Hemoglobin-mediated selenium export from red blood cells

On the basis of the fact that selenium from selenite binds to hemoglobin (Hb), we investigated the missing process in the selenium export from red blood cells (RBCs), i.e., the transfer of selenium bound to Hb to RBC membrane proteins. To elucidate the molecular events of the Hb-associated selenium export from RBC, a Hb–Se complex was synthesized from thiol-exchange of Cys-β93 in Hb with penicillamine-substituted glutathione selenotrisulfide, as a model of major metabolic intermediates, and then interactions between the Hb–Se complex and RBC inside-out vesicles (IOVs) were examined. Selenium bound to Hb was transferred to the IOV membrane on the basis of the intrinsic interactions between Hb and the cytoplasmic domains of band 3 protein (CDB3). The observed selenium transfer was inhibited by the pretreatments of IOVs with iodoacetamide and the α-chymotrypsin digestion, indicating that the Hb mediates the selenium transfer to the thiol groups of CDB3. In addition, it was found that deoxygenated Hb, with a high binding affinity for CDB3, more favorably transferred selenium to the IOV membranes than oxygenated Hb, with a low affinity. When selenium export from RBC to the plasma was examined by continuously introducing nitrogen gas, the selenium export rate was promoted with an increase in the rate of deoxygenated Hb. Overall, these data suggested that Hb could possibly play a role in the selenium export from RBC treated with selenite in an oxygen-linked fashion. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Properties of the basal calcium influx in human red blood cells

The basal ⁴⁵Ca²⁺ influx in human red blood cells (RBC) into intact RBC was measured. ⁴⁵Ca²⁺ was equilibrated with cells with t_1/2=15-20 s and the influx reached the steady state value in about 90-100 s and the steady state level was 1.5±0.2 μmol/l_{packed cells} (n=6) at 37 °C. The average value of the Ca²⁺ influx rate was 43.2±8.9 μmol/l_{packed cells} hour. The rate of the basal influx was pH-dependent with a pH optimum at pH 7.0 and on the temperature with the temperature optimum at 25 °C. The basal Ca²⁺ influx was saturable with Ca²⁺ up to 5 mmol/l but at higher extracellular Ca²⁺ concentrations caused further increase of basal Ca²⁺ influx. The ⁴⁵Ca²⁺ influx was stimulated by addition of submicromolar concentrations of phorbol esters (phorbol 12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu)) and forskolin. Uncoupler (3,3′,4′,5-tetrachloro-salicylanilide (TCS) 10⁻⁶-10⁻⁵ mol/l) inhibited in part the Ca²⁺ influx. The results show that the basal Ca²⁺ influx is mediated by a carrier and is under control of intracellular regulatory circuits. The effect of uncoupler shows that the Ca²⁺ influx is in part driven by the proton-motive force and indicates that the influx and efflux of Ca²⁺ are coupled via the RBC H⁺ homeostasis.     

Interrelations among Na and K content,cell volume,and buoyant density in human red blood cell populations

Summary This study establishes a method for determining the concentration of Na and K in single red blood cells from electron probe microanalysis of a cell's Na and K content. To this end, red blood cells were separated into subpopulations according to their buoyant density by means of bovine serum density gradient centrifugation. Cell water and Na+K contents were then determined in each fraction by conventional analytic methods with cell volume estimated from measurements of hematocrits and cell number. It was found that an inverse relationship obtains between the mean cell volume and buoyant cell density since cells increased in size as density decreased. Although the amount of hemoglobin per cell was found to slightly increase as cell density decreased, hemoglobin concentration showed the inverse relationship, indicating that buoyant cell density differences are primarily the result of differences in hemoglobin concentration. In confirmation of Funder and Wieth (Funder, J., Wieth, J.O. 1966.Scand. J. Lab. Invest. 18:167–180) cell water and cell volume was found to vary directly with the summed content of Na+K. Finally, by means of electron probe microanalysis of single cells, the cellular concentration of hemoglobin was found to vary inversely with the Na+K content, providing a quantitative basis for directly estimating cell volume, and thus ionic concentration, with this technique.     

Thermodynamic studies on oxygen binding by human red blood cells

Oxygen equilibrium curves have been measured on human normal red blood cells, at the temperatures of 20, 25, 30, 37 and 41 degrees C, and at pHs ranging from 6.8 to 8.2. The thermodynamical parameters have been determined for the four successive steps of oxygenation and for overall oxygenation, according to the Adair and MWC models [Monod J, Wyman J, Changeux JP. On the nature of allosteric transitions: a plausible model. J Mol Biol 1965;12:88-118]. The heat release appears to be nearly equal for the four steps. At the first three steps, the delta H change is counterbalanced by a nearly equivalent change of delta S, resulting in a rather small delta G value. delta G is greater at the fourth step, because of diminution of this enthalpy-entropy compensation phenomenon. The four steps are both enthalpy and entropy driven. According to the MWC model, the T to R transition is endothermic, and allosteric quaternary transition occurs at binding of the third oxygen. The average heat release increases by 2.8 kcal/mol when pH raises from 7.4 to 8.2, but flattens below pH 7.4. After correction for the heat of solution of oxygen and for the heat of proton release (referred to intracellular pH), an intrinsic heat for oxygenation of the heme of approximately--13 kcal/mol is obtained for the successive steps of oxygenation (at pH 7.4, 37 degrees C). These results are compared with those previously obtained for pigeon and trout red blood cells.     

Three-dimensional focusing of red blood cells in microchannel flows for bio-sensing applications

Three-dimensional (3D) focusing of particles in microchannels has been a long-standing issue in the design of biochemical/biomedical microdevices. Current microdevices for 3D cell or bioparticle focusing involve complex channel geometries in view of their fabrication because they require multiple layers and/or sheath flows. This paper proposes a simple method for 3D focusing of red blood cells (RBCs) in a single circular microcapillary, without any sheath flows, which is inspired from the fluid dynamics phenomenon in that a spherical particle lagging behind a Poiseuille flow migrates toward and along the channel axis. More explicitly, electrophoresis of RBCs superimposed on the pressure-driven flow is utilized to generate an RBC migration mode analogous to this phenomenon. A particle-tracking scheme with a sub-pixel resolution is implemented to spatially position red blood cells flowing through the channel, so that a probability density function (PDF) is constructed to evaluate the tightness of the cell focusing. Above a specific strength of the electric field, approximately 90% of the sheep RBCs laden in the flow are tightly focused within a beam diameter that is three times the cell dimension. Particle shape effect on the focusing is discussed by making comparisons between the RBCs and the spherical particles. The lateral migration velocity, predicted by an existing theoretical model, is in good agreement with the present experimental data. It is noteworthy that 3D focusing of non-spherical particles, such as RBCs, has been achieved in a circular microchannel, which is a significant improvement over previous focusing methodologies.     

Diffusional water permeability of mammalian red blood cells

An extensive programme of comparative nuclear magnetic resonance measurements of the membrane diffusional permeability for water (P_d) and of the activation energy (E_a,d) of this process in red blood cells (RBCs) from 21 mammalian species was carried out. On the basis of P_d, these species could be divided into three groups. First, the RBC's from humans, cow, sheep and “large” kangaroos (Macropus giganteus and Macropus rufus) had P_d values 5 × 10⁻³ cm/s at 25°C and 7 × 10⁻³ cm/s at 37°C. The RBCs from other marsupial species, mouse, rat, guinea pig and rabbit, had P_d values roughly twice higher, whereas echidna RBCs were twice lower than human RBCs. The value of E_a,d was in most cases correlated with the values of P_d. A value of E_a,d -26 kJ/mol was found for the RBCs from humans and the species having similar P_d values. Low values of E_a,d (ranging from 15 to 22 kJ/mol) appeared to be associated with relatively high values of P_d. The highest value of E_a,d (33 kJ/mol) was found in echidna RBCs. This points to specialized channels for water diffusion incorporated in membrane proteins; a relatively high water permeability of the RBC membrane could be due to a greater number of channel proteins. There are, however, situations where a very high water permeability of RBCs is associated with a high value of E_a,d (above 25 kJ/mol) as in the case of RBCs from mouse, rat and tree kangaroo. Moreover, it was found that P_d in different species was positively correlated to the RBC membrane phosphatidylcholine and negatively correlated to the sphingomyelin content. This suggests that in addition to the number of channel proteins, other factors are involved in the water permeability of the RBC membrane.     

Sugar moiety of cardiac glycosides is essential for the inhibitory action on the palytoxin-induced K+ release from red blood cells

Palytoxin (PTX), a highly toxic and sugar-containing substance isolated from Palythoa tuberculosa, caused K+ release from rabbit red blood cells. Cardiac glycosides, such as ouabain, convallatoxin, cymarin, digoxin and digitoxin, inhibited the PTX-induced K+ release. Their corresponding aglycones did not inhibit the K+ release, but antagonized the inhibitory effect of the glycosides. All these cardiotonic steroids equally inhibited the activity of (Na+ + K+)-ATPase prepared from hog cerebral cortex. These results suggest that the sugar moiety of the cardiac glycosides is important for the inhibitory effect on the K+ release induced by PTX and that the inhibition is not related to their inhibitory potency on the (Na+ + K+)-ATPase activity.     

Visualization study of motion and deformation of red blood cells in a microchannel with straight,divergent and convergent sections

The size of red blood cells (RBC) is on the same order as the diameter of microvascular vessels. Therefore, blood should be regarded as a two-phase flow system of RBCs suspended in plasma rather than a continuous medium of microcirculation. It is of great physiological and pathological significance to investigate the effects of deformation and aggregation of RBCs on microcirculation. In this study, a visualization experiment was conducted to study the microcirculatory behavior of RBCs in suspension. Motion and deformation of RBCs in a microfluidic chip with straight, divergent, and convergent microchannel sections have been captured by microscope and high-speed camera. Meanwhile, deformation and movement of RBCs were investigated under different viscosity, hematocrit, and flow rate in this system. For low velocity and viscosity, RBCs behaved in their normal biconcave disc shape and their motion was found as a flipping motion: they not only deformed their shapes along the flow direction, but also rolled and rotated themselves. RBCs were also found to aggregate, forming rouleaux at very low flow rate and viscosity. However, for high velocity and viscosity, RBCs deformed obviously under the shear stress. They elongated along the flow direction and performed a tank-treading motion.     

药物滥用者红细胞免疫功能的变化

通过检测红细胞C3b受体花环（RBC－C3bRR)、红细胞免疫复合物光环（RBC－ICR）、粘附增强因子活性（RFER）及抑制因子活性（RFIR）,观察了50例海洛因依赖者红细胞免疫功能的变化。结果表明,海洛因依赖者RBC－C3bRR、RFER明显下降（P＜0．01）,而RBC-ICR、RFIR则明显升高（P＜0．01）。本文对检测结果进行了初步探讨。     

PLC-dependent intracellular Ca2+ release was associated with C6-ceramide-induced inhibition of Na+ current in rat granule cells

In this report, the effects of C(6)-ceramide on the voltage-gated inward Na(+) currents (I(Na)), two types of main K(+) current [outward rectifier delayed K(+) current (I(K)) and outward transient K(+) current (I(A))], and cell death in cultured rat cerebellar granule cells were investigated. At concentrations of 0.01-100 microM, ceramide produced a dose-dependent and reversible inhibition of I(Na) without alteration of the steady-state activation and inactivation properties. Treatment with C(2)-ceramide caused a similar inhibitory effect on I(Na). However, dihydro-C(6)-ceramide failed to modulate I(Na). The effect of C(6)-ceramide on I(Na) was abolished by intracellular infusion of the Ca(2+)-chelating agent, 1,2-bis (2-aminophenoxy) ethane-N, N, N9, N9-tetraacetic acid, but was mimicked by application of caffeine. Blocking the release of Ca(2+) from the sarcoplasmic reticulum with ryanodine receptor blocker induced a gradual increase in I(Na) amplitude and eliminated the effect of ceramide on I(Na). In contrast, the blocker of the inositol 1,4,5-trisphosphate-sensitive Ca(2+) receptor did not affect the action of C(6)-ceramide. Intracellular application of GTPgammaS also induced a gradual decrease in I(Na) amplitude, while GDPbetaS eliminated the effect of C(6)-ceramide on I(Na). Furthermore, the C(6)-ceramide effect on I(Na) was abolished after application of the phospholipase C (PLC) blockers and was greatly reduced by the calmodulin inhibitors. Fluorescence staining showed that C(6)-ceramide decreased cell viability and blocking I(Na) by tetrodotoxin did not mimic the effect of C(6)-ceramide, and inhibiting intracellular Ca(2+) release by dantrolene could not decrease the C(6)-ceramide-induced cell death. We therefore suggest that increased PLC-dependent Ca(2+) release through the ryanodine-sensitive Ca(2+) receptor may be responsible for the C(6)-ceramide-induced inhibition of I(Na), which does not seem to be associated with C(6)-ceramide-induced granule neuron death.     

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.     

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.     

Studies on the methodology of the carboxyfluorescein assay and on the mechanism of liposome stabilization by red blood cells in vitro

The stability of small unilamellar liposomes was investigated in human blood, in vitro. Using the carboxyfluorescein technique, interaction between the dye, the detergent Triton X-100, and an as yet unidentified component of human serum grossly interferes with the experiment and necessitates the use of other detergents, preferably sodium deoxycholate. Separation of liposomes and blood cells by centrifugation induces a small leakage from the liposomes and can lead to an underestimation of the real liposome stability. Upon incubation with whole blood, intact liposomes are absorbed nonspecifically to erythrocytes and internalized by leukocytes, the extent and kinetics of the former process being insenstive to the presence of metabolic inhibitors. The stability of liposomes is significantly enhanced in whole blood or in serum containing washed erythrocytes. Similarly, liposome stability in serum could be augmented be presaturating the serum lipoproteins with excess phospholipid. Our work adds support to previous notions that stable liposomes with high affinities for certain blood-cell components might be developed as suitable carrier systems for drug targetting in pathological disorders within the blood stream.     

The effect of radiotherapy and chemotherapy on osmotic fragility of red blood cells and plasma levels of malondialdehyde in patients with breast cancer

BackgroundGamma radiation effects on the erythrocyte membrane from three different functional parts, lipid bilayer, cytoskeleton and protein components. When the red cell membrane is exposed to radiation, it loses its integrity and hemoglobin leaks out. In addition, irradiation leads to lipid peroxidation and the products of this process, leading to hemolysis. The aim of the present study was to measure osmotic fragility (OF) of red blood cells and malondialdehyde (MDA) levels as a marker of oxidative injury in breast cancer patients treated with radiation and chemotherapy.Materials and MethodsThe OF test was performed using different concentrations of a salt solution. The measurement of MDA was done with chemical methods.¹¹ The sampling was taken during three stages of treatment: first sample was taken before starting chemotherapy, the second sample was taken before radiation therapy and the third sample was taken after radiotherapy.ResultsNo statistically significant differences between levels of MDA in these three stages of treatment were observed. However, the comparison of mean levels of MDA showed an increase after radiotherapy. The OF rate did not show significant difference (P > 0.05) during the stages of treatment.ConclusionIn a standard treatment program of radiotherapy and chemotherapy lipid peroxidation level and OF do not significantly increase.     

Analysis of short RNAs in the malaria parasite and its red blood cell host

RNA interference (RNAi) is an RNA degradation process that involves short, double-stranded RNAs (dsRNA) as sequence specificity factors. The natural function of the RNAi machinery is to generate endogenous short double-stranded RNAs to regulate gene expression. It has been shown that treatment of Plasmodium falciparum, the etiologic agent of malaria, with dsRNA induces degradation of the corresponding microRNA (miRNA), yet typical RNAi-associated genes have not been identifiable in the parasite genome. To clarify this discrepancy we set out to clone short RNAs from P. falciparum-infected red blood cells and from purified parasites. We did not find any short RNA that was not a rRNA or tRNA fragment. Indeed, only known human miRNAs were isolated in parasite preparations indicating that very few if any short RNAs exist in P. falciparum. This suggests a different mechanism than classical RNAi in observations of dsRNA-mediated degradation. Of the human miRNAs identified, the human miRNA mir-451 accumulates at a very high level in both infected and healthy red blood cells. Interestingly, mir-451 was not detectable in a series of immortalised cell lines representing progenitor stages of all major blood lineages, suggesting that mir-451 may play a role in the differentiation of erythroid cells.     

Effect of carbonyl compounds on red blood cells deformability

The effect of Maillard reaction on red blood cells (RBC) deformability was investigated. Exposure of RBC to carbonyl compounds (dl-glyceraldehyde, glyoxal, glycolaldehyde, 3-deoxyglucosone, and d-glucose) leading to Maillard reaction caused a marked decrease in RBC deformability even at 1 mM level. The decrease rate depended on the kind of carbonyl compounds, in which both dl-glyceraldehyde and glyoxal significantly decreased the RBC deformability (p < 0.05). In addition, the decrease rate also differed among volunteers tested, indicating that the sensitivity against carbonyl compounds varies among them. In order to elucidate the mechanism of the decrease in RBC deformability, RBC was exposed to carbonyl compounds in the presence of aminoguanidine which is the inhibitor of AGE formation in Maillard reactions. Aminoguanidine inhibited the decrease in RBC deformability by dl-glyceraldehyde and glyoxal. When Hb which has a high reactivity with carbonyl compounds was incubated with those carbonyl compounds, dl-glyceraldehyde and glyoxal showed the high reactivity with Hb compared with other carbonyl compounds. These results indicate that Maillard reaction between RBC proteins and carbonyl compounds leads to the decrease in RBC deformability. On the other hand, generated by carbonyl compounds involved in lowering the deformability only to a negligible level.     

Comparative physiology and molecular analysis of carbonic anhydrase from the red blood cells of teleost fish

This study investigates the early evolution of vertebrate red blood cell (rbc) carbonic anhydrase (CA) by examining the physiological and molecular properties of rbc CA in teleost fish. When representatives of four different families of teleosts were compared, it was found that differences in overall rbc CA activity were due to different concentrations of CA, rather than differences in the enzymes kinetic properties. Additional molecular analysis of CA from the rbcs of rainbow trout provided further evidence that critical elements of the enzyme, such as the active site, have been highly conserved during vertebrate evolution. The active site of the trout CA differed from that of gar rbc CA at only two amino acid positions. The rainbow trout rbc CA sequence also showed high sequence homology with CA sequences from other fish tissues, and fits into an emerging group of fish CAs that are basal to mammalian CA I, II and III. Northern blot analysis of the tissue expression of the sequenced CA indicated that it is primarily found in the rbcs, but high amounts of cytosolic CA activity were also found in the gill, suggesting the presence of other cytosolic CA isozymes in this species.Abbreviations Az acetazolamide - CA carbonic anhydrase - MP maximum parsimony - NJ neighbour joining - RACE rapid amplification of cDNA ends - rbc red blood cellCommunicated by L.C.-H. Wang     

Effects of antioxidants and haemoglobin status on the t-butyl hydroperoxide-induced oxygen uptake by red blood cells

Oxygen uptake by erythrocytes exposed to t-butyl hydroperoxide (t-BHP) exhibited an induction period. The rate of oxygen consumption can be reduced by antioxidants and blood plasma. The induction time was not appreciably modified by the antioxidants tested, however, plasma increased it by a factor of two. The in vivo pretreatment with diethyl maleate (0.6 g kg-1) produced increased rates of oxygen uptake without changes in the induction period, while vitamin E (12.5 mg kg-1) elicited lower oxygen consumption rates and longer induction times, compared to those observed in cells from control rats upon addition of the hydroperoxide. These results suggest that the antioxidants tested on the t-BHP lipid peroxidation in erythrocyte suspensions act as inhibitors and/or retarders of the process. Furthermore, lipid peroxidation induced in these conditions seems to depend upon the haemoglobin status of the cells as oxygen uptake, malondialdehyde production and chemiluminescence were significantly higher in methaemoglobin-containing cells than in those containing oxyhaemoglobin.     

Inhibition of anion transport in the red blood cell membrane by anionic and non-anionic arginine-specific reagents

Arginine specific reagents are found to be powerful inhibitors of anion exchange in the red blood cell membrane. Some of these inhibitors such as cyclohexandione, phenylglyoxal and 2, 3-butandione are found to produce their inhibition by interacting covalently with band 3. In contrast to the action of these compounds, the inhibition caused by the phenylglyoxal derivative 4-hydroxy-3-nitrophenyl-glyoxal has been found to be completly reversible. In extending the studies on the mode of action of these compounds on sulfate exchange and to get some more information about their binding site, the degree of inhibition caused by different phenylglyoxal derivatives which have a similar core but differ in their substituent groups have been compared. The interaction between the binding sites of these compounds and other anion transport inhibitors have also been studied.