The aminoethylphosphonate-containing lipids of rumen protozoa

1. A method is presented for identifying and estimating the aminoethylphosphonate (ciliatine)-containing phospholipids in a complex mixture. 2. Evidence was obtained that the phospholipids of a pure culture of Entodinium caudatum and a mixed rumen protozoa sample contain diglyceride ciliatine, and a plasmalogen ciliatine was detected in the latter. 3. A ninhydrin-positive sphingolipid was isolated from rumen protozoa. Although chromatographically homogeneous on silica gel it contains two components, which were provisionally identified as ceramide ciliatine and ceramide phosphorylethanolamine. 4. A detailed phospholipid analysis of E. caudatum and rumen protozoa is presented. They contain no phosphatidylserine or cardiolipin, but an unidentified phosphoglyceride containing a zwitterionic amino acid is present.     

Signalling sphingomyelinases: which, where, how and why?

A major lipid signalling pathway in mammalian cells implicates the activation of sphingomyelinase (SMase), which upon cell stimulation hydrolyses the ubiquitous sphingophospholipid sphingomyelin to ceramide. This review summarizes our current knowledge on the nature and regulation of signalling SMase(s). Because of the controversy on the identity of this(these) phospholipase(s), the roles of various SMases in cell signalling are discussed. Special attention is also given to the subcellular site of action of signalling SMases and to the cellular factors that positively or negatively control their activity. These regulating agents include lipids (arachidonic acid, diacylglycerol and ceramide), kinases, proteases, glutathione and other proteins.     

Sphingomyelin-degrading pathways in human cells role in cell signalling

The ubiquitous sphingophospholipid sphingomyelin (SM) can be hydrolysed in human cells to ceramide by different sphingomyelinases (SMases). These enzymes exert a dual role, enabling not only the turnover of membrane SM and the degradation of exogenous (lipoprotein) SM, but also the signal-induced generation of the lipid second messenger ceramide. This review focuses on the function(s) of the different SMases in living cells. While both lysosomal and non-lysosomal pathways that ensure SM hydrolysis in intact cells can be distinguished, the precise contribution of each of these SM-cleaving enzymes to the production of ceramide as a signalling molecule remains to be clarified.     

Lipid composition of human serum lipoproteins

1. The lipid compositions of the low-density lipoproteins, the high-density lipoproteins and the ultracentrifugal residue of human serum are presented, with emphasis on certain lipoprotein classes and lipid components not previously described. 2. Except for the lipoproteins with the lowest and highest densities, there is a trend for stepwise successive increase or, respectively, decrease in the relative amounts of the main constituents of lipoproteins. 3. High-density lipoprotein-2 and high-density lipoprotein-3 have different amounts of certain lipids; high-density lipoprotein-2 has relatively more free cholesterol and sphingomyelin; high-density lipoprotein-3 has more free fatty acids, diglycerides and ceramide monohexosides. 4. All the lipoproteins contain hydrocarbons of the alkane series. The greatest amount, which averages 4.4% of total lipid extracted, is in the ultracentrifugal residue; n-alkanes comprise 18-50% of the hydrocarbons. 5. All the lipoproteins contain ceramide monohexosides. The highest relative contents of these glycolipids are in high-density lipoprotein-3 and in the ultracentrifugal residue. 6. The ultracentrifugal residue contains 55% of the total quantity of free fatty acids present in serum. The remaining free fatty acids are distributed among the other lipoprotein classes. 7. The choline-containing phospholipids (phosphatidylcholine, lysophosphatidylcholine and sphingomyelin) comprise about 90% of the phospholipids in all the lipoprotein classes except the low-density lipoprotein-2, which contains about 80% of these phospholipids. 8. The presence of a large amount of lysophosphatidylcholine in the ultracentrifugal residue and the successive decrease of sphingomyelin from the low-density lipoprotein-1 to the ultracentrifugal residue was confirmed. 9. The low-density lipoprotein-2 and the ultracentrifugal residue are characterized by relatively high contents of the lower glycerides.     

The isolation of a new,low-potential,iron-containing electron-transfer protein from rumen bacteria

A new low-potential iron-containing electron-transfer protein has been isolated from cell free extracts of rumen bacteria. This protein, like some bacterial ferredoxins, can 1) transfer electrons from illuminated spinach chloroplasts to NADP; 2) catalyze ferredoxin dependent reductive carboxylation reactions; 3) act as electron acceptor in the pyruvate lyase catalyzed reaction in which pyruvate is oxidized to acetyl-CoA and CO₂. Unlike ferredoxin, this low potential iron protein contains no acid-labile sulfide; contains tightly bound iron that is released only after prolonged hydrolysis with 6 N HCl at 105°C and with an optical spectrum different from that associated with the bacterial ferredoxins.     

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.     

Co-evolution of sphingomyelin and the ceramide transport protein CERT

Life creates many varieties of lipids. The choline-containing sphingophospholipid sphingomyelin (SM) exists ubiquitously or widely in vertebrates and lower animals, but is absent or rare in bacteria, fungi, protists, and plants. In the biosynthesis of SM, ceramide, which is synthesized in the endoplasmic reticulum, is transported to the Golgi region by the ceramide transport protein CERT, probably in a non-vesicular manner, and is then converted to SM by SM synthase, which catalyzes the reaction of phosphocholine transfer from phosphatidylcholine (PtdCho) to ceramide. Recent advances in genomics and lipidomics indicate that the phylogenetic occurrence of CERT and its orthologs is nearly parallel to that of SM. Based on the chemistry of lipids together with evolutionary aspects of SM and CERT, several concepts are here proposed. SM may serve as a chemically inert and robust, but non-covalently interactive lipid class at the outer leaflet of the plasma membrane. The functional domains and peptidic motifs of CERT are separated by exon units, suggesting an exon-shuffling mechanism for the generation of an ancestral CERT gene. CERT may have co-evolved with SM to bypass a competing metabolic reaction at the bifurcated point in the anabolism of ceramide. Human CERT is identical to the splicing variant of human Goodpasture antigen-binding protein (GPBP) annotated as an extracellular non-canonical serine/threonine protein kinase. The relationship between CERT and GPBP has also been discussed from an evolutionary aspect. Moreover, using an analogy of “compatible (or osmoprotective) solutes” that can accumulate to very high concentrations in the cytosol without denaturing proteins, choline phospholipids such as PtdCho and SM may act as compatible phospholipids in biomembranes. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

A novel biotinylated diazirinyl ceramide analogue for photoaffinity labeling

A novel photoreactive ceramide analogue, which contains (3-trifluoromethyl)phenyldiazirinyl lipid and biotinylated sphingosine, was synthesized. The probe was recognized as an antigen by anti-ceramide antibody and as a substrate for sphingolipid ceramide N-deacylase. These results indicate that the probe may be useful as a photoaffinity-biotinylating agent in sphingolipid studies.     

Structural analysis of sphingophospholipids derived from Sphingobacterium spiritivorum, the type species of genus Sphingobacterium

The unique feature of the genus Sphingobacterium is the presence of sphingophospholipids and ceramides, besides diacylglycerophospholipids. As major cellular lipid components, five kinds of sphingophospholipids were purified from Sphingobacterium spiritivorum ATCC 33861(T), the type species of genus Sphingobacterium. They were identified as ceramide phosphorylethanolamines (CerPE-1 and CerPE-2), ceramide phosphoryl-myo-inositols (CerPI-1 and CerPI-2), and ceramide phosphorylmannose (CerPM-1). The ceramide of CerPE-1, CerPI-1, and CerPM-1 was composed of 15-methylhexadecasphinganine (isoheptadeca sphinganine, iso-C17:0) and 13-methyltetradecanoic acid (isopentadecanoic acid, iso-C15:0), whereas that of CerPE-2 and CerPI-2 was composed of isoheptadeca sphinganine and 2-hydroxy-13-methyltetradecanoic acid (2-hydroxy isopentadecanoic acid, 2-OH iso-C15:0). These sphingophospholipids were also found in cellular lipids of Sphingobacterium multivorum ATCC 33613(T), Sphingobacterium mizutaii ATCC 33299(T), Sphingobacterium faecium IFO 15299(T), Sphingobacterium thalpophilum ATCC 43320(T), and Sphingobacterium antarcticum ATCC 51969(T). To our knowledge, the existence of CerPM-1 is a novel sphingophospholipid through eukaryotic and prokaryotic cells.     

A new 6-hydroxy-4-sphingenine-containing ceramide in human skin.

A new ceramide consisting of 6-hydroxysphingosine linked to a non-hydroxyacid was found in human epidermal lipid. This ceramide was sought because its fatty acid and sphingoid moieties are present in other combinations in human epidermal ceramides. To isolate the new ceramide, the mixture of ceramides in human epidermal lipid was first separated into fractions by thin-layer chromatography (TLC), and then each fraction was further purified by TLC after acetylation of all hydroxyl groups. TLC after acetylation revealed that one of the fractions isolated in the first TLC step contained two components, namely, the ceramide consisting of sphingosine linked to an alpha-hydroxyacid and an unknown ceramide. The new ceramide constituted about 9% of the total ceramides, and was shown by NMR spectroscopy to be N-acyl-6-hydroxysphingosine.     

Chemical analysis of and degradation studies on the cell wall lipopolysaccharide of Rhodopseudomonas capsulata

A lipopolysaccharide (LPS) has been isolated from the gram-negative photosynthetic bacterium Rhodopseudomonas capsulata. Chemical analysis revealed the presence of d-glucose, d-galactose, l-rhamnose, 3-O-methyl-l-rhamnose (l-acofriose), d-glucosamine, 2-keto-3-deoxyoctonate, and neuraminic acid. The LPS does not contain l-glycero-d-mannoheptose, a typical component of the LPS of enteric bacteria. Fatty acid analysis showed that, apart from lauric acid, two hydroxy fatty acids (hydroxycaproic and hydroxymyristic acids) are the main components. By hydrolysis in weak acid, the LPS has been separated into a polysaccharide part (degraded polysaccharide) and a lipid part (lipid A). Presumably the lipid A contains a glucosamine backbone. Whereas the OH-groups of glucosamine are esterified with lauric and hydroxycaproic acids, hydroxymyristic acid is linked to the amino group of the sugar. By separation of the degraded polysaccharide by gel filtration, a fraction has been isolated which inhibited hemagglutination in a system containing antiserum, obtained by immunization of rabbits with whole cells, and isolated LPS. This fraction, which includes the determinant group, contains the sugars glucose, rhamnose, and acofriose. A second fraction obtained in this way was found to be serologically inactive and is composed of glucose, galactose, neuraminic acid, and phosphate.     

1-(O-β-Glucosaminyl)-2,3-diglyceride in Bacillus megaterium

1. A lipid that contains glucosamine but not phosphorus has been isolated from Bacillus megaterium. It constitutes about 5% of the total lipid glucosaminide in this organism and can be distinguished chromatographically from 2'-(O-beta-glucosaminyl)phosphatidylglycerol and 3'-(O-beta-glucosaminyl)phosphatidylglycerol, which are also present. 2. The lipid contains glycerol, fatty acids and glucosamine in the molar proportion 1:2:1. The fatty acids are bound by an ester linkage and are similar to those found in other lipids of this organism. Partial acid hydrolysis or alkaline hydrolysis of the lipid yields 1-(O-beta-glucosaminyl)glycerol and degradation with nitrite yields 2,5-anhydromannose and diglyceride. 3. The lipid has been identified as 1-(O-beta-glucosaminyl)-2,3-diglyceride.     

Effect of Absence of Ciliate Protozoa From the Rumen on Microbial Activity and Growth of Lambs

A survey of the components of the rumen ciliate population in a series of adult sheep, raised in the Faculty of Agriculture, University of Alexandria, has shown that a mixture of Entodinium, Isotricha, Ophryoscolex, Diplodinium, and Polyplastron species was found in the rumen contents of Egyptian sheep; no Epidinium and a negligible number of Dasytricha ruminantium were also observed. The microbial population, reducing sugars, ammonia, volatile fatty acids (VFA) production, and growth rate of 14 lambs inoculated with whole rumen contents from a mature sheep were compared over a 6-month period with those of 13 lambs maintained under the same conditions, except that they were strictly isolated from other ruminants. Certain large oval organisms and large numbers of flagellates and Oscillospira were frequently observed in the rumen contents of the isolated lambs. The reducing sugars, ammonia, and VFA levels, measured before and at intervals after feeding, in the inoculated lambs showed a pronounced rise above the values found in the ciliate-free animals. The propionic acid-acetic acid ratio in the rumen contents of the faunated lambs was considerably higher than in the nonfaunated controls. The inoculated lambs grew faster than the isolated lambs. Differences in weight gain which ranged from 15 to 17% were statistically significant. The inoculated animals impressed the observers by their good appearance which was superior to that of the ciliate-free lambs. It was, therefore, concluded that the rumen ciliate protozoa are essential for the metabolism and growth of young lambs.     

Acetylene reduction by rumen microflora

Summary Acetylene reduction to ethylene by filtrates of rumen contents has been studied. The Km values for acetylene are comparable to those reported for nitrogenase enzymes from N₂ fixing bacteria. The enhancement of ethylene production from acetylene by phosphate and pyruvate suggests that the reduction was carried out by anaerobic microorganisms. Acetylene reduction occurred in the rumen only when a high nitrogen diet was fed to the sheep. Some microorganisms isolated from the rumen contents were grown anaerobically under N₂ gas on agar not supplemented with combined nitrogen. Methane production by filtrates of rumen contents was found to be inhibited by acetylene.     

Ceramide composition of the psoriatic scale

This paper investigates the ceramide composition of the psoriatic scale compared with that of normal human SC. A method was optimalized, based on TLC separation followed by densitometry, allowing the provision of good resolution and quantification of ceramide fractions from both normal and pathological specimens. Seven ceramide fractions were isolated and submitted to compositional analysis. The obtained results suggested a revisitation of previous ceramide designation. Therefore a simple classification is suggested, based on grouping ceramides carrying structural similarities under common codes. According to these rules, ceramides were grouped into five classes designated as: (1) Cer[EOS], which contains ester-linked fatty acids, ω-OH fatty acids and sphingosines; (2) Cer[NS], which contains non-OH fatty acids and sphingosines; (3) Cer[NP], which contains non-OH fatty acids and phytosphingosines; (4) Cer[AS], which contains α-OH fatty acids and sphingosines; (5) Cer[AP], which contains α-OH fatty acids and phytosphingosines. Analysis of ceramides from the psoriatic scale, compared to those from normal human SC, resulted in an impairment of the Cer[EOS] content as well as of the ceramides containing phytosphingosine, with concurrent increase in ceramides containing sphingosine, being the total amount maintained identical. Since one of the suggested pathways for phytosphingosine biosynthesis involves the water addition to the corresponding sphingosine double bond, we can speculate that the observed alterarion is due to a deranged water bioavailability, associated with psoriaris.     

Desulfovibrio of the sheep rumen.

A sulfate-reducing bacterium has been isolated in pure culture from sheep rumen contents. Its properties agree in all respects tested with those ascribed to Desulfovibrio desulfuricans. The populations observed (about 10(8)/ml) are sufficient to account for published rates of ruminal sulfide production.     

Isolation and Characterization of 2-Keto-3-Deoxyoctonate-Lipid A from a Heptose-Deficient Mutant of Escherichia coli

A heptose-deficient mutant of Escherichia coli has been isolated and from it a glycolipid, consisting of lipid A and 2-keto-3-deoxyoctonate (KDO), has been extracted with diisobutylketone-acetic acid-water. Based on beta-hydroxymyristic acid, the extractable glycolipid accounts for a major portion of the total lipid A in this mutant. A glycolipid, purified from the lipid extract by a combination of silicic acid and Sephadex LH-60 chromatography, contains glucosamine, phosphate, KDO, acetyl groups, and fatty acids in the following molar ratios: 1:2:2:1.7:5. These components account for over 80% of the lipid by weight. The fatty acid pattern of the glycolipid is typical of lipid A, the major component being beta-hydroxymyristic acid. The lipid also contains an amino sugar which appears to be 4-amino-4-deoxyarabinose. With the use of an ion-exchange paper chromatographic technique, gram-negative bacteria can be rapidly screened for the presence of this glycolipid. The mutant is believed to have a leaky defect in either biosynthesis of heptose or its incorporation into lipopolysaccharide. The lipopolysaccharide from the mutant contains only about a third as much heptose, glucose, and galactose as the parent CR34, a K-12 derivative. Chemical analysis and phage typing suggest that CR34 contains an incomplete core polysaccharide devoid of glucosamine.     

Gramicidin K, a new linear channel-forming gramicidin from Bacillus brevis

A new gramicidin has been isolated from a commercial mixture of gramicidins A, B, and C. This new molecule, designated gramicidin K, contains formyl and ethanolamine blocking groups, has a molecular weight approximately 20% higher than gramicidin A, and is strongly retained on reversed-phase liquid chromatographic columns. Gramicidin K can be resolved into two components, one of which contains tyrosine. In lipid bilayer membranes, both components form channels of considerably longer lifetime and somewhat lower conductance than gramicidin A. Gramicidin K appears to be a lipopeptide that consists of a fatty acyl chain attached to the ethanolamine of gramicidin A.     

Ceramide kinase, a novel lipid kinase. Molecular cloning and functional characterization

Ceramide-1-phosphate is a sphingolipid metabolite that has been implicated in membrane fusion of brain synaptic vesicles and neutrophil phagolysosome formation. Ceramide-1-phosphate can be produced by ATP-dependent ceramide kinase activity, although little is known of this enzyme because it has not yet been highly purified or cloned. Based on sequence homology to sphingosine kinase type 1, we have now cloned a related lipid kinase, human ceramide kinase (hCERK). hCERK encodes a protein of 537 amino acids that has a catalytic region with a high degree of similarity to the diacylglycerol kinase catalytic domain. hCERK also has a putative N-myristoylation site on its NH(2) terminus followed by a pleckstrin homology domain. Membrane but not cytosolic fractions from HEK293 cells transiently transfected with a hCERK expression vector readily phosphorylated ceramide but not sphingosine or other sphingoid bases, diacylglycerol or phosphatidylinositol. This activity was clearly distinguished from those of bacterial or human diacylglycerol kinases. With natural ceramide as a substrate, the enzyme had a pH optimum of 6.0-7.5 and showed Michaelis-Menten kinetics, with K(m) values of 187 and 32 microm for ceramide and ATP, respectively. Northern blot analysis revealed that hCERK mRNA expression was high in the brain, heart, skeletal muscle, kidney, and liver. A BLAST search analysis using the hCERK sequence revealed that putative ceramide kinases (CERKs) exist widely in diverse multicellular organisms including plants, nematodes, insects, and vertebrates. Phylogenetic analysis revealed that CERKs are a new class of lipid kinases that are clearly distinct from sphingosine and diacylglycerol kinases. Cloning of CERK should provide new molecular tools to investigate the physiological functions of ceramide-1-phosphate.     

Determination and physiological roles of the glycosylphosphatidylinositol lipid remodelling pathway in yeast

In the yeast Saccharomyces cerevisiae, glycosylphosphatidylinositol (GPI)‐anchored proteins play important roles in cell wall biogenesis/assembly and the formation of lipid microdomains. The lipid moieties of mature GPI‐anchored proteins in yeast typically contain either ceramide moieties or diacylglycerol. Recent studies have identified that the GPI phospholipase A₂ Per1p and O‐acyltransferase Gup1p play essential roles in diacylglycerol‐type lipid remodelling of GPI‐anchored proteins, while Cwh43p is involved in the remodelling of lipid moieties to ceramide. It has been generally proposed that phosphatidylinositol with diacylglycerol containing a C26 saturated fatty acid, which is generated by the sequential activity of Per1p and Gup1p, is converted to inositolphosphorylceramide by Cwh43p. In this report, we constructed double‐mutant strains defective in lipid remodelling and investigated their growth phenotypes and the lipid moieties of GPI‐anchored proteins. Based on our analyses of single‐ and double‐mutants of proteins involved in lipid remodelling, we demonstrate that an alternative pathway, in which lyso‐phosphatidylinositol generated by Per1p is used as a substrate for Cwh43p, is involved in the remodelling of GPI lipid moieties to ceramide when the normal sequential pathway is inhibited. In addition, mass spectrometric analysis of lipid species of Flag‐tagged Gas1p revealed that Gas1p contains ceramide moieties in its GPI anchor.