49 research outputs found

    Inhibition of Protein Kinase C-Driven Nuclear Factor-κB Activation: Synthesis, Structure−Activity Relationship, and Pharmacological Profiling of Pathway Specific Benzimidazole Probe Molecules

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    A unique series of biologically active chemical probes that selectively inhibit NF-κB activation induced by protein kinase C (PKC) pathway activators have been identified through a cell-based phenotypic reporter gene assay. These 2-aminobenzimidazoles represent initial chemical tools to be used in gaining further understanding on the cellular mechanisms driven by B and T cell antigen receptors. Starting from the founding member of this chemical series 1a (notated in PubChem as CID-2858522), we report the chemical synthesis, SAR studies, and pharmacological profiling of this pathway-selective inhibitor of NF-κB activation

    Oligomeric Structure of the MALT1 Tandem Ig-Like Domains

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    Mucosa-associated lymphoid tissue 1 (MALT1) plays an important role in the adaptive immune program. During TCR- or BCR-induced NF-κB activation, MALT1 serves to mediate the activation of the IKK (IκB kinase) complex, which subsequently regulates the activation of NF-κB. Aggregation of MALT1 is important for E3 ligase activation and NF-κB signaling.Unlike the isolated CARD or paracaspase domains, which behave as monomers, the tandem Ig-like domains of MALT1 exists as a mixture of dimer and tetramer in solution. High-resolution structures reveals a protein-protein interface that is stabilized by a buried surface area of 1256 Å(2) and contains numerous hydrogen and salt bonds. In conjunction with a second interface, these interactions may represent the basis of MALT1 oligomerization.The crystal structure of the tandem Ig-like domains reveals the oligomerization potential of MALT1 and a potential intermediate in the activation of the adaptive inflammatory pathway.This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1

    Modulation of the NF-κB Pathway by Bordetella pertussis Filamentous Hemagglutinin

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    Background Filamentous hemagglutinin (FHA) is a cell-associated and secreted adhesin produced by Bordetella pertussis with pro-apoptotic and pro-inflammatory activity in host cells. Given the importance of the NF-κB transcription factor family in these host cell responses, we examined the effect of FHA on NF-κB activation in macrophages and bronchial epithelial cells, both of which are relevant cell types during natural infection. Methodology/Principal Findings Exposure to FHA of primary human monocytes and transformed U-937 macrophages, but not BEAS-2B epithelial cells, resulted in early activation of the NF-κB pathway, as manifested by the degradation of cytosolic IκBα, by NF-κB DNA binding, and by the subsequent secretion of NF-κB-regulated inflammatory cytokines. However, exposure of macrophages and human monocytes to FHA for two hours or more resulted in the accumulation of cytosolic IκBα, and the failure of TNF-α to activate NF-κB. Proteasome activity was attenuated following exposure of cells to FHA for 2 hours, as was the nuclear translocation of RelA in BEAS-2B cells. Conclusions These results reveal a complex temporal dynamic, and suggest that despite short term effects to the contrary, longer exposures of host cells to this secreted adhesin may block NF-κB activation, and perhaps lead to a compromised immune response to this bacterial pathogen

    Macrophages inhibit Coxiella burnetii by the ACOD1 ‐itaconate pathway for containment of Q fever

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    Infection with the intracellular bacterium Coxiella (C.) burnetii can cause chronic Q fever with severe complications and limited treatment options. Here, we identify the enzyme cis-aconitate decarboxylase 1 (ACOD1 or IRG1) and its product itaconate as protective host immune pathway in Q fever. Infection of mice with C. burnetii induced expression of several anti-microbial candidate genes, including Acod1. In macrophages, Acod1 was essential for restricting C. burnetii replication, while other antimicrobial pathways were dispensable. Intratracheal or intraperitoneal infection of Acod1-/- mice caused increased C. burnetii burden, weight loss and stronger inflammatory gene expression. Exogenously added itaconate restored pathogen control in Acod1-/- mouse macrophages and blocked replication in human macrophages. In axenic cultures, itaconate directly inhibited growth of C. burnetii. Finally, treatment of infected Acod1-/- mice with itaconate efficiently reduced the tissue pathogen load. Thus, ACOD1-derived itaconate is a key factor in the macrophage-mediated defense against C. burnetii and may be exploited for novel therapeutic approaches in chronic Q fever

    Coronin-1A Links Cytoskeleton Dynamics to TCRαβ-Induced Cell Signaling

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    Actin polymerization plays a critical role in activated T lymphocytes both in regulating T cell receptor (TCR)-induced immunological synapse (IS) formation and signaling. Using gene targeting, we demonstrate that the hematopoietic specific, actin- and Arp2/3 complex-binding protein coronin-1A contributes to both processes. Coronin-1A-deficient mice specifically showed alterations in terminal development and the survival of αβT cells, together with defects in cell activation and cytokine production following TCR triggering. The mutant T cells further displayed excessive accumulation yet reduced dynamics of F-actin and the WASP-Arp2/3 machinery at the IS, correlating with extended cell-cell contact. Cell signaling was also affected with the basal activation of the stress kinases sAPK/JNK1/2; and deficits in TCR-induced Ca2+ influx and phosphorylation and degradation of the inhibitor of NF-κB (IκB). Coronin-1A therefore links cytoskeleton plasticity with the functioning of discrete TCR signaling components. This function may be required to adjust TCR responses to selecting ligands accounting in part for the homeostasis defect that impacts αβT cells in coronin-1A deficient mice, with the exclusion of other lympho/hematopoietic lineages

    Elektrochemicka redukce dusicnanovych iontu.

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    The present work deals with a method of NO_3 removal from drinking water based on a combination of ion exchange and electrochemical treatment of the regenerant solution. The main part focuses on the electrochemical reduction of NO_3 ions in a concentrated NaHCO_3 solution.Summary in EnglishAvailable from STL, Prague, CZ / NTK - National Technical LibrarySIGLECZCzech Republi

    Bovine blood derived macrophages are unable to control replication under hypoxic conditions

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    Background: Coxiella burnetii is a zoonotic pathogen, infecting humans, livestock, pets, birds and ticks. Domestic ruminants such as cattle, sheep, and goats are the main reservoir and major cause of human infection. Infected ruminants are usually asymptomatic, while in humans infection can cause significant disease. Human and bovine macrophages differ in their permissiveness for C. burnetii strains from different host species and of various genotypes and their subsequent host cell response, but the underlying mechanism(s) at the cellular level are unknown. Methods: C. burnetii infected primary human and bovine macrophages under normoxic and hypoxic conditions were analyzed for (i) bacterial replication by CFU counts and immunofluorescence; (ii) immune regulators by westernblot and qRT-PCR; cytokines by ELISA; and metabolites by gas chromatography-mass spectrometry (GC-MS). Results: Here, we confirmed that peripheral blood-derived human macrophages prevent C. burnetii replication under oxygen-limiting conditions. In contrast, oxygen content had no influence on C. burnetii replication in bovine peripheral blood-derived macrophages. In hypoxic infected bovine macrophages, STAT3 is activated, even though HIF1α is stabilized, which otherwise prevents STAT3 activation in human macrophages. In addition, the TNFα mRNA level is higher in hypoxic than normoxic human macrophages, which correlates with increased secretion of TNFα and control of C. burnetii replication. In contrast, oxygen limitation does not impact TNFα mRNA levels in C. burnetii-infected bovine macrophages and secretion of TNFα is blocked. As TNFα is also involved in the control of C. burnetii replication in bovine macrophages, this cytokine is important for cell autonomous control and its absence is partially responsible for the ability of C. burnetii to replicate in hypoxic bovine macrophages. Further unveiling the molecular basis of macrophage-mediated control of C. burnetii replication might be the first step towards the development of host directed intervention measures to mitigate the health burden of this zoonotic agent

    CaeA behaves like apoptotic inducers Ybh3 and human Bax in yeast.

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    <p>(A) CaeA expression induces oxidative stress. The graph represents the enhancement of positive YPH499 yeast cells expressing the indicated <i>C</i>. <i>burnetii</i> effector proteins from the corresponding <i>GAL1</i>-driven expression vectors, as determined by flow cytometry, expressed as times-fold over control cells transformed with the empty vector. The fluorochromes propidium iodide (white bars), rhodamine 123 (grey bars) and dihydroethidium (black bars), respectively monitor cell death, altered mitochondrial potential and intracellular ROS accumulation in the yeast populations. <i>GAL1</i>-driven <i>YBH3</i> overexpression on the same strain was used as a positive control. Data correspond to a representative experiment of triplicate replicas. (B) Flow cytometry histograms of representative experiments as in (A) on YPH499 transformants expressing the indicated proteins from the corresponding <i>GAL1</i>-driven expression vectors. The gating threshold is marked by a vertical line. The percentage of cells in the population beyond the gating threshold is shown for each histogram.</p

    Expression of <i>C</i>. <i>burnetii</i> T4BSS-translocated effectors in mammalian HeLa cells.

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    <p>(A) Immunofluorescence confocal images show the representative subcellular localization of the indicated GFP- or HA-tagged <i>C</i>. <i>burnetii</i> effector proteins (green) after ectopic expression in HeLa cells. For contrast, DAPI, Mitotracker, or anti-tubulin, anti-calnexin and anti-LAMP-1 antibodies, were used as marked. Scale bars represent 10mm. (B) Expression of the indicated GFP-tagged <i>C</i>. <i>burnetii</i> effectors was analyzed by immunoblot analysis. Protein extracts from transfected HeLa cells were separated by SDS-PAGE, transferred to PVDF membrane and probed with anti-GFP antibody and anti-tubulin as loading control. A representative immunoblot is shown out of three independent experiments with similar results.</p
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