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 mechanism of biotin uptake in human intestine has been well characterized and involves the human sodium-dependent multivitamin transporter (hSMVT), yet little is known about the molecular/transcriptional regulation of the system. Previous investigations cloned the 5' regulatory region of the hSMVT gene and identified the minimal promoter. To expand these investigations, we compared activity of the hSMVT promoter in three human intestinal epithelial cell lines (NCM460, Caco-2, and HuTu-80) and contrasted a renal epithelial cell line (HEK-293). We analyzed the role of putative cis-elements in regulating promoter activity and confirmed activity of the cloned hSMVT promoter in vivo. In vitro studies demonstrated that all cell lines utilized the same minimal promoter region, and mutation of specific cis-regulatory elements [Kruppel-like factor 4 (KLF-4) and activator protein-2 (AP-2)] led to a decrease in promoter activity in all intestinal cell types but not in renal cells. Using electrophoretic mobility shift assays, we identified two specific DNA/protein complexes. Using oligonucleotide competition and antibody supershift analysis, we determined that KLF-4 and AP-2 were involved in forming the complexes. In HEK-293 cells, overexpressing KLF-4 increased the endogenous hSMVT message levels threefold and activated a cotransfected hSMVT promoter-reporter construct. In vivo studies using hSMVT promoter-luciferase transgenic mice established physiological relevance and showed the pattern of hSMVT promoter expression to be similar to endogenous mouse SMVT mRNA expression. The results demonstrate, for the first time, the importance of KLF-4 and AP-2 in regulating the activity of the hSMVT promoter in the intestine and provide direct in vivo confirmation of hSMVT promoter activity.
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PMID:Regulation of the human biotin transporter hSMVT promoter by KLF-4 and AP-2: confirmation of promoter activity in vivo. 1713 99

Recent work has highlighted a role for PDK1 in adaptive immunity, however its contribution to innate immunity has not been addressed. We have investigated the role of PKB and PDK1 in IL-1beta-induced NF-kappaB activation. Over-expression of either in HCT 116 and HEK 293T cells, effected a reproducible NF-kappaB activation. This was validated in a one-hybrid assay utilizing Gal4-RelA and Gal4-luciferase assay. N-tosyl phenylalanyl chloromethyl ketone (TPCK), wortmannin and Ly294002 inhibited IL-1beta-induced NF-kappaB activation in both systems indicating involvement of the PI3K axis in this response. p65 (Rel A) Ser536 phosphorylation was not affected by the PI3K inhibitors but was dose-dependently attenuated by TPCK. Evaluation of IKK-associated activity using GST-p65 substrate phosphorylation in immune complex assays, revealed that whilst TPCK attenuated this, neither of the PI3K inhibitors had any effect. Furthermore whilst TPCK inhibited IL-1beta-induced p65 DNA binding, this was not apparent with either of wortmannin or Ly294002. Similarly, over-expression of PDK1 but not PKB resulted in promotion of p65 DNA binding. Using a p65-S536A reporter construct, we found inhibition of only PDK1 over-expression-induced, but not PKB over-expression-induced NF-kappaB activation. This was supported using biochemical analysis in which immunoprecipitated IKKgamma from IL-1beta-activated cells was unable to phosphorylate a p65-S536A substrate, confirming this as the dominant IKK-dependent site. In further support of a dissociated response, we observed an attenuation of the Ser177/181 IKK phosphorylation by TPCK but not in response to PI3K inhibition. Our data reveals for the first time that PDK1 and PKB may differentially activate NF-kappaB, and that TPCK may subserve a useful anti-inflammatory function by inhibiting IKKbeta.
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PMID:Investigation of interleukin 1beta-mediated regulation of NF-kappaB activation in colonic cells reveals divergence between PKB and PDK-transduced events. 1713 79

Phosphodiesterase (PDE) constitutes a superfamily of enzymes that catalyze the hydrolysis of cAMP and cGMP into their corresponding monophosphates and play an important role in diverse physiological functions. The present study provides a process for identifying PDE4 subtypes selective inhibitors using a reporter gene assay. Stable recombinant HEK-293 cell lines expressing high levels of PDE4A4B, PDE4B2A, and PDE4D3 subtypes individually were generated. Transient transfection of pCRE-Luc plasmid, harboring luciferase reporter gene under the control of cAMP response element (CRE)-binding sequence, into these stable recombinant cell lines followed by treatment with PDE4 inhibitor, resulted in a dose dependent increase in luciferase activity. This methods provide a novel, simple and sensitive assay for high throughput screening of PDE4 subtype selective inhibitors for treatment of asthma and COPD.
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PMID:A reporter gene assay for screening of PDE4 subtype selective inhibitors. 1733 28

On the basis of apparent molecular mass heterogeneity following reducing versus nonreducing SDS-PAGE, we determined that the beta-subunit of macaque (Macaca fascicularis) chorionic gonadotropin (mCG-beta) is more conformationally constrained than the beta-subunit of human chorionic gonadotropin (hCG-beta). The amino acid sequences of these two subunits are 81% identical. To determine the conformational variance source, which was not due to glycosylation differences, we generated a series of hCG-beta-mCG-beta chimeras and identified domains that contributed to CG-beta conformational freedom. We discovered that the CG-beta 54-101 domain contained a small subdomain, residues 74-77, that regulated the conformational freedom of the beta-subunit; i.e., when residues 74-77 were of macaque origin (PGVD), the mutated hCG-beta subunit displayed macaque-like conformational rigidity, and when residues 74-77 were of human origin (RGVN), the mutated mCG-beta subunit displayed human-like conformational freedom and microheterogeneity. Additionally, CG-beta N-terminal domain residues (8, 18, 42, and 46-48) were also found to influence CG-beta conformational freedom when residues 74-77 were of human but not macaque origin. The biological significance of the CG-beta conformational variance was tested using a biological assay that showed that the hCG-alpha-hCG-beta heterodimer facilitated human CG receptor-mediated cAMP-driven luciferase reporter gene activity in HEK cells nearly 1 order of magnitude more effectively than the hCG-alpha-mCG-beta chimera. Together, these data demonstrate that two essential amino acid residues within a four-amino acid subdomain regulated CG-beta conformational freedom and that a conformational difference between hCG-beta and mCG-beta was recapitulated in the context of receptor-mediated CG heterodimer signal transduction activation.
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PMID:A novel four-amino acid determinant defines conformational freedom within chorionic gonadotropin beta-subunits. 1735 49

Arachidonate 5-lipoxygenase is an enzyme encoded by the ALOX5 gene, and plays an important role in the synthesis of leukotrienes. These are inflammatory mediators, and have been involved in atherosclerosis and other pathological processes that require proinflammatory activities. Human and animal studies have suggested a role for the ALOX5 gene in atherosclerosis, including a significant association between a promoter polymorphism and a carotid intimal-medial thickness in response to dietary fat. This polymorphism was three- to six-tandem repeats of a Sp1/Egr1 binding motif (GGGCGG)(n), and the number of repeats has been linked with the amount of gene expression. We hypothesized that this ALOX5 polymorphism could influence the risk for myocardial infarction (MI). First, we analysed the effect of the four alleles on gene expression by transfecting the HEK-293 cell line with luciferase reporter-constructs. We found that luciferase activities are dependent on the number of the Sp1/Egr1 repeats, with the three and six repeats having the lowest and highest values. We genotyped 312 male MI survivors, aged < 55 years, and 376 healthy controls matched with patients for sex, age, and ethnicity. Ninety-six per cent of the patients were smokers, compared to only 42% among the controls (P < 0.001; OR = 31.84). The 55 + 56 repeat genotypes were less frequent in patients (55 = 56%, 56 = 0.6%) compared to controls (55 = 60%, 56 = 3%). However, these were non-significantly different frequencies. In addition, no difference in MI-onset age and biochemical values was found between the allele and genotypes. In conclusion, we confirmed the effect of the ALOX5-promoter polymorphism on gene expression, but our data did not support a significant effect of this functional variation on MI risk.
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PMID:A functional Sp1/Egr1-tandem repeat polymorphism in the 5-lipoxygenase gene is not associated with myocardial infarction. 1737 38

Gas-filled microbubbles have been successfully used as gene delivery reagents in combination with diagnostic ultrasound. Although shock wave exposure has been shown to transfect cells with naked DNA in vitro, it has not been tested whether the addition of microbubbles would augment DNA uptake under those conditions. Therefore, the aim of this study was to test the impact of microbubbles on transgene expression in vitro under shock wave exposure conditions. HEK 293 cells were treated with 60 or 120 pulses of shock waves at varying energy levels. Cells were mixed with either 100 microg/mL luciferase expressing plasmid DNA or with microbubbles that were produced with the same amount of this DNA. Cell death was evaluated after 1 h and transgene expression, after 24 h. In the presence of microbubbles, transgene expression was significantly higher (as much as 29-fold) relative to that obtained without microbubbles. Cells exposed to 120 pulses demonstrated higher transgene expression (as high as 2.7-fold) compared with cells exposed to 60 pulses. The use of microbubbles resulted in greater cell death, varying from 26% (low energy) to 78% (high energy). In conclusion, DNA-loaded microbubbles can significantly increase shock wave mediated gene transfer. However, this effect is associated with increased levels of cell destruction.
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PMID:Impact of microbubbles on shock wave-mediated DNA uptake in cells in vitro. 1738

The authors report the characterization of a novel cyclic adenosine monophosphate (cAMP)-responsive luciferase (Luc) reporter that exhibits optimal performance in high-throughput screens of agonist binding at G protein-coupled receptors (GPCRs). This reporter (RIP1-CRE-Luc) incorporates a nonpalindromic cAMP response element (CRE) originally identified within the 5' promoter of the rat insulin 1 gene (RIP1). When multimerized and fused to the coding sequence of firefly luciferase, the CRE of RIP1 allows for the efficient activation of luciferase expression by cAMP-elevating agents or by cAMP itself. Of primary importance is the demonstration that RIP1-CRE-Luc does not exhibit the relatively high levels of basal luciferase activity inherent to reporters incorporating the palindromic CRE first identified in the somatostatin gene promoter. Furthermore, studies of HEK cells expressing class II GPCRs for the cAMP-elevating hormones GLP-1, GIP, and glucagon demonstrate that RIP1-CRE-Luc affords a much wider dynamic range of activation upon exposure to agonist. Such properties of RIP1-CRE-Luc indicate its usefulness as a new and powerful tool for the identification of small-molecule compounds with receptor-stimulating actions or for the identification of constitutively active orphan receptors with cAMP-signaling properties.
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PMID:A novel cyclic adenosine monophosphate responsive luciferase reporter incorporating a nonpalindromic cyclic adenosine monophosphate response element provides optimal performance for use in G protein coupled receptor drug discovery efforts. 1750 37

In order to understand an overview of promoter activities intrinsic to primary DNA sequences in the human genome within a particular cell type, we carried out systematic quantitative luciferase assays of DNA fragments corresponding to putative promoters for 472 human genes which are expressed in HEK (human embryonic kidney epithelial) 293 cells. We observed the promoter activities of them were distributed in a bimodal manner; putative promoters belonging to the first group (with strong promoter activities) were designated as P1 and the latter (with weak promoter activities) as P2. The frequencies of the TATA-boxes, the CpG islands, and the overall G + C-contents were significantly different between these two populations, indicating there are two separate groups of promoters. Interestingly, similar analysis using 251 randomly isolated genomic DNA fragments showed that P2-type promoter occasionally occurs within the human genome. Furthermore, 35 DNA fragments corresponding to putative promoters of non-protein-coding transcripts (ncRNAs) shared similar features with the P2 in both promoter activities and sequence compositions. At least, a part of ncRNAs, which have been massively identified by full-length cDNA projects with no functional relevance inferred, may have originated from those sporadic promoter activities of primary DNA sequences inherent to the human genome.
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PMID:Intrinsic promoter activities of primary DNA sequences in the human genome. 1752 93

The secreted fragment of the amyloid precursor protein (sAPPalpha) generated following cleavage by alpha-secretase is an important mediator of cell function and is both neurotrophic and neuroprotective. HEK 293T cells have been stably integrated with a fragment of the APP gene to produce and secrete either sAPPalpha, or the alternative cleavage product sAPPbeta. Heparin binding domains on the proteins have been utilised to develop a one-step fast-performance-liquid-chromatography (FPLC) purification of sAPPs from the conditioned media. Immunoblotting analyses with a sAPP specific antibody coupled with highly sensitive silver staining techniques have validated the expression and purification strategy. Functional activity of the purified fragments was demonstrated by their ability to protect COS-7 and SH-SY5Y (neuroblastoma) cells against the adverse effects of glucose deprivation in a cell viability assay. The purified sAPPs also activated the NFkappaB transcription factor in COS-7 cells transfected with a luciferase reporter plasmid, with sAPPalpha the more potent activator as expected. The simple protocol to produce these mammalian expressed proteins will facilitate their use as potential neuropharmacological reagents in the elucidation of biochemical pathways modulated by sAPPs, and in the study of Alzheimer's disease mechanisms in general.
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PMID:Production, purification and functional validation of human secreted amyloid precursor proteins for use as neuropharmacological reagents. 1753 17

Stable prostacyclin analogues can signal through cell surface IP receptors or by ligand binding to nuclear peroxisome proliferator-activated receptors (PPARs). So far these agents have been reported to activate PPARalpha and PPARdelta but not PPARgamma. Given PPARgamma agonists and prostacyclin analogues both inhibit cell proliferation, we postulated that the IP receptor might elicit PPARgamma activation. Using a dual luciferase reporter gene assay in HEK-293 cells stably expressing the IP receptor or empty vector, we found that prostacyclin analogues only activated PPARgamma in the presence of the IP receptor. Moreover, the novel IP receptor antagonist, RO1138452, but not inhibitors of the cyclic AMP pathway, prevented activation. Likewise, the anti-proliferative effects of treprostinil observed in IP receptor expressing cells, were partially inhibited by the PPARgamma antagonist, GW9662. We conclude that PPARgamma is activated through the IP receptor via a cyclic AMP-independent mechanism and contributes to the anti-growth effects of prostacyclin analogues.
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PMID:IP receptor-dependent activation of PPARgamma by stable prostacyclin analogues. 1762 3


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