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In the presence of ciprofloxacin (CPFX), free radical adduct formation was demonstrated in rat cerebral microsomes using a spin trap α-(4-pyridyl-1-oxide)-N-tert-butyl-nitrone by electron spin reso...

International audience; Three heterotetranuclear complexes, [{RuII(bpy)2(Ln)}3MnII]8+ (bpy = 2,2′-bipyridine, n = 2, 4, 6), in which a MnII-tris-bipyridine-like centre is covalently linked to three RuII–tris-bipyridine-like moieties using bridging bis-bipyridine Ln ligands, have been synthesised and characterised. The electrochemical, photophysical and photochemical properties of these complexes have been investigated in CH3CN. The cyclic voltammograms of the three complexes exhibit two successive very close one-electron metal-centred oxidation processes in the positive potential region. The first, which is irreversible, corresponds to the MnII/MnIII redox system (Epa≈0.82 V vs Ag/Ag+ 0.01 M in CH3CN–0.1 M Bu4NClO4), whereas the second which is, reversible, is associated with the RuII/RuIII redox couple (E1/2≈0.91 V). In the negative potential region, three successive reversible four electron systems are observed, corresponding to ligand-based reduction processes. The three stable dimeric oxidized forms of the complexes, [Mn2III,IVO2{RuII(bpy)2(Ln)}4]11+, [Mn2IV,IVO2{RuII(bpy)2(Ln)}4]12+ and [Mn2IV,IVO2{RuIII(bpy)2(Ln)}4]16+ are obtained in fairly good yields by sequential electrolyses after consumption of respectively 1.5, 0.5 and 3 electrons per molecule of initial tetranuclear complexes. The formation of the di-µ-oxo binuclear complexes are the result of the instability of the {[RuII(bpy)2(Ln)]3MnIII}9+ species, which react with residual water, via a disproportionation reaction and the release of one ligand, [RuII(bpy)2(Ln)]2+. A quantitative yield can be obtained for these reactions if the electrochemical oxidations are performed in the presence of an added external base like 2,6-dimethylpyridine. Photophysical properties of these compounds have been investigated showing that the luminescence of the RuII-tris-bipyridine-like moieties is little affected by the presence of manganese within the tetranuclear complexes. A slight quenching of the excited states of the ruthenium moieties, which occurs by an intramolecular process, has been observed. Measurements made at low concentration (–5 M) indicate that some decoordination of Mn2+ arises in 1a–c. These measurements allow the calculation of the association constants for these complexes. Finally, photoinduced oxidation of the tetranuclear complexes has been performed by continuous photolysis experiments in the presence of a large excess of a diazonium salt, acting as a sacrificial oxidant. The three successive oxidation processes, MnII→MnIIIMnIV, MnIIIMnIV→MnIVMnIV and RuII→RuIII are thus obtained, the addition of 2,6-dimethylpyridine in the medium giving an essentially quantitative yield for the two first photo-induced oxidation steps as found for electrochemical oxidation.

International audience; The effects of growth hormone on the uridine diphosphate glucuronosyltransferase functional state, biophysical membrane parameters (order parameters and rotational correlation frequency) and the composition in phospholipids were studied in male rat hepatic microsomes. Sham-operated and hypophysectomized animals were injected with two different dosages of growth hormone, mimicking either the male or female growth hormone secretion pattern. Half the animals received thyroxine and cortisol in concentrations chosen to compensate for the lack of thyroid hormones and glucocorticoids in hypophysectomized rats. Growth hormone treatment resulted in a decrease in the latency (that gives a quantification of uridine diphosphate glucuronosyltransferase functional state) of the glucuronidation activities towards various substrates (testosterone, androsterone, bilirubin and 4-nitrophenol). This decrease with growth hormone treatment was particularly evident in hypophysectomized animals that had received cortisol and thyroxine supplementation treatment. These modifications were strongly correlated with modifications in the microsomal membrane lysophospholipid content and to a lower extent with microsomal membrane fatty acid composition. The cytosolic phospholipase A(2)-dependent increase in the lysophospholipid content in the endoplasmic reticulum is probably a major determinant in the regulation of the functional state of glucuronoyltransferases in response to high dosage growth hormone treatment.

Ciprofloxacin (CPFX) is a widely used fluoroquinolone antibiotic with a broad spectrum of activity. However, clinical experience has shown a possible incidence of undesirable adverse effects including gastrointestinal, skin, hepatic, and central nervous system (CNS) functions, and phototoxicity. Several examples in the literature data indicate that free radical formation might play a role in the mechanism of some of these adverse effects, including phototoxicity and cartilage defects. The purpose of this study is to investigate free radical formation during the metabolism of CPFX in hepatic microsomes using electron spin resonance (ESR) spectroscopy and spin trapping technique. We then investigate the effects of a cytochrome P450 inhibitor, SKF 525A, Trolox, and ZnCl2 on CPFX-induced free radical production. Our results show that CPFX induces free radical production in a dose- and time-dependent manner. The generation of 4-POBN/radical adduct is dependent on the presence of NADPH, CPFX, and active microsomes. Furthermore, free radical production is completely inhibited by SKF 525A, Trolox, or ZnCl2.

Erythrocyte membrane characteristics were compared in 15 normal women and 15 women with anorexia nervosa; the patients were studied at hospital admission and again after 1 month of refeeding. At admission, physical properties of erythrocyte membranes, studied with electron spin resonance spectrometry, significantly differed between the anorexic patients and the normal volunteers. Fluidity from the hydrophobic part of the erythrocyte membrane, estimated by the correlation frequency, was decreased in the patients. After 1 month of refeeding, fluidity increased. One of the possible mechanisms of the variation of membrane fluidity could be the effect of cholesterol on membrane structure. Increased cholesterol levels in anorexic subjects could reduce fluidity. These alterations in membrane fluidity could explain some of the neurobiological abnormalities observed in anorexia nervosa.

The fluidity of isolated chicken ventricular plasma membranes was investigated by ESR spectroscopy, using three doxylstearic spin labels (5-SASL, 10-SASL and 16-SASL), and by fluorescence anisotropy measurements, using DPH probe. The analysis of 2T1 splitting constant and rotational correlation frequency have shown differences, during in-ovo development, characterized by a rigidification phase with a maximum at 19 days of development, just before hatching, followed by a fluidification phase after birth. These phenomena were also improved by DPH fluorescence and dynamical analysis of the hydrophobic 16-SASL probe.

A novel analytical method has been developed to detect and quantify VX (O-ethyl S-(2(diisopropylamino) ethyl) (methylphosphonothioate)) in plasma using an LC–MS/MS technique. VX detection and quantification in plasma following percutaneous exposure represent a formidable challenge and it is an important part of the ongoing struggle against chemical warfare agents. Liquid–liquid extraction of VX from plasma was performed and it generated a recovery rate of approximately 65% followed by an LC–MS/MS analysis in a 100% organic phase. An Allure biphenyl column (Restek) was tested with detection limit at 0.5 pg/mL (5 μL injected). Initial application was focused on human skin grafted on nude mice as an experimental model with proper adjustments done for very small quantities of plasma.

Degradation of organophosphorus compounds was achieved in the presence of purified fungal laccase from Trametes versicolor and a small molecular weight redox mediator (ABTS). This laccase-mediator system (LMS) catalyzed degradation of VX, PhX and VR while had no apparent effect on CVX, ecothiophate or demeton. Inhibition of ABTS oxidation was shown with VX, PhX, VR and CVX. Results with CVX suggest either no degradation subsequent to interaction with the laccase active site or the formation of a new toxic compound. PhX degradation was also monitored by mass spectroscopy, a method that allowed us to identify certain intermediates formed during OP degradation. Altogether, results underline the importance of the OP nitrogen atom at beta-position and of its substituents, even though the intimate mechanism of laccase-catalyzed degradation is not yet known.

International audience; Three heterotetranuclear complexes [{RuII(bpy)2(LLn)}3FeII]8+ {bpy = 2,2'-bipyridine, n = 2, 4, 6; denoted [{Ru(LLn)}3Fe]8+}, in which one iron centre is complexed by three RuII-tris-bipyridine-like moieties containing covalently bridging bis-bipyridine LLn ligands, have been synthesised and characterised. The stability and the electrochemical, photophysical and photochemical properties of these complexes have been investigated in CH3CN. The cyclic voltammograms of all complexes exhibit two successive reversible oxidation processes in the positive region, corresponding to the FeII/FeIII and RuII/RuIII redox couples. These systems are clearly separated (ΔE1/2 300 mV), which indicates the absence of an electronic connection between the two subunits. In the negative region, three successive reversible four-electron systems are observed, corresponding to the ligand-based reduction processes. The two oxidized forms of the complexes [{RuII(LLn)}3FeIII]9+ and [{RuIII(LLn)}3FeIII]12+, which are obtained by two successive exhaustive electrolyses, are very stable. The [{Ru(LLn)}3Fe]8+ complexes are luminescent, which shows that the covalent linkage between the RuII-tris-bipyridine and FeII-tris-bipyridine subunits leads to an only partial quenching of the RuII* excited states by energy transfer to the FeII centre. The luminescence lifetime and quantum yield are found to be independent of the complexes concentration, thus indicating that the energy-transfer process is only due to an intramolecular electron-exchange mechanism. Quantitative photoinduced oxidation of the tetranuclear complexes has been performed by continuous photolysis experiments in the presence of a large excess of a diazonium salt, which plays the role of a sacrificial oxidant. Two successive oxidation processes (FeII FeIII and RuII RuIII) are observed.

In a previous study, the production of acetaldehyde and free radicals derived from ethanol was characterized in astrocytes in primary culture. In the present study, the effects of chronic exposure on the production of both compounds as well as on the main antioxidant system were compared with those of an acute exposure. This was done to better understand the different ways the brain reacts to these modes of exposure. Under these conditions, both a time-dependent increase in the accumulation of acetaldehyde and a decreased formation of the alpha-hydroxyethyl radical were shown. This was associated with increased activities of catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPX) and with decreased glutathione (GSH) content. These effects, which counteract reactive oxygen species (ROS) formation by stimulating the main enzymes of the antioxidant system, were also associated with the reduced amount of radicals derived from ethanol. This could be a beneficial effect, but this was counter-balanced by the increased rate of acetaldehyde accumulation, whose high toxicity is well known. All these effects underline the crucial role played by catalase which, on one hand converts hydrogen peroxide to water and, on the other hand, ethanol to acetaldehyde.

Formation of the alpha-hydroxyethyl radical (CH3 degree CHOH) has already been extensively demonstrated after ethanol metabolism in the liver. Despite favourable conditions, this formation in the brain has remained speculative since there is no direct experimental evidence in intact brain cells. In this preliminary study, the formation of such a radical was observed after exposure of astrocytes and astrocytic C6 glioma cells to ethanol. These cells were studied because astrocyte integrity is essential for normal growth and functioning of neurons. The free radicals were detected by EPR spectroscopy using the spin trapping technique. Astrocytes appeared to be more sensitive than the C6 cells to free radical formation as the intensity of the signal was higher after exposure of the astrocytes and increased with time, a fact not observed after exposure of the C6 cells.

The fluidity of isolated plasma and disc membranes from bovine rod outer segments has been compared by electron-spin resonance spectroscopy using the stearic acid spin labels 5-SASL and 16-SASL. This comparison is of interest, given the fact that the disc membranes arise by invagination from the plasma membrane. Analysis of the order parameter in the hydrophilic part of the membrane, and of rotational correlation time in the hydrophobic part, showed a difference of fluidity between these two types of membranes, with a higher fluidity in the disc membranes in both their hydrophilic and hydrophobic regions. For example, at a temperature of 37 degrees C, plasma and disc membranes exhibited values of 0.64 and 0.61 for the order parameter (S), and 9.1 and 14.5 s-1 for the rotational correlation frequency (v), respectively. These findings are discussed in relation to previously described differences in the biochemical compositions of the membranes.

The effects of dietary polyunsaturated fatty acid (PUFA) deficiency on intestinal brush border membrane (BBM) fluidity, lipid composition and 5'-nucleotidase activity were examined in piglets. Cholesterol/phospholipid and sphingomyelin (SM)/phosphatidylcholine (PC) ratios were unaffected. However, fluidity was decreased in the external regions and also tended to decrease in the core of the PUFA-deficient pig membrane lipid bilayer. Therefore, the change in the membrane physical properties seemed to be due to the large diet-induced alteration in the phospholipid (PL) fatty acid composition and to the concomitant decrease in PC and increase in phosphatidylserine levels. In the membrane total PL, the arachidonic acid level was slightly lowered, while linoleic, eicosapentaenoic and docosahexaenoic acid levels markedly decreased. PC was mainly concerned by the altered distribution of unsaturated fatty acids, but not SM. However, a significant decrease in (n − 6)/(n − 3) ratio occurred in the latter. These structural changes were associated with a higher 5'-nucleotidase activity in the intestinal BBM of PUFA-deficient as compared to control piglets.

ESR spectrometry with 5-, 7-, 10-, and 12-doxylstearate probes and a combined index considering separately the double-bond numbers of essential and nonessential fatty acids were used to investigate the structural role of the double bonds of polyunsaturated fatty esters in membrane phosphoglycerides. Purified brush border membrane vesicles were prepared from the jejunum of piglets receiving either high (HLA) or low (LLA) dietary levels of linoleic acid (18:2 n-6). In the LLA as compared to the HLA group, there were no significant modifications of (a) the relative contents of cholesterol, phospholipid, and protein and of (b) the phosphoglyceride class distribution, contrasting with very large changes in the fatty acid compositions of each phosphoglyceride. These changes were characterized by an increase in nonessential monoene and triene (18:1 n-9 and 20:3 n-9) and a decrease in essential diene (18:2 n-6) in LLA- as compared to HLA-fed piglets. The essential tetraene 20:4 n-6 remained rather constant despite an overall nonsignificant increase in the LLA group. The total double-bond number (TDBn) was not significantly affected, contrasting with the variations in the double-bond numbers of essential and nonessential fatty acids (DBn(EFA) and DBn(nonEFA), respectively). The combined DBn(EFA)/DBn(nonEFA) index was 1.7-3.3 times lower in LLA than in HLA membrane phospholipids. It was concluded that the diet was able to affect the double-bond distribution in the upper and inner half-parts of the membrane leaflet without changing the total number of double bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

One of the most powerful tools for the study of the molecular dynamics and molecular organization of biological membranes is the spin label method which uses stable free radicals (nitroxides) studied by electron spin resonance spectroscopy. The spectra are very dependent on the mobility and orientation of these probes in a magnetic field. They give information on the tumbling rate of molecules in liquids, the membrane microviscosity, the order parameter of the phospholipid fatty acid chains, the activation energy of their rotation and their flexibility. The method has interesting applications in the rheological domain; for example it enables measurement of the internal viscosity of erythrocytes and red blood cell orientation in a Poiseulle flow.

— Ultraviolet irradiation of photosensitive molecules like phenothiazine derivatives leads to the formation of short lived free radicals which are able to reduce stable nitroxide free radicals generally used as spin labels. The measurement of the electron spin resonance signal decay of nitroxides offers a tool for studying the photochemical reaction of phenothiazine derivatives in solution at room temperature, in a 10-5 to 10-2M concentration range. Analysis of the reaction mechanism shows that the paramagnetic nitroxide is an efficient quencher of the phenothiazine triplet state; this reaction was used to demonstrate the influence of the solvent, quenching by oxygen and the role of the chemical structure of six phenothiazine derivatives on their photoreactivity in solution.

Spectrin extracted from human red blood cells has been spin labeled in its dimeric and tetrameric forms with five different nitroxide derivatives of increasing chain length between their maleimide binding group and their nitroxide reporter group. Three molecules of spin label are bound per spectrin dimer. Electron spin resonance spectra show the simultaneous presence of strongly and weakly immobilized spin labels. Their relative proportion depends on the label length and is suddenly modified when it reaches 12 A This indicates the presence of cavities of approximately this size in the tertiary structure of spectrin in solution at 0°C. The conformation of spectrin varies greatly with temperature. Reversible changes occur between 0 and 35°C. At higher temperatures, partial denaturation is observed. Divalent cations (Mg 2+ and Ca 2+ ) stabilize spectrin in a more constrained conformation and protect it against thermal denaturation. The same behavior is observed when spin-labeled spectrin is reassociated with spectrin-depleted inside-out erythrocyte vesicles. When fatty acid spin labels are incorporated in the phospholipidic structure of these vesicles, the reassociation of spectrin does not change their electron spin resonance spectra. This result confirms the fact that spectrin interacts predominantly with proteins on erythrocyte membranes.

Biophysical abnormalities of the erythrocyte membrane in muscular dystrophies have been described by numerous authors. This work presents the results we have obtained on 23 subjects suffering from myotonic muscular dystrophy (MyD, Steinert disease) by the spin label method. Two types of fatty acid spin labels were used: 5-nitroxide stearic acid (5NS) and 16-nitroxide stearic acid (16NS) which probe the membranes respectively near their polar heads and in their hydrophobic core. We measured the classical order parameter, the saturation behaviour of the electron paramagnetic resonance signal, and the label apparent rotation correlation time as a function of the temperature, on fresh and in vitro stored red blood cells. With the 5NS label, no differences were found between controls and patients. With the 16NS label, a highly significant variation in the thermic behaviour of the membrane is observed in its hydrophobic and fluid core. This last result may suggest some similarities between the red blood cell membranes of adult MyD's and healthy children.

The membrane effects of ticlopidine on the erythrocyte membrane were explored by the spin label method at the proteic and phospholipidic levels. This spectroscopic study was completed by polyacrylamide gel electrophoresis of proteins, measurement of the protection against haemolysis and observation of the erythrocyte shape changes induced by the drug. Two types of effects have been observed. At concentrations higher than 5 × 10 −4 M, ticlopidine is a weak denaturating agent of the membrane proteins. At concentrations of pharmacological interest, the main effect of the drug is a protection against hypotonic haemolysis, and an increase in the fluidity of the membrane phospholipidic core. This last result could explain in part the interesting pharmacological effect of ticlopidine on various circulatory troubles.

The effect of dietary essential fatty acid (EFA) deficiency on the dynamic molecular organization of pig intestinal brush-border membrane (BBM) was studied using purified BBM vesicles. A 6 week dietary treatment of weaning piglets induced a typical EFA-deficient pattern in the lipid composition of both plasma and epithelial membranes. In pigs fed on the EFA-deficient diet, the plasma 20:3(n−9)/20:4(n−6) ratio progressively increased and reached a stable value after 3 weeks of experiment, whereas it remained low (less than 0.2) in controls. In the intestinal BBM, the cholesterol/protein, phospholipid/protein and consequently the cholesterol/phospholipid ratios, as well as the phospholipid class distribution, were unchanged. In particular, the sphingomyelin/phosphatidylcholine (SM/PC) molar ratio was not affected. However, the fatty acid composition of phospholipid main classes was markedly modified, leading to decreased lipid fluidity and to a large change in membrane protein behaviour with EFA deficiency. These findings could be interpreted in terms of reduced lipid-protein interactions. Moreover, the increasing gradient of fluidity which took place within the lipidic matrix from its surface was modified by the dietary treatment, as fluidity was lowered by EFA deficiency at different depths of the layer.

The erythrocyte membranes of mothers and sisters of boys suffering from Duchenne Muscular Dystrophy (DMD) have been studied by spin labelling. Two oxazolidine nitroxide derivatives of stearic acid were used. With the first of them (16 NS) which probes the hydrophobic part of the phospholipids, we measured the fluidity of the membrane as a function of temperature. The second nitroxide derivative (5 NS) probes the membrane near the phospholipid polar heads. The amplitude of the electron spin resonance signal was studied as a function of the spectrometer microwave power in order to determine the paramagnetic label saturation behaviour. No significant difference was observed between the control adult women and the carrier mothers. On the contrary, almost all the normal young premenarchial girls showed simultaneously a break in the fluidity vs. temperature plot of the 16 NS probe and a saturation phenomenon of the 5 NS label signal. In about 50% of the DMD boys' sisters, no break in the temperature plot nor saturation behaviour was observed. This corresponds to the theoretical repartition between normal and carrier girls if one admits that about 30% of the latter do not have any detectable membrane abnormality, as in the case of the creatine kinase (CK) test which shows about 30% of normal levels in carrier women. The study of the erythrocyte membrane in young girls can then be an useful complementary tool to detect DMD carriers.

The fluorescence anisotropy probe perylene and the spin-labels 5-doxylsterate and 16-doxylstearate were used to estimate the order and internal microviscosity of the pigeon erythrocyte membrane upon perturbation by cationic or neutral amphipathic drugs (chlorpromazine, methochlorpromazine, tetracaine, and octanol) and an anionic drug, octanoic acid. Both methods gave identical results. The fluidity changes were found to strictly correlate with those of adenylate cyclase activity in the presence of GTP when perturbed by the drugs [Salesse, R., & Garnier, J. (1979) Biochim. Biophys. Acta 554, 102-113]. The cationic or neutral drugs, in an intermediate range of concentration, decreased the degree of organization and the internal microviscosity of the lipids together with the activity of the adenylate cyclase. At a higher concentration they reincreased them up to or higher than their initial level before the final destruction of the membrane structure and functions. This concentration effect was time dependent with tetracaine. The quaternary amine methochlorpromazine acted as chlorpromazine only on open ghosts. On intact cells, it inhibited catecholamine receptors at higher concentration and monotonously decreased the order and microviscosity, as the anionic amphipath octanoic acid did. This is taken as evidence that the inner leaflet of the bilayer is the seat for the observed multiphasic changes of viscosity and the control of adenylate cyclase and catecholamine receptors. This could stem from either a preferential intercalation or a surface effect of the amphipaths in the inner leaflet of the membrane. Since the basal activity of adenylate cyclase was not affected in the presence of drugs, it may be inferred that the enzyme holds its activity but that its stimulation is modulated by the membrane physical state.

We have compared the binding parameters of native wheat germ agglutinin (WGA) and its succinylated form (SWGA) to rat lymphocytes. Scatchard plots were obtained with the fluoresceinated lectins in a concentration range of 10 nM to 0.1 mM. Association and dissociation rate parameters were also measured. The following differences were observed: at low concentration of WGA, binding is positively cooperative with a Hill coefficient of 1.75, whereas binding of SWGA is not. The numbers of high-affinity sites are respectively (2.5 +/- 0.8) X 10(6) and (6.4 +/- 1.3) X 10(5) for WGA and SWGA. Association constants were found to be (4.7 +/- 1.7) X 10(6) l mol-1 for WGA and (1.42 +/- 0.36) X 10(7) l mol-1 for SWGA, which is 35 times higher than for native WGA. Neuraminidase treatment decreases the Hill coefficient as well as the number of sites involved in the cooperative binding of native WGA. Equilibrium data were obtained at three temperatures to determine the thermodynamic parameters (delta H degree and delta S degree). These results are indicative of an oligomerization process dynamically formed at the membrane level before tight binding of the lectin to its receptors could occur.

In the preceding paper, we described the parallel effect of amphipathic drugs on the fluidity and adenylate cyclase activity of pigeon erythrocyte membrane. This parallelism was found when the cyclase activity was assayed in the presence of a guanyl nucleotide and with Mg-ATP as the substrate after a preincubation of the membrane with the drugs [Salesse, R., Garnier, J., Leterrier, F., Daveloose, D., & Viret, J. (1982) Biochemistry (preceding paper in this tissue)]. However, when the regulatory protein (N) and the catalytic unit (E) were precoupled by GppNHp or fluoride before the action of the drugs, the cyclic AMP production was never inhibited. Thus, the drug-induced fluidization appeared to interfere with the efficiency of the activating coupling between N and E. Chlorpromazine even enhanced the cyclase activity: if the catecholamine receptor (R) repressed the cyclase activity in the absence of hormone [Rodbell, M. (1980) Nature (London) 284, 17-22], the loss of R molecules with chlorpromazine would prevent this inhibition and lead to hyperactivity of the enzyme. On the other hand, the comparison between two states of the adenylate cyclase system, (1) N and E reversibly precoupled in the presence of GTP and (2) R, N, and E precoupled in the presence of GTP plus isoproterenol, showed no difference between the activity curves at various drug concentrations: this may be interpreted as a permanent coupling of R and N. The main control exerted by fluidity on the activity of the adenylate cyclase system would thus be at the level of the activating coupling between the N subunit and the catalytic unit in pigeon erythrocyte membrane.

The effect of the antimitotic drug vinblastine sulfate has been studied on fully hydrated dipalmitoylphosphatidylcholine (DPPC) liposomes in the temperature range 0 degrees to 60 degrees using differential scanning calorimetry and electron spin resonance spectroscopy with two fatty acid spin labels. In the gel phase, vinblastine interacts essentially with the DPPC polar heads and induces an important disorganization of the phospholipidic bilayer. The co-operativity of the main thermal transition is decreased. In the crystal-liquid phase, the drug penetrates inside the artificial membrane and induces the formation of domains which increased thermal stability. These effects are opposite to those observed with the drug isaxonine which is used to reduce the axonal degenerating effects due to vinblastine.

Using quantitative fluorimetry with fluoresceinated wheat germ agglutinin, we have been able to investigate in vivo gamma radiation-induced damage at the outer membrane level of rat splenic lymphocytes, namely damage to the glucosidic moieties of membrane glycoproteins and glycolipids. This paper demonstrates that below an irradiation level of 1 gray (Gy), removal of sialic acid is the major feature leading to new exposed specific binding sites for wheat germ agglutinin, since this lectin is specific for sialic acid and N-acetyl-D-glucosamine. Our studies also suggest that above 1 Gy of irradiation more internal damage occurs, since we observed a striking decrease in wheat germ agglutinin binding sites.

One of the important questions in biomembranes now is: Do the essential fatty acids (polyunsaturated fatty acids of the n-6 and n-3 series) play an original structural role in the arrangement of the lipid matrix capable, in particular, of triggering modifications of intrinsic protein activities? Preliminary results from our laboratories are presented in rat and piglet fed standard or essential fatty acid-deficient diets. The relative amounts of 18:2 (n-6) and 20:4 (n-6) in total fatty acids of hepatic microsome or enterocyte brush border membrane phospholipids are closely dependent on the type of diet (a globally decreasing effect with deficiency), whereas no differences were observed with relative amounts of cholesterol, phospholipids, and proteins. This effect of deficiency on membrane fatty acids has to be compared to the decreasing specific activities of microsome NADPH-cytochrome c reductase or aniline hydroxylase (studied in rat), to the increasing order of the structure of both membrane microsome and brush border lipid matrix (studied in both rat and piglet), and to the increasing mobility (or accessibility) of the membrane-protein surface-bonded spin-label (studied in the piglet brush border membrane), suggesting a probably defective protein-lipid fit in the case of deficiency. These results could favor conformational change in the whole membrane structure (i.e. proteins and lipids). The specificity of these effects remains to be assessed.

The structure of ajoene, a molecule extracted from garlic, has been studied by 1H-NMR and its interaction with model membranes by 1H-, 2H-, 31-P-NMR and ESR experiments. This study clearly shows that the ajoene molecule is located deep in the layer and is close to the interlayer medium. Moreover while NMR experiments show that the membrane structure is only slightly affected by the presence of ajoene, ESR experiments reveal significant modifications in phospholipid dynamics. This interaction, observed before with the phenothiazine derivative, promazine, results in an increase of the membrane fluidity in its hydrophobic part and could be related to clinical properties of ajoene.

A new spin-label method for the measurement of the internal microviscosity of erythrocyte is presented. The spin label used is 2,2',5,5'-tetramethyl-3-maleimidopyrrolidinyl-N-oxyl (MAL-5) which penetrates inside the red blood cell and binds covalently on cytoplasmic glutathione. After washing off the external label, 98% of the electron paramagnetic signal is due to the labelled glutathione. This signal allows one to measure the rotational correlation time of the label. A calibration curve established with spin-labelled glutathione in sucrose solutions of increasing viscosity is used to convert the measured rotation times into viscosity units. This method avoids the use of unphysiological salts like potassium ferricyanide, and permits the study of red blood cells in various suspension media. In normal human subjects, the mean value of microviscosity is 4.45 +/- 0.16 mPa . s at 20 degrees C in isotonic saline (25 subjects) and 6 +/- 0.25 mPa . s in plasma. The variations of microviscosity as a function of the osmolarity of the medium are explained according to a theoretical model taking into account the variations of the red blood cell volume and the viscometric properties of haemoglobin.

Red blood cells and freshly prepared erythrocyte membranes of 15 patients with Duchenne muscular dystrophy (DMD) as well as age-matched controls were studied by the spin label method. No significant modifications appeared for spin-labelled proteins of ghost membranes. With the two fatty acid spin labels, 5-nitroxide stearate and 16-nitroxide stearate, we have confirmed previous results of Sato et al. concerning the thermal behaviour of the erythrocyte membranes, i.e. no change near the polar part probed by 5-nitroxide stearate and a linearization of the fluidity versus temperature variation around 12 degrees C, as explored by 16-nitroxide stearate. Furthermore we studied in the whole erythrocyte the amplitude of the 5-nitroxide stearate electron spin resonance signal as a function of the microwave power. This saturation effect was observed in 12 out of 15 controls and only in 1 out of 13 DMD cases studied. In erythrocyte membranes labelled with 16-nitroxide stearate the penetration of the label inside membranes was statistically different between DMD and controls. These new findings furnish further arguments in favour of a structural alteration of the phospholipid organization of erythrocyte membranes in DMD. Associated together, these different sets of tests obtained by spin labelling permit good statistical discrimination between DMD and normal subjects.