Indole-inducible proteins in bacteria suggest membrane and oxidant toxicity

Oxidant toxicity of indole was demonstrated by the induction of alkylhydroperoxide reductase subunit C (AhpC) in Escherichia coli K12 and by the constitutive overproduction of AhpC in a variant of E. coli JM109 with enhanced resistance to indole. Oxidant toxicity was also indicated in an indole-adapted variant of Brevibacterium flavum by the indole-inducible overproduction of a novel 36-kDa protein with N-terminal sequence similarity to proteins involved in superoxide and singlet oxygen resistance. It is proposed that indole dissolved in membrane lipids, which caused membrane derangement and enabled direct interaction of redox-cycling isoprenoid quinones and dioxygen, resulting in the generation of superoxide. A direct indication of membrane derangement in E. coli may be the indole-inducible overproduction of spheroplast protein y (Spy).     

Superoxide dismutase in ruminal bacteria.

Of 13 species of anaerobic ruminal bacteria examined, 11 were found to contain measurable levels of superoxide dismutase activity. Four of five other strict anaerobic species studied for comparison were found to contain superoxide dismutase activity.     

Superoxide dismutase in ruminal bacteria

Of 13 species of anaerobic ruminal bacteria examined, 11 were found to contain measurable levels of superoxide dismutase activity. Four of five other strict anaerobic species studied for comparison were found to contain superoxide dismutase activity.     

Superoxide removal and radiation protection in bacteria

Previous work with procaryotic cells has identified one kind of lethal damage from ionizing radiation which occurs only within a specific range of low O2 concentrations, about 10(-6) to 10(-4) M. Within this range, protection can occur in three ways: through the enzymatic decomposition of hydrogen peroxide (H2O2) by added catalase, through the enzymatic degradation of superoxide anion radicals (.O2-) by added superoxide dismutase (SOD), and through scavenging hydroxyl radicals (.OH) by various additives. These results indicate that three radiolytic products, H2O2, .OH, and .O2- (and/or the conjugate acid, the perhydroxyl radical, .HO2) are involved in this single kind of radiation-induced damage. Although the radiolytic productions of H2O2 and .O2- are strongly enhanced in higher O2 concentrations, neither enzyme protects when these air-equilibrated bacteria are irradiated. These experiments address this apparent contradiction and focus on the specific issue of why the addition of SOD protects at low but not at high O2 concentrations. We propose that, at a given O2 concentration, .O2- (and/or .HO2) may either react (with some cellular component?) to cause damage or react (with itself) to form hydrogen peroxide (H2O2). The specific O2 concentration during irradiation would determine the relative rates of these competing reactions and therefore the O2 concentration itself would establish whether or not we will observe damage from .O2-.     

Superoxide dismutase and pulmonary ozone toxicity

Rats and mice were pre-exposed to low concentrations of ozone and subsequently challenged with higher concentrations of this gas. When the mortalities of pre-exposed and control animals were compared after a lethal challenge, there was a significant difference in ozone toxicity. The levels of superoxide dismutase in lung tissue of control and ozone tolerant rats or mice were not significantly different whether the results were expressed as units of activity per mg protein of lung tissue or as total units of enzyme activity per lung. Mice which were tolerant to ozone were not tolerant to oxygen. The data suggest that ozone tolerance is unrelated to an induction of superoxide dismutase. It is suggested that the induction of tolerance to oxygen and ozone involve biochemically distinct mechanisms.     

Paradoxical Relationship between Mn Superoxide Dismutase Deficiency and Radiation-Induced Cognitive Defects

Radiation therapy of the CNS, even at low doses, can lead to deficits in neurocognitive functions. Reduction in hippocampal neurogenesis is usually, but not always, associated with cognitive deficits resulting from radiation therapy. Generation of reactive oxygen species is considered the main cause of radiation-induced tissue injuries, and elevated levels of oxidative stress persist long after the initial cranial irradiation. Consequently, mutant mice with reduced levels of the mitochondrial antioxidant enzyme, Mn superoxide dismutase (MnSOD or Sod2), are expected to be more sensitive to radiation-induced changes in hippocampal neurogenesis and the related functions. In this study, we showed that MnSOD deficiency led to reduced generation of immature neurons in Sod2−/+ mice even though progenitor cell proliferation was not affected. Compared to irradiated Sod2+/+ mice, which showed cognitive defects and reduced differentiation of newborn cells towards the neuronal lineage, irradiated Sod2−/+ mice showed normal hippocampal-dependent cognitive functions and normal differentiation pattern for newborn neurons and astroglia. However, we also observed a disproportional decrease in newborn neurons in irradiated Sod2−/+ following behavioral studies, suggesting that MnSOD deficiency may render newborn neurons more sensitive to stress from behavioral trainings following cranial irradiation. A positive correlation between normal cognitive functions and normal dendritic spine densities in dentate granule cells was observed. The data suggest that maintenance of synaptic connections, via maintenance of dendritic spines, may be important for normal cognitive functions following cranial irradiation.     

Antimicrobial peptides increase tolerance to oxidant stress in Drosophila melanogaster

It is well appreciated that reactive oxygen species (ROS) are deleterious to mammals, including humans, especially when generated in abnormally large quantities from cellular metabolism. Whereas the mechanisms leading to the production of ROS are rather well delineated, the mechanisms underlying tissue susceptibility or tolerance to oxidant stress remain elusive. Through an experimental selection over many generations, we have previously generated Drosophila melanogaster flies that tolerate tremendous oxidant stress and have shown that the family of antimicrobial peptides (AMPs) is over-represented in these tolerant flies. Furthermore, we have also demonstrated that overexpression of even one AMP at a time (e.g. Diptericin) allows wild-type flies to survive much better in hyperoxia. In this study, we used a number of experimental approaches to investigate the potential mechanisms underlying hyperoxia tolerance in flies with AMP overexpression. We demonstrate that flies with Diptericin overexpression resist oxidative stress by increasing antioxidant enzyme activities and preventing an increase in ROS levels after hyperoxia. Depleting the GSH pool using buthionine sulfoximine limits fly survival, thus confirming that enhanced survival observed in these flies is related to improved redox homeostasis. We conclude that 1) AMPs play an important role in tolerance to oxidant stress, 2) overexpression of Diptericin changes the cellular redox balance between oxidant and antioxidant, and 3) this change in redox balance plays an important role in survival in hyperoxia.     

Superoxide as a signal for increase in intracellular pH

We examined the role of superoxide in the increase in intracellular pH (pHi) of human histiocytic leukemia U937 cells treated with 4 beta-phorbol-12,13-didecanoate (4 beta-PDD) or serum. 4 beta-PDD or serum induced a rapid increase in pHi, and antioxidants such as superoxide dismutase (SOD), vitamin E, and butylated hydroxyanisole (BHA) were found to inhibit the amiloride-sensitive increase in pHi induced by 4 beta-PDD. SOD inhibited the increase in pHi caused by serum, and essentially the same was found in concanavalin A-stimulated mouse thymocytes. Also, a superoxide-generating system, xanthine-xanthine oxidase (X-XOD), increased pHi of U937 cells as much as 4 beta-PDD or serum. From these findings, it appears that superoxide is the basis for the modulation of pHi.     

Genetic toxicity of procarbazine in bacteria and yeast.

Procarbazine [N-isopropyl-alpha-(2-methylhydrazino)-p-toluamide hydrochloride] is used to treat Hodgkin's disease. This compound was tested in vitro without and with S10 fraction from mice liver (microsomal assay) using Saccharomyces cerevisiae strain D7, Salmonella typhimurium (strains TA98, TA100, TA1535) and in vivo in Swiss albino mice (host-mediated assay) using D7. Procarbazine, without S10 fraction, is highly toxic and induced mitotic crossover, gene conversion, and reverse mutation in D7. It had a toxic effect on all the Salmonella strains; but did not induce reverse mutations at the histidine loci. Procarbazine, with S10 fraction, was less toxic and did not induce genetic effects in yeast or Salmonella. In the host-mediated assay, no genetic effects were seen.     

Superoxide dismutase biosynthesis by two thermophilic bacteria

Some high-molecular weight antioxidant defense system components of two thermophilic bacteria isolated from spa waters of Serbia (Yugoslavia) and identified as Bacillus stearothermophilus and Thermothrix sp. were studied. In addition to superoxide dismutase (SOD; EC 1.15.1.1), qualitative analyses demonstrated the presence of catalase (EC 1.11.1.6), peroxidases and oxidases in both bacterial strains. Cell-free extracts were subjected to nondenaturing polyacrylamide gel electrophoresis (PAGE) and SOD activity in the eluates of the corresponding bands was examined in the presence of several specific inhibitors. A slight decrease of SOD activity observed in the presence of 0.3 M potassium cyanide and its complete insensitivity to hydrogen peroxide (5 mM) and sodium azide (20 mM) action suggest that the enzyme occurring in the two thermophiles represents Mn SOD. A high SOD activity recorded in cell-free extracts strongly recommends these two bacterial strains as potential producers of this important antioxidant defense system component at industrial scale.     

Superoxide dismutase activities in Rhizobium phaseoli bacteria and bacteroids

Superoxide dismutase activity in free-living Rhizobium phaseoli is due to the presence of two different enzymes containing manganese or iron. Under usual culture conditions, the manganese-enzyme appears largely predominant but the induction of the iron-superoxide dismutase can be obtained by addition of methyl viologen to the culture media. The corresponding bacteroid, extracted from French-bean nodules, contains only a manganese-superoxide dismutase whose characteristics are similar to those of the bacterial enzyme. However, the activity of the microsymbiont is slightly lower than that of free-living cells. The presence of an active superoxide dismutase in the bacteroids suggests a significant formation of superoxide anion by their metabolism; this can be correlated with the existence of a large oxygen demand by the microsymbionts within the nodule, as suggested by their important oxygen uptake in vitro.     

Superoxide radicals and antiradical defence of propionic acid bacteria

Superoxide dismutase activity was demonstrated for 6 strains of 3 propionibacteria species. Rather high level of superoxide dismutase activity found in propionibacteria was in accordance with high level of catalase activity reported for propionibacteria previously. Both these activities were shown to have cytozolic localization. For the first time peroxidase activity was detected in gel-fractionated crude cell extracts of propionibacteria. The ability to produce superoxide radicals in NADH-dependent oxidation system was revealed for three strains of the bacteria. The level of superoxide production by the membrane particles of the propionic acid bacteria correlated with the levels of superoxide dismutase and catalase activities and was the lowest for Propionibacterium shermanii. The ability to perform monovalent oxygen reduction during succinate oxidation was not revealed. The intact cells of P. globosum, P. vannielii, P. shermanii apparently did not excrete superoxide radicals into culture fluid during respiration.     

Superoxide dismutase and catalase conjugated to polyethylene glycol increases endothelial enzyme activity and oxidant resistance

Covalent conjugation of superoxide dismutase and catalase with polyethylene glycol (PEG) increases the circulatory half-lives of these enzymes from less than 10 min to 40 h, reduces immunogenicity, and decreases sensitivity to proteolysis. Because PEG has surface active properties and can induce cell fusion, we hypothesized that PEG conjugation could enhance cell binding and association of normally membrane-impermeable enzymes. Incubation of cultured porcine aortic endothelial cells with 125I-PEG-catalase or 125I-PEG-superoxide dismutase produced a linear, concentration-dependent increase in cellular enzyme activity and radioactivity. Fluorescently labeled PEG-superoxide dismutase incubated with endothelial cells showed a vesicular localization. Mechanical injury to cell monolayers, which is known to stimulate endocytosis, further increased the uptake of fluorescent PEG-superoxide dismutase. Endothelial cell cultures incubated with PEG-superoxide dismutase and PEG-catalase for 24 h and then extensively washed were protected from the damaging effects of reactive oxygen species derived from exogenous xanthine oxidase as judged by two criteria: decreased release of intracellular 51Cr-labeled proteins and free radical-induced changes in membrane fluidity, measured by electron paramagnetic resonance spectroscopy of endothelial membrane proteins covalently labeled with 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl. Addition of PEG and PEG-conjugated enzymes perturbed the spin-label binding environment, indicative of producing an increase in plasma membrane fluidity. Thus, PEG conjugation to superoxide dismutase and catalase enhances cell association of these enzymes in a manner which increases cellular enzyme activities and provides prolonged protection from partially reduced oxygen species.