Lipid Extracts Of Membranes Research Articles

Enterococcus faecalis Glycolipids Modulate Lipoprotein-Content of the Bacterial Cell Membrane and Host Immune Response.

In this study, we investigated the impact of the cell membrane composition of E. faecalis on its recognition by the host immune system. To this end, we employed an E. faecalis deletion mutant (ΔbgsA) that does not synthesize the major cell membrane glycolipid diglycosyl-diacylglycerol (DGlcDAG). Proteomic analysis revealed that 13 of a total of 21 upregulated surface-associated proteins of E. faecalis ΔbgsA were lipoproteins. This led to a total lipoprotein content in the cell membrane of 35.8% in ΔbgsA compared to only 9.4% in wild-type bacteria. Increased lipoprotein content strongly affected the recognition of ΔbgsA by mouse macrophages in vitro with an increased stimulation of TNF-α production by heat-fixed bacteria and secreted antigens. Inactivation of the prolipoprotein diacylglycerol transferase (lgt) in ΔbgsA abrogated TNF-α induction by a ΔbgsA_lgt double mutant indicating that lipoproteins mediate increased activation of mouse macrophages by ΔbgsA. Heat-fixed ΔbgsA bacteria, culture supernatant, or cell membrane lipid extract activated transfected HEK cells in a TLR2-dependent fashion; the same was not true of wild-type bacteria. In mice infected intraperitoneally with a sublethal dose of E. faecalis we observed a 70% greater mortality in mice infected with ΔbgsA compared with wild-type-infected mice. Increased mortality due to ΔbgsA infection was associated with elevated plasma levels of the inflammatory cytokines TNF-α, IL-6 and MIP-2. In summary, our results provide evidence that an E. faecalis mutant lacking its major bilayer forming glycolipid DGlcDAG upregulates lipoprotein expression leading to increased activation of the host innate immune system and virulence in vivo.

Monoclonal Antibodies Targeting LecLex-Related Glycans with Potent Antitumor Activity.

To produce antitumor monoclonal antibodies (mAbs) targeting glycans as they are aberrantly expressed in tumors and are coaccessory molecules for key survival pathways. Two mAbs (FG88.2 and FG88.7) recognizing novel tumor-associated Lewis (Le) glycans were produced by immunizations with plasma membrane lipid extracts of the COLO205 cell line. Glycan array analysis showed that both mAbs bound Le(c)Le(x), di-Le(a), and Le(a)Le(x), as well as Le(a)-containing glycans. These glycans are expressed on both lipids and proteins. Both mAbs showed strong tumor reactivity, binding to 71% (147 of 208) of colorectal, 81% (155 of 192) of pancreatic, 54% (52 of 96) of gastric, 23% (62 of 274) of non-small cell lung, and 31% (66 of 217) of ovarian tumor tissue in combination with a restricted normal tissue distribution. In colorectal cancer, high FG88 glyco-epitope expression was significantly associated with poor survival. The mAbs demonstrated excellent antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), in addition to direct tumor cell killing via a caspase-independent mechanism. Scanning electron microscopy revealed antibody-induced pore formation. In addition, the mAbs internalized, colocalized with lysosomes, and delivered saporin that killed cells with subnanomolar potency. In vivo, the mAbs demonstrated potent antitumor efficacy in a metastatic colorectal tumor model, leading to significant long-term survival. The mAbs direct and immune-assisted tumor cell killing, pan-tumor reactivity, and potent in vivo antitumor efficacy indicate their potential as therapeutic agents for the treatment of multiple solid tumors. In addition, internalization of saporin conjugates and associated tumor cell killing suggests their potential as antibody drug carriers.

Lipid Librations at the Interface with the Na,K-ATPase

Transitions between conformational substates of membrane proteins can be driven by torsional librations in the protein that may be coupled to librational fluctuations of the lipid chains. Here, librational motion of spin-labeled lipid chains in membranous Na,K-ATPase is investigated by spin-echo electron paramagnetic resonance. Lipids at the protein interface are targeted by using negatively charged spin-labeled fatty acids that display selectivity of interaction with the Na,K-ATPase. Echo-detected electron paramagnetic resonance spectra from native membranes are corrected for the contribution from the bilayer regions of the membrane by using spectra from dispersions of the extracted membrane lipids. Lipid librations at the protein interface have a flat profile with chain position, whereas librational fluctuations of the bilayer lipids increase pronouncedly from C-9 onward, then flatten off toward the terminal methyl end of the chains. This difference is accounted for by increased torsional amplitude at the chain ends in bilayers, while the amplitude remains restricted throughout the chain at the protein interface with a limited lengthening in correlation time. The temperature dependence of chain librations at the protein interface strongly resembles that of the spin-labeled protein side chains, suggesting solvent-mediated transitions in the protein are driven by fluctuations in the lipid environment.

Molecular Model for the Solubilization of Membranes into Nanodisks by Styrene Maleic Acid Copolymers

A recent discovery in membrane research is the ability of styrene-maleic acid (SMA) copolymers to solubilize membranes in the form of nanodisks allowing extraction and purification of membrane proteins from their native environment in a single detergent-free step. This has important implications for membrane research because it allows isolation as well as characterization of proteins and lipids in a near-native environment. Here, we aimed to unravel the molecular mode of action of SMA copolymers by performing systematic studies using model membranes of varying compositions and employing complementary biophysical approaches. We found that the SMA copolymer is a highly efficient membrane-solubilizing agent and that lipid bilayer properties such as fluidity, thickness, lateral pressure profile, and charge density all play distinct roles in the kinetics of solubilization. More specifically, relatively thin membranes, decreased lateral chain pressure, low charge density at the membrane surface, and increased salt concentration promote the speed and yield of vesicle solubilization. Experiments using a native membrane lipid extract showed that the SMA copolymer does not discriminate between different lipids and thus retains the native lipid composition in the solubilized particles. A model is proposed for the mode of action of SMA copolymers in which membrane solubilization is mainly driven by the hydrophobic effect and is further favored by physical properties of the polymer such as its relatively small cross-sectional area and rigid pendant groups. These results may be helpful for development of novel applications for this new type of solubilizing agent, and for optimization of the SMA technology for solubilization of the wide variety of cell membranes found in nature.

Multi-lamellar organization of fully deuterated lipid extracts of yeast membranes.

Neutron scattering studies on mimetic biomembranes are currently limited by the low availability of deuterated unsaturated lipid species. In the present work, results from the first neutron diffraction experiments on fully deuterated lipid extracts from the yeast Pichia pastoris are presented. The structural features of these fully deuterated lipid stacks are compared with those of their hydrogenous analogues and with other similar synthetic systems. The influence of temperature and humidity on the samples has been investigated by means of small momentum-transfer neutron diffraction. All of the lipid extracts investigated self-assemble into multi-lamellar stacks having different structural periodicities; the stacking distances are affected by temperature and humidity without altering the basic underlying arrangement. At high relative humidity the deuterated and hydrogenous samples are similar in their multi-lamellar arrangement, being characterized by two main periodicities of ∼75 and ∼110 Å reflecting the presence of a large number of polar phospholipid molecules. Larger differences are found at lower relative humidity, where hydrogenous lipids are characterized by a larger single lamellar structure than that observed in the deuterated samples. In both cases the heterogeneity in composition is reflected in a wide structural complexity. The different behaviour upon dehydration can be related to compositional differences in the molecular composition of the two samples, which is attributed to metabolic effects related to the use of perdeuterated growth media.

Water Penetration Profile at the Protein-Lipid Interface in Na,K-ATPase Membranes

The affinity of ionized fatty acids for the Na,K-ATPase is used to determine the transmembrane profile of water penetration at the protein-lipid interface. The standardized intensity of the electron spin echo envelope modulation (ESEEM) from 2H-hyperfine interaction with D2O is determined for stearic acid, n-SASL, spin-labeled systematically at the C-n atoms throughout the chain. In both native Na,K-ATPase membranes from shark salt gland and bilayers of the extracted membrane lipids, the D2O-ESEEM intensities of fully charged n-SASL decrease progressively with position down the fatty acid chain toward the terminal methyl group. Whereas the D2O intensities decrease sharply at the n = 9 position in the lipid bilayers, a much broader transition region in the range n = 6 to 10 is found with Na,K-ATPase membranes. Correction for the bilayer population in the membranes yields the intrinsic D2O-intensity profile at the protein-lipid interface. For positions at either end of the chains, the D2O concentrations at the protein interface are greater than in the lipid bilayer, and the positional profile is much broader. This reveals the higher polarity, and consequently higher intramembrane water concentration, at the protein-lipid interface. In particular, there is a significant water concentration adjacent to the protein at the membrane midplane, unlike the situation in the bilayer regions of this cholesterol-rich membrane. Experiments with protonated fatty acid and phosphatidylcholine spin labels, both of which have a considerably lower affinity for the Na,K-ATPase, confirm these results.

The interaction of aurein 2.5 with fungal membranes.

Aurein 2.5 (GLFDIVKKVVGAFGSL-NH2) is an antimicrobial peptide, which was seen to have activity against Stachybotris chartarum, Penicillium roseopurpureum and Aspergillus flavus with minimum fungicidal concentrations in the range 250-500 μM. S. chartarum showed enhanced susceptibility to lysis as compared to P. roseopurpureum and A. flavus, (44, 26 and 28 % respectively). Monolayers formed from lipid membrane extracts derived from S. chartarum, P. roseopurpureum and A. flavus showed maximal surface pressure changes of 13.5, 10.3 and 10.2 mN m(-1) respectively. However, aurein 2.5 adopted similar levels of α-helical structure (circa 45 %) in the presence of vesicles formed from membrane lipid extracts derived from all three fungi. These data imply that differential activity is not due to targeting and membrane association but linked to the ability of the bound peptide to lyse the cells. At sterol levels mimetic of eukaryotic systems, high levels of α-helical structure (circa 50 %) were also observed and hence similar binding. However, enhanced sterol levels (>0.6) led to a reduction in monolayer membrane interaction, suggesting that the sterols influence efficacy. Consistent with this suggestion, thermodynamic analysis showed that the peptide was able to destabilise model fungal monolayers, as indicated by negative values of ∆Gmix.

Characterization of Thermoplasma Species Cultured from Sampling on Tangkuban Perahu, Indonesia

Archaea is an organisme with unique feature because of its ability to inhabit an extremophyle conditions. Our expeditions to Tangkuban Perahu, West Java aimed to obtain archaealstrains from the solfatara fields located in Domas crater. From the samples, we intended to culture Thermoplasma species growing around 55 °C below pH 2, which until now have not yet been fully characterized. We collected five samples from mud holes with temperatures from 52 °C to 57 °C and pH below 2. In serial cultures of up to 8 transfers in Freundt’s medium we grew tetraetherlipid synthesizing Thermoplasma species as documented by phase contrast microscope. Total membrane lipid extracts were analysed by thin layer chromatography; the pattern matched total lipid extracts from Thermoplasma acidophilum DSM 1728 membranes. For confirmation, 16S rDNA identification performing PCR and sequencing were carried-out. Analysis using BLAST showed T. acidophilum identities as the highest similarity of 99%, followed by T. volcanium, also with99% similarity (ANKF776908 and ANKF776909). This is the first report of culturing cell-wall-less thermoacidophilicarchaea,in particular Thermoplasma species in Indonesian laboratories.

Intake of Xylooligosaccharides Alters the Structural Organization of Liver Plasma Membrane Bilayer

Xylooligosaccharides (XOS) are non-digestible carbohydrate prebiotics that beneficially affect the host by selective stimulation of specific bacteria in the gastro-intestinal tract. The impact of XOS on gastrointestinal microflora and blood lipids is well known but the exact mechanism of action on liver membranes is still unclear. The organization of membrane lipids in domains is known to be important for the proper functioning of various receptors and mechanisms triggering cell signaling. In this study the influence of XOS-enriched diet on the lipid bilayer structure of rat liver plasma membrane was investigated. XOS intake caused a slight decrease of the fluidity of lipid extracts from liver plasma membranes compared to the controls. This observation was based on the increased generalized polarization (GP) and blue shifted emission spectra of Laurdan. The elevated amount of membrane sphingomyelin may be one possible reason for the reported effects. The micron-scale phase separation of the lipid extracts was also investigated by fluorescence microscopy. A different temperature of phase separation and domain pattern was observed in plasma membrane lipid extracts from XOS-fed animals. We presume that it could be assigned to the altered lipid composition of the membrane bilayer, in particular to the changes in the sphingomyelin/cholesterol ratio. All observed alterations are discussed in the light of the impact of XOS on human health and physiology.

The role of membrane fatty acid remodeling in the antitumor mechanism of action of 2-hydroxyoleic acid

The synthetic fatty acid 2-hydroxyoleic acid (2OHOA) is a potent antitumor drug that we rationally designed to regulate the membrane lipid composition and structure. The lipid modifications caused by 2OHOA treatments induce important signaling changes that end up with cell death (Terés et al., 2012 [1]). One of these regulatory effects is restoration of sphingomyelin levels, which are markedly lower in cancer cells compared to normal cells (Barceló‐Coblijn et al., 2011 [2]). In this study, we report another important regulatory effect of 2OHOA on cancer cell membrane composition: a large increase in 2OHOA levels, accounting for ~15% of the fatty acids present in membrane phospholipids, in human glioma (SF767 and U118) and lung cancer (A549) cells. Concomitantly, we observed marked reductions in oleic acid levels and inhibition of stearoyl-CoA desaturase. The impact of these changes on the biophysical properties of the lipid bilayer was evaluated in liposomes reconstituted from cancer cell membrane lipid extracts. Thus, 2OHOA increased the packing of ordered domains and decreased the global order of the membrane. The present results further support and extend the knowledge about the mechanism of action for 2OHOA, based on the regulation of the membrane lipid composition and structure and subsequent modulation of membrane protein-associated signaling.

Phosphatidylcholine Transfer Protein Attenuates High Fat Diet Induced Changes in Mitochondrial Membrane Phosphatidylcholine Composition

Phosphatidylcholine transfer protein (PC‐TP) is a phospholipid binding protein that is enriched in liver and catalyzes intermembrane exchange of phosphatidylcholines (PCs) in vitro. Because it associates with the mitochondria, we explored the hypothesis that PC‐TP regulates the distribution of PC molecular species in mitochondrial membranes. Pctp−/− and Pctp+/+ mice were fed chow or high fat diets. Liver mitochondria were purified and membrane lipid extracts were analyzed by tandem mass spectrometry. The distribution of PC species was determined by precursor ion scanning of m/z 184, which corresponds to the headgroup. In chow fed mice, PC‐TP expression did not influence the distribution of PCs. High fat fed Pctp+/+ mice exhibited a modest 21% decrease in mitochondrial 16:0–18:2 PC and a 27% increase in 16:0–20:4 PC. By contrast, high fat feeding of Pctp−/− mice led to more pronounced changes in the same PCs, with a 43% decrease in 16:0–18:2 PC and 34% increase in 16:0–20:4 PC. We conclude that under physiological conditions, PC‐TP expression is not a determinant of the PC composition of mitochondrial membranes. In response to dietary fat, PC‐TP expression mitigates the enrichment of mitochondria with polyunsaturated PCs, which may modulate biophysical properties of the membrane. Supported by NIH.

Evaluation of ion activation strategies and mechanisms for the gas-phase fragmentation of sulfoquinovosyldiacylglycerol lipids from Rhodobacter sphaeroides

Sulfoquinovosyldiacylglycerol (SQDG) lipids, found in plants and photosynthetic bacteria, can substitute for phospholipids under phosphate limiting conditions. Here, various low-energy ion activation strategies have been evaluated for the identification and characterization of deprotonated SQDG lipids from a crude membrane lipid extract of Rhodobacter sphaeroides, using collision-induced dissociation-tandem mass spectrometry (CID-MS/MS) in either a triple quadrupole mass spectrometer or in a hybrid quadrupole ion trap-multipole mass spectrometer coupled with high resolution/accurate mass analysis capabilities. In the triple quadrupole instrument, using energy resolved CID-MS/MS experiments, the SQDG head group specific product ion at m/z 225 (C6H9O7S−), rather than m/z 81 (SO3H−), was determined to provide the greatest sensitivity for SQDG lipid detection, and is therefore the preferred ‘fingerprint’ ion for the identification of this lipid class from within complex lipid mixtures when using precursor ion scan mode MS/MS experiments. A comparison of conventional ion trap CID-MS/MS and -MSn, with ‘low Q’ CID-MS/MS, pulsed Q dissociation (PQD)-MS/MS and higher energy collision induced dissociation (HCD)-MS/MS performed in an LTQ Orbitrap Velos mass spectrometer, revealed that HCD-MS/MS coupled with high resolution/accurate mass analysis represents the most sensitive, and perhaps most importantly the most specific strategy, for ion trap based identification and characterization of SQDG lipids, due to the ability to readily distinguish the SQDG head group specific product ion at m/z 225.0069 from other products that may be present at the same nominal m/z value. Finally, the mechanisms responsible for formation of each of the major product ions observed by low-energy CID-MS/MS of deprotonated SQDG lipids were elucidated using uniform H/D exchange, HCD-MS/MS and high resolution mass analysis. Formation of the m/z 225 ‘fingerprint’ ion occurs via a charge-remote cis-elimination reaction, likely involving transfer of a hydrogen from the hydroxyl group located on the C2 position of the sugar ring.

Sphingomyelin content of erythrocyte membranes is increased in patients with acute coronary syndrome

ObjectiveIntraplaque haemorrhage-mediated accumulation of erythrocytes has been recognised as a novel contributor in lipid core formation and unstable atherosclerotic plaques. We hypothesized that sphingomyelin content is increased in the erythrocytes...

Studies on Lactoferricin-derived Escherichia coli Membrane-active Peptides Reveal Differences in the Mechanism of N-Acylated Versus Nonacylated Peptides

To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis.

Structural organization of plasma membrane lipids isolated from cells cultured as a monolayer and in tissue-like conditions

Complementary biophysical approaches were used to study the structural organization of plasma membrane lipids obtained from fibroblasts cultured as two-dimensional (2D) monolayer and in tissue-like three-dimensional (3D) conditions. Fluorescence microscopy experiments demonstrated different domain patterns for 2D and 3D plasma membrane lipid extracts. ESR demonstrated that 3D lipid extract is characterized with lower order parameter than 2D in the deep hydrophobic core of the lipid bilayer. Higher cholesterol and sphingomyelin content in 3D extract, known to increase the order in the glycerophospholipid matrix, was not able to compensate higher fatty acid polyunsaturation of the phospholipids. The interfacial region of the bilayer was probed by the fluorescent probe Laurdan. A higher general polarization value for 3D extract was measured. It is assigned to the increased content of sphingomyelin, cholesterol, phosphatidylethanolamine and phosphatidylserine in the 3D membranes. These results demonstrate that cells cultured under different conditions exhibit compositional heterogeneity of the constituent lipids which determine different structural organization of the membranes.

Inclusion of Menaquinone in Lipid Membranes Decreases Susceptibility to Antimicrobial Peptides

Most studies on the interaction of antimicrobial peptides with lipid bilayers have used unsaturated, fluid-state phospholipids to model bacterial membranes. However, unsaturated lipids are rarely found in cell membranes of gram-(+) bacteria, including Staphylococcus aureus. To maintain cell membrane function, S. aureus uses fully saturated but terminally branched lipid acyl chains, which have a significantly lower phase transition temperature than their linear counterparts. We found that lipid vesicles composed of synthetic branched phospholipids are much more susceptible to attack by an amphipathic peptide than lipid vesicles composed of unsaturated phospholipids with the same headgroup composition. By contrast, natural membrane lipid extracts from S. aureus produce lipid vesicles that are as resistant to membrane-active peptides as those produced using synthetic, unsaturated lipids. We postulated that an unidentified component present in the S. aureus lipid extracts serves to stabilize the bacterial cytoplasmic membrane. A possible candidate is menaquinone, which participates in the bacterial electron transport chain but could, in addition, have a structural effect on the lipid bilayer. In the work presented here, we investigated the kinetics of dye efflux from lipid vesicles containing between 1 and 5 mol% menaquinone, induced by the antimicrobial peptide PMAP-23. PMAP-23 is a 23 amino acid, linear peptide of the cathelicidin family with the sequence RIIDLLWRVRRPQKPKFVTVWVR. We found that the presence of physiological concentrations of menaquinone had a notable effect on peptide-induce dye efflux.

Gel Domains in the Plasma Membrane of Saccharomyces cerevisiae: HIGHLY ORDERED, ERGOSTEROL-FREE, AND SPHINGOLIPID-ENRICHED LIPID RAFTS

The plasma membrane of Saccharomyces cerevisiae was studied using the probes trans-parinaric acid and diphenylhexatriene. Diphenylhexatriene anisotropy is a good reporter of global membrane order. The fluorescence lifetimes of trans-parinaric acid are particularly sensitive to the presence and nature of ordered domains, but thus far they have not been measured in yeast cells. A long lifetime typical of the gel phase (>30 ns) was found in wild-type (WT) cells from two different genetic backgrounds, at 24 and 30 °C, providing the first direct evidence for the presence of gel domains in living cells. To understand their nature and location, the study of WT cells was extended to spheroplasts, the isolated plasma membrane, and liposomes from total lipid and plasma membrane lipid extracts (with or without ergosterol extraction by cyclodextrin). It is concluded that the plasma membrane is mostly constituted by ordered domains and that the gel domains found in living cells are predominantly at the plasma membrane and are formed by lipids. To understand their composition, strains with mutations in sphingolipid and ergosterol metabolism and in the glycosylphosphatidylinositol anchor remodeling pathway were also studied. The results strongly indicate that the gel domains are not ergosterol-enriched lipid rafts; they are mainly composed of sphingolipids, possibly inositol phosphorylceramide, and contain glycosylphosphatidylinositol-anchored proteins, suggesting an important role in membrane traffic and signaling, and interactions with the cell wall. The abundance of the sphingolipid-enriched gel domains was inversely related to the cellular membrane system global order, suggesting their involvement in the regulation of membrane properties.

Haemoglobin-induced oxidative stress is associated with both endogenous peroxidase activity and H2O2 generation from polyunsaturated fatty acids

Patients with increased haemolytic haemoglobin (Hb) have 10–20-times greater incidence of cardiovascular mortality. The objective of this study was to evaluate the role of Hb peroxidase activity in LDL oxidation. The role of Hb in lipid peroxidation, H2O2 generation and intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was assessed using NaN3, a peroxidase inhibitor, catalase, a H2O2 decomposing enzyme and human umbilical vein endothelial cells (HUVECs), respectively. Hb induced H2O2 production by reacting with LDL, linoleate and cell membrane lipid extracts. Hb-induced LDL oxidation was inhibited by NaN3 and catalase. Furthermore, Hb stimulated ICAM-1 and VCAM-1 expression, which was inhibited by the antioxidant, probucol. Thus, the present study suggests that the peroxidase activity of Hb produces atherogenic, oxidized LDL and oxidized polyunsaturated fatty acids (PUFAs) in the cell membrane and reactive oxygen species (ROS) formation mediated Hb-induced ICAM-1 and VCAM-1 expression.

Noninvasive Measurements of Integrin Microclustering under Altered Membrane Cholesterol Levels

Reported herein is a method that can be used to study the role of cholesterol in the microclustering of a ubiquitous class of membrane receptors, termed integrins. Integrin microclustering was measured using a fluorescence resonance energy transfer assay that does not require direct attachment of fluorescent donors or acceptors onto the integrins, and thus minimizes unwanted perturbations to integrin clustering. Membrane cholesterol levels were reduced using methyl-β-cyclodextrin (mβCD), as confirmed by Amplex Red assays of total cellular lipid or plasma membrane lipid extract. Subsequent changes in integrin microclustering were measured in cells expressing wild-type (WT) or mutant integrins. Although less integrin microclustering was measured after 27% membrane cholesterol depletion in a cell line expressing WT integrins, there was no statistically significant change for cells expressing α-cytoplasmic integrin mutants after a 45% reduction in plasma membrane cholesterol, and a significant increase in clustering for cells expressing ligand-binding domain integrin mutants after a 57% decrease in membrane cholesterol. These results are explained by differences in WT and mutant integrin partitioning into lipid nanodomains. Restoration of original cholesterol levels was used to confirm that the measured changes in membrane properties were cholesterol-dependent. No correlations between lipid diffusion and integrin microclustering were measured by means of fluorescence recovery after photobleaching using a fluorescent lipid mimetic. Similar lipid diffusion coefficients were measured after cholesterol depletion, irrespective of the integrins being expressed.

Hemoglobin induced oxidative stress is associated with both endogenous peroxidase activity and H2O2 generation from polyunsaturated fatty acids

Patients with increased hemolytic hemoglobin (Hb) have 10–20 times greater incidence of cardiovascular mortality than the general population. In the presence of an exogenous source of H2O2, Hb is essential and responsible for the oxidation of low‐density lipoproteins (LDL). However, the role of Hb peroxidase activity in LDL oxidation remains elusive. Our study indicated that Hb could induce the formation of H2O2 by reacting with LDL, linoleic acid, and cell membrane lipid extracts. In the presence of NaN3 (a peroxidase inhibitor), endogenous peroxidase activity and subsequent Hb‐induced LDL oxidation was significantly inhibited and H2O2 concentrations accumulated. Furthermore, Hb was shown to dramatically stimulate the expression of intercellular adhesion molecule‐1 (ICAM‐1) and vascular cell adhesion molecule‐1 (VCAM‐1) in both a concentration‐ and time‐ dependent manner. Nevertheless, this stimulation was completely inhibited by a potent antioxidant, probucol. We found that 2, 2′azo‐bis‐(2‐amindinopropane) hydrochloride (AAPH) could also induce LDL oxidation and stimulate the expression of ICAM‐1 and VCAM‐1, but was not inhibited by NaN3. In conclusion, the present study suggests that the oxidative effect of Hb to produce atherogenic oxidized LDL and to induce the production of ICAM‐1 and VCAM‐1 are due to H2O2 formation as well as its endogenous peroxidase activity toward polyunsaturated fatty acids. The biochemical and physiologic significance of this study are discussed.