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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mitogen-activated protein (MAP) kinases represent a group of serine/threonine protein kinases playing a central role in signal transduction processes in eukaryotic cells. Using a strategy based on the complementation of the thermosensitive autolytic phenotype of slt2 null mutants, we have isolated a Candida albicans homolog of Saccharomyces cerevisiae MAP kinase gene SLT2 (MPK1), which is involved in the recently outlined PKC1-controlled signalling pathway. The isolated gene, named MKC1 (MAP kinase from C. albicans), coded for a putative protein, Mkc1p, of 58,320 Da that displayed all the characteristic domains of MAP kinases and was 55% identical to S. cerevisiae Slt2p (Mpk1p). The MKC1 gene was deleted in a diploid Candida strain, and heterozygous and homozygous strains, in both Ura+ and Ura- backgrounds, were obtained to facilitate the analysis of the function of the gene. Deletion of the two alleles of the MKC1 gene gave rise to viable cells that grew at 28 and 37 degrees C but, nevertheless, displayed a variety of phenotypic traits under more stringent conditions. These included a low growth yield and a loss of viability in cultures grown at 42 degrees C, a high sensitivity to thermal shocks at 55 degrees C, an enhanced susceptibility to caffeine that was osmotically remediable, and the formation of a weak cell wall with a very low resistance to complex lytic enzyme preparations. The analysis of the functions downstream of the MKC1 gene should contribute to understanding of the connection of growth and morphogenesis in pathogenic fungi.
Mol Cell Biol 1995 Apr
PMID:Functional characterization of the MKC1 gene of Candida albicans, which encodes a mitogen-activated protein kinase homolog related to cell integrity. 789 15

Cocaine can produce both positive and negative inotropy. The mechanism of cocaine-induced negative inotropy is poorly understood. In order to evaluate the inhibitory effect of cocaine on myocardial contractility, its action on potassium chloride-induced depolarization, release of calcium from sarcoplasmic reticulum, and sarcolemmal sodium-calcium exchange were studied. At a relatively high concentration (10(-3) M), cocaine significantly blocked an elevation of cytosolic calcium during potassium chloride-induced depolarization and significantly inhibited the release of calcium from sarcoplasmic reticulum by caffeine. In contrast, a much lower concentration of cocaine (10(-7) M) significantly reduced sarcolemmal sodium-calcium exchange. Our results suggest that the negative inotropic action of cocaine may be related to a concentration-dependent effect: higher concentrations may inhibit calcium release from sarcoplasmic reticulum and block calcium influx across the sarcolemma. In contrast, lower concentrations would lead to a positive inotropic effect because of an impaired sarcolemmal sodium-calcium exchanger.
J Mol Cell Cardiol 1994 Nov
PMID:Inhibitory effect of cocaine on calcium mobilization in cultured rat myocardial cells. 789 65

The effects of pressure-overload left ventricular hypertrophy on the Ca2+ release channel in sarcoplasmic reticulum (SR) were studied by [3H]ryanodine binding and 45Ca2+ flux measurements. The density of Ca2+ release channel in left ventricle determined by equilibrium [3H]ryanodine binding to whole homogenates was significantly lower in hypertrophy than sham (Bmax: 0.47 +/- 0.04 v 0.72 +/- 0.10 pmol/mg protein), whereas total number of Ca2+ release channels in whole left ventricle was similar in the two groups. Ryanodine binding to SR vesicles isolated by differential centrifugation was also similar in the two groups, but the SR yield was less in the hypertrophied left ventricle. The Ca2+ release channels in hypertrophied left ventricles showed a significantly increased sensitivity to Ca2+ release agonists (e.g. caffeine and doxorubicin), as characterized by the effects of these agonists on ryanodine binding to whole homogenates and Ca2+ release from isolated SR. These results indicate that pressure-overload left ventricular hypertrophy is associated with both qualitative and quantitative changes in Ca2+ release channel function.
J Mol Cell Cardiol 1994 Nov
PMID:Alteration of Ca2+ release channel function in sarcoplasmic reticulum of pressure-overload-induced hypertrophic rat heart. 789 73

Expression of the recombination activating genes, RAG-1 and RAG-2, in lymphocytes, has been shown to depend on second messenger systems. An increase in intracellular cAMP upon stimulation with caffeine increases RAG expression while activation of protein kinase C (PKC) with phorbol myristate acetate (PMA) results in decreased RAG expression. The stringent regulation of recombination appears to be partially dependent on protein kinase activities which, alone, are not likely to be sufficient to regulate recombinase activity. We provide evidence implicating a role for serine/threonine phosphatases in the signal transduction pathway which regulates RAG gene expression and consequently the recombination process in lymphocytes. The cell permeable tumor promoter, calyculin-A (CLA), which is a potent inhibitor of the type 1 and 2A serine/threonine protein phosphatases (PP1 and PP2A, respectively), was shown to upregulate the expression of RAG-1 and RAG-2 in pre-B as well as mature B- and T-lymphocyte cell lines. Although agents such as caffeine known to increase intracellular cAMP levels induce RAG expression, synergy between CLA and caffeine was not detected in pre-B cells. An in vivo assessment of recombination activity after transfection of pre-B cells with an extrachromosomal recombination vector revealed a moderate increase in recombinase activity which paralleled RAG expression after CLA stimulation. Although increased cAMP levels in pre-B cells has been associated with upregulation of RAG expression we found no such upregulation in a surface immunoglobulin M positive (sIgM+) cell line, WEHI-231, and a T cell receptor positive (TCR+) murine cell line, EL-4. Moreover, in these mature lymphocyte cell lines there was no evidence of synergy in the regulation of RAG-1 and RAG-2 mRNA upon stimulation with CLA and caffeine. These results suggest novel intracellular mechanisms for the upregulation of RAG gene expression and confirm a role for type 1 and 2A phosphatases in the control of RAG gene expression and recombinase activity in lymphocyte cell lines.
Mol Immunol 1995 Feb
PMID:RAG-1 and RAG-2 gene expression and V(D)J recombinase activity are enhanced by protein phosphatase 1 and 2A inhibition in lymphocyte cell lines. 789 93

The potential of 8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-[3H] methylxanthine ([3H]Kf17837S) as a highly selective antagonist radioligand for the adenosine A2A receptor was examined and compared with the properties of the adenosine A2A receptor agonist radioligand 2-[p-(2-[3H]carboxyethyl)phenethylamino]-5'-N-ethyl- carboxamidoadenosine ([3H]CGS21680). [3H]KF17837S specific binding to rat striatal membranes was saturable and reversible. Saturation studies showed that the binding of [3H]KF17837S occurred at a single site, with high affinity (Kd, 7.1 +/- 0.91 nM) and limited capacity (Bmax, 1.3 +/- 0.23 pmol/mg of protein). Adenosine receptor antagonist ligands competed with the binding of 1 nM [3H]KF17837S with the following order of activity: CGS15943 > KF17837S > N-[2-(dimethylamino)ethyl]-N-methyl- 4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)benzenesul fonamide > or = xanthine amine congener > 8-cyclopentyl-1,3-dipropylxanthine > 8-(noradamantan-3-yl)-1,3-dipropylxanthine > caffeine. Adenosine receptor agonists inhibited [3H] KF17837S binding in the following order: 5'-N-ethylcarboxamidoadenosine > or = CGS21680 > 2-phenylaminoadenosine > or = (R)- N6-phenylisopropyladenosine > N6-cyclopentyladenosine > (S)- N6-phenylisopropyladenosine. The Ki values of the antagonists for [3H]KF17837S binding and the rank order of potency were similar to those for [3H]CGS21680 binding. The affinities of the agonists were lower with [3H]KF17837S binding than with [3H] CGS21680 binding. However, a strong positive correlation (r = 0.98) was observed between the pharmacological profiles for these two radioligand assays. The inhibition curve for CGS21680 was best fitted to a two-component binding model and addition of GTP shifted the inhibition curve to the right, suggesting that [3H]KF17837S labeled two agonist coupling states. Other pharmacological agents had negligible affinities for the [3H]KF17837S binding site. Autoradiographic study of [3H]KF17837S binding using rat brain sections revealed that the binding site was highly enriched in the striatal region. These data indicate that [3H] KF17837S labels the adenosine A2A receptor in rat brain.
Mol Pharmacol 1994 Nov
PMID:Binding of [3H]KF17837S, a selective adenosine A2 receptor antagonist, to rat brain membranes. 796 67

Recent findings on the ryanodine receptor of vertebrates, a Ca-release channel protein for the caffeine- and ryanodine-sensitive Ca pools, are reviewed in this article. Three distinct genes, i.e., ryr1, ryr2, and ryr3, express different isoforms in specific locations: Ryr1 in skeletal muscle and Purkinje cells of cerebellum; Ryr2 in cardiac muscle and brain, especially cerebellum; Ryr3 in skeletal muscle of nonmammalian vertebrates, the corpus striatum, and limbic cortex of brain, smooth muscles, and the other cells in vertebrates. While only one isoform (Ryr1) is expressed in mammalian skeletal muscles, two isoforms (alpha- and beta-isoforms expressed by ryr1 and ryr3, respectively) are found in nonmammalian vertebrate skeletal muscles. Although the coexistence of two isoforms may merely be related to differentiation and specialization, the biological significance remains to be clarified. Ryanodine receptors in vertebrate skeletal muscles are believed to mediate two different modes of Ca release: Ca(2+)-induced Ca release and action potential-induced Ca release. All results obtained so far with any isoform of ryanodine receptor are related to Ca(2+)-induced Ca release and show very similar characteristics. Ca(2+)-induced Ca release, however, cannot be the underlying mechanism of Ca release on skeletal muscle activation. Susceptibility of the ryanodine receptor's ryanodine-binding activity to modification by physical factors, such as osmolality of the medium, might be related to action potential-induced Ca release. A hypothesis of molecular interaction in view of the plunger model of action potential-induced Ca release is discussed, suggesting that the model could be compatible with Ryr1 and Ryr3, but incompatible with Ryr2. The functional relevance of ryanodine receptor isoforms, especially Ryr3, in brain also remains to be clarified. Among ryr1 gene-related diseases, malignant hyperthermia was the first to be identified; however, there is still the possibility of involvement of the other genes. Central core disease has been added to the list recently. A molecular approach for the diagnosis and treatment of diseases is now in progress.
Crit Rev Biochem Mol Biol 1994
PMID:Role of ryanodine receptors. 800 96

Excitation contraction (EC) coupling in muscle cells involves the movement of calcium through the calcium release channel of the sarcoplasmic reticulum (SR) membrane known as the ryanodine receptor. We have recently shown that the novel second messenger, sphingosine, can block calcium release from skinned skeletal muscle fibers and from isolated skeletal muscle SR membranes (Sabbadini et al., J Biol Chem 267: 15475-15484, 1992). In this report, we demonstrate that sphingosine also inhibits calcium release from isolated canine cardiac SR membranes containing the ryanodine receptor when release is induced by caffeine, doxorubicin or by calcium. Sphingosine also prevents the augmentation of [3H]-ryanodine binding normally produced by caffeine and doxorubicin and exerts noncompetitive inhibition with regard to both releasing agents. Sphingosine significantly reduces in a dose-dependent manner [3H]-ryanodine binding to the high affinity site of the receptor and increases by several-fold the Kd for binding, which is consistent with a blocking action of sphingosine on the ryanodine receptor calcium channel. Sphingosine inhibits the extent of calcium-induced calcium release (CICR) and significantly shifts the threshold for CICR so that a higher level of trigger calcium is required to initiate CICR. The sphingosine inhibition of CICR is consistent with the near abolition of calcium dependent [3H]-ryanodine binding. HPLC analysis of cardiac sphingosine content indicates that sphingosine is present in the cardiac cell at moderately high levels (29.4 nmol/g wet wt for the entire cell and approximately 0.4 microM for the cytosol) which are sufficient to produce significant inhibition by sphingosine on calcium release and ryanodine binding. The data suggest that sphingosine acts on the cardiac ryanodine receptor by opposing the physiological stimulus (e.g. trigger calcium entering via the dihydropyridine receptor). We propose that sphingosine is produced by the T-tubule membranes and that sphingosine is released into the protected intracellular environment of the T-tubule/SR junction to negatively modulate calcium release. Consequently, it is possible that sphingosine is a physiologically relevant regulator of calcium levels in the heart.
J Mol Cell Cardiol 1994 Feb
PMID:Modulation of cardiac sarcoplasmic reticulum ryanodine receptor by sphingosine. 800 84

Two experiments were conducted to assess the effects of caffeine and casein phosphopeptides (CPPs). One experiment tested the ability of frozen-thawed epididymal spermatozoa from boar (A, B, C), of proven low in vitro fertilization rates, to penetrate pig follicular oocytes. The other experiment tested the ability of ejaculated spermatozoa to uptake Ca2+. In Experiment 1, oocytes matured in vitro were inseminated with spermatozoa (Boar A) in medium that contained 0, 2, 5, 10, 15, and 20 mM caffeine and CPPs (1 mg/ml), or in medium that contained the same caffeine concentrations without CPPs. When CPPs were added to the caffeine-containing medium, significantly higher penetration rates were obtained than when the oocytes were inseminated in the CPPs-free medium. When the oocytes were inseminated with the spermatozoa (Boar A, B, C) in medium that contained 5 mM caffeine and dephosphorylated CPPs (dCPP:1 mg/ml), the penetration rate was significantly lower than when the oocytes were inseminated with the spermatozoa in medium containing 5 mM caffeine and CPPs (1 mg/ml). In Experiment 2, the concentration of Ca2+ in ejaculated spermatozoa of proven low in vitro fertilization rates during incubation in the fertilization medium was determined with fluorescence, Fura2/AM. When the medium contained CPPs, the intracellular concentration of Ca2+ in spermatozoa increased with a peak of 113 nM after 90 min of incubation. The concentration of Ca2+ was gradually decreased in the medium without CPPs. However, addition of CPPs in the medium had no effect on the motility of spermatozoa in Experiments 1 and 2.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Reprod Dev 1994 Apr
PMID:Effects of caffeine and casein phosphopeptides on fertilization in vitro of pig oocytes matured in culture. 801 29

The role of intracellular signal transduction mechanisms in regulating the motility and metabolism of rat spermatozoa in undiluted caudal epididymal fluid (CEF) was examined. Samples of CEF containing immotile spermatozoa were exposed to drugs and other agents that either stimulate signal transduction pathways or mimic the action of their second messengers. Under these conditions, sperm motility in 25-30 nl of CEF was stimulated by calcium ions (Ca2+), N2,2'-O-dibutyrylguanosine 3':5'-cyclic monophosphate (dibutyryl cGMP), cyclic adenosine 3':5'-monophosphate (cAMP), N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (dibutyryl cAMP), 8-bromoadenosine 3':5'-cyclic monophosphate (8-bromo cAMP), caffeine, theophylline and bicarbonate ions (HCO3-). Other agents such as magnesium ions (Mg2+), veratridine, phospholipase C (PLC), ionophore A23187, 1,2-dioctenoyl-sn-glycerol (DAG), phorbol 12-myristate 13-acetate, phospholipase A2 (PLA2), arachidonic acid, and melittin did not significantly influence motility. In the presence of radiolabelled energy substrates, untreated (immotile) spermatozoa in samples of CEF utilised D-[U-14C]glucose and [1-14C]acetate as exogenous energy sources for oxidative metabolism. No detectable 14C-lactate was produced, and none of the drugs altered the rate of glycolytic or oxidative metabolism. The findings suggest that the motility of rat caudal epididymal spermatozoa is regulated by Ca2+ and the guanylate cyclase and adenylate cyclase pathways, but not through the PLC and PLA2 pathways. Also, their metabolism of exogenous substrate was uncoupled from the induction of motility, and their oxidative capacity exceeded the rate of flux of glucose-carbon through the glycolytic pathway.
Mol Reprod Dev 1994 May
PMID:Intracellular signal transduction mechanisms of rat epididymal spermatozoa and their relationship to motility and metabolism. 804 68

In previous studies we have shown highly significant increases in chromosome damage and sister chromatid exchanges in heroin addicts, particularly when caffeine and metabolic inhibitors are added to the medium. Using human HUT-78 T-cell cultures, we now find direct in vitro evidence of opiate-induced or opiate-promoted mutagenesis via several assay systems. First, with microgel electrophoresis (MGE), we observed graded, dose-dependent, significant increases (P < .0001) in the frequency of comet tails of fragmented DNA when cells were treated with morphine alone (5 x 10(-9) M up to 10(-7) M) or when co-treated with the more potent mutagen, ethylmethanesulfonate (EMS). There were also dose-dependent increases in the lengths and densities of the comet tails observed. These findings were confirmed by a series of MGE experiments in which several days of morphine exposure preceded a 2-hr pulse of EMS. Second, mutant frequency (MF) assays also indicated significant opiate effects. These studies required separate assessment of cloning efficiencies and the frequencies of TG-resistant, HPRT-deficient mutant clones under four test conditions: no treatment, morphine alone for 4 days, morphine plus EMS, and EMS alone. Prior to the treatment phase, aminopterin was used to eliminate background HPRT mutations. The medium was changed after the treatment phase, the cells were allowed to express mutant phenotypes, and then TG was added and resistant mutant clones counted after 16 days. The background MF level for controls and for cells treated with EMS alone were negligible at 5.12 x 10(-8) and 7.25 x 10(-8), respectively. In the cells treated with morphine alone or morphine plus EMS, MF levels increased very significantly (P < .001) by > 100-fold to 5.1 x 10(-6) and 7.0 x 10(-6), respectively. Cloning efficiency also decreased significantly with both morphine-exposed conditions. Preliminary analysis with the single strand conformational polymorphism (SSCP) procedure following 6-thioguanine (TG) selection, also confirmed the occurrence of Exon 3 mutants of the HPRT gene in cells exposed to morphine plus EMS. It appears that brief EMS exposure can be repaired, whereas, if morphine exposure persists through one or more cell cycles, direct or indirect mutagenesis is initiated.
Environ Mol Mutagen 1994
PMID:Detection of opiate-enhanced increases in DNA damage, HPRT mutants, and the mutation frequency in human HUT-78 cells. 812 82


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