Structure of Leishmania mexicana lipophosphoglycan.

Lipophosphoglycan (LPG) was isolated from the culture supernatant of Leishmania mexicana promastigotes and its structure elucidated by a combination of 1H NMR, fast atom bombardment mass spectrometry, methylation analysis, and chemical and enzymatic modifications. It consists of the repeating phosphorylated oligosaccharides PO4-6Gal beta 1-4Man alpha 1- and PO4-6[Glc beta 1-3]Gal beta 1-4Man alpha 1-, which are linked together in linear chains by phosphodiester linkages. Each chain of repeat units is linked to a phosphosaccharide core with the structure PO4-6Gal alpha 1-6Gal alpha 1-3Galf beta 1- 3[Glc alpha 1-PO4-6]Man alpha 1-3Man alpha 1-4GlcNH2 alpha 1-6 myo-inositol, where the myo-inositol residue forms the head group of a lyso-alkylphosphatidylinositol moiety. The nonreducing terminus of the repeat chains appear to be capped with the neutral oligosaccharides Man alpha 1-2Man, Man alpha 1-2Man alpha 1-2Man, or Man alpha 1-2[Gal beta 1-4]Man. Cellular LPG, isolated from promastigotes, has a very similar structure to the culture supernatant LPG. However, it differs from culture supernatant LPG in the average number of phosphorylated oligosaccharide repeat units (20 versus 28) and in alkyl chain composition. Although culture supernatant LPG contained predominantly C24:0 alkyl chains, cellular LPG contained approximately equal amounts of C24:0 and C26:0 alkyl chains. It is suggested that culture supernatant LPG is passively shed from promastigotes and that it may contribute significantly, but not exclusively, to the "excreted factor" used for serotyping Leishmania spp. Comparison of L. mexicana LPG with the LPGs of Leishmania major and Leishmania donovani indicate that these molecules are highly conserved but that species-specific differences occur in the phosphorylated oligosaccharide repeat branches and in the relative abundance of the neutral cap structures.     

Fast atom bombardment-mass spectrometric identification of molecular species of platelet-activating factor produced by stimulated human polymorphonuclear leukocytes

Fast atom bombardment mass spectrometry was used to identify molecular species of platelet-activating factor (PAF) produced by stimulated human neutrophilic polymorphonuclear leukocytes. Normal and reverse-phase high performance liquid chromatography were employed to separate the individual regions with PAF activity prior to mass spectrometric analysis. The following alkyl chain homologs of acetyl glyceryl ether phosphorylcholine (AGEPC) were found: C16:0, C17:0, C18:0 and C18:1. There was also evidence for the presence of the C15:0 homolog, as well as other species which have not yet been identified.     

Human spermatozoa produce C16-platelet-activating factor.

Recent data indicate that human spermatozoa produce platelet-activating factor as determined by the rabbit platelet [3H]serotonin release bioassay. In this report, we examined by fast atom bombardment/mass spectrometry the molecular species of platelet-activating factor generated by these germ cells. Extracted spermatozoal samples that contained platelet-activating factor bioactivity underwent straight-phase high-performance liquid chromatography, and fractions which coeluted with authentic C16- and C18-platelet-activating factor standards were subjected to fast atom bombardment/mass spectrometry. Our mass spectral data indicate that human spermatozoa synthesize C16-platelet-activating factor but not C18-platelet-activating factor.     

Synthesis, characterization, DNA binding study and biological activity against Leishmania mexicana of [Cu(dppz)2]BF4

[Cu(dppz)(2)]BF(4) complex has been synthesized by the reaction of [Cu(CH(3)CN)(4)]BF(4) and dipyrido[3,2-A:2',3'-c]phenazine (dppz) in a molar ratio of 1:2. The compound was characterized by fast atom bombardment mass spectrometry, 1H nuclear magnetic resonance, UV-Vis and IR spectroscopies. Absorption and viscometric studies carried out on the interaction of [Cu(dppz)(2)]BF(4) complex with calf thymus DNA suggested that the complex binds by intercalation. No covalent binding was observed. Additionally, the results obtained from electrophoresis showed nuclease activity. The biological activity of the complex was tested in vitro on Leishmania mexicana promastigote cultures. A leishmanicidal effect (LD(30)) was observed in 48 h at concentration of 41 nM. Preliminary studies of the ultrastructure of L. mexicana treated with a sublethal dose of the complex (IC(7)=4.1 nM) for 48 h showed an induction of cytoplasm disorganization, vacuolization and binucleated cells. These findings suggest that the leishmanicidal activity of the title complex could be associated with its interaction with the parasitic DNA.     

Characterization of glycosyl phosphatidylinositol lipids of parasitic protozoans: Leishmania mexicana mexicana promastigotes, Trypanosoma cruzi Peru epimastigotes and Tritrichomonas foetus

Glycosyl phosphatidylinositol lipids of cultured L.mex, mexicana LV732 promastigotes, T. cruzi Peru epimastigotes and Tritrichomonas foetus have been isolated and characterized using metabolic labelling and chromatographic and mass spectrometric (MS) techniques. TLC of the unsaponifiable lipid fractions of L. mex. mexicana and T. cruzi obtained from DEAE Sephadex A-25 followed by Iatrobead column chromatography showed three inositol phosphate-containing lipid components. [3H]myo-inositol, [3H]palmitic acid or H3 32PO4 lipid precursors were incorporated into these three lipid components. Fraction 2 (LM2 and TCP-2) comprises inositol phosphate ceramides. The other two fractions appear to contain mono-O-alkyl and di-O-alkyl glycerol inositol phosphates. Lyso-1-O-alkyl phosphatidylinositols could be cleaved by treatment of PI-specific phosphalipase C. The di-O-alkyl-phospho inositols of these parasites being the first dialkylglycerol lipids reported from eukaryotic membranes raises the possibility of chemotherapy for leishmaniasis and trypanosomiasis based upon functional impairment of alkyl ether lipids. Tritrichomonas foetus contains two major glycophosphosphingolipids, designated TF1 and TF2, which are metabolically labelled with [3H]myo-inositol and H3 32PO4. Both lipids contained ceramides. The major ceramide contains the 18:0 and 18:1 bases and 16:0 N-acyl group. The major glycolipid fraction (TF1) contains fucose linked to inositol diphosphate; one of the phosphates being linked to the ceramide moiety, and the other to ethanolamine. TF1 appears to be a novel class of glycophosphosphingolipid, which may be a part of a membrane anchor.     

Human medulloblastoma gangliosides

To establish a model system for the study of ganglioside metabolismof the human brain tumor, medulloblastoma, we have chemicallycharacterized the gangliosides of the Daoy cell line. Thesecells contain a high concentration of gangliosides (143 ±13 nmol LBSA/10⁸ cells). The major species have been structurallyconfirmed to be G_M2 (65.9%), G_M3 (13.0%), and G_Dla (10.3%).Isolation of individual gangliosides homogeneous in both carbohydrateand ceramide moieties by reversed-phase HPLC and analysis bynegative-ion fast atom bombardment collisionally activated dissociationtandem mass spectrometry have allowed us to unequivocally characterizeceramide structures. In the case of G_M2, 10 major ceramide subspecieswere identified: d18:1-hC16:0, d18:1-C16:0, d18:0-C16:0, d18:1-C18:0,d18:1-C20:0, d18:1-C22:0, d18:2-C24:1, d18: 1-C23:1, d18:1-C24:1,and d18:1-C24:0. Taken together with previous studies, thesefindings in human medullo-blastoma cells support the view thathigh expression and marked heterogeneity of ceramide structureare general characteristics of tumor gangliosides, moleculeswhich are shed by the tumor cells and which are biologicallyactive in vivo. medulloblastoma gangliosides ceramide structure HPLC mass spectrometry     

THE MAIN NONPOLAR CHLOROPHYLL c FROM EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) IS A CHLOROPHYLL c 2-MONOGALACTOSYLDIACYLGLYCERIDE ESTER: A MASS SPECTROMETRY STUDY

The main nonpolar chlorophyll c  -like pigment was extracted from Emiliania huxleyi (Lohm.) Hay et Mohler (strain CCMP 370) cultures and isolated by preparative column chromatography and HPLC. The pigment, whose visible spectrum closely resembled that of chlorophyll c  ₂, was studied by low-resolution fast atom bombardment mass spectrometry, showing a very high mass molecular ion (m/z 1313). The fragment ions, either in the direct spectrum or obtained by tandem mass spectrometry with collision-induced dissociation of the molecular ion, were compatible with the consecutive losses of two fatty acids (14:0 and 18:4), glycerol, and a hexose, leaving a chlorophyll c  ₂ backbone, suggesting the molecule consists of a chlorophyll c  ₂ residue linked, via an ester bond, to the sugar moiety of a monohexosyldiacylglycerol. The identities of the two fatty acid residues (14:0 and 18:4n-3) were subsequently corroborated by gas chromatography of the corresponding methyl esters. Chemical hydrolysis–derivatization–gas chromatography–mass spectrometry demonstrated the occurrence of glycerol and that galactose is the constituent sugar. The porphyrin obtained on acid hydrolysis showed chromatographic and visible spectral properties identical to pheoporphyrin c  ₂. This evidence led us to propose a tentative structure whose molecular formula, C₇₆H₉₆O₁₄N₄Mg, was supported by the values of exact mass measurements by high-resolution fast atom bombardment mass spectrometry. This novel structure represents the highest molecular weight natural chlorophyll described to date.     

Novel modification of glycosphingolipids by long-chain cyclic acetals: isolation and characterization of plasmalocerebroside from human brain.

A glycosphingolipid component of human brain, having long-chain cyclic acetals, has been isolated and characterized. This compound incorporates a novel type of natural glycan modification, in which a long-chain aliphatic aldehyde is conjugated through a cyclic acetal (plasmal) linkage to the galactosyl moiety of cerebroside. In addition to components normally observed by gas chromatography-mass spectrometry (GC-MS) following methanolysis of cerebroside (fatty acid methyl esters, methyl alpha- and beta-galactosides, sphingosine), this compound produced 16:0, 18:0, and 18:1 fatty aldehydes, unequivocally identified as their enol methyl ether derivatives. Results of positive ion fast atom bombardment mass spectrometry (FAB-MS) of the native compound, and GC-MS of partially methylated hexitol acetates derived from the permethylated derivative, were consistent with structures of galactocerebroside having 3,4- and 4,6-linked cyclic plasmal substituents, as shown. [formula: see text]     

Purine phosphoribosyltransferases of Leishmania mexicana mexicana and other flagellate protozoa

Amastigotes and cultured promastigotes of Leishmania mexicana mexicana and L. m. amazonensis, cultured promastigotes of L. donovani and L. tarentolae, and the culture forms of Crithidia fasciculata, Herpetomonas muscarum muscarum and H. m. ingenoplastis all possessed four phosphoribosyltransferase (PRTase) activities: adenine PRTase, hypoxanthine PRTase, guanine PRTase and xanthine PRTase. The enzymes of L. m. mexicana required divalent cations for activity; Mn2+ or Co2+ produced maximal activity in most cases. Hypoxanthine PRTase, guanine PRTase and xanthine PRTase from all organisms were sedimentable in part, suggesting that they may occur within glycosomes. The enzymes of L. m. mexicana cultured promastigotes were inhibited by a range of purine analogues.     

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.     

Lipid composition of PC12 pheochromocytoma cells: characterization of globoside as a major neutral glycolipid

We have studied the lipid composition of PC12 pheochromocytoma cells cultured in the presence and absence of nerve growth factor (NGF). Neutral and acidic lipid fractions were isolated by column chromatography on DEAE-Sephadex and analyzed by high-performance thin-layer chromatography (HPTLC). The total lipid concentration was approximately 220 micrograms/mg of protein, and the concentration of neutral glycolipids was 1.6-1.8 microgram/mg of protein for both NGF-treated and untreated cells. The neutral glycolipid fraction contained a major component, which accounted for approximately 80% of the total and which was characterized as globoside on the basis of HPTLC mobility, carbohydrate analysis, fast atom bombardment mass spectrometry, and mild acid hydrolysis. The major fatty acids of globoside were C16:0 (10%), C18:0 (16%), C22:0 (23%), C24:1 (17%), and C24:0 (24%). C18 sphingenine accounted for almost all of the long-chain bases. The other neutral glycolipids were tentatively identified as glucosylceramide (15%), lactosylceramide (4%), and globotriosylceramide (4.5%). The concentration of ganglioside sialic acid was approximately 0.34 and 0.18 microgram/mg of protein for cells grown in the presence and absence of NGF, respectively. Although there was an increase in ganglioside concentration in NGF-treated cells, NGF did not produce any differential effects on the relative proportions of the individual gangliosides. Several of the gangliosides appear to contain fucose, and one of these was tentatively identified as fucosyl-GM1. Brain-type gangliosides of the ganglio series were also detected by an HPTLC-immunostaining method. However, the fatty acid and long chain base compositions of PC12 cell gangliosides (and their TLC mobility) differ from those of brain gangliosides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of the major acidic glycosphingolipids from the liver of the English sole (Parophrys vetulus). Presence of a novel ganglioside with a Forssman antigen determinant

Acidic glycosphingolipids of the liver of English sole, Parophrys vetulus, have been isolated and characterized by 1H nuclear magnetic resonance spectroscopy, methylation analysis, and by direct probe electron-impact and fast atom bombardment mass spectrometry. In addition to the acidic glycosphingolipids with known structures (sulfatide, GM4, GM3, GM2, and GD1a), two fractions of a major monosialosylganglioside with TLC mobility slower than GM1 were isolated and characterized as having the following structure. (Formula:q see text). The structure represents a novel combination of a terminal Forssman disaccharide (GalNAc alpha 1----3GalNAc beta 1----3R) and a GM1 ganglioside core linked together. The identity of the terminal Forssman disaccharide was further established by TLC immunostaining with an anti-Forssman monoclonal antibody. This antibody showed strongly positive staining of the ganglioside only after removal of the sialic acid. Thus, the II3NeuAc residue inhibited antibody binding to the terminal disaccharide unit. Analysis of the ceramide moieties of both fractions indicated a predominance of 16:0, 22:1, 22:0, and 24:1 fatty acids in the faster migrating form and 16:0, 18:0, and 18:1 in the slower form in combination with d18:1 sphingosine.     

Synthesis and characterization of [Au(dppz)2]Cl3. DNA interaction studies and biological activity against Leishmania (L) mexicana

[Au(dppz)(2)]Cl(3) was synthesized by the reaction of HAuCl(4) in excess of the dypirido[3,2-a: 2,3-c]phenazine (dppz) ligand. This complex was characterized by elemental analysis, fast atom bombardment (FAB) mass, NMR, UV-visible and IR spectroscopies. DNA-gold complex interactions were studied by spectroscopic titrations, viscosity measurements and electrophoretical assays. These studies showed that the gold complex interacts with DNA by intercalation mode. These observations, led us to carry out biological tests on cultures of promastigotes of Leishmania (L) mexicana. [Au(dppz)(2)]Cl(3) induced a dose dependent antiproliferative activity with minimal inhibitory concentration (MIC) of 3.4nM and lethal doses LD(26) of 17nM for 48h. These findings suggest that a very potent leishmanicidal activity could be associated to the cellular processes involving parasite DNA, constituting a new promising chemotherapeutic alternative in the search for definitive leishmaniasis cure.     

Syngeneic monoclonal antibody against melanoma antigen with interspecies cross-reactivity recognizes GM3, a prominent ganglioside of B16 melanoma

It has previously been reported that a mouse (C57BL/6) monoclonal antibody, M2590, was established against syngeneic melanoma B16 cells, which was shown to react only with melanoma cells from various species but not with other tumor cells or normal tissues (Taniguchi, M., and Wakabayashi, S. (1984) Gann 75, 418-426). In the present study, the specificity of M2590 antibody was shown to be directed to a saccharide arrangement (NeuAc alpha 2-3Gal beta 1-4Glc (or -GlcNAc)) of gangliosides by three different assay systems including enzyme immunostaining on thin layer plates, sandwich radioimmunoassay, and enzyme-linked immunoadsorbent assays using a variety of glycolipids with known structures. Neither gangliosides having NeuGc terminus, including NeuGc alpha 2-3Gal beta 1-4Glc-ceramide and NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-ceramide, nor ganglio series gangliosides carrying NeuAc reacted with the antibody. An M2590 antibody-reactive antigen was isolated from B16 melanoma cells, and its structure was determined to be NeuAc alpha 2-3Gal beta 1-4Glc-ceramide by fast atom bombardment mass spectrometry, methylation analysis, and exoglycosidase treatment. The ceramide was composed of d18:1 as its long-chain base and C16:0, C24:1, and C24:0 as major fatty acids. The same ganglioside was also detected in the culture supernatant of the melanoma cells as shedding antigen.     

Aberrant fatty acyl alpha-hydroxylation in human neuroblastoma tumor gangliosides

Abnormalities of ganglioside structure characterize the neoplastic state, and aberrant glycosylation has been implicated as underlying many new tumor ganglioside structures. However, variations in ceramide structure can also result in novel tumor gangliosides. To address systematically this aspect of ganglioside metabolism, we have initiated a study of the structures of the ceramide species of an oligosaccharide-homogeneous human tumor-derived ganglioside, GM2. The ganglioside was isolated from neuroblastoma tissue and purified by normal-phase high pressure liquid chromatography. Marked ceramide heterogeneity was observed; 18 individual ceramide species of neuroblastoma GM2 were separated by reversed-phase high pressure liquid chromatography and collected. Their structures were determined by a combination of negative- and positive-ion fast atom bombardment mass spectrometry and collisionally activated dissociation tandem mass spectrometry of the underivatized gangliosides. The striking finding was the detection of alpha-hydroxylation of a significant fraction of each of the major fatty acid species (16:0, 18:0, 20:0, 22:0, and 24:1); alpha-hydroxylated species quantitatively represented almost one-fifth of the total tumor GM2 species. Fatty acyl hydroxylation was also detected in the ceramide of several other human tumor gangliosides. In contrast, as previously known, fatty acyl hydroxylation was not detected in the normal human brain gangliosides GM3, GM2, and GM1. We propose that aberrant fatty acid alpha-hydroxylation is a novel and sometimes quantitatively significant characteristic of human tumor ganglioside metabolism.     

Structural identification of glycerolipid molecular species isolated from cyanobacterium Synechocystis sp. PCC 6803 using fast atom bombardment tandem mass spectrometry

Our previous works have demonstrated that fast atom bombardment tandem mass spectrometry can be a valuable tool in determining the complete structure of glycoglycerolipids and glycerophospholipids. Collision-induced dissociation of sodium-adducted molecular ions ([M + Na]+ or [M - H + 2Na]+) generates diverse product ions informative on the double-bond position in fatty acyl groups as well as the polar head group and fatty acid composition. Here we report that this direct and rapid method can be applied to the structural determination of individual molecular species of each glycerolipid class purified from the total lipid extract of cyanobacterium Synechocystis sp. PCC 6803. Especially, based on the preference for the loss of the fatty acyl group positioned at the sn-2, it was proved that all of the molecular species of diacylglycerolipids contained a palmitoyl group exclusively at the sn-2 position. Additionally, lysoglycerolipids, monoacyl forms of four major membrane diacylglycerolipids, were first isolated together from a fresh extract. Using fast atom bombardment mass spectrometry and tandem mass spectrometry, it was found that each lysoglycerolipid had a molecular species with only palmitic acid as a fatty acyl group. Thus, these compounds could be synthesized by specific enzyme-catalyzed hydrolysis of the sn-1 fatty acyl group of the corresponding diacylglycerolipids.     

Comparison of 252californium plasma desorption and fast atom bombardment mass spectrometry for analysis of small peptides

The data obtained with 252Cf plasma desorption (PD) and fast atom bombardment mass spectrometry of eight tri-, tetra- and pentapeptides were compared. Good spectra were obtained with 1-10 nmol of peptide. In both techniques molecular weight information was obtained. The PD mass spectra are often dominated by the cationized molecular ions in contrast to the fast atom bombardment (FAB) mass spectra, where cationization is rarely observed. Amino acid content is reflected in the immonium ions equally well in both techniques. The fragmentation patterns observed with the two techniques are almost identical. However, practical sequencing of peptides based on either FAB or PD mass spectrometry of underivatized peptides alone is difficult. This is due to the unpredictable and sometimes absent cleavage yield at certain peptide bonds. Another difficulty is the many simultaneous fragmentation pathways. However, for many peptides enough information is present to allow sequence determination for at least a major part of the molecule.     

Gangliosides from the eggs of the sea urchin, Anthocidaris crassispina

NeuGc alpha 2-6Glc beta 1-1Cer (M5 ganglioside) and HSO3-8NeuGc alpha 2-6Glc beta 1-1Cer (T1 ganglioside) were purified by column chromatographies with DEAE-Sephadex A-25 and silicic acid from the eggs of the sea urchin, Anthocidaris crassispina. Their chemical structures were determined by gas-liquid chromatography, methylation analysis, enzymatic hydrolysis, negative-ion fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectroscopy. Long-chain base compositions of both gangliosides were almost identical: all the long-chain bases were phytosphingosines, and C18-phytosphingosine accounted for more than 95% of them. Fatty acid compositions were also very similar: the main fatty acids were 22:1, 23:1, 24:1, and their 2-hydroxylated forms, and the 2-hydroxy fatty acids amounted to 65.3 and 74.3% of the fatty acids in M5 and T1 gangliosides, respectively. Proton nuclear magnetic resonance spectroscopic study revealed a downfield-shifted H8 proton signal of NeuGc residue in T1 ganglioside, in agreement with the presence of sulfate ester at the C8 position.     

Sulfogalactosylceramides in motor and psycho-cognitive adult metachromatic leukodystrophy: relations between clinical, biochemical analysis and molecular aspects

Metachromatic leukodystrophy (MLD) is a human autosomal recessive lysosomal neurodegenerative disorder that results from the accumulation of sulfatides in the central and peripheral nervous system. It is due to the enzyme deficiency of the sulfatide sulfatase i.e. arylsulfatase A (ASA). During adolescence and/or adulthood, there are 2 clinical presentations. It may be that of a degenerative disease of the central nervous system with mainly spastic manifestations or a spino-cerebellar ataxia, or that of a psychosis. As several lines of evidence indicate that the psychotic form of MLD could be a model of psychosis, we decided to do a pluridisciplinary study on 11 psycho-cognitive cases involving mental and psychiatric testing, in comparison with 5 adult motor cases, a biochemical study with enzyme assays and quantitative mass spectrometry of urinary sulfatides, so as to determine whether there were biochemical particularities related to the psychotic forms. For quantitative mass spectrometry (MS), a non physiological sulfatide with C17:0 fatty acid was synthesized. The major sulfatide isoforms were present in the 2 clinical forms with the following fatty acids and sphingoid bases: C22:1/d18:1, and /or C22:0/d18:2 (m/z 862.5), C22:0 (OH)/d18:1 (m/z 878,5), C24:0/d18:1 and / or C24:0/C23:1(OH)/d18:2 (m/z 890,3), C24:0 (OH)/d18:1(m/z 906.5). We had shown previously that there were different ASA mutations in the psychiatric adult form (heterozygous I179S) versus the adult motor form (homozygous P426L). We show here that there were no relations with the level of ASA and with the mass spectrometric study of the sulfatide isoforms which were identical in the 2 clinical forms.     

Cerebroside of the dimorphic human pathogen, Candida albicans

Structural studies on the cerebroside isolated from the yeast form of a dimorphic pathogen, Candida albicans were carried out using fast atom bombardment mass spectrometry (FAB/MS), proton magnetic resonance spectrometry, gas chromatography-mass spectrometry and usual chemical methods. The component sugar was only glucose attached to ceramide in a beta-configuration. The major fatty acid was 2-hydroxystearic acid (62%). The predominant long chain base was identified as 9-methyl-C18-sphinga-4,8-dienine which is widely distributed in fungi and reported to be essential to the fruit-inducing activity of fungi. Therefore, the structure of the main molecular species of the cerebroside was determined to be N-2-hydroxystearoyl-1-O-beta-glucosyl-9-methyl-C18-sphinga-4 ,8-dienine. Cerebroside prepared from the mycelial form of C. albicans has the same structure.