Check out our New Publishers' Select for Free Articles

BBA - Biomembranes (v.1768, #3)

Editorial Board (pp. ii).

How important are transmembrane helices of bitopic membrane proteins? by Moti Zviling; Uzi Kochva; Isaiah T. Arkin (pp. 387-392).

The topology of a bitopic membrane protein consists of a single transmembrane helix connecting two extra-membranous domains. As opposed to helices from polytopic proteins, the transmembrane helices of bitopic proteins were initially considered as merely hydrophobic anchors, while more recent studies have begun to shed light on their role in the protein's function. Herein the overall importance of transmembrane helices from bitopic membrane proteins was analyzed using a relative conservation analysis. Interestingly, the transmembrane domains of bitopic proteins are on average, significantly more conserved than the remainder of the protein, even when taking into account their smaller amino acid repertoire. Analysis of highly conserved transmembrane domains did not reveal any unifying consensus, pointing to a great diversity in their conservation patterns. However, Fourier power spectrum analysis was able to show that regardless of the conservation motif, in most sequences a significant conservation moment was observed, in that one side of the helix was conserved while the other was not. Taken together, it may be possible to conclude that a significant proportion of transmembrane helices from bitopic membrane proteins participate in specific interactions, in a variety of modes in the plane of the lipid bilayer.

Characterization of liposomes coated with S-layer proteins from lactobacilli by A. Hollmann; L. Delfederico; G. Glikmann; G. De Antoni; L. Semorile; E.A. Disalvo (pp. 393-400).

The stability of liposomes coated with S-layer proteins from Lactobacillus brevis and Lactobacillus kefir was analyzed as a previous stage to the development of a vaccine vehicle for oral administration. The interactions of the different S-layer proteins with positively charged liposomes prepared with soybean lecithin or dipalmitoylphosphatidylcholine were studied by means of the variation of the Z potential at different protein–lipid ratios, showing that both proteins were able to attach in a greater extent to the surface of soybean lecithin liposomes. The capacity of these particles to retain carboxyfluorescein or calcein by exposure to bile salts, pancreatic extract, pH change and after a thermal shock showed that both S-layer proteins increased the stability of the liposomes in the same magnitude. The non-glycosylated protein from L. brevis protects more efficiently the liposomes at pH 7 than those from L. kefir even without treatment with glutaraldehyde.

Keywords: S-layer protein; Liposome; Lactobacilli; pH stress; Thermal shock; Bile and pancreatic bile effect; CF and calcein release

Differential effect of cross-linking the CD98 heavy chain on fusion and amino acid transport in the human placental trophoblast (BeWo) cell line by Paola Dalton; Helen C. Christian; Christopher W.G. Redman; Ian L. Sargent; C.A.R. Boyd (pp. 401-410).

CD98 (otherwise known as 4F2) is an integral membrane protein with multiple functions including amino acid transport, integrin activation, cell fusion and cell activation. The molecular mechanisms coordinating these multiple functions remain unclear. We have studied CD98 heavy chain (hc) function in a human placental trophoblast cell line (BeWo). We show that cross-linking of CD98hc by incubation of cells in the presence of functional monoclonal antibodies causes cellular re-distribution of the protein from the cytoplasm to the plasma membrane as measured by flow cytometry, western blotting and quantitative immuno-electron microscopy. The latter technique also indicated that CD98hc is trafficked between cell surface and cytoplasmic pools in vesicles. Increased cell surface CD98 correlates with increased cellular fusion in BeWo cells. In addition, we show reduced LAT 1 surface expression and neutral amino acid transport in the presence of the CD98 mabs. The results thus suggest that the function of CD98 in cell fusion is distinct from its role in cellular nutrient delivery.

Keywords: CD98; LAT 1; Cell fusion; Amino acid transport

Atomic force microscopy study of the antimicrobial action of Sushi peptides on Gram negative bacteria by A. Li; P.Y. Lee; B. Ho; J.L. Ding; C.T. Lim (pp. 411-418).

The antibacterial effect of the endotoxin-binding Sushi peptides against Gram-negative bacteria (GNB) is investigated in this study. Similar characteristics observed for Atomic force microscopy (AFM) images of peptide-treated Escherichia coli and Pseudomonas aeruginosa suggest that the Sushi peptides (S3) evoke comparable mechanism of action against different strains of GNB. The results also indicate that the Sushi peptides appear to act in three stages: damage of the bacterial outer membrane, permeabilization of the inner membrane and disintegration of both membranes. The AFM approach has provided vivid and detailed close-up images of the GNB undergoing various stages of antimicrobial peptide actions at the nanometer scale. The AFM results support our hypothesis that the S3 peptide perturbs the GNB membrane via the “carpet-model” and thus, provide important insights into their antimicrobial mechanisms.

Keywords: Gram negative bacteria; Antimicrobial Sushi peptide; Lipopolysaccharide; Membranolytic; Atomic force microscopy

Translocation and nuclear accumulation of monomer and dimer of HIV-1 Tat basic domain in triticale mesophyll protoplasts by Archana Chugh; François Eudes (pp. 419-426).

Cellular internalization of cell-penetrating peptide HIV-1 Tat basic domain (RKKRRQRRR) was studied in Triticale cv AC Alta mesophyll protoplasts. Fluorescently labeled monomer (Tat) and dimer (Tat₂) of Tat basic domain efficiently translocated through the plasma membrane of mesophyll protoplast and showed distinct nuclear accumulation within 10 min of incubation. Substitution of first arginine residue with alanine in Tat basic domain (M-Tat) severely reduced cellular uptake of the peptide (3.8 times less than Tat). Tat₂ showed greater cellular internalization than Tat (1.6 times higher). However, characteristics of cellular uptake remained same for Tat and Tat₂. Cellular internalization of Tat and Tat₂ was concentration dependent and non-saturable whereas no significant change in cellular uptake was observed even at higher concentrations of M-Tat. Low temperature (4 °C) remarkably increased cellular internalization of Tat as well as Tat₂ but M-Tat showed no enhanced uptake. Viability test showed that peptide treatment had no cytotoxic effect on protoplasts further indicating involvement of a common mechanism of peptide uptake at all the temperatures. Endocytic inhibitors nocodazole (10 μM), chloroquine (100 μM) and sodium azide (5 mM) did not show any significant inhibitory effect on cellular internalization of either Tat or Tat₂. These results along with stimulated cellular uptake at low temperature indicate that Tat peptide is internalized in the plant protoplasts in a non-endocytic and energy-independent manner. Competition experiments showed that non-labeled peptide did not inhibit or alter nuclear accumulation of fluorescent Tat or Tat₂ suggesting active transport to the nucleus was not involved. Studies in mesophyll protoplasts show that internalization pattern of Tat peptide is apparently similar to that observed in mammalian cell lines.

Keywords: Abbreviations; CPW; cell protoplast wash; cv; cultivar; dsRNA; double stranded RNA; EDTA; ethylenediaminetetracetate; FDA; Fluorescein diacetate; FITC; fluorescein isothiocyanate; HPLC; high performance liquid chromatography; MS; Murashige and Skoog; M-Tat; Mutated Tat; PTD; protein transduction domain; RT; room temperature; ssDNA; single stranded DNA; TAR; transactivation responsive region; t-Boc; tert-; butyloxycarbonylHIV-1 Tat basic domain; Cellular internalization; Plants; Triticale; Mesophyll protoplast

Development and characterization of magnetic cationic liposomes for targeting tumor microvasculature by Suman Dandamudi; Robert B. Campbell (pp. 427-438).

Cationic liposomes preferentially target tumor vasculature compared to vessels in normal tissues. The distribution of cationic liposomes along vascular networks is, however, patchy and heterogeneous. To target vessels more uniformly we combined the electrostatic properties of cationic liposomes with the strength of an external magnet. We report part I of development. We evaluated bilayer physical properties of our preparations. We investigated interaction of liposomes with target cells including the role of PEG (polyethylene-glycol), and determined whether magnetic cationic liposomes can respond to an external magnetic field. The inclusion of relatively high concentration of MAG-C (magnetite) at 2.5 mg/ml significantly increased the size of cationic liposomes from 105±26.64 to 267±27.43 nm and reduced the zeta potential from 64.55±16.68 to 39.82±5.26 mv. The phase transition temperature of cationic liposomes (49.97±1.34 °C) reduced with inclusion of MAG-C (46.05±0.21 °C). MAG-C cationic liposomes were internalized by melanoma (B16-F10 and HTB-72) and dermal endothelial (HMVEC-d) cells. PEG partially shielded cationic charge potential of MAG-C cationic liposomes, reduced their ability to interact with target cells in vitro, and uptake by major RES organs. Finally, application of external magnet enhanced tumor retention of magnetic cationic liposomes.

Keywords: Abbreviations; DMPC; 1,2-Dimyristoyl-phosphatidylcholine; DMTAP; 1,2-Dimyristoyl-trimethyl-ammonium propane; DMPE-PEG5000; 1,2-Dimyristoyl-phosphatidylethanolamine-polyethylene glycol; CHOL; Cholesterol; MAG-C; Fluid MAG-CS (magnetite, citric acid matrix); DPH; 1,6-diphenyl-1,3,5-hexatriene; DMEM; Dulbecco's Modified Eagle Medium; MEME; Eagles Minimum Essential Medium; EBM-2; Endothelial Cell Basal Medium-2; HMVEC-d; Human Dermal Microvascular Endothelial Cells; HMEC-1; Human Dermal Microvascular Endothelial Cells; HUVEC; Human Umbilical Vein Endothelial Cells; B16-F10; Murine melanoma; HTB-72; Human melanoma; SRB; Sulforhodamine BCancer; Melanoma; Tumor vasculature; Magnetic drug targeting; Magnetic cationic liposome; Physical characterization

Profilin binding to sub-micellar concentrations of phosphatidylinositol (4,5) bisphosphate and phosphatidylinositol (3,4,5) trisphosphate by Pierre D.J. Moens; Luis A. Bagatolli (pp. 439-449).

Profilin is a small (12–15 kDa) actin binding protein which promotes filament turnover. Profilin is also involved in the signaling pathway linking receptors in the cell membrane to the microfilament system within the cell. Profilin is thought to play critical roles in this signaling pathway through its interaction with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃] (P.J. Lu, W.R. Shieh, S.G. Rhee, H.L. Yin, C.S. Chen, Lipid products of phosphoinositide 3-kinase bind human profilin with high affinity, Biochemistry 35 (1996) 14027–14034). To date, profilin's interaction with polyphosphoinositides (PPI) has only been studied in micelles or small vesicles. Profilin binds with high affinity to small clusters of PI(4,5)P₂ molecules. In this work, we investigated the interactions of profilin with sub-micellar concentrations of PI(4,5)P₂ and PI(3,4,5)P₃. Fluorescence anisotropy was used to determine the relevant dissociation constants for binding of sub-micellar concentrations of fluorescently labeled PPI lipids to profilin and we show that these are significantly different from those determined for profilin interaction with micelles or small vesicles. We also show that profilin binds more tightly to sub-micellar concentrations of PI(3,4,5)P₃ ( K_D=720 μM) than to sub-micellar concentrations of PI(4,5)P₂ ( K_D=985 μM). Despite the low affinity for sub-micellar concentration of PI(4,5)P₂, profilin was shown to bind to giant unilamellar vesicles in presence of 0.5% mole fraction of PI(4,5)P₂ The implications of these findings are discussed.

Keywords: Fluorescence spectroscopy; Anisotropy; Lifetime; FRET; PIP; GUVAbbreviations; PI(4,5)P; ₂; phosphatidylinositol 4,5-bisphosphate; PI(3,4,5)P; ₃; phosphatidylinositol 3,4,5-trisphosphate; PPI; polyphosphoinositides; GUV; giant unilamellar vesicles

Effect of arbutin on the dipole potential and area per lipid of ester and ether phosphatidylcholine and phosphatidyl ethanolamine monolayers by Fabiana Lairion; E. Anibal Disalvo (pp. 450-456).

The present results report for the first time a systematic study of the effect of arbutin on the dipole potential of lipid membranes. The dipole potential and the area per lipid were measured in monolayers of dimyristoylphosphatidylcholine (DMPC), 1,2-di- O-tetradecyl- sn-glycero-3-phosphocholine (dietherPC), dimyristoylphosphatidylethanolamine (DMPE) and 1,2-di- O-tetradecyl- sn-glycero-3-phosphoethanolamine (dietherPE), spread on aqueous solutions of different concentrations of arbutin. The decrease of the dipole potential of DMPC, both in condensed and expanded monolayers, is parallel to an increase in the area per lipid. In contrast, for dietherPC, the area per lipid is not affected, in spite of the fact that arbutin is also able to decrease the dipole potential in a less drastic extent. In the case of DMPE, the response is similar to that observed with dietherPC: the dipole potential decreases, while the area per lipid remains unchanged. However, when the carbonyl groups are absent in phosphatidylethanolamine derivatives such as the dietherPE, the dipole potential is not affected by arbutin, with a small decrease in the area. The effect of arbutin on the dipole potential differs from that of sucrose, trehalose and phloretin and is congruent with previous results obtained by FTIR on its interaction with the CO groups. Arbutin binding is interpreted in terms of the exposure to water of the phosphate and carbonyl groups at the membrane interface of the different monolayers.

Keywords: Dimyristoylphosphatidylcholine (DMPC); 1,2-di-; O; -tetradecyl-; sn; -glycero-3-phosphocholine (dietherPC); Dimyristoylphosphatidylethanolamine (DMPE); 1,2-di-; O; -tetradecyl-; sn; -glycero-3-phosphoethanolamine (dietherPE); Monolayer; Arbutin; Dipole potential; Surface pressure; Area per lipid

Evidence that late-endosomal SNARE multimerization complex is promoted by transmembrane segments by Laura Mascia; Dieter Langosch (pp. 457-466).

Assembly of SNARE proteins into quaternary complexes is a critical step in membrane docking and fusion. Here, we have studied the influence of the transmembrane segments on formation of the late endosomal SNARE complex. The complex was assembled in vitro from full-length recombinant SNAREs and from mutants, where the transmembrane segments were either deleted or replaced by oligo-alanine sequences. We show that endobrevin, syntaxin 7, syntaxin 8, and vti1b readily form a complex. This complex forms a dimer as well as multimeric structures. Interestingly, the natural transmembrane segments accelerate the conversion of the quaternary complex to the dimeric form and are essential for multimerization. These in vitro results suggest that the transmembrane segments are responsible for supramolecular assembly of the endosomal SNARE complex.

Keywords: Abbreviations; BN-PAGE; blue native polyacrylamide gel electrophoresis; CHAPS; 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate; eb; endobrevin; GST; glutathione S-transferase; HRP; horseradish peroxidase; IPTG; isopropyl-1-thio-β-; d; -galactopyranoside; SNARE; soluble N-ethylmaleimide-sensitive factor attachment protein receptor; syx7; syntaxin 7; syx8; syntaxin 8; TMS; transmembrane segmentSNARE; Transmembrane segment; Multimer; Membrane fusion; Protein interaction

Location and dynamics of acyl chain NBD-labeled phosphatidylcholine (NBD-PC) in DPPC bilayers. A molecular dynamics and time-resolved fluorescence anisotropy study by Luís M.S. Loura; J.P. Prates Ramalho (pp. 467-478).

100-ns molecular dynamics simulations of fluid 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) bilayers, both pure and containing 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) acyl-chain labeled fluorescent analogs (C6-NBD-PC and C12-NBD-PC), are described. These molecules are widely used as probes for lipid structure and dynamics. The results obtained here for pure DPPC agree with both experimental and theoretical published works. We verified that the NBD fluorophore of both derivatives loops to a transverse location closer to the interface than to the center of the bilayer. Whereas this was observed previously in experimental literature works, conflicting transverse locations were proposed for the NBD group. According to our results, the maximum of the transverse distribution of NBD is located around the glycerol backbone/carbonyl region, and the nitro group is the most external part of the fluorophore. Hydrogen bonds from the NH group of NBD (mostly to glycerol backbone lipid O atoms) and to the nitro O atoms of NBD (from water OH groups) are continuously observed. Rotation of NBD occurs with ∼2.5–5 ns average correlation time for these probes, but very fast, unresolved reorientation motions occur in <20 ps, in agreement with time-resolved fluorescence anisotropy measurements. Finally, within the uncertainty of the analysis, both probes show lateral diffusion dynamics identical to DPPC.

Keywords: Fluorescence probes; NBD-labeled lipids; Molecular simulation; Membrane model system; Membrane penetration depth

Thermotropic and barotropic phase transitions in bilayer membranes of ether-linked phospholipids with varying alkyl chain lengths by Hitoshi Matsuki; Eri Miyazaki; Fumihiko Sakano; Nobutake Tamai; Shoji Kaneshina (pp. 479-489).

The bilayer phase transitions of a series of ether-linked phospholipids, 1,2-dialkylphosphatidylcholines containing linear saturated alkyl chain (C _n=12, 14, 16 and 18), were observed by differential scanning calorimetry (DSC) under ambient pressure and light-transmittance measurements under high pressure. The thermodynamic quantities of the pre- and main-transitions for the ether-linked PC bilayer membranes were calculated and compared with those of a series of ester-linked PCs, 1,2-diacylphosphatidylcholines. The thermodynamic quantities of the main transition for the ether-linked PC bilayers showed distinct dependence on alkyl-chain length and were slightly different from those of the ester-linked PC bilayers. From the comparison of thermodynamic quantities for the main transition between both PC bilayers, we revealed that the attractive interaction in the gel phase for the ether-linked PC bilayers is weaker than that for the ester-linked PC bilayers. Regarding the pretransition, although changes in enthalpy and entropy for both PC bilayers were comparable to each other, the volume changes of the ether-linked PC bilayers roughly doubled those of the ester-linked PC bilayers. The larger volume change results from the smallest partial molar volume of the ether-linked PC molecule in the interdigitated gel phase. Further, we constructed the temperature–pressure phase diagrams for the ether-linked PC bilayers by using the phase-transition data. The region of the interdigitated gel phase in the phase diagrams was extended by applying pressure and by increasing the alkyl-chain length of the molecule. Comparing the phase diagrams with those for the ester-linked PC bilayers, it was proved that the phase behavior of the ester-linked PC bilayers under high temperature and pressure is almost equivalent to that of the ether-linked PC bilayers in the vicinity of ambient pressure.

Keywords: Ether-linked phospholipid; Differential scanning calorimetry; Interdigitation; Phase transition; Phospholipid bilayer; Pressure

The detrimental effect of serum albumin on the re-spreading of a dipalmitoyl phosphatidylcholine Langmuir monolayer is counteracted by a fluorocarbon gas by Frédéric Gerber; Marie Pierre Krafft; Thierry F. Vandamme (pp. 490-494).

We have recently reported that fluorocarbon gases exhibit an effective fluidizing effect on Langmuir monolayers of dipalmitoyl phosphatidylcholine (DPPC), preventing them from crystallizing up to surface pressures of ∼40 mN m−¹, i.e. well above the DPPC's equilibrium surface pressure. We now report that gaseous perfluorooctyl bromide (gPFOB) promotes the re-spreading of DPPC Langmuir monolayers compressed on a bovine serum albumin (BSA)-containing sub-phase. The latter protein is known to maintain a concentration-dependent surface pressure that can exceed the re-spreading pressure of collapsed monolayers. This phenomenon was proposed to be responsible for lung surfactant inactivation. Compression/expansion isotherms and fluorescence microscopy experiments were carried out to assess the monolayers' physical state. We have found that, during expansion under gPFOB-containing air, the surface pressure of a DPPC monolayer on a BSA-containing sub-phase decreased to much lower values than when the DPPC monolayer was expanded in the presence of BSA under air (∼0 mN m−¹ vs. ∼7.5 mN m−¹ at 120 Å², respectively). Moreover, fluorescence images showed that, during expansion, the BSA-coupled DPPC monolayers, in contact with gPFOB, remained in the liquid-expanded state for surface pressures lower than 10 mN m−¹, whereas they were in a liquid-condensed semi-crystalline state, even at large molecular areas (120 Å²), when expanded under air. The re-incorporation of the PFOB molecules in the DPPC monolayer during expansion thus competes with the re-incorporation of BSA, thus preventing the latter from penetrating into the DPPC monolayer. We suggest that combinations of DPPC and a fluorocarbon gas may be useful in the treatment of lung conditions resulting from a deterioration of the native lung surfactant function due to plasma proteins, such as in the acute respiratory distress syndrome.

Keywords: Dipalmitoyl phosphatidylcholine; Fluorocarbon; Perfluorooctyl bromide; Serum albumin; Langmuir monolayer; Fluorescence microscopy; Lung surfactant; Acute respiratory distress syndrome

Activation of Archaeoglobus fulgidus Cu⁺-ATPase CopA by cysteine by Ying Yang; Atin K. Mandal; Luis M. Bredeston; F. Luis González-Flecha; José M. Argüello (pp. 495-501).

CopA, a thermophilic ATPase from Archaeoglobus fulgidus, drives the outward movement of Cu⁺ across the cell membrane. Millimolar concentration of Cys dramatically increases (≅800%) the activity of CopA and other P_IB-type ATPases ( Escherichia coli ZntA and Arabidopsis thaliana HMA2). The high affinity of CopA for metal (≅1 μM) together with the low Cu⁺–Cys K_D (<10−¹⁰M) suggested a multifaceted interaction of Cys with CopA, perhaps acting as a substitute for the Cu⁺ chaperone protein present in vivo. To explain the activation by the amino acid and further understand the mechanism of metal delivery to transport ATPases, Cys effects on the turnover and partial reactions of CopA were studied. 2–20 mM Cys accelerates enzyme turnover with little effect on CopA affinity for Cu⁺, suggesting a metal independent activation. Furthermore, Cys activates the p-nitrophenyl phosphatase activity of CopA, even though this activity is metal independent. Cys accelerates enzyme phosphorylation and the forward dephosphorylation rates yielding higher steady state phosphoenzyme levels. The faster dephosphorylation would explain the higher enzyme turnover in the presence of Cys. The amino acid has no significant effect on low affinity ATP K_m suggesting no changes in the E₁↔E₂ equilibrium. Characterization of Cu⁺ transport into sealed vesicles indicates that Cys acts on the cytoplasmic side of the enzyme. However, the Cys activation of truncated CopA lacking the N-terminal metal binding domain (N-MBD) indicates that activation by Cys is independent of the regulatory N-MBD. These results suggest that Cys is a non-essential activator of CopA, interacting with the cytoplasmic side of the enzyme while this is in an E1 form. Interestingly, these effects also point out that Cu⁺ can reach the cytoplasmic opening of the access path into the transmembrane transport sites either as a free metal or a Cu⁺–Cys complex.

Keywords: Copper; P; _1B; -type ATPases; Cysteine; CopA; Metal transport

Spontaneous, intervesicular transfer rates of fluorescent, acyl chain-labeled phosphatidylcholine analogs by Shelley M. Elvington; J. Wylie Nichols (pp. 502-508).

It was recently shown that the structure of the fluorophore attached to the acyl chain of phosphatidylcholine analogs determines their mechanism of transport across the plasma membrane of yeast cells (Elvington et al., J. Biol Chem. 280:40957, 2005). In order to gain further insight into the physical properties of these fluorescent phosphatidylcholine (PC) analogs, the rate and mechanism of their intervesicular transport was determined. The rate of spontaneous exchange was measured for PC analogs containing either NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl), Bodipy FL (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza- s-indacene), Bodipy 530 (4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza- s-indacene), or Bodipy 581 (4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza- s-indacene) attached to a five or six carbon acyl chain in the sn-2 position. The rate of transfer between phospholipid vesicles was measured by monitoring the increase in fluorescence as the analogs transferred from donor vesicles containing self-quenching concentrations to unlabeled acceptor vesicles. Kinetic analysis indicated that the transfer of each analog occurred by diffusion through the water phase as opposed to transfer during vesicle collisions. The vesicle-to-monomer dissociation rate constants differed by over four orders of magnitude: NBD-PC ( k_dis=0.115 s−¹; t_1/2=6.03 s); Bodipy FL-PC ( k_dis=5.2×10−⁴; t_1/2=22.2 min); Bodipy 530-PC ( k_dis=1.52×10−⁵; t_1/2=12.6 h); and Bodipy 581-PC ( k_dis=5.9×10−⁶; t_1/2=32.6 h). The large differences in spontaneous rates of transfer through the water measured for these four fluorescent PC analogs reflect their hydrophobicity and may account for their recognition by different mechanisms of transport across the plasma membrane of yeast.

Keywords: Abbreviations; Bodipy FL-PC; 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-; s; -indacene-3-pentanoyl)-1-hexadecanoyl-; sn; -glycero-3-phosphocholine; Bodipy 530-PC; 2-(4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-; s; -indacene-3-pentanoyl)-1-hexadecanoyl-; sn; -glycero-3-phosphocholine; Bodipy 581-PC; 2-(4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-; s; -indacene-3-pentanoyl)-1-hexadecanoyl-; sn; -glycero-3-phosphocholine; HBS; HEPES buffered saline; NBD; 7-nitrobenz-2-oxa-1,3-diazol-4-yl; NBD-PC; 1-myristoyl-2-[6-(NBD) aminocaproyl]-phosphatidylcholine; PC; phosphatidylcholine; POPC; 1-palmitoyl, 2-oleoyl phosphatidylcholinePhosphatidylcholine; Phospholipid; NBD; Bodipy; Fluorescence; Intervesicular transfer

Structure of the antimicrobial β-hairpin peptide protegrin-1 in a DLPC lipid bilayer investigated by molecular dynamics simulation by Himanshu Khandelia; Yiannis N. Kaznessis (pp. 509-520).

All atom molecular dynamics simulations of the 18-residue β-hairpin antimicrobial peptide protegrin-1 (PG-1, RGGRLCYCRRRFCVCVGR-NH₂) in a fully hydrated dilauroylphosphatidylcholine (DLPC) lipid bilayer have been implemented. The goal of the reported work is to investigate the structure of the peptide in a membrane environment (previously solved only in solution [R.L. Fahrner, T. Dieckmann, S.S.L. Harwig, R.I. Lehrer, D. Eisenberg, J. Feigon, Solution structure of protegrin-1, a broad-spectrum antimicrobial peptide from porcine leukocytes. Chemistry and Biology, 3 (1996) 543–550]), and to delineate specific peptide–membrane interactions which are responsible for the peptide's membrane binding properties. A novel, previously unknown, “kick” shaped conformation of the peptide was detected, where a bend at the C-terminal β-strand of the peptide caused the peptide backbone at residues 16–18 to extend perpendicular to the β-hairpin plane. This bend was driven by a highly persistent hydrogen-bond between the polar peptide side-chain of TYR7 and the unshielded backbone carbonyl oxygen atom of GLY17. The H-bond formation relieves the unfavorable free energy of insertion of polar groups into the hydrophobic membrane core. PG-1 was anchored to the membrane by strong electrostatic binding of the protonated N-terminus of the peptide to the lipid head group phosphate anions. The orientation of the peptide in the membrane, and its influence on bilayer structural and dynamic properties are in excellent agreement with solid state NMR measurements [S. Yamaguchi, T. Hong, A. Waring, R.I. Lehrer, M. Hong, Solid-State NMR Investigations of Peptide–Lipid Interaction and Orientation of a b-Sheet Antimicrobial Peptide, Protegrin, Biochemistry, 41 (2002) 9852–9862]. Importantly, two simulations which started from different initial orientations of the peptide converged to the same final equilibrium orientation of the peptide relative to the bilayer. The kick-shaped conformation was observed only in one of the two simulations.

New arrangement of proteins and lipids in the stratum corneum cornified envelope by O López; M. Cócera; P.W. Wertz; C. López-Iglesias; A. de la Maza (pp. 521-529).

A new arrangement of proteins and lipids of stratum corneum (SC) cornified envelope (CE) is proposed. The chemical analysis of CE revealed the presence of free fatty acids (FFA), ceramides (Cer), and important percentages of glutamic acid/glutamine (Glx) and serine (Ser) residues. The molecular structure of these components suggests the existence of covalent links not only between Cer and Glx but also between FFA and Ser. The protein distribution of extracellular surface of CE, i.e., the proteins that could be involved in the bonds with lipids, was studied using post- and pre-embedding immunolabeling electron microscopy. Some loricrin (protein rich in Ser) was detected in the outermost part of the CE protein layer. The external arrangement of some domains of this protein may give rise to form linkages with FFA, yielding further insight into the CE arrangement in which Cer–Glx bonds and FFA–Ser bonds would be involved. Although the importance of fatty acids in the cohesion and barrier function of SC has been widely demonstrated, their role could be associated not only to the presence of these lipids in the intercellular lamellae but also in the CE, in the same way that Cer.

Keywords: Abbreviations; CE; cornified envelope; Cer; ceramide; Chol; cholesterol; Cys; cisteine; FFA; free fatty acid; Gln; glutamine; Glu; glutamic acid; Gly; glycine; PBS; phosphate buffered solution; SC; stratum corneum; Ser; serine; TEM; transmission electron microscopy; TG1; transglutaminase 1; TLC-FID; thin layer chromatography coupled to a flame ionization detectorImmunolabeling-transmission electron microscopy; TLC-FID iatroscan; Ceramides; Loricrin

Transmembrane helices of membrane proteins may flex to satisfy hydrophobic mismatch by Philip L. Yeagle; Michael Bennett; Vincent Lemaître; Anthony Watts (pp. 530-537).

A novel mechanism for membrane modulation of transmembrane protein structure, and consequently function, is suggested in which mismatch between the hydrophobic surface of the protein and the hydrophobic interior of the lipid bilayer induces a flexing or bending of a transmembrane segment of the protein. Studies on model hydrophobic transmembrane peptides predict that helices tilt to submerge the hydrophobic surface within the lipid bilayer to satisfy the hydrophobic effect if the helix length exceeds the bilayer width. The hydrophobic surface of transmembrane helix 1 (TM1) of lactose permease, LacY, is accessible to the bilayer, and too long to be accommodated in the hydrophobic portion of a typical lipid bilayer if oriented perpendicular to the membrane surface. Hence, nuclear magnetic resonance (NMR) data and molecular dynamics simulations show that TM1 from LacY may flex as well as tilt to satisfy the hydrophobic mismatch with the bilayer. In an analogous study of the hydrophobic mismatch of TM7 of bovine rhodopsin, similar flexing of the transmembrane segment near the conserved NPxxY sequence is observed. As a control, NMR data on TM5 of lacY, which is much shorter than TM1, show that TM5 is likely to tilt, but not flex, consistent with the close match between the extent of hydrophobic surface of the peptide and the hydrophobic thickness of the bilayer. These data suggest mechanisms by which the lipid bilayer in which the protein is embedded modulates conformation, and thus function, of integral membrane proteins through interactions with the hydrophobic transmembrane helices.

Keywords: Transmembrane helice; NMR; Molecular dynamics; Helix tilt

The predicted transmembrane fragment 17 of the human multidrug resistance protein 1 (MRP1) behaves as an interfacial helix in membrane mimics by Michel Vincent; Jacques Gallay; Nadège Jamin; Manuel Garrigos; Béatrice de Foresta (pp. 538-552).

The human multidrug resistance protein MRP1 (or ABCC1) is one of the most important members of the large ABC transporter family, in terms of both its biological (tissue defense) and pharmacological functions. Many studies have investigated the function of MRP1, but structural data remain scarce for this protein. We investigated the structure and dynamics of predicted transmembrane fragment 17 (TM17, from Ala₁₂₂₇ to Ser₁₂₅₁), which contains a single Trp residue (W₁₂₄₆) involved in MRP1 substrate specificity and transport function. We synthesized TM17 and a modified peptide in which Ala₁₂₂₇ was replaced by a charged Lys residue. Both peptides were readily solubilized in dodecylmaltoside (DM) or dodecylphosphocholine (DPC) micelles, as membrane mimics. The interaction of these peptides with DM or DPC micelles was studied by steady-state and time-resolved Trp fluorescence spectroscopy, including experiments in which Trp was quenched by acrylamide or by two brominated analogs of DM. The secondary structure of these peptides was determined by circular dichroism. Overall, the results obtained indicated significant structuring (∼50% α-helix) of TM17 in the presence of either DM or DPC micelles as compared to buffer. A main interfacial location of TM17 is proposed, based on significant accessibility of Trp₁₂₄₆ to brominated alkyl chains of DM and/or acrylamide. The comparison of various fluorescence parameters including λ_max, lifetime distributions and Trp rotational mobility with those determined for model fluorescent transmembrane helices in the same detergents is also consistent with the interfacial location of TM17. We therefore suggest that TM17 intrinsic properties may be insufficient for its transmembrane insertion as proposed by the MRP1 consensus topological model. This insertion may also be controlled by additional constraints such as interactions with other TM domains and its position in the protein sequence. The particular pattern of behavior of this predicted transmembrane peptide may be the hallmark of a fragment involved in substrate transport.

Keywords: Abbreviations; MRP1 (or ABCC1); multidrug resistance protein 1; LTC; ₄; cysteinyl leukotriene C; ₄; E; ₂; 17βG; estradiol 17-(β-; d; -glucuronide); GSH; reduced glutathione; DM; dodecylmaltoside; BrDM; 7, 8-dibromododecylmaltoside; BrUM; 10, 11-dibromoundecanoylmaltoside; DPC; dodecylphosphocholine; NATA; N-acetyltryptophanamide; TOE; tryptophan octyl ester; DMSO; dimethylsulfoxide; MSD; membrane-spanning domain; TM; transmembrane; MEM; maximum entropy method; P3; K; ₂; WL; ₉; AL; ₉; K; ₂; A; P5; K; ₂; CLWL; ₇; AL; ₉; K; ₂; A; P7; K; ₂; CL; ₃; WL; ₅; AL; ₉; K; ₂; A; P9; K; ₂; CL; ₅; WL; ₃; AL; ₉; K; ₂; A; P11; K; ₂; CL; ₇; WLAL; ₉; K; ₂; A; P13; K; ₂; CL; ₉; WL; ₉; K; ₂; A; TM17; AGL-VGL-SVS-YSL-QVT-TYL-NWL-VRM-S; mTM17; KGL-VGL-SVS-YSL-QVT-TYL-NWL-VRM-SMRP1 (ABCC1) transmembrane helix 17; Aromatic residues; Steady-state and time-resolved fluorescence; Brominated detergents; Dodecylmaltoside and dodecylphosphocholine micelles; Multidrug resistance

Surface tension induced by sphingomyelin to ceramide conversion in lipid membranes by Iván López-Montero; Marisela Vélez; Philippe F. Devaux (pp. 553-561).

We have investigated the effect of sphingomyelin (SM) to ceramide enzymatic conversion on lipid bilayers using Giant Unilamellar Vesicles (GUVs). Sphingomyelinase was added externally to GUVs containing various proportions of SM. In situ asymmetrical SM conversion to ceramide reduced the area of one leaflet. In the absence of equilibration of all the lipids between the two leaflets, a mismatch between the two monolayers was generated. The tension generated by this mismatch was sufficient to trigger the formation of membrane defects and total vesicle collapse at relatively low percentage of SM (≈5% mol). The formation of nanometric size defects was visualised by AFM in supported bilayers. Vesicle rupture was prevented in two circumstances: (a) in GUVs containing a mixture of l_d and l_o domains and (b) in GUVs containing 5% lyso-phosphatidylcholine. In both cases, the accumulation of enough ceramide (at initial SM concentration of 10%) allowed the formation of ceramide-rich domains. The coupling between the two asymmetrical monolayers and the condensing effect produced by the newly formed ceramide generated a tension that could underlie the mechanism through which ceramide formation induces membrane modifications observed during the late stages of apoptosis.

Keywords: Ceramide; Sphingomyelinase; Membrane surface tension; Lipid flip-flop; Lipid asymmetry; Lipid scrambling

Orientational polarisability of lipid membrane surfaces by Gaëlle Le Goff; Mark F. Vitha; Ronald J. Clarke (pp. 562-570).

Here we present a fluorescence method based on the Stokes shift of the voltage-sensitive dye di-8-ANEPPS to quantify the orientational polarisability of lipid membrane surfaces, i.e. the polarisability due to molecular reorientation. Di-8-ANEPPS is already an established probe of membrane dipole potential. Its use, therefore, as a probe of both the dipole potential and orientational polarisability allows a direct comparison of these two properties in an identical region of the lipid bilayer. We applied the new technique on phosphatidylcholine vesicles to study the effects of different degrees of hydrocarbon saturation and of the incorporation of cholesterol and some of its oxidized derivatives. We found that lipids with unsaturated chains had a lower orientational polarisability than those with saturated chains. This could be explained by a reduction in membrane dipole potential as a result of a decrease in lipid packing density. Cholesterol derivatives were found to either increase or decrease the orientational polarisability depending on their molecular structure. The varying effects could be explained by antagonistic effects of the dipole potential and membrane order, which are both changed to varying degrees by the cholesterol derivatives and which lead to increases and decreases in orientational polarisability, respectively.

Keywords: Lipid vesicles; Voltage-sensitive styryl dyes; Fluorescence; Stokes shift; Cholesterol; Lippert plot; Solvent relaxation

Cellular entry pathway and gene transfer capacity of TAT-modified lipoplexes by Roosmarijn E. Vandenbroucke; Stefaan C. De Smedt; Joseph Demeester; Niek N. Sanders (pp. 571-579).

Several reports have shown a fast and efficient translocation of TAT-modified lipoplexes and particles into the cell cytoplasm. However, neither the uptake mechanism nor the biological effect of TAT-modified lipoplexes has been studied in detail. In this report we show that the increase in gene transfer of TAT-modified lipoplexes depends on the amount of cationic lipid in the lipoplexes and on the way TAT was coupled to the lipoplexes. We demonstrate that the cellular uptake of both TAT-modified and unmodified lipoplexes is very fast and, in contrast to previous publications, temperature-dependent. Additionally, after internalization TAT-modified as well as unmodified lipoplexes end up in lysosomal vesicles, indicating the involvement of clathrin-mediated endocytosis. Furthermore, chlorpromazine, a specific inhibitor of clathrin-dependent endocytosis, strongly inhibits the cellular uptake and biological activity of both the TAT-modified and unmodified lipoplexes. We also found that the uptake and biological activity of these lipoplexes are diminished when cholesterol in the cell membrane was bound by filipin, an inhibitor of the lipid-raft mediated pathway. Considering these data, we conclude that TAT-modified and unmodified lipoplexes are mainly internalized via a cholesterol-dependent clathrin-mediated pathway.

Keywords: Gene therapy; Endocytosis; Clathrin; Lipid rafts; Lysosome; TAT

The human non-gastric H,K-ATPase has a different cation specificity than the rat enzyme by Herman G.P. Swarts; Jan B. Koenderink; Peter H.G.M. Willems; Jan Joep H.H.M. De Pont (pp. 580-589).

The primary sequence of non-gastric H,K-ATPase differs much more between species than that of Na,K-ATPase or gastric H,K-ATPase. To investigate whether this causes species-dependent differences in enzymatic properties, we co-expressed the catalytic subunit of human non-gastric H,K-ATPase in Sf9 cells with the β₁ subunit of rat Na,K-ATPase and compared its properties with those of the rat enzyme (Swarts et al., J. Biol. Chem. 280, 33115–33122, 2005). Maximal ATPase activity was obtained with NH₄⁺ as activating cation. The enzyme was also stimulated by Na⁺, but in contrast to the rat enzyme, hardly by K⁺. SCH 28080 inhibited the NH₄⁺-stimulated activity of the human enzyme much more potently than that of the rat enzyme. The steady-state phosphorylation level of the human enzyme decreased with increasing pH, [K⁺], and [Na⁺] and nearly doubled in the presence of oligomycin. Oligomycin increased the sensitivity of the phosphorylated intermediate to ADP, demonstrating that it inhibited the conversion of E₁P to E₂P. All three cations stimulated the dephosphorylation rate dose-dependently. Our studies support a role of the human enzyme in H⁺/Na⁺ and/or H⁺/NH₄⁺ transport but not in Na⁺/K⁺ transport.

Keywords: H,K-ATPase; Cation-specificity; Ouabain; SCH 28080; Oligomycin

Effect of hydrogen peroxide production and the Fenton reaction on membrane composition of Streptococcus pneumoniae by Stella Pesakhov; Rachel Benisty; Noga Sikron; Zvi Cohen; Pavel Gomelsky; Inna Khozin-Goldberg; Ron Dagan; Nurith Porat (pp. 590-597).

As part of its aerobic metabolism, Streptococcus pneumoniae generates high levels of H₂O₂ by pyruvate oxidase (SpxB), which can be further reduced to yield the damaging hydroxyl radicals via the Fenton reaction. A universal conserved adaptation response observed among bacteria is the adjustment of the membrane fatty acids to various growth conditions. The aim of the present study was to reveal the effect of endogenous reactive oxygen species (ROS) formation on membrane composition of S. pneumoniae. Blocking carbon aerobic metabolism, by growing the bacteria at anaerobic conditions or by the truncation of the spxB gene, resulted in a significant enhancement in fatty acid unsaturation, mainly cis-vaccenic acid. Moreover, reducing the level of OH^· by growing the bacteria at acidic pH, or in the presence of an OH^· scavenger (salicylate), resulted in increased fatty acid unsaturation, similar to that obtained under anaerobic conditions. RT-PCR results demonstrated that this change does not originate from a change in mRNA expression level of the fatty acid synthase II genes. We suggest that endogenous ROS play an important regulatory role in membrane adaptation, allowing the survival of this anaerobic organism at aerobic environments of the host.

Keywords: Streptococcus pneumoniae; Membrane lipid; Hydrogen peroxide; Fenton reaction; Fatty acid

Structural correlates of antimicrobial efficacy in IL-8 and related human kinocidins by Nannette Y. Yount; Alan J. Waring; Kimberly D. Gank; William H. Welch; Deborah Kupferwasser; Michael R. Yeaman (pp. 598-608).

Chemokines are small (8–12 kDa) effector proteins that potentiate leukocyte chemonavigation. Beyond this role, certain chemokines have direct antimicrobial activity against human pathogenic organisms; such molecules are termed kinocidins. The current investigation was designed to explore the structure–activity basis for direct microbicidal activity of kinocidins. Amino acid sequence and 3-dimensional analyses demonstrated these molecules to contain iterations of the conserved γ-core motif found in broad classes of classical antimicrobial peptides. Representative CXC, CC and C cysteine-motif-group kinocidins were tested for antimicrobial activity versus human pathogenic bacteria and fungi. Results demonstrate that these molecules exert direct antimicrobial activity in vitro, including antibacterial activity of native IL-8 and MCP-1, and microbicidal activity of native IL-8. To define molecular determinants governing its antimicrobial activities, the IL-8 γ-core (IL-8γ) and α-helical (IL-8α) motifs were compared to native IL-8 for antimicrobial efficacy in vitro. Microbicidal activity recapitulating that of native IL-8 localized to the autonomous IL-8α motif in vitro, and demonstrated durable microbicidal activity in human blood and blood matrices ex vivo. These results offer new insights into the modular architecture, context-related deployment and function, and evolution of host defense molecules containing γ-core motifs and microbicidal helices associated with antimicrobial activity.

Keywords: Abbreviations; PF-4; platelet factor 4; PMP; platelet microbicidal protein; PBP; platelet basic protein; CTAP-3; connective tissue-activating peptide 3; β-TG; beta thromboglobulin; NAP-2; neutrophil activating peptide-2; RANTES; releasable upon activation, normal T cell expressed and secreted; CXC; cysteine-X-cysteine; CC; cysteine-cysteine; CXCR; CXC receptor; CCR; CC receptor; YNB; yeast nitrogen broth; QSAR; quantitative structure–activity relationship; CD; circular dichroism; F-moc; (9-fluorenyl-methyloxycarbonyl); TFA; trifluoroacetic acid; MALDI-TOF; matrix-assisted laser desorption ionization time-of-flight; PIPES; piperazine-; N; ,; N′; -bis[2-ethanesulfonic acid]; Da; daltons, and standard single letter codes for amino acidsAntimicrobial; Peptide; Structure; Chemokine

Modular determinants of antimicrobial activity in platelet factor-4 family kinocidins by Michael R. Yeaman; Nannette Y. Yount; Alan J. Waring; Kimberly D. Gank; Deborah Kupferwasser; Robert Wiese; Arnold S. Bayer; William H. Welch (pp. 609-619).

Mammalian platelets contain an array of antimicrobial peptides, termed platelet microbicidal proteins (PMPs). Human and rabbit PMPs include known chemokines, such as platelet factor-4 (hPF-4); PMP-1 is the rabbit orthologue of hPF-4. Chemokines that also exert direct antimicrobial activity have been termed kinocidins. A consensus peptide domain library representing mammalian PF-4 family members was analyzed to define structural domains contributing to antimicrobial activity against a panel of human pathogens. Secondary conformations were assessed by circular dichroism spectrometry, and molecular modeling was employed to investigate structural correlates of antimicrobial efficacy. Antimicrobial activity against isogenic peptide-susceptible or -resistant Staphylococcus aureus, Salmonella typhimurium, and Candida albicans strain pairs mapped to the C-terminal hemimer (38–74) and modular domains thereof (49–63 and 60–74). Increasing electrostatic charge and steric bulk were general correlates of efficacy. Structural data corroborated spatial distribution of charge, steric bulk and putative secondary structure with organism-specific efficacy. Microbicidal efficacies of the cPMP antimicrobial hemimer and C-terminal peptide (60–74) were retained in a complex human-blood biomatrix assay. Collectively, these results suggest that modular determinants arising from structural components acting independently and cooperatively govern the antimicrobial functions of PF-4 family kinocidins against specific target pathogens.

Keywords: Abbreviations; PF-4; platelet factor 4; PMP; platelet microbicidal protein; PBP; platelet basic protein; CTAP-3; connective tissue-activating peptide 3; β-TG; beta thromboglobulin; NAP-2; neutrophil activating peptide-2; CXC; cysteine–X–cysteine; CC; cysteine–cysteine; CXCR; CXC receptor; CCR; CC receptor; YNB; yeast nitrogen broth; CD; circular dichroism; F-moc (9-fluorenyl-methyloxycarbonyl); TFA; trifluoroacetic acid; MALDI-TOF; matrix-assisted laser desorption ionization time-of-flight; PIPES; piperazine-; N,N′; -bis`2-ethanesulfonic acid]; r; ²; linear correlation coefficient; Da; daltons and standard single letter codes for amino acidsAntimicrobial; Peptide; Structure; Platelet; Kinocidin; Chemokine

Phase behavior and dynamic heterogeneities in lipids: A coarse-grained simulation study of DPPC–DPPE mixtures by Brian Y. Wong; Roland Faller (pp. 620-627).

The coarse-grained Marrink-model for biomembrane simulation is used to study mixtures of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine (DPPE) at various concentrations and temperatures. At high temperatures close to ideal mixing is observed. In the low temperature ordered phase dynamic heterogeneities are identified under some conditions. These are correlated with heterogeneities in the local order and define local neighborhoods.

Keywords: DPPC; DPPE; Lipid mixtures; Molecular simulation

Shiga toxin B-subunit sequential binding to its natural receptor in lipid membranes by David G. Pina; Ludger Johannes; Miguel A.R.B. Castanho (pp. 628-636).

Shiga toxin B-subunit (STxB), a protein involved in the cell-binding and intracellular trafficking of Shiga holotoxin, binds to a specific glycolipid, the globotriaosyl ceramide (Gb₃). Tryptophan residues of STxB, located at the protein–membrane interface, allow one to study its interaction with model membranes by means of spectroscopic methods with no need for chemical derivatisation with a fluorophore. The protein emits maximally around 346 nm and a blue shift of about 8 nm, as well as the occurrence of changes in the emission fluorescence intensity spectra, is indicative of insertion and partition into the membrane. However, the interaction seems to take place without pentamer dissociation. Acrylamide quenching experiments confirm tryptophan residues become less exposed to solvent when in the presence of vesicles, and the use of lipophilic probes suggests that they are located in a shallow position near the water/membrane interface. Fluorescence intensity and lifetime measurements upon STxB titration with Gb₃-containing vesicles suggest a complex STxB/Gb₃ docking mechanism involving static quenching in the later stages. Based on our observations, a model of the protein–membrane interaction is proposed and the STxB membrane partition and binding constants were calculated.

Keywords: Abbreviations; STxB; Shiga toxin B-subunit; CD; circular dichroism; LUV; large unilamellar vesicles; Trp; tryptophan; Gal; galactose; Glc; glucose; Suc; sucrose; POPC; 1-palmitoyl-2-oleyl-; sn; -glycero-3-phosphocholine; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; POPG; 1-palmitoyl-2-oleyl-; sn; -glycero-3-[phospho-rac-(1-glycerol)]; 5NS; 5-doxyl-stearic acid; 16NS; 16-doxyl-stearic acidShiga toxin B-subunit; Globotriaosyl ceramide; Fluorescence; Lipid vesicles

Regulation of transporter expression in mouse liver, kidney, and intestine during extrahepatic cholestasis by Angela L. Slitt; Katryn Allen; Jennifer Morrone; Lauren M. Aleksunes; Chuan Chen; Jonathan M. Maher; José E. Manautou; Nathan J. Cherrington; Curtis D. Klaassen (pp. 637-647).

It is hypothesized that during cholestasis, the liver, kidney, and intestine alter gene expression to prevent BA accumulation; enhance urinary excretion of BA; and decrease BA absorption, respectively. To test this hypothesis, mice were subjected to either sham or bile-duct ligation (BDL) surgery and liver, kidney, duodenum, ileum, and serum samples were collected at 1, 3, 7, and 14 days after surgery. Serum total BA concentrations were 1–5 μmol/l in sham-operated mice and were elevated at 1, 3, 7, and 14 days after BDL, respectively. BDL decreased liver Ntcp, Oatp1a1, 1a5, and 1b2 mRNA expression and increased Bsep, Oatp1a4, and Mrp1–5 mRNA levels. In kidney, BDL decreased Oatp1a1 and increased Mrp1–5 mRNA levels. In intestine, BDL increased Mrp3 and Ibat mRNA levels in ileum. BDL increased Mrp1, 3, 4, and 5 protein expression in mouse liver. These data indicate that the compensatory regulation of transporters in liver, kidney, and intestine is unable to fully compensate for the loss of hepatic BA excretion because serum BA concentration remained elevated after 14 days of BDL. Additionally, hepatic and renal Oatp and Mrp genes are regulated similarly during extrahepatic cholestasis, and may suggest that transporter expression is regulated not to remove bile constituents from the body, but instead to remove bile constituents from tissues.

Keywords: Abbreviations; Ntcp, Slc10a1; Sodium taurocholate cotransporting polypeptide; Oatp, Slc21a; Organic anion transporting polypeptide; Ibat, Slc10a1; Ileal bile acid transporter; Bsep, abcb11; Bile salt-export pump; Mrp, Abcc; Multidrug resistance-associated protein; BDL; Bile duct ligation; CDCA; Chenodeoxycholic acidCholestasis; Bile-duct ligation; Multidrug resistance-associated protein; Organic anion transporting polypeptide; Transporter; Bile salt export pump

Binding of the dystrophin second repeat to membrane di-oleyl phospholipids is dependent upon lipid packing by Elisabeth Le Rumeur; Sandrine Pottier; Gregory Da Costa; Laurent Metzinger; Liza Mouret; Chantal Rocher; Marie Fourage; Corinne Rondeau-Mouro; Arnaud Bondon (pp. 648-654).

Dystrophin is the genetically deficient protein in Duchenne Muscular Dystrophy. Its C- and N-terminal ends interact with cytoskeletal and membrane proteins, establishing a link between the cytoskeleton and the extracellular matrix. In a previous study, we showed that there is an interaction between the second repeat of the rod domain and membrane phospholipids, which places tryptophan residues in close contact with the membrane. Here, we examine the binding of the dystrophin repeat-2 to small unilamellar vesicles with varying composition. We find that the protein binds predominantly to di-oleyl-phosphatidylserine. The binding as a function of increasing mol% of DOPS appears to be cooperative due to reduction of dimensionality, greatly enhanced in the absence of salts, and partly modulated by pH. Substituting small by large unilamellar vesicles induces a 30-fold lower affinity of the protein for the membrane phospholipids. However, modifying the packing of the acyl chains by introducing lipids such as phosphatidylethanolamine and cholesterol to the vesicle leads to an approximately 7-fold increase in affinity. Taken together, these results show that the binding involves electrostatic forces in addition to hydrophobic ones.

Keywords: Abbreviations; DOPC; dioleyl-phosphatidylcholine; DOPS; dioleyl-phosphatidylserine; DOPG; dioleyl-phosphatidylglycerol; DOPA; dioleyl-phosphatidic acid; DOPE; dioleyl-phosphatidylethanolamine; SUVs; small unilamellar vesicles; LUVs; large unilamellar vesicles; DYSR2; repeat 2 of dystrophin rod domainDystrophin; Spectrin repeat; Protein–lipid interaction; Membrane packing

Cholesterol depletion induces dynamic confinement of the G-protein coupled serotonin_1A receptor in the plasma membrane of living cells by Thomas J. Pucadyil; Amitabha Chattopadhyay (pp. 655-668).

Cholesterol is an essential constituent of eukaryotic membranes and plays a crucial role in membrane organization, dynamics, function, and sorting. It is often found distributed non-randomly in domains or pools in biological and model membranes and is thought to contribute to a segregated distribution of membrane constituents. Signal transduction events mediated by seven transmembrane domain G-protein coupled receptors (GPCRs) are the primary means by which cells communicate with and respond to their external environment. We analyzed the role of cholesterol in the plasma membrane organization of the G-protein coupled serotonin_1A receptor by fluorescence recovery after photobleaching (FRAP) measurements with varying bleach spot sizes. Our results show that lateral diffusion parameters of serotonin_1A receptors in normal cells are consistent with models describing diffusion of molecules in a homogenous membrane. Interestingly, these characteristics are altered in cholesterol-depleted cells in a manner that is consistent with dynamic confinement of serotonin_1A receptors in the plasma membrane. Importantly, analysis of ligand binding and downstream signaling of the serotonin_1A receptor suggests that receptor function is affected in a significantly different manner when intact cells or isolated membranes are depleted of cholesterol. These results assume significance in the context of interpreting effects of cholesterol depletion on diffusion characteristics of membrane proteins in particular, and cholesterol-dependent cellular processes in general.

Keywords: Abbreviations; 8-OH-DPAT; 8-hydroxy-2(di-N-propylamino)tetralin; EYFP; enhanced yellow fluorescent protein; FRAP; fluorescence recovery after photobleaching; GPCR; G-protein coupled receptor; IBMX; 3-isobutyl-1-methylxanthine; MβCD; methyl-β-cyclodextrinG-protein coupled receptor; Serotonin; _1A; receptor; Cholesterol; Membrane organization and dynamics; FRAP; Beam radius

Metal contaminants promote degradation of lipid/DNA complexes during lyophilization by Marion d.C. Molina; Thomas J. Anchordoquy (pp. 669-677).

Oxidation reactions represent an important degradation pathway of nucleic acid-based pharmaceuticals. To evaluate the role of metal contamination and chelating agents in the formation of reactive oxygen species (ROS) during lyophilization, ROS generation and the stability of lipid/DNA complexes were investigated. Trehalose-containing formulations were lyophilized with different levels of transition metals. ROS generation was examined by adding proxyl fluorescamine to the formulations prior to freeze-drying. Results show that ROS were generated during lyophilization, and both supercoil content and transfection rates decreased as the levels of metal-induced ROS increased. The experiments incorporating chelators demonstrated that some of these agents (e.g., DTPA, desferal) clearly suppress ROS generation, while others (e.g., EDTA) enhance ROS. Surprisingly, there was not a strong correlation of ROS generated in the presence of chelators with the maintenance of supercoil content. In this study, we demonstrated the adverse effects of the presence of metals (especially Fe²⁺) in nonviral vector formulations. While some chelators attenuate ROS generation and preserve DNA integrity, the effects of these additives on vector stability during lyophilization are difficult to predict. Further study is needed to develop potent formulation strategies that inhibit ROS generation and DNA degradation during lyophilization and storage.

Keywords: Nonviral vectors; Lyophilization; Metal contamination; Reactive oxygen species; Chelating agents; Gene delivery

Coencapsulation of irinotecan and floxuridine into low cholesterol-containing liposomes that coordinate drug release in vivo by Paul G. Tardi; Ryan C. Gallagher; Sharon Johnstone; Natashia Harasym; Murray Webb; Marcel B. Bally; Lawrence D. Mayer (pp. 678-687).

A liposomal delivery system that coordinates the release of irinotecan and floxuridine in vivo has been developed. The encapsulation of floxuridine was achieved through passive entrapment while irinotecan was actively loaded using a novel copper gluconate/triethanolamine based procedure. Coordinating the release rates of both drugs was achieved by altering the cholesterol content of distearoylphosphatidylcholine (DSPC)/distearoylphosphatidylglycerol (DSPG) based formulations. The liposomal retention of floxuridine in plasma after intravenous injection was dramatically improved by decreasing the cholesterol content of the formulation below 20 mol%. In the case of irinotecan, the opposite trend was observed where increasing cholesterol content enhanced drug retention. Liposomes composed of DSPC/DSPG/Chol (7:2:1, mole ratio) containing co-encapsulated irinotecan and floxuridine at a 1:1 molar ratio exhibited matched leakage rates for the two agents so that the 1:1 ratio was maintained after intravenous administration to mice. The encapsulation of irinotecan was optimal when copper gluconate/triethanolamine (pH 7.4) was used as the intraliposomal buffer. The efficiency of irinotecan loading was approximately 80% with a starting drug to lipid molar ratio of 0.1/1. Leakage of floxuridine from the liposomes during irinotecan loading at 50 °C complicated the ability to readily achieve the target 1:1 irinotecan/floxuridine ratio inside the formulation. As a result, a procedure for the simultaneous encapsulation of irinotecan and floxuridine was developed. This co-encapsulation method has the advantage over sequential loading in that extrusion can be performed in the absence of chemotherapeutic agents and the drug/drug ratios in the final formulation can be more precisely controlled.

Keywords: Liposomes; Irinotecan; Floxuridine; Coordinated release; Metal loading

Aquaporin-11 containing a divergent NPA motif has normal water channel activity by Kaya Yakata; Yoko Hiroaki; Kenichi Ishibashi; Eisei Sohara; Sei Sasaki; Kaoru Mitsuoka; Yoshinori Fujiyoshi (pp. 688-693).

Recently, two novel mammalian aquaporins (AQPs), AQPs 11 and 12, have been identified and classified as members of a new AQP subfamily, the “subcellular AQPs”. In members of this subfamily one of the two asparagine–proline–alanine (NPA) motifs, which play a crucial role in selective water conduction, are not completely conserved. Mouse AQP11 (mAQP11) was expressed in Sf9 cells and purified using the detergent Fos-choline 10. The protein was reconstituted into liposomes, which were used for water conduction studies with a stopped-flow device. Single water permeability ( pf) of AQP11 was measured to be 1.72±0.03×10−¹³ cm³/s, suggesting that other members of the subfamily with incompletely conserved NPA motifs may also function as water channels.

Keywords: Abbreviations; AQP; aquaporin; NPA motif; asparagine–proline–alanine motif; ER; endoplasmic reticulum; SIPs; small basic intrinsic proteins; DMPC; dimyristoylphosphatidylcholine; SuD; single channel densitySubcellular-aquaporin; Asparagine–proline–alanine (NPA)-motif; Baculovirus; Fos-choline 10; Stopped-flow

The role of lipid II in membrane binding of and pore formation by nisin analyzed by two combined biosensor techniques by Katrin Christ; Imke Wiedemann; Udo Bakowsky; Hans-Georg Sahl; Gerd Bendas (pp. 694-704).

Nisin, a peptide antibiotic, efficiently kills bacteria through a unique mechanism which includes inhibition of cell wall biosynthesis and pore formation in cytoplasmic membranes. Both mechanisms are based on interaction with the cell wall precursor lipid II which is simultaneously used as target and pore constituent. We combined two biosensor techniques to investigate the nisin activity with respect to membrane binding and pore formation in real time. Quartz crystal microbalance (QCM) allows the detection of nisin binding kinetics. The presence of 0.1 mol% lipid II strongly increased nisin binding affinity to DOPC ( k_D 2.68×10−⁷ M vs. 1.03×10−⁶ M) by a higher association rate. Differences were less pronounced while using negatively charged DOPG membranes. However, lipid II does not influence the absolute amount of bound nisin. Cyclic voltammetry (CV) data confirmed that in presence of 0.1 mol% lipid II, nanomolar nisin concentrations were sufficient to form pores, while micromolar concentrations were necessary in absence of lipid II. Both techniques suggested unspecific destruction of pure DOPG membranes by micromolar nisin concentrations which were prevented by lipid II. This model membrane stabilization by lipid II was confirmed by atomic force microscopy. Combined CV and QCM are valuable to interpret the role of lipid II in nisin activity.

Keywords: Atomic force microscopy (AFM); Cyclic voltammetry (CV); Quartz crystal microbalance (QCM); Lantibiotic; Lipid II; Nisin; Fluorescence recovery after photobleaching (FRAP)

Association of vasoactive intestinal peptide with polymer-grafted liposomes: Structural aspects for pulmonary delivery by Brigitte Stark; Paul Debbage; Fritz Andreae; Wilhelm Mosgoeller; Ruth Prassl (pp. 705-714).

A polymer-grafted liposomal formulation that has the potential to be developed for aerosolic pulmonary delivery of vasoactive intestinal peptide (VIP), a potent vasodilatory neuropeptide, is described. As VIP is prone to rapid proteolytic degradation in the microenvironment of the lung a proper delivery system is required to increase the half-life and bioavailability of the peptide. Here we investigate structural parameters of unilamellar liposomes composed of palmitoyl-oleoyl-phosphatidylcholine, lyso-stearyl-phosphatidylglycerol and distearyl-phosphatidyl-ethanolamine covalently linked to polyethylene glycol 2000, and report on VIP–lipid interaction mechanisms. We found that the cationic VIP is efficiently entrapped by the negatively charged spherical liposomes and becomes converted to an amphipathic α-helix. By fluorescence spectroscopy using single Trp-modified VIP we could show that VIP is closely associated to the membrane. Our data suggest that the N-terminal random-coiled domain is embedded in the interfacial headgroup region of the phospholipid bilayer. By doing so, neither the bilayer thickness of the lipid membrane nor the mobility of the phospholipid acyl chains are affected as shown by small angle X-ray scattering and electron spin resonance spectroscopy. Finally, in an ex vivo lung arterial model system we found that liposomal-associated VIP is recognized by its receptors to induce vasodilatory effects with comparable high relaxation efficiency as free VIP but with a significantly retarded dilatation kinetics. In conclusion, we have designed and characterized a liposomal formulation that is qualified to entrap biologically active VIP and displays structural features to be considered for delivery of VIP to the lung.

Keywords: Polyethlene glycolated liposome; Sterically stabilized liposome; Nanostructure; Amphipathic peptide; Peptide–lipid interaction

Effects of topology, length, and charge on the activity of a kininogen-derived peptide on lipid membranes and bacteria by Lovisa Ringstad; Lukasz Kacprzyk; Artur Schmidtchen; Martin Malmsten (pp. 715-727).

Effects of topology, length, and charge on peptide interactions with lipid bilayers was investigated for variants of the human kininogen-derived peptide HKH20 (HKHGHGHGKHKNKGKKNGKH) by ellipsometry, CD, fluorescence spectroscopy, and z-potential measurements. The peptides display primarily random coil conformation in buffer and at lipid bilayers, and their lipid interaction is dominated by electrostatics, the latter evidenced by higher peptide adsorption and resulting membrane rupture for an anionic than for a zwitterionic membrane, as well as by strongly reduced adsorption and membrane rupture at high ionic strength. At sufficiently high peptide charge density, however, electrostatic interactions contribute to reducing the peptide adsorption and membrane defect formation. Truncating HKH20 into overlapping 10 amino acid peptides resulted in essentially eliminated membrane rupture and in a reduced amount peptide charges pinned at the lipid bilayer. Finally, cyclic HKH20 was found to be less efficient than the linear peptide in causing liposome rupture, partly due to a lower adsorption. Analogous results were found regarding bactericidal effects .

Keywords: Adsorption; Antimicrobial; Bacteria; Ellipsometry; Liposome; Membrane; Peptide

Effects of freezing on membranes and proteins in LNCaP prostate tumor cells by Willem F. Wolkers; Saravana K. Balasubramanian; Emily L. Ongstad; Helena C. Zec; John C. Bischof (pp. 728-736).

Fourier transform infrared spectroscopy (FTIR) and cryomicroscopy were used to define the process of cellular injury during freezing in LNCaP prostate tumor cells, at the molecular level. Cell pellets were monitored during cooling at 2 °C/min while the ice nucleation temperature was varied between −3 and −10 °C. We show that the cells tend to dehydrate precipitously after nucleation unless intracellular ice formation occurs. The predicted incidence of intracellular ice formation rapidly increases at ice nucleation temperatures below −4 °C and cell survival exhibits an optimum at a nucleation temperature of −6 °C. The ice nucleation temperature was found to have a great effect on the membrane phase behavior of the cells. The onset of the liquid crystalline to gel phase transition coincided with the ice nucleation temperature. In addition, nucleation at −3 °C resulted in a much more co-operative phase transition and a concomitantly lower residual conformational disorder of the membranes in the frozen state compared to samples that nucleated at −10 °C. These observations were explained by the effect of the nucleation temperature on the extent of cellular dehydration and intracellular ice formation. Amide-III band analysis revealed that proteins are relatively stable during freezing and that heat-induced protein denaturation coincides with an abrupt decrease in α-helical structures and a concomitant increase in β-sheet structures starting at an onset temperature of approximately 48 °C.

Keywords: Abbreviations; FTIR; Fourier transform infrared spectroscopy; IIF; intracellular ice formation; Tm; membrane phase transition temperatureCryosurgery; Cryopreservation; FTIR; Membrane phase behavior; Prostate tumor cell; Protein denaturation

Pharmacokinetics of poly(hydroxyethyl-l-asparagine)-coated liposomes is superior over that of PEG-coated liposomes at low lipid dose and upon repeated administration by Birgit Romberg; Christien Oussoren; Cor J. Snel; Myrra G. Carstens; Wim E. Hennink; Gert Storm (pp. 737-743).

‘Stealth’ liposomes with a poly(ethylene glycol) (PEG) coating are frequently studied for drug delivery and diagnostic purposes because of their prolonged blood circulation kinetics. However, several recent reports have demonstrated that PEG-liposomes are rapidly cleared at single low lipid doses (<1 μmol/kg) and upon repeated administration (time interval between the injections 5 days–4 weeks). Recently, poly(amino acid)-based stealth liposome coatings have been developed as alternative to the PEG-coating. In this study, the pharmacokinetic behavior of liposomes coated with the poly(amino acid) poly(hydroxyethyl-l-asparagine) (PHEA) was evaluated at low lipid doses and upon repeated administration in rats. Blood circulation times and hepatosplenic localization of PHEA-liposomes were assessed after intravenous injection. When administered at a dose of 0.25 μmol/kg or less, PHEA-liposomes showed significantly longer blood circulation times than PEG-liposomes. A second dose of PHEA-liposomes 1 week after the first injection was less rapidly cleared from the circulation than a second dose of PEG-liposomes. Although the mechanisms behind these observations are still not clear yet, the use of PHEA-liposomes appears beneficial when single low lipid doses and/or repeated dosing schedules are being applied.

Keywords: Long-circulating liposomes; Pharmacokinetics; Poly(ethylene glycol); Poly(amino acid)s; Accelerated blood clearance; Repeated administration

Erratum to “Steroid structural requirements for interaction of ostreolysin, a lipid-raft binding cytolysin, with lipid monolayers and bilayers” [Biochim. Biophys. Acta 1758 (2006) 1662–1670] by Katja Rebolj; Nataša Poklar Ulrih; Peter Maček; Kristina Sepčić (pp. 744-744).

Corrigendum to “Substrate specificity of a chimera made from Xenopus SGLT1-like protein and rabbit SGLT1” [Biochim. Biophys. Acta 1758 (2006) 747–754] by Katsumi Nagata; Yoshio Hata (pp. 745-745).

Sections

Personal tools

BBA - Biomembranes (v.1768, #3)

Sections

Personal tools

Local resources

BBA - Biomembranes (v.1768, #3)