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891.
Bertrand L Parent S Caron M Legault M Joly E Angers S Bouvier M Brown M Houle B Ménard L 《Journal of receptor and signal transduction research》2002,22(1-4):533-541
In BRET2 (Bioluminescence Resonance Energy Transfer), a Renilla luciferase (RLuc) is used as the donor protein, while a Green Fluorescent Protein (GFP2) is used as the acceptor protein. In the presence of the cell permeable substrate DeepBlueC, RLuc emits blue light at 395 nm. If the GFP2 is brought into close proximity to RLuc via a specific biomolecular interaction, the GFP2 will absorb the blue light energy and reemit green light at 510nm. BRET2 signals are therefore easily determined by measuring the ratio of green over blue light (510/395nm) using appropriate dual channel luminometry instruments (e.g., Fusion Universal Microplate Analyzer, Packard BioScience). Since no light source is required for BRET2 assays, the technology does not suffer from high fluorescent background or photobleaching, the common problems associated with standard FRET-based assays. Using BRET2, we developed a generic G Protein-Coupled Receptor (GPCR) assay based on the observation that activation of the majority of GPCRs by agonists leads to the interaction of beta-arrestin (a protein that is involved in receptor desensitization and sequestration) with the receptor. We established a cell line stably expressing the GFP2:beta-arrestin 2 fusion protein, and showed that it can be used to monitor the activation of various transiently expressed GPCRs, in BRET2/arrestin assays. In addition, using the HEK 293/GFP2:beta-arrestin 2 cell line as a recipient, we generated a double-stable line co-expressing the vasopressin 2 receptor (V2R) fused to RLuc (V2R:RLuc) and used it for the pharmacological characterization of compounds in BRET2/arrestin assays. This approach yields genuine pharmacology and supports the BRET2/arrestin assay as a tool that can be used with recombinant cell lines to characterize ligand-GPCR interactions which can be applied to ligand identification for orphan receptors. 相似文献
892.
Odile Petsaris Fabien Miszczak Mireille Gicquel-Bruneau Agnès Perrin-Guyomard Florence Humbert Pascal Sanders Roland Leclercq 《Applied microbiology》2005,71(5):2796-2799
Nineteen E. faecium strains isolated from chicken caecum samples, collected in slaughterhouses and highly resistant to vancomycin or gentamicin, were coresistant to erythromycin, and/or tetracyclines, and/or streptogramins, and/or avilamycin. Multiple antibiotic resistance was related to the presence in various combinations of aac(6′)-aph(2"), erm(B), emtA, mef(A), tet(L), tet(M), and vanA genes. 相似文献
893.
van de Poll ML van Rotterdam W Gadellaa MM Jacobs-Oomen S van Zoelen EJ 《Experimental cell research》2005,304(2):630-641
EGF activates the ErbB1 receptor, but there appears only a limited correlation between its receptor binding affinity and mitogenic activity. This is indicated by our present observation that in cells with high ErbB1 expression, including SUM102 breast tumor cells, low affinity EGF/Notch chimeras have similarly high mitogenic activity as EGF, in spite of the fact that EGF is superior in inducing receptor tyrosine phosphorylation and p42/p44 MAP-kinase activity. However, as a result of receptor-mediated internalisation high-affinity ligands such as EGF are depleted much more rapidly from the extracellular medium than low-affinity EGF/Notch chimeras. As a consequence, the mitogenic activity of EGF on ErbB1 overexpressing cells is limited by substantial degradation of internalised ligand in the period before cells enter S-phase, a phenomenon that is not observed for low affinity mutant ligands. The mitogenic activity of EGF on ErbB1 overexpressing cells does therefore not only depend on the applied concentration but also on the total amount of ligand added, and is strongly underestimated when tested in a limited assay volume. No such dependence on the incubation volume was observed for EGF activity on cells with low ErbB1 expression levels and on cells for which EGF is growth inhibitory. 相似文献
894.
Interactions of TolB with the translocation domain of colicin E9 require an extended TolB box 下载免费PDF全文
Hands SL Holland LE Vankemmelbeke M Fraser L Macdonald CJ Moore GR James R Penfold CN 《Journal of bacteriology》2005,187(19):6733-6741
The mechanism by which enzymatic E colicins such as colicin E3 (ColE3) and ColE9 cross the outer membrane, periplasm, and cytoplasmic membrane to reach the cytoplasm and thus kill Escherichia coli cells is unique in prokaryotic biology but is poorly understood. This requires an interaction between TolB in the periplasm and three essential residues, D35, S37, and W39, of a pentapeptide sequence called the TolB box located in the N-terminal translocation domain of the enzymatic E colicins. Here we used site-directed mutagenesis to demonstrate that the TolB box sequence in ColE9 is actually larger than the pentapeptide and extends from residues 34 to 46. The affinity of the TolB box mutants for TolB was determined by surface plasmon resonance to confirm that the loss of biological activity in all except one (N44A) of the extended TolB box mutants correlates with a reduced affinity of binding to TolB. We used a PCR mutagenesis protocol to isolate residues that restored activity to the inactive ColE9 D35A, S37A, and W39A mutants. A serine residue at position 35, a threonine residue at position 37, and phenylalanine or tyrosine residues at position 39 restored biological activity of the mutant ColE9. The average area predicted to be buried upon folding (AABUF) was correlated with the activity of the variants at positions 35, 37, and 39 of the TolB box. All active variants had AABUF profiles that were similar to the wild-type residues at those positions and provided information on the size, stereochemistry, and potential folding pattern of the residues of the TolB Box. 相似文献
895.
Flexibility in the receptor-binding domain of the enzymatic colicin E9 is required for toxicity against Escherichia coli cells 下载免费PDF全文
Penfold CN Healy B Housden NG Boetzel R Vankemmelbeke M Moore GR Kleanthous C James R 《Journal of bacteriology》2004,186(14):4520-4527
The events that occur after the binding of the enzymatic E colicins to Escherichia coli BtuB receptors that lead to translocation of the cytotoxic domain into the periplasmic space and, ultimately, cell killing are poorly understood. It has been suggested that unfolding of the coiled-coil BtuB receptor binding domain of the E colicins may be an essential step that leads to the loss of immunity protein from the colicin and immunity protein complex and then triggers the events of translocation. We introduced pairs of cysteine mutations into the receptor binding domain of colicin E9 (ColE9) that resulted in the formation of a disulfide bond located near the middle or the top of the R domain. After dithiothreitol reduction, the ColE9 protein with the mutations L359C and F412C (ColE9 L359C-F412C) and the ColE9 protein with the mutations Y324C and L447C (ColE9 Y324C-L447C) were slightly less active than equivalent concentrations of ColE9. On oxidation with diamide, no significant biological activity was seen with the ColE9 L359C-F412C and the ColE9 Y324C-L447C mutant proteins; however diamide had no effect on the activity of ColE9. The presence of a disulfide bond was confirmed in both of the oxidized, mutant proteins by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The loss of biological activity of the disulfide-containing mutant proteins was not due to an indirect effect on the properties of the translocation or DNase domains of the mutant colicins. The data are consistent with a requirement for the flexibility of the coiled-coil R domain after binding to BtuB. 相似文献
896.
Toy-Miou-Leong M Cortes CL Beaudet A Rostène W Forgez P 《The Journal of biological chemistry》2004,279(13):12636-12646
Most G protein-coupled receptors are internalized after interaction with their respective ligand, a process that subsequently contributes to cell desensitization, receptor endocytosis, trafficking, and finally cell resensitization. Although cellular mechanisms leading to cell desensitization have been widely studied, those responsible for cell resensitization are still poorly understood. We examined here the traffic of the high affinity neurotensin receptor (NT1 receptor) following prolonged exposure to high agonist concentration. Fluorescence and confocal microscopy of Chinese hamster ovary, human neuroblastoma (CHP 212), and murine neuroblastoma (N1E-115) cells expressing green fluorescent protein-tagged NT1 receptor revealed that under prolonged treatment with saturating concentrations of neurotensin (NT) agonist, NT1 receptor and NT transiently accumulated in the perinuclear recycling compartment (PNRC). During this cellular event, cell surface receptors remained markedly depleted as detected by both confocal microscopy and (125)I-NT binding assays. In dividing cells, we observed that following prolonged NT agonist stimulation, NT1 receptors were removed from the PNRC, accumulated in dispersed vesicles inside the cytoplasm, and subsequently reappeared at the cell surface. This NT binding recovery allowed for constant cell sensitization and led to a chronic activation of mitogen-activated protein kinases p42 and p44. Under these conditions, the constant activation of NT1 receptor generates an oncogenic regulation. These observations support the potent role for neuropeptides, such as NT, in cancer progression. 相似文献
897.
Belnoue E Guettier C Kayibanda M Le Rond S Crain-Denoyelle AM Marchiol C Ziol M Fradelizi D Rénia L Viguier M 《Journal of immunology (Baltimore, Md. : 1950)》2004,173(8):4882-4888
Most types of cancer are difficult to eradicate, and some, like hepatocellular carcinoma, are almost always fatal. Among various interventions to improve the survival of patients with cancer, immunotherapy seems to hold some promises. However, it requires relevant animal models for preclinical development. In this study we report a new and relevant experimental model where liver tumors grow inside a nontumoral parenchyma of adult mice. This model is based on the intrasplenic injection in syngeneic recipient mice of hepatocytes from transgenic mice expressing SV40 large T oncogene specifically in the liver. Using this model where no apparent spontaneous cellular immune response was observed, immunization using a single injection of monoepitopic SV40 T Ag short peptide was sufficient to provoke liver tumor destruction, leading rapidly to complete remission. Tumor regression was associated with the induction of a long-lasting CD8+ T cell response, observed not only in the spleen but also, more importantly, in the tumoral liver. These results show the efficacy of peptide immunotherapy in the treatment of liver cancer. 相似文献
898.
Stenbak CR Ryu JH Leulier F Pili-Floury S Parquet C Hervé M Chaput C Boneca IG Lee WJ Lemaitre B Mengin-Lecreulx D 《Journal of immunology (Baltimore, Md. : 1950)》2004,173(12):7339-7348
Innate immune recognition of microbes is a complex process that can be influenced by both the host and the microbe. Drosophila uses two distinct immune signaling pathways, the Toll and immune deficiency (Imd) pathways, to respond to different classes of microbes. The Toll pathway is predominantly activated by Gram-positive bacteria and fungi, while the Imd pathway is primarily activated by Gram-negative bacteria. Recent work has suggested that this differential activation is achieved through peptidoglycan recognition protein (PGRP)-mediated recognition of specific forms of peptidoglycan (PG). In this study, we have further analyzed the specific PG molecular requirements for Imd activation through the pattern recognition receptor PGRP-LC in both cultured cell line and in flies. We found that two signatures of Gram-negative PG, the presence of diaminopimelic acid in the peptide bridge and a 1,6-anhydro form of N-acetylmuramic acid in the glycan chain, allow discrimination between Gram-negative and Gram-positive bacteria. Our results also point to a role for PG oligomerization in Imd activation, and we demonstrate that elements of both the sugar backbone and the peptide bridge of PG are required for optimum recognition. Altogether, these results indicate multiple requirements for efficient PG-mediated activation of the Imd pathway and demonstrate that PG is a complex immune elicitor. 相似文献
899.
Emergence of a CD4+CD28- granzyme B+, cytomegalovirus-specific T cell subset after recovery of primary cytomegalovirus infection 总被引:4,自引:0,他引:4
van Leeuwen EM Remmerswaal EB Vossen MT Rowshani AT Wertheim-van Dillen PM van Lier RA ten Berge IJ 《Journal of immunology (Baltimore, Md. : 1950)》2004,173(3):1834-1841
Cytotoxic CD4(+)CD28(-) T cells form a rare subset in human peripheral blood. The presence of CD4(+)CD28(-) cells has been associated with chronic viral infections, but how these particular cells are generated is unknown. In this study, we show that in primary CMV infections, CD4(+)CD28(-) T cells emerge just after cessation of the viral load, indicating that infection with CMV triggers the formation of CD4(+)CD28(-) T cells. In line with this, we found these cells only in CMV-infected persons. CD4(+)CD28(-) cells had an Ag-primed phenotype and expressed the cytolytic molecules granzyme B and perforin. Importantly, CD4(+)CD28(-) cells were to a large extent CMV-specific because proliferation was only induced by CMV-Ag, but not by recall Ags such as purified protein derivative or tetanus toxoid. CD4(+)CD28(-) cells only produced IFN-gamma after stimulation with CMV-Ag, whereas CD4(+)CD28(+) cells also produced IFN-gamma in response to varicella-zoster virus and purified protein derivative. Thus, CD4(+)CD28(-) T cells emerge as a consequence of CMV infection. 相似文献
900.