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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cold-water-fish pathogen Vibrio salmonicida expresses a functional bacterial luciferase but produces insufficient levels of its aliphatic-aldehyde substrate to be detectably luminous in culture. Our goals were to (i) better explain this cryptic bioluminescence phenotype through molecular characterization of the lux operon and (ii) test whether the bioluminescence gene cluster is associated with virulence. Cloning and sequencing of the V. salmonicida lux operon revealed that homologs of all of the genes required for luminescence are present: luxAB (luciferase) and luxCDE (aliphatic-aldehyde synthesis). The arrangement and sequence of these structural lux genes are conserved compared to those in related species of luminous bacteria. However, V. salmonicida strains have a novel arrangement and number of homologs of the luxR and luxI quorum-sensing regulatory genes. Reverse transcriptase PCR analysis suggests that this novel arrangement of quorum-sensing genes generates antisense transcripts that may be responsible for the reduced production of bioluminescence. In addition, infection with a strain in which the luxA gene was mutated resulted in a marked delay in mortality among Atlantic salmon relative to infection with the wild-type parent in single-strain challenge experiments. In mixed-strain competition between the luxA mutant and the wild type, the mutant was attenuated up to 50-fold. It remains unclear whether the attenuation results from a direct loss of luciferase or a polar disturbance elsewhere in the lux operon. Nevertheless, these findings document for the first time an association between a mutation in a structural lux gene and virulence, as well as provide a new molecular system to study Vibrio pathogenesis in a natural host.
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PMID:A novel lux operon in the cryptically bioluminescent fish pathogen Vibrio salmonicida is associated with virulence. 1727 25

Dengue viruses (DENV) have 5'-capped RNA genomes of (+) polarity and encode a single polyprotein precursor that is processed into mature viral proteins. NS2B, NS3 and NS5 proteins catalyse/activate enzyme activities that are required for key processes in the virus life cycle. The heterodimeric NS2B/NS3 is a serine protease required for processing. Using a high-throughput protease assay, we screened a small molecule chemical library and identified -200 compounds having > or = 50% inhibition. Moreover, NS3 exhibits RNA-stimulated NTPase, RNA helicase and the 5'-RNA triphosphatase activities. The NTPase and the 5'-RTPase activities of NS3 are stimulated by interaction with NS5. Moreover, the conserved, positively charged motif in DENV-2 NS3, 184RKRK, is required for RNA binding and modulates the RNA-dependent enzyme activities of NS3. To study viral replication, a variety of methods are used such as the in vitro RNA-dependent RNA polymerase assays that utilize lysates from DENV-2-infected mosquito or mammalian cells or the purified NS5 along with exogenous short subgenomic viral RNAs or the replicative intracellular membrane-bound viral RNAs as templates. In addition, a cell-based DENV-2 replicon RNA encoding a luciferase reporter is also used to examine the role of cis-acting elements within the 3' UTR and the RKRK motif in viral replication.
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PMID:Multiple enzyme activities of flavivirus proteins. 1731 55

The aim of the current study was to confirm that tenascin-C large splice variant (TNC320) stimulates matrix metalloproteinase-1 (MMP-1) expression and to elucidate molecular mechanisms underlying this activation. The analysis of gene expression in cultured cells grown under different conditions indicated significant increases of MMP-1 mRNA steady-state levels in the cells treated with TNC320 (200%) compared with TNC220 (100%) and bovine serum albumin (BSA), which served as controls. Gel electrophoresis results demonstrated augmented MMP-1 protein in cells cultured with TNC320, whereas slight up-regulation was noticed in cells treated with TNC220 or fibronectin. Reverse transcriptase polymerase chain reaction results demonstrated significantly higher levels of MMP-1 gene expression in TNC320 cultured cells than in all other treatment groups. The result was confirmed by examining the level of MMP-1 promoter transactivation by different extracellular proteins. Data demonstrated 30-fold activation of MMP-1 promoter by TNC320 treatment in comparison with other treatments (TNC220 or fibronectin) and BSA as a control. Both invasion and collagenase activity assays demonstrated a 3-fold difference in the cells treated with TNC320 in comparison with the control. MMP-1 was quantified by enzyme-linked immunosorbent assay as well. Experiments with constitutively active expression kinases indicated that MMP-1 expression induced by TNC320 was associated with mitogen-activated protein kinase (MAPK) cascade activation. Culture with TNC320 resulted in more than 2-fold activation of MMP1-luciferase in the presence of mitogen-activated protein kinase kinase kinase 1 and also 2-fold down-regulation in the presence of mitogen-activated protein kinase kinase 1. We hypothesize that tenascin-C stimulates invasion via up-regulation of MMP-1 expression through activation of MAPK cascade signaling.
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PMID:Molecular mechanism of tenascin-C action on matrix metalloproteinase-1 invasive potential. 1739 87

The development of effective antiviral drugs against hepatitis C virus (HCV) continues to be needed, since neither vaccines nor broadly effective therapeutic agents are available. HCV RNA-dependent RNA polymerase (NS5B) is strictly required for viral replication and thus represents an attractive target. Here, aqueous extracts from five traditional Chinese medicines were tested for their ability to inhibit NS5B activity by reporter assays using cell-based NS5B activity detecting systems. Among them, aqueous extract from Fructus Ligustri Lucidi exhibited a promising result, dose-dependent inhibition of the luciferase activity, an indicator of intracellular NS5B activity (p<0.001), in the absence of cytotoxicity. Further Quantitative RT-PCR assays and Western blot analysis showed aqueous extract from Fructus Ligustri Lucidi inhibited intracellular NS5B-catalyzed RNA synthesis dose-dependently (p<0.001) without affecting intracellular NS5B expression. Subsequent in vitro NS5B assays revealed that this extract could directly inhibit NS5B activity. Taken together, these results indicated that Fructus Ligustri Lucidi might offer a promising source of antiviral drugs against HCV NS5B. Purification of the active compound(s) and antiviral effect are clearly required in the future.
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PMID:Inhibition of HCV RNA-dependent RNA polymerase activity by aqueous extract from Fructus Ligustri Lucidi. 1753 44

Lung caner cells have a striking tendency to metastasize to bone. The chemokine stromal cell-derived factor-1 (SDF-1) is constitutively secreted by osteoblasts and bone marrow stromal cells and plays a key role for homing of hematopoietic cells to the bone marrow. Reverse transcriptase-polymerase chain reaction and flow cytometry studies demonstrated SDF-1 receptor (CXCR4) messenger RNA (mRNA) and surface expression of CXCR4 in lung cancer cell lines, especially higher in those with high invasiveness (A549) as compared with lower level in H928 cells and H1299 cells. SDF-1, osteoblast-conditioned medium (OBCM) and stromal cell-conditioned medium all induced the invasiveness of lung cancer cells. Matrix metalloproteinase (MMP)-9 small interfering RNA inhibited the SDF-1alpha- or OBCM-induced MMP-9 expression and thereby significantly inhibited the SDF-1alpha- or OBCM-induced cell invasion. Furthermore, mitogen-activated protein kinase kinase inhibitor PD98059 suppressed SDF-1alpha-induced MMP-9 mRNA expression. Transient transfection with dominant-negative extracellular signal-regulated kinase (ERK) mutant also showed that the ERK-signaling pathway was involved in SDF-1alpha-induced MMP-9 expression. Moreover, nuclear factor-kappaB (NF-kappaB) decoy oligodeoxynucleotide suppressed the MMP-9 promoter activity enhanced by SDF-1alpha. Both mitogen-activated protein kinase kinase inhibitor and ERK mutant also antagonized SDF-1alpha-induced NF-kappaB-driven luciferase promoter activity. Taken together, our results indicated that bone marrow-derived-SDF-1alpha enhances the invasiveness of lung cancer cells by increasing MMP-9 expression through the CXCR4/ERK/NF-kappaB signal transduction pathway.
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PMID:Involvement of matrix metalloproteinase-9 in stromal cell-derived factor-1/CXCR4 pathway of lung cancer metastasis. 1791 7

The aim of this study was to investigate the application of ultrasonic gas-filled liposomes in enhancing transfer for breast cancer-related antisense oligonucleotides in vitro. An antisense oligodeoxynucleotide (AS-ODN) sequence, HA2741, modified with luciferase reporter plasmid, was used in evaluating the enhancing effect of gas-filled liposomes for gene transfer in breast cancer cells. Some important factors on HA 2741 transfection efficiency, such as wave intensity, ultrasound duration, gas-filled liposome concentration, and HA2741 concentration, were tested, respectively. Transfection efficiency was detected by fluorescence microscopy. Cell viability was verified by propidium iodide assay. Reverse-transcriptase polymerase chain reaction and immunocytochemistry were used to detect the inhibitory effect of HA2741 on HER-2 expression. All the four factors (wave intensity, ultrasound duration, gas-filled liposome concentration, and HA2741 concentration) showed a positive effect on AS-ODN transfection efficiency. However, these factors had a negative effect on cell viability. Considering all the factors investigated, the maximum transfection efficiency with minimum cell viability achieved under 2% gas-filled liposome mixed with 80 nmol/L HA2741 for 30-second ultrasound exposure at -3.0 dB wave intensity, which gave an overall transfection efficiency exceeding 90% and a cell viability near 90%. Under controlled conditions, ultrasound-mediated AS-ODN transfer, enhanced by gas-filled liposomes, may represent an effective, safe avenue for cancer-related gene delivery.
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PMID:Application of ultrasonic gas-filled liposomes in enhancing transfer for breast cancer-related antisense oligonucleotides: an experimental study. 1898 10

Corticotropin-releasing hormone (CRH) is a key regulator of the mammalian stress response, mediating a wide variety of stress-associated behaviors including stress-induced inhibition of reproductive function. To investigate the potential direct action of CRH on pituitary gonadotrope function, we examined CRH receptor expression and second messenger signaling in alpha T3-1 cells, a murine gonadotrope-like cell line. Reverse transcriptase PCR (RT-PCR) studies demonstrated that alpha T3-1 cells express mRNA for the two CRH receptor subtypes, CRHR1 and CRHR2, with CRHR2alpha as the predominant CRHR2 isoform. Stimulation of the cells with CRH or urocortin (UCN) resulted in rapid, transient increases in the intracellular levels of cAMP that were completely blocked by the addition of alpha-helical CRH 9-41 or astressin, non-selective CRH receptor antagonists. Stimulation of the cells with CRHR2-specific ligands, urocortin 2 (UCN2) or urocortin 3 (UCN3), resulted in rapid increases in intracellular cAMP levels to 50-60% of the levels observed with UCN. Treatment with a selective CRHR2 antagonist, antisauvagine, completely blocked UCN3-mediated increases in cAMP and significantly reduced, but did not completely block UCN-mediated increases in cAMP, demonstrating that both CRHR1 and CRHR2 are functionally active in these gonadotrope-like cells. Finally, UCN treatment significantly increased the transcriptional activity of the glycoprotein hormone alpha-subunit promoter as assessed by alpha-luciferase transfection assays. Together, these results demonstrate the functional signaling of CRH receptors in alpha T3-1 cells, suggesting that CRH may also modulate pituitary gonadotrope function in vivo.
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PMID:Corticotropin-releasing hormone receptor expression and functional signaling in murine gonadotrope-like cells. 1900 30

Although flaviviruses cause significant human diseases, no antiviral therapy is currently available for clinical treatment of these pathogens. To identify flavivirus inhibitors, we performed a high-throughput screening of compound libraries using cells containing luciferase-reporting replicon of West Nile viruses (WNV). Five novel small molecular inhibitors of WNV were identified from libraries containing 96,958 compounds. The inhibitors suppress epidemic strain of WNV in cell culture, with EC(50) (50% effective concentration) values of <10microM and TI (therapeutic index) values of >10. Viral titer reduction assays, using various flaviviruses and nonflaviviruses, showed that the compounds have distinct antiviral spectra. Mode-of-action analysis showed that the inhibitors block distinct steps of WNV replication: four compounds inhibit viral RNA syntheses, while the other compound suppresses both viral translation and RNA syntheses. Biochemical enzyme assays showed that two compounds selectively inhibit viral RNA-dependent RNA polymerase (RdRp), while another compound specifically inhibits both RdRp and methyltransferase. The identified compounds could potentially be developed for treatment of flavivirus infections.
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PMID:Identification and characterization of inhibitors of West Nile virus. 1950 Dec 58

Innate immunity is part of the antiviral response. Interferon (IFN)-beta plays a leading role in this system. To investigate the influence of hepatitis C virus (HCV) on innate immunity, we examined the effect of viral proteins on IFN-beta induction. HepG2 cells were co-transfected with plasmids for seven HCV proteins (core protein, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) and the IFN-beta promoter luciferase. Toll-like receptor (TLR) 3 and Toll/IL-1 receptor domain-containing adapter inducing IFN-beta (TRIF) play key roles in dsRNA-mediated activation of interferon regulatory factor (IRF)-3 and IFN-beta; therefore, the participation of TLR3/TRIF in NS5B-mediated IFN induction was examined. Among seven HCV proteins, only NS5B, a viral RNA-dependent RNA polymerase (RdRp), activated the IFN-beta promoter. However, mutant NS5B without RdRp activity or template/primer association did not activate the IFN-beta promoter. Activation of the IFN-beta promoter by NS5B required the positive regulatory domain III, a binding sequence for IRF-3. Moreover, IRF-3 was phosphorylated by NS5B. Both inhibition of TLR3 expression by small interfering RNA and expression of the dominant negative form of TRIF significantly reduced NS5B-induced activation of IFN-beta. Of the six other HCV proteins, NS4A, NS4B, and NS5A efficiently inhibited this activation. HCV NS5B is a potent activator of the host innate immune system, possibly through TLR3/TRIF and synthesis of dsRNA. Meanwhile, NS4A, NS4B, and NS5A block IFN-beta induction by NS5B, which may contribute toward the persistence of this virus.
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PMID:Interferon-beta is activated by hepatitis C virus NS5B and inhibited by NS4A, NS4B, and NS5A. 1966 54

The hepatitis C virus (HCV) NS5B, a RNA-dependent RNA polymerase (RdRp), is an attractive target for anti-HCV agents. The major disadvantages of the commonly used polymerase inhibitor screening involving the assessment of in vitro RNA synthesis are that it is incapable of demonstrating the cellular permeability and the cytotoxicity of compounds. To overcome these limitations, we created the BHK-NS5B-FRLuc reporter cell line that carries stably transfected NS5B and a bicistronic reporter gene, (+)FLuc-(-)UTR-RLuc, which can be used to simultaneously measure cellular toxicity and intracellular RdRp activity. The (+)FLuc-(-)UTR-RLuc construct comprises the firefly luciferase (FLuc) gene and the Renilla luciferase (RLuc) gene in reverse orientation flanked by both negative strands of the HCV 5'- and 3'-untranslated regions (UTRs), in which FLuc and RLuc reporter proteins are regulated by host polymerase and functional NS5B polymerase, respectively. The reporter system was validated with specific agents against NS5B polymerization. Additionally, this assay was placed in 96-well plates and had a Z'-factor value of approximately 0.75, which is amenable for facilitating high-throughput screening operations. Notably, in combination with the structured-based virtual screening, an imidazole derivative compound was evaluated as a candidate HCV RdRp inhibitor.
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PMID:A cell-based reporter assay for inhibitor screening of hepatitis C virus RNA-dependent RNA polymerase. 2038 6


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