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

---

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

The human chromosome 21 AML1 gene is expressed predominantly in the hematopoietic system. In several leukemia-associated translocations AML1 is fused to other genes and transcription of the fused regions is mediated by upstream sequences that normally regulate the expression of AML1. The 5' genomic region of AML1 was cloned and sequenced. The two 5' untranslated regions (UTRs) previously identified in AML1 cDNAs were located in this region and the distance between them was established. The distal 5' UTR maps over 7 kb upstream of the proximal one. Using primer extension with mRNA, transcription start sites were identified at two distinct sites above these 5' uTRs. Sequence analysis revealed the absence of a TATA motif and the presence of Sp1, PU.1, Oct, CRE, Myb, Ets, and Ets-like binding sites in both upstream regions. Several initiator elements (Inr) that overlap the transcription start sites were also identified. These proximal and distal upstream regions and their deletion mutants were cloned in front of a luciferase reporter gene and used in transfection assays. We demonstrate that both upstream regions function as promoters in hematopoietic (Jurkat) and nonhematopoietic (HEK) cell lines. The activity of both promoters was orientation dependent and was enhanced, in a cell-type specific manner, by a heterologous enhancer sequence. These results indicate that additional control elements, either negative or positive, regulate the tissue-specific expression of AML1.
...
PMID:Expression of the human acute myeloid leukemia gene AML1 is regulated by two promoter regions. 870 Aug 62

We examined whether histamine could regulate cell proliferation and expression of the early response gene c-fos in HEK-293 cells stably transfected with the human H2 receptor (HEK-H2). Histamine stimulated [3H]thymidine incorporation [50% effective concentration (EC50) = 3.6 x 10(-6) M] in HEK-H2 cells in a cimetidine-sensitive manner and increased c-fos mRNA in a time-dependent fashion, reaching maximal induction after 30 min. Histamine induced luciferase activity in HEK-H2 cells transiently transfected with a construct containing the luciferase reporter gene (Luc) coupled to the serum response element (SRE) of the c-fos gene promoter (EC50 = 1.5 x 10(-6) M) or a plasmid containing the SRE core fragment (bases -320 to -298). The protein kinase C (PKC) inhibitor staurosporine and long-term pretreatment of HEK cells with phorbol ester inhibited the effect of histamine on PKC activation, SRE-Luc activity, and [3H]thymidine incorporation. We have demonstrated that activation of the human H2 receptor can lead to induction of c-fos gene transcription and cell proliferation through a PKC-dependent mechanism.
...
PMID:Activation of the human histamine H2 receptor is linked to cell proliferation and c-fos gene transcription. 943 11

We investigated the 5'-flanking promoter region of the rat somatostatin receptor subtype 3 (rSSTR3). Using a cDNA probe, genomic clones containing the 5'-flanking promoter region of the rSSTR3 gene were isolated. A sequence of 5.4 kb directly upstream from the start codon was analyzed and two introns were found in the 5' untranslated region (UTR) of the cDNA sequence. The transcriptional initiation site was determined by 5' rapid amplification of cDNA ends (RACE), primer extension and RNase protection analysis with cerebellar RNA. Two major transcriptional initiation sites were found at position 1040 (tsp1) and -856 (tsp2) relative to the translational initiation site. Like a number of other promoters of G-protein-coupled receptors, the rSSTR3 gene lacks TATA and CAAT motifs and includes G+C-rich regions. Functional analysis of the promoter region by transfecting rSSTR3 luciferase-reporter gene constructs into rat pituitary GH3 cells and HEK 293 cells indicated that a 107-bp region upstream of tsp2 was sufficient to drive transcription. Furthermore a 562-bp region at position -1304 to -1865 upstream of the ATG start codon exerted a negative regulatory effect on transcriptional activity.
...
PMID:Characterization of the 5'-flanking promoter region of the rat somatostatin receptor subtype 3 gene. 986 19

Monocytes respond to lipopolysaccharide (LPS) stimulation with a rapid expression of the tumor necrosis factor (TNF) gene. Upon repeated LPS stimulation there is, however, little production of TNF mRNA and protein; i.e., the cells are tolerant to LPS. Analysis of NF-kappaB proteins in gel shift assays demonstrated that the DNA binding activity that is induced by LPS stimulation in tolerant cells consists mainly of p50-p50 homodimers. Since p50 can bind to DNA but lacks a transactivation domain, this may explain the blockade of TNF gene expression. We now show that in the monocytic cell line Mono Mac 6, this inability to respond can be largely ascribed to NF-kappaB, since a reporter construct directed by a trimeric NF-kappaB motif is strongly transactivated by LPS stimulation of naive cells whereas LPS-tolerant cells exhibit only low activity. Also, Western blot analyses of proteins extracted from purified nuclei showed mobilization of threefold-higher levels of p50 protein in tolerant compared to naive cells, while mobilization of p65 was unaltered. Overexpression of p50 in HEK 293 cells resulted in a strong reduction of p65-driven TNF promoter activity at the levels of both luciferase mRNA and protein. These data support the concept that an upregulation of p50 is instrumental in LPS tolerance in human monocytes.
...
PMID:NF-kappaB1 (p50) is upregulated in lipopolysaccharide tolerance and can block tumor necrosis factor gene expression. 1008 86

TLR4 is a member of the recently identified Toll-like receptor family of proteins and has been putatively identified as Lps, the gene necessary for potent responses to lipopolysaccharide in mammals. In order to determine whether TLR4 is involved in lipopolysaccharide-induced activation of the nuclear factor-kappaB (NF-kappaB) pathway, HEK 293 cells were transiently transfected with human TLR4 cDNA and an NF-kappaB-dependent luciferase reporter plasmid followed by stimulation with lipopolysaccharide/CD14 complexes. The results demonstrate that lipopolysaccharide stimulates NF-kappaB-mediated gene expression in cells transfected with the TLR4 gene in a dose- and time-dependent fashion. Furthermore, E5531, a lipopolysaccharide antagonist, blocked TLR4-mediated transgene activation in a dose-dependent manner (IC50 approximately 30 nM). These data demonstrate that TLR4 is involved in lipopolysaccharide signaling and serves as a cell-surface co-receptor for CD14, leading to lipopolysaccharide-mediated NF-kappaB activation and subsequent cellular events.
...
PMID:Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction. 1019 38

Corticotropin releasing factor (CRF) receptors belong to the super-family of G protein-coupled receptors. These receptors are classified into two subtypes (CRF1 and CRF2). Both receptors are positively coupled to adenylyl cyclase but they have a distinct pharmacology and distribution in brain. Two isoforms belonging to the CRF2 subtype receptors, CRF2alpha and CRF2beta, have been identified in rat and man. The neuropeptides CRF and urocortin mediate their actions through this CRF G protein-coupled receptor family. In this report, we describe the pharmacological characterization of the recently identified hCRF2, receptor. We have used radioligand binding with [125I]-tyr0-sauvagine and a gene expression assay in which the firefly luciferase gene expression is under the control of cAMP responsive elements. Association kinetics of [125I]-tyr0-sauvagine binding to the hCRF2beta receptor were monophasic while dissociation kinetics were biphasic, in agreement with the kinetics results obtained with the hCRF2alpha receptor. Saturation binding analysis revealed two affinity states in HEK 293 cells with binding parameters in accord with those determined kinetically and with parameters obtained with the hCRF2alpha receptor. A non-hydrolysable GTP analog, Gpp(NH)p, reduced the high affinity binding of [125I]-tyr0-sauvagine to both hCRF2 receptor isoforms in a similar manner. The rank order of potency of CRF agonist peptides in competition experiments was identical for both hCRF2 isoforms (urocortin > sauvagine > urotensin 1 > r/hCRF > alpha-helical CRF(9-41) > oCRF). Similarly, agonist potency was similar for the two isoforms when studied using the luciferase gene reporter system. The peptide antagonist alpha-helical CRF(9-41) exhibited a non-competitive antagonism of urocortin-stimulated luciferase expression with both hCRF2 receptor isoforms. Taken together, these results indicate that the pharmacological profiles of the CRF2 splice variants are identical. This indicates that the region of the N-terminus that varies between the receptors is probably not important in the binding of peptide CRF receptor ligands or functional activation of the receptor.
...
PMID:Human CRF2 alpha and beta splice variants: pharmacological characterization using radioligand binding and a luciferase gene expression assay. 1021 82

For long-term gene expression in tissues, we constructed an Epstein-Barr virus (EBV) replicon-based plasmid, pEB, containing the latent viral DNA replication origin (oriP) and EBV nuclear antigen-1 (EBNA-1). When pEB was transferred to human cells (HeLa-S3, HEK 293 and FS 3) and rodent cells (BHK-21) using HVJ-cationic liposomes, luciferase expression was observed in those cells for at least 10 days. Luciferase activity was two to 10 times higher in those cell lines on and after day 3 post-transfection of pEBActLuc compared with plasmids without the EBV replicon sequence. Southern blot analysis showed that the pEB vector luciferase gene was maintained extrachromosomally in BHK-21 cells. In human cells, transformation was five to 20 times more efficient with pEBc than with pcDNA3, and 18-35% of the introduced EBV replicon plasmid was replicated autonomously. The luciferase gene or lacZ gene was introduced into mouse liver using HVJ-AVE liposomes. Luciferase gene expression was observed for at least 35 days in cells transfected with pEBActLuc, whereas it was not detected on day 14 in cells transfected with pActLuc, which lacks the EBV sequence. By the transfer of pEBActNlacF, the lacZ gene expression rate in hepatocytes was approximately 35 and 12% on days 7 and 35, respectively.
...
PMID:Sustained transgene expression in vitro and in vivo using an Epstein-Barr virus replicon vector system combined with HVJ liposomes. 1032 25

Mammalian cells are able to sense oxygen and regulate a number of genes in response to hypoxia. The transcription factor Hypoxia Inducible Factor-1 (HIF-1) was identified as an important key component of the hypoxia signaling pathway. HIF-1 is a heterodimer composed of two members of the basic helix-loop-helix transcription factor superfamily containing a PAS (PER-ARNT-SIM) domain: HIF-1alpha and HIF-1beta/ARNT. During the cloning by reverse transcriptase-polymerase chain reaction of the human HIF-1alpha subunit, we isolated two cDNA clones which corresponded to alternative splicing of the HIF-1alpha gene. Polymerase chain reaction analysis and sequencing revealed that both clones possessed three additional base pairs between exons 1 and 2. Also, one of them lacked 127 base pairs corresponding to exon 14. We demonstrate that the mRNA of this truncated form is expressed in several human cells lines and human skin but apparently not in rodents. When transfected in HEK 293 cells, the corresponding 736 amino acid protein (HIF-1alpha(736)) is regulated by hypoxia in a similar manner as the full-length HIF-1alpha (HIF-1alpha(FL)). In luciferase transfection assays, both recombinant proteins HIF-1alpha(736) and HIF-1alpha(FL) dimerize with HIF-1beta/ARNT and activate the VEGF promoter upon hypoxia. However, the shorter HIF-1alpha isoform is 3-fold less active than HIF-1alpha(FL), a result consistent with the lack of the C-terminal transactivation domain. As expected, this small isoform can compete with the endogenous and transfected full-length HIF-1alpha. Altogether, these results suggest that the HIF-1alpha(736) isoform modulates gene expression upon hypoxia.
...
PMID:Identification of alternative spliced variants of human hypoxia-inducible factor-1alpha. 1070 53

Heptahelical receptors that interact with heterotrimeric G proteins represent the largest family of proteins involved in signal transduction across biological membranes. Although these receptors generally were believed to be monomeric entities, a growing body of evidence suggests that they may form functionally relevant dimers. However, a definitive demonstration of the existence of G protein-coupled receptor (GPCR) dimers at the surface of living cells is still lacking. Here, using bioluminescence resonance energy transfer (BRET), as a protein-protein interaction assay in whole cells, we unambiguously demonstrate that the human beta(2)-adrenergic receptor (beta(2)AR) forms constitutive homodimers when expressed in HEK-293 cells. Receptor stimulation with the hydrophilic agonist isoproterenol led to an increase in the transfer of energy between beta(2)AR molecules genetically fused to the BRET donor (Renilla luciferase) and acceptor (green fluorescent protein), respectively, indicating that the agonist interacts with receptor dimers at the cell surface. Inhibition of receptor internalization did not prevent agonist-promoted BRET, demonstrating that it did not result from clustering of receptors within endosomes. The notion that receptor dimers exist at the cell surface was confirmed further by the observation that BS3, a cell-impermeable cross-linking agent, increased BRET between beta(2)AR molecules. The selectivity of the constitutive interaction was documented by demonstrating that no BRET occurred between the beta(2)AR and two other unrelated GPCR. In contrast, the well characterized agonist-dependent interaction between the beta(2)AR and the regulatory protein beta-arrestin could be monitored by BRET. Taken together, the data demonstrate that GPCR exist as functional dimers in vivo and that BRET-based assays can be used to study both constitutive and hormone-promoted selective protein-protein interactions.
...
PMID:Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). 1072 88

Nuclear factor I (NFI) proteins are DNA-binding transcription factors that participate in the tissue specific expression of various genes. They are encoded by four different genes (NFI-A, B, C, and X) each of which generates multiple isoforms by alternative RNA splicing. NFI-like binding sites have been identified in several genes preferentially expressed in olfactory receptor neurons. Our prior demonstration that NFI binds to these elements led to the hypothesis that NFI is involved in the regulation of these genes. To analyse the role of NFI in the regulation of olfactory neuron gene expression we have performed transient transfection experiments in HEK 293 cells using constructs that place luciferase expression under the control of an olfactory marker protein (OMP)-promoter fragment containing the NFI binding site. In vitro mutagenesis of this site revealed a negative modulation of luciferase expression by endogenous NFI proteins in HEK 293 cells. In addition, we have used in situ hybridization to analyse the tissue and cellular distribution of the four NFI gene transcripts during pre- and postnatal mouse development. We have simultaneously characterized the expression of Pax-6, and O/E-1, transcription factors known to regulate the phenotype of olfactory receptor neurons. We demonstrate that all of these transcription factors vary in specific spatio-temporal patterns during the development of the olfactory system. These data on NFI activity, and on transcription factor expression, provide a basis to understand the role of NFI in regulating gene expression in olfactory receptor neurons.
...
PMID:NFI in the development of the olfactory neuroepithelium and the regulation of olfactory marker protein gene expression. 1076 65


1 2 3 4 5 6 7 8 9 10 Next >>

---