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  • 1
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science Ltd
    Journal of neurochemistry 67 (1996), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Previous studies from this laboratory have shown that synthesis of GT3, the precursor of c series gangliosides, occurs in proximal Golgi compartments, as has been shown for the synthesis of GM3 and GD3, the precursors of a and b series gangliosides, respectively. In this work we studied whether the synthesis of GM3, GD3, and GT3 occurs in the same or in different compartments of the proximal Golgi. For this, we examined in retina cells (a) the effect of monensin, a sodium ionophore that affects mostly the trans Golgi and the trans Golgi network function, on the metabolic labeling of glycolipids from [3H]Gal by cultured cells from 7- and 10-day chick embryos and (b) the labeling in vitro of endogenous glycolipids of Golgi membrane preparations from 7-day embryos incubated with UDP-[3H]Gal. In (a), 1 µM monensin produced a twofold accumulation of radioactive glucosylceramide and a decrease to ∼50 and 20% of total ganglioside labeling in 7- and 10-day cells, respectively. At both ages, monensin produced a threefold accumulation of radioactive GM3 and an inhibition of 〉90% of GT3, GM1, GD1a, and GT1b synthesis. GD3 synthesis was inhibited ∼30 and 70%, respectively, in 7- and 10-day cells. In (b), 〉80% of the [3H]Gal was incorporated into endogenous glucosylceramide to form radioactive lactosylceramide. About 90% of [3H]Gal-labeled lactosylceramide was converted into GM3, and most of this in turn into GD3 when unlabeled CMP-NeuAc was also present in the incubation system. Under the same conditions, however, 〈5% of labeled GD3 was converted into GT3. Golgi membranes incubated with CMP-[3H]NeuAc incorporated ∼20% of [3H]NeuAc into endogenous GT3, and this percentage was not affected by 1 µM monensin. These results indicate that synthesis of GT3 is carried out in a compartment of the proximal Golgi different from those for lactosylceramide, GM3, and GD3 synthesis. Results from the experiments with monensin point to the cis/medial Golgi as the main compartment for coupled synthesis of lactosylceramide, GM3, and GD3 and to the trans Golgi as the main compartment for synthesis of GT3.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 49 (1987), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Cells from neural retina from 7-day chick embryos were cultured on polylysine-coated dishes up to 7 days. The small, round-shaped cells at seeding differentiated progressively, and after 4 days in vitro the majority had enlarged bodies and abundant processes. The content of protein and DNA was essentially unchanged during the entire period of culture. The incorporation of radioactivity from [3H]glucosamine into gangliosides declined slightly, reaching about 65% of the initial values at the end of the culture period. The proliferating activity measured by the incorporation of [3H]thymidine into DNA decreased to 10% or less of the initial value after 3 days in vitro. Almost at the same chronological times as in ovo, the synthesis of GD3 and of a ganglioside partially identified as GT3 decreased from 70 and 19% of the total incorporation into gangliosides in the first 20 h of culture to about 7 and 5%, respectively, after 3 days in vitro. Conversely, the synthesis of GDI a increased from about 6% at the beginning to about 70% at the end of the culture times. Immunocytochemical analyses of the expression of gangliotetraosyl gangliosides in cultured cells showed that these gangliosides appeared in the bodies and processes of cells having neuronal morphology; very little immunostaining of the scarce flattened cells, probably Müller cells, was to and. The results indicate that the changes in ganglioside metabolism, which lead to decreased synthesis of gangliosides lacking the galactosyl-N-acetyl-galactosaminyl disaccharide end and to increased synthesis of gangliotetraosyl gangliosides, occur in cells that in culture differentiate into neurons.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 52 (1989), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: The expression of gangliosides of the lactosylcer-amide (LC) and of the gangliotetraosylceramide (GTC) series on the surface of cells from the chick neural retina was investigated by double-color indirect immunofluorescence. GD3 was assumed to be representative of LC and was detected using a specific monoclonal antibody. GM1 was assumed to be representative of GTC and was detected using the binding of cholera toxin followed by the binding of cholera toxin antibodies. The expression of polysialosylated GTC (polysialosyl-GTC) was detected using the cholera toxin-cholera toxin antibody experimental approach, after conversion of polysialosyl-GTC to GM1 by treatment of the cells with neuraminidase. In retinas from 6-day-old embryos (R6), most cells (∼80%) expressed GD3 but not GTC. After culturing for 7 days, (R6+7), the expression of GTC was found confined to neuron-like cells; flat cells derived from Müller cells expressed GD3 but were negative for GTC expression. On the other hand, postmitotic Müller cells obtained from 13-day-old embryo (R13) or 1-day-old hatched chick retina (Rpl) expressed GD3, GM1, and polysialosyl-GTC but were unable to maintain the expression of these GTCs when kept in culture for several days. According to these results, retinal cells can be defined on the basis of their ganglioside expression as follows: (a) retinoblasts, by the expression of GD3; (b) postmitotic neuronal cells, by the expression of GTC; and (c) postmitotic Müller cells, by the expression of GD3 and GTC. In addition, it was concluded that in contrast to retinal neurons, which express GTC in vitro, flat, Müller-derived cells either lack (R6 cell cultures) or are unable to maintain (R13 cell cultures) the expression of GTC, observations suggesting a dependence of these cells on environmental conditions for expression of these gangliosides.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science Ltd
    Journal of neurochemistry 65 (1995), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Ganglioside GT3 is the precursor of c-series gangliosides. It is synthesized by sialylation of GD3 and is expressed in nervous tissue of birds and mammals at early stages of development. In this study we examined the sub-Golgi location of GT3 synthesis and the mechanism of its transport from the site of synthesis to the plasma membrane in chicken embryo retina cells in culture. Neural retina cells from 10-day-old chick embryo were cultured with [3H]galactose in the absence (control cells) or in the presence of 1 µg/ml brefeldin A (BFA). At the end of the labeling period, the fraction of labeled gangliosides transported to the plasma membrane was determined. For this, cells were treated with C. perfringens neuraminidase in conditions to desialylate only those gangliosides that were transported to the plasma membrane and consequently accessible to the enzyme. After neuraminidase treatment of cells, gangliosides were isolated, purified, and the pattern of radioactivity analyzed by HPTLC-fluorography. It was found that BFA blocked the synthesis of complex gangliosides without affecting the synthesis of GM3, GD3, and GT3. Furthermore, in BFA-treated cells, GM3, GD3, and GT3 were protected from the action of added neuraminidase, indicating an intracellular localization and, hence, an inhibition of their transport to the plasma membrane. The results indicate that synthesis of the first intermediates of a-, b-, and c- series gangliosides occurs in a proximal Golgi compartment and that the proximal Golgi-synthesized gangliosides (GM3, GD3, and GT3) use a transport mechanism that is dependent on ADP ribosylation factor and coatomer proteins.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Oxford UK : Blackwell Science Ltd.
    Journal of neurochemistry 74 (2000), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: GD3 synthase (Sial-T2) is a key enzyme of gangliosidesynthesis that, in concert with GM2 synthase (GAlNAc-T), regulates the ratioof a- and b-pathway gangliosides. In this work, we study the sub-Golgilocation of an epitope-tagged version of chicken Sial-T2 transfected to CHO-K1cells. The expressed protein was enzymatically active both in vitro and invivo and showed a molecular mass of ∼47 or ∼95 kDa on sodium dodecylsulfate-polyacrylamide gel electrophoresis in the presence or absence of,respectively, β-mercaptoethanol. The 95-kDa form of Sial-T2 was alsodetected if the protein was retained in the endoplasmic reticulum (ER) due toimpaired glycosylation, indicating that it was formed in the ER. Confocalimmunofluorescence microscopy showed Sial-T2 localized to the Golgi complexand, within the organelle, partially co-localizing with themannose-6-phosphate receptor, a marker of the trans-Golgi network(TGN). In cells treated with brefeldin A, a major fraction of Sial-T2redistributed to the ER, even under controlled expression to control formislocalization due to protein overloading. In experiments of incorporation ofsugars into endogenous acceptors of Golgi membranes in vitro, GD3 moleculesformed by incubation with CMP-NeuAc were converted to GD2 upon incubation withUDP-GalNAc. These results indicate that Sial-T2 localizes mainly to theproximal Golgi, although a fraction is located in the TGN functionally coupledto GalNAc-T. Consistent with this, most of the enzyme was in anendoglycosidase H (Endo-H)-sensitive, neuraminidase (NANase)-insensitive form.A minor secreted form lacking ∼40 amino acids was Endo-H-resistant andNANase-sensitive, indicating that the cells were able to processN-glycans to Endo-H-resistant forms. Taken together, the results of these biochemical and immunocytochemical experiments indicate that in CHO-K1 cells, most Sial-T2 localizes in the proximal Golgi and that a functional fraction is also present in the TGN.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1573-6903
    Keywords: Gangliosides ; GM1 synthase ; N-glycosylation ; glycolipid expression ; Golgi complex ; Tunicamycin ; N-glycan trimming
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Glycosyltransferase cDNAs contain a variable number of potential N-glycosylation sites. Here we examined the occupancy and relevance for the activity and intracellular trafficking of the only potential N-glycosylation site of the mouse β1,3galactosyltransferase (Gal-T2 or GA1/GM1/GD1b synthase) in Gal-T2 cDNA transfected CHO-K1 cells. Transfected cells synthesize a Golgi located active enzyme of 43 kDa whose N-glycan was metabolically labeled from [3H]mannose and was Endo-H sensitive. Inhibition of N-glycosylation by Tunicamycin or by point mutation of the N-glycosylation site resulted in the synthesis of a polypeptide of 40 kDa which lacked enzyme activity and was concentrated in the endoplasmic reticulum (ER). Inhibition of ER glucosidases by Castanospermine impaired the exit of a form of Gal-T2 having reduced enzyme activity from the ER. The N-terminal Gal-T2 domain (aa 1–52) was able to direct and to retain the green fluorescence protein in the Golgi complex. Taken together, these results indicate that Gal-T2 depends on N-glycosylation for its activity and for proper trafficking to, but not its retention in, the Golgi complex.
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    Neurochemical research 22 (1997), S. 455-461 
    ISSN: 1573-6903
    Keywords: Ganglioside-glycosyltransferases ; Golgi compartments ; retina cells ; ganglioside endogenous acceptors ; ganglioside metabolic relationships
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The relationship among lactosylceramide-(LacCer), GD3- and GM2-synthases and between the two last transferases and their common GM3 acceptor was investigated in intact Golgi membrane from chick embryo neural retina cells at early (8-days) and late (14 days) stages of the embryonic development. [3H]Gal was incorporated into endogenous glucosylceramide by incubation of Golgi membranes with UDP-[3H]Gal. Conversion of the synthesized [3H]Gal-LacCer into GM3, and of the latter into GD3, GM2 and GD2 was examined after a second incubation step with unlabeled CMP-NeuAc and/or UDP-GalNAc. With CMP-NeuAc, most [3H]Gal-LacCer was converted into GM3 in either 8- or 14- day membranes. However, while about 90% of GM3 was converted into GD3 in 8-day membranes, only about 25% followed this route in 14-day membranes. With CMP-NeuAc and UDP-GalNAc, about 90% of GM3 was used for synthesis of GM2 in 14-day membranes, while in 8-day membranes about 80% followed the route to GD3, and a part to GD2. Performing the second incubation step in the presence of increasing detergent concentrations showed that conversion of GM3 to GM2 was inhibited at concentrations lower than those required for inhibition of LacCer to GM3 conversion. Taken together, results indicate that transfer steps leading to synthesis of GM3, GD3, GM2 and GD2 from LacCer are functionally coupled in the Golgi membranes, and that GD3- and GM2-synthases compete in a common compartment for using a fraction of GM3 as substrate. In this competition, the relative activities of the transferases and their relative saturation with the respective donor sugar nucleotides, are important factors influencing conversion of GM3 toward either GD3 or GM2.
    Type of Medium: Electronic Resource
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Neurochemical research 25 (2000), S. 1-3 
    ISSN: 1573-6903
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 9
    ISSN: 1573-6903
    Keywords: Gangliosides ; ceramide ; glucosylceramide ; glycolipid synthesis topology ; glycosyltransferases ; Golgi complex compartments
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract It is known that ceramide (Cer), the precursor of sphingoglycolipids and of sphingomyelin, participates in events leading to activation of the apoptotic pathway, and per se or through conversion to glucosylceramide (GlcCer) modulates formation of neuritic processes in developing neurons. To learn about the fate of de novo synthesized Cer and GlcCer we examined, in Golgi membranes from chicken embryo neural retina cells, the metabolic relationships of endogenous Cer, GlcCer and lactosylceramide (LacCer). Incubation of the membranes with UDP-[3H]Glc revealed a pool of endogenous Cer useful for synthesis of GlcCer. Most of the GlcCer synthesized, however, was not used for synthesis of LacCer, indicating that it was functionally uncoupled from LacCer synthase. On the other hand, incubation with UDP-[3H]Gal revealed a pool of endogenous GlcCer that depending of the integrity of the membranes was functionally coupled to LacCer and ganglioside synthesis. These results indicate that most GlcCer formed in vitro from Cer is topologically segregated from the synthesis of LacCer. However, subfractionation in sucrose gradients of Golgi membranes labeled with both precursors failed to separate membranes enriched in [3H]GlcCer from those enriched in [3H]Gal-labeled LacCer. It is concluded that despite both transfer steps co-localize in the Golgi membranes, coupling of GlcCer synthesis to LacCer synthesis requires conditions not present in our in vitro assay. This suggests that a coupling activity exists that could be relevant for regulation of the cytoplasmic levels of Cer and GlcCer.
    Type of Medium: Electronic Resource
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  • 10
    ISSN: 1573-4919
    Keywords: neurospora crassa filaments ; intermediate filaments ; rat tongue epithelial polypeptide ; epithelial cell differentiation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract P59Nc is a polypeptide associated with bundles of cytoplasmic and nuclear filamentous structures of 8–10 nm of diameter in Neurospora crassa cells. It is immunologically unrelated to both higher and lower eucaryotic tubulin and actin proteins and is detected weakly by the anti IFA monoclonal antibody. We analyze here the immunological relationship between P59Nc and intermediate filament (IF) mammalian proteins by using anti P59Nc, anti keratin, anti vimentin and anti IFA antibodies. Anti P59Nc antibodies detected a 59 kDa polypeptide from rat and bovine tissues which copurifies with polypeptides of the IF family. Neither P59Nc nor the 59 kDa rat polypeptide were recognized by anti keratin or anti vimentin antibodies. Immunostaining of rat tongue sections with anti P59Nc antibodies showed that the 59 kDa rat polypeptide is present in the cortical cytoplasm of suprabasal epithelial cells. The results indicate that P59Nc shares common antigenic determinants with a still uncharacterized 59 kDa polypeptide from mammalian tissues which have extractability and immunological properties similar to those of IF polypeptides and shows a tissular distribution and a cellular localization similar but distinguishable from keratins.
    Type of Medium: Electronic Resource
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