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A mouse IgM monoclonal antibody, NUH2, was raised after immunization of mice with the disialoganglioside fraction of human colonic adenocarcinoma. This antibody reacts specifically with disialogangliosides having the Structure 1 shown below.

1. A technique has been developed for the measurement of kallikrein ‘production’ in rat renal cortical cells in suspension. 2. After preparative steps, column chromatography on DEAE-cellulose yielded a peak of a-N-tosyl-l-arginine methyl ester (Tos-Arg-OMe) esterase activity identical with kallikrein isolated from rat urine in respect of pH optimum, effects of inhibitors, biological activity and immunological properties. 3. The nutrient medium surrounding incubated cells contained measurable kallikrein activity, which was increased by aldosterone and decreased by spironolactone. 4. The results raised the possibility that kallikrein could be an aldosterone-induced protein.

A series of glycolipid antigens with Ley determinant (Fuc alpha 1----2Gal beta 1----4(Fuc alpha 1----3)GlcNAc) defined by monoclonal antibody AH6 (Abe, K., McKibbin, J. M., and Hakomori, S. (1983). J. Biol. Chem. 258, 11793-11797) have been detected in human colonic carcinoma cases. Three Ley-active components have been identified as follows. The simplest compound was characterized as Ley hexaosylceramide (lactodifucohexaosylceramide, III3FucIV2FucnLc4), which was previously isolated and was found as the major component in six out of eight cases of colonic adenocarcinoma but as only a very minor component in two cases. The second component was a very minor component in all eight cases, and its structure was identified by 1H NMR spectroscopy as an extended Ley (lactodifucooctaosylceramide, V3FucVI2FucnLc6; Structure 1 below). The third, major component, common in all eight cases, has been identified as trifucosyl Ley (lactotrifucononaosylceramide, III3FucV3FucVI2FucnLc6, Structure 2 below) based on 1H NMR spectroscopy, methylation analysis, and direct-probe electron-impact mass spectrometry. (formula; see text)

Immunization of mice with Ley-active trifucosylnonaosylceramide (III3FucV3FucVI2FucnLc6) isolated from human colonic adenocarcinoma (Nudelman, E., Levery, S. B., Kaizu, T., and Hakomori, S. (1986) J. Biol. Chem. 261, 11247-11253) followed by selection of hybridoma by positive reaction with this antigen and negative reaction with two other Ley antigens (III3FucIV2FucnLc4 and V3FucVI2FucnLc6) resulted in successful isolation of the hybridoma producing IgM antibody, termed KH1, specific to Ley-active trifucosylnonaosylceramide, which does not cross-react with Ley-active hexaosyl- or octaosylceramides (III3FucIV2FucnLc4 and V3FucVI2FucnLc6) without internal fucosyl substitution. The three-dimensional structure of the trifucosylnonaosylceramide was simulated based on previously published glycosidic torsion angles for fucosyl type 2 chain (Lex and Ley) and for GlcNAc beta 1----3Gal beta as predicted by hard sphere exo-anomeric calculations (Thøgersen, H., Lemieux, R. U., Bock, K., and Meyer, B. (1982) Can. J. Chem. 60, 44-57). The picture thus constructed showed a broad nonpolar area consisting of the hydrophobic surface of the pyranosyl ring and acetamido group of N-acetylglucosamine and three CH3 groups of L-fucose; this hydrophobic area is adjacent to a hydrophilic area. In analogy to the detailed structure of Leb or Ley involved in their interactions with antibodies and lectins (Spohr, U., Hindsgaul, O., and Lemieux, R. U. (1985) Can. J. Chem. 63, 2644-2652), such a wide hydrophobic area adjacent to a hydrophilic region could be recognized by the antibody KH1, as shown in the model illustrated in the text. Since the axis of ceramide, which is inserted in the lipid bilayer, is perpendicular to the plane of type 2 chain, the epitope recognized by the antibody KH1 is located at the external nonpolar surface of the carbohydrate chain that is overlaid on the lipid bilayer.

Mouse GM979 erythroleukemic cells were found to synthesize the branched form of erythroglycan, a large polylactosamine structure known to be attached to band 3 and possibly to other proteins on human erythrocytes. Total protein-derived oligosaccharides from GM979 cells, labeled metabolically with [3H]glucosamine, [3H]mannose, or [3H]galactose, were prepared by hydrazinolysis after extracting the lipids. The 3H-labeled oligosaccharides were fractionated on Sephadex G-50 revealing 10-25% of each labeled product as a high molecular weight fraction (Mr = 10,000). Digestion of this [3H]glucosamine fraction with endo-beta-galactosidase from Escherichia freundii, specific for the repeating structure of Gal beta (1 leads to 4)GlcNAc beta (1 leads to 3), resulted in the following four products upon Bio-Gel P-2 Gel filtration: 1) a disaccharide with the structure GlcNAc beta (1 leads to 3)Gal, 2) a trisaccharide with the structure Gal beta (1 leads to 4)GlcNAc beta (1 leads to 3) beta Gal, 3) a tetrasaccharide with the sequence Fuc alpha (1 leads to 2)Gal beta (1 leads to 4)GlcNAc beta (1 leads to 3)Gal, and 4) a large complex fragment which contained mannose, glucosamine, galactose, and fucose (presumably the protein linkage region). Methylation linkage analysis of the large complex fraction shows mainly the presence of 4-substituted and terminal N-acetylglucosamine; 3,6-substituted, 6-substituted, 2-substituted, and 2,3-substituted galactose. The GM979 cell erythroglycan is only 30% susceptible to endo-beta-galactosidase degradation probably because of the branched galactose residues, whereas the linear form of erythroglycan from human K562 cells is 60% susceptible. The branched residues in GM979 cell saccharides indicate that this mouse cell line bears an arborized erythroglycan-like glycopeptide similar to those found on human adult erythrocytes, and thus may be a source for the enzyme which transfers an N-acetylglucosamine residue to a 3-linked galactose to form a 3,6-disubstituted galactose.