Oxidant inhibition of epithelial active sodium transport

The purpose of this study was to quantify the effects of extracellularly generated partially reduced oxygen species on active sodium (NA⁺) transport across the ventral toad skin, a well-studied epithelium. Sections of skin from decapitated toads were mounted in an Ussing chamber, bathed on both sides with electrolyte solution containing 500 μM xanthine and bubbled continuously with room air. The tissues were short-circuited, and short circuit current (Isc) and tissue resistance (R_t were monitored continuously with an automatic voltage clamp apparatus. Fifteen mU/ml of xanthine oxidase (XO), either purchased from Calbiochem or purified from cream, were instilled in either the apical (mucosal) or basolateral (serosal) baths at t = 0 and T = 10 min. Hydrogen peroxide (H₂O₂) concentrations increased to 200 μM within the first 20 min and then decreased, reaching a value of 40 μM by 60 min. Mean [H₂O₂] was 90 μM. Instillation of XO in the apical bath resulted in a large decrease in Isc and an increase in R_t, their values being 43% and 160% of their corresponding controls 85 min after the first instillation. Addition of superoxide dismutase and catalase completely prevented these changes. Instillation of XO in the basolateral bath had no effect. Similar physiological responses were obtained using the Calbiochem XO or the purified XO, which contained no measurable protease activity. It was concluded that extracellularly generated partially reduced oxygen species may interfere with active Na⁺ transport by possibly damaging apical Na⁺ channel proteins.     

DNA Single Strand Breakage by H2O2 and Ferric or Cupric Ions: Its Modulation by Histidine

The role of histidine on DNA breakage induced by hydrogen peroxide (H₂O₂) and ferric ions or by H₂O₂ and cupric ions was studied on purified DNA. L-histidine slightly reduced DNA breakage by H₂O₂ and Fe³⁺ but greatly inhibited DNA breakage by H₂O₂ and Cu²⁺. However, only when histidine was present, the addition of EDTA to H₂O₂ and Fe³⁺ exhibited a bimodal dose response curve depending on the chelator metal ratio. The enhancing effect of histidine on the rate of DNA degradation by H₂O₂ was maximal at a chelator metal ratio between 0.2 and 0.5, and was specific for iron. When D-histidine replaced L-histidine, the same pattern of EDTA dose response curve was observed. Superoxide dismutase greatly inhibited the rate of DNA degradation induced by H₂O₂, Fe³⁺, EDTA and L-histidine involving the superoxide radical.

These studies suggest that the enhancing effect of histidine on the rate of DNA degradation by H₂O₂ and Fe³⁺ is mediated by an oxidant which could be a ferrous-dioxygen-ferric chelate complex or a chelate-ferryl ion.     

Seasonal water uptake and movement in root systems of Australian phraeatophytic plants of dimorphic root morphology: a stable isotope investigation

A natural abundance hydrogen stable isotope technique was used to study seasonal changes in source water utilization and water movement in the xylem of dimorphic root systems and stem bases of several woody shrubs or trees in mediterranean-type ecosystems of south Western Australia. Samples collected from the native treeBanksia prionotes over 18 months indicated that shallow lateral roots and deeply penetrating tap (sinker) roots obtained water of different origins over the course of a winter-wet/summer-dry annual cycle. During the wet season lateral roots acquired water mostly by uptake of recent precipitation (rain water) contained within the upper soil layers, and tap roots derived water from the underlying water table. The shoot obtained a mixture of these two water sources. As the dry season approached dependence on recent rain water decreased while that on ground water increased. In high summer, shallow lateral roots remained well-hydrated and shoots well supplied with ground water taken up by the tap root. This enabled plants to continue transpiration and carbon assimilation and thus complete their seasonal extension growth during the long (4–6 month) dry season. Parallel studies of other native species and two plantation-grown species ofEucalyptus all demonstrated behavior similar to that ofB. prionotes. ForB. prionotes, there was a strong negative correlation between the percentage of water in the stem base of a plant which was derived from the tap root (ground water) and the amount of precipitation which fell at the site. These data suggested that during the dry season plants derive the majority of the water they use from deeper sources while in the wet season most of the water they use is derived from shallower sources supplied by lateral roots in the upper soil layers. The data collected in this study supported the notion that the dimorphic rooting habit can be advantageous for large woody species of floristically-rich, open, woodlands and heathlands where the acquisition of seasonally limited water is at a premium.     

Chilling,oxidative stress and antioxidant responses inArabidopsis thaliana callus

Chilling ofArabidopsis thaliana (L.) Heynh. callus tissue to 4 °C led to conditions of oxidative stress, as indicated by increased levels of the products of peroxidative damage to cell membranes. Cellular H₂O₂ was also observed to increase initially upon chilling but by day 8 cellular levels had declined to below control levels. Although levels of catalase activity remained similar to those in unchilled tissue, activity of ascorbate peroxidase increased between days 4 and 8 of chilling to 4 °C. In callus held at 23 °C, levels of reduced glutathione remained static whereas they rose in callus held at 4 °C. Levels of oxidised glutathione were initially low but increased significantly by day 4 in the chilled callus. At 23 °C, however, levels of oxidised glutathione remained low. Between days 1 and 3 at 4 °C, levels of glutathione reductase activity increased but by day 8 glutathione reductase activity was similar to that in cells held at 23 °C. Exposure of callus to abscisic acid at 23 °C also led to increased activities of ascorbate peroxidase and glutathione reductase.Abbreviations ABA abscisic acid - GSH reduced glutathione - GSSG oxidised glutathione - TTC 2,35-triphenyltetrazolium chloride This work is supported by a grant from the Biotechnology and Biological Sciences Research Council.     

Mutual Contact of Adherent Polymorphonuclear Leukocytes Inhibits Their Generation of Superoxide

Superoxide generation by polymorphonuclear leukocytes (PMNs) in suspension, or adherent to glass or plastic, after stimulation with /V-formylmethionyl-leucyl-phenylalanine or phorbol myristate acetate was measured by cytochromec reduction and spin trapping. Amounts of superoxide generated by adherent PM Ns were inversely related to cell density. The generation of hydrogen peroxide was also inhibited at higher cell densities. In contrast to adherent cells, superoxide released by PMNs in suspension linearly increased with respect to cell number over a wider range. Microscopic observation indicated that the number of cells in mutual contact increased rapidly at cell densities higher than 4 × 10⁴ cells/cm², and inhibition of superoxide became apparent at higher cell densities. Mediators which could be released by PMNs, such as NO and adenosine, were not the cause of inhibition. Thesedatu suggest that mutual contact of PMNs suppresses their generation of superoxide. Survival rates of PMNs after stimulation increased at higher densities, indicating that the mutual contact-induced inhibition of superoxide generation by PMNs may be physiologically relevant at sites of inflammation.     

Biochemical diversity among sulfur-dependent, hyperthermophilic microorganisms

Abstract: Hyperthermophiles are a recently discovered group of microorganisms that grow at and above 90°C. They currently comprise over 20 different genera, and except for two novel bacteria, all are classified as Archaea. The majority of these organisms are obligately anaerobic heterotrophs that reduce elemental sulfur (S°) to H₂S. The best studied from a biochemical perspective are the archaeon, Pyrococcus furiosus , and the bacterium, Thermotoga maritima , both of which are saccharolytic. P. furiosus is thought to contain a new type of Entner-Doudoroff pathway for the conversion of carbohydrates ultimately to acetate, H₂ and CO₂. The pathway is independent of nicotinamide nucleotides and involves novel types of ferredoxin-linked oxidoreductases, one of which has tungsten, a rarely used element, as a prosthetic group. The only site of energy conservation is at the level of acetyl CoA, which in the presence of ADP and phosphate is converted to acetate and ATP in a single step. In contrast, T. maritima utilizes a conventional Embden-Meyerhof pathway for sugar oxidation. P. furiosus also utilizes peptides as a sole carbon and energy source. Amino acid oxidation is thought to involve glutamate dehydrogenase together with at least three types of novel ferredoxin-linked oxidoreductases which catalyze the oxidation of 2-ketoglutarate, aryl pyruvates and formaldehyde. One of these enzymes also utilizes tungsten. In P. furiosus , virtually all of the reductant that is generated during the catabolism of both carbohydrates and peptides is channeled to a cytoplasmic hydrogenase. This enzyme is now termed sulhydrogenase, as it reduces both protons to H₂ and S°(or polysulfide) to H₂S. S° reduction appears to lead to the conservation of energy in P. furiosus but not in T. maritima , although the mechanism by which this occurs is not known.     

Peptide crystal growth via sulfonate salt formation: The structure of bis-glycylglycine-1,5-naphthalenedisulfonate hydrate

Summary Continuing a line of investigations on methods for formation and growth of high-quality crystals of peptides, the glycylglycine sequence has been crystallized by evaporation methods as a salt with 1,5-naphthalenedisulfonic acid. The structure of the peptide is highly extended, and is conformationally quite similar to the structures which have been characterized for other zwitterionic and salt forms of this sequence. Thus, crystallization as a salt with this sulfonic acid has imposed no undue influence upon the molecular conformation. These results offer further indication that the preparation of peptide sulfonate salts, particularly with arene templates, may have broad general utility for crystallization of interesting sequences which until now have not been approachable in their zwitterionic forms.     

The oxidative stress response in Bacillus subtilis

Abstract Bacillus subtilis undergoes a typical bacterial stress response when exposed to low concentrations (0.1 mM) of hydrogen peroxide. Protection is thereby induced against otherwise lethal, challenge concentrations (10 mM) of this oxidant and a number of proteins are induced including the scavenging enzymes, catalase and alkyl hydroperoxide reductase, and a putative DNA binding and protecting protein. Induced protection against higher concentrations (10–30 mM) of hydrogen peroxide is eliminated in a catalase-deficient mutant. Both RecA and Spo0A influence the basal but not the induced resistance to hydrogen peroxide. A regulatory mutation has been characterized that affects the inducible phenotype and is constitutively resistant to high concentrations of hydrogen peroxide. This mutant constitutively overexpresses the proteins induced by hydrogen peroxide in the wild-type. The resistance of spores to hydrogen peroxide is partly attributable to binding of small acid soluble proteins by the spore DNA and partly to a second step which coincides with the depletion of the NADH pool, which may inhibit the generation of hydroxyl radicals from hydrogen peroxide.     

Hydrogen peroxide-mediated inactivation of microsomal cytochrome P450 during monooxygenase reactions

Cytochrome P450 can undergo inactivation following monooxygenase reactions in liver microsomes of untreated, phenobarbital and 3-methylcholanthrene-treated rats and rabbits. The acceleration of cytochrome P450 loss in the presence of catalase inhibitors (sodium azide, hydroxylamine) indicates that hydrogen peroxide is involved in hemoprotein degradation. It was revealed that cytochrome P450 is inactivated mainly by H₂O₂ formed through peroxy complex breakdown, whereas H₂O₂ formed via the dismutation of superoxide anions produces a slight inactivating effect. The hydrogen peroxide added outside or formed by a glucose-glucose oxidase system has less of an inactivating effect than H₂O₂ produced within the cytochrome P450 active center. Self-inactivation of cytochrome P450 during oxygenase reactions is highly specific. Other components of the monooxygenase system, such as cytochrome b₅, NADH- and NADPH-specific flavorproteins, undergo no inactivation. The alterations in phospholipid content and in the rate of lipid peroxidation were not observed as well. The inactivation of cytochrome P450 by H₂O₂ is the result of heme loss or destruction without cytochrome P420 formation. Such. a mechanism operates with different substrates and cytochrome P450 species catalyzing the partially coupled monooxygenase reactions.     

Generation of hydrogen peroxide,superoxide anion and the hydroxyl free radical from polyphenols and active benzene metabolites: Their possible role in mutagenesis

Benzene is strongly suspected of being an animal and human carcinogen, but the mechanisms by which it induces tumors of lymphoid and hematopoietic organs are unknown. Production of active oxygen species from benzene metabolites [hydroquinone (HQ), catechol and 1,2,4-benzenetriol (1,2,4-BT) and related polyphenols (resorcinol, pyrogallol and phloroglucinol) are investigated. Pyrogallol and 1,2,4-BT can produce H₂O₂, O ₂ ^– and^·OH simultaneously, and have powerful mutagenic potential. Resorcinol and phloroglucinol cannot produce all of the active oxygen species, and show no mutagenic effects. Catechol can produce H₂O₂, but cannot produce O ₂ ^– and^·OH, and has no mutagenic activity. These data strongly support the hypothesis that benzene metabolites can cause mutagenicity via the generation of oxygen radicals. Although HQ produces H₂O₂ only, and less than produced by pyrogallol and 1,2,4-BT, the mutagenicity of HQ is higher. The results indicate that HQ may act via another mechanism to cause mutagenicity. In the presence of trace metal ions, the reactivity of polyphenols is increased. The biological significance of these phenomena are investigated and discussed.