Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.22.56 (caspase-3)
35,750 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previous histopathologic results have suggested that one mechanism whereby hyperglycemia (HG) leads to exaggerated ischemic damage involves fragmentation of DNA. DNA fragmentation in normoglycemia (NG) and HG rats subjected to 30 minutes of forebrain ischemia was studied by terminal deoxynucleotidyl transferase mediated DNA nick-labeling (TUNEL) staining, by pulse-field gel electrophoresis (PFGE), and by ligation-mediated polymerase chain reaction (LM-PCR). High molecular weight DNA fragments were detected by PFGE, whereas low molecular weight DNA fragments were detected using LM-PCR techniques. The LM-PCR procedure was performed on DNA from test samples with blunt (without Klenow polymerase) and 3'-recessed ends (with Klenow polymerase). In addition, cytochrome c release and caspase-3 activation were studied by immunocytochemistry. Results show that HG causes cytochrome c release, activates caspase-3, and exacerbates DNA fragments induced by ischemia. Thus, in HG rats, but not in control or NGs, TUNEL-stained cells were found in the cingulate cortex, neocortex, thalamus, and dorsolateral crest of the striatum, where neuronal death was observed by conventional histopathology, and where both cytosolic cytochrome c and active caspase-3 were detected by confocal microscopy. In the neocortex, both blunt-ended and stagger-ended fragments were detected in HG, but not in NG rats. Electron microscopy (EM) analysis was performed in the cingulate cortex, where numerous TUNEL-positive neurons were observed. Although DNA fragmentation was detected by TUNEL staining and electrophoresis techniques, EM analysis failed to indicate apoptotic cell death. It is concluded that HG triggers a cell death pathway and exacerbates DNA fragmentation induced by ischemia.
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PMID:Hyperglycemia enhances DNA fragmentation after transient cerebral ischemia. 1133 67

Applying the recently developed DNA array technique to a murine stroke model, we found that the gene coding for RhoB, a member of the family of GTPases that regulate a variety of signal transduction pathways, is upregulated in ischemia-damaged neurons. RhoB immunoreactivity precedes DNA single-strand breaks and heralds the evolving infarct, making it an early predictor of neuronal death. Expression of RhoB colocalized with drastic rearrangement of the actin cytoarchitecture indicates a role for Rho in postischemic morphological changes. Apoptosis in a murine hippocampal cell line was also associated with an early increase in RhoB protein. Activation of caspase-3, a crucial step in apoptosis, could be inhibited by cytochalasin D, a substance that counteracts the actin-modulating activity of Rho GTPases, indicating that Rho proteins may have impact on injury-initiated neuronal signal transduction. Our findings make Rho GTPases potential targets for the development of drugs aimed at limiting neuronal death following brain damage.
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PMID:GTPase RhoB: an early predictor of neuronal death after transient focal ischemia in mice. 1135 85

We tested the hypothesis that combined use of trophic factors and caspase inhibitors increases brain resistance to ischaemia in mice. Intracerebroventricular administration of bFGF (>10 ng) 30 min after MCA occlusion decreased infarct size and neurological deficit in a dose-dependent manner following 2 h ischemia and reperfusion (20 h). Combined administration of the subthreshold doses of bFGF (3 ng) and caspase inhibitors (z-VAD.FMK, 27 ng or z-DEVD.FMK, 80 mg) reduced infarct volume by 60%, and reduced neurological deficit. Treatment with a subthreshold dose of bFGF (3 ng) extended the therapeutic window for z-DEVD.FMK (480 ng) from 1 to 3 h after reperfusion. Caspase-3 activity in the ischaemic brain was increased 30 min and 2 h after reperfusion but, was significantly reduced in bFGF-treated animals by 29 and 16%, respectively. Caspase-3 activity was not reduced by a direct bFGF effect because addition of bFGF (10 nM - 2 microM) did not decrease recombinant caspase-3 activity, in vitro. Our data show that combining caspase inhibitors and bFGF lengthens the treatment window for the second treatment, plus lowers the dosage requirements for neuroprotection. These findings are important because low doses of caspase inhibitors or bFGF reduce the possibility of side effects plus extend the short treatment window for ischaemic stroke.
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PMID:Synergistic protective effect of caspase inhibitors and bFGF against brain injury induced by transient focal ischaemia. 1137 50

Nuclear changes, including internucleosomal DNA fragmentation, are characteristic features of neuronal apoptosis resulting from transient cerebral ischemia and related brain insults for which the molecular mechanism has not been elucidated. Recent studies suggest that a caspase-3-mediated mechanism may be involved in the process of nuclear degradation in ischemic neurons. In this study, we cloned from rat brain a homolog cDNA encoding caspase-activated deoxyribonuclease (CAD)/DNA fragmentation factor 40 (DFF40), a 40 kDa nuclear enzyme that is activated by caspase-3 and promotes apoptotic DNA degradation. Subsequently, we investigated the role of CAD/DFF40 in the induction of internucleosomal DNA fragmentation in the hippocampus in a rat model of transient global ischemia and in primary neuronal cultures under ischemia-like conditions. At 8-72 hr after ischemia, CAD/DFF40 mRNA and protein were induced in the degenerating hippocampal CA1 neurons. CAD/DFF40 formed a heterodimeric complex in the nucleus with its natural inhibitor CAD (ICAD) and was activated after ischemia in a delayed manner (>24 hr) by caspase-3, which translocated into the nucleus and cleaved ICAD. Furthermore, an induced CAD/DFF40 activity was detected in nuclear extracts in both in vivo and in vitro models, and the DNA degradation activity of CAD/DFF40 was inhibited by purified ICAD protein. These results strongly suggest that CAD/DFF40 is the endogenous endonuclease that mediates caspase-3-dependent internucleosomal DNA degradation and related nuclear alterations in ischemic neurons.
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PMID:Caspase-activated DNase/DNA fragmentation factor 40 mediates apoptotic DNA fragmentation in transient cerebral ischemia and in neuronal cultures. 1142 95

The brain's response to ischemia, which helps determine clinical outcome after stroke, is regulated partly by competing genetic programs that respectively promote cell survival and delayed cell death. Many genes involved in this response have been identified individually or systematically, providing insights into the molecular basis of ischemic injury and potential targets for therapy. The development of microarray systems for gene expression profiling permits screening of large numbers of genes for possible involvement in biological or pathological processes. Therefore, we used an oligodeoxynucleotide-based microarray consisting of 374 human genes, most implicated previously in apoptosis or related events, to detect alterations in gene expression in the hippocampus of rats subjected to 15 minutes of global cerebral ischemia followed by up to 72 hours of reperfusion. We found 1.7-fold or greater increases in the expression of 57 genes and 1.7-fold or greater decreases in the expression of 34 genes at 4, 24, or 72 hours after ischemia. The number of induced genes increased from 4 to 72 hours, whereas the number of repressed genes decreased. The induced genes included genes involved in protein synthesis, genes mutated in hereditary human diseases, proapoptotic genes, antiapoptotic genes, injury-response genes, receptors, ion channels, and enzymes. We detected transcriptional induction of several genes implicated previously in cerebral ischemia, including ALG2, APP, CASP3, CLU, ERCC3, GADD34, GADD153, IGFBP2, TIAR, VEGF, and VIM, as well as other genes not so implicated. We also found coinduction of several groups of related genes that might represent functional modules within the ischemic neuronal transcriptome, including VEGF and its receptor, NRP1; the IGF1 receptor and the IGF1-binding protein IGFBP2; Rb, the Rb-binding protein E2F1, and the E2F-related transcription factor, TFDP1; the CACNB3 and CACNB4 beta-subunits of the voltage-gated calcium channel; and caspase-3 and its substrates, ACINUS, FEM1, and GSN. To test the hypothesis that genes identified through this approach might have roles in the pathophysiology of cerebral ischemia, we measured expression of the products of two induced genes not heretofore implicated in cerebral ischemia-GRB2, an adapter protein involved in growth-factor signaling pathways, and SMN1, which participates in RNA processing and is deleted in most cases of spinal muscular atrophy. Western analysis showed enhanced expression of both proteins in hippocampus at 24 to 72 hours after ischemia, and SMN1 was localized by immunohistochemistry to hippocampal neurons. These results suggest that microarray analysis of gene expression may be useful for elucidating novel molecular mediators of cell death and survival in the ischemic brain.
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PMID:Microarray analysis of hippocampal gene expression in global cerebral ischemia. 1145 15

The evolution of brain injury was examined in mice subjected to focal cerebral ischemia as induced by 30 min of intraluminar thread occlusion of the middle cerebral artery, followed by 3 h to 3 days of reperfusion. Metabolic dysfunctions were studied by 3H-leucine autoradiography for the measurement of cerebral protein synthesis and by regional ATP bioluminescent imaging. Metabolic changes were compared with responses of the genes c-fos, c-jun, heat-shock protein gene (hsp)72, p53-activated gene (pag)608 and caspase-3, which were investigated by in situ hybridization histochemistry and immunocytochemistry, and correlated with the degree of DNA fragmentation, as assessed by the terminal TdT-mediated dUTP-biotin nick end labeling method. Intraluminar thread occlusion led to a reproducible reduction of cerebral laser Doppler flow to 20-30% of control. Thread withdrawal was followed by a short-lasting post-ischemic hyperperfusion to approximately 120%. In non-ischemic control animals, fractional protein synthesis values of 0.81+/-0.26 and 0.94+/-0.23 were obtained. Thread occlusion resulted in a suppression of protein synthesis throughout the territory of the middle cerebral artery after 3 h of reperfusion (0.04+/-0.08 in caudate-putamen and 0.14+/-0.19 in somatosensory cortex, P<0.05). Protein synthesis partly recovered in the cortex after 24 h and 3 days (0.71+/-0.40 and 0.63+/-0.26, respectively), but remained suppressed in the caudate-putamen (0.14+/-0.22 and 0.28+/-0.28). Regional ATP levels did not show any major disturbances at the reperfusion times examined. Thread occlusion resulted in a transient increase of c-fos mRNA levels in ischemic and non-ischemic parts of the cortex and caudate-putamen at 3 h after ischemia, which suggests that spreading depressions were elicited in the tissue. At the same time, c-jun and hsp72 mRNAs were elevated only in ischemic brain areas showing inhibition of protein synthesis. C-fos and c-jun responses completely disappeared within 24 h of reperfusion. Hsp72 mRNA levels remained elevated in the cortex after 24 h, but decreased to basal values in the caudate-putamen. Twenty-four hours after reperfusion, pag608 and caspase-3 mRNA levels increased in the caudate-putamen, where protein synthesis rates were still reduced, and remained elevated even after 3 days. However, pag608 and caspase-3 mRNA levels did not increase in the cortex, where protein synthesis recovered. After 24 h and 3 days, functionally active p20 fragment of caspase-3 was detected in the caudate-putamen, closely associated with the appearance of DNA fragmented cells. Neither activated caspase-3 nor DNA fragmentation were noticed in the cortex.In summary, the suppression of protein synthesis is reversible in the ischemia-resistant cortex following 30 min of thread occlusion in mice, but persists in the vulnerable caudate-putamen. In the caudate-putamen, apoptotic programs are induced, closely in parallel with the manifestation of delayed cell death. Thus, the recovery of protein synthesis may be a major factor influencing tissue survival after transient focal ischemia.
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PMID:Relationship between metabolic dysfunctions, gene responses and delayed cell death after mild focal cerebral ischemia in mice. 1145 82

The effects of hypothermia on caspase-3 activation were investigated in the newborn rat brain after hypoxia-ischemia (HI). Intense caspase-3 activation was observed in the control brains after HI, but this activation was significantly reduced by postischemic hypothermia. These findings suggest that the inhibition of caspase-3 activation may be an interventional point underlying the neuroprotective effect of hypothermia in neonates.
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PMID:Post-ischemic hypothermia blocks caspase-3 activation in the newborn rat brain after hypoxia-ischemia. 1148 70

During the last few years, adenoviral gene transfer techniques have achieved increasing interest in the treatment of neurodegenerative diseases. However, gene therapy requires that delivered genes are translated into proteins. This may pose a problem in focal ischemia where protein synthesis is compromized. The present study was conducted to find out the feasibility of adenoviral GDNF and CNTF delivery in transient focal ischemia, as induced by 30 min of intraluminar middle cerebral artery (MCA) occlusion in mice. Injections of vehicle, of an adenoviral vector deleted in the E1 region (Ad-dE1) and of vectors expressing the GDNF (Ad-GDNF), CNTF (Ad-CNTF), or GFP (Ad-EGFP) gene from a CMV promoter were stereotactically placed in the dorsolateral striatum, i.e., the core of the MCA territory, and focal ischemia was induced seven days later. Thread occlusion resulted in disseminated injury of the striatum, but not the overlying cortex. The number of viable neurons was significantly increased after 1 and 3 days of reperfusion both in Ad-GDNF and Ad-CNTF as compared with vehicle or Ad-dE1-treated animals, whereas the number of injured cells was significantly reduced, as shown by cresyl violet staining, terminal transferase biotinylated-dUTP nick end-labeling (TUNEL), and immunocytochemistry for activated caspase-3. Interestingly, the protective effects of Ad-GDNF were similarly strong in areas of the striatum adjacent and remote of the adenoviral infusion site, while Ad-CNTF showed pronounced rescue effects in the surrounding, but rather little effects distant to the infusion. The present study demonstrates that adenoviral delivery of neurotrophic factors may be a useful tool for the treatment of focal ischemia.
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PMID:Adenovirus-mediated GDNF and CNTF pretreatment protects against striatal injury following transient middle cerebral artery occlusion in mice. 1149 30

Recent studies suggest that the degree of mitochondrial dysfunction in cerebral ischemia may be an important determinant of the final extent of tissue injury. Although loss of mitochondrial membrane potential (psi(m)), one index of mitochondrial dysfunction, has been documented in neurons exposed to ischemic conditions, it is not yet known whether astrocytes, which are relatively resistant to ischemic injury, experience changes in psi(m) under similar conditions. To address this, we exposed cortical astrocytes cultured alone or with neurons to oxygen-glucose deprivation (OGD) and monitored psi(m) using tetramethylrhodamine ethyl ester. Both neurons and astrocytes exhibited profound loss of psi(m) after 45-60 min of OGD. However, although this exposure is lethal to nearly all neurons, it is hours less than that needed to kill astrocytes. Astrocyte psi(m) was rescued during OGD by cyclosporin A, a permeability transition pore blocker, and (G)N-nitro-arginine, a nitric oxide synthase inhibitor. Loss of mitochondrial membrane potential in astrocytes was not accompanied by depolarization of the plasma membrane. Recovery of astrocyte psi(m) after reintroduction of O(2) and glucose occurred over a surprisingly long period (>1 hr), suggesting that OGD caused specific, reversible changes in astrocyte mitochondrial physiology beyond the simple lack of O(2) and glucose. Decreased psi(m) was associated with a cyclosporin A-sensitive loss of cytochrome c but not with activation of caspase-3 or caspase-9. Our data suggest that astrocyte mitochondrial depolarization could be a previously unrecognized event early in ischemia and that strategies that target the mitochondrial component of ischemic injury may benefit astrocytes as well as neurons.
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PMID:The mitochondrial permeability transition pore and nitric oxide synthase mediate early mitochondrial depolarization in astrocytes during oxygen-glucose deprivation. 1151 50

EPC-K1, L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-yl-hydrogen phosphate] potassium salt, is a novel antioxidant. In this study, we investigated a reduction of oxidative neuronal cell damage with EPC-K1 by immunohistochemical analysis for 8-hydroxy-2'-deoxyguanosine (8-OHdG) in rat brain with 60 min transient middle cerebral artery occlusion, in association with terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) and staining for total and active caspase-3. Treatment with EPC-K1 (20 mg kg(-1) i.v.) significantly reduced infarct size (p < 0.05) at 24 h of reperfusion. There were no positive cells for 8-OHdG and TUNEL in sham-operated brain, but numerous cells became positive for 8-OHdG, TUNEL and caspase-3 in the brains with ischemia. The number was markedly reduced in the EPC-K1 treated group. These reductions were particularly evident in the border zone of the infarct area, but the degree of reduction was less in caspase-3 staining than in 8-OHdG and TUNEL stainings. These results indicate EPC-K1 attenuates oxidative neuronal cell damage and prevents neuronal cell death.
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PMID:Attenuation of oxidative DNA damage with a novel antioxidant EPC-K1 in rat brain neuronal cells after transient middle cerebral artery occlusion. 1154 42


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