Cohabitation within mice of Salmonella typhimurium seqA mutant increases its virulence

The efficacy of a vaccine is conditioned by its capacity to elicit a protective immune response. The principal safety concerns of live vaccine are virulence reversion. The aim of this work was to evaluate the virulence of Salmonella typhimurium seqA mutant after cohabitation with mice. Our results indicated that LD50 of hosted strains were at least twofold lower than those of parental strains. Also, the in vivo competition assays have showed that the development of a systemic infection was most obvious for recovered strains than for control strains. In addition, the number of hosted mutants colonizing spleen and liver was relatively higher than control strains. Adhesion and invasion experiments were performed in order to compare the pathogenicity of Salmonella. For instance recovered-mutant attached to epithelial cells (KB cells) better than parental strains. According to these results, we report that in vivo adaptation of Salmonella typhimurium seqA mutants can increase their virulence.     

Effects of growth hormone on hypothalamic catalase and Cu/Zn superoxide dismutase

Age-associated changes in hypothalamic catalase activity and level, and Cu/Zn superoxide dismutase (Cu/Zn SOD) activity were examined in Ames dwarf mice with growth hormone (GH) deficiency and prolonged lifespan, in PEPCK-hGH transgenic mice with overexpression of GH and reduced lifespan, and compared to values measured in normal controls. Hypothalami from young (3-4 months), middle-aged (9-10 months), and old (19-23 months) male mice were examined using spectrophotometric assay and Western blot. In dwarf mice, Cu/Zn SOD and catalase activities declined with age, and were higher than the corresponding normal values in young and middle-aged groups. Catalase levels also declined with age, but were similar to values in normal controls. In GH transgenic mice, age-associated decline of both catalase and Cu/Zn SOD occurred earlier than in normal animals. Catalase levels and activities in transgenic animals were similar to controls, whereas Cu/Zn SOD activity was higher in transgenics than in normal mice. The present results suggest that dwarf mice, during early life, have enhanced hypothalamic free radical defenses, which may contribute to their extended lifespan. However, from the present results in GH transgenic mice, it is impossible to conclude whether early decline of hypothalamic catalase and Cu/Zn SOD in these animals represents a correlate of accelerated aging, or contributes to their reduced lifespan.     

Effect of hydrogen peroxide on the iron-containing superoxide dismutase of Escherichia coli

The iron-containing superoxide dismutase from Escherichia coli is inactivated by H2O2 to a limit of approximately 90%. When corrected for the H2O2-resistant portion, this inactivation was first order with respect to residual activity and exhibited a pseudo-first-order rate constant of 0.066 min-1 at 25 degrees C in 0.24 mM H2O2 at pH 7.8. The superoxide dismutase activity remaining after treatment with H2O2 differed from the activity of the native enzyme with respect to heat stability, inhibition by azide, and inactivation by light in the presence of rose bengal and by N-bromosuccinimide. The native and the H2O2-modified enzymes were indistinguishable by electrophoresis on polyacrylamide gels. Inactivation of the enzyme by H2O2 was accompanied by loss of tryptophan and some loss of iron, but there was no detectable loss of histidine or of other amino acids. H2O2 treatment caused changes in the optical spectrum of the enzyme. Inactivation of the enzyme by H2O2 depends upon the iron at the active site. Thus, the apoenzyme and the manganese-substituted enzyme were unaffected by H2O2. We conclude that reaction of H2O2 with the iron at the active site generates a potent oxidant capable of attacking tryptophan residues. A mechanism is proposed.     

Coimobilization of superoxide dismutase, catalase and peroxidase

For preparationing the polyenzyme antioxidant complex, containing superoxide dismutase (SOD), catalase and horseradish peroxidase (HRP), the different successivities of those enzymes co-immobilization were compared. The optimum successivity is provided by simultaneous co-immobilization of covalently bound HRP with the SOD and catalase. The catalytic enzyme activity and the catalase operational stability was kinetically characterized in various samples. For one sample, the influence of ascorbate, glutathione and ethanol on the catalase kinetic parameters was studied. A possible scheme of different processes at the H2O2 decomposition in the presence of co-immobilized SOD, catalase, HRP and the substrates-reductans was discussed.     

Role of catalase and superoxide dismutase in the yeast Saccharomyces cerevisiae response to hydrogen peroxide in exponential phase of growth

The role of catalase and superoxide dismutase (SOD) in response of the yeast Saccharomyces cerevisiae to oxidative stress induced by hydrogen peroxide in the middle-exponential phase has been investigated. It was shown that cell survival is significantly decreased after yeast exposure to hydrogen peroxide in the strains defective in cytosolic or peroxisomal catalases. Treatment of the wild-type cells with 0.5 mM H2O2 for 30 min causes an increase in the activity of catalase and superoxide dismutase, but the effect was not observed in all strains investigated. It was also shown that hydrogen peroxide leads to an increase in the activities of both catalases and Cu,Zn-containing SOD. The effect was cancelled by cycloheximide, an inhibitor of protein synthesis.     

CuZn superoxide dismutase, Mn superoxide dismutase, catalase and glutathione peroxidase in glutathione-deficient human fibroblasts

The effect of genetically determined glutathione deficiency on the fibroblast content of CuZn superoxide dismutase, Mn superoxide dismutase, catalase and glutathione peroxidase was investigated. No significant differences between glutathione-deficient and -proficient human fibroblasts were revealed. There was a large variation in the content of the investigated enzymes in fibroblasts grown and analysed on different occasions. Whereas the contents of CuZn superoxide dismutase, catalase and glutathione peroxidase did not deviate much from what has been found in other human cell-lines and tissues, the fibroblasts were found to contain exceptional amounts of Mn superoxide dismutase.     

Possible accumulation of non-active molecules of catalase and superoxide dismutase in S. cerevisiae cells under hydrogen peroxide induced stress

The effect of hydrogen peroxide on the activities of catalase and superoxide dismutase (SOD) in S. cerevisiae has been studied under different experimental conditions: various H₂O₂ concentrations, time exposures, yeast cell densities and media for stress induction. The yeast treatment with 0.25–0.50 mM H₂O₂ led to an increase in catalase activity by 2–3-fold. At the same time, hydrogen peroxide caused an elevation by 1.6-fold or no increase in SOD activity dependently on conditions used. This effect was cancelled by cycloheximide, an inhibitor of protein synthesis in eukaryotes. Weak elevation of catalase and SOD activities in cells treated with 0.25–0.50 mM H₂O₂ found in this study does not correspond to high level of synthesis of the respective enzyme molecules observed earlier by others. It is well known that exposure of microorganisms to low sublethal concentrations of hydrogen peroxide leads to the acquisition of cellular resistance to a subsequent lethal oxidative stress. Hence, it makes possible to suggest that S. cerevisiae cells treated with low sublethal doses of hydrogen peroxide accumulate non-active stress-protectant molecules of catalase and SOD to survive further lethal oxidant concentrations.     

Bactericidal effect of hydrogen peroxide on Salmonella typhimurium in liquid whole egg

The effect of hydrogen peroxide on Salmonella typhimurium in whole egg was evaluated. The bactericidal effects observed on the test organism at 5 degrees and 20 degrees C were found to be similar. There was a 99% kill in the presence of 0.5% and 1.0% H2O2. Addition of the test organism and H2O2 after pre-heating the egg material at 40 degrees C for 15 min caused a rapid kill which was 10,000-fold greater than that produced by H2O2 alone.     

Effect of hydrogen sulfide on the mutagenicity of hydrogen peroxide in Salmonella typhimurium strain TA102

In a previous paper, the main mutagenic compound isolated from the model reaction system D-fructose, DL-alanine and creatinine was tentatively identified as 4,8-DiMeIQx. Its mutagenic activity and spectral characteristics have now been compared with those of the isomer 5,8-DiMeIQx. The comparison clearly demonstrates that the isolated compound was indeed 4,8-DiMeIQx. This finding is in agreement with the hypothesis that sugars, amino acids and creatinine present in meat may be the precursors of the mutagenic imidazoquinolin- and imidazoquinoxalin-2-amines (IQ compounds).     

Bactericidal effect of hydrogen peroxide on Salmonella typhimurium in liquid whole egg

The effect of hydrogen peroxide on Salmonella typhimurium in whole egg was evaluated. The bactericidal effects observed on the test organism at 5° and 20°C were found to be similar. There was a 99% kill in the presence of 0°5% and 1°0% H₂O₂. Addition of the test organism and H₂O₂ after pre-heating the egg material at 40°C for 15 min caused a rapid kill which was 10000-fold greater than that produced by H₂O₂ alone.     

Effects of Greek legume plant extracts on xanthine oxidase,catalase and superoxide dismutase activities

Legumes are considered to have beneficial health implications, which have been attributed to their phytochemical content. Polyphenols are considered the most important phytochemical compounds extensively studied for their antioxidant properties. The aim of the present study was to examine the effects of potent antioxidant legume plant extracts on xanthine oxidase (XO), catalase (CAT) and superoxide dismutase (SOD) activities. XO exerts a dual role, as it is the major contributor of free radicals during exercise while it generates uric acid, the most potent antioxidant molecule in plasma. CAT and SOD are two of the main enzymes of the antioxidant defence of tissues. We demonstrate that the majority of the extracts inhibited XO activity, but they had no effect on CAT inhibition and SOD induction when used at low concentrations. These results imply that the tested extracts may be considered as possible source of novel XO inhibitors. However, we have shown that allopurinol administration, a known XO inhibitor, before exercise reduces performance and induces oxidative stress in rats. Considering the fact that the extracts examined had an inhibitory effect on XO activity, possibly posing a restriction in their characterization as antioxidants, phytochemical antioxidant administration before exercise should probably be reconsidered.     

The interaction between Cu(I) superoxide dismutase and hydrogen peroxide

The interaction between superoxide dismutase (SOD) and peroxide, under anaerobic conditions in the presence of an OH radical scavenger, formate, and an indicator, nitro blue tetrazolium, involves five reactions and an equilibrium: (table; see text) Reaction 3 occurs at a rate that is proportional to both peroxide and enzyme with no kinetic evidence for any intermediate peroxide-enzyme complex. Rate studies as a function of pH corroborate previously published work (Fuchs, H. J. R., and Borders, C. L., Jr. (1983) Biochem Biophys. Res. Commun. 116, 1107-1113; Blech, D. M., and Borders, C. L., Jr. (1983) Arch. Biochem. Biophys. 224, 579-586) suggesting that HO2-, and not H2O2, is the active species in this system: k(HO2- + superoxide dismutase-Cu+) = 2.6 x 10(3) M-1 s-1. Evidence is presented which suggests that HO2-, like O2-, reacts at rates that are affected by the electrostatic forces of the enzyme.     

Generation of superoxide radicals in alkaline solutions of hydrogen peroxide and the effect of superoxide dismutase on this system

Superoxide radicals in high concentrations were generated from alkaline H₂O₂ without using catalysts or irradiation. The dependence of the intensity and parameters of the superoxide radical EPR spectrum on pH, temperature, viscosity and H₂O₂ concentration were studied. The observed changes are explained on the base of matrix effects. The addition of superoxide dismutase to alkaline H₂O₂ led initially to a drop in the EPR spectrum intensity, followed by an increase in the concentration of superoxide radicals.     

New conjugates of superoxide dismutase/catalase mimetics with cyclodestrins

Mimetics of antioxidant enzymes such as superoxide dismutases (SOD) or catalases are reported as potential new drugs able to reduce oxidative stress damage. In particular, manganese(III) complexes of salen-type ligands have been studied as both SOD and catalase mimetics. In this paper, we report the synthesis of two novel conjugates of salen-type ligands with the β-cyclodextrin, the 6-deoxy-6-[(S-cysteamidopropyl(1,2-diamino)N,N′-bis(salicylidene))]-β-cyclodextrin and the 6-deoxy-6-[(S-cysteamidopropyl(1,2-diamino)N,N′-bis(3-methoxysalicylidene))]-β-cyclodextrin, their spectroscopic characterization, and the synthesis and the characterization of their manganese(III) complexes. The SOD-like activity of the metal complexes was investigated by the indirect Fridovich method. The catalase like activity was tested using a Clark-type oxygen electrode. The peroxidase activity was tested using the ABTS (2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)) assay. The glycoconjugation of salen-manganese(III) complexes yields compounds with enhanced SOD activity. These complexes also show catalase and peroxidase activities higher than the simple salen complexes (EUK 113 and EUK 108).     

Enhanced oligomerization of the alpha-synuclein mutant by the Cu,Zn-superoxide dismutase and hydrogen peroxide system

The alpha-synuclein is a major component of Lewy bodies that are found in the brains of patients with Parkinson's disease (PD). Also, two point mutations in this protein, A53T and A30P, are associated with rare familial forms of the disease. We investigated whether there are differences in the Cu,Zn-SOD and hydrogen peroxide system mediated-protein modification between the wild-type and mutant alpha-synucleins. When alpha-synuclein was incubated with both Cu,Zn-SOD and H2O2, then the amount of A53T mutant oligomerization increased relative to that of the wild-type protein. This process was inhibited by radical scavenger, spin-trapping agent, and copper chelator. These results suggest that the oligomerization of alpha-synuclein is mediated by the generation of the hydroxyl radical through the metal-catalyzed reaction. The dityrosine formation of the A53T mutant protein was enhanced relative to that of the wild-type protein. Antioxidant molecules, carnosine, and anserine effectively inhibited the wild-type and mutant proteins' oligomerization. Therefore, these compounds may be explored as potential therapeutic agents for PD patients. The present experiments, in part, may provide an explanation for the association between PD and the alpha-synuclein mutant.     

Effects of superoxide dismutase/catalase mimetics on life span and oxidative stress resistance in the housefly,Musca domestica

The purpose of this study was to determine whether superoxide dismutase/catalase mimetics lengthen the life span of the housefly, Musca domestica, as previously demonstrated for the nematode Caenorhabditis elegans. Various concentrations of Eukarion-8 or Eukarion-134 were administered via the drinking water and the effects on the life span of the flies and amounts of protein carbonyls were determined under normoxic and hyperoxic conditions. These SOD/catalase mimetics neither extended the life span of the flies nor attenuated the protein carbonyl content under normoxic conditions and shortened life span under hyperoxic conditions. Thus, the effect of these SOD/catalase mimetics on the life span of animals seem to be species-specific.     

Functional and crystallographic characterization of Salmonella typhimurium Cu,Zn superoxide dismutase coded by the sodCI virulence gene

The functional and three-dimensional structural features of Cu,Zn superoxide dismutase coded by the Salmonella typhimurium sodCI gene, have been characterized. Measurements of the catalytic rate indicate that this enzyme is the most efficient superoxide dismutase analyzed so far, a feature that may be related to the exclusive association of the sodCI gene with the most pathogenic Salmonella serotypes. The enzyme active-site copper ion is highly accessible to external probes, as indicated by quenching of the water proton relaxation rate upon addition of iodide. The shape of the electron paramagnetic resonance spectrum is dependent on the frozen or liquid state of the enzyme solution, suggesting relative flexibility of the copper ion environment. The crystal structure (R-factor 22.6%, at 2.3 A resolution) indicates that the dimeric enzyme adopts the quaternary assembly typical of prokaryotic Cu,Zn superoxide dismutases. However, when compared to the structures of the homologous enzymes from Photobacterium leiognathi and Actinobacillus pleuropneumoniae, the subunit interface of Salmonella Cu,Zn superoxide dismutase shows substitution of 11 out of 19 interface residues. As a consequence, the network of structural water molecules that fill the dimer interface cavity is structured differently from the other dimeric bacterial enzymes. The crystallographic and functional characterization of this Salmonella Cu,Zn superoxide dismutase indicates that structural variability and catalytic efficiency are higher in prokaryotic than in the eukaryotic homologous enzymes.