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R K Yu

Publications and source records attributed to R K Yu.

At least 217 records · Page 12Linked to original sources
PubMed1983

High-resolution proton NMR studies of gangliosides. 2. Use of two-dimensional nuclear Overhauser effect spectroscopy and sialylation shifts for determination of oligosaccharide sequence and linkage sites.

Homonuclear two-dimensional proton nuclear Overhauser effect (2-D-NOE) spectra have been obtained for asialo-GM2 (4) and gangliosides GM2 (8) and GM1 (9) at 500 MHz and 40 degrees C in Me2SO-d6-D2O (98:2 v/v). The anomeric protons of each oligosaccharide residue of 4, 8, and 9 are observed to NOE couple via intraresidue 1,3- and 1,5-diaxial interactions and interresidue interactions across the glycosidic linkages. The former couplings are used to confirm the H-3 and H-5 assignments for each residue. From the latter couplings the sequence and glycosidic linkage sites of all oligosaccharide residues, except the sialic acid residues, are revealed. Sialic acid attachment sites are determined for the monosialogangliosides (6-9) through consideration of sialylation-induced glycosidation shifts. Combination of the above sequence and linkage-site data with the monosaccharide composition, anomeric configurations, and characterization of the aglycon (information obtained via two-dimensional spin-echo J-correlated spectroscopy or 2-D-SECSY) allows complete assignment of monosialoganglioside primary structure, independent of other methods of structural analysis. Compared to conventional chemical and enzymatic methods, high-resolution two-dimensional proton nuclear magnetic resonance spectroscopy has the advantages of speed, sensitivity, and sample preservation.

Brain↗
PubMed1983

Comparison of the 13C-N.M.R. spectra of gangliosides GM1 with those of GM1-oligosaccharide and asialo-GM1.

The 13C-n.m.r. spectra of asialo-GM1 and GM1-oligosaccharide are completely assigned and compared to those previously found for intact GM1 and for the series GM4, GM3, GM2, GM1, GD1a, GD1b, and GT1b. Removal of the ceramide residue from GM1 liberated a free, reducing aldehyde group, which was reflected in a doubling of the 13C-n.m.r. signals assignable to the D-glucose residue because of alpha, beta equilibrium. The spectrum of asialo-GM1 lacks the resonances from the sialic acid residue, as expected; in addition, several resonances from the neutral gangliotetraglycosyl residue shifted to different field positions after removal of sialic acid from GM1. These resonances include that of C-4 of the inner beta-D-galactosyl residue, and C-1 of the 2-acetamido-2-deoxy-D-galactosyl residue that is near the site of attachment of the sialosyl residue. The differences between the chemical shifts of the carbon resonances of oligomeric and monomeric saccharides, termed linkage shifts, provide a quantitative assignment aid. They are approximately 1/3 of those for residues linked to sialic acid than those for residues linked to the neutral hexose chain. Correlations among linkage shifts for pairs of glycosidically-linked carbon atoms for asialo-GM1 and GM1-oligosaccharide were compared with those for the series of gangliosides GM4 to GT1b, and differences are noted for resonances for carbon atoms near the sialic acid residue. The spectrum of ganglioside GM1b, a positional isomer of GM1 whose 13C-n.m.r. spectrum has not yet been observed, is predicted.

Animals↗
PubMed1983

Ganglioside-basic protein interaction: protection of gangliosides against neuraminidase action.

The ability of acidic phospho- and sphingolipids to interact with basic proteins was studied by double diffusion analysis. The phospholipids, tri- and diphosphoinositide, and the sphingolipid, sulfatide, interacted with myelin basic protein as evidenced by precipitin line formation. Of the sialoglycosphingolipids (gangliosides) tested, only the myelin-specific monosialoganglioside, GM4, formed a precipitin line with myelin basic protein. In addition, myelin basic protein retarded the activity of Clostridium perfringens neuraminidase against GM4 and the disialoganglioside, GD1b. Examination of purified rat brain myelin suggested the presence of a neuraminidase activity intrinsic to myelin. This finding, in concert with ganglioside-myelin basic protein complexes which selectively protect against neuraminidase, may provide a physiological explanation for the simplified ganglioside pattern found in myelin.

Animals↗
PubMed1983

Chemical analysis of organotypic cultures of mouse spinal cord in normal, demyelinative, and nondemyelinative conditions.

Several biochemical parameters were analyzed in cultured embryonic mouse spinal cord during various stages of normal myelinogenesis or demyelination. In cultures demyelinated by exposure to anti-whole CNS tissue serum plus complement, the activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37) was decreased 70%, whereas in cultures that did not show morphological changes with complement-inactivated anti-CNS serum or anti-myelin basic protein serum, the activity was 30% lower than in control cultures. The lipid composition of these cultures was quantitated by means of high-performance thin-layer chromatography densitometry technique. Cultures with normal nutrient medium alone or with the addition of 5% normal rabbit serum plus 10% guinea pig serum had 30% of the total lipid content of that present in newborn mouse spinal cord of the corresponding age. There were, however, relatively more lysophospholipids, cholesterol esters, triglycerides, and free fatty acids and less phosphatidylethanolamine and galactolipids in cultures as compared with normal spinal cord. Explants demyelinated by exposure to anti-CNS serum plus complement demonstrated principally a 70% decrease in the content of galactolipids with respect to normal cultures. When complement was inactivated, total lipids increased 42% (with increases of 40-70% in individual lipids). Inclusion of anti-myelin basic protein serum plus complement in the medium produced no significant changes in the lipid composition of the cultures.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗
PubMed1983

Cellular localization of gangliosides in the developing mouse cerebellum: analysis using the weaver mutant.

The distribution of gangliosides was studied in the weaver (wv/wv) mutant mouse, where the vast majority of postmitotic granule cell neurons die prior to their differentiation. The wv mutation also shows a dosage effect, as granule cell migration is slowed or retarded in the +/wv heterozygotes. By correlating changes in ganglioside composition with the well-documented histological events that occur during cerebellar development in the normal (+/+), heterozygous (+/wv), and weaver (wv/wv) mutant mice, information was obtained on the cellular localization and function of gangliosides. Ganglioside GM1 may be enriched in granule cell growth cones and play an important role in neurite outgrowth. A striking accumulation of GM1, which may result from altered metabolism, occurred in the adult wv/wv mice. GD3 was heavily concentrated in undifferentiated granule cells, but was rapidly displaced by the more complex gangliosides during differentiation. GD1a became enriched in granule cells during formation of synaptic and dendritic membranes, whereas GT1a appeared enriched in Purkinje cell synaptic spines. A possible fucose-containing ganglioside was quantitated only in the wv/wv mice. Ganglioside GT1b became enriched in granule cells during synaptogenesis, whereas GQ1b became enriched in these cells after synaptogenesis. The concentrations of GT1b and especially GQ1b increased continuously with age. Our results provide further evidence for a differential cellular enrichment of gangliosides in the mouse cerebellum and also suggest that certain gangliosides may be differentially distributed within the membranes of these cells at various stages of development.

Aging↗
PubMed1983

Biochemical study of heterosis for brain myelin content in mice.

Heterosis (hybrid vigor) for brain myelin content has been examined in detail in (C57BL/6J x DBA/2J)F1 hybrid mice at 17 days of age. The amount of myelin isolated from the F1 hybrid brain is greater than that isolated from either parental strain. In addition, the total protein content in the myelin of the three genotypes showed the following trend: F1 greater than DBA greater than C57. However, no discernible differences in myelin protein compositions could be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of the whole brain for several myelin-associated constituents such as GM1 ganglioside, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase), 5'-nucleotidase, and carbonic anhydrase indicated that heterosis exists for these components. No heterosis was found for such nonmyelin constituents as gangliosides GD1a, GT, GQ, RNA, DNA, and choline acetyltransferase. A developmental study of the whole brain CNPase indicated that the heterotic effect was greatest during the most active period of myelination (17-30 days). We conclude that the heterotic effect is specific for myelin content and is probably the result of an accelerated myelin synthesis. The heterotic effect should have great potential as a new model for studying aspects of myelinogenesis.

Animals↗
PubMed1983

Isolation and characterization of the trisialogangliosides from bovine adrenal medulla.

Trisialogangliosides were isolated from bovine adrenal medulla by DEAE-Sephadex A-25 and Iatrobeads column chromatography. Their structures were elucidated by sugar analysis, neuraminidase digestion, and permethylation studies. The complete structures of trisialogangliosides, A to D, were identified as follows. A: GT1b, IV3NeuAc, II3 (NeuAc)2-GgOse4Cer. B: GT1b(NeuAc/NeuAc-NeuGc-); IV3NeuAc, II3 (NeuAc alpha 2-8 NeuGc-)GgOse4Cer. C: GT1b (NeuGc/NeuAc-NeuAc-); IV3NeuGc, II3 (NeuAc alpha 2-8 NeuAc-)GgOse4Cer. D: GT1b (NeuAc/NeuGc-NeuGc-); IV3NeuAc, II3 (NeuGc alpha 2-8 NeuGc-)GgOse4Cer. Gangliosides B, C, and D, which contain N-glycolylneuraminic acid, have not previously been reported in the literature.

Adrenal Medulla↗
PubMed1983

Analysis of brain lipids by high performance thin-layer chromatography and densitometry.

We have devised a high performance thin-layer chromatography (HPTLC) densitometry method to resolve the major lipid classes of brain tissue. We used DEAE-Sephadex column chromatography to separate the total lipid into neutral and acidic lipid fractions. The lipid fractions were then spotted on separate HPTLC plates and chromatographed in one dimension using two solvent systems. Quantitation was by in situ densitometry with absolute amounts of the lipid classes determined from co-chromatographed standards. An internal standard was also used to improve the precision. The individual lipid classes of rat whole brain, human brain gray and white matter, rat and bovine myelin, and bovine oligodendroglia were quantitated. Human brain phosphatidylethanolamine plasmalogen was also quantitated. Sensitivity was increased by using the cupric acetate charring reagent, which we found to be more sensitive than the conventional sulfuric acid-dichromate reagent. Total lipid (less than 400 micrograms) was quantitated from 5 mg of tissue wet weight. The limit of detection, on HPTLC, for the individual lipid classes was below 20 ng.

Animals↗
PubMed1982

Assignment of the carbon-13 nuclear magnetic resonance spectra of gangliosides GM4, GM3, GM2, GM1, GD1a, GD1b, and GT1b.

Complete 13C nuclear magnetic resonance assignments are presented for gangliosides in the series GM4, GM3, GM2, GM1, GD1a, GD1b, and GT1b. The gangliosides studied are related by the sequential addition of single saccharide residues. The structural relationships among these molecules were confirmed and subsequently utilized to provide the basis for a detailed investigation of 13C NMR oligomer-monomer shielding differences accompanying increasing oligosaccharide complexity. This gradual increase in complexity was reflected in the 13C NMR spectra and proved to be of significant value in the assignment task, resulting in the reassignment of four GM1 resonances from our previous work [Sillerud, L. O., Prestegard, J. H., Yu, R. K., Schafer, D. E., & Konigsberg, W. H. (1978) Biochemistry 17, 2619--2628]. The carboxyl-containing sialic acids in gangliosides have glycosidic linkage resonance shifts only approximately 30% as large as those found for neutral hexopyranosides; thus, care must be used in interpreting the 13C spectra of charge oligosaccharides. Secondary structural effects are also found to produce shifts in the resonances of the sialic acid adjacent to the GalNAc residue of GM2 and the more complex gangliosides, leading to inequivalence of the sialic acids in GD1a, GD1b, and GT1b.

Animals↗
PubMed1982

Disialogangliosides in bovine adrenal medulla.

Gangliosides were isolated from bovine adrenal medulla by DEAE-Sephadex A-25 and Iatrobeads column chromatography. The disialo fraction contained five major gangliosides, A to E; together they accounted for 4.6% of the total lipid-bound sialic acid. On the basis of sugar analysis, permethylation, mild acid hydrolysis and neuraminidase degradation studies, the structures of these five gangliosides are characterized as follows: (formula: see text). The major fatty acids of the disialogangliosides were C16:0, C18:0, C22:0, C23:0, C24:0, and C24:1, which were characteristic of extraneural gangliosides.

Adrenal Medulla↗
PubMed1982

In vitro biosynthesis of the tetrasialoganglioside GQ1b.

A sialyltransferase activity which catalyzes the synthesis of the tetrasialoganglioside GQ1b (N-acetylneuraminyl-N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl [N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosylceramide) from added trisialoganglioside GT1b (N-=acetylneuraminylgalactosyl-N-acetylgalactosaminyl [N-acetylneuraminyl-N-acetylneuraminyl]galactosylglucosylceramide) and CMP-N-acetyl[4-14C]neuraminic acid has been demonstrated using a membrane fraction of embryonic chick brain. Optimum enzymatic activity was obtained using the detergent Triton CF-54 at a pH of 6.6. Enzyme activity appeared unaffected by Ca2+, Mg2+, Mn2+, EDTA, or histone. A slight elevation in activity was seen in the presence of Hg2+. When the disialoganglioside GD1b (galactosyl-N-acetylgalactosaminyl [N-acetylneuraminyl-N-acetylneuraminyl]galactosylglucosylceramide) was used as the glycolipid substrate, approximately 15% of the radioactive label was found in GQ1b. When this GQ1b was subjected to a periodate oxidation-borohydride reduction, the distribution of radioactive label was consistent with GQ1b being the major tetrasialoganglioside product and that its synthesis could proceed via the sequence GD1b-GT1b-GQ1b.

Animals↗
PubMed1982

Gangliosides and proteins in developing chicken brain myelin.

Ganglioside and protein content of chicken brain myelin have been studied as a function of development and maturation. Ganglioside concentration remained relatively constant at approximately 225-266 micrograms sialic acid per 100 mg myelin, from 17-day-old embryos to 540-day-old adults. The ganglioside distributional pattern was also constant, GM1 and GM4 each accounting for approximately one-third of total sialic acid. These properties are contrasted with those of mouse and rat brain myelin which previously showed enrichment of GM1 at early stages of myelination and appearance of GM4 at a later stage of maturation. In an earlier study of the mouse total myelin gangliosides doubled in concentration between 3 and 5 weeks of age and full maturity. Myelin proteins of the chicken also remained relatively constant in concentration and distributional pattern during development, although the percentage of basic protein increased somewhat during the first few weeks after hatching. Correlations were observed between molar concentrations of basic protein and various gangliosides, particularly the monosialo types.

Aging↗
PubMed1982

Preparation of anti-GM4 antiserum and its assay by a solid-phase radioimmunoassay.

Anti-GM4 antiserum was prepared from rabbits by immunization with pure human brain GM4 ganglioside in complete Freund's adjuvant and methylated bovine serum albumin. None of the immunized animals developed any clinically apparent neurological dysfunction. The antiserum titer and specificity were analyzed by double immunodiffusion and a novel solid-phase radioimmunoassay (RIA). In the latter procedure, microtiter plate wells were coated first with the glycolipid antigen, followed by sequential application of antiserum and [125I]-Staphylococcal Protein A. The absorbed radioactivity in the well was then counted. Employing the RIA procedure, anti-GM4 antibody achieved a titer of 1:1600. The antiserum also exhibited a high degree of specificity to GM4; cross-reactivity with glycolipids of similar structure was negligible. The production of highly specific antiserum to GM4 and the feasibility of detecting antibodies to glycolipid antigens by a convenient solid-phase RIA should be useful to further study the biological and immunological roles of GM4 and other glycolipids in the central nervous system.

Animals↗