Effect of growth temperature on ether lipid biochemistry in Archaeoglobus fulgidus

The archaea are distinguished by their unique isoprenoid ether lipids, which typically consist of the sn-2,3-diphytanylglycerol diether or sn-2,3-dibiphytanyldiglycerol tetraether core modified with a variety of polar headgroups. However, many hyperthermophilic archaea also synthesize tetraether lipids with up to four pentacyclic rings per 40-carbon chain, presumably to improve membrane thermal stability at temperatures up to∼110 °C. This study aimed to correlate the ratio of tetraether to diether core lipid, as well as the presence of pentacyclic groups in tetraether lipids, with growth temperature for the hyperthermophilic archaeon, Archaeoglobus fulgidus. Analysis of the membrane core lipids of A. fulgidus using APCI–MS analysis revealed that the tetraether-to-diether lipid ratio increases from 0.3 ± 0.1 for cultures grown at 70°C to 0.9 ± 0.1 for cultures grown at 89°C. Thin-layer chromatography (TLC) followed by APCI–MS analysis provided evidence for no more than one pentacycle in the hydrocarbon chains of tetraether lipid from cultures grown at 70°C and up to 2 pentacycles in the tetraether lipid from cultures grown at higher temperatures. Analysis of the polar lipid extract using TLC and negative-ion ESI–MS suggested the presence of diether and tetraether phospholipids with inositol, glycosyl, and ethanolamine headgroup chemistry.     

Ether polar lipids of methanogenic bacteria: structures, comparative aspects, and biosyntheses.

Complete structures of nearly 40 ether polar lipids from seven species of methanogens have been elucidated during the past 10 years. Three kinds of variations of core lipids, macrocyclic archaeol and two hydroxyarchaeols, were identified, in addition to the usual archaeol and caldarchaeol (for the nomenclature of archaeal [archaebacterial] ether lipids, see the text). Polar head groups of methanogen phospholipids include ethanolamine, serine, inositol, N-acetylglucosamine, dimethyl- and trimethylaminopentanetetrol, and glucosaminylinositol. Glucose is the sole hexose moiety of glycolipids in most methanogens, and galactose and mannose have been found in a few species. Methanogen lipids are characterized by their diversity in phosphate-containing polar head groups and core lipids, which in turn can be used for chemotaxonomy of methanogens. This was shown by preliminary simplified analyses of lipid component residues. Core lipid analysis by high-pressure liquid chromatography provides a method of determining the methanogenic biomass in natural samples. There has been significant progress in the biosynthetic studies of methanogen lipids in recent years. In vivo incorporation experiments have led to delineation of the outline of the synthetic route of the diphytanylglycerol ether core. The mechanisms of biosynthesis of tetraether lipids and various polar lipids, and cell-free systems of either lipid synthesis, however, remain to be elucidated. The significance and the origin of archaeal ether lipids is discussed in terms of the lipid composition of bacteria living in a wide variety of environments, the oxygen requirement for biosynthesis of hydrocarbon chains, and the physicochemical properties and functions of lipids as membrane constituents.     

Two new phospholipids,hydroxyarchaetidylglycerol and hydroxyarchaetidylethanolamine,from the Archaea Methanosarcina barkeri

The structures of two new ether phospholipids of the methanogenic Archaea, Methanosarcina barkeri, were determined as hydroxyarchaetidylglycerol and hydroxyarchaetidylethanolamine by means of chemical, chromatographic and enzymatic analyses, and fast atom bombardment-mass spectrometry. These lipids are hydroxy diether analogs of phosphatidylglycerol and phosphatidylethanolamine, respectively, with β-hydroxyarchaeol (2-O-(3′-hydroxy)phytanyl-3-O-phytanyl-sn-glycerol) as a core lipid. In addition, two other ether phospholipids, usual archaetidylglycerol and archaetidylethanolamine, were also identified in the organism. The stereochemical structure of the unalkylated glycerophosphate of hydroxyarchaetidylglycerol and archaetidylglycerol was determined as sn-glycerol-3-phosphate by use of sn-glycerol-3-phosphate dehydrogenase. The stereochemical configuration of the glycerophosphoglycerol backbone of these lipids was a mirror image of that of diacylphosphatidylglycerol from the organisms of the domains Bacteria and Eucarya, and it was shared with extremely halophilic Archaea. These four phospholipids, in addition to five lipids that had already been reported, accounted for 88% of the total polar lipids of this organism.     

Monolayers of ether lipids from archaebacteria

The surface behavior of six different ether lipids from archaebacteria, based on condensation of glycerol or more complex polyols with two isoprenoid alcohols at 20 or 40 carbon atoms, was investigated in monolayers at the air-water interface.The compounds with no complex polar group (GD, GDGT, GDNT) form monolayers showing a reversible collapse at surface pressure as low as 22 dynes/cm. This collapse pressure decrease with temperature in such a way that the film tension remains constant. In condensed films, these molecules do not assume a completely upright position.Lipids with complex polar ends (HL, GLB, PLII) form films more stable to compression. Forcearea characteristics and surface moment values of HL monolayers are similar to those of analogous ester lipids with fatty acid chains. Monolayers of the two bipolar lipids, GLB and PLII, at room temperature present a more condensed state, probably due to the lateral cohesion between long alkyl chains, but a lower collapse pressure.For all bipolar lipids, the area expansion induced by temperature increase is larger than that of monopolar ones.Abbreviations GD Glycerol diether (2,3-di-O-phytanyl-sn-glycerol - GDGT Glycerol-dialkyl-glycerol tetraether - GDNT Glycerol-dialkyl-nonitol tetraether - GLB Glycolipid B - PLII Phospholipid II - HL Total lipid extract from Halobacterium halobium     

Composition, accumulation and utilization of yolk lipids in teleost fish

Lipid reserves in teleost eggs are stored in lipoprotein yolk and, in some species, a discrete oil globule. Lipoprotein yolk lipids are primarily polar lipids, especially phosphatidylcholine and phosphatidylethanolamine (PE), and are rich in (n–3) polyunsaturated fatty acids, especially 22:6(n–3) (docosahexaenoic acid, DHA). Oil consists of neutral lipids and is rich in monounsaturated fatty acids (MUFA). Egg lipids are derived from dietary fatty acid, fatty acid mobilized from reserves and possibly fatty acid synthesized de novo. There is selective incorporation of essential fatty acids, particularly DHA, into yolk lipids and discrimination against incorporation of 22:1(n–11). Lipid is delivered to the oocyte by vitellogenin, which is rich in polar lipids, and likely also by other lipoproteins, especially very low density lipoprotein, which is rich in triacylglycerol (TAG). All classes of lipid may be used as fuel during embryonic and larval development and MUFA are preferred fatty acids for catabolism by embryos. Catabolism of oil globules is frequently delayed until latter stages of development. In some species, DHA derived from hydrolysis of phospholipid may be conserved by transfer to the neutral lipid. Recent work has expanded knowledge of the role of DHA in membrane structure, especially in neural tissue, and molecular species analysis has indicated that PE containing sn-1 oleic acid is a prime contributor to membrane fluidity. The results of this type of study provide an explanation for the selection pressures that influence yolk lipid composition. Future work ought to expand knowledge of specific roles of individual fatty acids in embryos along with knowledge of the ecological physiology of ovarian recrudescence, environmental influences on vitellogenin and yolk lipid composition, and the control of yolk lipid accumulation and utilization.     

Lipid composition of positively buoyant eggs of reef building corals

Lipid composition of the eggs of three reef building corals, Acropora millepora, A. tenuis and Montipora digitata, were determined. Sixty to 70% of the egg dry weight was lipid, which consisted of wax esters (69.5–81.8%), triacylglycerols (1.1–8.4%) and polar lipids c/mainly phospholipids (11.9–13.2%). Montipora digitata also contained some polar lipids typical of the thylakoid membrane in chloroplasts, probably due to the presence of symbiotic zooxanthellae in the eggs. The wax esters appeared to be the major contributor to positive buoyancy of the eggs, and specific gravity of wax esters in A. millepora was estimated to be 0.92. Among the fatty acids of the wax esters, 34.9–51.3% was hexadecanoic acid (16:0) while the major fatty acids in polar lipids were octadecenoic acid (18:1), hexadecanoic acid (16:0), eicosapentaenoic acid (20:5) and eicosatetraenoic acid (20:4). The wax ester appears to be the main component of the 4.5 6.0 m diameter lipid droplets which fill most of the central mass of the coral eggs.     

Phenotypic Features of Ferroplasma acidiphilum Strains YT and Y-2

Earlier, we described a new family of mesophilic, strictly autotrophic Fe²⁺-oxidizing archaebacteria, Ferroplasmaceae, which belongs to the order Thermoplasmales and includes the genus Ferroplasma and the species F. acidiphilum (strain Y^T) [1]. The present work is concerned with a comparative study of phenotypic characteristics of the type strain Y and a new strain, F. acidiphilum Y-2, isolated from dense pulps during oxidation of gold-containing arsenopyrite/pyrite concentrates from the Bakyrchikskoe (Kazakhstan) and Olimpiadinskoe (Krasnoyarsk krai) ore deposits, respectively. The G+C content of DNA from strains Y^T and Y-2 comprised 35.1 and 35.2 mol %, respectively; the level of DNA–DNA homology between the strains was 84%. Restriction profiles of chromosomal DNA from both strains exhibited a similarity coefficient of 0.87. Genotypic characteristics of these strains indicate their affiliation to the same species. The cells of both strains are polymorphic and lack cell walls. Strains of F. acidiphilum oxidized ferrous iron and pyrite as the sole source of energy and fixed carbon dioxide as the sole carbon source. The strains required yeast extract as a growth factor. Optimum pH for cell growth ranged from 1.7 to 1.8; the temperature optima for the growth of strains Y^T and Y-2 were 34–36 and 40–42°, respectively. Comparative analysis of the total lipids revealed their close similarity in the strains; two glycophospholipids comprised 90% of the total lipids: lipid I, -D-glucopyranosylcaldarchaetidylglycerol (about 55%), and lipid II, trihexosylcaldarchaetidylglycerol (26%), whose isopranyl chains contained no cyclopentane rings. The carbohydrate fraction of lipid I hydrolysate contained only D-glucose, whereas hydrolysate of lipid II contained both D-glucose and D-galactose in a molar ratio of 2 : 1. Thus, it was established that the intraspecies phylogenetic divergence within F. acidiphilum is manifested in the two strains by different temperature optima against a background of similarity in other phenotypic properties.     

Lipids and fatty acids of two pelagic cottoid fishes (Comephorus spp) endemic to Lake Baikal

Matured females of two Lake Baikal endemic fish species, Comephorus baicalensis and Comephorus dybowski, have been investigated for lipid of the whole body and specific tissues (liver, muscles, ovaries), phospholipid classes and fatty acids of neutral and polar lipids. Total lipid in the body (38.9% fresh weight), liver (23.5%) and muscles (14.5%) of C. baicalensis were greater than those of C. dybowski (4.7, 8.7 and 2.6%, respectively); only their ovaries were similar (5.3 and 5.6% lipid, respectively). In both species, phosphatidylcholine and phosphatidylethanolamine were the major phospholipids, ranging from 60.7 to 75.1% of total phospholipid and 14.5–25.7%, respectively. In most cases, monounsaturated fatty acids (MUFA) were the major fatty acid group in C. baicalensis, whereas polyunsaturated fatty acids (PUFA) were the major group in C. dybowski. The MUFA 18:1(n-9) prevailed over other fatty acids in C. baicalensis and varied from 19% in polar lipids of muscles to 56.1% in neutral lipids of muscles. In polar lipid of C. dybowski, the PUFA 22:6(n-3) prevailed over other fatty acids in muscles and ovaries, while 16:0 dominated polar liver lipids and neutral lipids of all tissues. Other major fatty acids included 16:1(n-7), 18:1(n-7), and 20:5(n-3). Values of the (n-3)/(n-6) fatty acid ratio for neutral lipids of C. baicalensis (0.5–0.9) are well below the range of values characteristic either for marine or freshwater fish, while these values for polar lipids (1.6–1.8) are in the range typical of freshwater fish. Neutral lipid fatty acid ratios in C. dybowski (2.5–3.1) allow it to be assigned to freshwater fish, but polar lipids (2.8–3.7) leave it intermediary between freshwater and marine fish.     

A new set of regulatory molecules in plants: A plant phospholipid similar to platelet-activating factor stimulates protein kinase and proton-translocating ATPase in membrane vesicles

1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, an ether phospholipid from mammals known as platelet-activating factor (PAF), specifically stimulates proton transport in zucchini (Cucurbita pepo L.) microsomes (G.F.E. Scherer, 1985, Biochem. Biophys. Res. Commm. 133, 1160–1167). When plant lipids were analyzed by two-dimensional thin-layer chromatography a lipid was found with chromatographic properties very similar to the PAF (G.F.E. Scherer and B. Stoffel, 1987, Planta, 172, 127–130). This lipid was isolated from zucchini hypocotyls, red beet root, lupin root, maize seedlings and crude soybean phospholipids. It had biological activity similar to that of the PAF, based on phosphorus content, and stimulated the steady-state pH in zucchini hypocotyl microsomes about twofold. Other phospholipids, monoglyceride, diglyceride, triglyceride, oleic acid, phorbol ester, and 1-O-alkylglycerol did not stimulate proton transport. When microsomes were washed the PAF was ineffective but when soluble protein was added the PAF stimulation of H⁺ transport was reconstituted. The soluble protein responsible for the PAF-dependent stimulation of transport activity could be partially purified by diethylaminoethyl Sephacel column chromatography. In the same fractions where the PAF-dependent transport-stimulatory protien was found, a protein kinase was active. This protein kinase was stimulated twofold either by the PAF or by Ca²⁺. When Ca²⁺ was present the PAF did not stimulate protein-kinase activity. When either the PAF, protein kinase, or both were added to membranes isolated on a linear sucrose gradient, ATPase activity was stimulated up to 30%. Comparison with marker enzymes indicated the possibility that tonoplast and plasma-membrane H⁺-ATPase might be stimulated by the PAF and protein kinase. We speculate that a PAF-dependent protein kinase is involved in the regulation of proton transport in plants in vitro and in vivo.Abbreviations BTP 1,3-bis[tris(hydroxymethyl)-methylamino] propane - DEAE diethylaminoethyl - EGTA ethylene glycolbis(-aminoethyl ether)-N,N,N,N,-tetraacetic acid - Mes 2-(N-morpholino)ethanesulfonic acid - PAF platelet-activating - factor 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine     

Actin-induced perturbation of PS lipid–cholesterol interaction: A possible mechanism of cytoskeleton-based regulation of membrane organization

To obtain insight into the potential role of the cytoskeleton on lipid mixing behavior in plasma membranes, the current study explores the influence of physisorbed actin filaments (F-actin) on lipid–lipid phase separations in planar model membrane systems containing raft-mimicking lipid mixtures of well-defined compositions using a complementary experimental approach of epifluorescence microscopy, fluorescence anisotropy, wide-field single molecule fluorescence microscopy, and interfacial rheometry. In particular, we have explored the impact of F-actin on cholesterol (CHOL)–phospholipid interactions, which are considered important for the formation of CHOL-enriched lipid raft domains. By using epifluorescence microscopy, we show that physisorbed filamentous actin (F-actin) alters the domain size of lipid–lipid phase separations in the presence of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS) and cholesterol (CHOL). In contrast, no actin-induced modification in lipid–lipid phase separations is observed in the absence of POPS or when POPS is replaced by another anionic lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG). Wide-field single molecule fluorescence microscopy on binary lipid mixtures indicate that PS and PG lipids show similar electrostatic interactions with physisorbed actin filaments. Complementary fluorescence anisotropy experiments on binary PS lipid-containing lipid mixtures are provided to illustrate the actin-induced segregation of anionic lipids. The similarity of electrostatic interactions between actin and both anionic lipids suggests that the observed differences in actin-mediated perturbations of lipid phase separations are caused by distinct PS lipid–CHOL versus PG lipid–CHOL interactions. We hypothesize that the actin cytoskeleton and some peripheral membrane proteins may alter lipid–lipid phase separations in plasma membranes in a similar way by interacting with PS lipids.     

Arsenite as an Inducer of Lipid Peroxidation in Saccharomyces cerevisiae Cells

The ability of sodium arsenite at concentrations of 10^–2, 10^–4, and 10^–6 M to induce lipid peroxidation in Saccharomyces cerevisiae cells was studied. Arsenite at the concentrations 10^–2 and 10^–4 M enhanced lipid peroxidation and inhibited the growth of yeast cells. Enhanced lipid peroxidation likely induced oxidative damage to various cellular structures, which led to suppression of the metabolic activity of cells. Arsenite at the concentration 10^–6 M did not activate lipid peroxidation in cells. All of the tested arsenite concentrations inhibited the activity of -ketoglutarate dehydrogenase and pyruvate dehydrogenase in cells. The inference is made that the toxicity of arsenite may be related to its stimulating effect on intracellular lipid peroxidation.     

A monophasic extraction strategy for the simultaneous lipidome analysis of polar and nonpolar retina lipids

Lipid extraction using a monophasic chloroform/methanol/water mixture, coupled with functional group selective derivatization and direct infusion nano-ESI-high-resolution/accurate MS, is shown to facilitate the simultaneous analysis of both highly polar and nonpolar lipids from a single retina lipid extract, including low abundance highly polar ganglioside lipids, nonpolar sphingolipids, and abundant glycerophospholipids. Quantitative comparison showed that the monophasic lipid extraction method yielded similar lipid distributions to those obtained from established “gold standard” biphasic lipid extraction methods known to enrich for either highly polar gangliosides or nonpolar lipids, respectively, with only modest relative ion suppression effects. This improved lipid extraction and analysis strategy therefore enables detailed lipidome analyses of lipid species across a broad range of polarities and abundances, from minimal amounts of biological samples and without need for multiple lipid class-specific extractions or chromatographic separation prior to analysis.     

Lipoamino acids and sulfonoplipids in Cytophaga johnsonae Stanier strain C21 and six related species of Cytophaga

Cytophaga johnsonae Stanier strain C21 (C. johnsonae C21) contains phosphatidylethanolamine (PE), an unusual glycine-containing lipid (glycine lipid), and two kinds of unidentified lipid as major lipid components. One of the latter lipids was identified by chemical and physicochemical methods as iso-3-hydroxy fatty acid, -amide linked to ornithine and esterified to iso-nonhydroxy fatty acid (ornithine lipid). The other lipid was identified as a sulfonolipid by a tracer experiment using ³⁵S. PE, glycine lipid and sulfonolipid were found in all seven species of Cytophage examined, namely, C. huchinsonii, C. heparina, C. johnsonae C21, C. aquatilis, and three unidentified species of Cytophaga. However, ornithine lipid was found only in the latter five species. By contrast, a serine-containing lipid, which is a specific lipid component of Flavobacterium species, was not found in any species of Cytophaga examined. The possible use and significance of amino acid-containing lipids and sulfonolipids as chemosystematic markers of the Cytophaga species are discussed.     

Composition of the lipids of <Emphasis Type="Italic">Nanoarchaeum equitans</Emphasis> and their origin from its host <Emphasis Type="Italic">Ignicoccus</Emphasis> sp. strain KIN4/I

The contents and nature of the membrane lipids of Nanoarchaeum equitans and Ignicoccus sp. strain KIN4/I, grown at 90°C, and Ignicoccus sp. strain KIN4/I, cultivated at its lowest and highest growth temperatures (75°C and 95°C) were analyzed. Both organisms contained very simple and qualitatively identical assemblages of glycerol ether lipids, showing only differences in the amounts of certain components. LC–MS analyses of the total lipid extracts revealed that archaeol and caldarchaeol were the main core lipids. The predominant polar headgroups consisted of one or more sugar residues attached either directly to the core lipid or via a phosphate group. GC–MS analyses of hydrolyzed total lipid extracts revealed that the co-culture of N. equitans and Ignicoccus sp. strain KIN4/I, as well as Ignicoccus sp. strain KIN4/I grown at 90°C, contained phytane and biphytane in a ratio of approximately 4:1. Purified N. equitans cells and Ignicoccus sp. strain KIN4/I cultivated at 75°C and 95°C had a phytane to biphytane ratio of 10:1. Sugar residues were mainly mannose and small amounts of glucose. Consistent ¹³C fractionation patterns of isoprenoid chains of N. equitans and its host indicated that the N. equitans lipids were synthesized in the host cells.     

Development of resistance in slow-growing rhizobia to 1-naphthyl-N-methyl carbamate (sevin)

Summary Different strains of rhizobia (isolated fromLotus corniculatus andVigna unguiculata) andRhizobium meliloti adapt to sevin (50 g/ml). The number of transfers (20–31) and days of incubation (80–130) during which different strains of rhizobia develop resistance varied. The results of reversion of resistance to sevin, experiments showed that the resistance developed was stable. Rate of growth was faster in resistant strains but their final cell numbers were less than those of sensitive strains. Dehydrogenase activity increased with the development of resistance to sevin, except in strain D-467. With the development of resistance to sevin, total lipids and phospholipids decreased, glycolipids increased and neutral lipids varied. Presence of glycerol, sodium oleate and sodium acetate (known to stimulate lipid production) and flavin mononucleotide and wheat germ lipases (known to decrease lipid production) in the culture medium did not change the growth pattern and lipids of the sevin resistant and sensitive strains of rhizobia.     

Interaction of eosinophil granule major basic protein with synthetic lipid bilayers: A mechanism for toxicity

Summary Eosinophil granule major basic protein (MBP) is a potent toxin for mammalian cells and helminths, but the mechaism of its toxicity is not known. Here we tested whether MBP toxicity is exerted through its effect on the lipid bilayer of its targets. Liposomes prepared from synthetic phospholipids were used as targets for MBP and their properties examined by fluorescence and circular dichroism (CD) spectroscopy. MBP caused a change in the temperature transition profiles of acidic liposomes (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl serine or an equimolar mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid) and induced their aggregation as shown by fluorescence resonance energy transfer experiments. The CD spectra and fluorescence characteristics of MBP itself were altered by its interaction with acidic lipids. Blue shifts in the emission maxima of the Trp, and of the dimethylaminonaphthyl moiety in acrylodan-labeled MBP, and a reduction in the effectiveness of quenching of Trp fluorescence by acrylamide were observed in the presence of acidic lipids. None of these effects were noted with zwitterionic lipids. This MBP : lipid bilayer interaction resulted in fusion and lysis of liposomes as indicated by the fluorescent indicator calcein. The results demonstrate that MBP associates with acidic lipids and that it disrupts, aggregates, fuses, and lyses liposomes prepared from such lipids. Such interaction might account for its wide range of toxicity.Abbreviations used Acrylodan 6-acryloyl-2-dimethylam-inonaphthalene - CD circular dichroism - DMPA 1,2-dimyrist-oyl-sn-glycero-3-phosphatidic acid - DMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine - DPH 1,6-diphenyl-1,3,5-hexatriene - DTT dithiothreitol - FRET fluorescence resonance energy transfer - HEPES N-2-hydroxyethyl piperazine-N-2-ethane sulfonic acid - K _sv Stern-Volmer constant - K _q bimolecular quenching coefficient - _em emission wavelength - _ex excitation wavelength - MBP major basic protein - MOPC 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine - NBD-PE N-(7-nitro-2,1,3-benzo-xadiazol-4-yl)-phosphatidylethanolamine - nMBP native major basic protein - PBS phosphate-buffered saline - POPC 1-palmit-oyl-2-oleoyl-sn-glycero-3-phosphocholine - POPS 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl serine - raMBP reduced and alkylated major basic protein - RHO-PE rhodamine-phosphatidylethanolamine - Tes N-tris[hydroxymethyl]-methyl-2-amino-ethane-sulfonic acid - Tris tris[hydroxymethyl]-amino-methane We would like to thank Dr. Predrag J.K. Ilich for assistance with initial data analysis, Dr. Salah S. Sedarous for the lifetime data and for helpful discussions, Dr. S. Yu. Venyaminov for helpful discussions, Mr. Kenneth D. Peters and Mr. Peter J. Callahan for assistance with some of the illustrations, and Ms. Jill Wagner for performing the radioimmunoassays. We would also like to thank Ms. Jill Kappers for excellent secretarial work. This work was supported in part by a Fellowship grant from the American Heart Association, Minnesota Affiliate, and by grants from the National Institutes of Health AI 09728 and from the Mayo Foundation. RIA-G is a Fellow of the American Heart Association.     

Lipid composition of thermophilic moulds Acremonium alabamensis and Thermomucor indicae-seudaticae

The total lipid content of Acremonium alabamensis and Thermomucor indicae-seudaticae ranged 2.6–7.3 and 8.5–13.0% of dry mycelium, respectively during development. Neutral lipid fraction increased during growth while polar and phospholipids declined. Both moulds contained palmitic, oleic, linoleic and palmitoleic acids as major fatty acid components in lipids. Degree of unsaturation of lipids of A. alabamensis was greater than that of T. indicae-seudaticae. Neutral lipids were more unsaturated than the polar lipids. The ratio of unsaturation index of polar lipids to neutral lipids was either one or less than one. The principal phospholipids of these moulds were phosphatidyl choline, phosphatidyl ethanolamine and phosphatidic acid. However, phosphatidic acid was not found in very high amounts as observed in Humicola grisea var. thermoidea.     

The fluidity of plasma membranes from ethanol-treated rat liver

Summary Male Wistar rats were maintained for 35–40 days on a liquid diet containing 36% of calories as ethanol. Ethanol was replaced by carbohydrates in the isocaloric diet fed to control animals. The effect of ethanol consumption has been studied on the fluorescence polarization of rat liver plasma membranes and artificial lipid vesicles and on the lipid composition of the membranes. Fluorescence polarization in both membranes and vesicles was determined using DPH and TMA-DPH as fluorescence markers; from these data, the polarization term (r_o/r–1)^–1 and flow activation energy (E) were calculated. The ethanol consumption induces a more fluid environment within the membrane core of liver plasma membranes; the ethanol-fed rat membranes are more resistant to the in vitro effect of ethanol disordering the membrane structure. Vesicles obtained with lipids from either control membranes or ethanol-fed rat membranes were treated with ethanol and the changes in polarization paralleled to those exhibited by the membranes. The absence of phase transitions and of E changes was also shown in temperature-dependence studies. The lower cholesterol content found in ethanol-fed rat plasma membranes might be responsible for observed variations in the microviscosity.Abbreviation OG octyl -D-glucopyranoside     

Alterations in ether lipid metabolism and the consequences for the mouse lipidome

Alkylglycerol monooxygenase (AGMO) and plasmanylethanolamine desaturase (PEDS1) are enzymes involved in ether lipid metabolism. While AGMO degrades plasmanyl lipids by oxidative cleavage of the ether bond, PEDS1 exclusively synthesizes a specific subclass of ether lipids, the plasmalogens, by introducing a vinyl ether double bond into plasmanylethanolamine phospholipids. Ether lipids are characterized by an ether linkage at the sn-1 position of the glycerol backbone and they are found in membranes of different cell types. Decreased plasmalogen levels have been associated with neurological diseases like Alzheimer's disease. Agmo-deficient mice do not present an obvious phenotype under unchallenged conditions. In contrast, Peds1 knockout mice display a growth phenotype. To investigate the molecular consequences of Agmo and Peds1 deficiency on the mouse lipidome, five tissues from each mouse model were isolated and subjected to high resolution mass spectrometry allowing the characterization of up to 2013 lipid species from 42 lipid subclasses. Agmo knockout mice moderately accumulated plasmanyl and plasmenyl lipid species. Peds1-deficient mice manifested striking changes characterized by a strong reduction of plasmenyl lipids and a concomitant massive accumulation of plasmanyl lipids resulting in increased total ether lipid levels in the analyzed tissues except for the class of phosphatidylethanolamines where total levels remained remarkably constant also in Peds1 knockout mice. The rate-limiting enzyme in ether lipid metabolism, FAR1, was not upregulated in Peds1-deficient mice, indicating that the selective loss of plasmalogens is not sufficient to activate the feedback mechanism observed in total ether lipid deficiency.     

Role of phospholipids in the binding of bumetanide to the rabbit parotid Na/K/Cl cotransporter

Summary It was recently reported (Turner, R.J., George, J.N., 1990,J. Membrane Biol. 113:203–210) that the high affinity bumetanide binding site of the rabbit parotid Na/K/Cl cotransporter could be extracted from a basolateral membrane preparation from this gland using relatively low concentrations of the non-ionic detergent Triton X-100. At the detergent: protein ratios required for complete membrane solubilization bumetanide binding activity in this extract was lost but could be recovered by the addition of crude soybean lipids. In the present paper the ability of various purified lipids to restore high affinity bumetanide binding activity in detergent solubilized rabbit parotid basolateral membranes is studied. We show that the effect of exogenous lipid on the detergent-inactivated bumetanide binding site is to increase the affinity of binding without affecting the number of binding sites. Of the 11 lipid species tested, several relatively minor, negatively charged membrane phospholipids are the most effective in restoring binding activity (phosphatidylserine phosphatidylglycerol > phosphatidylinositol > cardiolipin). while the major mammalian plasma membrane lipid components phosphatidylcholine, phosphatidylethanolamine, sphingomyelin and cholesterol are without effect. In addition, we show that in the presence of these minor lipids the affinity of bumetanide binding is considerably increased over that observed in the native membrane (e.g.,K _d 0.06 m in membranes extracted with 0.3% Triton and treated with 0.15% wt/vol phosphatidylserine,vs. K _d 3 m in native basolateral membranes). This dramatic dependence of bumetanide binding affinity on the presence of certain lipid species suggests that the properties of the bumetanide binding proteinin situ may be quite dependent on the minor lipid content of the plasma membrane. This effect may account for the relatively large variations in bumetanide binding affinity observed from tissue to tissue.