Interactions of polymerizable phosphatidylcholine vesicles with blood components: relevance to biocompatibility

We have studied the biocompatibility properties of polymerizable phosphatidylcholine bilayer membranes, in the form of liposomes, with a view toward the eventual utilization of such polymerized lipid assemblies in drug carrier systems or as surface coatings for biomaterials. The SH-based polymerizable lipid 1,2-bis[1,2-(lipoyl)dodecanoyl]-sn-glycero-3-phosphocholine (dilipoyl lipid, DLL) and the methacryl-based lipid 1,2-bis[(methacryloyloxy)dodecanoyl]-sn-glycero-3-phosphocholine (dipolymerizable lipid, DPL) were studied in comparison to ‘conventional’ zwitterionic or charged phospholipids. We examined binding of serum proteins to liposomes and effects of liposomes on fibrin clot formation and on platelet aggregation. All types of liposomes tested bound complex mixtures of serum proteins with IgG being the most abundant bound component. DPL vesicles and anionic vesicles bound substantially more protein than other vesicle types. Polymerized DPL vesicles uniquely bound a protein of about 53 kDa which was not bound to other types of phosphatidylcholine liposomes. Likewise polymerized DPL vesicles, but not other types of phosphatidylcholine vesicles, caused a marked alteration in coagulation as measured by activated partial thromboplastin time (APTT) and prothrombin time (PT) tests; this effect was shown to be due to binding and depletion of clothing factor V by the DPL polymerized vesicles. Polymerized DPL liposomes and DLL liposomes in polymerized or nonpolymerized form, were without substantial effect on platelet aggregation. However, DPL nonpolymerized vesicles, while not causing aggregation, did impair ADP-induced aggregation of platelets. These studies suggest that SH based polymerizable lipids of the DLL type may be very suitable for in vivo use in the contexts of drug delivery systems or biomaterials development. Methacryloyl-based lipids of the DPL type seem to display interactions with the hemostatic process which militate against their in vivo utilization.     

Investigation on lipid asymmetry using lipid probes: Comparison between spin-labeled lipids and fluorescent lipids

Synthetic lipids with a nitroxide or a fluorescent probe have been extensively used during the last 30 years to determine the transmembrane diffusion of phospholipids in artificial or biological membranes. However, the relevance of data obtained with these modified lipids has sometimes been questioned. Beside possible artefacts introduced by the reporter probe, synthetic lipids used in cells often contain a short fatty acid chain in the sn-2 position, which gives them higher water solubility than naturally occurring lipids. In the present review, we have attempted to give a critical appraisal. Main strategies are recalled and important discoveries obtained with lipid probes on transmembrane lipid traffic in eukaryotic cells are briefly summarized. Examples of artefacts caused by lipid probes are given. Comparisons between data obtained by different techniques such as ESR and fluorescence allow us to emphasize the complementary character of the two approaches and more generally show the necessity to use several probes before drawing conclusions concerning endogenous lipids. In spite of these pitfalls, overall, lipid probes have provided a wealth of useful information that, to date, cannot be obtained with unlabeled lipids.     

Spreading and polymerization behavior of diacetylenic phospholipids at the gas-water interface

A variety of polymerizable lipids derived from hexacosa-10,12-diynoic acid and hexacosa-10,12-diyne-1-ol have been synthesized and spread at the gas/water interface. The measured surface pressure/area isotherms indicate that head group charge and bulkiness have strong influences on the area occupied per molecule. In the case of zwitterionic phospholipids additional area changes are brought about by alkaline and acidic subphases, which is probably due to an alteration of head group conformation. Condensed state diacetylenic lipid monolayers in a nitrogen atmosphere are polymerizable by UV irradiation. The polymerization reaction was monitored at the gas/water interface by the area change at constant surface pressure and the change of optical density in the visible region. As already observed for vesicle polymerization, single chain amphiphiles exhibit a different absorption behavior than asymmetric double chain amphiphiles of the phosphoglycerol type. The polymerized monolayers were more densely packed and more stable than their monomeric counterparts as indicated by the smaller areas and higher pressures reached before the collapse points.     

Effects of lipid physicochemical characteristics on interrelations between lipid composition and the mouse liver index

Interrelations between the liver index (the relative mass) and various parameters of the liver lipid composition in 9 groups of intact female SHK mice sacrificed at different Seasons and years groups were studied depending on physicochemical characteristics of their lipids. There were revealed correlations between the liver index and the following parameters of the lipid composition: relative content of cardiolipin, lysoforms of the phospholipid/phosphatidylcholine ratio in liver phospholipids, and the [serine]/[phospholipids] molar ratio. The direction of these correlations has been established to depend on the lipid physicochemical characteristics. In all cases, there is observed a direct correlation if lipids have prooxidant properties and antiperoxide activity and an inverse correlation if lipids are characterized by antioxidant and antiperoxide activities.     

Functional reconstitution of a membrane protein in a diacetylenic polymerizable lecithin

It is shown that polymerized diacetylenic lecithins may be used for the functional reconstitution of a membrane protein. Purple membrane patches isolated from Halobacterium halobium and liposomes of the polymerizable diacetylenic lecithin 1,2-bis(10,12 tricosadiynoyl)-sn-glycero-3-phosphocholine were sonicated together to form mixed vesicles highly enriched in the polymerizable lipid. A net inward proton flow on illumination as determined by the change of pH of the external medium demonstrated the stability of the vesicular form in this mixed lipid system as well as vectorial orientation of the bacteriorhodopsin in the bilayer. When bacteriorhodopsin was incorporated in non-polymerizable lipids, irradiation with ultraviolet light resulted in complete loss of function. In the diacetylenic lipids, the loss of function was slower than the increase in polymer concentration. This demonstrates the utility of the diacetylenic lecithin system for study of interactions between membrane proteins and polymerizable lipids, as well as its potential in the development of biosensors based on membrane proteins.     

THE ULTRASTRUCTURE OF LIPID-DEPLETED ROD PHOTORECEPTOR MEMBRANES

The structure of lipid-depleted retinal rod photoreceptor membranes was studied by means of electron microscopy. Aldehyde-fixed retinas were exhaustively extracted with acetone, chloroform-methanol, and acidified chloroform-methanol. The effect of prefixation on the extractability of lipids was evaluated by means of thin-layer chromatography and fatty acid analysis. Prefixation with glutaraldehyde rendered 38% of the phospholipids unextractable, while only 7% were unextractable after formaldehyde fixation. Embedding the retina in a lipid-retaining, polymerizable glutaraldehyde-urea mixture allows a comparison of the interaction of OsO₄ with lipid-depleted membranes and rod disk membranes which contain all their lipids. A decrease in electron density and a deterioration of membrane fine structure in lipid-depleted tissue are correlated with the extent of lipid extraction. These observations are indicative of the role of the lipid bilayer in the ultrastructural visualization of membrane structure with OsO₄. Negatively stained thin sections of extracted tissue reveal substructures in the lipid-depleted rod membranes. These substructures are probably the opsin molecules which are the major protein component of retinal rod photoreceptor membranes.     

The proteomics of lipid droplets: structure, dynamics, and functions of the organelle conserved from bacteria to humans

Lipid droplets are cellular organelles that consists of a neutral lipid core covered by a monolayer of phospholipids and many proteins. They are thought to function in the storage, transport, and metabolism of lipids, in signaling, and as a specialized microenvironment for metabolism in most types of cells from prokaryotic to eukaryotic organisms. Lipid droplets have received a lot of attention in the last 10 years as they are linked to the progression of many metabolic diseases and hold great potential for the development of neutral lipid-derived products, such as biofuels, food supplements, hormones, and medicines. Proteomic analysis of lipid droplets has yielded a comprehensive catalog of lipid droplet proteins, shedding light on the function of this organelle and providing evidence that its function is conserved from bacteria to man. This review summarizes many of the proteomic studies on lipid droplets from a wide range of organisms, providing an evolutionary perspective on this organelle.     

酵母线粒体的磷脂转运

线粒体是一种由两层膜包被的细胞器,其功能和结构的稳定性取决于线粒体膜上精确的磷脂组成及分布。线粒体膜上的大部分脂类物质由内质网合成,既而转运到线粒体。而部分脂类利用内质网上产生的前体,在线粒体内膜上合成。由此可见,线粒体膜脂的生物合成需要线粒体与内质网以及线粒体外膜(outer mitochondrial membrane, OMM)与内膜(inner mitochondrial membrane, IMM)之间进行大量的脂质转运。目前认为,这种运输过程既可在拴系因子(tether factors)形成的膜结合部位(membrane contact sites, MCSs)内发生,也可借助脂质转运蛋白(lipid transfer proteins, LTPs)完成。近年来,研究者以酵母为对象,建立了多种线粒体磷脂转运(phospholipid trafficking)的模型,这使人们初步理解了线粒体磷脂转运的机制。本综述总结了酵母线粒体磷脂转运的最新发现,并对这些磷脂转运的模型进行了讨论,以期为今后深入了解线粒体脂类代谢提供参考。     

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.     

An efficient synthesis of phosphatidylcholines

This article deals with two of the major steps involved in phospholipid synthesis: the preparation of the optically pure precursors, sn-glycero-3-phosphocholine (GPC) and -ethanolamine, from a convenient lipid source such as egg yolk, and acylation of hydroxyl groups present in those precursors involving an acid to yield the corresponding phospholipid. Phosphatidylcholines and phosphatidylethanolamines were separated from lipids extracted from egg yolk using low-pressure column chromatography. The advantages of this method include the use of smaller volumes of solvents and silica gel and reuse of adsorbent. Acylation of GPC is aided by ultrasound from a common laboratory bath cleaner. Ultrasound-assisted base-catalyzed esterification of GPC is accomplished between 2-6 hours providing a phospholipid in more than 80% yield. This scheme is particularly valuable in the synthesis of polymerizable phospholipids.     

Calorimetric and molecular mechanics studies of the thermotropic phase behavior of membrane phospholipids.

In this review, we summarize the results of recent studies on the main phase transition behavior of phospholipid bilayers using the combined approaches of molecular mechanics simulations and high-resolution differential scanning calorimetry. Following a brief overview of the phase transition phenomenon exhibited by the lipid bilayer, we begin with the review by showing how several structural parameters underlying various phospholipids including phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol are defined and determined. Specifically, these structural parameters are obtained with saturated lipids packed in the gel-state bilayer using computer-based molecular mechanics calculations. Then we proceed to present the calorimetric data obtained with the lipid bilayer composed of saturated phospholipids as it undergoes the gel-to-liquid-crystalline phase transition in excess water. The general equations that can correlate the gel-to-liquid-crystalline phase transition temperature (T(m)) of the lipid bilayer with the structural parameters of the lipid molecule constituting the lipid bilayer are subsequently presented. From these equations, two tables of predicated T(m) values for well over 400 molecular species of saturated phosphatidylcholine and saturated phosphatidylethanolamine are generated. We further review the structure and chain-melting behavior of a large number of sn-1 saturated/sn-2 unsaturated phospholipids. Two T(m)-diagrams are shown, from which the effects of the number and the position of one to five cis carbon-carbon double bonds on T(m) can be viewed simultaneously. Finally, in the last part of this review, simple molecular models that have been invoked to interpret the characteristic T(m) trends exhibited by lipid bilayers composed of unsaturated lipids with different numbers and positions of cis carbon-carbon double bonds as seen in the T(m)-diagram are presented.     

Membrane Lipid Co-Aggregation with α-Synuclein Fibrils

Amyloid deposits from several human diseases have been found to contain membrane lipids. Co-aggregation of lipids and amyloid proteins in amyloid aggregates, and the related extraction of lipids from cellular membranes, can influence structure and function in both the membrane and the formed amyloid deposit. Co-aggregation can therefore have important implications for the pathological consequences of amyloid formation. Still, very little is known about the mechanism behind co-aggregation and molecular structure in the formed aggregates. To address this, we study in vitro co-aggregation by incubating phospholipid model membranes with the Parkinson’s disease-associated protein, α-synuclein, in monomeric form. After aggregation, we find spontaneous uptake of phospholipids from anionic model membranes into the amyloid fibrils. Phospholipid quantification, polarization transfer solid-state NMR and cryo-TEM together reveal co-aggregation of phospholipids and α-synuclein in a saturable manner with a strong dependence on lipid composition. At low lipid to protein ratios, there is a close association of phospholipids to the fibril structure, which is apparent from reduced phospholipid mobility and morphological changes in fibril bundling. At higher lipid to protein ratios, additional vesicles adsorb along the fibrils. While interactions between lipids and amyloid-protein are generally discussed within the perspective of different protein species adsorbing to and perturbing the lipid membrane, the current work reveals amyloid formation in the presence of lipids as a co-aggregation process. The interaction leads to the formation of lipid-protein co-aggregates with distinct structure, dynamics and morphology compared to assemblies formed by either lipid or protein alone.     

Efficient gene transfection by bisguanylated diacetylene lipid formulations

We have previously shown that cationic cholesterol derivatives bearing guanidinium groups were efficient vectors for gene transfer. To further evaluate the potentiality of this novel class of cationic lipids, we undertook to study the transfection efficiency of guanidinium-based lipids with other hydrophobic moieties. Specifically, we synthesized a reagent where two guanidinium groups are linked to a diacetylene lipid which may provide the lipoplexes with favorable structural features. We report here that the cationic lipid bisguanidinium-diacetylene (BGDA) is highly efficient for in vitro gene transfection when formulated with dioleoylphosphatidyl ethanolamine (DOPE). We also show that liposomes composed of BGDA, DOPE, and a neutral diacetylene colipid, hydroxyethylenediacetylene (HEDA), are efficient for transfection. Thus, diacetylene-based lipids provide a novel scaffold for gene transfection and will be particularly useful for gaining new insights into the structure-activity relationships of the lipid/DNA complexes as they offer a means to study the effects of polymerizable domains.     

Purification and characterization of phthalanilide--lipid complexes from tissues

What appears to be a new class of phospholipids has been isolated from dog brain in the form of complexes with a substituted phthalanilide. The complexes were extracted by chloroform-methanol and purified by countercurrent distribution in solvent systems containing water, chloroform, methanol, and Freon 113. The binding of the phthalanilide congener to lipids has some ionic character. Cations such as H(+) or Ca(++) displaced the phthalanilide from its lipid complex. The pH for 50% displacement acid was about 3.8 and was independent of the purity of the complex. Thin-layer chromatography of the lipid yielded four subfractions of lipid, three of which were ninhydrin-positive and all of which yielded a group of unidentified ninhydrin-positive components on hydrolysis. Each lipid subfraction contained nitrogen, phosphorus, fatty acids, and glycerol but in different ratios. Of the known phospholipids containing nitrogen, none matches the composition and behavior of the lipids isolated as phthalanilide complexes. We have therefore concluded that the phthalanilides bind to a new class of phospholipids characterized by a high content of unidentified ninhydrin-positive components.     

Lipid peroxidation in rat uterus

Lipid peroxidation in rat uterus has been studied using NADPH- and ascorbate-induced systems. Lipid peroxidation in rat uterus is low as compared to rat liver. Uterus is more sensitive to ascorbate-induced lipid peroxidation than that induced by NADPH. Uterus contains lower amounts of phospholipids and has a lesser degree of unsaturation in lipids. Co-factor studies show that Fe2+ is more important for ascorbate-induced lipid peroxidation. Endometrium is more sensitive to ascorbate-induced lipid peroxidation than myometrium. It also contains more total lipids and phospholipids besides having a higher degree of unsaturation in the lipids as compared to myometrium. Among the subcellular fractions, mitochondria are more prone to ascorbate-induced lipid peroxidation, whereas microsomes are more sensitive to NADPH-induced lipid peroxidation. Uteri from old rats (24 months) and pregnant rats are more resistant to lipid peroxidation than those from 3-month-old control rats. Uterus of pregnant rats contains more factors which inhibit lipid peroxidation and also has a lesser degree of unsaturation in lipids compared with uterus of control rats. The possible consequences of the resistance of uterus to lipid peroxidation, especially during pregnancy and senescence, are discussed.     

Lipid composition of lysosomal multilamellar bodies of male mouse urine

Previous studies from our laboratory have shown that male C57BL/6J mice excrete into the urine multilamellar lysosomal bodies that contain specific neutral glycosphingolipids. These mice excrete approximately 20-30% of their kidney glycolipids each day. The significance and function of this secretion of multilamellar lysosomal organelles is unknown. To characterize these excreted bodies further, we report here their neutral lipid and phospholipid composition. The bodies were collected by differential centrifugation, extracted with chloroform-methanol, and lipids were fractionated into neutral lipids, glycolipids, and phospholipids. The neutral lipids consisted primarily of cholesterol, dolichol, and ubiquinone. The phospholipid fraction consisted primarily of a single molecular species of phosphatidylcholine. This lipid which comprises more than 90% of the total phospholipids was found to contain 16:0 ether and C22:6 n-3 fatty acid as determined by gas-liquid chromatography-mass spectrometry. The glycosphingolipids as reported previously consisted primarily of galabiosylceramides and globotriaosylceramides. This membrane lipid composition is different from any previously reported cellular organelle.     

Fatty-acid composition of lipids and physicochemical characteristics of membrane fractions and the membrane of endoplasmic reticulum of thymus and Pliss' lymphosarcoma

The paper is concerned with composition of neutral lipids and phospholipids, the regularity of lipid bilayer and structural reorganization of plasma membranes, and membranes of smooth and rough cell reticulum of thymus and Pliss lymphosarcoma are studied at linear and stationary growth phase. No qualitative differences are found in the fatty-acid composition of lipid membranes in normal and tumour cells. In plasma membranes of phospholipids and in membranes of smooth reticulum of tumour cells the unsaturated lipid component increases in the process of growth, the cholesterin/phospholipids ratio decreases, fluidity of the lipid bilayer diminishes and structural heterogeneity of these membranes rises while in membranes of rough reticulum the saturation of lipids increases, but the cholesterin/phospholipids ratio does not change. The temperatures of structural reorganization also does not change, which evidences for a less structural lability of membranes of rough reticulum as compared with other membranes.     

Etude comparee des lipides et de la fixation passive du calcium dans les racines et les fractions subcellulaires du Lupinus luteus et de la Vicia faba

In both lupin and broad bean, the root lipids contain paraffins, triglycerides, diglycerides, free fatty acids and polar lipids (phospholipids and galactolipids). The polar lipids and the triglycerides are the more abundant classes. The root galactolipids are mono- and di-galactosyldiglycerides; two steryl glycosides are also present. The phospholipids in both species are: phosphatidylinositol, phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, diphosphatidylglycerol and phosphatidic acid. This last phospholipid represents 8·3% of total lipid phosphorus in Lupinus against 2·3% in Vicia. The other acidic phospholipids represent 30·4% in Lupinus against 20·9% in Vicia. The lipids of Lupinus are rich in linolenic acid whereas those found in Vicia are richer in linoleic acid. The various subcellular fractions prepared from the roots of both species have an homogeneous lipid composition, reflecting exactly that of entire cells. The calcium passive fixation capacity in microsomes and mitochondria of Lupinus roots is more important than that in the same organelles of Vicia faba roots. Thus a relationship is suggested between the amount of phospholipids in membranes and the passive fixation of calcium.     

Interactions of dipalmitoylphosphatidylcholine with ceramide-based mixtures

The outermost layer of the skin, the stratum corneum (SC), acts as the natural physical barrier. The SC consists of corneocytes embedded in a crystalline lipid matrix consisting of ceramides, free fatty acids and cholesterol.Although phospholipids are frequently present in topical formulations, no detailed information is reported on the interactions between phospholipids and SC lipids. The aim of this study was to examine the interactions between a model phospholipid, dipalmitoylphosphatidylcholine (DPPC) and synthetic ceramide-based mixtures (referred to as SC lipids).(Perdeuterated) DPPC was mixed with SC lipids and the lipid organization and mixing properties were examined. The studies revealed that DPPC participates in the same lattice as SC lipids thereby enhancing a hexagonal packing. Even at a high DPPC level, no phase separated pure DPPC was observed.When a DPPC containing formulation is applied to the skin surface it must partition into the SC lipid matrix prior to any mixing with the SC lipids. To mimic this, DPPC was applied on top of a SC lipid membrane. DPPC applied in a liquid crystalline state was able to mix with the SC lipids and participated in the same lattice as the SC lipids. However, when DPPC was applied in a rippled gel-state very limited partitioning of DPPC into the SC lipid matrix occurred. Thus, when applied to the skin, liquid crystalline DPPC will have very different interactions with SC lipids than DPPC in a (rippled-)gel phase.     

Minimal Effect of Lipid Charge on Membrane Miscibility Phase Behavior in Three Ternary Systems

Giant unilamellar vesicles composed of a ternary mixture of phospholipids and cholesterol exhibit coexisting liquid phases over a range of temperatures and compositions. A significant fraction of lipids in biological membranes are charged. Here, we present phase diagrams of vesicles composed of phosphatidylcholine (PC) lipids, which are zwitterionic; phosphatidylglycerol (PG) lipids, which are anionic; and cholesterol (Chol). Specifically, we use DiPhyPG-DPPC-Chol and DiPhyPC-DPPG-Chol. We show that miscibility in membranes containing charged PG lipids occurs over similarly high temperatures and broad lipid compositions as in corresponding membranes containing only uncharged lipids, and that the presence of salt has a minimal effect. We verified our results in two ways. First, we used mass spectrometry to ensure that charged PC/PG/Chol vesicles formed by gentle hydration have the same composition as the lipid stocks from which they are made. Second, we repeated the experiments by substituting phosphatidylserine for PG as the charged lipid and observed similar phenomena. Our results consistently support the view that monovalent charged lipids have only a minimal effect on lipid miscibility phase behavior in our system.