Superoxide dismutase, catalase and nitrogenase activities of symbiotic Frankia (Alnus incana) in response to different oxygen tensions

Presence and activity of the enzymes superoxide dismutase (SOD) and catalase were studied in Frankia in symbiosis with Alnus incana (L.) Moench. Analysis on native PAGE gels indicated that symbiotic Frankia contained an FeSOD and catalase. The activity of the enzymes was in the same range as reported for cultured Frankia . Attempts to characterize SOD by western blots with antisera from Escherichia coli and Azotobacter vinelandii did not give clear-cut results with the antibodies used. Alnus incana plants were grown with the root system in 5, 10, 21 or 40% O₂ for up to 6 days. Nitrogenase activity, measured as ARA (acetylene reducing activity) dropped within 3 h when roots were exposed to low or high oxygen. At 40% O₂ ARA was almost completely lost while at 5 and 10% O₂ ARA decreased to 69 and 74% of the inital value, respectively, Nitrogenase activity recovered at ail oxygen tensions. Recovery rates resembled the continuous increase in ARA in plants continuosly kept at 21% O₂, and suggests that new vesicles with envelopes of appropriate thickness were formed. The ARA measurements confirm results from an earlier study where nitrogenase activity was measured as H₂ evolution. There was a tendency for increased SOD and catalase activities in Frankia from root systems exposed to 40% O₂ for 24 h but not earlier or later than this. When data from all experimental times were pooled. SOD activity increased significantly with increased oxygen tension whereas catalase activity decreased. Although ARA per plant varied with oxygen tension, there was no statistically significant correlation between ARA and SOD or between ARA and catalase. It seems that being linked to nitrogenase activity is only one role of SOD and catalase in this symbiotic Frankia .     

Chemiluminescence response and adherence of neutrophils to cultured endothelial cells–influence of immunoglobulin G

The effect of different concentrations (0.87, 4.35, 8.7, 17.5, 25 and 35 mg/mL) of intravenous immunoglobulin G (Endobulin®) on neutrophil–endothelial cell interaction was studied using an in vitro model of human umbilical cord vein endothelial cells and human neutrophils. Because adherence of neutrophils to endothelial cells is an essential component in inflammatory processes leading to endothelial cell injury the influence of immunoglobulin G on adherence has been investigated. A second aim of the present study was to determine changes in chemiluminescence response of neutrophils during adherence to endothelial cells. Production of oxygen-derived metabolites, measured by chemiluminescence response of neutrophils, decreased significantly in the presence of 8.7 mg immunoglobulin/mL test during coincubation of neutrophils and endothelial cells (p < 0.025). The adherence of neutrophils to endothelial cells was significantly decreased at a concentration of 8.7 mg immunoglobulin/mL test (p < 0.025). The present results indicate that this preparation of immunoglobulin G might exert a protective effect on neutrophil–endothelial cell interaction by decreasing adherence of neutrophils to endothelial cells and by scavenging reactive oxygen metabolites. Therefore, the current investigation points to a probable protective effect of immunoglobulin G in oxidative diseases, such as the adult respiratory distress syndrome.     

The superoxide anion and singlet molecular oxygen: their role in the microbicidal activity of the polymorphonuclear leukocyte

Superoxide dismutase was found to partially inhibit both chemiluminescence and nitroblue tetrazolium (NBT) reduction from intact human polymorphonuclear leukocytes. This capacity to reduce NBT was lost when the polymorphonuclear leukocytes were sonicated, but could be regained if exogenous NADPH (or NADH) was added to the system. Superoxide dismutase was found to inhibit this NADPH- and NADH-dependent NBT reduction. A mechanism is proposed that relates superoxide anion generation to the univalent reduction of O₂ by the activated NADPH (and NADH) oxidase. The relationship of superoxide anion production to NBT reduction, singlet molecular oxygen generation, and chemiluminescence is discussed.     

Responses of antioxidants in the lichen Ramalina lacera may serve as an early-warning bioindicator system for the detection of air pollution stress

The aim of this study was to identify, in the lichen Ramalina lacera, antioxidants that could provide indications of air pollution stress, and respond earlier than traditionally used structural/physiological parameters. The pollution-sensitive lichen R. lacera was transplanted from its relatively unpolluted natural habitat to two air-polluted sites for a period of up to 6 months. The superoxide dismutase and catalase activities, total water- and lipid-soluble low-molecular-weight antioxidant capacities and chlorophyll b/chlorophyll a ratios were assessed every 6 weeks. The earliest signs of oxidative stress were detected in the activities of fungal copper/zinc-superoxide dismutase, algal iron-superoxide dismutase and water-soluble low-molecular-weight antioxidants, which increased significantly as early as 42 days after exposure to pollution. Catalase activity increased in lichens transplanted to the polluted sites after 90 days. All activities decreased towards the end of the experiment. The impact of air pollution on R. lacera, using the traditionally employed parameter of chlorophyll b/chlorophyll a ratio, was only detected after 6 months of exposure to air pollution. Our results indicate that antioxidant parameters may serve as improved early-warning indicators of air pollution stress in lichens.     

Pyruvate relieves the necessity of high induction levels of catalase and enables Campylobacter jejuni to grow under fully aerobic conditions

Aims:  Several cases of campylobacteriosis reported worldwide seemingly conflict with the strict growth requirements and sensitivity to environmental stress of Campylobacter jejuni. In this study, the need for a micro‐aerobic environment [dissolved oxygen tension (DOT): 0·1–90%; 100% air saturation)] and the adaptive responses to oxygen stress were studied. Methods and Results:  The growth of C. jejuni in continuous culture was assessed under different DOT in the presence or absence of pyruvate. In a medium without pyruvate, continuous cultures of C. jejuni showed typically micro‐aerobic behaviour and cells were unable to grow under fully aerobic conditions. However in the presence of pyruvate (25 mmol l^?1), continuous cultures of C. jejuni were able to grow in a broad DOT range, varying from 0·1% to at least 90%, and the catalase activity was decreased. Conclusions:  Addition of pyruvate results in the decrease in the concentration of hydrogen peroxide, which enables C. jejuni to grow aerobically. Significance and Impact of the Study:  New information on the oxidative physiology of C. jejuni and its ability to grow aerobically in media supplemented with pyruvate is presented.     

Ultraweak light emission is a common response of bacterial cells to chemico-physical stress

Escherichia coli JM101 cells were subjected to pore-forming electric fields, irradiation with ultraviolet light or oxidative stress by either the lipoxygenase products 9- and 13-hydroperoxyoctadecadienoic acids (9- and 13-HPOD) or hydrogen peroxide. It was found that all chemico-physical stresses enhanced ultraweak light emission from the bacterial cells, the most effective treatment being electroporation (up to 20-fold increase in luminescence compared to the control value), followed by oxidative stress with 9- or 13-HPOD (up to 4-fold increase) and irradiation with UV light (up to 2.8-fold increase). Bacterial luminescence was always in the red edge of the spectrum and was paralleled by changes in membrane oxidative index and specific activity of catalase and superoxide dismutase. © 1998 John Wiley & Sons, Ltd.     

Microbial Strategies to Prevent Oxygen-Dependent Killing by Phagocytes

Microorganisms which are taken up by professional phagocytic cells of a host organism (e.g., by macrophages and polymorphonuclear leukocytes) encounter a series of antimicrobial events including confrontation with toxic oxygen species, derived mainly from the superoxide radical produced by phagocytic NADPH oxidase after uptake of the microorganism. Many microbes are susceptible to the oxygen-dependent phagocytic stress and are efficiently killed. The strategies of some microorganisms to bypass an encounter with the phagocytes' reactive oxygen species, and biochemical systems contributing to the microbes' resistance to killing by reactive oxygen species are outlined.     

Lipopolysaccharide-induced alterations in oxygen consumption and radical generation in endothelial cells

Oxygen consumption rate (OCR) and generation of superoxide and nitric oxide (NO) in mouse aortic endothelial cells (MAECs) treated with lipopolysaccharide (LPS) were studied. The OCR was determined in cell suspensions at 37 °C by electron paramagnetic resonance (EPR) spectroscopy. LPS significantly altered the OCR in a dose and time-dependent fashion. The OCR was significantly elevated immediately following the treatment of MAECs with LPS (5 and 10 μg/ml) and NADPH (100 μM) whereas the same was depressed 1 h after exposure to similar conditions of incubation. Under similar experimental conditions, superoxide generation was also determined by EPR spectroscopy and cytochrome c reduction assays. A marginal increase in the superoxide production was observed when the cells were treated with LPS and NADPH alone whereas the same was further enhanced significantly when the cells were treated with LPS and NADPH together. The increase in oxygen consumption and superoxide production caused by LPS was inhibited by diphenyleneiodonium (DPI), suggesting the involvement of NAD(P)H oxidase. A significant increase in the NO production by MAECs was noticed 1 h after treatment with LPS and was inhibited by L-NAME, further suggesting the involvement of nitric oxide synthase (NOS). Thus, on a temporal scale, LPS-induced alterations in oxygen consumption by MAECs may be under the control of dual regulation by NAD(P)H oxidase and NOS. (Mol Cell Biochem 278: 119–127, 2005)     

High glucose concentration affects the oxidant-antioxidant balance in cultured mouse podocytes

Hyperglycemia is well-recognized and has long-term complications in diabetes mellitus and diabetic nephropathy. In podocytes, the main component of the glomerular barrier, overproduction of reactive oxygen species (ROS) in the presence of high glucose induces dysfunction and increases excretion of albumin in urine. This suggests an impaired antioxidant defense system has a role in the pathogenesis of diabetic nephropathy. We studied expression of NAD(P)H oxidase subunits by Western blotting and immunofluorescence and the activities of the oxidant enzyme, NAD(P)H, and antioxidant enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT), in mouse podocytes cultured in a high glucose concentration (30 mM). We found long-term (3 and 5 days) exposure of mouse podocytes to high glucose concentrations caused oxidative stress, as evidenced by increased expression of Nox4 and activities of NAD(P)H oxidase (Δ 182%) and SOD (Δ 39%) and decreased activities of GPx (Δ -40%) and CAT (Δ -35%). These biochemical changes were accompanied by a rise in intracellular ROS production and accumulation of hydrogen peroxide in extracellular space. The role of Nox4 in ROS generation was confirmed with Nox4 siRNA. In conclusion, high glucose concentration affects the oxidant-antioxidant balance in mouse podocytes, resulting in enhanced generation of superoxide anions and its attenuated metabolism. These observations suggest free radicals may play an important role in the pathogenesis of diabetic nephropathy.     

Rickettsial pathogen uses arthropod tryptophan pathway metabolites to evade reactive oxygen species in tick cells

Reactive oxygen species (ROS) that are induced upon pathogen infection plays an important role in host defence. The rickettsial pathogen Anaplasma phagocytophilum, which is primarily transmitted by Ixodes scapularis ticks in the United States, has evolved many strategies to escape ROS and survive in mammalian cells. However, little is known on the role of ROS in A. phagocytophilum infection in ticks. Our results show that A. phagocytophilum and hemin induce activation of l ‐tryptophan pathway in tick cells. Xanthurenic acid (XA), a tryptophan metabolite, supports A. phagocytophilum growth in tick cells through inhibition of tryptophan dioxygenase (TDO) activity leading to reduced l ‐kynurenine levels that subsequently affects build‐up of ROS. However, hemin supports A. phagocytophilum growth in tick cells by inducing TDO activity leading to increased l ‐kynurenine levels and ROS production. Our data reveal that XA and kynurenic acid (KA) chelate hemin. Furthermore, treatment of tick cells with 3‐hydroxyl l ‐kynurenine limits A. phagocytophilum growth in tick cells. RNAi‐mediated knockdown of kynurenine aminotransferase expression results in increased ROS production and reduced A. phagocytophilum burden in tick cells. Collectively, these results suggest that l ‐tryptophan pathway metabolites influence A. phagocytophilum survival by affecting build up of ROS levels in tick cells.     

Retinol supplementation induces oxidative stress and modulates antioxidant enzyme activities in rat Sertoli cells

Recent intervention studies revealed that supplementation with retinoids resulted in a higher incidence of lung cancer. Recently the causal mechanism has begun to be clarified. We report here that retinol caused cellular oxidative stress and modulated superoxide dismutase, catalase and glutathione peroxidase activities. Retinol (7 μM) significantly increased TBARS, conjugated dienes, and hydroperoxide-initiated chemiluminescence in cultured Sertoli cells. In response to retinol treatment superoxide dismutase, catalase and glutathione peroxidase activities increased. TBARS content and catalase activities were decreased by a free radical scavenger. These findings suggest that retinol may induce oxidative stress and modulate antioxidant enzyme activities in Sertoli cells.     

Effect of prostaglandin E1 on chemiluminescence response and adherence of human polymorphonuclear leukocytes to human cultured endothelial cells of prostaglandin E1 treated polytraumatized patients

We investigated the effect of prostaglandin E1 on human polymorphonuclear leukocytes, in vivo. Polymorphonuclear leukocytes of a prostaglandin E1 and placebo study group were harvested and their function, as production of oxygen-derived metabolites and adherence to human cultured endothelial cells, was compared. Additionally, data obtained from polymorphonuclear leukocytes of a prostaglandin E1 and placebo group were compared with data obtained from polymorphonuclear leukocytes from 28 blood donors, who served as a control group. Production of oxygen-derived metabolites by polymorphonuclear leukocytes during contact with endothelial cells was measured by chemiluminescence. Chemiluminescence was significantly (p < 0.01) increased in the placebo group in comparison to the control group decreasing to values of control group after 6 d (post-trauma). Chemiluminescence response was not significantly suppressed in patients treated with prostaglandin E1 in comparison to the placebo group. Adherence of polymorphonuclear leukocytes (placebo group) to endothelial cells was significantly increased (p < 0.01) within the first 6 d post-trauma Following day 6, values were in the same range as values for the control group. Adherence was not significantly suppressed in patients treated with prostaglandin E1 in comparison to the placebo group. In conclusion, prostaglandin E1 at a dose of 20 ng/kg bw/min does not influence production of oxygenderived metabolites and adherence in polytraumatized patients in comparison to a placebo group. Additionally, production of oxygen-derived metabolites by polymorphonuclear leukocytes in response to endothelial cells is shown and it is evident that endothelial cells might influence production of oxygen derived metabolites by polymorphonuclear leukocytes.     

Nox4 overexpression activates reactive oxygen species and p38 MAPK in human endothelial cells

Nicotine adenine dinucleotide phosphate (NADPH) oxidase (Nox) complexes are the main sources of reactive oxygen species (ROS) formation in the vessel wall. We have used DNA microarray, real-time PCR and Western blot to demonstrate that the subunit Nox4 is the major Nox isoform in primary human endothelial cells; we also found high levels of NADPH oxidase subunit p22^phox expression. Nox4 was localized by laser scanning confocal microscopy within the cytoplasm of endothelial cells. Endothelial Nox4 overexpression enhanced superoxide anion formation and phosphorylation of p38 MAPK. Nox4 down-regulation by shRNA has in contrast to TGF-β no effect on p38 MAPK phosphorylation. We conclude that Nox4 is the major Nox isoform in human endothelial cells, and forms an active complex with p22^phox. The Nox4-containing complex mediates formation of reactive oxygen species and p38 MAPK activation. This is a novel mechanism of redox-sensitive signaling in human endothelial cells.     

Stress tolerance and reactive oxygen metabolism in the intertidal red seaweeds Mastocarpus stellatus and Chondrus crispus

Mastocarpus stellatus and Chondrus crispus are morphologically similar red seaweeds that co-occur on rocky intertidal seashores in the Northern Atlantic. Mastocarpus stellatus grows higher on the shore and is more tolerant of environmental stress, caused by factors such as freezing and desiccation, than C. crispus. Here we report a correlation between reactive oxygen metabolism and stress tolerance, which suggests that reactive oxygen metabolism may play a role in stress tolerance of intertidal red seaweeds. Mastocarpus stellatus scavenged added H₂O₂ slightly faster, and was more resistant to oxidative stress induced by addition of H₂O₂ and Rose Bengal, than C. crispus. These data were consistent with higher levels of ascorbate and β-carotene and higher activities of catalase and glutathione reductase, in M. stellatus. Tocopherol content and activities of superoxide dismutase and ascorbate peroxidase were similar in both species. Activities of reactive oxygen scavenging enzymes generally increased with tidal height in M. stellatus; this was, however, not a consistent trend in C. crispus.     

Bone marrow derived cells and reactive oxygen species in hypertrophy of contralateral kidney of transient unilateral renal ischemia-induced mouse

Abstract

Renal mass reduction, such as unilateral nephrectomy induces a compensatory hypertrophy of remaining renal mass in response to overload induced by reduction of functional renal parenchyma. In our recent study, we observed that the recovery of ischemic injured kidney following transient unilateral renal ischemia took longer time than that following transient bilateral renal ischemia, indicating that non-damaged kidney may affect the damaged kidney and vice versa. Here, we investigated whether transient and partial renal parenchymal injury by transient unilateral renal ischemia (UI) results in the hypertrophy of its contralateral kidney (CLK) and reactive oxygen species is associated with the hypertrophy. Thirty minutes of UI resulted in gradual increase in CLK weight over time. UI increased superoxide formation, but not lipid peroxidation in the CLK. After UI, a significant increase in the number of NADPH oxidase 2 (Nox2)-expressing cells and the level of Nox2 expression in the CLK was observed. In parallel with the increases in Nox2-expressing cells in CLKs, infiltration of bone marrow-derived cells (BMDC) increased in CLK. Treatments with Mn(III) Tetrakis(1-methyl-4-pyridyl) porphyrin (MnTMPyP, a superoxide dismutase (SOD) mimetic) and apocynin (a putative NADPH oxidase inhibitor) inhibited UI-induced hypertrophy of CLK along with reduction in Nox2-positive cell, BMDC, amount of Nox2 expression and superoxide formation. In conclusion, transient and partial renal mass reduction by UI resulted in the hypertrophy of CLK through increased ROS formation by infiltrated cells into the interstitium of CLK.     

MnSOD marks cord blood late outgrowth endothelial cells and accompanies robust resistance to oxidative stress

Cord blood is source of colony-forming progenitors to vascular endothelial cells for potential use in cell therapies. These cells-called blood late outgrowth endothelial cells (OECs)-have undergone endothelial differentiation, but appear to still possess functional properties different from mature endothelial cells. A large-scale comparative proteomics screen of cord blood OECs versus human vein endothelial cells (HUVECs) using two-dimensional gel electrophoresis and mass spectrometry identified specific expression of manganese superoxide dismutase (MnSOD), a key antioxidant enzyme expressed in the mitochondria, in OECs but not in HUVECs. Immunoblotting verified significant MnSOD levels in all OEC isolates tested and maintained throughout passaging. Endothelial function and cell survival/proliferation assays in the presence of high cytotoxic doses of the superoxide generator compound LY83583 showed OECs profoundly better protected against oxidative stress than HUVECs. Such cytoprotective levels of MnSOD cells could give therapeutic cell transplants a survival advantage in necrotic or ischemic conditions.     

The challenges of using fluorescent probes to detect and quantify specific reactive oxygen species in living cells

Background

Small molecule fluorescent probes are vital tools for monitoring reactive oxygen species in cells.

Scope of review

The types of probe available, the extent to which they are specific or quantitative and complications in interpreting results are discussed.

Major conclusions

Most commonly used probes (e.g. dihydrodichlorofluorescein, dihydrorhodamine) have some value in providing information on changes to the redox environment of the cell, but they are not specific for any one oxidant and the response is affected by numerous chemical interactions and not just increased oxidant generation. These probes generate the fluorescent end product by a free radical mechanism, and to react with hydrogen peroxide they require a metal catalyst. Probe radicals can react with oxygen, superoxide, and various antioxidant molecules, all of which influence the signal. Newer generation probes such as boronates act by a different mechanism in which nucleophilic attack by the oxidant on a blocking group releases masked fluorescence. Boronates react with hydrogen peroxide, peroxynitrite, hypochlorous acid and in some cases superoxide, so are selective but not specific. They react with hydrogen peroxide very slowly, and kinetic considerations raise questions about how the reaction could occur in cells.

General significance

Data from oxidant-sensitive fluorescent probes can provide some information on cellular redox activity but is widely misinterpreted. Recently developed non-redox probes show promise but are not generally available and more information on specificity and cellular reactions is needed. We do not yet have probes that can quantify cellular production of specific oxidants. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.