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Biomedical subjects

P H Fishman

Publications and source records attributed to P H Fishman.

At least 145 records · Page 8Linked to original sources
PubMed1976

Biosynthesis and function of gangliosides.

Gangliosides are unique acidic glycolipids that are selectively concentrated in the plasma membrane of cells. Surface labeling studies have demonstrated that at least a portion of the oligosaccharde chain of gangliosides extends beyond the hydrophe) is imbedded in the membrane bilayer. It is becoming increasingly apparent that gangliosides participate in the internalization of environmental signals elicited by cholera toxin and glycoprotein hormones such as thyrotropic hormone and chorionic gonadotropin as well as other substances such as interferon and possibly serotonin. The mechanism by which cholera toxin binds to a specific ganglioside receptor on the celraction of trophic agents with gangliosides. We would predict that analyogous phenomena involving gangliosides will be discovered in brain. The biosynthesis of gangliosides proceeds by the ordered sequential addition of sugars to the lipid moiety. These reactions are catalyzed by a cluster of membrane-bound glycosyltransferases. Any alteration in the activity or specificity of one of these enzymes will result in a dramatic change in the ganglioside pattern of an afflicted cell or organ. The drastic consequences that accompany abnormalities of ganglioside synthesis have been documented in a heritable metabolic disorder in vivo and in tumorigenic transformation of cells in vitro. In this article, we have attempted to unify these observations and to provide a reasonable interpretation of the role of gangliosides in mediating cell surface phenomena.

Animals↗
PubMed1976

Uptake and metabolism of gangliosides in transformed mouse fibroblasts. Relationship of ganglioside structure to choleragen response.

NCTC 2071 cells, transformed mouse fibroblasts, when grown in chemically defined medium, are deficient in gangliosides and do not respond to choleragen. The cells lack two biosynthetic enzymes, CMP-sialic acid:lactosylceramide sialyltransferase and UDP-galactose:GM2 (GalNAc-[AcNeu]-Gal-Glc-ceramide) galactosyltransferase, which are required for ganglioside synthesis. Following uptake of ganglioside GM1 (Gal-GalNAc-[AcNeu]-Gal-Glc-ceramide) from the medium, the cells respond to choleragen; however, they remain unresponsive following uptake of gangliosides GM2 (approximately 6 X 10(6) molecules/cell) and GM3 (AcNeu-Gal-Glc-ceramide) (approximately 2 X 10(6) molecules/cell). A response was observed when the cells had bound approximately 2 X 10(7) molecules of GM2/cell. After binding GD1a (AcNeu-Gal-GalNAc-[AcNeu]-Gal-Glc ceramide) (approximately 2 X 10(5) molecules/cell), cells exhibit some response to the toxin which can be attributed to enzymatic conversion of GD1a to GM1. A second line of NCTC 2071 cells which have 2.5 X 10(7) molecules of endogenous GM2/cell is slightly responsive to choleragen; adenosine 3':5'-monophosphate (cyclic AMP) levels rise 150%. However, when these cells have bound 4.4 X 10(4) molecules of GM1 per cell, cyclic AMP levels rise 7-fold following toxin treatment. GM1, which becomes functionally integrated into the cells, appears to be the natural receptor for choleragen and is 50 to 1000 times more effective than other gangliosides in eliciting a choleragen response.

Binding Sites↗
PubMed1976

A comparison of membrane glycoconjugates from mouse cells transformed by murine and primate RNA sarcoma viruses.

Kirsten murine sarcoma virus (Ki-MSV) transformed Balb/eT3 mouse cells (K-Balb) were found to have altered membrane glycoconjugates compared to normal Balb/3T3 cells. There were reduced amounts of mono- and disialogangliosides, GM1 and GD1a, and activity of the specific galactosyltransferase required for synthesis of these gangliosides was reduced to between 0 and 18.5% of normal in the several K-Balb clones examined. When fucose-labeled glycopeptides derived from the surfaces of Balb/3T3 and K-Balb cells were compared by gel filtration chromatography, the glycopeptides from the transformed cells were enriched in earlier eluting components. These differences were also observed when the glycopeptides were derived from the entire cell and were diminished when the surface or cellular glycopeptides from Balb/3T3 and K-Balb were digested with neuraminidase prior to chromatographic analysis. Changes in these membrane sialoglycolipids and sialoglycopeptides were not influenced by Rauscher leukemia virus infection. In marked contrast, these changes in membrane glycoconjugates were not observed in Wooley monkey sarcoma virus (WSV) transformed Balb/3T3 cells (W-Balb). Although W-Balb cells like K-Balb were transformed by tissue culture criteria, their ganglioside composition, galactosyltransferase activity, and glycopeptide patterns were similar to normal Balb/3T3. These findings have potential implications concerning the role of these complex carbohydrates in the phenotypic alterations of transformed cells.

Animals↗
PubMed1976

Choleragen-mediated release of trapped glucose from liposomes containing ganglioside GM1.

125I-Labeled choleragen was bound to liposomes containing galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide (GM1), but not in large amounts to ganglioside-free liposomes nor to those containing N-acetylneuraminylgalactosylglucosylceramide (GM3), N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide (GM2), or N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide (GD1a). Choleragen released trapped glucose only from GM1-liposomes. This choleragen-induced glucose release from GM1-liposomes was relatively rapid for the first few minutes, then continued more slowly. The amount of glucose released from liposomes in 30 min was dependent on both the GM1 content and choleragen concentration. Prior incubation of GM1-liposomes with anti-GM1 antiserum prevented the choleragen-dependent release of trapped glucose. After incubation of GM1-liposomes with choleragen, addition of anticholeragen antibodies and complement led to more extensive glucose release. Under these latter conditions a much smaller glucose release was observed also from liposomes containing GM1 or N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide in the absence of choleragen. These releases were attributed to naturally-occurring antiganglioside antibodies in the antiserum and complement. Ganglioside-free liposomes did not release glucose in response to anticholeragen and complement. It appears that choleragen in the absence of other proteins binds specifically to liposomes containing GM1 and can induce permeability changes.

Antigen-Antibody Reactions↗
PubMed1976

Relationship of gangliosides to the structure and function of thyrotropin receptors: their absence on plasma membranes of a thyroid tumor defective in thyrotropin receptor activity.

Plasma membranes derived from rat thyroid tumor (1-8R) which is unresponsive to thyrotropin but is responsive to dibutyryl adenosine 3':5'-cyclic monophosphate bind less than 20% of the [125I] thyrotropin which can be bound to plasma membranes from normal rat thyroids under conditions which optimize tumor membrane binding relative to normal thyroid membranes. In addition, the binding is different from thyrotropin binding to normal thyroid membranes both in its altered sensitivity to changes in hydrogen ion concentration and in a decreased sensitivity to competition by unlabeled thyrotropin. This reduced capacity to bind [125I] thyrotropin cannot be attributed to degradation of the hormone by membrane-associated proteases. Although the supernatant phase of the thyroid tumor homogenates contains a soluble component which inhibits [125I] thyrotropin binding to thyrotropin receptors on plasma membranes, its level is the same as in homogenates of normal thyroid tissue. Trypsin digestion does not expose thyrotropin receptors in a manner analogous to that seen in normal thyroid tissue. The major ganglioside in the tumor membranes is N-acetylneuraminylgalactosylglucosylceramide and the membranes lack the N-acetylgalactosaminyltransferase required for the synthesis of more complex gangliosides. In contrast, the normal rat thyroid membranes contain more complex gangliosides such as galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide and N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide as well as the glycosyltransferase activities required for their syntheses. Galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide can also be detected in normal membranes, but not in tumor membranes, by selective labeling with galactose oxidase (D-galactose: oxygen 6-oxidoreductase, EC 1.1.3.9) and [3H] sodium borohydride. These results support the hypothesis that gangliosides are important structural or functional components of thyrotropin receptors on thyroid plasma membranes.

Animals↗
PubMed1976

Thyrotropin-ganglioside interactions and their relationship to the structure and function of thyrotropin receptors.

Gangliosides inhibit 125I-labeled thyrotropin binding to the thyrotropin receptors on bovine thyroid plasma membranes, on guinea pig retro-orbital tissue plasma membranes, and on human adipocyte membranes. This inhibition by gangliosides is critically altered by the number and location of the sialic acid residues within the ganglioside structure, the efficacy of inhibition having the following order: GD1b greater than GT1 greater than GM1 greater than GM2 = GM3 greater than GD1a. The inhibition results from the interaction of thyrotropin and gangliosides, rather than the interaction of membrane and gangliosides. Fluorescence studies show that the inhibition is associated with a distinct conformational change of the thyrotropin molecule and that the progression from a "noninhibitory conformation" to an "inhibitory conformation" parallels exactly the order of effectiveness in inhibiting 125I-labeled thyrotropin binding. The ganglioside inhibition of 125I-labeled thyrotropin binding appears to be hormonally specific in that it is not affected by albumin, glucagon, insulin, prolactin, follicle-stimulating hormone, growth hormone, or corticotropin. The possibility that a ganglioside or ganglioside-like structure is a component of the thyrotropin receptor is suggested by the finding that gangliosides more complex than N-acetylneuraminylgalactosylglucosylceramide are present in bovine thyroid membranes in much higher quantities than have been previously found in extraneural tissue. The finding that the B component of cholera toxin, which also interacts with gangliosides, has a peptide sequence in common with the beta subunit of thyrotropin, suggests that thyrotropin and cholera toxin may be analogous in their mode of action on the membrane.

Adipose Tissue↗
PubMed1976

Functional incorporation of ganglioside into intact cells: induction of choleragen responsiveness.

NCTC 2071 cells are unable to synthesize the monosialoganglioside GM1. When grown in chemically defined medium these cells contained no detectable GM1 and did not accumulate 3': 5'-cyclic AMP in response to choleragen. Incubation of the cells with [3H]GM1 permitted quantification of ganglioside uptake which was dependent on time and concentration of [3H]GM1 in the medium. Responsiveness to choleragen was demonstrated with binding of as few as 17,000 molecules of [3H]GM1 per cell; a maximal response was observed with 10(5) molecules per cell. With increasing cellular content of GM1, the rate of rise in intracellular cyclic AMP in response to choleragen was increased. With greater than 1 X 10(5) molecules of GM1 per cell, the delay between addition of choleragen and the cyclic AMP response was inversely proportional to choleragen concentration; less than 250 molecules of choleragen per cell caused a significant increase in cyclic AMP after 8 hr of incubation. Although the responsiveness of intact cells to choleragen was dependent on GM1, choleragen activation of adenylate cyclase in homogenates with 0.6 mM NAD was independent of added ganglioside. These observations are consistent with the view that exogenous ganglioside GM1 can be functionally integrated into the surface membrane of intact cells and serve as the choleragen receptor. Furthermore, although exogenous GM1 is required for choleragen responsiveness in intact cells, the ganglioside does not play an obligatory role in cell homogenates, where the surface receptor can presumably be bypassed.

Adenylyl Cyclases↗
PubMed1976

Cholera toxin interactions with thyrotropin receptors on thyroid plasma membranes.

Unlabeled cholera toxin inhibits [125I]thyrotropin binding to thyrotropin receptors on thyroid plasma membranes. Maximal inhibition by cholera toxin does not exceed 40%, whereas unalbeled thyrotropin completely inhibits [125I]thyrotropin binding to these same membranes. Kinetic analyses of the binding data are compatible with the view that the cholera toxin decreases the number of receptor sites available to thyrotropin and that the mechanism by which the cholera toxin inhibits [125I]thyrotropin binding to these receptor sites involves both competitive and noncompetitive elements.

Adenylyl Cyclases↗
PubMed1975

Deficient Ganglioside Biosynthesis: a novel human sphingolipidosis.

An unusual lipid storage disese is chracterized by the accumulation of hematoside (Gms3) in the patient's liver and brain. In contrast to the other sphingoliidoses, the accumulation of Gm3 is not the result of a defective catabolic reaction, but is the first disorder caused by deficiency in ganglioside biosynthesis to be described in man.

Brain↗
PubMed1975

Altered ganglioside biosynthesis in mouse cell cultures following transformation with chemical carcinogens and x-irradiation.

Chemicaly and x-ray-transformed subclones of BALB/c 3T3 mouse embryo cells were found to have reduced amounts of the mono- and disialogangliosides galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (Gm1) and N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (Gd1a), and increased amounts of N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (Gm2). The activity of the enzyme UDP-Gal:Gm2 galactosyltransferase was reduced to between 2.7 and 14.3% of normal in the transformed clones. Other ganglioside glycosyltransferase activities were unaffected. This enzymatic change was consistent with the observed alteration in ganglioside pattern in the transformed cells. The residual galactosyltransferase activity in the transformed cells was kinetically similar to the normal enzyme, suggesting that transformation alters ganglioside biosynthesis by blocking enzyme synthesis at the translational or transcriptional levels.

Animals↗
PubMed1975

Morphological changes in cultured mammalian cells: prevention by the calcium ionophore A23187.

The morphological changes induced by butyrate in HeLa cells and by monobutyryl or dibutyryl cAMP in CHO cells are prevented by micromolar concentrations of the divalent cation ionophore A23187. The ionophore is unable to prevent such changes in medium from which calcium is omitted. At slightly higher (but nontoxic) concentrations, the ionophore inhibits the butyrate-mediated induction of the ganglioside biosynthetic enzyme, sialyltransferase, in HeLa. In CHO, sialyltransferase activity is normally high and not altered by any of the compounds tested.

Alkaline Phosphatase↗
PubMed1975

Morphological alterations and ganglioside sialyltransferase activity induced by small fatty acids in HeLa cells.

Incubation of HeLa cells in the presence of millimolar concentrations of propionate, butyrate, or pentanoate increases the specific activity of CMP-sialic acid:lactosylceramide sialyltransferase 7-20-fold within 24 h. Longer-chain saturated fatty acids or acetate are much less effective, decanoate showing no induction. Unsaturated fatty acid analogs of butyrate and other compounds are ineffective. Only the three most effective compounds also produce characteristic smooth extended cell processes in HeLa cells. Butyrate (5 mM) induces the sialyltransferase after a 4-h lag, producing maximum specific activity by 24 h. The amount of sialyl-lactosylceramide, the glycolipid product of the enzyme, increases during that time 3.5 times more than in control cultures. No other glycosphingolipid enzyme is significantly altered by butyrate exposure. The cellular shape changes occur 2-3 h later than the increase of sialyltransferase activity, and both processes require the continuous presence of inducer and the synthesis of RNA and protein but not the synthesis of DNA or the presence of serum.

Blood Proteins↗