Effects of monovalent cations on red cell shape and size

Human erythrocytes were incubated in isotonic solutions of different monovalent cations. The apparent size of the red cells measured on scanning electron microscopic pictures decreases in the order Li+ greater than Na+ = K+ greater than Rb+. These differences in size are abolished after pretreatment with trypsin, which removes a large part of the charges associated with membrane glycoproteins. Shape alterations are also observed. Normal biconcave shapes are visible after Na+ or K+ incubation, whereas Li+ leads to flabby, flattened cells with a certain tendency to crenation, and Rb+ causes more pronounced biconcavity with a certain tendency to cupping. The overall effects of pretreatment with trypsin are similar to those of Li+. Our results provide evidence that the electrostatic repulsion of glycoproteins and other charged membrane components may play an essential role in maintaining red cell shape.     

Effects of membrane reference state on shape memory of a red blood cell

By using a three-dimensional continuum model, we simulate the shape memory of a red blood cell after the remove of external forces. The purpose of this study is to illustrate the effect of membrane reference state on cell behavior during the recovery process. The reference state of an elastic element is the geometry with zero stress. Since the cell membrane is composed of cytoskeleton and lipid bilayer, both the reference states of cytoskeleton (RSC) and lipid bilayer (RSL) are considered. Results show that a non-spherical RSC can result in shape memory. The energy barrier due to non-spherical RSC is determined by the ratio of the equator length to the meridian length of the RSC. Thus different RSCs can have similar energy barrier and leading to identical recovery response. A series of simulations of more intermediate RSCs show that the recovery time scale is inversely proportional to the energy barrier. Comparing to spherical RSL, a spheroid RSL contributes to the energy barrier and recovery time. Furthermore, we observe a folding recovery due to the biconcave RSL which is different from the tank treading recovery. These results may motivate novel numerical and experimental studies to determine the exact RSC and RSL.     

Biophysical responses of red cell-membrane systems to very low concentrations of inorganic mercury

Changes in red blood cellsize, deformability, andosmotic fragility are indicators of altered condition and/or altered regulatory processes at the whole cell and membrane levels. An agent, such as HgCl₂, that brings about specific changes of this kind can therefore serve as a selective probe of such cell condition and regulatory state. Conversely, for a health-threatening agent “active” in this way, the cell-membrane responses serve to clarify the more fundamental bases of its toxicity, as well as to permit identification and characterization of its early and low-level actions on living systems. Taking advantage of recent advances in the technique of “resistive pulse spectroscopy,” we present a coordinated study of these three interrelated biophysical properties for the interactions of HgCl₂ with human red cells. We thereby are able to extend previous studies of this kind into domains of shorter time (instantaneous exposures), lower level exposures (down to 10⁻⁹ M, well below the level of acute human toxicity), as well as to additional kinds of responses (e.g., “dynamic osmotic hemolysis”). For conditions ranging from 10⁻⁴ to 10⁻⁹ M in HgCl₂, for instantaneous to 90-min-incubated exposures, for medium osmolarities from 120 to 300, the matrix of observed cell responses includes relative swelling as well as shrinkage, changes in deformability, and both enhancement of and protection against osmotic hemolysis. Some unexpected short-term effects of time and temperature of storage of blood cell stock samples, with respect to increasing and decreasing osmotic fragility, are also reported. These apparently disparate results are interpreted in terms of mercury interactions with cell and membrane SH groups, and a reasonable rationale is presented for most of the responses in terms of disruption of passive and active Na⁺−K⁺, gradient controls, plus interactions with cellular proteins.     

Resistance to the Brownian movement of red blood cells on flat horizontal surfaces

The movements of red blood cells (RBC), suspended in plasma, on plastic, glass, rhodium metal plate, siliconized glass, and siliconized rhodium were recorded on cinéfilm and analyzed. Values for the drag coefficient were calculated, using Einstein's theory of Brownian movement, and compared with the theoretical Stokes' hydrodynamic drag. The difference between the computed and Stokes' values gave the frictional coefficient or resistance resulting from the interaction of the cells, with the test surface. Of the three uncoated test surfaces, plastic was found to have the least interaction with the RBC. The frictional coefficient for plastic was found to be 1.75×10⁻⁷ N s m⁻¹ compared with a value of 2.82×10⁻⁷ N s m⁻¹ for rhodium metal, which had the largest interaction. Upon siliconization of the test surfaces, the interaction decreased by 40%. Reduction in the pH of the suspending plasma increased the interaction between the cells and the uncoated test surfaces, but the pH effect of diminished when the surfaces were siliconized.     

Relationship between the shape and the membrane potential of human red blood cells

Summary Microscopic observations of isotonic suspensions of human red blood cells demonstrate that cell shape is unaltered when the transmembrane electrical potential, orE _m , is set in the range –85 to +10 mV with valinomycin at varied external K⁺, or K _o .E _m was measured with the fluorescent potentiometric indicator, diS-C₃(5), as calibrated by a pH method. Repeating Glaser's experiments in which echinocytosis was attributed to hyperpolarization, we found that at low ionic strength the pH-dependent effects of amphotericin B appear to be unrelated toE _m . The effects of increased intracellular Ca²⁺, or Ca _o , on echinocytosis and onE _m are separable. With Ca ionophore A23187 half-maximal echinocytosis occurs at greater Ca _o than that which induces the half-maximal hyperpolarization associated with Ca-induced K⁺ conductance (Gardos effect). Thus, cells hyperpolarized by increased Ca _o remain discoidal when Ca is below the threshold for echinocytosis. With A23187 and higher Ca _o , extensive echinocytosis occurs in cells which are either hyperpolarized or at their resting potential. The Ca-activation curve for echinocytosis is left-shifted by low K _o , a new observation consistent with increased DIDS-sensitive uptake of⁴⁵Ca by hyperpolarized cells. These results support the following conclusions: (1) the shape and membrane potential of human red blood cells are independent under the conditions studied; (2) in cells treated with A23187, the Gardos effect facilitates echinocytosis by increasing Ca.     

Membrane and cytoplasmic resistivity properties of normal and sickle red blood cells

The cytoplasmic resistivities and membrane breakdown potentials of normal (AA), sickle-cell-trait (AS), and sickle (SS) red blood cells have been measured by the biophysical methodology of resistive pulse spectroscopy over a range of osmolalities. At isotonicity, the average membrane breakdown potentials are virtually identical for the three types of cells occurring at about 1150 V/cm. Average isotonic cytoplasmic resistivities are somewhat higher for the SS cells (166.7±7.49 ohm-cm) compared to the AA (147.6±1.98 ohm-cm) or AS cells (148.7±1.79 ohm-cm). As medium osmolality is varied, the differences in resistive properties become enlarged, especially at very low and very high osmolalities. At high osmolalities, both types of sickle cells show a large increase in internal resistivity compared to the normals; at low osmolality, the SS samples exhibit a distinctly different membrane breakdown characteristic, decreasing in this parameter, whereas the other two groups increase. Of the 15 SS samples tested, three displayed much higher cytoplasmic resistivities at isotonicity: 218.2±5.25 ohm-cm, compared to an average of 153.5±3.46 ohm-cm for the other 12. The relationship between these high resistivities and the subfraction of irreversibly sickled cells in the sample is discussed.     

Influence of increased membrane cholesterol on membrane fluidity and cell function in human red blood cells

Cholesterol and phospholipid are the two major lipids of the red cell membrane. Cholesterol is insoluble in water but is solubilized by phospholipids both in membranes and in plasma lipoproteins. Morever, cholesterol exchanges between membranes and lipoproteins. An equilibrium partition is established based on the amount of cholesterol relative to phospholipid (C/PL) in these two compartments. Increases in the C/PL of red cell membranes have been studied under three conditions: First, spontaneous increases in vivo have been observed in the spur red cells of patients with severe liver disease; second, similar red cell changes in vivo have been induced by the administration of cholesterol-enriched diets to rodents and dogs; third, increases in membrane cholesterol have been induced in vitro by enriching the C/PL of the lipoprotein environment with cholesterol-phospholipid dispersions (liposomes) having a C/PL of >1.0. In each case, there is a close relationship between the C/PL of the plasma environment and the C/PL of the red cell membrane. In vivo, the C/PL mole ratio of red cell membranes ranges from a normal value of 0.9–1.0 to values which approach but do not reach 2.0. In vitro, this ratio approaches 3.0. Cholesterol enrichment of red cell membranes directly influences membrane lipid fluidity, as assessed by the rotational diffusion of hydrophobic fluorescent probes such as diphenyl hexatriene (DPH). A close correlation exists between increases in red cell membrane C/PL and decreases in membrane fluidity over the range of membrane C/PL from 1.0 to 2.0; however, little further change in fluidity occurs when membrane C/PL is increased to 2.0–3.0. Cholesterol enrichment of red cell membranes is associated with the transformation of cell contour to one which is redundant and folded, and this is associated with a decrease in red cell filterability in vitro. Circulation in vivo in the presence of the slpeen further modifies cell shape to a spiny, irregular (spur) form, and the survival of cholesterol-rich red cells is decreased in the presence of the spleen. Although active Na-K transport is not influenced by cholesterol enrichment of human red cells, several carrier-mediated transport pathways are inhibited. We have demonstrated this effect for the cotransport of Na + K and similar results have been obtained by others in studies of organic acid transport and the transport of small neutral molecules such as erythritol and glycerol. Thus, red cell membrane C/PL is sensitive to the C/PL of the plasma environment. Increasing membrane C/PL causes a decrease in membrane fluidity, and these changes are associated with a reduction in membrane permeability, a distortion of cell contour and filterability and a shortening of the survival of redcells in vivo.     

Effect of dietary versus pharmacological correction of hypertriglyceridemia on red blood cell membrane sodium/lithium countertransport activity

An elevated red blood cell Na/Li countertransport (Na/Li CT) is often associated with high blood pressure and metabolic abnormalities. Recent studies suggested that a reduction in serum TG levels is associated with a decrease in Na/Li CT activity. However, it is still unclear if this phenomenon could be originated from systemic metabolic alterations or from modifications of the membrane dynamic properties. Aim of the present study was to investigate whether dietary or pharmacological TG lowering therapy might have a different effect on Na/Li CT activity and related metabolic parameters. Twenty normotensive hyper-TG patients were recruited from the Lipid outpatient Clinic: they had a baseline Na/Li CT activity significantly higher compared with age- and BMI-matched normolipidemic controls (386 ± 33 vs 274 ± 39 umol/l RBC/h, p < 0.05). The patients were randomly prescribed one of the following two-months treatment: Group 1)-triglyceride lowering diet; Group 2)-lipid lowering drug (Gemfibrozil 600 mg b.i.d.). Na/Li CT and metabolic and anthropometric variables were measured at baseline and after 1 and 2 months of treatment. At the end of intervention, there was in both groups a significant and comparable fall in plasma triglyceride (group 1: −2.61 ± 0.73 mmol/l p < 0.01; group 2: −4.29 ± 1.20 mmol/l p < 0.01). In the diet-treated group there were, in addition small but significant reductions in body weight (−3.7 ± 0.8 kg p < 0.01), fasting glucose (−0.36 ± 0.14 mmol/l p < 0.05) and insulin levels (−2. ± 0.5 mUl/l, p < 0.01), while no such changes were observed in the fibrate treated patients. Na/Li CT activity was significantly and comparably reduced at the end of treatment in both groups (group 1: −97 ± 28 umol/l cell/h, p < 0.01; group 2: −89 ± 30 umol/l cell/h, p < 0.01). In conclusion, these results indicate that the decrease in Na/Li CT associated with both dietary and drug treatment of hypertriglyceridemia is to be traced to a direct effect of plasma TG concentration on this transport system (probably as a result of modification in the membrane lipid environment) rather than to changes in plasma insulin levels or insulin resistance.     

Effects of ovarian hormones on cell membranes in the rat uterus

Freeze-fracture techniques have been used to study tight junctions on the lateral plasma membrane of cells of the luminal epithelium of the rat uterus under various hormonal regimes. Tight junctions from ovariectomized control rats extended some 0.5 μm down the lateral membrane and the junctional strands often formed a network of closely packed, circular compartments. Following treatment of rats with estrogen for 3 days the tight junctional regional still extended 0.5 μm down the lateral membrane, but the strands ran more parallel to the apical surface. They did not enclose circular compartments. After treatment with progesterone, either alone or with estrogen in such a way as to condition the ovariectomized uterus for implantation, a third pattern of junctional organization emerged. In these animals the junctional region extended 1.1 μm down the lateral membrane and the strands frequently crosslinked, enclosing compartments of varying and irregular size and shape. Our observations suggest that ovarian hormones could regulate the contents of the uterine lumen by altering the structure extent of the tight junctions which connect the epithelial cells enclosing the lumen.     

Effects of dietary tin and aluminum on selenium utilization by adult males

The main purpose of these studies was to determine whether the amounts of tin and aluminum that can enter foods during processing and storage are sufficient to affect the utilization of selenium by human subjects. Two 40-day balance studies were conducted. The eight adult males who participated in the first study lost significantly more selenium in their feces when fed a test diet containing 50 mg tin daily than when fed the control diet containing 0.1 mg tin daily. During the first study subjects tended to excrete less selenium in the urine when fed the test diet rather than the control diet. In the second study, the dietary treatments (5 and 125 mg aluminum daily) had no effect on the excretion and apparent retention of selenium by eight adult males.     

Fish oil diet affects on oxidative senescence of red blood cells linked to degeneration of spleen cells in mice

The effect of dietary polyunsaturated fatty acids and α-tocopherol supplementation on erythrocyte lipid peroxidation and immunocompetent cells in mice was studied comparatively using seven dietary oils (15% oil/diet, w/w) including fish oil rich in eicosapentaenoic acid (EPA, 20:5, n–3) and docosahexaenoic acid (DHA, 22:6, n–3). A 43% increase in spleen weight, about twice as many spleen cells and no change in the subpopulations of spleen cells, as well as a significant depression of mitogen-induced blastogenesis of both T and B cells in the spleen were observed in mice fed fish oil for 30 days in comparison with soybean oil diet-fed mice. In the fish oil diet-fed mice, membranous lipid hydroperoxide (hydroperoxides of phosphatidylcholine and phosphatidylethanolamine) accumulation as a marker of oxidative senescence in red blood cells (RBC) was 2.7–3.5 times higher than that in mice fed soybean oil, although there was no difference in the plasma phosphatidylcholine hydroperoxide concentration. In spite of the supplementation of α-tocopherol to up to 10 times the level in the basal diet, the degeneration of spleen cells and the stimulated oxidative senescence of RBC found by the fish oil feeding could not be prevented. The results suggest that oral intake of excess polyunsaturated fatty acids, i.e. EPA and DHA, in a fish oil diet can lead to acceleration of membrane lipid peroxidation resulting in RBC senescence linked to the lowering of immune response of spleen cells, and that supplementation of α-tocopherol as antioxidant does not always effectively prevent such oxidative degeneration as observed in spleen cells and RBC in vivo.     

Functional impairment of MSC induced by transient warming events: Correlation with loss of adhesion and altered cell size

Background

We recently showed that transient warming effects decreased the functional and adhesion properties of mesenchymal stromal cells (MSC) while post-thaw viability remained high. In an attempt to better predict functional impairment of cryopreserved MSC, we further analysed the correlation between viability, immunosuppressive activity and adhesion of cells exposed or not to warming events.

Adaptive modifications in membranes of halotolerant and halophilic microorganisms

Halotolerant and halophilic microorganisms can grow in (hyper)saline environments, but only halophiles specifically require salt. Genotypic and phenotypic adaptations are displayed by halophiles; the halotolerants adapt phenotypically, but it is not established whether they show genotypic adaptation. This paper reviews the various strategies of haloadaptation of membrane proteins and lipids by halotolerant and halophilic microorganisms. Moderate halophiles and halotolerants adapt their membrane lipid composition by increasing the proportion of anionic lipids, often phosphatidylglycerol and/or glycolipids, which in the moderately halophilic bacteriumVibrio costicola appears to be part of an osmoregulatory response to minimize membrane stress at high salinities. Extreme halophiles possess typical archaebacterial ether lipids, which are genotypically adapted by having additional substitutions with negatively-charged residues such as sulfate. In contrast to the lipids, it is less clear whether membrane proteins are haloadapted, although they may be more acidic; very few depend on salt for their activity.     

In vivo and in vitro effects of aluminum treatment on rat liver mitochondrial function

This study examines the effect on mitochondrial respiration and permeability of in vivo and in vitro aluminium (Al) exposure. Rats were treated intraperitoneally with AlCl₃ to achieve serum and liver Al concentrations comparable to those seen in Al-related disorders. Mitochondria isolated from Al-treated rats had higher (p<0.01) Al concentration, lower (p<0.05) state 3 respiration, respiratory control (RCR), and ADP/O ratio (succinate substrate), and greater passive swelling in 100 mM KCl or 200 mM NH₄NO₃ than controls. The in vitro addition of Al (0–180 μM) to mitochondria from normal rats also decreased (p<0.01) state 3 respiration, RCR, and ADP/O and stimulated passive swelling in KCl and NH₄NO₃ at 42–180 μM Al. These studies show that Al depresses mitochondrial energy metabolism and increases membrane permeability. The toxicity associated with Al may be related to its effect on mitochondria.     

Kinetics of virus adsorption to single cells using fluorescent membrane probes and multiparameter flow cytometry

Different genetic stains of avian RNA tumor virus (ATV) were labeled with the fluorescent membrane probe R-18 (rhodamine conjugated to a hydrocarbon chain) and cellular receptors for virus infection were analyzed on a rapid, single-cell basis by a multiparameter cell sorter. Chicken cells genetically susceptible to various R-18 ATV were found to adsorb much more virus, as measured by increased fluorescent binding, than did genetically resistant chicken cells. Virus binding to receptor sites could be saturated with increased concentrations of labeled virus. This binding could be altered by removal of the polycation, polybrene, indicating the important influence of electrostatic forces. Correlated time measurements of virus binding to single cells were taken with these fluorescence measurements allowing for a minute-to-minute study of the kinetics of viral adsorption to resistant and susceptible cells. The ratio of fluorescence (proportional to the number of virions bound per cell) to light scatter (proportional to cell surface area) on a cell-to-cell basis was analyzed to examine the heterogeneity in fluorescent virion bound per unit cell surface area within a given cell type. With these calculations, it was found that a large amount, but not all, of observed fluorescence heterogeneity merely reflects differences in cell surface areas. However, there are significant differences in viral receptor site densities within this supposedly homogeneous population of cells. This study represents a successful application of fluorescent membrane probes and flow cytometry to the study of cellular responses to viral infection at the single-cell level. Sine large numbers of cells can be examined rapidly, small subpopulations of live virally susceptible or resistant cells can be cloned by multiparameter cell sorting.     

Interaction of the exported malaria protein Pf332 with the red blood cell membrane skeleton

Intra-erythrocytic Plasmodium falciparum malaria parasites synthesize and export numerous proteins into the red blood cell (RBC) cytosol, where some bind to the RBC membrane skeleton. These interactions are responsible for the altered antigenic, morphological and functional properties of parasite-infected red blood cells (IRBCs). Plasmodium falciparum protein 332 (Pf332) is a large parasite protein that associates with the membrane skeleton and who's function has recently been elucidated. Using recombinant fragments of Pf332 in in vitro interaction assays, we have localised the specific domain within Pf332 that binds to the RBC membrane skeleton to an 86 residue sequence proximal to the C-terminus of Pf332. We have shown that this region partakes in a specific and saturable interaction with actin (K_d = 0.60 µM) but has no detectable affinity for spectrin. The only exported malaria protein previously known to bind to actin is PfEMP3 but here we demonstrate that there is no competition for actin-binding between PfEMP3 and Pf332, suggesting that they bind to different target sequences in actin.     

稻草覆盖和香根草篱对红壤水稳性团聚体组成及有机碳含量的影响

基于植物篱和秸秆覆盖控制红壤坡耕地水土流失的长期定位试验,研究香根草篱(H)、稻草覆盖(M)、香根草篱+稻草覆盖(HM)水保措施下红壤水稳性团聚体组成及有机碳分布特征。结果表明:与常规等高农作模式(CK)相比,草篱、稻草覆盖、草篱+稻草覆盖模式下土壤总有机碳含量提高0.07—2.42 g/kg。草篱对土壤团聚体组成及其结合有机碳的影响在篱内效果显著,随着与草篱距离增大影响减弱。草篱和稻草覆盖对土壤团聚体组成和结合有机碳含量的影响不同,草篱主要增加2 mm水稳性团聚体含量及其结合有机碳含量,稻草覆盖增加0.25 mm水稳性团聚体含量及其结合有机碳含量。综合来看,草篱和稻草覆盖相结合对改善坡面土壤结构作用稳定。土壤有机碳含量较高时,土壤总有机碳含量与粒径2mm的大团聚体有机碳含量呈显著正相关(r=0.659);随着有机碳含量降低,土壤总有机碳含量与土壤0.25—0.053 mm和0.053 mm微小团聚体碳含量相关性逐渐增大。