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Daniela C S Damico - One of the best experts on this subject based on the ideXlab platform.
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lmrtx a basic pla2 d49 purified from Lachesis muta rhombeata snake venom with enzymatic related antithrombotic and anticoagulant activity
Toxicon, 2012Co-Authors: Daniela C S Damico, Edson Antunes, T Vassequisilva, Frank Denis Torreshuaco, A C C Nerydiez, R C G De Souza, S L Da Silva, Cristina P Vicente, Camila B Mendes, Claudio C WerneckAbstract:Abstract A basic phospholipase A 2 (LmrTX) isoform was isolated from Lachesis muta rhombeata snake venom and partially characterized. The venom was fractionated by molecular exclusion chromatography in ammonium bicarbonate buffer followed by reverse-phase HPLC on a C-5 Discovery ® Bio Wide column. From liquid chromatography–electrospray ionization/mass spectrometry, the molecular mass of LmrTX was measured as 14.277.50 Da. The amino acid sequence showed a high degree of homology between PLA 2 LmrTX from L. muta rhombeata and other PLA 2 from snake venoms, like CB1 and CB2 from Crotalus durissus terrificus ; LmTX-I and LmTX-II from Lachesis muta muta . LmrTX had PLA 2 activity in the presence of a synthetic substrate and alkylation of histidine residues significantly inhibited ( P 2 from L. muta rhombeata venom, caused a change in the occlusion time to 99 ± 10 min with doses of 7.5 μg/mice. Additionally, LmrTX showed the anticoagulant activity in vitro and ex vivo and prolonging the time aggregation in wash platelet induced by ADP and Thrombin.
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inflammatory oedema induced by Lachesis muta muta surucucu venom and lmtx i in the rat paw and dorsal skin
Toxicon, 2009Co-Authors: Tatiane Ferreira, Enilton A Camargo, Maria Teresa C P Ribela, Daniela C S Damico, Sergio Marangoni, Edson Antunes, Gilberto De Nucci, Elen C T LanducciAbstract:The ability of crude venom and a basic phospholipase A2 (LmTX-I) from Lachesis muta muta venom to increase the microvascular permeability in rat paw and skin was investigated. Crude venom or LmTX-I were injected subplantarly or intradermally and rat paw oedema and dorsal skin plasma extravasation were measured. Histamine release from rat peritoneal mast cell was also assessed. Crude venom or LmTX-I induced dose-dependent rat paw oedema and dorsal skin plasma extravasation. Venom-induced plasma extravasation was inhibited by the histamine H1 antagonist mepyramine (6 mg/kg), histamine/5-hydroxytriptamine antagonist cyproheptadine (2 mg/kg), cyclooxygenase inhibitor indomethacin (5 mg/kg), nitric oxide synthesis inhibitor L-NAME (100 nmol/site), tachykinin NK1 antagonist SR140333 (1 nmol/site) and bradykinin B2 receptor antagonist Icatibant (0.6 mg/kg). Platelet-activating factor (PAF) antagonist PCA4248 (5 mg/kg) had no effect. LmTX-I-induced skin extravasation was inhibited by cyproheptadine, mepyramine, indomethacin and PCA4248, while L-NAME and SR140333 had no effect. Additionally, both Lachesis muta muta venom and LmTX-I concentration-dependently induced histamine release from rat mast cells. In conclusion, Lachesis muta muta venom and LmTX-I increase microvascular permeability by mechanisms involving in vivo mast cell activation and arachidonic acid metabolites. Additionally, crude venom-induced responses also involve substance P, nitric oxide and bradykinin release, whether LmTX-I-induced responses involve PAF. 2008 Published by Elsevier Ltd.
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biochemical and enzymatic characterization of two basic asp49 phospholipase a2 isoforms from Lachesis muta muta surucucu venom
Biochimica et Biophysica Acta, 2005Co-Authors: Daniela C S Damico, Gilberto De Nucci, Jose C Novello, Sergio Lilla, Luis Alberto Poncesoto, Flavia Vischi Winck, Sergio MarangoniAbstract:Abstract Two basic phospholipase A2 (PLA2) isoforms were isolated from Lachesis muta muta snake venom and partially characterized. The venom was fractionated by molecular exclusion chromatography in ammonium bicarbonate buffer followed by reverse-phase HPLC on a C-18 μ-Bondapack column and RP-HPLC on a C-8 column. From liquid chromatography-electrospray ionization/mass spectrometry, the molecular mass of the two isoforms LmTX-I and LmTX-II was respectively measured as 14,245.4 and 14,186.2 Da. The pI was respectively estimated to be 8.7 and 8.6 for LmTX-I and LmTX-II, as determined by two-dimensional electrophoresis. The two proteins were sequenced and differentiated from each other by a single amino acid substitution, Arg65 (LmTX-I) → Pro65 (LmTX-II). The amino acid sequence showed a high degree of homology between PLA2 isoforms from Lachesis muta muta and other PLA2 snake venoms. LmTX-I and LmTX-II had PLA2 activity in the presence of a synthetic substrate and showed a minimum sigmoidal behaviour; with maximal activity at pH 8.0 and 35–45 °C. Full PLA2 activity required Ca2+ and was respectively inhibited by Cu2+ and Zn2+ in the presence and absence of Ca2+. Crotapotin from Crotalus durissus cascavella rattlesnake venom significantly inhibited (P
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neurotoxic and myotoxic actions from Lachesis muta muta surucucu whole venom on the mouse and chick nerve muscle preparations
Web Science, 2005Co-Authors: Daniela C S Damico, Sergio Marangoni, Lilian G F Bueno, Lea Rodriguessimioni, Maria Alice Da Cruzhofling, Jose C NovelloAbstract:Lachesis genus is one of the less studied among others from Viperidae's genera, mainly due to difficulties in obtaining the venom. Accidents by Lachesis snakes cause severe envenoming syndrome, eventually leading victims to shock. This work is part of a comprehensive study aimed at studying the venom and its effects. Herein the neurotoxicity and myotoxicity of L. muta muta venom were investigated on mouse phrenic nerve-diaphragm (PNDp) and chick biventer cervicis (BCp) preparations. For both preparations the time required to venom produces 50% neuromuscular blockade was indirectly concentration-dependent, being for PNDp: 117.6±6.5 min (20 μg/ml), 70.1±8.6 min (50 μg/ml) and 43.6±3.8 min (100 μg/ml), and for BCp: 28±1.8 min (50 μg/ml), 30.4±2.3 min (10 μg/ml), 50.4±4.3 min (5 μg/ml) and 75.2±0.7 min (2 μg/ml), (n=5/dose). In BCp, a venom dose of 50 μg/ml significantly reduced contractures elicited by exogenous acetylcholine (55 μM) and KCl (20 mM), as well as increased the release of creatine kinase (442.7±39.8 IU/l in controls vs 4322.6±395.2 IU/l, after 120 min of venom incubation (P<0.05). Quantification of myonecrosis in BCp indicated the doses 50 and 10 μg/ml as significantly myotoxic affecting 59.7±6.2%, and 20.8±1.2% of fibers, respectively, whereas 5 and 2 μg/ml that affected 13.5±0.8% and 5.4±0.6% of fibers, were considered weakly- and non-myotoxic, respectively. We concluded that there are neurotoxins present in the venom, the concentration of which governs its pre- (if low) or postsynaptic (if high) activity. Since myotoxicity in the avian preparation is negligible at lower venom doses, but not neurotoxicity, we suggest that this effect may contribute minimum to the venom neurotoxic effect. The BCp is more sensible than PNDp to Lachesis m. muta venom.
Guillermo Leon - One of the best experts on this subject based on the ideXlab platform.
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contributions of the snake venoms of bothrops asper crotalus simus and Lachesis stenophrys to the paraspecificity of the central american polyspecific antivenom polival icp
Toxicon, 2018Co-Authors: Gabriela Solano, Ricardo Estrada, Aaron Gomez, Greivin Corrales, Danilo Chacon, Guillermo LeonAbstract:Abstract PoliVal-ICP antivenom is produced from plasma of horses immunized toward the venoms of Bothrops asper , Crotalus simus and Lachesis stenophrys . The antibody response induced by these venoms confers PoliVal-ICP the capacity to neutralize the venoms of the most important Central American viperids, including not only homologous venoms (i.e., venoms used as immunogen), but many heterologous venoms (i.e., venoms not used as immunogen). In this work, the individual contributions of homologous venoms to the paraspecificity of PoliVal-ICP were inferred from the capacity of experimental monospecific antivenoms toward venoms of B. asper (anti-Ba), C. simus (anti-Cs) and L. stenophrys (anti-Ls), and an experimental polyspecific antivenom (anti-Ba/Cs/Ls) to neutralize the lethality induced by different venoms in mice. It was found that all antivenoms neutralized their corresponding homologous venoms. Moreover, the anti-Ba antivenom cross-neutralized the venoms of Agkistrodon howardgloydi , Atropoides picadoi , Bothriechis lateralis , Bothriechis supraciliaris and Porthidium ophryomegas ; the anti-Cs antivenom cross-neutralized the venoms of B. lateralis , B. supraciliaris , Cerrophidion sasai and Porthidium nasutum ; and the anti-Ls antivenom cross-neutralized the venoms of B. lateralis , B. supraciliaris , C. sasai and Lachesis melanocephala . All venoms neutralized by any monospecific antivenom were also neutralized by the anti-Ba/Cs/Ls antivenom. Venoms of Atropoides mexicanus , Bothriechis nigroviridis and Bothriechis schlegelii were not neutralized by any experimental antivenom, thus explaining the limitations of PoliVal-ICP to neutralize these venoms. Consequently, an enlargement of the neutralization scope of PoliVal-ICP could be achieved by including these venoms in the group of those used as immunogens.
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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
Toxicon, 2017Co-Authors: Cynthia Arroyo, Jose Maria Gutierrez, Sergio Solano, Maria Herrera, Alvaro Segura, Ricardo Estrada, Mariangela Vargas, Mauren Villalta, Guillermo LeonAbstract: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.
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Lachesis stenophrys venom reduces the equine antibody response towards bothrops asper venom used as co immunogen in the production of polyspecific snake antivenom
Toxicon, 2015Co-Authors: Cynthia Arroyo, Jose Maria Gutierrez, Sergio Solano, Maria Herrera, Alvaro Segura, Ricardo Estrada, Mariangela Vargas, Mauren Villalta, Guillermo LeonAbstract: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.
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neutralization of bothrops mattogrossensis snake venom from bolivia experimental evaluation of llama and donkey antivenoms produced by caprylic acid precipitation
Toxicon, 2010Co-Authors: Gil Patrick Fernandez, Jose Maria Gutierrez, Maria Herrera, Alvaro Segura, Williams Velasco, Gabriela Solano, Guillermo LeonAbstract: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.
Jose Maria Gutierrez - One of the best experts on this subject based on the ideXlab platform.
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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
Toxicon, 2017Co-Authors: Cynthia Arroyo, Jose Maria Gutierrez, Sergio Solano, Maria Herrera, Alvaro Segura, Ricardo Estrada, Mariangela Vargas, Mauren Villalta, Guillermo LeonAbstract: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.
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cross reactivity antivenomics and neutralization of toxic activities of Lachesis venoms by polyspecific and monospecific antivenoms
PLOS Neglected Tropical Diseases, 2017Co-Authors: Marvin Madrigal, Davinia Pla, Libia Sanz, Elexandra Barboza, Cynthia Arroyoportilla, Carlos Correanetto, Jose Maria Gutierrez, Alberto Alapegiron, Marietta Floresdiaz, Juan J CalveteAbstract:Background Bothrops, Crotalus and Lachesis represent the most medically relevant genera of pitvipers in Central and South America. Similarity in venom phenotype and physiopathological profile of envenomings caused by the four nominal Lachesis species led us to hypothesize that an antivenom prepared against venom from any of them may exhibit paraspecificity against all the other congeneric taxa. Methods To assess this hypothesis, in this work we have applied antivenomics and immunochemical methods to investigate the immunoreactivity of three monovalent antivenoms and two polyvalent antivenoms towards the venoms from different geographic populations of three different Lachesis species. The ability of the antivenoms to neutralize the proteolytic, hemorrhagic, coagulant, and lethal activities of the seven Lachesis venoms was also investigated. Results A conspicuous pattern of immunorecognition and cross-neutralization for all effects was evident by the polyspecific antivenoms, indicating large immunoreactive epitope conservation across the genus during more than 10 million years since the Central and South American bushmasters diverged. Conclusions Despite the broad geographic distribution of Lachesis, antivenoms against venoms of different species are effective in the neutralization of congeneric venoms not used in the immunization mixture, indicating that they can be used equivalently for the clinical treatment of any lachesic envenoming. General significance This study demonstrates that antivenoms raised against venom of different Lachesis species are indistinctly effective in the neutralization of congeneric venoms not used in the immunization mixture, indicating that antivenoms against conspecific venoms may be used equivalently for the clinical treatment of envenomings caused by any bushmaster species.
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cambios en el patron electroforetico del veneno de la serpiente cascabel muda Lachesis muta stenophrys almacenado bajo diferentes condiciones
Revista De Biologia Tropical, 2016Co-Authors: Jose Antonio Gene, Jose Maria Gutierrez, Bruno Lomonte, Luis CerdasAbstract:Liquid and lyophilized samples of Lachesis muta venom were stored at different temperatures and for different periods of time, and analyzed by polyacrylamide gel electrophoresis (PAGE) and immunoelectrophoresis. Only slight variations were evident when three pools of freeze-dried venom, that had been kept at -30 degrees C for several months were compared with fresh venom. These results suggest that L. muta venom is not altered drastically when stored under these conditions.
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Lachesis stenophrys venom reduces the equine antibody response towards bothrops asper venom used as co immunogen in the production of polyspecific snake antivenom
Toxicon, 2015Co-Authors: Cynthia Arroyo, Jose Maria Gutierrez, Sergio Solano, Maria Herrera, Alvaro Segura, Ricardo Estrada, Mariangela Vargas, Mauren Villalta, Guillermo LeonAbstract: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.
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snake venomics of Lachesis muta rhombeata and genus wide antivenomics assessment of the paraspecific immunoreactivity of two antivenoms evidence the high compositional and immunological conservation across Lachesis
Journal of Proteomics, 2013Co-Authors: Davinia Pla, Marvin Madrigal, Libia Sanz, Alberto Alapegiron, Marietta Floresdiaz, Vitelbina Nunez, Pedro Molinasanchez, Virginia Zorita, Vicente Andres, Jose Maria GutierrezAbstract:Abstract We report the proteomic analysis of the Atlantic bushmaster, Lachesis muta rhombeata, from Brazil. Along with previous characterization of the venom proteomes of L. stenophrys (Costa Rica), L. melanocephala (Costa Rica), L. acrochorda (Colombia), and L. muta muta (Bolivia), the present study provides the first overview of the composition and distribution of venom proteins across this wide-ranging genus, and highlights the remarkable similar compositional and pharmacological profiles across Lachesis venoms. The paraspecificity of two antivenoms, produced at Instituto Vital Brazil (Brazil) and Instituto Clodomiro Picado (Costa Rica) using different conspecific taxa in the immunization mixtures, was assessed using genus-wide comparative antivenomics. This study confirms that the proteomic similarity among Lachesis sp. venoms is mirrored in their high immunological conservation across the genus. The clinical and therapeutic consequences of genus-wide venomics and antivenomics investigations of Lachesis venoms are discussed. Biological significance The proteomics characterization of L. m. rhombeata venom completes the overview of Lachesis venom proteomes and confirms the remarkable toxin profile conservation across the five clades of this wide-ranging genus. Genus-wide antivenomics showed that two antivenoms, produced against L. stenophrys or L. m. rhombeata, exhibit paraspecificity towards all other congeneric venoms. Our venomics study shows that, despite the broad geographic distribution of the genus, monospecific antivenoms may achieve clinical coverage for any Lachesis sp. envenoming.
M Richardson - One of the best experts on this subject based on the ideXlab platform.
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the complete amino acid sequence of a thrombin like enzyme gyroxin analogue from venom of the bushmaster snake Lachesis muta muta
FEBS Letters, 1993Co-Authors: Arinos Magalhaes, Beatriz Campos Brasil Da Fonseca, Carlos Ribeiro Diniz, John Gilroy, M RichardsonAbstract:The complete amino acid sequence of a thrombin-like enzyme with gyroxin activity isolated from the venom of the bushmaster snake Lachesis muta muta was determined by automated and DABITC/PITC microsequencing of the intact protein; fragments derived from it by separate cleavages with cyanogen bromide, iodosobenzoic acid and hydroxylamine; and peptides resulting from enzymatic digestions with trypsin, pepsin, chymotrypsin, and elastase. The protein, which is composed of 228 residues, contains four putative sites of N-linked glycosylation and exhibits significant sequence similarities with other serine proteases reported from snake venoms.
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the complete amino acid sequence of the haemorrhagic factor lhfii a metalloproteinase isolated from the venom of the bushmaster snake Lachesis muta muta
FEBS Letters, 1991Co-Authors: Eladio F Sanchez, C R Diniz, M RichardsonAbstract:The complete amino acid sequence the haemorrhagic agent LHFII, a Zn and Ca containing metalloproteinase isolated from the venom of the Bushmaster snake Lachesis muta muta was determined by automated and DABITC/PITC microsequencing of the intact protein, fragments derived by cleavage with cyanogen bromide, and peptides resulting from enzymatic digestions with trypsin and the protease from S. aureas V8. The protein is composed of 200 residues and exhibits considerable sequence homology with the haemorrhagic toxins from a number of other snake venoms, and some metalloproteinases in the region of the putative Zn-binding sites.
Sergio Marangoni - One of the best experts on this subject based on the ideXlab platform.
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inflammatory oedema induced by Lachesis muta muta surucucu venom and lmtx i in the rat paw and dorsal skin
Toxicon, 2009Co-Authors: Tatiane Ferreira, Enilton A Camargo, Maria Teresa C P Ribela, Daniela C S Damico, Sergio Marangoni, Edson Antunes, Gilberto De Nucci, Elen C T LanducciAbstract:The ability of crude venom and a basic phospholipase A2 (LmTX-I) from Lachesis muta muta venom to increase the microvascular permeability in rat paw and skin was investigated. Crude venom or LmTX-I were injected subplantarly or intradermally and rat paw oedema and dorsal skin plasma extravasation were measured. Histamine release from rat peritoneal mast cell was also assessed. Crude venom or LmTX-I induced dose-dependent rat paw oedema and dorsal skin plasma extravasation. Venom-induced plasma extravasation was inhibited by the histamine H1 antagonist mepyramine (6 mg/kg), histamine/5-hydroxytriptamine antagonist cyproheptadine (2 mg/kg), cyclooxygenase inhibitor indomethacin (5 mg/kg), nitric oxide synthesis inhibitor L-NAME (100 nmol/site), tachykinin NK1 antagonist SR140333 (1 nmol/site) and bradykinin B2 receptor antagonist Icatibant (0.6 mg/kg). Platelet-activating factor (PAF) antagonist PCA4248 (5 mg/kg) had no effect. LmTX-I-induced skin extravasation was inhibited by cyproheptadine, mepyramine, indomethacin and PCA4248, while L-NAME and SR140333 had no effect. Additionally, both Lachesis muta muta venom and LmTX-I concentration-dependently induced histamine release from rat mast cells. In conclusion, Lachesis muta muta venom and LmTX-I increase microvascular permeability by mechanisms involving in vivo mast cell activation and arachidonic acid metabolites. Additionally, crude venom-induced responses also involve substance P, nitric oxide and bradykinin release, whether LmTX-I-induced responses involve PAF. 2008 Published by Elsevier Ltd.
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biochemical and enzymatic characterization of two basic asp49 phospholipase a2 isoforms from Lachesis muta muta surucucu venom
Biochimica et Biophysica Acta, 2005Co-Authors: Daniela C S Damico, Gilberto De Nucci, Jose C Novello, Sergio Lilla, Luis Alberto Poncesoto, Flavia Vischi Winck, Sergio MarangoniAbstract:Abstract Two basic phospholipase A2 (PLA2) isoforms were isolated from Lachesis muta muta snake venom and partially characterized. The venom was fractionated by molecular exclusion chromatography in ammonium bicarbonate buffer followed by reverse-phase HPLC on a C-18 μ-Bondapack column and RP-HPLC on a C-8 column. From liquid chromatography-electrospray ionization/mass spectrometry, the molecular mass of the two isoforms LmTX-I and LmTX-II was respectively measured as 14,245.4 and 14,186.2 Da. The pI was respectively estimated to be 8.7 and 8.6 for LmTX-I and LmTX-II, as determined by two-dimensional electrophoresis. The two proteins were sequenced and differentiated from each other by a single amino acid substitution, Arg65 (LmTX-I) → Pro65 (LmTX-II). The amino acid sequence showed a high degree of homology between PLA2 isoforms from Lachesis muta muta and other PLA2 snake venoms. LmTX-I and LmTX-II had PLA2 activity in the presence of a synthetic substrate and showed a minimum sigmoidal behaviour; with maximal activity at pH 8.0 and 35–45 °C. Full PLA2 activity required Ca2+ and was respectively inhibited by Cu2+ and Zn2+ in the presence and absence of Ca2+. Crotapotin from Crotalus durissus cascavella rattlesnake venom significantly inhibited (P
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neurotoxic and myotoxic actions from Lachesis muta muta surucucu whole venom on the mouse and chick nerve muscle preparations
Web Science, 2005Co-Authors: Daniela C S Damico, Sergio Marangoni, Lilian G F Bueno, Lea Rodriguessimioni, Maria Alice Da Cruzhofling, Jose C NovelloAbstract:Lachesis genus is one of the less studied among others from Viperidae's genera, mainly due to difficulties in obtaining the venom. Accidents by Lachesis snakes cause severe envenoming syndrome, eventually leading victims to shock. This work is part of a comprehensive study aimed at studying the venom and its effects. Herein the neurotoxicity and myotoxicity of L. muta muta venom were investigated on mouse phrenic nerve-diaphragm (PNDp) and chick biventer cervicis (BCp) preparations. For both preparations the time required to venom produces 50% neuromuscular blockade was indirectly concentration-dependent, being for PNDp: 117.6±6.5 min (20 μg/ml), 70.1±8.6 min (50 μg/ml) and 43.6±3.8 min (100 μg/ml), and for BCp: 28±1.8 min (50 μg/ml), 30.4±2.3 min (10 μg/ml), 50.4±4.3 min (5 μg/ml) and 75.2±0.7 min (2 μg/ml), (n=5/dose). In BCp, a venom dose of 50 μg/ml significantly reduced contractures elicited by exogenous acetylcholine (55 μM) and KCl (20 mM), as well as increased the release of creatine kinase (442.7±39.8 IU/l in controls vs 4322.6±395.2 IU/l, after 120 min of venom incubation (P<0.05). Quantification of myonecrosis in BCp indicated the doses 50 and 10 μg/ml as significantly myotoxic affecting 59.7±6.2%, and 20.8±1.2% of fibers, respectively, whereas 5 and 2 μg/ml that affected 13.5±0.8% and 5.4±0.6% of fibers, were considered weakly- and non-myotoxic, respectively. We concluded that there are neurotoxins present in the venom, the concentration of which governs its pre- (if low) or postsynaptic (if high) activity. Since myotoxicity in the avian preparation is negligible at lower venom doses, but not neurotoxicity, we suggest that this effect may contribute minimum to the venom neurotoxic effect. The BCp is more sensible than PNDp to Lachesis m. muta venom.
