PC Lipids Research Articles

Stability of Asymmetric Lipid Bilayers Assessed by Molecular Dynamics Simulations

The asymmetric insertion of amphiphiles into biological membranes compromises the balance between the inner and outer monolayers. As a result, area expansion of the receiving leaflet and curvature strain may lead to membrane permeation, shape changes, or membrane fusion events. We have conducted both atomistic and coarse-grained molecular dynamics simulations of dipalmitoyl-phosphatidylcholine (DPPC) bilayers to study the effect of an asymmetric distribution of lipids between the two monolayers on membrane stability. Highly asymmetric lipid bilayers were found to be surprisingly stable within the submicrosecond time span of the simulations. Even the limiting case of a monolayer immersed in water ruptured spontaneously only after at least 20 ns simulation. A thermal shock could destabilize these kinetically trapped states. We also studied mixed systems composed of DPPC and short tail diC(8)PC lipids, showing that the presence of the cone-shaped short tail lipid facilitates the release of tension in the asymmetric systems via formation of a transmembrane pore. Thus, asymmetric area expansion and curvature stress cooperate to yield bilayer disruption. It appears that, although asymmetric area expansion destabilizes the bilayer structure, the activation energy for transmonolayer lipid re-equilibration is increased. Such a large kinetic barrier can be reduced by lipids with positive spontaneous curvature. These effects are important at the onset of bilayer destabilization phenomena, such as lipid pore formation and membrane fusion, and should be considered for the mechanism of induction of such processes by peptides and proteins.

Omega‐3‐producing fat‐1 transgenic mice display reduced complex I‐associated hydrogen peroxide production and enzyme activity

The fat‐1 gene of C. elegans encodes a desaturase, which can convert n‐6 to n‐3 fatty acids. A transgenic mouse that constitutively expresses this gene was used as a mammalian model of increased tissue n‐3 fatty acids. The aim of the study was to evaluate the influence of changes in membrane fatty acid composition on the lipid‐protein interaction, specifically on the production of H2O2 and electron transport chain enzyme activities. Liver mitochondria were isolated by differential centrifugation and the activities of the electron transport chain enzymes determined spectrophotometrically, while H2O2 production was determined fluorimetrically. Fatty acid analyses of mitochondrial membranes were undertaken on Percoll‐purified mitochondria, and performed by Lipomics Technology, Inc. The results indicate that the transgenic mice show a decreased H2O2 production at complex I, as well as decreased complex I enzyme activity. Total H2O2 production was also decreased when mitochondria were respiring on succinate or succinate/glutamate/malate. Fatty acid analysis showed an increased n‐3/n‐6 ratio in mitochondrial PC, PE and LyPC lipid classes, with arachidonic acid (n‐6) decreased and eicosapentaenoic acid (n‐3) increased in all phospholipid classes. The results indicate that decreased complex I‐linked ROS production may provide some protection against oxidative stress in membranes with high n‐3 levels.

Perturbation of DPPC/POPG bilayers by the N-terminal helix of lung surfactant protein SP-B: a 2H NMR study

SP-B(8-25) is a synthetic peptide comprising the N-terminal helix of the essential lung surfactant protein SP-B. Rat lung oxygenation studies have shown that SP-B(8-25) retains some of the function of full-length SP-B. We have used deuterium nuclear magnetic resonance ((2)H-NMR) to examine the influence of SP-B(8-25) on the mixing properties of saturated PC and unsaturated PG lipids in model mixed lipid bilayers containing dipalmitoylphosphatidylcholine (DPPC) and palmitoyl-oleoyl-phosphatidylglycerol (POPG), in a molar ratio of 7:3. In the absence of the peptide, (2)H-NMR spectra of DPPC/POPG mixtures, with one or the other lipid component deuterated, indicate coexistence of large liquid crystal and gel domains over a range of about 10 degrees C through the liquid crystal to gel transition of the bilayer. Addition of SP-B(8-25) has little effect on the width of the transition but the spectra through the transition range cannot be resolved into distinct liquid crystal and gel spectral components suggesting that the peptide interferes with the tendency of the DPPC and POPG lipid components in this mixture to phase separate near the bilayer transition temperature. Quadrupole echo decay observations suggest that the peptide may also reduce differences in the correlation times for local reorientation of the two lipids. These observations suggest that SP-B(8-25) promotes a more thorough mixing of saturated PC and unsaturated PG components and may be relevant to understanding the behaviour of lung surfactant material under conditions of lateral compression which might be expected to enhance the propensity for saturated and unsaturated surfactant lipid components to segregate.

Membrane Protein Frustration: Protein Incorporation into Hydrophobic Mismatched Binary Lipid Mixtures

Bacteriophage M13 major coat protein was reconstituted in different nonmatching binary lipid mixtures composed of 14:1PC and 22:1PC lipid bilayers. Challenged by this lose-lose situation of hydrophobic mismatch, the protein-lipid interactions are monitored by CD and site-directed spin-label electron spin resonance spectroscopy of spin-labeled site-specific single cysteine mutants located in the C-terminal protein domain embedded in the hydrophobic core of the membrane (I39C) and at the lipid-water interface (T46C). The CD spectra indicate an overall α-helical conformation irrespective of the composition of the binary lipid mixture. Spin-labeled protein mutant I39C senses the phase transition in 22:1PC, in contrast to spin-labeled protein mutant T46C, which is not affected by the transition. The results of both CD and electron spin resonance spectroscopy clearly indicate that the protein preferentially partitions into the shorter 14:1PC both above and below the gel-to-liquid crystalline phase transition temperature of 22:1PC. This preference is related to the protein tilt angle and energy penalty the protein has to pay in the thicker 22:1PC. Given the fact that in Escherichia coli, which is the host for M13 bacteriophage, it is easier to find shorter 14 carbon acyl chains than longer 22 carbon acyl chains, the choice the M13 coat protein makes seems to be evolutionary justified.

Lateral Stress and Spontaneous Curvature in Mixed Membranes

A key aspect of membrane mechanics is the balance of attractive and repulsive forces involving lipid head groups and acyl chains [1]. In particular, demethylation of PC lipids and addition of polyunsaturated chains [2] have been shown to alter substantially the activity of membrane receptors [3, 4] and of ion channels. How to best describe membrane forces? An appealing theoretical description is the lateral stress profile, which is easily calculated from computer simulations. However the lateral stress profile is not yet accessible experimentally.

NADPH Oxidase-dependent Generation of Lysophosphatidylserine Enhances Clearance of Activated and Dying Neutrophils via G2A

Exofacial phosphatidylserine (PS) is an important ligand mediating apoptotic cell clearance by phagocytes. Oxidation of PS fatty acyl groups (oxPS) during apoptosis reportedly mediates recognition through scavenger receptors. Given the oxidative capacity of the neutrophil NADPH oxidase, we sought to identify oxPS signaling species in stimulated neutrophils. Using mass spectrometry analysis, only trace amounts of previously characterized oxPS species were found. Conversely, 18:1 and 18:0 lysophosphatidylserine (lyso-PS), known bioactive signaling phospholipids, were identified as abundant modified PS species following activation of the neutrophil oxidase. NADPH oxidase inhibitors blocked the production of lyso-PS in vitro, and accordingly, its generation in vivo by activated, murine neutrophils during zymosan-induced peritonitis was absent in mice lacking a functional NADPH oxidase (gp91phox-/-). Treatment of macrophages with lyso-PS enhanced the uptake of apoptotic cells in vitro, an effect that was dependent on signaling via the macrophage G2A receptor. Similarly, endogenously produced lyso-PS also enhanced the G2A-mediated uptake of activated PS-exposing (but non-apoptotic) neutrophils, raising the possibility of non-apoptotic mechanisms for removal of inflammatory cells during resolution. Finally, antibody blockade of G2A signaling in vivo prolonged zymosan-induced neutrophilia in wild-type mice, whereas having no effect in gp91phox-/- mice where lyso-PS are not generated. Taken together, we show that lyso-PS are modified PS species generated following activation of the NADPH oxidase and lyso-PS signaling through the macrophage G2A functions to enhance existing receptor/ligand systems for optimal resolution of neutrophilic inflammation.

Assessment of hypercoagulability markers and lipid levels in postmenopausal women undergoing either oral or transdermal hormone replacement therapy

This study investigated the effect of either oral or transdermal hormone replacement therapy (HRT) on haemostatic, fibrinolytic and lipid profiles in a group of Brazilian women 3 months after beginning treatment by comparing these results with those obtained immediately before HRT. Plasma levels of TAT, DDi, F1+2, PC, PS, AT, PAI-1 and serum lipids were determined in blood samples collected from 24 women undergoing oral HRT and from 11 women undergoing transdermal HRT. Significant increases in DDi and F1+2 plasma levels were observed after 3 months of oral HRT, while PS levels decreased. After transdermal HRT, a significant decrease was observed only for AT levels. After 3 months of oral HRT and in the absence of major genetic and acquired risk factors, women displayed a predisposition for activation of blood coagulation, and an increased activity of the fibrinolytic system. Oral HRT seemed to be more effective in predisposing haemostatic changes as compared to transdermal.

Comparison of DPPC and DPPG Environments in Pulmonary Surfactant Models

Deuterium nuclear magnetic resonance was used to monitor lipid acyl-chain orientational order in suspensions of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) containing Ca 2+ and the lung surfactant proteins SP-A and SP-B separately and together. To distinguish between protein-lipid interactions involving the PC and PG lipid headgroups and to examine whether such interactions might influence spatial distribution of lipids within the bilayer, acyl chains on either the DPPC or the DPPG component of the mixture were deuterated. The lipid components of the resulting mixtures were thus either DPPC- d 62/DPPG (7:3) or DPPC/DPPG- d 62 (7:3), respectively. SP-A had little effect on DPPC- d 62 chain order but did narrow the temperature range over which DPPG- d 62 ordered at the liquid-crystal-to-gel transition. No segregation of lipid components was seen for temperatures above or below the transition. Near the transition, though, there was evidence that SP-A promoted preferential depletion of DPPG from liquid crystalline domains in the temperature range over which gel and liquid crystal domains coexist. SP-B lowered average chain order of both lipids both above and below the main transition. The perturbations of chain order by SP-A and SP-B together were smaller than by SP-B alone. This reduction in perturbation of the lipids by the additional presence of SP-A likely indicated a strong interaction between SP-A and SP-B. The competitive lipid-lipid, lipid-protein, and protein-protein interactions suggested by these observations presumably facilitate the reorganization of surfactant material inherent in the transformation from lamellar bodies to a functional surfactant layer.

Expression of 25 Human ABC Transporters in the Yeast Pichia pastoris and Characterization of the Purified ABCC3 ATPase Activity

Human ATP-binding cassette (ABC) transporters comprise a family of 48 membrane-spanning transport proteins, many of which are associated with genetic diseases or multidrug resistance of cancers. In this study, we present a comprehensive approach for the cloning, expression, and purification of human ABC transporters in the yeast Pichia pastoris. We analyzed the expression of 25 proteins and demonstrate that 11 transporters, including ABCC3, ABCB6, ABCD1, ABCG1, ABCG4, ABCG5, ABCG8, ABCE1, ABCF1, ABCF2, and ABCF3, were expressed at high levels comparable to that of ABCB1 (P-glycoprotein). As an example of the purification strategy via tandem affinity chromatography, we purified ABCC3 (MRP3) whose role in the transport of anticancer drugs, bile acids, and glucuronides has been controversial. The yield of ABCC3 was 3.5 mg/100 g of cells in six independent purifications. Purified ABCC3, activated with PC lipids, exhibited significant ATPase activity with a Vmax of 82 +/- 32 nmol min-1 mg-1. The ATPase activity was stimulated by bile acids and glucuronide conjugates, reaching 170 +/- 28 nmol min-1 mg-1, but was not stimulated by a variety of anticancer drugs. The glucuronide conjugates ethinylestradiol-3-glucuronide and 17beta-estradiol-17-glucuronide stimulated the ATPase with relatively high affinities (apparent Km values of 2 and 3 microM, respectively) in contrast to bile acids (apparent Km values of >130 microM), suggesting that glucuronides are the preferred substrates for this transporter. Overall, the availability of a purification system for the production of large quantities of active transporters presents a major step not only toward understanding the role of ABCC3 but also toward future structure-function analysis of other human ABC transporters.

Buckling of Lipid Tubules in Shrinking Liquid Droplets

Self-assembled hollow lipid tubules are interesting and potentially useful supramolecular structures. Here, we study the deformation of lipid tubules of 1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) trapped inside liquid droplets on glass substrates. The interface tension of the shrinking liquid droplets exerts a compression force on the ends of the trapped lipid tubules, and causes them to buckle. This provides a method to measure their mechanical properties. The Young's modulus of the DC8,9PC lipid tubules is estimated to approximately 1.07 GPa. As the strain energy of the buckled tubules builds up, they poke through the interface of shrinking liquid droplets and then adhere onto glass substrates to form looplike shapes.

Equine Sperm Membrane Phase Behavior: The Effects of Lipid-Based Cryoprotectants1

The plasma membrane of sperm can undergo lipid phase separation during freezing, resulting in irreversible damage to the cell. The objective of our study was to examine the membrane phase behavior of equine spermatozoa in the absence and presence of lipid-based cryoprotectants. Biophysical properties of sperm membranes were investigated with Fourier-transform infrared spectroscopy. Compared to fresh untreated sperm, postthaw untreated sperm showed extensive lipid phase separation and rearrangement. In contrast, postthaw sperm that were cryopreserved in egg phosphatidylcholine (egg PC)- or soy phosphatidylcholine (soy PC)-based diluents showed similar lipid phase behavior to that of fresh, untreated sperm. Studies with a deuterium-labeled PC lipid (POPCd-31) suggest that exogenous lipid from the diluents are strongly associated with the sperm membrane, and scanning electron microscopy images of treated sperm show the presence of lipid aggregates on the membrane surface. Thus, the exogenous lipid does not appear to be integrated into the sperm membrane after cryopreservation. When compared to a standard egg-yolk-based diluent (INRA 82), the soy and egg PC media preserved viability and motility equally well in postthaw sperm. A preliminary fertility study determined that sperm cryopreserved in the soy PC-based medium were capable of fertilization at the same rate as sperm frozen in the conventional INRA 82 medium. Our results show that pure lipid-based diluents can prevent membrane damage during cryopreservation and perform as well as a standard egg-yolk-based diluent in preserving sperm viability, motility, and fertility.

P0115 PP INVERSE ASSOCIATION BETWEEN TRANS ISOMERIC AND LONG-CHAIN POLYUNSATURATED FATTY ACIDS IN ERYTHROCYTE MEMBRANE LIPIDS IN EXPECTING WOMEN

Introduction: Inverse associations were reported between values of long-chain polyunsaturated fatty acids (LC-PUFAs) and values of trans fatty acids both in cord blood plasma lipids and in umbilical blood vessel wall lipids (Decsi et al., Am J Clin Nutr 74: 364–368, 2001 and Lipids 37: 959–965, 2002). It was speculated that the inverse association seen at birth should have its origin in the maternal fatty acid status, but the question has not been explicitly addressed as yet. Methods: Fatty acid composition of erythrocyte membrane phospatidylcholine lipids was determined by high- resolution capillary gas-liquid chromatography at the 20th week of gestation in expecting women living in Germany (n = 23), Hungary (n = 34) and Spain (n = 44). Results: The sum of trans isomeric fatty acids was significantly (P <0.01) higher in Spanish (0.71 [0.32], % wt/wt, median [IQR]) than in German (0.49 [0.24]) and Hungarian (0.56 [0.34]) mothers. No correlation was found between trans isomers and LC-PUFA in erythrocyte membrane lipids in German and Hungarian mothers. In Spanish mothers, there was no correlation between trans fatty acids versus linoleic acid (15.96 [3.90]) and alphalinolenic acid (0.08 [0.11]) (table). In contrast, trans hexadecenoic acid (C16:1t, 0.20 [0.15]), trans octadecenoic acid (C18:1t, 0.36 [0.18]) and the sum of trans isomeric fatty acids were significantly inversely correlated to both arachidonic acid (7.92 [4.37]) and docosahexaenoic acid (2.50 [1.76]) (table).Table 1: Correlation coefficients between fatty acids in PC lipids in expecting womenConclusion: 1. In this study, significant inverse associations were found between LC-PUFA and trans fatty acids in erythrocyte membrane lipids in expecting women. 2. This observation supports the concept that maternal trans fatty acid intake may be inversely associated to the LC-PUFA status of the foetus. 3. Among three groups of women investigated, the inverse association between trans fatty acids and LC-PUFA was seen only in the group with the highest erythrocyte membrane trans fatty acid values.

Structure and Fluctuations of Charged Phosphatidylserine Bilayers in the Absence of Salt

Using x-ray diffraction and NMR spectroscopy, we present structural and material properties of phosphatidylserine (PS) bilayers that may account for the well documented implications of PS headgroups in cell activity. At 30°C, the 18-carbon monounsaturated DOPS in the fluid state has a cross-sectional area of 65.3 Å 2 which is remarkably smaller than the area 72.5 Å 2 of the DOPC analog, despite the extra electrostatic repulsion expected for charged PS headgroups. Similarly, at 20°C, the 14-carbon disaturated DMPS in the gel phase has an area of 40.8 Å 2 vs. 48.1 Å 2 for DMPC. This condensation of area suggests an extra attractive interaction, perhaps hydrogen bonding, between PS headgroups. Unlike zwitterionic lipids, stacks of PS bilayers swell indefinitely as water is added. Data obtained for osmotic pressure versus interbilayer water spacing for fluid phase DOPS are well fit by electrostatic interactions calculated for the Gouy-Chapman regime. It is shown that the electrostatic interactions completely dominate the fluctuational pressure. Nevertheless, the x-ray data definitively exhibit the effects of fluctuations in fluid phase DOPS. From our measurements of fluctuations, we obtain the product of the bilayer bending modulus K C and the smectic compression modulus B. At the same interbilayer separation, the interbilayer fluctuations are smaller in DOPS than for DOPC, showing that B and/or K C are larger. Complementing the x-ray data, 31P-chemical shift anisotropy measured by NMR suggest that the DOPS headgroups are less sensitive to osmotic pressure than DOPC headgroups, which is consistent with a larger K C in DOPS. Quadrupolar splittings for D 2O decay less rapidly with increasing water content for DOPS than for DOPC, indicating greater perturbation of interlamellar water and suggesting a greater interlamellar hydration force in DOPS. Our comparisons between bilayers of PS and PC lipids with the same chains and the same temperature enable us to focus on the effects of these headgroups on bilayer properties.

Effect of Hydrophobic Surfactant Proteins SP-B and SP-C on Binary Phospholipid Monolayers: II. Infrared External Reflectance-Absorption Spectroscopy

In situ external reflection infrared spectroscopy at the air-water interface was used to study the influence on phospholipid structure of an endogenous mixture of the two hydrophobic surfactant proteins, SP-B and SP-C, which are thought to play pivotal roles in the adsorption and function of pulmonary surfactant. Mixtures studied were 1:1, 2:1, and 7:1 (mol:mol) DPPC-d 62:DPPG, and 7:1 DPPC-d 62:DOPG, alone and in the presence of 0.5–10 wt % mixed SP-B/C purified chromatographically from calf lung surfactant extract. Perdeuteration of DPPC produced a shift in vibrational frequencies so that it could be differentiated spectroscopically from the phosphoglycerol component in the surface monolayer. CH 2 antisymmetric and symmetric stretching bands (∼2920 and 2852 cm −1) along with the analogous CD 2 stretching bands (∼2194 and 2089 cm −1) were analyzed, and band heights and peak wavenumber positions were assessed as a function of monolayer surface pressure. Small, near-physiological contents of 1–2 wt % SP-B/C typically produced the maximum observed spectroscopic effects, which were abolished at high protein contents of 10 wt %. Analysis of CH 2 and CD 2 stretching bands and C-H/C-D band height ratios indicated that SP-B/C affected PC and PG lipids differently within the surface monolayer. SP-B/C had preferential interactions with DPPG in 1:1, 2:1, and 7:1 DPPC-d 62:DPPG films that increased its acyl chain order. SP-B/C also interacted specifically with DOPG in 7:1 DPPC-d 62:DOPG monolayers, but in this case an increase in CH 2 band heights and peak wavenumber positions indicated a further disordering of the already fluid DOPG acyl chains. CD 2 band height and peak wavenumber analysis indicated that SP-B/C had no significant effect on the structure of DPPC-d 62 chains in 7:1 films with DPPG or DOPG, and had only a slight tendency to increase the acyl chain order in 1:1 films of DPPC-d 62:DPPG. SP-B/C had no significant effect on DPPC-d 62 structure in films with DOPG. Infrared results also indicated that interactions involving SP-B/C and lipids led to exclusion of PC and PG lipids from the compressed interfacial monolayer, in agreement with our previous report on the phase morphology of lipid monolayers containing 1 wt % SP-B/C.

X-ray diffraction and foam film investigations of PC head group interaction in water/ethanol mixtures

The influence of ethanol on single phospholipid monolayers at the water/air interface and in foam films has been investigated. Grazing incidence X-ray diffraction investigations (GIXD) of Langmuir monolayers from 1,2-distearoyl-phosphatidylcholine (DSPC) spread on water subphases with different amounts of ethanol were performed. The thickness and free specific energy of formation of foam films stabilized by 1,2-dimyristoyl-phosphatidylcholine (DMPC) at different concentrations of ethanol in the film forming dispersions were measured. The GIXD investigations show that the tilt angle of the alkyl chains in the PC lipid monolayer decreases with increasing concentration of ethanol caused by a decrease of the diameter of the head groups. With increasing ethanol content of the solution also the thickness of the aqueous core of PC lipid foam films decreases. We assume that ethanol causes a decreasing probability for the formation of hydrogen bonds of water molecules to the PC head groups. The distinct difference between the effects of ethanol on lipid bilayers as described in the literature and on monolayers and foam films found in this study is discussed. Whereas PC monolayers at the water/air interface become unstable above 25 vol.% ethanol, the PC foam films are stable up to 50 vol.% ethanol. This is related to the decrease of the surface excess energy per lipid molecule by the interaction between the two film surfaces.

Quantitative determination of phospholipid compositions by ESI-MS: effects of acyl chain length, unsaturation, and lipid concentration on instrument response

Electrospray ionization-mass spectrometry (ESI-MS) is a very promising tool for the analysis of phospholipid compositions, but is hampered by the fact that not all molecular species are detected with equal efficiency. We studied this and other issues that need to be taken into account to obtain truly quantitative compositional data. The key findings were as follows: First, the instrument response for both saturated and unsaturated phospholipid species decreased with increasing acyl chain length. This effect became increasingly prominent with increasing overall lipid concentration. Second, the degree of acyl chain unsaturation also had a significant effect on instrument response. At the highest concentration studied (10 pmol/microl), polyunsaturated species gave 40% higher intensity than the fully saturated ones. The effect of unsaturation diminished and nearly disappeared with progressive dilution. Third, the instrument response for the different head group classes varied markedly depending on the infusion solvent used. Notably, inclusion of ammonia in the infusion solvent eliminated sodium adduct formation in the positive ion mode, thus greatly simplifying the interpretation of the spectra. The fact that instrument response is dependent on many structural features, overall lipid concentration, solvent composition, and instrument settings makes it necessary to include several internal standards for each phospholipid class to obtain accurate data. Preferably, both unsaturated and saturated standards should be used. Finally, we quantified the major phospholipid classes of BHK cells using ESI-MS. The data agreed closely with those obtained with thin-layer chromatography and phosphorus analysis. This study indicates that quantitative compositional data can be obtained with ESI-MS, provided that proper attention is paid to experimental details, particularly the choice of internal standards.

Nonequilibrium Lipid Domain Growth in the Gel−Fluid Two-Phase Region of a DC16PC−DC22PC Lipid Mixture Investigated by Monte Carlo Computer Simulation, FT-IR, and Fluorescence Spectroscopy

The nonequilibrium ordering dynamics of lipid domains in the gel−fluid coexistence region of an equimolar DC16PC−DC22PC lipid mixture has been studied by means of Monte Carlo computer simulation, fluorescence, and Fourier transform infrared spectroscopy. The results reveal that the nonequilibrium phase separation process after a sudden temperature or pressure quench of the binary mixture from the one-phase fluid region into the two-phase gel−fluid coexistence region has a strong influence on the lateral membrane organization on different length scales. This is manifested as the formation of a heterogeneous lateral bilayer structure composed of long-living gel and fluid lipid domains characterized by a relaxation time on the order of hours. In the early time stage of the phase separation process a distinct local lipid structure of ordered DC16PC lipids is formed at the dynamically changing network of domain boundaries. Our combined theoretical and experimental investigations suggest that nonequilibrium eff...

Water Permeability and Mechanical Strength of Polyunsaturated Lipid Bilayers

Micropipette aspiration was used to test mechanical strength and water permeability of giant-fluid bilayer vesicles composed of polyunsaturated phosphatidylcholine PC lipids. Eight synthetic-diacyl PCs were chosen with 18 carbon chains and degrees of unsaturation that ranged from one double bond (C18:0/1, C18:1/0) to six double bonds per PC molecule (diC18:3). Produced by increasing pipette pressurization, membrane tensions for lysis of single vesicles at 21°C ranged from ∼9 to 10 mN/m for mono- and dimono-unsaturated PCs (18:0/1, 18:1/0, and diC18:1) but dropped abruptly to ∼5 mN/m when one or both PC chains contained two cis-double bonds (C18:0/2 and diC18:2) and even lower ∼3 mN/m for diC18:3. Driven by osmotic filtration following transfer of individual vesicles to a hypertonic environment, the apparent coefficient for water permeability at 21°C varied modestly in a range from ∼30 to 40 μm/s for mono- and dimono-unsaturated PCs. However, with two or more cis-double bonds in a chain, the apparent permeability rose to ∼50 μm/s for C18:0/2, then strikingly to ∼90 μm/s for diC18:2 and ∼150 μm/s for diC18:3. The measurements of water permeability were found to scale exponentially with the reduced temperatures reported for these lipids in the literature. The correlation supports the concept that increase in free volume acquired in thermal expansion above the main gel-liquid crystal transition of a bilayer is a major factor in water transport. Taken together, the prominent changes in lysis tension and water permeability indicate that major changes occur in chain packing and cohesive interactions when two or more cis-double bonds alternate with saturated bonds along a chain.

In vitro evaluation and characterization of newly designed alkylamidophospholipid analogues as anti-human immunodeficiency virus type 1 agents.

Our laboratories first reported two novel classes of complex synthetic lipids, including alkylamidophosphocholines (PC lipid; CP-51) and alkylamidophosphate ester-linked lipid-AZT conjugates (lipid-AZT conjugates; CP-92), with selective and potent activity against human immunodeficiency virus type 1 (HIV-1). To extend these observations, we synthesized additional PC lipids and lipid-AZT conjugates (INK and INK-AZT conjugate) to evaluate their structure-activity relationships by testing for selectivity against infectious wild-type (wt) and drug-resistant HIV-1 replication, virus fusogenic activity and toxicity for mouse bone marrow cells. PC lipid compounds with medium chain lengths at positions 1 and 2 gave an improved selective index (SI). INK-3, with 12 and 8 carbons and INK-15, with 10 and 12 carbons were among the most selective when evaluated in CEM-SS cells. INK-14, a lipid-AZT conjugate where AZT replaced the choline in PC lipid INK-3, gave the highest SI of > 1250 against both infectious wt HIV-1 replication in CEM-SS cells and a clinical isolate in peripheral blood leukocytes. Notably, the PC lipid compounds INK-3 and INK-15, but not the lipid-AZT conjugate INK-14, were potent inhibitors of matched pairs of AZT-sensitive and AZT-resistant HIV-1 clinical isolates. INK-3 also inhibited replication of HIV-2 and TIBO-resistant HIV-1, and inhibited HIV-1-mediated fusogenic activity by 78, 41 and 9% in a dose-dependent manner. The TC50 for mouse bone marrow cells was > 100 micrograms/ml for INK-3 compared to 9.15-14.17 micrograms/ml for CP-51 and 0.142-0.259 microgram/ml for AZT. These data suggest that optimum PC lipid compounds are significantly less toxic than AZT and have high potential as novel therapeutic agents for AIDS.

20S human proteasomes bind with a specific orientation to lipid monolayers in vitro

20S Proteasomes are non-lysosomal, high molecular weight proteinases implicated in the degradation of misfolded proteins and several short-lived regulatory proteins. They have a well established role, as the core of the 26S proteasome complex, in the ubiquitin-dependent proteolytic pathway and in antigen processing. While correctly folded proteins are not degraded by the 20S proteasome, unfolding, for example by oxidation, may render them degradable. The 20S proteasome is a 700-kDa cylindrical particle, composed of 14 subunits of molecular masses 20–35 kDa. There is evidence that 20S proteasomes are in close proximity to or associate with the endoplasmic reticulum and nuclear and plasma membranes in vivo. To better understand the lipid association of 20S proteasomes in vitro, we used a lipid monolayer system as a simple model system for biological membranes. The structure and orientation of the monolayer lipid bound 20S proteasomes has been determined by electron microscopy. 20S proteasomes associated in an ‘end-on’ configuration specifically on PI lipid monolayers forming large arrays, with their channels opposite the lipid headgroups. On ER and Golgi lipid films 20S proteasones were oriented in the same way as on the PI lipid film but were monodisperse. Protein molecules were randomly oriented in the presence of PA, PG, PS, PC and mitochondrial lipid monolayers. We show that 20S proteasomes bind to phospholipids in vitro in a preferred orientation which places the proteasome channel perpendicular to the membrane.