Effect of ionic milieu to the free radicals generated by phenizine methosulphate (PMS)

The effect of free radicals generated by PMS was studied for membrane damage in the presence of different ions in the erythrocyte model. The degree of membrane damage depended on the quality of ionic composition in the incubation medium. We supposed that the degree of membrane damage depends on the average life and concentration and/or reactivity of the free radicals generated. For control of this supposition free radicals were generated by PMS in the presence of Sodium-di-thionite in isosmotic, waterly systems with different ionic composition. At different time intervals the concentration of free radicals was measured by the ESR method. It seams that concentration of radicals depends on the qualitative composition of ionic milieu. The increase of the average life of free radicals generated by PMS is accompanied by decrease in their reactivity. This is reflected by a moderate membrane damage.     

Axisymmetric optical-trap measurement of red blood cell membrane elasticity

The elastic properties of the cell membrane play a crucial role in determining the equilibrium shape of the cell, as well as its response to the external forces it experiences in its physiological environment. Red blood cells are a favored system for studying membrane properties because of their simple structure: a lipid bilayer coupled to a membrane cytoskeleton and no cytoplasmic cytoskeleton. An optical trap is used to stretch a red blood cell, fixed to a glass surface, along its symmetry axis by pulling on a micron-sized latex bead that is bound at the center of the exposed cell dimple. The system, at equilibrium, shows Hookean behavior with a spring constant of 1.5×10(-6)?N/m over a 1-2 μm range of extension. This choice of simple experimental geometry preserves the axial symmetry of the native cell throughout the stretch, probes membrane deformations in the small-extension regime, and facilitates theoretical analysis. The axisymmetry makes the experiment amenable to simulation using a simple model that makes no a priori assumption on the relative importance of shear and bending in membrane deformations. We use an iterative relaxation algorithm to solve for the geometrical configuration of the membrane at mechanical equilibrium for a range of applied forces. We obtain estimates for the out-of-plane membrane bending modulus B≈1×10(-19)?Nm and an upper limit to the in-plane shear modulus H<2×10(-6)?N/m. The partial agreement of these results with other published values may serve to highlight the dependence of the cell's resistance to deformation on the scale and geometry of the deformation.     

蜂毒溶血肽对鸡红细胞及膜的生化作用

本文采用荧光分光光度、薄层层析、原子吸收、荧光显微图像等多种生化技术,系统研究了蜂毒肽作用于鸡红细胞及膜的生化机理。结果表明：蜂毒肽影响红细胞膜上及胞内两种酶的功能。它抑制膜Na⁺-K⁺-ATPase活性,导致胞内外离子转运异常,K⁺浓度失衡;它也抑制细胞内葡萄糖-6-磷酸脱氢酶活性,其正电区域干扰胞内带负电小分子的作用,影响红细胞正常代谢。蜂毒肽干扰膜中阴离子通道的转运功能,使细胞渗透压改变,引起膨胀而溶血。蜂毒肽对有核红细胞核内DNA没有作用,与其他抗微生物多肽作用的靶向不同。据此认为,抗菌蛋白类抗生素对细菌作用的生化机理与传统抗生素不同,这是细菌对其不易产生耐药性的重要原因。     

In vitro effects of some drugs on human red blood cell glucose-6-phosphate dehydrogenase enzyme activity

In vitro effects of omeprazole, morphine sulphate, remifentanyl, ketamine and vankomycin were investigated on human red blood cell glucose-6 phosphate dehydrogenase (G-6PD) (E.C. 1.1.1.49) enzyme activity purified from human red blood cell by 2', 5'ADP-Sepharose 4B affinity gel. The obtained I50 values of omeprazole, morphine, remifentanil, ketamine and vankomycin were 3.24, 43.58, 97.6, 64.16 and 0.903 mM, respectively and the Ki constants for omeprazole, morphine and vankomycin were 8.22 +/- 2.055, 25.93 +/- 6.482, and 2.71 +/- 0.677 mM, respectively and they were non competitive inhibitors.     

In vitro interaction of mutagenic chromium (VI) with red blood cells

The interaction of mutagenic Cr(VI) with red blood cells has been studied by ESR spectroscopy. Signals of two Cr(V) species are observed almost immediately after contacting red cells with chromate(VI) aqueous solution at pH 7.4. The signal at go = 1.985, which decays within one hour, is attributed to a Cr(V) complex formed by glutathione due its reducing and chelating ability. The other signal at go = 1.979, which is distinctly more persistent, may indicate that some immobilization of the formed Cr(V) ions takes place on the macromolecular cell components, e.g. glycoproteins.     

In vitro effect of hydrostatic pressure exposure on hydroxyl radical production in fish red muscle

The effects of hydrostatic pressure on reactive oxygen species (ROS) production have been studied in vitro on fish red muscle fibres. In the eel, Anguilla anguilla, previous studies have shown that hydrostatic pressure acclimatization improves oxidative phosphorylation efficiency together with a supposed concomitant decrease in electron leak and ROS production. In order to test the hypothesis of an electron leak decrease under pressure, hydroxyl radical (HO*) production and oxygen consumption were measured on fish red muscle fibres directly exposed to hydrostatic pressure. Experiments were performed under two conditions--atmospheric pressure and hydrostatic pressure (16.1 MPa)--on eel and trout (which exhibit low- and high-pressure sensitivity, respectively). This work has permitted, first, the validation of an indirect HO* measurement (in vitro) on fish red muscle and the documentation of reference values for fish. Second, at atmospheric pressure, results show higher oxygen consumption for trout (+40%) than for eel which is accompanied by higher HO* production (+90%); in addition, both species present a positive relationship between HO* production and oxygen consumption. Hydrostatic pressure exposure reverses this relationship for eel but not for trout. These preliminary results only partially verify the proposed hypothesis and further experiments are needed.     

In vitro effects of wastewater treatment plant effluent on sea bass red blood cells

Red blood cells of marine fish have been used in suitable biological assays to study the (eco)toxicity of wastewater treatment plant effluents. The aim of the present work was to draw upon their more relevant effects on cell hemolysis, ATP content, osmotic resistance and cell volume regulation. Following physico-chemical treatment, treatment plant effluents showed a residual toxicity resulting from multiple impairment of cell metabolism and structures. The earliest and most sensitive effects were related to the regulation of intracellular osmotic pressure leading to decreased cell water volume. Such effects were also observed following short-term incubation in 10-fold diluted effluent. Other damages were found following incubation in non-diluted effluent. Membrane structure was affected leading to increased osmotic resistance. Later, a decrease of the intracellular ATP level was found, followed by hemolysis. The presence of glucose in the incubation medium lessened the fall in ATP content and hemolysis in the treated cells but also in control cells.     

Modulation of free radical stress in human red blood cell membrane by forskolin and the prospects for treatment of cardiovascular disease and diabetes.

This study has examined the effect of oxidants and elevated levels of glucose on membrane lipid peroxidation of human red blood calls (RBC). Washed RBC incubated with varying concentrations of glucose at 37 degrees C for 24 hrs. showed a significant increased membrane lipid peroxidation when compared with control RBC. Addition of ferrous sulphate and ascorbic acid which are known oxidants caused greater rise in lipid peroxides compared to elevated level of glucose alone. Pre-treatment of RBC with varying doses of forskolin (4-70 microg) was associated with significantly smaller rise in lipid peroxides in a dose dependent manner. Forskolin effect was comparable with the antioxidant effect of other drugs such as vitamin E, trimetazidine, ginkocer, probucol. It is possible that forskolin at higher concentrations may have a greater antioxidant effect than other antioxidant drug. However more studies, especially animal experiments, and trials in human would be necessary to confirm our findings.     

The effect of brief physical exercise on free radical scavenging enzyme systems in human red blood cells

Eleven sedentary male students were studied, using a bicycle ergometer, for 30 min at about 75% of their maximal oxygen uptake, to observe the effects of brief physical exercise on free radical scavenging system enzymes of the erythrocytes. Of the enzymes examined, only total glutathione reductase activity showed a significant elevation immediately after exercise and appeared to remain high at 30 min after exercise. The results suggest that acute physical exercise has some effects on red blood cell glutathione reductase activity, which is related primarily to maintenance of reduced glutathione.     

Non-invasive in situ simultaneous measurement of multi-parameter mechanical properties of red blood cell membrane

The purpose of this study was to develop a new dynamic image analyzing technique that will give us the ability to measure the viscoelastic parameters of individual living red blood cells non-invasively, in situ and in real time. With this technique, the bending modulus Kc, the shear elasticity μ and their ratio ε were measured under different temperatures, oxygen partial pressures and osmotic pressures. The results not only show the effects of external conditions on mechanical properties of cell membranes including deformability,flexibility, adhesive ability and plasticity, but also demonstrate that the technique can be used to measure cell membrane parameters continuously under several physiological and pathological conditions.     

In vitro effects of 50 Hz magnetic fields on oxidatively damaged rabbit red blood cells

The aim of this study was to investigate the effects of 50 Hz magnetic fields (0.2–0.5 mT) on rabbit red blood cells (RBCs) that were exposed simultaneously to the action of an oxygen radical-generating system, Fe(II)/ascorbate. Previous data obtained in our laboratory showed that the exposure of rabbit erythrocytes or reticulocytes to Fe(II)/ascorbate induces hexokinase inactivation, whereas the other glycolytic enzymes do not show any decay. We also observed depletion of reduced glutathione (GSH) content with a concomitant intracellular and extracellular increase in oxidized glutathione (GSSG) and a decrease in energy charge. In this work we investigated whether 50 Hz magnetic fields could influence the intracellular impairments that occur when erythrocytes or reticulocytes are exposed to this oxidant system, namely, inactivation of hexokinase activity, GSH depletion, a change in energy charge, and hemoglobin oxidation. The results obtained indicate that a 0.5 mT magnetic field had no effect on intact RBCs, whereas it increased the damage in an oxidatively stressed erythrocyte system. In fact, exposure of intact erythrocytes incubated with Fe(II)/ascorbate to a 0.5 mT magnetic field induced a significant further decay in hexokinase activity (about 20%) as well as a twofold increase in methemoglobin production compared with RBCs that were exposed to the oxidant system alone. Although further studies will be needed to determine the physiological implications of these data, the results reported in this study demonstrate that the effects of the magnetic fields investigated are able to potentiate the cellular damage induced in vitro by oxidizing agents. Bioelectromagnetics 18:125–131, 1997. © 1997 Wiley-Liss, Inc.     

Differential effects of arylating and oxidizing analogs of N-acetyl-p-benzoquinoneimine on red blood cell membrane proteins

Incubation of human red blood cell membranes (white ghosts) with N-acetyl-p-benzoquinone imine (NAPQI), a toxic metabolite of acetaminophen, or with either an arylating or an oxidizing analog of NAPQI, resulted in the inhibition of membrane ion transporting systems and the modification of cytoskeletal proteins. NAPQI and 2,6-dimethyl-NAPQI, which primarily arylates protein thiols, inhibited the calmodulin-activated Ca pump ATPase activity, the basal (calmodulin-independent) Ca pump ATPase activity and the Na,K pump ATPase activity. In contrast, 3,5-dimethyl-NAPQI, which primarily oxidizes protein thiols, caused selective inhibition of the calmodulin-activated Ca pump ATPase activity. Sodium dodecyl sulfate gel electrophoresis of red blood cell (RBC) membrane proteins revealed that NAPQI and 2,6-dimethyl-NAPQI, but not 3,5-dimethyl-NAPQI, decreased the intensity of band 3 corresponding to the anion transporter, whereas NAPQI as well as 2,6-dimethyl-NAPQI, and to a lesser extent 3,5-dimethyl-NAPQI, caused a decrease of cytoskeletal protein bands, including spectrin, actin, and bands 4.1 and 4.2. These modifications were associated with increased formation of high molecular weight protein aggregates that did not enter the gel. Treatment of 3,5-dimethyl-NAPQI-exposed ghosts with the reducing agent dithiothreitol (DTT), resulted in the recovery of the affected cytoskeletal protein bands. Conversely, the modifications caused by NAPQI and 2,6-dimethyl-NAPQI were only partially reversed by DTT treatment. Taken together our results suggest that NAPQI and its two analogs modified ion transporting systems and cytoskeletal proteins by reacting with protein thiols. Both oxidation and arylation of protein thiols can alter the functional properties of important RBC membrane proteins. Of the two reactions, arylation appeared to be the less specific and more damaging event.     

Effect of hydroperoxides on red blood cell membrane mechanical properties

We investigate the effect of oxidative stress on red blood cell membrane mechanical properties in vitro using detailed analysis of the membrane thermal fluctuation spectrum. Two different oxidants, the cytosol-soluble hydrogen peroxide and the membrane-soluble cumene hydroperoxide, are used, and their effects on the membrane bending elastic modulus, surface tension, strength of confinement due to the membrane skeleton, and 2D shear elastic modulus are measured. We find that both oxidants alter significantly the membrane elastic properties, but their effects differ qualitatively and quantitatively. While hydrogen peroxide mainly affects the elasticity of the membrane protein skeleton (increasing the membrane shear modulus), cumene hydroperoxide has an impact on both membrane skeleton and lipid bilayer mechanical properties, as can be seen from the increased values of the shear and bending elastic moduli. The biologically important implication of these results is that the effects of oxidative stress on the biophysical properties, and hence the physiological functions, of the cell membrane depend on the nature of the oxidative agent. Thermal fluctuation spectroscopy provides a means of characterizing these different effects, potentially in a clinical milieu.     

Inhibitory effect of resveratrol on free radical generation in blood platelets

Resveratrol (3,4',5-trihydroxystilbene), a compound found in many plants, has been shown to prevent coronary heart diseases and to exert a variety of antiinflammatory and anticancerogenic effects. It is effective in lowering the level of serum lipids and in inhibiting platelet aggregation. We evaluated the effect of trans-resveratrol on the production of free radicals in pig blood platelets and showed that resveratrol inhibited the production of different reactive oxygen species (O2*-, H2O2, singlet oxygen and organic radicals) measured by the luminol-dependent chemiluminescence in resting platelets (P < 0.05). Resveratrol inhibited also the generation of radicals in platelets activated by thrombin (P < 0.05). Treatment of platelets with resveratrol at concentrations of 6.25 and 12.5 microg/ml caused a statistically insignificant increase in the production of O2*- in these cells, as measured by reduction of cytochrome c; however, at higher doses (25, 50 and 100 microg/ml) resveratrol distinctly reduced the generation of O2*- in platelets (P < 0.05). We suggest that free radicals play an important role in the reduced reactivity of blood platelets induced by resveratrol.     

Interaction of flavonoids with red blood cell membrane lipids and proteins: antioxidant and antihemolytic effects

Plant flavonoids are emerging as potent therapeutic drugs effective against a wide range of free radical mediated diseases. Hence their interactions with cell membranes, which generally serve as targets for lipid peroxidation, are of enormous interest. Here we report in vitro studies, via absorption and fluorescence spectroscopy, on the effects of several flavonoids (namely fisetin, quercetin, chrysin, morin, and 3-hydroxyflavone, 3-HF) in goat RBC membranes. Owing to the presence of functionally relevant membrane protein components embedded in the lipid bilayer RBC ghosts provide a more realistic system for exploring drug actions in biomembranes than simpler membrane models like phosphatidylcholine liposomes used in our previous studies [e.g. B. Sengupta, A. Banerjee, P.K. Sengupta, FEBS Lett. 570 (2004) 77-81]. Here, we demonstrate that binding of the flavonoids to the RBC membranes significantly inhibits lipid peroxidation, and at the same time enhances their integrity against hypotonic lysis. Interestingly, the antioxidant and antihemolytic activities are found to be crucially dependent on the locations of the flavonoids in the membrane matrix as revealed by fluorescence studies. Furthermore, we observe that FRET (from membrane protein tryptophans to flavonoids) occurs with significant efficiency indicating that the flavonoid binding sites lie in close proximity to the tryptophan residues in the ghost membrane proteins.     

Theory of non-Newtonian viscosity of red blood cell suspension: effect of red cell deformation

The effects of the deformation of red blood cells on non-Newtonian viscosity of a concentrated red cell suspension are investigated theoretically. To simplify the problem an elastic spherical shell filled with an incompressible Newtonian fluid is considered as a model of a normal red cell. The equation of the surface of the shell suspended in a steady simple shear flow is calculated on the assumption that the deformation from a spherical shape is very small. The relative viscosity of a concentrated suspension of such particles is obtained based on the "free surface cell" method proposed by Happel. It is shown that the relative viscosity decreases as the shear rate increases.     

In vitro effect of free bile acids on the bile canalicular membrane phospholipids in the rat.

Liver cell plasma membranes of male rats were isolated and separated into two fractions, one rich in bile canalicular membranes (BCM) and the other comprising the rest of the plasma membrane (PM). Aliquots of BCM, PM, and microsomes were incubated with deoxycholic, chenodeoxycholic, or cholic acid at bile acid - membrane phospholipid mole ratios up to 100, and the phospholipid solubilization from the PM and from microsomes was linear and apparently nonselective, while that from BCM was biphasic and distinctly selective. Phosphatidyl choline and phosphatidyl ethanolamine made up 90% of the phospholipids solubilized from the BCM at a bile acid - membrane phospholipid mole ratio sufficient to solubilize about 50% of the total phospholipids of the BCM. Of particular interest was the observation that the molecular species and fatty acid composition of the phospholipids solubilized from the BCM under these experimental conditions were similar to those of bile obtained from the same animal, and were quite unlike those solubilized at higher bile acid - phospholipids mole ratios. The data are discussed in terms of the mechanism of the biliary secretion of phospholipids.     

Susceptibility of tamarin (Saguinus labiatus) red blood cell membrane lipids to oxidative stress: implications for wasting marmoset syndrome

Captive Callitrichids frequently suffer a fatal wasting disease, wasting marmoset syndrome (WMS), of unexplained cause. This paper describes studies on the erythrocytes from animals in a breeding colony of tamarins (Saguinus labiatus), in which deaths from anaemia and wasting were occurring, to seek evidence for biochemical changes which could lead to oxidative damage and premature cell lysis. In only one animal of 33 studied did the red blood cell lipids show an increased susceptibility to oxidative damage. This animal, with some degree of certainty, could be diagnosed as having WMS. It was concluded that evidence for a primary deficiency of antioxidants as a cause of unexplained deaths, or WMS, in the colony could not at present be substantiated.