Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 7 de 7
Filter
Add more filters










Database
Language
Publication year range
1.
Scand J Gastroenterol ; 45(11): 1307-19, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20695836

ABSTRACT

OBJECTIVE: Norovirus is the most common cause of acute gastroenteritis in humans worldwide. Typical symptoms are vomiting, nausea and severe watery diarrhea. Because of the lack of cell lines susceptible to human norovirus infection, pathomechanisms and replication cycle are largely unknown. Here, we address the issue of how norovirus infection could lead to epithelial barrier dysfunction. MATERIAL AND METHODS: Expression of the non-structural norovirus protein p20 in the epithelial cell line HT-29/B6 was activated through a tetracycline sensitive promoter. Tight junction proteins were studied by Western blot and confocal laser scanning microscopy. Apoptoses were detected in TUNEL stainings. Epithelial restitution was monitored by conductance scanning after induction of single cell lesions. RESULTS: Changes in the expression or localization of the tight junction proteins occludin and/or claudin-1, -2,- 3, -4, -5, -7 and -8 could be ruled out to mediate epithelial barrier modulation. Cell motility was also unaltered by p20. Investigation of epithelial apoptosis revealed an accumulation of apoptic cells in epithelial monolayers after induction of p20 expression. In epithelial cell restitution assays, an arrest was identified in p20 expressing cells. Fluorescence microscopy revealed an inability for condensation and redistribution of cellular actin, which led to a reduced transepithelial electrical resistance. CONCLUSIONS: Functional data for norovirus protein p20 suggest a role in modulation of the actin cytoskeleton leading to barrier dysfunction through impairment of restitution of epithelial defects.


Subject(s)
Caliciviridae Infections/genetics , Cytoskeleton/metabolism , Gene Expression Regulation, Viral , Norovirus/metabolism , RNA, Messenger/genetics , Viral Core Proteins/genetics , Viral Nonstructural Proteins/metabolism , Actins/metabolism , Apoptosis , Blotting, Western , Caliciviridae Infections/metabolism , Caliciviridae Infections/pathology , Cytoskeleton/virology , HT29 Cells , Humans , In Situ Nick-End Labeling , Microscopy, Confocal , Microscopy, Fluorescence , Viral Core Proteins/biosynthesis
2.
J Gen Virol ; 90(Pt 5): 1190-1201, 2009 May.
Article in English | MEDLINE | ID: mdl-19264656

ABSTRACT

Rhesus monkey rhadinovirus (RRV) is a gamma-2 herpesvirus related to the human Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus 8). This study identified an alternatively spliced gene at the right side of the RRV genome (strain 17577) between open reading frame 75 and the terminal repeat region. Of its eight exons, the first seven encoded up to 12 transmembrane domains, whilst the eighth exon encoded a predicted C-terminal cytoplasmic domain. Structurally and positionally, this RRV gene therefore resembles the K15 gene of KSHV; it was provisionally named RK15 to avoid confusion with other RRV17577 genes. In ectopic expression studies, the 55 kDa RK15 protein isoform activated the JNK and NF-kappaB pathways, like the 45 kDa KSHV K15-encoded protein isoform. In contrast to K15, which activates angiogenic and inflammatory cytokines such as interleukin (IL)-8, IL-6 and CCL20, the range of cellular transcripts activated by the RRV K15 homologue was much more restricted, but included IL-6, IL-8 and FGF21. These data suggest functional differences between terminal membrane proteins at the right end of the genomes of Old World primate gamma-2 herpesviruses.


Subject(s)
Herpesvirus 8, Human/genetics , Rhadinovirus/genetics , Alternative Splicing , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Cloning, Molecular , Gene Expression Regulation, Viral/physiology , Genes, Viral , Humans , MAP Kinase Kinase 4/metabolism , Macaca mulatta , Mitogen-Activated Protein Kinase Kinases/metabolism , Molecular Sequence Data , NF-kappa B/metabolism , Rhadinovirus/classification , Rhadinovirus/metabolism , Sequence Homology , Transcription, Genetic , Viral Proteins
3.
Gastroenterology ; 129(2): 550-64, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16083712

ABSTRACT

BACKGROUND & AIMS: Ulcerative colitis (UC) is characterized by a Th2 immune response with inflammation and epithelial barrier dysfunction. So far, Th2 cytokines have not been shown to directly influence epithelial barrier function. METHODS: Lamina propria mononuclear cells (LPMCs) were stimulated and interleukin (IL)-13 was measured by enzyme-linked immunosorbent assay. Functional IL-13 and IL-4 effects were studied on HT-29/B6 colonic epithelial cells in Ussing chambers and by conductance scanning. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays. IL-13/IL-4 receptors were analyzed by reverse-transcription polymerase chain reaction and immunofluorescence. Western blotting combined with immunofluorescence was used to detect tight junction proteins. Furthermore, restitution velocity was measured. Finally, mucosal biopsy specimens from patients with UC were compared with cultured cells for these features. RESULTS: LPMCs from patients with UC produced large amounts of IL-13 (985 +/- 73 pg/mL), much more than from controls or patients with Crohn's disease. IL-13Ralpha1 and IL-4Ralpha receptors were present in HT-29/B6 cells and colonic epithelial cells of control patients and patients with UC. IL-13 had a dose-dependent effect on transepithelial resistance of HT-29/B6 monolayers (reduction to 60% +/- 4%), whereas IL-4 had no effect. This was due to an increased number of apoptotic cells (5.6-fold +/- 0.9-fold) and an increased expression of the pore-forming tight junction protein claudin-2 to 295% +/- 37%, both of which contributed equally. Finally, epithelial restitution velocity decreased from 15.1 +/- 0.6 to 10.6 +/- 0.5 microm/h after treatment with IL-13. Parallel changes were observed in human samples, with an increase in claudin-2 expression to 956% +/- 252%. CONCLUSIONS: IL-13 was identified as an important effector cytokine in UC that impairs epithelial barrier function by affecting epithelial apoptosis, tight junctions, and restitution velocity.


Subject(s)
Apoptosis/immunology , Colitis, Ulcerative/immunology , Cytokines/metabolism , Interleukin-13/metabolism , Tight Junctions/immunology , Adult , Apoptosis/physiology , Biomarkers/analysis , Biopsy, Needle , Blotting, Western , Cell Proliferation , Cells, Cultured , Colitis, Ulcerative/pathology , Crohn Disease/immunology , Crohn Disease/pathology , Cytokines/analysis , Epithelial Cells/cytology , Female , Humans , Immunohistochemistry , Interleukin-13/analysis , Intestinal Mucosa/immunology , Intestinal Mucosa/pathology , Male , Middle Aged , Probability , Prognosis , Risk Factors , Sampling Studies , Sensitivity and Specificity , Th2 Cells/immunology
4.
Biochem Biophys Res Commun ; 314(4): 1001-7, 2004 Feb 20.
Article in English | MEDLINE | ID: mdl-14751232

ABSTRACT

Assembly of tight junctions at the most apical part of the lateral cell membrane is a key event in the differentiation of polarized epithelial cells. Claudin-2, a transmembrane protein involved in tight junction strand formation, has turned out to play a crucial role for the paracellular barrier function by opening pores for small cations. Physiological and pathological variations of epithelial barrier function are accompanied by differential expression of tight junction proteins. Therefore, we characterized molecular mechanisms regulating claudin-2 gene expression. Genomic DNA containing the transcription start point of human claudin-2 was isolated and functionally characterized by reporter gene assays. Activity of the claudin-2 promoter was elevated in mouse mammary epithelial C57 cells expressing Wnt-1. LEF-1, a nuclear effector of the Wnt signaling pathway which is involved in the regulation of cell differentiation and polarization, was found to bind directly to the claudin-2 promoter as revealed by electrophoretic mobility shift assays. Expression of LEF-1 and beta-catenin both enhanced claudin-2 promoter activity. This increase was reduced after mutation of LEF-1 binding sites within the claudin-2 promoter. Furthermore, claudin-2 promoter activity was found to be enhanced by the TCF-4/beta-catenin transcription complex. Therefore, we conclude that gene expression mediated by the promoter of the human tight junction protein claudin-2 is regulated by factors involved in Wnt signaling. Moreover, a functional crosstalk between Wnt signaling and transcriptional activation related to caudal-related homeobox (Cdx) proteins could be demonstrated in the regulation of claudin-2 promoter-mediated gene expression.


Subject(s)
Gene Expression Regulation/physiology , Homeodomain Proteins/physiology , Membrane Proteins/genetics , Promoter Regions, Genetic , Proto-Oncogene Proteins/metabolism , Signal Transduction , Transcriptional Activation/physiology , Zebrafish Proteins , Base Sequence , Cell Line , Claudins , DNA , Electrophoretic Mobility Shift Assay , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Proto-Oncogene Proteins/physiology , Wnt Proteins , Wnt1 Protein
5.
Vet Microbiol ; 98(2): 81-8, 2004 Feb 04.
Article in English | MEDLINE | ID: mdl-14741119

ABSTRACT

The rep gene of Porcine circovirus type 1 directs the synthesis of two proteins. The full-length protein Rep is 312 amino acids in size, the spliced variant Rep' is truncated (168 aa) and exon 2 is frame-shifted. Replication of PCV1 DNA depends on synthesis of both proteins. Rep and Rep' bind in vitro to double-stranded DNA fragments comprising part of the origin of replication of PCV1, but the minimal binding sites of the two proteins are distinct. Rep protein represses the promoter of the rep gene by binding to the two inner hexamers H1 and H2. Although Rep' binds to the same sequence, it does not influence Prep. Twelve hours after PCV1 infection, similar amounts of rep and rep' were detected by real-time PCR, but later on, the ratio of the two transcripts varied. Both proteins are co-localised in the nucleus and formation of homo- and heteromeric complexes has been observed. When a replication assay was performed, in which Rep and Rep' protein of PCV1 was used to replicate the origin of PCV1 and PCV2, the rep gene products were found to initiate replication at both origins of replication.


Subject(s)
Circovirus/physiology , Amino Acid Sequence , Animals , Base Sequence , Circovirus/genetics , Circovirus/growth & development , DNA, Viral/chemistry , DNA, Viral/genetics , Genome, Viral , Molecular Sequence Data , Phylogeny , Viral Proteins/chemistry , Viral Proteins/genetics , Virus Replication
6.
Biochem Biophys Res Commun ; 298(5): 657-66, 2002 Nov 15.
Article in English | MEDLINE | ID: mdl-12419305

ABSTRACT

Occludin is an integral membrane protein located at the tight junctions of epithelial cells. Multiple domains of occludin are involved in the regulation of paracellular permeability as well as in the targeting of the protein to the tight junction. In this study, different occludin variants were identified on the mRNA level. Four differentially spliced occludin-specific mRNA transcripts were detected. Expression of the resulting proteins revealed an altered subcellular distribution and a loss of co-localization with zonula occludens protein ZO-1 in the tight junction for two of the four splice variants. Our findings demonstrate that the fourth transmembrane domain of occludin is important for targeting occludin to the tight junction. Loss of the fourth transmembrane domain leads to a relocation of the C-terminal domain to the extracellular space. The structural diversity of natural occludin variants is further increased by an additional promoter and transcription start giving rise to an alternative exon 1. Gene expression mediated by this promoter is influenced by the pro-inflammatory cytokine tumor necrosis factor alpha.


Subject(s)
Membrane Proteins/genetics , Tight Junctions/genetics , Tight Junctions/metabolism , Alternative Splicing , Amino Acid Sequence , Base Sequence , Cell Line , DNA, Complementary/genetics , Exons , Gene Expression , Genes, Reporter , Green Fluorescent Proteins , Humans , Introns , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Molecular Sequence Data , Occludin , Promoter Regions, Genetic , Protein Structure, Tertiary , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Subcellular Fractions/metabolism
7.
J Gen Virol ; 83(Pt 11): 2743-2751, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12388810

ABSTRACT

Porcine circovirus type 1 (PCV1) contains two major open reading frames encoding the replication initiator proteins, Rep and Rep', and the structural protein, Cap. The promoters of these two genes (P(cap) and P(rep)) have been mapped. P(cap) is located within the rep open reading frame (nt 1328-1252). P(rep) has been mapped to the intergenic region immediately upstream of the rep gene (nt 640-796) and overlaps the origin of replication of PCV1. Although binding of both rep gene products to a fragment containing P(rep) and the overlapping origin of replication has been reported, only the full-length Rep protein repressed P(rep), while the spliced isoform Rep' did not. P(rep) repression is mediated by binding of the Rep protein to the two inner hexamers, H1 and H2, located in the origin of PCV1, whereas binding of Rep to hexamers H3 and H4 was not necessary. Use of Rep mutants indicated that the conserved rolling-circle replication domain II as well as the P loop are essential for repression of P(rep). In contrast to P(rep), transcription of P(cap) was not influenced by viral proteins. Additionally, the ratio of the rep and rep' transcripts was analysed. Twelve hours after transfection of PK15 cells with an infectious clone of PCV1, similar amounts of both transcripts were detected, but later the amount of the two transcripts varied, indicating a balanced expression of the two rep transcripts.


Subject(s)
Capsid Proteins/genetics , Circovirus/genetics , DNA Helicases/genetics , DNA-Binding Proteins , Gene Expression Regulation, Viral , Trans-Activators/genetics , Transcription, Genetic , Viral Proteins/genetics , Animals , Base Sequence , Cell Line , Chromosome Mapping , DNA Helicases/metabolism , Genes, Viral , Molecular Sequence Data , Mutagenesis , Promoter Regions, Genetic , Swine , Trans-Activators/metabolism , Viral Proteins/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...