Biosynthesis of Glycosyldiglycerides in Mycobacterium smegmatis

A particulate enzyme preparation from Mycobacterium smegmatis catalyzes the transfer of [(14)C]galactose from uridine 5'-diphosphate (UDP)-[(14)C]galactose and of [(14)C]glucose from UDP-[(14)C]glucose into chloroform-soluble products. The radioactive neutral lipids were purified by passage through diethylaminoethyl-cellulose, followed by thin-layer chromatography. When UDP-glucose was used as substrate, two major radioactive lipids were obtained; one had a hexose-glucose-glycerol ratio of 1:1:1. The second product had a hexose-glycerol ratio of 2:1 and, in addition to glucose, contained lesser amounts of mannose and galactose. With UDP-galactose as substrate, two radioactive products were observed that were chromatographically indistinguishable from the [(14)C]glucosyl-labeled mono- and diglycosyldiglyceride. Palmitate and oleate were the predominant fatty acid constituents in these lipids and were present in equimolar amounts in all of the products examined. The products have thus been identified as monoglycosyldiglyceride and a diglycosyldiglyceride containing glucose as the major hexose along with mannose and galactose. Properties of the galactosyl and glucosyl transferases are described.     

Ether lipids in the cell membrane of Mycoplasma fermentans.

Two new ether lipids, 1-O-alkyl/alkenyl-2-O-acyl-glycero-3-phosphocholine and its lyso form, 1-O-alkyl/alkenyl-glycero-3-phosphocholine, were identified in the cell membrane of Mycoplasma fermentans using chemical analyses, GLC-MS, MALDI-TOF MS, and 1D and 2D NMR spectroscopy. The lipids are heterogeneous with respect to both acyl and alkyl/alkenyl residues. The acyl residues at position 2 of glycerol are hexadecanoyl and octadecanoyl in a molar ratio of 3.6 : 1 with a trace amount of octadecenoyl. The alkyl/alkenyl residues at position 1 of glycerol are hexadecyl (78%), octadecyl (7%), octadecenyl (14%), and hexadecenyl (traces). In the octadecenyl residue, the double bond has a cis configuration and is located at either position 1' (plasmalogen-type lipid) or 9' in a ratio approximately 1 : 1. This is the first report of the presence of alkyl and vinyl (alk-1'-enyl) ether lipids in the cell membrane of aerobically grown mycoplasmas. Lipids of this type have been found in some Gram-positive bacteria, thus supporting the hypothesized close taxonomical relationship of these bacteria to mycoplasmas. The ether lipids of M. fermentans are structurally similar to platelet activating factor; it was demonstrated that the 2-O-acetylated lyso form lipid can mimic platelet-activating factor activity in isolated perfused and ventilated rat lungs.     

Replacement of the aliphatic chains of Clostridium acetobutylicum by exogenous fatty acids: regulation of phospholipid and glycolipid composition.

The membrane lipid aliphatic chains of Clostridium acetobutylicum ATCC 4259 have been extensively modified by growth in biotin-free medium containing vitamin-free casein hydrolysate supplemented with either elaidic acid, oleic acid, or mixtures of palmitic and oleic acids. Growth with elaidic acid resulted in polar lipids containing 88.6% 18:1 acyl chains and 94.5% 18:1 ether-linked chains. Growth with oleic acid resulted in comparable levels of enrichment of the lipids with 18:1 chains and C19 chains containing cyclopropane rings. When cells were grown with mixtures of palmitic and oleic acids, the ether-linked chains of the plasmalogens were greater than or equal to 64% 18:1 plus C19 chains containing cyclopropane rings at all ratios of oleic to palmitic acid in the medium. The acyl chains reflected the palmitic acid content of the medium more closely. Marked changes were observed in both phospholipid and glycosyldiglyceride compositions as the lipid acyl and ether-linked chains became more enriched with unsaturated and cyclopropane chains. The ratio of the glycerol acetal of plasmenylethanolamine to phosphatidylethanolamine increased, the ratio of cardiolipin to phosphatidylglycerol decreased, and the ratio of diglycosyldiglyceride to monoglycosyldiglyceride increased. However, the monoglycosyldiglyceride/diglycosyldiglyceride ratio was lower for cells grown on 100% oleic acid than for cells grown on 60 or 80% oleic acid. In the membranes of cells grown on 100% oleic acid, the ratio of glycolipids to phospholipids was lower than that found in cells grown on 60% oleic acid. These results indicate that C. acetobutylicum regulates its polar lipid composition in a complex manner involving phospholipids and glycosyldiglycerides. These changes can affect the equilibria between those lipids that form bilayers and those lipids that tend to form nonlamellar phases when enriched with unsaturated aliphatic chains. Phosphoglycolipids of unknown structure were also observed in cells grown either with biotin or with fatty acids. The content of the most abundant phosphoglycolipid also varied with the degree of unsaturation of the cellular lipids.     

Phase behavior of ethanolamine plasmalogen

In the present study the phase behavior of multilamellar dispersions of 1-O-(1′-alkenyl)-2-oleoyl-glycerophosphoethanolamine (ethanolamine plasmalogen), 1-O-alkyl-2-oleoyl-glycerophosphoethanolamine and 1-acyl-2-oleoyl-glycerophosphoethanolamine was compared using differential scanning calorimetry (DSC) and ³¹P-NMR. The three compounds differed only in the type of bonding (vinyl ether, alkyl ether or acyl ester) linking the aliphatic moiety to position 1 of sn-glycerol.The gel to liquid-crystalline phase transition temperature as determined by DSC was lowest for ethanolamine plasmalogen (26°C) and was similar for the alkylacyl and diacyl analogs (29.5° and 30°C, respectively). Enthalpies of the G → L phase transition were not significantly different for the three phospholipids tested.Ethanolamine plasmalogen undergoes the lamellar to hexagonal phase transition at 30°C, the analogous alkylacyl-glycerophosphoethanolamine(alkylacyl-GPE) and diacyl-GPE at 53°C and 69°C, respectively. Thus, an alkenyl ether bond in position 1 of sn-glycerol, the structural characteristic of plasmalogens, effectively stabilizes the hexagonal H_II arrangement of ethanolamine glycerophospholipids, while it has relatively little effect on destabilization of the lamellar gel state.     

Chemical structure of the O-polysaccharide isolated from Pectobacterium atrosepticum SCRI 1039

The lipopolysaccharide (LPS) of the bacterium Pectobacterium atrosepticum SCRI 1039 was hydrolyzed and the products were separated. A study of the obtained O-polysaccharide by means of chemical methods, GLC, GLC–MS, and NMR spectroscopy allowed us to identify a branched polymer with a pentasaccharide repeating unit of the structure shown below, in which the fucose residue was partially O-acetylated at C-2, C-3 or C-4.     

Plasmalogens and O-alkylglycerophospholipids in aquatic animals

1. Qualitative and quantitative distribution of ether phospholipids was studied in several aquatic organisms. 2. Brain, nerve and gills of these animals were rich in ether glycerophospholipids. 3. 1-alkenyl ethers were in general more abundant than alkyl ones. The alkenyl ethers were mainly components of PE in all aquatic animals. Those of PS occurred in mollusca only. 4. Alkyl ether moieties appeared to be essentially located in PE and PC. 5. This review suggests that ether phospholipids may play an important role in the permeability properties of biological membranes.     

The cerebro-hepato-renal (Zellweger) syndrome. Impaired de novo biosynthesis of plasmalogens in cultured skin fibroblasts

In tissues of patients with the cerebro-hepato-renal (Zellweger) syndrome the plasmalogen content is very low. In order to study the biosynthesis of plasmalogens, skin fibroblasts of Zellweger patients, controls and heterozygotes, and amniotic fluid cells of controls were cultured in a medium supplemented with [1-14 C]hexadecanol or 1-O-[9,10-3H2]octadecylglycerol. The incorporation of 14C-label into the alkenyl moiety of plasmalogens was strongly reduced in Zellweger patients as compared to controls. The low concentration of 14C-labeled plasmalogens was not compensated for by an elevated levels of 14C-labeled alkyl phospholipids. Hexadecanol was partly oxidized to fatty acid in all cell lines and the incorporation of 14C-labeled fatty acid into phospholipids was comparable for patients and controls. [3H]Alkylglycerol was incorporated into plasmalogens with the same efficiency in Zellweger patients as in controls. These results indicate that only the reaction(s) involved in the introduction of the ether bond in the process of plasmalogen synthesis are deficient in Zellweger patients. The results also suggest that the hexadecanol incorporation patterns can be used for the (prenatal) diagnosis of the Zellweger syndrome.     

Enhancement of chlorophyll a fluorescence yield, low-temperature F685/F730 fluorescence emission ratio, and electron transport rate by ether phospholipids (platelet activating factor and analogs) in isolated chloroplasts

Acetyl glyceryl ether phosphorylcholine (1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine; or platelet activating factor (PAF)), when incubated with chloroplasts or subchloroplast fractions derived from stroma or grana lamellae, induces a drastic increase in the low-temperature fluorescence emission ratio F685/F730 (77 degree K). The molecular structure requirement for the effect to be elicited is the ether bond and a long C chain at the C-1 position of glycerol, a short C chain at C-2 (or the lyso form), and a large polar head at C-3, the potent effector being PAF C-16. The effect is more pronounced in grana-derived fractions. PAF also induces an increase in the chlorophyll alpha fluorescence yield, enhances the association of chlorophyll in the supramolecular pigment-protein complexes of the thylakoid (especially those of Photosystem II), and enhances electron transfer from 1,5-diphenyl carbazide to 2,6-dichlorophenol. These effects are attributed to alteration of the Photosystem II unit organization via the incorporation/intercalation in the grana of the wedge-shaped PAF.     

Structural determinants of the packing and electrostatic behavior of unsaturated phosphoglycerides

Docosahexaenoic acid-containing phosphoglycerides accumulate preferentially in membranes of the retina, brain, and spermatozoa, but the functional significance of this largely remains to be determined. Previously we compared the physical properties of homogeneous monolayers of these and other phosphoglyceride species to obtain insights into their physiological roles. Particularly noteworthy were the unusually low dipole moments of species having sn-2-docosahexaenoyl chains. In this study, we have investigated the electrostatic and lateral packing properties of related phosphoglycerides and found that: 1), The dipole moment-lowering effect of the docosahexaenoyl group arises from its having a Z double bond at chain position n-3. 2), The large dipole moment-lowering effects at sn-1 of an ether bond to an alkyl or a 1Z alkenyl chain and that of a sn-2-esterified n-3 fatty acid are additive. 3), The 1Z double bond in an alkenyl chain lowers the molecular area of a phosphoglyceride and, concomitantly, makes it less compressible. 4), Ethanolamine-containing phosphoglycerides are generally less compressible than their corresponding choline analogs. Our data showing that relatively small lipid structural changes markedly alter lipid physical properties in fluid phases underscores the need to study the function of peripheral and integral membrane proteins in the presence of appropriate lipid species.     

Stereochemical specificity of the biosynthesis of the alkyl ether bond in alkyl ether lipids.

The stereochemical course of the formation of the alkyl ether bond in alkyl ether lipids was investigated through the synthesis of stereospecifically labeled acyl R- or S-[1-3H]dihydroxyacetone 3-phosphate (DHAP) starting from L-glyceraldehyde. It was demonstrated directly that the formation of the alkyl ether bond results in the stereospecific exchange of the pro-R C-1 hydrogen of DHAP with a proton of water. The configuration of the hydrogen that is retained on C-1 after formation of the alkyl ether bond was also investigated. The alkyl ether lipid was degraded, and the DHAP backbone isolated as glycerol, converted to DHAP via glycerol 3-phosphate and treated with either aldolase or triose phosphate isomerase. The results demonstrated that the retained hydrogen on C-1, which was pro-S in the starting substrate, was pro-S in the product alkyl ether.     

Structure-activity relationships of ganoderma acids from Ganoderma lucidum as aldose reductase inhibitors

A series of lanostane-type triterpenoids, known as ganoderma acids were isolated from the fruiting body of Ganoderma lucidum. Some of these compounds were identified as active inhibitors of the in vitro human recombinant aldose reductase. To clarify the structural requirement for inhibition, some structure–activity relationships were determined. Our structure–activity studies of ganoderma acids revealed that the OH substituent at C-11 is an important feature and the carboxylic group in the side chain is essential for the recognition of aldose reductase inhibitory activity. Moreover, double bond moiety at C-20 and C-22 in the side chain contributes to improving aldose reductase inhibitory activity. In the case of ganoderic acid C2, all of OH substituent at C-3, C-7 and C-15 is important for potent aldose reductase inhibition. These results provide an approach to understanding the structural requirements of ganoderma acids from G. lucidum for aldose reductase inhibitor. This understanding is necessary to design a new-type of aldose reductase inhibitor.     

Novel Taxa-4(20),12-diene and 2(3→20)Abeotaxane from Needles of Taxus canadensis

A novel 6/8/6-membered taxane with a rare C-12(13)-double bond and rare 2(3→20)abeotaxane were isolated from the needles of Taxus canadensis. Their structures were characterized as 7β,9α,10β-triacetoxytaxa-4(20),12-diene-2α,5α,11β-triol (1) and 2α,7β,10β-triacetoxy-5α-hydroxy-2(3→20)abeotaxa-4(20),11-diene-9,13-dione (2) on the basis of 1D and 2D spectroscopic data. 1 is the first example of a natural taxane without substitution at both C-13 and C-14.     

Determination of choline and ethanolamine plasmalogens in human plasma by HPLC using radioactive triiodide (1-) ion (125I3-)

For the purpose of developing highly sensitive and convenient determination of plasmalogens, the high-performance liquid chromatography (HPLC) method using radioactive iodine ((125)I) was investigated. Radioactive triiodide (1-) ion ((125)I(3)(-)), which is an actual iodine form capable of reacting with vinyl ether bond ([bond]CH(2)[bond]O[bond]CH[double bond]CH[bond]) of plasmalogens, could be safely and efficiently produced by oxidizing a commercial radioactive sodium iodine (Na(125)I) with hydrogen peroxide (H(2)O(2)) under acid condition (pH 5.5-6.0), which is called iodine-125 reagent. I(3)(-) specifically reacted with plasmalogens at the molar ratio of 1:1 in methanol, and 1 or 2 mol of plasmalogens was involved in the binding with iodine per iodine atom, resulting in the formation of stable iodine-binding phospholipids. The HPLC system with Diol column and acetonitrile/water as a mobile phase was available for separating iodine-binding phospholipids from nonbinding free iodine and for separately eluting iodine-binding phospholipids derived from choline and ethanolamine plasmalogens. Using iodine-125 reagent (1.85 MBq/ml), plasmalogens were detectable at high sensitivity of 10,000-15,000 cpm/nmol, which is more than 1000-fold higher sensitivity than the classical determination with nonradioactive iodine. Plasmalogen concentrations in human plasma were measured with the HPLC system and determined as, on average, 129.1+/-31.3 microM (n=8) in a 1.2 content ratio of choline to ethanolamine plasmalogens, a concentration that nearly agrees with the value reported previously.     

Structure-Activity Relationships of Abscisic Acid Analogs Based on the Induction of Freezing Tolerance in Bromegrass (Bromus inermis Leyss) Cell Cultures

The induction of freezing tolerance in bromegrass (Bromus inermis Leyss) cell culture was used to investigate the activity of absisic acid (ABA) analogs. Analogs were either part of an array of 32 derived from systematic alterations to four regions of the ABA molecule or related, pure optical isomers. Alterations were made to the functional group at C-1 (acid replaced with methyl ester, aldehyde, or alcohol), the configuration at C-2, C-3 (cis double bond replaced with trans double bond), the bond order at C-4, C-5 (trans double bond replaced with a triple bond), and ring saturation (C-2′, C-3′ double bond replaced with a single bond so that the C-2′ methyl and side chain were cis). All deviations in structure from ABA reduced activity. A cis C-2, C-3 double bond was the only substituent absolutely required for activity. Overall, acids and esters were more active than aldehydes and alcohols, cyclohexenones were more active than cyclohexanones, and dienoic and acetylenic analogs were equally active. The activity associated with any one substituent was, however, markedly influenced by the presence of other substituents. cis, trans analogs were more active than their corresponding acetylenic analogs unless the C-1 was an ester. Cyclohexenones were more active than cyclohexanones regardless of oxidation level at C-1. An acetylenic side chain decreased the activity of cyclohexenones but increased the activity of cyclohexanones relative to their cis, trans counterparts. Trends suggested that for activity the configuration at C-1′ has to be the same as in (S)-ABA, in dihydro analogs the C-2′-methyl and the side chain must be cis, small positional changes of the 7′-methyl are tolerable, and the C-1 has to be at the acid oxidation level.     

Structural characterization of the polar lipids of Clostridium novyi NT. Further evidence for a novel anaerobic biosynthetic pathway to plasmalogens

A study of the polar lipids of Clostridium novyi NT has revealed the presence of phosphatidylethanolamine (PE) and cardiolipin as major phospholipids with smaller amounts of phosphatidylglycerol (PG), lysyl-PG and alanyl-PG. Other minor phospholipids included phosphatidic acid, CDP-diacylglycerol, phosphatidylserine (PS) and phosphatidylthreonine (PT). PE, PG and amino acyl PG were present in both the diacyl and alk-1'-enyl acyl (plasmalogen) forms and cardiolipin plasmalogens were found to contain one or two alk-1'-enyl chains. In contrast, the precursor lipids phosphatidic acid, CDP-diacylglycerol and PS were present almost exclusively as diacyl phospholipids. These findings are consistent with the hypothesis that plasmalogens are formed from diacylated phospholipids at a late stage of phospholipid formation in Clostridium species. This novel pathway contrasts with the route in animals in which a saturated ether bond is formed at an early stage of plasmalogen biosynthesis and the alk-1-enyl bond is formed by an aerobic mechanism.     

Antifeedant effects of azadirachtin and structurally related compounds on lepidopterous larvae

The antifeedant activity of azadirachtin, azadirachtin-derivatives and related limonoids was assessed in choice and no-choice bioassays against four species of Lepidoptera: Spodoptera littoralis, Spodoptera frugiperda, Heliothis virescens and Heliothis armigera. The choice bioassay showed that the feeding behaviour of S. littoralis was affected by more of the compounds than that of either S. frugiperda or H. virescens. H. armigera was the least affected. Azadirachtin and dihydroazadirachtin were the most potent of the 40 compounds tested. The results showed that hydrogenation of the C-22,23 double bond did not decrease antifeedant activity and the nature of the substitutes at C-1, C-3 and C-11 were important. Molecules with bulky substitutes at either C-22 or C-23 were usually ineffective antifeedants as were compounds lacking an epoxide. Compounds recorded as active antifeedants in the choice bioassay were not always as active in the no-choice test. The value of the bioassays in assessing the mode of action of the compounds is discussed.

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Résumé L'activité phagodissuadante de l'azadirachtine, de ses dérivés et des limonoïdes voisins sur 4 espèces de lépidoptères: Spodoptera littoralis, S. frugiperda, Heliothis virescens et H. armigera a été évaluée par des expériences avec et sans choix. Les expériences de choix ont montré que le comportement alimentaire de S. littoralis était modifié par plus de substances que celui de S. frugiperda ou H. virescens. Celui de H. armigera était le moins modifié. Les 2 substances les plus puissantes parmi les 40 examinées, ont été l'azadirachtine et le dihydroazadirachtine. Ces résultats montrent que l'hydrogénation de la double liaison C-22,23 ne réduit pas l'activité phagodissuadante et que la nature des substitutions en C-1, C-3 et C-11 sont importantes. Les molécules avec des substitutions volumineuses en C-22 ou C-23 sont généralement des phagodissuadants aussi inefficaces que ceux ayant perdu un époxide. Les substances notées comme phagodissuadants actifs dans les expériences de choix ne sont pas toujours aussi actives en absence de choix. La valeur des tests dans l'évaluation du mode d'action des substances est discuté.

     

Three iridoid glycosides from Viburnum furcatum

Three new bitter iridoid glycosides having an 8,10,11-oxygen substituted iridoid skeleton with an isovaleryl moiety at C-1, have been isolated from the ether and ethyl acetate soluble fractions of the leaves of Viburnum furcatum. Two of them had a glucose moiety at C-11 of the iridoid skeleton and a p-coumaroyl group linked to C-6 of the sugar, and they were found to be geometrical isomers about the double bond of the p-coumaroyl2 moiety. The third one was characterized as a alloside of the same aglycone.     

Age-related changes of the endogenous cardiolipin and plasmalogens of guinea pig kidney and their in vitro hydrolysis by endogenous phospholipases: a thin layer chromatographic analysis in conjunction with densitometric measurement

The phosphoglycerides profile of guinea pig kidney, fetal, young adult, and aged, and their in vitro response to the endogenous lipolytic enzymes, mainly in the phospholipase group were determined by TLC technology in conjunction with densitometric measurement. Changes in phosphoglycerides profile subsequent to in vitro incubation of these tissues at pH 7.4, and 38 degrees C for 45 min and prior to phospholipid extraction has provided evidence relating to their respective lipolytic enzymes capabilities and age. These changes are mainly related to endogenous cardiolipin (CL), alkenyl phospholipids (phosphatidyl ethanolamine and phosphatidyl choline) and their endogenous deacylation to their respective lyso derivatives monolysocardiolipin (MLCL), lyso alkenyl phosphatidyl ethanolamine (LPE), and lyso alkenyl phosphatidyl choline (LPC) by endogenous phospholipases. The hydrolysis of the plasmalogen confirms the action of endogenous PLA(2) on sn-2 fatty acids of these compounds.     

Proton and carbon-13 nuclear magnetic resonance spectroscopy of diastereoisomeric 3- and 17 beta-tetrahydropyranyl ether derivatives of estrone and estradiol

Protection of 3- and 17 beta-hydroxyl groups of estrone and estradiol as tetrahydropyranyl ether derivatives led to mixtures of 2'(R)- and 2'(S)-diastereoisomers which were separated by crystallization (3-tetrahydropyranyl ethers), or by thin-layer chromatography (17-tetrahydropyranyl ethers), and characterized by 1H and 13C nuclear magnetic resonance (NMR). Assignments for NMR signals of estradiol 3,17 beta-ditetrahydropyranyl ether were facilitated by comparison with those of its 15 zeta, 16 zeta-dideuterio analog and by 2D 1H-13C heteroshift correlation experiments. Diastereoisomers of 3-tetrahydropyranyl ether derivatives could be identified through the 13C NMR doublet signals of the anomeric C-2' and the aromatic C-4 carbon atoms in CDCl3. Diastereoisomers of 17-tetrahydropyranyl ether derivatives were recognized from characteristic modifications of 1H NMR signals of H-2', H-6', H-1, H-17, and 18-CH3 protons as well as from the 13C NMR doublet signals corresponding to C-2', C-4', C-6', C-12, C-13, C-16, and C-17 carbon atoms. Low-temperature experiments showed a splitting of the C-2', C-6', and C-17 13C NMR signals of each of the two 17-tetrahydropyranyl ether isomers. The downfield signal (equatorial conformer) of the three resulting doublets was more intense for the 17-tetrahydropyranyl ether 2'(S)-isomer, whereas the upfield signal (axial conformer) was more intense for the 2'(R)-isomer.     

Incorporation of ω6 polyunsaturated fatty acids into phospholipids of the crab Carcinus maenas

The incorporation in vivo of ¹⁴C-18:2 ω6 and ³H-20:4 ω6 fatty acids in phospholipids isolated from gills, hepatopancreas and hemolymph of the crab Carcinus maenas was analysed. PC was the most heavily labelled phospholipid from these ω6-unsaturated fatty acids and appeared to play an important part in the phospholipids metabolism in Crustaceans. The pathway of fatty acids synthesis in phospholipids of C. maenas seems to be similar to those described for mammals. It is at the level of tissue Pl of C. maenas that the renewal of the 20:4 ω6 fatty acid is the most important. It is suggested that the rapid reorganization of phospholipid molecular species composition in the crab is checked by deacylation—reacylation cycle.