Biomedical subjects

P H Fishman

Publications and source records attributed to P H Fishman.

At least 73 records · Page 4Linked to original sources

PubMed1986

Intravitreal liposome-encapsulated gentamicin in a rabbit model. Prolonged therapeutic levels.

The authors investigated the effect of liposome encapsulation on the pharmacokinetics of gentamicin after intravitreal injection in albino rabbits, using immunofluorescent assay. Gentamicin was encapsulated into liposomes of phosphatidylcholine, phosphatidic acid, and alpha-tocopherol. The final liposomal suspension contained gentamicin, 10 mg/ml, 95% encapsulated. One eye of each rabbit received an intravitreal injection (100 mg gentamicin per 0.1 ml) of either liposome-encapsulated gentamicin (LEG) or gentamicin in phosphate-buffered saline (Gs). Equilibrium dialysis separated free from encapsulated drug in vitreous samples. The peak free drug concentration was significantly less (P less than .01, unpaired t-test) with LEG than with Gs. Concentrations of free and total gentamicin were significantly greater (P less than .05) with LEG than with Gs at 24, 72, 120, and 192 hr. LEG gave a twofold increase in area under the drug concentration time curve for total drug, and a 1.5-fold increase for free drug when compared to Gs.

Animals↗

PubMed1985

Direct evidence that endogenous GM1 ganglioside can mediate thymocyte proliferation.

The B subunit of cholera toxin, which is multivalent and binds exclusively to a specific ganglioside, GM1, was mitogenic for rat thymocytes. When exposed to the B subunit, the cells proliferated, as measured by 3H-labeled thymidine incorporation. Mitogenesis depended on the direct interaction of the B subunit with GM1 on the surface of the cells. This demonstrates that endogenous plasma membrane gangliosides can mediate proliferation in lymphocytes.

Adenylyl Cyclases↗

PubMed1985

Gangliosides do not move from apical to basolateral plasma membrane in cultured epithelial cells.

Both qualitative and quantitative approaches were used to ascertain whether gangliosides, incorporated into the apical plasma membrane of cultured epithelial cells from kidney of toad (A6) and dog (MDCK), were able to redistribute past the tight junctions to the basolateral membrane. The apical surfaces of confluent epithelia were exposed to rhodaminyl gangliosides and the distribution of the inserted gangliosides was assessed qualitatively by fluorescence microscopy. All of the fluorescence was confined to the apical surface for at least 1 h after the fluorescent gangliosides had become incorporated; none appeared on the basolateral surface. These observations were confirmed by incubating the cells with anti-rhodamine antibodies and 125I-labeled protein A. In order to quantitate further the ganglioside distribution, binding assays were performed using 125I-labeled cholera toxin, which binds specifically to ganglioside GM1. Exogenous GM1 added to the apical membrane was not detected on the basolateral membrane 4 h after its incorporation even though there was extensive disappearance of the inserted ganglioside, presumably through endocytosis. To directly examine the behaviour of endogenous gangliosides, the apical surface of the epithelial cells was exposed to bacterial neuraminidase, which hydrolyzes more complex gangliosides to GM1. The cells exhibited a 10-fold increase in binding of cholera toxin to their apical surface, but no increase in binding to their basolateral surface. Thus, no cellular pathways for movement from apical to basolateral plasma membrane appear to be available for implanted or endogenous gangliosides.

Animals↗

PubMed1985

Phorbol esters and beta-adrenergic agonists mediate desensitization of adenylate cyclase in rat glioma C6 cells by distinct mechanisms.

Exposure of rat glioma C6 cells to either isoproterenol or 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in desensitization of isoproterenol-stimulated adenylate cyclase activity. After either treatment, the affinity of beta-receptors for isoproterenol was reduced. Thus, desensitization by TPA or isoproterenol appeared to involve an "uncoupling" of the beta-receptor from the stimulatory regulatory component (Ns) of adenylate cyclase. The activity of Ns, assayed by reconstitution of S49 cyc- adenylate cyclase activity, was found to be unchanged after desensitization. The activity of beta-receptors was measured by inactivating Ns and the catalytic component of adenylate cyclase in C6 membranes and fusing them with membranes lacking beta-receptors. Receptors from isoproterenol-treated C6 cells were less active in "coupling" to the foreign adenylate cyclase than receptors from untreated cells, whereas receptors from TPA-treated cells were fully active. This unexpected latter result was explored further. Lysates from C6 cells were centrifuged on linear sucrose density gradients and the gradient fractions assayed for beta-receptor binding activity. Most of the receptors were recovered in a "heavy" plasma membrane peak but some receptors also appeared in a "light" membrane peak. After treatment of the cells with isoproterenol or TPA, the proportion of receptors in the light peak increased. Prior treatment of the cells with concanavalin A prevented the increase in light receptors caused by isoproterenol or TPA. In addition, the concanavalin A treatment prevented the desensitization of adenylate cyclase caused by TPA but not that caused by isoproterenol. Finally, desensitization of adenylate cyclase was reversed by polyethylene glycol-induced fusion of membranes from cells treated with TPA but not isoproterenol. We conclude that beta-agonists and phorbol esters desensitize adenylate cyclase by distinct mechanisms. Agonists cause a reduction in the functional activity of the beta-receptors followed by a segregation of the receptors into a light membrane fraction devoid of Ns. Phorbol esters do not alter the activity of the receptors but do cause their segregation.

Adenylyl Cyclases↗

PubMed1985

Monoclonal IgM in a patient with paraproteinemic polyneuropathy binds to gangliosides containing disialosyl groups.

Monoclonal IgM kappa from a patient with polyneuropathy associated with paraproteinemia was found to bind to several polysialogangliosides. Binding of IgM to gangliosides was shown by enzyme-linked immunosorbent assay (ELISA) and by overlaying thin-layer chromatograms of human brain and peripheral nerve gangliosides with the patient's serum followed by radioiodinated goat antihuman IgM. The latter technique showed that the IgM paraprotein reacted with a number of gangliosides. In an ELISA the IgM paraprotein reacted strongly with GD2, GD3, GD1b, and GT1b, but not with GM1, GM3, and GD1a. Thus, the epitope for the patient's IgM paraprotein appears to involve the disialosyl configurations.

Brain Chemistry↗

PubMed1985

Down-regulation of gonadotropin and beta-adrenergic receptors by hormones and cyclic AMP.

Loss of gonadotropin receptors in murine Leydig tumor cells and of beta-adrenergic receptors in rat glioma C6 cells occurred following exposure of the cells to human chorionic gonadotropin and isoproterenol, respectively. Down-regulation of receptors was mimicked in part by other agents that elevated cyclic AMP levels in the cells such as cholera toxin and dibutyryl cyclic AMP. Whereas agonist-mediated receptor loss was rapid and almost total, down-regulation by cyclic AMP was slower and less extensive. Down-regulation of receptors did not appear to be accompanied by loss of the regulatory and catalytic components of adenylate cyclase. Hormone-mediated down-regulation was preceded by desensitization of hormone-stimulated adenylate cyclase. In contrast, there was no evidence that cyclic AMP caused desensitization. Finally, loss of receptors induced either by agonists or cyclic AMP required protein synthesis as cycloheximide inhibited down-regulation. We conclude that down-regulation of receptors in these cells is a complex process involving both cyclic AMP-independent and -dependent events.

Animals↗

PubMed1985

Alterations in the blood-aqueous barrier of the rabbit eye after neodymium: YAG photodisruption.

We studied the effects of the Nd:YAG laser on the blood-aqueous barrier following photodisruption of the anterior capsule, posterior capsule, and mid-vitreous of the albino rabbit eye with fluorophotometric techniques using albumin-bound fluorescein. After photo-disruption of the anterior capsule, fluorescence appeared in the anterior chamber at 30 minutes, peaked at two hours, and decreased almost to baseline values by 24 hours. No fluorescence was noted at any time in contralateral control eyes or in eyes receiving photo-disruption of the posterior capsule or mid-vitreous.

Animals↗

PubMed1985

Fluorescent gangliosides as probes for the retention and organization of fibronectin by ganglioside-deficient mouse cells.

Ganglioside-deficient transformed mouse fibroblasts (NCTC 2071A cells), which grow in serum-free medium, synthesize fibronectin but do not retain it on the cell surface. When fluorescent derivatives of gangliosides, containing either rhodamine or Lucifer yellow CH attached to the sialic acid residues, were added to the culture medium, the cells incorporated the derivatives and their surfaces became highly fluorescent. When the cells were stained with anti-fibronectin antibodies and a fluorescent second antibody, fibrillar strands of fibronectin were observed to be attached to the cell surface, with partial coincidence of the patterns of direct ganglioside fluorescence and indirect fibronectin immunofluorescence at the cell surface. When the cells were exposed to bacterial neuraminidase during the time of ganglioside insertion, similar patterns of fluorescence were observed. Because the fluorescent gangliosides are resistant to the enzyme, these results suggest that neuraminidase-sensitive endogenous glycoconjugates were not involved in the ganglioside-mediated retention and organization of endogenous fibronectin. After cells were exposed to exogenous chicken fibronectin, most of the fibronectin was attached to the substratum and only a few fibrils were attached to the cells. When exogenous gangliosides were included in the incubation, there was a striking increase in cell-associated exogenous fibronectin, which was highly organized into a fibrillar network. Conversely, cells incubated for 18 h with exogenous unmodified gangliosides exhibited a highly organized network of endogenously derived fibronectin. Upon further incubation of the cells for 2 h with fluorescent gangliosides, there was considerable co-distribution of the fluorescent gangliosides with the fibronectin network as revealed by immunofluorescence. Our results support the concept that gangliosides can mediate the attachment of fibronectin to the cell surface and its organization into a fibrillar network.

Animals↗

PubMed1985

Fate of tetanus toxin bound to the surface of primary neurons in culture: evidence for rapid internalization.

We examined the nature of the tetanus toxin receptor in primary cultures of mouse spinal cord by ligand blotting techniques. Membrane components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose sheets, which were overlaid with 125I-labeled tetanus toxin. The toxin bound only to material at or near the dye front, which was lost when the cells were delipidated before electrophoresis. Gangliosides purified from the lipid extract were separated by thin-layer chromatography and the chromatogram was overlaid with 125I-toxin. The toxin bound to gangliosides corresponding to GD1b and GT1b. Similar results were obtained with brain membranes; thus, gangliosides rather than glycoproteins appear to be the toxin receptors both in vivo and in neuronal cell cultures. To follow the fate of tetanus toxin bound to cultured neurons, we developed an assay to measure cell-surface and internalized toxin. Cells were incubated with tetanus toxin at 0 degree C, washed, and sequentially exposed to antitoxin and 125I-labeled protein A. Using this assay, we found that much of the toxin initially bound to cell surface disappeared rapidly when the temperature was raised to 37 degrees C but not when the cells were kept at 0 degree C. Some of the toxin was internalized and could only be detected by our treating the cells with Triton X-100 before adding anti-toxin. Experiments with 125I-tetanus toxin showed that a substantial amount of the toxin bound at 0 degree C dissociated into the medium upon warming of the cells. Using immunofluorescence, we confirmed that some of the bound toxin was internalized within 15 min and accumulated in discrete structures. These structures did not appear to be lysosomes, as the cell-associated toxin had a long half-life and 90% of the radioactivity released into the medium was precipitated by trichloroacetic acid. The rapid internalization of tetanus toxin into a subcellular compartment where it escapes degradation may be important for its mechanism of action.

Animals↗

PubMed1985

Effect of inoculum size on the induction of endophthalmitis in aphakic rabbit eyes.

A reproducible animal model is necessary to examine the use of antimicrobial agents for prophylaxis and treatment of bacterial endophthalmitis. We determined the minimum inoculum size of S. aureus and P. aeruginosa that consistently produced endophthalmitis when injected into aphakic rabbit eyes immediately following surgery. Both anterior chamber and intravitreal injections were examined. For S. aureus, an intravitreal inoculum of 19.3 +/- 7.5 CFU and an anterior chamber inoculum of 50.5 +/- 4.0 CFU were required. For P. aeruginosa, an intravitreal inoculum of 5.5 +/- 2.6 CFU and an anterior chamber inoculum of 97.5 +/- 10.7 CFU consistently produced a fulminant infection. Lower inocula of both bacteria produced endophthalmitis in both locations, but the effect was inconsistent.

Animals↗

PubMed1984

A sensitive enzymatic assay for determination of cholesterol in lipid extracts.

A procedure for the determination of free and total cholesterol in lipid extracts is described. The method for free cholesterol employs cholesterol oxidase to generate H2O2 and peroxidase to catalyze the reaction of H2O2 with o-dianisidine to yield a colored product. For the determination of total cholesterol, cholesterol ester hydrolase is included.

Animals↗

PubMed1984

Antibodies against human chorionic gonadotropin convert the deglycosylated hormone from an antagonist to an agonist.

Chemical deglycosylation of human chorionic gonadotropin (hCG) produced an antagonist (DG-hCG) that specifically bound to hCG receptors but was no longer able to stimulate adenylate cyclase in the murine Leydig tumor cell line, MLTC-1. DG-hCG was restored to an agonist by incubating cells or membranes having the bound analogue with antibodies against hCG (anti-hCG). In the presence of anti-hCG, cyclic AMP accumulation and adenylate cyclase activity were stimulated over DG-hCG alone. There was no accumulation of cyclic AMP when the cells were exposed to anti-hCG alone or DG-hCG and normal serum or anti-hCG first then DG-hCG. Several different batches of anti-hCG were effective but their activity did not correlate with their affinity for DG-hCG or hCG. The effect of anti-hCG on DG-hCG activity was dose- and time-dependent. Maximal stimulation of cyclic AMP was achieved with antisera dilutions of 1:200 or less. When DG-hCG-treated cells were exposed to anti-hCG at 37 degrees C, there was a 10-min lag. The lag was eliminated when the cells were exposed to the antibodies at 4 degrees C for 3 h and then warmed to 37 degrees C. Adenylate cyclase was also activated when Fab fragments prepared by papain digestion of anti-hCG were used, whereas Fc fragments were not effective. Thus, the divalency of the anti-hCG is not the critical factor in the mechanism of antibody action. Our results suggest that anti-hCG converts DG-hCG from an antagonist to an agonist possibly by altering the conformation of the modified hormone.

1-Methyl-3-isobutylxanthine↗

PubMed1984

The role of gangliosides in the interaction of human chorionic gonadotropin and cholera toxin with murine Leydig tumor cells.

A clonal line of murine Leydig tumor cells (MLTC-1) bound both human chorionic gonadotropin (hCG) and cholera toxin (CT) with high affinity and accumulated cyclic AMP in response to either effector. The major cellular ganglioside was GM3 with small amounts of GM2, GM1, and GD1a. The gangliosides became labeled when the cells were grown in medium containing [3H] galactose or were exposed to galactose oxidase or NaIO4 followed by NaB3H4. CT specifically protected GM1 from surface labeling whereas hCG did not protect any gangliosides from being labeled. When the cells were exposed to sialidase, surface GD1a was eliminated, and GM1 increased with a corresponding increase in CT binding. When sialidase-treated cells were first incubated with the B component of CT, binding and action of CT was blocked. The cells, however, retained their ability to bind and respond to hCG. Addition of purified gangliosides to the medium effectively inhibited the binding and action of CT but not hCG. The cells incorporated the exogenous gangliosides and exhibited increased binding of and responsiveness to CT but not hCG. Both hCG- and CT-receptor complexes were extracted from the cells with nonionic detergent and analyzed by sucrose gradient centrifugation. The hCG-receptor complex had an apparent molecular weight of 190,000 whereas the CT-receptor complex sedimented only slightly faster than CT itself. MLTC-1 gangliosides were separated on thin layer chromatograms which were overlayed with either iodinated CT or hCG. The toxin bound to a ganglioside corresponding to GM1 whereas the hormone did not bind to any of the gangliosides. When the cells were incubated overnight with hCG, they lost their hCG receptors but exhibited an increase in CT binding and gangliosides. Our results indicate that GM1 is the specific receptor for CT whereas gangliosides are not involved in the binding and action of hCG.

Animals↗

PubMed1984

Effect of drugs and temperature on biosynthesis and transport of glycosphingolipids in cultured neurotumor cells.

Neuroblastoma and glioma cells were grown in the presence of [3H]galactose, and the incorporation of 3H into gangliosides and the transport of newly synthesized gangliosides to the cell surface were examined under different experimental conditions. A variety of drugs, including inhibitors of protein synthesis and energy metabolism, modulators of the cytoskeleton and the ionophore monensin, had no effect on the transport of newly synthesized GD1a in neuroblastoma cells. Only low temperature effectively blocked translocation to the plasma membrane. Monensin, however, had marked effects on the biosynthesis of gangliosides and neutral glycosphingolipids. Whereas incorporation of 3H into complex glycosphingolipids was reduced, labeling of glucosylceramide was increased in cells exposed to monensin. In addition, biosynthesis of the latter glycolipid was less susceptible to low temperatures than that of more complex ones. Previous studies have implicated the Golgi apparatus as the predominant site of glycosylation of gangliosides. As monensin has been reported to interfere with the Golgi apparatus, our results indicate that glucosylceramide may be synthesized at a site that is separate from the site where further glycosylation occurs. Once synthesis of a ganglioside is completed, transport of the molecule to the cell surface proceeds under conditions of cytoskeletal disruption, energy depletion and ionic inbalance , but not low temperature.

Animals↗

PubMed1984

Induction of catecholamine-responsive adenylate cyclase in HeLa cells by sodium butyrate. Evidence for a more efficient stimulatory regulatory component.

HeLa cells, when exposed to 5 mM sodium butyrate, increased their responsiveness to isoproterenol and their number of beta-receptors. As untreated HeLa cells have a substantial number of receptors but respond poorly to isoproterenol, the effect of butyrate could be due to quantitative or qualitative changes in beta-receptors or other components of the adenylate cyclase system. Receptors were analyzed by membrane/membrane and membrane/cell fusion techniques. HeLa donor membranes, treated to inactivate regulatory and catalytic components of adenylate cyclase, were fused with Fc cells, which lack beta-receptors. Isoproterenol-stimulated adenylate cyclase activity in the fusates was proportional to the number of receptors present. There appeared to be only quantitative but not qualitative differences in beta-receptors from control and butyrate-treated HeLa. Prostaglandin E1 receptors from neuroblastoma cell membranes were similarly coupled to HeLa adenylate cyclase. The hybrid prostaglandin E1-stimulated activity was lower when acceptor membranes were from control HeLa than when they were from butyrate-treated HeLa cells. These results suggested that butyrate was altering the ability of the regulatory component to interact with receptors. HeLa membranes were extracted with sodium cholate and the extracts used to reconstitute effector-stimulated adenylate cyclase activity in S49 cyc- membranes, which lack a functional regulatory component. Whereas extracts from control and butyrate-treated HeLa were equally effective in restoring NaF-stimulated activity in cyc- membranes, extracts from control HeLa were less efficient in reconstituting isoproterenol- and prostaglandin E1-stimulated activities. We conclude that the poor response of control HeLa to beta-agonists is due to a limited activity of the regulatory component but not the receptor. Butyrate induces quantitative changes in the receptor and qualitative changes in the regulatory component that facilitate its ability to couple to receptors but do not alter its ability to interact with the catalytic component of adenylate cyclase.

Adenosine Diphosphate Ribose↗

PubMed1984

Down-regulation of gonadotropin receptors in a murine Leydig tumor cell line.

The murine Leydig tumor cell line, MLTC-1, has specific cell surface receptors for human chorionic gonadotropin (hCG), which are coupled to adenylate cyclase. When the cells were exposed to hCG, there was a loss of these receptors (down-regulation), which was both dose- and time-dependent. Down-regulation was inhibited by lowering the temperature, removing bound hormone or blocking protein synthesis with cycloheximide. Down-regulation was found to be a biphasic event. The initial phase was dependent upon the binding of hormone and had a half-time of approximately 3 h. The second phase occurred around 8 h after exposing the cells to the hormone and was apparently independent of bound hCG. It was related to increases in cyclic AMP since it could be mimicked by incubating the cells with choleragen or dibutyryl cyclic AMP and isobutylmethylxanthine. The reappearance of hormone receptors began approximately 24-32 h after the initial exposure to hCG and was complete within 48 h. Adenylate cyclase activity in membranes from control and down-regulated cells responded equally well to Mn2+ and NaF indicating that neither the catalytic or regulatory component (G/F) of the adenylate cyclase system had been lost during downregulation. Cholate extracts of control and down-regulated cells also were equally effective at reconstituting isoproterenol-stimulated adenylate cyclase activity in S49 cyc-membranes (which lack a functional G/F). Thus, down-regulation did not impair the ability of G/F to couple receptors to the catalytic component of adenylate cyclase.

1-Methyl-3-isobutylxanthine↗

PubMed1984

Functional alteration of the beta-adrenergic receptor during desensitization of mammalian adenylate cyclase by beta-agonists.

Exposure of several mammalian cell lines to isoproterenol resulted in a desensitization of the beta-adrenergic receptor-adenylate cyclase system in membranes isolated from the cells. Under the experimental conditions chosen, desensitization was accompanied by a minimal loss of beta-receptors. The cells tested included HeLa, S49 cyc- lymphoma, and rat glioma C6. The functional activity of the beta-receptors was determined by coupling them to a foreign adenylate cyclase by membrane fusion. The donor membranes were treated to inactivate the regulatory and catalytic components of adenylate cyclase. The acceptor membranes were from Friend erythroleukemic cells (Fr cells), which lack beta-receptors, and HeLa cells treated overnight with isoproterenol to eliminate their receptors. The fused membranes were assayed for agonist-stimulated activity, which was always reduced when the donor beta-receptors were from the desensitized membranes. The desensitization appeared to be specific for beta-receptors, as the activity of other receptors and cyclase components was not altered. By fusing HeLa membranes with intact Fr cells, we directly measure the intrinsic activity of native and desensitized beta-receptors. For an equal amount of transferred beta-receptors, the activity was 40%-50% lower when the donor membranes were from desensitized cells. Our results clearly indicate that desensitization mediated by a beta-agonist in mammalian cells results in a functional alteration of the beta-receptor.

Adenylyl Cyclases↗

PubMed1984

Incorporation of fluorescent gangliosides into human fibroblasts: mobility, fate, and interaction with fibronectin.

Rhodamine- and fluorescein-labeled gangliosides were used as probes to investigate the distribution, dynamics, and fate of plasma membrane-bound gangliosides on cultured human fibroblasts. When sparse cultures of fibroblasts were incubated with the fluorescent ganglioside derivatives, their surfaces became highly fluorescent. The fluorescent gangliosides were taken up by the cells in a time- and temperature-dependent manner and were not removed from the cell surface by trypsin or serum. Thus, the gangliosides appeared to be stably incorporated into the lipid bilayer of the plasma membrane. Fluorescent photobleaching recovery measurements showed that the inserted gangliosides were free to diffuse in the plane of the membrane with a high diffusion coefficient of approximately 10(-8) cm2/s. When the ganglioside-treated cells were washed and incubated in fresh medium, the surface gangliosides became internalized with time, and localized in the perinuclear region of the fibroblasts. In dense cultures of fibroblasts, a large fraction of the fluorescent gangliosides were organized in a fibrillar network and were immobile on the time scale of fluorescent photobleaching recovery measurements. Using antifibronectin antibodies and indirect immunofluorescence, these gangliosides were found to co-distribute with fibrillar fibronectin. Thus, exogenous gangliosides appear to be stably inserted into the lipid bilayer of the plasma membrane and to diffuse freely in its plane as well as form a less mobile state with the fibrillar networks of fibronectin associated with the cells.

Antibodies↗