Cytochrome c oxidase is decreased in Alzheimer's disease platelets

Cytochrome c oxidase (COX) activity reportedly is reduced in Alzheimer's disease (AD) brain and platelets. The reasons for the defect in either tissue are unknown, but its presence in a non-degenerating tissue suggests it is not simply a consequence of neurodegeneration. We now offer confirmation of the AD platelet COX defect. Compared to age-matched controls, in mitochondria isolated from AD platelets there was a 15% decrease in COX activity despite the fact that COX subunits were present at normal levels. Platelet ATP levels were diminished in AD (from 11.33 +/- 0.52 to 9.11 +/- 0.72 nmol/mg), while reactive oxygen species (ROS) were increased (from 97.03 +/- 25.9 to 338.3 +/- 100 K/mg). Platelet membrane fluidity, Vitamin E, and cholesterol content were similar between groups. We conclude that COX catalytic activity is indeed diminished in AD platelet mitochondria, does not result from altered membrane fluidity, and is associated with ROS overproduction and ATP under-production.     

Measurement of platelet membrane fluidity in patients with Binswanger's disease or Alzheimer's disease

目的和方法：探讨血小板在Ｂｉｎｓｗａｎｇｅｒ病（ＢＤ）发病中的作用，用荧光探针测定１１例ＢＤ患者、９例Ａｌｚｈｅｉｍｅｒ病（ＡＤ）患者和６例健康对照者血小板膜的流动性（ＰＭＦ），以颈内静脉和肘静脉血ＰＭＦ的差值作为反映脑循环中ＰＭＦ变化的指标。结果：ＢＤ和ＡＤ患者的ＰＭＦ的均值显著低于健康对照组，但只有ＢＤ患者颈内静脉血ＰＭＦ显著低于肘静脉血，ＢＤ患者ＰＭＦ差值与ＨＤＳ积分具有显著相关。结论：ＢＤ病人脑循环中ＰＭＦ降低可能是ＢＤ的发病因素之一。     

肺癌患者红细胞膜物理特性实验研究

本文测量了肺癌患者红细胞可变形性，红细胞膜流动性和通透性，并将其与正常人血样本进行比较，结果表明，与正常人相比肺癌患者红细胞可变形性、红细胞膜流动性与膜阴离子通透性皆降低。     

An electron spin resonance (ESR) study of lymphocyte membrane fluidity in patients with bronchial asthma and atopic dermatitis]

The lymphocyte membrane fluidity of patients with allergic diseases was measured by electron spin resonance (ESR), and the effect of the epinephrine stimulation on the membrane fluidity was examined. The peripheral lymphocytes were obtained from 15 patients with bronchial asthma and atopic dermatitis (28.7 +/- 9.9 years old, 10 females and 5 males) and 11 healthy adults (30.5 +/- 4.6 years old, 2 females and 9 males). Lymphocyte membranes were spin-labeled with 5-doxyl-stearic acid. Before and after the stimulation of epinephrine of the final concentrations at 10(-5) and 10(-4) mol/l, ESR spectra of the outer membranes were analyzed to evaluate the membrane fluidity. The membrane fluidity of the intact lymphocytes of allergic patients was significantly decreased in comparison to healthy controls. Although the epinephrine stimulation increased the lymphocyte membrane fluidity, the increase in fluidity was less in allergic patients than in healthy controls. There are various receptors on the surface of the lymphocyte membranes, and changes of the membrane fluidity have an influence on their functions. The results in this study elucidate the decreased fluidity of lymphocyte membrane in patients with bronchial asthma and atopic dermatitis, and suggest that the functions of the membrane receptors might be impaired.     

Intracellular membranes are more fluid in platelets of Alzheimer's disease patients.

We studied 12 patients with probable Alzheimer's disease versus age- and sex-matched healthy control subjects. Platelets were subfractionated into intracellular membranes and plasma membranes, and steady-state anisotropy of diphenylhexatriene was measured on the preparations as an index of membrane fluidity. Fluidity was higher in intracellular membranes from platelets of Alzheimer's patients compared to controls (P = 0.016). However, no difference was observed in purified plasma membrane's fluidity from the same patients versus controls. Neither the platelet counts, platelet volumes, percent of larger platelets, nor the amount of internal membrane protein per platelet were different between groups. There was no correlation between intracellular membrane anisotropy and severity of dementia as measured on the Mini-Mental State Exam. The results extend previous studies suggesting that there is an intracellular membrane alteration in platelets in Alzheimer's disease.     

Change of membrane fluidity of rat neutrophils accompanying Escherichia coli inoculation

Membrane fluidity of rat neutrophils was studied following Escherichia coli inoculation, and characteristic changes were observed. Membrane fluidity was assessed by the excimer-forming lipid technique using pyrenedecanoic acid and flow cytometry and expressed as the fluorescence intensity ratios of excimer and monomer pyrenedecanoic acid (IE/IM ratio). High IE/IM ratios indicated high membrane fluidity. The IE/IM ratio of rat neutrophils (0.50 +/- 0.048) increased after E. coli inoculation, reaching a maximum of almost 1.00 after 10-20 min and then returning to its starting value. Intravenous injection of heat-killed E. coli or E. coli-conditioned culture supernatants into rats induced a rapid increase of IE/IM ratios, which returned to initial levels after 20 min. The effect on membrane fluidity of in vitro neutrophil incubation with E. coli, heat-killed E. coli, or E. coli-conditioned culture supernatants was similar to that observed in vivo. Addition of 5 mM ethylenediaminetetraacetic acid (EDTA) did not affect neutrophil membrane fluidity. Addition of either 5 micrograms/ml cytochalasin B or 10(-5) M colchicine did not directly affect neutrophil membrane fluidity but did block the change observed following incubation with bacteria.     

Dynamic microstructure of plasma and mitochondrial membranes from bullfrog myocardium--a nanosecond time-resolved fluorometric study

The viscosity of the phospholipid bilayer and the wobbling angle of the phospholipid molecules of mitochondrial membranes and plasma membranes from bullfrog myocardium were measured with a nanosecond time-resolved fluorometer using pulsed excitation of a hydrophobic fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). The mean +/- S.D. in the viscosity of the mitochondrial and plasma membranes was 0.54 +/- 0.9 and 0.37 +/- 0.03 P, respectively, at 30 degrees C. The wobbling angle of phospholipid molecules was 42 +/- 1 and 47 +/- 1 degree, respectively. Cholesterol content was lower in mitochondria (6.4 micrograms/mg protein) than in plasma membranes (43.7 micrograms/mg protein) but phosphatidylethanolamine concentration was higher in mitochondria (31.8%) than in plasma membranes (27.3%). Cardiolipin was contained only in mitochondria. The results of these lipid analyses appear consistent with the measurements of membrane viscosity and phospholipid wobbling angle. When the results are compared with those from a previous study on the erythrocyte membranes from bullfrogs, viscosity is found to increase in the order mitochondrial membranes less than plasma membranes less than erythrocyte membranes. The complex requirements of biomembranes of organelles performing different functions appear to be met by the particular dynamic microstructure of the biomembrane. The effect of membrane viscosity on oxygen diffusion through membranes is discussed.     

Effect of Blood Lipoproteins and Apolipoproteins A-I,C, and E on the Microviscosity of Erythrocyte Membranes

We studied in vitro modifying effect of high-density lipoproteins, low-density lipoproteins, very-low-density lipoproteins, and apolipoproteins A-I, C, and E on membrane structure of rat erythrocytes. Incubation of erythrocyte membranes with lipoproteins was accompanied by significant changes in the behavior of fluorescent probe pyrene in the hydrophobic membrane region. The regulatory effect of lipoproteins was probably realized via exchange of lipid components between these particles and erythrocyte membrane. Apolipoproteins probably had membranotropic activity. Apolipoproteins A-I, C, and E had various effects on biophysical properties of the lipid phase in erythrocyte membranes.     

Merozoite release from Plasmodium falciparum-infected erythrocytes involves the transfer of DiIC₁₆ from infected cell membrane to Maurer's clefts

Merozoite release from infected erythrocytes is a complex process, which is still not fully understood. Such process was characterised at ultra-structural level in this work by labelling erythrocyte membrane with a fluorescent lipid probe and subsequent photo-conversion into an electron-dense precipitate. A lipophilic DiIC₁₆ probe was inserted into the infected erythrocyte surface and the transport of this phospholipid analogue through the erythrocyte membrane was followed up during 48 h of the asexual erythrocyte cycle. The lipid probe was transferred from infected erythrocyte membranes to Maurer’s clefts during merozoite release, thereby indicating that these membranes remained inside host cells after parasite release. Fluorescent structures were never observed inside infected erythrocytes preceding merozoite exit and merozoites released from infected erythrocyte were not fluorescent. However, specific precipitated material was localised bordering the parasitophorous vacuole membrane and tubovesicular membranes when labelled non-infected erythrocytes were invaded by merozoites. It was revealed that lipids were interchangeable from one membrane to another, passing from infected erythrocyte membrane to Maurer’s clefts inside the erythrocyte ghost, even after merozoite release. Maurer’s clefts became photo-converted following merozoite release, suggesting that these structures were in close contact with infected erythrocyte membrane during merozoite exit and possibly played some role in malarial parasite exit from the host cell.     

Evaluation of antiepileptic drug effect on membrane fluidity

Many drugs and chemicals have been shown to induce modifications of the physicochemical properties of cellular membranes. In this study we investigated the changes in fluidity of erythrocyte membrane from epileptic patients under different pharmacological treatments, with respect to healthy controls, by using trimethylammonium-1,6-diphenyl-1,3,5-hexatriene (TMA-DPH) fluorescence polarization. The increase in TMA-DPH fluorescence polarization values observed in epileptic patients indicated a decrease in membrane fluidity. Since the analysis of erythrocyte membrane composition did not reveal significant differences between the two groups studied, a correlation with membrane lipoperoxide content was tried, as different drugs and chemicals elicit in vivo alterations resulting in peroxidation of membrane lipids. Therefore the presence of peroxidation products in the blood and the possible correlation with membrane lipoperoxide were studied. Although a direct causal linkage cannot be proved we can hypothesize that exogenous compounds such as antiepileptic drugs could modify membrane fluidity by increasing membrane lipid peroxidation. Moreover the increase of peroxidative products in the blood could indicate that the peroxidative damage might propagate through the formation of new free radical species. The possibility of using erythrocyte membrane as a model system to analyze antiepileptic drug side effects is advanced.     

Age and membrane fluidity

Male rats aged 1, 9 and 19 months were used to study changes in membrane fluidity with age, employing the fluorescence polarization technique with 1,6-diphenyl-1,3,5-hexatriene (DPH) as the fluorescent probe. The intestinal microvillus membranes derived from the 19-month-old rats were found to possess lower fluidity than that observed with the membranes derived from the younger animals. The decrease in fluidity with age was also reflected in a corresponding increase in the gel-to-liquid crystalline transition temperature. Only small insignificant changes with age, were observed in the fluidity of the red blood cell membrane.     

Effect of zinc on carp (Cyprinus carpio L.) erythrocytes.

The effect of zinc exposure on some properties of the carp erythrocyte membrane was studied in vitro. Red blood cells plasma membranes were separated from other cellular membranes using a combination of differential and density gradient centrifugation. The purity of obtained plasma membrane preparations was determined by measuring the activity of the marker enzymes. Electrophoretic patterns of the main erythrocyte membrane proteins excluded their degradation during the isolation and purification procedure. Carp erythrocyte membranes, obtained from cells previously incubated with increasing ZnSO4 concentrations, were used to elucidate the effect of zinc ions on their physical and biochemical properties. Using fluorescent probes: 12-AS and TMA-DPH, we found that zinc ions reduced the fluidity of the lipid bilayer, both in the middle and near the aqueous interface. Moreover, it was observed that zinc had no significant influence neither on the Na,K-ATPase activity nor on the thiol groups content in the erythrocyte membrane. We also detected that incubation of erythrocytes with zinc lead to the marked decrease of hemolytic resistance of the cells. Our studies demonstrate that zinc at higher concentrations may be toxic to carp erythrocytes causing changes in the membrane fluidity and hemolytic resistance.     

Abnormal erythrocyte anion exchange in Alzheimer disease

CONTEXT: Several abnormalities have been described in red blood cells of patients with Alzheimer disease (AD), but to date none of these has been confirmed by a second, independent study. Erythrocyte anion exchange has been reported to be abnormal in AD; we have developed a new technique for measuring anion exchange. OBJECTIVES: To confirm the abnormality of erythrocyte anion exchange in AD and to determine whether the phenomenon has potential for clinical utility. DESIGN: Comparison of patients with probable AD to age-matched controls. SETTING: University hospital and ambulatory clinic. METHODS: Chloride-bicarbonate exchange was measured in erythrocyte ghosts resealed with a fluorescent probe of chloride concentration. RESULTS: Erythrocyte anion exchange is abnormal in AD. This difference appears in citrate but not EDTA anticoagulant. Mahalanobis's generalized distance between the 2 populations is 1.7, and a discriminant function derived from our technique classifies 82% of the study population in accordance with the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association criteria. Receiver operating characteristic analysis demonstrates the possibility of choosing cutoffs with high sensitivity and specificity. CONCLUSIONS: Measurement of red blood cell anion exchange may be useful in classifying patients with AD. The dependence of this phenomenon on anticoagulant suggests the involvement of platelet activation or complement fixation.     

Erythrocyte membrane fluidity and haemoglobin haemoporphyrin conformation: features revealed in patients with heart failure

This study examined the possible involvement of abnormal erythrocyte oxygen (O₂) transport in the pathogenesis of heart failure. Haemoglobin (Hb) haemoporphyrin conformation was assessed by Raman spectroscopy (RS) of blood samples, whereas membrane fluidity was estimated at depths of 0.6–0.8 and 2.2 nm by electron-paramagnetic resonance spectroscopy of erythrocytes loaded with spin-labeled 5-doxylstearic acid and 16-doxylstearic acid, respectively. The fluidity of erythrocyte membranes from patients with heart failure was decreased in the area near the membrane surface and remained unchanged in the deeper hydrophobic membrane regions. The same differences were also detected in healthy controls subjected to chronic high-altitude hypoxia. RS demonstrated that in heart failure the total content of Hb–ligand complexes and the relative content of Hb–nitric oxide (NO) complexes with cleaved Fe²⁺-globin bond was decreased, whereas content of Hb–NO complexes with preserved Fe²⁺-globin bond was increased. We propose that this phenomenon contributes to the reduced O₂ tissue supply seen in patients with heart failure.     

Membrane biophysical studies of lymphocytes and erythrocytes in manic-depressive illness

Recent research suggests that manic-depressive illness is associated with a membrane abnormality which is detectable in peripheral tissues. Using fluorescence spectroscopy, the cellular membrane dynamics of intact erythrocytes and lymphocytes from manic-depressive patients and controls were studied in a double blind fashion. A cross-sectional analysis of membrane dynamics was obtained by using fluorescent probes with known affinity for specific regions of erythrocyte membranes. This preliminary study demonstrates alterations in the hydrocarbon region of erythrocyte membranes and the cell surface of lymphocytes in patients with manic-depressive illness. These abnormalities appear to be independent of clinical symptomatology and medication. The membrane abnormality demonstrated by fluorescence spectroscopy may provide clues to the molecular pathophysiology in manic-depressive illness, as well as a method of diagnosis in presymptomatic patients.     

中重度烧伤早期红细胞膜流动性的实验研究和临床观察

采用ＤＰＨ荧光探针标记、荧光测定和光镜观察烧伤大鼠中重度烫伤早期红细胞膜流动性、血浆过氧化脂质含量、红细胞异形率的动态变化，以及不同的处理方法对其影响。同时对同期１０例中重度烧伤病进行观察，发现中重度烧伤早期红细胞膜流动性明显下降，血浆脂质过氧化物增多，红细胞异形率增高，这些改变与烧伤面积成正相关，提示烧伤后氧自由基是损伤红细胞膜流动性的因素之一。应用抗自由基药物有保护红细胞膜结构和功能的作用，有     

Food restriction and membrane fluidity.

The fluidity of the erythrocyte membrane derived from growing rats raised under restricted food intake was found to be higher than the fluidity of the respective membranes from ad libitum fed animals. Considering the apparent relationship between the decrease in membrane fluidity and aging, the results point to the possible beneficial effects of food restriction at a young age.     

VIP-sensitive adenylate cyclase, guanylate cyclase, muscarinic receptors, choline acetyltransferase and acetylcholinesterase, in brain tissue afflicted by Alzheimer's disease/senile dementia of the Alzheimer type

Biochemical parameters were determined in autopsy material from several brain regions of thirteen patients with Alzheimer's disease/senile dementia of Alzheimer type (AD/SDAT) (mean age 75 years) and from brains of ten age-matched controls (mean age 76 years). Choline acetyltransferase specific activity was significantly lower in the nucleus caudatus, putamen, left thalamus, hippocampus and the cortex from gyrus hippocampus and the temporal lobe in AD/SDAT, acetylcholinesterase specific activity was significantly lower in the hippocampus, parietal and left frontal lobe in AD/SDAT samples than in corresponding samples from aged-matched controls. A compensatory increase of muscarinic receptors was found in the nucleus caudatus and left frontal lobe samples in AD/SDAT. Guanylate cyclase activity was not significantly altered in AD/SDAT in the brain regions examined. The basal, non-stimulated activity of adenylate cyclase was significantly (p less than 0.05) elevated in hippocampus samples from AD/SDAT patients and the enzyme activity stimulated by the vasoactive intestinal polypeptide VIP (2 microM) or forskolin (10 microM) was also elevated in AD/SDAT although not significantly.     

High cholesterol affects platelet APP processing in controls and in AD patients

Alzheimer disease (AD) is characterised by a decrease of platelet Amyloid Precursor Protein forms ratio (APPr), which parallels symptoms' severity. Recent studies have suggested that cholesterol might play a role in the pathophysiology of AD by modulating Abeta production. Aim of this study was to evaluate the relationship between serum cholesterol levels and platelet APP processing in controls and AD. Sixty AD patients and 45 age-matched controls (CTRL) were investigated. Neuropsychological assessment, cholesterol dosage and APP forms' evaluation were performed on each subject. CTRL showed lower serum cholesterol levels compared to AD (P<0.01) and higher mean APPr scores (P<0.0001). Hypercholesterolaemic AD patients showed lower APPr scores compared to normocholesterolaemic AD patients matched for disease severity (0.31+/-0.16 versus 0.45+/-0.28; P<0.05), since the early stage of the disease. In AD, cholesterol levels influence APPr independently of disease severity. These findings confirm the association between cholesterol and AD, and suggest that in vivo cholesterol affects APP processing by interfering with its maturation.     

The movement of fluorescent endocytic tracers in Plasmodium falciparum infected erythrocytes.

The fluorescent lipophilic probe 1,1'-dihexadecyl-3-3'-3-3'- tetramethylindocarbocyanine (diIC16) inserted in the red cell surface, functioned as a non-exchangeable lipid marker which was not metabolised or toxic in plasmodial cultures. Invasion by Plasmodium falciparum resulted in the internalisation of the lipid, suggesting the uptake of red cell membrane components during parasite entry. The fluorescent lipid was not transported from red cell to parasite membranes at subsequent stages of development, but label in the erythrocyte-derived parasitophorous vacuole was eventually detected in daughter merozoites. Fluorescent dextrans of 10 kDa in the extracellular medium were also not internalised during intraerythrocytic parasite growth. The results support that with the exception of the invasion step, plasmodial infection does not induce endocytosis in the erythrocyte membrane. Despite the lack of endocytosis, both D and L stereoisomers of the head group blocked phospholipid analogue N-4-nitrobenzo-2-oxa-1,3-diazoledipalmitoyl phosphatidylethanolamine (N-NBD-PE) inserted in the erythrocyte membrane, were internalised by mature infected erythrocytes. Lipid internalisation occurred by a non head group dependent parasite mechanism, which could account for the stage-specific uptake of phospholipids observed in mature infected erythrocytes. We were unable to detect the transport of carbocyanine dyes and N-NBD-PE from intracellular parasites back to the erythrocyte membrane. Additionally, the carbocyanine dyes were not transferred between adjacent organisms in a double infected red cell. The data argue for the absence of bulk membrane lipid transport between individual parasites and their host cell bilayer in an infected erythrocyte.