Electron spin resonance studies of an animal model of human congenital myotonia: Increased erythrocyte membrane fluidity in rats with 20,25-diazacholesterol-induced myotonia

Summary Electron spin resonance experiments have been performed on erythrocyte membranes from rats with myotonia induced by treatment with 20,25-diazacholesterol. The results suggest that erythrocyte membranes in this animal model of human congenital myotonia possess a highly significantly increased surface membrane fluidity compared to that of controls. Alterations in the physical state of membrane proteins were not apparent. These findings, also present in human congenital myotonia [Butterfield, Chesnut, Roses & Appel, 1976,Nature (London)263:159; Butterfield, 1977 (Submitted for publication)], strengthen the concepts that increased membrane fluidity is associated with the presence of myotonia and that congenital myotonia may be a diffuse membrane disease.     

Membrane fluidity and myotonia: Effects of cholesterol and desmosterol on erythrocyte membrane fluidity in rats with 20,25-diazacholesterol-induced myotonia and on phospholipid liposomes

Previous spin-label and electromyographic experiments with rats fed 20,25-diazacholesterol, an inhibitor of the biosynthetic conversion of desmostero[ to cholesterol, demonstrated an increased erythrocyte membrane fluidity and myotonia, a prolonged muscle contraction upon stimulation. The current studies with rats showed normal erythrocyte fluidity in animals fed 20,25-diazacholesterol but maintained on a high-cholesterol diet and no myotonia. Studies of model membrane systems composed of phospholipid vesicles containing desmosterol, cholesterol, or both demonstrated that desmosterol increased membrane lipid fluidity relative to cholesterol, suggesting that in 20,25-diazacholesterol-induced myotonia, in which desmosterot accounts for 85% of the plasma sterol, the increased membrane fluidity previously observed in erythrocytes and sarcolemma m this animal model of human congenital myotonia may be due to desmosterol.     

Electron spin resonance investigations of membrane proteins in erythrocytes in muscle diseases. Duchenne and myotonic muscular dystrophy and congenital myotonia.

Comparison of electron spin resonance spectra of spin labeled erythrocyte membranes from patients with the dystrophic conditions Duchenne and myotonic muscular dystrophy with those of normal controls suggests that alterations in membrane protein conformation and/or organization are present in these disease states. These protein alterations are not apparent in the non-dystrophic disease congenital myotonia. The results suggest a correlation between changes in the physical state of proteins in membranes with the presence of dystrophy. In addition, the present results from erythrocytes lend support for the concept of a generalized membrane defect in these diseases.     

Electron spin resonance investigations of membrane proteins in erythrocytes in muscle diseases. Duchenne and myotonic muscular dystrophy and congenital myotonia

Comparison of electron spin resonance spectra of spin labeled erythrocyte membranes from patients with the dystrophic conditions Duchenne and myotonic muscular dystrophy with those of normal controls suggests that alterations in membrane protein conformation and/or organization are present in these disease states. These protein alterations are not apparent in the nondystrophic disease congenital myotonia. The results suggest a correlation between changes in the physical state of protein in membranes with the presence of dystrophy. In addition, the present results from erythrocytes lend support for the concept of a generalized membrane defect in these diseases.     

Electron paramagnetic resonance and saturation transfer electron paramagnetic resonance studies on erythrocytes from goats with and without heritable myotonia

Summary Erythrocytes from myotonic goats, an animal model of heritable myotonia, and normal goats were studied using electron paramagnetic resonance (EPR) and saturation transfer electron paramagnetic resonance (ST-EPR) spin labeling techniques. Three fatty acid spin labels with the nitroxide moiety at progressively greater distances from the carboxyl group were used to monitor different regions within the erythrocyte membrane. Since spin labels have been shown to induce hemolytic and morphologic alterations in erythrocytes, conditions for minimizing these alterations were first defined by hemolysis studies and scanning electron microscopy. Using these defined conditions for our studies we observed no significant differences in any of the EPR or ST-EPR parameters for normal and myotomic goat erythrocytes with any of the fatty acid spin labels used. Our results do not support the theory that myotonia is the result of a generalized membrane defect characterized by increased membrane fluidity as determined by fatty acid spin labels.     

竹红菌甲素对红细胞膜和几种磷脂脂质体膜的流动...

In this paper, the photodamage of Hypocrellin A to the fluidity of human erythrocyte membranes and some kinds of membranes of phospholipid liposomes was investigated by measuring the changes in fluorescence polarization of the membranes. The results showed that the photosensitization effect of HA caused the decrease of membrane fluidity of the phospholipid (DPPC, DPPC/DPPE, phospholipid of erythrocyte membranes) liposomes. The DPPC and DPPC/DPPE liposomes were more sensitive to the damage than the phospholipid liposomes of erythrocyte membranes. To human erythrocyte membranes, the photodamage effect of HA caused its fluidity first increased and then, with the increment of illumination time, decreased. To spectrin-depleted and trypsin-treated erythrocyte membranes, this kind of change in fluidity was inhibited. All of the results indicated that phospholipids and proteins play different roles in the photodamage of HA to the fluidity of membranes. Membrane proteins, especially spectrin, were the key factor involved in the changes of the fluidity.     

竹红菌甲素对红细胞膜和几种磷脂脂质体膜的流动性的光敏损伤

本文以荧光探针为手段,通过测量膜偏振度的变化,探讨了竹红菌甲素光敏作用对红细胞膜和几种磷脂脂质体膜的流动性的损伤。结果表明,甲素光敏作用使不同种类的磷脂(DPPC,DPPC/DPPE,红细胞膜磷脂)脂质体的流动性增加,其对光敏作用的敏感程度为红细胞膜磷脂脂质体显著小于DPPC/DPPE脂质体及DPPC脂质体。对红细胞膜来说,甲素光敏作用使其流动性呈现先降低而后增加的现象。去除膜上的spectrin以及用胰蛋白酶处理可使这种流动性变化的幅度受到抑制。据此,我们认为,膜磷脂,膜蛋白对甲素光敏作用中膜流动性的变化有着不同的影响,膜蛋白,特别是spectrin,是其中极重要的因素。     

Membrane fluidity of platelets and erythrocytes in patients with Alzheimer's disease and the effect of small amounts of aluminium on platelet and erythrocyte membranes

The membrane fluidity of platelet and erythrocyte membranes in 10 Alzheimer's disease patients and 9 age-matched controls was studied. The platelet membranes of patients with Alzheimer's disease were found to be significantly more fluid than those of controls (p<0.02). However, erythrocyte membranes of Alzheimer patients were less fluid (more viscous) than those of controls (p<0.05). On further investigation of platelet and erythrocyte membranes obtained from healthy volunteers, the fluidity was found to change with increasing aluminium concentrations. When aluminium ammonium sulphate (0.01–10 M) was added to membrane suspensions, the fluidity of platelet membranes was increased, whereas the fluidity of erythrocyte membranes was decreased (i.e. the microviscosity was increased).     

The influence of metmyoglobin and ferrylmyoglobin on the human erythrocyte membrane

Preliminary experiments revealed that ferrylmyoglobin decayed more slowly in the absence than in the presence of intact erythrocytes and erythrocyte membranes. This suggested the existence of interactions between FerrylMb and the erythrocyte membrane. Subsequent studies examined the influence of FerrylMb on the membrane of intact erythrocytes and on isolated erythrocyte membranes. The incubation of intact erythrocytes with FerrylMb did not influence their osmotic fragility or the fluidity of their membranes; the level of peroxidation of the membrane lipids increased only slightly (there was only a slight increase in the level of membrane lipid peroxidation). The activity of acetylcholinesterase significantly increased after 15 minutes of incubation, whereas longer incubation did not lead to any changes in the activity of this enzyme. The incubation of isolated erythrocyte membranes with FerrylMb resulted in an increase in their fluidity and a significant rise in the level of lipid peroxidation.     

Electron spin resonance, hematological, and deformability studies of erythrocytes from patients with Huntington's disease.

Electron spin resonance, hematologic, and deformability studies of erythrocytes from patients with Huntington's disease have been performed A decreased deformability of Huntington's disease erythrocytes compared to normal controls was demonstrated. No difference in erythrocyte hematologic indices, osmotic fragility, reticulocyte counts, or intracellular Na+ concentration was found. Huntington's disease serum had no demonstrable effect on electron spin resonance parameters of a protein-specific spin label attached to membrane proteins in control erythrocytes compared to the effect of control serum. This finding suggests that under the conditions employed no serum component or circulating factor is responsible for the changes in the physical state of membrane proteins in Huntington's disease erythrocytes (Butterfield, D.A., Oeswein, J.Q. and Markesbery, W.R. (1977) Nature 267, 453--455). No alteration in lipid fluidity of Huntington's disease erythrocyte membranes could be discerned suggesting that the underlying molecular defect in Huntington's disease involves a membrane protein. The results of the present studies on erythrocytes strongly support the concept that Huntington's disease is associated with a generalized membrane abnormality.     

Structure-activity investigation of the alteration of the physical state of the skeletal network of proteins in human erythrocyte membranes induced by 9-amino-1,2,3,4-tetrahydroacridine

The oral administration of 9-amino-1,2,3,4-tetrahydroacridine (THA) is purported to increase the mental function of Alzheimer's disease patients (Summers et al. (1986) N. Engl. J. Med. 315, 1241-1245). Numerous erythrocyte membrane proteins are known to be identical or highly similar to neuronal proteins. In a previous study (Butterfield and Palmieri [1990) Free Radical Res. Commun., in press), we showed that THA greatly increased skeletal protein-protein interactions in erythrocyte membranes as monitored by a spin label specifically bound to membrane proteins. In this report, a structure-activity study has been performed to determine which THA structural components are involved in its effect on the physical state of human erythrocyte membrane skeletal proteins. The results imply that both the planarity of the molecule and the amino group at the 9-position of the parent acridine molecule are important in the mechanism of interaction with membrane proteins.     

Fluorescence polarization study on the increase of membrane fluidity of human erythrocyte ghosts induced by synthetic water-soluble polymers

The effect of water-soluble polymers on the membrane fluidity of human erythrocyte ghosts was investigated and was compared with that of concanavalin A by means of the fluorescence polarization technique. 8-Anilino-1-naphthalene sulfonic acid sodium salt and 1,6-diphenyl-1,3,5-hexatriene were used as probe molecules. The membrane fluidity was increased by the addition of polycations with concentrations of less than $\text{2 · 10}{}^{\mathrm{?3}}$ wt% 60 min after mixing. The fluidity changes were affected by the chemical structure (hydrophobicity, charge density, etc.) of polycations. Thus, the membrane fluidity increased markedly with increasing charge density on the chain backbone of polycations. On the other hand, nonionic polymers such as poly(ethylene glycol) and $\text{poly}\text{(N-}\text{vinyl}\text{-2-}\text{pyrrolidone}$) changed the membrane fluidity in a biphasic manner. That is, the fluidity of human erythrocyte ghost was temporarily increased and then decrease. For example, 20 wt% of poly(ethylene glycol) gave a maximum fluidity 15 min after mixing with erythrocyte ghosts. A similar fluidity change was observed by adding concanavalin A. Such fluidity changes were not observed when lipid bilayer vesicles were used instead of cell membranes. These results suggested that the increase of membrane fluidity resulted from the intramembraneous aggregation of membrane-bound proteins which was induced by the added polymers. Cell agglutination was also induced by the addition of a large amount of polymers. This agglutination was considered to be due to the intermembraneous aggregation of membrane-bound proteins.     

Alterations of human erythrocyte membrane fluidity by oxygen-derived free radicals and calcium

Two possible reasons for the structural alterations of cell membranes caused by free radicals are lipid peroxidation and an increase in the intracellular calcium ion concentration. To characterize the alterations in membrane molecular dynamics caused by oxygen-derived free radicals and calcium, human erythrocytes were spin-labeled with 5-doxyl stearic acid, and alterations in membrane fluidity were quantified by electron spin resonance oxidase (0.07 U/mL) decreased membrane fluidity, and the addition of superoxide dismutase and catalase inhibited the effect on membrane fluidity of the hypoxanthine-xanthine oxidase system. Hydrogen peroxide (0.1 and 1 nM) also decreased membrane fluidity and caused alterations to erythrocyte morphology. In addition, a decrease in membrane fluidity was observed in erythrocytes incubated with 2.8 mM CaCl₂. On the other hand, incubation of erythrocytes with calcium-free solution decreased the changes in membrane fluidity caused by hydrogen peroxide.

These results suggest that changes in membrane fluidity are directly due to lipid peroxidation and are indirectly the result of increased intracellular calcium concentration. We support the hypothesis that alterations of the biophysical properties of membranes caused by free radicals play an important role in cell injury, and that the accumulation of calcium amplifies the damge to membranes weakened by free radicals.     

Fluidity of natural membranes and phosphatidylserine and ganglioside dispersions. Effect of local anesthetics, cholesterol and protein.

The microviscosity of artificial lipid membranes and natural membranes was measured by the fluorescence polarization technique employing perylene as the probe. Lipid dispersions composed of brain gangliosides exhibited greater microviscosity than phosphatidylserine (268 cP vs 173 cP, at 25 degrees C). Incorporation of cholesterol (30-50%) increased the microviscosity of lipid phases by 200-500 cP. Cholesterol's effect on membrane fluidity was completely reversed by digitonin but not by amphotericin B. Incorporation of membrane proteins into lipid vesicles gave varying results. Cytochrome b5 did not alter membrane fluidity. However, myelin proteolipid produced an apparent increase in microviscosity, but this effect might be due to partitioning of perylene between lipid and protein binding sites since tha latter have a higher fluorescence anisotropy than the lipid. The local anesthetics tetracain and butacaine increased the fluidity of lipid dispersions, natural membranes and intact ascites tumor cell membranes. The effect of anesthetics appears to be due to an increased disordering of lipid structure. The fluidity of natural membranes at 25 degrees C varied as follows: polymorphonuclear leukocytes, 335 cP; bovine brain myelin, 270 cP; human erythrocyte, 180 cP; rat liver microsomes, 95 cP; rat liver mitochondria, 90 cP. In most cases the microviscosity of natural membranes reflects their cholesterol: phospholipid ratio. The natural variations in fluidity of cellular membranes probably reflect important functional requirements. Similarly, the effects of some drugs which alter membrane permeability may be the result of their effects on membrane fluidity.     

山莨菪碱对人红细胞膜蛋白及膜脂运动的影响

本文采用自旋标记顺磁共振波谱技术,研究了山茛菪碱对人红细胞膜蛋白和膜脂运动的影响.结果表明:用马来酰亚胺标记的人红细胞膜,加入山茛菪碱后,其顺磁共振波谱中强、弱固定化作用谱的峰值比增大,膜蛋白的运动受到限制.山茛菪碱对红细胞膜脂的作用部位主要在极性头部,并影响膜脂的流动性.本文还对山茛菪碱与红细胞膜作用的可能机制进行了讨论.     

巴氏碳球C60光激发对红细胞膜流动性的影响

巴氏碳球Ｃ_（６０）光激发对红细胞膜流动性的影响黄文栋,钱凯先,唐海琼（浙江大学生物科学与技术系,杭州３１００２７）李文铸（渐江大学物理系,杭州３１００２７）关键词Ｃ_（６０）;光激发;红细胞膜;荧光偏振;膜流动性Ｃ６０是Ｋｒｏｔｏ等人［１］于１９８５...     

Effects of intramembrane particle aggregation on erythrocyte membrane fluidity: an electron spin resonance study in normal and in dystrophic subjects

Mobilization and aggregation of intramembrane particles (IMPs) are physiological events observed in various cells. In erythrocyte membranes, aggregation of IMPs can be induced by the exposure of partially desprectrinized erythrocyte membranes to acidic pH. We investigated the association between IMPs aggregation, protein mobility, and membrane fluidity in erythrocyte membranes of healthy controls and Duchenne muscular dystrophy (DMD) patients by using electron spin resonance and specific spin labels for membrane proteins and lipids. In erythrocyte membranes of control subjects, the partial spectrin removal induced a decreased segmental motion of protein spin label indicating an increase of protein-protein interactions. Stearic acid spin labels 5- and 16-(N-oxyl-4,4'-dimethyloxazolidine) showed that the treatment induces an increase of membrane fluidity. In DMD patients, both treated and untreated erythrocyte membranes showed changes of membrane fluidity when compared to those of the controls. Our results suggest that defects in the interactions between skeletal proteins and/or between membrane and skeleton components may contribute to the alterations of erythrocyte membranes in DMD.     

Effects of weight loss on erythrocyte membrane composition and fluidity in overweight and moderately obese women

A previous study showed chemical and physical impairment of the erythrocyte membrane of overweight and moderately obese women. The present study investigated the effects of a low-calorie diet (800 kcal/day deficit for 8 weeks) on erythrocyte membrane properties in 70 overweight and moderately obese (body mass index, 25-33 kg/m²) normotensive, nondiabetic women. At the end of dietary intervention, 24.3% of women dropped out, 45.7% lost less than 5% of their initial weight (Group I) and only 30% of patients lost at least 5% of their initial body weight (Group II). Group I showed no significant changes in erythrocyte membrane composition and function. The erythrocyte membranes of Group II showed significant reductions in malondialdehyde, lipofuscin, cholesterol, sphingomyelin, palmitic acid and nervonic acid and an increase in di-homo-γ-linolenic acid, arachidonic acid and membrane fluidity. Moreover, Group II showed an improvement in total cholesterol, low-density lipoprotein cholesterol, glycemia and insulin resistance. These changes in erythrocyte membrane composition could reflect a virtuous cycle resulting from the reduction in insulin resistance associated with increased membrane fluidity that, in turn, results in a sequence of metabolic events that concur to further improve membrane fluidity.     

Spectroscopic characterization of vesicle formation on heated human erythrocytes and the influence of the antiviral agent amantadine

EPR investigations on the vesiculation process of heated human erythrocytes were performed, using different fatty acid spin labels. Spectrin denaturation and vesiculation do not influence the fluidity of the lipid phase of the remaining membrane of human erythrocytes: Vesicles released differ in chemical composition as well as in the lipid fluidity of their membrane from the intact human erythrocyte membrane. A reduced cholesterol-to-phospholipid ratio and a depletion of spectrin was found. By changing the ionic concentration of the suspension medium an effect on membrane spectra and on vesicle release was established. The adamantane derivative amantadine causes fluidization of the human erythrocyte membrane and inhibits vesicle release. Based on these results, a model for the mechanism by which adamantane-like molecules could interact with membranes is proposed.     

Interaction of tacrine and velnacrine with neocortical synaptosomal membranes: Relevance to Alzheimer's disease

The acridine-based, potential Alzheimer's disease therapeutic agents, tacrine and velnacrine, were incubated with rat or gerbil neocortical synaptosomal membranes. Electron paramagnetic resonance employing a protein-specific spin label was used to monitor this interaction. Analogous to their effects in erythrocyte membranes [Butterfield and Rangachari (1992) Biochem. Biophys. Res. Commun. 185: 596–603], in the present studies both agents decreased segmental motion of spin labeled synaptosomal membrane proteins, consistent with increased cytoskeletal protein-protein interactions (0.001相似文献