- Email: [email protected]
44. Leishmanicidal Effects of a Phospholipase A2 Isolated from Crotalus viridis viridis Snake Venom
Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248
44. Leishmanicidal Effects of a Phospholipase A2 Isolated from Crotalus viridis viridis Snake Venom Camila M. Adade 1, S. Fernandes Anne Cristine 1, O. Carvalho Ana Lúcia 2, Russolina B. Zingali 2, Thais Souto-Padrón 1 1 Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil 2 Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil E-mail address: [email protected] (C.M. Adade).
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45. Carbon-13 and Nitrogen-15 Turnover in Serum of Bubaline Donors of Biological Material for Medical Use Daniela A. Fossato da Silva 1, Natália P. Biscola 1, Renato M.F. Souza 1, Denis A. Caetano 1, Juliana C. Denadai 2, Maria M.P. Sartori 2, Evandro T. da Silva 2, Carlos Ducatti 2, Cyntia L. Martins 3, André M. Jorge 3, Lucilene D. dos Santos 1, Rui S. Ferreira Junior 1, Benedito Barraviera 1 1 Centro de Estudos de Venenos e Animais Peçonhentos (CEVAP), Faculdade de Medicina, Univ. Estadual Paulista, Botucatu, SP, Brazil 2 Centro de Isótopos Estáveis (CIE), Univ. Estadual Paulista, Botucatu, SP, Brazil 3 Faculdade de Medicina Veterinária e Zootecnia, Univ. Estadual Paulista, Botucatu, SP, Brazil E-mail address: [email protected] (D.A. Fossato da Silva).
Background: Treatment of Leishmaniasis, caused by Leishmania sp., a disease which afflicts millions of people worldwide, is based on drugs as amphotericin B, pentavalent antimonial and pentamidine, which exhibit toxic effects and limited efficacy. Therefore the search for new drugs is a lining research to be exploited. Several toxins have been used as therapeutic agents and pharmacological tools for drug development. Snake venom phospholipases, exhibited different in vitro effects such as modulate cell proliferation, bactericidal activity and Plasmodium falciparum inhibition growth. Crotalus viridis viridis (Cvv) phospholipase A2 (PLA2) is a 14 kDa, non-neurotoxic, basic single-chain myotoxin, previously described. This work is based in the Cvv venom PLA2, and its effects over L. amazonensis promastigotes. Methods: The crude venom extract was loaded onto to a reverse phase analytical (C8) column using a high performance liquid chromatographer. A linear gradient of water/acetonitrile with 0.1% trifluoroacetic acid was used. The peak contained the isolated PLA2 (confirmed by SDSPAGE and mass spectrometry) was collected, lyophilized, resuspended in distilled water and its protein content measured. L. amazonensis promastigotes were incubated in Schneider medium, with 0.3125 to 10 mcg/ ml PLA2, at 24oC, and the effect on the cells proliferation was evaluated by counting with a Neubauer chamber, up to 72 h. The parameter used to estimate proliferation inhibition was the IC50, which corresponds to the drug concentration that inhibited 50% cell growth. Parasites viability was assessed by flow cytometry using propidium iodide (PI) labeling, and the morphological alterations were examined by light microscopy through Giemsa staining. Results: The treatment caused a significant inhibition (32% to 82%) in the parasites growth, dosis- and timedependent, soon the first 24 hours. The data obtained allowed us to estimate the IC50/ 24 h of 2.50 1.42 mcg/ ml, decreasing to 0.77 0.5 mcg/ ml after the final 72 h. The morphological alterations presented by treated parasites were rounded and unusual cell body shapes, with loss of membrane integrity, corroborated by the 96.6% of PI-positive labeling. Discussion: We presented the employee of Cvv PLA2 against L. amazonensis promastigotes, which inhibited the parasites in vitro growth. Further studies by electron microscopy are underway in order to detect the intracellular targets and the PLA2 effects over amastigotes forms. Conclusions: This work presented that Cvv PLA2 can be object of further research for a new leishmanicidal agent, as we have looked forward to achieving. Financial Support: CAPES, CNPq and FAPERJ.
Background: Toxicology is a fascinating area for potential bioprospect molecules in the development of new drugs. The synergism between the animal toxins and biological materials can be used to formulate a new compound. In this regard, the fibrin sealant produced by CEVAP (Centro de Estudos de Venenos e Animais Peçonhentos) is part of this strategy. The knowledge of the assimilation of carbon and nitrogen isotopes in different tissues or fractions shows the turnover, which suggests the incorporation of diet as a function of feeding time. The traceability involves biological samples that reflect the diet, allowing detection of the inclusion of animal meal in the final product. This method may prevent the transmission of diseases such as Creutzfeldt-Jakob disease, characteristic of human prion, which is also transmitted by the action on the medical use of drugs or implants. The aim was to evaluate the d13C and d15N turnover in serum of bubaline for the certification of "green buffalo" as a donor of biological material in the development of products for medical use. Methods: Three young buffaloes (Murrah) were evaluated, during 117 days, under two different composition of isotopic feeding: 1) Diet with vegetable protein source, constituted by C3 e C4 photosynthetic plants species (0-21 days); 2) Diet with animal protein source, constituted by bovine meat and bone meal (22-117 days). Isotopic ratios of carbon (13C/12C) and nitrogen (15N/14N) were measured in a Mass Spectrometer Delta V Advantage Isotope Ratio MS. To quantitatively measure the speed of isotopes replacement, after determining the time interval, we used the exponential function of time (turnover), expressed by equation 1: d &13C/15N (t) ¼ d & 13C/15N (f) þ [d & 13C/15N (i) - d & 13C/15N (f)] e-kt.. The half life of Carbon-13 and Nitrogen-15, provided 50% of each diet (t ¼ T), was calculated by the equation 2: T ¼ ln 2/k. Results: Isotopic values of carbon-13 were initially expressed by d13C ¼ -12,93 0,20&, reaching values of d13C ¼ -11,83 0,16& to 117 days. For nitrogen-15, the values ranged from d15N ¼ 7,43 0,45& to d15N ¼ 8,68 0,24&. The half-life values were T ¼ 11.7 days for carbon-13 and T ¼ 14.6 days for nitrogen-15. Conclusion: The evaluation time of 95 days is sufficient to incorporate the isotopic signal of diet supplemented with animal protein, fundamental for the certification of "green buffalo" such a donor of biological material.
Keywords: PLA2, Crotalus viridis viridis, Leishmania sp., Leishmaniasis 10.1016/j.toxicon.2012.04.045
Keywords: animal toxins, stable isotopes, fibrin sealant 10.1016/j.toxicon.2012.04.046
44. Leishmanicidal Effects of a Phospholipase A2 Isolated from Crotalus viridis viridis Snake Venom Camila M. Adade 1, S. Fernandes Anne Cristine 1, O. Carvalho Ana Lúcia 2, Russolina B. Zingali 2, Thais Souto-Padrón 1 1 Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil 2 Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil E-mail address: [email protected] (C.M. Adade).
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45. Carbon-13 and Nitrogen-15 Turnover in Serum of Bubaline Donors of Biological Material for Medical Use Daniela A. Fossato da Silva 1, Natália P. Biscola 1, Renato M.F. Souza 1, Denis A. Caetano 1, Juliana C. Denadai 2, Maria M.P. Sartori 2, Evandro T. da Silva 2, Carlos Ducatti 2, Cyntia L. Martins 3, André M. Jorge 3, Lucilene D. dos Santos 1, Rui S. Ferreira Junior 1, Benedito Barraviera 1 1 Centro de Estudos de Venenos e Animais Peçonhentos (CEVAP), Faculdade de Medicina, Univ. Estadual Paulista, Botucatu, SP, Brazil 2 Centro de Isótopos Estáveis (CIE), Univ. Estadual Paulista, Botucatu, SP, Brazil 3 Faculdade de Medicina Veterinária e Zootecnia, Univ. Estadual Paulista, Botucatu, SP, Brazil E-mail address: [email protected] (D.A. Fossato da Silva).
Background: Treatment of Leishmaniasis, caused by Leishmania sp., a disease which afflicts millions of people worldwide, is based on drugs as amphotericin B, pentavalent antimonial and pentamidine, which exhibit toxic effects and limited efficacy. Therefore the search for new drugs is a lining research to be exploited. Several toxins have been used as therapeutic agents and pharmacological tools for drug development. Snake venom phospholipases, exhibited different in vitro effects such as modulate cell proliferation, bactericidal activity and Plasmodium falciparum inhibition growth. Crotalus viridis viridis (Cvv) phospholipase A2 (PLA2) is a 14 kDa, non-neurotoxic, basic single-chain myotoxin, previously described. This work is based in the Cvv venom PLA2, and its effects over L. amazonensis promastigotes. Methods: The crude venom extract was loaded onto to a reverse phase analytical (C8) column using a high performance liquid chromatographer. A linear gradient of water/acetonitrile with 0.1% trifluoroacetic acid was used. The peak contained the isolated PLA2 (confirmed by SDSPAGE and mass spectrometry) was collected, lyophilized, resuspended in distilled water and its protein content measured. L. amazonensis promastigotes were incubated in Schneider medium, with 0.3125 to 10 mcg/ ml PLA2, at 24oC, and the effect on the cells proliferation was evaluated by counting with a Neubauer chamber, up to 72 h. The parameter used to estimate proliferation inhibition was the IC50, which corresponds to the drug concentration that inhibited 50% cell growth. Parasites viability was assessed by flow cytometry using propidium iodide (PI) labeling, and the morphological alterations were examined by light microscopy through Giemsa staining. Results: The treatment caused a significant inhibition (32% to 82%) in the parasites growth, dosis- and timedependent, soon the first 24 hours. The data obtained allowed us to estimate the IC50/ 24 h of 2.50 1.42 mcg/ ml, decreasing to 0.77 0.5 mcg/ ml after the final 72 h. The morphological alterations presented by treated parasites were rounded and unusual cell body shapes, with loss of membrane integrity, corroborated by the 96.6% of PI-positive labeling. Discussion: We presented the employee of Cvv PLA2 against L. amazonensis promastigotes, which inhibited the parasites in vitro growth. Further studies by electron microscopy are underway in order to detect the intracellular targets and the PLA2 effects over amastigotes forms. Conclusions: This work presented that Cvv PLA2 can be object of further research for a new leishmanicidal agent, as we have looked forward to achieving. Financial Support: CAPES, CNPq and FAPERJ.
Background: Toxicology is a fascinating area for potential bioprospect molecules in the development of new drugs. The synergism between the animal toxins and biological materials can be used to formulate a new compound. In this regard, the fibrin sealant produced by CEVAP (Centro de Estudos de Venenos e Animais Peçonhentos) is part of this strategy. The knowledge of the assimilation of carbon and nitrogen isotopes in different tissues or fractions shows the turnover, which suggests the incorporation of diet as a function of feeding time. The traceability involves biological samples that reflect the diet, allowing detection of the inclusion of animal meal in the final product. This method may prevent the transmission of diseases such as Creutzfeldt-Jakob disease, characteristic of human prion, which is also transmitted by the action on the medical use of drugs or implants. The aim was to evaluate the d13C and d15N turnover in serum of bubaline for the certification of "green buffalo" as a donor of biological material in the development of products for medical use. Methods: Three young buffaloes (Murrah) were evaluated, during 117 days, under two different composition of isotopic feeding: 1) Diet with vegetable protein source, constituted by C3 e C4 photosynthetic plants species (0-21 days); 2) Diet with animal protein source, constituted by bovine meat and bone meal (22-117 days). Isotopic ratios of carbon (13C/12C) and nitrogen (15N/14N) were measured in a Mass Spectrometer Delta V Advantage Isotope Ratio MS. To quantitatively measure the speed of isotopes replacement, after determining the time interval, we used the exponential function of time (turnover), expressed by equation 1: d &13C/15N (t) ¼ d & 13C/15N (f) þ [d & 13C/15N (i) - d & 13C/15N (f)] e-kt.. The half life of Carbon-13 and Nitrogen-15, provided 50% of each diet (t ¼ T), was calculated by the equation 2: T ¼ ln 2/k. Results: Isotopic values of carbon-13 were initially expressed by d13C ¼ -12,93 0,20&, reaching values of d13C ¼ -11,83 0,16& to 117 days. For nitrogen-15, the values ranged from d15N ¼ 7,43 0,45& to d15N ¼ 8,68 0,24&. The half-life values were T ¼ 11.7 days for carbon-13 and T ¼ 14.6 days for nitrogen-15. Conclusion: The evaluation time of 95 days is sufficient to incorporate the isotopic signal of diet supplemented with animal protein, fundamental for the certification of "green buffalo" such a donor of biological material.
Keywords: PLA2, Crotalus viridis viridis, Leishmania sp., Leishmaniasis 10.1016/j.toxicon.2012.04.045
Keywords: animal toxins, stable isotopes, fibrin sealant 10.1016/j.toxicon.2012.04.046