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Query: UMLS:C0022116 (ischemia)
 91,303 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Housekeeping genes like glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin are often used as internal standards for quantitative RNA analysis. In our study we analyzed the relative expression level of GAPDH and beta-actin as well as of the 18S rRNA and the Poly (A)+ RNA in growing collateral arteries in a rabbit model of arteriogenesis which is not associated with ischemia. Relative quantitation of the housekeeping genes displayed a significant upregulation of the beta-actin- and GAPDH mRNA during the first 24 h of vessel growth. For day 3 our results revealed an even stronger upregulation of the beta-actin mRNA (140%) but a significant downregulation of the GAPDH mRNA (50% of control). The 18S rRNA, however, showed for the same periods only minor alterations compared to the Poly (A)+ RNA. From these results we conclude that the 18S rRNA, but not the GAPDH- or beta-actin mRNA is an appropriate internal control for relative quantitation of gene expression under conditions of cell proliferation in growing vessels.
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PMID:Differential expression of GAPDH and beta3-actin in growing collateral arteries. 1219 Jan 13

Cellular homeostatic adaptation to cerebral ischemia is complex and contains changes in receptor mediated gene expression and signaling pathways. The proteins of the immediate early genes c-Fos and c-Jun are thought to be involved in coupling neuronal excitation to target gene expression, due to formation of heterodimers and binding to the AP1 promotor region. We used an in vitro model to compare ischemia induced c-Fos and c-Jun expression in rat neuronal cell cultures and nerve growth factor (NGF) differentiated PC 12 cells. Since activation of glutamate receptors is known to mediate ischemic injury we determined the effect of the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist MK 801 on c-Fos and c-Jun expression in both cell culture systems during ischemia. Neuron rich cultures and NGF differentiated PC 12 cells were exposed to sublethal in vitro ischemia using an hypoxic chamber flushed with argon/CO2 (95 %/5%). C-Fos and c-Jun mRNA expression was analyzed by competitive reverse transcription-polymerase chain reaction using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal standard. One hour of in vitro ischemia significantly increased c-Fos and c-Jun mRNA levels in both cell culture systems. In neuron rich cultures a 10-fold (c-Fos) and 7-fold (c-Jun) mRNA increase was observed. The mRNA rise was less pronounced in PC 12 cells (5.5-fold and 2-fold) for c-Fos and c-Jun, respectively. The addition of MK 801 significantly reduced the expression of c-Fos and c-Jun mRNA in neuronal cultures, whereas no effect was detectable in PC 12 cells. Since MK 801 failed to reduce the c-Fos and c-Jun expression in NGF differentiated PC 12 cells different signaling pathways may initiate c-Fos and c-Jun expression in both cell culture systems.
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PMID:MK 801 attenuates c-Fos and c-Jun expression after in vitro ischemia in rat neuronal cell cultures but not in PC 12 cells. 1239 13

Oxidative stress results from an oxidant/antioxidant imbalance, an excess of oxidants, or a depletion of antioxidants. A considerable body of recent evidence suggests that oxidative stress and exaggerated production of reactive oxygen species play a major role in several aspects of septic shock and ischemia and reperfusion. Initiation of lipid peroxidation, direct inhibition of mitochondrial respiratory chain enzymes, inactivation of glyceraldehyde-3-phosphate dehydrogenase, inhibition of membrane Na /K adenosine triphosphatase activity, inactivation of membrane sodium channels, and other oxidative protein modifications contribute to the cytotoxic effect of reactive oxygen species. In addition, reactive oxygen species are potent triggers of DNA strand breakage, with subsequent activation of the nuclear enzyme poly-adenosine 5'-diphosphate ribosyl synthetase, and eventual severe energy depletion of the cells. Pharmacologic evidence suggests that the peroxynitrite-poly-adenosine 5'-diphosphate ribosyl polymerase pathway contributes to the cellular injury in shock and endothelial injury. Treatment with superoxide dismutase mimetics, which selectively mimic the catalytic activity of the human superoxide dismutase enzymes, has been shown to prevent the cellular energetic failure associated with shock and ischemia-reperfusion and to prevent tissue damage associated with these conditions. In this article, we will briefly review the role of superoxide in septic shock and ischemia-reperfusion injury. We hope to present evidence to support the potential development of superoxide dismutase mimetics as novel and effective agents in the area of critical care medicine.
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PMID:Therapeutic potential of superoxide dismutase mimetics as therapeutic agents in critical care medicine. 1254 74

Oxidative stress or signaling is widely implicated in apoptosis, ischemia and mitogenesis. Previously, our group reported that the hydrogen peroxide (H2O2)-dependent activation of phospholipase D2 (PLD2) in PC12 cells is involved in anti-apoptotic effect. However, the precise mechanism of PLD2 activation by H2O2 was not revealed. To find H2O2-dependent PLD2-regulating proteins, we immunoprecipitated PLD2 from PC12 cells and found that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) coimmunoprecipitated with PLD2 upon H2O2 treatment. This interaction was found to be direct by in vitro reconstitution of purified GAPDH and PLD2. In vitro studies also indicated that PLD2-associated GAPDH was modified on its reactive cysteine residues. Koningic acid, an alkylator of GAPDH on catalytic cysteine residue, also increased interaction between the two proteins in vitro and enhanced PLD2 activity in PC12 cells. Blocking H2O2-dependent modification of GAPDH with 3-aminobenzamide resulted in the inhibition of the GAPDH/PLD2 interaction and attenuated H2O2-induced PLD2 activation in PC12 cells. From the results, we suggest that H2O2 modifies GAPDH on its catalytic cysteine residue not only to inactivate the dehydrogenase activity of GAPDH but also to endow GAPDH with the ability to bind PLD2 and the resulting association is involved in the regulation of PLD2 activity by H2O2.
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PMID:Hydrogen peroxide induces association between glyceraldehyde 3-phosphate dehydrogenase and phospholipase D2 to facilitate phospholipase D2 activation in PC12 cells. 1275 82

From conventional relative gene expression analyses (Northern blotting, in situ hybridization, and RT-PCR), it has been reported that the expression of control genes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin, used as references may be affected by ischemia. Therefore, we extended searching and evaluation at the mRNA level of transcripts whose expression levels were not changed by cerebral ischemia, using a high-density oligonucleotide array and statistical analysis in a rat global cerebral ischemia and reperfusion model. We added a hyperthermic factor and localization factor to ischemia and identified transcripts with a stable expression level under conditions even more disadvantageous than ischemia only. Screening of more than 8,000 transcripts with the Rat Genome U34A array yielded 28 transcripts, which we listed and classified according to their expression level. Widely used control genes, GAPDH and beta-actin, were not included, although cyclophilin A was included. In addition, we conducted a functional classification based on gene ontology. Under the functional classification of the 28 transcripts, many genes tended to be associated with metabolism. In conclusion, use of several transcripts is recommended, such as those we identified, as references in the analysis of gene expression in pathological models of ischemia.
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PMID:Screening for control genes in rat global cerebral ischemia using high-density oligonucleotide array. 1511 23

Oxidative stress results from an oxidant/antioxidant imbalance, an excess of oxidants and/or a depletion of antioxidants. A considerable body of recent evidence suggests that oxidant stress plays a major role in several aspects of acute and chronic inflammation and is the subject of this review. Immunohistochemical and biochemical evidence demonstrate the significant role of reactive oxygen species (ROS) in acute and chronic inflammation. Initiation of lipid peroxidation, direct inhibition of mitochondrial respiratory chain enzymes, inactivation of glyceraldehyde-3-phosphate dehydrogenase, inhibition of membrane Na+/K+ ATP-ase activity, inactivation of membrane sodium channels, and other oxidative protein modifications contribute to the cytotoxic effect of ROS. All these toxicities are likely to play a role in the pathophysiology of shock, inflammation and ischemia and reperfusion. (2) Treatment with either peroxynitrite decomposition catalysts, which selectively inhibit peroxynitrite, or with SODm's, which selectively mimic the catalytic activity of the human superoxide dismutase (SOD) enzymes, have been shown to prevent in vivo the delayed tissue injury and the cellular energetic failure associated with inflammation. ROS (e.g., superoxide, peroxynitrite, hydroxyl radical and hydrogen peroxide) are all potential reactants capable of initiating DNA single strand breakage, with subsequent activation of the nuclear enzyme poly (ADP ribose) synthetase (PARS), leading to eventual severe energy depletion of the cells, and necrotic-type cell death. Antioxidant treatment inhibits the activation of PARS, and prevents the organ injury associated with acute and chronic inflammation.
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PMID:Potential therapeutic effect of antioxidant therapy in shock and inflammation. 1513 12

Because we were interested in assessing glucose-mediated regulation of the activity of sarcolemmal ATP-sensitive K(+) channels (K(ATP) channels) (which are closed by physiological levels of intracellular ATP and serve to couple intracellular metabolism with the membrane excitability in the heart) during ischemia, we performed experiments designed to test whether high extracellular glucose would have effects on sarcolemmal K(ATP) channels per se. Surprisingly, we found that high extracellular glucose (50 mmol/l) activates sarcolemmal K(ATP) channels in isolated guinea pig cardiomyocytes. To activate K(ATP) channels, glucose had to be transported into cardiomyocytes and subjected to glycolysis. The activation of these channels was independent of ATP production and intracellular ATP levels. The effect of glucose on sarcolemmal K(ATP) channels was mediated by the catalytic activity of glyceraldehyde-3-phosphate dehydrogenase and consequent generation of 1,3-bisphosphoglycerate. The 1,3-bisphosphoglycerate (20 mmol/l), an intermediate product of glycolysis, directly targeted and activated K(ATP) channels, despite physiological levels of intracellular ATP (5 mmol/l). We conclude that glucose, so far exclusively viewed as a metabolic fuel in the heart important only during ischemia/hypoxia, may serve a signaling role in the nonstressed myocardium by producing an agent that regulates cardiac membrane excitability independently of high-energy phosphates.
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PMID:High glucose regulates the activity of cardiac sarcolemmal ATP-sensitive K+ channels via 1,3-bisphosphoglycerate: a novel link between cardiac membrane excitability and glucose metabolism. 1567 96

In conventional relative gene expression analysis (Northern blotting, RT-PCR, and in situ hybridization), housekeeping genes such as the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin genes, whose expression levels are considered stable, have been used as control genes for normalization of RNA quantitation. However, it has been reported that the expression levels of these two control genes are affected by ischemia. Therefore, we have been searching for novel control genes whose expression levels are stable in a mouse model of transient forebrain ischemia. Using the GeneChip Mu6500 array set, we monitored the expression levels of approximately 6000 murine genes in the mouse hippocampus during 24 h of ischemia-reperfusion. To select stable genes, we applied the restricted criterion of a 1.5-fold change in expression level as the threshold. By adding statistical analysis with this criterion, we identified 10 genes as candidates for control genes from the GeneChip data. In this criterion, GAPDH and beta-actin genes were not included in the 10 genes as candidates for control genes. The present findings might be relevant to the use of control genes in quantitation of RNA, particularly in the study of mouse transient forebrain ischemia.
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PMID:Screening for control genes in mouse hippocampus after transient forebrain ischemia using high-density oligonucleotide array. 1671

Infarct size is a good predictor of the neurological outcome following stroke. Estimation of infarct size in the early phase following experimental stroke depends on the availability of reliable techniques that can distinguish ischemic from nonischemic tissue. The objective of this study was to provide a simple and robust method for reliable delineation of the ischemic infarct area in fresh frozen cryosections from mice subjected to focal cerebral ischemia. Mice were subjected to permanent middle cerebral artery (MCA) occlusion and euthanised after 30 min, 1, 2, 4, 6, 12 and 24 h. The size of the developing infarct was compared in parallel series of sections in situ hybridized for mRNA encoding the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or stained with toluidine blue (TB). The infarct was clearly delineated in GAPDH mRNA in situ hybridized sections as soon as 4 h after MCA occlusion. Infarct size was similar at 4 and 6 h in GAPDH mRNA in situ hybridized sections. Sections hybridized for GAPDH mRNA showed significantly larger infarcts than sections stained with TB after 6 h but not after 24 h of ischemia. Analysis of in situ hybridized sections revealed changes in neuronal GAPDH mRNA in areas prone to undergo degeneration 30 min to 1 h after MCA occlusion, thereby preceding visible pycnosis in TB-stained sections. The results showed that in situ hybridization for GAPDH mRNA was a reliable method and superior to TB staining for precise infarct delineation prior to 6 h of permanent MCA occlusion.
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PMID:Glyceraldehyde-3-phosphate dehydrogenase versus toluidine blue as a marker for infarct volume estimation following permanent middle cerebral artery occlusion in mice. 1672 6

In addition to their role in physiological activities, ionotropic glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs) play an important role in neuronal death, especially that following ischemic insults. In this study, we examined the effect of single (SI) and twice repeated (RI)-4-vessel occlusion-ischemia on rat performance in the 8-armed radial maze test. Moreover, the effects of SI and RI on the AMPARs subunits glutamate receptor (GluR) 1 and GluR2 flip and flop variants composition in the CA1 subregion of the hippocampus were investigated using RT-PCR, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and expressed as their ratios to the latter. The results showed that SI and RI impaired the maze performance by decreasing correct choices and increasing the error choices, but RI increased error choices to a greater extent than the SI. The SI reduced only GluR1 flip/GAPDH on day 1. The SI did not alter ratios of GluR2 variants to those of GluR1. On the other hand, the RI decreased GluR2 flip and flop variants after 1 and 3 days, respectively, whereas after 7 days, it increased the flip variant of both GluR1 and GluR2. Moreover, the RI reduced ratios of GluR2 variants to those of GluR1. These results reveal the differential effects of the SI and RI on memory and expression of the AMPARs subunits GluR1 and GluR2 and their flip and flop variants in the CA1.
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PMID:Comparison of single- and repeated-ischemia-induced changes in expression of flip and flop splice variants of AMPA receptor subtypes GluR1 and GluR2 in the rats hippocampus CA1 subregion. 1725 88


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