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:1.14.14.3 (luciferase)
38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) plays a critical role in the contractile performance of cardiac and slow-twitch skeletal muscle by restoring cytosolic calcium to low resting levels during the contractile cycle. We have previously shown that SERCA2 expression in the heart is altered by a number of pathophysiological stimuli. In an effort to define molecular mechanisms regulating expression of the SERCA2 gene in cardiac muscle cells, deletions of a 1460-bp promoter fragment were generated and inserted into a luciferase reporter plasmid. Promoter constructs were transiently transfected into embryonic cardiocytes and skeletal muscle cell lines Sol 8 and C2C12 in vitro and injected into adult myocardium in vivo. Results demonstrate that sequences from the transcription start site to -284 are both necessary and sufficient for high-level transcription of the reporter gene in differentiating muscle cells and in fetal cardiocytes in culture. We further demonstrate that this promoter fragment is highly active in vivo when injected into rat hearts, suggesting that the same regulatory elements are functional in vivo as well as in vitro. The region of the gene from -284 to -658 exerts a modest positive effect in cardiocytes and Sol 8 myotubes but exerts a negative effect in C2C12 fast skeletal muscle cells. This initial analysis of transcriptional regulation of the SERCA2 gene will serve as a foundation for the study of alterations of expression of the gene in pathological conditions.
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PMID:Characterization of promoter elements of the rabbit cardiac sarcoplasmic reticulum Ca(2+)-ATPase gene required for expression in cardiac muscle cells. 837 Jan 20

The human Na,K-ATPase beta 1 subunit gene promoter activity is stimulated by thyroid hormone (T3) in the human intestinal Caco-2 cells. To identify potential cis-acting transcriptional regulatory elements involved in this process, chimeric plasmids containing varying lengths of the 5' flanking region of the human beta 1 Na,K-ATPase gene linked to the firefly luciferase reporter gene were introduced into Caco-2 cells by transient transfection. Analysis of T3-regulated luciferase activity of cells carrying these plasmids, and subsequent use of site-directed mutagenesis revealed that a region from -459 to -438 (relative to the transcriptional start site) is required for the induction of the beta 1 Na,K-ATPase gene by T3. An oligonucleotide containing this sequence from -465 to -433 confers T3 responsiveness to a heterologous promoter. Gel mobility shift assays showed specific binding of nuclear proteins of Caco-2 cells to this region and immunoreactive T3 receptor was identified in one of these complexes. These data demonstrate that there is a cis-acting thyroid hormone responsive element in the 5' flanking region of the human beta 1 Na,K-ATPase gene and induction of transcription of this gene by T3 involves specific binding of the thyroid hormone receptor to the TRE located at position -459 to -438.
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PMID:Identification of a functional thyroid hormone response element in the upstream flanking region of the human Na,K-ATPase beta 1 gene. 839 3

Na,K-ATPase (Na,K-pump) plays an important role in the regulation of intracellular ion composition. The purpose of this study is to determine whether Na+ regulates the levels of mRNA coding for Na,K-ATPase alpha and beta subunits in cultured neonatal rat cardiocytes. We measured intracellular Na+ levels ([Na+]i) in cardiocytes using a Na(+)-sensitive fluorescence dye (SBFI). 1 mM ouabain caused a significant increase in [Na+]i in cardiocytes; from 12.8 +/- 0.3 to 28.8 +/- 1.8 mM. Exposure of cardiocytes to 1 mM ouabain resulted in a three- to fourfold increase in alpha 1, alpha 2, and alpha 3 mRNA accumulation, and an approximate two-fold increase in beta 1 mRNA accumulation. A maximum elevation was reached at 60 min in both cases. The ouabain-induced alpha 1 mRNA accumulation was still observed in the Ca(2+)-free culture medium. Exposure of cardiocytes to 10 microM monensin in the absence of extracellular Ca2+ also resulted in a threefold increase in alpha 1 mRNA accumulation. The increased alpha 1 mRNA expression by 1 mM ouabain was associated with a fourfold increase in alpha 1 subunit protein accumulation. Transfection experiments with chimeric plasmids containing 5'-flanking sequences of alpha 1, alpha 2, and alpha 3 isoform genes and a luciferase reporter gene revealed that 1 mM ouabain caused a twofold increase in luciferase activity in each alpha system. These results suggest that Na+ directly regulates Na,K-ATPase gene expression in cardiocytes. The transfection study further supports the premise that Na(+)-responsive elements are located within the 5'-flanking sequences of each alpha isoform gene.
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PMID:Regulation of Na,K-adenosine triphosphatase gene expression by sodium ions in cultured neonatal rat cardiocytes. 840 40

We have purified to apparent homogeneity a 66-kDa protein from rabbit reticulocyte lysate which is associated with hsp 70. Our characterization of this 66-kDa protein demonstrates that its physiological role is to promote the recycling of hsp 70 by catalyzing the dissociation of hsp 70-bound ADP in exchange for ATP. We have therefore termed the 66-kDa protein RF-hsp 70, a recycling factor for hsp 70. RF-hsp 70 promotes stoichiometric binding of ATP to hsp 70, and it increases about 5-fold the rate of dissociation of hsp 70.ADP in the presence of ATP. This process represents adenine nucleotide exchange, since dissociation of ADP does not occur unless ATP is added; dATP, GTP, and ITP cannot substitute for ATP. The mechanism of action of RF-hsp 70 is to lower the KD of hsp 70 for ATP about 6-7-fold to a value that is close to the KDof hsp 70 for ADP. RF-hsp 70 also stimulates the ATPase activity of hsp 70, including the 42-kDa amino-terminal portion of hsp 70 generated by chymotrypsin, demonstrating that RF-hsp 70 interacts with that part of hsp 70 known to contain the ATP/ADP binding site. Confirming its recycling function, RF-hsp 70 stimulates about 7-10-fold the ability of hsp 70 to reactivate heat-denatured firefly luciferase. In addition, RF-hsp 70 acts catalytically to recycle hsp 70, since, at 0.2 times the molar concentration of hsp 70, RF-hsp 70 increases the rate of renaturation of luciferase by hsp 70 about 3-4-fold. The action of RF-hsp 70 is also partially species-specific since it is most effective with rabbit reticulocyte hsp 70, less effective with bovine brain hsp 70, even less effective with human hsp 70, and ineffective with broad bean hsp 70.
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PMID:Purification and characterization of a 66-kDa protein from rabbit reticulocyte lysate which promotes the recycling of hsp 70. 866 20

Lipophilic inhibitors such as general anaesthetics or drugs can conceivably act by displacing boundary lipid molecules that are required by many functional membrane proteins. The resulting lipid-protein mismatch has been analyzed previously in terms of multiple site kinetics (Sandermann H. (1993) Biochim. Biophys. Acta 1150, 130-133). Expressions for kinetic cooperativity are now derived, and data for the inhibition of dog kidney Na+,K+-ATPase and Escherichia coli lactose permease by organic solvents are presented and analyzed. Half-maximal inhibitor concentrations were without diagnostic value because they were within the general range of critical solvent concentrations known for general anaesthesia and several membraneous and non-membraneous systems, as well as two specific liposomal parameters. The kinetic cooperativity of inhibition was of much higher diagnostic value because the cooperativity values for the solvent inhibition of Na+,K+-ATPase and lactose permease were characteristic for the lipid displacement mechanism, in contrast to cooperativity values of protein kinase C and luciferase. The latter enzymes are known not to require a boundary lipid layer, so that the degree of kinetic cooperativity provides a new diagnostic tool to distinguish between modes of action of lipophilic inhibitors.
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PMID:Induction of lipid-protein mismatch by xenobiotics: kinetic cooperativity. 867 87

The effects of the human DnaJ homolog, Hsp40, on the ATPase and chaperone functions of the constitutively expressed Hsp70 homolog, Hsc70, were analyzed. Hsp40 stimulates the hydrolysis of ATP by Hsc70, causing a approximately 7-fold increase in its steady-state ATPase activity. In contrast to the prokaryotic Hsp70 system, ATP-hydrolysis and not the release of bound ADP is the rate-limiting step in the overall ATPase cycle of mammalian Hsc70. The ability to activate the Hsc70 ATPase is partially preserved in a deletion mutant containing the J-domain and the G/F region of Hsp40 but not in a deletion mutant that contains the J-domain alone. As a result of its ATPase stimulating activity, addition of Hsp40 allows Hsc70 to bind peptide in the presence of ATP, whereas in the absence of Hsp40, peptide is efficiently released upon ATP binding to Hsc70. The functional cooperation of Hsp40 with Hsc70 is essential to ensure the ATP hydrolysis-dependent binding of aggregation-sensitive denatured polypeptides, such as thermally denatured firefly luciferase and chemically denatured rhodanese. Binding of these proteins results in the formation of ternary complexes of Hsc70, Hsp40, and substrates. Hsc70 and Hsp40 cooperate with further factors in protein renaturation, as demonstrated by the finding that luciferase, thermally denatured in the presence of Hsc70, Hsp40, and ATP, refolds upon addition of rabbit reticulocyte cytosol. Our results indicate that Hsp40 has a critical regulatory function in the Hsc70 ATPase cycle that is required for the efficient loading of peptide substrate onto Hsc70.
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PMID:Regulation of the heat-shock protein 70 reaction cycle by the mammalian DnaJ homolog, Hsp40. 870 58

Infection of quiescent cells with the DNA tumor virus simian virus 40 induces expression of the cellular thymidine kinase (TK) gene a minimum of 10- to 20-fold, and this induction depends upon the viral protein large T antigen (T-Ag). To define both human TK promoter elements and T-Ag functional domains required for transcriptional induction, we have established a system in which stable Rat-1 transfectants harboring TK promoter-luciferase hybrid genes are infected with recombinant adenoviruses expressing either wild-type or mutant forms of T-Ag and luciferase expression is measured as an indicator of promoter activity. The results show that (i) a 135-bp TK promoter fragment is activated 10- to 15-fold by viral infection; (ii) this activation is the result of both T-Ag-dependent and -independent mechanisms; (iii) the T-Ag pRb family-binding domain, but not the p53-binding, helicase, or ATPase domain, is required for activation; and (iv) activation is severely diminished with a TK promoter fragment in which E2F-like-binding sites have been removed. These data demonstrate a requirement for both an E2F-related factor and a pRb family member in activation of the TK promoter by T-Ag. This contrasts with the promiscuous activation of many cellular and viral genes by T-Ag, which is independent of its ability to bind pRb.
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PMID:Activation of the human thymidine kinase (TK) promoter by simian virus 40 large T antigen requires both the T antigen pRb family-binding domain and TK promoter sequences resembling E2F-binding sites. 870 58

The association of peroxisomes with cytoskeletal structures was investigated both by electron microscopy and by kinetic analysis of peroxisome movement. The morphological studies indicated distinct interactions of peroxisomes with microtubules and frequently revealed multiple contact sites. The kinetic approach utilised microinjection and import of fluorescein-labeled luciferase in order to mark and track peroxisomes in vivo. Peroxisomal motility was analysed by time-lapse imaging and fluorescence microscopy. According to their movement peroxisomes were classified into two groups. Group 1 peroxisomes comprising the majority of organelles at 37 degrees C moved slowly with an average velocity of 0.024 +/- 0.012 micron/second whereas the movement of group 2 peroxisomes, 10-15% of the total population, was saltatory exhibiting an average velocity of 0.26 +/- 0.17 micron/second with maximal values of more than 2 microns/second. Saltations were completely abolished by the microtubule-depolymerising drug nocodazole and were slightly reduced by about 25% by cytochalasin D which disrupts the actin microfilament system. Double fluorescence labeling of both peroxisomes and microtubules revealed peroxisome saltations linked to distinct microtubule tracks. Cellular depletion of endogenous levels of NTPs as well as the use of 5'-adenylylimidodiphosphate, a nonhydrolysable ATP analog, applied to a permeabilised cell preparation both completely blocked peroxisomal movement. These data suggest an ATPase dependent, microtubule-based mechanism of peroxisome movement. Both the intact and the permeabilised cell system presented in this paper for the first time allow kinetic measurements on peroxisomal motility and thus will be extremely helpful in the biochemical characterisation of the motor proteins involved.
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PMID:Microtubule-based peroxisome movement. 871 75

Previous biochemical and genetic studies have demonstrated the universal conservation of the DnaK (Hsp70) chaperone machine. Its three members, DnaK, DnaJ, and GrpE, in Escherichia coli work synergistically to promote protein protection, disaggregation, and import into the various organelles. In the mitochondria of Saccharomyces cerevisiae the three corresponding members are designated as Ssc1p, Mdj1p, and Mge1p, respectively. The MGE1 gene was previously cloned by us and others, and its product has been shown to be absolutely essential for protein transport into mitochondria and hence cell viability. To better understand its biological role, we have proceeded to overexpress and purify the mature Mge1p in E. coli through the construction of the appropriate vector clone. Mge1p has been shown to functionally substitute for its E. coli GrpE counterpart in a variety of its biological functions, including suppression of the bacterial temperature-sensitive phenotype of the grpE280 mutation, formation of a stable complex with DnaK, stimulation of DnaK's ATPase activity, and the refolding of denatured luciferase by the DnaK/DnaJ chaperone proteins. Thus, the function of the GrpE homologues appears to be highly conserved across the biological kingdoms.
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PMID:Purification and biochemical properties of Saccharomyces cerevisiae's Mge1p, the mitochondrial cochaperone of Ssc1p. 879 29

We have previously demonstrated in vitro actin movement at nanomolar adenosine triphosphate (ATP) levels using heavy meromyosin from skeletal muscle. In the present work we tested whether the motility at nonomolar ATP-concentrations could be supported by cardiac myosin as well. Actomyosin (skeletal actin and bovine ventricular myosin) was pretreated in the in vitro motility assay with 1 mM ATP; subsequently, the ATP level was reduced by multiple rigor-solution washes. By the final rigor-solution wash, the ATP concentration, monitored by the luciferin-luciferase assay, dropped to the order of 100 nM. Even at this low ATP level actin-filament movement remained in evidence. This was in marked contrast to the situation where ATP concentration was gradually increased from zero; in the latter, filament movement began only as ATP levels exceeded 1-2 microM. The difference indicates that potential energy is stored during the initial ATP treatment, and utilized later as the free ATP falls below micromolar levels. Although the velocity of cardiac myosin-supported movement was only one fourth of that of skeletal myosin, both myosins supported actin movement down to similar ATP concentrations. The similarity in response of the two myosins to ATP implies a similar degree of potential energy storage. Given the significantly different specific ATPase activities, however, it appears that the mechanism of potential energy storage and release involves factors different from those involved in the release of chemical energy by the myosin ATPase.
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PMID:Persisting in vitro actin motility at nanomolar adenosine triphosphate levels: comparison of skeletal and cardiac myosins. 886 77


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