Increase In Protein Carbonyl Content Research Articles

Activation of proteasome by insulin-like growth factor-I may enhance clearance of oxidized proteins in the brain

The insulin-like growth factor type 1 (IGF-I) plays an important role in neuronal physiology. Reduced IGF-I levels are observed during aging and this decrease may be important to age-related changes in the brain. We studied the effects of IGF-I on total protein oxidation in brain tissues and in cell cultures. Our results indicate that in frontal cortex the level of oxidized proteins is significantly reduced in transgenic mice designed to overproduce IGF-I compared with wild-type animals. The frontal cortex of IGF-I-overproducing mice exhibited high chymotrypsin-like activity of the 20S and 26S proteasomes. The proteasome can also be activated in response to IGF-I in cell cultures. Kinetic studies revealed peak activation of the proteasome within 15 min following IGF-I stimulation. The effects of IGF-I on proteasome were not observed in R − cells lacking the IGF-I receptor. Experiments using specific kinase inhibitors suggested that activation of proteasome by IGF-I involves phosphatidyl inositol 3-kinase and mammalian target of rapamycin signaling. IGF-I also attenuated the increase in protein carbonyl content induced by proteasome inhibition. Thus, appropriate levels of IGF-I may be important for the elimination of oxidized proteins in the brain in a process mediated by activation of the proteasome.

Mice lacking the glutamate-cysteine ligase modifier subunit are susceptible to myocardial ischaemia–reperfusion injury

Glutamate-cysteine ligase (GCL), a rate-limiting enzyme for glutathione (GSH) synthesis, is composed of catalytic and modifier subunits. This study examined the pathogenic role of GCL modifier subunits (GCLM) in myocardial ischaemia-reperfusion (I/R) injury using mice lacking the GCLM (GCLM(-/-)). The GCLM(-/-)mice had an increase in myocardial I/R injury and apoptosis in ischaemic myocardium compared with GCLM(+/+) mice. There was a decrease in mitochondrial glutathione (GSH) levels in ischaemic myocardium that was more pronounced in GCLM(-/-) mice than in GCLM(+/+) mice (12 vs. 55% of baseline GCLM(+/+), respectively). The ESR signal intensity of the dimethyl-1-pyrroline-N-oxide-hydroxyl radical adducts in ischaemic myocardium was higher in GCLM(-/-) mice than in GCLM(+/+) mice. Hypoxia-reoxygenation induced greater mitochondrial damage in cultured cardiomyocytes from GCLM(-/-) mice than from GCLM(+/+) mice, as evidenced by a reduced membrane potential and increased protein carbonyl content in isolated mitochondria, together with enhanced cytochrome c translocation into the cytosol. Administration of GSH ethyl-ester attenuated myocardial I/R injury and reversed the mitochondrial damage in parallel with the mitochondrial GSH restoration in the myocardium or the cardiomyocytes of GCLM(-/-) mice. GCLM(-/-) mice were susceptible to myocardial I/R injury partly through an increased vulnerability of mitochondria to oxidative damage owing to mitochondrial GSH reduction.

Effect of a gastrin-releasing peptide receptor antagonist and a proton pump inhibitor association in an animal model of gastritis

It has been proposed that reactive oxygen species play a causative role of gastric mucosal damage induced by increased gastric secretion. Gastrin-releasing peptide is a typical neuropeptide that stimulates acid secretion by release of gastrin. In the present work we have investigated the mechanism of indomethacin (IDM)-induced gastric ulcer caused by ROS and determined the effects of a selective gastrin-releasing peptide receptor antagonist, RC-3095, alone and in association with omeprazole (OM) and compared it with an established antioxidant compound N-acetyl cysteine (NAC). Adult male Wistar rats were pre-treated for 7 days with OM, RC-3095, NAC, both drugs and water (control). The animals were then submitted to fasting for 24 h; IDM was administered. Rats were killed 6 h after that and the stomachs were used for evaluation of macroscopic damage and oxidative stress parameters. Our results showed that IDM increased mitochondrial superoxide production; OM and RC-3095 alone did not prevent such effect, but the combination of these drugs was effective. TBARS assay revealed that IDM-induced lipid peroxidation in gastric tissue and that OM and RC-3095, alone or in combination, prevented this effect with superior action that NAC. Finally, we verified that IDM increased protein carbonyl content and that this effect was prevented RC-3095, alone or in combination with OM, being similar to standard antioxidant. The present results support the view that, besides the inhibition of acid secretion, the protective effects exerted by OM and RC-3095 against IDM-induced gastric damage can be ascribed to a reduction of gastric oxidative injury.

(+)-Catechin with Cu2+ Induces Protein Modifications via Reactive Oxygen Species-independent Pathway

Catechins show various biological activities, for which beneficial as well as adverse effects have been observed. Unfavorable properties of catechins are often attributed to reactive oxygen species (ROS) production. Our aim was to investigate whether catechins affect protein via ROS generation. We examined the effects on bovine serum albumin (BSA) of (+)-catechin with Cu 2+ as well as ascorbate with Cu 2+ .T he ascorbate/Cu 2+ system is a metalcatalyzed oxidation (MCO) system and can produce ROS. Both systems altered spectroscopic properties of albumin, increased protein carbonyl content and resulted in several conformational changes. Catalase and hydroxyl radical scavengers markedly suppressed protein modifications by the ascorbate/Cu 2+ system but not those by the (+)-catechin/Cu 2+ system. As far as we observed, no protein alteration induced by the (+)-catechin/Cu 2+ system correlated with ROS formation.

Hypoxic Stress Exacerbates Hyperoxia-Induced Lung Injury in a Neonatal Mouse Model of Bronchopulmonary Dysplasia

Background: Premature infants with lung injury often experience intermittent episodes of hypoxemia. Objective: This study investigates whether intermittent hypoxemia exacerbates oxidative stress and lung injury in neonatal mice in a hyperoxia-induced model of bronchopulmonary dysplasia (BPD). Methods: For the BPD model, 3-day-old C57Bl/6J mice were exposed to hyperoxia (65% O₂) for 4 weeks (O₂ group) or to hyperoxia and intermittent (10 min daily) hypoxia (O₂ + H group). Upon completion of O₂ or O₂ + H exposure, the degree of pulmonary alveolarization and granulocytic infiltration were examined. The severity of oxidative injury in lungs was defined by tissue glutathione and protein carbonyl content. Data were compared to those in naïve mice and mice subjected only to intermittent hypoxia. Results: Hyperoxia-exposed mice exhibited a dramatic (p < 0.0001) decrease of alveolarization, significantly increased granulocytic infiltration (p < 0.0001) and increased protein carbonyl content (p = 0.04) compared to naïve mice. However, O₂ + H mice demonstrated significantly (p = 0.03) fewer alveoli compared to their O₂ counterparts. This was associated with a significantly (p = 0.02) decreased pulmonary total/oxidized glutathione ratio and a significant (p = 0.03) elevation of protein carbonyl content. Conclusions: Thus, intermittent hypoxic stress during hyperoxic induction of BPD in mice potentiates oxidative stress in lung tissue and exacerbates alveolar developmental arrest.

Biomarkers of oxidative stress in the fungal strain Humicola lutea under copper exposure

The fungal strain Humicola lutea 103 was used as a model organism to examine the relationship between copper toxicity and oxidative stress in low eukaryotes such as filamentous fungi. Spores or submerged cultures were treated with different copper concentrations and the oxidative stress-inducing agent paraquat (PQ). Oxidative stress biomarkers such as reactive oxygen species (ROS), cyanide-resistant respiration, protein carbonyls, reserve carbohydrates, and antioxidant defence were identified in cells treated or not treated with either copper ions or PQ. Copper inhibited the growth and conidiospore formation of H. lutea 103 in a concentration-dependent manner. This treatment also resulted in increased superoxide anion radical formation. Copper stress was furthermore accompanied by transient accumulation of trehalose and glycogen, as well as increased protein carbonyl content. Compared to control cultures, copper-treated mycelia demonstrated a marked increase in the activity of protective enzymes (superoxide dismutase, catalase, and glucose-6-phosphate dehydrogenase). These increased antioxidant enzyme activities were blocked by inhibitors of protein synthesis, suggesting that de novo enzyme formation was involved. Biomarker response to the heavy metal was similar to treatment with known ROS generators such as PQ. The observed hyper-oxidative status and increased oxidative damage suggest a relationship between acute metal treatment and oxidative stress in fungal cells.

Antioxidant effects of melatonin in rats during chronic exposure to hyperbaric oxygen

In addition to its beneficial effects, hyperbaric oxygen (HBO) exposure causes some detrimental effects via oxidative stress. Previous experimental studies showed that melatonin is a useful agent to block single session HBO-induced oxidative stress. In the present study, we investigated the antioxidant effect of exogenously administered as well as endogenously produced melatonin in lung and brain tissues of rats exposed to long term HBO. The HBO procedure was set as daily exposures to 2.5 ATA of oxygen for 1 hr and a total of 10 sessions. Twenty-eight male Sprague-Dawley rats were divided into four groups as follows: control, daytime HBO, daytime HBO plus melatonin (5 mg/kg), nighttime HBO. Tissue oxidative/antioxidant status was examined by determining the protein carbonyl content as a criteria for oxidative stress and the activities of the antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). HBO exposure for 10 days caused significant increases in protein carbonyl content and SOD levels of lung and brain, but GSH-Px activities remained unaffected. The increases in protein carbonyls were blocked by exogenously administered melatonin and in part by nighttime exposure to darkness whereas the increase of SOD activity was only impeded by endogenously produced melatonin in brain tissue. Lung SOD activity was augmented by endogenous melatonin. In conclusion, melatonin blocks long-term HBO-induced cumulative oxidative stress as indicated changes in protein carbonyls. Both exogenously injected and physiologically secreted melatonin has this potential. The effects of HBO-exposure and melatonin on the activities of the antioxidative enzymes are less clear.

Study on the ability of 1,2,3,4‐tetrahydropapaveroline to cause oxidative stress: Mechanisms and potential implications in relation to parkinson's disease

Tetrahydropapaveroline (THP) is a compound derived from dopamine monoamine oxidase-mediated metabolism, particularly present in the brain of parkinsonian patients receiving L-dopa therapy, and is capable of causing dopaminergic neurodegeneration. The aim of this work was to evaluate the potential of THP to cause oxidative stress on mitochondrial preparations and to gain insight into the molecular mechanisms responsible for its neurotoxicity. Our data show that THP autoxidation occurs with a continuous generation of hydroxyl radicals (*OH) and without the involvement of the Fenton reaction. The presence of ascorbate enhances this process by establishing a redox cycle, which regenerates THP from its quinolic forms. It has been shown that the production of *OH is not affected by the presence of either ferrous or ferric iron. Although THP does not affect lipid peroxidation, it is capable of reducing the high levels of thiobarbituric acid-reactive substances obtained in the presence of ascorbate and/or iron. However, THP autoxidation in the presence of ascorbate causes both an increase in protein carbonyl content and a reduction in protein-free thiol content. THP also increases protein carbonyl content when the autoxidation occurs in the presence of iron. The remarkable role played by ascorbate in the production of oxidative stress by THP autoxidation is of particular interest.

Propionic acid induces convulsions and protein carbonylation in rats

Propionic acid (PA) accumulates in patients with propionic acidemia, an inherited metabolic disorder caused by the deficiency of propionyl-CoA carboxylase activity that is clinically characterized by neurological dysfunction, including seizures. However, it is not known whether PA causes seizures in experimental animals. In the current study, we investigated whether intrastriatal injection of PA (0.6–6 μmol) causes seizures and alters protein carbonyl content in the striatum of adult rats. The injection of PA caused the appearance of seizures and increased protein carbonyl content in injected and noninjected striata. PA-induced seizures and increased protein carbonylation in the striatum were prevented by the injection of MK-801 (3 nmol/0.5 μL). Our results suggest that PA causes seizures and oxidative damage by NMDA receptor-mediated mechanisms. The involvement of NMDA receptors in the pathogenesis of propionic acidemia is suggested.

Exposure of Lemna minor to Arsenite: Expression Levels of the Components and Intermediates of the Ubiquitin/Proteasome Pathway

In animal cells, arsenite has been reported to cause sulfhydryl depletion, generate reactive oxygen species and increase the level of large ubiquitin-protein conjugates. Plant viability tests and DNA laddering experiments have shown that Lemna minor remains viable after exposure to 50 microM NaAsO(2) for periods of at least 6 h. However, protein metabolism is affected in two major ways: the synthesis of an array of stress proteins, which confer thermotolerance; and an increase in the amount of large ubiquitin-protein conjugates, particularly evident after 2-3 h of stress, indicative of a role for the ubiquitin/proteasome pathway. This outcome is primarily attributed to an increased availability of protein substrates during arsenite treatment for three main reasons: an increase in protein carbonyl content after 1-2 h of stress; moderate increments in the transcript levels of the sequences coding for the ubiquitin pathway components chosen as markers (polyubiquitin, E1 and E2, and the beta subunit and the ATPase subunits of the 26S proteasome); the observed increase in ubiquitin conjugates does not depend on de novo protein synthesis. This study is the first report on the involvement of the ubiquitin/proteasome pathway in response to arsenite in plants. In addition, it addresses the simultaneous expression of selected genes encoding the various components of the pathway. The results suggest that in plants, unlike in animals, the response to a relatively low level of arsenite does not induce apoptotic cell death. As a whole, the response to arsenite apparently involves a conjugation of salvage and proteolytic machineries, including heat shock protein synthesis and the ubiquitin/proteasome pathway.

Coordinated response of goldfish antioxidant defenses to environmental stress

Different kinds of oxidative stress cause responses of antioxidant defenses which often act in concert. In previous works, some relationships have been found between oxidative stress markers and antioxidant enzyme activities in goldfish treated with different levels of oxygen or heat shock. This study aimed to check whether or not there are general patterns of relationships between antioxidant enzyme activities and oxidative stress indices in goldfish tissues, regardless of the stressor. For this, goldfish were treated with different concentrations of iron sulphate, 20 or 500 μM, as well as limestone water for 7 days. Both iron ions and limestone water led to a pH shift. Therefore, complex effects of iron ions and/or a pH shift on levels of oxidative stress indices and antioxidant enzyme activities in goldfish liver and kidney were investigated. Experimental conditions resulted in increased protein carbonyl content by 1.5–1.9-fold. Externally added iron ions did not change lipid peroxide levels in the liver but decreased them in the kidney, while levels of thiobarbituric acid reactive substances (TBARS) were elevated by 1.4–2.5-fold. Limestone water raised levels of both lipid peroxidation products in the liver. The treatment affected activities of superoxide dismutase and catalase only slightly, but activities of glutathione-associated enzymes, glutathione- S-transferase (GST), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PDH) were lowered in many cases. G6PDH activities correlated inversely with protein carbonyl levels ( R 2 = 0.77–0.97), suggesting possible inactivation of the enzymes due to their carbonylation. Levels of lipid peroxidation products had a positive correlation to activities of catalase in the liver and GR in the kidney ( R 2 = 0.83), indicating possible up-regulation of the enzymes by these products. A negative link between TBARS levels and GST activities may reflect the involvement of GST in the detoxification of lipid peroxide products. The main conclusions are: (i) experimental conditions resulted in increased levels of protein carbonyls and end products of lipid peroxidation (TBARS); (ii) under oxidative stress, some enzymes can be inactivated due to oxidation; (iii) lipid peroxidation products seem to be involved in up-regulation of some antioxidant enzymes.

Aortic coarctation induces oxidative stress in rat tissues

The purpose of this study was to evaluate the induction of oxidative stress in heart and erythrocytes from rats with abdominal aorta coarctation (Coa) compared with sham-operated normotensive controls (Sham). The group of Coa animals developed myocardial hypertrophy, showing heart homogenates markedly increased levels of reduced glutathione (48%), lipid peroxidation (148%) and activation of superoxide dismutase and glutathione peroxidase (189% and 37%, respectively), compared with controls. Other oxidative stress indicators were also altered in erythrocytes from Coa rats: increased protein carbonyl content (141%) and total glutathione level (349%) were determined. Inactivation of the antioxidant enzymes catalase (27%), superoxide dismutase (58%) and glutathione peroxidase (25%) was observed in erythrocytes from the Coa group. Taken jointly our results provide strong evidence for the production of oxidative stress in heart and erythrocytes from aortic coarcted rats.

Vertebrate freezing survival: Regulation of the multicatalytic proteinase complex and controls on protein degradation

The wood frog , Rana sylvatica, survives weeks of whole body freezing during winter hibernation, expressing numerous metabolic adaptations that deal not only with freezing but with its consequences including organ ischemia and cellular dehydration. The present study analyzes the 20s multicatalytic proteinase (MCP) complex from skeletal muscle to determine how protein degradation is managed in the ischemic frozen state . MCP was partially purified and assayed fluorometrically using three AMC-labeled substrates to compare multiple states: control (5 °C acclimated), 24 h frozen at −2.5 °C, 4 or 8 h thawed at 5 °C, 8 h anoxia, and 40% dehydration. MCP from frozen frogs showed significantly different K m and V max values compared with controls; e.g., K m Z-LLE-AMC increased by 45% during freezing and 52% under anoxia whereas V max decreased by 40%. After thawing, K m was restored and V max rose by 2.2-fold. Incubations promoting protein kinase or phosphatase action on MCP showed that phosphatase treatment strongly increased V max implicating reversible phosphorylation in MCP regulation during freeze–thaw. Western blotting showed a 36% decrease in MCP protein in muscle from frozen frogs. The 20s MCP preferentially degrades oxidatively-damaged proteins and evidence of impaired function during freezing came from a 1.4-fold increase in protein carbonyl content in muscle and liver during freezing. Ubiquitin and ubiquitin conjugate levels were unchanged in muscle but changed markedly in liver during freeze–thaw.

Enhanced oxygen releasing capacity and oxidative stress in diabetes mellitus and diabetes mellitus-associated cardiovascular disease: A comparative study

Background Macrovascular disease, especially cardiovascular accounts for most of the mortality in patients with type 2 diabetes mellitus. We compared oxidative stress, thermal stability and oxygen releasing capacity of hemoglobin in patients with type 2 diabetes mellitus and diabetes-associated cardiovascular disease. Methods The study was performed on 38 control subjects, 31 diabetics, 36 diabetics with cardiovascular complications, and 33 non-diabetic cardiovascular subjects. Results Enhanced oxidative stress was shown by an increased protein carbonyl content observed both in plasma and in hemolysate of the diseased samples in type 2 diabetes and diabetes-associated cardiovascular diseased patients. Altered levels of cytoprotective enzymes were shown by decreased catalase activity, increased glutathione reductase activity and unaltered superoxide dismutase activity. Peroxidative activity of diseased hemoglobin was much higher compared to healthy controls indicating possible structural changes in pathologic hemoglobin molecule as a result of disease induced oxidative stress. This result is in good agreement with the observation that thermal stability of pathologic hemoglobin was also found to be less compared to control subjects. Enhanced oxygen releasing capacity of tetrameric oxyhemoglobin was monitored in presence of the drug Trifluoperazine in pathologic red blood cells, maximum increment being noticed in diabetic cardiovascular diseased subjects. Conclusion Hyperglycemia-induced oxidative stress was responsible to affect the thermal stability and oxygen releasing capacity of hemoglobin and the effect is more pronounced in diabetes-associated cardiovascular disease.

The presence of ascorbate induces expression of brain derived neurotrophic factor in SH-SY5Y neuroblastoma cells after peroxide insult, which is associated with increased survival

Oxidative stress and free radical production have been implicated in Alzheimer's disease, where low levels of the antioxidant vitamin C (ascorbate) have been shown to be associated with the disease. In this study, neuroblastoma SH-SY5Y cells were treated with hydrogen peroxide in the presence of ascorbate in order to elucidate the mechanism(s) of protection against oxidative stress afforded by ascorbate. Protein oxidation, glutathione levels, cell viability and the effects on the proteome and its oxidized counterpart were monitored. SH-SY5Y cells treated with ascorbate prior to co-incubation with peroxide showed increased viability in comparison to cells treated with peroxide alone. This dual treatment also caused an increase in protein carbonyl content and a decrease in glutathione levels within the cells. Proteins, extracted from SH-SY5Y cells that were treated with either ascorbate or peroxide alone or with ascorbate prior to peroxide, were separated by two-dimensional gel electrophoresis and analyzed for oxidation. Co-incubation for 24 hours decreased the number of oxidised proteins (e.g. acyl CoA oxidase 3) and induced brain derived neurotrophic factor (BDNF) expression. Enhanced expression of BDNF may contribute to the protective effects of ascorbate against oxidative stress in neuronal cells.

The oxidative effect of prolonged CO2 pneumoperitoneum on renal tissue of rats

Despite its advantages, laparoscopic donor nephrectomy is associated with prolonged operation time, which could potentially increase oxidative stress in the graft. We performed the first experimental, randomized, controlled study with blind assessment of outcome to address this possibility. Wistar-Albino rats were randomized into three groups. The animals in the control group were subjected to a sham operation under anesthesia; the animals in the other two groups were subjected to CO(2) pneumoperitoneum (Pp) for 120 and 240 min, respectively. The kidneys were removed at the end of each experiment. The concentrations of protein carbonyl and sulfhydryl (SH) groups and the activities of superoxide dismutase (SOD) were measured in renal tissue samples as markers of oxidative stress. Renal tissue samples were also evaluated histopathologically using light microscopy. Exposure to 120 min of Pp significantly increased the finding of oxidative stress in renal tissue samples, with an increase in protein carbonyl content and a decrease in protein sulfhydryls and tissue (SOD) activities. When exposure to Pp was prolonged from 120 min to 240 min, Pp associated oxidative stress was found to be increased. These changes occurred in the absence of light microscopical evidence of overt tissue damage. In an experimental model resembling laparoscopic donor nephrectomy, we found that exposure of pneumoperitoneum prolonged from 120 min to 240 min acts as an additive factor with respect to causing increased oxidative stress in renal tissue. Because these effects imply subtle tissue injury that may contribute to the chronic demise of renal grafts obtained laparoscopically, avoiding the use of Pp if possible and keeping operation time less than 120 min during laparoscopic donor nephrectomy appear to be advisable.

S-allylmercaptocysteine scavenges hydroxyl radical and singlet oxygen in vitro and attenuates gentamicin-induced oxidative and nitrosative stress and renal damage in vivo.

BackgroundOxidative and nitrosative stress have been involved in gentamicin-induced nephrotoxicity. The purpose of this work was to study the effect of S-allylmercaptocysteine, a garlic derived compound, on gentamicin-induced oxidative and nitrosative stress and nephrotoxicity. In addition, the in vitro reactive oxygen species scavenging properties of S-allylmercaptocysteine were studied.ResultsS-allylmercaptocysteine was able to scavenge hydroxyl radicals and singlet oxygen in vitro. In rats treated with gentamicin (70 mg/Kg body weight, subcutaneously, every 12 h, for 4 days), renal oxidative stress was made evident by the increase in protein carbonyl content and 4-hydroxy-2-nonenal, and the nitrosative stress was made evident by the increase in 3-nitrotyrosine. In addition, gentamicin-induced nephrotoxicity was evident by the: (1) decrease in creatinine clearance and in activity of circulating glutathione peroxidase, and (2) increase in urinary excretion of N-acetyl-β-D-glucosaminidase, and (3) necrosis of proximal tubular cells. Gentamicin-induced oxidative and nitrosative stress and nephrotoxicity were attenuated by S-allylmercaptocysteine treatment (100 mg/Kg body weight, intragastrically, 24 h before the first dose of gentamicin and 50 mg/Kg body weight, intragastrically, every 12 h, for 4 days along gentamicin-treatment).ConclusionIn conclusion, S-allylmercaptocysteine is able to scavenge hydroxyl radicals and singlet oxygen in vitro and to ameliorate the gentamicin-induced nephrotoxicity and oxidative and nitrosative stress in vivo.

Role of Thioredoxin Peroxidase in Aging of Stationary Cultures of Saccharomyces cerevisiae

A soluble protein from Saccharomyces cerevisiae acts as a peroxidase but requires a NADPH-dependent thioredoxin system and was named thioredoxin peroxidase (TPx). The role of TPx in aging of stationary cultures of S. cerevisiae was investigated in a wild-type strain and a mutant yeast strain in which the tsa gene that encodes TPx was disrupted by homologous recombination. The occurrence of oxidative stress during aging of stationary cultures of the yeast has been proposed. Comparison of 5-day-old (young) stationary cultures of S. cerevisiae and of cultures aged for 3 months (old) revealed decreased viability, increased generation of reactive oxygen species, modulation of cellular redox status, and increased cellular oxidative damage reflected by increased protein carbonyl content and lipid peroxidation. The magnitude of this stress was augmented in yeast mutant lacking TPx. These results suggest that TPx may play a direct role in cellular defense against aging of stationary cultures presumably, functioning as an antioxidant enzyme.

Novel Antioxidant Role of Alcohol Dehydrogenase E from Escherichia coli

Alcohol dehydrogenase E (AdhE) is an Fe-enzyme that, under anaerobic conditions, is involved in dissimilation of glucose. The enzyme is also present under aerobic conditions, its amount is about one-third and its activity is only one-tenth of the values observed under anaerobic conditions. Nevertheless, its function in the presence of oxygen remained ignored. The data presented in this paper led us to propose that the enzyme has a protective role against oxidative stress. Our results indicated that cells deleted in adhE gene could not grow aerobically in minimal media, were extremely sensitive to oxidative stress and showed division defects. In addition, compared with wild type, mutant cells displayed increased levels of internal peroxides (even higher than those found in a Delta katG strain) and increased protein carbonyl content. This pleiotropic phenotype disappeared when the adhE gene was reintroduced into the defective strain. The purified enzyme was highly reactive with hydrogen peroxide (with a Ki of 5 microM), causing inactivation due to a metal-catalyzed oxidation reaction. It is possible to prevent this reactivity to hydrogen peroxide by zinc, which can replace the iron atom at the catalytic site of AdhE. This can also be achieved by addition of ZnSO4 to cell cultures. In such conditions, addition of hydrogen peroxide resulted in reduced cell viability compared with that obtained without the Zn treatment. We therefore propose that AdhE acts as a H2O2 scavenger in Escherichia coli cells grown under aerobic conditions.

Mutation or Overexpression of a Terminal Oxidase Leads to a Cell Division Defect and Multiple Antibiotic Sensitivity in Pseudomonas aeruginosa

Mutation of the cyanide-insensitive terminal oxidase of Pseudomonas aeruginosa leads to pleiotropic effects. A cio mutant and strains, including the wild-type, carrying the cioAB genes on a multicopy plasmid were temperature-sensitive and had a cell division defect, leading to the formation of non-septate, multinucleated filaments. Such strains of this intrinsically antibiotic-resistant bacterium were more sensitive to a range of antibiotics including chloramphenicol, beta-lactams, quinolones, aminoglycosides, and macrolides. The effect of cio mutation on Deltap-dependent accumulation of chloramphenicol suggested that antibiotic sensitivity resulted from loss of or damage to a multidrug efflux pump. The ability of reducing agents and catalase to suppress the temperature-sensitive phenotype and of catalase to partially suppress antibiotic sensitivity suggested that increased levels of reactive oxygen species might be the cause of the observed phenotypes. Consistent with this was the increased sensitivity of strains to H(2)O(2) and their increased protein carbonyl content, an indicator of oxidative protein modification. The temperature-dependent synthesis of a specific catalase was absent in the cio mutant and in strains carrying multiple plasmid-borne copies of cioAB. We propose that reduced catalase levels result in oxidative modification and consequent loss of function of proteins involved in a range of cellular functions. How mutation or overexpression of the cyanide-insensitive terminal oxidase leads to a loss of catalase activity is unknown at present.