The antioxidant status during maturation of reticulocytes to erythrocytes in type 2 diabetics

Free radical-induced lipid peroxidation has been associated with numerous disease processes including diabetes mellitus. The extent of lipid peroxidation (LPO) and antioxidant defense system [i.e., levels of glutathione (GSH), glucose-6-phosphate dehydrogenase (G6PDH), glutathione reductase (GR), glutathione peroxidase (GPx), glutathione-S-transferase (GST), and catalase (CAT)] were evaluated in reticulocytes and erythrocytes of type 2 diabetic males and age-matched controls. Type 2 diabetics have shown increased lipid peroxidation and decreased levels of GSH, GR, GPx, G6PDH, and GST both in reticulocytes and erythrocytes compared to controls, indicating the presence of oxidative stress and defective antioxidant systems in these patients. CAT activity is found to be enhanced in both the reticulocytes and erythrocytes of diabetics, with a greater percentage enhancement in reticulocytes. The extent of increase in lipid peroxidation is greater in erythrocytes compared to reticulocytes in these patients. Furthermore, the maturation of reticulocytes to erythrocytes resulted in decreased GSH and decreased activities of all antioxidant enzymes (except CAT) both in normals and type 2 diabetes individuals, indicating decreased scavenging capacity as reticulocytes mature to erythrocytes. These maturational alterations are further intensified in type 2 diabetics. The present study reveals that the alterations in lipid peroxidation and antioxidant system lean toward early senescence of erythrocytes in type 2 diabetic patients.     

Human erythrocyte recycling of ascorbic acid: relative contributions from the ascorbate free radical and dehydroascorbic acid

Recycling of ascorbic acid from its oxidized forms helps to maintain the vitamin in human erythrocytes. To determine the relative contributions of recycling from the ascorbate radical and dehydroascorbic acid, we studied erythrocytes exposed to a trans-membrane oxidant stress from ferricyanide. Ferricyanide was used both to induce oxidant stress across the cell membrane and to quantify ascorbate recycling. Erythrocytes reduced ferricyanide with generation of intracellular ascorbate radical, the concentrations of which saturated with increasing intracellular ascorbate and which were sustained over time in cells incubated with glucose. Ferricyanide also generated dehydroascorbic acid that accumulated in the cells and incubation medium to concentrations much higher than those of the radical, especially in the absence of glucose. Ferricyanide-stimulated ascorbate recycling from dehydroascorbic acid depended on intracellular GSH but was well maintained at the expense of intracellular ascorbate when GSH was severely depleted by diethylmaleate. This likely reflects continued radical reduction, which is not dependent on GSH. Erythrocyte hemolysates showed both NAD- and NADPH-dependent ascorbate radical reduction. The latter was partially due to thioredoxin reductase. GSH-dependent dehydroascorbate reduction in hemolysates, which was both direct and enzyme-dependent, was greater than that of the radical reductase activity but of lower apparent affinity. Together, these results suggest an efficient two-tiered system in which high affinity reduction of the ascorbate radical is sufficient to remove low concentrations of the radical that might be encountered by cells not under oxidant stress, with back-up by a high capacity system for reducing dehydroascorbate under conditions of more severe oxidant stress.     

Ascorbate-and iron-driven redox activity of Dp44mT and Emodin facilitates peroxidation of micelles and bicelles

BackgroundIron (Fe)-induced oxidative stress leads to reactive oxygen species that damage biomembranes, with this mechanism being involved in the activity of some anti-cancer chemotherapeutics.MethodsHerein, we compared the effect of the ligand, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), or the potential ligand, Emodin, on Fe-catalyzed lipid peroxidation in cell membrane models (micelles and bicelles). These studies were performed in the presence of hydrogen peroxide (H₂O₂) and the absence or presence of ascorbate.ResultsIn the absence of ascorbate, Fe(II)/Emodin mixtures incubated with H₂O₂ demonstrated slight pro-oxidant properties on micelles versus Fe(II) alone, while the Fe(III)-Dp44mT complex exhibited marked antioxidant properties. Examining more physiologically relevant phospholipid-containing bicelles, the Fe(II)- and Fe(III)-Dp44mT complexes demonstrated antioxidant activity without ascorbate. Upon adding ascorbate, there was a significant increase in the peroxidation of micelles and bicelles in the presence of unchelated Fe(II) and H₂O₂. The addition of ascorbate to Fe(III)-Dp44mT substantially increased the peroxidation of micelles and bicelles, with the Fe(III)-Dp44mT complex being reduced by ascorbate to the Fe(II) state, explaining the increased reactivity. Electron paramagnetic resonance spectroscopy demonstrated ascorbyl radical anion generation after mixing ascorbate and Emodin, with signal intensity being enhanced by H₂O₂. This finding suggested Emodin semiquinone radical formation that could play a role in its reactivity via ascorbate-driven redox cycling. Examining cultured melanoma cells in vitro, ascorbate at pharmacological levels enhanced the anti-proliferative activity of Dp44mT and Emodin.Conclusions and general significanceAscorbate-driven redox cycling of Dp44mT and Emodin promotes their anti-proliferative activity.     

Protein and lipid oxidation of banked human erythrocytes: role of glutathione

In banked human erythrocytes (RBCs), biochemical and functional changes are accompanied with vesiculation and reduced in vivo survival. We hypothesized that some of these changes might have resulted from oxidative modification of membrane lipids, proteins, or both as a result of atrophy of the antioxidant defense system(s). In banked RBCs, we observed a time-dependent increase in protein clustering, especially band 3; carbonyl modification of band 4.1; and malondialdehyde, a lipid peroxidation product. Examination of the antioxidative defense system showed a time-dependent decline in glutathione (GSH) concentration and glutathione-peroxidase (GSH-PX) activity, with a concomitant increase in extracellular GSH, cysteine, and homocysteine, and unchanged catalase activity. When subjected to acute oxidant stress by exposure to ferric/ascorbic acid or tert-butylhydroperoxide (tert-BHT), catalase activity showed a steeper decline compared with GSH-PX. The results demonstrate that GSH and GSH-PX appear to provide the primary antioxidant defense in stored RBCs, and their decline, concurrent with an increase in oxidative modifications of membrane lipids and proteins, may destabilize the membrane skeleton, thereby compromising RBC survival.     

Lipid peroxidation and antioxidant enzyme activities in erythrocytes of type I and II alcoholics

Reduced and oxidized glutathione (GSH and GSSG), protein-bound glutathione, lipid peroxidation and antioxidant enzyme activities were determined in the erythrocyte lysates and membranes of type I and II alcoholics in order to clarify the effect of age-of-onset and the duration of the alcohol consumption on erythrocyte oxidant and antioxidant status. The osmotic fragility and susceptibility of the erythrocytes to haemolysis were also determined. Erythrocyte lipid peroxidation was significantly increased but, GSH and protein-bound GSH, GSH/GSSG ratio and antioxidant enzyme activities were markedly decreased in the erythrocytes of the alcoholic subgroups. Erythrocyte count and haemoglobin content in the blood of alcoholics were found to be decreased in accordance with the finding that erythrocytes were more fragile and less resistant to haemolysis particularly in type II alcoholics. The present study showed that ethanol-induced oxidative stress in erythrocytes can lead to haemolysis and membrane-specific injuries in erythrocytes of the alcoholic subtypes.     

Effect of 50 Hz, 0.2 mT magnetic fields on RBC properties and heart functions of albino rats

In this work the effect of sinusoidal 50 Hz, 0.2 mT magnetic fields on the red blood cells (RBCs) and heart functions of Albino rats were investigated. Twenty-four male Albino rats were equally divided into four groups, A, B, C, and D. Animals from groups B were continuously exposed to the magnetic field for 15 days; and groups C and D, for 30 days. Group A was used as control. Animals from group D were kept after exposure to the magnetic field for a period of 45 days for delayed effect studies. The osmotic fragility and shape of RBCs' membrane and hemoglobin (Hb) structure tests were carried out for all groups. The dielectric relaxation of Hb molecules was measured in the frequency range of 0.1-10 MHz and the dielectric increment (Deltaepsilon), relaxation time (tau), molecular radius (r), and Cole-Cole parameter (alpha) were calculated for all groups. The ECG was measured for all animals before and after exposure to the magnetic field. The results indicated that exposure of the animals to 50 Hz, 0.2 mT magnetic fields resulted in the decrease of RBCs membrane elasticity and permeability and changes in the molecular structure of Hb. The ECG of the exposed animals was considerably altered. The data also indicated that there was no sign of repair in the newly generated RBCs structure and the ECG after removing the animals from the magnetic field, which indicates that the blood generating system was severely injured. The injuries in the heart of the animals were attributed to the loss of some physiological functions of the RBCs as a result of exposures of the rats to the magnetic field.     

Effect of magnetic fields on antioxidative defense and fitness-related traits of Baculum extradentatum (insecta, phasmatodea)

This study aimed to determine the effect of magnetic fields on the antioxidative defense and fitness-related traits of Baculum extradentatum. Following exposure to magnetic fields, antioxidative defense (superoxide dismutase (SOD), catalase (CAT) activities, and total glutathione (GSH) content) and fitness-related traits (egg mortality, development dynamics, and mass of nymphs) were monitored in nymphs. The experimental groups were: control (kept out of influence of the magnets), a group exposed to a constant magnetic field (CMF) of 50 mT, and a group exposed to an alternating magnetic field (AMF) of 50 Hz, 6 mT. We found increased SOD and CAT activities in animals exposed to constant and AMFs, whereas GSH activity was not influenced by experimental magnetic fields. No differences were found in egg mortality between control and experimental groups. Significant differences in the time of development between the control and the CMF group were observed, as well as between the CMF and the AMF group. No differences were found in the mass of the nymphs between the three experimental groups. In conclusion, CMF and AMF have the possibility to modulate the antioxidative defense and some of the fitness-related traits in B. extradentatum.     

Electrophoretic characterization and subcellular distribution of hexokinase isoenzymes in red blood cells of rabbits

The isoenzyme pattern of hexokinase in rabbit red cells (erythrocytes, fetal erythrocytes and reticulocytes) were determined by means of agarose gel and disc electrophoresis. One duplicated hexokinase (4a and 4b according to the IUPAC-nomenclature) was detected in rabbit erythrocytes as also described for human erythrocytes. Besides the isoenzymes 4a and 4b reticulocytes also contain hexokinase 2 and 3 like rabbit and rat liver. The high KM glucose phosphorylating enzyme, hexokinase 1 could be demonstrated only under specific conditions in the reticulocytes during the initial stage of the anemia. After the fractionation of reticulocyte homogenates the total hexokinase activity was recovered in the mitochondria and cytosol to nearly equal amounts as revealed by the distribution of markers. Hexokinase 2 and 3 were detectable in reticulocytes and in isolated mitochondria only after the addition of certain dissociating agents. In contrast to the tightly bound mitochondrial hexokinases 2 and 3 the type 4a and 4b are more loosely bound and exhibit a bilocal distribution between mitochondria and cytosol of reticulocytes.     

Enhancement of the hydrolysis activity of F0F1‐ATPases using 60 Hz magnetic fields

The effects of extremely low frequency (ELF) magnetic fields on membrane F₀F₁‐ATPase activity have been studied. When the F₀F₁‐ATPase was exposed to 60 Hz magnetic fields of different magnetic intensities, 0.3 and 0.5 mT magnetic fields enhanced the hydrolysis activity, whereas 0.1 mT exposure caused no significant changes. Even if the F₀F₁‐ATPase was inhibited by N,N‐dicyclohexylcarbodiimide, its hydrolysis activity was enhanced by a 0.5 mT 60 Hz magnetic field. Moreover, when the chromatophores which were labeled with F‐DHPE were exposed to a 0.5 mT, 60 Hz magnetic field, it was found that the pH of the outer membrane of the chromatophore was unchanged, which suggested that the magnetic fields used in this work did not affect the activity of F0. Taken together, our results show that the effects of magnetic fields on the hydrolysis activity of the membrane F₀F₁‐ATPases were dependent on magnetic intensity and the threshold intensity is between 0.1 and 0.3 mT, and suggested that the F1 part of F₀F₁‐ATPase may be an end‐point affected by magnetic fields. Bioelectromagnetics 30:663–668, 2009. © 2009 Wiley‐Liss, Inc.     

Glutathione loading prevents free radical injury in red blood cells after storage

We have previously demonstrated that the loss of glutathione (GSH) and GSH-peroxidase (GSH-PX) in banked red blood cells (RBCs) is accompanied by oxidative modifications of lipids, proteins and loss of membrane integrity^[1]. The objective of this study was to determine whether artificial increases in antioxidant (GSH) or antioxidant enzyme (catalase) content could protect membrane damage in the banked RBCs following an oxidant challenge. RBCs stored at 1–6°C for 0, 42 and 84 days in a conventional additive solution (Adsol®) were subjected to oxidative stress using ferric/ascorbic acid (Fe/ASC) before and after enriching them with GSH or catalase using a hypotonic lysis-isoosmotic resealing procedure. This lysis-resealing procedure in the presence of GSH/catalase raised intracellular GSH and catalase concentrations 4–6 fold, yet produced only a small reduction in mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentrations (MCHC). Indicators of oxidative stress and membrane integrity were measured, including acetylcholinesterase (AChE) activity, GSH concentration, phosphatidylserine (PS) externalization (prothrombin-converting activity) and transmembrane lipid movements (¹⁴C-lyso phosphatidylcholine flip-flop and PS transport). GSH-enrichment protected AChE activity in fresh (0 day) and stored (42 and 84 days) RBCs from Fe/ASC oxidation by 10, 23 and 26%, respectively, compared with not-enriched controls. Following oxidative stress, the rate of transbilayer lipid flip-flop did not increase in fresh cells, but increased 9.3% in 42-day stored cells. Phosphatidylserine exposure, as measured by prothrombinase activity, increased 2.4-fold in fresh and 5.2-fold in 42-day stored cells exposed to Fe/ASC. Previous studies have shown that 42-day storage causes a moderate decrease in PS transport (～ 50 %), whereas transport rates declined by up to 75% in stored RBCs when challenged with Fe/ASC. GSH-enrichment prevented the increase in passive lipid flip-flop and the increase in prothrombinase activity, but offered no protection against oxidative damage of PS transport. In contrast to these effects, catalase-enrichment failed to protect GSH levels and AChE activity upon oxidative stress. Membrane protein thiol oxidation was assessed by labeling reactive protein thiols with 5-acetalamidofluorescein followed by immunoblotting with antifluorescein antibodies. Significant oxidation of membrane proteins was confirmed by a greater loss of thiols in stored RBCs than in fresh RBCs. These results demonstrate that it may be possible to prevent storage-mediated loss of AChE, increased lipid flip-flop, and increased PS exposure, by maintaining or increasing GSH levels of banked RBCs.     

Glutathione loading prevents free radical injury in red blood cells after storage

We have previously demonstrated that the loss of glutathione (GSH) and GSH-peroxidase (GSH-PX) in banked red blood cells (RBCs) is accompanied by oxidative modifications of lipids, proteins and loss of membrane integrity^[1]. The objective of this study was to determine whether artificial increases in antioxidant (GSH) or antioxidant enzyme (catalase) content could protect membrane damage in the banked RBCs following an oxidant challenge. RBCs stored at 1-6°C for 0, 42 and 84 days in a conventional additive solution (Adsol®) were subjected to oxidative stress using ferric/ascorbic acid (Fe/ASC) before and after enriching them with GSH or catalase using a hypotonic lysis-isoosmotic resealing procedure. This lysis-resealing procedure in the presence of GSH/catalase raised intracellular GSH and catalase concentrations 4-6 fold, yet produced only a small reduction in mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentrations (MCHC). Indicators of oxidative stress and membrane integrity were measured, including acetylcholinesterase (AChE) activity, GSH concentration, phosphatidylserine (PS) externalization (prothrombin-converting activity) and transmembrane lipid movements (¹⁴C-lyso phosphatidylcholine flip-flop and PS transport). GSH-enrichment protected AChE activity in fresh (0 day) and stored (42 and 84 days) RBCs from Fe/ASC oxidation by 10, 23 and 26%, respectively, compared with not-enriched controls. Following oxidative stress, the rate of transbilayer lipid flip-flop did not increase in fresh cells, but increased 9.3% in 42-day stored cells. Phosphatidylserine exposure, as measured by prothrombinase activity, increased 2.4-fold in fresh and 5.2-fold in 42-day stored cells exposed to Fe/ASC. Previous studies have shown that 42-day storage causes a moderate decrease in PS transport (∼ 50 %), whereas transport rates declined by up to 75% in stored RBCs when challenged with Fe/ASC. GSH-enrichment prevented the increase in passive lipid flip-flop and the increase in prothrombinase activity, but offered no protection against oxidative damage of PS transport. In contrast to these effects, catalase-enrichment failed to protect GSH levels and AChE activity upon oxidative stress. Membrane protein thiol oxidation was assessed by labeling reactive protein thiols with 5-acetalamidofluorescein followed by immunoblotting with antifluorescein antibodies. Significant oxidation of membrane proteins was confirmed by a greater loss of thiols in stored RBCs than in fresh RBCs. These results demonstrate that it may be possible to prevent storage-mediated loss of AChE, increased lipid flip-flop, and increased PS exposure, by maintaining or increasing GSH levels of banked RBCs.     

Inactivation of Yeast Glutathione Reductase by Fenton Systems: Effect of Metal Chelators, Catecholamines and Thiol Compounds

Oxygen radical generating systems, namely, Cu(II)/ H₂O₂, Cu(II)/ascorbate, Cu(II)/NAD(P)H, Cu(II)/ H₂O₂/catecholamine and Cu(II)/H₂O₂/SH-compounds irreversibly inhibited yeast glutathione reductase (GR) but Cu(II)/H₂O₂ enhanced the enzyme diaphorase activity. The time course of GR inactivation by Cu(II)/H₂O₂ depended on Cu(II) and H₂O₂ concentrations and was relatively slow, as compared with the effect of Cu(II)/ascorbate. The fluorescence of the enzyme Tyr and Trp residues was modified as a result of oxidative damage. Copper chelators, catalase, bovine serum albumin and HO^˙ scavengers prevented GR inactivation by Cu(II)/H₂O₂ and related systems. Cysteine, N-acetylcysteine, N-(2-dimercaptopropi-onylglycine and penicillamine enhanced the effect of Cu(II)/H₂O₂ in a concentration- and time-dependent manner. GSH, Captopril, dihydrolipoic acid and dithiotreitol also enhanced the Cu(II)/H₂O₂ effect, their actions involving the simultaneous operation of pro-oxidant and antioxidant reactions. GSSG and try-panothione disulfide effectively protected GR against Cu(II)/H₂O₂ inactivation. Thiol compounds prevented GR inactivation by the radical cation ABTS*⁺. GR inactivation by the systems assayed correlated with their capability for HO* radical generation. The role of amino acid residues at GR active site as targets for oxygen radicals is discussed.     

Erythrocyte enrichment in hematopoietic progenitor cell cultures based on magnetic susceptibility of the hemoglobin

Using novel media formulations, it has been demonstrated that human placenta and umbilical cord blood-derived CD34+ cells can be expanded and differentiated into erythroid cells with high efficiency. However, obtaining mature and functional erythrocytes from the immature cell cultures with high purity and in an efficient manner remains a significant challenge. A distinguishing feature of a reticulocyte and maturing erythrocyte is the increasing concentration of hemoglobin and decreasing cell volume that results in increased cell magnetophoretic mobility (MM) when exposed to high magnetic fields and gradients, under anoxic conditions. Taking advantage of these initial observations, we studied a noninvasive (label-free) magnetic separation and analysis process to enrich and identify cultured functional erythrocytes. In addition to the magnetic cell separation and cell motion analysis in the magnetic field, the cell cultures were characterized for cell sedimentation rate, cell volume distributions using differential interference microscopy, immunophenotyping (glycophorin A), hemoglobin concentration and shear-induced deformability (elongation index, EI, by ektacytometry) to test for mature erythrocyte attributes. A commercial, packed column high-gradient magnetic separator (HGMS) was used for magnetic separation. The magnetically enriched fraction comprised 80% of the maturing cells (predominantly reticulocytes) that showed near 70% overlap of EI with the reference cord blood-derived RBC and over 50% overlap with the adult donor RBCs. The results demonstrate feasibility of label-free magnetic enrichment of erythrocyte fraction of CD34+ progenitor-derived cultures based on the presence of paramagnetic hemoglobin in the maturing erythrocytes.     

Rabbit red blood cell hexokinase:intracellular distribution during reticulocytes maturation

Summary The intracellular localization and isozyme distribution of hexokinase were studied during rabbit reticulocyte maturation and aging. In reticulocytes 50% of the enzyme was particulate while in the mature erythrocytes all the hexokinase activity was soluble. The bound enzyme co-sediments with mitochondria and by column chromatography it was found to be hexokinase Ia. The cytosol of reticulocytes contains hexokinase Ia (38%) and hexokinase Ib (62%) while the mature erythrocytes contain only hexokinase Ia. The amount of bound hexokinase decreases very quickly during cell maturation and aging as was shown by following in vivo reticulocyte maturation or by analysis of hexokinase compartmentation in cells of different ages, obtained by density gradient ultracentrifugations. A role for this intracellular distribution of hexokinase is suggested.     

Ascorbic acid blunts oxidant stress due to menadione in endothelial cells

Endothelial cells are exposed to potentially damaging reactive oxygen species generated both within the cells and in the bloodstream and underlying vessel wall. In this work, we studied the ability of ascorbic acid to protect cultured human-derived endothelial cells (EA.hy926) from oxidant stress generated by the redox cycling agent menadione. Menadione caused intracellular oxidation of dihydrofluorescein, which required the presence of D-glucose in the incubation medium, and was inhibited by intracellular ascorbate and desferrioxamine. At concentrations of 100 microM and higher, menadione depleted the cells of both GSH and ascorbate, and ascorbate loading partially prevented the decrease in GSH due to menadione. Menadione increased L-arginine uptake by the cells, but inhibited endothelial nitric oxide synthase, an effect that was prevented by acute loading with ascorbate. Ascorbate blunts menadione-induced oxidant stress in EA.hy926 cells, which may help to preserve nitric oxide synthase activity under conditions of excessive oxidant stress.     

Copper-specific damage in human erythrocytes exposed to oxidative stress

Ascorbate and complexes of Cu(II) and Fe(III) are capable of generating significant levels of oxygen free radicals. Exposure of erythrocytes to such oxidative stress leads to increased levels of methemoglobin and extensive changes in cell morphology. Cu(II) per mole is much more effective than Fe(III). However, isolated hemoglobin is oxidized more rapidly and completely by Fe(III)- than by Cu(II)-complexes. Both Fe(III) and Cu(II) are capable of inhibiting a number of the key enzymes of erythrocyte metabolism. The mechanism for the enhanced activity of Cu(II) has not been previously established. Using intact erythrocytes and hemolysates we demonstrate that Cu(II)-, but not Fe(III)-complexes in the presence of ascorbate block NADH-methemoglobin reductase. Complexes of Cu(II) alone are not inhibitory. The relative inability of Fe(III)-complexes and ascorbate to cause methemoglobin accumulation is not owing to Fe(III) association with the membrane, or its failure to enter the erythrocytes. The toxicity of Cu(II) and ascorbate appears to be a result of site-specific oxidative damage of erythrocyte NADH-methemoglobin reductase and the enzyme's subsequent inability to reduce the oxidized hemoglobin.     

Iron transport mechanisms in reticulocytes and mature erythrocytes

The mechanism of iron transport into erythroid cells was investigated using rabbit reticulocytes and mature erythrocytes incubated with ⁵⁹Fe-labelled Fe(II) in isotonic sucrose or in solutions in which the sucrose was replaced with varying amounts of isotonic NaCl or KCl. Iron uptake was inhibited at all concentrations of NaCl, in a concentration-dependent manner, but with KCl inhibition occurred only at concentrations up to 10 mM. Higher KCl concentrations stimulated iron uptake to the cytosol of the cells, but inhibited its incorporation into heme. This effect became more marked as the iron concentration was raised. It was found that KCl inhibits iron incorporation into heme and stimulates iron uptake by mature erythrocytes, as well as by reticulocytes. It is concluded that erythroid cells can take up nontransferrin-bound Fe(II) by two mechanisms. One is a high-affinity mechanism that is limited to reticulocytes, saturates at a low iron concentration, and is inhibited by metabolic inhibitors. The other is a low-affinity process that is found in both reticulocytes and erythrocytes, becomes more prominent at higher iron concentrations, and is stimulated by KCl, as well as RbCl, LiCl, CsCl, and choline Cl. The KCl stimulation is inhibited by amiloride, but not by metabolic inhibitors, and its operation is not dependent on changes in cell volume or membrane potential, but it does require the presence of a permeant extracellular anion. Iron uptake by this process appears to occur by facilitated transport and is possibly assoicated with exchange of Na⁺. A further aspect of this study was a comparison of iron uptake by reticulocytes from Fe(II)-sucrose and Fe(II)-ascorbate using a variety of incubation conditions. No major differences were observed. © 1995 Wiley-Liss, Inc.     

The isolation of reticulocyte-free human red blood cells

We depleted reticulocytes from erythrocytes of both sickle cell disease (SCD) subjects and healthy controls by four methods: fluorescence-activated cell sorting (FACS), Miltenyi immunomagnetic depletion (MACS), a combination of these methods (FACS + MACS) and Percoll density separation. The efficiency of these methods was assessed by new methylene blue staining and manual enumeration of the reticulocytes. FACS sorted erythrocytes from reticulocytes based on size and granularity, as well as the absence of dsDNA staining. MACS depleted reticulocytes from erythrocytes based on the immunoaffinity to CD36 and CD71. Reticulocytes from healthy controls were depleted to 相似文献   

CD and MCD spectroscopic studies of the two Dps miniferritin proteins from Bacillus anthracis: role of O2 and H2O2 substrates in reactivity of the diiron catalytic centers

DNA protection during starvation (Dps) proteins are miniferritins found in bacteria and archaea that provide protection from uncontrolled Fe(II)/O radical chemistry; thus the catalytic sites are targets for antibiotics against pathogens, such as anthrax. Ferritin protein cages synthesize ferric oxymineral from Fe(II) and O(2)/H(2)O(2), which accumulates in the large central cavity; for Dps, H(2)O(2) is the more common Fe(II) oxidant contrasting with eukaryotic maxiferritins that often prefer dioxygen. To better understand the differences in the catalytic sites of maxi- versus miniferritins, we used a combination of NIR circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field MCD (VTVH MCD) to study Fe(II) binding to the catalytic sites of the two Bacillus anthracis miniferritins: one in which two Fe(II) react with O(2) exclusively (Dps1) and a second in which both O(2) or H(2)O(2) can react with two Fe(II) (Dps2). Both result in the formation of iron oxybiomineral. The data show a single 5- or 6-coordinate Fe(II) in the absence of oxidant; Fe(II) binding to Dps2 is 30× more stable than Dps1; and the lower limit of K(D) for binding a second Fe(II), in the absence of oxidant, is 2-3 orders of magnitude weaker than for the binding of the single Fe(II). The data fit an equilibrium model where binding of oxidant facilitates formation of the catalytic site, in sharp contrast to eukaryotic M-ferritins where the binuclear Fe(II) centers are preformed before binding of O(2). The two different binding sequences illustrate the mechanistic range possible for catalytic sites of the family of ferritins.     

RNA oxidation catalyzed by cytochrome c leads to its depurination and cross-linking, which may facilitate cytochrome c release from mitochondria

Growing evidence indicates that RNA oxidation is correlated with a number of age-related neurodegenerative diseases, and RNA oxidation has also been shown to induce dysfunction in protein synthesis. Here we study in vitro RNA oxidation catalyzed by cytochrome c (cyt c)/H(2)O(2) or by the Fe(II)/ascorbate/H(2)O(2) system. Our results reveal that the products of RNA oxidation vary with the oxidant used. Guanosine residues are preferentially oxidized by cyt c/H(2)O(2) relative to the Fe(II)/ascorbate/H(2)O(2) system. GC/MS and LC/MS analyses demonstrated that the guanine base was not only oxidized but also depurinated to form an abasic sugar moiety. Results from gel electrophoresis and HPLC analyses show that RNA formed a cross-linked complex with cyt c in an H(2)O(2) concentration-dependent manner. Furthermore, when cyt c was associated with liposomes composed of cardiolipin/phosphatidylcholine, and incubated with RNA and H(2)O(2), it was found cross-linked with the oxidized RNA and dissociated from the liposome. Results of the quantitative analysis indicate that the release of the cyt c from the liposome is facilitated by the formation of an RNA-cyt c cross-linked complex. Thus, RNA oxidation may facilitate the release of cyt c from the mitochondrial membrane to induce apoptosis in response to oxidative stress.