RESUMO
Clostridium botulinum types C and D cause animal botulism by the production of serotype-specific or mosaic botulinum neurotoxin (BoNT). The D/C mosaic BoNT (BoNT/DC), which is produced by the isolate from bovine botulism in Japan, exhibits the highest toxicity to mice among all BoNTs. In contrast, rats appeared to be very resistant to BoNT/DC in type C and D BoNTs and their mosaic BoNTs. We attempted to characterize the enzymatic and receptor-binding activities of BoNT/DC by comparison with those of type C and D BoNTs (BoNT/C and BoNT/D). BoNT/DC and D showed similar toxic effects on cerebellar granule cells (CGCs) derived from the mouse, but the former showed less toxicity to rat CGCs. In recombinant murine-derived vesicle-associated membrane protein (VAMP), the enzymatic activities of both BoNTs to rat isoform 1 VAMP (VAMP1) were lower than those to the other VAMP homologues. We then examined the physiological significance of gangliosides as the binding components for types C and D, and mosaic BoNTs. BoNT/DC and C were found to cleave an intracellular substrate of PC12 cells upon the exogenous addition of GM1a and GT1b gangliosides, respectively, suggesting that each BoNT recognizes a different ganglioside moiety. The effect of BoNT/DC on glutamate release from CGCs was prevented by cholera toxin B-subunit (CTB) but not by a site-directed mutant of CTB that did not bind to GM1a. Bovine adrenal chromaffin cells appeared to be more sensitive to BoNT/DC than to BoNT/C and D. These results suggest that a unique mechanism of receptor binding of BoNT/DC may differentially regulate its biological activities in animals.
Assuntos
Toxinas Botulínicas/toxicidade , Clostridium botulinum/metabolismo , Neurotoxinas/toxicidade , Glândulas Suprarrenais/citologia , Animais , Toxinas Botulínicas/classificação , Toxinas Botulínicas/metabolismo , Bovinos , Cerebelo/citologia , Células Cromafins/efeitos dos fármacos , Feminino , Gangliosídeos/metabolismo , Camundongos , Neurotoxinas/classificação , Neurotoxinas/metabolismo , Células PC12 , Ligação Proteica , Ratos , Proteínas Recombinantes , Especificidade da EspécieRESUMO
BACKGROUND AND AIMS: Previous studies have shown that transgenic rice plants overexpressing YK1, which possesses dihydroflavonol-4-reductase (DFR) activity, showed biotic and abiotic stress tolerance. High throughput profiles of metabolites have also been shown in such transgenic plants by Fourier transform ion cyclotron mass spectrometry. In this study, capillary electrophoresis mass spectrometry analysis (CE/MS) was employed to identify precise metabolites such as organic acids, amino acids and sugars. METHODS: Using CE/MS, we analysed several metabolites of glycolysis, the tricarboxylic acid (TCA) cycle and the pentose phosphate pathway. In addition, the concentrations of sugars and ion were quantified. KEY RESULTS: In YK1 (DFR)-overexpressing plants, the concentrations of cis-aconitate, isocitrate and 2-oxoglutarate were higher in leaves, whereas those of fructose-1,6-bisphosphate and glyceraldehyde-3-phosphate were lower in roots. In seeds, the amounts of free amino acids and metals were altered, whereas sugars in seeds were kept constant. In YK1 calli, an approx. 3-fold increase in glutathione was observed, whereas the activities of glutathione peroxidase and glutathione reductase were concomitantly increased. CONCLUSIONS: The overexpression of YK1 (DFR) was associated with slight changes in the amounts of several metabolites analysed in whole plants, whilst glutathione derivatives were substantially increased in suspension-cultured cells.
Assuntos
Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Regulação da Expressão Gênica de Plantas , Oryza/genética , Oryza/metabolismo , Aminoácidos/metabolismo , Glutationa/metabolismo , Oryza/enzimologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Sementes/genética , Sementes/metabolismoRESUMO
The maize Hm1 gene encoding the NADPH-dependent HC-toxin reductase is capable of detoxifying HC-toxin of fungus Cochliobolus carbonum. Here, we conducted the metabolic and biochemical analysis in transgenic rice plants overexpressing an HC-toxin reductase-like gene in rice (YK1 gene). Methods employing NADPH oxidation and capillary electrophoresis mass spectrometry analysis confirmed that YK1 possessed dihydroflavonol-4-reductase activity in vitro and in vivo. The overexpression of YK1 in both suspension-cultured cells and rice plants increased NAD(H) and NADP(H) levels by causing an increase in NAD synthetase and NAD kinase activities. Activity changes in enzymes that require NAD(P) as coenzymes were also noted in rice cells ectopically expressing YK1, where the cell death caused by hydrogen peroxide and bacterial disease was down-regulated. Thus, a strategy was proposed that the combination of dihydroflavonol-4-reductase activity and the elevated level of NAD(P)H pool may confer the prevention of induced cell death in planta.