参考文献

This study analyzes the gene repertoire coding for antibodies to an evolutionary novel immunogenic carbohydrate antigen in mice. The alpha-gal epitope (Gal alpha 1-3Gal beta 1-4GlcNAc-R) is an autoantigen,abundantly expressed in wild type mice,but absent in alpha 1,3galactosyltransferase knock-out (KO) mice,where it can induce the production of the anti-Gal antibody. Hybridoma clones secreting anti-Gal were isolated from different mice and their immunoglobulin genes were analyzed. All anti-Gal clones were found to be encoded by the heavy chain gene VH22.1 and light chain gene VK5.1. Moreover,one 'forbidden' anti-Gal clone,produced in a wild type mouse,was also encoded by VH 22.1 and VK 5.1. The genes coding for the different anti-Gal clones were found to contain somatic mutations and different CDR3 domains. These data imply that a highly restricted gene usage combined with junctional diversity and somatic mutations can generate new antibodies that have not been produced in the course of the evolution of a species. View Publication

Cytokines are important in the regulation of haematopoiesis and immune responses,and can influence lymphocyte development. Here we have identified a class I cytokine receptor that is selectively expressed in lymphoid tissues and is capable of signal transduction. The full-length receptor was expressed in BaF3 cells,which created a functional assay for ligand detection and cloning. Conditioned media from activated human CD3+ T cells supported proliferation of the assay cell line. We constructed a complementary DNA expression library from activated human CD3+ T cells,and identified a cytokine with a four-helix-bundle structure using functional cloning. This cytokine is most closely related to IL2 and IL15,and has been designated IL21 with the receptor designated IL21 R. In vitro assays suggest that IL21 has a role in the proliferation and maturation of natural killer (NK) cell populations from bone marrow,in the proliferation of mature B-cell populations co-stimulated with anti-CD40,and in the proliferation of T cells co-stimulated with anti-CD3. View Publication

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The imidazoquinoline R-848,originally identified as a highly effective antiviral agent,has recently been shown to be capable of potent B lymphocyte activation. The B cell-activating properties of R-848 are strikingly similar to the effects of the CD40 ligand CD154. The present study demonstrates that this similarity extends to the intracellular signaling pathways triggered by the compound,although both overlapping and distinct mechanisms of signaling were seen. Like CD40 ligation,R-848 stimulated activation of the stress-activated protein kinases c-Jun kinase and p38 and activated the NF-kappaB family of transcription factors. Both R-848- and CD40-mediated B cell differentiation were dependent upon NF-kappaB activation,although the relative importance of individual NF-kappaB family members appeared to differ between R-848- and CD40-mediated signals. Both signals were partially dependent upon induction of TNF-alpha and IL-6,and the cytoplasmic adaptor molecule TNF receptor-associated factor 2 is involved in both R-848- and CD40-mediated differentiation. View Publication

BACKGROUND Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase,the activity of which is inhibited by a variety of extracellular stimuli including insulin,growth factors,cell specification factors and cell adhesion. Consequently,inhibition of GSK-3 activity has been proposed to play a role in the regulation of numerous signalling pathways that elicit pleiotropic cellular responses. This report describes the identification and characterisation of potent and selective small molecule inhibitors of GSK-3. RESULTS SB-216763 and SB-415286 are structurally distinct maleimides that inhibit GSK-3alpha in vitro,with K(i)s of 9 nM and 31 nM respectively,in an ATP competitive manner. These compounds inhibited GSK-3beta with similar potency. However,neither compound significantly inhibited any member of a panel of 24 other protein kinases. Furthermore,treatment of cells with either compound stimulated responses characteristic of extracellular stimuli that are known to inhibit GSK-3 activity. Thus,SB-216763 and SB-415286 stimulated glycogen synthesis in human liver cells and induced expression of a beta-catenin-LEF/TCF regulated reporter gene in HEK293 cells. In both cases,compound treatment was demonstrated to inhibit cellular GSK-3 activity as assessed by activation of glycogen synthase,which is a direct target of this kinase. CONCLUSIONS SB-216763 and SB-415286 are novel,potent and selective cell permeable inhibitors of GSK-3. Therefore,these compounds represent valuable pharmacological tools with which the role of GSK-3 in cellular signalling can be further elucidated. Furthermore,development of similar compounds may be of use therapeutically in disease states associated with elevated GSK-3 activity such as non-insulin dependent diabetes mellitus and neurodegenerative disease. View Publication

The application of multi-parameter flow cytometry for the assessment of T-cell and cytokine functioning has been used by several groups for studying human and mouse samples,although little has been reported for the rat. Here we report the optimisation of immunofluorescent staining for cell surface and intracellular antigens using three-colour flow cytometric analysis to measure the frequency of rat CD3(+)4(+) T-cells that produce IFN-gamma,IL-4 and IL-10. In vitro stimulation of IFN-gamma production required incubation of splenocytes with PMA and ionomycin in the presence of the protein transport inhibitor brefeldin A for 6 h. Three stimulation protocols for IL-4 and IL-10 production were evaluated. In vitro priming of splenic T-cells with antibodies against CD3 and CD28 and recombinant cytokines (IL-2 and IL-4) for 5 days followed by restimulation with PMA and ionomycin was required to stimulate cells to produce either IL-4 or IL-10. Brefeldin A was found to be a more suitable protein transport inhibitor than monensin. This method will be useful for analysing the nature of individual rat cytokine-producing cells in a variety of experimental model systems. View Publication

The paired box transcription factor Pax7 was isolated by representational difference analysis as a gene specifically expressed in cultured satellite cell-derived myoblasts. In situ hybridization revealed that Pax7 was also expressed in satellite cells residing in adult muscle. Cell culture and electron microscopic analysis revealed a complete absence of satellite cells in Pax7(-/-) skeletal muscle. Surprisingly,fluorescence-activated cell sorting analysis indicated that the proportion of muscle-derived stem cells was unaffected. Importantly,stem cells from Pax7(-/-) muscle displayed almost a 10-fold increase in their ability to form hematopoietic colonies. These results demonstrate that satellite cells and muscle-derived stem cells represent distinct cell populations. Together these studies suggest that induction of Pax7 in muscle-derived stem cells induces satellite cell specification by restricting alternate developmental programs. View Publication

There is evidence that the total cellular content of placental cord blood (PCB) grafts is related to the speed of engraftment,though the total nucleated cell (TNC) dose is not a precise predictor of the time of neutrophil or platelet engraftment. It is important to understand the reasons for the quantitative association and to improve the criteria for selecting PCB grafts by using indices more precisely predictive of engraftment. The posttransplant course of 204 patients who received grafts evaluated for hematopoietic colony-forming cell (CFC) content among 562 patients reported previously were analyzed using univariate and multivariate life-table techniques to determine whether CFC doses predicted hematopoietic engraftment speed and risk for transplant-related events more accurately than the TNC dose. Actuarial times to neutrophil and platelet engraftment were shown to correlate with the cell dose,whether estimated as TNC or CFC per kilogram of recipient's weight. CFC association with the day of recovery of 500 neutrophils/microL,measured as the coefficient of correlation,was stronger than that of the TNC (R = -0.46 and -0.413,respectively). In multivariate tests of speed of platelet and neutrophil engraftment and of probability of posttransplantation events,the inclusion of CFC in the model displaced the significance of the high relative risks associated with TNC. The CFC content of PCB units is associated more rigorously with the major covariates of posttransplantation survival than is the TNC and is,therefore,a better index of the hematopoietic content of PCB grafts. (Blood. 2000;96:2717-2722) View Publication

Incubation of skeletal muscle with 5-aminoimidazole-4carboxamide ribonucleoside (AICAR),a compound that activates 5'-AMP-activated protein kinase (AMPK),has been demonstrated to stimulate glucose transport and GLUT4 translocation to the plasma membrane. In this study,we characterized the AMPK cascade in 3T3-L1 adipocytes and the response of glucose transport to incubation with AICAR. Both isoforms of the catalytic alpha-subunit of AMPK are expressed in 3T3-L1 adipocytes,in which AICAR stimulated AMPK activity in a time- and dose-dependent fashion. AICAR stimulated 2-deoxy-D-glucose transport twofold and reduced insulin-stimulated uptake to 62% of the control transport rate dose-dependently,closely correlating with the activation of AMPK. AICAR also inhibited insulin-stimulated GLUT4 translocation,assessed using the plasma membrane lawn assay. The effects of AICAR on insulin-stimulated glucose transport are not mediated by either adenosine receptors or nitric oxide synthase and are mediated downstream of phosphatidylinositol 3'-kinase stimulation. We propose that in contrast to skeletal muscle,in which AMPK stimulation promotes glucose transport to provide ATP as a fuel,AMPK stimulation inhibits insulin-stimulated glucose transport in adipocytes,inhibiting triacylglycerol synthesis,to conserve ATP under conditions of cellular stress. Investigation of the mode of action of AICAR and AMPK may,therefore,give insight into the mechanism of insulin action. View Publication

The bis-indole indirubin is an active ingredient of Danggui Longhui Wan,a traditional Chinese medicine recipe used in the treatment of chronic diseases such as leukemias. The antitumoral properties of indirubin appear to correlate with their antimitotic effects. Indirubins were recently described as potent (IC(50): 50-100 nm) inhibitors of cyclin-dependent kinases (CDKs). We report here that indirubins are also powerful inhibitors (IC(50): 5-50 nm) of an evolutionarily related kinase,glycogen synthase kinase-3beta (GSK-3 beta). Testing of a series of indoles and bis-indoles against GSK-3 beta,CDK1/cyclin B,and CDK5/p25 shows that only indirubins inhibit these kinases. The structure-activity relationship study also suggests that indirubins bind to GSK-3 beta's ATP binding pocket in a way similar to their binding to CDKs,the details of which were recently revealed by crystallographic analysis. GSK-3 beta,along with CDK5,is responsible for most of the abnormal hyperphosphorylation of the microtubule-binding protein tau observed in Alzheimer's disease. Indirubin-3'-monoxime inhibits tau phosphorylation in vitro and in vivo at Alzheimer's disease-specific sites. Indirubins may thus have important implications in the study and treatment of neurodegenerative disorders. Indirubin-3'-monoxime also inhibits the in vivo phosphorylation of DARPP-32 by CDK5 on Thr-75,thereby mimicking one of the effects of dopamine in the striatum. Finally,we show that many,but not all,reported CDK inhibitors are powerful inhibitors of GSK-3 beta. To which extent these GSK-3 beta effects of CDK inhibitors actually contribute to their antimitotic and antitumoral properties remains to be determined. Indirubins constitute the first family of low nanomolar inhibitors of GSK-3 beta to be described. View Publication