C8-glycosphingolipids preferentially insert into tumor cell membranes and promote chemotherapeutic drug uptake

Insufficient drug delivery into tumor cells limits the therapeutic efficacy of chemotherapy. Co-delivery of liposome-encapsulated drug and synthetic short-chain glycosphingolipids (SC-GSLs) significantly improved drug bioavailability by enhancing intracellular drug uptake. Investigating the mechanisms underlying this SC-GSL-mediated drug uptake enhancement is the aim of this study. Fluorescence microscopy was used to visualize the cell membrane lipid transfer intracellular fate of fluorescently labeled C₆-NBD-GalCer incorporated in liposomes in tumor and non-tumor cells. Additionally click chemistry was applied to image and quantify native SC-GSLs in tumor and non-tumor cell membranes. SC-GSL-mediated flip-flop was investigated in model membranes to confirm membrane-incorporation of SC-GSL and its effect on membrane remodeling. SC-GSL enriched liposomes containing doxorubicin (Dox) were incubated at 4 °C and 37 °C and intracellular drug uptake was studied in comparison to standard liposomes and free Dox.SC-GSL transfer to the cell membrane was independent of liposomal uptake and the majority of the transferred lipid remained in the plasma membrane. The transfer of SC-GSL was tumor cell-specific and induced membrane rearrangement as evidenced by a transbilayer flip-flop of pyrene-SM. However, pore formation was measured, as leakage of hydrophilic fluorescent probes was not observed. Moreover, drug uptake appeared to be mediated by SC-GSLs. SC-GSLs enhanced the interaction of doxorubicin (Dox) with the outer leaflet of the plasma membrane of tumor cells at 4 °C. Our results demonstrate that SC-GSLs preferentially insert into tumor cell plasma membranes enhancing cell intrinsic capacity to translocate amphiphilic drugs such as Dox across the membrane via a biophysical process.     

Heat-activated liposome targeting to streptavidin-coated surfaces

There is a great need of improved anticancer drugs and corresponding drug carriers. In particular, liposomal drug carriers with heat-activated release and targeting functions are being developed for combined hyperthermia and chemotherapy treatments of tumors. The aim of this study is to demonstrate the heat-activation of liposome targeting to biotinylated surfaces, in model experiments where streptavidin is used as a pretargeting protein. The design of the heat-activated liposomes is based on liposomes assembled in an asymmetric structure and with a defined phase transition temperature. Asymmetry between the inside and the outside of the liposome membrane was generated through the enzymatic action of phospholipase D, where lipid head groups in the outer membrane leaflet, i.e. exposed to the enzyme, were hydrolyzed. The enzymatically treated and purified liposomes did not bind to streptavidin-modified surfaces. When activation heat was applied, starting from 22 °C, binding of the liposomes occurred once the temperature approached 33 ± 0.5 °C. Moreover, it was observed that the asymmetric structure remained stable for at least 2 weeks. These results show the potential of asymmetric liposomes for the targeted binding to cell membranes in response to (external) temperature stimulus. By using pretargeting proteins, this approach can be further developed for personalized medicine, where tumor-specific antibodies can be selected for the conjugation of pretargeting agents.     

Binding of peptides corresponding to the carboxy-terminal region of human-β-defensins-1–3 with model membranes investigated by isothermal titration calorimetry

Human-β-defensins HBD-1–3 are important components of the innate immune system. Synthetic peptides Phd-1–3 with a single disulphide bond, spanning the cationic C-terminal region of HBD-1–3, have antimicrobial activity. The interaction of Phd-1–3 with model membranes was investigated using isothermal titration calorimetry (ITC) and steady-state fluorescence polarization to understand the biophysical basis for the mechanism of antimicrobial action. Calorimetric titration of POPE:POPG (7:3) vesicles with peptides at 25 °C and 37 °C showed complex profiles with two distinct regions of heat changes. The data indicate binding of Phd-1–3 at 37 °C to both negative and zwitterionic lipid vesicles is exothermic with low enthalpy values (ΔH ~ ? 1.3 to ? 2.8 kcal/mol) as compared to amphipathic helical antibacterial peptides. The adsorption of peptides to negatively charged lipid membranes is modulated by electrostatic interactions that are described by surface partition equilibrium model using Gouy–Chapman theory. However, this model could not explain the isotherms of peptide binding to zwitterionic lipid vesicles. Fluorescence polarization of TMA-DPH (1-[4-(trimethylammonio) phenyl]-6-phenyl-1,3,5-hexatriene) and DPH (1,6-diphenyl-1,3,5-hexatriene) located in the head group and acyl chain region respectively, indicates that the peptides interact with interfacial region of negatively charged membranes. Based on the results obtained, we conclude that adsorption of cationic peptides Phd-1–3 on lipid surface do not result in conformational change or pore formation. It is proposed that interaction of Phd-1–3 with the negatively charged lipid head group causes membrane destabilization, which in turn affects the efficient functioning of cytoplasmic membrane proteins in bacteria, resulting in cell death.     

Partitioning of perfluorooctanoate into phosphatidylcholine bilayers is chain length-independent

The chain length dependence of the interaction of PFOA, a persistent environmental contaminant, with dimyristoyl- (DMPC), dipalmitoyl- (DPPC) and distearoylphosphatidylcholine (DSPC) was investigated using steady-state fluorescence anisotropy spectroscopy, differential scanning calorimetry (DSC) and dynamic light scattering (DLS). PFOA caused a linear depression of the main phase transition temperature T_m while increasing the width of the phase transition of all three phosphatidylcholines. Although PFOA's effect on T_m and the transition width decreased in the order DMPC > DPPC > DSPC, its relative effect on the phase behavior was largely independent of the phosphatidylcholine. PFOA caused swelling of DMPC but not DPPC and DSPC liposomes at 37 °C in the DLS experiments, which suggests that PFOA partitions more readily into bilayers in the fluid phase. These findings suggest that PFOA's effect on the phase behavior of phosphatidylcholines depends on the cooperativity and state (i.e., gel versus liquid phase) of the membrane. DLS experiments are also consistent with partial liposome solubilization at PFOA/lipid molar ratios > 1, which suggests the formation of mixed PFOA–lipid micelles.     

Lipid-mediated regulation of pore-forming activity of syringomycin E by thyroid hormones and xanthene dyes

The effects of dipole modifiers, thyroid hormones (thyroxine and triiodothyronine) and xanthene dyes (Rose Bengal, phloxineB, erythrosin, eosinY and fluorescein) on the pore-forming activity of the lipopeptide syringomycin E (SRE) produced by Pseudomonas syringae were studied in a model bilayer. Thyroxine does not noticeably influence the steady-state number of open SRE channels (N_op), whereas triiodothyronine decreases it 10-fold at − 50 mV. Rose Bengal, phloxine B and erythrosin significantly increase N_op by 350, 100 and 70 times, respectively. Eosin Y and fluorescein do not practically affect the pore-forming activity of SRE. Recently, we showed that hormones decrease the dipole potential of lipid bilayers by approximately 60 mV at 50 μM, while Rose Bengal, phloxine B and erythrosin at 2.5 μM reduce the membrane dipole potential by 120, 80 and 50 mV, respectively. In the present study using differential scanning microcalorimetry, confocal fluorescence microscopy, the calcein release technique and measurements of membrane curvature elasticity, we show that triiodothyronine strongly affects the fluidity of model membranes: its addition leads to a significant decrease in the temperature and cooperativity of the main phase transition of DPPC, calcein leakage from DOPC vesicles, fluidization of solid domains in DOPC/DPPC liposomes, and promotion of lipid curvature stress. Thyroxine exerts a weaker effect. Xanthene dyes do not influence the phase transition of DPPC. Despite the decrease in the dipole potential, thyroid hormones modulate SRE channels predominantly via the elastic properties of the membrane, whereas the xanthene dyes Rose Bengal, phloxine B and erythrosine affect SRE channels via bilayer electrostatics.     

Formation of asymmetric vesicles via phospholipase D-mediated transphosphatidylation

Most biomembranes have an asymmetric structure with regard to phospholipid distribution between the inner and outer leaflets of the lipid bilayers. Control of the asymmetric distribution plays a pivotal role in several cellular functions such as intracellular membrane fusion and cell division. The mechanism by which membrane asymmetry and its alteration function in these transformation processes is not yet clear. To understand the significance of membrane asymmetry on trafficking and metabolism of intracellular vesicular components, a system that experimentally reproduces the asymmetric nature of biomembranes is essential. Here, we succeeded in obtaining asymmetric vesicles by means of transphosphatidylation reactions with phospholipase D (PLD), which acts exclusively on phosphatidylcholine (PC) present in the outer leaflet of vesicles. By treating PC vesicles with PLD in the presence of 1.7 M serine and 0.3 M ethanolamine, we obtained asymmetric vesicles that are topologically similar to intracellular vesicles containing phosphatidylserine and phosphatidylethanolamine in the cytosolic leaflet. PLD and other unwanted compounds could be removed by trypsin digestion followed by dialysis. Our established technique has a great advantage over conventional methods in that asymmetric vesicles can be provided at high yield and high efficiency, which is requisite for most physicochemical assays.     

Channel-forming activity of syringomycin E in two mercury-supported biomimetic membranes

The lipodepsipeptide syringomycin E (SR-E) interacts with two mercury-supported biomimetic membranes, which consist of a self-assembled phospholipid monolayer (SAM) and of a tethered bilayer lipid membrane (tBLM) separated from the mercury surface by a hydrophilic tetraethyleneoxy (TEO) spacer that acts as an ionic reservoir. SR-E interacts more rapidly and effectively with a SAM of dioleoylphosphatidylserine (DOPS) than with one of dioleoylphosphatidylcholine (DOPC). The proximal lipid monolayer of the tBLM has no polar head region, being linked to the TEO spacer via an ether bond, while the distal monolayer consists of either a DOPC or a DOPS leaflet. The ion flow into or out of the spacer through the lipid bilayer moiety of the tBLM was monitored by potential step chronocoulometry and cyclic voltammetry. With the distal monolayer bathed by aqueous 0.1 M KCl and 0.8 μM SR-E, an ion flow in two stages was monitored with DOPC at pH 3 and 5.4 and with DOPS at pH 3, while a single stage was observed with DOPS at pH 5.4. This behavior was compared with that already described at conventional bilayer lipid membranes. The sigmoidal shape of the chronocoulometric charge transients points to an aggregation of SR-E monomers forming an ion channel via a mechanism of nucleation and growth. The ion flow is mainly determined by potassium ions, and is inhibited by calcium ions. The contribution to the transmembrane potential from the distal leaflet depends more on the nature of the lipid than that of the ion channel.     

Effects of resveratrol on membrane biophysical properties: relevance for its pharmacological effects

The current study gathers a range of spectrophotometric and spectrofluorimetric techniques to systematically monitor the effects of resveratrol (trans-3,5,4′-trihydrostilbene) on the biophysical properties of membrane model systems consisting of unilamellar liposomes of phosphatidylcholine (DPPC) with the ultimate goal of relating these effects with some of the well documented pharmacological properties of this compound, and clarifying some controversial results reported on the literature.Physiological conditions have been pursued, such as a buffered pH control with adjusted ionic strength similar to the blood plasma conditions (pH 7.4, I = 0.1 M) and the study at different membrane physical states (gel phase and fluid phase) for the assessment of resveratrol-membrane: aqueous partition coefficient by derivative spectroscopy. Results obtained by fluorescence quenching and anisotropy studies indicate that resveratrol has a membrane fluidizing effect and is able to permeate the membrane even in the gel phase. These results mirror the well described antioxidant effect of resveratrol, since antioxidants have to reach peroxidised rigid membranes and increase membrane fluidity in order to interact more efficiently with lipid radicals in the disordered lipid bilayer. Location of resveratrol pointed also to a membrane distribution that is favourable for scavenging the lipid radicals and was elucidated using probes positioned at different membrane depths suggesting that this compound penetrates into the acyl membrane region but also positions its polar hydroxyl group near the headgroup region of the membrane.     

Phase state and surface topography of palmitoyl-ceramide monolayers

In cell biology (and in many biophysical) studies there is a natural tendency to consider ceramide as a highly condensed, solid-type lipid conferring rigidity and close packing to biomembranes. In the present work we advanced the understanding of the phase behavior of palmitoyl-ceramide restricted to a planar interface using Langmuir monolayers under strictly controlled and known surface packing conditions. Surface pressure–molecular area isotherms were complemented with molecular area–temperature isobars and with observations of the surface topography by Brewster Angle Microscopy. The results described herein indicate that palmitoyl-ceramide can exhibit expanded, as well as condensed phase states. Formation of three phases was found, depending on the surface pressure and temperature: a solid (1.80 nm thick), a liquid-condensed (1.73 nm thick, likely tilted) and a liquid-expanded (1.54 nm thick) phase over the temperature range 5–62 °C. A large hysteretic behavior is observed for the S phase monolayer that may indicate high resistance to domain boundary deformation. A second (or higher) order S → LC phase transition is observed at about room temperature while a first order LC → LE transition occurs in a range of temperature encompassing the physiological one (observed above 30 °C at low surface pressure). This phase behavior broadens the view of ceramide as a type of lipid not-always-rigid but able to exhibit polymorphic properties.     

Lipid asymmetry in DLPC/DSPC-supported lipid bilayers: a combined AFM and fluorescence microscopy study

A fundamental attribute of cell membranes is transmembrane asymmetry, specifically the formation of ordered phase domains in one leaflet that are compositionally different from the opposing leaflet of the bilayer. Using model membrane systems, many previous studies have demonstrated the formation of ordered phase domains that display complete transmembrane symmetry; but there have been few reports on the more biologically relevant asymmetric membrane structures. Here we report on a combined atomic force microscopy and fluorescence microscopy study whereby we observe three different states of transmembrane symmetry in phase-separated supported lipid bilayers formed by vesicle fusion. We find that if the leaflets differ in gel-phase area fraction, then the smaller domains in one leaflet are in registry with the larger domains in the other leaflet and the system is dynamic. In a presumed lipid flip-flop process similar to Ostwald ripening, the smaller domains in one leaflet erode away whereas the large domains in the other leaflet grow until complete compositional asymmetry is reached and remains stable. We have quantified this evolution and determined that the lipid flip-flop event happens most frequently at the interface between symmetric and asymmetric DSPC domains. If both leaflets have identical area fraction of gel-phase, gel-phase domains are in registry and are static in comparison to the first state. The stability of these three DSPC domain distributions, the degree of registry observed, and the domain immobility have biological significance with regards to maintenance of lipid asymmetry in living cell membranes, communication between inner leaflet and outer leaflet, membrane adhesion, and raft mobility.     

Mixing of oxidized and bilayer phospholipids

Composition and phase dependence of the mixing of 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), with the oxidized phospholipid, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) were investigated by characterizing the aggregation states of DPPC/PGPC and DOPC/PGPC using a fluorescence quenching assay, dynamic light scattering, and time-resolved fluorescence quenching in the temperature range 5–60 °C. PGPC forms 3.5 nm radii micelles of aggregation number 33. In the gel phase, DPPC and PGPC fuse to form mixed vesicles for PGPC molar fraction, X_PGPC ≤ 0.3 and coexisting vesicles and micelles at higher X_PGPC. Data suggest that liquid phase DPPC at 50 °C forms mixed vesicles with segregated or hemi fused DPPC and PGPC for X_PGPC ≤ 0.3. At 60 °C, DPPC and PGPC do not mix, but form coexisting vesicles and micelles. DOPC and PGPC do not mix in any proportion in the liquid phase. Two dissimilar aggregates of the sizes of vesicles and PGPC micelles were observed for all X_PGPC for T ≥ 22 °C. DOPC–PGPC and DPPC–PGPC mixing is non-ideal for X_PGPC > 0.3 in both gel and fluid phases resulting in exclusion of PGPC from the bilayer. Formation of mixed vesicles is favored in the gel phase but not in the liquid phase for X_PGPC ≤ 0.3. For X_PGPC ≤ 0.3, aggregation states change progressively from mixed vesicles in the gel phase to component segregated mixed vesicles in the liquid phase close to the chain melting transition temperature to separated coexisting vesicles and micelles at higher temperatures.     

Polydimethylsiloxane modified chitosan. Part III: Preparation and characterization of hybrid membranes

The paper deals with the synthesis of organic–inorganic hybrid membranes, Hy, obtained by simultaneous grafting and crosslinking of chitosan with epoxy-terminated polydimethylsiloxane and γ-glycidoxypropyltrimethoxysilane. Porous membranes, HyP, were also obtained by acid decomposition, at different temperatures (25 and 50 °C), of calcium carbonate porogenic agent trapped inside the material. As proved by electron and atomic force microscopy, the non-porous membrane is a phase segregated material with spherical domains (10–40 μm) of silica core covered by hydrophobic siloxane in a hydrophilic chitosan matrix. The porous membranes showed different morphologies with irregular circular pores of 10–30 μm diameters for the membranes obtained at lower temperature, while the membranes prepared at 50 °C tend to adopt a plan-parallel porosity. The water contact angles of hybrid membranes (78°) and pure chitosan membranes (72°) indicated a lower hydrophilic character of modified chitosan. As a result of the crosslinking and of increased hydrophobicity, the hybrid membranes were characterized by a smaller water swelling degree (about 30%) as compared to pure chitosan membrane (700%). However, the presence of the pores in HyP membranes determined an increase of the water adsorption (maximum swelling degree, about 100%). The hybrid membranes possess a slightly higher thermal stability as compared to chitosan (first initial decomposition temperature, 147 and 175 °C for chitosan and hybrid membranes, respectively), but a lower one as compared to pure polydimethylsiloxane. The high storage modulus of chitosan (about 5.1 × 10⁹ Pa at 20 °C) is decreased by about one order of magnitude by the introduction of the highly flexible polysiloxane and the hybrid membranes are more flexible.     

Structural adaptations of proteins to different biological membranes

To gain insight into adaptations of proteins to their membranes, intrinsic hydrophobic thicknesses, distributions of different chemical groups and profiles of hydrogen-bonding capacities (α and β) and the dipolarity/polarizability parameter (π*) were calculated for lipid-facing surfaces of 460 integral α-helical, β-barrel and peripheral proteins from eight types of biomembranes. For comparison, polarity profiles were also calculated for ten artificial lipid bilayers that have been previously studied by neutron and X-ray scattering. Estimated hydrophobic thicknesses are 30–31 Å for proteins from endoplasmic reticulum, thylakoid, and various bacterial plasma membranes, but differ for proteins from outer bacterial, inner mitochondrial and eukaryotic plasma membranes (23.9, 28.6 and 33.5 Å, respectively). Protein and lipid polarity parameters abruptly change in the lipid carbonyl zone that matches the calculated hydrophobic boundaries. Maxima of positively charged protein groups correspond to the location of lipid phosphates at 20–22 Å distances from the membrane center. Locations of Tyr atoms coincide with hydrophobic boundaries, while distributions maxima of Trp rings are shifted by 3–4 Å toward the membrane center. Distributions of Trp atoms indicate the presence of two 5–8 Å-wide midpolar regions with intermediate π* values within the hydrocarbon core, whose size and symmetry depend on the lipid composition of membrane leaflets. Midpolar regions are especially asymmetric in outer bacterial membranes and cell membranes of mesophilic but not hyperthermophilic archaebacteria, indicating the larger width of the central nonpolar region in the later case. In artificial lipid bilayers, midpolar regions are observed up to the level of acyl chain double bonds.     

Characterization of lipid rafts in human platelets using nuclear magnetic resonance: A pilot study

Lipid microdomains (‘lipid rafts’) are plasma membrane subregions, enriched in cholesterol and glycosphingolipids, which participate dynamically in cell signaling and molecular trafficking operations. One strategy for the study of the physicochemical properties of lipid rafts in model membrane systems has been the use of nuclear magnetic resonance (NMR), but until now this spectroscopic method has not been considered a clinically relevant tool. We performed a proof-of-concept study to test the feasibility of using NMR to study lipid rafts in human tissues. Platelets were selected as a cost-effective and minimally invasive model system in which lipid rafts have previously been studied using other approaches. Platelets were isolated from plasma of medication-free adult research participants (n=13) and lysed with homogenization and sonication. Lipid-enriched fractions were obtained using a discontinuous sucrose gradient. Association of lipid fractions with GM1 ganglioside was tested using HRP-conjugated cholera toxin B subunit dot blot assays. ¹H high resolution magic-angle spinning nuclear magnetic resonance (HRMAS NMR) spectra obtained with single-pulse Bloch decay experiments yielded spectral linewidths and intensities as a function of temperature. Rates of lipid lateral diffusion that reported on raft size were measured with a two-dimensional stimulated echo longitudinal encode-decode NMR experiment. We found that lipid fractions at 10–35% sucrose density associated with GM1 ganglioside, a marker for lipid rafts. NMR spectra of the membrane phospholipids featured a prominent ‘centerband’ peak associated with the hydrocarbon chain methylene resonance at 1.3 ppm; the linewidth (full width at half-maximum intensity) of this ‘centerband’ peak, together with the ratio of intensities between the centerband and ‘spinning sideband’ peaks, agreed well with values reported previously for lipid rafts in model membranes. Decreasing temperature produced decreases in the 1.3 ppm peak intensity and a discontinuity at ~18 °C, for which the simplest explanation is a phase transition from L_d to L_o phases indicative of raft formation. Rates of lateral diffusion of the acyl chain lipid signal at 1.3 ppm, a quantitative measure of microdomain size, were consistent with lipid molecules organized in rafts. These results show that HRMAS NMR can characterize lipid microdomains in human platelets, a methodological advance that could be extended to other tissues in which membrane biochemistry may have physiological and pathophysiological relevance.     

Phase diagrams and lipid domains in multicomponent lipid bilayer mixtures

Understanding the phase behavior of biological membranes is helped by the study of more simple systems. Model membranes that have as few as 3 components exhibit complex phase behavior that can be well described, providing insight for biological membranes. A number of different studies are in agreement on general findings for some compositional phase diagrams, in particular, those that model the outer leaflet of animal cell plasma membranes. These model mixtures include cholesterol, together with one high-melting lipid and one low-melting lipid. An interesting finding is of two categories of such 3-component mixtures, leading to what we term Type I and Type II compositional phase diagrams. The latter have phase regions of macroscopic coexisting domains of {Lα + Lβ + Lo} and of {Lα + Lo}, with domains resolved under the light microscope. Type I mixtures have the same phase coexistence regions, but the domains seem to be nanoscopic. Type I mixtures are likely to be better models for biological membranes.     

Recent progress on lipid lateral heterogeneity in plasma membranes: From rafts to submicrometric domains

The concept of transient nanometric domains known as lipid rafts has brought interest to reassess the validity of the Singer–Nicolson model of a fluid bilayer for cell membranes. However, this new view is still insufficient to explain the cellular control of surface lipid diversity or membrane deformability. During the past decades, the hypothesis that some lipids form large (submicrometric/mesoscale vs nanometric rafts) and stable (> min vs s) membrane domains has emerged, largely based on indirect methods. Morphological evidence for stable submicrometric lipid domains, well-accepted for artificial and highly specialized biological membranes, was further reported for a variety of living cells from prokaryot es to yeast and mammalian cells. However, results remained questioned based on limitations of available fluorescent tools, use of poor lipid fixatives, and imaging artifacts due to non-resolved membrane projections. In this review, we will discuss recent evidence generated using powerful and innovative approaches such as lipid-specific toxin fragments that support the existence of submicrometric domains. We will integrate documented mechanisms involved in the formation and maintenance of these domains, and provide a perspective on their relevance on membrane deformability and regulation of membrane protein distribution.     

Arachidonic acid is important for efficient use of light by the microalga Lobosphaera incisa under chilling stress

The oleaginous microalga Lobosphaera incisa (Trebouxiophyceae, Chlorophyta) contains arachidonic acid (ARA, 20:4 n − 6) in all membrane glycerolipids and in the storage lipid triacylglycerol. The optimal growth temperature of the wild-type (WT) strain is 25 °C; chilling temperatures (≤ 15 °C) slow its growth. This effect is more pronounced in the delta-5-desaturase ARA-deficient mutant P127, in which ARA is replaced with dihomo-γ-linolenic acid (DGLA, 20:3 n − 6). In nutrient-replete cells grown at 25 °C, the major chloroplast lipid monogalactosylglycerol (MGDG) was dominated by C18/C16 species in both strains. Yet ARA constituted over 10% of the total fatty acids in the WT MGDG as a component of C20/C18 and C20/C20 species, whereas DGLA was only a minor component of MGDG in P127. Both strains increased the percentage of 18:3 n − 3 in membrane lipids under chilling temperatures. The temperature downshift led to a dramatic increase in triacylglycerol at the expense of chloroplast lipids. WT and P127 showed a similarly high photochemical quantum yield of photosystem II, whereas non-photochemical quenching (NPQ) and violaxanthin de-epoxidation were drastically higher in P127, especially at 15 °C. Fluorescence anisotropy measurements indicated that ARA-containing MGDG might contribute to sustaining chloroplast membrane fluidity upon dropping to the chilling temperature. We hypothesize that conformational changes in chloroplast membranes and increased rigidity of the ARA-deficient MGDG of P127 at chilling temperatures are not compensated by trienoic fatty acids. This might ‘lock’ violaxanthin de-epoxidase in the activated state causing high constitutive NPQ and alleviate the risk of photodamage under chilling conditions in the mutant.     

Reactive oxygen species mediated modifications in Bacillus subtilis lipid membrane to improve protein productivities

The overall objective of this work was to investigate the modifications that occur in Bacillus subtilis lipid membrane during induced oxidative stress caused by reactive oxygen species (ROS), via an electrophysiological approach. Further, based on the results, we have developed and demonstrated a novel strategy to enhance specific enzyme production. Electrical parameters such as phase angle (θ), impedance (Z), capacitance (C) and breakdown voltage of reconstituted bilayer lipid membrane (BLM) composed of lipids extracted from non-stressed, mildly stressed (2.5 mM H₂O₂) and strongly stressed (2.5 mM H₂O₂ with 100 μM FeSO₄) B. subtilis were compared. Strongly stressed BLM showed lower values of θ (10°), Z (0.4 Mohm), and breakdown voltage (100 mV) in comparison with those observed for non-stressed BLM, i.e., 30°, 0.5 Mohm and 250 mV, respectively. The capacitance of strongly stressed BLM, however, was higher (2.28 nF) compared to that of the non-stressed BLM (0.4 nF). These results suggest that under strongly stressed conditions, the lipids were loosely packed that resulted in a more permeable BLM. The higher permeability seems to result, unexpectedly, from a higher unsaturated fatty acid (UFA) synthesis and membrane incorporation (UFA fraction increased by 227%), and expectedly, from increased lipid peroxidation (increased by nearly 200%) in the BLM. A strategy that is based on increased membrane permeability due to induced ROS, enhanced specific amylase and protease production under oxidative stress by 62% and 137%, respectively.     

Effects of aqueous ozone on Pseudomonas syringae viability and ice nucleating activity

This study reports the impact of different ozone treatments on a Pseudomonas syringae strain known for its ice nucleation activity (INA). Ozone is a very powerful germicidal agent used for water treatment. The effect of ozone on viability and on cultivability of P. syringae was determined by flow cytometry analysis and by plate counting respectively. The impact of ozone on the outer membrane using the INA as marker was investigated by the drop freezing technique.The destruction curve followed a shoulder pattern with a slight reduction in population with CT values between 0 and 8 min. For an initial population of 9.3 log CFU mL^?1, the cultivability was lost starting at 14 min and a loss of viability was observed after 16 min of ozone treatment at 0.45 mg L^?1. Microscopic observations at this point revealed whole but aggregated bacilli. INA decreased after 8 min of ozone treatment but did not disappear. This decrease could be due to the progressive disruption of ice nucleating sites in the outer membrane. It was however partially restored after long storage at 4 °C of dead cells treated for 16 min.     

riDOM,a cell penetrating peptide. Interaction with phospholipid bilayers

Melittin is an amphipathic peptide which has received much attention as a model peptide for peptide–membrane interactions. It is however not suited as a transfection agent due to its cytolytic and toxicological effects. Retro-inverso-melittin, when covalently linked to the lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (riDOM), eliminates these shortcomings. The interaction of riDOM with phospholipid membranes was investigated with circular dichroism (CD) spectroscopy, dynamic light scattering, ζ-potential measurements, and high-sensitivity isothermal titration calorimetry. riDOM forms cationic nanoparticles with a diameter of ~ 13 nm which are well soluble in water and bind with high affinity to DNA and lipid membranes. When dissolved in bilayer membranes, riDOM nanoparticles dissociate and form transient pores. riDOM-induced membrane leakiness is however much reduced compared to that of authentic melittin. The secondary structure of the ri-melittin is not changed when riDOM is transferred from water to the membrane and displays a large fraction of β-structure. The ³¹P NMR spectrum of the nanoparticle is however transformed into a typical bilayer spectrum. The Gibbs free energy of riDOM binding to bilayer membranes is − 8.0 to − 10.0 kcal/mol which corresponds to the partition energy of just one fatty acyl chain. Half of the hydrophobic surface of the riDOM lipid extension with its 2 oleic acyl chains is therefore involved in a lipid–peptide interaction. This packing arrangement guarantees a good solubility of riDOM both in the aqueous and in the membrane phase. The membrane binding enthalpy is small and riDOM binding is thus entropy-driven.