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BBA - Biomembranes (v.1768, #7)

Editorial Board (pp. ii).

The ergosterol biosynthesis inhibitor zaragozic acid promotes vacuolar degradation of the tryptophan permease Tat2p in yeast by Katsue Daicho; Hironori Maruyama; Asuka Suzuki; Masaru Ueno; Masahiro Uritani; Takashi Ushimaru (pp. 1681-1690).

Ergosterol is the yeast functional equivalent of cholesterol in mammalian cells. Deletion of the ERG6 gene, which encodes an enzyme catalyzing a late step of ergosterol biosynthesis, impedes targeting of the tryptophan permease Tat2p to the plasma membrane, but does not promote vacuolar degradation. It is unknown whether similar features appear when other steps of ergosterol biogenesis are inhibited. We show herein that the ergosterol biosynthesis inhibitor zaragozic acid (ZA) evoked massive vacuolar degradation of Tat2p, accompanied by a decrease in tryptophan uptake. ZA inhibits squalene synthetase (SQS, EC 2.5.1.21), which catalyzes the first committed step in the formation of cholesterol/ergosterol. The degradation of Tat2p was dependent on the Rsp5p-mediated ubiquitination of Tat2p and was not suppressed by deletions of VPS1, VPS27, VPS45 or PEP12. We will discuss ZA-mediated Tat2p degradation in the context of lipid rafts.

Keywords: Ergosterol; Zaragozic acid; Rapamycin; Tryptophan; Tat2p

Ouabain activates signaling pathways associated with cell death in human neuroblastoma by Andrey Kulikov; Alexander Eva; Ulrike Kirch; Alexander Boldyrev; Georgios Scheiner-Bobis (pp. 1691-1702).

Cardiotonic steroids (CTS) like ouabain are not only specific inhibitors of the sodium pump (Na⁺,K⁺-ATPase), they also can influence various cytosolic signaling events in a hormone-like manner. In the neuroblastoma cell line SH-SY5Y ouabain triggers multiple signaling pathways. Within 30 min of incubation with 1 or 10 μM ouabain, SH-SY5Y cells generate reactive oxygen species to a level approximately 50% above control and show a modest but significant elevation in cytosolic [Ca²⁺] of about 25%. After 6 h of exposure, ouabain stimulates a series of anti-apoptotic actions in SH-SY5Y cells, including concentration-dependent phosphorylation of Erk1/2, Akt, and Bad. Nevertheless, at the same time this CTS also induces a series of events that inhibit retinoic acid–induced neuritogenesis and promote cell death. Both of these latter phenomena are possibly associated with the observed ouabain-induced reduction in the abundance of the anti-apoptotic proteins Bcl-XL and Bcl-2. In addition, ouabain treatment results in cytochrome c release into the cytosol and induces activation of caspase 3, events that point towards the stimulation of apoptotic pathways that are probably enhanced by the stimulation of p53 phosphorylation at Ser15 also observed in this study. These pathways may eventually lead to cell death: treatment with 10 nM ouabain results in a 20% decrease in cell number after 4 days of incubation and treatment with 1 μM ouabain decreases cells number by about 75%. The results obtained here emphasize the importance of further research in order to elucidate the various signalling cascades triggered by ouabain and possibly other CTS that are used in the treatment of heart failure and to identify their primary receptor(s).

Keywords: Ouabain; Cell signalling; Na; ⁺; ,K; ⁺; -ATPase; Apoptosis; Neuroblastoma; p53

Ouabain activates signaling pathways associated with cell death in human neuroblastoma by Andrey Kulikov; Alexander Eva; Ulrike Kirch; Alexander Boldyrev; Georgios Scheiner-Bobis (pp. 1691-1702).

Keywords: Ouabain; Cell signalling; Na; ⁺; ,K; ⁺; -ATPase; Apoptosis; Neuroblastoma; p53

Lipid contribution to the affinity of antigen association with specific antibodies conjugated to liposomes by Melvin E. Klegerman; Shaoling Huang; Devang Parikh; Janet Martinez; Sasha M. Demos; Hayat A. Onyuksel; David D. McPherson (pp. 1703-1716).

Immunoliposomes, directed to clinically relevant cell-surface molecules with antibodies, antibody fragments or peptides, are used for site-specific diagnostic evaluation or delivery of therapeutic agents. We have developed intrinsically echogenic liposomes (ELIP) covalently linked to fibrin(ogen)-specific antibodies and Fab fragments for ultrasonic imaging of atherosclerotic plaques. In order to determine the effect of liposomal conjugation on the molecular dynamics of fibrinogen binding, we studied the thermodynamic characteristics of unconjugated and ELIP-conjugated antibody molecules. Utilizing radioimmunoassay and enzyme-linked immunosorbent assay protocols, binding affinities were derived from data obtained at three temperatures. The thermodynamic functions Δ H°, Δ G° and Δ S° were determined from van't Hoff plots and equations of state. The resultant functions indicated that both specific and nonspecific associations of antibody molecules with fibrinogen occurred through a variety of molecular interactions, including hydrophophic, ionic and hydrogen bonding mechanisms. ELIP conjugation of antibodies and Fab fragments introduced a characteristic change in both Δ H° and Δ S° of association, which corresponded to a variable contribution to binding by phospholipid gel–liquid crystal phase transitions. These observations suggest that a reciprocal energy transduction, affecting the strength of antibody–antigen binding, may be a singular characteristic of immunoliposomes, having utility for optimization and further development of the technology.

Keywords: Immunoliposome; Radioimmunoassay; ELISA; Thermodynamics; Phospholipid phase transition

Conformational behavior of oxygenated mycobacterial mycolic acids from Mycobacterium bovis BCG by Masumi Villeneuve; Mizuo Kawai; Motoko Watanabe; Yutaka Aoyagi; Yukio Hitotsuyanagi; Koichi Takeya; Hiroaki Gouda; Shuichi Hirono; David E. Minnikin; Hiroo Nakahara (pp. 1717-1726).

Phase diagrams of Langmuir monolayers of oxygenated mycolic acids, i.e. methoxy mycolic acid (MeO-MA), ketomycolic acid (Keto-MA), and artificially obtained deoxo-mycolic acid (deoxo-MA) from Mycobacterium bovis BCG were obtained by thermodynamic analysis of the surface pressure ( π) vs. average molecular area ( A) isotherms. At lower temperatures and lower surface pressures, both Keto- and MeO-MAs formed rigid condensed monolayers where each MA molecule was considered to be in a 4-chain form, in which the three carbon chain segments due to bending of the 3-hydroxy aliphatic carboxylate chain and the 2-side chain were in compact parallel arrangement. At higher temperatures and surface pressures, MeO-MA and deoxo-MA tended to take stretched-out conformations in which the 3-hydroxy aliphatic carboxylate chain was more or less in an extended form, but Keto-MA retained the original 4-chain structure. The thickness measurement of the monolayers in situ by ellipsometry at different π values and temperatures supported the above conclusions derived from the phase diagrams. The enthalpy changes associated with the phase transitions of MeO-MA and deoxo-MA implied that the MeO-MA needed larger energy to change from a compact conformation to an extended one, possibly and partly due to the dehydration of the methoxy group from water surface involved. Molecular dynamics studies of MA models derived from Monte Carlo calculations were also performed, which confirmed the conformational behavior of MAs suggested by the thermodynamic studies on the Langmuir monolayers.

Keywords: Mycobacterium bovis; BCG; Oxygenated mycolic acid; Phase diagram; Thermodynamics; Langmuir monolayers

Modeling P2Y receptor–Ca²⁺ response coupling in taste cells by Ilya V. Fedorov; Olga A. Rogachevskaja; Stanislav S. Kolesnikov (pp. 1727-1740).

Here we elaborated an analytical approach for the simulation of dose–response curves mediated by cellular receptors coupled to PLC and Ca²⁺ mobilization. Based on a mathematical model of purinergic Ca²⁺ signaling in taste cells, the analysis of taste cells responsiveness to nucleotides was carried out. Consistently with the expression of P2Y₂ and P2Y₄ receptors in taste cells, saturating ATP and UTP equipotently mobilized intracellular Ca²⁺. Cellular responses versus concentration of BzATP, a P2Y₂ agonist and a P2Y₄ antagonist, implicated high and low affinity BzATP receptors. Suramin modified the BzATP dose–response curve in a manner that suggested the low affinity receptor to be weakly sensitive to this P2Y antagonist. Given that solely P2Y₂ and P2Y₁₁ are BzATP receptors, their high sensitivity to suramin is poorly consistent with the suramin effects on BzATP responses. We simulated a variety of dose–response curves for different P2Y receptor sets and found that the appropriate fit of the overall pharmacological data was achievable only with dimeric receptors modeled as P2Y₂/P2Y₄ homo- and heterodimers. Our computations and analytical analysis of experimental dose–response curves raise the possibility that ATP responsiveness of mouse taste cells is mediated by P2Y₂ and P2Y₄ receptors operative mostly in the dimeric form.

Keywords: P2Y receptor; Intracellular Ca; ²⁺; Mathematical model; Taste cell

Modeling P2Y receptor–Ca²⁺ response coupling in taste cells by Ilya V. Fedorov; Olga A. Rogachevskaja; Stanislav S. Kolesnikov (pp. 1727-1740).

Keywords: P2Y receptor; Intracellular Ca; ²⁺; Mathematical model; Taste cell

New liver cell mutants defective in the endocytic pathway by Richard J. Stockert; Barry Potvin; Sangeeta Nath; Allan W. Wolkoff; Pamela Stanley (pp. 1741-1749).

To isolate mutant liver cells defective in the endocytic pathway, a selection strategy using toxic ligands for two distinct membrane receptors was utilized. Rare survivors termed trafficking mutants (Trf2–Trf7) were stable and more resistant than the parental HuH-7 cells to both toxin conjugates. They differed from the previously isolated Trf1 HuH-7 mutant as they expressed casein kinase 2 α″ (CK2α″) which is missing from Trf1 cells and which corrects the Trf1 trafficking phenotype. Binding of¹²⁵I-asialoorosomucoid (ASOR) and cell surface expression of asialoglycoprotein receptor (ASGPR) were reduced approximately 20%–60% in Trf2–Trf7 cells compared to parental HuH-7, without a reduction in total cellular ASGPR. Based on¹²⁵I-transferrin binding, cell surface transferrin receptor activity was reduced between 13% and 88% in the various mutant cell lines. Distinctive phenotypic traits were identified in the differential resistance of Trf2–Trf7 to a panel of lectins and toxins and to UV light-induced cell death. By following the endocytic uptake and trafficking of Alexa₄₈₈-ASOR, significant differences in endosomal fusion between parental HuH-7 and the Trf mutants became apparent. Unlike parental HuH-7 cells in which the fusion of endosomes into larger vesicles was evident as early as 20 min, ASOR endocytosed into the Trf mutants remained within small vesicles for up to 60 min. Identifying the biochemical and genetic mechanisms underlying these phenotypes should uncover novel and unpredicted protein–protein or protein–lipid interactions that orchestrate specific steps in membrane protein trafficking.

Keywords: Trafficking mutant; Endocytosis; Asialoglycoprotein receptor; HuH-7 cells, casein kinase 2 α″; Glycoprotein–toxin selection; Apoptosis

New liver cell mutants defective in the endocytic pathway by Richard J. Stockert; Barry Potvin; Sangeeta Nath; Allan W. Wolkoff; Pamela Stanley (pp. 1741-1749).

Keywords: Trafficking mutant; Endocytosis; Asialoglycoprotein receptor; HuH-7 cells, casein kinase 2 α″; Glycoprotein–toxin selection; Apoptosis

Interaction of insecticides with mammalian P-glycoprotein and their effect on its transport function by K. Sreeramulu; Ronghua Liu; Frances J. Sharom (pp. 1750-1757).

We studied the effects of four commonly used insecticides (methylparathion, endosulfan, cypermethrin and fenvalerate) on P-glycoprotein isolated from multidrug-resistant cells. All the pesticides stimulated P-glycoprotein ATPase activity, with maximum stimulation of up to 213% in a detergent-solubilized preparation, and up to 227% in reconstituted liposomes. The ATPase stimulation profiles were biphasic, displaying lower stimulation, and in the case of methylparathion, inhibition of activity, at higher insecticide concentrations. Quenching of the intrinsic Trp fluorescence of purified P-glycoprotein was used to quantitate insecticide binding; the estimated K_d values fell in the range 4–6 μM. Transport of the fluorescent substrate tetramethylrosamine (TMR) into proteoliposomes containing P-glycoprotein was monitored in real time. The TMR concentration gradient generated by the transporter was collapsed by the addition of insecticides, and prior addition of these compounds prevented its formation. The rate of TMR transport was inhibited in a saturable fashion by all the compounds, indicating that they compete with the substrate for membrane translocation. Taken together, these data suggest that the insecticides bind to Pgp with high affinity and effectively block drug transport. Inhibition of Pgp by pesticides may compromise its ability to clear xenobiotics from the body, leading to a higher risk of toxicity.

Keywords: P-glycoprotein (ABCB1); Insecticides; ATPase activity; Substrate transport; Fluorescence quenching; Proteoliposomes

Interaction of insecticides with mammalian P-glycoprotein and their effect on its transport function by K. Sreeramulu; Ronghua Liu; Frances J. Sharom (pp. 1750-1757).

Keywords: P-glycoprotein (ABCB1); Insecticides; ATPase activity; Substrate transport; Fluorescence quenching; Proteoliposomes

Molecular organization of surfactin–phospholipid monolayers: Effect of phospholipid chain length and polar head by O. Bouffioux; A. Berquand; M. Eeman; M. Paquot; Y.F. Dufrêne; R. Brasseur; M. Deleu (pp. 1758-1768).

Mixed monolayers of the surface-active lipopeptide surfactin-C₁₅ and various lipids differing by their chain length (DMPC, DPPC, DSPC) and polar headgroup (DPPC, DPPE, DPPS) were investigated by atomic force microscopy (AFM) in combination with molecular modeling (Hypermatrix procedure) and surface pressure-area isotherms. In the presence of surfactin, AFM topographic images showed phase separation for each surfactin–phospholipid system except for surfactin–DMPC, which was in good agreement with compression isotherms. On the basis of domain shape and line tension theory, we conclude that the miscibility between surfactin and phospholipids is higher for shorter chain lengths (DMPC>DPPC>DSPC) and that the polar headgroup of phospholipids influences the miscibility of surfactin in the order DPPC>DPPE>DPPS. Molecular modeling data show that mixing surfactin and DPPC has a destabilizing effect on DPPC monolayer while it has a stabilizing effect towards DPPE and DPPS molecular interactions. Our results provide valuable information on the activity mechanism of surfactin and may be useful for the design of surfactin delivery systems.

Keywords: Langmuir film; Atomic force microscopy; Molecular modeling; Miscibility; Stabilizing effect; Nanoscale resolution

Peptide degradation is a critical determinant for cell-penetrating peptide uptake by Caroline Palm; Mala Jayamanne; Marcus Kjellander; Mattias Hällbrink (pp. 1769-1776).

Cell-penetrating peptide mediated uptake of labels appears to follow an equilibrium-like process. However, this assumption is only valid if the peptides are stabile. Hence, in this study we investigate intracellular and extracellular peptide degradation kinetics of two fluorescein labeled cell-penetrating peptides, namely MAP and penetratin, in Chinese hamster ovarian cells. The degradation and uptake kinetics were assessed by RP-HPLC equipped with a fluorescence detector. We show that MAP and penetratin are rapidly degraded both extracellularly and intracellularly giving rise to several degradation products. Kinetics indicates that intracellularly, the peptides exist in (at least) two distinct pools: one that is immediately degraded and one that is stabile. Moreover, the degradation could be decreased by treating the peptides with BSA and phenanthroline and the uptake was significantly reduced by cytochalasin B, chloroquine and energy depletion. The results indicate that the extracellular degradation determines the intracellular peptide concentration in this system and therefore the stability of cell-penetrating peptides needs to be evaluated.

Keywords: Abbreviations; CPP; cell-penetrating peptide; PTD; protein transduction domain; fl-HPLC; fluorescence-HPLC; MAP; model amphipathic peptide; PBS; phosphate buffered saline; IC; intracellular; EC; extracellular; CHO; Chinese hamster ovarianCell-penetrating peptide; Protein transduction domains; Uptake; Degradation; Endocytosis inhibitor

Peptide degradation is a critical determinant for cell-penetrating peptide uptake by Caroline Palm; Mala Jayamanne; Marcus Kjellander; Mattias Hällbrink (pp. 1769-1776).

The phosphatase activity of the plasma membrane Ca²⁺ pump. Activation by acidic lipids in the absence of Ca²⁺ increases the apparent affinity for Mg²⁺ by Luciana R. Mazzitelli; Hugo P. Adamo (pp. 1777-1783).

The purified PMCA supplemented with phosphatidylcholine was able to hydrolyze pNPP in a reaction media containing only Mg²⁺ and K⁺. Micromolar concentrations of Ca²⁺ inhibited about 75% of the pNPPase activity while the inhibition of the remainder 25% required higher Ca²⁺ concentrations. Acidic lipids increased 5–10 fold the pNPPase activity either in the presence or in the absence of Ca²⁺. The activation by acidic lipids took place without a significant change in the apparent affinities for pNPP or K⁺ but the apparent affinity of the enzyme for Mg²⁺ increased about 10 fold. Thus, the stimulation of the pNPPase activity of the PMCA by acidic lipids was maximal at low concentrations of Mg²⁺. Although with differing apparent affinities vanadate, phosphate, ATP and ADP were all inhibitors of the pNPPase activity and their effects were not significantly affected by acidic lipids. These results indicate that (a) the phosphatase function of the PMCA is optimal when the enzyme is in its activated Ca²⁺ free conformation (E2) and (b) the PMCA can be activated by acidic lipids in the absence of Ca²⁺ and the activation improves the interaction of the enzyme with Mg²⁺.

Keywords: Abbreviations; pPMCA; plasma membrane Ca; ²⁺; pump from pig erythrocytes; pNPP; p-nitrophenylphosphate; EGTA; ethylene glycol bis(b-aminoethyl ether)-; N,N,N′,N′; -tetraacetic acid; PC; phosphatidylcholine; PS; phosphatidylserine; BE; lipid extract from bovine brain; CaM; calmodulin; EP; phosphoenzymePlasma membrane Ca; ²⁺; pump; Ca; ²⁺; ATPase; Phospatese; P-ATPase; Acidic lipids; Mg; ²⁺; pNPP; PMCA; pNPPase

The mitochondrial ryanodine receptor in rat heart: A pharmaco-kinetic profile by Beth A. Altschafl; Gisela Beutner; Virendra K. Sharma; Shey-Shing Sheu; Héctor H. Valdivia (pp. 1784-1795).

A protein discovered within inner mitochondrial membranes (IMM), designated as the mitochondrial ryanodine receptor (mRyR), has been recognized recently as a modulator of Ca²⁺ fluxes in mitochondria. The present study provides fundamental pharmacological and electrophysiological properties of this mRyR. Rat cardiac IMM fused to lipid bilayers revealed the presence of a mitochondrial channel with gating characteristics similar to those of classical sarcoplasmic reticulum RyR (SR-RyR), but a variety of other mitochondrial channels obstructed clean recordings. Mitochondrial vesicles were thus solubilized and subjected to sucrose sedimentation to obtain mRyR-enriched fractions. Reconstitution of sucrose-purified fractions into lipid bilayers yielded Cs⁺-conducting, Ca²⁺-sensitive, large conductance (500–800 pS) channels with signature properties of SR-RyRs. Cytosolic Ca²⁺ increased the bursting frequency and mean open time of the channel. Micromolar concentrations of ryanodine induced the appearance of subconductance states or inhibited channel activity altogether, while Imperatoxin A (IpTx_a), a specific activator of RyRs, reversibly induced the appearance of distinct subconductance states. Remarkably, the cardiac mRyR displayed a Ca²⁺ dependence of [³H]ryanodine binding curve similar to skeletal RyR (RyR1), not cardiac RyR (RyR2). Overall, the mRyR displayed elemental attributes that are present in single channel lipid bilayer recordings of SR-RyRs, although some exquisite differences were also noted. These results therefore provide the first direct evidence that a unique RyR occurs in mitochondrial membranes.

Keywords: Mitochondria; Ryanodine receptor; Planar lipid bilayer; Single channel recording; Imperatoxin A; Calcium

The mitochondrial ryanodine receptor in rat heart: A pharmaco-kinetic profile by Beth A. Altschafl; Gisela Beutner; Virendra K. Sharma; Shey-Shing Sheu; Héctor H. Valdivia (pp. 1784-1795).

Keywords: Mitochondria; Ryanodine receptor; Planar lipid bilayer; Single channel recording; Imperatoxin A; Calcium

Aggregation and hemi-fusion of anionic vesicles induced by the antimicrobial peptide cryptdin-4 by Jason E. Cummings; T. Kyle Vanderlick (pp. 1796-1804).

We show that cryptdin-4 (Crp4), an antimicrobial peptide found in mice, induces the aggregation and hemi-fusion of charged phospholipid vesicles constructed of the anionic lipid POPG and the zwitterionic lipid POPC. Hemi-fusion is confirmed with positive total lipid-mixing assay results, negative inner monolayer lipid-mixing assay results, and negative results from contents-mixing assays. Aggregation, as quantified by absorbance and dynamic light scattering, is self-limiting, creating finite-sized vesicle assemblies. The rate limiting step in the formation process is the mixing of juxtaposed membrane leaflets, which is regulated by bound peptide concentration as well as vesicle radius (with larger vesicles less prone to hemi-fusion). Bound peptide concentration is readily controlled by total peptide concentration and the fraction of anionic lipid in the vesicles. As little as 1% PEGylated lipid significantly reduces aggregate size by providing a steric barrier for membrane apposition. Finally, as stable hemi-fusion is a rare occurrence, we compare properties of Crp4 to those of many peptides known to induce complete fusion and lend insight into conditions necessary for this unusual type of membrane merger.

Keywords: Peptide; Aggregation; Hemi-fusion; Cryptdin-4; Defensin; Vesicles

Aggregation and hemi-fusion of anionic vesicles induced by the antimicrobial peptide cryptdin-4 by Jason E. Cummings; T. Kyle Vanderlick (pp. 1796-1804).

Keywords: Peptide; Aggregation; Hemi-fusion; Cryptdin-4; Defensin; Vesicles

PEG molecular weight and lateral diffusion of PEG-ylated lipids in magnetically aligned bicelles by Ronald Soong; Peter M. Macdonald (pp. 1805-1814).

Lateral diffusion coefficients of PEG-ylated lipids with three different molecular weight PEG groups (1000, 2000 and 5000) were measured in magnetically-aligned bicelles using the stimulated echo (STE) pulsed field gradient (PEG)¹H nuclear magnetic resonance (NMR) method. At concentrations below the PEG “mushroom-to-brush” transition, all three PEG-ylated lipids exhibited unrestricted lateral diffusion, with lateral diffusion coefficients comparable to those of corresponding non-PEG-ylated lipids and independent of PEG molecular weight. At concentrations above this transition, lateral diffusion slowed progressively with increasing concentration of PEG-ylated lipid as a result of surface crowding. As well, the lateral diffusion coefficients exhibited a pronounced decrease with increasing PEG group molecular weight and a diffusion-time dependence indicative of obstructed diffusion. We conclude that, while lateral diffusion of PEG-ylated lipids within lipid bilayers is determined primarily by the hydrophobic anchoring group, when crowding at the lipid bilayer surface becomes significant, the size of the extra-membranous domain, in this case the PEG group, can influence lateral diffusion, leading to decreased diffusivity with increasing size and producing obstructed diffusion at high crowding. These findings imply that similar considerations will pertain to lateral diffusion of membrane proteins with large extra-membranous domains.

Keywords: Lateral diffusion; PEG-ylated lipid; Magnetically aligned bicelle; Pulsed field gradient NMR; Obstructed diffusion

PEG molecular weight and lateral diffusion of PEG-ylated lipids in magnetically aligned bicelles by Ronald Soong; Peter M. Macdonald (pp. 1805-1814).

Keywords: Lateral diffusion; PEG-ylated lipid; Magnetically aligned bicelle; Pulsed field gradient NMR; Obstructed diffusion

Comparative transport efficiencies of urea analogues through urea transporter UT-B by Dan Zhao; N.D. Sonawane; Marc H. Levin; Baoxue Yang (pp. 1815-1821).

Expression of urea transporter UT-B confers high urea permeability to mammalian erythrocytes. Erythrocyte membranes also permeate various urea analogues, suggesting common transport pathways for urea and structurally similar solutes. In this study, we examined UT-B-facilitated passage of urea analogues and other neutral small solutes by comparing transport properties of wildtype to UT-B-deficient mouse erythrocytes. Stopped-flow light-scattering measurements indicated high UT-B permeability to urea and chemical analogues formamide, acetamide, methylurea, methylformamide, ammonium carbamate, and acrylamide, each with P_s>5.0×10−⁶ cm/s at 10 °C. UT-B genetic knockout and phloretin treatment of wildtype erythrocytes similarly reduced urea analogue permeabilities. Strong temperature dependencies of formamide, acetamide, acrylamide and butyramide transport across UT-B-null membranes ( E_a>10 kcal/mol) suggested efficient diffusion of these amides across lipid bilayers. Urea analogues dimethylurea, acryalmide, methylurea, thiourea and methylformamide inhibited UT-B-mediated urea transport by >60% in the absence of transmembrane analogue gradients, supporting a pore-blocking mechanism of UT-B inhibition. Differential transport efficiencies of urea and its analogues through UT-B provide insight into chemical interactions between neutral solutes and the UT-B pore.

Keywords: Urea; Urea analogue; Erythrocyte; Urea transporter; UT-B

Comparative transport efficiencies of urea analogues through urea transporter UT-B by Dan Zhao; N.D. Sonawane; Marc H. Levin; Baoxue Yang (pp. 1815-1821).

Keywords: Urea; Urea analogue; Erythrocyte; Urea transporter; UT-B

Regulation of intestinal hPepT1 (SLC15A1) activity by phosphodiesterase inhibitors is via inhibition of NHE3 (SLC9A3) by Catriona M.H. Anderson; David T. Thwaites (pp. 1822-1829).

The H⁺-coupled transporter hPepT1 (SLC15A1) mediates the transport of di/tripeptides and many orally-active drugs across the brush-border membrane of the small intestinal epithelium. Incubation of Caco-2 cell monolayers (15 min) with the dietary phosphodiesterase inhibitors caffeine and theophylline inhibited Gly–Sar uptake across the apical membrane. Pentoxifylline, a phosphodiesterase inhibitor given orally to treat intermittent claudication, also decreased Gly–Sar uptake through a reduction in capacity ( V_max) without any effect on affinity ( K_m). The reduction in dipeptide transport was dependent upon both extracellular Na⁺ and apical pH but was not observed in the presence of the selective Na⁺/H⁺ exchanger NHE3 (SLC9A3) inhibitor S1611. Measurement of intracellular pH confirmed that caffeine was not directly inhibiting hPepT1 but rather having an indirect effect through inhibition of NHE3 activity. NHE3 maintains the H⁺-electrochemical gradient which, in turn, acts as the driving force for H⁺-coupled solute transport. Uptake of β-alanine, a substrate for the H⁺-coupled amino acid transporter hPAT1 (SLC36A1), was also inhibited by caffeine. The regulation of NHE3 by non-nutrient components of diet or orally-delivered drugs may alter the function of any solute carrier dependent upon the H⁺-electrochemical gradient and may, therefore, be a site for both nutrient–drug and drug–drug interactions in the small intestine.

Keywords: PepT1; Dipeptide transport; Na; ⁺; /H; ⁺; exchange; NHE3; Intestinal absorption; Drug transport

Regulation of intestinal hPepT1 (SLC15A1) activity by phosphodiesterase inhibitors is via inhibition of NHE3 (SLC9A3) by Catriona M.H. Anderson; David T. Thwaites (pp. 1822-1829).

Keywords: PepT1; Dipeptide transport; Na; ⁺; /H; ⁺; exchange; NHE3; Intestinal absorption; Drug transport

Decrease of elastic moduli of DOPC bilayers induced by a macrolide antibiotic, azithromycin by N. Fa; L. Lins; P.J. Courtoy; Y. Dufrêne; P. Van Der Smissen; R. Brasseur; D. Tyteca; M.-P. Mingeot-Leclercq (pp. 1830-1838).

The elastic properties of membrane bilayers are key parameters that control its deformation and can be affected by pharmacological agents. Our previous atomic force microscopy studies revealed that the macrolide antibiotic, azithromycin, leads to erosion of DPPC domains in a fluid DOPC matrix [A. Berquand, M. P. Mingeot-Leclercq, Y. F. Dufrene, Real-time imaging of drug-membrane interactions by atomic force microscopy, Biochim. Biophys. Acta 1664 (2004) 198-205.]. Since this observation could be due to an effect on DOPC cohesion, we investigated the effect of azithromycin on elastic properties of DOPC giant unilamellar vesicles (GUVs). Microcinematographic and morphometric analyses revealed that azithromycin addition enhanced lipid membranes fluctuations, leading to eventual disruption of the largest GUVs. These effects were related to change of elastic moduli of DOPC, quantified by the micropipette aspiration technique. Azithromycin decreased both the bending modulus ( k_c, from 23.1±3.5 to 10.6±4.5 k_B T) and the apparent area compressibility modulus ( K_app, from 176±35 to 113±25 mN/m). These data suggested that insertion of azithromycin into the DOPC bilayer reduced the requirement level of both the energy for thermal fluctuations and the stress to stretch the bilayer. Computer modeling of azithromycin interaction with DOPC bilayer, based on minimal energy, independently predicted that azithromycin (i) inserts at the interface of phospholipid bilayers, (ii) decreases the energy of interaction between DOPC molecules, and (iii) increases the mean surface occupied by each phospholipid molecule. We conclude that azithromycin inserts into the DOPC lipid bilayer, so as to decrease its cohesion and to facilitate the merging of DPPC into the DOPC fluid matrix, as observed by atomic force microscopy. These investigations, based on three complementary approaches, provide the first biophysical evidence for the ability of an amphiphilic antibiotic to alter lipid elastic moduli. This may be an important determinant for drug: lipid interactions and cellular pharmacology.

Keywords: Micropipette; Giant vesicle; Bending modulus; Area compressibility modulus; Azithromycin; DOPC

Phosphatidylcholine and sphingomyelin containing an elaidoyl fatty acid can form cholesterol-rich lateral domains in bilayer membranes by Anders Björkbom; Bodil Ramstedt; J. Peter Slotte (pp. 1839-1847).

Elaidic acid is a trans-fatty acid found in many food products and implicated for having potentially health hazardous effects in humans. Elaidic acid is readily incorporated into membrane lipids in vivo and therefore affects processes regulating membrane physical properties. In this study the membrane properties of sphingomyelin and phosphatidylcholine containing elaidic acid ( N-E-SM and PEPC) were determined in bilayer membranes with special emphasis on their interaction with cholesterol and participation in ordered domain formation. In agreement with previous studies the melting temperatures were found to be about 20 °C lower for the elaidoyl than for the corresponding saturated lipids. The trans-unsaturation increased the polarity at the membrane–water interface as reported by Laurdan fluorescence. Fluorescence quenching experiments using cholestatrienol as a probe showed that both N-E-SM and PEPC were incorporated in lateral membrane domains with sterol and saturated lipids. At low temperatures the elaidoyl lipids were even able to form sterol-rich domains without any saturated lipids present in the bilayer. We conclude from this study that the ability of N-E-SM and PEPC to form ordered domains together with cholesterol and saturated phospho- and sphingolipids in model membranes indicates that they might have an influence on raft formation in biological membranes.

Keywords: Abbreviations; 7SLPC; 1-palmitoyl-2-stearoyl-(7-doxyl)-; sn; -glycero-3-phosphocholine; CTL; cholestatrienol; β-CyD; β-cyclodextrin; DPH; 1,6-diphenyl-1,3,5-hexatriene; HDL; high-density lipoprotein; Laurdan; 6-lauroyl-2-(; N,N; -dimethylamino)naphthalene; LDL; low-density lipoprotein; NMR; nuclear magnetic resonance; N; -E-SM; d; -; erythro-N; -elaidoyl-sphingomyelin; N; -O-SM; d; -; erythro-N; -oleoyl-sphingomyelin; N; -S-SM; d; -; erythro-N; -stearoyl-sphingomyelin; PC; phosphatidylcholine; PEPC; 1-palmitoyl-2-elaidoyl-; sn; -glycero-3-phopshocholine; PGalCer; N; -Palmitoyl-galactosylceramide; PGlcCer; N; -palmitoyl-glucosylceramide; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phopshocholine; PSPC; 1-palmitoyl-2-stearoyl-; sn; -glycero-3-phopshocholine; SM; sphingomyelin; T; _m; gel–liquid crystalline transition temperature; tPA; trans; -parinaric acidPhosphatidylcholine; Sphingomyelin; Cholestatrienol; Trans; -parinaric acid; Fluorescence quenching; Laurdan

Diverse effects of phospholipids on lipoprotein sorting and ATP hydrolysis by the ABC transporter LolCDE complex by Shigehiko Miyamoto; Hajime Tokuda (pp. 1848-1854).

The LolCDE complex of Escherichia coli releases outer membrane-specific lipoproteins from the inner membrane. Lipoproteins with Asp at +2 remain in the inner membrane since this residue functions as a LolCDE avoidance signal depending on phosphatidylethanolamine. We examined the effects of other phospholipids on lipoprotein sorting in proteoliposomes reconstituted with LolCDE and various synthetic phospholipids. The lipoprotein release and ATP hydrolysis were both low at 2 mM Mg²⁺ but very high at 10 mM Mg²⁺ in proteoliposomes containing cardiolipin alone. However, the Lol avoidance function was abolished at 10 mM Mg²⁺, and the release of lipoproteins with Asp at +2 was as efficient as that of outer membrane-specific lipoproteins. The addition of phosphatidylethanolamine to cardiolipin stimulated the ATP hydrolysis and increased the Lol avoidance function of Asp at +2 at 2 mM Mg²⁺. The addition of phosphatidylglycerol to cardiolipin nearly completely inhibited the release of lipoproteins with Asp at +2 even at 10 mM Mg²⁺, while that of outer membrane-specific lipoproteins was not. Taken together, these results indicate that three major phospholipids of E. coli differently affect lipoprotein sorting and the activity of LolCDE.

Keywords: ABC transporter; Phospholipid; Reconstitution; Lipoprotein; LolCDE; Sorting signal

Diverse effects of phospholipids on lipoprotein sorting and ATP hydrolysis by the ABC transporter LolCDE complex by Shigehiko Miyamoto; Hajime Tokuda (pp. 1848-1854).

Keywords: ABC transporter; Phospholipid; Reconstitution; Lipoprotein; LolCDE; Sorting signal

Surface-active properties of the antitumour ether lipid 1- O-octadecyl-2- O-methyl- rac-glycero-3-phosphocholine (edelfosine) by Jon V. Busto; Jesús Sot; Félix M. Goñi; Faustino Mollinedo; Alicia Alonso (pp. 1855-1860).

The surface activity and interaction with lipid monolayers and bilayers of the antitumour ether lipid 1- O-octadecyl-2- O-methyl- rac-glycero-3-phosphocholine (edelfosine) have been studied. Edelfosine is a surface-active soluble amphiphile, with critical micellar concentrations at 3.5 μM and 19 μM in water. When the air–water interface is occupied by a phospholipid, edelfosine becomes inserted in the phospholipid monolayer, increasing surface pressure. This increase is dose-dependent, and reaches a plateau at ca. 2 μM edelfosine bulk concentration. The ether lipid can become inserted in phospholipid monolayers with initial surface pressures of up to 33 mN/m, which ensures its capacity to become inserted into cell membranes. Upon interaction with phospholipid vesicles, edelfosine exhibits a weak detergent activity, causing release of vesicle contents to a low extent (<5%), and a small proportion of lipid solubilization. The weak detergent properties of edelfosine can be related to its very low critical micellar concentrations. Its high affinity for lipid monolayers combined with low lytic properties support the use of edelfosine as a clinical drug. The surface-active properties of edelfosine are similar to those of other “single-chain” lipids, e.g. lysophosphatidylcholine, palmitoylcarnitine, or N-acetylsphingosine.

Keywords: Antitumour lipid; Edelfosine; Surface activity; Lipid monolayers; Critical micellar concentration

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BBA - Biomembranes (v.1768, #7)