Opposite Leaflet Research Articles

Cardiolipin Prevents Membrane Translocation and Permeabilization by Daptomycin

Daptomycin is an acidic lipopeptide antibiotic that, in the presence of calcium, forms oligomeric pores on membranes containing phosphatidylglycerol. It is clinically used against various Gram-positive bacteria such as Staphylococcus aureus and Enterococcus species. Genetic studies have indicated that an increased content of cardiolipin in the bacterial membrane may contribute to bacterial resistance against the drug. Here, we used a liposome model to demonstrate that cardiolipin directly inhibits membrane permeabilization by daptomycin. When cardiolipin is added at molar fractions of 10 or 20% to membranes containing phosphatidylglycerol, daptomycin no longer forms pores or translocates to the inner membrane leaflet. Under the same conditions, daptomycin continues to form oligomers; however, these oligomers contain only close to four subunits, which is approximately half as many as observed on membranes without cardiolipin. The collective findings lead us to propose that a daptomycin pore consists of two aligned tetramers in opposite leaflets and that cardiolipin prevents the translocation of tetramers to the inner leaflet, thereby forestalling the formation of complete, octameric pores. Our findings suggest a possible mechanism by which cardiolipin may mediate resistance to daptomycin, and they provide new insights into the action mode of this important antibiotic.

The Antibacterial Activity of Cassia fistula Organic Extracts.

Background:Cassia fistula, is a flowering plant and a member of Fabaceae family. Its leaves are compound of 4 - 8 pairs of opposite leaflets. There are many Cassia species around the world which are used in herbal medicine.Objectives:This study was designed to examine in vitro anti-bacterial activity of methanolic and ethanolic extracts of C. fistula native to Khuzestan, Iran.Materials and Methods:The microbial inhibitory effect of methanolic and ethanolic extracts of C. fistula was tested on 3 Gram positive: Bacillus cereus, Staphylococcus aureus and S. epidermidis and 5 Gram negative: Salmonella Typhi, Kelebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Proteus mirabilis bacterial species using disc diffusion method at various concentrations. The minimum inhibitory and bactericidal concentrations (MIC and MBC) were measured by the tube dilution assay.Results:The extract of C. fistula was effective against B. cereus, S. aureus, S. epidermidis, E. coli and K. pneumoniae. The most susceptible microorganisms to ethanolic and methanolic extracts were E. coli and K. pneumoniae, respectively. Also B. cereus and S. aureus showed the least sensitivity to ethanolic and methanolic extracts, respectively. The MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) of ethanolic extracts against S. aureus, E. coli, S. epidermidis and K. pneumoniae were also determined.Conclusions:With respect to the obtained results and regarding to the daily increase of the resistant microbial strains to the commercial antibiotics, it can be concluded that these extracts can be proper candidates of antibacterial substance against pathogenic bacterial species especially S. aureus, E. coli, K. pneumoniae and S. epidermidis.

The double orifice valve technique to treat tricuspid valve incompetence

A straightforward tricuspid valve (TV) repair technique was used to treat either moderate or severe functional (normal valve with dilated annulus) or for primary/organic (Ebstein's anomaly, leaflet retraction/tethering and chordal malposition/tethering, with annular dilatation) TV incompetence, and its long-term outcome assessed. A double-orifice valve technique was employed in 91 patients (mean age 52.6 ± 23.2 years; median age 56 years; range: 0.6-82 years) with severe tricuspid regurgitation. Among the patients, three had post-transplant iatrogenic chordal rupture, five had infective endocarditis, 11 had mitral valve insufficiency, 23 had Ebstein's anomaly, and 47 had isolated severe TV incompetence. The basic principle was to reduce the distance between the coapting leaflets, wherein the most mobile leaflet could coapt to the opposite leaflet, by creating two orifices, ensuring valve competence. The TV repair was performed through a median sternotomy or right anterior thoracotomy in the fifth intercostal space under cardiopulmonary bypass. The degree and extent of creating a double-valve orifice was determined by considering the minimal body surface area (BSA)-related acceptable TV diameter. Repair was accomplished by passing pledgeted mattress sutures from the middle of the true anterior annulus to a spot on the opposite septal annulus, located approximately two-thirds of the length of the septal annulus to avoid injury to the bundle of His. The annular apposition divides the TV into a larger anterior and a smaller posterior orifices, enabling valve closure, on both sides. In adults, the diameter of the anterior valve orifice should be 23-25 mm, and the posterior orifice 15-18 mm; thus, the total valve orifice area is 5-6 cm2. In children, the total valve orifice should be a standard deviation of 1.7 mm for a BSA of <1. 0m2, and 1.5 mm for a BSA of >1.0m2. During a mean follow up of 8.7 ± 1.34 years (median 10 years; range: 1.5-25.9 years) there have been no reoperations for TV insufficiency or stenosis. Reoperations on three patients (mean age 42.5 ± 8.7 years) were indicated for aortic valve replacement at 14 months postoperatively (n = 1) and for assist device implantation (n = 2) who eventually underwent heart transplant at 18 and 20 months after TV repair, respectively. The cumulative 12-year survival rate was 86.9%. This double-orifice technique is technically a straightforward repair to abolish TV incompetence with highly satisfactory results, particularly in patients with severe annular dilatation or with leaflet and chordal tethering. In the present series, the technique provided no pitfalls (if the location of the conduction system was borne in mind), requiring only a gentle placement of sutures. It also led to no residual regurgitation or reoperation during the follow up period.

Three-dimensional Echocardiography Is Not Essential for Intraoperative Assessment of Mitral Regurgitation

The problem is not what you see on intraoperative echocardiography; it is what you do not see. Intraoperative transesophageal echocardiography and 3-dimensional (3D) echocardiography provide useful information for the surgeon performing mitral valve surgery, but they are not indispensable. We should consider the outstanding results of the pioneer of mitral valve repair, Professor Alain Carpentier. A report of very late results (>20 years) of the earliest series (1970–1984) of mitral valve repair is informative.1 Even without the availability of intraoperative transesophageal echocardiography, the late results were outstanding. At 20 years, freedom from reoperation was 96.9% for patients with posterior leaflet prolapse and 86.2% for patients with anterior prolapse. At the last follow-up (median, 17 years) 15% had moderate or worse mitral regurgitation. An experienced mitral valve surgeon can achieve durable high-quality results without any echocardiography, so 3D echocardiography is not essential. Response by Tsang and Lang on p 658 What is essential for mitral valve surgery? The fundamental elements from the surgical standpoint are the same whether the procedure is performed through a sternotomy or a minimally invasive incision or is robotically assisted. Achieving good surgical exposure is paramount.2 Numerous surgical techniques, including the type and location of venous cannulation, the atrial incision, retraction of the atrial wall, sutures to help rotate the valve and improve visualization, and occasionally the use of an instrument pushing from outside the heart to facilitate exposure of commissures or the anterior leaflet, are routine practice to optimally visualize the valve. Unless the surgeon is expert in the first step of the procedure, results will be compromised. A perfect 3D echocardiography image of the valve will not compensate for poor exposure. Although real-time 3D color echocardiography is useful, there is nothing more realistic than real-time 3D color visualization through 2.5-power loupes by the …

The additional value of live/real-time three-dimensional transesophageal echocardiography over two-dimensional transesophageal echocardiography for assessing mitral regurgitation with eccentric jets

BackgroundTwo-dimensional transesophageal echocardiography (2D TEE) Doppler color flow imaging is the gold standard for assessing the severity of mitral regurgitation (MR). Severe MR with very eccentric jet may involve more than one mitral leaflet lesion and can be missed by 2D TEE. The purpose of this study was to assess the usefulness and the incremental value of real-time three-dimensional (RT 3D) TEE over 2D TEE findings in the evaluation of patients suffering eccentric MR with more than one mitral leaflet lesion. MethodsIntraoperative 2D TEE and RT 3D TEE examinations were performed on 168 patients with Carpenter II MR who underwent surgery. MR was defined as either central, free-standing eccentric jet or very eccentric jet. 2D TEE and RT 3D TEE finding were compared with surgical findings. ResultsOf these 168 MR patients, 25 patients (14.9%) had central jets and 143 patients (85.1%) had eccentric jets. Among 143 patients with eccentric jets, 47 patients (32.9%) had free-standing eccentric MR jets, and 96 (67.1%) patients had very eccentric jets. 3D TEE diagnosed the severity and location of MR lesions correctly in all patients; this was not the case with 2D TEE, which had significant diagnostic misses in nine patients (9.4%, p < 0.001) having MR with very eccentric jets. These nine patients had lesions on both mitral leaflets, which were missed during 2D TEE examination owing to the highly turbulent flows produced by very eccentric jets from one mitral leaflet lesion and impinging the opposite mitral leaflet lesion. ConclusionThe severity of MR with very eccentric jet was more accurately and comprehensively assessed by 3D TEE than by 2D TEE. Therefore, intraoperative RT 3D TEE provides incremental information that is generally superior to 2D TEE in patients with complex MR due to very eccentric jets.

Limited Perturbation of a DPPC Bilayer by Fluorescent Lipid Probes: A Molecular Dynamics Study

The properties of lipid bilayer nanometer-scale domains could be crucial for understanding cell membranes. Fluorescent probes are often used to study bilayers, yet their effects on host lipids are not well understood. We used molecular dynamics simulations to investigate perturbations in a fluid DPPC bilayer upon incorporation of three indocarbocyanine probes: DiI-C18:0, DiI-C18:2, or DiI-C12:0. We find a 10-12% decrease in chain order for DPPC in the solvation shell nearest the probe but smaller effects in subsequent shells, indicating that the probes significantly alter only their local environment. We also observe order perturbations of lipids directly across from the probe in the opposite leaflet. Additionally, the DPPC headgroup phosphorus-to-nitrogen vector of lipids nearest the probe exhibits preferential orientation pointing away from the DiI. We show that, while DiI probes perturb their local environment, they do not strongly influence the average properties of "nanoscopic" domains containing a few hundred lipids.

Interaction at the Membrane Midplane Mediates Interleaflet Coupling

Transmembrane signaling implies that peripheral protein binding to one leaflet be detected by the opposite leaflet. Even without involvement of raft lipids we showed (Horner et. al. Biophys. J. 2009) that, peripheral binding of charged molecules (poly-lysine, PLL) to planar lipid bilayers is detected at the other side. Addition of PLL to both sides of the membrane sandwiched lipids between two PLL molecules, indicating a formed nanodomain. As the 2dim surface tension between the monolayers is thought to be of comparable size as the line tension in one of the monolayers, interactions at the membrane midplane don't explain the existence of small nano domains. We tested this assumption by measuring the individual lipid mobility's in both leaflets of a free standing planar lipid bilayers and developed a model. The adsorption of a polypeptide decreased lipid mobility of the adjacent (lower) monolayer. This was revealed by fluorescence correlation spectroscopy (FCS). Depending on the size of the polypeptide, lipid diffusion was either slower or faster than that of the polypeptide. Although only one lipid type was used in both monolayers, lipid mobility's were not equal. The polypeptide decelerated lipid movement in the distant monolayer to a much smaller degree than it did affect lipid mobility in the adjacent monolayer. Based on these observations we propose a model which suggests that the coupling between monolayers is due to friction at the membrane midplane. The interlayer friction coefficient seems to be much larger than the in-layer friction coefficient which explains the existence of small nano-domains.

Acyl Chain Length and Saturation Modulate Interleaflet Coupling in Asymmetric Bilayers: Effects on Dynamics and Structural Order

A long-standing question about membrane structure and function is the degree to which the physical properties of the inner and outer leaflets of a bilayer are coupled to one another. Using our recently developed methods to prepare asymmetric vesicles, coupling was investigated for vesicles containing phosphatidylcholine (PC) in the inner leaflet and sphingomyelin (SM) in the outer leaflet. The coupling of both lateral diffusion and membrane order was monitored as a function of PC and SM acyl chain structure. The presence in the outer leaflet of brain SM, which decreased outer-leaflet lateral diffusion, had little effect upon lateral diffusion in inner leaflets composed of dioleoyl PC (i.e., diffusion was only weakly coupled in the two leaflets) but did greatly reduce lateral diffusion in inner leaflets composed of PC with one saturated and one oleoyl acyl chain (i.e., diffusion was strongly coupled in these cases). In addition, reduced outer-leaflet diffusion upon introduction of outer-leaflet milk SM or a synthetic C24:0 SM, both of which have long interdigitating acyl chains, also greatly reduce diffusion of inner leaflets composed of dioleoyl PC, indicative of strong coupling. Strikingly, several assays showed that the ordering of the outer leaflet induced by the presence of SM was not reflected in increased lipid order in the inner leaflet, i.e., there was no detectable coupling between inner and outer leaflet membrane order. We propose a model for how lateral diffusion can be coupled in opposite leaflets and discuss how this might impact membrane function.

How to do the double orifice valve technique to treat tricuspid valve incompetence

A straightforward tricuspid valve repair technique is illustrated here, which may be employed either for functional (normal valve with dilated annulus) or for primary or organic (Ebstein's anomaly, leaflet retraction/tethering and chordal malposition/tethering, with annular dilatation) tricuspid valve incompetence and for moderate and severe degrees of incompetence, without any residual regurgitation or reoperation. The basic principle is to reduce the distance between the coapting leaflets in a manner in which the most mobile leaflet, which is usually the anterior, could coapt to the opposite leaflet, by creating two orifices, ensuring valve competence. Additionally, it reduces annular dilatation and lessens tricuspid valve leaflet tethering, thereby stabilizing the annulus and increasing leaflet coaptation.

Meet Me on the Other Side: Trans-Bilayer Modulation of a Model Voltage-Gated Ion Channel Activity by Membrane Electrostatics Asymmetry

While it is accepted that biomembrane asymmetry is generated by proteins and phospholipids distribution, little is known about how electric changes manifested in a monolayer influence functional properties of proteins localized on the opposite leaflet. Herein we used single-molecule electrophysiology and investigated how asymmetric changes in the electrostatics of an artificial lipid membrane monolayer, generated oppositely from where alamethicin - a model voltage-gated ion channel - was added, altered peptide activity. We found that phlorizin, a membrane dipole potential lowering amphiphile, augmented alamethicin activity and transport features, whereas the opposite occurred with RH-421, which enhances the monolayer dipole potential. Further, the monolayer surface potential was decreased via adsorption of sodium dodecyl sulfate, and demonstrated that vectorial modification of it also affected the alamethicin activity in a predictive manner. A new paradigm is suggested according to which asymmetric changes in the monolayer dipole and surface potential extend their effects spatially by altering the intramembrane potential, whose gradient is sensed by distantly located peptides.

Interaction of 7-Nitrobenz-2-oxa-1,3-diazol-4-yl-Labeled Fatty Amines with 1-Palmitoyl, 2-Oleoyl-sn-glycero-3-phosphocholine Bilayers: A Molecular Dynamics Study

A complete homologous series of fluorescent 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled fatty amines of varying alkyl chain length, NBD-C(n), inserted in 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers, was studied using atomistic molecular dynamics (MD) simulations. For all amphiphiles, the NBD fluorophore locates near the glycerol backbone/carbonyl region of POPC and establishes stable hydrogen bonding with POPC ester oxygen atoms. Small differences observed in the transverse location of the fluorophore correlate with other calculated parameters and with small discrepancies recently measured in the photophysical properties of the molecules. The longer-chained NBD-C(n) amphiphiles show significant mass density near the bilayer midplane, and the chains of these derivatives interdigitate to some extent the opposite bilayer leaflet. This phenomenon leads to a slower lateral diffusion for the longer-chained derivatives (n > 12). Effects of these amphiphiles on the structure and dynamics of the host lipid were found to be relatively mild, in comparison with acyl-chain-labeled NBD probes. The molecular details obtained by this work allow the rationalization of the nonmonotonic behavior, recently obtained experimentally, for the photophysical parameters of the amphiphiles and the kinetic and thermodynamic parameters for their interaction with the POPC membranes.

Exchange of Gramicidin between Lipid Bilayers: Implications for the Mechanism of Channel Formation and Gating

The canonical mechanism of gramicidin (gA) channel formation is a transmembrane dimerization of non-conducting subunits that reside in opposite bilayer leaflets. The channels do not open and close per se; rather they appear and disappear. This basic monomer-dimer mechanism is supported by conductance-concentration studies, fluorescence measurements, conductance relaxation experiments using voltage, and pressure and light-inactivation experiments to perturb the monomer-dimer equilibrium. Recently this mechanism was challenged by Jones et al. (BJ 98:1486, 2010). Using lipid vesicle-incorporated gA under conditions where vesicle fusion could be controlled, Jones et al. proposed that gA single-channel current transitions result from closed-open transitions in long-lived bilayer-spanning dimers. A key assumption in these experiments is that gA monomers do not partition between vesicles; see Kemp and Wenner (ABB 176:547, 1976) and Bruggemann and Kayalar (PNAS 83:4276, 1986). To explore this issue further, we added gA-containing 1,2-dierucoyl-sn-glycero-3-phosphocholine (DC22:1PC) vesicles to one or both sides of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) planar bilayers. This protocol produced gA activity that was 10-fold higher when gA-containing vesicles were added to both sides of the bilayer, as compared to one side. We also found that when gA-free DC22:1PC vesicles were added to both sides of planar DOPC bilayers pre-incubated with gA, channel activity would reduce about 10-fold. We conclude that gA subunits can exchange between lipid vesicles and planar bilayers. We similarly could demonstrate the exchange of gA between lipid vesicles using a fluorescence assay for gA channel activity (Ingólfsson and Andersen, ADDT 4:427, 2010). We conclude that the parsimonious interpretation of the results of Jones et al. is that their channel activity is due to gA exchange between the vesicles and the planar bilayer, and that gA channels form by the canonical mechanism- transbilayer association of non-conducting subunits.

Tabaroa, a new genus of Leguminosae tribe Brongniartieae from Brazil

A new genus of Leguminosae in tribe Brongniartieae is proposed based on a new species endemic to the Caatinga of Bahia state. The new genus is named as Tabaroa L. P. Queiroz, G. P. Lewis & M. F. Wojc. and the new species as Tabaroa caatingicola L. P. Queiroz, G. P. Lewis & M. F. Wojc. A phylogenetic study of the Brongniartieae based on nuclear rDNA ITS and plastid matK sequences supports a closer relationship between Tabaroa and Harpalyce Moc. & Sesse than to the two South American genera Poecilanthe Benth. and Cyclolobium Benth., which are more similar morphologically. Optimisation of selected morphological characters on one of the most parsimonious trees indicates that the sessile ovary and the explosive pollen presentation are putative synapomorphies of the Tabaroa-Harpalyce clade. The genus Tabaroa may be diagnosed by the absence of peltate glandular trichomes; leaves imparipinnate, exstipellate and with opposite leaflets; flowers sessile, not resupinate, grouped in panicles; anthers apiculate; and fruit indehiscent. The only known species inhabits areas of arboreal caatinga on sandy soil in southwestern Bahia, near the boundaries of Dom Basilio and Livramento de Nossa Senhora municipalities.

Protein-Lipid Interaction and Domain Formation in Asymmetric Membranes

The lateral organization of lipids and receptors in cellular membranes is connected to a multitude of biological and pathological processes. The role of sphingolipid- and cholesterol-rich lipid “rafts” as membrane domains that control protein-protein interaction has recently attracted much attention. While the model membrane approach has yielded fundamental insights into cell membrane structure and function, the majority of these studies did not take into account the very important asymmetry between the composition of the inner and the outer leaflets of cell membranes. Therefore, certain key questions remain still unanswered: for example, how is the partition of membrane components in raft domains affected by the presence of non-raft lipids in the inner leaflet? To answer questions of this type we have developed a new method based upon cyclodextrin-induced lipid exchange to create giant unilamellar vesicles (GUVs) featuring the same type of asymmetry encountered in biological membranes. These vesicles can be produced with a very large yield, while avoiding use of organic solvents. The asymmetry of the bilayer can be confirmed by leaflet-targeted Fluorescence Correlation Spectroscopy (FCS). The use of asymmetric GUVs as novel model bilayers should allow the investigation of the principles underlying communication between raft-like domains in opposite leaflets of the bilayer, as well as investigation of the origin of lipid and membrane protein affinity for rafts.

Coupled Diffusion of Peripherally Bound Peptides along the Outer and Inner Membrane Leaflets

Transmembrane signaling implies that peripheral protein binding to one leaflet be detected by the opposite leaflet. Therefore, protein recruitment into preexisting cholesterol and sphingolipid rich platforms may be required. However, no clear molecular picture has evolved about how these rafts in both leaflets are connected. By using planar lipid bilayers, we show that the peripheral binding of a charged molecule (poly-lysine, PLL) is detected at the other side of the bilayer without involvement of raft lipids. The diffusion coefficient, D P, of PLL differed by a factor of √2 when PLL absorbed to one or to both leaflets of planar membranes. Fluorescence correlation spectroscopy showed that the changes of the lipid diffusion coefficient, D M, were even more pronounced. Although D M remained larger than D P on PLL binding to the first membrane leaflet, D M dropped to D P on PLL binding to both leaflets, which indicated that the lipids sandwiched between two PLL molecules had formed a nanodomain. Due to its small area of ∼20 nm 2 membrane electrostriction or leaflet interaction at bilayer midplane can only make a small contribution to interleaflet coupling. The tendency of the system to maximize the area where the membrane is free to undulate seems to be more important. As a spot with increased bending stiffness, the PLL bound patch in one leaflet attracts a stiffening additive on the other leaflet. That is to say, instead of suppressing undulations in two spots, two opposing PLL molecules migrate along a membrane at matching positions and suppress these undulations in a single spot. The gain in undulation energy is larger than the energy required for the alignment of two small PLL domains in opposite leafs and their coordinated diffusion. We propose that this type of mechanical interaction between two membrane separated ligands generally contributes to transmembrane signaling.

Breakdown of Charged Lipid Asymmetry as a Result of Lipidic Pore Formation

Negatively charged lipids are usually located in the inner leaflet of the plasma membrane. Their appearance in the outer leaflet is known to correlate with several physiological and pathological conditions in cells. Understanding how membrane lipids lose their asymmetric transmembrane distribution and achieve their nonrandom distribution in cells is a key challenge in cell biology. Negatively charged lipids do not spontaneously exchange between the two leaflets of a lipid bilayer because the polar headgroups cannot readily cross the hydrophobic membrane interior. We hypothesized that the formation of a transient hydrophilic lipidic pore in the membrane leads to diffusive translocation of negatively charged lipids through the pore to the opposite membrane leaflet. To test this hypothesis, we established a variation of the inner field compensation technique for time resolved measurements of membrane boundary potentials in asymmetric bilayer lipid membranes formed by the Montal-Mueller method. External application of electric fields across the bilayer induced transient conductive states. We observed fast transitions between these different conductance levels, reflecting opening and closing of meta-stable lipidic pores. Comparison of the capacitance minimization potential for different asymmetric membranes before and after pore formation confirmed negatively charged lipids transfer across the bilayer. We also constructed a model governing lipid flow rate based on pore analysis and lipid lateral diffusion rate. Together, our study provides a new tool to monitor loss of membrane asymmetry and our results indicate that lipid transfer can occur through lipidic pores.

Interaction of Ethanol with Biological Membranes: The Formation of Non-bilayer Structures within the Membrane Interior and their Significance

To gain a better understanding of how ethanol affects biological membranes, we have performed a series of atomic-scale molecular dynamics simulations of phospholipid membranes in aqueous solution with ethanol, whose concentration was varied from 2.5 to 30 mol % (lipid-free basis). At concentrations below the threshold value of approximately 12 mol % (30.5 v/v %) ethanol induces expansion of the membrane, accompanied by a drop in the membrane thickness as well as disordering and enhanced interdigitation of lipid acyl chains. These changes become more pronounced with increase in ethanol concentration, but the bilayer structure of the membrane is maintained. Above the threshold concentration the appearance of multiple transient defects in the lipid/water interface eventually gives rise to desorption and assembly of some of the lipids into non-bilayer structures within the membrane interior. These structures, being small and irregular, resemble inverted micelles and have a long-lived character. Furthermore, formation of the non-bilayer structures is accompanied by mixing of lipids that belong to the opposite membrane leaflets, thereby leading to irreversible changes in the membrane structure. Remarkably, this observation of the formation of non-bilayer structures within the membrane interior, being in good agreement with experimental data, is found to be robust with respect to both the simulation conditions (the system size and the presence of salt) and the type of lipids (phosphatidylcholine and phosphatidylethanolamine). We discuss the significance of these non-bilayer structures in relation with the well-known ability of ethanol to promote membrane hemifusion as well as with the possible role of the micelle-like structures as a delivery system for polar solutes and ions. The ethanol-induced "damage" to the bilayer structure also suggests that strong alcoholic beverages ( approximately 40 v/v %) might be potentially hazardous to the epithelial tissues of the human body (such as lips, mouth, throat, gullet, and stomach) that come in direct contact with high-concentrations of ethanol.

Membrane Transporters for Anions That Use a Relay Mechanism

A new type of synthetic membrane transporter is described and shown to operate in vesicles by a relay mechanism. The transporter structure is a phosphatidylcholine derivative with a urea group appended to the end of its sn-2 acyl chain. The urea can bind a chloride ion at the membrane surface via hydrogen bonds and then relay it through the bilayer interior to an acceptor molecule located in the opposite membrane leaflet. Three phosphatidylcholine derivatives were studied and transport rates increased with transporter affinity for chloride. The results of various controls studies are consistent with an anion countertransport process using a relay mechanism and a kinetically active aggregate of two or four transporter molecules. Transport is inhibited if the transporter resides in only one leaflet of the membrane, if the bilayer is too thick, and if the counteranion is sulfate dianion. The expected favorable formulation properties of these amphiphilic compounds should facilitate efforts to transform them into tools for biomedical research and perhaps as therapeutic agents.

Lipid bilayer curvature and pore formation induced by charged linear polymers and dendrimers: the effect of molecular shape.

We performed molecular dynamics (MD) simulations of multiple copies of poly- l-lysine (PLL) and charged polyamidoamine (PAMAM) dendrimers in dimyristoylphosphatidylcholine (DMPC) bilayers with explicit water using the coarse-grained model developed by Marrink et al. ( J. Chem. Theory Comput. 2008, 4, 819 ). Membrane disruption is enhanced at higher concentrations and charge densities of both spheroidally shaped dendrimers and linear PLL polymers, in qualitatively agreement with experimental studies by Hong et al. (Bioconjugate Chem. 2006, 17, 728 ). However, larger molecular size enhances membrane disruption and pore formation only for dendrimers and not for the linear PLL. Despite more intimate electrostatic interactions of linear molecules than are possible for spheroidal dendrimers, only the dendrimers were found to perforate membranes, apparently because they cannot spread onto a single leaflet, and so must penetrate the bilayer to get favorable electrostatic interactions with head groups on the opposite leaflet. These results indicate that a relatively rigid spheroidal shape is more efficient than a flexible linear shape in increasing membrane permeability. These results compare favorably with experimental findings.

Transmembrane localization of cis-isomers of zeaxanthin in the host dimyristoylphosphatidylcholine bilayer membrane

The effects of the 9- cis and 13- cis isomers of zeaxanthin on the molecular organization and dynamics of dimyristoylphosphatidylcholine (DMPC) membranes were investigated using conventional and saturation recovery EPR observations of the 1-palmitoyl-2-(14-doxylstearoyl)phosphatidylcholine (14-PC) spin label. The results were compared with the effects caused by the all- trans isomer of zeaxanthin. Effects on membrane fluidity, order, hydrophobicity, and the oxygen transport parameter were monitored at the center of the fluid phase DMPC membrane. The local diffusion-solubility product of oxygen molecules (oxygen transport parameter) in the membrane center, studied by saturation-recovery EPR, decreased by 47% and 27% by including 10 mol% 13- cis and 9- cis zeaxanthin, respectively; whereas, incorporation of all- trans zeaxanthin decreased this parameter by only 11%. At a zeaxanthin-to-DMPC mole ratio of 1:9, all investigated isomers decreased the membrane fluidity and increased the alkyl chain order in the membrane center. They also increased the hydrophobicity of the membrane interior. The effects of these isomers of zeaxanthin on the membrane properties mentioned above increase as: all- trans < 9- cis ≤ 13- cis. Obtained results suggest that the investigated cis-isomers of zeaxanthin, similar to the all- trans isomer, are located in the membrane interior, adopting transmembrane orientation with the polar terminal hydroxyl groups located in the opposite leaflets of the bilayer. However, the existence of the second pool of cis-zeaxanthin molecules located in the one leaflet and anchored by the terminal hydroxyl groups in the same polar headgroup region cannot be completely ruled out.