Ultrasound-assisted fabrication of acoustically active,erythrocyte membrane “bubbles”

In this communication, we report an ultrasound-assisted method, utilising human red blood cell (RBC) or erythrocyte membranes, to produce acoustically active “bubbles”, intended for vasculature imaging. The resulting RBC membrane bubbles have an average size of 1.5 μm with a generally spherical morphology, altered internal aqueous compartment contents, and small gas-containing protrusions or “pockets” in between the membrane bilayer. We also found that this method produced some nanobubbles (200–400 nm diameter), due to the shedding of lipid components from the RBC membranes to compensate for the membrane structural changes. In vitro ultrasound imaging showed that RBC membrane bubbles had comparable ultrasound contrast enhancement as the standard DEFINTY^TM microbubble preparation (~13% v/v) and lower concentrations of this standard contrast agent. This current technology demonstrate a new and important application of ultrasound and of RBC membranes, having inherent biocompatibility, as potential material for the development of new types of ultrasound imaging agents, without the use of additional lipid components and pre-made microbubbles.     

Video fluorescence microscopic techniques to monitor local lipid and phospholipid molecular order and organization in cell membranes during hypoxic injury

Digitized video microscopy is rapidly finding uses in a number of fields of biological investigation because it allows quantitative assessment of physiological functions in intact cells under a variety of conditions. In this review paper, we focus on the rationale for the development and use of quantitative digitized video fluorescence microscopic techniques to monitor the molecular order and organization of lipids and phospholipids in the plasma membrane of single living cells. These include (1) fluorescence polarization imaging microscopy, used to measure plasma membrane lipid order, (2) fluorescence resonance energy transfer (FRET) imaging microscopy, used to detect and monitor phospholipid domain formation, and (3) fluorescence quenching imaging microscopy, used to spatially map fluid and rigid lipid domains. We review both the theoretical as well as practical use of these different techniques and their limits and potential for future developments, and provide as an illustrative example their application in studies of plasma membrane lipid order and topography during hypoxic injury in rat hepatocytes. Each of these methods provides complementary information; in the case of hypoxic injury, they all indicated that hypoxic injury leads to a spatially and temporally heterogeneous alteration in lipid order, topography, and fluidity of the plasma membrane. Hypoxic injury induces the formation of both fluid and rigid lipid domains; the formation of these domains is responsible for loss of the plasma membrane permeability barrier and the onset of irreversible injury (cell death). By defining the mechanisms which lead to alterations in lipid and phospholipid order and organization in the plasma membrane of hypoxic cells, potential sites of intervention to delay, prevent, or rescue cells from hypoxic injury have been identified. Finally, we briefly discuss fluorescence lifetime imaging microscopy (FLIM) and its potential application for studies monitoring local lipid and phospholipid molecular order and organization in cell membranes.     

Monitoring of the Proton Electrochemical Gradient in Reconstituted Vesicles: Quantitative Measurements of Both Transmembrane Potential and Intravesicular pH by Ratiometric Fluorescent Probes

Proteoliposomes carrying reconstituted yeast plasma membrane H⁺-ATPase in their lipid membrane or plasma membrane vesicles are model systems convenient for studying basic electrochemical processes involved in formation of the proton electrochemical gradient (Δμ_H ⁺) across the microbial or plant cell membrane. Δψ- and pH-sensitive fluorescent probes were used to monitor the gradients formed between inner and outer volume of the reconstituted vesicles. The Δψ-sensitive fluorescent ratiometric probe oxonol VI is suitable for quantitative measurements of inside-positive Δψ generated by the reconstituted H⁺-ATPase. Its Δψ response can be calibrated by the K⁺/valinomycin method and ratiometric mode of fluorescence measurements reduces undesirable artefacts. In situ pH-sensitive fluorescent probe pyranine was used for quantitative measurements of pH inside the proteoliposomes. Calibration of pH-sensitive fluorescence response of pyranine entrapped inside proteoliposomes was performed with several ionophores combined in order to deplete the gradients passively formed across the membrane. Presented model system offers a suitable tool for simultaneous monitoring of both components of the proton electrochemical gradient, Δψ and ΔpH. This approach should help in further understanding how their formation is interconnected on biomembranes and even how transport of other ions is combined to it.     

The Susceptibility of Non-UV Fluorescent Membrane Dyes to Dynamical Properties of Lipid Membranes

Fluorescence spectroscopy and microscopy are powerful techniques to detect dynamic properties in artificial and natural lipid membrane systems. Unfortunately, most fluorescent dyes that sense dynamically relevant membrane parameters are UV sensitive. Their major disadvantage is a high susceptibility to fluorescence bleaching. Additionally, the risk for hazardous damages in biological components generally increases with decreasing excitation wavelength. Therefore the use of non-UV–sensitive membrane dyes would provide significant advantage, particularly for applications in fluorescence microscopy, which usually implies high local excitation intensities. We applied steady-state fluorescence spectroscopy techniques to several UV and non-UV membrane dyes to detect and compare dynamically relevant excitation and emission characteristics. Small unilamellar liposomes (composed of egg yolk phosphatidylcholine) served as a model system for biological membranes. The dynamic properties of the membranes were varied by two independent parameters: the intrinsic cholesterol content (0–50 mol%) and temperature (10–50°C). We tested four non-UV–sensitive membrane dyes: 9-diethylamino-5H-benzophenoxazine-5-one (Nile Red), 4-(dicyanovinyl)julolidine (DCVJ), N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide (FM 4-64), and 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiIC₁₈). We also tested three derivatives of DiIC₁₈: DiIC₁₆ and DiIC₁₂ differ in acyl chain length and Fast-DiIC₁₈ provides double bonds between hydrocarbon atoms. The spectral results were compared to established fluorescence characteristics of four UV membrane dyes: the anisotropy of 1-6-phenyl-1,3,5,-hexatrien (DPH), two derivatives of DPH (TMA-DPH and COO^–-DHP), and the generalized polarization of 6-dodecanoyl-2-dimethyl-aminonaphthalene (Laurdan). Our results indicate that the tested non-UV dyes do not reveal dynamically relevant membrane parameters in a direct manner. However, spectral characteristics make DiIC₁₈, Nile Red, and DCVJ promising probes for the microscopic detection of lateral lipid organization, an indirect indicator of membrane dynamics. In particular, DiIC₁₈ showed very selective shifts in the emission spectra at defined temperatures and cholesterol contents that have not been reported elsewhere.     

Dynamics of nonspecific adsorption of insulin to erythrocyte membranes

Molecules may arrive at targets (receptors, enzymes, etc.) localized on a membrane surface by first adsorbing onto the surface and then surface diffusing to the targets. The flux rate of molecules arriving at targets via this mechanism depends on the surface diffusion coefficient of the molecules and, in some circumstances, on the adsorption/desorption kinetics. The technique of total internal reflection with fluorescence recovery after photobleaching (TIR-FRAP) was used here to study these rate parameters of fluorescein-labeled insulin (f-insulin) interacting with erythrocyte ghosts. Ghosts were adhered to polylysine coated slides for TIR illumination. Some ghosts became flattened and unsealed on the polylysine so that both extracellular and cytoplasmic sides of the membrane were openly exposed to the solution. An aluminum thin film between the polylysine and the fused silica of a slide quenched background fluorescence from f-insulin adsorbed directly onto the polylysine. An interference fringe pattern from two intersecting and totally internally reflecting laser beams provided surface-selective excitation with a spatial variation of illumination intensity across a ghost for surface diffusion measurements. Measured characteristic values of desorption rate constants ranged from 0.043 to 270 s^–1. According to a preexisting theoretical model, the largest desorption rate constant in this range would result in some increase in the total flux rate to a perfect sink target due to capture from the surface, provided that the surface diffusion coefficient was about 10^–8 cm²/s. However, based on TIR-FRAP measurements on our system, we estimate that the surface diffusion coefficient is less than about 5×10^–10 cm²/s. The combination of novel techniques presented here may prove valuable to other workers seeking to make diffusive and chemical kinetic rate parameter measurements of biomolecules at biological cell membranes.     

Diffusion Coefficients of Several Rhodamine Derivatives as Determined by Pulsed Field Gradient–Nuclear Magnetic Resonance and Fluorescence Correlation Spectroscopy

Rhodamine derivatives are popular, photostable fluorophores that are used in a number of fluorescent based techniques, including fluorescence correlation spectroscopy (FCS). Indeed, in FCS, both rhodamine 6G (R6G) and rhodamine 110 (R110) are used as calibration standards to determine the dimensions of the instrument confocal volume. In spite of a requirement for precise values of the diffusion coefficients, literature values are scarce and vary over an order of magnitude. In this paper, the diffusion coefficients of four rhodamine fluorophores (rhodamine 6G (R6G), rhodamine B (RB), rhodamine 123 (R123), rhodamine 110 (R110)) were determined by pulsed field gradient nuclear magnetic resonance (PFG-NMR) spectrometry and then validated by comparison with fluorescence correlation spectroscopy. With the objective of validating the FCS calibration, diffusion coefficients of several dextrans and a polystyrene nanoparticle were also determined and compared with literature values or theoretical values that were based upon the Stoke–Einstein equation. The work presented here lead us to conclude that the diffusion coefficients for R6G and R110 have generally been underestimated in the literature. We propose revised values of 4.4 × 10⁻¹⁰ m² s⁻¹ for R110 and 4.0 × 10⁻¹⁰ m² s⁻¹ for R6G. Using the revised D value for R110 to calibrate the FCS instrument, diffusion coefficients have then been systematically determined for different conditions of pH, ionic strength and concentration. To correct for differences due to solvent effects (D₂O vs. H₂O), an isotopic correction factor, of 1.23, was determined from both FCS and from the solvent auto-diffusion coefficients obtained by NMR.     

Fluorescence of Supported Phospholipid Bilayers Recorded in a Conventional Horizontal-Beam Spectrofluorometer

Supported phospholipid bilayers are a convenient model of cellular membranes in studies of membrane biophysics and protein-lipid interactions. Traditionally, supported lipid bilayers are formed on a flat surface of a glass slide to be observed through fluorescence microscopes. This paper describes a method to enable fluorescence detection from the supported lipid bilayers using standard horizontal-beam spectrofluorometers instead of the microscopes. In the proposed approach, the supported lipid bilayers are formed on the inner optical surfaces of the standard fluorescence microcell. To enable observation of the bilayer absorbed on the cell wall, the microcell is placed in a standard fluorometer cell holder and specifically oriented to expose the inner cell walls to both excitation and emission channels with a help of the custom cell adaptor. The signal intensity from supported bilayers doped with 1 % (mol) of rhodamine-labeled lipid in the standard 3-mm optical microcell was equivalent to fluorescence of the 70–80 nM reference solution of rhodamine recorded in a commercial microcell adaptor. Because no modifications to the instruments are required in this method, a variety of steady-state and time-domain fluorescence measurements of the supported phospholipid bilayers may be performed with the spectral resolution using standard horizontal-beam spectrofluorometers.     

Laurdan Spectrum Decomposition as a Tool for the Analysis of Surface Bilayer Structure and Polarity: a Study with DMPG,Peptides and Cholesterol

The highly hydrophobic fluorophore Laurdan (6-dodecanoyl-2-(dimethylaminonaphthalene)) has been widely used as a fluorescent probe to monitor lipid membranes. Actually, it monitors the structure and polarity of the bilayer surface, where its fluorescent moiety is supposed to reside. The present paper discusses the high sensitivity of Laurdan fluorescence through the decomposition of its emission spectrum into two Gaussian bands, which correspond to emissions from two different excited states, one more solvent relaxed than the other. It will be shown that the analysis of the area fraction of each band is more sensitive to bilayer structural changes than the largely used parameter called Generalized Polarization, possibly because the latter does not completely separate the fluorescence emission from the two different excited states of Laurdan. Moreover, it will be shown that this decomposition should be done with the spectrum as a function of energy, and not wavelength. Due to the presence of the two emission bands in Laurdan spectrum, fluorescence anisotropy should be measured around 480 nm, to be able to monitor the fluorescence emission from one excited state only, the solvent relaxed state. Laurdan will be used to monitor the complex structure of the anionic phospholipid DMPG (dimyristoyl phosphatidylglycerol) at different ionic strengths, and the alterations caused on gel and fluid membranes due to the interaction of cationic peptides and cholesterol. Analyzing both the emission spectrum decomposition and anisotropy it was possible to distinguish between effects on the packing and on the hydration of the lipid membrane surface. It could be clearly detected that a more potent analog of the melanotropic hormone α-MSH (Ac-Ser¹-Tyr²-Ser³-Met⁴-Glu⁵-His⁶-Phe⁷-Arg⁸-Trp⁹-Gly¹⁰-Lys¹¹-Pro¹²-Val¹³-NH₂) was more effective in rigidifying the bilayer surface of fluid membranes than the hormone, though the hormone significantly decreases the bilayer surface hydration.     

A New Fluorescent Squaraine Probe for the Measurement of Membrane Polarity

The present study was undertaken to evaluate the sensitivity of newly synthesized squaraine dye 1 to the changes in lipid bilayer physical properties and compared it with the well-known dye 2. Partitioning of the dye 1 into lipid bilayer was found to be followed by significant increase of its fluorescence intensity and red-shift of emission maximum, while intensity of the dye 2 fluorescence increased only slightly on going from aqueous to lipidic environment. This suggests that dye 1 is more sensitive to the changes in membrane properties as compared to dye 2. Partition coefficients of the dye 1 have been determined for the model membranes composed of zwitterionic phospholipid phosphatidylcholine (PC) and its mixtures with positively charged detergent cetyltrimethylammonium bromide (CTAB), anionic phospholipid cardiolipin (CL), and sterol (Chol). The spectral responses of the dye 1 in different liposome media proved to correlate with the increase of bilayer polarity induced by Chol and CL or its decrease caused by CTAB. It was concluded that dye 1 can be used as fluorescent probe for examining membrane-related processes.     

Reaction Rate of Small Diffusing Molecules on a Cylindrical Membrane

Biomembranes consist of a lipid bi-layer into which proteins are embedded to fulfill numerous tasks in localized regions of the membrane. Often, the proteins have to reach these regions by simple diffusion. Motivated by the observation that IP₃ receptor channels (IP₃R) form clusters on the surface of the endoplasmic reticulum (ER) during ATP-induced calcium release, the reaction rate of small diffusing molecules on a cylindrical membrane is calculated based on the Smoluchowski approach. In this way, the cylindrical topology of the tubular ER is explicitly taken into account. The problem can be reduced to the solution of the diffusion equation on a finite cylindrical surface containing a small absorbing hole. The solution is constructed by matching appropriate ‘inner’ and ‘outer’ asymptotic expansions. The asymptotic results are compared with those from numerical simulations and excellent agreement is obtained. For realistic parameter sets, we find reaction rates in the range of experimentally measured clustering rates of IP₃R. This supports the idea that clusters are formed by a purely diffusion limited process.     

A Versatile Method for Determining the Molar Ligand-Membrane Partition Coefficient

A novel method for the quantitative assessment of the membrane partitioning of a ligand from the aqueous phase is described, demonstrated here with the thoroughly studied antipsychotic chlorpromazine (CPZ). More specifically, collisional quenching of the fluorescence of a pyrene labeled fluorescent lipid analog 1-palmitoyl-2[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC) by CPZ was utilized, using 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine and -serine (POPC and POPS) liposomes as model membranes. The molar partition coefficient is obtained from two series of titrations, one with constant [phospholipid] and increasing [drug] and the other with constant [drug] and varying total [phospholipid], the latter further comprising of large unilamellar vesicles (LUVs) of POPC/POPS/PPDPC at a constant concentration of 10 μM and indicated concentrations of POPC/POPS LUVs. Notably, the approach described is generic and can be employed in screening for the membrane partitioning of compounds, providing that a suitable fluorescence parameter can be incorporated into one population of liposomes utilized as model membranes.     

Numerical and experimental study of excited light and auto-fluorescence diffusion in teeth

Excited light and corresponding intrinsic fluorescence diffusion inside teeth tissue are an essential problem for light-based carious lesion detection. Based on finite element numerical analysis of diffusion equation, the photon density distribution of both excited light and autofluorescence of 2D premolar teeth model is obtained. The dependence of excited light and autofluorescence density distribution inside the teeth model on the scattering coefficient of enamel (5–25 mm⁻¹) and dentine (100–140 mm⁻¹) is numerically simulated and analyzed. The fitted results reveal that fluorescence intensity decreases exponentially. Optical penetration depth and fluorescence relative depth declined with the increment of scattering coefficient of enamel. And the dentine had the opposite effect. Finally, the experiment of measurement of fluorescence intensity on the teeth surface is conducted and the result is compared with the numerical computation.     

Kinetics of epidermal growth factor/receptor binding on cells measured by total internal reflection/fluorescence recovery after photobleaching

Total internal reflection fluorescence (TIRF) microscopy is used to measure the dissociation kinetic rate of fluorescein-labeled epidermal growth factor from its specific receptors on the surface of intact but mildly fixed A431 human epidermoid cells in culture. Prior applications of TIRF microscopy have been limited to nonreceptor binding or to model membrane systems. The evanescent field excites fluorescence selectively at the surface of the cell proximal to the coverslip. Prismless epiillumination TIR is employed to avoid space limitations and is achieved by passing the excitation laser beam through a high (1.4)-aperture objective so that the light is incident at the glass/water interface beyond the critical angle. Long-term focus is maintained by a special feedback system. Of the possible effects that can influence the time course of the postbleach fluorescence recoveries—the EGF/receptor dissociation ratek ₂, the bulk solution diffusion rate of EGF, and the cell surface motion of the receptors—we infer that the dissociation ratek ₂ dominates. Several fitting schemes are compared and indicate the presence of a multiplicity of values fork ₂, ranging from about 0.05 to 0.004 s^–1, with an average value of about 0.012 s^–1. These results compare well with values previously obtained by radiolabel/washing techniques. The significance of the results in terms of kinetic models and the advantages of the TIRF technique for these sorts of measurements are discussed.     

Conformational differences of oxytocin and vasopressin as observed by fluorescence anisotropy decays and transient effects in collisional quenching of tyrosine fluorescence

We used gigahertz frequency-domain fluorometry to examine the tyrosyl fluorescence intensity and anisotropy decays of the single-tyrosine cyclic peptide hormones oxytocin and vasopressin. Acrylamide quenching and a distance-dependent quenching model for collisional quenching were used to evaluate the extent of tyrosyl exposure to the quencher and to provide increased resolution of the picosecond anisotropy decays. Analysis of the intensity decays using a lifetime distribution model shows different distributions for oxytocin and vasopressin. We found that the tyrosyl fluorescence of lysine-vasopressin, as revealed both by the lifetime Stern-Volmer plots and from the quenching analysis, is quenched more effectively than oxytocin. ForN-acetyltyrosinamide (NATyrA), oxytocin, and lysine-vasopressin, we recovered apparent diffusion coefficients for quenching of 4.7×10^–6, 0.44×10^–6, and 4.3×10^–6 cm²/s, respectively, the lower value for oxytocin suggesting a shielded environment for its tyrosyl residue. Tyrosyl anisotropy decays were recovered by global analysis of progressively quenched samples. Compared with oxytocin, vasopressin displayed a longer correlation time for overall rotational diffusion and a higher amplitude for picosecond segmented motions of its tyrosyl residue. All the data are consistent with a more extended and flexible solution structure for vasopressin than for oxytocin.Dedicated to Professor Alfons Kawski on the occasion of his 65th birthday.     

Water and lipid diffusion MRI using chemical shift displacement-based separation of lipid tissue (SPLIT)

PurposeTo obtain water and lipid diffusion-weighted images (DWIs) simultaneously, we devised a novel method utilizing chemical shift displacement-based separation of lipid tissue (SPLIT) imaging.Materials and methodsSingle-shot diffusion echo-planar imaging without fat suppression was used and the imaging parameters were optimized to separate water and lipid DWIs by chemical shift displacement of the lipid signals along the phase-encoding direction. Using the optimized conditions, transverse DWIs at the maximum diameter of the right calf were scanned with multiple b-values in five healthy subjects. Then, apparent diffusion coefficients (ADCs) were calculated in the tibialis anterior muscle (TA), tibialis bone marrow (TB), and subcutaneous fat (SF), as well as restricted and perfusion-related diffusion coefficients (D and D*, respectively) and the fraction of the perfusion-related diffusion component (F) for TA.ResultsWater and lipid DWIs were separated adequately. The mean ADCs of the TA, TB, and SF were 1.56 ± 0.03 mm²/s, 0.01 ± 0.01 mm²/s, and 0.06 ± 0.02 mm²/s, respectively. The mean D*, D, and F of the TA were 13.7 ± 4.3 mm²/s, 1.48 ± 0.05 mm²/s, and 4.3 ± 1.6%, respectively.ConclusionSPLIT imaging makes it possible to simply and simultaneously obtain water and lipid DWIs without special pulse sequence and increases the amount of diffusion information of water and lipid tissue.     

Relative quantification of cellular sections with molecular depth profiling ToF-SIMS imaging

We report the use of secondary ion mass spectrometry (SIMS) imaging to quantify the relative difference in the amount of lipid between two sections, the plasma membrane and the cytoplasm, of single cells from two different populations. Cells were each labeled with lipophillic dyes, frozen, fractured and analyzed in a ToF-SIMS mass spectrometer equipped with a 40 keV C₆₀⁺ ion source. In addition to identifying cells from separate populations, the lipophilic dyes can be used as a marker for the outer leaflet of the cell membrane and therefore as a depth finder. Here, we show that it is possible to compare the amount of lipids with particular headgroups in the cell membrane of a treated cell to the membrane of a control cell. Following erosion of the cell membranes, the amount of the two specific lipid head groups in the cytoplasm of the treated cell can be compared to those lipids in a control cell. Here we take the first step in this experimental design and display the ability to analyze multiple sections of frozen cells following a single fracture.     

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.     

Flexoelectricity of lyotropics and biomembranes

Summary Flexoelectric properties of the following lyotropic systems are described: monolayers, bilayers, lamellar lipid-water phases, black lipid membranes and biomembranes. Lipid layers (one-component and mixed) and lipid-protein layers are considered. Different molecular mechanisms (dipolar and quadrupolar) at free and blocked flip-flop and at free and blocked lateral diffusion are discussed in details. Surface potential measurements in monolayers and diamagnetic anisotropy of bilayers are used to evaluate the contribution of the different mechanisms. The area flexoelectric coefficient is typically −5·10⁻¹¹ statC. Biomembranes with free lateral diffusion of the integral proteins (conical and dipolar ones) would exhibit dipolar flexoelectricity, while those with blocked lateral diffusion (high protein content) would exhibit quadrupolar flexoelectricity. The flexoelectric coefficient of biomembranes seems to be one order of magnitude higher than that of the protein-free bilayers and both positive and negative signs are possible. Some mechano-electrical phenomena in membrane systems are discussed in connection to the flexoelectricity. Paper presented at the ?Meeting on Lyotropics and Related Fields?, held in Rende, Cosenza, September 13–18, 1982.     

On the Coupling Between Surface Charge and Hydration in Biomembranes: Experiments with 3-Hydroxyflavone Probes

The newly synthesized 3-hydroxyflavone derivative [2-(4-N,N-diethylaminophenyl)-3-hydroxy-6-chromonyl](N,N-dimethyl-octyl) ammonium bromide (F2) together with already used 4-dimethylamino-3-HF (F) are found to be extremely sensitive to the effects of preferential hydration in model solvent system. This property is explored in the study of phospholipid vesicles of different composition made of neutral, cationic, and anionic lipids. We observe an extremely high level of response of both F and F2 fluorescence emission spectra to the surface charge of the vesicles: the N^* form is strongly favored with less positively charged and more negatively charged membrane surface. The strong red-edge effects, which are almost independent of the lipid composition demonstrate the immobility of the probe environment on the time scale of fluorescence emission and suggest the static nature of hydration effects.     

Toy model that explains the regulation of cholesterol on lipid rafts

Cholesterol, as a common lipid on mammalian cell membranes, plays an important role in the formation of lipid rafts. Recent experiments suggest that the strength of cholesterol’s regulation on lipid rafts can be affected by the length of the unsaturated phospholipid acyl chain on the membrane. In order to understand this observation, a simplified toy model containing three different molecules is proposed in this paper, where the tail length of phospholipids is considered. This model shows the regulation of membrane cholesterol on the phase separation of the lipid mixture and the formation of nano-domains, and also suggests that the configuration entropy of phospholipid tails is an essential factor.