Michiji Komoto

Characterization of neutral glycosphingolipids in rat lens

Neutral glycosphingolipids were purified from non-cataractous lenses of Sprague-Dawley rats by a combination of solvent extraction, Folchs partition, and column chromatography using DEAE-Sephadex and Iatrobeads. Six major GSLs from monohexosylceramide to pentahexosylceramide were identified by sugar composition analysis, methylation analysis and glycosidase digestion. Structural relationships among the six neutral glycosphingolipids revealed metabolic pathways leading to the synthesis of Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1 ceramide (IV3Gal alpha nLc4), instead of a Lewis(x) glycolipid (Gal beta 1- 4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1 ceramide, III3FucnLc4), from neolactotetraosylceramide (nLc4), together with isoglobotriaosylceramide (iGb3). The alpha-galactosyl epitope, Gal alpha 1-3Gal-R, is evolutionarily conserved in many types of cells of non-primate mammals, prosimians and New World monkeys, but not in those of Old World monkeys or humans. This evolution-related difference in carbohydrate epitopes suggests different cell-to-cell attachments, which may be mediated through cell surface glycosphingolipids, between rat and human lenses.

Electrical myotonia and cataract in X-linked muscular dystrophy (mdx) mouse

An X chromosome-linked mouse mutant (mdx) has been investigated as an animal model of Duchennes muscular dystrophy, and has been found to have the same defect of dystrophin in the muscle surface membrane. Intracellular recordings from the mdx mouse hemidiaphragm preparations revealed low resting membrane potentials and electrical myotonia which occurred at the time of microelectrode insertion and withdrawal. Electrical myotonia of the mdx mouse was observed in 30-50% of the impaled muscle fibers at low temperature, which decreased to only 7.8% at 37 degrees C. Electrical myotonia of mdx mice was not abolished by (+)-tubocurarine. Though there was no behavioral myotonia in mdx mice, repetitive bursts of action potentials in mdx mice were based on the abnormalities of the muscle membrane since neuromuscular blockade did not abolish the repetitive bursts. Also close observation of the lenses of mdx mice revealed cataracts from the newborn stage to the adult age. Slit lamp examination of the lenses of the mdx mice revealed nuclear cataracts followed by anterior subcapsular cataract as they grew. The cataract of mdx mice is different from that of myotonic dystrophy which is usually posterior subcapsular.

Identification and synthetic pathway of sialyl-Lewisx-containing neolacto-series gangliosides in lens tissues. l. Characterization of gangliosides in human senile cataractous lens

Human lens accumulates gangliosides in association with aging and senile cataract progression. In this study we purified and characterized five major gangliosides in human cataractous lenses. Structural analyses and immunological studies revealed the presence of ganglio-series gangliosides, GM3, GM2, GM1 and GD1a, and a sialyl-Lewisx-containing neolacto-series ganglioside, NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1ceramide (IV3NeuAcIII3FucnLc4). Slow-moving gangliosides, although minor components, were also found to have sialyl-Lewisx-related structures, based on anti-Lewisx antiserum binding to their asialo forms. However, sialyl-paragloboside, a possible precursor of the sialyl-Lewisx ganglioside, was not identified.

Age-dependent changes in monkey lenticular gangliosides

The content, composition, and distribution of gangliosides were examined in the lenses of normal rhesus monkeys aged 6-16 years. Gangliosides were isolated by organic solvent extraction. DEAE-Sephadex ion-exchange column chromatography, and thin-layer chromatography (TLC). Ganglioside contents determined by the thiobarbituric acid method increased in the lens with aging. TLC analysis of gangliosides showed a much more complex pattern with aging, and the predominant gangliosides were tentatively identified as GM3, GM1, and GD1a. Individual lenticular gangliosides were identified by TLC-immunostaining procedures using anti-GM1 and anti-asialoGM1 antisera.

Identification and synthetic pathway of sialyl-Lewisx-containing neolacto-series gangliosides in lens tissues. 2. Enzymatic synthesis of sialyl-Lewisx gangliosides in monkey and rat lenses

In Japanese monkey lenses, 3H-labeled fucose and N-acetylneuraminic acid were enzymatically transferred to neolactotetraosylceramide (nLc4) and III 3 FucnLc4, respectively, suggesting the presence of a synthetic pathway of IV3 NeuAcIII3 FucnLc4 via III3 FucnLc4 in monkey lenses. Six rat strains, Wistar, Sprague-Dawley and pigmented strains, contained sialyl-Lewis(x) gangliosides in non-cataractous lenses in a strain-specific manner. Glycosyltransferase assay revealed that the transfer of 3H-labeled fucose to nLc4 occurred in all the strains, but that the transfer of 3H-labeled N-acetylneuraminic acid to III3 FucnLc4 was strain-specific. These results suggested that sialyl-Lewis(x) gangliosides were generally synthesized from neolactotetraosylceramide via Lewis(x) glycolipid (III3 FucnLc4) in lens tissues, differing from other tissues. Combining our results, we propose two synthetic pathways of sialyl-Le(x)- containing neolacto-series gangliosides and A-pathway ganglio-series gangliosides in human senile cataractous lens: one to sialyl-Lewis(x) gangliosides from nLc4 via Lewis(x) glycolipid, and the other to GD1a from GM3, via GM2 and GM1.