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Second messengers involved in the signal transduction pathway leading to induction of the plasminogen activator inhibitor (PAI-1) have not yet been well characterized. This study focuses on the mechanisms of regulation of PAI-1 expression by reactive oxygen species (ROS) in human endothelial cells. Inhibition of the tumor necrosis factor α (TNFα)-induced expression of PAI-1 by antioxidant N-acetyl-L-cysteine (NAC) indicated redox-sensitive mechanisms involved in the signalling pathway. Because TNFα induces PAI-1 production in endothelial cells, and NAC attenuated this response, we attempted to investigate the possible involvement of ROS in the activation of PAI-1 by TNFα. Upregulation of PAI-1 expression in endothelial cells by the stimulation with TNFα (50ng/ml) or H2O2 (10-200µM), observed by measurement of the antigen and mRNA levels, was reversed in the presence of NAC (20mM). The stimulatory effect of ROS was detected also at the level of the PAI-1 promoter in endothelial cells transfected with plasmid p800 LUC containing a PAI-1 promoter fragment (+71 to -800). The PAI-1 promoter activity was increased in the presence of ROS, and was suppressed by up to 75% in the presence of antioxidants [1], On the basis of this study we can conclude that reactive oxygen species play an important role in a cytokine-induced activation of PAI-1 expression, and may act as a signal transduction messenger.
The plasma membrane is a specialised multi-component structure with inter- and intracellular signalling functions. Ca2+ plays a crucial role in cellular physiology, and an ATP-driven plasma membrane calcium pump (PMCA) plays the greatest role in the maintenance of a low free Ca2+ concentration in the cytoplasm. The enzyme is coded by four separate genes (PMCA 1-4), and, due to alternative splicing, more than 20 variants can exist. PMCA 1 and 4 isoforms are present in almost all tissues, whereas PMCA 2 and 3 are found in more specialised cell types. The variants differ primarily in their regulatory regions, thus the modulation of calcium pump activity strongly depends on the isoform and the membrane composition. The unique function of PMCA isoforms was confirmed using the practical experimental models - a rat pheochromocytoma cell line, a human neuroblastoma cell line, or, more recently, knockout mice. In addition, based on the finding that PMCA could interact with several specific signaling proteins, it was concluded that its location in defined sites of the cell membrane could be a prerequisite for efficient intercellular communication.
Plasma membrane Ca2+-ATPase (PMCA), encoded by four separate genes, constitutes a high affinity system extruding Ca2+ outside the cell. The nerve growth factor-treated PC12 cell line possesses all four main PMCA isoforms. To evaluate the potential role of PMCA isoforms in the differentiation process, we transiently suppressed the expression of PMCA2 and 3 using the antisense oligonucleotides. In the transfected PC12 cells, we observed morphological changes, slowed neurite extension and diminished survival of the cells. The apparent transport activity and affinity of the calcium pump to Ca2+ were lower in the cells with suppressed PMCA2 and 3 isoforms than in the control cells. Moreover, in the transfected PC12 plasma membranes, the calcium pump was insensitive to stimulation by calmodulin. These findings suggest that PMCA2 and 3 isoforms may be involved in developmental and differentiation processes.
Elevated levels of vascular endothelial growth factor (VEGF) are observed in conditions with vessel and neuron damage or pathological arborization and can therefore be detected in chronic inflammatory process, cardiovascular disease and depression. Hyperlipidemia and cigarette smoking are two factors that have been implicated in endothelial damage. The high comorbidity between cardiovascular disease and major depression disorder (MDD) prompted us to study the effect of cigarette smoking, hyperlipidemia and statin treatment on the VEGFA mRNA and protein expression levels measured in MDD patients. We analyzed 38 MDD patients and 38 healthy control individuals and observed that the MDD group had a significantly higher VEGFA mRNA level and protein serum concentration (P=0.001; P<0.001, respectively). We found no significant association between VEGFA expression at the mRNA or protein level and cigarette smoking, hyperlipidemia or treatment with statins (P>0.05). Interestingly, patients who had attempted suicide had a lower VEGF serum level compared with patients who had not attempted suicide. The translational value of this finding remains unknown. A higher VEGF concentration may play a potentially significant role in the pathogenesis of depression, and the expression level appears to be unaffected by additional factors.
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