Biomedical subjects

R K Yu

Publications and source records attributed to R K Yu.

At least 127 records · Page 7Linked to original sources

PubMed1990

Direct assay of glycosphingolipid glycosyltransferase activities on thin-layer chromatogram.

A modified method for the determination of glycosphingolipid glycosyltransferase activity using high-performance thin-layer chromatographic (HPTLC) plates has been developed. An acceptor glycosphingolipid was chromatographed on an HPTLC plate and was incubated with an enzyme mixture and an appropriate radioactive sugar nucleotide. After incubation, the plate was washed with phosphate buffer and 2% Tween 80. The radiolabeled reaction product was scrapped off the plate and the radioactivity determined using a liquid scintillation counter or, alternatively, the plate was exposed to an X-ray film to reveal the radioactive product. We have used this assay method to determine the activities of rat brain cytidine 5'-monophosphate-N-acetylneuraminic acid: LacCer-, GM3-, GM1-, or GD3-sialyltransferases. This method is sensitive, fast, and reliable and is capable of assaying simultaneously the activities of glycosyltransferases with multiple acceptor specificity. It should be useful in monitoring the enzyme activities present in various column fractions during chromatographic fractionation of glycosyltransferases with different substrate specificities.

Animals↗

PubMed1990

Purification to homogeneity of GD3 synthase and partial purification of GM3 synthase from rat brain.

A CMP-sialic acid: GM3 sialyltransferase (GD3 synthase) and a CMP-sialic acid: LacCer sialyltransferase (GM3 synthase) have been purified 10,000- and 3,000-fold, respectively, from the Triton X-100 extract of rat brain. The two enzymes were purified and resolved by affinity chromatography on two successive CDP-Sepharose columns by NaCl gradient elution. Final purification of GD3 synthase was achieved by specific elution from a 'GM3 acid'-Sepharose column with buffer containing GM3. Sodium dodecylsulfate-gel electrophoresis of GD3 synthase revealed a single major protein band with an apparent molecular weight of 55,000.

Animals↗

PubMed1990

Autoimmune mechanisms in peripheral neuropathies.

In certain patients with demyelinating neuropathy and plasma cell dyscrasia, there are IgM monoclonal antibodies that recognize a carbohydrate epitope shared by myelin-associated glycoprotein (MAG) and at least two acidic glycolipids in the peripheral nervous system (PNS). The structures of the two acidic lipids have been elucidated as a new class of glycosphingolipids, termed sulfoglucuronyl glycolipids (SGGLs). SGGLs have been demonstrated to be present in myelin, axolemma, and other glia-related membranes in PNS of several animal species, as well as in human dorsal root ganglia and sympathetic ganglia. In rabbits sensitized with sulfoglucuronyl paragloboside (SGPG), a major SGGL in PNS, antibodies developed with reactivities toward SGPG and MAG. The animals also showed moderate weakness, a slowed nerve conduction velocity, and evidence of conduction block. Recently we also found SGPG in rat brain microvessels. This finding supports our hypothesis that autoantibodies may first interact with endothelial cell-bound antigens and that this might change the permeability of the blood-brain or blood-nerve barrier to permit the entry of these autoantibodies into the nervous system. Our data are consistent with the concept that an autoimmune response against the sulfoglucuronyl residue may participate in the pathogenesis of immune-mediated neuropathy.

Animals↗

PubMed1990

Cerebellar ganglioside abnormalities in pcd mutant mice.

The distribution of cerebellar gangliosides was studied in Purkinje cell degeneration (pcd/pcd) mutant mice at postnatal days 25, 30, 50, and 150. These mutants lose the majority of Purkinje cells between 18 and 50 days of age. A reactive gliosis accompanies Purkinje cell loss and a partial loss of granule cells occurs in pcd/pcd mice older than p50. Purkinje cell loss is associated with significant reductions in cerebellar weight and ganglioside concentration. This neuronal loss was also developmentally correlated with reductions of gangliosides (GT1a/LD1 and GT1b and with elevations of GD3. These results agree with previous findings in other cerebellar mutants that GT1a/LD1 and GT1b are concentrated in Purkinje cells and that GD3 is enriched in reactive glial cells. A slight, but significant, reduction in GD1a concentration occurred only in older pcd/pcd mice, consistent with previous findings in weaver and staggerer mice that GD1a is enriched in mature granule cells. The findings with pcd/pcd and other neurological mutants indicate that certain gangliosides can serve as cell-surface markers for monitoring changes in cerebellar cytoarchitecture that accompany development or disease.

Animals↗

PubMed1990

Sulfate incorporation into peripheral nerve endoneurial glycolipids after crush and permanent transection injury.

The sulfation of peripheral nerve glycolipids was examined at 35 days after both crush injury or permanent transection of the adult rat sciatic nerve by in vitro incorporation of [35S]sulfate into endoneurial slices. These experimental models of neuropathy are characterized by the presence and absence of both axonal regeneration and subsequent myelin assembly. Although the sulfo-glucuronosyl glycosphingolipids (SGGLs) were not detected by alpha-napthol reagent after HPTLC separation of the total acidic lipid extract, fluorographic analysis after sulfate incorporation revealed a 4.7-fold increase in [35S]sulfate in the sulfo-glucuronosyl paragloboside (SGPG) and a 3.5-fold increase in the sulfo-glucuronosyl-lactosaminosyl paragloboside (SGLPG) after the crush injury compared to permanent transection. These [35S]sulfate-labeled lipids were identified by comigration after HPTLC separation by immunostaining with specific IgM monoclonal antibodies from a patient with demyelinating neuropathy and plasma cell dyscrasia. Enhanced incorporation of sulfate in the crushed nerves was also observed in the sulfatides and in several unknown lipids migrating between GM2 and GM3, between GM1, and GM2, slightly above the origin, and at the origin. Since previous studies (Yao and Poduslo: J Neurochem 50:630-638, 1988) have shown [35S]sulfate incorporation, but not [3H]Gal or [3H]Glc, into sulfatides at 35 days after transection, it is possible that the sulfation observed in the present studies does not represent de novo biosynthesis but rather sulfation of an endogenous pool of glycolipids that results from the nerve injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Sugars↗

PubMed1990

Glycosphingolipids in the cerebrospinal fluid of patients with multiple sclerosis.

Glycosphingolipids in cerebrospinal fluid (CSF) of individual patients with multiple sclerosis (MS) were analyzed using a glycolipid-overlay technique. The ganglioside composition of CSF of non-MS patients was characterized by an abundance of polysialo species, including GT1b and GQ1b. This pattern is completely different from that of human white or gray matter, in which mono- and disialogangliosides predominate. Increased levels of GM1, either associated with or without increases of other gangliosides, such as GD1a, were observed in 16% of the patients with MS (6 of 37 cases: 1 of 15 progressive progressive stage, 4 of 16 progressive stationary stage, and 1 of 6 relapsing stage). The concentration of GD3 was increased in 23% (3 of 13 cases), whereas 1 of 13 cases (8%) showed a dramatic increase of sulfoglucuronyl paragloboside (SGPG) associated with a high level of GD3. These changes may reflect the cellular changes associated with the known pathological lesions in MS, which are characterized by demyelination, gliosis, and/or remyelination with oligodendrocytic proliferation.

Cholera Toxin↗

PubMed1990

Sulfated glucuronyl paragloboside in rat brain microvessels.

In patients with neuropathy associated with paraproteinemia, there are monoclonal immunoglobulin M antibodies reacting with myelin-associated glycoprotein and sulfated glucuronyl glycolipids. There are indications that the monoclonal antibodies may be responsible for these neuropathies. However, the mechanism by which the antibodies gain access to the nervous tissue, which is separated by the blood-brain barrier or blood-nerve barrier, is still unknown. In this study, we examined the presence of the sulfated glucuronyl glycolipid antigens on brain endothelial cells. Microvessels were isolated from adult Lewis rat brain cortex. Sulfated glucuronyl paragloboside (SGPG) was detected in the acidic lipid fraction by a TLC immunostaining method. Immunofluorescence studies showed positive staining on the surface of microvessels. In addition, SGPG could be detected in the cultured endothelial cells of human umbilical vein. These findings suggest that the endothelial cells contain antigenic sites for interaction with the autoantibodies. This type of interaction may result in damages to the endothelial cell function and may be responsible for changes in the blood-brain barrier permeability and the ensuing penetration of large molecules, such as immunoglobulins, into the endoneurial space.

Animals↗

PubMed1990

Heterosis for brain cerebroside synthesis in mice.

The (C57BL/6J X DBA/2J) F1 or B6D2F1 hybrid mice are known to exhibit a transitory hypermyelinating activity compared with their parental strains B6 and D2. These mice exhibit an elevated accumulation of cerebrosides in the brain which can be explained by an increase in their synthesis. Analysis of the two major cerebroside species indicated that the elevated content of total cerebrosides in the cerebellum and cerebrum of B6D2F1, as well as D2B6F1 hybrids, reflected an increased accumulation of the hydroxylated species. The UDP-galactose:ceramide galactosyltransferase (CgalT) activities in B6, D2 and hybrid mice were studied using as substrates alpha-hydroxy fatty acid (HFA)-containing ceramides or normal fatty acid (NFA)-containing ceramides (HFA- and NFA-CgalT activities, respectively). Both CgalT activities were found to be about 2 times higher in the cerebellum than in the cerebrum for all the strains studied. Before 20 days of age, the HFA-CgalT activities in B6D2F1, D2B6F1 and D2 mice were higher than in B6. However, at 20 days, there was no difference between B6 and D2 while the HFA-CgalT activity in the hybrids remained about 20% higher than in the parental strains. In contrast, no strain differences could be detected for the NFA-CgalT activity at all ages. The data suggest that the increased synthesis of brain cerebrosides in the B6D2F1 and D2B6F1 strains of mice could be largely accounted for by an increased HFA-CgalT activity.

Animals↗

PubMed1989

Glycosphingolipids in Bomirski transplantable melanomas in hamsters.

The glycosphingolipid compositions of Bomirski melanomas at different stages of differentiation, including Ab amelanotic melanoma (fast growing), Ma melanotic melanoma (slow growing), and MI hypomelanotic melanoma (slow growing), were studied. The total concentration of lipid-bound sialic acid in Ab amelanotic melanoma was found to be much lower than those in Ma and MI melanomas (0.8 micrograms versus 1.4 micrograms and 1.4 micrograms/mg of dry tissue, respectively). The ganglioside patterns in melanoma tissues were composed mainly of three components, which were confirmed as NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer (GM3), acetyl1-9-O-NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer (9-O-acetyl-GD3), and NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer(GD3) by structural analysis and monoclonal antibody detections. However, the relative ratios of these gangliosides expressed in the different types of melanomas were completely different. The MI melanoma tissues contained GM3 as the predominant species (greater than 90% of the total gangliosides) with very little of GD3 and 9-O-acetyl-GD3 gangliosides (less than 2% of the total gangliosides). In contrast, Ab amelanotic melanomas contained mainly 9-O-acetyl-GD3 (greater than 27%) and GD3 (greater than 51%) with lesser amounts of GM3. However, Ma melanoma had intermediate levels of GM3, GD3, and 9-O-acetyl GD3. The MI and Ma melanomas also contained monohexosylceramide (GL1) (about 60% as Gal beta 1-1'Cer and 40% as Glc beta 1-1'Cer in Ma and 30% as Gal beta 1-1'Cer and 70% as Glc beta 1-1'Cer in MI) and Gal beta 1-4Glc beta 1-1'Cer as the predominant neutral glycosphingolipid species. In contrast, Ab melanoma tissues contained more GalNAc beta 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1'Cer (Gb5), Gal alpha 1-4Gal beta 1-4Glc beta 1-1'Cer (Gb3), and GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1'Cer (Gb4) than MI and Ma melanomas. Our data suggest that the expression of glycosphingolipids in hamster melanoma cells may be closely related to cell growth and the degree of differentiation, with slow growing, highly differentiated cells expressing GM3 and GL1, and fast growing, undifferentiating cells having a preponderance of GD3, 9-O-acetyl-GD3, Gb5, Gb3, and Gb4.

Animals↗

PubMed1989

High-performance liquid chromatography of sialooligosaccharides and gangliosides.

Glycans were cleaved from gangliosides and separated by high-performance liquid chromatography (HPLC). The columns were packed with bonded stationary phases made of microparticulate, macroporous silica with serotonin, phenylpropanolamine or tryptamine as the biogenic amine ligate. The ganglioside oligosaccharides were eluted in the order of increasing number of sialic acid residues in the molecule and their retention decreased with the ionic strength of the mobile phase. Best selectivity was obtained in the pH range from 3.0 to 4.0. The two major sialic acids, N-acetylneuraminic and N-glycolylneuraminic acids, were separated by lectin affinity chromatography using an HPLC column packed with silica-bound wheat germ agglutinin and 10 mM phosphate buffer, pH 4.0, as the eluent. Throughout this study, isocratic elution was used and the column effluent was monitored at 195 nm.

Chromatography, High Pressure Liquid↗

PubMed1989

High resolution proton NMR studies of gangliosides. Structure of two types of GD3 lactones and their reactivity with monoclonal antibody R24.

Ganglioside GD3 was converted at room temperature to two stable lactones, denoted as GD3 lactones I and II. The reaction sequence was presumed to be GD3----GD3 lactone I----GD3 lactone II based on the time course of their production. Lactone I behaved as a monosialoganglioside and lactone II as a neutral species. The two lactones were isolated by DEAE-Sephadex column chromatography. The positions of the inner ester linkages were investigated by two-dimensional J-correlated proton NMR spectroscopy. An ester linkage was most likely formed between the carboxyl group of the external sialic acid residue and C9-OH of the internal sialic acid residue in lactone I. In addition to this ester linkage, a second ester linkage between the carboxyl group of the internal sialic acid and C2-OH of the galactose residue was likely formed in lactone II. The structural changes induced by lactonization were further examined by their reactivity with the monoclonal antibody R24 (Puckel, C. S., Lloyd, K. O., Travassos, L. R., Dippold, W. G., Oettgen, H. F., and Old, L. J. (1982) J. Exp. Med. 155, 1133-1147), which reacted with GD3. R24 was found to bind weakly to GD3 lactone I, but not to GD3 lactone II. The results suggest that the monoclonal antibody requires both sialic acid residues for high affinity binding, and the complete lactonization results in a loss of negative charges and/or a change in the overall conformation of the oligosaccharide moiety which may account for the loss of binding.

Animals↗

PubMed1989

Accumulation of unique globo-series glycolipids in PC 12h pheochromocytoma cells.

In a previous paper, we reported the presence of a unique globo-series glycolipid as one of the major neutral glycolipid: Gal alpha 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1' Cer, in the subcloned PC 12h pheochromocytoma cells (Ariga, T., Yu, R. K., Scarsdale, J. N., Suzuki, M., Kuroda, Y., Kitagawa, H., and Miyatake, T. (1988) Biochemistry 27, 5335-5340). Recently we found that the subcloned PC 12h cells accumulated other unusual neutral glycolipids. In order to characterize these glycolipids, PC 12h cells were subcutaneously transplanted into rats. The induced tumor tissue accumulated four minor neutral glycolipids, which were purified by droplet counter-current, Iatrobeads column, and preparative thin-layer chromatographies. These glycolipid structures were determined by fast atom bombardment-mass spectrometry, proton nuclear magnetic resonance spectroscopy, permethylation study, and sequential degradation with various exoglycosidases to be as follows: A, Fuc alpha 1-2Gal alpha 1-3Gal alpha 1- 4Gal beta 1-4Glc beta 1-1'Cer; B, GalNAc beta 1-3Gal alpha 1-3Gal alpha 1- 4Gal beta 1-4Glc beta 1-1'Cer; C, Gal alpha 1-3Gal alpha 1-3Gal alpha 1- 4Gal beta 1-4Glc beta 1-1'Cer; and D, Gal alpha 1-3Gal alpha 1-3Gal alpha 1- 3Gal alpha 1-4Gal beta 1-4Glc beta 1-1'Cer. Glycolipids A and B were tentatively characterized in normal rat small intestine (Breimer, M. E., Hansson, G. C., Karlsson, K.-A., and Leffler, H. (1982) J. Biol. Chem. 257, 557-568; Angstrom, J., Breimer, M. E., Falk, K.-E., Hansson, G. C., Karlsson, K.-A., and Leffler, H. (1982) J. Biol. Chem. 257, 682-688). Glycolipids C and D have not been reported in the literature.

Adrenal Gland Neoplasms↗

PubMed1989

Ganglioside and lipid composition of bulk-isolated rat and bovine oligodendroglia.

We have examined the ganglioside composition of 30-day and 60-day postnatal rat oligodendroglia, adult bovine oligodendroglia, gray matter, white matter, and myelin and also the total lipid composition of the oligodendroglial preparations. The ganglioside patterns of rat and bovine oligodendroglia, as previously found for human oligodendroglia, were more complex than those of myelin. These data indicate that oligodendroglial perikarya can synthesize many brain type gangliosides, not all of which are incorporated into the compact myelin. Alternatively, the ganglioside composition of myelin may be altered in situ by the myelin-associated neuraminidase. In these two species, as in human, GM4 appears specific to oligodendroglia and myelin, while GD3 and GM3 are enriched in oligodendroglia but not myelin. In bovine oligodendrocytes GD3 is the major ganglioside. The total lipid concentration, as well as the percentage of cholesterol, sphingomyelin, phosphatidylinositol, and phosphatidylserine, differ for 30- and 60-day-old rat oligodendroglia and may be developmentally correlated with changes in myelin composition during myelinogenesis. There are also marked differences in the lipid composition of bovine oligodendroglia compared to rat oligodendroglia, with the former having more galactolipid and less ethanolamine phosphoglycerides.

Animals↗

PubMed1989

Isolation and characterization of ganglioside 9-O-acetyl-GD3 from bovine buttermilk.

Bovine buttermilk contains a unique ganglioside, 9-O-acetyl-GD3. In order to isolate large quantities of this ganglioside, a simplified isolation scheme which consists of several ion-exchange and silica gel column chromatographic procedures was devised. The isolated 9-O-acetyl-GD3 was characterized on the basis of its thin-layer chromatographic behavior, its immunoreactivity with a specific monoclonal antibody, JONES, and by conversion to authentic GD3 by mild base treatment.

Ammonium Hydroxide↗

PubMed1989

Interaction and fusion of unilamellar vesicles containing cerebrosides and sulfatides induced by myelin basic protein.

The effects of myelin basic protein on the aggregation, lipid bilayer merging, intercommunication of aqueous compartments and leakage of small unilamellar vesicles of egg phosphatidylcholine containing different proportions of galactocerebroside and sulfatide were investigated. This was performed employing light scattering, absorbance changes and fluorescence assays (resonance energy transfer, Terbium/dipicolinic acid assay and carboxyfluorescein release). The apposition of membranes rapidly induced by myelin basic protein is enhanced by sulfatide but reduced by galactocerebroside compared to vesicles of egg phosphatidylcholine alone. On the other hand, the presence of either glycosphingolipid in the membrane interferes with the induction by myelin basic protein of lipid bilayer merging, subsequent fusion and changes of the membrane permeability. Our results support an important modulation by sulfatide and galactocerebroside on the interactions among membranes induced by myelin basic protein, depending on the relative proportions of the glycosphingolipids and phosphatidylcholine.

Cerebrosides↗

PubMed1989

Effects of inducers of differentiation on protein kinase C and CMP-N-acetylneuraminic acid:lactosylceramide sialyltransferase activities of HL-60 leukemia cells.

Exposure of HL-60 leukemia cells to either 12-O-tetradecanoylphorbol-13-acetate (TPA), dimethylsulfoxide (DMSO), exogenous gangliosides GM3, GM1, or bovine brain ganglioside mixture (BBG) resulted in a marked inhibition of the growth of cells. The order of the inhibitory potency was TPA greater than GM3 greater than DMSO greater than BBG greater than GM1. In contrast, sulfatides were without effect on cellular replication. Treatment of HL-60 cells with TPA or GM3 induced differentiation along the monocyte/macrophage lineage, while treatment with DMSO induced maturation along the granulocytic pathway. These effects were accompanied by more than a twofold increase in protein kinase C (PKC) activity. In contrast, treatment with GM1, BBG, or sulfatides caused only a relatively small increase in PKC activity. The activity of CMP-N-acetylneuraminic acid:lactosylceramide sialyltransferase (ST1), a key enzyme for membrane gangliosides synthesis, in HL-60 cells was also influenced by the exposure to TPA, GM3, DMSO, GM1, or sulfatides. The inducers of differentiation, TPA and DMSO, caused an increase in ST1 activity, whereas GM3, which also induced cellular differentiation, inhibited ST1 activity, perhaps through the action of end-product inhibition. The non-inducers of differentiation, GM1 and sulfatides, also increased the activity of ST1, but to a much lesser extent. The findings suggest that the direct or indirect modulation of PKC activity by some of these agents may be involved, at least in part, in the regulation of cellular growth and differentiation. Furthermore, it is conceivable that differences in PKC activity may be responsible for the changes in ST1 activity associated with cell differentiation and proliferation.

Cell Differentiation↗

PubMed1988

Thermodynamic-geometric correlations for the morphology of self-assembled structures of glycosphingolipids and their mixtures with dipalmitoylphosphatidylcholine.

The morphology of aqueous dispersions of five neutral glycosphingolipids (GalCer, GlcCer, LacCer, asialo-GM2, asialo-GM1), sulfatide, and five gangliosides (GM3, GM2, GM1, GD1a and GT1b) and their mixtures with dipalmitoylphosphatidylcholine was studied by negative staining electron microscopy. The morphological features are interpreted on the basis of thermodynamic and geometric constraints previously studied in these systems (Maggio, B (1985) Biochim. Biophys. Acta 815, 245-258). The correlation between the theoretical predictions and the experimental findings are in reasonable agreement. Small changes in the molecular parameters of the individual glycosphingolipids or in their proportion in mixtures with dipalmitoylphosphatidylcholine bring about remarkable variations on the type of structure formed, its radius of curvature and thermodynamic stability.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1988

Accumulation of a globo-series glycolipid having Gal alpha 1-3Gal in PC12h pheochromocytoma cells.

In a previous paper, we reported the presence of globoside as a major neutral glycolipid in PC12 pheochromocytoma cells [Ariga, T., Macala, L. J., Saito, M., Margolis, R. K., Greene, L. A., Margolis, R. U., & Yu, R. K. (1988) Biochemistry 27, 52-58]. Recently, we found that subcloned PC12h cells accumulated another unusual neutral glycolipid. In order to characterize this glycolipid, PC12h cells were subcutaneously transplanted into rats. The induced tumor tissue accumulated two major neutral glycolipids, which were purified by Iatrobeads column and preparative thin-layer chromatographies. One of the glycolipids was found to be globoside, and the other had a globotriaosyl structure with an additional terminal Gal alpha 1-3 residue. Its structure was determined by fast atom bombardment mass spectrometry, two-dimensional proton nuclear magnetic resonance spectrometry (2D NMR), permethylation study, sequential degradation with exoglycosidase, and mild acid hydrolysis to be Gal(alpha 1-3)Gal(alpha 1-4)Gal(beta 1-4)Glc(beta 1-1')Cer.

Adrenal Gland Neoplasms↗