Recurrent decreases of erythrocyte electrophoretic mobility in rats exposed to 1.0 R whole-body x-radiation

Circulating erythrocytes from rats were examined up to 30 weeks post whole-body exposures of 1.0 R for alterations in the expression of net negative surface charge as measured by whole-cell microelectrophoresis in saline sorbitol. Erythrocyte electrophoretic mobility was determined in an apparatus composed of a horizontal transilluminated cylindrical chamber, equipped with a reversible, blacked platinum electrode, immersed in a water bath maintained at 25.0 +/- 1.0 degree C (Rank Brothers). In two separate experiments, recurrent decreases in the expression of net negative surface charge occurred at 10, 17, and 30 weeks post-irradiation. At these times distributional analyses of recorded erythrocyte electrophoretic mobility (EEPM) values revealed a skewing of the normally distributed EEPM population values to lower EEPM. Total sialic acid content released from hydrolyzed erythrocyte membrane preparations revealed no significant differences between erythrocytes from sham and irradiated animals. In vivo post-irradiation labeling of erythrocytes with diisopropyl-[32P] phosphorofluoridate at 4 and 33 weeks (separate experiments) indicated only a minor abbreviated erythrocyte life span at 33 weeks. Therefore, effects from low dose (1.0 R) whole-body irradiation would appear to include a recurrent defect in the expression of the net negative surface charge.     

Analysis of electrophoretic mobility data for human erythrocytes according to sublayer models

An attempt was made to analyze the electrophoretic mobility data of human erythrocytes in media of different pH values and ionic strengths through cell surface models in which the surface charge layer consists of several ion-penetrable sublayers with a uniform charge distribution in each sublayer. As a result, the three-sublayer model was found to explain the mobility data much better than the two-sublayer model in a wide range of ionic strength at all pH values studied.     

Early electrochemical events in lectin-lymphocyte interaction

Concanavalin A, at extremely low concentrations, will produce significant increases in the electrophoretic mobility of murine splenic T lymphocytes. It has been established that the alteration in cellular surface charge is mediated by a factor produced by those lymphocytes that have reacted directly with con A. We originally conjectured that the mobility change might be the consequence of an alteration in the distribution of the charged moieties of membrane glycoproteins. The results of experiments conducted at low temperature raise some questions about this mechanism. Further experiments have been performed to establish the nature of the physicochemical alterations in the peripheral zone of the factor-stimulated lymphocytes that are manifest as changes in cellular surface charge. The results of these studies indicate that, subsequent to the interaction of T lymphocytes with con A, there is a reduction in the number of positively charged amino groups effective at the electrophoretic surface of the cells.     

Electrostatic repulsion among erythrocytes in tube flow, demonstrated by the thickness of marginal cell-free layer

Electrostatic repulsion among erythrocytes in flow was evaluated through measurement of the thickness of the marginal cell-free layer in narrow glass tubes of 20-50 microns in inner diameter. To reduce the electrostatic repulsive force, due mainly to sialic acid of the membrane glycoproteins, human erythrocytes were treated with neuraminidase. The surface negative charge of the erythrocytes, as determined from the electrophoretic mobility using free-flow electrophoresis, was found to be proportional to the sialic acid content. When erythrocytes with decreased sialic acid content flowed through narrow tubes, the thickness of cell-free layer determined using an image processor increased even in the absence of erythrocyte aggregation in the suspension. The effect was more pronounced at acidic pH. The addition of Dextran T-70 (70,400 Mol. Wt.) further increased the cell-free layer thickness due to erythrocyte aggregation. Thus, reducing the negative charge density on the erythrocyte surface by itself accelerates the axial accumulation of erythrocytes in flow due to the decreased electrostatic repulsive force between the cells, even in the absence of erythrocyte aggregation.     

Electrophoretic detection of reversible chlorpromazine · HCl binding at the human erythrocyte surface

The binding of chlorpromazine · HCl at the human erythrocyte surface has been detected through its effect on cellular electrophoretic mobility. Incubation of erythrocytes (approx. 5 · 10⁶/ml) in 23 μM chlorpromazine · HCl resulted in a reduction of negative electrophoretic mobility from the control value of $\text{?1.11 ± 0.01}$ (μm · s^?1)/(V · cm^?1) to $\text{?1.00 ± 0.02}$ (μm · s^?1)/(V · cm^?1) (pH 7.2, ionic strength 0.155). This mobility change was completely reversed when chlorpromazine · HCl was removed by centrifugal washing. Increasing the drug concentration to 70μM did not affect the mobility change, indicating saturation of the electrophoretically detectable drug binding sites over chlorpromazine · HCl concentration range studied here. The effect of the 23 μM chlorpromazine · HCl on electrophoretic mobility was also measured in isotonic media of reduced ionic strength. The drug-induced reduction in negative surface charge density was found to be independent of ionic strength over the range 0.155 (Debye length, 0.8 nm) to 0.00310 (Debye length, 5.7 nm).Fixation of erythrocytes with glutaraldehyde affected neither the normal electrophoretic mobility of discocytes nor the reduced electrophoretic mobility of chlorpromazine · HCl-induced stomatocytes. When these stomatocytes were first fixed with glutaraldehyde, then washed free of chlorpromazine · HCl, they retained the stomatocyte form while regaining a normal control electrophoretic mobility. Conversely, when discocytes fixed in that form were treated with chlorpromazine · HCl, they showed the same mobility change as did fixed or unfixed stomatocytes. The drug-induced mobility change is therefore independent of the shape change, but reflects a contribution to cellular surface charge density from the membrane-bound chlorpromazine · HCl molecules. From the charge reduction, it is estimated that about 10⁶ chlorpromazine · HCl molecules are bound at the electrokinetic cell surface and occupy approximately 0.4% of the total surface area.     

Association of spectrin with its membrane attachment site restricts lateral mobility of human erythrocyte integral membrane proteins

Interactions between spectrin and the inner surface of the human erythrocyte membrane have been implicated in the control of lateral mobility of the integral membrane proteins. We report here that incubation of “leaky” erythrocytes with a water-soluble proteolytic fragment containing the membrane attachment site for spectrin achieves a selective and controlled dissociation of spectrin from the membrane, and increases the rate of lateral mobility of fluorescein isothiocyanate-labeled integral membrane proteins (> 70% of label in band 3 and PAS-1). Mobility of membrane proteins is measured as an increase in the percentage of uniformly fluorescent cells with time after fusion of fluorescent with nonfluorescent erythrocytes by Sendai virus. The cells are permeable to macromolecules since virus-fused erythrocytes lose most of their hemoglobin. The membrane attachment site for spectrin has been solubilized by limited proteolysis of inside-out erythrocyte vesicles and has been purified (V). Bennett, J Biol Chem 253:2292 (1978). This 72,000-dalton fragment binds to spectrin in solution, competitively inhibits association of ³²P-spectrin with inside-out vesicles with a K_i of 10^?7M, and causes rapid dissociation of ³²P-spectrin from vesicles. Both acid-treated 72,000-dalton fragment and the 45,000 dalton-cytoplasmic portion of band 3, which also was isolated from the proteolytic digest, have no effect on spectrin binding, release, or membrane protein mobility. The enhancement of membrane protein lateral mobility by the same polypeptide that inhibits binding of spectrin to inverted vesicles and displaces spectrin from these vesicles provides direct evidence that the interaction of spectrin with protein components in the membrane restricts the lateral mobility of integral membrane proteins in the erythrocyte.     

Modeling the free solution electrophoretic mobility of short DNA fragments

Boundary element methods are used to model the free solution electrophoretic mobility of short DNA fragments. The Stern surfaces of the DNA fragments are modeled as plated cylinders that reproduce translational and rotational diffusion constants. The solvent-accessible and ion-accessible surfaces are taken to be coincident with the Stern surface. The mobilities are computed by solving simultaneously the coupled Navier–Stokes, Poisson, and ion-transport equations. The equilibrium electrostatics are treated at the level of the full Poisson–Boltzmann equation and ion relaxation is included. For polyions as highly charged as short DNA fragments, ion relaxation is substantial. At .11 M KCl, the simulated mobilities of a 20 base pair DNA fragment are in excellent agreement with experiment. At .04 M Tris acetate, pH = 8.0, the simulated mobilities are about 10–15% higher than experimental values and this discrepancy is attributed to the relatively large size of the Tris counterion. The length dependence of the mobility at .11 M KCl is also investigated. Earlier mobility studies on lysozyme are reexamined in view of the present findings. In addition to electrophoretic mobilities, the effective polyion charge measured in steady state electrophoresis and its relationship to the preferential interaction parameter γgG is briefly considered. © 1998 John Wiley & Sons, Inc. Biopoly 46: 359–373, 1998     

Surface charge of mammalian neurones as revealed by microelectrophoresis

Summary The surface charge of isolated rat dorsal root ganglion neurones was studied by microelectrophoresis technique. The increase of Ca concentration caused greater reduction of the electrophoretic mobility compared to that produced by an equivalent amount of divalent organic cations, dimethonium or hexamethonium. No charge reversal for Ca concentrations up to 80 mM was observed. These data fit the suggestion that two anion groups of the outer membrane surface can bind one Ca ion with apparent binding constant of about 50 M^–1. In solutions of low pH the electrophoretic mobility of cells decreased corresponding to titration of acidic groups with apparent pK=4.2. Trypsin treatment in mild conditions markedly reduced the surface charge: however, neuraminidase and hyaluronidase did not change it. N-bromosuccinimide (a specific reagent for carboxylic groups of proteins) decreased the electrophoretic mobility about 60%. However, no increase of the surface charge after the action of specific reagents for amino groups (2,4,6-trinitrobenzenesulfonic acid and maleic anhydride) was observed. It was shown that the surface charge depends also on the intracellular metabolism. If 1 mM dibutyryl cAMP or theophilline was added to the culture medium (thus, raising the concentration of cAMP inside the cell) the surface charge increased. This effect developed slowly and reached its maximum on the third day of incubation. Treatment of cells by 5 mM tolbutamide (an inhibitor of some protein kinases) did not change cell mobility. Addition of 5 mM N-ethylmaleimide (an inhibitor of adenylate cyclase) to the culture medium produced some decrease of the surface charge. On the basis of data obtained it is suggested that the charge of the outer membrane surface of neurones studied is mainly determined by carboxylic groups of membrane proteins, and changes in intracellular cAMP concentration influence the synthesis and reconstruction of these membrane components.     

肠淋巴液引流对失血性休克大鼠红细胞流变性的影响

目的:观察肠淋巴液引流对失血性休克大鼠红细胞流变性指标以及血液黏度的作用。方法:Wistar雄性大鼠均分为假休克组、休克组(复制失血性休克模型)、引流组(复制失血性休克模型,自低血压1 h引流休克肠淋巴液)。在低血压3 h或相应时间,经腹主动脉取血,检测红细胞参数、红细胞电泳、红细胞沉降率(ESR)以及血液黏度,计算红细胞聚集指数、红细胞变形指数。结果:与假休克组比较,休克组红细胞数量、红细胞比积(HCT)、血红蛋白(Hb)、平均红细胞血红蛋白浓度(MCHC)、红细胞电泳率与迁移率、红细胞变形指数、全血黏度、全血低切与高切相对黏度和还原黏度显著降低,休克组平均红细胞体积、红细胞电泳时间、ESR、血沉方程K值与校正K值、红细胞聚集性指数、血浆黏度显著升高;引流组MCHC、红细胞电泳率与迁移率、全血黏度、全血低切与高切还原黏度均显著降低,引流组红细胞体积分布宽度(RDW-SD)显著增加。同时,引流组HCT、RDW-SD、红细胞变形指数、全血黏度、全血低切与高切相对黏度显著高于休克组;ESR、血沉方程K值与校正K值、红细胞聚集性指数、血浆黏度显著低于休克组。结论:休克肠淋巴液引流可改善失血性休克大鼠红细胞流变行为,从而改善血液流变性。     

An experimental test of new theoretical models for the electrokinetic properties of biological membranes. The effect of UO2++ and tetracaine on the electrophoretic mobility of bilayer membranes and human erythrocytes

For a large smooth particle with charges at the surface, the electrophoretic mobility is proportional to the zeta potential, which is related to the charge density by the Gouy-Chapman theory of the diffuse double layer. This classical model adequately describes the dependence of the electrophoretic mobility of phospholipid vesicles on charge density and salt concentration, but it is not applicable to most biological cells, for which new theoretical models have been developed. We tested these new models experimentally by measuring the effect of UO2++ on the electrophoretic mobility of model membranes and human erythrocytes in 0.15 M NaCl at pH 5. We used UO2++ for these studies because it should adsorb specifically to the bilayer surface of the erythrocyte and should not change the density of fixed charges in the glycocalyx. Our experiments demonstrate that it forms high-affinity complexes with the phosphate groups of several phospholipids in a bilayer but does not bind significantly to sialic acid residues. As observed previously, UO2++ adsorbs strongly to egg phosphatidylcholine (PC) vesicles: 0.1 mM UO2++ changes the zeta potential of PC vesicles from 0 to +40 mV. It also has a large effect on the electrophoretic mobility of vesicles formed from mixtures of PC and the negative phospholipid phosphatidylserine (PS): 0.1 mM UO2++ changes the zeta potential of PC/PS vesicles (10 mol % PS) from -13 to +37 mV. In contrast, UO2++ has only a small effect on the electrophoretic mobility of either vesicles formed from mixtures of PC and the negative ganglioside GM1 or erythrocytes: 0.1 mM UO2++ changes the apparent zeta potential of PC/GM1 vesicles (17 mol % GM1) from -11 to +5 mV and the apparent zeta potential of erythrocytes from -12 to -4 mV. The new theoretical models suggest why UO2++ has a small effect on PC/GM1 vesicles and erythrocytes. First, large groups (e.g., sugar moieties) protruding from the surface of the PC/GM1 vesicles and erythrocytes exert hydrodynamic drag. Second, charges at the surface of a particle (e.g., adsorbed UO2++) exert a smaller effect on the mobility than charges located some distance from the surface (e.g., sialic acid residues).     

The Effect of Contrast Medium SonoVue<Superscript>®</Superscript> on the Electric Charge Density of Blood Cells

The effect of contrast medium SonoVue® on the electric charge density of blood cells (erythrocytes and thrombocytes) was measured using a microelectrophoretic method. We examined the effect of adsorbed H⁺ and OH^? ions on the surface charge of erythrocytes or thrombocytes. Surface charge density values were determined from electrophoretic mobility measurements of blood cells performed at various pH levels. The interaction between solution ions and the erythrocyte’s or thrombocyte’s surface was described by a four-component equilibrium model. The agreement between the experimental and theoretical charge variation curves of the erythrocytes and thrombocytes was good at pH 2–9. The deviation observed at a higher pH may be caused by disregarding interactions between the functional groups of blood cells.     

Seasonal activity of frog erythrocytes by data of electrophoretic mobility

The peculiarities of frog erythrocyte electrophoretic mobility, coupled to the seasonal course of temperatures, have been studied. At the periods of anabiosis and of burst of hemopoiesis, in the vascular bed there increases the portion of functionally young erythrocytes (up to 22%) with increased values of the cell membrane surface charge. Preparation to winter is accompanied by a rise of the number of circulating functionally worn-down blood cells (up to 60%) with low values of the superficial charge and low mobility in electrical field. Use of the cell microelectrophoresis method of evaluation of seasonal activity of frog erythrocytes allows obtaining objective data about the cellular surface charge and its depending functional cell activity without submitting the erythrocytes to modifying actions.     

Leishmania mexicana amazonensis: Surface charge of amastigote and promastigote forms

The surface charge of Leishmania mexicana amazonensis was evaluated by means of the binding of colloidal iron hydroxyde particles at pH 1.8 and cationized ferritin particles at pH 7.2 to the cell surface, visualizated by electron microscopy and by direct measurements of the electrophoretic mobility of cells suspended in solutions of different pH. The following forms of the parasite were analysed: amastigotes (surrounded or not by the membrane of the endocytic vacuole, isolated from lesions), transitional forms, and infective (5 passages) and noninfective (176 passages) promastigotes. The results obtained indicate that the surface of L. m. amazonensis contains both negatively and positively charged dissociating groups and that changes occur in the surface charge during amastigote-promastigote transformation. Treatment of the parasite with neuraminidase significantly reduced the electrophoretic mobility of the cells. Neuraminidase-treated cells recovered their normal electrophoretic mobility when incubated for 8 hr in fresh culture medium by a process that is inhibited by puromycin.     

Detection of Differences in Surface-charge-associated Properties of Cells by Partition in Two-polymer Aqueous Phase Systems

WHEN aqueous solutions of two polymers are mixed in certain proportions they may form two-phase systems^1,2 which can be buffered and used to partition and separate cells, particles and macromolecules by countercurrent distribution (CCD). Partition generally depends on polymer composition and concentration, the ionic composition and the charge sign of the material being partitioned. Such systems have been used to separate erythrocytes from white cells and erythrocytes on the basis of age. Changes in the surface properties of cells resulting from enzyme treatment or storage have also been demonstrated by this means³. Higher cell partition often accompanies increasing electrophoretic mobility which suggests that surface charge may be an important factor in partitioning^4–6. An apparent exception to this is the increased partition of stored human erythrocytes as compared with fresh⁷, as opposed to the mean electrophoretic mobility of both cell populations which remain identical⁸.     

Changes in the cell surface of human diploid fibroblasts during cellular aging.

The electrophoretic mobility of 13 human diploid cell strains, TIG-1, TIG-2, TIG-3, TIG-7, WI-38, IMR-90, MRC-5, MRC-9, TIG-1H, TIG-1L, TIG-2M, TIG-2B, and TIG-3S, which were established from different tissues of human embryos, was studied at different passages. The net negative surface charge of the cells was characteristic for each cell strain and decreased significantly during the in vitro aging of the cells. The decrease in the net negative charge of the cells correlated well with the decrease in cell density throughout the life span of the cells. A strict linear correlation between the electrophoretic mobility and the number of cells harvested at each passage was obtained for all the human diploid cell strains. Moreover, almost the same linear regression coefficient of the cells was obtained among these cell strains. Therefore, the net negative surface charge of human diploid cell strains could serve as a cell surface marker for in vitro cellular aging.     

On the two electrophoretic forms of the Human superoxide dismutase A in the homozygote SOD A1

Human superoxide dismutase A of the common phenotype SOD A1 is present in two electrophoretic forms in erythrocytes. These forms are charge isomers, easily permuting one into the other. Oxidation and reduction of the copper, partial or complete saturation of the dimers with copper, and presence of thiol groups on the enzyme do not seem to be involved.     

Postnatal changes in canine erythrocyte pyruvate kinase isozymes

The isozyme pattern of pyruvate kinase in canine erythrocytes changes following birth. These changes have been followed by electrophoretic, immunologic, and kinetic measurements of the isozymes. At birth, a mixture of isozymes is present consisting of the M₂ isozyme and hybrid molecules containing M₂ and R subunits. With increasing animal age, the content of M₂ subunits decreases and the content of R subunits increases. At 6 months of age, the isozyme pattern is indistinguishable from that of adult erythrocytes which contain only the R tetramer. We conclude that there is a switch in erythrocyte pyruvate kinase gene expression during the first 6 months of postnatal life. The existence of hybrid molecules during the switch indicates that both M₂ and R genes are expressed within each erythroid precursor cell. The developmental changes in erythrocyte pyruvate kinase are consistent with the role of this enzyme in the regulation of the oxygen-transport function of canine hemoglobin by 2,3-diphosphoglycerate in the postnatal period.This research was supported by Public Health Service Grant HD-10595.     

On the mechanism of binding of human lymphocyte products to guinea pig macrophages

When lymphocytes from a majority of patients with cancer are incubated with encephalitogenic factor, a lymphocyte product is released that reduces the anodic electrophoretic mobilities of guinea pig macrophages and fixed, tanned sheep erythrocytes. Although these reactions are not specific for cancer, it is distinctly possible that in patients with cancer, products from stimulated lymphocytes are capable of altering the surfaces of the patients' own macrophages, thereby modifying the course of their disease. In this paper, we attempt to elucidate some mechanisms for the binding of lymphocyte products to macrophages, such as occurs in the macrophage electrophoretic mobility (MEM) test, since this may be of general interest. Binding of lymphocyte product to macrophages has been monitored by measurements of their electrophoretic mobilities and by electron microscopic determination of the density of binding of electron-dense, cationic colloidal iron hydroxide particles to their surfaces. The results show that the lymphocyte products reduce the net surface negativity of the macrophages by (coulombic) binding of this net positively charged material to sialic acids at the macrophage surface. Product-binding can be prevented by prior treatment of the macrophages with neuraminidase. It appears that only a minority of sialic acids are involved in the binding process, which occurs without demonstrable blocking of adjacent sialic acids or redistribution of such sites over the macrophage surface. Parallel experiments with fixed tanned erythrocytes also suggest that binding of lymphocyte products is not solely determined by surface sialic acids, although it cannot occur without them.     

Regulation of rat liver NADP+-isocitrate dehydrogenase during aging

In an attempt to understand the mechanism of aging in relation to the differences in enzyme regulation, the induction and kinetic properties of NADP⁺ -isocitrate dehydrogenase of the liver of immature (6 weeks), mature (13 weeks), adult (33 weeks) and old (85 weeks) female rats were studied. The specific activity of the cytoplasmic and mitochondrial NADP+ -isocitrate dehydrogenase increased up to the adult age (33 weeks) and decreased in the old rats (85 weeks). Overiectomy decreased and estradiol administration induced activity of both the mitochondrial and eytoplasmic enzyme in the liver ol immature, mature and adult rats but had no significant effect in old rats. However, the activity of mitochondrial NADP⁺ -isocitrate dehydrogenase decreased and eytoplasmic NADP+ -isocitrate dehydrogenase increased following ovariectomy in old rats (85 weeks). Hormone-mediated induction of enzyme activity was actinomycin D sensitive. The Km for isocitrate and NADP, Ki value for oxalomalate, heat stability and electrophoretic mobility of the purified enzyme from the cytosol fraction of the liver of immature and old rats were similar. It can he concluded that the enzyme does not change structurally with age. Part of this work was presented at the 48th Annual General Meeting of the Society of Biological Chemist, India, 1979.     

Application of continuous zone electrophoresis to preparative separation of proteins

A comprehensive study of the application of continuous zone electrophoresis to preparative separation of proteins in free solution is presented. First, the influence of electric field strength, buffer residence time in the chamber, sample flow rate, and sample concentration on separation resolution and throughput were studied. Using multiple injections of sample into the electrophoresis chamber, a throughput of 500 mg protein/h was achieved for partially purified model proteins. Experiments on Escherichia coli crude extracts yielded a fivefold purification of beta-galactosidase along with a simultaneous separation of proteins from cell debris in a single step. Experiments correlating the electrophoretic mobility in continuous electrophoresis with the elution behavior in ion-exchange chromatography were performed on more than a dozen proteins which conclusively showed that separation of proteins in continuous zone electrophoresis is governed by net surface charge. Based on these results, the fraction numbers in which the proteins eluted could be correctly predicted. Proteins and enzymes with differences >0.5 M elution molarities in ion-exchange chromatography were separated by continuous zone electrophoresis on a preparative scale (mg/h or g/h) with >90% recovery. This corresponds to a preparative scale separation of proteins and enzymes which differ in apparent electrophoretic mobility by only 0.70 x 10(-5) cm(2)/V . s. (c) 1993 John Wiley & Sons, Inc.