RESUMO
NADPH is a key reductant carrier that maintains internal redox and antioxidant status, and that links biosynthetic, catabolic and signalling pathways. Plants have a mitochondrial external NADPH oxidation pathway, which depends on Ca2+ and pH in vitro, but concentrations of Ca2+ needed are not known. We have determined the K0.5(Ca2+) of the external NADPH dehydrogenase from Solanum tuberosum mitochondria and membranes of E. coli expressing Arabidopsis thaliana NDB1 over the physiological pH range using O2 and decylubiquinone as electron acceptors. The K0.5(Ca2+) of NADPH oxidation was generally higher than for NADH oxidation, and unlike the latter, it depended on pH. At pH 7.5, K0.5(Ca2+) for NADPH oxidation was high (≈100 µM), yet 20-fold lower K0.5(Ca2+) values were determined at pH 6.8. Lower K0.5(Ca2+) values were observed with decylubiquinone than with O2 as terminal electron acceptor. NADPH oxidation responded to changes in Ca2+ concentrations more rapidly than NADH oxidation did. Thus, cytosolic acidification is an important activator of external NADPH oxidation, by decreasing the Ca2+-requirements for NDB1. The results are discussed in relation to the present knowledge on how whole cell NADPH redox homeostasis is affected in plants modified for the NDB1 gene.
Assuntos
Arabidopsis/enzimologia , Cálcio/metabolismo , Citosol/metabolismo , Mitocôndrias/enzimologia , NADPH Desidrogenase/metabolismo , Solanum tuberosum/enzimologia , Elétrons , Concentração de Íons de Hidrogênio , NADP/metabolismo , Oxirredução , Quinonas/metabolismoRESUMO
In the present study, the in vitro cytotoxicity of daidzein was evaluated in human BEL-7402, A549, HeLa, HepG-2 and MG-63 cancer cell lines. BEL-7402 cells were sensitive to daidzein treatment, with an IC50 value of 59.7±8.1 µM. Daidzein showed no cytotoxic activity toward A549, HeLa, HepG-2 and MG-63 cells. Daidzein increased the levels of reactive oxygen species (ROS) and induced a decrease in mitochondrial membrane potential. Morphological and comet assays showed that daidzein effectively induced apoptosis in BEL-7402 cells. Additionally, daidzein caused cell cycle arrest at the G2/M phase in the BEL-7402 cell line. Daidzein downregulated the expression of Bcl-2, Bcl-x and Baid proteins and upregulated the levels of Bim protein in the BEL-7402 cells. The results demonstrated that daidzein induced BEL-7402 cell apoptosis through an ROS-mediated mitochondrial dysfunction pathway.
Assuntos
Apoptose/efeitos dos fármacos , Isoflavonas/farmacologia , Fitoestrógenos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Western Blotting , Caspases/biossíntese , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaio Cometa , Citometria de Fluxo , Humanos , Técnicas In Vitro , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/biossínteseRESUMO
Cytosolic NADPH can be directly oxidized by a calcium-dependent NADPH dehydrogenase, NDB1, present in the plant mitochondrial electron transport chain. However, little is known regarding the impact of modified cytosolic NADPH reduction levels on growth and metabolism. Nicotiana sylvestris plants overexpressing potato (Solanum tuberosum) NDB1 displayed early bolting, whereas sense suppression of the same gene led to delayed bolting, with consequential changes in flowering time. The phenotype was dependent on light irradiance but not linked to any change in biomass accumulation. Whereas the leaf NADPH/NADP(+) ratio was unaffected, the stem NADPH/NADP(+) ratio was altered following the genetic modification and strongly correlated with the bolting phenotype. Metabolic profiling of the stem showed that the NADP(H) change affected relatively few, albeit central, metabolites, including 2-oxoglutarate, glutamate, ascorbate, sugars, and hexose-phosphates. Consistent with the phenotype, the modified NDB1 level also affected the expression of putative floral meristem identity genes of the SQUAMOSA and LEAFY types. Further evidence for involvement of the NADPH redox in stem development was seen in the distinct decrease in the stem apex NADPH/NADP(+) ratio during bolting. Additionally, the potato NDB1 protein was specifically detected in mitochondria, and a survey of its abundance in major organs revealed that the highest levels are found in green stems. These results thus strongly suggest that NDB1 in the mitochondrial electron transport chain can, by modifying cell redox levels, specifically affect developmental processes.
Assuntos
Proteínas Mitocondriais/metabolismo , NADPH Desidrogenase/metabolismo , Nicotiana/enzimologia , Oxirredução , Proteínas de Plantas/metabolismo , Flores/genética , Flores/crescimento & desenvolvimento , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas Mitocondriais/genética , NADPH Desidrogenase/genética , Proteínas de Plantas/genética , Caules de Planta/enzimologia , Caules de Planta/genética , Caules de Planta/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Solanum tuberosum/genética , Nicotiana/genética , Nicotiana/crescimento & desenvolvimentoRESUMO
The modified Cry1Ac was expressed in transgenic tobacco plants. To allow secretion of the Cry1Ac protein into the intercellular space, the signal peptide sequence of potato proteinase inhibitor II (pinII) was N-terminally fused to the Cry1Ac encoding region. Expression of Cry1Ac in transgenic tobacco plants was assayed with ELISA. The results showed that pinII signal peptide sequence enhanced the expression of Cry1Ac protein and led to the secretion of the Cry1Ac protein in transgenic tobacco plants. GFP gene was also fused to the signal peptide sequence and transformed to tobacco. The results of fluorescent detection showed that GFP had localized in the apoplast of transgenic plants.