Summary
Phosphatidylinositol 3‐kinase (PI 3‐kinase) and its target protein kinase B (Akt) are involved in various processes including internalization, chemotaxis and proliferation. We analysed the ...activation of Akt in J774 macrophages infected with virulent (pYV+) or avirulent (pYV−) Yersinia enterocolitica. During the early stage of infection with pYV+ and pYV− bacteria, Akt and its targets, glycogen synthase kinase 3 (GSK‐3) and forkhead transcription factor (FKHRL1), became phosphorylated. This phosphorylation induction was inhibited by wortmannin and thus dependent on PI 3‐kinase. When infection was carried out with pYV+ bacteria but not with pYV− bacteria, Akt and its targets became dephosphorylated at later time points. Using single knock‐out mutants in bacterial effector genes, we have determined that the tyrosine phosphatase YopH was responsible for the inactivation of the PI 3‐kinase cascade. In macrophages, this inactivation correlated with the downregulation of mRNA coding for monocyte chemoattractant protein 1 (MCP‐1), suggesting that YopH inhibits recruitment of macrophages to lymph nodes. We also analysed the effects of Y. enterocolitica infection on the proliferation of T lymphocytes. Consistent with the observation that YopH inactivated the Akt pathway, YopH inhibited PI 3‐kinase‐dependent secretion of interleukin 2 and proliferation. These data reveal a new effect of YopH in Yersinia pathogenesis.
One major question in molecular biology is whether the spatial distribution of observed molecules is random or organized in clusters. Indeed, this analysis gives information about molecules' ...interactions and physical interplay with their environment. The standard tool for analyzing molecules' distribution statistically is the Ripley's K function, which tests spatial randomness through the computation of its critical quantiles. However, quantiles' computation is very cumbersome, hindering its use. Here, we present an analytical expression of these quantiles, leading to a fast and robust statistical test, and we derive the characteristic clusters' size from the maxima of the Ripley's K function. Subsequently, we analyze the spatial organization of endocytic spots at the cell membrane and we report that clathrin spots are randomly distributed while clathrin-independent spots are organized in clusters with a radius of 2 mm, which suggests distinct physical mechanisms and cellular functions for each pathway.
One major question in molecular biology is whether the spatial distribution of observed molecules is random or organized in clusters. Indeed, this analysis gives information about molecules' ...interactions and physical interplay with their environment. The standard tool for analyzing molecules' distribution statistically is the Ripley's K function, which tests spatial randomness through the computation of its critical quantiles. However, quantiles' computation is very cumbersome, hindering its use. Here, we present an analytical expression of these quantiles, leading to a fast and robust statistical test, and we derive the characteristic clusters' size from the maxima of the Ripley's K function. Subsequently, we analyze the spatial organization of endocytic spots at the cell membrane and we report that clathrin spots are randomly distributed while clathrin-independent spots are organized in clusters with a radius of 2 mm, which suggests distinct physical mechanisms and cellular functions for each pathway.
An in frame gene fusion containing the coding region for mature beta-lactamase and the 3'-end of hylA encoding the haemolysin secretion signal, was constructed under the control of a lac promoter. ...The resulting 53 kDa hybrid protein was specifically secreted to the external medium in the presence of the haemolysin translocator proteins, HlyB and HlyD. The specific activity of the beta-lactamase portion of the secreted protein (measured by the hydrolysis of penicillin G), approximately 1 U/microgram protein, was close to that of authentic, purified TEM-beta-lactamase. This is an important example of a hybrid protein that is enzymatically active, and secreted via the haemolysin pathway. Previous studies have indicated that haemolysin is secreted directly into the medium, bypassing the periplasm, to which beta-lactamase is normally targeted. This study indicated, therefore, that normal folding of an active beta-lactamase, can occur, at least when fused to the HlyA C-terminus, without the necessity of entering the periplasm. Despite the secretion of approximately 5 micrograms/ml levels of the active beta-lactamase fusion into the medium, there was maximally only a 50% detectable increase in the LD50 for resistance to ampicillin at the individual cell level. This result suggests that, normally, resistance to ampicillin requires a high concentration of the enzyme close to killing targets, i.e. in the periplasm, in order to achieve significant levels of protection.
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