RESUMO
We show that ethyl 2-oxo-2H-chromene-3-carboxylate (EOCC), a synthetic coumarin, irreversibly inhibits phospholipase A(2) (sPLA2) from Crotalus durissus ruruima venom (sPLA2r) with an IC(50) of 3.1 +/- 0.06 nmol. EOCC strongly decreased the V(max) and K(m), and it virtually abolished the enzyme activity of sPLA2r as well as sPLA2s from other sources. The edema induced by sPLA2r + EOCC was less than that induced by sPLA2r treated with p-bromophenacyl bromide, which was more efficient at neutralizing the platelet aggregation activity of native sPLA2r. Native sPLA2r induced platelet aggregation of 91.54 +/- 9.3%, and sPLA2r + EOCC induced a platelet aggregation of 18.56 +/- 6.5%. EOCC treatment also decreased the myotoxic effect of sPLA2r. Mass spectrometry showed that EOCC formed a stable complex with sPLA2r, which increased the mass of native sPLA2r from 14,299.34 Da to 14,736.22 Da. Moreover, the formation of this complex appeared to be involved in the loss of sPLA2r activity. Our results strongly suggest that EOCC can be used as a pharmacological agent against the sPLA2 in Crotalus durissus sp. venom as well as other sPLA2s.
Assuntos
Antivenenos/farmacologia , Cumarínicos/farmacologia , Venenos de Crotalídeos/enzimologia , Crotalus/fisiologia , Edema/prevenção & controle , Inibidores de Fosfolipase A2 , Agregação Plaquetária/efeitos dos fármacos , Animais , Edema/induzido quimicamente , Inibidores Enzimáticos/farmacologia , Masculino , Fosfolipases A2/farmacologia , Ratos , Ratos WistarRESUMO
Snake venom proteins from the C-type lectin family have very distinct biological activities despite their highly conserved primary structure, which is homologous to the carbohydrate recognition region of true C-type lectins. We purified a lectin-like protein (BmLec) from Bothrops moojeni venom and investigated its effect on platelet aggregation, insulin secretion, antibacterial activity, and isolated kidney cells. The BmLec was purified using two chromatographic steps: affinity chromatography and reverse phase high performance liquid chromatography (HPLC). BmLec showed a dose-dependent platelet aggregation and significantly decreased the bacterial growth rate in approximately 15%. During scanning electron microscopy, the profile of Xanthomonas axonopodis pv. passiflorae treated with lectin disclosed a high vesiculation and membrane rupture. BmLec induced a strong and significant increase in insulin secretion at 2.8 and 16.7 mM glucose concentrations, and this effect was seen in the presence of EGTA in both experiments. BmLec (10 ìg/mL) increased the perfusion pressure, renal vascular resistance and urinary flow. The glomerular filtration rate and percentages of sodium, potassium and chloride tubular transport were reduced at 60 minutes of perfusion. Renal alterations caused by BmLec were completely inhibited by indomethacin in all evaluated parameters. In conclusion, the C-type lectin isolated from Bothrops moojeni affected platelet aggregation, insulin secretion, antibacterial activity and isolated kidney function.(AU)
Assuntos
Humanos , Animais , Lectinas Tipo C/análise , Serpentes/classificação , Venenos de Serpentes/análise , Insulina/análise , Antibacterianos/biossínteseRESUMO
Snake venom proteins from the C-type lectin family have very distinct biological activities despite their highly conserved primary structure, which is homologous to the carbohydrate recognition region of true C-type lectins. We purified a lectin-like protein (BmLec) from Bothrops moojeni venom and investigated its effect on platelet aggregation, insulin secretion, antibacterial activity, and isolated kidney cells. The BmLec was purified using two chromatographic steps: affinity chromatography and reverse phase high performance liquid chromatography (HPLC). BmLec showed a dose-dependent platelet aggregation and significantly decreased the bacterial growth rate in approximately 15 percent. During scanning electron microscopy, the profile of Xanthomonas axonopodis pv. passiflorae treated with lectin disclosed a high vesiculation and membrane rupture. BmLec induced a strong and significant increase in insulin secretion at 2.8 and 16.7 mM glucose concentrations, and this effect was seen in the presence of EGTA in both experiments. BmLec (10 µg/mL) increased the perfusion pressure, renal vascular resistance and urinary flow. The glomerular filtration rate and percentages of sodium, potassium and chloride tubular transport were reduced at 60 minutes of perfusion. Renal alterations caused by BmLec were completely inhibited by indomethacin in all evaluated parameters. In conclusion, the C-type lectin isolated from Bothrops moojeni affected platelet aggregation, insulin secretion, antibacterial activity and isolated kidney function.
Assuntos
Animais , Bothrops , Venenos de Crotalídeos , Insulina , Rim , Lectinas Tipo C/isolamento & purificação , Agregação Plaquetária , Cromatografia Líquida de Alta Pressão/métodosRESUMO
ABSTRACT Recently, generalized blight symptoms were observed in tomato leaves of the types Salad and Italian, at two commercial fields located in Bragança Paulista and Mogi Guaçú, SP, Brazil. The presence of bacterial exudation was verified in observations of infected tissues under an optical microscope. Isolation attempts allowed the recovery of rod-shaped, Gram-negative bacteria, in white colonies that produced fluorescent pigment in Kings B medium. Bacterial isolates were submitted to LOPAT tests and classified into group III (+ +); consequently, they were identified as Pseudomonas cichorii. These results were corroborated by indirect immunofluorescence tests, using antisera produced for the type strain of P. cichorii. This bacterium causes diseases in several crops of economic importance, and had not yet been observed on tomatoes in Brazil. Bacterial isolates have been deposited with the Phytobacteria Culture Collection of the Instituto Biológico, under accession numbers IBSBF 2309 and IBSBF 2323.
RESUMO Recentemente, em dois campos comerciais de tomateiro dos tipos Salada e Italiano, localizados em Bragança Paulista e Mogi Guaçú, SP, foram observados sintomas de queima generalizada nas folhas. Em observações, ao microscópio óptico, de tecidos infectados, foi constatada a presença de exsudação bacteriana. Desses tecidos, foram isoladas bactérias em formato bastonete, Gramnegativas, com colônias de coloração branca e produtoras de pigmento fluorescente em meio B de King. Isolados bacterianos foram submetidos aos testes LOPAT, sendo enquadrados no grupo III (+ +) e, portanto, identificados como sendo Pseudomonas cichorii. Esses resultados foram corroborados por testes serológicos de imunofluorescência indireta, com antissoros produzidos para linhagem tipo de P. cichorii. Esta bactéria causa doença em várias culturas de importância econômica e ainda não havia sido constatada no Brasil na cultura de tomateiro. Os isolados bacterianos foram depositados na Coleção de Culturas de Fitobactérias do Instituto Biológico, sob os números de acesso IBSBF 2309 e IBSBF 2323.
RESUMO
The venom of Crotalus durissus terrificus was fractionated by reverse-phase HPLC to obtain crotapotins (F5 and F7) and PLA2 (F15, F16, and F17) of high purity. The phospholipases A2 (PLA2S) and crotapotins showed antimicrobial activity against Xanthomonas axonopodis pv. passiflorae, although the unseparated crotoxin did not. The F17 of the PLA2 also revealed significant anticoagulant activity, althrough for this to occur the presence of Glu 53 and Trp 61 is important. The F17 of the PLA2 showed allosteric behavior in the presence of a synthetic substrate. The amino acid sequence of this PLA2 isoform, determined by automatic sequencing, was HLLQFNKMLKFETRK NAVPFYAFGCYCGWGGQRRPKDATDRCCFVHDCCYEKVTKCNTKWDFYRYSLKSGY ITCGKGTWCKEQICECDRVAAECLRRSLSTYKNEYMFYPDSRCREPSETC. Analysis showed that the sequence of this PLA2 isoform differed slightly from the amino acid sequence of the basic crotoxin subunit reported in the literature. The homology with other crotalid PLA2 cited in the literature varied from 60% to 90%. The pL was estimated to be 8.15, and the calculated molecular weight was 14664.14 as determined by Tricine SDS-PAGE, two-dimensional electrophoresis, and MALDI-TOFF. These results also suggested that the enzymatic activity plays an important role in the bactericidal effect of the F17 PLA2 as well as that of anticoagulation, although other regions of the molecule may also be involved in this biological activity.