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B Obrink

Publications and source records attributed to B Obrink.

At least 37 records · Page 2Linked to original sources
PubMed1994

Distribution of C-CAM in developing oral tissues.

C-CAM is a cell surface glycoprotein that is involved in cell adhesion and may play a role in histogenesis and organogenesis. It is a member of the carcinoembryonic antigen (CEA) gene family, which is a subfamily of the immunoglobulin gene superfamily. We have analyzed the expression of C-CAM during normal and disturbed craniofacial development in the mouse by immunohistochemistry and in situ hybridization. Developmental disturbances were induced by retinoic acid (RA) treatment of pregnant mice. Normal and malformed fetuses were examined on days 14, 15, 16, 17 and 18 of gestation. The expression of C-CAM was detected first at day 16. With age, the signal became gradually stronger. C-CAM was detected in the epithelia of both ectodermal and mesodermal origin, including oral and respiratory epithelia, epithelia of the developing vessels, glands and their ducts. In the RA-treated fetuses, the expression of C-CAM was higher in the epithelium of the oral cavity than in that of the nasal cavity, with a distinct borderline between differentiating nasal and oral epithelium of the palatal shelves. However, the submucosal nasal glands and ducts showed higher expression than oral glands in both normal and RA-treated mice. The expression of C-CAM did not differ significantly between control and RA-treated animals. The presence of C-CAM in all proliferating craniofacial epithelia indicates that this molecule may play an important role in development.

Adenosine Triphosphatases↗
PubMed1994

Differential regulation of C-CAM isoforms in epithelial cells.

C-CAM is a Ca(2+)-independent cell adhesion molecule (CAM) that mediates intercellular adhesion of isolated rat hepatocytes. It is widely distributed in epithelia, where its presence both at lateral cell borders and on apical cell surfaces suggests that it may have diverse biological functions. Two major isoforms, C-CAM1 and C-CAM2, which differ in the lengths of their cytoplasmic domains, have been identified. The lack of suitable in vitro systems has so far prevented a detailed study of the physiological role of C-CAM in epithelia. We now report on the identification, biochemical characterization and functional analysis of C-CAM isoforms in the established epithelial cell line NBT II, derived from a chemically induced carcinoma of rat bladder. C-CAM in NBT II cells is a 110-115 kDa cell surface glycoprotein located predominantly at sites of cell-cell contact but also present on the apical cell surface. Northern blotting analysis revealed the presence of both C-CAM1 and C-CAM2, with the major transcripts for both isoforms present within the 4.0 kb size range. The dissociation of NBT II cell colonies by anti-C-CAM antibodies indicated that at least one function of C-CAM in these cells is to mediate intercellular adhesion. The maintenance of extensive cell-cell contacts and the expression of C-CAM at the contact sites in cells grown in low Ca2+ medium suggested that, like its counterpart in hepatocytes, C-CAM in NBT II cells may be a Ca(2+)-independent cell-cell adhesion molecule. The co-localization and coordinate reorganization of both C-CAM and actin by anti-C-CAM antibodies indicated that these two proteins were associated and suggested that interactions with the cytoskeleton may be important for the regulation of C-CAM function. The specific upregulation of C-CAM1 in cells induced to undergo epithelial to mesenchymal-like transitions (EMT) by the serum substitute Ultroser G suggested that C-CAM isoforms are important modulators of the adhesive properties of these cells.

Actins↗
PubMed1993

Transmembrane neural cell-adhesion molecule (NCAM), but not glycosyl-phosphatidylinositol-anchored NCAM, down-regulates secretion of matrix metalloproteinases.

During embryogenesis interactions between cells and extracellular matrix play a central role in the modulation of cell motility, growth, and differentiation. Modulation of matrix structure is therefore crucial during development; extracellular matrix ligands, their receptors, extracellular proteinases, and proteinase inhibitors all participate in the construction, maintenance, and remodeling of extracellular matrix by cells. The neural cell-adhesion molecule (NCAM)-negative rat glioma cell line BT4Cn secretes substantial amounts of metalloproteinases, as compared with its NCAM-positive mother cell line BT4C. We have transfected the BT4Cn cell line with cDNAs encoding the human NCAM-B and -C isoforms. We report here that the expression of transmembrane NCAM-B, but not of glycosyl-phosphatidylinositol-linked NCAM-C, induces a down-regulation of 92-kDa gelatinase (matrix metalloproteinase 9) and interstitial collagenase (matrix metalloproteinase 1), indicating that cellular expression of the recognition molecule NCAM regulates the metabolism of the surrounding matrix.

Animals↗
PubMed1993

Evidence for calmodulin binding to the cytoplasmic domains of two C-CAM isoforms.

C-CAM (cell-CAM 105) is a transmembrane cell adhesion molecule, belonging to the immunoglobulin superfamily. It is expressed in epithelia, vessel endothelia and leukocytes, and mediates intercellular adhesion in rat hepatocytes by homophilic binding. Two major isoforms (C-CAM1 and C-CAM2) that differ in their cytoplasmic domains occur. A previous study demonstrated that C-CAM can bind calmodulin in a Ca(2+)-dependent manner. In this study we have expressed the cytoplasmic domains of C-CAM1 and C-CAM2 in fusion proteins and measured calmodulin binding by a gel overlay assay, using 125I-labelled calmodulin. Our results indicate that the cytoplasmic domains of both C-CAM1 and C-CAM2 can bind calmodulin.

Adenosine Triphosphatases↗
PubMed1993

Different isoforms and stock-specific variants of the cell adhesion molecule C-CAM (cell-CAM 105) in rat liver.

C-CAM is a cell adhesion molecule of the immunoglobulin superfamily with homophilic binding properties. Here we used the polymerase chain reaction to isolate clones of C-CAM from a rat liver cDNA library. Sequence analyses identified two major isoforms, C-CAM1 and C-CAM2, which differed in their 3' ends. C-CAM2 lacked a sequence of 53 nucleotides that was present in C-CAM1. This causes a frame shift and new stop codons, which gives rise to cytoplasmic domains of different sizes in the two isoforms (10 versus 71 amino-acid residues). In addition, all the clones had a different nucleotide and deduced amino-acid sequence (variant b) in the most N-terminal of the four immunoglobulin-like domains, compared to a previously published C-CAM sequence (variant a). Northern-blot analyses with specific oligonucleotide probes demonstrated that two different rat stocks expressed either variant a or variant b. Northern-blot analyses of rat liver and lung also showed that at least five different C-CAM transcripts are produced. Two major mRNA size classes of 4.0 kb and 6.0 kb, and one minor class of 3.0 kb were found. Both the 4.0-kb and 3.0-kb messenger classes reacted with two different probes that could distinguish between C-CAM1 and C-CAM2, while the 6.0-kb population only reacted with the probe selective for C-CAM1. Taken together these data demonstrate the existence of four different protein-coding sequences of rat liver C-CAM (C-CAM1 a and b, and C-CAM2 a and b). We suggest that both allelic variation and alternative splicing may contribute to the isoform-expression pattern of C-CAM in rats.

Amino Acid Sequence↗
PubMed1992

Decreased expression of E-cadherin and increased invasive capacity in EBV-LMP-transfected human epithelial and murine adenocarcinoma cells.

The EBV-encoded membrane protein LMP is one of 9 viral proteins regularly expressed in virally immortalized B lymphocytes. It is expressed in EBV-carrying lymphoblastoid cell lines of normal origin and in the majority of the poorly differentiated nasopharyngeal carcinomas, but not in Burkitt lymphomas. LMP has been reported to transform rodent fibroblasts, to inhibit epithelial differentiation and to alter morphology and cytokeratin expression in an in vitro immortalized human keratinocyte cell-line, RHEK-I. We now report that an LMP-transfected mouse mammary carcinoma line, SHG, exhibits a similar morphological change to that previously described in the LMP-transfected RHEK-I. In the LMP-transfected RHEK-I and SHG cells, we observed a decreased expression of the calcium-dependent adhesion molecule E-cadherin. The LMP-transfected RHEK-I cells were capable of invading type-I collagen gels while the control cells were not. The LMP-transfected SHG cells showed a significantly higher invasive capacity than the original cell line.

Adenocarcinoma↗
PubMed1992

C-CAM (Cell-CAM 105) is a calmodulin binding protein.

C-CAM (Cell-CAM 105) is a transmembrane cell adhesion molecule belonging to the immunoglobulin superfamily. It mediates intercellular adhesion of rat hepatocytes and occurs in various isoforms in several epithelia, vessel endothelia and leukocytes. We now report that purified liver C-CAM interacts specifically with calmodulin. Binding was observed both when 125I-labeled C-CAM was used in a dot-blot assay and when 125I-labeled calmodulin was used in a gel overlay assay. Experiments with protease-generated peptides indicated that calmodulin bound to the cytoplasmic domain of C-CAM. Analyses of whole liver membranes demonstrated that C-CAM is one of five major proteins that bind calmodulin in a calcium-dependent manner.

Adenosine Triphosphatases↗
PubMed1991

C-CAM (cell-CAM 105)--a member of the growing immunoglobulin superfamily of cell adhesion proteins.

Cell recognition and adhesion, being of prime importance for the formation and integrity of tissues, are mediated by cell adhesion molecules, which can be divided into several distinct protein superfamilies. The cell adhesion molecule C-CAM (cell-CAM 105) belongs to the immunoglobulin superfamily, and more specifically is a member of the carcinoembryonic antigen (CEA) gene family. C-CAM can mediate adhesion between hepatocytes in vitro in a homophilic, calcium-independent binding reaction. The molecule, which occurs in various isoforms, is expressed in liver, several epithelia, vessel endothelia, platelets and granulocytes and its expression is dynamically regulated under various physiological and pathological conditions. It is proposed that C-CAM in different cells and tissues plays different functional roles, where the common denominator is membrane-membrane binding.

Adenosine Triphosphatases↗
PubMed1991

Cell-cell contacts mediated by E-cadherin (uvomorulin) restrict invasive behavior of L-cells.

L-cells were cotransfected with plasmids coding for mouse E-cadherin (uvomorulin) and the neophosphotransferase gene, and stable transfectants expressing E-cadherin at the cell surface were selected and cloned. Control transfection was done with the neophosphotransferase gene alone. The invasive migration of transfected and untransfected L-cells into three-dimensional collagen gels was then analyzed. L-cells not expressing E-cadherin migrated efficiently into the gels, whereas invasion of the E-cadherin-expressing L-cells was restricted in a cell density dependent manner. At sparse density, when the cells exhibited little cell-cell contacts, no difference was observed between the level of invasion of the cadherin-expressing cells and the control cells. However, with increasing cell density, decreasing amounts of the cadherin-expressing cells but increasing amounts of the control cells migrated into the gels. At confluent density hardly any cadherin-expressing cells were able to migrate into the gels. The inhibition of the invasion of the cadherin-expressing cells could be reverted if confluent cells were cultured in the presence of monoclonal antibodies against E-cadherin. Since the expression of E-cadherin did not influence the invasive mobility of single cells, these results indicate that E-cadherin-mediated cell-cell contacts inhibited invasive cellular migration. Time-lapse videoscopy and studies of cell migration from a monolayer into a cell-free area demonstrated that the restricted invasion could be explained by contact inhibition of cell movement of the cadherin-expressing cells.

Animals↗
PubMed1990

The cell adhesion molecule Cell-CAM 105 is an ecto-ATPase and a member of the immunoglobulin superfamily.

Cell-CAM 105 (C-CAM), a cell adhesion molecule in rat hepatocytes, was digested with trypsin, and peptides were isolated and sequenced by Edman degradation. The sequences of 4 peptides agreed with different regions of rat liver ecto-ATPase. Detailed biochemical analyses confirmed the identity between C-CAM and the ecto-ATPase. C-CAM/ecto-ATPase is a transmembrane protein having 4 immunoglobulin-like domains in the extracellular portion, demonstrating membership of the immunoglobulin superfamily. The ATPase activity suggests that ATP might influence cell adhesion, which would explain the inhibitory effect of exogenously added ATP on adhesion of several cell types.

Adenosine Triphosphatases↗
PubMed1990

Comparison and functional characterization of C-CAM, glycoprotein IIb/IIIa and integrin beta 1 in rat platelets.

Three adhesion-related proteins in rat platelets were compared, namely C-CAM, gpIIb/IIIa, and integrin beta 1. These three proteins behaved as distinct components as judged by both biochemical and immunological analyses. GpIIb/IIIa bound to a GRGDSPC-peptide, but neither beta 1-containing integrins nor C-CAM had any affinity either for this peptide or for a large cell-binding fragment of fibronectin. C-CAM and integrin beta 1 behaved differently when platelets were labeled with 125I, solubilized by detergent, and immunoprecipitated. Significant amounts of labeled C-CAM was precipitated when the platelets were first solubilized with detergent and then 125I-labeled. Almost no labeled C-CAM could be precipitated when intact platelets, that were unactivated or activated by ADP, were labeled. In contrast, labeled integrin beta 1 was immunoprecipitated when unactivated platelets were surface-labeled. However, when platelets that were activated by ADP and calcium ions were labeled almost no labeled integrin beta 1 could be immunoprecipitated. These data indicate 1) that C-CAM in intact platelets is inaccessible to surface-labeling and 2) that beta 1 integrin is less accessible to surface-labeling after platelet activation in the presence of calcium ions.

Adenosine Triphosphatases↗
PubMed1990

C-CAM (cell-CAM 105) is an adhesive cell surface glycoprotein with homophilic binding properties.

C-CAM (Cell-CAM 105) is a cell surface glycoprotein that is involved in cell-cell adhesion of rat hepatocytes in vitro. To elucidate the adhesion mechanism the binding properties of purified C-CAM were investigated. Using proteins immobilized on nitrocellulose it was found that radiolabeled C-CAM bound to C-CAM but not to a variety of other proteins. Partitioning in Triton X-114 showed that C-CAM has hydrophobic properties. In accordance with this, C-CAM was effectively incorporated into phosphatidylcholine liposomes by dialysis from octylglucoside-containing solutions. The C-CAM-containing liposomes bound specifically to isolated hepatocytes. This binding was blocked by Fab fragments of anti-C-CAM antibodies. Furthermore, preincubation of hepatocytes with anti-C-CAM antibodies followed by washing of the cells blocked binding of C-CAM-containing liposomes. At increasing C-CAM contents in the reconstituted liposomes a marked self-aggregation of the liposomes occurred. This aggregation was blocked by Fab fragments of anti-C-CAM antibodies and by alkaline pH. After neutralization a rapid reaggregation occurred. Neither C-CAM binding to C-CAM immobilized on nitrocellulose nor C-CAM-liposome aggregation required calcium ions. Liposomes reconstituted with C-CAM-depleted membrane glycoproteins did not self-aggregate or bind to hepatocytes. Thus, it is concluded that C-CAM can bind specifically to C-CAM in a homophilic binding reaction that does not require calcium. Accordingly, C-CAM has the potential of directly mediating cell-cell adhesion via C-CAM-C-CAM binding between adjacent cells.

Adenosine Triphosphatases↗
PubMed1990

Expression of cellCAM-105 in the apical surface of rat uterine epithelium is controlled by ovarian steroid hormones.

Affinity-purified antibodies to cellCAM-105, an adhesive cell surface glycoprotein, were used in immunohistochemical investigations of rat uteri at various functional stages: (i) the oestrous, pro-oestrous, metoestrous, and dioestrous stages of the oestrous cycle, (ii) Days 1-8 of normal pregnancy, (iii) delayed implantation, (iv) 18 h after oestrogen reactivation from delay of implantation, and (v) juvenile rats, and normal ovariectomized adults, respectively, before and after experimental injection of progesterone and/or oestrogen. CellCAM-105 was present in the apical zones of the luminal and glandular epithelium cells in a stage-specific and hormone-dependent manner. The results indicate that: (1) steroid hormones are essential for the expression of cellCAM-105 in the uterine epithelial cells; (2) progesterone induces cellCAM-105 expression in the glandular epithelium, and oestrogen induces cellCAM-105 expression in the luminal epithelium; (3) progesterone induces down-regulation of cellCAM-105 from the surface of the uterine luminal epithelium of juvenile rats; (4) cellCAM-105 is absent in the luminal epithelial cells but present in the glandular epithelial cells of the rat uterus at the time of blastocyst implantation.

Adenosine Triphosphatases↗
PubMed1989

Structural assessment of the N-linked oligosaccharides of cell-CAM 105 by lectin-agarose affinity chromatography.

The N-linked oligosaccharides of cell-CAM 105, a glycoprotein involved in the intercellular adhesion between rat hepatocytes, were studied by sequential lectin-agarose affinity chromatography of desialylated, [14C]-labelled glycopeptides. These glycopeptides were obtained by extensive pronase digestion followed by N-[14C]acetylation of the peptide moieties and desialylation by mild acid hydrolysis. Assuming that all glycopeptides were radiolabelled to the same specific radioactivity, Concanavalin A-Sepharose chromatography indicated that the majority of the glycans (84%) were of the complex-type of which approximately half were bi-antennary structures. The remainder of the glycans comprised oligomannose-type structures and/or incomplete bi-antennary structures. Pisum sativum lectin-agarose chromatography revealed that part of the bi-antennary glycans contained a fucose residue alpha(1-6)-linked to the N-acetylglucosamine which is attached to asparagine. Furthermore, the presence of tri-, and tetra- and/or tri'-antennary complex-type glycans was demonstrated by chromatography on immobilized Phaseolus vulgaris leukoagglutinating phytohemagglutinin and Aleuria aurantia lectin (AAL). AAL-agarose chromatography furthermore indicated the presence of alpha(1-3)-linked fucose in part of these glycopeptides, whereas no alpha(1-6)-linked fucose could be detected in these structures. The degree of beta-galactosylation of the complex-type glycans was investigated by chromatography on Ricinus communis agglutinin-agarose. The results indicated that only part of the bi-antennary glycans were completely beta-galactosylated. Similarly, at least three beta-galactose residues were present in only a part of the tri-, and tetra- and/or tri'-antennary glycans.

Adenosine Triphosphatases↗
PubMed1989

Distribution and dynamics of cell surface-associated cellCAM 105 in cultured rat hepatocytes.

The cellular location of cellCAM 105 was studied by indirect immunofluorescence microscopy of primary rat hepatocytes grown in monolayer culture. Staining corresponding to cellCAM 105 was seen both in cell-cell contact areas and on the upper surfaces of the cells. In the cell-cell contact areas the antigen was not accessible to the antibodies unless the cells were either permeabilized with detergent or incubated in a calcium-free medium. Removal of calcium from the medium caused the cells to separate from each other. Within a few minutes wide intercellular clefts were formed, and upon further incubation the cells became stellate-shaped and finally remained in contact with each other only via thin cellular processes. These processes were cellCAM 105-positive and at sites where they attached to the bodies of the contracted cells a granular fluorescence pattern appeared. After 24-48 h of culture, intercellular channels resembling bile canaliculi were sometimes formed in the hepatocyte monolayers. The membranes of these intercellular channels were stained for cellCAM 105. After culture for several days the hepatocytes lost their polygonal shape and gradually acquired a more fibroblast-like morphology. This morphological change was accompanied by a decrease in cellCAM 105-specific fluorescence, both in the cell-cell contact areas and on the free cell surfaces.

Animals↗
PubMed1989

Cell-surface location and molecular properties of cell-CAM 105 in intestinal epithelial cells.

Cell-CAM 105 is involved in intercellular adhesion of isolated rat hepatocytes in vitro. In addition to liver, cell-CAM 105 occurs in several different epithelia, in platelets, and in granulocytes. In this paper we present a detailed analysis of cell-CAM 105 in the small intestinal mucosa. Isolated rat intestinal epithelial cells and brush-borders were investigated by indirect immunofluorescence. A strong fluorescence occurred in the brush-border region and a much weaker staining was seen in the lateral cell surfaces. The brush-border staining was heterogeneous and concentrated to the periphery where brush-border microvilli from adjacent cells are in contact with each other. It is suggested that cell-CAM 105 might mediate binding between the outer surfaces of neighboring microvilli. Immunoblotting and electrophoretic analyses of the biochemical properties of intestinal cell-CAM 105 demonstrated significant differences compared with cell-CAM 105 isolated from liver. Intestinal cell-CAM 105 was smaller than liver cell-CAM 105 under reducing conditions, whereas it was larger than liver cell-CAM 105 under non-reducing conditions. Chemical reduction decreased the size of intestinal cell-CAM 105, but increased the size of liver cell-CAM 105. Our interpretation of these data is that intestinal cell-CAM 105 occurs as a part of a macromolecular complex. This interpretation was supported by electrophoretic analyses of intestinal cell-CAM 105 isolated by immunoaffinity chromatography on anti-cell-CAM antibodies. In addition to cell-CAM 105, this material contained several other proteins of lower molecular weight than cell-CAM 105. These data suggest that intestinal cell-CAM 105 participates in cell-surface interactions that may regulate the structure and function of the apical brush-border regions of the intestinal epithelial cells.

Adenosine Triphosphatases↗
PubMed1988

The cell-surface expression of the cell adhesion molecule cellCAM 105 in rat fetal tissues and regenerating liver.

In the present investigation we have used a sensitive immunohistochemical technique to study the appearance and cell-surface distribution of cellCAM 105 in rat fetal tissues and in regenerating liver. CellCAM 105 is an integral membrane glycoprotein that is involved in cell-cell adhesion of mature rat hepatocytes in vitro. In 12-day-old rat fetuses no cellCAM 105 was detected. CellCAM 105 then appeared on Day 13 in megakaryocytes of the fetal liver, on Day 16 in the liver parenchyme, and on Day 17 in the epithelial cells of the proximal kidney tubules and of the small intestinal mucosa. In the liver parenchyme cellCAM 105 first appeared in immature bile canaliculi. During Days 19-21 a significant staining also occurred on the contiguous sides of the hepatocytes, which at that time became closely associated when the blood-forming cells disappeared. This surface staining then gradually disappeared and 2-3 weeks after birth cellCAM 105 was expressed in the bile canalicular area which is typical of mature hepatocytes. In regenerating liver the amount of cellCAM 105 decreases to a minimum 2-3 days post-hepatectomy, then increases and reaches the normal concentration 10-15 days post-hepatectomy [Odin and Obrink (1986) Exp. Cell Res. 164, 103-114]. The cell-surface distribution of cellCAM 105 also changed, and on Days 3-5 post-hepatectomy it appeared on all faces of the hepatocytes which then were closely associated without obvious sinusoids in between. This staining pattern then slowly changed toward the normal pattern of mature liver, which appeared about 15 days post-hepatectomy. A theoretical analysis of the mode of hepatocyte cell division during liver regeneration suggested that the surface of the postmitotic hepatocytes should become unpolarized with respect to macromolecular composition. This is in agreement with the observed surface distribution of cellCAM 105. The results support the hypothesis that cell-surface interactions mediated by cellCAM 105 might contribute to the regular organization of hepatocytes in the normal, mature liver plates.

Animals↗