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R K Yu

Publications and source records attributed to R K Yu.

At least 73 records · Page 4Linked to original sources
PubMed1996

Neuritogenesis, not receptor expression, of NG108-15 cells can be modulated by monosialoganglioside GM1.

In this study, involvement of gangliosides in neurite outgrowth and receptor expression of the neuroblastoma X glioma hybrid NG108-15 cloned cells was investigated. Monosialoganglioside GM1 (100 microM) and disialoganglioside GD1a (100 microM) were applied to the culture medium at different concentrations of fetal bovine serum, 1-10%, with or without addition of dibutyryl adenosine 3',5'-cyclic monophosphate (500 microM). In some experiments, 5 mg/ml of cholera toxin B was added to the media to block endogenous GM1. The results indicated that GM1 had an influence on cell proliferation and neuritogenesis but did not induce muscarinic receptor expression of NG108-15 cells.

Animals↗
PubMed1995

Analysis of the antibody response to immunization with purified O-acetyl GD3 gangliosides in patients with malignant melanoma.

Gangliosides expressed in malignant melanoma are potential targets for immunotherapy. Immunization of melanoma patients with vaccines containing purified GM2 ganglioside has resulted in induction of GM2 antibodies, and high titers of GM2 antibodies have correlated with increased survival. Melanoma ganglioside 9-O-acetyl GD3 is another candidate for ganglioside vaccine construction because of its limited expression in normal human tissues. As purification of 9-O-acetyl GD3 from human melanoma (9-O-acetylated on the terminal sialic acid) is not practical for broad application, we investigated the antibody response of melanoma patients to O-acetyl GD3 from several additional sources: hamster melanoma (7-O-acetyl GD3), bovine buttermilk (mixture of 7-O-acetyl GD3, 9-O-acetyl GD3 and 7,9-di-O-acetyl GD3) and chemically modified GD3 from bovine brain (9-O-acetylated on the subterminal sialic acid). Only immunization with the buttermilk-derived O-acetyl GD3 preparation resulted in consistent production of IgM antibodies. However, the induced antibodies reacted with the immunogen and with 7-O-acetyl GD3 derived from hamster melanoma but not with 9-O-acetyl GD3 or human melanoma cells expressing 9-O-acetyl GD3 on their cell surface. In contrast, all O-acetyl GD3 derivatives used for immunization were recognized by murine MAbs that reacted with 9-O-acetyl GD3, and immunization of mice with buttermilk-derived O-acetyl GD3 resulted in the production of antibodies that reacted with human melanoma cells expressing 9-O-acetyl GD3. Apparently, the human and murine immune systems preferentially recognize different epitopes on these molecules.

Adjuvants, Immunologic↗
PubMed1995

Glycosphingolipid composition of murine neuroblastoma cells: O-acetylesterase gene downregulates the expression of O-acetylated GD3.

We have studied the glycosphingolipid composition in an F-11 neuroblastoma cell line originated from hybridization of a mouse neuroblastoma cell line (N18TG-2) with rat dorsal root ganglion cells. The total lipid-bound glucose of F-11 cells was estimated to be 0.28 micrograms/mg of protein and the total lipid-bound sialic acid was 0.82 micrograms/mg of protein. The major neutral glycosphingolipids were Gb4 (37% of the total neutral glycosphingolipids), Gb3 (15%), LacCer (21%), and GlcCer (15%). The major gangliosides were found to be GM3 (37% of the total gangliosides), GD3 (27%), O-acetylated GD3 (18%), and GD1a (4%), with trace amounts of GD2. The unusually high concentration of O-acetylated GD3 is consistent with its putative role as a tumor marker. Immunocytochemical localization studies of GD3 and O-acetylated GD3, examined by mouse monoclonal antibodies R24 and D1.1, respectively, revealed that the cell bodies and processes were all positively stained. To elucidate the role of O-acetylated GD3 in tumorigenesis, we transfected F-11 cells with the O-acetylesterase gene from influenza C virus. Compared with the original cell line, the transfected cells showed a dramatic increase in the level of GD3 (150% of that in the control cells) and a significant decrease of the concentration of O-acetylated GD3 (27% of control cells). In addition, the transfected F-11 cells exhibited a morphology different from the parental cells with enlarged cell bodies and elongated neurites. We conclude that alteration of ganglioside composition, particularly the expression of GD3 and O-acetylated GD3, may be associated with the morphological changes observed in this cell line.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗
PubMed1995

Regulation of glycolipid synthesis in HL-60 cells by antisense oligodeoxynucleotides to glycosyltransferase sequences: effect on cellular differentiation.

Treatment of the human promyelocytic leukemia cell line HL-60 with antisense oligodeoxynucleotides to UDP-N-acetylgalactosamine:beta-1,4-N-acetylgalactosaminyl-transferase (GM2-synthase; EC 2.4.1.92) and CMP-sialic acid:alpha-2,8-sialyltransferase (GD3-synthase; EC 2.4.99.8) sequences effectively down-regulated the synthesis of more complex gangliosides in the ganglioside synthetic pathways after GM3, resulting in a remarkable increase in endogenous GM3 with concomitant decreases in more complex gangliosides. The treated cells underwent monocytic differentiation as judged by morphological changes, adherent ability, and nitroblue tetrazolium staining. These data provide evidence that the increased endogenous ganglioside GM3 may play an important role in regulating cellular differentiation and that the antisense DNA technique proves to be a powerful tool in manipulating glycolipid synthesis in the cell.

Base Sequence↗
PubMed1995

Interleukin 1 beta up-regulates the expression of sulfoglucuronosyl paragloboside, a ligand for L-selectin, in brain microvascular endothelial cells.

Treatment of cultured bovine brain microvascular endothelial cells (BMECs) with interleukin 1 beta (IL-1 beta), an inflammatory cytokine, was shown to induce the accumulation of sulfoglucuronosyl paragloboside (SGPG), a glycolipid bearing the HNK-1 epitope. This resulted in the attachment of a greater number of human lymphocytes to the treated than to the untreated BMEC monolayers. Attachment of human lymphocytes to the IL-1 beta-activated BMEC cells could be blocked either by incubation of the human lymphocytes with an anti-L-selectin antibody or by application of an anti-SGPG antibody to the BMECs. These results suggest that SGPG may act as an important ligand for L-selectin for the regulation of the attachment of activated lymphocytes and their subsequent invasion into the nervous system parenchyma in inflammatory disorders of the central and peripheral nervous systems.

Animals↗
PubMed1995

Characterization of sialidase activity in mouse synaptic plasma membranes and its age-related changes.

Sialidase activity in synaptic plasma membranes (SPM) isolated from C57BL/6 mouse brain was examined using exogenous ganglioside substrates. The enzyme activity directed toward GM3 showed sharp pH dependency with optimal pH of 4.0, and was greatly enhanced by Triton CF-54, Nonidet P-40 or CHAPS. The apparent Km and Vmax values for enzyme activity in SPM were 11 microM and 164 pmol/mg protein/min, respectively. Examination of sialidase activities in subcellular fractions of brain tissues showed the enrichment of enzyme activity in SPM prepared from either young adult or senescent mice. Substrate specificity of SPM sialidase was compared with that of myelin sialidase using delipidated, solubilized enzyme preparations. The SPM sialidase hydrolyzed GD1a more effectively as compared with the myelin enzyme. While SPM sialidase could hydrolyze GM1, the hydrolytic rate by the SPM enzyme was significantly lower than that by the myelin enzyme. The sialidase activity in SPM decreased with increasing age; activity was highest between the ages of 4-7 months, decreased to a relatively constant level between 13-25 months, and reached its lowest level at 31 months. These results demonstrate that SPM contain a distinct sialidase activity which is regulated in an age-dependent manner.

Age Factors↗
PubMed1995

Accumulation of isogloboside and ganglio-N-tetraosyl ceramide having blood group B determinant in the hepatomas of female LEC rats.

We have studied the neutral glycolipid composition of spontaneous hepatomas in LEC female rats. Neutral lipid fractions were isolated and purified by column chromatographies on DEAE-Toyopearl 650(M) and Iatrobeads. The neutral glycolipid fraction contained 3.2 to 4.4 micrograms lipid-bound glucose (Glc) per mg protein, and consisted of isogloboside (iso-Gb4, 50.8% of total neutral glycolipids) and IV3Gal, IV2Fuc, GgOse4Cer (asialo-BGM1, 13.5%) as the major neutral glycolipids and Gb3 and iso-Gb3 (9.2%), GlcCer (7.2%), LacCer (6.1%) as the other species. The structure of iso-Gb4 was elucidated by gas-liquid chromatography (GLC), permethylation study, liquid secondary ion (LSI) mass spectrometry, and nuclear Overhauser enhancement spectroscopy (NOESY) and that for asialo-BGM1 by GLC, LSI mass spectrometry, and high-performance thin-layer chromatography (HPTLC)-overlay method using anti-asialo-BGM1 antibody. Isogloboside and asialo-BGM1 which are found in negligible amounts in normal liver tissues may represent excellent markers for studying tumor metastasis and cellular adhesion.

ABO Blood-Group System↗
PubMed1995

Anti-sulfoglucuronyl paragloboside IgM antibodies in amyotrophic lateral sclerosis.

We report here our results on IgM anti-sulfated glucuronyl paragloboside (SGPG) antibodies in sera from patients with amyotrophic lateral sclerosis (ALS). Studies by enzyme linked immunosorbent assay on 72 ALS sera showed IgM polyclonal reactivity towards SGPG in 25 cases. The titer was high in 16 cases. Thin-layer chromatography immuno-overlay showed that reactivity with SGPG was associated to reactivity towards GM1 in five cases and to GM1 and GD1b in one case. Anti-SGPG reactivity was not found in controls and in multifocal motor neuropathy with conduction blocks, in contrast to anti-GM1 antibodies. The presence of anti-SGPG antibodies in ALS patients sera raise again the question of autoimmunity in this pathology.

Amyotrophic Lateral Sclerosis↗
PubMed1995

Characterization of sialyltransferase-IV activity and its involvement in the c-pathway of brain ganglioside metabolism.

To characterize the sialyltransferase-IV activity in brain tissues, the activities of GM1b-, GD1a-, GT1b-, and GQ1c-synthases in adult cichlid fish and rat brains were examined using GA1, GM1, GD1b, or a cod brain ganglioside mixture as the substrate. The GD1a-synthase activity in the total membrane fraction from cichlid fish brain required divalent cations such as Mg2+ or Mn2+ and Triton CF-54 for its full activity. The Vmax value was 1,340 pmol/mg of protein/h at an optimal pH of 6.5, whereas the apparent Km values for CMP-sialic acid and GM1 were 172 and 78 microM, respectively. Cichlid fish and rat brains also contained GM1b-, GT1b-, and GQ1c-synthase activities. The ratio of GM1b-, GD1a-, and GT1b-synthase activities in fish brain was 1.00:0.89:1.13, respectively, and in rat brain 1.00:0.60:0.63. Incubation of fish brain membranes with a cod brain ganglioside mixture, which contains GT1c, and [3H]CMP-sialic acid produced radiolabeled GQ1c. It is interesting that the adult rat brain also contains an appreciable level of GQ1c-synthase activity despite its very low concentrations of c-series gangliosides. The GD1a- or GQ1c-synthase activity in fish and rat brain was inhibited specifically by coincubation with the glycolipids that serve as the substrates for other sialyltransferase-IV reactions. Thus, the GD1a-synthase activity was inhibited by GA1 and GD1b, but not by LacCer, GM3, or GD3. In a similar manner, the synthesis of GQ1c was suppressed by GA1, GM1, and GD1b, but not by LacCer, GM3, or GD3.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Effect of nerve growth factor and forskolin on glycosyltransferase activities and expression of a globo-series glycosphingolipid in PC12D pheochromocytoma cells.

The glycosphingolipid (GSL) composition of cells changes dramatically during cellular differentiation. Nerve growth factor (NGF) or forskolin (FRK) are known to induce cellular differentiation including process formation in PC12 pheochromocytoma cells. In this respect, we present the NGF/FRK-dependent regulation of glycosyltransferase activities and the corresponding GSL expression in PC12D cells. After treatment of PC12D cells with NGF or FRK, the cell processes, including varicoses and growth cones, became strongly immunoreactive with an antibody against a unique globo-series neutral GSL, Gal alpha 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1'Cer (GalGb3), and the activity of GalGb3-synthase increased significantly. Other glycosyltransferase activities, including GM1 containing blood group B determinant (BGM1)-, GM3-, GD1a-, and GM2-synthases, also increased significantly upon NGF treatment, but the immunoreactivity against BGM1 did not show any appreciable change. For the parent PC12 cells, NGF/FRK treatment significantly increased the percentage of anti-GalGb3 positive cells and induced some immunoreactive cell processes. Because the parent PC12 cells do not express appreciable amounts of GalGb3, and because PC12D cells are considered to be more differentiated than the parent PC12 cells, the expression of GalGb3 and the increase of GalGb3-synthase activity may be closely related to the cellular differentiation process in this cell line.

Animals↗
PubMed1995

Regulation of sialyltransferase activities by phosphorylation and dephosphorylation.

The composition of tissue gangliosides is thought to result mainly from the active regulation and selective expression of specific enzymes responsible for their metabolism. In the last few years, we have purified several rat brain sialyltransferases to homogeneity; the availability of these highly purified enzymes enabled us to investigate their regulation and expression at the molecular level. Thus, we studied the regulation of sialyltransferase activities, in particular, CMP-NeuAc:GM1 and CMP-NeuAc:LacCer sialyltransferases by a phosphorylation/dephosphorylation mechanism. Protein kinase C was added to a standard enzyme assay mixture containing [gamma-32P]ATP, and the activity of the enzyme was measured after various incubation times. We found that treatment of several sialyltransferases by protein kinase C decreased their activities in a time-dependent manner. Analyses of 32P-labeled amino acids revealed that the major phosphorylation site of CMP-NeuAc:GM1 alpha 2-->3 sialyltransferase (ST-IV) was serine and that for CMP-NeuAc:LacCer alpha 2-->3 sialyltransferase (ST-I) was primarily threonine. Partial recovery of the enzyme activity could be achieved by treatment of the phosphorylated sialyltransferases with rat brain protein phosphatase. We conclude that the activities of sialyltransferases can be modulated by protein kinase C and protein phosphatase and this may represent a potential regulatory mechanism for ganglioside biosynthesis.

Adenosine Triphosphate↗
PubMed1994

Modulation by gangliosides of the lamellar-inverted micelle (hexagonal II) phase transition in mixtures containing phosphatidylethanolamine and dioleoylglycerol.

We studied the effect of gangliosides GD1a and GM1 on the lamellar-to-hexagonal II phase transition of mixtures of dioleoylphosphatidylethanolamine/dioleoylphosphatidyl choline, 3:1, and of transphosphatidylated phosphatidylethanolamine with dioleoylglycerol by high-sensitivity differential scanning calorimetry, 31P-NMR, and pyrene fluorescence of a phosphatidylcholine probe. Gangliosides had a dual effect. Below 1 mol % ganglioside the hexagonal II phase transition was affected but still occurred at lower temperature than in the absence of gangliosides. The presence of between 1 and 2 mol % gangliosides increased the temperature for formation of the hexagonal II phase and progressively decreased its cooperativity. Above 3 mol % gangliosides totally inhibited the formation of both the temperature-induced and composition-induced hexagonal phase, probably by opposing the geometric distortions necessary for the inverted micellar structures.

Diglycerides↗
PubMed1994

Oligodendroglial cyclic AMP response element-binding protein: a member of the CREB family of transcription factors.

Several laboratories have shown that cyclic AMP (cAMP) plays an important role in inducing oligodendrocyte differentiation and myelin synthesis. Our previous results have shown that oligodendrocytes contain a nuclear protein that binds to the DNA sequence TGACGTCA or cAMP response element (CRE) known to be involved in the transcriptional regulation of cAMP-responsive genes. In this report the oligodendroglial CRE-binding protein was further identified by using two different antibodies which specifically recognize the CRE-binding protein known as CREB. In DNA-shift assays CREB-1(X-12) antibody interacted with the CRE-protein complexes resulting in further retardation ("super shift") of the mobility of the bands in the gels. Immunoprecipitation of oligodendroglial nuclear extracts with CREB(240) antibody prior to the DNA binding assays resulted in a lack of formation of CRE-protein complexes. In addition immunoreaction with CREB(240) antibody identified the CRE-binding species as a 45 kDa phosphoprotein. Immunocytochemical staining with CREB(240) antibody in oligodendrocytes from 10-, 14-, and 18-day-old and adult rats indicated that this protein is expressed before the appearance of myelin basic protein (MBP) which was used as a marker of myelin synthesis. Collectively, these observations support our previous results and indicate that the oligodendroglial CRE-binding protein species is highly homologous to the CREB protein. The developmental expression of this CREB protein supports the idea of a possible role during the early stages of oligodendrocyte differentiation preceding the peak of myelin synthesis in rat CNS.

Animals↗
PubMed1994

Modulation of the activity of Clostridium perfringens neuraminidase by the molecular organization of gangliosides in monolayers.

The activity of Clostridium perfringens neuraminidase against gangliosides GM3, GD1a and GM1 was studied in lipid monolayers at the air-buffer solution interface. The enzyme activity assay against pure ganglioside monolayers is based on the markedly different molecular packing areas of the substrate gangliosides and the resulting product glycosphingolipids. This allows to control and monitor the surface pressure and the ganglioside intermolecular organization (cross-sectional packing areas and dipole potentials) in a continuous manner during the catalytic process. It was found that the rate and the extent of the enzymatic reaction depended markedly on the lateral surface pressure. In general, the activity of neuraminidase against GM3 and GD1a was higher at lower surface pressure. This corresponded to larger intermolecular spacings among the ganglioside molecules. Both the activity and the extent of the reaction against GM3 were higher than toward GD1a. GM1 could not be degraded by the enzyme, irrespective of the surface pressure but the enzyme could interact with this ganglioside. A latency period, longer for GM3 than for GD1a, was observed prior to the onset of rapid degradation; this indicates that pre-catalytic steps are occurring at the interface before effective ganglioside degradation takes place. The latency period, the total amount of ganglioside degraded, and the velocity of the reaction varied with the surface pressure in different manners. Our data indicate that the different steps of the catalytic reaction occurring at the surface (i.e., substrate recognition and interfacial adsorption, catalysis, maximum extent of substrate conversion) are independently regulated by the molecular organization of the substrate gangliosides.

Clostridium perfringens↗
PubMed1994

Regulation by gangliosides and sulfatides of phospholipase A2 activity against dipalmitoyl- and dilauroylphosphatidylcholine in small unilamellar bilayer vesicles and mixed monolayers.

The modulation by gangliosides GM1 and GD1a, and sulfatide (Sulf) of the activity of porcine pancreatic phospholipase A2 was studied with small unilamellar vesicles of dipalmitoylphosphatidylcholine (L-dpPC) and lipid monolayers of dilauroylphosphatidylcholine (L-dlPC). The presence of Sulf always led to an increase of the maximum rate of the enzymatic reaction, irrespective on whether the vesicles were above, in the range of, or below the bilayer transition temperature. Sulf did not modify the latency period for the reaction that is observed at the bilayer transition temperature. Gangliosides inhibited the maximum rate of enzymatic activity bilayer vesicles in the gel phase but the effect was complex. When the reaction was carried out at a temperature within the range of the bilayer phase transition, the gangliosides inhibited the maximal rate of the reaction in proportion to their content in the bilayer. However, at the same time the latency period observed with vesicles of pure phospholipid at this temperature was shortened in proportion to the mole fraction of gangliosides in the bilayer. At temperatures above the bilayer phase transition, gangliosides stimulated the activity of PLA2. Preincubation of the enzyme with Sulf or gangliosides did not affect the activity against bilayer vesicles of pure substrate. These glycosphingolipids did not modify the rate or extent of desorption of the enzyme from the interface, nor the pre-catalytic steps for the interfacial activation of PLA2, or the enzyme affinity for the phospholipid substrate. Also, the activity of the enzyme was not altered irreversibly by glycosphingolipids. Our results indicate that Sulf and gangliosides modulate the catalytic activity of PLA2 at the interface itself, beyond the initial steps of enzyme adsorption and activation, probably through modifications of the intermolecular organization and surface electrostatics of the phospholipid substrate.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1994

Glycolipid composition of human cataractous lenses. Characterization of Lewisx glycolipids.

We have studied the glycolipid composition of human cataractous lenses. Neutral and acidic lipid fractions were isolated by column chromatographies on DEAE-Sephadex and Iatrobeads. The neutral glycolipid fraction and acidic glycolipid fraction contained 0.6-0.9 micrograms of lipid-bound glucose (Glc) per mg of protein and 0.8-1.3 micrograms of lipid-bound sialic acid (NeuAc) per mg of protein, respectively. The neutral glycolipid fraction was found to contain LacCer (39.0% of total neutral glycolipids), Gb3 (16.2%), Gb4 (1.1%), nLc4 (5.0%), X (29.0%), and Y (9.2%). The acidic lipid fraction was found to contain mainly GM3 (33.1% of the total ganglioside fraction), GM1 (8.3%), LM1 (7.3%), GD1a (16.0%), and G (30.1%). The structures of neutral glycolipids X and Y and ganglioside G were elucidated by high performance thin-layer chromatography overlay method of glycolipids, gas-liquid chromatography, proton NMR spectrometry, and liquid secondary ion mass spectrometry as follows: 1) X, Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1'Cer, III3FucnLc4 (Lex); 2) Y, Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4(Fuc alpha 1- 3)GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1'Cer, V3FucIII3FucnLc6; and 3) G, NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3 Gal-beta 1-4Glc beta 1-1'Cer, IV3NeuAcIII3FucnLc4 (sialosyl-Le(x)). A minor neutral glycolipid Z was isolated and tentatively characterized as GlcNAc beta 1-3?Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1'Cer (GlcNAc-Le(x)), suggesting that it may be the precursor of glycolipid Y. The major long-chain base of these human cataract glycolipids was C18:0 sphingosine (sphinganine). The major fatty acids were C16:0, C24:1 and C24:0, and monounsaturated fatty acids accounted for 40-55% of the total fatty acids.

Aged↗
PubMed1994

GD3 vaccines for melanoma: superior immunogenicity of keyhole limpet hemocyanin conjugate vaccines.

Cell surface gangliosides show altered patterns of expression as a consequence of malignant transformation and have therefore been of interest as potential targets for immunotherapy, including vaccine construction. One obstacle has been that some of the gangliosides that are overexpressed in human cancers are poorly immunogenic in humans. A case in point is GD3, a prominent ganglioside of human malignant melanoma. Using an approach that has been effective in the construction of bacterial carbohydrate vaccines, we have succeeded in increasing the immunogenicity of GD3 in the mouse by conjugating the ganglioside with immunogenic carriers. Several conjugation methods were used. The optimal procedure involved ozone cleavage of the double bond of GD3 in the ceramide backbone, introducing an aldehyde group, and coupling to aminolysyl groups of proteins by reductive amination. Conjugates were constructed with a synthetic multiple antigenic peptide expressing repeats of a malarial T-cell epitope, outer membrane proteins of Neisseria meningitidis, cationized bovine serum albumin, keyhole limpet hemocyanin, and polylysine. Mice immunized with these conjugates showed a stronger antibody response to GD3 than mice immunized with unconjugated GD3. The strongest response was observed in mice immunized with the keyhole limpet hemocyanin conjugate of the GD3 aldehyde derivative and the adjuvant QS-21. These mice showed not only a long-lasting high-titer IgM response but also a consistent high-titer IgG response (predominantly IgG1), indicating recruitment of T-cell help, although the titers of IgM and IgG antibodies following booster immunizations were not as high as they are in the response to classical T-cell-dependent antigens. This method is applicable to other gangliosides, and it may be useful in the construction of immunogenic ganglioside vaccines for the immunotherapy of human cancers expressing gangliosides on their cell surface.

Adjuvants, Immunologic↗