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C A Buck

Publications and source records attributed to C A Buck.

At least 55 records · Page 3Linked to original sources
PubMed1989

Identification and characterization of cell-substratum adhesion receptors on cultured human endothelial cells.

A series of immunological approaches was utilized to identify the molecules involved in cell-substratum adhesion of human endothelial cells (EC) derived from adult large vessels, fat capillaries, and umbilical veins. A polyclonal antibody prepared against partially purified extracellular matrix receptors disrupted adhesion of EC to a wide variety of substrates and identified four groups of glycoproteins migrating with apparent Mr of 150, 125, 110, and 95 kD in immunoprecipitation experiments. Specific monoclonal antibodies identified these proteins as members of the Integrin family of extracellular matrix receptors and included the alpha and beta chains of the fibronectin receptor (alpha 5/beta 1), a collagen receptor (alpha 2 beta 1), a multifunctional receptor that binds to fibronectin, collagen, and laminin (alpha 3/beta 1), as well as a receptor related to platelet IIb/IIIa (alpha v/beta 3). To directly test the importance of these molecules in cell-substratum adhesion, these proteins were purified by a combination of ion exchange, lectin affinity, and immunoaffinity chromatography and used to block the biological activity of the adhesion-disrupting polyclonal antibody. Immunofluorescence experiments further supported the role of these glycoproteins in adhesion. The GPIIb/IIIa-like receptor localized to well-formed adhesion plaques on EC plated on fibrinogen, but not on fibronectin, laminin, or type IV collagen. Receptors containing the beta 1 subunit were visualized as discontinuous fibrils which colocalized with fibronectin fibrils and actin stress fibers.

Adult↗
PubMed1988

Myoblast migration specifically inhibited in the chick embryo by grafted CSAT hybridoma cells secreting an anti-integrin antibody.

We report a teratological method in which mouse hybridoma cells are grafted into a chick host. CSAT (Cell Substratum ATtachment) hybridoma was used. It produces an antibody directed against the avian integrin complex. The grafts were performed during the second and third days of incubation either at the level of the somites or in the coelom of the chick embryo. The anomalies were revealed by means of a monoclonal antibody that recognizes myogenic cells as soon as they become committed in the myotome. When embryos were grafted at the level of the somites, body wall muscles failed to develop on the side of the graft only. After coelomic grafting, total agenesis of abdominal muscles was induced. The anomalies were specific since the engraftment of three control hybridoma clones induced no change in muscle formation. These control hybridomas produce antibodies directed against the same molecular complex but not against the same epitope as CSAT. The injection of hybridoma cells in an embryo appears as a method of general interest to determine the long-term consequences of perturbing a specific developmental process.

Animals↗
PubMed1987

Soluble 80-kd fragment of cell-CAM 120/80 disrupts cell-cell adhesion.

Calcium-dependent cell adhesion molecules (CAMs) mediate intercellular adhesion in epithelial cells and in preimplantation mammalian embryos. One of these molecules, cell-CAM 120/80, is found on cells as a 120-kd membrane glycoprotein and as a soluble 80-kd species in conditioned culture medium [Damsky et al: Cell 34:455, 1983]. We have purified to homogeneity the soluble 80-kd fragment of cell-CAM 120/80 by using monoclonal antibody affinity chromatography. We have shown that the purified molecule can disrupt cell-cell adhesion in cultured epithelial cells, thus indicating that it is directly involved in the adhesive process. In addition, we have further characterized both the 120-kd cell-associated molecule and its 80-kd fragment, including N-terminal sequence analysis.

Amino Acid Sequence↗
PubMed1987

Cell surface receptors for extracellular matrix molecules.

Table 2 lists most of the extracellular matrix and related receptors identified to date. The wide range of binding affinities of these receptors for their ligands may be important to their function. The affinity of integrins for fibronectin is moderate, with a dissociation constant in the micromolar range. This affinity level leads to relatively rapid dissociation and reformation of receptor-ligand complexes. Thus changes in component concentration can shift binding equilibria within minutes (the time scale of many biologic phenomena) and change the number or organization of adhesive complexes. This type of interaction would be useful in motile cells, in which adhesions must form and dissociate rapidly. In contrast, the affinity of the 68-kDa laminin receptor for its ligand is three orders of magnitude higher. Such levels of affinity would be useful in stabilizing tissue. Members of the integrin family appear to recognize an RGD sequence on the ligands to which they bind. Since there are many ligands containing the RGD sequence, the question of specificity arises. Avian integrin shows little specificity and appears to behave as a multifunctional, promiscuous receptor for extracellular matrix molecules. Figure 1 summarizes our current view of the structural and functional features of avian integrin. In contrast, the mammalian receptors for vitronectin and fibronectin are specific for their respective ligands. More than one of these receptors may be present simultaneously on a cell surface, e.g. fibroblasts express receptors for fibronectin, laminin, and vitronectin at the same time. This multiplicity of receptors provides potential mechanisms for generating the adhesive differences among cells believed to play a prominent role in morphogenesis. Further adhesive differences may stem from the formation of different combinations of various alpha- and beta-subunits expressed in the cell. The mechanism of regulation of adhesive interactions with the extracellular matrix is only beginning to be explored. There are several levels at which this regulation might occur. Integrin appears to be more regionalized in more developed cells that are integral parts of tissue structures. Changes in receptor distribution could alter the relative strength of adhesive interactions. In certain instances, avian integrin disappears, or its concentration is reduced, e.g. during the development of embryonic lung (Chen et al 1986) and erythroid cells (Patel & Lodish 1985). Post-translational modifications provide yet another mechanism for regulating integrin-ligand binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Vasculogenesis in the early quail blastodisc as studied with a monoclonal antibody recognizing endothelial cells.

QH1, a monoclonal antibody that recognizes quail endothelial and haemopoietic cells, was applied to quail blastodiscs in toto, in order to analyse by immunofluorescence the emergence of the vascular tree. The first endothelial cells were detected in the area opaca at the headfold stage and in the area pellucida at the 1-somite stage. Single cells then interconnected progressively, especially in the anterior intestinal portal and along the somites building up the linings of the heart and dorsal aortas. This study demonstrates that endothelial cells differentiate as single entities 4 h earlier in development than hitherto detected and that the vascular network forms secondarily. The horseshoe shape of the extraembryonic area vasculosa is also a secondary acquisition. A nonvascularized area persists until later (at least the 14-somite stage) in the region of the regressing primitive streak.

Animals↗
PubMed1987

Integrin, a transmembrane glycoprotein complex mediating cell-substratum adhesion.

A monoclonal antibody, CSAT, which inhibits the adhesion of chick cells to substrata coated with fibronectin, laminin and vitronectin, has been used to identify a cell surface receptor required for cell-substratum adhesion. This receptor, termed integrin, is found on the ventral surface of cells in close contact adhesion sites, at the periphery of adhesion plaques and beneath stress fibres. It is a heterodimer consisting of non-covalently linked alpha and beta subunits. Integrin binds directly to laminin, fibronectin and vitronectin with dissociation constants in the micromolar range. The binding of integrin to matrix molecules is sensitive to peptides carrying the cell-binding sequence Arg-Gly-Asp and requires heteromeric integrity. Integrin also binds directly to the cytoskeleton-associated protein talin. Thus, integrin has the properties of a transmembrane molecule capable of bringing extracellular matrix and cytoskeleton-associated molecules in proper juxtaposition to form adhesion structures. The integrin beta subunit is phosphorylated following Rous sarcoma virus transformation. Phosphorylation alters the ability of the receptor to bind extracellular matrix molecules as well as talin, suggesting a mechanism for the alteration of cellular adhesive and morphological properties following malignant transformation. A major phosphorylation site is on the cytoplasmic domain of the beta subunit. Synthetic peptides homologous with this region of integrin inhibit integrin-talin binding. The gene for the beta subunit of integrin has been sequenced. Its structure is consistent with the membrane-spanning properties of the receptor. Integrin is structurally and serologically related to adhesion receptors from mammalian tumour cells, fibroblasts, platelets and lymphocytes. It appears to be a member of a supergene family of receptors involved in cellular adhesive interactions. Antibody and peptide inhibition experiments have suggested a role for integrin and integrin-like molecules in cell migration, neurite extension, neural differentiation, histogenesis and embryonic development in Drosophila. Thus, integrin appears representative of a set of evolutionarily conserved, biologically important adhesive molecules.

Animals↗
PubMed1986

Integrin (the CSAT antigen): functionality requires oligomeric integrity.

Integrin, the cell-substrate attachment (CSAT) antigen, is a complex of integral membrane glycoproteins whose apparent function is to mediate cell-substratum adhesion by serving as a transmembrane link between the extracellular matrix and elements of the cytoskeleton. Previous attempts to separate the members of this complex under nondenaturing conditions have been successful. We have now produced a monoclonal antibody "G" that is specific for the lower molecular mass cysteine-rich band 3 of the complex. Using an antibody affinity column containing this monoclonal antibody, it is possible to dissociate integrin into two fractions, one containing band 3, the other containing bands 1 plus 2. Neither fraction will by itself bind fibronectin, laminin, or talin. However, when the fractions are combined, the reconstituted integrin elutes from a gel filtration column in the same position as the native complex, and binding activity to these molecules returns. Further, it is shown by gel filtration that the recognition site for the adhesion-disrupting monoclonal antibodies CSAT and JG22 is on band 3, supporting the contention that integrin is an oligomer. The data presented here is consistent with integrin being either a mixture of heterodimers, each with a common subunit and reacting with a particular extracellular matrix molecule, or a single heterotrimer capable of binding to several different extracellular matrix molecules.

Animals↗
PubMed1985

Cell surface glycoproteins mediate compaction, trophoblast attachment, and endoderm formation during early mouse development.

Early mouse embryos undergo several morphogenetic processes, such as compaction, trophoblast attachment, and endoderm formation that can be studied in vitro. Several polyspecific and monospecific antisera have been used to perturb these processes in a nontoxic, reversible fashion. One of the antibody-defined molecules, cell CAM 120/80, promotes epithelial cell adhesion, embryo compaction, and endoderm formation. The results suggest the presence of another such molecule(s) involved in these same processes. Evidence is also presented that another set of antibody-defined molecules, GP 140, involved in attachment of somatic cells to the substrate, mediates trophoblast attachment of the mouse blastocyst. The possible role of these molecules in governing the processes leading to cell lineages in the mouse embryo is discussed.

Animals↗
PubMed1985

A monoclonal antibody identifies a glycoprotein complex involved in cell-substratum adhesion.

The monoclonal antibody CSAT has been reported to perturb the adhesion of chick embryo cells to their substratum (Neff et al. [19]). Evidence is presented here that the antigen recognized by this monoclonal antibody is comprised of three membrane glycoproteins. The antigen is released from cells with non-ionic detergent and purified by monoclonal antibody affinity chromatography. When analysed by SDS-PAGE under non-reducing conditions, the antigen resolves into three components of apparent molecular weights 160 000 (band 1), 135000 (band 2), and 110 000 (band 3). Following reduction of each component, bands 1 and 2 migrate at slightly lower apparent molecular weights, while band 3 migrates at a higher apparent molecular weight, suggesting that band 3 has an internal disulfide bond. All three bands differ from one another as determined by peptide mapping and by immunologic cross-reactivity. It is postulated that the three glycoproteins function as a complex that plays a central role in cell-substratum adhesion.

Amino Acids↗
PubMed1985

Distribution of the cell substratum attachment (CSAT) antigen on myogenic and fibroblastic cells in culture.

Previous studies (Neff et al., 1982, J. Cell. Biol. 95:654-666; Decker et al., 1984. J. Cell. Biol. 99:1388-1404) have described a monoclonal antibody (CSAT Mab) directed against a complex of three integral membrane glycoproteins of 120,000-160,000 mol wt (CSAT antigen [ag]) involved in the cell matrix adhesion of myoblasts and fibroblasts. In localization studies on fibroblasts presented here, CSAT ag has a discrete, well-organized distribution pattern. It co-aligns with portions of stress fibers and is enriched at the periphery of, but not directly beneath vinculin-rich focal contacts. In this last location, it co-distributes with fibronectin, consistent with the suggestion that the CSAT ag participates in the mechanism by which fibroblasts attach to fibronectin. In prefusion myoblasts, which are rapidly detached by CSAT Mab, CSAT ag is distributed diffusely as are vinculin, laminin, and fibronectin. After fusion, myotubes become more difficult to detach with CSAT Mab. The CSAT ag and vinculin are organized in a much more discrete pattern on the myotube surface, becoming enriched at microfilament bundle termini and in lateral lamellae which appear to attach myotubes to the substratum. These results suggest that the organization of CSAT ag-adhesive complexes on the surface of myogenic cells can affect the stability of their adhesive contacts. We conclude from the sum of the studies presented that, in both myogenic and fibroblastic cells, the CSAT ag is localized in sites expected of a surface membrane mediator of cell adhesion to extracelluon of CSAT ag-adhesive complexes on the surface of myogenic cells can affect the stability of their adhesive contacts. We conclude from the sum of the studies presented that, in both myogenic and fibroblastic cells, the CSAT ag is localized in sites expected of a surface membrane mediator of cell adhesion to extracellular matrix. The results from studies that use fibroblasts in particular suggest the involvement of CSAT ag in the adhesion of these cells to fibronectin.

Actins↗
PubMed1983

Identification and purification of a cell surface glycoprotein mediating intercellular adhesion in embryonic and adult tissue.

An antiserum against material shed into serum-free medium by MCF-7 human mammary carcinoma cells (anti-SFM II) disrupts cell-cell interactions in murine mammary tumor epithelial cells (MMTE). We now report purification of an 80 kd glycoprotein (GP80) from SFM of MCF-7 mammary carcinoma cells that blocks the activity of anti-SFM II. Anti-SFM II also inhibits compaction of eight-cell mouse embryos, and purified GP80 blocks this reaction. An antiserum against purified GP80 (anti-GP80) has all adhesion-disrupting activities displayed by anti-SFM II. It recognizes one band at 80 kd in SFM and a 120 kd band in detergent extracts of epithelial but not fibroblastic cells. In immunofluorescence studies it is restricted to sites of cell-cell interaction in cultured epithelial cells. Thus a cell surface glycoprotein of 120 kd, the medium form of which is approximately 80 kd, which is neither species nor tissue specific, is expressed at early stages of mammalian development and is found on epithelia.

Animals↗
PubMed1982

Integral membrane glycoproteins related to cell-substratum adhesion in mammalian cells.

Broad spectrum antisera have been raised against surface membrane-derived material from baby hamster kidney cells and mouse mammary tumor epithelial cells. These antisera disrupt cell-substratum adhesion in their respective cell types. Using an antibody neutralization (blocking) assay, adhesion-related glycoproteins have been isolated from non-ionic detergent extracts of each cell type. The purified material in each case consisted of a restricted population of glycoproteins of approximately 120,000-160,000 Mr. Purified material from each system blocked the disruption of adhesion induced by the heterologous antiserum on either cell type. The antisera were capable of disrupting cell-substratum adhesion of a large number of cell types and species sources. In addition, antibody blocking activity could be detected from partially purified extracts of several adult hamster cell types and a variety of cultured cell types. Thus, in addition to having similar substratum-associated glycoproteins ((eg, fibronectin) and cytoskeleton-associated proteins (eg, alpha-actinin and vinculin) cells from different species and tissue sources appear to have a relatively conserved class of integral membrane glycoproteins involved in cell substratum-adhesion.

Animals↗
PubMed1982

Expression of adhesion-related membrane components in adherent versus nonadherent hamster melanoma cells.

The existence of integral membrane components that are involved in cell-substratum adhesion has been postulated. Using an immunochemical approach developed in this laboratory, we provide further evidence for the role in cell-substratum adhesion of integral membrane glycoproteins within a molecular weight region of 120,000-140,000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of material enriched approximately 100-fold in adhesion-related components revealed the 120,000-140,000 Mr glycoproteins in an adherent hamster melanoma cell line. These glycoproteins are greatly reduced in a nonadherent variant. Induction of adhesion in these cells by exposure to BudR is accompanied by re-expression of the surface adhesion antigens.

Animals↗
PubMed1981

Membrane glycoproteins involved in cell--substratum adhesion.

A combination of immunological and biochemical methods were used to identify surface membrane components involved in cell-substratum adhesion. Broad-spectrum antiserum, prepared against surface membranes from hamster cells, induced reversible rounding and detachment of hamster fibroblasts from a substratum in vitro. This phenomenon was inhibited by Nonidet P-40 extracts of hamster cells. Therefore, an antibody neutralization assay was developed to detect the presence of antigen during the fractionation of Nonidet P-40 extracts of cells. After two differential precipitation steps, anion exchange chromatography, and sequential lectin affinity chromatography, a fraction greatly enriched in ability to block antiserum-induced changes in cell adhesion and appearance was isolated. Analysis of this fraction by NaDodSO4/polyacrylamide gel electrophoresis revealed a highly restricted group of glycoproteins with Mr approximately 140,000. A lectin-purified glycoprotein fraction was used to raise a higher titer antiserum that was able to induce reversible rounding and detachment of cells from a substratum and, when immobilized on an antibody affinity column, was able to bind and release material capable of blocking antiserum-induced cell rounding. These methods have allowed us to focus attention on a restricted group of glycoproteins that are integral constituents of the surface membrane and which play some as yet undetermined role in the process of cell--substratum adhesion.

Animals↗
PubMed1981

Manipulation of cell-cell and cell-substratum interactions in mouse mammary tumor epithelial cells using broad spectrum antisera.

Two antisera were raised in goats against material shed by two different mammary epithelial cell lines into serum-free culture medium. These antisera, when added to the medium of intact, growing mouse mammary tumor cells in the absence of complement, cause distinct and dramatic alterations in cell morphology and adhesiveness. One antiserum (anti-SFM I) causes mouse mammary tumor epithelial cells to round and detach from the substratum. Treatment with the other antiserum (anti-SFM II) does not affect cell-substratum interactions, but causes the cells to convert from an epitheloid to a fibroblastic morphology. Statistical analysis of transmission electron micrographs of control and antibody-treated cells indicates that treatment with anti-SFM II is associated with a substantial reduction in the extent of intercellular junctions, particularly desmosomes. To identify the components with which the two antisera interact, nonionic detergent extracts of mouse mammary tumor cells were fractionated, and the ability of various fractions to block the morphological effects of either antiserum was determined. The whole Nonidet P40 (NP40) extract of the epithelial cells blocked the effects of both antisera. After the extract was subjected to ion exchange and lectin affinity chromatography, two separate fractions were obtained. One fraction blocks and anti-SFM I induced rounding and detachment of cells from the substratum. The second fraction blocks the effects of both antisera. The isolation of the former fraction, which has highly restricted number of components, represents a significant first step toward identifying the surface membrane molecule(s) involved in cell-substratum adhesion in epithelial cells.

Animals↗
PubMed1980

Glycosaminoglycans and other carbohydrate groups bound to proteins of control and transformed cells.

The membrane glycoproteins from control (BHK21/C13) and Rous sarcoma virus-transformed (C13/B4) baby hamster kidney cells labeled with D-[14C]- or D-[3H]glucosamine, respectively, were purified by means of polyacrylamide electrophoresis and gel electrofocusing. The homogeneity of the isolated glycoproteins was demonstrated by analysis of the NH2-terminal peptides. Some purified glycoproteins were found to be hybrid molecules in terms of the type of oligosaccharides they bear. The majority of the oligosaccharides (approximately 90%) bound on thee glycoproteins are N-glycosidically linked (Mr approximately 3000 to 5000). Another 5% appears to be small groups linked O-glycosidically to several adjacent or closely spaced amino acid residues. The remainder (5%) of the carbohydrate groups appears to be small, covalently bound glycosaminoglycans. This is the first report of hybrid molecules bearing glycosaminoglycans in the cell surface. The ratio of the types of oligosaccharides varies among different glycoproteins. There is slightly more glycosaminoglycan present on glycoproteins from malignant cells. A remarkably complex but similar array of N-glyucosidically linked oligosccharides is bound to different individual membrane glycoproteins. Each individual polypeptide must contain only a small number of the total observed carbohydrate groups, i.e. the carbohydrate groups on individual polypeptides are grossly heterogeneous. This implies that purification is based largely on the characteristics of the polypeptide, and that overall charge and size of the carbohydrate groups are relatively constant in a single population of glycoproteins. Our results suggest that the differences between the carbohydrate groups derived from glycoproteins from control and transformed cells are mainly quantitative.

Animals↗
PubMed1980

Comparison of glycopeptides from control and virus-transformed baby hamster kidney fibroblasts.

Glucosamine-labeled glycopeptides from control and virus-transformed BHK fibroblasts were characterized by size, lectin affinity, charge, and composition. As already demonstrated, on the basis of elution position on a column of Sephadex G-50, transformed cells contained a greater proportion of large glycopeptides than did control cells. Transformed cells also contained a larger proportion of glycopeptides which do not bind to Con A-Sepharose. By sequential chromatography on Sephadex G-50, Con A-Sepharose, and DEAE-Sephadex, approximately 40 individual peaks were partially or completely resolved. If sialic acid was removed from the glycopeptides prior to analysis by ion-exchange chromatography, 95% of the glycopeptides from control cells and 85% of the glycopeptides from transformed cells were no longer bound by DEAE-Sephadex. It was concluded that the DEAE-Sephadex elution properties of the glycopeptides are determined almost entirely by the sialic acid content of the molecules. A comparison of the profiles of control and transformed cell glycopeptides simultaneously eluting from columns of DEAE-Sephadex revealed that the differences between the two cells were largely quantitative; however, the possibility of the existence of qualitative differences as well cannot be excluded. In particular, there was one component present on the surface of transformed cells that was virtually absent in control cells. It was degraded by nitrous acid hydrolysis and heparinase and appeared to be heparan sulfate like material. After fractionation, each isolated glycopeptide population was analyzed for carbohydrate and, in some cases, amino acid content. The apparently larger glycopeptides, group A, the dominant population in transformed cells, were found to contain 3 to 4 mannose residues/glycopeptide when the sugars were normalized to sialic acid content. On the basis of the same criteria, group B glycopeptides contained 4-6 mannose residues/glycopeptide. The carbohydrate and amino acid compositions of the glycopeptides from transformed cells were, with a few exceptions, similar to those from control cells. Some isolated glycopeptides appeared to contain both O-glycosidic anad N-glycosidic linkages on the same oligopeptide.

Amino Acids↗