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)

Excess nitric oxide (NO) induces apoptosis in some cell types including macrophages; however, the cascade of NO-mediated apoptosis is not fully understood. We investigated the initial steps of NO-mediated apoptosis in mouse macrophage-like RAW 264.7 cells. When cells were treated with bacterial lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma), NO-mediated apoptosis occurred. Under these conditions, p53 accumulation was not observed, indicating that DNA damage is not the main trigger of NO-mediated apoptosis. On the other hand, mRNA and protein for CHOP, a transcription factor known to be induced by endoplasmic reticulum (ER) stress, were induced. The CHOP induction by LPS/IFN-gamma treatment preceded cytochrome c release from mitochondria. In addition, p90ATF6, an ER membrane-bound transcription factor involved in ER stress response, was cleaved to its active soluble form p50ATF6, which was transported to nucleus and bound to the ER stress response element of the CHOP gene. In the luciferase reporter assay, both the CHOP-binding element of the Rous sarcoma virus long terminal repeat and ER stress response element of the CHOP gene were activated by LPS/IFN-gamma treatment. When RAW 264.7 cells or COS-7 cells were transfected with expression plasmids for CHOP, p90ATF6, or p50ATF6, cell death was observed. In addition, apoptosis induced by p50ATF6 was prevented by a CHOP dominant negative form as well as by an ATF6 dominant negative form, and LPS/IFN-gamma-induced apoptosis was prevented by the CHOP dominant negative form. Peritoneal macrophages from CHOP knockout mice showed resistance to NO-induced apoptosis. These results indicate that the ER stress pathway involving ATF6 and CHOP plays a key role in NO-mediated apoptosis in macrophages.
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PMID:Nitric oxide-induced apoptosis in RAW 264.7 macrophages is mediated by endoplasmic reticulum stress pathway involving ATF6 and CHOP. 1180 88

Recent reports demonstrate that the rat GnRH promoter is activated in an episodic fashion in immortalized GnRH neurons, but little information is available on molecular processes that contribute to this phenomenon. In this study, we dissected the regions of the rat GnRH promoter that mediate these effects by testing a series of 5' deletion luciferase reporter constructs on the pattern of photonic emissions from single, living GT1-7 GnRH neuronal cells. Deletion analysis revealed that the region -2012/-1597 that contains the neuron-specific enhancer (NSE) was required for the elaboration of pulses of GnRH promoter activity. The importance of this region was supported by observations that episodic reporter activity could be transferred to a neutral nonpulsatile promoter (Rous sarcoma virus, RSV(180)). Immunoneutralization of Oct-1 as well as mutation of an octamer binding site located at -1787/-1783 (AT-a site) blocked the pulsatile GnRH promoter activity in GT1-7 neuronal cells. Taken together, our findings indicate that episodic GnRH gene expression is a promoter-dependent phenomenon, which is mediated by Oct-1 interaction with regulatory elements in the NSE region.
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PMID:Episodic activation of the rat GnRH promoter: role of the homeoprotein oct-1. 1219 45

The folate receptor type alpha (FR-alpha) is a promising tumor marker and target. Here, we investigate the mechanistic basis for the tumor specificity and vast overexpression of FR-alpha. Among representative FR-alpha-positive (HeLa and JAR) and FR-alpha-negative (MG63, Caki1, and HT3) cell lines, the transcription rates of the endogenous FR-alpha gene, as well as the FR-alpha promoter activity, were relatively weak and comparable, but the FR-alpha transcript was abundant only in total RNA and nuclear RNA from the FR-alpha-positive cells. Rous sarcoma virus (RSV) promoter-driven expression of the FR-alpha gene was 7 to 30 times greater in the FR-alpha-positive than in FR-alpha-negative cells, both at the protein and mRNA levels, independently of intron sequences. Through the use of chimeric FR-alpha/FR-beta cDNAs, the above pattern of FR-alpha expression was attributed to a 60-bp sequence in the FR-alpha open reading frame. This sequence element, when placed in the 5' untranslated region of RSV promoter-luciferase, decreased the reporter expression approximately 7- to 20-fold in FR-alpha-negative cells (MG63, Caki1, HT3, BG1, and MCF7) relative to FR-alpha-positive cells (HeLa, JAR, and JEG3). Substitution of this FR-alpha element in FR-beta increased the in vivo degradation rate of the transcript in the nuclei of MG63 cells but not in the nuclei of HeLa cells or in the cytosol of MG63 or HeLa cells. The results reveal an efficient mechanism by which a novel sequence element causes differential transcript degradation in the nucleus to ensure narrow tissue specificity for a gene (e.g., that for FR-alpha) whose transcription is weak and relatively nonselective. FR-alpha exhibited constitutive mRNA and protein synthesis during the cell cycle and a slow protein turnover, presumably ensuring a high steady-state level of the receptor in cells that could override the nuclear mRNA instability determinant.
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PMID:mRNA instability in the nucleus due to a novel open reading frame element is a major determinant of the narrow tissue specificity of folate receptor alpha. 1261 90

A central issue in gene delivery systems is choosing promoters that will direct defined and sustainable levels of gene expression. Pantropic retroviral vectors provide a means to insert genes into either somatic or germline cells. In this study, we focused on somatic cell infection by evaluating the activity of 3 promoters inserted by vectors into fish cell lines and fish skin using pantropic retroviruses. In bluegill and zebrafish cell lines, the highest levels of luciferase expression were observed from the 5' murine leukemia virus long terminal repeat of the retroviral vector. The Rous sarcoma virus long terminal repeat and cytomegalovirus early promoter, as internal promoters, generated lower levels of luciferase. Luciferase reporter vectors infected zebrafish skin, as measured by the presence of viral DNA, and expressed luciferase. We infected developing walleye dermal sarcomas with retroviral vectors to provide an environment with enhanced cell proliferation, a condition necessary for integration of the provirus into the host genome. We demonstrated a 4-fold to 7-fold increase in luciferase gene expression in tumor tissue over infections in normal walleye skin.
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PMID:Reporter gene expression in fish following cutaneous infection with pantropic retroviral vectors. 1496 3

Dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEAS), the representative sex steroid precursors, are postulated to have antiinflammatory effects, although the molecular background remains unknown. In this study, we examined the effects of these sex steroid precursors on cytokine-induced, nuclear factor-kappaB (NF-kappaB)-mediated transcription. The HuH7 human hepatocyte cell line was stably transfected with an NF-kappaB-luciferase reporter gene or transiently transfected with other representative response elements-luciferase fusion genes, and the effects of DHEA/DHEAS on proinflammatory cytokine-induced transcription were estimated by luciferase assay. The results showed that DHEA/DHEAS potently inhibited TNF-alpha-induced NF-kappaB-dependent transcription in a time- and dose-dependent manner. The effect was more obvious for DHEAS than for DHEA, and both steroids preferentially inhibited the cytokine-stimulated rather than basal NF-kappaB-mediated transcription. Similar effects were observed in activator protein-1-dependent but not constitutive Rous sarcoma virus promoter-dependent transcription. Two major downstream products of the sex steroid precursors, estradiol and testosterone, had no effect, indicating that the observed suppressive effect is not mediated by these metabolites. In contrast, glucocorticoids showed inhibitory effects on both basal and stimulated transcription and had an additive effect with DHEAS, suggesting the independent mechanisms of action of these steroid hormones. Finally, DHEAS eliminated hydroxyradical-induced activation of NF-kappaB-dependent transcription as well. Altogether, these results suggest that DHEA/DHEAS have an antiinflammatory effect in such a way that they inhibit proinflammatory cytokine-stimulated, NF-kappaB-mediated transcription, at least partly through their antioxidant properties.
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PMID:Dehydroepiandrosterone-sulfate inhibits nuclear factor-kappaB-dependent transcription in hepatocytes, possibly through antioxidant effect. 1524 Jun 30

Ectopic protein expression in mammalian cells is a valuable tool to analyze protein functions. Increasingly, inducible promoters are being used for regulated gene expression. Here, we compare expression maxima, induction rates, and "leakiness" of the following promoter systems: (I) two tetracycline-responsive Tet systems (Tet-On, Tet-Off), (II) the glucocorticoid-responsive mouse mammary tumor virus promoter (MMTVprom), (III) the ecdysone-inducible promoter (EcP), and (IV) the T7 promoter/T7 RNA polymerase system (T7P). The systems were analyzed by expressing an enhanced green fluorescent protein (EGFP) luciferase fusion reporter protein in transiently transfected cells. Expression was assessed qualitatively by fluorescence microscopy of the EGFP component and quantitatively by measuring the enzymatic activity of the luciferase component of the fusion protein. Basal expression levels ("leakiness") were ranked Tet-On>Tet-Off>MMTVprom>EcP>T7P. Induction rates were EcP>MMTVprom>T7P>Tet-Off>Tet-On. Expression maxima were ranked. Tet-On>Tet-Off>MMTVprom>EcP>T7P. To increase T7-promoter-mediated expression we inserted an internal ribosomal entry site element into the T7 expression cassette. In presence of T7 RNA polymerase this modified T7 promoter achieved expression levels of 42% of a Rous Sarcoma virus promoter, while keeping basal expression extremely low.
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PMID:Comparative analysis of inducible expression systems in transient transfection studies. 1546 49

Somatolactin (SL) is a pituitary hormone belonging to the growth hormone-prolactin family and is produced in the intermediate lobe of teleosts. The SL gene was isolated from a sea bream genomic library and found to be composed of 5 exons distributed within a 9-kb length of DNA. Sequence analysis of the proximal promoter region showed the presence of a classical TATA box located 59 bp upstream from the initial start ATG codon, 5 consensus sequences corresponding to the Pit-1 binding element, and a putative CREB site. In CHO cells cotransfected with the DNA from 2 plasmids, one encoding sea bream Pit-1 under Rous sarcoma virus long terminal repeat regulation and one encoding the SL promoter driving the expression of luciferase, Pit-1 was found to enhance the expression of luciferase. Only one Pit-1 binding site was necessary for enhancement. Analysis by immunoblots of in vitro culture of pituitaries of Sparus aurata showed that several agents, including estradiol, verapamil, and phorbol myristate acetate, had different inhibitory effects on SL and growth hormone released to the culture medium.
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PMID:Genomic structure and functional analysis of promoter region of somatolactin gene of sea bream (Sparus aurata). 1554 52

To optimize vectors for salivary gland gene transfer, we screened viral [cytomegalovirus (CMV; human immediate early), Rous sarcoma virus (RSV), simian virus 40, and Moloney murine leukemia virus long terminal repeat] and mammalian [elongation factor 1alpha (EF1alpha), cytokeratin 18 (K18), cytokeratin 19 (K19), kallikrein (Kall), and amylase (AMY), all human, and rat aquaporin-5 (rAQP5), and derivative elements] promoters driving luciferase activity in vitro and in vivo. In adenoviral vectors, the CMV promoter showed highest activity, with the EF1alpha and RSV promoters slightly less powerful, in rat submandibular glands (SMGs). The K18 2.5-kb, K19 3.0-kb, and rAQP5 0.4-kb and Kall promoters had intermediate activity, while the AMY promoter exhibited lowest activity. To localize transgene expression, enhanced green fluorescence protein was used. The CMV, RSV, EF1alpha, K18 2.5-kb, K19 3.0-kb, rAQP5 0.4-kb, and AMY promoters were not cell-type specific in SMGs; however, the Kall promoter was primarily active in ductal cells. These data will facilitate optimal expression cassette design for salivary gland gene transfer.
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PMID:Evaluation of viral and mammalian promoters for use in gene delivery to salivary glands. 1609 14

Fifteen luciferase plasmid constructs driven by various promoters including cytomegalovirus (CMV), Rous sarcoma virus (RSV), human serum albumin (SA), alpha-1 antitrypsin (AAT), cytochrome P450 CYP1A2, CYP2C9, CYP2C18, CYP2D6, CYP3A4, mouse CYP2b10, human amyloid precursor protein (APP), chicken beta actin (ACT), nuclear factor kappa B (NFkappaB), and heat shock protein 70 (HS) promoters were hydrodynamically introduced into mouse hepatocytes, and the level and persistence of luciferase gene expression were examined. Eight hours post-gene transfer, the CMV and AAT promoters showed the highest activity, followed by the CYP2D6, HS, and RSV promoters which were slightly less active. The human serum albumin promoter exhibited the lowest activity among the promoters examined. The time course of gene expression showed a two-phase decline in luciferase activity with a rapid phase within the first 5-7 days and a slower decline thereafter. Results from Southern and Northern blot analyses revealed a good correlation between the decline of luciferase activity and the decrease in mRNA level, suggesting promoter silencing as the possible mechanism for the observed transient luciferase gene expression. Inclusion of EBN1 and oriP sequences of Epstein-Barr virus into the plasmid extended the period of active transcription for about one week. These results provide important information concerning the role of promoters in regulating transgene expression and for the proper design of plasmids for gene expression and gene therapy.
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PMID:Evaluation of viral and mammalian promoters for driving transgene expression in mouse liver. 1631 30

Avian leukosis virus subgroup J (ALV-J) was first isolated from meat-producing chickens that had developed myeloid leukosis. However, ALV-J infections associated with hemangiomas have occurred in egg-producing (layer) flocks in China. In this study, we identified an ALV-J layer isolate (HLJ13SH01) as a recombinant of ALV-J and a Rous sarcoma virus Schmidt-Ruppin B strain (RSV-SRB), which contained the RSV-SRB 5'-LTR and the other genes of ALV-J. Replication kinetic testing indicated that the HLJ13SH01 strain replicated faster than other ALV-J layer isolates in vitro. Sequence analysis indicated that the main difference between the two isolates was the 5'-LTR sequences, particularly the U3 sequences. A 19 nt insertion was uniquely found in the U3 region of the HLJ13SH01 strain. The results of a Dual-Glo luciferase assay revealed that the 19 nt insertion in the HLJ13SH01 strain increased the enhancer activity of the U3 region. Moreover, an additional CCAAT/enhancer element was found in the 19 nt insertion and the luciferase assay indicated that this element played a key role in increasing the enhancer activity of the 5'-U3 region. To confirm the potentiation effect of the 19 nt insertion and the CCAAT/enhancer element on virus replication, three infectious clones with 5'-U3 region variations were constructed and rescued. Replication kinetic testing of the rescued viruses demonstrated that the CCAAT/enhancer element in the 19 nt insertion enhanced the replication capacity of the ALV-J recombinant in vitro.
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PMID:An avian leukosis virus subgroup J isolate with a Rous sarcoma virus-like 5'-LTR shows enhanced replication capability. 2527 57


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