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Author (up) Baylis, H.A.; Furuichi, T.; Yoshikawa, F.; Mikoshiba, K.; Sattelle, D.B. file  url
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  Title Inositol 1,4,5-trisphosphate receptors are strongly expressed in the nervous system, pharynx, intestine, gonad and excretory cell of Caenorhabditis elegans and are encoded by a single gene (itr-1) Type Journal Article
  Year 1999 Publication Journal of Molecular Biology Abbreviated Journal J Mol Biol  
  Volume 294 Issue 2 Pages 467-476  
  Keywords Amino Acid Sequence; Animals; Animals, Genetically Modified; Binding Sites; Caenorhabditis elegans/*genetics; Calcium Channels/*genetics/*metabolism; Cell Membrane/genetics/metabolism; Conserved Sequence; Gene Expression Profiling; Gonads/metabolism; Helminth Proteins/*genetics/*metabolism; Inositol 1,4,5-Trisphosphate Receptors; Intestines/metabolism; Molecular Sequence Data; Nervous System/metabolism; Pharynx/metabolism; RNA, Messenger; Receptors, Cytoplasmic and Nuclear/*genetics/*metabolism; Rectum/cytology/metabolism  
  Abstract Inositol 1,4,5-trisphosphate (InsP3) activates receptors (InsP3Rs) that mediate intracellular Ca(2+ )release, thereby modulating intracellular calcium signals and regulating important aspects of cellular physiology and gene expression. To further our understanding of InsP3Rs we have characterised InsP3Rs and the InsP3R gene, itr-1, from the model organism Caenorhabditis elegans. cDNAs encoding InsP3Rs were cloned enabling us to: (a) identify three putative transcription start sites that result in alternative mRNA 5' ends: (b) detect alternative splicing at three sites and: (c) determine the full genomic organisation of the itr-1 gene. The InsP3R protein (ITR-1) is approximately 42 % identical with known InsP3Rs and possesses conserved structural features. When the putative InsP3 binding domain was expressed in Escherichia coli, specific binding of InsP3 was detected. Using antibodies against ITR-1 we detected a protein of 220 kDa in C. elegans membranes. These antibodies and itr-1::GFP (green fluorescent protein) reporter constructs were used to determine the expression pattern of itr-1 in C. elegans. Strong expression was observed in the intestine, pharynx, nerve ring, excretory cell and gonad. These results demonstrate the high degree of structural and functional conservation of InsP3Rs from nematodes to mammals and the utility of C. elegans as a system for studies on InsP3R mediated signalling.  
  Call Number Serial 309  
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Author (up) Birchall, P.S.; Fishpool, R.M.; Albertson, D.G. file  url
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  Title Expression patterns of predicted genes from the C. elegans genome sequence visualized by FISH in whole organisms Type Journal Article
  Year 1995 Publication Nature Genetics Abbreviated Journal Nat Genet  
  Volume 11 Issue 3 Pages 314-320  
  Keywords Animals; Base Sequence; Caenorhabditis/cytology/*genetics; *Gene Expression; *Genome; Helminth Proteins/genetics; In Situ Hybridization, Fluorescence/*methods; Molecular Sequence Data; Muscle Proteins/genetics; RNA, Messenger/analysis  
  Abstract More than 10 megabases of contiguous genome sequence have been submitted to the databases by the Caenorhabditis elegans Genome Sequencing Consortium. To characterize the genes predicted from the sequence, we have developed high resolution FISH for visualization of mRNA distributions in whole animals. The high resolution and sensitivity afforded by the use of directly fluorescently labelled probes and confocal imaging permitted mRNA distributions to be recorded at the cellular and subcellular level. Expression patterns were obtained for 8 out of 10 genes in an initial test set of predicted gene sequences, indicating that FISH is an effective means of characterizing predicted genes in C. elegans.  
  Call Number Serial 172  
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Author (up) Bourne, G.L.; Grainger, D.J. file  url
openurl 
  Title Development and characterisation of an assay for furin activity Type Journal Article
  Year 2011 Publication Journal of Immunological Methods Abbreviated Journal J Immunol Methods  
  Volume 364 Issue 1-2 Pages 101-108  
  Keywords Anoxia/diagnosis/genetics/*metabolism; Antibodies/immunology/*metabolism; Biochemistry/methods; Cell Extracts/chemistry; Furin/*genetics/immunology/*metabolism; Gene Expression Regulation, Enzymologic; Hep G2 Cells; Humans; Immunomodulation; *Immunosorbent Techniques; RNA, Messenger/*analysis; Reference Standards; Sensitivity and Specificity; Transforming Growth Factor beta/immunology/metabolism  
  Abstract Furin is a serine endoprotease that is responsible for the proteolytic processing of proteins within the secretory pathway, including cytokines, hormones, integrins, other proteases, and also pathogen-derived proteins. It is likely that the level of furin activity determines the extent of processing of these substrates. Furin is ubiquitously expressed across all tissues, at low levels, but can be induced in response to environmental cues such as hypoxia and cytokine stimulation. However, all studies to date that have investigated furin expression have been limited to analysis of furin mRNA; there has been no assay sensitive enough to quantify endogenous furin. Though activity-based assays have been described for furin-like enzyme activity, we demonstrate that these assays are dominated by the activity of other enzymes and cannot be used to approximate furin activity. A sensitive and specific assay for furin activity was therefore developed and characterised, using an antibody capture step to immobilise furin from whole cell lysates. Furin activity is quantified relative to that of recombinant active furin protein, to allow estimation of active furin protein concentration. The assay has a minimum detection limit of 0.006 nM; sensitive enough to determine the furin activity of many of the cell lines tested. The specificity of the assay was demonstrated by genetic modulation of furin expression. Furthermore, the assay was used to demonstrate that the cytokine transforming growth factor beta (TGF-beta) stimulates increased furin activity in HepG2 cells, confirming and extending previous reports that TGF-beta increases furin expression, and adding to the mounting body of evidence that cellular furin activity can be modulated.  
  Call Number Serial 525  
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Author (up) Chen, X.; Qian, Y.; Yan, F.; Tu, J.; Yang, X.; Xing, Y.; Chen, Z. file  url
openurl 
  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) Cleary, M.D.; Meiering, C.D.; Jan, E.; Guymon, R.; Boothroyd, J.C. file  url
openurl 
  Title Biosynthetic labeling of RNA with uracil phosphoribosyltransferase allows cell-specific microarray analysis of mRNA synthesis and decay Type Journal Article
  Year 2005 Publication Nature Biotechnology Abbreviated Journal Nat Biotechnol  
  Volume 23 Issue 2 Pages 232-237  
  Keywords Animals; Gene Expression Profiling/*methods; Gene Expression Regulation/*physiology; Humans; Metabolic Clearance Rate; Oligonucleotide Array Sequence Analysis/*methods; Pentosyltransferases/chemistry/*metabolism; RNA, Messenger/chemistry/*genetics/*metabolism; Signal Transduction/physiology; Staining and Labeling/methods; Toxoplasma/genetics/metabolism; Transcription Factors/*metabolism; Transcriptional Activation/*physiology  
  Abstract Standard microarrays measure mRNA abundance, not mRNA synthesis, and therefore cannot identify the mechanisms that regulate gene expression. We have developed a method to overcome this limitation by using the salvage enzyme uracil phosphoribosyltransferase (UPRT) from the protozoan Toxoplasma gondii. T. gondii UPRT has been well characterized because of its application in monitoring parasite growth: mammals lack this enzyme activity and thus only the parasite incorporates (3)H-uracil into its nucleic acids. In this study we used RNA labeling by UPRT to determine the roles of mRNA synthesis and decay in the control of gene expression during T. gondii asexual development. We also used this approach to specifically label parasite RNA during a mouse infection and to incorporate thio-substituted uridines into the RNA of human cells engineered to express T. gondii UPRT, indicating that engineered UPRT expression will allow cell-specific analysis of gene expression in organisms other than T. gondii.  
  Call Number Serial 1344  
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