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- Forsberg, Maria, et al.
(författare)
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Differential effects of invasion by and phagocytosis of Salmonella typhimurium on apoptosis in human macrophages : potential role of Rho–GTPases and Akt
- 2003
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Ingår i: Journal of Leukocyte Biology. - : Oxford University Press (OUP). - 0741-5400 .- 1938-3673. ; 74:4, s. 620-629
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Tidskriftsartikel (refereegranskat)abstract
- In addition to direct activation of caspase-1 and induction of apoptosis by SipB, invasive Salmonella stimulates multiple signaling pathways that are key regulators of host cell survival. Nevertheless, little is known about the relative contributions of these pathways to Salmonella-mediated death of macrophages. We studied human monocytic U937 cells and found that apoptosis was induced by invading wild-type Salmonella typhimurium but not by phagocytosed, serum-opsonized, noninvasive Salmonella mutants. Pretreating U937 cells with inhibitors of tyrosine kinases or phosphatidylinositol-3 kinase (PI-3K) completely blocked phagocytosis of opsonized Salmonella mutants but did not affect invasion by wild-type Salmonella or the apoptosis caused by invasion. However, pretreatment with GGTI-298, a geranylgeranyltransferase-1 inhibitor that prevents prenylation of Cdc42 and Rac1, suppressed Salmonella-induced apoptosis by ∼70%. Transduction of Tat fusion constructs containing dominant-negative Cdc42 or Rac1 significantly inhibited Salmonella-induced cell death, indicating that the cytotoxicity of Salmonella requires activation of Cdc42 and Rac. In contrast to phagocytosis of opsonized bacteria, invasion by S. typhimurium stimulated Cdc42 and Rac1, regardless of the activities of tyrosine- or PI-3K. Moreover, Salmonella infection activated Akt protein in a tyrosine-kinase or PI-3K-dependent manner, and a reduced expression of Akt by antisense transfection rendered the cells more sensitive to apoptosis induced by opsonized Salmonella. These results indicate that direct activation of Cdc42 and Rac1 by invasive Salmonella is a prerequisite of Salmonella-mediated death of U937 cells, whereas the simultaneous activation of Akt by tyrosine kinase and PI-3K during receptor-mediated phagocytosis protects cells from apoptosis.
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| 3. |
- Gustafsdottir, Sigrun M, et al.
(författare)
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Use of Proximity Ligation to Screen for Inhibitors of Interactions between Vascular Endothelial Growth Factor A and Its Receptors
- 2008
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 54:7, s. 1218-1225
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Improved methods are required to screen drug candidates for their influences on protein interactions. There is also a compelling need for miniaturization of screening assays, with attendant reductions in reagent consumption and assay costs. METHODS: We used sensitive, miniaturized proximity ligation assays (PLAs) to monitor binding of vascular endothelial growth factor A (VEGF-A) to 2 of its receptors, VEGFR-1 and VEGFR-2. We measured the effects of proteins and low molecular weight compounds capable of disrupting these interactions and compared the results with those obtained by immunoblot analysis. We analyzed 6 different inhibitors: a DNA aptamer, a mixed DNA/RNA aptamer, a monoclonal VEGF-A neutralizing antibody, a monoclonal antibody directed against VEGFR-2, a recombinant competitive protein, and a low molecular weight synthetic molecule. RESULTS: The PLAs were successful for monitoring the formation and inhibition of VEGF-A-receptor complexes, and the results correlated well with those obtained by measuring receptor phosphorylation. The total PLA time is just 3 hours, with minimal manual work and reagent additions. The method allows evaluation of the apparent affinity [half-maximal inhibitory concentration (IC(50))] from a dose-response curve. CONCLUSIONS: The PLA may offer significant advantages over conventional methods for screening the interactions of ligands with their receptors. The assay may prove useful for parallel analyses of large numbers of samples in the screening of inhibitor libraries for promising agents. The technique provides dose-response curves, allowing IC(50) values to be calculated.
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