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There is no known treatment for fatty liver,a ubiquitous cause of chronic liver disease. However,because it is associated with hyperinsulinemia and insulin-resistance,insulin-sensitizing agents might be beneficial. To evaluate this possibility,insulin-resistant ob/ob mice with fatty livers were treated with metformin,an agent that improves hepatic insulin-resistance. Metformin improved fatty liver disease,reversing hepatomegaly,steatosis and aminotransferase abnormalities. The therapeutic mechanism likely involves inhibited hepatic expression of tumor necrosis factor (TNF) alpha and TNF-inducible factors that promote hepatic lipid accumulation and ATP depletion. These findings suggest a mechanism of action for metformin and identify novel therapeutic targets in insulin-resistant states. View Publication

The specificities of 28 commercially available compounds reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases have been examined against a large panel of protein kinases. The compounds KT 5720,Rottlerin and quercetin were found to inhibit many protein kinases,sometimes much more potently than their presumed targets,and conclusions drawn from their use in cell-based experiments are likely to be erroneous. Ro 318220 and related bisindoylmaleimides,as well as H89,HA1077 and Y 27632,were more selective inhibitors,but still inhibited two or more protein kinases with similar potency. LY 294002 was found to inhibit casein kinase-2 with similar potency to phosphoinositide (phosphatidylinositol) 3-kinase. The compounds with the most impressive selectivity profiles were KN62,PD 98059,U0126,PD 184352,rapamycin,wortmannin,SB 203580 and SB 202190. U0126 and PD 184352,like PD 98059,were found to block the mitogen-activated protein kinase (MAPK) cascade in cell-based assays by preventing the activation of MAPK kinase (MKK1),and not by inhibiting MKK1 activity directly. Apart from rapamycin and PD 184352,even the most selective inhibitors affected at least one additional protein kinase. Our results demonstrate that the specificities of protein kinase inhibitors cannot be assessed simply by studying their effect on kinases that are closely related in primary structure. We propose guidelines for the use of protein kinase inhibitors in cell-based assays. View Publication

Cyclic AMP (cAMP) and cyclic GMP (cGMP) are key second messengers involved in a multitude of cellular events. From the wealth of synthetic analogs of cAMP and cGMP,only a few have been explored with regard to their therapeutic potential. Some of the first-generation cyclic nucleotide analogs were promising enough to be tested as drugs,for instance N(6),O(2)'-dibutyryl-cAMP and 8-chloro-cAMP (currently in clinical Phase II trials as an anticancer agent). Moreover,8-bromo and dibutyryl analogs of cAMP and cGMP have become standard tools for investigations of biochemical and physiological signal transduction pathways. The discovery of the Rp-diastereomers of adenosine 3',5'-cyclic monophosphorothioate and guanosine 3',5'-cyclic monophosphorothioate as competitive inhibitors of cAMP- and cGMP-dependent protein kinases,as well as subsequent development of related analogs,has proven very useful for studying the molecular basis of signal transduction. These analogs exhibit a higher membrane permeability,increased resistance against degradation,and improved target specificity. Furthermore,better understanding of signaling pathways and ligand/protein interactions has led to new therapeutic strategies. For instance,Rp-8-bromo-adenosine 3',5'-cyclic monophosphorothioate is employed against diseases of the immune system. This review will focus mainly on recent developments in cyclic nucleotide-related biochemical and pharmacological research,but also highlights some historical findings in the field. 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

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

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

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

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 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

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

Trichostatin A (TSA) and trapoxin (TPX) are potent inhibitors of histone deacetylases (HDACs). TSA is proposed to block the catalytic reaction by chelating a zinc ion in the active-site pocket through its hydroxamic acid group. On the other hand,the epoxyketone is suggested to be the functional group of TPX capable of alkylating the enzyme. We synthesized a novel TPX analogue containing a hydroxamic acid instead of the epoxyketone. The hybrid compound cyclic hydroxamic acid-containing peptide (CHAP) 1 inhibited HDAC1 at low nanomolar concentrations. The HDAC1 inhibition by CHAP1 was reversible as it was by TSA,in contrast to the irreversible inhibition by TPX. CHAP with an aliphatic chain length of five,which corresponded to that of acetylated lysine,was stronger than those with other lengths. These results suggest that TPX is a substrate mimic and that the replacement of the epoxyketone with the hydroxamic acid converted TPX to an inhibitor chelating the zinc like TSA. Interestingly,HDAC6,but not HDAC1 or HDAC4,was resistant to TPX and CHAP1,whereas TSA inhibited these HDACs to a similar extent. HDAC6 inhibition by TPX at a high concentration was reversible,probably because HDAC6 is not alkylated by TPX. We further synthesized the counterparts of all known naturally occurring cyclic tetrapeptides containing the epoxyketone. HDAC1 was highly sensitive to all these CHAPs much more than HDAC6,indicating that the structure of the cyclic tetrapeptide framework affects the target enzyme specificity. These results suggest that CHAP is a unique lead to develop isoform-specific HDAC inhibitors. View Publication