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)

Human intestinal mucin genes MUC3A and MUC3B are members of a membrane mucin gene family residing at chromosome 7q22. In this paper, we utilized genomic and cDNA cloning to elucidate the sequence of the 5'-region of the MUC3A gene including the gene promoter and the amino terminus coding sequence. Following its 21-residue signal peptide, the amino terminus of the mucin consists of a 233-residue Thr-, Ser-, and Pro-rich nonrepetitive sequence that is contiguous with its hypervariable domain of 375-residue repeats. RNase protection analysis and 5'-GeneRacer PCR indicated that MUC3A gene transcripts initiate from multiple start sites along a region spanning approximately 180 bases. The 5'-flanking region of the gene had promoter activity when fused to a luciferase reporter gene in all of the tested cell lines. This region contained binding sites for several transcription factors, including those implicated in the regulation of intestinal genes, but lacked a cognate TATA box. These features of the gene promoter may enable the gene to be expressed at variable levels in several cell types with different repertoires of transcription factors. We also utilized 5'-GeneRacer PCR to determine the sequence of the 5'-terminus of the MUC3B message. The amino termini of the MUC3A and MUC3B mucins are 91% conserved at the amino acid level. Thus, MUC3A and MUC3B have highly conserved amino and carboxyl termini, suggesting a recent duplication of the entire ancestral gene. It remains to be determined whether other members of the 7q22 membrane mucin gene family have amino-terminal domains similar to MUC3A and MUC3B.
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PMID:Initiation of transcription of the MUC3A human intestinal mucin from a TATA-less promoter and comparison with the MUC3B amino terminus. 1295 10

gamma-Glutamyl hydrolase (GGH) plays a central role in folate metabolism and antifolate action. Increased GGH activity has been found in rat hepatoma cells resistant to the cancer drug methotrexate (MTX). The aim of this study was to identify polymorphisms in the GGH gene that modulate GGH activity and that may affect methotrexate resistance. Exons of the human gamma-glutamyl hydrolase (hGGH) gene were amplified by polymerase chain reaction (PCR) from breast cancer tissue and leukemia cell lines. Single-stranded conformational polymorphism (SSCP) analysis was performed, and PCR products containing different patterns were cloned and sequenced. Six single nucleotide polymorphisms (SNPs) were identified, at bases -401C>T, -354G>T, -124T>G, +16T>C, +452C>T, and +1102A>G, relative to the A of the translation start codon being considered as +1. The SNP at +16, which changes codon -19 (relative to the start of the mature hGGH protein) in the endoplasmic reticulum targeting sequence of hGGH protein from cysteine to arginine, has previously been identified in this laboratory. The SNP at +452 changes the conserved hGGH protein codon 127 from threonine to isoleucine. The functions of SNPs in the promoter of the hGGH gene were studied by site-directed mutagenesis of a 516-bp region of the hGGH gene promoter in a luciferase reporter vector and transfection into HepG2 and MCF-7 cells. All of the promoter polymorphisms enhanced the production of luciferase compared to the wild-type hGGH gene promoter in HepG2 cells, and -401C>T and -124T>G enhanced luciferase expression in MCF-7 cells, suggesting that polymorphisms in the hGGH gene promoter may increase expression of hGGH protein.
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PMID:Identification of single nucleotide polymorphisms in the human gamma-glutamyl hydrolase gene and characterization of promoter polymorphisms. 1459 82

Substitution of a methionine residue at position 79 in poliovirus protein 3A with valine or threonine caused defective viral RNA synthesis, manifested as delayed onset and reduced yield of viral RNA, in HeLa cells transfected with a luciferase-containing replicon. Viruses containing these same mutations produced small or minute plaques that generated revertants upon further passage, with either wild-type 3A sequences or additional nearby compensating mutations. Translation and polyprotein processing were not affected by the mutations, and 3AB proteins containing the altered amino acids at position 79 showed no detectable loss of membrane-binding activity. Analysis of individual steps of viral RNA synthesis in HeLa cell extracts that support translation and replication of viral RNA showed that VPg uridylylation and negative-strand RNA synthesis occurred normally from mutant viral RNA; however, positive-strand RNA synthesis was specifically reduced. The data suggest that a function of viral protein 3A is required for positive-strand RNA synthesis but not for production of negative strands.
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PMID:Strand-specific RNA synthesis defects in a poliovirus with a mutation in protein 3A. 1461 Jan 90

Mammalian cells chronically exposed to ionizing radiation (IR) induce stress response with a tolerance to the subsequent cytotoxicity of IR. Although p53 is well documented in IR response, the signaling network causing p53 activation in chronic IR remains to be identified. Using breast carcinoma MCF+FIR cells that showed a transient radioresistance after exposure chronically to fractionated IR (FIR), the present study shows that the basal DNA binding and transcriptional activity of p53 was elevated by FIR. p53-controlled luciferase activity was strikingly induced ( approximately 7.9-fold) with little enhancement of p53/DNA binding activity ( approximately 1.3-fold). The phosphorylated p53 (Thr 55) was increased in the cytoplasm and nucleus of MCF+FIR but not in the sham-FIR control cells. On the contrary, the sham-FIR control MCF-7 cells showed a low p53 luciferase transcription ( approximately 3-fold) but a striking enhancement of p53/DNA binding (12-fold) after 5 Gy of IR. To determine the signaling elements regulating p53 activity, DNA microarray of MCF+FIR using sham-FIR MCF-7 cells as a reference demonstrated that the mRNA of p21, MDM2, and p14ARF was up-regulated. Time course Western blot analysis, however, showed no difference in p21 induction. In contrast, MDM2 that was absent in control cells and was predominantly induced by IR was not induced in MCF+FIR cells. In agreement with MDM2 inhibition, MDM2-inhibitory protein p14ARF was increased in MCF+FIR cells. In summary, these results demonstrate that up-regulation of p14ARF paralleled with MDM2 inhibition contributes to p53 accumulation in the nucleus and causes a high responsiveness of p53 in chronic IR-treated breast cancer cells.
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PMID:p53 activation in chronic radiation-treated breast cancer cells: regulation of MDM2/p14ARF. 1472 28

Alkylphospholipids (ALKs) are a novel class of antitumor agents with an unknown mechanism of action. The first ALK tested in the clinic, miltefosine, has been approved recently in Europe for the local treatment of patients with cutaneous metastasis. Perifosine, the only available oral ALK, is being studied currently in human cancer clinical trials. We have shown previously that perifosine induces p21(waf1/cip1) in a p53-independent fashion and that induction of p21(waf1/cip1) is required for the perifosine-induced cell cycle arrest because cell lines lacking p21(waf1/cip1) are refractory to perifosine. In this report, we investigated the mechanism by which perifosine induces p21(waf1/cip1) protein expression. We observed that perifosine induces the accumulation of p21(waf1/cip1) mRNA without affecting p21(waf1/cip1) mRNA stability. Using several p21(waf1/cip1) promoter-driven luciferase reporter plasmids, we observed that perifosine activates the 2.4-kb full-length p21(waf1/cip1) promoter as well as a p21 promoter construct lacking p53-binding sites, suggesting that perifosine activates the p21(waf1/cip1) promoter independent of p53. The minimal p21 promoter region required for perifosine-induced p21 promoter activation contains four consensus Sp1-binding sites. Mutations in each particular Sp1 site block perifosine-induced p21(waf1/cip1) expression. Moreover, we showed that perifosine activates the mitogen-activated protein/extracellular signal-regulated kinase pathway, and this activation promotes the phosphorylation of Sp1 in known mitogen-activated protein kinase residues (threonine 453 and 739), thereby leading to increased Sp1 binding and enhanced p21(waf1/cip1) transcription. These results represent a novel mechanism by which alkylphospholipids modulate transcription, and may contribute to the discovery of new signal transduction pathways crucial for normal and neoplastic cell cycle control.
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PMID:Transcriptional activation of p21(waf1/cip1) by alkylphospholipids: role of the mitogen-activated protein kinase pathway in the transactivation of the human p21(waf1/cip1) promoter by Sp1. 1474 93

We delineate a mechanism by which dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin or TCDD)-mediated formation of the aryl hydrocarbon receptor (AhR) DNA binding complex is disrupted by a single mutation at the conserved AhR tyrosine 9. Replacement of tyrosine 9 with the structurally conservative phenylalanine (AhRY9F) abolished binding to dioxin response element (DRE) D, E, and A and abrogated DRE-driven gene induction mediated by the AhR with no effect on TCDD binding, TCDD-induced nuclear localization, or ARNT heterodimerization. The speculated role for phosphorylation at tyrosine 9 was also examined. Anti-phosphotyrosine immunoblotting could not detect a major difference between the AhRY9F mutant and wild-type AhR, but a basic isoelectric point shift was detected by two-dimensional gel electrophoresis of AhRY9F. However, an antibody raised to recognize only phosphorylated tyrosine 9 (anti-AhRpY9) confirmed that AhR tyrosine 9 is not a phosphorylated residue required for DRE binding. Kinase assays using synthetic peptides corresponding to the wild-type and mutant AhR residues 1-23 demonstrated that a tyrosine at position 9 is important for substrate recognition at serine(s)/threonine(s) within this sequence by purified protein kinase C (PKC). Also, compared with AhRY9F, immunopurified full-length wild-type receptor was more rapidly phosphorylated by PKC. Furthermore, co-treatment of AhR-deficient cells that expressed AhRY9F and a DRE-driven luciferase construct with phorbol 12-myristate 13-acetate and TCDD resulted in a 30% increase in luciferase activity compared with AhRY9F treated with TCDD alone. Overall, AhR tyrosine 9, which is not a phosphorylated residue itself but is required for DNA binding, appears to play a crucial role in AhR activity by permitting proper phosphorylation of the AhR.
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PMID:The aryl hydrocarbon receptor (AhR) tyrosine 9, a residue that is essential for AhR DNA binding activity, is not a phosphoresidue but augments AhR phosphorylation. 1497 34

The serine/threonine phosphatase calcineurin is an important regulator of calcium-activated intracellular responses in eukaryotic cells. In higher eukaryotes, calcium/calmodulin-mediated activation of calcineurin facilitates direct dephosphorylation and nuclear translocation of the transcription factor nuclear factor of activated T-cells (NFAT). Recently, controversy has surrounded the role of calcineurin in mediating skeletal muscle cell hypertrophy. Here we examined the ability of calcineurin-deficient mice to undergo skeletal muscle hypertrophic growth following mechanical overload (MOV) stimulation or insulin-like growth factor-1 (IGF-1) stimulation. Two distinct models of calcineurin deficiency were employed: calcineurin Abeta gene-targeted mice, which show a approximately 50% reduction in total calcineurin, and calcineurin B1-LoxP-targeted mice crossed with a myosin light chain 1f cre knock-in allele, which show a greater than 80% loss of total calcineurin only in skeletal muscle. Calcineurin Abeta-/- and calcineurin B1-LoxP(fl/fl)-MLC-cre mice show essentially no defects in muscle growth in response to IGF-1 treatment or MOV stimulation, although calcineurin Abeta-/- mice show a basal defect in total fiber number in the plantaris and a mild secondary reduction in growth, consistent with a developmental defect in myogenesis. Both groups of gene-targeted mice show normal increases in Akt activation following MOV or IGF-1 stimulation. However, overload-mediated fiber-type switching was dramatically impaired in calcineurin B1-LoxP(fl/fl)-MLC-cre mice. NFAT-luciferase reporter transgenic mice failed to show a correlation between IGF-1- or MOV-induced hypertrophy and calcineurin-NFAT-dependent signaling in vivo. We conclude that calcineurin expression is important during myogenesis and fiber-type switching, but not for muscle growth in response to hypertrophic stimuli.
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PMID:Genetic loss of calcineurin blocks mechanical overload-induced skeletal muscle fiber type switching but not hypertrophy. 1508 23

Many proteins and pathways of pharmaceutical interest impinge on ubiquitin ligases or their substrates. The cyclin-dependent kinase (Cdk) inhibitor p27, for example, is polyubiquitylated in a cell cycle-dependent manner by a ubiquitin ligase complex containing the F-box protein Skp2. Regulated turnover of p27 is due, at least partly, to its phosphorylation by Cdk2 on threonine 187, which generates a Skp2-binding site. We made a p27-luciferase (p27Luc) fusion protein and show here that its abundance, like that of p27, is regulated by Skp2 in a cell cycle-dependent manner. As predicted, p27Luc levels increased after blocking Cdk2 activity with inhibitory proteins, peptides or small interfering RNA (siRNA). Accumulation of p27Luc in response to Cdk2 inhibitory drugs (flavopiridol and R-roscovitine) was demonstrable in human tumor cells in vivo using noninvasive bioluminescent imaging. In theory, the approach described here could be used to develop bioluminescent reporters for any drug target that directly or indirectly affects the turnover of a ubiquitin ligase substrate.
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PMID:Bioluminescent imaging of Cdk2 inhibition in vivo. 1512 51

Diesel exhaust particles (DEP) induce intense inflammatory and allergic immune responses. The epidermal cells receive much exposure to DEP, and are an important source of pro-inflammatory cytokines and other inflammatory mediators. Transcription factors, such as nuclear factor kappa B (NF-kappaB) and activator protein 1 (AP-1), regulate the expression of these mediators. We hypothesize that the transcription factors are target of DEP action. The current study sought to determine whether DEP-activated NF-kappaB and AP-1 in a mouse epidermal cell line, JB6 P(+) cells. Using stable transfectants of JB6 P(+) cells expressing NF-kappaB or AP-1 luciferase reporter constructs, we demonstrated that exposure to DEP at a non-cytotoxic concentration significantly enhanced the transactivation of NF-kappaB, but not AP-1. Furthermore, DEP promoted phosphorylation of Akt, a substrate of phosphatidylinositol 3-kinase (PI3K), on Ser-473 and Thr-308 in a PI3K-dependent manner, and enhanced phosphorylation of down-stream p70/p85 S6 kinases (p70/p85S6K) as well as glycogen synthase kinase-3beta (GSK-3beta). Blockage of PI3K activation eliminated DEP-stimulated NF-kappaB transactivation. Although SAPK/JNK pathway was modestly activated by DEP, it was not involved in NF-kappaB transactivation. DEP had little effect on the phosphorylation of ERKs and p38 MAPK. Thus, DEP-induced transactivation of NF-kappaB is mediated by PI3K/Akt signaling pathway.
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PMID:Activation of nuclear factor kappa B by diesel exhaust particles in mouse epidermal cells through phosphatidylinositol 3-kinase/Akt signaling pathway. 1513 Jul 73

The small molecule UCN-01 is a cyclin-dependent kinase (CDK) modulator shown to have antiproliferative effects against several in vitro and in vivo cancer models currently being tested in human clinical trials. Although UCN-01 may inhibit several serine-threonine kinases, the exact mechanism by which it promotes cell cycle arrest is still unclear. We have reported previously that UCN-01 promotes G(1)-S cell cycle arrest in a battery of head and neck squamous cancer cell lines. The arrest is accompanied by an increase in both p21(waf1/cip1) and p27(kip1) CDK inhibitors leading to loss in G(1) CDK activity. In this report, we explore the role and the mechanism for the induction of these endogenous CDK inhibitors. We observed that p21 was required for the cell cycle effects of UCN-01, as HCT116 lacking p21 (HCT116 p21(-/-)) was refractory to the cell cycle effects of UCN-01. Moreover, UCN-01 promoted the accumulation of p21 at the mRNA level in the p53-deficient HaCaT cells without increase in the p21 mRNA half-life, suggesting that UCN-01 induced p21 at the transcriptional level. To study UCN-01 transcriptional activation of p21, we used several p21(waf1/cip1) promoter-driven luciferase reporter plasmids and observed that UCN-01 activated the full-length p21(waf1/cip1) promoter and a construct lacking p53 binding sites. The minimal promoter region required for UCN-01 (from -110 bp to the transcription start site) was the same minimal p21(waf1/cip1) promoter region required for Ras enhancement of p21(waf1/cip1) transcription. Neither protein kinase C nor PDK1/AKT pathways were relevant for the induction of p21 by UCN-01. In contrast, the activation of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase mitogen-activated protein kinase pathways was required for p21 induction as UCN-01 activated this pathway, and genetic or chemical MEK inhibitors blunted p21 accumulation. These results demonstrated for the first time that p21 is required for UCN-01 cell cycle arrest. Moreover, we showed that the accumulation of p21 is transcriptional via activation of the MEK pathway. This novel mechanism, by which UCN-01 exerts its antiproliferative effect, represents a promising strategy to be exploited in future clinical trials.
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PMID:UCN-01-induced cell cycle arrest requires the transcriptional induction of p21(waf1/cip1) by activation of mitogen-activated protein/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase pathway. 1515 Jan 22


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