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Embryonic stem (ES) cells are permanent pluripotent stem cell lines established from pre-implantation mouse embryos. There is currently great interest in the potential therapeutic applications of analogous cells derived from human embryos. The isolation of ES cells is commonly presented as a straightforward transfer of cells in the early embryo into culture. In reality,however,continuous expansion of pluripotent cells does not occur in vivo,and in vitro is the exception rather than the norm. Both genetic and epigenetic factors influence the ability to derive ES cells. We have tracked the expression of a key marker and determinant of pluripotency,the transcription factor Oct-4,in primary cultures of mouse epiblasts and used this to assay the effect of experimental manipulations on the maintenance of a pluripotent cell compartment. We find that expression of Oct-4 is often lost prior to overt cytodifferentiation of the epiblast. The rate and extent of Oct-4 extinction varies with genetic background. We report that treatment with the MAP kinase/ERK kinase inhibitor PD98059,which suppresses activation of the mitogen-activated protein kinases Erk1 and Erk2,results in increased persistence of Oct-4-expressing cells. Oct-4 expression is also relatively sustained in cultures of diapause embryos and of isolated inner cell masses. Combination of all three conditions allowed the derivation of germline-competent ES cells from the normally refractory CBA mouse strain. These findings suggest that the genesis of an ES cell is a relatively complex process requiring epigenetic modulation of key gene expression over a brief time-window. Procedures that extend this time-window and/or directly regulate the critical genes should increase the efficiency of ES cell derivation. View Publication

Exposure of murine bone marrow (BM) cells to ionizing radiation (IR; 4 Gy) resulted in textgreater95% inhibition of the frequency of various day types of cobblestone area-forming cells in association with the induction of apoptosis in hematopoietic stem cell alike cells (Lin(-) ScaI(+) c-kit(+) cells; IR: 64.8 +/- 0.4% versus control: 20.4 +/- 0.5%; P textless 0.001) and progenitors (Lin(-) ScaI(-) c-kit(+) cells; IR: 46.2 +/- 1.4% versus control: 7.8 +/- 0.5%; P textless 0.001). Incubation of murine BM cells with busulfan (BU; 30 micro M) for 6 h also inhibited the cobblestone area-forming cell frequency but failed to cause a significant increase in apoptosis in these two types of hematopoietic cells. After 5 weeks of long-term BM cell culture,33% and 72% of hematopoietic cells survived IR- and BU-induced damage,respectively,as compared with control cells,but they could not form colony forming units-granulocyte macrophages. Moreover,these surviving cells expressed an increased level of senescence-associated beta-galactosidase,p16(Ink4a),and p19(Arf). These findings suggest that IR inhibits the function of hematopoietic stem cell alike cells and progenitors primarily by inducing apoptosis,whereas BU does so mainly by inducing premature senescence. In addition,induction of premature senescence in BM hematopoietic cells also contributes to IR-induced inhibition of their hematopoietic function. Interestingly,the induction of hematopoietic cell senescence by IR,but not by BU,was associated with an elevation in p53 and p21(Cip1/Waf1) expression. This suggests that IR induces hematopoietic cell senescence in a p53-p21(Cip1/Waf1)-dependent manner,whereas the induction of senescence by BU bypasses the p53-p21(Cip1/Waf1) pathway. 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

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

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

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The action of retinoids on gene regulation is mediated by three distinct nuclear retinoic acid receptor (RAR) subtypes called RAR alpha,beta and gamma. Since RAR gamma is predominantly expressed in adult skin,specific ligands for this subtype could (i) represent valuable tools to evaluate the biological role of RAR gamma in skin and (ii) provide therapeutic entities with a higher therapeutic index at lower teratogenic risk. Using in vitro binding studies and a functional transactivation assay,we have identified three compounds with high RAR gamma selectivity. View Publication

Retinoic acid (RA) exerts its pleiotropic effects on cell growth and differentiation through the activation of a family of transcription factors-the RA receptors (RARs). Three subtypes of these receptors exist,RAR alpha,RAR beta,and RAR gamma. The receptors are differentially expressed in different cell types and stages of development,suggesting that they may regulate different sets of genes. We have identified a synthetic retinoid with the characteristics of a selective RAR alpha antagonist. This antagonist counteracts RA effects on HL-60 cell differentiation and on B-lymphocyte polyclonal activation. Beyond its potential practical relevance,this and other specific antagonists will be useful to dissect the RAR system and to assign to one given receptor each of the many RA-regulated functions. View Publication

1. The mechanism of Ca2+ release from intracellular stores was studied in defolliculated Xenopus laevis oocytes by measuring whole-cell currents using the two-electrode voltage-clamp method. 2. The extracellular application of ionomycin,a selective Ca2+ ionophore,evoked an inward current consisting of a spike-like fast component followed by a long-lasting slow component with few superimposed current oscillations (fluctuations). The ionomycin response occurred in a dose-dependent manner and was dependent on Cl-. 3. No apparent refractory period was observed for repetitively evoked small ionomycin responses when the concentration of ionomycin was low (0.1 microM). In contrast,a larger ionomycin response (1 microM),consisting of fast and slow components,was followed by refractory period. Washing for 50-90 min was necessary for full recovery of the ionomycin response. 4. The response to ionomycin was suppressed by the extracellular application of acetoxymethyl ester of bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA AM,1-10 microM),a membrane-permeable intracellular Ca2+ chelator. 5. The ionomycin response was not affected by pertussis toxin (PTX,0.3-2.0 microgram/ml),a blocker of guanine nucleotide-binding regulatory proteins (G proteins). In contrast,the response to acetylcholine (ACh),which is known to occur via a G protein,was suppressed by PTX. 6. The fast component was not affected by removing Ca2+ from the bathing medium or by replacing extracellular Ca2+ with Ba2+ or Mn2+ (all of these solutions were supplemented with 2 mM EGTA),whereas the slow component was suppressed. 7. Injection of inositol 1,4,5-trisphosphate (IP3) following a response to extra-cellularly applied ionomycin did not evoke an appreciable membrane current. In contrast,ionomycin evoked a small inward current when it was applied after an inward-current response evoked by IP3 injection,whereas a second injection of IP3 did not evoke any appreciable current. 8. The results indicate that (a) ionomycin releases Ca2+ from its intracellular stores without the involvement of G proteins,resulting in activation of Ca(2+)-activated Cl- channels,(b) ionomycin mainly acts on the same intracellular Ca2+ stores as IP3,and (c) entry of Ca2+ from outside the cell considerably contributes to the slow component of the ionomycin response,whereas its fast component is predominantly dependent on the release of Ca2+ from the intracellular stores. View Publication

Mice deficient in early B cell factor (EBF) are blocked at the progenitor B cell stage prior to immunoglobulin gene rearrangement. The EBF-dependent block in B cell development occurs near the onset of B-lineage commitment,which raises the possibility that EBF may act instructively to specify the B cell fate from uncommitted,multipotential progenitor cells. To test this hypothesis,we transduced enriched hematopoietic progenitor cells with a retroviral vector that coexpressed EBF and the green fluorescent protein (GFP). Mice reconstituted with EBF-expressing cells showed a near complete absence of T lymphocytes. Spleen and peripheral blood samples were textgreater95 and 90% GFP+EBF+ mature B cells,respectively. Both NK and lymphoid-derived dendritic cells were also significantly reduced compared with control-transplanted mice. These data suggest that EBF can restrict lymphopoiesis to the B cell lineage by blocking development of other lymphoid-derived cell pathways. View Publication

The effects of the agonist enantiomer S(-)Bay K 8644 on gating charge of L-type Ca channels were studied in single ventricular myocytes. From a holding potential (Vh) of -40 mV,saturating (250 nm) S(-)Bay K shifted the half-distribution voltage of the activation charge (Q1) vs. V curve -7.5 +/- 0.8 mV,almost identical to the shift produced in the Ba conductance vs. V curve (-7.7 +/- 2 mV). The maximum Q1 was reduced by 1.7 +/- 0.2 nC/microF,whereas Q2 (charge moved in inactivated channels) was increased in a similar amount (1.4 +/- 0.4 nC/microF). The steady-state availability curves for Q1,Q2,and Ba current showed almost identical negative shifts of -14.8 +/- 1.7 mV,-18.6 +/- 5.8 mV,and -15.2 +/- 2.7 mV,respectively. The effects of the antagonist enantiomer R(+)BayK 8644 were also studied,the Q1 vs. V curve was not significantly shifted,but Q1max (Vh = -40 mV) was reduced and the Q1 availability curve shifted by -24.6 +/- 1.2 mV. We concluded that: a) the left shift in the Q1 vs. V activation curve produced by S(-)BayK is a purely agonistic effect; b) S(-)BayK induced a significantly larger negative shift in the availability curve than in the Q1 vs. V relation,consistent with a direct promotion of inactivation; c) as expected for a more potent antagonist,R(+)Bay K induced a significantly larger negative shift in the availability curve than did S(-)Bay K. View Publication

The hemopoietic stem cell (HSC) compartment encompasses cell subsets with heterogeneous proliferative and developmental potential. Numerous CD34(-) cell subsets that might reside at an earlier stage of differentiation than CD34(+) HSCs have been described and characterized within human umbilical cord blood (UCB). We identified a novel subpopulation of CD34(-)CD133(-)CD7(-)CD45(dim)lineage (lin)(-) HSCs contained within human UCB that were endowed with low but measurable extended long-term culture-initiating cell activity. Exposure of CD34(-)CD133(-)CD7(-)CD45(dim)lin(-) HSCs to stem cell factor preserved cell viability and was associated with the following: 1) concordant expression of the stem cell-associated Ags CD34 and CD133,2) generation of CFU-granulocyte-macrophage,burst-forming unit erythroid,and megakaryocytic aggregates,3) significant extended long-term culture-initiating cell activity,and 4) up-regulation of mRNA signals for myeloperoxidase. At variance with CD34(+)lin(-) cells,CD34(-)CD133(-)CD7(-)CD45(dim)lin(-) HSCs maintained with IL-15,but not with IL-2 or IL-7,proliferated vigorously and differentiated into a homogeneous population of CD7(+)CD45(bright)CD25(+)CD44(+) lymphoid progenitors with high expression of the T cell-associated transcription factor GATA-3. Although they harbored nonclonally rearranged TCRgamma genes,IL-15-primed CD34(-)CD133(-)CD7(-)CD45(dim)lin(-) HSCs failed to achieve full maturation,as manifested in their CD3(-)TCRalphabeta(-)gammadelta(-) phenotype. Conversely,culture on stromal cells supplemented with IL-15 was associated with the acquisition of phenotypic and functional features of NK cells. Collectively,CD34(-)CD133(-)CD7(-)CD45(dim)lin(-) HSCs from human UCB displayed an exquisite sensitivity to IL-15 and differentiated into lymphoid/NK cells. Whether the transplantation of CD34(-)lin(-) HSCs possessing T/NK cell differentiation potential may impact on immunological reconstitution and control of minimal residual disease after HSC transplantation for autoimmune or malignant diseases remains to be determined. View Publication