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BBA - Biomembranes (v.1778, #1)

Editorial Board (pp. ii).

Acknowledgement (pp. iii-vii).

Membrane interactions of hemoglobin variants, HbA, HbE, HbF and globin subunits of HbA: Effects of aminophospholipids and cholesterol by Poppy Datta; Sudipa Chakrabarty; Amit Chakrabarty; Abhijit Chakrabarti (pp. 1-9).

The interaction of hemoglobin with phospholipid bilayer vesicles (liposomes) has been analyzed in several studies to better understand membrane–protein interactions. However, not much is known on hemoglobin interactions with the aminophospholipids, predominantly localized in the inner leaflet of erythrocytes, e.g., phosphatidylserine (PS), phosphatidylethanolamine (PE) in membranes containing phosphatidylcholine (PC). Effects of cholesterol, largely abundant in erythrocytes, have also not been studied in great details in earlier studies. This work therefore describes the study of the interactions of different hemoglobin variants HbA, HbE and HbF and the globin subunits of HbA with the two aminophospholipids in the presence and absence of cholesterol. Absorption measurements indicate preferential oxidative interaction of HbE and alpha-globin subunit with unilamellar vesicles containing PE and PS compared to normal HbA. Cholesterol was found to stabilize such oxidative interactions in membranes containing both the aminophospholipids. HbE and alpha-globin subunits were also found to induce greater leakage of membrane entrapped carboxyfluorescein (CF) using fluorescence measurements. HbE was found to induce fusion of membrane vesicles containing cholesterol and PE when observed under electron microscope. Taken together, these findings might be helpful in understanding the oxidative stress-related mechanism(s) involved in the premature destruction of erythrocytes in peripheral blood, implicated in the hemoglobin disorder, HbE/beta-thalassemia.

Keywords: Abbreviations; HbA; adult hemoglobin; HbF; fetal hemoglobin; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PS; phosphatidylserine; Chol; cholesterol; DMPC; dimyristoylphosphatidylcholine; DMPE; dimyristoylphosphatidylethanolamine; DMPS; dimyristoylphosphatidylserine; DOPC; dioleoylphosphatidylcholine; DOPE; dioleoylphosphatidylethanolamine; CF; 6-carboxyfluorescein; SUV; small unilamellar vesicles; PMB; p; -hydroxymercuribenzoic acid sodium salt; S.E.M.; standard error of the mean; TEM; transmission electron microscopyHbE variant; Globin subunit; Aminophospholipid; Hemoglobin autoxidation; Membrane fusion

Transmembrane localization of cis-isomers of zeaxanthin in the host dimyristoylphosphatidylcholine bilayer membrane by Justyna Widomska; Witold K. Subczynski (pp. 10-19).

The effects of the 9- cis and 13- cis isomers of zeaxanthin on the molecular organization and dynamics of dimyristoylphosphatidylcholine (DMPC) membranes were investigated using conventional and saturation recovery EPR observations of the 1-palmitoyl-2-(14-doxylstearoyl)phosphatidylcholine (14-PC) spin label. The results were compared with the effects caused by the all- trans isomer of zeaxanthin. Effects on membrane fluidity, order, hydrophobicity, and the oxygen transport parameter were monitored at the center of the fluid phase DMPC membrane. The local diffusion-solubility product of oxygen molecules (oxygen transport parameter) in the membrane center, studied by saturation-recovery EPR, decreased by 47% and 27% by including 10 mol% 13- cis and 9- cis zeaxanthin, respectively; whereas, incorporation of all- trans zeaxanthin decreased this parameter by only 11%. At a zeaxanthin-to-DMPC mole ratio of 1:9, all investigated isomers decreased the membrane fluidity and increased the alkyl chain order in the membrane center. They also increased the hydrophobicity of the membrane interior. The effects of these isomers of zeaxanthin on the membrane properties mentioned above increase as: all- trans<9- cis≤13- cis. Obtained results suggest that the investigated cis-isomers of zeaxanthin, similar to the all- trans isomer, are located in the membrane interior, adopting transmembrane orientation with the polar terminal hydroxyl groups located in the opposite leaflets of the bilayer. However, the existence of the second pool of cis-zeaxanthin molecules located in the one leaflet and anchored by the terminal hydroxyl groups in the same polar headgroup region cannot be completely ruled out.

Keywords: cis; -Zeaxanthin; Xanthophyll; Carotenoid; Lipid bilayer; Spin labeling; EPR

Structural determinants for partitioning of lipids and proteins between coexisting fluid phases in giant plasma membrane vesicles by Prabuddha Sengupta; Adam Hammond; David Holowka; Barbara Baird (pp. 20-32).

The structural basis for organizational heterogeneity of lipids and proteins underlies fundamental questions about the plasma membrane of eukaryotic cells. A current hypothesis is the participation of liquid ordered (Lo) membrane domains (lipid rafts) in dynamic compartmentalization of membrane function, but it has been difficult to demonstrate the existence of these domains in live cells. Recently, giant plasma membrane vesicles (GPMVs) obtained by chemically induced blebbing of cultured cells were found to phase separate into optically resolvable, coexisting fluid domains containing Lo-like and liquid disordered (Ld)-like phases as identified by fluorescent probes. In the present study, we used these GPMVs to investigate the structural bases for partitioning of selected lipids and proteins between coexisting Lo-like/Ld-like fluid phases in compositionally complex membranes. Our results with lipid probes show that the structure of the polar headgroups, in addition to acyl chain saturation, can significantly affect partitioning. We find that the membrane anchor of proteins and the aggregation state of proteins both significantly influence their distributions between coexisting fluid phases in these biological membranes. Our results demonstrate the value of GPMVs for characterizing the phase preference of proteins and lipid probes in the absence of detergents and other perturbations of membrane structure.

Keywords: Lipid raft; Liquid order; Liquid disorder; GPMVs; Detergent-resistant; Membranes; RBL mast cell

Transepithelial transport of hesperetin and hesperidin in intestinal Caco-2 cell monolayers by Shoko Kobayashi; Soichi Tanabe; Masanori Sugiyama; Yutaka Konishi (pp. 33-41).

The cell permeability of hesperetin and hesperidin, anti-allergic compounds from citrus fruits, was measured using Caco-2 monolayers. In the presence of a proton gradient, hesperetin permeated cells in the apical-to-basolateral direction at the rate ( Jap→_bl) of 10.43±0.78 nmol/min/mg protein, which was more than 400-fold higher than that of hesperidin (0.023±0.008 nmol/min/mg protein). The transepithelial flux of hesperidin, both in the presence or absence of a proton gradient, was nearly the same and was inversely correlated with the transepithelial electrical resistance (TER), indicating that the transport of hesperidin was mainly via paracellular diffusion. In contrast, the transepithelial flux of hesperetin was almost constant irrespective of the TER. Apically loaded NaN₃ or carbonyl cyanide m-chlorophenylhydrazone (CCCP) decreased the Jap→_bl of hesperetin, in the presence of proton gradient, by one-half. In the absence of a proton gradient, both Jap→_bl and Jbl→_ap of hesperetin were almost the same (5.75±0.40 and 5.16±0.73 nmol/min/mg protein). Jbl→_ap of hesperetin in the presence of a proton gradient was lower than Jbl→_ap in the absence of a proton gradient. Furthermore, Jbl→_ap in the presence of a proton gradient remarkably increased upon addition of NaN₃ specifically to the apical side. These results indicate that hesperetin is absorbed by transcellular transport, which occurs mainly via proton-coupled active transport, and passive diffusion. Thus, hesperetin is efficiently absorbed from the intestine, whereas hesperidin is poorly transported via the paracellular pathway and its transport is highly dependent on conversion to hesperetin via the hydrolytic action of microflora. We have given novel insight to the absorption characteristics of hesperetin, that is proton-coupled and energy-dependent polarized transport.

Keywords: Abbreviations; AC; altepillin C; CCCP; carbonyl cyanide; m; -chlorophenylhydrazone; DMEM; Dulbecco's modified Eagle's medium; DMSO; dimethyl sulfoxide; ECD; electrochemical detector; Gly-Sar; glycylsarcosine; HBSS; Hanks' balanced salt solution; PEPT; peptide transporter; SGLT; sodium-dependent; d; -glucose cotransporter; TER; transepithelial electrical resistance; MCT; monocarboxylic acid transporter; MRP; multidrug resistance-associated proteinHesperetin; Hesperidin; Transcellular active transport; Transcellular passive diffusion; Caco-2

Uptake of 3,4-methylenedioxymethamphetamine and its related compounds by a proton-coupled transport system in Caco-2 cells by Kenji Kuwayama; Hiroyuki Inoue; Tatsuyuki Kanamori; Kenji Tsujikawa; Hajime Miyaguchi; Yuko Iwata; Seiji Miyauchi; Naoki Kamo; Tohru Kishi (pp. 42-50).

3,4-Methylenedioxymethamphetamine (MDMA) is an illegal amphetamine-type stimulant (ATS) that is abused orally in the form of tablets for recreational purposes. The aim of this work is to investigate the absorption mechanism of MDMA and other related compounds that often occur together in ATS tablets, and to determine whether such tablet components interact with each other in intestinal absorption. The characteristics of MDMA uptake by the human intestinal epithelial Caco-2 cell line were investigated. The Michaelis constant and the maximal uptake velocity at pH 6.0 were 1.11 mM and 13.79 nmol/min/mg protein, respectively, and the transport was electroneutral. The initial uptake rate was regulated by both intra- and extracellular pH. MDMA permeation from the apical to the basolateral side was inferior to that in the reverse direction, and a decrease in apical pH enhanced MDMA permeation from the basolateral to the apical side. These facts indicate that this transport system may be an antiporter of H⁺. However, under physiological conditions, the proton gradient cannot drive the MDMA uptake because it is inwardly directed. Large concentration differences of MDMA itself drive this antiporter. Various compounds with similar amine moieties inhibited the uptake, but substrates of organic cation transporters (OCT1–3) and an H⁺-coupled efflux antiporter, MATE, were not recognized.

Keywords: MDMA; Amphetamine-type stimulant; Transport; Caco-2 cells; Intestinal absorption

Enhanced lymph node retention of subcutaneously injected IgG1-PEG₂₀₀₀-liposomes through pentameric IgM antibody-mediated vesicular aggregation by S. Moein Moghimi; Majid Moghimi (pp. 51-55).

An efficient strategy for enhancing the lymph node deposition of rapidly drained liposomes from the interstitial injection site is described. Subcutaneously injected small-sized immuno-poly(ethyleneglycol)-liposomes (immuno-PEG-liposomes), containing 10 mol% mPEG₃₅₀–phospholipid and 1 mol% PEG₂₀₀₀–phospholipid in their bilayer and where IgG1 is coupled to the distal end of PEG₂₀₀₀, not only drain rapidly from the interstitial spaces into the initial lymphatic system, but also accumulate efficiently among the lymph nodes draining the region when compared with non-PEG-bearing immunoliposomes where IgG is directly coupled to the phospholipid. Liposome deposition among the draining lymph nodes, however, was further enhanced dramatically following an adjacent subcutaneous injection of a pentameric IgM against the surface attached IgG molecules (IgM:IgG, 10:1) without compromising vesicle drainage from the interstitium. This is suggested to arise either as a result of formation of large immuno-aggregates within the lymphatic vessels with subsequent transport to and trapping among the regional lymph nodes and/or following IgM binding to Fc receptors of the lymph node sinus macrophages forming a platform for subsequent trapping of drained IgG-coupled liposomes. This lymph node targeting approach may be amenable for the design and surface engineering of any rapidly drained nanoparticulate system bearing peptides and proteins that can be aggregated with a desired monoclonal pentameric IgM.

Keywords: IgM; Immunoliposome; Lymph node; Lymphatic system; Macrophage; Targeting

Accelerated internalization of junctional membrane proteins (connexin 43, N-cadherin and ZO-1) within endocytic vacuoles: An early event of DDT carcinogenicity by Céline Fiorini; Jérome Gilleron; Diane Carette; Astrid Valette; Anne Tilloy; Stephan Chevalier; Dominique Segretain; Georges Pointis (pp. 56-67).

Stability of cell-to-cell interactions and integrity of junctional membrane proteins are essential for biological processes including cancer prevention. The present study shows that DDT, a non-genomic carcinogen used at a non-cytotoxic dose (1 μM), rapidly disrupted the cell–cell contacts and concomitantly induced the formation of cytoplasmic vacuoles close to the plasma membrane in the SerW3 Sertoli cell line. High-resolution deconvolution microscopy reveals that this vacuolization process was clathrin-dependent since a hyperosmotic media (0.2 M sucrose) blocked rhodamine–dextran endocytosis. In response to DDT, junctional proteins such as Cx43, N-Cadherin and ZO-1 were internalized and present in vacuoles. In Cx43-GFP transfected cells, time lapse videomicroscopy demonstrates that DDT rapidly enhanced fragmentation of the gap junction plaques and abolished the gap junction coupling without major modification of Cx43 phosphorylation status. Repeated exposure to DDT resulted in chronic gap junction coupling injury. The present results demonstrate that one of the early effect of DDT is to interfere with the plasma membrane and to perturb its function, specifically its ability to establish cell–cell junctions that are essential for tissue homeostasis and control of cell proliferation and differentiation. Such an alteration may play a specific role during carcinogenesis.

Keywords: DDT; Endocytosis; Junctional proteins; Connexin 43; N-cadherin; ZO-1; Carcinogen; Sertoli cell

Functional characterization of the Saccharomyces cerevisiae ABC-transporter Yor1p overexpressed in plasma membranes by Ioana Grigoras; Myriam Lazard; Pierre Plateau; Sylvain Blanquet (pp. 68-78).

Yor1p, a Saccharomyces cerevisiae plasma membrane ABC-transporter, is associated to oligomycin resistance and to rhodamine B transport. Here, by using the overexpressing strain Superyor [A. Decottignies, A.M. Grant, J.W. Nichols, H. de Wet, D.B. McIntosh, A. Goffeau, ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p, J. Biol. Chem. 273 (1998) 12612–12622], we show that Yor1p also confers resistance to rhodamine 6G and to doxorubicin. In addition, Yor1p protects cells, although weakly, against tetracycline, verapamil, eosin Y and ethidium bromide. The basal ATPase activity of the overexpressed form of Yor1p was studied in membrane preparations. This activity is quenched upon addition of micromolar amounts of vanadate. V_max and K_m values of ∼0.8 s−¹ and 50±8 μM are measured. Mutations of essential residues in the nucleotide binding domain 2 reduces the activity to that measured with a Δ yor1 strain. ATP hydrolysis is strongly inhibited by the addition of potential substrates of the transporter. Covalent reaction of 8-azido-[α-³²P]ATP with Yor1p is not sensitive to the presence of excess oligomycin. Thus, competition of the drug with ATP binding is unlikely. Finally, we inspect possible hypotheses accounting for substrate inhibition, rather than stimulation, of ATP hydrolysis by the membrane preparation.

Keywords: Abbreviations; ABC; ATP binding cassette; TMD; Transmembrane domain; NBD; Nucleotide binding domain; P-gp; P-glycoprotein; CFTR; Cystic fibrosis transmembrane conductance regulator; MRP; Multidrug resistance associated protein; p-CMPS; p-chloromercuriphenylsulfonate; DCC; N,N'; -di-cyclohexylcarbodiimide; NEM; N-ethylmaleimide; DTT; 1–4 dithiothreitol; BSA; Bovine serum albumine; EDTA; Ethylenediaminetetraacetic acid; PNK; Polynucleotide 5′-hydroxyl-kinase; MIC; Minimal inhibitory concentrationYor1p; ABC transporter; Plasma membranes; ATPase activity

Positional dependence of non-native polar mutations on folding of CFTR helical hairpins by Hania Wehbi; Geneviève Gasmi-Seabrook; Mei Y. Choi; Charles M. Deber (pp. 79-87).

Mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) cause CF disease by altering the biosynthesis, maturation, folding and ion conductance of this protein. Our laboratory has focused on expression and structural analysis of the CFTR transmembrane (TM) domains using two-TM segments (i.e., helix–loop–helix constructs) which we term ‘helical hairpins’; these represent the minimal model of tertiary contacts between two helices in a membrane. Previous studies on a library of TM3/4 hairpins of the first CFTR TM domain suggested that introduction of non-native polar residues into TM4 can compromise CFTR function through side chain-side chain H-bonding interactions with native Q207 in TM3 [Choi, M. Y., Cardarelli, L., Therien, A. G., and Deber, C. M. Non-native interhelical hydrogen bonds in the cystic fibrosis transmembrane conductance regulator domain modulated by polar mutations, Biochemistry 43 (2004) 8077–8083]. In the present work, we combine gel shift assays with a series of NMR experiments for comparative structural characterization of the wild type TM3/4 hairpin and its mutants V232D, I231D, Q207N/V232E. Over 95% of the backbone resonances of a¹⁵N,¹³C-labelled V232D-TM3/4 construct in the membrane-mimetic environment of perfluorooctanoate (PFO) micelles were successfully assigned, and the presence and boundaries of helical segments within TM3 and TM4 were defined under these conditions. Comparative analysis of¹⁵N and¹H chemical shift variations among HSQC spectra of WT-, V232D-, I231D- and Q207N/V232E-TM3/4 indicated that hairpin conformations vary with the position of a polar mutation (i.e., V232D and I231D vs. WT), but remain similar when hairpins with identically-positioned polar partners are compared (i.e., V232D vs. Q207N-V232E). The overall findings suggest that a polar mutation in a TM helix can potentially distort native interfacial packing determinants in membrane proteins such as CFTR, with consequences that may lead to disease.

Keywords: Abbreviations; CF; Cystic fibrosis; CFTR; Cystic fibrosis transmembrane conductance regulator; TM; Transmembrane; TMD; Transmembrane domain; TM3/4; Helical hairpin including residues 194–241 of CFTR; WT; Wild type; V232D-TM3/4; TM3/4 construct with mutation of Val to Asp at position 232; I231D-TM3/4; TM3/4 construct with mutation of Ile to Asp at position 231; Q207N/V232E-TM3/4; TM3/4 construct with mutation of Gln to Asn at position 207 and Val to Glu at position 232; PFO; Perfluorooctanoate; DPC; Dodecylphosphocholine; SDS; Sodium dodecylsulfate; CD; Circular dichroism; H-bond; Hydrogen bond; HSQC; Heteronuclear single quantum coherenceCFTR; Transmembrane segment; Helical hairpin; NMR spectroscopy; Membrane protein; Helix–helix interaction

PKA-dependent activation of the vascular smooth muscle isoform of K_ATP channels by vasoactive intestinal polypeptide and its effect on relaxation of the mesenteric resistance artery by Yang Yang; Yun Shi; Shouli Guo; Shuang Zhang; Ningren Cui; Weiwei Shi; Daling Zhu; Chun Jiang (pp. 88-96).

Vasoactive intestinal polypeptide (VIP) is a potent vasodilator and has been successfully used to alleviate hypertension. Consistently, disruption of VIP gene in mice leads to hypertension. However, its downstream targets in the vascular regulation are still not well demonstrated. To test the hypothesis that the vascular smooth muscle isoform of K_ATP channels is a downstream target of the VIP signaling, we performed the studies on the Kir6.1/SUR2B channel expressed in HEK293 cells. We found that the channel was strongly activated by VIP. Through endogenous VIP receptors, the channel activation was reversible and dependent on VIP concentrations with the midpoint-activation concentration ∼10 nM. The channel activation was voltage-independent and could be blocked by K_ATP channel blocker glibenclamide. In cell-attached patches, VIP augmented the channel open-state probability with modest suppression of the single channel conductance. The VIP-induced Kir6.1/SUR2B channel activation was blocked by PKA inhibitor RP-cAMP. Forskolin, an adenylyl cyclase activator, activated the channel similarly as VIP. The effect of VIP was further evident in the native tissues. In acutely dissociated mesenteric vascular smooth myocytes, VIP activated the K_ATP currents in a similar manner as in HEK293 cells. In endothelium-free mesenteric artery rings, VIP produced concentration-dependent vasorelaxation that was attenuated by glibenclamide. These results therefore indicate that the vascular isoform (Kir6.1/SUR2B) of K_ATP channels is a target of VIP. The channel activation relies on the PKA pathway and produces mesenteric arterial relaxation.

Keywords: VIP; K; ⁺; channel; Kir6.1; Antagonist; Second messenger; Vascular tone

Using³¹P MAS NMR to monitor a gel phase thermal disorder transition in sphingomyelin/cholesterol bilayers by Alison L. Costello; Todd M. Alam (pp. 97-104).

The impact of low cholesterol concentrations on an egg sphingomyelin bilayer is investigated using³¹P magic angle spinning (MAS) NMR spectroscopy. The magnitude of the isotropic³¹P MAS NMR line width is used to monitor the main gel to liquid crystalline phase transition, along with a unique gel phase pretransition. In addition, the³¹P chemical shift anisotropy (CSA) and spin–spin relaxation times ( T₂), along with the effects of spinning speed, proton decoupling and magnetic field strength, are reported. The variation of this unique gel phase thermal pretransition with the inclusion of 5 through 21 mol% cholesterol is presented and discussed.

Keywords: Abbreviations; SM; sphingomyelin; PC; phosphatidylcholine; Chol; cholesterol; MAS NMR; magic-angle spinning nuclear magnetic resonance; DSC; differential scanning calorimetry; CSA; chemical shift anisotropy; FWHM; full width at half maximumSolid-state nuclear magnetic resonance; Sphingomyelin bilayer; Magic-angle spinning; Phase transition; Cholesterol; Gel phase

Differential solubilization of inner plasma membrane leaflet components by Lubrol WX and Triton X-100 by Jean-Louis Delaunay; Michelyne Breton; Germain Trugnan; Michèle Maurice (pp. 105-112).

A commonly-used method for analysing raft membrane domains is based on their resistance to extraction by non-ionic detergents at 4 °C. However, the selectivity of different detergents in defining raft membrane domains has been questioned. We have compared the lipid composition of detergent-resistant membranes (DRMs) obtained after Triton X-100 or Lubrol WX extraction in MDCK cells in order to understand the differential effect of these detergents on membranes and their selectivity in solubilizing or not proteins. Both Lubrol and Triton DRMs were enriched with cholesterol over the lysate, thus exhibiting characteristics consistent with the properties of membrane rafts. However, the two DRM fractions differed considerably in the ratio between lipids of the inner and outer membrane leaflets. Lubrol DRMs were especially enriched with phosphatidylethanolamine, including polyunsaturated species with long fatty acyl chains. Lubrol and Triton DRMs also differed in the amount of raft transmembrane proteins and raft proteins anchored to the cytoplasmic leaflet. Our results suggest that the inner side of rafts is enriched with phosphatidylethanolamine and cholesterol, and is more solubilized by Triton X-100 than by Lubrol WX.

Keywords: Detergent; Raft; Cholesterol; Phosphatidylethanolamine; Inner membrane leaflet

Interactions of the dipeptide paralysin β-Ala-Tyr and the aminoacid Glu with phospholipid bilayers by Ioanna Kyrikou; Nikolas P. Benetis; Petros Chatzigeorgiou; Maria Zervou; Kyriakos Viras; Constantine Poulos; Thomas Mavromoustakos (pp. 113-124).

Existing evidence points out that the biological activity of β-Ala-Tyr may in part related to its interactions with the cell membranes. For comparative reasons the effects of Glu were also examined using identical techniques and conditions. In order to examine their thermal and dynamic effects on membrane bilayers a combination of DSC, Raman and solid state NMR spectroscopy on DPPC/water model membranes were applied and the results were compared. DSC data showed that Glu perturbs to a greater degree the model membrane compared to β-Ala-Tyr. Thus, alteration of the phase transition temperature and half width of the peaks, abolishment of the pretransition and influence on the enthalpy of the phase transition were more pronounced in the Glu loaded bilayers. Raman spectroscopy showed that incorporation of Glu in DPPC/water bilayers increased the order in the bilayers in contrast to the effect of the dipeptide. Several structural and dynamical properties of the DPPC multilamellar bilayers with and without the dipeptide or Glu were compared using high resolution C-13 MAS (Magic Angle Spinning) spectra and spectral simulations of inhomogeneously broadened, stationary P-31 NMR lineshapes measured under CP (Cross-polarization) conditions. These methods revealed that the aminoacid Glu binds in the close realm of the phosphate in the hydrophilic headgroup of DPPC while β-Ala-Tyr is located more deeply inside the hydrophobic zone of the bilayer. The P-31 NMR simulations indicated restricted fast rotary motion of the phospholipids about their long axes in the organized bilayer structure. Finally, by the applied methodologies it is concluded that the two molecules under study exert dissimilar thermal and dynamic effects on lipid bilayers, the Glu improving significantly the packing of the lipids in contrast to the smaller and opposite effect of the dipeptide.

Keywords: Paralysin; β; -Ala-Tyr; C-13 MAS NMR; CP P-31 NMR broadline, differential scanning calorimetry DSC; DPPC/water bilayer; Raman spectroscopy; The aminoacid neurotransmitter Glutamate (; l; -Glu)

Membrane raft actin deficiency and altered Ca²⁺-induced vesiculation in stomatin-deficient overhydrated hereditary stomatocytosis by D. Katie Wilkinson; E. Jane Turner; Edward T. Parkin; Ashley E. Garner; Penny J. Harrison; Mark Crawford; Gordon W. Stewart; Nigel M. Hooper (pp. 125-132).

In overhydrated hereditary stomatocytosis (OHSt), the membrane raft-associated stomatin is deficient from the erythrocyte membrane. We have investigated two aspects of raft structure and function in OHSt erythrocytes. First, we have studied the distribution of other membrane and cytoskeletal proteins in rafts by analysis of detergent-resistant membranes (DRMs). In normal erythrocytes, 29% of the actin was DRM-associated, whereas in two unrelated OHSt patients the DRM-associated actin was reduced to <10%. In addition, there was a reduction in the amount of the actin-associated protein tropomodulin in DRMs from these OHSt cells. When stomatin was expressed in Madin–Darby canine kidney cells, actin association with the membrane was increased. Second, we have studied Ca²⁺-dependent exovesiculation from the erythrocyte membrane. Using atomic force microscopy and proteomics analysis, exovesicles derived from OHSt cells were found to be increased in number and abnormal in size, and contained greatly increased amounts of the raft proteins flotillin-1 and -2 and the calcium binding proteins annexin VII, sorcin and copine 1, while the concentrations of stomatin and annexin V were diminished. Together these observations imply that the stomatin–actin association is important in maintaining the structure and in modulating the function of stomatin-containing membrane rafts in red cells.

Keywords: Abbreviations; AFM; atomic force microscopy; DRM; detergent-resistant membrane; DTT; dithiothreitol; MALDI-TOF; matrix-assisted laser desorption ionisation time-of-flight; MDCK; Madin–Darby canine kidney; PBS; phosphate-buffered saline; OHSt; overhydrated hereditary stomatocytosis; TBS; Tris-buffered salineActin; Detergent-resistant membrane; Erythrocyte; Membrane raft; Stomatin

Interaction of ferredoxin:NADP⁺ oxidoreductase with model membranes by Joanna Grzyb; Mariusz Gagoś; Wiesław I. Gruszecki; Monika Bojko; Kazimierz Strzałka (pp. 133-142).

The ferredoxin:NADP⁺ oxidoreductase (FNR) is a plant enzyme, catalyzing the last step of photosynthetic linear electron transport, and involved also in cyclic electron transport around photosystem I. In this study we present the first evidence of FNR (isolated from spinach and from wheat) interaction directly with a model membrane without the mediation of any additional protein. The monomolecular layer technique measurements showed a significant increase in surface pressure after the injection of enzyme solution beneath a monolayer consisting of chloroplast lipids: monogalactosyldiacylglycerol or digalactosyldiacylglycerol. An ATR FTIR study revealed also the presence of FNR in a bilayer composed of these lipids. The secondary structure of the protein was significantly impaired by lipids, as with a pH-induced shift. The stabilization of FNR in the presence of lipids leads to an increase in the rate of NADPH-dependent reduction of dibromothymoquinone catalyzed by the enzyme. The biological significance of FNR–membrane interaction is discussed.

Keywords: Ferredoxin:NADP+ oxidoreductase; Fourier transform infrared spectroscopy; Isoform; Model membrane; Monomolecular lipid layer; Photosynthesis

Different oxidized phospholipid molecules unequally affect bilayer packing by Francesco M. Megli; Luciana Russo (pp. 143-152).

The aim of this study was to gain more detailed knowledge about the effect of the presence of defined oxidized phospholipid molecules in phospholipid bilayers. After chromatographic and mass spectrometry analysis, the previously used product of the Fenton reaction with unsaturated lecithins proved to consist of a plethora of oxidatively modified lecithins, unuseful either for the detailed study of the effects brought about in the bilayer or as the source of defined oxidized phospholipid molecules. The latter, particularly 2-(ω-carboxyacyl)- and 2-( n-hydroperoxyacyl)-lecithins, can be more conveniently prepared by chemical or enzymatic synthesis rather than by chemical or physical oxidation. The effect of those molecules and of commercially available 12-hydroxy-stearic and dodecanedioic acid was studied in planar supported phospholipid bilayers (SPBs) by use of EPR spectrometry. The SPBs also contained 2-(5-doxylstearoyl)-lecithin as the spin probe, and the EPR spectral anisotropy loss, indicative of bilayer disordering, was measured as a function of the molar percentage of oxidized lipid. Most oxidized lipid molecules examined in this study were able to induce bilayer disordering, while hydroperoxyl group-bearing acyl chains appeared to be much less effective. It is concluded that the effects of different oxidized phospholipids on phospholipid bilayer structure cannot be generalized, as happens with batch-oxidized phospholipids, and that the use of defined oxidized phospholipid molecular species for membrane oxidative stress guarantees a more reliable and detailed response.

Keywords: Phospholipid bilayer; Lipoperoxidation; EPR; Spin labeling; Bilayer disordering; EPR spectral anisotropy

Cell surface phosphorylation by a novel ecto-protein kinase: A key regulator of cellular functions in spermatozoa by Debjani Nath; Arunima Maiti; Gopal C. Majumder (pp. 153-165).

Since 1976 many studies have been reported on the occurrence and functional significance of ecto-protein kinases in a variety of cell types although their precise biochemical identity is largely unknown. This study reports for the first time purification to apparent homogeneity of an ecto-protein kinase (ecto-CIK) and some of its characteristics using caprine sperm as the cell model. The ecto-CIK is a unique membrane-specific serine/threonine protein kinase. It is a strongly basic 115 kDa protein made up of two subunits: 63 and 55 kDa. The ecto-kinase undergoes a remarkable lateral movement on the outer cell surface culminating in capping on the sperm acrosomal tip. MPS, its major protein substrate is also located on the acrosomal tip. Both ecto CIK and MPS serve as potential regulators of flagellar motility. This novel enzyme appears to be major kinase responsible for the reported regulation of mammalian cellular functions by modulating phosphorylation of the membrane-bound proteins.

Liquid–liquid immiscibility in model membranes activates secretory phospholipase A₂ by Kerstin Wagner; Bernard Desbat; Gerald Brezesinski (pp. 166-174).

Secretory phospholipase A₂ (sPLA₂) hydrolyzes phosphatidylcholines (PC) within lipid bilayers to produce lyso-PC and a fatty acid, which can act as signaling molecule in biological membranes. The activity of sPLA₂ depends on the membrane structure. Bilayer defects, curvature, and gel–fluid micro-heterogeneity are known to activate sPLA₂. Here, we investigate if liquid–liquid immiscibility within model membranes is sufficient for sPLA₂ activation. The onset of the hydrolytic activity of cobra-venom sPLA₂ towards mixed monolayers of dimyristoyl-PC (DMPC)/cholesterol 2:1 (mol/mol) has been determined using infrared reflection–absorption spectroscopy (IRRAS) and polarization-modulated (PM-) IRRAS. The lag phase of sPLA₂ activity increases exponentially with rising surface pressures starting at 12 mN/m. This indicates that enzyme activation is hampered at higher surface pressures. Below 12 mN/m, no lag phase is observed, and sPLA₂ is efficiently activated. The surface pressure that is critical for sPLA₂ activation correlates with the critical miscibility pressure according to the phase diagram of DMPC and cholesterol. Thus, coexisting, liquid-phase domains provide sufficient boundaries to activate sPLA₂. Moreover, liquid–liquid immiscibility is an activating mechanism for sPLA₂ that also applies to biological membranes under physiological conditions because the corresponding bilayer structure is associated with that of membrane rafts.

Keywords: Dimyristoylphosphatidylcholine (DMPC); Cholesterol; Monolayer structure; Phase separation; Infrared reflection–absorption spectroscopy (IRRAS); Lipolytic enzyme activation

Kinetics of cholesterol extraction from lipid membranes by methyl-β-cyclodextrin—A surface plasmon resonance approach by Mojca Podlesnik Beseničar; Andrej Bavdek; Aleš Kladnik; Peter Maček; Gregor Anderluh (pp. 175-184).

The kinetics of cholesterol extraction from cellular membranes is complex and not yet completely understood. In this paper we have developed an experimental approach to directly monitor the extraction of cholesterol from lipid membranes by using surface plasmon resonance and model lipid systems. Methyl-β-cyclodextrin was used to selectively remove cholesterol from large unilamellar vesicles of various compositions. The amount of extracted cholesterol is highly dependent on the composition of lipid membrane, i.e. the presence of sphingomyelin drastically reduced and slowed down cholesterol extraction by methyl-β-cyclodextrin. This was confirmed also in the erythrocyte ghosts system, where more cholesterol was extracted after erythrocytes were treated with sphingomyelinase. We further show that the kinetics of the extraction is mono-exponential for mixtures of 1,2-dioleoyl- sn-glycero-3-phosphocholine and cholesterol. The kinetics is complex for ternary lipid mixtures composed of 1,2-dioleoyl- sn-glycero-3-phosphocholine, bovine brain sphingomyelin and cholesterol. Our results indicate that the complex kinetics observed in experiments with cells may be the consequence of lateral segregation of lipids in cell plasma membrane.

Keywords: Abbreviations; CHO; cholesterol; CD; cyclodextrin; DOPC; 1,2-Dioleoyl-; sn; -Glycero-3-Phosphocholine; LUV; large unilamellar vesicles; MBCD; methyl-β-cyclodextrin; NBD-CHO; 25-(N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino)-27-norcholesterol; RU; response units; SM; sphingomyelin; SPR; surface plasmon resonanceLipid membrane; Cholesterol extraction; Cyclodextrin; Surface plasmon resonance; Biacore

Sphingomyelinase generation of ceramide promotes clustering of nanoscale domains in supported bilayer membranes by Ira; Linda J. Johnston (pp. 185-197).

The effects of ceramide incorporation in supported bilayers prepared from ternary lipid mixtures which have small nanoscale domains have been examined using atomic force and fluorescence microscopy. Both direct ceramide incorporation in vesicles used to prepare the supported bilayers and enzymatic hydrolysis of SM by sphingomyelinase were compared for membranes prepared from 5:5:1 DOPC/sphingomyelin/cholesterol mixtures. Both methods of ceramide incorporation resulted in enlargement of the initial small ordered domains. However, enzymatic ceramide generation led to a much more pronounced restructuring of the bilayer to give large clusters of domains with adjacent areas of a lower phase. The individual domains were heterogeneous with two distinct heights, the highest of which is assigned to a ceramide-rich phase which is hypothesized to occur via ceramide flip-flop to the lower leaflet with formation of a raised domain due to negative membrane curvature. A combination of AFM and fluorescence showed that the bilayer restructuring starts rapidly after enzyme addition, with formation of large clusters of domains at sites of high enzyme activity. The clustering of domains is accompanied by redistribution of fluid phase to the periphery of the domain clusters and there is a continued slow evolution of the bilayer over a period of an hour or more after the enzyme is removed. The relevance of the observed clustering of small nanoscale domains to the postulated coalescence of raft domains to form large signaling platforms is discussed.

Keywords: Abbreviations; AFM; atomic force microscopy; chol; cholesterol; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholine; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; ESM; egg sphingomyelin; GUV; giant unilamellar vesicles; PC; phosphatidylcholine; SMase; sphingomyelinase; TIRF; total internal reflection fluorescence; TR-DHPE; Texas Red® 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium saltAtomic force microscopy; Bilayer; Ceramide; Membrane; Total internal reflection fluorescence

Cholesterol and anionic phospholipids increase the binding of amyloidogenic transthyretin to lipid membranes by Xu Hou; Adam Mechler; Lisandra L. Martin; Marie-Isabel Aguilar; David H. Small (pp. 198-205).

Deposition of transthyretin (TTR) amyloid is a pathological hallmark of familial amyloidotic polyneuropathy (FAP). Recently we showed that TTR binds to membrane lipids via electrostatic interactions and that membrane binding is correlated with the cytotoxicity induced by amyloidogenic TTR. In the present study, we examined the role of lipid composition in membrane binding of TTR by a surface plasmon resonance (SPR) approach. TTR bound to lipid bilayers through both high- and low-affinity interactions. Increasing the mole fraction of cholesterol in the bilayer led to an increase in the amount of high-affinity binding of an amyloidogenic mutant (L55P) TTR. In addition, a greater amount of L55P TTR bound with high affinity to membranes made from anionic phospholipids, phosphatidylglycerol (PG) and phosphatidylserine (PS), than to membranes made from zwitterionic phospholipid phosphatidylcholine (PC). The anionic phospholipids (PS and PG) promoted the aggregation of L55P TTR by accelerating the nucleation phase of aggregation, whereas the zwitterionic phospholipid PC had little effect. These results suggest that cholesterol and anionic phospholipids may be important for TTR aggregation and TTR-induced cytotoxicity.

Keywords: Abbreviations; Aβ; β-amyloid protein; AFM; atomic force microscopy; DLS; dynamic light scattering; ER; endoplasmic reticulum; FAP; familial amyloidotic polyneuropathy; HOPG; highly oriented pyrolytic graphite; K; _D; dissociation constant; PC; dimyristoyl-; l; -α-phosphatidylcholine; PE; dimyristoyl-; l; -α-phosphatidylethanolamine; PG; dimyristoyl-; l; -α-phosphatidylglycerol; PrP; prion protein; PS; dimyristoyl-; l; -α-phosphatidylserine; RU; response unit; SM; sphingomyelin; SPR; surface plasmon resonance; SUVs; small unilamellar vesicles; T; ₄; thyroxine; TTR; transthyretin; VGCCs; voltage-gated Ca; ²⁺; -channels; WT; wild-typeTransthyretin (TTR); Cholesterol; Phospholipid; Aggregation; Amyloid; Binding

Relevance of the N-terminal NLS-like sequence of the prion protein for membrane perturbation effects by Kamila Oglęcka; Pontus Lundberg; Mazin Magzoub; L.E. Göran Eriksson; Ülo Langel; Astrid Gräslund (pp. 206-213).

We investigated the nuclear localization-like sequence KKRPKP, corresponding to the residues 23–28 in the mouse prion protein (mPrP), for its membrane perturbation activity, by comparing effects of two mPrP-derived peptides, corresponding to residues 1–28 (mPrPp(1–28)) and 23–50 (mPrPp(23–50)), respectively. In erythrocytes, mPrPp(1–28) induced ∼60% haemoglobin leakage after 30 min, whereas mPrPp(23–50) had negligible effects. In calcein-entrapping, large unilamellar vesicles (LUVs), similar results were obtained. Cytotoxicity estimated by lactate dehydrogenase leakage from HeLa cells, was found to be ∼12% for 50 μM mPrPp(1–28), and ∼1% for 50 μM mPrPp(23–50). Circular dichroism spectra showed structure induction of mPrPp(1–28) in the presence of POPC:POPG (4:1) and POPC LUVs, while mPrPp(23–50) remained a random coil. Membrane translocation studies on live HeLa cells showed mPrPp(1–28) co-localizing with dextran, suggesting fluid-phase endocytosis, whereas mPrPp(23–50) hardly translocated at all. We conclude that the KKRPKP-sequence is not sufficient to cause membrane perturbation or translocation but needs a hydrophobic counterpart.

Keywords: Abbreviations; NLS; Nuclear Localization Sequence; PrP; Prion Protein; PrP; ^C; cellular isoform of PrP; PrP; ^Sc; scrapie isoform of PrP; mPrPp(1–28); residues 1–28 of the mouse PrP sequence; mPrPp(23–50); residues 23–50 of the mouse PrP sequence; bPrPp(1–30); residues 1–30 of the bovine PrP sequence; CD; Circular Dichroism; LUVs; Large Unilamellar Vesicles; hRBC; Human Red Blood Cell; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-[phospho-; rac; -(1-glycerol)] sodium salt; LDH; Lactate Dehydrogenase; CPP; Cell-Penetrating Peptide; PTD; Protein Transduction Domain; HeLa cells; Henrietta Lacks cells; CHO cells; Chinese Hamster Ovary cells; TSE; Transmissible Spongiform EncephalopathiesPrion protein; Membrane translocation; Calcein leakage; Endosomal escape; NLS-like sequence; Haemoglobin leakage

Local and translational dynamics in DNA–lipid assemblies monitored by solid-state and diffusion NMR by Cecília Leal; Dick Sandström; Pernilla Nevsten; Daniel Topgaard (pp. 214-228).

The influence of electrostatic interactions on the dynamic properties of complexes containing DNA and mixtures of cationic- (DDA) and zwitterionic (DLPC) lipids are studied by means of NMR. The systems are arranged in lamellar membrane stacks intercalated by DNA molecules. This is confirmed by³¹P-NMR, where a superposition of an axially symmetric powder pattern arising from the phospholipid membrane and an asymmetric tensor due to DNA can be fitted to the experimentally observed lineshape. The local mobility and order is assessed using two solid-state NMR techniques applicable to samples with natural isotopic abundance: WIdeline SEparation (WISE) and Separated Local Field (SLF) spectroscopy. Both experiments yield highly resolved¹³C spectra in the direct dimension. The indirect dimension contains information about molecular dynamics through the¹H dipolar linewidth (WISE) or the¹H–¹³C dipolar coupling constant (SLF). The experiments suggest that DNA is static while it induces an increased disorder in the hydrocarbon chains as compared to the parent lipid case. DDA chain order is more affected than DLPC due to the attractive electrostatic interaction between DNA and the cationic lipid. Translational dynamics of the lipids and the water was measured with the Pulsed Field Gradient STimulated Echo (PFG STE) technique. The influence of lamellar domain size and the angular dependence of the diffusion coefficients and nuclear relaxation times on the results of the PFG STE experiments are discussed. The local water diffusion coefficient is reduced by a factor four from the value of bulk water, and increases as the DLPC content is increased. We observe two lipid components with an order of magnitude difference in diffusion coefficients in the DNA:DDA:DLPC precipitate and these are assigned to DLPC (fast) and DDA (slow). Cationic lipid (DDA) diffusion is decreasing a factor of 2 when DLPC is added to the pure DNA:DDA system, indicating DNA-induced lipid segregation within the bilayer and the transition from locally 2D to 1D diffusion of the DDA. The results show that DNA–lipid electrostatic interactions reduce the long-range lipid mobility but locally enhance the hydrocarbon chain dynamics by perturbing the preferred lipid packing.

Keywords: DNA-lipid assemblies; Membrane local mobility; Membrane local order; Lateral diffusion; Domain size; Domain formation; Solid-state NMR; Diffusion NMR

Amphipathic α-helical peptide, HP (2–20), and its analogues derived from Helicobacter pylori: Pore formation mechanism in various lipid compositions by Seong-Cheol Park; Mi-Hyun Kim; Mohammed Akhter Hossain; Song Yub Shin; Yangmee Kim; Lorenzo Stella; John D. Wade; Yoonkyung Park; Kyung-Soo Hahm (pp. 229-241).

In a previous study, we determined that HP(2–20) (residues 2–20 of parental HP derived from the N-terminus of Helicobacter pylori Ribosomal Protein L1) and its analogue, HPA3, exhibit broad-spectrum antimicrobial activity. The primary objective of the present study was to gain insight into the relevant mechanisms of action using analogues of HP(2–20) together with model liposomes of various lipid compositions and electron microscopy. We determined that these analogues, HPA3 and HPA3NT3, exert potent antibacterial effects in low-salt buffer and antifungal activity against chitin-containing fungi, while having little or no hemolytic activity or cytotoxicity against mammalian cell lines. Our examination of the interaction of HP(2–20) and its analogues with liposomes showed that the peptides disturb both neutral and negatively-charged membranes, as demonstrated by the release of encapsulated fluorescent markers. The release of fluorescent markers induced by HP(2–20) and its analogues was inversely related to marker size. The pore created by HP(2–20) shows that the radius is approximately 1.8 nm, whereas HPA3, HPA3NT3, and melittin have apparent radii between 3.3 and 4.8 nm. Finally, as shown by electron microscopy, the liposomes and various microbial cells treated with HPA3 and HPA3NT3 showed oligomerization and blebbing similar to that seen with melittin, while HP(2–20) exhibited flabbiness. These results suggest that HP(2–20) may exert its antibiotic effects through a small pore (about 1.8 nm), whereas HPA3 and HPA3NT3 formed pores of a size consistent with those formed by melittin.

Keywords: Abbreviations; BS; ³; Bis(sulfosuccinimidyl)suberate; CH; cholesterol; CL; cardiolipin; DCC; dicyclohexylcarbodiimide; Dis-C; ₃; -5, 3,3′; diethylthio-dicarbocyanine iodide; FITC-D; fluorescein isothiocyanate dextrans; HOBT; 1-hydroxy benzotriazole; LPS; lipopolysaccharide; NMP; N-methyl-2-pyrrolidone; PC; egg yolk; l; -α-phosphatidylcholine; PE; l; -α-phosphatidylethanolamine; PG; l; -α-phosphatidyl-; dl; -glycerol; PS; Phosphatidylserine; SM; SphingomyelinHP(2–20); Mechanism of action; Fluorescent markers; Oligomerization; Pore-forming

Adhesion characteristics and stability assessment of lectin-modified liposomes for site-specific drug delivery by Heike Bakowsky; Thomas Richter; Carsten Kneuer; Dick Hoekstra; Ulrich Rothe; Gerd Bendas; Carsten Ehrhardt; Udo Bakowsky (pp. 242-249).

Carbohydrate moieties of the cellular glycocalyx have been suggested to play an important role in biological recognition processes during pathologic conditions, such as inflammation and cancer. Herein, we describe lectin-modified liposomes which might have potential for site-specific drug delivery during the therapy of such diseases. Specific interactions of plain (i.e., unmodified) and PEGylated, lectin-grafted liposomes with model membranes were investigated under real-time flow conditions using a quartz crystal microbalance. In addition, the morphology of the liposomal systems was assessed by atomic force microscopy. Plain liposomes exhibited only unspecific adhesion to glycolipid membranes and had a tendency to coalesce. The degree of membrane interaction was significantly increased when plain liposomes were modified with the lectin, Concanavalin A. However, vesicle fusion also markedly increased as a result of lectin modification. Additional PEGylation of liposomes reduced unspecific adhesion phenomena, as well as coalescence. Moreover, our studies enabled us to establish quartz crystal microbalance and atomic force microscopy as powerful and complementary methods to characterize adhesion properties of targeted drug delivery systems.

Keywords: Concanavalin A; Quartz crystal microbalance; Atomic force microscopy; Drug targeting; PEGylation; Glycocalyx

uPA binding increases UPAR localization to lipid rafts and modifies the receptor microdomain composition by Macarena Sahores; Alessandro Prinetti; Gustavo Chiabrando; Francesco Blasi; Sandro Sonnino (pp. 250-259).

UPAR is a GPI anchored protein, which is found in both lipid rafts and in more fluid regions of the plasma membrane. We have studied the role of the ligand uPA on uPAR localization and on the composition of the lipid membrane microdomains. We have analyzed the glycosphingolipid environment of uPAR in detergent resistant membrane (DRM) fractions prepared by cell lysis with 1% Triton X-100 and fractionated by sucrose gradient centrifugation obtained from HEK293-uPAR cells. The uPAR specific lipid membrane microdomain has been separated from the total DRM fraction by immunoprecipitation with an anti-uPAR specific antibody under conditions that preserve membrane integrity. We have also tested uPA-induced ERK phosphorylation in the presence of methyl-β-cyclodextrin, which is known to disrupt lipid rafts by sequestering cholesterol from such domains. Our results show that uPAR is partially associated with DRM and this association is increased by ligands, is independent of the catalytic activity of uPA, and is required for intracellular signalling. In the absence of ligands, uPAR experiences a lipid environment very similar to that of total DRM, enriched in sphingomyelin and glycosphingolipids. However, after treatment of cells with uPA or ATF the lipid environment is strongly impoverished of neutral glycosphingolipids.

Keywords: Abbreviations: Ganglioside and glycosphingolipid nomenclature is in accordance with Svennerholm, 1980 , and the IUPAC-IUBMB recommendations .; GlcCer; β-Glc-(1-1)-Cer; LacCer; β-Gal-(1-4)-β-Glc-(1-1)-Cer; GM3; II; ³; Neu5AcLacCer, α-Neu5Ac-(2-3)-β-Gal-(1-4)-β-Glc-(1-1)-Cer; GM2; II; ³; Neu5AcGgOse; ₃; Cer, β-GalNAc-(1-4)-[α-Neu5Ac-(2-3)]-β-Gal-(1-4)-β-Glc-(1-1)-Cer; GM1; II; ³; Neu5AcGgOse; ₄; Cer, β-Gal-(1-3)-β-GalNAc-(1-4)-[α-Neu5Ac-(2-3)]-β-Gal-(1-4)-β-Glc-(1-1)-Cer; Cer; Ceramide,; N; -acyl-sphingosine; Sph; Sphingosine, (2; S; ,3; R; ,4; E; )-2-amino-1,3-dihydroxy-octadecene; [1-; ³; H]sphingosine; (2; S; ,3; R; ,4; E; )-2-amino-1,3-dihydroxy-[1-; ³; H]octadecene; PE; Phosphatidylethanolamine; SM; Sphingomyelin; SL; Sphingolipids; 2D-HPTLC; Two-dimensional high-performance thin layer chromatography; uPA; Urokinase plasminogen activator; uPAR; Urokinase receptor; ATF; Amino terminal fragment of uPA; TfR; Transferrin receptor; DRM; Detergent-resistant membranes; DS; Detergent-soluble material; GPI; Glycosylphosphatidylinositol; TX-100; Triton X-100; CHO; Cholesterol; CD; Methyl-β-cyclodextrinUrokinase receptor; Lipid raft; Immunoprecipitation; Urokinase

Pore formation by the Bordetella adenylate cyclase toxin in lipid bilayer membranes: Role of voltage and pH by Oliver Knapp; Elke Maier; Jiří Mašín; Peter Šebo; Roland Benz (pp. 260-269).

The bifunctional adenylate cyclase toxin (ACT or CyaA) of Bordetella pertussis invades target cells via transport through the cytoplasmic membrane. The membrane potential represents thereby an important factor for the uptake in vivo. Previous studies demonstrated that adenylate cyclase (AC) delivery into cells requires a negative membrane potential inside the cells. The results of lipid bilayer experiments with ACT presented here indicated that two different types of pore-like structures are formed by ACT dependent on the orientation of the electrical potential across the membranes. Pore formation at a positive potential at the cis side of the membranes, the side of the addition of the toxin, was fast and its conductance had a defined size, whereas at negative potential the pores were not defined, had a reduced pore-forming activity and a very short lifetime. Fluctuations inserted at positive potentials showed asymmetric current–voltage relationships for positive and negative voltages. Positive potentials at the cis side resulted in an increasing current, whereas at negative potentials the current decreased or remained at a constant level. Calcium ions enhanced the voltage dependence of the ACT pores when they were added to the cis side. The single-pore conductance was strongly affected by the variation of the pH value and increased in 1M KCl with increasing pH from about 4 pS at pH 5 to about 60 pS at pH 9. The ion selectivity remained unaffected by pH. Experiments with ACT mutants revealed, that the adenylate cyclase (AC) and repeat (RT) domains were not involved in voltage and pH sensing.

Keywords: Abbreviations; CyaA, ACT; adenylate cyclase toxin of; Bordetella pertussis; HlyA; α-hemolysin of; Escherichia coli; RTX; Repeats in Toxin; G; conductance, i.e. current divided by voltageAdenylate cyclase toxin; ACT; CyaA; Voltage; pH; Channel information; Calcium; Bordetella pertussis; Lipid bilayer

Functional analysis of phenolsulfonphthalein transport system in Long–Evans Cinnamon rats by Shirou Itagaki; Makoto Chiba; Masaki Kobayashi; Mitsuru Sugawara; Michiya Kobayashi; Takeshi Hirano; Ken Iseki (pp. 270-275).

It has been reported that the transport function for organic anions on the kidney is maintained in multidrug resistance-associated protein 2 (Mrp2)-deficient rats. Different from Mrp2-deficient rats, Long–Evans Cinnamon (LEC) rats have impaired urinary excretion of Mrp2-substrate, phenolsulfonphthalein (PSP). PSP is transported by the potential-sensitive urate transport system in rat brush-border membranes. We analyzed the function of PSP transport system in LEC rats. Unlike Long–Evans Agouti (LEA) rats, the initial uptake of PSP and urate into the renal brush-border membrane vesicles of LEC rats were not significantly enhanced in the presence of positive intravesicular potential, suggesting that the potential-sensitive urate transport system is impaired in LEC rats. LEC rats should be useful for elucidating the potential-sensitive urate transport system in rats at the molecular level.

Keywords: LEC rats; Urinary excretion; Transporter; Organic anion; Urate

Blistering of supported lipid membranes induced by Phospholipase D, as observed by real-time atomic force microscopy by Karim El Kirat; Vincent Duprès; Yves F. Dufrêne (pp. 276-282).

Phospholipase D from Streptomyces chromofuscus (PLDSc) is a soluble enzyme known to be activated by the phosphatidic acid (PA)–calcium complexes. Despite the vast body of literature that has accumulated on this enzyme, the exact mechanism of activation remains poorly understood. In this work, we report the first observation of PLDSc activity in real time and at nanometer resolution using atomic force microscopy (AFM). AFM images of continuous and patchy dipalmitoylphosphatidylcholine (DPPC) bilayers were recorded, prior and after incubation with PLDSc. For continuous bilayers, the enzyme induced important morphological alterations; holes corresponding to the bilayer thickness were created, while an additional elevated phase, about 2.5 nm high, was observed. This bilayer blistering is believed to be due to the production of the negatively charged lipid PA that would cause localized repulsions between the bilayer and the underlying mica surface. By contrast, these elevated domains were not seen on patchy bilayers incubated with the enzyme. Instead, the shapes of DPPC patches were strongly deformed by enzyme activity and evolved into melted morphologies. These results point to the importance of lipid packing on PLD activity and illustrate the potential of AFM for visualizing remodeling enzymatic activities.

Keywords: Atomic force microscopy; Lipid bilayers; Phospholipase D; Phosphatidic acid; Membrane blistering; Anionic lipid

Activation of a polyvalent cation-sensing receptor decreases magnesium transport via claudin-16 by Akira Ikari; Chiaki Okude; Hayato Sawada; Yohei Sasaki; Yasuhiro Yamazaki; Junko Sugatani; Masakuni Degawa; Masao Miwa (pp. 283-290).

Renal magnesium is mainly reabsorbed by a paracellular pathway in the thick ascending limb of Henle. The expression of claudin-16 increased magnesium transport in Madin–Darby canine kidney (MDCK) cells. Little is known about the regulatory mechanism of magnesium transport via claudin-16. Here we examined the effect of a polyvalent cation-sensing receptor (CaSR) on the intracellular distribution of and transport of magnesium by claudin-16. FLAG-tagged claudin-16 was stably expressed in MDCK cells using a Tet-OFF system. The activation of CaSR by magnesium, calcium, neomycin, and gadolinium did not affect the expression of FLAG-tagged claudin-16, CaSR, or ZO-1, a tight junctional scaffolding protein. These activators decreased the phosphoserine level of FLAG-tagged claudin-16 and the association of FLAG-tagged claudin-16 with ZO-1. The activation of CaSR induced a decrease in PKA activity. Immunofluorescence microscopy revealed that FLAG-tagged claudin-16 is distributed at the cell–cell border under unstimulated conditions, whereas it translocates to the intracellular compartment, mainly lysosome, with the activation of CaSR. In contrast, the distribution of ZO-1 was unaffected by the activation. The expression of FLAG-tagged claudin-16 increased transepithelial electrical resistance (TER) and transepithelial magnesium transport without affecting FITC-dextran (MW 4000) flux. The activation of CaSR decreased TER and magnesium transport, which were recovered by co-treatment with dibutyryl cAMP, a membrane-permeable cAMP analogue. Taken together, CaSR activation may decrease PKA activity, resulting in a decrease in phosphorylated claudin-16, the translocation of claudin-16 to lysosome and a decrease in magnesium reabsorption.

Keywords: Abbreviations; CaSR; polyvalent cation-sensing receptor; MDCK; Madin–Darby canine kidney; PKA; protein kinase A; TAL; thick ascending limb; TER; transepithelial electrical resistanceClaudin-16; PKA; Phosphorylation; Polyvalent cation-sensing receptor

Interaction between chitosan and bovine lung extract surfactants by Ningxi Kang; Zdenka Policova; Gelareh Bankian; Michael L. Hair; Yi Y. Zuo; A. Wilhelm Neumann; Edgar J. Acosta (pp. 291-302).

The interaction between a cationic polyelectrolyte, chitosan, and an exogenous bovine lung extract surfactant (BLES) was studied using dynamic compression/expansion cycles of dilute BLES preparations in a Constrained Sessile Drop (CSD) device equipped with an environmental chamber conditioned at 37 °C and 100% R.H. air. Under these conditions, dilute BLES preparations tend to produce variable and relatively high minimum surface tensions. Upon addition of “low” chitosan to BLES ratios, the minimum surface tension of BLES–chitosan preparations were consistently low (i.e. <5 mJ/m²), and the resulting surfactant monolayers (adsorbed at the air–water interface) were highly elastic and stable. However, the use of “high” chitosan to BLES ratios induced the collapse of the surfactant monolayer at high minimum surface tensions (i.e. >15 mJ/m²). The zeta potential of the lung surfactant aggregates in the subphase suggests that chitosan binds to the anionic lipids (phosphatidyl glycerols) in BLES, and that this binding is ultimately responsible for the changes in the surface activity (elasticity and stability) of these surfactant–polyelectrolyte mixtures. Furthermore the transition from “low” to “high” chitosan to BLES ratios correlates with the flocculation and de-flocculation of surfactant aggregates in the subphase. It is proposed that the aggregation/segregation of “patches” of anionic lipids in the surfactant monolayer produced at different chitosan to BLES ratios explains the enhancing/inhibitory effects of chitosan. These observations highlight the importance of electrostatic interactions in lung surfactant systems.

Keywords: Polyelectrolyte; Surface tension; Zeta potential; Respiratory distress syndrome

Amphotericin B induces interdigitation of apolipoprotein stabilized nanodisk bilayers by Thanh-Son Nguyen; Paul M.M. Weers; Vincent Raussens; Zhen Wang; Gang Ren; Todd Sulchek; Paul D. Hoeprich Jr.; Robert O. Ryan (pp. 303-312).

Amphotericin B nanodisks (AMB-ND) are ternary complexes of AMB, phospholipid and apolipoprotein organized as discrete nanometer scale disk-shaped bilayers. In gel filtration chromatography experiments, empty ND lacking AMB elute as a single population of particles with a molecular weight in the range of 200 kDa. AMB-ND formulated at a 4:1 phospholipid:AMB weight ratio separated into two peaks. One peak eluted at the position of control ND lacking AMB while the second peak, containing all of the AMB present in the original sample, eluted in the void volume. When ND prepared with increased AMB were subjected to gel filtration chromatography an increased proportion of phospholipid and apolipoprotein was recovered in the void volume with AMB. Native gradient gel electrophoresis corroborated the gel filtration chromatography data and electron microscopy studies revealed an AMB concentration-dependent heterogeneity in ND particle size. Stability studies revealed that introduction of AMB into ND decreases the ability of apoA-I to resist denaturation. Atomic force microscopy experiments showed that AMB induces compression of ND bilayer thickness while infrared spectroscopy analysis revealed that the presence of AMB does not induce extreme lipid disorder or alter the mean angle of the molecular axis along fatty acyl chains of ND phospholipids. Taken together the results are consistent with AMB-induced bilayer interdigitation, a phenomenon that likely contributes to AMB-dependent pore formation in susceptible membranes.

Keywords: Amphotericin B; Membrane; Nanodisk; Interdigitation; Atomic force microscopy; Infrared spectroscopy

Glycyrrhetinic acid as inhibitor or amplifier of permeability transition in rat heart mitochondria by Valentina Battaglia; Anna Maria Brunati; Cristina Fiore; Carlo Alberto Rossi; Mauro Salvi; Elena Tibaldi; Mario Palermo; Decio Armanini; Antonio Toninello (pp. 313-323).

Glycyrrhetinic acid (GE), a hydrolysis product of glycyrrhizic acid, one of the main constituents of licorice root, is able, depending on its concentration, to prevent or to induce the mitochondrial permeability transition (MPT) (a phenomenon related to oxidative stress) in rat heart mitochondria (RHM). In RHM, below a threshold concentration of 7.5 μM, GE prevents oxidative stress and MPT induced by supraphysiological Ca²⁺ concentrations. Above this concentration, GE induces oxidative stress by interacting with a Fe–S centre of Complex I, thus producing ROS, and amplifies the opening of the transition pore, once again induced by Ca²⁺. GE also inhibits Ca²⁺ transport in RHM, thereby preventing the oxidative stress induced by the cation. However, the reduced amount of Ca²⁺ transported in the matrix is sufficient to predispose adenine nucleotide translocase for pore opening. Comparisons between observed results and the effects of GE in rat liver mitochondria (RLM), in which the drug induces only MPT without exhibiting any protective effect, confirm that it interacts in a different way with RHM, suggesting tissue specificity for its action. The concentration dependence of the opposite effects of GE, in RHM but not RLM, is most probably due to the existence of a different, more complex, pathway by means of which GE reaches its target. It follows that high GE concentrations are necessary to stimulate the oxidative stress capable of inducing MPT, because of the above effect, which prevents the interaction of low concentrations of GE with the Fe–S centre. The reported results also explain the mechanism of apoptosis induction by GE in cardiomyocytes.

Keywords: Abbreviations; AdNT; adenine nucleotide translocase; AIF; apoptosis inducing factor; BHT; butylhydroxytoluene; BKA; bongkrekic acid; CsA; cyclosporin A; ER; endoplasmic reticulum; GE; glycyrrhetinic acid; MPT; mitochondrial permeability transition; PARP; poly (ADP-ribose) polymerase; RHM; rat heart mitochondria; RLM; rat liver mitochondria; ROS; reactive oxygen species; TPP; ⁺; tetraphenylphosphonium; 11HSD; 11-hydroxysteroid dehydrogenase; Δ; Ψ; membrane potentialGlycyrrhetinic acid; Mitochondria; Permeability transition; Heart

Structural stability of GlcV, the nucleotide binding domain of the glucose ABC transporter of Sulfolobus solfataricus by Monika G. Pretz; Chris van der Does; Sonja-Verena Albers; Gea Schuurman-Wolters; Arnold J.M. Driessen (pp. 324-333).

GlcV is the nucleotide binding domain of the ABC-type glucose transporter of the hyperthermoacidophile Sulfolobus solfataricus. GlcV consists of two domains, an N-terminal domain containing the typical nucleotide binding-fold and a C-terminal β-barrel domain with unknown function. The unfolding and structural stability of the wild-type (wt) protein and three mutants that are blocked at different steps in the ATP hydrolytic cycle were studied. The G144A mutant is unable to dimerize, while the E166A and E166Q mutants are defective in ATP hydrolysis and dimer dissociation. Unfolding of the wt GlcV and G144A GlcV occurred with a single transition, whereas the E166A and E166Q mutants showed a second transition at a higher melting temperature indicating an increased stability of the ABCα/β subdomain. The structural stability of GlcV was increased in the presence of nucleotides suggesting that the transition corresponds to the unfolding of the NBD domain. Unfolding of the C-terminal domain appears to occur at temperatures above the unfolding of the NBD which coincides with the aggregation of the protein. Analysis of the domain organization of GlcV by trypsin digestion demonstrates cleavage of the NBD domain into three fragments, while nucleotides protect against proteolysis. The cleaved GlcV protein retained the ability to bind nucleotides and to dimerize. These data indicate that the wt GlcV NBD domain unfolds as a single domain protein, and that its stability is modified by mutations in the glutamate after the Walker B motif and by nucleotide binding.

Keywords: Abbreviations; ABC; ATP binding cassette; DSC; differential scanning calorimetry; GdnCl; Guandiniumchloride; NBD; nucleotide binding domain; wt; wild-type; AMP-PNP; adenosine-(β,γ-imido)triphosphateABC transporters; Nucleotide binding; Differential scanning calorimetry; Protein unfolding; Dimerization

Site-directed mutagenesis investigation of coupling properties of metal ion transport by DCT1 by Yaniv Nevo (pp. 334-341).

DCT1 (NRAMP2, DMT1, slc11a2) is a member of the NRAMP family and functions as general metal ion transporter in mammals; defective DCT1 causes anemia. The driving force for metal ion transport is protonmotive force, where protons are transported in the same direction as metal ions. The stoichiometry between metal ion and proton varies under different conditions due to mechanistic proton slip. To better understand this phenomenon, we performed site-directed mutagenesis of DCT1 and analyzed the mutants by measurement of metal ion uptake activity and electrophysiology in Xenopus laevis oocytes. A single reciprocal mutation, I144F, between DCT1 and the homologous yeast transporter Smf1p located in putative transmembrane domain 2 abolished the metal ion transport activity of DCT1, significantly increased the slip currents, and generated sodium slip currents. A double mutation adding F227I in transmembrane domain 4 to I144F in transmembrane domain 2 restored the uptake activity of DCT1 and reduced the slip currents. These results demonstrate the importance of these regions in coupling of metal ions and protons as well as the possible proximity of I144 and F227 in the folded structure of DCT1.

Keywords: NRAMP; Metal ion; Proton; Coupling; Slip; Anemia

Lipocalin-interacting-membrane-receptor (LIMR) mediates cellular internalization of β-lactoglobulin by Maria Fluckinger; Petra Merschak; Martin Hermann; Thomas Haertlé; Bernhard Redl (pp. 342-347).

β-Lactoglobulin (BLG) is a member of the lipocalin protein family and a major food-borne allergen in humans. Numerous in vitro studies have suggested a role for BLG in molecular transport processes; however, its physiological role remains enigmatic. A cellular receptor for BLG has been proposed, but has not yet been identified. Here we show that human LIMR, known to act as an endocytic receptor for lipocalin-1, also binds bovine BLG and mediates its cellular uptake. The specificity of this interaction is corroborated by a complete block of cellular uptake of BLG in the presence of LIMR antibodies or LIMR downregulation by antisense RNA. Furthermore, heterologous expression of human LIMR in insect cells mediates cellular internalization of FITC-BLG. Since LIMR is highly expressed in the human intestine, it might also function in the uptake of food-borne BLG.

Keywords: Abbreviations; BLG; β-lactoglobulin; b; bovine; Lcn-1; lipocalin-1 (also called tear lipocalin, or von Ebner's gland protein); Lcn-2; lipocalin-2 (also called Ngal); LIMR; lipocalin-interacting-membrane- receptor; FITC; fluorescein-isothiocyanateβ-Lactoglobulin; Endocytosis; Receptor; Lipocalin; LIMR

Laurdan fluorescence spectroscopy in the thylakoid bilayer: The effect of violaxanthin to zeaxanthin conversion on the galactolipid dominated lipid environment by Anna Szilágyi; Eva Selstam; Hans-Erik Åkerlund (pp. 348-355).

Laurdan (6-lauroyl-2-dimethylaminonaphthalene) fluorescence spectroscopy has been applied to probe the physical status of the thylakoid membrane upon conversion of violaxanthin to zeaxanthin. So far, only phospholipid-dominated membranes have been studied by this method and hereby we report the first use of laurdan in mono- and digalactosyldiacylglycerol-dominated membrane systems. The generalised polarisation (GP) of laurdan was used as a measure of the structural effect of xanthophyll cycle pigments in isolated spinach ( Spinacia oleracea) thylakoids and in model membrane vesicles composed of chloroplast galactolipids. Higher GP values indicate a membrane in a more ordered structure, whereas lower GP values point to a membrane in a less ordered fluid phase. The method was used to probe the effect of violaxanthin and zeaxanthin in thylakoid membranes at different temperatures. At 4, 25 and 37 °C the GP values for dark-adapted thylakoids in the violaxanthin-form were 0.55, 0.28 and 0.26. After conversion of violaxanthin to zeaxanthin, at the same temperatures, the GP values were 0.62, 0.36 and 0.34, respectively. GP values increased gradually upon conversion of violaxanthin to zeaxanthin. Similar results were obtained in the liposomal systems in the presence of these xanthophyll cycle pigments. We conclude from these results that the conversion of violaxanthin to zeaxanthin makes the thylakoid membrane more ordered.

Keywords: Abbreviations; DGDG; digalactosyldiacylglycerol; DMSO; dimethyl sulfoxide; DPPC; dipalmitoyl-phosphatidylcholine; EPR; electron paramagnetic resonance; GP; general polarisation; H; _II; inverted hexagonal phase; HPLC; high performance liquid chromatography; laurdan; 6-lauroyl-2-dimethylaminonaphtalene; MES; 2-(N-morpholino)ethanesulfonic acid; MGDG; monogalactosyldiacylglycerol; MOPS; 3-(N-morpholino)propanesulfonic acid; NPQ; non-photochemical quenching; PG; phosphatidylglycerol; VDE; violaxanthin de-epoxidase; XC; xanthophyll cycleLaurdan; Fluorescence; Thylakoid membrane; Galactolipids; Xanthophyll cycle; Zeaxanthin

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BBA - Biomembranes (v.1778, #1)