Effect of angiotensin II non-peptide AT(1) antagonist losartan on phosphatidylethanolamine membranes

Losartan was found to affect both the thermotropic behavior and molecular mobility of dimyristoyl- and dipalmitoyl-phosphatidylcholine membranes (Theodoropoulou and Marsh, Biochim. Biophys. Acta 1461 (1999) 135-146). At low concentrations, the antagonist is located close to the interfacial region of the phosphatidylcholine bilayer while at high mole fractions it inserts deeper in the bilayers. In the present study, we investigated the interactions of losartan with phosphatidylethanolamine membranes using differential scanning calorimetry (DSC), electron spin resonance (ESR) and 31P nuclear magnetic resonance (NMR) spectroscopy. DSC showed that the antagonist affected the thermotropic transitions of dimyristoyl-, dipalmitoyl- and dielaidoyl-phosphatidylethanolamine membranes (DMPE, DPPE and DEPE, respectively). ESR spectroscopy showed that the interaction of losartan with phosphatidylethanolamine membranes is more superficial than in the case of phosphatidylcholine bilayers. Additionally, losartan increased the spin-spin broadening of 12-PESL spin labels in the gel phase of DMPE and DPPE membranes, while in the case of DEPE membranes the opposite effect was observed. (31)P-NMR showed that the antagonist stabilizes the fluid lamellar phase of DEPE membranes relative to the hexagonal H(II) phase. Our results show that losartan affects the thermotropic behavior of phosphatidylethanolamine membranes, while the molecular mobility of the membranes is not affected greatly. Furthermore, its interactions with phosphatidylethanolamine membranes are more superficial than with phosphatidylcholine bilayers.     

Non-ideal mixing and fluid–fluid immiscibility in phosphatidic acid–phosphatidylethanolamine mixed bilayers

Differential scanning calorimetry (DSC) was used to study the miscibility of phosphatidic acids (PAs) with phosphatidylethanolamines (PEs) as a function of chain length (n = 14, 16) and degree of ionization of PAs at pH 4, pH 7, and pH 12. Phase diagrams were constructed using temperature data for onset and end of the phase transition obtained from the direct simulation of the heat-capacity curves. The phase diagrams were analyzed by simulations of the coexistence curves utilizing a four-parameter regular solution model. For PA–PE mixtures, the non-ideality parameters are a function of composition indicating non-symmetric non-ideal mixing behavior. At pH 7, where the PA component is negatively charged, the systems DMPA:DMPE and DPPA:DPPE have positive non-ideality parameters ρ ₁ in both phases, indicating a preferred aggregation of like molecules. In contrast, DMPA:DPPE and DPPA:DMPE mixtures had negative ρ ₁ values. Measurements at pH 4 showed that mixed pair formation is favored when PA is protonated. At pH 12 where PA is doubly charged, highly positive ρ _l1 parameters are obtained for the liquid-crystalline phase except for the system DPPA:DPPE (ρ ₁ < 0). This indicates clustering of like molecules and possibly domain formation in the liquid-crystalline phase. DPPA:DMPE at pH 12 even shows a miscibility gap in the liquid-crystalline phase. Obviously, despite the presence of doubly charged PA a fluid–fluid immiscibility is induced.     

Differential scanning calorimetry of chain-melting phase transitions of N-acylphosphatidylethanolamines.

Phosphatidylethanolamines in which the polar headgroup is N-acylated by a long-chain fatty acid (N-acyl PEs) are present in many plasma membranes under normal conditions, and their content increases dramatically in response to membrane stress in a variety of organisms. The thermotropic phase behavior of a homologous series of saturated N-acyl PEs, in which the length of the N-acyl chain is equal to that of the O-acyl chains attached at the glycerol backbone, has been investigated by differential scanning calorimetry (DSC). All fully hydrated N-acyl PEs with even chain lengths from C-12 to C-18 exhibit sharp endothermic chain-melting phase transitions in the absence of salt and in 1 M NaCl. Cooperative chain-melting is demonstrated directly by the temperature dependence of the electron spin resonance spectra from probe phospholipids bearing a spin label group in the acyl chain. The calorimetric transition enthalpy and the transition entropy obtained from DSC depend approximately linearly on the chain length with incremental values per CH2 group that exceed those of normal diacyl phosphatidylethanolamines, but to an extent that underrepresents the additional N-acyl chain. A thermodynamic model is constructed for the chain-length dependences and end effects of the calorimetric quantities, which includes a deficit proportional to the difference in O-acyl and N-acyl chain lengths for nonmatched chains, as is found and justified structurally for mixed-chain diacyl phospholipids. From data on the chain-length dependence of N-acyl diC16PEs, it is then deduced that the N-acyl chains are less well packed than the O-acyl chains and, from the data on the matched-chain N-acyl PEs, that the O-acyl chain packing is similar to that in normal diacyl PEs. The gel-to-fluid phase transition temperatures of the N-acyl PEs in the absence of salt are practically the same as those of the normal diacyl PEs of the corresponding chain lengths, although the transition enthalpies and entropies are appreciably greater, indicating entropy-enthalpy compensation. In 1 M NaCl, the transition temperatures are 3-4.5 degrees higher than in the absence of salt, representing the contribution of the electrostatic surface potential of the N-acyl PEs.     

Interaction of N-myristoylethanolamine with cholesterol investigated in a Langmuir film at the air-water interface

The dramatic increase in the content of N-acylethanolamines (NAEs) having different acyl chains in various tissues when subjected to stress has resulted in significant interest in investigations on these molecules. Previous studies suggested that N-myristoylethanolamine (NMEA) and cholesterol interact to form a 1:1 (mol/mol) complex. In studies reported here, pressure-area isotherms of Langmuir films at the air-water interface have shown that at low fractions of cholesterol, the average area per molecule is lower than that predicted for ideal mixing, whereas at high cholesterol content the observed molecular area is higher, with a cross-over point at the equimolar composition. A plausible model that can explain these observations is the following: addition of small amounts of cholesterol to NMEA results in a reorientation of the NMEA molecules from the tilted disposition in the crystalline state to the vertical and stabilization of the intermolecular interactions, leading to the formation of a compact monolayer film, whereas at the other end of the composition diagram, addition of small amounts of NMEA to cholesterol leads to a tilting of the cholesterol molecules resulting in an increase in the average area per molecule. In Brewster angle microscopy experiments, a stable and bright homogeneous condensed phase was observed at a relatively low applied pressure of 2 mN.m(-1) for the NMEA:Chol. (1:1, mol/mol) mixture, whereas all other samples required significantly higher pressures (>10 mN.m(-1)) to form a homogeneous condensed phase. These observations are consistent with the formation of a 1:1 stoichiometric complex between NMEA and cholesterol and suggest that increase in the content of NAEs under stress may modulate the composition and dynamics of lipid rafts in biological membranes, resulting in alterations in signaling events involving them, which may be relevant to the putative cytoprotective and stress-combating ability of NAEs.     

Influence of retinoids on phosphatidylethanolamine lipid polymorphism.

The interaction of all-trans-retinoic acid and all-trans-retinol with dielaidoylphosphatidylethanolamine has been studied by differential scanning calorimetry and 31P-NMR spectroscopy. Increasing concentrations of all-trans-retinoic acid up to a mol fraction of 0.09 were found to induce shifts to lower temperatures of both the L beta to L alpha and L alpha to hexagonal-HII phase transitions, with a slight decrease in the enthalpy change of the transitions. At higher concentrations no further effects on the transitions were observed, and this is interpreted as indicative of a limited miscibility of retinoic acid with the phospholipid. 31P-NMR spectroscopy confirmed that the L alpha to hexagonal-HII phase transition was shifted to lower temperatures in the presence of retinoic acid. On the other hand increasing concentrations of all-trans-retinol up to a mol fraction of 0.166, induced a progressive shift of the L beta to L alpha and the L alpha to hexagonal-HII phase transitions to lower temperatures. At higher concentrations the main gel to liquid-crystalline phase transition was further displaced to lower temperatures and the lamellar to hexagonal-HII phase transition was not observed in the thermograms. 31P-NMR spectroscopy indicated that retinol was able of inducing the phospholipid to adopt the hexagonal-HII phase at temperatures even below the main gel to liquid-crystalline phase transition temperature of the pure phospholipid.     

Landau theory of two-component phospholipid bilayers. I. Phosphatidylcholine/phosphatidylethanolamine mixtures

Priest's phenomenological model (Mol. Cryst. Liq. Cryst. 60 (1980) 167.) on one- and two-component PC bilayers is extended here. We constructed a new excess free energy term in the state function to describe the thermodynamic properties of the two-component phospholipid bilayers where the chain lengths and the polar heads of the components can be different simultaneously. By means of this generalized state function, we can calculate the phase diagrams of DPPC/DPPE, DMPC/DMPE, DMPC/DPPE, DPPC/DMPE and DSPC/DMPE mixtures. We obtained complete miscibility both in the liquid crystalline and in the gel phase if the chain lengths of the components were the same. If the chain length of the PE component was longer than that of the PC component, we obtained a peritectic system. A eutectic system was obtained in the reverse case. The results of the model were compared with the experimental data available. Applying the quasichemical approximation, we determined the molecular meaning of the phenomenological model parameters. Namely, sigma and gamma are proportional to the sublimation heat of the CH2 group in the long-chain alkanes and to the hydrogen-bonding energy between the polar heads of the ethanolamines; otherwise the model resulted in--1.94 kcal/mol per CH2 for the sublimation heat and --1.4 kcal/mol for the hydrogen-bond energy.     

Packing characteristics of two-component bilayers composed of ester- and ether-linked phospholipids.

The miscibility properties of ether- and ester-linked phospholipids in two-component, fully hydrated bilayers have been studied by differential scanning calorimetry (DSC) and Raman spectroscopy. Mixtures of 1,2-di-O-hexadecyl-rac-glycero-3-phosphocholine (DHPC) with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DHPE) and of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) with 1,2-di-O-hexadecyl-sn-glycero-3-phosphoethanolamine (DHPE) have been investigated. The phase diagram for the DPPC/DHPE mixtures indicates that these two phospholipids are miscible in all proportions in the nonrippled bilayer gel phase. In contrast, the DHPC/DPPE mixtures display two regions of gel phase immiscibility between 10 and 30 mol% DPPE. Raman spectroscopic measurements of DHPC/DPPE mixtures in the C-H stretching mode region suggest that this immiscibility arises from the formation of DHPC-rich interdigitated gel phase domains with strong lateral chain packing interactions at temperatures below 27 degrees C. However, in the absence of interdigitation, our findings, and those of others, lead to the conclusion that the miscibility properties of mixtures of ether- and ester-linked phospholipids are determined by the nature of the phospholipid headgroups and are independent of the character of the hydrocarbon chain linkages. Thus it seems unlikely that the ether linkage has any significant effect on the miscibility properties of phospholipids in biological membranes.     

Effect of acyl chain composition on salt-induced lamellar to inverted hexagonal phase transitions in cardiolipin

Salt-induced fluid lamellar (L alpha) to inverted hexagonal (HII) phase transitions have been studied in diphosphatidylglycerols (cardiolipins) with different acyl chain compositions, using 31P nuclear magnetic resonance (NMR) spectroscopy. Cardiolipins with four myristoyl chains, tetramyristoyl cardiolipin (TMCL), and with four oleoyl chains, tetraoleoyl cardiolipin (TOCL), were synthesized chemically. TMCL was found to undergo a thermotropic lamellar gel to lamellar liquid-crystalline phase transition at 33-35 degrees C. This lipid exhibited an axially symmetric 31P-NMR spectrum corresponding to a lamellar phase at all NaCl concentrations between 0 and 6 M. In the case of TOCL, formation of an HII phase was induced by salt concentrations of 3.5 M NaCl or greater. These observations, taken together with earlier findings that bovine heart cardiolipin aqueous dispersions adopt an HII phase at salt concentrations of 1.5 M NaCl or greater, indicate that increasing unsaturation and length of the acyl chains favour formation of the HII phase in diphosphatidylglycerols.     

On the miscibility of cardiolipin with 1,2-diacyl phosphoglycerides: binary mixtures of dimyristoylphosphatidylethanolamine and tetramyristoylcardiolipin

The thermotropic phase behavior and organization of model membranes composed of binary mixtures of the quadruple-chained, anionic phospholipid tetramyristoylcardiolipin (TMCL) with the double-chained zwitterionic phospholipid dimyristoylphosphatidylethanolamine (DMPE) were examined by a combination of differential scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy. After equilibration at low temperature, DSC thermograms exhibited by binary mixtures of TMCL and DMPE containing < 80 mol DMPE exhibit a fairly energetic lower temperature endotherm and a highly energetic higher temperature endotherm. As the relative amount of TMCL in the mixture decreases, the temperature, enthalpy and cooperativity of the lower temperature endotherm also decreases and is not calorimetrically detectable when the TMCL content falls below 20 mol%. In contrast, the temperature of the higher temperature endotherm increases as the proportion of TMCL decreases, but the enthalpy and cooperativity both decrease and the transition endotherms become multimodal. The FTIR spectroscopic results indicate that the lower temperature endotherm corresponds to a lamellar crystalline (L(c)) to lamellar gel (L(β)) phase transition and that the higher temperature transition involves the conversion of the L(β) phase to the lamellar liquid-crystalline (L(α)) phase. Moreover, the FTIR spectroscopic signatures observed at temperatures below the onset of the L(c)/L(β) phase transitions are consistent with the coexistence of structures akin to a TMCL-like L(c) phase and the L(β) phase, and with the relative amount of the TMCL-like L(c) phase increasing progressively as the TMCL content of the mixture increases. These latter observations suggest that the TMCL and DMPE components of these mixtures are poorly miscible at temperatures below the L(β)/L(α) phase transition temperature. Poor miscibility of these two components is also suggested by the complexity of the DSC thermograms observed at the L(β)/L(α) phase transitions of these mixtures and with the complex relationship between their L(β)/L(α) phase transition temperatures and the composition of the mixture. Overall, our data suggests that TMCL and DMPE may be intrinsically poorly miscible across a broad composition range, notwithstanding the homogeneity of the fatty acid chains of the two components and the modest (~10 °C) difference between their L(β)/L(α) phase transition temperatures.     

Phase behavior of ethanolamine plasmalogen

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

Endogenous unsaturated C18 N-acylethanolamines are vanilloid receptor (TRPV1) agonists

The endogenous C18 N-acylethanolamines (NAEs) N-linolenoylethanolamine (18:3 NAE), N-linoleoylethanolamine (18:2 NAE), N-oleoylethanolamine (18:1 NAE), and N-stearoylethanolamine (18:0 NAE) are structurally related to the endocannabinoid anandamide (20:4 NAE), but these lipids are poor ligands at cannabinoid CB(1) receptors. Anandamide is also an activator of the transient receptor potential (TRP) vanilloid 1 (TRPV(1)) on primary sensory neurons. Here we show that C18 NAEs are present in rat sensory ganglia and vascular tissue. With the exception of 18:3 NAE in rat sensory ganglia, the levels of C18 NAEs are equal to or substantially exceed those of anandamide. At submicromolar concentrations, 18:3 NAE, 18:2 NAE, and 18:1 NAE, but not 18:0 NAE and oleic acid, activate native rTRPV(1) on perivascular sensory nerves. 18:1 NAE does not activate these nerves in TRPV(1) gene knock-out mice. Only the unsaturated C18 NAEs elicit whole cell currents and fluorometric calcium responses in HEK293 cells expressing hTRPV(1). Molecular modeling revealed a low energy cluster of U-shaped unsaturated NAE conformers, sharing several pharmacophoric elements with capsaicin. Furthermore, one of the two major low energy conformational families of anandamide also overlaps with the cannabinoid CB(1) receptor ligand HU210, which is in line with anandamide being a dual activator of TRPV(1) and the cannabinoid CB(1) receptor. This study shows that several endogenous non-cannabinoid NAEs, many of which are more abundant than anandamide in rat tissues, activate TRPV(1) and thus may play a role as endogenous TRPV(1) modulators.     

Binary phase diagram of hydrated dimyristoylglycerol-dimyristoylphosphatidylcholine mixtures

The thermotropic phase behavior of binary mixtures of dimyristoylphosphatidylcholine with dimyristoyl glycerol (DMPC-DMG) has been studied in aqueous dispersion by using differential scanning calorimetry and spin label electron spin resonance spectroscopy. Phase identifications have been made by means of ³¹P nuclear magnetic resonance spectroscopy and x-ray diffraction. The binary phase diagram of DMPC-DMG mixtures displays three regions corresponding to the existence of compounds (C1 and C2, respectively) with approximately 1:1 and 1:2 mol/mol DMPC:DMG stoichiometries. The first region displays immiscibility between DMPC and C1 in the low temperature lamellar phase and miscibility of the components in the fluid phase that is lamellar. The second region displays immiscibility between C1 and C2 in the low temperature phase that is lamellar, whereas the fluid phase is of the inverted hexagonal type (H_II). The third region displays immiscibility between C2 and DMG in the low temperature phase that is lamellar, whereas the fluid phase is isotropic. The presence of immiscible DMG in the low temperature phase of the third region is indicated by hysteresis in the temperature scans corresponding to conversion between the stable and metastable crystalline polymorphs. Analysis of the first region of the phase diagram using regular solution theory further demonstrates the existence of a DMPC:DMG complex with approximately 1:1 stoichiometry and provides parameters for the nonideality of mixing in the fluid phase.     

MSI-78, an analogue of the magainin antimicrobial peptides,disrupts lipid bilayer structure via positive curvature strain

In this work, we present the first characterization of the cell lysing mechanism of MSI-78, an antimicrobial peptide. MSI-78 is an amphipathic alpha-helical peptide designed by Genaera Corporation as a synthetic analog to peptides from the magainin family. (31)P-NMR of mechanically aligned samples and differential scanning calorimetry (DSC) were used to study peptide-containing lipid bilayers. DSC showed that MSI-78 increased the fluid lamellar to inverted hexagonal phase transition temperature of 1,2-dipalmitoleoyl-phosphatidylethanolamine indicating the peptide induces positive curvature strain in lipid bilayers. (31)P-NMR of lipid bilayers composed of MSI-78 and 1-palmitoyl-2-oleoyl-phosphatidylethanolamine demonstrated that the peptide inhibited the fluid lamellar to inverted hexagonal phase transition of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine, supporting the DSC results, and the peptide did not induce the formation of nonlamellar phases, even at very high peptide concentrations (15 mol %). (31)P-NMR of samples containing 1-palmitoyl-2-oleoyl-phosphatidylcholine and MSI-78 revealed that MSI-78 induces significant changes in the bilayer structure, particularly at high peptide concentrations. At lower concentrations (1-5%), the peptide altered the morphology of the bilayer in a way consistent with the formation of a toroidal pore. Higher concentrations of peptide (10-15%) led to the formation of a mixture of normal hexagonal phase and lamellar phase lipids. This work shows that MSI-78 induces significant changes in lipid bilayers via positive curvature strain and presents a model consistent with both the observed spectral changes and previously published work.     

The formation of multilamellar vesicles from saturated phosphatidylcholines and phosphatidylethanolamines: morphology and quasi-elastic light scattering measurements

The aggregation properties of diacyl phosphatidylcholines (PC) and phosphatidylethanolamines (PE), with linear symmetrical saturated chains, were characterized at temperatures below and above the lipid solid to fluid transition. PEs in the solid state form bundles of closely apposed flat bilayer stacks which at the solid to fluid transition temperature fold into closed multilamellar vesicles. On the other hand, PCs in the solid state form extended multilayer sheets. At the solid to fluid transition, multilamellar vesicles appear to "bud off" from the surface. Quasi-elastic light scattering (QELS) measurements indicated that for the PEs, bundle size is independent of acyl chain length n, but that the sizes of vesicles which form at the solid to fluid transition are positively correlated with n. The results of temperature jump experiments showed that once the transition temperature was reached, vesicle formation was largely complete within 30 s.     

A correlation between lipid domain shape and binary phospholipid mixture composition in free standing bilayers: A two-photon fluorescence microscopy study

Giant unilamellar vesicles (GUVs) composed of different phospholipid binary mixtures were studied at different temperatures, by a method combining the sectioning capability of the two-photon excitation fluorescence microscope and the partition and spectral properties of 6-dodecanoyl-2-dimethylamino-naphthalene (Laurdan) and Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (N-Rh-DPPE). We analyzed and compared fluorescence images of GUVs composed of 1,2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DLPC/DPPC), 1, 2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-distearoyl-sn-glycero-3-phosphocholine (DLPC/DSPC), 1, 2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-diarachidoyl-sn-glycero-3-phosphocholine (DLPC/DAPC), 1, 2-dimyristoyl-sn-glycero-3-phosphocholine/1, 2-distearoyl-sn-glycero-3-phosphocholine (DMPC/DSPC) (1:1 mol/mol in all cases), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine/1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPE/DMPC) (7:3 mol/mol) at temperatures corresponding to the fluid phase and the fluid-solid phase coexistence. In addition, we studied the solid-solid temperature regime for the DMPC/DSPC and DMPE/DMPC mixtures. From the Laurdan intensity images the generalized polarization function (GP) was calculated at different temperatures to characterize the phase state of the lipid domains. We found a homogeneous fluorescence distribution in the GUV images at temperatures corresponding to the fluid region for all of the lipid mixtures. At temperatures corresponding to phase coexistence we observed concurrent fluid and solid domains in the GUVs independent of the lipid mixture. In all cases the lipid solid domains expanded and migrated around the vesicle surface as we decreased the temperature. The migration of the solid domains decreased dramatically at temperatures close to the solid-fluid-->solid phase transition. For the DLPC-containing mixtures, the solid domains showed line, quasicircular, and dendritic shapes as the difference in the hydrophobic chain length between the components of the binary mixture increases. In addition, for the saturated PC-containing mixtures, we found a linear relationship between the GP values for the fluid and solid domains and the difference between the hydrophobic chain length of the binary mixture components. Specifically, at the phase coexistence temperature region the difference in the GP values, associated with the fluid and solid domains, increases as the difference in the chain length of the binary mixture component increases. This last finding suggests that in the solid-phase domains, the local concentration of the low melting temperature phospholipid component increases as the hydrophobic mismatch decreases. At the phase coexistence temperature regime and based on the Laurdan GP data, we observe that when the hydrophobic mismatch is 8 (DLPC/DAPC), the concentration of the low melting temperature phospholipid component in the solid domains is negligible. This last observation extends to the saturated PE/PC mixtures at the phase coexistence temperature range. For the DMPC/DSPC we found that the nonfluorescent solid regions gradually disappear in the solid temperature regime of the phase diagram, suggesting lipid miscibility. This last result is in contrast with that found for DMPE/DMPC mixtures, where the solid domains remain on the GUV surface at temperatures corresponding to that of the solid region. In all cases the solid domains span the inner and outer leaflets of the membrane, suggesting a strong coupling between the inner and outer monolayers of the lipid membrane. This last finding extends previous observations of GUVs composed of DPPE/DPPC and DLPC/DPPC mixtures (, Biophys. J. 78:290-305).     

Structural characterization of cationic DODAB bilayers containing C24:1 β-glucosylceramide

The effect of 5 mol%, 9 mol%, and 16 mol% of C24:1 β-glucosylceramide (βGlcCer) on the structure of cationic DODAB bilayers was investigated by means of differential scanning calorimetry (DSC), electron spin resonance (ESR) spectroscopy and fluorescence microscopy. βGlcCer is completely miscible with DODAB at all fractions tested, since no domains were observed in fluorescence microscopy or ESR spectra. The latter showed that βGlcCer destabilized the gel phase of DODAB bilayers by decreasing the gel phase packing. As a consequence, βGlcCer induced a decrease in the phase transition temperature and cooperativity of DODAB bilayers, as seen in DSC thermograms. ESR spectra also showed that βGlcCer induced an increase in DODAB fluid phase order and/or rigidity. Despite their different structures, a similar effect of loosening the gel phase packing and turning the fluid phase more rigid/organized has also been observed when low molar fractions of cholesterol were incorporated in DODAB bilayers. The structural characterization of mixed membranes made of cationic lipids and glucosylceramides may be important for developing novel immunotherapeutic tools such as vaccine adjuvants.     

The influence of Ca2+ on the lateral lipid distribution and phase transition in phosphatidylethanolamine/phosphatidylserine vesicles

The lateral lipid distribution within dipalmitoylphosphatidylethanolamine (DPPE)/dipalmitoylphosphatidylserine (DPPS) vesicle membranes was investigated under the influence of Ca²⁺ using a lipid cross-linking method. To characterize the phase transition in DPPE/DPPS vesicles and to correlate the different phase states of the membrane lipids with the obtained lipid distribution ESR measurements using a fatty acid spin label were carried out. It is shown that Ca²⁺ has a significant influence on the lateral lipid distribution within the fluid phase of the membrane lipids; instead of a slight alternating lipid arrangement in absence of Ca²⁺ due to the electrostatic interaction between the DPPS headgroups after addition of Ca²⁺ a lateral cluster structure is characteristic of the fluid phase.     

Involvement of acid ceramidase in the degradation of bioactive N-acylethanolamines

Bioactive N-acylethanolamines (NAEs) include palmitoylethanolamide, oleoylethanolamide, and anandamide, which exert anti-inflammatory, anorexic, and cannabimimetic actions, respectively. The degradation of NAEs has been attributed to two hydrolases, fatty acid amide hydrolase and NAE acid amidase (NAAA). Acid ceramidase (AC) is a lysosomal enzyme that hydrolyzes ceramide (N-acylsphingosine), which resembles NAAA in structure and function. In the present study, we examined the role of AC in the degradation of NAEs. First, we demonstrated that purified recombinant human AC can hydrolyze various NAEs with lauroylethanolamide (C12:0-NAE) as the most reactive NAE substrate. We then used HEK293 cells metabolically labeled with [¹⁴C]ethanolamine, and revealed that overexpressed AC lowered the levels of ¹⁴C-labeled NAE. As analyzed with liquid chromatography-tandem mass spectrometry, AC overexpression decreased the amounts of different NAE species. Furthermore, suppression of endogenous AC in LNCaP prostate cells by siRNA increased the levels of various NAEs. Lastly, tissue homogenates from mice genetically lacking saposin D, a presumable activator protein of AC, showed much lower hydrolyzing activity for NAE as well as ceramide than the homogenates from wild-type mice. These results demonstrate the ability of AC to hydrolyze NAEs and suggest its physiological role as a third NAE hydrolase.     

Orientation and lipid-peptide interactions of gramicidin A in lipid membranes: polarized attenuated total reflection infrared spectroscopy and spin-label electron spin resonance

Gramicidin A was incorporated at a peptide/lipid ratio of 1:10 mol/mol in aligned bilayers of dimyristoyl phosphatidylcholine (DMPC), phosphatidylserine (DMPS), phosphatidylglycerol (DMPG), and phosphatidylethanolamine (DMPE), from trifluoroethanol. Orientations of the peptide and lipid chains were determined by polarized attenuated total reflection infrared spectroscopy. Lipid-peptide interactions with gramicidin A in DMPC bilayers were studied with different spin-labeled lipid species by using electron spin resonance spectroscopy. In DMPC membranes, the orientation of the lipid chains is comparable to that in the absence of peptide, in both gel and fluid phases. In gel-phase DMPC, the effective tilt of the peptide exceeds that of the lipid chains, but in the fluid phase both are similar. For gramicidin A in DMPS, DMPG, and DMPE, the degree of orientation of the peptide and lipid chains is less than in DMPC. In the fluid phase of DMPS, DMPG, and DMPE, gramicidin A is also less well oriented than are the lipid chains. In DMPE especially, gramicidin A is largely disordered. In DMPC membranes, three to four lipids per monomer experience direct motional restriction on interaction with gramicidin A. This is approximately half the number of lipids expected to contact the intramembranous perimeter of the gramicidin A monomer. A selectivity for certain negatively charged lipids is found in the interaction with gramicidin A in DMPC. These results are discussed in terms of the integration of gramicidin A channels in lipid bilayers, and of the interactions of lipids with integral membrane proteins.     

Phase diagrams of pseudo-binary phospholipid systems. I. Influence of the chain length differences on the miscibility properties of cephaline/cephaline/water systems

The miscibility properties of homologous cephalines (PEs) were studied by means of differential scanning calorimetry (DSC). The phase diagrams of 5 pseudo-binary cephaline/cephaline/water systems (50% water) are discussed. In the high temperature L alpha-phase, all the homologous cephalines of fatty acid chain length from C = 12 to C = 18 were completely miscible. On the other hand in the low temperature L beta-phase, a miscibility gap occurred in dependence on the differences of the acyl chain lengths. Further, a distinct succession of the phase diagram types was observed according to increasing chain length differences of the PEs: complete miscibility (systems di-(C12:O)-PE/di-(C14:O)-PE/H2O; di-(C14:O)-PE/di-C(16:O)-PE/H2O)----peritectic mixing behaviour (systems di-(C12:O)-PE/di-(C16:O)-PE/H2O; di-(C14:O)-PE/di-(C18:O)-PE/H2O)----eutectic mixing behaviour (system di-(C12:O)-PE/di-(C18:O)-PE/H2O). The change in the type of phase diagram from azeotropic to semi-azeotropic and from semi-azeotropic to eutectic is interpreted by means of the Landau theory.