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Biomedical subjects

G M Edelman

Publications and source records attributed to G M Edelman.

At least 253 records · Page 14Linked to original sources
PubMed1979

Cell-free synthesis and segregation of beta 2-microglobulin.

beta2-Microglobulin has been synthesized in vitro by using a rabbit reticulocyte lysate system and mRNA from the mouse tumor cell line EL4. The molecule is synthesized as a precursor with an NH2-terminal extension of 19 amino acids: Ser-X-Ser-Val-X-Leu-Val-Phe-Leu-Val-Leu-Val-Ser-Leu-X-Gly-Leu-Tyr-X. The processing and segregation of this peripheral membrane protein are directly comparable to those of secretory proteins and integral membrane proteins: addition of dog pancreas microsomal membranes during translation caused conversion to the processed chain, but addition of membranes after synthesis did not; only the processed chain sedimented with the membrane vesicles and was protected from proteolysis by the vesicles; and processing of nascent beta 2-microglobulin was blocked by competitive inhibitors that prevent processing and segregation of secretory and integral membrane proteins. These results suggest that the signal sequences of secretory proteins, integral membrane proteins, and peripheral membrane proteins have a common function and a common receptor on the cytoplasmic face of dog pancreas microsomal membranes. This system also provides a means for studying in vitro the expression and function of the major histocompatibility antigens that are associated with beta 2-microglobulin on cell surfaces.

Amino Acid Sequence↗
PubMed1978

Binding and functional properties of concanavalin A and its derivatives. II. A proteolytic product with saccharide-binding activity.

Limited digestion of the intact subunit of concanavalin A (Mr = 26,000) with trypsin followed by affinity chromatography on Sephadex G-100 has yielded a highly purified product designated here as Tn-Con A. Chemical studies have shown that Tn-Con A is composed of several components: a large fragment (Tn I, Mr = 19,000) spanning residues 1 to 172, and lower molecular weight polypeptides that are noncovalently associated with Tn I to form the active molecule. The molecular weight of Tn-Con A at pH 7 was 90,000, suggesting that, like native concanavalin A, it was a tetramer at physiological pH. Equilibrium dialysis experiments showed that Tn-Con A bound 1 molecule of alpha-methyl-D-glucoside/22,000 g atoms of protein and therefore that four saccharides are bound by the tetrameric molecule. Tn-Con A and native concanavalin A competed for the same receptors on the lymphocyte surface. Moreover, Tn-Con A was mitogenic for both mouse and human lymphocytes with dose-response curves similar to those of the native lectin. All of these results indicate that tryptic hydrolysis of concanavalin A produces a fragmented molecule retaining the saccharide-binding, subunit association, and mitogenic capacity of the native protein.

Amino Acids↗
PubMed1978

Binding and functional properties of concanavalin A and its derivatives. III. Interactions with indoleacetic acid and other hydrophobic ligands.

The binding of concanavalin A to various structures via hydrophobic interactions has been studied using a variety of physicochemical assays. It was found that concanavalin A binds to nonpolar compounds such as the plant auxin beta-indoleacetic acid and its structural analogue tryptophan and that this binding is independent of the saccharide-binding activity normally associated with the lectin. The results of equilibrium dialysis experiments on the binding of beta-indoleacetic acid were consistent with the presence of a single weak binding site per subunit of protein, having an association constant of about 7 X 10(2) M-1. Competition experiments using various nonpolar compounds such as o-iodobenzoic acid suggested that this hydrophobic binding site is located in the same cavity which binds the iodine-containing ligand as shown by x-ray crystallography. Concanavalin A also binds to lipid vesicles composed of dipalmitoylphosphatidylcholine or 12-O-tetradecanoyl phorbol-13-O-acetate. This binding to lipid membranes raises the possibility that the synergistic effects of concanavalin A and tetradecanoyl phorbol acetate on lymphocyte mitogenesis may be due in part to an interaction between lectin and the phorbol ester.

Concanavalin A↗
PubMed1978

Inactivation and chemical alteration of mating factor alpha by cells and spheroplasts of yeast.

Mating factor alpha isolated from yeast culture filtrates was radiolabeled by lactoperoxidase-catalyzed iodination, with full retention of biological activity. The (125)I-labeled alpha factor bound at low levels to cells of both mating types (a and alpha) but not to spheroplasts. Despite the low level of binding, large quantities of alpha factor activity were lost by incubation with a cells and a spheroplasts, but not with alpha or a/alpha diploid cells. The amount of activity removed from the culture medium was much larger than the amount of (125)I-labeled alpha factor bound to the cells and was correlated with the appearance of radiolabeled derivatives separable by thin-layer chromatography. Upon removal of the cell wall of alpha and a/alpha cells, the spheroplasts acquired the ability to remove alpha factor activity from culture medium, to generate derivatives of alpha factor, and to respond to alpha factor by a morphological alteration resembling the response of a cells. These findings raise the possibility that the specific enzyme capable of altering alpha factor, possibly a peptidase, is associated with both a and alpha cells but is masked by the alpha cell wall. This suggestion is consistent with the observation that the alpha factor activities of G(1) arrest and cell elongation were blocked by preincubation of a cells with the protease inhibitor Trasylol.

Journal Article↗
PubMed1978

Changes in the three-dimensional structure of concanavalin A upon demetallization.

When the Mn(2+) and Ca(2+) ions normally present in concanavalin A are removed, the protein becomes incapable of binding saccharides. To explore the structural differences between the native and demetallized forms and their effects on the saccharide-binding properties of the protein, we have refined and compared the crystal structures of both forms. Refinement, carried out by automated difference Fourier methods, has revealed a number of differences between the two structures as well as minor differences between the two crystallographically independent monomers in the demetallized structure. Significant differences between the holo- and apoproteins are confined to the region where the metals are bound. These differences include a reorganization and disordering of the loop, consisting of residues 7-25, that contains all of the direct metal ligands of the protein. In some molecules, the side chain of arginine-228 appears to move into the metal-binding region, possibly compensating in part for the absence of the metal's positive charge. The cis peptide observed in the native protein at alanine-207 is apparently not present in the demetallized protein. The conformational differences affect many of the residues currently thought to be involved in the specific binding of saccharides.

Apoproteins↗
PubMed1978

Assay for early cytoplasmic effects of the src gene product of Rous sarcoma virus.

When microinjected into normal fibroblasts, cytoplasmic extracts of cells transformed by Rous sarcoma virus caused dissolution of microfilament bundles. This activity was not found in extracts of normal cells. The maximum effect was seen within 30 min of injection, and the activity could still be measured after a 10-fold dilution of the cytoplasmic extracts (14 mg/ml original protein concentration). The activity was trypsin sensitive and was destroyed by boiling, but was not RNase sensitive. Protein synthesis was not required for the disruption of actin-containing stress fibers by the injected activity. Microinjected cytoplasts prepared from normal 3T3 cells also showed dissolution of microfilament bundles, indicating that the cell nucleus was not required for expression of activity. Extracts made from fibroblasts transformed by Rous sarcoma virus having a temperature-sensitive mutation in the src gene were also temperature sensitive in the microinjection assay. Thus, the activity of extracts from cells infected with src mutant virus, but not from cells infected with wild-type virus, was destroyed either by in vitro incubation of the extract at the nonpermissive temperature before injection or by incubation of recipient cells at the nonpermissive temperature after injection. We conclude that the microinjection assay can detect a cytoplasmic activity coded for by the src gene of Rous sarcoma virus and that an early direct or indirect target of the src gene product is the cytoskeleton and cell motility system. This result is discussed in relation to the hypothesis that submembranous arrays of microfilaments, microtubules, and their associated proteins interact with cell surface receptors to form a surface modulating assembly that functions as a key regulator of cell growth.

Actins↗
PubMed1978

Adhesion among neural cells of the chick embryo. III. Relationship of the surface molecule CAM to cell adhesion and the development of histotypic patterns.

We have previously identified a molecule (named cell adhesion molecule [CAM]) that is involved in the in vitro aggregation of neural cells from chick embryos. In the present report, specific anti-CAM antibodies have been used to demonstrated that CAM is localized in neural tissues, and is associated with the plasma membrane of retinal cells and neurites. Furthermore, it has been shown by antibody absorption techniques that the decreased adhesiveness of cultured retinal cells obtained originally from older embryos is correlated with a decrease in the density or accessibility of cell adhesion molecules on the surface of these cells. The central role of CAM in neural cell aggregation has been established by the observation that anti-CAM Fab' fragments inhibit adhesion between neural cells in a variety of assays. To investigate the function of CAM and cell adhesion in developing tissues, aggregates of retinal cells that are capable of forming histotypic patterns in vitro were cultured in the presence and absence of anti-CAM Fab'. The Fab' was found to inhibit sorting out of cell bodies and neurites and to decrease the number of membrane-membrane contacts, suggesting that CAM is associated with cell-cell, cell-neurite, and neurite-neurite interactions.

Animals↗
PubMed1978

Adhesion among neural cells of the chick embryo. IV. Role of the cell surface molecule CAM in the formation of neurite bundles in cultures of spinal ganglia.

The cell adhesion molecule (CAM) is involved in adhesion among embryonic retinal and brain cells and has been detected in a variety of neural tissues. This paper describes the use of spinal ganglion cultures and specific anti-CAM antibodies to determine the distribution of CAM on plasma membranes of nerve processes, and to assess the results of perturbation of its function during the growth of neurites from ganglia. The results indicate that CAM is distributed over the entire surface of nerve processes, and that specific anti-CAM Fab' fragments alter the morphology of neurite outgrowth. In particular, it was observed that anti-CAM inhibits formation of nerve bundles, so that the ganglion becomes surrounded by a tangled net of fine processes. Growth cone functions, such as neurite elongation, motility, and attachment to the substratum, did not appear to be affected by the antibody. These studies suggest that one of the major functions of CAM is to mediate side-to-side adhesion between neurites to form fascicles, and raise the possibility that this molecule serves a key role in embryogenesis of nerve tissues.

Animals↗
PubMed1977

Adhesion among neural cells of the chick embryo. I. An immunological assay for molecules involved in cell-cell binding.

An immunologically based method for the quantitative assay of molecules involved in cell adhesion is described. Three observations served as a basis for this assay: (a) cells obtained by trypsinization of retinal tissue aggregated rapidly, provided they had been allowed to recover in culture from the dissociation process; (b) treatment of chick retinal cells with Fab' fragments from rabbit antibodies against these cells prevented their aggregation; and (c) incubation of these antibody fragments with antigens released by retinal cells in culture neutralized their ability to inhibit aggregation. The amount of neutralizing antigen was determined by measuring the rates of cell aggregation in the presence and absence of antibody and antigen using a particle counter. Although adhesion was inhibited by anti-retinal cell antibodies, it was not affected by lectins or anti-carbohydrate antibodies that also were bound to the cell surface. Together, the results suggest that the inhibition involved blockade or inactivation of particular cell surface molecules and that the retinal cell antigens capable of neutralizing the antibodies represented these molecules or their fragments. In the accompanying paper, we describe the use of this assay for the purification from culture supernatants of a cell surface molecule involved in cell to cell adhesion.

Animals↗
PubMed1977

Adhesion among neural cells of the chick embryo. II. Purification and characterization of a cell adhesion molecule from neural retina.

The aggregation of cells from dissociated neural retinas of chick embryos can be inhibited by antibodies prepared against whole retinal cells. In order to identify the antigens involved, substances released by retinal tissues in culture were tested for their ability to neutralize specifically the inhibition by antibody of cell adhesion. Using this assay, three active polypeptides from the culture supernatant were purified 500-fold by gel filtration and polyacrylamide gel electrophoresis. Rabbit antibodies prepared against these purified supernatant activities inhibited cell adhesion and reacted only with the three polypeptides. Immunoprecipitation by the specific antibodies of 3H-labeled proteins from a detergent extract of embryonic retinal cell membranes yielded a polypeptide having a Mr of 140,000 in sodium dodecyl sulfate. This precipitation was inhibited in the presence of the three culture supernatant polypeptides that had activity, suggesting that they contained antigenic determinants in common with the 140,000 Mr surface component. They therefore represent all or parts of this cell surface molecule that were released into solution during tissue culture. The data are consistent with the hypothesis that the 140,000 Mr polypeptide is intimately involved in initial adhesion among neural cells.

Animals↗
PubMed1977

Synthesis and distribution of H-2 antigens in preimplantation mouse embryos.

Synthesis of H-2 antigens by preimplantation mouse embryos is first detectable at the late blastocyst stage. These antigens were detected using immune precipitation assays of extracts of embryos labeled by incorporation of radioactive amino acids but not by surface iodination. Experiments using isolated inner cell massess and trophoblast vesicles indicate that it is the cells of the inner cell mass that synthesize these antigens. H-2 antigens were not detected in either early blastocysts or at earlier cleavage stages.

Animals↗
PubMed1977

The specific antigen-binding cell populations of individual fetal mouse spleens: repertoire composition, size, and genetic control.

In order to analyze the genetic and physiological basis of controls affecting the generation of the repertoire of antigen-binding cells in fetal mice, we have measured the numbers of spleen cells specific for each of four antigens as a function of the total numbers of nucleated and Ig-bearing cells in inbred, hybrid, and random bred fetuses. For each of the two inbred strains BALB/c and CBA/J, the proportion of nucleated cells specific for a given antigen was the same for all individuals of the strain at the 18th day of gestation. The proportion did vary from antigen to antigen, however, and for each antigen the proportion of specific cells observed in CBA/J fetuses was approximately four times that observed in BALB/c fetuses. This difference appeared to be due to a difference between the two strains in the relative size of the repertoire of antigen-binding spleen cells at this stage of development, inasmuch as the frequency of Ig-bearing spleen cells in CBA/J fetuses was likewise approximately four times that observed in BALB/c fetuses. In random bred Swiss-L fetal mice at the 18th day of gestation, the proportion of cells specific for a given antigen varied significantly from one individual to the next. The ratio of proportions of the two antigens observed was constant from individual to individual, however, and this constant ratio differed significantly from the ratio observed for the same two antigens in fetal BALB/c and CBA/J inbred mice. These data suggest that the ontogeny of the repertoire of antigen-binding cells in fetal mice is subject to at least two independent sets of controls, one affecting the relative size of the repertoire in the spleen, and the other affecting the distribution of antigen-binding specificities within that repertoire. Analysis of repertoire size and composition in the spleens of hybrid fetuses confirmed the observation that the two parameters are controlled independently, and suggested further that the control of repertoire size in these fetuses is due to the action of one or a few closely-linked autosomal Mendelian genes. These data are consistent with models for the origin of antibody diversity in which the genes coding for the full repertoire of antibodies are generated somatically from a small number of germ-line genes early in development and in the absence of any strong positive or negative selection with respect to antigenic specificity.

Animals↗
PubMed1977

Joint recognition by cytotoxic T cells of inactivated Sendai virus and products of the major histocompatibility complex.

Cytotoxic T cells specific for Sendai virus were generated by culturing murine spleen cells in vitro together with UV-inactivated Sendai virus. In vivo immunization of donor mice with UV-inactivated Sendai virus resulted in an in vitro secondary response of increased magnitude. Cytotoxic activity was demonstrated in a short-term 51Cr-release assay, using syngeneic tumor cells which had been coated with inactivated Sendai virus by incubation at 4 degrees C for 30 min. The lysis of Sendai virus-coated target cells was restricted by the H-2 haplotype of the target cells, suggesting that the H-2 genes of the target cell contributed to the specificity of the lysis. Kinetic experiments showed that susceptibility to lysis by cytotoxic T cells specific for Sendai virus appeared within 30 min after coating target cells with inactivated virus. Furthermore, there was no detectable synthesis of new proteins in cells treated with UV-inactivated Sendai virus. For these reasons, we suggest that neither viral replication nor the synthesis of new proteins are necessary for the production of the antigen recognized by cytotoxic cells specific for Sendai virus. We infer that the virus-specific component on the target cells is probably a preformed virion antigen adsorbed onto or integrated into the cell membrane. These results imply that, if the cytotoxic T cell recognizes a single antigenic determinant specified both by viral and H-2 genes, this determinant is formed by the physical association of H-2 and Sendai virus antigens rather than by their alteration during the processes of synthesis.

Animals↗
PubMed1977

Isolation by cell-column chromatography of immunoglobulins specific for cell surface carbohydrates.

A new method of affinity chromatography using glutaraldehyde-fixed cells immobilized on Sephadex beads has been used to isolate immunoglobulins (Ig's) specific for cell surface glycoproteins. Ig's that specifically bound and agglutinated the same cells as those originally fixed on the columns were isolated from nonimmune sera of various species. Periodate treatment of the cell-columns and the free cells destroyed their ability to bind the Ig's, and the binding of the Ig's to untreated cells was inhibited by monosaccharides such as D-galactose and sialic acid. The binding of antibodies directed against cell surfaces obtained by immunizing animals with the same mouse tumor cell lines used on the columns (P388 and EL4) was not inhibited by various saccharides. Surface glycoproteins obtained from the mouse tumor cells by immunoprecipitation with the column-isolated Ig's yielded specific electrophoretic patterns that differed from those obtained using Ig's from the sera of rabbits immunized with the tumor cells. The data suggest that the Ig's isolated by cell-column chromatography were directed against carbohydrates, probably those in terminal positions of the polysaccharide portions of the tumor cell surface glycoproteins. Column-isolated Ig's specific for carbohydrates were also useful in studies of cell interactions in nonmammalian systems including Dictyostelium discoideum and Saccharomyces cerevisiae. The cell-column method appears to be adaptable to the isolation of a variety of molecules in addition to antibodies.

Agglutinins↗