In vivo and in vitro influence of etretinate on erythrocyte membrane fluidity.

The molecular mechanisms underlying the action of synthetic retinoids have been studied intensively, but they are not fully understood yet. It is well known that retinoids exert their effects on gene expression via the retinoic acid receptor. Some observations suggest that the main aromatic retinoid etretinate (Tigason) exerts its therapeutic effect in psoriasis also through an action on the cell membrane. In this paper, we present the results of previously unreleased experiments (when Tigason was still in use) concerning the in vivo and in vitro influence of etretinate on erythrocyte membrane fluidity in psoriatic patients. Erythrocytes from healthy subjects and topically treated psoriatics were chosen as control groups. Membrane fluidity was measured by the electron paramagnetic resonance (EPR) spin-labelling technique. Erythrocytes from psoriatic patients had lower membrane fluidity, a lower antioxidant activity and a greater susceptibility to peroxidation than those from healthy subjects. After treatment with etretinate, a significant increase in erythrocyte membrane fluidity and in antioxidant activity as well as a decrease in lipid peroxidation were observed in erythrocytes from patients. Local therapy of psoriatic lesions had no influence on the improvement in membrane fluidity and antioxidant activity of erythrocytes. Incubation of erythrocytes from healthy controls and topically treated psoriatics with etretinate in vitro confirmed its fluidizing effect on erythrocyte membranes. These data may indicate that two mechanisms lead to an increase in erythrocyte membrane fluidity in psoriatic patients treated with Tigason: the first one, indirect, by improvement of the antioxidant defence system and cell protection against lipid peroxidation, and the second one, by a direct fluidizing effect of etretinate on the erythrocyte membrane.

Erythrocyte membrane fluidity changes in psoriasis: an EPR study.

The aim of the study was to find the cause of membrane fluidity decrease in psoriasis, observed by other authors, in different types of cells and its clinical consequences. To this end, we have examined the influence of different clinical and biochemical factors on erythrocyte membrane fluidity changes in psoriatic patients. Membrane fluidity was studied by the electron paramagnetic resonance spin-labeling method. The data revealed that the decrease of membrane fluidity corresponded with exacerbation of skin lesions. The results clearly indicate that the increased lipid peroxidation may be the essential mechanism of membrane fluidity decrease in psoriasis.

Relaxation of water protons in highly concentrated aqueous protein systems studied by 1H NMR spectroscopy.

Concentrated Aqueous Protein Systems, Proton Relaxation Times, Slow Chemical Exchange In this paper we present proton spin-lattice (T1) and spin-spin (T2) relaxation times measured vs. concentration, temperature, pulse interval (tauCPMG) as well as 1H NMR spectral measurements in a wide range of concentrations of bovine serum albumin (BSA) solutions. The anomalous relaxation behaviour of the water protons, similar to that observed in mammalian lenses, was found in the two most concentrated solutions (44% and 46%). The functional dependence of the spin-spin relaxation time vs. tauCPMG pulse interval and the values of the motional activation parameters obtained from the temperature dependencies of spin-lattice relaxation times suggest that the water molecule mobility is reduced in these systems. The slow exchange process on the T2 time scale is proposed to explain the obtained data. The proton spectral measurements support the hypothesis of a slow exchange mechanism in the highest concentrated solutions. From the analysis of the shape of the proton spectra the mean exchange times between bound and bulk water proton groups (tauex) have been estimated for the range of the highest concentrations (30%-46%). The obtained values are of the order of milliseconds assuring that the slow exchange condition is fulfilled in the most concentrated samples.

In vitro effects of ozone on human erythrocyte membranes: an EPR study.

The effects of ozone at different concentrations (10, 30, 45 g/m3) on fluidity and thermotropic properties of erythrocyte membranes were investigated by EPR using two spin probes: 5-doxylstearic acid (5-DSA) and 16-doxylstearic acid (16-DSA). The effect of ozone on the erythrocyte membrane fluidity was a dose-dependent process. The ozone at concentration of 10 g/m3 caused rigidization of the membrane while at concentration of 45 g/m3 increased fluidity both on the surface and in the deeper hydrocarbon region of the membrane. Temperature transitions close to the polar heads region (monitored by 5-DSA) were not sensitive to an increase in ozone concentration. In the case of 16-DSA, low temperature thermotropic transition (around 20 degrees C) gradually decreased with the increase of ozone concentration. High temperature transition (around 40 degrees C) significantly differed at the ozone concentration of 10 g/m3 and 45 g/m3, being higher and lower, respectively, as compared to untreated cells. For the ozone concentration of 45 g/m3 the disappearance of the low temperature break and the appearance of two breaks at 37 degrees C and 16 degrees C were observed.

Evidence of surface diffusion of water molecules on proteins of rabbit lens by 1H NMR relaxation measurements.

In this work, we propose a relaxation model for the interpretation of NMR proton spinlattice and spin-spin relaxation times of mammalian lenses. The framework for this model is based on nuclear magnetic spin-lattice relaxation measurements as a function of temperature at different Larmor frequencies for whole rabbit lenses and fragments of the lens. According to this model, two different dynamic processes of the water molecules determine the relaxation behaviour, namely rotational diffusion and translational surface diffusion. These dynamic processes in conjunction with a two site exchange model give a good explanation of all the measured relaxation data. From the experimental data, we were able to obtain the activation parameters for rotational and translational diffusion of bound lens water. Correlation times of 2.1 x 10(-11) sec and 2.5 x 10(-9) sec and activation energies of 20.5 kJ/mol and 22.5 kJ/mol respectively were found at 308K. At low Larmor frequencies (< or = 100 MHz) the longitudinal relaxation is mainly determined by translational surface diffusion of bound water with a mean square displacement of 1.5 nm, whereas at higher frequencies (> or = 300 MHz), rotational diffusion is the main relaxation mechanism. The spin-spin relaxation is determined by translational diffusion over the whole frequency range and therefore shows only a very small dispersion. By our model it is possible to explain: 1) the strikingly large difference between the T1 value and the T2A and T2B values observed in the lens and 2) the different values of the activation energies measured at different fields for the lens.

1H NMR and calorimetric measurements on rabbit eye lenses.

The dynamic properties of water molecules in the rabbit lens were studied by proton nuclear magnetic resonance line shape analysis, measurements of relaxation times as a function of temperature and calorimetric measurements. The experiments prove, as already suggested by other authors, that there are two types of water in the lens of rabbit eyes, namely bound unfreezable hydration water and bulk freezable water. Line shape analysis and relaxometry showed, that this two types of water exist in two different environments, which may be identified as the nucleus and the cortex of the lens. The line shape analysis showed furthermore that water molecules in the rabbit lens has a common spin lattice relaxation time (T1), but two different transverse relaxation times (T2A and T2B). The tentative model of fast water exchange on the T1 time scale and slow water exchange on the T2 time scale, was used to explain experimental proton relaxation data of the rabbit lens. An estimation for this exchange rate kex by comparing it to the relaxation times is given (T1(-1) << kex << T1(-1)). It has also been shown by a calorimetric measurements, that the lenses can be easily under-cooled to temperatures well below the freezing point of water. The achievable maximum undercooling temperature of the lens is a function of the cooling rate KC, therefore it has to be considered as an experimentally adjustable parameter which is not characteristic for the investigated sample. Thus it must be noted that any previous discussions about the specific value of the temperature of water crystallisation in biological systems need to be carefully reconsidered.

Components of light scattered by eye lens.

Using the angular dependence of intensities of light scattered on sections of bovine lenses, we have determined correlation lengths related to the scattering samples. The correlations were calculated on the basis of random density and orientation fluctuation theory. The lenses were classified by means of an instrument for the measurement of transmission and the unscattered component of light. The correlation lengths are compared with dimensions of aggregates.

IR spectra of lens crystallins.

The IR technique has been applied to investigate secondary structure of the crystallins from the normal bovine eye. Crystallins have been isolated by column chromatography. IR spectra were recorded for the solid phase of proteins. From these spectra, especially amide I, amide II and amide V bands, the presence of alpha-helix, beta-sheet, beta-chain and unordered structures is stated. It was elucidated that alpha-crystallins are present mainly in a beta-sheet conformation but they also contain a considerable quantity of alpha-helix and a slight quantity of unordered and beta-chain forms. In beta H-crystallins, alpha-helix and, in a lesser percentage, beta-structures predominante. beta-sheet, alpha-helix and a low content of beta-chain forms are present in beta L-crystallins. In gamma-crystallins all forms secondary structure have been found, with predominance of beta-sheet and alpha-helix forms. A satisfactory agreement has been noticed between the forms of secondary structures in crystallins investigated by the IR technique and the results obtained by means of other methods. In conclusion IR spectroscopy has been suggested to be applied to observe crystallin structure during formation and development of a cataract.

Studies of aqueous humor proteins in rabbits after posterior chamber lens implantation.

In 13 rabbits, 1 month to 1 year after posterior chamber lens implantation/polymethylmetacrylate/, the level of aqueous humor proteins was evaluated and the proteins separation in polyacylamide gel was performed. The studies were also carried out in unoperated eyes of the same animals and control group was composed of the eyes before surgery. It was found that in pseudophakic eyes an increased level of proteins remained during the whole year/the highest one month after surgery, slowly decreasing afterwards/, with the appearance of additional fractions. The moderate increase of the proteins concentration was also observed in unoperated eyes. The increase of aqueous humor proteins in pseudophakic eyes indicates that the presence of polymethyl metacrylate is not completely indifferent to the eyeball in spite of the suggestions derived from the clinical observations.

Measurements of proton relaxation time T2 on cattle eyes lenses.

A simple two phase model does not explain the temperature dependence of T1 relaxation time in lenses as biological systems. Therefore, a distribution of correlation times of water particles has to be assumed by a certain distribution of the water protein binding energy. As a consequence, from the temperature dependence of T1 relaxation time, the activation energy of water molecules in the lens cannot be evaluated directly without the knowledge of the distribution width. This problem can be solved by T2 measurements in lenses. From the slope of T2 as a function of temperature, mean activation energy can be calculated independently on the distribution width. Measurements were performed on lenses originating from 5-7 years old cows, 2-year old bull-calfs and a 12-year old bull in the temperature range -30 to +105 degrees C. It could be demonstrated that about 80% of water behaves as liquid-like water with an activation energy 14 +/- 4 kJ/mol corresponding to the value of free water. The remaining water (about 20%) is bound to the protein with an activation energy of 20 +/- 5 kJ/mol. At 42 degrees C the protein denaturation process starts in the eye lens and will be completed by 70 degrees C, yielding a protein bound-water complex.