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Remarkably,apoptosis was induced by exposing peritoneal resident macrophages (PRM) of C3H mice,but not other strains of mice,to ionizing radiation. The molecular mechanism of this strain-specific apoptosis in PRM was studied. The apoptosis elicited in C3H mouse PRM 4 h after exposure was effectively blocked by proteasome inhibitors. Irradiation-induced disruption of mitochondrial transmembrane potential and the release of cytochrome c into the cytosol were also suppressed by a proteasome inhibitor but not by a caspase inhibitor. To determine whether the apoptosis occurred due to a depletion of antiapoptotic proteins,Bcl-2 family proteins were examined. Irradiation markedly decreased the level of Mcl-1,but not Bcl-2,Bcl-X(L),Bax,A1,or cIAP1. Mcl-1's depletion was suppressed by a proteasome inhibitor but not by a caspase inhibitor. The amount of Mcl-1 was well correlated with the rate of apoptosis in C3H mouse PRM exposed to irradiation and not affected by irradiation in radioresistant B6 mouse PRM. Irradiation increased rather than decreased the Mcl-1 mRNA expression in C3H mouse PRM. On the other hand,Mcl-1 protein synthesis was markedly suppressed by irradiation. Global protein synthesis was also suppressed by irradiation in C3H mouse PRM but not in B6 mouse PRM. The down-regulation of Mcl-1 expression with Mcl-1-specific small interfering RNA or antisense oligonucleotide significantly induced apoptosis in both C3H and B6 mouse PRM without irradiation. It was concluded that the apoptosis elicited in C3H mouse PRM by ionizing radiation was attributable to the depletion of Mcl-1 through radiation-induced arrest of global protein synthesis. View Publication

Neutropenia and neutrophil dysfunction are common in many diseases,although their etiology is often unclear. Previous views held that there was a single ER enzyme,glucose-6-phosphatase-alpha (G6Pase-alpha),whose activity--limited to the liver,kidney,and intestine--was solely responsible for the final stages of gluconeogenesis and glycogenolysis,in which glucose-6-phosphate (G6P) is hydrolyzed to glucose for release to the blood. Recently,we characterized a second G6Pase activity,that of G6Pase-beta (also known as G6PC),which is also capable of hydrolyzing G6P to glucose but is ubiquitously expressed and not implicated in interprandial blood glucose homeostasis. We now report that the absence of G6Pase-beta led to neutropenia; defects in neutrophil respiratory burst,chemotaxis,and calcium flux; and increased susceptibility to bacterial infection. Consistent with this,G6Pase-beta-deficient (G6pc3-/-) mice with experimental peritonitis exhibited increased expression of the glucose-regulated proteins upregulated during ER stress in their neutrophils and bone marrow,and the G6pc3-/- neutrophils exhibited an enhanced rate of apoptosis. Our results define a molecular pathway to neutropenia and neutrophil dysfunction of previously unknown etiology,providing a potential model for the treatment of these conditions. View Publication

Vascular remodeling,rather than vasoconstriction,is believed to account for high vascular resistance in severe pulmonary arterial hypertension (PAH). We have found previously that acute Rho kinase inhibition nearly normalizes PAH in chronically hypoxic rats that have no occlusive neointimal lesions. Here we examined whether Rho kinase-mediated vasoconstriction was also important in a rat model of severe occlusive PAH. Adult rats were exposed to chronic hypoxia ( approximately 10% O(2)) after subcutaneous injection of the vascular endothelial growth factor receptor inhibitor SUGEN 5416. Hemodynamic measurements were made in anesthetized rats after 2 weeks of hypoxia (early group) and 3 weeks of hypoxia plus 2 weeks of normoxia (late group). Both groups developed PAH,with greater severity in the late group. In the early group,intravenous fasudil was more effective than intravenous bradykinin,inhaled NO,or intravenous iloprost in reducing right ventricular systolic pressure. Despite more occlusive vascular lesions,fasudil also markedly reduced right ventricular systolic pressure in late-stage rats. Blood-perfused lungs from late-stage rats showed spontaneous vasoconstriction,which was reversed partially by the endothelin A receptor blocker BQ123 and completely by fasudil or Y-27632. Phosphorylation of MYPT1,a downstream target of Rho kinase,was increased in lungs from both groups of rats,and fasudil (intravenous) reversed the increased phosphorylation in the late group. Thus,in addition to structural occlusion,Rho kinase-mediated vasoconstriction is an important component of severe PAH in SUGEN 5416/hypoxia-exposed rats,and PAH can be significantly reduced in the setting of a severely remodeled lung circulation if an unconventional vasodilator is used. View Publication

Fasudil,a Rho-kinase inhibitor,may improve insulin signaling. However,its long-term effect on metabolic abnormalities and its preventive effect on diabetic nephropathy are still unknown. We assessed these effects of fasudil in insulin-resistant diabetic rats,comparing them with those of an angiotensin II receptor blocker,olmesartan. Male Otsuka Long-Evans Tokushima fatty (OLETF) and Long-Evans Tokushima Otsuka,non-diabetic control,rats at 15 weeks of age were used. OLETF rats were randomized to receive a low or a high dose of fasudil or olmesartan for 25 weeks. To examine the therapeutic effects after the development of diabetes,OLETF rats at 30 weeks of age were given fasudil for 10 weeks. Administration of high-dose fasudil completely suppressed the development of diabetes,obesity,and dyslipidemia and increased serum adiponectin levels in OLETF rats. High-dose olmesartan also decreased hemoglobin A1c and increased serum adiponectin. There was a significant correlation between hemoglobin A1c and serum adiponectin or free fatty acid levels. The treatment with high-dose fasudil ameliorated proteinuria,glomerulosclerosis,renal interstitial fibrosis,and macrophage infiltration in OLETF rats. Olmesartan,even at the low dose,suppressed renal complications. The treatment with fasudil after the development of diabetes improved the metabolic abnormalities in OLETF rats,but could not suppress the progression of nephropathy. We conclude that the long-term treatment with fasudil prevents the development of diabetes,at least in part,by improving adipocyte differentiation in insulin-resistant diabetic rats. Early use of fasudil may prevent diabetic nephropathy. View Publication

BACKGROUND AND OBJECTIVES: Chemokines are soluble mediators involved in angiogenesis,cellular growth control and immunomodulation. In the present study we investigated the effects of various chemokines on proliferation of acute myelogenous leukemia (AML) cells and constitutive chemokine release by primary AML cells. DESIGN AND METHODS: Native human AML cells derived from 68 consecutive patients were cultured in vitro. We investigated AML cell proliferation (3H-thymidine incorporation,colony formation),chemokine receptor expression,constitutive chemokine release and chemotaxis of normal peripheral blood mononuclear cells. RESULTS: Exogenous chemokines usually did not have any effect on AML blast proliferation in the absence of hematopoietic growth factors,but when investigating growth factor-dependent (interleukin 3 + granulocyte-macrophage colony-stimulating factor + stem cell factor) proliferation in suspension cultures the following patient subsets were identified: (i) patients whose cells showed chemokine-induced growth enhancement (8 patients); (ii) divergent effects on proliferation (15 patients); and (iii) no effect (most patients). These patient subsets did not differ in chemokine receptor expression,but,compared to CD34- AML cells,CD34+ cells showed higher expression of several receptors. Chemokines also increased the proliferation of clonogenic AML cells from the first subset of patients. Furthermore,a broad constitutive chemokine release profile was detected for most patients,and the following chemokine clusters could be identified: CCL2-4/CXCL1/8,CCL5/CXCL9-11 (possibly also CCL23) and CCL13/17/22/24/CXCL5 (possibly also CXCL6). Only the CCL2-4/CXCL1/8 cluster showed significant correlations between corresponding mRNA levels and NFkB levels/activation. The chemotaxis of normal immunocompetent cells for patients without constitutive chemokine release was observed to be decreased. INTERPRETATION AND CONCLUSIONS: Differences in chemokine responsiveness as well as chemokine release contribute to patient heterogeneity in AML. Patients with AML can be classified into distinct subsets according to their chemokine responsiveness and chemokine release profile. View Publication

We have found that a fungal strain,Talaromyces wortmannin KY12420,produces a potent inhibitor of smooth muscle myosin light chain kinase (MLCK). This active product,designated as MS-54,was isolated and purified from the culture broth of the fungus and identified as wortmannin. The inhibition of MLCK by wortmannin was prevented by a high concentration of ATP. The activity of the catalytic domain,which was disclosed by partial tryptic digestion,was also inhibited by wortmannin. These results suggest that wortmannin acts at or near to the catalytic site of the enzyme. It was shown clearly by kinetic analyses,preincubation studies,and dialysis experiments that the inhibitory action of wortmannin on MLCK was irreversible. Under the condition of preincubation for 3 min,0.3 microM wortmannin inhibited the activity of MLCK,while 10 microM wortmannin had no effect on the activities of cAMP-dependent protein kinase,cGMP-dependent protein kinase,and calmodulin-dependent protein kinase II,and had little effect on protein kinase C activity. These data expressed clearly the marked selectivity of the compound for MLCK. Furthermore,wortmannin also inhibited both the phosphorylation of myosin light chain and the contraction in rat thoracic aorta stimulated with KCl,which indicates the effectiveness of the compound in the cellular level as an MLCK inhibitor. View Publication

The hemopoietic microenvironment consists of a diverse repertoire of cells capable of providing signals that influence hemopoietic stem cell function. Although the role of osteoblasts and vascular endothelial cells has recently been characterized,the function of the most abundant cell type in the bone marrow,the adipocyte,is less defined. Given the emergence of a growing number of adipokines,it is possible that these factors may also play a role in regulating hematopoiesis. Here,we investigated the role of adiponectin,a secreted molecule derived from adipocytes,in hemopoietic stem cell (HSC) function. We show that adiponectin is expressed by components of the HSC niche and its receptors AdipoR1 and AdipoR2 are expressed by HSCs. At a functional level,adiponectin influences HSCs by increasing their proliferation,while retaining the cells in a functionally immature state as determined by in vitro and in vivo assays. We also demonstrate that adiponectin signaling is required for optimal HSC proliferation both in vitro and in long term hemopoietic reconstitution in vivo. Finally we show that adiponectin stimulation activates p38 MAPK,and that inhibition of this pathway abrogates adiponectin's proliferative effect on HSCs. These studies collectively identify adiponectin as a novel regulator of HSC function and suggest that it acts through a p38 dependent pathway. View Publication

Sirtuins are NAD(+)-dependent protein deacetylases and are emerging as molecular targets for the development of pharmaceuticals to treat human metabolic and neurological diseases and cancer. To date,several sirtuin inhibitors and activators have been identified,but the structural mechanisms of how these compounds modulate sirtuin activity have not yet been determined. We identified suramin as a compound that binds to human SIRT5 and showed that it inhibits SIRT5 NAD(+)-dependent deacetylase activity with an IC(50) value of 22 microM. To provide insights into how sirtuin function is altered by inhibitors,we determined two crystal structures of SIRT5,one in complex with ADP-ribose,the other bound to suramin. Our structural studies provide a view of a synthetic inhibitory compound in a sirtuin active site revealing that suramin binds into the NAD(+),the product,and the substrate-binding site. Finally,our structures may enable the rational design of more potent inhibitors. View Publication

IL-17F and IL-17A are members of the IL-17 pro-inflammatory cytokine family. IL-17A has been implicated in the pathogenesis of autoimmune diseases. IL-17F is a disulfide-linked dimer that contains a cysteine-knot motif. We hypothesized that IL-17F and IL-17A could form a heterodimer due to their sequence homology and overlapping pattern of expression. We evaluated the structure of recombinant IL-17F and IL-17A proteins,as well as that of natural IL-17F and IL-17A derived from activated human CD4+ T cells,by enzyme-linked immunosorbent assay,immunoprecipitation followed by Western blotting,and mass spectrometry. We find that both IL-17F and IL-17A can form both homodimeric and heterodimeric proteins when expressed in a recombinant system,and that all forms of the recombinant proteins have in vitro functional activity. Furthermore,we find that in addition to the homodimers of IL-17F and IL-17A,activated human CD4+ T cells also produce the IL-17F/IL-17A heterodimer. These data suggest that the IL-17F/IL-17A heterodimer may contribute to the T cell-mediated immune responses. View Publication

Although studies with primary lymphocytes are almost always conducted in CO(2) incubators maintained at atmospheric oxygen levels (atmosO(2); 20%),the physiological oxygen levels (physO(2); 5%) that cells encounter in vivo are 2-4 times lower. We show here that culturing primary T cells at atmosO(2) significantly alters the intracellular redox state (decreases intracellular glutathione,increases oxidized intracellular glutathione),whereas culturing at physO(2) maintains the intracellular redox environment (intracellular glutathione/oxidized intracellular glutathione) close to its in vivo status. Furthermore,we show that CD3/CD28-induced T cell proliferation (based on proliferation index and cell yield) is higher at atmosO(2) than at physO(2). This apparently paradoxical finding,we suggest,may be explained by two additional findings with CD3/CD28-stimulated T cells: (i) the intracellular NO (iNO) levels are higher at physO(2) than at atmosO(2); and (ii) the peak expression of CD69 is significantly delayed and more sustained at physO(2) that at atmosO(2). Because high levels of intracellular NO and sustained CD69 tend to down-regulate T cell responses in vivo,the lower proliferative T cell responses at physO(2) likely reflect the in vitro operation of the natural in vivo regulatory mechanisms. Thus,we suggest caution in culturing primary lymphocytes at atmosO(2) because the requisite adaptation to nonphysiological oxygen levels may seriously skew T cell responses,particularly after several days in culture. View Publication

We analyzed 112 patients with high-risk acute myeloid leukemia (61 in complete remission [CR]; 51 in relapse),who received human leukocyte-antigen (HLA)-haploidentical transplants from natural killer (NK) alloreactive (n = 51) or non-NK alloreactive donors (n = 61). NK alloreactive donors possessed HLA class I,killer-cell immunoglobulin-like receptor (KIR) ligand(s) which were missing in the recipients,KIR gene(s) for missing self recognition on recipient targets,and alloreactive NK clones against recipient targets. Transplantation from NK-alloreactive donors was associated with a significantly lower relapse rate in patients transplanted in CR (3% versus 47%) (P textgreater .003),better event-free survival in patients transplanted in relapse (34% versus 6%,P = .04) and in remission (67% versus 18%,P = .02),and reduced risk of relapse or death (relative risk versus non-NK-alloreactive donor,0.48; 95% CI,0.29-0.78; P textgreater .001). In all patients we tested the missing ligand" model which pools KIR ligand mismatched transplants and KIR ligand-matched transplants from donors possessing KIR(s) for which neither donor nor recipient have HLA ligand(s). Only transplantation from NK-alloreactive donors is associated with a survival advantage." View Publication

Embryonic stem cells (ESCs) represent an important research tool and a potential resource for regenerative medicine. Generally,ESCs are cocultured with a supportive feeder cell layer of murine embryonic fibroblasts,which maintain the ESCs' capacity for self-renewal and block spontaneous differentiation. These cumbersome conditions,as well as the risk of xenobiotic contamination of human ESCs grown on murine embryonic fibroblasts,make it a priority to develop chemically defined methods that can be safely used for the expansion of ESCs. Using a high-throughput,cell-based assay,we identified the small molecule IQ-1 that allows for the Wnt/beta-catenin-driven long-term expansion of mouse ESCs and prevents spontaneous differentiation. We demonstrate that IQ-1,by targeting the PR72/130 subunit of the serine/threonine phosphatase PP2A,prevents beta-catenin from switching coactivator usage from CBP to p300. The increase in beta-catenin/CBP-mediated transcription at the expense of beta-catenin/p300-mediated transcription is critical for the maintenance of murine stem cell pluripotency. View Publication