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BBA - Biomembranes (v.1715, #1)
Shape relaxations in a fluid supported membrane during hydrolysis by phospholipase A2 by Uffe Bernchou Jensen; Adam Cohen Simonsen (pp. 1-5).
The behavior of a fluid supported membrane during hydrolysis by phospholipase A2 is for the first time visualized by time-resolved fluorescence imaging. After a lag phase, hydrolysis proceeds from the boundary of existing holes and via nucleation of new holes. During subsequent hydrolysis, the shape of the membrane boundary is determined both by hydrolysis and by shape relaxations due to the action of line tension. This is manifested by the appearance of Rayleigh instabilities in membrane rims and by an effect analogous to domain coarsening in phase transitions in which membrane holes decay when they are within a certain distance from larger and expanding holes.
Keywords: Phospholipase A; 2; Fluid supported membrane; Fluorescence microscopy; Line tension
Membrane association and activity of 15/16-membered peptide antibiotics: Zervamicin IIB, ampullosporin A and antiamoebin I by T.N. Kropacheva; E.S. Salnikov; H.-H. Nguyen; S. Reissmann; Z.A. Yakimenko; A.A. Tagaev; T.V. Ovchinnikova; J. Raap (pp. 6-18).
Permeabilization of the phospholipid membrane, induced by the antibiotic peptides zervamicin IIB (ZER), ampullosporin A (AMP) and antiamoebin I (ANT) was investigated in a vesicular model system. Membrane-perturbing properties of these 15/16 residue peptides were examined by measuring the K+ transport across phosphatidyl choline (PC) membrane and by dissipation of the transmembrane potential. The membrane activities are found to decrease in the order ZER>AMP>>ANT, which correlates with the sequence of their binding affinities. To follow the insertion of the N-terminal Trp residue of ZER and AMP, the environmental sensitivity of its fluorescence was explored as well as the fluorescence quenching by water-soluble (iodide) and membrane-bound (5- and 16-doxyl stearic acids) quenchers. In contrast to AMP, the binding affinity of ZER as well as the depth of its Trp penetration is strongly influenced by the thickness of the membrane (diC16:1PC, diC18:1PC, C16:0/C18:1PC, diC20:1PC). In thin membranes, ZER shows a higher tendency to transmembrane alignment. In thick membranes, the in-plane surface association of these peptaibols results in a deeper insertion of the Trp residue of AMP which is in agreement with model calculations on the localization of both peptide molecules at the hydrophilic–hydrophobic interface. The observed differences between the membrane affinities/activities of the studied peptaibols are discussed in relation to their hydrophobic and amphipathic properties.
Keywords: Abbreviations; ZER; zervamicin IIB; AMP; ampullosporin A; ANT; antiamoebin I; ALA; alamethicin; 5(16)-doxyl SA; 5(16)-doxyl stearic acid; PC; phospatidyl choline; DPoPC (diC; 16:1; PC; 1,2-dipalmitoleoyl-; sn; -glycero-3-phosphocholine; POPC (C; 16:0; /C; 18:1; PC); 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; DOPC (diC; 18:1; PC); 1,2-dioleoyl-; sn; -glycero-3-phosphocholine; DEiPC (diC; 20:1; PC); 1,2-dieicosenoyl-; sn; -glycero-3-phosphocholine; LUV; large unilamellar vesicle; CD; circular dichroism; SA; surface associated; TM; transmembranePeptaibol; Bilayer; Conductance; Channel formation; Transmembrane; Amphipathicity
Nateglinide uptake by a ceftibuten transporter in the rat kidney brush-border membrane by Masaki Kobayashi; Yoshitaka Saito; Shirou Itagaki; Takeshi Hirano; Ken Iseki (pp. 19-24).
Nateglinide, a novel oral hypoglycemic agent, possesses a carbonyl group and a peptide-type bond in its structure. We previously reported that nateglinide transport occurs via a single system that may be identical to the ceftibuten/H+ cotransport system by the rat small intestine. We speculated that the absorption system present on the intestinal epithelium may be similar to that found on the renal tubular epithelium. The aim of this study was to characterize the transporters on the apical side of the kidney that may contribute to the reabsorption of ceftibuten and nateglinide. The uptake of nateglinide by rat renal brush-border membranes is associated with an H+-coupled transport system. Ceftibuten competitively inhibited H+-dependent nateglinide uptake. In contrast, Gly-Sar, cephradine and cephalexin had no effect on nateglinide uptake. Nateglinide competitively inhibited H+-driven transporter-mediated ceftibuten uptake. We conclude that nateglinide transport occurs via a single system that is H+-dependent and may be identical to the ceftibuten/H+ cotransport system.
Keywords: Nateglinide; Kidney; Ceftibuten; Brush-border membrane vesicle
Identification and characterization of PorH, a new cell wall channel of Corynebacterium glutamicum by Peter Hünten; Noelia Costa-Riu; Dieter Palm; Friedrich Lottspeich; Roland Benz (pp. 25-36).
The cell wall of Corynebacterium glutamicum contains the cation-selective channel (porin) PorA C.glut and the anion-selective channel PorB C.glut for the passage of hydrophilic solutes. Lipid bilayer experiments with organic solvent extracts of whole C. glutamicum cells cultivated in minimal medium suggested that also another cation-selective channel-forming protein, named PorH C.glut, is present in C. glutamicum. The protein was purified to homogeneity by fast-protein liquid chromatography across a HiTrap-Q column. The pure protein had an apparent molecular mass of about 12 kDa on SDS-PAGE. Western blot analysis suggested that the cell wall channel is presumably formed by protein oligomers. The purified protein forms cation-selective channels with an average single-channel conductance of about 2.5 nS in 1 M KCl in the lipid bilayer assay. The PorH C.glut protein was partially sequenced, and based on the resulting amino acid sequence, the corresponding gene, designated as porH C.glut, was identified in the published genome sequence of C. glutamicum ATCC13032. PorH C.glut contains only the inducer methionine but no N-terminal extension, which suggests that the export and assembly of the protein follow a yet unknown pathway. PorH C.glut is coded in the bacterial chromosome by a gene that is localized in the vicinity of porA C.glut, within a putative operon of 13 genes. RT-PCR revealed that both porins are cotranscribed. They coexist according to immunological detection experiments in the cell wall of C. glutamicum together with PorB C.glut and PorC C.glut.
Keywords: Abbreviations; LDAO; N; ,; N; -dimethyldodecylamine-; N; -oxide; Genapol; oligoethylenglycol-monoalkylether; PC; diphytanoyl phosphatidylcholine; nS; nanosiemensCell wall channel; Mycolic acid; Porin; Lipid bilayer membrane; Corynebacterium glutamicum
Preparation and in vivo evaluation of PEGylated spherulite formulations by Pierre Simard; Didier Hoarau; Mohamed Nabil Khalid; Emmanuelle Roux; Jean-Christophe Leroux (pp. 37-48).
Spherulites are multilamellar vesicles obtained by shearing a lamellar phase of lipids and surfactants. They consist of concentric bilayers of amphiphiles alternating with layers of aqueous medium in which hydrophilic drugs can be sequestered with high yield. To be useful for drug targeting applications, spherulites should be small and long circulating. The objectives of this work were threefold. First, the spherulite size was optimized to obtain a mean diameter of less than 300 nm. Second, the vesicle composition was adjusted to minimize in vitro leakage of internal content. Third, the spherulites were coated with 1,2-distearoyl- sn-glycero-3-phosphatidylethanolamine- N-[methoxy poly(ethylene glycol)] (DSPE-PEG) to impart them with a long half-life. Then, the PEGylated spherulites (Phospholipon 90G/Solutol HS15/cholesterol/DSPE-PEG 2000 or 5000) were loaded with 1-β-d-arabinofuranosylcytosine (ara-C) and injected intravenously to rats. They were compared to uncoated spherulites and to an ara-C solution. The surface-modified vesicles exhibited long circulation times with areas under the blood concentration vs. time curve exceeding by 3.1- to 6.9-fold that of uncoated spherulites. Similarly, blood levels of ara-C encapsulated in PEGylated vesicles were higher than those of the controls, but they did not parallel the carrier pharmacokinetics. Two hours post-injection, most of the drug was cleared from the systemic circulation, reflecting rapid leakage of ara-C from the vesicles.
Keywords: Spherulite; Liposome; Ara-C; Biodistribution
N-acyl phosphatidylethanolamines affect the lateral distribution of cholesterol in membranes by Bohdana Térová; Gitte Petersen; Harald S. Hansen; J. Peter Slotte (pp. 49-56).
N-Acyl phosphatidylethanolamines are negatively charged phospholipids, which are naturally occurring albeit at low abundance. In this study, we have examined how the amide-linked acyl chain affected the membrane behavior of the N-acyl-1-palmitoyl-2-oleoyl- sn-glycero-3-phosphatidylethanolamine ( N-acyl-POPE) or N-acyl-dipalmitoyl- sn-glycero-3-phosphatidylethanolamine ( N-acyl-DPPE), and how the molecules interacted with cholesterol. The gel→liquid crystalline transition temperature of sonicated N-acyl phosphatidylethanolamine vesicles in water correlated positively with the number of palmitic acyl chains in the molecules. Based on diphenylhexatriene steady state anisotropy measurements, the presence of 33 mol% cholesterol in the membranes removed the phase transition from N-oleoyl-POPE bilayers, but failed to completely remove it from N-palmitoyl-DPPE and N-palmitoyl-POPE bilayers, suggesting rather weak interaction of cholesterol with the N-saturated NAPEs. The rate of cholesterol desorption from mixed monolayers containing N-palmitoyl-DPPE and cholesterol (1:1 molar ratio) was much higher compared to cholesterol/DPPE binary monolayers, suggesting a weak cholesterol interaction with N-palmitoyl-DPPE also in monolayers. In bilayer membranes, both N-palmitoyl-POPE and N-palmitoyl-DPPE failed to form sterol-rich domains, and in fact appeared to displace sterol from sterol/ N-palmitoyl-sphingomyelin domains. The present data provide new information about the effects of saturated NAPEs on the lateral distribution of cholesterol in NAPE-containing membranes. These findings may be of relevance to neural cells which accumulate NAPEs during stress and cell injury.
Keywords: Abbreviations; 7SLPC; 1-palmitoyl-2-stearoyl-(7-doxyl)-; sn; -glycero-3-phosphocholine; ββ-CyD; β-cyclodextrin; CTL; cholesta-5,7,9(11)-trien-3-beta-ol; DPH; 1,6-diphenyl-1,3,5-hexatriene; NAPE; N; -acyl phosphatidylethanolamine; N; -acyl-POPE; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phospho(; N; -acyl)ethanolamine; N; -acyl-DPPE; 1,2-dipalmitoyl-; sn; -glycero-3-phospho(; N; -acyl)ethanolamine; PSM; d; -; erythro-N-; palmitoyl-sphingomyelin; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPE; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphoethanolamine; tParSM; d; -; erythro-N-trans; -parinoyl-sphingomyelinCholesterol; Cholesterol desorption; Cholestatrienol; Membrane domain; Quenching
The relationship between folate transport activity at low pH and reduced folate carrier function in human Huh7 hepatoma cells by Rongbao Zhao; Marie Hanscom; I. David Goldman (pp. 57-64).
Transport of folates and antifolates in both hepatocytes and Huh7 human hepatoma cells is characterized by a low-pH optimum. Studies were undertaken to determine the extent to which this transport activity is mediated by the reduced folate carrier (RFC) in Huh7 human hepatoma cells. RFC expression was ablated by chemical mutagenesis and antifolate selective pressure with PT632 resulting in the PT632R subline in which RFC mRNA could not be detected. Methotrexate (MTX) influx in these cells at pH 7.4 was reduced by 70%, leaving substantial residual RFC-independent influx while influx of MTX and folic acid at pH 5.5 was not significantly decreased. The influx Kt for folic acid and MTX at pH 5.5 in PT632R cells was 0.36 and 1.5 μM, respectively. The affinity of the low pH transporter in PT632R cells was highest for pemetrexed ( Ki=140 nM), very low for PT632 ( Ki=77 μM), and was stereospecific for the natural isomer (6S) of 5-formyltetrahydrofolate. In Huh7 cells transiently transfected with an RFC siRNA, RFC expression was reduced by 60% resulting in a 40% decrease in MTX influx at pH 7.4 but only a very small (5%) reduction in MTX or folic acid influx at pH 5.5. These data indicate that MTX transport in Huh7 cells at neutral pH is mediated largely by RFC while at pH 5.5 the predominant route of transport is independent of RFC.
Keywords: Abbreviations; 5-CHO-THF; 5-formyltetrahydrofolate; DHFR; dihydrofolate reductase; MTX; methotrexate; RFC; reduced folate carrier; PT523; Nα-(-4-amino-4-deoxypteroyl)-Nδ-hemiphthaloyl-1-ornithine; PT632; 5,8-dideaza analog of PT523Folate transport; Folic acid; Methotrexate; Pemetrexed; Acidic pH
Specific volumes of unsaturated phosphatidylcholines in the liquid crystalline lamellar phase by Bernd W. Koenig; Klaus Gawrisch (pp. 65-70).
The specific volumes of seven 1,2-diacyl- sn-glycero-3-phosphocholines with symmetric, unbranched acyl chains containing one, four, or six cis double bonds per chain, or with a saturated sn-1 chain and one, four, or six cis double bonds in the sn-2 chain were determined by the neutral buoyancy method. Experiments were conducted in the liquid crystalline lamellar phase over the temperature range from 5 to 35 °C. It is demonstrated that the molecular volume of phosphatidylcholines can be well approximated as the sum of a constant volume of the polar lipid head region and the temperature-dependent volumes of hydrocarbon chainCH2,CH, and terminal CH3 groups. A linear dependence of chain segment volumes on temperature was observed. A self-consistent set of partially temperature-dependent volumes is obtained that allows prediction of phosphatidylcholine molecular volumes within very tight error margins.
Keywords: Abbreviations; 16:0, 18:1 PC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; 18:0, 18:1 PC; 1-stearoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; 18:0, 20:4 PC; 1-stearoyl-2-arachidonoyl-; sn; -glycero-3-phosphocholine; 18:0, 22:6 PC; 1-stearoyl-2-docosahexaenoyl-; sn; -glycero-3-phosphocholine; 18:1, 18:1 PC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholine; 20:4, 20:4 PC; 1,2-diarachidonoyl-; sn; -glycero-3-phosphocholine; 22:6, 22:6 PC; 1,2-didocosahexaenoyl-; sn; -glycero-3-phosphocholine; BHT; butylated hydroxytoluene; DTPA; diethylenetriaminepentaacetic acidSpecific volume; Lipid; Polyunsaturated fatty acid; DHA; Neutral buoyancy method