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P H Fishman

Publications and source records attributed to P H Fishman.

At least 91 records · Page 5Linked to original sources
PubMed1984

Direct visualization of redistribution and capping of fluorescent gangliosides on lymphocytes.

Fluorescent derivatives of gangliosides were prepared by oxidizing the sialyl residues to aldehydes and reacting them with fluorescent hydrazides. When rhodaminyl gangliosides were incubated with lymphocytes, the cells incorporated them in a time- and temperature-dependent manner. Initially, the gangliosides were evenly distributed on the cell surface but were redistributed into patches and caps by antirhodamine antibodies. When the cells were then stained with a second antibody or protein A labeled with fluorescein, the fluorescein stain revealed the coincident movement of both the gangliosides and the antirhodamine antibodies. When the cells were treated with both rhodamine and Lucifer yellow CH-labeled gangliosides, the antirhodamine antibodies induced patching and capping of both fluorescent gangliosides but had no effect on cells incubated only with Lucifer yellow CH-labeled gangliosides. In addition, capping was observed on cells exposed to cholera toxin, antitoxin antibodies, and rhodamine-labeled protein A, indirectly showing the redistribution of endogenous ganglioside GM1, the cholera toxin receptor. By incorporating Lucifer yellow CH-labeled GM1 into the cells and inducing capping as above, we were able to demonstrate directly the coordinate redistribution of the fluorescent GM1 and the toxin. When the lymphocytes were stained first with Lucifer yellow CH-labeled exogenous ganglioside GM3, which is not a toxin receptor, there was co-capping of endogenous GM1 (rhodamine) and exogenous GM3 (Lucifer yellow CH). These results suggest that gangliosides may self-associate in the plasma membrane which may explain the basis for ganglioside redistribution and capping.

Animals↗
PubMed1984

Desensitization of catecholamine-stimulated adenylate cyclase and down-regulation of beta-adrenergic receptors in rat glioma C6 cells. Role of cyclic AMP and protein synthesis.

When exposed to the beta-agonist (-)-isoproterenol, rat glioma C6 cells exhibited a time-and concentration-dependent reduction in isoproterenol responsiveness (desensitization) and a loss of beta-adrenergic receptors (down-regulation). Other agents, such as dibutyryl cyclic AMP, isobutylmethylxanthine, and cholera toxin, all of which elevate intracellular cyclic AMP levels, also induced receptor down-regulation but at a much slower rate than isoproterenol. Loss of beta-receptors was detected with intact cells, cell lysates, and cell membranes. Receptor loss was accompanied by a reduction in isoproterenol-stimulated cyclic AMP production and adenylate cyclase activity. For a given amount of receptor loss, this reduction was much greater with isoproterenol than with other agents. In addition, the concentration of isoproterenol required for half-maximal stimulation of cyclic AMP production was increased in cells treated with isoproterenol but not with isobutylmethylxanthine or dibutyryl cyclic AMP. The affinity of beta-receptors for the agonist was also lower in membranes from cells treated with isoproterenol but not the other agents. Prior treatment of the cells with cycloheximide inhibited receptor loss by isoproterenol but did not prevent desensitization or reduced affinity of beta-receptors for the agonist. Cycloheximide also blocked the loss of receptors induced by dibutyryl cyclic AMP and, in addition, prevented a reduction in agonist-stimulated adenylate cyclase activity. We propose that desensitization is mediated in rat glioma C6 cells only by agonists and is not dependent on either cyclic AMP or protein synthesis. Down-regulation can be induced both by agonists and by cyclic AMP and does depend on protein synthesis. Thus, desensitization and down-regulation can occur independently.

1-Methyl-3-isobutylxanthine↗
PubMed1984

Iontophoresis of gentamicin into aphakic rabbit eyes. Sustained vitreal levels.

The authors evaluated the intraocular penetration of gentamicin (50 mg/ml) into aphakic rabbit eyes following anodal iontophoresis (0.75 mA for 10 min). Gentamicin levels were determined at 0.5, 4, 8, 16, and 24 hrs after iontophoresis (n = 6 eyes for each time) using an agar diffusion bioassay. Peak levels of 72.04 +/- 6.1 (means +/- SE) micrograms/ml for the corneas and 77.8 +/- 3.0 micrograms/ml for the aqueous humor were obtained at 30 min after iontophoresis. The peak vitreous level was 10.4 +/- 0.4 micrograms/ml, which was found at 16 hrs after iontophoresis. Therapeutic levels of 6.2 +/- 3.0 micrograms/ml were still present in the vitreous humor 24 hrs after iontophoresis. Iontophoresis appears to be an effective noninvasive method for delivering therapeutic levels of gentamicin into ocular tissues and fluids of the aphakic rabbit eye.

Animals↗
PubMed1983

Deglycosylated human chorionic gonadotropin. An antagonist to desensitization and down-regulation of the gonadotropin receptor-adenylate cyclase system.

Human chorionic gonadotropin (hCG) was deglycosylated with anhydrous HF and compared with native hCG for binding and biological activity. The deglycosylated hormone (DG-hCG) had the same affinity as hCG for gonadotropin receptors in murine Leydig tumor cells (MLTC-1) but was less than 1% as potent as hCG in stimulating cyclic AMP production in these cells. Exposure of MLTC-1 cells for 30 min to hCG caused a desensitization of hCG-stimulated adenylate cyclase activity, whereas DG-hCG did not induce desensitization even after 4 h. hCG induced down-regulation of hCG receptors; by 4 h, 40% of the receptors had disappeared, whereas there was no receptor loss in cells exposed to DG-hCG for the same time. By 6 h, receptor down-regulation began to occur in the DG-hCG-treated cells and could be mimicked by exposing the cells to dibutyryl cyclic AMP or cholera toxin. Thus, the small increase in cyclic AMP generated by DG-hCG appears to result in some loss of receptors. Cells were incubated with iodinated hCG or DG-hCG for 30 min, washed, and incubated in fresh medium. Both bound ligands were degraded as measured by disappearance of cell-associated radioactivity and appearance of trichloroacetic acid-soluble label in the medium. The half-lives were 3 and 6 h for hCG and DG-hCG, respectively. Our results indicate that DG-hCG in contrast to hCG does not cause either rapid desensitization of hCG-stimulated adenylated cyclase or rapid down-regulation of hCG receptors. Therefore, receptor occupancy alone is insufficient to induce these phenomena.

Adenylyl Cyclases↗
PubMed1983

Binding of choleragen and anti-ganglioside antibodies to gangliosides incorporated into preformed liposomes.

Exogenously added gangliosides were taken up and incorporated into liposomes just as they are incorporated into cells. Ganglioside GM1 was rapidly taken up by liposomes containing dimyristoyl- or dipalmitoylphosphatidylcholine, cholesterol and dicetyl phosphate. When incubated with a wide range of GM1 concentrations for 18 h, the liposomes incorporated about 10% of the added ganglioside. The rate of GM1 uptake by preformed liposomes was both time- and temperature-dependent. The liposomes also incorporated other gangliosides to a similar extent. The GM1 taken up by preformed liposomes was predominantly located on the outer surface of the liposomes and did not appear to be internalized into the inner half of the lipid bilayer. Liposomes containing GM1 added after liposome formation bound as many anti-GM1 antibodies and as much choleragen as liposomes having GM1 added during the formation of the lipid bilayers. Thus, preformed liposomes sensitized by incubation with GM1 are a good model system for studying the interactions of antibodies and toxins with membrane-associated gangliosides.

Antibodies↗
PubMed1983

Fluorescence labeling of cell surface glycoconjugates with Lucifer yellow CH.

Lucifer yellow CH, a fluorescent hydrazide, reacted with cell surface glycoconjugates on murine thymocytes oxidized with NaIO4 or galactose oxidase. Surface labeling was monitored by fluorescence microscopy, spectrophotometry and by flow microfluorometry. No labeling was detected in unoxidized cells and fluorescence was reduced by neuraminidase and by trypsin. Lucifer yellow CH derivatives of gangliosides also were prepared and incorporated into the surface membranes of thymocytes.

Animals↗
PubMed1983

Degradation of choleragen bound to cultured human fibroblasts and mouse neuroblastoma cells.

125I-choleragen bound to human fibroblasts was degraded slowly with a t1/2 of 2-3 days; the radiolabel in bound 125I-choleragen was present in both the A and B subunits. During degradation, radiolabel was lost more rapidly from the 125I-A1 (t1/2 approximately 2 days) than from the 125I-B peptides (t1/2 greater than 5 days). 125I-Choleragen bound to neuroblastoma cells showed a considerably shorter t1/2 for both the 125I-A1 and 125I-B peptides; as with the fibroblasts, radiolabel was lost more rapidly from the 125I-A1 than from the 125I-B peptides. The continued presence of choleragen in the fibroblasts and neuroblastoma cells was associated with a prolonged activation of adenylate cyclase. In addition, fibroblasts, previously exposed to toxin and then washed free of unbound choleragen, only slowly recovered their ability to bind 125I-choleragen with a t1/2 of 7 days. Fibroblasts exposed to choleragen also showed evidence of persistent toxin on the surface based on the ability of the cells to bind antitoxin, antisubunit A or antisubunit B antibodies followed by 3H-protein A. It appears that choleragen remains persistently bound to fibroblasts, is degraded at a slow rate, and may prevent the binding of new toxin molecules to the fibroblast. The relatively slow degradation of toxin by fibroblasts may explain the prolonged activation of adenylate cyclase by toxin. The loss of 125I-toxin binding capacity following incubation with toxin may result from continued presence of toxin subunits on the cell surface.

Adenylyl Cyclases↗
PubMed1983

Exogenous gangliosides enhance the interaction of fibronectin with ganglioside-deficient cells.

The major cell-surface glycoprotein fibronectin mediates a variety of cellular adhesive interactions that have been reported to be competitively inhibited by gangliosides. These effects suggest a possible function of gangliosides as receptors for fibronectin. To test this hypothesis more directly, we examined the interaction of endogenous fibronectin with a ganglioside-deficient cell line, NCTC 2071. These cells, which grow in serum-free medium, synthesized fibronectin. The fibronectin did not bind to these cells, but instead bound diffusely to the culture substratum. When the cells were cultured in medium containing ganglioside, the fibronectin became bound to the cell surface in fibrillar strands. The order of effectiveness of purified gangliosides was GT1b greater than GD1a greater than GM1 greater than GM2 greater than GM3. The effect with mixed gangliosides was accompanied by a restoration of cellular capacity to bind and to respond to cholera toxin. Treatment of the cells with several phospholipids did not alter fibronectin binding. Our results support the hypothesis that gangliosides can help mediate the binding of fibronectin to fibroblasts.

Animals↗
PubMed1983

Mechanism of action of glycopeptide hormones and cholera toxin: what is the role of ADP-ribosylation?

The cultured murine Leydig tumor cell line MLTC-1 and the normal rat thyroid strain FRTL have adenylate cyclase activities that are stimulated by human chorionic gonadotropin (hCG) and thyrotropin, respectively. Both cell types also respond to choleragen. Activation of adenylate cyclase in membranes by choleragen required NAD whereas stimulation of the enzyme by hormones did not. With [alpha-32P]NAD as a donor, ADP-ribosylation of membrane proteins was determined under the same conditions used to assay adenylate cyclase activity. Under these conditions, choleragen, but not the hormones, caused the ADP-ribosylation of subunits of the regulatory component (G/F) of adenylate cyclase in both FRTL and MLTC-1 membranes. In the absence of any effectors, several membrane proteins became labeled but the hormones did not cause the specific labeling of these or any other membrane proteins. Pretreatment of intact MLTC-1 cells with hCG did not block the ability of choleragen to ADP-ribosylate G/F in isolated membranes; labeling was actually enhanced in a manner related to the length of exposure to hCG. In contrast, pretreatment of the cells with choleragen inhibited ADP-ribosylation of G/F by the toxin in isolated membranes. Extracts of membranes from untreated, hCG-treated, and choleragen-treated MLTC-1 cells were used to reconstitute adenylate cyclase activity in membranes from the cyc- variant of S49 lymphoma cells which lacks a functional G/F. Toxin but not hormone treatment caused an increase in the basal activity of adenylate cyclase in the reconstituted system. Our results indicate that ADP-ribosylation of the regulatory component of adenylate cyclase is required for choleragen action but not for hormone action.

Adenosine Diphosphate Ribose↗
PubMed1983

Endocytosis of exogenous GM1 ganglioside and cholera toxin by neuroblastoma cells.

Cholera toxin (CT) covalently linked to horseradish peroxidase (HRP) is a specific cytochemical marker for its receptor, the monosialoganglioside GM1. The binding and endocytosis of exogenous [3H]GM1 by cultured murine neuroblastoma cells (line 2A [CCl-131] ), which contain predominantly GM3, was examined by quantitative electron microscope autoradiography. The relationship between exogenous receptor, [3H]GM1, and CT HRP was studied in double labeling experiments consisting of autoradiographic demonstration of [3H]GM1 and cytochemical visualization of HRP. Exogenous [3H]GM1 was not degraded after its endocytosis by cells for 2 h at 37 degrees C. Quantitative studies showed similar grain density distributions in cells treated with [3H]GM1 alone and in cells treated with [3H]GM1 followed by CT-HRP. Qualitative studies conducted in double labeling experiments showed autoradiographic grains over the peroxidase-stained plasma membrane, lysosomes, and vesicles at the trans aspect of the Golgi apparatus. The findings indicate that exogenous glycolipid is associated with the plasmid membrane of deficient cells and undergoes endocytosis. The quantitative ultra-structural autoradiographic studies are consistent with the hypothesis that the spontaneous endocytosis of exogenous [3H]GM1 controls the subsequent uptake of CT-HRP.

Animals↗
PubMed1983

Uptake and metabolism of exogenous gangliosides by cultured cells: effect of choleragen on the turnover of GM1.

When added to the culture medium, 3H-labeled GM1 (tritiated predominantly in the terminal galactose residue) was taken up by murine NCTC 2071 and rat glioma C6 cells, both of which are GM1-deficient. Upon incubating the labeled cells in fresh medium, the cell-associated GM1 was metabolized by the cells with a half-life of 1 to 2 days. Some of the GM1 was converted to GD1a but the bulk of the label appeared in the medium as degradation products. When GM1 labeled in the sialic acid or lipid portion of the molecule was utilized, GM2 also was detected with time in the cells and only a small fraction of the radioactivity was detected in the medium. The rat glioma C6 cells appeared unable to degrade the GM2 that they accumulated; this was demonstrated directly by incubating the cells with labeled GM2. The uptake and subsequent metabolism of GM1 was observed over a wide range of GM1 concentrations (10(-8) to 10(-4) M). The GM1-treated cells initially bound more iodinated choleragen than did untreated cells; but with time, binding capacity decreased. When GM1-treated cells were transferred to fresh medium in the presence of excess choleragen, the amount of cell-associated GM1 remained relatively constant for several days; the conversion of GM1 to GD1a also was blocked. Although labeled GM3 and GD1b also were taken up by the cells, choleragen had no effect on their subsequent metabolism. Choleragenoid, the binding subunit of choleragen, also inhibited GM1 metabolism without activating adenylate cyclase. These results indicate that exogenous gangliosides taken up by cultured cells are metabolized and that choleragen, which binds with high affinity to GM1, specifically prevents the metabolism of this ganglioside.

Animals↗
PubMed1982

Mechanism of action of cholera toxin on intact cells. Generation of A1 peptide and activation of adenylate cyclase.

When intact mouse neuroblastoma NB cells were incubated with choleragen at 4 degrees C, washed, and incubated at 37 degrees C, activation of adenylate cyclase occurred rapidly after a delay of 15 min. The cells were incubated under the same conditions with 125I-labeled toxin, lysed, and solubilized with sodium dodecyl sulfate under mild conditions. Soluble proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence of dithiol reductants to separate labeled toxin products. Initially, only 0.1 to 0.2% of the cell-associated radioactivity migrated on the gels as the A1 peptide of choleragen. After a 15-min delay, the amount of A1 peptide increased rapidly with time and paralleled the activation of adenylate cyclase. Similar results were observed with human skin fibroblasts, Friend erythroleukemic cells, and II3-alpha-N-acetylneuraminosyl-gangliotetraosylceramide-treated rat glioma C6 cells. When toxin-treated NB cells were incubated at increasing temperatures, generation of A1 peptide and activation of adenylate cyclase increased in parallel. Both processes were prevented by incubation of cells at 4 or at 37 degrees C in the presence of anticholeragen antibodies. These results indicate that there is delay both in the formation of A1 peptide and in the activation of adenylase cyclase in intact cells. As A1 is believed to be the catalytically active component of choleragen, it is suggested that the lag period may be related in part to the time required to generate A1 peptide from choleragen.

Adenylyl Cyclases↗
PubMed1982

Biosynthesis and localization of gangliosides in cultured cells.

Mouse neuroblastoma N18 cells contain a homologous series of gangliosides (GM3, GM2, GM1, and GD1a) which constitute a biosynthetic pathway. When added to the culture medium, tritium-labeled palmitate, galactose, and N-acetylmannosamine were incorporated into these gangliosides. Incorporation of [3H]galactose into all four gangliosides was detected by 5 min and continued at essentially linear rates for several hours. When the cells were treated with Vibrio cholerae neuraminidase, the amounts of GM3 and GD1a were reduced from 72% to 85%; there was a severalfold increase in GM1 and no change in GM2. In spite of these large alterations in cellular ganglioside composition, there was no change in the rate of [3H]galactose incorporation into the gangliosides. A large proportion of GM3 and GD1a also was accessible to neuraminidase in neuroblastoma NB41A, Friend erythroleukemic, and rat glioma C6 cells. N18, NB41A, and Friend cells bound large amounts of 125I-labeled cholera toxin with high affinity. At saturation, the ratio of GM1 content to toxin bound for the three cell lines was between 5.5 and 7. When treated with neuraminidase, the cells bound more toxin in correspondence to the increase in GM1 content. As each toxin molecule has five binding sites, these results suggest that most of the GM1 in these cells is on the surface. Our results indicate that the sequential glycosylation of one ganglioside to form the next higher homologue involves a very small pool of intermediates and that the bulk of the gangliosides are on the cell surface.

Animals↗
PubMed1982

Translocation of newly synthesized gangliosides to the cell surface.

A new method was developed to follow the translocation of gangliosides from their site of synthesis within the cell to the plasma membrane. Cultured mouse neuroblastoma N18 and rat glioma C6 cells were labeled for increasing times with D- [1-3H]galactose and then subjected to mild oxidation with NaIO4. Under the conditions chosen, oxidation was essentially restricted to cell-surface sialic acid residues, which were converted to derivatives with an aldehyde function. The labeled gangliosides were isolated from the cells and reacted with dinitrophenylhydrazine to form dinitrophenyl (DNP) derivatives of the oxidized gangliosides. The DNP-gangliosides then were separated from their unmodified counterparts by thin-layer chromatography. Thus, the rate of labeling of surface gangliosides was distinguished from the rate of labeling of total gangliosides. Our results indicated that the transfer of gangliosides from the site of synthesis to the cell surface required approximately 20 min and that newly synthesized gangliosides appeared to be transported to the plasma membrane at a constant rate. No essential differences were found in the rates of translocation of different ganglioside species by N18 cells or between gangliosides of N18 and C6 cells.

Animals↗
PubMed1982

Detergent extraction of cholera toxin and gangliosides from cultured cells and isolated membranes.

Choleragen, when bound to various cultured cells, resisted extraction by Triton X-100 under conditions which retained the cytoskeletal framework of the cells. The resistance (greater than 75% of the bound toxin) was observed in Friend erythroleukemic, mouse neuroblastoma N18 and NB41A and rat glioma C6 cells even though the different cells varied over 1000-fold in the number of toxin receptors. The extent of extraction did not depend on whether the cells were in monolayer culture of in suspension or whether choleragen was found at 0 or 37 degrees C. A similar resistance to extraction was also observed in membranes isolated from toxin-treated cells. Using more drastic conditions and other non-ionic detergents, 90% of the bound choleragen was solubilized from cells and membranes. When rat glioma C6 cells, which bind only small amounts of choleragen, were incubated with the ganglioside GM1, toxin binding was increased and the bound toxin was also resistant to extraction. When these cells were incubated with [3H]GM1, up to 70% of the cell-associated GM1 was extracted under the mild conditions. When the Gm1-labeled cells were incubated with choleragen or its B (binding) component, there was a significant reduction in the solubilization of GM1. Similar results were obtained with isolated membranes. When choleragen-receptor complexes were isolated from N18 cells labeled with [3H] galactose by immunoadsorption, only labeled GM1 was specifically recovered. These results suggest that it is the choleragen-ganglioside complex that is resistant to detergent extraction.

Animals↗
PubMed1982

Role of membrane gangliosides in the binding and action of bacterial toxins.

Gangliosides are complex glycosphingolipids that contain from one to several residues of sialic acid. They are present in the plasma membrane of vertebrate cells with their oligosaccharide chains exposed to the external environment. They have been implicated as cell surface receptors and several bacterial toxins have been shown to interact with them. Cholera toxin, which mediates its effects on cells by activating adenylate cyclase, bind with high affinity and specificity to ganglioside GM1. Toxin-resistant cells which lack GM1 can be sensitized to cholera toxin by treating them with GM1. Cholera toxin specifically protects GM1 from cell surface labeling procedures and only GM1 is recovered when toxin-receptor complexes are isolated by immunoadsorption. These results clearly demonstrate that GM1 is the specific and only receptor for cholera toxin. Although cholera toxin binds to GM1 on the external side of the plasma membrane, it activates adenylate cyclase on the cytoplasmic side of the membrane by ADP-ribosylation of the regulatory component of the cyclase. GM1 in addition to functioning as a binding site for the toxin appears to facilitate its transmembrane movement. The heat-labile enterotoxin of E. coli is very similar to cholera toxin in both form and function and can also use GM1 as a cell surface receptor. The potent neurotoxin, tetanus toxin, has a high affinity for gangliosides GD1b and GT1b and binds to neurons which contain these gangliosides. It is not yet clear whether these gangliosides are the physiological receptors for tetanus toxin. By applying the techniques that established GM1 as the receptor for cholera toxin, the role of gangliosides as receptors for tetanus toxin as well as physiological effectors may be elucidated.

Adenosine Diphosphate Ribose↗