Radiation-Induced Changes Associated with Obliteration of Brain AVMs after Repeat Radiosurgery.

BACKGROUND AND PURPOSE: Radiation-induced changes can occur after stereotactic radiosurgery for brain AVMs, potentially causing symptomatic complications. We evaluated the incidence of such changes and the efficacy of repeat gamma knife radiosurgery for incompletely obliterated AVMs. MATERIALS AND METHODS: We retrospectively evaluated 150 patients who underwent gamma knife radiosurgery for AVMs between 2002 and 2020; twenty-five underwent further radiosurgical procedures for incompletely obliterated AVMs. We recorded the median margin doses at the first (median, 20 Gy; range, 12-23 Gy; AVM volume, 0.026-31.3 mL) and subsequent procedures (median, 18 Gy; range, 12-23 Gy; AVM volume, 0.048-9.2 mL). RESULTS: After the first treatment, radiologic radiation-induced changes developed in 48 (32%) patients, eight of whom had symptomatic changes. After repeat gamma knife radiosurgery, 16 of 25 patients achieved complete AVM obliteration (64%). The development of radiation-induced changes after the first treatment was significantly associated with successful obliteration by subsequent radiosurgery (OR = 24.0, 95% CI 1.20-483, P = .007). Radiation-induced changes occurred in only 5 (20%) patients who underwent a second gamma knife radiosurgery, one of whom experienced transient neurologic deficits. Between the first and repeat gamma knife radiosurgery procedures, there was no significant difference in radiologic and symptomatic radiation-induced changes (P = .35 and P = 1.0, respectively). CONCLUSIONS: Radiation-induced changes after the first gamma knife radiosurgery were associated with AVM obliteration after a repeat procedure. The risk of symptomatic radiation-induced changes did not increase with retreatment. When the first procedure fails to achieve complete AVM obliteration, a favorable outcome can be achieved by a repeat gamma knife radiosurgery, even if radiation-induced changes occur after the first treatment.

Characterization of blood group ABO(H)-active gangliosides in type AB erythrocytes and structural analysis of type A-active ganglioside variants in type A human erythrocytes.

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:A-active ganglioside II: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.

Inhibition of Vero cell cytotoxic activity in Escherichia coli O157:H7 lysates by globotriaosylceramide, Gb3, from bovine milk.

In order to clarify the presence and verotoxin (VT) inhibitory activity of globotriaosylceramide (Gb3) in bovine milk, we analyzed neutral glycosphingolipids (GSLs) from bovine milk and investigated the inhibitory effect of bovine milk Gb3 on the cytotoxicity of VT2. Five species of neutral GSLs, designated as N-1, N-2, N-3, N-4, and N-5, were separated on thin-layer chromatography (TLC). N-1, N-2, and N-3 showed the same mobility as glucosylceramide, lactosylceramide, and Gb3 on the TLC plate, respectively. N-4 and N-5 GSLs migrated below globoside on the TLC plate. N-3 GSL having the same TLC mobility as Gb3 from bovine milk was immunologically identified as Gb3 by monoclonal antibody against Gb3, anti-CD77 monoclonal antibody. Furthermore, the effect of bovine milk Gb3 on VT2-induced cytotoxicity was investigated. We found that treatment of VT2 with bovine milk Gb3 can reduce the cytotoxic effect of VT2.

Isolation and characterization of a novel Forssman active acidic glycosphingolipid with branched isoglobo-, ganglio-, and neolacto-series hybrid sugar chains.

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

[Color Doppler signal enhancement with SH/TH-508 in pancreatic tumors].

In this report, we showed the efficacy of a new contrast agent (SH/TA-508, Schering AG, Germany) for color Doppler imaging of the pancreatic tumors. In pancreatic ductal cancer, no enhancement of the lesion was observed, but vascular invasion by cancer became to be easily evaluated. On the other hand, hypervascular tumors such as islet cell tumor and cystadenocarcinoma, were increased in color Doppler signals of vessels by SH/TA-508. We concluded that SH/TA-508 was useful for evaluating the vascular invasion by pancreatic cancer as well as vascularity of hypervascular mass and solid component of cystic neoplasma.

Sulfatide is expressed in both erythrocytes and platelets of bovine origin.

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.

Isolation and characterization of gangliosides from Theileria sergenti.

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.

Ceramide induces apoptosis via CPP32 activation.

Although both ceramide and interleukin-1beta 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 anti-Fas- 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.

Expression and localization of Lewis(x) glycolipids and GD1a ganglioside in human glioma cells.

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.

Glycosphingolipid composition of murine neuroblastoma cells: O-acetylesterase gene downregulates the expression of O-acetylated GD3.

We have studied the glycosphingolipid composition in an F-11 neuroblastoma cell line originated from hybridization of a mouse neuroblastoma cell line (N18TG-2) with rat dorsal root ganglion cells. The total lipid-bound glucose of F-11 cells was estimated to be 0.28 micrograms/mg of protein and the total lipid-bound sialic acid was 0.82 micrograms/mg of protein. The major neutral glycosphingolipids were Gb4 (37% of the total neutral glycosphingolipids), Gb3 (15%), LacCer (21%), and GlcCer (15%). The major gangliosides were found to be GM3 (37% of the total gangliosides), GD3 (27%), O-acetylated GD3 (18%), and GD1a (4%), with trace amounts of GD2. The unusually high concentration of O-acetylated GD3 is consistent with its putative role as a tumor marker. Immunocytochemical localization studies of GD3 and O-acetylated GD3, examined by mouse monoclonal antibodies R24 and D1.1, respectively, revealed that the cell bodies and processes were all positively stained. To elucidate the role of O-acetylated GD3 in tumorigenesis, we transfected F-11 cells with the O-acetylesterase gene from influenza C virus. Compared with the original cell line, the transfected cells showed a dramatic increase in the level of GD3 (150% of that in the control cells) and a significant decrease of the concentration of O-acetylated GD3 (27% of control cells). In addition, the transfected F-11 cells exhibited a morphology different from the parental cells with enlarged cell bodies and elongated neurites. We conclude that alteration of ganglioside composition, particularly the expression of GD3 and O-acetylated GD3, may be associated with the morphological changes observed in this cell line.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of blood-group-ABO(H)-active glycosphingolipids in type-AB human erythrocytes.

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 alpha-N-acetylhexosaminidase or alpha-galactosidase, a branched hybrid-type molecule with both A and B determinants was not detected.

Production of monoclonal antibodies directed to Hanganutziu-Deicher active gangliosides, N-glycolylneuraminic acid-containing gangliosides.

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.

Glycolipid composition of human cataractous lenses. Characterization of Lewisx glycolipids.

We have studied the glycolipid composition of human cataractous lenses. Neutral and acidic lipid fractions were isolated by column chromatographies on DEAE-Sephadex and Iatrobeads. The neutral glycolipid fraction and acidic glycolipid fraction contained 0.6-0.9 micrograms of lipid-bound glucose (Glc) per mg of protein and 0.8-1.3 micrograms of lipid-bound sialic acid (NeuAc) per mg of protein, respectively. The neutral glycolipid fraction was found to contain LacCer (39.0% of total neutral glycolipids), Gb3 (16.2%), Gb4 (1.1%), nLc4 (5.0%), X (29.0%), and Y (9.2%). The acidic lipid fraction was found to contain mainly GM3 (33.1% of the total ganglioside fraction), GM1 (8.3%), LM1 (7.3%), GD1a (16.0%), and G (30.1%). The structures of neutral glycolipids X and Y and ganglioside G were elucidated by high performance thin-layer chromatography overlay method of glycolipids, gas-liquid chromatography, proton NMR spectrometry, and liquid secondary ion mass spectrometry as follows: 1) X, Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1'Cer, III3FucnLc4 (Lex); 2) Y, Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4(Fuc alpha 1- 3)GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1'Cer, V3FucIII3FucnLc6; and 3) G, NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3 Gal-beta 1-4Glc beta 1-1'Cer, IV3NeuAcIII3FucnLc4 (sialosyl-Le(x)). A minor neutral glycolipid Z was isolated and tentatively characterized as GlcNAc beta 1-3?Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1'Cer (GlcNAc-Le(x)), suggesting that it may be the precursor of glycolipid Y. The major long-chain base of these human cataract glycolipids was C18:0 sphingosine (sphinganine). The major fatty acids were C16:0, C24:1 and C24:0, and monounsaturated fatty acids accounted for 40-55% of the total fatty acids.

Characterization of a hamster melanoma-associated ganglioside antigen as 7-O-acetylated disialoganglioside GD3.

We previously reported a hamster animal model of melanoma in which the tumor tissue expresses gangliosides GM3, GD3, and O-acetyl GD3. This ganglioside pattern is similar to that in human melanomas (Ren, S., A. Slominski, and R. K. Yu. 1989 Cancer Res. 49: 7051). In this study, we isolated and purified these gangliosides using chloroform-methanol extraction, Folch partition, chromatographies on DEAE-Sephadex A-25, and Iatrobeads columns. The yields of gangliosides GM3, GD3, and O-acetyl GD3 were 44.1 mg, 19.6 mg, and 9 mg per 100 g of Ma melanotic melanoma tissues, respectively. The structures of these gangliosides were characterized by periodate oxidation, gas chromatographic (GC) analysis, fast-atom bombardment-mass spectrometry (FAB-MS), and nuclear magnetic resonance (NMR) studies. The structure of hamster melanoma O-acetyl GD3 is different from the 9-O-acetyl GD3 previously reported in human melanoma. The major fatty acids of this ganglioside are C16:0, C18:0, C20:0, C22:0, and C24:0 and the long-chain base is C18-sphingosine.

Fucosyl-GM1 in human sensory nervous tissue is a target antigen in patients with autoimmune neuropathies.

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.

Isolated bovine spinal motoneurons have specific ganglioside antigens recognized by sera from patients with motor neuron disease and motor neuropathy.

The gangliosides GM1 and GD1b have recently been reported to be potential target antigens in human motor neuron disease (MND) or motor neuropathy. The mechanism for selective motoneuron and motor nerve impairment by the antibodies directed against these gangliosides, however, is not fully understood. We recently investigated the ganglioside composition of isolated bovine spinal motoneurons and found that the ganglioside pattern of the isolated motoneurons was extremely complex. GM1, GD1a, GD1b, and GT1b, which are major ganglioside components of CNS tissues, were only minor species in motoneurons. Among the various ganglioside species in motoneurons, several were immunoreactive to sera from patients with MND and motor neuropathy. One of these gangliosides was purified from bovine spinal cord and characterized as N-glycolylneuraminic acid-containing GM1 [GM1(NeuGc)] by compositional analysis, fast atom bombardment mass spectra, and the use of specific antibodies. Among seven sera with anti-GM1 antibody activities, five sera reacted with GM1(NeuGc) and two did not. Two other gangliosides, which were recognized by another patient's serum, appeared to be specific for motoneurons. We conclude that motoneurons contained, in addition to the known ganglioside antigens GM1 and GD1b, other specific ganglioside antigens that could be recognized by sera from patients with MND and motor neuropathy.

O-acetylated gangliosides in bovine buttermilk. Characterization of 7-O-acetyl, 9-O-acetyl, and 7,9-di-O-acetyl GD3.

Three O-acetylated gangliosides, G1, G2, and G3, were purified from bovine buttermilk by using chloroform/methanol extraction, Folch partitioning, chromatography on DEAE-Sephadex A-25, and Iatrobeads columns. The final yields of gangliosides G1, G2, and G3 were 2 mg, 37 mg, and 40 mg per 1.7 kg of the buttermilk powder, respectively. On the basis of immunostaining on high performance thin layer chromatography with specific monoclonal antibodies, mild alkaline treatment, gas-liquid chromatographic analysis, fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance studies, G1 and G2 are characterized as O-acetylated GD3 and G3 as O-acetylated GT3, and the structures of these gangliosides are as follows: [formula: see text] The major fatty acids of these gangliosides were C18:0, C22:0, C23:0, and C24:0, and the long chain base was C18-sphingosine.

Blood group A-active glycosphingolipids analysis by the combination of TLC-immunostaining assay and TLC/SIMS mass spectrometry.

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.

Mono-sulfated globotetraosylceramide from human kidney.

A novel sulfated glycosphingolipid that belongs to "globo-series" was isolated from human kidney. This lipid was purified from a pooled kidney preparation by chloroform-methanol extraction, mild alkaline treatment, DEAE-Sephadex and silicic acid column chromatographies, and preparative TLC. The structure and the properties were studied by IR spectroscopy, proton NMR spectroscopy, negative secondary ion-mass spectrometry, solvolysis, periodate oxidation, compositional and methylation analyses, monoclonal antibodies, and a sulfatide-binding protein. From the results of the above analyses, the structure of this glycolipid was proposed to be HSO3-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1ceramide. This sulfated lipid reacted with a monoclonal anti-SSEA-3 (stage-specific embryonic antigen-3) (MC-631) (Kannagi, R., Cochran, N.A., Ishigami, F., Hakomori, S., Andrews, P.W., Knowles, B.B., & Solter, D. (1983) EMBO J. 2, 2355-2361), whose epitope is R-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-R', on TLC and solid-phase radioimmunoassay. This lipid also bound to the 125I-labeled sulfatide-binding protein, thrombospondin. The yield of this sulfated glycolipid was 34 pmol/g of tissue, which was about 0.028, 0.16, and 18 mol% of galactosyl- and lactosylceramide sulfates, and globopentosylceramide sulfate (Nagai, K.-i., Roberts, D.D., Toida, T., Matsumoto, H., Kushi, Y., Handa, S., & Ishizuka, I. (1989) J. Biol. Chem. 264, in press), respectively, in human kidney.