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Mouse embryonic stem (ES) cells cultured as aggregates and exposed to retinoic acid are induced to express multiple phenotypes normally associated with neurons. A large percentage of treated aggregates produce a rich neuritic outgrowth. Dissociating the induced aggregates with trypsin and plating the cells as a monolayer results in cultures in which a sizable percentage of the cells have a neuronal appearance. These neuron-like cells express class III beta-tubulin and the neurofilament M subunit. Induced cultures express transcripts for neural-associated genes including the neurofilament L subunit,glutamate receptor subunits,the transcription factor Brn-3,and GFAP. Levels of neurofilament L and GAD67 and GAD65 transcripts rise dramatically upon induction. Physiological studies show that the neuron-like cells generate action potentials and express TTX-sensitive sodium channels,as well as voltage-gated potassium channels and calcium channels. We conclude that a complex system of neuronal gene expression can be activated in cultured ES cells. This system should be favorable for investigating some of the mechanisms that regulate neuronal differentiation. View Publication

Sphingolipids,particularly gangliosides,are enriched in neuronal membranes where they have been implicated as mediators of various regulatory events. We recently provided evidence that sphingolipid synthesis is necessary to maintain neuronal growth by demonstrating that in hippocampal neurons,inhibition of ceramide synthesis by Fumonisin B1 (FB1) disrupted axonal outgrowth (Harel,R. and Futerman,A. H. (1993) J. Biol. Chem. 268,14476-14481). We now analyze further the relationship between neuronal growth and sphingolipid metabolism by examining the effect of an inhibitor of glucosylceramide synthesis,D-threo-1-phenyl-2-decanoylamino-3-morpholino-1- propanol (PDMP) and by examining the effects of both FB1 and PDMP at various stages of neuronal development. No effects of FB1 or PDMP were observed during the first 2 days in culture,but by day 3 axonal morphology was significantly altered,irrespective of the time of addition of the inhibitors to the cultures. Cells incubated with FB1 or PDMP had a shorter axon plexus and less axonal branches. FB1 appeared to cause a retraction of axonal branches between days 2 and 3,although long term incubation had no apparent effect on neuronal morphology or on the segregation of axonal or dendritic proteins. In contrast,incubation of neurons with conduritol B-epoxide,an inhibitor of glucosylceramide degradation,caused an increase in the number of axonal branches and a corresponding increase in the length of the axon plexus. A direct correlation was observed between the number of axonal branch points per cell and the extent of inhibition of either sphingolipid synthesis or degradation. These results suggest that sphingolipids play an important role in the formation or stabilization of axonal branches. View Publication

The AMP-activated protein kinase (AMPK) is believed to protect cells against environmental stress (e.g. heat shock) by switching off biosynthetic pathways,the key signal being elevation of AMP. Identification of novel targets for the kinase cascade would be facilitated by development of a specific agent for activating the kinase in intact cells. Incubation of rat hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) results in accumulation of the monophosphorylated derivative (5-aminoimidazole-4-carboxamide ribonucleoside; ZMP) within the cell. ZMP mimics both activating effects of AMP on AMPK,i.e. direct allosteric activation and promotion of phosphorylation by AMPK kinase. Unlike existing methods for activating AMPK in intact cells (e.g. fructose,heat shock),AICAR does not perturb the cellular contents of ATP,ADP or AMP. Incubation of hepatocytes with AICAR activates AMPK due to increased phosphorylation,causes phosphorylation and inactivation of a known target for AMPK (3-hydroxy-3-methylglutaryl-CoA reductase),and almost total cessation of two of the known target pathways,i.e. fatty acid and sterol synthesis. Incubation of isolated adipocytes with AICAR antagonizes isoprenaline-induced lipolysis. This provides direct evidence that the inhibition by AMPK of activation of hormone-sensitive lipase by cyclic-AMP-dependent protein kinase,previously demonstrated in cell-free assays,also operates in intact cells. AICAR should be a useful tool for identifying new target pathways and processes regulated by the protein kinase cascade. View Publication

Thrombopoietin (TPO),a lineage-specific cytokine affecting the proliferation and maturation of megakaryocytes from committed progenitor cells,is believed to be the major physiological regulator of circulating platelet levels. Recently we have isolated a cDNA encoding a ligand for the murine c-mpl protooncogene and shown it to be TPO. By employing a murine cDNA probe,we have isolated a gene encoding human TPO from a human genomic library. The TPO locus spans over 6 kb and has a structure similar to that of the erythropoietin gene (EPO). Southern blot analysis of human genomic DNA reveals a hybridization pattern consistent with a single gene locus. The locus was mapped by in situ hybridization of metaphase chromosome preparations to chromosome 3q26-27,a site where a number of chromosomal abnormalities associated with thrombocythemia in cases of acute myeloid leukemia have been mapped. A human TPO cDNA was isolated by PCR from kidney mRNA. The cDNA encodes a protein with 80% identity to previously described murine TPO and is capable of initiating a proliferative signal to murine interleukin 3-dependent BaF3 cells expressing the murine or human TPO receptor. View Publication

We have cloned the FP receptor from rat corpus luteum and human uterus cDNA libraries,respectively. The coding DNA sequence in the rat cDNA is 1101 bp and is similar to the mouse cDNA coding for a receptor protein of 366 amino acids. The human sequence shows a 5 bp deficiency in the 3' region,truncating the coding sequence to 359 amino acids. Northern blot analysis indicates highest expression in the ovary. Cell lines have been established giving stable expression of the FP receptor. Activation of the cloned FP receptor gave an increase in intracellular calcium,indicating signaling via phospholipase C-mediated phosphoinositide turnover. Using [3H]PGF2 alpha,binding of PGs showed the rank order of fluprostenol textgreater PhXA70 textgreater PGF2 alpha textgreater or = PhXA85 textgreater PGD2 textgreater PGE2. View Publication

In fura-2-loaded ECV304 cells ionomycin elicited a saturable biphasic change in intracellular Ca2+ concentration ([Ca2+]i),where the initial phase represented mobilization of intracellular stores and the sustained component represented Ca2+ influx. To examine whether ionomycin could stimulate influx via a store-dependent mechanism. Mn2+ entry was monitored by the quenching of fura-2 fluorescence: influx was enhanced even after ionomycin wash-out,provided that internal stores were not refilled with Ca2+. Moreover,the maximal rate of histamine-stimulated Mn2+ entry was unaffected by ionomycin,suggesting a common route of entry. The Ca(2+)-entry blocker SK&F 96365 inhibited both the ionomycin-induced Mn2+ entry and the sustained [Ca2+]i response to the ionophore (leaving the initial peak [Ca2+]i response unaffected). In other experiments,although addition of ionomycin further increased the plateau phase induced by 100 microM histamine,the increase was completely abolished by pretreatment with the store Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA). Furthermore,in store-depleted cells,re-addition of 1 mM extracellular Ca2+ (in the presence of CPA plus histamine) led to a rapid rise in [Ca2+]i,dependent on Ca2+ influx,with kinetics that were not enhanced by ionomycin. These data suggest that ionomycin acts primarily at the level of the internal Ca2+ stores,so that,at the concentrations used here (textless or = 1 microM),it increases Ca2+ (and Mn2+) influx via activation of endogenous entry pathways and not by plasmalemmal translocation. View Publication

An efficient system was developed that induced the differentiation of embryonic stem (ES) cells into blood cells of erythroid,myeloid,and B cell lineages by coculture with the stromal cell line OP9. This cell line does not express functional macrophage colony-stimulating factor (M-CSF). The presence of M-CSF had inhibitory effects on the differentiation of ES cells to blood cells other than macrophages. Embryoid body formation or addition of exogenous growth factors was not required,and differentiation was highly reproducible even after the selection of ES cells with the antibiotic G418. Combined with the ability to genetically manipulate ES cells,this system will facilitate the study of molecular mechanisms involved in development and differentiation of hematopoietic cells. View Publication

Individually,transforming growth factor beta (TGF beta) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) alter the growth and differentiation of normal and transformed osteoblast-like (OB) cells. Although recent evidence suggests interactions between TGF beta and 1,25(OH)2D3 may occur,little is known of the individual or combined effects of these hormones on the expression of the osteoblast phenotype at the cytochemical and biochemical levels in normal human OB (hOB) cells. Primary cultures of hOBs were treated with TGF beta (0.001-10 ng/ml) and 1,25(OH)2D3 (0.1 pM-100 nM) either alone or in combination. TGF beta and 1,25(OH)2D3 stimulated spindle-shaped cells to become stellate in appearance and increased the number of cytoplasmic processes. TGF beta increased 3H-thymidine incorporation and 1,25(OH)2D3 reduced this effect. Conversely,procollagen type-I synthesis and secretion were increased in a dose-dependent manner in the presence of TGF beta but were not significantly affected in the presence of 1,25(OH)2D3. TGF beta and 1,25(OH)2D3 each marginally increased alkaline phosphatase (ALP) activity,but the combination synergistically increased ALP activity in a dose- and time-dependent manner at the cytochemical and biochemical level (three to tenfold over vehicle controls; n = 12). In contrast,TGF beta reduced 1,25(OH)2D3-stimulated osteocalcin secretion. These data suggest that TGF beta stimulates hOB cells to actively produce collagen matrix and proliferate. The combination of TGF beta and 1,25(OH)2D3,however,produces a synergistic increase in ALP activity and maintenance of collagen synthesis. 1,25(OH)2D3 stimulation may induce cells to advance to an endstage where cell proliferation is reduced and osteocalcin expression is promoted. Interactions between TGF beta and 1,25(OH)2D3 may represent important steps in the regulation of osteoblast differentiation and matrix production. View Publication

The epithelial glycoprotein 40 (EGP40,also known as GA733-2,ESA,KSA,and the 17-1A antigen),encoded by the GA-733-2 gene,is expressed on the baso-lateral cell surface in most human simple epithelia. The protein is also expressed in the vast majority of carcinomas and has attracted attention as a tumor marker. The function of the protein is unknown. We demonstrate here that EGP40 is an epithelium-specific intercellular adhesion molecule. The molecule mediates,in a Ca(2+)-independent manner,a homophilic cell-cell adhesion of murine cells transfected with the complete EGP40 cDNA. Two murine cell lines were tested for the effects of EGP40 expression: fibroblastic L cells and dedifferentiated mammary carcinoma L153S cells. The expression of the EGP40 protein causes morphological changes in cultures of transfected cells--increasing intercellular adhesion of the transfectants--and has a clear effect on cell aggregating behavior in suspension aggregation assays. EGP40 directs sorting in mixed cell populations,in particular,causes segregation of the transfectants from the corresponding parental cells. EGP40 expression suppresses invasive colony growth of L cells in EHS-matrigel providing tight adhesions between cells in growing colonies. EGP40 can thus be considered a new member of the intercellular adhesion molecules. In its biological behavior EGP40 resembles to some extent the molecules of the immunoglobulin superfamily of cell adhesion molecules (CAMs),although no immunoglobulin-like repeats are present in the EGP40 molecule. Certain structural similarities in general organization of the molecule exist between EGP40 and the lin-12/Notch proteins. A possible role of this adhesion molecule in formation of architecture of epithelial tissues is discussed. To reflect the function of the molecule the name Ep-CAM for EGP40 seems appropriate. View Publication

A novel series of 3,4-dihydroxychalcones was synthesized to evaluate their effects against 5-lipoxygenase and cyclooxygenase. Almost all compounds exhibited potent inhibitory effects on 5-lipoxygenase with antioxidative effects,and some also inhibited cyclooxygenase. The 2',5'-disubstituted 3,4-dihydroxychalcones with hydroxy or alkoxy groups exhibited optimal inhibition of cyclooxygenase. We found that 2',5'-dimethoxy-3,4-dihydroxychalcone (37; HX-0836) inhibited cyclooxygenase to the same degree as flufenamic acid and 5-lipoxygenase,more than quercetin. Finally,these active inhibitors of 5-lipoxygenase inhibited arachidonic acid-induced mouse ear edema more than phenidone. View Publication

There is a medical need for an agent with the positive effects of estrogen on bone and the cardiovascular system,but without the negative effects on reproductive tissue. Raloxifene (LY139481 HCI) is a benzothiophene derivative that binds to the estrogen receptor and inhibits the effects of estrogen on the uterus. In an ovariectomized (OVX) rat model we investigated the effects of raloxifene on bone loss (induced by estrogen deficiency),serum lipids,and uterine tissue. After oral administration of raloxifene for 5 wk (0.1-10 mg/kg per d) to OVX rats,bone mineral density in the distal femur and proximal tibia was significantly greater than that observed in OVX controls (ED50 of 0.03-0.3 mg/kg). Serum cholesterol was lower in the raloxifene-treated animals,which had a minimal effective dose of 0.1 mg/kg and an approximate oral ED50 of 0.2 mg/kg. The effects of raloxifene on bone and serum cholesterol were comparable to those of a 0.1-mg/kg per d oral dose of ethynyl estradiol. Raloxifene diverged dramatically from estrogen in its lack of significant estrogenic effects on uterine tissue. Ethynyl estradiol produced a marked elevation in a number of uterine histologic parameters (e.g.,epithelial cell height,stromal eosinophilia). These data suggest that raloxifene has promise as an agent with beneficial bone and cardiovascular effects in the absence of significant uterine effects. View Publication

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