Biomedical subjects

Y K Levine

Publications and source records attributed to Y K Levine.

At least 19 recordsLinked to original sources

PubMed2004

Field-induced alignment of a smectic-A phase: a time-resolved x-ray diffraction investigation.

The field-induced alignment of a smectic-A phase is, in principle, a complicated process involving the director rotation via the interaction with the field and the layer rotation via the molecular interactions. Time-resolved nuclear magnetic resonance spectroscopy has revealed this complexity in the case of the director alignment, but provides no direct information on the motion of the layers. Here we describe a time-resolved x-ray diffraction experiment using synchrotron radiation to solve the challenging problem of capturing the diffraction pattern on a time scale which is fast in comparison with that for the alignment of the smectic layers. We have investigated the alignment of the smectic-A phase of 4-octyl-4(')-cyanobiphenyl by a magnetic field. The experiment consists of creating a monodomain sample of the smectic-A phase by slow cooling from the nematic phase in a magnetic field with a flux density of 7 T. The sample is then turned quickly through an angle phi(0) about an axis parallel to the x-ray beam direction but orthogonal to the field. A sequence of two-dimensional small angle x-ray diffraction patterns are then collected at short time intervals. Experiments were carried out for different values of phi(0), and at different temperatures. The results show that the alignment behavior changes fundamentally when phi(0) exceeds 45 degrees, and that there is a sharp change in the alignment process when the temperature is less than 3 degrees C below the smectic-A-nematic transition. The results of the x-ray experiments are in broad agreement with the NMR results, but reveal major phenomena concerning the maintenance of the integrity of the smectic-A layer structure during the alignment process.

Journal Article↗

PubMed1999

Translational diffusion of flexible lipid chains in a Langmuir monolayer: a dynamic Monte Carlo study.

We report a computer simulation study of the lateral diffusion of conformationally disordered lipid molecules in a monolayer structure. The simulations were carried out with dynamic Monte Carlo methods, employing two different representations of the internal motions of the lipid chains. The classical Cohen-Turnbull theory is found to provide a good description of the simulated lateral diffusion coefficients at moderate densities. The substantial deviations found at low densities are attributed to the small density fluctuations needed to create the free volume required for the lateral diffusion process.

Computer Simulation↗

PubMed1998

The Dutch-Belgian beamline at the ESRF.

A brief description is given of the design principles and layout of the Dutch-Belgian beamline at the ESRF. This beamline optimizes the use of the available bending-magnet radiation fan by splitting the beam into two branches, each accommodating two experimental techniques.

Journal Article↗

PubMed1998

On the use of the phase memory time T2 for the quantitative characterization of the rotational motions of proteins in lipid bilayer systems.

Numerical simulations of the echo responses from a nitroxide label rigidly attached to a large protein undergoing ultraslow rotational motions in a lipid bilayer are presented. The echoes are formed by the application of Hahn, COSY, and 2D-ELDOR sequences utilizing both soft and hard microwave pulses. The simulations address the question of whether the echo responses elicited by these sequences are affected by restricted angular excursions of the long axis of the protein relative to the normal to the bilayer plane. The results indicate that all three pulse sequences yield the same quantitative motional information regardless of the nature of the microwave pulses and there is no theoretical reason for preferring one sequence above the others.

Algorithms↗

PubMed1996

Rotational motion of Ca-ATPase monitored by electron spin echoes.

Electron spin echoes are used to study the dynamics of different aggregational forms of spin-labeled Ca-ATPase in the sarcoplasmic reticulum membrane. The 2D-ESE measurements are sensitive to motions on the microsecond time scale. The motional information is extracted from the variation of the echo decays across the CW-ESR absorption spectrum. The motional contribution to the decays is described by assuming that the Ca-ATPase molecule is perfectly oriented along the normal to the membrane surface and only undergoes rotational motion about its long axis. The echo-amplitude decays have been evaluated in the time domain by solving the Bloch equations for the stochastic spin Hamiltonian on making use of stochastic trajectories for the orientational behavior of the spin-labeled protein. This approach provides a useful insight into the information provided by the 2D-ESE measurements and affords a direct comparison of the results obtained with different experimental techniques. It is shown that the 2D-ESE technique monitors the orientational motions of dimers or larger aggregates of Ca-ATPase molecules whose rotational correlation times vary between 200 microseconds and 1 ms for the temperature range between 37 and 4 degrees C.

Animals↗

PubMed1996

High-resolution confocal microscopy using synchrotron radiation.

A confocal scanning light microscope coupled to the Daresbury Synchrotron Radiation Source is described. The broad spectrum of synchrotron radiation and the application of achromatic quartz/CaF2 optics allows for confocal imaging over the wavelength range 200-700 nm. This includes UV light, which is particularly suitable for high-resolution imaging. The results of test measurements using 290-nm light indicate that a lateral resolution better than 100 nm is obtained. An additional advantage of the white synchrotron radiation is that the excitation wavelength can be chosen to match the absorption band of any fluorescent dye. The availability of UV light for confocal microscopy enables studies of naturally occurring fluorophores. The potential applications of the microscope are illustrated by the real-time imaging of hormone traffic using the naturally occurring oestrogen coumestrol. (The IUPAC name for coumestrol is 3,9-dihydroxy-6H-benzofurol[3,2-c][1]benzo-pyran-6-one (Chem. Abstr. Reg. No. 479-13-0). The trivial name will be used throughout this paper.

Animals↗

PubMed1995

Quantitative pH imaging in cells using confocal fluorescence lifetime imaging microscopy.

The pH-sensitive probe carboxy SNAFL-1 can be used for imaging using ratiometric and fluorescence lifetime techniques. The former method suffers from the drawback that quantitative pH imaging in cells requires a time-consuming and cumbersome calibration procedure. In contrast, straightforward calibrations in buffer suffice for fluorescence lifetime imaging. This is illustrated here by a comparative study of the two techniques under different controlled conditions. The effect of probe concentration, protein concentration, and hydrophobicity, the contents of damaged cells and living cells on the emission ratio, and the fluorescence lifetime of carboxy SNAFL-1 were studied. The results clearly demonstrate that the fluorescence lifetime imaging technique is more convenient than the ratiometric method for pH determination.

Animals↗

PubMed1994

A computer simulation study of the relation between lipid and probe behaviour in bilayer systems.

Computer simulations are presented of the behaviour of elongated probe molecules anchored to the interface of lipid bilayers above the phase transition of the hydrocarbon chains. The simulations thus mimic the behaviour of the fluorescent probe 1-(4-(trimethylammonio)phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and Cholestane spin label in lipid systems. In contrast to any experimental technique the simulations follow the behaviour of both the lipid molecules and the probe within the bilayer structure. Thus, the relation between the behaviour of the probe molecules and the order and dynamics of the lipid chains can be studied in detail. We find that the presence of probe molecules, at the low concentrations used experimentally, causes only a marginal perturbation in the intrinsic properties of the lipid chains. The simulations presented support the conventional prescription for describing the orientational behaviour of probe molecules in lipid bilayers in terms of a local effective orienting potential. They indicate, however, that the potential arises from the confinement of the probe molecules between long segments of lipid chains in elongated free-volume cavities within the bilayer structure. In this sense the orienting potential concept needs to be refined in order to take into account the combined effect of the restricted free rattling motions of the probes within the free-volume cavities and the orientations of the cavities themselves relative to the normal to the bilayer plane. The time scale of the motions of the cavities within the bilayer is determined by the rotational motions of long segments of the lipid chains. These observations justify the use of rigid probe molecules such as TMA-DPH and Cholestane spin labels for monitoring the orientational order and dynamics in lipid bilayer systems.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1994

A fluorescence depolarization study of the orientational distribution of crossbridges in muscle fibres.

A fluorescence depolarization study of the orientational distribution of crossbridges in dye-labelled muscle fibres is presented. The characterization of this distribution is important since the rotation of crossbridges is a key element in the theory of muscle contraction. In this study we exploited the advantages of angle-resolved experiments to characterize the principal features of the orientational distribution of the crossbridges in the muscle fibre. The directions of the transition dipole moments in the frame of the dye and the orientation and motion of the dye relative to the crossbridge determined previously were explicitly incorporated into the analysis of the experimental data. This afforded the unequivocal determination of all the second and fourth rank order parameters. Moreover, this additional information provided discrimination between different models for the orientational behaviour of the crossbridges. Our results indicate that no change of orientation takes place upon a transition from rigor to relaxation. The experiments, however, do no rule out a conformational change of the myosin S1 during the transition.

Animals↗

PubMed1994

[Application of confocal laser microscopy in studying the effect of epidermal growth factor on the seminiferous epithelium].

Effects of epidermal growth factor (EGF) on pH transients in aggregates of Sertoli cells and germinal cells have been investigated with confocal microscopy using a fluorescent pH sensitive indicator. In some Sertoli cells EGF caused a rapid rise in the pH whereas other Sertoli cells did not respond to EGF. Some Sertoli cells showed a delayed response which coincidated with a similar pH change in neighbouring germinal cells. Since isolated germinal cells never showed a pH response after exposure to EGF, we have concluded that some Sertoli cells may communicate with germinal cells via gap junctions.

Animals↗

PubMed1992

The orientation of transition moments of dye molecules used in fluorescence studies of muscle systems.

Fluorescence and phosphorescence depolarization techniques can provide information on orientational order and rotational motion of crossbridges in muscle fibres. However the depolarization experiment monitors the orientation and motion of the crossbridges indirectly. The changes in depolarization arise from a change in the orientation of the transition dipoles of the dye attached to the crossbridge. In order to extract the physiologically relevant orientations from the data it is therefore necessary to characterize the orientation of the dye molecule relative to the crossbridge and the orientation of the transition moments in the frame of the dyes. The dyes 1,5-I-AEDANS and eosin-5-maleimide are commonly used for labelling the crossbridge in muscle fibres. The orientations of the absorption and fluorescence emission dipoles of these two dyes in the molecular frame were determined. Angle resolved fluorescence depolarization experiments on the dyes, macroscopically aligned in a stretched polymer matrix of poly vinyl alcohol, were carried out. The data were analyzed in terms of an orientational distribution of the dye molecules in the film and the orientations of the absorption and emission dipoles in the frame of the dye molecule. Experimental data, obtained from a given sample at different excitation wavelengths, were analyzed simultaneously in a global target approach. This leads to a reduction in the number of independent parameters optimized by the non-linear least squares procedure.

Animals↗

PubMed1990

Effects of steroid molecules on the dynamical structure of dioleoylphosphatidylcholine and digalactosyldiacylglycerol bilayers.

The ESR spectra of cholestane spin labels (CSL) in dioleoylphosphatidylcholine (DOPC) bilayers containing 20 wt% of cholesterol, 7-dehydrocholesterol, beta-sitosterol, stigmasterol and lanosterol exhibit a marked similarity, thus indicating that these steroids induced the same effects on the lipid bilayer over the temperature range 21-55 degrees C. The incorporation of these steroids into the DOPC bilayers enhances the orientational order of the CSL molecules at every temperature studied, but only induces a pronounced slow-down in their rotational motions at temperatures above 35 degrees C. Similar results were obtained in DOPC/ergosterol multilamellar liposomes, but the changes are now less pronounced than in the other five DOPC/steroid systems. In contrast, the addition of stigmasterol to digalactosyldiacylglycerol (DGDG) bilayers appears to increase the order parameter mean value of P2, without affecting the diffusion coefficients. Furthermore, the incorporation of 7-dehydrocholesterol to DGDG bilayers causes a large enhancement in the orientational order, but has only a small effect on D perpendicular of the CSL molecules. Importantly, this latter effect appears to be independent of temperature. The marked changes in the rates of the rotational motion brought about by the addition of steroids, contrasts with the lack of a significant effect of unsaturation on the bilayer dynamics reported by us previously (Korstanje et al. (1989), Biochim. Biophys. Acta 980, 225-233, and 982, 196-204).

Chemical Phenomena↗

PubMed1989

Reorientational dynamics in lipid vesicles and liposomes studied with ESR: effects of hydration, curvature and unsaturation.

Electron spin resonance experiments were carried out on 3-doxyl-5 alpha-cholestane spin-label (CSL) molecules embedded in multilamellar liposomes and small unilamellar vesicles (SUVs) of palmitoyloleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC) and dilinoleoylphosphatidylcholine (DLPC). The experimental spectra were analyzed by a numerical solution of the stochastic Liouville equation. Effects of temperature, presence of unsaturated bonds and high bilayer curvature on the dynamic behaviour of the lipid molecules were studied. Our results, combined with results from planar multibilayers with a varying hydration rate (Korstanje et al. (1989) Biochim. Biophys. Acta 980, 225-233), give a consistent picture of the orientational order and rotational dynamics of CSL molecules embedded in lipid matrices with various geometrical configurations. Increase of hydration or temperature reduces molecular ordering and increases molecular dynamics. In highly curved vesicle configurations, SUVs, molecular order is found to be lower than in multilamellar liposomes. In contrast, rotational motion is not affected by increase of curvature. In all lipid configurations studied, increase of the number of unsaturated bonds in the fatty acid chains reduces molecular ordering. We find, however, no effect of unsaturation on the rotational mobility of the CSL probe molecules. These results clearly show that changes in molecular orientational order and reorientational dynamics have to be considered separately, and that they are not necessarily correlated as implied by the common concept of membrane fluidity. Comparing our results with data from a motional narrowing analysis shows that the latter approach seriously overestimates the rate of molecular reorientation.

Electron Spin Resonance Spectroscopy↗

PubMed1989

Slow-motion ESR study of order and dynamics in oriented lipid multibilayers: effects of unsaturation and hydration.

Electron spin resonance (ESR) experiments were carried out on 3-doxyl-5 alpha-cholestane spin-label (CSL) molecules embedded in macroscopically oriented multibilayers of dimyristoylphosphatidylcholine (DMPC), palmitoyloleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC) and dilinoleoylphosphatidylcholine (DLPC). For these lipids we studied the effects of temperature, hydration and unsaturation on the orientational order parameters and rotational motions of the probe molecules in the liquid crystalline phase. The experimental ESR spectra were simulated by a numerical solution of the stochastic Liouville equation (SLE) for the density matrix of a spin-label molecule. This allows extraction of detailed information about both molecular order and rotational dynamics. The data show that, in our temperature range, the lipid systems are in the slow-motion regime, thereby precluding a motional narrowing interpretation. This is illustrated by a simple model calculation which shows that a fast-motion interpretation seriously overestimates the order parameters. We have compared our results with data obtained independently from angle-resolved fluorescence depolarization (AFD) experiments on oriented bilayers in which 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) molecules were used as fluorescent probes (Deinum et al., (1988) Biochemistry 27, 852-860). It is found that the orientational order and the rotational dynamics obtained with both techniques agree well. This shows that the probe molecules do not perturb the local bilayer structure to any large extent and that they indeed reflect the intrinsic behaviour of the lipid molecules. Upon increase in temperature or hydration, we observe faster reorientational motion and lower molecular ordering. In contrast, we do not find any systematic effect of unsaturation on molecular reorientational motion. Our results indicate that changes in membrane molecular order and reorientational dynamics have to be considered separately and are not necessarily correlated as implied by the common concept of membrane fluidity.

Electron Spin Resonance Spectroscopy↗

PubMed1989

The fidelity of response by 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene in time-resolved fluorescence anisotropy measurements on lipid vesicles. Effects of unsaturation, headgroup and cholesterol on orientational order and reorientational dynamics.

Time-resolved fluorescence anisotropy measurements on 1-[4-(tri-methylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) molecules in lipid vesicles of palmitoyloleoylphosphatidylcholine (POPC), PC extracted from egg yolk (EggPC), dioleoyl-PC (DOPC), dilinoleoyl-PC (DLPC), phosphatidylglycerol extracted from egg yolk (EggPG), dioleoyl-PG (DOPG), sulfoquinovosyldiacylglycerol (SQDG) and digalactosyl-DG (DGDG) with and without cholesterol are presented. The observed intensity decay curves are analyzed simultaneously in terms of the Brownian rotational diffusion model. The analysis thus yields the isotropic fluorescence decay, the initial anisotropy r (0), the order parameters mean value of P2 and mean value of P4 as well as the diffusion coefficient of the long molecular axis. It is shown that increasing unsaturation in the acyl chains of the PC lipids results in an increase in the rotational diffusion rates of the probes and a decrease in the order parameter mean value of P2. However, the value of mean value of P4 remains unchanged. The corresponding orientational distribution function of the probes is bimodal, with fractions lying preferentially parallel and perpendicular to the local vesicle surface. Surprisingly, the fraction of probe molecules lying with their long axes parallel to the bilayer surface increases with increasing unsaturation with a concomitant narrowing in the width of the distribution of the fraction lying perpendicular to it. As expected, cholesterol is found to increase the order parameters in all the systems and to suppress the tendency of the molecules to lie parallel to the bilayer surface. Furthermore, the rotational diffusion coefficients of the probes is found to increase in all the systems except for DLPC. Interestingly, the effects of unsaturation on the reorientational dynamics of TMA-DPH molecules in the vesicle systems are opposite to those found in the corresponding planar multibilayers (Deinum et al. 1988), whereas the same cholesterol effect is observed for the two systems. Nevertheless, the TMA-DPH molecules exhibit higher diffusion coefficients in the vesicle than in the planar multibilayer systems. In addition, a unimodal distribution of the probe molecules is found in the multibilayer systems. The differences between the two systems are ascribed to the differences in the radius of curvature and the hydration of the bilayers. Lastly we rationalize the bimodal distribution of the TMA-DPH molecules in the vesicles in terms of their observed partition between the lipid and aqueous phases.

Cholesterol↗

PubMed1989

The membrane fluidity concept revisited by polarized fluorescence spectroscopy on different model membranes containing unsaturated lipids and sterols.

Quantitative analysis of time-resolved anisotropy measurements of DPH or TMA-DPH in lipid vesicles yields more than one mathematically correct solution. The solutions differ with respect to the average orientation and to the reorientational dynamics of the probe molecules in the bilayer. This leads to quite opposite results regarding the effects of cholesterol on membrane fluidity. One solution predicts an increase in fluidity, the other a decrease. Angle-resolved fluorescence depolarization (AFD) measurements of probes in oriented lipid bilayers enable determination of the average orientation of the probes in the bilayer and, if the fluorescence decay function is known, of the reorientational dynamics. Analysis of AFD measurements of DPH and TMA-DPH show that increasing unsaturation leads to a decrease in molecular order and a decrease in reorientational dynamics (= fluidity) of the probes. At temperatures above the phase transition of the lipids, the addition of cholesterol causes an increase in molecular order and an increase in reorientational dynamics (= fluidity). The plant sterol stigmaterol, which is structurally closely related to cholesterol, has different effects than cholesterol. The effects vary with the structure of the surrounding lipids. The membrane fluidity concept as it was originally proposed by Chapman attempts to describe the structural and dynamic properties of lipids in a membrane using one single parameter indicated as 'membrane fluidity'. Our results show that it is necessary to distinguish between structural parameters describing molecular order and motion parameters describing molecular dynamics, thus supporting a similar suggestion by Seelig and Seelig. In order to be useful, the membrane fluidity concept has to be limited to the parameters describing molecular dynamics.

Cholesterol↗

PubMed1989

Order and dynamics in the lamellar L alpha and in the hexagonal HII phase. Dioleoylphosphatidylethanolamine studied with angle-resolved fluorescence depolarization.

Fluorescence depolarization techniques are used to determine the molecular order and reorientational dynamics of the probe molecule TMA-DPH embedded in the lamellar L alpha and the hexagonal HII phases of lipid/water mixtures. The thermotropically induced L alpha----HII phase transition of the lipid DOPE is used to obtain macroscopically aligned samples in the hexagonal HII phase at 45 degrees C from samples prepared in the lamellar L alpha phase at 7 degrees C. The interpretation of angle-resolved fluorescence depolarization experiments on these phases, within the framework of the rotational diffusion model, yields the order parameters (P2) and (P4), and the diffusion constants for the reorientational motions. The reorientational motion rates of the TMA-DPH molecules in the hexagonal HII phase are comparable with those in the lamellar L alpha phase. Furthermore, the lateral diffusion of the probe molecule on the surface of the lipid/water cylinder in the hexagonal phase is found to be considerably slower than the reorientational motion.

Liposomes↗

PubMed1988

Orientational properties of biological pigments in ordered systems studied with polarized light: photosynthetic pigment-protein complexes in membranes.

A discussion is presented of the problems involved in the interpretation of linear dichroism and fluorescence depolarization experiments on macroscopically ordered membrane systems. Particular attention has been paid to ordered membranes containing photosynthetic pigment-protein complexes, but the mathematical treatment can equally well be applied to other systems. The information about the orientational properties of the pigments is obtained by the application of the theories developed for the characterization of the molecular orientational order in liquid-crystalline materials. It is shown that while linear dichroism only yields the order parameter S mu of the absorption transition moment, fluorescence depolarization experiments yield in addition the order parameter Sv of the emission transition moment as well as three orientational correlation functions of the two transition moments. It is argued that in general the latter information can only be obtained on utilizing a number of experimental scattering geometries. In particular, the merits of angle-resolved experiments are illustrated.

Biophysical Phenomena↗