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S Roseman

Publications and source records attributed to S Roseman.

At least 109 records · Page 6Linked to original sources
PubMed1982

Studies on the intercellular adhesion of rat and chicken hepatocytes. Conditions for stimulation by liver plasma membranes.

A new assay was developed for measuring the stimulation of intercellular adhesion of rat hepatocytes by rat liver plasma membranes. Aggregates formed in the presence of the membranes are separated from single cells by filtration, and the number of cells in the aggregates is determined by their lactate dehydrogenase content. The formation of aggregates was specifically stimulated by rat liver plasma membranes and the rate of aggregate formation was proportional to the quantity of added membranes. The effects of divalent cations on the initial rates of rat and chicken hepatocytes adhesion were also examined. Optimal rates of adhesion of rat hepatocytes were obtained in the presence of physiological levels of Mg2+, and adhesion was inhibited by Ca2+, whereas optimal rates of chicken hepatocyte adhesion were obtained in the presence of physiological levels of Ca2+. Plasma membrane stimulation of hepatocyte adhesion also showed the same divalent ion requirements with the respective cell types. The stimulatory activity in the rat plasma membranes determined by the filter assay was found to be sensitive both to trypsin digestion and to mild periodate oxidation. The activity was also completely resistant to vigorous reductive alkylation. These results taken together suggest that the rat plasma membrane stimulatory activity is associated with a glycoprotein(s).

Animals↗
PubMed1982

Molecular cloning of the crr gene and evidence that it is the structural gene for IIIGlc, a phosphocarrier protein of the bacterial phosphotransferase system.

Sugar substrates of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) normally prevent bacterial cells from utilizing sugars that are not substrates of this system (diauxic growth, "the glucose effect"). We have previously shown that this type of PTS-mediated repression can be completely reversed by a single mutation, designated crr. Two lines of evidence are presented in this report showing that crr is the structural gene for IIIGlc, one of the proteins of the PTS. First, homogeneous IIIGlc was isolated from wild-type and a crr- mutant of Salmonella typhimurium, and the proteins were compared. The preparations of IIIGlc were indistinguishable except as follows: IIIGlc from the mutant showed only 2-3% of the activity of the wild-type IIIGlc in its ability to act as a phosphocarrier protein in the in vitro phosphorylation of methyl alpha-glucoside. In addition, under certain conditions, the two proteins exhibited different behavior on gel filtration columns and in polyacrylamide gel electrophoresis. The second line of evidence was obtained by cloning the Escherichia coli crr gene, which has an estimated minimum length of 0.6 kilobase, into a high-copy-number plasmid as part of a 1.3-kilobase fragment. The plasmid transforms E. coli crr- to crr+ strains and simultaneously directs the synthesis of IIIGlc.

Carrier Proteins↗
PubMed1981

Differential uptake of D-galactosyl- and D-glucosyl-neoglycoproteins by isolated rat hepatocytes.

Bovine serum albumin was modified with 2-imino-2-methoxyethyl 1-thioglycopyranosides of galactose and glucose (Gal-AI-BSA, Glc-AI-BSA, respectively). Both types of neoglycoproteins were taken up by isolated rat hepatocytes. Gal-AI-BSA and Glc-AI-BSA inhibited the uptake of each other, and also inhibited the uptake of asialo-orosomucoid as expected from the results obtained with rabbit liver membranes (Stowell, C. P., and Lee, Y. C. (1978) J. Biol. Chem. 253, 6107-6110). Gal-neoglycoproteins of bovine serum albumin containing other linking groups between the galactose moiety and protein were found to be as effective in inhibition of the uptake of asialo-orosomucoid as Gal-AI-BSA, indicating that the method of carbohydrate attachment had little influence on endocytosis of galactose-terminated neoglycoproteins. In contrast, comparable Glc-neoglycoproteins (other than Glc-AI-BSA) were weak inhibitors. It thus appears that the positively charged amidino group present in Glc-AI-BSA contributes to the uptake of this neoglycoprotein by rat hepatocytes.

Animals↗
PubMed1981

Quantitative analysis of intercellular adhesive specificity in freshly explanted and cultured cells.

A new method is presented for the quantitative analysis of intercellular adhesive specificity. In this assay, two cell types are mixed, one unlabeled and the other labeled with the fluorescent dye, fluorescamine [4-phenylspiro(feran-2[3H],1'-phthalan)-3,3'-dione]. The resulting aggregates are analyzed by fluorescence microscopy to determine the number of labeled and unlabeled cells per aggregate. Random (nonspecific) aggregation was characterized by a binomial distribution, and adhesive specificity was accordingly quantified by the deviation (as determined by a chi-square test) from the calculated binomial distribution. The labeling procedure was simple and rapid, and experiments with 18 different cell types showed that it did not affect cell viability, morphology, rate and extent of adhesion, plating efficiency, and the capability of myogenic cells to undergo terminal differentiation. Most important, assays with morphologically identifiable cell pairs indicated that the fluorescent label neither induced apparent nor destroyed existing adhesive specificity. The most pronounced adhesive specificities were observed with freshly explanted cells from adult tissues and also with mixtures of simian virus 40-transformed and nontransformed BALB/c 3T3 cells. A glucosamine-6-phosphate N-acetylase-deficient mutant 3T3 line (AD6), however, aggregated randomly with parental 3T3 cells. Lectin-resistant mutant Chinese hamster ovary (CHO) cells displayed marginal adhesive specificity when mixed with normal CHO cells.

Adipose Tissue↗
PubMed1979

Kinetics of adhesion and de-adhesion of Chlamydomonas gametes.

In medium with low nitrogen content, vegetative strains of the unicellular biflagellate alga Chlamydomonas reinhardi form gametes. Mating type plus (mt+) and mating type minus (mt-) gametes adhere via their flagella to give aggregates in which the gametes eventually fuse to form zygotes. A quantitative assay has now been developed which measures aggregation and fusion by use of a Coulter electronic particle counter to determine loss of single gametes as they form aggregates in suspension. Determination of the rate and extent of cell fusion by microscopy agrees with the results obtained with the more rapid and convenient Coulter counter assay. By use of the assay it was found that aggregation and fusion occur at the same rate and to the same extent at 12 degrees C and 25 degrees C. Flagella from one of the mating types can specifically substitute for the corresponding live gametes; more than 70% of the gametes were aggregated and the extent of aggregation was proportional to the number of flagella added, until the ratio of cells to flagella exceeded 2. At 22 degrees C, in the flagella/gamete mixtures, adhesion was complete in less than 5 min, but at 5 to 10 min, gametes began to de-adhere from the clusters and, depending on the number of flagella added, essentially all of the gametes detached from the aggregates in 10 to 50 min. The gametes in such mixtures were fully competent to aggregate again, whereas the flagella recovered from such mixtures were shown by use of a radioactive flagella-binding assay to be inactive with fresh gametes. Inactivation of the flagella was temperature-dependent, was not catalyzed by soluble factors, and required adhesion of flagella to gametes of the opposite mating type. The potential physiological functions of the de-adhesion process are discussed.

Cell Adhesion↗
PubMed1978

Adhesion of chicken hepatocytes to polyacrylamide gels derivatized with N-acetylglucosamine.

Complex carbohydrates on the surfaces of eukaryotic cells are thought to participate in a wide variety of cell-cell interactions. A model system has therefore been developed to study these processes. In the present experiments, the ability of chicken hepatocytes to recognize and adhere to sugars covalently linked to polyacrylamide gels was investigated. The gels were snythesized by two methods. Type I gels were prepared from a co-polymer of an active ester of acrylic acid (N-succinimidyl acrylate), acrylamide, and bisacrylamide. The "activated" polyacrylamide gel was then treated with the desired ligand containing an amino group, such as 6-aminohexyl O- or S-glycoside. Type II gels were formed by treating similar ligands with acryloyl chloride, followed by co-polymerization of the resulting N-substituted acrylamide with acrylamide and N,N'-methylenebisacrylamide. These polyacrylamide derivatives offer many advantages for studies with intact cells. They are not toxic to any cell type studied, can be cast in any desired shape, are transparent and stable over a wide range of pH values, and contain no cationic and low to negligible levels of anionic charge (charged groups can be introduced if desired), and the polyacrylamide matrix is stable to common biological agents such as bacteria and enzymes. In addition, type I gels can be synthesized using a broad range of molecules containing amino groups, such as glycopeptides, proteins, etc. The hepatocytes were prepared by collagenase perfusion of intact chicken livers. The rate and extent of adhesion of the cells to the derivatized gels was determined by measuring lactate dehydrogenase in these cells. This enzyme was also used to assay viability and cell "leakiness." At 37 degrees C, 70 to 100% of the cells adhered within 60 min to gels derivatized with N-acetylglucosamine, i.e. gels derivatized with 6-aminohexyl 2-acetamido-2-deoxy-beta-D-glucopyranoside (or the corresponding thioglycoside). By contrast, less than 5% of the cells adhered to polyacrylamide or to gels derivatized with 6-aminohexanol or the 6-aminohexyl glycosides of beta-D-glucose, beta-D-galactose, alpha-D-mannose, beta-D-maltose, beta-D-melibiose, beta-D-cellobiose, and (alpha or beta)-D-lactose. Kinetic studies with the chicken hepatocytes and N-acetylglucosamine gels showed that cell-gel binding was dependent upon Ca2+ and was decreased at low temperatures. Binding was inhibited by N-acetylglucosamine or by glycosides of this sugar, the most effective inhibitor being orosomucoid (alpha1-acid glycoprotein) pretreated with sialidase and beta-galactosidase. The cell surface receptor(s) involved in this interaction is not known, but may be related or identical to the chicken liver binding protein described by Lunney and Ashwell (Lunney, J., and Ashwell, G. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 341--343). The present results suggest that this model system should prove useful in delineating cell surface interactions with carbohydrates.

Acetylglucosamine↗
PubMed1978

Specific adhesion of rat hepatocytes to beta-galactosides linked to polyacrylamide gels.

Rat hepatocytes, isolated by a collagenase perfusion technique, specifically bind to polyacrylamide gel containing covalently immobilized 6-aminohexyl beta-D-galactopyranosyl groups. Less than 5% of these cells bind to polyacrylamide or to gels with the following covalently linked ligands: 6-aminohexanol, or the 6-aminohexyl D-pyranosides of alpha-mannose, beta-glucose, beta-2-acetamido-2-deoxyglucose, beta-cellobiose, beta-maltose, or beta-melibiose. Cell binding to beta-D-galactoside gels occurs after a lag period at 37 degrees and 65 to 100% (depending on the cell preparation) of the cells adhere. The duration of the lag period is inversely related to the beta-D-galactoside content of the gel but preincubation of the cells at 37 degrees reduces the lag period. Cell-gel binding is a threshold phenomenon. Adhesion of cells to gels does not occur when the glycoside concentration is less than about 900 nmol per cm2 x 0.25 mm thick gel piece. Above this critical concentration, cell-gel binding occurs and becomes maximal when the concentration is increased by only 20%. If these in vitro results apply to cellular interactions in vivo, they suggest that slight changes in the levels of cell surface or extracellular matrix carbohydrates may profoundly influence the behavior of neighboring cells.

Acrylamides↗
PubMed1977

Periplasmic space in Salmonella typhimurium and Escherichia coli.

The volume of the periplasmic space in Escherichia coli and Salmonella typhimurium cells was measured. This space, in cells grown and collected under conditions routinely used in work with these bacteria, was shown to comprise from 20 to 40% of the total cell volume. Further studies were conducted to determine the osmotic relationships between the periplasm, the external milieu, and the cytoplasm. Results showed that there is a Donnan equilibrium between the periplasm and the extracellular fluid, and that the periplasm and cytoplasm are isoosmotic. In minimal salts medium, the osmotic strength of the cell interior was estimated to be approximately 300 mosM, with a net pressure of approximately 3.5 atm being applied to the cell wall. A corollary of these findings was that an electrical potential exists across the outer membrane. This potential was measured by determining the distributions of Na+ and Cl- between the periplasm and the cell exterior. The potential varied with the ionic strength of the medium; for cells in minimal salts medium it was approximately 30 mV, negative inside.

Acetylglucosamine↗