Inhibition of the mitochondrial respiratory components (Complex I and Complex III) as stimuli to induce oxidative damage in Oryza sativa L. under thiocyanate exposure.

Repression of the electron transport in mitochondria can result in an increase of reactive oxygen species (ROS) in plant cells. This study was to clarify inhibition of the mitochondrial respiratory components (Complex I and Complex III) as stimuli to induce oxidative damage in Oryza sativa L. under exogenous SCN- exposure with special emphasis on lipid peroxidation, protein modification, and DNA damage at the biochemical and molecular levels. Our results showed that enzymatic activity and gene expression of cytochrome c reductase (Complex III) in roots and shoots of rice seedlings were significantly repressed by SCN- exposure, where significant inhibition of NADH dehydrogenase (Complex I) was only detected in shoots, suggesting that Complex III was the main target attacked by SCN- ligand in rice roots, and both components were arrested in shoots. ROS analysis in tissues indicated that SCN- exposure caused significant accumulation of H2O2 and O2-•, increased malondialdehyde (MDA) and carbonyl content in rice materials in a dose-dependent manner. Similarly, a remarkable elevation of electrolyte leakage was observed in rice tissue samples. The comet assay indicated a positive correlation between DNA damage and external SCN- exposure. In conclusion, oxidative burst generated from the inhibitions of the electron transport in mitochondria in rice seedlings under SCN- exposure can cause lipid peroxidation, protein modification and DNA damage, eventually decreasing fresh weight of rice seedlings.

Knockdown of electron transfer flavoprotein ß subunit reduced TGF-ß-induced α-SMA mRNA expression but not COL1A1 in fibroblast-populated three-dimensional collagen gel cultures.

BACKGROUND: The inhibition of transforming growth factor ß (TGF-ß)-induced myofibroblast differentiation is a key objective for the treatment of hypertrophic scarring. We previously reported that knockdown of the electron transfer flavoprotein ß subunit (ETFB) reduced mechanoregulated cell number in fibroblast-populated collagen gel cultures [1]. OBJECTIVE: To characterize the effects of ETFB knockdown, we investigated gel contraction, TGF-ß-induced collagen, α-SMA mRNA expression and stress fiber formation. METHODS: Fibroblasts were transfected with negative control or ETFB-specific siRNAs and embedded in collagen gels in an attached or detached condition. The gel contraction assay was performed in three different concentrations of collagen (0.5, 1.0 or 1.5mg/mL) and was analyzed by measuring the changes in the gel area throughout the culture period. The attached collagen gel culture was performed in the presence of rTGF-ß and the mRNA levels of α-SMA and COL1A1 were measured by qRT-PCR. The effect of ETFB knockdown on proliferation and stress fiber organization in monolayer cultures was investigated by conducting AlamarBlue assays and phalloidin staining. RESULTS: The transfection of ETFB siRNA did not alter gel contraction compared to the negative control in all collagen concentrations. When the cells were treated with TGF-ß under mechanical stress conditions, ETFB knockdown attenuated α-SMA mRNA expression to a level comparable to that observed in the absence of TGF-ß. However, no inhibitory effect on COL1A1 mRNA levels was observed. The AlamarBlue assay indicated that the knockdown had no effect on the proliferation of cells cultured on plastic. Phalloidin staining of a monolayer culture showed that ETFB knockdown weakened the stress fiber organization induced by rTGF-ß. CONCLUSION: ETFB knockdown can affect TGF-ß-induced tissue remodeling and/or fibrotic processes in vitro.

Binding of the human "electron transferring flavoprotein" (ETF) to the medium chain acyl-CoA dehydrogenase (MCAD) involves an arginine and histidine residue.

The interaction between the "electron transferring flavoprotein" (ETF) and medium chain acyl-CoA dehydrogenase (MCAD) enables successful flavin to flavin electron transfer, crucial for the beta-oxidation of fatty acids. The exact biochemical determinants for ETF binding to MCAD are unknown. Here we show that binding of human ETF, to MCAD, was inhibited by 2,3-butanedione and diethylpyrocarbonate (DEPC) and reversed by incubation with free arginine and hydroxylamine respectively. Spectral analyses of native ETF vs modified ETF suggested that flavin binding was not affected and that the loss of ETF activity with MCAD involved modification of one ETF arginine residue and one ETF histidine residue respectively. MCAD and octanoyl-CoA protected ETF against inactivation by both 2,3-butanedione and DEPC indicating that the arginine and histidine residues are present in or around the MCAD binding site. Comparison of exposed arginine and histidine residues among different ETF species, however, indicates that arginine residues are highly conserved but that histidine residues are not. These results lead us to conclude that this single arginine residue is essential for the binding of ETF to MCAD, but that the single histidine residue, although involved, is not.