Structural state of erythrocyte membranes in a human with Alzheimer’s disease

The structural state of erythrocyte membranes collected from patients with Alzheimer’s disease (AD) was studied. The structural state was characterized by the degree of hemolysis and the content of malonic dialdehyde (MDA), as measures of the peroxide oxidation of lipids (POL), and by the microviscosity of the lipid bilayer. The microviscosity of the membranes was measured by the EPR spin probe method, with 2,2,6,6-tetramethyl-4-capryloyl-oxypiperidine-1-oxyl (lipid probe) and 5,6-benzo-2,2,6,6-tetramethyl-1,2,3,4-tetrahydro-gamma-carboline-3-oxyl (protein probe). It was established that all patients with AD feature an increased fluidity of both the lipid and near-protein areas of erythrocyte membranes. Based on the POL intensity, three groups of patients were identified. The first group was characterized by an increased POL intensity. This group featured a high degree of hemolysis and a high MDA content both before and after incubation, with increases in both these parameters being observed upon incubation. The second and third groups were characterized by a low POL intensity. The second group exhibited a low degree of hemolysis and no increment upon incubation, with the MDA/PH ratio being much higher than unity. The third group was characterized by an increased degree of mechanical hemolysis and by decreases in the degree of homolysis and the MDA content upon incubation.     

Permeability Studies of Redox-Sensitive Nitroxyl Spin Probes Through Lipid Membranes Using an L-Band ESR Spectrometer

Electron spin resonance (ESR) studies were carried out for 2 mM ¹⁴N-labeled deuterated 3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl (MC-PROXYL) and 3-carboxy-2,2,5,5,-tetramethyl-pyrrolidin-1-oxyl (carboxy-PROXYL) in pure water and various concentrations of liposomal solution by using an L-band ESR spectrometer. The ESR parameters, such as the line width, hyperfine coupling constant, g-factor, rotational correlation time and partition parameter, were reported for the samples. The changes in the line width were observed for ¹⁴N-labeled deuterated MC-PROXYL and carboxy-PROXYL in liposomal solution. The hyperfine coupling constant was observed for both nitroxyl spin probes. The permeable and impermeable nature of nitroxyl radicals was demonstrated using the ESR L-band spectra. The rotational correlation time increases with increasing concentration of liposome. The partition parameter for ¹⁴N-labeled deuterated MC-PROXYL in liposomal solution increases with increasing concentration of liposome, which reveals that the nitroxyl spin probe permeates into lipid membrane. The lipid peaks were observed for 2 mM ¹⁴N-labeled deuterated MC-PROXYL in 200, 300 and 400 mM liposomal concentration. The lipid peaks were not observed for ¹⁴N-labeled deuterated carboxy-PROXYL. These results indicate the permeable and impermeable nature of ¹⁴N-labeled deuterated nitroxyl spin probe.     

用荧光探针法研究茶叶花粉黄酮对氧自由基致鼠红细胞膜氧化损伤的保护作用

应用1,6-二苯基-1,3,5-已三烯(DPH)为荧光探针,超氧阴离子自由基和羟基自由基致鼠红细胞膜氧化损伤为模型,研究了茶叶花粉黄酮对鼠红细胞膜氧化损伤的影响.结果表明,超氧阴离子自由基和羟基自由基均能引起鼠红细胞膜脂质过氧化反应,脂质过氧化产物丙二醛含量显著升高,膜脂流动性下降.将鼠红细胞膜预先用茶叶花粉黄酮处理后,膜脂的MDA含量明显下降,呈现剂量与效应关系,膜脂流动性显著提高,表明茶叶花粉黄酮对超氧阴离子自由基和羟基自由基引起的鼠红细胞膜的氧化损伤有保护作用.     

An ESR, Mass Spectrometry and Fluorescence Microscopy Approach to Study the Stearic Acid Derivatives Anchoring in Cells

Lateral phase separations in biological membranes are of great interest, making electron spin resonance (ESR) spectroscopy combined with spin labeling a nondestructive and sensitive technique for the study of lipid rafts. It is currently accepted that the spin probe is localized on the plasma membrane. However, no study confirms this hypothesis. Herein, we report, for the first time, an accurate multispectral method for the quantification of the lipid spin label presence in every subcellular fraction. Cells were incubated with the 5-DOXYL stearic acid derivative and then subfractionated. Results of our multimodal spectroscopy approach ubiquitously demonstrate that the ESR spin label presence only sets in the plasma membranes.     

Protein–membrane interactions investigated with surface-induced fluorescence attenuation

Research on protein–membrane interactions has been undeveloped due to the lack of proper techniques to detect the position of proteins at membranes because membranes are usually only about 4-nm thick. We have recently developed a new method named surface-induced fluorescence attenuation(SIFA) to track both vertical and lateral kinetics of a single labelling dye in supported lipid bilayers. It takes advantage of strong interaction between a light-emitting dye and a partially reflecting surface. By applying the technique to membrane proteins being fluorescently labelled at different residues, here we show that SIFA can measure not only the insertion depth of a dye inside a lipid bilayer, but also the position of a dye in solution near the surface. SIFA can therefore be used to study membrane proteins of various types.     

Fluorescence Study of Lipid Bilayer Interactions of Eu(III) Coordination Complexes

The interaction between Eu(III) tris-β-diketonato coordination complexes (EC), displaying antitumor activity, and lipid vesicles composed of zwitterionic lipid phosphatidylcholine has been studied using fluorescence spectroscopy techniques. To characterize EC-membrane binding, several fluorescent probes, including pyrene, Prodan and 1,6-diphenyl-1,3,5-hexatriene, have been employed. It has been found that EC display effective partitioning into lipid phase, giving rise to structural modifications of both polar and nonpolar lipid bilayer regions, viz. enhancement of membrane hydration and increase in tightness of lipid chain packing. The fact that EC accumulating in lipid bilayer are incapable of inducing significant disruption of membrane structural integrity creates strong prerequisites for development of liposomal nanocarriers of these potential antitumor drugs. Such a possibility is also corroborated by the observation that EC membrane incorporation does not prevent lipid bilayer partitioning of long-wavelength squaraine dyes which represent promising candidates for visualization of liposome biodistribution.     

The New Fluorescent Membrane Probe Ahba: A Comparative Study with the Largely Used Laurdan

Lipid bilayers have been largely used as model systems for biological membranes. Hence, their structures, and alterations caused on them by biological active molecules, have been the subject of many studies. Accordingly, fluorescent probes incorporated into lipid bilayers have been extensively used for characterizing lipid bilayer fluidity and/or polarity. However, for the proper analysis of the alterations undergone by a membrane, a comprehensive knowledge of the fluorescent properties of the probe is fundamental. Therefore, the present work compares fluorescent properties of a relative new fluorescent membrane probe, 2-amino-N-hexadecyl-benzamide (Ahba), with the largely used probe 6-dodecanoyl-N,N-dimethyl-2-naphthylamine (Laurdan), using both static and time resolved fluorescence. Both Ahba and Laurdan have the fluorescent moiety close to the bilayer surface; Ahba has a rather small fluorescent moiety, which was shown to be very sensitive to the bilayer surface pH. The main goal was to point out the fluorescent properties of each probe that are most sensitive to structural alterations on a lipid bilayer. The two probes were incorporated into bilayers of the well-studied zwitterionic lipid dimyristoyl phosphatidylcholine (DMPC), which exhibits a gel-fluid transition around 23 °C. The system was monitored between 5 and 50 °C, hence allowing the study of the two different lipid structures, the gel and fluid bilayer phases, and the transition between them. As it is known, the fluorescent emission spectrum of Laurdan is highly sensitive to the bilayer gel-fluid transition, whereas the Ahba fluorescence spectrum was found to be insensitive to changes in bilayer structure and polarity, which are known to happen at the gel-fluid transition. However, both probes monitor the bilayer gel-fluid transition through fluorescence anisotropy measurements. With time-resolved fluorescence, it was possible to show that bilayer structural variations can be monitored by Laurdan excited state lifetimes changes, whereas Ahba lifetimes were found to be insensitive to bilayer structural modifications. Through anisotropy time decay measurements, both probes could monitor structural bilayer changes, but the limiting anisotropy was found to be a better parameter than the rotational correlation time. It is interesting to have in mind that the relatively small fluorophore of Ahba (o-Abz) could possibly be bound to a phospholipid hydrocarbon chain, not disturbing much the bilayer packing and being a sensitive probe for the bilayer core.     

Spin labeling studies of the interactions of potentially useful therapeutic agents for the treatment of Alzheimer’s disease with cytoskeletal proteins in erythrocyte ghosts and brain synaptosomal membranes

Alzheimer’s disease (AD) is the major dementing disorder of the elderly with four million victims of this disease in the United States. The molecular basis of AD remains unknown, but the biochemical and biophysical state of cytoskeletal proteins is reportedly altered in AD cortical neurons. No approved effective therapy for AD is available. We have used electron spin resonance (ESR) and a protein-specific spin label to investigate the interactions of three potential therapeutic agents in AD: tacrine, velnacrine, and acetylcarnitine, with cytoskeletal proteins in erythrocyte membranes. Further, we report for the first time the effects of tacrine on the physical state of membrane proteins in brain neocortex synaptosomal membranes. All three agents lead to decreased segmental motion and increased cytoskeletal protein-protein interactions in erythrocyte membranes in order of effectiveness: tacrine > velnacrine > acetylcarnitine. In addition, we have synthesized N-methylacridinium methosulfate which has a positive charge on the opposite side of the molecule relative to tacrine. This former agent gave a less pronounced diminution of the relevant ESR parameter than that caused by tacrine, implying that the orientation of the molecule with its interaction site is important in the increased cytoskeletal protein-protein interactions induced by tacrine. With synaptosomal membranes tacrine also significantly decreased segmental motion of membrane proteins. Our ESR results on erythrocyte and brain membranes suggest that in addition to their biochemical effects, these potential AD therapeutic agents function to strengthen cytoskeletal protein-protein interactions. These results are discussed with reference to possible molecular mechanisms involving cytoskeletal proteins in AD.     

电子自旋共振(ESR)技术在生物和医学中的应用

电子自旋共振(electron spin resonance,ESR)是检测自由基最直接最有效的方法,是自由基生物学和医学不可缺少的重要研究技术. 作者综述了ESR、自旋标记、自旋捕集和ESR 成像技术的最新发展及ESR技术在细胞膜、蛋白质结构和一些重大疾病如心脏病、老年痴呆症、帕金森综合症和中风等疾病研究及辐射损伤和植物疾病研究中的应用.     

Laurdan in Fluid Bilayers: Position and Structural Sensitivity

Laurdan (2-dimethylamino-6-lauroylnaphthalene) is a hydrophobic fluorescent probe widely used in lipid systems. This probe was shown to be highly sensitive to lipid phases, and this sensitivity related to the probe microenvironment polarity and viscosity. In the present study, Laurdan was incorporated in 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG), which has a phase transition around 41°C, and DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), which is in the fluid phase at all temperatures studied. The temperature dependence of Laurdan fluorescent emission was analyzed via the decomposition into two gaussian bands, a short- and a long-wavelength band, corresponding to a non-relaxed and a water-relaxed excited state, respectively. As expected, Laurdan fluorescence is highly sensitive to DPPG gel–fluid transition. However, it is shown that Laurdan fluorescence, in DLPC, is also dependent on the temperature, though the bilayer phase does not change. This is in contrast to the rather similar fluorescent emission obtained for the analogous hydrophilic probe, Prodan (2-dimethylamino-6-propionylnaphthalene), when free in aqueous solution, over the same range of temperature. Therefore, Laurdan fluorescence seems to be highly dependent on the lipid bilayer packing, even for fluid membranes. This is supported by Laurdan fluorescence anisotropy and spin labels incorporated at different positions in the fluid lipid bilayer of DLPC. The latter were used both as structural probes for bilayer packing, and as Laurdan fluorescence quenchers. The results confirm the high sensitivity of Laurdan fluorescence emission to membrane packing, and indicate a rather shallow position for Laurdan in the membrane.     

Enhancement of ultrasonically induced cell damage by phthalocyanines in vitro

In this work, erythrocytes from carp were used as a nucleated cell model to test the hypothesis that the phthalocyanines (zinc - ZnPc and chloroaluminium -AlClPc) enhance ultrasonically induced damage in vitro. In order to confirm and complete our earlier investigation, the influence of ultrasound (US) and phthalocyanines (Pcs) on unresearched cellular components, was studied. Red blood cells were exposed to 1 MHz continuous ultrasound wave (0.61 and/or 2.44 W/cm²) in the presence or absence of phthalocyanines (3 μM). To identify target cell damage, we studied hemolysis, membrane fluidity and morphology of erythrocytes. To demonstrate the changes in the fluidity of plasma membrane we used the spectrofluorimetric methods using two fluorescence probes: 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5,-hexatriene (TMA-DPH) and 1,6-diphenyl-1,3,5-hexatriene (DPH). The effect of US and Pcs on nucleated erythrocytes morphology was estimated on the basis of microscopic observation.The enhancement of ultrasonically induced membrane damage by both phthalocyanines was observed in case of hemolysis, and membrane surface fluidity, in comparison to ultrasound. The authors also observed changes in the morphology of erythrocytes. The obtained results support the hypothesis that the Pcs enhance ultrasonically induced cell damage in vitro.Furthermore, the influence of ultrasound on phthalocyanines (Pcs) in medium and in cells was tested. The authors observed changes in the phthalocyanines absorption spectra in the medium and the increase in the intensity of phthalocyanines fluorescence in the cells. These data can suggest changes in the structure of phthalocyanines after ultrasound action.     

Study of plasma membrane heterogeneity using a phosphatidylcholine derivative of 1,6-diphenyl-1,3,5-hexatriene [2-(3-(diphenylhexatriene)propanoyl)-3-palmitoyl-l-α-phosphatidylcholine]

The fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) and DPH derivatives have been used to characterize structural and physicochemical properties of specific membrane domains. Steady-state and fluorescence decay measurements of three probes, DPH (1,6-diphenyl-1,3,5-hexatriene), TMA-DPH [1-(4-trimethyl-ammonium-phenyl)-6-phenyl-1,3,5-hexatriene], and a phosphatidylcholine derivative of DPH, DPH-pPC [2-(3-(diphenylhexatriene)propanoyl)-3-pamitoyl-L--phosphatidyl choline], have been performed in erythrocyte membranes and in lymphocyte plasma membranes. The steady-state fluorescence polarization of the three probes showed a similar trend in both membranes. In fact either in erythrocyte or in lymphocyte plasma membranes the fluorescence polarization values of DPH-pPC and TMA-DPH were similar, but significantly higher with respect to DPH. A better characterization of erythrocyte and lymphocyte plasma membranes was possible by using fluorescence decay measurements. The data suggest the possible use of different DPH derivatives to characterize specific domains in biological membranes.     

Influence of Nature of Functional Groups on Interaction of Tetrasubstituted at Lower Rim <Emphasis Type="Italic">p</Emphasis>-<Emphasis Type="Italic">tert</Emphasis>-Butyl Thiacalix[4]arenes in 1,3-Alternate Configuration with Model Lipid Membranes

The influence of p-tert-butyl thiacalix[4]arenes (1,3-alternate) functionalized by N-propyl morpholine, N,N-dipropyliden-amine and aminodiacetate fragments on dynamic structure of liposomal membranes prepared from dipalmitoyl phosphatidylcholine was investigated by means of electron spin resonance spin-probe technique and Fourier transform infrared spectroscopy. Nuclear magnetic resonance and transmission electron microscopy techniques were applied to characterize the interacting systems. The obtained results have shown that all studied calixarenes interact with polar domains of bilayer. Depending on functional groups and hydrophobic/hydrophilic properties of calixarenes they can dip into bilayer, locate close to the surface of bilayer or form bridges between positively charged groups of adjacent lipid molecules, thus assisting to disordering or putting in order the lipid molecules.     

Fluorescence evidence for cholesterol regular distribution in phosphatidylcholine and in sphingomyelin lipid bilayers

Our previous studies indicated that sterols (including cholesterol and dehydroergosterol) can be regularly distributed into hexagonal superlattices in the plane of liquid-crystalline phosphatidylcholine bilayers. It was suggested that regular and irregular regions coexist in the membrane. In the present study, we report supporting evidence for our sterol regular distribution model. We have examined the fractional concentration dependencies of dehydroergosterol (a naturally occurring cholesterol analogue) fluorescence intensity and lifetime in various phosphatidylcholine and sphingomyelin bilayers. Fluorescence intensity and lifetime dips have been observed at specific sterol mole fractions. At those mole fractions, the acrylamide quenching rate constant of dehydroergosterol fluorescence reaches a local maximum. Those mole fractions match the critical sterol mole fractions at which sterol molecules are expected to be regularly distributed into hexagonal superlattices. The results support the idea that the sterols in the regular region are embedded in the bilayer less deep than those in the irregular regions. We have also examined the fractional cholesterol concentration dependencies of diphenylhexatriene (DPH) fluorescence intensity, lifetime, and polarization in DMPC vesicles. DPH fluorescence intensity and polarization also exhibit distinct dips and peaks, respectively, at critical sterol mole fractions for hexagonal superlattices. However, DPH lifetime changes little with sterol mole fraction. As a comparison, the fluorescence properties of DHE and DPH behave differently in response to the formation of sterol regular distribution. Furthermore, finding evidence for sterol regular distribution in both phosphatidylcholine and sphingomyelin membranes raises the possibility that sterol regular distribution may occur within phospholipid/cholesterol enriched domains of real biological membranes.     

Wetting of phospholipid membranes on hydrophilic surfaces - Concepts towards self-healing membranes

We report on the wetting behavior of phospholipid membranes on solid surfaces immersed in aqueous solution. Using fluorescence microscopy, the spreading velocity of fluid bilayers advancing from a lipid source is investigated. The kinetic spreading coefficient was measured as a function of temperature for pure DMPC membranes and as a function of charge density and cholesterol content for binary membranes. A theoretical model for the membrane flow is presented, which takes into account the liquid crystalline bilayer architecture of the lipid membrane. The spreading power results from the membrane-solid VdW interaction and is dissipated in hydrodynamic shear flow as well as by inter-monolayer friction within the bilayer. The frictional drag causes a dynamic tension gradient in the spreading membrane, which is manifested by a single exponential decay of the fluorescence intensity profile along the spreading direction. Obstacles are shown to act as pinning centers deforming the advancing line interface. However, no depinning was observed, since the centers are circumflown without abrupt relaxation. Received 6 November 1998     

New diphenylhexatriene derivatives as fluorescent membrane probes: Partitioning properties

Three new diphenylhexatriene derivatives, two phospholipids and one single-chain amphiphilic molecule, have been synthesized and considered as probes for measuring membrane fluidity by fluorescence anisotropy. The possibility of using these probes to determine specifically fluidity of inner leaflets of cellular plasma membranes was inferred from their partitioning properties between gel and liquid crystalline phases of phospholipid vesicles of binary composition.     

Investigation of acetone-butanol-ethanol (ABE) fermentation by fluorescence

Photophysical techniques have potential for the development of optical sensors in monitoring and controlling fermentors. In the particular case of acetone-butanol-ethanol (ABE) fermentation, carried out by bacteria of the speciesClostridium acetobutylicum, we have developed two studies based on fluorescence spectroscopy. First, we measured the intrinsic fluorescence of NADH related to bacteria metabolism, leading to a linear relationship between the NADH specific fluorescence and the specific rate of butyric acid production. At the same time, we have correlated enzymatic activities (acetate kinase, butyrate kinase, acetoacetate decarboxylase) with NADH specific fluorescence. Second, we studied the fluorescence polarization of extrinsic DPH (1,6-diphenyl-1,3,5-hexatriene) related to membrane fluidity. A simultaneous increase in both DPH anisotropy (order parameter increase) and butanol production is observed. Even though these results seem contradictory, because of the well-known fluidizing effect of butanol on lipids, they can be explained by a homeoviscous response ofC. acetobutylicum to the presence of butanol during fermentation. Thus the apparent changes in fluidity could be the result of the adaptative membrane alteration.     

几种含羟基化合物对红细胞膜流动性的影响

利用氮氧自由基自旋标记物 CAT-16 和 5-DSA 较为详尽地探讨了两类含羟基化合物 - 羟胺衍生物与苯酚取代物对红细胞膜流动性变化的影响. 实验结果表明：当非双亲性的小分子苯酚取代物插入红细胞膜内,膜的整体有序性降低和流动性增加;羟胺衍生物可作为促氧化剂诱导产生过氧化自由基,促使膜内磷脂间发生化学偶联,从而降低生物膜的内部流动性. 在红细胞膜的表面,标记物分子运动基本不受偶联影响,综合效应来自小分子插入导致的膜流动性增加.     

Does membrane lipid profile explain chilling sensitivity and membrane lipid phase transition of spermatozoa and oocytes?

Ram, fowl and bee spermatozoa, and oocytes of cows and zebrafish were used to study lipid membrane profiles, chilling sensitivity and lipid-phase transitions. The integrity of the membranes was determined by carboxyfluorescein diacetate (CFDA) staining following exposure for 15 minutes to low temperatures. Ram and fowl spermatozoa showed different degrees of loss of membrane integrity. Surprisingly, bee spermatozoa did not show any sensitivity to chilling, and their membranes remained intact down to 0 degree C. In bovine oocytes (at the GV stage) chilling injury was very severe at 16 degree C (membrane integrity decreased by 50%). Lipid phase transition (LPT) and membrane fluidity, which were evaluated by Fourier transform infrared (FTIR) microscopy, and fluorescence polarisation, showed phase transitions at the same temperatures as caused damage (between 30 and 12 degree C). The membrane lipid profiles showed high concentrations of polyunsaturated fatty acids (PUFA) in cold-sensitive ram spermatozoa and zebrafish oocytes, but the ratio between PUFA and saturated fatty acids was highest in cold-resistant bee spermatozoa and lowest in cold-sensitive bovine oocytes. These results suggest a close relationship among cold susceptibility, lipid phase transition and lipids profile in animal gametes.     

Fluorescence lifetimes of diphenylhexatriene-containing probes reflect local probe concentrations: Application to the measurement of membrane fusion

An important process in the life of a cell is fusion between cellular membranes. This is the process by which two cellular compartments surrounded by different membranes join to become a single compartment surrounded by a single membrane, without significant loss of compartment contents. To demonstrate fusion, the cell biophysicist must demonstrate all three critical aspects of the process: (1) mixing of membrane components, (2) mixing of compartment contents; and (3) retention of compartment contents. Most commonly, accomplishing this involves the use of fluorescence probes. The general theme to the methods described involves some form of concentration-dependent quenching. An unique method developed in our laboratory utilizes the concentration dependence of the fluorescence lifetime of a phosphatidylcholine containing carboxyethyl diphenylhexatriene at position 2 and palmitic acid at position 1 of glycerol (DPHpPC). The fluorescence lifetime of this molecule and that of its parent fluorophore diphenylhexatriene (DPH) shorten dramatically as their two-dimensional concentrations in a membrane increase. This lifetime quenching can be described by dimer formation that reduces the symmetry of the DPH excited state. This phenomenon allows one to use the fluorescence lifetime to gain insight into the local concentration of probe in microscopic regions of a membrane. One application of this is in distinguishing lipid transfer between the outer leaflets of two contacting membrane bilayers from fusion between these membranes that leads to mixing of lipids in both the inner and outer leaflets of the membrane bilayers. This allows a single measurement to demonstrate fusion between membrane pairs.Abbreviations PEG poly(ethylene glycol) - Na₂EDTA ethyiene-diamine-tetraacedic acid, disodium salt - LUV large, unilamellar vesicles made by rapid extrusion technique - DPH 1,6-diphenyl-trans-1,3,5-hexatriene - DPHpPC 1-palmitoyl-2-[[[2-[4- (phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]oxy]carbonyl]-3-sn-phosphatidylcholine - DPPC 1,2-dipalmitoyl-3-sn-phosphatidylcholine - PA palmitic acid - NBD-PE N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-PE - Rh-PE N-(lissamine Rhodamine B sulfoyl)-PE - R₁₈ octadecyl Rhodamine B chloride - ANTS 1-aminonaphthalene-3,6,8-trisulfonic acid - DPX N,N-p-xylylene-bis(pyradinium bromide)