RESUMO

Snake venoms are rich sources of active proteins that have been employed in the diagnosis and treatment of health disorders and antivenom therapy. Developing countries demand fast economical downstream processes for the purification of this biomolecule type without requiring sophisticated equipment. We developed an alternative, simple and easy to scale-up method, able to purify simultaneously protease and phospholipase A2 toxins from Bothrops alternatus venom. It comprises a multiple-step partition procedure with polyethylene-glycol/phosphate aqueous two-phase systems followed by a gel filtration chromatographic step. Two single bands in SDS-polyacrylamide gel electrophoresis and increased proteolytic and phospholipase A2 specific activities evidence the homogeneity of the isolated proteins.(AU)

Assuntos

RESUMO

Cross-neutralization of Crotalus durissus terrificus venom coagulant activity was tested using bivalent horse antivenom against Bothrops alternatus and Bothrops diporus venoms. Our in vitro and in vivo experiments showed that bothropic antivenom neutralizes the thrombin-like activity of crotalic snake venom and this cross-reaction was demonstrated by immunoassays either with whole venom or a purified thrombin-like enzyme. These results suggest common antigenic properties and, consequently, similar molecular structure among venom thrombin-like enzymes. Besides, they provide information that could be further used in the development of new antivenom formulations.(AU)

Assuntos

RESUMO

Snake venoms are rich sources of active proteins that have been employed in the diagnosis and treatment of health disorders and antivenom therapy. Developing countries demand fast economical downstream processes for the purification of this biomolecule type without requiring sophisticated equipment. We developed an alternative, simple and easy to scale-up method, able to purify simultaneously protease and phospholipase A2 toxins from Bothrops alternatus venom. It comprises a multiple-step partition procedure with polyethylene-glycol/phosphate aqueous two-phase systems followed by a gel filtration chromatographic step. Two single bands in SDS-polyacrylamide gel electrophoresis and increased proteolytic and phospholipase A2 specific activities evidence the homogeneity of the isolated proteins.

Assuntos

RESUMO

Cross-neutralization of Crotalus durissus terrificus venom coagulant activity was tested using bivalent horse antivenom against Bothrops alternatus and Bothrops diporus venoms. Our in vitro and in vivo experiments showed that bothropic antivenom neutralizes the thrombin-like activity of crotalic snake venom and this cross-reaction was demonstrated by immunoassays either with whole venom or a purified thrombin-like enzyme. These results suggest common antigenic properties and, consequently, similar molecular structure among venom thrombin-like enzymes. Besides, they provide information that could be further used in the development of new antivenom formulations.

Assuntos

RESUMO

The therapeutic effects of Argentine propolis ear drop formulation on canine otitis externa were evaluated. Forty-eight dogs with symptoms of otitis externa were randomly assigned to double-blinded, controlled clinical trial to evaluate the efficacy of topical formulation with propolis versus a topical placebo in the treatment of otitis externa. The propolis preparation and placebo were administrated into both external ear canals, twice daily for 14 days. Throughout the study, clinical examination and microbiological analysis of dogs ear exudates were made. The most frequent microorganisms isolated in culture media were: Malassezia pachydermatis (54.2 percent), Staphylococcus aureus (43.8 percent), coagulase-negative Staphylococcus (25.0 percent), Pseudomonas aeruginosa (20.8 percent), Candida albicans (18.8 percent), Proteus mirabilis (16.7 percent), Streptococcus spp. (16.7 percent), Enteroccocus faecalis (12.5 percent), Escherichia coli (12.5 percent), Staphylococcus intermedius (6.3 percent), Klebsiella spp. (4.2 percent), andCandida glabrata (2.1 percent). Whereas the control group did not recover from the infectious ear disease, the propolis preparation exhibited antimicrobial activity against most of the microorganisms isolated from samples of the treated group. In addition, no propolis-adverse effects were observed. This allowed propolis-treated patients to show a significant improvement of the clinical parameters. Thus, this new Argentine propolis ear drop formulation may be used for topical treatment of otitis externa in dogs.(AU)

Os efeitos terapêuticos da formulação em gotas óticas de própolis procedentes da Argentina foram avaliados no tratamento da otite externa canina. Quarenta e oito cães com sintomas de otite externa foram distribuídos aleatoriamente em ensaio clínico duplo-cego controlado para avaliar a eficácia da formulação tópica com a própolis contra um placebo tópico no tratamento da otite externa. A preparação de própolis e placebo foi administrada em ambos os canais da orelha externa, duas vezes por dia, durante 14 dias. Ao longo do estudo, os cães foram submetidos a exame físico e à análise microbiológica de exsudatos auriculares. Os mais frequentes microrganismos isolados em meios de cultura foram: Malassezia pachydermatis (54,2 por cento), Staphylococcus aureus (43,8 por cento), Staphylococcus coagulase-negativo (25,0 por cento), Pseudomonas aeruginosa (20,8 por cento), Candida albicans (18,8 por cento), Proteus mirabilis (16,7 por cento), Streptococcus spp.(16,7 por cento), Enterococcus faecalis (12,5 por cento), Escherichia coli (12,5 por cento), Staphylococcus intermedius (6,3 por cento), Klebsiella spp.(4,2 por cento) e Candida glabrata (2,1 por cento). A preparação de própolis apresentou atividade antimicrobiana contra a maioria dos microrganismos isolados de amostras do grupo de tratamento, sendo que os do grupo-controle não se recuperaram da doença infecciosa auricular, e não foram observados efeitos adversos à própolis. Isso permitiu aos pacientes tratados com própolis melhora significativa dos parâmetros clínicos. Essa nova formulação da própolis argentina para o ouvido apresenta potencial utilidade no tratamento tópico da otite externa em cães.(AU)

Assuntos

RESUMO

The therapeutic effects of Argentine propolis ear drop formulation on canine otitis externa were evaluated. Forty-eight dogs with symptoms of otitis externa were randomly assigned to double-blinded, controlled clinical trial to evaluate the efficacy of topical formulation with propolis versus a topical placebo in the treatment of otitis externa. The propolis preparation and placebo were administrated into both external ear canals, twice daily for 14 days. Throughout the study, clinical examination and microbiological analysis of dogs ear exudates were made. The most frequent microorganisms isolated in culture media were: Malassezia pachydermatis (54.2 percent), Staphylococcus aureus (43.8 percent), coagulase-negative Staphylococcus (25.0 percent), Pseudomonas aeruginosa (20.8 percent), Candida albicans (18.8 percent), Proteus mirabilis (16.7 percent), Streptococcus spp. (16.7 percent), Enteroccocus faecalis (12.5 percent), Escherichia coli (12.5 percent), Staphylococcus intermedius (6.3 percent), Klebsiella spp. (4.2 percent), andCandida glabrata (2.1 percent). Whereas the control group did not recover from the infectious ear disease, the propolis preparation exhibited antimicrobial activity against most of the microorganisms isolated from samples of the treated group. In addition, no propolis-adverse effects were observed. This allowed propolis-treated patients to show a significant improvement of the clinical parameters. Thus, this new Argentine propolis ear drop formulation may be used for topical treatment of otitis externa in dogs.

Os efeitos terapêuticos da formulação em gotas óticas de própolis procedentes da Argentina foram avaliados no tratamento da otite externa canina. Quarenta e oito cães com sintomas de otite externa foram distribuídos aleatoriamente em ensaio clínico duplo-cego controlado para avaliar a eficácia da formulação tópica com a própolis contra um placebo tópico no tratamento da otite externa. A preparação de própolis e placebo foi administrada em ambos os canais da orelha externa, duas vezes por dia, durante 14 dias. Ao longo do estudo, os cães foram submetidos a exame físico e à análise microbiológica de exsudatos auriculares. Os mais frequentes microrganismos isolados em meios de cultura foram: Malassezia pachydermatis (54,2 por cento), Staphylococcus aureus (43,8 por cento), Staphylococcus coagulase-negativo (25,0 por cento), Pseudomonas aeruginosa (20,8 por cento), Candida albicans (18,8 por cento), Proteus mirabilis (16,7 por cento), Streptococcus spp.(16,7 por cento), Enterococcus faecalis (12,5 por cento), Escherichia coli (12,5 por cento), Staphylococcus intermedius (6,3 por cento), Klebsiella spp.(4,2 por cento) e Candida glabrata (2,1 por cento). A preparação de própolis apresentou atividade antimicrobiana contra a maioria dos microrganismos isolados de amostras do grupo de tratamento, sendo que os do grupo-controle não se recuperaram da doença infecciosa auricular, e não foram observados efeitos adversos à própolis. Isso permitiu aos pacientes tratados com própolis melhora significativa dos parâmetros clínicos. Essa nova formulação da própolis argentina para o ouvido apresenta potencial utilidade no tratamento tópico da otite externa em cães.

Assuntos

RESUMO

Due to variability of venom components from the same species of snakes that inhabit different regions, particular properties of the venom of Crotalus durissus terrificus that inhabits the North-East of Argentina were studied. Gyroxin, a thrombin-like enzyme, was isolated from this venom by gel filtration and affinity chromatography, it was found to be homogeneous according to SDS-PAGE, with a molecular weight of 33 kDa. [quot ]Gyroxin syndrome[quot ] in mice was tested and it showed changes in the animal behavior, confirming that the isolated thrombin-like enzyme is gyroxin. Effects of this enzyme and the crude venom on mice plasmatic fibrinogen levels were determined. The mice plasma fibrinogen decreased rapidly until incoagulability during the first hour after thrombin-like enzyme injection, then reaching its normal level 10 hours after injection; whereas crude venom resulted in a 60% decrease of the mice plasma fibrinogen, reaching its normal level after the same period of time. After 1 hour of gyroxin inoculation, intravascular coagulation was observed in histological cuttings of lung, cardiac muscle and liver. The isolated enzyme showed strong hydrolyzing activity on fibrinogen and fibrin in vitro, whereas the crude venom exhibited weak hydrolyzing activity on both substrates. It is probable that this very low activity is due to the low percentage of the enzyme in the crude venom. Decreasing of plasmatic fibrinogen levels may be due to either the coagulant or hydrolyzing actions of the enzyme.

Teniendo en cuenta la variabilidadde los componentes del veneno de serpientes de una misma especie que habitan regiones diferentes, se decidióestudiar las propiedades particulares del veneno de Crotalus durissus terrificus que habita el nordeste de Argentina, Giroxina, una enzima con actividad trombínica, fue aislada del veneno por cromatografía de filtración por gel y de afinidad; se comprobó su homogeneidad y se determinó su peso molecular, 33 kDa, por SDSPAGE. Se ensayó el síndrome giroxina en ratones, los que mostraron cambios en el comportamiento, confirmandoque la enzima tipo trombina aislada es giroxina. Se evaluó la acción de esta enzima sobre los niveles de fibrinógeno plasmático en ratones, comparándola con la del veneno crudo. Se comprobó que la enzima provoca una disminución de los niveles plasmáticos de fibrinógeno hasta la incoagulabilidad, durante la primer hora de inoculación, mientras que el veneno entero produjo una reducción del nivel plasmático en un 60%; sin embargo, en ambos casos, se evidenció una rápida reposición de fibrinógeno plasmático, alcanzando sus valores normales en un plazo de 10 horas. Se observó coagulación intravascular con la administración de giroxina una hora después de la inoculación, evidenciados en estudios histológicos de tejido pulmonar, cardíaco y hepático. En ensayos realizados in vitro, la enzima aislada mostró capacidad de degradar fibrinógeno como así también coágulos de fibrina, mientras que el veneno exhibió débil actividad hidrolítica sobre ambos sustratos. Es probable que esta baja actividad sea debida a la baja concentración de la enzima en el veneno. La disminución de los niveles de fibrinógeno plasmático observado en ratones se debería a la acción coagulante de la enzima, sin embargo no se descarta que también contribuya a este proceso una acción hidrolítica sobrefibrinógeno y fibrina por parte de la enzima.

Assuntos

RESUMO

Due to variability of venom components from the same species of snakes that inhabit different regions, particular properties of the venom of Crotalus durissus terrificus that inhabits the North-East of Argentina were studied. Gyroxin, a thrombin-like enzyme, was isolated from this venom by gel filtration and affinity chromatography, it was found to be homogeneous according to SDS-PAGE, with a molecular weight of 33 kDa. [quot ]Gyroxin syndrome[quot ] in mice was tested and it showed changes in the animal behavior, confirming that the isolated thrombin-like enzyme is gyroxin. Effects of this enzyme and the crude venom on mice plasmatic fibrinogen levels were determined. The mice plasma fibrinogen decreased rapidly until incoagulability during the first hour after thrombin-like enzyme injection, then reaching its normal level 10 hours after injection; whereas crude venom resulted in a 60% decrease of the mice plasma fibrinogen, reaching its normal level after the same period of time. After 1 hour of gyroxin inoculation, intravascular coagulation was observed in histological cuttings of lung, cardiac muscle and liver. The isolated enzyme showed strong hydrolyzing activity on fibrinogen and fibrin in vitro, whereas the crude venom exhibited weak hydrolyzing activity on both substrates. It is probable that this very low activity is due to the low percentage of the enzyme in the crude venom. Decreasing of plasmatic fibrinogen levels may be due to either the coagulant or hydrolyzing actions of the enzyme.(AU)

Teniendo en cuenta la variabilidadde los componentes del veneno de serpientes de una misma especie que habitan regiones diferentes, se decidióestudiar las propiedades particulares del veneno de Crotalus durissus terrificus que habita el nordeste de Argentina, Giroxina, una enzima con actividad trombínica, fue aislada del veneno por cromatografía de filtración por gel y de afinidad; se comprobó su homogeneidad y se determinó su peso molecular, 33 kDa, por SDSPAGE. Se ensayó el síndrome giroxina en ratones, los que mostraron cambios en el comportamiento, confirmandoque la enzima tipo trombina aislada es giroxina. Se evaluó la acción de esta enzima sobre los niveles de fibrinógeno plasmático en ratones, comparándola con la del veneno crudo. Se comprobó que la enzima provoca una disminución de los niveles plasmáticos de fibrinógeno hasta la incoagulabilidad, durante la primer hora de inoculación, mientras que el veneno entero produjo una reducción del nivel plasmático en un 60%; sin embargo, en ambos casos, se evidenció una rápida reposición de fibrinógeno plasmático, alcanzando sus valores normales en un plazo de 10 horas. Se observó coagulación intravascular con la administración de giroxina una hora después de la inoculación, evidenciados en estudios histológicos de tejido pulmonar, cardíaco y hepático. En ensayos realizados in vitro, la enzima aislada mostró capacidad de degradar fibrinógeno como así también coágulos de fibrina, mientras que el veneno exhibió débil actividad hidrolítica sobre ambos sustratos. Es probable que esta baja actividad sea debida a la baja concentración de la enzima en el veneno. La disminución de los niveles de fibrinógeno plasmático observado en ratones se debería a la acción coagulante de la enzima, sin embargo no se descarta que también contribuya a este proceso una acción hidrolítica sobrefibrinógeno y fibrina por parte de la enzima. (AU)

Assuntos