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BBA - Biomembranes (v.1712, #1)
Effects of lipid composition on the membrane activity and lipid phase behaviour of Vibrio sp. DSM14379 cells grown at various NaCl concentrations by TjaÅ¡a DanevÄ?iÄ?; Leif Rilfors; Janez Å trancar; Göran Lindblom; David Stopar (pp. 1-8).
The membrane lipid composition of living cells generally adjusts to the prevailing environmental and physiological conditions. In this study, membrane activity and lipid composition of the Gram-negative bacterium Vibrio sp. DSM14379, grown aerobically in a peptone-yeast extract medium supplemented with 0.5, 1.76, 3, 5 or 10% (w/v) NaCl, was determined. The ability of the membrane to reduce a spin label was studied by EPR spectroscopy under different salt concentrations in cell suspensions labeled with TEMPON. For lipid composition studies, cells were harvested in a late exponential phase and lipids were extracted with chloroform–methanol–water, 1:2:0.8 (v/v). The lipid polar head group and acyl chain compositions were determined by thin-layer and gas–liquid chromatographies.31P-NMR spectroscopy was used to study the phase behaviour of the cell lipid extracts with 20 wt.% water contents in a temperature range from −10 to 50 °C. The results indicate that the ability of the membrane to reduce the spin label was highest at optimal salt concentrations. The composition of both polar head groups and acyl chains changed markedly with increasing salinity. The fractions of 16:0, 16:1 and 18:0 acyl chains increased while the fraction of 18:1 acyl chains decreased with increasing salinity. The phosphatidylethanolamine fraction correlated inversely with the lysophosphatidylethanolamine fraction, with phosphatidylethanolamine exhibiting a minimum, and lysophosphatidylethanolamine a maximum, at the optimum growth rate. The fraction of lysophosphatidylethanolamine was surprisingly high in the lipid extracts. This lipid can form normal micellar and hexagonal phases and it was found that all lipid extracts form a mixture of lamellar and normal isotropic liquid crystalline phases. This is an anomalous behaviour since the nonlamellar phases formed by total lipid extracts are generally of the reversed type.
Keywords: Abbreviations; PG; phosphatidylglycerol; PE; phosphatidylethanolamine; lyso-PE; lysophosphatidylethanolamine; DPG; diphosphatidylglycerol; TLC; thin-layer chromatography; GLC; gas–liquid chromatography; 16:0, hexadecanoic acid; 18:0, octadecanoic acid; 14:1; cis; -9-tetradecenoic acid; 16:1; cis; -9-hexadecenoic acid; 18:1; cis; -9-octadecenoic acidLipid composition; Phase behaviour; Salt effect; Lysolipid; Vibrio; sp.
Biological and polymeric self-assembled hybrid systems: Structure and properties of thylakoid/polyelectrolyte complexes by A.A. Dementiev; A.A. Baikov; V.V. Ptushenko; G.B. Khomutov; A.N. Tikhonov (pp. 9-16).
A novel hybrid system composed of biological components and synthetic polymer, thylakoid/polycation complex, has been formed and studied. Effects of complex formation on the structure, electrostatics and functioning of thylakoid membranes have been examined. Thylakoids from bean leaves were used to form complexes with polycation polyallylamine hydrochloride (PAAH) in two systems: (i) thylakoid/polycation complexes formed in an aqueous bulk phase, and (ii) immobilized thylakoid/polycation planar complexes. Immobilized on a solid substrate surface, thylakoid/polycation complexes were prepared using layer-by-layer stepwise alternate adsorption technique, i.e., via the sequential alternate adsorption of thylakoids and polycation molecules. The morphology of built up structures was investigated by scanning electron microscopy. Light-induced electron transport in chloroplasts was studied by the electron paramagnetic resonance (EPR) method. Spin probe technique was employed to study the structural and electrostatic characteristics of thylakoid membranes. We have found that efficiency of light-induced electron transport in thylakoid membranes and membrane structure were not changed noticeably by PAAH binding to thylakoids in a wide range of PAAH concentrations. The data obtained indicate the physiologically-soft character of polycation interactions with thylakoid membranes and demonstrate effectiveness of interfacial self-assembly approach to fabrication of complex planar functional nanostructures from biological components and synthetic polymers.
Keywords: Abbreviations; EPR; electron paramagnetic resonance; CAT; 9; N; ,; N; -dimethyl-; N; -nonyl-tempoylammonium bromide; CTAB; cetyltrimethylammonium bromide; PAAH; polyallylamine hydrochloride; PEI; polyethylenimine; PSS; polystyrene sulfonate; PSI and PSII; photosystems I and II, respectively; S.E.M.; scanning electron microscopy; 5-SASL; 5-doxylstearate; 16-MeSASL; 16-doxylstearate methyl esterThylakoid membranes; Electron transport; Polyelectrolytes; Layer-by-layer deposition; EPR
Studies on canthaxanthin in lipid membranes by Agnieszka Sujak; Janina Gabrielska; Justyna Milanowska; Piotr Mazurek; Kazimierz Strzałka; Wiesław I. Gruszecki (pp. 17-28).
Polar carotenoid pigment – canthaxanthin – has been found to interfere with the organization of biological membranes, in particular of the retina membranes of an eye of primates. The organization of lipid membranes formed with dipalmitoylphosphatidylcholine (DPPC) and egg yolk phosphatidylcholine containing canthaxanthin was studied by means of several techniques including: electronic absorption spectroscopy, linear dichroism, X-ray diffractometry,1H-NMR spectroscopy and FTIR spectroscopy. It appears that canthaxanthin present in the lipid membranes at relatively low concentration (below 1 mol% with respect to lipid) modifies significantly physical properties of the membranes. In particular, canthaxanthin (i) exerts restrictions to the segmental molecular motion of lipid molecules both in the headgroup region and in the hydrophobic core of the bilayer, (ii) promotes extended conformation of alkyl lipid chains, (iii) modifies the surface of the lipid membranes (in particular in the gel state, Lβ´) and promotes the aggregation of lipid vesicles. It is concluded that canthaxanthin incorporated into lipid membranes is distributed among two pools: one spanning the lipid bilayer roughly perpendicularly to the surface of the membrane and one parallel to the membrane, localized in the headgroup region. The population of the horizontal fraction increases with the increase in the concentration of the pigment in the lipid phase. Such a conclusion is supported by the linear dichroism analysis of the oriented lipid multibilayers containing canthaxanthin: The mean angle between the dipole transition moment and the axis normal to the plane of the membrane was determined as 20±3° at 0.5 mol% and 47±3° at 2 mol% canthaxanthin. The analysis of the absorption spectra of canthaxanthin in the lipid phase and1H-NMR spectra of lipids point to the exceptionally low aggregation threshold of the pigment in the membrane environment (∼1 mol%). All results demonstrate a very strong modifying effect of canthaxanthin with respect to the dynamic and structural properties of lipid membranes.
Keywords: Abbreviations; CAN; canthaxanthin; DPPC; dipalmitoylphosphatidylcholine; EYPC; egg yolk phosphatidylcholine; NMR; nuclear magnetic resonance; PCS; photon correlation spectroscopy; FTIR; Fourier transform infrared absorption spectroscopyCanthaxanthin; Biomembrane; Carotenoid pigment; Lipid membrane; Molecular aggregation; Retinopathy
AFM characterization of solid-supported lipid multilayers prepared by spin-coating by G. Pompeo; M. Girasole; A. Cricenti; F. Cattaruzza; A. Flamini; T. Prosperi; J. Generosi; A. Congiu Castellano (pp. 29-36).
Lipids are the principal components of biologically relevant structures as cellular membranes. They have been the subject of many studies due to their biological relevance and their potential applications. Different techniques, such as Langmuir–Blodgett and vesicle-fusion deposition, are available to deposit ordered lipid films on etched surfaces. Recently, a new technique of lipid film deposition has been proposed in which stacks of a small and well-controlled number of bilayers are prepared on a suitable substrate using a spin-coater.We studied the morphological properties of multi-layers made of cationic and neutral lipids (DOTAP and DOPC) and mixtures of them using dynamic mode atomic force microscopy (AFM). After adapting and optimizing, the spin-coating technique to deposit lipids on a chemically etched Silicon (1,0,0) substrate, a morphological nanometer-scale characterization of the aforementioned samples has been provided. The AFM study showed that an initial layer of ordered vesicles is formed and, afterward, depending on details of the spin-coating preparation protocol and to the dimension of the silicon substrate, vesicle fusion and structural rearrangements of the lipid layers may occur.The present data disclose the possibility to control the lipid's structures by acting on spin-coating parameters with promising perspectives for novel applications of lipid films.
Keywords: AFM; Tapping mode; Lipid film; Spin-coating
Self-association of glutamic acid-rich fusion peptide analogs of influenza hemagglutinin in the membrane-mimic environments: Effects of positional difference of glutamic acids on side chain ionization constant and intra- and inter-peptide interactions deduced from NMR and gel electrophoresis measurements by Ding-Kwo Chang; Shu-Fang Cheng; Chi-Hui Lin; EricAssen B. Kantchev; Cheng-Wei Wu (pp. 37-51).
Two glutamic acid-rich fusion peptide analogs of influenza hemagglutinin were synthesized to study the organization of the charged peptides in the membranous media. Fluorescence and gel electrophoresis experiments suggested a loose association between the monomers in the vesicles. A model was built which showed that a positional difference of 3, 7 and 4, 8 results in the exposure of Glu3 and Glu7 side chains to the apolar lipidic core. Supportive results include: first, p Ka values of two pH units higher than reference value in aqueous medium for Glu3 and Glu7 CγH, whereas the deviation of p Ka from the reference value for Glu4 and Glu8 CγH is substantially smaller; second, Hill coefficients of titration shift of these protons indicate anti-cooperativity for Glu3 and Glu7 side chain protons but less so for Glu4 and Glu8, implying a strong electrostatic interaction between Glu3 and Glu7 possibly resulting from their localization in an apolar environment; third, positive and larger titration shift for NH of Glu3 is observed compared to that of Glu4, suggesting stronger hydrogen bond between the NH and the carboxylic group of Glu3 than that of Glu4, consistent with higher degree of exposure to hydrophobic medium for the side chain of Glu3.
Keywords: Charged residue; Membrane insertion; Hydrogen bond; Rhodamine self-quenching; Fluorescence resonance energy transfer; Self-assembly; Proton titration shift; Ionization constant shift
The effect of a hyposmotic shock and purinergic agonists on K+(Rb+) efflux from cultured human breast cancer cells by I.F. Gow; J. Thomson; J. Davidson; D.B. Shennan (pp. 52-61).
The effect of a hyposmotic shock and extracellular ATP on the efflux of K+(Rb+) from human breast cancer cell lines (MDA-MB-231 and MCF-7) has been examined. A hyposmotic shock increased the fractional efflux of K+(Rb+) from MDA-MB-231 cells via a pathway which was unaffected by Cl− replacement. Apamin, charybdotoxin or removing extracellular Ca2+ had no effect on volume-activated K+(Rb+) efflux MDA-MB-231 cells. An osmotic shock also stimulated K+(Rb+) efflux from MCF-7 cells but to a much lesser extent than found with MDA-MB-231 cells. ATP-stimulated K+(Rb+) efflux from MDA-MB-231 cells in a dose-dependent fashion but had little effect on K+(Rb+) release from MCF-7 cells. ATP-stimulated K+(Rb+) efflux was only inhibited slightly by replacing Cl− with NO3−. Removal of external Ca2+ during treatment with ATP reduced the fractional efflux of K+(Rb+) in a manner suggesting a role for cellular Ca2+ stores. Charybdotoxin, but neither apamin nor iberiotoxin, inhibited ATP-stimulated K+(Rb+) release from MDA-MB-231 cells. Suramin inhibited the ATP-activated efflux of K+(Rb+). UTP also stimulated K+(Rb+) efflux from MDA-MB-231 cells whereas ADP, AMP and adenosine were without effect. A combination of an osmotic shock and ATP increased the fractional efflux of K+(Rb+) to a level greater than the sum of the individual treatments. It appears that the hyposmotically-activated and ATP-stimulated K+ efflux pathways are separate entities. However, there may be a degree of ‘crosstalk’ between the two pathways.
Keywords: Hyposmotic shock; Purinergic agonist; Breast cancer cell
Functional studies with membrane-bound and detergent-solubilized α2-adrenergic receptors expressed in Sf9 cells by Saurabh Sen; Veli-Pekka Jaakola; Päivi Pirilä; Moshe Finel; Adrian Goldman (pp. 62-70).
A chip-based biosensor technology using surface plasmon resonance (SPR) was developed for studying the interaction of ligands and G protein-coupled receptors (GPCRs). GPCRs, the fourth largest superfamily in the human genome, are the largest class of targets for drug discovery.We have expressed the three subtypes of α2-adrenergic receptor (α2-AR), a prototypical GPCR as functional fusion proteins in baculovirus-infected insect cells. The localization of the expressed receptor was observed in intracellular organelles, as detected by eGFP fluorescence. In addition, the deletion mutants of α2B-AR, with a deletion in the 3rd intracellular loop, exhibited unaltered Kd values and enhanced stability, thus making them more promising candidates for crystallization. SPR demonstrated that small molecule ligands can bind the detergent-solubilized receptor, thus proving that α2-AR is active even in a lipid-free environment. The Kd values obtained from the biosensor analysis and traditional ligand binding studies correlate well with each other. This is the first demonstration of the binding of a small molecule to the detergent-solubilized state of α2-ARs and interaction of low-molecular mass-ligands in real time in a label-free environment. This technology will also allow the development of high throughput platform for screening a large number of compounds for generation of leads.
Keywords: Abbreviations; α; 2; -AR; human α; 2; -adrenergic receptor; α; 2A; , α; 2B; and α; 2C; , subtypes of human α; 2; -adrenergic receptor; CHAPS; 3-[(3-cholamidopropyl)-dimethylammonio]propanesulfonate; EDC; ethyl-3(3-dimethylamino) propyl carbodiimide; GFP; green fluorescent protein from; Aequorea victoria; GPCR; G protein-coupled receptor; G protein; guanine nucleotide-binding regulatory protein; HA; hemagglutinin; HRP; horseradish peroxidase; HTS; high throughput screening; K; d; dissociation constant; NHS; N; -hydroxysuccinimide; P20; polysorbate 20; PVDF; polyvinylidene difluoride; PBS; phosphate-buffered saline; SFV; Semliki Forest virus; SPR; surface plasmon resonanceG protein-coupled receptor; Baculovirus; Over-expression; Localization; Spodoptera frugiperda; Sf9 cell; Surface plasmon resonance
Membrane insertion of betaine/GABA transporter during hypertonic stress correlates with nuclear accumulation of TonEBP by Stephen A. Kempson; Jeffrey A. Beck; Philip E. Lammers; J. Scott Gens; Marshall H. Montrose (pp. 71-80).
MDCK cells stably transfected with betaine/GABA transporter tagged with EGFP (EGFP–BGT) were used to study plasma membrane insertion of EGFP–BGT. Adaptive response to hypertonicity requires nuclear migration of TonEBP. Confocal microscopy showed that after 6 h hypertonicity, the nuclear/cytoplasmic ratio of TonEBP fluorescence was increased to 2.4 compared to 1.4 in isotonic controls ( P<0.001). The ratio in hypertonic cells was reduced by the proteasome inhibitor MG-132 in a dose-dependent way. Inhibition was 50% at 3 μM. After 6 h, hypertonicity expressed EGFP–BGT was localized in the plasma membrane, but there was no change in total EGFP–BGT abundance compared to isotonic controls. In contrast, EGFP–BGT remained mostly intracellular when 3 μM MG-132 was included in the hypertonic medium. The transport function of EGFP–BGT was studied as Na+-dependent uptake of [3H]GABA. This was not changed by MG-132 in isotonic controls, but MG-132 produced dose-dependent inhibition of hypertonic upregulation of Na+/GABA cotransport. Inhibition was 80% at 3 μM MG-132. Transport likely reflects membrane insertion of EGFP–BGT and there was a positive correlation ( P<0.05) between Na+/GABA cotransport and the N/C ratio of TonEBP. Results are consistent with a role for TonEBP-mediated transcription in synthesis of additional proteins required for membrane insertion of EGFP–BGT protein.
Keywords: MDCK cells; Betaine; Kidney; Stable transfections; MG-132
ESR as a valuable tool for the investigation of the dynamics of EPC and EPC/cholesterol liposomes containing a carboranyl-nucleoside intended for BNCT by Simona Rossi; Raymond F. Schinazi; Giacomo Martini (pp. 81-91).
Electron Spin Resonance (ESR) spectroscopy of long-chain nitroxides (5-, 7-, and 16-doxyl stearic acid) has been used to evaluate the perturbations induced by β-5- o-carboranyl-2′-deoxyuridine (CDU) on the structure and dynamics of egg phosphatidylcholine (EPC) and EPC/cholesterol liposomes. Loaded liposomes are intended for the use in Boron Neutron Capture Therapy (BNCT). From a detailed analysis of the motional and order parameters determining the ESR line shape as a function of temperature and of CDU content in liposomes, an increased order and a hindered motion of the phospholipid membranes resulted in the presence of increasing CDU concentration. This occurred particularly at the liposome surface level. Both higher ordering and increased rigidity of the membrane lipids were the result of the constraints exerted by the embedded carboranyl-nucleoside.
Keywords: Spectroscopy; Phosphatidylcholine; Cholesterol liposome
Effect of deuterium oxide on the thermodynamic quantities associated with phase transitions of phosphatidylcholine bilayer membranes by Hitoshi Matsuki; Hiroko Okuno; Fumihiko Sakano; Masataka Kusube; Shoji Kaneshina (pp. 92-100).
The bilayer phase transitions of three kinds of phospholipids, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) and dihexadecylphosphatidylcholine (DHPC), in deuterium oxide (D2O) and hydrogen oxide (H2O) were observed by differential scanning calorimetry (DSC) under ambient pressure and light-transmittance measurements under high pressure. The DSC measurements showed that the substitution of H2O by D2O affected the pretransition temperatures and the main-transition enthalpies of all PC bilayers. The temperature–pressure phase diagrams for these PC bilayer membranes in both solvents were constructed by use of the data of light-transmittance measurements. Regarding the main transition of all PC bilayer membranes, there was no appreciable difference between the transition temperatures in D2O and H2O under high pressure. On the other hand, the phase transitions among the gel phases including the pretransition were significantly affected by the solvent substitution. The thermodynamic quantities of phase transitions for the PC bilayer membranes were evaluated and the differences in thermodynamic properties by the water substitution were considered from the difference of interfacial-free energy per molecule in the bilayer in both solvents. It was proved that the substitution of H2O by D2O causes shrinkage of the molecular area of phospholipid at bilayer interface due to the difference in bond strength between deuterium and hydrogen bonds and produces the great influence on the bilayer phase with the smaller area. Further, the induction of bilayer interdigitation in D2O turned out to need higher pressures than in H2O.
Keywords: Deuterium oxide; Differential scanning calorimetry; Interdigitation; Phase transition; Phospholipid bilayer; Pressure
Detergent-like properties of magainin antibiotic peptides: A31P solid-state NMR spectroscopy study by Burkhard Bechinger (pp. 101-108).
31P solid-state NMR spectroscopy has been used to investigate the macroscopic phase behavior of phospholipid bilayers in the presence of increasing amounts of magainin antibiotic peptides. Addition of >1 mol% magainin 2 to gel-phase DMPC or liquid crystalline POPC membranes respectively, results in31P NMR spectra that are characterized by the coexistence of isotropic signals and line shapes typical for phospholipid bilayers. The isotropic signal intensity is a function of temperature and peptide concentration. At peptide concentrations >4 mol% of the resulting phospholipid31P NMR spectra are characteristic of magnetically oriented POPC bilayers suggesting the formation of small disk-like micelles or perforated sheets. In contrast, addition of magainin to acidic phospholipids results in homogenous bilayer-type31P NMR spectra with reduced chemical shift anisotropies. The results presented are in good agreement with the interfacial insertion of magainin helices with an alignment parallel to the surface of the phospholipid bilayers. The resulting curvature strain results in detergent-like properties of the amphipathic helical peptides.
Keywords: Abbreviations; NMR; nuclear magnetic resonance; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphatidylcholine; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphatidylcholine; POPS; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphatidylserine; PI; diacyl-phosphatidylinositol; CD; circular dichroism; FTIR; Fourier transform infraredMagainin; Lipid membrane; Polypeptide–lipid interactions; 31; P solid-state NMR; Phase transition; Phosphatidylcholine; Phase diagram; Detergent; Peptidergent