Dephosphorylation of the catenins p120 and p100 in endothelial cells in response to inflammatory stimuli. (33/155046)

Inflammatory mediators such as histamine and thrombin increase the tight-junction permeability of endothelial cells. Tight-junction permeability may be independently controlled, but is dependent on the adherens junction, where adhesion is achieved through homotypic interaction of cadherins, which in turn are associated with cytoplasmic proteins, the catenins. p120, also termed p120(cas)/p120(ctn), and its splice variant, p100, are catenins. p120, originally discovered as a substrate of the tyrosine kinase Src, is also a target for a protein kinase C-stimulated pathway in epithelial cells, causing its serine/threonine dephosphorylation. The present study shows that pharmacological activation of protein kinase C stimulated a similar pathway in endothelial cells. Activation of receptors for agents such as histamine (H1), thrombin and lysophosphatidic acid in the endothelial cells also caused serine/threonine dephosphorylation of p120 and p100, suggesting physiological relevance. However, protein kinase C inhibitors, although blocking the effect of pharmacological activation of protein kinase C, did not block the effects due to receptor activation. Calcium mobilization and the myosin-light-chain-kinase pathway do not participate in p120/p100 signalling. In conclusion, endothelial cells possess protein kinase C-dependent and -independent pathways regulating p120/p100 serine/threonine phosphorylation. These data describe a new connection between inflammatory agents, receptor-stimulated signalling and pathways potentially influencing intercellular adhesion in endothelial cells.  (+info)

Purinogen is not an endogenous substrate used in endothelial cells during substrate deprivation. (34/155046)

Porcine aortic endothelial cells (PAEC) are known to be metabolically robust. They are capable of surviving extended periods of complete lack of exogenous substrate, and purine release has been shown to be significantly up-regulated. The endogenous substrates used during substrate deprivation, as well as the sources responsible for the increased purine release, have not been completely identified. We tested the possibility that a phosphoglyceroyl-ATP-containing polymer, purinogen, might support PAEC hibernation induced by lack of exogenous substrate. This involved isolation of the acid-insoluble fraction of PAEC, which was presumed to contain purinogen, and analysis by HPLC and 31P NMR. No evidence supporting the presence of triphosphate-containing compounds (purinogen) was found. Similar results were obtained in the rat heart. The majority of the products in the acid-insoluble, alkaline-treated fraction were identified as RNA degradation products (2'- and 3'-nucleoside monophosphates). A [14C]adenosine labelling experiment showed that incorporation of adenosine into the acid-insoluble fraction was almost completely prevented after inhibition of RNA synthesis with actinomycin D. Furthermore, RNA isolated from PAEC and subsequently treated with alkali showed a profile that was almost identical with the HPLC profile of the acid-insoluble fraction. Finally, substrate-free incubation of the cells did not quantitatively or qualitatively influence the distribution of acid-insoluble derivatives. We conclude that PAEC survival during the absence of exogenous substrate is not supported by purinogen but rather by some other, yet-to-be-identified, endogenous substrate.  (+info)

Salmonella typhimurium and lipopolysaccharide stimulate extracellularly regulated kinase activation in macrophages by a mechanism involving phosphatidylinositol 3-kinase and phospholipase D as novel intermediates. (35/155046)

Activation of the extracellularly regulated kinase (ERK) pathway is part of the early biochemical events that follow lipopolysaccharide (LPS) treatment of macrophages or their infection by virulent and attenuated Salmonella strains. Phagocytosis as well as the secretion of invasion-associated proteins is dispensable for ERK activation by the pathogen. Furthermore, the pathways used by Salmonella and LPS to stimulate ERK are identical, suggesting that kinase activation might be solely mediated by LPS. Both stimuli activate ERK by a mechanism involving herbimycin-dependent tyrosine kinase(s) and phosphatidylinositol 3-kinase. Phospholipase D activation and stimulation of protein kinase C appear to be intermediates in this novel pathway of MEK/ERK activation.  (+info)

Borrelia burgdorferi spirochetes induce mast cell activation and cytokine release. (36/155046)

The Lyme disease spirochete, Borrelia burgdorferi, is introduced into human hosts via tick bites. Among the cell types present in the skin which may initially contact spirochetes are mast cells. Since spirochetes are known to activate a variety of cell types in vitro, we tested whether B. burgdorferi spirochetes could activate mast cells. We report here that freshly isolated rat peritoneal mast cells or mouse MC/9 mast cells cultured in vitro with live or freeze-thawed B. burgdorferi spirochetes undergo low but detectable degranulation, as measured by [5-3H] hydroxytryptamine release, and they synthesize and secrete the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha). In contrast to findings in previous studies, where B. burgdorferi-associated activity was shown to be dependent upon protein lipidation, mast cell TNF-alpha release was not induced by either lipidated or unlipidated recombinant OspA. This activity was additionally shown to be protease sensitive and surface expressed. Finally, comparisons of TNF-alpha-inducing activity in known low-, intermediate-, and high-passage B. burgdorferi B31 isolates demonstrated passage-dependent loss of activity, indicating that the activity is probably plasmid encoded. These findings document the presence in low-passage B. burgdorferi spirochetes of a novel lipidation-independent activity capable of inducing cytokine release from host cells.  (+info)

Vibrio parahaemolyticus thermostable direct hemolysin modulates cytoskeletal organization and calcium homeostasis in intestinal cultured cells. (37/155046)

Vibrio parahaemolyticus is a marine bacterium known to be the leading cause of seafood gastroenteritis worldwide. A 46-kDa homodimer protein secreted by this microorganism, the thermostable direct hemolysin (TDH), is considered a major virulence factor involved in bacterial pathogenesis since a high percentage of strains of clinical origin are positive for TDH production. TDH is a pore-forming toxin, and its most extensively studied effect is the ability to cause hemolysis of erythrocytes from different mammalian species. Moreover, TDH induces in a variety of cells cytotoxic effects consisting mainly of cell degeneration which often leads to loss of viability. In this work, we examined the cellular changes induced by TDH in monolayers of IEC-6 cells (derived from the rat crypt small intestine), which represent a useful cell model for studying toxins from enteric bacteria. In experimental conditions allowing cell survival, TDH induces a rapid transient increase in intracellular calcium as well as a significant though reversible decreased rate of progression through the cell cycle. The morphological changes seem to be dependent on the organization of the microtubular network, which appears to be the preferential cytoskeletal element involved in the cellular response to the toxin.  (+info)

Enhanced adhesion of Pasteurella multocida to cultured turkey peripheral blood monocytes. (38/155046)

Capsular hyaluronic acid (HA) mediates adhesion of serogroup A strains of Pasteurella multocida to elicited turkey air sac macrophages (TASM). In contrast, freshly isolated turkey peripheral blood monocytes (TPBM) do not bind serogroup A strains. Following culture of TPBM for 6 days in chamber slides, adhesion of the bacteria to TPBM increased gradually. Incubation in chamber slides coated with entactin-collagen IV-laminin (ECL) attachment matrix or exposure to phorbol myristate acetate (PMA) further enhanced the adhesion of P. multocida to TPBM. Addition of HA, but not Arg-Gly-Asp peptide, to TPBM culture inhibited bacterial adherence similarly to the inhibition previously reported for TASM. Exposure of TPBM to monoclonal antibody directed against HA-binding cell surface proteoglycan (CD44) decreased binding of P. multocida. Collectively, these findings indicate that P. multocida adhesion to TPBM is mediated by capsular HA and can be increased by culture on ECL attachment matrix or PMA exposure. Additionally, the findings suggest that the capsular mucopolysaccharide of serogroup A strains of P. multocida recognizes an isoform of CD44 expressed on cultured TPBM.  (+info)

Infection of human endothelial cells with Chlamydia pneumoniae stimulates transendothelial migration of neutrophils and monocytes. (39/155046)

We have previously shown that different isolates of Chlamydia pneumoniae display heterogeneity in the in vitro stimulation of chemokines and adhesion molecules from infected human endothelial cells. In the present study, we examined the ability of different isolates of C. pneumoniae to promote transendothelial migration of neutrophils and monocytes. Human umbilical vein endothelial cells (HUVEC) were infected with low (<15)-passage C. pneumoniae isolates A-03, PS-32, and BR-393 and high (>40)-passage isolates BAL-16, TW-183, and T-2634, and levels of neutrophil and monocyte transendothelial migration were determined following 24 h of infection. Compared to mock-infected controls, significant increases in neutrophil migration were observed in response to most C. pneumoniae isolates examined (P < 0.001). Levels of monocyte migration were significantly increased in response to TW-183 and T-2634 (P < 0.001). Serial passage (>40 times) of the three low-passage isolates in HEp-2 cell cultures prior to infection of HUVEC generally resulted in the promotion of higher levels of neutrophil and monocyte transendothelial migration. These findings were compatible with differences observed in the extent of interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) stimulation between low- and high-passage A-03, PS-32, and BR-393. As opposed to C. pneumoniae, infection with C. trachomatis L2 caused only a slight increase in neutrophil transendothelial migration, which correlated with the lack of measurable IL-8 levels by this species. However, significant levels of monocyte migration were induced in response to C. trachomatis L2 despite a lack of measurable MCP-1 stimulation. C. trachomatis serovars A and E also failed to induce IL-8 and MCP-1 production in HUVEC. Results from this study indicate that the passage history of C. pneumoniae may play a role in the divergence of stimulatory activities observed among isolates in human endothelial cells. In addition, the differences observed between this organism and C. trachomatis suggest that the upregulation of IL-8 and MCP-1 in endothelial cells may be unique to C. pneumoniae.  (+info)

The virulence plasmid-encoded impCAB operon enhances survival and induced mutagenesis in Shigella flexneri after exposure to UV radiation. (40/155046)

Upon exposure to UV radiation, Shigella flexneri SA100 displayed survival and mutation frequencies comparable to those of Escherichia coli AB1157, which contains a functional UmuDC error-prone DNA repair system. Survival of SA100 after UV irradiation was associated with the presence of the 220-kb virulence plasmid, pVP. This plasmid encodes homologues of ImpA and ImpB, which comprise an error-prone DNA repair system encoded on plasmid TP110 that was initially identified in Salmonella typhimurium, and ImpC, encoded upstream of ImpA and ImpB. Although the impB gene was present in representatives of all four species of Shigella, not all isolates tested contained the gene. Shigella isolates that lacked impB were more sensitive to UV radiation than isolates that contained impB. The nucleotide sequence of a 2.4-kb DNA fragment containing the imp operon from S. flexneri SA100 pVP was 96% identical to the imp operon from the plasmid TP110. An SA100 derivative with a mutation in the impB gene had reduced survival following UV irradiation and less UV-induced mutagenesis relative to the parental strain. We also found that S. flexneri contained a chromosomally encoded umuDC operon; however, the umuDC promoter was not induced by exposure to UV radiation. This suggests that the imp operon but not the umuDC operon contributes to survival and induced mutagenesis in S. flexneri following exposure to UV radiation.  (+info)