Nuclear Receptors: 364 (Methods in Enzymology)
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Nuclear receptors NRs are ligand-inducible transcription factors that regulate diverse functions as a superfamily of crucial medical significance. Because of their involvement in many physiological and pathological processes, the development of methods to infer the different NR subfamilies has become an important goal in biomedical research. In this paper we introduce a sequence-based computational approach-Support Vector Machine to classify the 19 subfamilies of NRs. We use 4-tuple residue composition instead of dipeptide composition to encode the NR sequences.
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Methods for Detecting Domain Interactions in Nuclear Receptors
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This process is experimental and the keywords may be updated as the learning algorithm improves. S9, B and E. Along these lines, reduced regulatory cKO donor T cell numbers were found in spleen fig. S9F and mesenteric lymph nodes fig. S9G of recipient mice, suggesting that the disturbed protective functions of cKO T cells resulted from reduced in vivo expansion. S9H , there was no difference in Il1b expression fig. Representative pictures are shown. E:T, effector:target. An important effector function of virus-specific T cells is the killing of virus-infected cells and, thereby, virus clearance.
Accordingly, a massive reduction of virus particles was seen in the spleen, liver, small intestine, and serum of LCMV-infected control mice at day 10 after infection Fig. The lack of viral clearance was also obvious when detecting LCMV-infected cells in liver tissue using immunofluorescence. While control mice almost completely cleared the virus from liver tissue at day 10 after infection, cKO mice still contained high numbers of virus-producing hepatocytes Fig. As LCMV clearance is critically dependent on perforin-mediated cytotoxicity 27 , we analyzed Prf1 and Gzmb mRNA expression in the spleen of control and cKO mice, revealing an infection-induced increase in both mouse lines Fig.
This indicates that impaired expression of cytotoxic effector molecules is not responsible for the inability of cKO T cells to clear LCMV. The expression and role of LRH-1 has been well described in various endodermal tissues, while considerably less is known about its expression and role in other tissues, specifically the hematopoietic system. The first indirect report investigating the LRH-1 expression in pancreatic tumors, where they accidentally observed LRH-1—positive tumor-infiltrating leukocytes, came from Benod and colleagues However, besides these few studies, the expression and role of LRH-1 in the hematopoietic system is currently unexplored.
One of the underlying reasons seems to be that steady-state levels are extremely low; thus, LRH-1 expression and function in immune cells had been largely ignored. We observed that expression levels in unstimulated thymocytes and mature T cells are about to times lower than those observed in the intestine and the liver fig.
S1, A to D. However, LRH-1 expression in T cells appears to be rather dynamic. Activation of T cells by mitogenic stimuli strongly induces LRH-1 promoter activity, expression, and function Fig. LRH-1 induction by mitogenic stimuli correlates well with the up-regulation of cell cycle—regulating genes, i. This is in line with the established role of LRH-1 in the transcriptional control of these cyclin genes and the regulation of proliferation of intestinal epithelial stem cells and tumor cells Supporting a suggested role of LRH-1 in cell cycle progression, we observed a profound inhibition of activation-induced proliferation when LRH-1 was either genetically deleted or pharmacologically inhibited Fig.
Reduced proliferation was observed not only in vitro but also during homeostatic expansion in vivo fig. S7, A and B and the pathogenesis of experimental colitis Fig. This proliferation deficiency is unlikely a consequence of improper T cell activation, as LRH-1—deficient T cells readily up-regulated early activation markers Fig. Given the previously described role of LRH-1 in the transcriptional regulation of cell cycle—related genes, e.
As the CD4 promoter becomes activated already at the double-positive stage during thymic maturation, the LRH-1 gene is already deleted at this stage of T cell development fig. S4, A to D. S4, F to H. Thus, costimulatory signals distinct from B7-CD28 interaction may provide compensatory signals, enabling LRH-1—independent proliferation. S9, A to G.
A somewhat different situation was observed in the LCMV infection model. In marked contrast, LRH-1—deficient cytotoxic T cells failed to control viral spreading. While in control animals LCMV titers were reduced at day 10 after infection by several logs and barely detectable, viral titers in mice with defective LRH-1 expression remained at a high level Fig. Given that virus-specific T cells appeared to expand normally, this difference is difficult to reconcile.
However, gene expression analysis revealed that granzyme B and perforin are, at least on an mRNA level, induced upon viral infection and equally expressed in control and cKO mice Fig. While we have recently identified the cytotoxic effector molecule Fas ligand as a target gene of LRH-1 16 , it is unlikely that impaired Fas ligand expression could account for inefficient viral clearance, as perforin 27 but not Fas ligand 29 is critical for controlling LCMV spreading.
Furthermore, we confirmed that T cells from both virus-infected control and cKO mice killed LCMV peptide—presenting target cells equally well, suggesting normal cytotoxic effector functions in both mouse lines. Another possibility is that cKO T cells are more readily exhausted and thereby cannot control viral expansion.
An interesting aspect of our study is its potential translational application. Although no endogenous ligands for LRH-1 have been identified yet, a number of pharmacological inhibitors with high specificity have been developed.
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The fact that transient inhibition of LRH-1 with two inhibitors that act in different mechanisms [3d2 Fig. The use of LRH-1 inhibitors in relatively low doses could permit the efficient inhibition of T cell mediated immunopathologies while not affecting vital functions of other tissues. While studying the role of LRH-1 in the transcriptional control of Fas ligand, we were already able to provide proof of principle for this concept. Injection of the lectin ConA into mice leads to rapid T cell activation and Fas ligand—dependent damage of the liver, which was strongly reduced after administration of 3d2 The liver seems to tolerate 3d2 relatively well, as only a minimal increase in 3d2-induced serum transaminases was observed.
In summary, we have described a novel role for LRH-1 in T cell development, proliferation, and effector functions.
Animals were housed in individually ventilated cages at the central animal facility of the University of Konstanz. The Cre-reporter mTmG transgenic mice were provided by U. Throughout all experiments, cKO animals were compared to their corresponding floxed littermate controls. Basler, University of Konstanz mice were bred on a Ly5. All experiments were performed in well round-bottom plates Greiner in technical triplicates with , cells per well in complete culture medium unless otherwise stated.
Cell culture media and supplements were purchased from Sigma-Aldrich, if not otherwise indicated.here
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Cells have been routinely tested for the absence of mycoplasma. The 1. Luciferase assays were carried out as described previously Single-cell suspensions from the spleen, thymus, and lymph nodes were prepared by manual disruption between frosted glass slides. ConA-activated mouse splenocytes were prepared as a source of T cell blasts and cultured as described Primers were designed to span exon-exon junctions.
Basler University of Konstanz. Data were analyzed using FlowJo software TreeStar. For immunohistochemistry, organs were immediately embedded in O. Tissue-Tek cryosection medium and shock frozen on dry ice. Fiji software 37 was used for image processing. Isolated T cells were cultured in the presence or absence of dexamethasone Sigma-Aldrich , etoposide Enzo Life Sciences , staurosporine Sigma-Aldrich , plate bound anti-CD3 antibody, or solvent control dimethyl sulfoxide or ethanol for 8 hours.
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In some experiments, the pharmacological LRH-1 inhibitor 3d2 20 , control substance cd7 20 both synthesized by ChemBridge Corporation , or SR 21 were added 30 min before T cell activation. For 3 H-thymidine incorporation assays, cells were pulsed with 0. For cell cycle profile analysis, wild-type splenocytes were cultured for indicated time points before nuclear DNA content was analyzed as described earlier After 14 days, the spleen and serum were isolated for further analysis of antibody production by ELISA and proliferation of antigen-specific T cells by in vitro restimulation and 3 H-thymidine incorporation.