技术资料

In many mammalian cells brefeldin A interferes with mechanisms that keep the Golgi appartus separate from the endoplasmic reticulum. The earliest effect of brefeldin A is release of the coat protein beta-COP from the Golgi. This release is blocked by pretreatment with GTP-gamma S or AlF4- (ref. 12). The AlF4- ion activates heterotrimeric G proteins but not proteins of the ras superfamily,suggesting that a heterotrimeric G protein might control membrane transfer from the endoplasmic reticulum to the Golgi. We report here that mastoparan,a peptide that activates heterotrimeric G proteins,promotes binding of beta-COP to Golgi membranes in vitro and antagonizes the effect of brefeldin A on beta-COP in perforated cells and on isolated Golgi membranes. This inhibition is greatly diminished if cells are pretreated with pertussis toxin before perforation. Thus,a heterotrimeric G protein of the Gi/Go subfamily regulates association of coat components with Golgi membranes. View Publication

OVERVIEW: All current antipsychotic medications work by binding to Gi-coupled dopamine (DA) D2 receptors. Such medications are thought to affect cellular function primarily by decreasing DA-mediated regulation of intracellular cyclic adenosine monophosphate (cAMP).However,several studies indicate that cAMP signal transduction abnormalities in schizophrenia may not be limited to D2-containing cells. The current study examines the potential of using non-receptor-based agents that modify intracellular signal transduction as potential antipsychotic medications. METHODS: The indirect DA agonist amphetamine has been used to model the auditory sensory processing deficits in schizophrenia. Such pharmacologically induced abnormalities are reversed by current antipsychotic treatments. This study examines the ability of the phosphodiesterase-4 inhibitor,rolipram,to reverse amphetamine-induced abnormalities in auditory-evoked potentials that are characteristic of schizophrenia. RESULTS: Rolipram reverses amphetamine-induced reductions in auditory-evoked potentials. CONCLUSION: This finding could lead to novel approaches to receptor-independent treatments for schizophrenia. View Publication

Adult T-cell leukemia (ATL) caused by human T-cell leukemia virus type 1 (HTLV-1) infection,occurs in 2% to 4% of the HTLV-1 carriers with a long latent period,suggesting that additional alterations participate in the development of ATL. To characterize and identify novel markers of ATL,we examined the expression profiles of more than 12 000 genes in 8 cases of acute-type ATL using microarray. One hundred ninety-two genes containing interleukin 2 (IL-2) receptor alpha were up-regulated more than 2-fold compared with CD4(+) and CD4(+)CD45RO(+) T cells,and tumor suppressor in lung cancer 1 (TSLC1),caveolin 1,and prostaglandin D2 synthase showed increased expression of more than 30-fold. TSLC1 is a cell adhesion molecule originally identified as a tumor suppressor in the lung but lacks its expression in normal or activated T cells. We confirmed ectopic expression of the TSLC1 in all acute-type ATL cells and in 7 of 10 ATL- or HTLV-1-infected T-cell lines. Introduction of TSLC1 into a human ATL cell line ED enhanced both self-aggregation and adhesion ability to vascular endothelial cells. These results suggested that the ectopic expression of TSLC1 could provide a novel marker for acute-type ATL and may participate in tissue invasion,a characteristic feature of the malignant ATL cells. View Publication

Most polyreactive and antinuclear antibodies are removed from the human antibody repertoire during B cell development. To elucidate how B cell receptor (BCR) signaling may regulate human B cell tolerance,we tested the specificity of recombinant antibodies from single peripheral B cells isolated from patients suffering from X-linked agammaglobulinemia (XLA). These patients carry mutations in the Bruton's tyrosine kinase (BTK) gene that encode an essential BCR signaling component. We find that in the absence of Btk,peripheral B cells show a distinct antibody repertoire consistent with extensive secondary V(D)J recombination. Nevertheless,XLA B cells are enriched in autoreactive clones. Our results demonstrate that Btk is essential in regulating thresholds for human B cell tolerance. View Publication

GATA-2 is an essential transcription factor in the hematopoietic system that is expressed in hematopoietic stem cells (HSCs) and progenitors. Complete deficiency of GATA-2 in the mouse leads to severe anemia and embryonic lethality. The role of GATA-2 and dosage effects of this transcription factor in HSC development within the embryo and adult are largely unexplored. Here we examined the effects of GATA-2 gene dosage on the generation and expansion of HSCs in several hematopoietic sites throughout mouse development. We show that a haploid dose of GATA-2 severely reduces production and expansion of HSCs specifically in the aorta-gonad-mesonephros region (which autonomously generates the first HSCs),whereas quantitative reduction of HSCs is minimal or unchanged in yolk sac,fetal liver,and adult bone marrow. However,HSCs in all these ontogenically distinct anatomical sites are qualitatively defective in serial or competitive transplantation assays. Also,cytotoxic drug-induced regeneration studies show a clear GATA-2 dose-related proliferation defect in adult bone marrow. Thus,GATA-2 plays at least two functionally distinct roles during ontogeny of HSCs: the production and expansion of HSCs in the aorta-gonad-mesonephros and the proliferation of HSCs in the adult bone marrow. View Publication

Acute myeloid leukemia (AML) cells are characterized by a constitutive and abnormal activation of the nuclear factor-kappaB (NF-kappaB) transcription factor. This study,conducted in vitro on 18 patients,shows that targeting the IKB kinase 2 (IKK2) kinase with the specific pharmacologic inhibitor AS602868 to block NF-kappaB activation led to apoptosis of human primary AML cells. Moreover,AS602868 potentiated the apoptotic response induced by the current chemotherapeutic drugs doxorubicin,cytarabine,or etoposide (VP16). AS602868-induced cell death was associated with rupture of the mitochondrial transmembrane potential and activation of cellular caspases. NF-kappaB inhibition did not affect normal CD34+ hematopoietic precursors,suggesting that it could represent a new adjuvant strategy for AML treatment. View Publication

A new entity of acute leukemia coexpressing CD4(+)CD56(+) markers without any other lineage-specific markers has been identified recently as arising from lymphoid-related plasmacytoid dendritic cells (pDCs). In our laboratory,cells from a patient with such CD4(+)CD56(+) lineage-negative leukemia were unexpectedly found to also express the myeloid marker CD33. To confirm the diagnosis of pDC leukemia despite the CD33 expression,we demonstrated that the leukemic cells indeed exhibited pDC phenotypic and functional properties. In 7 of 8 other patients with CD4(+)CD56(+) pDC malignancies,we were able to confirm that the tumor cells expressed CD33 although with variable expression levels. CD33 expression was shown by flow cytometry,reverse transcriptase-polymerase chain reaction,and immunoblot analysis. Furthermore,CD33 monoclonal antibody stimulation of purified CD4(+)CD56(+) leukemic cells led to cytokine secretion,thus confirming the presence of a functional CD33 on these leukemic cells. Moreover,we found that circulating pDCs in healthy individuals also weakly express CD33. Overall,our results demonstrate that the expression of CD33 on CD4(+)CD56(+) lineage-negative cells should not exclude the diagnosis of pDC leukemia and underline that pDC-specific markers should be used at diagnosis for CD4(+)CD56(+) malignancies. View Publication

To investigate the role of transforming growth factor-beta 1 (TGF beta) in bone metabolism,the effects of this agent on the differentiation characteristics of human bone cells were studied in vitro. Human bone cells were isolated from femoral head samples by collagenase digestion. Differentiation characteristics included alkaline phosphatase activity,osteocalcin production,and mRNA levels for alkaline phosphatase,type I alpha 2-procollagen,and osteocalcin. The effect of TGF beta on alkaline phosphatase was not constant,but varied with the incubation conditions. At high cell density and in the presence of serum,TGF beta decreased alkaline phosphatase activity. However,at low cell density and under serum-free conditions,TGF beta stimulated alkaline phosphatase activity. The addition of 1,25(OH)2 vitamin D3 also stimulated alkaline phosphatase. The combination of the two agents gave a greater increase in activity than the sum of the activities when the two agents were given alone. The percentage of cells that stain positively for alkaline phosphatase changed in parallel with the change in specific activity. The percentage of positive cells increased from 17% to 64%,while the specific activity increased from 22 to 169 mU/mg protein. To investigate the mechanism of this stimulation,mRNA levels were measured at 24 hours. Individually,TGF beta and 1,25(OH)2D3 increased message levels for alkaline phosphatase and type I procollagen,but the greatest effect was produced by the combination of the two factors. 1,25(OH)2D3 increased osteocalcin mRNA levels,but TGF beta markedly inhibited this stimulation. TGF beta also inhibited production of osteocalcin by the human bone cells. TGF beta appears to modulate differentiation of human bone cells in combination with 1,25(OH)2D3 and other factors. View Publication

Mesenchymal stem cells (MSCs) are capable of down-regulating alloimmune responses and promoting the engraftment of hematopoietic stem cells. MSCs may therefore be suitable for improving donor-specific tolerance induction in solid-organ transplantation. Cells from cadaveric vertebral bone marrow (V-BM),aspirated iliac crest-BM,and peripheral blood progenitor cells were compared. Cells were characterized by flow cytometry and colony assays. MSCs generated from V-BM were assayed for differentiation capacity and immunomodulatory function. A median 5.7 x 10(8) nucleated cells (NCs) were recovered per vertebral body. The mesenchymal progenitor,colony-forming unit-fibroblast,frequency in V-BM (11.6/10(5) NC,range: 6.0-20.0) was considerably higher than in iliac crest-BM (1.4/10(5) NC,range: 0.4-2.6) and peripheral blood progenitor cells (not detectable). MSC generated from V-BM had the typical MSC phenotype (CD105(pos)CD73(pos)CD45(neg)CD34(neg)),displayed multilineage differentiation potential,and suppressed alloreactivity in mixed lymphocyte reactions. V-BM may be an excellent source for MSC cotransplantation approaches. View Publication

Leukocyte trafficking to sites of inflammation follows a defined temporal pattern,and evidence suggests that initial neutrophil transendothelial migration modifies endothelial cell phenotype. We tested the hypothesis that preconditioning of human umbilical vein endothelial cells (HUVEC) by neutrophils would also modify the subsequent transendothelial migration of T lymphocytes across cytokine-stimulated HUVEC in an in vitro flow assay. Using fluorescence microscopy,preconditioning of HUVEC by neutrophils was observed to significantly reduce the extent of subsequent stromal cell-derived factor-1alpha (SDF-1alpha [CXCL12])-mediated T lymphocyte transendothelial migration,without reducing accumulation. In contrast,recruitment of a second wave of neutrophils was unaltered. Conditioned medium harvested after transendothelial migration of neutrophils or supernatants from stimulated neutrophils mediated a similar blocking effect,which was negated using a specific neutrophil elastase inhibitor. Furthermore,T lymphocyte transendothelial migration was inhibited by treatment of HUVEC with purified neutrophil elastase,which selectively cleaved the amino terminus of HUVEC-bound SDF-1alpha,which is required for its chemotactic activity. The reduction in T lymphocyte transendothelial migration was not observed using a different chemokine,ELC (CCL19),and was not reversed by replenishment of SDF-1alpha,indicating endothelial retention of the inactivated chemokine. In summary,transmigrating neutrophils secrete localized elastase that is protected from plasma inhibitors,and thereby modulate trafficking of other leukocyte subsets by altering the endothelial-associated chemotactic activities. View Publication

Virus-specific CD8(+) T cells are associated with declining viremia in acute human immunodeficiency virus (HIV)1 infection,but do not correlate with control of viremia in chronic infection,suggesting a progressive functional defect not measured by interferon gamma assays presently used. Here,we demonstrate that HIV-1-specific CD8(+) T cells proliferate rapidly upon encounter with cognate antigen in acute infection,but lose this capacity with ongoing viral replication. This functional defect can be induced in vitro by depletion of CD4(+) T cells or addition of interleukin 2-neutralizing antibodies,and can be corrected in chronic infection in vitro by addition of autologous CD4(+) T cells isolated during acute infection and in vivo by vaccine-mediated induction of HIV-1-specific CD4(+) T helper cell responses. These data demonstrate a loss of HIV-1-specific CD8(+) T cell function that not only correlates with progressive infection,but also can be restored in chronic infection by augmentation of HIV-1-specific T helper cell function. This identification of a reversible defect in cell-mediated immunity in chronic HIV-1 infection has important implications for immunotherapeutic interventions. View Publication

Previously,a small molecule,purmorphamine,was identified that selectively induces osteogenesis in multipotent mesenchymal progenitor cells. In order to gain insights into the mechanism of action of purmorphamine,high-density oligonucleotide microarrays were used to profile gene expression in multipotent mesenchymal progenitor cells treated with either purmorphamine or bone morphogenetic protein-4 (BMP-4). In contrast to BMP-4 treatment,purmorphamine activates the Hedgehog (Hh) signaling pathway,resulting in the up- and downregulation of its downstream target genes,including Gli1 and Patched. Moreover,the known Hh signaling antagonists,cyclopamine and forskolin,completely block the osteogenesis and Glimediated transcription induced by purmorphamine. These results demonstrate that purmorphamine is a small molecule agonist of Hedgehog signaling,and it may ultimately be useful in the treatment of bone-related disease and neurodegenerative disease. View Publication