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1.
Gut Microbes ; 2(3): 167-72, 2011.
Article in English | MEDLINE | ID: mdl-21637033

ABSTRACT

Helicobacter pylori infection is the most common bacterial infection worldwide and is strongly associated with gastric oncogenesis. Recently, we discovered that the H. pylori protein CagA, a risk factor for carcinogenesis, consists of two distinct membrane-targeting domains. The C-terminal membrane-binding domain induces host cell responses associated with a high oncogenic potential. The N-terminal membrane-targeting domain, however, localizes to a different membrane substructure at the site of newly formed cell-cell contacts thereby diminishing the effects of C-terminal signaling motifs on host cell physiology. This inhibitory function may allow H. pylori to establish a colonization niche in the host by maintaining the host epithelial architecture and thus decreasing the oncogenic potential as a side effect. From a bacterial standpoint, however, its main purpose maybe is to translocate the CagA protein via the type IV secretion apparatus into host epithelial cells. 


Subject(s)
Antigens, Bacterial/chemistry , Antigens, Bacterial/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Helicobacter Infections/microbiology , Helicobacter pylori/physiology , Animals , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Bacterial Translocation , Cell Line , Epithelial Cells/microbiology , Helicobacter pylori/chemistry , Helicobacter pylori/genetics , Humans , Protein Structure, Tertiary , Protein Transport
2.
J Biol Chem ; 286(11): 8999-9008, 2011 Mar 18.
Article in English | MEDLINE | ID: mdl-21212271

ABSTRACT

The Helicobacter pylori protein CagA (cytotoxin-associated gene A) is associated with an increased risk for gastric cancer formation. After attachment to epithelial cells, the bacteria inject CagA via a type IV secretion apparatus into host cells, where it exerts its biological activity. Host cell responses to intracellular CagA have been linked exclusively to signaling motifs in the C terminus of the CagA protein. Little is known about the functional role of the remaining CagA protein. Using transgenic expression of CagA mutants in epithelial cells, we were able to identify a novel CagA inhibitory domain at the N terminus consisting of the first 200 amino acids. This domain localizes to cell-cell contacts and increases the rate and strength of cell-cell adhesion in epithelial cells. Thus, it compensates for the loss of cell-cell adhesion induced by the C terminus of the CagA protein. Consistent with its stabilizing role on cell-cell adhesion, the CagA N terminus domain reduces the CagA-induced ß-catenin transcriptional activity in the nucleus. Furthermore, it inhibits apical surface constriction and cell elongations, host cell phenotypes induced by the C terminus in polarized epithelia. Therefore, our study suggests that CagA contains an intrinsic inhibitory domain that reduces host cell responses to CagA, which have been associated with the formation of cancer.


Subject(s)
Antigens, Bacterial/metabolism , Bacterial Proteins/metabolism , Epithelial Cells/microbiology , Helicobacter pylori/physiology , Host-Pathogen Interactions , Signal Transduction , Amino Acid Motifs , Animals , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Cell Line , Cell Nucleus/genetics , Cell Nucleus/metabolism , Dogs , Epithelial Cells/metabolism , Mutation , Protein Structure, Tertiary , Risk Factors , Stomach Neoplasms/genetics , Stomach Neoplasms/metabolism , Stomach Neoplasms/microbiology , Transcription, Genetic/genetics , beta Catenin/biosynthesis , beta Catenin/genetics
3.
Radiat Environ Biophys ; 50(1): 155-66, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21104263

ABSTRACT

High doses of ionising radiation damage the heart by an as yet unknown mechanism. A concern for radiological protection is the recent epidemiological data indicating that doses as low as 100-500 mGy may induce cardiac damage. The aim of this study was to identify potential molecular targets and/or mechanisms involved in the pathogenesis of low-dose radiation-induced cardiovascular disease. The vascular endothelium plays a pivotal role in the regulation of cardiac function and is therefore a potential target tissue. We report here that low-dose radiation induced rapid and time-dependent changes in the cytoplasmic proteome of the human endothelial cell line EA.hy926. The proteomes were investigated at 4 and 24 h after irradiation at two different dose rates (Co-60 gamma ray total dose 200 mGy; 20 mGy/min and 190 mGy/min) using 2D-DIGE technology. Differentially expressed proteins were identified, after in-gel trypsin digestion, by MALDI-TOF/TOF tandem mass spectrometry, and peptide mass fingerprint analyses. We identified 15 significantly differentially expressed proteins, of which 10 were up-regulated and 5 down-regulated, with more than ±1.5-fold difference compared with unexposed cells. Pathways influenced by the low-dose exposures included the Ran and RhoA pathways, fatty acid metabolism and stress response.


Subject(s)
Endothelial Cells/diagnostic imaging , Endothelial Cells/metabolism , Proteome/metabolism , Apoptosis/radiation effects , Cell Proliferation/radiation effects , Cytosol/metabolism , Cytosol/radiation effects , Dose-Response Relationship, Drug , Endothelial Cells/cytology , Gene Expression Profiling , Humans , Proteomics , Radiography , Time Factors
4.
PLoS One ; 5(12): e14325, 2010 Dec 14.
Article in English | MEDLINE | ID: mdl-21179475

ABSTRACT

BACKGROUND: E-cadherin is a major component of adherens junctions. Impaired expression of E-cadherin in the small intestine and colon has been linked to a disturbed intestinal homeostasis and barrier function. Down-regulation of E-cadherin is associated with the pathogenesis of infections with enteropathogenic bacteria and Crohn's disease. METHODS AND FINDINGS: To genetically clarify the function of E-cadherin in intestinal homeostasis and maintenance of the epithelial defense line, the Cdh1 gene was conditionally inactivated in the mouse intestinal epithelium. Inactivation of the Cdh1 gene in the small intestine and colon resulted in bloody diarrhea associated with enhanced apoptosis and cell shedding, causing life-threatening disease within 6 days. Loss of E-cadherin led cells migrate faster along the crypt-villus axis and perturbed cellular differentiation. Maturation and positioning of goblet cells and Paneth cells, the main cell lineage of the intestinal innate immune system, was severely disturbed. The expression of anti-bacterial cryptidins was reduced and mice showed a deficiency in clearing enteropathogenic bacteria from the intestinal lumen. CONCLUSION: These results highlight the central function of E-cadherin in the maintenance of two components of the intestinal epithelial defense: E-cadherin is required for the proper function of the intestinal epithelial lining by providing mechanical integrity and is a prerequisite for the proper maturation of Paneth and goblet cells.


Subject(s)
Cadherins/physiology , Cell Cycle Proteins/genetics , Gene Expression Regulation , Paneth Cells/cytology , Animals , Cadherins/biosynthesis , Cdh1 Proteins , Cell Cycle Proteins/metabolism , Cell Death , Colon/metabolism , Crohn Disease/metabolism , Epithelial Cells/metabolism , Goblet Cells/metabolism , Homeostasis , Homozygote , Intestine, Small/metabolism , Mice , Mice, Transgenic
5.
Nucleic Acids Res ; 38(6): 1853-65, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20040573

ABSTRACT

The Mre11/Rad50/Xrs2 (MRX) complex in Saccharomyces cerevisiae has well-characterized functions in DNA double-strand break processing, checkpoint activation, telomere length maintenance and meiosis. In this study, we demonstrate an involvement of the complex in the base excision repair (BER) pathway. We studied the repair of methyl-methanesulfonate-induced heat-labile sites in chromosomal DNA in vivo and the in vitro BER capacity for the repair of uracil- and 8-oxoG-containing oligonucleotides in MRX-deficient cells. Both approaches show a clear BER deficiency for the xrs2 mutant as compared to wildtype cells. The in vitro analyses revealed that both subpathways, long-patch and short-patch BER, are affected and that all components of the MRX complex are similarly important for the new function in BER. The investigation of the epistatic relationship of XRS2 to other BER genes suggests a role of the MRX complex downstream of the AP-lyases Ntg1 and Ntg2. Analysis of individual steps in BER showed that base recognition and strand incision are not affected by the MRX complex. Reduced gap-filling activity and the missing effect of aphidicoline treatment, an inhibitor for polymerases, on the BER efficiency indicate an involvement of the MRX complex in providing efficient polymerase activity.


Subject(s)
DNA Repair , DNA-Binding Proteins/physiology , Endodeoxyribonucleases/physiology , Exodeoxyribonucleases/physiology , Saccharomyces cerevisiae Proteins/physiology , Epistasis, Genetic , Gene Deletion , Hot Temperature , Methyl Methanesulfonate/toxicity , Mutation , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics
6.
DNA Repair (Amst) ; 7(9): 1563-77, 2008 Sep 01.
Article in English | MEDLINE | ID: mdl-18599383

ABSTRACT

Xrs2 is a member of the MRX complex (Mre11/Rad50/Xrs2) in Saccharomyces cerevisiae. In this study we demonstrate the important role of the MRX complex and in more detail of Xrs2 for the repair of radiation-induced chromosomal double-strand breaks by pulsed field gel electrophoresis. By using a newly designed in vivo plasmid-chromosome recombination system, we could show that gap repair efficiency and the association with crossovers were reduced in the MRX null mutants, but repair accuracy was unaffected. For these processes, an intact Mre11-binding domain of Xrs2 is crucial, whereas the FHA- and BRCT-domains as well as the Tel1-binding domain of Xrs2 are dispensable. Obviously, the Mre11-binding domain of the Xrs2 protein is crucial for the analysed functions and our results suggest a new role of the MRX complex for the formation of crossovers. Analysis of double mutants showed that the phenotype of the Deltaxrs2 null mutant concerning the crossover frequency is dominant over the phenotypes of Deltasrs2 and Deltasgs1 null mutants. Thus, the complex seems to be involved in early steps of double-strand break and gap repair, and we propose that it has a regulatory role for the selection of homologous recombination pathways.


Subject(s)
Crossing Over, Genetic , DNA Breaks, Double-Stranded , DNA Repair , Saccharomyces cerevisiae Proteins/physiology , Plasmids , Saccharomyces cerevisiae/genetics
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