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BBA - Biomembranes (v.1778, #10)

Editorial Board (pp. ii).

Current perspectives in pulmonary surfactant — Inhibition, enhancement and evaluation by Yi Y. Zuo; Ruud A.W. Veldhuizen; A. Wilhelm Neumann; Nils O. Petersen; Fred Possmayer (pp. 1947-1977).

Pulmonary surfactant (PS) is a complicated mixture of approximately 90% lipids and 10% proteins. It plays an important role in maintaining normal respiratory mechanics by reducing alveolar surface tension to near-zero values. Supplementing exogenous surfactant to newborns suffering from respiratory distress syndrome (RDS), a leading cause of perinatal mortality, has completely altered neonatal care in industrialized countries. Surfactant therapy has also been applied to the acute respiratory distress syndrome (ARDS) but with only limited success. Biophysical studies suggest that surfactant inhibition is partially responsible for this unsatisfactory performance. This paper reviews the biophysical properties of functional and dysfunctional PS. The biophysical properties of PS are further limited to surface activity, i.e., properties related to highly dynamic and very low surface tensions. Three main perspectives are reviewed. (1) How does PS permit both rapid adsorption and the ability to reach very low surface tensions? (2) How is PS inactivated by different inhibitory substances and how can this inhibition be counteracted? A recent research focus of using water-soluble polymers as additives to enhance the surface activity of clinical PS and to overcome inhibition is extensively discussed. (3) Which in vivo, in situ, and in vitro methods are available for evaluating the surface activity of PS and what are their relative merits? A better understanding of the biophysical properties of functional and dysfunctional PS is important for the further development of surfactant therapy, especially for its potential application in ARDS.

Keywords: Abbreviations; ADSA; axisymmetric drop shape analysis; AFM; atomic force microscopy; ALI; acute lung injury; ARDS; acute respiratory distress syndrome; BLES; bovine lipid extract surfactant; CBS; captive bubble surfactometer; CSD; constrained sessile drop; DPPC; dipalmitoyl phosphatidylcholine; DPPG; dipalmitoyl phosphatidylglycerol; LE; liquid-expanded; LWB; Langmuir–Wilhelmy balance; PBS; pulsating bubble surfactometer; PC; phosphatidylcholine; PD; pendant drop; PEG; polyethylene glycol; PG; phosphatidylglycerol; PL; phospholipid(s); PMPC; palmitoyl–myristoyl phosphatidylcholine; POPC; palmitoyl–oleoyl phosphatidylcholine; POPG; palmitoyl–oleoyl phosphatidylglycerol; PS; pulmonary surfactant; RDS; respiratory distress syndrome; SP; surfactant protein; TC; tilted-condensed; TEM; transmission electron microscopy; T; _m; main transition temperature; π; surface pressure(s); π; _e; equilibrium spreading pressure; γ; surface tension(s); γ; _eq; equilibrium surface tension; γ; _min; and; γ; _max; minimum and maximum surface tensions (upon film oscillation), respectivelyPulmonary surfactant; Phospholipid; Surface activity; Surfactant inhibition; Water-soluble polymer; Tensiometry

The ADP and ATP transport in mitochondria and its carrier by Martin Klingenberg (pp. 1978-2021).

Different from some more specialised short reviews, here a general although not encyclopaedic survey of the function, metabolic role, structure and mechanism of the ADP/ATP transport in mitochondria is presented. The obvious need for an “old fashioned” review comes from the gateway role in metabolism of the ATP transfer to the cytosol from mitochondria. Amidst the labours, 40 or more years ago, of unravelling the role of mitochondrial compartments and of the two membranes, the sequence of steps of how ATP arrives in the cytosol became a major issue. When the dust settled, a picture emerged where ATP is exported across the inner membrane in a 1:1 exchange against ADP and where the selection of ATP versus ADP is controlled by the high membrane potential at the inner membrane, thus uplifting the free energy of ATP in the cytosol over the mitochondrial matrix. Thus the disparate energy and redox states of the two major compartments are bridged by two membrane potential responsive carriers to enable their symbiosis in the eukaryotic cell. The advance to the molecular level by studying the binding of nucleotides and inhibitors was facilitated by the high level of carrier (AAC) binding sites in the mitochondrial membrane. A striking flexibility of nucleotide binding uncovered the reorientation of carrier sites between outer and inner face, assisted by the side specific high affinity inhibitors. The evidence of a single carrier site versus separate sites for substrate and inhibitors was expounded. In an ideal setting principles of transport catalysis were elucidated. The isolation of intact AAC as a first for any transporter enabled the reconstitution of transport for unravelling, independently of mitochondrial complications, the factors controlling the ADP/ATP exchange. Electrical currents measured with the reconstituted AAC demonstrated electrogenic translocation and charge shift of reorienting carrier sites. Aberrant or vital para-functions of AAC in basal uncoupling and in the mitochondrial pore transition were demonstrated in mitochondria and by patch clamp with reconstituted AAC. The first amino acid sequence of AAC and of any eukaryotic carrier furnished a 6-transmembrane helix folding model, and was the basis for mapping the structure by access studies with various probes, and for demonstrating the strong conformation changes demanded by the reorientation mechanism. Mutations served to elucidate the function of residues, including the particular sensitivity of ATP versus ADP transport to deletion of critical positive charge in AAC. After resisting for decades, at last the atomic crystal structure of the stabilised CAT–AAC complex emerged supporting the predicted principle fold of the AAC but showing unexpected features relevant to mechanism. Being a snapshot of an extreme abortive “c-state” the actual mechanism still remains a conjecture.

Keywords: Abbreviations; AAC; ADP/ATP carrier; BHM; bovine heart mitochondria; SMP; submitochondrial particles; ATR; atractyloside; CAT; carboxyatractyloside; BKA; bongkrekic acid; AdN; adenine nucleotides; T; ATP; D; ADP; PP; _i; pyrophosphate; MCF; mitochondrial carrier family; TMH; transmembrane helix; C-loop; cytosol loop; M-loop; matrix loop; ITF; induced transition fit; SBCGP; single binding center gated pore; EAM; eosine maleimide; HTS; hydroxylapatite; PL; phospholipid; CL; cardiolipin; FA; fatty acid; LAPAO; lauroyl propyl aminoxide; LDAO; lauroyl diamine oxide; PEG; polyethylene glycol; PheG; phenylglyoxal; Fo; formycin; CrK; creatine kinase; CrP; creatine phosphate; CyP; cyclophilin; CsA; cyclosporin; HFS; hyperfine splitting; VDAC; voltage dependent anion channel; NEM; N; -ethyl maleimide; DTNB; 2,2′ dinitro dithiodibenzoate; DAN; 1,5 dimethylamino-naphtoyl; N; naphtoylADP/ATP transport; Mitochondrial carrier; Biomembrane; Transport mechanism; Carrier structure

Effect of sphingomyelinase treatment on ligand binding activity of human serotonin_1A receptors by Md. Jafurulla; Thomas J. Pucadyil; Amitabha Chattopadhyay (pp. 2022-2025).

The serotonin_1A receptor is an important member of the G-protein coupled receptor family, and is involved in the generation and modulation of a variety of cognitive, behavioral, and developmental functions. We have monitored the ligand binding of the human serotonin_1A receptor stably expressed in CHO cells (termed CHO-5-HT_1AR) following treatment with sphingomyelinase (SMase), an enzyme that specifically catalyzes the hydrolysis of sphingomyelin into ceramide and phosphorylcholine. Our results show, for the first time, that the specific ligand binding activity of the serotonin_1A receptor in membranes isolated from CHO-5-HT_1AR cells is increased upon sphingomyelinase treatment. Saturation binding analysis reveals increase in binding affinity of the receptor under these conditions. This is accompanied by a reduction in membrane order, as monitored by fluorescence anisotropy of the membrane probe 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) in intact cells. These results represent the first report on the effect of sphingomyelinase treatment on the ligand binding activity of this important neurotransmitter receptor.

Keywords: Serotonin; _1A; receptor; Sphingomyelin; Sphingomyelinase; Ligand binding activity; Fluorescence anisotropy

Gramicidin A-based peptide vector for intracellular protein delivery by Tatiana B. Stoilova; Sergey I. Kovalchuk; Natalya S. Egorova; Andrey Y. Surovoy; Vadim T. Ivanov (pp. 2026-2031).

The development of the peptide-based vectors for the intracellular delivery of biologically active macromolecules has opened new prospects of their application in research and therapy. Earlier the amphipathic cell-penetrating peptide (CPP) Pep-1 was reported to mediate cellular uptake of proteins without covalent binding to them. In this work we studied the ability of a series of membrane-active amphipathic peptides, based on the gramicidin A sequence, to transport a model protein across the eukaryotic cell membrane. Among them the positively charged Cys-containing peptide P10C demonstrated the most effective β-galactosidase intracellular delivery. Besides, this peptide was shown to form noncovalent associates with β-galactosidase as judged from electrophoresis and enzymatic activity assays. In addition, a series of new gramicidin analogues were prepared and the effect of N-terminus modification of gramicidin on the protein transduction efficiency was studied.

Keywords: Abbreviations; CPP; cell-penetrating peptide; PTD; protein transduction domain; NLS; nuclear localization sequence; SV-40; simian virus 40; Tat; CPP, derived from HIV-1; β-Gal; β-galactosidase; oNPG; o; -nitrophenil-β-; d; -galactoside; X-Gal; 5-bromo-4-chloro-3-indolyl-β-; d; -galactoside; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DMEM; Dulbecco's Modified Eagle's Medium; FBS; fetal bovine serum; PBS; phosphate-buffered saline solution; HPLC; high performance liquid chromatography; HOBt; 1-hydroxybenzotriazole; DIC; diisopropylcarbodiimide; Fmoc; fluorenylmethoxycarbonyl; MALDI-TOF; matrix-assisted laser desorption ionization time-of-flight; PAGE; polyacrylamide gel electrophoresisGramicidin A; Cell-penetrating peptide; Protein delivery; Amphipathic peptides

Molecular weight dependence of the depletion attraction and its effects on the competitive adsorption of lung surfactant by Patrick C. Stenger; Stephen G. Isbell; Joseph A. Zasadzinski (pp. 2032-2040).

Albumin competes with lung surfactant for the air–water interface, resulting in decreased surfactant adsorption and increased surface tension. Polyethylene glycol (PEG) and other hydrophilic polymers restore the normal rate of surfactant adsorption to the interface, which re-establishes low surface tensions on compression. PEG does so by generating an entropic depletion attraction between the surfactant aggregates and interface, reducing the energy barrier to adsorption imposed by the albumin. For a fixed composition of 10 g/L (1% wt.), surfactant adsorption increases with the 0.1 power of PEG molecular weight from 6 kDa–35 kDa as predicted by simple excluded volume models of the depletion attraction. The range of the depletion attraction for PEG with a molecular weight below 6 kDa is less than the dimensions of albumin and there is no effect on surfactant adsorption. PEG greater than 35 kDa reaches the overlap concentration at 1% wt. resulting in both decreased depletion attraction and decreased surfactant adsorption. Fluorescence images reveal that the depletion attraction causes the surfactant to break through the albumin film at the air–water interface to spread as a monolayer. During this transition, there is a coexistence of immiscible albumin and surfactant domains. Surface pressures well above the normal equilibrium surface pressure of albumin are necessary to force the albumin from the interface during film compression.

Keywords: Competitive adsorption; Pulmonary surfactant; Inactivation; Inhibition; Polymers; Depletion attraction

Oxidized phospholipids as potential molecular targets for antimicrobial peptides by Juha-Pekka Mattila; Karen Sabatini; Paavo K.J. Kinnunen (pp. 2041-2050).

The effects of oxidatively modified phospholipids on the association with model biomembranes of four antimicrobial peptides (AMPs), temporin B and L, indolicidin, and LL-37(F27W) were studied by Langmuir balance and fluorescence spectroscopy. In keeping with previous reports the negatively charged phospholipid phosphatidylglycerol (PG) enhanced the intercalation of all four peptides into lipid monolayers and liposomal bilayers under low ionic strength conditions. Interestingly, similar effect was observed for 1-palmitoyl-2-(9′-oxo-nonanoyl)- sn-glycero-3-phosphocholine (PoxnoPC), a zwitterionic oxidized phospholipid bearing an aldehyde function at the end of its truncated sn-2 acyl chain. Instead, the structurally similar 1-palmitoyl-2-azelaoyl- sn-glycero-3-phosphocholine (PazePC) containing a carboxylic moiety was less efficient in promoting the membrane association of these peptides. Physiological saline reduced the binding of the above peptides to membranes containing PG, whereas interactions with PoxnoPC were found to be insensitive to ionic strength. Notably, membrane intercalation of temporin L, the most surface active of the above peptides could be into PoxnoPC containing monolayers was strongly attenuated by methoxyamine, suggesting the importance of Schiff base formation between peptide amino groups and the lipid aldehyde function. PoxnoPC and similar aldehyde bearing oxidatively modified phospholipids could represent novel molecular targets for AMPs.

Keywords: Abbreviations; AMP; antimicrobial peptide; LUV; large unilamellar vesicle; oxPL; oxidized phospholipid; PazePC; 1-palmitoyl-2-azelaoyl-; sn; -glycero-3-phosphocholine; PoxnoPC; 1-palmitoyl-2-(9′-oxo-nonanoyl)-; sn; -glycero-3-phosphocholine; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DMPG; 1,2-dimyristoyl-; sn; -glycero-3-phospho-; rac; -1-glycerol; PC; phosphatidylcholine; PG; phosphatidylglycerol; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phospho-; rac; -1-glycerol; RFI; relative fluorescence intensity; ROS; reactive oxygen species; K; _SV; Stern–Volmer quenching constant; X; _Y; mole fraction of compound Y; π; surface pressure; π; ₀; initial surface pressure; π; _c; critical packing pressure; Δ; π; change of surface pressureMonolayer penetration; Peptide–lipid interactions; Tryptophan fluorescence; Reactive oxygen species

Physico-chemical and biophysical study of the interaction of hexa- and heptaacyl lipid A from Erwinia carotovora with magainin 2-derived antimicrobial peptides by Satoshi Fukuoka; Jörg Howe; Jorg Andra Jörg Andrä; Thomas Gutsmann; Rossle Manfred Rössle; Klaus Brandenburg (pp. 2051-2057).

The neutralization of endotoxin structures such as the active ‘endotoxic principle’ lipid A by suitable compounds has been shown to be a key step in the treatment of infectious diseases, in particular in the case of Gram-negative bacteria which frequently may lead to the septic shock syndrome. An effective antimicrobial peptide, originally found in the skin of an African frog, is magainin 2. Here, the interaction of magainin 2-amide and a peptide derived thereof, M2V, with chemically defined and homogeneous hexaacyl and heptaacyl lipids A isolated from LPS of Erwinia carotovora, was investigated. By using Fourier-transform infrared spectroscopy, the gel to liquid crystalline phase transition of the acyl chains of lipid A and the conformation of their phosphate groups due to peptide binding was investigated. The former parameter was also determined by using differential scanning calorimetry. The electrophoretic mobility of lipid A aggregates under the influence of the peptides was studied to determine the Zeta potential, and small-angle X-ray scattering was applied for the elucidation of the types of aggregate structures in the absence and presence of the peptides. The lipid A-induced cytokine production in human mononuclear cells shows that the ability of the two peptides to inhibit a tumor necrosis factor-α production correlates with characteristic changes of the biophysical parameters. These are much stronger expressed for the peptide M2V than for magainin 2-amide, which apparently is connected with the higher number of positive as well as more hydrophobic amino acids, leading to a stronger amphiphilicity necessary to neutralize the amphiphilic lipid A aggregates.

Keywords: Abbreviations; AMP; antimicrobial peptide; LPS; lipopolysaccharide; LBP; lipopolysaccharide-binding protein; MNC; human mononuclear cells; M2; magainin 2-amide; M2V; magainin 2-amide derived peptide; SAXS; small-angle X-ray scattering; PMB; polymyxin BLipopolysaccharide; Lipid A; Antimicrobial peptide; Endotoxin neutralization; Cytokine

Nanoscale membrane activity of surfactins: Influence of geometry, charge and hydrophobicity by Grégory Francius; Samuel Dufour; Magali Deleu; Michel Paquot; Marie-Paule Mingeot-Leclercq; Dufrene Yves F. Dufrêne (pp. 2058-2068).

We used real-time atomic force microscopy (AFM) to visualize the interactions between supported lipid membranes and well-defined surfactin analogs, with the aim to understand the influence of geometry, charge and hydrophobicity. AFM images of mixed dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers recorded after injection of cyclic surfactin at 1 mM, i.e. well-above the critical micelle concentration, revealed a complete solubilization of the bilayers within 30 min. A linear analog having the same charge and acyl chains was able to solubilize DOPC, but not DPPC, and to promote redeposition leading eventually to a new bilayer. Increasing the charge of the polar head or the length of the acyl chains of the analogs lead to the complete solubilization of both DOPC and DPPC, thus to a stronger membrane activity. Lastly, we found that at low surfactin concentrations (40 µM), DPPC domains were always resistant to solubilization. These data demonstrate the crucial role played by geometry, charge and hydrophobicity in modulating the membrane activity (solubilization, redeposition) of surfactin. Also, this study suggests that synthetic analogs are excellent candidates for developing new surfactants with tunable, well-defined properties for medical and biotechnological applications.

Keywords: AFM; Analogs; Micelles; Supported lipid membranes; Surfactin

The pre-transmembrane region of the HCV E1 envelope glycoprotein by Perez-Berna Ana J. Pérez-Berná; Angela Bernabeu; Miguel R. Moreno; Guillen Jaime Guillén; Villalain José Villalaín (pp. 2069-2080).

The previously identified membranotropic regions of the HCV E1 envelope glycoprotein, a class II membrane fusion protein, permitted us to identify different sequences which might be implicated in viral membrane fusion, membrane interaction and/or protein–protein binding. HCV E1 glycoprotein presents a membrano-active region immediately adjacent to the transmembrane segment, which could be involved in membrane destabilization similarly to the pre-transmembrane domains of class I fusion proteins. Consequently, we have carried out a study of the binding and interaction with the lipid bilayer of a peptide corresponding to segment 309–340, peptide E1_PTM, as well as the structural changes which take place in both the peptide and the phospholipid molecules induced by the binding of the peptide to the membrane. Here we demonstrate that peptide E1_PTM strongly partitions into phospholipid membranes, interacts with negatively-charged phospholipids and locates in a shallow position in the membrane. These data support its role in HCV-mediated membrane fusion and suggest that the mechanism of membrane fusion elicited by class I and II fusion proteins might be similar.

Keywords: Abbreviations; 5NS; 5-Doxyl-stearic acid; 16NS; 16-Doxyl-stearic acid; BPS; Bovine brain; l; -α-phosphatidylserine; CF; 5-Carboxyfluorescein; Chol; Cholesterol; di-8-ANEPPS; 4-(2-(6-(Dioctylamino)-(2-naphthalenyl)-(ethenyl)-1-(3-sulfopropyl)-pyridinium inner salt; DMPA; 1,2-Dimyristoyl-; sn; -glycero-phosphatidic acid; DMPC; 1,2-Dimyristoyl-; sn; -glycero-phosphatidylcholine; DMPG; 1,2-Dimyristoyl-; sn; -glycero-phosphatidylglycerol; DMPS; 1,2-Dimyristoyl-; sn; -glycero-3-phosphatidylserine; DPH; 1,6-Diphenyl-1,3,5-hexatriene; EPA; Egg; l; -α-phosphatidic acid; EPC; Egg; l; -α-phosphatidylcholine; EPG; Egg; l; -α-phosphatidylglycerol; ER; Endoplasmic reticulum; FD10/20; Fluorescein isothiocyanate dextran with an average molecular weight of 10,000/20,000; FPE; fluorescein-phosphatidylethanolamine; HCV; Hepatitis C virus; HIV; Human immunodeficiency virus; LPC; Lyso-α-phosphatidylcholine; LUV; Large Unilamellar Vesicles; MLV; Multilamellar Vesicles; NBD-PE; N; -(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-; sn; -glycero-phosphoethanolamine; N-RhB-PE; Lissamine™ rhodamine B 1,2-dihexadecanoyl-; sn; -glycero-3-phosphoethanolamine; PTM; Pre-transmembrane domain; SM; Egg sphingomyelin; T; _m; Temperature of the gel-to-liquid crystalline phase transition; TM; Transmembrane domain; TMA-DPH; 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene; TPE; Egg trans-esterified; l; -α-phosphatidylethanolaminePeptide–lipid interaction; E1 glycoprotein; HCV; Pre-transmembrane

Probing the steric barrier of nonionic surfactant vesicles with melittin by Richard D. Harvey; M. Jayne Lawrence; Tam T. Bui (pp. 2081-2090).

The role of the surface polymer brush of nonionic surfactant vesicles (NSV) in inhibiting interactions with small membrane-perturbing molecules was investigated using the bee venom peptide melittin as a probe. The interaction between melittin and NSV was compared with that of distearoylphosphatidylcholine (DSPC) vesicles and sterically stabilised liposomes (SSL) containing 5 mol% pegylated distearoylphosphatidylethanolamine (DSPE.E₄₄). The degree of melittin interaction with the various vesicles was determined by measuring peptide binding and folding, using intrinsic tryptophan fluorescence and circular dichroism respectively, in addition to monitoring the release of encapsulated carboxyfluorescein dye. NSV composed of 1,2-di- O-octadecyl- rac-glyceryl-3-(ω-dodecaethylene glycol) (2C₁₈E₁₂) showed a strong affinity for melittin, whilst exhibiting ~50% less bound peptide than SSL. 2C₁₈E₁₂:Chol vesicles showed reduced melittin interaction, in a manner consistent with Chol incorporation into DSPC vesicles. These results are discussed with respect to the effect of Chol on the in-plane order of 2C₁₈E₁₂ bilayers and consequent attenuation of hydrophobic interactions with the peptide. NSV formed from equimolar mixtures of polyoxyethylene- n-stearoyl ethers C₁₈E₂ and C₁₈E₂₀ showed a greater interaction with melittin than 2C₁₈E₁₂. However, replacing C₁₈E₂₀ with C₁₈E₁₀ was sufficient to achieve an attenuation of melittin interaction similar to that observed in 2C₁₈E₁₂:Chol vesicles. This indicates that the presence of surface polymer brush alone may confer resistance to melittin, provided hydrophobic interactions between the peptide and the vesicles can be minimised, through improved in-plane bilayer order.

Keywords: Abbreviations; PEG; polyethylene glycol; EPC; 1,2-Diacyl-; sn; -glycero-3-phosphocholine from egg yolk; DSPC; disteroylphosphatidylcholine; DSPE.E; ₄₄; 1,2-Distearoyl-; sn; -glycero-3-phosphoethanolamine-; N; -[methoxy(polyethylene glycol)-2000]; Chol; cholesterol; 2C; ₁₈; E; ₁₂; 1,2-di-; O; -octadecyl-; rac; -glyceryl-3-(; ω; -dodecaethylene glycol); C; ₁₈; E; ₂; polyoxyethylene-2-stearoyl ether; C; ₁₈; E; ₁₀; polyoxyethylene-10-stearoyl ether; C; ₁₈; E; ₂₀; polyoxyethylene-20-stearoyl ether; 5(6) carboxyfluorescein; (CF); CD; circular dichroism; R; _i; lipid:peptide ratioNiosome; Poly(ethylene glycol); Drug delivery; Melittin; Membrane perturbation; Molecular packing

The effects of lipids on the structure of the eukaryotic cytolysin equinatoxin II: A synchrotron radiation circular dichroism spectroscopic study by Andrew J. Miles; Alison Drechsler; Katarina Kristan; Gregor Anderluh; Raymond S. Norton; B.A. Wallace; Frances Separovic (pp. 2091-2096).

Synchrotron radiation circular dichroism (SRCD) spectroscopy studies of the eukaryotic pore-forming protein equinatoxin II (EqtII) were carried out in solution and in the presence of micelles or small unilamellar vesicles (SUV) of different lipid composition. The SRCD structural data was correlated with calcein leakage from SUV and with partitioning of EqtII to liposomes, and micelles, according to haemolysis assays. The structure of EqtII in water and dodecylphosphocholine micelles as determined by SRCD was similar to the values calculated from crystal and solution structures of the protein, and no changes were observed with the addition of sphingomyelin (SM). SM is required to trigger pore formation in biological and model membranes, but our results suggest that SM alone is not sufficient to trigger dissociation of the N-terminal helix and further structural rearrangements required to produce a pore. Significant changes in conformation of EqtII were detected with unsaturated phospholipid (DOPC) vesicles when SM was added, but not with saturated phospholipids (DMPC), which suggests that not only is membrane curvature important, but also the fluidity of the bilayer. The SRCD data indicated that the EqtII structure in the presence of DOPC:SM SUV represents the ‘bound’ state and the ‘free’ state is represented by spectra for DOPC or DOPC:Chol vesicles, which correlates with the high lytic activity for SUV of DOPC:SM. The SRCD results provide insight into the lipid requirements for structural rearrangements associated with EqtII toxicity and lysis.

Keywords: Cytolysin; Synchrotron radiation circular dichroism (SRCD) spectroscopy; Secondary structure; Protein–lipid interactions; Equinatoxin II; Phospholipid membrane

Implication of connexins 40 and 43 in functional coupling between mouse cardiac fibroblasts in primary culture by Claire Louault; Najate Benamer; Jean-François Faivre; Daniel Potreau; Jocelyn Bescond (pp. 2097-2104).

Cardiac fibroblasts contribute to the structure and function of the myocardium. However their involvement in electrophysiological processes remains unclear; particularly in pathological situations when they proliferate and develop fibrosis. We have identified the connexins involved in gap junction channels between fibroblasts from adult mouse heart and characterized their functional coupling. RT-PCR and Western blotting results show that mRNA and proteins of connexin40 and connexin43 are expressed in cultured cardiac fibroblasts, while Cx45 is not detected. Analysis of gap junctional communications established by these connexins with the gap-FRAP technique demonstrates that fibroblasts are functionally coupled. The time constant of permeability, k, calculated from the fluorescence recovery curves between cell pairs is 0.066±0.005 min−¹ ( n=65). Diffusion analysis of Lucifer Yellow through gap junction channels with the scrape-loading method demonstrates that when they are completely confluent, a majority of fibroblasts are coupled forming an interconnecting network over a distance of several hundred micrometers. These data show that cardiac fibroblasts express connexin40 and connexin43 which are able to establish functional communications through homo and/or heterotypic junctions to form an extensive coupled cell network. It should then be interesting to study the conditions to improve efficiency of this coupling in pathological conditions.

Keywords: Cardiac fibroblast; Connexin40; Connexin43; Gap junction communication

Dipalmitoylphosphatidylcholine membranes modified with carotenoid pigment lutein: Experiment versus Monte Carlo simulation study of the membrane organization by Witold Okulski; Agnieszka Sujak; Wiesław I. Gruszecki (pp. 2105-2118).

Organization of bilayer lipid membrane formed with dipalmitoylphosphatidylcholine and containing a xanthophyll pigment lutein was studied by both the Monte Carlo simulation and UV-Vis absorption spectroscopy. The simulations were based on ten-state Pink model. The proposed model consisted of two monolayers represented by a two-dimensional triangular lattice with vacancies. The orientation and aggregation state of lutein, obtained from the analysis of the spectroscopic measurements, were used to calibrate intermolecular interactions in the model. In accordance with the experimental data, the model allows two orientations of lutein molecules: one spanning the membrane and the other parallel to its plane. The influence of the intermolecular interactions on the main phase transition as well as on the aggregation of lutein molecules is discussed. The analysis of the model enables us to learn about molecular mechanisms that govern the effects of lutein on the membrane properties as well as the effects of the lipid matrix on lutein organization in the membrane. A concept is discussed according to which increasing domination of parallel lutein orientation, observed at high temperatures, can protect the membrane against penetration by water molecules and reactive oxygen species and against loss of the membrane compactness, especially in the regions of oxidized acyl chains.

Keywords: Abbreviations; DPPC; dipalmitoylphosphatidylcholine; LUT; lutein; LUT; _⊥; lutein spanning the membrane; LUT; _||; lutein oriented parallel to the surface of the lipid membraneCarotenoid; Lutein; Lipid membrane; Absorption spectroscopy; Monte Carlo simulation; Molecular aggregate

Degradation of lyophilized lipid/DNA complexes during storage: The role of lipid and reactive oxygen species by Marion d.C. Molina; Thomas J. Anchordoquy (pp. 2119-2126).

The presence of trace amounts of metal ions in nonviral vector formulations can significantly affect the stability of lipid/DNA complexes (lipoplexes) during acute freeze-drying. The goal of the present study was to evaluate the generation of reactive oxygen species (ROS) in dried formulations of lipoplexes and in their individual components (lipid or naked DNA). The experiments were conducted in the presence or absence of a transition metal (Fe²⁺). Lipoplexes and their individual components were formulated in trehalose and subjected to lyophilization and stored for a period of up to 2 months at +60 °C. Physico-chemical characteristics and biological activity were evaluated at different time intervals. Generation of ROS during storage was determined by adding a fluorescence probe to the formulations prior to freeze-drying. We also monitored the formation of thiobarbituric reactive substances (TBARS). Our results show that ROS and TBARS form during storage in the dried state. Our findings also suggest that degradation is more rapid in the presence of lipid, even in the absence of metal. We also showed that dried naked DNA formulations are more stable without the lipid component. Effective strategies are then needed to minimize the formation and accumulation of oxidative damage of lipoplexes during storage.

Keywords: Nonviral vectors; Lyophilization; Metal contamination; Reactive oxygen species; Thiobarbituric acid-reactive substances; Gene delivery

Structural and functional properties of peptides based on the N-terminus of HIV-1 gp41 and the C-terminus of the amyloid-beta protein by Larry M. Gordon; Alex Nisthal; Andy B. Lee; Sepehr Eskandari; Piotr Ruchala; Chun-Ling Jung; Alan J. Waring; Patrick W. Mobley (pp. 2127-2137).

Given their high alanine and glycine levels, plaque formation, α-helix to β-sheet interconversion and fusogenicity, FP (i.e., the N-terminal fusion peptide of HIV-1 gp41; 23 residues) and amyloids were proposed as belonging to the same protein superfamily. Here, we further test whether FP may exhibit ‘amyloid-like’ characteristics, by contrasting its structural and functional properties with those of Aβ(26–42), a 17-residue peptide from the C-terminus of the amyloid-beta protein responsible for Alzheimer's. FTIR spectroscopy, electron microscopy, light scattering and predicted amyloid structure aggregation (PASTA) indicated that aqueous FP and Aβ(26–42) formed similar networked β-sheet fibrils, although the FP fibril interactions were weaker. FP and Aβ(26–42) both lysed and aggregated human erythrocytes, with the hemolysis-onsets correlated with the conversion of α-helix to β-sheet for each peptide in liposomes. Congo red (CR), a marker of amyloid plaques in situ, similarly inhibited either FP- or Aβ(26–42)-induced hemolysis, and surface plasmon resonance indicated that this may be due to direct CR-peptide binding. These findings suggest that membrane-bound β-sheets of FP may contribute to the cytopathicity of HIV in vivo through an amyloid-type mechanism, and support the classification of HIV-1 FP as an ‘amyloid homolog’ (or ‘amylog’).

Keywords: Abbreviations; HIV-1; Human Immunodeficiency Virus, Type 1; HIV-2; Human Immunodeficiency Virus, Type 2; gp41; glycoprotein 41,000 of HIV-1; FP; fusion peptide (23-residues) at the N-terminus of HIV-1 gp41 (LAV; _1a; strain); SIV; simian immunodeficiency virus; gp32; glycoprotein 32,000 of HIV-2/SIV; Aβ; amyloid-beta protein; Aβ(1–40) or Aβ(1–42); amyloid-beta protein residues 1–40 or 1–42; Aβ(26–42); C-terminal peptide 26–42 of amyloid-beta protein; DLS; dynamic light scattering; TEM; transmission electron microscopy; PIRA; parallel β-sheet, in register arrangement; PASTA; prediction of amyloid structure aggregation; PBS; phosphate-buffered saline, Phosphate buffer, 10 mM sodium phosphate buffer, pH 7.4; HBS-EP buffer; 10 mM Hepes, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005%, surfactant P20; DMSO; dimethyl sulfoxide; HFIP; hexafluoroisopropanol; SDS; sodium dodecyl sulfate; TFE; trifluoroethanol; RBC; red blood cells; POPG; 1-palmitoyl-2-oleoyl phosphatidylglycerol; LUV; large unilamellar vesicles; POPC; 1-palmitoyl-2-oleoylphosphatidylcholine; POPG; 1-palmitoyl-2-oleoylphosphatidylglycerol; P/L; peptide/lipid molar ratio; CD; circular dichroism; FTIR; Fourier transform infrared; ATR; attenuated-total-reflectance; 2D-NMR; two dimensional Nuclear Magnetic Resonance; IR; ₅₀; inhibitory ratio (IR) of the agent concentration to that the membrane-active agent that induces 50% inhibition; IR; ₁₀₀; inhibitory ratio (IR) of the agent concentration to that the membrane-active agent that induces 100% inhibition; ID; ₅₀; inhibitor concentration that yields 50% inhibition for a stated dose of membrane-active agent; PrP; prion protein; HPrP; human prion protein; BPrP; bovine prion protein; MD; molecular dynamics; SPR; surface plasmon resonance; CR; Congo redFourier transform infrared; Electron microscopy; Light scattering; Aggregation; Hemolysis; Prion; Fibrils; Congo red; Surface plasmon resonance; AIDS

Function of taurine transporter (Slc6a6/TauT) as a GABA transporting protein and its relevance to GABA transport in rat retinal capillary endothelial cells by Masatoshi Tomi; Ayumi Tajima; Masanori Tachikawa; Ken-ichi Hosoya (pp. 2138-2142).

The purpose of this study was to identify the uptake mechanism of γ-aminobutyric acid (GABA) via taurine transporter (Slc6a6/TauT) and its relationship with GABA transport at the inner BRB. Rat Slc6a6/TauT-transfected HeLa cells exhibited Na⁺-, Cl⁻-, and concentration-dependent [³H]GABA uptake with a K_m of 1.5 mM. Taurine, β-alanine, and GABA markedly inhibited Slc6a6/TauT-mediated uptake of [³H]GABA. The uptake of [³H]GABA by a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) was Na⁺-, Cl⁻-, and concentration-dependent with a K_m of 2.0 mM. This process was more potently inhibited by substrates of Slc6a6/TauT, taurine and β-alanine, than those of GABA transporters, GABA and betaine. In the presence of taurine, there was competitive inhibition with a K_i of 74 μM. [³H]Taurine also exhibited competitive inhibition with a K_i of 1.8 mM in the presence of GABA. In conclusion, rat Slc6a6/TauT has the ability to use GABA as a substrate and Slc6a6/TauT-mediated GABA transport appears to be present at the inner BRB.

Keywords: γ-Aminobutyric acid; Taurine transporter; Carrier-mediated transport; Blood-retinal barrier; Retinal capillary endothelial cell

A novel catalytic mechanism for ATP hydrolysis employed by the N-terminal nucleotide-binding domain of Cdr1p, a multidrug ABC transporter of Candida albicans by Versha Rai; Manisha Gaur; Antresh Kumar; Sudhanshu Shukla; Sneha Sudha Komath; Rajendra Prasad (pp. 2143-2153).

Although essentially conserved, the N-terminal nucleotide-binding domain (NBD) of Cdr1p and other fungal transporters has some unique substitutions of amino acids which appear to have functional significance for the drug transporters. We have previously shown that the typical Cys193 in Walker A as well as Trp326 and Asp327 in the Walker B of N-terminal NBD (NBD-512) of Cdr1p has acquired unique roles in ATP binding and hydrolysis. In the present study, we show that due to spatial proximity, fluorescence resonance energy transfer (FRET) takes place between Trp326 of Walker B and MIANS [2-(4-maleimidoanilino) naphthalene-6-sulfonic acid] on Cys193 of Walker A motif. By exploiting FRET, we demonstrate how these critical amino acids are positioned within the nucleotide-binding pocket of NBD-512 to bind and hydrolyze ATP. Our results show that both Mg²⁺ coordination and nucleotide binding contribute to the formation of the active site. The entry of Mg²⁺ into the active site causes the first large conformational change that brings Trp326 and Cys193 in close proximity to each other. We also show that besides Trp326, typical Glu238 in the Q-loop also participates in coordination of Mg²⁺ by NBD-512. A second conformational change is induced when ATP, but not ADP, docks into the pocket. Asn328 does sensing of the γ-phosphate of the substrate in the extended Walker B motif, which is essential for the second conformational change that must necessarily precede ATP hydrolysis. Taken together our results imply that the uniquely placed residues in NBD-512 have acquired critical roles in ATP catalysis, which drives drug extrusion.

Keywords: Abbreviations; ABC; ATP binding cassette; MFS; M; ajor; F; acilitator; S; uperfamily; ATP; adenosine triphosphate; DTE; dithioerythritol; FRET; fluorescence resonance energy transfer; MIANS; 2′-(4′-maleimidylanilino)naphthalene-6-sulfonic acid; NBD; nucleotide-binding domain; TMD; transmembrane domain; NEM; N; -ethylmaleimide; NBS; N; -bromosuccinimideATP binding cassette; ATP hydrolysis; Fluorescence resonance energy transfer; Multidrug resistance; Nucleotide-binding domain

Cholesterol's decoupling effect on membrane partitioning and permeability revisited: Is there anything beyond Fick's law of diffusion? by Andreas Missner; Andreas Horner; Peter Pohl (pp. 2154-2156).

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

Keywords: Weak acids; Scanning electrochemical microscopy; Membrane transport; Planar bilayers

Thallium(III)-mediated changes in membrane physical properties and lipid oxidation affect cardiolipin–cytochrome c interactions by Lis del Carmen Puga Molina; Sandra Viviana Verstraeten (pp. 2157-2164).

Trivalent thallium (Tl(III)) is a highly toxic heavy metal through not completely understood mechanisms. Previously, we demonstrated that Tl(III) causes mitochondrial depolarization in PC12 cells leading to a decrease in cell viability. Given the role of the phospholipid cardiolipin (CL) in mitochondrial events, we evaluated in vitro the short- (2 min) and long- (60 min) time effects of Tl(III) (1–75 μM) on CL-containing membranes physical properties, and the consequences on cytochrome c binding to CL. After 2 min of incubation, Tl(III) significantly decreased liposome surface potential, lipid packing, and hydration of phosphatidylcholine:CL liposomes, while CL pK₂ decreased from 9.8 to 8.2. The magnitude of these changes was even higher after 60 min of incubation. While no Tl(III) was found bound to membranes, Tl(I) was present in the samples. Accordingly, significant oxidative damage to both CL fatty acids and polar headgroup was observed. Cytochrome c binding to CL was decreased in Tl(III)-treated liposomes. The present results indicate that Tl(III) interaction with CL-containing membranes affected their physical properties, caused lipid oxidation and CL hydrolysis, and resulted in a decrease of cytochrome c binding. If occurring in vivo, these effects of Tl(III) could partially account for mitochondrial dysfunction in cells exposed to this metal.

Keywords: Abbreviations; 10-PyrPC; 1-hexadecanoyl-2-(1-pyrenedecanoyl)-; sn; -glycero-3-phosphocholine; 16-AP; 16-(9-anthroyloxy) palmitic acid; 6-AS; 6-(9-anthroyloxy) stearic acid; ANS; 8-anilino-1-naphthalene-sulfonic acid; CL; cardiolipin; GP; generalized polarization; HHC; 4-heptadecyl-7-hydroxycoumarin; HPTLC; high performance thin layer chromatography; Laurdan; 6-dodecanoyl-2-dimethylaminonaphthalene; PC; phosphatidylcholine; PG; phosphatidylglycerol; TBARS; 2-thiobarbituric acid reactive substancesThallium; Liposomes; Cardiolipin; Membrane fluidity; Cytochrome; c; Lipid oxidation

Psychotropic drugs interfere with the tight coupling of polyphosphoinositide cycle metabolites in human platelets: A result of receptor-independent drug intercalation in the plasma membrane? by Ramadhan Oruch; Erlend Hodneland; Ian F. Pryme; Holm Holmsen (pp. 2165-2176).

Incubation of platelets with increasing concentrations of thrombin produced large amounts of phosphatidic acid (PA) and distinct changes in phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP₂), prominent metabolites in the polyphosphoinositide (PPI) cycle. The relation between normalized PA and PIP or PIP₂ levels in such thrombin-treated platelets from 22 normal donors gave a very similar pattern, suggesting tight control of the metabolites in the polyphosphoinositide (PPI) cycle. Prochlorperazine (PCP), trifluoperazine (TFP), haloperidol (HPD), quetiapine (QTP), pimozide (PMZ) and clozapine (CLO) interfered with this tight coupling produced by treating platelets with increasing thrombin concentrations. All drugs decreased the formation of PA at a given thrombin concentration, a decrease that varied greatly among platelets from different donors. This made it difficult to treat the PIP/PA and PIP₂/PA relationships with ordinary, descriptive statistics. The data were therefore subjected to regression analysis using polynomials of second or first degree and gave the interference ranking order: PCP>TFP≫PMZ=HPD>CLO>QTP. All six drugs increased the mean molecular area of monolayers of dipalmitoyl phosphatidylserine on pure water at 37 °C by 20–50%, while they had little effect on monolayers of dipalmitoyl phosphatidylcholine. These results suggest that the drugs are membrane-active and may intercalate in biomembranes containing negatively charged phospholipids. Since human platelets do not contain D₂ receptors, the interference with the tight coupling of PPI cycle metabolites was not receptor-mediated. We suggest that the drugs are intercalated in the plasma membrane and alter the relative, spatial positioning of phospholipid-consuming enzymes and thereby alter the velocities of the enzyme-catalyzed reactions. Such intercalation could be part of the side effects of the drugs and may explain their psychotropic action(s).

Keywords: Abbreviations; CPZ; chlorpromazine; CLO; clozapine; DMSO; dimethylsulfoxide; DPPC; 1,2-dipalmitoyl phosphatidylcholine; DPPS; 1,2-dipalmitoyl phosphatidylserine; GFP; gel-filtered platelets; HPD; haloperidol; mma; mean molecular area (in Å; ²; ); Π; (Greek Pi); surface pressure (in mN/m); PA; phosphatidic acid; PC; phosphatidylcholine; PCP; prochlorperazine; PI; phosphatidylinositol; PIP; phosphatidyl-4-phosphate; PIP; ₂; phosphatidylinositol-4,5-; bis; phosphate; PMZ; pimozide; PPI; polyphosphoinositide; PS; phosphatidylserine; QTP; quetiapine; TFP; trifluoperazinePlatelet; Polyphosphoinositide metabolism; Thrombin; Phenothiazines; Psychotropic drugs; Intercalation

Cholesterol domains in cationic lipid/DNA complexes improve transfection by Long Xu; Thomas J. Anchordoquy (pp. 2177-2181).

The interaction between the cationic lipid DOTAP and cholesterol is examined in high cholesterol formulations by differential scanning calorimetry (DSC). Preparation of liposomes above 66 mol% cholesterol results in formulations that exhibit a calorimetric transition for anhydrous cholesterol at 38–40 °C. The enthalpy of this transition progressively increases at higher cholesterol contents, and is not detected below 66 mol% cholesterol. Furthermore, the enthalpy changes indicate that the composition of the non-domain forming portion containing DOTAP saturated with cholesterol is relatively constant above 66 mol% cholesterol. Greater transfection efficiency in the presence of 50% serum is observed at the formulations with high cholesterol contents where anhydrous cholesterol domains are detected by DSC. Although formulations possessing higher cholesterol exhibited a greater resistance to serum-induced aggregation, maintenance of small particle size does not appear to be responsible for the enhanced transfection efficiency. Additional studies quantifying albumin binding suggest that cholesterol domains in the lipid/DNA complex do not bind protein, and this may enable these moieties to enhance transfection by facilitating membrane fusion.

Keywords: Cholesterol domain; Transfection; Microdomain; Gene delivery; Liposome

Peak intensity analysis as a method for estimation of fluorescent probe binding to artificial and natural nanoparticles: Tetramethylrhodamine uptake by isolated mitochondria by I.V. Perevoshchikova; D.B. Zorov; Y.N. Antonenko (pp. 2182-2190).

A modified version of fluorescence correlation spectroscopy (FCS) closely related to the photon counting histogram (PCH) method, which is used in the case of a mixture of molecules with similar diffusion coefficients, was applied here for analyzing the binding of the potential-sensitive dye tetramethylrhodamine ethyl ester, TMRE, to isolated mitochondria both in energized and deenergized states. Fluorescence time traces of suspensions of TMRE-doped mitochondria representing sequences of peaks of different intensity appeared to be similar to those of fluorescent beads and TMRE-doped latex particles. The experimental data were obtained under stirring conditions which increased the number of events by about three orders of magnitude thus substantially enhancing the resolution of the method. The statistics of the brightness of identical fluorescent particles reflecting their random walk through the confocal volume was described by a simple analytical equation which enabled us to perform the peak intensity analysis (PIA) of TMRE-doped mitochondria. The validity of PIA was tested with fluorescent beads of different sizes and TMRE-doped latex particles. Mitochondrial energization in the presence of TMRE led to the increase in the number and the intensity of the peaks in fluorescence time traces, the PIA of which allowed us to determine mitochondrial membrane potential and additionally a number of mitochondrial particles per ml of the suspension. The value of the membrane potential on a single mitochondrion was estimated to be about 180 mV in agreement with the data related to mitochondrial suspensions. Importantly, the PIA method required less than 1 microgram of mitochondrial protein per measurement.

Keywords: Abbreviations; FCS; fluorescence correlation spectroscopy; PCH; photon counting histogram; TMRE; tetramethylrhodamine ethyl ester; DNP; 2,4-dinitrophenol; Δ; φ; electrical potential on the inner mitochondrial membrane; PIA; peak intensity analysisMitochondria; Fluorescence correlation spectroscopy; Photon counting histogram; Tetramethylrhodamine ethyl ester

Comparative calorimetric and spectroscopic studies of the effects of cholesterol and epicholesterol on the thermotropic phase behaviour of dipalmitoylphosphatidylcholine bilayer membranes by David A. Mannock; Maria Y.T. Lee; Ruthven N.A.H. Lewis; Ronald N. McElhaney (pp. 2191-2202).

We carried out comparative differential scanning calorimetric and Fourier transform infrared spectroscopic studies of the effects of cholesterol (Chol) and epicholesterol (EChol) on the thermotropic phase behaviour and organization of dipalmitoylphosphatidylcholine (DPPC) bilayers. EChol is an epimer of Chol in which the axially oriented hydroxyl group of C3 of Chol is replaced by an equatorially oriented hydroxyl group, resulting in a different orientation of the hydroxyl group relative to sterol fused ring system. Our calorimetric studies indicate that the incorporation of EChol is more effective than Chol is in reducing the enthalpy of the pretransition of DPPC. EChol is also initially more effective than Chol in reducing the enthalpies of both the sharp and broad components of the main phase transition of DPPC. However, at higher EChol concentrations (~30–50 mol%), EChol becomes less effective than Chol in reducing the enthalpy and cooperativity of the main phase transition, such that at sterol concentrations of 50 mol%, EChol does not completely abolish the cooperative hydrocarbon chain-melting phase transition of DPPC, while Chol does. However, EChol does not appear to form a calorimetrically detectable crystallite phase at higher sterol concentrations, suggesting that EChol, unlike Chol, may form dimers or lower order aggregates at higher sterol concentrations. Our spectroscopic studies demonstrate that EChol incorporation produces more ordered gel and comparably ordered liquid-crystalline bilayers compared to Chol, which are characterized by increased hydrogen bonding in the glycerol backbone region of the DPPC bilayer. These and other results indicate that monomeric EChol is less miscible in DPPC bilayers than is Chol at higher sterol concentrations, but perturbs their organization to a greater extent at lower sterol concentrations, probably due primarily to the larger effective cross-sectional area of the EChol molecule. Nevertheless, EChol does appear to produce a lamellar liquid-ordered phase in DPPC bilayers.

Keywords: Abbreviations; Chol; cholesterol; EChol; epicholesterol; Lano; lanosterol; PC; phosphatidylcholine; SpM; sphingomyelin; PE; phosphatidylethanolamine; DPPC; dipalmitoylphosphatidylcholine; DMPC; dimyristoylphosphatidylcholine; OPPC; 1-oleoyl-2-palmitoylphosphatidylcholine; MLVs; multilamellar vesicles; LUVs; large unilamellar vesicles; SUV; small unilamellar vesicles; DSC; differential scanning calorimetry; NMR; nuclear magnetic resonance; ESR; electron spin resonance; FTIR; Fourier transform infrared; T; _p; pretransition temperature; T; _m; main transition temperature; Δ; H; transition enthalpy; Δ; T; _1/2; , width of the phase transition at half height; inversely related to the cooperativity of the phase transition; L; _β; ′ and L; _β; lamellar gel phases with titled and untilted hydrocarbon chains, respectively; P; _β; ′; rippled gel phase with tilted hydrocarbon chains; L; _α; or L; _d; lamellar liquid-crystalline or liquid-disordered phase; L; _o; lamellar liquid-ordered phase; H; _II; inverted hexagonal phaseCholesterol; Epicholesterol; Phosphatidylcholine; Sterol–phospholipid interactions; Phospholipid bilayer membranes; Thermotropic phase behaviour; Differential scanning calorimetry; Fourier transform infrared spectroscopy

Heterologous expression of human Neuromedin U receptor 1 and its subsequent solubilization and purification by Hongyan Xia; Lihong Liu; Christoph Reinhart; Hartmut Michel (pp. 2203-2209).

Human Neuromedin U receptor 1 (hNmU-R1) is a member of G protein-coupled receptor family. For structural determination of hNmU-R1, the production of hNmU-R1 in milligram amounts is a prerequisite. Here we reported two different eukaryotic expression systems, namely, Semliki Forest virus (SFV)/BHK-21 and baculovirus/ Spodoptera frugiperda (Sf9) cell systems for overproduction of this receptor. In the SFV-based expression system, hNmU-R1 was produced at a level of 5 pmol receptor/mg membrane protein and the yield could be further increased to 22 pmol receptor/mg membrane protein by supplementation with 2% dimethyl sulfoxide (DMSO). Around 8 pmol receptor/mg membrane protein could be achieved in baculovirus-infected Sf9 cells. The recombinant hNmU-R1 from SFV- and baculovirus-based systems was functional, with a K_d value of [¹²⁵I] NmU-23 (rat) similar to that from transiently transfected COS-7 cells, where hNmU-R1 was first identified. With the aid of 1% n-dodecyl-β-d-maltoside (LM)/0.25% cholesteryl hemisuccinate (CHS), the yield of functional hNmU-R1 could reach 80%. The recombinant receptor from Sf9 cells was purified to homogeneity. The specific binding of the purified receptor to [¹²⁵I] NmU-23 (rat) indicated that the receptor is bioactive. This is the first report of successful solubilization and purification of hNmU-R1, and will enable functional and structural studies of the hNmU-R1.

Keywords: Abbreviations; BHK-21; Baby hamster kidney; BCIP; 5-Bromo-4-Chloro-3-Indolyl Phosphate; p; -Toluidinium; CHS; Cholesteryl hemisuccinate; HEGA-10; Decanoyl-; N; -hydoxyethylglucamide; DMSO; Dimethyl sulfoxide; DMEM; Dulbecco's Modified Eagle's Medium; E. coli; Escherichia coli; FCS; Fetal calf serum; FOS-12; FOS-choline-12; FOS-14; FOS-choline-14; FOS-16; FOS-choline-16; GPCR; G protein-coupled receptor; hNmU-R1; human Neuromedin U receptor 1; MOI; Multiplicity of Infection; LM; n; -Dodecyl-β-; d; -maltoside; NTA; Ni-nitrilotriacetic acid; NBT; Nitro-Blue Tetrazolium Chloride; OG; Octyl-β-; d; -glucopyronoside; PMSF; Phenymethylsulfonylfluoride; SFV; Semliki Forest Virus; Sf9; Spodoptera frugiperda; TSA; Trichostatin AGPCR; Membrane protein; Expression; Purification; hNmU-R1

Melittin–Lipid interaction: A comparative study using liposomes, micelles and bilayerdisks by Anna Lundquist; Per Wessman; Adrian R. Rennie; Katarina Edwards (pp. 2210-2216).

Comparison of melittin interaction with liposomes, bilayer disks and micelles showed that melittin binding to lipid aggregates is largely dictated by the amount of highly curved areas in the aggregates. The PEG-stabilised bilayer disks were characterised by a combination of small angle neutron scattering, cryo-transmission electron microscopy and dynamic light scattering. Importantly, the theoretically foreseen partial segregation of the lipid components, important for maintaining the structure of the bilayer disk, was confirmed. Steady state fluorescence spectroscopy indicated that melittin mainly resides at the rim of the bilayer disks. Results of the present study help increase the understanding of the mechanisms behind, and the physico-chemical factors affecting, melittin–lipid interaction. We suggest that bilayer disks, due to their stable structure, constitute interesting vehicles for transport of peptides that have high propensity to associate with lipid surfaces of high curvature.

Keywords: Bilayer disk; PEG–lipid; Micelle; Liposome; Neutron scattering; Melittin

Coincident exposure of phosphatidylethanolamine and anionic phospholipids on the surface of irradiated cells by Andrei Marconescu; Philip E. Thorpe (pp. 2217-2224).

The major anionic phospholipid, phosphatidylserine (PS), and the neutral phospholipid, phosphatidylethanolamine (PE), are largely confined to the inner leaflet of the plasma membrane bilayer in mammalian cells under normal conditions. This asymmetry is lost when cells undergo apoptosis, become activated, or are exposed to irradiation, reactive oxygen species or certain drugs. It is not known whether exposure of anionic phospholipids (APLs) and PE occurs simultaneously or in the same region of the plasma membrane. Here we examined the coincidence of exposure of APLs and PE on the surface of bovine aortic endothelial cells and NS0 myeloma cells after irradiation. The cells were irradiated (5 Gy) and stained for APLs and PE using liposomes coated with either an Fab′ fragment of a PS-binding antibody (bavituximab) or a PE-binding peptide (duramycin). Using live cell imaging and flow cytometry, we showed that irradiation leads to synchronous externalization of APLs and PE. The time course of appearance of APLs and PE on the cell surface was the same and the two phospholipid types remained colocalized over time. Distinct patches double positive for APLs and PE were visible. Larger areas of APLs and PE appeared to have detached from the cytoskeleton to form membrane blebs which protruded and drifted on the cell surface. We conclude that APLs and PE coincidently appear on the external leaflet of the plasma membrane of cells after irradiation. Probably, this is because PE and the major APL, PS, share common regulatory mechanisms of translocation.

Keywords: Anionic phospholipids; Phosphatidylserine; Phosphatidylethanolamine; Membrane blebs; Irradiation; Apoptosis

Fluorescence and thermotropic studies of the interactions of PEI-cholesterol based PEI-chol lipopolymers with dipalmitoyl phosphatidylcholine membranes by Santanu Bhattacharya; Avinash Bajaj (pp. 2225-2233).

Numerous PEI derived polymers have been explored for their use in gene delivery. Nine PEI-chol lipopolymers based on cholesterol grafting on three polyethyleneimines (PEI) of different molecular weights have been synthesized. Firstly their aggregation behavior has been studied using transmission electron microscopy and then their interactions withl-α-dipalmitoyl phosphatidylcholine (DPPC) membranes have been examined using fluorescence anisotropy and differential scanning calorimetry (DSC). These PEI-chol lipopolymers are found to quench the chain motion of the acyl chains of DPPC, when incorporated in membranes, depending upon the cholesterol grafting on PEI. These PEI-chol lipopolymers act as filler molecules in membranes. Electron microscopy shows the different aggregation behavior of these new PEI-chol lipopolymers depending upon the molecular weight of PEI and percentage of cholesterol grafting. Detailed analysis of the fluorescence anisotropy and DSC data indicate that the nature of perturbation induced by PEI-chol lipopolymers is dependent upon the molecular weight of the PEI used and the % of cholesterol grafting on PEI. In general, PEI-chol lipopolymers rigidify the liquid-crystalline phase of the membranes without any noticeable effect on the gel phase unlike natural cholesterol, which is known to fluidize the gel phase of the membranes.

Keywords: Liposomes; PEI-chol lipopolymers; Cholesterol; PEI; Membranes; Thermotropic properties; Anisotropy

Partitioning of amino-acid analogues in a five-slab membrane model by Durba Sengupta; Jeremy C. Smith; G. Matthias Ullmann (pp. 2234-2243).

The positional preferences of the twenty amino-acid residues in a phospholipid bilayer are investigated by calculating the solvation free energy of the corresponding side chain analogues using a five-slab continuum electrostatic model. The side-chain analogues of the aromatic residues tryptophan and tyrosine are found to partition in the head-group region, due to compensation between the increase of the non-polar component of the solvation free energy at the boundary with the aqueous region and the decrease in the electrostatic component. The side chain analogue of phenylalanine differs from the other aromatic molecules by being able to partition in both the head-group region and the membrane core. This finding is consistent with experimental findings of the position of phenylalanine in membrane helices. Interestingly, the charged side-chain analogues of arginine and lysine are shown to prefer the head-group region in an orientation that allows the charged moiety to interact with the aqueous layer. The orientation adopted is similar to the “snorkelling” effect seen in lysine and arginine residues in membrane helices. In contrast, the preference of the charged side-chain analogues of histidine (protonated) and aspartate (deprotonated) for the aqueous layer is shown to be due to a steep decrease in the electrostatic component of the solvation free energy at the boundary to the aqueous region. The calculations allow an understanding of the origins of side chain positioning in membranes and are thus useful in understanding membrane–protein:lipid thermodynamics.

Keywords: Amino-acid positional preference; Membrane partitioning; Snorkeling; Five-slab membrane model; Poisson–Boltzmann electrostatics

Comparison of the interaction of tomatine with mixed monolayers containing phospholipid, egg sphingomyelin, and sterols by Barry W. Walker; Nathan Manhanke; Keith J. Stine (pp. 2244-2257).

The interaction of the glycoalkaloid tomatine with monolayers of a phospholipid (dimyristoylphosphatidylcholine, DMPC), and sphingolipid (egg sphingomyelin), and cholesterol is compared. Using measurements of the surface pressure response as a function of the subphase concentration of tomatine, interfacial binding constants are estimated for mixed monolayers of DMPC and cholesterol and for those of egg sphingomyelin and cholesterol of mole ratio 7:3. The binding constants obtained suggest a stronger interaction of tomatine with DMPC and cholesterol mixed monolayers, reflecting easier displacement of cholesterol from its interaction with DMPC than from its interaction with egg sphingomyelin. Mixtures of tomatine and cholesterol are found to spread directly at the water–air interface and form stable monolayers, suggesting that cholesterol holds tomatine at the interface despite the absence of observed monolayer behavior for tomatine alone. The interaction of tomatine with DMPC and cholesterol monolayers is found to exhibit a pH dependence in agreement with previously reported results for its interaction with liposomes; in particular, the interaction is much less at pH 5 than at pH 7 or pH 9. It is found that while tomatine interacts strongly with monolayers containing sitosterol, it does not interact with monolayers containing sitosterol glucoside. The response of monolayers of varying composition of DMPC and cholesterol to tomatine is also examined. Brewster angle microscopy (BAM) reveals further evidence for formation of suspected islands of tomatine+cholesterol complexes upon interaction with mixed monolayers of lipid and sterol.

Keywords: Phospholipid; Sphingomyelin; Cholesterol; Sterol; Tomatine; Glycoalkaloid; Monolayer

Reconstitution into liposomes of the B°-like glutamine-neutral amino acid transporter from renal cell plasma membrane by Francesca Oppedisano; Cesare Indiveri (pp. 2258-2265).

Na⁺ dependent [³H]glutamine uptake was found in liposomes reconstituted with solubilized rat kidney brush border in the presence of intraliposomal K⁺. The reconstituted system was optimised with respect to the critical parameters of the cyclic detergent removal procedure, i.e., the detergent used for the solubilization, the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. Time dependent [³H]glutamine accumulation in proteoliposomes occurred only in the presence of external Na⁺and internal K⁺. The transporter showed low if there is any tolerance towards the substitution of Na⁺ or K⁺ for other cations. Valinomycin strongly stimulated the transport indicating that it is electrogenic. Intraliposomal glutamine had no effect. From the dependence of the transport rate on the Na⁺ concentration cooperativity index close to 1 was derived, indicating that 1 Na⁺ should be involved in the cotransport with glutamine. The electrogenicity of the transport originated from the Na⁺ transport. Optimal rate of 0.1 mM [³H]glutamine uptake was found in the presence of 50 mM intraliposomal K-gluconate. At higher K-gluconate concentrations the transport rate decreased. The activity of the reconstituted transporter was pH dependent with optimal function in the range pH 6.5–7.0. [³H]glutamine (and [³H]leucine) uptake was inhibited by all the neutral but not by the positively or negatively charged amino acids. The sulfhydryl reagents HgCl₂, mersalyl, p-hydroxymercuribenzoate and the substrate analogue 2-aminobicyclo[2,2,1]heptane-2-carboxylate strongly inhibited the transporter, whereas the amino acid analogue α-(methylamino)isobutyrate had no effect. The inhibition by mersalyl was protected by the presence of the substrate. On the basis of the Na⁺ dependence, the electrogenic transport mode and the specificity towards the amino acids, the reconstituted transporter was classified as B°-like.

Keywords: Abbreviations; pOHMB; p; -hydroxymercuribenzoate; MTSET; [2-(trimethylammonium)ethyl] methanethiosulfonate; C; _x; E; _y; alkyl(x)-poly(y)oxyethylene ether; MeAIB; α-(methylamino)isobutyric acid; PLP; pyridoxal-5-phosphate; BCH; 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid; NEM; N; -ethylmaleimide; NPM; N; -phenylmaleimidePlasma membrane; Transport; Liposome; Reconstitution; Glutamine; Amino acid; B°

An infrared spectroscopic based method to measure membrane permeance in liposomes by Changfeng Chen; Carl P. Tripp (pp. 2266-2272).

A new method for studying membrane permeance in liposomes is described. The method uses liposomes fabricated to contain IR probe molecules with CN moieties in combination with attenuated total reflection—Fourier transform infrared (ATR-FTIR) spectroscopy. The liposomes are adsorbed on a TiO₂ coated ATR crystal and remain intact to flowing aqueous solutions. A change in permeance is determined by monitoring the time dependent decrease in the intensity of a band due to CN groups. It is shown that the transport of the probe molecule depends on the size of the probe molecule and the structure of the liposome membrane. A much clearer molecular understanding of membrane permeance is obtained when the information derived from transport of the probe molecules is combined with the membrane packing arrangement determined from the infrared bands due to the lipids.

Keywords: Membrane permeance; Liposome; ATR-FTIR; IR probe; Molecular details; CN band

Efficacy of external tetraethylammonium block of the KcsA potassium channel: Molecular and Brownian dynamics studies by David Bisset; Shin-Ho Chung (pp. 2273-2282).

Blockade of the KcsA potassium channel by externally applied tetraethylammonium is investigated using molecular dynamics calculations and Brownian dynamics simulations. In KcsA, the aromatic rings of four tyrosine residues located just external to the selectivity filter create an attractive energy well or a binding cage for a tetraethylammonium molecule. We first investigate the effects of re-orienting the four tyrosine residues such that the centers of the aromatic rings face the tetraethylammonium molecule directly. Then, we systematically move the residues inward in both orientations so that the radius of the binding cage formed by them becomes smaller. For each configuration, we construct a one-dimensional free energy profile by bringing in a tetraethylammonium molecule from the external reservoir toward the selectivity filter. The free energy profile is then converted to a one-dimensional potential energy profile, taking the available space between the tyrosine residues and the tetraethylammonium molecule into account. Incorporating this potential energy profile into the Brownian dynamics algorithm, we determine the conductance properties of the channel under various conditions, construct the current-tetraethylammonium-concentration curve and compare it with the experimentally determined inhibitory constant k_i for externally applied tetraethylammonium. We show that the experimentally determined binding affinity for externally applied tetraethylammonium can be replicated when each of the four tyrosine residues is moved inward by about 0.7 Å, irrespective of orientation of their aromatic rings.

Keywords: Potassium ion channel; Tetraethylammonium; TEA; KcsA; Brownian dynamics; Conductance; Ion permeation

Roles of membrane structure and phase transition on the hyperosmotic stress survival of Geobacter sulfurreducens by Vishard Ragoonanan; Jason Malsam; Daniel R. Bond; Alptekin Aksan (pp. 2283-2290).

Geobacter sulfurreducens is a δ-proteobacterium bacteria that has biotechnological applications in bioremediation and as biofuel cells. Development of these applications requires stabilization and preservation of the bacteria in thin porous coatings on electrode surfaces and in flow-through bioreactors. During the manufacturing of these coatings the bacteria are exposed to hyperosmotic stresses due to dehydration and the presence of carbohydrates in the medium. In this study we focused on quantifying the response of G. sulfurreducens to hyperosmotic shock and slow dehydration to understand the hyperosmotic damage mechanisms and to develop the methodology to maximize the survival of the bacteria. We employed FTIR spectroscopy to determine the changes in the structure and the phase transition behavior of the cell membrane. Hyperosmotic shock resulted in greatly decreased membrane lipid order in the gel phase and a less cooperative membrane phase transition. On the other hand, slow dehydration resulted in increased membrane phase transition temperature, less cooperative membrane phase transition and a small decrease in the gel phase lipid order. Both hyperosmotic shock and slow dehydration were accompanied by a decrease in viability. However, we identified that in each case the membrane damage mechanism was different. We have also shown that the post-rehydration viability could be maximized if the lyotropic phase change of the cell membrane was eliminated during dehydration. On the other hand, lyotropic phase change during re-hydration did not affect the viability of G. sulfurreducens. This study conclusively shows that the cell membrane is the primary site of injury during hyperosmotic stress, and by detailed analysis of the membrane structure as well as its thermodynamic transitions it is indeed possible to develop methods in a rational fashion to maximize the survival of the bacteria during hyperosmotic stress.

Keywords: Osmotic stress; FTIR; Phase transition; Lyotropic; Dehydration

Enhanced activity of horseradish peroxidase in Langmuir–Blodgett films of phospholipids by Thais F. Schmidt; Luciano Caseli; Tapani Viitala; Osvaldo N. Oliveira Jr. (pp. 2291-2297).

The immobilization of enzymes in nanostructured films has potential applications, e.g. in biosensing, for which the activity may not only be preserved, but also enhanced if optimized conditions are identified. Optimization is not straightforward because several requirements must be fulfilled, including a suitable matrix and film-forming technique. In this study, we show that horseradish peroxidase (HRP) has its activity enhanced when immobilized in Langmuir–Blodgett (LB) films, in conjunction with dipalmitoylphosphatidylglycerol (DPPG). Incorporation of HRP into a DPPG monolayer at the air–water interface was demonstrated with compression isotherms, and Polarization-Modulation Infrared Reflection Absorption Spectroscopy (PM-IRRAS). From the PM-IRRAS data, we inferred that HRP was not denatured when adsorbed on a pre-formed, low pressure DPPG monolayer. A change in orientation was induced by the phospholipid matrix, with the amide CO and NH groups from HRP being oriented perpendicular to the surface, parallel to the DPPG acyl chains, i.e. the α-helix was inserted into the monolayer. The mixed DPPG-HRP monolayer could be transferred onto solid supports, to which HRP activity was ca. 23% higher than in solution. The control of molecular architecture and choice of a suitable phospholipid matrix allowed HRP-containing LB films to be used in sensing peroxide.

Keywords: Cell membrane; Enzyme activity; Horseradish peroxidase; Langmuir–Blodgett film; Langmuir monolayer; Air–water interface

Micelle formation in the presence of photosystem I by Muh Frank Müh ⁎; Athina Zouni (pp. 2298-2307).

The correlation between membrane protein solubilisation and detergent aggregation in aqueous solution is studied for a series of n-alkyl-β-d-maltosides (C_xG₂ with x=10, 11, 12 being the number of carbon atoms in the alkyl chain) using the trimeric photosystem I core complex (PSIcc) of oxygenic photosynthesis from Thermosynechococcus elongatus as model protein. While protein solubilisation is monitored via the turbidity of the solution, the aggregation behavior of the detergent is probed via the fluorescence spectrum of the polycyclic aromatic hydrocarbon pyrene. In addition, changes of the fluorescence spectrum of PSIcc in response to formation of the detergent belt surrounding its hydrophobic surface are investigated. Solubilisation of PSIcc and aggregation of detergent into micelles or belts are found to be strictly correlated. Both processes are complete at the critical solubilisation concentration (CSC) of the detergent, at which the belts are formed. The CSC depends on the concentration of the membrane protein, [prot], and is related to the critical micelle concentration (CMC) by the empirical law ln(CSC/CMC)= n¯₀ [prot], where the constant n¯₀ = (2.0±0.3) μM⁻¹ is independent of the alkyl chain length x. Formation of protein-free micelles below the CSC is not observed even for x=10, where a significant excess of detergent is present at the CSC. This finding indicates an influence of PSIcc on micelle formation that is independent of the binding of detergent to the hydrophobic protein surface. The role of the CSC in the optimisation of membrane protein crystallisation is discussed.

Keywords: Abbreviations; bRC; bacterial reaction centre; Chl; a; chlorophyll; a; CMC; critical micelle concentration; CSC; critical solubilisation concentration; C; _x; G; ₂; n; -alkyl-β-; d; -maltoside with; x; carbon atoms in the; n; -alkyl chain; PEG; polyethylene glycol; PSIcc; trimeric photosystem I core complex; PSIIcc; photosystem II core complexCrystallisation; Detergent; Fluorescence; Membrane protein; Pyrene

Toroidal pores formed by antimicrobial peptides show significant disorder by Durba Sengupta; Hari Leontiadou; Alan E. Mark; Siewert-Jan Marrink (pp. 2308-2317).

A large variety of antimicrobial peptides have been shown to act, at least in vitro, by poration of the lipid membrane. The nanometre size of these pores, however, complicates their structural characterization by experimental techniques. Here we use molecular dynamics simulations, to study the interaction of a specific class of antimicrobial peptides, melittin, with a dipalmitoylphosphatidylcholine bilayer in atomic detail. We show that transmembrane pores spontaneously form above a critical peptide to lipid ratio. The lipid molecules bend inwards to form a toroidally shaped pore but with only one or two peptides lining the pore. This is in strong contrast to the traditional models of toroidal pores in which the peptides are assumed to adopt a transmembrane orientation. We find that peptide aggregation, either prior or after binding to the membrane surface, is a prerequisite to pore formation. The presence of a stable helical secondary structure of the peptide, however is not. Furthermore, results obtained with modified peptides point to the importance of electrostatic interactions in the poration process. Removing the charges of the basic amino-acid residues of melittin prevents pore formation. It was also found that in the absence of counter ions pores not only form more rapidly but lead to membrane rupture. The rupture process occurs via a novel recursive poration pathway, which we coin the Droste mechanism.

Keywords: Antimicrobial peptide; Melittin; Molecular dynamics simulation; Toroidal pore

Extracellular calcium- and magnesium-mediated regulation of passive calcium transport across Caco-2 monolayers by Sarah L. Davies; Claire E. Gibbons; Martin C. Steward; Donald T. Ward (pp. 2318-2324).

The calcium-sensing receptor (CaR) is expressed on intestinal epithelial serosal membrane and in Caco-2 cells. In renal epithelium, CaR expressed on the basolateral membrane acts to limit excess tubular Ca²⁺ reabsorption. Therefore, here we investigated whether extracellular calcium (Ca_o²⁺) can regulate active or passive⁴⁵Ca²⁺ transport across differentiated Caco-2 monolayers via CaR-dependent or CaR-independent mechanisms. Raising the Ca_o²⁺ concentration from 0.8 to 1.6 mM increased transepithelial electrical resistance (TER) and decreased passive Ca²⁺ permeability but failed to alter active Ca²⁺ transport. The Ca_o²⁺ effect on TER was rapid, sustained and concentration-dependent. Increasing basolateral Mg²⁺ concentration increased TER and inhibited both passive and active Ca²⁺ transport, whereas spermine and the CaR-selective calcimimetic NPS R-467 were without effect. We conclude that small increases in divalent cation concentration elicit CaR-independent increases in TER and inhibit passive Ca²⁺ transport across Caco-2 monolayers, most probably through a direct effect on tight junction permeability. Whilst it is known that the complete removal of Ca_o²⁺ lowers TER, here we show that Ca_o²⁺ addition actually increases TER in a concentration-dependent manner. Therefore, such Ca_o²⁺-sensitivity could modulate intestinal solute transport including the limiting of excess Ca²⁺ absorption.

Keywords: Calcium; Magnesium; Epithelial transport; Intestine

Influence of antimicrobial peptides on the formation of nonlamellar lipid mesophases by Andrea Hickel; Sabine Danner-Pongratz; Heinz Amenitsch; Gabor Degovics; Michael Rappolt; Karl Lohner; Georg Pabst (pp. 2325-2333).

We have studied the influence of four antimicrobial peptides of different secondary and ternary structure – melittin (Mel), protegrin-1 (PG-1), peptidyl-glycylleucine-carboxyamide (PGLa), and gramicidin S (GS) – on the lamellar-to-nonlamellar transition of palmitoyloleoyl phosphatidylethanolamine (POPE) applying differential scanning calorimetry and small-angle X-ray diffraction. None of the peptides studied led to the formation of an inverted hexagonal phase observed for pure POPE at high temperatures. Instead either cubic or lamellar phases were stabilized to different degrees. GS was most effective in inducing a cubic phase, whereas Mel fully stabilized the lamellar phase. The behavior of POPE in the presence of PG-1 and PGLa was intermediate to GS and Mel. In addition to the known role of membrane elasticity we propose two mechanisms, which cause stabilization of the lamellar phase: electrostatic repulsion and lipid/peptide pore formation. Both mechanisms prevent transmembrane contact required to form either an inverted hexagonal phase or fusion pores, as precursors of the cubic phase.

Keywords: Lipid/peptide interaction; Pore formation; Membrane electrostatics; Phase transition; X-ray diffraction; Differential scanning calorimetry

Can membrane-bound carotenoid pigment zeaxanthin carry out a transmembrane proton transfer? by Kamila Kupisz; Agnieszka Sujak; Magdalena Patyra; Kazimierz Trebacz; Wiesław I. Gruszecki (pp. 2334-2340).

Polar carotenoid pigment zeaxanthin (β,β-carotene-3,3′-diol) incorporated into planar lipid membranes formed with diphytanoyl phosphatidylcholine increases the specific electric resistance of the membrane from ca. 4 to 13×10⁷ Ω cm² (at 5 mol% zeaxanthin with respect to lipid). Such an observation is consistent with the well known effect of polar carotenoids in decreasing fluidity and structural stabilization of lipid bilayers. Zeaxanthin incorporated into the lipid membrane at 1 mol% has very small effect on the overall membrane resistance but facilitates equilibration of the transmembrane proton gradient, as demonstrated with the application of the H⁺-sensitive antimony electrodes. Relatively low changes in the electrical potential suggest that the equilibration process may be associated with a symport/antiport activity or with a transmembrane transfer of the molecules of acid. UV–Vis linear dichroism analysis of multibilayer formed with the same lipid–carotenoid system shows that the transition dipole moment of the pigment molecules forms a mean angle of 21° with respect to the axis normal to the plane of the membrane. This means that zeaxanthin spans the membrane and tends to have its two hydroxyl groups anchored in the opposite polar zones of the membrane. Detailed FTIR analysis of β-carotene and zeaxanthin indicates that the polyene chain of carotenoids is able to form weak hydrogen bonds with water molecules. Possible molecular mechanisms responsible for proton transport by polyenes are discussed, including direct involvement of the polyene chain in proton transfer and indirect effect of the pigment on physical properties of the membrane.

Keywords: Carotenoid; Xanthophyll pigment; Zeaxanthin; Proton transport; Weak hydrogen bond; Biomembrane

Membrane interactions and lipid binding of casein oligomers and early aggregates by Miri Sokolovski; Tania Sheynis; Sofiya Kolusheva; Raz Jelinek (pp. 2341-2349).

Caseins constitute the main protein components in mammalian milk and have critical functions in calcium transport and prevention of protein aggregation. Fibrillation and aggregation of κ-casein, a phenomenon which has only recently been detected, might be associated with malfunctions of milk secretion and amyloidosis phenomena in the mammary glands. This study employs a newly-designed chromatic biomimetic vesicle assay to investigate the occurrence and the parameters affecting membrane interactions of casein aggregates and the contribution of individual casein members to membrane binding. We show that physiological casein colloids exhibit membrane activity, as well as early globular aggregates of κ-casein, a prominent casein isoform. Furthermore, inhibition of κ-casein fibrillation through complexation with α_S-casein and β-casein, respectively, was found to go hand in hand with induction of enhanced membrane binding; these data are important in the context of casein biology since in secreted milk κ-casein is found only in assemblies containing also α_S-casein and β-casein. The chromatic experiments, complemented by transmission electron microscopy analysis and fluorescence quenching assays, also revealed significantly higher affinity early spherical aggregates of k-casein to anionic phosphatidylglycerol-lipids, as compared to zwitterionic phospholipids. Overall, this study suggests that lipid interactions play important roles in maintaining the essential physiological functions of caseins in mammalian milk.

Keywords: Abbreviations; PDA; polydiacetylene; DMPA; dimyristoylphosphatidic acid; DMPC; dimyristoylphosphatydilcholine; DMPG; dimyristoylphosphatidylglycerol; DMPS; dimyristoylphosphatidylserine; SDS; sodium dodecyl sulfate; ThT; Thioflavin T; NBD-PE; N; -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)1,2-dihexadecanoyl-snglycero-3-phosphoethanolamineCasein fibrils; Casein; Pre-fibril oligomers; Amyloidogenic protein; Membrane interaction; Polydiacetylene

Lanolin-derived lipid mixtures mimic closely the lipid composition and organization of vernix caseosa lipids by Robert Rissmann; Marion H.M. Oudshoorn; Elise Kocks; Wim E. Hennink; Maria Ponec; Joke A. Bouwstra (pp. 2350-2360).

The aim of the present study was to use semi-synthetic lipid mixtures to mimic the complex lipid composition, organization and thermotropic behaviour of vernix caseosa (VC) lipids. As VC shows multiple protecting and barrier supporting properties before and after birth, it is suggested that a VC substitute could be an innovative barrier cream for barrier deficient skin. Lanolin was selected as the source of the branched chain sterol esters and wax esters — the main lipid classes of VC. Different lipid fractions were isolated from lanolin and subsequently mixed with squalene, triglycerides, cholesterol, ceramides and fatty acids to generate semi-synthetic lipid mixtures that mimic the lipid composition of VC, as established by high-performance thin-layer chromatography. Differential scanning calorimetry and Fourier transform infrared spectroscopy investigations revealed that triglycerides play an important role in the (lateral) lipid organization and thermotropic behaviour of the synthetic lipid mixtures. Excellent resemblance of VC lipids was obtained when adding unsaturated triglycerides. Moreover, these lipid mixtures showed similar long range ordering as VC. The optimal lipid mixture was evaluated on tape-stripped hairless mouse skin in vivo. The rate of barrier recovery was increased and comparable to VC lipid treatment.

Keywords: Abbreviations; CER; ceramides; CHOL; cholesterol; DIOL; dihydroxy wax esters; DSC; differential scanning calorimetry; FFA; free fatty acids, FTIR, Fourier transform infrared spectroscopy, HPTLC, high-performance thin-layer chromatography; SAXD; small-angle X-ray diffraction; SC; stratum corneum; SE/WE; sterol esters and wax esters; SSLM; semi-synthetic lipid mixture; SQ; squalene; TEWL; transepidermal water loss; TG; triglycerides; VC; vernix caseosaLipid phase transition; Differential scanning calorimetry; Small-angle X-ray diffraction; Branched fatty acids; Barrier recovery

Transmembrane domain of EphA1 receptor forms dimers in membrane-like environment by Elena O. Artemenko; Natalya S. Egorova; Alexander S. Arseniev; Alexey V. Feofanov (pp. 2361-2367).

Eph receptor tyrosine kinases (RTKs) are activated by a ligand-mediated dimerization in the plasma membrane and subjected to clusterization at a high local density of receptors and their membrane-anchored ligands. Interactions between transmembrane domains (TMDs) were recognized to assist to the ligand-binding extracellular domains in the dimerization of some RTKs, whereas a functional role of Eph-receptor TMDs remains unknown. We have studied a propensity of EphA1-receptor TMDs (TMA1) to self-association in membrane-mimetic environment. Dimerization of TMA1 in SDS environment was revealed by SDS-PAGE and confirmed by FRET analysis of the fluorescently labeled peptide ( K_d=7.2±0.4 μM at 1.5 mM SDS). TMA1 dimerization was also found in 1,2-dimyristoyl- sn-glycero-3-phosphocholine liposomes (Δ G=−15.4±0.5 kJ/mol). Stability of TMA1 dimers is comparable to the reported earlier stability of TMD dimers of fibroblast growth factor receptor 3 and tenfold weaker than the stability of TMD dimers of glycophorin A possessing high propensity to dimerization. Our results suggest that EphA1-receptor TMD contribute to the dimerization-mediated receptor activation. An assumed role of the TMD interactions is the efficient signal transduction due to TMD-driving mutual orientation of kinase domains in dimers, while a relatively low force of the TMD interactions does not prevent a ligand-controlled regulation of the receptor dimerization.

Keywords: EphA1 receptor; Transmembrane domain; Dimerization; FRET; SDS-PAGE

Spontaneous formation of DPPC monolayers at aqueous/vapor interfaces and the impact of charged surfactants by Süleyman Z. Can; Chia Fang Chang; Robert A. Walker (pp. 2368-2377).

Surface tensiometry and vibrational sum-frequency spectroscopy were used to examine the structure and organization in phospholipid monolayers at the aqueous/vapor interface in the absence and in the presence of simple, charged surfactants. 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) was the phospholipid employed in these studies and surfactants included sodium dodecyl sulfate (SDS) and dodecyl trimethyl ammonium bromide (DTAB). DPPC spontaneously spreads on a pure water (pH=5.5) surface to form monolayers as evidenced by an equilibrium spreading pressure (ESP) of 7.9±2.3 mN/m and a clearly resolved vibrational spectrum. Low concentrations of surfactants inhibit the spreading of DPPC and result in significantly lower ESP values. Anionic and cationic surfactants at higher concentrations have opposite effects on monolayer organization; SDS creates well-organized monolayers while DTAB leads to poor organization of lipid molecules. Surface-specific vibrational spectra showed that high concentrations of charged surfactants (≥100 µM) lead to accumulation of net surface charges as evidenced by destructive and constructive interferences. Selectively deuterating surfactants results in changes in vibrational band intensities and phases enabling assignment of relative orientations of equivalent functional groups belonging to the lipid and surfactant.

Keywords: Dipalmitoyl phosphatidylcholine (DPPC); Sodium dodecyl sulfate (SDS); Dodecyl trimethyl ammonium bromide (DTAB); Mixed monolayer; Surface pressure isotherm; Vibrational sum-frequency spectroscopy

Functional ABCG1 expression induces apoptosis in macrophages and other cell types by László Seres; Judit Cserepes; N. Barry Elkind; Daniel Torocsik Dániel Törőcsik; László Nagy; Balázs Sarkadi; László Homolya (pp. 2378-2387).

The expression of the ATP-binding cassette transporter ABCG1 is greatly increased in macrophages by cholesterol loading via the activation of the nuclear receptor LXR. Several recent studies demonstrated that ABCG1 expression is associated with increased cholesterol efflux from macrophages to high-density lipoprotein, suggesting an atheroprotective role for this protein. Our present study uncovers an as yet not described cellular function of ABCG1. Here we demonstrate that elevated expression of human ABCG1 is associated with apoptotic cell death in macrophages and also in other cell types. We found that overexpression of the wild type protein results in phosphatidyl serine (PS) translocation, caspase 3 activation, and subsequent cell death, whereas neither the inactive mutant variant of ABCG1 (ABCG1_K124M) nor the ABCG2 multidrug transporter had such effect. Induction of ABCG1 expression by LXR activation in Thp1 cells and in human monocyte-derived macrophages was accompanied by a significant increase in the number of apoptotic cells. Thyroxin and benzamil, previously identified inhibitors of ABCG1 function, selectively prevented ABCG1-promoted apoptosis in transfected cells as well as in LXR-induced macrophages. Collectively, our results suggest a causative relationship between ABCG1 function and apoptotic cell death, and may offer new insights into the role of ABCG1 in atherogenesis.

Keywords: ABC transporter; Macrophage; LXR-ligand; Annexin V binding; Atherosclerosis

Oxidative stress causes membrane phospholipid rearrangement and shedding from RBC membranes—An NMR study by Inna Freikman; Johnny Amer; Jack S. Cohen; Israel Ringel; Eitan Fibach (pp. 2388-2394).

Nuclear Magnetic Resonance (NMR) spectroscopy was used to investigate the relationship between oxidative stress experienced by RBCs and their phospholipid content and shedding. Using¹H-NMR, we demonstrated a higher lactate/pyruvate ratio, an indicator of oxidative stress, in normal RBCs treated with oxidants ( t-butylhydroxyperoxide and H₂O₂) as well as in β-thalassemic RBCs. Using³¹P-NMR, we found 30% more phosphatidylcholine (PC), and unexpectedly, 35% less phosphatidylserine (PS) in the thalassemic RBCs. PS was decreased by treatment with oxidants and increased by anti-oxidants (vitamin C and N-acetyl cysteine); PC showed the opposite behavior. Thalassemic RBCs incubated in phosphate buffered saline produced more PS in the supernatant than normal RBCs. Anti-oxidants reduced the PS in the supernatant while oxidants increased it. Plasma of thalassemic patients contained 2.6-fold and 1.8-fold more PS and PC, respectively, than normal plasma. These results indicate that the decreased PS in RBCs resulted from increased shedding. The nature of the shed PS was studied by purifying and analyzing membranous microparticles from the plasma and RBC supernatants. More PS was found in microparticles purified from thalassemic plasma and RBC supernatants (5.6- and 4.8-fold, respectively) than in their normal counterparts. However, the bulk (80–90%) of the shed PS was not associated with microparticles. The significance of PS shedding for RBC survival needs further clarification.

Keywords: Abbreviations; BHP; t; -butylhydroxyperoxide; MP; microparticles; NAC; N; -acetyl cysteine; NEM; N; -ethylmaleimide; NMR; Nuclear Magnetic Resonance; PBS; phosphate buffered saline; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PE-plas; phosphatidylethanolamine-plasmalogen; PI; phosphatidylinositol; PL; phospholipids; PS; phosphatidylserine; RBC; red blood cell; S.D.; standard deviation; TMP; Trimethylphosphate; Vit.C; vitamin CMembrane phospholipids; Oxidative stress; Thalassemia; RBC; NMR

A novel agonist effect on the nicotinic acetylcholine receptor exerted by the anticonvulsive drug Lamotrigine by Ana Sofía Vallés; Ingrid Garbus; Silvia S. Antollini; Francisco J. Barrantes (pp. 2395-2404).

The anticonvulsive drug Lamotrigine (LTG) is found to activate adult muscle nicotinic acetylcholine receptors (AChR). Single-channel patch-clamp recordings showed that LTG (0.05–400 μM) applied alone is able to open AChR channels. [¹²⁵I]α-bungarotoxin-binding studies further indicate that LTG does not bind to the canonical ACh-binding sites. Fluorescence experiments using the probe crystal violet demonstrate that LTG induces the transition from the resting state to the desensitized state of the AChR in the presence of excess α-bungarotoxin, that is, when the agonist site is blocked. Allosterically-potentiating ligands or the open-channel blocker QX-314 exhibited a behavior different from that of LTG. We conclude that LTG activates the AChR through a site that is different from those of full agonists/competitive antagonists and allosterically-potentiating ligands, respectively.

Keywords: Lamotrigine; AChR; APL; αBTX; Agonist

Modulation of a membrane lipid lamellar–nonlamellar phase transition by cationic lipids: A measure for transfection efficiency by Boris G. Tenchov; Li Wang; Rumiana Koynova; Robert C. MacDonald (pp. 2405-2412).

Synthetic cationic lipids can be used as DNA carriers and are regarded to be the most promising non-viral gene carriers. For this investigation, six novel phosphatidylcholine (PC) cationic derivatives with various hydrophobic moieties were synthesized and their transfection efficiencies for human umbilical artery endothelial cells (HUAEC) were determined. Three compounds with relatively short, myristoleoyl or myristelaidoyl 14:1 chains exhibited very high activity, exceeding by ∼10 times that of the reference cationic derivative dioleoyl ethylPC (EDOPC). Noteworthy, cationic lipids with 14:1 hydrocarbon chains have not been tested as DNA carriers in transfection assays previously. The other three lipids, which contained oleoyl 18:1 and longer chains, exhibited moderate to weak transfection activity. Transfection efficiency was found to correlate strongly with the effect of the cationic lipids on the lamellar-to-inverted hexagonal, L_α→H_II, phase conversion in dipalmitoleoyl phosphatidylethanolamine dispersions (DPoPE). X-ray diffraction on binary DPoPE/cationic lipid mixtures showed that the superior transfection agents eliminated the direct L_α→H_II phase transition and promoted formation of an inverted cubic phase between the L_α and H_II phases. In contrast, moderate and weak transfection agents retained the direct L_α→H_II transition but shifted to higher temperatures than that of pure DPoPE, and induced cubic phase formation at a later stage. On the basis of current models of lipid membrane fusion, promotion of a cubic phase by the high-efficiency agents may be considered as an indication that their high transfection activity results from enhanced lipoplex fusion with cellular membranes. The distinct, well-expressed correlation established between transfection efficiency of a cationic lipid and the way it modulates nonlamellar phase formation of a membrane lipid could be useful as a criterion to assess the quality of lipid carriers and for rational design of new and superior nucleotide delivery agents.

Keywords: Cationic lipid; Lipofection; Lipoplex; Phase transition; Inverted hexagonal phase; Cubic phase

Multidrug resistance-associated protein 1 as a major mediator of basal and apoptotic glutathione release by Rosemarie Marchan; Christine L. Hammond; Nazzareno Ballatori (pp. 2413-2420).

The proteins responsible for reduced glutathione (GSH) export under both basal conditions and in cells undergoing apoptosis have not yet been identified, although recent studies implicate some members of the multidrug resistance-associated protein family (MRP/ABCC) in this process. To examine the role of MRP1 in GSH release, the present study measured basal and apoptotic GSH efflux in HEK293 cells stably transfected with human MRP1. MRP1-overexpressing cells had lower intracellular GSH levels and higher levels of GSH release, under both basal conditions and after apoptosis was induced with either Fas antibody or staurosporine. Despite the enhanced GSH efflux in MRP1-overexpressing cells, intracellular GSH levels were not further depleted when cells were treated with Fas antibody or staurosporine, suggesting an increase in GSH synthesis. MRP1-overexpressing cells were also less susceptible to apoptosis, suggesting that the stable intracellular GSH levels may have protected cells from death. Overall, these results demonstrate that basal and apoptotic GSH release are markedly enhanced in cells overexpressing MRP1, suggesting that MRP1 plays a key role in these processes. The enhanced GSH release, with a concurrent decrease of intracellular GSH, appears to be necessary for the progression of apoptosis.

Keywords: Glutathione; Multidrug resistance-associated proteins; MRP1; Apoptosis

Two new methods for preparing a unique stratum corneum substitute by Daniël Groen; Gert S. Gooris; Maria Ponec; Joke A. Bouwstra (pp. 2421-2429).

Stratum corneum lipids play an important role in the barrier function of the skin. An in vitro permeation model consisting of synthetic lipids has previously been developed to replace human stratum corneum (SC) in permeation studies. This model is referred to as the stratum corneum substitute (SCS). In order to improve its reproducibility and to increase the efficiency in preparing the SCS, two new preparation methods are developed. Subsequently the properties of the SCS prepared by the various methods, i.e. the manual airbrush method, the rotor airbrush method and the linomat method, are investigated. The results show that the SCS prepared with the various methods share the properties of a uniform lipid composition and lipid distribution. Furthermore, irrespective of the preparation method, the lipids form crystalline lamellar phases, mimicking the lipid organization and orientation in human SC. As a result, permeation profiles of benzoic acid through SCS are very similar to human SC. The rotor method increases the efficiency and reproducibility of the manual airbrush method, while the linomat method reduces the lipid loss during preparation and results in SCS with a more uniform membrane thickness. In conclusion, the linomat method was chosen as the preferred method for preparing the substitute.

Keywords: Stratum corneum; In vitro; Model; Lipids; Ceramides; Substitute

Characterization of antimicrobial peptide activity by electrochemical impedance spectroscopy by William K. Chang; William C. Wimley; Peter C. Searson; Kalina Hristova; Mikhail Merzlyakov (pp. 2430-2436).

Electrochemical impedance spectroscopy performed on surface-supported bilayer membranes allows for the monitoring of changes in membrane properties, such as thickness, ion permeability, and homogeneity, after exposure to antimicrobial peptides (AMPs). We show that two model cationic peptides, very similar in sequence but different in activity, induce dramatically different changes in membrane properties as probed by impedance spectroscopy. Moreover, the impedance results excluded the “barrel-stave” and the “toroidal pore” models of AMP mode of action, and are more consistent with the “carpet” and the “detergent” models. The impedance data provide important new insights about the kinetics and the scale of the peptide action which currently are not addressed by the “carpet” and the “detergent” models. The method presented not only provides additional information about the mode of action of a particular AMP, but offers a means of characterizing AMP activity in reproducible, well-defined quantitative terms.

Keywords: Antimicrobial peptides; Cationic peptides; Electrochemical impedance spectroscopy; Planar supported bilayer; Membrane electrochemical properties; Peptide–membrane interaction

Spermine biphasically affects N-type calcium channel currents in adult dorsal root ganglion neurons of the rat by Ilaria Cino; Alessandro Formenti (pp. 2437-2443).

Spermine (Spe) is a polyamine co-secreted with neurotransmitters. In this work its effects on N-type Ca²⁺ channel (Ca_V2.2) have been studied on adult sensory neurons of the rat by means of whole-cell patch-clamp. Spe exerted biphasic effects when added to the external solution: at 500 μM decreased N-type Ca²⁺ channel currents, reducing the maximum whole-cell conductance, shifting the activation curve to the right on the voltage axes and decreasing its slope; conversely, at lower concentration (500 nM) Spe induced completely opposite effects. In 62% of the neurons the inhibitory effects were accompanied by a slowing down of the activation kinetics relieved by a conditioning pre-pulse to +50 mV. The biphasic effects and their rapid onset and offset time course may be explained if multiple sites of action with a different affinity for Spe are present directly on the channel. The effects of Spe on HVA Ca²⁺ currents were strongly dependent on [Ca²⁺]_ext, high [Ca²⁺] powerfully reducing Spe effects. This may be explained if we take into account that as Spe has four positive charges at physiological pH; it may compete with divalent cations for some negatively charged regulatory sites. In these experiments, Spe was effective at concentrations possibly reached in physiological conditions.

Keywords: N-type Ca; ²⁺; channel; Spermine; Polyamine; Calcium current; Sensory neuron; Adult rat

Identification of the initial binding sites of α_s2-casein f(183–207) and effect on bacterial membranes and cell morphology by Iván López-Expósito; Lourdes Amigo; Isidra Recio (pp. 2444-2449).

The aim of this work was to identify the initial binding sites to the bacterial membranes of the antimicrobial peptide α_s2-casein f(183–207) and also to acquire further insight into membrane permeabilization of this peptide. Furthermore, cell morphology was studied by transmission electron microscopy. In all the experiments, bovine LFcin was employed as a comparison. Results showed that initial binding sites of α_s2-casein f(183–207) peptide were lipoteichoic acid in Gram-positive bacteria and lipopolysaccharide in Gram-negative. The peptide was able to permeabilize the outer and inner membranes. Moreover, the α_s2-casein peptide f(183–207) generated pores in the outer membrane of Gram-negative bacteria and in the cell wall of Gram-positive bacteria. In the Gram-negative bacteria, f(183–207) originated cytoplasm condensation, and in the Gram-positive bacteria the cytoplasmic content leaked into the extracellular medium. Furthermore, the experiments of inner and outer membrane permeabilization performed with LFcin-B showed that this peptide also has the ability to permeabilize both the inner and outer membranes.

Keywords: Antimicrobial peptides; Binding sites; Permeabilization; α; _s2; -casein; Milk-derived bioactive peptides

Specific adsorption of functionalized colloids at the surface of living cells: A quantitative kinetic analysis of the receptor-mediated binding by C. Poirier; D. van Effenterre; B. Delord; L. Johannes; D. Roux (pp. 2450-2457).

This paper presents a statistical experimental study of the adsorption of colloids onto the plasma membrane of living cells mediated by specific ligand–receptor interactions. The colloids consist of lipid multilamellar liposomes (spherulites) functionalized by Shiga toxin B-subunit (STxB), while cells are cervix carcinoma epithelial cells expressing the Shiga toxin receptor, the glycolipid globotriaosyl ceramide (Gb3). The specificity of the colloid adsorption is demonstrated using both confocal microscopy and flow cytometry, while a thorough cytometry study on living cells allows characterizing the kinetics of this specific adsorption. The final number of bound colloids and the characteristic adsorption time are shown to depend on bulk concentration, as expected for a thermodynamic equilibrium. However, the colloids appear to be irreversibly attached to the membrane. We interpret this apparent irreversibility as the result of a progressive recruitment of receptors. The methodology used here, whereby microscopic mechanisms are deduced from direct quantitative measurements on living cells, might allow the optimization of drug delivery systems or the quantification of virus infectivity.

Keywords: Shiga toxin; Spherulite; Flow cytometry; Fluorescence microscopy; Specific binding; Kinetic adsorption

Corrigendum to “Monoterpenes affect chlorodiazepoxide–micelle interaction through micellar dipole potential modifications” [Biochim. Biophys. Acta 1616 (2003) 112–120] by Anahí V. Turina; María A. Perillo (pp. 2458-2458).

Corrigendum to “Surface-active properties of the antitumour ether lipid 1- O-octadecyl-2- O-methyl- rac-glycero-3-phosphocholine (edelfosine)” [Biochim. Biophys. Acta 1768 (2007) 1855–1860] by Jon V. Busto; Jesús Sot; Goni Félix M. Goñi; Faustino Mollinedo; Alicia Alonso (pp. 2459-2459).

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BBA - Biomembranes (v.1778, #10)