Infection of myeloid dendritic cells with Listeria monocytogenes leads to the suppression of T cell function by multiple inhibitory mechanisms

Myeloid dendritic cells (DC) and macrophages play an important role in pathogen sensing and antimicrobial defense. In this study we provide evidence that myeloid DC respond to infection with Listeria monocytogenes with simultaneous induction of multiple stimulatory and inhibitory molecules. However, the overall impact of infected DC during T cell encounter results in suppression of T cell activation, indicating that inhibitory pathways functionally predominate. Inhibitory activity of infected DC is effected mainly by IL-10 and cyclooxygenase 2-mediated mechanisms, with soluble CD25 acting as an IL-2 scavenger as well as by the products of tryptophan catabolism. These inhibitory pathways are strictly TNF-dependent. In addition to direct infection, DC bearing this regulatory phenotype can be induced in vitro by a combination of signals including TNF, TLR2, and prostaglandin receptor ligation and by supernatants derived from the infected cells. Both infection-associated DC and other in vitro-induced regulatory DC are characterized by increased resistance to infection and enhanced bactericidal activity. Furthermore, myeloid DC expressing multiple regulatory molecules are identified in vivo in granuloma during listeriosis and tuberculosis. Based on the in vivo findings and the study of in vitro models, we propose that in granulomatous infections regulatory DC may possess dual function evolved to protect the host from disseminating infection via inhibition of granuloma destruction by T cells and control of pathogen spreading.     

RIP2 regulates growth and differentiation of normal myoblasts and of rhabdomyosarcoma cells

RIP2 is an important regulator of myoblast proliferation and differentiation. We have previously demonstrated that in the myoblast cell line C2C12 and in primary myoblasts, downregulation of rip2 gene expression is a prerequisite for differentiation. To further study the role of rip genes in myogenesis, we compared expression patterns of rip1–4 in two myoblast cell lines, C2C12 and C2F3, after the induction of differentiation. These two cell lines are derived from the same clonal origin, but differ with respect to their differentiation behaviour: specifically, the differentiation process is slower and more incomplete in C2F3 cells. When analyzing cells up to 4 days after the induction of differentiation, we found no downregulation of rip2 gene expression in C2F3 cells, which might be linked to the low differentiation potential of these cells. In addition, in contrast to C2C12 cells, the rip3 gene was not expressed in C2F3 cells. To further study the role of rip genes in the regulation of myoblast growth and differentiation, we analyzed expression patterns of rip1–4 in rhabdomyosarcoma cell lines. We found that in these cells, rip2 expression was not downregulated after the induction of differentiation. Furthermore, in contrast to normal myoblasts, they did not express the rip3 and rip4 genes. Thus, we focused on the functional role of RIP2 in rhabdomyosarcoma cells. Inhibition of rip2 gene expression in C2C12 and in rhabdomyosarcoma cells using specific siRNAs led to decreased proliferation and promoted the differentiation process of these cells. These data indicate that differential expression of rip genes can be associated with abnormal growth and differentiation behaviour of skeletal myoblasts.     

The RGD integrin binding site in human L1-CAM is important for nuclear signaling

L1 cell adhesion molecule (L1-CAM) is a transmembrane cell adhesion molecule initially defined as a promigratory molecule in the developing nervous system. L1 is also overexpressed in a variety of human carcinomas and is associated with bad prognosis. In carcinoma cell lines L1 augments cell motility and metastasis, tumor growth in nude mice and induces expression of L1-dependent genes. It is not known whether L1-signaling requires ligand binding. The RGD motif in the sixth Ig domain of L1 is a binding site for integrins. In the present study we analyzed the role of RGDs in L1-signaling using site-directed mutagenesis combined with antibody blocking studies. We observed that L1-RGE expressing HEK293 cells showed reduced cell–cell binding, cell motility, invasiveness and tumor growth in NOD/SCID mice. The RGE-mutation impaired L1-dependent gene regulation and antibodies to αvβ5 integrin had similar effects. Mutant L1 was unable to translocate to the nucleus. Our findings highlight the importance of the RGD site in L1 for human tumors and suggest that nuclear signaling of L1 is dependent on integrins.     

Microbial community shifts as a response to efficient degradation of chlorobenzene under hypoxic conditions

Limitations in the availability of oxygen restrict aerobic biodegradation of chloroaromatic compounds in groundwater ecosystems. In this context the activity of ring-cleaving chlorocatechol dioxygenases (CC12O) is crucial for effective mineralization. Previously we demonstrated that oxygen-related enzyme characteristics of CC12O can vary widely among the Proteobacteria (Balcke et al. submitted). Here, we investigated how strains with different ability to transform intermediary 3-chlorocatechol integrate into biodegradation of chlorobenzene (CB) under low or high oxygen availability. Pseudomonas veronii UFZ B549 and Acidovorax facilis UFZ B530, which had differing oxygen affinities for CC12O, were mixed together at different proportions (20:80; 80:20), and compared for degradation of chlorobenzene under oxic (215 μM O2) and hypoxic (11 μM O2) conditions. Changes in community composition in binary mixed cultures were determined and compared with an indigenous groundwater community, cultivated under comparable conditions. Community shifts were determined by FISH (fluorescent in situ hybridization) in our model system and SSCP (single stranded conformation polymorphism) fingerprinting in the groundwater community, as well as by analysis of respiratory quinones of taxonomic value. Hypoxia led to enrichment of Acidovoracae in the groundwater and binary cultures. Under hypoxic conditions cis,cis-2-chloromuconate released to the medium by A. facilis allowed for concomitant growth of P. veronii, although its low-affinity type CC12O would not imply growth on CB. Vice versa, increasing abundance of P. veronii induced intermediary 3-chlorocatechol accumulation, which was reduced by growth of A. facilis. Thus, reduced oxygen availability caused syntrophic rather than competitive interactions.     

Esterase Autodisplay: Enzyme Engineering and Whole-Cell Activity Determination in Microplates with pH Sensors

Among the GDSL family of serine esterases/lipases is a group of bacterial enzymes that posses C-terminal extensions involved in outer membrane anchoring or translocation. ApeE from Salmonella enterica serovar Typhimurium, a member of this group, has been expressed in Escherichia coli and was resistant to protease digestion when the protease was added to whole cells, indicating a periplasmic localization. The five consensus blocks conserved within all GDSL esterases were identified in ApeE by multiple sequence alignment and separated from the C-terminal extension. The DNA sequence spanning the four invariant residues Ser, Gly, Asn, and His, and hence representing the catalytic domains of ApeE, was amplified by PCR and fused in frame to the transport domains of the autodisplay system. The resulting artificial esterase, called EsjA, was overexpressed in the cell envelope of E. coli and was shown to be active by the use of α-naphthyl acetate (α-NA) as a substrate in an in-gel activity stain after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Surface exposure of EsjA was indicated by its accessibility to protease added to whole cells. The esterase activity of whole cells displaying EsjA was determined by a pH agar assay and by the use of microplates with integrated pH-dependent optical sensors. α-NA, α-naphthyl butyrate, and α-naphthyl caproate were used as substrates, and it turned out that the substrate preferences of artificial EsjA were altered in comparison to original ApeE. Our results indicate that autodisplay of esterase in combination with pH sensor microplates can provide a new platform technology for the screening of tailor-made hydrolase activities.     

Untersuchungen über die Rolle und Bedeutung der Chinasäure in höheren Pflanzen

Ohne Zusammenfassung     

Untersuchungen über die Skelettsubstanz der Fruchtkörper der Myxomyceten und die Beziehung des Plastins zur Bildung derselben

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Impacts of coral bleaching on pH and oxygen gradients across the coral concentration boundary layer: a microsensor study

Coral Reefs - Reef-building corals are surrounded by complex microenvironments (i.e. concentration boundary layers) that partially isolate them from the ambient seawater. Although the presence of...