Identification of a novel hepraglycosylceramide with two fucose residues and a terminal hexosamine.

A polar fucose-containing glycosphingolipid fraction isolated from dog small intestine has been characterized by mass spectrometry of intact methylated, and methylated and reduced (LiAlH4) glycolipid. The native fraction, which was homogenous on thin-layer chromatography, was shown after methylation to be a mixture of two compounds. One was identified as a hexaglycoslyceramide with the following composition and sequence: fucose-hexose(fucose)-hexosamine-hexose-hexose-ceramide, with a terminal saccharide structure similar to blood group Leb determinants. The second compound was a novel heptaglycosyceramide with the sequence: hexosamine(fucose)-hexose-tfucose)-hexosamine-hexose-hexose-ceramide. This glycolipid was also detected in human small intestine and pancreas. The dog intestinal fraction had phytosphingosine as its major base and contained almost exclusively 2-hydroxy fatty acids (16 : 0--24 : 0). The fraction of human pancreas differed in having spingosine as its major base and normal fatty acids (16 : 0--24 :0) as major acids.     

Glucosylceramide having a novel tri-unsaturated long-chain base from the spermatozoa of the starfish, Asterias amurensis

Glucosylceramide (Glc beta 1-1Cer) was isolated from the spermatozoa of the starfish, Asterias amurensis. The long-chain bases of the glycolipid consisted of dihydroxy (d18:2, d18:3, d19:3, and d22:2), and trihydroxy (t22:1) types. Long-chain aldehydes derived from them were analyzed mainly by proton nuclear-magnetic resonance to determine the detailed structures. Two of the tri-unsaturated bases were identified as (4E,8E,10E)-2-amino-4,8,10-octadecatriene-1,3-di ol (d18:3) and (4E,8E,10E)-2-amino-9-methyl-4,8,10-octadecatriene+ ++-1,3-diol (d19:3), which is a novel base. Both d22:2 and t22:1 had a cis double bond at the C9 or C13 position. All fatty acids were 2-hydroxylated (C14-C25): Most of them were saturated and unbranched. About 10% was mono-unsaturated and unbranched (C22-C25), while saturated but branched (iso- and anteiso-types) C15-C18 acids were found as minor components. The main fatty acids, which summed up to more than 93% of the fatty acids in the glucosylceramide, were n-14h:0, n-15h:0, n-16h:0, n-17h:0, n-18h:0, and n-24h:1.     

The lipid composition of the electric organ of the ray, Torpedo marmorata, with specific reference to sulfatides and Na+-K+-ATPase.

The lipids from the electric organ of the ray, Torpedo marmorata, have been isolated and characterized. The major lipids were cholesterol, choline phospholipids, ethanolamine phospholipids, and sphingomyelins. The major fatty acids of ethanolamine phospholipids were 18:1, 18:0, 22:6, and 20:4. More than 50% of the acids in choline phospholipids were 16:0. The sphingomyelins consisted of five major ceramide species, all with sphingosine and the fatty acids 14:0, 15:0, 16:0, 22:1, and 24:1. The fatty acid 15:0 was mostly branched (n-2), a fatty acid earlier identified in sphingomyelins of the rectal gland of spiny dogfish. All long-chain bases were dihydroxy bases with a small percentage of branched chains. Sulfatides (cerebroside sulfate) made up the largest glycolipid fraction. The polar moiety wase galactose-3-sulfate. The fatty acids were normal and 2-hydroxy; the homologue 24:1 was the most abundant in both types of fatty acids. Most fatty acids were higher homologues of mono-unsaturated acids, but normal 18:0 fatty acid was also found. The long-chain bases were both dihydroxy and trihydroxy, with very small amounts of branched chains. The two major ceramide species of sulfatides were sphingosine combined with normal and hydroxy 24:1 fatty acids, respectively. Smaller amounts of trihydroxy base (18:0) were found linked to hydroxy 24:1 fatty acid, but not to its normal homologue. The cerebrosides contained the two major species mentioned above but lacked the trihydroxy base-hydroxy fatty acid species. The ratio of the activity of Na+-K+-dependent ATPase (EC 3.6.1.3) and the concentration of sulfatides was similar to ratios found for other tissues with normal and increased Na+ and K+ transporting capacity. The significance of this finding is discussed.     

Main structures of the Forssman glycolipid hapten and a Leb-like glycolipid of dog small intestine, as revealed by mass spectrometry. Difference in ceramide structure related to tissue localization.

Two glycolipids of dog small intestine, one with Forssman activity and one with Leb-like activity, have been characterized by mass spectrometry of methylated, and methylated and reduced (LiAlH4) derivatives. The Forssman glycolipid was conclusively shown to be a pentaglycosylceramide with the carbohydrate sequence hexosamine-hexosamine-hexose-hexose-hexose-ceramide, and with sphingosine (dihydroxy base) as major long-chain base and normal fatty acids as the only fatty acids. The Leb-like glycolipid was a hexaglycosyl-ceramide with sequence fucose-hexose-[fucose-] hexosamine-hexose-hexose-ceramide and with phytosphingosine (trihydroxy base) as major long-chain base and only 2-hydroxy fatty acids as fatty acids. The difference of two hydroxy groups in the ceramide between the two glycolipids may be related to a different tissue localization. As shown by immunofluorescense study the Forssman activity was associated with the lamina propria and the Leb-like activity to the glandular epithelium of dog small intestine.     

The glycosphingolipid receptor for Vibrio trachuri in the red sea bream intestine is a GM4 ganglioside which contains 2-hydroxy fatty acids

Three major glycosphingolipids (tentatively designated IGL-1, 2, and 3) were isolated from the intestine of red sea bream (Pagrus major) and were subjected to a TLC-overlay assay with (35)S-labeled Vibrio trachuri which causes vibriosis of fish. The bacteria adhered to IGL-2, which was determined to be a GM4 ganglioside (NeuAcalpha2-3Galbeta1-ceramide). The fatty acid portion of IGL-2 was composed of 2-hydroxy C22:0, C24:0, and C24:1, in addition to the non-hydroxy C16:0 and C18:0, while the sphingoid base was composed exclusively of sphingenine (d18:1). Among glycosphingolipids tested, V. trachuri adhered to GM4 the most strongly followed by adherence to GM3 and GalCer, but the bacteria did not adhere to GM1a, GM2, LacCer, or GlcCer. V. trachuri was found to aggregate with the erythrocytes coated with GM4, but not with those coated with GM1a or GM2, thus indicating that specific adhesion occurs on intact cells. Interestingly, the dynamics for adhesion of V. trachuri to glycosphingolipids was defined by the structure of not only the sugar moiety but also the ceramide moiety, since the bacteria adhered to GM4 which contained 2-hydroxy fatty acids much more strongly than to that which contained non-hydroxy fatty acids.     

Chemical and biological evidence for base propenals as the major source of the endogenous M1dG adduct in cellular DNA

The endogenous DNA adduct, M(1)dG, has been shown to arise in vitro in reactions of dG with malondialdehyde (MDA), a product of both lipid peroxidation and 4'-oxidation of deoxyribose in DNA, and with base propenals also derived from deoxyribose 4'-oxidation. We now report the results of cellular studies consistent with base propenals, and not MDA, as the major source of M1dG under biological conditions. As a foundation for cellular studies, M1dG, base propenals, and MDA were quantified in purified DNA treated with oxidizing agents known to produce deoxyribose 4'-oxidation. The results revealed a consistent pattern; Fe2+-EDTA and gamma-radiation generated MDA but not base propenals or M1dG, whereas bleomycin and peroxynitrite (ONOO-) both produced M1dG as well as base propenals with no detectable MDA. These observations were then assessed in Escherichia coli with controlled membrane levels of polyunsaturated fatty acids (PUFA). ONOO- treatment (2 mm) of cells containing no PUFA (defined medium with 18:0/stearic acid) produced 6.5 M1dG/10(7) deoxynucleotides and no detectable lipid peroxidation products, including MDA, as compared with 3.8 M1dG/10(7) deoxynucleotides and 0.07 microg/ml lipid peroxidation products with control cells grown in a mixture of fatty acids (0.5% PUFA) mimicking Luria-Bertani medium. In cells grown with linoleic acid (18:2), the level of PUFA rose to 54% and the level of MDA rose to 0.14 microg/ml, whereas M1dG fell to 1.4/10(7) deoxynucleotides. Parallel studies with gamma-radiation revealed levels of MDA similar to those produced by ONOO- but no detectable M1dG. These results are consistent with base propenals as the major source of M1dG in this model cell system.     

Fatty acids and long-chain bases of gangliosides of human gastrointestinal mucosa

The fatty acid and long-chain base composition of five major gangliosides from human stomach and small and large intestine mucosa were analyzed with gas chromatography. All the gangliosides greatly resembled each other in the fatty acid pattern. The main fatty acids were C_16:0, C_18:0 and C_24:0. No hydroxy fatty acids could be detected. In all the gangliosides 4-sphingenine was the predominant long-chain base (70–75%). About 15% of the long-chain bases had 20 carbon atoms in their chain. No trihydroxy long-chain bases could be detected.     

Dietary regulation of mammary lipogenesis in lactating rats.

The proportion of medium-chain fatty acids (C8:0, C10:0 and C12:0) in rat milk increased significantly between day 4 and day 8 of lactation and for the remainder of lactation these acids comprised 40-50mol% of the total fatty acids. The milk fatty acid composition from day 8 was markedly dependent on the presence of dietary fat and altered to include the major fatty acids of the fats (peanut oil, coconut oil and linseed oil). The distribution of fatty acids made within the gland, however, was independent of dietary lipid and C8:0, C10:0 and C12:0 acids accounted for over 70% of the fatty acids made. The rates of lipogenesis in both the mammary gland and liver determined in vivo after the administration of 3H2O were affected by the presence of dietary lipid. In the mammary gland the rate for rats fed a diet containing peanut oil for 7 days was only one fifth that for rats fed a fat-free diet. Coconut oil also suppressed lipogenesis. Both dietary fats also suppressed lipogenesis in the liver.     

Lipids of higher fungi. III. The fatty acids and 2-hydroxy fatty acids in some species of Basidiomycetes

Total fatty acids derived from 12 species of mushrooms were separated into fatty acid and 2-hydroxy fatty acid fractions (FA and HFA), and analyzed quantitatively. The HFA content varied from 0 to 22% of total fatty acids. The major fatty acids were 16:0, 18:0, 18:1, 18:2, and the major 2-hydroxy fatty acids were h16:0, h18:0, h22:0, h24:0. The predominant HFA in the mushrooms investigated had chain-lengths greater than 20 C-atoms, and were derived from sphingolipids — ceramides and cerebrosides.     

Incorporation of exogenous fatty acids into phospholipids by cultured hamster fibroblasts. Effect of SV40 transformation

In situ incorporation of two saturated (palmitic, 16:0; stearic, 18:0) and three unsaturated fatty acids (oleic, 18:1; linoleic, 18:2; arachidonic, 20:4) into the four major phospholipids, sphingomyelin, PC, PI and PE, was followed. Transformed cells incorporated unsaturated fatty acids more rapidly, whereas no significant differences were found concerning saturated fatty acids. In vitro determination of phospholipid acylation showed that incorporation of coenzyme A-activated forms of two saturated fatty acids (16:0 and 18:0) and one unsaturated fatty acid (18:1) into phospholipids was increased in transformed cells. Comparison of results obtained in situ and in vitro strongly suggests that incorporation of fatty acids into phospholipids in cultured cells is not limited by acyltransferase activities.     

Lipids and fatty acids of a moderately halophilic bacterium, No. 101.

The extractable and bound lipids of a moderately halophilic gram-negative rod, strain No. 101 (wild type) grown in a medium containing 2 M NaC1, were examined. The extractable lipids were separated into at least 8 components by using thin-layer chromatography. The major phospholipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and an unidentified phosphoglycolipid in the whole cells, cell envelopes and outer membrane preparations, commonly. Judging from mild alkaline hydrolysis and exzymatic treatment with phospholipase A2, C and D, the unidentified phosphoglycolipid possessing Pi, glycerol, fatty acids and glucose in a molar ratio of 1 : 2 : 2 : 1, appeared likely to be a glucosyl derivative of phosphatidylglycerol. No glucuronic acid containing lipid was detected. The exractable lipid composition varied greatly with the concentrations of NaC1 in the medium and the stages of bacterial growth. The most characteristic phosphoglycolipid in this organism increased up to 25% of the total phospholipids with the addition of 1% glucose in the medium. The major fatty acids of the extractable lipids were C16:0, C16:1, C18:0, C18:1 and cyclopropanoic C17 and C19 acids and these compositions were very similar for each phospholipid. The cyclopropanoic fatty acids predominated as growth proceeded. The fatty acids of the bound lipids comprised a high concentration of 3-hydroxydodecanoic acid. The esterified fatty acids of the lipopolysaccharide molecule seemed to contain a wide variety of hydroxy and non-hydroxy shorter chain fatty acids, while the amide-linked fatty acids consisted almost entirely of 3-hydroxydodecanoic acid.     

Analysis of Glucocerebrosides of Rye (Secale cereale L. cv Puma) Leaf and Plasma Membrane

Glucocerebrosides of whole rye (Secale cerale L. cv Puma) leaf and plasma membrane were analyzed using gas chromatography-mass spectrometry and gas chromatography following hydrolysis or as intact molecules purified by reverse-phase high performance liquid chromatography. Fatty acids of acid-hydrolyzed leaf and plasma membrane glucocerebrosides consisted of >98 weight percent saturated and monounsaturated 2-hydroxy fatty acids which contained 16 to 26 carbon atoms. The major fatty acids detected were 2-hydroxynervonic acid (24:1h), 2-hydroxylignoceric acid (24:0h), 2-hydroxyerucic acid (22:1h), and 2-hydroxybehenic acid (22:0h). Long-chain bases of alkaline-hydrolyzed glucocerebrosides consisted primarily of cis-trans isomers of the trihydroxy base 4-hydroxysphingenine (t18:1) and the dihydroxy base sphingadienine (d18:2) with lesser amounts of 4-hydroxysphinganine (t18:0) and isomers of sphingenine (d18:1). Intact, underivatized glucocerebroside molecular species of rye leaf and plasma membrane were separated into more than 30 molecular species using reverse-phase HPLC. The molecular species composition of leaf and plasma membrane were quantitatively and qualitatively similar. The major molecular species was 24:1h-t18:1 which constituted nearly 40 weight percent of leaf and plasma membrane extracts. Several other species including 22:1h-t18:1, 24:1h-t18:1 (isomer), 22:0h-t18:1, 24:1h-d18:2, and 24:0h-t18:1 each comprised 4 to 8% of the total. It is anticipated that the high performance liquid chromatography procedure developed in this study to separate intact, underivatized lipid molecular species will be useful in future studies of the physical properties and biosynthesis of plant glucocerebrosides.     

Reliable identification of Prevotella and Butyrivibrio spp. from rumen by fatty acid methyl ester profiles

Data for bacterial identification were provided by culturing anaerobic bacteria under standardized conditions followed by extraction and methylation of cellular long-chain fatty acids and gas chromatographic analysis. The databases of fatty acid methyl ester (FAMEs) profiles for two predominant ruminal genera,Prevotella andButyrivibrio, were created. Major long-chain cellular fatty acids found in the 23 analyzedPrevotella strains were 15:0 (anteiso), 15:0, 15:0 (iso) and 16:0. The strains ofPrevotella could be well identified on species level by the characteristic ratios among major fatty acids and by acids unique fatty for each species. The 45Butyrivibrio strains were grouped into 4 major and 2 minor groups according to FAMEs profiles. The major fatty acids for the bulk of theButyrivibrio strains were 14:0, 15:1, 16:0 and 16:0 (iso). This groups corresponded to those based on 16S rDNA sequences.     

Ceramides of human normal and cataractous lens.

Ceramides were quantitatively isolated from human normal and cataractous lens by solvent extraction, silicic acid chromatography, thin-layer chromatography, and gas-liquid chromatography. Only two species of ceramides with normal fatty acids were detected. In the mature cataracts, there was an increase in palmitate and nervonate at the expense of the other fatty acids. Due to the increase of 24 : 1, the ratio of 24 : 1/24 : 0 increased significantly from normals to cataracts. Sphinganine was the major long-chain base, but 4-sphingenine was also present. The total amount of ceramides in the immature and mature cataracts was 1.8 and 3.0 times higher than the normals of the same age group. Such an increase does not seem to be the result of an age-dependent process.     

Occurrence of long-chain fatty acids and glycolipids in the cell-envelope fractions of baker''s yeast

1. The total yield of fatty acids from the whole envelopes was markedly higher than that obtained from the ordinary cell walls. In both samples the major fatty acids were C(16) and C(18) acids. 2. The whole envelopes contained C(18) acids and long-chain (C(19)-C(26)) fatty acids, in a higher proportion than did the ordinary cell walls. Fifteen fatty acids with more than 18 carbon atoms were identified, among which 2-hydroxy-C(26:0) and C(26:0) acids predominated. 3. A complex sphingolipid containing inositol, phosphorus and mannose was isolated from the whole cell envelopes. The main fatty acids of this lipid were 2-hydroxy-C(26:0) and C(26:0) acids. It was concluded that this sphingolipid is present both in the ordinary cell wall and in the plasma membrane of baker's yeast. 4. The neutral lipids amounted to over 50% and the glycerophosphatides to about 30% of the total fatty acid content of the whole envelope. The major fatty acids in these lipids were C(16:1), C(18:1) and C(16:0) acids. The proportion of fatty acids with more than 18 carbon atoms was lowest in the neutral lipids, whereas the neutral glycolipids contained the highest percentage of these fatty acids. Acidic glycolipids amounted to 14% of the total fatty acid content of the whole envelope. The presence of a cerebroside sulphate in this lipid fraction was demonstrated, whereas the high content of 2-hydroxy-C(26:0) acid found is caused by the complex inositol- and mannose-containing sphingolipid.     

Isolation and characterization of a neutral glycosphingolipid from the epimastigote form of Trypanosoma mega

A glycosphingolipid fraction from Trypanosoma mega was isolated after acetylation and was further purified on a silicic acid column. Final purification was by preparative thin-layer chromatography. The carbohydrate components of the glycolipid were fucose and galactose in approximately equimolar amounts. The neutral glycolipid of T. mega has a sphingosine base composition that consists of sphingosine and traces of dihydrosphingosine. Fatty acids forming amide groups with the sphingosine bases were analyzed by gas-liquid chromatography-mass spectrometry and are a mixture of normal and alpha-hydroxy fatty acids. Normal C16:0, C18:0, and 2-hydroxy C18:0 are the predominant fatty acids.     

Structural components of sphingophosphonolipids from the ciliated protozoan, Tetrahymena pyriformis WH-14.

1. Two sphingophosphonolipids were isolated from the lipids of the ciliated protozoan, Tetrahymena pyriformis WH-14. They were ceramide N-methyl-2-aminoethylphosphonate (CMAEP) and ceramide 2-aminoethylphosphonate (CAEP), in yields of 0.05 mg/g and 1.74 mg/g dry cells, respectively. 2. Two chromatographically distinguishable CAEP species were found, a slow-moving major component and a minor component which moved faster; the slow-moving one contained primarily hydroxy fatty acids, while in the other one nonhydroxy fatty acids were predominant. However, their long-chain base constituents were similar. 3. The major fatty acids of CAEP were 2-hydroxy acids with carbon numbers of 16 to 19, which were almost exclusively iso-types. The fatty acids of CMAEP consisted mainly of palmitic, iso-octadecanoic, and 2-hydroxy iso-heptadecanoic acids. 4. The long-chain bases were dominated by C16, C17, and C19 iso-4-sphingenine homologs.     

Effect of growth phase on the content and composition of ceramides of the hydrocarbon-assimilating yeast Candida lipolytica.

Candida lipolytica yeast was grown batchwise on n-hexadecane as the carbon and energy source. Ceramides were quantitatively isolated from total lipids of exponential and stationary phase cells by a combination of column chromatography and preparative high-performance thin-layer chromatography. After acid methanolysis their composition was analyzed by gas-liquid chromatography. The ceramide content of the exponential phase cells was two times higher than the one of the stationary phase cells. The composition of long-chain base moiety of ceramides did not change significantly during the growth. In both growth phases 19-phytosphingosine was the major long-chain base. However, the fatty acid composition of ceramides changed greatly during the growth. In the exponential growth phase, ceramides contained predominantly fatty acids greater than 20 carbon atoms, while fatty acids shorter than 20 atoms predominated in ceramides of the stationary phase, 16:0 being the main one. In the exponential growth phase fatty acid moiety of ceramides was characterized by unusually high degree of unsaturation and relatively high proportion of odd-numbered fatty acids. However, the proportion of both, unsaturated and odd-numbered fatty acid decreased significantly in ceramides of the stationary phase. The unexpected finding was the absence of fatty acid hydroxylation of ceramides in the exponential phase cells and unusually low degree of hydroxylation in the stationary phase.     

Differentiation of free-living Anabaena and Nostoc cyanobacteria on the basis of fatty acid composition.

The cellular fatty acids of free-living, nitrogen-fixing cyanobacteria belonging to the genera Anabaena and Nostoc were analyzed to differentiate the genera. The fatty acid compositions of 10 Anabaena strains and 10 Nostoc strains that were grown for 12 days on BG-11o medium were determined by gas-liquid chromatography-mass spectroscopy. Of the 53 fatty acids detected, 17 were major components; the average level for each of these 17 fatty acids was at least 0.9% of the total fatty acids (in at least one of the genera). These fatty acids included (with mean percentages in the Anabaena and Nostoc strains, respectively) the saturated fatty acids 16:0 (30.55 and 23.23%) and 18:0 (0.77 and 1.27%); several unsaturated fatty acids, including 14:1 cis-7 (2.50 and 0.11%), 14:1 cis-9 (3.10 and 3.41%), a polyunsaturated 16-carbon (sites undetermined) fatty acid with an equivalent chain length of 15.30 (1.20 and 1.03%), 16:4 cis-4 (0.95 and 0.87%), 16:3 cis-6 (2.16 and 1.51%), 16:1 cis-7 (1.44 and 0.36%), 16:1 cis-9 (6.53 and 18.76%), 16:1 trans-9 (4.02 and 1.35%), 16:1 cis-11 (1.62 and 0.42%), 18:2 cis-9 (10.16 and 12.44%), 18:3 cis-9 (18.19 and 17.25%), 18:1 cis-9 (4.01 and 5.10%), and 18:1 trans-9 (0.92 and 1.94%); and the branched-chain fatty acids iso-16:0 (2.50 and 1.14%) and iso-15:1 (0.34 and 2.05%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereospecific analysis of major glycerolipids of Phycomyces blakesleeanus sporangiophores and mycelium.

The positional distribution of fatty acids was determined in the major groups of glycerolipids from the mycelium and sporangiophores of the fungus Phycomyces blakesleeanus. At the sn-1 positions of the triacylglycerols, in both regions of the fungus, greater than 65% of the fatty acids were 16:0 and 18:1. At the sn-2 positions of the triacylglycerols, 18:1, 18:2 and 18:3 comprised greater than 85% of the sporangial fatty acids and more than 90% of the mycelial fatty acids. Positions sn-3 of the triacylglycerols, from both regions of the fungus, contained approximately 40% of 16:0, approximately 30% of 18:2, and the largest proportions of 18:3 (21%) in the triacyglycerols. The major phosphoglycerides of P. blakesleeanus mycelium and sporangiophores are phosphatidylcholine and phosphatidylethanolamine, and more than 85% of the fatty acids at the sn-1 positions of these phosphatides consisted of 16:0, 18:2, and 18:3. The sn-2 positions of phosphatidylcholine and phosphatidylethanolamine contained approximately 98% unsaturated fatty acids. In the phosphoglycerides of both regions of the fungus, 18:2 and 18:3 constituted greater than 85% of the total fatty acids. Although the mycelium and sporangiophores of P. blakesleeanus had different morphological and physiological characteristics, the major glycerolipids of the two regions had similar stereospecific distributions of fatty acids.