The photoprotein pholasin as a luminescence substrate for detection of superoxide anion radicals and myeloperoxidase activity in stimulated neutrophils

Pholasin, the photoprotein of the common piddock Pholas dactylus, emits an intense luminescence upon oxidation. The contribution of superoxide anion radicals and myeloperoxidase (MPO) to Pholasin luminescence in stimulated neutrophils was investigated. Data on Pholasin luminescence were compared with results of superoxide anion radical generation detected by the cytochrome c test as well as with the release of elastase and MPO. In N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated neutrophils, most of the luminescence is caused by superoxide anion radicals, whereas MPO shows only a small effect as shown by coincubation with superoxide dismutase (SOD) as well as potassium cyanide (KCN), an inhibitor of MPO. However, both, O₂^- and MPO contribute to light emission in fMLP/cytochalasin B and phorbol myristoyl acetate (PMA) stimulated cells. Thus, the kinetics of O₂^- generation and MPO release can be very well detected by Pholasin luminescence in stimulated neutrophils.

Degranulation of azurophilic granules was assessed using an ELISA test kit for released MPO or detection of elastase activity with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide in the supernatant of stimulated cells. Both approaches revealed concurrently similar results concerning the amount and kinetics of enzyme release with data of Pholasin luminescence. Both, cytochrome c measurements and Pholasin luminescence indicate that fMLP/cytochalasin B and PMA stimulated neutrophils produce more O₂^- than fMLP stimulated cells. Thus, Pholasin luminescence can be used to detect, sensitively and specifically, O₂^- production and MPO release from stimulated neutrophils.     

Effect of microtubule-disrupting agents on superoxide production in human polymorphonuclear leukocytes

We explored the effects of compounds known or proposed to affect microtubule functions on superoxide (O₂⁻) production in human polymorphonuclear leukocytes stimulated by N-formyl-methionyl-phenylalanine (f-Met-Phe), calcium ionophore A23187 and phorbol myristate acetate. F-Met-Phe-induced O₂⁻ production was markedly potentiated not only by microtubule-disrupting agents, including colchicine, vincristine, vinblastine, nocodazole, podophyllotoxin and griseofulvin, but also deuterium oxide (²H₂O), which is proposed to stabilize microtubules, and not affected by lumicolchicine. Ionophore A23187-induced O₂⁻ production was not influenced by colchicine, and markedly enhanced by ²H₂O, whereas phorbol myristate acetate-induced O₂⁻ production was not influenced by colchicine, and slightly inhibited by ²H₂O. ²H₂O did not counteract the effects of colchicine and vice versa. Dibutyryl cyclic AMP and prostaglandin E₁ inhibited O₂⁻ production stimulated by f-Met-Phe and ionophore A23187, whereas phorbol myristate acetate-induced O₂⁻ production was strongly resistant to the inhibitory effect of these agents. The enhancing effect of colchicine and ²H₂O on f-Met-Phe-induced O₂⁻ production was abolished by dibutyryl cyclic AMP. Colchicine promoted concanavalin A cap formation, and ²H₂O produced cancanavalin A patch formation, whereas dibutyryl cyclic AMP did not affect the distribution of concanavalin A receptors. In addition, ²H₂O and dibutyryl cyclic AMP did not interfere with the colchicine-induced concanavalin A cap formation. These findings suggest that f-Met-Phe, ionophore A23187 and phorbol myristate acetate may activate the oxidative metabolism of human polymorphonuclear leukocytes through different mechanisms, and that microtubule-disrupting agents, ²H₂O and cyclic AMP agonists may affect the different steps of the activating system of NAD(P)H oxidase.     

Oxidative metabolism of chicken polymorphonuclear leucocytes during phagocytosis

Summary The oxidative response to phagocytosis by chicken polymorphonuclear leucocytes was investigated as compared to guinea pig polymorphonuclear leucocytes.The polymorphs from both species respond to phagocytosis with an increased oxygen consumption, an increased generation of O₂ ^– and H₂O₂, and an increased oxidation of glucose through the hexose monophosphate shunt. The rate of oxygen consumption, and generation of O₂ ^– and H₂O₂ by phagocytosing chicken polymorphonuclear leucocytes is considerably lower than with phagocytosing guinea pig polymorphonuclear leucocytes. By contrast, the extent of hexose monophosphate shunt stimulation in chicken polymorphs is comparable to that of guinea pig polymorphs. Evidence is presented suggesting that H₂O₂ is preferentially degraded in chicken cells through the glutathione cycle, whereas catalase and myeloperoxidase are the two main H₂O₂ degrading enzymes in guinea pig cells.The 20,000 g fraction of the postnuclear supernatant of chicken polymorphs contains a cyanide-insensitive NADPH oxidizing activity which is stimulated during phagocytosis. Similar properties for the NADPH oxidizing activity of guinea pig polymorphs have been previously reported.It is concluded that the metabolic burst of phagocytosing chicken polymorphonuclear leucocytes is qualitatively similar to that of guinea pig polymorphonuclear leucocytes, but the latter cells are more active in all the biochemical parameters that have been measured. The difference in the H₂O₂ degradation pathways between the two species is accounted for by the lack of myeloperoxidase and catalase in chicken polymorphs.     

Superoxide Anion-Generating Activities of Macrophages as Studied by Using Cytochalasin E and Lectins as Synergistic Stimulants for Superoxide Release

Treatment of macrophages with cytochalasin E in combination with a lectin was found to stimulate the generation of superoxide anions (O₂^-) very efficiently. The macrophages stimulated with concanavalin A, phytohemagglutinin or wheat germ agglutinin released superoxide, but cells pretreated with cytochalasin E released much greater amounts of superoxide, without notable lag time, upon stimulation with the lectin. Wheat germ agglutinin was found to be the most efficient stimulant among the lectins tested. Superoxide generation in guinea pig macrophages was shown to be dependent largely on cytoplasmic glucose metabolism and to some extent on mitochondrial respiration, since the superoxide release was largely but not totally inhibited by 2-deoxyglucose and to a lesser extent by antimycin A or KCN. The method presented is sensitive and allows rapid assay of the superoxide-generating activity with only 1–5 × 10⁵ macrophages for a single determination. In application of this technique, elevation of the superoxide-generating activity was shown with macrophages elicited by chemical inflammation or those obtained from mice after treatment with tubercle bacilli.     

Activation of superoxide production and differential exocytosis in polymorphonuclear leukocytes by cytochalasins A,B, C,D and E: Effects of various ions

All of the common cytochalasins activate superoxide anion release and exocytosis of β-N-acetylglucosaminidase and lysozyme from guinea-pig polymorphonuclear leukocytes (neutrophils) incubated in a buffered sucrose medium. Half-maximal activation of both processes is produced by approx. 2 μM cytochalasin A, C >μM cytochalasin B ? 4–5 μM cytochalasin D, E. While maximal rates of O₂^? release and extents of exocytosis require extracellular calcium (1–2 mM), replacing sucrose with monovalent cation chlorides is inhibitory to neutrophil activation by cytochalasins. Na⁺, K⁺ or choline inhibited either cytochalasin B- or E-stimulated O₂^? production with IC₅₀ values of 5–10 mM and inhibition occurs whether Cl^?, NO₃^? or SCN^? is the anion added with Na⁺ or K⁺. Release of β-N-acetylglucosaminidase in control or cytochalasin B-stimulated cells is inhibited by NaCl (IC₅₀ ≈ 10 mM), while cytochalasin E-stimulated exocytosis is reduced less and K⁺ or choline chloride are ineffective in inhibiting either cytochalasin B- or E-stimulated exocytosis. Release of β-glucuronidase, myeloperoxidase or acid phosphatase from neutrophils incubated in buffered sucrose is not stimulated by cytochalasin B. Stimulation of either O₂^? or β-N-acetylglucosaminidase release by low concentrations of cytochalasin A is followed by inhibition of each at higher concentrations. It appears that all cytochalasins can activate both NAD(P)H oxidase and selective degranulation of neutrophils incubated in salt-restricted media and that differential inhibition of these two processes by monovalent cations and/or anions is produced at some step(s) subsequent to cytochalasin interaction with the cell.     

Macrophage-mediated cytolysis of erythrocytes in the guinea pig. II. Role of oxidative burst products in macrophage cytotoxicity activated by lectins and phorbol ester derivatives

Guinea pig peritoneal macrophages (GPPM) exhibited enhanced production of O₂^? and H₂O₂, and cytolytic activity toward erythrocytes, in response to reagents such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), its methylated derivative 4-O-MeTPA, Con A, wheat germ agglutinin (WGA), and opsonized zymosan. In order to examine the possible role of oxidative burst products such as O₂^? and H₂O₂ in the cytolytic process, we used reagents and enzymes which influence the balance of O₂^? and H₂O₂ outside and inside the GPPM cells. Macrophage-mediated cytolysis (MMC) of erythrocytes in the presence of the activators and modulators was assessed by ⁵¹Cr release assay. MMC activated by TPA and 4-O-MeTPA was inhibited by scavengers of H₂O₂ such as catalase and α-tocopherol, and was augmented by the catalase inhibitor 3-amino-1,2,4-triazole, and by horseradish peroxidase. TPA- and 4-O-MeTPA-activated MMC was only partially inhibited by the O₂^? scavenger cytochrome c and the enzyme superoxide dismutase and unaffected by cytochalasin D (an inhibitor of phagocytosis). MMC activated by the lectins Con A and WGA was unaffected by the scavengers and enzymes used, but markedly inhibited by cytochalasin D. Activation of MMC by TPA, WGA, and phagocytosis of opsonized zymosan, as well as O₂^? and H₂O₂ generation triggered by these reagents, were markedly inhibited by chlorpromazine. The results indicate that GPPM-mediated cytolysis activated by lectins, phorbol ester derivatives, and phagocytosis of opsonized zymosan, is dependent on the generation of oxidative burst products, mainly H₂O₂. TPA- or 4-O-MeTPA-activated MMC is mainly an extracellular event, while lectin-activated MMC may take place within the macrophages.     

Hydrogen peroxide increases the phagocytic function of human neutrophils by calcium mobilisation

We have studied the effect of exogenous administration of hydrogen peroxide (H₂O₂) on phagocytic activity of human neutrophils. The treatment of cells with increasing concentrations of H₂O₂ evoke a significant elevation of phagocytic function assayed as phagocytic index, percentage and efficiency; and was similar to that induced by the calcium mobilising agonist formyl-methionyl-leucyl-phenylalanine (fMLP). This stimulatory effect was reduced by pre-treatment of neutrophils with catalase and abolished in neutrophils loaded with the intracellular calcium quelator dimethyl BAPTA. In the absence of extracellular calcium, treatment of cells with H₂O₂ resulted in a increase in [Ca²⁺] _i , indicating the release of calcium from intracellular stores. H₂O₂ abolished the typical calcium release stimulated by the physiological agonist fMLP, while depletion of agonist-sensitive calcium pools by fMLP was able to prevent H₂O₂-induced calcium release. We conclude that H₂O₂ induces calcium release from agonist-sensitive stores and consequently increase the phagocytosis process.     

Fungal elicitors induce a transient release of active oxygen species from cultured spruce cells that is dependent on Ca2+ and protein-kinase activity

Cell-wall components from the ectomycorrhizal fungi Amanita muscaria and Hebeloma crustuliniforme and from the spruce pathogen Heterobasidion annosum elicited a transient release of active oxygen species from cultured spruce cells (Picea abies (L.) Karst.). Since the detection of active oxygen was suppressed by catalase, H₂O₂ was assumed to be the prevailing O₂ species. On the other hand, superoxide dismutase enhanced the concentration of detectable H₂O₂ indicating that the superoxide anion was formed before dismutating to H₂O₂. The elicitors induced the formation of active oxygen in a dose-dependent manner. Interestingly, elicitors from mycorrhizal fungi had a lower H₂O₂-inducing activity than equal amounts of cell-wall preparations from the pathogen H. annosum. In Ca²⁺-depleted medium the production of active oxygen by elicitor-treated spruce cells was suppressed. Additionally, the ionophore A 23187 induced active oxygen formation in a medium with Ca²⁺ but not in a Ca²⁺-depleted medium. Furthermore, the protein-kinase inhibitor staurosporine inhibited the oxidative burst. At a concentration of 34 nM the effect was diminished to 50%. From these results it is suggested that the release of active oxygen species from cultured spruce cells triggered by cell-wall-derived fungal elicitors depends on external Ca²⁺ and a protein-kinase activity. In these respects the effect shows similarities with the well-studied respiratory burst of mammalian neutrophils.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - KP_i potassium phosphate This work was supported by grants from Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie.     

Singlet Oxygen and Other Reactive Oxygen Species are Involved in Regulation of Release of Iron-Binding Chelators from Scenedesmus cells

Freshly-added iron only slightly affected the growth of iron-sufficient cells of the green alga Scenedesmus incrassatulus Bohl, strain R-83, but induced accumulation of malondialdehyde (MDA) in cells and excretion of MDA in the medium. These effects were stronger in response to Fe²⁺ as compared to Fe³⁺, but Fe³⁺ induced the release of more iron-binding chelators from these cells than Fe²⁺. Fe³⁺ added either in dark or in light induced release of equal concentrations of iron-complexing agents, part of which formed strong chelates with iron in the medium. Exogenously added hydrogen peroxide inhibited iron-induced release of chelators but the effect was removed by addition of the hydroxyl radical scavenger dimethylsulfoxide (DMSO). Malondialdehyde also inhibited the release of chelators. Release of chelators was induced in the absence of iron salts by photoexcited chlorophyll (Chl). The Chl-induced release was efficiently inhibited by singlet oxygen scavengers such as dimethylfuran, -carotene, sodium azide and vitamin B₆, and stimulated in D₂O or DMSO. Exogenously added catalase inhibited the release more than added superoxide dismutase. The Fe³-induced release of chelators was also inhibited by scavengers of singlet oxygen, but was not affected by sodium azide and by ethanol. Hence both H₂O₂ and singlet oxygen were involved in induction of chelator release in the absence of iron in light. The induction of chelator release by iron in dark involved H₂O₂, but not singlet oxygen.     

Light-dependent hydrogen evolution by Scenedesmus

Summary The effect of glucose and the uncoupler Cl-CCP upon hydrogen production was studied in adapted cells of Scenedesmus obliquus D₃. Cl-CCP at 10^-5M concentration completely inhibited the evolution of H₂ in the dark and increased the apparent rate of H₂ evolution in the light. At 10^-5M Cl-CCP, photosynthesis and photoreduction by anaerobically adapted algae were only temporarily inhibited; O₂ evolution reappeared after approximately 1 hr of illumination if CO₂ was present. Increasing the Cl-CCP concentration to 5 x 10^-5M led to a maximum rate of photohydrogen production and fully inhibited H₂ evolution, photoreduction and dark H₂ evolution. H₂ evolution was accompanied by a release of varying amounts of CO₂ in the light, as well as in the dark. Dark CO₂ production was stimulated by Cl-CCP. H₂ evolution in the light was stimulated by adding glucose to autotrophically grown cells or by growing the cells heterotrophically with glucose; starvation had an opposite effect. Adapted cells released ¹⁴CO₂ from the 3 and/or 4 position of specifically labeled glucose, indicating that degradation occurred via the Embden-Meyerhof pathway. The amount of H₂ released by autotrophically grown cells was the same either with continuous illumination or with short periods of light, followed by darkness. Scenedesmus mutant No. 11, which is unable to evolve O₂ was not inhibited in its capacity to evolve H₂ in the light. These data indicate that the evolution of H₂ in the light by adapted Scenedesmus depends upon the degradation of organic material and does not require the production of free O₂ by photosystem II.The following abbreviations are used: Cl-CCP = carbonyl cyanide m-chlorophenylhydrazone; DCMU = 3-(3,4-dichlorophenyl)-1,1-dimethylurea, DNP = 2,4-dinitrophenol.This work was supported by contract AT-(40-1)-2687 from the U.S. Atomic Energy Commission.     

P2X4 Activation Modulates Volume-sensitive Outwardly Rectifying Chloride Channels in Rat Hepatoma Cells

Volume-sensitive outwardly rectifying (VSOR) Cl⁻ channels are critical for the regulatory volume decrease (RVD) response triggered upon cell swelling. Recent evidence indicates that H₂O₂ plays an essential role in the activation of these channels and that H₂O₂ per se activates the channels under isotonic isovolumic conditions. However, a significant difference in the time course for current onset between H₂O₂-induced and hypotonicity-mediated VSOR Cl⁻ activation is observed. In several cell types, cell swelling induced by hypotonic challenges triggers the release of ATP to the extracellular medium, which in turn, activates purinergic receptors and modulates cell volume regulation. In this study, we have addressed the effect of purinergic receptor activation on H₂O₂-induced and hypotonicity-mediated VSOR Cl⁻ current activation. Here we show that rat hepatoma cells (HTC) exposed to a 33% hypotonic solution responded by rapidly activating VSOR Cl⁻ current and releasing ATP to the extracellular medium. In contrast, cells exposed to 200 μm H₂O₂ VSOR Cl⁻ current onset was significantly slower, and ATP release was not detected. In cells exposed to either 11% hypotonicity or 200 μm H₂O₂, exogenous addition of ATP in the presence of extracellular Ca²⁺ resulted in a decrease in the half-time for VSOR Cl⁻ current onset. Conversely, in cells that overexpress a dominant-negative mutant of the ionotropic receptor P2X4 challenged with a 33% hypotonic solution, the half-time for VSOR Cl⁻ current onset was significantly slowed down. Our results indicate that, at high hypotonic imbalances, swelling-induced ATP release activates the purinergic receptor P2X4, which in turn modulates the time course of VSOR Cl⁻ current onset in a extracellular Ca²⁺-dependent manner.     

Release of elicitors from rice blast spores under the action of reactive oxygen species

Effects of reactive oxygen species (ROS) on the release of putative elicitors from spores of rice blast causal fungus Magnaporthe grisea (Hebert) Barr were studied. While studying the influence of exogenous ROS, the spores were germinated for 5 h in the presence of 50 μM H₂O₂ and then treated with catalase to decompose hydrogen peroxide. The spore germination fluid was then boiled to inactivate catalase. When the resulting diffusate was applied onto rice (Oryza sativa L.) leaves, it caused necroses and stimulated superoxide (O₂⁻) production. Both effects were observed with the resistant rice cultivar but not with the cultivar susceptible to the fungal strain. The susceptible cultivar did not acquire resistance to challenge with fungal spores, which were applied one day after the treatment. The fractionation of the spore diffusate showed that both low- and high-molecular compounds (mol wt < 3 kD and >3 kD, respectively) should be present in combination to induce O₂⁻ production by leaves. The diffusates from spores germinated in water also caused necroses and stimulated O₂⁻ generation, though to a weaker extent than diffusates from spores germinated in H₂O₂. The effect of diffusates from spores germinated in water was abolished by catalase or superoxide dismutase added initially to the spore suspension. The results suggest that germinating spores of M. grisea are able to release elicitors and this ability depends on ROS formation by spores. Presumably, the yield of elicitors is increased additionally if fungus M. grisea is stressed or subjected to exogenous ROS. The described phenomena may be involved in incompatibility mechanisms.     

Effects of H2O2 on the growth, secretion, and metabolism of hybridoma cells in culture

Summary The effect of low concentrations of hydrogen peroxide (H₂O₂) (5 × 10⁻⁷−9.5 × 10⁻⁷ M) on cell growth and antibody production was investigated with murine hybridoma cells (Mark 3 and anti-hPL) in culture. Cell growth, measured by flow cytometry with morphological parameters, was significantly stimulated by H₂O₂ (8 × 10⁻⁷ M) but H₂O₂ concentration of 7 × 10⁻⁶ M and above increased cell death. H₂O₂ stimulation of antibody production was nonsignificant. The metabolism of cells treated with 8 × 10⁻⁷ or 1 × 10⁻⁵ M H₂O₂ was similar to that of the control in terms of glucose and glutamine consumption, lactate and ammonia production, and amino acid concentrations in the medium. The concentrations of lactate dehydrogenase, a marker of cell death, in test and control cells were similar. However, concentrations of intracellular free radicals measured by flow cytometry with dihydrorhodamine 123 (DHR 123) and dichlorofluorescein diacetate (DCFH-DA) as fluorochromes were different. The reactive oxygen species content of cells in 8 × 10⁻⁷ M H₂O₂ was similar to that of the controls, but there was a sudden, marked production of superoxide anions (detected with DHR 123) and H₂O₂ or peroxides (detected with DCFH-DA) by cells incubated with 1 × 10⁻⁵ M H₂O₂ which increased with increasing H₂O₂ until cell death.     

Temperature shift-induced changes in the antioxidant enzyme system of cyanobacteriumSynechocystis PCC 6803

The 24 h effect of low (20°C) and high (43°C) temperature on the antioxidant enzyme activities and lipid peroxidation was investigated in intact cells of the cyanobacteriumSynechocystis PCC 6803 grown at 36°C. At low temperature treated cells, the superoxide dismutase, catalase and glutathione peroxidase activities were significantly higher and the protein content lower than in high temperature treated cells. The increase of hydroxyl free radical level and malonyldialdehyde formation, when algal cells were exposed to low temperature, were due to the stimulated production of superoxide radicals O₂ ⁻ and hydrogen peroxide (H₂O₂).     

Leukocytic functions in burn-injured patients

Anergy associated with an increase in suppressor helper T cell (T_c) ratio and a decrease in natural killer (NK) is one main cause of death following thermal injury (Tl). Recently, in vitro studies have shown that LTB₄ can induce human T_c to exert suppressor cell activity, and incubation of lymphocytes with LTB₄ for 24 hours significantly suppressed NK cell activity. Thus, we undertook an investigation of both AA metabolism and immunologic response in 20 patients who suffered 40–90% total body surface area (TBSA) burns. Cyclooxygenase (CO:RIA) and lipoxygenase (LO;HPLC det.) metabolites and superoxide (O₂^.−) production were measured in stimulated polymorphonuclear cells (PMNL) (A 23187 ± AA for icosanoid release; phorbol myristate acetate for O₂^.− production). Lyso-paf-acether (P-LPA) was measured in plasma samples. Ca²⁺-dependent K⁺ permeability in PMNL was measured by the cell K⁺ release induced by A 23187. T_c and T_c subsets were determined using monoclonal antibodies (OKT₃₊, OKT₄₊ and OKT₈₊). A biphasic sequential release of the different substances (leukocytic icosanoids and O₂^.− was monitored: increase ( 36–48 h after Tl) and decrease ( 72 h after Tl). The increase in AA stimulation was more transient than that of O₂^.−. The decline in the release of AA metabolites and O₂^.− production was associated with the anergic phase (decrease OKT₄₊/OKT₈₊ ratio) and with the clinical outcome of the patients. The decrease in LTB₄ and other LO metabolites could explain the impairment of neutrophil chemotaxis. Ca²⁺-dependent K⁺ permeability increased early up to 2 or 3 times normal.In order to go further with the mechanism of inhibition of LTB₄ and O₂^.− release, the effect of Tl plasma was assayed on normal leukocytes: a 10 min incubation with such plasma was sufficient to abolish LTB₄ secretion. A less important inhibition was observed with O₂^.− release (−32%) and Ca²⁺-dependent K⁺ permeability (−30%). Plasma inhibition seems to be due to a thermolabile factor(s) [protein(s): “suppressive factor(s) of membrane activation ”SFMA] which is (are) under active investigation using gel-filtration chromatography and fast protein liquid chromotography (FPLC). Among the SFMAs, certain acute phase proteins could play a key role: i.e., incubation (10 min) of normal PMNL with ceruloplasmin (1 mg/ml) abolished LO products and O₂^.− release.     

Cofilin undergoes rapid dephosphorylation in stimulated neutrophils and translocates to ruffled membranes enriched in products of the NADPH oxidase complex. Evidence for a novel cycle of phosphorylation and dephosphorylation

 Neutrophils contain a 21-kDa phosphoprotein that undergoes rapid dephosphorylation upon stimulation of these cells with the chemoattractant N-fMet-Leu-Phe (fMLP), activators of protein kinase C [e.g., 4β-phorbol 12-myristate 13-acetate (PMA)] or the calcium ionophore A23187. This phosphoprotein was identified as the non-muscle form of cofilin by peptide sequencing and immunoblotting with specific antibodies. Evidence is presented that in neutrophils cofilin is regulated by a continual cycle of phosphorylation and dephosphorylation, and that the phosphatase undergoes activation during cell stimulation. Experiments with a wide variety of antagonists further suggested that the protein kinase that participates in these reactions may be a novel enzyme. The kinetics of cofilin dephosphorylation in neutrophils stimulated with fMLP or PMA were very similar to those observed for superoxide (O₂ ^–) release. Immunofluorescent studies revealed that cofilin was present thouroughout the cytosol of resting neutrophils and underwent rapid translocation to the F-actin-rich, ruffled membranes of stimulated cells. Cytochemical analysis further revealed that the ruffled membranes also contained large amounts of hydrogen peroxide (H₂O₂), a product of the O₂ ^–/H₂O₂-generating activity of stimulated neutrophils (NADPH oxidase). Cofilin is therefore well placed to participate in the continual polymerization and depolymerization of F-actin that is thought to give rise to the oscillatory pattern of H₂O₂ production observed under certain conditions. Accepted: 22 April 1997     

Effects of iron chelates on the transferrin-free culture of rat dermal fibroblasts through active oxygen generation

Summary Effects of nonchelating and chelating agents at 10 mM on the serum-free culture of rat dermal fibroblasts were investigated. A strong iron-chelating agent, iminodiacetic acid (IDA), and a weak one, dihydroxyethylglycine (DHEG), decreased iron permeation into preconfluent fibroblasts. A weak iron-chelating agent, glycylglycine (GG), a nonchelating agent, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), and human apotransferrin (10 μg/ml) increased the permeation with time. Iron may be essential for survival of fibroblasts because subconfluent fibroblasts exposed to 100 μM FeSO₄ in combination with transferrin, HEPES, or GG significantly decreased to release lactate dehydrogenase into the medium. Superoxide dismutase and dimethyl sulfoxide blocked the enzyme release, suggesting that superoxide and hydroxyl radical induce cellular damage but hydrogen peroxide (H₂O₂) generated by superoxide dismutation does not. GG significantly reduced H₂O₂ cytotoxicity. DHEG acted as a potent promoter of the iron-stimulated cellular damage if ascorbate or H₂O₂ was added to the medium. FeSO₄ and FeCl₃ (50 to 100 μM) individually combined with IDA maximally promoted fibroblast proliferation. Ascorbate increased formation of thiobarbituric acid-reactive substances from deoxyribose in the medium supplemented with FeSO₄ and either IDA or DHEG. Conversely, ascorbate decreased the formation in the medium with FeSO₄ and with or without other agents. Fibroblast proliferation may thus be stimulated through the active oxygen generation mediated by a redox-cycling between Fe³⁺ and Fe²⁺, which are dissolved in the medium at a high concentration, rather than through delivery of iron into the cells.     

Mutations by near-ultraviolet radiation in Escherichia coli strains lacking superoxide dismutase

Om wild-type Escherichia coli, near-ultraviolet radiation (NUV) was only weakly mutagenic. However, in an allelic mutant strain (sodA sodB) that lacks both Mn- and Fe-superoxide dismutase (SOD) and assumed to have excess superoxide anion (O₂⁻), NUV induced a 9-fold increase in mutation above the level that normally occurs in this double mutant. When a sodA sodB double mutant contained a plasmid carrying katG⁺ HP-I catalase), mutation by NUV was reduced to wild-type (sodA⁺sodB⁺) levels. Also, in the sodA sodB xthA triple mutant, which lacks exonuclease III (exoIII) in addition to SOD, the mutations frequency by NUV was reduced to wild-type levels. This synergistic action of NUV and O₂⁻ suggested that pre-mutational lesions occur, with exoIII converting these lesions to stable mutants. Exposure to H₂O₂ induced a 2.8 fold increase in mutations in sodA sodB double mutants, but was reduced to control levels when a plasmid carrying katG⁺ was introduced. These results suggest that NUV, in addition to its other effects on cells, increases mutations indirectly by increasing the flux of OH^. radicals, possibly by generating excess H₂O₂.     

Effect of 6,9-deepoxy-6,9-(phenylimino)-delta 6,8-prostaglandin 1(1) (U-60,257), an inhibitor of leukotriene synthesis, on human neutrophil function

A23187, a calcium ionophore, stimulated a time-dependent generation of 5(S), 12(R)-dihydroxy-6,8,10,14-eicosatetraenoic acid (leukotriene B₄), production of superoxide anion (O₂^?) and release of granule-associated β-glucuronidase and lysozyme by human neutrophils. Leukotriene B₄ also elicited the selective release of granule enzymes from cytochalasin B-treated neutrophils. U-60,257, a recently identified inhibitor of leukotriene (LT) C₄ and D₄ synthesis, caused a dose-related (1–10 μM) suppression of LTB₄ production by A23187-activated neutrophils. Degranulation and O₂^? generation by neutrophils exposed to A23187 and the chemotactic oligopeptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), were also inhibited with U-60,257.     

Permeability of bilayer lipid membranes for superoxide (O2) radicals

Egg yolk phosphatidylcholine monolamellar liposomes (1000 Å in diameter) loaded with cytochrome c were placed into an external solution, in which superoxide radicals, O₂⁻, were generated by a xanthine-xanthine oxidase system. The penetration of the superoxide radicals across the liposomal membrane was detected by cytochrome c reduction in the inner liposome compartment. The effects of modifiers and temperature on this process were studied. The permeability of liposomal membrane for O₂⁻(P′_O2⁻ = (7.6 ± 0.3) · 10^-8 cm/s), or HO₂^• (P′_HO2⁻ = 4.9 · 10^-4 cm/s) were determined. The effect of the transmembrane electric potential (K⁺ concentration gradient, valinomycin) on the permeability of liposomal membranes for O₂⁻ were investigated. It was found that O₂⁻ can penetrate across liposomal membrane in an uncharged form. The feasibility of penetration of superoxide radicals through liposomal membrane, predominantly via anionic channels, was demonstrated by the use of an intramolecular cholesterol-amphotericin B complex.