Loss and recovery of nitrogenase in Abuts incana nodules exposed to low oxygen and low temperature

The possibility that respiration limits oxygen access to nitrogenase was tested by artificially upsetting the balance between oxygen consumption (respiration) and oxygen influx (diffusion). Argon treatment of the nodulated root system on intact plants stopped in vivo nitrogenase activity almost completely. Upon return to air, nitrogenase activity was very low and recovered gradually to full activity after about 5 h. In vitro measurements on nodule homogenates indicated that active nitrogenase was lost upon the shift from low (argon) to normal (air) oxygen. Maintenance of nodulated root systems at low temperature (2°C) inhibited both respiration and in vivo nitrogenase activity. Upon return to normal temperature (22°C), oxygen uptake recovered very rapidly, but nitrogenase activity recovered only gradually to full activity after about 5 to 6 h. Again, loss of active nitrogenase could, at least partly, explain the reduced in vivo nitrogenase activity. The effects from a temporarily impaired balance between oxygen consumption and oxygen influx thus point to the importance of respiration for limiting oxygen access to nitrogenase.     

Reversible and irreversible inactivation of cellular nitrogenase upon oxygen stress in Azotobacter vinelandii growing in oxygen controlled continuous culture

Azotobacter vinelandii growing in oxygen controlled chemostat culture was subjected to sudden increases of ambient oxygen concentrations (oxygen stress) after adaptation to different oxygen concentrations adjustable with air (100% air saturation corresponds to 225±14 M O₂). Inactivations of cellular nitrogenase during stress (switch off) as well as after release of stress (switch on) were evaluated in vivo as depending on stress duration and stress height (pO₂). Switch off was at its final extent within 1 min of stress. The extent of switch off, however, increased with stress height and was complete at pO₂ between 8–10% air saturation irrespective of different oxygen concentrations the organisms were adapted to before stress, indicating that switch off is adaptable. Inactivation of nitrogenase measurable after switch on represents irreversible loss of activity. Irreversible inactivation was at its characteristic level within less than 3 min of stess and at a pO₂ of less than 1% air saturation. The level of irreversible inactivation increased linearly with the oxygen concentration the organisms were adapted to before stress. Thus adaptation of cells to increased oxygen concentrations did not prevent increased susceptibility of nitrogenase to irreversible inhibition during oxygen stress. The fast response of irreversible inactivation at low stress heights suggests that it takes place already during stress. Thus switch off comprised both a reversible and an irreversible phase. The data showed that reversible inactivation of nitrogenase was less susceptible to oxygen stress than irreversible inactivation. A basic pre-requisite of the hypothesis of respiratory protection of nitrogenase, i.e. the proposed relationship between respiratory activities and the protection of nitrogenase from irreversible inhibition by oxygen, was not supported by the results of this report.     

Protective effects of fluvastatin against reactive oxygen species induced DNA damage and mutagenesis

Oxidative stress may be an important factor in the development of diabetic complications. Advanced glycation end-products have drown attention as potential sources of oxidative stress in diabetes. We investigated the protective effects of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on oxidative DNA damage from reactive oxygen species or advanced glycation end-products in vitro, as well as effects of main fluvastatin metabolites and other inhibitors of the same enzyme, pravastatin and simvastatin. Protective effects were assessed in terms of the DNA breakage rate in a single-stranded phage DNA system in vitro. DNA was exposed to either reactive oxygen species or advanced glycation end-products. Fluvastatin and its metabolites showed a strong protective effect comparable to those seen with thiourea and mannitol, though pravastatin and simvastatin did not exert clear protective effects. Furthermore, fluvastatin reduced the mutagenesis by reactive oxygen species or advanced glycation end-products in Salmonella typhimurium test strains. Both pravastatin and simvastatin still lacked protective activity. Fluvastatin and its metabolites protect against oxidative DNA damage and may reduce risk of consequent diabetic complications.     

Genotoxicity of chloroquine in rat liver cells: Protective role of free radical scavengers

The genotoxic effect of chloroquine (CQ), a 4-aminoquinoline antimalarial drug was investigated in rat liver cells using the alkaline comet assay. Chloroquine (0–1000 μmol/L) significantly increased DNA strand breaks of rat liver cells dose-dependently. Rat liver cells exposed to CQ (100–500 μmol/L) and treated with endonuclease III and formamidopyrimidine-DNA glycosylase, the bacterial DNA repair enzymes that recognize oxidized pyrimidine and purine, respectively, showed greater DNA damage than those not treated with the enzymes, providing evidence that CQ induced oxidation of purines and pyrimidines. Treatment of cells with 5 mmol/L N-acetylcysteine, an intracellular reactive oxygen species (ROS) scavenger, and 100 μmol/L and 250 μmol/L deferoxamine, an established iron chelator, significantly decreased the CQ-induced strand breaks and base oxidation, respectively. Similarly, the formation of DNA strand breaks and oxidized bases was prevented by vitamin C (10 μmol/L) (a water-soluble antioxidant), quercetin (50 μmol/L) (an antioxidant flavonoid), and kolaviron (30 μmol/L and 90 μmol/L) (an antioxidant and a liver hepatoprotective phytochemical). The results indicate that the genotoxicity of CQ in rat liver cells might involve ROS and that free radical scavengers may elicit protective effects in these cells.     

Protective systems against activated oxygen species compared in normal and fully habituated nonorganogenic sugarbeet calluses

Summary The levels of the water-soluble reductants ascorbic acid and glutathione and the activities of the enzymatic antioxidants superoxide dismutase, catalase, ascorbate peroxidase, monodehydroascorbate and dehydroascorbate reductases and glutathione reductase were determined in a fully habituated nonorganogenic sugarbeet callus line (considered a neoplasm) compared with a normal hormone-dependent callus of the same plant. Ascorbic acid was not recovered from either of the two calluses, irrespective of the technique used. Glutathione was titrated at a slightly higher level in the normal callus. Catalase activity was almost nonexistent in the habituated callus. The other enzymes (superoxide dismutase, glutathione reductase, monodehydroascorbate reductase, dehydroascorbate reductase, and ascorbate peroxidase) were found to have higher activities in the habituated callus. The results are interpreted as a higher protection of the neoplastic habituated cells against oxygen-free radicals and hydroperoxide-dependent oxidations. Such strong scavenging properties of the habituated cell line could explain previous results already reported, namely the stimulation of cell division at the expense of cell differentiation.     

Protective mechanisms against peptide and protein peroxides generated by singlet oxygen

Reaction of certain amino acids, peptides, and proteins with singlet oxygen yields substrate-derived peroxides. Recent studies have shown that these species are formed within intact cells and can inactivate key cellular enzymes. This study examines potential mechanisms by which cells might remove or detoxify such peroxides. It is shown that catalase, horseradish peroxidase, and Cu/Zn superoxide dismutase do not react rapidly with these peroxides. Oxymyoglobin and oxyhemoglobin, but not the met (Fe3+) forms of these proteins, react with peptide but not protein, peroxides with oxidation of the heme iron. Glutathione peroxidase, in the presence of reduced glutathione (GSH) rapidly removes peptide, but not protein, peroxides, consistent with substrate size being a key factor. Protein thiols, GSH, other low-molecular-weight thiols, and the seleno-compound ebselen react, in a nonstoichiometric manner, with both peptide and protein peroxides. Cell lysate studies show that thiol consumption and peroxide removal occur in parallel; the stoichiometry of these reactions suggests that thiol groups are the major direct, or indirect, reductants for these species. Ascorbic acid and some derivatives can remove both the parent peroxides and radicals derived from them, whereas methionine and the synthetic phenolic antioxidants Probucol and BHT show little activity. These studies show that cells do not have efficient enzymatic defenses against protein peroxides, with only thiols and ascorbic acid able to remove these materials; the slow removal of these species is consistent with protein peroxides playing a role in cellular dysfunction resulting from oxidative stress.     

Protective effects of green tea polyphenol against reactive oxygen species-induced oxidative stress in cultured rat calvarial osteoblast

The injurious effects of reactive oxygen species on osteoblasts and the potential protective role played by green tea polyphenols (GtPP) were investigated using primarily cultured rat calvarial osteoblasts. Oxidative stress was induced in cultured osteoblasts, either by adding 100 mmol/L H2O2 or by the action of 40 U/L xanthine oxidase (XO) in the presence of xanthine (250 micromol/L). After incubation, the cellular viability, function and morphology were evaluated. Both treatments produced a significant reduction in osteoblast viability, as assessed by a two-colored fluorescence staining method combined with flow cytometric analysis and MTT assay. A significant reduction in the alkaline phosphatase activity was observed after H2O2 addition, whereas XO did not have the same effect. On the microscopic observations, the morphological changes and intracellular ultrastructural damages were remarkably induced by both treatments. The H2O2-induced alterations were prevented by pre-incubating the osteoblasts with 200 microg/ml GtPP for 1 h. When the oxidative stress was induced by XO, the cellular viability and morphology was also maintained at the same polyphenol concentration. These results demonstrate that GtPP can act as a biological antioxidant in a cell culture experimental model and protect cells from oxidative stress-induced toxicity.     

What triggers the regulation of nitrogenase activity in forage legume nodules after defoliation?

The dramatic decrease in nitrogenase activity after the defoliation of forage legumes has been recognized for a long time; however, the underlying mechanisms are not understood yet. The impact of current photosynthesis can be excluded. The precise role of carbohydrate availability is still unclear and remains to be established. From current knowledge we can conclude that, after defoliation, nitrogenase activity in legume nodules is down-regulated by a variable oxygen diffusion resistance. The triggering elements are not known; there is, however, increasing evidence that the plant's demand for symbiotically fixed nitrogen plays an important role. The possibility is here discussed that, after defoliation, a nitrogen feedback mechanism regulates nitrogenase activity through a variable oxygen diffusion resistance in the nodules.     

Photoinhibition of Photosystem I damages both reaction centre proteins PSI-A and PSI-B and acceptor-side located small Photosystem I polypeptides

Photoinhibition of Photosystem I at chilling temperatures was investigated. Illumination of barley and cucumber leaves at 4°C induced a lowered Photosystem I activity. In barley, the reaction centre proteins PSI-A and PSI-B were both partially degraded as was the nuclear-encoded PSI-D polypeptide. Barley leaves infiltrated with KCN to increase oxidative stress, showed increased photoinhibition of Photosystem I, including reduced photochemical activity and marked degradation of several Photosystem I polypeptides. The most rapid and pronounced degradation was found in the PSI-D and PSI-E polypeptides exposed at the Photosystem I acceptor side. The PSI-A, -B, -C, -G, -H, -K and -L polypeptides were less extensively damaged. No damage of the lumenally oriented PSI-F and -N polypeptides was detected. The elevated photoinhibition of Photosystem I seen in KCN treated barley is most likely induced by a combination of increased active oxygen due to inhibited scavenging and increased accumulation of reducing power due to inhibition of the Calvin cycle. In barley, photo-inactivation of Photosystem I closely followed the degradation of PSI-A and PSI-B. Illumination of cucumber resulted in a pronounced loss of activity and appearance of specific PSI-A and PSI-B degradation products whereas the total PSI-A/B degradation was small. The PSI-A/B degradation identified in barley is interpreted to reflect a physiologically relevant process being part of a repair cycle, whereas the much smaller PSI-A/B degradation observed in cucumber is interpreted to represent an irreversible damage induced far below the temperature tolerance for cucumber.     

O2 regulation and O2‐limitation of nitrogenase activity in root nodules of pea and lupin

The gas exchange characteristics of intact attached nodulated roots of pea (Pisum sativum cv. Finale X) and lupin (Lupinus albus cv. Ultra) were studied under a number of environmental conditions to determine whether or not the nodules regulate resistance to oxygen diffusion. Nitrogenase activity (H₂ evolution) in both species was inhibited by an increase in rhizosphere pO₂ from 20% to 30%, but recovered within 30 min without a significant increase in nodulated root respiration (CO₂ evolution). These data suggest that the nodules possess a variable barrier to O₂ diffusion. Also, nitrogenase activity in both species declined when the roots were either exposed to an atmosphere of Ar:O₂ or when the shoots of the plants were excised. These declines could be reversed by elevating rhizosphere pO₂, indicating that the inhibition of nitrogenase activity resulted from an increase in gas diffusion resistance and consequent O₂-limitation of nitrogenase-linked respiration. These results indicate that nodules of pea and lupin regulate their internal O₂ concentration in a manner similar to nodules of soybean, despite the distinct morphological and biochemical differences that exist between the nodules of the 3 species. Experiments in which total nitrogenase activity (TNA = H₂ production in Ar:O₂) in pea and lupin nodules was monitored while rhizosphere pO₂ was increased gradually to 100%, showed that the resistance of the nodules to O₂ diffusion maintains nitrogenase activity at about 80% of its potential activity (PNA) under normal atmospheric conditions. The O₂-limitation coefficient of nitrogenase (OLC_N= TNA/PNA) declined significantly with prolonged exposure to Ar:O₂ or with shoot excision. Together, these results indicate a significant degree of O₂-limitation of nitrogenase activity in pea and lupin nodules, and that yields may be increased by realizing full potential activity.     

Estimating excess post-exercise oxygen consumption using multiple linear regression in healthy Korean adults: a pilot study

[Purpose]This pilot study aimed to develop a regression model to estimate the excess post-exercise oxygen consumption (EPOC) of Korean adults using various easy-to-measure dependent variables.[Methods]The EPOC and dependent variables for its estimation (e.g., sex, age, height, weight, body mass index, fat-free mass [FFM], fat mass, % body fat, and heart rate_sum [HR_sum]) were measured in 75 healthy adults ( 31 males, 44 females). Statistical analysis was performed to develop an EPOC estimation regression model using the stepwise regression method.[Results]We confirmed that FFM and HR_sum were important variables in the EPOC regression models of various exercise types. The explanatory power and standard errors of estimates (SEE) for EPOC of each exercise type were as follows: the continuous exercise (CEx) regression model was 86.3% (R²) and 85.9% (adjusted R²), and the mean SEE was 11.73 kcal, interval exercise (IEx) regression model was 83.1% (R²) and 82.6% (adjusted R²), while the mean SEE was 13.68 kcal, and the accumulation of short-duration exercise (AEx) regression models was 91.3% (R²) and 91.0% (adjusted R²), while the mean SEE was 27.71 kcal. There was no significant difference between the measured EPOC using a metabolic gas analyzer and the predicted EPOC for each exercise type.[Conclusion]This pilot study developed a regression model to estimate EPOC in healthy Korean adults. The regression model was as follows: CEx = -37.128 + 1.003 × (FFM) + 0.016 × (HR_sum), IEx = -49.265 + 1.442 × (FFM) + 0.013 × (HR_sum), and AEx = -100.942 + 2.209 × (FFM) + 0.020 × (HR_sum).     

Glycinebetaine protects plants against abiotic stress: mechanisms and biotechnological applications

Various compatible solutes enable plants to tolerate abiotic stress, and glycinebetaine (GB) is one of the most-studied among such solutes. Early research on GB focused on the maintenance of cellular osmotic potential in plant cells. Subsequent genetically engineered synthesis of GB-biosynthetic enzymes and studies of transgenic plants demonstrated that accumulation of GB increases tolerance of plants to various abiotic stresses at all stages of their life cycle. Such GB-accumulating plants exhibit various advantageous traits, such as enlarged fruits and flowers and/or increased seed number under non-stress conditions. However, levels of GB in transgenic GB-accumulating plants are relatively low being, generally, in the millimolar range. Nonetheless, these low levels of GB confer considerable tolerance to various stresses, without necessarily contributing significantly to cellular osmotic potential. Moreover, low levels of GB, applied exogenously or generated by transgenes for GB biosynthesis, can induce the expression of certain stress-responsive genes, including those for enzymes that scavenge reactive oxygen species. Thus, transgenic approaches that increase tolerance to abiotic stress have enhanced our understanding of mechanisms that protect plants against such stress.     

Heterotrophic metabolism and regulation of uptake hydrogenase activity in symbiotic cyanobacteria

The H₂ uptake activity of three cyanobionts isolated fromCycas revoluta, C. circinalis andazolla filiculoides was shown to be related primarily to the growth rate and independent of the main mode of carbon nutrition. Significant H₂ uptake was found in the coralloid roots ofCycas revoluta andZamia furfuracea (3 and 22 times higher than the respective C₂H₂ reduction activities). The results attained allow us to conclude that in cyanobacteria, in contrast to most nitrogen-fixing heterotrophs, uptake hydrogenase activity is not repressed by carbon substrates and that cyanobacteria in association seem to be endowed with sufficient H₂ uptake capacity to recover all of the H₂ released during the process of N₂-fixation.     

Interaction with magnesium and ADP stabilizes both components of nitrogenase from Klebsiella pneumoniae against urea denaturation

The nitrogenase enzyme of Klebsiella pneumoniae consists of two separable proteins, each with multiple subunits and one or more oxygen sensitive metallocenters. The wild-type nitrogenase proteins are stable to electrophoresis in high concentrations of urea under anaerobic conditions. Addition of Mg+2 and ADP greatly increases the stability of the smaller Fe protein (from <4 to >6 M for full unfolding), an effect directly analogous to stabilization in p21ras induced by Mg+2 and GDP. Stabilization by Mg+2 is slight for the holo MoFe protein (from approximately 1.5 to approximately 2.4 M) but more dramatic for the apo protein form of the MoFe protein accumulated by certain Fe protein (nifH gene) mutants. The potent product inhibitor of nitrogenase function, MgADP, increases stability of the MoFe protein more than Mg+2 alone, to approximately 3.6 M, showing that nucleotides interact with the MoFe protein. Mutations of the nifM gene result in slower accumulation of less stable Fe protein, indicating that NifM is involved in correct folding of the Fe protein. Mutationally altered proteins are often difficult to purify for study because of their inherent instability, low expression level, or oxygen lability. Crude extracts of 11 different mutants of Fe protein (nifH gene) were examined by transverse urea gradient gels to rapidly screen for stabilizing interactions in the presence or absence of substrate or inhibitor analogs. Amino acid alterations D44N and R188C, at the interface of the dimer, in the vicinity of the nucleotide binding site(s), have significantly lower stability than the wild-type enzyme in the absence of Mg+2 but comparable stability in its presence, showing the importance of Mg+2 in the subunit interactions. Mutations N163S and E266K, in which residues normally involved in hydrogen bonding far from the active site were altered, are more labile than the wild-type even with Mg+2 added. Seven other mutants, though nonfunctional, did not appear altered in stability compared to the wild-type.     

The effect of quenchers on the chemiluminescence of luminol and lucigenin

The chemiluminescence of luminol and lucigenin is often used to detect the production of reactive oxygen derivatives by phagocytic cells. Also, several quenchers and enzyme inhibitors are used to determine which oxygen derivatives are responsible for the observed effects. In the present work we have assessed the reliability of dimethylthiourea and cysteamine (OH^. quenchers), desferrioxamine (iron chelator) and diethyldithiocarbamate (superoxide dismutase inhibitor). They all react with CIO^? and are also strong inhibitors of the luminescence of luminol catalysed by horseradish peroxidase (HRP); cysteamine and diethyldithiocarbamate also react with H₂O₂. NaN₃ is an inhibitor of myeloperoxidase and a quencher of singlet O₂, but we found that under certain conditions it can amplify the the luminescence of luminol triggered by CIO^? or Fenton's reagent. A complex of copper and penicillamine that had been proposed as an $ {\rm O}_{\rm 2} ^{\bar .} $ quencher, quenches all luminescent reactions studied. On the other hand, we were able to confirm the relative specificity of other quenchers: taurine for CIO^?, benzoate for OH^. and mannitol for both OH^. and ‘crypto-OH^.’.     

In search of the mechanism of nitrate inhibition of nitrogenase activity in legume nodules: Recent developments

The mechanisms involved in the inhibition of nitrogenase activity in legume nodules by nitrate is unclear. This paper reviews and evaluates proposed mechanisms of this inhibition. Emphasis is placed on recent developments, which suggest that nitrate causes an O₂ limitation of nitrogenase activity. Several mechanisms that involve a nitrate-induced increase in resistance to O₃ diffusion in the nodule cortex are discussed.     

Salt stress induces a decrease in the oxygen uptake of soybean nodules and in their permeability to oxygen diffusion

The effects of short-term NaCl-salinity on nodules of soybean ( Glycine max L. cv. Kingsoy) were studied on hydroponically-grown plants. Both acetylene reducing activity (ARA) and nodule respiration (O₂ uptake and CO₂ evolution) were immediately inhibited, and the stimulation of them by rising the external partial pressure of O₂ (pO₂) was diminished by the application of 0.1 M NaCl in the nutrient solution. The permeability of the nodule to O₂ diffusion, estimated by O₂ consumption or CO₂ evolution, was significantly lower in the stressed nodules than in the cootrol ones. The respiratory quotient of intact nodules and the ethanol production of excised nodules were increased by low pO₂ and by salt stress. These data confirm that in salt-stressed soybean nodules, O₂ availability is reduced and fermentative pathways are stimulated.