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The Ly-49 family consists of at least nine members,of which Ly-49A and C have been found to be NK-cell inhibitory receptors specific for class I MHC. The functions of other Ly-49 molecules are still unclear. Further analysis of Ly-49 is complicated by the cross-reactivities of some anti-Ly-49 antibodies initially thought to be specific for individual Ly-49 molecules. Studies on the role of Ly-49 in hybrid resistance as well as on allelic exclusion are also complicated by our recent finding that a novel Ly-49CB6 gene is the likely allelic form of Ly-49CBALB as opposed to a previously reported highly related but distinct gene in B6 mice. In cell-cell binding assays,only Ly-49A and C show significant binding to class I MHC. Ly-49A and C also bind some polysaccharides,and carbohydrates on class I MHC seem to be important for its binding to Ly-49. However,this interaction involves not only the carbohydrate recognition domain of Ly-49 but also a part of the stalk region,suggesting that both carbohydrates and peptide backbone of class I MHC may be recognized by Ly-49. It is likely that additional Ly-49 molecules yet to be identified function as NK-inhibitory receptors specific for class I MHC. View Publication

MUC-1 mucin is considered to be aberrantly glycosylated in breast,ovary,and other carcinomas in comparison with mucin from corresponding normal tissues. In order to clarify these differences in glycosylation,we have compared the O-linked carbohydrate chains from MUC-1 immunoprecipitated from [3H]GlcN-labeled breast epithelial cell lines (MMSV1-1,MTSV1-7,and HB-2) derived from cells cultured from human milk,with three breast cancer cell lines (MCF-7,BT-20,and T47D). Analysis by high pH anion chromatography showed that the normal cell lines had a higher ratio of GlcN/GalN and more complex oligosaccharide profiles than the cancer cell lines. Structural analyses were carried out on the oligosaccharides from MTSV1-7 and T47D MUC-1,and the following structures were proposed. MUC-1 from T47D had rather a simple glycosylation pattern,with NeuAcalpha2-3Galbeta1-3GalNAc-ol,Galbeta1-3GalNAc-ol,and GalNAc-ol predominating; in contrast,MUC-1 from MTSV1-7 had more complex structures,including a number of disialo,core 2 species,i.e. NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-6[NeuAcalpha2 -3Galbeta1-3]GalNAc- ol and NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-6[NeuAcalpha2 -3Galbeta1-4GlcNAcbet a1-3Galbeta1-3]GalNAc-ol. Double-labeling experiments with [3H]GlcN and 14C-aminoacids and analysis of GalNAc or GalNAc-ol:protein ratios in MUC-1 showed that there was also a significant difference in the degree of glycosylation of the mucin between the two cell types. We conclude that MUC-1 from breast cancer cell lines has simpler,and fewer,carbohydrate chains than MUC-1 from normal breast epithelial cells,and that these differences,combined or separately,explain the differential tumor specificity of some MUC-1 antibodies and T cells. View Publication

The murine heat-stable antigen (HSA) is a glycosyl-phosphatidylinositol-linked cell surface protein which has been implicated in cellular adhesion processes,the co-stimulation of CD4+ T cells,and B cell memory. We have recently demonstrated a significant reduction in pro-B and pre-B lymphocytes in transgenic mice that overexpress HSA. We now report that cross-linking HSA with the M1/69 monoclonal antibody induces the apoptosis of cultured B cell precursors in a stomal cell and cytokine-independent manner and that sensitivity to HSA-mediated cell death increases with developmental maturity. The cross-linking of HSA does not induce apoptosis in mature splenic B cells,but instead inhibits their ability to proliferate in response to anti-CD40 + IL-4. Taken together,these data implicate HSA as a potent negative regulator of B cell development and activation. View Publication

The mechanisms involved in the regulation of vasculogenesis still remain unclear in mammals. Totipotent embryonic stem (ES) cells may represent a suitable in vitro model to study molecular events involved in vascular development. In this study,we followed the expression kinetics of a relatively large set of endothelial-specific markers in ES-derived embryoid bodies (EBs). Results of both reverse transcription-polymerase chain reaction and/or immunofluorescence analysis show that a spontaneous endothelial differentiation occurs during EBs development. ES-derived endothelial cells express a full range of cell lineage-specific markers: platelet endothelial cell adhesion molecule (PECAM),Flk-1,tie-1,tie-2,vascular endothelial (VE) cadherin,MECA-32,and MEC-14.7. Analysis of the kinetics of endothelial marker expression allows the distinction of successive maturation steps. Flk-1 was the first to be detected; its mRNA is apparent from day 3 of differentiation. PECAM and tie-2 mRNAs were found to be expressed only from day 4,whereas VE-cadherin and tie-1 mRNAs cannot be detected before day 5. Immunofluorescence stainings of EBs with antibodies directed against Flk-1,PECAM,VE-cadherin,MECA-32,and MEC-14.7 confirmed that the expression of these antigens occurs at different steps of endothelial cell differentiation. The addition of an angiogenic growth factor mixture including erythropoietin,interleukin-6,fibroblast growth factor 2,and vascular endothelial growth factor in the EB culture medium significantly increased the development of primitive vascular-like structures within EBs. These results indicate that this in vitro system contains a large part of the endothelial cell differentiation program and constitutes a suitable model to study the molecular mechanisms involved in vasculogenesis. View Publication

To understand the mechanism of the sequential restriction of multipotency of stem cells during development,we have established culture conditions that allow the differentiation of neuroepithelial precursor cells from embryonic stem (ES) cells. A highly enriched population of neuroepithelial precursor cells derived from ES cells proliferates in the presence of basic fibroblast growth factor (bFGF). These cells differentiate into both neurons and glia following withdrawal of bFGF. By further differentiating the cells in serum-containing medium,the neurons express a wide variety of neuron-specific genes and generate both excitatory and inhibitory synaptic connections. The expression pattern of position-specific neural markers suggests the presence of a variety of central nervous system (CNS) neuronal cell types. These findings indicate that neuronal precursor cells can be isolated from ES cells and that these cells can efficiently differentiate into functional post-mitotic neurons of diverse CNS structures. View Publication

beta 1 Integrins are known to regulate terminal differentiation and morphogenesis in the adult epidermis. We have investigated their role in the embryonic development of keratinocytes by comparing the differentiation of wild-type and beta 1-null mouse embryonal stem (ES) cells. By 12-15 days in culture,differentiation of embryonic or simple epithelial cells occurred in both ES cell populations,as detected by expression of keratins 8,18,and 19. From 21 days,expression of keratins 10 and 14 and of the cornified envelope precursor involucrin indicated that some of the wild-type cells had differentiated into keratinocytes. In contrast,keratinocyte markers were not expressed in beta 1-null cultures. The beta 1-null cells failed to express the alpha 2 and alpha 3 integrin subunits on the cell surface,consistent with the association of these a subunits with beta 1. Furthermore,alpha 6 and beta 4 expression was reduced in the beta 1-null cultures. Although beta 1-null ES cells failed to undergo differentiation into keratinocytes in vitro,they did form keratinocyte cysts expressing alpha 6 beta 4,keratins 1 and 14,and involucrin when allowed to form teratomas by subcutaneous injection in mice; furthermore,beta 1-null keratinocytes were found in the epidermis of a wild-type/beta 1-null chimeric mouse. As judged by immunofluorescence microscopy,extracellular matrix assembly was severely impaired in beta 1-null ES cell cultures,but not in the teratomas or chimeric mouse skin. We therefore speculate that the failure of beta 1-null cells to differentiate into keratinocytes in vitro may reflect an inability to assemble a basement membrane. View Publication

The inhibitory and bactericidal activities of anacardic acid and totarol,alone and in combination with methicillin,were investigated against methicillin-resistant Staphylococcus aureus (MRSA). The growth of two MRSA strains was inhibited by 6 x 25 microg ml-1 of anacardic acid and 0 x 78 microg ml-1 of totarol. The time-kill curve study showed that these two compounds were bactericidal against MRSA. Anacardic acid killed MRSA cells more rapidly than totarol,and no viable cells were detected after being exposed to 6 x 25 microg ml-1 of anacardic acid for 6 h. Anacardic acid showed bactericidal activity against MRSA at any stage of growth,and also even when cell division was inhibited by chloramphenicol. In the combination studies,the minimal inhibitory concentration (MIC) of methicillin was lowered from 800 to 1 x 56 microg ml-1 for MRSA ATCC 33591,and from 800 to 6 x 25 microg ml-1 for MRSA ATCC 33592,by combining with 1/2 x MIC of anacardic acid. The time-kill curves demonstrated synergistic bactericidal activities for these combinations. View Publication

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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

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

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