Starvation-induced autophagocytosis paradoxically decreases the susceptibility to oxidative stress of the extremely oxidative stress-sensitive NIT insulinoma cells

Glucose and amino acid starvation of cells in culture generally enhances their sensitivity to oxidative stress. This is explained by compensatory autophagocytosis, which results in increased amounts of lysosomal low-molecular-weight, redox-active iron, due to the degradation of metallo-proteins, with a potential increase in iron-catalyzed, intralysosomal oxidative reactions. Such reactions diminish the stability of lysosomal membranes, with resultant leakage of hydrolytic enzymes into the cytosol and ensuing cellular degeneration, often of apoptotic type. However, starvation of NIT insulinoma cells, which are normally remarkably sensitive to oxidative stress, actually attenuated the sensitivity to such stress. We found that starved NIT cells rapidly synthesized ferritin. Moreover, ferritin was found to be autophagocytosed, and the lysosomes were stabilized, as assayed by the acridine orange relocation test. We hypothesize that compensatory autophagocytosis during starvation increases the cytosolic pool of redox-active iron, as a reflection of enhanced transportation of low-molecular-weight iron from autophagic lysosomes to the cytosol, resulting in ferritin induction. The newly formed ferritin would, in turn, become autophagocytosed and bind redox-active lysosomal iron in a non-redox-active form. We also suggest that the proposed mechanism may be a way for oxidative stress-sensitive cells to compensate partly for their failing capacity to degrade hydrogen peroxide before it leaks into the acidic vacuolar apparatus and induces intralysosomal oxidative stress. The insulin-producing beta cell may belong to this type of cells.     

The effect of cetiedil on red cell membrane permeability

We report the effects of cetiedil, a new antisickling agent, on red cell membrane permeability. With fresh red cells containing normal levels of intracellular ATP, cetiedil increases membrane permeability to both sodium and potassium. With drug concentrations from 100 to 500 microM, net sodium gain exceeds net potassium loss, and the cells quickly swell. Changes are identical with normal and sickle red cells. Membrane permeability returns to normal after washing the cells in buffer free of cetiedil. In the absence of phosphate, ouabain potentiates the cetiedil effect. With external phosphate present, the effect of cetiedil is also enhanced, but ouabain is without effect. Our findings support the idea that the antisickling effect of cetiedil observed in vitro is secondary to cell swelling.     

EPR determination of low molecular weight iron content applied to whole rat hepatocytes

Electron paramagnetic resonance (EPR) has been described as suitable for the evaluation of low molecular weight (LMW) iron in liver homogenates after chelation by desferrioxamine. LMW iron is a highly toxic iron species incriminated in free radical production. The first aim of the study was to evaluate the conditions of EPR application for LMW iron content determination in whole rat hepatocytes. For this purpose, LMW iron was simultaneously quantified by EPR and by atomic absorption spectrometry, EPR determination of LMW iron needed a preincubation of hepatocyte cultures with the iron chelator for at least on hr. Deferiprone as LMW iron chelator was revealed to be more suited than desferrioxamine. Secondly, we showed the applicability of this methods for evaluating the prooxidant status during an oxidative stress. As an example, oxidative stress induced by ethanol in hepatocytes was studied during inflammatory circumstances, well-known to lead to nitric oxide production. In hepatocyte cultures supplemented with ethanol, an evaluation of LMW iron content was observed in cells. But when nitric oxide donors or a supplementation constituted of lipopolysaccharide and gamma-interferon, able to induce nitric oxide synthase, were added, LMW iron content decreased. Thus EPR determination of LMW iron content in whole hepatocytes could give some insight about the mechanism of induction or inhibition of a oxidative stress.     

Red blood cell indices, cation content, and membrane cation transports

In sickle cell disease, in the homozygous state, the increased heterogeneity of erythrocytes results mainly from membrane defects secondary to Hb S polymerization and the increased survival of F cells. The density distribution curve, using phthalate esters or the red blood cell indices measured with the H*3 system, are useful methods for the hematological follow-up of patients under specific therapies. The methods evaluating the red blood cell cation contents and the abnormal membrane potassium transport pathways are also described, in order to evaluate agents which can restore normal hemoglobin concentration and water content in dehydrated sickle cells.     

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Cytosolic low molecular weight phosphotyrosine-phosphatase shows dephosphorylating activity of the band 3 protein. Increased phosphorylation of this protein increases membrane rigidity and resistance to invasion of red blood cells by malarial parasites. This observation may explain the negative association previously reported by our group between the high activity *C allele of cytosolic low molecular weight phosphotyrosine-phosphatase and past malarial morbidity.     

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An iron-mediated oxidative stress caused by an increase of the intracellular pool of low molecular weight complex of iron (LMWC) can be observed with iron overloading or ethanol metabolism. The aim of this study was to determine whether nitric oxide (NO) behaved as a pro-oxidant or an antioxidant in such an iron-mediated oxidative stress in rat hepatocytes. The cells were set up in primary cultures and incubated with lipopolysaccharide (LPS) and gamma-interferon (IFN) for 18 hours to induce NO synthase and to trigger NO production. Then 20 micromol/L iron or 50 mmol/L ethanol were added. Oxidative stress was evaluated by measuring lipoperoxidation using two markers: malondialdehyde (MDA) and conjugated dienes. Simultaneously, NO production was followed by the quantitation of nitrites in the culture medium, dinitrosyl iron complexes (DNICs) and mononitrosyl iron complexes (MNICs) in intact hepatocytes. DNIC and MNIC, evaluated by electron paramagnetic resonance (EPR), corresponded to NO bound to iron-containing molecules and to free NO, respectively. In cultures preincubated with LPS and IFN before iron or ethanol addition, a net decrease of lipid peroxidation induced by either NO, iron, or ethanol was noted. Moreover, an elevation of iron-bound NO and a decrease of free NO were observed in these cultures compared with the cultures incubated with only LPS and IFN. These data support the idea that there is a relationship between the changes of NO pool and the inhibition of oxidative stress. In addition, using N(G)-monomethyl-L-arginine (L-NMMA), a NO synthase inhibitor, NO was shown to be involved in the inhibition of oxidative stress induced by iron or ethanol. Addition of the chelator of LMWC iron, deferiprone, was followed by the inhibition of the increase of iron-bound NO and the reincrease of lipid peroxidation extent, which was as high as in cultures incubated only with LPS and IFN. Thus LMWC iron appeared to be involved also in the inhibition of oxidative stress induced by NO. All the results favor the conclusion that NO acts as an antioxidant in iron-mediated oxidative stress in rat hepatocytes. NO reacted with LMWC iron to form inactive iron complexes unable to induce oxidative stress in rat hepatocytes. Thus NO played a critical role in protecting the liver from oxidative stress.     

Plasma and red cell lipids in sickle cell disease

Lipids, in particular phospholipids, are essential components of membrane systems, and the measurement of phospholipids and cholesterol in plasma and tissues is helpful in diagnosis. Phospholipids represent about 60 to 70% of total red cell (RBC) lipids, while about 25% is free cholesterol. Lipids in RBC are present in a dynamic state of equilibrium, and the RBC have the capacity for rapid exchange of lipids with plasma in several ways. The present study examined the cholesterol and phospholipid levels of plasma and erythrocytes in male patients with sickle cell anemia and in healthy male individuals of comparable age. This was performed with a view to detecting possible differences that might be related to some of the RBC abnormalities which accompany the disease. The results show that plasma lipids are significantly reduced in patients with sickle cell anemia and that RBC cholesterol was higher in sickle cell patients than in normal subjects.     

Aspirin increases ferritin synthesis in endothelial cells: a novel antioxidant pathway

Aspirin has recently been shown to increase endothelial resistance to oxidative damage. However, the mechanism underlying aspirin-induced cytoprotection is still unknown. Using cultured cells, the present study investigates the effect of aspirin on the expression of ferritin, a cytoprotective protein that sequesters free cytosolic iron, the main catalyst of oxygen radical formation. In bovine pulmonary artery endothelial cells, aspirin at low antithrombotic concentrations (0.03 to 0.3 mmol/L) induced the synthesis of ferritin protein in a time- and concentration-dependent fashion up to 5-fold over basal levels, whereas ferritin H (heavy chain) mRNA remained unaltered. Aspirin-induced cytoprotection from hydrogen peroxide toxicity was mimicked by exogenous iron-free apoferritin but not iron-loaded ferritin, demonstrating the antioxidant function of newly synthesized ferritin under these conditions. Ferritin induction by aspirin was specific in that other nonsteroidal anti-inflammatory drugs such as salicylic acid, indomethacin, or diclofenac failed to alter ferritin protein levels. Aspirin-induced ferritin synthesis was abrogated in the presence of the iron chelator desferrioxamine, pointing to an interaction of aspirin with iron-responsive activation of ferritin translation. Together, our results suggest induction of ferritin as a novel mechanism by which aspirin may prevent endothelial injury in cardiovascular disease, eg, during atherogenesis.     

Removal of erythrocyte membrane iron in vivo ameliorates the pathobiology of murine thalassemia

Abnormal deposits of free iron are found on the cytoplasmic surface of red blood cell (RBC) membranes in beta-thalassemia. To test the hypothesis that this is of importance to RBC pathobiology, we administered the iron chelator deferiprone (L1) intraperitoneally to beta-thalassemic mice for 4 wk and then studied RBC survival and membrane characteristics. L1 therapy decreased membrane free iron by 50% (P = 0.04) and concomitantly improved oxidation of membrane proteins (P = 0.007), the proportion of RBC gilded with immunoglobulin (P = 0.001), RBC potassium content (P < 0.001), and mean corpuscular volume (P < 0.001). Osmotic gradient ektacytometry confirmed a trend toward improvement of RBC hydration status. As determined by clearance of RBC biotinylated in vivo, RBC survival also was significantly improved in L1-treated mice compared with controls (P = 0.007). Thus, in vivo therapy with L1 removes pathologic free iron deposits from RBC membranes in murine thalassemia, and causes improvement in membrane function and RBC survival. This result provides in vivo confirmation that abnormal membrane free iron deposits contribute to the pathobiology of thalassemic RBC.     

Anionic polysaccharides inhibit adhesion of sickle erythrocytes to the vascular endothelium and result in improved hemodynamic behavior

The abnormal adherence of sickle red blood cells (SS RBC) to vascular endothelium may play an important role in vasoocclusion in sickle cell anemia. Thrombospondin (TSP), unusually large molecular weight forms of von Willebrand factor, and laminin are known to enhance adhesion of SS RBC. Also, these endothelial proteins bind to sulfated glycolipids and this binding is inhibited by anionic polysaccharides. Reversible sickling may expose normally cryptic membrane sulfatides that could mediate this adhesive interaction. In this study, we have investigated the effect of anionic polysaccharides, in the presence or absence of TSP, on SS RBC adhesion to the endothelium, using cultured human umbilical vein endothelial cells (HUVEC) (for the adhesion assay) and the ex vivo mesocecum of the rat (for hemodynamic evaluation). The baseline adhesion (ie, without added TSP) of SS RBC to HUVEC was most effectively inhibited by high molecular weight dextran sulfate (HDS), whereas low molecular weight dextran sulfate (LDS) and the glycosaminoglycan chondroitin sulfate A (CSA) also had significant inhibitory effects. Heparin was mildly effective whereas other glycosaminoglycans (chondroitin sulfates B and C, heparan sulfate, and fucoidan) were ineffective. Similarly, HDS and CSA resulted in an improved hemodynamic behavior of SS RBC. Soluble TSP caused significant increases in SS RBC adhesion and in the peripheral resistance. Both HDS and CSA prevented TSP-enhanced adhesion and hemodynamic abnormalities. Thus, anionic polysaccharides can inhibit SS RBC-endothelium interaction in the presence or absence of soluble TSP. These agents may interact with RBC membrane component(s) and prevent TSP-mediated adhesion of SS RBC to the endothelium.     

The secondary alcohol metabolite of doxorubicin irreversibly inactivates aconitase/iron regulatory protein-1 in cytosolic fractions from human myocardium

Anticancer therapy with doxorubicin (DOX) is limited by severe cardiotoxicity, presumably reflecting the intramyocardial formation of drug metabolites that alter cell constituents and functions. In a previous study, we showed that NADPH-supplemented cytosolic fractions from human myocardial samples can enzymatically reduce a carbonyl group in the side chain of DOX, yielding a secondary alcohol metabolite called doxorubicinol (DOXol). Here we demonstrate that DOXol delocalizes low molecular weight Fe(II) from the [4Fe-4S] cluster of cytoplasmic aconitase. Iron delocalization proceeds through the reoxidation of DOXol to DOX and liberates DOX-Fe(II) complexes as ultimate by-products. Under physiologic conditions, cluster disassembly abolishes aconitase activity and forms an apoprotein that binds to mRNAs, coordinately increasing the synthesis of transferrin receptor but decreasing that of ferritin. Aconitase is thus converted into an iron regulatory protein-1 (IRP-1) that causes iron uptake to prevail over sequestration, forming a pool of free iron that is used for metabolic functions. Conversely, cluster reassembly converts IRP-1 back to aconitase, providing a regulatory mechanism to decrease free iron when it exceeds metabolic requirements. In contrast to these physiologic mechanisms, DOXol-dependent iron release and cluster disassembly not only abolish aconitase activity, but also affect irreversibly the ability of the apoprotein to function as IRP-1 or to reincorporate iron within new Fe-S motifs. This damage is mediated by DOX-Fe(II) complexes and reflects oxidative modifications of -SH residues having the dual role to coordinate cluster assembly and facilitate interactions of IRP-1 with mRNAs. Collectively, these findings describe a novel mechanism of cardiotoxicity, suggesting that intramyocardial formation of DOXol may perturb the homeostatic processes associated with cluster assembly or disassembly and the reversible switch between aconitase and IRP-1. These results may also provide a guideline to design new drugs that mitigate the cardiotoxicity of DOX.     

Viability and function of stored sickle erythrocytes

Functional and metabolic characteristics of fresh and three-week stored erythrocytes from patients with sickle cell anemia were compared. The storage-related changes in ATP, 2,3-DPG, and P50 in sickle erythrocytes were similar to those in control (HbA) red blood cells. After storage in CPD, sickle erythrocytes maintained significantly higher levels of 2,3-DPG (mean 2.20 +/- 0.73 mM/ml RBC) than did control cells (mean 0.36 +/- 0.13 mM/ml RBC). The posttransfusion recovery and survival of stored SS erythrocytes in autologous recipients and in an animal test system were at least as good as those before storage. Tolerance of the storage lesion by sickle erythrocytes is probably related to their young mean cell age. These results also suggest that the option of autotransfusion should be explored for selected patients with sickle cell disease in special clinical settings.     

Social anxiety in children with anxiety disorders: relation with social and emotional functioning

BACKGROUND: Oxygen radicals have been implicated as important mediators in the early pathogenesis of acute pancreatitis, but the mechanism by which they produce pancreatic tissue injury remains unclear. We have, therefore, investigated the effects of oxygen radicals on isolated rat pancreatic acinar cells as to the ultrastructure, cytosolic Ca2+ concentration and energy metabolism. METHODS: Acinar cells were exposed to an oxygen radical-generating system consisting of xanthine oxidase, hypoxanthine and chelated iron ions. Cell injury was assessed by LDH release and electron microscopy. Cytosolic Ca2+ levels and mitochondrial membrane potential were determined by flow cytometry; adenine nucleotide concentrations by HPLC. Mitochondrial dehydrogenase activity was measured by spectrophotometric assay. RESULTS: Oxygen radicals damaged the plasma membrane as shown by a 6-fold LDH increase in the incubation medium within 180 min. At the ultrastructural level, mitochondria were the most susceptible to oxidative stress. In correlation to the pronounced mitochondrial damage, the mitochondrial dehydrogenase activity declined by 70%, whereas the mitochondrial membrane potential was enhanced by 27% after 120 min. Together this may cause the 85% decrease in the ATP concentration and the corresponding increase in ADP/AMP observed in parallel. In addition, an immediate 26% increase in cytosolic Ca2+ was found, a change which could be inhibited by BAPTA, reducing cellular damage. CONCLUSION: Cytosolic Ca2+ synergizes with oxygen radicals causing alterations of the ultrastructure and energy metabolism of acinar cells which might contribute to the cellular changes found in early stages of acute pancreatitis.     

Localization and seizure-regulation of integrin beta 1 mRNA in adult rat brain

Lipofuscin (age pigment) is a brown-yellow, electron-dense, autofluorescent material that accumulates progressively over time in lysosomes of postmitotic cells, such as neurons and cardiac myocytes. The exact mechanisms behind this accumulation are still unclear. This review outlines the present knowledge of age pigment formation, and considers possible mechanisms responsible for the increase of lipofuscin with age. Numerous studies indicate that the formation of lipofuscin is due to the oxidative alteration of macromolecules by oxygen-derived free radicals generated in reactions catalyzed by redox-active iron of low molecular weight. Two principal explanations for the increase of lipofuscin with age have been suggested. The first one is based on the notion that lipofuscin is not totally eliminated (either by degradation or exocytosis) even at young age, and, thus, accumulates in postmitotic cells as a function of time. Since oxidative reactions are obligatory for life, they would act as age-independent enhancers of lipofuscin accumulation, as well as of many other manifestations of senescence. The second explanation is that the increase of lipofuscin is an effect of aging, caused by an age-related enhancement of autophagocytosis, a decline in intralysosomal degradation, and/or a decrease in exocytosis.     

Characterization of two homologous yeast genes that encode mitochondrial iron transporters

Two different yeast genes were identified that when overexpressed suppressed the low iron growth defect of a mutation in the endoplasmic reticulum iron binding enzyme methyl sterol oxidase. These genes were determined to be novel and highly related. The deduced amino acid sequences indicated that both were membrane proteins having two identical histidine-rich motifs. The predicted proteins, while not ABC transporters, are homologous to a widely distributed family of transition metal transporters present in all kingdoms. Subcellular fractionation and fluorescence microscopy localized these gene products to mitochondria. Based on this result we term these genes Mitochondrial Fe Transporters (MFT). Cells with disruptions in both genes show a growth defect on low iron medium, suggesting that these genes have redundant function and can affect cytosolic iron levels. Measurement of mitochondrial iron in cells grown in iron-rich medium overexpressing MFT1 or MFT2 show a 2-5-fold increase in iron compared with mitochondria from control cells. These results suggest that the mitochondria may act as a reservoir for iron that can be mobilized and used for cytosolic purposes.     

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A series of permeability thresholds to Ca2+ metabolites and macromolecules, occurring at different times when cells are attacked by complement, has been established by imaging HeLa cells transiently expressing a recombinant cytosolic fusion protein of firefly luciferase and aequorin (luciferase-aequorin) to measure changes in ATP and cytosolic free Ca2+. Nuclear fluorescence of propidium was used as a measure of permeability to small molecules, and luciferase activity imaged to assess lysis. The rise in cytosolic free Ca2+ observed after C9 attack preceded by at least 60 s both the increase in propidium fluorescence, measured in single cells, and the decrease in ATP monitored by luciferase light emission. These effects were dependent on the concentration of C9. At concentrations of C9 up to 4 micrograms/ml no loss of luciferase-aequorin protein was detected at the end of the experiment. Thus the membrane integrity of the cells remained intact, even though the cells were permeable to propidium. These results confirmed our earlier observations that propidium permeability in cells attacked by complement was not a reliable measure of cell death. They also show that it is vital to take account of cellular heterogeneity if the mechanisms by which cells respond to membrane pore former attack are to be correctly interpreted.     

Iron-balance and dose-response studies of the oral iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1) in iron-loaded patients with sickle cell disease

Several life-threatening complications of the common disorder sickle cell disease require management with red blood cell transfusions and, hence, long-term iron-chelating therapy. The efficacy of the oral iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1) has not previously been determined in patients with sickle cell disease. We compared the efficacy of L1 to that of standard-dose subcutaneous deferoxamine in four regularly transfused patients with homozygous sickle cell disease, who had evidence of severe iron overload and a history of poor compliance with deferoxamine. Determination of 24-hour urinary iron excretion conducted over 5 days immediately after transfusion showed that the mean daily urinary iron excretion induced by L1 at 75 mg/kg/d (0.48 +/- 0.23 mg/kg) was equivalent to that induced by deferoxamine at 50 mg/kg/d (0.39 +/- 0.06 mg/kg). In two of three patients studied, a significant (P < .025) increase in mean daily urinary iron excretion was achieved when the dose of L1 was increased to 100 mg/kg/d. Total iron balance studies, which quantitated both urinary and stool iron excretion on L1 and deferoxamine, determined that mean total daily iron excretion induced by deferoxamine (0.88 +/- 0.05 mg/kg) was significantly greater (P < .05) than that induced by L1 (0.53 +/- 0.17 mg/kg), attributable to the significantly greater stool iron excretion during deferoxamine treatment (0.50 +/- 0.16 mg/kg/d) compared with that measured during L1 treatment (0.12 +/- 0.08 mg/kg/d, P < .01). Stool iron excretion accounted for a significantly greater percentage of total iron excretion during deferoxamine treatment (59% +/- 20%) than during L1 treatment (23% +/- 14%, P < .01). These iron balance studies are the first to compare total iron excretion induced by L1 with that achieved by deferoxamine. They demonstrate that the mean total daily iron excretion during L1 treatment (0.53 +/- 0.17 mg/kg) is sufficient to maintain net negative iron balance in most regularly transfused patients with sickle cell disease. Because long-term compliance with L1 has been shown previously to be superior to that with deferoxamine in patients with homozygous beta-thalassemia, the use of L1 should increase the long-term effectiveness of iron chelation in patients with sickle cell disease.     

Lysosomal heterogeneity between and within cells with respect to resistance against oxidative stress

The prevailing opinion on lysosomal endurance is that, as long as the cells are still alive, these organelles are generally quite stable and, thus, do not induce cell damage by leaking their numerous powerful hydrolytic enzymes to the cytosol. We suggest that this opinion is basically wrong and consider that many lysosomes are quite vulnerable, especially to oxidative stress. Moreover, we suggest that cellular degeneration, including apoptosis as well as necrosis, follows upon lysosomal disruption. We have found differing stability of lysosomal membranes to oxidative stress, not only among different cell types, but also between cells of the same type and between lysosomes of individual cells. We suggest that cellular resistance to oxidative stress is mainly a function of three parameters: (i) the capacity to degrade hydrogen peroxide before it reaches, and may diffuse into, the acidic vacuolar compartment; (ii) the resistance to reactive oxygen species of lysosomal membranes; and (iii) the intralysosomal amounts of redox-active, low molecular weight iron. Iron-catalysed intralysosomal reactions, if pronounced enough, result in peroxidation and destabilization of the lysosomal membrane. Owing to differences in the cellular synthesis of hydrogen peroxide-degrading enzymes, degree of autophagocytotic degradation of iron-containing metalloproteins, lysosomal localization within the cytoplasm and intralysosomal iron chelation, the above three parameters may vary between both different and similar cells and between lysosomes of individual cells as well, explaining their observed variability with respect to resistance against oxidative stress.     

Oxidative damage does not alter membrane phospholipid asymmetry in human erythrocytes

Oxidant-induced damage has been proposed to be the underlying mechanism for loss of membrane phospholipid asymmetry in the erythrocyte membrane. In sickle cell disease, thalassemia, and diabetes as well as in senescent erythrocytes, an apparent correlation between oxidative damage and loss of phosphatidylserine asymmetry has been reported. In the present study, erythrocytes were subjected to various levels of oxidative stress and/or sulfhydryl modifying agents. The transmembrane location of phosphatidylserine (PS) was assessed by FITC-conjugated annexin V labeling and the PS-dependent prothrombinase assay. Transbilayer movement of spin-labeled PS was used to determine aminophospholipid translocase activity. Our data show that cells did not expose PS as the result of oxidative stress induced by phenylhydrazine, hydrogen peroxide, tert-butyl hydroperoxide, cumene hydroperoxide, or sulfhydryl modification by N-ethylmaleimide (NEM) and diamide, even under conditions that led to severe cellular damage and impairment of aminophospholipid translocase activity. In contrast, the increase of intracellular calcium induced by treatment with calcium and ionophore A23187 leads to a rapid scrambling of the lipid bilayer and the exposure of PS, which can be exacerbated by the inhibition of aminophospholipid translocase activity. Oxidation of the cells with hydrogen peroxide or phenylhydrazine did not affect A23187-induced uptake of calcium, but partly inhibited calcium-induced membrane scrambling. In conclusion, oxidative damage of erythrocytes does not induce exposure of phosphatidylserine on the membrane surface, but can interfere with both aminophospholipid translocase activity and calcium-induced randomization of membrane phospholipids.     

Developmental expression of thrombin in zebrafish embryos: a novel model to study hemostasis

Oxidative stress causes modification of cellular macromolecules and leads to cell damage. The objective of this study was to identify protein modifications that relate to thiol groups in human red blood cells under oxidative stress. With t-butyl hydroperoxide (t-BH) treatment, results of isoelectric focusing (IEF) analysis showed that two dithiothreitol-reversible modifications are observed, one toward the cathode and the other to the anode. Protein change toward the cathode was demonstrated to be hemoglobin oxidation, which gains a net positive charge, based on the same focus on IEF gels as hemoglobin and methemoglobin and molecular weight analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Otherwise, the change toward the anode was the result of mixed disulfide formation between GSH and protein thiols. Based on the results of molecular weight analysis and its reversion from methemoglobin, protein formed mixed disulfides with GSH were also regarded as hemoglobin. As red blood samples were treated with diamide or GSSG, in addition to the mixed disulfides observed in t-BH-treated cells, additional hemoglobin-GSH mixed disulfide appeared. But the disappearance of this diamide-induced additional mixed disulfide by treating cells with t-BH after diamide treatment suggests that the increase of negative charges from GSH are offset by ferrohemoglobin oxidation to ferrihemoglobin. Additionally, other dithiothreitol-reversible modifications of one cell membrane protein, spectrin, were also observed from the formation of high molecular weight molecules as detected by SDS-PAGE. Results indicate that protein thiols in human red blood cells are susceptible to modification under oxidative stress. IEF analysis provides a useful tool to measure methemoglobin and hemoglobin GSH mixed disulfide formation.