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Several monosialogangliosides containing the type A-active epitope have been detected in type A erythrocytes on immunological analysis with a monoclonal antibody, and three of them were purified by repeated silica bead column chromatography and by scraping from the TLC plate. Two of these A-active gangliosides were characterized by methylation analysis by GC/MS, negative SIMS, MALDI-TOF/MS, proton nuclear magnetic resonance spectroscopy, and immunological assays, and their structures were concluded to be as follows. A-active ganglioside I: Download : Download full-size image A-active ganglioside II: Download : Download full-size image The reactivity of the purified gangliosides to the anti-A monoclonal antibodies (mAbs) exhibited enhancement after removal of the sialic acid. Therefore, the sialic residue has been shown to inhibit the binding to the terminal A-active epitope through the formation of an immune complex. To confirm the presence of A- (including S-A-I, -II and -III) and B-active gangliosides, the reactivity of anti-A and -B mAbs were investigated using total gangliosides from type A, -B and -AB erythrocytes on TLC plate. The results were that the gangliosides from types A and AB showed positive reaction to anti-A mAbs, whereas in the anti-B mAbs binding the gangliosides from types B and AB were positive. Thus, it revealed that A-active gangliosides were present in type A and -AB, and B-active gangliosides in types B and AB. As there was no difference in respective gangliosides on type AB erythrocytes of 22 individuals, both A- and B-active gangliosides are equally present in type AB erythrocytes. The biological significance of these A- and B-active ganglioside variants remains vague at present. As these molecules exhibit different reactivities to the anti-A mAbs, it is very likely that they can regulate the antigenicity of the A-epitope on the cell surface.

To investigate a pathway to apoptosis which may involve ceramides and to elucidate the minimum structure which leads to apoptosis, we synthesized several novel acylamides. Although the four synthesized compounds were different in structure from C2-ceramide, they caused Jurkat cells to undergo apoptosis. The most effective of them was N-myristoyl- d -alaninol ( d -MA), as shown by DNA fragmentation (detected with propidium iodide) and a decrease in the mitochondrial transmembrane potential (ΔΨm) (detected with rhodamine 123). Nevertheless, peripheral blood leukocytes exhibited no change after d -MA exposure, like after C2-ceramide or anti-Fas antibody treatment. The DNA fragmentation and ΔΨm caused by d -MA were blocked by a caspase-3 specific inhibitor as in the case of anti-Fas antibody stimulation. Quantification of ceramides by metabolic labeling with [14C]palmitic acid and HPTLC showed no increases in the ceramide levels on stimulation with d -MA, C2-ceramide or anti-Fas antibodies. Furthermore, d -MA had an apoptosis-inducing effect on an anti-Fas-resistant subline of Jurkat cells. These data suggest that d -MA may cause apoptosis of Jurkat cells without distinct ceramide formation and that this apoptotic pathway is very comparable, i.e. not identical, to that induced by anti-Fas antibodies.

In a recent study of the ganglioside profiles of human head and neck squamous cell carcinomas versus normal tissue, one unidentified GX ganglioside was found exclusively in tumor extracts, migrating between GM1 and GD3 by thin-layer chromatography. To determine the chemical structure of this ganglioside which accounted for 3-8% of the total gangliosides, the lipid samples were pooled and separated by high-pressure liquid chromatography to obtain individual ganglioside species purified to homogeneity. The tumor-associated GX ganglioside was analyzed by gas-liquid chromatography, mass spectrometry and immunostaining on thin-layer plates with mouse monoclonal antibodies after enzymatic cleavage. The data allowed the identification of GX ganglioside as GalNAc-GM1 that has been reported as a very minor brain ganglioside in humans. Thus, GalNAc-GM1 is a specific tumor-associated ganglioside in human head and neck squamous cell carcinomas that could be potentially valuable for clinicians.

We studied ganglioside expression in 12 human metastatic brain tumors metastasized from colon (4), renal (3), lung (2), esophagus (1), pancreas (1), and mammary (1) carcinomas. GM3 was the major common ganglioside expressed in brain metastatic tumor tissues, and GT1b was also present in all the metastatic brain tumor tissues. The latter was identified by TLC-immunostaining and characterized structurally by secondary ion mass spectrometry combined with 'Far-Eastern blot'. The immunohistochemical analysis of frozen tissue sections confirmed localization of GT1b in the tumor cell membrane or cytosol. GT1b was shown to be expressed both in the primary colon carcinoma and the metastasis of a single patient by immunohistochemical procedure. In systemic carcinomas without brain metastasis, GM3 was a common major component, but no GT1b was detected. These findings indicate that GT1b is a brain metastasis-associated ganglioside. We speculate that the presence of GT1b would be a useful marker for estimating metastatic potentials to the brain.

Glycosphingolipids of head and neck carcinomas from six tumor-bearing patients were analyzed and compared to those of normal tissue from similar areas. The total glycosphingolipid content and the lipid-bound sialic acid were much higher in carcinomas than in normal tissue. Major neutral glycolipids were glucosylceramide, lactosylceramide, trihexosylceramide and paragloboside. Sulfatides were seen only in extracts from normal tissue which also showed a rather simple ganglioside pattern with #GM3 and GD3 as major species, whereas tumors showed additional species such as GM2 and GD2, along with a strong increase in LM1, GM1, GD1a and GT1b.

Serum antibodies from 8 (13%) of 62 patients with the acute Guillain-Barré syndrome (GBS) and 1 of 3 patients with the Miller Fisher syndrome (MFS) recognized a minor ganglioside in bovine and human brain trisialoganglioside fractions. The ganglioside antigen migrated between GD1a and GD1b on thin-layer chromatograms. The structure of this ganglioside was established to be GT1a by thin-layer chromatography blotting and mass spectrometry. GT1a a ganglioside was also detected in human and bovine peripheral nerves by thin-layer chromatogram immunostaining. Serum from the GBS patients had IgM, IgG, or IgA antibodies against GT1a detectable by enzyme-linked immunosorbent assay (ELISA). Serum from the MFS patient also had elevated levels of IG against GT1a. None of the sera from 43 patients with other neurological diseases or from 24 healthy controls reacted with GT1a. Sera from 6 of 8 GBS patients with anti-Gt1a antibodies also reacted with GQ1b. There was no difference in the incidence of anti-GT1a immunoglobulins in acute GBS patients with or without oculomotor abnormalities. Levels of anti-GT1a antibodies correlated temporally wit clinical symptoms in GBS patients. Although the incidence of dysphagia was slightly higher in GBS patients with anti-GT1a antibodies than in those without, the number of patients studied may have been too small to detect an association between anti-GT1a antibodies and an a specific clinical variant of GBS. Our data demonstrate that a proportion of GBS patients have antibodies against GT1a ganglioside and suggest that these antibodies may play a role in the pathogenesis of neuropathy in GBS.

One of the big problems in xenotransplantation from pigs to humans is the hyperacute immune reaction due to the carbohydrate epitope of Galα1-3Gal. Based on the porcine α-1, 3-galactosyltransferase cDNA sequence, several antisense oligonucleotide DNAs (20-base pair phosphorothioates) were chemically synthesized and used to suppress the expression of the Galα1-3Gal carbohydrate epitope on the surface of a porcine kidney cell line. One of the antisense oligonucleotide cDNAs including the stop colon of the sequences, caused a significant 30 to 35% decrease in the level of expression compared to in untreated cells. However, the treatment had no effect on cell growth and exhibited no toxicity. In contrast, the corresponding sense oligonucleotide or other antisense oligonucleotides containing the 5'-start colon had no effect on the carbohydrate expression. The binding of cells to human serum was investigated after the effective antisense oligonucleotide treatment. Cells thus treated were less reactive to human IgG or IgM. This evidence strongly supported that natural antibodies contained in human serum became less reactive with these cells due to suppression of Galα1-3Gal carbohydrate epitope expression on the cell surface by antisense oligonucleotide treatment. These findings provide a basis for and a means of genetic manipulation of porcine α-1, 3-galactosyltransferase for future xenotransplantation studies.

Mycoplasma fermentans is thought to be a pathogen of rheumatoid arthritis or a cofactor of AIDS (acquired immunodeficiency syndrome). To elucidate the possible involvement of membrane constituents in the pathogenesis of these diseases, we studied its lipid components. Several alkali labile glycophospholipids were detected and named glycoglycerophospholipids (GGPLs). Previously, we purified and determined the structure of one of them as 6'-O-phosphocholine-alpha-glucopyranosyl-(1'-3)-1,2-diacyl-sn-glycerol (GGPL-I). The present paper describes the purification and structural characterization of GGPL-III, the major GGPL of M. fermentans using 1H-, 13C- and 31P-nuclear magnetic resonance spectroscopy, and mass-spectroscopy as 1"-phosphocholine,2"-amino dihydroxypropane-3"-phospho-6'-alpha-glucopyranosyl-(1'-3)-1,2-dia cyl-glycerol.

Leukemia inhibitory factor (LIF) is a cytokine involved in hematopoiesis, neuropoiesis, and embryogenesis. Transcriptional activation of various genes occurs subsequent to LIF signal transduction in its target cells. Using the mRNA differential display method, a LIF-inducible gene was isolated from LIF-stimulated M1 murine myeloid leukemia cells. By DNA sequencing, this gene turned out to be gp49B1, which has been reported as an inhibitory signaling receptor to attenuate mast cell activation. Because gp49B1 expression was limited to the uterus of a pregnant mouse, its uterine expression was examined especially in relation to LIF expression during pregnancy. gp49B1 was expressed specifically on day 4.0 of pregnancy, as was LIF, and the site of the most abundant expression of LIF and gp49B1 mRNA was the luminal epithelium of the uterine endometrium. These findings suggest that the gp49B1 expression in the uterine endometrium is induced just before implantation by paracrine and/or autocrine effects of LIF. Considering its function as an inhibitory signaling receptor on mast cells, a possible role for gp49B1 on the surface of the uterine endometrium as an immunoreceptor that allows blastocyst attachment is proposed.

After treatment of melanomas with anti-GD2 monoclonal antibody (MAb) (14G2a), some patients develop sensorimotor demyelinating polyneuropathy with and without the syndrome of inappropriate antidiuretic hormone (SIADH). To clarify what causes the neurotoxicity of anti-GD2 MAb, we investigated the immunohistochemical localization of GD2 in the human nervous system. Anti-GD2 MAb (14G2a) reacted with the myelin sheaths in the peripheral nerves as well as with the pituicyte cytoplasm in the posterior lobe of the pituitary gland. We assume that the binding of anti-GD2 MAb to peripheral nerve myelin and the pituicytes in the posterior pituitary causes sensorimotor demyelinating neuropathy and SIADH.

Cumulative evidence supports the theory that anti-ganglioside antibodies function in the development of Guillain-Barré syndrome (GBS). Some patients have developed GBS after the administration of monosialoganglioside extracted from bovine brain. To clarify the pathogenesis of GBS associated with and without administration of the monosialoganglioside fraction, we investigated serum antibodies to the minor monosialogangliosides GM1b and GM1 alpha in patients with GBS and in control patients. GM1b and GM1 alpha were recognized specifically by the IgG antibody from the GBS patients. Twelve of 20 GBS patients who had high IgG anti-GM1b antibody titers had a preceding gastrointestinal infection. To evaluate the hypothesis that GM1b could be an immunogen, we determined whether a GM1b epitope was present in Campylobacter jejuni isolated from a patient with GBS associated with anti-GM1b antibody. Immunostaining with the monoclonal anti-GM1b antibody indicated that the lipopolysaccharide of the C. jejuni strain has the GM1b epitope. We speculate that an injection of bovine GM1 fraction that contains GM1b, as well as infection by an agent that bears the GM1b epitope, induces production of the anti-GM1b antibody which functions in the development of GBS in some patients.

A novel sulfated glycosphingolipid containing a sulfated galactosyl residue was isolated from bovine erythrocyte ghosts, and purified to homogeneity by column chromatography on DEAE-Sephadex and silica beads. Structural characterization included compositional analyses, permethylation studies, proton nuclear magnetic resonance (NMR) spectroscopy, negative secondary ion mass spectrometry (SIMS), solvolysis and immunostaining on thin-layer chromatogram. As a result, the structure of this glycolipid is proposed as HSO3-Gal beta 1-1 Cer. The ceramide portion contained d18:1, d18:0 and t18:0, and the predominant fatty acid consisted of palmitate and palmitate with a hydroxy group, as deduced by both compositional analysis and negative SIMS mass spectrometry. The component of this glycosphingolipid probably originates from erythrocytes and platelets as indicated by the results of flow cytometry analysis using Sulph I monoclonal antibody. The yield of galactosyl sulfatide was about 0.37 mg/kg wet bovine erythrocyte membranes, about three times that of human kidney. Our results strongly suggest that galactosylceramide sulfate on erythroid cells may play an important biological role in cell to cell interaction and recognition.

N-Acetylgalactosaminyl GD1a (GalNAc-GD1a) is a proposed target molecule for serum antibody in some patients with Guillain-Barre syndrome (GBS) (Kusunoki et al., 1994). We examined autoantibody to GalNAc-GD1a in sera from 58 GBS patients. Eight GBS patients had high IgG anti-GalNAc-GD1a antibody titers, 3 of whom also had high IgM anti-GalNAc-GD1a antibody titers. These 8 patients had experienced gastrointestinal infection before the onset of their neurological symptoms. Campylobacter jejuni was isolated from 4 of them. An absorption test indicated the presence of the GalNAc-GD1a epitope in lipopolysaccharides of C. jejuni. Sera that had anti-GalNAc-GD1a antibody reacted with several acidic glycolipids in bovine peripheral nerve, one of which was identified as N-acetylgalactosaminyl GM1b (GalNAc-GM1b). Serum binding to GalNAc-GM1b was decreased by absorption with GalNAc-GD1a. The presence of GalNAc-GM1b as well as GalNAc-GD1a has been reported in human peripheral nerves. We assume that C. jejuni, which bears the [GalNAc beta 1-4 (NeuAc alpha 2-3) Gal beta 1-3 GalNAc beta 1-] epitope, is the immunogen and that the glycoconjugates with the epitope are target molecules for the autoantibody in peripheral nerves of some GBS patients.

Although both ceramide and interleukin-1β converting enzyme (ICE) family proteases are key molecules during apoptosis, their relationship remains to be elucidated. We report here that cell-permeable ceramide induced cleavage and activation of CPP32, a Ced-3/ICE-like protease, but not ICE. Ceramide-induced apoptosis of Jurkat cells was blocked by the CPP32-specific tetrapeptide inhibitor DEVD-CHO, but not by the ICE inhibitor YVAD-CHO. Furthermore, variant Jurkat cells with defective CPP32 activation were resistant to both antiFas- and ceramide-induced apoptosis. These results indicate that CPP32 activation is required for ceramide-induced apoptosis, and suggest sphingomyelin-ceramide pathway functions upstream of CPP32.

Unlike CNS myelin, human peripheral nerve myelin has the acidic glycosphingolipids sialosyl paragloboside (SPG), sialosyl lactosaminyl paragloboside (SLPG), and sulfated glucuronyl paragloboside (SGPG). To elucidate the pathogenesis of Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating neuropathy (CIDP), we investigated the autoantibodies to peripheral nerve molecules in patients with these diseases and compared the frequency of the autoantibodies with that of autoantibody to GM1 which is present in both the CNS and PNS. The report of Sheikh et al. (Ann. Neurol. 1995; 38: 350) that Campylobacter jejuni bears the SGPG epitope led us to study whether sera from patients with GBS subsequent to C. jejuni enteritis have anti-SGPG antibody; but, high anti-SGPG antibody titers were not found in the GBS patients from whom C. jejuni was isolated. Although the frequency of the anti-SPG, anti-SLPG and anti-SGPG antibodies were lower than that of the anti-GM1 antibody in GBS, 5 patients with demyelinating GBS had high IgG anti-SPG antibody titers. IgG anti-SPG antibody may function in the development of demyelinating GBS. We found that 6 CIDP patients had elevated IgM anti-SGPG antibody titers. Immunoelectrophoresis failed to detect IgM M-protein in 3 of the patients. IgM anti-SGPG antibody could be a diagnostic marker for a subgroup of CIDP with or without paraprotein.

The gangliosides of Theileria sergenti piroplasms were isolated and analyzed by thin-layer chromatography (TLC) and TLC immunostaining test. Four species of gangliosides, designated as G-1, G-2, G-3, and G-4, were separated on TLC. G-1, G-2, G-3, and G-4 ganglioside showed the same mobility as GM3, sialosylparagloboside (SPG), i-active ganglioside, and I-active ganglioside on the TLC plate, respectively. In order to characterize the molecular species of gangliosides from T. sergenti, G-1, G-2, G-3, and G-4 gangliosides were purified and tested by TLC immunostaining test with monoclonal antibodies against gangliosides. G-1 ganglioside had reactivity to anti-GM3 monoclonal antibody. G-2 gave reaction with monoclonal antibody to SPG containing N-glycolylneuraminic acid (NeuGc). G-3 showed reactivity to the anti-i-active ganglioside (NeuGc) monoclonal antibody. G-4 was recognized by the monoclonal antibody which reacts with I-active ganglioside (NeuGc). In addition, sialic acid moiety of the gangliosides from T. sergenti piroplasms was also analyzed. N-acetylneuraminic acid-containing gangliosides were hardly detectable in T. sergenti piroplasms. Gangliosides from T. sergenti (G-1, G-2, G-3, and G-4) carried only NeuGc as their sialic acid moiety. These results suggest that G-1, G-2, G-3, and G-4 gangliosides are GM3 (NeuGc) [NeuGc alpha 2-3Gal beta 1-4Glc beta 1-Cer], SPG (NeuGc) [NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer], i-active ganglioside (NeuGc) [NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer], and I-active ganglioside(NeuGc) [NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3 (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6) Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer], respectively.

Previously, we have reported the occurrence of novel phosphocholine-containing glycoglycerolipids (GGPLs: GGPL-I and GGPL-III) in human helper T-cell culture (MT-4 cell line) (Matsuda et al, Glycoconjugate J. 10: 340). However, the GGPLs disappeared from the MT-4 cells after treatment with an antimycoplasma agent. This disappearance suggested the involvement of microorganisms in the GGPL expression. In this paper, we show that the novel lipids are components of Mycoplasma fermentans itself. The supernatant fluid of the antimycoplasma agent-untreated MT-4 cell culture produced mycoplasma-like colonies on PPLO agar plates, and PCR and immunological methods revealed the presence of M. fermentans. GGPLs were expressed again in the treated MT-4 cells after infection with the isolated M. fermentans. The isolated M. fermentans had glycoglycerolipids corresponding to GGPL-I and GGPL-III. Thin-layer chromatography-mass spectrometry and immunological analyses showed that these glycoglycerolipids which were derived from the isolated M. fermentans were identical with GGPL-I and GGPL-III previously obtained. This is the first report that shows mycoplasma has phosphocholine-containing glycoglycerolipids.

Sera from patients with Guillain-Barre syndrome (GBS) frequently have anti-GM1 antibody. We earlier showed that an lipopolysaccharides (LPS) from Campylobacter jejuni (PEN 19) isolated from a GBS patient has a GM1 ganglioside-like structure. Aspinall et al. (Biochemistry, 61 (1994) 335–337) reported that OH 4382 has an LPS that bears a GD3 ganglioside-like structure and that OH 4384 has an LPS that bears a GT1a-like structure; both strains were isolated from patients with GBS. They also suggested a GM1-like structure is present in the LPSs from OH 4384, but failed to show the presence in the LPSs from OH 4382. To clarify the pathogenesis of GBS after infection by C. jejuni (PEN 19), we investigated the carbohydrate structures of the three strains by thin-layer chromatography immunostaining with cholera toxin and monoclonal anti-ganglioside antibodies. We found that both OH 4382 and OH 4384 have an LPS with the GM1 epitope as well as one with the GT1a or GD3 epitope.

Syntheses of both stereoisomers of α-d-glucopyranosylglycerolipids enabled us to confirm the structure and the (2S)-configuration of a new glycoglycerolipid isolated from HTLV-I infected T-cell lines.

There is a strong association between Guillain-Barré syndrome (GBS) and Penner's serotype 19 (PEN 19) of Campylobacter jejuni. Sera from patients with GBS after C. jejuni infection have autoantibodies to GM1 ganglioside in the acute phase of the illness. Our previous work has suggested that GBS results from an immune response to cross-reactive antigen between lipopolysaccharide (LPS) of the Gram-negative bacterium and membrane components of peripheral nerves. To clarify the pathogenesis of GBS, we have investigated whether GM1-oligosaccharide structure is present in the LPS of C. jejuni (PEN 19) that was isolated from a GBS patient. After extraction of the LPS, the LPS showing the binding activity of cholera toxin, that specifically recognizes the GM1-oligosaccharide was purified by a silica bead column chromatography. Gas-liquid chromatography-mass spectrometric analysis has shown that the purified LPS contained Gal, GalNAc, and NeuAc, which are sugar components of GM1 ganglioside. 1H NMR methods [Carr-Purcell-Meiboom-Gill (CPMG), total correlation spectroscopy (TOCSY), and nuclear Overhauser effect spectroscopy (NOESY)] have revealed that the oligosaccharide structure [Gal beta 1-3 GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta] protrude from the LPS core. This terminal structure [Gal beta 1-3GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta] is identical to the terminal tetrasaccharide of the GM1 ganglioside. This is the first study to demonstrate the existence of molecular mimicry between nerve tissue and the infectious agent that elicits GBS.

A murine monoclonal antibody, AD117m, was produced by fusion of murine myeloma cells with spleen cells obtained from mice immunized with intact OMC-4 (a human adenocarcinoma cell line derived from uterine cervix). The hybridoma that produced AD117m was selected from five colonies showing strong reactivity with OMC-4 but no reactivity with OMC-1 (a human squamous cell carcinoma cell line from uterine cervix). Since antigenic activity was reduced by treatment of the cells with periodate and enhanced by treatment of them with sialidase, the epitope of the antigen seemed to be expressed as a carbohydrate moiety. The AD117m-defined antigen was found not only in the glycoproteins and glycolipids of OMC-4 cells but also in a human meconium neutral glycolipid fraction. We purified the antigen from glycolipids of human meconium, and its structure was characterized by methylation analysis, mass-spectrometry, and sequential glycosidase treatment. The antigen structure was concluded as Galβ1-3GlcNAcβ1-3Galβ1-4Glcβ1-1Cer (lactotetraosylceramide). Immunohistochemical study using formalin-fixed and paraffin-embedded tissue sections showed that in the uterine cervix, all cases of adenocarcinomawere stained with AD117m (20/20), but AD117m reacted in only two out of ten cases of squamous cell carcinoma. In an effusion cytological study, AD117m was found to distinguish metastatic carcinoma cells from reactive mesothelial cells, and showed increased specificity and sensitivity when compared with antiCEA antibody. These observations indicate that AD117m is able to improve the differential diagnosis between reactive mesothelial cells and metastatic carcinoma cells in effusion cytology.

A novel sulfoglycosphingolipid based on the isoglobo-series core structure was isolated from rat kidney and purified by column chromatographies with DEAE-Sephadex and silica beads. The structure was characterized by solvolysis, compositional analysis, proton NMR spectroscopy, Fourier-transform infrared spectroscopy, methylation analysis and liquid secondary ion mass spectrometry (LSIMS). The characteristic fragment ions for a sulfate and a sulfated N-acetylhexosamine were observed in LSIMS spectra. The two-dimensional chemical-shift-correlated spectroscopy (COSY) and nuclear Overhauser enhancement spectroscopy experiments evidenced the presence of a 3-O-sulfated N-acetylgalactosamine and a Gal alpha 1-3Gal structure in the molecule. The major ceramide consisted of 4-hydroxysphinganine linked to a C24 nonhydroxy fatty acid, deduced from both compositional analysis and LSIMS. From the above results, the following structure was established for this glycolipid: HSO3-3GalNAc beta 1-3Gal alpha 1-3Gal beta 1-4Glc beta 1-1Cer, isoglobotetraosylceramide (iGb4Cer) IV3-sulfate. Rat kidney also contained globotetraosylceramide (Gb4Cer) IV3-sulfate which has a carbohydrate core identical to that from human kidney. The yields of iGb4Cer IV3-sulfate and Gb4Cer IV3-sulfate were 0.27 and 0.07 nmol/g wet tissue, respectively.

Three monosialogangliosides containing the NeuAc alpha 2-6Gal structure have been detected in human meconium by immunological analysis using a monoclonal antibody, MSG-15, and purified by repeated silica beads column chromatography. One was previously shown to be NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer. The remaining two were characterized by proton NMR, fast atom bombardment mass spectrometry, methylation analysis by gas chromatography-mass spectrometry, and immunological studies, and their structures were concluded to be as follows. [formula: see text] The second ganglioside has the same structure that was isolated from bovine buttermilk (Takamizawa, K., Iwamori, M., Mutai, M., and Nagai, Y. (1986) J. Biol. Chem. 261, 5625-5630), and this is the first description of the occurrence of the ganglioside with the branched structure with two N-acetyllactosamines linked to lactosylceramide via beta 1-6 and beta 1-3 in human linked to lactosylceramide via beta 1-6 and beta 1-3 in human tissues. The third ganglioside is a novel ganglioside with blood group I-type and a NeuAc alpha 2-6Gal structure.

Six major acidic glycosphingolipids were isolated from human amnion using DEAE Sephadex A-25 and silica beads column chromatography. The structures of these glycosphingolipids were determined by methylation analysis, TLC immunostaining and/or negative ion FAB-MS, and were concluded to be II3 alpha NeuAcLacCer(GM3), IV3 alpha NeuAcnLc4-Cer (sialyl[alpha 2-3]paragloboside), IV6 alpha NeuAcnLc4Cer (sialyl[alpha 2-6]paragloboside), IV3 alpha NeuAcIII4 alpha FucLc4Cer (sialyl Lea), VI3 alpha NeuAcnLc6Cer (i-ganglioside) and II3 alpha (NeuAc alpha 2----8NeuAc)LacCer (GD3). In addition, several minor glycosphingolipids were detected with specific monoclonal antibodies, including glycolipids with NeuAc alpha 2-3Gal beta 1-4GlcNAc-beta 1- or NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1- determinant. Our results show that the glycosphingolipids of human amnion are characterized by having mainly type II chain analogues and onco-fetal antigens.

Ganglioside-hydrolyzing sialidase activity was solubilized from rat brain particulate fraction by using Triton X-100 plus sodium deoxycholate. When chromatographed on AH-Sepharose 4B, the solubilized activity was resolved into two peaks, which were designated sialidases I and II in order of elution. The two sialidases were purified by using sequential chromatographies on Octyl-Sepharose CL-4B, Phenyl-Sepharose CL-4B, and Sephadex G-200. Sialidase II was purified further by Mono Q-FPLC. Overall purification was 450- and 2,150-fold, for sialidases I and II, respectively. Purified sialidases I and II were maximally active at near pH 5.0 and exhibited M = 70,000 by gel filtration. Sialidase I hydrolyzed gangliosides but scarcely other substrates including 4-methylumbelliferyl-NeuAc (4MU-NeuAc). Sialidase II hydrolyzed oligosaccharides, glycoproteins, and 4MU-NeuAc although gangliosides appeared to be preferential substrates. Sialidase II cleaved GM2 much faster than sialidase I. An antibody raised in rabbits against sialidase I reacted with only sialidase I and an antibody against sialidase II reacted with only sialidase II. A subcellular distribution study suggested sialidase I in the synaptosomal membrane and sialidase II in the synaptosomal and lysosomal membranes, and this was verified by using the above antibodies.

Blood group A-active glycosphingolipids from human erythrocyte membranes were identified by the combination of thin-layer chromatography and matrix-assisted secondary ion mass spectrometry (TLC/SIMS). Partially purified lipid extracts were chromatographed by TLC and then blood group A-active glycolipids were detected by TLC-immunostaining assay using anti-A antibody. The parts of the plates which contained the same Rf area as anti-A positive spots were cut out and subjected to direct SIMS analysis. The TLC/SIMS spectra were quite similar to those obtained by ordinary SIMS. Detailed information, such as molecular weight, molecular species, ceramide portion, and oligosaccharide sequence, was obtained. Also, peracetylated blood group A-active glycolipids were analyzed in a similar manner. After the position of A-active glycolipids on a TLC plate was confirmed by in situ deacetylation and TLC-immunostaining, acetylated A-active glycolipids were also analyzed by the TLC/SIMS. Enhanced sensitivity was obtained with peracetylated glycolipids. Consequently, small amounts of unpurified bioactive glycolipids can be readily analyzed by TLC/SIMS.

We investigated the lipopolysaccharide (LPS) from Penner's serotype 19 (PEN 19) of C. jejuni that had been isolated from a patient with the Guillain-Barre syndrome (GBS). The LPS was extracted from the bacterium and separated by a silica beads column chromatography. One homogeneous fraction showing reactivity with the patient's serum, rabbit anti-G M1 antibody, and also with cholera toxin, which binds oligosaccharide of G M1 , was obtained

We generated a rat monoclonal antibody (mAb W#10) with the ability to neutralize human immunodeficiency virus type 1IIIB (HIV-1IIIB) infection. The epitope recognized by mAb W#10 was defined as R-I-Q-R-G-P-G by enzyme-linked immunosorbent assay (ELISA) with the use of synthetic peptides. The filamentous phage clones displaying random 15-amino-acid peptides on the amino terminus of the pIII coat protein reacting with mAb W#10 were identified with affinity and immunological selection procedures. Thirteen out of 16 selected phage clones contained the G-X-G-R-X-F sequence in the coat protein region representing significant homology to a part of conserved G-P-G-R-A-F sequence in the V3 loop of various HIV-1 strains. In addition, the phage clones included the G-X-G sequence in the sequence detected by synthetic peptides as the recognition site. The selected phage clones were stained by mAb W#10 specifically and were able to compete with mAb binding to cells expressing viral antigens.

Equine kidney and spleen contain a Forssman active glycosphingolipid, and the structure of this glycolipid has been reported to be that of a globopentaosylceramide (GalNAcalpha-1,3GalNAcbeta-1,3Galalpha-1, 4Galbeta-1,4Glcbeta-1,1'Ceramide). We found that equine kidney contains several other anti-Forssman antibody-reactive glycosphingolipids. One of these acidic Forssman active glycosphingolipids was isolated and characterized by means of NMR, mass spectrometry, permethylation studies, and TLC-immunostaining. This glycolipid contains three moles of galactose, one mole of glucose, three moles of N-acetylgalactosamine, one mole of N-acetylglucosamine, and one mole of N-acetylneuraminic acid, and is stained on TLC with anti-Forssman antibodies and anti-GM2 ganglioside antibodies. HOHAHA and ROESY experiments and permethylation studies showed this glycolipid oligosaccharide to be branched at the innermost galactose; one chain has an isoglobo structure with a terminal Forssman disaccharide and the other chain is branched through the linkage of N-acetylglucosaminebeta-1,6 to the inner galactose. The nonreducing end of the GM2 trisaccharide is linked to this glucosamine. The structure of the oligosaccharide of the glycolipid presented here is a novel type, having branched isoglobo-, ganglio-, and neolacto-series oligosaccharides. Mass spectrometric analyses indicated the ceramide moiety of the glycolipid to be composed predominantly of hydroxy fatty acids (C20:0, C22:0, C23:0, C24:0, and C25:0) and hydroxysphinganine. GalNAcalpha-1,3GalNAcbeta-1,3Galalpha-1,3[GalNAcbet a-1, 4(NeuAcalpha-2,3)Galbeta-1,4GlcNAcbeta-1,6]Galbeta+ ++-1,4Glcbeta-1, 1'Ceramide

To clarify the pathogenesis of Guillain-Barre syndrome (GBS) after parenteral injections of bovine brain gangliosides, we searched for new molecules in bovine brain gangliosides recognized by sera from GBS patients. Gangliosides fractionated in a Q-Sepharose column were used as the antigens, and the binding of serum IgG or IgM was examined by thin-layer chromatography/immunostaining. Fourteen of 175 serum samples from the patients reacted with the monosialoganglioside fraction 2. In the neutral solvent system, a band in this fraction migrated with N-acetylneuraminic acid-containing GM1 [GM1(NeuAc)], whereas in the alkaline solvent system it migrated slower. This suggested that the band was N-glycolylneuraminic acid-containing GM1 [GM1(NeuGc)]. In both solvent systems, its mobility was almost the same as that of authentic GM1(NeuGc) from mouse liver. Secondary ion mass spectrometry showed that the ganglioside's structure was consistent with that of GM1(NeuGc). IgG anti-GM1(NeuGc) antibodies in sera from the GBS patients were significantly absorbed by GM1(NeuAc), indicative that the anti-GM1(NeuGc) antibodies cross-react with GM1(NeuAc). N-Glycolylneuraminic acid-containing gangliosides are so highly immunogenic in humans that the injection of GM1(NeuGc) could induce the production of IgG anti-GM1(NeuGc) antibody, which cross-reacts with GM1(NeuAc).

We describe the use of a phage-displayed random pentadecamer peptide library for searching glycosphingolipid mimicking peptides. Two phage clones (AD-1 and AD-2) were selected by biopanning using monoclonal antibody AD117m, directed to lactotetraosylceramide (Lc4Cer). The amino acid sequences of the selected clones showed high homology (VPPXFXXXY) in 9-mer. Three phage clones were selected by using monoclonal antibody H11, directed to neolactotetraosylceramide (nLc4Cer), the linkage isomer of Lc4Cer, and the displayed amino acid sequences were compared. One of these peptides showed the same amino acid sequence as that of AD-2 except for one amino acid substitution. Pentadecamer, 9-mer and point mutated 9-mer peptides were synthesized on the basis of the displayed amino acid sequences. Binding activity of the peptides to the monoclonal antibodies or Ricinus communis lectin showed that 9-mer peptides are enough to mimic the epitope carbohydrate structure. Furthermore, six of the synthesized peptides inhibited Jack bean β-galactosidase activity towards nLc4Cer at a high concentration of the enzyme, whereas at lower enzyme concentrations some peptides showed potent activation of the enzyme activity. This is the first report of carbohydrate mimicking peptides which modulate glycosidase activity.

Guillain-Barre syndrome (GBS) is characterized as an acute symmetrically progressive inflammatory neuropathy. Plasma exchange elicits a beneficial response (1). Since antibodies against gangliosides are commonly detected in patients with GBS, gangliosides are considered to be the target antigens of anti-neural antibodies. There is a close association between GBS and antecedent infection with a Gram-negative bacterium, Campylobacter jejuni,which causes acute gastroenteritis in human. The bacterium which is frequently isolated from GBS patients was serotyped as Penner type 19 (PEN 19). Sera from GBS patients after C. jejuniinfection contain antibody against GM1 in the acute phase of illness (2,3). On the other hand, papers concerning that the carbohydrate structures of lipopolysaccharides (LPS) of C. jejunishow striking similarities with those of gangliosides have been accumulated (4–6). On the basis of this back ground, we examined the presence of molecular mimicry between carbohydrate structure of LPS of C. jejuni(PEN 19) and that of GM1 ganglioside to clarify the molecular mechanism which produces anti-GMl antibodies as well as the pathogenic significance of the bacterium in eliciting GBS (7).

The glycosphingolipid compositions of rat mammary tumour cell lines with different metastatic potentials for the lung [a parental tumour cell line (MTC) and its subclones MTLn2 (a non metastatic subclone) and MTLn3 (a subclone with high metastatic potential to the lung)] were studied using a newly developed TLC blotting/secondary ion mass spectrometry system and crude glycosphingolipids obtained from 0.5-1 x 10(7) cells of each cell line. GM3 and GM2 were the major components of the MTC cell line, but they were very minor components in the MTLn2 and MTLn3 cell lines, GDla being the major ganglioside HexNAc-fucosyl-GMla was found in the MTLn2 cells by the TLC blotting/SIMS method, and the terminal sugar linkage was shown to be a blood group A-type structure by immunostaining. These findings suggest that the ganglioside is a novel type of blood group A-active ganglioside, GalNAc alpha 1-3(fuc alpha 1 -2)GMla. No blood group A-active lipid was present in MTLn3 cells, whereas Hex-GMla and neutral glycosphingolipids with more than 5 sugar residues were.

A simple assay method for bacterial binding to glycosphingolipids on a polyvinylidene difluoride (PVDF) membrane has been developed. Glycosphingolipids were separated on a high-performance thin-layer chromatography (HPTLC) plate and transferred onto a PVDF membrane by TLC blotting [Taki, T., Handa, S., and Ishikawa, D. (1994) Anal. Biochem. 221, 312–316]. The PVDF membrane was blocked with phosphate-buffered saline containing 4% casein and 0.1% methionine and overlaid with 35 S-labeled Escherichia coli possessing K99 fimbriae ( E. coli K99) at 37°C for more than 2 h. Binding of the 35 S-labeled E. coli K99 was detected with a bioimaging analyzer. Radioactivities were located on the bands corresponding to N -glycolylneuraminic acid containing glycosphingolipids such as sialylparagloboside, GM2, and GM3 with hydroxy fatty acid in ceramide moiety, and a weak binding was detected on the band of N -acetylneuraminylparagloboside. Furthermore, an in situ mass spectrometric analysis of the ligand glycosphingolipids on the membrane was demonstrated. The present method has several advantages compared with the overlay binding assay on the HPTLC plate as follows: (i) the method is simple and rapid; (ii) the membrane is easy to handle; (iii) binding is clear with low background; (iv) a small amount of [ 35 S]methionine is required; and (v) sensitive ligand characterization can be done by in situ mass spectrometric analysis.

A novel mono-sulfated glycosphingolipid based on the gangliotriaose core structure was isolated from rat kidney. The isolation procedure involved extraction of lipids with chloroform/methanol, mild alkaline methanolysis, column chromatographies with anion exchangers and silica beads. The structure was characterized by compositional analysis, FTIR spectroscopy, methylation analysis, 1H-NMR spectroscopy and negative-ion liquid secondary ion mass spectrometry (LSMIS) using the intact glycolipid and its desulfation product. The two dimensional chemical shift correlated spectroscopy provided information on the sugar sequence as well as anomeric configurations, and indicated the presence of a 3-O-sulfated N-acetylgalactosamine within the molecule. Negative-ion LSIMS with high- and low-energy collision-induced dissociation defined the sugar sequence and ceramide composition, confirming the presence of a sulfated N-acetylgalactosamine at the non-reducing terminus. From these results, the complete structure was proposed to be HSO3-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer (Gg3Cer III3-sulfate, SM2b).

We analysed the glycolipid composition of glioma cells (N-370 FG cells), which are derived from a culture of transformed human fetal glial cells. The neutral and acidic glycolipid fractions were isolated by column chromatography on DEAE-Sephadex and analysed by high-performance thin-layer chromatography (HPTLC). The neutral glycolipid fraction contained 1.6 micrograms of lipid-bound glucose/galactose per mg protein and consisted of GlcCer (11.4% of total neutral glycolipids), GalCer (21.5%), LacCer (21.4%), Gb4 (21.1%), and three unknown neutral glycolipids (23%). These unknown glycolipids were characterized as Lewis(x) (fucosylneolactonorpentaosyl ceramide; Le(x)), difucosylneolactonorhexaosyl ceramide (dimeric Le(x)), and neolactonorhexaosyl ceramide (nLc6) by an HPTLC-overlay method for glycolipids using specific mouse anti-glycolipid antibodies against glycolipid and/or liquid-secondary ion (LSI) mass spectrometry. The ganglioside fraction contained 0.6 micrograms of lipid-bound sialic acid per mg protein with GD1a as the predominant ganglioside species (83% of the total gangliosides) and GM3, GM2, and GM1 as minor components. Trace amounts of sialyl-Le(x) and the complex type of sialyl-Le(x) derivatives were also present. Immunocytochemical studies revealed that GD1a and GalCer were primarily localized on the surface of cell bodies. Interestingly, Le(x) glycolipids and sialyl-Le(x) were localized not only on the cell bodies but also on short cell processes. Especially, sialyl-Le(x) glycolipid was located on the tip of fine cellular processes. The unique localization of the Le(x) glycolipids suggests that they may be involved in cellular differentiation and initiation of cellular growth in this cell line.

Several underivatized mono- and bis-sulfated glycosphingolipids having gangliotriaose or gangliotetraose core structure were analyzed by negative liquid secondary ion mass spectrometry (LSIMS) with high- and low-energy collision-induced dissociation (CID). In the normal negative LSIMS spectra, each mono-sulfated glycolipid gave abundant [M − H]− ions and each bis-sulfated glycolipid gave abundant [M + Na − 2H]− ions as well as the hydrogen sulfate anion [OSO3H]−. In high-energy CID spectra of the deprotonated molecule, only ions containing a sulfate ester were clearly observed. When a sulfate was present on the non-reducing terminal saccharide residue, a series of ions corresponding to sulfated mono- to tetra-saccharides, resulting from sequential cleavage of glycosidic bonds, were observed. If the sulfate was attached to an internal hexose of the sugar chain, the product ions corresponding to the non-sulfated, non-reducing terminal residue were absent. In contrast, the low-energy CID resulted in extremely simple spectra that contained only one or two major product ions characteristic of each sulfated glycolipid. These results provided clear information on the overall sugar and ceramide compositions, and allowed saccharide structures differing in location and number of sulfate esters to be distinguished.

We have examined ganglioside compositions and the presence of sulfated glucuronyl glycolipids of immortalized motor neuron-like cell lines, neuroblastoma-spinal cord (NSC) hybrid cell lines established by fusing mouse neuroblastoma N18TG2 with motor neuron-enriched embryonic spinal cord cells. Among NSC cell lines, only NSC-34 aggregates acetylcholine receptors on co-cultured myotube and expresses a receptor for S-laminin, a neuromuscular junction specific basal lamina protein. GM2, which is only a minor ganglioside component of CNS, was the major component in NSC-34 occupying almost 75% of total gangliosides, whereas GD1a and GM3 were major species in the parental N18TG2, which had only 8.5% GM2. These results indicated that NSC lines have unique ganglioside pattern that is distinctive from other nervous tissues, and this pattern, especially that of NSC-34 cells, might reflect the characteristics of mouse spinal motor neuron gangliosides. Sulfated glucuronyl paragloboside was demonstrated to be present in N18TG2, however, it could not be detected in either of NSC cell lines. Even though the pathogenesis of amyotrophic lateral sclerosis remains unknown, autoimmunological participation has been suggested. Because high-titered antibody against GM2 has been observed in a patient with amyotrophic lateral sclerosis-like disease, GM2 which is possibly expressed on the surface of motor neurons might serve as a potential target antigen in this disorder.

Neutral glycolipids in Folch's upper phase were isolated from human erythrocyte membranes of 22 individuals with blood type AB. On immunostaining by TLC with anti-A IgG, all reactive glycolipids in type A corresponded to reactive glycolipids in type-AB erythrocytes. With anti-B IgM, all reactive glycolipids in type-B erythrocytes also corresponded to reactive glycolipids in type-AB erythrocytes. By comparison of the reactivity to that of the anti-A and anti-B antibodies, it was found that, in type-AB erythrocytes, all glycolipids reactive with either one of the anti-A or anti-B antibodies were detected in both type-A and type-B erythrocytes, and that A-active glycolipids had higher Rf values than B-active glycolipids on TLC plates. A series of glycolipids reactive with both antibodies were purified from the Folch's upper neutral glycolipid fraction of erythrocyte membranes by column chromatography, and was characterized by TLC-immunostaining and negative secondary-ion mass spectrometry. The results strongly suggested that A-active and B-active carbohydrate chain epitopes existed separately as glycolipid molecules in blood-type-AB erythrocytes. It was also confirmed that these phenotypes observed in erythrocyte membranes were exhibited by blood-group-active glycosphingolipids in the small intestine of blood-type-AB individuals. Furthermore, upon treatment of fractions obtained from silicic acid column chromatography with α-N -acetylhexosaminidase or α-galactosidase, a branched hybrid-type molecule with both A and B determinants was not detected.

We have established three kinds of monoclonal antibodies against gangliosides containing N-glycolylneuraminic acid (NeuGc) by immunization of BALB/c mice with the purified gangliosides inserted into liposomes comprising Salmonella minnesota R595 lipopolysaccharides, and fusion of spleen cells with a mouse myeloma cell line. One monoclonal antibody, SHS-1, which was generated by immunizing mice with purified i-active ganglioside(NeuGc), reacted specifically with the i-active ganglioside(NeuGc) used as an immunogen. Structurally related gangliosides, such as GM3(NeuGc), sialosylparagloboside (SPG) (NeuGc), or I-active ganglioside(NeuGc), corresponding gangliosides [GM3 containing N-acetylneuraminic acid (NeuAc), SPG(NeuAc), i-active ganglioside(NeuAc), and I-active ganglioside(NeuAc)], other gangliosides, or neutral glycosphingolipid (GSL) were not recognized by the monoclonal antibody. These findings indicate that the SHS-1 monoclonal antibody may be specific for NeuGc-containing i-active ganglioside. On the other hand, the other two monoclonal antibodies, MSG-1 and SPS-20, which were generated by immunizing mice with purified ganglioside GM3(NeuGc) and SPG(NeuGc), respectively, showed cross-reactivity to structurally related gangliosides. The MSG-1 monoclonal antibody exhibited reactivity to ganglioside GM3(NeuAc). The SPS-20 monoclonal antibody also cross-reacted with SPG(NeuAc), i-active ganglioside(NeuGc), and i-active ganglioside(NeuAc). Neither MSG-1 nor SPS-20 reacted with corresponding gangliosides, other gangliosides, or neutral GSLs tested. Using the SHS-1 antibody specific for i-active ganglioside(NeuGc), we studied the expression of NeuGc-containing antigen in human colon cancer tissue. An NeuGc-containing glycoconjugate was detected in the colon cancer tissue.

Mycoplasma fermentans is thought to be a pathogen of rheumatoid arthritis or cofactor of AIDS. A novel phosphocholine-containing glycoglycerophospholipid named GGPL-I was isolated from a M. fermentans-infected human helper T-cell culture. It was revealed that GGPL-I is a lipid component of the M. fermentans and a major immunological determinant. The GGPL-I was purified by DEAE-Sephadex column chromatography and repeated Iatrobeads column chromatography. The purified glycophospholipid was subjected to structural characterization by thin-layer chromatography, Fourier-transform infrared spectrometry, liquid secondary ion mass spectrometry, and nuclear magnetic resonance spectroscopy. Its structure was determined to be as follows: 6'-O-phosphocholine-alpha-glucopyranosyl-(1'-3)-1,2-diacyl-sn-glycerol. This glycoglycerophospholipid is unique in containing phosphocholine, which is attached to C-6 of glucose. The stereospecific numbering (sn) of naturally occurring GGPL-I was determined through comparison with chemically synthesized compounds.

A new and simple method for purifying glycosphingolipids and phospholipids by using "TLC blotting" was established. Glycosphingolipids separated by two-dimensional thin-layer chromatography (TLC) were made visible with primuline reagent, and then bands were marked with a drawing colored pencil. The glycosphingolipids that separated on the HPTLC plate were transferred by TLC blotting to a polyvinylidene difluoride membrane together with the color marks. The marked areas were excised after which their glycosphingolipids were extracted and monitored by TLC. By this method, 20 glycosphingolipids showing homogeneous bands on a HPTLC plate were isolated from the neutral glycosphingolipid fraction of human meconium. Moreover, 10 kinds of acidic glycosphingolipids were purified as homogeneous bands from the bovine acidic glycosphingolipid fraction. The yields of glycosphingolipids (13 different ones) ranged from 68 to 92%, the mean value being 82.3%. The glycosphingolipids were confirmed to be purified as intact forms by mass spectrometric analysis and chromatographic mobilities on a HPTLC plate. The same procedure could also be used to purify phospholipids.

We isolated Campylobacter jejuni from 2 patients with Fisher's syndrome subsequent to enteritis. Crude lipopolysaccharide fractions were extracted from the bacteria and separated by thin-layer chromatography. Monoclonal antibodies to GQ1b ganglioside (GMR13 and 7F5) reacted with both lipopolysaccharide fractions, indicating that the lipopolysaccharides bear the GQ1b epitope. This is the first report of molecular mimicry between neural tissue components and the antecedent infectious agents of Fisher's syndrome.

A new method for assaying the activity of the enzyme that catalyzes the formation of a cancer-associated glycolipid, paragloboside (nLc4Cer), from lactotriaosylceramide (Lc3Cer) and UDP-galactose has been developed that is based on a time-resolved fluoroimmunoassay (TRFIA) with a Europium (Eu)-chelate-labeled antibody. The substrate, Lc3Cer, immobilized on a microtiter plate, was incubated with UDP-galactose, MnCl2, Triton CF-54, and the enzyme. The content of the incubation product, nLc4Cer, was determined by the TRFIA with anti-nLc4Cer monoclonal antibody H-11 as the first antibody and Eu-labeled anti-mouse IgM antibody as the second one. The lower limit of detection of nLc4Cer was estimated to be 0.2 pmol. This method was used to detect the galactosyltransferase activity in sera from patients with colorectal cancer or benign colorectal adenomas and from healthy subjects of a reference sample group. The reference interval was 0-0.25 pmol/25 microliters serum/2 h. Activity was significantly greater in patients with colorectal cancer than in those with colorectal benign adenoma (P0.05) and the subjects of the reference sample group (P0.01).

The carbohydrate structures of lipopolysaccharides (LPSs) of Campylobacter jejuni strains belonging to Penner's serotypes (PEN) 1, 2, 4, 19, 23, and 36 were studied by thin-layer chromatography and immunostaining with several monoclonal antiganglioside antibodies. Anti-GM1 and anti-GD1a antibodies reacted with the LPSs of PEN 1, 4, and 19. Aspinall et al. (G. O. Aspinall, A. G. McDonald, T. S. Raju, H. Pang, A. P. Moran, and J. L. Penner. Eur. J. Biochem. 213:1017-1027, 1993) recently reported that the LPS of PEN 4 has a GD1a ganglioside-like structure rather than a GM1-like structure. We found that the LPS fraction of C. jejuni (PEN 4) has an LPS that bears a GM1 epitope as well as an LPS that bears a GD1a epitope.

The electrophoretic mobilities of the cells of malignant lymphosarcoma cell line RAW117-P and its variant H10 with a highly metastatic property to the liver have been measured at various ionic strengths. The cells of parental cell line (RAW117-P) show higher mobility values in magnitude than those of its variant line (RAW117-H10) in the whole range of electrolyte concentration measured. We have also measured the sialic acid amount carried by cells of both lines. The content of sialic acids in RAW117-H10 cells is observed to be about 27% less than that in RAW117-P cells. The mobility data obtained have been analyzed by a novel mobility formula for colloidal particles with ion-penetrable surface charge layers. The observed mobility difference between RAW117-P cells and RAW117-H10 cells is found to be due to the difference in friction exerted by the cell surface layers on the liquid flow around the cells between these two types of cells and to the difference in fixed-charge density in their surface layers, which is caused by the 27% decrease in sialic acid content. A possible explanation for this mobility difference between these two types of cells is given.

Two acidic glycosphingolipids were isolated and purified from rainbow trout ovarian fluid. They were designated as ovarian fluid gangliosides ofg-2a and ofg-2b. Both of these glycolipids were found to contain glucose, galactose, and N-acetylgalactosamine in a molar ratio of 1:2:1, but they differ by the presence of 2 mol of deaminated neuraminic acid (KDN; 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) in ofg-2a and 1 mol each of KDN and 9-O-acetyl-KDN in ofg-2b. On the basis of composition analysis, methylation analysis, mild acid hydrolysis, fast atom bombardment mass spectrometry (FABMS), 400-MHz 1H nuclear magnetic resonance spectroscopy, and immunochemical analysis using a monoclonal antibody (mAb.kdn3G), the complete structures of these gangliosides were determined to be KDN alpha 2-->3Gal beta 1-->3GalNAc beta 1-->4(KDN alpha 2-->3)Gal beta 1-->4Glc beta 1-->Cer for ofg-2a [(KDN)GD1a] and 9-O-AcKDN alpha 2-->3Gal beta 1-->3GalNAc beta 1-->4(KDN alpha 2-->3)Gal beta 1-->4Glc beta 1-->Cer for ofg-2b [(KDN)GD1a(OAc+)]. The ceramide moieties (Cer) in both ofg-2a [(KDN)GD1a] and ofg-2b [(KDN)GD1a(OAc+)] were found by combining of the results from fatty acid analysis and FABMS measurements to be made up of 4-sphingenine and mainly a C24:1 fatty acyl chain (nervonate). The structures of ofg-2a and ofg-2b are novel, and they represent the second example of naturally occurring KDN-gangliosides. Mild acid hydrolysis of both ofg-2a and ofg-2b resulted in formation of (KDN)GM1a.

Several gangliosides of human nervous tissues have been reported to be potential target antigens in autoimmune neuropathies. To explain the diversity of clinical symptoms in patients with antiganglioside antibodies, we have searched for ganglioside antigens that are specific to individual nervous tissues such as motoneurons, peripheral motor nerves, and sensory nerves. Although the major ganglioside compositions were not different among human peripheral motor and sensory nerves, fucosyl-GM1 was found to be expressed in sensory nervous tissue but not in spinal cord, motor nerve, and sympathetic ganglia. Sera from several patients with sensory nerve involvement also reacted with fucosyl-GM1 as well as GM1. Thus, fucosyl-GM1 may be a responsible target antigen for developing sensory symptoms in some patients with autoimmune neuropathies.