Simulations of simple Bovine and Homo sapiens outer cortex ocular lens membrane models with a majority concentration of cholesterol

The lipid composition of bovine and human ocular lens membranes has been probed, and a variety of lipids have been found including phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin (SM), and cholesterol (CHOL) with cholesterol being present in particularly high concentrations. In this study, we use the all-atom CHARMM36 force field to simulate binary, ternary, and quaternary mixtures as models of the ocular lens. High concentration of cholesterol, in combination with different and varying diversity of phospholipids (PL) and sphingolipids (SL), affect the structure of the ocular lens lipid bilayer. The following analyses were done for each simulation: surface area per lipid, component surface area per lipid, deuterium order parameters (S_CD), electron density profiles (EDP), membrane thickness, hydrogen bonding, radial distribution functions, clustering, and sterol tilt angle distribution. The S_CD show significant bilayer alignment and packing in cholesterol-rich bilayers. The EDP show the transition from liquid crystalline to liquid ordered with the addition of cholesterol. Hydrogen bonds in our systems show the tendency for intramolecular interactions between cholesterol and fully saturated lipid tails for less complex bilayers. But with an increased number of components in the bilayer, the acyl chain of the lipids becomes a less important characteristic, and the headgroup of the lipid becomes more significant. Overall, cholesterol is the driving force of membrane structure of the ocular lens membrane where interactions between cholesterol, PL, and SL determine structure and function of the biomembrane. The goal of this work is to develop a baseline for further study of more physiologically realistic ocular lens lipid membranes.This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo.     

Melatonin Enhances Tamoxifen's Ability to Prevent the Reduction in Microsomal Membrane Fluidity Induced by Lipid Peroxidation

The indoleamine melatonin and the synthetic antiestrogenic drug tamoxifen seem to have similar mechanisms in inhibiting the growth of estrogen receptor positive breast cancer cells. In this study, we compared the ability of these molecules, alone and in combination, in stabilizing microsomal membranes against free radical attack. Hepatic microsomes were obtained from male rats and incubated with or without tamoxifen (50–200 μm), melatonin (1 mm) or both; lipid peroxidation was induced by addition of FeCl₃, NADPH and ADP. After oxidative damage, membrane fluidity, measured by fluorescence polarization techniques, decreased whereas malonaldehyde (MDA) and 4-hydroxyalkenals (4-HDA) concentrations increased. Incubation of the microsomes with tamoxifen prior to exposure to free radical generating processes inhibited, in a dose-dependent manner, the increase in membrane rigidity and the rise in MDA+4-HDA levels. When melatonin was added, the efficacy of tamoxifen in preventing membrane rigidity was enhanced. Thus, the IC₅₀s for preventing membrane rigidity and for inhibiting lipid peroxidation obtained for tamoxifen in the presence of melatonin were lower than those obtained with tamoxifen alone. Moreover, tamoxifen (50–200 μm) in the presence of melatonin reduced basal membrane fluidity and MDA+4-HDA levels in microsomes. These synergistic effects of tamoxifen and melatonin in stabilizing biological membranes may be important in protecting membranes from free radical damage. Received: 7 July 1997/Revised: 12 November 1997     

Altering in vivo membrane sterol composition affects the activity of the cyclic lipopeptides tolaasin and sessilin against Pythium

Cyclic lipopeptides (CLiPs) are secondary metabolites produced by a variety of bacteria. These compounds show a broad range of antimicrobial activities; therefore, they are studied for their potential applications in agriculture and medicine. It is generally assumed that the primary target of the CLiPs is the cellular membrane, where they can permeabilize the lipid bilayer. Model membrane systems are commonly used to investigate the effect of lipid composition on the permeabilizing activity of CLiPs, but these systems do not represent the full complexity of true biological membranes. Here, we introduce a novel method that uses sterol-auxotrophic oomycetes to investigate how the activity of membrane-active compounds is influenced by alterations in membrane sterol composition. More specifically, we investigated how ergosterol, cholesterol, beta-sitosterol and stigmasterol affect the activity of the structurally related Pseudomonas-derived CLiPs tolaasin and sessilin against the oomycete Pythium myriotylum. Both compounds were effective against oomycetes, although tolaasin was considerably more active. Interestingly, tolaasin and sessilin effects were similarly reduced by the presence of sterols, with cholesterol showing the highest reduction of activity.     

Cholesterol's decoupling effect on membrane partitioning and permeability revisited: Is there anything beyond Fick's law of diffusion?

In general, Fick's law of diffusion describes membrane permeation of hydrophobic or amphiphilic molecules. In contrast to this, Thomae et al. recently identified the volume ratio between barrier and aqueous compartments as important additional determinants of membrane permeability (P_m) [A.V. Thomae, T. Koch, C. Panse, H. Wunderli-Allenspach, and S.D. Kramer, Comparing the lipid membrane affinity and permeation of drug-like acids: the intriguing effects of cholesterol and charged lipids, Pharm. Res. 24 (2007) 1457-1472.]. This new theory was supported by the striking observation that low concentrations of cholesterol increased P_m of salicylic acid. As Fick's law is of fundamental importance to all membrane transport processes, we reinvestigated this phenomenon. We measured the electrophoretic mobility of vesicles and used electrochemical scanning microscopy to study the adsorption of the SA anion to lipid vesicular bilayers and SA transport through planar lipid bilayers, respectively. As predicted by Fick's law, P_m of SA decreased continuously with increasing cholesterol content. Thomae et al. made the contrasting artifactual observation because their kinetic approach lacked the required time resolution and led to an underestimation of P_m by five orders of magnitude. We conclude that there is nothing beyond Fick's law of diffusion. It is still valid.     

TRH-receptor mobility and function in intact and cholesterol-depleted plasma membrane of HEK293 cells stably expressing TRH-R-eGFP

Here we investigated the effect of disruption of plasma membrane integrity by cholesterol depletion on thyrotropin-releasing hormone receptor (TRH-R) surface mobility in HEK293 cells stably expressing TRH-R-eGFP fusion protein (VTGP cells). Detailed analysis by fluorescence recovery after photobleaching (FRAP) in bleached spots of different sizes indicated that cholesterol depletion did not result in statistically significant alteration of mobile fraction of receptor molecules (M_f). The apparent diffusion coefficient (D_app) was decreased, but this decrease was detectable only under the special conditions of screening and calculation of FRAP data. Analysis of mobility of receptor molecules by raster image correlation spectroscopy (RICS) did not indicate any significant difference between control and cholesterol-depleted cells. Results of our FRAP and RICS experiments may be collectively interpreted in terms of a “membrane fence” model which regards the plasma membrane of living cells as compartmentalized plane where lateral diffusion of membrane proteins is limited to restricted areas by cytoskeleton constraints. Hydrophobic interior of plasma membrane, studied by steady-state and time-resolved fluorescence anisotropy of hydrophobic membrane probe DPH, became substantially more “fluid” and chaotically organized in cholesterol-depleted cells. Decrease of cholesterol level impaired the functional coupling between the receptor and the cognate G proteins of G_q/G₁₁ family.In conclusion: the presence of an unaltered level of cholesterol in the plasma membrane represents an obligatory condition for an optimum functioning of TRH-R signaling cascade. The decreased order and increased fluidity of hydrophobic membrane interior suggest an important role of this membrane area in TRH-R–G_q/G₁₁α protein coupling.     

Towards the mechanism and comparative effect of diphenyl diselenide, diphenyl ditelluride and ebselen under various pathophysiological conditions in rat's kidney preparation

As an extension of our previous work we not only evaluated the relationship between acidosis and lipid peroxidation in rat's kidney homogenate, but also determined for the first time the potential anti-oxidant activity of diphenyl diselenide, diphenyl ditelluride and ebselen at a range of pH values (7.4–5.4). Because of the pH dependency of iron redox cycling, pH and iron need to be well controlled and for the reason we tested a number of pH values (from 7.4 to 5.4) to get a closer idea about the role of iron under various pathological conditions. Acidosis increased rate of lipid peroxidation in the absence Fe (II) in kidney homogenates especially at pH 5.4. This higher extent of lipid peroxidation can be explained by; the mobilized iron which may come from reserves where it is weakly bound. Addition of iron (Fe) chelator desferoxamine (DFO) to reaction medium completely inhibited the peroxidation processes at all studied pH values including acidic values (5.8–5.4). In the presence of Fe (II) acidosis also enhanced detrimental effect of Fe (II) especially at pH (6.4–5.4). Diphenyl diselenide significantly protected lipid peroxidation at all studied pH values, while ebselen offered only a small statistically non-significant protection. The highest anti-oxidant potency was observed for diphenyl ditelluride. These differences in potencies were explained by the mode of action of these compounds using their catalytic anti-oxidant cycles. However, changing the pH of the reaction medium did not alter the anti-oxidant activity of the tested compounds. This study provides evidence for acidosis catalyzed oxidative stress in kidney homogenate and for the first time anti-oxidant potential of diphenyl diselenide and diphenyl ditelluride not only at physiological pH but also at a range of acidic values.     

Impact of cholesterol and sphingomyelin on intrinsic membrane permeability

Transwell experiments with Caco-2 or MDCK cells are the gold standard for determining the intestinal permeability of chemicals. The intrinsic membrane permeability (P₀), that can be extracted from these experiments, might be comparable to P₀ measured in black lipid membrane (BLM) experiments and P₀ predicted by the solubility-diffusion model. Unfortunately, the overlap between experimental P_{0,Caco-2/MDCK} and P_0,BLM data is very small. So far, differences between both approaches have been attributed to the cholesterol and sphingomyelin content of cell membranes, but the database is too sparse to thoroughly test this theory. To create a diverse dataset, we measured P_0,BLM of ten chemicals in BLM experiments using DPhPC and DPhPC/cholesterol/sphingomyelin membranes. The results were compared to predicted BLM data and experimental Caco-2/MDCK data obtained from literature. While P_0,BLM of all chemicals was well predicted by the solubility-diffusion model, P_{0,Caco-2/MDCK} was only predictable for rather hydrophilic compounds with logarithmic hexadecane/water partition coefficients below ?0.5. The effect of cholesterol and sphingomyelin on P_0,BLM was negligibly small.     

Impact of oxygen stress and energy availability on membrane stability of plant cells

This article reviews the relationship between the energy status of plant cells under O₂ stress (e.g. waterlogging) and the maintenance of membrane intactness, using information largely derived from suspension cultures of anoxia‐intolerant potato cells. Energy‐related parameters measured were fermentation end‐products (ethanol, lactate, alanine), respiratory rate, ATP, adenylate energy charge, nitrate reductase activity and biomass. ATP synthesis rates were calculated from the first four parameters. Reactive oxygen species were estimated from H₂O₂ and superoxide levels, and the enzymatic detoxification potential from the activity levels of catalase and superoxide dismutase. Structure‐related parameters were total fatty acids, free fatty acids (FFAs), lipid hydroperoxides, total phospholipids, N‐acylphosphatidylethanolamine (NAPE) and cell viability. The following issues are addressed in this review: (1) what is the impact of anoxia on membrane lipids and how does this relate to energy status; (2) does O₂ per se play a role in these changes; (3) under which conditions and to what extent does lipid peroxidation occur upon re‐aeration; and (4) can the effects of re‐aeration be distinguished from those of anoxia? The emerging picture is a reappraisal of the relative contributions of anoxia and re‐aeration. Two successive phases (pre‐lytic and lytic) characterize potato cells under anoxia. They are connected by a threshold in ATP production rate, below which membrane lipids are hydrolysed to FFAs, and NAPE increases. Since lipid peroxidation occurs only when cells are reoxygenated during the lytic phase, its biological relevance in an already damaged system is questionable.Key words: Acorus calamus L., energy shortage, free fatty acids, lipid peroxidation, lipolytic acyl hydrolase, lipoxygenase, membrane intactness, N‐acylphosphatidylethanolamine, O₂ stress, reactive oxygen species, Solanum tuberosum L.     

Phenolic contents and antioxidant activity of ethanolic extract of Capparis spinosa

Caper plant (Capparis spinosa) extracts have been associated with diverse biological activities including anti-oxidant properties. In this work, we characterized the hydro-ethanolic extract obtained from C. spinosa leaves [hydroethanolic extract of C. spinosa (HECS)] by analyzing the content in anti-oxidant compounds such as polyphenols, flavonoids and anthocyanins. Further, we evaluated HECS antioxidant activities in vitro using bleaching of 1,1-diphenyl-2-picrylhydrazyl radical and ABTS test as well as by pretreatment of HeLa cells exposed to Fe²⁺ or H₂O₂. Our findings indicate that HECS contains high amount of total phenolic compounds and high levels of flavonoids and anthocyanins. Furthermore, HECS exhibited antioxidant activity in both chemical and biological tests. Specially, pretreatment of HeLa cells with different concentrations of the extract conferred protection against lipid peroxidation and modulated activities of two antioxidant enzymes, SOD and catalase. These results revealed HECS antioxidant effects and suggest that C. spinosa leaves are a potential source of natural antioxidant molecules with possible applications in industry and medicine.     

Experimental approaches to free radicals and ageing

Abstract

Probucol, a clinically used cholesterol lowering and antioxidant drug, was investigated for possible protection against lipid peroxidation and DNA damage induced by iron nitrilotriacetate (Fe-NTA) plus hydrogen peroxide (H₂O₂). Fe-NTA is a potent nephrotoxic agent and induces acute and subacute renal proximal tubular necrosis by catalyzing the decomposition of H₂O₂-derived production of hydroxyl radicals, which are known to cause lipid peroxidation and DNA damage. Fe-NTA is associated with a high incidence of renal adenocarcinoma in rodents. Lipid peroxidation and DNA damage are the principal manifestation of Fe-NTA induced toxicity, which could be mitigated by probucol. Incubation of renal microsomal membrane and/or calf thymus DNA with H₂O₂ (40 mM) in the presence of Fe-NTA (0.1 mM) induces renal microsomal lipid peroxidation and DNA damage to about 2.4-fold and 5.9-fold, respectively, as compared to control (P < 0.05). Induction of renal microsomal lipid peroxidation and DNA damage was inhibited by probucol in a concentration-dependent manner. In lipid peroxidation protection studies, probucol treatment showed a concentration-dependent inhibition (10–34% inhibition; P <0.05) of Fe-NTA plus H₂O₂-induced lipid peroxidation as measured by thiobarbituric acid reacting species' (TBARS) formation in renal microsomes. Similarly, in DNA damage protection studies, probucol treatment also showed a concentration-dependent strong inhibition (36–71% inhibition; P < 0.05) of DNA damage. From these studies, it was concluded that probucol inhibits peroxidation of microsomal membrane lipids and DNA damage induced by Fe-NTA plus H₂O₂. However, because the lipid peroxidation and DNA damage studied here are regarded as early markers of carcinogenesis, we suggest that probucol may be developed as a cancer chemopreventive agent against renal carcinogenesis and other adverse effects of Fe-NTA exposure in experimental animals, in addition to being a cholesterol-lowering drug, useful for the control of hypercholestrolemia.     

Lipid lateral diffusion and membrane heterogeneity

The pulsed field gradient (pfg)-NMR method for measurements of translational diffusion of molecules in macroscopically aligned lipid bilayers is described. This technique is proposed to have an appreciable potential for investigations in the field of lipid and membrane biology. Transport of molecules in the plane of the bilayer can be successfully studied, as well as lateral phase separation of lipids and their dynamics within the bilayer organizations. Lateral diffusion coefficients depend on lipid packing and acyl chain ordering and investigations of order parameters of perdeuterated acyl chains, using ²H NMR quadrupole splittings, are useful complements. In this review we summarize some of our recent achievements obtained on lipid membranes. In particular, bilayers exhibiting two-phase coexistence of liquid disordered (l_d) and liquid ordered (l_o) phases are considered in detail. Methods for obtaining good oriented lipid bilayers, necessary for the pfg-NMR method to be efficiently used, are also briefly described. Among our major results, besides determinations of l_d and l_o phases, belongs the finding that the lateral diffusion is the same for all components, independent of the molecular structure (including cholesterol (CHOL)), if they reside in the same domain or phase in the membrane. Furthermore, quite unexpectedly CHOL seems to partition into the l_dand l_o phases to roughly the same extent, indicating that CHOL has no strong preference for any of these phases, i.e. CHOL seems to have similar interactions with all of the lipids. We propose that the lateral phase separation in bilayers containing one high-T_m and one low-T_m lipid together with CHOL is driven by the increasing difficulty of incorporating an unsaturated or prenyl lipid into the highly ordered bilayer formed by a saturated lipid and CHOL, i.e. the phase transition is entropy driven to keep the disorder of the hydrocarbon chains of the unsaturated lipid.     

Strobilanthes crispus attenuates renal carcinogen, iron nitrilotriacetate (Fe-NTA)-mediated oxidative damage of lipids and DNA

This study was aimed to evaluate the effect of Strobilanthes crispus extract for possible protection against lipid peroxidation and DNA damage induced by iron nitrilotriacetate (Fe-NTA) and hydrogen peroxide (H₂O₂). Fe-NTA is a potent nephrotoxic agent and induces acute and subacute renal proximal tubular necrosis by catalyzing the decomposition of H₂O₂-derived production of hydroxyl radicals, which are known to cause lipid peroxidation and DNA damage. Incubation of postmitochondrial supernatant and/or calf thymus DNA with H₂O₂ (40 mM) in the presence of Fe-NTA (0.1 mM) induces lipid peroxidation and DNA damage to about 2.3-fold and 2.9-fold, respectively, as compared to control (P < 0.05). In lipid peroxidation protection studies, S. crispus treatment showed a dose-dependent inhibition (45–53% inhibition, P < 0.05) of Fe-NTA and H₂O₂ induced lipid peroxidation. Similarly, in DNA damage protection studies, S. crispus treatment also showed a dose-dependent inhibition (18–30% inhibition, P < 0.05) of DNA damage. In addition, the protection was closely related to the content of phenolic compounds as evident by S. crispus extract showing the value of 124.48 mg/g total phenolics expressed as gallic acid equivalent (GAE, mg/g of extract). From these studies, it is concluded that S. crispus inhibits peroxidation of membrane lipids and DNA damage induced by Fe-NTA and H₂O₂ and possesses the potential to be used to treat or prevent degenerative diseases where oxidative stress is implicated.     

The antioxidant action of ketoconazole and related azoles: comparison with tamoxifen and cholesterol

The azole antifungal drug ketoconazole was found to inhibit Fe(III)-ascorbate dependent lipid peroxidation using either rat liver microsomes or ox-brain phospholipid liposomes as the substrate. It also inhibited microsomal peroxidation induced by the Fe(III)-ADP/NADPH system. The related azoles, miconazole and clotrimazole, were much weaker inhibitors than ketoconazole. Ketoconazole was approximately equipotent with the triphenylethylene anticancer drug tamoxifen in the microsomal system and was almost as effective as 4-hydroxytamoxifen in the liposomal system. Ketoconazole introduced into phospholipid liposomes during their preparation inhibited Fe(III)-ascorbate induced lipid peroxidation to a greater extent than similarly introduced cholesterol, ergosterol or tamoxifen. Miconazole and clotrimazole were again poor inhibitors of lipid peroxidation in this system. These antioxidant effects of ketoconazole may be due to membrane stabilization in the systems used. The implications of our findings for the clinical applications of these drugs are discussed.     

Effect of dietary antioxidants and phenobarbital pretreatment on microsomal lipid peroxidation and activation by carbon tetrachloride.

The effect of the dietary antioxidants, vitamin E and selenium, and the effect of phenobarbital pretreatment on $\text{in}$$\text{vitro}$ NADPH-dependent microsomal lipid peroxidation and the activation of microsomal lipid peroxidation by CCl₄ were studied. The rate of microsomal lipid peroxidation decreased as a function of dietary anti-oxidant, while the degree of CCl₄ activation increased. Phenobarbital pretreatment diminished the antioxidant inhibition of microsomal lipid peroxidation found with microsomes from rats fed high levels of antioxidant. Phenobarbital pretreatment lowered the extent of lipid peroxidation as measured by malonaldehyde production but had little effect on the rate of lipid peroxidation as measured by oxygen uptake. The kinetics of lipid peroxidation and the stoichiometry of the reaction were assessed as a function of dietary antioxidant.The findings suggest that at low microsomal antioxidant concentrations, the lipid peroxidation reaction occurs at a maximal rate dependent upon some rate-limiting step, such as the reduction of Fe⁺³, which is unaffected by CCl₄ addition. Conversely, at high microsomal antioxidant concentrations, the antioxidant termination reactions appear to determine the overall reaction rate.     

Glucose promotes membrane cholesterol crystalline domain formation by lipid peroxidation

Oxidative damage to vascular cell membrane phospholipids causes physicochemical changes in membrane structure and lipid organization, contributing to atherogenesis. Oxidative stress combined with hyperglycemia has been shown to further increase the risk of vascular and metabolic diseases. In this study, the effects of glucose on oxidative stress-induced cholesterol domain formation were tested in model membranes containing polyunsaturated fatty acids and physiologic levels of cholesterol. Membrane structural changes, including cholesterol domain formation, were characterized by small angle X-ray scattering (SAXS) analysis and correlated with spectrophotometrically-determined lipid hydroperoxide levels. Glucose treatment resulted in a concentration-dependent increase in lipid hydroperoxide formation, which correlated with the formation of highly-ordered cholesterol crystalline domains (unit cell periodicity of 34 Å) as well as a decrease in overall membrane bilayer width. The effect of glucose on lipid peroxidation was further enhanced by increased levels of cholesterol. Treatment with free radical-scavenging agents inhibited the biochemical and structural effects of glucose, even at elevated cholesterol levels. These data demonstrate that glucose promotes changes in membrane organization, including cholesterol crystal formation, through lipid peroxidation.     

Lipid peroxidation in hemoglobin-containing liposomes. Effects of membrane phospholipid composition and cholesterol content

The effects of phospholipid-oxidation state and vesicle composition on lipid peroxidation in hemolysate-containing liposomes (hemosomes) were studied by the thiobarbituric acid assay. Liposomes (hemosomes) were prepared from egg phosphatidylcholine (PC) with either low (PC0.08) or high (PC0.66) oxidation indices reflecting low and high conjugated diene/lipid hydroperoxy contents. Thiobarbituric acid reactivity was negligible over 6 h at 38 degrees C in buffer-containing (control) liposomes prepared from PC0.08, whereas it was slightly increased in those prepared from PC0.66. Encapsulated hemolysate had no effect in PC0.08 liposomes, but significantly increased thiobarbituric acid reactivity in those prepared from PC0.66. Inclusion of either phosphatidylethanolamine or phosphatidylinositol in the membrane further increased lipid peroxidation in hemosomes prepared from PC0.66, whereas phosphatidic acid and phosphatidylserine were inhibitory. Inclusion of cholesterol in the membrane had no effect in PC0.66 hemosomes, but significantly inhibited lipid peroxidation in the presence of phosphatidylethanolamine or phosphatidylinositol. The effects of phosphatidic acid and cholesterol were dose-dependent. Co-incorporation of cholesterol and phosphatidic acid or phosphatidylserine in the membrane resulted in almost complete elimination of hemoglobin (Hb)-induced lipid peroxidation. Lysophosphatidic acid had similar effect as phosphatidic acid, whereas lysophosphatidylserine exerted inhibition only in the presence of phosphatidylethanolamine. The rate of lipid peroxidation showed no correlation with the amount of encapsulated Hb, neither with the oxidation indices nor the polyunsaturated fatty acid contents of negatively charged phospholipids. The above findings suggest a possible role for the high cholesterol content and preferential localization of phosphatidylserine in the inner bilayer leaflet of erythrocyte membrane in protecting against Hb-induced lipid peroxidation in the membrane.     

Could cholesterol bound to haemoglobin be a missing link for the occasional inverse relationship between superoxide dismutase and glutathione peroxidase activities?

The concept of an anti-oxidant defence system as a means to prevent oxidative cell damage implies balanced activities of anti-oxidant defence enzymes. As well as positive correlations between anti-oxidant enzyme activities in human erythrocytes, it has been observed that sometimes when glutathione peroxidase activity is increased, CuZn-superoxide dismutase activity is decreased. In our current study we have examined the plasma lipid profile and the anti-oxidant defence enzymes in erythrocytes from humans, pigs, and bulls. We found that a negative correlation existed between CuZn-superoxide dismutase and glutathione peroxidase activities in human erythrocytes when the concentrations of both plasma triglycerides and total cholesterol were high. This correlation was also found in pig erythrocytes, but not in bull erythrocytes. We propose that cholesterol could affect membrane lipid peroxidation and superoxide generation in erythrocytes via the recently found fraction of cholesterol bound to haemoglobin, termed haemoglobin-cholesterol.     

Cholesterol affects spectrin-phospholipid interactions in a manner different from changes resulting from alterations in membrane fluidity due to fatty acyl chain composition

We previously showed that erythrocyte and brain spectrins bind phospholipid vesicles and monolayers prepared from phosphatidylethanolamine and phosphatidylserine and their mixtures with phosphatidylcholine (Review: A.F. Sikorski, B. Hanus-Lorenz, A. Jezierski, A. R. Dluzewski, Interaction of membrane skeletal proteins with membrane lipid domain, Acta Biochim. Polon. 47 (2000) 565). Here, we show how changes in the fluidity of the phospholipid monolayer affect spectrin-phospholipid interaction. The presence of up to 10%-20% cholesterol in the PE/PC monolayer facilitates the penetration of the monolayer by both types of spectrin. For monolayers constructed from mixtures of PI/PC and cholesterol, the effect of spectrins was characterised by the presence of two maxima (at 5 and 30% cholesterol) of surface pressure for erythroid spectrin, and a single maximum (at 20% cholesterol) for brain spectrin. The binding assay results indicated a small but easily detectable decrease in the affinity of erythrocyte spectrin for FAT-liposomes prepared from a PE/PC mixture containing cholesterol, and a 2- to 5-fold increase in maximal binding capacity (B_max) depending on the cholesterol content. On the other hand, the results from experiments with a monolayer constructed from homogenous synthetic phospholipids indicated an increase in Δπ change with the increase in the fatty acyl chain length of the phospholipids used to prepare the monolayer. This was confirmed by the results of a pelleting experiment. Adding spectrins into the subphase of raft-like monolayers constructed from DOPC, SM and cholesterol (1/1/1) induced an increase in surface pressure. The Δπ change values were, however, much smaller than those observed in the case of a natural PE/PC (6/4) monolayer. An increased binding capacity for spectrins of liposomes prepared from a “raft-like” mixture of lipids could also be concluded from the pelleting assay. In conclusion, we suggest that the effect of membrane lipid fluidity on spectrin-phospholipid interactions is not simple but depends on how it is regulated, i.e., by cholesterol content or by the chemical structure of the membrane lipids.     

The influence of different membrane components on the electrical stability of bilayer lipid membranes

A good understanding of cell membrane properties is crucial for better controlled and reproducible experiments, particularly for cell electroporation where the mechanism of pore formation is not fully elucidated. In this article we study the influence on that process of several constituents found in natural membranes using bilayer lipid membranes. This is achieved by measuring the electroporation threshold (V_th) defined as the potential at which pores appear in the membrane. We start from highly stable 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) membranes (V_th ∼ 200 mV), and subsequently add therein other phospholipids, cholesterol and a channel protein. While the phospholipid composition has a slight effect (100 mV ≤ V_th ≤ 290 mV), cholesterol gives a concentration-dependent effect: a slight stabilization until 5% weight (V_th ∼ 250 mV) followed by a noticeable destabilization (V_th ∼ 100 mV at 20%). Interestingly, the presence of a model protein, α-hemolysin, dramatically disfavours membrane poration and V_th shows a 4-fold increase (∼ 800 mV) from a protein density in the membrane of 24 × 10^− 3 proteins/μm². In general, we find that pore formation is affected by the molecular organization (packing and ordering) in the membrane and by its thickness. We correlate the resulting changes in molecular interactions to theories on pore formation.     

Nonallergenic urushiol derivatives inhibit the oxidation of unilamellar vesicles and of rat plasma induced by various radical generators

Urushiols consist of an o-dihydroxybenzene (catechol) structure and an alkyl chain of 15 or 17 carbons in the 3-position of a benzene ring and are allergens found in the family Anacardiaceae. We synthesized various veratrole (1,2-dimethoxybenzene)-type and catechol-type urushiol derivatives that contained alkyl chains of various carbon atom lengths, including –H, –C₁H₃, –C₅H₁₁, –C₁₀H₂₁, –C₁₅H₃₁, and –C₂₀H₄₁, and investigated their contact hypersensitivities and antioxidative activities. 3-Decylcatechol and 3-pentadecylcatechol displayed contact hypersensitivity, but the other compounds did not induce an allergic reaction, when the ears of rats were sensitized by treatment with the compounds every day for 20 days. Catechol-type urushiol derivatives (CTUDs) exerted very high radical-scavenging activity on the 1,1-diphenyl-2-picrylhydrazyl radical and inhibited lipid peroxidation in a methyl linoleate solution induced by 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN). However, veratrole-type urushiol derivatives did not scavenge or inhibit lipid peroxidation. CTUDs also acted as effective inhibitors of lipid peroxidation of the egg yolk phosphatidylcholine large unilamellar vesicle (PC LUV) liposome system induced by various radical generators such as AMVN, 2,2′-azobis(2-amidino-propane) dihydrochloride, and copper ions, although their efficiencies differed slightly. In addition, CTUDs suppressed formation of cholesteryl ester hydroperoxides in rat blood plasma induced with copper ions. CTUDs containing more than five carbon atoms in the alkyl chain showed excellent lipophilicity in a n-octanol/water partition experiment. These compounds also exhibited high affinities to the liposome membrane using the ultrafiltration method of the PC LUV liposome system. Therefore, CTUDs seem to act as efficient antioxidative compounds against membranous lipid peroxidation owing to their localization in the phospholipid bilayer. These results suggest that nonallergenic CTUDs act as antioxidants to protect against oxidative damage of cellular and subcellular membranes.