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)

Site-specific S-glutathionylation is emerging as a novel mechanism by which S-nitrosoglutathione (GSNO) may modify functionally important protein thiols. Here, we show that GSNO-Sepharose mimicks site-specific S-glutathionylation of the transcription factors c-Jun and p50 by free GSNO in vitro. Both c-Jun and p50 were found to bind to immobilized GSNO through the formation of a mixed disulphide, involving a conserved cysteine residue located in the DNA-binding domains of these transcription factors. Furthermore, we show that c-Jun, p50, glycogen phosphorylase b, glyceraldehyde-3-phosphate dehydrogenase, creatine kinase, glutaredoxin and caspase-3 can be precipitated from a mixture of purified thiol-containing proteins by the formation of a mixed-disulphide bond with GSNO-Sepharose. With few exceptions, protein binding to this matrix correlated well with the susceptibility of the investigated proteins to undergo GSNO- but not diamide-induced mixed-disulphide formation in vitro. Finally, it is shown that covalent GSNO-Sepharose chromatography of HeLa cell nuclear extracts results in the enrichment of proteins which incorporate glutathione in response to GSNO treatment. As suggested by DNA-binding assays, this group of nuclear proteins include the transcription factors activator protein-1, nuclear factor-kappaB and cAMP-response-element-binding protein. In conclusion, we introduce GSNO-Sepharose as a probe for site-specific S-glutathionylation and as a novel and potentially useful tool to isolate and identify proteins which are candidate targets for GSNO-induced mixed-disulphide formation.
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PMID:Novel application of S-nitrosoglutathione-Sepharose to identify proteins that are potential targets for S-nitrosoglutathione-induced mixed-disulphide formation. 1088 Mar 56

Nitric oxide (NO) and its derivative, peroxynitrite (ONOO-), inhibit mitochondrial respiration, and this inhibition may contribute to both the physiological and cytotoxic actions of NO. Nanomolar concentrations of NO rapidly and reversibly inhibited cytochrome oxidase in competition with oxygen, as shown with isolated cytochrome oxidase, mitochondria, brain nerve terminals and cells. Cultured astrocytes and macrophages activated (by cytokines and endotoxin) to express the inducible form of NO synthase produced up to 1 microM NO, and inhibited their own respiration and that of co-incubated cells via reversible NO inhibition of cytochrome oxidase. NO-induced inhibition of respiration in brain nerve terminals resulted in rapid glutamate release, which might contribute to the neurotoxicity of NO. NO inhibition of cytochrome oxidase is reversible; however, incubation of cells with NO donors for 4 hours resulted in an inhibition of complex I, which was reversible by light and thiol reagents and may be due to nitrosylation of thiols in complex I. NO also caused the acute inhibition of catalase, stimulation of hydrogen peroxide production by mitochondria, and reaction with hydrogen peroxide on superoxide dismutase to produce peroxynitrite. Peroxynitrite inhibited complexes I, II and V (the ATP synthase), aconitase, creatine kinase, and increases the proton leak in isolated mitochondria. Peroxynitrite also caused opening of the permeability transition pore, resulting in the release of cytochrome c, which might then trigger apoptosis. Hypoxia/ischaemia also resulted in an acute reversible inhibition of cytochrome oxidase. Heart ischaemia caused the release of cytochrome c from mitochondria into the cytosol, and at the same time caspase-3-like-protease activity was activated in the cytoplasm. Addition of cytochrome c to non-ischaemic cytosol also caused activation of this protease activity, suggesting that caspase activation and consequent apoptosis is at least partly a result of this cytochrome c release.
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PMID:Nitric oxide, cytochrome c and mitochondria. 1098 53

Recent studies have suggested that apoptosis and necrosis share common features in their signaling pathway and that apoptosis requires intracellular ATP for its mitochondrial/apoptotic protease-activating factor-1 suicide cascade. The present study was, therefore, designed to examine the role of intracellular energy levels in determining the form of cell death in cardiac myocytes. Neonatal rat cardiac myocytes were first incubated for 1 h in glucose-free medium containing oligomycin to achieve metabolic inhibition. The cells were then incubated for another 4 h in similar medium containing staurosporine and graded concentrations of glucose to manipulate intracellular ATP levels. Under ATP-depleting conditions, the cell death caused by staurosporine was primarily necrotic, as determined by creatine kinase release and nuclear staining with ethidium homodimer-1. However, under ATP-replenishing conditions, staurosporine increased the percentage of apoptotic cells, as determined by nuclear morphology and DNA fragmentation. Caspase-3 activation by staurosporine was also ATP dependent. However, loss of mitochondrial transmembrane potential (DeltaPsi(m)), Bax translocation, and cytochrome c release were observed in both apoptotic and necrotic cells. Moreover, cyclosporin A, an inhibitor of mitochondrial permeability transition, attenuated staurosporine-induced apoptosis and necrosis through the inhibition of DeltaPsi(m) reduction, cytochrome c release, and caspase-3 activation. Our data therefore suggest that staurosporine induces cell demise through a mitochondrial death signaling pathway and that the presence of intracellular ATP favors a shift from necrosis to apoptosis through caspase activation.
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PMID:Important role of energy-dependent mitochondrial pathways in cultured rat cardiac myocyte apoptosis. 1155 54

The relationship between human sperm maturity and apoptosis is of interest because of the persistence of immature sperm in ejaculates in spite of various apoptotic processes during spermatogenesis. We assessed sperm maturity by HspA2 chaperone levels, and plasma membrane maturity by sperm binding to immobilized hyaluronic acid (HA). We also utilized objective morphometry. Sperm were stained with three antibody combinations: active caspase-3/creatine kinase (CK, a marker of cytoplasmic retention), caspase-3/the antiapoptotic Bcl-(XL), and CK/Bcl-(XL). In semen, 13% of sperm stained with CK, caspase-3 or Bcl-(XL), and 28% had stained with two markers. In the mature HA-bound sperm fraction, <4% were single- or double-stained. Regarding sperm regions, CK staining, whether alone or as double staining, occurred in the head and midpiece (15-20%), whereas caspase-3 and Bcl-(XL) were primarily (>80% of sperm) in the midpiece. Morphometrical attributes of clear, single- and double-stained sperm, in line with their more pronounced maturation arrest, showed an incremental increase in head size (due to cytoplasmic retention) and shorter tail length. We hypothesize that during faulty sperm development, three alternatives may occur: (i) elimination of aberrant germ cells by apoptosis; (ii) in surviving immature cells, caspase-3 is activated, and in response the antiapoptotic Bcl-(XL), and perhaps HspA2, provide protection; (iii) in a third type of immature sperm, in addition to the CK, caspase-3 and Bcl-(XL) expression, there are related manifestations of increased head size and shorter tail length. Thus, immature sperm may vary in the type of developmental arrest and in protection mechanisms for apoptosis. These variations are likely to explain the persistence of immature sperm in the ejaculate.
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PMID:Cellular maturity and apoptosis in human sperm: creatine kinase, caspase-3 and Bcl-XL levels in mature and diminished maturity sperm. 1504 2

Mechanosensitive cation channels (MSC) are ubiquitous in eukaryotic cell types. However, the physiological functions of MSC in several tissues remain in question. In this study we have investigated the role of MSC in skeletal myogenesis. Treatment of C2C12 myoblasts with gadolinium ions (MSC blocker) inhibited myotube formation and the myogenic index in differentiation medium (DM). The enzymatic activity of creatine kinase (CK) and the expression of myosin heavy chain-fast twitch (MyHCf) in C2C12 cultures were also blocked in response to gadolinium. Treatment of C2C12 myoblasts with gadolinium ions did not affect the expression of either cyclin A or cyclin D1 in DM. Other inhibitors of MSC such as streptomycin and GsTMx-4 also suppressed the expression of CK and MyHCf in C2C12 cultures. The inhibitory effect of gadolinium ions on myogenic differentiation was reversible and independent of myogenic cell type. Real-time-polymerase chain reaction analysis revealed that inhibition of MSC decreases the expression of myogenic transcription factors MyoD, myogenin, and Myf-5. Furthermore, the activity of skeletal alpha-actin promoter was suppressed on MSC blockade. Treatment of C2C12 myoblasts with gadolinium ions prevented differentiation-associated cell death and inhibited the cleavage of poly (ADP-ribose) polymerase and activation of caspase-3. On the other hand, delivery of active caspase-3 protein to C2C12 myoblasts reversed the inhibitory effect of gadolinium ions on myogenesis. Our data suggest that inhibition of MSC suppresses myogenic differentiation by inhibiting the caspase-3 activity and the expression of myogenic regulatory factors.
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PMID:Inhibition of mechanosensitive cation channels inhibits myogenic differentiation by suppressing the expression of myogenic regulatory factors and caspase-3 activity. 1631 42

We previously showed that C-phycocyanin (PC), an antioxidant biliprotein pigment of Spirulina platensis (a blue-green alga), effectively inhibited doxorubicin-induced oxidative stress and apoptosis in cardiomyocytes. Here we investigated the cardioprotective effect of PC against ischemia-reperfusion (I/R)-induced myocardial injury in an isolated perfused Langendorff heart model. Rat hearts were subjected to 30 min of global ischemia at 37 degrees C followed by 45 min of reperfusion. Hearts were perfused with PC (10 microM) or Spirulina preparation (SP, 50 mg/l) for 15 min before the onset of ischemia and throughout reperfusion. After 45 min of reperfusion, untreated (control) hearts showed a significant decrease in recovery of coronary flow (44%), left ventricular developed pressure (21%), and rate-pressure product (24%), an increase in release of lactate dehydrogenase and creatine kinase in coronary effluent, significant myocardial infarction (44% of risk area), and TdT-mediated dUTP nick end label-positive apoptotic cells compared with the preischemic state. PC or SP significantly enhanced recovery of heart function and decreased infarct size, attenuated lactate dehydrogenase and creatine kinase release, and suppressed I/R-induced free radical generation. PC reversed I/R-induced activation of p38 MAPK, Bax, and caspase-3, suppression of Bcl-2, and increase in TdT-mediated dUTP nick end label-positive apoptotic cells. However, I/R also induced activation of ERK1/2, which was enhanced by PC treatment. Overall, these results for the first time showed that PC attenuated I/R-induced cardiac dysfunction through its antioxidant and antiapoptotic actions and modulation of p38 MAPK and ERK1/2.
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PMID:C-phycocyanin protects against ischemia-reperfusion injury of heart through involvement of p38 MAPK and ERK signaling. 1637 83

We investigated whether low-pressure reperfusion may attenuate postischemic contractile dysfunction, limits necrosis and apoptosis after a prolonged hypothermic ischemia, and inhibits mitochondrial permeability transition-pore (MPTP) opening. Isolated rats hearts (n = 72) were exposed to 8 h of cold ischemia and assigned to the following groups: 1) reperfusion with low pressure (LP = 70 cmH(2)O) and 2) reperfusion with normal pressure (NP = 100 cmH(2)O). Cardiac function was assessed during reperfusion using the Langendorff model. Mitochondria were isolated, and the Ca(2+) resistance capacity (CRC) of the MPTP was determined. Malondialdehyde (MDA) production, caspase-3 activity, and cytochrome c were also assessed. We found that functional recovery was significantly improved in LP hearts with rate-pressure product averaging 30,380 +/- 1,757 vs. 18,000 +/- 1,599 mmHg/min in NP hearts (P < 0.01). Necrosis, measured by triphenyltetrazolium chloride staining and creatine kinase leakage, was significantly reduced in LP hearts (P < 0.01). The CRC was increased in LP heart mitochondria (P < 0.01). Caspase-3 activity, cytochrome c release, and MDA production were reduced in LP hearts (P < 0.001 and P < 0.01). This study demonstrated that low-pressure reperfusion after hypothermic heart ischemia improves postischemic contractile dysfunction and attenuates necrosis and apoptosis. This protection could be related to an inhibition of mitochondrial permeability transition.
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PMID:Controlled reperfusion after hypothermic heart preservation inhibits mitochondrial permeability transition-pore opening and enhances functional recovery. 1679 30

EPO (erythropoietin) has recently been shown to have protective actions upon the myocardium; however, the direct effects of EPO upon cardiac contractile and secretory functions are unknown and the signalling mechanisms are not well defined. In the present study, we provide the first evidence of direct cardiac contractile actions of EPO. In isolated perfused Sprague-Dawley rat hearts, a 30 min infusion of EPO significantly increased contractility in a dose-dependent fashion (maximal change 18+/-2% with 1 unit/ml EPO; P<0.005 compared with vehicle). Perfusate ET-1 (endothelin-1) increased transiently during EPO infusion, and the ET(A/)ET(B) antagonist bosentan abolished the inotropic response to EPO. BNP (B-type natriuretic peptide) secretion (28+/-8%; P<0.05) and nuclear transcription factor GATA-4 DNA-binding activity (51%; P<0.05) were both significantly increased by EPO and blocked by bosentan. In a model of global ischaemic injury, delivery of 1 unit/ml EPO during reperfusion significantly attenuated creatine kinase release (28+/-12%; P<0.05) and significantly improved contractile recovery (P<0.001), independent of ET(A) blockade. Apoptotic indices [assessed by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling)/cleaved caspase-3-positive cells] were significantly decreased (P<0.01) by 1 unit/ml EPO during reperfusion alone, coincident with significantly increased phosphorylation of myocardial JAK2 (Janus kinase 2) and STAT3 (signal transducer and activator of transcription 3). Thus EPO directly enhances cardiac contractility and BNP secretion and alleviates ischemia/reperfusion injury via ET-1-dependent and -independent mechanisms respectively.
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PMID:Direct cardiac actions of erythropoietin (EPO): effects on cardiac contractility, BNP secretion and ischaemia/reperfusion injury. 1791 23

The receptor for advanced glycation end-products (RAGE) has been implicated in the pathogenesis of ischemia-reperfusion (I/R) injury in the isolated perfused heart. To test the hypothesis that RAGE-dependent mechanisms modulated responses to I/R in a murine model of transient occlusion and reperfusion of the left anterior descending coronary artery (LAD), we subjected male homozygous RAGE(-/-) mice and their wild-type age-matched littermates to 30 min of occlusion of the LAD followed by reperfusion. At 48 h of reperfusion, hematoxylin and eosin staining revealed significantly larger infarct size in wild-type versus RAGE(-/-) mice. Contractile function, as evaluated by echocardiography 48 h after reperfusion, revealed that fractional shortening was significantly higher in RAGE(-/-) versus wild-type mice. Plasma levels of creatine kinase were markedly decreased in RAGE(-/-) versus wild-type animals. Integral to the impact of RAGE deletion on diminished myocardial damage after infarction was significantly decreased apoptosis in the heart, as assessed by TUNEL staining, release of cytochrome c, and caspase-3 activity. Experiments investigating the impact of RAGE on early signaling pathways influencing myocardial ischemic injury revealed attenuation of JNK and STAT5 phosphorylation in RAGE(-/-) mouse hearts versus robust activation observed in wild-type mice upon ischemia and reperfusion. Solidifying the link to RAGE, these experiments revealed that infarction stimulated the rapid production of advanced glycation end-products in the heart. Thus, we tested the effect of ligand decoy soluble RAGE (sRAGE). Administration of sRAGE protected the myocardium from ischemic damage, similar to the effects observed in RAGE(-/-) mouse hearts. Taken together, these data implicate RAGE and its ligands in the pathogenesis of I/R injury and identify JNK and STAT signal transduction as central downstream effector pathways of the ligand-RAGE axis in the heart subjected to I/R injury.
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PMID:RAGE modulates myocardial injury consequent to LAD infarction via impact on JNK and STAT signaling in a murine model. 1824 63

Human cytochrome P-450 (CYP)2J2 is abundant in heart and active in biosynthesis of epoxyeicosatrienoic acids (EETs). Recently, we demonstrated that these eicosanoid products protect myocardium from ischemia-reperfusion injury. The present study utilized transgenic (Tr) mice with cardiomyocyte-specific overexpression of human CYP2J2 to investigate protection toward toxicity resulting from acute (0, 5, or 15 mg/kg daily for 3 days, followed by 24-h recovery) or chronic (0, 1.5, or 3.0 mg/kg biweekly for 5 wk, followed by 2-wk recovery) doxorubicin (Dox) administration. Acute treatment resulted in marked elevations of serum lactate dehydrogenase and creatine kinase levels that were significantly greater in wild-type (WT) than CYP2J2 Tr mice. Acute treatment also resulted in less activation of stress response enzymes in CYP2J2 Tr mice (catalase 750% vs. 300% of baseline, caspase-3 235% vs. 165% of baseline in WT vs. CYP2J2 Tr mice). Moreover, CYP2J2 Tr hearts exhibited less Dox-induced cardiomyocytes apoptosis (measured by TUNEL) compared with WT hearts. After chronic treatment, comparable decreases in body weight were observed in WT and CYP2J2 Tr mice. However, cardiac function, assessed by measurement of fractional shortening with M-mode transthoracic echocardiography, was significantly higher in CYP2J2 Tr than WT hearts after chronic Dox treatment (WT 37 +/- 2%, CYP2J2 Tr 47 +/- 1%). WT mice also had larger increases in beta-myosin heavy chain and cardiac ankryin repeat protein compared with CYP2J2 Tr mice. CYP2J2 Tr hearts had a significantly higher rate of Dox metabolism than WT hearts (2.2 +/- 0.25 vs. 1.6 +/- 0.50 ng.min(-1).100 microg protein(-1)). In vitro data from H9c2 cells demonstrated that EETs attenuated Dox-induced mitochondrial damage. Together, these data suggest that cardiac-specific overexpression of CYP2J2 limited Dox-induced toxicity.
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PMID:Overexpression of CYP2J2 provides protection against doxorubicin-induced cardiotoxicity. 1942 16


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