Increased mitochondrial oxidative stress in the Sod2 (+/−) mouse results in the age-related decline of mitochondrial function culminating in increased apoptosis

To determine the importance of mitochondrial reactive oxygen species toxicity in aging and senescence, we analyzed changes in mitochondrial function with age in mice with partial or complete deficiencies in the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD). Liver mitochondria from homozygous mutant mice, with a complete deficiency in MnSOD, exhibited substantial respiration inhibition and marked sensitization of the mitochondrial permeability transition pore. Mitochondria from heterozygous mice, with a partial deficiency in MnSOD, showed evidence of increased proton leak, inhibition of respiration, and early and rapid accumulation of mitochondrial oxidative damage. Furthermore, chronic oxidative stress in the heterozygous mice resulted in an increased sensitization of the mitochondrial permeability transition pore and the premature induction of apoptosis, which presumably eliminates the cells with damaged mitochondria. Mice with normal MnSOD levels show the same age-related mitochondrial decline as the heterozygotes but occurring later in life. The premature decline in mitochondrial function in the heterozygote was associated with the compensatory up-regulation of oxidative phosphorylation enzyme activity. Thus mitochondrial reactive oxygen species production, oxidative stress, functional decline, and the initiation of apoptosis appear to be central components of the aging process.

Two Structurally Similar Maize Cytosolic Superoxide Dismutase Genes, Sod4 and Sod4A, Respond Differentially to Abscisic Acid and High Osmoticum1

The maize (Zea mays) superoxide dismutase genes Sod4 and Sod4A are highly similar in structure but each responds differentially to environmental signals. We examined the effects of the hormone abscisic acid (ABA) on the developmental response of Sod4 and Sod4A. Although both Sod4 and Sod4A transcripts accumulate during late embryogenesis, only Sod4 is up-regulated by ABA and osmotic stress. Accumulation of Sod4 transcript in response to osmotic stress is a consequence of increased endogenous ABA levels in developing embryos. Sod4 mRNA is up-regulated by ABA in viviparous-1 mutant embryos. Sod4 transcript increases within 4 h with ABA not only in developing embryos but also in mature embryos and in young leaves. Sod4A transcript is up-regulated by ABA only in young leaves, but neither Sod4 nor Sod4A transcripts changed in response to osmotic stress. Our data suggest that in leaves Sod4 and Sod4A may respond to ABA and osmotic stress via alternate pathways. Since the Sod genes have a known function, we hypothesize that the increase in Sod mRNA in response to ABA is due in part to ABA-mediated metabolic changes leading to changes in oxygen free radical levels, which in turn lead to the induction of the antioxidant defense system.

Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts.

Inflammatory processes in chronic rejection remain a serious clinical problem in organ transplantation. Activated cellular infiltrate produces high levels of both superoxide and nitric oxide. These reactive oxygen species interact to form peroxynitrite, a potent oxidant that can modify proteins to form 3-nitrotyrosine. We identified enhanced immunostaining for nitrotyrosine localized to tubular epithelium of chronically rejected human renal allografts. Western blot analysis of rejected tissue demonstrated that tyrosine nitration was restricted to a few specific polypeptides. Immunoprecipitation and amino acid sequencing techniques identified manganese superoxide dismutase, the major antioxidant enzyme in mitochondria, as one of the targets of tyrosine nitration. Total manganese superoxide dismutase protein was increased in rejected kidney, particularly in the tubular epithelium; however, enzymatic activity was significantly decreased. Exposure of recombinant human manganese superoxide dismutase to peroxynitrite resulted in a dose-dependent (IC50 = 10 microM) decrease in enzymatic activity and concomitant increase in tyrosine nitration. Collectively, these observations suggest a role for peroxynitrite during development and progression of chronic rejection in human renal allografts. In addition, inactivation of manganese superoxide dismutase by peroxynitrite may represent a general mechanism that progressively increases the production of peroxynitrite, leading to irreversible oxidative injury to mitochondria.

Formate is the hydrogen donor for the anaerobic ribonucleotide reductase from Escherichia coli.

During anaerobic growth Escherichia coli uses a specific ribonucleoside-triphosphate reductase (class III enzyme) for the production of deoxyribonucleoside triphosphates. In its active form, the enzyme contains an iron-sulfur center and an oxygen-sensitive glycyl radical (Gly-681). The radical is generated in the inactive protein from S-adenosylmethionine by an auxiliary enzyme system present in E. coli. By modification of the previous purification procedure, we now prepared a glycyl radical-containing reductase, active in the absence of the auxiliary reducing enzyme system. This reductase uses formate as hydrogen donor in the reaction. During catalysis, formate is stoichiometrically oxidized to CO2, and isotope from [3H]formate appears in water. Thus E. coli uses completely different hydrogen donors for the reduction of ribonucleotides during anaerobic and aerobic growth. The aerobic class I reductase employs redox-active thiols from thioredoxin or glutaredoxin to this purpose. The present results strengthen speculations that class III enzymes arose early during the evolution of DNA.

Endogenous intracellular glutathionyl radicals are generated in neuroblastoma cells under hydrogen peroxide oxidative stress.

We report the detection of endogenous intracellular glutathionyl (GS.) radicals in the intact neuroblastoma cell line NCB-20 under oxidative stress. Spin-trapping and electron paramagnetic resonance (EPR) spectroscopic methods were used for monitoring the radicals. The cells incubated with the spin trap 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) were challenged with H2O2 generated by the enzymic reaction of glucose/glucose oxidase. These cells exhibit the EPR spectrum of the GS. radical adduct of DMPO (DMPO-.SG) without exogenous reduced glutathione (GSH). The identity of this radical adduct was confirmed by observing hyperfine coupling constants identical to previously reported values in in vitro studies, which utilized known enzymic reactions, such as horseradish peroxidase and Cu/Zn superoxide dismutase, with GSH and H2O2 as substrates. The formation of the GS. radicals required viable cells and continuous biosynthesis of GSH. No significant effect on the resonance amplitude by the addition of a membrane-impermeable paramagnetic broadening agent indicated that these radicals were located inside the intact cell. N-Acetyl-L-cysteine (NAC)-treated cells produced NAC-derived free radicals (NAC.) in place of GS. radicals. The time course studies showed that DMPO-.SG formation exhibited a large increase in its concentration after a lag period, whereas DMPO-NAC. formation from NAC-treated cells did not show this sudden increase. These results were discussed in terms of the limit of antioxidant enzyme defenses in cells and the potential role of the GS. radical burst in activation of the transcription nuclear factor NF-kappa B in response to oxidative stress.

Cytochrome c(551) from Starkeya novella - Characterization, spectroscopic properties, and phylogeny of a diheme protein of the SoxAX family

Cytochromes from the SoxAX family have a major role in thiosulfate oxidation via the thiosulfate-oxidizing multi-enzyme system (TOMES). Previously characterized SoxAX proteins from Rhodovulum sulficlophilum and Paracoccus pantotrophus contain three heme c groups, two of which are located on the SoxA subunit. In contrast, the SoxAX protein purified from Starkeya novella was found to contain only two heme groups. Mass spectrometry showed that a disulfide bond replaced the second heme group found in the diheme SoxA subunits. Apparent molecular masses of 27,229 +/- 10.3 Da and 20,258.6 +/- 1 Da were determined for SoxA and SoxX with an overall mass of 49.7 kDa, indicating a heterodimeric structure. Optical redox potentiometry found that the two heme cofactors are reduced at similar potentials (versus NHE) that are as follows: + 133 mV (pH 6.0); + 104 mV (pH 7.0); +49 (pH 7.9) and +10 mV (pH 8.7). EPR spectroscopy revealed that both ferric heme groups are in the low spin state, and the spectra were consistent with one heme having a His/Cys axial ligation and the other having a His/Met axial ligation. The His/Cys ligated heme is present in different conformational states and gives rise to three distinct signals. Amino acid sequencing was used to unambiguously assign the protein to the encoding genes, soxAX, which are part of a complete sox gene cluster found in S. novella. Phylogenetic analysis of soxA- and soxX-related gene sequences indicates a parallel development of SoxA and SoxY, with the diheme and monoheme SoxA sequences located on clearly separated branches of a phylogenetic tree.

A numerical approach to modeling the catalytic voltammetry of surface-confined redox enzymes

A finite difference method for simulating voltammograms of electrochemically driven enzyme catalysis is presented. The method enables any enzyme mechanism to be simulated. The finite difference equations can be represented as a matrix equation containing a nonlinear sparse matrix. This equation has been solved using the software package Mathematica. Our focus is on the use of cyclic voltammetry since this is the most commonly employed electrochemical method used to elucidate mechanisms. The use of cyclic voltammetry to obtain data from systems obeying Michaelis-Menten kinetics is discussed, and we then verify our observations on the Michaelis-Menten system using the finite difference simulation. Finally, we demonstrate how the method can be used to obtain mechanistic information on a real redox enzyme system, the complex bacterial molybdoenzyme xanthine dehydrogenase.

Human sulfotransferases and their role in chemical metabolism

Sulfonation is an important reaction in the metabolism of numerous xenobiotics, drugs, and endogenous compounds. A supergene family of enzymes called sulfotransferases (SULTs) catalyze this reaction. In most cases, the addition of a sulfonate moiety to a compound increases its water solubility and decreases its biological activity. However, many of these enzymes are also capable of bioactivating procarcinogens to reactive electrophiles. In humans three SULT families, SULT1, SULT2, and SULT4, have been identified that contain at least thirteen distinct members. SULTs have a wide tissue distribution and act as a major detoxification enzyme system in adult and the developing human fetus. Nine crystal structures of human cytosolic SULTs have now been determined, and together with site-directed mutagenesis experiments and molecular modeling, we are now beginning to understand the factors that govern distinct but overlapping substrate specificities. These studies have also provided insight into the enzyme kinetics and inhibition characteristics of these enzymes. The regulation of human SULTs remains as one of the least explored areas of research in the field, though there have been some recent advances on the molecular transcription mechanism controlling the individual SULT promoters. Interindividual variation in sulfonation capacity may be important in determining an individual's response to xenobiotics, and recent studies have begun to suggest roles for SULT polymorphism in disease susceptibility. This review aims to provide a summary of our present understanding of the function of human cytosolic sulfotransferases.

Kidney dysfunction and hypertension: Role for cadmium, P450 and heme oxygenases?

Cadmium (Cd) is a metal toxin of continuing worldwide concern. Daily intake of Cd, albeit in small quantities, is associated with a number of adverse health effects which are attributable to distinct pathological changes in a variety of tissues and organs. In the present review, we focus on its renal tubular effects in people who have been exposed environmentally to Cd at levels below the provisional tolerable intake level set for the toxin. We highlight the data linking such low-level Cd intake with tubular injury, altered abundance of cytochromes P450 (CYPs) in the kidney and an expression of a hypertensive phenotype. We provide updated knowledge on renal and vascular effects of the eicosanoids 20-hydroxyeicosatetraenoic acid (20-HETE) and eicosatrienoic acids (EETs), which are biologically active metabolites from arachidonate metabolism mediated by certain CYPs in the kidney. We note the ability of Cd to elicit oxidative stress and to alter metal homeostasis notably of zinc which may lead to augmentation of the defense mechanisms involving induction of the antioxidant enzyme heme oxygenase-1 (HO-1) and the metal binding protein metallothionein (MT) in the kidney. We hypothesize that renal Cd accumulation triggers the host responses mediated by HO-I and MT in an attempt to protect the kidney against injurious oxidative stress and to resist a rise in blood pressure levels. This hypothesis predicts that individuals with less active HO-1 (caused by the HO-1 genetic polymorphisms) are more likely to have renal injury and express a hypertensive phenotype following chronic ingestion of low-level Cd, compared with those having more active HO-1. Future analytical and molecular epidemiologic research should pave the way to the utility of induction of heme oxygenases together with dietary antioxidants in reducing the risk of kidney injury and hypertension in susceptible people.

Effect of polymorph derived oxidants on IgG in relation to rheumatoid factor binding

Immunoglobulin G from rheumatoid patients is denatured around the hinge region. This has been proposed as an explanation for the presence of circulating autoantibodies to IgG in these patients. It has previously been suggested that oxygen radicals (OR) derived from activated polymorphs may play a role in denaturation in vivo. Using sera from rheumatoid patients and age-matched controls in a modified ELISA technique, we have investigated the potential for polyclonal rheumatoid factors (RF) to bind to OR denatured IgG. Three model systems were used to generate OR in vitro: (a) purified PMN s activated by the cell surface stimulant PMA, (b) radiolysis of IgG in solution to generate specifically the superoxide radical and, in a separate system, the hydroxyl radical, (OH.), (c) purified myeloperoxide in the presence of H2O2 and halide ions. Results: 1. The binding of both IgA and IgM RF s to PMN denatured IgG increased dose dependently for seropositive sera only. 2. The OH. radical but not the superoxide radical significantly increased the binding of IgA and M RF, again only for seropositive sera. 3. The myeloperoxidase enzyme system did not increase RF binding. 4. IgG incubated with elastase was not found to be a better antigen than native IgG. These results indicate that IgG is denatured by OR released from activated PMN, thereby producing an antigen for polyclonal RF s.

An ideal ocular nutritional supplement?

The role of nutritional supplementation in prevention of onset or progression of ocular disease is of interest to health care professionals and patients. The aim of this review is to identify those antioxidants most appropriate for inclusion in an ideal ocular nutritional supplement, suitable for those with a family history of glaucoma, cataract, or age-related macular disease, or lifestyle factors predisposing onset of these conditions, such as smoking, poor nutritional status, or high levels of sunlight exposure. It would also be suitable for those with early stages of age-related ocular disease. Literature searches were carried out on Web of Science and PubMed for articles relating to the use of nutrients in ocular disease. Those highlighted for possible inclusion were vitamins A, B, C and E, carotenoids beta-carotene, lutein, and zeaxanthin, minerals selenium and zinc, and the herb, Ginkgo biloba. Conflicting evidence is presented for vitamins A and E in prevention of ocular disease; these vitamins have roles in the production of rhodopsin and prevention of lipid peroxidation respectively. B vitamins have been linked with a reduced risk of cataract and studies have provided evidence supporting a protective role of vitamin C in cataract prevention. Beta-carotene is active in the prevention of free radical formation, but has been linked with an increased risk of lung cancer in smokers. Improvements in visual function in patients with age-related macular disease have been noted with lutein and zeaxanthin supplementation. Selenium has been linked with a reduced risk of cataract and activates the antioxidant enzyme glutathione peroxidase, protecting cell membranes from oxidative damage while zinc, although an essential component of antioxidant enzymes, has been highlighted for risk of adverse effects. As well as reducing platelet aggregation and increasing vasodilation, Gingko biloba has been linked with improvements in pre-existing field damage in some patients with normal tension glaucoma. We advocate that vitamins C and E, and lutein/zeaxanthin should be included in our theoretically ideal ocular nutritional supplement.

The production of clinical dextran using membrane processes

Clinical dextran is used as a blood volume expander. The British Pharmacopeia (BP) specification for this product requires the amount of dextran below 12,000 MW and above 98,000 MW to be strictly controlled. Dextran is presently fractionated industrially using ethanol precipitation. The aim of this work was to develop an ultrafiltration system which could replace the present industrial process. Initially these molecular weight (MW) bands were removed using batch ultrafiltration. A large number of membranes were tested. The correct BP specification could be achieved using these membranes but there was a significant loss of saleable material. To overcome this problem a four stage ultrafiltration cascade (UFC) was used. This work is the first known example of a UFC being used to remove both the high and low MW dextran. To remove the high MW material it was necessary to remove 90% of the MW distribution and retain the remaining 10%. The UFC significantly reduced the amount of dialysate required. To achieve the correct specification below 12,000 MW, the UFC required only 2.5 - 3.0 diavolumes while the batch system required 6 - 7. The UFC also improved the efficiency of the fractionation process. The UFC could retain up to 96% of the high MW material while the batch system could only retain 82.5% using the same number of diavolumes. On average the UFC efficiency was approximately 10% better than the equivalent batch system. The UFC was found to be more predictable than the industrial process and the specification of the final product was easier to control. The UFC can be used to improve the fractionation of any polymer and also has several other potential uses including enzyme purification. A dextransucrase bioreactor was also developed. This preliminary investigation highlighted the problems involved with the development of a successful bioreactor for this enzyme system.

A study of some constituents in human female saliva

Changes in the concentration of some constituents in women's saliva during the menstrual cycle were studied. Saliva was used because it is easier to collect than other body fluids and is continuously available for analysis. Glucose, the enzyme 17-Acetyl-D-glucosaminidase (NAG) and Calcium which are saliva constituents and belong to three different chemical groups were selected for the study. Several analytical techniques were investigated. The fluorometric assay procedure was found to be the best because of its specificity and sensitivity for the estimation of these constituents. resides the fluorametric method a spectrophotometric method was used in the NAG determination and an atomic absorption method in the calcium estimation. Glucose was estimated by an enzymatic method. This is based on the reaction of glucose with the enzymes glucose oxidase and peroxidase to yield hydrogen peroxide, which in turn oxidises a non-fluorescent substrate, p-hydroxyphenylacetic acid, to a highly fluorescent product. The saliva samples in this determination had to be centrifuged at high speed, heated in a boiling water bath, centrifuged again and then treated with a mixture of cation and anion resins to remove the substances that inhibited the enzyme system. In the determination of the NAG activity the saliva samples were diluted with citric acid/phosphate buffer, and then centrifuged at high speed. The assay was based on the enzymic hydrolysis of the non-fluorescent substrate 4-Methyl-umbelli1eryl-p-D-glucosaminide to the highly fluorescent 4-Methyl-umbelliferone• Calcium was estimated by a fluorometric procedure based upon the measurement of the fluorescence produced by the complex formed between calcein blue and calcium, at pH 9 - 13. From the results obtained from the analysis of saliva samples of several women it was found that glucose showed a significant increase in its level around the expected time of ovulation. This was found in seven cycles out of ten. Similar results were found with the enzyme NAG. No significant change in the calcium levels was observe& at any particular time of the cycle. The levels of the glucose, the activity of the enzyme NAG and the concentration of the calcium were found to change daily, and to differ from one subject to another and in the same subject from cycle to cycle. The increase observed it salivary glucose levels and the enzyme NAG activity could be monitored to predict the time of ovulation.

Metabolic and morphological alterations induced by proteolysis-inducing factor from Walker tumour-bearing rats in C2C12 myotubes

BACKGROUND: Patients with advanced cancer suffer from cachexia, which is characterised by a marked weight loss, and is invariably associated with the presence of tumoral and humoral factors which are mainly responsible for the depletion of fat stores and muscular tissue. METHODS: In this work, we used cytotoxicity and enzymatic assays and morphological analysis to examine the effects of a proteolysis-inducing factor (PIF)-like molecule purified from ascitic fluid of Walker tumour-bearing rats (WF), which has been suggested to be responsible for muscle atrophy, on cultured C2C12 muscle cells. RESULTS: WF decreased the viability of C2C12 myotubes, especially at concentrations of 20-25 mug.mL-1. There was an increase in the content of the pro-oxidant malondialdehyde, and a decrease in antioxidant enzyme activity. Myotubes protein synthesis decreased and protein degradation increased together with an enhanced in the chymotrypsin-like enzyme activity, a measure of functional proteasome activity, after treatment with WF. Morphological alterations such as cell retraction and the presence of numerous cells in suspension were observed, particularly at high WF concentrations. CONCLUSION: These results indicate that WF has similar effects to those of proteolysis-inducing factor, but is less potent than the latter. Further studies are required to determine the precise role of WF in this experimental model. © 2008 Yano et al; licensee BioMed Central Ltd.

Fatty acid composition, antioxidants and lipid oxidation in chicken breasts from different production regimes

Chicken breast from nine products and from the following production regimes: conventional (chilled and frozen), organic and free range, were analysed for fatty acid composition of total lipids, preventative and chain breaking antioxidant contents and lipid oxidation during 5 days of sub-ambient storage following purchase. Total lipids were extracted with an optimal amount of a cold chloroform methanol solvent. Lipid compositions varied, but there were differences between conventional and organic products in their contents of total polyunsaturated fatty acids and n-3 and n-6 fatty acids and n-6:n-3 ratio. Of the antioxidants, a-tocopherol content was inversely correlated with lipid oxidation. The antioxidant enzyme activities of catalase, glutathione peroxidase and glutathione reductase varied between products. Modelling with partial least squares regression showed no overall relationship between total antioxidants and lipid data, but certain individual antioxidants showed a relationship with specific lipid fractions.