技术资料

Bay K8644 increased unidirectional Ca2+ influx and produced tension development in rabbit aorta. Both responses could be evoked in the tissue maximally stimulated with norepinephrine. When the arterial rings were maximally activated by high K+ depolarization,Bay K8644 was without effect. The tension evoked by high K+ and Bay K8644 was more sensitive to the dihydropyridine Ca2+ antagonist PY108-068 than norepinephrine induced tension. These results indicate that Bay K8644 activates only potential operated Ca2+ channels which are opened by high K+ depolarization. View Publication

In HeLa cells which have been exposed to 5 mM sodium butyrate for 21 h,the level of histone acetylation is greatly increased as compared to control cells (Riggs,M.G.,Whittaker,R.G.,Neumann,J.R.,and Ingram,V.R. (1977) Nature 268,462-464). Our experiments indicate that the increase in the relative amounts of multiacetylated forms of histones H4 and H3 following butyrate treatment is the result of an inhibition of histone deacetylase activity. View Publication

Sinefungin (A9145) and a related metabolite,A9145C,were found to be potent inhibitors of Newcastle disease virion and vaccinia virion mRNA(guanine-7-)-methyltransferase and vaccinia virion mRNA(nucleoside-2'-)-methyltransferase. Both Sinefungin and A9145C were competitive inhibitors of these S-adenosyl-L-methionine-dependent enzymes having inhibition constants substantially less than S-adenosyl-L-homocysteine. These compounds also inhibited plaque formation by vaccinia virus in mouse L-cells. View Publication

This report describes the establishment directly from normal preimplantation mouse embryos of a cell line that forms teratocarcinomas when injected into mice. The pluripotency of these embryonic stem cells was demonstrated conclusively by the observation that subclonal cultures,derived from isolated single cells,can differentiate into a wide variety of cell types. Such embryonic stem cells were isolated from inner cell masses of late blastocysts cultured in medium conditioned by an established teratocarcinoma stem cell line. This suggests that such conditioned medium might contain a growth factor that stimulates the proliferation or inhibits the differentiation of normal pluripotent embryonic cells,or both. This method of obtaining embryonic stem cells makes feasible the isolation of pluripotent cells lines from various types of noninbred embryo,including those carrying mutant genes. The availability of such cell lines should made possible new approaches to the study of early mammalian development. View Publication

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To study the role of different cytokine combinations on the proliferation and differentiation of highly purified primitive progenitor cells,a serum-free liquid culture system was used in combination with phenotypic and functional analysis of the cells produced in culture. CD34+ CD45RAlo CD71lo cells,purified from umbilical cord blood by flow cytometry and cell sorting,were selected for this study because of their high content of clonogenic cells (34%),particularly multipotent progenitors (CFU-MIX,12% of all cells). Four cytokine combinations were tested: (1) mast cell growth factor (MGF; a c-kit ligand) and interleukin-6 (IL-6); (2) MGF,IL-6,IL-3,and erythropoietin (Epo); (3) MGF,IL-6,granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-3 fusion protein (FP),macrophage colony-stimulating factor (M-CSF),and granulocyte-CSF (G-CSF); and (4) MGF,IL-6,FP,M-CSF,G-CSF,and Epo. Maximum numbers of erythroid progenitors (BFU-E,up to 55-fold increase) and mature erythroid cells were observed in the presence of MGF,IL-6,IL-3,and Epo,whereas maximum levels of myeloid progenitors (CFU-C,up to 70-fold increase) and mature myeloid cells were found in cultures supplemented with MGF,IL-6,FP,M-CSF,and G-CSF. When MGF,IL-6,FP,M-CSF,G-CSF,and Epo were present,maximum levels of both erythroid and myeloid progenitors and their progeny were observed. These results indicate that specific cytokine combinations can act directly on primitive hematopoietic cells resulting in significant expansion of progenitor cell numbers and influencing their overall patterns of proliferation and differentiation. Furthermore,the observations presented in this study suggest that the cytokine combinations used were unable to bias lineage commitment of multipotent progenitors,but rather had a permissive effect on the development of lineage-restricted clonogenic cells. View Publication

Lysophosphatidic acid (LPA; 1-acyl-sn-glycero-3-phosphate) is a platelet-derived lipid mediator that activates its own G-protein-coupled receptor to trigger phospholipase C-mediated Ca2+ mobilization and other effector pathways in numerous cell types. In this study we have examined the structural features of LPA that are important for activation of the Ca(2+)-mobilizing receptor in human A431 carcinoma cells,which show an EC50 for oleoyl-LPA as low as 0.2 nM. When the acyl chain at the sn-1 position is altered,the rank order of potency is oleoyl-LPA textgreater arachidonoyl-LPA textgreater linolenoyl-LPA textgreater linoleoyl-LPA textgreater stearoyl-LPA = palmitoyl-LPA textgreater myristoyl-LPA. The shorter-chain species,lauroyl- and decanoyl-LPA,show little or no activity. Ether-linked LPA (1-O-hexadecyl-sn-glycero-3-phosphate) is somewhat less potent than the corresponding ester-linked LPA; its stereoisomer is about equally active. Deletion of the glycerol backbone causes a 1000-fold decrease in potency. Replacement of the phosphate group in palmitoyl-LPA by a hydrogen- or methyl-phosphonate moiety results in complete loss of activity. A phosphonate analogue with a methylene group replacing the oxygen at sn-3 has strongly decreased activity. All three phosphonate analogues induce cell lysis at doses textgreater 15 microM. Similarly,the methyl and ethyl esters of palmitoyl-LPA are virtually inactive and become cytotoxic at micromolar doses. None of the LPA analogues tested has antagonist activity. Sphingosine 1-phosphate,a putative messenger with some structural similarities to LPA,elicits a transient rise in intracellular [Ca2+] only at micromolar doses; however,cross-desensitization experiments indicate that sphingosine 1-phosphate does not act through the LPA receptor. The results indicate that,although many features of the LPA structure are important for optimal activity,the phosphate group is most critical,suggesting that this moiety is directly involved in receptor activation. View Publication

Cytosolic aldehyde dehydrogenase (ALDH),an enzyme responsible for oxidizing intracellular aldehydes,has an important role in ethanol,vitamin A,and cyclophosphamide metabolism. High expression of this enzyme in primitive stem cells from multiple tissues,including bone marrow and intestine,appears to be an important mechanism by which these cells are resistant to cyclophosphamide. However,although hematopoietic stem cells (HSC) express high levels of cytosolic ALDH,isolating viable HSC by their ALDH expression has not been possible because ALDH is an intracellular protein. We found that a fluorescent aldehyde,dansyl aminoacetaldehyde (DAAA),could be used in flow cytometry experiments to isolate viable mouse and human cells based on their ALDH content. The level of dansyl fluorescence exhibited by cells after incubation with DAAA paralleled cytosolic ALDH levels determined by Western blotting and the sensitivity of the cells to cyclophosphamide. Moreover,DAAA appeared to be a more sensitive means of assessing cytosolic ALDH levels than Western blotting. Bone marrow progenitors treated with DAAA proliferated normally. Furthermore,marrow cells expressing high levels of dansyl fluorescence after incubation with DAAA were enriched for hematopoietic progenitors. The ability to isolate viable cells that express high levels of cytosolic ALDH could be an important component of methodology for identifying and purifying HSC and for studying cyclophosphamide-resistant tumor cell populations. View Publication

Under appropriate conditions in culture,embryonic stem cells will differentiate and form embryoid bodies that have been shown to contain cells of the hematopoietic,endothelial,muscle and neuronal lineages. Many aspects of the lineage-specific differentiation programs observed within the embryoid bodies reflect those found in the embryo,indicating that this model system provides access to early cell populations that develop in a normal fashion. Recent studies involving the differentiation of genetically altered embryonic stem cells highlight the potential of this in vitro differentiation system for defining the function of genes in early development. View Publication

The available techniques for directed gene manipulation in the mouse are unprecedented in any multicellular organism and make the mouse an invaluable tool for unraveling all aspects of mammalian biology. To realize fully the potential of these genetic tools requires that phenotypic analysis be efficient,rapid,and complete. Genetic chimeras and mosaics,in which mutant cells are mixed with wild-type cells,can be used to augment standard analysis of intact mutant animals and alleviate the time required and the expense involved in generating and maintaining multiple strains of mutant mice. View Publication

Acute lymphoblastic leukaemia (ALL) is the most common cancer of childhood. Despite the progress achieved in its treatment,20% of cases relapse and no longer respond to chemotherapy. The most common phenotype of ALL cells share surface antigens with very early precursors of B cells and are therefore believed to originate from this lineage. Characterization of the growth requirement of ALL cells indicated that they were dependent on various cytokines,suggesting paracrine and/or autocrine growth regulation. Because many cytokines induce tyrosine phosphorylation in lymphoid progenitor cells,and constitutive tyrosine phosphorylation is commonly observed in B-lineage leukaemias,attempts have been made to develop protein tyrosine kinase (PTK) blockers of leukaemia cell growth. Here we show that leukaemic cells from patients in relapse have constitutively activated Jak-2 PTK. Inhibition of Jak-2 activity by a specific tyrosine kinase blocker,AG-490,selectively blocks leukaemic cell growth in vitro and in vivo by inducing programmed cell death,with no deleterious effect on normal haematopoiesis. View Publication

A 145-kDa tyrosine-phosphorylated protein that becomes associated with Shc in response to multiple cytokines has been purified from the murine hemopoietic cell line B6SUtA1. Amino acid sequence data were used to clone the cDNA encoding this protein from a B6SUtA1 library. The predicted amino acid sequence encodes a unique protein containing an N-terminal src homology 2 domain,two consensus sequences that are targets for phosphotyrosine binding domains,a proline-rich region,and two motifs highly conserved among inositol polyphosphate 5-phosphatases. Cell lysates immunoprecipitated with antiserum to this protein exhibited both phosphatidylinositol 3,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate polyphosphate 5-phosphatase activity. This novel signal transduction intermediate may serve to modulate both Ras and inositol signaling pathways. Based on its properties,we suggest the 145-kDa protein be called SHIP for SH2-containing inositol phosphatase. View Publication