脂质体重组和脂蛋白体在植物生物膜研究中的应用

脂质体是磷脂在一定条件下在水中形成的由脂质双分子层组成的内部为水相的闭合囊泡。在推动生物膜的研究进展中,它作为模式系统起着非常重要的作用,能用于研究膜蛋白的性质和功能;膜脂和膜蛋白的相互关系;膜的电化学性质等。近年来脂质体重组技术开始引入到植物学研究领域,用于对植物膜蛋白的研究。本文简要介绍了脂质体的制备和脂酶体重组的方法及其在植物生物膜研究中的应用。     

Determination of the size distribution of liposomes by SEC fractionation,and PCS analysis and enzymatic assay of lipid content

In this study, small liposomes obtained by high-pressure homogenization were fractionated according to their particle sizes by size exclusion chromatography (SEC). The subfractions were analyzed by photon correlation spectroscopy (PCS) as well as enzymatic phosphatidylcholine (PC) assay for their particle sizes and lipid contents, respectively. For small egg PC-liposomes, a size range of 15 nm to 60 nm was found, with 80% of the vesicles being smaller than 30 nm in size. This is in contradiction to a mean size of 85±32 nm as indicated by PCS without fractionation. The PCS technique appears to underestimate very small particles below 30 nm if (few) bigger particles are present. The PCS particle size analysis of unfractionated hydrogenated egg PC/cholesterol-liposomes (2:1, mole/mole) by PCS did not yield any significant results. On fractionation, however, a particle size range of 40 nm to 120 nm was determined in a reproducible manner. Our results indicate that the combination of size exclusion fractionation with subsequent photon correlation spectroscopic particle size analysis and enzymatic PC assay can give both more detailed and more reliable insight into the particle size distribution of small liposomes than PCS alone. Published: May 15, 2002.     

Structural investigation of biological material in aqueous environment by means of infrared-ATR spectroscopy

Infrared attenuated total reflection (ATR) spectroscopy may be used to investigate biological material (e.g., membranes, proteins, erythrocytes etc.) under biological conditions provided that adhesion of the sample can be achieved in aqueous environment. Uncharged lipid multilayer model membranes can be attached by hydrophobic interaction when hydrophobic internal reflection plates (e.g., ZnSe, CdTe) are used. However, if an electric field is applied across the menbrane, germanium reflection elements would be preferred because of their low electric resistance (50 cm). This material can also be used if cells or proteins are linked chemically to the ATR plate because of the hydrophilic surface which is similar to that of glass and, thus, enables chemical modification by silanization. It has turned out that good adhesion of uncharged and negatively charged model membranes to germanium plates is achieved when they are coated with a monomolecular layer of aminopropylsilane. There is some evidence that erythrocytes remain more stable when adsorbed to a polymerized aminosilane coating (organic silanization) rather than to the corresponding monolayer (aqueous silanization). Negatively charged germanium surfaces have been obtained by succinylation of the aminosilane coating. Furthermore it has been demonstrated that proteins can be bound to the aminosilane coating by means of carbodiimide. Immobilized acetylcholinesterase was still enzymatically active.     

Scar-driven shape-changes of virus capsids

We propose that certain patterns (scars) could be relevant to extend the classic Caspar and Klug construction for icosahedrally-shaped virus capsids. These scars are theoretically and numerically predicted to be formed by electrons arranged on a sphere to minimize the repulsive Coulomb potential (the Thomson problem) and are experimentally found in spherical crystals formed by self-assembled polystyrene beads (an instance of the generalized Thomson problem). Scars could be produced on the capsid at an intermediate stage of its evolution and the release of the bending energy present in scars into stretching energy could allow for shape-changes. The conjecture can be tested in experiments and/or in numerical simulations.     

Modeling protein-mediated morphology in biomembranes

The equilibrium theory for lipid membranes is used to describe the structure of nuclear pores and the membrane shapes accompanying endocytosis. The commonly used variant of the theory contains a fixed parameter called the spontaneous curvature which accounts for asymmetry in the bending response of the membrane. This is replaced here by a variable distribution of spontaneous curvature representing the influence of attached proteins. The required adjustments to the standard theory are described and the resulting model is applied to the study of membrane morphology at the cites of protein-assisted nuclear pore formation and endocytosis.     

A templating route to nanoporous chitosan materials

Nanoporous chitosan materials in both membrane and bulk form with pore diameter ranges of 10–50 nm were prepared by a novel emulsion-mediated templating method. Considering the biocompatibility and versatility of chitosan, the nanoporous chitosan material is expected to have a wide variety of applications in the biotechnological and biomaterials areas.     

Oxidative Stability of Polyunsaturated Fatty Acid in Phosphatidylcholine Liposomes

Synthesized PCs containing docosahexaenoic acid (DHA), arachidonic acid (AA), linoleic acid (LA), and palmitic acid (PA) at known positions in the glycerol moiety were oxidized in liposomes, bulk, and organic solvent. In bulk and organic solvent, the oxidative stability of PC decreased with increasing degrees of unsaturation. However, the degree of unsaturation had little effect on the stability of PC in liposomes. The oxidative stability of PC in liposomes would be affected by the chemical reactivity based on the degree of unsaturation and by the conformation of fatty acyl component in PC bilayers. When the oxidative stability of 1-PA-2-LA-PC or 1-PA-2-AA-PC was compared with that of a 1:1 (mol ratio) mixture of 1,2-diPA-PC+1,2-diLA-PC, or 1,2-diPA-PC+1,2-diAA-PC, respectively, the former PC was more oxidatively stable than that of the latter PC mixture in all oxidation systems, although the degree of unsaturation of 1-PA-2-PUFA-PC was the same as that of the corresponding mixture of diPA-PC+diPUFA-PC. The higher oxidative stability of 1-PA-2-PUFA-PC than that of a corresponding mixture of diPA-PC+diPUFA-PC in liposomes was suggested to be due to the different conformation of PC bilayers and the different rate of hydrogen abstraction by free radicals from intermolecular and intramolecular acyl groups.     

Structural descriptions of ligands in their binding site of integral membrane proteins at near physiological conditions using solid-state NMR

Using solid-state NMR approaches, it is now possible to define the structure and dynamics of binding for a small, isotopically (²H, ¹³C, ¹⁹F, ¹⁵N) labelled ligand, prosthetic group or solute in its binding site of a membrane-bound protein at near physiological conditions in natural membrane fragments or in reconstituted complexes. Studies of oriented membranes permit the orientational bond vectors of labelled groups to be determined to good precision, as shown for retinal in bacteriorhodopsin and bovine rhodopsin. Using novel magic angle spinning NMR methods on membrane dispersions, high-resolution NMR spectra can be obtained. Dipolar couplings can be reintroduced into the spectrum of labelled ligands in their binding sites of membrane-bound proteins to give interatomic distances to high precision (±0.5 Å). Relaxation and cross-polarization data give estimates for the kinetics for on-off rates for binding. In addition, chemical shifts can be measured directly to help provide details of the binding environment for a bound ligand, as shown for analogues of drugs used in peptic ulcer treatment in the gastric ATPase, and for acetylcholine in the acetylcholine receptor.     

General Mathematical Frame for Open or Closed Biomembranes (Part I): Equilibrium Theory and Geometrically Constraint Equation

This paper aims at constructing a general mathematical frame for the equilibrium theory of open or closed biomembranes. Based on the generalized potential functional, the equilibrium differential equation for open biomembrane (with free edge) or closed one (without boundary) is derived. The boundary conditions for open biomembranes are obtained. Besides, the geometrically constraint equation for the existence, formation and disintegration of open or closed biomembranes is revealed. The physical and biological meanings of the equilibrium differential equation and the geometrically constraint equation are discussed. Numerical simulation results for axisymmetric open biomembranes show the effectiveness and convenience of the present theory.     

Effect of antibiotic amphotericin B on structural and dynamic properties of lipid membranes formed with egg yolk phosphatidylcholine

Amphotericin B (AmB) is a popular antibiotic applied in treatment of deep-seated mycotic infections. The mode of action of AmB is based upon interactions with biomembranes but exact binding properties of the antibiotic to the lipid membranes still remain obscure. Effect of incorporation of AmB into egg yolk phosphatidylcholine membranes in the concentration range from 0.01 to 5 mol% on structural and dynamic properties of lipid bilayers was studied with application of small-angle neutron scattering, X-ray diffractometry and Fourier-transform infrared spectroscopy (FTIR). The results of the experiments show that AmB is located predominantly in the headgroup region of the membranes at concentrations below 1 mol%. The process of AmB aggregation, at concentrations above 1 mol%, is associated with ordering effect within the acyl chain region and therefore indicates incorporation of AmB into the hydrophobic membrane core.     

Vesicular Phospholipid Gels: A Technology Platform

Vesicular phospholipid gels (VPGs) represent semi-solid phospholipid dispersions. Their morphology is truly vesicular with aqueous compartments both within the core of the vesicles and in-between the vesicles. VPGs are suited to carry both hydrophilic, amphiphilic and lipophilic drugs. Their drug load is stable since there is no concentration gradient between the vesicles' core and the surrounding water phase. VPGs are suited to release drugs in a controlled manner, and thus may serve as depot implants. When blended with excess aqueous medium VPGs are easily converted into small-sized liposome (SUV) dispersions showing high encapsulation efficiencies for all kinds of drugs. VPG-formulations with various cytostatic drugs have been tested successfully in human xenografts. Obviously, the vesicles protect the drugs from premature metabolic inactivation and/or elimination and guide them to solid tumors with enhanced vascular permeability (passive targeting).

Furthermore, when mounted on a filter support, VPGs represent a tight diffusion barrier suitable for screening of oral drug permeability, as demonstrated by a set of 21 drug compounds. Permeability values were shown to fit well with human absorption in vivo, indicating that the model is suited for rapid screening of passive transport properties of new chemical entities.     

The behavior of the fluorescence lifetime and polarization of oxonol potential-sensitive extrinsic probes in solution and in beef heart submitochondrial particles

Summary The fluorescence polarization and lifetime of the extrinsic potential-sensitive probes oxonols V and VI have been investigated both for the dyes free in aqueous and ethanol solutions and in the presence of beef heart submitochondrial particles under resting and energy-transducing conditions. The emission lifetime of the dyes appears to be inversely related to the solvent dielectric constant and increases as the solvent is changed from an aqueous medium to ethanol to the biological membrane. The fluorescence decay curve becomes biphasic in the presence of the membrane preparation and consists of a faster decaying component, the lifetime of which is the same as that of the probe in aqueous solution and of a slower decaying component. The longer lived component suffers an uncoupler-sensitive decrease in lifetime when ATP is added to the medium. The decrease in lifetime of the longer lived species is accompanied by large depolarizations of the dye fluorescence. These observations are consistent with a redistribution-type mechanism for the energy-dependent spectral changes involving the movement of probe from the aqueous phase to the membrane vesicles. The rotational relaxation time of oxonols V and VI is increased by over an order of magnitude when these dyes associate with the membrane. This observation is consistent with a previously developed model for the location of the dyes in the bilayer in which the side chains serve as anchors, preventing the rapid tumbling of the probe in the membrane.     

Assembly of polymer/lipid composite films on solids based on hairy rod LB-films

The present work deals with the assembly of multilayers or rod-like polymers with hydrophobic side chains (called hairy rods) and their potential application as ultrathin polymer cushions for the build-up of self healing supported membranes on various solids (Si/SiO₂-wafer, gold covered substrates). Three types of hairy rods were studied: Isopentyl cellulose (IPC), phtalocyaniatopolysiloxane with mixed alkane side chains (PCPS) and trimethylsilane cellulose (TMCS). Detailed analysis of the thickness of supported multilayers as a function of the number of deposited monolayers with ellipsometry, near infrared surface plasmon resonance (NIR-SPR), a quartz crystal microbalance (QCM) and reflection interference contrast microscopy (RICM), show that the basic building blocks of hairy rod multilayers are bilayers with the hydrophobic surfaces of the monolayers facing each other. Continuous and stable firms of hairy rods can be deposited if the hydrophobicities of the solid surface and the monolayer are matched. It is demonstrated by lateral diffusion measurements (using photobleaching techniques) that continuous phospholipid bilayers can be deposited onto multilayers of rigid rods of TMCS after hydrophilization by cleavage of trimethylsilane side chains in HCl-vapour, while stable lipid monolayers can be deposited onto hydrophobic surfaces of rigid rod layers. NIR-SPR allows the observation of double band reflectivity curves at interfaces separating different surface layers and thus offers the possibility of differential detection of ligand binding at the interface of differently functionalized domains. Received: 2 February 1996 / Accepted: 28 October 1996     

细胞内膜泡运输中拴系因子的功能

膜泡运输是不同细胞器间进行物质传递的基本方式,分为4个重要步骤：囊泡的出芽、转运、拴系和融合。在此过程中,有许多相关因子参与调控,如包被蛋白、Rab蛋白、拴系因子、SM蛋白和SNARE等。拴系因子在运输囊泡和靶位膜发生接触的最初阶段起重要调控作用,多数拴系因子形成大的多亚基复合体发挥功能。目前,关于拴系因子的功能已经有了一定的了解,在此,我们对酵母、哺乳动物以及植物细胞中的已知拴系因子的特点和功能进行了概述。     

CRAC motif peptide of the HIV-1 gp41 protein thins SOPC membranes and interacts with cholesterol

This study uses low-angle (LAXS) and wide-angle (WAXS) X-ray synchrotron scattering, volume measurements and thin layer chromatography to determine the structure and interactions of SOPC, SOPC/cholesterol mixtures, SOPC/peptide and SOPC/cholesterol/peptide mixtures. N-acetyl-LWYIK-amide (LWYIK) represents the naturally-occurring CRAC motif segment in the pretransmembrane region of the gp41 protein of HIV-1, and N-acetyl-IWYIK-amide (IWYIK), an unnatural isomer, is used as a control. Both peptides thin the SOPC bilayer by ∼ 3 Å, and cause the area/unit cell (peptide + SOPC) to increase by ∼ 9 Å² from the area/lipid of SOPC at 30 °C (67.0 ± 0.9 Å²). Model fitting suggests that LWYIK's average position is slightly closer to the bilayer center than IWYIK's, and both peptides are just inside of the phosphate headgroup. Both peptides increase the wide-angle spacing d of SOPC without cholesterol, whereas with 50% cholesterol LWYIK increases d but IWYIK decreases d. TLC shows that LWYIK is more hydrophobic than IWYIK; this difference persists in peptide/SOPC 1:9 mole ratio mixtures. Both peptides counteract the chain ordering effect of cholesterol to roughly the same degree, and both decrease K_C, the bending modulus, thus increasing the SOPC membrane fluidity. Both peptides nucleate crystals of cholesterol, but the LWYIK-induced crystals are weaker and dissolve more easily.     

Molecular Dynamics Investigation of Bond Ordering of Unsaturated Lipids in Monolayers

Molecular dynamics simulations of three model lipid monolayers of 2,3-diacyl-D-glycerolipids, that contained stearoyl (18:0) in the position 3 and oleoyl (18:9cis), linoleoyl (18:26cis), or linolenoyl (18:33cis) in the position 2, have been carried out. The simulation systems consisted of 24 lipid molecules arranged in a rectangular simulation cell, with periodic boundary conditions in the surface plane. 1 nanosecond simulations were performed at T = 295 K. C-C and C-H bond order parameter profiles and the bond orientation distributions about the monolayer normal have been calculated. The relation of the distributions to the order parameters was analyzed in terms of maxima and widths of the distributions. The cis double bond order parameter is found to be higher than those of adjacent single C-C bonds. The widths of the two distributions of C-H bonds of the cis double bond segment in di- and triunsaturated molecules are much smaller than that obtained for methylene group located between the double bonds. The bond orientation distribution function widths depend on both the segment location in the chain and the segment chemical structure.     

Immobilized-biomembrane affinity chromatography for binding studies of membrane proteins

Analyses of specific interactions between solutes and a membrane protein can serve to characterize the protein. Frontal affinity chromatography of an interactant on a column containing the membrane protein immobilized in a lipid environment is a simple and robust approach for series of experiments with particular protein molecules. Regression analysis of the retention volumes at a series of interactant concentrations shows the affinity of the protein for the interactant and the amount of active binding sites. The higher the affinity, the fewer sites are required to give sufficient retention. Competition experiments provide the affinities of even weakly binding solutes and the non-specific retention of the primary interactant. Hummel and Dreyer size-exclusion chromatography allows complementary analyses of non-immobilized membrane materials. Analyses of the human facilitative glucose transporter GLUT1 by use of the inhibitor cytochalasin B (radioactively labeled) and the competitive substrate D-glucose (non-labeled) showed that GLUT1 interconverted between two states, exhibiting one or two cytochalasin B-binding sites per two GLUTI monomers, dependent on the membrane composition and environment. Similar analyses of a nucleoside transporter, a photosynthetic reaction center, nicotinic acetylcholine receptors and a P-glycoprotein, alternative techniques, and immobilized-liposome chromatographic approaches are presented briefly.     

Binding of antibiotic amphotericin B to lipid membranes: a 1H NMR study

The (1)H NMR technique was applied to study binding of AmB, an antifungal drug, to lipid membranes formed with egg yolk phosphatidylcholine. The analysis of (1)H NMR spectra of liposomes, containing also cholesterol and ergosterol (at 40 mol%), shows that AmB binds preferentially to the polar headgroups. Such a binding restricts molecular motion of the choline fragment in the hydrophilic region at the surface of liposomes but increases the segmental motional freedom in the hydrophobic core. The same effects are also observed in the sterol-containing membranes, except that the effect on the hydrophobic core was exclusively observed in the membranes containing ergosterol.     

Characterisation of phosphatidylcholine/phosphatidylinositol sonicated vesicles. Effects of phospholipid composition on vesicle size

Phosphatidylinositol (PI), dipalmitoylphosphatidylcholine (DPPC) and mixed lipid (DPPC plus PI) sonicated vesicles have been prepared covering a range of composition. The vesicles were characterised by gel filtration, electron microscopy and photon correlation spectroscopy. The dimensions of the vesicles as measured by electron microscopy were in good accord with those obtained from photon correlation spectroscopy measurements. The number average diameters of the vesicles increase on increasing the PI content and range from approx. 30–80 nm as the weight % of PI is increased from 0 to 100. Gel filtration on Sepharose 4B columns gave anomalous results indicating that PI-containing vesicles were retarded on the gel possibly due to an interaction between the inositol headgroup and the gel matrix. Electrophoretic measurements on multilamellar vesicles show that the surface charge density increases with the PI content of the vesicles upto 50 weight % PI and remains constant thereafter. The radii of sonicated vesicles also increase with PI content which reflects a decreasing liposome curvature with increasing surface charge density.