Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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The effect of ketoconazole on the fusion of L6 myoblasts was studied. Ketoconazole was a potent inhibitor of myoblast fusion at concentrations as low as 0.1 microM, but fusion was restored when the inhibitor was removed. The inhibitor resulted in decreased binding of conA and WGA to cell surface oligosaccharides showing that it was inhibiting N-linked cell surface glycoproteins. Inhibition of fusion by ketoconazole was accompanied by reduced creatine phosphokinase activities showing that it is affecting biochemical differentiation. Incorporation of labelled mannose from GDP-mannose into lipid-sugar and lipid-oligosaccharide complexes involved in the synthesis of N-linked oligosaccharides was also inhibited by ketoconazole, but the inhibition was reversed by addition of exogenous dolichol phosphate to the incorporation mixture. The main conclusion from these studies was that ketoconazole inhibited fusion of L6 myoblasts by affecting the synthesis of dolichol-phosphate required for the synthesis of lipid-oligosaccharides needed for the synthesis of fusogenic cell surface N-linked glycoproteins.
Mol Cell Biochem 1990 Feb 09
PMID:Studies on the effect of ketoconazole on the fusion of L6 myoblasts. 230 83

We have demonstrated earlier that the per sperm creatine-N-phosphotransferase (CK) activity was increased in oligospermic vs. normospermic men. The increased sperm CK activity is related to higher concentrations of cellular CK, which may indicate a defect of cytoplasmic extrusion during spermatogenesis. In the present work, we examined whether in spermatozoa, similar to muscle, there is a change in the synthesis of B-CK and M-CK isoforms during cellular differentiation. In 109 normospermic and 50 oligospermic specimens (sperm concentrations 60.6 +/- 3.7 vs. 8.8 +/- 1.3 million sperm/ml; all values expressed as mean +/- SEM), the relative concentrations of the M-CK isoform (M-CK/M-CK + B-CK) were 27.2% +/- 2.1% vs. 6.7% +/- 0.9% (P less than 0.001). The per sperm CK activities showed comparable differences (0.21 +/- 0.02 vs. 0.89 +/- 0.1 CK IU/100 million sperm; P less than 0.001) in the two groups, and there was a close correlation between per sperm CK activities and M-CK concentrations (R = 0.69, P less than 0.001, N = 159). This indicates that the loss of cytoplasm and the commencement of M-CK isoform synthesis are related events during the last phase of spermatogenesis, also that the incidence of spermatozoa with incomplete cellular maturation is higher in oligospermic specimens. In characterizing the M-CK, we found that sperm (unlike muscle tissue) lack the MB hybrid of CK dimers. However, in the presence of muscle M-CK, the muscle-sperm MB-CK hybrid has formed. Thus in sperm and muscle the M-CK isoforms are structurally different, whereas the B-CKs are apparently homologous.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Reprod Dev 1990 Mar
PMID:Spermatogenesis-related change in the synthesis of the creatine kinase B-type and M-type isoforms in human spermatozoa. 233 74

Myocardial ischemia leads to the damage of cellular membranes and release of intracellular enzymes. We studied the influence of the prostacyclin analog, iloprost, on alterations in membrane phospholipid content and composition in rat myocardium during ischemia. Infusion of iloprost (100 ng/kg/min) or its vehicle started 20 min after coronary artery ligation, and the hearts were analyzed after 6 h. Myocardial creatine kinase activity was significantly reduced by approximately 25% in the ischemic areas of hearts from rats receiving vehicle. This reduction in myocardial creatine kinase activity was totally abolished by infusion of iloprost. Total phospholipid content was significantly reduced by 10% in ischemic areas of hearts obtained from vehicle infused animals. Iloprost infusion also prevented the loss of total phospholipids in the ischemic areas. The data show that coronary artery ligation is associated with a significant loss of total membrane phospholipids in ischemic regions of rat myocardium, characterized by significant decreases in phosphatidylcholine, phosphatidylethanolamine and cardiolipin. The decrease in cardiac phosphatidylcholine and phosphatidylethanolamine content was prevented by iloprost, whereas the decrease in cardiolipin content was unaltered. Infusion of the prostacyclin analog iloprost almost totally inhibited the ischemia induced loss of phospholipids, suggesting that this may be an important component of its cytoprotective mechanism of action.
J Mol Cell Cardiol 1987 Mar
PMID:Protective actions of a stable prostacyclin analog in ischemia induced membrane damage in rat myocardium. 243 96

To assess whether the administration of the stable prostacyclin-mimetic ZK 36374 (iloprost) protects the myocardium in a dose-dependent manner against ischaemia and reperfusion, isolated rabbit hearts were infused with three different concentrations of iloprost: 2.7, 27 and 270 nM. Diastolic and developed pressures were monitored; coronary effluent was collected and assayed for creatine phosphokinase (CPK) activity and for noradrenaline concentration; mitochondria were harvested and assayed for respiratory activity; ATP production and calcium content and tissue concentration of adenosine triphosphate (ATP) and creatine phosphate (CP) were determined. Treatment with iloprost altered neither developed pressure under normoxic conditions nor the rate and extent of depletion of ATP and CP during ischaemia. The ischaemic-induced deterioration of mitochondrial function, however, was attenuated. On reperfusion, hearts treated with iloprost recovered better than the untreated hearts with respect to left ventricular performance, replenishment of ATP and CP stores and mitochondrial function. The reperfusion-induced mitochondrial calcium overload and release of CPK and of noradrenaline were also significantly reduced. The effect of iloprost was dose-dependent. The lower concentration (2.7 nM) failed to modify ischaemic and reperfusion damage. The best protective effect was found at 27 nM. An increase of the dose to 270 nM did not result in further protection. It is concluded that iloprost infusion provides a dose-dependent protection of the heart against some of the deleterious effects of ischaemia and reperfusion and, in particular, prevents mitochondrial calcium overload and maintains mitochondrial function. Because this protection occurred in the absence of negative inotropic effect during normoxia or of a coronary dilatory effect during ischaemia, it cannot be attributed to an energy sparing effect or to improvement of oxygen delivery. Therefore, alternative mechanisms of action are to be considered.
J Mol Cell Cardiol 1988 Dec
PMID:Protective effect of a prostacyclin-mimetic on the ischaemic-reperfused rabbit myocardium. 247 Sep 8

Reoxygenation of isolated rat cardiac myocytes following a period of hypoxia and substrate deprivation resulted in a 1.5-2-fold increase in the total Ca2+ content which could be inhibited by 1 microM antimycin A or ruthenium red (50% inhibition at 2.5 microM). This increase in Ca2+ content was not accompanied by any release of creatine kinase into the medium. Treatment of reoxygenated cells with digitonin also resulted in an antimycin A-sensitive increase in Ca2+ but this was inhibited by a lower concentration of ruthenium red (50% inhibition at 0.25 microM) and was associated with a substantial release of creatine kinase from the cells. It is concluded that the reoxygenation-stimulated increase in Ca2+ is dependent on functioning mitochondria and does not occur as a result of physical damage to the sarcolemma. In a parallel series of experiments, the effects of antimycin A and ruthenium red on the reoxygenation-induced increase in Ca2+ and release of cytosolic contents in the perfused heart (the oxygen paradox) were also investigated. As was observed with the isolated myocytes, each of the compounds significantly reduced the magnitude of the Ca2+ increase that occurred on reoxygenation: the compounds also reduced the extent of release of cell contents in the perfused heart. The implications of these results for the series of events occurring on reoxygenation of the hypoxic myocardium are discussed.
J Mol Cell Cardiol 1989 Oct
PMID:Hypoxia-reoxygenation induced increase in cellular Ca2+ in myocytes and perfused hearts: the role of mitochondria. 247 60

In the preceding report (Kelvin, D.J., G. Simard, H.H. Tai, T.P. Yamaguchi, and J.A. Connolly. 1989. J. Cell Biol. 108:159-167) we demonstrated that pertussis toxin (PT) blocked proliferation and induced differentiation in BC3H1 muscle cells. In the present study, we have used PT to examine specific growth factor signaling pathways that may regulate these processes. Inhibition of [3H]thymidine by PT in 20% FBS was reversed in a dose-dependent fashion by purified fibroblast growth factor (FGF). In 0.5% FBS, the normally induced increase in creatine kinase (CK) activity was blocked by FGF in both the presence and absence of PT. Similar results were obtained with purified epidermal growth factor (EGF). We subsequently examined the effect of a family of growth factors linked to inositol lipid hydrolysis and found that thrombin, like FGF, would increase [3H]thymidine incorporation and block CK synthesis. However, PT blocked thymidine incorporation induced by thrombin, and blocked the inhibition of CK turn-on in 0.5% FBS by thrombin. The ras oncogene, a G protein homologue, has previously been shown to block muscle cell differentiation in C2 muscle cells (Olson, E.N., G. Spizz, and M.A. Tainsky. 1987. Mol. Cell. Biol. 7:2104-2111); we have characterized a BC3H1 cell line, BCT31, which we transfected with the val12 oncogenic Harvey ras gene. This cell line did not express CK in response to serum deprivation. Whereas [3H]thymidine incorporation was inhibited by 70-80% by increasing doses of PT in control cells, BCT31 cells were only inhibited by 15-20%. ADP ribosylation studies indicate this PT-insensitivity is not because of the lack of a PT substrate in this cell line. Furthermore, PT could not induce CK expression in BCT31 cells as it did in parental cells. We conclude that there are at least two distinct growth factor pathways that play a key role in regulating proliferation and differentiation in BC3H1 muscle cells, one of which is PT sensitive, and postulate that a G protein is involved in transducing signals from the thrombin receptor. We believe that ras functions in the transduction of growth factor signals in the nonPT-sensitive pathway or downstream from the PT substrate in the second pathway.
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PMID:Growth factors, signaling pathways, and the regulation of proliferation and differentiation in BC3H1 muscle cells. II. Two signaling pathways distinguished by pertussis toxin and a potential role for the ras oncogene. 249 22

Bovine chromogranin A (CGA) was purified by three steps of column chromatography to a single-band purity in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antibody against this preparation was purified by a CGA-coupled Sepharose column, and F(ab')2 and Fab' of the antibody IgG were prepared by enzymatic digestion. An enzyme immunoassay (EIA) system was developed with the F(ab')2 immobilized on polystyrene balls and the Fab' labeled with beta-D-galactosidase. The EIA was able to detect 1 pg of CGA and was three to four orders more sensitive compared with any radioimmunoassay systems hitherto reported. Several neural acidic proteins (dopamine beta-hydroxylase, neuron specific enolase, S-100a protein, and brain-type creatine kinase) showed no cross-reaction. Using this EIA, CGA was detected in all regions of bovine central nervous system. CGA concentrations were within a narrow range, being lowest in the cerebellar white matter and highest in the putamen (17.3 and 78.1 ng/mg protein, respectively). The concentrations were extremely low compared to the concentration in the adrenal medulla (205,000 ng/mg protein). The highly sensitive EIA system should be useful for studies of materials containing very small amounts of CGA.
J Mol Neurosci 1989
PMID:Highly sensitive enzyme immunoassay for bovine chromogranin A: application for studies of regional distribution in bovine central nervous system. 251 59

Administration of adrenergic agonists induced c-fos mRNA in the salivary glands of the mouse and in the heart of the mouse, rat, and hamster (Barka et al., 1986, Mol. Cell Biol. 6, 2984-2989; 1987; Oncogene 1, 439-443). To further analyze transcriptional and post-transcriptional control of c-fos expression by adrenergic receptors and the putative role of fos in replication and differentiation pathways, we have examined c-fos expression in BC3H1 cells, a tumor-derived nonfusing muscle cell line. BC3H1 cells possess alpha 1- and beta 2-adrenergic receptors as well as receptors for histamine and acetylcholine. Furthermore, rapidly proliferating BC3H1 cells undergo differentiation toward muscle phenotype when exposed to low serum-containing culture media. Both alpha- and beta-adrenergic agonists and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate caused a rapid, transient increase in the steady-state level of c-fos mRNA. This induction was essentially independent of whether the cells were in the proliferative, relatively quiescent, or differentiated state. Protein synthesis inhibitors cycloheximide and anisomycin also increased markedly the concentration of c-fos mRNA, and in the presence of anisomycin c-fos mRNA was superinduced by the alpha-adrenergic agonist norepinephrine. Run-on transcription assays indicated that the c-fos gene is expressed in both proliferating and differentiated cells, although the steady-state levels of c-fos mRNA were low, or even undetectable, in such cells. The adrenergic agonists and the tumor promoter stimulated the transcription of the c-fos gene in both proliferating and differentiated cells. This stimulation, however, was modest, two- to three-fold compared to controls, in contrast to the marked elevation of the level of c-fos mRNA they caused. Neither the proliferation nor the expression of muscle type creatine kinase activity was influenced by adrenergic agonists. It is suggested that activation of the c-fos gene is a consequence of adrenoreceptor stimulation in diverse cell types, and thus it is involved in pleiotropic cellular responses to adrenergic agonists. Catecholamines may be one of the physiologic regulators of the c-fos gene.
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PMID:Adrenergic regulation of c-fos expression in cultured BC3H1 muscle cells. 255 27

On reoxygenation of ischemic or hypoxic hearts a sudden release of cytosolic enzymes coupled with hypercontraction and cell injury occurs, which has been termed the "oxygen paradox". We have attempted to imitate this phenomenon in cultured chick myocytes to try to find the cause of this sudden enzyme release. During 4 hours of normoxic perfusion (pH 7.4) monolayer cultures of chick embryonic myocytes retain their normal morphology, beat rhythmically, and show no release of creatine kinase (CK) into the perfusate. Hypoxic perfusion (O2 less than or equal to 0.25 microliter/ml) stops cell contraction (15-20 min) and causes "blebbing" of the sarcolemma (20-30 min). Membrane blebs increase in size and number with continuing hypoxia and eventually the cells become irreversibly damaged. Perfusion at pH 7.4 leads to a release of CK shortly after membrane damage occurs (30-40 min), with peak enzyme levels at 60-90 min. Reoxygenation after 120 min hypoxia does not exacerbate release. Hypoxic perfusion at pH 7.0 suppresses the release of CK from the cells despite extensive membrane blebbing. Normoxic perfusion at pH 7.4 after 100 min hypoxia (pH 7.0) causes an efflux of enzyme from the irreversibly injured cells. This can be prevented by reoxygenating the cells at pH 7.0 and stimulated by raising the pH of the hypoxic perfusate to 7.4. Shorter hypoxic periods (30 mins) at pH 7.0 followed by normoxic perfusion at pH 7.4 lead to a sudden large efflux of CK, arrhythmic contractions and hypercontraction of myofilaments, i.e. the typical symptoms of the "oxygen paradox". Thus changes in external pH can influence the release of intracellular enzymes during hypoxia and reoxygenation.
J Mol Cell Cardiol 1989 Oct
PMID:Enzyme release from chick myocytes during hypoxia and reoxygenation: dependence on pH. 258 22

The protection of angiotensin converting enzyme (ACE) inhibitors, captopril and ramiprilat, against free radical-mediated myocardial injury were studied in isolated working rat hearts. Free radicals were generated by electrolysis of Krebs-Henseleit solution with 10 mA direct current for 1 min. Both captopril (360 mumol/l) and ramiprilat (12.5 mumol/l) significantly reduced the decrease of left ventricle dP/dt'max, coronary flow (CF), myocardial superoxide dismutase (SOD) and creatine kinase (CK) activities and the elevation of S-T segment of epicardial ECG as well as the rise of myocardial malondialdehyde (MDA) content caused by electrolysed perfusate. Captopril afforded a dose-dependent protection on cardiac functions with various concentrations of 45, 90, 180 and 360 mumol/l. Iloprost (30 nmol/l), a stable mimetic of prostacyclin, could also alleviate free radical-mediated myocardial injuries. All the beneficial effects of ramiprilat (12.5 mumol/l) were abolished by the administration of indomethacin (5 mumol). In contrast, captopril (90 mumol/l) still exhibited significant protective effects after indomethacin (9 mumol) was administered, though these protective effects were insignificantly weakened. In order to assess the role of sulfhydryl (-SH) group in the effects of captopril, a SH-containing drug S8 and a disulfide DG4, both are deficient in ACE inhibitory properties in vitro, were examined. Data showed that S8 (180 mumol/l) provided a significant protection while DG4 showed no protective effect. It is concluded that ACE inhibitors can protect against free radical-induced myocardial damage. Ramiprilat, a non-SH-containing ACE inhibitor, inhibits free radical-induced damages mainly by stimulation of prostacyclin synthesis and/or release. In addition to this effect, captopril, a SH-containing ACE inhibitor, may exert additional anti-free radical effects by a mechanism which is probably related to the sulfhydryl group.
J Mol Cell Cardiol 1989 Dec
PMID:Captopril and ramiprilat protect against free radical injury in isolated working rat hearts. 269 63


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