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Biomedical subjects

P H Fishman

Publications and source records attributed to P H Fishman.

At least 127 records · Page 7Linked to original sources
PubMed1979

Choleragen (cholera toxin): a bacterial lectin.

Choleragen (cholera toxin) agglutinated erythrocytes and liposomes containing the toxin receptor, galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide (ganglioside GM1). Cells that had been exposed to GM1 were agglutinated, but agglutination was not observed when cells had been exposed to other gangliosides (GM2, GM3, GD1a, GD1b). Choleragen-dependent agglutination of liposomes was slightly less specific, because liposomes containing either GM1 or GD1b, but neither GM2, GD1a, nor GM3 were agglutinated. The oligosaccharide isolated from GM1 inhibited both the agglutination of cells and liposomes containing GM1 and the binding of choleragen to liposomes containing GM1. Galactose and sialic acid were less effective inhibitors of liposomal agglutination and did not inhibit cellular agglutination or binding of choleragen to liposomes. Liposomal agglutination was dependent on choleragen concentration and occurred with the B but not the A protomer of choleragen. These results suggest that choleragen, through its binding to the oligosaccharide portion of a glycolipid, exhibits lectinlike activity, which results in agglutination of liposomes and erythrocytes.

Agglutination↗
PubMed1979

Ganglioside inhibition of fibronectin-mediated cell adhesion to collagen.

Fibronectin mediates the adhesion of cells to collagen by first binding to the collagen substrate, followed by attachment of the cells to the fibronectin-collagen complex. Bovine brain gangliosides were found to block fibronectin-mediated cell adhesion to collagen in a concentration-dependent manner. The gangliosides did not block the binding of fibronectin to collagen but did prevent the attachment of the cells to the fibronectin-collagen complex. Of the individual gangliosides tested, GT1 and GD1a were the most effective inhibitors followed by GD1b greater than GM1 greater than GM2; GM3 was not an inhibitor. The inhibition of cell adhesion also was observed with the oligosaccharide portion of the gangliosides, but not with ceramides or with a variety of free sugars or glycosaminoglycans. Mild periodate oxidation of mixed gangliosides or of GD1a modified their sialic acid residues and the oxidized gangliosides were no longer inhibitory; subsequent reduction with NaBH4 did not restore the inhibitory activity of the modified gangliosides. These results suggest that specific gangliosides or related sialic acid-containing glycoconjugates on the cell surface may act as the receptors for fibronectin.

Animals↗
PubMed1979

Gangliosides sensitize unresponsive fibroblasts to Escherichia coli heat-labile enterotoxin.

Chemically transformed mouse fibroblasts did not raise their cyclic AMP level in response to Escherichia coli heat-labile enterotoxin. These fibroblasts did, however, incorporate exogenous mono-, di-, and trisialogangliosides. After the uptake of monosialoganglioside galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (GM1), the cells responded to E. coli heat-labile enterotoxin. The di- and trisialogangliosides were considerably less effective. GM1, the putative cholera toxin (choleragen) receptor, has been implicated previously as the receptor for E. coli heat-labile enterotoxin based on the ability of the free ganglioside to inhibit the effects of toxin. This investigation establishes that the ganglioside, when incorporated into fibroblasts, serves a functional role in mediating the responsiveness to the toxin.

Animals↗
PubMed1978

Effect of thyroid phospholipids on the interaction of thyrotropin with thyroid membranes.

Various lipids extracted from bovine thyroid glands were tested for their ability to affect the binding of 125I-labeled thyrotropin to bovine thyroid membranes. The most potent inhibitors were the acidic phospholipids in the order cardiolipin greater than phosphatidylglycerol greater than phosphatidylinositol greater than phosphatidylserine. Other phospholipids, neutral lipids, and neutral glycolipids were ineffective. As reported previously [mullin, B. R., Pacuszka, T., Lee, G., Kohn, L. D., Brady, R. O. & Fishman, P. H. (1978) Science 199, 77--79], thyroid gangliosides also inhibited thyrotropin binding but not as effectively as phospholipids. In addition, the mode of action of these two classes of acidic lipids was different. When thyroid membranes were preincubated with the phospholipids and then separated by centrifugation, their ability to bind thyrotropin was still diminished. In contrast, gangliosides appear to interact with the hormone and not with the membranes. The effect of phospholipids on thyroid membranes was further examined by incubating the membranes with phospholipase A. The treated membranes now bound more labeled hormone. These results suggest that certain acidic phospholipids, which are present in only small amounts in thyroid membranes, influence the state of the thyrotropin receptor and its ability to bind thyrotropin.

Animals↗
PubMed1978

Interaction of human chorionic gonadotropin with membrane components of rat testes.

Previous studies demonstrating that gangliosides interacted with thyrotropin and human chorionic gonadotropin (hCG) suggested that gangliosides participate in the transduction of the hormonal message across the target cell membrane. As a continuation of these investigations, we examined the effects of down-regulation of hCG receptors on the interaction of hCG with rat testis membrane components. Rat testes contained a complex ganglioside pattern that did not appear to change qualitatively or quantitatively after the injection of hCG into the animals, although testis membranes from hCG-treated rats lost their capacity to bind (125)I-labeled hCG ((125)I-hCG). Gangliosides extracted from the testes of control and treated animals were equally effective inhibitors of (125)I-hCG binding to testis membranes. However, inhibition of binding was observed only under conditions (pH 6.0, low ionic strength) such that unlabeled hCG (>2500-fold excess) did not block (125)I-hCG binding, and (125)I-hCG bound similarly to testis membranes from control and treated rats. Under conditions such that hCG binding was specific (blocked by 250-fold excess of unlabeled hCG), testis gangliosides were noninhibitory. Liposomes containing gangliosides from the testes of control or hCG-treated rats bound similar small amounts of (125)I-hCG. These same liposomes bound 50 and 1000 times more thyrotropin and cholera toxin, respectively, than hCG. Oligosaccharides derived from gangliosides did not inhibit (125)I-hCG binding to testis membranes nor did they alter the fluorescence of hCG conjugated with fluorescent probes, whereas the gangliosides themselves were inhibitory and enhanced the fluorescence intensity of the hCG derivatives. Exposure of testis membranes from hCG-treated rats to 4 M MgCl(2), which displaces bound hCG [Chen, Y.-D. I. & Payne, A. H. (1977) Biochem. Biophys. Res. Commun. 74, 1589-1596], did not restore their ability to bind (125)I-hCG. When membranes were solubilized with Triton X-100, a solubilized receptor was detected from testis membranes of control but not hCG-treated rats. These findings and the absence of demonstrable changes in the composition or quantity of rat testis gangliosides when hCG receptors are down-regulated suggest that gangliosides do not represent the primary binding determinants of hCG receptors.

Animals↗
PubMed1978

Effect of serum on ganglioside uptake and choleragen responsiveness of transformed mouse fibroblasts.

NCTC 2071 cells, transformed mouse fibroblasts, did not respond to choleragen when grown in chemically defined medium. When grown in medium containing 10 percent fetal calf serum, however, the cells accumulated cyclic AMP upon exposure to the toxin. Gangliosides isolated from the fetal calf serum were as effective as whole serum in inducing choleragen responsiveness in the cells. The putative choleragen receptor, the monosialo-ganglioside GM1, could not be detected by chemical analysis in cells exposed to serum. 3H-Labeled GM1 was detected in these cells, however, following sequential exposure to galactose oxidase and sodium borotritide. Thus, uptake of minute amounts of GM1 from serum by these cells sensitized them to choleragen.

Blood Physiological Phenomena↗
PubMed1978

Similar content of phospholipids and gangliosides in normal and homozygous familial hypercholesterolemia fibroblasts.

The cellular content of total and individual phospholipids and gangliosides was measured in fibroblasts cultured from four normal subjects, three patients with lysosomal lipid storage diseases, and two subjects with homozygous familial hypercholesterolemia. Measurements were made on cells grown in medium containing fetal calf serum under conditions in which normal cells derive cholesterol for cell growth from low density lipoprotein present in the fetal calf serum, whereas familial hypercholesterolemia homozygote cells, which lack cell surface low density lipoprotein receptors, derive cholesterol from endogenous synthesis. No difference was observed in the cellular content of total or individual phospholipids and gangliosides in the normal and familial hypercholesterolemia homozygote cells. In contrast, cells from a patient with Niemann-Pick disease and a patient with Sandhoff disease showed elevations in the content of sphingomyelin and complex gangliosides, respectively.

Adult↗
PubMed1977

Synthesis and uptake of gangliosides by choleragen-responsive human fibroblasts.

Human fibroblasts, cultured in medium containing 10% fetal calf serum, responded dramatically to choleragen with an increase in cyclic adenosine monophosphate content to greater than 48 times basal levels. Analysis of these cells for gangliosides indicated that the major ganglioside was N-acetylneuraminylgalactosylglucosylceramide (GM3) with trace amounts (less than or equal to 100 pmol/mg of protein) of other gangliosides including GM1, the putative choleragen receptor. Although the cells contained three glycosyltransferases required for ganglioside synthesis, the N-acetylgalactosaminyltransferase activity necessary for the conversion of GM3 to more complex gangliosides was not detected. When the cells were grown in medium containing [14C]galactose or N-acety[3H]mannosamine, however, all of the gangliosides became labeled, indicating that the cells can synthesize complex gangliosides. Although fetal calf serum contains gangliosides including GM1, [3H]GM1 was taken up poorly from the growth medium and uptake at the rate observed could have accounted for less than 2% of the GM1 content of the cells. When the cells were incubated in chemically defined medium containing [3H]GM1 at the concentrations present in fetal calf serum, rapid uptake of the ganglioside occurred and the total GM1 content of the cells increased threefold in less than 3 h. Thus, although the cells are capable of binding exogenous gangliosides, the gangliosides in fetal calf serum are in a form not readily available to the cells.

Bacterial Toxins↗
PubMed1977

Enzymatic and chemical oxidation of gangliosides in cultured cells: effects of choleragen.

Cell surface glycolipids of normal human fibroblasts and NCTC2071 cells (transformed mouse fibroblasts) were labeled by incubating the intact cells with either galactose oxidase or sodium periodate, followed by reduction of the oxidized sugar residues with NaB3H4. In intact human fibroblasts, incorporation of 3H was increased with increasing time of exposure to galactose oxidase prior to treatment with NaB3H4. Following limited exposure to galactose oxidase, more label was incorporated into the larger glycolipids. Although labeling of the monosialoganglioside GM1 was maximal by 16 h, not all of the GM1 in the intact cells appeared to be accessible to galactose oxidase, since 10 to 12 times more GM1 was labeled when cells were disrupted before incubation with the enzyme. The human fibroblasts contained approximately 8 X 10(6) molecules of GM1 per cell. Maximal binding of choleragen (5 X 10(5) molecules of [125I]choleragen per cell) completely prevented cholevented oxidation of GM1 in intact fibroblasts by galactose oxidase but only partially protected the sialic acid moiety of GM1 from oxidation by periodate. Choleragen had little effect on the enzymatic or chemical oxidation of other glycolipids. NCTC 2071 cells do not contain endogenous GM1 but incorporate exogenous GM1 from the culture medium. When bound to NCTC 2071 cells, exogenous GM1 was protected by choleragen from oxidation by galactose oxidase or whether endogenous or taken up from the incubation medium, are, after interaction with choleragen, less accessible to oxidation by periodate or galactose oxidase.

Animals↗
PubMed1977

Effect of the A and B protomers of choleragen on release of trapped glucose from liposomes containing or lacking ganglioside GM1.

Liposomes containing trapped glucose were used to examine the interaction of the A and B protomers of choleragen with ganglioside GM1 and lipid model membranes. The B protomer (choleragenoid) was as effective as choleragen in causing release of trapped glucose from liposomes containing GM1; the A protomer did not release glucose from GM1 liposomes. Neither choleragen nor the A or B protomers caused release of trapped glucose from glycolipid-free liposomes. Anti-choleragen and complement, however, caused release of trapped glucose from ganlioside-free liposomes previously incubated with the A protomer but not from those incubated with the B protomer or choleragen. These results suggest that the A protomer, but not intact choleragen or B protomer, bound to ganglioside-free liposomes. Presumably, the A protomer must be freed of the constraints present in the intact choleragen in order to interact with the liposomal model membrane system.

Bacterial Proteins↗
PubMed1977

Effect of gangliosides and substrate analogues on the hydrolysis of nicotinamide adenine dinucleotide by choleragen.

Choleragen and its A protomer catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide. NADase activity was inhibited by gangliosides GM1 (galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide), GM2 (N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide), GM3 (N-acetylneuraminyl-galactosylglucosylceramide), and GD1a (N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-E1N-acetylneuraminyl]-galactosylglucosylceramide). These gangliosides also increased the intensity of the tryptophanyl fluorescence of the isolated A protomer (lambda max = 328 nm). GM1 but not GM2, GM3, and GD1a caused a "blue shift" in the fluorescence spectrum of the B protomer. These results are consistent with other evidence that the specificity of GM1 as the choleragen receptor resides in its carbohydrate moiety. The NADase activity of choleragen was similar to that of diphtheria toxin previously described [J. Kandel, R. J. Collier & D. W. Chung (1974) J. Biol. Chem. 249, 2088-2097]. As with diphtheria toxin, analogues of NAD were inhibitory, adenine being the most effective. Significant inhibition was also noted with adenosine, AMP, ADP-ribose, nicotinamide, nicotinamide mononucleotide, and NADP. NADP was hydrolyzed only slowly by choleragen. In the NADase reaction catalyzed by diphtheria toxin, water serves as an acceptor for the ADP-ribose moiety of NAD in lieu of the natural acceptor molecule, which is elongation factor II (Kandel et al., 1974). It seems probable that the natural protein acceptor for ADP-ribose in the reaction catalyzed by choleragen is adenylate cyclase or a protein component of a cyclase complex that regulates enzymatic activity.

Adenine↗