Michel Bouchy

Evolution of fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) during the labelling of living cells☆

Abstract Detailed information on the environment of the fluorescent lipid probe 1,6-diphenyl-1,3,5-hexatriene (DPH) during its interaction with a living cellular system was obtained by following the changes in emission parameters of the probe on the nanosecond time scale. Changes in the steady-state anisotropy r , the anisotropy r ∞ at long time, the rotational relaxation time φ and the lifetime τ were recorded during the early course of DPH uptake in murine-transformed cells. Results are compatible with a model assuming that the plasma membrane and the cytoplasm constitute two compartments, with some DPH translocation from one compartment to another. In short term incubation, fluorescence parameters of DPH characterize the plasma membrane. In long term incubation, most fluorescent signals are due to DHP embedded in intracellular lipids which are in a more fluid state than membranous lipids.

Kinetics of partly diffusion-controlled reactions. XVI: The use of liquid state physics for fluorescence quenching models

Abstract Theories of partly diffusion-controlled reaction kinetics are re-investigated, taking into account the existence of a non-uniform radial distribution function. This allows us to partially explain some discrepancies occurring when molecular emission spectroscopy experiments are carried out using continuous or pulsed excitation.

Structural changes of liver microsomes in rat during neonatal life: Influence on the glucuronidation rates of various substrates

The Vmax of the membrane bound UDP-glucuronosyltransferase (UDP-GT) towards group 1 substrates (4-nitrophenol, 2-naphthol) was particularly higher in young rats than in adults. On the contrary, activity towards group 2 substrates such as borneol or testosterone was not detectable in fetus liver. The developmental pattern of UDP-GT was related to changes in lipid composition of microsomes, namely in the content in lysophosphatidylcholine which rose at birth. The phospholipid-cholesterol molar ratio also increased 2 fold from the 16th day of fetal life to the 4th day after birth. Measurement of the steady state anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) as well as determination of the order parameter S and the rotation cone angle of the fluorescent probe strongly suggested an increase in membrane fluidity in rat liver microsomes during ontogenesis.

Kinetics of partly diffusion controlled reactions. VII — Pyrene excimer formation in erythrocyte membranes

Abstract Intermolecular excimer forming systems such as pyrene have been proposed as probes for the microviscosity measurement of model and biological membranes. However, our experimental study of pyrene excimer formation in erythrocyte membranes and the theoretical calculations show that it is quite difficult to get the diffusion coefficient of pyrene in membranes and therefore the microviscosity of its environment.

Kinetics of partly diffusion controlled reactions

We propose a simple kinetic model in which two types of processes between two reactants occur: diffusional processes for distances greater than σ′, the “reaction” distance, and static quenching for distances smaller than σ′. This model might be useful in the studies of diffusion in membrane systems.AbstractБыла предложена простая кинетическая модель, заключающая в себе два типа процессов, протекающих между двумя реагентми: диффузионные процессы для расстояний, превышающих расстояние “реакции” σ′, и статическое гашение для расстояний, меньших σ′. Эта модель может быть использована при исслеловании диффузии в мембранных системах.

On diffusion in organized assemblies and in biological membranes.

The following paper is a brief presentation of problems related to the concepts of diffusion coefficient D and so-called viscosity eta used to characterize the cohesion of biological membranes. The first approach to this problem is a recall of the definition of D and eta in liquids. It appears that the models developed with exogenous probes to account for the diffusion-viscosity relationship are not verified in membranes. The existence of complex diffusional mechanisms, the influence of the size of the probe are presented. The results of a model calculation suggest that there is no direct correlation other than great simplifications, between the diffusion coefficient and viscosity. The calculations are then extended to actual biological assemblies and the influence of proteins on the motion of the probe considered. The limitations of the methods involving exogenous probes for determining the cohesion of biological membranes are discussed.

Kinetics of partly diffusion controlled reactions, IX. Cage effect in the photolysis of benzyl chloride in the liquid phase. First results

The cage recombination of free benzyl and chloride radicals after their formation via photolysis of benzyl chloride is interpreted using a model for partly diffusion controlled reactions. A new kinetic model is presented which takes into account the noncontact of the interacting species at the moment of their formation. There is good agreement between the results observed and the model presented.AbstractРекомбинация в клетке свободных бензильного и хлоридното радикалов, после образования их фотолизом бензил хлорида, была интерпретирована на основе модели реакции, частично контролируемой диффузией. Предлагается новая кинетическая модель, которая принимает во внимание отсутствие контакта между реагирующими частицами при их образовании. Было получено хорошее согласие между наблюдаемыми результатами и приводимой моделью.

Membrane labelling by fluorescent probes: incorporation of TMA-DPH in erythrocyte membranes.

A study of the labelling of isolated resealed erythrocyte membranes by TMA-DPH has been carried out. A quantitative study shows that saturation appears to take place when increasing the relative quantity of probe bulk concentration to membrane concentration; this is readily interpreted by a simple incorporation model with a limited number of sites in the membrane. A qualitative study shows that an increase in the labelling leads to an evolution of the probe fluorescence properties; the existence of different types of sites is involved in the interpretation but the system is too complex to allow it to be represented by a simple model. As a consequence of this study, care has to be taken in labelling biological material so as to avoid excessive probe incorporation.

Erythrocyte membranes alteration in a shear stress measured by fluorescence anisotropy

An experimental setup has been designed to allow fluorescence anisotropy measurements on labeled cell membranes under shear stress. An important change is observed when increasing the shear stress and varying the experimental parameters indicates that a decrease in membrane cohesion leads to a subsequent increase in the membrane alteration under shear stress. A model has been developed that shows, in agreement with experiment, that the effect observed is mainly the result of the alteration of the membrane, elongation, and orientation with respect to the fluid flow, which can be estimated.

Kinetics of partly diffusion-controlled reactions. Part 23.—The case of ionic reactions

Diffusion-controlled reactions have been investigated for many years, and have been modelled for the case where Brownian particles move in a medium of hard spheres. We here present models based upon liquid physics concepts and known interaction potentials for cases where the transport phenomenon is due to a random walk hampered by the electrostatic potential and screen effect.Our model assumes the diffusing particles to be spheres, of identical radius and with a central charge. A ‘continuum’ model is developed which combines the radial configurational function of hard spheres and the Debye–Huckel interaction potential. The solution of a classical integro-differential system then allows us to compute the apparent rate constant of the reaction.This model is applied to the interpretation of the fluorescence quenching of fluorescein ions by iodide ions, a well known system for which several unsatisfactory models (especially static or non-diffusional quenching) have been proposed. The use of the present model, where coupling between diffusion and reactivity is taken into account, makes it possible to find a satisfactory agreement with experiment. However, theoretical refinements are yet required for a better understanding of the phenomenon occurring at low relative permittivity.