Since de‐novo synthesis of pyrimidine nucleotides is coupled to the mitochondrial respiratory chain (RC) via dehydroorotic acid dehydrogenase (DHODH), respiratory chain dysfunction should impair pyrimidine synthesis. To investigate this, we used specific RC inhibitors, Antimycin A and Rotenone, to treat primary human keratinocytes and 143B cells, a human osteosarcoma cell line, in culture. This resulted in severe impairment of de novo pyrimidine nucleotide synthesis. The effects of RC inhibition were not restricted to pyrimidine synthesis, but concerned purine nucleotides, too. While the total amount of purine nucleotides was not diminished, they were significantly broken down from triphosphates to monophosphates, reflecting impaired mitochondrial ATP regeneration. The effect of Rotenone was similar to that of Antimycin A. This was surprising since Rotenone inhibits complex I of the respiratory chain, which is upstream of ubiquinone where DHODH interacts with the RC. In order to avoid unspecific effects of Rotenone, we examined the consequences of a mitochondrial DNA mutation that causes a specific complex I defect. The effect was much less pronounced than with Rotenone, suggesting that complex I inhibiton cannot fully explain the marked effect of Rotenone on pyrimidine nucleotide synthesis. 相似文献
31P-NMR studies of intact functional rat liver mitochondria at 37°C demonstrate that the large majority (?95%) of endogenous phospholipids exhibit motional properties consistent with bilayer structure. This property is unaffected by oxidative phosphorylation processes or the presence of Ca2+. 相似文献
Researchers working with fission yeast conduct protein extraction widely and frequently, but this includes the handling of glass beads, and hence is laborious and cumbersome, especially when dealing with a large number of samples. Here we describe a rapid and reliable method for preparing protein extract from fission yeast, one which is applicable to routine western blotting. 相似文献
Novel cationic dimethylaminopyridine derivatives of pentacyclic triterpenes were previously described to promote mitochondrial depolarization and cell death in breast and melanoma cell lines. The objective of this work was to further investigate in detail the mechanism of mitochondrial perturbations, correlating those effects with breast cancer cell responses to those same agents. Initially, a panel of tumor and non-tumor cell lines was grown in high-glucose or glucose-free glutamine-containing media, the later forcing cells to synthesize ATP by oxidative phosphorylation only. Cell proliferation, cell cycle, cell death and mitochondrial membrane polarization were evaluated. Inhibition of cell proliferation was observed, accompanied by an arrest in the G1-cell cycle phase, and importantly, by loss of mitochondrial membrane potential. On a later time-point, caspase-9 and 3 activation were observed, resulting in cell death. For the majority of test compounds, we determined that cell toxicity was augmented in the galactose media. To investigate direct evidences on mitochondria isolated rat liver mitochondria were used. The results showed that the compounds were strong inducers of the permeability transition pore. Confirming our previous results, this work shows that the novel DMAP derivatives strongly interact with mitochondria, resulting in pro-apoptotic signaling and cell death. 相似文献
The dipeptidyl peptidase 4 inhibitor vildagliptin (VLD), a widely used anti‐diabetic drug, exerts favourable effects on vascular endothelium in diabetes. We determined for the first time the improving effects of VLD on mitochondrial dysfunction in diabetic mice and human umbilical vein endothelial cells (HUVECs) cultured under hyperglycaemic conditions, and further explored the mechanism behind the anti‐diabetic activity. Mitochondrial ROS (mtROS) production was detected by fluorescent microscope and flow cytometry. Mitochondrial DNA damage and ATP synthesis were analysed by real time PCR and ATPlite assay, respectively. Mitochondrial network stained with MitoTracker Red to identify mitochondrial fragmentation was visualized under confocal microscopy. The expression levels of dynamin‐related proteins (Drp1 and Fis1) were determined by immunoblotting. We found that VLD significantly reduced mtROS production and mitochondrial DNA damage, but enhanced ATP synthesis in endothelium under diabetic conditions. Moreover, VLD reduced the expression of Drp1 and Fis1, blocked Drp1 translocation into mitochondria, and blunted mitochondrial fragmentation induced by hyperglycaemia. As a result, mitochondrial dysfunction was alleviated and mitochondrial morphology was restored by VLD. Additionally, VLD promoted the phosphorylation of AMPK and its target acetyl‐CoA carboxylase in the setting of high glucose, and AMPK activation led to a decreased expression and activation of Drp1. In conclusion, VLD improves endothelial mitochondrial dysfunction in diabetes, possibly through inhibiting Drp1‐mediated mitochondrial fission in an AMPK‐dependent manner. 相似文献
Context: Yes-associated protein (Yap) has been linked to several cardiovascular disorders, but the role of this protein in septic cardiomyocytes is not fully understood.
Objective: The aim of our study was to explore the influence of Yap in septic cardiomyopathy in vivo and in vitro.
Materials and methods: In the current study, Yap transgenic mice and Yap adenovirus-mediated gain-of-function assays were used in an LPS-established septic cardiomyopathy model. Mitochondrial function and mitochondrial fission were determined through western blotting, immunofluorescence analysis and ELISA.
Results: Our results demonstrated that Yap expression was downregulated by LPS, whereas Yap overexpression sustained cardiac function and attenuated cardiomyocyte death. The functional exploration revealed that LPS treatment induced cardiomyocyte mitochondrial stress, as manifested by mitochondrial superoxide overproduction, cardiomyocyte ATP deprivation, and caspase-9 apoptosis activation. Furthermore, we demonstrated that LPS-mediated mitochondrial damage was controlled by mitochondrial fission. However, Yap overexpression reduced mitochondrial fission and therefore improved mitochondrial function. A molecular investigation revealed that Yap overexpression inhibited mitochondrial fission by reversing ERK activity, and the inhibition of the ERK pathway promoted DRP1 upregulation and thereby mediated mitochondrial fission activation in the presence of Yap overexpression.
Conclusions: Overall, our results suggest that the cause of septic cardiomyopathy appears to be connected with Yap downregulation. The overexpression of Yap can attenuate myocardial inflammation injury through the reduction of DRP1-related mitochondrial fission in an ERK pathway activation-dependent manner. 相似文献