Scientific Resources

We have systematically optimized the concentrations of 20 components of a previously published serum-free medium (Brewer and Cotman,Brain Res 494: 65-74,1989) for survival of rat embryonic hippocampal neurons after 4 days in culture. This serum-free medium supplement,B27,produced neuron survival above 60%,independent of plating density above 160 plated cells/mm2. For isolated cells (textless 100 cells/mm2),survival at 4 days was still above 45%,but could be rescued to the 60% level at 40 cells/mm2 by simply applying a coverslip on top of the cells. This suggests a need for additional trophic factors. High survival was achieved with osmolarity lower than found in Dulbecco's Modified Eagle's Medium (DMEM),and by reducing cysteine and glutamine concentrations and by the elimination of toxic ferrous sulphate found in DME/F12. Neurobasal is a new medium that incorporates these modifications to DMEM. In B27/Neurobasal,glial growth is reduced to less than 0.5% of the nearly pure neuronal population,as judged by immunocytochemistry for glial fibrillary acidic protein and neuron-specific enolase. Excellent long-term viability is achieved after 4 weeks in culture with greater than 90% viability for cells plated at 640/mm2 and greater than 50% viability for cells plated at 160/mm2. Since the medium also supports the growth of neurons from embryonic rat striatum,substantia nigra,septum,and cortex,and neonatal dentate gyrus and cerebellum (Brewer,in preparation),support for other neuron types is likely. B27/Neurobasal should be useful for in vitro studies of neuronal toxicology,pharmacology,electrophysiology,gene expression,development,and effects of growth factors and hormones. View Publication

An obligatory step in retroviral growth is the integration of a DNA copy of the viral RNA into the genomic DNA of the host. Recombinant human immunodeficiency virus type I (HIV-1) integrase (IN) expressed in Escherichia coli efficiently catalyzes the overall in vitro integration reaction,namely the processing of the LTR ends and the strand transfer reaction. Using the 3' end of synthetic oligonucleotides which match the termini of the HIV-1 U5 LTR as substrate and supercoiled pSP65 DNA as target,we have investigated the effect of the polyanionic drug suramin on the catalytic activity of the IN protein. It was found that at stoichiometric suramin to protein ratios,suramin displays a strong inhibitory effect on both the processing and strand transfer reactions. This inhibitory effect is related to the decrease of IN protein binding efficiency to the LTR end DNA fragment. View Publication

Analysis of [3H]phorbol-12,13-dibutyrate (PDBu) binding was performed with protein kinase C (PKC)-alpha,-beta 1,-gamma,-delta,-epsilon,-eta,and -zeta produced in Sf9 insect cells using the baculovirus expression system. With the exception of PKC-zeta,all of the PKC isozymes bound [3H]PDBu with high affinity (Kd textless 1 nM),either in the presence or in the absence of calcium. Scatchard analysis using 100% phosphatidylserine vesicles revealed slightly lower affinity for the calcium-independent isozymes (PKC-delta,-epsilon,and -eta) than for the calcium-dependent isozymes (PKC-alpha,-beta,and -gamma). Competition for [3H]PDBu binding by different classes of PKC activators showed that 12-deoxyphorbol esters,mezerein,and octahydromezerein likewise possessed lower affinity for the calcium-independent isozymes. The mezerein analog thymeleatoxin was the most marked example,being almost 20-fold less potent for binding to PKC-epsilon and -eta than to PKC-beta 1. In contrast,the indole alkaloids (-)-indolactam V and (-)-octylindolactam V and the postulated endogenous activator 1,2-diacylglycerol bound with similar affinities to all of the PKC isoforms,suggesting that different residues/configurations in the binding sites of the different PKC isozymes might be involved in interaction with the pharmacophore of the activators. The seven PKC isozymes also showed clearly different substrate specificities with exogenous peptide and protein substrates. The heterogeneous behavior of the different members of the PKC family with ligands and substrates may contribute to the heterogeneity of PKC-mediated pathways at the cellular level. View Publication

A cDNA clone coding for a functional human prostanoid FP receptor has been isolated from a uterus cDNA library. The human FP receptor consists of 359 amino acid residues with a predicted molecular mass of 40,060,and has the seven putative transmembrane domains characteristic of G-protein-coupled receptors. Challenge of Xenopus oocytes expressing the FP receptor with 10 nM of either prostaglandin (PG) F2 alpha or the selective FP-receptor agonist fluprostenol resulted in an elevation in intracellular Ca2+. Radioreceptor binding studies using membranes prepared from mammalian COS cells transfected with the FP receptor cDNA showed that the rank order of potency for prostaglandins and prostaglandin analogs in competition for [3H]PGF2 alpha specific binding sites was as predicted for the FP receptor,with PGF2 alpha approximately fluprostenol textgreater PGD2 textgreater PGE2 textgreater U46619 textgreater iloprost. In summary,we have cloned the human prostanoid FP receptor which is functionally coupled to the Ca2+ signalling pathway. 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

Most primitive hematopoietic cells appear to be normally quiescent in vivo,whereas their leukemic counterparts in patients with chronic myeloid leukemia (CML) are maintained in a state of rapid turnover. This difference is also seen in the long-term culture system,where control of primitive hematopoietic progenitor proliferation is mediated by interactions of these cells with marrow-derived mesenchymal cells of the fibroblast lineage. We now show that exogenous addition of macrophage inflammatory protein 1 alpha (MIP-1 alpha) to normal long-term cultures can reversibly and specifically block the activation of primitive" (high proliferative potential)� 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

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

Pluripotent embryonic stem cells (ESC,ES cells) of line D3 were differentiated in vitro and via embryo-like aggregates (embryoid bodies) of defined cell number into spontaneously beating cardiomyocytes. By using RT-PCR technique,alpha- and beta-cardiac myosin heavy chain (MHC) genes were found to be expressed in embryoid bodies of early to terminal differentiation stages. The exclusive expression of the beta-cardiac MHC gene detected in very early differentiated embryoid bodies proved to be dependent on the number of ES cells developing in the embryoid body. Cardiomyocytes enzymatically isolated from embryoid body outgrowths at different stages of development were further characterized by immunocytological and electrophysiological techniques. All cardiomyocytes appeared to be positive in immunofluorescence assays with monoclonal antibodies against cardiac-specific alpha-cardiac MHC,as well as muscle-specific sarcomeric myosin heavy chain and desmin. The patch-clamp technique allowed a more detailed characterization of the in vitro differentiated cardiomyocytes which were found to represent phenotypes corresponding to sinusnode,atrium or ventricle of the heart. The cardiac cells of early differentiated stage expressed pacemaker-like action potentials similar to those described for embryonic cardiomyocytes. The action potentials of terminally differentiated cells revealed shapes,pharmacological characteristics and hormonal regulation inherent to adult sinusnodal,atrial or ventricular cells. In cardiomyocytes of intermediate differentiation state,action potentials of very long duration (0.3-1 s) were found,which may represent developmentally controlled transitions between different types of action potentials. Therefore,the presented ES cell differentiation system permits the investigation of commitment and differentiation of embryonic cells into the cardiomyogenic lineage in vitro. View Publication

The cell line described here was established for a 50-year-old male patient with rapidly progressive non-Hodgkin's lymphoma whose marrow was diffusely infiltrated with large granular lymphocytes (LGL). Immunophenotyping of marrow blasts and peripheral lymphocytes was positive for CD56,CD2 and CD7,and negative for CD3. Cytotoxicity of peripheral blood mononuclear cells at an effector: target (E:T) cell ratio of 50:1 was 79% against K562 cells and 48% against Daudi cells. To establish the line,cells from the peripheral blood were placed into enriched alpha medium containing 12.5% fetal calf serum,12.5% horse serum,10(-4) M beta-mercaptoethanol and 10(-6) M hydrocortisone. Growth of the line (termed NK-92) is dependent on the presence of recombinant IL-2 and a dose as low as 10 U/ml is sufficient to maintain proliferation. Conversely,cells die within 72 h when deprived of IL-2; IL-7 and IL-12 do not maintain long-term growth,although IL-7 induces short-term proliferation measured by 3H-thymidine incorporation. None of the other cytokines tested (IL-1 alpha,IL-6,TNF-alpha,IFN-alpha,IFN-gamma) supported growth of NK-92 cells which have the following characteristics: surface marker positive for CD2,CD7,CD11a,CD28,CD45,CD54,CD56bright; surface marker negative for CD1,CD3,CD4,CD5,CD8,CD10,CD14,CD16,CD19,CD20,CD23,CD34,HLA-DR. DNA analysis showed germline configuration for T-cell receptor beta and gamma genes. CD25 (p55 IL-2 receptor) is expressed on about 50% of all cells when tested at 100 U/ml of IL-2 and its expression correlates inversely with the IL-2 concentration. The p75 IL-2 receptor is expressed on about half of the cells at low density irrespective of the IL-2 concentration. NK-92 cells kill both K562 and Daudi cells very effectively in a 4 h51-chromium release assay (84 and 86% respectively,at an E:T cell ratio of 5:1). The cell line described here thus displays characteristics of activated NK-cells and could be a valuable tool to study their biology. View Publication

The FLT3/FLK2 receptor tyrosine kinase is closely related to two receptors,c-Kit and c-Fms,which function with their respective ligands,Kit ligand and macrophage colony-stimulating factor to control differentiation of haematopoietic and non-haematopoietic cells. FLT3/FLK2 is thought to be present on haematopoietic stem cells and found in brain,placenta and testis. We have purified to homogeneity and partially sequenced a soluble form of the FLT3/FLK2 ligand produced by mouse thymic stromal cells. We isolated several mouse and human complementary DNAs that encode polypeptides with identical N termini and different C termini. Some variants contain hydrophobic transmembrane segments,suggesting that processing may be required to release soluble ligand. The purified ligand enhances the response of mouse stem cells and a primitive human progenitor cell population to other growth factors such as interleukins IL-3 and IL-6 and to granulocyte-macrophage colony-stimulating factor,and also stimulates fetal thymocytes. 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