Genetically regulated expression of UDP-N-acetylgalactosamine: GM3(NeuGc) N-acetylgalactosaminyltransferase [EC 2.4.1.92] activity in mouse liver

GM2 containing NeuGc was a major ganglioside in the liver of mouse strains such as BALB/c, DBA/2, C3H/He, and C57BL/10, whereas WHT/Ht mouse liver did not contain GM2(NeuGc) but contained GM3(NeuGc) as a major ganglioside. Since GM3(NeuGc) is a biosynthetic precursor of GM2(NeuGc), WHT/Ht liver was considered to lack the ability to synthesize GM2(NeuGc) from GM3(NeuGc) (Hashimoto, Y., et al. (1983) J. Biochem. 93, 895-901). In this study we measured the activity of UDP-N-acetylgalactosamine : GM3(NeuGc) N-acetylgalactosaminyltransferase in the liver of BALB/c, WHT/Ht, and their progeny. The transferase activity in the microsomal fraction of BALB/c liver was 2.10 +/- 0.32 X 10(-5) units/mg protein (means +/- S.D.), whereas no activity was detected in that of WHT/Ht liver, F1 hybrids between BALB/c and WHT/Ht expressed GM2(NeuGc) as well as the enzyme activity, the level of which was almost half that in BALB/c liver 1.10 +/- 0.12 X 10(-5) units/mg protein). The backcross generation of F1 to WHT/Ht segregated into two groups with respect to expression of GM2(NeuGc) and the transferase activity: 11 of the 21 mice analyzed expressed both GM2(NeuGc) and the transferase activity (1.28 +/- 0.18 X 10(-5) units/mg protein), whereas the rest expressed neither. These results suggest that the expression of GM2(NeuGc) is directly regulated by the activity of UDP-N-acetylgalactosamine: GM3(NeuGc) N-acetylgalactosaminyltransferase in mouse liver.     

Genetic regulation of GM4(NeuAc) expression in mouse erythrocytes

The polymorphic expression of GM4(NeuAc), GM3(NeuGc), GM2(NeuGc), and GM1(NeuGc) was found in erythrocytes of inbred strains of mice [Nakamura, K. et al. (1988) J. Biochem. 103, 201-208]. In this paper, we report the results of genetic analysis of the expression of GM4(NeuAc) and GM2(NeuGc). Ganglioside analysis of the progeny obtained on mating between BALB/c mice [GM4 (+)] and WHT/Ht or C57BL/6 mice [both GM4 (-)] indicated that the expression of GM4(NeuAc) is an autosomal dominant trait, and that WHT/Ht and C57BL/6 mice carry a defect on a single autosomal gene. We named this gene Gsl-4. On quantitative determination of galactosylceramide (GalCer), which is the biosynthetic precursor of GM4(NeuAc), the content of GalCer was found to be quite low in WHT/Ht erythrocytes, compared with in BALB/c erythrocytes. On analysis of GM4(NeuAc) and GalCer in 92 backcross mice produced on mating between BALB/c and WHT/Ht mice, it was found that 45 GM4(+) mice apparently expressed a detectable amount of GalCer and that 47 GM4(-) mice expressed an almost undetectable amount of GalCer. These results suggest that Gsl-4 controls the expression of GM4(NeuAc) by regulating the content of GalCer. Linkage analysis of Gsl-4 and the gene controlling GM2(NeuGc) in erythrocytes indicated that the two genes are not genetically linked. Comparison of the ganglioside expression in liver and erythrocytes of the same backcross mice suggested that the gene controlling GM2(NeuGc) expression in the liver (Ggm-2) is also responsible for the expression of GM2(NeuGc) in erythrocytes.     

Two pathways for GM2(NeuGc) expression in mice: genetic analysis

We have reported that WHT/Ht mice express neither GM2(NeuGc) nor GM1(NeuGc) in the liver or erythrocytes due to a defect on the Ggm-2 gene, which was demonstrated to control the activity of UDP-GalNAc:GM3(NeuGc) N-acetylgalactosaminyltransferase in mouse liver, and, in addition, WHT/Ht mice do not express a detectable amount of GM2(NeuGc) but do express GM1(NeuGc) in tissues other than the liver and erythrocytes, such as the spleen, thymus, heart, lung, kidney, and testis [Nakamura et al. (1988) J. Biochem. 103, 201-208]. In order to determine whether the phenotype of WHT/Ht mice exhibiting an undetectable amount of GM2(NeuGc) in these tissues is genetically controlled or not, we analyzed the expression of gangliosides in the progeny obtained on backcross mating between (BALB/c X WHT/Ht)F1 and WHT/Ht mice, and in a GM2(NeuGc) congenic mouse, WHT.C. Concerning the expression of GM2(NeuGc) in the liver, lung, and kidney, 102 backcross mice could be segregated into two types. One type expressed a detectable amount of GM2(NeuGc) in the liver, lung, and kidney, and the other type did not. The ratio of the numbers of mice exhibiting these two types was 42: 60, indicating that the two phenotypes were genetically determined by the involvement of a single autosomal gene. Recombination as to GM2(NeuGc) expression in the liver, lung, and kidney was not detected among the 102 backcross mice. Analysis of the GM2(NeuGc) congenic mouse indicated that a detectable amount of GM2(NeuGc) was expressed in the liver, erythrocytes, lung, kidney, heart, spleen, and small intestine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic polymorphism of ganglioside expression in mouse organs

In previous studies it was demonstrated that there are three variations as to the expression of liver gangliosides in inbred strains of mice; the first group expresses GM3(NeuGc) as a major component, the second group, GM2(NeuGc), and the third group, GM2(NeuGc), GM1 (NeuGc), and GD1a(NeuGc). In the present study, we attempted to determine which organs, if any, exhibit the same polymorphic variations as those observed in the liver. Thus, the gangliosides in spleen, thymus, heart, lung, kidney, testis, and erythrocytes, as well as those in liver, were examined using a TLC-mapping technique or by one-dimensional TLC. WHT/Ht, BALB/c, and ICR mice, which are typical strains as to the polymorphic expression of liver gangliosides, were used for the analysis. The presence of GM1 was confirmed by not only chemical detection on TLC plates but also with a TLC-immunostaining procedure using choleragenoid. These comparative studies indicated that only erythrocytes exhibited the same polymorphic variations of ganglioside expression as those in the liver, but the other six organs showed specific patterns which were not polymorphic. In addition to this, there were the following two interesting findings. Firstly, WHT/Ht mice, in which GM2(NeuGc) and GM1(NeuGc) are not expressed in the liver and erythrocytes, did not express a detectable amount of GM2(NeuGc) but expressed GM1(NeuGc) in all the other organs. Secondly, marked polymorphic variation was found in the expression of GM4(NeuAc) in the erythrocytes.     

Genetic control of glycolipid expression

A polymorphic variation of sialic acid species of sialosyllactosylceramide was found in dog erythrocytes. The analysis of the glycolipids in the erythrocytes of the individual dogs in a family of a Japanese breed of dog, Shiba-Inu, showed that the expression of sialosyllactosylceramide containing N-glycolylneuraminic acid was an autosomal dominant trait over the expression of that containing N-acetylneuraminic acid. Polymorphic variations of major liver gangliosides were also found in various strains of inbred mice. The strains were classified into three groups; the first group possessed only II3 NeuGc-LacCer, the second group possessed II3NeuGc-GgOse3Cer in addition to II3NeuGc-LacCer and the third group possessed II3NeuGc-GgOse4Cer and II3NeuGc,IV3NeuGc-GgOse4Cer as well as the above two gangliosides. By subjecting mice of these three groups to genetic analysis, the strain of the first group (WHT/Ht mice) was demonstrated to be a recessive homozygote which had a single autosomal defective gene making it unable to express N-acetylgalactosaminyltransferase activity to produce II3NeuGc-GgOse3Cer. The strains of the second group (BALB/c and C57BL/10 mice) were also demonstrated to be recessive homozygotes which had a single autosomal defective gene making them unable to express high enough level of galactosyltransferase activity to produce II3NeuGc-GgOse4Cer. By the analysis of gangliosides and the enzyme activity of H-2 congenic mice and mice produced by a mating, this defective gene controlling the expression of II3NeuGc-GgOse4-Cer through the regulation of the transferase activity was demonstrated to be linked to H-2 complex on chromosome 17.     

Structural characterization andin vivo immunosuppressive activity of neuroblastoma GD2

Shedding of neuroblastoma gangliosides is positively correlated with tumour progression in patients with neuroblastoma. In assessing the biological activity of these ganglioside molecules, we recently found that total human neuroblastoma gangliosides inhibit cellular immune responses. Here, we have studied the major neuroblastoma ganglioside, G_D2. G_D2 was purified by high performance liquid chromatography and structurally characterized by mass spectrometry. Immunoregulatory effects of G_D2 in vivo were then determined in an established murine model. G_D2 significantly downregulated the local cellular immune response to an allogeneic cell challenge; the usual increase in mass of the lymph node draining the injection site was reduced by 88%, from 1.52 to 0.19 mg (control versus G_D2-treated mice;p<0.01). In parallel, lymphocyte recovery from each node was also reduced from 2.4 to 1.2×10⁶ cells, and lymphocyte DNA synthesis was reduced to half of the control level. These results show that certain shed tumour gangliosides, such as G_D2, function as intercellular signalling molecules, downregulate the cellular immune response, and may thereby enhance tumour formation and progression.     

Susceptibility of infant mice to F5 (K99)E. coli infection: Differences in glycosyltransferase activities in intestinal mucosa of inbred CBA and DBA/2 strains

EnterotoxigenicEscherichia coli (ETEC) strains expressing F5 (K99) fimbriae cause diarrhoea in the young animal through adhesion to specific sialoglycolipids of the small intestine surface. We studied here an infant mouse diarrhoea model, as CBA infant mice are susceptible to F5-positive ETEC infection, whereas DBA/2 ones are resistant. In an attempt to determine an enzymatic basis for susceptibility and resistance, we investigated the intestine ganglioside pattern in relation to the activity of glycosyltransferases responsible for the globo- and ganglio-series. We observed that the intestine of susceptible CBA infant mice displayed a characteristic sialoglycolipid pattern containing mainly the F5 receptors. The two murine strains differed in the relative activities of galactosyltransferases (GbOse₃Cer and GM1 synthases),N-acetylgalactosylaminyltransferases (GA2 and GM2 synthases) and sialyltransferases (GM3 and GD3 synthases). An elevated GM3-synthase activity was observed in the intestine of susceptible CBA infant mice, at the age of high susceptibility. Hence, we conclude that the marked specificity of mouse type correlated with susceptibility and resistance to F5-positive ETEC infection which could be controlled through the regulation of glycosyltransferase activities.Abbreviations NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - Glc glucose - GalNAc N-acetylgalactosamine - Gal galactose - Car ceramide - LacCer lactosylceramide (Galß-4Glcß1-1Cer) - GA2 asialo-GM2 (GgOse₃Cer) - GA1 asialo-GM1 (GgOse₄Cer) - NeuAc/NeuGc-GMla II³ NeuAc/NeuGc-GgOse₄Cer - NeuAc/NeuGc-GM1a IV³ NeuAc/NeuGc-GgOse₄Cer - NeuAc/NeuGc-GM2 II³ NeuAc/neuGc-GgOse₃Cer - NeuAc/NeuGc-GM3, II³ NeuAc/NeuGc-LacCer; NeuAc/NeuGc-GD1a, IV³ NeuAc/NeuGc, II³ NeuAc/NeuGc-GgOse₄Cer; NeuAc/NeuGc-GD1b II³ (NeuAc/NeuGc)₂-GgOse₄Cer - NeuAc/NeuGc-GD1c IV³ (NeuAc/NeuGc)₂-GgOse₄Cer - NeuAc/NeuGc-GD2, II³ (NeuAc/NeuGc)₂-GgOse₃Cer; NeuAc/NeuGc-GD3, II³ (NeuAc/NeuGc)₂-Lac Cer; NeuAc/NeuGcGT1a IV³ (NeuAc/NeuGc)₂, II³ NeuAc/NeuGc-GgOse₄Cer - NeuAc/neuGc-GT1b IV³ NeuAc/NeuGc, II³ (NeuAc/NeuGc)₂-GgOse₄Cer - NeuAc/NeuGc-GT1c II³ (NeuAc/NeuGc)₃-GgOse₄Cer; NeuAc/NeuGc-GT2, II³ (NeuAc/NeuGc)₃-GgOse₃Cer - NeuAc/NeuGc-GT3 II³ (NeuAc/NeuGc)₃-Lac Cer - NeuAc/NeuGc-GQ1b IV³ (NeuAc/NeuGc)₂, II³ (NeuAc/NeuGc)₂-GgOse₄Cer - NeuAc/NeuGc-GQ1c IV³ NeuAc/NeuGc, II³ (NeuAc/NeuGc)₃-GgOse₄Cer - NeuAc/NeuGc-GP1c IV³ (NeuAc/NeuGc)₂, II³ (NeuAc/NeuGc)₃-GgOse₄Cer - GD, GT and GQ di-, tri- and tetra-sialoglangliosides. NeuGc-SPG, IV³ NeuGc-nLcOse₄Cer. Glycosyltransferases assayed in this work areN-acetylgalactosaminyltransferases - UDP-GalNAc lactosylceramide 1-4N-acetylgalactosaminyltransferase or GA2 synthase (EC 2.4.1-) and UDP-GalNAc:(N-acetylneuraminyl)-lactosylceramide 1-4N-acetylgalactosaminyltransferase or GM2 synthase (EC 2.4.1.92) - sialyltransferases CMP-N-acetylneuraminate: lactosylceramide 2–3 sialyltransferase (sialyltransferases I and IV) or GM3 synthase (EC 2.4.99.-) and CMP-N-acetylneuraminate:(N-acetylneuraminyl) lactosylceramide 2-8 sialyltransferase (sialyltransferase II) or GD3 synthase (EC 24.99.8) - galactosyltransferases UDP-galactose:N-acetylgalactosaminyl-(N-acetylneuraminyl) lactosylceramide 1-3 galactosyltransferase (galactosyltransferase II) or GM1a synthase (EC 2.4.1.62) and UDP-galactose:lactosylceramide 1-4 galactosyltransferase or GbOse₃Cer synthase (EC 2.4.1-)     

Fucosyl-GM1 — A ganglioside associated with small cell lung carcinomas

Characterization of monosialogangliosides of a small cell lung carcinoma showed a unique composition. The tumour contained G_M2 and Fucosyl-G_M1 (Fuc-G_M1) with 2-hydroxy fatty acids as major ganglioside components. Three out of four other small cell carcinomas analysed contained also Fuc-G_M1 as a characteristic ganglioside. Fuc-G_M1 is suggested to be a small-cell lung carcinoma associated ganglioside antigen.Nomenclature: The gangliosides have been designated according to Svennerholm [25] G_M3 II³NeuAc-Lac-Cer - G_D3 II³(NeuAc)₂-LacCer - G_M2 II³NeuAc-GgOse₃Cer - G_M1 II³NeuAc-GgOse₄Cer - Fuc-G_M1 FuclV²Neu-AcII³-GgOse₄Cer - 3-L_M1 IV³NeuAc-nLcOse₄Cer - 6-L_M1 IV⁶NeuAc-nLcOse₄Cer     

Substrate specificities of a bacterial sialidase and rat liver ganglioside GM3 sialyltransferase

Sialidases cleave off sialic acid residues from the oligosaccharide chain of gangliosides in their catabolic pathway while sialyltransferases transfer sialic acid to the growing oligosaccharide moiety in ganglioside biosynthesis. Ganglioside G_M3 is a common substrate for both types of enzymes, for sialidase acting on ganglioside G_M3 as well as for ganglioside G_D3 synthase. Therefore, it is possible that both enzymes recognize similar structural features of the sialic acid moiety of their common substrate, ganglioside G_M3. Based on this idea we used a variety of G_M3 derivatives as glycolipid substrates for a bacterial sialidase (Clostridium perfringens) and for G_D3 synthase (of rat liver Golgi vesicles). This study revealed that those G_M3 derivatives that were poorly degraded by sialidase also were hardly recognized by sialyltransferase (G_D3 synthase). This may indicate similarities in the substrate binding sites of these enzymes.     

Expression of GM1 and GD1a in liver of wild mice

Wild mice are divided into two groups with different ganglioside compositions in the liver. Most Japanese and a few Chinese wild mice have GM2(NeuGc) as a major ganglioside, whereas all wild mice caught at other places distributed all over the world other than Japan and China express GM1(NeuGc) and GD1a(NeuGc) in addition to GM2(NeuGC). We recently reported that inbred strains of laboratory mice were also grouped into the same two types based on the ganglioside composition in the liver, and that the expression of GM1(NeuGc) and GD1a(NeuGc) was regulated by a gene located at the left outside the H-2 complex on chromosome 17 (Hashimoto, Y., Suzuki, A., Yamakawa, T., Miyashita, N., & Moriwaki, K. (1983) J. Biochem. 94, 2049-2054). The present study suggests that oriental wild mice would be a donor of a defective gene for expression of GM1(NeuGc) and GD1a(NeuGc) in mice of laboratory stocks which are commonly used for biochemical and immunological studies, such as C57BL/6, C57BL/10, BALB/c, DBA/2, C3H/He, and CBA mice.     

A single autosomal gene controlling the expression of the extended globoglycolipid carrying SSEA-1 determinant is responsible for the expression of two extended globogangliosides

Two extended globogangliosides, designated as Z1 and Z2, were purified from the kidney of DBA/2 mice. By means of GLC, 1H-NMR spectroscopy, negative-ion fast atom bombardment mass spectrometry, methylation analysis, and enzymatic digestion, the structures of Z1 and Z2 were determined to be NeuGc alpha 2-3Gal beta 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-Cer and NeuGc alpha 2-8NeuGc alpha 2-3Gal beta 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-Cer, respectively. Since Z1 and Z2 were not detectable in the kidney of C57BL/10 and 6, BALB/c, and WHT/Ht mice, the mode of genetic control on Z1 and Z2 expression was examined by mating experiments between C57BL/10 or BALB/c and DBA/2. The results indicated that the expression of Z1 and Z2 is a recessive phenotype and that DBA/2 mice carry a single autosomal recessive gene. In the previous paper, we reported that DBA/2 mice do not express GL-Y (Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-6(Gal beta 1-3)Gb4Cer) but express GL-X (Gal beta 1-3Gb4Cer) in the kidney (J. Biochem. 101, 553-562 (1987)), and that a single autosomal defective gene responsible for the defective GL-Y expression was identified by genetic analysis (J. Biochem. 101, 563-568 (1987)).(ABSTRACT TRUNCATED AT 250 WORDS)     

Significant inhibition of hybridoma cells by exogenous application of ganglioside G M3, a possible modulator of cell growthin vitro

Gangliosides of the mouse-rat hybridoma cell line 187.1, which secretes an antibody against -light chain of mouse IgG, were isolated and structurally characterized by biochemical and immunological methods (overlay technique), and fast atom bombardment-mass spectrometry. Exclusively G _M3, substituted with C₂₄₁ and C₁₆₀ fatty acid and C₁₈₁ sphingosine, was found in this B cell derived cell line. A G _M3 (NeuGc) to G _M3(NeuAc) ratio (80 to 20), was characteristic for 187.1 cells, and absolute G _M3 amounts of about 0.3 mg 10^–9 viable cells were determined. Exogenous application of G _M3, which has been isolated from large cell preparations, to 187.1 cells showed growth inhibition in a concentration dependent manner. Using the MTT-assay and the [³H]thymidine incorporation assay, the cells exhibited a strong reduction in metabolic and proliferative activity, respectively, after exposure of cells to G _M3. G _M3 was applied in concentrations between 3M and 30M, giving evidence for strong inhibitory effects at 30M G _M3 and less but significant suppression after application of G _M3 concentrations lower than 20M. No cellular response was observed at the lowest concentration (3M) used in this study. Hybridoma cells as well as other cell types like fibroblasts, muscle cells and endothelial cells, are in general characterized by high expression of the G _M3 ganglioside, which is known to act as a modulator of cellular growth in monolayer cultures of adherent cells. Since gangliosides are released to the culture medium by cell lysis, i.e. cell death, and/or by active membrane shedding, the results obtained in this study suggest a growth regulatory role of G _M3 in high density hybridoma cell cultures.Abbreviations DMB 1,2-diamino-4,5-methylenedioxybenzene - FAB-MS fast atom bombardment-mass spectrometry - GSL(s) glycosphingolipid(s) - HPLC high performance liquid chromatography - HPTLC high performance thin layer chromatography - MTT 3,(4,5 dimethylthiazol-2-yl)2,5 diphenyl tetrazolium bromide - NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - PBS phosphate buffered saline The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations (1977) and the nomenclature of Svennerholm (1963). Lactosylceramide or LacCer, Galß1–4Glcß1-1Cer; gangliotriaosylceramide or GgOse₃Cer; GalNAcß1–4Galß1–4Glcß1-1 Cer; gangliotetraosylceramide or GgOse₄Cer, Galß1–3GalNAcß1–4Galß1–4Glcß1-1Cer; G _M3(NeuAc), II³NeuAc-LacCer; G _M3(NeuGc), II³NeuGc-LacCer; G _M2(NeuGc), II³NeuGc-GgOse₃Cer; G _M1 or G _M1a, II³NeuAc-GgOse₄Cer; G _M1b, IV³NeuAc-GgOse₄Cer.     

Genetically controlled IgM hyporesponsiveness to aK. pneumoniae polysaccharide

Examination of the immune response to aKlebsiella pneumoniae polysaccharide (K47-PS) has revealed that BALB/c mice demonstrate only a very weak primary response to this antigen. The low response does not result from either a peculiar dose response curve for BALB/c mice or from differing optimal antigen concentrations for high and low responder mice. Genetic analysis indicates that this variability of response is explicable assuming two alleles at a single locus; high responsiveness is dominant. Variability of response is probably not linked to theH-2 complex since the low and high responder mice, BALB/c and B10.D2/Sn new line, respectively, share the sameH-2 haplotype (H- 2_d). Tests of F₂s, backcrosses, and appropriate congenics have not shown evidence of linkage to sex, the albinism gene, the genes controlling coat color (agouti, black, brown), or the allotype-constant-region genes. The hyporesponsiveness is apparent only in the primary (IgM) response; hyperimmunization evokes similar antibody titers in high and low responding strains.     

Regulation of the production of murine IgG2a by an H-2-linked gene and other unlinked genes

Alleles of at least two loci (rig-1 and Rig-2) regulate the levels of serum immunoglobulin of the Igh-1b class and allotype in BALB/c Ig^b (BAB/14) and (BALB/c × BAB/14)F₁ mice. The combined effect of the BALB/c alleles at these two loci is to lower Igh-1b levels significantly below those observed in other strains and below their own levels of Igh-1a in allotype heterozygous mice. The rig-1 locus is closely linked to or within the H-2 complex. Two alleles have been defined: rig-1 ^d and rig-1 ^b in H-2 ^d and H-2 ^b haplotypes, respectively. Homozygous rig-1 ^d d animals heterozygous for the BALB/c Rig-2 allele(s) have very low levels of Igh-1b. The designation of Rig-2 is provisional since it has not been mapped or defined as a single locus.     

A sialidase-susceptible ganglioside, IV3 alpha(NeuGc alpha 2-8NeuGc)-Gg4Cer, is a major disialoganglioside in WHT/Ht mouse thymoma and thymocytes.

The disialogangliosides of WHT/Ht mouse thymomas, which were obtained by subcutaneous transplantation of a thymoma that developed spontaneously in a WHT/Ht mouse, were purified and characterized. From the results of sugar-composition analysis, a permethylation study, enzymatic hydrolysis followed by TLC-immunostaining, negative-ion fast atom bombardment mass spectrometry (FAB/MS), and 1H-NMR spectroscopy, the structure of one of the five purified disialogangliosides was determined to be IV3 alpha(NeuGc alpha 2-8NeuGc)-Gg4Cer. The other 4 disialogangliosides were tentatively characterized on the basis of sialidase treatment followed by TLC-immunostaining with cholera toxin B subunit and anti-Gg4Cer antibody to be IV alpha(NeuAc alpha-NeuGc)-Gg4Cer, IV alpha(NeuGc alpha-NeuAc)-Gg4Cer, IV alpha NeuAc,II3 alpha NeuAc-Gg4Cer, and IV alpha NeuGc,II3 alpha NeuGc-Gg4Cer. In addition, another component exhibiting one spot on TLC was a mixture of IV alpha NeuGc,II3 alpha NeuAc-Gg4Cer and IV alpha NeuAc,II3 alpha NeuGc-Gg4Cer. Then the occurrence of these gangliosides in WHT/Ht mouse thymocytes was examined. As one of two major disialogangliosides, the thymocytes contained IV3 alpha(NeuGc alpha 2-8NeuGc)-Gg4Cer, which was characterized with a mass spectrum and mass chromatograms obtained by micro high-performance liquid chromatography-FAB/MS. The other major disialoganglioside was tentatively characterized to be II3 alpha-(NeuGc alpha-NeuGc)-Gg4Cer by sialidase treatment followed by TLC-immunostaining. A sialidase-susceptible monosialoganglioside, IV3 alpha NeuGc-Gg4Cer [GM1b(NeuGc)], had been reported to be characteristic of mouse immune tissues [Nakamura, K. et al. (1988) J. Biochem, 103, 201-208]. Taken together, the results suggest that the pathway from Gg4Cer to IV3 alpha(NeuGc alpha 2-8NeuGc)-Gg4Cer through GM1b(NeuGc) is quite active in mouse immune tissues.     

Monoclonal antibody directed to a Hanganutziu-Deicher active ganglioside, GM2 (NeuGc)

Spleen cells from NZB mouse immunized with a membrane fraction of rabbit thymus tissue were fused with BALB/c 6-thioguanine-resistant myeloma cells, P3-X63-Ag8.653. One hybridoma clone (Y-2-HD-1) produced IgM immunoglobulin that bound to an N-glycolylneuraminic acid-containing GM2 ganglioside, GM2(NeuGc), which is known to be a Hanganutziu-Deicher antigen. The specificity of the Y-2-HD-1 monoclonal antibody was examined, using authentic glycosphingolipids structurally related to GM2(NeuGc), by means of an enzyme-linked immunosorbent assay and thin-layer chromatography/enzyme immunostaining, respectively. The monoclonal antibody was found to be highly specific to GM2(NeuGc) and the epitope was a non-reducing terminal GalNAc beta 1-4[NeuGc alpha 2-3]Gal structure. This monoclonal antibody (Y-2-HD-1) bound to native mouse erythrocytes, in which GM2(NeuGc) is a major ganglioside. These results indicate that GM2(NeuGc) is located on the surface of mouse erythrocytes.     

An H-2-restricted CML target antigen controlled by a gene linked to theH-2 complex

(B10 × BALB/c)F₁ anti B10.D2/n effector cells obtained after in vitro restimulation of spleen cells from in vivo primed mice react in the CML assay with B10.D2/n target cells and target cells from certain otherH-2D _d carrying strains. The gene controlling the antigen involved maps proximal toH-2K.     

Preparation of the tritiated molecular forms of gangliosides with homogeneous long chain base composition

Gangliosides G_M2, G_M1 and G_D1b were radiolabelled at C-6 of the terminal galactose orN-acetylgalactosamine by the galactose oxidase/[³H]NaBH₄ method; gangliosides G_M2, G_M1, Fuc-G_M1 and G_D1a were radiolabelled at C-3 of the long chain base by the 2,3-dichloro-5,6-dicyanobenzoquinone/[³H]NaBH₄ method.By application of an original HPLC procedure, eight different molecular species were prepared from each labelled ganglioside. Each of these species was characterized by the presence of one of the following long chain bases:erythro C18 sphingosine,threo C18 sphingosine,erythro C18 sphinganine,threo C18 sphinganine,erythro C20 sphingosine,threo C20 sphingosine,erythro C20 sphinganine andthreo C20 sphinganine.From G_D1b only the species containing theerythro forms of long chain bases were obtained.The individual molecular species were more than 99% homogeneous and had a radiopurity better than 99%. The molecular species of the same ganglioside, radiolabelled at C-3 of the long chain base, had identical specific radioactivity, namely 1.17, 1.25, 0.85 and 1.28 Ci/mmol for G_M2, G_M1, Fuc-G_M1 and G_D1a respectively. The molecular species of the same ganglioside, radiolabelled at C-6 of terminal galactose orN-acetylgalactosamine, had similar specific radioactivity, namely 1.34–1.40, 1.44–1.51, 1.37–1.44 Ci/mmol for G_M2, G_M1 and G_D1b respectively.     

Murine responses to (Tyr-Glu-Ala-Gly)n: II. H-2 and non-H-2 gene effects

Mice of the H-2^b haplotype responded to the sequential polymer poly(Tyr-Glu-Ala-Gly) in the in vitro T-cell proliferative assay, irrespective of whether they were homozygous or heterozygous at the H-2^b locus. The antibody responses of the H-2^b congenic mice to this polymer were variable, with A.BY and BALB.B showing responses better than those of C57BL/6 and C57BL/10 strains. The antibody responses of the F₁ progeny of (responder × nonresponder) strains of mice to this polymer are generally lower than the responder parents. F₁ mice with C57BL/10 background were the poorest responders. Studies with F₂ mice and backcross progenies of selective breeding of high and low antibody responder (C57BL/6 × BALB/c) F₁ to high responder C57BL/6 mice indicated that both non-H-2 genes and H-2 gene dosage effects influenced the magnitude of the humoral antibody responses. Animals having low responder non-H-2 background and only half the dosage of the responder immune response genes has greatly diminished antibody responses.     

Teratocarcinoma transplantation rejection loci: Genetic localization of the Gt-1 locus on chromosome 17 and the expression of alternate alleles

A series of congenic mice on the BALB/c genetic background have been employed to localize a teratocarcinoma transplantation rejection locus, Gt-1, to the K side of the H-2 locus on chromosome 17. Previous studies have placed the Gt-1 ^sv allele about 8 centimorgans away from the H-2 ^b or H-2 ^bv1 locus. Teratocarcinomas derived from 129/sv mice, Gt-1 ^sv (H-2K ^bv1/H-2D ^bv1), are rejected by BALB/c (H-2K ^d/H-2D^d) and BALB-G mice (H-2K ^d/H2D-^b, but form tumors in BALB-B (H-2K ^b/H2D ^b) and BALB/5R5 mice (H-2K ^b/H2D ^d). In the reciprocal tumor-rejection test, a BALB/c teratocarcinoma was rejected by immunized BALB·B mice, but formed tumors in the immunized isogenic BALB/c mouse. These studies demonstrate the reciprocal expression of two Gt-1 alleles, one Gt-1 ^c, in BALB/c mice, and the other, Gt-1 ^sv, in the congenic BALB·B mice. Shedlovsky and co-workers have placed the Gt-1 locus in a similar location on the K side of the H-2 locus on chromosome 17.