Characterization of a mammalian peroxiredoxin that contains one conserved cysteine

A new type of peroxidase enzyme, named thioredoxin peroxidase (TPx), that reduces H2O2 with the use of electrons from thioredoxin and contains two essential cysteines was recently identified. TPx homologs, termed peroxiredoxin (Prx), have also been identified and include several proteins, designated 1-Cys Prx, that contain only one conserved cysteine. Recombinant human 1-Cys Prx expressed in and purified from Escherichia coli has now been shown to reduce H2O2 with electrons provided by dithiothreitol. Furthermore, human 1-Cys Prx transiently expressed in NIH 3T3 cells was able to remove intracellular H2O2 generated in response either to the addition of exogenous H2O2 or to treatment with platelet-derived growth factor. The conserved Cys47-SH group was shown to be the site of oxidation by H2O2. Thus, mutation of Cys47 to serine abolished peroxidase activity. Moreover, the oxidized intermediate appears to be Cys-SOH. In contrast to TPx, in which one of the two conserved cysteines is oxidized to Cys-SOH and then immediately reacts with the second conserved cysteine of the second subunit of the enzyme homodimer to form an intermolecular disulfide, the Cys-SOH of 1-Cys Prx does not form a disulfide. Neither thioredoxin, which reduces the disulfide of TPx, nor glutathione, which reduces the Cys-SeOH of oxidized glutathione peroxidase, was able to reduce the Cys-SOH of 1-Cys Prx and consequently could not support peroxidase activity. Human 1-Cys Prx was previously shown to exhibit a low level of phospholipase A2 activity at an acidic pH; the enzyme was thus proposed to be lysosomal, and Ser32 was proposed to be critical for lipase function. However, the mutation of Ser32 or Cys47 has now been shown to have no effect on the lipase activity of 1-Cys Prx, which was also shown to be a cytosolic protein. Thus, the primary cellular function of 1-Cys Prx appears to be to reduce peroxides with the use of electrons provided by an as yet unidentified source; the enzyme therefore represents a new type of peroxidase.     

Effect of tumour necrosis factor-alpha on proliferation, activation and protein synthesis of rat hepatic stellate cells

BACKGROUND/AIMS: Hepatic stellate cells represent the principal matrix-synthesising cells of damaged liver and are targets of a number of cytokines currently under investigation. The study analyses the effects of tumour necrosis factor-alpha and interferon-gamma on proliferation, "activation" and protein synthesis of hepatic stellate cells. METHODS: Primary cultures of hepatic stellate cells were exposed to tumour necrosis factor-alpha and interferon-gamma. Cell proliferation was studied by 3H-thymidine and bromo-deoxy-uridine incorporation. Protein synthesis was analysed using immunoprecipitation, Western- and Northern blotting techniques. RESULTS: Proliferation of hepatic stellate cells was reduced by tumor necrosis factor-alpha and interferon-gamma, while "activation" of hepatic stellate cells as assessed by expression of smooth muscle alpha-actin and of TGF-beta/activin type I receptor was induced by tumour necrosis factor-alpha but downregulated by interferon-gamma. Tumour necrosis factor-alpha increased the synthesis of distinct extracellular matrix proteins, particularly of fibronectin and tenascin, but decreased collagen type III expression. In contrast, interferon-gamma reduced the synthesis of all connective tissue proteins tested. Among the protease inhibitors, interferon-gamma induced C1-esterase inhibitor synthesis, while tumour necrosis factor-alpha stimulated plasminogen activator inhibitor type 1 production. CONCLUSIONS: Tumour necrosis factor-alpha and interferon-gamma decrease proliferation of hepatic stellate cells, while "activation" of hepatic stellate cells and synthesis of proteins involved in matrix metabolism are regulated in a differential, cytokine-specific manner, suggesting that both cytokines play an important role in liver repair.     

Reactions of bovine liver catalase with superoxide radicals and hydrogen peroxide

The oxidized intermediates generated upon exposure of bovine liver catalase to hydrogen peroxide (H2O2) and superoxide radical (O2-) fluxes were examined with UV-visible spectrophotometry. H2O2 and O2- were generated by means of glucose/glucose oxidase and xanthine/xanthine oxidase systems. Serial overlay of absorption spectra in the Soret (350-450 nm) and visible (450-700 nm) regions showed that three oxidized intermediates, namely Compounds I, II and III, can be observed upon exposure of catalase to enzymatically generated H2O2 and O2-. Compound I is formed during the reaction of native enzyme with H2O2 and disappears in two ways: (i) via the catalytic reaction with H2O2 to restore native catalase and (ii) via the reaction with O2- to form Compound II. At low H2O2 concentrations (< 4.8 x 10(-9) M H2O2), Compound II reverts towards the native state mainly in a direct one-step reaction, whereas at higher H2O2 concentrations the pathway of Compound II back to the native enzyme involves Compound III. Formation of the latter from Compound II and H2O2 is irreversible and the rate constant of this reaction is 6.1 +/- 0.2 x 10(4) M-1 s-1. The formation of Compound III through the direct reaction of O2- with native enzyme has also been observed. Depending on the experimental conditions, the inactivation of catalase by O2- can be due to accumulation of Compound II ("slow" inhibition) or to the formation of Compound III ("rapid" inhibition) part of which leads to a dead end product. Formation of Compound III and of this dead end product are responsible for the irreversible inactivation in presence of an excess of H2O2.     

Nursing practice and patient care in a changing home healthcare environment

In this study, lenses of autopsy eyeballs, anterior capsules including lens epithelium taken during operation for cortical cataract, after cataract tissue obtained at the time of operation, and Elschnig's pearles and Soemmerring's ring from autopsy eye-balls were examined for a variety of factors, such as growth factors, cytokines, bioactive substance factors, cytoskeleton proteins and extracellular matrices by immunocytohistochemistry. Preoperative lens epithelium expressed epidermal growth factor (EGF), EGF-receptor (R), fibroblast growth factor (FGF), FGF-R, interleukin (IL)-1-RII, tumor necrosis factor-alpha (TNF-alpha), plasminogen activator inhibitor type-1 (PAI-1), keratin and laminine. In addition to the above factors, opacified fibrous capsule in after cataract expressed transforming growth factor-beta (TGF-beta), insulin-like growth factor-II (IGF-II), platelet derived growth factor-AB (PDGF-AB), IL-6, prostaglandin-E2 (PG-E2), alpha smooth muscle actin, fibronectin, and I and III to VI type collagen. Elschnig's pearls expressed FGF-R, TNF-alpha, and laminin. Soemmerring's ring expressed EGF, FGF, FGF-R, IL-1-RII, keratin, tissue-PA, and PAI-1.     

Effects of free radical scavengers on cytokine actions on islet cells

We investigated the effect of free radical scavengers on the actions of cytokines on islet cells. Interferon-gamma and tumor necrosis factor-alpha reduced the nicotinamide adenine dinucleotide content of mouse islet cells; the combination of interferon-gamma (4 x 10(5) U/l) and tumor necrosis factor-alpha (4 x 10(5) U/l) caused nicotinamide adenine dinucleotide reduction by approximately 40%. Dimethyl urea and dimethyl sulfoxide prevented the decrease, whereas superoxide dismutase, catalase, and mannitol were not effective. Dimethyl urea and dimethyl sulfoxide protected islet cells from the synergistic cytotoxic action of interferon-gamma and tumor necrosis factor-alpha. Major histocompatibility complex class II antigen induction by interferon-gamma and tumor necrosis factor-alpha was also inhibited by dimethyl urea and dimethyl sulfoxide, but not by superoxide dismutase, catalase and mannitol. Since superoxide dismutase of a membrane-penetrable form attenuated the class II antigen induction, the inefficiency of superoxide dismutase, catalase and mannitol may be attributable to their inability to penetrate islet cells. These results suggest that the intracellular generation of free oxygen radicals is involved in islet cell cytotoxicity and class II molecule expression by interferon-gamma and tumor necrosis factor-alpha, and that nicotinamide adenine dinucleotide reduction may be associated with islet cell dysfunction caused by the cytokines.     

Protection of the arterial internal elastic lamina by inhibition of the renin-angiotensin system in the rat

To endow biomaterials with the ability to regulate cell functions such as proliferation, differentiation, and apoptosis, growth factor proteins were covalently immobilized. The proteins were immobilized on various matrices using different chemical methods. It was shown that insulin and epidermal growth factor stimulated cellular functions even after immobilization. Pattern-immobilization of growth factor proteins clearly demonstrated the stimulation by immobilized proteins. In other words, this type of stimulation by non-diffusional growth factors enabled us to regulate tissue formation with artificial biomaterials. The stimulation was enhanced by coimmobilization with adhesion factors. These stimulations due to the immobilized growth factors may mimic juxtacrine stimulation of membrane-anchored growth factors such as heparin-binding epidermal growth factor, transforming growth factor-alpha, and tumor necrosis factor-alpha.     

Study of the effects of thermotherapy in benign prostatic hypertrophy

The role(s) of protein kinases in the regulation of G protein-dependent activation of phosphatidylinositol-specific phospholipase C by tumor necrosis factor-alpha was investigated in the osteoblast cell line MC3T3-E1. We have previously reported the stimulatory effects of tumor necrosis factor-alpha and A1F4-, an activator of G proteins, on this phospholipase pathway documented by a decrease in mass of PI and release of diacylglycerol. In this study, we further explored the mechanism(s) by which the tumor necrosis factor or A1F4(-)-promoted breakdown of phosphatidylinositol and the polyphosphoinositides by phospholipase C is regulated. Tumor necrosis factor-alpha was found to elicit a 4-5-fold increase in the formation of [3H]inositol-1,4-phosphate and [3H]inositol-1,4,5-phosphate; and a 36% increase in [3H]inositol-1-phosphate within 5 min in prelabeled cells. [3H]inositol-4-phosphate, a metabolite of [3H]inositol-1,4-phosphate and [3H]inositol-1,4,5-phosphate, was found to be the predominant phosphoinositol product of tumor necrosis factor-alpha and A1F4(-)-activated phospholipase C hydrolysis after 30 min. In addition, the preincubation of cells with pertussis toxin decreased the tumor necrosis factor-induced release of inositol phosphates by 53%. Inhibitors of protein kinase C, including Et-18-OMe and H-7, dramatically decreased the formation of [3H]inositol phosphates stimulated by either tumor necrosis factor-alpha or A1F4- by 90-100% but did not affect basal formation. The activation of cAMP-dependent protein kinase, or protein kinase A, by the treatment of cells with forskolin or 8-BrcAMP augmented basal, tumor necrosis factor-alpha and A1F4(-)-induced [3H]inositol phosphate formation. Therefore, we report that protein kinases can regulate tumor necrosis factor-alpha-initiated signalling at the cell surface in osteoblasts through effects on the coupling between receptor, G-protein and phosphatidylinositol-specific phospholipase C.     

Glutathione peroxidase degrades intracellular hydrogen peroxide and thereby inhibits intracellular protein iodination in thyroid epithelium

Protein iodination in the thyroid is largely confined to the surface of the epithelium. Intracellular iodine binding is insignificant. We have tested our hypothesis that the key mechanism in the control of intracellular iodination is the control of the intracellular availability of H2O2. The sites of iodination were identified by locating bound radioiodine in electron microscopic autoradiographs, produced from porcine thyroid epithelium grown on filter in Transwell bicameral culture chambers. Autoradiographs obtained after standard incubations with 125I for 15 min to 3 h were all characterized by concentrations of autoradiographic grains along the external surface of the plasma membrane and very few grains over the cytoplasm. The presence of 10 microM H2O2 in the incubation medium resulted in a drastically changed labeling pattern now showing a dissemination of grains over the entire cytoplasm. Epithelia with elevated GSH peroxidase activity produced autoradiographs showing the same restriction of grains to the cell surface as controls; this pattern was the same in the absence and presence of H2O2 (up to 10 microM). Cultures with subnormal GSH peroxidase activity presented cytoplasmic labeling both in the absence and presence of H2O2. In conclusion, iodine binding in filter-cultured thyroid epithelium under normal conditions is an extracellular process located at the cell surface. When H2O2 is available intracellularly, iodination takes place in the cytoplasm, evidently catalyzed by intracellular thyroperoxidase. Normally, this iodination is prevented by cytosolic GSH peroxidase that effectively degrades H2O2 and thus controls intracellular iodination. The observations should be applicable to the thyroid in vivo.     

An essential role for ectodomain shedding in mammalian development

The ectodomains of numerous proteins are released from cells by proteolysis to yield soluble intercellular regulators. The responsible protease, tumor necrosis factor-alpha converting enzyme (TACE), has been identified only in the case when tumor necrosis factor-alpha (TNFalpha) is released. Analyses of cells lacking this metalloproteinase-disintegrin revealed an expanded role for TACE in the processing of other cell surface proteins, including a TNF receptor, the L-selectin adhesion molecule, and transforming growth factor-alpha (TGFalpha). The phenotype of mice lacking TACE suggests an essential role for soluble TGFalpha in normal development and emphasizes the importance of protein ectodomain shedding in vivo.     

A heritable component for external apical root resorption in patients treated orthodontically

Double-stimulation was used to demonstrate that, in a T lymphocytic cell line (CEM), phorbol myristate acetate (PMA) rapidly induced NF-kappa B through a signaling pathway which did not involve reactive oxygen species (ROS) and was different from the activation triggered by either H2O2 or tumor necrosis factor-alpha (TNF-alpha). Since these latter compounds were known to activate NF-kappa B translocation in a redox-sensitive way, we have demonstrated that NF-kappa B activation by PMA was resistant to antioxidant N-acetyl-L-cysteine (NAC) and sensitive to kinase inhibitors staurosporine and H7 while activation by H2O2 or TNF-alpha were not.     

Cobalt-mediated generation of reactive oxygen species and its possible mechanism

Electron spin resonance spin trapping was utilized to investigate free radical generation from cobalt (Co) mediated reactions using 5,5-dimethyl-1-pyrroline (DMPO) as a spin trap. A mixture of Co with water in the presence of DMPO generated 5,5-dimethylpyrroline-(2)-oxy(1) DMPOX, indicating the production of strong oxidants. Addition of superoxide dismutase (SOD) to the mixture produced hydroxyl radical (.OH). Catalase eliminated the generation of this radical and metal chelators, such as desferoxamine, diethylenetriaminepentaacetic acid or 1,10-phenanthroline, decreased it. Addition of Fe(II) resulted in a several fold increase in the .OH generation. UV and O2 consumption measurements showed that the reaction of Co with water consumed molecular oxygen and generated Co(II). Since reaction of Co(II) with H2O2 did not generate any significant amount of .OH radicals, a Co(I) mediated Fenton-like reaction [Co(I) + H2O2-->Co(II) + .OH + OH-] seems responsible for .OH generation. H2O2 is produced from O2.- via dismutation, O2.- is produced by one-electron reduction of molecular oxygen catalyzed by Co. Chelation of Co(II) by biological chelators, such as glutathione or beta-ananyl-3-methyl-L-histidine alters, its oxidation-reduction potential and makes Co(II) capable of generating .OH via a Co(II)-mediated Fenton-like reaction [Co(II) + H2O2-->Co(III) + .OH + OH-]. Thus, the reaction of Co with water, especially in the presence of biological chelators, glutathione, glycylglycylhistidine and beta-ananyl-3-methyl-L-histidine, is capable of generating a whole spectrum of reactive oxygen species, which may be responsible for Co-induced cell injury.     

Site-specific DNA damage induced by NADH in the presence of copper(II): role of active oxygen species

Oxidative DNA damage by NAD(P)H in the presence of metal ions has been characterized by using 32P 5' end-labeled DNA fragments obtained from human p53 tumor suppressor gene and c-Ha-ras-1 protooncogene. NADH, as well as other endogenous reductants, induced DNA damage in the presence of Cu(II). The order of inducing effect on Cu(II)-dependent DNA damage was ascorbate > reduced glutathione (GSH) > NADH > NADPH. Although NADH caused no or little DNA damage in the presence of Fe(III)-EDTA, the addition of H2O2 induced the DNA damage. The Cu(II)-mediated DNA damage induced by NADH was inhibited by catalase and bathocuproine, a Cu(I)-specific chelator; but not by scavengers of hydroxyl free radical (.OH), suggesting the involvement of active species derived from hydrogen peroxide (H2O2) and Cu(I) rather than .OH. The predominant cleavage sites were thymine residues located 5' and/or 3' to guanine. The cleavage pattern was similar to that induced by Cu(II) plus GSH, Cu(II) plus ascorbate, or Cu(I) plus H2O2. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine by NADH increased with its concentration in the presence of Cu(II). UV-visible spectroscopy indicated the facilitation of reduction of Cu(II) by NADH under some conditions. ESR spin-trapping experiments and mass spectrometry showed that the carbon-centered radical was formed during the reaction of NADH with Cu(II). These results suggest that optimal molar ratios of DNA/metal ion yield copper with a high redox potential which catalyzes NADH autoxidation to NAD. being further oxidized to NAD+ with generation of superoxide radical and that H2O2 reacts with Cu(I) to form active oxygen species such as copper(I)-peroxide complex causing DNA damage.     

Kinetic studies on the removal of extracellular hydrogen peroxide by cultured fibroblasts

To investigate the function of antioxidant enzymes in intact cells, we examined the removal of extracellular H2O2 by cultured fibroblasts (IMR-90). H2O2 concentration dependence of the reaction rate was interpreted as that the process involves two kinetically different reactions (referred to as reactions 1 and 2). Reaction 1 was characterized by a relatively low Km value (about 40 microM), and reaction 2 by linear dependence of the rate up to 500 microM H2O2. The magnitude of reaction 1 was reduced by treatment of the cells with diethyl maleate or 6-amino-nicotinamide, while reaction 2 was inhibited by 3-amino-1,2,4-triazole treatment. It was concluded that reactions 1 and 2 are principally due to GSH peroxidase and catalase, respectively. The values of kinetic parameters were estimated by curve-fitting, and it was inferred that 80 to 90% of H2O2 is decomposed by GSH peroxidase at H2O2 concentrations lower than 10 microM. The contribution of catalase increases with the increase in H2O2 concentration. The intact cells showed a low catalase activity (about 15%), as compared with the activity found in the solubilized cells. The low catalase activity was ascribed to the latency of the enzyme caused by localization in peroxisomes. Fibroblasts also removed intracellular H2O2 generated by menadione. Treatment with diethyl maleate greatly impaired the H2O2-removing capability and caused H2O2 efflux into the medium.     

Mechanism of horseradish peroxidase catalyzed epinephrine oxidation: obligatory role of endogenous O2- and H2O2

Horseradish peroxidase (HRP) catalyzes cyanide sensitive oxidation of epinephrine to adrenochrome at physiological pH in the absence of added H2O2 with concurrent consumption of O2. Both adrenochrome formation and O2 consumption are significantly inhibited by catalase, indicating a peroxidative mechanism as a major part of oxidation due to intermediate formation of H2O2. Sensitivity to superoxide dismutase (SOD) also indicates involvement of O2- in the oxidation. Although SOD-mediated H2O2 formation should continue epinephrine oxidation through a peroxidative mechanism, low catalytic turnover, on the contrary, indicates that O2- takes part in a vital reaction to form an intermediate for adrenochrome formation and O2 consumption. Generation of O2- is evidenced by ferricytochrome c reduction sensitive to SOD. On addition of H2O2, both adrenochrome formation and O2 consumption are further increased due to reaction of molecular oxygen with some intermediate oxidation product. Peroxidative oxidation proceeds by one-electron transfer generating o-semiquinone and similar free radicals which when stabilized with Zn2+ or spin-trap, alpha-phenyl-tert-butylnitrone (PBN), inhibit adrenochrome formation and O2 consumption. The free radicals thus favor reduction of O2 rather than the disproportionation reaction. Spectral studies indicate that, during epinephrine oxidation in the presence of catalase, HRP remains in the ferric state absorbing at 403 nm. This suggests that HRP catalyzes epinephrine oxidation by its oxidase activity through Fe3+/Fe2+ shuttle consuming O2, where the rate of reduction of ferric HRP with epinephrine is slower than subsequent oxidation of ferrous HRP by O2 to form compound III. Compound III was not detected spectrally because of its quick reduction to the ferric state by epinephrine or its subsequent oxidation product. In the absence of catalase, peroxidative cycles predominate when HRP still remains in the ferric state through the transient formation of compounds I and II not detectable spectrally. Among various mono- and dihydroxyl aromatic donors tested, only epinephrine shows the oxidase reaction. Binding studies indicate that epinephrine interferes with the binding of CN-, SCN-, and guaiacol indicating that HRP preferentially binds epinephrine near the heme iron close to the anion or aromatic donor binding site to catalyze electron transfer for oxidation. HRP thus initiates epinephrine oxidation by its oxidase activity generating O2- and H2O2. Once H2O2 is generated, the peroxidative cycle continues with the consumption of O2, through the intermediate formation of O2- and H2O2 which play an obligatory role in subsequent cycles of peroxidation.     

Exercise, dietary obesity, and growth in the rat

Four regimens: high-fat diet, exercised (I); chow, exercised (II); high-fat sedentary (III); and chow, sedentary (IV) were initiated in 35-day-old male rats. Growth was exponential in I and II and exponential progressing to rectilinear in III and IV. The exponential model predicted the decreasing rank order in asymptotic weight to be: III, IV, I, II. Body composition data (9 components) showed rank order in masses of fat and the fat-free body mass compartment (FFBM) to be the same as for asymptotic live weight. The rectilinear growth mode probably reflected fat accretion. High-fat diet increased and treadmill exercise decreased FFBM, the latter being reversible. These effects depended on regimen initiations by the 5-7th wk of age. During growth, masses of H2O, muscle, and skin increased as functions of body size; bone as a function of age; and heart, liver, gut, testevity, and diet. Growth in body size was expressed more precisely with FFBM, instead of live weight, as the index of size.