Unsaturated Phosphatidylcholine Research Articles

An Extension and Further Validation of an All-Atomistic Force Field for Biological Membranes.

Biological membranes are versatile in composition and host intriguing molecular processes. In order to be able to study these systems, an accurate model Hamiltonian or force field (FF) is a necessity. Here, we report the results of our extension of earlier developed all-atomistic FF parameters for fully saturated phospholipids that complements an earlier parameter set for saturated phosphatidylcholine lipids (J. Phys. Chem. B, 2012, 116, 3164-3179). The FF, coined Slipids (Stockholm lipids), now also includes parameters for unsaturated phosphatidylcholine and phosphatidylethanolamine lipids, e.g., POPC, DOPC, SOPC, POPE, and DOPE. As the extended set of parameters is derived with the same philosophy as previously applied, the resulting FF has been developed in a fully consistent manner. The capabilities of Slipids are demonstrated by performing long simulations without applying any surface tension and using the correct isothermal-isobaric (NPT) ensemble for a range of temperatures and carefully comparing a number of properties with experimental findings. Results show that several structural properties are very well reproduced, such as scattering form factors, NMR order parameters, thicknesses, and area per lipid. Thermal dependencies of different thicknesses and area per lipid are reproduced as well. Lipid diffusion is systematically slightly underestimated, whereas the normalized lipid diffusion follows the experimental trends. This is believed to be due to the lack of collective movement in the relatively small bilayer patches used. Furthermore, the compatibility with amino acid FFs from the AMBER family is tested in explicit transmembrane complexes of the WALP23 peptide with DLPC and DOPC bilayers, and this shows that Slipids can be used to study more complex and biologically relevant systems.

Comparison of Ternary Bilayer Mixtures with Asymmetric or Symmetric Unsaturated Phosphatidylcholine Lipids by Coarse Grained Molecular Dynamics Simulations

We studied the phase behavior of various ternary bilayer mixtures composed of cholesterol, an unsaturated lipid, and a fully saturated lipid, by means of molecular dynamics simulations of the MARTINI coarse grain model. We aimed at comparing lateral organization and local properties of bilayers containing phosphatidylcholine (PC) lipids, either with two unsaturated tails (symmetric), or one unsaturated and one saturated tail (asymmetric), as the low-melting component of the mixture. The number of unsaturations per chain was systematically varied in both classes of unsaturated lipids, to account for its consequences in segregation. In the asymmetric unsaturated PCs, the saturated tail was kept identical to the hydrophobic chains of the fully saturated lipid component. Membranes with a symmetric or an asymmetric unsaturated lipid, with the same kind of unsaturated chain, show different phase behavior. Symmetric polyunsaturated PCs set the separation in two phases. Instead, the asymmetric polyunsaturated lipids induced nonideal mixing of components in single-phase bilayers. A significative drop of temperature, within the accessible temperature range, enhances the segregation in mixtures with the more unsaturated asymmetric PC, but still within a single phase. This different phase behavior between membranes with symmetric and asymmetric unsaturated PCs is also observed for lipids with the same total number of unsaturations. On the other hand, the degree of unsaturation per se enhances the segregation, by increasing the composition fluctuations in single-phase membranes with asymmetric PC lipids, and raising the line tension in the two-phase bilayer mixtures with symmetric polyunsaturated PCs. Dynamic clusters of unsaturated asymmetric lipids can be identified. The clusters show no correlation between leaflets, as observed for the phase domains in mixtures with the symmetric polyunsaturated PCs. Interestingly, we found that asymmetric PC lipids have a preferential orientation such that their saturated tails increase their density toward the periphery of the clusters, facing regions enriched in the fully saturated lipids and cholesterol. The degree of unsaturation increases the cluster size and also enhances the anisotropy of the orientation. The surface density of cholesterol follows a gradient that favors its interaction with the saturated tails. Such gradients in composition lead to gradients in order parameters, such as the conformational order and the area of the tails, which increases away from the unsaturated lipid clusters. We compared, in addition, differences in hydrophobic length mismatch between acyl chains of the low-melting and high-melting components, in mixtures containing either symmetric or asymmetric unsaturated lipids.

Inter-Leaflet Coupling and Domain Formation in Asymmetric Giant Unilamellar Vesicles

Cellular membranes exhibit an asymmetric distribution of lipids across the two leaflets of the bilayer. While the sphingolipid-rich outer (exoplasmic) leaflet has a lipid composition prone to lateral segregation and domain formation, the situation in the sphingolipid-poor inner (cytosolic) leaflet is unclear. The interaction between the two leaflets (i.e. the inter-leaflet coupling), although involved in several biological processes, is not well understood. We recently developed a highly reproducible solvent-free method for preparing asymmetric giant unilamellar vesicles (GUV). The method is fast, has a high yield, can be used with a wide variety of lipids and is compatible with the reconstitution of transmembrane proteins.The asymmetry of the GUVs was investigated using fluorescence correlation spectroscopy (FCS) in order to probe dynamics of lipids in the two leaflets. We have shown that introduction of sphingomyelin exclusively into the outer leaflet of GUVs composed of an unsaturated phosphatidylcholine (PC) results in a much larger decrease in outer leaflet lateral diffusion compared to the effect on the inner leaflet. Furthermore, by systematically comparing different PCs, we found that the degree to which the inner leaflet dynamics is coupled to that in the outer leaflet depends on the length and saturation of the acyl chains and, possibly, the interaction between the lipids’ terminal methyl groups at the bilayer mid-plane. In more detail, preliminary studies indicate that the degree of coupling increases both when: i) the PC has one saturated acyl chain (independently from its Tm) or ii) the SM is asymmetric and interdigitates into the opposing leaflet. These findings suggest how cells might modulate the inter-leaflet coupling - and thus the flow of information through the membrane - by simply varying its lipid composition.

Thermodynamic and structural insight into the underlying mechanisms of the phosphatidylcholine liposomes – casein associates co-assembly and functionality

The combination of data obtained from isothermal mixing calorimetry and light scattering allowed us to reveal the relationships between the character of the interactions of casein (β-casein and sodium caseinate (SCN) particles) with phosphatidylcholine (PC) liposomes and their specific properties, such as, the hydrophilic-lipophilic balance of the surface properties, the size, and the architecture. The size distribution diagrams, obtained by dynamic light scattering for the particles of pure PC, β-casein and their complexes, indicated the involvement of both the protein and liposome particles in the complex formation at pH 5.5 and low ionic strength (0.001 M). As this took place, the data of the extraction of the free PC from the aqueous solutions of the complex particles by diethyl ether showed about 90% of the binding of the PC liposomes by the β-casein particles. As a result of the interactions, the complex particles showed markedly higher values of their density, calculated on the basis of the light scattering data, as compared to the ones, which were inherent to the pure protein particles. The generality of the importance of such a structural parameter as the density of the complex (protein + PC) particles for their function as protectors against oxidation for the unsaturated PC was supported by the data obtained for the complex particles formed between SCN and PC liposomes at pH 5.5, 40 °C and the low ionic strength (0.001 M). In addition, the importance of both the density and the architecture of the complex (protein + PC) particles for their susceptibility to the proteolysis in the gastrointestinal tract was illustrated by the example of the proteolysis of the complex (β-casein + PC) particles in the simulated gastrointestinal conditions in vitro.

Variations in the Condensing Effect of Cholesterol on Saturated versus Unsaturated Phosphatidylcholines at Low and High Sterol Concentration

In this work, we have investigated the condensing and ordering effect induced by cholesterol on phosphatidylcholines (PCs). To perform the studies systematically, for the experiments we have selected phospholipids differing only in the number of cis monounsaturated chains (1,2-distearoyl-sn-glycero-3-phosphocholine--DSPC, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine--SOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine--DOPC) or in the length (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine--POPC vs SOPC) of sn-1 acyl chain. Because the cholesterol concentration in mammalian membranes can be as high as 70 mol % of total lipids, the investigations were performed in a wide range of the sterol content. The results of the Langmuir monolayer experiments evidence that the relation between the structure of hydrophobic part of PC and the magnitude of the effects induced by cholesterol found at lower sterol content is different from that observed at higher sterol concentration. At a lower concentration of sterol (up to 30%), the condensing effect of cholesterol is stronger on saturated DSPC than on PCs containing monounsaturated chain(s), which is consistent with the conclusions drawn by other authors. However, at higher sterol content (≥50%), saturated DSPC is less susceptible to the influence of sterol than the investigated unsaturated PCs. To explain these irregularities, we have considered the strength of van der Waals interactions as well as the influence of sterol on the tilt of polar heads of PCs. It was also found that in the whole range of sterol concentration the ordering effect is stronger on saturated DSPC as compared to unsaturated phospholipids. However, at lower sterol content (up to 30%) the ordering effect induced on unsaturated PCs is rather weak, and the ordering does not change drastically in comparison with pure PCs film.

Pulmonary Surfactant Phosphatidylglycerol Inhibits Mycoplasma pneumoniae-stimulated Eicosanoid Production from Human and Mouse Macrophages

Mycoplasma pneumoniae is a human pathogen causing respiratory infections that are also associated with serious exacerbations of chronic lung diseases. Membranes and lipoproteins from M. pneumoniae induced a 4-fold increase in arachidonic acid (AA) release from RAW264.7 and a 2-fold increase in AA release from primary human alveolar macrophages. The bacterial lipoprotein mimic and TLR2/1 agonist Pam3Cys and the TLR2/6 agonist MALP-2 produced effects similar to those elicited by M. pneumoniae in macrophages by inducing the phosphorylation of p38(MAPK) and p44/42(ERK1/2) MAP kinases and cyclooxygenase-2 (COX-2) expression. M. pneumoniae induced the generation of prostaglandins PGD(2) and PGE(2) from RAW264.7 cells and thromboxane B(2) (TXB(2)) from human alveolar macrophages. Anti-TLR2 antibody completely abolished M. pneumoniae-induced AA release and TNFα secretion from RAW264.7 cells and human alveolar macrophages. Disruption of the phosphorylation of p44/42(ERK1/2) or inactivation of cytosolic phospholipase A(2)α (cPLA(2)α) completely inhibited M. pneumoniae-induced AA release from macrophages. The minor pulmonary surfactant phospholipid, palmitoyl-oleoyl-phosphatidylglycerol (POPG), antagonized the proinflammatory actions of M. pneumoniae, Pam3Cys, and MALP-2 by reducing the production of AA metabolites from macrophages. The effect of POPG was specific, insofar as saturated PG, and saturated and unsaturated phosphatidylcholines did not have significant effect on M. pneumoniae-induced AA release. Collectively, these data demonstrate that M. pneumoniae stimulates the production of eicosanoids from macrophages through TLR2, and POPG suppresses this pathogen-induced response.

The Role of Domains and Proteins in the Function of Lung Surfactant

Lung surfactant forms a thin film at the gas exchange interface in alveoli. The film reduces the surface tension which is necessary for breathing. The film consists of a monolayer at the air/water interface connected with bilayer reservoirs in water. Lung surfactant is composed of a mixture of saturated and unsaturated, zwitterionic and anionic lipids; surfactant proteins B (SP-B) and C are associated with the interface. Surfactant proteins facilitate adsorption of lipids to the interface likely via stalk-like intermediates. The proteins are also believed to induce monolayer collapse by creating nucleation sites/fluidizing effect. Lipid components in monolayer segregate into domains of coexisting phases, which is suggested to increase monolayer stability. However, the exact mechanisms of these effects are still not fully understood.We study the role of phase coexistence and proteins in the function of lung surfactant. Molecular dynamics simulations with the coarse-grained model MARTINI are employed. We simulate mixtures of saturated and unsaturated phosphatidylcholine and phosphatidylglycerol lipids, cholesterol and SP-B proteins. The model of SP-B is built based on homology with the saposin family and was shown previously to mediate early stages of vesicle fusion [S Baoukina, DP Tieleman, Biophys J, 2010]. We reproduce phase separation into liquid-expanded (LE) and liquid-condensed (LC) or liquid ordered (Lo) phases in monolayers. SP-B partitions into the LE phase and prevents formation of the LC phase at conditions close to the phase transition. The protein also enhances lipid de-mixing. SP-B dimers induce bilayer folds in monolayers at positive surface tensions below the equilibrium spreading value. Monolayer collapse is initiated from the LE phase, and the composition of bilayer folds differs from the monolayer. SP-B anchors monolayers and bilayers and promotes lipid transfer.

Relationships Between Bilayer Phase and Equilibration Rates of Patman and Laurdan

Patman is a fluorescent membrane probe related to laurdan. The structural distinctions between the two probes are the lengths of the aliphatic tails (eleven carbons in laurdan and fifteen in patman) and the presence of a trimethylammonium forming a positively-charged head on patman. Studies exploring patman as a probe to detect membrane properties during apoptosis revealed that the two edges of the emission spectrum (435 and 500 nm) stabilize at different rates as the probe binds to the cell membrane. To test whether these differences represent dissimilarities in probe binding to ordered and disordered domains, experiments were conducted to monitor patman equilibration with bilayers composed of various mixtures of saturated and unsaturated phosphatidylcholines at temperatures above, at, and below the main thermotropic phase transition. In general, patman equilibrated more rapidly with bilayers in the liquid disordered phase than in the solid ordered phase. With solid phase membranes, the fluorescence stabilized faster at 435 nm than at 500 nm. Similar yet more subtle results occurred in the lipid disordered phase. In contrast, the situation was reversed at the phase transition temperature; equilibration was faster at 500 nm than at 435 nm. To determine whether these results reflected specific properties of patman, the experiments were repeated with laurdan, and several distinctions were observed. First, equilibration with solid phase lipids was faster than for patman and not different from equilibration with the fluid phase. Second, differences in rates between the two wavelengths were less than with patman for solid phase membranes but greater than with patman for melted bilayers. Third, at the phase transition temperature, equilibration rates favored 435 nm over 500 nm, the opposite of the result obtained with patman. Computer simulations were used to assist with interpretation of these results.

Inter-Leaflet Coupling and Domain Formation in Asymmetric Giant Unilamellar Vesicles

Cell membranes exhibit an asymmetric distribution of lipids across the two leaflets of the bilayer. While the sphingolipid-rich outer (exoplasmic) leaflet has a lipid composition prone to lateral segregation and domain formation, the situation in the sphingolipid-poor inner (cytosolic) leaflet is unclear. The interaction between the two leaflets, although involved in several biological processes, is not well understood. Although both supported and free-standing asymmetric bilayers have been previously produced, such approaches were based on a leaflet-by-leaflet assembly of the bilayer and, therefore, possibly incompatible with the reconstitution of transmembrane proteins. A highly reproducible solvent-free method that is fast, has a high yield and can be used with a wide variety of lipids and membrane proteins, is still needed. Here, we introduce a simple lipid exchange method to obtain stable asymmetric giant unilamellar vesicles (GUV) that satisfies the above-mentioned requirements. The asymmetry of the GUVs is investigated using fluorescence correlation spectroscopy (FCS) in order to probe dynamics of lipids in the two leaflets. It is shown that introduction of up to 50% sphingomyelin into the outer leaflet of GUV composed of an unsaturated phosphatidylcholine (PC) in the inner leaflet results in a larger decrease in outer leaflet lateral diffusion than inner leaflet lateral diffusion. The degree to which the inner leaflet later diffusion decreases depends on the identity of the inner leaflet PC. We also show that a transmembrane protein can be incorporated into the asymmetric GUV and its diffusion probed by FCS. Thus, this approach can be used to investigate the molecular mechanisms behind inter-leaflet coupling and the effect of membrane asymmetry on transmembrane proteins.

Glycomics meets lipidomics—associations of N-glycans with classical lipids, glycerophospholipids, and sphingolipids in three European populations

Recently, high-throughput technologies have been made available which allow the measurement of a broad spectrum of glycomics and lipidomics parameters in many samples. The aim of this study was to apply these methods and investigate associations between 46 glycan and 183 lipid traits measured in blood of 2041 Europeans from three different local populations (Croatia - VIS cohort; Sweden - NSPHS cohort; Great Britain - ORCADES cohort). N-glycans have been analyzed with High Performance Liquid Chromatography (HPLC) and lipids with Electrospray Ionization Tandem Mass Spectrometry (ESI-MS/MS) covering sterol lipids, glycerolipids, glycerophospholipids and sphingolipids in eight subclasses. Overall, 8418 associations were calculated using linear mixed effect models adjusted for pedigree, sex, age and multiple testing. We found 330 significant correlations in VIS. Pearson's correlation coefficient r ranged from -0.27 to 0.34 with corresponding p-values between 1.45 × 10(-19) and 4.83 × 10(-6), indicating statistical significance. A total of 71 correlations in VIS could be replicated in NSPHS (r = [-0.19; 0.35], p = [4.16 × 10(-18); 9.38 × 10(-5)]) and 31 correlations in VIS were also found in ORCADES (r = [-0.20; 0.24], p = [2.69 × 10(-10); 7.55 × 10(-5)]). However, in total only 10 correlations between a subset of triantennary glycans and unsaturated phosphatidylcholine, saturated ceramide, and sphingomyelin lipids in VIS (r = [0.18; 0.34], p = [2.98 × 10(-21); 1.69 × 10(-06)]) could be replicated in both NSPHS and ORCADES. In summary, the results show strong and consistent associations between certain glycans and lipids in all populations, but also population-specific correlations which may be caused by environmental and genetic differences. These associations point towards potential interactive metabolic pathways.

Atomic-scale lateral heterogeneity and dynamics of two-component lipid bilayers composed of saturated and unsaturated phosphatidylcholines

Studies of lateral heterogeneity in cell membranes are important since they help to understand the physical origin of lipid domains and rafts. The simplest membrane mimics are hydrated bilayers composed of saturated and unsaturated lipids. While their atomic structural details resist easy experimental characterization, important insight can be gained via computer modeling. We present the results of all-atom molecular dynamics simulations for a series of fluid dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) bilayers. Lateral arrangement of lipids in these systems is not random and reveals small geometrical and hydrophobic clusters on the surface. Being “sharp” inside the bilayer, lateral heterogeneity is very “fuzzy” on the surface. This picture is highly dynamic – lifetimes of clusters are ∼1 ns. In the binary system, DPPC acts as an “order-preferring” agent, which efficiently modulates behavior of DOPC. Bilayer properties are tuned in a wide range by the chemical nature and relative content of lipids. The impact that the micro-heterogeneity may have on formation of lateral domains in response to external signals is discussed. Understanding of such effects creates a basis for rational design of artificial membranes with predefined properties.

EPR investigation of clomipramine interaction with phosphatidylcholine membranes in presence and absence of cholesterol

The effects of tricylic antidepressant clomipramine (CLO) on the membrane properties of saturated dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine as well as on unsaturated egg yolk phosphatidylcholine liposomes were investigated by the electron paramagnetic resonance spin-labeling technique, in combination with the simulation of the spectra, taking into account that the membrane is heterogeneous and composed of the regions with different fluidity characteristics. Different spin labels, monitoring membrane properties in the upper and inner parts of the membrane, were used. In general, two spectral components, having different motional characteristics, were detected in all liposomes investigated. In liposomes with saturated chains, CLO decreased the phase-transition temperature, disordered the membrane, and increased polarity in the upper part of the membrane. However, less impact was observed in liposomes with unsaturated chains. In dipalmitoyl phosphatidylcholine liposomes, it also induced molecular rearrangements near the pretransition temperature. The presence of 30 mol% cholesterol increased the fluidizing effect of CLO and modified the lateral diffusion of nitroxide in the inner part of the membrane. A unique anomalous increase in diffusion of nitroxide, dependent on CLO concentration, was detected in the temperature region where the phosphatidylcholine membrane without cholesterol experiences the phase transitions. Since the changes in the central part of the membrane were even more pronounced than in the upper part of the membrane, it could be concluded that CLO incorporates into the membrane with its hydrophobic ring parallel to the phospholipid chains.

Molecular volumes of phospholipids and glycolipids in membranes

Data on the molecular volumes of phospholipids and glycolipids in membranes are collected together in order to determine the contributions from the component groups, for as wide a range of lipids as possible, including sphingolipids. Wherever possible, the volumes of the methylene groups in the lipid chains are established from the dependence on chain length at fixed temperature in a given phase. In this way, it is also possible to determine the constant contribution from cis double bonds in the chains of monoenoic unsaturated phosphatidylcholines, and the volume of the branched methyl groups in isoacyl phosphatidylcholines. Issues concerning separation of contributions from the polar head groups from those of the chain terminal methyl groups are discussed. Molecular volumes of lipids in crystals are also analysed to provide information on head-group packing that can be compared with the situation in membranes, and used to set limits on the relative contributions from polar groups and terminal methyl groups. Comparisons are made with volumetric analyses based on diffraction studies of bilayers of single lipids. The parameters derived can be used to estimate molecular volumes of lipids for which dilatometric or densitometric data are lacking. Lipid volumes are determining parameters for lipid dynamics, membrane partitioning and permeation of solutes, and are essential quantities for the structural analysis of lipid membranes.

177 Kinome analysis in renal cell carcinoma

In this data article we provide topologies and force field parameters files for molecular dynamics simulations of lipids in the OPLS-aa force field using the GROMACS package. This is the first systematic parameterization of lipid molecules in this force field. Topologies are provided for four phosphatidylcholines: saturated DPPC, mono-cis unsaturated POPC and DOPC, and mono-trans unsaturated PEPC. Parameterization of the phosphatidylcholines was achieved in two steps: first, we supplemented the OPLS force field parameters for DPPC with new parameters for torsion angles and van der Waals parameters for the carbon and hydrogen atoms in the acyl chains, as well as new partial atomic charges and parameters for torsion angles in the phosphatidylcholine and glycerol moieties [1]. Next, we derived parameters for the cis and trans double bonds and the neighboring them single bonds [2]. Additionally, we provide GROMACS input files with parameters describing simulation conditions (md.mdp), which are strongly recommended to be used with these lipids models. The data are associated with the research article “Cis and trans unsaturated phosphatidylcholine bilayers: a molecular dynamics simulation study” [2] and provided as supporting materials.

176 Is there a role of DNA methylation in Estrogen Receptor alpha (ERa) expression in Breast Cancer (BC)?

In this data article we provide topologies and force field parameters files for molecular dynamics simulations of lipids in the OPLS-aa force field using the GROMACS package. This is the first systematic parameterization of lipid molecules in this force field. Topologies are provided for four phosphatidylcholines: saturated DPPC, mono-cis unsaturated POPC and DOPC, and mono-trans unsaturated PEPC. Parameterization of the phosphatidylcholines was achieved in two steps: first, we supplemented the OPLS force field parameters for DPPC with new parameters for torsion angles and van der Waals parameters for the carbon and hydrogen atoms in the acyl chains, as well as new partial atomic charges and parameters for torsion angles in the phosphatidylcholine and glycerol moieties [1]. Next, we derived parameters for the cis and trans double bonds and the neighboring them single bonds [2]. Additionally, we provide GROMACS input files with parameters describing simulation conditions (md.mdp), which are strongly recommended to be used with these lipids models. The data are associated with the research article “Cis and trans unsaturated phosphatidylcholine bilayers: a molecular dynamics simulation study” [2] and provided as supporting materials.

The Arabidopsis acbp1acbp2 double mutant lacking acyl‐CoA‐binding proteins ACBP1 and ACBP2 is embryo lethal

*In Arabidopsis thaliana, the amino acid sequences of membrane-associated acyl-CoA-binding proteins ACBP1 and ACBP2 are highly conserved. We have shown previously that, in developing seeds, ACBP1 accumulates in the cotyledonary cells of embryos and ACBP1 is proposed to be involved in lipid transfer. We show here by immunolocalization, using ACBP2-specific antibodies, that ACBP2 is also expressed in the embryos at various stages of seed development in Arabidopsis. *Phenotypic analyses of acbp1 and acbp2 single mutants revealed that knockout of either ACBP1 or ACBP2 alone did not affect their life cycle as both single mutants exhibited normal growth and development similar to the wild-type. However, the acbp1acbp2 double mutant was embryo lethal and was also defective in callus induction. *On lipid and acyl-CoA analyses, the siliques, but not the leaves, of the acbp1 mutant accumulated galactolipid monogalactosyldiacylglycerol and 18:0-CoA, but the levels of most polyunsaturated species of phospholipid, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine, declined. *As recombinant ACBP1 and ACBP2 bind unsaturated phosphatidylcholine and acyl-CoA esters in vitro, we propose that ACBP1 and ACBP2 are essential in lipid transfer during early embryogenesis in Arabidopsis.

Effect of pressure on the bilayer phase transitions of asymmetric lipids with an unsaturated acyl chain in sn-1 position

The bilayer phase transitions of a series of mixed-chain phosphatidylcholines (PCs) with an unsaturated chain in the sn-1 position and saturated chain with different lengths in the sn-2 position, 1-oleoyl-2-myristoyl-PC (OMPC), 1-oleoyl-2-palmitoyl-PC (OPPC) and 1-oleoyl-2-stearoyl-PC (OSPC), were observed by differential scanning calorimetry (DSC) under ambient pressure and light transmittance measurements under high pressure. All the PC bilayer membranes exhibited not only the main transition from the lamellar gel (Lβ) phase to the liquid crystalline (Lα) phase but also the transition from the lamellar crystal (Lc) to the Lβ (or Lα) phase. The thermodynamic quantities of the transition between the Lc and Lα phases for bilayers of unsaturated mixed-chain PCs were compared with those of the corresponding mixed-chain PCs with saturated acyl chains such as 1-stearoyl-2-myristoyl-PC (SMPC), 1-stearoyl-2-palmitoyl-PC (SPPC) and 1,2-distearoyl-PC (DSPC). It turned out that the partial molar quantities of the unsaturated mixed-chain PC bilayers in the Lc phase are larger than those of the saturated mixed-chain PCs bilayers. The increase in the partial molar quantities in the Lc phase by the introduction of a cis double bond into the sn-1 acyl chain is attributable to the loose packing of the saturated and unsaturated acyl chains in the Lc phase.

Lipid Peroxyl Radicals Mediate Tyrosine Dimerization and Nitration in Membranes

Protein tyrosine dimerization and nitration by biologically relevant oxidants usually depend on the intermediate formation of tyrosyl radical ((*)Tyr). In the case of tyrosine oxidation in proteins associated with hydrophobic biocompartments, the participation of unsaturated fatty acids in the process must be considered since they typically constitute preferential targets for the initial oxidative attack. Thus, we postulate that lipid-derived radicals mediate the one-electron oxidation of tyrosine to (*)Tyr, which can afterward react with another (*)Tyr or with nitrogen dioxide ((*)NO(2)) to yield 3,3'-dityrosine or 3-nitrotyrosine within the hydrophobic structure, respectively. To test this hypothesis, we have studied tyrosine oxidation in saturated and unsaturated fatty acid-containing phosphatidylcholine (PC) liposomes with an incorporated hydrophobic tyrosine analogue BTBE (N-t-BOC l-tyrosine tert-butyl ester) and its relationship with lipid peroxidation promoted by three oxidation systems, namely, peroxynitrite, hemin, and 2,2'-azobis (2-amidinopropane) hydrochloride. In all cases, significant tyrosine (BTBE) oxidation was seen in unsaturated PC liposomes, in a way that was largely decreased at low oxygen concentrations. Tyrosine oxidation levels paralleled those of lipid peroxidation (i.e., malondialdehyde and lipid hydroperoxides), lipid-derived radicals and BTBE phenoxyl radicals were simultaneously detected by electron spin resonance spin trapping, supporting an association between the two processes. Indeed, alpha-tocopherol, a known reactant with lipid peroxyl radicals (LOO(*)), inhibited both tyrosine oxidation and lipid peroxidation induced by all three oxidation systems. Moreover, oxidant-stimulated liposomal oxygen consumption was dose dependently inhibited by BTBE but not by its phenylalanine analogue, BPBE (N-t-BOC l-phenylalanine tert-butyl ester), providing direct evidence for the reaction between LOO(*) and the phenol moiety in BTBE, with an estimated second-order rate constant of 4.8 x 10(3) M(-1) s(-1). In summary, the data presented herein demonstrate that LOO(*) mediates tyrosine oxidation processes in hydrophobic biocompartments and provide a new mechanistic insight to understand protein oxidation and nitration in lipoproteins and biomembranes.

Composition, structure and substrate properties of reconstituted discoidal HDL with apolipoprotein A-I and cholesteryl ester

To investigate the influence of lipid unsaturation and neutral lipid on the maturation of high density lipoproteins, the discoidal complexes of apoA-I, phosphatidylcholine and cholesteryl ester (CE) were prepared. Saturated dipalmitoylphosphatidylcholine (DPPC) and unsaturated palmitoyllinoleoylphosphatidylcholine (PLPC), palmitoyloleoylphosphatidylcholine (POPC), and fluorescent probe cholesteryl 1-pyrenedecanoate (CPD) that forms in a diffusion- and concentration-dependent manner short-lived dimer of unexcited and excited molecules (excimer) were used. The apoA-I/DPPC/CPD complexes were heterogeneous by size, composition and probe location. CPD molecules incorporated more efficiently into larger complexes and accumulated in a central part of the discs. The apoA-I/POPC(PLPC)/CPD were also heterogeneous, however, probe molecules distributed preferentially into smaller complexes and accumulated at disc periphery. The kinetics of CPD transfer by recombinant cholesteryl ester transfer protein (CETP) to human plasma LDL is well described by two-exponential decay, the fast component with a shorter transfer time being more populated in PLPC compared to DPPC complexes. The presence of CE molecules in discoidal HDL results in particle heterogeneity. ApoA-I influences the CETP activity modulating the properties of apolipoprotein–phospholipid interface. This may include CE molecules accumulation in the boundary lipid in unsaturated phosphatidylcholine and cluster formation in the bulk bilayer in saturated phosphatidylcholine.

Mixed micelle formation with phosphatidylcholines: The influence of surfactants with different molecule structures

The number of mixed micellar (MM) drug products being introduced into the commercial pharmaceutical market is very limited although there is need for alternative dosage forms for poorly soluble active drug substances. While known systems are composed of phosphatidylcholine and bile salts, it was the aim of this study to investigate if alternative surfactants are able to form isotropically clear solutions over a broad range of concentrations and at higher ratios of phosphatidylcholine (PC). It was a particular challenge of this work to find a MM system with a unimodal particle size distribution since it is known that surfactants often form vesicles with phospholipids instead of MM. The theoretical approach behind this work was the transfer of the packing parameter concept, which describes the molecular association of one amphiphilic species, to the organisation behaviour of two different amphiphilic species (water-insoluble phospholipid + surfactant leading to MM). Therefore the influence of the surfactant molecular geometry on the ability to form MM with phospholipids was investigated. A homologous series of two different surfactant classes, namely polyglycerol esters and sucrose esters, with a large hydrophilic head region leading to a smaller packing parameter were analysed regarding their ability to form clear MM solutions with PC. For comparison, surfactants with no strictly defined partition between a polar head and a non-polar tail (e.g. Poloxamer 188) were tested. Decaglycerol laurate and especially sucrose laurate (SL) were superior compared to all other tested surfactants with respect to their ability to form clear solutions with hydrogenated PC (hPC) at a higher ratio and over a broad range of concentrations while unsaturated PC showed an inferior performance to form MM. The favourite MM system composed of SL with 0.5 weight fractions of hPC formed about 20 nm sized MM in a concentration range of 1.0–80 mg/mL and showing a unimodal particle size distribution with a PDI value <0.1. The results of the study have shown that the transferred packing parameter concept is applicable to the tested surfactants to describe their ability forming mixed micelles with PC.