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BBA - Biomembranes (v.1768, #6)

Editorial Board (pp. ii).

Use of cyclodextrins to manipulate plasma membrane cholesterol content: Evidence, misconceptions and control strategies by Raphael Zidovetzki; Irena Levitan (pp. 1311-1324).

The physiological importance of cholesterol in the cell plasma membrane has attracted increased attention in recent years. Consequently, the use of methods of controlled manipulation of membrane cholesterol content has also increased sharply, especially as a method of studying putative cholesterol-enriched cell membrane domains (rafts). The most common means of modifying the cholesterol content of cell membranes is the incubation of cells or model membranes with cyclodextrins, a family of compounds, which, due to the presence of relatively hydrophobic cavity, can be used to extract cholesterol from cell membranes. However, the mechanism of this activity of cyclodextrins is not completely established. Moreover, under conditions commonly used for cholesterol extraction, cyclodextrins may remove cholesterol from both raft and non-raft domains of the membrane as well as alter the distribution of cholesterol between plasma and intracellular membranes. In addition, other hydrophobic molecules such as phospholipids may also be extracted from the membranes by cyclodextrins. We review the evidence for the specific and non-specific effects of cyclodextrins and what is known about the mechanisms for cyclodextrin-induced cholesterol and phospholipid extraction. Finally, we discuss useful control strategies that may help to verify that the observed effects are due specifically to cyclodextrin-induced changes in cellular cholesterol.

Keywords: Membrane cholesterol; Membrane rafts; Cyclodextrin

Transport and transporters in the endoplasmic reticulum by Miklós Csala; Paola Marcolongo; Beáta Lizák; Silvia Senesi; Éva Margittai; Rosella Fulceri; Judit É. Magyar; Angelo Benedetti; Gábor Bánhegyi (pp. 1325-1341).

Enzyme activities localized in the luminal compartment of the endoplasmic reticulum are integrated into the cellular metabolism by transmembrane fluxes of their substrates, products and/or cofactors. Most compounds involved are bulky, polar or even charged; hence, they cannot be expected to diffuse through lipid bilayers. Accordingly, transport processes investigated so far have been found protein-mediated. The selective and often rate-limiting transport processes greatly influence the activity, kinetic features and substrate specificity of the corresponding luminal enzymes. Therefore, the phenomenological characterization of endoplasmic reticulum transport contributes largely to the understanding of the metabolic functions of this organelle. Attempts to identify the transporter proteins have only been successful in a few cases, but recent development in molecular biology promises a better progress in this field.

Keywords: Abbreviations; ER; endoplasmic reticulum; SER; smooth ER; RER; rough ER; EST; expressed sequence tags; DIDS; 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid; NEM; N-ethylmaleimide; G6Pase; glucose 6-phosphatase; PDI; protein disulfide isomerase; G6P; glucose 6-phosphate; H6PDH; hexose 6-phosphate dehydrogenase; 11β-HSD1; 11β-hydroxysteroid dehydrogenase type 1; GSD1; glycogen storage disease type 1; G6PT; G6P translocase; vG6PT; variant G6P translocase; SR; sarcoplasmic reticulum; NaPi or NPT; sodium/phosphate transporter; UDP; uridine diphosphate; UDP-Glc; UDP-glucose; UDP-GlcNAc; UDP-N-acetylglucosamine; UDP-Gal; UDP-galactose; UDP-GalNAc; UDP-N-acetylgalactosamine; UDP-Xyl; UDP-xylose; NSTs; nucleotide sugar transporters; SLC35; solute carrier family 35; Glc; glucose Man, mannose; GlcNAc; N-acetylglucosamine; UGGT; UDP-Glc glycoprotein glucosyltransferase; UGTrel1 (SLC35B1); UDP-Gal transporter related protein 1; AtUTr1; Arabidopsis thaliana; UDP-Gal/UDP-Glc transporter; UDP-GlcA; UDP-glucuronic acid; SITS; 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid; DEPC; diethyl pyrocarbonate; UGTrel7 (SLC35D1); UDP-Gal transporter related protein 7; UGT; UDP-glucuronosyltransferase; Ero1p; endoplasmic reticulum oxidoreductin 1 protein; Erv1p; essential for respiration and viability 1 protein; Fmo1p; flavin-containing monooxygenase; Flc; flavin carrier; CoA; coenzyme-A; Ac-CoA; acetyl-CoA; AT-1; Ac-CoA transporter; STS; steroid sulfatase; ARSC; arylsulfatase C; ES; estrone sulfatase; GSH; glutathione; GSSG; glutathione disulfide; RyR1; ryanodine receptor type 1; MHC; major histocompatibility complex; TAP; transporter associated with antigen processing; HEPES; 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acidNucleotide sugar transporters; Solute carrier family 35; Glucose 6-phosphate translocase; TAP; Translocon; Permeability

Transport capabilities of eleven gram-positive bacteria: Comparative genomic analyses by Graciela L. Lorca; Ravi D. Barabote; Vladimir Zlotopolski; Can Tran; Brit Winnen; Rikki N. Hvorup; Aaron J. Stonestrom; Elizabeth Nguyen; Li-Wen Huang; David S. Kim; Milton H. Saier Jr. (pp. 1342-1366).

The genomes of eleven Gram-positive bacteria that are important for human health and the food industry, nine low G+C lactic acid bacteria and two high G+C Gram-positive organisms, were analyzed for their complement of genes encoding transport proteins. Thirteen to 18% of their genes encode transport proteins, larger percentages than observed for most other bacteria. All of these bacteria possess channel proteins, some of which probably function to relieve osmotic stress. Amino acid uptake systems predominate over sugar and peptide cation symporters, and of the sugar uptake porters, those specific for oligosaccharides and glycosides often outnumber those for free sugars. About 10% of the total transport proteins are constituents of putative multidrug efflux pumps with Major Facilitator Superfamily (MFS)-type pumps (55%) being more prevalent than ATP-binding cassette (ABC)-type pumps (33%), which, however, usually greatly outnumber all other types. An exception to this generalization is Streptococcus thermophilus with 54% of its drug efflux pumps belonging to the ABC superfamily and 23% belonging each to the Multidrug/Oligosaccharide/Polysaccharide (MOP) superfamily and the MFS. These bacteria also display peptide efflux pumps that may function in intercellular signalling, and macromolecular efflux pumps, many of predictable specificities. Most of the bacteria analyzed have no pmf-coupled or transmembrane flow electron carriers. The one exception is Brevibacterium linens, which in addition to these carriers, also has transporters of several families not represented in the other ten bacteria examined. Comparisons with the genomes of organisms from other bacterial kingdoms revealed that lactic acid bacteria possess distinctive proportions of recognized transporter types (e.g., more porters specific for glycosides than reducing sugars). Some homologues of transporters identified had previously been identified only in Gram-negative bacteria or in eukaryotes. Our studies reveal unique characteristics of the lactic acid bacteria such as the universal presence of genes encoding mechanosensitive channels, competence systems and large numbers of sugar transporters of the phosphotransferase system. The analyses lead to important physiological predictions regarding the preferred signalling and metabolic activities of these industrially important bacteria.

Keywords: Lactic acid bacteria; Transport proteins; Genomic analyses; Energetics

Influence of poly(ethylene glycol) grafting density and polymer length on liposomes: Relating plasma circulation lifetimes to protein binding by Nancy Dos Santos; Christine Allen; Anne-Marie Doppen; Malathi Anantha; Kelly A.K. Cox; Ryan C. Gallagher; Goran Karlsson; Katarina Edwards; Gail Kenner; Lacey Samuels; Murray S. Webb; Marcel B. Bally (pp. 1367-1377).

The incorporation of poly(ethylene glycol) (PEG)-conjugated lipids in lipid-based carriers substantially prolongs the circulation lifetime of liposomes. However, the mechanism(s) by which PEG-lipids achieve this have not been fully elucidated. It is believed that PEG-lipids mediate steric stabilization, ultimately reducing surface-surface interactions including the aggregation of liposomes and/or adsorption of plasma proteins. The purpose of the studies described here was to compare the effects of PEG-lipid incorporation in liposomes on protein binding, liposome-liposome aggregation and pharmacokinetics in mice. Cholesterol-free liposomes were chosen because of their increasing importance as liposomal delivery systems and their marked sensitivity to protein binding and aggregation. Specifically, liposomes containing various molecular weight PEG-lipids at a variety of molar proportions were analyzed for in vivo clearance, aggregation state (size exclusion chromatography, quasi-elastic light scattering, cryo-transmission and freeze fracture electron microscopy) as well as in vitro and in vivo protein binding. The results indicated that as little as 0.5 mol% of 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE) modified with PEG having a mean molecular weight of 2000 (DSPE-PEG₂₀₀₀) substantially increased plasma circulation longevity of liposomes prepared of 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). Optimal plasma circulation lifetimes could be achieved with 2 mol% DSPE-PEG₂₀₀₀. At this proportion of DSPE-PEG₂₀₀₀, the aggregation of DSPC-based liposomes was completely precluded. However, the total protein adsorption and the protein profile was not influenced by the level of DSPE-PEG₂₀₀₀ in the membrane. These studies suggest that PEG-lipids reduce the in vivo clearance of cholesterol-free liposomal formulations primarily by inhibition of surface interactions, particularly liposome-liposome aggregation.

Keywords: Abbreviations; ANOVA; analysis of variance; AUC; area-under-the-curve; BCA; bicinchoninic acid; CH; cholesterol; CHE; cholesteryl hexadecyl ether; DDP; didodecylphosphate; DOPE; 1,2-dioleoyl-; sn; -glycero-3-phosphatidylethanolamine; DSPC; 1,2-distearoyl-; sn; -glycero-3-phosphatidylcholine; DSPE; 1,2-distearoyl-; sn; -glycero-3-phosphatidylethanol-amine; ³; [H]; tritium radiolabel; HEPES; N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]; HBS; HEPES buffered saline, pH 7.4; LUV; large unilamellar vesicle; MPS; mononuclear phagocytic system; PAGE; polyacrylamide gel electrophoresis; P; _B; protein binding (μmol protein/μmol lipid); PC; phosphatidylcholine; PE; phosphatidylethanolamine; PEG; poly(ethylene glycol); PK; pharmacokinetic; QELS; quasielastic light scattering; SDS; sodium dodecyl sulphate; T; c; phase transition temperatureCholesterol-free; Liposomes; PEG; Protein binding; Plasma elimination

Modulation of succinate transport in Hep G2 cell line by PKC by Piyarat Srisawang; Atip Chatsudthipong; Varanuj Chatsudthipong (pp. 1378-1388).

The cellular uptake of the tricarboxylic acid cycle (TCA) intermediates is very important for cellular metabolism. However, the transport pathways for these intermediates in liver cells are not well characterized. We have examined the transport of succinate and citrate in the human hepatoma cell line Hep G2 and found that it exhibited a higher rate of succinate compared to citrate transport, which was sodium dependent. Comparison of the transport properties of Hep G2 to that of human retinal pigment epithelial (HRPE) cells transfected with human sodium dicarboxylate transporters, hNaDC-1, hNaDC-3, and hNaCT indicated that Hep G2 cells express a combination of hNaDC-3 and hNaCT. Short period activation of protein kinase C (PKC) by phorbol 12-myristate, 13-acetate (PMA) and α-adrenergic receptor agonist, phenylephrine (PE), downregulated sodium-dependent succinate transport presumably via hNaDC-3. The inhibition by PMA was partially prevented by cytochalasin D, suggesting that PKC reduces the hNaDC-3 activity, at least in part, by increased endocytosis. In contrast, activation of PKA by both forskolin and epidermal growth factor (EGF) had no effect on succinate transport. Our results suggest that Hep G2 cells provide a useful model for studies of di- and tricarboxylate regulation of human liver.

Keywords: Succinate; Citrate; Hep G2; Dicarboxylate; Protein kinase C; Protein kinase A

The binding of advanced glycation end products to cell surfaces can be measured using bead-reconstituted cellular membrane proteins by Annett Schmitt; Joachim Nöller; Johannes Schmitt (pp. 1389-1399).

Advanced glycation end products (AGEs) that arise from the reaction of sugars with protein side chains are supposed to be involved in the pathogenesis of several diseases and therefore the effects of AGEs on cells are the objective of numerous investigations. Although different cellular responses to AGEs can be measured in cell culture studies, knowledge about the nature of AGE-binding and the involved cell surface receptors is poor. The measurement of AGE-binding to cell surfaces bears the potential to gain a deeper understanding about the nature of AGE-binding to cell surface proteins and could be applied as a preliminary test before performing cell culture studies on AGE effects. Herein, a new material and method for the detection of AGE-binding to cell surfaces is introduced, which has the potential to facilitate the detection of binding. In the present paper, the detection of AGE-binding to cell surface proteins using an artificial system of cellular membrane proteins reconstituted on beads (TRANSIL CaCo-2) is described. The binding of a BSA-AGE derived from a 37 °C incubation with 500 mM Glc (BSA-Glc 500) and the corresponding control to this artificial system was compared with the binding to intact cells and was found to be in good agreement. Additionally, the K_d for the binding of the BSA-Glc 500 used in the study to CaCo-2 surfaces was determined using FITC-labelled samples in a flow cytometric approach. Competitive binding studies were performed using a set of non-labelled BSA-AGEs to compete with FITC-labelled BSA-Glc 500 for the cell surface binding sites. The binding was found to be inhibited to different extends, virtually depending on the degree of arginine modifications within the modified protein used for competition. Additionally, the effects of all AGEs used in the study on CaCo-2 cells was measured using the detection of reactive oxygen species (ROS), which are known to be induced as a primary result of AGE-receptor binding. The induction of ROS was found to linearly correlate to the capacity of the individual AGE to displace FITC-labelled BSA-Glc 500 in competitive binding studies. Therefore, the data indicate, that at least in case of CaCo-2 cells the detection of cell surface binding can serve as a reliable preliminary test for a potential cell-damaging effect of AGEs.

Keywords: Abbreviations; AGEs; advanced glycation end products; DMEM; Dubelco's modified Eagle's medium; Egg PC; egg yolk phosphatidylcholine; FCS; foetal calf serum; FITC; fluorescein isothiocyanate; Glc; glucose; H; ₂; DCFDA; dichlorodihydrofluorescein diacetate; MALDI-TOF MS; matrix-assisted laser desorption ionization time-of-flight mass spectrometry; MG; methyl glyoxal; RAGE; receptor for AGEs; RFU; relative fluorescence units; ROS; reactive oxygen speciesAdvanced glycation end products; Receptor for AGEs; Binding constant; Induction of reactive oxygen species; ROS

Fungicidal effect of pleurocidin by membrane-active mechanism and design of enantiomeric analogue for proteolytic resistance by Hyun Jun Jung; Yoonkyung Park; Woo Sang Sung; Bo Kyoung Suh; Juneyoung Lee; Kyung-Soo Hahm; Dong Gun Lee (pp. 1400-1405).

Pleurocidin (Ple) is a 25-residue peptide which is derived from the skin mucous secretion of the winter flounder ( Pleuronectes americanus). In this study, we investigated antifungal effects and its mode of action of Ple on human pathogenic fungi. Ple showed potent antifungal activity with low hemolytic activity. To investigate the antifungal mechanisms of Ple, the cellular localization and membrane interaction of Ple were examined. Protoplast regeneration and membrane-disrupting activity by DPH-labeled membrane support the idea, that Ple exerts fungicidal activity against the human pathogenic fungus Candida albicans with the disruption of a plasma membrane. To aim for which was the application of a therapeutic agent, we designed a synthetic enantiomeric peptide composed of all-d-amino acids to enhance proteolytic resistance. The synthetic all-d-Ple also displayed two-fold more potent antifungal activity than that of all-l-Ple, and its antifungal activity showed proteolytic resistance against various proteases. Therefore, these results suggest a therapeutic potential of all-d-Ple with regard to its proteolytic resistance against human fungal infections.

Keywords: Pleurocidin; Antimicrobial peptide; Fungicidal activity; Membrane disrupting activity; Protease resistance

Selectivity signatures of three isoforms of recombinant T-type Ca²⁺ channels by Aleksandr Shcheglovitov; Platon Kostyuk; Yaroslav Shuba (pp. 1406-1419).

Voltage-gated Ca²⁺ channels (VGCCs) are recognized for their superb ability for the preferred passage of Ca²⁺ over any other more abundant cation present in the physiological saline. Most of our knowledge about the mechanisms of selective Ca²⁺ permeation through VGCCs was derived from the studies on native and recombinant L-type representatives. However, the specifics of the selectivity and permeation of known recombinant T-type Ca²⁺-channel α1 subunits, Ca_v3.1, Ca_v3.2 and Ca_v3.3, are still poorly defined. In the present study we provide comparative analysis of the selectivity and permeation Ca_v3.1, Ca_v3.2, and Ca_v3.3 functionally expressed in Xenopus oocytes. Our data show that all Ca_v3 channels select Ca²⁺ over Na⁺ by affinity. Ca_v3.1 and Ca_v3.2 discriminate Ca²⁺, Sr²⁺ and Ba²⁺ based on the ion's effects on the open channel probability, whilst Ca_v3.3 discriminates based on the ion's intrapore binding affinity. All Ca_v3s were characterized by much smaller difference in the K_D values for Na⁺ current blockade by Ca²⁺ ( K_D1∼6 μM) and for Ca²⁺ current saturation ( K_D2∼2 mM) as compared to L-type channels. This enabled them to carry notable mixed Na⁺/Ca²⁺ current at close to physiological Ca²⁺ concentrations, which was the strongest for Ca_v3.3, smaller for Ca_v3.2 and the smallest for Ca_v3.1. In addition to intrapore Ca²⁺ binding site(s) Ca_v3.2, but not Ca_v3.1 and Ca_v3.3, is likely to possess an extracellular Ca²⁺ binding site that controls channel permeation. Our results provide novel functional tests for identifying subunits responsible for T-type Ca²⁺ current in native cells.

Keywords: Cloned T-type Ca; ²⁺; channels; Ca; _v; 3.1; Ca; _v; 3.2; Ca; _v; 3.3; Xenopus; oocytes; Selectivity; Permeation

PrP106–126 amide causes the semi-penetrated poration in the supported lipid bilayers by Jian Zhong; Wenfu Zheng; Lixin Huang; Yuankai Hong; Lijun Wang; Yang Qiu; Yinlin Sha (pp. 1420-1429).

A major hallmark of prion diseases is the cerebral amyloid accumulation of the pathogenic PrP^Sc, an abnormally misfolded, protease-resistant, and β-sheet rich protein. PrP106–126 is the key domain responsible for the conformational conversion and aggregation of PrP. It shares important physicochemical characteristics with PrP^Sc and presents similar neurotoxicity as PrP^Sc. By combination of fluorescence polarization, dye release assay and in situ time-lapse atomic force microscopy (AFM), we investigated the PrP106–126 amide interacting with the large unilamellar vesicles (LUVs) and the supported lipid bilayers (SLBs). The results suggest that the interactions involve a poration-mediated process: firstly, the peptide binding results in the formation of pores in the membranes, which penetrate only half of the membranes; subsequently, PrP106–126 amide undergoes the poration-mediated diffusion in the SLBs, represented by the formation and expansion of the flat high-rise domains (FHDs). The possible mechanisms of the interactions between PrP106–126 amide and lipid membranes are proposed based on our observations.

Keywords: Abbreviations; PrP; ^Sc; scrapie PrP protein; PrP; ^c; cellular PrP protein; PrP106–126, K; ¹⁰⁶; TNMKHMAGAAAAGAVVGGLG; ¹²⁶; numbered according to the human PrP sequence; PrP106–126 amide; C-terminal amidation of PrP106–126; SLBs; supported lipid bilayers; AFM; atomic force microscopy; DPH; 1,6-diphenyl-1,3,5-hexatriene; LUVs; large unilamellar vesicles; MLVs; multilamellar vesicles; ANTS; 1-aminonaphthalene-3,6,8-trisulfonic acid; DPX; N; ,; N′; -p-xylylenebis (pyridinium) bromide; POPC; 1-Palmitoyl-2-Oleoyl-; sn; -Glycero-3-Phosphocholine; Chl; cholesterol; PBS; phosphate-buffered saline; HBS; HEPES-buffered saline; FHDs; flat high-rise domainsIn situ time-lapse AFM; Supported lipid bilayers (SLBs); PrP106–126 amide; Semi-penetrated poration; Flat high-rise domains (FHDs)

A novel lipid-based drug carrier targeted to the non-parenchymal cells, including hepatic stellate cells, in the fibrotic livers of bile duct ligated rats by Joanna E. Adrian; Jan A.A.M. Kamps; Gerrit L. Scherphof; Dirk K.F. Meijer; Anne-miek van Loenen-Weemaes; Catharina Reker-Smit; Peter Terpstra; Klaas Poelstra (pp. 1430-1439).

In fibrotic livers, collagen producing hepatic stellate cells (HSC) represent a major target for antifibrotic therapies. We designed liposomes with surface-coupled mannose 6-phosphate (M6P) modified human serum albumin (HSA) to target HSC via the M6P receptor. In this study we determined the pharmacokinetics and target specificity of M6P-HSA-liposomes in a rat model of liver fibrosis. Ten minutes after injection of [³H]-M6P-HSA-liposomes 90% of the dose has cleared the circulation. The blood elimination of these liposomes was counteracted by free M6P-HSA and polyinosinic acid, a competitive inhibitor of scavenger receptors. The M6P-HSA-liposomes accumulated in HSC. However, also Kupffer cells and endothelial cells contributed to the uptake of M6P-HSA-liposomes in the fibrotic livers. Polyinosinic acid inhibited the accumulation of the liposomes in Kupffer cells and liver endothelial cells, but not in HSC. PCR analysis revealed that cultured HSC express scavenger receptors. This was confirmed by Western blotting, although activation of HSC diminishes scavenger receptor protein expression. In conclusion, in a rat model for liver fibrosis M6P-HSA-liposomes can be efficiently targeted to non-parenchymal cells, including HSC. M6P receptors and scavenger receptors are involved in the cellular recognition of these liposomes, allowing multiple pharmacological interference in different pathways involved in the fibrosis.

Keywords: Hepatic stellate cells; Targeted liposomes; Liver fibrosis; Non-parenchymal cells; Mannose 6-phosphate receptor; Scavenger receptor

Role of hydrophobic residues in the voltage sensors of the voltage-gated sodium channel by Saïd Bendahhou; Andrias O. O'Reilly; Hervé Duclohier (pp. 1440-1447).

The role of hydrophobic residues in voltage sensors S4 of voltage-sensitive ion channels is less documented than that of charged residues. We performed alanine-substitution of branched-sidechain residues contiguous to the third, fourth and fifth positively charged residues in S4s of the first three domains of the sodium channel expressed in HEK cells. These locations were selected because they are close to the arginines and lysines important in gating. Mutations in the first two domains (DIS4 and DIIS4) altered steady-state activation curves. In DIIIS4, the mutation L1131A next to the third arginine greatly slowed inactivation in a manner similar to that for substitutions of charged residues in DIVS4, whereas the mutation L1137A next to the fifth arginine preserved wild-type behaviour. Homology models of domain III, based on the structure of a crystallized mammalian potassium channel, shows that L1131 is located at the interface between S3 and S4 helices, whereas L1137, on the opposite side of S4, does not interact with the voltage sensor. The two mutated residues are closer to each other in domains I and II than in domain III, as may be corroborated by their different electrophysiological effects.

Keywords: Voltage sensors; Activation–inactivation coupling; Heterologous expression; Electrophysiology; Molecular modelling

Chemical specificity in short-chain fatty acids and their analogues in increasing osmotic fragility in rat erythrocytes in vitro by Hitoshi Mineo; Hiroshi Hara (pp. 1448-1453).

We examined the role of the chemical specificity of short-chain fatty acids (SCFAs) and their derivatives in increasing osmotic fragility (OF) in rat red blood cells (RBCs). Except for formic acid, normal SCFAs with 2 to 8 carbons increased the OF in rat RBCs with increasing number of hydrocarbons in a dose-dependent manner. Replacement of the carboxylic group with sulfonic group inhibited, but did not abolish, the SCFA-mediated increase in OF. Introduction of another carboxylic group (dicarboxylic acids) completely abolished the SCFA-mediated increase in OF. Transformation of the hydrocarbon chains in SCFAs from straight to branched or cyclic chains affected the degree of the OF-increasing effect. Introduction of double- or triple-carbon bonds to the hydrocarbon chain in parent SCFAs did not affect the increase in OF. Both hydrophilic (carboxylic group) and hydrophobic elements (hydrocarbons) at opposite sides of a molecule were required to affect the RBC membrane, and the size and form of hydrophobic element were important factors in determining the SCFA-mediated increase in OF. The hydrocarbon chains probably enter the plasma membrane, with the hydrophilic carboxylic base remaining outside of the membrane, and interact with phospholipid in cell membrane and disturb the structure of lipid layer resulting in the increase in OF in the rat RBCs.

Keywords: SCFA; RBC; Erythrocyte; Membrane; Osmotic fragility; Rat

Physical properties of the lipid bilayer membrane made of calf lens lipids: EPR spin labeling studies by Justyna Widomska; Marija Raguz; James Dillon; Elizabeth R. Gaillard; Witold K. Subczynski (pp. 1454-1465).

The physical properties of a membrane derived from the total lipids of a calf lens were investigated using EPR spin labeling and were compared with the properties of membranes made of an equimolar 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol (POPC/Chol) mixture and of pure POPC. Conventional EPR spectra and saturation–recovery curves show that spin labels detect a single homogenous environment in all three membranes. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are practically identical in lens lipid and POPC/Chol membranes, but differ drastically from profiles in pure POPC membranes. In both lens lipid and POPC/Chol membranes, the lipids are strongly immobilized at all depths, which is in contrast to the high fluidity of the POPC membrane. Hydrophobicity and oxygen transport parameter profiles in lens lipid and POPC/Chol membranes have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. At this position, hydrophobicity increases from the level of methanol to the level of hexane, and the oxygen transport parameter increases by a factor of 2–3. These profiles in POPC membranes are bell-shaped. It is concluded that the high level of cholesterol in lens lipids makes the membrane stable, immobile, and impermeable to both polar and nonpolar molecules.

Keywords: Lens lipid; Calf; Oxygen transport; Cholesterol; Membrane; EPR

Study of the interaction of an α-helical transmembrane peptide with phosphatidylcholine bilayer membranes by means of densimetry and ultrasound velocimetry by Peter Rybar; Roland Krivanek; Tomas Samuely; Ruthven N.A.H. Lewis; Ronald N. McElhaney; Tibor Hianik (pp. 1466-1478).

We applied precise densimetry and ultrasound velocimetry methods to study the interaction of a synthetic α-helical transmembrane peptide, acetyl-K₂-L₂₄-K₂-amide (L₂₄), with model bilayer lipid membranes. The large unilamellar vesicles (LUVs) utilized were composed of a homologous series of n-saturated diacylphosphatidylcholines (PCs). PCs whose hydrocarbon chains contained from 13 to 16 carbon atoms, thus producing phospholipid bilayers of different thicknesses and gel to liquid-crystalline phase transition temperatures. This allowed us to analyze how the difference between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer influences the thermodynamical and mechanical properties of the membranes. We showed that the incorporation of L₂₄ decreases the temperature and cooperativity of the main phase transition of all LUVs studied. The presence of L₂₄ in the bilayer also caused an increase of the specific volume and of the volume compressibility in the gel state bilayers. In the liquid crystalline state, the peptide decreases the specific volume at relatively higher peptide concentration (mole ratio L₂₄:PC=1:50). The overall volume compressibility of the peptide-containing lipid bilayers in the liquid-crystalline state was in general higher in comparison with pure membranes. There was, however, a tendency for the volume compressibility of these lipid bilayers to decrease with higher peptide content in comparison with bilayers of lower peptide concentration. For one lipid composition, we also compared the thermodynamical and mechanical properties of LUVs and large multilamellar vesicles (MLVs) with and without L₂₄. As expected, a higher cooperativity of the changes of the thermodynamical and mechanical parameters took place for MLVs in comparison with LUVs. These results are in agreement with previously reported DSC and²H NMR spectroscopy study of the interaction of the L₂₄ and structurally related peptides with phosphatidylcholine bilayers. An apparent discrepancy between²H NMR spectroscopy and compressibility data in the liquid crystalline state may be connected with the complex and anisotropic nature of macroscopic mechanical properties of the membranes. The observed changes in membrane mechanical properties induced by the presence of L₂₄ suggest that around each peptide a distorted region exists that involves at least 2 layers of lipid molecules.

Keywords: Abbreviations; L; ₂₄; acetyl-K; ₂; -L; ₂₄; -K; ₂; -amide; P; ₂₄; acetyl-K; ₂; -G-L; ₂₄; -K; ₂; -A-amide; LUV; large unilamellar vesicle; MLV; large multilamellar vesicle; FTIR; Fourier transform infrared; PC; phosphatidylcholine; DMPC; dimyristoylphosphatidylcholine; DPPC; dipalmitoylphosphatidylcholine; DSC; differential scanning calorimetry; T; _m; gel to liquid-crystalline phase transition temperatureα-helical transmembrane peptide; Phosphatidylcholine bilayers; Lipid–peptide interaction; Densimetry; Ultrasound velocimetry; Volume compressibility

Rapamycin stimulates arginine influx through CAT2 transporters in human endothelial cells by Rossana Visigalli; Amelia Barilli; Ovidio Bussolati; Roberto Sala; Gian C. Gazzola; Alessandro Parolari; Elena Tremoli; Alexandra Simon; Ellen I. Closs; Valeria Dall'Asta (pp. 1479-1487).

In endothelial cells Tumor Necrosis Factor-α (TNFα) stimulates arginine transport through the increased expression of SLC7A2/CAT2 transcripts. Here we show that also rapamycin, an inhibitor of mTOR kinase, stimulates system y⁺-mediated arginine uptake in human endothelial cells derived from either saphenous (HSVECs) or umbilical veins (HUVECs). When used together with TNFα, rapamycin produces an additive stimulation of arginine transport in both cell models. These effects are observed also upon incubation with AICAR, a stimulator of Adenosine-Monophosphate-dependent-Protein Kinase (AMPK) that produces a rapamycin-independent inhibition of the mTOR pathway. Rapamycin increases the V_max of high affinity arginine transport and causes the appearance of a low affinity component that is particularly evident if the treatment is carried out in the presence of TNFα. RT-qPCR studies have demonstrated that these kinetic changes correspond to the induction of both the high affinity transporter CAT2B and the low affinity isoform CAT2A. Western blot and immunocytochemical analyses indicate that, consistently, the expression of CAT2 proteins is also stimulated under the same conditions. These changes are associated with an increase of the intracellular arginine concentration but with a decrease of NO production. Thus, our data suggest that mTOR activity is associated with the repression of CAT2 expression at mRNA and protein level.

Keywords: Abbreviations; AICAR; 5-aminoimidazole-4-carboxamide-1-b-; d; -ribofuranosyl 3′-5′-cyclic-monophosphate; AU; Arbitrary Unit; CAA; Cationic Amino Acid; CLSM; Confocal Laser Scanning Microscopy; DAN; 2,3-diaminonaphtalene; DTT; Dithiothreitol; EBSS; Earle's Balanced Salt Solution; FBS; Fetal Bovine Serum; GAPDH; Glyceraldehyde-3-phosphate dehydrogenase; HSVECs; Human Saphenous Vein Endothelial Cells; HUVECs; Human Umbilical Vein Endothelial Cells; M199; Medium 199; NEM; N-ethylmaleimide; TNFα; Tumor Necrosis Factor-αmTOR; CAT transporter; SLC7A gene; Arginine; System y; ⁺

Inhibition of malaria parasite blood stages by tyrocidines, membrane-active cyclic peptide antibiotics from Bacillus brevis by Marina Rautenbach; N. Maré Vlok; Marietjie Stander; Heinrich C. Hoppe (pp. 1488-1497).

Tyrothricin, a complex mixture of antibiotic peptides from Bacillus brevis, was reported in 1944 to have antimalarial activity rivalling that of quinine in chickens infected with Plasmodium gallinaceum. We have isolated the major components of tyrothricin, cyclic decapeptides collectively known as the tyrocidines, and tested them against the human malaria parasite Plasmodium falciparum using standard in vitro assays. Although the tyrocidines differ from each other by conservative amino acid substitutions in only three positions, their observed 50% parasite inhibitory concentrations (IC₅₀) spanned three orders of magnitude (0.58 to 360 nM). Activity correlated strictly with increased apparent hydrophobicity and reduced total side-chain surface area and the presence of ornithine and phenylalanine in key positions. In contrast, mammalian cell toxicity and haemolytic activities of the respective peptides were considerably less variable (2.6 to 28 μM). Gramicidin S, a structurally analogous antimicrobial peptide, was less active (IC₅₀=1.3 μM) and selective than the tyrocidines. It exerted its parasite inhibition by rapid and selective lysis of infected erythrocytes as judged by fluorescence and light microscopy. The tyrocidines, however, did not cause an overt lysis of infected erythrocytes, but an inhibition of parasite development and life-cycle progression.

Keywords: Malaria; P. falciparum; Tyrocidine; Selective inhibition; Cyclic peptide antibiotic; Bacillus brevis

Thermogenic activity of Ca²⁺-ATPase from skeletal muscle heavy sarcoplasmic reticulum: The role of ryanodine Ca²⁺ channel by Ana Paula Arruda; Mariana Nigro; Gaya M. Oliveira; Leopoldo de Meis (pp. 1498-1505).

The sarcoplasmic reticulum Ca²⁺ ATPase 1 (SERCA 1) is able to handle the energy derived from ATP hydrolysis in such a way as to determine the parcel of energy that is used for Ca²⁺ transport and the fraction that is converted into heat. In this work we measured the heat production by SERCA 1 in the two sarcoplasmic reticulum (SR) fractions: the light fraction (LSR), which is enriched in SERCA and the heavy fraction (HSR), which contains both the SERCA and the ryanodine Ca²⁺ channel. We verified that although HSR cleaved ATP at faster rate than LSR, the amount of heat released during ATP hydrolysis by HSR was smaller than that measured by LSR. Consequently, the amount of heat released per mol of ATP cleaved (ΔH^cal) by HSR was lower compared to LSR. In HSR, the addition of 5 mM Mg²⁺ or ruthenium red, conditions that close the ryanodine Ca²⁺ channel, promoted a decrease in the ATPase activity, but the amount of heat released during ATP hydrolysis remained practically the same. In this condition, the ΔH^cal values of ATP hydrolysis increased significantly. Neither Mg²⁺ nor ruthenium red had effect on LSR. Thus, we conclude that heat production by SERCA 1 depends on the region of SR in which the enzyme is inserted and that in HSR, the ΔH^cal of ATP hydrolysis by SERCA 1 depends on whether the ryanodine Ca²⁺ channel is opened or closed.

Keywords: Ca; ²⁺; -ATPase; Heavy sarcoplasmic reticulum; Heat production; ATP hydrolysis; Ryanodine Ca; ²⁺; -channel

Substitution of the leucine zipper sequence in melittin with peptoid residues affects self-association, cell selectivity, and mode of action by Wan Long Zhu; Yun Mi Song; Yoonkyung Park; Ka Hyon Park; Sung-Tae Yang; Jae Il Kim; Il-Seon Park; Kyung-Soo Hahm; Song Yub Shin (pp. 1506-1517).

Melittin (ME), a non-cell-selective antimicrobial peptide, contains the leucine zipper motif, wherein every seventh amino acid is leucine or isolucine. Here, we attempted to generate novel cell-selective peptides by substituting amino acids in the leucine zipper sequence of ME with peptoid residues. We generated a series of ME analogues by replacing Leu-6, Lue-13 and Ile-20 with Nala, Nleu, Nphe, or Nlys, and we examined their secondary structure, self-association activity, cell selectivity and mode of action. Circular dichroism spectroscopy indicated that the substitutions disrupt the α-helical structure of ME in micelles of sodium dodecyl sulfate and on negatively charged and zwitterionic phospholipid vesicles. Substitution by Nleu, Nphe, or Nlys but not Nala disturbed the self-association in an aqueous environment, interaction with zwitterionic membranes, and toxicity to mammalian cells of ME but did not affect the interaction with negatively charged membranes or antibacterial activity. Notably, peptides with Nphe or Nlys substitution had the highest therapeutic indices, consistent with their lipid selectivity. In addition, all of peptoid residue-containing ME analogues had little or no ability to induce membrane disruption, membrane depolarization and lipid flip-flop. Taken together, our studies indicate that substitution of the leucine zipper motif in ME with peptoid residues increases its selectivity against bacterial cells by impairing self-association activity and changes its mode of antibacterial action from membrane-targeting mechanism to possible intracellular targeting mechanism. Furthermore, our ME analogues especially those with Nleu, Nphe, or Nlys substitutions, may be therapeutically useful antimicrobial peptides.

Keywords: Abbreviations; CD; circular dichroism; C; ₆; -NBD-PE; 1-palmitoyl-2-[6-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino]capryl]-; l; -α-phosphatidylethanolamine; CFUs; colony-forming units; DiSC; ₃; -5; 3,3′-dipropylthiadicarbocyanine iodide; EYPC; egg yolk; l; -α-phosphatidylcholine; EYPE; egg yolk; l; -α-phosphatidylethanolamine; EYPG; egg yolk; l; -α-phosphatidyl-; dl; -glycerol; Fmoc; fluoren-9-yl-methoxycarbonyl; hRBCs; human red blood cells; LUVs; large unilamellar vesicles; MALDI-TOF MS; matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry; MIC; minimal inhibitory concentration; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; PBS; phosphate-buffered saline; RP-HPLC; reversed-phase high performance liquid chromatography; SDS; sodium dodecyl sulfate; SUVs; small unilamellar vesiclesAntimicrobial peptide; Cell selectivity; Leucine zipper; Melittin; Peptoid residue; Self-association

Nanosized bilayer disks: Attractive model membranes for drug partition studies by Emma Johansson; Anna Lundquist; Shusheng Zuo; Katarina Edwards (pp. 1518-1525).

Stable nanosized bilayer disks were prepared from either 1,2-distearoyl- sn-glycero-3-phosphocholine (DSPC) and cholesterol, or lipid mixtures with a composition reflecting that of the porcine brush border membrane. Two different polyethylene glycol (PEG)-grafted lipids, the negatively charged 1,2-distearoyl- sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-5000] (DSPE-PEG₅₀₀₀) and the neutral N-palmitoyl-sphingosine-1-[succinyl (methoxy (polyethylene glycol) 5000] (Ceramide-PEG₅₀₀₀), were used to stabilize the disks. The disks were employed as model membranes in drug partition studies based on a fast chromatography method. Results show that the lipid composition, as well as the choice of PEG-lipid, have an important influence on the partition behavior of charged drugs. Comparative studies using multilamellar liposomes indicate that bilayer disks have the potential to generate more accurate partition data than do liposomes. Further, initial investigations using bacteriorhodopsin suggest that membrane proteins can be reconstituted into the bilayer disks. This fact further strengthens the potential of the bilayer disk as an attractive model membrane.

Keywords: Bilayer disk; Drug partitioning; Liposome; Model membrane; PEG-lipid; Phospholipid; Bacteriorhodopsin

Galactosyl headgroup interactions control the molecular packing of wheat lipids in Langmuir films and in hydrated liquid-crystalline mesophases by C. Bottier; J. Géan; F. Artzner; B. Desbat; M. Pézolet; A. Renault; D. Marion; V. Vié (pp. 1526-1540).

The behavior of the two major galactolipids of wheat endosperm, mono- (MGDG) and di-galactosyldiacylglycerol (DGDG) was studied in aqueous dispersion and at the air/liquid interface. The acyl chains of the pure galactolipids and their binary equimolar mixture are in the fluid or liquid expanded phase. SAXS measurements on liquid-crystalline mesophases associated with the electron density reconstructions show that the DGDG adopts a lamellar phase L_α with parallel orientation of the headgroups with respect to the plane of the bilayer, whereas MGDG forms an inverse hexagonal phase H_II with a specific organization of galactosyl headgroups. The equimolar mixture shows a different behavior from those previously described with formation of an Im3m cubic phase. In comparing monolayers composed of the pure galactolipids and their equimolar mixtures, PM-IRRAS spectra show significant differences in the optical properties and orientation of galactosyl groups with respect to the interface. Furthermore, Raman and FTIR spectroscopies show that the acyl chains of the galactolipid mixture are more ordered compared to those of the pure components. These results suggest strong interactions between MGDG and DGDG galactosyl headgroups and these specific physical properties of galactolipids are discussed in relation to their biological interest in wheat seed.

Keywords: Abbreviations; AFM; atomic force microscopy; ATR; attenuated total reflectance; CPK; Corey–Pauling–Koltun; DGDG; 1,2-di-; O; -acyl-3-; O; -(β-; d; -galactopyranosyl-1,6-β-; d; -galactopyranosyl)-; sn; -glycerol; DMPC; 1,2-di-myristoyl-3-phosphorylcholine-; sn; -glycerol; DOPC; 1,2-di-oleoyl-3-phosphorylcholine-; sn; -glycerol; FT-IR; Fourier transform infrared spectroscopy; LB film; Langmuir–Blodgett film; MGDG; 1,2-di-; O; -acyl-3-; O; -(β-; d; -galactopyranosyl)-; sn; -glycerol; PM-IRRAS; polarization modulation-infrared reflexion-absorption spectroscopy; SAXS; small-angle X-ray scattering; WAXS; wide-angle X-ray scatteringGalactolipid; Air/liquid interface; Glycosyl headgroup organization; Small-angle X-ray scattering; Phase behavior; Wheat endosperm

Electron spin-echo studies of spin-labelled lipid membranes and free fatty acids interacting with human serum albumin by Francesco De Simone; Rita Guzzi; Luigi Sportelli; Derek Marsh; Rosa Bartucci (pp. 1541-1549).

Human serum albumin (HSA) is an abundant plasma protein that transports fatty acids and also binds a wide variety of hydrophobic pharmacores. Echo-detected (ED) EPR spectra and D₂O-electron spin echo envelope modulation (ESEEM) Fourier-transform spectra of spin-labelled free fatty acids and phospholipids were used jointly to investigate the binding of stearic acid to HSA and the adsorption of the protein on dipalmitoyl phosphatidylcholine (DPPC) membranes. In membranes, torsional librations are detected in the ED-spectra, the intensity of which depends on chain position at low temperature. Water penetration into the membrane is seen in the D₂O-ESEEM spectra, the intensity of which decreases greatly at the middle of the membrane. Both the chain librational motion and the water penetration are only little affected by adsorption of serum albumin at the DPPC membrane surface. In contrast, both the librational motion and the accessibility of the chains to water are very different in the hydrophobic fatty acid binding sites of HSA from those in membranes. Indeed, the librational motion of bound fatty acids is suppressed at low temperature, and is similar for the different chain positions, at all temperatures. Correspondingly, all segments of the bound chains are accessible to water, to rather similar extents.

Keywords: Electron spin resonance; Electron spin echo envelope modulation (ESEEM); Echo-detected spectra; Water accessibility; Librational motion

Counterion-mediated membrane penetration: Cationic cell-penetrating peptides overcome Born energy barrier by ion-pairing with phospholipids by Elin K. Esbjörner; Per Lincoln; Bengt Nordén (pp. 1550-1558).

Arginine-rich cell-penetrating peptides (CPPs) can enter cells non-endocytotically, despite that transport of charge across a membrane should be formally associated with an extremely high Born energy barrier. We studied partitioning of several derivatives of the CPP penetratin in a water–octanol two-phase system in presence of natural phospholipids to explore if solvation by ion-pairing to hydrophobic counter-ions may serve as a mechanism for cell internalisation. We demonstrate that anionic lipids can aid peptide partitioning into octanol. Particularly efficient partitioning into octanol is observed with an arginine-rich penetratin compared to a lysine-rich derivative. Substituting tryptophans for phenylalanines results in poor partitioning into octanol, due to decreased overall peptide hydrophobicity. Partitioning into octanol is dependent of phospholipid type and the peptides induced structural changes in the lipid assemblies found in octanol. Attachment of carboxyfluorescein as a model cargo was found to enhance peptide partitioning into octanol. We discuss our results with respect to theoretical electrostatic energies, empirical hydrophobicity scales and in terms of implications for CPP uptake mechanisms. An important improvement of the theoretical transfer energies is obtained when, instead of singular ions, the insertion of ion-paired dipolar species is considered.

Keywords: Arginine; Tryptophan; Octanol; Partitioning; Cargo transport

The interaction of the brominated flame retardant: Tetrabromobisphenol A with phospholipid membranes by Oluseye A. Ogunbayo; Karina T. Jensen; Francesco Michelangeli (pp. 1559-1566).

Tetrabromobisphenol A (TBBPA) is one of the most widely used members of the family of brominated flame retardants (BFRs). BFRs, including TBBPA have been shown to be widely distributed within the environment and there is growing evidence of their bio-accumulation within animals and man. Toxicological studies have shown that TBBPA can be harmful to cells by modulating a number of cell signalling processes. In this study, we employed fluorescence spectroscopy and differential scanning calorimetry to investigate the interaction of TBBPA with phospholipid membranes, as this is the most likely route for it to influence membrane-associated cellular processes. TBBPA readily and randomly partitions throughout all regions of the phospholipid bilayer with high efficacy {partition coefficient (Log K_p)=5.7±0.7}. A decrease in membrane fluidity in both liquid-crystalline and gel-phase membranes was detected at concentrations of TBBPA as low as 2.5 μM. TBBPA also decreases the phase transition temperature of dipalmitoyl phoshatidylcholine (DPPC) membranes and broadened transition peaks, in a fashion similar to that for cholesterol. TBBPA, however, also prefers to partition into membrane regions not too highly enriched with cholesterol. Our findings therefore suggests that, the toxic effects of TBBPA, may at least in part, be due to its lipid membrane binding/perturbing effects, which in turn, could influence biological processes involving cell membranes.

Keywords: Brominated flame retardant; Tetrabromobisphenol A; Phospholipid; Membrane fluidity; Fluorescence spectroscopy; Differential scanning calorimetry

Oligomeric behavior of the RND transporters CusA and AcrB in micellar solution of detergent by David Stroebel; Véronique Sendra; Dominique Cannella; Kerstin Helbig; Dietrich H. Nies; Jacques Covès (pp. 1567-1573).

We have used analytical ultracentrifugation to explore the oligomeric states of AcrB and CusA in micellar solution of detergent. These two proteins belong to the resistance, nodulation and cell division (RND) family of efflux proteins that are involved in multiple drug and heavy metal resistance. Only the structure of AcrB has been determined so far. Although functional RND proteins should assemble as trimers as AcrB does, both AcrB and CusA form a mixture of quaternary structures (from monomer to heavy oligomer) in detergent solution. The distribution of the oligomeric states was studied as a function of different parameters: nature and concentration of the detergent, ionic strength, pH, protein concentration. This pseudo-heterogeneity does not hamper the crystallization of AcrB as a homotrimer.

Keywords: Abbreviations; AUC; analytical ultracentrifugation; DDM; n; -dodecyl-; β; -; d; -maltopyranoside; FC14; n; -tetradecylphosphocholine; FTIR; Fourier transform infrared spectroscopy; RND; resistance nodulation and cell divisionRND transporter; Analytical ultracentrifugation; Detergent; Crystallization; Membrane protein; Oligomer

Addition of a small hydrophobic segment from the head region to an amphipathic leucine zipper like motif of E. coli toxin hemolysin E enhances the peptide-induced permeability of zwitterionic lipid vesicles by Sharada Prasad Yadav; Aqeel Ahmad; Jimut Kanti Ghosh (pp. 1574-1582).

To find out the sequence requirement of the H-205 peptide, containing an amphipathic leucine zipper motif corresponding to the amino acid (a.a.) region 205–234 of hemolysin E (HlyE) to induce efficient permeation in zwitterionic lipid vesicles, the peptide was extended at the N-terminal after the addition of seven amino acids from the predicted transmembrane region in the head domain of the protein-toxin. The new peptide, H-198 (a.a. 198–234) and a scrambled mutant peptide of the same size were synthesized, fluorescently labeled and characterized functionally and structurally. The results showed that H-198 induced significantly higher permeation in the zwitterionic PC/Chol lipid vesicles than its shorter version, H-205. H-198 formed large aggregates in the PC/Chol vesicles unlike H-205 and also adopted more helical structure in the membrane mimetic environments compared to that of H-205. Fluorescence energy transfer experiments by flow cytometry indicated that only H-198 but not its mutant or H-205 oligomerized in the zwitterionic lipid vesicles, while in the negatively charged lipid vesicles both H-198 and H-205 formed oligomeric assembly. The results suggest a probable role of the hydrophobic residues of the head domain of HlyE in inducing permeability in the zwitterionic lipid vesicles by the peptide derived from the a.a. 198–234 of the toxin.

Keywords: Membrane-interaction of hemolysin E; Membrane permeability; Zwitterionic and negatively charged lipid vesicles; Membrane-assembly of hemolysin E-derived peptides; Peptide–lipid interaction; Fluorescence and flow cytometric studies

Conformational changes of chicken liver bile acid-binding protein bound to anionic lipid membrane are coupled to the lipid phase transitions by María Belén Decca; Massimiliano Perduca; Hugo L. Monaco; Guillermo G. Montich (pp. 1583-1591).

Chicken liver bile acid-binding protein (L-BABP) binds to anionic lipid membranes by electrostatic interactions and acquires a partly folded state [Nolan, V., Perduca, M., Monaco, H., Maggio, B. and Montich, G. G. (2003) Biochim. Biophys. Acta 1611, 98–106]. We studied the infrared amide I′ band of L-BABP bound to dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidylglycerol (DMPG) and palmitoyloleoylphosphatidylglycerol (POPG) in the range of 7 to 60 °C. Besides, the thermotrophic behaviour of DPPG and DMPG was studied in the absence and in the presence of bound-protein by differential scanning calorimetry (DSC) and infrared spectra of the stretching vibration of methylene and carbonyl groups. When L-BABP was bound to lipid membranes in the liquid-crystalline state (POPG between 7 and 30 °C) acquired a more unfolded conformation that in membranes in the gel state (DPPG between 7 and 30 °C). Nevertheless, this conformational change of the protein in DMPG did not occur at the temperature of the lipid gel to liquid-crystalline phase transition detected by infrared spectroscopy. Instead, the degree of unfolding in the protein was coincident with a phase transition in DMPG that occurs with heat absorption and without change in the lipid order.

Keywords: Abbreviations; FABP; fatty acid-binding protein; L-BABP; chicken liver bile acid-binding protein; DPPG; 1-palmitoyl-2-palmitoyl -; sn; -glycero-3-phosphoglycerol; DMPG; 1-myristoyl-2-oleoyl-; sn; -glycero-3-phosphoglycerol; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphoglycerol; FT-IR; Fourier transform infrared; DSC; differential scanning calorimetry; LUV; large unilamellar vesicleChicken liver bile acid-binding protein; Lipid membranes; FT-IR; DSC; Protein conformation; Lipid phase transition

Comparative analysis of P2Y₄ and P2Y₆ receptor architecture in native and transfected neuronal systems by Nadia D’Ambrosi; Monia Iafrate; Elena Saba; Patrizia Rosa; Cinzia Volonté (pp. 1592-1599).

Although extensive studies provided molecular and pharmacological characterization of metabotropic P2Y receptors for extracellular nucleotides, little is still known about their quaternary structure. By the use of transfected cellular systems and SDS-PAGE, in our previous work we established the propensity of P2Y₄ receptor to form dimeric interactions. Here we focused on endogenously expressed P2Y₄ and P2Y₆ subtypes, comparing their oligomeric complexes under Blue Native (BN) gel electrophoresis. We provided evidence that P2Y₄ and P2Y₆ receptors form high order complexes in native neuronal phenotypes and that the oligomers can be disaggregated down to the dimeric P2Y₄ or to the dimeric and monomeric P2Y₆ receptor. Moreover, dimeric P2Y₄ and monomeric P2Y₆ proteins display selective microdomain partitioning in lipid rafts from specialized subcellular compartments such as synaptosomes. Ligand activation by UTP shifted the oligomerization of P2Y₆ but not of P2Y₄ receptor, as analysed by BN electrophoresis. Finally, whereas transfected P2Y₄ and P2Y₆ proteins homo-interact and posses the appropriate domains to associate with all P2Y_1,2,4,6,11 subtypes, in naive PC12 cells the endogenous P2Y₄ forms hetero-oligomers only with the P2Y₆ subunit. In conclusion, our results indicate that quaternary structure distinguishing P2Y₄ from P2Y₆ receptors might be crucial for specific ligand activation, membrane partitioning and consequent functional regulation.

Keywords: Abbreviations; BN; Blue Native; DM; N-dodecyl-β-maltoside; DTT; dithiothreitol; FCS; foetal calf serum; GPCRs; G protein-coupled receptors; HDAC; histone deacetylase; PAGE; polyacrylamide gel electrophoresis; SDS; sodium dodecyl sulphate; SNAP; synaptosome-associated proteinGPCR; Purinergic receptor; Dimerization; Lipid raft; Synaptosomes

Sequence of occurring damages in yeast plasma membrane during dehydration and rehydration: Mechanisms of cell death by Hélène Simonin; Laurent Beney; Patrick Gervais (pp. 1600-1610).

Yeasts are often exposed to variations in osmotic pressure in their natural environments or in their substrates when used in fermentation industries. Such changes may lead to cell death or activity loss. Although the involvement of the plasma membrane is strongly suspected, the mechanism remains unclear. Here, the integrity and functionality of the yeast plasma membrane at different levels of dehydration and rehydration during an osmotic treatment were assessed using various fluorescent dyes. Flow cytometry and confocal microscopy of cells stained with oxonol, propidium iodide, and lucifer yellow were used to study changes in membrane polarization, permeabilization, and endocytosis, respectively. Cell volume contraction, reversible depolarization, permeabilization, and endovesicle formation were successively observed with increasing levels of osmotic pressure during dehydration. The maximum survival rate was also detected at a specific rehydration level, of 20 MPa, above which cells were strongly permeabilized. Thus, we show that the two steps of an osmotic treatment, dehydration and rehydration, are both involved in the induction of cell death. Permeabilization of the plasma membranes is the critical event related to cell death. It may result from lipidic phase transitions in the membrane and from variations in the area-to-volume ratio during the osmotic treatment.

Keywords: Plasma membrane; Osmotic stress; Saccharomyces cerevisiae; Flow cytometry; Confocal microscopy

Fructans from oat and rye: Composition and effects on membrane stability during drying by Dirk K. Hincha; David P. Livingston III; Ramaswamy Premakumar; Ellen Zuther; Nicolai Obel; Constança Cacela; Arnd G. Heyer (pp. 1611-1619).

Fructans have been implicated in the abiotic stress tolerance of many plant species, including grasses and cereals. To elucidate the possibility that cereal fructans may stabilize cellular membranes during dehydration, we used liposomes as a model system and isolated fructans from oat ( Avena sativa) and rye ( Secale cereale). Fructans were fractionated by preparative size exclusion chromatography into five defined size classes (degree of polymerization (DP) 3 to 7) and two size classes containing high DP fructans (DP>7 short and long). They were characterized by high performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The effects of the fructans on liposome stability during drying and rehydration were assessed as the ability of the sugars to prevent leakage of a soluble marker from liposomes and liposome fusion. Both species contain highly complex mixtures of fructans, with a DP up to 17. The two DP>7 fractions from both species were unable to protect liposomes, while the fractions containing smaller fructans were protective to different degrees. Protection showed an optimum at DP 4 and the DP 3, 4, and 5 fractions from oat were more protective than all other fractions from both species. In addition, we found evidence for synergistic effects in membrane stabilization in mixtures of low DP with DP>7 fructans. The data indicate that cereal fructans have the ability to stabilize membranes under stress conditions and that there are size and species dependent differences between the fructans. In addition, mixtures of fructans, as they occur in living cells may have protective properties that differ significantly from those of the purified fractions.

Keywords: Desiccation; Fructan; Liposome; Oat (; Avena sativa; ); Rye (; Secale cereale; )

Isolation and characterization of lipids strictly associated to PSII complexes: Focus on cardiolipin structural and functional role by A. Ventrella; L. Catucci; G. Mascolo; A. Corcelli; A. Agostiano (pp. 1620-1627).

In this work, lipid extracts from spinach membrane fragments enriched in Photosystem II (PSII) and from spinach PSII dimers were analyzed, by means of Thin Layer Chromatography (TLC) and Electro-Spray Ionization Mass Spectrometry. Cardiolipin found in association with PSII was isolated and purified by preparative TLC, then characterized by mass and mass–mass analyses. Cardiolipin structures with four unsaturated C18 acyl chains and variable saturation degrees were evidenced. Structural and functional effects of different phospholipids on PSII complexes were investigated by Fluorescence, Resonance Light Scattering and Oxygen Evolution Rate measurements. An increment of PSII thermal stability was observed in the presence of cardiolipin and phosphatidylglycerol.

Keywords: Abbreviations; CL; Cardiolipin; Chl; Chlorophyll; DCBQ; 2,5-dichloro-p-benzoquinone; DM; n-dodecyl-β-; d; -maltoside; ESI-MS; Electro-Spray Ionization Mass Spectrometry; ESI-MS-MS; Electro-Spray Ionization Mass–Mass Spectrometry; LHCI; Light Harvesting Complex I; LHCII; Light Harvesting Complex II; MNCB; aqueous buffer containing [2-N-morpholine]ethane-sulphonic acid (25 mM), NaCl (10 mM), CaCl; ₂; (5 mM) and NaHCO; ₃; (10 mM); OG; n-octyl-β-; d; -glucopyranoside; OER; Oxygen Evolution Rate; PA; Phosphatidic acid; PG; Phosphatidylglycerol; PI; Phosphatidylinositol; PSI; Photosystem I; PSII; Photosystem II; Qq-TOF; Quadrupole quadrupole time of flight; RC; Reaction Centre; RLS; Resonance Light Scattering; SQDG; Sulfoquinovosyldiacylglycerol; TLC; Thin Layer ChromatographyPhotosystem II; Cardiolipin; Electro-Spray Ionization Mass Spectrometry; Resonance Light Scattering; Fluorescence Emission; Oxygen Evolution Rate

Characterization of the conformational and orientational dynamics of ganglioside GM1 in a dipalmitoylphosphatidylcholine bilayer by molecular dynamics simulations by Ronak Y. Patel; Petety V. Balaji (pp. 1628-1640).

The structure and dynamics of a single GM1 (Gal5-β1,3-GalNAc4-β1,4-(NeuAc3-α2,3)-Gal2-β1,4-Glc1-β1,1-Cer) embedded in a DPPC bilayer have been studied by MD simulations. Eleven simulations, each of 10 ns productive run, were performed with different initial conformations of GM1. Simulations of GM1-Os in water and of a DPPC bilayer were also performed to delineate the effects of the bilayer and GM1 on the conformational and orientational dynamics of each other. The conformation of the GM1 headgroup observed in the simulations is in agreement with those reported in literature; but the headgroup is restricted when embedded in the bilayer. NeuAc3 is the outermost saccharide towards the water phase. Glc1 and Gal2 prefer a parallel, and NeuAc3, GalNac4 and Gal5 prefer a perpendicular, orientation with respect to the bilayer normal. The overall characteristics of the bilayer are not affected by the presence of GM1; however, GM1 does influence the DPPC molecules in its immediate vicinity. The implications of these observations on the specific recognition and binding of GM1 embedded in a lipid bilayer by exogenous proteins as well as proteins embedded in lipids have been discussed.

Keywords: Abbreviations; Cer; Ceramide; DMPC; Dimyristoylphosphatidylcholine; DPPC; Dipalmitoylphosphatidylcholine; DPPE; Dipalmitoylphosphatidylethanolamine; EPC; Egg yolk phosphatidylcholine; FRAP; Fluorescence recovery after photobleaching; Gal; Galactose; GalNAc; 2-Deoxy-2-amino-N-acetylgalactosamine; Glc; Glucose; GM1; Gal-β1,3-GalNAc-β1,4-(NeuAc-α2,3)-Gal-β1,4-Glc-β1,1-Cer; GM1-Os; Oligosaccharide headgroup of GM1; MD; Molecular dynamics; NeuAc; N-Acetylneuraminic acid; POPC; Palmitoyloleoylphosphatidylcholine; SASA; Solvent accessible surface areaGlycolipids; Cell surface recognition; Lipid environment; DPPC bilayer; Local perturbation of bilayer; Conformational restriction

Plasma membrane calcium pump activity is affected by the membrane protein concentration: Evidence for the involvement of the actin cytoskeleton by Laura Vanagas; Rolando C. Rossi; Ariel J. Caride; Adelaida G. Filoteo; Emanuel E. Strehler; Juan Pablo F.C. Rossi (pp. 1641-1649).

Plasma membrane calcium pumps (PMCAs) are integral membrane proteins that actively expel Ca²⁺ from the cell. Specific Ca²⁺-ATPase activity of erythrocyte membranes increased steeply up to 1.5–5 times when the membrane protein concentration decreased from 50 μg/ml to 1 μg/ml. The activation by dilution was also observed for ATP-dependent Ca²⁺ uptake into vesicles from Sf9 cells over-expressing the PMCA 4b isoform, confirming that it is a property of the PMCA. Dilution of the protein did not modify the activation by ATP, Ca²⁺ or Ca²⁺-calmodulin. Treatment with non-ionic detergents did not abolish the dilution effect, suggesting that it was not due to resealing of the membrane vesicles. Pre-incubation of erythrocyte membranes with Cytochalasin D under conditions that promote actin polymerization abolished the dilution effect. Highly-purified, micellar PMCA showed no dilution effect and was not affected by Cytochalasin D. Taken together, these results suggest that the concentration-dependent behavior of the PMCA activity was due to interactions with cytoskeletal proteins. The dilution effect was also observed with different PMCA isoforms, indicating that this is a general phenomenon for all PMCAs.

Keywords: PMCA; Calmodulin; Calcium; Membrane; Cytoskeleton; Cytochalasin D

Molecular rearrangement in POR macrodomains as a reason for the blue shift of chlorophyllide fluorescence observed after phototransformation by Katalin Solymosi; László Smeller; Margareta Ryberg; Christer Sundqvist; Judit Fidy; Béla Böddi (pp. 1650-1658).

The activation energy and activation volume of the spectral blue shift subsequent to protochlorophyllide phototransformation (called Shibata shift in intact leaves) were studied in prolamellar body (PLB) and prothylakoid-(PT)-enriched membrane fractions prepared from dark-grown wheat ( Triticum aestivum, L.) leaves. The measurements were done at 20, 30 and 40 °C and at various pressure values. The activation energy values were 181±8 kJ mol−¹ and 188±6 kJ mol−¹ for the PLBs and the PTs, respectively. The pressure stabilized the structure of the NADPH:protochlorophyllide oxidoreductase (POR) macrodomains; it prevented or slowed down the blue shift. There were no significant differences between the activation volumes of PLBs and PTs at 30 or 40 °C giving values around 100–125 ml mol−¹ which correspond to changes in the tertiary structure of proteins but also resemble the volume changes occurring during the disaggregation of protein dimers or oligomers, or during dissociation of peripheral membrane proteins from membranes. The small differences in the activation parameters of PLBs and PTs indicate that molecular rearrangements inside the POR macrodomains are the primary reasons of the fluorescence blue shift; however, their lipid microenvironment must be also important in the initialization of the shift.

Keywords: Abbreviations; Pchlide; protochlorophyllide; Chlide; chlorophyllide; PLB; prolamellar body; PT; prothylakoid; POR; NADPH:protochlorophyllide oxidoreductaseProtochlorophyllide; Shibata shift; Prolamellar body; Prothylakoid; Activation volume; Activation energy

Structure of the C-terminal domain of the pro-apoptotic protein Hrk and its interaction with model membranes by Angela Bernabeu; Jaime Guillén; Ana J. Pérez-Berná; Miguel R. Moreno; José Villalaín (pp. 1659-1670).

The protein harakiri (Hrk) is a pro-apoptotic BH3-only protein which belongs to the Bcl-2 family. Hrk appears associated to the mitochondrial outer membrane, apparently by a putative transmembrane domain, where it exerts its function. In this work we have identified a 27mer peptide supposed to be the putative membrane domain of the protein at the C-terminal region, and used infrared and fluorescence spectroscopies to study its secondary structure as well as to characterize its effect on the physical properties of phospholipid model membranes. The results presented here showed that the C-terminal region of Hrk adopts a predominantly α-helical structure whose proportion and destabilization capability varied depending on phospholipid composition. Moreover it was found that the orientation of the α-helical component of this C-terminal Hrk peptide was nearly perpendicular to the plane of the membrane. These results indicate that this domain is able of inserting into membranes, where it adopts a transmembrane α-helical structure as well as it considerably perturbs the physical properties of the membrane.

Keywords: Abbreviations; 5NS; 5-Doxyl-stearic acid; 16NS; 16-Doxyl-stearic acid; ANTS; 8-Aminonaphtalene-1,3,5-trisulfonic acid; ATR; attenuated total reflection; BPS; Bovine phosphatidylserine; DMPA; 1,2-Dimyristoyl-; sn; -glycerophosphatidic acid; DMPC; 1,2-Dimyristoyl-; sn; -glycerophosphatidylcholine; DMPC; _d; 1,2-Dimyristoyl-; d54; -; sn; -glycero-3-phosphatidylcholine; DMPG; 1,2-Dimyristoyl-; sn; -glycerophosphatidylglycerol; DMPS; 1,2-Dimyristoyl-; sn; -glycero-3-phosphatidylserine; DPH; 1,6-Diphenyl-1,3,5-hexatriene; DPX; p-Xylene-bis-pyridiniumbromide; EPA; Egg yolk; l; -α-phosphatidic acid; EPC; Egg yolk; l; -α-phosphatidylcholine; EPG; Egg yolk; l; -α-phosphatidylglycerol; IR; Fourier transform infrared spectroscopy; LUV; Large unilamellar vesicles; MLV; Multilamellar vesicles; OMM; outer mitochondrial membrane; BPI; Bovine phosphatidylinositol.; POPE; 1-Palmitoyl-2-oleoyl-; sn; -glycero-3-phosphoethanolamine; T; _m; Temperature of the gel-to-liquid crystalline phase transition; TMA-DPH; 1-(4-Trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene; TMCL; 1,1′,2,2′-Tetramyristoyl-CardiolipinHarakiri; Fluorescence; Infrared; Lipid–protein interaction

Downregulation of the reduced folate carrier transport activity by phenobarbital-type cytochrome P450 inducers and protein kinase C activators by Sandra Halwachs; Carsten Kneuer; Walther Honscha (pp. 1671-1679).

The sodium dependent reduced folate carrier (Rfc1; Slc19a1) provides the major route for cellular uptake of reduced folates and antifolate drugs such as methotrexate (MTX) into various tissues. Despite its essential role in folate homeostasis and cancer treatment, little is known about Rfc1 regulation. A barbiturate recognition box, which as yet has only been found in the promoter region of xenobiotic metabolizing enzymes, particularly those of the CYP450 enzyme family, was predicted in the 5′ untranslated region of rat rfc1 cDNA. We have therefore investigated the regulation of Rfc1 by phenobarbital (PB)-type CYP450 inducers on the functional, transcriptional and translational level in a suitable in vitro model for rat liver. A decrease of >75% in substrate uptake was observed following treatment (48 h) with 1–10 times therapeutic plasma concentrations of PB-type CYP450 inducers like PB, carbamazepine, chlorpromazine, clotrimazole and with 0.1–1 ng/ml of the constitutive androstane receptor agonist TCPOBOP. This was not associated with reduced mRNA and protein levels. Further mechanistic investigations revealed that short-term treatment (2 h) of cells with protein phosphatase 1/2A inhibitor okadaic acid (80.5 ng/ml) and proteinkinase C inducer phorbol 12-myristate 13-acetate (PMA; 0.62 μg/ml) almost abolished Rfc1 mediated MTX uptake. Finally, the reduction in Rfc1 activity caused by PB, TCPOBOP and PMA was reversed by simultaneous incubation with the specific PKC inhibitor bisindolylmaleimide (BIM; 21 ng/ml). These results demonstrate that clinically relevant concentrations of PB-type CYP450 inducers cause a significant PKC-dependent reduction in Rfc1 uptake activity at the posttranscriptional level.

Keywords: Abbreviations; ALL; acute lymphoblastic leukemia; BHQ; black hole quencher; BIM; bisindolylmaleimide, bZIP, leucine-rich dimerization domain; DB-cAMP; dibutyryl-3′,5′cyclo-adenosinmonophosphate; cDNA; complementary DNA; CAR; constitutive androstane receptor; DMEM; Dulbecco's modified Eagle's medium; dNTP; 2′-desoxynucleoside-5′-triphosphate; FAM; 6-carboxyfluoresceine; HPCT; hepatocytoma; HRP; horseradish peroxidase; IC; inhibition constant; OA; okadaic acid; OATP; organic anion transporting polypeptide; PMA; phorbol-12-myristate-13-acetate; PBS; phosphate-buffered saline; RLU; relative light unit; SDS; sodiumdodecylsulfate; SEAP; secretory alkaline phosphatase; SLC; solute carrier; Sp; simian virus 40 protein; taq; thermus aquaticus; TCPOBOP; 1,4-bis[2-(3,5-dichloropyridyloxy)]-benzene; TexRed; Texas RedTransport; Regulation; CYP450 inducer; Proteinkinase C; Phenobarbital; Methotrexate

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BBA - Biomembranes (v.1768, #6)

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BBA - Biomembranes (v.1768, #6)