom was incubated with a monoclonal antibody against a 60 kDa haemorrhagic factor. These results suggest that susceptibility to Bothrops atrox venom is genetically dependent but MHC independent; that IL-6, IL-10, TNF-alpha, IFN-gamma and NO can be involved in the mediation of tissue damage; and that the major venom component inducers of the lesions are haemorrhagins. PMID:9883969

  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. The Triterpenoid Betulin Protects against the Neuromuscular Effects of Bothrops jararacussu Snake Venom In Vivo.

    PubMed

    Ferraz, Miriéle Cristina; de Oliveira, Jhones Luiz; de Oliveira Junior, Joel Reis; Cogo, José Carlos; Dos Santos, Márcio Galdino; Franco, Luiz Madaleno; Puebla, Pilar; Ferraz, Helena Onishi; Ferraz, Humberto Gomes; da Rocha, Marisa Maria Teixeira; Hyslop, Stephen; San Feliciano, Arturo; Oshima-Franco, Yoko

    2015-01-01

    We confirmed the ability of the triterpenoid betulin to protect against neurotoxicity caused by Bothrops jararacussu snake venom in vitro in mouse isolated phrenic nerve-diaphragm (PND) preparations and examined its capability of in vivo protection using the rat external popliteal/sciatic nerve-tibialis anterior (EPSTA) preparation. Venom caused complete, irreversible blockade in PND (40 μg/mL), but only partial blockade (~30%) in EPSTA (3.6 mg/kg, i.m.) after 120 min. In PND, preincubation of venom with commercial bothropic antivenom (CBA) attenuated the venom-induced blockade, and, in EPSTA, CBA given i.v. 15 min after venom also attenuated the blockade (by ~70% in both preparations). Preincubation of venom with betulin (200 μg/mL) markedly attenuated the venom-induced blockade in PND; similarly, a single dose of betulin (20 mg, i.p., 15 min after venom) virtually abolished the venom-induced decrease in contractility. Plasma creatine kinase activity was significantly elevated 120 min after venom injection in the EPSTA but was attenuated by CBA and betulin. These results indicate that betulin given i.p. has a similar efficacy as CBA given i.v. in attenuating the neuromuscular effects of B. jararacussu venom in vivo and could be a useful complementary measure to antivenom therapy for treating snakebite.

  16. Screening of Bothrops snake venoms for L-amino acid oxidase activity

    SciTech Connect

    Pessati, M.L.; Fontana, J.D.; Guimaraes, M.F.

    1995-12-31

    Toxins, enzymes, and biologically active peptides are the main components of snake venoms from the genus Bothrops. Following the venom inoculation, the local effects are hemorrhage, edema, and myonecrosis. Nineteen different species of Brazilian Bothrops were screened for protein content and L-amino acid oxidase activity. B. cotiara, formerly found in the South of Brazil, is now threatened with extinction. Its venom contains a highly hemorrhagic fraction and, as expected from the deep yellow color of the corresponding lyophilized powder, a high L-amino acid oxidase (LAO) activity was also characterized. Flavin adenine dinucleotide (FAD) is its associate coenzyme. B. cotiara venom LAO catalyzed the oxidative deamination of several L-amino acids, and the best substrates were methionine, leucine, tryptophan, and phenylalanine, hence, its potential application for the use in biosensors for aspartame determination and for the removal of amino acids from plasma. High levels for LAO were also found in other species than B. cotiara. In addition, the technique of isoelectric focusing (IEF) was employed as a powerful tool to study the iso- or multi-enzyme distribution for LAO activity in the B. cotiara snake venom.

  17. 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. (c) 2010 Elsevier Ltd. All rights reserved.

  18. Purification and biological effects of C-type lectin isolated from Bothrops insularis venom.

    PubMed

    Braga, Marcus Davis Machado; Martins, Alice Maria Costa; Amora, Daniela Nascimento; de Menezes, Dalgimar Beserra; Toyama, Marcos Hikari; Toyama, Daniela Oliveira; Marangoni, Sergio; Barbosa, Paulo Sérgio Ferreira; de Sousa Alves, Renata; Fonteles, Manassés Claudino; Monteiro, Helena Serra Azul

    2006-06-15

    Bothrops insularis is a snake from Queimada Grande Island, which is an island located about 20 miles away from the southeastern coast of Brazil. Compared to other Brazilian species of Bothrops, the toxinology of B. insularis is still poorly understood. Its C-type lectin is involved in several biological processes including anticoagulant and platelet-modulating activities. We purified the C-type lectin (BiLec) from Bothrops insularis venom and investigated its effect in the isolated kidney. BiLec was purified after two chromatographic steps; firstly, the whole venom was submitted to an HPLC molecular exclusion chromatography followed by a second purification through affinity chromatography. B. insularis lectin (BiLec) was studied as to its effect on the renal function of isolated perfused rat kidneys with the use of six Wistar rats. The concentration of 10mug/mL increased perfusion pressure (PP; control(60)=108.27+/-4.9; BiLec(60)=112.9+/-5.4 mmHg; *p<0.05) and renal vascular resistance (RVR; control(60)=5.38+/-0.51; BiLec(60)=6.01+/-0.57 mmHg; *p<0.05). The urinary flow reduced significantly at 90 and 120 min of perfusion (UF; control(120)=0.160+/-0.020; BiLec(120)=0.082+/-0.008 mL g(-1) min(-1); *p<0.05). Glomerular filtration rate (GFR; control(120)=0.697+/-0.084; BiLec(120)=0.394+/-0.063 mL g(-1) min(-1); *p<0.05) diminished only at 120 min. BiLec did not change the percentage of sodium (TNa(+)), potassium (TK(+)) and chloride tubular transport (TCl(-)). The histological alterations probably reflected direct injury on glomerular and tubular renal cells, as demonstrated by the rise in permeability of glomerular endothelial cells, revealed by the presence of a proteinaceous material in the Bowman space. We postulate that the C-type lectin B. insularis promoted its effects probably through interactions with endothelial cells or through the release of other mediators by tubular, mesangial and endothelial cells.

  19. Estudio de la influencia de la refrigeracion con aire de forma natural e inducida en el comportamiento de instalaciones fotovoltaicas

    NASA Astrophysics Data System (ADS)

    Mazon Hernandez, Rocio

    panels are analysed to compare and select the best configuration. The presented research provides a deep knowledge of how they work as well as information and results for an improvement in future designs of building integrated photovoltaic systems. Este estudio se centra en analizar la influencia negativa de la temperatura en la produccion electrica de paneles fotovoltaicos al estar emplazados sobre cubierta de acero, como sucede en naves industriales y sobre un invernadero. Se estudian diferentes configuraciones que permitan refrigerar los paneles, reduciendo su temperatura y mejorar su rendimiento. Para abordar este problema, se han construido dos instalaciones experimentales, fieles a plantas solares en funcionamiento. Una instalacion engloba dos paneles fotovoltaicos sobre estructura fija al suelo. Uno de los paneles esta integrado sobre una superficie paralela y metalica. Entre ambas superficies existe un espacio que posibilita circular aire, permitiendo refrigerar el panel por conveccion natural, o conveccion forzada impulsando el aire con un ventilador. El otro panel, libre por su cara posterior y se ha considerado de referencia. Se ha estudiado el comportamiento del panel integrado sobre cubierta para diferentes secciones de aire y velocidades inducidas, comparandolo con el panel de referencia. Se ha desarrollado un modelo experimental que nos permite determinar la temperatura del panel en funcion de las variables que influyen en su refrigeracion. Adicionalmente, se han analizado los datos de una planta solar en funcionamiento, con paneles de igual caracteristicas, obteniendo correlaciones entre la temperatura del panel y las variables electricas y comparandolos con las obtenidas en la instalacion experimental. La segunda instalacion experimental reproduce parte de una instalacion solar sobre un invernadero, formada por cuatro paneles fotovoltaicos colocados sobre el plastico del invernadero, existiendo un canal divergente entre ambas superficies. Se estudia la

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

    PubMed

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

    2015-01-01

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

  1. Cloning and functional expression of secreted phospholipases A(2) from Bothrops diporus (Yarará Chica).

    PubMed

    Yunes Quartino, Pablo Javier; Barra, José Luis; Fidelio, Gerardo Daniel

    2012-10-19

    Bothrops diporus is a very common viper in Argentina. At present, no complete sequence of secreted phospholipase A(2) (sPLA(2)) from this snake has been reported. We have cloned two sPLA(2) isoenzymes as well as a putative sPLA(2)-like myotoxin from venom gland. The two sPLA(2) were expressed as inclusion bodies in Escherichia coli with an N-terminal tag of ubiquitin. After in vitro renaturation and cleavage step, using an ubiquitin specific peptidase, the recombinants exhibited sPLA(2) activity when analyzed by means of Langmuir dilauroylphosphatidylcholine monolayers as substrate. Both enzymes have a similar surface pressure-activity profile when compared with non-recombinant purified isoforms. To our knowledge, this is the first time that analysis of optimal lateral pressure of substrate monolayers by using the surface barostat technique is performed on recombinant sPLA(2)s.

  2. Crystal structure and molecular dynamics studies of L-amino acid oxidase from Bothrops atrox.

    PubMed

    Feliciano, Patricia R; Rustiguel, Joane K; Soares, Ricardo O S; Sampaio, Suely V; Cristina Nonato, M

    2017-03-15

    L-amino acid oxidases (LAAOs) are dimeric flavoproteins that catalyze the deamination of L-amino acid to α-keto acid, producing ammonia and hydrogen peroxide. In this study, we report the crystal structure and molecular dynamics simulations of LAAO from the venom of Bothrops atrox (BatroxLAAO). BatroxLAAO presents several biological and pharmacological properties with promising biomedical applications. BatroxLAAO structure contains the highly conserved structural pattern of LAAOs comprising a FAD-binding domain, substrate-binding domain and helical domain, and a dimeric arrangement that can be stabilized by zinc. Also, molecular dynamics results show an asymmetric behavior, and a direct communication between FAD- and substrate-binding domains of counterpart subunits. These findings shed light on the structural role of dimerization to catalytic mechanism of SV-LAAOs.

  3. Local and systemic biochemical alterations induced by Bothrops atrox snake venom in mice

    PubMed Central

    de Souza, Carlos AT; Kayano, Anderson M; Setúbal, Sulamita S; Pontes, Adriana S; Furtado, Juliana L; Kwasniewski, Fábio H; Zaqueo, Kayena D; Soares, Andreimar M; Stábeli, Rodrigo G; Zuliani, Juliana P

    2012-01-01

    The local and systemic alterations induced by Bothrops atrox snake venom (BaV) injection in mice were studied. BaV induced superoxide production by migrated neutrophils, mast cell degranulation and phagocytosis by macrophages. Moreover, BaV caused hemorrhage in dorsum of mice after 2hr post- injection. Three hours post-injection in gastrocnemius muscle, we also observed myonecrosis, which was assessed by the determination of serum and tissue CK besides the release of urea, but not creatinine and uric acid, indicating kidney alterations. BaV also induced the release of LDH and transaminases (ALT and AST) indicating tissue and liver abnormalities. In conclusion, the data indicate that BaV induces events of local and systemic importance. PMID:23487552

  4. Local and systemic biochemical alterations induced by Bothrops atrox snake venom in mice.

    PubMed

    de Souza, Carlos At; Kayano, Anderson M; Setúbal, Sulamita S; Pontes, Adriana S; Furtado, Juliana L; Kwasniewski, Fábio H; Zaqueo, Kayena D; Soares, Andreimar M; Stábeli, Rodrigo G; Zuliani, Juliana P

    2012-01-01

    The local and systemic alterations induced by Bothrops atrox snake venom (BaV) injection in mice were studied. BaV induced superoxide production by migrated neutrophils, mast cell degranulation and phagocytosis by macrophages. Moreover, BaV caused hemorrhage in dorsum of mice after 2hr post- injection. Three hours post-injection in gastrocnemius muscle, we also observed myonecrosis, which was assessed by the determination of serum and tissue CK besides the release of urea, but not creatinine and uric acid, indicating kidney alterations. BaV also induced the release of LDH and transaminases (ALT and AST) indicating tissue and liver abnormalities. In conclusion, the data indicate that BaV induces events of local and systemic importance.

  5. In vitro cleavage of bioactive peptides by peptidases from Bothrops jararaca venom and its neutralization by bothropic antivenom produced by Butantan Institute: Major contribution of serine peptidases.

    PubMed

    Kuniyoshi, Alexandre Kazuo; Kodama, Roberto Tadashi; Moraes, Luis Henrique Ferreira; Duzzi, Bruno; Iwai, Leo Kei; Lima, Ismael Feitosa; Cajado-Carvalho, Daniela; Portaro, Fernanda Vieira

    2017-10-01

    In Brazil, envenomation by Bothrops pitvipers is responsible for over 73% of snakebites, and their venom is a rich source of proteolytic enzymes. Most studies have demonstrated that Bothrops jararaca venom acts on macromolecular substrates, causing an imbalance in the victim's hemostatic system. In contrast, fewer studies have examined the proteolytic activity on small molecules such as peptides. In this study, we used a set of bioactive peptides (insulin B chain, Met-enkephalin, Leu-enkephalin, neuropeptide Y, peptide YY, pancreatic polypeptide, substance P and somatostatin) to identify new peptide substrates for the metallopeptidases and serine peptidases from the B. jararaca venom. The majority of these peptides were substrates for the venom, but neuropeptide Y and pancreatic polypeptide presented higher hydrolyses rates. Although most of the peptides were simultaneously substrates for both classes of proteases, serine peptidases were the most active. Substance P was an exclusive substrate for metallopeptidases, while somatostatin was a selective substrate for serine peptidases. The neutralizing efficacy of the bothropic antivenom produced by the Butantan Institute was also assessed and found to totally prevent substance P hydrolysis, whereas somatostatin cleavage was not inhibited. Thus, the antivenom effectively inhibited metallopeptidase activity, but did not neutralize some of the serine peptidases. These results indicate that, in addition to cleaving proteins, the proteolytic enzymes from this venom also hydrolyze bioactive peptides, and this peptidase activity could effectively contribute to some of the many dire manifestations of envenomation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Phylogenetic analysis of β-defensin-like genes of Bothrops, Crotalus and Lachesis snakes.

    PubMed

    Correa, Poliana G; Oguiura, Nancy

    2013-07-01

    Defensins are components of the vertebrate innate immune system; they comprise a diverse group of small cationic antimicrobial peptides. Among them, β-defensins have a characteristic β-sheet-rich fold plus six conserved cysteines with particular spacing and intramolecular bonds. They have been fully studied in mammals, but there is little information about them in snakes. Using a PCR approach, we described 13 β-defensin-like sequences in Bothrops and Lachesis snakes. The genes are organized in three exons and two introns, with exception of B.atrox_defensinB_01 which has only two exons. They show high similarities in exon 1, intron 1 and intron 2, but exons 2 and 3 have undergone accelerated evolution. The theoretical translated sequences encode a pre-β-defensin-like molecule with a conserved signal peptide and a mature peptide. The signal peptides are leucine-rich and the mature β-defensin-like molecules have a size around 4.5 kDa, a net charge from +2 to +11, and the conserved cysteine motif. Phylogenetic analysis was done using maximum parsimony, maximum likelihood and Bayesian analyses, and all resulted in similar topologies with slight differences. The genus Bothrops displayed two separate lineages. The reconciliation of gene trees and species tree indicated eight to nine duplications and 23 to 29 extinctions depending on the gene tree used. Our results together with previously published data indicate that the ancestral β-defensin-like gene may have three exons in vertebrates and that their evolution occurred according to a birth-and-death model.

  7. Differential efficiency of simvastatin and lipoic acid treatments on Bothrops jararaca envenomation-induced acute kidney injury in mice.

    PubMed

    Barone, Juliana Marton; Alponti, Rafaela Fadoni; Frezzatti, Rodrigo; Zambotti-Villela, Leonardo; Silveira, Paulo Flávio

    2011-01-01

    Snake bite accidents by Bothrops genus is an important public health issue in Brazil and one of its most serious complications is the acute kidney injury (AKI). Here we evaluated the effects of Bothrops jararaca venom (vBj) and the treatments with lipoic acid (LA) and simvastatin (SA) on renal function, aminopeptidase (AP) activities and renal redox status. Primordial events for establishment of AKI by vBj were hyperuricemia, hypercreatinemia, urinary hyperosmolality, renal oxidative stress and reduction of hematocrit and protein content in the membrane of renal cortex and medulla and in the plasma. In the renal cortex and medulla the changes caused by vBj in soluble and membrane-bound AP activities had a similar pattern. The beneficial effects of LA and SA on envenomed mice were similar on the hyperuricemia, renal oxidative stress and reduction of hematocrit. LA mitigated the hypercreatinemia, but exacerbated the urinary urea and creatinine, whereas SA mitigated the decrease of plasma urea, urinary hyperosmolality and hypercreatinuria induced by vBj. The beneficial effects of LA and especially of SA on renal effects of vBj open a new perspective for clinical investigations of these drugs as coadjuvant agents in the serotherapy of Bothrops envenomation.

  8. Unmasking Snake Venom of Bothrops leucurus: Purification and Pharmacological and Structural Characterization of New PLA2 Bleu TX-III

    PubMed Central

    Marangoni, Fábio André; Ponce-Soto, Luis Alberto; Marangoni, Sergio; Landucci, Elen Cristina Teizem

    2013-01-01

    Bleu TX-III was isolated from Bothrops leucurus snake venom on one-step analytical chromatography reverse phase HPLC, was homogeneous on SDS-PAGE, and was confirmed by Q-Tof Ultima API ESI/MS (TOF MS mode) mass spectrometry in 14243.8 Da. Multiple alignments of Bleu TX-III show high degree of homology with basic PLA2 myotoxins from other Bothrops venoms. Our studies on local and systemic myotoxicity “in vivo” reveal that Bleu TX-III is myotoxin with local but not systemic action due to the decrease in the plasmatic CK levels when Bleu TX-III is administrated by intravenous route in mice (dose 1 and 5 μg). And at a dose of 20 μg myotoxin behaves like a local and systemic action. Bleu TX-III induced moderate marked paw edema, evidencing the local increase in vascular permeability. The inflammatory events induced in the mice (I.M.) were investigated. The increase in the levels of IL-1, IL-6, and TNF-α was observed in the plasma. It is concluded that Bleu TX-III induces inflammatory events in this model. The enzymatic phospholipid hydrolysis may be relevant to these phenomena. Bothrops leucurus venom is still not extensively explored, and the knowledge of its toxins separately through the study of structure/function will contribute for a better understanding of its action mechanism. PMID:23509815

  9. Functional proteomic analyses of Bothrops atrox venom reveals phenotypes associated with habitat variation in the Amazon.

    PubMed

    Sousa, Leijiane F; Portes-Junior, José A; Nicolau, Carolina A; Bernardoni, Juliana L; Nishiyama-Jr, Milton Y; Amazonas, Diana R; Freitas-de-Sousa, Luciana A; Mourão, Rosa Hv; Chalkidis, Hipócrates M; Valente, Richard H; Moura-da-Silva, Ana M

    2017-04-21

    Venom variability is commonly reported for venomous snakes including Bothrops atrox. Here, we compared the composition of venoms from B. atrox snakes collected at Amazonian conserved habitats (terra-firme upland forest and várzea) and human modified areas (pasture and degraded areas). Venom samples were submitted to shotgun proteomic analysis as a whole or compared after fractionation by reversed-phase chromatography. Whole venom proteomes revealed a similar composition among the venoms with predominance of SVMPs, CTLs, and SVSPs and intermediate amounts of PLA2s and LAAOs. However, when distribution of particular isoforms was analyzed by either method, the venom from várzea snakes showed a decrease in hemorrhagic SVMPs and an increase in SVSPs, and procoagulant SVMPs and PLA2s. These differences were validated by experimental approaches including both enzymatic and in vivo assays, and indicated restrictions in respect to antivenom efficacy to variable components. Thus, proteomic analysis at the isoform level combined to in silico prediction of functional properties may indicate venom biological activity. These results also suggest that the prevalence of functionally distinct isoforms contributes to the variability of the venoms and could reflect the adaptation of B. atrox to distinct prey communities in different Amazon habitats. In this report, we compared isoforms present in venoms from snakes collected at different Amazonian habitats. By means of a species venom gland transcriptome and the in silico functional prediction of each isoform, we were able to predict the principal venom activities in vitro and in animal models. We also showed remarkable differences in the venom pools from snakes collected at the floodplain (várzea habitat) compared to other habitats. Not only was this venom less hemorrhagic and more procoagulant, when compared to the venom pools from the other three habitats studied, but also this enhanced procoagulant activity was not efficiently

  10. Purification and biological effects of L-amino acid oxidase isolated from Bothrops insularis venom.

    PubMed

    Braga, Marcus Davis Machado; Martins, Alice Maria Costa; Amora, Daniela Nascimento; de Menezes, Dalgimar Beserra; Toyama, Marcos Hikari; Toyama, Daniela Oliveira; Marangoni, Sergio; Alves, Claudênio Diógenes; Barbosa, Paulo Sérgio Ferreira; de Sousa Alves, Renata; Fonteles, Manassés Claudino; Monteiro, Helena Serra Azul

    2008-02-01

    Bothrops insularis is a snake from Ilha da Queimada Grande, an island located about 20 miles away from the Southeastern coast of Brazil. Compared with other Brazilian species of Bothrops, the toxinology of B. insularis is still poorly understood, and so far, no fraction from this venom with amino acid oxidase activity had been isolated or its biological activity tested. We investigated the biochemical and biological effects of one l-amino acid oxidase enzyme isolated from B. insularis snake venom (BiLAO), which was purified using HPLC and sequence grade. We also evaluated the renal effects induced by BiLAO. Chromatographic profile of B. insularis whole venom disclosed seven main fractions (I, II, III, IV, V, VI and VII) and the main LAO enzymatic activity was detected in fraction II. The group treated with BiLAO showed a decrease in perfusion pressure (C(120)=110.28+/-3.69; BiLAO(120)=82.2+/-5.6 mmHg*); renal vascular resistance (C(120)=5.48+/-0.53; BiLAO(120)=4.12+/-0.42 mmHg/mL/g/min*), urinary flow (C(120)=0.160+/-0.020; BiLAO(120)=0.064+/-0.012 mL/g/min*), glomerular filtration rate (C(120)=0.697+/-0.084; BiLAO(120)=0.176+/-0.017 mL/g/min*), sodium (C(120)=79.76+/-0.56; BiLAO(120)=65.39+/-6.19%*), potassium (C(120)=69.94+/-6.86; BiLAO(120)=60.26+/-2.24%*) and chloride tubular reabsortion (C(120)=78.53+/-2.33; BiLAO(120)=64.58+/-6.68%*). Acute tubular necrosis foci were observed in the group treated with the LAO fraction of the B. insularis snake venom. Some findings have the same morphological aspect of apoptosis, more evident cortically; otherwise, reversible degenerative phenomena represented by hydropic ballooning with extensive cytoplasmic vacuolization and discontinuity of the cell brush borders in the proximal tubular epithelium were observed; furthermore, necrotic detachment of these cells into the tubular lumina, and increased amount of protein deposits in the distal and proximal tubules were observed. In conclusion, the slowness of blood flow and of

  11. Effects of photobiomodulation therapy on Bothrops moojeni snake-envenomed gastrocnemius of mice using enzymatic biomarkers.

    PubMed

    Dourado, Doroty Mesquita; Matias, Rosemary; Barbosa-Ferreira, Marcos; da Silva, Baldomero Antonio Kato; de Araujo Isaias Muller, Jéssica; Vieira, Willians Fernando; da Cruz-Höfling, Maria Alice

    2017-08-01

    Bothropic venom contains a range of biologically active substances capable of causing severe local and systemic envenoming symptomatology within its victims. The snake anti-venom is effective against systemic effects but has no neutralizing effect against the fast developing local effects. Herein, mice gastrocnemius injected with Bothrops moojeni venom (40 μg/kg) or saline solution were irradiated with HeNe (632.8 nm) and GaAs (904 nm) lasers (daily energy density of 4 J/cm(2); 0.03/0.21 power density; 0.07/0.16 spot size; 1.2/0.04 total energy, 1 cm off contact, for HeNe and GaAs lasers, respectively) and euthanized in periods ranging from 3 h to 21 days. Blood biochemistry for creatine kinase (CK), alkaline phosphatase (ALP), acid phosphatase (AP), lactate dehydrogenase (LDH), aspartate transaminase (AST), and myoglobin and histopathological analysis, for assessing the degree of myonecrosis and regeneration of gastrocnemius, were done at every time interval. GaAs laser promoted faster photobiomodulation therapy (PBMT) effects, and the GaAs group exhibited a better clinical outcome than the HeNe group. Within the GaAs group, the serum levels of CK, LDH, AP, AST, and myoglobin, which were increased by the physiological effects of the venom, were reduced to initial baseline before snake envenomation in less time than those irradiated by the HeNe laser. However, the group receiving irradiation from the HeNe laser returned the levels of ALP activity to baseline faster than those of the GaAs group. Histopathological analysis revealed enhanced muscle regeneration in mice groups treated with both lasers. PBM promoted by GaAs and HeNe showed well-developed centrally nucleate regenerating cells and an increased number of newly formed blood vessels when compared to unirradiated muscle. We therefore suggest that GaAs had the best outcomes likely derived from a deeper penetrating longer wavelength. We conclude that PMBT is a promising, non-invasive approach to be

  12. Purification and variability in thrombin-like activity of Bothrops atrox venom from different geographic regions.

    PubMed

    Cavinato, R A; Remold, H; Kipnis, T L

    1998-02-01

    Bothrops atrox snake venoms from two different Amazon regions, i.e., Manaus, AM (3 degrees 0.6'40"S; 60 degrees 0.1'6.0"W) and Tucurui, PA (3 degrees 0.42'30"S; 49 degrees 0.41'45"W), were analyzed with respect to the thrombin-like activity component by elution profile on gel-filtration and reverse phase HPLC chromatography, electrophoretic mobility on SDS-PAGE, and enzymatic activity on fibrinogen. Despite some individual discrepancies among venom specimens, the thrombin-like activity present in the Manaus pool was eluted earlier compared with the Tucurui pool but its enzymatic specific activity on thrombin was lower (s.a. = 6.0) than that observed in the Tucurui pool (s.a. = 134.0). However, the electrophoretic mobilities of the pools were similar, with most protein bands being concentrated around three main regions, i.e., protein bands with an apparent mr of 100 kDa, of 38-37 kDa and 30 kDa. However, no significant differences were observed in amidolytic activity on the synthetic substrate Tos-Gly-Pro-Arg-pNa, and there was no correlation between thrombin-like and amidolytic activities. A 32 kDa protein endowed with thrombin-like activity and specific activity of 2444 recognized and neutralized by horse anti-B. atrox antivenom, was purified by the successive use of gel filtration, electrofocusing and reverse phase HPLC.

  13. Individual variability in the venom proteome of juvenile Bothrops jararaca specimens.

    PubMed

    Dias, Gabriela S; Kitano, Eduardo S; Pagotto, Ana H; Sant'anna, Sávio S; Rocha, Marisa M T; Zelanis, André; Serrano, Solange M T

    2013-10-04

    Snake venom proteomes/peptidomes are highly complex and subject to ontogenetic changes. Individual variation in the venom proteome of juvenile snakes is poorly known. We report the proteomic analysis of venoms from 21 juvenile specimens of Bothrops jararaca of different geographical origins and correlate it with the evaluation of important venom features. Individual venoms showed similar caseinolytic activities; however, their amidolytic activities were significantly different. Rather intriguingly, plasma coagulant activity showed remarkable variability among the venoms but not the prothrombin-activating activity. LC-MS analysis showed significant differences between venoms; however, an interesting finding was the ubiquitous presence of the tripeptide ZKW, an endogenous inhibitor of metalloproteinases. Electrophoretic profiles of proteins submitted to reduction showed significant variability in total proteins, glycoproteins, and in the subproteomes of proteinases. Moreover, identification of differential bands revealed variation in most B. jararaca toxin classes. Profiles of venoms analyzed under nonreducing conditions showed less individual variability and identification of proteins in a conserved band revealed the presence of metalloproteinases and l-amino acid oxidase as common components of these venoms. Taken together, our findings suggest that individual venom proteome variability in B. jararaca exists from a very early animal age and is not a result of ontogenetic and diet changes.

  14. Irradiation of the Crude Venom of Bothrops jararacussu to Obtain Toxoid

    NASA Astrophysics Data System (ADS)

    Ferreira, Camila G.; Avalloni, Tânia M.; Oshima-Franco, Yoko; de J. Oliveira, Sara; de Oliveira, José M.; Cogo, José C.

    2011-08-01

    The aim of this work was to reduce the toxicity of Bothrops jararacussu venom using gamma-rays of low-energy coming from a source of Americium-241 (E = 59.6 keV and 3.7×109 Bq of activity) in order to obtain a toxoid. The radiation dose that each sample received was controlled by exposure time of the venom to the radiation beam. Mouse nerve phrenic-diaphragm preparation was used for testing the loss of venom toxicity, since the venom causes an irreversible neuromuscular blockade. In this condition, the several samples of irradiated venom, when assayed in neuromuscular preparation showed that with a dose of 0.051 Gy the paralysis caused by the irradiated venom was of 91%, at 0.360 Gy was of 79%, at 1.662 Gy was of 50% and at 2.448 Gy was of 42%. Therefore, it can be concluded that the irradiation model was able to induce a progressive loss of the venom toxicity.

  15. Dynamic Rearrangement in Snake Venom Gland Proteome: Insights into Bothrops jararaca Intraspecific Venom Variation.

    PubMed

    Augusto-de-Oliveira, César; Stuginski, Daniel R; Kitano, Eduardo S; Andrade-Silva, Débora; Liberato, Tarcísio; Fukushima, Isabella; Serrano, Solange M T; Zelanis, André

    2016-10-07

    We carried out an analysis of the venom gland proteome of Bothrops jararaca taking into account two distinct phases of its ontogenetic development (i.e., newborn and adult) and the marked sexual dimorphism recently reported on its venom proteome. Proteomic data analysis showed a dynamic rearrangement in the proteome landscape of B. jararaca venom gland upon development and gender-related changes. Differentially expressed proteins covered a number of biological pathways related to protein synthesis, including proteins associated with transcription and translation, which were found to be significantly higher expressed in the newborn venom gland. Our results suggest that the variation in the expression levels of cellular proteins might give rise to an even higher variation in the levels of the expressed toxins. Upon aging, the venom gland proteome repertoire related to the protein synthesis together with ecological traits would have an impact on the toxin repertoire, which, in the case of B. jararaca species, would enable the species to deal with different prey types during its lifespan. Proteomic data are available via ProteomeXchange with identifier PXD004186.

  16. Variability in expression of Bothrops insularis snake venom proteases: an ontogenetic approach.

    PubMed

    Zelanis, André; de Souza Ventura, Janaina; Chudzinski-Tavassi, Ana Marisa; de Fátima Domingues Furtado, Maria

    2007-05-01

    Bothrops insularis is a threatened snake endemic to Queimada Grande Island, southern coast of São Paulo, Brazil, and the occurrence of sexual abnormalities in males, females and intersexes (females with functional ovaries and rudimentary hemipenis) has been reported in this population. The aim of this study was to identify ontogenetic shifts in protease expression of offspring of captive-bred B. insularis. Three neonates from a single litter were maintained at the facilities of Laboratory of Herpetology, Institute Butantan, for 41 months. The snakes were individually milked and venoms were analyzed both by SDS-PAGE, under reducing conditions, and for biochemical activities. The venoms from the mother and from a pool of adult specimens were used as references. In regard to the electrophoretic patterns, common bands were identified mainly between 14 and 50 kDa among snakes. The occurrence of proteolytic activity was noticed predominantly between 27 and 45 kDa in zymograms. Inhibitory assays with 1,10-phenantroline (10 mM) and PMSF (5 mM) showed that venoms possessed both metalloproteases and serine proteases. Venoms of young specimens showed a higher coagulant activity than those of adults, especially upon factors X and II. All venoms presented fibrino(geno)lytic activity, degrading Aalpha and Bbeta chains of fibrinogen, and lysing fibrin plate. These findings can reflect important individual, ontogenetic and sexual differences on venom composition and are likely correlated with diet habits of this species.

  17. Population dynamics of the critically endangered golden lancehead pitviper, Bothrops insularis: stability or decline?

    PubMed

    Guimarães, Murilo; Munguía-Steyer, Roberto; Doherty, Paul F; Martins, Marcio; Sawaya, Ricardo J

    2014-01-01

    Little is known about vital rates of snakes generally because of the difficulty in collecting data. Here we used a robust design mark-recapture model to estimate survival, behavioral effects on capture probability, temporary emigration, abundance and test the hypothesis of population decline in the golden lancehead pitviper, Bothrops insularis, an endemic and critically endangered species from southeastern Brazil. We collected data at irregular intervals over ten occasions from 2002 to 2010. Survival was slightly higher in the wet season than in the dry season. Temporal emigration was high, indicating the importance of accounting for this parameter both in the sampling design and modeling. No behavioral effects were detected on capture probability. We detected an average annual population decrease (λ = 0.93, CI = 0.47-1.38) during the study period, but estimates included high uncertainty, and caution in interpretation is needed. We discuss the potential effects of the illegal removal of individuals and the implications of the vital rates obtained for the future persistence and conservation of this endemic, endangered species.

  18. Systemic alterations induced by a Bothrops alternatus hemorrhagic metalloproteinase (baltergin) in mice.

    PubMed

    Gay, C C; Maruñak, S L; Teibler, P; Ruiz, R; Acosta de Pérez, O C; Leiva, L C

    2009-01-01

    Systemic alterations induced by a Bothrops alternatus hemorrhagin, named baltergin, a 55kDa fibrinogenolytic metalloproteinase isolated from venom of north-eastern Argentina specimens, were studied in mice. It caused macroscopic hemorrhagic spots in lungs which was injected intravenously with a minimum pulmonary hemorrhagic dose of 10microg. Histological observations of lungs showed mainly hemorrhagic areas, evidenced by the presence of erythrocytes in the alveolar spaces, congestion and increase of thickness of alveolar septum due to polymorphonuclear infiltrate and mononuclear cells. Neither macroscopic hemorrhage in other organs nor histological alterations in heart and cerebrum/cerebellum were observed at doses assayed. However, kidney and liver were mildly affected. Kidney examination revealed congestion, subcapsular hemorrhage with local capsule detachment, inflammatory infiltrate and degeneration of tubular cells. Congestion of blood vessels and hydropic degeneration of hepatocytes were observed in liver. Besides, baltergin was able to further hydrolyze type IV collagen. Although the enzyme showed to be less lethal than whole venom, it induced severe pulmonary bleeding and affected kinder and liver in minor grade. In conclusion, baltergin is able to alter the integrity of capillary vessels and simultaneously, to interfere on the hemostatic system. Thus, this metalloproteinase contribute markedly to systemic alterations characteristic of B. alternatus envenomations.

  19. Functional characterization of fibrinolytic metalloproteinases (colombienases) isolated from Bothrops colombiensis venom.

    PubMed

    Girón, María E; Guerrero, Belsy; Salazar, Ana María; Sánchez, Elda E; Alvarez, Marco; Rodríguez-Acosta, Alexis

    2013-11-01

    Researchers trying to improve the safety and efficacy of fibrinolytic therapy have been isolating fibrinolytic enzymes from snake venoms. Two fibrinolytic enzymes, colombienases 1 and 2, with significant activity have been isolated from the venom of Bothrops colombiensis. The colombienases were characterized for various biological activities which include hemorrhagic, fibrinogenolytic, proteolytic, hemolytic, edematogenic and cytotoxic. Colombienases directly acted on fibrin by degrading fibrinogen Aα and Bβ chains without activating the fibrinolytic system (plasminogen/plasmin), additionally colombienase-2 degraded fibrinogen γ chains as well as the fibronectin molecule. Laminin and type IV collagen were colombienases resistant. Gelatin-zymogram activity was present in B. colombiensis venom (BcV) bands between 64 and 148 kDa. All activities were abolished by metalloproteinases inhibitors. Both enzymes lacked hemorrhagic, hemolytic, cytotoxic, plasminogen activator and coagulant activities. Both colombienases had direct fibrino(geno)lytic activity without other toxic side effects including in vivo hemorrhaging, which could be promising in terms of therapeutic potential as thrombolytic agents. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Chemical constituents of the bark of Dipteryx alata vogel, an active species against Bothrops jararacussu venom.

    PubMed

    Puebla, Pilar; Oshima-Franco, Yoko; Franco, Luiz M; Santos, Marcio G Dos; Silva, Renata V da; Rubem-Mauro, Leandro; Feliciano, Arturo San

    2010-11-12

    The effect of four sub-extracts prepared from the lyophilized hydroalcoholic bark of Dipteryx alata (Leguminosae-Papilionoideae) dissolved in a methanol-water (80:20) mixture through a liquid-liquid partition procedure has been investigated against the neuromuscular blockade of the venom of the snake Bothrops jararacussu. The active CH₂Cl₂ sub-extract has been extensively analyzed for its chemical constituents, resulting in the isolation of four lupane-type triterpenoids: lupeol, lupenone, 28-hydroxylup-20(29)-en-3-one, betulin, nine isoflavonoids: 8-O-methylretusin, 7-hydroxy-5,6,4'-trimethoxyisoflavone, afrormosin, 7-hydroxy-8,3',4'-trimethoxyisoflavone, 7,3'-dihydroxy-8,4'-dimethoxyisoflavone, odoratin, 7,8,3'-trihydroxy-4'-methoxyisoflavone, 7,8,3'-trihydroxy-6,4'-dimethoxyisoflavone, dipteryxin, one chalcone: isoliquiritigenin, one aurone: sulfuretin and three phenolic compounds: vanillic acid, vanillin, and protocatechuic acid. Their chemical structures were elucidated on the basis of spectroscopic analysis, including HRMS, 1D- and 2D-NMR techniques.

  1. Bmoo FIBMP-I: A New Fibrinogenolytic Metalloproteinase from Bothrops moojeni Snake Venom

    PubMed Central

    Torres, F. S.; Rates, B.; Gomes, M. T. R.; Salas, C. E.; Pimenta, A. M. C.; Oliveira, F.; Santoro, M. M.; de Lima, M. E.

    2012-01-01

    A new fibrinogenolytic metalloproteinase (Bmoo FIBMP-I) was purified from Bothrops moojeni snake venom. This enzyme was isolated through a combination of three chromatographic steps (ion-exchange, molecular exclusion, and affinity chromatography). Analyses by reverse phase chromatography, followed by mass spectrometry, showed the presence of enzyme isoforms with average molecular mass of 22.8 kDa. The SDS-PAGE analyses showed a single chain of 27.6 kDa, in the presence and absence of reducing agent. The protein has a blocked N-terminal. One of the peptides obtained by enzymatic digestion of a reduced and S-alkylated isoform was completely sequenced by mass spectrometry (MS/MS). Bmoo FIBMP-I showed similarity with hemorrhagic factor and several metalloproteinases (MP). This enzyme degraded Aα-chain faster than the Bβ-chain and did not affect the γ-chain of bovine fibrinogen. The absence of proteolytic activity after treatment with EDTA, together with the observed molecular mass, led us to suggest that Bmoo FIBMP-I is a member of the P-I class of the snake venom MP family. Bmoo FIBMP-I showed pH-dependent proteolytic activity on azocasein, but was devoid of coagulant, defibrinating, or hemorrhagic activities. The kinetic parameters of proteolytic activity in azocasein were determined (Vmax = 0.4596 Uh−1nmol−1 ± 0.1031 and Km = 14.59 mg/mL ± 4.610). PMID:23762636

  2. Cotiarinase is a novel prothrombin activator from the venom of Bothrops cotiara.

    PubMed

    Kitano, Eduardo S; Garcia, Thalita C; Menezes, Milene C; Tashima, Alexandre K; Zelanis, André; Serrano, Solange M T

    2013-08-01

    Snake venom serine proteinases (SVSPs) may affect hemostatic pathways by specifically activating components involved in coagulation, fibrinolysis and platelet aggregation or by unspecific proteolytic degradation. In this study, we purified and characterized an SVSP from Bothrops cotiara venom, named cotiarinase, which generated thrombin upon incubation with prothrombin. Cotiarinase was isolated by a two-step procedure including gel-filtration and cation-exchange chromatographies and showed a single protein band with a molecular mass of 29 kDa by SDS-polyacrylamide gel electrophoresis under reducing conditions. Identification of cotiarinase by mass spectrometric analysis revealed peptides that matched sequences of viperid SVSPs. Cotiarinase did not show fibrinogen-clotting, platelet-aggregating, fibrinogenolytic and factor X activating activities. Upon incubation with prothrombin the generation of thrombin was detected using the peptide substrate d-Phe-Pip-Arg-pNA. Moreover, mass spectrometric identification of prothrombin fragments generated by cotiarinase in the absence of co-factors (phospholipids, factor Va, factor Xa and Ca(2+) ions), indicated the limited proteolysis of this protein to release prothrombin 1, fragment 1 and thrombin. Cotiarinase is a novel SVSP that acts on prothrombin to release active thrombin that does not match any group of the current classification of snake venom prothrombin activators.

  3. A rational protocol for the successful crystallization of l-amino-acid oxidase from Bothrops atrox

    PubMed Central

    Alves, Raquel Melo; Feliciano, Patricia Rosa; Sampaio, Suely Vilela; Nonato, Maria Cristina

    2011-01-01

    Despite the valuable contributions of robotics and high-throughput approaches to protein crystallization, the role of an experienced crystallographer in the evaluation and rationalization of a crystallization process is still crucial to obtaining crystals suitable for X-ray diffraction measurements. In this work, the difficult task of crystallizing the flavoenzyme l-amino-acid oxidase purified from Bothrops atrox snake venom was overcome by the development of a protocol that first required the identification of a non-amorphous precipitate as a promising crystallization condition followed by the implementation of a methodology that combined crystallization in the presence of oil and seeding techniques. Crystals were obtained and a complete data set was collected to 2.3 Å resolution. The crystals belonged to space group P21, with unit-cell parameters a = 73.64, b = 123.92, c = 105.08 Å, β = 96.03°. There were four protein subunits in the asymmetric unit, which gave a Matthews coefficient V M of 2.12 Å3 Da−1, corresponding to 42% solvent content. The structure has been solved by molecular-replacement techniques. PMID:21505245

  4. Hemostatic effects of recombinant DisBa-01, a disintegrin from Bothrops alternatus.

    PubMed

    Kauskot, Alexandre; Cominetti, Marcia R; Ramos, Oscar H P; Bechyne, Iga; Renard, Jean-Marie; Hoylaerts, Marc F; Crepin, Michel; Legrand, Chantal; Selistre-de-Araujo, Heloisa S; Bonnefoy, Arnaud

    2008-05-01

    A monomeric RGD-disintegrin was recently identified from a cDNA library from the venom gland of Bothrops alternatus. The corresponding 12 kDa-recombinant protein, DisBa-01, specifically interacted with alpha(v)beta3 integrin and displayed potent anti-metastatic and anti-angiogenic properties. Here, the interaction of DisBa-01 with platelet alphaIIb beta3 integrin and its effects on hemostasis and thrombosis were investigated. DisBa-01 bound to Chinese Hamster Ovary (CHO) cells expressing beta3 or alphaIIb beta3 and promoted their adhesion and the adhesion of resting platelets onto glass coverslips. The disintegrin inhibited the binding of FITC-fibrinogen and FITC-PAC-1 to ADP-stimulated platelets and inhibited ADP-, TRAP- and collagen-induced aggregation of murine, rabbit or human platelets. In a flow chamber assay, DisBa-01 inhibited and reverted platelet adhesion to immobilized fibrinogen. DisBa-01 inhibited the phosphorylation of FAK following platelet activation. The intravenous injection of DisBa-01 in C57Bl6/j mice, prolonged tail bleeding time as well as thrombotic occlusion time in mesenteric venules and arterioles following vessel injury with FeCl3. In conclusion, DisBa-01 antagonizes the platelet alphaIIb beta3 integrin and potently inhibits thrombosis.

  5. Inhibition of Bothrops jararacussu venom activities by Plathymenia reticulata Benth extracts

    PubMed Central

    Farrapo, Nicole M; Silva, Gleidy AA; Costa, Karine N; Silva, Magali G; Cogo, José C; Belo, Cháriston A Dal; dos Santos, Márcio G; Groppo, Francisco C; Oshima-Franco, Yoko

    2011-01-01

    Hexane (HEX), dichloromethane (DM), ethyl acetate (EA) and methanol (M) extracts (0.1, 0.2 and 0.4mg/ml) were obtained via Soxhlet from Plathymenia reticulata barks (Pr). These extracts were evaluated against the myotoxicity (58%) and the irreversible in vitro neuromuscular blockade of Bothrops jararacussu (Bjssu) venom (40μg/ml) in a mouse phrenic-nerve diaphragm preparation, by using light-microscopy and conventional myographic techniques. Thin layer chromatography (TLC) was used to access the basic composition of extracts. The efficacy of the extracts was analyzed by Student's t-test or repeated measures ANOVA. The significance level was set at 5%. The Pr extracts showed a higher polyphenols content (3.75%), from which tannins take part, around 20 times more than flavonoids content (0.16%). Qualitatively, via TLC, DM and EA extracts showed higher tannins concentration than the HEX and M extracts. Pharmacologically, at 0.4mg/ml, DM was more effective (92 ± 6.2%) than EA (81.3 ±10%) = HEX, 77.2 ±4.7%) > M (54 ±10%) against the toxic effects of the venom. Morphologically, DM extract preserved intact 52.8% of the muscle fibers in the presence of the venom. We concluded that P. reticulata extracts are able to inhibit toxic effects of B. jararacussu venom, whose protective mechanism could be mediated by tannins. PMID:22331992

  6. Chromatographic behavior of Bothrops erythromelas phospholipase and other venom constituents on Superdex 75.

    PubMed

    Aird, Steven D

    2004-11-01

    In a chromatographic method modification intended to preserve protease activity in Bothrops erythromelas venom, 2 mM CaCl2 was added to the gel filtration buffer [50mM Tris/HCl/150mM NaCl (pH 8.0)], in lieu of an equimolar portion of NaCl. This minor compositional change induced significant differences in the venom elution profile on Superdex 200. For this reason, the influence of buffer composition on chromatographic behavior was investigated using an analytical Superdex 75 HR 10/30 column. Phospholipase (PLA) was used as a marker because Naja atra PLA had previously been observed to interact hydrophobically with this resin. PLA elution volumes generally increased as buffer pH decreased. Addition of 20% acetonitrile to the Tris buffer with CaCl2, reduced hydrophobic interaction of the PLA so significantly that its elution was non-overlapping in the two buffers. Other venom constituents, including bradykinin-potentiating peptides and probable hemorrhagic metalloproteases, were similarly affected. Buffer calcium, bound by vicinal dextran hydroxyl groups, appears to retard elution of this acidic PLA.

  7. Effect of heparin and antivenom on skeletal muscle damage produced by Bothrops jararacussu venom.

    PubMed

    Calil-Elias, S; Martinez, A M B; Melo, P A

    2002-04-01

    We examined the effect of treatment with heparin and polyvalent antivenom on mice muscle Extensor digitorum longus (EDL) regeneration, after damage induced by injection of Bothrops jararacussu crude venom over the muscle of the right posterior limb. The mice were separated into groups and each group received treatment, by intravenous route with either high molecular weight heparin (H), low molecular weight heparin (LMWH), polyvalent antivenom (PAV) or with the combination of PAV plus H or PAV plus LMWH at 15 minutes and 4 hours after the injection of the venom. Myotoxicity was measured by the increase in plasma creatine kinase (CK) activity at two hours after the injection of the venom. The histological changes in EDL at 1, 3, 7 and 21 days after the injection of the venom were analyzed by light microscopy. In each group the normal and regenerated muscle fibers were quantified using Scion Image computer program. We also evaluated in vitro, the influence of these substances in the proteolytic and phospholipase activities of the venom. Heparins decreased the proteolytic activity of the venom but did not affect its phospholipase activity. However the PAV antagonized both activities. PAV and its combinations showed antimyotoxic activity, according to the magnitude of CK plasma levels. At 21 days the regeneration was observed in all animals, also in those that received only the venom. All treatments, except LMWH, promote a significant increase in the number of muscle fibers.

  8. Neutralization of Bothrops alternatus regional venom pools and individual venoms by antivenom: a systematic comparison.

    PubMed

    de Roodt, Adolfo Rafael; Lanari, Laura Cecilia; de Oliveira, Vanessa Costa; Laskowicz, Rodrigo Daniel; Stock, Roberto Pablo

    2011-06-01

    In this study we report that variation in lethality, hemorrhagic potency and procoagulation between individual samples of Bothrops alternatus venom from a single region, and variation between regional pools at the national level are comparable in range. Furthermore, the range of relative neutralization potencies of individual venoms within a region overlaps, and sometimes exceeds, the range of neutralization of regional venom pools throughout the country. Thus, the potency of neutralization of a national venom pool is poorly predictive of the potencies of neutralization of its constituent regional venom pools and, furthermore, the potency of neutralization of a regional venom pool is poorly predictive of the potencies of neutralization of its individual venom constituents. The efficiencies of neutralization of each of these effects (lethality, hemorrhage and procoagulation) were not significantly related to each other and did not correlate to the corresponding toxic potency of each venom or venom pool. Some implications of these findings are discussed in the context of the distinction between experimental quantitation of antivenom potency and the amount of antivenom that might be actually required to successfully treat two apparently comparable B. alternatus envenomations.

  9. An in-depth snake venom proteopeptidome characterization: Benchmarking Bothrops jararaca.

    PubMed

    Nicolau, Carolina A; Carvalho, Paulo C; Junqueira-de-Azevedo, Inácio L M; Teixeira-Ferreira, André; Junqueira, Magno; Perales, Jonas; Neves-Ferreira, Ana Gisele C; Valente, Richard H

    2017-01-16

    A large-scale proteomic approach was devised to advance the understanding of venom composition. Bothrops jararaca venom was fractionated by OFFGEL followed by chromatography, generating peptidic and proteic fractions. The latter was submitted to trypsin digestion. Both fractions were separately analyzed by reversed-phase nanochromatography coupled to high resolution mass spectrometry. This strategy allowed deeper and joint characterizations of the peptidome and proteome (proteopeptidome) of this venom. Our results lead to the identification of 46 protein classes (with several uniquely assigned proteins per class) comprising eight high-abundance bona fide venom components, and 38 additional classes in smaller quantities. This last category included previously described B. jararaca venom proteins, common Elapidae venom constituents (cobra venom factor and three-finger toxin), and proteins typically encountered in lysosomes, cellular membranes and blood plasma. Furthermore, this report is the most complete snake venom peptidome described so far, both in number of peptides and in variety of unique proteins that could have originated them. It is hypothesized that such diversity could enclose cryptides, whose bioactivities would contribute to envenomation in yet undetermined ways. Finally, we propose that the broad range screening of B. jararaca peptidome will facilitate the discovery of bioactive molecules, eventually leading to valuable therapeutical agents.

  10. 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.

  11. Hyperbaric oxygen therapy after Bothrops lanceolatus snake bites in Martinique: a brief report.

    PubMed

    Hochedez, P; Thomas, L; Mehdaoui, H

    2010-01-01

    Every year 10 to 20 cases of snake bites are reported on the Caribbean island of Martinique. The only snake involved, Bothrops lanceolatus, is endemic on the island, and its bite may lead to systemic multifocal thrombotic complications in the'absence of the monospecific antivenom. Between January 1988 and January 2009, more than 250 snake bites have been reported, and five patients were treated with hyperbaric oxygen (HBO2) therapy for local complications. The patients were male, bitten on the leg or the hand, and presented with severe complications such as necrotizing soft tissue infections, compartment syndrome or abscesses despite prompt wound care and administration of antivenomous serum. Outcomes were favorable for these five patients, except for one who was left with a functional defect of the hand. Although snake bites are not part of the currently recommended indications for HBO2 therapy, local complications, namely compartment syndrome, necrotizing soft tissue infections and enhancement of healing in selected problem wounds, are approved uses of HBO2 therapy as defined by the Hyperbaric Oxygen Therapy Committee and would benefit from prospective studies.

  12. Action of two phospholipases A2 purified from Bothrops alternatus snake venom on macrophages.

    PubMed

    Setúbal, S S; Pontes, A S; Furtado, J L; Xavier, C V; Silva, F L; Kayano, A M; Izidoro, L F M; Soares, A M; Calderon, L A; Stábeli, R G; Zuliani, J P

    2013-02-01

    The in vitro effects of BaltTX-I, a catalytically inactive Lys49 variant of phospholipase A2 (PLA2), and BaltTX-II, an Asp49 catalytically active PLA2 isolated from Bothrops alternatus snake venom, on thioglycollate-elicited macrophages (TG-macrophages) were investigated. At non-cytotoxic concentrations, the secretory PLA2 BaltTX-I but not BaltTX-II stimulated complement receptor-mediated phagocytosis. Pharmacological treatment of TG-macrophages with staurosporine, a protein kinase C (PKC) inhibitor, showed that this kinase is involved in the increase of serum-opsonized zymosan phagocytosis induced by BaltTX-I but not BaltTX-II secretory PLA2, suggesting that PKC may be involved in the stimulatory effect of this toxin in serum-opsonized zymosan phagocytosis. Moreover, BaltTX-I and -II induced superoxide production by TG-macrophages. This superoxide production stimulated by both PLA2s was abolished after treatment of cells with staurosporine, indicating that PKC is an important signaling pathway for the production of this radical. Our experiments showed that, at non-cytotoxic concentrations, BaltTX-I may upregulate phagocytosis via complement receptors, and that both toxins upregulated the respiratory burst in TG-macrophages.

  13. Cardiovascular responses to Bothrops alternatus (Urutu) snake venom in anesthetized dogs.

    PubMed

    Dias, Lourdes; Rodrigues, Mariana A P; Smaal, Adriana; Rennó, André L; Mello, Sueli M; Moreno, Heitor; Hyslop, Stephen

    2012-09-01

    The cardiovascular responses to Bothrops alternatus snake venom in anesthetized dogs were investigated. Venom (0.3 mg/kg, i.v.) markedly decreased arterial blood pressure, coronary perfusion pressure, and cardiac output (CO) after 5 min, with progressive recovery of the first two parameters to pre-venom levels after 3 h; CO showed little recovery. There was an abrupt, sustained decrease in left and right ventricular systolic work and stroke volume but no significant changes in heart rate, electrocardiogram, and pulmonary hemodynamics; systemic vascular resistance increased from 1 h onwards. A venom dose of 1 mg/kg produced more pronounced cardiovascular alterations, with a progressive decrease to death. There were no significant changes in blood gas (pO(2), pCO(2), HCO(3), SBC, and SBE) and metabolic (pH, lactate, glucose, creatine kinase activity, Na(+), and K(+)) parameters, although there was a transitory increase in plasma lactate dehydrogenase 2 min after the lower venom dose. There were no cardiac histological alterations, but microaneurysms and epithelial desquamation were seen in renal tubules. Circulating venom concentrations (determined by ELISA) decreased rapidly after administration, but venom was still detectable after 4 h. These results show that in dogs, B. alternatus venom produces marked hypotension and a direct cardiac action, with few metabolic alterations.

  14. 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

  15. Differences between renal effects of venom from two Bothrops jararaca populations from southeastern and southern Brazil.

    PubMed

    Jorge, Roberta Jeane Bezerra; Jorge, Antônio Rafael Coelho; de Menezes, Ramon Róseo Paula Pessoa Bezerra; Mello, Clarissa Perdigão; Lima, Danya Bandeira; Silveira, João Alison de Moraes; Alves, Natacha Teresa Queiroz; Marinho, Aline Diogo; Ximenes, Rafael Matos; Corrêa-Netto, Carlos; Gonçalves Machado, Larissa; Zingali, Russolina Benedeta; Martins, Alice Maria Costa; Monteiro, Helena Serra Azul

    2017-01-01

    Components from animal venoms may vary according to the snake's age, gender and region of origin. Recently, we performed a proteomic analysis of Bothrops jararaca venom from southern (BjSv) and southeastern (BjSEv) Brazil, showing differences in the venom composition, as well as its biological activity. To continue the study, we report in this short communication the different effects induced by the BjSEv and BjSv on isolated kidney and MDCK renal cells. BjSEv decreased perfusion pressure (PP) and renal vascular resistance (RVR) and increased urinary flow (UF) and glomerular filtration rate (GFR), while BjSv did not alter PP and RVR and reduced UF and GFR. Both types of venom, more expressively BjSEv, reduced %TNa(+), %TK(+) and %Cl(-). In MDCK cells, the two types of venom showed cytotoxicity with IC50 of 1.22 μg/mL for BjSv and 1.18 μg/mL for BjSEv and caused different profiles of cell death, with BjSv being more necrotic. In conclusion, we suggest that BjSv is more nephrotoxic than BjSEv. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Abarema cochliacarpos extract decreases the inflammatory process and skeletal muscle injury induced by Bothrops leucurus venom.

    PubMed

    Saturnino-Oliveira, Jeison; Santos, Daiana Do Carmo; Guimarães, Adriana Gibara; Santos Dias, Antônio; Tomaz, Marcelo Amorim; Monteiro-Machado, Marcos; Estevam, Charles Santos; De Lucca Júnior, Waldecy; Maria, Durvanei Augusto; Melo, Paulo A; Araújo, Adriano Antunes de Souza; Santos, Márcio Roberto Viana; Almeida, Jackson Roberto Guedes da Silva; Oliveira, Rita de Cássia Meneses; Pereira de Oliveira, Aldeidia; Quintans Júnior, Lucindo José

    2014-01-01

    Snakebites are a public health problem, especially in tropical countries. However, treatment with antivenom has limited effectiveness against venoms' local effects. Here, we investigated the ability of Abarema cochliacarpos hydroethanolic extract (EAc) to protect mice against injection of Bothrops leucurus venom. Swiss mice received perimuscular venom injection and were subsequently treated orally with EAc in different doses. Treatment with EAc 100, 200, and 400 mg/kg reduced the edema induced by B. leucurus in 1%, 13%, and 39%, respectively. Although lower doses showed no antihypernociceptive effect in the Von Frey test, the higher dose significantly reduced hyperalgesia induced by the venom. Antimyotoxic activity of EAc was also observed by microscopy assessment, with treated muscles presenting preserved structures, decreased edema, and inflammatory infiltrate as compared to untreated ones. Finally, on the rotarod test, the treated mice showed better motor function, once muscle fibers were preserved and there were less edema and pain. Treated mice could stand four times more time on the rotating rod than untreated ones. Our results have shown that EAc presented relevant activities against injection of B. leucurus venom in mice, suggesting that it can be considered as an adjuvant in the treatment of envenomation.

  17. Bmoo FIBMP-I: A New Fibrinogenolytic Metalloproteinase from Bothrops moojeni Snake Venom.

    PubMed

    Torres, F S; Rates, B; Gomes, M T R; Salas, C E; Pimenta, A M C; Oliveira, F; Santoro, M M; de Lima, M E

    2012-01-01

    A new fibrinogenolytic metalloproteinase (Bmoo FIBMP-I) was purified from Bothrops moojeni snake venom. This enzyme was isolated through a combination of three chromatographic steps (ion-exchange, molecular exclusion, and affinity chromatography). Analyses by reverse phase chromatography, followed by mass spectrometry, showed the presence of enzyme isoforms with average molecular mass of 22.8 kDa. The SDS-PAGE analyses showed a single chain of 27.6 kDa, in the presence and absence of reducing agent. The protein has a blocked N-terminal. One of the peptides obtained by enzymatic digestion of a reduced and S-alkylated isoform was completely sequenced by mass spectrometry (MS/MS). Bmoo FIBMP-I showed similarity with hemorrhagic factor and several metalloproteinases (MP). This enzyme degraded Aα-chain faster than the Bβ-chain and did not affect the γ-chain of bovine fibrinogen. The absence of proteolytic activity after treatment with EDTA, together with the observed molecular mass, led us to suggest that Bmoo FIBMP-I is a member of the P-I class of the snake venom MP family. Bmoo FIBMP-I showed pH-dependent proteolytic activity on azocasein, but was devoid of coagulant, defibrinating, or hemorrhagic activities. The kinetic parameters of proteolytic activity in azocasein were determined (V max = 0.4596 Uh(-1)nmol(-1) ± 0.1031 and K m = 14.59 mg/mL ± 4.610).

  18. Abarema cochliacarpos Extract Decreases the Inflammatory Process and Skeletal Muscle Injury Induced by Bothrops leucurus Venom

    PubMed Central

    Saturnino-Oliveira, Jeison; Santos, Daiana Do Carmo; Guimarães, Adriana Gibara; Santos Dias, Antônio; Tomaz, Marcelo Amorim; Monteiro-Machado, Marcos; Estevam, Charles Santos; Lucca Júnior, Waldecy De; Maria, Durvanei Augusto; Melo, Paulo A.; Araújo, Adriano Antunes de Souza; Santos, Márcio Roberto Viana; Almeida, Jackson Roberto Guedes da Silva; Oliveira, Rita de Cássia Meneses; Pereira de Oliveira, Aldeidia; Quintans Júnior, Lucindo José

    2014-01-01

    Snakebites are a public health problem, especially in tropical countries. However, treatment with antivenom has limited effectiveness against venoms' local effects. Here, we investigated the ability of Abarema cochliacarpos hydroethanolic extract (EAc) to protect mice against injection of Bothrops leucurus venom. Swiss mice received perimuscular venom injection and were subsequently treated orally with EAc in different doses. Treatment with EAc 100, 200, and 400 mg/kg reduced the edema induced by B. leucurus in 1%, 13%, and 39%, respectively. Although lower doses showed no antihypernociceptive effect in the Von Frey test, the higher dose significantly reduced hyperalgesia induced by the venom. Antimyotoxic activity of EAc was also observed by microscopy assessment, with treated muscles presenting preserved structures, decreased edema, and inflammatory infiltrate as compared to untreated ones. Finally, on the rotarod test, the treated mice showed better motor function, once muscle fibers were preserved and there were less edema and pain. Treated mice could stand four times more time on the rotating rod than untreated ones. Our results have shown that EAc presented relevant activities against injection of B. leucurus venom in mice, suggesting that it can be considered as an adjuvant in the treatment of envenomation. PMID:25136627

  19. 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

  20. Biological and biochemical characterization of new basic phospholipase A(2) BmTX-I isolated from Bothrops moojeni snake venom.

    PubMed

    Calgarotto, Andrana K; Damico, Daniela C S; Ponce-Soto, L A; Baldasso, Paulo A; Da Silva, Saulo L; Souza, Gustavo H M F; Eberlin, Marcos N; Marangoni, Sergio

    2008-06-15

    BmTX-I, an Asp49 phospholipase A(2), was purified from Bothrops moojeni venom after only one chromatographic step using reverse-phase HPLC on mu-Bondapak C-18 column. A molecular mass of 14238.71Da was determined by MALDI-TOF mass spectrometry. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The BmTX-I PLA(2) had a sequence of 121 residues of amino acids: DLWQFNKMIK KEVGKLPFPF YGAYGCYCGW GGRGEKPKDG TDRCCFVHDC CYKKLTGCPK WDDRYSYSWK DITIVCGEDL PCEEICECDR AAAVCFYENL GTYNKKYMKH LKPCKKADYP C and pI value 7.84, and showed a high degree of homology with basic Asp49 PLA(2) myotoxins from other Bothrops venoms. BmTX-I presented PLA(2) activity in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 35-45 degrees C. Maximum PLA(2) activity required Ca(2+) and in the presence of Mg(2+), Cd(2+) and Mn(2+) it was reduced in presence or absence of Ca(2+). Crotapotin from Crotalus durissus colillineatus rattlesnake venom has significantly inhibited (P<0.05) the enzymatic activity of BmTX-I. In vitro, the whole venom and BmTX-I caused a blockade of the neuromuscular transmission in young chick biventer cervicis preparations in a similar way to other bothrops species. In mice, BmTX-I and the whole venom-induced myonecrosis and a systemic interleukin-6 response upon intramuscular injection. Edema-forming activity was also analyzed through injection of the venom and the purified BmTX-I into the subplantar region of the right footpad. Since BmTX-I exert a strong proinflammatory effect; the enzymatic phospholipids hydrolysis might be relevant for these phenomena.

  1. First record of Porocephalus cf. clavatus (Pentastomida: Porocephalida) as a parasite on Bothrops asper (Squamata: Viperidae) in Costa Rica.

    PubMed

    Alvarado, G; Sánchez-Monge, A

    2015-11-01

    Pentastomids are parasites that infect respiratory cavities of vertebrates, they are pretty common but poorly known in wildlife veterinary. A Bothrops asper snake (Garman, 1884) was captured in the Caribbean region of Costa Rica and had its lung infested with pentastomids, identified as ca Porocephalus clavatus (Wyman, 1845). This represents the first record of Porocephalus (Humboldt, 1812) on B. asper as well as P. cf. clavatus in Costa Rica. Further studies are needed to clarify their taxonomic position, images and scanning electron microscopy photographs (SEM) of the specimens are given.

  2. Kinetics of venom and antivenom serum and clinical parameters and treatment efficacy in Bothrops alternatus envenomed dogs.

    PubMed

    Jacome, Do; Melo, M M; Santos, M M B; Heneine, L G D

    2002-12-01

    Dogs envenomed with non-lethal doses of Bothrops alternatus venom received standard antivenom therapy, im injections of flunixin meglumine, or topical treatmentwith aqueous Curcuma longa plant extract. Biodistribution of the venom and antivenom were determined by ELISA. There was no significant difference in the efficacy of antivenom and plant extract on local effects; flunixin treatment had lower efficacy. Distribution of the venom was similar with all 3 treatments. Serum levels of the antivenom reached maximum 2-4 h after administration and were not detected after the 5th d.

  3. Structural bases for a complete myotoxic mechanism: crystal structures of two non-catalytic phospholipases A2-like from Bothrops brazili venom.

    PubMed

    Fernandes, Carlos A H; Comparetti, Edson J; Borges, Rafael J; Huancahuire-Vega, Salomón; Ponce-Soto, Luis Alberto; Marangoni, Sergio; Soares, Andreimar M; Fontes, Marcos R M

    2013-12-01

    Bothrops brazili is a snake found in the forests of the Amazonian region whose commercial therapeutic anti-bothropic serum has low efficacy for local myotoxic effects, resulting in an important public health problem in this area. Catalytically inactive phospholipases A2-like (Lys49-PLA2s) are among the main components from Bothrops genus venoms and are capable of causing drastic myonecrosis. Several studies have shown that the C-terminal region of these toxins, which includes a variable combination of positively charged and hydrophobic residues, is responsible for their activity. In this work we describe the crystal structures of two Lys49-PLA2s (BbTX-II and MTX-II) from B. brazili venom and a comprehensive structural comparison with several Lys49-PLA2s. Based on these results, two independent sites of interaction were identified between protein and membrane which leads to the proposition of a new myotoxic mechanism for bothropic Lys49-PLA2s composed of five different steps. This proposition is able to fully explain the action of these toxins and may be useful to develop efficient inhibitors to complement the conventional antivenom administration. © 2013.

  4. Acute kidney injury caused by Crotalus and Bothrops snake venom: a review of epidemiology, clinical manifestations and treatment.

    PubMed

    Albuquerque, Polianna L M M; Jacinto, Camilla N; Silva Junior, Geraldo B; Lima, Juliana B; Veras, Maria do Socorro B; Daher, Elizabeth F

    2013-01-01

    Ophidic accidents are an important public health problem due to their incidence, morbidity and mortality. An increasing number of cases have been registered in Brazil in the last few years. Several studies point to the importance of knowing the clinical complications and adequate approach in these accidents. However, knowledge about the risk factors is not enough and there are an increasing number of deaths due to these accidents in Brazil. In this context, acute kidney injury (AKI) appears as one of the main causes of death and consequences for these victims, which are mainly young males working in rural areas. Snakes of the Bothrops and Crotalus genera are the main responsible for renal involvement in ophidic accidents in South America. The present study is a literature review of AKI caused by Bothrops and Crotalus snake venom regarding diverse characteristics, emphasizing the most appropriate therapeutic approach for these cases. Recent studies have been carried out searching for complementary therapies for the treatment of ophidic accidents, including the use of lipoic acid, simvastatin and allopurinol. Some plants, such as Apocynaceae, Lamiaceae and Rubiaceae seem to have a beneficial role in the treatment of this type of envenomation. Future studies will certainly find new therapeutic measures for ophidic accidents.

  5. ACUTE KIDNEY INJURY CAUSED BY Crotalus AND Bothrops SNAKE VENOM: A REVIEW OF EPIDEMIOLOGY, CLINICAL MANIFESTATIONS AND TREATMENT

    PubMed Central

    Albuquerque, Polianna L.M.M.; Jacinto, Camilla N.; Silva, Geraldo B.; Lima, Juliana B.; Veras, Maria do Socorro B.; Daher, Elizabeth F.

    2013-01-01

    SUMMARY Ophidic accidents are an important public health problem due to their incidence, morbidity and mortality. An increasing number of cases have been registered in Brazil in the last few years. Several studies point to the importance of knowing the clinical complications and adequate approach in these accidents. However, knowledge about the risk factors is not enough and there are an increasing number of deaths due to these accidents in Brazil. In this context, acute kidney injury (AKI) appears as one of the main causes of death and consequences for these victims, which are mainly young males working in rural areas. Snakes of the Bothrops and Crotalus genera are the main responsible for renal involvement in ophidic accidents in South America. The present study is a literature review of AKI caused by Bothrops and Crotalus snake venom regarding diverse characteristics, emphasizing the most appropriate therapeutic approach for these cases. Recent studies have been carried out searching for complementary therapies for the treatment of ophidic accidents, including the use of lipoic acid, simvastatin and allopurinol. Some plants, such as Apocynaceae, Lamiaceae and Rubiaceae seem to have a beneficial role in the treatment of this type of envenomation. Future studies will certainly find new therapeutic measures for ophidic accidents. PMID:24037282

  6. Isolation and biochemical characterization of a new thrombin-like serine protease from Bothrops pirajai snake venom.

    PubMed

    Zaqueo, Kayena D; Kayano, Anderson M; Simões-Silva, Rodrigo; Moreira-Dill, Leandro S; Fernandes, Carla F C; Fuly, André L; Maltarollo, Vinícius G; Honório, Kathia M; da Silva, Saulo L; Acosta, Gerardo; Caballol, Maria Antonia O; de Oliveira, Eliandre; Albericio, Fernando; Calderon, Leonardo A; Soares, Andreimar M; Stábeli, Rodrigo G

    2014-01-01

    This paper presents a novel serine protease (SP) isolated from Bothrops pirajai, a venomous snake found solely in Brazil that belongs to the Viperidae family. The identified SP, named BpirSP-39, was isolated by three chromatographic steps (size exclusion, bioaffinity, and reverse phase chromatographies). The molecular mass of BpirSP-39 was estimated by SDS-PAGE and confirmed by mass spectrometry (39,408.32 Da). The protein was able to form fibrin networks, which was not observed in the presence of serine protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF). Furthermore, BpirSP-39 presented considerable thermal stability and was apparently able to activate factor XIII of the blood coagulation cascade, unlike most serine proteases. BpirSP-39 was capable of hydrolyzing different chromogenic substrates tested (S-2222, S-2302, and S-2238) while Cu(2+) significantly diminished BspirSP-39 activity on the three tested substrates. The enzyme promoted platelet aggregation and also exhibited fibrinogenolytic, fibrinolytic, gelatinolytic, and amidolytic activities. The multiple alignment showed high sequence similarity to other thrombin-like enzymes from snake venoms. These results allow us to conclude that a new SP was isolated from Bothrops pirajai snake venom.

  7. Isolation and Biochemical Characterization of a New Thrombin-Like Serine Protease from Bothrops pirajai Snake Venom

    PubMed Central

    Zaqueo, Kayena D.; Kayano, Anderson M.; Simões-Silva, Rodrigo; Moreira-Dill, Leandro S.; Fernandes, Carla F. C.; Fuly, André L.; Maltarollo, Vinícius G.; Honório, Kathia M.; da Silva, Saulo L.; Acosta, Gerardo; Caballol, Maria Antonia O.; de Oliveira, Eliandre; Albericio, Fernando; Calderon, Leonardo A.; Soares, Andreimar M.; Stábeli, Rodrigo G.

    2014-01-01

    This paper presents a novel serine protease (SP) isolated from Bothrops pirajai, a venomous snake found solely in Brazil that belongs to the Viperidae family. The identified SP, named BpirSP-39, was isolated by three chromatographic steps (size exclusion, bioaffinity, and reverse phase chromatographies). The molecular mass of BpirSP-39 was estimated by SDS-PAGE and confirmed by mass spectrometry (39,408.32 Da). The protein was able to form fibrin networks, which was not observed in the presence of serine protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF). Furthermore, BpirSP-39 presented considerable thermal stability and was apparently able to activate factor XIII of the blood coagulation cascade, unlike most serine proteases. BpirSP-39 was capable of hydrolyzing different chromogenic substrates tested (S-2222, S-2302, and S-2238) while Cu2+ significantly diminished BspirSP-39 activity on the three tested substrates. The enzyme promoted platelet aggregation and also exhibited fibrinogenolytic, fibrinolytic, gelatinolytic, and amidolytic activities. The multiple alignment showed high sequence similarity to other thrombin-like enzymes from snake venoms. These results allow us to conclude that a new SP was isolated from Bothrops pirajai snake venom. PMID:24719874

  8. Mechanism of the cytotoxic effect of l-amino acid oxidase isolated from Bothrops alternatus snake venom.

    PubMed

    Ribeiro, Patrícia H; Zuliani, Juliana P; Fernandes, Carla F C; Calderon, Leonardo A; Stábeli, Rodrigo G; Nomizo, Auro; Soares, Andreimar M

    2016-11-01

    BaltLAAO-I, an L-amino acid oxidase isolated from Bothrops alternatus, is a glycoprotein enzyme with a pI-5.3, 15% sugar and a related molecular mass of 66,000Da in its monomeric form, and 123,000Da in its dimeric form. The objective of this study is to describe the cytotoxicity activity induced by BaltLAAO-I isolated from Bothrops alternatus venom and its possible mechanism of action on tumor cells. Our results clearly depict that BaltLAAO-I has a strong selective cytotoxic activity on tumor cell lines (JURKAT, SK-BR-3 and B16F10). On the other hand, the results show low cytotoxicity on human peripheral blood mononuclear cells. Furthermore, our findings demonstrate that BaltLAAO-I induces the apoptosis of tumor cell lines through a cytotoxic activity exerted by a generation of reactive oxygen intermediates. All in all, the data indicate that LAAOs exert a selective cytotoxic role on tumor cells, demonstrating a great potential for future use in clinical therapy. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Purification and Biochemical Characterization of Three Myotoxins from Bothrops mattogrossensis Snake Venom with Toxicity against Leishmania and Tumor Cells

    PubMed Central

    de Moura, Andréa A.; Kayano, Anderson M.; Oliveira, George A.; Setúbal, Sulamita S.; Ribeiro, João G.; Barros, Neuza B.; Nicolete, Roberto; Moura, Laura A.; Fuly, Andre L.; Nomizo, Auro; da Silva, Saulo L.; Fernandes, Carla F. C.; Zuliani, Juliana P.; Stábeli, Rodrigo G.; Soares, Andreimar M.; Calderon, Leonardo A.

    2014-01-01

    Bothrops mattogrossensis snake is widely distributed throughout eastern South America and is responsible for snakebites in this region. This paper reports the purification and biochemical characterization of three new phospholipases A2 (PLA2s), one of which is presumably an enzymatically active Asp49 and two are very likely enzymatically inactive Lys49 PLA2 homologues. The purification was obtained after two chromatographic steps on ion exchange and reverse phase column. The 2D SDS-PAGE analysis revealed that the proteins have pI values around 10, are each made of a single chain, and have molecular masses near 13 kDa, which was confirmed by MALDI-TOF mass spectrometry. The N-terminal similarity analysis of the sequences showed that the proteins are highly homologous with other Lys49 and Asp49 PLA2s from Bothrops species. The PLA2s isolated were named BmatTX-I (Lys49 PLA2-like), BmatTX-II (Lys49 PLA2-like), and BmatTX-III (Asp49 PLA2). The PLA2s induced cytokine release from mouse neutrophils and showed cytotoxicity towards JURKAT (leukemia T) and SK-BR-3 (breast adenocarcinoma) cell lines and promastigote forms of Leishmania amazonensis. The structural and functional elucidation of snake venoms components may contribute to a better understanding of the mechanism of action of these proteins during envenomation and their potential pharmacological and therapeutic applications. PMID:24724078

  10. Purification and biochemical characterization of three myotoxins from Bothrops mattogrossensis snake venom with toxicity against Leishmania and tumor cells.

    PubMed

    de Moura, Andréa A; Kayano, Anderson M; Oliveira, George A; Setúbal, Sulamita S; Ribeiro, João G; Barros, Neuza B; Nicolete, Roberto; Moura, Laura A; Fuly, Andre L; Nomizo, Auro; da Silva, Saulo L; Fernandes, Carla F C; Zuliani, Juliana P; Stábeli, Rodrigo G; Soares, Andreimar M; Calderon, Leonardo A

    2014-01-01

    Bothrops mattogrossensis snake is widely distributed throughout eastern South America and is responsible for snakebites in this region. This paper reports the purification and biochemical characterization of three new phospholipases A2 (PLA2s), one of which is presumably an enzymatically active Asp49 and two are very likely enzymatically inactive Lys49 PLA2 homologues. The purification was obtained after two chromatographic steps on ion exchange and reverse phase column. The 2D SDS-PAGE analysis revealed that the proteins have pI values around 10, are each made of a single chain, and have molecular masses near 13 kDa, which was confirmed by MALDI-TOF mass spectrometry. The N-terminal similarity analysis of the sequences showed that the proteins are highly homologous with other Lys49 and Asp49 PLA2s from Bothrops species. The PLA2s isolated were named BmatTX-I (Lys49 PLA2-like), BmatTX-II (Lys49 PLA2-like), and BmatTX-III (Asp49 PLA2). The PLA2s induced cytokine release from mouse neutrophils and showed cytotoxicity towards JURKAT (leukemia T) and SK-BR-3 (breast adenocarcinoma) cell lines and promastigote forms of Leishmania amazonensis. The structural and functional elucidation of snake venoms components may contribute to a better understanding of the mechanism of action of these proteins during envenomation and their potential pharmacological and therapeutic applications.

  11. Ontogenetic variations in the venom proteome of the Amazonian snake Bothrops atrox.

    PubMed

    Guércio, Rafael A P; Shevchenko, Anna; Shevchenko, Andrej; López-Lozano, Jorge L; Paba, Jaime; Sousa, Marcelo V; Ricart, Carlos A O

    2006-05-11

    Bothrops atrox is responsible for the majority of snakebite accidents in the Brazilian Amazon region. Previous studies have demonstrated that the biological and pharmacological activities of B. atrox venom alter with the age of the animal. Here, we present a comparative proteome analysis of B. atrox venom collected from specimens of three different stages of maturation: juveniles, sub-adults and adults. Optimized conditions for two-dimensional gel electrophoresis (2-DE) of pooled venom samples were achieved using immobilized pH gradient (IPG) gels of non-linear 3-10 pH range during the isoelectric focusing step and 10-20% gradient polyacrylamide gels in the second dimension. Software-assisted analysis of the 2-DE gels images demonstrated differences in the number and intensity of spots in juvenile, sub-adult and adult venoms. Although peptide mass fingerprinting (PMF) failed to identify even a minor fraction of spots, it allowed us to group spots that displayed similar peptide maps. The spots were subjected to a combination of tandem mass spectrometry and Mascot and MS BLAST database searches that identified several classes of proteins, including metalloproteinases, serine proteinases, lectins, phospholipases A2, L-amino oxidases, nerve growth factors, vascular endothelial growth factors and cysteine-rich secretory proteins. The analysis of B. atrox samples from specimens of different ages by 2-DE and mass spectrometry suggested that venom proteome alters upon ontogenetic development. We identified stage specific and differentially expressed polypeptides that may be responsible for the activities of the venom in each developmental stage. The results provide insight into the molecular basis of the relation between symptomatology of snakebite accidents in humans and the venom composition. Our findings underscore the importance of the use of venoms from individual specimen at various stages of maturation for the production of antivenoms.

  12. Snake venomics of Bothrops punctatus, a semiarboreal pitviper species from Antioquia, Colombia.

    PubMed

    Fernández Culma, Maritza; Andrés Pereañez, Jaime; Núñez Rangel, Vitelbina; Lomonte, Bruno

    2014-01-01

    Bothrops punctatus is an endangered, semi-arboreal pitviper species distributed in Panamá, Colombia, and Ecuador, whose venom is poorly characterized. In the present work, the protein composition of this venom was profiled using the 'snake venomics' analytical strategy. Decomplexation of the crude venom by RP-HPLC and SDS-PAGE, followed by tandem mass spectrometry of tryptic digests, showed that it consists of proteins assigned to at least nine snake toxin families. Metalloproteinases are predominant in this secretion (41.5% of the total proteins), followed by C-type lectin/lectin-like proteins (16.7%), bradykinin-potentiating peptides (10.7%), phospholipases A2 (93%), serine proteinases (5.4%), disintegrins (38%), L-amino acid oxidases (3.1%), vascular endothelial growth factors (17%), and cysteine-rich secretory proteins (1.2%). Altogether, 6.6% of the proteins were not identified. In vitro, the venom exhibited proteolytic, phospholipase A2, and L-amino acid oxidase activities, as well as angiotensin-converting enzyme (ACE)-inhibitory activity, in agreement with the obtained proteomic profile. Cytotoxic activity on murine C2C12 myoblasts was negative, suggesting that the majority of venom phospholipases A2 likely belong to the acidic type, which often lack major toxic effects. The protein composition of B. punctatus venom shows a good correlation with toxic activities here and previously reported, and adds further data in support of the wide diversity of strategies that have evolved in snake venoms to subdue prey, as increasingly being revealed by proteomic analyses.

  13. Thrombelastographic characterization of the thrombin-like activity of Crotalus simus and Bothrops asper venoms.

    PubMed

    Nielsen, Vance G; Boyer, Leslie V; Redford, Daniel T; Ford, Paul

    2017-04-01

    : Annually, thousands suffer venomous snake-bite from Crotalus simus and Bothrops asper vipers in central and South America. The goals of the present study were to generally characterize the thrombin-like effects of venom from these snakes in human plasma with viscoelastic methods. Human plasma was exposed to the venom of three different C. simus subspecies and venoms obtained from B. asper vipers located in three different locations in Mexico. To characterize the factor X-activating and thrombin-like activity of these venoms, plasma (normal or factor XIII deficient) was pretreated with a variety of additives (e.g., heparin) in the absence or presence of calcium prior to exposure to 2.0 μg/ml of each viper's venom. These profiles were compared with plasma without venom that had contact activation of coagulation. Coagulation kinetics were determined with thrombelastography. All venoms had thrombin-like activity, with C. s. simus creating a slow growing, weak clot that was likely mediated by metalloproteinases. In contrast, B. asper venoms had rapid onset of coagulation and a high velocity of thrombus growth. Further, B. asper venom activity was calcium-independent, activated prothrombin, activated factor XIII, and independently polymerized fibrinogen. The viscoelastic methods used were able to differentiate subspecies of C. simus and specimens of B. asper, and provide insight into the mechanisms by which the venoms acted on plasma. These methods may be useful in the profiling of similar venoms and perhaps can assist in the assessment of interventions designed to treat envenomation (e.g., antivenom).

  14. Bothrops jararaca venom metalloproteinases are essential for coagulopathy and increase plasma tissue factor levels during envenomation.

    PubMed

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

    2014-05-01

    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. 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. 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 bleeding manifestations in severely-envenomed patients.

  15. Cell adhesion molecules involved in the leukocyte recruitment induced by venom of the snake Bothrops jararaca.

    PubMed Central

    Zamuner, Stella R; Teixeira, Catarina F P

    2002-01-01

    It has been shown that Bothrops jararaca venom (BjV) induces a significant leukocyte accumulation, mainly neutrophils, at the local of tissue damage. Therefore, the role of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1), LECAM-1, CD18, leukocyte function-associated antigen-1 (LFA-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1) on the BjV-induced neutrophil accumulation and the correlation with release of LTB4, TXA2, tumor necrosis factor-alpha, interleukin (IL)-1 and IL-6 have been investigated. Anti-mouse LECAM-1, LFA-1, ICAM-1 and PECAM-1 monoclonal antibody injection resulted in a reduction of 42%, 80%, 66% and 67%, respectively, of neutrophil accumulation induced by BjV (250 microg/kg, intraperitoneal) injection in male mice compared with isotype-matched control injected animals. The anti-mouse CD18 monoclonal antibody had no significant effect on venom-induced neutrophil accumulation. Concentrations of LTB(4), TXA(2), IL-6 and TNF-alpha were significant increased in the peritoneal exudates of animals injected with venom, whereas no increment in IL-1 was detected. This results suggest that ICAM-1, LECAM-1, LFA-1 and PECAM-1, but not CD18, adhesion molecules are involved in the recruitment of neutrophils into the inflammatory site induced by BjV. This is the first in vivo evidence that snake venom is able to up-regulate the expression of adhesion molecules by both leukocytes and endothelial cells. This venom effect may be indirect, probably through the release of the inflammatory mediators evidenced in the present study. PMID:12581499

  16. A C-Type Lectin from Bothrops jararacussu Venom Disrupts Staphylococcal Biofilms

    PubMed Central

    Klein, Raphael Contelli; Fabres-Klein, Mary Hellen; de Oliveira, Leandro Licursi; Feio, Renato Neves; Malouin, François; Ribon, Andréa de Oliveira Barros

    2015-01-01

    Bovine mastitis is a major threat to animal health and the dairy industry. Staphylococcus aureus is a contagious pathogen that is usually associated with persistent intramammary infections, and biofilm formation is a relevant aspect of the outcome of these infections. Several biological activities have been described for snake venoms, which led us to screen secretions of Bothrops jararacussu for antibiofilm activity against S. aureus NRS155. Crude venom was fractionated by size-exclusion chromatography, and the fractions were tested against S. aureus. Biofilm growth, but not bacterial growth, was affected by several fractions. Two fractions (15 and 16) showed the best activities and were also assayed against S. epidermidis NRS101. Fraction 15 was identified by TripleTOF mass spectrometry as a galactose-binding C-type lectin with a molecular weight of 15 kDa. The lectin was purified from the crude venom by D-galactose affinity chromatography, and only one peak was observed. This pure lectin was able to inhibit 75% and 80% of S. aureus and S. epidermidis biofilms, respectively, without affecting bacterial cell viability. The lectin also exhibited a dose-dependent inhibitory effect on both bacterial biofilms. The antibiofilm activity was confirmed using scanning electron microscopy. A pre-formed S. epidermidis biofilm was significantly disrupted by the C-type lectin in a time-dependent manner. Additionally, the lectin demonstrated the ability to inhibit biofilm formation by several mastitis pathogens, including different field strains of S. aureus, S. hyicus, S. chromogenes, Streptococcus agalactiae, and Escherichia coli. These findings reveal a new activity for C-type lectins. Studies are underway to evaluate the biological activity of these lectins in a mouse mastitis model. PMID:25811661

  17. 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

  18. Venom from the snake Bothrops asper Garman. Purification and characterization of three phospholipases A2

    PubMed Central

    Anagón, Alejandro C.; Molinar, Ricardo R.; Possani, Lourival D.; Fletcher, Paul L.; Cronan, John E.; Julia, Jordi Z.

    1980-01-01

    The water-soluble venom of Bothrops asper Garman (San Juan Evangelista, Veracruz, México) showed 15 polypeptide bands on polyacrylamide-gel electrophoresis. This material exhibited phospholipase, hyaluronidase, N-benzoyl-l-arginine ethyl hydrolase, N-benzoyl-l-tyrosine ethyl hydrolase and phosphodiesterase activity, but no alkaline phosphatase or acid phosphatase activity. Fractionation on Sephadex G-75 afforded seven protein fractions, which were apparently less toxic than the whole venom (LD50=4.3μg/g mouse wt.). Subsequent separation of the phospholipase-positive fraction (II) on DEAE-cellulose with potassium phosphate buffers (pH7.55) gave several fractions, two being phospholipase-positive (II.6 and II.8). These fractions were further purified on DEAE-cellulose columns with potassium phosphate buffers (pH8.6). Fraction II.8.4 was rechromatographed in the same DEAE-cellulose column, giving a pure protein designated phospholipase 1. The fraction II.6.3 was further separated by gel disc electrophoresis yielding two more pure proteins designated phospholipase 2 and phospholipase 3. Analysis of phospholipids hydrolysed by these enzymes have shown that all three phospholipases belong to type A2. Amino acid analysis has shown that phospholipase A2 (type 1) has 97 residues with a calculated mol.wt. of 10978±11. Phospholipase A2 (type 2) has 96 residues with a mol.wt. of 10959±11. Phospholipase A2 (type 3) has 266 residues with 16 half-cystine residues and a calculated mol.wt of 29042±31. Automated Edman degradation showed the N-terminal sequence to be: Asx-Leu-Trp-Glx-Phe-Gly-Glx-Met-Met-Ser-Asx-Val- Met-Arg-Lys-Asx-Val-Val-Phe-Lys-Tyr-Leu- for phospholipase A2 (type 2). ImagesFig. 1. PMID:7387631

  19. A Transcriptomic View of the Proteome Variability of Newborn and Adult Bothrops jararaca Snake Venoms

    PubMed Central

    Zelanis, André; Andrade-Silva, Débora; Rocha, Marisa M.; Furtado, Maria F.; Serrano, Solange M. T.; Junqueira-de-Azevedo, Inácio L. M.; Ho, Paulo Lee

    2012-01-01

    Background Snake bite is a neglected public health problem in communities in rural areas of several countries. Bothrops jararaca causes many snake bites in Brazil and previous studies have demonstrated that the pharmacological activities displayed by its venom undergo a significant ontogenetic shift. Similarly, the venom proteome of B. jararaca exhibits a considerable variation upon neonate to adult transition, which is associated with changes in diet from ectothermic prey in early life to endothermic prey in adulthood. Moreover, it has been shown that the Brazilian commercial antibothropic antivenom, which is produced by immunization with adult venom, is less effective in neutralizing newborn venom effects. On the other hand, venom gland transcripts of newborn snakes are poorly known since all transcriptomic studies have been carried out using mRNA from adult specimens. Methods/Principal Findings Here we analyzed venom gland cDNA libraries of newborn and adult B. jararaca in order to evaluate whether the variability demonstrated for its venom proteome and pharmacological activities was correlated with differences in the structure of toxin transcripts. The analysis revealed that the variability in B. jararaca venom gland transcriptomes is quantitative, as illustrated by the very high content of metalloproteinases in the newborn venom glands. Moreover, the variability is also characterized by the structural diversity of SVMP precursors found in newborn and adult transcriptomes. In the adult transcriptome, however, the content of metalloproteinase precursors considerably diminishes and the number of transcripts of serine proteinases, C-type lectins and bradykinin-potentiating peptides increase. Moreover, the comparison of the content of ESTs encoding toxins in adult male and female venom glands showed some gender-related differences. Conclusions/Significance We demonstrate a substantial shift in toxin transcripts upon snake development and a marked decrease in the

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

    PubMed Central

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

    2011-01-01

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

  1. Systemic effects induced by the venom of the snake Bothrops caribbaeus in a murine model.

    PubMed

    Herrera, Cristina; Rucavado, Alexandra; Warrell, David A; Gutiérrez, José María

    2013-03-01

    Snakebite envenoming by Bothrops caribbaeus, an endemic viperid from the Lesser Antillean island of Saint Lucia, is clinically characterized by local tissue damage and systemic thrombosis that can lead to cerebral, myocardial or pulmonary infarctions and venous thromboses. Systemic effects (lethality, pulmonary hemorrhage, thrombocytopenia and coagulopathy) induced by intravenous (i.v.) administration of B. caribbaeus venom were studied in mice. The role of snake venom metalloproteinases (SVMPs) in these systemic alterations was assessed by inhibition with the chelating agent calcium disodium ethylenediaminetetraacetic acid (CaNa(2)EDTA). A snake C-type lectin-like (snaclec) and a type P-III hemorrhagic SVMP were isolated and characterized from this venom, and the effect of venom and the isolated snaclec on human platelet aggregation was studied in vitro. Results indicate that SVMPs play an important role in the overall toxicity of B. caribbaeus venom, being responsible for systemic hemorrhage and lethality, but not thrombocytopenia, whereas the isolated snaclec is involved in the thrombocytopenic effect. Both venom and snaclec induce platelet aggregation/agglutination. Moreover, the snaclec binds directly to glycoprotein Ib (GPIb) and induces agglutination in washed fixed platelets. On the other hand, B. caribbaeus venom hydrolyzed fibrinogen in vitro and induced a partial drop of fibrinogen levels with an increase in fibrin/fibrinogen degradation products (FDP) levels in vivo. The negative result for D-dimer (DD) in plasma is consistent with the lack of microscopic evidence of pulmonary thrombosis and endothelial cell damage. Likewise, no increments in plasma sE-selectin levels were detected. The absence of thrombosis in this murine model suggests that this effect may be species-specific. Copyright © 2012 Elsevier Ltd. All rights reserved.

  2. BbrzSP-32, the first serine protease isolated from Bothrops brazili venom: Purification and characterization.

    PubMed

    Zaqueo, Kayena D; Kayano, Anderson M; Domingos, Thaisa F S; Moura, Laura A; Fuly, André L; da Silva, Saulo L; Acosta, Gerardo; Oliveira, Eliandre; Albericio, Fernando; Zanchi, Fernando B; Zuliani, Juliana P; Calderon, Leonardo A; Stábeli, Rodrigo G; Soares, Andreimar M

    2016-05-01

    Snake venom toxins are related not only in detention, death and the promotion of initial digestion of prey but also due to their different biochemical, structural and pharmacological effects they can result in new drugs. Among these toxins snake venom serine proteases (SVSPs) should be highlighted because they are responsible for inducing changes in physiological functions such as blood coagulation, fibrinolysis, and platelet aggregation. This article presents the first serine protease (SP) isolated from Bothrops brazili: BbrzSP-32. The new SP showed 36 kDa of relative molecular mass and its absolute mass was confirmed by mass spectrometry as 32,520 Da. It presents 79.48% identity when compared to other SVSPs and was able to degrade the α-chain of fibrinogen, in in vitro models, because of this it is considered a SVTLE-A. It showed dose-dependent activity in the process of degradation of fibrin networks demonstrating greater specificity for this activity when compared to its thrombolytic action. BbrzSP-32 demonstrated proteolytic activity on gelatin and chromogenic substrates for serine proteases and thrombin-like enzymes (S-2288 and S-2238 respectively), besides having coagulant activity on human plasma. After pre-incubation with PMSF and benzamidine the coagulant and proteolytic activities on the S-2288 and S-2238 substrates were reduced. BbrzSP-32 shows stability against pH and temperature variations, demonstrating optimum activity between 30 and 40 °C and in the pH range 7.5 to 8.5. A new SP with potential biotechnological application was isolated. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. A prothrombin activator from Bothrops erythromelas (jararaca-da-seca) snake venom: characterization and molecular cloning.

    PubMed

    Silva, Márcia B; Schattner, Mirta; Ramos, Celso R R; Junqueira-de-Azevedo, Inácio L M; Guarnieri, Míriam C; Lazzari, María A; Sampaio, Claudio A M; Pozner, Roberto G; Ventura, Janaina S; Ho, Paulo L; Chudzinski-Tavassi, Ana M

    2003-01-01

    A novel prothrombin activator enzyme, which we have named 'berythractivase', was isolated from Bothrops erythromelas (jararaca-da-seca) snake venom. Berythractivase was purified by a single cation-exchange-chromatography step on a Resource S (Amersham Biosciences) column. The overall purification (31-fold) indicates that berythractivase comprises about 5% of the crude venom. It is a single-chain protein with a molecular mass of 78 kDa. SDS/PAGE of prothrombin after activation by berythractivase showed fragment patterns similar to those generated by group A prothrombin activators, which convert prothrombin into meizothrombin, independent of the prothrombinase complex. Chelating agents, such as EDTA and o -phenanthroline, rapidly inhibited the enzymic activity of berythractivase, like a typical metalloproteinase. Human fibrinogen A alpha-chain was slowly digested only after longer incubation with berythractivase, and no effect on the beta- or gamma-chains was observed. Berythractivase was also capable of triggering endothelial proinflammatory and procoagulant cell responses. von Willebrand factor was released, and the surface expression of both intracellular adhesion molecule-1 and E-selectin was up-regulated by berythractivase in cultured human umbilical-vein endothelial cells. The complete berythractivase cDNA was cloned from a B. erythromelas venom-gland cDNA library. The cDNA sequence possesses 2330 bp and encodes a preproprotein with significant sequence similarity to many other mature metalloproteinases reported from snake venoms. Berythractivase contains metalloproteinase, desintegrin-like and cysteine-rich domains. However, berythractivase did not elicit any haemorrhagic response. These results show that, although the primary structure of berythractivase is related to that of snake-venom haemorrhagic metalloproteinases and functionally similar to group A prothrombin activators, it is a prothrombin activator devoid of haemorrhagic activity. This is a feature

  4. Snakebites by Bothrops spp in children in Campinas, São Paulo, Brazil.

    PubMed

    Bucaretchi, F; Herrera, S R; Hyslop, S; Baracat, E C; Vieira, R J

    2001-01-01

    From January, 1984 to March, 1999, 73 children under 15 y old (ages 1-14 y, median 9 y) were admitted after being bitten by snakes of the genus Bothrops. Twenty-six percent of the children were classified as mild envenoming, 50.7% as moderate envenoming and 20.6% as severe envenoming. Two patients (2.7%) showed no signs of envenoming. Most of the patients presented local manifestations, mainly edema (94.5%), pain (94.5%) ecchymosis (73.9%) and blisters (11%). Local and/or systemic bleeding was observed in 28.8% of the patients. Before antivenom (AV) administration, blood coagulation disorders were observed in 60.7% (incoagulable blood in 39.3%) of the 56 children that received AV only in our hospital. AV early reactions, most of which were considered mild, were observed in 44.6% of these cases (in 15/30 patients not pretreated and in 10/26 patients pretreated with hydrocortisone and histamine H1 and H2 antagonists). The main clinical complications observed were local infection (15.1%), compartment syndrome (4.1%), gangrene (1.4%) and acute renal failure (1.4%). No deaths were recorded. There were no significant differences with regard to severity of envenoming versus the frequency of blood coagulation disorders among the three categories of envenoming (p = 0.75) or in the frequency of patients with AV early reactions between the groups that were and were not pretreated (p = 0.55). The frequency of local infection was significantly greater in severe cases (p < 0.001). Patients admitted more than 6 h after the bite had a higher risk of developing severe envenoming (p = 0.04).

  5. Snake venomics of Bothrops punctatus, a semiarboreal pitviper species from Antioquia, Colombia

    PubMed Central

    Fernández Culma, Maritza; Andrés Pereañez, Jaime; Núñez Rangel, Vitelbina

    2014-01-01

    Bothrops punctatus is an endangered, semi-arboreal pitviper species distributed in Panamá, Colombia, and Ecuador, whose venom is poorly characterized. In the present work, the protein composition of this venom was profiled using the ‘snake venomics’ analytical strategy. Decomplexation of the crude venom by RP-HPLC and SDS-PAGE, followed by tandem mass spectrometry of tryptic digests, showed that it consists of proteins assigned to at least nine snake toxin families. Metalloproteinases are predominant in this secretion (41.5% of the total proteins), followed by C-type lectin/lectin-like proteins (16.7%), bradykinin-potentiating peptides (10.7%), phospholipases A2 (93%), serine proteinases (5.4%), disintegrins (38%), L-amino acid oxidases (3.1%), vascular endothelial growth factors (17%), and cysteine-rich secretory proteins (1.2%). Altogether, 6.6% of the proteins were not identified. In vitro, the venom exhibited proteolytic, phospholipase A2, and L-amino acid oxidase activities, as well as angiotensin-converting enzyme (ACE)-inhibitory activity, in agreement with the obtained proteomic profile. Cytotoxic activity on murine C2C12 myoblasts was negative, suggesting that the majority of venom phospholipases A2 likely belong to the acidic type, which often lack major toxic effects. The protein composition of B. punctatus venom shows a good correlation with toxic activities here and previously reported, and adds further data in support of the wide diversity of strategies that have evolved in snake venoms to subdue prey, as increasingly being revealed by proteomic analyses. PMID:24498576

  6. Effects of Bothrops asper Snake Venom on Lymphatic Vessels: Insights into a Hidden Aspect of Envenomation

    PubMed Central

    Mora, Javier; Mora, Rodrigo; Lomonte, Bruno; Gutiérrez, José María

    2008-01-01

    Background Envenomations by the snake Bothrops asper represent a serious medical problem in Central America and parts of South America. These envenomations concur with drastic local tissue pathology, including a prominent edema. Since lymph flow plays a role in the maintenance of tissue fluid balance, the effect of B. asper venom on collecting lymphatic vessels was studied. Methodology/Principal Findings B. asper venom was applied to mouse mesentery, and the effects were studied using an intravital microscopy methodology coupled with an image analysis program. B. asper venom induced a dose-dependent contraction of collecting lymphatic vessels, resulting in a reduction of their lumen and in a halting of lymph flow. The effect was reproduced by a myotoxic phospholipase A2 (PLA2) homologue isolated from this venom, but not by a hemorrhagic metalloproteinase or a coagulant thrombin-like serine proteinase. In agreement with this, treatment of the venom with fucoidan, a myotoxin inhibitor, abrogated the effect, whereas no inhibition was observed after incubation with the peptidomimetic metalloproteinase inhibitor Batimastat. Moreover, fucoidan significantly reduced venom-induced footpad edema. The myotoxic PLA2 homologue, known to induce skeletal muscle necrosis, was able to induce cytotoxicity in smooth muscle cells in culture and to promote an increment in the permeability to propidium iodide in these cells. Conclusions/Significance Our observations indicate that B. asper venom affects collecting lymphatic vessels through the action of myotoxic PLA2s on the smooth muscle of these vessels, inducing cell contraction and irreversible cell damage. This activity may play an important role in the pathogenesis of the pronounced local edema characteristic of viperid snakebite envenomation, as well as in the systemic biodistribution of the venom, thus representing a potential therapeutical target in these envenomations. PMID:18923712

  7. Bothrops protease A, a unique highly glycosylated serine proteinase, is a potent, specific fibrinogenolytic agent.

    PubMed

    Paes Leme, A F; Prezoto, B C; Yamashiro, E T; Bertholim, L; Tashima, A K; Klitzke, C F; Camargo, A C M; Serrano, S M T

    2008-08-01

    The hemostatic system is the major target of snake venom serine proteinases (SVSPs) that act on substrates of the coagulation, fibrinolytic and kallikrein-kinin systems. Bothrops protease A (BPA), the most glycosylated SVSP, is a non-coagulant, thermostable enzyme. A cDNA encoding BPA showed that the protein has a calculated molecular mass of 25 409 Da, implying that approximately 62% of its molecular mass as assessed by sodium dodecylsulfate polyacrylamide gel electrophoresis (67 kDa) is due to carbohydrate moieties. Here we show that BPA is a potent fibrinogenolytic agent in vitro, as it readily degraded human and rat fibrinogen at a very low enzyme concentration. Partially N-deglycosylated BPA (p-N-d-BPA) generated similar fibrinogen products, but with enhanced fibrinogenolytic activity. In vivo, injection of 0.75 nmoles of BPA in rats completely avoided thrombus formation induced by stasis in the vena cava, or by endothelium injury in the jugular vein. Moreover, it decreased the fibrinogen plasma level and prolonged the recalcification time. Cleavage of fibrinogen in human and rat plasma was observed with native BPA and p-N-d-BPA by electrophoresis followed by western blot using an anti-fibrinogen antibody. BPA did not cause unspecific degradation of plasma proteins and did not cleave isolated albumin, vitronectin and fibronectin at the same concentration used with fibrinogen. Serine proteinase inhibitors failed to inhibit BPA, probably due to steric hindrance caused by its huge carbohydrate moieties. To the best of our knowledge, this investigation underscores a new, thermostable, specific defibrinogenating agent that may have an application in the prevention of thrombus formation.

  8. Annual changes in seminal variables of golden lanchead pitvipers (Bothrops insularis) maintained in captivity.

    PubMed

    Silva, K B; Zogno, M A; Camillo, A B; Pereira, R J G; Almeida-Santos, S M

    2015-12-01

    Bothrops insularis is an endemic and critically endangered snake with an estimated population of 2000 individuals restricted to Queimada Grande Island, in southeastern Brazil. Brazilian researchers established a captive breeding program for the species that includes the application of assisted reproductive technologies. The present study, therefore, aimed to evaluate semen samples from captive B. insularis throughout the year to ascertain seasonal differences in semen traits as well as correlations with body size and weight. Eighteen males with snout-vent length (SVL) ranging from 43.5 to 73.7 cm were collected at quarterly basis between August 2012 and May 2013. Macroscopic analysis revealed semen volumes ranging from 0.5 to 6.0 μL with samples featuring whitish to yellowish color and creamy and thick consistency. Viable sperm was obtained from all males indicating that individuals with SVL equal to or greater than 43.5 cm are sexually developed. However, adult and immature males (estimated by SVL) exhibited different seasonal profiles for motility and progressive motility. Adult males had a decrease in sperm motility and progressive motility during summer and spring, respectively, whereas the same variables did not vary throughout the year in immature snakes. Sperm concentration in all individuals was less (0.5 × 10(9) μL) during the winter, but no seasonal fluctuations were detected in semen volume. These findings are of particular importance to the development of reproductive tools such as male selection, artificial insemination and sperm freezing for the genetic management of this critically endangered snake. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. 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

  10. Snake venomics of the lancehead pitviper Bothrops asper: geographic, individual, and ontogenetic variations.

    PubMed

    Alape-Girón, Alberto; Sanz, Libia; Escolano, José; Flores-Díaz, Marietta; Madrigal, Marvin; Sasa, Mahmood; Calvete, Juan J

    2008-08-01

    We report the comparative proteomic characterization of the venoms of adult and newborn specimens of the lancehead pitviper Bothrops asper from two geographically isolated populations from the Caribbean and the Pacific versants of Costa Rica. The crude venoms were fractionated by reverse-phase HPLC, followed by analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The two B. asper populations, separated since the late Miocene or early Pliocene (8-5 mya) by the Guanacaste Mountain Range, Central Mountain Range, and Talamanca Mountain Range, contain both identical and different (iso)enzymes from the PLA 2, serine proteinase, and SVMP families. Using a similarity coefficient, we estimate that the similarity of venom proteins between the two B. asper populations may be around 52%. Compositional differences between venoms among different geographic regions may be due to evolutionary environmental pressure acting on isolated populations. To investigate venom variability among specimens from the two B. asper populations, the reverse-phase HPLC protein profiles of 15 venoms from Caribbean specimens and 11 venoms from snakes from Pacific regions were compared. Within each B. asper geographic populations, all major venom protein families appeared to be subjected to individual variations. The occurrence of intraspecific individual allopatric variability highlights the concept that a species, B. asper in our case, should be considered as a group of metapopulations. Analysis of pooled venoms of neonate specimens from Caribbean and Pacific regions with those of adult snakes from the same geographical habitat revealed prominent ontogenetic changes in both geographical populations. Major ontogenetic changes appear to be a shift from a PIII-SVMP-rich to a PI-SVMP-rich venom and the secretion in adults of a distinct set of PLA 2 molecules than in

  11. Suramin counteracts the haemostatic disturbances produced by Bothrops jararaca snake venom.

    PubMed

    Fernandes, Renato S; Assafim, Mariane; Arruda, Emerson Z; Melo, Paulo A; Zingali, Russolina B; Monteiro, Robson Q

    2007-06-01

    Snakebite accidents produced by Bothrops jararaca typically results in haemostatic changes including pro- and anticoagulant disturbs as well as interference with platelets. Suramin is a hexasulfonated naphthylurea derivative that was recently characterized as a thrombin inhibitor (Monteiro et al., 2004. Suramin interaction with human alpha-thrombin: inhibitory effects and binding studies. Int. J. Biochem. Cell Biol. 36(10), 2077-2085). Here, we evaluated the ability of suramin to counteract some of the haemostatic disturbs produced by B. jararaca venom. In vitro assays showed that suramin inhibited venom-induced hydrolysis of a number of synthetic substrates: S-2238, S-2266, S-2302 and S-2288, being this ability more prominent towards the thrombin substrate S-2238 (IC(50)=4.3 microM). It was also observed that suramin impaired the fibrinogen clotting induced by B. jararaca venom (IC(50)=124 microM). Accordingly, increasing concentrations of suramin progressively delayed venom-induced plasma clotting, with complete inhibition attained at concentrations above 1.0 mM. In addition, the platelet-aggregating properties of B. jararaca venom were inhibited by suramin in a dose-dependent fashion (IC(50)=127 microM). Suramin showed no effect in the in vivo hemorrhagic effect of venom in mouse skin. The in vivo effect of suramin was further tested using a previously established venous thrombosis model in rats induced by intravenous administration of B. jararaca venom combined with stasis. Venom doses of 100 microg/kg produced 100% of thrombus incidence (10.6+/-1.7 mg). On the other hand, previous administration of suramin partially inhibited thrombus formation. Thus, 12.5 or 25 mg/kg of suramin decreased thrombus weight by 24% and 40%, respectively. Remarkably, co-administration of 3 microL/kg of antibothropic serum (which has no effect on thrombus formation) and 12.5 mg/kg of suramin decreased thrombus weight by 75%, suggesting a synergic effect. Altogether, we demonstrate

  12. Edema induced by Bothrops asper (Squamata: Viperidae) snake venom and its inhibition by Costa Rican plant extracts.

    PubMed

    Badilla, Beatriz; Chaves, Fernando; Mora, Gerardo; Poveda, Luis J

    2006-06-01

    We tested the capacity of leaf (Urera baccifera, Loasa speciosa, Urtica leptuphylla, Chaptalia nutans, and Satureja viminea) and root (Uncaria tomentosa) extracts to inhibit edema induced by Bothrops asper snake venom. Edema-forming activity was studied plethysmographically in the rat hind paw model. Groups of rats were injected intraperitoneally with various doses of each extract and, one hour later, venom was injected subcutaneously in the right hind paw. Edema was assessed at various time intervals. The edematogenic activity was inhibited in those animals that received an injection U. tomentosa, C. nutans or L. speciosa extract. The extract of U. baccifera showed a slight inhibition of the venom effect. Extract from S. viminea and, to a lesser extent that of U. leptuphylla, induced a pro-inflammatory effect, increasing the edema at doses of 250 mg/kg at one and two hours.

  13. Description of Serpentirhabdias atroxi n. sp. (Nematoda: Rhabdiasidae), a parasite of Bothrops atrox (Linnaeus) (Reptilia: Serpentes: Viperidae) in Brazilian Amazonia.

    PubMed

    Kuzmin, Yuriy; Giese, Elane Guerreiro; Melo, Francisco Tiago de Vasconcelos; da Costa, Paulo André Ferreira Borges; Maschio, Gleomar Fabiano; dos Santos, Jeannie Nascimento

    2016-01-01

    A new lung-dwelling nematode species is described from the common lancehead Bothrops atrox (Linnaeus) in the Brazilian Amazon Region. The species is assigned to the genus Serpentirhabdias Tkach, Kuzmin & Snyder, 2014 based on the presence of six lips arranged in two lateral groups, the absence of prominent cuticular inflations, and lung parasitism in snakes. Serpentirhabdias atroxi n. sp. differs from other species of the genus mainly by details of the morphology of the anterior end: cuticularised ring surrounding the anterior part of the buccal cavity and six minute onchia present in the oesophastome. Serpentirhabdias atroxi n. sp. is the seventh species of the genus known from the Neotropical Realm and the second species described from viperid snakes.

  14. 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

    2016-04-04

    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.

  15. ESI-MS/MS identification of a bradykinin-potentiating peptide from Amazon Bothrops atrox snake venom using a hybrid Qq-oaTOF mass spectrometer.

    PubMed

    Coutinho-Neto, Antonio; Caldeira, Cleópatra A S; Souza, Gustavo H M F; Zaqueo, Kayena D; Kayano, Anderson M; Silva, Rodrigo S; Zuliani, Juliana P; Soares, Andreimar M; Stábeli, Rodrigo G; Calderon, Leonardo A

    2013-02-18

    A bradykinin-potentiating peptide (BPP) from Amazon Bothrops atrox venom with m/z 1384.7386 was identified and characterized by collision induced dissociation (CID) using an ESI-MS/MS spectra obtained in positive ion mode on a hybrid Qq-oaTOF mass spectrometer, Xevo G2 QTof MS (Waters, Manchester, UK). De novo peptide sequence analysis of the CID fragmentation spectra showed the amino acid sequence ZKWPRPGPEIPP, with a pyroglutamic acid and theoretical monoisotopic m/z 1384.7378, which is similar to experimental data, showing a mass accuracy of 0.6 ppm. The peptide is homologous to other BPP from Bothrops moojeni and was named as BPP-BAX12.

  16. Synergism between baltergin metalloproteinase and Ba SPII RP4 PLA2 from Bothrops alternatus venom on skeletal muscle (C2C12) cells.

    PubMed

    Bustillo, Soledad; Gay, Claudia C; García Denegri, María E; Ponce-Soto, Luis A; Bal de Kier Joffé, Elisa; Acosta, Ofelia; Leiva, Laura C

    2012-02-01

    Acute muscle damage, myonecrosis, is one of the main characteristics of envenoming by Bothrops genus. In this in vitro study we investigated the role of a metalloproteinase (baltergin) and an acidic phospholipase A2 (Ba SPII RP4) in the cytotoxicity exhibited by Bothrops alternatus venom. Baltergin metalloproteinase purified from the venom exerted a toxic effect on C2C12 myoblast cells (CC50: 583.34 μg/mL) which involved morphological alterations compatible with apoptosis/anoikis. On the contrary, the most abundant PLA2 isolated from this venom did not exhibit cytotoxicity at times and doses tested. However, when myoblasts were treated with both enzymes together, synergic activity was demonstrated. Neutralization of the venom with specific antibodies (IgG anti-baltergin and IgG anti-PLA2) confirmed this synergism. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Biochemical Characterization, Action on Macrophages, and Superoxide Anion Production of Four Basic Phospholipases A2 from Panamanian Bothrops asper Snake Venom

    PubMed Central

    Rueda, Aristides Quintero; Rodríguez, Isela González; Arantes, Eliane C.; Setúbal, Sulamita S.; Calderon, Leonardo de A.; Zuliani, Juliana P.; Stábeli, Rodrigo G.; Soares, Andreimar M.

    2013-01-01

    Bothrops asper (Squamata: Viperidae) is the most important venomous snake in Central America, being responsible for the majority of snakebite accidents. Four basic PLA2s (pMTX-I to -IV) were purified from crude venom by a single-step chromatography using a CM-Sepharose ion-exchange column (1.5 × 15 cm). Analysis of the N-terminal sequence demonstrated that pMTX-I and III belong to the catalytically active Asp49 phospholipase A2 subclass, whereas pMTX-II and IV belong to the enzymatically inactive Lys49 PLA2s-like subclass. The PLA2s isolated from Panama Bothrops asper venom (pMTX-I, II, III, and IV) are able to induce myotoxic activity, inflammatory reaction mainly leukocyte migration to the muscle, and induce J774A.1 macrophages activation to start phagocytic activity and superoxide production. PMID:23509779

  18. Lachesis muta muta venom: immunological differences compared with Bothrops atrox venom and importance of specific antivenom therapy.

    PubMed

    Colombini, M; Fernandes, I; Cardoso, D F; Moura-da-Silva, A M

    2001-05-01

    Lachesis muta muta and Bothrops atrox snakes are responsible for most accidents occurring in the Amazon. The clinical features of the accidents are similar; however, there are still controversies about the efficacy of Bothrops antivenoms for treating L. m. muta accidents. In this work, we evaluated the antigenic cross-reactivity between these venoms using polyclonal and monoclonal antibodies and the efficacy of B. atrox and L. m. muta experimental antivenoms in cross-neutralizing the main toxic activities of each venom. Electrophoretic patterns differed consistently between the species. However, antigenic cross-reactivity was extensive except for a few bands. Several species-specific monoclonal antibodies were obtained by immunization of Balb/c mice with L. m. muta whole venom or B. atrox and L. m. muta specific antigens. The monoclonal antibodies specific to L. m. muta recognized different bands of this venom and the antibodies specific to B. atrox recognized a complex pattern on whole venom by Western blotting. These antibodies are important tools for developing an immunoassay able to discriminate patients bitten by these snakes. The experiments involving cross-neutralization of the main activities of the venoms showed that hemorrhage and blood incoagulability induced by B. atrox venom were similarly neutralized by both B. atrox and L. m. muta antivenoms. However, B. atrox antivenom partially neutralized the hemorrhage and completely failed in neutralizing coagulopathy induced by L. m. muta venom. Therefore, antigenic variation between B. atrox and L. m. muta venoms does occur and the use of specific antivenom is suggested for patients bitten by Lachesis snakes.

  19. Inhibitory effects of ascorbic acid, vitamin E, and vitamin B-complex on the biological activities induced by Bothrops venom.

    PubMed

    Oliveira, Carlos Henrique de Moura; Assaid Simão, Anderson; Marcussi, Silvana

    2016-01-01

    Natural compounds have been widely studied with the aim of complementing antiophidic serum therapy. The present study evaluated the inhibitory potential of ascorbic acid and a vitamin complex, composed of ascorbic acid, vitamin E, and all the B-complex vitamins, on the biological activities induced by snake venoms. The effect of vitamins was evaluated on the phospholipase, proteolytic, coagulant, and fibrinogenolytic activities induced by Bothrops moojeni (Viperidae), B. jararacussu, and B. alternatus snake venoms, and the hemagglutinating activity induced by B. jararacussu venom. The vitamin complex (1:5 and 1:10 ratios) totally inhibited the fibrinogenolytic activity and partially the phospholipase activity and proteolytic activity on azocasein induced by the evaluated venoms. Significant inhibition was observed in the coagulation of human plasma induced by venoms from B. alternatus (1:2.5 and 1:5, to vitamin complex and ascorbic acid) and B. moojeni (1:2.5 and 1:5, to vitamin complex and ascorbic acid). Ascorbic acid inhibited 100% of the proteolytic activities of B. moojeni and B. alternatus on azocasein, at 1:10 ratio, the effects of all the venoms on fibrinogen, the hemagglutinating activity of B. jararacussu venom, and also extended the plasma coagulation time induced by all venoms analyzed. The vitamins analyzed showed relevant in vitro inhibitory potential over the activities induced by Bothrops venoms, suggesting their interaction with toxins belonging to the phospholipase A2, protease, and lectin classes. The results can aid further research in clarifying the possible mechanisms of interaction between vitamins and snake enzymes.

  20. 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

  1. Preliminary assessment of Hedychium coronarium essential oil on fibrinogenolytic and coagulant activity induced by Bothrops and Lachesis snake venoms

    PubMed Central

    2014-01-01

    Background The search for new inhibitors of snake venom toxins is essential to complement or even replace traditional antivenom therapy, especially in relation to compounds that neutralize the local effects of envenomations. Besides their possible use as alternative to traditional antivenom therapy, some plant species possess bioactive secondary metabolites including essential oils, which can be extracted from weeds that are considered substantial problems for agriculture, such as Hedychium coronarium. Methods The essential oils of leaves and rhizomes from H. coronarium were extracted by hydrodistillation, and their potential inhibitory effects on the coagulant and fibrinogenolytic activities induced by the venoms of Lachesis muta, Bothrops atrox and Bothrops moojeni were analyzed. Citrated human plasma was used to evaluate the clotting time whereas changes in fibrinogen molecules were visualized by electrophoresis in polyacrylamide gel. The experimental design used for testing coagulation inhibition was randomized in a 3 × 2 factorial arrangement (concentration × essential oils), with three replications. The essential oils were compared since they were extracted from different organs of the same botanical species, H. coronarium. Results The results suggest that the oils interact with venom proteases and plasma constituents, since all oils evaluated, when previously incubated with venoms, were able to inhibit the clotting effect, with less inhibition when oils and plasma were preincubated prior to the addition of venoms. Conclusions Thus, after extensive characterization of their pharmacological and toxicological effects, the essential oils can be used as an alternative to complement serum therapy, especially considering that these plant metabolites generally do not require specific formulations and may be used topically immediately after extraction. PMID:26413083

  2. Aqueous leaf extract of Jatropha gossypiifolia L. (Euphorbiaceae) inhibits enzymatic and biological actions of Bothrops jararaca snake venom.

    PubMed

    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

  3. Neutralization of pharmacological and toxic activities of bothrops snake venoms by Schizolobium parahyba (Fabaceae) aqueous extract and its fractions.

    PubMed

    Vale, Luis Henrique F; Mendes, Mirian M; Hamaguchi, Amélia; Soares, Andreimar M; Rodrigues, Veridiana M; Homsi-Brandeburgo, Maria Inês

    2008-07-01

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

  4. Bothrops asper snake venom and its metalloproteinase BaP-1 activate the complement system. Role in leucocyte recruitment.

    PubMed Central

    Farsky, S H; Gonçalves, L R; Gutiérrez, J M; Correa, A P; Rucavado, A; Gasque, P; Tambourgi, D V

    2000-01-01

    The venom of the snake Bothrops asper, the most important poisonous snake in Central America, evokes an inflammatory response, the mechanisms of which are not well characterized. The objectives of this study were to investigate whether B. asper venom and its purified toxins--phospholipases and metalloproteinase--activate the complement system and the contribution of the effect on leucocyte recruitment. In vitro chemotaxis assays were performed using Boyden's chamber model to investigate the ability of serum incubated with venom and its purified toxins to induce neutrophil migration. The complement consumption by the venom was evaluated using an in vitro haemolytic assay. The importance of complement activation by the venom on neutrophil migration was investigated in vivo by injecting the venom into the peritoneal cavity of C5-deficient mice. Data obtained demonstrated that serum incubated with crude venom and its purified metalloproteinase BaP-1 are able to induce rat neutrophil chemotaxis, probably mediated by agent(s) derived from the complement system. This hypothesis was corroborated by the capacity of the venom to activate this system in vitro. The involvement of C5a in neutrophil chemotaxis induced by venom-activated serum was demonstrated by abolishing migration when neutrophils were pre-incubated with antirat C5a receptor antibody. The relevance of the complement system in in vivo leucocyte mobilization was further demonstrated by the drastic decrease of this response in C5-deficient mice. Pre-incubation of serum with the soluble human recombinant complement receptor type 1 (sCR 1) did not prevent the response induced by the venom, but abolished the migration evoked by metalloproteinase-activated serum. These data show the role of the complement system in bothropic envenomation and the participation of metalloproteinase in the effect. Also, they suggest that the venom may contain other component(s) which can cause direct activation of C5a. PMID:11200361

  5. Analgesic Effect of Photobiomodulation on Bothrops Moojeni Venom-Induced Hyperalgesia: A Mechanism Dependent on Neuronal Inhibition, Cytokines and Kinin Receptors Modulation

    PubMed Central

    Oliveira, Victoria Regina da Silva; Toniolo, Elaine Flamia; Feliciano, Regiane dos Santos; da Silva Jr., José Antonio; Zamuner, Stella Regina

    2016-01-01

    Background Envenoming induced by Bothrops snakebites is characterized by drastic local tissue damage that involves an intense inflammatory reaction and local hyperalgesia which are not neutralized by conventional antivenom treatment. Herein, the effectiveness of photobiomodulation to reduce inflammatory hyperalgesia induced by Bothrops moojeni venom (Bmv), as well as the mechanisms involved was investigated. Methodology/Principal Findings Bmv (1 μg) was injected through the intraplantar route in the right hind paw of mice. Mechanical hyperalgesia and allodynia were evaluated by von Frey filaments at different time points after venom injection. Low level laser therapy (LLLT) was applied at the site of Bmv injection at wavelength of red 685 nm with energy density of 2.2 J/cm2 at 30 min and 3 h after venom inoculation. Neuronal activation in the dorsal horn spinal cord was determined by immunohistochemistry of Fos protein and the mRNA expression of IL-6, TNF-α, IL-10, B1 and B2 kinin receptors were evaluated by Real time-PCR 6 h after venom injection. Photobiomodulation reversed Bmv-induced mechanical hyperalgesia and allodynia and decreased Fos expression, induced by Bmv as well as the mRNA levels of IL-6, TNF-α and B1 and B2 kinin receptors. Finally, an increase on IL-10, was observed following LLLT. Conclusion/Significance These data demonstrate that LLLT interferes with mechanisms involved in nociception and hyperalgesia and modulates Bmv-induced nociceptive signal. The use of photobiomodulation in reducing local pain induced by Bothropic venoms should be considered as a novel therapeutic tool for the treatment of local symptoms induced after bothropic snakebites. PMID:27749899

  6. Effect of monospecific antibodies against baltergin in myotoxicity induced by Bothrops alternatus venom from northeast of Argentina. Role of metalloproteinases in muscle damage.

    PubMed

    Gay, Carolina; Maruñak, Silvana; Teibler, Pamela; Leiva, Laura; Acosta, Ofelia

    2013-03-01

    Myotoxicity, one of the most relevant local manifestations in envenomation by Bothrops genus, may result from a direct action of myotoxins or be due to an indirect vascular degeneration and ischemia. Baltergin, a snake venom metalloproteinase (SVMP), isolated from Bothrops alternatus venom has been used to obtain monospecific IgG, in order to determine the relative role of toxin in myotoxicity induced by whole venom. Bothrops diporus venom, another medical relevant genus of the northeastern region of Argentina, was also studied. Anti-baltergin IgG was able to neutralize completely the hemorrhagic activity of B. alternatus venom at an antibodies:venom ratio of 30:1 (w:w). However, mice injected with B. diporus venom showed a small spot remaining even at the highest ratio of IgG:venom assayed (50:1; w:w). Specific antibodies were efficient to neutralize the myotoxicity of B. alternatus venom at ratio 30:1 (w:w) but did not neutralize the same effects in B. diporus venom. Anti-baltergin polyclonal antibodies were useful tools for revealing the central role of SVMPs in the development of myotoxicity of B. alternatus venom, as well as, helping to suggest indirectly presence of potent myotoxic phospholipases A2 (PLA2s) in B. diporus venom. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Preliminary X-ray crystallographic studies of BthTX-II, a myotoxic Asp49-phospholipase A{sub 2} with low catalytic activity from Bothrops jararacussu venom

    SciTech Connect

    Corrêa, L. C.; Marchi-Salvador, D. P.; Cintra, A. C. O.; Soares, A. M.

    2006-08-01

    A myotoxic Asp49-PLA{sub 2} with low catalytic activity from B. jararacussu (BthTX-II) was crystallized in the monoclinic crystal system; a complete X-ray diffraction data set was collected and a molecular-replacement solution was obtained. The oligomeric structure of BthTX-II resembles those of the Asp49-PLA{sub 2} PrTX-III and all bothropic Lys49-PLA{sub 2}s. For the first time, a complete X-ray diffraction data set has been collected from a myotoxic Asp49-phospholipase A{sub 2} (Asp49-PLA{sub 2}) with low catalytic activity (BthTX-II from Bothrops jararacussu venom) and a molecular-replacement solution has been obtained with a dimer in the asymmetric unit. The quaternary structure of BthTX-II resembles the myotoxin Asp49-PLA{sub 2} PrTX-III (piratoxin III from B. pirajai venom) and all non-catalytic and myotoxic dimeric Lys49-PLA{sub 2}s. In contrast, the oligomeric structure of BthTX-II is different from the highly catalytic and non-myotoxic BthA-I (acidic PLA{sub 2} from B. jararacussu). Thus, comparison between these structures should add insight into the catalytic and myotoxic activities of bothropic PLA{sub 2}s.

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

    PubMed

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

    2010-01-01

    Various toxins isolated from Bothrops snake venoms induce inflammatory reactions and have been claimed to contribute to the severity of local symptoms present in this envenomation. Notwithstanding, the relative participation of serine proteases, metalloproteases and phospholipases A(2) in the inflammatory reaction produced by crude Bothrops venoms is poorly understood. Herein, crude Bothrops jararaca venom was treated with phenylmethanesulfonyl fluoride (PMSF), 1,10-phenanthroline (oPhe), or p-bromophenacyl-bromide (p-BPB) to inhibit those classes of enzymes, respectively, and inflammatory parameters were evaluated and compared to those induced by the control crude venom. The intensity of edema and hyperalgesia/allodynia was remarkably reduced in animals administered with oPhe-treated venom. Leukocyte-endothelium interactions (LEI), such as adhesion and migration of leukocytes, were also modified at 2h and 24h. Edema and LEI parameters induced by p-BPB-treated venom were similar to those observed with the control venom, but hyperalgesia/allodynia was significantly lower. Inflammatory parameters induced by PMSF-treated venom were similar to those induced by the crude venom, except for a mild reduction in edema intensity. Our results indicate that metalloproteases have a pivotal role in the inflammatory reactions induced by B. jararaca venom, and phospholipases A(2) and serine proteases have a minor role. Copyright 2009 Elsevier Ltd. All rights reserved.

  9. Lachesis stenophrys venom reduces the equine antibody response towards Bothrops asper venom used as co-immunogen in the production of polyspecific snake antivenom.

    PubMed

    Arroyo, Cynthia; Solano, Sergio; Herrera, María; Segura, Álvaro; Estrada, Ricardo; Vargas, Mariángela; Villalta, Mauren; Gutiérrez, José María; León, Guillermo

    2015-09-01

    The anti-bothropic activity of an antivenom prepared from the plasma of horses immunized with Bothrops asper venom (anti-B antivenom) was compared with a similar formulation produced from the plasma of horses immunized with a mixture of B. asper and Lachesis stenophrys venoms (anti-BL antivenom). Likewise, a comparison between the anti-lachesic activity of the anti-BL antivenom and a similar formulation prepared from horses immunized only with L. stenophrys venom (anti-L antivenom) was performed. The anti-BL antivenom had lower concentration of anti-bothropic antibodies than the anti-B antivenom. This difference was associated to a lower response towards all components of B. asper venom, but particularly towards some D49-phospholipases A2 (PLA2s) and PIII-metalloproteinases. Consequently, the anti-BL antivenom was less effective neutralizing lethal, coagulant, defibrinogenating, PLA2, and myotoxic activities of B. asper venom. On the other hand, anti-BL and anti-L antivenoms showed similar concentration of anti-lachesic antibodies, and similar capacity to recognize the HPLC fractions of L. stenophrys venom and to neutralize lethal, coagulant, proteolytic, hemorrhagic, PLA2 and myotoxic activities induced by this venom. It is concluded that, when used as co-immunogens, the venom of L. stenophrys reduces the antibody response towards B. asper venom, whereas the latter does not affect the anti-lachesic response. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Influence of phospholipasic inhibition on neuromuscular activity of Bothrops fonsecai snake venom.

    PubMed

    Schezaro-Ramos, Raphael; Collaço, Rita de Cássia O; Randazzo-Moura, Priscila; Rocha, Thalita; Cogo, José Carlos; Rodrigues-Simioni, Léa

    2017-05-01

    Bothrops fonsecai (B. fonsecai), a pitviper endemic to southeastern Brazil, has a venom mainly composed by snake venom phospholipases (PLA2) and metalloproteases, compounds that could interfere with neuromuscular junction in vitro. In this work, we investigated the role of PLA2 in the myotoxicity and neuromuscular blockade caused by B. fonsecai venom using different procedures frequently associated with PLA2 activity inhibition: 24 °C bath temperature, Ca(2+) - Sr(2+) replacement and chemical modification with p-bromophenacyl bromide (p-BPB). Mice extensor digitorum longus preparations (EDL) were incubated with usual or modified Tyrode solution (prepared with Ca(2+) or Sr(2+) respectively) at 24 °C or 37 °C (as controls) and in addition of B. fonsecai venom (100 μg/mL) alone or after its incubation with buffer (24 h, 23 °C) on the absence (alkylation control) and presence of p-BPB; all muscle were processed for histological analysis. The PLA2, proteolytic and amidolytic activities under the same conditions (24 °C or 37 °C, Ca(2+) - Sr(2+) replacement, absence or presence p-BPB) were also assessed. The B. fonsecai venom caused total neuromuscular blockade after 100 min of incubation, in Ca(2+) Tyrode solution at 37 °C (usual conditions); on Sr(2+) Tyrode solution (37 °C) the twitch height were 31.7 ± 7.4% of basal, and at 24 °C (Ca(2+) Tyrode solution) were 53.6 ± 7.0% of basal. The alkylation of PLA2 with p-BPB promoted a great blockade decrease at 100 min of incubation (88.7 ± 5.7% of basal), but it was also observed on alkylation control preparations (66.2 ± 6.6%). The venom produced 50% of blockade at 40.5 ± 5.9 min, in Ca(2+) Tyrode solution at 37 °C. The protocols delayed the time for 50% blockade: 105.7 ± 7.1 min (at 24 °C, in Ca(2+) Tyrode solution) and 71.1 ± 9.0 min (at 37 °C, in Sr(2+) Tyrode solution). Regarding p-BPB incubation and alkylation control preparations, 50% of blockade was not reached

  11. Renal and antibacterial effects induced by myotoxin I and II isolated from Bothrops jararacussu venom.

    PubMed

    Barbosa, P S F; Martins, A M C; Havt, A; Toyama, Daniela O; Evangelista, J S A M; Ferreira, D P P; Joazeiro, P P; Beriam, Luis O S; Toyama, Marcos H; Fonteles, M C; Monteiro, H S A

    2005-09-15

    Bothrops jararacussu myotoxin I (BthTx-I; Lys 49) and II (BthTX-II; Asp 49) were purified by ion-exchange chromatography and reverse phase HPLC. In this work we used the isolated perfused rat kidney method to evaluate the renal effects of B. jararacussu myotoxins I (Lys49 PLA2) and II (Asp49 PLA2) and their possible blockage by indomethacin. BthTX-I (5 microg/ml) and BthTX-II (5 microg/ml) increased perfusion pressure (PP; ct120=110.28+/-3.70 mmHg; BthTX I=171.28+/-6.30*mmHg; BthTX II=175.50+/-7.20*mmHg), renal vascular resistance (RVR; ct120=5.49+/-0.54 mmHg/ml.g(-1)min(-1); BthTX I=8.62+/-0.37*mmHg/ml g(-1)min(-1); BthTX II=8.9+/-0.36*mmHg/ml g(-1)min(-1)), urinary flow (UF; ct(120)=0.14+/-0.01ml g(-1)min(-1); BthTX I=0.32+/-0.05*ml g(-1)min(-1); BthTX II=0.37+/-0.01*ml g(-1)min(-1)) and glomerular filtration rate (GFR; ct120=0.72+/-0.10 ml g(-1)min(-1); BthTX I=0.85+/-0.13*ml g(-1)min(-1); BthTX II=1.22+/-0.28*ml g(-1)min(-1)). In contrast decreased the percent of sodium tubular transport (%TNa(+); ct(120)=79,76+/-0.56; BthTX I=62.23+/-4.12*; BthTX II=70.96+/-2.93*) and percent of potassium tubular transport (%TK(+);ct120=66.80+/-3.69; BthTX I=55.76+/-5.57*; BthTX II=50.86+/-6.16*). Indomethacin antagonized the vascular, glomerular and tubular effects promoted by BthTX I and it's partially blocked the effects of BthTX II. In this work also evaluated the antibacterial effects of BthTx-I and BthTx-II against Xanthomonas axonopodis. pv. passiflorae (Gram-negative bacteria) and we observed that both PLA2 showed antibacterial activity. Also we observed that proteins Also we observed that proteins chemically modified with 4-bromophenacyl bromide (rho-BPB) decrease significantly the antibacterial effect of both PLA2. In conclusion, BthTx I and BthTX II caused renal alteration and presented activity antimicrobial. The indomethacin was able to antagonize totally the renal effects induced by BthTx I and partially the effects promoted by BthTx II, suggesting involvement of

  12. Evaluation of the antichagasic activity of batroxicidin, a cathelicidin-related antimicrobial peptide found in Bothrops atrox venom gland.

    PubMed

    Mello, Clarissa Perdigão; Lima, Danya Bandeira; Menezes, Ramon Róseo Paula Pessoa Bezerra de; Bandeira, Izabel Cristina Justino; Tessarolo, Louise Donadello; Sampaio, Tiago Lima; Falcão, Claudio Borges; Rádis-Baptista, Gandhi; Martins, Alice Maria Costa

    2017-05-01

    Antimicrobial peptides (AMPs) are potential alternatives to conventional antibiotics, as they have a fast mode of action, a low likelihood of resistance development and can act in conjunction with existing drug regimens. We report in this study the effects of batroxicidin (BatxC), a cathelicidin-related AMP from Bothrops atrox venom gland, over Trypanosoma cruzi, a protozoan that causes Chagas' disease. BatxC inhibited all T. cruzi (Y strain: benznidazole-resistant) developmental forms, with selectivity index of 315. Later, separate flow cytometry assays showed T. cruzi cell labeling by 7-aminoactinomycin D, the increase in reactive oxygen species and the loss of mitochondrial membrane potential when the parasite was treated with BatxC, which are indication of necrosis. T. cruzi cell death pathway by a necrotic mechanism was finally confirmed by scanning electron microscopy which observed loss of cell membrane integrity. In conclusion, BatxC was able to inhibit T. cruzi, with high selectivity index, by inducing necrosis.

  13. Venom of Bothrops asper from Mexico and Costa Rica: intraspecific variation and cross-neutralization by antivenoms.

    PubMed

    Segura, Alvaro; Herrera, María; Villalta, Mauren; Vargas, Mariángela; Uscanga-Reynell, Alfredo; de León-Rosales, Samuel Ponce; Jiménez-Corona, María Eugenia; Reta-Mares, José Francisco; Gutiérrez, José María; León, Guillermo

    2012-01-01

    Bothrops asper is the species that induces the highest incidence of snakebite envenomation in southern Mexico, Central America and parts of northern South America. The intraspecies variability in HPLC profile and toxicological activities between the venoms from specimens collected in Mexico (Veracruz) and Costa Rica (Caribbean and Pacific populations) was investigated, as well as the cross-neutralization by antivenoms manufactured in these countries. Venoms differ in their HPLC profiles and in their toxicity, since venom from Mexican population showed higher lethal and defibrinogenating activities, whereas those from Costa Rica showed higher hemorrhagic and in vitro coagulant activities. In general, antivenoms were more effective in the neutralization of homologous venoms. Overall, both antivenoms effectively neutralized the various toxic effects of venoms from the two populations of B. asper. However, antivenom raised against venom from Costa Rican specimens showed a higher efficacy in the neutralization of defibrinogenating and coagulant activities, thus highlighting immunochemical differences in the toxins responsible for these effects associated with hemostatic disturbances in snakebite envenoming. These observations illustrate how intraspecies venom variation may influence antivenom neutralizing profile. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. Intraspecific differences in the immunochemical reactivity and neutralization of venom from Argentinean Bothrops (Rhinocerophis) alternatus by specific experimental antivenoms.

    PubMed

    Lanari, Laura Cecilia; Alagón, Alejandro; Olvera, Alejandro; Costa de Oliveira, Vanessa; Laskowicz, Rodrigo Daniel; Boyer, Leslie; Lago, Néstor Rubén; Alejandro, Alagon; de Roodt, Adolfo Rafael

    2014-07-01

    The venoms of Bothrops (Rhinocerophis) alternatus (B.a.) from different regions of Argentina have shown biochemical, toxicological and immunological variations. Considering these variations, we produced nine experimental antisera (rabbit, IgG) against venoms from snakes of nine different regions and a pool of venom, comprised of equal amounts of venoms from each region. The immunologic studies (ELISA, Westernblot) showed significant cross reactivity among all regional antivenoms with all regional venoms, with no significant differences regarding the specificity of the immunogens used for the production of antivenom. Neutralization of hemorrhage was variable (although all the antivenoms neutralized this activity in all venoms) and the neutralization of coagulant and phospholipase activities were evident in all cases. Some antivenoms neutralized toxic activities that were absent or very low in the venoms used as immunogen, on other non-homologous venoms (e.g. thrombin like activity). Despite the different toxic potencies of regional venoms, antivenoms developed using venoms of snakes from a particular region showed high immunochemical reactivity and cross-neutralizing capacity on snake venoms from different and distant regions, in occasions over those of the homologous antivenoms. These findings could be used to improve the generation of pools of venoms for the production of antivenoms. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Isolation and characterization of two new non-hemorrhagic metalloproteinases with fibrinogenolytic activity from the mapanare (Bothrops colombiensis) venom.

    PubMed

    Girón, María E; Rodríguez-Acosta, Alexis; Salazar, Ana María; Sánchez, Elda E; Galán, Jacob; Ibarra, Carlos; Guerrero, Belsy

    2013-01-01

    Colombienases are acidic, low molecular weight metalloproteinases (Mr of 23,074.31 Da colombienase-1 and 23,078.80 Da colombienase-2; pI of 6.0 and 6.2, respectively) isolated from Bothrops colombiensis snake venom. The chromatographic profile in RP-HPLC and its partial sequence confirmed its high homogeneity. Both colombienases present fibrino(geno)lytic activity, but did not show any hemorrhagic, amidolytic, plasminogen activator or coagulant activities, and no effect on platelet aggregation induced by collagen or ADP. Both enzymes were strongly active on fibrinogen Aα chains followed by the Bβ chains, and colombienases-2, at high doses, also degraded the γ chains. This activity was stable at temperatures ranging between 4 and 37 °C, with a maximum activity at 25 °C, and at pHs between 7 and 9. The homology demonstrated by the comparison of sequences, with zinc-dependent metalloproteinases, as well as the metal chelant effects on, confirmed that the colombienases were metalloproteinases, particularly to α-fibrinogenases belonging to the P-I class of SVPMs (20-30 kDa), which contain only the single-domain proteins. The biological characteristics of the colombienases confer a therapeutic potential, since they contain a high fibrino(geno)lytic activity, devoid of hemorrhagic activity. These metalloproteinases might be explored as thrombolytic agents given that they dissolve fibrin clots or prevent their formation.

  16. Comparison of the adjuvant activity of aluminum hydroxide and calcium phosphate on the antibody response towards Bothrops asper snake venom.

    PubMed

    Olmedo, Hidekel; Herrera, María; Rojas, Leonardo; Villalta, Mauren; Vargas, Mariángela; Leiguez, Elbio; Teixeira, Catarina; Estrada, Ricardo; Gutiérrez, José María; León, Guillermo; Montero, Mavis L

    2014-01-01

    The adjuvanticity of aluminum hydroxide and calcium phosphate on the antibody response in mice towards the venom of the snake Bothrops asper was studied. It was found that, in vitro, most of the venom proteins are similarly adsorbed by both mineral salts, with the exception of some basic phospholipases A2, which are better adsorbed by calcium phosphate. After injection, the adjuvants promoted a slow release of the venom, as judged by the lack of acute toxicity when lethal doses of venom were administered to mice. Leukocyte recruitment induced by the venom was enhanced when it was adsorbed on both mineral salts; however, venom adsorbed on calcium phosphate induced a higher antibody response towards all tested HPLC fractions of the venom. On the other hand, co-precipitation of venom with calcium phosphate was the best strategy for increasing: (1) the capacity of the salt to couple venom proteins in vitro; (2) the venom ability to induce leukocyte recruitment; (3) phagocytosis by macrophages; and (4) a host antibody response. These findings suggest that the chemical nature is not the only one determining factor of the adjuvant activity of mineral salts.

  17. Isolation, amino acid sequence and biological characterization of an "aspartic-49" phospholipase A₂ from Bothrops (Rhinocerophis) ammodytoides venom.

    PubMed

    Clement, Herlinda; Costa de Oliveira, Vanessa; Zamudio, Fernando Z; Lago, Néstor R; Valdez-Cruz, Norma A; Bérnard Valle, Melisa; Hajos, Silvia E; Alagón, Alejandro; Possani, Lourival D; de Roodt, Adolfo R

    2012-12-01

    A phospholipase enzyme was separated by chromatography from the venom of the snake Bothrops (Rhinocerophis) ammodytoides and characterized. The experimentally determined molecular weight was 13,853.65 Da, and the full primary structure was determined by Edman degradation and mass spectrometry analysis. The enzyme contains 122 amino acids residues closely stabilized by 7 disulfide bridges with an isoelectric point of 6.13. Sequence comparison with other known secretory PLA2 shows that the enzyme isolated belongs to the group II, presenting an aspartic acid residue at position 48 (numbered by convention as Asp49) of the active site, and accordingly displaying enzymatic activity. The enzyme corresponds to 3% of the total mass of the venom. The enzyme is mildly toxic to mice. The intravenous LD₅₀ of this phospholipase in CD-1 mice was around 6 μg/g of mouse body weight (more exactly 117 μg/mouse of 20 g) and the minimal mortal dose (MMD) was estimated to be close to 10 μg/g. In contrast, the LD₅₀ of the venom was circa 2 μg/g mouse body weight. Toxicological analyses of the purified enzyme were performed in vitro and in vivo using experimental animals (mice and rats). The enzyme at high doses caused pulmonary congestion, intraperitoneal bleeding, inhibition of clot retraction and muscle tissue alterations with increasing of creatine kinase levels.

  18. Neutralising ability of Terminalia fagifolia extract (Combretaceae) against the in vitro neuromuscular effects of Bothrops jararacussu venom.

    PubMed

    Tribuiani, Natália; Tavares, Marylu Oliveira; Santana, Monique Neves; Fontana Oliveira, Isadora Caruso; Amaral Filho, Jorge do; Silva, Magali Glauzer; Dos Santos, Marcio Galdino; Cogo, José Carlos; Floriano, Rafael Stuani; Cogo-Müller, Karina; Oshima-Franco, Yoko

    2017-03-02

    The ability of Terminalia fagifolia hydroalcoholic extract (Tf-HE) to neutralise the paralysis and myotoxicity induced by Bothrops jararacussu venom was assayed using mouse phrenic nerve-diaphragm (PND) preparation and two varieties of chick biventer cervicis (BC) preparations. Tf-HE 100 μg/mL and 500 μg/mL were tested against 40 and 200 μg of venom/mL in PND and BC preparations, respectively, using pre- and post-venom incubation treatments. The effects of Tf-HE against the myotoxicity caused by venom were evaluated via histological analysis (PND) and creatine kinase (CK) release (BC). Tf-HE was able to reverse the venom paralysis in both preparation types. The contractures to exogenous ACh in BC preparations showed that Tf-HE may act on extrinsic, preserving those intrinsic postsynaptic receptors. There was a positive correlation between CK and morphological changes. The high non-hemolytic saponin content can explain the Tf-HE efficacy against the toxic effects of B. jararacussu venom in vertebrate neuromuscular preparations.

  19. An unexpected cell-penetrating peptide from Bothrops jararaca venom identified through a novel size exclusion chromatography screening.

    PubMed

    Sciani, Juliana Mozer; Vigerelli, Hugo; Costa, André Santos; Câmara, Diana Aparecida Dias; Junior, Paulo Luiz-de-Sá; Pimenta, Daniel Carvalho

    2017-01-01

    Efficient drug delivery systems are currently one of the greatest challenges in pharmacokinetics, and the transposition of the gap between in vitro candidate molecule and in vivo test drug is, sometimes, poles apart. In this sense, the cell-penetrating peptides (CPP) may be the bridge uniting these worlds. Here, we describe a technique to rapidly identify unlabeled CPPs after incubation with liposomes, based on commercial desalting (size exclusion) columns and liquid chromatography-MS/MS, for peptide de novo sequencing. Using this approach, we found it possible to identify one new CPP - interestingly, a classical bradykinin-potentiating peptide - in the peptide-rich low molecular mass fraction of the Bothrops jararaca venom, which was also able to penetrate live cell membranes, as confirmed by classical approaches employing fluorescence-labeled analogues of this CPP. Moreover, both the labeled and unlabeled CPPs caused no metabolic, cell-cycle or morphologic alterations, proving to be unmistakably cargo deliverers and not drugs themselves. In sum, we have developed and validated a method for screening label-free peptides for CPP activity, regardless of their biological origin, which could lead to the identification of new and more efficient drug delivery systems. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

  20. Effect of perimuscular injection of Bothrops jararacussu venom on plasma creatine kinase levels in mice: influence of dose and volume.

    PubMed

    Calil-Elias, S; Thattassery, E; Martinez, A M B; Melo, P A

    2002-10-01

    The effect of dose and volume of a perimuscular injection of Bothrops jararacussu venom on myonecrosis of skeletal muscle was studied in mice. An increase of the venom dose (0.25 to 2.0 micro g/g) at a given volume (50 micro l) resulted in an increase in plasma creatine kinase (CK) levels 2 h after injection. Plasma CK activity increased from the basal level of 129.27 +/- 11.83 (N = 20) to 2392.80 +/- 709.43 IU/l (N = 4) for the 1.0 micro g/g dose. Histological analysis of extensor digitorum longus muscle 4 h after injection showed lesion of peripheral muscle fibers, disorganization of the bundles or the complete degeneration of muscle fibers. These lesions were more extensive when higher doses were injected. Furthermore, an increase in volume (12.5 to 100 micro l) by dilution of a given dose (0.5 micro g/g) also increased plasma CK levels from 482.31 +/- 122.79 to 919.07 +/- 133.33 IU/l (N = 4), respectively. These results indicate that care should be taken to standardize volumes and sites of venom injections.

  1. Evaluation of three Brazilian antivenom ability to antagonize myonecrosis and hemorrhage induced by Bothrops snake venoms in a mouse model.

    PubMed

    da Silva, Noelson M V; Arruda, Emerson Z; Murakami, Yugo L B; Moraes, Raphael A M; El-Kik, Camila Z; Tomaz, Marcelo A; Fernandes, Fabrício F A; Oliveira, Clayton Z; Soares, Andreimar M; Giglio, Jose R; Melo, Paulo A

    2007-08-01

    Despite preventing death after snakebites, there is little evidence that polyvalent antivenoms (PAVs) protect against myotoxicity and local damages. We evaluated antibothropic Brazilian PAVs from three manufacturers against the myotoxicity and hemorrhagic activity of Bothrops jararacussu and B. jararaca venoms, respectively, by using two protocols: preincubation of PAVs with venom, and i.v. pretreatment with PAVs, prior to the venom inoculation. In this investigation, we used doses of PAVs ranging from 0.4 to 4.0mL/mg of venom equivalent up to 10 times the amount recommended by the producers for the clinical practice in Brazil. In our preincubation protocol in vivo, PAVs antagonized myotoxicity of B. jararacussu venom by 40-95%, while our pretreatment protocol antagonized myotoxic activity by 0-60%. Preincubation of antivenoms with B. jararaca venom antagonized its hemorrhagic activity by 70-95%, while pretreatment antagonized hemorrhagic activity by 10-50%. Although all PAVs demonstrated partial antagonism against both venoms, the magnitude of these effects varied greatly among the manufactures. The results suggest that the current clinical doses of these PAVs may have negligible antimyotoxic effect.

  2. Effect of BjcuL, a lectin isolated from Bothrops jararacussu, on human peripheral blood mononuclear cells.

    PubMed

    Pires, Weverson Luciano; de Castro, Onassis Boeri; Kayano, Anderson Makoto; da Silva Setúbal, Sulamita; Pontes, Adriana Silva; Nery, Neriane Monteiro; Paloschi, Mauro Valentino; Dos Santos Pereira, Soraya; Stábeli, Rodrigo Guerino; Fernandes, Carla Freire Celedônio; Soares, Andreimar Martins; Zuliani, Juliana Pavan

    2017-06-01

    BjcuL is a C-type lectin with specificity for the binding of β-d-galactose units isolated from Bothrops jararacussu venom. It triggers cellular infiltration in post capillary venules, increases edema and vascular permeability in murine models, contributes to in vitro neutrophil activation and modulates macrophage functional activation towards an M1 state. The purpose of this study was to investigate the effect of BjcuL on human peripheral blood mononuclear cells (PBMCs) activation with a focus on PBMCs proliferation and inflammatory mediators release. Results showed that BjcuL is not toxic to PBMCs, that BjcuL inhibits PBMCs proliferation and that it stimulates PBMCs to produce superoxide anion and hydrogen peroxide, primarily via lymphocyte stimulation, but does not stimulate the production of nitric oxide and PGE2. These results demonstrate that BjcuL has an immunomodulatory effect on PBMCs. Further studies are needed to confirm the immunomodulatory effect of BjcuL, to elucidate the molecular mechanisms of action responsible for its effects and to determine its potential application as an immunopharmacological and biotechnological tool. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Crystallization and preliminary X-ray diffraction analysis of three myotoxic phospholipases A2 from Bothrops brazili venom

    PubMed Central

    Fernandes, Carlos A. H.; Gartuzo, Elaine C. G.; Pagotto, Ivan; Comparetti, Edson J.; Huancahuire-Vega, Salomón; Ponce-Soto, Luis Alberto; Costa, Tássia R.; Marangoni, Sergio; Soares, Andreimar M.; Fontes, Marcos R. M.

    2012-01-01

    Two myotoxic and noncatalytic Lys49-phospholipases A2 (braziliantoxin-II and MT-II) and a myotoxic and catalytic phospholipase A2 (braziliantoxin-III) from the venom of the Amazonian snake Bothrops brazili were crystallized. The crystals diffracted to resolutions in the range 2.56–2.05 Å and belonged to space groups P3121 (braziliantoxin-II), P6522 (braziliantoxin-III) and P21 (MT-II). The structures were solved by molecular-replacement techniques. Both of the Lys49-phospholipases A2 (braziliantoxin-II and MT-II) contained a dimer in the asymmetric unit, while the Asp49-phospholipase A2 braziliantoxin-III contained a monomer in its asymmetric unit. Analysis of the quaternary assemblies of the braziliantoxin-II and MT-II structures using the PISA program indicated that both models have a dimeric conformation in solution. The same analysis of the braziliantoxin-III structure indicated that this protein does not dimerize in solution and probably acts as a monomer in vivo, similar to other snake-venom Asp49-phospholipases A2. PMID:22869126

  4. Molecular cloning and structural modelling of gamma-phospholipase A2 inhibitors from Bothrops atrox and Micrurus lemniscatus snakes.

    PubMed

    Picelli, Carina G; Borges, Rafael J; Fernandes, Carlos A H; Matioli, Fabio M; Fernandes, Carla F C; Sobrinho, Juliana C; Holanda, Rudson J; Ozaki, Luiz S; Kayano, Anderson M; Calderon, Leonardo A; Fontes, Marcos R M; Stábeli, Rodrigo G; Soares, Andreimar M

    2017-10-01

    Phospholipases A2 inhibitors (PLIs) produced by venomous and non-venomous snakes play essential role in this resistance. These endogenous inhibitors may be classified by their fold in PLIα, PLIβ and PLIγ. Phospholipases A2 (PLA2s) develop myonecrosis in snake envenomation, a consequence that is not efficiently neutralized by antivenom treatment. This work aimed to identify and characterize two PLIs from Amazonian snake species, Bothrops atrox and Micrurus lemniscatus. Liver tissues RNA of specimens from each species were isolated and amplified by RT-PCR using PCR primers based on known PLIγ gene sequences, followed by cloning and sequencing of amplified fragments. Sequence similarity studies showed elevated identity with inhibitor PLIγ gene sequences from other snake species. Molecular models of translated inhibitors' gene sequences resemble canonical three finger fold from PLIγ and support the hypothesis that the decapeptide (residues 107-116) may be responsible for PLA2 inhibition. Structural studies and action mechanism of these PLIs may provide necessary information to evaluate their potential as antivenom or as complement of the current ophidian accident treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. BbMP-1, a new metalloproteinase isolated from Bothrops brazili snake venom with in vitro antiplasmodial properties.

    PubMed

    Kayano, Anderson M; Simões-Silva, Rodrigo; Medeiros, Patrícia S M; Maltarollo, Vinícius G; Honorio, Kathia M; Oliveira, Eliandre; Albericio, Fernando; da Silva, Saulo L; Aguiar, Anna Caroline C; Krettli, Antoniana U; Fernandes, Carla F C; Zuliani, Juliana P; Calderon, Leonardo A; Stábeli, Rodrigo G; Soares, Andreimar M

    2015-11-01

    This study describes the biochemical and functional characterization of a new metalloproteinase named BbMP-1, isolated from Bothrops brazili venom. BbMP-1 was homogeneous on SDS-PAGE, presented molecular mass of 22,933Da and pI 6.4. The primary structure was partially elucidated with high identity with others metalloproteinases from Viperidae venoms. The enzymatic activity on azocasein was evaluated in different experimental conditions (pH, temperature). A significant reduction in enzyme activity after exposure to chelators of divalent cations (EDTA), reducing agents (DTT), pH less than 5.0 or temperatures higher than 45 °C was observed. BbMP-1 showed activity on fibrinogen degrading Aα chain quickly and to a lesser extent the Bβ chain. Also demostrated to be weakly hemorrhagic, presenting however, significant myotoxic and edematogenic activity. The in vitro activity of BbMP-1 against Plasmodium falciparum showed an IC50 of 3.2 ± 2.0 μg/mL. This study may help to understand the pathophysiological effects induced by this group of toxin and their participation in the symptoms observed in cases of snake envenomation. Moreover, this result is representative for this group of proteins and shows the biotechnological potential of BbMP-1 by the demonstration of its antiplasmodial activity. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Purification and Characterization of BmooAi: A New Toxin from Bothrops moojeni Snake Venom That Inhibits Platelet Aggregation

    PubMed Central

    Ribeiro de Queiroz, Mayara; Mamede, Carla Cristine N.; de Morais, Nadia Cristina G.; Cortes Fonseca, Kelly; Barbosa de Sousa, Bruna; Migliorini, Thaís M.; Pereira, Déborah Fernanda C.; Stanziola, Leonilda; Calderon, Leonardo A.; Simões-Silva, Rodrigo; Martins Soares, Andreimar; de Oliveira, Fábio

    2014-01-01

    In this paper, we describe the purification/characterization of BmooAi, a new toxin from Bothrops moojeni that inhibits platelet aggregation. The purification of BmooAi was carried out through three chromatographic steps (ion-exchange on a DEAE-Sephacel column, molecular exclusion on a Sephadex G-75 column, and reverse-phase HPLC chromatography on a C2/C18 column). BmooAi was homogeneous by SDS-PAGE and shown to be a single-chain protein of 15,000 Da. BmooAi was analysed by MALDI-TOF Spectrometry and revealed two major components with molecular masses 7824.4 and 7409.2 as well as a trace of protein with a molecular mass of 15,237.4 Da. Sequencing of BmooAi by Edman degradation showed two amino acid sequences: IRDFDPLTNAPENTA and ETEEGAEEGTQ, which revealed no homology to any known toxin from snake venom. BmooAi showed a rather specific inhibitory effect on platelet aggregation induced by collagen, adenosine diphosphate, or epinephrine in human platelet-rich plasma in a dose-dependent manner, whereas it had little or no effect on platelet aggregation induced by ristocetin. The effect on platelet aggregation induced by BmooAi remained active even when heated to 100°C. BmooAi could be of medical interest as a new tool for the development of novel therapeutic agents for the prevention and treatment of thrombotic disorders. PMID:24971359

  7. Purification and characterization of BmooAi: a new toxin from Bothrops moojeni snake venom that inhibits platelet aggregation.

    PubMed

    de Queiroz, Mayara Ribeiro; Mamede, Carla Cristine N; de Morais, Nadia Cristina G; Fonseca, Kelly Cortes; de Sousa, Bruna Barbosa; Migliorini, Thaís M; Pereira, Déborah Fernanda C; Stanziola, Leonilda; Calderon, Leonardo A; Simões-Silva, Rodrigo; Soares, Andreimar Martins; de Oliveira, Fábio

    2014-01-01

    In this paper, we describe the purification/characterization of BmooAi, a new toxin from Bothrops moojeni that inhibits platelet aggregation. The purification of BmooAi was carried out through three chromatographic steps (ion-exchange on a DEAE-Sephacel column, molecular exclusion on a Sephadex G-75 column, and reverse-phase HPLC chromatography on a C2/C18 column). BmooAi was homogeneous by SDS-PAGE and shown to be a single-chain protein of 15,000 Da. BmooAi was analysed by MALDI-TOF Spectrometry and revealed two major components with molecular masses 7824.4 and 7409.2 as well as a trace of protein with a molecular mass of 15,237.4 Da. Sequencing of BmooAi by Edman degradation showed two amino acid sequences: IRDFDPLTNAPENTA and ETEEGAEEGTQ, which revealed no homology to any known toxin from snake venom. BmooAi showed a rather specific inhibitory effect on platelet aggregation induced by collagen, adenosine diphosphate, or epinephrine in human platelet-rich plasma in a dose-dependent manner, whereas it had little or no effect on platelet aggregation induced by ristocetin. The effect on platelet aggregation induced by BmooAi remained active even when heated to 100°C. BmooAi could be of medical interest as a new tool for the development of novel therapeutic agents for the prevention and treatment of thrombotic disorders.

  8. Activation of J77A.1 macrophages by three phospholipases A2 isolated from Bothrops atrox snake venom.

    PubMed

    Furtado, Juliana L; Oliveira, George A; Pontes, Adriana S; Setúbal, Sulamita da S; Xavier, Caroline V; Lacouth-Silva, Fabianne; Lima, Beatriz F; Zaqueo, Kayena D; Kayano, Anderson M; Calderon, Leonardo A; Stábeli, Rodrigo G; Soares, Andreimar M; Zuliani, Juliana P

    2014-01-01

    In the present study, we investigated the in vitro effects of two basic myotoxic phospholipases A2 (PLA2), BaTX-I, a catalytically inactive Lys-49 variant, and BaTX-II, a catalytically active Asp-49, and of one acidic myotoxic PLA2, BaPLA2, a catalytically active Asp-49, isolated from Bothrops atrox snake venom, on the activation of J774A.1 macrophages. At noncytotoxic concentrations, the toxins did not affect the adhesion of the macrophages, nor their ability to detach. The data obtained showed that only BaTX-I stimulated complement receptor-mediated phagocytosis. However, BaTX-I, BaTX-II, and BaPLA2 induced the release of the superoxide anion by J774A.1 macrophages. Additionally, only BaTX-I raised the lysosomal volume of macrophages after 15 min of incubation. After 30 min, all the phospholipases increased this parameter, which was not observed within 60 min. Moreover, BaTX-I, BaTX-II, and BaPLA2 increased the number of lipid bodies on macrophages submitted to phagocytosis and not submitted to phagocytosis. However, BaTX-II and BaPLA2 induced the release of TNF-α by J774A.1 macrophages. Taken together, the data show that, despite differences in enzymatic activity, the three toxins induced inflammatory events and whether the enzyme is acidic or basic does not seem to contribute to these effects.

  9. Heparin at low concentration acts as antivenom against Bothrops jararacussu venom and bothropstoxin-I neurotoxic and myotoxic actions

    PubMed Central

    Rostelato-Ferreira, Sandro; Leite, Gildo Bernardo; Cintra, Adélia Cristina Oliveira; da Cruz-Höfling, Maria Alice; Rodrigues-Simioni, Léa; Oshima-Franco, Yoko

    2010-01-01

    Heparin has been shown to antagonize myotoxic effects of crotaline venoms. Here a very low heparin concentration (LHC) was examined in its ability to antagonize the neurotoxic/myotoxic effects of Bothrops jararacussu venom and its phospholipase A2 myotoxin, bothropstoxin-I (BthTX-I), in an in vitroz nerve-muscle preparation and in mice gastrocnemius. Normalization of results was done by assays with commercial antibothropic antivenom (CBA). LHC (1IU/ml) added to the incubation bath reduced by 4- and 4.5-fold (vs 2.8- and 2.5-fold by CBA) the neuromuscular paralysis, by 5.4 and 4.4-fold (vs 2.5- and 13.3-fold by CBA) the percentage of fibers damaged and by 6- and 1.7-fold (vs 30- and 1.6-fold by CBA) the CK activity induced by B. jararacussu and BthTX-I, respectively. Protamine sulphate added 15min after the incubation of the preparation with LHC+venom, avoided the LHC neutralizing effect against venom neurotoxicity. This strongly attests that given the polycationic nature of protamine, it probably complexed with the polyanionic heparin making it unattainable for binding to basic components of venom, reducing toxicity. Since heparin antagonism is generally stronger against venom effects than is myotoxin we discuss that other venom components than the BthTX-I are likely target for the antagonism promoted by the polyanionic heparin. PMID:21544183

  10. Crystallization and preliminary X-ray diffraction analysis of three myotoxic phospholipases A2 from Bothrops brazili venom.

    PubMed

    Fernandes, Carlos A H; Gartuzo, Elaine C G; Pagotto, Ivan; Comparetti, Edson J; Huancahuire-Vega, Salomón; Ponce-Soto, Luis Alberto; Costa, Tássia R; Marangoni, Sergio; Soares, Andreimar M; Fontes, Marcos R M

    2012-08-01

    Two myotoxic and noncatalytic Lys49-phospholipases A(2) (braziliantoxin-II and MT-II) and a myotoxic and catalytic phospholipase A(2) (braziliantoxin-III) from the venom of the Amazonian snake Bothrops brazili were crystallized. The crystals diffracted to resolutions in the range 2.56-2.05 Å and belonged to space groups P3(1)21 (braziliantoxin-II), P6(5)22 (braziliantoxin-III) and P2(1) (MT-II). The structures were solved by molecular-replacement techniques. Both of the Lys49-phospholipases A(2) (braziliantoxin-II and MT-II) contained a dimer in the asymmetric unit, while the Asp49-phospholipase A(2) braziliantoxin-III contained a monomer in its asymmetric unit. Analysis of the quaternary assemblies of the braziliantoxin-II and MT-II structures using the PISA program indicated that both models have a dimeric conformation in solution. The same analysis of the braziliantoxin-III structure indicated that this protein does not dimerize in solution and probably acts as a monomer in vivo, similar to other snake-venom Asp49-phospholipases A(2).

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

    PubMed

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

    2015-03-01

    Here we evaluate the effects of BpLec, a C-type lectin isolated from Bothrops pauloensis snake venom, on Toxoplasma gondii parasitism. BpLec (0.195-12.5 μg/mL) did not interfere with HeLa (host cell) viability by MTT assay, whereas higher doses decreased viability and changed HeLa morphology. In addition, the host cell treatment before infection did not influence adhesion and proliferation indexes. BpLec did not alter T. gondii tachyzoite viability, as carried out by trypan blue exclusion, but decreased both adhesion and parasite replication, when tachyzoites were treated before infection. Galactose (0.4 M) inhibited the BpLec effect on adhesion assays, suggesting that BpLec probably recognize some glycoconjugate from T. gondii membrane. Additionally, we performed cytokine measurements from supernatants collected from HeLa cells infected with T. gondii tachyzoites previously treated with RPMI or BpLec. MIF and IL-6 productions by HeLa cells were increased by BpLec treatment. Also, TGF-β1 secretion was diminished post-infection, although this effect was not dependent on BpLec treatment. Taken together, our results show that BpLec is capable of reducing T. gondii parasitism after tachyzoite treatment and may represent an interesting tool in the search for parasite antigens involved in these processes. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Clinical trial of two antivenoms for the treatment of Bothrops and Lachesis bites in the north eastern Amazon region of Brazil.

    PubMed

    Pardal, Pedro Pereira de Oliveira; Souza, Suzana Medeiro; Monteiro, Maria Rita de Cássia da Costa; Fan, Hui Wen; Cardoso, João Luiz Costa; França, Francisco Oscar Siqueira; Tomy, Sandra Corrallo; Sano-Martins, Ida S; de Sousa-e-Silva, Maria Cristina Cirillo; Colombini, Mônica; Kodera, Nancy F; Moura-da-Silva, Ana Maria; Cardoso, Diva Ferreira; Velarde, David Toledo; Kamiguti, Aura S; Theakston, Robert David; Warrell, David A

    2004-01-01

    The efficacies of specific Bothrops atrox-Lachesis and standard Bothrops-Lachesis antivenoms were compared in the north eastern Amazon region of Brazil. The main aim was to investigate whether a specific antivenom raised against the venom of B. atrox, the most important Amazon snake species from a medical point of view, was necessary for the treatment of patients in this region. Seventy-four patients with local and systemic effects of envenoming by Bothrops or Lachesis snakes were randomly allocated to receive either specific (n = 38) or standard (n = 36) antivenoms. In 46 cases (24 in the standard antivenom group, 22 in the other) the snake was identified either by enzyme immunoassay or by examination of the dead snake, as B. atrox in 45, L. muta in one. Patients were similar in all clinical and epidemiological respects before treatment. Results indicated that both antivenoms were equally effective in reversing all signs of envenoming detected both clinically and in the laboratory. Venom-induced haemostatic abnormalities were resolved within 24 h after the start of antivenom therapy in most patients. The extent of local complications, such as local skin necrosis and secondary infection, was similar in both groups. There were no deaths. The incidence of early anaphylactic reactions was 18% and 19%, respectively for specific and standard antivenoms; none was life-threatening. Measurement of serum venom concentrations by enzyme immunoassay (EIA) confirmed that both antivenoms cleared venom antigenaemia effectively. EIA also revealed that one patient had been bitten by Lachesis muta, although the clinical features in this case were not distinctive.

  13. Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments

    PubMed Central

    Prado, Nidiane D. R.; Pereira, Soraya S.; da Silva, Michele P.; Morais, Michelle S. S.; Kayano, Anderson M.; Moreira-Dill, Leandro S.; Luiz, Marcos B.; Zanchi, Fernando B.; Fuly, André L.; E. F. Huacca, Maribel; Fernandes, Cleberson F.; Calderon, Leonardo A.; Zuliani, Juliana P.; Soares, Andreimar M.; Stabeli, Rodrigo G.; F. C. Fernandes, Carla

    2016-01-01

    Antivenoms, produced using animal hyperimmune plasma, remains the standard therapy for snakebites. Although effective against systemic damages, conventional antivenoms have limited efficacy against local tissue damage. Additionally, the hypersensitivity reactions, often elicited by antivenoms, the high costs for animal maintenance, the difficulty of producing homogeneous lots, and the instability of biological products instigate the search for innovative products for antivenom therapy. In this study, camelid antibody fragments (VHH) with specificity to Bothropstoxin I and II (BthTX-I and BthTX-II), two myotoxic phospholipases from Bothrops jararacussu venom, were selected from an immune VHH phage display library. After biopanning, 28 and 6 clones recognized BthTX-I and BthTX-II by ELISA, respectively. Complementarity determining regions (CDRs) and immunoglobulin frameworks (FRs) of 13 VHH-deduced amino acid sequences were identified, as well as the camelid hallmark amino acid substitutions in FR2. Three VHH clones (KF498607, KF498608, and KC329718) were capable of recognizing BthTX-I by Western blot and showed affinity constants in the nanomolar range against both toxins. VHHs inhibited the BthTX-II phospholipase A2 activity, and when tested for cross-reactivity, presented specificity to the Bothrops genus in ELISA. Furthermore, two clones (KC329718 and KF498607) neutralized the myotoxic effects induced by B. jararacussu venom, BthTX-I, BthTX-II, and by a myotoxin from Bothrops brazili venom (MTX-I) in mice. Molecular docking revealed that VHH CDRs are expected to bind the C-terminal of both toxins, essential for myotoxic activity, and to epitopes in the BthTX-II enzymatic cleft. Identified VHHs could be a biotechnological tool to improve the treatment for snake envenomation, an important and neglected world public health problem. PMID:27028872

  14. Antibodies to a fragment of the Bothrops moojenil-amino acid oxidase cross-react with snake venom components unrelated to the parent protein.

    PubMed

    Stábeli, Rodrigo Guerino; Magalhães, Lívia Maria Pimenta; Selistre-de-Araujo, Heloísa S; Oliveira, Eduardo Brandt

    2005-09-01

    It is widely accepted that immunological cross-reactivity of snake venoms is mediated by antibodies that recognize venom components bearing either amino acid sequence homology or similar biological functions. However, here we demonstrate that polyspecific Bothrops antivenom is a source of cross-reactive antibodies that interact with venom proteins of distinctive primary structures and biological functions. The homoserine lactone derivative of the undecapeptide IQRWSLDKYAM (Ile1-Hse11), excised from the l-amino acid oxidase (LAAO) of the Bothrops moojeni venom, was the ligand of an affinity resin used to isolate specific anti-Ile1-Hse11 antibodies which were instrumental in revealing immunological cross-reactivity among unrelated venom proteins. We examined the extent of the cross-reactivity of these antibodies by probing electroblots of venoms from representative snakes of genera Bothrops, Lachesis, Crotalus and Micrurus, and by unambiguous structural characterization of the affinity-purified proteins of B. moojeni venom recovered from an agarose-anti-Ile1-Hse11 column. Our results indicate that all venoms tested had at least three reactive components toward anti-Ile1-Hse11 antibodies, among which we identified two serine proteases, one phospholipase A2 homologue, and LAAO. We hypothesize that the cross-reactivity of the anti-Ile1-Hse11 antibodies to unrelated venom proteins derives from their mechanism of antigen recognition, whereby complementarity is achieved through reciprocal conformational adaptation of the reacting molecules. Also, we believe these findings have implications both in the development of improved antivenoms and the preparation of immunochemical reagents for diagnostic and scientific investigation purposes in the field of snake venoms.

  15. Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments.

    PubMed

    Prado, Nidiane D R; Pereira, Soraya S; da Silva, Michele P; Morais, Michelle S S; Kayano, Anderson M; Moreira-Dill, Leandro S; Luiz, Marcos B; Zanchi, Fernando B; Fuly, André L; Huacca, Maribel E F; Fernandes, Cleberson F; Calderon, Leonardo A; Zuliani, Juliana P; Pereira da Silva, Luiz H; Soares, Andreimar M; Stabeli, Rodrigo G; Fernandes, Carla F C

    2016-01-01

    Antivenoms, produced using animal hyperimmune plasma, remains the standard therapy for snakebites. Although effective against systemic damages, conventional antivenoms have limited efficacy against local tissue damage. Additionally, the hypersensitivity reactions, often elicited by antivenoms, the high costs for animal maintenance, the difficulty of producing homogeneous lots, and the instability of biological products instigate the search for innovative products for antivenom therapy. In this study, camelid antibody fragments (VHH) with specificity to Bothropstoxin I and II (BthTX-I and BthTX-II), two myotoxic phospholipases from Bothrops jararacussu venom, were selected from an immune VHH phage display library. After biopanning, 28 and 6 clones recognized BthTX-I and BthTX-II by ELISA, respectively. Complementarity determining regions (CDRs) and immunoglobulin frameworks (FRs) of 13 VHH-deduced amino acid sequences were identified, as well as the camelid hallmark amino acid substitutions in FR2. Three VHH clones (KF498607, KF498608, and KC329718) were capable of recognizing BthTX-I by Western blot and showed affinity constants in the nanomolar range against both toxins. VHHs inhibited the BthTX-II phospholipase A2 activity, and when tested for cross-reactivity, presented specificity to the Bothrops genus in ELISA. Furthermore, two clones (KC329718 and KF498607) neutralized the myotoxic effects induced by B. jararacussu venom, BthTX-I, BthTX-II, and by a myotoxin from Bothrops brazili venom (MTX-I) in mice. Molecular docking revealed that VHH CDRs are expected to bind the C-terminal of both toxins, essential for myotoxic activity, and to epitopes in the BthTX-II enzymatic cleft. Identified VHHs could be a biotechnological tool to improve the treatment for snake envenomation, an important and neglected world public health problem.

  16. Poor efficacy of preemptive amoxicillin clavulanate for preventing secondary infection from Bothrops snakebites in the Brazilian Amazon: A randomized controlled clinical trial.

    PubMed

    Sachett, Jacqueline A G; da Silva, Iran Mendonça; Alves, Eliane Campos; Oliveira, Sâmella S; Sampaio, Vanderson S; do Vale, Fábio Francesconi; Romero, Gustavo Adolfo Sierra; Dos Santos, Marcelo Cordeiro; Marques, Hedylamar Oliveira; Colombini, Mônica; da Silva, Ana Maria Moura; Wen, Fan Hui; Lacerda, Marcus V G; Monteiro, Wuelton M; Ferreira, Luiz C L

    2017-07-01

    Secondary bacterial infections from snakebites contribute to the high complication rates that can lead to permanent function loss and disabilities. Although common in endemic areas, routine empirical prophylactic use of antibiotics aiming to prevent secondary infection lacks a clearly defined policy. The aim of this work was to estimate the efficacy of amoxicillin clavulanate for reducing the secondary infection incidence in patients bitten by Bothrops snakes, and, secondarily, identify risk factors for secondary infections from snakebites in the Western Brazilian Amazon. This was an open-label, two-arm individually randomized superiority trial to prevent secondary infection from Bothrops snakebites. The antibiotic chosen for this clinical trial was oral amoxicillin clavulanate per seven days compared to no intervention. A total of 345 patients were assessed for eligibility in the study period. From this total, 187 accomplished the inclusion criteria and were randomized, 93 in the interventional group and 94 in the untreated control group. All randomized participants completed the 7 days follow-up period. Enzyme immunoassay confirmed Bothrops envenoming diagnosis in all participants. Primary outcome was defined as secondary infection (abscess and/or cellulitis) until day 7 after admission. Secondary infection incidence until 7 days after admission was 35.5% in the intervention group and 44.1% in the control group [RR = 0.80 (95%CI = 0.56 to 1.15; p = 0.235)]. Survival analysis demonstrated that the time from patient admission to the onset of secondary infection was not different between amoxicillin clavulanate treated and control group (Log-rank = 2.23; p = 0.789).Secondary infections incidence in 7 days of follow-up was independently associated to fibrinogen >400 mg/dL [AOR = 4.78 (95%CI = 2.17 to 10.55; p<0.001)], alanine transaminase >44 IU/L [AOR = 2.52 (95%CI = 1.06 to 5.98; p = 0.037)], C-reactive protein >6.5 mg/L [AOR = 2.98 (95%CI = 1.40 to 6.35; p = 0

  17. Cytotoxic action in myoblasts and myotubes (C2C12) and enzymatic characterization of a new phospholipase A2 isoform (Bj-V) from Bothrops jararacussu venom.

    PubMed

    Bonfim, Vera Luis; Ponce-Soto, Luis Alberto; Novello, Jose Camilo; Marangoni, Sergio

    2006-01-01

    A new PLA2 Bj-V from Bothrops jararacussu (14039.49 Da determined by MALDI-TOF mass spectrometry) was isolated in only one chromatographic step by HPLC ion-exchange and its purity was confirmed by reverse phase. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The N-terminal sequence (DLWQFGQMIL KETGKIPFPY YGAYGCYCGW GGRGGKPKDG TDRCCYVHD...) showed a high degree of homology with basic D49 PLA2 myotoxins from other Bothrops venoms. Bj V showed discrete sigmoidal enzymatic behavior, with maximal activity at pH 8.4 and 35-40 degrees C. Full PLA2 activity required Ca2+ (10 mM) and there was little catalytic activity in the presence of 1 mM Ca2+. The addition of Mn2+ or Mg2+ (10 mM) in the presence of low (1 mM) Ca2+ slightly increased the enzyme activity, whereas Zn2+ and Cu2+ (10 mM) diminished the activity. The substitution of Ca2+ for Mg2+ or Cu2+ also reduced the enzymatic activity. Bj V had PLA2 activity and produced cytotoxicity in murine C2C12 skeletal muscle myoblasts and myotubes. The isolation of these isoforms Bj-IV [1] and Bj-V (described herein) found in a fraction previously described as homogeneous shows us the importance of optimization in purification techniques in order to better understand their biological behavior.

  18. Contribution of mast cells to the oedema induced by Bothrops moojeni snake venom and a pharmacological assessment of the inflammatory mediators involved.

    PubMed

    Galvão Nascimento, Neide; Sampaio, Marlos Cortez; Amaral Olivo, Renata; Teixeira, Catarina

    2010-01-01

    The ability of Bothrops moojeni venom (BmV) to induce oedema in mice, the involvement of principal inflammatory mediators and mast cells (MCs) were investigated. The intraplantar injection of BmV (0.3-6 microg/paw) caused a dose- and time-dependent oedema with a peak between 30 and 60 min after venom injection (0.3-1 microg/paw), disappearing within 24h. Either MCs granule inhibition or depletion by cromoglycate or C48/80, respectively, markedly reduced BmV-induced oedema. MCs depletion by imatinib also reduced oedema. Intraperitoneal BmV injection (2.5-10 microg/site) induced MCs degranulation and release of PGD(2). Treatment with promethazine, cimetidine or thioperamide, histamine H1, H2 and H3/H4 receptor antagonists, respectively, markedly reduced the initial phase of oedema. Combined treatment with these antagonists further reduced, but not abrogated oedema. Indomethacin or eterocoxib (cyclooxygenase inhibitors) reduced oedema until 180 min, whereas zileuton (lipoxygenase inhibitor) affected this event until 60 min. Dexamethazone caused a long lasting reduction of oedema. However, L-NAME and aminoguanidine (NO synthase inhibitors) significantly increased BmV-induced oedema. In conclusion, BmV induces oedema, mediated by MCs degranulation, histamine by H1, H2, H3/H4 receptors, prostaglandins and leukotrienes, and down-regulated by NO. Partial neutralization of oedema was observed even when polyspecific bothropic antivenom was injected immediately after venom. Copyright 2009 Elsevier Ltd. All rights reserved.

  19. 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).

  20. Local inflammatory events induced by Bothrops atrox snake venom and the release of distinct classes of inflammatory mediators.

    PubMed

    Moreira, Vanessa; Dos-Santos, Maria Cristina; Nascimento, Neide Galvão; Borges da Silva, Henrique; Fernandes, Cristina Maria; D'Império Lima, Maria Regina; Teixeira, Catarina

    2012-07-01

    Bothrops atrox is responsible for most accidents involving snakes in the Brazilian Amazon and its venom induces serious systemic and local effects. The local effects are not neutralized effectively by commercial antivenoms, resulting in serious sequelae in individuals bitten by this species. This study investigates the local inflammatory events induced in mice by B. atrox venom (BaV), such as vascular permeability, leukocyte influx and the release of important inflammatory mediators such as cytokines, eicosanoids and the chemokine CCL-2, at the injection site. The effect of BaV on cyclooxygenase (COX-1 and COX-2) expression was also investigated. The results showed that intraperitoneal (i.p.) injection of BaV promoted a rapid and significant increase in vascular permeability, which reached a peak 1 h after venom administration. Furthermore, BaV caused leukocyte infiltration into the peritoneal cavity between 1 and 8 h after i.p. injection, with mononuclear leukocytes (MNs) predominating in the first 4 h, and polymorphonuclear leukocytes (PMNs) in the last 4 h. Increased protein expression of COX-2, but not of COX-1, was detected in leukocytes recruited in the first and fourth hours after injection of BaV. The venom caused the release of eicosanoids PGD₂, PGE₂, TXA₂ and LTB₄, cytokines TNF-α, IL-6, IL-10 and IL-12p70, but not IFN-γ, and chemokine CCL-2 at different times. The results show that BaV is able to induce an early increase in vascular permeability and a leukocyte influx to the injection site consisting mainly of MNs initially and PMNs during the later stages. These phenomena are associated with the production of cytokines, the chemokine CCL-2 and eicosanoids derived from COX-1 and COX-2. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. Isotopic change in the tissues of Bothrops atrox in captivity collected from environments of the eastern Amazon

    NASA Astrophysics Data System (ADS)

    Martinez, M. G.; Chalkidis, H. D.; Amazonas, D. R.; da Silva, A. M.; De Oliveira, R., Jr.; Camargo, P. B.

    2013-12-01

    The Bothrops atrox is little studied because it is sympatric Amazonian animals, and very little is known about the ecology and natural history of this species. It has a generalist diet and the distribution of this species is very wide. The adult animals forage mostly on the ground, while the younger animals prefer to stay on the vegetation. They are easily find in the rainy months in areas near lakes and seasonally flooded and are difficult to find in the driest months, a period where there is less availability of preys in these environments. Due to its aggressiveness, is considered one of the most feared snakes in South America and in the eastern Amazon, being responsible for the largest number of snakebites in the region. Through stable isotope carbon-13 and nitrogen-15, is intended to characterize the variations of the feeding habits of these collected animals in different environments and also when they are kept in captivity, feeding the animal's bioterium. The serpents were collected in environments with different land uses, such as native forest, savannah, pasture and have been brought to the serpentarium Integrated College Tapajos (FIT), being retained in order to Samplings throughout the experiment with feeding mice's own bioterium. When these snakes came from different locations, samples were collected scales and blood (T0), before receiving the new supply (captive), and every time we fed the mice the vivarium, new tissue samples were collected, (T1, T2, T3) to exchange all the nature of food for the food captivity.Based on the results of δ13C and δ15N, the samples collected in the tissues of snakes of different environments (nature and captivity), it was observed that changes in food sources reflect changes in tissues (blood and scales), also reflecting the production of poison different periods of turnover of absorbed material in those tissues, contributing to the study of animal ecology and behavior in relation to habitat.

  2. Bothrops lanceolatus (Fer de lance) venom induces oedema formation and increases vascular permeability in the mouse hind paw.

    PubMed

    de Araújo, A L; de Souza, A O; da Cruz-Höfling, M A; Flores, C A; Bon, C

    2000-02-01

    The ability of snake venoms to increase vascular permeability and to induce oedema through the release of pharmacologically active substances is well known. We have studied the oedema and vascular permeability induced by Bothrops lanceolatus venom in male Swiss white mice. Paw oedema was induced by the subplantar injection of B. lanceolatus venom (125-1000 ng/paw) and was quantified as the increase in paw weight. Changes in vascular permeability were assessed by measuring the amount of Evans blue dye extravasation. The oedema and the increase in vascular permeability were maximal within 2 h and had resolved after 24 h. The administration of the vasodilator iloprost (20 ng/paw) immediately after B. lanceolatus venom potentiated the oedema and the increase in vascular permeability by approximately four-fold. Pretreating the mice with indomethacin, dexamethasone, NDGA or BW A4C inhibited the venom-induced oedema and the increase in vascular permeability. In contrast, histamine, serotonin and PAF-acether antagonists (mepyramine, cyproheptadine and WEB 2086, respectively) were ineffective. Histological examination showed that B. lanceolatus venom (250 ng and 500 ng/paw) caused thickening of the inner dermal layers which was accompanied by extensive intercellular spaces indicative of oedema. In addition, there was a marked infiltration of inflammatory cells, particularly neutrophils, into the underlying muscle layer. The latter, however, remained morphologically unaffected during the 3 h of observation. Venom doses larger than 500 ng/paw produced intense haemorrhage. These results indicate that B. lanceolatus venom induces oedema and increases vascular permeability in the mouse hind paw. The principal mediators of this inflammatory response are cyclooxygenase and lipoxygenase products.

  3. 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.

  4. Structural and binding studies of a C-type galactose-binding lectin from Bothrops jararacussu snake venom.

    PubMed

    Sartim, Marco A; Pinheiro, Matheus P; de Pádua, Ricardo A P; Sampaio, Suely V; Nonato, M Cristina

    2017-02-01

    BJcuL is a snake venom galactoside-binding lectin (SVgalL) isolated from Bothrops jararacussu and is involved in a wide variety of biological activities including triggering of pro-inflammatory response, disruption of microbial biofilm structure and induction of apoptosis. In the present work, we determined the crystallographic structure of BJcuL, the first holo structure of a SVgalL, and introduced the fluorescence-based thermal stability assay (Thermofluor) as a tool for screening and characterization of the binding mechanism of SVgalL ligands. BJcuL structure revealed the existence of a porous and flexible decameric arrangement composed of disulfide-linked dimers related by a five-fold symmetry. Each monomer contains the canonical carbohydrate recognition domain, a calcium ion required for BJcuL lectinic activity and a sodium ion required for protein stabilization. BJcuL thermostability was found to be induced by calcium ion and galactoside sugars which exhibit hyperbolic saturation profiles dependent on ligand concentration. Serendipitously, the gentamicin group of aminoglycoside antibiotics (gAGAs) was also identified as BJcuL ligands. On contrast, gAGAs exhibited a sigmoidal saturation profile compatible with a cooperative mechanism of binding. Thermofluor, hemagglutination inhibition assay and molecular docking strategies were used to identify a distinct binding site in BJcuL localized at the dimeric interface near the fully conserved intermolecular Cys86-Cys86 disulfide bond. The hybrid approach used in the present work provided novel insights into structural behavior and functional diversification of SVgaLs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. The role of indomethacin and tezosentan on renal effects induced by Bothrops moojeni Lys49 myotoxin I.

    PubMed

    Barbosa, P S F; Martins, A M C; Alves, R S; Amora, D N; Martins, R D; Toyama, Marcos H; Havt, A; Nascimento, N R F; Rocha, V L C; Menezes, D B; Fonteles, M C; Monteiro, H S A

    2006-06-15

    Renal changes determined by Lys49 myotoxin I (BmTx I), isolated from Bothrops moojeni are well known. The scope of the present study was to investigate the possible mechanisms involved in the production of these effects by using indomethacin (10 microg/mL), a non-selective inhibitor of cyclooxygenase, and tezosentan (10 microg/mL), an endothelin antagonist. By means of the method of mesenteric vascular bed, it has been observed that B. moojeni myotoxin (5 microg/mL) affects neither basal perfusion pressure nor phenylephrine-preconstricted vessels. This fact suggests that the increase in renal perfusion pressure and in renal vascular resistance did not occur by a direct effect on renal vasculature. Isolated kidneys from Wistar rats, weighing 240-280 g, were perfused with Krebs-Henseleit solution. The infusion of BmTx-I increased perfusion pressure, renal vascular resistance, urinary flow and glomerular filtration rate. Sodium, potassium and chloride tubular transport was reduced after addition of BmTx-I. Indomethacin blocked the effects induced by BmTx-I on perfusion pressure and renal vascular resistance, however, it did not revert the effect on urinary flow and sodium, potassium and chloride tubular transport. The alterations of glomerular filtration rate were inhibited only at 90 min of perfusion. The partial blockade exerted by indomethacin treatment showed that prostaglandins could have been important mediators of BmTx-I renal effects, but the participation of other substances cannot be excluded. The blockage of all renal alterations observed after tezosentan treatment support the hypothesis that endothelin is the major substance involved in the renal pathophysiologic alterations promoted by the Lys49 PLA(2) myotoxin I, isolated from B. moojeni. In conclusion, the rather intense renal effects promoted by B. moojeni myotoxin-I were probably caused by the release of renal endothelin, interfering with the renal parameters studied.

  6. 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

  7. Anti-platelet aggregation activity of two novel acidic Asp49-phospholipases A2 from Bothrops brazili snake venom.

    PubMed

    Sobrinho, Juliana C; Kayano, Anderson M; Simões-Silva, Rodrigo; Alfonso, Jorge J; Gomez, Ana F; Gomez, Maria C Vega; Zanchi, Fernando B; Moura, Laura A; Souza, Vivian R; Fuly, André L; de Oliveira, Eliandre; da Silva, Saulo L; Almeida, José R; Zuliani, Juliana P; Soares, Andreimar M

    2017-09-23

    Phospholipases A2 (PLA2s) are important enzymes present in snake venoms and are related to a wide spectrum of pharmacological effects, however the toxic potential and therapeutic effects of acidic isoforms have not been fully explored and understood. Due to this, the present study describes the isolation and biochemical characterization of two new acidic Asp49-PLA2s from Bothrops brazili snake venom, named Braziliase-I and Braziliase-II. The venom was fractionated in three chromatographic steps: ion exchange, hydrophobic interaction and reversed phase. The isoelectric point (pI) of the isolated PLA2s was determined by two-dimensional electrophoresis, and 5.2 and 5.3 pIs for Braziliase-I and II were observed, respectively. The molecular mass was determined with values ​​of 13,894 and 13,869Da for Braziliase-I and II, respectively. Amino acid sequence by Edman degradation and mass spectrometry completed 87% and 74% of the sequences, respectively for Braziliase-I and II. Molecular modeling of isolated PLA2s using acid PLA2BthA-I-PLA2 from B. jararacussu template showed high quality. Both acidic PLA2s showed no significant myotoxic activity, however they induced significant oedematogenic activity. Braziliase-I and II (100μg/mL) showed 31.5% and 33.2% of cytotoxicity on Trypanosoma cruzi and 26.2% and 19.2% on Leishmania infantum, respectively. Braziliase-I and II (10μg) inhibited 96.98% and 87.98% of platelet aggregation induced by ADP and 66.94% and 49% induced by collagen, respectively. The acidic PLA2s biochemical and structural characterization can lead to a better understanding of its pharmacological effects and functional roles in snakebites pathophysiology, as well as its possible biotechnological applications as research probes and drug leads. Copyright © 2017. Published by Elsevier B.V.

  8. Hemorrhagic, coagulant and fibrino(geno)lytic activities of crude venom and fractions from mapanare (Bothrops colombiensis) snakes.

    PubMed

    Girón, María E; Salazar, Ana M; Aguilar, Irma; Pérez, John C; Sánchez, Elda E; Arocha-Piñango, Carmen L; Rodríguez-Acosta, Alexis; Guerrero, Belsy

    2008-01-01

    Bothrops colombiensis venom from two similar geographical locations were tested for their hemostatic functions and characterized by gel-filtration chromatography and SDS-PAGE electrophoresis. The snakes were from Caucagua and El Guapo towns of the Venezuelan state of Miranda. Fibrino(geno)lytic, procoagulant, hemorrhagic, lethal activities, gel-filtration chromatography and SDS-PAGE profiles were analyzed and compared for both venoms. The highest hemorrhagic activity of 5.3 mug was seen in El Guapo venom while Caucagua venom had the lowest LD(50) of 5.8 mg/kg. Both venoms presented similar thrombin-like activity. El Guapo showed a factor Xa-like activity two times higher than Caucagua. Differences were observed in kallikrein-like and t-PA activities, being highest in El Guapo. Caucagua venom showed the maximum fibrin lysis. Both crude venom runs on Sephadex G-100 chromatography gave fraction SII with the high fibrinolytic activity. Proteases presented in SII fractions and eluted from Benzamidine-Sepharose (not bound to the column) provoked a fast degradation of fibrinogen alpha chains and a slower degradation of beta chains, which could possibly be due to a higher content of alpha fibrinogenases in these venoms. The fibrinogenolytic activity was decreased by metalloprotease inhibitors. The results suggested that metalloproteases in SII fractions were responsible for the fibrinolytic activity. The analysis of samples for fibrin-zymography of SII fractions showed an active band with a molecular mass of approximately 30 kDa. These results reiterate the importance of using pools of venoms for antivenom immunization, to facilitate the neutralization of the maximum potential number of toxins.

  9. Crystal structure of a phospholipase A2 from Bothrops asper venom: Insights into a new putative "myotoxic cluster".

    PubMed

    Salvador, Guilherme H M; Dos Santos, Juliana I; Lomonte, Bruno; Fontes, Marcos R M

    2017-02-01

    Snake venoms from the Viperidae and Elapidae families often have several phospholipases A2 (PLA2s), which may display different functions despite having a similar structural scaffold. These proteins are considered an important target for the development of drugs against local myotoxic damage because they are not efficiently neutralized by conventional serum therapy. PLA2s from these venoms are generally divided into two classes: (i) catalytic PLA2s (or Asp49-PLA2s) and (ii) non-catalytic PLA2-like toxins (or Lys49-PLA2s). In many Viperidae venoms, a subset of the basic Asp49-PLA2s displays some functional and structural characteristics of PLA2-like proteins and group within the same phylogenetic clade, but their myotoxic mechanism is still largely unknown. In the present study, we have crystallized and solved the structure of myotoxin I (MT-I), a basic myotoxic Asp49-PLA2 isolated from Bothrops asper venom. The structure presents a dimeric conformation that is compatible with that of previous dimers found for basic myotoxic Asp49-PLA2s and Lys49-PLA2s and has been confirmed by other biophysical and bioinformatics techniques. This arrangement suggests a possible cooperative action between both monomers to exert myotoxicity via two different sites forming a putative membrane-docking site (MDoS) and a putative membrane disruption site (MDiS). This mechanism would resemble that proposed for Lys49-PLA2s, but the sites involved appear to be situated in a different region. Thus, as both sites are close to one another, they form a "myotoxic cluster", which is also found in two other basic myotoxic Asp49-PLA2s from Viperidae venoms. Such arrangement may represent a novel structural strategy for the mechanism of muscle damage exerted by the group of basic, Asp49-PLA2s found in viperid snake venoms.

  10. Neutralisation of the pharmacological activities of Bothrops alternatus venom by anti-PLA2 IgGs.

    PubMed

    Garcia Denegri, María E; Maruñak, Silvana; Todaro, Juan S; Ponce-Soto, Luis A; Acosta, Ofelia; Leiva, Laura

    2014-08-01

    Basic phospholipases A2 (PLA2) are toxic and induce a wide spectrum of pharmacological effects, although the acidic enzyme types are not lethal or cause low lethality. Therefore, it is challenging to elucidate the mechanism of action of acidic phospholipases. This study used the acidic non-toxic Ba SpII RP4 PLA2 from Bothrops alternatus as an antigen to develop anti-PLA2 IgG antibodies in rabbits and used in vivo assays to examine the changes in crude venom when pre-incubated with these antibodies. Using Ouchterlony and western blot analyses on B. alternatus venom, we examined the specificity and sensitivity of phospholipase A2 recognition by the specific antibodies (anti-PLA2 IgG). Neutralisation assays using a non-toxic PLA2 antigen revealed unexpected results. The (indirect) haemolytic activity of whole venom was completely inhibited, and all catalytically active phospholipases A2 were blocked. Myotoxicity and lethality were reduced when the crude venom was pre-incubated with anti-PLA2 immunoglobulins. CK levels in the skeletal muscle were significantly reduced at 6 h, and the muscular damage was more significant at this time-point compared to 3 and 12 h. When four times the LD50 was used (224 μg), half the animals treated with the venom-anti PLA2 IgG mixture survived after 48 h. All assays performed with the specific antibodies revealed that Ba SpII RP4 PLA2 had a synergistic effect on whole-venom toxicity. IgG antibodies against the venom of the Argentinean species B. alternatus represent a valuable tool for elucidation of the roles of acidic PLA2 that appear to have purely digestive roles and for further studies on immunotherapy and snake envenoming in affected areas in Argentina and Brazil.

  11. Aqueous two-phase systems: A simple methodology to obtain mixtures enriched in main toxins of Bothrops alternatus venom.

    PubMed

    Gomez, Gabriela; Leiva, Laura; Nerli, Bibiana Beatriz

    2016-08-01

    Phospholipase A2 (PLA2) and protease (P) are enzymes responsible of myotoxic, edematogenic and hemostasis disorder effects observed in the envenomation by Bothrops alternatus pitviper. Their partitioning coefficient (Kp) in different polyethyleneglycol/potassium phosphate aqueous two-phase systems (ATPSs) was determined in order to both achieve a better understanding of the partitioning mechanism and define optimal conditions for toxin isolation. Polyethyleneglycols (PEGs) of molecular weights 1000; 3350; 6000 and 8000; different temperatures (5, 20 and 37 °C) and phase volume ratios of 0.5; 1 and 2 were assayed. PLA2 partitioned preferentially to the top phase while P mainly distributed to the bottom phase. Either entropically- or enthalpically-driven mechanisms were involved in each case (PLA2 and P). The aqueous two-phase system formed by PEG of MW 3350 (12.20% wt/wt) and KPi pH 7.0 (11.82% wt/wt) with a volume ratio of one and a load of 1.25 mg of venom/g of system showed to be the most efficient to recover both enzymes. It allowed obtaining the 72% of PLA2 in the top phase with a purification factor of 2 and the 82% of P at the bottom phase simultaneously. A further adsorption batch step with DEAE-cellulose was used to remove satisfactorily the PEG from the top phase and recover the active PLA2. The proposed methodology is simple, inexpensive, and only requires professionals trained in handling basic laboratory equipment. It could be easily adoptable by developing countries in which the snakebite accidents cause considerable morbidity and mortality.

  12. Activation of J77A.1 Macrophages by Three Phospholipases A2 Isolated from Bothrops atrox Snake Venom

    PubMed Central

    Furtado, Juliana L.; Oliveira, George A.; Pontes, Adriana S.; Setúbal, Sulamita da S.; Xavier, Caroline V.; Lacouth-Silva, Fabianne; Lima, Beatriz F.; Zaqueo, Kayena D.; Kayano, Anderson M.; Calderon, Leonardo A.; Stábeli, Rodrigo G.; Soares, Andreimar M.; Zuliani, Juliana P.

    2014-01-01

    In the present study, we investigated the in vitro effects of two basic myotoxic phospholipases A2 (PLA2), BaTX-I, a catalytically inactive Lys-49 variant, and BaTX-II, a catalytically active Asp-49, and of one acidic myotoxic PLA2, BaPLA2, a catalytically active Asp-49, isolated from Bothrops atrox snake venom, on the activation of J774A.1 macrophages. At noncytotoxic concentrations, the toxins did not affect the adhesion of the macrophages, nor their ability to detach. The data obtained showed that only BaTX-I stimulated complement receptor-mediated phagocytosis. However, BaTX-I, BaTX-II, and BaPLA2 induced the release of the superoxide anion by J774A.1 macrophages. Additionally, only BaTX-I raised the lysosomal volume of macrophages after 15 min of incubation. After 30 min, all the phospholipases increased this parameter, which was not observed within 60 min. Moreover, BaTX-I, BaTX-II, and BaPLA2 increased the number of lipid bodies on macrophages submitted to phagocytosis and not submitted to phagocytosis. However, BaTX-II and BaPLA2 induced the release of TNF-α by J774A.1 macrophages. Taken together, the data show that, despite differences in enzymatic activity, the three toxins induced inflammatory events and whether the enzyme is acidic or basic does not seem to contribute to these effects. PMID:24592395

  13. Poor Regenerative Outcome after Skeletal Muscle Necrosis Induced by Bothrops asper Venom: Alterations in Microvasculature and Nerves

    PubMed Central

    Hernández, Rosario; Cabalceta, Carmen; Saravia-Otten, Patricia; Chaves, Alessandra; Gutiérrez, José María; Rucavado, Alexandra

    2011-01-01

    Background Viperid snakebite envenoming is characterized by prominent local tissue damage, including muscle necrosis. A frequent outcome of such local pathology is deficient skeletal muscle regeneration, which causes muscle dysfunction, muscle loss and fibrosis, thus provoking permanent sequelae that greatly affect the quality of life of patients. The causes of such poor regenerative outcome of skeletal muscle after viperid snakebites are not fully understood. Methodology/Principal Findings A murine model of muscle necrosis and regeneration was adapted to study the effects of the venom and isolated toxins of Bothrops asper, the medically most important snake in Central America. Gastrocnemius muscle was injected with either B. asper venom, a myotoxic phospholipase A2 (Mtx), a hemorrhagic metalloproteinase (SVMP), or saline solution. At various time intervals, during one month, tissue samples were collected and analyzed by histology, and by immunocytochemical and immunohistochemical techniques aimed at detecting muscle fibers, collagen, endothelial cells, myoblasts, myotubes, macrophages, TUNEL-positive nuclei, and axons. A successful regenerative response was observed in muscle injected with Mtx, which induces myonecrosis but does not affect the microvasculature. In contrast, poor regeneration, with fibrosis and atrophic fibers, occurred when muscle was injected with venom or SVMP, both of which provoke necrosis, microvascular damage leading to hemorrhage, and poor axonal regeneration. Conclusions/Significance The deficient skeletal muscle regeneration after injection of B. asper venom is likely to depend on the widespread damage to the microvasculature, which affects the removal of necrotic debris by phagocytes, and the provision of nutrients and oxygen required for regeneration. In addition, deficient axonal regeneration is likely to contribute to the poor regenerative outcome in this model. PMID:21629691

  14. Effects of neutrophil depletion in the local pathological alterations and muscle regeneration in mice injected with Bothrops jararaca snake venom

    PubMed Central

    Teixeira, Catarina F P; Chaves, Fernando; Zamunér, Stella R; Fernandes, Cristina M; Zuliani, Juliana P; Cruz-Hofling, María Alice; Fernandes, Irene; Gutiérrez, José María

    2005-01-01

    In order to study the role of neutrophils in the acute local pathological alterations induced by Bothrops jararaca snake venom, and in the process of skeletal muscle regeneration that follows, an experimental model was developed in mice pretreated with either an anti-mouse granulocyte rat monoclonal immunoglobulin G, which induces a profound neutropenia, or an isotype-matched control antibody. B. jararaca venom induced prominent haemorrhage and oedema, but only a moderate myonecrosis. No significant differences were observed in the extent of local haemorrhage, oedema and myonecrosis between neutropenic and control mice, suggesting that neutrophils do not play a determinant role in the acute pathological alterations induced by B. jararaca venom in this experimental model. Moreover, no differences were observed in skeletal muscle regeneration between these two experimental groups. In both the cases, limited areas of myonecrosis were associated with a drastic damage to the microvasculature and a scarce inflammatory infiltrate, with the consequent lack of removal of necrotic debris during the first week, resulting in a poor regenerative response at this time interval. Subsequently, a similar regenerative process occurred in both groups, and by 30 days, necrotic areas were substituted by groups of small regenerating muscle fibres. It is suggested that the drastic effect exerted by B. jararaca venom in the microvasculature precludes an effective access of inflammatory cells to necrotic areas, thereby compromising an effective removal of necrotic debris; this explains the poor regenerative response observed during the first week and the fact that there were no differences between neutropenic and control mice. As neutropenia in this model lasted only 7 days, the successful regenerative process observed at 30 days is associated with revascularization of necrotic regions and with a successful removal by phagocytes of necrotic debris in both groups. PMID:15810982

  15. Muscle Tissue Damage Induced by the Venom of Bothrops asper: Identification of Early and Late Pathological Events through Proteomic Analysis

    PubMed Central

    Herrera, Cristina; Macêdo, Jéssica Kele A.; Feoli, Andrés; Escalante, Teresa; Rucavado, Alexandra; Gutiérrez, José María; Fox, Jay W.

    2016-01-01

    The time-course of the pathological effects induced by the venom of the snake Bothrops asper in muscle tissue was investigated by a combination of histology, proteomic analysis of exudates collected in the vicinity of damaged muscle, and immunodetection of extracellular matrix proteins in exudates. Proteomic assay of exudates has become an excellent new methodological tool to detect key biomarkers of tissue alterations for a more integrative perspective of snake venom-induced pathology. The time-course analysis of the intracellular proteins showed an early presence of cytosolic and mitochondrial proteins in exudates, while cytoskeletal proteins increased later on. This underscores the rapid cytotoxic effect of venom, especially in muscle fibers, due to the action of myotoxic phospholipases A2, followed by the action of proteinases in the cytoskeleton of damaged muscle fibers. Similarly, the early presence of basement membrane (BM) and other extracellular matrix (ECM) proteins in exudates reflects the rapid microvascular damage and hemorrhage induced by snake venom metalloproteinases. The presence of fragments of type IV collagen and perlecan one hour after envenoming suggests that hydrolysis of these mechanically/structurally-relevant BM components plays a key role in the genesis of hemorrhage. On the other hand, the increment of some ECM proteins in the exudate at later time intervals is likely a consequence of the action of endogenous matrix metalloproteinases (MMPs) or of de novo synthesis of ECM proteins during tissue remodeling as part of the inflammatory reaction. Our results offer relevant insights for a more integrative and systematic understanding of the time-course dynamics of muscle tissue damage induced by B. asper venom and possibly other viperid venoms. PMID:27035343

  16. Flow cytometric characterization of peripheral blood leukocyte populations of 3 neotropical snake species: Boa constrictor, Bothrops jararaca, and Crotalus durissus.

    PubMed

    Carvalho, Marcelo P N; Queiroz-Hazarbassanov, Nicolle G T; Massoco, Cristina O; Rossi, Silmara; Sant'Anna, Sávio S; Catão-Dias, José L; Grego, Kathleen F

    2016-06-01

    The reptilian immune system is represented by innate, humoral, and cell-mediated mechanisms, involving different types of blood leukocytes. The development of optimized methods for the advanced study of origin and function of reptilian blood leukocytes is needed. The purpose of the study was to optimize leukocyte density gradient isolation protocols from snake peripheral blood samples, and characterize recovered cells by flow cytometry based on size and internal complexity for a qualitative and semi-quantitative assessment of leukocyte populations in one boa (Boa constrictor), and 2 viper species (Bothrops jararaca, Crotalus durissus). Blood samples from 30 snakes (10 from each species, 5 males and 5 females) were collected in tubes with sodium heparin. Fresh blood was centrifuged with either ficoll-paque PLUS or percoll density gradients for leukocyte isolation. Flow cytometric leukocyte gates were defined based on size (forward scatter [FSC]) and internal complexity (side scatter [SSC]). Relative leukocyte differential counts after sorting the cells in these gates in one snake for each species were compared to conventional light microscopic differential counts on unsorted isolated leukocytes. There was no statistical difference in the relative leukocyte populations, including heterophils, azurophils, and small and large lymphocytes between samples isolated by ficoll or percoll. Four leukocyte gates were identified based on their location in FSC/SSC cytograms. The relative leukocyte differential counts after sorting in single animals showed some agreement with the light microscopy differential count on unsorted cells. Based on FSC and SSC, 4 distinct leukocyte populations were found in ficoll or percoll density gradient isolated leukocytes from peripheral blood from boa and viper species. Further optimization of the technique should allow the performance of functional assays. © 2016 American Society for Veterinary Clinical Pathology.

  17. High throughput screening of bradykinin-potentiating peptides in Bothrops moojeni snake venom using precursor ion mass spectrometry.

    PubMed

    Menin, Laure; Perchuć, Anna; Favreau, Philippe; Perret, Frédéric; Michalet, Sophie; Schöni, Reto; Wilmer, Marianne; Stöcklin, Reto

    2008-06-01

    Snake venoms are known to be an extensive source of bioactive peptides. Bradykinin-potentiating peptides (BPPs) are inhibitors of the angiotensin-converting enzyme that have already been identified in the venom of many snake, scorpion, spider and batrachian species. Their most characteristic structural features are an invariable N-terminal pyroglutamate residue (pGlu or Z) and two consecutive proline residues at the C-terminus. Fragmentation of BPPs by collision-induced dissociation during electrospray tandem mass spectrometry analysis (ESI-MS/MS) generates a predominant signal at m/z 213.1 corresponding to the y-ion of the terminal Pro-Pro fragment. In addition, signals at m/z 226.1 and 240.1 that correspond to the b ions of the N-terminus pGlu-Asn and pGlu-Lys, respectively, can often be observed. Based on these structural determinants, the present work describes an original methodology for the discovery of BPPs in natural extracts using liquid chromatography coupled to ESI-MS/MS operated in precursor ion-scan mode. The venom of the Bothrops moojeni snake was used as a model and the methodology was applied for subsequent structural analysis of the identified precursors by tandem mass spectrometry on quadrupole-time-of-flight (Q-TOF) and matrix-assisted laser-desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) instruments. More than 40 peptides below 2500 Da could be detected, among them 20 were shown to belong to the BPP-like family including the related tripeptides pGlu-Lys-Trp and pGlu-Asn-Trp. A total of 15 new sequences have been identified using this approach.

  18. A novel bradykinin potentiating peptide isolated from Bothrops jararacussu venom using catallytically inactive oligopeptidase EP24.15.

    PubMed

    Rioli, Vanessa; Prezoto, Benedito C; Konno, Katsuhiro; Melo, Robson L; Klitzke, Clécio F; Ferro, Emer S; Ferreira-Lopes, Mônica; Camargo, Antonio C M; Portaro, Fernanda C V

    2008-05-01

    Characterization of the peptide content of venoms has a number of potential benefits for basic research, clinical diagnosis, development of new therapeutic agents, and production of antiserum. Here, we use a substrate-capture assay that employs a catalytically inactive mutant of thimet oligopeptidase (EC 3.4.24.15; EP24.15) to identify novel bioactive peptides in Bothrops jararacussu venom. Of the peptides captured with inactive EP24.15 and identified by mass spectrometry, three were previously identified bradykinin-potentiating peptides (BPP),

  19. Angiogenenic effects of BpLec, a C-type lectin isolated from Bothrops pauloensis snake venom.

    PubMed

    Castanheira, Letícia Eulalio; Lopes, Daiana Silva; Gimenes, Sarah Natalie Cirilo; Deconte, Simone Ramos; Ferreira, Bruno Antônio; Alves, Patricia Terra; Filho, Luiz Ricardo Goulart; Tomiosso, Tatiana Carla; Rodrigues, Renata Santos; Yoneyama, Kelly Aparecida Geraldo; Araújo, Fernanda de Assis; Rodrigues, Veridiana de Melo

    2017-09-01

    The present work reports the effects of a C-type lectin (BpLec) isolated from Bothrops pauloensis snake venom upon in vitro and in vivo angiogenesis models. Initially, we noted that BpLec was not cytotoxic to endothelial cells (tEnd) in doses up to 40μg/mL, but lower doses (2.5μg/mL, 5μg/mL, 10μg/mL and 20μg/mL) reduced tEnd cells adhesion to some extracellular matrix proteins and inhibited the in vitro vessel formation in Matrigel assay stimulated by bFGF. β-galactosides (d-lactose, N-acetyl-d-galactosamine and d-galactose) at 400mM reversed the effect of BpLec on tEnd cells adhesion, whereas d-galactose (400mM) partially reversed BpLec property of inhibiting vessel formation by tEnd cells in Matrigel. In vivo assays showed that BpLec increased hemoglobin content and capillary vessels number in polyether-polyurethane sponge discs subcutaneously implanted into dorsal skin mice. Additionally, BpLec also reduced collagen deposition and did not induce a pro-inflammatory response, as demonstrated by the decreased the secretion of some inflammatory cytokines, whereas myeloperoxidase (MPO) and N-acetylglucosaminidase (NAG) activities were not altered by BpLec. Taken together, our results indicate that BpLec might represent an interesting angiogenesis and inflammatory modulator that could also be used for searching possible therapeutic targets involved in these processes. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Snakebites and ethnobotany in the northwest region of Colombia. Part III: neutralization of the haemorrhagic effect of Bothrops atrox venom.

    PubMed

    Otero, R; Núñez, V; Barona, J; Fonnegra, R; Jiménez, S L; Osorio, R G; Saldarriaga, M; Díaz, A

    2000-11-01

    Thirty-one of 75 extracts of plants used by traditional healers for snakebites, had moderate or high neutralizing ability against the haemorrhagic effect of Bothrops atrox venom from Antioquia and Chocó, north-western Colombia. After preincubation of several doses of every extract (7.8-4000 microg/mouse) with six minimum haemorrhagic doses (10 microg) of venom, 12 of them demonstrated 100% neutralizing capacity when the mixture was i.d. injected into mice (18-20 g). These were the stem barks of Brownea rosademonte (Caesalpiniaceae) and Tabebuia rosea (Bignoniaceae); the whole plants of Pleopeltis percussa (Polypodiaceae), Trichomanes elegans (Hymenophyllaceae) and Senna dariensis (Caesalpiniaceae); rhizomes of Heliconia curtispatha (Heliconiaceae); leaves and branches of Bixa orellana (Bixaceae), Philodendron tripartitum (Araceae), Struthanthus orbicularis (Loranthaceae) and Gonzalagunia panamensis (Rubiaceae); the ripe fruits of Citrus limon (Rutaceae); leaves, branches and stem of Ficus nymphaeifolia (Moraceae). Extracts of another 19 species showed moderate neutralization (21-72%) at doses up to 4 mg/mouse, e.g. the whole plants of Aristolochia grandiflora (Aristolochiaceae), Columnea kalbreyeriana (Gesneriaceae), Sida acuta (Malvaceae), Selaginella articulata (Selaginellaceae) and Pseudoelephantopus spicatus (Asteraceae); rhizomes of Renealmia alpinia (Zingiberaceae); the stem of Strychnos xinguensis (Loganiaceae); leaves, branches and stems of Hyptis capitata (Lamiaceae), Ipomoea cairica (Convolvulaceae), Neurolaena lobata (Asteraceae), Ocimum micranthum (Lamiaceae), Piper pulchrum (Piperaceae), Siparuna thecaphora (Monimiaceae), Castilla elastica (Moraceae) and Allamanda cathartica (Apocynaceae); the macerated ripe fruits of Capsicum frutescens (Solanaceae); the unripe fruits of Crescentia cujete (Bignoniaceae); leaves and branches of Piper arboreum (Piperaceae) and Passiflora quadrangularis (Passifloraceae). When the extracts were independently administered

  1. The inhibitory potential of the condensed-tannin-rich fraction of Plathymenia reticulata Benth. (Fabaceae) against Bothrops atrox envenomation.

    PubMed

    de Moura, Valéria Mourão; da Silva, Wania Cristina Rodrigues; Raposo, Juliana D A; Freitas-de-Sousa, Luciana A; Dos-Santos, Maria Cristina; de Oliveira, Ricardo Bezerra; Veras Mourão, Rosa Helena

    2016-05-13

    Ethnobotanical studies have shown that Plathymenia reticulata Benth. (Fabaceae) has been widely used in cases of snake envenomation, particularly in Northern Brazil. In light of this, the aim of this study was to evaluate the inhibitory potential of the condensed-tannin-rich fraction obtained from the bark of P. reticulata against the main biological activities induced by Bothrops atrox venom (BaV). The chemical composition of the aqueous extract of P. reticulata (AEPr) was first investigated by thin-layer chromatography (TLC) and the extract was then fractionated by column chromatography on Sephadex LH-20. This yielded five main fractions (Pr1, Pr2, Pr3, Pr4 and Pr5), which were analyzed by colorimetry to determine their concentrations of total phenolics, total tannins and condensed tannins and to assess their potential for blocking the phospholipase activity of BaV. The Pr5 fraction was defined as the fraction rich in condensed tannins (CTPr), and its inhibitory potential against the activities of the venom was evaluated. CTPr was evaluated in different in vivo and in vitro experimental protocols. The in vivo protocols consisted of (1) pre-incubation (venom:CTPr, w/w), (2) pre-treatment (orally administered) and (3) post-treatment (orally administered) to evaluate the effect on the hemorrhagic and edematogenic activities of BaV; in the in vitro protocol the effect on phospholipase and coagulant activity using pre-incubation in both tests was evaluated. There was statistically significant inhibition (p<0.05) of hemorrhagic activity by CTPr when the pre-incubation protocol was used [55% (1:5, w/w) and 74% (1:10, w/w)] and when pre-treatment with doses of 50 and 100mg/kg was used (19% and 13%, respectively). However, for the concentrations tested, there was no statistically significant inhibition in the group subjected to post-treatment administered orally. CTPr blocked 100% of phospholipase activity and 63.3% (1:10, w/w) of coagulant activity when it was pre

  2. 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

  3. Structural and Functional Studies of a Bothropic Myotoxin Complexed to Rosmarinic Acid: New Insights into Lys49-PLA2 Inhibition

    PubMed Central

    dos Santos, Juliana I.; Cardoso, Fábio F.; Soares, Andreimar M.; dal Pai Silva, Maeli; Gallacci, Márcia; Fontes, Marcos R. M.

    2011-01-01

    Snakebite envenoming is an important public health problem in many tropical and subtropical countries, and is considered a neglected tropical disease by the World Health Organization. Most severe cases are inflicted by species of the families Elapidae and Viperidae, and lead to a number of systemic and local effects in the victim. One of the main problems regarding viperidic accidents is prominent local tissue damage whose pathogenesis is complex and involves the combined actions of a variety of venom components. Phospholipases A2 (PLA2s) are the most abundant muscle-damaging components of these venoms. Herein, we report functional and structural studies of PrTX-I, a Lys49-PLA2 from Bothops pirajai snake venom, and the influence of rosmarinic acid (RA) upon this toxin's activities. RA is a known active component of some plant extracts and has been reported as presenting anti-myotoxic properties related to bothopic envenomation. The myotoxic activity of Lys49-PLA2s is well established in the literature and although no in vivo neurotoxicity has been observed among these toxins, in vitro neuromuscular blockade has been reported for some of these proteins. Our in vitro studies show that RA drastically reduces both the muscle damage and the neuromuscular blockade exerted by PrTX-I on mice neuromuscular preparations (by ∼80% and ∼90%, respectively). These results support the hypothesis that the two effects are closely related and lead us to suggest that they are consequences of the muscle membrane-destabilizing activity of the Lys49-PLA2. Although the C-terminal region of these proteins has been reported to comprise the myotoxic site, we demonstrate by X-ray crystallographic studies that RA interacts with PrTX-I in a different region. Consequently, a new mode of Lys49-PLA2 inhibition is proposed. Comparison of our results with others in the literature suggests possible new ways to inhibit bothropic snake venom myotoxins and improve serum therapy. PMID:22205953

  4. 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

  5. Poor efficacy of preemptive amoxicillin clavulanate for preventing secondary infection from Bothrops snakebites in the Brazilian Amazon: A randomized controlled clinical trial

    PubMed Central

    Sachett, Jacqueline A. G.; da Silva, Iran Mendonça; Alves, Eliane Campos; Oliveira, Sâmella S.; do Vale, Fábio Francesconi; dos Santos, Marcelo Cordeiro; Marques, Hedylamar Oliveira; Colombini, Mônica; da Silva, Ana Maria Moura; Wen, Fan Hui; Lacerda, Marcus V. G.; Monteiro, Wuelton M.

    2017-01-01

    Background Secondary bacterial infections from snakebites contribute to the high complication rates that can lead to permanent function loss and disabilities. Although common in endemic areas, routine empirical prophylactic use of antibiotics aiming to prevent secondary infection lacks a clearly defined policy. The aim of this work was to estimate the efficacy of amoxicillin clavulanate for reducing the secondary infection incidence in patients bitten by Bothrops snakes, and, secondarily, identify risk factors for secondary infections from snakebites in the Western Brazilian Amazon. Methods and findings This was an open-label, two-arm individually randomized superiority trial to prevent secondary infection from Bothrops snakebites. The antibiotic chosen for this clinical trial was oral amoxicillin clavulanate per seven days compared to no intervention. A total of 345 patients were assessed for eligibility in the study period. From this total, 187 accomplished the inclusion criteria and were randomized, 93 in the interventional group and 94 in the untreated control group. All randomized participants completed the 7 days follow-up period. Enzyme immunoassay confirmed Bothrops envenoming diagnosis in all participants. Primary outcome was defined as secondary infection (abscess and/or cellulitis) until day 7 after admission. Secondary infection incidence until 7 days after admission was 35.5% in the intervention group and 44.1% in the control group [RR = 0.80 (95%CI = 0.56 to 1.15; p = 0.235)]. Survival analysis demonstrated that the time from patient admission to the onset of secondary infection was not different between amoxicillin clavulanate treated and control group (Log-rank = 2.23; p = 0.789).Secondary infections incidence in 7 days of follow-up was independently associated to fibrinogen >400 mg/dL [AOR = 4.78 (95%CI = 2.17 to 10.55; p<0.001)], alanine transaminase >44 IU/L [AOR = 2.52 (95%CI = 1.06 to 5.98; p = 0.037)], C-reactive protein >6.5 mg/L [AOR = 2

  6. Crystallization and preliminary X-ray diffraction analysis of myotoxin I, a Lys49-phospholipase A{sub 2} from Bothrops moojeni

    SciTech Connect

    Marchi-Salvador, D. P.; Silveira, L. B.; Soares, A. M.

    2005-10-01

    A new myotoxic Lys49-phospholipase from B. moojeni has been crystallized and X-ray diffraction data were collected to 2.18 Å resolution. Preliminary analysis indicates the presence of four molecules in the asymmetric unit, leading to a possible new oligomeric structure for Lys49-PLA{sub 2}s. A new myotoxic Lys49-phospholipase A{sub 2} isolated from Bothrops moojeni snake venom has been crystallized. The crystals diffracted to 2.18 Å resolution using a synchrotron-radiation source and belong to space group C2. The unit-cell parameters are a = 56.8, b = 125.0, c = 64.7 Å, β = 105.5°. Preliminary analysis indicates the presence of four molecules in the asymmetric unit. This may suggest a new quaternary structure for this Lys49-phospholipase A{sub 2} in contrast to the dimeric and monomeric structures solved so far for this class of proteins.

  7. Snake population venomics and antivenomics of Bothrops atrox: Paedomorphism along its transamazonian dispersal and implications of geographic venom variability on snakebite management.

    PubMed

    Calvete, Juan J; Sanz, Libia; Pérez, Alicia; Borges, Adolfo; Vargas, Alba M; Lomonte, Bruno; Angulo, Yamileth; Gutiérrez, José María; Chalkidis, Hipócrates M; Mourão, Rosa H V; Furtado, M Fatima D; Moura-Da-Silva, Ana M

    2011-04-01

    We describe two geographically differentiated venom phenotypes across the wide distribution range of Bothrops atrox, from the Colombian Magdalena Medio Valley through Puerto Ayacucho and El Paují, in the Venezuelan States of Amazonas and Orinoquia, respectively, and São Bento in the Brazilian State of Maranhão. Colombian and Venezuelan venoms show an ontogenetic toxin profile phenotype whereas Brazilian venoms exhibit paedomorphic phenotypes. Venoms from each of the 16 localities sampled contain both population-specific toxins and proteins shared by neighboring B. atrox populations. Mapping the molecular similarity between conspecific populations onto a physical map of B. atrox range provides clues for tracing dispersal routes that account for the current biogeographic distribution of the species. The proteomic pattern is consistent with a model of southeast and southwest dispersal and allopatric fragmentation northern of the Amazon Basin, and trans-Amazonian expansion through the Andean Corridor and across the Amazon river between Monte Alegre and Santarém. An antivenomic approach applied to assess the efficacy towards B. atrox venoms of two antivenoms raised in Costa Rica and Brazil using Bothrops venoms different than B. atrox in the immunization mixtures showed that both antivenoms immunodepleted very efficiently the major toxins (PIII-SVMPs, serine proteinases, CRISP, LAO) of paedomorphic venoms from Puerto Ayacucho (Venezuelan Amazonia) through São Bento, but had impaired reactivity towards PLA(2) and P-I SVMP molecules abundantly present in ontogenetic venoms. The degree of immunodepletion achieved suggests that each of these antivenoms may be effective against envenomations by paedomorphic, and some ontogenetic, B. atrox venoms. Copyright © 2010 Elsevier B.V. All rights reserved.

  8. Inhibitory Effects of Hydroethanolic Leaf Extracts of Kalanchoe brasiliensis and Kalanchoe pinnata (Crassulaceae) against Local Effects Induced by Bothrops jararaca Snake Venom

    PubMed Central

    Fernandes, Júlia Morais; Félix-Silva, Juliana; da Cunha, Lorena Medeiros; Gomes, Jacyra Antunes dos Santos; Siqueira, Emerson Michell da Silva; Gimenes, Luisa Possamai; Lopes, Norberto Peporine; Soares, Luiz Alberto Lira; Fernandes-Pedrosa, Matheus de Freitas; Zucolotto, Silvana Maria

    2016-01-01

    The species Kalanchoe brasiliensis and Kalanchoe pinnata, both known popularly as “Saião,” are used interchangeably in traditional medicine for their antiophidic properties. Studies evaluating the anti-venom activity of these species are scarce. This study aims to characterize the chemical constituents and evaluate the inhibitory effects of hydroethanolic leaf extracts of K. brasiliensis and K. pinnata against local effects induced by Bothrops jararaca snake venom. Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography coupled with Diode Array Detection and Electrospray Mass Spectrometry (HPLC-DAD-MS/MS) were performed for characterization of chemical markers of the extracts from these species. For antiophidic activity evaluation, B. jararaca venom-induced paw edema and skin hemorrhage in mice were evaluated. In both models, hydroethanolic extracts (125–500 mg/kg) were administered intraperitoneally in different protocols. Inhibition of phospholipase enzymatic activity of B. jararaca was evaluated. The HPLC-DAD-MS/MS chromatographic profile of extracts showed some particularities in the chemical profile of the two species. K. brasileinsis exhibited major peaks that have UV spectra similar to flavonoid glycosides derived from patuletin and eupafolin, while K. pinnata showed UV spectra similar to flavonoids glycosides derived from quercetin and kaempferol. Both extracts significantly reduced the hemorrhagic activity of B. jararaca venom in pre-treatment protocol, reaching about 40% of inhibition, while only K. pinnata was active in post-treatment protocol (about 30% of inhibition). In the antiedematogenic activity, only K. pinnata was active, inhibiting about 66% and 30% in pre and post-treatment protocols, respectively. Both extracts inhibited phospholipase activity; however, K. pinnata was more active. In conclusion, the results indicate the potential antiophidic activity of Kalanchoe species against local effects induced by B. jararaca

  9. Isolation and functional characterization of a new acidic PLA(2) Ba SpII RP4 of the Bothrops alternatus snake venom from Argentina.

    PubMed

    Garcia Denegri, María E; Acosta, Ofelia C; Huancahuire-Vega, Salomón; Martins-de-Souza, Daniel; Marangoni, Sergio; Maruñak, Silvana L; Teibler, Gladys P; Leiva, Laura C; Ponce-Soto, Luis A

    2010-08-01

    An acidic protein with phospholipase A(2) activity was purified to homogeneity from the venom of the Northeast Argentinian viperid Bothrops alternatus by two chromatographic steps: a conventional gel filtration on Sephadex G-75 and reversed phase on C18 HPLC column. A molecular mass of 14185.48 Da was determined by mass spectrometry, displaying a homodimer conformation. The kinetic assay demonstrated a catalytically active phospholipase A(2) in correspondence with Asp49 PLA(2) group. The enzyme designated Ba SpII RP4 contains an amino acid composition of 121 residues and a calculated theoretical pI value of 4.88. Amino acid sequence alignments with other Bothrops PLA(2) revealed a high degree of homology sequence (90-56%). Ba SpII RP4 did not show myotoxic activity upon muscular fibers at doses up to 100 microg i.m. route injection or lethal response when it was i.p. injected at the hightest dose of 200 microg. This toxin generates slight biological activities like paw edema inflammation and a delay in the clotting time, although Ba SpII RP4 exhibited catalytic activity. The primary amino acid sequence, determined a quadruple-time of flight (Q-TOF) hybrid mass spectrometer Q-TOF Ultima from Micromass (Manchester, UK) equipped with a nano Zspray source operating in a positive ion mode and tandem mass spectrum, an ESI/MS mass spectrum (TOF MS mode) "de novo amino acid sequencing", also provides more database about the small group of the non-myotoxic PLA(2)s isolated up to the present. Copyright 2010 Elsevier Ltd. All rights reserved.

  10. An isoflavone from Dipteryx alata Vogel is active against the in vitro neuromuscular paralysis of Bothrops jararacussu snake venom and bothropstoxin I, and prevents venom-induced myonecrosis.

    PubMed

    Ferraz, Miriéle C; Yoshida, Edson H; Tavares, Renata V S; Cogo, José C; Cintra, Adélia C O; Dal Belo, Cháriston A; Franco, Luiz M; dos Santos, Márcio G; Resende, Flávia A; Varanda, Eliana A; Hyslop, Stephen; Puebla, Pilar; San Feliciano, Arturo; Oshima-Franco, Yoko

    2014-05-06

    Snakebite is a neglected disease and serious health problem in Brazil, with most bites being caused by snakes of the genus Bothrops. Although serum therapy is the primary treatment for systemic envenomation, it is generally ineffective in neutralizing the local effects of these venoms. In this work, we examined the ability of 7,8,3'-trihydroxy-4'-methoxyisoflavone (TM), an isoflavone from Dipteryx alata, to neutralize the neurotoxicity (in mouse phrenic nerve-diaphragm preparations) and myotoxicity (assessed by light microscopy) of Bothrops jararacussu snake venom in vitro. The toxicity of TM was assessed using the Salmonella microsome assay (Ames test). Incubation with TM alone (200 μg/mL) did not alter the muscle twitch tension whereas incubation with venom (40 μg/mL) caused irreversible paralysis. Preincubation of TM (200 μg/mL) with venom attenuated the venom-induced neuromuscular blockade by 84% ± 5% (mean ± SEM; n = 4). The neuromuscular blockade caused by bothropstoxin-I (BthTX-I), the major myotoxic PLA2 of this venom, was also attenuated by TM. Histological analysis of diaphragm muscle incubated with TM showed that most fibers were preserved (only 9.2% ± 1.7% were damaged; n = 4) compared to venom alone (50.3% ± 5.4% of fibers damaged; n = 3), and preincubation of TM with venom significantly attenuated the venom-induced damage (only 17% ± 3.4% of fibers damaged; n = 3; p < 0.05 compared to venom alone). TM showed no mutagenicity in the Ames test using Salmonella strains TA98 and TA97a with (+S9) and without (-S9) metabolic activation. These findings indicate that TM is a potentially useful compound for antagonizing the neuromuscular effects (neurotoxicity and myotoxicity) of B. jararacussu venom.

  11. Immune cells and mediators involved in the inflammatory responses induced by a P-I metalloprotease and a phospholipase A2 from Bothrops atrox venom.

    PubMed

    Menaldo, Danilo L; Bernardes, Carolina P; Zoccal, Karina F; Jacob-Ferreira, Anna L; Costa, Tássia R; Del Lama, Maria P F M; Naal, Rose M Z G; Frantz, Fabiani G; Faccioli, Lúcia H; Sampaio, Suely V

    2017-05-01

    Bothrops envenomations can promote severe inflammatory responses by inducing edema, pain, leukocyte recruitment and release of chemical mediators by local cells. In the present study, two toxins from Bothrops atrox venom (the P-I metalloprotease Batroxase and the acidic phospholipase A2 BatroxPLA2) were evaluated in relation to their inflammatory effects induced in vivo and in vitro, mainly focusing on the participation of different immune cells and inflammatory mediators. Both toxins mainly promoted acute inflammatory responses with significant recruitment of neutrophils in the early hours (1-4h) after administration into the peritoneal cavity of C57BL/6 mice, and increased infiltration of mononuclear cells especially after 24h. Among the mediators induced by both toxins are IL-6, IL-10 and PGE2, with Batroxase also inducing the release of L-1β, and BatroxPLA2 of LTB4 and CysLTs. These responses pointed to possible involvement of immune cells such as macrophages and mast cells, which were then evaluated in vitro. Mice peritoneal macrophages stimulated with Batroxase produced significant levels of IL-6, IL-1β, PGE2 and LTB4, whereas stimulus with BatroxPLA2 induced increases of IL-6, PGE2 and LTB4. Furthermore, both toxins were able to stimulate degranulation of RBL-2H3 mast cells, but with distinct concentration-dependent effects. Altogether, these results indicated that Batroxase and BatroxPLA2 promoted local and acute inflammatory responses related to macrophages and mast cells and to the production of several mediators. Our findings should contribute for better understanding the different mechanisms of toxicity induced by P-I metalloproteases and phospholipases A2 after snakebite envenomations. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Inhibitory Effects of Hydroethanolic Leaf Extracts of Kalanchoe brasiliensis and Kalanchoe pinnata (Crassulaceae) against Local Effects Induced by Bothrops jararaca Snake Venom.

    PubMed

    Fernandes, Júlia Morais; Félix-Silva, Juliana; da Cunha, Lorena Medeiros; Gomes, Jacyra Antunes Dos Santos; Siqueira, Emerson Michell da Silva; Gimenes, Luisa Possamai; Lopes, Norberto Peporine; Soares, Luiz Alberto Lira; Fernandes-Pedrosa, Matheus de Freitas; Zucolotto, Silvana Maria

    2016-01-01

    The species Kalanchoe brasiliensis and Kalanchoe pinnata, both known popularly as "Saião," are used interchangeably in traditional medicine for their antiophidic properties. Studies evaluating the anti-venom activity of these species are scarce. This study aims to characterize the chemical constituents and evaluate the inhibitory effects of hydroethanolic leaf extracts of K. brasiliensis and K. pinnata against local effects induced by Bothrops jararaca snake venom. Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography coupled with Diode Array Detection and Electrospray Mass Spectrometry (HPLC-DAD-MS/MS) were performed for characterization of chemical markers of the extracts from these species. For antiophidic activity evaluation, B. jararaca venom-induced paw edema and skin hemorrhage in mice were evaluated. In both models, hydroethanolic extracts (125-500 mg/kg) were administered intraperitoneally in different protocols. Inhibition of phospholipase enzymatic activity of B. jararaca was evaluated. The HPLC-DAD-MS/MS chromatographic profile of extracts showed some particularities in the chemical profile of the two species. K. brasileinsis exhibited major peaks that have UV spectra similar to flavonoid glycosides derived from patuletin and eupafolin, while K. pinnata showed UV spectra similar to flavonoids glycosides derived from quercetin and kaempferol. Both extracts significantly reduced the hemorrhagic activity of B. jararaca venom in pre-treatment protocol, reaching about 40% of inhibition, while only K. pinnata was active in post-treatment protocol (about 30% of inhibition). In the antiedematogenic activity, only K. pinnata was active, inhibiting about 66% and 30% in pre and post-treatment protocols, respectively. Both extracts inhibited phospholipase activity; however, K. pinnata was more active. In conclusion, the results indicate the potential antiophidic activity of Kalanchoe species against local effects induced by B. jararaca snake

  14. Biochemical characterization and pharmacological properties of new basic PLA2 BrTX-I isolated from Bothrops roedingeri (Roedinger's Lancehead) Mertens, 1942, snake venom.

    PubMed

    Gomes Heleno, Mauricio Aurelio; Baldasso, Paulo Aparecido; Ponce-Soto, Luis Alberto; Marangoni, Sérgio

    2013-01-01

    BrTX-I, a PLA2, was purified from Bothrops roedingeri venom after only one chromatographic step using reverse-phase HPLC on μ-Bondapak C-18 column. A molecular mass of 14358.69 Da was determined by MALDI-TOF mass spectrometry. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The total amino acid sequence was obtained using SwissProt database and showed high amino acid sequence identity with other PLA2 from snake venom. The amino acid composition showed that BrTX-I has a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA2. BrTX-I presented PLA2 activity and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0, 35-45°C, and required Ca(2+). In vitro, the whole venom and BrTX-I caused a neuromuscular blockade in biventer cervicis preparations in a similar way to other Bothrops species. BrTX-I induced myonecrosis and oedema-forming activity analyzed through injection of the purified BrTX-I in mice. Since BrTX-I exerts a strong proinflammatory effect, the enzymatic phospholipid hydrolysis might be relevant for these phenomena; incrementing levels of IL-1, IL-6, and TNF α were observed at 15 min, 30 min, one, two, and six hours postinjection, respectively.

  15. Complications of Bothrops, Porthidium, and Bothriechis snakebites in Colombia. A clinical and epidemiological study of 39 cases attended in a university hospital.

    PubMed

    Otero, Rafael; Gutiérrez, Johnayro; Beatriz Mesa, María; Duque, Edison; Rodríguez, Orlando; Luis Arango, Jorge; Gómez, Francisco; Toro, Alvaro; Cano, Fidel; María Rodríguez, Libia; Caro, Erika; Martínez, José; Cornejo, William; Mariano Gómez, Luis; Luis Uribe, Francisco; Cárdenas, Silvia; Núñez, Vitelbina; Díaz, Abel

    2002-08-01

    The clinical and epidemiological features, as well as complications presented by 39 patients with Bothrops, Porthidium and Bothriechis snakebites, are described. Patients were admitted during 1 year in 25 hospitals of Antioquia and Chocó and then, they were transferred to the Hospital Universitario San Vicente de Paúl in Medellín, 30 of them because of the presence of complications, eight because of lack of antivenoms and another one because of the desire of his relatives. Thirty--one (79.5%) of the patients were male, 13 (33.3%) children, 59% of them were bitten at the lower extremities, the majority (74.4%) by Bothrops asper. Twenty-one (53.8%) of the patients were initially attended by traditional healers and sought medical attention at the local hospitals after 2h in 87.2% of the cases. Edema (100%), hemorrhage (74.4%), blistering (38.5%) and necrosis (38.5%), were the local signs of envenomation, while blood coagulation alteration (79.5%), hematuria (74.4%), gingival bleeding (43.6%), hypovolemic shock (23.1%) and oliguria (23.1%), were the systemic signs of envenomation. The final grade of envenomation was severe in 29 patients (74.4%). Thirty patients (76.9%) had one or more complications of the envenomation: acute renal failure (ARF), 15 (38.5%); soft-tissue infection, 12 (30.8%); central nervous system (CNS) hemorrhage, 5 (12.8%); compartment syndrome, 3 (7.7%); soft--tissue hematomas, 6 (15.4%); and Abruptio placentae, one (2.6%). There were four deaths (10.3%), two from ARF and two from cerebral hemorrhage. Fourteen other patients (35.9%) had sequelae. The onset of serotherapy after 2h of the bite was associated with the occurrence of ARF and CNS hemorrhage (p=0.02), as well as the risk of death and sequelae (RR=2.5).

  16. Biochemical Characterization and Pharmacological Properties of New Basic PLA2 BrTX-I Isolated from Bothrops roedingeri (Roedinger's Lancehead) Mertens, 1942, Snake Venom

    PubMed Central

    Gomes Heleno, Mauricio Aurelio; Baldasso, Paulo Aparecido; Ponce-Soto, Luis Alberto; Marangoni, Sérgio

    2013-01-01

    BrTX-I, a PLA2, was purified from Bothrops roedingeri venom after only one chromatographic step using reverse-phase HPLC on μ-Bondapak C-18 column. A molecular mass of 14358.69 Da was determined by MALDI-TOF mass spectrometry. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The total amino acid sequence was obtained using SwissProt database and showed high amino acid sequence identity with other PLA2 from snake venom. The amino acid composition showed that BrTX-I has a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA2. BrTX-I presented PLA2 activity and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0, 35–45°C, and required Ca2+. In vitro, the whole venom and BrTX-I caused a neuromuscular blockade in biventer cervicis preparations in a similar way to other Bothrops species. BrTX-I induced myonecrosis and oedema-forming activity analyzed through injection of the purified BrTX-I in mice. Since BrTX-I exerts a strong proinflammatory effect, the enzymatic phospholipid hydrolysis might be relevant for these phenomena; incrementing levels of IL-1, IL-6, and TNFα were observed at 15 min, 30 min, one, two, and six hours postinjection, respectively. PMID:23509747

  17. Degenerative and regenerative changes in murine skeletal muscle after injection of venom from the snake Bothrops asper: a histochemical and immunocytochemical study.

    PubMed Central

    Arce, V.; Brenes, F.; Gutiérrez, J. M.

    1991-01-01

    The degenerative and regenerative changes in murine skeletal muscle after injection of Bothrops asper venom were studied by histological, lectin histochemical and immunocytochemical techniques. According to our observations, the process was divided into four main stages: (a) During the first 3 days prominent degenerative events took place in skeletal muscle fibres, capillaries, arteries, veins and intramuscular nerves. An inflammatory infiltrate was abundant after the first day and removal of necrotic material was well advanced by the third day. (b) Muscle regeneration was evident by the fourth day. From 4 to 6 days there were two populations of regenerating muscle fibres, one of apparently normal fibres located in areas where capillary vessels were abundant, and another population of groups of regenerative fibres showing signs of degeneration. This second type of fibre was predominant in areas where the number of capillaries was greatly reduced. (c) One and 2 weeks after envenomation areas of small regenerative fibres of normal morphology and areas of degenerating regenerative fibres were observed. The latter were abundant in regions of dense fibrotic tissue and scarce capillaries. (d) Finally, at 4 and 8 weeks after envenomation there were both areas of fibrosis and areas where regenerating muscle fibres predominated. However, the diameter of these fibres was abnormally small, an indication that they may have been atrophic fibres. It is suggested that muscle regeneration is partially impaired after myonecrosis induced by Bothrops asper venom, probably due to the damage induced by this venom on muscle microvasculature and nerves. Images Fig. 4 Fig. 5 Fig. 1 Fig. 2 Fig. 3 Fig. 6 Fig. 8 PMID:1707650

  18. 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

  19. Effects of N-acetyl-L-cysteine on redox status and markers of renal function in mice inoculated with Bothrops jararaca and Crotalus durissus terrificus venoms.

    PubMed

    Barone, Juliana Marton; Frezzatti, Rodrigo; Silveira, Paulo Flavio

    2014-03-01

    Renal dysfunction is an important aggravating factor in accidents caused by Crotalus durissus terrificus (Cdt) and Bothrops jararaca (Bj) bites. N-acetyl-l-cysteine (NAC) is well known as a nephroprotective antioxidant with low toxicity. The present study investigated the effects of NAC on redox status and markers of renal function in mice that received vehicle (controls) or venoms (v) of Cdt and Bj. In controls NAC promoted hypercreatinemia, hypouremia, hyperosmolality with decreased urea in urine, hyperproteinuria, decreased protein and increased dipeptidyl peptidase IV (DPPIV) in membrane-bound fraction (MF) from renal cortex (RC) and medulla (RM). NAC ameliorated or normalized altered creatinuria, proteinemia and aminopeptidase (AP) acid in MF, AP basic (APB) in soluble fraction (SF), and neutral AP in SF and MF from RC and RM in vBj envenomation. NAC ameliorated or normalized altered neutral AP in SF from RC and RM, and DPPIV and protein in MF from RC in vCdt envenomation. NAC ameliorated or restored renal redox status respectively in vCdt and vBj, and normalized uricemia in both envenomations. These data are promising perspectives that recommend the clinical evaluation of NAC as potential coadjuvant in the anti venom serotherapy for accidents with these snake's genera. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Isolation: analysis and properties of three bradykinin-potentiating peptides (BPP-II, BPP-III, and BPP-V) from Bothrops neuwiedi venom.

    PubMed

    Ferreira, L A; Galle, A; Raida, M; Schrader, M; Lebrun, I; Habermehl, G

    1998-04-01

    In the course of systematic investigations on low-molecular-weight compounds from the venom of Crotalidae and Viperidae, we have isolated and characterized at least three bradykinin-potentiating peptides (BPP-II, BPP-III, and BPP-V) from Bothrops neuwiedi venom by gel filtration on Sephadex G-25 M, Sephadex G-10 followed by HPLC. The peptides showed bradykinin-potentiating action on isolated guinea-pig ileum, for which the BPP-V was more active than of BPP-II, and BPP-III, rat arterial blood pressure, and a relevant angiotensin-converting enzyme (ACE) competitive inhibiting activity. The kinetic studies showed a Ki of the order of 9.7 x 10(-3) microM to BPP-II, 7 x 10(-3) microM to BPP-III, and 3.3 x 10(-3) microM to BPP-V. The amino acid sequence of the BPP-III has been determined to be pGlu-Gly-Gly-Trp-Pro-Arg-Pro-Gly-Pro-Glu-Ile-Pro-Pro, and the amino acid compositions of the BPP-II and BPP-V by amino acid analysis were 2Glu-2Gly-1Arg-4Pro-1Ile and 2Glu-2Gly-1Ser-3Pro-2Val-1Ile, with molecular weight of 1372, 1046, and 1078, respectively.

  1. Extracts of Renealmia alpinia (Rottb.) MAAS Protect against Lethality and Systemic Hemorrhage Induced by Bothrops asper Venom: Insights from a Model with Extract Administration before Venom Injection

    PubMed Central

    Patiño, Arley Camilo; Quintana, Juan Carlos; Gutiérrez, José María; Rucavado, Alexandra; Benjumea, Dora María; Pereañez, Jaime Andrés

    2015-01-01

    Renealmia alpinia (Rottb.) MAAS, obtained by micropropagation (in vitro) and wild forms have previously been shown to inhibit some toxic activities of Bothrops asper snake venom if preincubated before injection. In this study, assays were performed in a murine model in which extracts were administered for three days before venom injection. R. alpinia extracts inhibited lethal activity of B. asper venom injected by intraperitoneal route. Median Effective Dose (ED50) values were 36.6 ± 3.2 mg/kg and 31.7 ± 5.4 mg/kg (p > 0.05) for R. alpinia wild and in vitro extracts, respectively. At a dose of 75 mg/kg, both extracts totally inhibited the lethal activity of the venom. Moreover, this dose prolonged survival time of mice receiving a lethal dose of venom by the intravenous route. At 75 mg/kg, both extracts of R. alpinia reduced the extent of venom-induced pulmonary hemorrhage by 48.0% (in vitro extract) and 34.7% (wild extract), in agreement with histological observations of lung tissue. R. alpinia extracts also inhibited hemorrhage in heart and kidneys, as evidenced by a decrease in mg of hemoglobin/g of organ. These results suggest the possibility of using R. alpinia as a prophylactic agent in snakebite, a hypothesis that needs to be further explored. PMID:25941768

  2. Coagulant thrombin-like enzyme (barnettobin) from Bothrops barnetti venom: molecular sequence analysis of its cDNA and biochemical properties.

    PubMed

    Vivas-Ruiz, Dan E; Sandoval, Gustavo A; Mendoza, Julio; Inga, Rosalina R; Gontijo, Silea; Richardson, Michael; Eble, Johannes A; Yarleque, Armando; Sanchez, Eladio F

    2013-07-01

    The thrombin-like enzyme from Bothrops barnetti named barnettobin was purified. We report some biochemical features of barnettobin including the complete amino acid sequence that was deduced from the cDNA. Snake venom serine proteases affect several steps of human hemostasis ranging from the blood coagulation cascade to platelet function. Barnettobin is a monomeric glycoprotein of 52 kDa as shown by reducing SDS-PAGE, and contains approx. 52% carbohydrate by mass which could be removed by N-glycosidase. The complete amino acid sequence was deduced from the cDNA sequence. Its sequence contains a single chain of 233 amino acid including three N-glycosylation sites. The sequence exhibits significant homology with those of mammalian serine proteases e.g. thrombin and with homologous TLEs. Its specific coagulant activity was 251.7 NIH thrombin units/mg, releasing fibrinopeptide A from human fibrinogen and showed defibrinogenating effect in mouse. Both coagulant and amidolytic activities were inhibited by PMSF. N-deglycosylation impaired its temperature and pH stability. Its cDNA sequence with 750 bp encodes a protein of 233 residues. Indications that carbohydrate moieties may play a role in the interaction with substrates are presented. Barnettobin is a new defibrinogenating agent which may provide an opportunity for the development of new types of anti-thrombotic drugs.

  3. P-I class metalloproteinase from Bothrops moojeni venom is a post-proline cleaving peptidase with kininogenase activity: insights into substrate selectivity and kinetic behavior.

    PubMed

    Okamoto, Débora N; Kondo, Marcia Y; Oliveira, Lilian C G; Honorato, Rodrigo V; Zanphorlin, Leticia M; Coronado, Monika A; Araújo, Mariana S; da Motta, Guacyara; Veronez, Camila L; Andrade, Sheila S; Oliveira, Paulo S L; Arni, Raghuvir K; Cintra, Adelia C O; Sampaio, Suely V; Juliano, Maria A; Juliano, Luiz; Murakami, Mário T; Gouvea, Iuri E

    2014-03-01

    Snake venom metalloproteinases (SVMPs) belonging to P-I class are able to hydrolyze extracellular matrix proteins and coagulation factors triggering local and systemic reactions by multiple molecular mechanisms that are not fully understood. BmooMPα-I, a P-I class SMVP from Bothrops moojeni venom, was active upon neuro- and vaso-active peptides including angiotensin I, bradykinin, neurotensin, oxytocin and substance P. Interestingly, BmooMPα-I showed a strong bias towards hydrolysis after proline residues, which is unusual for most of characterized peptidases. Moreover, the enzyme showed kininogenase activity similar to that observed in plasma and cells by kallikrein. FRET peptide assays indicated a relative promiscuity at its S2-S'2 subsites, with proline determining the scissile bond. This unusual post-proline cleaving activity was confirmed by the efficient hydrolysis of the synthetic combinatorial library MCA-GXXPXXQ-EDDnp, described as resistant for canonical peptidases, only after Pro residues. Structural analysis of the tripeptide LPL complexed with BmooMPα-I, generated by molecular dynamics simulations, assisted in defining the subsites and provided the structural basis for subsite preferences such as the restriction of basic residues at the S2 subsite due to repulsive electrostatic effects and the steric impediment for large aliphatic or aromatic side chains at the S1 subsite. These new functional and structural findings provided a further understanding of the molecular mechanisms governing the physiological effects of this important class of enzymes in envenomation process.

  4. Prognostic significance of clinical grading of patients envenomed by Bothrops lanceolatus in Martinique. Members of the Research Group on Snake Bite in Martinique.

    PubMed

    Thomas, L; Tyburn, B; Ketterlé, J; Biao, T; Mehdaoui, H; Moravie, V; Rouvel, C; Plumelle, Y; Bucher, B; Canonge, D; Marie-Nelly, C A; Lang, J

    1998-01-01

    The correlation between clinical grading of patients bitten by Bothrops lanceolatus and the subsequent development of their envenoming was examined. Severity of envenoming was graded using a 1-4 scale (minor to major). Patients were classified into 2 groups according to the time elapsed between bite and treatment with a specific purified equine F(ab')2 antivenom. The late/no treatment group (n = 33) was characterized by a systemic thrombotic complication rate of 14/33 (42.4%) leading to 4 deaths, which increased with the maximum severity assessed on the first day following the bite (P = 0.003). However, infarctions could develop in patients who presented initially with signs of moderate envenoming, normal blood clotting and low serum levels of venom antigens. No such complication of fatality occurred in the early (0.5-6 h) treatment group (n = 70). Multiple regression analysis showed that duration of stay in hospital in this group increased with the length of the snake (P = 0.017), venom antigenaemia (P = 0.016), initial grading (P < 0.001), and with the need for surgical debridement (n = 10/70, P < 0.001). Outcome was correlated with initial severity of envenoming. However, the only factor with a positive prognostic significance for the individual envenomed patient was the early infusion of specific antivenom, which led to 100% recovery in our series.

  5. Alkylation of histidine residues of Bothrops jararacussu venom proteins and isolated phospholipases A2: a biotechnological tool to improve the production of antibodies.

    PubMed

    Guimarães, C L S; Andrião-Escarso, S H; Moreira-Dill, L S; Carvalho, B M A; Marchi-Salvador, D P; Santos-Filho, N A; Fernandes, C A H; Fontes, M R M; Giglio, J R; Barraviera, B; Zuliani, J P; Fernandes, C F C; Calderón, L A; Stábeli, R G; Albericio, F; da Silva, S L; Soares, A M

    2014-01-01

    Crude venom of Bothrops jararacussu and isolated phospholipases A2 (PLA2) of this toxin (BthTX-I and BthTX-II) were chemically modified (alkylation) by p-bromophenacyl bromide (BPB) in order to study antibody production capacity in function of the structure-function relationship of these substances (crude venom and PLA2 native and alkylated). BthTX-II showed enzymatic activity, while BthTX-I did not. Alkylation reduced BthTX-II activity by 50% while this process abolished the catalytic and myotoxic activities of BthTX-I, while reducing its edema-inducing activity by about 50%. Antibody production against the native and alkylated forms of BthTX-I and -II and the cross-reactivity of antibodies to native and alkylated toxins did not show any apparent differences and these observations were reinforced by surface plasmon resonance (SPR) data. Histopathological analysis of mouse gastrocnemius muscle sections after injection of PBS, BthTX-I, BthTX-II, or both myotoxins previously incubated with neutralizing antibody showed inhibition of the toxin-induced myotoxicity. These results reveal that the chemical modification of the phospholipases A2 (PLA2) diminished their toxicity but did not alter their antigenicity. This observation indicates that the modified PLA2 may provide a biotechnological tool to attenuate the toxicity of the crude venom, by improving the production of antibodies and decreasing the local toxic effects of this poisonous substance in animals used to produce antivenom.

  6. Investigating possible biological targets of Bj-CRP, the first cysteine-rich secretory protein (CRISP) isolated from Bothrops jararaca snake venom.

    PubMed

    Lodovicho, Marina E; Costa, Tássia R; Bernardes, Carolina P; Menaldo, Danilo L; Zoccal, Karina F; Carone, Sante E; Rosa, José C; Pucca, Manuela B; Cerni, Felipe A; Arantes, Eliane C; Tytgat, Jan; Faccioli, Lúcia H; Pereira-Crott, Luciana S; Sampaio, Suely V

    2017-01-04

    Cysteine-rich secretory proteins (CRISPs) are commonly described as part of the protein content of snake venoms, nevertheless, so far, little is known about their biological targets and functions. Our study describes the isolation and characterization of Bj-CRP, the first CRISP isolated from Bothrops jararaca snake venom, also aiming at the identification of possible targets for its actions. Bj-CRP was purified using three chromatographic steps (Sephacryl S-200, Source 15Q and C18) and showed to be an acidic protein of 24.6kDa with high sequence identity to other snake venom CRISPs. This CRISP was devoid of proteolytic, hemorrhagic or coagulant activities, and it did not affect the currents from 13 voltage-gated potassium channel isoforms. Conversely, Bj-CRP induced inflammatory responses characterized by increase of leukocytes, mainly neutrophils, after 1 and 4h of its injection in the peritoneal cavity of mice, also stimulating the production of IL-6. Bj-CRP also acted on the human complement system, modulating some of the activation pathways and acting directly on important components (C3 and C4), thus inducing the generation of anaphylatoxins (C3a, C4a and C5a). Therefore, our results for Bj-CRP open up prospects for better understanding this class of toxins and its biological actions.

  7. Complete amino-acid sequence, crystallization and preliminary X-ray diffraction studies of leucurolysin-a, a nonhaemorrhagic metalloproteinase from Bothrops leucurus snake venom

    PubMed Central

    Ferreira, Rodrigo Novaes; Rates, Breno; Richardson, Michael; Guimarães, Beatriz Gomes; Sanchez, Eládio Oswaldo Flores; de Castro Pimenta, Adriano Monteiro; Nagem, Ronaldo Alves Pinto

    2009-01-01

    Leucurolysin-a (leuc-a) is a class P-I snake-venom metalloproteinase isolated from the venom of the South American snake Bothrops leucurus (white-tailed jararaca). The mature protein is composed of 202 amino-acid residues in a single polypeptide chain. It contains a blocked N-terminus and is not glycosylated. In vitro studies revealed that leuc-a dissolves clots made either from purified fibrinogen or from whole blood. Unlike some other venom fibrinolytic metalloproteinases, leuc-a has no haemorrhagic activity. Leuc-a was sequenced and was crystallized using the hanging-drop vapour-diffusion technique. Crystals were obtained using PEG 6000 or PEG 1500. Diffraction data to 1.80 and 1.60 Å resolution were collected from two crystals (free enzyme and the endogenous ligand–protein complex, respectively). They both belonged to space group P212121, with very similar unit-cell parameters (a = 44.0, b = 56.2, c = 76.3 Å for the free-enzyme crystal). PMID:19652343

  8. Morphometric and phylogenetic analyses of Serpentirhabdias viperidicus n. sp. (Nematoda: Rhabdiasidae) from the lancehead snake Bothrops moojeni Hoge, 1966 (Reptilia: Serpentes: Viperidae) in Brazil.

    PubMed

    Morais, D H; Aguiar, A; Müller, M I; Narciso, R B; da Silva, L A F; da Silva, R J

    2017-05-01

    Serpentirhabdias viperidicus n. sp. (Nematoda: Rhabdiasidae) is described from the lungs of the 'Brazilian lancehead' Bothrops moojeni (Hoge, 1966) from the savannah in São Paulo State, Brazil. The new species is the eighth species of Serpentirhabdias described in the Neotropical region, and differs from other species mainly by a combination of characters: lips slightly notable, presence of fine striations at posterior ends, presence of two parallel lines with intercalated pores, a pore-shaped phasmid situated at the level of the anal aperture and another two in the posterior half of the tail. It is the first species of Serpentirhabdias reported in this snake host and the second species of this genus found parasitizing South American viperidian snakes. Molecular phylogenetic analysis using ribosomal (ITS and 28S partial) genes confirms Serpentirhabdias viperidicus n. sp. as a new species that clustered in the Serpentirhabdias clade, sister taxon to Serpentirhabdias fuscovenosa and Serpentirhabdias elaphe. This is the first description of Serpentirhabdias species from Brazil using molecular approaches and morphological characters to confirm the monophyly of this recent genus.

  9. Alkylation of Histidine Residues of Bothrops jararacussu Venom Proteins and Isolated Phospholipases A2: A Biotechnological Tool to Improve the Production of Antibodies

    PubMed Central

    Guimarães, C. L. S.; Andrião-Escarso, S. H.; Moreira-Dill, L. S.; Carvalho, B. M. A.; Marchi-Salvador, D. P.; Santos-Filho, N. A.; Fernandes, C. A. H.; Fontes, M. R. M.; Giglio, J. R.; Barraviera, B.; Zuliani, J. P.; Fernandes, C. F. C.; Calderón, L. A.; Stábeli, R. G.; Albericio, F.; da Silva, S. L.; Soares, A. M.

    2014-01-01

    Crude venom of Bothrops jararacussu and isolated phospholipases A2 (PLA2) of this toxin (BthTX-I and BthTX-II) were chemically modified (alkylation) by p-bromophenacyl bromide (BPB) in order to study antibody production capacity in function of the structure-function relationship of these substances (crude venom and PLA2 native and alkylated). BthTX-II showed enzymatic activity, while BthTX-I did not. Alkylation reduced BthTX-II activity by 50% while this process abolished the catalytic and myotoxic activities of BthTX-I, while reducing its edema-inducing activity by about 50%. Antibody production against the native and alkylated forms of BthTX-I and -II and the cross-reactivity of antibodies to native and alkylated toxins did not show any apparent differences and these observations were reinforced by surface plasmon resonance (SPR) data. Histopathological analysis of mouse gastrocnemius muscle sections after injection of PBS, BthTX-I, BthTX-II, or both myotoxins previously incubated with neutralizing antibody showed inhibition of the toxin-induced myotoxicity. These results reveal that the chemical modification of the phospholipases A2 (PLA2) diminished their toxicity but did not alter their antigenicity. This observation indicates that the modified PLA2 may provide a biotechnological tool to attenuate the toxicity of the crude venom, by improving the production of antibodies and decreasing the local toxic effects of this poisonous substance in animals used to produce antivenom. PMID:24901004

  10. Ability of six Latin American antivenoms to neutralize the venom of mapaná equis (Bothrops atrox) from Antioquia and Chocó (Colombia).

    PubMed

    Otero, R; Nuñez, V; Osorio, R G; Gutiérrez, J M; Giraldo, C A; Posada, L E

    1995-06-01

    This investigation compared the ability of six Latin American antivenoms (monovalent antibothropic INS, Santafé de Bogotá; polyvalent INS; polyvalent probiol, Santafé de Bogotá; antibothropic Instituto Butantan, IB, São Paulo, Brazil; polyvalent Instituto Clodomiro Picado, ICP, San José, Costa Rica; polyvalent MYN, Mexico) to neutralize various pharmacological and enzymatic effects of Bothrops atrox venom from Antioquia and Chocó, north-west of Colombia. Our results demonstrated conspicuous differences in the ability of the six antivenoms. In terms of neutralization of lethality, the highest efficacy was observed in the polyvalent INS and the lowest in the polyvalent MYN antivenom. All antivenoms were highly effective in the neutralization of hemorrhage, polyvalent INS and probiol being the highest. In the neutralization of edema-forming activity, the most effective antivenom was the polyvalent (ICP); monovalent (INS) and polyvalent (MYN) were the least effective. All antivenoms were effective in the neutralization of the myotoxic activity of B. atrox venom, the most effective being the polyvalent (INS) and antibothropic (IB). Defibrinating activity was neutralized by all antivenoms; polyvalent (MYN) showed the lowest efficiency. Polyvalent (ICP) antivenom had the highest neutralizing ability against the indirect hemolytic effect of B. atrox venom; polyvalent (MYN) did not neutralize this enzymatic activity. Overall, the polyvalent antivenom (INS) showed the highest neutralizing ability.

  11. A Lys49-PLA2 myotoxin of Bothrops asper triggers a rapid death of macrophages that involves autocrine purinergic receptor signaling

    PubMed Central

    Tonello, F; Simonato, M; Aita, A; Pizzo, P; Fernández, J; Lomonte, B; Gutiérrez, J M; Montecucco, C

    2012-01-01

    Lys49-PLA2 myotoxins, an important component of various viperid snake venoms, are a class of PLA2-homolog proteins deprived of catalytic activity. Similar to enzymatically active PLA2 (Asp49) and to other classes of myotoxins, they cause severe myonecrosis. Moreover, these toxins are used as tools to study skeletal muscle repair and regeneration, a process that can be very limited after snakebites. In this work, the cytotoxic effect of different myotoxins, Bothrops asper Lys49 and Asp49-PLA2, Notechis scutatus notexin and Naja mossambica cardiotoxin, was evaluated on macrophages, cells that have a key role in muscle regeneration. Only the Lys49-myotoxin was found to trigger a rapid asynchronous death of mouse peritoneal macrophages and macrophagic cell lines through a process that involves ATP release, ATP-induced ATP release and that is inhibited by various purinergic receptor antagonists. ATP leakage is induced also at sublytical doses of the Lys49-myotoxin, it involves Ca2+ release from intracellular stores, and is reduced by inhibitors of VSOR and the maxi-anion channel. The toxin-induced cell death is different from that caused by high concentration of ATP and appears to be linked to localized purinergic signaling. Based on present findings, a mechanism of cell death is proposed that can be extended to other cytolytic proteins and peptides. PMID:22764102

  12. Crystallization and preliminary X-ray diffraction analysis of a myotoxic Lys49-PLA{sub 2} from Bothrops jararacussu venom complexed with p-bromophenacyl bromide

    SciTech Connect

    Marchi-Salvador, D. P.; Fernandes, C. A. H.; Amui, S. F.; Soares, A. M.

    2006-06-01

    A non-catalytic and myotoxic Lys49-PLA{sub 2} from B. jararacussu venom was crystallized with BPB inhibitor and X-ray diffraction data were collected. Preliminary analysis indicates that the ligand is bound to the His48 residue. Structure determination may provide insights into the myotoxic and cytotoxic mechanisms of Lys49-PLA{sub 2}s. For the first time, a non-catalytic and myotoxic Lys49-PLA{sub 2} (BthTX-I from Bothrops jararacussu venom) has been crystallized with BPB inhibitor. X-ray diffraction data were collected and electron-density calculations showed that the ligand is bound to the His48 residue. BthTX-I with His48 chemically modified by BPB shows strongly reduced myotoxic and cytotoxic activities. This suggests a biological correlation between the modification of His48, which is associated with catalytic activity of PLA{sub 2}s, and other toxicological activities of Lys49-PLA{sub 2}s.

  13. Geographical variability of the venoms of four populations of Bothrops asper from Panama: Toxicological analysis and neutralization by a polyvalent antivenom.

    PubMed

    Vélez, Sara María; Salazar, Marcos; Acosta de Patiño, Hildaura; Gómez, Leandra; Rodriguez, Abdiel; Correa, David; Saldaña, Julio; Navarro, Deyvi; Lomonte, Bruno; Otero-Patiño, Rafael; Gutiérrez, José María

    2017-04-06

    Bothrops asper is the medically most important venomous snake in Central America. In Panama, the country having the highest incidence of snakebites in Latin America, B. asper is widely distributed throughout the country and is responsible for the vast majority of snakebites. This study was performed to analyze whether there are variations in the toxicological profile and in some biochemical parameters between the venoms of B. asper from four different regions in Panama. The venoms showed a similar profile of lethal, hemorrhagic, in vitro coagulant, defibrinogenating, edema-forming, myotoxic and indirect hemolytic activities, with subtle quantitative variations between samples of some regions. The venoms also had similar SDS-PAGE patterns and reverse phase HPLC profiles. A polyvalent antivenom manufactured in Costa Rica, and regularly used in Panama, was effective in the neutralization of lethal activity of the venoms of the four populations, with Mean Effective Doses (ED50) ranging from 5.98 to 9.72 mg venom/mL antivenom. In agreement, a widespread pattern of cross-reactivity between this antivenom and the four venoms was observed by immunoblotting. Overall, results highlight the lack of marked differences between the venoms of the various populations of B. asper in Panama, and that the antivenom from Costa Rica is effective in neutralizing lethality.

  14. Crystallization and preliminary X-ray diffraction studies of BmooPLA2-I, a platelet-aggregation inhibitor and hypotensive phospholipase A2 from Bothrops moojeni venom

    PubMed Central

    Salvador, Guilherme H. M.; Marchi-Salvador, Daniela P.; Silveira, Lucas B.; Soares, Andreimar M.; Fontes, Marcos R. M.

    2011-01-01

    Phospholipases A2 (PLA2s) are enzymes that cause the liberation of fatty acids and lysophospholipids by the hydrolysis of membrane phospholipids. In addition to their catalytic action, a wide variety of pharmacological activities have been described for snake-venom PLA2s. BmooPLA2-I is an acidic, nontoxic and catalytic PLA2 isolated from Bothrops moojeni snake venom which exhibits an inhibitory effect on platelet aggregation, an immediate decrease in blood pressure, inducing oedema at a low concentration, and an effective bactericidal effect. BmooPLA2-I has been crystallized and X-ray diffraction data have been collected to 1.6 Å resolution using a synchrotron-radiation source. The crystals belonged to space group C2221, with unit-cell parameters a = 39.7, b = 53.2, c = 89.2 Å. The molecular-replacement solution of BmooPLA2-I indicated a monomeric conformation, which is in agreement with nondenaturing electrophoresis and dynamic light-scattering experiments. A comparative study of this enzyme with the acidic PLA2 from B. jararacussu (BthA-I) and other toxic and nontoxic PLA2s may provide important insights into the functional aspects of this class of proteins. PMID:21821890

  15. Cross-reactivity and cross-immunomodulation between venoms of the snakes Bothrops asper, Crotalus simus and Lachesis stenophrys, and its effect in the production of polyspecific antivenom for Central America.

    PubMed

    Arroyo, Cynthia; Solano, Sergio; Segura, Álvaro; Herrera, María; Estrada, Ricardo; Villalta, Mauren; Vargas, Mariángela; Gutiérrez, José María; León, Guillermo

    2017-08-09

    A mixture of the venoms of Bothrops asper, Crotalus simus and Lachesis stenophrys is used as immunogen to produce the polyspecific Central American antivenom (PoliVal-ICP). In this work, we studied the ability of each of these venoms to modulate the antibody response induced by the other two venoms included in the immunization mixture. For that, equine monospecific, bispecific and polyspecific antivenoms were prepared and compared regarding their ability to neutralize the phospholipase A2, coagulant and lethal activities of each venom, and their anti-venom antibodies concentration. Results indicate that there is low cross-reactivity and cross-neutralization between venoms of B. asper, C. simus and L. stenophrys, hence justifying the use of all of them as immunogens for the production of the Central American antivenom. It was also found that the venom of B. asper reduces the anti-crotalic response while the venom of C. simus does not affect the anti-bothropic response. On the other hand, the venoms of B. asper and C. simus increase the anti-lachesic response, and L. stenoprhys venom reduced both the anti-bothropic and anti-crotalic responses. On the basis of these results, the immunization strategy can be adjusted by preventing or taking advantage of cross-immunomodulation between venoms, in order to maximize the antibody response towards all venoms. Immune responses can be improved by injecting horses with several immunogen mixtures, composed by one or two of the three venoms, and administering them at different times during the immunization, eventually generating a high titer against the three venoms. Our results suggest that addressing the issue of immunomodulation by venoms might improve antivenom manufacture worldwide. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Do snakes represent the principal predatory threat to callitrichids? Fatal attack of a viper (Bothrops leucurus) on a common marmoset (Callithrix jacchus) in the Atlantic Forest of the Brazilian Northeast.

    PubMed

    Ferrari, Stephen F; Beltrão-Mendes, Raone

    2011-07-01

    A juvenile common marmoset (Callithrix jacchus) was attacked by a whitetail lancehead viper (Bothrops leucurus) while playing with other group members close to the ground at a site in northeastern Brazil. The attack was almost immediately fatal, but the viper was unable to ingest the body of the marmoset. After approximately 10 min, during which it attempted to swallow the marmoset a number of times, the viper moved away, abandoning the body. While raptors are the principal predators of callitrichids, this record reinforces the relative vulnerability of these primates to snakes in comparison with other platyrrhines, although the small number of recorded events precludes a more definitive analysis of the phenomenon.

  17. Comparison of the effect of Crotalus simus and Crotalus durissus ruruima venoms on the equine antibody response towards Bothrops asper venom: implications for the production of polyspecific snake antivenoms.

    PubMed

    Dos-Santos, Maria Cristina; Arroyo, Cynthia; Solano, Sergio; Herrera, María; Villalta, Mauren; Segura, Alvaro; Estrada, Ricardo; Gutiérrez, José María; León, Guillermo

    2011-02-01

    Antivenoms are preparations of immunoglobulins purified from the plasma of animals immunized with snake venoms. Depending on the number of venoms used during the immunization, antivenoms can be monospecific (if venom from a single species is used) or polyspecific (if venoms from several species are used). In turn, polyspecific antivenoms can be prepared by purifying antibodies from the plasma of animals immunized with a mixture of venoms, or by mixing antibodies purified from the plasma of animals immunized separately with single venom. The suitability of these strategies to produce polyspecific antibothropic-crotalic antivenoms was assessed using as models the venoms of Bothrops asper, Crotalus simus and Crotalus durissus ruruima. It was demonstrated that, when used as co-immunogen, C. simus and C. durissus ruruima venoms exert a deleterious effect on the antibody response towards different components of B. asper venom and in the neutralization of hemorrhagic and coagulant effect of this venom when compared with a monospecific B. asper antivenom. Polyspecific antivenoms produced by purifying immunoglobulins from the plasma of animals immunized with venom mixtures showed higher antibody titers and neutralizing capacity than those produced by mixing antibodies purified from the plasma of animals immunized separately with single venom. Thus, despite the deleterious effect of Crotalus sp venoms on the immune response against B. asper venom, the use of venom mixtures is more effective than the immunization with separate venoms for the preparation of polyspecific bothropic-crotalic antivenoms. Copyright © 2010 Elsevier Ltd. All rights reserved.

  18. Snake venomics and antivenomics of Bothrops atrox venoms from Colombia and the Amazon regions of Brazil, Perú and Ecuador suggest the occurrence of geographic variation of venom phenotype by a trend towards paedomorphism.

    PubMed

    Núñez, Vitelbina; Cid, Pedro; Sanz, Libia; De La Torre, Pilar; Angulo, Yamileth; Lomonte, Bruno; Gutiérrez, José María; Calvete, Juan J

    2009-11-02

    The venom proteomes of Bothrops atrox from Colombia, Brazil, Ecuador, and Perú were characterized using venomic and antivenomic strategies. Our results evidence the existence of two geographically differentiated venom phenotypes. The venom from Colombia comprises at least 26 different proteins belonging to 9 different groups of toxins. PI-metalloproteinases and K49-PLA(2) molecules represent the most abundant toxins. On the other hand, the venoms from Brazilian, Ecuadorian, and Peruvian B. atrox contain predominantly PIII-metalloproteinases. These toxin profiles correlate with the venom phenotypes of adult and juvenile B. asper from Costa Rica, respectively, suggesting that paedomorphism represented a selective trend during the trans-Amazonian southward expansion of B. atrox through the Andean Corridor. The high degree of crossreactivity of a Costa Rican polyvalent (Bothrops asper, Lachesis stenophrys, Crotalus simus) antivenom against B. atrox venoms further evidenced the close evolutionary kinship between B. asper and B. atrox. This antivenom was more efficient immunodepleting proteins from the venoms of B. atrox from Brazil, Ecuador, and Perú than from Colombia. Such behaviour may be rationalized taking into account the lower content of poorly immunogenic toxins, such as PLA(2) molecules and PI-SVMPs in the paedomorphic venoms. The immunological profile of the Costa Rican antivenom strongly suggests the possibility of using this antivenom for the management of snakebites by B. atrox in Colombia and the Amazon regions of Ecuador, Perú and Brazil.

  19. Portable exhausters POR-004 SKID B, POR-005 SKID C, POR-006 SKID D storage plan

    SciTech Connect

    Nelson, O.D.

    1997-09-04

    This document provides a storage plan for portable exhausters POR-004 SKID B, POR-005 SKID C, AND POR-006 SKID D. The exhausters will be stored until they are needed by the TWRS (Tank Waste Remediation Systems) Saltwell Pumping Program. The storage plan provides criteria for portable exhauster storage, periodic inspections during storage, and retrieval from storage.

  20. A comparative analysis of the clotting and fibrinolytic activities of the snake venom (Bothrops atrox) from different geographical areas in Venezuela.

    PubMed

    Salazar, Ana Maria; Rodriguez-Acosta, Alexis; Girón, Maria E; Aguilar, Irma; Guerrero, Belsy

    2007-01-01

    Venom constitution within the same snake species can present considerable geographical variations. Bothrops atrox venoms were obtained from adult snakes captured at different geographical locations: Parguasa (Bolívar state); Puerto Ayacucho 1, Serranía del Cuao and Puerto Ayacucho 2 (Amazon state). The coagulant and fibrinolytic activities of these venoms were compared. Amidolytic activity of crude snake venom was measured by a micromethod designed in our laboratory. Coagulant activity on plasma and fibrinogen due to thrombin-like activity in venoms was also determined. Crude snake venom fibrinolytic activity by the fibrin plate method was assayed. Chromatographic studies were developed on Protein-Pack 300 column. Polyacrylamide gel electrophoresis was carried out under reduced conditions. After SDS-PAGE of samples, the fibrin-zymography was tested on agarose-fibrin plates. The results demonstrated several differences among B. atrox venoms from different geographical areas. Chromatograms and SDS-PAGE profiles indicated that venoms from the same species presented differences in the molecular mass of their components. The procoagulant activity depended on the utilized method (amidolytic versus clotting). Parguasa and Puerto Ayacucho 2 venoms presented procoagulant activity for both methods. Furthermore, Parguasa venom had also the highest hemorrhagic activity and the lowest LD50. In relation to the fibrinolytic activity, Puerto Ayacucho 1 venom was the most active, equally for fibrin plates as for the amidolytic method (t-PA like). This venom had the lowest coagulant activity, which induced us to think that probably its procoagulant activity was interfered by its fibrinolytic activity.

  1. Comparison of venoms from wild and long-term captive Bothrops atrox snakes and characterization of Batroxrhagin, the predominant class PIII metalloproteinase from the venom of this species.

    PubMed

    Freitas-de-Sousa, L A; Amazonas, D R; Sousa, L F; Sant'Anna, S S; Nishiyama, M Y; Serrano, S M T; Junqueira-de-Azevedo, I L M; Chalkidis, H M; Moura-da-Silva, A M; Mourão, R H V

    2015-11-01

    Comparisons between venoms from snakes kept under captivity or collected at the natural environment are of fundamental importance in order to obtain effective antivenoms to treat human victims of snakebites. In this study, we compared composition and biological activities of Bothrops atrox venom from snakes collected at Tapajós National Forest (Pará State, Brazil) or maintained for more than 10 years under captivity at Instituto Butantan herpetarium after have been collected mostly at Maranhão State, Brazil. Venoms from captive or wild snakes were similar except for small quantitative differences detected in peaks correspondent to phospholipases A2 (PLA2), snake venom metalloproteinases (SVMP) class PI and serine proteinases (SVSP), which did not correlate with fibrinolytic and coagulant activities (induced by PI-SVMPs and SVSPs). In both pools, the major toxic component corresponded to PIII-SVMPs, which were isolated and characterized. The characterization by mass spectrometry of both samples identified peptides that matched with a single PIII-SVMP cDNA characterized by transcriptomics, named Batroxrhagin. Sequence alignments show a strong similarity between Batroxrhagin and Jararhagin (96%). Batroxrhagin samples isolated from venoms of wild or captive snakes were not pro-coagulant, but inhibited collagen-induced platelet-aggregation, and induced hemorrhage and fibrin lysis with similar doses. Results suggest that in spite of environmental differences, venom variability was detected only among the less abundant components. In opposition, the most abundant toxin, which is a PIII-SVMP related to the key effects of the venom, is structurally conserved in the venoms. This observation is relevant for explaining the efficacy of antivenoms produced with venoms from captive snakes in human accidents inflicted at distinct natural environments. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  2. Structural and functional properties of BaTX, a new Lys49 phospholipase A2 homologue isolated from the venom of the snake Bothrops alternatus.

    PubMed

    Ponce-Soto, Luis Alberto; Lomonte, Bruno; Gutiérrez, José María; Rodrigues-Simioni, Lea; Novello, José Camillo; Marangoni, Sergio

    2007-04-01

    BaTX PLA(2), a K49 phospholipase A(2) homologue was purified from Bothrops alternatus venom after two chromatographic steps, molecular exclusion on Superdex 75 and reverse phase HPLC on mu-Bondapack C-18. A molecular mass of 13898.71 Da was determined by MALDI-TOF mass spectrometry. The amino acid composition showed that BaTX has a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA(2). The complete amino acid sequence of BaTX PLA(2) contains 121 residues, resulting in a calculated pI value of 8.63. This sequence shows high identity values when compared to other K49 PLA(2)s isolated from the venoms of viperid snakes. Lower identity is observed in comparison to D49 PLA(2)s. The sequence was SLFELGKMIL QETGKNPAKS YGAYYCYCGW GGQGQPKDAT DRCCYVHKCC YKKLTGCNPK KDRYSYSWKD KTIVCGENNS CLKELCECDK AVAICLRENL NTYNKKYRYY LKPLCKKADA C. In mice, BaTX induced myonecrosis and edema, upon intramuscular or subcutaneous injections, respectively. The LD(50) of BaTX was 7 mug/g body weight, by intravenous route. In vitro, the toxin caused a potent blockade of neuromuscular transmission in young chicken biventer cervicis preparations. The blockage 50% was achieved at a concentration of 0.03 microM: 40+/-0.4 min and 0.07 microM: 35+/-0.3 min. Moreover, this protein induced a rapid cytolytic effect upon mouse skeletal muscle myoblasts in culture. Thus, the combined structural and functional information obtained identify BaTX as a new member of the K49 PLA(2) family, which presents the typical bioactivities described for such proteins.

  3. BmajPLA2-II, a basic Lys49-phospholipase A2 homologue from Bothrops marajoensis snake venom with parasiticidal potential.

    PubMed

    Grabner, Amy N; Alfonso, Jorge; Kayano, Anderson M; Moreira-Dill, Leandro S; Dos Santos, Ana Paula de A; Caldeira, Cleópatra A S; Sobrinho, Juliana C; Gómez, Ana; Grabner, Fernando P; Cardoso, Fabio F; Zuliani, Juliana Pavan; Fontes, Marcos R M; Pimenta, Daniel C; Gómez, Celeste Vega; Teles, Carolina B G; Soares, Andreimar M; Calderon, Leonardo A

    2017-09-01

    Snake venoms contain various proteins, especially phospholipases A2 (PLA2s), which present potential applications in diverse areas of health and medicine. In this study, a new basic PLA2 from Bothrops marajoensis with parasiticidal activity was purified and characterized biochemically and biologically. B. marajoensis venom was fractionated through cation exchange followed by reverse phase chromatographies. The isolated toxin, BmajPLA2-II, was structurally characterized with MALDI-TOF (Matrix-assisted laser desorption/ionization-time of flight) mass spectrometry, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by two-dimensional electrophoresis, partial amino acid sequencing, an enzymatic activity assay, circular dichroism, and dynamic light scattering assays. These structural characterization tests presented BmajPLA2-II as a basic Lys49 PLA2 homologue, compatible with other basic snake venom PLA2s (svPLA2), with a tendency to form aggregations. The in vitro anti-parasitic potential of B. marajoensis venom and of BmajPLA2-II was evaluated against Leishmania infantum promastigotes and Trypanosoma cruzi epimastigotes, showing significant activity at a concentration of 100μg/mL. The venom and BmajPLA2-II presented IC50 of 0.14±0.08 and 6.41±0.64μg/mL, respectively, against intraerythrocytic forms of Plasmodium falciparum with CC50 cytotoxicity values against HepG2 cells of 43.64±7.94 and >150μg/mL, respectively. The biotechnological potential of these substances in relation to leishmaniasis, Chagas disease and malaria should be more deeply investigated. Copyright © 2017. Published by Elsevier B.V.

  4. Increase of the cytotoxic effect of Bothrops jararacussu venom on mouse extensor digitorum longus and soleus by potassium channel blockers and by Na(+)/K(+)-ATPase inhibition.

    PubMed

    Tomaz, Marcelo A; Fernandes, Fabrício F A; El-Kik, Camila Z; Moraes, Raphael A M; Calil-Elias, Sabrina; Saturnino-Oliveira, Jeison; Martinez, Ana Maria B; Ownby, Charlotte L; Melo, Paulo A

    2008-09-15

    We investigated the myotoxicity of Bothrops jararacussu crude venom and other cytolytic agents on mouse isolated extensor digitorum longus (EDL) and soleus (SOL) muscles, which present distinct properties: EDL is a fast-twitch, white muscle with predominantly glycolytic fibers, while SOL is slow-twitch, red muscle with predominantly oxidative fibers. Muscles were exposed to B. jararacussu crude venom (25 microg/ml) and other crotaline venoms (Agkistrodon contortrix laticinctus; Crotalus viridis viridis; Crotalus durissus terrificus) at the same concentration. Basal creatine kinase (CK) release to bathing solution was 0.43+/-0.06 for EDL and 0.29+/-0.06 for SOL (U g(-)(1) h(-)(1), n=36 for each muscle). Sixty minutes after exposure to B. jararacussu venom, EDL presented higher increase in the rate of CK release than SOL, respectively, 13.2+/-1.5 and 2.9+/-0.7 U g(-)(1)h(-)(1), n=10-12. Muscle denervation, despite decreasing CK content, did not affect sensitivities to B. jararacussu venom. Ouabain and potassium channel blockers (TEA; clotrimazole; glibenclamide) increased the rate of CK release by B. jararacussu in EDL and SOL muscles, decreasing and almost abolishing the different sensitivity. When we exposed EDL or SOL muscles to Naja naja, Apis mellifera venoms (25 microg/ml), or Triton X-100 (0.01%), they showed similar rate of CK release. Our present data suggest that a mechanism involving intracellular calcium regulation or potassium channels may participate in the different sensitivity of EDL and SOL to B. jararacussu venom.

  5. Differential transcript profile of inhibitors with potential anti-venom role in the liver of juvenile and adult Bothrops jararaca snake.

    PubMed

    Gomes, Cícera Maria; de Morais-Zani, Karen; Lu, Stephen; Buarque, Diego de Souza; Braz, Glória Regina Cardoso; Grego, Kathleen Fernandes; Tanaka, Aparecida Sadae; Tanaka-Azevedo, Anita Mitico

    2017-01-01

    Snakes belonging to the Bothrops genus are vastly distributed in Central and South America and are responsible for most cases of reported snake bites in Latin America. The clinical manifestations of the envenomation caused by this genus are due to three major activities-proteolytic, hemorrhagic and coagulant-mediated by metalloproteinases, serine proteinases, phospholipases A2 and other toxic compounds present in snake venom. Interestingly, it was observed that snakes are resistant to the toxic effects of its own and other snake's venoms. This natural immunity may occur due the absence of toxin target or the presence of molecules in the snake plasma able to neutralize such toxins. In order to identify anti-venom molecules, we construct a cDNA library from the liver of B. jararaca snakes. Moreover, we analyzed the expression profile of four molecules-the already known anti-hemorrhagic factor Bj46a, one gamma-phospholipase A2 inhibitor, one inter-alpha inhibitor and one C1 plasma protease inhibitor-in the liver of juvenile and adult snakes by qPCR. The results revealed a 30-fold increase of gamma-phospholipase A2 inhibitor and a minor increase of the inter-alpha inhibitor (5-fold) and of the C1 inhibitor (3-fold) in adults. However, the Bj46a factor seems to be equally transcribed in adults and juveniles. The results suggest the up-regulation of different inhibitors observed in the adult snakes might be a physiological adaptation to the recurrent contact with their own and even other snake's venoms throughout its lifespan. This is the first comparative analysis of ontogenetic variation of expression profiles of plasmatic proteins with potential anti-venom activities of the venomous snake B. jararaca. Furthermore, the present data contributes to the understanding of the natural resistance described in these snakes.

  6. Differential transcript profile of inhibitors with potential anti-venom role in the liver of juvenile and adult Bothrops jararaca snake

    PubMed Central

    Gomes, Cícera Maria; de Morais-Zani, Karen; Lu, Stephen; Buarque, Diego de Souza; Braz, Glória Regina Cardoso; Grego, Kathleen Fernandes

    2017-01-01

    Background Snakes belonging to the Bothrops genus are vastly distributed in Central and South America and are responsible for most cases of reported snake bites in Latin America. The clinical manifestations of the envenomation caused by this genus are due to three major activities—proteolytic, hemorrhagic and coagulant—mediated by metalloproteinases, serine proteinases, phospholipases A2 and other toxic compounds present in snake venom. Interestingly, it was observed that snakes are resistant to the toxic effects of its own and other snake’s venoms. This natural immunity may occur due the absence of toxin target or the presence of molecules in the snake plasma able to neutralize such toxins. Methods In order to identify anti-venom molecules, we construct a cDNA library from the liver of B. jararaca snakes. Moreover, we analyzed the expression profile of four molecules—the already known anti-hemorrhagic factor Bj46a, one gamma-phospholipase A2 inhibitor, one inter-alpha inhibitor and one C1 plasma protease inhibitor—in the liver of juvenile and adult snakes by qPCR. Results The results revealed a 30-fold increase of gamma-phospholipase A2 inhibitor and a minor increase of the inter-alpha inhibitor (5-fold) and of the C1 inhibitor (3-fold) in adults. However, the Bj46a factor seems to be equally transcribed in adults and juveniles. Discussion The results suggest the up-regulation of different inhibitors observed in the adult snakes might be a physiological adaptation to the recurrent contact with their own and even other snake’s venoms throughout its lifespan. This is the first comparative analysis of ontogenetic variation of expression profiles of plasmatic proteins with potential anti-venom activities of the venomous snake B. jararaca. Furthermore, the present data contributes to the understanding of the natural resistance described in these snakes. PMID:28462021

  7. Cross-neutralization of the neurotoxicity of Crotalus durissus terrificus and Bothrops jararacussu venoms by antisera against crotoxin and phospholipase A2 from Crotalus durissus cascavella venom.

    PubMed

    Beghini, Daniela G; da Cruz-Höfling, Maria Alice; Randazzo-Moura, Priscila; Rodrigues-Simioni, Léa; Novello, José Camilo; Hyslop, Stephen; Marangoni, Sérgio

    2005-11-01

    We have previously demonstrated that rabbit antisera raised against crotoxin from Crotalus durissus cascavella venom (cdc-crotoxin) and its PLA2 (cdc-PLA2) neutralized the neurotoxicity of this venom and its crotoxin. In this study, we examined the ability of these antisera to neutralize the neurotoxicity of Crotalus durissus terrificus and Bothrops jararacussu venoms and their major toxins, cdt-crotoxin and bothropstoxin-I (BthTX-I), respectively, in mouse isolated phrenic nerve-diaphragm preparations. Immunoblotting showed that antiserum to cdc-crotoxin recognized cdt-crotoxin and BthTX-I, while antiserum to cdc-PLA2 recognized cdt-PLA2 and BthTX-I. ELISA corroborated this cross-reactivity. Antiserum to cdc-crotoxin prevented the neuromuscular blockade caused by C. d. terrificus venom and its crotoxin at a venom/crotoxin:antiserum ratio of 1:3. Antiserum to cdc-PLA2 also neutralized the neuromuscular blockade caused by C. d. terrificus venom or its crotoxin at venom or toxin:antiserum ratios of 1:3 and 1:1, respectively. The neuromuscular blockade caused by B. jararacussu venom and BthTX-I was also neutralized by the antisera to cdc-crotoxin and cdc-PLA2 at a venom/toxin:antiserum ratio of 1:10 for both. Commercial equine antivenom raised against C. d. terrificus venom was effective in preventing the neuromuscular blockade typical of B. jararacussu venom (venom:antivenom ratio of 1:2), whereas for BthTX-I the ratio was 1:10. These results show that antiserum produced against PLA2, the major toxin in C. durissus cascavella venom, efficiently neutralized the neurotoxicity of C. d. terrificus and B. jararacussu venoms and their PLA2 toxins.

  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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Snakebites and ethnobotany in the northwest region of Colombia: Part II: neutralization of lethal and enzymatic effects of Bothrops atrox venom.

    PubMed

    Otero, R; Núñez, V; Jiménez, S L; Fonnegra, R; Osorio, R G; García, M E; Díaz, A

    2000-08-01

    Twelve of 74 ethanolic extracts of plants used by traditional healers for snakebites in the northwest region of Colombia, were active against lethal effect of Bothrops atrox venom when they were i.p. injected into mice (18-20 g). After preincubation of sublethal doses of every extract (0.5-4.0 mg/mouse) with 1.5 i.p. lethal dose 50% (LD50) (99.3 microg) of venom, seven of them demonstrated 100% neutralizing capacity within 48 h. These were the stem barks of Brownea rosademonte (Caesalpiniaceae) and Tabebuia rosea (Bignoniaceae); rhizomes of Renealmia alpinia (Zingiberaceae) and Heliconia curtispatha (Heliconiaceae); the whole plants of Pleopeltis percussa (Polypodiaceae) and Trichomanes elegans (Hymenophyllaceae); and the ripe fruits of Citrus limon (Rutaceae). The other five extracts showing partial neutralization (45-80%; 10-30% survival rate in the control group receiving the venom alone; P<0.05) were: leaves, branches and stem of Costus lasius (Costaceae); the whole plant of Sida acuta (Malvaceae); rhizomes of Dracontium croatii (Araceae); leaves and branches of Bixa orellana (Bixaceae) and Struthanthus orbicularis (Loranthaceae). When the extracts were independently administered per oral or i.p. route 60 min before an i.m. venom injection (204 microg=1.5 i.m. LD50), C. limon, T. elegans, B. orellana and T. rosea extracts had partial and significant neutralizing capacity against B. atrox venom lethal effect. C. limon extract was also partially effective when it was administered either i.v. 15 min before or i.p. 5 min after an i.m. venom injection. Three of the 12 extracts with anti-lethal effect (C. limon, D. croatii and S. acuta) were devoid of antiphospholipase A2 activity, when they were tested against one minimum indirect hemolytic dose of B. atrox venom (2 microg) in agarose-erythrocyte-egg yolk gels.

  10. Neutralization of the edema-forming, defibrinating and coagulant effects of Bothrops asper venom by extracts of plants used by healers in Colombia.

    PubMed

    Núñez, V; Otero, R; Barona, J; Saldarriaga, M; Osorio, R G; Fonnegra, R; Jiménez, S L; Díaz, A; Quintana, J C

    2004-07-01

    We determined the neutralizing activity of 12 ethanolic extracts of plants against the edema-forming, defibrinating and coagulant effects of Bothrops asper venom in Swiss Webster mice. The material used consisted of the leaves and branches of Bixa orellana (Bixaceae), Ficus nymphaeifolia (Moraceae), Struthanthus orbicularis (Loranthaceae) and Gonzalagunia panamensis (Rubiaceae); the stem barks of Brownea rosademonte (Caesalpiniaceae) and Tabebuia rosea (Bignoniaceae); the whole plant of Pleopeltis percussa (Polypodiaceae) and Trichomanes elegans (Hymenophyllaceae); rhizomes of Renealmia alpinia (Zingiberaceae), Heliconia curtispatha (Heliconiaceae) and Dracontium croatii (Araceae), and the ripe fruit of Citrus limon (Rutaceae). After preincubation of varying amounts of each extract with either 1.0 microg venom for the edema-forming effect or 2.0 microg venom for the defibrinating effect, the mixture was injected subcutaneously (sc) into the right foot pad or intravenously into the tail, respectively, to groups of four mice (18-20 g). All extracts (6.2-200 microg/mouse) partially neutralized the edema-forming activity of venom in a dose-dependent manner (58-76% inhibition), with B. orellana, S. orbicularis, G. panamensis, B. rosademonte, and D. croatii showing the highest effect. Ten extracts (3.9-2000 microg/mouse) also showed 100% neutralizing ability against the defibrinating effect of venom, and nine prolonged the coagulation time induced by the venom. When the extracts were administered either before or after venom injection, the neutralization of the edema-forming effect was lower than 40% for all extracts, and none of them neutralized the defibrinating effect of venom. When they were administered in situ (sc at the same site 5 min after venom injection), the neutralization of edema increased for six extracts, reaching levels up to 64% for C. limon.

  11. Plants used to treat snakebites in Santarém, western Pará, Brazil: an assessment of their effectiveness in inhibiting hemorrhagic activity induced by Bothrops jararaca venom.

    PubMed

    de Moura, Valéria Mourão; Freitas de Sousa, Luciana A; Cristina Dos-Santos, Maria; Almeida Raposo, Juliana Divina; Evangelista Lima, Aline; de Oliveira, Ricardo Bezerra; da Silva, Milton Nascimento; Veras Mourão, Rosa Helena

    2015-02-23

    The poor distribution and limited availability of antivenoms in Brazil have led to greater use of plants to treat snakebites. Very often such plants are the only alternative available to riverside communities. Direct questionnaire-based interviews were conducted with members of the Cucurunã, São Pedro and Alter do Chão communities in Santarém, Pará, Brazil. For each of the 12 most frequently mentioned species aqueous extracts were prepared and the phytochemical profiles determined by thin layer chromatography. The concentrations of phenolic compounds (tannins and flavonoids) in the aqueous extracts were determined by colorimetric assays. To assess inhibition of the hemorrhagic activity of Bothrops jararaca venom, solutions containing the venom mixed with aqueous extracts in the ratios 1:12 and 1:48 were tested (w/w). SDS-PAGE and Western blot were used to assess the action of the extracts on Bothrops jararaca venom. In all, 24 plants belonging to 19 families were mentioned in the survey as being used to treat snakebites. Leaves (84%), seeds (60.9%) and inner bark (53%) were cited as the most frequently used parts in folk remedies, which were usually prepared in the form of a decoction (62.5%), tincture (45%) or maceration (22.5%). Hemorrhage induced by Bothrops jararaca venom was completely inhibited by aqueous extracts of Bellucia dichotoma, Connarus favosus, Plathymenia reticulata and Philodendron megalophyllum, which had a high phenolic content and contained condensed and hydrolyzable tannins. The results of SDS-PAGE showed that some venom protein bands were not visible when the venom was preincubated with the extracts that had completely inhibited hemorrhagic activity of the venom. Western blot showed that the extracts did not have any enzymatic action on the proteins in the venom as it failed to detect low-molecular-weight bands, which are indicative of possible enzymatic cleavage. Traditional use of plants to treat snakebites is a common practice in the

  12. Comparative study of the efficacy and safety of two polyvalent, caprylic acid fractionated [IgG and F(ab')2] antivenoms, in Bothrops asper bites in Colombia.

    PubMed

    Otero-Patiño, Rafael; Segura, Alvaro; Herrera, María; Angulo, Yamileth; León, Guillermo; Gutiérrez, José María; Barona, Jacqueline; Estrada, Sebastián; Pereañez, Andrés; Quintana, Juan Carlos; Vargas, Leidy J; Gómez, Juan Pablo; Díaz, Abel; Suárez, Ana María; Fernández, Jorge; Ramírez, Patricia; Fabra, Patricia; Perea, Monica; Fernández, Diego; Arroyo, Yobana; Betancur, Dalila; Pupo, Lady; Córdoba, Elkin A; Ramírez, C Eugenio; Arrieta, Ana Berta; Rivero, Alcides; Mosquera, Diana Carolina; Conrado, Nectty Lorena; Ortiz, Rosina

    2012-02-01

    The efficacy and safety of two polyvalent horse-derived antivenoms in Bothrops asper envenomings were tested in a randomized, double-blind, clinical trial performed in Colombia. Both antivenoms were manufactured from the same pool of hyperimmune plasma. Antivenom A was made of F(ab')2 fragments, generated by pepsin digestion and caprylic acid precipitation, whereas antivenom B consisted of whole IgG molecules produced by caprylic acid precipitation followed by ion-exchange chromatography. Besides the different nature of the active substance, antivenom B had higher protein concentration, slightly higher turbidity and aggregate content. No significant differences were observed in the efficacy of antivenoms. Both halted local and systemic bleeding (P = 0.40) within 6-12 h of treatment in 100% of the cases, and restored blood coagulation (P = 0.87) within 6-24 h in 84.7% of patients, and within 48 h in all of them, in agreement with restoration of plasma fibrinogen concentration. Venom concentrations in serum dropped significantly (P < 0.001), to very low levels, 1 h after antivenom infusion. Nevertheless, eight patients (11.1%), four for each antivenom, presented recurrence of venom antigenaemia at different times, from 6 to 96 h, with clinical significance (recurrent coagulopathy) only in one group B patient (2.9%). Serum creatine kinase (CK) activity was increased, as a consequence of local myonecrosis. There was no significant difference (P = 0.51) in the incidence of early adverse reactions to antivenom administration (28.9% for patients of group A and 20.6% for patients of group B), most of the reactions being mild, mainly cutaneous. The most frequent complications were cellulitis (16.7%), abscess formation (5.6%), acute renal failure (8.3%), and compartmental syndrome (5.6%). In conclusion, IgG and F(ab')2 antivenoms, prepared by caprylic acid fractionation, presented similar efficacy and safety profiles for the treatment of B. asper envenomings in Colombia

  13. Increments in cytokines and matrix metalloproteinases in skeletal muscle after injection of tissue-damaging toxins from the venom of the snake Bothrops asper.

    PubMed Central

    Rucavado, Alexandra; Escalante, Teresa; Teixeira, Catarina F P; Fernándes, Cristina María; Diaz, Cecilia; Gutiérrez, José María

    2002-01-01

    Envenomations by the snake Bothrops asper are characterized by prominent local tissue damage (i.e. myonecrosis), blistering, hemorrhage and edema. Various phospholipases A2 and metalloproteinases that induce local pathological alterations have been purified from this venom. Since these toxins induce a conspicuous inflammatory response, it has been hypothesized that inflammatory mediators may contribute to the local pathological alterations described. This study evaluated the local production of cytokines and matrix metalloproteinases (MMPs) as a consequence of intramuscular injections of an Asp-49 myotoxic phospholipase A2 (myotoxin III (MT-III)) and a P-I type hemorrhagic metalloproteinase (BaP1) isolated from B. asper venom. Both enzymes induced prominent tissue alterations and conspicuous increments in interleukin (IL)-1beta, IL-6 and a number of MMPs, especially gelatinase MMP-9, rapidly after injection. In contrast, no increments in tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma were detected. In agreement, MT-III and BaP1 did not induce the synthesis of TNF-alpha by resident peritoneal macrophages in vitro. Despite the conspicuous expression of latent forms of MMPs in muscle, evidenced by zymography, there were no increments in activated MMP-2 and only a small increase in activated MMP-9, as detected by a functional enzymatic assay. This suggests that MMP activity was regulated by a highly controlled activation of latent forms and, probably, by a concomitant synthesis of MMP inhibitors. Since no hemorrhage nor dermonecrosis were observed after injection of MT-III, despite a prominent increase in MMP expression, and since inflammatory exudate did not enhance hemorrhage induced by BaP1, it is suggested that endogenous MMPs released in the tissue are not responsible for the dermonecrosis and hemorrhage characteristic of B. asper envenomation. Moreover, pretreatment of mice with the peptidomimetic MMP inhibitor batimastat did not reduce myotoxic nor

  14. A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom.

    PubMed

    Castro, J M A; Oliveira, T S; Silveira, C R F; Caporrino, M C; Rodriguez, D; Moura-da-Silva, A M; Ramos, O H P; Rucavado, A; Gutiérrez, J M; Magalhães, G S; Faquim-Mauro, E L; Fernandes, I

    2014-09-01

    BaP1 is a P-I class snake venom metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomings by Bothrops asper, a medically important snake species in Central America and parts of South and North America. The main treatment for these accidents is the passive immunotherapy using antibodies raised in horses. In order to obtain more specific and batch-to-batch consistent antivenons, recombinant antibodies are considered a good option compared to animal immunization. We constructed a recombinant single chain variable fragment (scFv) from a monoclonal antibody against BaP1 (MABaP1) formerly secreted by a hybridoma clone. This recombinant antibody was cloned into pMST3 vector in fusion with SUMO protein and contains VH and VL domains linked by a flexible (G4S)3 polypeptide (scFvBaP1). The aim of this work was to produce scFvBaP1 and to evaluate its potential concerning the neutralization of biologically important activities of BaP1. The cytoplasmic expression of this construct was successfully achieved in C43 (DE3) bacteria. Our results showed that scFvBaP1-SUMO fusion protein presented an electrophoretic band of around 43 kDa from which SUMO alone corresponded to 13.6 kDa, and only the scFv was able to recognize BaP1 as well as the whole venom by ELISA. In contrast, neither an irrelevant scFv anti-LDL nor its MoAb partner recognized it. BaP1-induced fibrinolysis was significantly neutralized by scFvBaP1, but not by SUMO, in a concentration-dependent manner. In addition, scFvBaP1, as well as MaBaP1, completely neutralized in vivo hemorrhage, muscle necrosis, and inflammation induced by the toxin. Docking analyses revealed possible modes of interaction of the recombinant antibody with BaP1. Our data showed that scFv recognized BaP1 and whole B. asper venom, and neutralized biological effects of this SVMP. This scFv antibody can be used for understanding the molecular mechanisms of neutralization of SVMPs, and for exploring the potential of

  15. The potential of aqueous extracts of Bellucia dichotoma Cogn. (Melastomataceae) to inhibit the biological activities of Bothrops atrox venom: A comparison of specimens collected in the states of Pará and Amazonas, Brazil.

    PubMed

    de Moura, Valéria Mourão; de Souza, Luana Yamille Andrade; da Costa Guimarães, Noranathan; Dos Santos, Ilia Gilmara Carvalho; de Almeida, Patrícia Danielle Oliveira; de Oliveira, Ricardo Bezerra; Mourão, Rosa Helena Veras; Dos-Santos, Maria Cristina

    2017-01-20

    The effectiveness of aqueous extract of Bellucia dichotoma Cogn. (Melastomataceae) specimems collected in Santarém, PA, against some biological activities of Bothrops atrox venom (BaV) has been scientifically proven. Here, we analyzed the components and assessed the anti-snakebite potential of aqueous extracts of bark of B. dichotoma collected in Manaus, AM, (AEBd-MAO) and Santarém, PA, (AEBd-STM), both in Brazil. The phytochemical profiles of the aqueous extracts were identified using thin layer chromatography (TLC), and the concentrations of phenolics were determined by colorimetric assay. The inhibitory potential of the extracts was tested against the phospholipase A2, coagulant and gelatinolytic activities of BaV in vitro and its defibrinating and edema-inducing activities in vivo. Interaction between BaV and the extracts was investigated using SDS-Page electrophoresis and Western blotting. Extract cytotoxicity and antioxidant potential were assessed using the human fibroblast cell line MRC-5 and the DPPH assay in cell culture, respectively. While there was no difference between the phytochemical profiles of the extracts, AEBd-MAO had higher concentrations of total phenolics, total tannins and hydrolysable tannins. The extracts inhibited 100% of the phospholipase and coagulant activity of BaV when pre-incubated. Without pre-incubation, however, there was no reduction in phospholipase activity, although significant inhibition of coagulant activity was observed. In the doses used in folk medicine, without pre-incubation, both extracts inhibited 100% of the coagulant activity of BaV. In vivo, the extracts were unable to inhibit the defibrinating activity of the venom but were effective in inhibiting its edema-inducing activity. In the profiles of the extracts pre-incubated with BaV, not all the protein bands revealed by SDS-PAGE and Western blot were observed. Both extracts had a high antioxidant potential and neither had a cytotoxic effect. Although the

  16. Efficacy and safety of two whole IgG polyvalent antivenoms, refined by caprylic acid fractionation with or without beta-propiolactone, in the treatment of Bothrops asper bites in Colombia.

    PubMed

    Otero, Rafael; León, Guillermo; Gutiérrez, José María; Rojas, Gustavo; Toro, María Fabiola; Barona, Jacqueline; Rodríguez, Verónica; Díaz, Abel; Núñez, Vitelbina; Quintana, Juan Carlos; Ayala, Shirley; Mosquera, Diana; Conrado, Lesdy L; Fernández, Diego; Arroyo, Yobana; Paniagua, Carlos A; López, Mercedes; Ospina, Carlos E; Alzate, Claudia; Fernández, Jorge; Meza, Jazmín J; Silva, Juan F; Ramírez, Patricia; Fabra, Patricia E; Ramírez, Eugenio; Córdoba, Elkin; Arrieta, Ana B; Warrell, David A; Theakston, R David G

    2006-12-01

    The efficacy and safety of two whole IgG polyvalent antivenoms (A and B) were compared in a randomised, blinded clinical trial in 67 patients systemically envenomed by Bothrops asper in Colombia. Both antivenoms were fractionated by caprylic acid precipitation and had similar neutralising potencies, protein concentrations and aggregate contents. Antivenom B was additionally treated with beta-propiolactone to lower its anticomplementary activity. Analysing all treatment regimens together, there were no significant differences between the two antivenoms (A=34 patients; B=33 patients) in the time taken to reverse venom-induced bleeding and coagulopathy, to restore physiological fibrinogen concentrations and to clear serum venom antigenaemia. Blood coagulability was restored within 6-24 h in 97% of patients, all of whom had normal coagulation and plasma fibrinogen levels 48 h after the start of antivenom treatment. Two patients (3.0%) had recurrent coagulopathy and eight patients suffered recurrence of antigenaemia within 72 h of treatment. None of the dosage regimens of either antivenom used guaranteed resolution of venom-induced coagulopathy within 6 h, nor did they prevent recurrences. A further dose of antivenom at 6 h also did not guarantee resolution of coagulopathy within 12-24 h in all patients. The incidence of early adverse reactions (all mild) was similar for both antivenoms (15% and 24%; P>0.05).

  17. Interaction between TNF and BmooMP-Alpha-I, a Zinc Metalloprotease Derived from Bothrops moojeni Snake Venom, Promotes Direct Proteolysis of This Cytokine: Molecular Modeling and Docking at a Glance

    PubMed Central

    Silva, Maraisa Cristina; Lopes Silva, Tamires; Silva, Murilo Vieira; Mota, Caroline Martins; Santiago, Fernanda Maria; Fonseca, Kelly Cortes; Oliveira, Fábio; Mineo, Tiago Wilson Patriarca; Mineo, José Roberto

    2016-01-01

    Tumor necrosis factor (TNF) is a major cytokine in inflammatory processes and its deregulation plays a pivotal role in several diseases. Here, we report that a zinc metalloprotease extracted from Bothrops moojeni venom (BmooMP-alpha-I) inhibits TNF directly by promoting its degradation. This inhibition was demonstrated by both in vitro and in vivo assays, using known TLR ligands. These findings are supported by molecular docking results, which reveal interaction between BmooMP-alpha-I and TNF. The major cluster of interaction between BmooMP-alpha-I and TNF was confirmed by the structural alignment presenting Ligand Root Mean Square Deviation LRMS = 1.05 Å and Interactive Root Mean Square Deviation IRMS = 1.01 Å, this result being compatible with an accurate complex. Additionally, we demonstrated that the effect of this metalloprotease on TNF is independent of cell cytotoxicity and it does not affect other TLR-triggered cytokines, such as IL-12. Together, these results indicate that this zinc metalloprotease is a potential tool to be further investigated for the treatment of inflammatory disorders involving TNF deregulation. PMID:27447669

  18. A randomized blinded clinical trial of two antivenoms, prepared by caprylic acid or ammonium sulphate fractionation of IgG, in Bothrops and Porthidium snake bites in Colombia: correlation between safety and biochemical characteristics of antivenoms.

    PubMed

    Otero, R; Gutiérrez, J M; Rojas, G; Núñez, V; Díaz, A; Miranda, E; Uribe, A F; Silva, J F; Ospina, J G; Medina, Y; Toro, M F; García, M E; León, G; García, M; Lizano, S; De La Torre, J; Márquez, J; Mena, Y; González, N; Arenas, L C; Puzón, A; Blanco, N; Sierra, A; Espinal, M E; Lozano, R

    1999-06-01

    A randomized blinded clinical trial was performed in 53 patients bitten by Bothrops sp. and Porthidium sp. in Antioquia and Chocó, Colombia, in order to compare the efficacy and safety of two antivenoms made of whole IgG obtained by either ammonium sulphate (monovalent anti-B. atrox) or caprylic acid (polyvalent) fractionation. Additionally, antivenoms were compared by electrophoretic and chromatographic analyses and anticomplementary activity in vitro. With a protocol of 2, 4 and 6 antivenom vials for the treatment of mild, moderate and severe envenomings, respectively, both antivenoms were equally efficient to neutralize the most relevant signs of envenoming and to clear serum venom levels in patients from the first hour and later on. Three patients with severe envenoming and initially treated with less than six vials on admission had persistent or recurrent venom antigenemia within 12-48 h. Monovalent antivenom fractionated by ammonium sulphate precipitation had higher amounts of protein aggregates and nonimmunoglobulin proteins than polyvalent antivenom fractionated by caprylic acid precipitation. Both antivenoms presented anticomplementary activity in vitro, being higher in the monovalent product. In agreement, monovalent antivenom induced a significantly higher incidence of early antivenom reactions (52%) than polyvalent antivenom (25%).

  19. Glycolic acid inhibits enzymatic, hemorrhagic and edema-inducing activities of BaP1, a P-I metalloproteinase from Bothrops asper snake venom: insights from docking and molecular modeling.

    PubMed

    Pereañez, Jaime Andrés; Patiño, Arley Camilo; Rey-Suarez, Paola; Núñez, Vitelbina; Henao Castañeda, Isabel Cristina; Rucavado, Alexandra

    2013-09-01

    Glycolic acid (GA) (2-Hydroxyethanoic acid) is widely used as chemical peeling agent in Dermatology and, more recently, as a therapeutic and cosmetic compound in the field of skin care and disease treatment. In this work we tested the inhibitory ability of glycolic acid on the enzymatic, hemorrhagic and edema-inducing activities of BaP1, a P-I metalloproteinase from Bothrops asper venom, which induces a variety of toxic actions. Glycolic acid inhibited the proteolytic activity of BaP1 on azocasein, with an IC₅₀ of 1.67 mM. The compound was also effective at inhibiting the hemorrhagic activity of BaP1 in skin and muscle in experiments involving preincubation of enzyme and inhibitor prior to injection. When BaP1 was injected i.m. and then, at the same site, different concentrations of glycolic acid were administered at either 0 or 5 min, 7 mM solutions of the inhibitor partially abrogated hemorrhagic activity when administered at 0 min. Moreover, glycolic acid inhibited, in a concentration-dependent manner, edema-forming activity of BaP1 in the footpad. In order to have insights on the mode of action of glycolic acid, UV-vis and intrinsic fluorescence studies were performed. Results of these assays suggest that glycolic acid interacts directly with BaP1 and chelates the Zn²⁺ ion at the active site. These findings were supported by molecular docking results, which suggested that glycolic acid forms hydrogen bonds with residues Glu143, Arg110 and Ala111 of the enzyme. Additionally, molecular modeling results suggest that the inhibitor chelates Zn²⁺, with a distance of 3.58 Å, and may occupy part of substrate binding cleft of BaP1. Our results suggest that glycolic acid is a candidate for the development of inhibitors to be used in snakebite envenomation.

  20. Moojenactivase, a novel pro-coagulant PIIId metalloprotease isolated from Bothrops moojeni snake venom, activates coagulation factors II and X and induces tissue factor up-regulation in leukocytes.

    PubMed

    Sartim, Marco A; Costa, Tassia R; Laure, Helen J; Espíndola, Milena S; Frantz, Fabiani G; Sorgi, Carlos A; Cintra, Adélia C O; Arantes, Eliane C; Faccioli, Lucia H; Rosa, José C; Sampaio, Suely V

    2016-05-01

    Coagulopathies following snakebite are triggered by pro-coagulant venom toxins, in which metalloproteases play a major role in envenomation-induced coagulation disorders by acting on coagulation cascade, platelet function and fibrinolysis. Considering this relevance, here we describe the isolation and biochemical characterization of moojenactivase (MooA), a metalloprotease from Bothrops moojeni snake venom, and investigate its involvement in hemostasis in vitro. MooA is a glycoprotein of 85,746.22 Da, member of the PIIId group of snake venom metalloproteases, composed of three linked disulfide-bonded chains: an N-glycosylated heavy chain, and two light chains. The venom protease induced human plasma clotting in vitro by activating on both blood coagulation factors II (prothrombin) and X, which in turn generated α-thrombin and factor Xa, respectively. Additionally, MooA induced expression of tissue factor (TF) on the membrane surface of peripheral blood mononuclear cells (PBMC), which led these cells to adopt pro-coagulant characteristics. MooA was also shown to be involved with production of the inflammatory mediators TNF-α, IL-8 and MCP-1, suggesting an association between MooA pro-inflammatory stimulation of PBMC and TF up-regulation. We also observed aggregation of washed platelets when in presence of MooA; however, the protease had no effect on fibrinolysis. Our findings show that MooA is a novel hemostatically active metalloprotease, which may lead to the development of coagulopathies during B. moojeni envenomation. Moreover, the metalloprotease may contribute to the development of new diagnostic tools and pharmacological approaches applied to hemostatic disorders.

  1. Evolution of NADPH-cytochrome P450 oxidoreductases (POR) in Apiales - POR 1 is missing.

    PubMed

    Andersen, Trine Bundgaard; Hansen, Niels Bjørn; Laursen, Tomas; Weitzel, Corinna; Simonsen, Henrik Toft

    2016-05-01

    The NADPH-dependent cytochrome P450 oxidoreductase (POR) is the obligate electron donor to eukaryotic microsomal cytochromes P450 enzymes. The number of PORs within plant species is limited to one to four isoforms, with the most common being two PORs per plant. These enzymes provide electrons to a huge number of different cytochromes P450s (from 50 to several hundred within one plant). Within the eudicotyledons, PORs can be divided into two major clades, POR 1 and POR 2. Based on our own sequencing analysis and publicly available data, we have identified 45 PORs from the angiosperm order Apiales. These were subjected to a phylogenetic analysis along with 237 other publicly available (NCBI and oneKP) POR sequences found within the clade Asterids. Here, we show that the order Apiales only harbor members of the POR 2 clade, which are further divided into two distinct subclades. This is in contrast to most other eudicotyledon orders that have both POR 1 and POR 2. This suggests that through gene duplications and one gene deletion, Apiales only contain members of the POR 2 clade. Three POR 2 isoforms from Thapsia garganica L., Apiaceae, were all full-length in an Illumina root transcriptome dataset (available from the SRA at NCBI). All three genes were shown to be functional upon reconstitution into nanodiscs, confirming that none of the isoforms are pseudogenes. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Portable exhauster POR-007/Skid E and POR-008/Skid F storage plan

    SciTech Connect

    Nelson, O.D.

    1998-07-25

    This document provides storage requirements for 1,000 CFM portable exhausters POR-O07/Skid E and POR-008/Skid F. These requirements are presented in three parts: preparation for storage, storage maintenance and testing, and retrieval from storage. The exhauster component identification numbers listed in this document contain the prefix POR-007 or POR-008 depending on which exhauster is being used.

  3. Consequences of POR mutations and polymorphisms

    PubMed Central

    Miller, Walter L.; Agrawal, Vishal; Sandee, Duanpen; Tee, Meng Kian; Huang, Ningwu; Choi, Ji Ha; Morrissey, Kari; Giacomini, Kathleen M.

    2015-01-01

    P450 oxidoreductase (POR) transports electrons from NADPH to all microsomal cytochrome P450 enzymes, including steroidogenic P450c17, P450c21 and P450aro. Severe POR mutations A287P (in Europeans) and R457H (in Japanese) cause the Antley-Bixler skeletal malformation syndrome (ABS) plus impaired steroidogenesis (causing genital anomalies), but the basis of ABS is unclear. We have characterized the activities of ~40 POR variants, showing that assays based on P450c17 activities, but not cytochrome c assays, correlate with the clinical phenotype. The human POR gene is highly polymorphic: the A503V sequence variant, which decreases P450c17 activities to ~60%, is found on ~28% of human alleles. A promoter polymorphism (~8% of Asians and ~13% of Caucasians) at −152 reduces transcriptional activity by half. Screening of 35 POR variants showed that most mutants lacking activity with P450c17 or cytochrome c also lacked activity to support CYP1A2 and CYP2C19 metabolism of EOMCC (a fluorogenic non-drug substrate), although there were some remarkable differences: Q153R causes ABS and has ~30% of wild-type activity with P450c17 but had 144% of WT activity with CYP1A2 and 284% with CYP2C19. The effects of POR variants on CYP3A4, which metabolizes nearly 50% of clinically used drugs, was examined with multiple, clinically-relevant drug substrates, showing that A287P and R457H dramatically reduce drug metabolism, and that A503V variably impairs drug metabolism. The degree of activity can vary with the drug substrate assayed, as the drugs can influence the conformation of the P450. POR is probably an important contributor to genetic variation in both steroidogenesis and drug metabolism. PMID:21070833

  4. Consequences of POR mutations and polymorphisms.

    PubMed

    Miller, Walter L; Agrawal, Vishal; Sandee, Duanpen; Tee, Meng Kian; Huang, Ningwu; Choi, Ji Ha; Morrissey, Kari; Giacomini, Kathleen M

    2011-04-10

    P450 oxidoreductase (POR) transports electrons from NADPH to all microsomal cytochrome P450 enzymes, including steroidogenic P450c17, P450c21 and P450aro. Severe POR mutations A287P (in Europeans) and R457H (in Japanese) cause the Antley-Bixler skeletal malformation syndrome (ABS) plus impaired steroidogenesis (causing genital anomalies), but the basis of ABS is unclear. We have characterized the activities of ∼40 POR variants, showing that assays based on P450c17 activities, but not cytochrome c assays, correlate with the clinical phenotype. The human POR gene is highly polymorphic: the A503V sequence variant, which decreases P450c17 activities to ∼60%, is found on ∼28% of human alleles. A promoter polymorphism (∼8% of Asians and ∼13% of Caucasians) at -152 reduces transcriptional activity by half. Screening of 35 POR variants showed that most mutants lacking activity with P450c17 or cytochrome c also lacked activity to support CYP1A2 and CYP2C19 metabolism of EOMCC (a fluorogenic non-drug substrate), although there were some remarkable differences: Q153R causes ABS and has ∼30% of wild-type activity with P450c17 but had 144% of WT activity with CYP1A2 and 284% with CYP2C19. The effects of POR variants on CYP3A4, which metabolizes nearly 50% of clinically used drugs, was examined with multiple, clinically relevant drug substrates, showing that A287P and R457H dramatically reduce drug metabolism, and that A503V variably impairs drug metabolism. The degree of activity can vary with the drug substrate assayed, as the drugs can influence the conformation of the P450. POR is probably an important contributor to genetic variation in both steroidogenesis and drug metabolism. Copyright © 2010. Published by Elsevier Ireland Ltd.

  5. A comparison of the ability of Bellucia dichotoma Cogn. (Melastomataceae) extract to inhibit the local effects of Bothrops atrox venom when pre-incubated and when used according to traditional methods.

    PubMed

    Mourão de Moura, Valéria; Serra Bezerra, Adrielle N; Veras Mourão, Rosa Helena; Varjão Lameiras, Juliana L; Almeida Raposo, Juliana D; Luckwu de Sousa, Rafael; Boechat, Antônio Luiz; Bezerra de Oliveira, Ricardo; de Menezes Chalkidis, Hipocrátes; Dos-Santos, Maria Cristina

    2014-07-01

    Bellucia dichotoma Cogn. (Melastomataceae) is one of various plant species used in folk medicine in the west of the state of Pará, Brazil, to treat snake bites. Many studies have been carried out to evaluate the effectiveness of anti-snake bite plants, but few of these use the same preparation methods and doses as those traditionally used by the local populations. This study therefore compared inhibition of the main local effects of B. atrox venom (BaV) by aqueous extract of B. dichotoma (AEBd) administered according to traditional methods and pre-incubated with BaV). The concentrations of phenolic compounds (tannins and flavonoids) in AEBd were determined by colorimetric assays. The effectiveness of AEBd in inhibiting the hemorrhagic and edematogenic activities of BaV was evaluated in mice in four different experimental in vivo protocols: (1) pre-incubation (venom:extract, w/w); (2) pre-treatment (p.o.); (3) post-treatment (p.o.); and (4) AEBd (p.o.) in combination with Bothrops antivenom (BA) (i.v.). To assess in vitro inhibition of BaV phospholipase A₂ activity, the pre-incubation method or incorporation of AEBd or BA in agarose gels were used. The effect of AEBd on BaV was determined by SDS-PAGE, zymography and Western blot. Colorimetric assays revealed higher concentrations of (condensed and hydrolyzable) tannins than flavonoids in AEBd. Hemorrhagic activity was completely inhibited using the pre-incubation protocol. However, with pre-treatment there was no significant inhibition for the concentrations tested, and with the post-treatment only the 725 mg/kg dose of AEBd was able to inhibit 40.5% (p = 0.001) of the hemorrhagic activity of BaV. Phospholipase A₂ activity was only inhibited when AEBd was pre-incubated with BaV. BaV-induced edema was completely inhibited with pre-incubation (p < 0.05) and significantly reduced (p < 0.05) with pre- and post-treatment (p.o.) for the concentrations tested. The reduction in local edema was even greater when AEBd

  6. Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System

    PubMed Central

    Gorasia, Dhana G.; Veith, Paul D.; Hanssen, Eric G.; Glew, Michelle D.; Sato, Keiko; Yukitake, Hideharu; Nakayama, Koji; Reynolds, Eric C.

    2016-01-01

    The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32–36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component. PMID:27509186

  7. Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System.

    PubMed

    Gorasia, Dhana G; Veith, Paul D; Hanssen, Eric G; Glew, Michelle D; Sato, Keiko; Yukitake, Hideharu; Nakayama, Koji; Reynolds, Eric C

    2016-08-01

    The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32-36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component.

  8. Hydrogen-atom transfer in reactions of organic radicals with [Co(II)(por)]* (por = porphyrinato) and in subsequent addition of [Co(H)(por)] to olefins.

    PubMed

    de Bruin, Bas; Dzik, Wojciech I; Li, Shan; Wayland, Bradford B

    2009-01-01

    The mechanisms for hydrogen-atom transfer from the cyanoisopropyl radical (*)C(CH(3))(2)CN to [Co(II)(por)](*) (yielding [Co(III)(H)(por)] and CH(2)=C(CH(3))(CN); por = porphyrinato) and the insertion of vinyl acetate (CH(2)=CHOAc) into the Co-H bond of [Co(H)(por)] (giving [Co(III){CH(OAc)CH(3)}(por)]) were investigated by DFT calculations. The results are compared with experimental data. These reactions are relevant to catalytic chain transfer (CCT) in radical polymerization of olefins mediated by [Co(II)(por)](*), the formation and homolysis of organo-cobalt complexes that mediate living radical polymerization of vinyl acetate, and cobalt-mediated hydrogenation of olefins. Hydrogen transfer from (*)C(CH(3))(2)CN to [Co(II)(por)](*) proceeds via a single transition state that has structural features resembling the products [Co(H)(por)] and CH(2)=C(CH(3))CN. The separated radicals approach to form a close-contact radical pair and then pass through the transition state for hydrogen-atom transfer to form [Co(III)(H)(por)] and CH(2)=C(CH(3))CN. This process provides a very low overall barrier for the hydrogen-atom transfer reaction (DeltaG(double dagger) = +3.8 kcal mol(-1)). The reverse reaction corresponding to the addition of [Co(H)(por)] to CH(2)=C(CH(3))CN has a low barrier (DeltaG(double dagger) = +8.9 kcal mol(-1)) as well. Insertion of vinyl acetate into the Co-H bond of [Co(III)(H)(por)] also proceeds over a low barrier (DeltaG(double dagger) = +11.4 kcal mol(-1)) hydrogen-transfer step from [Co(III)(H)(por)] to a carbon atom of the alkene to produce a close-contact radical pair. Dissociation of the radical pair, reorientation, and radical-radical coupling to form an organo-cobalt complex are the culminating steps in the net insertion of an olefin into the Co-H bond. The computed energies obtained for the hydrogen-atom transfer reactions from (*)C(CH(3))(2)CN to [Co(II)(por)](*) and from [Co(H)(por)] to olefins, as well as the organo-cobalt bond homolysis

  9. The PorX Response Regulator of the Porphyromonas gingivalis PorXY Two-Component System Does Not Directly Regulate the Type IX Secretion Genes but Binds the PorL Subunit.

    PubMed

    Vincent, Maxence S; Durand, Eric; Cascales, Eric

    2016-01-01

    The Type IX secretion system (T9SS) is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion or cell surface exposition of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY, and PorX encode typical two-component system (TCS) sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN, and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we demonstrate that PorX does not bind T9SS gene promoters and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS.

  10. The PorX Response Regulator of the Porphyromonas gingivalis PorXY Two-Component System Does Not Directly Regulate the Type IX Secretion Genes but Binds the PorL Subunit

    PubMed Central

    Vincent, Maxence S.; Durand, Eric; Cascales, Eric

    2016-01-01

    The Type IX secretion system (T9SS) is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion or cell surface exposition of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY, and PorX encode typical two-component system (TCS) sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN, and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we demonstrate that PorX does not bind T9SS gene promoters and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS. PMID:27630829

  11. System design description for portable 1,000 CFM exhauster Skids POR-007/Skid E and POR-008/Skid F

    SciTech Connect

    Nelson, O.D.

    1998-07-25

    The primary purpose of the two 1,000 CFM Exhauster Skids, POR-007-SKID E and POR-008-SKID F, is to provide backup to the waste tank primary ventilation systems for tanks 241-C-106 and 241-AY-102, and the AY-102 annulus in the event of a failure during the sluicing of tank 241-C-106 and subsequent transfer of sluiced waste to 241-AY-102. This redundancy is required since both of the tank ventilation systems have been declared as Safety Class systems.

  12. Identification of Porphyromonas gingivalis proteins secreted by the Por secretion system.

    PubMed

    Sato, Keiko; Yukitake, Hideharu; Narita, Yuka; Shoji, Mikio; Naito, Mariko; Nakayama, Koji

    2013-01-01

    The Gram-negative bacterium Porphyromonas gingivalis possesses a number of potential virulence factors for periodontopathogenicity. In particular, cysteine proteinases named gingipains are of interest given their abilities to degrade host proteins and process other virulence factors such as fimbriae. Gingipains are translocated on the cell surface or into the extracellular milieu by the Por secretion system (PorSS), which consists of a number of membrane or periplasmic proteins including PorK, PorL, PorM, PorN, PorO, PorP, PorQ, PorT, PorU, PorV (PG27, LptO), PorW and Sov. To identify proteins other than gingipains secreted by the PorSS, we compared the proteomes of P. gingivalis strains kgp rgpA rgpB (PorSS-proficient strain) and kgp rgpA rgpB porK (PorSS-deficient strain) using two-dimensional gel electrophoresis and peptide-mass fingerprinting. Sixteen spots representing 10 different proteins were present in the particle-free culture supernatant of the PorSS-proficient strain but were absent or faint in that of the PorSS-deficient strain. These identified proteins possessed the C-terminal domains (CTDs), which had been suggested to form the CTD protein family. These results indicate that the PorSS is used for secretion of a number of proteins other than gingipains and that the CTDs of the proteins are associated with the PorSS-dependent secretion. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  13. 'Carbene radicals' in Co(II)(por)-catalyzed olefin cyclopropanation.

    PubMed

    Dzik, Wojciech I; Xu, Xue; Zhang, X Peter; Reek, Joost N H; de Bruin, Bas

    2010-08-11

    The mechanism of cobalt(II)-porphyrin-mediated cyclopropanation of olefins with diazoesters was studied. The first step--reaction of cobalt(II)-porphyrin with ethyl diazoacetate (EDA)--was examined using EPR and ESI-MS techniques. EDA reacts with cobalt(II)-porphyrin to form a 1:1 Co(por)(CHCOOEt) adduct that exists as two isomers: the 'bridging carbene' C' in which the 'carbene' is bound to the metal and the pyrrolic nitrogen of the porphyrin that has a d(7) configuration on the metal, and the 'terminal carbene' C in which the 'carbene' behaves as a redox noninnocent ligand having a d(6) cobalt center and the unpaired electron residing on the 'carbene' carbon atom. The subsequent reactivities of the thus formed 'cobalt carbene radical' with propene, styrene, and methyl acrylate were studied using DFT calculations. The calculations suggest that the formation of the carbene is the rate-limiting step for the unfunctionalized Co(II)(por) and that the cyclopropane ring formation proceeds via a stepwise radical process: Radical addition of the 'carbene radical' C to the C=C double bonds of the olefins results in formation of the gamma-alkyl radical intermediates D. Species D then easily collapse in almost barrierless ring-closure reactions (TS3) to form the cyclopropanes. This radical mechanism readily explains the high activity of Co(II)(por) species in the cyclopropanation of electron-deficient olefins such as methyl acrylate.

  14. Arabidopsis protochlorophyllide oxidoreductase A (PORA) restores bulk chlorophyll synthesis and normal development to a porB porC double mutant.

    PubMed

    Paddock, Troy N; Mason, Mary E; Lima, Daniel F; Armstrong, Gregory A

    2010-03-01

    In angiosperms the strictly light-dependent reduction of protochlorophyllide to chlorophyllide is catalyzed by NADPH:protochlorophyllide oxidoreductase (POR). The Arabidopsis thaliana genome encodes three structurally related but differentially regulated POR genes, PORA, PORB and PORC. PORA is expressed primarily early in development-during etiolation, germination and greening. In contrast, PORB and PORC are not only expressed during seedling development but also throughout the later life of the plant, during which they are responsible for bulk chlorophyll synthesis. The Arabidopsis porB-1 porC-1 mutant displays a severe xantha (highly chlorophyll-deficient) phenotype characterized by smaller prolamellar bodies in etioplasts and decreased thylakoid stacking in chloroplasts. Here we have demonstrated the ability of an ectopic PORA overexpression construct to restore prolamellar body formation in the porB-1 porC-1 double mutant background. In response to illumination, light-dependent chlorophyll production, thylakoid stacking and photomorphogenesis are also restored in PORA-overexpressing porB-1 porC-1 seedlings and adult plants. An Arabidopsis porB-1 porC-1 double mutant can therefore be functionally rescued by the addition of ectopically expressed PORA, which suffices in the absence of either PORB or PORC to direct bulk chlorophyll synthesis and normal plant development.

  15. Click-chemistry approach to study mycoloylated proteins: Evidence for PorB and PorC porins mycoloylation in Corynebacterium glutamicum.

    PubMed

    Issa, Hanane; Huc-Claustre, Emilie; Reddad, Thamila; Bonadé Bottino, Nolwenn; Tropis, Maryelle; Houssin, Christine; Daffé, Mamadou; Bayan, Nicolas; Dautin, Nathalie

    2017-01-01

    Protein mycoloylation is a recently identified, new form of protein acylation. This post-translational modification consists in the covalent attachment of mycolic acids residues to serine. Mycolic acids are long chain, α-branched, β-hydroxylated fatty acids that are exclusively found in the cell envelope of Corynebacteriales, a bacterial order that includes important genera such as Mycobacterium, Nocardia or Corynebacterium. So far, only 3 mycoloylated proteins have been identified: PorA, PorH and ProtX from C. glutamicum. Whereas the identity and function of ProtX is unknown, PorH and PorA associate to form a membrane channel, the activity of which is dependent upon PorA mycoloylation. However, the exact role of mycoloylation and the generality of this phenomenon are still unknown. In particular, the identity of other mycoloylated proteins, if any, needs to be determined together with establishing whether such modification occurs in Corynebacteriales genera other than Corynebacterium. Here, we tested whether a metabolic labeling and click-chemistry approach could be used to detect mycoloylated proteins. Using a fatty acid alkyne analogue, we could indeed label PorA, PorH and ProtX and determine ProtX mycoloylation site. Importantly, we also show that two other porins from C. glutamicum, PorB and PorC are mycoloylated.

  16. Click-chemistry approach to study mycoloylated proteins: Evidence for PorB and PorC porins mycoloylation in Corynebacterium glutamicum

    PubMed Central

    Issa, Hanane; Huc-Claustre, Emilie; Reddad, Thamila; Bonadé Bottino, Nolwenn; Tropis, Maryelle; Houssin, Christine; Daffé, Mamadou; Bayan, Nicolas

    2017-01-01

    Protein mycoloylation is a recently identified, new form of protein acylation. This post-translational modification consists in the covalent attachment of mycolic acids residues to serine. Mycolic acids are long chain, α-branched, β-hydroxylated fatty acids that are exclusively found in the cell envelope of Corynebacteriales, a bacterial order that includes important genera such as Mycobacterium, Nocardia or Corynebacterium. So far, only 3 mycoloylated proteins have been identified: PorA, PorH and ProtX from C. glutamicum. Whereas the identity and function of ProtX is unknown, PorH and PorA associate to form a membrane channel, the activity of which is dependent upon PorA mycoloylation. However, the exact role of mycoloylation and the generality of this phenomenon are still unknown. In particular, the identity of other mycoloylated proteins, if any, needs to be determined together with establishing whether such modification occurs in Corynebacteriales genera other than Corynebacterium. Here, we tested whether a metabolic labeling and click-chemistry approach could be used to detect mycoloylated proteins. Using a fatty acid alkyne analogue, we could indeed label PorA, PorH and ProtX and determine ProtX mycoloylation site. Importantly, we also show that two other porins from C. glutamicum, PorB and PorC are mycoloylated. PMID:28199365

  17. Por La Vida intervention model for cancer prevention in Latinas.

    PubMed

    Navarro, A M; Senn, K L; Kaplan, R M; McNicholas, L; Campo, M C; Roppe, B

    1995-01-01

    Our goal was to describe the development and implementation of an intervention on cancer prevention for Latinas in San Diego, Calif. Thirty-six lay community workers ("consejeras") were recruited and trained to conduct educational group sessions. Each consejera recruited approximately 14 peers from the community to participate in the program (total number = 512). Half of the consejeras were randomly assigned to a control group, in which they participated in an equally engaging program entitled "Community Living Skills." Implementation of the intervention was assessed by qualitative and quantitative methods. Preintervention and postintervention self-report information was obtained from project participants on access to health care services, cancer knowledge, preventive measures, and previous cancer-screening examinations. Base-line data suggest that lack of knowledge, costs of cancer-screening tests, and the lack of a regular health care provider are the major obstacles against obtaining cancer-screening tests. Predisposing factors, such as fear and embarrassment, also constitute barriers to getting regular cervical cancer screening. Preliminary analysis indicates that the Por La Vida intervention increases use of cancer-screening tests in comparison to a community living skills control group. Universal access to health care would remove some of the major financial barriers to cancer screening. The Por La Vida program attempts to overcome the substantial barriers by reaching out to low-income Latinas and by providing information regarding the availability, acceptability, and preventive nature of cancer-screening tests.

  18. 21 CFR 864.8100 - Bothrops atrox reagent.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... levels to aid in the evaluation of disseminated intravascular coagulation (nonlocalized clotting in the blood vessels) in patients receiving heparin therapy (the administration of the anticoagulant heparin in...

  19. Acceptance test report for portable exhauster POR-007/Skid E

    SciTech Connect

    Kriskovich, J.R.

    1998-07-24

    This document describes Acceptance Testing performed on Portable Exhauster POR-007/Skid E. It includes measurements of bearing vibration levels, pressure decay testing, programmable logic controller interlocks, high vacuum, flow and pressure control functional testing. The purpose of Acceptance testing documented by this report was to demonstrate compliance of the exhausters with the performance criteria established within HNF-0490, Rev. 1 following a repair and upgrade effort at Hanford. In addition, data obtained during this testing is required for the resolution of outstanding Non-conformance Reports (NCR), and finally, to demonstrate the functionality of the associated software for the pressure control and high vacuum exhauster operating modes provided for by W-320. Additional testing not required by the ATP was also performed to assist in the disposition and close out of receiving inspection report and for application design information (system curve). Results of this testing are also captured within this document.

  20. Compton imaging with the PorGamRays spectrometer

    NASA Astrophysics Data System (ADS)

    Judson, D. S.; Boston, A. J.; Coleman-Smith, P. J.; Cullen, D. M.; Hardie, A.; Harkness, L. J.; Jones, L. L.; Jones, M.; Lazarus, I.; Nolan, P. J.; Pucknell, V.; Rigby, S. V.; Seller, P.; Scraggs, D. P.; Simpson, J.; Slee, M.; Sweeney, A.; PorGamRays Collaboration

    2011-10-01

    The PorGamRays project aims to develop a portable gamma-ray detection system with both spectroscopic and imaging capabilities. The system is designed around a stack of thin Cadmium Zinc Telluride (CZT) detectors. The imaging capability utilises the Compton camera principle. Each detector is segmented into 100 pixels which are read out through custom designed Application Specific Integrated Circuits (ASICs). This device has potential applications in the security, decommissioning and medical fields. This work focuses on the near-field imaging performance of a lab-based demonstrator consisting of two pixelated CZT detectors, each of which is bonded to a NUCAM II ASIC. Measurements have been made with point 133Ba and 57Co sources located ˜35 mm from the surface of the scattering detector. Position resolution of ˜20 mm FWHM in the x and y planes is demonstrated.

  1. Suppression of cytochrome P450 reductase (POR) expression in hepatoma cells replicates the hepatic lipidosis observed in hepatic POR-null mice.

    PubMed

    Porter, Todd D; Banerjee, Subhashis; Stolarczyk, Elzbieta I; Zou, Ling

    2011-06-01

    Cytochrome P450 reductase (POR) is a microsomal electron transport protein essential to cytochrome P450-mediated drug metabolism and sterol and bile acid synthesis. The conditional deletion of hepatic POR gene expression in mice results in a marked decrease in plasma cholesterol levels counterbalanced by the accumulation of triglycerides in lipid droplets in hepatocytes. To evaluate the role of cholesterol and bile acid synthesis in this hepatic lipidosis, as well as the possible role of lipid transport from peripheral tissues, we developed a stable, small interfering RNA (siRNA)-mediated cell culture model for the suppression of POR. POR mRNA and protein expression were decreased by greater than 50% in McArdle-RH7777 rat hepatoma cells 10 days after transfection with a POR-siRNA expression plasmid, and POR expression was nearly completely extinguished by day 20. Immunofluorescent analysis revealed a marked accumulation of lipid droplets in cells by day 15, accompanied by a nearly 2-fold increase in cellular triglyceride content, replicating the lipidosis seen in hepatic POR-null mouse liver. In contrast, suppression of CYP51A1 (lanosterol demethylase) did not result in lipid accumulation, indicating that loss of cholesterol synthesis is not the basis for this lipidosis. Indeed, addition of cholesterol to the medium appeared to augment the lipidosis in POR-suppressed cells, whereas removal of lipids from the medium reversed the lipidosis. Oxysterols did not accumulate in POR-suppressed cells, discounting a role for liver X receptor in stimulating triglyceride synthesis, but addition of chenodeoxycholate significantly repressed lipid accumulation, suggesting that the absence of bile acids and loss of farnesoid X receptor stimulation lead to excessive triglyceride synthesis.

  2. Suppression of Cytochrome P450 Reductase (POR) Expression in Hepatoma Cells Replicates the Hepatic Lipidosis Observed in Hepatic POR-Null Mice

    PubMed Central

    Banerjee, Subhashis; Stolarczyk, Elzbieta I.; Zou, Ling

    2011-01-01

    Cytochrome P450 reductase (POR) is a microsomal electron transport protein essential to cytochrome P450-mediated drug metabolism and sterol and bile acid synthesis. The conditional deletion of hepatic POR gene expression in mice results in a marked decrease in plasma cholesterol levels counterbalanced by the accumulation of triglycerides in lipid droplets in hepatocytes. To evaluate the role of cholesterol and bile acid synthesis in this hepatic lipidosis, as well as the possible role of lipid transport from peripheral tissues, we developed a stable, small interfering RNA (siRNA)-mediated cell culture model for the suppression of POR. POR mRNA and protein expression were decreased by greater than 50% in McArdle-RH7777 rat hepatoma cells 10 days after transfection with a POR-siRNA expression plasmid, and POR expression was nearly completely extinguished by day 20. Immunofluorescent analysis revealed a marked accumulation of lipid droplets in cells by day 15, accompanied by a nearly 2-fold increase in cellular triglyceride content, replicating the lipidosis seen in hepatic POR-null mouse liver. In contrast, suppression of CYP51A1 (lanosterol demethylase) did not result in lipid accumulation, indicating that loss of cholesterol synthesis is not the basis for this lipidosis. Indeed, addition of cholesterol to the medium appeared to augment the lipidosis in POR-suppressed cells, whereas removal of lipids from the medium reversed the lipidosis. Oxysterols did not accumulate in POR-suppressed cells, discounting a role for liver X receptor in stimulating triglyceride synthesis, but addition of chenodeoxycholate significantly repressed lipid accumulation, suggesting that the absence of bile acids and loss of farnesoid X receptor stimulation lead to excessive triglyceride synthesis. PMID:21368239

  3. Acceptance test report for portable exhauster POR-008/Skid F

    SciTech Connect

    Kriskovich, J.R.

    1998-07-24

    Portable Exhauster POR-008 was procured via HNF-0490, Specification for a Portable Exhausted System for Waste Tank Ventilation. Prior to taking ownership, acceptance testing was performed at the vendors. However at the conclusion of testing a number of issues remained that required resolution before the exhausters could be used by Project W-320. The purpose of acceptance testing documented by this report was to demonstrate compliance of the exhausters with the performance criteria established within HNF-O49O, Rev. 1 following a repair and upgrade effort at Hanford. In addition, data obtained during this testing is required for the resolution of outstanding Non-conformance Reports (NCR), and finally, to demonstrate the functionality of the associated software for the pressure control and high vacuum exhauster operating modes provided for by W-320. Additional testing not required by the ATP was also performed to assist in the disposition and close out of receiving inspection report and for application design information (system curve). Results of this testing are also captured within this document.

  4. Prenatal Diagnosis of Antley-Bixler Syndrome and POR Deficiency.

    PubMed

    Oldani, Elena; Garel, Catherine; Bucourt, Martine; Carbillon, Lionel

    2015-12-16

    Prenatal diagnosis of severe bone diseases is challenging and requires complete and precise analysis of fetal anomalies to guide genetic investigation and parental counselling. We report a rare case of Antley-Bixler syndrome prenatally diagnosed at 26 weeks' gestation by ultrasound and computed tomography in a 28-year-old woman with a history of early termination of pregnancy for "malposition of the inferior limbs". The prenatal ultrasound scan showed severe femoral bowing and frontal bossing. Taking into account the high probability of a recurrent severe skeletal disorder, a computed tomography (CT) scan was proposed. CT findings revealed bilateral femora deformation, craniosynostosis, severe midface hypoplasia, and radiohumeral synostosis. These anomalies strongly suggested Antley-Bixler syndrome. Sequencing of the POR gene in the fetus and the parents revealed compound heterozygous mutations in exon 9 and intron 7, both inherited from each parent, and this finding allowed genetic counseling. The first step in the proper prenatal diagnosis of fetal bone disorders is the precise analysis of ultrasonographic images. However, when a severe fetal inherited disorder is strongly suspected in late mid-trimester, CT may be discussed and usefully contribute to diagnosis and prognosis assessment.

  5. [Cloning and prokaryotic expression of the outer membrane protein gene PorB of Neisseria gonorrhoeae].

    PubMed

    Wang, Yan; Zhang, Lei; Zhang, Li; Wang, Han

    2011-07-01

    To construct a fused expression vector of the outer membrane protein gene PorB of Neisseria gonorrhoeae, express the fusion protein in the prokaryotic system, and obtain a gene recombination protein, for the purpose of preparing the ground for further research on the pathopoiesis and immune protective response of PorB. A pair of primers were designed according to the known sequence of the PorB gene, and the PorB gene was amplified by PCR from the genome of Neisseria gonorrhoeae 29403 and cloned into the prokaryotic expression plasmid pGEX-4T-1 to generate pGEX-4T-PorB recombinants. The recombinant plasmid pGEX4T-PorB was transferred into competent cells E. coli BL21. After confirmed by restriction endonuclease digestion, PCR and DNA sequencing analysis, the recombinant protein was induced to express by isopropyl-beta-D-thiogalactoside (IPTG), and examined by SDS-PAGE and Western blotting. Restriction endonuclease digestion, PCR amplification and DNA sequencing analysis showed that the PorB gene of 1 047 bp was amplified from Neisseria gonorrhoeae DNA, and the recombinant plasmid pGEX-4T-PorB was successfully constructed and highly expressed in E. coli. The prokaryotic expression vector of pGEX-4T-PorB was successfully constructed and efficiently expressed in the prokaryotic system, which has provided a basis for further study on the biological activity of the PorB protein, as well as animal immune experiment and detection of Neisseria gonorrhoeae, and its application as a mucosal immune vaccine.

  6. P450 (Cytochrome) Oxidoreductase Gene (POR) Common Variant (POR*28) Significantly Alters CYP2C9 Activity in Swedish, But Not in Korean Healthy Subjects.

    PubMed

    Hatta, Fazleen H M; Aklillu, Eleni

    2015-12-01

    CYP2C9 enzyme contributes to the metabolism of several pharmaceuticals and xenobiotics and yet displays large person-to-person and interethnic variation. Understanding the mechanisms of CYP2C9 variation is thus of immense importance for personalized medicine and rational therapeutics. A genetic variant of P450 (cytochrome) oxidoreductase (POR), a CYP450 redox partner, is reported to influence CYP2C9 metabolic activity in vitro. We investigated the impact of a common variant, POR*28, on CYP2C9 metabolic activity in humans. 148 healthy Swedish and 146 healthy Korean volunteers were genotyped for known CYP2C9 defective variant alleles (CYP2C9*2, *3). The CYP2C9 phenotype was determined using a single oral dose of 50 mg losartan. Excluding oral contraceptive (OC) users and carriers of 2C9*2 and *3 alleles, 117 Korean and 65 Swedish were genotyped for POR*5, *13 and *28 using Taqman assays. The urinary losartan to its metabolite E-3174 metabolic ratio (MR) was used as an index of CYP2C9 metabolic activity. The allele frequency of the POR*28 variant allele in Swedes and Koreans was 29% and 44%, respectively. POR*5 and *13 were absent in both study populations. Considering the CYP2C9*1/*1 genotypes only, the CYP2C9 metabolic activity was 1.40-fold higher in carriers of POR*28 allele than non-carriers among Swedes (p = 0.02). By contrast, no influence of the POR*28 on CYP2C9 activity was found in Koreans (p = 0.68). The multivariate analysis showed that ethnicity, POR genotype, and smoking were strong predictors of CYP2C9 MR (p < 0.05). This is the first report to implicate the importance of POR*28 genetic variation for CYP2C9 metabolic activity in humans. These findings contribute to current efforts for global personalized medicine and using medicines by taking into account pharmacogenetic and phenotypic variations.

  7. Gliding motility and Por secretion system genes are widespread among members of the phylum bacteroidetes.

    PubMed

    McBride, Mark J; Zhu, Yongtao

    2013-01-01

    The phylum Bacteroidetes is large and diverse, with rapid gliding motility and the ability to digest macromolecules associated with many genera and species. Recently, a novel protein secretion system, the Por secretion system (PorSS), was identified in two members of the phylum, the gliding bacterium Flavobacterium johnsoniae and the nonmotile oral pathogen Porphyromonas gingivalis. The components of the PorSS are not similar in sequence to those of other well-studied bacterial secretion systems. The F. johnsoniae PorSS genes are a subset of the gliding motility genes, suggesting a role for the secretion system in motility. The F. johnsoniae PorSS is needed for assembly of the gliding motility apparatus and for secretion of a chitinase, and the P. gingivalis PorSS is involved in secretion of gingipain protease virulence factors. Comparative analysis of 37 genomes of members of the phylum Bacteroidetes revealed the widespread occurrence of gliding motility genes and PorSS genes. Genes associated with other bacterial protein secretion systems were less common. The results suggest that gliding motility is more common than previously reported. Microscopic observations confirmed that organisms previously described as nonmotile, including Croceibacter atlanticus, "Gramella forsetii," Paludibacter propionicigenes, Riemerella anatipestifer, and Robiginitalea biformata, exhibit gliding motility. Three genes (gldA, gldF, and gldG) that encode an apparent ATP-binding cassette transporter required for F. johnsoniae gliding were absent from two related gliding bacteria, suggesting that the transporter may not be central to gliding motility.

  8. Impact of POR*28 on the pharmacokinetics of tacrolimus and cyclosporine A in renal transplant patients.

    PubMed

    Elens, Laure; Hesselink, Dennis A; Bouamar, Rachida; Budde, Klemens; de Fijter, Johannes W; De Meyer, Martine; Mourad, Michel; Kuypers, Dirk R J; Haufroid, Vincent; van Gelder, Teun; van Schaik, Ron H N

    2014-02-01

    The P450 oxidoreductase (POR)*28 variant allele has been associated with altered cytochrome P450 3A enzyme activities. Both CYP3A5 and CYP3A4 are involved in the metabolism of calcineurin inhibitors and recent data show that POR*28 may explain part of the variability observed in tacrolimus (Tac) pharmacokinetics. The aim of this study was to investigate the impact of the POR*28 allele on Tac and cyclosporine A (CsA) immunosuppressive therapies. Kidney transplant recipients receiving either Tac (n = 184) or CsA (n = 174), participating in a prospective multicenter trial, were genotyped for POR*28, CYP3A4*22, and CYP3A5*3. CYP3A5 expressers that were carriers of at least 1 POR*28 allele had a 16.9% decrease in dose-adjusted predose concentrations when compared CYP3A5 expressers that carried the POR*1/*1 genotype (P = 0.03), indicating an increased CYP3A5 activity for POR*28 carriers. In CYP3A5, nonexpressers carrying 2 POR*28 alleles, a 24.1% (confidence interval95% = -39.4% to -4.9%; P = 0.02) decrease in dose-adjusted predose concentrations was observed for Tac, suggesting higher CYP3A4 activity. For CsA, POR*28/*28 patients not expressing CYP3A5 and not carrying the CYP3A4*22 decrease-of-function allele showed 15% lower CsA dose-adjusted predose concentrations (P = 0.01), indicating also increased CYP3A4 activity. In both cohorts (ie, Tac and CsA), the POR*28 allele was neither associated with the incidence of delayed graft function nor with biopsy-proven acute rejection. These results were further confirmed in 2 independent cohorts. Our results show that the POR*28 allele is associated with increased in vivo CYP3A5 activity for Tac in CYP3A5 expressers, whereas POR*28 homozygosity was associated with a significant higher CYP3A4 activity in CYP3A5 nonexpressers for both Tac and CsA.

  9. Successful Mnemonics for "por"/"para" and Affirmative Commands with Pronouns.

    ERIC Educational Resources Information Center

    Mason, Keith

    1992-01-01

    Two mnemonic devices, "4A Rule" and "PERFECT," are described to simplify the learning of two grammar points: the placement of object pronouns with respect to commands and the distinction between "por" and "para." (five references) (LB)

  10. Crystallographic analysis of Neisseria meningitidis PorB extracellular loops potentially implicated in TLR2 recognition

    PubMed Central

    Kattner, Christof; Toussi, Deana; Zaucha, Jan; Wetzler, Lee M.; Rüppel, Nadine; Zachariae, Ulrich; Massari, Paola; Tanabe, Mikio

    2014-01-01

    Among all Neisseriae species, N. meningitidis and N. gonorrhoeae are the only human pathogens, causative agents of bacterial meningitis and gonorrhoea, respectively. PorB, a pan-Neisseriae trimeric porin that mediates diffusive transport of essential molecules across the bacterial outer membrane, is also known to activate host innate immunity via Toll-like receptor 2 (TLR2)-mediated signaling. The molecular mechanism of PorB binding to TLR2 is not known, but it has been hypothesized that electrostatic interactions contribute to ligand/receptor binding. Strain-specific sequence variability in the surface-exposed loops of PorB which are potentially implicated in TLR2 binding, may explain the difference in TLR2-mediated cell activation in vitro by PorB homologs from the commensal N. lactamica and the pathogen N. meningitidis. Here, we report a comparative structural analysis of PorB from N. meningitidis serogroup B strain 8765 (63% sequence homology with PorB from N. meningitidis serogroup W135) and a mutant in which amino acid substitutions in the extracellular loop 7 lead to significantly reduced TLR2-dependent activity in vitro. We observe that this mutation both alters the loop conformation and causes dramatic changes of electrostatic surface charge, both of which may affect TLR2 recognition and signalling. PMID:24361688

  11. An investigation of exploitation versus exploration in GBEA optimization of PORS 15 and 16 Problems

    SciTech Connect

    Koch, Kaelynn

    2012-01-01

    It was hypothesized that the variations in time to solution are driven by the competing mechanisms of exploration and exploitation.This thesis explores this hypothesis by examining two contrasting problems that embody the hypothesized tradeoff between exploration and exploitation. Plus one recall store (PORS) is an optimization problem based on the idea of a simple calculator with four buttons: plus, one, store, and recall. Integer addition and store are classified as operations, and one and memory recall are classified as terminals. The goal is to arrange a fixed number of keystrokes in a way that maximizes the numerical result. PORS 15 (15 keystrokes) represents the subset of difficult PORS problems and PORS 16 (16 keystrokes) represents the subset of PORS problems that are easiest to optimize. The goal of this work is to examine the tradeoff between exploitation and exploration in graph based evolutionary algorithm (GBEA) optimization. To do this, computational experiments are used to examine how solutions evolve in PORS 15 and 16 problems when solved using GBEAs. The experiment is comprised of three components; the graphs and the population, the evolutionary algorithm rule set, and the example problems. The complete, hypercube, and cycle graphs were used for this experiment. A fixed population size was used.

  12. Molecular characterisation of Porcine rubulavirus (PorPV) isolates from different outbreaks in Mexico.

    PubMed

    Cuevas-Romero, S; Rivera-Benítez, J F; Blomström, A-L; Ramliden, M; Hernández-Baumgarten, E; Hernández-Jáuregui, P; Ramírez-Mendoza, H; Berg, M

    2016-02-01

    Since the report of the initial outbreak of Porcine rubulavirus (PorPV) infection in pigs, only one full-length genome from 1984 (PorPV-LPMV/1984) has been characterised. To investigate the overall genetic variation, full-length gene nucleotide sequences of current PorPV isolates were obtained from different clinical cases of infected swine. Genome organisation and sequence analysis of the encoded proteins (NP, P, F, M, HN and L) revealed high sequence conservation of the NP protein and the expression of the P and V proteins in all PorPV isolates. The V protein of one isolate displayed a mutation that has been implicated to antagonise the antiviral immune responses of the host. The M protein indicated a variation in a short region that could affect the electrostatic charge and the interaction with the membrane. One PorPV isolate recovered from the lungs showed a mutation at the cleavage site (HRKKR) of the F protein that could represent an important factor to determine the tissue tropism and pathogenicity of this virus. The HN protein showed high sequence identity through the years (up to 2013). Additionally, a number of sequence motifs of very high amino acid conservation among the PorPV isolates important for polymerase activity of the L protein have been identified. In summary, genetic comparisons and phylogenetic analyses indicated that three different genetic variants of PorPV are currently spreading within the swine population, and a new generation of circulating virus with different characteristics has begun to emerge.

  13. Por Secretion System-Dependent Secretion and Glycosylation of Porphyromonas gingivalis Hemin-Binding Protein 35

    PubMed Central

    Shoji, Mikio; Sato, Keiko; Yukitake, Hideharu; Kondo, Yoshio; Narita, Yuka; Kadowaki, Tomoko; Naito, Mariko; Nakayama, Koji

    2011-01-01

    The anaerobic Gram-negative bacterium Porphyromonas gingivalis is a major pathogen in severe forms of periodontal disease and refractory periapical perodontitis. We have recently found that P. gingivalis has a novel secretion system named the Por secretion system (PorSS), which is responsible for secretion of major extracellular proteinases, Arg-gingipains (Rgps) and Lys-gingipain. These proteinases contain conserved C-terminal domains (CTDs) in their C-termini. Hemin-binding protein 35 (HBP35), which is one of the outer membrane proteins of P. gingivalis and contributes to its haem utilization, also contains a CTD, suggesting that HBP35 is translocated to the cell surface via the PorSS. In this study, immunoblot analysis of P. gingivalis mutants deficient in the PorSS or in the biosynthesis of anionic polysaccharide-lipopolysaccharide (A-LPS) revealed that HBP35 is translocated to the cell surface via the PorSS and is glycosylated with A-LPS. From deletion analysis with a GFP-CTD[HBP35] green fluorescent protein fusion, the C-terminal 22 amino acid residues of CTD[HBP35] were found to be required for cell surface translocation and glycosylation. The GFP-CTD fusion study also revealed that the CTDs of CPG70, peptidylarginine deiminase, P27 and RgpB play roles in PorSS-dependent translocation and glycosylation. However, CTD-region peptides were not found in samples of glycosylated HBP35 protein by peptide map fingerprinting analysis, and antibodies against CTD-regions peptides did not react with glycosylated HBP35 protein. These results suggest both that the CTD region functions as a recognition signal for the PorSS and that glycosylation of CTD proteins occurs after removal of the CTD region. Rabbits were used for making antisera against bacterial proteins in this study. PMID:21731719

  14. In silico studies of outer membrane of Neisseria meningitidis por a: its expression and immunogenic properties.

    PubMed

    Behrouzi, Ava; Bouzari, Saeid; Siadat, Seyed Davar; Irani, Shiva

    2014-01-01

    Neisseria meningitidis is a major causative agent of bacterial septicemia and meningitis in humans. Currently, there are no vaccines to prevent disease caused by strains of N.meningitidis serogroup B. The Class 1 Outer Membrane Protein (OMP) has been named porA which is a cation selective transmembrane protein of 45 KDa that forms trimeric pore in the meningococcal outer membrane. PorA from serogroup B N. meningitidis was cloned into prokaryotic expression vector pBAD-gIIIA. Recombinant protein was expressed with arabinose and affinity purified by Ni-NTA agarose, SDS-PAGE and western blotting were performed for protein determination and verification. BALB/c mice were immunized subcutaneously with purified rPorA together with alum adjuvant. Serum antibody responses to serogroups B N.meningitidis were determined by ELISA. Serum IgG response significantly increased in the group immunized with rPorA together with alum adjuvant in comparison with control groups. These results suggest that rPorA can be a potential vaccine candidate for serogroup B N.meningitidis.

  15. In Silico Studies of Outer Membrane of Neisseria Meningitidis Por A: Its Expression and Immunogenic Properties

    PubMed Central

    Behrouzi, Ava; Bouzari, Saeid; Siadat, Seyed Davar; Irani, Shiva

    2014-01-01

    Neisseria meningitidis is a major causative agent of bacterial septicemia and meningitis in humans. Currently, there are no vaccines to prevent disease caused by strains of N.meningitidis serogroup B. The Class 1 Outer Membrane Protein (OMP) has been named porA which is a cation selective transmembrane protein of 45 KDa that forms trimeric pore in the meningococcal outer membrane. PorA from serogroup B N. meningitidis was cloned into prokaryotic expression vector pBAD-gIIIA. Recombinant protein was expressed with arabinose and affinity purified by Ni-NTA agarose, SDS-PAGE and western blotting were performed for protein determination and verification. BALB/c mice were immunized subcutaneously with purified rPorA together with alum adjuvant. Serum antibody responses to serogroups B N.meningitidis were determined by ELISA. Serum IgG response significantly increased in the group immunized with rPorA together with alum adjuvant in comparison with control groups. These results suggest that rPorA can be a potential vaccine candidate for serogroup B N.meningitidis. PMID:25317403

  16. Impact of POR*28 on the clinical pharmacokinetics of CYP3A phenotyping probes midazolam and erythromycin.

    PubMed

    Elens, Laure; Nieuweboer, Annemieke J M; Clarke, Stephen J; Charles, Kellie A; de Graan, Anne-Joy M; Haufroid, Vincent; van Gelder, Teun; Mathijssen, Ron H J; van Schaik, Ron H N

    2013-03-01

    P450 oxidoreductase (POR) is essential for cytochrome P450 (CYP) activity in humans. The POR*28 allele (A503V) has been shown to impact on in-vitro CYP-mediated metabolism, including CYP3A isoenzymes. The aim of the present study was to determine the in vivo impact of the POR*28 allele on the pharmacokinetics of the classic CYP3A phenotyping probes midazolam and erythromycin. Whereas midazolam is metabolized by both CYP3A4 and CYP3A5, erythromycin is exclusively oxidized by CYP3A4. To assess CYP3A activity, 108 cancer patients received midazolam and 45 others underwent the erythromycin breath test. Patients were genotyped for POR*28, CYP3A4*22 and CYP3A5*3. In patients expressing CYP3A5, POR*28 carriers showed 45% lower midazolam metabolic ratios compared with POR*1/*1 patients (P<0.001). This is in line with a lower CYP3A5 activity toward midazolam for POR*28 carriers. In CYP3A5 nonexpressers, POR*28 had no influence on midazolam pharmacokinetics. For erythromycin, POR*28 carriership did not influence its metabolism. Our data show that the POR*28 allele is associated with a lower in vivo CYP3A5 activity, but has no effects on CYP3A4-mediated erythromycin and midazolam metabolism.

  17. PorA Represents the Major Cell Wall Channel of the Gram-Positive Bacterium Corynebacterium glutamicum

    PubMed Central

    Costa-Riu, Noelia; Burkovski, Andreas; Krämer, Reinhard; Benz, Roland

    2003-01-01

    The cell wall of the gram-positive bacterium Corynebacterium glutamicum contains a channel (porin) for the passage of hydrophilic solutes. The channel-forming polypeptide PorA is a 45-amino-acid acidic polypeptide with an excess of four negatively charged amino acids, which is encoded by the 138-bp gene porA. porA was deleted from the chromosome of C.glutamicum wild-type strain ATCC 13032 to obtain mutant ATCC 13032ΔporA. Southern blot analysis demonstrated that porA was deleted. Lipid bilayer experiments revealed that PorA was not present in the cell wall of the mutant strain. Searches within the known chromosome of C. glutamicum by using National Center for Biotechnology Information BLAST and reverse transcription-PCR showed that no other PorA-like protein is encoded on the chromosome or is expressed in the deletion strain. The porA deletion strain exhibited slower growth and longer growth times than the C. glutamicum wild-type strain. Experiments with different antibiotics revealed that the susceptibility of the mutant strain was much lower than that of the wild-type C. glutamicum strain. The results presented here suggest that PorA represents a major hydrophilic pathway through the cell wall and that C. glutamicum contains cell wall channels which are not related to PorA. PMID:12896997

  18. Hexacoordinate oxy-globin models Fe(Por)(NH3)(O2) react with NO to form only the nitrato analogs Fe(Por)(NH3)(η1-ONO2), even at ~100 K.

    PubMed

    Kurtikyan, Tigran S; Ford, Peter C

    2010-12-07

    The oxy-globin models Fe(Por)(NH(3))(O(2)), prepared by sequential reactions of O(2) ((18)O(2)) and NH(3) with thin porous layers of Fe(II)(Por), react with NO ((15)NO) at 80-100 K to form only the low-spin nitrato complexes Fe(Por)(NH(3))(η(1)-ONO(2)), thus implying that peroxynitrite intermediates, if formed, must undergo very facile isomerization to the nitrato analog.

  19. Oral administration of recombinant Neisseria meningitidis PorA genetically fused to H. pylori HpaA antigen increases antibody levels in mouse serum, suggesting that PorA behaves as a putative adjuvant

    PubMed Central

    Vasquez, Abel E; Manzo, Ricardo A; Soto, Daniel A; Barrientos, Magaly J; Maldonado, Aurora E; Mosqueira, Macarena; Avila, Anastasia; Touma, Jorge; Bruce, Elsa; Harris, Paul R; Venegas, Alejandro

    2015-01-01

    The Neisseria meningitidis outer membrane protein PorA from a Chilean strain was purified as a recombinant protein. PorA mixed with AbISCO induced bactericidal antibodies against N. meningitidis in mice. When PorA was fused to the Helicobacter pylori HpaA antigen gene, the specific response against H. pylori protein increased. Splenocytes from PorA-immunized mice were stimulated with PorA, and an increase in the secretion of IL-4 was observed compared with that of IFN-γ. Moreover, in an immunoglobulin sub-typing analysis, a substantially higher IgG1 level was found compared with IgG2a levels, suggesting a Th2-type immune response. This study revealed a peculiar behavior of the purified recombinant PorA protein per se in the absence of AbISCO as an adjuvant. Therefore, the resistance of PorA to proteolytic enzymes, such as those in the gastrointestinal tract, was analyzed, because this is an important feature for an oral protein adjuvant. Finally, we found that PorA fused to the H. pylori HpaA antigen, when expressed in Lactococcus lactis and administered orally, could enhance the antibody response against the HpaA antigen approximately 3 fold. These observations strongly suggest that PorA behaves as an effective oral adjuvant. PMID:25750999

  20. Oral administration of recombinant Neisseria meningitidis PorA genetically fused to H. pylori HpaA antigen increases antibody levels in mouse serum, suggesting that PorA behaves as a putative adjuvant.

    PubMed

    Vasquez, Abel E; Manzo, Ricardo A; Soto, Daniel A; Barrientos, Magaly J; Maldonado, Aurora E; Mosqueira, Macarena; Avila, Anastasia; Touma, Jorge; Bruce, Elsa; Harris, Paul R; Venegas, Alejandro

    2015-01-01

    The Neisseria meningitidis outer membrane protein PorA from a Chilean strain was purified as a recombinant protein. PorA mixed with AbISCO induced bactericidal antibodies against N. meningitidis in mice. When PorA was fused to the Helicobacter pylori HpaA antigen gene, the specific response against H. pylori protein increased. Splenocytes from PorA-immunized mice were stimulated with PorA, and an increase in the secretion of IL-4 was observed compared with that of IFN-γ. Moreover, in an immunoglobulin sub-typing analysis, a substantially higher IgG1 level was found compared with IgG2a levels, suggesting a Th2-type immune response. This study revealed a peculiar behavior of the purified recombinant PorA protein per se in the absence of AbISCO as an adjuvant. Therefore, the resistance of PorA to proteolytic enzymes, such as those in the gastrointestinal tract, was analyzed, because this is an important feature for an oral protein adjuvant. Finally, we found that PorA fused to the H. pylori HpaA antigen, when expressed in Lactococcus lactis and administered orally, could enhance the antibody response against the HpaA antigen approximately 3 fold. These observations strongly suggest that PorA behaves as an effective oral adjuvant.

  1. Variation in the Neisseria lactamica porin, and its relationship to meningococcal PorB.

    PubMed

    Bennett, Julia S; Callaghan, Martin J; Derrick, Jeremy P; Maiden, Martin C J

    2008-05-01

    One potential vaccine strategy in the fight against meningococcal disease involves the exploitation of outer-membrane components of Neisseria lactamica, a commensal bacterium closely related to the meningococcus, Neisseria meningitidis. Although N. lactamica shares many surface structures with the meningococcus, little is known about the antigenic diversity of this commensal bacterium or the antigenic relationships between N. lactamica and N. meningitidis. Here, the N. lactamica porin protein (Por) was examined and compared to the related PorB antigens of N. meningitidis, to investigate potential involvement in anti-meningococcal immunity. Relationships among porin sequences were determined using distance-based methods and F(ST), and maximum-likelihood analyses were used to compare the selection pressures acting on the encoded proteins. These analyses demonstrated that the N. lactamica porin was less diverse than meningococcal PorB and although it was subject to positive selection, this was not as strong as the positive selection pressures acting on the meningococcal porin. In addition, the N. lactamica porin gene sequences and the protein sequences of the loop regions predicted to be exposed to the human immune system were dissimilar to the corresponding sequences in the meningococcus. This suggests that N. lactamica Por, contrary to previous suggestions, may have limited involvement in the development of natural immunity to meningococcal disease and might not be effective as a meningococcal vaccine component.

  2. An Analysis of Interlanguage Development Over Time: Part 1, "por" and "para".

    ERIC Educational Resources Information Center

    Guntermann, Gail

    1992-01-01

    The first part of a larger planned investigation, this study examines the use of "por" and "para" by nine Peace Corps volunteers in oral interviews at the end of training and roughly one year later, to trace their acquisition over time, in two learning contexts. (24 references) (LB)

  3. The Acquisition of Lexical Meaning in a Study Abroad Context: The Spanish Prepositions "por" and "para."

    ERIC Educational Resources Information Center

    Lafford, Barbara A.; Ryan, John M.

    1995-01-01

    Examination of the development of form/function relations of the prepositions "por" and "para" at different levels of proficiency in the interlanguage of study-abroad students in Granada, Spain, revealed "noncanonical" as well as "canonical" uses of these prepositions. The most common noncanonical uses were…

  4. Methodology for obtaining stakeholder assessments of obesity policy options in the PorGrow project.

    PubMed

    Stirling, A; Lobstein, T; Millstone, E

    2007-05-01

    The Policy Options for Responding to the Growing Challenge of Obesity Research Project (PorGrow) study provided a unique opportunity to develop a large-scale application of a semi-quantitative technique for exploring interviewees' views on options to tackle obesity, using multi-criteria mapping. This 'heuristic' approach utilizes the advantages of a structured interviews framework by predefining a set of options for appraisal, while leaving interviewees free to select their own criteria for making their judgements. Additional information can be gleaned from the interview transcripts and related materials to set the appraisals in their policy context, and allowing interviewees to express their views on the options presented and their own appraisals. The PorGrow study team agreed a predefined set of 20 options for appraisal, and interviewed sets of stakeholders representing more than 20 aspects of policy development in each of the nine participating countries. The details of the methodology adopted are set out in this paper.

  5. ATP for the portable 500 CFM exhauster POR-004 skid B

    SciTech Connect

    Keller, C.M.

    1997-05-06

    This Acceptance Test Plan is for a 500 CFM Portable Exhauster POR-004 to be used for saltwell pumping. The Portable Exhauster System will be utilized to eliminate potential flammable gases that may exist within the dome space of the tank. This Acceptance Plan will test and verify that the exhauster meets the specified design criteria, safety requirements, operations requirements, and will provide a record of the functional test results.

  6. ATP for the portable 500 CFM exhauster POR-005 skid C

    SciTech Connect

    Keller, C.M.

    1997-06-27

    This Acceptance Test Plan is for a 500 CFM Portable Exhauster POR-005 to be used for saltwell pumping. The Portable Exhauster System will be utilized to eliminate potential flammable gases that may exist within the dome space of the tank. This Acceptance Plan will test and verify that the exhauster meets the specified design criteria, safety requirements, operations requirements, and will provide a record of the functional test results.

  7. Delayed diagnosis of disorder of sex development (DSD) due to P450 oxidoreductase (POR) deficiency.

    PubMed

    Koika, Vasiliki; Armeni, Anastasia K; Georgopoulos, Neoklis A

    2016-04-01

    A 36-year old man, operated on for cryptorchidism at the age of 8 years, was referred to the Outpatient Clinic of Reproductive Endocrinology for investigation of infertility. Clinical examination revealed ambiguous genitalia: penis 4-5 cm, testicular volume 2-3 ml, hypospadias, hypertrophic foreskin and scrotum bifida. Mild hypertension was confirmed. No skeletal malformations were detected. Hormonal and electrolytic determinations as well as semen analysis were conducted. PCR of the coding regions of 17-hydroxylase/17,20 lyase (P450c17) and of P450 oxidoreductase (POR) genes was also performed. Normal levels of electrolytes, low levels of androgens, high levels of gonadotropins and 17-hydroxyprogesterone as well as azoospermia were detected. Karyotype was shown to be 46,XY. Both hCG and ACTH stimulation significantly increased 17-hydroxyprogesterone with no increase in androgens. The diagnosis was congenital adrenal hyperplasia with apparent combined P450c17 and P450c21 deficiency due to mutations in the POR gene. Sequencing of the POR gene revealed: one deletion in exon 12 (Del 1696_1698delGTC >del531Valine) and one missense mutation in exon 7 (A259G) as well as two polymorphisms: rs1057868 (C/T A503V) and rs1057870 (G/A S572S) in exons 12 and 13, respectively. No nucleotide changes were detected in the 8 exons of P450c17. Molecular findings were consistent with the diagnosis of P450 oxidoreductase deficiency. Despite this severe deficiency, skeletal malformations simulating Antley-Bixler syndrome, which usually characterize the most severe forms, were not confirmed. This discrepancy could be attributed to the differential impact of a POR variant on each one of the P450 enzymes.

  8. ATP for the portable 500 CFM exhauster POR-006 skid D

    SciTech Connect

    Keller, C.M.

    1997-07-29

    This Acceptance Test Plan is for a 500 CFM Portable Exhauster POR-006 to be used for saltwell pumping. The Portable Exhauster System will be utilized to eliminate potential flammable gases that may exist within the dome space of the tank. This Acceptance Plan will test and verify that the exhauster meets the specified design criteria, safety requirements, operations requirements, and will provide a record of the functional test results.

  9. A Lack of Significant Effect of POR*28 Allelic Variant on Tacrolimus Exposure in Kidney Transplant Recipients.

    PubMed

    Jannot, Anne-Sophie; Vuillemin, Xavier; Etienne, Isabelle; Buchler, Mathias; Hurault de Ligny, Bruno; Choukroun, Gabriel; Colosio, Charlotte; Thierry, Antoine; Vigneau, Cécile; Moulin, Bruno; Rerolle, Jean-Philippe; Heng, Anne-Elizabeth; Subra, Jean-Francois; Legendre, Christophe; Beaune, Philippe; Loriot, Marie Anne; Thervet, Eric; Pallet, Nicolas

    2016-04-01

    POR*28 is a recently newly described allelic variant of the cytochrome P450 oxidoreductase (POR), which might be associated with an increased metabolic activity of P450 cytochromes (CYP) 3A5 and 3A4. Consequently, carriers of at least 1 allele of this polymorphism could require increased calcineurin inhibitors doses to reach the target residual concentrations (C0). The objective of this study was to test whether the allelic variant of POR, which is associated with an increased metabolic activity of CYP3A, impacts tacrolimus (Tac) pharmacokinetics. We tested this hypothesis in a population of 229 kidney transplant recipients (KTR) from a large, multicenter, prospective and randomized study. We have analyzed the association between POR*28 genotype and the proportion of individuals reaching the target Tac residual concentration (Tac C0) 10 days after transplantation. We have also measured the association between POR*28 and the Tac C0, and adjusted Tac C0 (Tac C0/Tac dose) over time using generalized mixed linear models. Ten days after transplantation, there was no difference of frequencies of KTR within the target range of Tac C0 (C0 10-15 ng/mL) according to the POR*28 genotype (P = 0.8). The mean Tac C0 at day 10 in the POR*1/*1 group was 15.3 ± 9.7 ng/mL compared with 15.7 ± 7.8 ng/mL in the POR*1/*28 group and 14.2 ± 6.8 ng/mL, in the POR*28/*28 group, P = 0.8. The adjusted Tac C0 was not associated with POR*28 genotype over time (random effects model, P = 0.9). When restricted to KTR expressing CYP3A5, POR*28 genotype did not impact the proportion of individuals within the Tac C0 target range neither the adjusted Tac C0 (random effects model, P = 0.1). POR*28 does not significantly influence Tac pharmacokinetic parameters in a large cohort of KTR. This study does not confirm recent findings indicating that POR*28 carriers require more Tac to reach target C0.

  10. P450 oxidoreductase *28 (POR*28) and tacrolimus disposition in pediatric kidney transplant recipients--a pilot study.

    PubMed

    Gijsen, Violette M G J; van Schaik, Ron H N; Soldin, Offie P; Soldin, Steven J; Nulman, Irena; Koren, Gideon; de Wildt, Saskia N

    2014-04-01

    Both age and CYP3A5 genotype are important determinants of tacrolimus disposition in pediatric kidney transplant recipients. In a recent study in adults, POR*28 was associated with increased dosing requirements early after transplant of CYP3A5-expressing kidney transplant recipients. The authors aimed to evaluate the additional contribution of POR*28 to early tacrolimus disposition in pediatric kidney transplant recipients. Retrospective data of 43 pediatric kidney transplant recipients up to 14 days posttransplant were evaluated on tacrolimus dose and tacrolimus predose blood concentrations. Recipient POR*28 and CYP3A5 genotype were determined. CYP3A5 expressers carrying at least 1 POR*28 allele had on average 18.3% lower tacrolimus predose concentrations and 20.2% lower concentration/dose ratios compared with CYP3A5 expressers with POR*1/*1 genotype (P = 0.002 and P = 0.001, respectively). In CYP3A5 nonexpressers, tacrolimus disposition did not significantly differ between POR genotypes. In this small cohort of pediatric kidney transplant recipients, POR*28 genotype seems to explain part of the variability found in tacrolimus disposition, in addition to age and CYP3A5 genotype. This finding should be validated in a larger population, and it would be worthwhile to evaluate the clinical impact of this genotype.

  11. Two NADPH: Protochlorophyllide Oxidoreductase (POR) Isoforms Play Distinct Roles in Environmental Adaptation in Rice.

    PubMed

    Kwon, Choon-Tak; Kim, Suk-Hwan; Song, Giha; Kim, Dami; Paek, Nam-Chon

    2017-12-01

    NADPH: protochlorophyllide oxidoreductase (POR) is an essential enzyme that catalyzes the photoreduction of protochlorophyllide to chlorophyllide, which is ultimately converted to chlorophyll in developing leaves. Rice has two POR isoforms, OsPORA and OsPORB. OsPORA is expressed in the dark during early leaf development; OsPORB is expressed throughout leaf development regardless of light conditions. The faded green leaf (fgl) is a loss-of-function osporB mutant that displays necrotic lesions and variegation in the leaves due to destabilized grana thylakoids, and has increased numbers of plastoglobules in the chloroplasts. To investigate whether the function of OsPORA can complement that of OsPORB, we constitutively overexpressed OsPORA in fgl mutant. In the 35S:OsPORA/fgl (termed OPAO) transgenic plants, the necrotic lesions of the mutant disappeared and the levels of photosynthetic pigments and proteins, as well as plastid structure, were recovered in developing leaves under natural long days in the paddy field and under short days in an artificially controlled growth room. Under constant light conditions, however, total chlorophyll and carotenoid levels in the developing leaves of OPAO plants were lower than those of wild type. Moreover, the OPAO plants exhibited mild defects in mature leaves beginning at the early reproductive stage in the paddy field. The physiological function of OsPORB in response to constant light or during reproductive growth cannot be completely replaced by constitutive activity of OsPORA, although the biochemical functions of OsPORA and OsPORB are redundant. Therefore, we suggest that the two OsPORs have differentiated over the course of evolution, playing distinct roles in the adaptation of rice to the environment.

  12. Genetic polymorphisms and haplotypes of por, encoding cytochrome p450 oxidoreductase, in a Japanese population.

    PubMed

    Saito, Yoshiro; Yamamoto, Noboru; Katori, Noriko; Maekawa, Keiko; Fukushima-Uesaka, Hiromi; Sugimoto, Daisuke; Kurose, Kouichi; Sai, Kimie; Kaniwa, Nahoko; Sawada, Jun-Ichi; Kunitoh, Hideo; Ohe, Yuichiro; Yoshida, Teruhiko; Matsumura, Yasuhiro; Saijo, Nagahiro; Okuda, Haruhiro; Tamura, Tomohide

    2011-01-01

    Cytochrome P450 oxidoreductase (POR) transfers electrons from NADPH to all microsomal cytochrome P450 (CYP) enzymes and is necessary for microsomal CYP activities. In this study, to find genetic variations and to elucidate the haplotype structures of POR, we comprehensively screened the genetic variations in the 5'-flanking region, all the exons and their flanking introns of POR for 235 Japanese subjects. Seventy-five genetic variations including 26 novel ones were found: 7 were in the 5'-flanking region, 2 in the 5'-untranslated region (5'-UTR, non-coding exon 1), 16 in the coding exons (10 nonsynonymous and 6 synonymous), 45 in the introns, 4 in the 3'-UTR and 1 in the 3'-flanking region. Of these, 4 novel nonsynonymous variations, 86C>T (T29M), 1648C>T (R550W), 1708C>T (R570C) and 1975G>A (A659T), were detected with allele frequencies of 0.002. We also detected known nonsynonymous SNPs 683C>T (P228L), 1237G>A (G413S), 1453G>A (A485T), 1508C>T (A503V), 1510G>A (G504R) and 1738G>C (E580Q) with frequencies of 0.002, 0.009, 0.002, 0.434, 0.002 and 0.002, respectively. Based on the linkage disequilibrium (LD) profiles, the analyzed region could be divided into two LD blocks. For Blocks 1 and 2, 14 and 46 haplotypes were inferred, respectively, and 2 and 6 common haplotypes found in more than 0.03 frequencies accounted for more than 81% of the inferred haplotypes. This study provides fundamental and useful information for the pharmacogenetic studies of drugs metabolized by CYPs in the Japanese population.

  13. Zinc finger nuclease knock-out of NADPH:cytochrome P450 oxidoreductase (POR) in human tumor cell lines demonstrates that hypoxia-activated prodrugs differ in POR dependence.

    PubMed

    Su, Jiechuang; Gu, Yongchuan; Pruijn, Frederik B; Smaill, Jeff B; Patterson, Adam V; Guise, Christopher P; Wilson, William R

    2013-12-27

    Hypoxia, a ubiquitous feature of tumors, can be exploited by hypoxia-activated prodrugs (HAP) that are substrates for one-electron reduction in the absence of oxygen. NADPH:cytochrome P450 oxidoreductase (POR) is considered one of the major enzymes responsible, based on studies using purified enzyme or forced overexpression in cell lines. To examine the role of POR in HAP activation at endogenous levels of expression, POR knock-outs were generated in HCT116 and SiHa cells by targeted mutation of exon 8 using zinc finger nucleases. Absolute quantitation by proteotypic peptide mass spectrometry of DNA sequence-confirmed multiallelic mutants demonstrated expression of proteins with residual one-electron reductase activity in some clones and identified two (Hko2 from HCT116 and S2ko1 from SiHa) that were functionally null by multiple criteria. Sensitivities of the clones to 11 HAP (six nitroaromatics, three benzotriazine N-oxides, and two quinones) were compared with wild-type and POR-overexpressing cells. All except the quinones were potentiated by POR overexpression. Knocking out POR had a marked effect on antiproliferative activity of the 5-nitroquinoline SN24349 in both genetic backgrounds after anoxic exposure but little or no effect on activity of most other HAP, including the clinical stage 2-nitroimidazole mustard TH-302, dinitrobenzamide mustard PR-104A, and benzotriazine N-oxide SN30000. Clonogenic cell killing and reductive metabolism of PR-104A and SN30000 under anoxia also showed little change in the POR knock-outs. Thus, although POR expression is a potential biomarker of sensitivity to some HAP, identification of other one-electron reductases responsible for HAP activation is needed for their rational clinical development.

  14. Zinc Finger Nuclease Knock-out of NADPH:Cytochrome P450 Oxidoreductase (POR) in Human Tumor Cell Lines Demonstrates That Hypoxia-activated Prodrugs Differ in POR Dependence*

    PubMed Central

    Su, Jiechuang; Gu, Yongchuan; Pruijn, Frederik B.; Smaill, Jeff B.; Patterson, Adam V.; Guise, Christopher P.; Wilson, William R.

    2013-01-01

    Hypoxia, a ubiquitous feature of tumors, can be exploited by hypoxia-activated prodrugs (HAP) that are substrates for one-electron reduction in the absence of oxygen. NADPH:cytochrome P450 oxidoreductase (POR) is considered one of the major enzymes responsible, based on studies using purified enzyme or forced overexpression in cell lines. To examine the role of POR in HAP activation at endogenous levels of expression, POR knock-outs were generated in HCT116 and SiHa cells by targeted mutation of exon 8 using zinc finger nucleases. Absolute quantitation by proteotypic peptide mass spectrometry of DNA sequence-confirmed multiallelic mutants demonstrated expression of proteins with residual one-electron reductase activity in some clones and identified two (Hko2 from HCT116 and S2ko1 from SiHa) that were functionally null by multiple criteria. Sensitivities of the clones to 11 HAP (six nitroaromatics, three benzotriazine N-oxides, and two quinones) were compared with wild-type and POR-overexpressing cells. All except the quinones were potentiated by POR overexpression. Knocking out POR had a marked effect on antiproliferative activity of the 5-nitroquinoline SN24349 in both genetic backgrounds after anoxic exposure but little or no effect on activity of most other HAP, including the clinical stage 2-nitroimidazole mustard TH-302, dinitrobenzamide mustard PR-104A, and benzotriazine N-oxide SN30000. Clonogenic cell killing and reductive metabolism of PR-104A and SN30000 under anoxia also showed little change in the POR knock-outs. Thus, although POR expression is a potential biomarker of sensitivity to some HAP, identification of other one-electron reductases responsible for HAP activation is needed for their rational clinical development. PMID:24196959

  15. Comparison of Serologic and Genetic porB-Based Typing of Neisseria gonorrhoeae: Consequences for Future Characterization

    PubMed Central

    Unemo, Magnus; Olcén, Per; Albert, Jan; Fredlund, Hans

    2003-01-01

    Due to temporal changes in the epidemiology of gonorrhea, a precise characterization of Neisseria gonorrhoeae is essential. In the present study genetic heterogeneity in the porB genes of N. gonorrhoeae was examined, and serovar determination was compared to porB gene sequencing. Among 108 N. gonorrhoeae isolates, phylogenetic analysis of the entire porB alleles (924 to 993 bp) identified 87 unique sequences. By analyzing only the four to six most heterogeneous porB gene regions (174 to 363 bp), 86 out of these 87 genetic variants were identified. Consequently, analysis of shorter highly variable regions of the porB gene generates high-level discriminatory ability as well as fast, objective, reproducible, and portable data for epidemiological characterization of N. gonorrhoeae. Regarding putative antigenic epitopes of PorB for Genetic Systems monoclonal antibodies (MAbs), some of the previous findings were confirmed, but new findings were also observed. For several of the MAbs, however, the precise amino acid residues of PorB critical for single-MAb reactivity were difficult to identify. In addition, repeated serovar determination of 108 N. gonorrhoeae isolates revealed discrepancies for 34 isolates, mostly due to nonreproducible reactivity with single MAbs. Thus, the prospects of a genetic typing system with congruent translation of the serovar determination seem to be limited. In conclusion, analysis of short highly variable regions of the porB gene could form the basis for a fast molecular epidemiological tool for the examination of emergence and transmission of N. gonorrhoeae strains within the community. PMID:12958238

  16. Improved purification of native meningococcal porin PorB and studies on its structure/function.

    PubMed

    Massari, Paola; King, Carol A; MacLeod, Heather; Wetzler, Lee M

    2005-12-01

    The outer membrane protein PorB of Neisseria meningitidis is a pore-forming protein which has various effects on eukaryotic cells. It has been shown to (1) up-regulate the surface expression of the co-stimulatory molecule CD86 and of MHC class II (which are TLR2/MyD88 dependent and related to the porin's immune-potentiating ability), (2) be involved in prevention of apoptosis by modulating the mitochondrial membrane potential, and (3) form pores in eukaryotic cells. As an outer membrane protein, its native trimeric form isolation is complicated by its insoluble nature, requiring the presence of detergent throughout the whole procedure, and by its tight association with other outer membrane components, such as neisserial LOS or lipoproteins. In this study, an improved chromatographic purification method to obtain an homogeneous product free of endotoxin and lipoprotein is described, without loss of any of the above-mentioned properties of the porin. Furthermore, we have investigated the requirement of the native trimeric structure for the porin's activity. Inactivation of functional PorB trimers into non-functional monomers was achieved by incubation on ice. Thus, routine long- and medium-term storage at low temperature may be a cause of porin inactivation.

  17. Diagnóstico diferencial en la encefalitis por anticuerpos contra el receptor NMDA

    PubMed Central

    González-Valcárcel, J.; Rosenfeld, M.R.; Dalmau, J.

    2011-01-01

    Resumen Introducción La encefalitis por anticuerpos contra el receptor de NMDA (NMDAR) suele desarrollarse como un síndrome característico de evolución multifásica y diagnóstico diferencial amplio. Pacientes Presentamos a 2 pacientes diagnosticadas de encefalitis por anticuerpos NMDAR con un cuadro clínico típico, pero que inicialmente señaló otras etiologías. Discusión La afectación frecuente de pacientes jóvenes con manifestaciones psiquiátricas prominentes indica frecuentemente otras consideraciones diagnósticas; las más frecuentes son las encefalitis virales, los procesos psiquiátricos y el síndrome neuroléptico maligno. Varios síndromes previamente definidos de manera parcial o descriptiva en adultos y pacientes pediátricos probablemente eran casos de encefalitis anti-NMDAR. Conclusiones La encefalitis anti-NMDAR debe considerarse en pacientes jóvenes con manifestaciones psiquiátricas subagudas, movimientos anormales y alteraciones autonómicas. La caracterización clínica e inmunológica de esta enfermedad ha llevado a la identificación de nuevos anticuerpos que afectan a procesos de memoria, aprendizaje, conducta y psicosis. PMID:20964986

  18. Effect of SPM-based cleaning POR on EUV mask performance

    NASA Astrophysics Data System (ADS)

    Choi, Jaehyuck; Lee, Han-shin; Yoon, Jinsang; Shimomura, Takeya; Friz, Alex; Montgomery, Cecilia; Ma, Andy; Goodwin, Frank; Kang, Daehyuk; Chung, Paul; Shin, Inkyun; Cho, H.

    2011-11-01

    EUV masks include many different layers of various materials rarely used in optical masks, and each layer of material has a particular role in enhancing the performance of EUV lithography. Therefore, it is crucial to understand how the mask quality and patterning performance can change during mask fabrication, EUV exposure, maintenance cleaning, shipping, or storage. The fact that a pellicle is not used to protect the mask surface in EUV lithography suggests that EUV masks may have to undergo more cleaning cycles during their lifetime. More frequent cleaning, combined with the adoption of new materials for EUV masks, necessitates that mask manufacturers closely examine the performance change of EUV masks during cleaning process. We have investigated EUV mask quality and patterning performance during 30 cycles of Samsung's EUV mask SPM-based cleaning and 20 cycles of SEMATECH ADT exposure. We have observed that the quality and patterning performance of EUV masks does not significantly change during these processes except mask pattern CD change. To resolve this issue, we have developed an acid-free cleaning POR and substantially improved EUV mask film loss compared to the SPM-based cleaning POR.

  19. Multiple active site residues are important for photochemical efficiency in the light-activated enzyme protochlorophyllide oxidoreductase (POR).

    PubMed

    Menon, Binuraj R K; Hardman, Samantha J O; Scrutton, Nigel S; Heyes, Derren J

    2016-08-01

    Protochlorophyllide oxidoreductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide), an essential, regulatory step in chlorophyll biosynthesis. The unique requirement of the enzyme for light has provided the opportunity to investigate how light energy can be harnessed to power biological catalysis and enzyme dynamics. Excited state interactions between the Pchlide molecule and the protein are known to drive the subsequent reaction chemistry. However, the structural features of POR and active site residues that are important for photochemistry and catalysis are currently unknown, because there is no crystal structure for POR. Here, we have used static and time-resolved spectroscopic measurements of a number of active site variants to study the role of a number of residues, which are located in the proposed NADPH/Pchlide binding site based on previous homology models, in the reaction mechanism of POR. Our findings, which are interpreted in the context of a new improved structural model, have identified several residues that are predicted to interact with the coenzyme or substrate. Several of the POR variants have a profound effect on the photochemistry, suggesting that multiple residues are important in stabilizing the excited state required for catalysis. Our work offers insight into how the POR active site geometry is finely tuned by multiple active site residues to support enzyme-mediated photochemistry and reduction of Pchlide, both of which are crucial to the existence of life on Earth.

  20. Effects of heme precursors on CYP1A2 and POR expression in the baculovirus/Spodoptera frugiperda system.

    PubMed

    Lu, Huiyuan; Ma, Jun; Liu, Nian; Wang, Shoulin

    2010-05-01

    CYP1A2 and NADPH-CYP450 oxidoreductase (POR) were expressed in the baculovirus/Spodoptera frugiperda (sf9) system. The aim of this study was to investigate the effects of heme precursors on the expression of CYP1A2 and POR. The heme precursors [δ-Aminolaevulinic Acid (5-ALA), Fe(3+) and hemin] were introduced into the system to evaluate their effects on the expression of CYP1A2, POR and their co-expression. All the proteins were identified using immunoblotting, CO-difference spectroscopy, or cytochrome c assay. In the present study, functional CYP1A2 and POR were successfully expressed in the baculovirus/sf9 system, and both of them showed high activities. Co-addition of 5-ALA and Fe(3+) significantly improved expression of CYP1A2 by about 50% compared with the addition of 5-ALA, Fe(3+) or hemin alone. Either co-addition of 5-ALA and Fe(3+) or addition of 5-ALA or Fe(3+) alone improved the POR expression level 2 fold and its activity 7-10 fold compared with control (no addition). However, unlike CYP1A2, there was no difference between the co-addition and addition of these heme precursors alone. Different ratios of BvCYP1A2 to BvPOR also affected the co-expression of CYP1A2 and POR, with a 3:1 ratio of BvCYP1A2 / BvPOR significantly increasing their co-expression. Surprisingly, the addition of 0.1 mM 5-ALA or Fe(3+) alone, but not their co-addition, could significantly improve the CYP1A2 and POR co-expression (P < 0.05). 5-ALA and Fe(3+) increased the expression of CYP1A2 and POR in a baculovirus/sf9 system, but the pattern of their expression was different between their expression alone and co-expression.

  1. Reconstrução tridimensional de arcos magnéticos por tomografia

    NASA Astrophysics Data System (ADS)

    Simões, P. J. A.; Costa, J. E. R.

    2003-08-01

    Uma explosão solar é uma variação súbita do brilho que ocorre nas regiões ativas da atmosfera solar. Estas regiões são constituídas por um plasma magnetizado com intensa indução magnética e em cenários bem complexos como visto recentemente através de experimentos embarcados em satélites operando instrumentos em raios X moles e ultra-violeta distante. A energia magnética, que pode ser armazenada por um período de horas até dias em configurações magnéticas estressadas, é subitamente lançada na atmosfera solar e transferida para partículas como elétrons, prótons e núcleos pesados, que são acelerados e/ou aquecidos, produzindo radiação eletromagnética. A proposta final deste projeto é determinar as características espaciais de alta resolução da emissão e polarização girossincrotrônica de explosões solares em ambientes complexos de campos magnéticos. Os recentes resultados da emissão difusa em EUV apresentado pelos satélites TRACE e SOHO dos arcos magnéticos conectando as diferentes polaridades magnéticas sobre as regiões ativas possibilitam novas abordagens sobre o papel do campo magnético na emissão em rádio. Nesta etapa apresentamos os resultados da reconstrução da geometria tridimensional das linhas de força destes arcos utilizando técnicas tomográficas, a partir de imagens de alta resolução espacial obtidas pelo instrumento EIT (Extreme ultraviolet Imaging Telescope), além da modelagem das induções magnéticas por um campo dipolar e as densidades de partículas aceleradas. Utilizamos para a reconstrução geométrica, imagens tomadas em vários ângulos dos arcos devido à rotacão solar. Com estes resultados, daremos continuidade ao projeto, com os cálculos da transferência radiativa nos modos ordinário e extraordinário de propagação da radiação girossincrotrônica de explosões solares.

  2. Intermitência alfvênica gerada por caos na atmosfera solar e no vento solar

    NASA Astrophysics Data System (ADS)

    Rempel, E. L.; Chian, A. C.-L.; Macau, E. E. N.; Rosa, R. R.

    2003-08-01

    Dados medidos no vento solar rápido proveniente dos buracos coronais revelam que os plasmas no meio interplanetário são dominados por flutuações Alfvênicas, caracterizadas por uma alta correlação entre as variações do campo magnético e da velocidade do plasma. As flutuações exibem muitas características esperadas em turbulência magneto-hidrodinâmica totalmente desenvolvida, tais como intermitência e espectros contínuos. Contudo, os mecanismos responsáveis pela evolução de turbulência Alfvênica intermitente não são completamente compreendidos. Neste trabalho a teoria de caos é usada para explicar como sistemas Alfvênicos, modelados pela equação Schrödinger não-linear derivativa e pela equação Kuramoto-Sivashinsky, podem se tornar fortemente caóticos à medida em que parâmetros do plasma são variados. Pequenas perturbações no parâmetro de dissipação podem fazer com que o sistema mude bruscamente de um regime periódico, ou fracamente caótico, para um regime fortemente caótico. As séries temporais das flutuações do campo magnético nos regimes fortemente caóticos exibem comportamento intermitente, em que fases laminares ou fracamente caóticas são interrompidas por fortes estouros caóticos. É mostrado que o regime fortemente caótico é atingido quando as soluções periódicas ou fracamente caóticas globalmente estáveis interagem com soluções do sistema que são fortemente caóticas, mas globalmente instáveis. Estas soluções globalmente instáveis são conjuntos caóticos não-atrativos conhecidos como selas caóticas, e são responsáveis pelos fortes estouros nos regimes intermitentes. Selas caóticas têm sido detectadas experimentalmente em uma grande variedade de sistemas, sendo provável que elas desempenhem um papel importante na turbulência intermitente observada em plasmas espaciais.

  3. Geometry sensing through POR1 regulates Rac1 activity controlling early osteoblast differentiation in response to nanofiber diameter.

    PubMed

    Higgins, A M; Banik, B L; Brown, J L

    2015-02-01

    Bone grafting procedures in the United States rely heavily upon autografts and allografts, which are donor-dependent, cause donor site pain, and can transmit disease. Synthetic bone grafts can reduce these risks; however, synthetics lack the bone differentiating (osteoinductive) abilities of auto- and allografts. Achieving innate osteoinductive properties of synthetics through surface modifications is currently under investigation. This study focuses on nanofibers, with emphasis on how fiber diameter and the potential curvature sensor POR1 affect the activation of the signaling molecules Rac1 and Arf1, and leading to expression of alkaline phosphatase (ALP), an osteoinductive marker. Diameters of 0.1, 0.3, and 1.0 μm were compared against a flat control. The highest level of Rac1 activation was achieved on the smallest fibers (0.1 μm), a trend that was lost in POR1 knockdowns. This supports the hypothesis that on small nanofibers, POR1 favorably binds to highly curved cell membranes, which allows Rac1 to subsequently dissociate and activate. When the curvature is insufficient to bind POR1, POR1 binds to inactive Rac1 and competitively inhibits its activation. Arf1 activation followed an opposite trend, with the largest nanofibers exhibiting the highest activity. This trend reinforces the known interaction between Rac1 and Arf1 through the GIT-PIX complex, an Arf1 GAP and Rac1 GEF, respectively. Large, (1.0 μm), nanofibers demonstrated the highest ALP activity, indicating that ALP expression is inversely dependent on Rac1 activation. Knockdown of POR1 resulted in increased ALP activity across the substrates but without regard to the curvature sensing trend seen previously. Thus, POR1 senses curvature and increases Rac1 activity, which negatively regulates bone differentiation.

  4. Interaction of nitrogen bases with iron-porphyrin nitrito complexes Fe(Por)(ONO) in sublimed solids.

    PubMed

    Kurtikyan, Tigran S; Hovhannisyan, Astghik A; Gulyan, Gurgen M; Ford, Peter C

    2007-08-20

    The reactions of the nitrogen Lewis bases (B) 1-methylimidazole (1-MeIm), pyridine (Py), and NH3 as gases with sublimed layers containing the 5-coordinate nitrito iron(III)-porphyrinato complexes Fe(Por)(eta1-ONO) (1) are described (Por = meso-tetraphenyl-porphyrinato or meso-tetra-p-tolyl-porphyrinato dianions). In situ FTIR and optical spectra are used to characterize the formation of the 6-coordinate nitro complexes formed by the reaction of 1 with B = 1-MeIm, Py, or NH3. These represent the first examples of 6-coordinate amino-nitro complexes with sterically unprotected iron-porphyrins. The interaction of ammonia with Fe(Por)(ONO) at 140 K initially led to the nitrito species Fe(Por)(NH3)(eta1-ONO), and this species isomerized to the nitro complexes Fe(Por)(NH3)(eta1-NO2) upon warming to 180 K. When the latter were warmed to room temperature under intense pumping, the initial nitrito complexes Fe(Por)(eta1-ONO) were restored. Assignments of vibrational frequencies for the coordinated nitro group in 6-coordinate iron-porphyrin complexes are given and confirmed using 15N-labeled nitrogen dioxide to identify characteristic infrared bands. For M(Por)(B)(NO2) complexes (M = Fe or Co), an inverse correlation between the net charge transfer from the axial ligand B to the nitro group and the value of Deltanu = nua(NO2) - nus(NO2) is proposed. These observations are discussed in the context of growing interest in potential physiological roles of nitrite ion reactions with ferro- and ferri-heme proteins.

  5. Using the PORS Problems to Examine Evolutionary Optimization of Multiscale Systems

    SciTech Connect

    Reinhart, Zachary; Molian, Vaelan; Bryden, Kenneth

    2013-01-01

    Nearly all systems of practical interest are composed of parts assembled across multiple scales. For example, an agrodynamic system is composed of flora and fauna on one scale; soil types, slope, and water runoff on another scale; and management practice and yield on another scale. Or consider an advanced coal-fired power plant: combustion and pollutant formation occurs on one scale, the plant components on another scale, and the overall performance of the power system is measured on another. In spite of this, there are few practical tools for the optimization of multiscale systems. This paper examines multiscale optimization of systems composed of discrete elements using the plus-one-recall-store (PORS) problem as a test case or study problem for multiscale systems. From this study, it is found that by recognizing the constraints and patterns present in discrete multiscale systems, the solution time can be significantly reduced and much more complex problems can be optimized.

  6. Fabrication of por-Si/SnO{sub x} nanocomposite layers for gas microsensors and nanosensors

    SciTech Connect

    Bolotov, V. V. Korusenko, P. M.; Nesov, S. N.; Povoroznyuk, S. N.; Roslikov, V. E.; Kurdyukova, E. A.; Sten'kin, Yu. A.; Shelyagin, R. V.; Knyazev, E. V.; Kan, V. E.; Ponomareva, I. V.

    2011-05-15

    Two-phase nanocomposite layers based on porous silicon and nonstoichiometric tin oxide were fabricated by various methods. The structure, as well as elemental and phase composition, of the obtained nanocomposites were studied using transmission and scanning electron microscopy, Raman spectroscopy, Auger electron spectroscopy, and X-ray photoelectron spectroscopy. The results obtained confirm the formation of nanocomposite layers with a thickness as large as 2 {mu}m thick and SnO{sub x} stoichiometry coefficients x = 1.0-2.0. Significant tin diffusion into the porous silicon matrix with D{sub eff} Almost-Equal-To 10{sup -14} cm{sup 2} s{sup -1} was observed upon annealing at 770 K. Test sensor structures based on por-Si/SnO{sub x} nanocomposite layers grown by magnetron deposition showed fairly high stability of properties and sensitivity to NO{sub 2}.

  7. Impact of PPARA and POR polymorphisms on tacrolimus pharmacokinetics and new-onset diabetes in kidney transplant recipients.

    PubMed

    Kurzawski, Mateusz; Malinowski, Damian; Dziewanowski, Krzysztof; Droździk, Marek

    2014-08-01

    Recent efforts have been made to identify genetic markers of CYP3A4 enzymatic activity within genes encoding for regulatory elements. The aim of the current study was to investigate the impact of polymorphism of PPARA and POR genes on tacrolimus (TAC) dose-adjusted trough concentration and risk of new-onset diabetes after transplantation (NODAT). A total of 241 White kidney transplant patients were genotyped for three functional single nucleotide polymorphisms: rs1057868 (*28) in POR, rs4253728:G>A, and rs4823613:A>G in PPARA. No significant genotype-dependent differences in TAC dose-adjusted trough concentration were observed for either POR or PPARA variants. No significant differences in the incidence of NODAT were observed between patients stratified by PPARA and POR genotypes. The frequency of NODAT among PPARA rs4253728 AA homozygotes (42%) was higher compared with heterozygotes (22%) and GG homozygotes (19%), but the difference was not significant. Testing TAC-medicated renal transplant recipients for POR and PPARA variants seems to have limited clinical application.

  8. POR*28 SNP is associated with lipid response to atorvastatin in children and adolescents with familial hypercholesterolemia.

    PubMed

    Drogari, Euridiki; Ragia, Georgia; Mollaki, Vasiliki; Elens, Laure; Van Schaik, Ron H N; Manolopoulos, Vangelis G

    2014-12-01

    In children and adolescents with familial hypercholesterolemia (FH) pharmacotherapy with statins is the cornerstone in the current regimen to reduce low-density lipoprotein cholesterol (LDLc) and premature coronary heart disease risk. There is, however, a great interindividual variation in response to therapy, partially attributed to genetic factors. The polymorphic enzyme POR transfers electrons from NADPH to CYP450 enzymes including CYP3A, which metabolize atorvastatin. POR*28 polymorphism is associated with increased CYP3A enzyme activity. We analyzed the association of POR*28 allele with response to atorvastatin. One hundred and five FH children and adolescents treated with atorvastatin at doses 10-40 mg were included in the study. Total cholesterol (TChol) and LDLc were measured at baseline and after 6 months of treatment. POR*28 allele was analyzed with TaqMan assay. CYP3A4*22, CYP3A5*3 and SLCO1B1 521T>C and 388A>G genotypes were also determined with TaqMan or PCR-RFLP methods. POR*28 carriers had significantly lower percent mean reduction of TChol (33.1% in *1/*1, 29.8% in *1/*28 and 25.9% in *28/*28 individuals, p = 0.045) and of LDLc (43.9% in *1/*1, 40.9% in *1/*28 and 30.8% in *28/*28 individuals, p = 0.013). In multivariable linear regression adjusted for confounding factors, POR*28 genotypes, additionally to baseline cholesterol level, accounted for an estimated 8.3% and 7.3% of overall variability in % TChol and LDLc reduction (β: 4.05; 95% CI: 1.73-6.37; p = 0.001 and β: 5.08; 95% CI: 1.62-8.54; p = 0.004, respectively). CYP3A4*22, CYP3A5*3 and SLCO1B1 521T>C and 388A>G polymorphisms were not associated with lipid reductions and did not modify the effect of POR*28 on atorvastatin response. In children with FH, carriage of POR*28 allele is associated with reduced effect of atorvastatin on TChol and LDLc and therefore identifies FH children that may require higher atorvastatin doses to achieve full therapeutic benefits. Additional studies in

  9. Interactions between CYP3A5*3 and POR*28 polymorphisms and lipid lowering response with atorvastatin.

    PubMed

    Wei, Kan-kan; Zhang, Li-rong

    2015-09-01

    The polymorphic enzyme P450 oxidoreductase (POR) transfers electrons from nicotinamide adenine dinucleotide phosphate (NADPH) to cytochrome P450 (CYP) 3A enzyme s, which metabolize atorvastatin. This suggests that variations in the CYP3A5 and POR genes may influence the response to statins. We aimed to investigate the association and interactions between CYP3A5*3 and POR*28 polymorphisms and the lipid-lowering effects of atorvastatin in a Chinese population. Genotypes were determined by polymerase chain reaction (PCR) with restriction fragment length polymorphism analysis and by PCR with direct sequencing analysis for 179 hyperlipidaemic patients treated with atorvastatin 20 mg once daily for 4 weeks. Serum levels of triglycerides (TGs), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were determined before and after treatment. For the 179 patients (including 100 males), the variant allele frequencies of CYP3A5*3 and POR*28 were 73.75 and 41.62 %, respectively. Among all patients, no significant association was found between CYP3A5*3 polymorphisms and TG, TC, LDL-C and HDL-C levels before and after treatment with 20 mg of atorvastatin daily for 4 weeks. Homozygotes for the POR*28 T allele showed a significantly lower mean concentration of LDL-C than homozygotes for the POR*28 C allele (POR*28 TT vs POR*28 CC: 2.46 ± 0.37 vs 2.69 ± 0.49 mmol/L, P = 0.019) after treatment with atorvastatin 20 mg daily for 4 weeks. After adjustment for age, sex and body mass index, CYP3A5 non-expressors who were POR*28 wild-type homozygotes showed significantly higher mean TC and LDL-C levels than those who were POR*28 variant homozygotes, both at baseline (*3/CC vs *3/TT; TC: 7.30 ± 0.73 vs 6.94 ± 0.36 mmol/L, P = 0.026; LDL-C: 3.88 ± 0.70 vs 3.47 ± 0.46 mmol/L, P = 0.009) and after atorvastatin treatment (*3/CC vs *3/TT; TC: 6.03 ± 0.64 vs 5.69 ± 0.34 mmol/L, P = 0

  10. Refolding by high pressure of a toxin containing seven disulfide bonds: bothropstoxin-1 from Bothrops jararacussu.

    PubMed

    Balduino, Keli N; Spencer, Patrick J; Malavasi, Natalia V; Chura-Chambi, Rosa M; Lemke, Laura S; Morganti, Ligia

    2011-07-01

    Aggregation is a serious obstacle for recovery of biologically active heterologous proteins from inclusion bodies (IBs) produced by recombinant bacteria. E. coli transformed with a vector containing the cDNA for Bothropstoxin-1 (BthTx-1) expressed the recombinant product as IBs. In order to obtain the native toxin, insoluble and aggregated protein was refolded using high hydrostatic pressure (HHP). IBs were dissolved and refolded (2 kbar, 16 h), and the effects of protein concentration, as well as changes in ratio and concentration of oxido-shuffling reagents, guanidine hydrochloride (GdnHCl), and pH in the refolding buffer, were assayed. A 32% yield (7.6 mg per liter of bacterial culture) in refolding of the native BthTx-1 was obtained using optimal conditions of the refolding buffer (Tris-HCl buffer, pH 7.5, containing 3 mM of a 2:3 ratio of GSH/GSSG, and 1 M GdnHCl). Scanning electron microscopy (SEM) showed that that disaggregation of part of IBs particles occurred upon compression and that the morphology of the remaining IBs, spherical particles, was not substantially altered. Dose-dependent cytotoxic activity of high-pressure refolded BthTx-1 was shown in C2C12 muscle cells.

  11. 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

  12. Bothrops jararaca venom gland secretory cells in culture: Effects of noradrenaline on toxin production and secretion.

    PubMed

    Viana, Luciana Godoy; Valente, Richard Hemmi; Heluany, Cíntia Scucuglia; Souza-Imberg, Andreia; Luna, Milene Schmidt; Perales, Jonas; Yamanouye, Norma

    2017-07-01

    Primary culture of snake venom gland secretory cells could be a good model to study the mechanism(s) of toxin(s) production. These cells can produce and secrete venom to the medium with a hemorrhagic activity comparable to that induced by venom collected from snakes. Production of new venom is triggered by the sympathetic outflow, through the release of noradrenaline, but the importance of this neurotransmitter on toxin synthesis has not been addressed. This work led to the identification and comparison of the toxin panel produced by cultured secretory cells, during a 12-day time-course analysis, as well as to the effects of noradrenaline on the process. The results showed that in our culture model the synthesis of new toxins is asynchronous, mimicking data previously published from proteomic analyses of venom glands harvested from animal experimentation. Furthermore, noradrenaline did regulate the synthesis and/or secretion of venom toxins over the analyzed period. Finally, we demonstrated that snake venom metalloproteinases present in these cultured cells secretome were mostly in their zymogen forms; consequently, processing occurs after secretion to the gland lumen. Overall, the data support the use of venom gland secretory cells as a reliable model to investigate the mechanism(s) of toxin(s) synthesis and secretion. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. [Control of Aedes aegypti breeding sites with the program Recicla por tu bienestar in Merida, Mexico].

    PubMed

    Barrera-Pérez, Mario A; Pavía-Ruz, Norma; Mendoza-Mezquita, Jorge Eduardo; Torres-Arcila, Nerio; Hernández-Hernández, Ramón; Castro-Gamboa, Francisco; Geded-Moreno, Eduardo; Cohuo-Rodríguez, Azael; Medina-Barreiro, Anuar; Koyoc-Cardeña, Edgar; Gómez-Dantés, Héctor; Kroeger, Axel; Vázquez-Prokopec, Gonzalo; Manrique-Saide, Pablo

    2015-01-01

    To determine the importance of Ae. aegypti breeding-sites in Merida;to evaluate the impact of Recicla por tu bienestar (RxB, a recycling program) on the reduction of breeding sites and the perception of participants. The relative importance for pupae production of the different types of breeding-sites was determined. Pre-and post-RxB entomological surveys were performed in participant neighborhoods to evaluate the impact on total containers and positive breeding-sites. A survey on the perception of participating people about dengue prevention and control and RxB was applied. Buckets/pots and "small diverse items" were the most important breeding-sites. RxB had a significant impact in the reduction of total containers (IRR = 0.74), positive containers (IRR = 0.33) and the risk of a house being positive for Ae. aegypti (OR = 0.41). All the interviewed participants referred RxB as needed and most consider it useful. RxB should be considered as a good practice for the dengue vector control.

  14. Seguridad del paciente en Radioterapia Intraoperatoria: Impacto de los elementos controlados por el Radiofisico

    NASA Astrophysics Data System (ADS)

    Tarjuelo, Juan Lopez

    Introduccion: En la administracion de la radioterapia intervienen profesionales y equipos de tratamiento, por lo que existe el riesgo de error y se precisa que dicho equipamiento funcione conforme a lo esperado. A los radiofisicos les corresponde participar en las actividades de garantia o aseguramiento de la calidad, incluyendo el control de calidad de los equipos, y en la evaluacion de los riesgos asociados. La radioterapia intraoperatoria (RIO) es una tecnica radioterapica de intensificacion de dosis, altamente selectiva, dirigida a volumenes anatomicos restringidos durante el tratamiento quirurgico oncologico, basada en la administracion de una dosis absorbida alta por medio de un haz de electrones tras el examen visual directo del lecho tumoral. Como incorporar los ultimos avances en el refuerzo de la seguridad en radioterapia es una tarea ambiciosa y compleja, resulta mas concreta y de inmediata aplicacion su introduccion en la RIO. El objetivo es analizar los elementos que reducen los riesgos y aumentan la seguridad en la RIO y su dosimetria, y valorar la funcion del radiofisico en esta labor. Material y metodos: Se emplearon el planificador Radiance de GMV y el acelerador lineal de los tratamientos de RIO Elekta Precise, controlado con el verificador diario de haces Daily QA Check 1090 y medido con las camaras de ionizacion PPC 40, FC65-G y FC65-P de PTW-Freiburg, a su vez verificadas con fuentes radiactivas adecuadas de estroncio-90 modelos CDP y CDC de IBA Dosimetry. Se realizo un analisis de modos de fallo y efectos (failure mode and effect analysis, FMEA) con el fin de identificar los elementos que forman la RIO y aplicar las herramientas necesarias para la minimizacion de los riesgos y la mejora de la seguridad en la tecnica. Se estudiaron las verificaciones diarias de dicho acelerador Precise con el control estadistico de procesos (statistical process control, SPC) y se simularon intervenciones para devolverlo al estado llamado en control. El SPC

  15. Mujeres felices por ser saludables: a breast cancer risk reduction program for Latino women.

    PubMed

    Fitzgibbon, Marian L; Gapstur, Susan M; Knight, Sara J

    2003-05-01

    Breast cancer is the most commonly diagnosed cancer and the most common cause of cancer mortality among Latino women. Several behavioral factors such as early detection and dietary practices could help decrease morbidity and mortality associated with breast cancer in this population. Unfortunately, there are few data regarding the efficacy of health-related interventions for young Latino women. Mujeres Felices por ser Saludables is a randomized intervention project designed to assess breast cancer risk reduction behavior among Latino women ages 20-40 years. The primary objectives of the project were to determine whether an 8-month integrated dietary/breast health intervention could lead to a greater reduction in dietary fat, increase in dietary fiber, increase in the frequency and proficiency of breast self examination (BSE), and reduction in anxiety related to BSE compared to controls. Herein we describe the overall design of the project and present baseline characteristics of the 256 randomized women. Our results suggest that the average daily intake of dietary fat (percentage of total energy) was slightly below 30% (percentage of total energy) among the women randomized. While over half of these women reported that they practice BSE, and few reported anxiety related to BSE, less than 27% of women were proficient in the recommended BSE technique. There are few data on the dietary and breast health behaviors of young low-acculturated Latino women. This study documents the feasibility of recruiting, randomizing, and obtaining both baseline dietary and breast health data on this unique and underserved population.

  16. Unidas por la Vida (United for Life): implementing a culturally-tailored, community-based, family-oriented lifestyle intervention.

    PubMed

    Sorkin, Dara H; Biegler, Kelly A; Peyreda, Margarita; Kilgore, David; Dow, Emily; Ngo-Metzger, Quyen

    2013-01-01

    Unidas por la Vida, a behavioral weight-loss program, was developed for use among low-income, Mexican-American women with diabetes and their overweight/obese adult daughters. The program leverages community resources in a partnership between primary care and community-based organizations. This paper describes the program's implementation, lessons learned, and implications for sustainability.

  17. [Professor Frantisek Por MD and Professor Robert Klopstock MD, students at Budapest and Prague Faculties of Medicine].

    PubMed

    Mydlík, M; Derzsiová, K

    2010-11-01

    Professor Frantisek Por MD and Professor Robert Klopstock MD were contemporaries, both born in 1899, one in Zvolen, the other in Dombovar, at the time of Austro-Hungarian Monarchy. Prof. Por attended the Faculty of Medicine in Budapest from 1918 to 1920, and Prof. Klopstock studied at the same place between 1917 and 1919. From 1920 until graduation on 6th February 1926, Prof. Por continued his studies at the German Faculty of Medicine, Charles University in Prague. Prof. Klopstock had to interrupt his studies in Budapest due to pulmonary tuberculosis; he received treatment at Tatranske Matliare where he befriended Franz Kafka. Later, upon Kafka's encouragement, he changed institutions and continued his studies at the German Faculty of Medicine, Charles University in Prague, where he graduated the first great go. It is very likely that, during their studies in Budapest and Prague, both professors met repeatedly, even though their life paths later separated. Following his graduation, Prof. Por practiced as an internist in Prague, later in Slovakia, and from 1945 in Kosice. In 1961, he was awarded the title of university professor of internal medicine at the Faculty of Medicine, Pavol Jozef Safarik University in Kosice, where he practiced until his death in 1980. Prof. Klopstock continued his studies in Kiel and Berlin. After his graduation in 1933, he practiced in Berlin as a surgeon and in 1938 left for USA. In 1962, he was awarded the title of university professor of pulmonary surgery in NewYork, where he died in 1972.

  18. jPOR: An ImageJ macro to quantify total optical porosity from blue-stained thin sections

    NASA Astrophysics Data System (ADS)

    Grove, Clayton; Jerram, Dougal A.

    2011-11-01

    A fast and effective method has been developed to measure total optical porosity (TOP) of blue resin-impregnated thin sections. This utilises a macro file (jPOR.txt) for ImageJ, which can be used on digital photomicrographs of thin sections. The method requires no specialised scientific equipment and can be run entirely using free to download software. Digital images are acquired from blue resin-impregnated thin sections using a conventional film scanner in the present study, though the technique can be applied to any high resolution colour digital acquired by different means (e.g., flat bed scanning, digital capture). Images are preprocessed using a newly developed custom 8-bit palette and analysed for porosity in ImageJ using the simple to use jPOR macro. Our method rapidly calculates TOP for batches of images with or without the option of user adjustment. Results are compared with conventional methods (e.g., to point counting), and tested with several users to estimate any user variability. jPOR provided comparable results to more time-consuming point counting, but with significantly less "counting error" and less interoperator variability than published point counting studies. The jPOR macro has been integrated into a macro tool set that can be configured to be run on ImageJ start up.

  19. Functional POR A503V is associated with the risk of bladder cancer in a Chinese population

    PubMed Central

    Xiao, Xue; Ma, Gaoxiang; Li, Shushu; Wang, Meilin; Liu, Nian; Ma, Lan; Zhang, Zhan; Chu, Haiyan; Zhang, Zhengdong; Wang, Shou-Lin

    2015-01-01

    Human cytochrome P450 oxidoreductase (POR) plays important roles in the metabolism of exogenous carcinogens and endogenous sterol hormones. However, few studies have explored the association between POR variants and the risk of bladder cancer. In this study, we first sequenced all 16 POR exons among 50 randomly selected controls, and found three variants, rs1135612, rs1057868 (A503V) and rs2228104, which were then assessed the relation to risk of bladder cancer in a case-control study of 1,050 bladder cancer cases and 1,404 cancer-free controls in a Chinese population. People with A503V TT genotype have a decreased risk of bladder cancer in a recessive model (TT vs. CC/CT, OR = 0.73, 95% CI = 0.57–0.93), which was more pronounced among elderly male, non-smoking, subjects. Especially, A503V TT genotype showed a protective effect in the invasive tumor stage. Functional analysis revealed that A503V activity decreased in cytochrome c reduction (50.5 units/mg vs. 135.4 units/mg), mitomycin C clearance (38.3% vs. 96.8%), and mitomycin C-induced colony formation (78.0 vs 34.3 colonies per dish). The results suggested that POR A503V might decrease the risk of bladder cancer by reducing its metabolic activity, and should be a potential biomarker for predicting the susceptibility to human bladder cancer. PMID:26123203

  20. Global Microlending in Education Reform: Enseñá Por Argentina and the Neoliberalization of the Grassroots

    ERIC Educational Resources Information Center

    Friedrich, Daniel S.

    2010-01-01

    This article examines the workings and underlying assumptions behind Enseñá por Argentina (Teach for Argentina), one specific program that takes part in the larger and expanding network of Teach for All, by thinking about the ways in which a global push for redefining teaching and teacher education encounters local characteristics and histories,…

  1. Functional POR A503V is associated with the risk of bladder cancer in a Chinese population.

    PubMed

    Xiao, Xue; Ma, Gaoxiang; Li, Shushu; Wang, Meilin; Liu, Nian; Ma, Lan; Zhang, Zhan; Chu, Haiyan; Zhang, Zhengdong; Wang, Shou-Lin

    2015-06-30

    Human cytochrome P450 oxidoreductase (POR) plays important roles in the metabolism of exogenous carcinogens and endogenous sterol hormones. However, few studies have explored the association between POR variants and the risk of bladder cancer. In this study, we first sequenced all 16 POR exons among 50 randomly selected controls, and found three variants, rs1135612, rs1057868 (A503V) and rs2228104, which were then assessed the relation to risk of bladder cancer in a case-control study of 1,050 bladder cancer cases and 1,404 cancer-free controls in a Chinese population. People with A503V TT genotype have a decreased risk of bladder cancer in a recessive model (TT vs. CC/CT, OR = 0.73, 95% CI = 0.57-0.93), which was more pronounced among elderly male, non-smoking, subjects. Especially, A503V TT genotype showed a protective effect in the invasive tumor stage. Functional analysis revealed that A503V activity decreased in cytochrome c reduction (50.5 units/mg vs. 135.4 units/mg), mitomycin C clearance (38.3% vs. 96.8%), and mitomycin C-induced colony formation (78.0 vs 34.3 colonies per dish). The results suggested that POR A503V might decrease the risk of bladder cancer by reducing its metabolic activity, and should be a potential biomarker for predicting the susceptibility to human bladder cancer.

  2. Global Microlending in Education Reform: Enseñá Por Argentina and the Neoliberalization of the Grassroots

    ERIC Educational Resources Information Center

    Friedrich, Daniel S.

    2010-01-01

    This article examines the workings and underlying assumptions behind Enseñá por Argentina (Teach for Argentina), one specific program that takes part in the larger and expanding network of Teach for All, by thinking about the ways in which a global push for redefining teaching and teacher education encounters local characteristics and histories,…

  3. Rescue of cytochrome P450 oxidoreductase (Por) mouse mutants reveals functions in vasculogenesis, brain and limb patterning linked to retinoic acid homeostasis.

    PubMed

    Ribes, Vanessa; Otto, Diana M E; Dickmann, Leslie; Schmidt, Katy; Schuhbaur, Brigitte; Henderson, Colin; Blomhoff, Rune; Wolf, C Roland; Tickle, Cheryll; Dollé, Pascal

    2007-03-01

    Cytochrome P450 oxidoreductase (POR) acts as an electron donor for all cytochrome P450 enzymes. Knockout mouse Por(-/-) mutants, which are early embryonic (E9.5) lethal, have been found to have overall elevated retinoic acid (RA) levels, leading to the idea that POR early developmental function is mainly linked to the activity of the CYP26 RA-metabolizing enzymes (Otto et al., Mol. Cell. Biol. 23, 6103-6116). By crossing Por mutants with a RA-reporter lacZ transgene, we show that Por(-/-) embryos exhibit both elevated and ectopic RA signaling activity e.g. in cephalic and caudal tissues. Two strategies were used to functionally demonstrate that decreasing retinoid levels can reverse Por(-/-) phenotypic defects, (i) by culturing Por(-/-) embryos in defined serum-free medium, and (ii) by generating compound mutants defective in RA synthesis due to haploinsufficiency of the retinaldehyde dehydrogenase 2 (Raldh2) gene. Both approaches clearly improved the Por(-/-) early phenotype, the latter allowing mutants to be recovered up until E13.5. Abnormal brain patterning, with posteriorization of hindbrain cell fates and defective mid- and forebrain development and vascular defects were rescued in E9.5 Por(-/-) embryos. E13.5 Por(-/-); Raldh2(+/-) embryos exhibited abdominal/caudal and limb defects that strikingly phenocopy those of Cyp26a1(-/-) and Cyp26b1(-/-) mutants, respectively. Por(-/-); Raldh2(+/-) limb buds were truncated and proximalized and the anterior-posterior patterning system was not established. Thus, POR function is indispensable for the proper regulation of RA levels and tissue distribution not only during early embryonic development but also in later morphogenesis and molecular patterning of the brain, abdominal/caudal region and limbs.

  4. Determinacion del error sistematico del momentum de muones producidos por interacciones neutrino-nucleon en el detector MINER$\

    SciTech Connect

    Diaz Bautista, Gonzalo A.

    2015-11-29

    El Modelo Estandar describe todas las partculas observadas en el naturaleza hasta el momento as como las caractersticas que gobiernan a las interacciones fundamentales entre ellas. En especial es posible identicar a las interacciones electromagnetica y debil, las cuales bajo determinadas condiciones de temperatura y energa pueden ser descritas a traves de una sola teora que engloba a ambas. A esta teora se le denomina electrodebil y tiene como nalidad caracterizar las propiedades de la interaccion maniesta a partir de la mezcla de las interacciones electromagnetica y debil, la que tambien lleva como nombre interaccion electrodebil. Particularmente, los neutrinos son de especial interes ya que, por un lado, interactuan por medio de la interaccion debil muy raramente en comparacion con otras partculas y, por el otro, no son acertadamente descritos por el Modelo Estandar. Por medio de observaciones experimentales que demostraban que los neutrinos cambian de sabor al propagarse, fenomeno llamado oscilaciones de neutrinos, se pudo llegar a la conclusion de que la implicancia de este fenomeno da como consecuencia que los neutrinos efectivamente s tienen masa, algo que entra en contradiccion con la descripcion inicial del Modelo Estandar, el cual los describe como partculas sin masa. Es de esta manera que las oscilaciones de neutrinos han sido y siguen siendo en la actualidad objeto de interes en la Fsica de Altas Energas tanto teorica como experimental. A n de poder realizar mediciones precisas de oscilaciones de neutrinos, los experimentos encargados de estas mediciones deben tratar de reducir sus incertidumbres en lo posible. Una de estas proviene de la caracterizacion de las secciones de choque de los neutrinos cuando interactuan con la materia, particularmente los nucleones al interior de los nucleos atomicos. El experimento MINERA esta orientado, entre otras cosas, a hacer una correcta caracterizacion de secciones de choque neutrino-nucleon por medio del estudio de

  5. Inversor Resonante de Tres Elementos L-LC con Caracteristica Cortocircuitable para Aplicaciones de Calentamiento por Induccion

    NASA Astrophysics Data System (ADS)

    Espi Huerta, Jose Miguel

    Los generadores de calentamiento por induccion son puentes inversores con carga resonante, cuya mision es basicamente crear una corriente sinusoidal de gran amplitud sobre la "bobina de caldeo", que forma parte del tanque resonante. En el interior de esta bobina se introduce la pieza que se desea calentar. EI campo magnetico creado induce corrientes superficiales (corrientes de Foucault) sobre la pieza, que producen su calentamiento. Los tanques resonantes (tambien llamados osciladores) utilizados en la actualidad son el resonante serie y el resonante paralelo. Aunque ya desde hace algun tiempo se vienen construyendo generadores de alta potencia basados en estos dos osciladores, el exito nunca ha. sido completo en ninguno de los dos casos. Tal y como se explica en la introduccion de esta memoria, los puentes inversores utilizados deben operar sobre una carga inductiva (corriente retrasada) para evitar el fenomeno de la recuperacion inversa de sus diodos y la consiguiente ruptura de los transistores. De la restriccion topologica anterior se deduce que el generador paralelo debe conmutar a frecuencias inferiores a la resonancia, y el serie a frecuencias superiores. A esta restriccion topologica hay que unir otra que es exclusiva del calentamiento por induccion: La corriente por la bobina de caldeo debe ser sinusoidal. De no ser asi, resultaria imposible disponer toda la potencia de calentamiento sobre la pieza en el espesor requerido por la aplicacion. Como consecuencia, los inversores no pueden operar por debajo de la frecuencia de resonancia del oscilador, pues en ese caso se amplifican los armonicos de orden superior de la tension/corriente de entrada situados sobre la resonancia, con la consiguiente distorsion de la corriente de salida. La conjuncion de las dos restricciones anteriores obligan al inversor paralelo a funcionar a la frecuencia de resonancia del oscilador. Esto imposibilita un control por variacion de frecuencia, regulandose la potencia desde la

  6. The amino acid sequence of Neisseria lactamica PorB surface-exposed loops influences Toll-like receptor 2-dependent cell activation.

    PubMed

    Toussi, Deana N; Carraway, Margaretha; Wetzler, Lee M; Lewis, Lisa A; Liu, Xiuping; Massari, Paola

    2012-10-01

    Toll-like receptors (TLRs) play a major role in host mucosal and systemic defense mechanisms by recognizing a diverse array of conserved pathogen-associated molecular patterns (PAMPs). TLR2, with TLR1 and TLR6, recognizes structurally diverse bacterial products such as lipidated factors (lipoproteins and peptidoglycans) and nonlipidated proteins, i.e., bacterial porins. PorB is a pan-neisserial porin expressed regardless of organisms' pathogenicity. However, commensal Neisseria lactamica organisms and purified N. lactamica PorB (published elsewhere as Nlac PorB) induce TLR2-dependent proinflammatory responses of lower magnitude than N. meningitidis organisms and N. meningitidis PorB (published elsewhere as Nme PorB). Both PorB types bind to TLR2 in vitro but with different apparent specificities. The structural and molecular details of PorB-TLR2 interaction are only beginning to be unraveled and may be due to electrostatic attraction. PorB molecules have significant strain-specific sequence variability within surface-exposed regions (loops) putatively involved in TLR2 interaction. By constructing chimeric recombinant PorB loop mutants in which surface-exposed loop residues have been switched between N. lactamica PorB and N. meningitidis PorB, we identified residues in loop 5 and loop 7 that influence TLR2-dependent cell activation using HEK cells and BEAS-2B cells. These loops are not uniquely responsible for PorB interaction with TLR2, but NF-κB and MAP kinases signaling downstream of TLR2 recognition are likely influenced by a hypothetical "TLR2-binding signature" within the sequence of PorB surface-exposed loops. Consistent with the effect of purified PorB in vitro, a chimeric N. meningitidis strain expressing N. lactamica PorB induces lower levels of interleukin 8 (IL-8) secretion than wild-type N. meningitidis, suggesting a role for PorB in induction of host cell activation by whole bacteria.

  7. Thermal properties and phase composition of water for frozen rocks of Por-Bajin island (Republic of Tuva, Russia)

    NASA Astrophysics Data System (ADS)

    Aleksyutina, Daria; Motenko, Rimma; Buldovich, Sergey; Koshurnikov, Andrey; Prosuncov, Kirill

    2010-05-01

    We investigated current permafrost state, thermal properties and phase composition of water for frozen rocks of Por-Bajin Island (Tere-Hol Lake, south-east part of Tuva republic, Russia). Rocks of the island are represented by dispersing grounds in permafrost state. The architectural monument Por-Bajin fortress (the middle of the VIII century) located at the island can be destructed due to thawing of frozen rocks resulted by water level rising in the surrounding Por-Bajin lake. Seasonally thawed layer on the island varies from 1.1 to 1.8 meters based on drilling observations from 13 drill holes the. Measured in the field the thermal conductivity indicates the low values in seasonally thawed layer (from 0.14 to 0.19 Wm-1K-1). Twenty most typical for the studied area frozen handsamples were collected from several holes in the depth interval from 1.5 to 10.15 meters. The humidity of sampled specimens varies from 13 to 270 percents and density varies from 1.21 to 2.17 g/cm3. Investigations of grain-size composition, thermal properties and liquid phase composition were undertaken using laboratory methods. The grain-size composition of Por-Bajin grounds varies from clays to gritstones and gravelites with clay-silt matrix. Phase composition of water in the frozen grounds was studied by contact and cryoscopic methods. Thermal properties were investigated by the I-st type regular mode method (a-calorimeter). Based on obtained data computer model allowed to forecast the evolution of permafrost at the island and predict the destiny of Por-Bajin monument. This work was supported by grant from President of the Russian Federation NSh - 4078.2008.5

  8. Encefalitis por anticuerpos contra el receptor de NMDA: experiencia con seis pacientes pediátricos. Potencial eficacia del metotrexato

    PubMed Central

    Bravo-Oro, Antonio; Abud-Mendoza, Carlos; Quezada-Corona, Arturo; Dalmau, Josep; Campos-Guevara, Verónica

    2016-01-01

    Introducción La encefalitis por anticuerpos contra el receptor de N-metil-D-aspartato (NMDA) es una entidad cada vez más diagnosticada en edad pediátrica. A diferencia de los adultos, en muchos casos no se asocia a tumores y las manifestaciones iniciales en niños más frecuentes son crisis convulsivas y trastornos del movimiento, mientras que en los adultos predominan las alteraciones psiquiátricas. Casos clínicos Presentamos seis casos pediátricos confirmados con anticuerpos contra la subunidad NR1 del receptor de NMDA en suero y líquido cefalorraquídeo. Cinco de los casos comenzaron con crisis convulsivas como manifestación clínica inicial antes de desarrollar el cuadro clásico de esta entidad. En todos los casos se utilizaron esteroides como primera línea de tratamiento, con los que sólo se observó control de las manifestaciones en uno, por lo que el resto de los pacientes requirió inmunomoduladores de segunda línea. Todos los pacientes recibieron metotrexato como tratamiento inmunomodulador para evitar recaídas y la evolución fue a la mejoría en todos ellos. Conclusiones En nuestra serie de pacientes con encefalitis por anticuerpos contra el receptor de NMDA, ninguno se asoció a tumores. Todos los casos recibieron metotrexato por lo menos durante un año, no observamos eventos adversos clínicos ni por laboratorio, ni hubo secuelas neurológicas ni recaídas durante el tratamiento. Aunque es una serie pequeña y es deseable incrementar el número y tiempo de evolución, consideramos el metotrexato una excelente alternativa como tratamiento inmunomodulador para esta patología. PMID:24150952

  9. The effect of essential oil on heart rate and blood pressure among solus por aqua workers.

    PubMed

    Chuang, Kai-Jen; Chen, Hua-Wei; Liu, I-Jung; Chuang, Hsiao-Chi; Lin, Lian-Yu

    2014-07-01

    Aromatherapy is widely used around the world for stress relief. Whether exposure to essential oil increases the risk of cardiovascular events is still unknown. The purpose of this study was to investigate the effect of essential oil on heart rate and blood pressure among solus por aqua (spa) workers. We recruited 100 healthy workers from various spa centres in Taipei, Taiwan. Between July and August of 2010, three repeated measurements - resting heart rate (HR), systolic blood pressure (SBP), and diastolic blood pressure (DBP) - were taken of each spa worker in our study room. Participants were exposed to essential oil vapour generated from an ultrasonic atomizer in the study room for two consecutive hours. The total volatile organic compound (VOC) level in the study room was measured during the study period. We used a linear mixed-effect model to determine the association between the total VOC level and the participants' HR, SBP, and DBP. For the times from 15 to 60 min after start of exposure, we found that the VOC level was significantly associated with reduced 15-min mean BP and HR. After exposure for more than 1 hour, from 75 to 120 min after start of exposure, we found that the VOC levels were associated with increased 15-min mean BP and HR. Exposure to essential oil for 1 hour was found to be an effective method of relaxation, as indicated by decreases in the HR and BP. Prolonged exposure for longer than 1 hour to essential oils may be harmful to cardiovascular health among spa workers. © The European Society of Cardiology 2012.

  10. The Sangre Por Salud Biobank: Facilitating Genetic Research in an Underrepresented Latino Community.

    PubMed

    Shaibi, Gabriel; Singh, Davinder; De Filippis, Eleanna; Hernandez, Valentina; Rosenfeld, Bill; Otu, Essen; Montes de Oca, Gregorio; Levey, Sharon; Radecki Breitkopf, Carmen; Sharp, Richard; Olson, Janet; Cerhan, James; Thibodeau, Stephen; Winkler, Erin; Mandarino, Lawrence

    2016-01-01

    The Sangre Por Salud (Blood for Health; SPS) Biobank was created for the purpose of expanding precision medicine research to include underrepresented Latino patients. It is the result of a unique collaboration between Mayo Clinic and Mountain Park Health Center, a federally qualified community health center in Phoenix, Arizona. This report describes the rationale, development, implementation, and characteristics of the SPS Biobank. Latino adults (ages 18-85 years) who were active patients within Mountain Park Health Center's internal medicine practice in Phoenix, Ariz., and had no history of diabetes were eligible. Participants provided a personal and family history of chronic disease, completed a sociodemographic, psychosocial, and behavioral questionnaire, underwent a comprehensive cardiometabolic risk assessment (anthropometrics, blood pressure and labs), and provided blood samples for banking. Laboratory results of cardiometabolic testing were returned to the participants and their providers through the electronic health record. During the first 2 years of recruitment into the SPS Biobank, 2,335 patients were approached and 1,432 (61.3%) consented to participate; 1,354 (94.5%) ultimately completed all requisite questionnaires and medical evaluations. The cohort is primarily Spanish-speaking (72.9%), female (73.3%), with a mean age of 41.3 ± 12.5 years. Most participants were born outside of the US (77.9%) and do not have health insurance (77.5%). The prevalence of overweight (35.5%) and obesity (45.0%) was high, as was previously unidentified prediabetes (55.9%), type 2 diabetes (7.4%), prehypertension (46.8%), and hypertension (16.2%). The majority of participants rated their health as good to excellent (72.1%) and, as a whole, described their overall quality of life as high (7.9/10). Collaborative efforts such as the SPS Biobank are critical for ensuring that underrepresented minority populations are included in precision medicine initiatives and biomedical

  11. Prevalencia y tamizaje del Trastorno por Déficit de Atención con Hiperactividad en Costa Rica

    PubMed Central

    Weiss, Nicholas T.; Schuler, Jovita; Monge, Silvia; McGough, James J.; Chavira, Denise; Bagnarello, Monica; Herrera, Luis Diego; Mathews, Carol A.

    2015-01-01

    Resumen La investigación tuvo como propósito estimar la prevalencia del Trastorno por Déficit de Atención con Hiperactividad (TDAH) en Costa Rica y determinar si la versión en español del cuestionario Swanson Nolan and Pelham Scale IV (SNAP-IV) es un instrumento de tamizaje útil en una población de niños y niñas escolares costarricenses. El instrumento fue entregado a padres y maestros de 425 niños entre 5 y 13 años de edad (promedio = 8.8). Todos fueron evaluados con el instrumento Swanson, Kotkin, Agler, M-Flynn and Pelham Scale (SKAMP). Su diagnóstico fue confirmado con una entrevista clínica. La sensibilidad y la especificidad del SNAP-IV fueron evaluadas como predictores de criterios de diagnóstico según el DSM-IV. La prevalencia puntual en la muestra del TDAH fue del 5%. El tamizaje más preciso lo hizo el SNAP-IV completado por el maestro en un corte de 20%, con una sensibilidad de 96% y una especificidad de un 82%. La sensibilidad de los instrumentos completados por los padres fue más baja que aquella de los maestros. El SNAP-IV completado por las maestras con un corte aislando el 20% de los mayores puntajes categorizó correctamente a un 87% de los sujetos. PMID:22432094

  12. Racism in digital era: Development and initial validation of the Perceived Online Racism Scale (PORS v1.0).

    PubMed

    Keum, Brian TaeHyuk; Miller, Matthew J

    2017-04-01

    The purpose of this study was to develop the Perceived Online Racism Scale (PORS) to assess perceived online racist interpersonal interactions and exposure to online racist content among people of color. Items were developed through a multistage process involving a comprehensive literature review, focus-groups, qualitative data collection, and survey of online racism experiences. Based on a sample of 1,023 racial minority participants, exploratory and confirmatory factor analyses provided support for a 30-item bifactor model accounted by the general factor and the following 3 specific factors: (a) personal experience of racial cyber-aggression, (b) vicarious exposure to racial cyber-aggression, and (c) online-mediated exposure to racist reality. The PORS demonstrated measurement invariance across racial/ethnic groups in our sample. Internal reliability estimates for the total and subscale scores of the PORS were above .88 and the 4-week test-retest reliability was adequate. Limitations and future directions for research are discussed. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

  13. The PorB porin from commensal Neisseria lactamica induces Th1 and Th2 immune responses to ovalbumin in mice and is a potential immune adjuvant.

    PubMed

    Liu, Xiuping; Wetzler, Lee M; Massari, Paola

    2008-02-06

    Porins from pathogenic Neisseriae are among several bacterial products with immune adjuvant activity. Neisseria meningitidis (Nme) PorB, has been shown to induce immune cells activation in a TLR2-dependent manner and acts as a vaccine immune adjuvant. The PorB porin from Neisseria lactamica (Nlac), a common nasopharyngeal commensal, shares significant structural and functional similarities with Nme PorB. In this work we ask whether the immune adjuvant ability of porins from pathogenic Neisserial strains is a characteristic shared with porins from non-pathogenic Neisserial species or whether it is unique for bacterial products derived from microorganisms capable of inducing inflammation and disease. We evaluated the potential immune adjuvant effect of Nlac PorB in mice using ovalbumin (OVA) as a prototype antigen. Immunization with Nlac PorB/OVA induced high OVA-specific IgG and IgM titers compared to OVA alone, similar to other adjuvants such as Nme PorB and alum. High titers of IgG1 and IgG2b were detected as well as production of IL-4, IL-10, IL-12 and INF-gamma in response to Nlac PorB, consistent with induction of both a Th1-type and a Th2-type immune response. OVA-specific proliferation was also determined in splenocytes from Nlac PorB/OVA-immunized mice. In addition, B cell activation in vitro and cytokine production in response to Nlac PorB was found to be mediated by TLR2, in a similar manner to Nme PorB.

  14. Chlorophyll Synthesis in a Deetiolated (det340) Mutant of Arabidopsis without NADPH-Protochlorophyllide (PChlide) Oxidoreductase (POR) A and Photoactive PChlide-F655.

    PubMed Central

    Lebedev, N.; Van Cleve, B.; Armstrong, G.; Apel, K.

    1995-01-01

    Chlorophyll (Chl) synthesis in Arabidopsis is controlled by two light-dependent NADPH-protochlorophyllide (PChlide) oxidoreductases (PORs), one (POR A) that is active transiently in etiolated seedlings at the beginning of illumination and another (POR B) that also operates in green plants. The function of these two enzymes during the light-induced greening of dark-grown seedlings has been studied in the wild type and a deetiolated (det340) mutant of Arabidopsis. One of the consequences of the det mutation is that POR A is constitutively down-regulated, and therefore, synthesis of the POR A enzyme is shut off. When grown in the dark, the det340 mutant lacks POR A and the photoactive PChlide-F655 species but maintains the second PChlide reductase, POR B. Previously, photoactive PChlide-F655 has often been considered to be the only PChlide form that leads to Chl formation. Despite its deficiency in POR A and photoactive PChlide-F655, the det340 mutant is able to green when placed in the light. Chl accumulation, however, proceeds abnormally. At the beginning of illumination, seedlings of det340 mutants are extremely susceptible to photooxidative damage and accumulate Chl only at extremely low light intensities. They form core complexes of photosystems I and II but are almost completely devoid of light-harvesting structures. The results of this study demonstrate that in addition to the route of Chl synthesis that has been studied extensively in illuminated dark-grown wild-type plants, a second branch of Chl synthesis exists that is driven by POR B and does not require POR A. PMID:12242369

  15. CYP2C9*2 allele increases risk for hypoglycemia in POR*1/*1 type 2 diabetic patients treated with sulfonylureas.

    PubMed

    Ragia, G; Tavridou, A; Elens, L; Van Schaik, R H N; Manolopoulos, V G

    2014-01-01

    It is previously shown that carriers of the defective allele CYP2C9*3 that leads to impaired sulfonylurea metabolism are at increased sulfonylurea-induced hypoglycemia risk due to diminished drug metabolism, whereas no effect of CYP2C9*2 allele was found. Recently, a polymorphism in P450 oxidoreductase (POR) gene, assigned as POR*28 allele, was associated with increased CYP2C9 activity. The aim of this study was to assess i) the effect of POR*28 allele on sulfonylurea-induced hypoglycemia risk and ii) the association of CYP2C9*2 allele with hypoglycemia risk in non-carriers of POR*28 allele. The study group consisted of 176 patients with diagnosed type 2 diabetes mellitus (T2DM) treated with sulfonylureas, of whom 92 patients had experienced at least one drug-associated hypoglycemic event (cases), while 84 had never experienced a hypoglycemic event (controls). POR*28 allele was detected by use of real-time TaqMan PCR. POR*28 allele was not associated with sulfonyl-urea-induced hypoglycemia. In POR*1/*1 patients, CYP2C9*1/*2 genotype was more common in cases than in controls (32.7 vs. 14.3%, p=0.041). In a model adjusted for age, BMI, duration of T2DM and renal function, and POR*1/*1 entered as a selection variable, CYP2C9*2 allele increased the hypoglycemia risk in response to sulfonylurea (odds ratio: 3.218, p=0.031). In conclusion, our results suggest that POR*28 allele is masking the association of CYP2C9*2 allele with sulfonyl-urea-induced hypoglycemia. Therefore, POR*28 allele is an important source of CYP2C9 activity variability and combined with CYP2C9 gene poly-morphisms may explain individual variability in the effect of sulfonylureas.

  16. Flavobacterium johnsoniae PorV is required for secretion of a subset of proteins targeted to the type IX secretion system.

    PubMed

    Kharade, Sampada S; McBride, Mark J

    2015-01-01

    Flavobacterium johnsoniae exhibits gliding motility and digests many polysaccharides, including chitin. A novel protein secretion system, the type IX secretion system (T9SS), is required for gliding and chitin utilization. The T9SS secretes the cell surface motility adhesins SprB and RemA and the chitinase ChiA. Proteins involved in secretion by the T9SS include GldK, GldL, GldM, GldN, SprA, SprE, and SprT. Porphyromonas gingivalis has orthologs for each of these that are required for secretion of gingipain protease virulence factors by its T9SS. P. gingivalis porU and porV have also been linked to T9SS-mediated secretion, and F. johnsoniae has orthologs of these. Mutations in F. johnsoniae porU and porV were constructed to determine if they function in secretion. Cells of a porV deletion mutant were deficient in chitin utilization and failed to secrete ChiA. They were also deficient in secretion of the motility adhesin RemA but retained the ability to secrete SprB. SprB is involved in gliding motility and is needed for formation of spreading colonies on agar, and the porV mutant exhibited gliding motility and formed spreading colonies. However, the porV mutant was partially deficient in attachment to glass, apparently because of the absence of RemA and other adhesins on the cell surface. The porV mutant also appeared to be deficient in secretion of numerous other proteins that have carboxy-terminal domains associated with targeting to the T9SS. PorU was not required for secretion of ChiA, RemA, or SprB, indicating that it does not play an essential role in the F. johnsoniae T9SS.

  17. Polymorphisms of POR, SULT2A1 and HSD11B1 in children with premature adrenarche.

    PubMed

    Utriainen, Pauliina; Laakso, Saila; Jääskeläinen, Jarmo; Voutilainen, Raimo

    2012-09-01

    Premature adrenarche (PA) refers to an earlier than normal increase in adrenocortical androgen production. The pathogenesis of PA remains largely unknown. We hypothesized that common polymorphisms at P450 oxidoreductase (POR), steroid sulfotransferase (SULT2A1), or 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1) genes could contribute to the polygenic pathogenesis of PA. We performed a case-control study on the polymorphisms rs1057868 at POR, rs182420 at SULT2A1, and rs12086634 at HSD11B1. The study cohort comprised 73 prepubertal children with PA (defined by clinical signs) and 97 age- and gender-matched healthy controls from a Finnish Caucasian population. Genotype distributions and clinical and metabolic phenotypes were determined. The genotype distributions of the polymorphisms were similar between the study groups. No variant was associated with alterations in serum adrenal steroid concentrations. The minor C variant at SULT2A1 was associated with higher serum sex hormone binding globulin (SHBG) concentrations (T/T, n=64 vs T/C&C/C, n=33; mean 94 vs 116 nmol/L; P=.001) and a trend for lower dehydroepiandrosterone sulfate/dehydroepiandrosterone ratios in the controls (P=.06), and with higher plasma total cholesterol concentrations in the PA subjects (T/T, n=42 vs T/C&C/C, n=31; 4.0 vs 4.6 mmol/L; P<.001). The minor G variant at HSD11B1 was associated with lower plasma triglyceride concentration in the controls (T/T, n=65 vs T/G&G/G, n=32; 0.61 vs 0.49 mmol/L; P=.013). Common polymorphisms at POR, SULT2A1 or HSD11B1 were not associated with PA in a Finnish Caucasian population. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. The POR rs1057868–rs2868177 GC-GT diplotype is associated with high tacrolimus concentrations in early post-renal transplant recipients

    PubMed Central

    Liu, Shu; Chen, Rong-xin; Li, Jun; Zhang, Yu; Wang, Xue-ding; Fu, Qian; Chen, Ling-yan; Liu, Xiao-man; Huang, Hong-bing; Huang, Min; Wang, Chang-xi; Li, Jia-li

    2016-01-01

    Aim: Cytochrome P450 oxidoreductase (POR) is the only flavoprotein that donates electrons to all microsomal P450 enzymes (CYP), and several POR SNPs have been shown to be important contributors to altered CYP activity or CYP-mediated drug metabolism. In this study we examined the association between 6 POR SNPs and tacrolimus concentrations in Chinese renal transplant recipients. Methods: A total of 154 renal transplant recipients were enrolled. Genotyping of CYP3A5*3 and 6 POR SNPs was performed. All patients received a triple immunosuppressive regimen comprising tacrolimus, mycophenolate mofetil and prednisone. Dose-adjusted tacrolimus trough concentrations were obtained on d 7 (C0D7/D) after transplantation when steady-state concentration of tacrolimus was achieved (dosage had been unchanged for more than 3 d). Results: Tacrolimus C0D7/D in CYP3A5*3/*3/ POR rs1057868–rs2868177 GC-GT diplotype carriers was 1.62- and 2.72-fold higher than those in CYP3A5*3/*3/ POR rs1057868–rs2868177 GC-GT diplotype non-carriers and CYP3A5*1 carriers (220.17±48.09 vs 135.69±6.86 and 80.84±5.27 ng/mL/mg/kg, respectively, P<0.0001). Of CYP3A5*3/*3/ POR rs1057868-rs2868177GC-GT diplotype carriers, 85.71% exceeded the upper limit of the target range (8 ng/mL), which was also significantly higher compared with the latter two groups (14.29% and 0.00%, respectively, P<0.0001). The CYP3A5*3 and POR rs1057868–rs2868177 GC-GT diplotype explained 31.7% and 5.7%, respectively, of the inter-individual variability of tacrolimus C0D7/D, whereas the POR rs1057868–rs2868177 GC-GT diplotype could explain 10.9% of the inter-individual variability of tacrolimus C0D7/D in CYP3A5 non-expressers. Conclusion: The CYP3A5*3 and POR rs1057868–rs2868177 GC-GT diplotype accounted for the inter-individual variation of tacrolimus C0D7/D. Genotyping of POR rs1057868–rs2868177 diplotypes would help to differentiate initial tacrolimus dose requirements and to achieve early target C0 ranges in Chinese

  19. The POR rs1057868-rs2868177 GC-GT diplotype is associated with high tacrolimus concentrations in early post-renal transplant recipients.

    PubMed

    Liu, Shu; Chen, Rong-Xin; Li, Jun; Zhang, Yu; Wang, Xue-Ding; Fu, Qian; Chen, Ling-Yan; Liu, Xiao-Man; Huang, Hong-Bing; Huang, Min; Wang, Chang-Xi; Li, Jia-Li

    2016-09-01

    Cytochrome P450 oxidoreductase (POR) is the only flavoprotein that donates electrons to all microsomal P450 enzymes (CYP), and several POR SNPs have been shown to be important contributors to altered CYP activity or CYP-mediated drug metabolism. In this study we examined the association between 6 POR SNPs and tacrolimus concentrations in Chinese renal transplant recipients. A total of 154 renal transplant recipients were enrolled. Genotyping of CYP3A5*3 and 6 POR SNPs was performed. All patients received a triple immunosuppressive regimen comprising tacrolimus, mycophenolate mofetil and prednisone. Dose-adjusted tacrolimus trough concentrations were obtained on d 7 (C0D7/D) after transplantation when steady-state concentration of tacrolimus was achieved (dosage had been unchanged for more than 3 d). Tacrolimus C0D7/D in CYP3A5*3/*3/ POR rs1057868-rs2868177 GC-GT diplotype carriers was 1.62- and 2.72-fold higher than those in CYP3A5*3/*3/ POR rs1057868-rs2868177 GC-GT diplotype non-carriers and CYP3A5*1 carriers (220.17±48.09 vs 135.69±6.86 and 80.84±5.27 ng/mL/mg/kg, respectively, P<0.0001). Of CYP3A5*3/*3/ POR rs1057868-rs2868177GC-GT diplotype carriers, 85.71% exceeded the upper limit of the target range (8 ng/mL), which was also significantly higher compared with the latter two groups (14.29% and 0.00%, respectively, P<0.0001). The CYP3A5*3 and POR rs1057868-rs2868177 GC-GT diplotype explained 31.7% and 5.7%, respectively, of the inter-individual variability of tacrolimus C0D7/D, whereas the POR rs1057868-rs2868177 GC-GT diplotype could explain 10.9% of the inter-individual variability of tacrolimus C0D7/D in CYP3A5 non-expressers. The CYP3A5*3 and POR rs1057868-rs2868177 GC-GT diplotype accounted for the inter-individual variation of tacrolimus C0D7/D. Genotyping of POR rs1057868-rs2868177 diplotypes would help to differentiate initial tacrolimus dose requirements and to achieve early target C0 ranges in Chinese renal transplant recipients.

  20. Clinical Neisseria gonorrhoeae isolate with a N. meningitidis porA gene and no prolyliminopeptidase activity, Sweden, 2011: danger of false-negative genetic and culture diagnostic results.

    PubMed

    Golparian, D; Johansson, E; Unemo, M

    2012-03-01

    We describe a Neisseria gonorrhoeae strain, found in Sweden in 2011, that harbours a N. meningitidis porA gene causing false-negative results in PCRs targeting the gonococcal porA pseudogene. Furthermore, the strain had no prolyliminopeptidase (PIP) activity that many commercial biochemical kits for species verification in culture rely on. Enhanced awareness of the spread of such strains and screening for them can be crucial.

  1. Monitoring Shifts in the Conformation Equilibrium of the Membrane Protein Cytochrome P450 Reductase (POR) in Nanodiscs*

    PubMed Central

    Wadsäter, Maria; Laursen, Tomas; Singha, Aparajita; Hatzakis, Nikos S.; Stamou, Dimitrios; Barker, Robert; Mortensen, Kell; Feidenhans'l, Robert; Møller, Birger Lindberg; Cárdenas, Marité

    2012-01-01

    Nanodiscs are self-assembled ∼50-nm2 patches of lipid bilayers stabilized by amphipathic belt proteins. We demonstrate that a well ordered dense film of nanodiscs serves for non-destructive, label-free studies of isolated membrane proteins in a native like environment using neutron reflectometry (NR). This method exceeds studies of membrane proteins in vesicle or supported lipid bilayer because membrane proteins can be selectively adsorbed with controlled orientation. As a proof of concept, the mechanism of action of the membrane-anchored cytochrome P450 reductase (POR) is studied here. This enzyme is responsible for catalyzing the transfer of electrons from NADPH to cytochrome P450s and thus is a key enzyme in the biosynthesis of numerous primary and secondary metabolites in plants. Neutron reflectometry shows a coexistence of two different POR conformations, a compact and an extended form with a thickness of 44 and 79 Å, respectively. Upon complete reduction by NADPH, the conformational equilibrium shifts toward the compact form protecting the reduced FMN cofactor from engaging in unspecific electron transfer reaction. PMID:22891242

  2. Monitoring shifts in the conformation equilibrium of the membrane protein cytochrome P450 reductase (POR) in nanodiscs.

    PubMed

    Wadsäter, Maria; Laursen, Tomas; Singha, Aparajita; Hatzakis, Nikos S; Stamou, Dimitrios; Barker, Robert; Mortensen, Kell; Feidenhans'l, Robert; Møller, Birger Lindberg; Cárdenas, Marité

    2012-10-05

    Nanodiscs are self-assembled ∼50-nm(2) patches of lipid bilayers stabilized by amphipathic belt proteins. We demonstrate that a well ordered dense film of nanodiscs serves for non-destructive, label-free studies of isolated membrane proteins in a native like environment using neutron reflectometry (NR). This method exceeds studies of membrane proteins in vesicle or supported lipid bilayer because membrane proteins can be selectively adsorbed with controlled orientation. As a proof of concept, the mechanism of action of the membrane-anchored cytochrome P450 reductase (POR) is studied here. This enzyme is responsible for catalyzing the transfer of electrons from NADPH to cytochrome P450s and thus is a key enzyme in the biosynthesis of numerous primary and secondary metabolites in plants. Neutron reflectometry shows a coexistence of two different POR conformations, a compact and an extended form with a thickness of 44 and 79 Å, respectively. Upon complete reduction by NADPH, the conformational equilibrium shifts toward the compact form protecting the reduced FMN cofactor from engaging in unspecific electron transfer reaction.

  3. Structural and functional probing of PorZ, an essential bacterial surface component of the type-IX secretion system of human oral-microbiomic Porphyromonas gingivalis.

    PubMed Central

    Lasica, Anna M.; Goulas, Theodoros; Mizgalska, Danuta; Zhou, Xiaoyan; de Diego, Iñaki; Ksiazek, Mirosław; Madej, Mariusz; Guo, Yonghua; Guevara, Tibisay; Nowak, Magdalena; Potempa, Barbara; Goel, Apoorv; Sztukowska, Maryta; Prabhakar, Apurva T.; Bzowska, Monika; Widziolek, Magdalena; Thøgersen, Ida B.; Enghild, Jan J.; Simonian, Mary; Kulczyk, Arkadiusz W.; Nguyen, Ky-Anh; Potempa, Jan; Gomis-Rüth, F. Xavier

    2016-01-01

    Porphyromonas gingivalis is a member of the human oral microbiome abundant in dysbiosis and implicated in the pathogenesis of periodontal (gum) disease. It employs a newly described type-IX secretion system (T9SS) for secretion of virulence factors. Cargo proteins destined for secretion through T9SS carry a recognition signal in the conserved C-terminal domain (CTD), which is removed by sortase PorU during translocation. Here, we identified a novel component of T9SS, PorZ, which is essential for surface exposure of PorU and posttranslational modification of T9SS cargo proteins. These include maturation of enzyme precursors, CTD removal and attachment of anionic lipopolysaccharide for anchorage in the outer membrane. The crystal structure of PorZ revealed two β-propeller domains and a C-terminal β-sandwich domain, which conforms to the canonical CTD architecture. We further documented that PorZ is itself transported to the cell surface via T9SS as a full-length protein with its CTD intact, independently of the presence or activity of PorU. Taken together, our results shed light on the architecture and possible function of a novel component of the T9SS. Knowledge of how T9SS operates will contribute to our understanding of protein secretion as part of host-microbiome interactions by dysbiotic members of the human oral cavity. PMID:27883039

  4. Influence of various polymorphic variants of cytochrome P450 oxidoreductase (POR) on drug metabolic activity of CYP3A4 and CYP2B6.

    PubMed

    Chen, Xuan; Pan, Li Qiang; Naranmandura, Hua; Zeng, Su; Chen, Shu Qing

    2012-01-01

    Cytochrome P450 oxidoreductase (POR) is known as the sole electron donor in the metabolism of drugs by cytochrome P450 (CYP) enzymes in human. However, little is known about the effect of polymorphic variants of POR on drug metabolic activities of CYP3A4 and CYP2B6. In order to better understand the mechanism of the activity of CYPs affected by polymorphic variants of POR, six full-length mutants of POR (e.g., Y181D, A287P, K49N, A115V, S244C and G413S) were designed and then co-expressed with CYP3A4 and CYP2B6 in the baculovirus-Sf9 insect cells to determine their kinetic parameters. Surprisingly, both mutants, Y181D and A287P in POR completely inhibited the CYP3A4 activity with testosterone, while the catalytic activity of CYP2B6 with bupropion was reduced to approximately ~70% of wild-type activity by Y181D and A287P mutations. In addition, the mutant K49N of POR increased the CLint (Vmax/Km) of CYP3A4 up to more than 31% of wild-type, while it reduced the catalytic efficiency of CYP2B6 to 74% of wild-type. Moreover, CLint values of CYP3A4-POR (A115V, G413S) were increased up to 36% and 65% of wild-type respectively. However, there were no appreciable effects observed by the remaining two mutants of POR (i.e., A115V and G413S) on activities of CYP2B6. In conclusion, the extent to which the catalytic activities of CYP were altered did not only depend on the specific POR mutations but also on the isoforms of different CYP redox partners. Thereby, we proposed that the POR-mutant patients should be carefully monitored for the activity of CYP3A4 and CYP2B6 on the prescribed medication.

  5. Influence of Various Polymorphic Variants of Cytochrome P450 Oxidoreductase (POR) on Drug Metabolic Activity of CYP3A4 and CYP2B6

    PubMed Central

    Naranmandura, Hua; Zeng, Su; Chen, Shu Qing

    2012-01-01

    Cytochrome P450 oxidoreductase (POR) is known as the sole electron donor in the metabolism of drugs by cytochrome P450 (CYP) enzymes in human. However, little is known about the effect of polymorphic variants of POR on drug metabolic activities of CYP3A4 and CYP2B6. In order to better understand the mechanism of the activity of CYPs affected by polymorphic variants of POR, six full-length mutants of POR (e.g., Y181D, A287P, K49N, A115V, S244C and G413S) were designed and then co-expressed with CYP3A4 and CYP2B6 in the baculovirus-Sf9 insect cells to determine their kinetic parameters. Surprisingly, both mutants, Y181D and A287P in POR completely inhibited the CYP3A4 activity with testosterone, while the catalytic activity of CYP2B6 with bupropion was reduced to approximately ∼70% of wild-type activity by Y181D and A287P mutations. In addition, the mutant K49N of POR increased the CLint (Vmax/Km) of CYP3A4 up to more than 31% of wild-type, while it reduced the catalytic efficiency of CYP2B6 to 74% of wild-type. Moreover, CLint values of CYP3A4-POR (A115V, G413S) were increased up to 36% and 65% of wild-type respectively. However, there were no appreciable effects observed by the remaining two mutants of POR (i.e., A115V and G413S) on activities of CYP2B6. In conclusion, the extent to which the catalytic activities of CYP were altered did not only depend on the specific POR mutations but also on the isoforms of different CYP redox partners. Thereby, we proposed that the POR-mutant patients should be carefully monitored for the activity of CYP3A4 and CYP2B6 on the prescribed medication. PMID:22719896

  6. Análise dos Conceitos Astronômicos Apresentados por Professores de Algumas Escolas Estaduais Brasileiras

    NASA Astrophysics Data System (ADS)

    Voelzke, Marcos Rincon; Gonzaga, Edson Pereira

    2011-12-01

    A razão para o desenvolvimento deste trabalho baseia-se no fato de que muitos professores da Educação Básica (EB) não lidam com conceitos relacionados à astronomia, e quando o fazem eles simplesmente seguem livros didáticos que podem conter erros conceituais. Como é de conhecimento geral a astronomia é um dos conteúdos a serem ensinados na EB fazendo parte dos Parâmetros Curriculares Nacionais e das Propostas Curriculares do Estado de São Paulo, mas é um fato, que vários pesquisadores apontam, a existência de muitos problemas no ensino da astronomia. Com o propósito de minimizar algumas dessas deficiências foi realizado um trabalho de pesquisa com a utilização de questionários pré e pós pesquisa, para tanto foi desenvolvido um Curso de Extensão Universitária para professores da Diretoria de Ensino Regional (DE) que abrange Mauá, Ribeirão Pires e Rio Grande da Serra (no Estado de São Paulo) com os seguintes objetivos: levantar concepções alternativas; subsidiar os professores por meio de palestras, debates e workshops, e verificar o sucesso da aprendizagem após o curso, adotando-se como referência, para a análise dos resultados, os dicionários de Língua Portuguesa (FERREIRA, 2004) e Enciclopédico de Astronomia e Astronáutica (MOURĀO, 1995). Portanto, dezesseis questões foram aplicadas antes e após o curso, assim pode-se verificar após a pesquisa que 100,0% dos professores sabiam os nomes das fases da Lua, 97,0% entenderam que o Sistema Solar é composto por oito planetas, 78,1% foram capazes de explicar como ocorre um eclipse lunar, um eclipse solar e um solstício, 72,7% sabiam como explicar a ocorrência das estações do ano; 64,5% explicaram corretamente a ocorrência do equinócio, 89,7% foram capazes de definir adequadamente o termo cometa; 63,6% definiram asteróide, 54,5% meteoro, 58,1% galáxia, e 42,4% planeta. Os resultados obtidos indicam uma aprendizagem significativa por parte dos participantes.

  7. The genetic polymorphisms of POR*28 and CYP3A5*3 significantly influence the pharmacokinetics of tacrolimus in Chinese renal transplant recipients.

    PubMed

    Zhang, Jing-Jing; Liu, Shuai-Bing; Xue, Ling; Ding, Xiao-Liang; Zhang, Hua; Miao, Li-Yan

    2015-09-01

    The aims of this study were to assess the influence of the polymorphism of cytochrome P450 oxidoreductase (POR) as well as other relevant genes (CYP3A4, CYP3A5, ABCB1) on individual variability of tacrolimus pharmacokinetics and perform population pharmacokinetic analysis of tacrolimus in Chinese renal transplant recipients. Tacrolimus trough whole blood concentrations and clinical details were retrospectively collected from 83 renal recipients. CYP3A4*1G, CYP3A5*3, and ABCB1 C3435T were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), POR*28 and CYP3A4*22 were genotyped by sequencing method. Population pharmacokinetic analysis was performed using NONMEM program. The significant influences of CYP3A5*3, CYP3A4*1G, and POR*28 polymorphisms on tacrolimus dose-adjusted trough concentrations (C0/D) were observed in 83 renal recipients. Subgroup analysis showed that POR*28 polymorphisms significantly decreased tacrolimus C0/D by 1.50 - 1.84-fold (p < 0.05) in patients who were CYP3A5 expressers (CYP3A5*1 carriers, n = 46), while similar results could not be obtained from CYP3A5 non-expressers (CYP3A5*3/*3 carriers, n = 37). Additionally, population pharmacokinetic analysis identified that the combined genotype of CYP3A5-POR was the only covariant for the apparent clearance of tacrolimus (CL/F). The study demonstrated that the POR*28 C>T mutation could decrease the C0/D of tacrolimus in renal recipients who were CYP3A5 expressers. The population pharmacokinetic model showed that the combined genotype of CYP3A5-POR was associated with the CL/F of tacrolimus which might provide references for personalized use of tacrolimus in clinic.

  8. Reactions of nitrogen oxides with the five-coordinate Fe(III)(porphyrin) nitrito intermediate Fe(Por)(ONO) in sublimed solids.

    PubMed

    Kurtikyan, Tigran S; Hovhannisyan, Astghik A; Hakobyan, Manya E; Patterson, James C; Iretskii, Alexei; Ford, Peter C

    2007-03-28

    Detailed experimental studies are described for reactions of several nitrogen oxides with iron porphyrin models for heme/NxOy systems. It is shown by FTIR and optical spectroscopy and by isotope labeling experiments that reaction of small increments of NO2 with sublimed thin layers of the iron(II) complex Fe(Por) (Por = meso-tetraphenylporphyrinato dianion, TPP, or meso-tetra-p-tolylporphyrinato dianion, TTP) leads to formation of the 5-coordinate nitrito complexes Fe(Por)(eta1-ONO) (1), which are fairly stable but very slowly decompose under vacuum giving mostly the corresponding nitrosyl complexes Fe(Por)(NO). Further reaction of 1 with new NO2 increments leads to formation of the nitrato complex Fe(Por)(eta2-O2NO) (2). The interaction of NO with 1 at low temperature involves ligand addition to give the nitrito-nitrosyl complexes Fe(Por)(eta1-ONO)(NO) (3); however, these isomerize to the nitro-nitrosyl analogs Fe(Por)(eta1-NO2)(NO) (4) upon warming. Experiments with labeled nitrogen oxides argue for an intramolecular isomerization ("flipping") mechanism rather than one involving dissociation and rebinding of NO2. The Fe(III) centers in the 6-coordinate species 3 and 4 are low spin in contrast to 1, which appears to be high-spin, although DFT computations of the porphinato models Fe(P)(nitrite) suggest that the doublet nitro species and the quartet and sextet nitrito complexes are all relatively close in energy. The nitro-nitrosyl complex 4 is stable under an NO atmosphere but decomposes under intense pumping to give a mixture of the ferrous nitrosyl complex Fe(Por)(NO) and the ferric nitrito complex Fe(Por)(eta1-ONO) indicating the competitive dissociation of NO and NO2. Hence, loss of NO from 4 is accompanied with nitro --> nitrito isomerization consistent with 1 being the more stable of the 5-coordinate NO2 complexes of iron porphyrins.

  9. Crystallization and preliminary X-ray analysis of the C-terminal fragment of PorM, a subunit of the Porphyromonas gingivalis type IX secretion system

    PubMed Central

    Stathopulos, Julien; Cambillau, Christian; Cascales, Eric; Roussel, Alain; Leone, Philippe

    2015-01-01

    PorM is a membrane protein involved in the assembly of the type IX secretion system (T9SS) from Porphyromonas gingivalis, a major bacterial pathogen responsible for periodontal disease in humans. The periplasmic domain of PorM was overexpressed in Escherichia coli and purified. A fragment of the purified protein was obtained by limited proteolysis. Crystals of this fragment belonged to the tetragonal space group P43212. Native and MAD data sets were recorded to 2.85 and 3.1 Å resolution, respectively, using synchrotron radiation. PMID:25615973

  10. Most but not all laboratories can detect the recently emerged Neisseria gonorrhoeae porA mutants - results from the QCMD 2013 N. gonorrhoeae external quality assessment programme.

    PubMed

    Luijt, D; Di Lorenzo, C; van Loon, A M; Unemo, M

    2014-02-27

    We describe the results of the Quality Control for Molecular Diagnostics 2013 Neisseria gonorrhoeae external quality assessment programme that included an N. gonorrhoeae strain harbouring an N. meningitidis porA gene which causes false-negative results in molecular diagnostic assays targeting the gonococcal porA pseudogene. Enhanced awareness of the international transmission of such gonococcal strains is needed to avoid false-negative results in both in-house and commercial molecular diagnostic assays used in laboratories worldwide, but particularly in Europe.

  11. "Estudio tribologico de aceros para moldes. Aplicacion al moldeo por inyeccion de polibutilentereftalato reforzado con fibra de vidrio"

    NASA Astrophysics Data System (ADS)

    Martinez Mateo, Isidoro Jose

    Mould materials for injection moulding of polymers and polymer-matrix composites represent a relevant industrial economic sector due to the large quantity of pieces and components processed. The material selection for mould manufacturing, its composition and heat treatment, the hardening procedures and machining and finishing processes determine the service performance and life of the mould. In the first part of the present study, the relationship between the hardness and microstructure and the wear resistance of mould steels from large blocks has been studied by pin-on-disc tests, studying the main wear mechanisms. In order to determine the surface damage on mould steels under real injection conditions, different commercial steels have been studied by measuring the variation of surface roughness with the number of injected pieces with different reinforcement percentages and different mould geometries, by using optical profilometry and scanning electron microscopy techniques. It was important to determine the variation of surface roughness of the moulded pieces with the number of injection operations. The materials used were polybutyleneterephthalate pure and reinforced with either 20% or 50% glass fibre. For the different mould designs, the evolution of the glass fibre orientation with injection flow has been determined by image analysis and related to roughness changes and surface damage, both of the composite parts and of the mould steel surface. Finally, the abrasion resistance of the composite parts has been studied by scratch tests as a function of the number of injected parts and of the scratch direction with respect to injection flow and glass fibre orientation. Los materiales para moldes de inyeccion de polimeros y materiales compuestos representan un sector economicamente muy relevante debido al gran aumento del numero de componentes fabricados a partir de materiales polimericos obtenidos mediante moldeo por inyeccion. La seleccion del material para la

  12. Palivizumab outcomes registry data from Spain: Infección Respiratoria Infantil por Virus Respiratorio Sincitial (IRIS) Study Group.

    PubMed

    Carbonell-Estrany, Xavier

    2003-02-01

    Respiratory syncytial virus (RSV) is the leading cause of lower respiratory illness in children <2 years of age. Severe RSV infection requiring hospitalization is linked to gestational age, chronic cardiopulmonary conditions and immunosuppression. The Infección Respiratoria Infantil por Virus Respiratorio Sincitial (IRIS) Study group in Spain conducted two pivotal epidemiologic studies establishing that serious RSV illness among premature infants was responsible for high rehospitalization rates (approximately 13%). RSV lower respiratory tract illness also correlated with prolonged hospital stay and more intensive care unit admissions. In Europe recent availability of palivizumab, a humanized monoclonal antibody to RSV, is a major therapeutic advancement directed against prevention of lower respiratory tract infection secondary to this viral pathogen. To ensure proper and optimal usage of palivizumab, the IRIS group, in conjunction with the Spanish Neonatology Group, developed prophylaxis guidelines for neonates. Palivizumab prophylaxis is strongly recommended in premature infants < or =28 weeks gestation or those affected with chronic lung disease. Additionally, palivizumab is recommended for infants with a gestational age of 29 to 32 weeks, without evidence of chronic lung disease and who are <6 months old at the onset of the RSV season. It was thought that slightly older premature infants (33 to 35 weeks gestational age) should be assessed on an individual basis to determine whether prophylaxis is warranted. The IRIS Study Group is currently determining the effectiveness of these recommendations by measuring the incidence of RSV-related hospital admissions in infants born at < or =32 weeks gestational age who are receiving palivizumab prophylaxis.

  13. Community-based education in nutrition and cancer: the Por La Vida Cuidándome curriculum.

    PubMed

    Navarro, A M; Rock, C L; McNicholas, L J; Senn, K L; Moreno, C

    2000-01-01

    The Por La Vida (PLV) intervention model relies on community lay health advisors trained to conduct education sessions among members of their existing social networks. PLV Cuidándome was funded by the NCI to develop, implement, and evaluate the PLV model with respect to nutrition and cancer prevention, as well as early detection of breast and cervical cancers. The target population is the Latino community, for which substantial barriers to health care access exist. This article presents the curriculum that guides the sessions and describes its development, which was based on semi-structured interviews with Latina lay-health community workers to explore relevant attitudes and behaviors. Also key to the process was the work of the educational materials committee, whose members offered community representation as well as expertise in nutritional sciences, educational technologies, and community-based health promotion interventions and research. The 12-session curriculum's goal was to increase both the variety and the quality of fruits and/or vegetables consumed. It included information about consumption of fiber and fat in the importance of balance between energy intake and physical activity. The program has been well received. An ongoing study examines how it enhances nutrition and cancer prevention.

  14. Avidity maturation following vaccination with a meningococcal recombinant hexavalent PorA OMV vaccine in UK infants.

    PubMed

    Longworth, Emma; Borrow, Ray; Goldblatt, David; Balmer, Paul; Dawson, Maureen; Andrews, Nick; Miller, Elizabeth; Cartwright, Keith

    2002-06-07

    To date, there are no data assessing the utility of avidity indices as a surrogate marker for the induction of immunological memory following meningococcal serogroup B outer membrane vesicle (OMV) vaccination. We studied infants who had been immunized with three doses of a recombinant hexavalent PorA OMV vaccine at ages 2-4 months, together with a fourth dose at age 12-18 months. A control group had received a single dose of the same vaccine at age 12-18 months. As previously reported, serum bactericidal antibody (SBA) titres increased after each of the first three doses, with a significant increase observed from 6 months post third dose to 1 month post fourth dose. The geometric mean avidity indices (GMAI), against strain H44/76 OMVs, increased from 1 month post first dose to 1 month post third dose. Significant increases in GMAI were observed at 6 months post third dose and again following the fourth dose. At 32-42 months of age, though the SBA titres had returned to post first dose levels, the GMAI remained elevated. No increase in avidity was observed in the control group. Antibody avidity indices are useful laboratory markers for the priming of immunological memory following vaccination with meningococcal serogroup B OMV vaccines.

  15. Spectroscopic and kinetic characterization of the light-dependent enzyme protochlorophyllide oxidoreductase (POR) using monovinyl and divinyl substrates.

    PubMed

    Heyes, Derren J; Kruk, Jerzy; Hunter, C Neil

    2006-02-15

    The enzyme POR [Pchlide (protochlorophyllide) oxidoreductase] catalyses the reduction of Pchlide to chlorophyllide, which is a key step in the chlorophyll biosynthesis pathway. This light-dependent reaction has previously been studied in great detail but recent reports suggest that a mixture of MV (monovinyl) and DV (divinyl) Pchlides may have influenced some of these properties of the reaction. Low-temperature absorbance and fluorescence spectroscopy have revealed several spectral differences between MV and DV Pchlides, which were purified from a Rhodobacter capsulatus strain that was shown to contain a mixture of the two pigments. A thorough steady-state kinetic characterization using both Pchlide forms demonstrates that neither pigment appears to affect the kinetic properties of the enzyme. The reaction has also been monitored following illumination at low temperatures and was shown to consist of an initial photochemical step followed by four 'dark' steps for both pigments. However, minor differences were observed in the spectral properties of some of the intermediates, although the temperature dependency of each step was nearly identical for the two pigments. This work provides the first detailed kinetic and spectroscopic study of this unique enzyme using biologically important MV and DV substrate analogues. It also has significant implications for the DV reductase enzyme, which is responsible for converting DV pigments into their MV counterparts, and its position in the sequence of reactions that comprise the chlorophyll biosynthesis pathway.

  16. Examination of P-OR bridging bond orders in phosphate monoesters using (18)O isotope shifts in 31P NMR.

    PubMed

    Sorensen-Stowell, Kerensa; Hengge, Alvan C

    2005-06-10

    Evidence indicates that phosphate monoesters undergo hydrolysis by a loose transition state with extensive bond fission to the leaving group. It has been proposed that part of the high dependence of the rate on the leaving group pKa (betalg = -1.23) arises from weaker ester bonds in the reactants as the pKa of the leaving group decreases, on the basis of X-ray structures and calculations. In contrast, IR and Raman studies suggest that the leaving group has little effect on the length of the P-OR bridging bond in solution. To gather additional data on this issue, we have used (18)O isotopic shifts in 31P NMR to monitor the bond order of P-O bonds in a range of phosphate esters with different leaving groups. Using this technique, we have been able to evaluate whether significant changes are observed in the P-O bond orders for the bridging and nonbridging positions of methyl, ethyl, phenethyl, propargyl, phenyl, and p-nitrophenyl phosphate using [(16)O(18)O] labeled species in deuterium oxide. The results indicate that the bridging and nonbridging bond orders to phosphorus in phosphate monoesters are not significantly altered by differences in the pKa of the leaving group or by the counterion of the phosphate ester dianion.

  17. P(O)R2-directed Pd-catalyzed C-H functionalization of biaryl derivatives to synthesize chiral phosphorous ligands.

    PubMed

    Hu, Rong-Bin; Wang, Hong-Li; Zhang, Hong-Yu; Zhang, Heng; Ma, Yan-Na; Yang, Shang-Dong

    2014-01-01

    Chiral phosphorus ligands have been widely used in transition metal-catalyzed asymmetric reactions. Herein, we report a new synthesis approach of chiral biaryls containing a phosphorus moiety using P(O)R2-directed Pd-catalyzed C-H activation; the functionalized products are produced with good enantioselectivity.

  18. Interaction between variants in the CYP2C9 and POR genes and the risk of sulfonylurea-induced hypoglycaemia: A GoDARTS Study.

    PubMed

    Dujic, Tanja; Zhou, Kaixin; Donnelly, Louise A; Leese, Graham; Palmer, Colin N A; Pearson, Ewan R

    2017-06-28

    Data on the association of CYP2C9 genetic polymorphisms with sulfonylurea (SU)-induced hypoglycaemia (SH) are inconsistent. Recent studies showed that variants in the P450 oxidoreductase (POR) gene could affect CYP2C9 activity. In this study, we explored the effects of POR*28 and combined CYP2C9*2 and CYP2C9*3 genotypes on SH and the efficacy of SU treatment in type 2 diabetes. A total of 1770 patients were included in the analysis of SU efficacy, assessed as the combined outcome of the HbA1c reduction and the prescribed SU daily dose. Sixty-nine patients with severe SH were compared with 311 control patients. The number of CYP2C9 deficient alleles was associated with nearly three-fold higher odds of hypoglycaemia (OR, 2.81; 95% CI, 1.30-6.09; P = .009) and better response to SU treatment (β, -0.218; SE, 0.074; P = .003) only in patients carrying the POR*1/*1 genotype. Our results indicate that interaction between CYP2C9 and POR genes may be an important determinant of efficacy and severe adverse effects of SU treatment. © 2017 The Authors. Diabetes, Obesity and Metabolism published by John Wiley & Sons Ltd.

  19. Adult Student Retention and Achievement with Language-Based Modular Materials. POR FIN: Program Organizing Related Family Instruction in the Neighborhood.

    ERIC Educational Resources Information Center

    Bexar County School Board, San Antonio, TX.

    The goal of the POR FIN research design was to develop a language-based curriculum emphasizing the audiolingual approach and integrating academic and social-functioning subject matter. The modular curriculum is designed so that each lesson is independent and complete in itself, and provides a high degree of motivation, retention, and achievement…

  20. El proceso hacia la integracion de la equidad por genero al curriculo.(The Process of the Integration of Gender Equity in the Curriculum.)

    ERIC Educational Resources Information Center

    Rivera-Bermudez, Carmen D.

    "El Proyecto Colaborativo de Equidad por Genero en la Educacion," or the Collaborative Project for Gender Equity in Education, was undertaken in Puerto Rico between 1990 and 1992 to study how to facilitate the integration of gender equity themes in the curriculum through the direct action of participating teachers. A study examined the…

  1. Recombinant PorA, the major outer membrane protein of Campylobacter jejuni, provides heterologous protection in an adult mouse intestinal colonization model.

    PubMed

    Islam, Anjum; Raghupathy, Raj; Albert, M John

    2010-11-01

    Immunity against Campylobacter jejuni, a major food-borne pathogen causing diarrhea, is largely serotype specific. The major outer membrane protein (MOMP) of C. jejuni, PorA, is a common antigen with the potential to provide broad protection. Adult BALB/c mice were orally immunized with a recombinant glutathione S-transferase (GST) fused to PorA prepared from Campylobacter jejuni C31 (O:6,7) (GST-PorA) combined with a modified heat-labile enterotoxin of Escherichia coli as an adjuvant and later orally challenged with C31 strain or three heterologous strains: 48 (O:19), 75 (O:3), and 111 (O:1,44). Protection from colonization with the challenge organism was studied by fecal screening daily for 9 days. Serum and intestinal lavage fluid antibodies against the vaccine and Sarkosyl-purified MOMP from C31 were measured by using an enzyme-linked immunosorbent assay. The vaccine produced robust antibody responses against both antigens in serum and secretion. Since strain C31 was a poor colonizer, homologous protection could not be studied. The protective efficacies of heterologous strains were 43% (for strain 48, P < 0.001), 29% (for strain 75, P < 0.005), and 42% (for strain 111, P < 0.001) for the 9-day period compared to control mice given phosphate-buffered saline. Thus, PorA provided appreciable protection against colonization with heterologous serotypes.

  2. Cell Growth Defect Factor1/CHAPERONE-LIKE PROTEIN OF POR1 Plays a Role in Stabilization of Light-Dependent Protochlorophyllide Oxidoreductase in Nicotiana benthamiana and Arabidopsis[C][W

    PubMed Central

    Lee, Jae-Yong; Lee, Ho-Seok; Song, Ji-Young; Jung, Young Jun; Reinbothe, Steffen; Park, Youn-Il; Lee, Sang Yeol; Pai, Hyun-Sook

    2013-01-01

    Angiosperms require light for chlorophyll biosynthesis because one reaction in the pathway, the reduction of protochlorophyllide (Pchlide) to chlorophyllide, is catalyzed by the light-dependent protochlorophyllide oxidoreductase (POR). Here, we report that Cell growth defect factor1 (Cdf1), renamed here as CHAPERONE-LIKE PROTEIN OF POR1 (CPP1), an essential protein for chloroplast development, plays a role in the regulation of POR stability and function. Cdf1/CPP1 contains a J-like domain and three transmembrane domains, is localized in the thylakoid and envelope membranes, and interacts with POR isoforms in chloroplasts. CPP1 can stabilize POR proteins with its holdase chaperone activity. CPP1 deficiency results in diminished POR protein accumulation and defective chlorophyll synthesis, leading to photobleaching and growth inhibition of plants under light conditions. CPP1 depletion also causes reduced POR accumulation in etioplasts of dark-grown plants and as a result impairs the formation of prolamellar bodies, which subsequently affects chloroplast biogenesis upon illumination. Furthermore, in cyanobacteria, the CPP1 homolog critically regulates POR accumulation and chlorophyll synthesis under high-light conditions, in which the dark-operative Pchlide oxidoreductase is repressed by its oxygen sensitivity. These findings and the ubiquitous presence of CPP1 in oxygenic photosynthetic organisms suggest the conserved nature of CPP1 function in the regulation of POR. PMID:24151298

  3. The influence of CYP3A, PPARA, and POR genetic variants on the pharmacokinetics of tacrolimus and cyclosporine in renal transplant recipients.

    PubMed

    Lunde, Ingrid; Bremer, Sara; Midtvedt, Karsten; Mohebi, Beata; Dahl, Miriam; Bergan, Stein; Åsberg, Anders; Christensen, Hege

    2014-06-01

    Tacrolimus (Tac) and cyclosporine (CsA) are mainly metabolized by CYP3A4 and CYP3A5. Several studies have demonstrated an association between the CYP3A5 genotype and Tac dose requirements. Recently, CYP3A4, PPARA, and POR gene variants have been shown to influence CYP3A metabolism. The present study investigated potential associations between CYP3A5*3, CYP3A4*22, PPARA c.209- 1003G>A and c.208+3819A>G, and POR*28 alleles and dose-adjusted concentrations (C/D) of Tac and CsA in 177 renal transplant patients early post-transplant. All patients (n=177) were genotyped for CYP3A4*22, CYP3A5*3, POR*28, PPARA c.209-1003G>A, and PPARA c.208+3819A>G using real-time polymerase chain reaction (PCR) and melting curve analysis with allelespecific hybridization probes or PCR restriction fragment length polymorphisms (RFLP) methods. Drug concentrations and administered doses were retrospectively collected from patient charts at Oslo University Hospital, Rikshospitalet, Norway. One steady-state concentration was collected for each patient. We confirmed a significant impact of the CYP3A5*3 allele on Tac exposure. Patients with POR*28 and PPARA variant alleles demonstrated 15 % lower (P=0.04) and 19 % higher (P=0.01) Tac C0/D respectively. CsA C2/D was 53 % higher among CYP3A4*22 carriers (P=0.03). The results support the use of pre-transplant CYP3A5 genotyping to improve initial dosing of Tac, and suggest that Tac dosing may be further individualized by additional POR and PPARA genotyping. Furthermore, initial CsA dosing may be improved by pre-transplant CYP3A4*22 determination.

  4. Compound heterozygosity of a paternal submicroscopic deletion and a maternal missense mutation in POR gene: Antley-bixler syndrome phenotype in three sibling fetuses.

    PubMed

    Tzetis, Maria; Konstantinidou, Anastasia; Sofocleous, Christalena; Kosma, Konstantina; Mitrakos, Anastasios; Tzannatos, Christina; Kitsiou-Tzeli, Sofia

    2016-07-01

    Antley-Bixler syndrome (ABS) is an exceptionally rare craniosynostosis syndrome that can be accompanied by disordered steroidogenesis, and is mainly caused by mutations in the POR gene, inherited in an autosomal recessive manner. Here we report the prenatal and postmortem findings of three sibling fetuses with ABS as a result of compound heterozygosity of a paternal submicroscopic deletion and a maternal missense mutation in the POR gene. Prenatal ultrasound and postmortem examination were performed in three sibling fetuses with termination of pregnancy at 22, 23, and 17 weeks of gestation, respectively. Molecular analysis of fetus 2 and 3 included (a) bidirectional sequencing of exon 8 of the POR gene after amplification of the specific locus by polymerase chain reaction, to detect single nucleotide variants (SNVs) and (b) high resolution comparative genomic hybridization (CGH) positive single nucleotide polymorphism array CGH (aCGH) analysis to detect copy number variants (CNVs), copy neutral areas of loss of heterozygosity and uniparental disomy. The diagnosis of ABS was suggested by the postmortem examination findings. The combination of the POR gene molecular analysis and aCGH revealed a compound heterozygous genotype of a maternal SNV (p.A287P) and a paternal CNV (NC_000007.13:g.(?_75608488)_(75615534_?)del). To the best of our knowledge, these sibling fetuses add to the few reported cases of ABS, caused by a combination of a SNV and a CNV in the POR gene. The detailed description of the pathologic and radiographic findings of second trimester fetuses affected with ABS adds novel knowledge concerning the early ABS phenotype, in lack of previous relevant reports. Birth Defects Research (Part A) 106:536-541, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  5. Clinical safety evaluation of F(ab')₂ antivenom (Crotalus durissus-Bothrops asper) administration in dogs.

    PubMed

    Woods, Craig; Young, David

    2011-10-01

    Antivenoms consisting of selective antigen binding antibody fragments, or F(ab')(2), are becoming more popular in human and veterinary medicine, owing to their preferred kinetics, tissue distribution, and removal of the Fc binding portion of IgG. Consequences of antivenom administration can include acute and delayed reactions, dependent, in part, on the antivenom's donor source, purity, and composition. This study evaluated an equine-derived polyvalent F(ab')(2) pit viper antivenom in healthy dogs of various size, age, and breed under controlled conditions. Dogs were allocated into 6 treatment groups (n = 10 per group) based on weight (3 weight groups) and dose (2 dose groups) and administered F(ab')(2) antivenom over 1 hour by IV infusion. Dogs were observed for adverse events at 3, 6, 12, and 24 hours after administration and blood was collected for CBC and serum biochemistry before and at 24 hours postadministration. No abnormalities were noted in the 3-vial group. In the 6-vial group, 3 dogs developed mild self-limiting edema around the head or neck, suggestive of a type 1 hypersensitivity, while another dog vomited and developed mild hypocalcemia at 24 hours, reflecting a 13% reaction rate at high doses. However, no dogs exhibited clinical signs of hypocalcemia or had any severe adverse events. CBC remained normal in all groups. This study demonstrated that the polyvalent F(ab')(2) pit viper antivenom is well tolerated in dogs given large doses in a short period of time. © Veterinary Emergency and Critical Care Society 2011.

  6. Effects of a low-level semiconductor gallium arsenide laser on local pathological alterations induced by Bothrops moojeni snake venom.

    PubMed

    Aranha de Sousa, Elziliam; Bittencourt, José Adolfo Homobono Machado; Seabra de Oliveira, Nayana Keyla; Correia Henriques, Shayanne Vanessa; dos Santos Picanço, Leide Caroline; Lobato, Camila Pena; Ribeiro, José Renato; Pereira, Washington Luiz Assunção; Carvalho, José Carlos Tavares; da Silva, Jocivânia Oliveira

    2013-10-01

    Antivenom therapy has been ineffective in neutralizing the tissue damage caused by snakebites. Among therapeutic strategies to minimize effects after envenoming, it was hypothesized that a low level laser would reduce complications and reduce the severity of local snake venom effects. In the current study, the effect of a low-level semiconductor gallium arsenide (GaAs) laser on the local pathological alterations induced by B. moojeni snake venom was investigated. The experimental groups consisted of five male mice, each administered either B. moojeni venom (VB), B. moojeni venom + antivenom (VAV), B. moojeni venom + laser (VL), B. moojeni venom + antivenom + laser (VAVL), or sterile saline solution (SSS) alone. Paw oedema was induced by intradermal administration of 0.05 mg kg(-1) of B. moojeni venom and was expressed in mm of directly induced oedema. Mice received by subcutaneous route 0.20 mg kg(-1) of venom for evaluating nociceptive activity and the time (in seconds) spent in licking and biting the injected paw was taken as an indicator of pain response. Inflammatory infiltration was determined by counting the number of leukocytes present in the gastrocnemius muscle after venom injection (0.10 mg kg(-1)). For histological examination of myonecrosis, venom (0.10 mg kg(-1)) was administered intramuscularly. The site of venom injection was irradiated by the GaAs laser and some animals received antivenom intraperitoneally. The results indicated that GaAs laser irradiation can help in reducing some local effects produced by the B. moojeni venom in mice, stimulating phagocytosis, proliferation of myoblasts and the regeneration of muscle fibers.

  7. Estimaciones de Prevalencia del VIH por Género y Grupo de Riesgo en Tijuana, México: 2006

    PubMed Central

    Iñiguez-Stevens, Esmeralda; Brouwer, Kimberly C.; Hogg, Robert S.; Patterson, Thomas L.; Lozada, Remedios; Magis-Rodriguez, Carlos; Elder, John P.; Viani, Rolando M.; Strathdee, Steffanie A.

    2010-01-01

    OBJETIVO Estimar la prevalencia del VIH en adultos de 15-49 años de edad en Tijuana, México - en la población general y en subgrupos de riesgo en el 2006. METODOS Se obtuvieron datos demográficos del censo Mexicano del 2005, y la prevalencia del VIH se obtuvo de la literatura. Se construyó un modelo de prevalencia del VIH para la población general y de acuerdo al género. El análisis de sensibilidad consistió en estimar errores estándar del promedio-ponderado de la prevalencia del VIH y tomar derivados parciales con respecto a cada parámetro. RESULTADOS La prevalencia del VIH es 0.54%(N = 4,347) (Rango: 0.22%–0.86%, (N = 1,750–6,944)). Esto sugiere que 0.85%(Rango: 0.39%–1.31%) de los hombres y 0.22%(Rango: 0.04%–0.40%) de las mujeres podrían ser VIH-positivos. Los hombres que tienen sexo con hombres (HSH), las trabajadoras sexuales usuarias de drogas inyectables (MTS-UDI), MTS-noUDI, mujeres UDI, y los hombres UDI contribuyeron las proporciones más elevadas de personas infectadas por el VIH. CONCLUSIONES El número de adultos VIH-positivos entre subgrupos de riesgo en la población de Tijuana es considerable, marcando la necesidad de enforcar las intervenciones de prevención en sus necesidades específicas. El presente modelo estima que hasta 1 en cada 116 adultos podrían ser VIH-positivos. PMID:19685824

  8. Results of Mujeres Felices por ser Saludables: a dietary/breast health randomized clinical trial for Latino women.

    PubMed

    Fitzgibbon, Marian L; Gapstur, Susan M; Knight, Sara J

    2004-10-01

    Data are limited on the efficacy of health-focused interventions for young, low-acculturated Latino women. Because breast cancer is the most commonly diagnosed cancer and the most common cause of cancer mortality in this population, combined interventions that address both early detection and dietary patterns could help reduce both morbidity and mortality associated with breast cancer in this underserved population. Mujeres Felices por ser Saludables was randomized intervention study designed to assess the efficacy of an 8-month combined dietary and breast health intervention to reduce fat and increase fiber intake as well as to increase the frequency and proficiency of breast self-examination (BSE) and reduce anxiety related to BSE among Latinas. Blocked randomization in blocks of 6 was used to randomize 256 20- to 40-year-old Latinas to the intervention (n = 127) or control group (n = 129). The intervention group attended an 8-month multicomponent education program designed specifically for low-acculturated Latinas. The control group received mailed health education material on a schedule comparable to the intervention. A total of 195 women (76.2%) completed both the baseline and 8-month follow-up interviews. The intervention and control groups were similar on baseline sociodemographic characteristics. At the 8-month follow up, the intervention group reported lower dietary fat (P < .001) and higher fiber intake (p = .06); a higher proportion reported practicing BSE at the recommended interval (p < .001) and showed improved BSE proficiency (p < .001) compared to the control group. BSE-related anxiety was low for both groups at baseline, and no difference in reduction was observed. This project provides a successful model for achieving dietary change and improving breast health behavior in young, low-acculturated Latinas.

  9. Relación entre la orientación de Nebulosas Planetarias y el campo magnético galáctico

    NASA Astrophysics Data System (ADS)

    Weidmann, W.; Díaz, R. J.; Carranza, G.

    Un estudio detallado de las orientaciones diferenciales de nebulosas planetarias en todo el cielo sugiere fuertemente una relación con la dirección de los campos magnéticos de origen galáctico, determinados en la polarización de estrellas de campo. Esto da consistencia a la hipótesis de que la orientación de las nebulosas planetarias ovaladas es inducida, al menos parcialmente, por el campo magnético general de la Vía Láctea.

  10. Study of the interaction mechanisms between absorbed NO{sub 2} and por-Si/SnO{sub x} nanocomposite layers

    SciTech Connect

    Bolotov, V. V.; Kan, V. E. Makushenko, R. K.; Biryukov, M. Yu.; Ivlev, K. E.; Roslikov, V. E.

    2013-10-15

    The interaction mechanisms between NO{sub 2} molecules and the surface of por-Si/SnO{sub x} nanocomposites obtained by magnetron deposition and chemical vapor deposition (CVD) are studied by infrared absorption spectroscopy and electron paramagnetic resonance methods. The observed increase in the free carrier concentration in the por-Si/SnO{sub x} nanocomposite layers is explained by a change in the charge state of P{sub b} centers due to the formation of neutral 'surface defect-adsorbed NO{sub 2} molecule' complexes with free carrier generation in the crystallite bulk. In the nanocomposite layers grown by the CVD method, the increase in the free hole concentration during NO{sub 2} adsorption is much less pronounced in comparison with the composite grown by magnetron deposition, which is caused by the competing interaction channel of NO{sub 2} molecules with electrically neutral P{sub b} centers.

  11. Adjuvant effects elicited by novel oligosaccharide variants of detoxified meningococcal lipopolysaccharides on Neisseria meningitidis recombinant PorA protein: a comparison in mice.

    PubMed

    Mehta, Ojas H; Norheim, Gunnstein; Hoe, J Claire; Rollier, Christine S; Nagaputra, Jerry C; Makepeace, Katherine; Saleem, Muhammad; Chan, Hannah; Ferguson, David J P; Jones, Claire; Sadarangani, Manish; Hood, Derek W; Feavers, Ian; Derrick, Jeremy P; Pollard, Andrew J; Moxon, E Richard

    2014-01-01

    Neisseria meningitidis lipopolysaccharide (LPS) has adjuvant properties that can be exploited to assist vaccine immunogenicity. The modified penta-acylated LPS retains the adjuvant properties of hexa-acylated LPS but has a reduced toxicity profile. In this study we investigated whether two modified glycoform structures (LgtE and IcsB) of detoxified penta-acylated LPS exhibited differential adjuvant properties when formulated as native outer membrane vesicles (nOMVs) as compared to the previously described LgtB variant. Detoxified penta-acylated LPS was obtained by disruption of the lpxL1 gene (LpxL1 LPS), and three different glycoforms were obtained by disruption of the lgtB, lgtE or icsB genes respectively. Mice (mus musculus) were immunized with a recombinant PorA P1.7-2,4 (rPorA) protein co-administered with different nOMVs (containing a different PorA serosubtype P1.7,16), each of which expressed one of the three penta-acylated LPS glycoforms. All nOMVs induced IgG responses against the rPorA, but the nOMVs containing the penta-acylated LgtB-LpxL1 LPS glycoform induced significantly greater bactericidal activity compared to the other nOMVs or when the adjuvant was Alhydrogel. Compared to LgtE or IcsB LPS glycoforms, these data support the use of nOMVs containing detoxified, modified LgtB-LpxL1 LPS as a potential adjuvant for future meningococcal protein vaccines.

  12. IgG antibody subclass responses determined by immunoblot in infants' sera following vaccination with a meningococcal recombinant hexavalent PorA OMV vaccine.

    PubMed

    Martin, S; Sadler, F; Borrow, R; Dawson, M; Fox, A; Cartwright, K

    2001-08-14

    The introduction of meningococcal serogroup C conjugate vaccines into the UK immunisation schedule has led to the decline of serogroup C disease in those vaccinated but there is no imminent vaccine solution for serogroup B disease. The PorA outer membrane protein (OMP) is a potential serogroup B vaccine candidate and an outer membrane vesicle (OMV) vaccine containing six different PorA OMPs (each representing a different serosubtype) has been evaluated in phase II trials with encouraging results. Little is known about the IgG subclass response to the various antigens contained within this vaccine. These responses are important due to the different half-lives and complement fixing abilities of these antibodies. In this study, immunoblotting was undertaken with infants' sera following either three or four doses of vaccine, and OMVs from six isogenic meningococcal strains differing only in their PorA serosubtype. Following either three or four doses of the vaccine, IgG(3) and IgG(1) subclass antibodies were induced to all six of the isogenic strains, although sera collected after four doses of vaccine showed stronger antibody levels. IgG(3) was found in more sera than IgG(1). For both sets of sera, the two isogenic strains expressing P1.5,2 and P1.5(c),10 induced stronger IgG subclass antibody responses than the other four meningococcal strains. The recombinant hexavalent PorA OMV vaccine stimulates both IgG(1) and IgG(3) subclass antibodies, the subclasses that are most effective in activating the complement system.

  13. Corynebacterium jeikeium jk0268 Constitutes for the 40 Amino Acid Long PorACj, Which Forms a Homooligomeric and Anion-Selective Cell Wall Channel

    PubMed Central

    Norouzy, Amir; Schulz, Robert; Nau, Werner M.; Kleinekathöfer, Ulrich; Tauch, Andreas; Benz, Roland

    2013-01-01

    Corynebacterium jeikeium, a resident of human skin, is often associated with multidrug resistant nosocomial infections in immunodepressed patients. C. jeikeium K411 belongs to mycolic acid-containing actinomycetes, the mycolata and contains a channel-forming protein as judged from reconstitution experiments with artificial lipid bilayer experiments. The channel-forming protein was present in detergent treated cell walls and in extracts of whole cells using organic solvents. A gene coding for a 40 amino acid long polypeptide possibly responsible for the pore-forming activity was identified in the known genome of C. jeikeium by its similar chromosomal localization to known porH and porA genes of other Corynebacterium strains. The gene jk0268 was expressed in a porin deficient Corynebacterium glutamicum strain. For purification temporarily histidine-tailed or with a GST-tag at the N-terminus, the homogeneous protein caused channel-forming activity with an average conductance of 1.25 nS in 1M KCl identical to the channels formed by the detergent extracts. Zero-current membrane potential measurements of the voltage dependent channel implied selectivity for anions. This preference is according to single-channel analysis caused by some excess of cationic charges located in the channel lumen formed by oligomeric alpha-helical wheels. The channel has a suggested diameter of 1.4 nm as judged from the permeability of different sized hydrated anions using the Renkin correction factor. Surprisingly, the genome of C. jeikeium contained only one gene coding for a cell wall channel of the PorA/PorH type found in other Corynebacterium species. The possible evolutionary relationship between the heterooligomeric channels formed by certain Corynebacterium strains and the homooligomeric pore of C. jeikeium is discussed. PMID:24116064

  14. Astronomy in the Classroom: Why? (Spanish Title: Astronomía en la Clase: ¿Por Qué?) Astronomia na Sala de Aula: Por Quê?

    NASA Astrophysics Data System (ADS)

    Daros Gama, Leandro; Bagdonas Henrique, Alexandre

    2010-07-01

    There are many discussions about the relevance of the topics covered in classes. One subject in particular is the focus of this essay: astronomy. In what sense and to what extent it would be worth to teach it in science or other kind of classes? In this paper we discuss some aspects of the advantages of dealing with this area of knowledge in schools, taking into account the epistemological and axiological dimensions of astronomy, in light of the vision of science as an intelligent dialogue with the world (Bachelard), in addition to the "problematization" knowledge of Paulo Freire. We propose that in fact the Astronomy does not need to be seen as just a new set of contents to be taught, but appears as a set of motivational contents for historical-philosophical discussions, and permit the discussion of concepts of other disciplines. Numerosas discusiones se están llevando a cabo acerca de la pertinencia de los temas tradicionalmente tratados en las clases. Uno de los temas, en particular, es el foco de este ensayo: la astronomía. ¿En qué sentido y en qué medida sería conveniente tratarla en clase, ya sea en clases de ciencias naturales, específicamente en las de astronomía o asignaturas afines? Elaboramos en este artículo algunos aspectos de las ventajas de tratar esta área del conocimiento en las escuelas, teniendo en cuenta las dimensiones epistemológica y axiológica de la astronomía, a la luz de la visión de la ciencia como un diálogo inteligente con el mundo (Bachelard), además de la propuesta del conocimiento "problematizador" de Paulo Freire. Proponemos que en realidad la astronomía no tiene por qué ser vista sólo como un nuevo conjunto de contenidos que se enseñan, sino que aparece como un conjunto de temas de motivación para el debate histórico-filosófico y para permitir la discusión de los conceptos típicos de otras disciplinas. Muitas discussões vêm acontecendo sobre a relevância dos temas abordados em sala de aula. Um tema, em

  15. [Optimization of tri-expression of human CYP3A4 with POR and cyt b5 in Sf 9 cells].

    PubMed

    Xie, Zhangming; Liu, Wenhui; Xu, Yingchun; Chen, Shuqing

    2013-01-01

    To investigate the optimal conditions of tri-expression of CYP3A4, POR and cyt b5 in Sf 9 cells. The Sf 9 cells expressing CYP3A4, POR and cyt b5 were cultured in shaker flasks. The optimized conditions, including the temperature and rotation speed, the culture volume, the amount of surfactant and the culture time were studied. The expressed products in microsomes were used to metabolize the testosterone and their metabolic activity was determined. When the temperature and rotation speed of the shaker were 27 degree and 90 r/min, the cell density and culture volume were 5X105 cells/ml and 80-120 ml per 250 ml shaker flasks, respectively. When Pluronic F-68 was 0.1% and the culture time was 72 h, the condition was most suitable for culture of Sf 9 cells and expression of targeted proteins. When the ratio of the volume of three added viruses was 1:1:1, the expression condition was optimal, under which the Km, Vmax, and CLint for testosterone metabolism were 119.6 μmol/L,0.52 μmol/(min*g protein) and 4.34 ml/(min*g protein), respectively. The conditions of tri-expressing of CYP3A4, POR and cyt b5 have been optimized in the study and the product CYP3A4 is obtained with higher metabolic activity.

  16. Obtención de la curva de luz en la ocultación de 35 Sgr por Júpiter el 6 de marzo de 1996

    NASA Astrophysics Data System (ADS)

    Paolantonio, S.; Duffard, R.; Carranza, G.

    La ocultación de la estrella de quinta magnitud 35 Sgr por Júpiter, se produjo el 6 de Marzo de 1996 a las 13 hs. TU. El objetivo era medir el cambio del flujo de la estrella en el ingreso y egreso por el limbo del planeta. Con estos datos se pueden determinar parámetros físicos del planeta (radio, eccentricidad) y de su atmósfera (escala de altura, temperatura, densidad, presión) Para lograr ésto se programó la cámara CCD TH 7896 1024 x 1025 instalada en el telescopio de 1.54 m de Bosque Alegre con el objetivo de lograr 2 imágenes por segundo. De esta forma se obtuvieron 2100 imágenes de la inmersión y otras tantas de la emersión. Hubo que tener grandes precauciones para evitar la saturación del CCD ya que la observación se realizó de día. En este momento las imágenes se encuentran en el Department of Planetary Sciences, Lunar and Planetary Laboratory, University of Arizona, para su reducción.

  17. Pincharse sin infectarse: estrategias para prevenir la infección por el VIH y el VHC entre usuarios de drogas inyectables

    PubMed Central

    MATEU-GELABERT, P.; FRIEDMAN, S.; SANDOVAL, M.

    2011-01-01

    Resumen Objetivo Desde principios de los noventa, en la ciudad de Nueva York se han implementado con éxito programas para reducir la incidencia del virus de la inmunodeficiencia humana (VIH) y, en menor medida, del virus de la hepatitis C (VHC). A pesar de ello, aproximadamente el 70% de los usuario de drogas inyectables (UDI) están infectados por el VHC. Queremos investigar cómo el 30% restante se las ha arreglado para no infectarse. El Staying safe (nombre original del estudio) explora los comportamientos y mecanismos que ayudan a evitar la infección por el VHC y el VIH a largo plazo. Material y métodos Hemos utilizado el concepto de «desviación positiva» aplicado en otros campos de salud pública. Estudiamos las estrategias, prácticas y tácticas de prevención de aquellos UDI que, viviendo en contextos de alta prevalencia, se mantienen sin infectar por VIH y el VHC, a pesar de haberse inyectado heroína durante años. Los resultados preliminares presentados en este artículo incluyen el análisis de las entrevistas realizadas a 25 UDI (17 doble negativos, 3 doble positivos y 5 con infección por el VHC y sin infección por el VIH). Se usaron entrevistas semiestructuradas que exploraban con detalle la historia de vida de los sujetos, incluyendo su consumo de drogas, redes sociales, contacto con instituciones, relaciones sexuales y estrategias de protección y vigilancia. Resultados La intencionalidad es importante para no infectarse, especialmente durante períodos de involución (períodos donde hay un deterioro económico y/o social que llevan al que se inyecta a situaciones de mayor riesgo). Presentamos tres dimensiones independientes de intencionalidad que conllevan comportamientos que pueden ayudar a prevenir la infección: a) evitar «el mono» (síntomas de abstención) asegurando el acceso a la droga; b) «llevarlo bien» para no convertirse en un junkie y así evitar la «muerte social» y la falta de acceso a los recursos, y c) seguir sin

  18. CYP3A5*3 and POR*28 genetic variants influence the required dose of tacrolimus in heart transplant recipients.

    PubMed

    Lesche, Dorothea; Sigurdardottir, Vilborg; Setoud, Raschid; Oberhänsli, Markus; Carrel, Thierry; Fiedler, Georg M; Largiadèr, Carlo R; Mohacsi, Paul; Sistonen, Johanna

    2014-12-01

    After heart transplantation (HTx), the interindividual pharmacokinetic variability of immunosuppressive drugs represents a major therapeutic challenge due to the narrow therapeutic window between over-immunosuppression causing toxicity and under-immunosuppression leading to graft rejection. Although genetic polymorphisms have been shown to influence pharmacokinetics of immunosuppressants, data in the context of HTx are scarce. We thus assessed the role of genetic variation in CYP3A4, CYP3A5, POR, NR1I2, and ABCB1 acting jointly in immunosuppressive drug pathways in tacrolimus (TAC) and ciclosporin (CSA) dose requirement in HTx recipients. Associations between 7 functional genetic variants and blood dose-adjusted trough (C0) concentrations of TAC and CSA at 1, 3, 6, and 12 months after HTx were evaluated in cohorts of 52 and 45 patients, respectively. Compared with CYP3A5 nonexpressors (*3/*3 genotype), CYP3A5 expressors (*1/*3 or *1/*1 genotype) required around 2.2- to 2.6-fold higher daily TAC doses to reach the targeted C0 concentration at all studied time points (P ≤ 0.003). Additionally, the POR*28 variant carriers showed higher dose-adjusted TAC-C0 concentrations at all time points resulting in significant differences at 3 (P = 0.025) and 6 months (P = 0.047) after HTx. No significant associations were observed between the genetic variants and the CSA dose requirement. The CYP3A5*3 variant has a major influence on the required TAC dose in HTx recipients, whereas the POR*28 may additionally contribute to the observed variability. These results support the importance of genetic markers in TAC dose optimization after HTx.

  19. Spatial and stimulus-type tuning in the LEC, MEC, POR, PrC, CA1, and CA3 during spontaneous item recognition memory.

    PubMed

    Beer, Zachery; Chwiesko, Caroline; Kitsukawa, Takashi; Sauvage, Magdalena M

    2013-12-01

    According to the "two streams" hypothesis, the lateral entorhinal (LEC) and the perirhinal (PrC) cortices process information related to items (a "what" stream), the postrhinal (POR) and the medial entorhinal cortices (MEC) process spatial information (a "where" stream), and both types of information are integrated in the hippocampus (HIP). However, within the framework of memory function, only the HIP is reliably shown to preferentially process spatial information, and the PrC items' features. In contrast, the role of the LEC and MEC in memory is virtually unexplored, and conflicting results emerge for the POR. Moreover, the specific contribution of the hippocampal subfields CA1 and CA3 to spatial and non-spatial memory is not thoroughly understood. To investigate which of these areas is specifically tuned to spatial demands or stimulus identity (odor or object), we assessed the pattern of activation of these areas during recognition memory by detecting the immediate-early gene Arc, commonly used as a marker of neuronal activation. We report that all MTL areas were recruited during the spatial and the non-spatial tasks. However, the LEC, MEC, POR, and CA1 were activated to a comparable level in spatial and non-spatial tasks, while the PrC was tuned to stimulus-type, not spatial demands, and CA3 to spatial demands but not stimulus-type. Results are discussed within the frame of a recent model suggesting that the MTL could be segregated in terms of memory processes, such as recollection and familiarity, rather than information content. Copyright © 2013 Wiley Periodicals, Inc.

  20. Cytochrome P450 oxidoreductase deficiency caused by R457H mutation in POR gene in Chinese: case report and literature review.

    PubMed

    Bai, Yang; Li, Jinhui; Wang, Xiaoli

    2017-03-14

    Cytochrome P450 oxidoreductase deficiency (PORD) is a rare disease exhibiting a variety of clinical manifestations. It can be difficult to differentiate with other diseases such as 21-hydroxylase deficiency (21-OHD), polycystic ovary syndrome (PCOS) and Antley-Bixler syndrome (ABS). Nearly 100 cases of PORD have been reported worldwide. However, the genetic characters and clinical management are still unclear, especially in China. In this study, we report a 27-year-old female Chinese patient who first presented with amenorrhea and recurrence of large ovary cyst. She was misdiagnosed with PCOS and non-classical 21-OHD due to ovary cysts and elevated 17-hydroxy-progesterone. The patient's complaining of a mild difficulty of bending the metacarpophalangeal joints reminded us to consider PORD, which usually presents with skeletal deformities and sexual dysfunction. The diagnosis of PORD was confirmed by genetic analyses, which showed the patient harboring a homozygous missense mutation in the POR gene (R457H) and her parents carrying the heterozygous mutation. The patient was treated with low-dose corticosteroids and estrogen/progesterone sequential therapy, and her ovarian cyst gradually reduced with regular menstruation in the follow-up. Moreover, the clinical and genetic characteristics of 104 previously reported PORD cases were also summarized and analyzed. PORD is a very rare disease which can be easily misdiagnosed in mild cases. Clinicians should keep in mind of this disease in patients with sexual dysfunction, especially combined with special skeletal deformities. Our data could provide a consciously understanding of this disease for clinic practicers. Low-dose corticosteroids combined with estrogen/progesterone sequential therapy will be effective in PORD patients with recurrence of large ovary cyst. The fact that the reported PORD patients in China carrying an identical variant R457H in POR gene also give us a viewpoint that R457H mutation in POR gene maybe

  1. Preclinical safety and immunogenicity evaluation of a nonavalent PorA native outer membrane vesicle vaccine against serogroup B meningococcal disease.

    PubMed

    Kaaijk, Patricia; van Straaten, Ineke; van de Waterbeemd, Bas; Boot, Elmieke P J; Levels, Lonneke M A R; van Dijken, Harry H; van den Dobbelsteen, Germie P J M

    2013-02-04

    An improved nonavalent PorA native outer membrane vesicle vaccine was developed with intrinsic adjuvating activity due to presence of less-toxic (lpxL1) LPS. In the present study, the safety and immunogenicity of this next-generation NonaMen vaccine were evaluated following repeated vaccination in rabbits and mice. A repeated-dose toxicology study was performed in rabbits. Immunogenicity of next-generation NonaMen was evaluated by determining the serum bactericidal antibody (SBA) titers against meningococcal serogroup B strains containing several PorA subtypes. Release of the pro-inflammatory cytokine, interleukin-6 (IL-6), by the human monocytic cell line (MM6) was measured to estimate pyrogenic activity. No toxicologically relevant findings were noted in vaccinated rabbits receiving plain next-generation NonaMen. In agreement, next-generation NonaMen induced reduced amounts of the pro-inflammatory cytokine, IL-6, released by human monocyte cell line. In both rabbits and mice, next-generation NonaMen induced high SBA titers against all tested MenB strains regardless of whether or not aluminium phosphate adjuvant is used. The data suggest that next-generation NonaMen is a safe vaccine with the potential to develop a broadly protective immune response and encourage the start of the first clinical studies. Copyright © 2012 Elsevier Ltd. All rights reserved.

  2. A survey of homopteran species (Auchenorrhyncha) from coffee shrubs and poró and laurel trees in shaded coffee plantations, in Turrialba, Costa Rica.

    PubMed

    Rojas, L; Godoy, C; Hanson, P; Hilje, L

    2001-01-01

    A survey of homopteran species (Auchenorryncha) was conducted in coffee plantations with no shade (C), and in those with shade of either poró (Erythrina poeppigiana) (CP) or poró plus laurel (Cordia alliodora) (CPL), in Turrialba, Costa Rica. A total of 130 species in ten families were collected, dominated by Cicadellidae (82 species). Species richness was highest in the CP system (88), followed by CPL (74) and C systems (60). Five most common species for all systems were Fusigonalia lativittata, Hebralebra nicaraguensis, Neocoelidia sp., Oliarus sp. and Clastoptera sp. Diversification of the coffee agroecosystem favors some species while limiting others, and have no effect on the majority of species. Thus, only F. lativittata, Neocoelidia sp. and Scaphytopius ca. latidens were well represented in all systems, but were more abundant in coffee shrubs. Additionally, the following were the dominant species in each system: Graphocephala sp. 1 (C), F. lativittata (CP) and H. nicaraguensis (CPL). Four species abundant on laurel trees, including H. nicaraguensis, appeared almost exclusively on these tree species. Species similarity was highest on the CP and CPL systems (51% of the species in common), followed by the C and CP (39%) and the C and CPL systems (38%). These findings show that even disturbed systems can harbor many insect species, so that they deserve attention from conservation advocates and biologists.

  3. A Fast Real-Time Polymerase Chain Reaction Method for Sensitive and Specific Detection of the Neisseria gonorrhoeae porA Pseudogene

    PubMed Central

    Hjelmevoll, Stig Ove; Olsen, Merethe Elise; Sollid, Johanna U. Ericson; Haaheim, Håkon; Unemo, Magnus; Skogen, Vegard

    2006-01-01

    Ever since the advent of molecular methods, the diagnostics of Neisseria gonorrhoeae has been troubled by false negative and false positive results compared with culture. Commensal Neisseria species and Neisseria meningitidis are closely related to N. gonorrhoeae and may cross-react when using molecular tests comprising too-low specificity. We have devised a real-time polymerase chain reaction (PCR), including an internal amplification control, that targets the N. gonorrhoeae porA pseudogene. DNA was automatically isolated on a BioRobot M48. Our subsequent PCR method amplified all of the different N. gonorrhoeae international reference strains (n = 34) and N. gonorrhoeae clinical isolates (n = 176) but not isolates of the 13 different nongonococcal Neisseria species (n = 68) that we tested. Furthermore, a panel of gram-negative bacterial (n = 18), gram-positive bacterial (n = 23), fungal (n = 1), and viral (n = 4) as well as human DNA did not amplify. The limit of detection was determined to be less than 7.5 genome equivalents/PCR reaction. In conclusion, the N. gonorrhoeae porA pseudogene real-time PCR developed in the present study is highly sensitive, specific, robust, rapid and reproducible, making it suitable for diagnosis of N. gonorrhoeae infection. PMID:17065426

  4. Prevalencia y factores de riesgo para infecciones del tracto urinario de inicio en la comunidad causadas por Escherichia coli productor de betalactamasas de espectro extendido en Colombia

    PubMed Central

    Blanco, Victor M.; Maya, Juan J.; Correa, Adriana; Perenguez, Marcela; Muñoz, Juan S.; Motoa, Gabriel; Pallares, Christian J.; Rosso, Fernando; Matta, Lorena; Celis, Yamile; Garzon, Martha; Villegas, y María V.

    2016-01-01

    RESUMEN Introducción Las infecciones del tracto urinario (ITU) son frecuentes en la comunidad. Sin embargo, la información de aislamientos resistentes en este contexto es limitada en Latinoamérica. Este estudio tiene como objetivo determinar la prevalencia y los factores de riesgo asociados con ITU de inicio en la comunidad (ITU-IC) causadas por Escherichia coli productor de betalactamasas de espectro extendido (BLEE) en Colombia. Materiales y métodos Entre agosto y diciembre de 2011 se realizó un estudio de casos y controles en 3 instituciones de salud de tercer nivel en Colombia. Se invitó a participar a todos los pacientes admitidos a urgencias con diagnóstico probable de ITU-IC, y se les pidió una muestra de orina. En los aislamien-tos de E. coli se realizaron pruebas confirmatorias para BLEE, susceptibilidad antibiótica, caracterización molecular (PCR en tiempo real para genes bla, repetitive element palindromic PCR [rep-PCR], multilocus sequence typing [MLST] y factores de virulencia por PCR). Se obtuvo información clínica y epidemiológica, y posteriormente se realizó el análisis estadístico. Resultados De los 2.124 pacientes seleccionados, 629 tuvieron un urocultivo positivo, en 431 de estos se aisló E. coli, 54 fueron positivos para BLEE y 29 correspondieron a CTX-M-15. La mayoría de los aislamientos de E. coli productor de BLEE fueron sensibles a ertapenem, fosfomicina y amikacina. La ITU complicada se asoció fuertemente con infecciones por E. coli productor de BLEE (OR = 3,89; IC 95%: 1,10–13,89; p = 0,03). E. coli productor de CTX-M-15 mostró 10 electroferotipos diferentes; de estos, el 65% correspondieron al ST131. La mayoría de estos aislamientos tuvieron 8 de los 9 factores de virulencia analizados. Discusión E. coli portador del gen blaCTX-M-15 asociado al ST131 sigue siendo frecuente en Colombia. La presencia de ITU-IC complicada aumenta el riesgo de tener E. coli productor de BLEE, lo cual debe tenerse en cuenta para ofrecer

  5. Effect of CYP3A4*22, POR*28, and PPARA rs4253728 on sirolimus in vitro metabolism and trough concentrations in kidney transplant recipients.

    PubMed

    Woillard, Jean-Baptiste; Kamar, Nassim; Coste, Sandra; Rostaing, Lionel; Marquet, Pierre; Picard, Nicolas

    2013-12-01

    Recent studies have identified new candidate polymorphisms in the genes related to CYP3A activity or calcineurin inhibitor dose requirements in kidney transplant recipients. These genes and polymorphisms are CYP3A4 (cytochrome P450, family 3, subfamily A, polypeptide 4) (rs35599367-C>T; *22); POR [P450 (cytochrome) oxidoreductase] (rs1057868-C>T; *28); and PPARA (peroxisome proliferator-activated receptor alpha) (rs4253728-G>A). We investigated the impact of these polymorphisms on sirolimus (SRL) in vitro hepatic metabolism, SRL trough concentrations (C0), and SRL adverse events in kidney transplant recipients. The clinical study included 113 stable kidney transplant patients switched from a calcineurin inhibitor to SRL (SRL C0 measured at 1, 3, and 6 months thereafter). We investigated SRL metabolism in vitro using human liver microsomes derived from individual donors (n = 31). Microsomes and patients were genotyped by use of Taqman® allelic discrimination assays. The effects of polymorphisms and covariates were studied using multilinear regression imbedded in linear mixed-effect models or logistic regressions. In vitro, the CYP3A4*22 allele resulted in approximately 20% lower metabolic rates of SRL (P = 0.0411). No significant association was found between CYP3A4, CYP3A5, or PPARA genotypes and SRL dose, C0, or C0/dose in kidney transplant patients. POR*28 was associated with a minor but significant decrease in SRL log-transformed C0 [CT/TT vs CC, β = -0.15 (0.05); P = 0.0197] but this did not have any impact on the dose administered, which limited the relevance of the finding. After adjustment for nongenetic covariates and correction for false discovery finding, none of the single-nucleotide polymorphisms tested showed significant association with SRL adverse events. These recently described polymorphisms do not seem to substantially influence the pharmacokinetics of SRL or the occurrence of SRL adverse events in kidney transplant recipients.

  6. Variation in P450 oxidoreductase (POR) A503V and flavin-containing monooxygenase (FMO)-3 E158K is associated with minor alterations in nicotine metabolism, but does not alter cigarette consumption.

    PubMed

    Chenoweth, Meghan J; Zhu, Andy Z X; Sanderson Cox, Lisa; Ahluwalia, Jasjit S; Benowitz, Neal L; Tyndale, Rachel F

    2014-03-01

    The rates of nicotine metabolism differ widely, even after controlling for genetic variation in the major nicotine-metabolizing enzyme, CYP2A6. Genetic variants in an additional nicotine-metabolizing enzyme, flavin-containing monooxygenase (FMO)-3, and an obligate microsomal CYP-supportive enzyme, cytochrome P450 oxidoreductase (POR), were investigated. We examined the impact of FMO3 E158K and POR A503V before and after stratifying by CYP2A6 metabolism group. In 130 nonsmokers of African descent who received 4 mg oral nicotine, FMO3 158K trended toward slower nicotine metabolism in reduced CYP2A6 metabolizers (P=0.07) only, whereas POR 503V was associated with faster CYP2A6 activity (nicotine metabolite ratio) in normal (P=0.03), but not reduced, CYP2A6 metabolizers. Neither FMO3 158K nor POR 503V significantly altered the nicotine metabolic ratio (N=659), cigarette consumption (N=667), or urine total nicotine equivalents (N=418) in smokers of African descent. Thus, FMO3 E158K and POR A503V are minor sources of nicotine metabolism variation, insufficient to appreciably alter smoking.

  7. Effect of CYP3A4*1G, CYP3A5*3, POR*28, and ABCB1 C3435T on the pharmacokinetics of nifedipine in healthy Chinese volunteers.

    PubMed

    Wang, Xiao-Fei; Yan, Liang; Cao, Hui-Min; Wei, Lu-Man; Yang, Wei-Hong; Zhang, Sheng-Jun; Zhang, Li-Rong

    2015-09-01

    Nifedipine is a calcium channel blocker that is widely used in the treatment of cardiovascular disease. However, significant individual variances in the disposition of nifedipine have been reported, and genetic factors are considered to play an important role. The aim of the present study was to investigate the effect of CYP3A4*1G, CYP3A5*3, ABCB1-C3435T, and POR*28 genetic polymorphisms on nifedipine pharmacokinetics in healthy Chinese volunteers. 45 healthy Chinese volunteers enrolled in this study received a single oral dose of 90 mg nifedipine after providing written informed consent. Volunteers were genotyped for CYP3A4*1G, CYP3A5*3, POR*28, and ABCB1-C3435T. The blood concentrations of nifedipine were determined by high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method. There were significant differences of AUC00-∞ and AUC0-48h in the different CYP3A5*3 genotype groups (p = 0.043 and p = 0.048, respectively). The CYP3A5*3 GG group and POR*28 CT/TT group were found to have lower AUC00-∞ and Cmax compared with the POR*28 CC group (p = 0.046 and p = 0.002, respectively). In addition, the POR*28 CT/TT group was found to have longer t1/2 but lower Cmax than the CYP3A4*1G GG group (p = 0.032 and p = 0.002, respectively) as well as the CYP3A4*1G GG and the CYP3A5*3 GG group (p = 0.038 and p = 0.036, respectively) compared with the POR*28 CC group. No significant associations were found between CYP3A4*1G/ABCB1-C3435T polymorphism and pharmacokinetics of nifedipine. Both CYP3A5*3 and POR*28 polymorphisms are found to be associated with the difference in disposition of nifedipine; POR*28 is considered to have an impact on CYP3A4 activity.

  8. Unique combined penA/mtrR/porB mutations and NG-MAST strain types associated with ceftriaxone and cefixime MIC increases in a 'susceptible' Neisseria gonorrhoeae population.

    PubMed

    Thakur, S D; Starnino, S; Horsman, G B; Levett, P N; Dillon, J R

    2014-06-01

    To determine which mutations in penA, mtrR and porB are implicated in increasing minimum MICs of ceftriaxone and cefixime in a susceptible gonococcal population and to ascertain associations with gonococcal strain types (STs). One hundred and forty-six Neisseria gonorrhoeae isolates formed two extended-spectrum cephalosporin susceptibility groups: group 1 isolates with cefixime and ceftriaxone MICs of 0.0005-0.016 mg/L; and group 2 isolates with cefixime MICs of 0.03-0.125 mg/L (n = 24) and ceftriaxone MICs of 0.03-0.06 mg/L (n = 23). Mutation patterns in penicillin-binding protein 2 (PBP2; penA), multiple transfer resistance repressor (MtrR; mtrR) and porin B (PorB; porB) were ascertained by DNA sequence and bioinformatic analysis. STs were determined using N. gonorrhoeae multiantigen sequence typing (NG-MAST). Most isolates carried PBP2 mutation pattern IX (D345a, F504L, A510V, A516G and P551L; 50/146, 34.2%), a G45D substitution in MtrR (37.7%) and a wild-type (WT) sequence for PorB (43.2%). Group 2 gonococcal isolates were significantly associated with: penA pattern IX; dual mutations in the promoter (A-) and DNA dimerization domain (H105Y) of MtrR; and G120K;A121D substitutions in PorB. There were 50 combined penA/mtrR/porB mutation patterns, with corresponding patterns I/WT/WT and IX/G45D/G120K;A121D predominating. Gonococci susceptible to ceftriaxone and cefixime were significantly associated with NG-MAST ST 25 (33/36; 92%) and the combined penA/mtrR/porB mutation pattern I/WT/WT. No combined mutation pattern or specific ST was associated with elevated ceftriaxone MICs. NG-MAST ST 3654 was significantly associated with the pattern IX/G45D/G120K;A121D and cefixime group 2 isolates. Specific single or combined mutation patterns in penA, mtrR and porB and specific STs were associated with differences in susceptibility to ceftriaxone and cefixime. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial

  9. Concepciones y concepciones alternativas de estudiantes universitarios/as de biologia y futuros maestros/as de Ciencia de escuela secundaria sobre la teoria de evolucion biologica por seleccion natural

    NASA Astrophysics Data System (ADS)

    Morales Ramos, Egda M.

    La teoria de evolucion biologica (TEB) por seleccion natural es uno de los conceptos unificadores mas importantes del curriculo de Biologia. En Puerto Rico se han hecho pocas investigaciones que abunden sobre las concepciones y concepciones alternativas (CA) que tienen los estudiantes universitarios/as de Biologia y los maestros/as de Ciencia del nivel secundario sobre esta teoria. La politica publica educativa actual establece mediante documentos normativos como los Estandares de contenido y Expectativas de grado del Programa de Ciencias [Puerto Rico Core Standards] la ensenanza de esta teoria. Sin embargo, no se encontraron preguntas sobre la seleccion natural en los ejercicios de practica provistos por el Departamento de Educacion para las pruebas estandarizadas lo cual puede influir para que no se ensene adecuadamente. Las preguntas de investigacion fueron 1. ¿Cuales son las concepciones y concepciones alternativas de estudiantes universitarios/as y de los futuros maestros y maestras de Ciencia sobre la TEB? 2. ¿Cuales conceptos que seleccionan los estudiantes universitarios/as y los futuros maestros y maestras de Ciencia sobre la TEB coinciden con lo aceptado como valido por la comunidad cientifica? y 3. ¿Como comparan las respuestas de la prueba original. v. Entendiendo el cambio biologico que mide concepciones y CA sobre la TEB por seleccion natural, con las de la traducida al idioma espanol? Se utilizo el metodo cuantitativo con un diseno de investigacion transversal por encuesta. La tecnica principal para recopilar los datos fue una prueba con doce items, que formo parte de un instrumento para el cual se recopilaron diversas fuentes de evidencia acerca de su validez. Las muestras estuvieron formadas por 69 estudiantes de Ciencias Naturales y por 16 estudiantes futuros maestros y maestras del nivel secundario de la UPR-RP. Se utilizaron estadisticas descriptivas, analisis de Ji cuadrado y se calcularon los coeficientes alfa de Cronbach y de Spearman

  10. 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.

  11. Bifurcación de las soluciones de vientos impulsados por radiación en estrellas Be: formación de líneas

    NASA Astrophysics Data System (ADS)

    Curé, M.; Rial, D.; Cidale, L.; Venero, R.

    Se ha estudiado la topología de la ecuación hidrodinámica no-lineal que describe el perfil de velocidades de vientos impulsados por radiación en estrellas tempranas. Al aplicar este modelo a estrellas Be se encuentra que existen dos tipos De soluciones: la estándar, que describe el viento polar, y una nueva, que describe un viento más denso y lento y que explicaría el disco que se encuentra alrededor de estos objetos. Existe una región de transición en donde ambas soluciones coexisten (bifurcación}). Ambas soluciones satisfacen en esta región las mismas condiciones de borde. Para estas dos soluciones se han obtenido los perfiles de líneas de hidrógeno del visible y del IR, resolviendo el transporte de radiación en el ``comoving frame". Para la solución estándar, se obtienen perfiles con componentes en emisión, mientras que para la nueva solución se obtienen perfiles en absorción. Se comparan cualitativamente los resultados con las observaciones.

  12. Weight of ABCB1 and POR genes on oral tacrolimus exposure in CYP3A5 nonexpressor pediatric patients with stable kidney transplant.

    PubMed

    Almeida-Paulo, G N; Dapía García, I; Lubomirov, R; Borobia, A M; Alonso-Sánchez, N L; Espinosa, L; Carcas-Sansuán, A J

    2017-01-17

    Tacrolimus (TAC) is highly effective for the prevention of acute organ rejection. However, its clinical use may be challenging due to its large interindividual pharmacokinetic variability, which can be partially explained by genetic variations in TAC-metabolizing enzymes and transporters. The aim of this study was to evaluate the influence of genetic and clinical factors on TAC pharmacokinetic variability in 21 stable pediatric renal transplant patients. This study was nested in a previous Prograf to Advagraf conversion clinical trial. CYP3A5, ABCB1 and two POR genotypes were assessed by real-time PCR. The impact on TAC pharmacokinetics of individual genetic variants on CYP3A5 nonexpressors was evaluated by genetic score. Explicative models for TAC AUC0-24h, Cmax and Cmin after Advagraf were developed by linear regression. The built genetic scores explain 13.7 and 26.5% of the total AUC0-24h and Cmin total variability, respectively. Patients genetic information should be considered to monitorizate and predict TAC exposure.The Pharmacogenomics Journal advance online publication, 17 January 2017; doi:10.1038/tpj.2016.93.

  13. Relación masa-radio para estrellas enanas blancas y la interpretación de recientes mediciones hechas por Hipparcos

    NASA Astrophysics Data System (ADS)

    Panei, J. A.; Althaus, L. G.; Benvenuto, O. G.

    Recientes mediciones de la masa y el radio hechas por Hipparcos de las estrellas enanas blancas 40 Eri B y Procyon B (Shipman, H. & Provencal, J. - ApJ. 1998, 494, 759), sugieren un núcleo compuesto de hierro para dichas estrellas, en lugar de carbono y oxígeno como predice la teoría standard de evolución estelar. Para interpretar estas observaciones, presentamos aquí, relaciones masa-radio para configuraciones degeneradas a temperatura finita para distintas composiciones químicas centrales. Para tal fin hemos calculado secuencias evolutivas de enanas blancas utilizando el código de evolución estelar, desarrollado en el Observatorio de La Plata. Dicho código resuelve las ecuaciones de estructura y evolución estelar mediante la técnica de relajación de Henyey, y esta basado en una descripción física muy detallada y actualizada.

  14. Population pharmacokinetic approach to evaluate the effect of CYP2D6, CYP3A, ABCB1, POR and NR1I2 genotypes on donepezil clearance.

    PubMed

    Noetzli, Muriel; Guidi, Monia; Ebbing, Karsten; Eyer, Stephan; Wilhelm, Laurence; Michon, Agnès; Thomazic, Valérie; Stancu, Ioana; Alnawaqil, Abdel-Messieh; Bula, Christophe; Zumbach, Serge; Gaillard, Michel; Giannakopoulos, Panteleimon;