Peroxynitrite rapidly permeates phospholipid membranes

Peroxynitrite (ONOO−) is a potent oxidant implicated in a number of pathophysiological processes. The activity of ONOO− is related to its accessibility to biological targets before its spontaneous decomposition (t1/2 ≈ 1 s at pH 7.4, 37°C). Using model phospholipid vesicular systems and manganese porphyrins as reporter molecules, we demonstrated that ONOO− freely crosses phospholipid membranes. The calculated permeability coefficient for ONOO− is ≈8.0 × 10−4 cm⋅s−1, which compares well with that of water and is ≈400 times greater than that of superoxide. We suggest that ONOO− is a significant biological effector molecule not only because of its reactivity but also because of its high diffusibility.

A unique phospholipid organization in bovine erythrocyte membranes

Ruminant erythrocytes are remarkable for their choline-phospholipid anomalies; namely, low or absent phosphatidylcholine (PC) along with high sphingomyelin levels. Here, we report another anomaly in bovine erythrocytes that affects aminophospholipids: phosphatidylethanolamine (PE) shows an extreme asymmetry, with only 2% of the total present in the outer leaflet. Furthermore, we found that phospholipase A2, an enzyme located on the external surface of the erythrocytes, shows higher activity against PC than against PE. In addition, we observed that acylation of PE is by far the most important biosynthetic event in this system. We propose that deacylation of PE and PC by phospholipase A2 to generate lysocompounds, followed by selective reacylation of lyso-PE in the inner leaflet, can account for the compositional and architectural peculiarities of bovine erythrocyte membranes.

Chemical specificity and physical properties of the lipid bilayer in the regulation of protein kinase C by anionic phospholipids: evidence for the lack of a specific binding site for phosphatidylserine.

The association of protein kinase C (PKC) with membranes was found not to be specific for phosphatidyl-L-serine (PS). In particular, a synthetic phospholipid, dansyl-phosphatidylethanolamine, proved to be fully functional in the association of PKC with lipid bilayers and in mediating the interaction of this enzyme with diacylglycerol. Dansyl-phosphatidylethanolamine was also able to activate the enzyme in a Ca2+-dependent fashion. Differences in the ability to bind and activate PKC observed for an array of anionic lipids were not larger than alterations caused by changes in acyl chain composition. Thus, although different lipids interact to different extents with PKC, there are no specific binding sites for the PS headgroup on the enzyme. We found that lipids with a greater tendency to form inverted phases increased the binding of PKC to bilayers. However, these changes in lipid structure cannot be considered separately from the miscibility of lipid components in the membrane. For pairs of lipids with similar acyl chains, the dependence on PS concentration is sigmoidal, while for dissimilar acyl chains there is much less dependence of binding on PS concentration. The results can be explained in terms of differences in the lateral distribution of components in the membrane.

Molecular dynamics study of MscL interactions with a curved lipid bilayer

Mechanosensitivity is a ubiquitous sensory mechanism found in living organisms. The simplest known mechanotransducing mechanism is found in bacteria in the form of the mechanosensitive membrane channel of large conductance, MscL. This channel has been studied extensively using a variety of methods at a functional and structural level. The channel is gated by membrane tension in the lipid bilayer alone. It serves as a safety valve protecting bacterial cells against hypoosmotic shock. MscL of Escherichia coli embedded in bilayers composed of asymmetric amounts of single-tailed and double-tailed lipids has been shown to gate spontaneously, even in the absence of membrane tension. To gain insight into the effect of the lipid membrane composition and geometry on MscL structure, a fully solvated, all-atom model of MscL in a stress-free curved bilayer composed of double- and single-tailed lipids was studied using a 9.5-ns molecular dynamics simulation. The bilayer was modeled as a domed structure accommodating the asymmetric composition of the monolayers. During the course of the simulation a spontaneous restructuring of the periplasmic loops occurred, leading to interactions between one of the loops and phospholipid headgroups. Previous experimental studies of the role of the loops agree with the observation that opening starts with a restructuring of the periplasmic loop, suggesting an effect of the curved bilayer. Because of limited resources, only one simulation of the large system was performed. However, the results obtained suggest that through the geometry and composition of the bilayer the protein structure can be affected even on short timescales.

Effect of incorporating cholesterol into DDA:TDB liposomal adjuvants on Bilayer properties, biodistribution, and immune responses

Cholesterol is an abundant component of mammalian cell membranes and has been extensively studied as an artificial membrane stabilizer in a wide range of phospholipid liposome systems. In this study, the aim was to investigate the role of cholesterol in cationic liposomal adjuvant system based on dimethyldioctadecylammonium (DDA) and trehalose 6,6'-dibehenate (TDB) which has been shown as a strong adjuvant system for vaccines against a wide range of diseases. Packaging of cholesterol within DDA:TDB liposomes was investigated using differential scanning calorimetery and surface pressure-area isotherms of lipid monolayers; incorporation of cholesterol into liposomal membranes promoted the formation of a liquid-condensed monolayer and removed the main phase transition temperature of the system, resulting in an increased bilayer fluidity and reduced antigen retention in vitro. In vivo biodistribution studies found that this increase in membrane fluidity did not alter deposition of liposomes and antigen at the site of injection. In terms of immune responses, early (12 days after immunization) IgG responses were reduced by inclusion of cholesterol; thereafter there were no differences in antibody (IgG, IgG1, IgG2b) responses promoted by DDA:TDB liposomes with and without cholesterol. However, significantly higher levels of IFN-gamma were induced by DDA:TDB liposomes, and liposome uptake by macrophages in vitro was also shown to be higher for DDA:TDB liposomes compared to their cholesterol-containing counterparts, suggesting that small changes in bilayer mechanics can impact both cellular interactions and immune responses. © 2013 American Chemical Society.

Effect of incorporating cholesterol into DDA:TDB liposomal adjuvants on Bilayer properties, biodistribution, and immune responses

Cholesterol is an abundant component of mammalian cell membranes and has been extensively studied as an artificial membrane stabilizer in a wide range of phospholipid liposome systems. In this study, the aim was to investigate the role of cholesterol in cationic liposomal adjuvant system based on dimethyldioctadecylammonium (DDA) and trehalose 6,6'-dibehenate (TDB) which has been shown as a strong adjuvant system for vaccines against a wide range of diseases. Packaging of cholesterol within DDA:TDB liposomes was investigated using differential scanning calorimetery and surface pressure-area isotherms of lipid monolayers; incorporation of cholesterol into liposomal membranes promoted the formation of a liquid-condensed monolayer and removed the main phase transition temperature of the system, resulting in an increased bilayer fluidity and reduced antigen retention in vitro. In vivo biodistribution studies found that this increase in membrane fluidity did not alter deposition of liposomes and antigen at the site of injection. In terms of immune responses, early (12 days after immunization) IgG responses were reduced by inclusion of cholesterol; thereafter there were no differences in antibody (IgG, IgG1, IgG2b) responses promoted by DDA:TDB liposomes with and without cholesterol. However, significantly higher levels of IFN-gamma were induced by DDA:TDB liposomes, and liposome uptake by macrophages in vitro was also shown to be higher for DDA:TDB liposomes compared to their cholesterol-containing counterparts, suggesting that small changes in bilayer mechanics can impact both cellular interactions and immune responses. © 2013 American Chemical Society.

Channel-forming activity of syringopeptin 25 A in mercury-supported phospholipid monolayers and negatively charged bilayers

Interactions of the cationic lipodepsipeptide syringopeptin 25 A (SP25A) with mercury-supported dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS) and dioeleoylphosphatidic acid (DOPA) self-assembled monolayers (SAMs) were investigated by AC voltammetry in 0.1 M KCl at pH 3, 5.4 and 6.8. SP25A targets and penetrates the DOPS SAM much more effectively than the other SAMs not only at pH 6.8, where the DOPS SAM is negatively charged, but also at pH 3, where it is positively charged just as SP25A. Similar investigations at tethered bilayer lipid membranes (tBLMs) consisting of a thiolipid called DPTL anchored to mercury, with a DOPS, DOPA or DOPC distal monolayer on top of it, showed that, at physiological transmembrane potentials, SP25A forms ion channels spanning the tBLM only if DOPS is the distal monolayer. The distinguishing chemical feature of the DOPS SAM is the ionic interaction between the protonated amino group of a DOPS molecule and the carboxylate group of an adjacent phospholipid molecule. Under the reasonable assumption that SP25A preferentially interacts with this ion pair, the selective lipodepsipeptide antimicrobial activity against Gram-positive bacteria may be tentatively explained by its affinity for similar protonated amino-carboxylate pairs, which are expected to be present in the peptide moieties of peptidoglycan strands.

Removal Of Glyphosate Herbicide From Water Using Biopolymer Membranes.

Enormous amounts of pesticides are manufactured and used worldwide, some of which reach soils and aquatic systems. Glyphosate is a non-selective herbicide that is effective against all types of weeds and has been used for many years. It can therefore be found as a contaminant in water, and procedures are required for its removal. This work investigates the use of biopolymeric membranes prepared with chitosan (CS), alginate (AG), and a chitosan/alginate combination (CS/AG) for the adsorption of glyphosate present in water samples. The adsorption of glyphosate by the different membranes was investigated using the pseudo-first order and pseudo-second order kinetic models, as well as the Langmuir and Freundlich isotherm models. The membranes were characterized regarding membrane solubility, swelling, mechanical, chemical and morphological properties. The results of kinetics experiments showed that adsorption equilibrium was reached within 4 h and that the CS membrane presented the best adsorption (10.88 mg of glyphosate/g of membrane), followed by the CS/AG bilayer (8.70 mg of glyphosate/g of membrane). The AG membrane did not show any adsorption capacity for this herbicide. The pseudo-second order model provided good fits to the glyphosate adsorption data on CS and CS/AG membranes, with high correlation coefficient values. Glyphosate adsorption by the membranes could be fitted by the Freundlich isotherm model. There was a high affinity between glyphosate and the CS membrane and moderate affinity in the case of the CS/AG membrane. Physico-chemical characterization of the membranes showed low values of solubility in water, indicating that the membranes are stable and not soluble in water. The SEM and AFM analysis showed evidence of the presence of glyphosate on CS membranes and on chitosan face on CS/AG membranes. The results showed that the glyphosate herbicide can be adsorbed by chitosan membranes and the proposed membrane-based methodology was successfully used to treat a water sample contaminated with glyphosate. Biopolymer membranes therefore potentially offer a versatile method to eliminate agricultural chemicals from water supplies.

Determination of chemical shielding tensor of an indole carbon and application of tryptophan orientation of a membrane peptide

Using tryptophan C-13-enriched at the C-4 (C epsilon(3)) of the indole, the orientation of the C epsilon(3) chemical shift tensor relative to the C epsilon(3)-H dipolar axis was determined from the C-13 chemical shift/C-13-H-1 dipolar 2D NMR powder pattern. The principal values obtained were 208, 137 and 15 ppm with sigma(33) perpendicular to the indole plane, and sigma(11) (least shielded direction) 5 degrees off the C epsilon(3)-H bond toward C xi(3). The side off the C epsilon(3)-H bond was determined by comparing the reduced chemical shift anisotropies obtained by solid-state NMR and from molecular dynamics calculations of [4-C-13] tryptophans in gramicidin A aligned in phospholipid membranes. (C) 1999 Elsevier Science B.V. All rights reserved.

Dermaseptin 01 as antimicrobial peptide with rich biotechnological potential: study of peptide interaction with membranes containing Leishmania amazonensis lipid-rich extract and membrane models

This article addresses the interactions of the synthetic antimicrobial peptide dermaseptin 01 (GLWSTIKQKGKEAAIAAA-KAAGQAALGAL-NH(2), DS 01) with phospholipid (PL) monolayers comprising (i) a lipid-rich extract of Leishmania amazonensis (LRE-La), (ii) zwitterionic PL (dipalmitoylphosphatidylcholine, DPPC), and (iii) negatively charged PL (dipalmitoylphosphatidylglycerol, DPPG). The degree of interaction of DS 01 with the different biomembrane models was quantified from equilibrium and dynamic liquid-air interface parameters. At low peptide concentrations, interactions between DS 01 and zwitterionic PL, as well as with the LRE-La monolayers were very weak, whereas with negatively charged PLs the interactions were stronger. For peptide concentrations above 1 mu g/ml, a considerable expansion of negatively charged monolayers occurred. In the case of DPPC, it was possible to return to the original lipid area in the condensed phase, suggesting that the peptide was expelled from the monolayer. However, in the case of DPPG, the average area per lipid molecule in the presence of DS 01 was higher than pure PLs even at high surface pressures, suggesting that at least part of DS 01 remained incorporated in the monolayer. For the LRE-La monolayers, DS 01 also remained in the monolayer. This is the first report on the antiparasitic activity of AMPs using Langmuir monolayers of a natural lipid extract from L. amazonensis. Copyright (C) 2011 European Peptide Society and John Wiley & Sons, Ltd.

Porphyrin-phospholipid interaction and ring metallation depending on the phospholipid polar head type

The interaction between a hydrophobically modified 5,10,15,20-tetrakis(4-N-tetradecyl-pyridyl) porphyrin and three phospholipids: two negatively charged, DMPA (the sodium salt of dimyristoyl-sn-glycero-phosphatidyl acid) and DMPG (the sodium salt of 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]) and a zwitterionic DMPC (dimyristoyl-sn-glycero-phosphatidylcholine), were studied by means of surface pressure isotherms and spectroscopic methods. The interaction results in partial or total metallation of the porphyrin with zinc ions in the presence of negatively charged phospholipids, as attested by UV-vis and luminescence spectroscopy of the transferred films. In the presence of the zwitterionic phospholipid no insertion of zinc ion in the porphyrin ring is detected. These results are relevant for the understanding of photosensitizer-lipid-carrier binding for use in photodynamic therapy. (C) 2010 Elsevier Inc. All rights reserved.

The interaction of bothropstoxin-I (Lys49-PLA(2)) with liposome membranes

Bothropstoxin-I (BthTx-I) is a Lys49-PLA(2) from the venom of the snake Bothrops jararacussu, which permeabilizes biological and artificial membranes by a mechanism independent of lipid hydrolysis. This mechanism has been investigated by studying the interaction of nine single tryptophan BthTx-I mutants with negatively charged phospholipid membranes. Changes in the solvent exposure of the tryptophan in each mutant were evaluated comparing the rate of chemical modification (k(mod)) by bromosuccinamide with the maximum intrinsic tryptophan fluorescence emission wavelength (lambda(max)) in buffer and in the presence of 10% DMPA/90% DPPC liposomes. No changes in lambda(max). were observed, whereas k(mod) values for tryptophans at positions 7, 10, 31 and 125 were significantly reduced in the presence of lipids, suggesting that bound phospholipid decreases solvent accessibility at these positions. Since the half-lives of the fluorescence and chemical modification effects differ by at least six orders of magnitude, these results suggest that the bound phospholipid may interact with multiple locations on the protein surface over micro- to millisecond timescales. (C) 2009 Elsevier Ltd. All rights reserved.

Insights on calcium-independent phospholipid membrane damage by Lys49-PLA(2) using tryptophan scanning mutagenesis of bothropstoxin-I from Bothrops jararacussu

Bothropstoxin-I (BthTx-I) is a homodimerie Lys49-PLA(2) from the venom of the snake Bothrops jararacussu, which lacks hydrolytic activity against phospholipid substrates, yet permeabilizes membranes by a Ca2+- independent mechanism. The interaction of the BthTx-I with model membranes has been studied by intrinsic tryptophan fluorescence emission (ITFE) spectroscopy. Nine separate mutants have been created each with a unique tryptophan residue located at a different position in the interfacial recognition site (IRS) of the protein. The rapid and efficient Ca2+-independent membrane damage against unilamellar liposomes composed of DPPC/DMPA in a 9:1 molar ratio was unaffected by these substitutions. Binding studies revealed low protein affinity for these liposomes and no changes were observed in the ITFE properties. In contrast, the binding of all mutants to DPPC/DMPA liposomes in a 1:1 molar ratio was stronger, and was correlated with altered ITFE properties. The blue-shifted emission spectra and increased emission intensity of mutants at positions 31, 67 and 115-117 in the interface recognition surface of the protein suggest these regions are partially inserted into the membrane. These results are consistent with a model for the Ca2+-independent membrane damaging mechanism that involves a transient interaction of the protein with the outer phospholipid leaflet of the target membrane. (C) 2007 Elsevier Masson SAS. All rights reserved.

Structural effects of lipophilic methotrexate conjugates on model phospholipid biomembranes

Lipophilic conjugates of the antitumor drug methotrexate (MTX) with lipoamino acids (LAAs) have been previously described as a tool to enhance MTX passive entrance into cells, overcoming a form of transport resistance which makes tumour cells insensitive to the antimetabolite. A knowledge of the mechanisms of interaction of such lipophilic derivatives with cell membranes could be useful for planning further lipophilic MTX derivatives with an optimal antitumour activity. To this aim, a calorimetric study was undertaken using a biomembrane model made from synthetic 1,2-dipalmitoyl-glycero-3-phosphocholine (DPPC) multilamellar liposomes. The effects of MTX and conjugates on the phase transition of liposomes were investigated using differential scanning calorimetry. The interaction of pure MTX with the liposomes was limited to the outer part of the phospholipid bilayers, due to the polar nature of the drug. Conversely, its lipophilic conjugates showed a hydrophobic kind of interaction, perturbing the packing order of DPPC bilayers. In particular, a reduction of the enthalpy of transition from the gel to the liquid crystal phase of DPPC membranes was observed. Such an effect was related to the structure and mole fraction of the conjugates in the liposomes. The antitumour activity of MTX conjugates was evaluated against cultures of a CCRF-CEM human leukemic T-cell line and a related MTX resistant sub-line. The in vitro cell growth inhibitory activity was higher for bis(tetradecyl) conjugates than for both the other shorter- and longer-chain derivatives. The biological effectiveness of the various MTX derivatives correlated very well with the thermotropic effects observed on the phase transition of DPPC biomembranes. (C), 2001 Elsevier Science B.V All rights reserved.

Basic biochemical investigations as rationale for the design of original antimalarial drugs. An example of phospholipid metabolism

The future of antimalarial chemotherapy is particulary alarming in view of the spread of parasite cross-resistances to drugs that are not even structurally related. Only the availability of new pharmacological models will make it possible to select molecules with novel mechanisms of action, thus delaving resistance and allowing the development of new chemotherapeutic strategies. We reached this objective in mice. Our approach is hunged on fundamental and applied research begun in 1980 to investigate to phospholipid (PL) metabolism of intraerythrocytic Plasmodium. This metabolism is abundant, specific and indispensable for the production of Plasmodium membranes. Any drug to interfere with this metabolism blocks parasitic development. The most effective interference yet found involves blockage of the choline transporter, which supplies Plasmodium with choline for the synthesis of phosphatidylcholine, its major PL, this is a limiting step in the pathway. The drug sensitivity thereshold is much lower for the parasite, which is more dependent on this metabolism than host cells. The compounds show in vitro activity against P. falciparum at 1 to 10 nM. They show a very low toxicity against a lymphblastoid cell line, demonstrating a total abscence of correlation between growth inhibition of parasites and lymphoblastoid cells. They show antimalarial activity in vivo, in the P. berghei or P. chabaudi/mouse system, at doses 20-to 100-fold lower than their in acute toxicity limit. The bioavailability of a radiolabeled form of the product seemed to be advantageous (slow blood clearance and no significant concentration in tissues). Lastly, the compounds are inexpensive to produce. They are stable and water-soluble.