参考文献

Increasing evidence suggests that the deposition of amyloid plaques,composed primarily of the amyloid-beta protein (Abeta),within the cerebrovasculature is a frequent occurrence in Alzheimer's disease and may play a significant role in disease progression. Accordingly,the pathogenic mechanisms by which Abeta can alter vascular function may have therapeutic implications. Despite observations that Abeta elicits a number of physiological responses in endothelial cells,ranging from alteration of protein expression to cell death,the Abeta species accountable for these responses remains unexplored. In the current study,we show that isolated soluble Abeta aggregation intermediates activate human brain microvascular endothelial cells for both adhesion and subsequent transmigration of monocyte cells in the absence of endothelial cell death and monolayer disruption. In contrast,unaggregated Abeta monomer and mature Abeta fibril fail to induce any change in endothelial adhesion or transmigration. Correlations between average Abeta aggregate size and observed increases in adhesion illustrate that smaller soluble aggregates are more potent activators of endothelium. These results support previous studies demonstrating heightened neuronal activity of soluble Abeta aggregates,including Abeta-derived diffusible ligands,oligomers,and protofibrils,and further show that soluble aggregates also selectively exhibit activity in a vascular cell model. View Publication

There are a number of leukemogenic protein-tyrosine kinases (PTKs) associated with leukemic transformation. Although each is linked with a specific disease their functional activity poses the question whether they have a degree of commonality in their effects upon target cells. Exon array analysis of the effects of six leukemogenic PTKs (BCR/ABL,TEL/PDGFRbeta,FIP1/PDGFRalpha,D816V KIT,NPM/ALK,and FLT3ITD) revealed few common effects on the transcriptome. It is apparent,however,that proteome changes are not directly governed by transcriptome changes. Therefore,we assessed and used a new generation of iTRAQ tagging,enabling eight-channel relative quantification discovery proteomics,to analyze the effects of these six leukemogenic PTKs. Again these were found to have disparate effects on the proteome with few common targets. BCR/ABL had the greatest effect on the proteome and had more effects in common with FIP1/PDGFRalpha. The proteomic effects of the four type III receptor kinases were relatively remotely related. The only protein commonly affected was eosinophil-associated ribonuclease 7. Five of six PTKs affected the motility-related proteins CAPG and vimentin,although this did not correspond to changes in motility. However,correlation of the proteomics data with that from the exon microarray not only showed poor levels of correlation between transcript and protein levels but also revealed alternative patterns of regulation of the CAPG protein by different oncogenes,illustrating the utility of such a combined approach. View Publication

Recently,a number of clinical trials used either mesenchymal stem cells (MSCs) or natural killer (NK) cells in an attempt to improve the effectiveness of hematopoietic stem cell transplantation (HSCT). In view of the relevant role of both MSCs and NK cells in HSCT,we have recently explored the result of possible interactions between the 2 cell types. We found that activated NK cells could kill MSCs,whereas MSCs strongly inhibited interleukin-2 (IL-2)-induced NK-cell proliferation. In this study,we further analyzed the inhibitory effect exerted by MSCs on NK cells. We show that MSCs not only inhibit the cytokine-induced proliferation of freshly isolated NK cells but also prevent the induction of effector functions,such as cytotoxic activity and cytokine production. Moreover,we show that this inhibitory effect is related to a sharp down-regulation of the surface expression of the activating NK receptors NKp30,NKp44,and NKG2D. Finally,we demonstrate that indoleamine 2,3-dioxygenase and prostaglandin E2 represent key mediators of the MSC-induced inhibition of NK cells. View Publication

Mesenchymal stem cells (MSC) establish close interactions with bone marrow sinusoids in a putative perivascular niche. These vessels contain a large storage pool of mature nonproliferating neutrophils. Here,we have investigated the effects of human bone marrow MSC on neutrophil survival and effector functions. MSC from healthy donors,at very low MSC:neutrophil ratios (up to 1:500),significantly inhibited apoptosis of resting and interleukin (IL)-8-activated neutrophils and dampened N-formyl-l-methionin-l-leucyl-l-phenylalanine (f-MLP)-induced respiratory burst. The antiapoptotic activity of MSC did not require cell-to-cell contact,as shown by transwell experiments. Antibody neutralization experiments demonstrated that the key MSC-derived soluble factor responsible for neutrophil protection from apoptosis was IL-6,which signaled by activating STAT-3 transcription factor. Furthermore,IL-6 expression was detected in MSC by real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Finally,recombinant IL-6 was found to protect neutrophils from apoptosis in a dose-dependent manner. MSC had no effect on neutrophil phagocytosis,expression of adhesion molecules,and chemotaxis in response to IL-8,f-MLP,or C5a. These results support the following conclusions: (a) in the bone marrow niche,MSC likely protect neutrophils of the storage pool from apoptosis,preserving their effector functions and preventing the excessive or inappropriate activation of the oxidative metabolism,and (b) a novel mechanism whereby the inflammatory potential of activated neutrophils is harnessed by inhibition of apoptosis and reactive oxygen species production without impairing phagocytosis and chemotaxis has been identified. View Publication

By assessing the contribution of deregulated E2F activity to erythroid defects in Rb null mice,we have identified E2f-2 as being upregulated in end-stage red cells,where we show it is the major pRb-associated E2f and the predominant E2f detected at key target gene promoters. Consistent with its expression pattern,E2f-2 loss restored terminal erythroid maturation to Rb null red cells,including the ability to undergo enucleation. Deletion of E2f-2 also extended the life span of Rb null mice despite persistent defects in placental development,indicating that deregulated E2f-2 activity in differentiating erythroblasts contributes to the premature lethality of Rb null mice. We show that the aberrant entry of Rb null erythroblasts into S phase at times in differentiation when wild-type erythroblasts are exiting the cell cycle is inhibited by E2f-2 deletion. E2f-2 loss induced cell cycle arrest in both wild-type and Rb null erythroblasts and was associated with increased DNA double-strand breaks. These results implicate deregulated E2f-2 in the cell cycle defects observed in Rb null erythroblasts and reveal a novel role for E2f-2 during terminal red blood cell differentiation. The identification of a tissue-restricted role for E2f-2 in erythropoiesis highlights the nonredundant nature of E2f transcription factor activities in cell growth and differentiation. View Publication

OBJECTIVE Metformin is an antidiabetic drug commonly used to treat type 2 diabetes. The aim of the study was to determine whether metformin regulates hepatic gluconeogenesis through the orphan nuclear receptor small heterodimer partner (SHP; NR0B2). RESEARCH DESIGN AND METHODS We assessed the regulation of hepatic SHP gene expression by Northern blot analysis with metformin and adenovirus containing a constitutive active form of AMP-activated protein kinase (AMPK) (Ad-AMPK) and evaluated SHP,PEPCK,and G6Pase promoter activities via transient transfection assays in hepatocytes. Knockdown of SHP using siRNA SHP was conducted to characterize the metformin-induced inhibition of hepatic gluconeogenic gene expression in hepatocytes,and metformin-and adenovirus SHP (Ad-SHP)-mediated hepatic glucose production was measured in B6-Lep(ob/ob) mice. RESULTS Hepatic SHP gene expression was induced by metformin,5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR),and Ad-AMPK. Metformin-induced SHP gene expression was abolished by adenovirus containing the dominant negative form of AMPK (Ad-DN-AMPK),as well as by compound C. Metformin inhibited hepatocyte nuclear factor-4alpha-or FoxA2-mediated promoter activity of PEPCK and G6Pase,and the inhibition was blocked with siRNA SHP. Additionally,SHP knockdown by adenovirus containing siRNA SHP inhibited metformin-mediated repression of cAMP/dexamethasone-induced hepatic gluconeogenic gene expression. Furthermore,oral administration of metformin increased SHP mRNA levels in B6-Lep(ob/ob) mice. Overexpression of SHP by Ad-SHP decreased blood glucose levels and hepatic gluconeogenic gene expression in B6-Lep(ob/ob) mice. CONCLUSIONS We have concluded that metformin inhibits hepatic gluconeogenesis through AMPK-dependent regulation of SHP. View Publication

BACKGROUND AND PURPOSE: Glycogen synthase kinase-3 (GSK-3) affects neuropathological events associated with Alzheimeŕs disease (AD) such as hyperphosphorylation of the protein,tau. GSK-3beta expression,enzyme activity and tau phosphorylated at AD-relevant epitopes are elevated in juvenile rodent brains. Here,we assess five GSK-3beta inhibitors and lithium in lowering phosphorylated tau (p-tau) and GSK-3beta enzyme activity levels in 12-day old postnatal rats. EXPERIMENTAL APPROACH: Brain levels of inhibitors following treatment in vivo were optimized based on pharmacokinetic data. At optimal doses,p-tau (Ser(396)) levels in brain tissue was measured by immunoblotting and correlated with GSK-3beta enzyme activities in the same tissues. Effects of GSK inhibitors on p-tau,GSK-3beta activities and cell death were measured in a human neuronal cell line (LUHMES). KEY RESULTS: Lithium and CHIR98014 reduced tau phosphorylation (Ser(396)) in the cortex and hippocampus of postnatal rats,while Alsterpaullone and SB216763 were effective only in hippocampus. AR-A014418 and Indirubin-3'-monoxime were ineffective in either brain region. Inhibition of p-tau in brain required several-fold higher levels of GSK inhibitors than the IC(50) values obtained in recombinant or cell-based GSK-3beta enzyme activity assays. The inhibitory effect on GSK-3beta activity ex vivo correlated with protection against cell death and decrease of p-tau- in LUHMES cells,using low microM inhibitor concentrations. CONCLUSIONS AND IMPLICATIONS: Selective small-molecule inhibitors of GSK-3 reduce tau phosphorylation in vivo. These findings corroborate earlier suggestions that GSK-3beta may be an attractive target for disease-modification in AD and related conditions where tau phosphorylation is believed to contribute to disease pathogenesis. View Publication

The present study explored the sensitivity of leukaemic blasts derived from 30 acute myeloid leukaemia (AML) patients to Bortezomib. Bortezomib induced apoptosis of primary AML blasts: 18/30 AMLs were clearly sensitive to the proapoptotic effects of Bortezomib,while the remaining cases were moderately sensitive to this molecule. The addition of tumour necrosis factor-related-apoptosis-inducing ligand,when used alone,did not induce apoptosis of AML blasts and further potentiated the cytotoxic effects of Bortezomib. The majority of AMLs sensitive to Bortezomib showed immunophenotypic features of the M4 and M5 French-American-British classification subtypes and displayed myelomonocytic features. All AMLs with mutated FLT3 were in the Bortezomib-sensitive group. Biochemical studies showed that: (i) Bortezomib activated caspase-8 and caspase-3 and decreased cellular FLICE [Fas-associated death domain (FADD)-like interleukin-1beta-converting enzyme]-inhibitory protein (c-FLIP) levels in AML blasts; (ii) high c-FLIP levels in AML blasts were associated with low Bortezomib sensitivity. Finally,analysis of the effects of Bortezomib on leukaemic cells displaying high aldehyde dehydrogenase activity suggested that this drug induced in vitro killing of leukaemic stem cells. The findings of the present study,further support the development of Bortezomib as an anti-leukaemic drug and provide simple tools to predict the sensitivity of AML cells to this drug. View Publication

BACKGROUND: ALDH-bright (ALDH(br)) cell populations sorted from freshly collected umbilical cord blood (UCB) on the basis of their high aldehyde dehydrogenase (ALDH) activity are highly enriched for HPC. HPC with low ALDH activity (ALDH(dim)) are primarily short-term progenitors,whereas progenitors that initiate long-term cultures or establish long-term grafts in xenograft models are ALDH(br). We examined the multilineage hematopoietic and platelet progenitor activities of ALDH(br) cells recovered from cryopreserved UCB units typically employed in the practice of clinical transplantation. METHODS: Frozen UCB units were thawed,washed,immunomagnetically depleted of cells expressing glycophorin A and CD14,reacted for flow cytometric detection of ALDH,and sorted to yield ALDH(br) and ALDH(dim) populations. We measured surface Ag expression and viability of cells in the ALDH(br) and ALDH(dim) populations by flow cytometry and hematopoietic (CFC-H) and megakaryocytic (CFC-Mk) colony-forming cells in each population. RESULTS: ALDH(br) populations isolated from thawed UCB cells were highly enriched for CD34(+) and CD133(+) cells. Flow-sorted ALDH(br) populations were enriched 1116-fold in CFC-H,10-fold in multilineage GEMM colonies and 2015-fold in CFC-Mk compared with the ALDH(dim) population. All progenitors giving rise to large Mk colonies were derived from ALDH(br) populations. DISCUSSION: ALDH(br) populations recovered from thawed,banked UCB with the method we describe have HPC activity and may be useful in the clinic to facilitate reconstitution of erythroid,myeloid and megakaryocytic blood elements. View Publication

The mechanisms that regulate hematopoietic stem cell (HSC) fate decisions between proliferation and multilineage differentiation are unclear. Members of the Wnt family of ligands that activate the canonical Wnt signaling pathway,which utilizes beta-catenin to relay the signal,have been demonstrated to regulate HSC function. In this study,we examined the role of noncanonical Wnt signaling in regulating HSC fate. We observed that noncanonical Wnt5a inhibited Wnt3a-mediated canonical Wnt signaling in HSCs and suppressed Wnt3a-mediated alterations in gene expression associated with HSC differentiation,such as increased expression of myc. Wnt5a increased short- and long-term HSC repopulation by maintaining HSCs in a quiescent G(0) state. From these data,we propose that Wnt5a regulates hematopoiesis by the antagonism of the canonical Wnt pathway,resulting in a pool of quiescent HSCs. View Publication