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W R Springer

Publications and source records attributed to W R Springer.

At least 19 recordsLinked to original sources
PubMed1997

Quantification of integrin subunits on human prostatic cell lines--comparison of nontumorigenic and tumorigenic lines.

BACKGROUND: We set out to quantify integrin subunits on the surface of several prostate cell lines, including two nontumorigenic lines, in order to assess their role in tumorigenicity and metastasis. METHODS: Flow cytometry was used to estimate the amounts of each subunit by changes in mean fluorescence intensity from control antibody. An in vitro Matrigel (Collaborative Biomedical Products, Bedford, MA) assay was used to determine invasiveness. RESULTS: Profiles of each cell line were developed using the change in subunit mean fluorescence intensity normalized to the beta 1-subunit. The alpha 4-subunit is only expressed on nontumorigenic cells. These same cells were unable to invade Matrigel. CONCLUSIONS: Comparison of nontumorigenic and cancerous lines suggests that a loss of the alpha 4-subunit correlates with the acquisition of tumorigenicity and perhaps metastatic potential. The ability to quantify expression of integrin subunits on prostate cell lines allows the determination of regulation by factors responsible for growth, tumorigenicity, and/or metastasis.

Antibodies, Monoclonal↗
PubMed1993

Antibodies specific for gp40 inhibit cell-cell adhesion by cross-linking the protein on the surface of Dictyostelium purpureum.

We have previously suggested a role for gp40 in cell-cell adhesion in Dictyostelium purpureum from the fact that antibodies specific for this protein inhibited adhesion in an in vitro assay [Springer: Dev Biol 133:447-455, 1989]. To further confirm the role mutants lacking the protein were isolated and characterized. To our surprise, the mutants had normal adhesive properties both in vitro and in situ. These results lead us to the conclusion that gp40 is not necessary for the cell-cell adhesions observed and may not be a molecule which directly participates in these adhesions. When studied further, we found that adhesion-inhibitory antibodies were only effective as divalent IgG. Monovalent Fab fragments of the same antibodies could not inhibit adhesion. The inhibitory antibodies also caused the cells to remain rounded and incapable of attaching to plastic surfaces. We conclude that when divalent antibodies specific for gp40 cross-link this protein on the cell surface a cytoskeletal change prevents them from attaching to substratum as well as to other cells, thereby inhibiting cell-cell adhesion. We suggest that an alternative mechanism for inhibition of cell-cell adhesion by divalent antibodies exists and should be considered before proposing a direct role for a protein in adhesion.

Animals↗
PubMed1991

A method for quantifying radioactivity associated with protein in silver-stained polyacrylamide gels.

A method is described in which individual proteins labeled with weak beta-emitting radionuclides, separated by polyacrylamide gel electrophoresis, and stained with silver are released from the gel by the use of the periodate soluble cross-linking agent N,N'-dialyltartardiamide. The radioactivity can then be quantitated using liquid scintillation counting. The method is shown to be insensitive to reasonable variations in the intensity of staining as well as the gel volume over a practical range of gel slices. Recovery from the gel is extremely good with 93% of the counts associated with 14C-labeled proteins of known radioactive concentration being recovered. Analysis of a complex mixture of 3H-labeled proteins indicates resolution similar to that obtainable by autoradiography without the problems associated with quenching of autoradiographic signal by the staining procedure. The method is used to determine the amount of fucose and mannose incorporated into a putative cell adhesion protein during development of the cellular slime mold Dictyostelium purpureum.

Autoradiography↗
PubMed1990

An anticarbohydrate monoclonal antibody inhibits cell-cell adhesion in many species of Dictyostelium but not of Polysphondylium.

The anticarbohydrate monoclonal antibody d-41 inhibits the adhesion of aggregating cells, as measured by an in vitro assay, in every species of Dictyostelium tested but in none of the species from the genus Polysphondylium. Although d-41 binds significantly to the surface of cells from both genera, the ability to inhibit adhesion correlates with the binding of the antibody to a few, mostly developmentally regulated, membrane-associated proteins in each of the species affected. Previous work in D. discoideum and D. purpureum have shown that the major d-41-b binding proteins from these species at this time in development are directly involved in the adhesion process. Therefore, the presence of the epitope on these proteins in the other species of Dictyostelium implicates them in the adhesion mechanism. The function of the carbohydrates containing the epitope is yet to be determined.

Antibodies, Monoclonal↗
PubMed1989

Developmentally regulated cell-cell adhesion in Dictyostelium purpureum is mediated by a glycoprotein synthesized in nonadhesive cells.

Upon starvation the cellular slime mold, Dictyostelium purpureum, develops a form of cell-cell adhesion aiding in the formation of large multicellular aggregates, which are capable of further differentiation. The molecule that mediates this adhesion is a glycoprotein of Mr approximately 40,000. The protein shares a common carbohydrate epitope with another well-characterized cell adhesion molecule from Dictyostelium discoideum, contact sites A, but the polypeptides to which it is attached differ for each species. Although mediating a developmental form of adhesiveness, the protein is synthesized in vegetative cells at a time when they do not adhere. Most of the vegetative protein is associated with cell membranes and appears to be on the surface of these cells. The protein is compared to other cell adhesion molecules from other species of cellular slime molds, and possible explanations for its inability to function in vegetative cells are discussed.

Amino Acids↗
PubMed1985

Protein-linked oligosaccharide implicated in cell-cell adhesion in two Dictyostelium species.

Monoclonal antibody d-41, previously shown to block in vitro cell-cell adhesion in aggregating Dictyostelium discoideum, also blocks adhesion in aggregating D. purpureum. In both species the antibody reacts with proteins with Mr approximately 80,000, 37,000, and 27,000, presumed to be glycoproteins since the d-41 epitope is destroyed by periodate oxidation but unaffected by extensive Pronase digestion. Polyclonal antibodies raised against the mixture of d-41 reactive glycoproteins that had been purified by immunoaffinity chromatography are potent inhibitors of D. discoideum adhesion, and adhesion-blocking activity is neutralized extensively and equivalently by each of the purified glycoproteins from D. discoideum with which d-41 reacts. In contrast, polyclonal antibodies raised against the same purified glycoproteins after they had been oxidized with periodate do not block cell-cell adhesion although they react with the glycoproteins with Mr approximately 80,000, 37,000, and 27,000 and bind as extensively to the surface of aggregating D. discoideum cells as do the adhesion-blocking polyclonal antibodies. When taken together, these results raise the possibility that some component of the d-41 binding oligosaccharide participates in cell-cell adhesion.

Animals↗
PubMed1985

Adhesion mutants of Dictyostelium discoideum lacking the saccharide determinant recognized by two adhesion-blocking monoclonal antibodies.

A mutant of Dictyostelium discoideum, strain HL260, was isolated based on its failure to bind d-41, a monoclonal antibody that blocks developmentally regulated cell-cell adhesion. The mutant fails to normally acquire cell-cell adhesion as assayed with cells shaken in 10 mM EDTA, but aggregates and and constructs fruiting bodies. Other mutant strains, HL216 and HL220, previously shown to have impaired cell-cell adhesion, also lack the determinant that binds d-41. The three strains all carry mutations in a gene designated mod B, which directs a post-translational modification of several developmentally regulated D. discoideum glycoproteins. Diploids formed between independent mod B mutant haploid strains also lack this determinant and show marked impairment of cell-cell adhesion in EDTA, indicating that mutations in mod B, rather than other mutations not shared by the haploid strains, are related to the adhesion defect. The results are consistent with other evidence that an oligosaccharide carried on several developmentally regulated glycoproteins plays an essential role in EDTA-resistant cell-cell adhesion in D. discoideum. However, this type of adhesion is not essential for morphogenesis in that the only defect detected thus far in mod B mutant strains is that they construct relatively smaller fruiting bodies that contain fewer spores.

Animals↗
PubMed1985

Receptor for the cell binding site of discoidin I.

Discoidin I, a developmentally regulated lectin in Dictyostelium discoideum, has been implicated in cell-substratum adhesion and ordered cell migration during aggregation. This depends on the cell binding site of discoidin I, which is distinct from its carbohydrate binding site. We have isolated a receptor for the cell binding site by affinity chromatography. The receptor binds immobilized discoidin I in the presence of 0.3 M galactose and can be eluted with gly-arg-gly-asp-his-asp, a synthetic peptide the sequence of which is found in discoidin I, and which blocks cell migration into aggregates. The receptor is a developmentally regulated cell-surface glycoprotein of apparent Mr approximately 67,000. Univalent antibodies specific for this glycoprotein block aggregation.

Adhesiveness↗
PubMed1984

Discoidin I is implicated in cell-substratum attachment and ordered cell migration of Dictyostelium discoideum and resembles fibronectin.

All three forms of discoidin I, an endogenous N-acetylgalactosamine-binding lectin from D. discoideum, contain the amino acid sequence gly-arg-gly-asp also found in fibronectin and implicated in its attachment to cells. Synthetic peptides containing these and adjacent amino acids of discoidin I block organized streaming during aggregation of D. discoideum and, at higher concentrations, block cell attachment and spreading on a plastic surface and formation of fruiting bodies. Pure discoidin I (with or without N-acetylgalactosamine) and univalent anti-discoidin I also block formation of streams during aggregation. Two mutants of D. discoideum with low levels of discoidin I apparently reflect the deficiency of this endogenous lectin by failing to form streams or to spread on plastic and by a partial failure to enter aggregates. Together, the results indicate that discoidin I functions like fibronectin to promote cell attachment and spreading as well as ordered cellular migration during morphogenesis.

Cell Movement↗
PubMed1983

Monoclonal antibodies block cell-cell adhesion in Dictyostelium discoideum.

Of 39 monoclonal antibodies that bind the cell surface of aggregating Dictyostelium discoideum, 4 block 76-98% of cell-cell adhesion measured in an in vitro assay. The active antibodies all bind in the range of 10(6) antigenic sites/cell surface and react with more than one material on nitrocellulose blots prepared after polyacrylamide gel electrophoresis of whole aggregating cells in sodium dodecyl sulfate. Active antibodies can by grouped into two classes, each with two very similar members. Class I binds several molecules that are prominent in aggregating cells but scarce or undetectable in vegetative cells, blocks cell adhesion only in the presence of EDTA, and has no detectable effect on cell morphology. Class II binds a wide range of molecules present in both vegetative and aggregating cells, inhibits adhesion as well in the absence as in the presence of EDTA, and reversibly alters cell shape.

Adhesiveness↗
PubMed1982

Evidence for another cell-adhesion molecule in Dictyostelium discoideum.

We raised a rabbit antiserum that completely blocked cell-cell adhesion of aggregating Dictyostelium discoideum cells in an in vitro assay. All adhesion-blocking activity of this antiserum was adsorbed with a D. discoideum fraction containing molecules having molecular weights as high as about 10(6), even after the material had been extensively digested with Pronase. The properties of this fraction indicate that the antigenic determinants in this macromolecule are saccharide residues. Antigen-rich material is found on vegetative cells but accumulates on or around differentiating D. discoideum cells as they aggregate. The cell surface of an aggregating cell contains about 5 X 10(5) antigenic sites. Antigen is also abundant in the medium of D. discoideum cells starved in suspension, which proved the most convenient starting material for its purification. Like several other macromolecules already discovered in D. discoideum by using this immunological approach, the material identified here may play a direct or indirect role in cell-cell adhesion and merits tentative consideration as a cell-adhesion molecule.

Cell Adhesion↗
PubMed1981

Endogenous lectins in chickens and slime molds: transfer from intracellular to extracellular sites.

Endogenous lectins in both cellular slime molds and chicken tissues have been localized primarily intracellularly, in contrast with the predominantly extracellular localization of the glycoproteins, glycolipids, and glycosaminoglycans with which they might interact. Here we present evidence that lectins in both of these organisms may be externalized and become associated with the cell surface and/or extracellular materials. In chicken intestine, chicken-lactose-lectin-II is shown to be localized in the secretory granules of the goblet cells, along with mucin, and to be secreted onto the intestinal surface. In embryonic muscle, chicken-lactose-lectin-I is shown to be externalized with differentiation, ultimately becoming localized on the surface of myotubes and in the extracellular spaces. In a cellular slime mold, Dictyostelium purpureum, externalization of lectin is elicited by either polyvalent glycoproteins that bind the small amount of endogenous cell surface lectin, or by slime mold or plant lectins that bind unoccupied complementary cell surface oligosaccharides. These results suggest that externalization of endogenous lectin may be a response to specific external signals. We conclude that lectins are frequently held in intracellular reserves awaiting release for specific external functions.

Animals↗
PubMed1980

Endogenous cell surface lectin in Dictyostelium: quantitation, elution by sugar, and elicitation by divalent immunoglobulin.

The amount of total endogenous cellular and cell surface lectin in aggregating Dictyostelium purpureum was determined by a number of immunochemical techniques. The results show that of the 5 x 10(6) molecules of the lectin (called purpurin) per aggregating cell only about 2% (1 x 10(5) molecules) is present on the cell surface. Cell surface purpurin can be specially eluted by lactose, which indicates that it is held to the surface by its carbohydrate-binding site. The eluted purpurin is replaced on the cell surface within 45 min. Estimates of cell surface purpurin made by binding of specific immunoglobulin to the cells at 4 degrees C indicate that a much larger amount, about 1 x 10(6) molecules, becomes associated with the cell surface in the presence of this divalent ligand. In contrast, univalent antibody fragments do not have this effect.

Antibodies, Fungal↗
PubMed1980

Cell adhesion molecules: detection with univalent second antibody.

Identification of cell surface molecules that play a role in cell-cell adhesion (here called cell adhesion molecules) has been achieved by demonstrating the inhibitory effect of univalent antibodies that bind these molecules in an in vitro assay of cell-cell adhesion. A more convenient reagent, intact (divalent) antibody, has been avoided because it might agglutinate the cells rather than blocking cell-cell adhesion. In this report, we show that intact rabbit immunoglobulin directed against certain cell surface molecules of Dictyostelium discoideum blocks cell-cell adhesion when the in vitro assay is performed in the presence of univalent goat anti-rabbit antibody. Under appropriate experimental conditions, the univalent second antibody blocks agglutination induced by the rabbit antibody without significantly interfering with its effect on cell-cell adhesion. This method promises to be useful for screening monoclonal antibodies raised against potential cell adhesion molecules because: (a) it allows for the screening of large numbers of antibody samples without preparation of univalent fragments; and (b) it requires much less antibody because of the greater affinity of divalent antibodies for antigens.

Antibodies, Anti-Idiotypic↗