Showing papers by "Kazuo Ishii published in 1990"

Accumulation of gangliosides with N-acetylneuraminosyl(alpha 2-6)lactosamine structure in primary human hepatoma.

Abstract: Gangliosides of hepatomas have been analyzed by using a monoclonal antibody directed to N-acetylneuraminosyl(alpha 2-6)lactoneotetraosylceramide (sialyl(alpha 2-6)paragloboside), which was prepared by injecting the monosialoganglioside fraction of human meconium into BALB/c mice. The monoclonal antibody, named MSG-15, was found to bind sialyl(alpha 2-6)paragloboside, but it failed to react with other gangliosides, including N-acetylneuraminosyl(alpha 2-3)lactoneotetraosylceramide (sialyl (alpha 2-3)paragloboside) and "Ii"-type gangliosides. MSG-15 was found to recognize NeuAc alpha 2-6Gal beta structure of the ganglioside. Gangliosides obtained from human hepatomas were analyzed by immunostaining on high-performance thin-layer chromatography plates using the monoclonal antibody MSG-15. All primary hepatoma samples used in this study (nine samples) were found to contain sialyl(alpha 2-6)paragloboside, which accounted for 13-31% of the monosialoganglioside fractions in the hepatomas. Furthermore, MSG-15 recognized several monosialogangliosides in addition to sialyl(alpha 2-6)paragloboside. These gangliosides apparently also contain a terminal NeuAc alpha 2-6Gal beta structure. Other ganglioside fractions obtained from hepatoma and meconium were immunostained on thin layer chromatography plates with MSG-15. Additionally, another monoclonal antibody (H-11), which recognizes terminal lactosamine structure, was used to immunostain these fractions after sialidase treatment. Bands stained with both monoclonal antibodies showed similar mobilities to each other in the di- and trisialoganglioside fractions as well as monosialoganglioside fraction. In control liver, GM3 ganglioside accounted for 92% of monosialoganglioside fraction, and sialyl(alpha 2-6)paragloboside accounted for less than 1% of the fraction. Immunohistochemical study by using MSG-15 in tissue sections from hepatocellular carcinoma and normal liver tissues demonstrated that only hepatocellular carcinoma cells gave a positive reaction. These results suggest that the biosynthetic pathway of gangliosides containing NeuAc alpha 2-6Gal beta 1-4GlcNAc beta structure is activated in hepatoma cells.

A simple and specific assay of glycosyltransferase and glycosidase activities by an enzyme-linked immunosorbent assay method, and its application to assay of galactosyltransferase activity in sera from patients with cancer.

Abstract: A simple, sensitive, and specific assay method for glycosyltransferase and glycosidase activities has been established by means of an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody, H-11 directed to lactoneotetraosylceramide (nLc4Cer). Enzyme activity was determined by assaying the amount of reaction product, nLc4Cer with the ELISA method. For the assay of galactosyltransferase activity, lactotriaosylceramide (Lc3Cer) immobilized on a 96-well microtiter plate was incubated with bovine milk galactosyltransferase in cacodylate buffer (pH 6.8) containing Triton CF-54, Mn2+, and UDP-galactose. Optimum incubation conditions for the enzyme were determined. Glycosidase activity was also assayed by the ELISA method by using Clostridium perfringens sialidase and neolacto-series gangliosides as substrates, and the substrate specificities towards the gangliosides were examined. By this method, 3-100 pmol of reaction product could be determined. The assay method has several advantages as follows: 1, the method is simple; 2, separation of the reaction product is not required; 3, quantification and identification of the reaction product were done simultaneously; 4, naturally occurring substrates are available (especially for glycosidase); 5, many samples can be assayed in one microplate; 6, sensitivity is very high. The present method was applied for the detection of galactosyltransferase in human sera. Significant elevations of the galactosyltransferase levels were observed in the sera from cancer patients. The formation of nLc4Cer was confirmed by employing the TLC-immunostaining method for bands of Lc3Cer after incubation of the bands with serum and cofactors on an HPTLC plate.