Inhibition of monosodium urate monohydrate-mediated hemolysis by vitamin E

Microcrystals of monosodium urate monohydrate(MSUM)induce cytolysis and hemolysis inerythrocytes.In this report,we studied the effect of vitamin E on MSUM-mediated hemolysis in humanerythrocytes.Vitamin E significantly inhibited hemolysis induced by MSUM.The hydroxyl group in thechromanol ring of vitamin E is dispensable for protecting erythrocytes against hemolysis induced by MSUM,indicating that the inhibitory effect of vitamin E is not due to its antioxidant properties.However,both thechromanol ring and the isoprenoid side chain are important for vitamin E to suppress MSUM-induced hemolysis.Our current study suggests that vitamin E inhibits hemolysis induced by MSUM as a membrane stabilizer.     

Differential modulation by AMPA of signals from red- and green-sensitive cones in carp retinal luminosity-type horizontal cells

Intracellular recordings were made from luminosity-type horizontal cells (LHCs) in the isolated superfused carp retina and the effect of AMPA (α-amino-3-hydroxy-5-methylisoxa-zole-4-propionic acid), a glutamate receptor agonist, on these cells was studied. AMPA suppressed the responses of LHCs driven by red-sensitive (R-) cones whereas it potentiated the responses driven by green-sensitive (G-) cones. The AMPA effect could be completely blocked by GYKI 53655, a specific AMPA receptor antagonist, indicating the exclusive involvement of AMPA-preferring receptors. The AMPA effect persisted in the presence of picrotoxin (PTX) or di-hydrokainic acid (DHK), suggesting that the feedback from LHCs onto cones and glutamate transporters on cones may not be involved. It is suggested that there may exist different AMPA receptor subtypes with distinct characteristics on LHCs, which mediate signal transfer from R- and G-cones to LHCs, respectively.     

Effect and analysis of phenolic compounds during somatic embryogenesis induction in Feijoa sellowiana Berg

Summary. The effect of phenolic compounds on somatic embryogenesis in Feijoa sellowiana was analysed. The results showed that caffeic acid (140–560 μM) significantly increased somatic embryogenesis induction compared with the control. The presence of phloridzin, even at lower concentrations (11.5 μM), or caffeic acid or phloroglucinol at concentrations greater than 140.0 and 197.5 μM, respectively, inhibited somatic embryo development beyond the globular stage. When somatic embryos were transferred to the germination medium, the highest rates of germination (81.9%) were obtained with embryos induced in the presence of phloroglucinol (79.0 μM). At all concentrations tested, somatic embryos induced in medium containing phloroglucinol germinated at higher rates than those induced in the presence of caffeic acid. Histological and ultrastructural studies showed that somatic embryos were formed in close association with phenolic-rich cells which, in more advanced stages of development, formed a zone isolating the embryo from the maternal tissue. A comparative analysis of total phenolic content indicated that phenolics reached a peak by the third week of culture, independently of the medium used. However, after that period, the amount of phenolic compounds was significantly higher in explants cultured in the presence of phloroglucinol than in those cultured in the control or in caffeic acid-containing medium. Attempts to identify the type of phenolic compounds showed that flavan-3-ols and gallic acid derivatives were mainly produced in phloroglucinol-containing medium, whereas flavanones and dihydroflavonols were also present in medium containing caffeic acid. Flavones were the main phenols detected in the control. The ways in which phenolic compounds may affect somatic embryogenesis are discussed. Correspondence: J. M. Canhoto, Departamento de Botanica, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, Cal?ada Martim de Freitas, 3001-455 Coimbra, Portugal.     

Oxidative Stress and Antioxidant Responses in Young Leaves of Mulberry Plants Grown Under Nitrogen, Phosphorus or Potassium Deficiency

The aim of this study was to associate the generation of reactive oxygen species （ROS） with Induced antloxidant responses and disturbed cellular redox environment in the nitrogen-（N）, phosphorus-（P）, or potassium-（K） deftcient mulberry （Morus alba L. var. Kanva-2） plants. The indicators of oxidative stress and cellular redox environment and antioxldant defense-related parameters were analyzed. Oeficlency of N, P or K suppressed growth, accelerated senescence, and decreased concentrations of chloroplastic pigments and glutathione. Lipid peroxidation and activities of superoxide dismutase, ascorbate peroxidase and glutathione reductase were also increased in these N, P, or K deprived plants. Concentration of hydrogen peroxide Increased in plants deficient in N or P. Oeficlency of N or P particularly altered the cellular redox environment as indicated by changes in the redox couples, namely ascorbic acid/total ascorbate decreased in P-, glutathione sulfydryl/total glutathione decreased in N-, and Increased in P-deficient plants. Activity staining of native gels for superoxide dismutase revealed Increased activity as Indicated by Increased intensity of bands, and induction of few new isoforms in P- and K-deficient plants. Oifferences in the patterns of superoxide dismutase isoforms and redox status （ascorbic acid/total ascorbate and glutathlone sulfydryl/total glutathione） Indicate that N-, P-, or K-deficiency altered antioxidant responses to varying extents in mulberry plants.     

Effect of membrane-partitioned n-alcohols and fatty acids on pore-forming activity of a sea anemone toxin

Equinatoxin II, a 19.8 kDa pore-forming toxin from the sea anemone Actinia equina, was examined for hemolytic activity and permeabilization of small unilamellar lipid vesicles (SUV) in the presence of increasing amounts of n-alcohols (methanol to n-octanol) and fatty acids (palmitic and palmitoleic acid). We observed an enhancement of toxin activity which was dependent on the concentration of the membrane partitioning additive. An exception was palmitic acid which exerted a bimodal role. While at low bulk concentrations it increased toxin-induced hemolysis, above 3 μM bulk concentration it was inhibitory; in neither case was it efficient in promoting release of the fluorescent marker calcein from SUV. The increased permeabilization activity was correlated with an increase in the amount of toxin bound as indicated by changes in the intrinsic toxin fluorescence. In the case of n-alcohols, at least, these effects appeared to depend on the actual amount of alcohol present inside the membrane rather than on its specific chemical nature. This suggests that the observed effects could be due to changes of the biophysical properties of the lipid bilayer, such as thickness, lipid acyl-chain ordering, and dielectric constant induced by the partitioned additives. Received: 27 March 1996 / Accepted: 10 October 1996     

Changes in Growth and Nitrogen Assimilation in Maize Plants Induced by NaCl and Growth Regulators

Experiments were conducted to determine the interactive effects of salinity and certain growth regulators on growth and nitrogen assimilation in maize (Zea mays L. cv. GS-2). 100 mM NaCl inhibited the biomass accumulation, chlorophyll and carotenoid contents in leaves, nitrate content and uptake and nitrate reductase activity. The application of kinetin, ascorbic acid and 10 and 50 μM abscisic acid in the first experiment and 50 and 100 μM abscisic acid in the second experiment induced a substantial increase in the above parameters, the effect was highest with abscisic acid in salinized as well as non-salinized plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of the Hemolytic Properties of an Extract from Phaeocystis globosa Scherffel

Phaeocystis globosa Scherffel, an organism that causes harmful algal blooms, is a genus of the family Prymnesiophyta (or Haptophyta) with eurythermal and euryhaline characteristics. P. globosa has been confirmed to produce hemolytic substances, which are a mixture of liposaccharides. In the present study, the hemolytic properties of extract of P. globosa are analyzed further. The effects of temperature, pH,different divalent cations, and membrane lipids on extract-induced hemolysis are discussed, as is the possible hemolytic mechanism. The results of the present study showed that the hemolytic activity of the extract was approximately 127.1 hemolytic units (HU)/L. The hemolytic reaction became fastest and a 50% decrease in absorbance was induced at 30 min at 37℃, and at pH 7.0; Hg^2 was the strongest inhibitor of the hemolysis compared with the other divalent cations and many membrane lipids, except for phosphatidic acid, inhibited the hemolytic activity to different degrees. These results suggest that the toxin may make pores in the surface of red blood cells and that Hg^2 either combines with the hemolysin or closes the pores,hence inhibiting its further hemolytic reaction. The toxin probably has no specific membrane receptor in the red blood cell membrane.     

Myocyte injury by hemin

Summary Effects of free hemin on myocardium were investigated using a model of neonatal myocyte primary cultures. Cells were subjected to free hemin at concentrations up to 20 μM and equilibrated for 5 h at 37° C. Distribution of hemin in media, cell sarcolemma, and cell interior was evaluated. Time-dependent reduction in beating rate was monitored throughout the entire concentration range of administrated hemin. With time and in a hemin concentration-dependent manner, arrhythmic beatings which were followed by loss of contractility were observed. In parallel, morphologic changes appeared from granulation to complete loss of cell integrity. At the concentration range studied, hemin also induced a biphasic release of cytosolic enzymes. In the first phase, the fraction of enzyme released was dependent of the ratio of hemin:cells and was correlated with the amount of nonviable cells as monitored by a trypan blue test. In the second phase, the fraction of released enzyme was much larger than that of nonviable cells. The data are interpreted as an indication of complete loss of cytosolic content due to sarcolemma damage in first phase and partial damage to cell interior in the prolonged second phase. It is concluded that in similarity with other amphipathic molecules, free hemin is toxic to the myocardium.     

Epidermal growth factor and insulin-like growth factor-1 protect MDA-231 cells from death induced by actinomycin D: The involvement of growth factors in drug resistance

Summary In the present study, we investigated the ability of epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and insulin to protect the human breast cancer cell line MDA-231 from death induced by the antitumor drug actinomycin D (ACT-D). ACT-D is an inhibitor of RNA and protein synthesis, and its cytotoxicity may result due to continuous depletion in some vital protein molecules. Cell death was induced in the MDA-231 cells by either continuous exposure to a low dose of ACT-D (0.2μg/ml), or by a short-time exposure to a high dose of ACT-D (2μg/ml) and further culturing in the absence of the drug. Cell death was evaluated by the trypan blue dye exclusion test, the release of lactic dehydrogenase into the culture medium, and the depletion in the cellular ATP content. EGF and IGF-1, each at an optimal concentration of 20 ng/ml, enhanced substantially survival of cells exposed either to a low or a high dose of ACT-D. The combination of EGF (10 ng/ml) and IGF-1 (10 ng/ml) had an additive survival effect, which proposes that each of the growth factors enhanced survival by a distinct pathway. Insulin up to 40 ng/ml had no effect on cell survival. Pretreatment of the cells for 1 to 5 h with EGF and IGF-1 protected cells from the cytotoxic effect of ACT-D. Exposure of the cells to 2μg/ml of ACT-D for 1 h resulted in a drastic inhibition in uridine incorporation and only in a slight inhibition in leucine incorporation. Further incubation in the absence of ACT-D resulted in a continuous decrease in uridine and in leucine incorporation, either in the absence or presence of the growth factors. However, EGF and IGF-1, but not insulin, attenuated significantly this continuous decrease. We assume that EGF and IGF-1 protect cell viability by a mechanism that maintains a critical level of some vital protein molecule above the critical level at which cells die. Our finding that EGF and IGF-1 induced resistance to ACT-D suggests that growth factors may be involved in the mechanism of drug resistance.     

抗坏血酸增强氯高铁血红素诱导的溶血

研究抗坏血酸对氯高铁血红素所诱导的红细胞溶血的影响.红细胞溶血采用在540 nm处测定 上清液血红蛋白吸光度的方法;红细胞巴比妥酸反应产物 (TBARS)测定采用Stocks 等建立的方法;高铁血红蛋白的测定采用Sezebeni等报道的方法.结果表明：抗坏血酸显著增强氯高铁血红素所诱导的溶血.尽管氯高铁血红素本身并不影响红细胞TBARS和高铁血红蛋白的水平,但是,氯高铁血红素和抗坏血酸一起诱导红细胞TBARS和高铁血红蛋白含量的增加;过氧化氢酶显著地抑制抗坏血酸增强氯高铁血红素诱导红细胞的溶血、TBARS和高铁血红蛋白的生成;氢氧自由基淬灭剂显著地抑制抗坏血酸增强氯高铁血红素诱导红细胞溶血.由上述可得到如下 结论：抗坏血酸增加氯高铁血红素诱导的红细胞氧化压力与H₂O₂有关;氢氧自由基可能是抗坏血酸增强氯高铁血红素诱导红细胞溶血的原因;抗坏血酸在氯高铁血红素存在时,可以作为一个亲氧化剂而非一个抗氧化剂.     

Desferrioxamine protects human red blood cells from hemin-induced hemolysis

Hemin binding to red cell membranes, its effect on red cell hemolysis, and it interaction with desferrioxamine (DFO) in these processes were investigated. DFO interacted with hemin via the iron moiety. Blockage of the binding groups in DFO prevented interaction of DFO with hemin, implying the importance of the hydroxamic acid groups in DFO-hemin interactions. Since hemolysis is a result of hemin association with the membrane components, its binding in the presence and absence of DFO was studied. DFO strongly inhibited hemin-induced lysis in a concentration-dependent manner. With 50 microM hemin, 1 mM DFO completely inhibited lysis. Preincubation of ghost membranes with DFO (1 mM) inhibited binding of hemin (50 microM) to membranes by 42%. After ghost membranes were preincubated with hemin (50 microM), the addition of DFO (1 mM) removed 20% of the membrane-bound hemin. It is suggested that DFO may have an important role in alleviating the hemin-induced deleterious effects on the red cell membrane, especially in hemolytic anemias associated with unstable, autoxidized hemoglobins.     

Long-term intercalation of residual hemin in erythrocyte membranes distorts the cell

The effect of long-term incubation of residual globin-free hemin on whole red blood cell and isolated cytoskeletal proteins was studied. Hemin at concentrations found in pathological red cells was inserted to fresh erythrocytes. Increased hemolysis developed in the hemin-containing cells after a few days at 37 degrees C and after about four weeks at 4 degrees C. Since lipid and hemoglobin peroxidation did not depend on the presence of hemin, time-dependent effects on the cytoskeleton proteins were studied. Observations were: (1) spectrin and protein 4.1 exhibited a time-dependent increasing tendency to undergo hemin-induced peroxidative crosslinking. (2) The ability of the serum proteins, albumin and hemopexin, to draw hemin from spectrin, actin and protein 4.1 decreased with time of incubation with hemin. These results were attributed to time-dependent hemin-induced denaturation of the cytoskeletal proteins. Albumin taken as a control for physiological hemin trap was unaffected by hemin. Small amounts of hemo-spectrin (2-5%) were analyzed in circulating normal cells, and this in vivo hemo-spectrin also failed to release hemin. It was concluded that slow accumulation of hemin, a phenomenon increased in pathological cells, is a toxic event causing erythrocyte destruction.     

Effect of second-messenger modulators in K-562 cell differentiation: dual action of calcium/phospholipid-dependent protein kinase in the process of differentiation

We investigated the roles of second messengers in K-562 cell differentiation induced by either commitment-inducing agents (Ara-C, thymidine), or a noncommitment-inducing agent (hemin). Cell differentiation induced by both types of agents was inhibited by dbc-AMP, staurosporine, and H-7. In contrast, OAG enhanced hemin-induced cell differentiation, but it inhibited that due to Ara-C or thymidine. When K-562 cells were incubated with 4 x 10(-6)M hemin or 2 x 10(-7)M Ara-C for 2 days, an increase of epsilon-mRNA occurred. The addition of cycloheximide (1 microgram/ml) completely blocked this change, suggesting that de novo protein synthesis was necessary for the increase of epsilon-mRNA. Simultaneous treatment with Ara-C and cycloheximide for 2 days did not block either the increase of epsilon-mRNA or that of benzidine-positive cells, which were measured after 5 days of further incubation without additives. This suggested that the process of Ara-C-induced K-562 cell differentiation could be divided into two steps, i.e., a commitment step and a phenotypic expression step, and that the commitment step was at least partly resistant to cycloheximide. We investigated the roles of second messengers in each step. Our results suggested that PKC may act as a negative regulator of commitment step and as a positive regulator of the phenotypic expression. This may explain the differing effects of OAG on hemin- and Ara-C-induced K-562 cell differentiation.     

Rapid mtDNA deletion by oxidants in rat liver mitochondria after hemin exposure

The amounts of superoxide and hydrogen peroxide generated by mitochondria under physiological conditions can be enhanced by cellular stress. This study tested the hypothesis that the response to hemin-induced stress, which includes heme oxygenase-1 (HO-1) induction, predisposes to oxidative damage of mitochondrial DNA (mtDNA). Hepatic mitochondria from control, hemin-, and CO-exposed rats were incubated with tert-butyl hydroperoxide (tert-BH) or the NO donor 1,2,3,4-oxatriazolium, 5-amino-3- (3,4-dichlorophenyl)-chloride (GEA 3162). Mitochondrial total and oxidized glutathione (GSH and GSSG), total and free iron, and 8-oxo-7, 8-dihydro-2' deoxyguanosine (8-OHdG) were determined with and without oxidants. As expected, oxidation by tert-BH induced significant GSH depletion and increased amounts of free iron and 8-OhdG. Oxidant exposure rapidly produced a large mtDNA deletion involving the coding regions for cytochrome c oxidase (COX 1) and NADH dehydrogenase (ND1 and ND2). Hemin and CO greatly exacerbated susceptibility to the deletion of mtDNA by tert-BH, and this was attenuated by preincubation with GSH methyl ester. Analysis of mitochondria-associated proteins Bax and Bcl-xl in hemin- and CO-exposed rats showed significant responses, revealing interactions with apoptotic pathways. Thus, hemin-induced mitochondrial events sensitize a specific region of the mitochondrial genome to deletion, which is related to depletion of GSH and is not explained by effects of CO. This mtDNA damage is associated with altered expression of mitochondrial cell death proteins, thereby suggesting a novel mechanism for systemic or environmental pro-oxidants to influence apoptosis.     

Hemin-Induced Membrane Sulfhydryl Oxidation: Possible Involvement of Thiyl Radicils

Sublytic levels (μM) of hemin destabilized RBC membrane as indicated by ghost fragmentation pattern using a laser viscodiffractometer. Furthermore, electron microscopic study shows that 5μM of hemin induced echinocytic transformation whereas higher hemin concentration (40μM) induced spherocytic transformation. In addition, hemin oxidized sulfhydryl groups in a dose dependent fashion and Electron Spin Resonance study suggests that such oxidation may involve a thiyl radical. Moreover, sulfhydryl compounds enhanced hemin-induced lipid peroxidation. Desferroxamine could prevent hemin-induced sulfhydryl oxidation as well as hemin-induced decrease in membrane stability. In contrast, vitamin E could effectively prevent hemin-induced lipid peroxidation but could not prevent hemin-mediated membrane destabilization.     

Role of ERK activation in growth and erythroid differentiation of K562 cells

Inhibition of signaling through Ras in BCR-ABL-positive pluripotent K562 cells leads to apoptosis and spontaneous differentiation. However, Ras-induced activation of the mitogen-activated protein kinase ERK has been suggested to play a critical role in either growth or differentiation in different model systems. We studied the role of ERK activation in the growth-promoting and anti-apoptotic effect of Ras and its involvement in hemin-induced nonterminal erythroid differentiation using the BCR-ABL-positive K562 cell line as a model. K562 cells were stably transfected with ERK1 or the dominant inhibitory mutant of ERK1 (ERK1-KR). Overexpression of ERK1-KR inhibited cell growth with an approximately fourfold increase in doubling time and induced apoptosis in K562 cells. Incubation with the MEK1 inhibitor UO126 inhibited cell growth and induced apoptosis in K562 cells in a dose-dependent manner as well. In the presence of exogenously added hemin, K562 cells differentiate into erythroblasts, as indicated by the production of large amounts of fetal hemoglobin. We examined the activation of MAP kinases during hemin-induced differentiation. The ERK1 and 2 activity increased within 2 h post hemin treatment and remained elevated for 24-48 h. During this time, fetal hemoglobin synthesis also increases from 0.8 to 10 pg/cell. There was no activation of JNK or p38 protein kinases. The hemin-induced accumulation of hemoglobin was inhibited in ERK1-KR overexpressing cells and was enhanced in the wild-type ERK1 transfectants. Our results suggest that ERK activation is involved in both growth and hemin-induced erythroid differentiation in the BCR-ABL-positive K562 cell line.     

The effect of peroxynitrite and some antioxidants on the rate of osmotic hemolysis of bovine erythrocytes

Bovine erythrocytes treated with peroxynitrite (ONOO(-)), a cytotoxic species formed in vivo via the reaction of nitric oxide (NO(.)) and the superoxide anion (O2(-.)), show an increased rate of hemolysis on sudden osmotic stress. The increase in the rate was peroxynitrite concentration dependent. In the presence of some antioxidants (uric acid, ascorbic acid, glutathione, melatonin and albumin), this effect was significantly lower, with ascorbic acid as the most efficient antioxidant.     

Clotrimazole as an amplifier of hemin-induced damage to human erythrocytes. Antioxidant-independent protective effects of flavonoids

This study is focused on the effect of the antifungal drug clotrimazole (CLT), also possessing anti-malarial and anticancer activities, on hemin-induced hemolysis and changes in ion permeability and filter-ability of human erythrocytes. In the presence of 10 μM clotrimazole, the hemolytic response of erythrocytes to exogenous hemin at concentrations as low as 2–8 μM was considerably potentiated and their filterability, as measured by passing them through a 5-μm nuclepore filter, dropped sharply. Flavonoids quercetin (Q) and taxifolin (DHQ), unlike the standard antioxidant Trolox, abolished the effects of clotrimazole, suggesting that protection of hemin-treated erythrocytes by flavonoids is not related to their antioxidant properties.     

Inhibition of free radical-induced erythrocyte hemolysis by 2-O-substituted ascorbic acid derivatives

Inhibitory effects of 2-O-substituted ascorbic acid derivatives, ascorbic acid 2-glucoside (AA-2G), ascorbic acid 2-phosphate (AA-2P), and ascorbic acid 2-sulfate (AA-2S), on 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative hemolysis of sheep erythrocytes were studied and were compared with those of ascorbic acid (AA) and other antioxidants. The order of the inhibition efficiency was AA-2S> or =Trolox=uric acid> or =AA-2P> or =AA-2G=AA>glutathione. Although the reactivity of the AA derivatives against AAPH-derived peroxyl radical (ROO(*)) was much lower than that of AA, the derivatives exerted equal or more potent protective effects on AAPH-induced hemolysis and membrane protein oxidation. In addition, the AA derivatives were found to react per se with ROO(*), not via AA as an intermediate. These findings suggest that secondary reactions between the AA derivative radical and ROO(*) play a part in hemolysis inhibition. Delayed addition of the AA derivatives after AAPH-induced oxidation of erythrocytes had already proceeded showed weaker inhibition of hemolysis compared to that of AA. These results suggest that the AA derivatives per se act as biologically effective antioxidants under moderate oxidative stress and that AA-2G and AA-2P may be able to act under severe oxidative stress after enzymatic conversion to AA in vivo.