Spin labeling of human spectrin. Effects of temperature, divalent cations and reassociation with erythrocyte membrane.

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 degrees C. The conformation of spectrin varies greatly with temperature. Reversible changes occur between 0 and 35 degrees C. At higher temperatures, partial denaturation is observed. Divalent cations (Mg2+ and Ca2+) 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.

Modification of fluidity and lipid-protein relationships in pig intestinal brush-border membrane by dietary essential fatty acid deficiency.

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.

A new spin label method for the measurement of erythrocyte internal microviscosity.

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.

Effect of in vivo gamma-irradiation on the binding of wheat germ agglutinin on lymphocyte plasma membranes.

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.

Simultaneous changes in lipid composition, fluidity and enzyme activity in piglet intestinal brush border membrane as affected by dietary polyunsaturated fatty acid deficiency.

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.

Microsomal metabolism of ciprofloxacin generates free radicals.

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.

Nutrition and biomembranes: additional information concerning the incidence of dietary polyunsaturated fatty acids on membrane organization and biological activity.

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.

Interaction of ticlopidine with the erythrocyte membrane.

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 x 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.

Interaction of vinblastine sulfate with artificial phospholipid membranes. A study by differential scanning calorimetry and spin labeling.

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.

Ajoene, the antiplatelet compound derived from garlic, specifically inhibits platelet release reaction by affecting the plasma membrane internal microviscosity.

Ajoene (E,Z-4,5,9-trithiadodeca-1,6,11-triene 9-oxide), a product of the rearrangement of allicin (a major component of raw garlic), has been shown to be a potent inhibitor of platelet aggregation in vitro through inhibition of granule release and fibrinogen binding. Our present study further elaborates on this inhibitory action, through studies of the effect of ajoene on the earliest steps of platelet activation. The transducing mechanism involved in thrombin-induced platelet activation was not modified by the drug as indicated by a normal breakdown of phosphatidylinositol 4,5,bisphosphate and normal production of phosphatidic acid. Likewise, the agonist-induced phosphorylation of myosin light chain (P20) and of the 43 kD protein (P43) were not impaired by ajoene. Under the same conditions, however, ajoene (100 microM) produced a strong inhibition of the thrombin-induced release of dense body and alpha-granule constituents. Electron spin resonance studies of the effect of ajoene on some physico-chemical properties of the platelet plasma membrane (intact platelets), as well as on artificial lipid membranes, indicated that ajoene increased mobility of the fatty acid spin label 16 nitroxide stearate. This suggests the existence of a decreased microviscosity of the most internal region within the lipid bilayer membrane, without affecting the outer hydrophilic moieties of the bilayer. As a whole, these results suggest that the effect of ajoene on the release reaction must be, in part, due to physical modification of the bilayer, which impairs the fusion of the granules and plasma membrane, a prerequisite for exocytosis.

Free radical production after exposure of astrocytes and astrocytic C6 glioma cells to ethanol. Preliminary results.

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.

A spin label study of the erythrocyte membranes in Duchenne muscular dystrophy.

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.

Physical state abnormality of erythrocyte membrane in myotonic dystrophy: a spin label study.

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.

A spin label study of the erythrocyte membrane in mothers and sisters of patients suffering from Duchenne muscular dystrophy.

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.

A spin label study of the membrane effect of various psychoactive drugs in human erythrocytes.

The effect of psychoactive agents with different clinical actions: three sedative neuroleptics (trifluoperazine, alimemazine tartrate, chlorpromazine), an anticholinergic agent (trihexyphenidyl hydrochloride), two tricyclic antidepressants (imipramine, desipramine) and lithium carbonate on the rotational correlation frequency (V+) of the spin label 16NS has been comparatively investigated in whole human erythrocytes. V+ was about 40% increased by the three neuroleptics, the anticholinergic agent and the antidepressant molecules at 0.2 mM. By contrast, lithium did not induce any significant change in V+ at the same concentrations. It can be suggested that the increase in "membrane fluidity", observed with a wide variety of drugs, is a non specific effect, unrelated to the psychotropic action, that can be ascribed to the amphiphilic properties of the tested drugs.

Modifications of erythrocyte membrane fluidity from patients with anorexia nervosa before and after refeeding.

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.

Effects of chronic ethanol exposure on acetaldehyde and free radical production by astrocytes in culture.

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.

[Study by EPR of structural modifications on liposomes induced by two metabolites of modafinil]. / Etude par RPE des modifications structurales induites sur les liposomes par deux métabolites du modafinil.

Awaking properties of modafinil are well known at this time, but its action mode is still hypothetic. Our contribution is limited to the study of the membrane structural modifications carried by this drug and two metabolites. By EPR spectroscopy, we have studied the two metabolites which are distinguished by acid function for the first one and sulfone function for the second one. These two metabolites have no waking properties when they are administrated to animals but one of these two metabolites has the same behaviour than modafinil on membrane action.

Interaction of per 3,6-anhydro-alpha cyclodextrins (alpha 36CD) and lead-alpha 36CD complex with biological systems.

The interactions of per (3,6 anhydro) alpha cyclodextrin (alpha 36CD) and of lead-alpha 36CD complex with biological systems were tested by NMR, ESR and electronic microscopy using erythrocytes and model membranes. It was found that the haemolytic activity of alpha 36CD alone was seven fold lower than that of natural alpha cyclodextrin (evaluated by the concentration inducing 50% haemolysis, DH50 = 35 mM). Conversely, the formation of the complex resulted in an increase of haemolytic properties, with DH50 of 1 mM. The mechanism proposed was an increased membrane diffusion by endocytosis of the complex, leading to higher amounts of intracellular lead.