Bilayer Order Research Articles

Synthesis of methotrexate ?,?-bis(amides) and correlation of thermotropic and DPPC biomembrane interaction parameters with their anticancer activity

The interaction of MTX and some aliphatic his(amide) derivatives with synthetic 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) biomembranes was investigated. The drug-membrane model interaction was carried out by differential scanning calorimetry. Anticancer activity of MIX his(amides) was evaluated on cultures of a human leukemic cell line (CCRF-CEM) in comparison with MTX. Compounds were tested at a concentration ranging between 10 nM and 1 μM. The MTX is able to interact with the outer part of the phospholipid bilayers due to its polar nature. Results showed that the amide derivatives of MTX, presenting a marked lipophilic character, are able to interact with the hydrophobic core of the DPPC bilayers, thus perturbing the packing order of the phospholipid bilayers. Particularly, a reduction of the enthalpy values linked to the transition from the gel state to the liquid crystal state of DPPC membranes was observed. This effect is a function of the type and molar fraction of the various compounds. The in vitro antitumor activity on leukemic CCRF-CEM cells was higher for MTX-bis(tetradecylamide) than for the other derivatives. The biological effectiveness of the various MTX derivatives correlates very well with the enthalpy of the transition of drug-loaded DPPC biomembranes.

Direct atomic force microscopic evidence of hydrogen bonding interaction in phosphatidic acid Langmuir-Blodgett bilayer

Molecularly resolved atomic force microscopic images of phosphatidic acid Langmuir-Blodgett bilayers show that phosphate groups in polar region of the films are packing in a distorted hexagonal organization with long-range orientational and positional order. Intermolecular hydrogen bonding interactions, which should be responsible for the ordering and stability of bilayers, are visualized directly between adjacent phosphate groups in the polar region of the bilayer. Some adjacent phosphatidic acid molecules link each other through the formation of intermolecular hydrogen bonds between phosphate groups in polar region to form local supramolecules, which provide the bilayer's potential as a functionized film in the investigation on the lateral conductions of protons in the biological bilayers.

The lipid peroxidation product, 4-hydroxy-2-trans-nonenal, alters the conformation of cortical synaptosomal membrane proteins.

Alzheimer's disease (AD) is widely held to be a disorder associated with oxidative stress due, in part, to the membrane action of amyloid beta-peptide (A beta). A beta-associated free radicals cause lipid peroxidation, a major product of which is 4-hydroxy-2-trans-nonenal (HNE). We determined whether HNE would alter the conformation of synaptosomal membrane proteins, which might be related to the known neurotoxicity of A beta and HNE. Electron paramagnetic resonance spectroscopy, using a protein-specific spin label, MAL-6 (2,2,6,6-tetramethyl-4-maleimidopiperidin-1-oxyl), was used to probe conformational changes in gerbil cortical synaptosomal membrane proteins, and a lipid-specific stearic acid label, 5-nitroxide stearate, was used to probe for HNE-induced alterations in the fluidity of the bilayer domain of these membranes. Synaptosomal membranes, incubated with low concentrations of HNE, exhibited changes in protein conformation and bilayer order and motion (fluidity). The changes in protein conformation were found to be concentration- and time-dependent. Significant protein conformational changes were observed at physiologically relevant concentrations of 1-10 microM HNE, reminiscent of similar changes in synaptosomal membrane proteins from senile plaque- and A beta-rich AD hippocampal and inferior parietal brain regions. HNE-induced modifications in the physical state of gerbil synaptosomal membrane proteins were prevented completely by using excess glutathione ethyl ester, known to protect neurons from HNE-caused neurotoxicity. Membrane fluidity was found to increase at higher concentrations of HNE (50 microM). The results obtained are discussed with relevance to the hypothesis of A beta-induced free radical-mediated lipid peroxidation, leading to subsequent HNE-induced alterations in the structure and function of key membrane proteins with consequent neurotoxicity in AD brain.

Effect of inhalation anaesthetics on the phase behaviour, permeability and order of phosphatidylcholine bilayers

We have used differential scanning calorimetry and fluorescence anisotropy measurements to investigate the effect of five inhalation anaesthetics of diverse chemical structure (halothane, enflurane, n-pentane, chloroform and diethylether) on the phase behaviour of liposomes prepared from dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC), respectively. The incorporation of these anaesthetics induced a decrease of the phase transition temperature and/or a broadening of the phase transition peak depending on the transverse localisation of the investigated anaesthetic. At high anaesthetic concentrations we observed the disappearance of the pretransition peak and the appearance of a shoulder on the main phase transition peak due to the domain formation of the anaesthetics. An anaesthetic induced carboxyfluorescein efflux from the vesicle lumen was completed within a few minutes after the addition of the anaesthetics, probably resulting from a transient formation of membrane holes. All results are discussed with regard to the physicochemical properties of the anaesthetics applied.

Narrow bandpass multilayer x-ray monochromator

We propose a method to reduce the bandpass of soft x-ray multilayer Bragg reflectors. It consists in etching down to the substrate a multilayer mirror having a large number of bilayers in order to get a lamellar grating with a multilayer bar width representing only 10%–30% of the grating period. The grating is used at the zeroth diffraction order. Once designed, this monochromator was tested with the Lα,β spectra of iron and copper.

Interactions of thionin from Pyrularia pubera with dipalmitoylphosphatidylglycerol large unilamellar vesicles.

The peptide toxin thionin from Pyrularia pubera binds to dipalmitoylphosphatidylglycerol (DPPG) large unilamellar vesicles as shown by an increase in the intensity and blue-shift of the fluorescence emission spectrum of the single tryptophan residue of the protein. The magnitude of these fluorescence changes increased with temperature near the thermotropic phase transition of DPPG (about 40 degrees C). Fluorescent probes sensitive to the structure and dynamics of the membrane were used to assess the effect of thionin binding on bilayer properties. The fluorescence emission spectra of Prodan, Patman, and Laurdan all showed spectral changes consistent with an increase in bilayer polarity at temperatures below the DPPG phase transition but a decrease in polarity at higher temperatures. Fluorescence polarization experiments and the ratio of monomer-to-excimer fluorescence of the probe 1,3-bis(1-pyrene)propane suggested that thionin increases the bilayer order above the transition temperature. Differential scanning calorimetry revealed that thionin broadens the transition and either increases or decreases the melting temperature depending on the concentration of the peptide. Taken together, the data are consistent with at least three distinct interactions of thionin with the bilayer: (1) thionin bound electrostatically to the bilayer surface; (2) tryptophan of the bound thionin inserted into the bilayer; (3) high-order aggregates of thionin-bound vesicles.

Dipolar Recoupling in MAS NMR: A Probe for Segmental Order in Lipid Bilayers

Novel two-dimensional magic-angle spinning (2D MAS) NMR experiments designed to measure the magnitudes and signs of 13C−1H dipolar interactions in fluid phase lipid bilayers are presented. MAS is e...

Interaction of Zn2+Ions with Phospholipid Multilayers

Abstract Phospholipids are large natural amphipathic molecules which, in contact with water, assemble into higher molecular aggregates. The most relevant one is the bilayer for its relation to the structure, properties and functions of cell membranes. Given their structural complexity phospholipid bilayers are usually used as models for studying how biologically relevant chemicals affect the structure and functions of biomembranes. Zn is a trace element essential for the functional integrity of cell membranes. It is also a common contaminant of sea waters. Therefore, it was thought of interest to study the interaction of Zn2- ions with phospholipid bilayers in order to understand the way they interact with cell membranes. With this aim Zn2+, in a wide range of concentrations, was made to interact with multibilayers of the phospholipids dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE). This study was performed by X-ray diffraction. Besides, the interaction of Zn2+ with D...

The effects of cholesterol inclusion on the vesicular membranes of cationic lipids

Small unilamellar vesicles formed from four cationic lipids in the absence and the presence of varying amounts of cholesterol were studied using fluorescence polarization and 1H-NMR techniques. The fluorescence polarization data clearly indicate that the packing order in the cationic lipid bilayers are affected by inclusion of cholesterol. Importantly, this effect exists also with a cationic lipid that is devoid of any formal linkage region where the interaction of the lipid with cholesterol through hydrogen bonding is not feasible. The interactions of cholesterol with different types of cationic lipids in excess water have also been examined in multilamellar dispersions using proton magnetic resonance spectroscopy. In all the cases, the methylene proton linewidths in the NMR spectra respond to the addition of cholesterol to vesicles. Hydrophobic association of the lipid and cholesterol imposes restriction on the chain (CH 2) n motions, leaving the terminal CH 3 groups relatively mobile. On the basis of energy-minimized structural models, a rationale of the cholesterol-cationic lipid assembly has also been presented.

Onset and evolution of the tilted smectic antiphase in a polar liquid-crystal binary mixture.

High-resolution x-ray diffraction studies of a binary mixture of the n=8 and n=10 homologs of alkoxyphenyl nitrobenzoyloxy benzoate ${\mathrm{DB}}_{\mathit{n}}$${\mathrm{ONO}}_{2}$ (52.6 mole % ${\mathrm{DB}}_{10}$${\mathrm{ONO}}_{2}$) have been performed at the onset of the tilted smectic antiphase (Sm-C\ifmmode \tilde{}\else \~{}\fi{}) upon cooling from the smectic-${\mathit{A}}_{1}$ phase. Fluctuations of the two competing smectic orders are found to be related to monolayer and partial bilayer ordering. With decreasing temperature, the period of the antiphase in-plane modulation increases while the smectic layer normal tilts at an angle of \ifmmode\pm\else\textpm\fi{}7\ifmmode^\circ\else\textdegree\fi{} with respect to the director. This tilt partially recovers upon the transition to the smectic-${\mathit{A}}_{2}$ phase. \textcopyright{} 1996 The American Physical Society.

Influence of the redox state of ubiquinones and plastoquinones on the order of lipid bilayers studied by fluorescence anisotropy of diphenylhexatriene and trimethylammonium diphenylhexatriene

The measurements of diphenylhexatriene (DPH) and trimethylammonium diphenylhexatriene (TMA-DPH) fluorescence anisotropy in egg yolk lecithin (EYL) and of DPH anisotropy in dipalmitoylphosphatidylcholine (DPPC) liposomes containing different concentrations of oxidized and reduced ubiquinone (UQ) and plastoquinone (PQ) homologues have been performed. All the oxidized UQ homologues strongly induced ordering of EYL membrane structure, whereas in DPPC liposomes, above the phase transition temperature, the most pronounced effect showed UQ-4. PQ-2 and PQ-9 were less effective than the corresponding ubiquinones in this respect. The reduced forms of UQ and PQ homologues increased the order of membrane lipids to a smaller extent than the corresponding quinones both in the interior of the membrane and closer to its surface. Nevertheless, the investigated prenylquinols showed stronger increase in the membrane order than α-tocopherol or α-tocopherol acetate, which could be connected with binding of prenylquinol head groups to phospholipid molecules by hydrogen bonds. The strong ordering influence of ubiquinones on the membrane structure was attributed to methoxyl groups of the UQ quinone rings.

Perturbation of artificial and biological membranes by organic compounds of aliphatic, alicyclic and aromatic structure

Aliphatic, alicyclic and aromatic hydrocarbons interact with biological membranes. Until now little has been known about their mode of interaction with the membrane bilayer and membrane integral proteins with toxic effects to cells. The lipid theory hypothesis explains the toxic effects by the organic solvent-induced disorder in the lipid bilayer, which indirectly affects the function of membrane-embedded proteins. The extent of bilayer perturbations is ascribed to the solvent accumulation in the bilayer, which is related only to the lipophilicity of the molecule, independent of the chemical structure. In this study the fluidizing effects of aliphatic, alicyclic and aromatic hydrocarbons were compared. Membrane fluidity changes were estimated from the pyrene excimer formation, using pyrene and pyrene derivatives to label specifically the localization of solvent molecules in the transverse plane of the bilayer. Liposomal, microsomal and synaptosomal membrane preparations were evaluated because proteins and cholesterol, as natural membrane components, increase the bilayer order and reduce the organic solvent membrane/buffer partition. In the concentration range investigated, only the aromatic solvents disorder the lipid bilayer, with the greatest perturbation in the centre of the bilayer. These results are related to structural properties of the organic solvents investigated.

Palmitoyl Carnitine Increases the Transmeningeal Flux of Hydrophilic but Not Hydrophobic Compounds In Vitro

Increasing drug penetration through the spinal meninges should improve epidural analgesia/anesthesia by increasing the rate and extent to which epidurally administered drugs redistribute to the spinal cord. Palmitoyl carnitine has been shown to improve drug penetration through the intestinal mucosa. The purpose of this study was to determine whether palmitoyl carnitine improves drug penetration through the spinal meninges. The transmeningeal flux of morphine, mannitol, bupivacaine, and sufentanil was determined through the spinal meninges of Macacca nemestrina monkeys before and after addition of palmitoyl carnitine. Flux was determined using a previously established in vitro diffusion cell method. Palmitoyl carnitine significantly increased the transmeningeal flux of mannitol, morphine, and bupivacaine by 942 +/- 102%, 401 +/- 43%, and 12 +/- 4%, respectively. However, palmitoyl carnitine had no significant effect on the transmeningeal flux of sufentanil. The penetration-enhancing effect of palmitoyl carnitine was shown to depend on the dose of palmitoyl carnitine added and on the octanol:buffer distribution coefficient of the study drugs but not on the concentration of the study drug. The mechanism by which palmitoyl carnitine increases transmeningeal flux is unclear but may be a result of palmitoyl carnitine's ability to decrease packing order of lipid bilayers in the arachnoid mater cell membranes. Regardless of the mechanism, palmitoyl carnitine's ability to selectively increase the transmeningeal flux of hydrophilic compounds in vitro offers the possibility of improving the spinal bioavailability of this group of epidurally administered drugs in vivo.

New insights into the gating mechanisms of cardiac ryanodine receptors revealed by rapid changes in ligand concentration.

We have developed a novel technique for incorporation of sheep cardiac sarcoplasmic reticulum (SR) Ca(2+)-release channels into planar phospholipid bilayers in order to investigate changes in [Ca2+] on a physiological time scale and have investigated whether the rate of change of cytosolic [Ca2+] has a direct effect on the gating of the cardiac SR Ca(2+)-release channel. Vesicles of heavy SR were incorporated into planar phospholipid bilayers painted on glass pipettes, and an established technique for rapid solution exchanges at excised membrane patches was modified to allow solution changes to be made at the bilayer within 10 ms. For a given change in [Ca2+], we demonstrate that the open probability (Po) is similar whether the cytosolic [Ca2+] is increased rapidly (10 ms) or slowly (1 s) and appears to be no different from the Po measured under steady state conditions that were recorded by using conventional bilayer techniques. We also demonstrate that no desensitization or inactivation occurs at -40 mV when the channel is activated by Ca2+ alone or in the presence of other channel activators, ATP or EMD 41000. However, at +40 mV, rapid channel activation followed by inactivation was observed. The probability of such voltage-dependent inactivation appears to depend on the mechanism of channel activation.

Effects of ethanol on lipid bilayers containing cholesterol, gangliosides, and sphingomyelin.

The influence of lipid composition on the response of bilayers to ethanol binding was investigated with 2H NMR spectroscopy. The bilayers were composed of various combinations of the lipids most often found in neural cell membranes: phosphatidylcholines (PCs), gangliosides, sphingomyelin, and cholesterol. The PCs, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), were chain-perdeuterated to allow the response of bilayer order to ethanol to be monitored at all positions through the depth of the bilayer interior. All bilayers were investigated in the lamellar liquid-crystalline (L alpha) phase. The results from the de-Paked NMR spectra demonstrate that ethanol binding in the lipid-water interface [Barry, J. A., & Gawrisch, K. (1994) Biochemistry 33, 8082-8088] alters order parameter profiles in the bilayer interior differently for the various lipid mixtures. The presence of 10 mol % brain gangliosides enhanced the disordering effect of ethanol and altered the response of the order profile along the PC chains. This effect was apparently caused by sugar-ethanol interactions in the oligosaccharide head group. The impact of the ceramide moiety of brain sphingomyelin (50 mol % in DMPC) was negligible. In bilayers containing cholesterol, the binding of ethanol and its effects on the hydrocarbon interior were found to reflect the phase transition to the liquid-ordered phase at about 25 mol % cholesterol [Thewalt, J. L., & Bloom, M. (1992) Biophys. J. 63, 1176-1181]. Results from the quadrupolar splittings for deuterated ethanol (CH3CD2OH) bound to cholesterol-containing bilayers showed that ethanol binding decreased with increasing amounts of cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydration and order in lipid bilayers.

The relationship between membrane lipid bilayer hydration and acyl chain order was investigated using time-resolved fluorescence spectroscopy. The degree of hydration in the head group region was assessed from fluorescence lifetime data along with fluorescence intensity measurements in D2O, relative to H2O buffer, using N-(5-dimethylaminonaphthalene-1-sulfonyl)dipalmitoylphosphatidylethan ola mine (dansyl-PE). The degree of hydration in the acyl chain region was estimated from its effect on the fluorescence lifetime of 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl] carbonyl]-3-sn-phosphatidylcholine (DPH-PC), and acyl chain order was determined from time-resolved anisotropy measurements of the DPH-PC. Comparisons of sn-2 unsaturation with sn-1,2 diunsaturation in phosphatidylcholine (PC) bilayers with the same number of double bonds/PC revealed a marked difference in interchain hydration and acyl chain order but little difference in terms of head group hydration. For diunsaturated dioleoyl-PC (DOPC) bilayers with two double bonds/PC, the DPH-PC fluorescence lifetime data indicated a greater level of interchain hydration than 1-palmitoyl-2-docosahexaenoyl-PC (PDPC) with six double bonds/sn-2 chain. By contrast, the head group hydration for DOPC was markedly less than for PDPC. A similar lack of correlation of effects on the two regions of the bilayer was found with cholesterol, it having opposite effects on interchain and head group hydration. When DPH-PC fluorescence lifetime data for bilayers composed of a range of different lipids was plotted as a function of acyl chain order, a strong correlation of interchain hydration with acyl chain order was revealed that was independent of lipid composition.(ABSTRACT TRUNCATED AT 250 WORDS)

Spontaneous translocation of a polymer across a curved membrane.

The translocation of a hydrophobic polymer across a curved bilayer membrane has been studied using Monte Carlo methods. It is found that for curved membranes the polymer crosses spontaneously and almost irreversibly from the side of lower curvature to the side of higher curvature. This phenomenon can be understood based upon the curvature-induced difference of lipid fluctuations between the two halves of the bilayer. The difference of fluctuations drives the polymer across the bilayer in order to maximize the conformational entropy of the polymer.

Monte Carlo study of lipid membranes: Simulation of diparmitoylphosphatidylcholine bilayers in gel and liquid‐crystalline phases

AbstractThe statistical properties of the bilayer membranes of diparmitoylphosphatidylcholine (DPPC) in the gel and liquid‐crystal phases were studied by Monte Carlo (MC) simulation using potential functions of the Lennard‐Jones, the simple Coulomb, and the bond torsion. The simulation was undertaken on a two‐dimensional periodic condition imposed on the bilayer model consisting of faithfully described molecules. The structure and ordering of the model bilayers accorded well with experiments, and the segment order parameters were in agreement with those of the nuclear magnetic resonance (NMR) experiments. The two kinds of lipid chains of DPPC do not equivalently behave in the bilayers, and chain 2 has lower ordering than chain 1. The order parameters of the first eight segments of chain 2 in the liquid‐crystal model are particularly small and are roughly constant. From electron density analysis, it has been observed that the liquid‐crystal bilayer has about one excess water molecule per one lipid molecule in comparison with the gel bilayer. The energy difference between the two bilayer models, taking account of the water contribution, is consistent with the latent heat of the phase transition. © 1995 by John Wiley & Sons, Inc.

Stages of the bilayer-micelle transition in the system phosphatidylcholine-C12E8 as studied by deuterium- and phosphorous-NMR, light scattering, and calorimetry

The perturbation of phospholipid bilayer membranes by a nonionic detergent, octaethyleneglycol mono-n-dodecylether (C12E8), was investigated by 2H- and 31P-NMR, static and dynamic light scattering, and differential scanning calorimetry. Preequilibrated mixtures of the saturated phospholipids 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), and 1,2-dilauroyl-sn-glycero-3-phosphorylcholine (DLPC) with the detergent were studied over a broad temperature range including the temperature of the main thermotropic phase transition of the pure phospholipids. Above this temperature, at a phospholipid/detergent molar ratio 2:1, the membranes were oriented in the magnetic field. Cooling of the mixtures below the thermotropic phase transition temperatures of the pure phospholipids led to micelle formation. In mixtures of DPPC and DMPC with C12E8, a narrow calorimetric signal at the onset temperature of the solubilization suggested that micelle formation was related to the disorder-order transition in the phospholipid acyl chains. The particle size changed from 150 nm to approximately 7 nm over the temperature range of the bilayer-micelle transition. The spontaneous orientation of the membranes at high temperatures enabled the direct determination of segmental order parameters from the deuterium spectra. The order parameter profiles of the phospholipid acyl chains could be attributed to slow fluctuations of the whole membrane and to detergent-induced local perturbations of the bilayer order. The packing constraints in the mixed bilayers that eventually lead to bilayer solubilization were reflected by the order parameters of the interfacial phospholipid acyl chain segments and of the phospholipid headgroup. These results are interpreted in terms of the changing average shape of the component molecules. Considering the decreasing cross sectional areas in the acyl chain region and the increasing hydration of the detergent headgroups, the bilayer-micelle transition is the result of an imbalance in the chain and headgroup repulsion. A neutral or pivotal plane can be defined on the basis of the temperature dependence of the interfacial quadrupolar splittings.

Dynamical order and disorder in lipid bilayers

Various order and disorder phenomena in lipid bilayers are considered as they arise due to the very many-particle character of the bilayer. Particular attention is paid to dynamically maintained order in terms of lateral density- and compositional fluctuations that lead to dynamic heterogeneity, local structure, and lipid-domain formation on length scales of 10–1000 Å. The influence of cholesterol and various drugs on the local structure is described. A discussion is presented of the possible role played by lipid order and disorder phenomena for the functional dynamics of membranes.