http://hzi.openrepository.com/hzi HZI captures, stores, indexes, preserves, and distributes digital research material. Search the Channel search http://hzi.openrepository.com/hzi/simple-search http://hdl.handle.net/10033/94233 Title: Chivosazoles A and F, cytostatic macrolides from myxobacteria, interfere with actin.<br/><br/>Authors: Diestel, Randi; Irschik, Herbert; Jansen, Rolf; Khalil, Mohammed W; Reichenbach, Hans; Sasse, Florenz<br/><br/>Abstract: Chivosazoles A and F, isolated from Sorangium cellulosum, showed high antiproliferative activity with different mammalian cell lines including human cancer cells. The chivosazoles caused a delay in G2/M phase of the cell cycle, and treated cells often contained two nuclei. By labeling F-actin it was shown that the actin cytoskeleton of the cells starts to break down after a few minutes of treatment. In vitro polymerization assays with purified G-actin revealed that the chivosazoles inhibit actin polymerization and also cause a depolymerization of pyrene-labeled F-actin microfilaments prepared in vitro. Chivosazoles are new tools for the investigation of issues concerning the actin cytoskeleton and they have a different mode of action from the known microfilament-disrupting compounds like rhizopodin and cytochalasin D. http://hdl.handle.net/10033/94173 Title: Haplotypes of the porcine peroxisome proliferator-activated receptor delta gene are associated with backfat thickness.<br/><br/>Authors: Meidtner, Karina; Schwarzenbacher, Hermann; Scharfe, Maren; Severitt, Simone; Blöcker, Helmut; Fries, Ruedi<br/><br/>Abstract: BACKGROUND: Peroxisome proliferator-activated receptor delta belongs to the nuclear receptor superfamily of ligand-inducible transcription factors. It is a key regulator of lipid metabolism. The peroxisome proliferator-activated receptor delta gene (PPARD) has been assigned to a region on porcine chromosome 7, which harbours a quantitative trait locus for backfat. Thus, PPARD is considered a functional and positional candidate gene for backfat thickness. The purpose of this study was to test this candidate gene hypothesis in a cross of breeds that were highly divergent in lipid deposition characteristics. RESULTS: Screening for genetic variation in porcine PPARD revealed only silent mutations. Nevertheless, significant associations between PPARD haplotypes and backfat thickness were observed in the F2 generation of the Mangalitsa x Piétrain cross as well as a commercial German Landrace population. Haplotype 5 is associated with increased backfat in F2 Mangalitsa x Piétrain pigs, whereas haplotype 4 is associated with lower backfat thickness in the German Landrace population. Haplotype 4 and 5 carry the same alleles at all but one SNP. Interestingly, the opposite effects of PPARD haplotypes 4 and 5 on backfat thickness are reflected by opposite effects of these two haplotypes on PPAR-delta mRNA levels. Haplotype 4 significantly increases PPAR-delta mRNA levels, whereas haplotype 5 decreases mRNA levels of PPAR-delta. CONCLUSION: This study provides evidence for an association between PPARD and backfat thickness. The association is substantiated by mRNA quantification. Further studies are required to clarify, whether the observed associations are caused by PPARD or are the result of linkage disequilibrium with a causal variant in a neighbouring gene. http://hdl.handle.net/10033/94153 Title: Probiotic Escherichia coli Nissle 1917 inhibits leaky gut by enhancing mucosal integrity.<br/><br/>Authors: Ukena, Sya N; Singh, Anurag; Dringenberg, Ulrike; Engelhardt, Regina; Seidler, Ursula; Hansen, Wiebke; Bleich, André; Bruder, Dunja; Franzke, Anke; Rogler, Gerhard; Suerbaum, Sebastian; Buer, Jan; Gunzer, Florian; Westendorf, Astrid M<br/><br/>Abstract: BACKGROUND: Probiotics are proposed to positively modulate the intestinal epithelial barrier formed by intestinal epithelial cells (IECs) and intercellular junctions. Disruption of this border alters paracellular permeability and is a key mechanism for the development of enteric infections and inflammatory bowel diseases (IBDs). METHODOLOGY AND PRINCIPAL FINDINGS: To study the in vivo effect of probiotic Escherichia coli Nissle 1917 (EcN) on the stabilization of the intestinal barrier under healthy conditions, germfree mice were colonized with EcN or K12 E. coli strain MG1655. IECs were isolated and analyzed for gene and protein expression of the tight junction molecules ZO-1 and ZO-2. Then, in order to analyze beneficial effects of EcN under inflammatory conditions, the probiotic was orally administered to BALB/c mice with acute dextran sodium sulfate (DSS) induced colitis. Colonization of gnotobiotic mice with EcN resulted in an up-regulation of ZO-1 in IECs at both mRNA and protein levels. EcN administration to DSS-treated mice reduced the loss of body weight and colon shortening. In addition, infiltration of the colon with leukocytes was ameliorated in EcN inoculated mice. Acute DSS colitis did not result in an anion secretory defect, but abrogated the sodium absorptive function of the mucosa. Additionally, intestinal barrier function was severely affected as evidenced by a strong increase in the mucosal uptake of Evans blue in vivo. Concomitant administration of EcN to DSS treated animals resulted in a significant protection against intestinal barrier dysfunction and IECs isolated from these mice exhibited a more pronounced expression of ZO-1. CONCLUSION AND SIGNIFICANCE: This study convincingly demonstrates that probiotic EcN is able to mediate up-regulation of ZO-1 expression in murine IECs and confer protection from the DSS colitis-associated increase in mucosal permeability to luminal substances. http://hdl.handle.net/10033/94096 Title: Bifidobacterial enolase, a cell surface receptor for human plasminogen involved in the interaction with the host.<br/><br/>Authors: Candela, Marco; Biagi, Elena; Centanni, Manuela; Turroni, Silvia; Vici, Manuela; Musiani, Francesco; Vitali, Beatrice; Bergmann, Simone; Hammerschmidt, Sven; Brigidi, Patrizia<br/><br/>Abstract: The interaction with the host plasminogen/plasmin system represents a novel component in the molecular cross-talk between bifidobacteria and human host. Here, we demonstrated that the plasminogen-binding bifidobacterial species B. longum, B. bifidum, B. breve and B. lactis share the key glycolytic enzyme enolase as a surface receptor for human plasminogen. Enolase was visualized on the cell surface of the model strain B. lactis BI07. The His-tagged recombinant protein showed a high affinity for human plasminogen, with an equilibrium dissociation constant in the nanomolar range. By site-directed mutagenesis we demonstrated that the interaction between the B. lactis BI07 enolase and human plasminogen involves an internal plasminogen-binding site homologous to that of pneumococcal enolase. According to our data, the positively charged residues Lys-251 and Lys-255, as well as the negatively charged Glu-252, of the B. lactis BI07 enolase are crucial for plasminogen binding. Acting as a human plasminogen receptor, the bifidobacterial surface enolase is suggested to play an important role in the interaction process with the host. http://hdl.handle.net/10033/94093 Title: SiaA and SiaD are essential for inducing autoaggregation as a specific response to detergent stress in Pseudomonas aeruginosa.<br/><br/>Authors: Klebensberger, Janosch; Birkenmaier, Antoinette; Geffers, Robert; Kjelleberg, Staffan; Philipp, Bodo<br/><br/>Abstract: Cell aggregation is a stress response and serves as a survival strategy for Pseudomonas aeruginosa strain PAO1 during growth with the toxic detergent Na-dodecylsulfate (SDS). This process involves the psl operon and is linked to c-di-GMP signalling. The induction of cell aggregation in response to SDS was studied. Transposon and site-directed mutagenesis revealed that the cupA-operon and the co-transcribed genes siaA (PA0172) and siaD (PA0169) were essential for SDS-induced aggregation. While siaA encodes a putative membrane protein with a HAMP and a PP2C-like phosphatase domain, siaD encodes a putative diguanylate cyclase involved in the biosynthesis of c-di-GMP. Complementation studies uncovered that the loss of SDS-induced aggregation in the formerly isolated spontaneous mutant strain N was caused by a non-functional siaA allele. DNA-microarray analysis of SDS-grown cells revealed consistent activation of eight genes, including cupA1, with known or presumptive important functions in cell aggregation in the parent strain compared with non-aggregating siaA and siaD mutants. A siaAD-dependent increase of cupA1 mRNA levels in SDS-grown cells was also shown by Northern blots. These results clearly demonstrate that SiaAD are essential for inducing cell aggregation as a specific response to SDS and suggest that they are responsible for perceiving and transducing SDS-related stress. http://hdl.handle.net/10033/92942 Title: Bifidobacterial enolase, a cell surface receptor for human plasminogen involved in the interaction with the host.<br/><br/>Authors: Candela, Marco; Biagi, Elena; Centanni, Manuela; Turroni, Silvia; Vici, Manuela; Musiani, Francesco; Vitali, Beatrice; Bergmann, Simone; Hammerschmidt, Sven; Brigidi, Patrizia<br/><br/>Abstract: The interaction with the host plasminogen/plasmin system represents a novel component in the molecular cross-talk between bifidobacteria and human host. Here, we demonstrated that the plasminogen-binding bifidobacterial species B. longum, B. bifidum, B. breve and B. lactis share the key glycolytic enzyme enolase as a surface receptor for human plasminogen. Enolase was visualized on the cell surface of the model strain B. lactis BI07. The His-tagged recombinant protein showed a high affinity for human plasminogen, with an equilibrium dissociation constant in the nanomolar range. By site-directed mutagenesis we demonstrated that the interaction between the B. lactis BI07 enolase and human plasminogen involves an internal plasminogen-binding site homologous to that of pneumococcal enolase. According to our data, the positively charged residues Lys-251 and Lys-255, as well as the negatively charged Glu-252, of the B. lactis BI07 enolase are crucial for plasminogen binding. Acting as a human plasminogen receptor, the bifidobacterial surface enolase is suggested to play an important role in the interaction process with the host. http://hdl.handle.net/10033/92920 Title: The mycolyltransferase 85A, a putative drug target of Mycobacterium tuberculosis: development of a novel assay and quantification of glycolipid-status of the mycobacterial cell wall.<br/><br/>Authors: Elamin, Ayssar A; Stehr, Matthias; Oehlmann, Wulf; Singh, Mahavir<br/><br/>Abstract: The enzymes of the antigen 85 complex (Ag85A, B, and C) possess mycolyltransferase activity and catalyze the synthesis of the most abundant glycolipid of the mycobacterial cell wall, the cord factor. The cord factor (trehalose 6,6'-dimycolate, TDM) is essential for the integrity of the mycobacterial cell wall and pathogenesis of the bacillus. Thus, TDM biosynthesis is regarded as a potential drug target for control of Mycobacterium tuberculosis infections. Trehalose 6,6'-dimycolate (TDM) is synthesized from two molecules of trehalose-6'-monomycolate (TMM) by antigen 85A. We report here a novel enzyme assay using the natural substrate TMM. The novel colorimetric assay is based on the quantification of glucose from the degradation of trehalose, which is the product from catalytic activity of antigen 85A. Using the new assay, K(m) and K(cat) were determined with values of 129.6+/-8.1 microM and 65.4+/-4.1 min(-1), respectively. This novel assay is also suitable for robust high-throughput screening (HTS) for compound library screening against mycolyltransferase (antigen 85A). The assay is significantly faster and more convenient to use than all assays currently in use. The assay has a very low coefficient of variance (0.04) in 96-well plates and shows a Z' factor of 0.67-0.73, indicating the robustness of the assay. In addition, this new assay is highly suitable for the quantification of total TMM of the mycobacterial cell envelope. http://hdl.handle.net/10033/92901 Title: Ligand-mediated dimerization of the Met Receptor tyrosine kinase by the bacterial invasion protein InlB.<br/><br/>Authors: Ferraris, Davide M; Gherardi, Ermanno; Di, Ying; Heinz, Dirk W; Niemann, Hartmut H<br/><br/>Abstract: The Listeria monocytogenes surface protein InlB mediates bacterial invasion into host cells by activating the human receptor tyrosine kinase Met. So far, it is unknown how InlB or the physiological Met ligand hepatocyte growth factor/scatter factor causes Met dimerization, which is considered a prerequisite for receptor activation. We determined two new structures of InlB, revealing a recurring, antiparallel, dimeric arrangement, in which the two protomers interact through the convex face of the leucine-rich repeat domain. The same contact is found in one structure of the InlB-Met complex. Mutations disrupting the interprotomeric contact of InlB reduced its ability to activate Met and downstream signaling. Conversely, stabilization of this crystal contact by two intermolecular disulfide bonds generates a constitutively dimeric InlB variant with exceptionally high signaling activity, which can stimulate cell motility and cell division. These data demonstrate that the signaling-competent InlB-Met complex assembles with 2:2 stoichiometry around a back-to-back InlB dimer, enabling the direct contact between the stalk region of two Met molecules. http://hdl.handle.net/10033/92849 Title: Characterization of the interferon-producing cell in mice infected with Listeria monocytogenes.<br/><br/>Authors: Stockinger, Silvia; Kastner, Renate; Kernbauer, Elisabeth; Pilz, Andreas; Westermayer, Sandra; Reutterer, Benjamin; Soulat, Didier; Stengl, Gabriele; Vogl, Claus; Frenz, Theresa; Waibler, Zoe; Taniguchi, Tadatsugu; Rülicke, Thomas; Kalinke, Ulrich; Müller, Mathias; Decker, Thomas<br/><br/>Abstract: Production of type I interferons (IFN-I, mainly IFNalpha and IFNbeta) is a hallmark of innate immune responses to all classes of pathogens. When viral infection spreads to lymphoid organs, the majority of systemic IFN-I is produced by a specialized "interferon-producing cell" (IPC) that has been shown to belong to the lineage of plasmacytoid dendritic cells (pDC). It is unclear whether production of systemic IFN-I is generally attributable to pDC irrespective of the nature of the infecting pathogen. We have addressed this question by studying infections of mice with the intracellular bacterium Listeria monocytogenes. Protective innate immunity against this pathogen is weakened by IFN-I activity. In mice infected with L. monocytogenes, systemic IFN-I was amplified via IFN-beta, the IFN-I receptor (IFNAR), and transcription factor interferon regulatory factor 7 (IRF7), a molecular circuitry usually characteristic of non-pDC producers. Synthesis of serum IFN-I did not require TLR9. In contrast, in vitro-differentiated pDC infected with L. monocytogenes needed TLR9 to transcribe IFN-I mRNA. Consistent with the assumption that pDC are not the producers of systemic IFN-I, conditional ablation of the IFN-I receptor in mice showed that most systemic IFN-I is produced by myeloid cells. Furthermore, results obtained with FACS-purified splenic cell populations from infected mice confirmed the assumption that a cell type with surface antigens characteristic of macrophages and not of pDC is responsible for bulk IFN-I synthesis. The amount of IFN-I produced in the investigated mouse lines was inversely correlated to the resistance to lethal infection. Based on these data, we propose that the engagement of pDC, the mode of IFN-I mobilization, as well as the shaping of the antimicrobial innate immune response by IFN-I differ between intracellular pathogens. http://hdl.handle.net/10033/91244 Title: Identification of a PA-binding peptide with inhibitory activity against influenza A and B virus replication.<br/><br/>Authors: Wunderlich, Kerstin; Mayer, Daniel; Ranadheera, Charlene; Holler, Anne-Sophie; Mänz, Benjamin; Martin, Arnold; Chase, Geoffrey; Tegge, Werner; Frank, Ronald; Kessler, Ulrich; Schwemmle, Martin<br/><br/>Abstract: There is an urgent need for new drugs against influenza type A and B viruses due to incomplete protection by vaccines and the emergence of resistance to current antivirals. The influenza virus polymerase complex, consisting of the PB1, PB2 and PA subunits, represents a promising target for the development of new drugs. We have previously demonstrated the feasibility of targeting the protein-protein interaction domain between the PB1 and PA subunits of the polymerase complex of influenza A virus using a small peptide derived from the PA-binding domain of PB1. However, this influenza A virus-derived peptide did not affect influenza B virus polymerase activity. Here we report that the PA-binding domain of the polymerase subunit PB1 of influenza A and B viruses is highly conserved and that mutual amino acid exchange shows that they cannot be functionally exchanged with each other. Based on phylogenetic analysis and a novel biochemical ELISA-based screening approach, we were able to identify an influenza A-derived peptide with a single influenza B-specific amino acid substitution which efficiently binds to PA of both virus types. This dual-binding peptide blocked the viral polymerase activity and growth of both virus types. Our findings provide proof of principle that protein-protein interaction inhibitors can be generated against influenza A and B viruses. Furthermore, this dual-binding peptide, combined with our novel screening method, is a promising platform to identify new antiviral lead compounds.