Noriyuki Kasai

The monoclonal antibody A2B5 is specific to ganglioside GQ1c

The neural specific monoclonal antibody A2B5 was found to interact with GQ1c but not with Gq1b, nor did it interact with other glycolipids such as GM1, GD1a, GD1b, GT1a, GT1b and GA1. Since GQ1c is enriched in embryonic chicken brains but not in adult chicken brains, this antibody should be useful as a tool in assessing the role of GQ1c in neuronal maturation.

In situ immunological determination of basic carbohydrate structures of gangliosides on thin-layer plates

An immunological method for the determination of the basic carbohydrate structure of gangliosides by using a thin-layer chromatographic immunostaining technique was developed. After high-performance thin-layer chromatography of gangliosides, the chromatogram is treated with a 0.4% polyisobutylmethacrylate solution. Arthrobacter ureafaciens neuraminidase is then applied to the separated gangliosides in situ on the chromatographic plate. This procedure will remove both external and internal sialic acid residues from the core oligosaccharide backbone. The resulting glycolipid products are then incubated with anti-Gg4 serum and 125I-staphylococcal protein A, successively, and exposed to an X-ray film. Through a highly specific binding, the anti-Gg4 antibody detects only those gangliosides having the oligosaccharide backbone of Gg4.

Anti-glycolipid antibodies and their immune complexes in multiple sclerosis

Antibody titers against myelin constituents in sera and CSF of patients with multiple sclerosis (MS) were examined by a solid-phase radioimmunoassay. Anti-GM4 and anti-galactocerebroside antibody titers were significantly elevated in the CSF of MS patients, but not anti-GM1 and anti-myelin basic protein antibodies. In sera of MS patients, the titers of antibodies against these myelin constituents were not elevated. Total IgG level was also significantly elevated in the CSF, but not in the sera of MS patients. Immune complexes from the CSF of MS patients were dissociated by acid-ultrafiltration and assayed for antibodies to GM4, GM1, and galactocerebroside. Anti-GM4 and antigalactocerebroside antibody titers were significantly enhanced after acid dissociation and ultrafiltration. These data suggest that antibodies of the IgG class against GM4 and galactocerebroside are present in CSF of MS patients, and a significant number of them exist as immune complexes with their corresponding glycolipid antigens.

A convenient method for the preparation of asialo-GM1

A convenient and efficient procedure has been devised for the large-scale preparation of asialo-GM1 from bovine brain gangliosides. The procedure relies on the complete desialylation of brain ganglio-sides, consisting primarily of GM1, GD1a, GD1b and GT1b, by mild formic acid hydrolysis (0.1 N, 100 C; 2 hr). Following the hydrolysis step, asialo-GM1 can be isolated and purified by Folch partitioning and Iatrobeads column chromatography, with an overall yield of more than 50%.

Human tumor necrosis factor increases the resistance against Listeria infection in mice

The resistance in mice against Listeria infection was augmented by treatment with recombinant human tumor necrosis factor (TNF). To elucidate this phenomenon, we examined the effect of TNF on macrophage activation. TNF-treated macrophages had listericidal activity in vitro and superoxide anion production. In addition, macrophage migration was inhibited in the presence of TNF. Therefore, activation of macrophages by TNF was similar to activation by macrophage-activating factor or macrophage-migration-inhibitory factor.

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 Freunds 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.

Occurrence of autoimmune antibodies to liver microsomal proteins in association with fulminant hepatitis in the LEC strain of rats

The Long Evans Cinnamon (LEC) rat, which has been established as a strain showing hereditary hepatitis and hepatic carcinoma, was found to possess autoimmune antibodies to liver microsomal proteins, particularly to a protein with the molecular weight of 56kD. The antibodies also recognized a protein(s) in liver microsomes from Long Evans Agouti and Sprague-Dawley rats. About 42 and 15 percent of respective female and male LEC rats died within a week after acute hepatitis; sera from all of the animals contained the antibodies. About 43 and 0 percent of the surviving female and male LEC rats possessed the antibodies, respectively. These results suggest that the autoantibodies occur in association with acute lethal hepatitis in the LEC rats.

Preparation and specificity of avian anti-GM2(NeuGc) ganglioside antiserum

Antibodies to N-glycolyl neuraminic acid-containing GM2 ganglioside, GM2(NeuGc), were prepared by immunizing chickens. The specificity of the antibodies was examined by the double immunodiffusion test and solid-phase radioimmunoassay (RIA). One(C-4) of two antisera produced did not cross-react with GM3(NeuGc) but the other(C-3) did as assessed by the double immunodiffusion test. In RIA, the antibody activity of C-4 antiserum was detected only in the IgG fraction. Specificity of the serum was examined using authentic glycolipids which were structurally related to GM2(NeuGc). The antiserum showed a high specificity for the homologous ganglioside by either an RIA or an inhibition assay. This antiserum is a useful tool for the detection of GM2(NeuGc) in human and animal tissues under normal and/or disease condition.

A high frequency of induction of chromosome aberrations in the bone marrow cells of LEC strain rats by X-irradiation

LEC strain rats, which have been known to develop hereditarily spontaneous fulminant hepatitis 4 to 5 months after birth, are highly sensitive to whole-body X-irradiation when compared to WKAH strain rats. The present results showed that the frequencies of all types of chromosome aberrations induced by X-irradiation in the bone marrow cells of LEC rats were approximately 2- to 3-fold higher than those of WKAH rats, though no significant difference was observed in the frequency of spontaneous chromosome aberrations between LEC and WKAH rats.

Accumulation of isogloboside and ganglio-N-tetraosyl ceramide having blood group B determinant in the hepatomas of female LEC rats

We have studied the neutral glycolipid composition of spontaneous hepatomas in LEC female rats. Neutral lipid fractions were isolated and purified by column chromatographies on DEAE-Toyopearl 650(M) and Iatrobeads. The neutral glycolipid fraction contained 3.2 to 4.4 micrograms lipid-bound glucose (Glc) per mg protein, and consisted of isogloboside (iso-Gb4, 50.8% of total neutral glycolipids) and IV3Gal, IV2Fuc, GgOse4Cer (asialo-BGM1, 13.5%) as the major neutral glycolipids and Gb3 and iso-Gb3 (9.2%), GlcCer (7.2%), LacCer (6.1%) as the other species. The structure of iso-Gb4 was elucidated by gas-liquid chromatography (GLC), permethylation study, liquid secondary ion (LSI) mass spectrometry, and nuclear Overhauser enhancement spectroscopy (NOESY) and that for asialo-BGM1 by GLC, LSI mass spectrometry, and high-performance thin-layer chromatography (HPTLC)-overlay method using anti-asialo-BGM1 antibody. Isogloboside and asialo-BGM1 which are found in negligible amounts in normal liver tissues may represent excellent markers for studying tumor metastasis and cellular adhesion.