技术资料

Fanconi Anemia (FA) is an autosomal recessive disorder characterized by cellular hypersensitivity to DNA cross-linking agents. Recent studies suggest that FA proteins share a common pathway with BRCA proteins. To study the in vivo role of the FA group A gene (Fanca),gene-targeting techniques were used to generate Fanca(tm1Hsc) mice in which Fanca exons 1-6 were replaced by a beta-galactosidase reporter construct. Fanca(tm1.1Hsc) mice were generated by Cre-mediated removal of the neomycin cassette in Fanca(tm1Hsc) mice. Fanca(tm1.1Hsc) homozygotes display FA-like phenotypes including growth retardation,microphthalmia and craniofacial malformations that are not found in other Fanca mouse models,and the genetic background affects manifestation of certain phenotypes. Both male and female mice homozygous for Fanca mutation exhibit hypogonadism,and homozygous females demonstrate premature reproductive senescence and an increased incidence of ovarian cysts. We showed that fertility defects in Fanca(tm1.1Hsc) homozygotes might be related to a diminished population of primordial germ cells (PGCs) during migration into the gonadal ridges. We also found a high level of Fanca expression in pachytene spermatocytes. Fanca(tm1Hsc) homozygous males exhibited an elevated frequency of mispaired meiotic chromosomes and increased apoptosis in germ cells,implicating a role for Fanca in meiotic recombination. However,the localization of Rad51,Brca1,Fancd2 and Mlh1 appeared normal on Fanca(tm1Hsc) homozygous meiotic chromosomes. Taken together,our results suggest that the FA pathway plays a role in the maintenance of reproductive germ cells and in meiotic recombination. View Publication

The signal transducer Stat5 plays a key role in the regulation of hematopoietic differentiation and hematopoietic stem cell function. To evaluate the effects of Stat5 signaling in the earliest hematopoietic progenitors,we have generated an embryonic stem cell line in which Stat5 signaling can be induced with doxycycline. Ectopic Stat5 activation at the point of origin of the hematopoietic lineage (from day 4 to day 6 of embryoid body differentiation) significantly enhances the number of hematopoietic progenitors with colony-forming potential. It does so without significantly altering total numbers or apoptosis of hematopoietic cells,suggesting a cell-intrinsic effect of Stat5 on either the developmental potential or clonogenicity of this population. From day-6 embryoid bodies,under the influence of Stat5 signaling,a population of semiadherent cells can be expanded on OP9 stromal cells that is comprised of primitive hematopoietic blast cells with ongoing,mainly myeloid,differentiation. When these cells are injected into lethally irradiated mice,they engraft transiently in a doxycycline-dependent manner. These results demonstrate that the hematopoietic commitment of embryonic stem cells may be augmented by a Stat5-mediated signal,and highlight the utility of manipulating individual components of signaling pathways for engineering tissue-specific differentiation of stem cells. View Publication

Chemokine receptors on T cells are frequently categorized as functioning either in immune system homeostasis within lymphoid organs,or in peripheral inflammation. CXCR3 is in the latter category and is reported to be expressed selectively on Th1 cells. We found that CXCR3 was expressed in vivo on newly activated tonsillar CD4(+) T cells. Using CD4(+) T cells from cord blood,we found that CXCR3 was induced by cellular activation in vitro independently of the cytokine milieu,although on resting cells,expression was maintained preferentially on those that had been activated in type 1 conditions. In inflamed tonsils,CXCR3(+)CD4(+) T cells were localized around and within germinal centers. The inference that CXCR3 has a role in germinal center reactions was supported by the finding that the CXCR3 ligand CXC chemokine ligand 9 was expressed in a pattern demarcating a subset of germinal centers both in tonsil and in lymph nodes from an HIV-infected individual. We next investigated the role of CXCR3 on peripheral effector/memory CD4(+) T cells by comparing its pattern of expression with that of CCR5,another Th1-cell associated chemokine receptor. Analysis of cells directly from peripheral blood and after activation in vitro suggested that CXCR3 expression preceded that of CCR5,supporting a model of sequential induction of chemokine receptors during CD4(+) T cell differentiation. Taken together,our data show that CXCR3 can be expressed at all stages of CD4(+) T cell activation and differentiation,bridging central function in lymphoid organs and effector function in peripheral tissues. View Publication

View Publication

Deamination of the nucleoside analogues ARA-C and 5-AZA-CdR by CR deaminase results in a loss of antileukemic activity. To prevent the inactivation of these analogues,inhibitors of CR deaminase may prove to be useful agents. In the present study we investigated the effects of the deaminase inhibitors Zebularine,5-F-Zebularine,and diazepinone riboside on the deamination of CR,ARA-C,and 5-AZA-CdR using highly purified human CR deaminase (EC 3.5.4.5). These inhibitors produced a competitive type of inhibition with each substrate,the potency of which followed the patterns diazepinone riboside greater than 5-F-Zebularine and THU greater than Zebularine. 5-AZA-CdR was more sensitive than ARA-C to the inhibition produced by these deaminase inhibitors. The inhibition constants for diazepinone riboside lay in the range of 5-15 nM,suggesting that this inhibitor could be an excellent candidate for use in combination chemotherapy with either ARA-C or 5-AZA-CdR in patients with leukemia. View Publication

To directly study the biological properties of purified hematopoietic colony-forming cell precursors,cells with a CD34+ CD45RAlo CD71lo phenotype were purified from human bone marrow using density separation and fluorescence-activated cell sorting,and were cultured in serum-free culture medium supplemented with various cytokines. In the presence of interleukin 3 (IL-3),IL-6,erythropoietin,and mast cell growth factor (a c-kit ligand),cell numbers increased approximately 10(6)-fold over a period of 4 wk,and the percentage of cells that expressed transferrin receptors (CD71) increased from less than 0.1% at day 0 to greater than 99% at day 14. Interestingly,the absolute number of CD34+ CD71lo cells did not change during culture. When CD34+ CD71lo cells were sorted from expanded cultures and recultured,extensive cell production was repeated,again without significant changes in the absolute number of cells with the CD34+ CD71lo phenotype that were used to initiate the (sub)cultures. These results document that primitive hematopoietic cells can generate progeny without an apparent decrease in the size of a precursor cell pool. View Publication

A wide variety of membrane transformations important in intracellular transport are inhibited by the fungal metabolite brefeldin A (refs 1-4),implying that the target for this drug is central to the formation and maintenance of subcellular compartments. Brefeldin A added to cells causes the rapid and reversible dissociation of a Golgi-associated peripheral membrane protein (M(r) 110,000) which was found to be identical to one of the subunits of the coat of Golgi-derived (non-clathrin) coated vesicles,beta-COP,implying that brefeldin A prevents transport by blocking the assembly of coats and thus the budding of enclosed vesicles. In addition to the coatomer (a cytosol-derived complex of seven polypeptide chains,one of which is beta-COP),the non-clathrin (COP) coat of Golgi-derived vesicles contains stoichiometric amounts of a small (M(r) approximately 20,000) GTP-binding protein,the ADP-ribosylation factor (ARF). Binding of ARF to Golgi membranes is necessary before coatomer/beta-COP can bind these membranes (ref. 12; and D. J. Palmer et al.,manuscript submitted),so the primary effect of brefeldin A seems to be on the reaction responsible for ARF binding. Indeed,like beta-COP,ARF is dissociated from the Golgi complex by treatment with brefeldin A and brefeldin A prevents ARF from associating in vitro,but the mechanism of this action by brefeldin A has been unclear. Here we report the discovery of an enzyme in a Golgi-enriched fraction that catalyses guanine nucleotide (GDP-GTP) exchange on ARF-1 protein,and which is inhibited by brefeldin A. We suggest that activation of ARF proteins for membrane localization by compartmentalized exchange enzymes is in general the first committed step in membrane transformation pathways. View Publication

Vertebrate body axis extension involves progressive generation and subsequent differentiation of new cells derived from a caudal stem zone; however,molecular mechanisms that preserve caudal progenitors and coordinate differentiation are poorly understood. FGF maintains caudal progenitors and its attenuation is required for neuronal and mesodermal differentiation and to position segment boundaries. Furthermore,somitic mesoderm promotes neuronal differentiation in part by downregulating Fgf8. Here we identify retinoic acid (RA) as this somitic signal and show that retinoid and FGF pathways have opposing actions. FGF is a general repressor of differentiation,including ventral neural patterning,while RA attenuates Fgf8 in neuroepithelium and paraxial mesoderm,where it controls somite boundary position. RA is further required for neuronal differentiation and expression of key ventral neural patterning genes. Our data demonstrate that FGF and RA pathways are mutually inhibitory and suggest that their opposing actions provide a global mechanism that controls differentiation during axis extension. View Publication

In mammals,the continuous production of hematopoietic cells (HCs) is sustained by a small number of hematopoietic stem cells (HSCs) residing in the bone marrow. Early HSC activity arises in the aorta-gonad mesonephros region,within cells localized to the ventral floor of the major blood vessels,suggesting that the first HSCs may be derived from cells capable of giving rise to the hematopoietic system and to the endothelial cells of the vasculature. TIE1 (TIE) and TIE2 (TEK) are related receptor tyrosine kinases with an embryonic expression pattern in endothelial cells,their precursors,and HCs,suggestive of a role in the divergence and function of both lineages. Indeed,gene targeting approaches have shown that TIE1,TIE2,and ligands for TIE2,the angiopoietins,are essential for vascular development and maintenance. To explore possible roles for these receptors in HCs,we have examined the ability of embryonic cells lacking both TIE1 and TIE2 to contribute to developmental and adult hematopoiesis by generating chimeric animals between normal embryonic cells and cells lacking these receptors. We show here that TIE receptors are not required for differentiation and proliferation of definitive hematopoietic lineages in the embryo and fetus; surprisingly,however,these receptors are specifically required during postnatal bone marrow hematopoiesis. View Publication

In a patient with refractory anemia with excess blasts (RAEB),a somatic mutation of mitochondrial transfer RNA(Leu(UUR)) was detected in bone marrow cells. Heteroduplex analysis indicated that 40% to 50% of mitochondrial DNA (mtDNA) molecules in the bone marrow (BM) carried the novel G3242A mutation. The proportion of mutant mtDNA was higher in CD34(+) cells than in the unfractionated sample. Surprisingly,the mutation was not detectable by heteroduplex analysis in the peripheral blood (PB). However,PB CD34(+) cells selected by immunomagnetic beads harbored the mutation with a proportion of approximately 50%. In hematopoietic colony assays,CD34(+) cells from BM and PB yielded only colonies with wild-type mtDNA. These results indicate that the mtDNA mutation in CD34(+) cells was associated with a maturation defect. Mitochondrial tRNA mutations impair mitochondrial protein synthesis,thereby causing dysfunction of the mitochondrial respiratory chain. We propose that this effect contributed to ineffective hematopoiesis in our patient. View Publication

Human immunodeficiency virus type 1 (HIV-1) isolates from 20 chronically infected patients who participated in a structured treatment interruption (STI) trial were studied to determine whether viral fitness influences reestablishment of viremia. Viruses derived from individuals who spontaneously controlled viremia had significantly lower in vitro replication capacities than viruses derived from individuals that did not control viremia after interruption of antiretroviral therapy (ART),and replication capacities correlated with pre-ART and post-STI viral set points. Of note,no clinically relevant improvement of viral loads upon STI occurred. Virus isolates from controlling and noncontrolling patients were indistinguishable in terms of coreceptor usage,genetic subtype,and sensitivity to neutralizing antibodies. In contrast,viruses from controlling patients exhibited increased sensitivity to inhibition by chemokines. Sensitivity to inhibition by RANTES correlated strongly with slower replication kinetics of the virus isolates,suggesting a marked dependency of these virus isolates on high coreceptor densities on the target cells. In summary,our data indicate that viral fitness is a driving factor in determining the magnitude of viral rebound and viral set point in chronic HIV-1 infection,and thus fitness should be considered as a parameter influencing the outcome of therapeutic intervention in chronic infection. View Publication

The CCAAT/enhancer binding protein alpha (C/EBPalpha) is an essential transcription factor for granulocytic differentiation. C/EBPalpha mutations are found in approximately 8% of acute myeloid leukemia (AML) patients. Most of these mutations occur in the N-terminal coding region,resulting in a frame shift and the enhanced translation of a dominant-negative 30-kDa protein,which may be responsible for the differentiation block observed in AML. To test this hypothesis,we introduced a cDNA encoding an N-terminal mutated C/EBPalpha (mut10) into primary hematopoietic progenitors using a retroviral vector. Expression of mut10 in human CD34+ cord blood cells dramatically inhibited differentiation of both myeloid and erythroid lineages. Immunohistochemical analysis demonstrated coexpression of both myeloid and erythroid markers in the immature transformed cells. Surprisingly,mut10 did not block myelocytic differentiation in murine progenitors but did alter their differentiation kinetics and clonogenicity. Experiments were performed to confirm that the differential effect of mut10 on murine and human progenitors was not due to species-specific differences in C/EBPalpha protein sequences,expression levels,or inefficient targeting of relevant cells. Taken together,our results underline the intrinsic differences between hematopoietic controls in mouse and human and support the hypothesis that mutations in CEBPA are critical events in the disruption of myeloid differentiation in AMLs. View Publication