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Biomedical subjects

G M Edelman

Publications and source records attributed to G M Edelman.

At least 289 records · Page 16Linked to original sources
PubMed1976

Identification of murine leukemia viral antigens detected on mouse cells by H-2 alloantisera.

H-2 alloantisera have been previously reported to contain antibodies against murine leukemia viral antigens, but the nature of the viral antigens on mouse cells which interact with these antibodies has not been established. We have found that H-2 alloantisera recognize components of molecular weight 70 000-80 000 mouse lymphocytes and leukemia cells. These components were also detected by a goat antiserum against the murine leukemia virus (MuLV) glycoprotein (gp 70) and are therefore closely related to or identical with that viral protein. Although most H-2 alloantisera detected gp 70-like molecules on lymphocytes and leukemia cells from a great variety of mouse strains, only one H-2 alloantiserum was found to interact with a gp 70 component on cells from C57BL/10 and C57BL/6 mice. Animals such as C57BL/10 mice that lacked the component reacting with most H-2 alloantisera showed increased serum levels of anti-MuLV antibodies after injection of B10.A spleen cells having a gp 70 component detectable by other H-2 alloantisera. In contrast, strains with cells reactive to antiviral antibodies in the H-2 alloantisera had low responses to MuLV antigens after a similar immunization procedure. Serum levels of anti-MuLV antibodies in both groups of mice, however, were increased after injection of Freund's adjuvant. These observations suggest that anti-MuLV antibodies in mouse alloantisera may arise from a response to viral antigens on the immunizing cells and general stimulation of the immune system.

Animals↗
PubMed1976

Subunit structure, cell surface orientation, and partial amino-acid sequences of murine histocompatibility antigens.

Detergent and papain solubilized murine histocompatibility (H-2) antigens have been compared by gel exclusion chromatography, ultracentrifugation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and amino-acid sequence analysis. From these data, we propose a molecular model for the H-2 antigens that includes the size and arrangement of the subunits on the cell surface and in solution, and we provide evidence for the orientation of these molecules on the cell surface. Detergent solubilized H-2 antigens (molecular weight 116,000) consist of two disulfide-linked heavy chains (46,000 daltons) and two monocovalently associated light chains (12,000 daltons). Alkylation with iodoacetamide prior to extraction prevented the formation of a disulfide linkage between the two heavy chains. A water-soluble 51,000-dalton molecule (Fs) consisting of a 39,000-dalton fragment (FH) of the heavy chain and one intact light chain was obtained by papain digestion of cells or detergent extracts. Therefore, the disulfide linkage between the heavy chains is located in the remaining membrane-associated portion (Fm). Amino-acid sequence analysis of the FH fragment of H-2Kb by radiochemical techniques showed that it is identical to the detergent solubilized H-2Kb heavy chain in eight positions for the three amino acids tested. These data indicate that the fragment FH derives from the amino-terminus of the heavy chain and suggest that it projects outward from the cell surface, while the carboxyl-terminal region is associated with the plasma membrane. The described amino-terminal sequence data have been found constant in H-2Kb, H-2Kd, H-2Kk, H-2Db, and H-2Dd gene products. These data support the hypothesis that the K and D products of the major histocompatibility antigen complex have evolved by gene duplication.

Amino Acid Sequence↗
PubMed1976

Acitivity of yeast extracts in cell-free stimulation of DNA replication.

Extracts of the cytoplasm of disrupted spheroplasts of Saccharomyces cerevisiae (bakers' yeast) stimulated DNA synthesis in a cell-free system consisting of nuclei from spleen cells of the frog Xenopus laevis, The stimulation required Mg++8 ATP, and the deoxynucleoside triphosphates, was saturated by an excess of nuclei or extract, and had kinetics resembling those obtained previously with extracts from mammalian and avian cells. After addition of the yeast extract, replication "eyes" were formed in the DNA from the nucleochromatin of the frog, suggesting that the extract stimulated the initiation of DNA replication. The activity was susceptible to heat, was nondialyzable, and was abrogated by tryptic digestion. Temperature-sensitive mutants of the cell division cycle (cdcmutants 4,7,8, and 28) grown at permissive temperature (23 degrees) yielded extracts that were capable of stimulating DNA replication. When the cells were incubated for one generation at the nonpermissive temperature (36 degrees), their extracts showed very low or no activity. All of these mutants are deficient in events of the dependent pathway leading to initiation of DNA synthesis in the yeast cell cycle. A ts mutant, cdc10, deficient in the separate pathway for cytokinesis, showed little or no loss of activity at the nonpermissive temperature. These data indicate that the "initiation" activity, as assayed in vitro, is subject to control in the yeast cell cycle, and its appearance may be one of the terminal events in the pathway leading to DNA synthesis. The finding that extracts from yeast cells can stimulate DNA synthesis in nucleochromatin from frog cells, and the fact that the cdc mutants 4,7,8, and 28 describe a dependent pathway terminating in development of "initiation" activity, are in accord with the hypothesis that the function of proteins in the dependent pathways of the cell cycle is conserved during evolution.

Adenosine Triphosphate↗
PubMed1976

Mechanisms of adhesion among cells from neural tissues of the chick embryo.

In order to analyze the molecular mechanisms of cell adhesion during development, proteins on the surface of chick embryonic neural cells were compared with proteins released after placing these cells in culture. One of the components released into culture, F1 (molecular weight, Mr 140,000), was derived by proteolytic cleavage of a cell surface precursor with a molecular weight of at least 240,000. Another protein, F2, recovered from culture as a dimer (Mr 1110,000), appeared to be a product of limited proteolytic cleavage of F1. Cells in retinal tissue possessed a surface protein of Mr 150,000 that also appeared to be derived by limited proteolytic cleavage of the cell surface precursor. Antibodies to F2 interacted with determinants on the cell surface protein of Mr 150,000, and specifically prevented homologous and heterologous binding among dissociated retinal and brain cells. In contrast, antibodies to F1 failed to prevent cell-cell adhesion and did not crossreact with F2. These data suggest that the cell surface protein of Mr 150,000 generated by limited proteolysis is involved in adhesion of both retinal and brain cells. Cell-cell binding of both retinal and brain cells varied as a function of developmental age and brain cells acquired their binding properties at an earlier time than retinal cells. Similar results were obtained in experiments on the binding of retinal and brain cells of different ages to nylon fibres coated with antibodies to F2. The results of the molecular and cellular experiments are incorporated in a model for cell adhesion invoking both proteolytic activation and modulation of cell surface ligands.

Age Factors↗
PubMed1976

Monovalent derivatives of concanavalin A.

Monovalent dimers of concanavalin A (Con A) have been prepared by a combination of succinylation and photoaffinity labeling. Partial derivatization of native Con A using the photoaffinity label, p-azidophenyl-alpha-D-mannopyranoside, followed by affinity chromatography yielded a fraction that consisted of dimers with a single saccharide-binding site at pH 5. These monovalent dimers formed divalent tetramers at pH 7. In order to achieve a monovalent dimer at this pH, the divalent tetramers were succinylated by previously developed methods. Ultracentrifugation, equilibrium dialysis, and chromatographic experiments indicated that the resultant preparations consisted mainly of monovalent dimers which showed subunit exchange to yield about 15% divalent dimers after 12 hr at physiological pH. Freshly prepared material failed to agglutinate sheep erythrocytes at concentrations 500-fold higher than native tetravalent Con A. In addition, they showed saturating dose-response curves of mitogenic stimulation of mouse splenic lymphocytes. These curves resembled those of divalent succinyl-Con A but not those of the native molecule. Further development of methods for preparing stable monovalent derivatives of Con A should allow a refined analysis of the effects of lectin valence at the cell surface.

Affinity Labels↗
PubMed1976

Temperature-sensitive changes in surface modulating assemblies of fibroblasts transformed by mutants of Rous sarcoma virus.

The hypothesis that surface modulating assemblies containing microfilaments and microtubules and altered after cellular transformation was tested on cells infected with temperature-sensitive mutants of avian sarcoma virus. Untransformed cells (mouse 3T3 and chick fibroblasts), cells transformed by simian virus 40 (SV 3T3), and chick fibroblasts infected with Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV-A-infected cells) were first compared for differences in microfilament and microtubule patterns after treatment with fluorescein-labeled antibodies to actin and tubulin. Transformed cells showed disappearance of ordered stress microfilaments and thickened or diffuse alterations of microtubular arrays. At restrictive temperatures (41 degrees), chick fibroblasts infected with a temperature-sensitive mutant (ts 68) of Rous sarcoma virus showed normal patterns of stress fialments and radial microtubular arrays originating in 1 or 2 centrioles. At permissive temperatures (37 degrees), these patterns were disordered and resembled those of SR-RSV-A-infected cells. After a shift from 41 degrees to 37 degrees, the changes in microtubules were observed in the majority of cells within 1 hr. These changes were reversible and did not result from the inability of tubulin to polymerize. In ts 68-infected cells at permissive temperatures, concanavalin A induced much less surface modulation (inhibition of receptor mobility) than at restrictive temperatures. These results suggest that cellular transformation alters both the structure and function of surface modulating assemblies and prompt the hypothesis that products of viral transforming genes may affect these assemblies with a consequent loss of growth control.

Actins↗
PubMed1976

Initiation of replication in chromosomal DNA induced by extracts from proliferating cells.

Addition of an extract prepared from a proliferating cell line to nuclei isolated from resting tissues such as frog liver and spleen resulted in the stimulation of DNA synthesis as assayed by [3H]dTTP incorporation. This stimulated incorporation of [3H]dTTP required ATP and depended on Mg2+ and deoxynucleoside triphosphates. Pulse-chase experiments showed that the synthesis of DNA in this system was discontinuous, resulting in the appearance of approximately 4S fragments and their ligation to yield higher molecular weight DNA. In addition, electron microscopic analysis of the DNA molecules from the reaction mixture showed that the frequency of replication "eyes" in the extract-stimulated reaction was 10-fold higher than that observed in controls. All of these results strongly suggest that the extract stimulated initiation of DNA replication in the chromatin of normally resting cells. Preliminary characterization by dialysis, heating, and enzyme treatments indicated that the activity is associated with one or more proteins of high molecular weight (greater than 50,000). Comparison of the levels of stimulatory activity in extracts from various mammalian and avian sources showed that the activity was present in cells proliferating either in vivo or in tissue culture. In contrast, extracts from normally resting tissues and cells had no activity. The level of activity present did not appear to be directly related to the levels of DNA polymerase. These results suggest a use for this system in studying regulation of the initiation of DNA synthesis and control of the various phases of the cell cycle.

Adenosine Triphosphate↗
PubMed1976

Some new views of the cell surface.

In recent years, it has become possible to investigate the cell surface of normal and tumor cells in new ways, and a number of lines of evidence obtained by various investigators have provided important new views of the surface of mammalian cells. Recent work has, for example, revealed the structure of the histocompatibility antigens, which appear to be ubiquitous on mammalian cells of different types. The dependency on these antigens of lymphocyte interactions with viruses suggests a new hypothesis: histocompatibility molecules may serve as cell surface adaptors for the recognition of foreign antigens such as viruses by T cell receptors. Contrary to older notions, the molecules that make up the cell surface, including the histocompatibility antigens, are not fixed but move freely in the cell membrane, which is a bilayer of lipid. It has been shown in several laboratories that cross-linking of cell surface proteins or "receptors" such as antibodies leads to clustering of these mobile receptor molecules into patches which are subsequently collected as a cap at one pole of the cell. The interactions among different cells and signaling from the surface of each cell to the interior depend upon the structure and physico-chemical interactions of such receptors and on their linkage to structures underlying the cell membrane. Work in our laboratory has produced consistent evidence that the motion and distribution of cell surface molecules are under control of an intracellular system consisting of a network of special proteins. We have found, for example, that mitogenic lectins such as concanavalin A bind to the carbohydrate portions of surface receptors and cross-link them. This binding to some receptors leads to inhibition of motion of all other receptors on the cell surface, suggesting that the original cross-linkage affects a general network responsible for anchoring cell surface receptors. Explorations of the nature of this network suggest that it consists in part of assemblies below the surface membrane containing microfilaments and microtubules.

Animals↗
PubMed1975

Frequency and avidity of specific antigen-binding cells in developing mice.

In order to analyze the development of antibody diversity in which the genes coding for the antigen-specific cells we have compared the binding of diverse antigens by cells in the fetal, neonatal, and adult mouse. Although the numbers of antigen-binding cells present in fetuses and young animals were smaller than in adults, no restriction could be detected in the varity of specificities expressed in the fetuses, either with respect to the kinds of antigens bound, or to the range of avidities of binding. Cells specific for each of the 11 antigens tested could be detected in the fetus only in the last 4 days before birth, at which time they appeared both in the liver and in the spleen. In all cases, these cells disappeared both in the liver and in the spleen. In all cases, these cells disappeared from the liver within a day of birth, but continued to increase in number in the spleen until adulthood...

Animals↗
PubMed1975

Isolation of lectins of different specificities on a single affinity adsorbent.

Affinity chromatography on Sepharose-fetuin columns was used in a single step procedure to isolate the lectins concanavalin A, Favin, phytohemagglutinin, wheat germ agglutinin, and Limulus hemagglutinin. New lectins with unknown binding specificities were also purified by the same procedure from extracts of small California white beans, Idaho red beans, and white pea beans. The purified lectins exhibited different cell surface mapping properties on erythrocytes, lymphocytes, and sperm cells. It was particularly striking that neither 131I-labeled concanavalin nor 125I-labeled wheat germ agglutinin had any effect on the binding of the other to mouse spleen cells. In accord with this observation, gel electrophoretic analysis of radiolabeled lymphocyte receptors for these two lecithins yielded different patterns. These results indicate that highly purified lectins prepared by affinity chromatography on the same adsorbent can possess strikingly different binding specificities for cell surface receptors.

Agglutination Tests↗
PubMed1975

The covalent and three-dimensional structural of concanavalin A. I. Amino acid sequence of cyanogen bromide fragments F1 and F2.

Concanavalin A is a lectin composed of identical subunits, each containg 237 amino acid residues. The complete amino acid sequence of the first 129 residues of the polypeptide chain has been determined by analysis of peptides obtained from digests of CNBr Fragments F1 (residues 1 to 42) and F2 (residues 40 to 129). Correlation of the chemical sequence with x-ray crystallographic results indicates that Fragment F1 contains all of the protein ligands for the binding of 2 metal ions, Mn2+ and Ca2+, and that Fragment F2 contains many of the residues involved in the interactions of the subunits to form dimers and tetramers. The site of cleavage of the polypeptide chain to yield the naturally occurring Fragments A1 and A2 has also been identified as the peptide bond between residues 118 and 119.

Amino Acid Sequence↗
PubMed1975

The covalent and three-dimensional structure of concanavalin A. II. Amino acid sequence of cyanogen bromide fragment F3.

The amino acid sequence of the COOH-terminal CNBr fragment, F3 (residues 130 to 237), of concanavalin A has been established, completing the determination of the covalent structure of this lectin. Analysis of the chemical sequence showed that the distribution of charged residues is generally more dense in the NH2-terminal half of the polypeptide chain than in the COOH-terminal portion and that in the latter region there is a linear stretch composed of many hydrophobic residues. Correlation with x-ray crystallographic results indicates that the hydrophobic region is located in the interior of the molecule, and that it forms a part of a deep cavity which is the binding site for the inhibitor, beta-(o-iodophenyl)-D-glucopyranoside. In conjunction with the three-dimensional structure, the amino acid sequence reported here provides new data for analysis of variables involved in predicting the three-dimensional folding of proteins from the primary structure. The sequence of concanavalin A is the first determined for a lectin and it serves as a reference structure for comparisons with other lectins.

Amino Acid Sequence↗
PubMed1975

The covalent and three-dimensional structure of concanavalin A. III. Structure of the monomer and its interactions with metals and saccharides.

The three-dimensional structure of the lectin concanavalin A (Con A) has been determined at 2.0-A resolution by x-ray diffraction analysis. The protomers are ellipsoidal domes of dimensions 42 times 40 times 39 A. Folding of the polypeptide backbone is dominated by the presence of two antiparallel pleated sheets, a twisted sheet of seven strands passing through the center of the molecule and a bowed sheet of six strands which forms the back surface of the monomer. Manganese and calcium ions bind to the protein at adjoining sites to form a binuclear complex of two octahedra sharing a common edge. The ligands for each metal ion are four groups from the NH2-terminal region of the protein and 2 water molecules. The binding site for the inhibitor beta-(o-iodophenyl)-D-glucopyranoside is in a deep cavity which contains distinct hydrophobic and hydrophilic binding subsites. Studies of the binding of beta-(o-iodophenyl)-D-glucopyranoside to Con A in the crystalline state and in solution have indicated that the binding behavior of the protein is somewhat different in the two states.

Amino Acid Sequence↗