Glycerophospholipid polyunsaturation modulates resveratrol action on biomimetic membranes.

The phytoalexin resveratrol has received increasing attention for its potential to prevent oxidative damages in human organism. To shed further light on molecular mechanisms of its interaction with lipid membranes we study resveratrol influence on the organisation and mechanical properties of biomimetic lipid systems composed of synthetic phosphatidylcholines with mixed aliphatic chains and different degree of unsaturation at sn-2 position (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC, and 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine, PDPC). High-sensitivity isothermal titration calorimetric measurements reveal stronger spontaneous resveratrol association to polyunsaturated phosphatidylcholine bilayers compared to the monounsaturated ones resulting from hydrophobic interactions, conformational changes of the interacting species and desolvation of molecular surfaces. The latter is supported by the results from Laurdan spectroscopy of large unilamellar vesicles providing data on hydration at the glycerol backbones of glycerophospholipides. Higher degree of lipid order is reported for POPC membranes compared to PDPC. While resveratrol mostly enhances the hydration of PDPC membranes, increasing POPC dehydration is reported upon treatment with the polyphenol. Dehydration of the polyunsaturated lipid bilayers is measured only at the highest phytoalexin content studied (resveratrol/lipid 0.5 mol/mol) and is less pronounced than the effect reported for POPC membranes. The polyphenol effect on membrane mechanics is probed by thermal shape fluctuation analysis of quasispherical giant unilamellar vesicles. Markedly different trend of the bending elasticity with increasing resveratrol concentration is reported for the two types of phospholipid bilayers studied. POPC membranes become more rigid in the presence of resveratrol, whereas PDPC-containing bilayers exhibit softening at lower concentrations of the polyphenol followed by a slight growth without bilayer stiffening even at the highest resveratrol content explored. The new data on the structural organization and membrane properties of resveratrol-treated phosphatidylcholine membranes may underpin the development of future liposomal applications of the polyphenol in medicinal chemistry.

Interaction of KLAKLAK-NH2 and Analogs with Biomimetic Membrane Models.

BACKGROUND: Specifically designed peptide mimetics offer higher selectivity regarding their toxicity to mammalian cells. In addition to the α-helix conformation, the specific activity is related to the peptide's ability to penetrate the cell membrane. The alterations in lipid membrane properties were addressed in the presence of the peptide KLAKLAK-NH2 and analogs containing ß-alanine, strengthening the antibacterial activity and/or naphtalimide with proven anticancer properties. METHODS: The molecular interactions of the peptide mimetics with POPC bilayers were studied using FTIR-ATR spectroscopy. The thermal shape fluctuation analysis of quasispherical unilamellar vesicles was applied to probe the membrane bending elasticity. The impedance characteristics of bilayer lipid membranes were measured using fast Fourier-transform electrochemical impedance spectroscopy. RESULTS: A lateral peptide association with the membrane is reported for ß-alanine-containing peptides. The most pronounced membrane softening is found for the NphtG-KLßAKLßAK-NH2 analog containing both active groups that corroborate with the indications for 1,8-naphthalimide penetration in the lipid hydrophobic area obtained from the FTIR-ATR spectra analysis. The ß-alanine substitution induces strong membrane-rigidifying properties even at very low concentrations of both ß-alanine-containing peptides. CONCLUSIONS: The reported results are expected to advance the progress in tailoring the pharmacokinetic properties of antimicrobial peptides with strengthened stability towards enzymatic degradation. The investigation of the nonspecific interactions of peptides with model lipid membranes is featured as a useful tool to assess the antitumor and antimicrobial potential of new peptide mimetics.

Structural Changes Induced by Resveratrol in Monounsaturated and Polyunsaturated Phosphatidylcholine-Enriched Model Membranes.

Resveratrol (Resv) is considered to exert a beneficial impact due to its radical scavenger, anti-microbial and anti-inflammatory properties through several mechanisms that could include its interaction with the cell plasma membrane. To address this issue, we investigated the influence of Resv on membrane lipid order and organization in large unilamellar vesicles composed of different lipids and ratios. The studied lipid membrane models were composed of phosphatidylcholine (PC) species (either palmitoyl-docosahexaenoyl phosphatidylcholine (PDPC) or palmitoyl-oleoyl phosphatidylcholine (POPC)), sphingomyelin (SM) and cholesterol (Chol). This study found that the addition of Resv resulted in complex membrane reorganization depending on the degree of fatty acid unsaturation at the sn-2 position, and the Lipid/Resv and SM/Chol ratios. Resv rigidified POPC-containing membranes and increased liquid-ordered (Lo) domain formation in 40/40/20 POPC/SM/Chol mixtures as this increase was lower at a 33/33/34 ratio. In contrast, Resv interacted with PDPC/SM/Chol mixtures in a bimodal manner by fluidizing/rigidifying the membranes in a dose-dependent way. Lo domain formation upon Resv addition occurred via the following bimodal mode of action: Lo domain size increased at low Resv concentrations; then, Lo domain size decreased at higher ones. To account for the variable effect of Resv, we suggest that it may act as a "spacer" at low doses, with a transition to a more "filler" position in the lipid bulk. We hypothesize that one of the roles of Resv is to tune the lipid order and organization of cell plasma membranes, which is closely linked to important cell functions such as membrane sorting and trafficking.

Impact of Truncated Oxidized Phosphatidylcholines on Phospholipase A2 Activity in Mono- and Polyunsaturated Biomimetic Vesicles.

The interplay between inflammatory and redox processes is a ubiquitous and critical phenomenon in cell biology that involves numerous biological factors. Among them, secretory phospholipases A2 (sPLA2) that catalyze the hydrolysis of the sn-2 ester bond of phospholipids are key players. They can interact or be modulated by the presence of truncated oxidized phosphatidylcholines (OxPCs) produced under oxidative stress from phosphatidylcholine (PC) species. The present study examined this important, but rarely considered, sPLA2 modulation induced by the changes in biophysical properties of PC vesicles comprising various OxPC ratios in mono- or poly-unsaturated PCs. Being the most physiologically active OxPCs, 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) have been selected for our study. Using fluorescence spectroscopy methods, we compared the effect of OxPCs on the lipid order as well as sPLA2 activity in large unilamellar vesicles (LUVs) made of the heteroacid PC, either monounsaturated [1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)], or polyunsaturated [1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC)] at a physiological temperature. The effect of OxPCs on vesicle size was also assessed in both the mono- and polyunsaturated PC matrices. Results: OxPCs decrease the membrane lipid order of POPC and PDPC mixtures with PGPC inducing a much larger decrease in comparison with POVPC, indicative that the difference takes place at the glycerol level. Compared with POPC, PDPC was able to inhibit sPLA2 activity showing a protective effect of PDPC against enzyme hydrolysis. Furthermore, sPLA2 activity on its PC substrates was modulated by the OxPC membrane content. POVPC down-regulated sPLA2 activity, suggesting anti-inflammatory properties of this truncated oxidized lipid. Interestingly, PGPC had a dual and opposite effect, either inhibitory or enhancing on sPLA2 activity, depending on the protocol of lipid mixing. This difference may result from the chemical properties of the shortened sn-2-acyl chain residues (aldehyde group for POVPC, and carboxyl for PGPC), being, respectively, zwitterionic or anionic under hydration at physiological conditions.

Erythrocyte Membrane Biophysical Changes Mediated by Pooled Immunoglobulin G and Hematin: Electrokinetic and Lipid Peroxidation Studies.

Pooled Immunoglobulin G (IgG), hematin and the membrane-disruptive amphipathic peptide melittin have received attention as powerful biomacromolecules for biomedical and pharmacology applications. Their action on surface properties, oxidation status and epifluorescence properties measured in vitro provide useful information about the functional activity of upper biomacromolecules in erythrocytes in vivo. The hemolysis of erythrocyte membranes, as well as changes in hematocrit and the morphology of erythrocytes, was investigated here via fluorescence microscopy using FITC-concanavalin A binding to cells. The effect of melittin on the membrane capacitance and resistance of model lipid bilayers was probed via electrochemical impedance spectroscopy. Lipid bilayer capacitance was higher in the presence of 0.10 g/L melittin compared to that in the control, which is likely related to bilayer thinning and alterations of the dielectric permittivity of melittin-treated membranes. The biomolecule interactions with red blood cells were probed in physiological media in which the surface of erythrocyte membranes was negatively charged. Surface parameters of erythrocytes are reported upon IgG/hematin and IgG/melittin treatment. Pooled IgG in the presence of melittin, preincubated IgG/hematin preparations promoted a significant decrease in the electrokinetic potential of erythrocytes (Rh-positive). A malondialdehyde (MDA) assay revealed a high rate of lipid peroxidation in erythrocytes treated with IgG/hematin or IgG/melittin preparations. This finding might be a result of pooled IgG interactions with the hematin molecule and the subsequent conformational changes in the protein molecule altering the electrokinetic properties of the erythrocyte membrane related to the Rh group type of erythrocytes. The pooled IgG and hematin are reported to have important consequences for the biophysical understanding of the immunopathological mechanisms of inflammatory, autoimmune and antibody-mediated pathological processes.

Valorphins alter physicochemical characteristics of phosphatidylcholine membranes: Datasets on lipid packing, bending rigidity, specific electrical capacitance, dipole potential, vesicle size.

Endogenous hemorphins are being intensively investigated as therapeutic agents in neuropharmacology, and also as biomarkers in mood regulation, inflammation and oncology. The datasets collected herein report physicochemical parameters of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes in the presence of VV-hemorphin-5 (Val-Val-Tyr-Pro-Trp-Thr-Gln) and analogues, modified at position 1 and 7 by the natural amino acid isoleucine or the non-proteinogenic 2-aminoisobutyric, 2,3-diaminopropanoic or 2,4-diaminobutanoic amino acids. These peptides have been previously screened for nociceptive activity and were chosen accordingly. The present article contains fluorescence spectroscopy data of Laurdan- and di-8-ANEPPS- labelled large unilamellar vesicles (LUV) providing the degree of hydration and dipole potential of lipid bilayers in the presence of VV-hemorphin-5 analogues. Lipid packing is accessible from Laurdan intensity profiles and generalized polarization datasets reported herein. The data presented on fluorescence intensity ratios of di-8-ANEPPS dye provide dipole potential values of phosphatidylcholine-valorphin membranes. Vesicle size and electrophoretic mobility datasets included refer to the effect of valorphins on the size distribution and ζ -potential of POPC LUVs. Investigation of physicochemical properties of peptides such as diffusion coefficients and heterogeneous rate constant relates to elucidation of transport mechanisms in living cells. Voltammetric data of valorphins are presented together with square-wave voltammograms of investigated peptides for calculation of their heterogeneous electron transfer rate constants. Datasets from the thermal shape fluctuation analysis of quasispherical 'giant' unilamellar vesicles (GUV) are provided to quantify the influence of hemorphin incorporation on the membrane bending elasticity. Isothermal titration calorimetric data on the thermodynamics of peptide-lipid interactions and the binding affinity of valorphin analogues to phosphatidylcholine membranes are reported. Data of frequency-dependent deformation of GUVs in alternating electric field are included together with the values of the specific electrical capacitance of POPC-valorphin membranes. The datasets reported in this article can underlie the formulation and implementation of peptide-based strategies in pharmacology and biomedicine.

Interaction of new VV-hemorphin-5 analogues with cell membrane models.

New analogues of the endogenous heptapeptide VV-hemorphin-5 (valorphin) synthesised by amino acid replacement allow for tailoring the peptide activity in vivo. Investigation of hemorphin-induced alterations of lipid bilayers' physicochemical parameters unravels membrane-mediated mechanisms of interaction with cells and subcellular structures. We studied the effect of modified valorphins with nociceptive activity on the structure, mechanical and electrical properties of lipid membrane models. Lower bending rigidity and higher specific capacitance of phosphatidylcholine bilayers were found in the presence of VV-hemorphin-5 analogues. Peptide partition constants for the transfer from the aqueous solution into the membrane were determined by isothermal titration calorimetry. It was found that the inclusion of non-proteinogenic acids with different number of methylene groups lead to alterations of hemorphin-membrane binding. The highest membrane affinity was obtained for a hemorphin derivative with dose-dependent variable effects on visceral nociception in mice. The valorphin analogue with the most pronounced anti-nociceptive effect in vivo induced the highest dipole and zeta potential change without significantly affecting the lipid packing at glycerol level in phosphatidylcholine bilayers.

Surface Properties of Synaptosomes in the Presence of L-Glutamic and Kainic Acids: In Vitro Alteration of the ATPase and Acetylcholinesterase Activities.

Morphologically and functionally identical to brain synapses, the nerve ending particles synaptosomes are biochemically derived membrane structures responsible for the transmission of neural information. Their surface and mechanical properties, measured in vitro, provide useful information about the functional activity of synapses in the brain in vivo. Glutamate and kainic acid are of particular interest because of their role in brain pathology (including causing seizure, migraine, ischemic stroke, aneurysmal subarachnoid hemorrhage, intracerebral hematoma, traumatic brain injury and stroke). The effects of the excitatory neurotransmitter L-glutamic acid and its agonist kainic acid are tested on Na+, K+-ATPase and Mg2+-ATPase activities in synaptic membranes prepared from the cerebral cortex of rat brain tissue. The surface parameters of synaptosome preparations from the cerebral cortex in the presence of L-glutamic and kainic acids are studied by microelectrophoresis for the first time. The studied neurotransmitters promote a significant increase in the electrophoretic mobility and surface electrical charge of synaptosomes at 1-4 h after isolation. The measured decrease in the bending modulus of model bimolecular membranes composed of monounsaturated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine provides evidence for softer membranes in the presence of L-glutamate. Kainic acid does not affect membrane mechanical stability even at ten-fold higher concentrations. Both the L-glutamic and kainic acids reduce acetylcholinesterase activity and deviation from the normal functions of neurotransmission in synapses is presumed. The presented results regarding the modulation of the enzyme activity of synaptic membranes and surface properties of synaptosomes are expected by biochemical and biophysical studies to contribute to the elucidation of the molecular mechanisms of neurotransmitters/agonists' action on membranes.

Dielectric Properties of Phosphatidylcholine Membranes and the Effect of Sugars.

Simple carbohydrates are associated with the enhanced risk of cardiovascular disease and adverse changes in lipoproteins in the organism. Conversely, sugars are known to exert a stabilizing effect on biological membranes, and this effect is widely exploited in medicine and industry for cryopreservation of tissues and materials. In view of elucidating molecular mechanisms involved in the interaction of mono- and disaccharides with biomimetic lipid systems, we study the alteration of dielectric properties, the degree of hydration, and the rotational order parameter and dipole potential of lipid bilayers in the presence of sugars. Frequency-dependent deformation of cell-size unilamellar lipid vesicles in alternating electric fields and fast Fourier transform electrochemical impedance spectroscopy are applied to measure the specific capacitance of phosphatidylcholine lipid bilayers in sucrose, glucose and fructose aqueous solutions. Alteration of membrane specific capacitance is reported in sucrose solutions, while preservation of membrane dielectric properties is established in the presence of glucose and fructose. We address the effect of sugars on the hydration and the rotational order parameter for 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC) and 1-stearoyl-2-oleoyl-sn-glycero-3- phosphocholine (SOPC). An increased degree of lipid packing is reported in sucrose solutions. The obtained results provide evidence that some small carbohydrates are able to change membrane dielectric properties, structure, and order related to membrane homeostasis. The reported data are also relevant to future developments based on the response of lipid bilayers to external physical stimuli such as electric fields and temperature changes.

Polylysine effect on thylakoid membranes.

Thylakoid membranes of photosynthetic plant chloroplasts are involved in a wide range of energy producing pathways. Their stacking can be employed in order to provide increased surface area for biocatalytic purposes. Here we probe the aggregate formation of higher plants' thylakoids using low-molecular poly-l-lysine as an electrostatic polymer linker in low ionic strength media. Microelectrophoresis, actinic light scattering, millisecond-delayed fluorescence and free radical production of thylakoid membranes are measured and analyzed in the presence of the cationic polypeptide to track its influence on the surface electrical properties, the electron-transport processes and the proton gradient accumulation across membranes. Enhanced proton gradient in polylysine-treated thylakoids is obtained upon illumination due to alterations of the proton intake across the membrane resulting from the non-specific electrostatic interactions of the cationic polypeptide with thylakoids. We report lower rates of lipid peroxidation in polylysine-treated thylakoids measured both in the dark and under illumination in salt-free medium. The gained insight on the effect of polycations on photosynthetic membranes may be used in future developments of thylakoid-based approaches for energy transfer applications.

Synthesis, characterization and anticonvulsant activity of new azobenzene-containing VV-hemorphin-5 bio photoswitch.

A novel analog of VV-hemorphin-5 containing azobenzene moiety has been synthesized and investigated for anticonvulsant activity in relation to its E → Z photophysical properties activated by long wavelength light at 365 nm. The synthesis was achieved by a modified SPPS by Fmoc-dimerization strategy. The electrochemical behavior before and after UV illumination was investigated using different voltammetric modes. The number of electrons transferred, heterogenic rate constant and diffusion coefficient for E- and Z-isomers were also evaluated. Revealing the governing principles involved in signaling and nerve pulse propagation requires the detailed characterization of the electrical properties of cell membranes. For probing the effect of synthesized azo-peptide on the membrane electrical properties, we measured the specific capacitance of lipid bilayers, representing a basic physical model of biomembranes with their simple reproducibility in laboratory conditions at controlled membrane composition and physicochemical parameters of the surrounding aqueous medium. Our results have shown reduced membrane capacitance in the presence of the azo-peptide, thus providing evidences for possible alterations in the dielectric permittivity of the bilayer. The (Val-Val-Tyr-Pro-Trp-Thr-Gln)2Azo peptide was explored also in vivo for preliminary anticonvulsant activity by using the 6-Hz seizure test and pentylenetetrazol (PTZ) seizure test in mice. The Z-isomer has exhibited higher potency compared to E-isomer most pronouncedly in the 6 Hz test for psychomotor seizures where the compound had activity at all three tested doses. It was found that the Z-isomer decrease the latency for onset of clonic seizures induced by PTZ. These results demonstrate that the Z-isomer deserves further evaluation in other screening tests for anticonvulsant activity.

Digital holographic microscopy as a tool to study the thermal shape fluctuations of lipid vesicles.

The bending elasticity modulus of lipid membranes is obtained by applying for the first time, to the best of our knowledge, a novel experimental technique based on digital holographic microscopy. The fluctuations of the radius with time were extracted by tracking and measuring the optical thickness at the vesicle poles. The temporal autocorrelation function of the vesicle diameter computed for each of the studied vesicles was then fitted with the theoretical expression to deduce the membrane's tension and bending constant. For the bending elasticity modulus of SOPC bilayers, the value of (0.93 ± 0.03) × 10(-12) erg was obtained. This result is in accordance with values previously obtained by means of other conventional methods for the same type of lipid membrane in the presence of sugar molecules in aqueous medium. The obtained results encourage the future development of the digital holographic microscopy as a technique suitable for the measurement of the bending elasticity of lipid membranes.

Phospholipase A2-Induced Remodeling Processes on Liquid-Ordered/Liquid-Disordered Membranes Containing Docosahexaenoic or Oleic Acid: A Comparison Study.

Vesicle cycling, which is an important biological event, involves the interplay between membrane lipids and proteins, among which the enzyme phospholipase A2 (PLA2) plays a critical role. The capacity of PLA2 to trigger the budding and fission of liquid-ordered (L(o)) domains has been examined in palmitoyl-docosahexaenoylphosphatidylcholine (PDPC) and palmitoyl-oleoylphosphatidylcholine (POPC)/sphingomyelin/cholesterol membranes. They both exhibited a L(o)/liquid-disordered (L(d)) phase separation. We demonstrated that PLA2 was able to trigger budding in PDPC-containing vesicles but not POPC ones. The enzymatic activity, line tension, and elasticity of the membrane surrounding the L(o) domains are critical for budding. The higher line tension of Lo domains in PDPC mixtures was assigned to the greater difference in order parameters of the coexisting phases. The higher amount of lysophosphatidylcholine generated by PLA2 in the PDPC-containing mixtures led to a less-rigid membrane, compared to POPC. The more elastic L(d) membranes in PDPC mixtures exert a lower counteracting force against the L(o) domain bending.

Registration and analysis of the shape fluctuations of nearly spherical lipid vesicles.

The analysis of shape fluctuations of giant nearly spherical lipid vesicles observed via optical microscopy is one of the widely used methods for the determination of the bending elasticity of lipid membranes. Although the method has been used already for three decades, the values of this material constant, obtained by different groups for membranes of the same composition, in identical conditions, differ significantly. The aim of the present work is the development of the method, enabling us to avoid the influence of artifacts on the value of the measured bending modulus. This is achieved by rejection of some images of the vesicle or the whole vesicle when they do not satisfy the requirements (selection criteria) of the applied theory. The bending modulus of 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphocholine lipid membranes is determined via the advanced method described here. The results are compared with the values in the literature and their difference is discussed.

Micro-macro link in rheology of erythrocyte and vesicle suspensions.

We report on the rheology of dilute suspensions of red blood cells (RBC) and vesicles. The viscosity of RBC suspensions reveals a previously unknown signature: it exhibits a pronounced minimum when the viscosity of the ambient medium is close to the value at which the transition from tank-treading to tumbling occurs. This bifurcation is triggered by varying the viscosity of the ambient fluid. It is found that the intrinsic viscosity of the suspension varies by about a factor of 4 in the explored parameter range. Surprisingly, this significant change of the intrinsic viscosity is revealed even at low hematocrit (5%). We suggest that this finding may be used to detect blood flow disorders linked to pathologies that affect RBC shape and mechanical properties. This opens future perspectives on setting up new diagnostic tools, with great efficiency even at very low hematocrit. Investigations are also performed on giant vesicle suspensions, and compared to RBCs.

Alamethicin influence on the membrane bending elasticity.

We investigate the bending elasticity of lipid membranes with the increase of the alamethicin concentrations in the membrane via analysis of the thermally induced shape fluctuations of quasi-spherical giant vesicles. Our experimental results prove the strong influence of alamethicin molecules on the bending elasticity of diphytanoyl phosphatidylcholine and dilauroyl phosphatidylcholine membranes even in the range of very low peptide concentrations (less than 10(-3) mol/mol in the membrane). The results presented in this work, testify to the peripheral orientation of alamethicin molecules at low peptide concentrations in the membrane for both types of lipid bilayers. An upper limit of the concentration of the peptide in the membrane is determined below which the system behaves as an ideal two-dimensional solution and the peptide molecules have a planar orientation in the membrane.

Permeability and the hidden area of lipid bilayers.

The passive water permeability of a lipid vesicle membrane was studied, related to the hydrostatic (not osmotic) pressure difference between the inner and the outer side of the vesicle in a water environment without additives. Each pressure difference was created by sucking a vesicle into a micropipette at a given sucking pressure. The part of the membrane sucked into the micropipette (the projection length) was measured as a function of time. The time dependence can be divided into two intervals. We put forward the idea that smoothing of membrane defects, accompanied by an increase of the membrane area, takes place during the initial time interval, which results in a faster increase of the projection length. In the second time interval the volume of the vesicle decreases due to the permeability of its membrane and the increase of the projection length is slower. The hidden area and the water permeability of a typical lipid bilayer were estimated. The measured permeability, conjugated to the hydrostatic pressure difference, is an order of magnitude higher than the known value of the permeability, conjugated to the osmotic pressure difference. A hypothesis, based on pore formation, is proposed as an explanation of this experimental result.