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Author (up) Baylis, H.A.; Vazquez-Manrique, R.P. file  url
  Title Genetic analysis of IP3 and calcium signalling pathways in C. elegans Type Journal Article
  Year 2012 Publication Biochimica et Biophysica Acta Abbreviated Journal Biochim Biophys Acta  
  Volume 1820 Issue 8 Pages 1253-1268  
  Keywords Animals; Caenorhabditis elegans--genetics, metabolism, physiology; Caenorhabditis elegans Proteins--genetics, metabolism; Calcium Signaling; Inositol 1,4,5-Trisphosphate Receptors--genetics, metabolism; Inositol Phosphates--physiology; Mutagenesis; Phenotype; Protein Interaction Maps; RNA Interference; Reverse Genetics  
  Abstract BACKGROUND: The nematode, Caenorhabditis elegans is an established model system that is particularly well suited to genetic analysis. C. elegans is easily manipulated and we have an in depth knowledge of many aspects of its biology. Thus, it is an attractive system in which to pursue integrated studies of signalling pathways. C. elegans has a complement of calcium signalling molecules similar to that of other animals. SCOPE OF REVIEW: We focus on IP3 signalling. We describe how forward and reverse genetic approaches, including RNAi, have resulted in a tool kit which enables the analysis of IP3/Ca2+ signalling pathways. The importance of cell and tissue specific manipulation of signalling pathways and the use of epistasis analysis are highlighted. We discuss how these tools have increased our understanding of IP3 signalling in specific developmental, physiological and behavioural roles. Approaches to imaging calcium signals in C. elegans are considered. MAJOR CONCLUSIONS: A wide selection of tools is available for the analysis of IP3/Ca2+ signalling in C. elegans. This has resulted in detailed descriptions of the function of IP3/Ca2+ signalling in the animal's biology. Nevertheless many questions about how IP3 signalling regulates specific processes remain. GENERAL SIGNIFICANCE: Many of the approaches described may be applied to other calcium signalling systems. C. elegans offers the opportunity to dissect pathways, perform integrated studies and to test the importance of the properties of calcium signalling molecules to whole animal function, thus illuminating the function of calcium signalling in animals. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signalling.  
  Call Number Serial 258  
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Author (up) Chen, X.; Qian, Y.; Yan, F.; Tu, J.; Yang, X.; Xing, Y.; Chen, Z. file  url
  Title 5'-triphosphate-siRNA activates RIG-I-dependent type I interferon production and enhances inhibition of hepatitis B virus replication in HepG2.2.15 cells Type Journal Article
  Year 2013 Publication European Journal of Pharmacology Abbreviated Journal Eur J Pharmacol  
  Volume 721 Issue 1-3 Pages 86-95  
  Keywords Base Sequence; DEAD-box RNA Helicases/*metabolism; DNA Replication/genetics; Hep G2 Cells; Hepatitis B Antigens/genetics/metabolism; Hepatitis B virus/*genetics/*physiology; Humans; Immunity, Innate; Interferon Type I/*biosynthesis/genetics; Polyphosphates/*chemistry; RNA Interference; RNA, Messenger/genetics; RNA, Small Interfering/chemistry/*genetics; Transcription, Genetic/genetics; Virus Replication/*genetics; 3-(4,5)-dimethylthiahiazol-2-y1)-2,5-diphenytetrazolium bromide; 3p-siRNA; 5â²-Triphosphated siRNA; 5â²-triphosphated siRNA; BF-siRNA; Ciap; Elisa; Hbv; HBV e antigen; HBV s antigen; HBeAg; HBsAg; Hcc; HepG2.2.15 cells; Hepatitis B virus; Ifn; Ifnî±/β; Mtt; NC-siRNA; Od; Prr; Rig-I; RNA interference; RNAi; Rt-Pcr; Tlr; bifunctional siRNA; calf intestine alkaline phosphatase; double strand DNA; double strand RNA; dsDNA; dsRNA; enzyme-linked immunosorbent assay; hepatitis B virus; hepatocellular carcinoma; interferon; negative control siRNA; optical density; pathogen-recognition receptor; retinoic acid-inducible gene I; reverse transcription PCR; siRNA; single strand RNA; small interfering RNA; ssRNA; toll-like receptor  
  Abstract Hepatitis B virus (HBV) infection often results in acute or chronic viral hepatitis and other liver diseases including cirrhosis and hepatocellular carcinoma. Current therapies for HBV usually have severe side effects and can cause development of drug-resistant mutants. An alternative and safe immunotherapeutic approach for HBV infection is urgently needed for effective anti-HBV therapy. In this study, we propose a new strategy for anti-HBV therapy that activates type-I interferon (IFN) antiviral innate immunity through stimulating pattern-recognition receptors with RNA interference (RNAi) using a 5'-end triphosphate-modified small interfering RNA (3p-siRNA). We designed and generated a 3p-siRNA targeting overlapping region of S gene and P gene of the HBV genome at the 5'-end of pregenomic HBV RNA. Our results demonstrated that 3p-siRNA induced a RIG-I-dependent antiviral type-I IFN response when transfected into HepG2.2.15 cells that support HBV replication. The 3p-siRNA significantly inhibited HBsAg and HBeAg secretion from HepG2.2.15 cells in a RIG-I-dependent manner, and the antiviral effect of 3p-siRNA was superior to that of siRNA. Furthermore, 3p-siRNA had more pronounced inhibition effects on the replication of HBV DNA and the transcription of mRNA than that of siRNA. Finally, 3p-siRNA displayed antiviral activity with long-term suppression of HBV replication. In conclusion, our findings suggest that 3p-siRNA could act as a powerful bifunctional antiviral molecule with potential for developing a promising therapeutic against chronic HBV infection.  
  Call Number Serial 1013  
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Author (up) Cram, E.J.; Shang, H.; Schwarzbauer, J.E. file  url
  Title A systematic RNA interference screen reveals a cell migration gene network in C. elegans Type Journal Article
  Year 2006 Publication Journal of Cell Science Abbreviated Journal J Cell Sci  
  Volume 119 Issue Pt 23 Pages 4811-4818  
  Keywords Animals; Animals, Genetically Modified; Caenorhabditis elegans/embryology/*genetics; Cell Movement/*genetics; *Gene Regulatory Networks; Genes, Helminth; Gonads/embryology; Phenotype; *RNA Interference  
  Abstract Cell migration is essential during embryonic development and tissue morphogenesis. During gonadogenesis in the nematode Caenorhabditis elegans, migration of the distal tip cells forms two U-shaped gonad arms. Malformation results if the distal tip cells stop prematurely or follow an aberrant path, and abnormalities are easily visualized in living nematodes. Here we describe the first comprehensive in vivo RNA interference screen for genes required for cell migration. In this non-biased screen, we systematically analyzed 16,758 RNA-interference depletion experiments by light microscopy and identified 99 genes required for distal tip cell migration. Genetic and physical interaction data connect 59 of these genes to form a cell migration gene network that defines distal tip cell migration in vivo.  
  Call Number Serial 1705  
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Author (up) Marimani, M.D.; Ely, A.; Buff, M.C.R.; Bernhardt, S.; Engels, J.W.; Arbuthnot, P. file  url
  Title Inhibition of hepatitis B virus replication in cultured cells and in vivo using 2'-O-guanidinopropyl modified siRNAs Type Journal Article
  Year 2013 Publication Bioorganic & Medicinal Chemistry Abbreviated Journal Bioorg Med Chem  
  Volume 21 Issue 20 Pages 6145-6155  
  Keywords Animals; Cell Line, Tumor; Cells, Cultured; Guanidines/chemistry/*pharmacology; Hepatitis B virus/drug effects/genetics/*physiology; Humans; Mice; Organophosphorus Compounds/chemistry/pharmacology; RNA Interference; RNA, Small Interfering/genetics/*pharmacology; Transfection; Virus Replication/*drug effects/genetics; 2â²-O-Guanidinopropyl; Hbv; RNAi; siRNAs  
  Abstract Silencing hepatitis B virus (HBV) gene expression with exogenous activators of the RNA interference (RNAi) pathway has shown promise as a new mode of treating infection with the virus. However, optimizing efficacy, specificity, pharmacokinetics and stability of RNAi activators remains a priority before clinical application of this promising therapeutic approach is realised. Chemical modification of synthetic short interfering RNAs (siRNAs) provides the means to address these goals. This study aimed to assess the benefits of incorporating nucleotides with 2'-O-guanidinopropyl (GP) modifications into siRNAs that target HBV. Single GP residues were incorporated at nucleotide positions from 2 to 21 of the antisense strand of a previously characterised effective antiHBV siRNA. When tested in cultured cells, siRNAs with GP moieties at selected positions improved silencing efficacy. Stability of chemically modified siRNAs in 80% serum was moderately improved and better silencing effects were observed without evidence for toxicity or induction of an interferon response. Moreover, partially complementary target sequences were less susceptible to silencing by siRNAs with GP residues located in the seed region. Hydrodynamic co-injection of siRNAs with a replication-competent HBV plasmid resulted in highly effective knock down of markers of viral replication in mice. Evidence for improved efficacy, reduced off target effects and good silencing in vivo indicate that GP-modifications of siRNAs may be used to enhance their therapeutic utility.  
  Call Number Serial 1014  
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Author (up) Schenone, M.; Dancik, V.; Wagner, B.K.; Clemons, P.A. file  url
  Title Target identification and mechanism of action in chemical biology and drug discovery Type Journal Article
  Year 2013 Publication Nature Chemical Biology Abbreviated Journal Nat Chem Biol  
  Volume 9 Issue 4 Pages 232-240  
  Keywords Animals; Biomarkers, Pharmacological/chemistry/*metabolism; *Drug Discovery; *Drug Evaluation, Preclinical; *High-Throughput Screening Assays; Humans; Isotope Labeling; Mass Spectrometry; Molecular Targeted Therapy; Phenotype; RNA Interference; Reverse Genetics; Saccharomyces cerevisiae/drug effects/genetics/metabolism; Small Molecule Libraries/chemistry/*metabolism/pharmacology; Validation Studies as Topic  
  Abstract Target-identification and mechanism-of-action studies have important roles in small-molecule probe and drug discovery. Biological and technological advances have resulted in the increasing use of cell-based assays to discover new biologically active small molecules. Such studies allow small-molecule action to be tested in a more disease-relevant setting at the outset, but they require follow-up studies to determine the precise protein target or targets responsible for the observed phenotype. Target identification can be approached by direct biochemical methods, genetic interactions or computational inference. In many cases, however, combinations of approaches may be required to fully characterize on-target and off-target effects and to understand mechanisms of small-molecule action.  
  Call Number Serial 1592  
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Author (up) Sirma, H.; Kumar, M.; Meena, J.K.; Witt, B.; Weise, J.M.; Lechel, A.; Ande, S.; Sakk, V.; Guguen-Guillouzo, C.; Zender, L.; Rudolph, K.-L.; Gunes, C. file  url
doi  openurl
  Title The promoter of human telomerase reverse transcriptase is activated during liver regeneration and hepatocyte proliferation Type Journal Article
  Year 2011 Publication Gastroenterology Abbreviated Journal Gastroenterology  
  Volume 141 Issue 1 Pages 326-37, 337.e1-3  
  Keywords Animals; Binding Sites; Cell Differentiation; *Cell Proliferation; Cells, Cultured; Chromatin Immunoprecipitation; E2F2 Transcription Factor/metabolism; E2F7 Transcription Factor/metabolism; Gene Expression Regulation, Enzymologic; Genes, Reporter; Hepatectomy; Hepatocytes/*enzymology; Humans; Lac Operon; Liver/*enzymology/surgery; *Liver Regeneration; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; *Promoter Regions, Genetic; RNA Interference; Regulatory Elements, Transcriptional; Retinoblastoma Protein/metabolism; Telomerase/*genetics/metabolism; Time Factors; *Transcriptional Activation  
  Abstract BACKGROUND & AIMS: Telomerase activity has not been detected in healthy human liver biopsy samples, but it is up-regulated in most human liver tumors. It is not clear whether telomerase is activated in response to acute or chronic liver injury. Telomerase activity is closely associated with expression of its catalytic subunit, telomerase reverse transcriptase (TERT). We analyzed the activity of the human TERT (hTERT) promoter during liver regeneration in vivo and hepatocyte proliferation in vitro. METHODS: We used hTERTp-lacZ transgenic mice, which contain an 8.0-kilobase pair fragment of the hTERT gene promoter, to study the role of TERT in liver regeneration following partial hepatectomy. As an in vitro model, we used the HepaRG cell line as a new model system for human hepatocyte proliferation and differentiation. RESULTS: Activity of the hTERT promoter increased significantly after partial hepatectomy; it was also induced in hepatocytes, based on immunohistologic analysis. Similar to the in vivo results, telomerase activity and hTERT expression were up-regulated in proliferating HepaRG cells and repressed in response to growth arrest and differentiation. Promoter mapping revealed that a proximal 0.3-kilobase pair fragment contains all elements necessary for regulation of hTERT in HepaRG cells. We identified E2F2 and E2F7 as transcription factors that control the differential expression of hTERT in proliferating hepatocytes, in vitro and in vivo. CONCLUSIONS: hTERT is induced in hepatocytes during liver regeneration, indicating a functional role for telomerase in human liver.  
  Call Number Serial 430  
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Author (up) Thongthae, N.; Payungporn, S.; Poovorawan, Y.; T-Thienprasert, N.P. file  url
  Title A rational study for identification of highly effective siRNAs against hepatitis B virus Type Journal Article
  Year 2014 Publication Experimental and Molecular Pathology Abbreviated Journal Exp Mol Pathol  
  Volume 97 Issue 1 Pages 120-127  
  Keywords 2',5'-Oligoadenylate Synthetase/genetics; Algorithms; Base Sequence; Gene Expression Regulation; Hep G2 Cells/virology; Hepatitis B virus/*genetics; Humans; Luciferases/genetics/metabolism; Molecular Sequence Data; NF-kappa B/genetics; Promoter Regions, Genetic; RNA, Small Interfering/chemistry/*genetics; Regulatory Sequences, Nucleic Acid; STAT1 Transcription Factor/genetics; Thermodynamics; Effective siRNAs; Hbv Pre; Hepatitis B virus; RNA interference; siRNA predicting program  
  Abstract RNA interference (RNAi) is a powerful gene knockdown technique used for study gene function. It also potentially provides effective agents for inhibiting infectious and genetic diseases. Most of RNAi studies employ a single siRNA designing program and then require large-scale screening experiments to identify functional siRNAs. In this study, we demonstrate that an assembly of results generated from different siRNA designing programs could provide clusters of predicting sites that aided selection of potent siRNAs. Based on the clusters, three siRNA target sites were selected on a conserved RNA region of hepatitis B virus (HBV), known as HBV post-transcriptional regulatory element (HBV PRE) at nucleotide positions 1317-1337, 1357-1377 and 1644-1664. All three chosen siRNAs driven by H1 promoter were highly effective and could drastically decrease expression of HBV transcripts (core, surface and X) and surface protein without induction of interferon response and cell cytotoxicity in liver cancer cell line (HepG2). Based on prediction of secondary structures, the silencing effects of siRNAs were less effective against a loop sequence of the mRNA target with hairpin structure. In summary, we demonstrate an effectual approach for identification of functional siRNAs. Moreover, highly potent siRNAs identified here may serve as novel agents for development of nucleic acid-based HBV therapy.  
  Call Number Serial 1012  
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Author (up) Van Baelen, K.; Vanoevelen, J.; Callewaert, G.; Parys, J.B.; De Smedt, H.; Raeymaekers, L.; Rizzuto, R.; Missiaen, L.; Wuytack, F. file  url
  Title The contribution of the SPCA1 Ca2+ pump to the Ca2+ accumulation in the Golgi apparatus of HeLa cells assessed via RNA-mediated interference Type Journal Article
  Year 2003 Publication Biochemical and Biophysical Research Communications Abbreviated Journal Biochem Biophys Res Commun  
  Volume 306 Issue 2 Pages 430-436  
  Keywords Calcium/metabolism; Calcium-Transporting ATPases/chemistry/*metabolism/*physiology; Cell Membrane/metabolism; Endoplasmic Reticulum/metabolism; Enzyme Inhibitors/pharmacology; Genetic Vectors; Golgi Apparatus/*metabolism; HeLa Cells; Humans; Immunoblotting; Immunohistochemistry; Kinetics; Microscopy, Fluorescence; *RNA Interference; Recombinant Proteins/metabolism; Thapsigargin/pharmacology; Transfection  
  Abstract The secretory-pathway Ca(2+)-ATPase SPCA1 is a thapsigargin-insensitive intracellular Ca(2+) pump found mostly in the Golgi compartment. We have explored the contribution of this Ca(2+) pump to cytosolic Ca(2+) signaling in HeLa cells by using RNA-mediated interference to disrupt its expression. Removal of SPCA1 was confirmed by immunofluorescence with specific anti-SPCA1 antibodies. Measurements of the free Ca(2+) concentration in the lumen of the Golgi apparatus by specifically targeting the Ca(2+)-sensitive luminescent protein aequorin to this organelle revealed that endogenous SPCA1 was responsible for Ca(2+) uptake in a subfraction of the Golgi apparatus. HeLa cells lacking SPCA1 could still set up baseline Ca(2+) spiking when stimulated with histamine, indicating that the SPCA1-containing Ca(2+) store was not absolutely needed to set up these oscillations. However, baseline Ca(2+) oscillations occurred less frequently than in control cells, pointing to a contribution of SPCA1 in the shaping of the cytosolic Ca(2+) signal in HeLa cells.  
  Call Number Serial 120  
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