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 cDNA encoding PDE10A (phosphodiesterase 10A) was cloned and a stable recombinant HEK-293 (human embryonic kidney-293) cell line expressing high levels of PDE10A was generated. Transient transfection of pCRE-Luc plasmid, harbouring the luciferase reporter gene under the control of CRE (cAMP-response element)-binding sequence, into the stable recombinant cell line, followed by treatment with PDE10 inhibitor, resulted in a dose-dependent increase in luciferase activity. This method provides a simple and sensitive cell-based assay for screening of PDE10 inhibitors for development of novel therapeutics for the treatment of neurological disorders.
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PMID:Development of a cell-based assay for screening of phosphodiesterase 10A (PDE10A) inhibitors using a stable recombinant HEK-293 cell line expressing high levels of PDE10A. 1764 Jan 73

In the present study we investigated promoter regions of the PHGPx [phospholipid hydroperoxide GPx (glutathione peroxidase)] gene and transcription factors involved in TNFalpha (tumour necrosis factor alpha)-induced up-regulation of PHGPx in non-differentiated HL60 cells. Non-differentiated HL60 cells displayed up-regulation of non-mitochondrial and mitochondrial PHGPx mRNA in response to TNFalpha stimulation. The promoter activity was up-regulated by TNFalpha stimulation in cells transfected with a luciferase reporter vector encoding the region from -282 to -123 of the human PHGPx gene compared with the non-stimulated control. The up-regulated promoter activity was effectively abrogated by a mutation in the C/EBP (CCAAT/enhancer-binding protein)-binding sequence in this region. ChIP (chromatin immunoprecipitation) assays demonstrated that C/EBPepsilon bound to the -247 to -34 region in HL60 cells, but C/EBPalpha, beta, gamma and delta did not. The binding of C/EBPepsilon to the promoter region was increased in HL60 cells stimulated with TNFalpha compared with that of the non-stimulated control. An increased binding of nuclear protein to the C/EBP-binding sequence was observed by EMSA (electrophoretic mobility-shift assay) in cells stimulated with TNFalpha, and it was inhibited by pre-treatment with an anti-C/EBPepsilon antibody, but not with other antibodies. The C/EBPepsilon mRNA was expressed in PMNs (polymorphonuclear cells), non-differentiated HL60 cells and neutrophil-like differentiated HL60 cells displaying TNFalpha-induced up-regulation of PHGPx mRNA, but not in macrophage-like differentiated HL60 cells, HEK-293 cells (human embryonic kidney-293 cells) and other cell lines exhibiting no up-regulation. The up-regulation of PHGPx mRNA, however, was detected in HEK-293 cells overexpressing C/EBPepsilon as a result of TNFalpha stimulation. These results indicate that C/EBPepsilon is a critical transcription factor in TNFalpha-induced up-regulation of PHGPx expression.
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PMID:Identification of a responsible promoter region and a key transcription factor, CCAAT/enhancer-binding protein epsilon, for up-regulation of PHGPx in HL60 cells stimulated with TNF alpha. 1768 22

The mammalian TLRs (Toll-like receptors) mediate the rapid initial immune response to pathogens through recognition of pathogen-associated molecular patterns. The pathogen pattern to which TLR8 responds is ssRNA (single-stranded RNA) commonly associated with ssRNA viruses. TLR8 also responds to small, purine-like molecules including the imidazoquinoline IRMs (immune-response modifiers). The IRMs include molecules that selectively activate TLR7, selectively activate TLR8 or non-selectively activate both TLR7 and TLR8. Using HEK-293 cells (human embryonic kidney cells) stably expressing an NF-kappaB (nuclear factor kappaB)/luciferase promoter-reporter system as a model system, we have examined the regulation of TLR8 using the non-selective TLR7/8 agonist, 3M-003. Using conservative tyrosine to phenylalanine site-directed mutation, we show that of the 13 tyrosine residues resident in the cytosolic domain of TLR8, only three appear to be critical to TLR8 signalling. Two of these, Tyr898 and Tyr904, reside in the Box 1 motif and the third, Tyr1048, lies in a YXXM putative p85-binding motif. TLR8 is tyrosine-phosphorylated following 3M-003 treatment and TLR8 signalling is inhibited by tyrosine kinase inhibitors. Treatment with 3M-003 results in the association of the p85 regulatory subunit of PI3K (phosphoinositide 3-kinase) with TLR8 and this association is inhibited by tyrosine to phenylalanine mutation of either the YXXM or Box 1 motifs. As a further consequence of activation by 3M-003, TLR8 is modified to yield both higher and lower molecular mass species. These species include a monoubiquitinated form as deduced from ubiquitin peptide sequencing by HPLC/MS/MS (tandem MS).
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PMID:The covalent modification and regulation of TLR8 in HEK-293 cells stimulated with imidazoquinoline agonists. 1786 34

Phosphodiesterases (PDEs) constitute a superfamily of enzymes that plays an important role in signal transduction by catalysing the hydrolysis of cAMP and cGMP. cDNA encoding PDE7A1 subtype was cloned and a stable recombinant HEK 293 cell line expressing high levels of PDE7A1 was generated. Transient transfection of pCRE-Luc plasmid, harboring luciferase reporter gene into the stable recombinant cell line and subsequent treatment with PDE7 inhibitor, resulted in a dose-dependent increase in luciferase activity. This method provides a simple and sensitive cell-based assay for screening of PDE7 selective inhibitors for the treatment of T cell mediated diseases.
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PMID:Cloning, stable expression of human phosphodiesterase 7A and development of an assay for screening of PDE7 selective inhibitors. 1795 31

Tryptophan hydroxylase-2 (TPH2) is a recently identified TPH isoform responsible for neuronal serotonin (5-HT) synthesis, and TPH2 polymorphisms are associated with a range of behavioral traits and psychiatric disorders. This study characterized cis-acting elements and three common polymorphisms (-703G/T, -473T/A, and 90A/G) in the 5' regulatory region of human TPH2 by using luciferase reporter assay, quantitative real-time PCR, and electrophoretic mobility shift assay (EMSA). The core promoter of human TPH2 was localized to the region between -107 and +7, and the segment of +8 to +53 within the 5'-UTR was found to exert a potent inhibitory effect on gene expression at both transcriptional and post-transcriptional levels. In both RN46A and HEK-293 cell lines, the TTA (-703T/-473T/90A) haplotype of the three polymorphisms showed the lowest gene expression compared with other haplotypes, and the -703G/T and -473T/A polymorphisms tended to exert a synergic effect on gene expression dependent upon the sequence of the 5'-UTR. In RN46A, the 90A/G polymorphism significantly increased luciferase activity and mRNA level irrespective of the other two polymorphisms, while in HEK-293 cells the effect of 90A/G was dependent on the alleles at loci -703 and -473. EMSA showed that all the three polymorphisms potentially alter DNA-protein interactions, while the 90A/G polymorphism predictably alters the 5'-UTR secondary structure of mRNA and influences RNA-protein interactions. In conclusion, our present study demonstrates that both the 5'-UTR and common polymorphisms (especially the 90A/G) in the 5' regulatory region of human TPH2 have a significant impact on gene expression.
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PMID:Functional characterization of the human TPH2 5' regulatory region: untranslated region and polymorphisms modulate gene expression in vitro. 1797 1

Activation of the innate immune system by bacterial DNA and DNA of other invertebrates represents a pathogen recognition mechanism. In this study we investigated macrophage responses to DNA from the intestinal protozoan parasite Entamoeba histolytica. E. histolytica genomic DNA was purified from log-phase trophozoites and tested with the mouse macrophage cell line RAW 264.7. RAW cells treated with E. histolytica DNA demonstrated an increase in levels of tumor necrosis factor alpha (TNF-alpha) mRNA and protein production. TNF-alpha production was blocked by pretreatment with chloroquine or monensin. In fact, an NF-kappaB luciferase reporter assay in HEK cells transfected with human TLR9 demonstrated that E. histolytica DNA signaled through Toll-like receptor 9 (TLR9) in a manner similar to that seen with CpG-ODN. Immunofluorescence assays confirmed NF-kappaB activation in RAW cells, as seen by nuclear translocation of the p65 subunit. Western blot analysis demonstrated mitogen-activated protein kinase activation by E. histolytica DNA. E. histolytica DNA effects were abolished in MYD88-/- mouse-derived macrophages. In the context of disease, immunization with E. histolytica DNA protected gerbils from an E. histolytica challenge infection. Taken together, these results demonstrate that E. histolytica DNA is recognized by TLR9 to activate macrophages and may provide an innate defense mechanism characterized by the induction of the inflammatory mediator TNF-alpha.
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PMID:Toll-like receptor 9-dependent macrophage activation by Entamoeba histolytica DNA. 1798 4

AMP-activated protein kinase (AMPK) has been identified as a regulator of gene transcription, increasing mitochondrial proteins of oxidative metabolism as well as hexokinase expression in skeletal muscle. In mice, muscle-specific knockout of LKB1, a component of the upstream kinase of AMPK, prevents contraction- and 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR)-induced activation of AMPK in skeletal muscle, and the increase in hexokinase II protein that is normally observed with chronic AICAR activation of AMPK. Since previous reports show a cAMP response element in the promoter region of the hexokinase II gene, we hypothesized that the cAMP-response element (CRE) binding protein (CREB) family of transcription factors could be targets of AMPK. Using radioisotopic kinase assays, we found that recombinant and rat liver and muscle AMPK phosphorylated CREB1 at the same site as cAMP-dependent protein kinase (PKA). AMPK was also found to phosphorylate activating transcription factor 1 (ATF1), CRE modulator (CREM), and CREB-like 2 (CREBL2), but not ATF2. Treatment of HEK-293 cells stably transfected with a CREB-driven luciferase reporter with AICAR increased luciferase activity approximately threefold over a 24-h time course. This increase was blocked with compound C, an AMPK inhibitor. In addition, AICAR-induced activation of AMPK in incubated rat epitrochlearis muscles resulted in an increase in both phospho-acetyl-CoA carboxylase and phospho-CREB. We conclude that CREB and related proteins are direct downstream targets for AMPK and are therefore likely involved in mediating some effects of AMPK on expression of genes having a CRE in their promoters.
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PMID:AMP-activated protein kinase phosphorylates transcription factors of the CREB family. 1806 5

Growth differentiation factor-9 (GDF9) is an oocyte secreted paracrine factor essential for mammalian ovarian folliculogenesis. Like other members of the transforming growth factor-beta (TGFbeta) superfamily, GDF9 is synthesized as a prepropeptide which needs processing by furin-like proteases to result in an active mature protein. We have previously characterized a preparation of unpurified recombinant mouse GDF9 which is bioactive as produced by human embryonic kidney 293T (HEK-293T) cells. However, we find that unpurified recombinant human GDF9 (hGDF9) produced by HEK-293T cells is not bioactive. Purified recombinant hGDF9 is bioactive and here we report the characterization of this protein. We find that the purified untagged mature region of hGDF9 is active in transcriptional reporter assays specific for Smad3/4 in human granulosa-luteal (hGL) cells. We also demonstrate the use of a BMP (Smad1/5) responsive (BRE-luciferase) adenovirus in primary cultures of hGL cells to detect BMP responses. Using this adenovirus we find that purified human GDF9 does not activate the Smad1/5 pathway. Purified hGDF9 mature region activated the Smad3 pathway also in the FSH responsive human granulosa tumor cell line KGN. Primary cultures of rat granulosa cells responded to purified hGDF9 with an increase in DNA synthesis as measured by [3H]-thymidine uptake. Here we also report that the inclusion of a C-terminal affinity purification tag destroys GDF9 bioactivity. This study is the first characterization of purified biologically active human GDF9 and as such is of importance for studies on human fertility, and efforts aimed at treating infertility conditions.
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PMID:Characterization of recombinant human growth differentiation factor-9 signaling in ovarian granulosa cells. 1816 87

Advanced glycation end-products (AGEs) of DNA are formed spontaneously by the reaction of carbonyl compounds such as sugars, methylglyoxal or dihydroxyacetone in vitro and in vivo. Little is known, however, about the biological consequences of DNA AGEs. In this study, a method was developed to determine the parameters that promote DNA glycation in cultured cells. For this purpose, the formation rate of N2-carboxyethyl-2'-deoxyguanosine (CEdG), a major DNA AGE, was measured in cultured hepatic stellate cells by liquid chromatography (LC)-MS/MS. In resting cells, a 1.7-fold increase of CEdG formation rate was observed during 14 days of incubation. To obtain insights into the functional consequences of DNA glycation, CEdG was introduced into a luciferase reporter gene vector and transfected into human embryonic kidney (HEK 293 T) cells. Gene activity was determined by chemiluminescence of the luciferase. Thus, CEdG adducts led to a dose-dependent and highly significant decrease in protein activity, which is caused by loss of functionality of the luciferase in addition to reduced transcription of the gene. When the CEdG-modified vector was transformed into Escherichia coli, a loss of ampicillin resistance was observed in comparison to transformation with the unmodified plasmid. These results indicate that CEdG accumulates in the genomic DNA of resting cells, which could lead to diminished protein activity.
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PMID:Analysis and biological relevance of advanced glycation end-products of DNA in eukaryotic cells. 1821 62

Nur77 is one member of the nuclear receptor superfamily. As a transcription factor, Nur77 participates in a variety of biological processes, including T cell development, inflammatory responses, steroid hormone synthesis, and hepatic glucose metabolism. It typically acts via binding to the Nur77 responsive element (NBRE) in the promoter regions of its target genes. In the present study, we identified a novel Nur77-regulated gene, alpha1-antichymotrypsin/SerpinA3, via an approach combining computational prediction and wet-laboratory validations. First, we identified 483 candidate genes via a human genome-wide scan for NBREs in their proximal promoters. Three out of 14 function-associated genes were further identified to be transactivated by Nur77 in luciferase reporter gene assays in HEK 293T cells. The transactivation assay proved that the NBRE (-182 to -175) in the SerpinA3 promoter region is a novel Nur77-dependent functional motif in HEK 293T and HepG2 cells. Electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated that Nur77 physically associates with the SerpinA3 promoter region both in vitro and in vivo. Nur77 overexpression and RNA interference-mediated Nur77 gene knockdown analysis confirmed that SerpinA3 is indeed a novel Nur77-targeted gene. These data may throw light on the function of Nur77 in inflammatory responses and acute-phase reactions as well as the development of Alzheimer's disease.
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PMID:Alpha 1-antichymotrypsin/SerpinA3 is a novel target of orphan nuclear receptor Nur77. 1824 59


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