Oxidative burst and electrolyte leakage induced by sulfhydryl blockers and by membrane permeabilizing reagents in different organs ofEgeria densa

Summary Sulfhydryl blockers, such as N-ethylmaleimide, iodoacetate and heavy metals induce a transitory stimulation of O₂ consumption and H₂O₂ production (oxidative burst) and a rapid release of electrolytes in leaves of various aquatic plants. The correlation between these two responses to N-ethylmaleimide or to Ag⁺ in separate organs and stages of leaf development was investigated inEgeria densa. Only adult leaves were able to respond to the sulfhydryl blockers with an oxidative burst, whereas this response was absent in immature growing leaves and in stem and root segments. In N-ethyl-maleimide- as well as in Ag⁺-treated adult leaves the oxidative burst was constantly associated with a relevant electrolyte leakage. These data are consistent with a model in which the SH reagent would first interact with a plasmalemma protein, leading to an increase in passive permeability to ions and to the activation of an oxidative enzyme of the type of the superoxide synthase described for granulocytes. In its turn, active-oxygen species produced by the activated oxidase might further damage the plasma membrane, increasing its passive permeability. Digitonin and nystatin, two reagents known to cause a permeabilization of lipid membranes, induced in adultE. densa leaves a transient increase in the rate of O₂ consumption and H₂O₂ production and an electrolyte leakage very similar to those induced by sulfhydryl blockers. These effects, however, were not influenced by the flavin analogues diphenylene iodonium and quinacrine, and were partially inhibited by the presence of CN^– and salicylhydroxamic acid, thus suggesting the involvement of a different oxidase in the oxidative burst elicited by these reagents.Abbreviations BTP 1,3-bis-tris(hydroxymethyl)methylaminopropane - CCCP carbonylcyanide-chlorophenylhydrazone - DCMU 3-(3,4 dichloropheny 1)-1,1-dimethylurea - DPI diphenylene iodonium - NEM N-ethylmaleimide - QO₂ O₂ uptake     

Paxillus involutus mycorrhiza attenuate NaCl-stress responses in the salt-sensitive hybrid poplar Populus×canescens

In order to characterise the effect of ectomycorrhiza on Na⁺-responses of the salt-sensitive poplar hybrid Populus × canescens, growth and stress responses of Paxillus involutus (strain MAJ) were tested in liquid cultures in the presence of 20 to 500 mM NaCl, and the effects of mycorrhization on mineral nutrient accumulation and oxidative stress were characterised in mycorrhizal and non-mycorrhizal poplar seedlings exposed to 150 mM NaCl. Paxillus involutus was salt tolerant, showing biomass increases in media containing up to 500 mM NaCl after 4 weeks growth. Mycorrhizal mantle formation on poplar roots was not affected by 150 mM NaCl. Whole plant performance was positively affected by the fungus because total biomass was greater and leaves accumulated less Na⁺ than non-mycorrhizal plants. Energy dispersive X-ray microanalysis using transmission electron microscopy analysis of the influence of mycorrhization on the subcellular localisation of Na⁺ and Cl⁻ in roots showed that the hyphal mantle did not diminish salt accumulation in root cell walls, indicating that mycorrhization did not provide a physical barrier against excess salinity. In the absence of salt stress, mycorrhizal poplar roots contained higher Na⁺ and Cl⁻ concentrations than non-mycorrhizal poplar roots. Paxillus involutus hyphae produced H₂O₂ in the mantle but not in the Hartig net or in pure culture. Salt exposure resulted in H₂O₂ formation in cortical cells of both non-mycorrhizal and mycorrhizal poplar and stimulated peroxidase but not superoxide dismutase activities. This shows that mature ectomycorrhiza was unable to suppress salt-induced oxidative stress. Element analyses suggest that improved performance of mycorrhizal poplar under salt stress may result from diminished xylem loading of Na⁺ and increased supply with K⁺.     

Redox-related peroxidative responses evoked by methyl-jasmonate in axenically cultured aeroponic sunflower (Helianthus annuus L.) seedling roots

Methyl-jasmonate (MeJA) has been proposed to be involved in the evocation of defense reactions, as the oxidative burst in plants, substituting the elicitors or enhancing their effect. 48 h dark- and sterilely cultured (axenic) aeroponic sunflower seedling roots excised and treated with different concentrations of MeJA showed a strong and quick depression of the H(+) efflux rate, 1.80 microM MeJA totally stopping it for approximately 90 min and then reinitiating it again at a lower rate than controls. These results were wholly similar to those obtained with nonsterilely cultured roots and have been interpreted as mainly based on H(+) consumption for O(2)(*-) dismutation to H(2)O(2). Also K(+) influx was strongly depressed by MeJA, even transitorily reverting to K(+) efflux. These results were consistent with those associated to the oxidative burst in plants. MeJA induced massive H(2)O(2) accumulation in the middle lamella and intercellular spaces of both the root cap cells and the inside tissues of the roots. The native acidic extracellular peroxidase activity of the intact (nonexcised) seedling roots showed a sudden enhancement (by about 52%) after 5 min of MeJA addition, maintained for approximately 15 min and then decaying again to control rates. O(2) uptake by roots gave similar results. These and other results for additions of H(2)O(2) or horseradish peroxidase, diphenylene iodonium, and sodium diethyldithiocarbamate trihydrate to the reaction mixture with roots were all consistent with the hypothesis that MeJA induced an oxidative burst, with the generation of H(2)O(2) being necessary for peroxidase activity. Results with peroxidase activity of the apoplastic fluid were in accordance with those of the whole root. Finally, MeJA enhanced NADH oxidation and inhibited hexacyanoferrate(III) reduction by axenic roots, and diphenylene iodonium cancelled out these effects. Redox activities by CN(-)- preincubated roots were also studied. All these results are consistent with the hypothesis that MeJA enhanced the NAD(P)H oxidase of a redox chain linked to the oxidative burst, so enhancing the generation of O(2)(*-) and H(2)O(2), O(2) uptake, and peroxidase activity by roots.     

The elicitor-induced oxidative burst in cultured chickpea cells drives the rapid insolubilization of two cell wall structural proteins

Elicitation of cultured chickpea cells caused rapid insolubilization of two cell wall structural proteins, p190, a putative hydroxyproline-rich glycoprotein and p80, a putative proline-rich protein. This process appeared to result from an H₂O₂-mediated oxidative cross-linking mechanism and was initiated within 5 min and complete within 20 min. Further, elicitation of cells induced a rapid, transient generation of H₂O₂ (oxidative burst), with an onset after 5 min and a maximum H₂O₂-release after 20 min, as measured by a luminol-dependent chemiluminescence assay. Both chemiluminescence and protein insolubilization were suppressed by exogenous application of catalase or diphenylene iodonium, an inhibitor of plasma-membrane NADPH oxidase, respectively. In contrast, exogenous H₂O₂ mimicked the effect of the elicitor, suggesting that the putative oxidative crosslinking of the proteins depends directly on H₂O₂ from the oxidative burst. The peroxidase inhibitor salicylhydroxamic acid blocked both the elicitor- and the exogenous-H₂O₂-stimulated insolubilization, indicating that a peroxidase activity downstream of H₂O₂-supply is required. The protein kinase inhibitor staurosporine blocked the elicitation of the oxidative burst and protein insolubilization. In contrast, the protein phosphatase 2A inhibitor cantharidin accelerated, potentiated and extended the elicited oxidative burst. Cantharidin even stimulated the responses in the absence of the elicitor. The competitive effect of both inhibitors confirms that a coordinated activation of (i) protein kinase(s) and (ii) counteracting protein phosphates(s) is a poised signal transduction step for the induction of an NADPH-oxidase-dependent oxidative burst, which drives the putative peroxidase-catalyzed cross-linking of the cell wall proteins.Abbreviations DPI diphenylene iodonium - Ext-1 extensin-1 - gE1 anti-glycosylated extensin-1 antibodies - HRGP hydroxyp-roline-rich glycoprotein - LDC luminol-dependent chemiluminescence - POD peroxidase - PA polyacrylamide - PRP proline-rich proteins - SHAM salicylhydroxamic acid Financial support by Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie is gratefully acknowledged. We thank Dr. C.J. Lamb (Salk Institute, La Jolla, Calif., USA) and Dr. L.A. Staehelin (University of Colorado, Boulder, Colo., USA) for their kind gifts of antibodies.     

Induction of alkalinization and an oxidative burst by low doses of cycloheximide in soybean cells

Soybean (Glycine max L. Merr.) Cell-suspension cultures inoculated with avirulent Pseudomonas syringae pv. glycinea bacteria generated a sustained oxidative burst 3–6 h after the infection. The H₂O₂ production was not dependent on protein biosynthesis but, surprisingly, cycloheximide itself was a very strong inducer of the oxidative burst and of the alkalinization measured in the cell culture medium. Both responses were activated in a very similar manner by inhibitors of protein phosphatases, implicating a phosphorylation change evoked by cycloheximide as a trigger for the elicitation. The activation of the oxidative burst was totally blocked by the kinase inhibitor K252a. The alkalinization response preceded the oxidative burst. The generation of H₂O₂ depleted the medium of H⁺ but the expected alkalinization of about one pH-unit did not occur. The H₂O₂ production by the plasma membrane oxidase must therefore be charge-compensated, likely via H⁺-channel activity. Received: 4 October 1997 / Accepted: 12 May 1998     

Cadmium-induced early changes in O2 •−, H2O2 and antioxidative enzymes in soybean (Glycine max L.) leaves

Cadmium-induced initial changes in the production of reactive oxygen species (ROS) and antioxidant mechanism were investigated in soybean (Glycine max L. cv. Don Mario 4800 RR) leaves. Whole plants (WP) and plants without roots (PWR) were exposed to 0.0, 10.0 and 40.0 μM Cd for 0, 4, 6 and 24 h. Compared to PWR, a higher level of endogenous Cd in WP was associated with a lower oxidative stress measured in terms of lipid peroxidation. Furthermore, O₂ ^•− content decreased in the leaves of Cd-treated WP, whereas it increased in those of Cd-treated PWR. Although O₂ ^•− accumulation in PWR was associated with a decrease in superoxide dismutase (SOD) activity, O₂ ^•− diminution in WP leaves was not related to any increase in SOD activity. H₂O₂ content increased in the leaves of both Cd-treated WP and PWR, and it was concomitant with a corresponding decline in catalase (CAT) and ascorbate peroxidase (APX) activities. When diphenyl iodonium (DPI), an inhibitor of NADPH oxidase, was added, H₂O₂ content remained unchanged in Cd-treated WP, suggesting that NADPH oxidase does not participate in the early hours of Cd toxicity. Taken together, our results showed that early ROS evolution and oxidative damage were different in WP and PWR. This suggests that the response in soybean leaves during the early hours of Cd toxicity is probably modulated by the root.     

Protoplast ion fluxes: their measurement and variation with time, position and osmoticum

The ability to measure directly individual protoplast ion fluxes is a valuable addition to patch clamp and other techniques when using protoplasts to study membrane transporters. Before interpreting observations on protoplasts in terms of behaviour of intact cells and tissues, some methodological questions should be addressed. These include effects of space and time variations of transporter activities over the membrane, the osmotic dependence of specific ion transporters and the effect of the regenerating cell wall. In this study net H⁺ and Ca²⁺ fluxes were measured from individual corn (Zea mays L.) coleoptile protoplasts using a non-invasive microelectrode technique for ion flux measurements. For Ca²⁺, the flux distribution was almost symmetrical, ranging ±30 nmol · m⁻² · s⁻¹ around zero. For H⁺ it was skewed towards efflux ranging from −100 to +10 nmol · m⁻² · s⁻¹. The distribution of H⁺ fluxes through the protoplast surface was a complex mosaic which changed with time, sometimes showing oscillations. These flux variations with time and position around the surface, apparently driven by endogenous mechanisms, may be relevant to protoplast pH homeostasis. When the new cell wall was partially regenerated on the next day, the correlation between H⁺ and Ca²⁺ fluxes increased, which is consistent with the weak-acid Donnan-Manning model of cell wall ion exchange. Received: 11 June 1997 / Accepted: 10 July 1997     

A polarographic, oxygen-selective, vibrating-microelectrode system for the spatial and temporal characterisation of transmembrane oxygen fluxes in plants

 A simple procedure is described for the fabrication of micrometer to nanometer-scale platinum electrodes to be used in a vibrating oxygen-selective system. The electrode was prepared by etching a fine platinum wire and insulating it with an electrophoretic paint. The dimensions allowed this electrode to be used with the “vibrating probe technique” in exploratory studies aimed at mapping and measuring the patterns of net influxes as well as effluxes of oxygen in Olea europaea L. leaves and roots with spatial and temporal resolutions of a few microns and a few seconds, respectively. The magnitude and spatial localisation of O₂ influxes in roots was characterised by two distinct peaks. The first, in the division zone, averaged 38 ± 5 nmol m⁻² s⁻¹; the second, in the elongation region, averaged 68 ± 6 nmol m⁻² s⁻¹. Long-term records of oxygen influx in the elongation region of the root showed an oscillatory regime characterised by a fast oscillation with periods of about 8–9 min. In leaves, the system allowed the measurement of real-time changes in O₂ evolution following changes in light. Furthermore, it was possible to obtain “topographical” images of the photosynthetically generated oxygen diffusing through different stomata from a region of the leaf of 120 μm × 120 μm. The combination of topographic and electrochemical information at the micrometer scale makes the system an efficient tool for studying biological phenomena involving oxygen diffusion. Received: 12 November 1999 / Accepted: 1 February 2000     

Astaxanthin prevents in vitro auto-oxidative injury in human lymphocytes

Upon mitogen sensitization, lymphocytes undergo proliferation by oxyradical-based mechanisms. Through continuous resting–restimulation cycles, lymphocytes accumulate auto-induced oxidative lesions which lead to cell dysfunction and limit their viability. Astaxanthin (ASTA) is a nutritional carotenoid that shows notable antioxidant properties. This study aims to evaluate whether the in vitro ASTA treatment can limit oxyradical production and auto-oxidative injury in human lymphocytes. Activated lymphocytes treated with 5 μM ASTA showed immediate lower rates of O₂^•−/H₂O₂ production whilst NO^• and intracellular Ca²⁺ levels were concomitantly enhanced (≤4 h). In long-term treatments (>24 h), the cytotoxicity test for ASTA showed a sigmoidal dose–response curve (LC50 = 11.67 ± 0.42 μM), whereas higher activities of superoxide dismutase and catalase in 5 μM ASTA-treated lymphocytes were associated to significant lower indexes of oxidative injury. On the other hand, lower proliferative scores of ASTA lymphocytes might be a result of diminished intracellular levels of pivotal redox signaling molecules, such as H₂O₂. Further studies are necessary to establish the ASTA-dose compensation point between minimizing oxidative damages and allowing efficient redox-mediated immune functions, such as proliferation, adhesion, and oxidative burst.     

Exchanges of oxygen, carbon dioxide, nitrogen and water in the caecilian Dermophis mexicanus

Oxygen consumption was measured in five Dermophis mexicanus and averaged (±SEM) 0.047 ± 0.004 ml O₂ g⁻¹ h⁻¹. Carbon dioxide production averaged 0.053 ± 0.005 ml CO₂ g⁻¹ h⁻¹ in the same five animals 1 week later. This metabolic rate is similar to metabolic rates of other Gymnophionans but lower than metabolic rates reported for Anurans and Urodeles. Total nitrogen excretion averaged 1.37 μmol N g⁻¹ h⁻¹ which is higher than that found for other amphibians. Of this, 82.5% (1.13 μmol N g⁻¹ h⁻¹) was in the form of urea while 17.5% (0.24 μmol N g⁻¹ h⁻¹) was in the form of NH₃ + NH⁺ ₄. Such ureotelism is typical of terrestrial amphibians like D. mexicanus. Osmotic water flux averaged 0.0193 ml g⁻¹ h⁻¹ in control (sham injected) animals and was not significantly altered by injection of either arginine vasotocin or mesotocin. This osmotic flux is similar to osmotic fluxes found for other terrestrial amphibians. The combined data suggest that metabolism in D. mexicanus is, like most other Gymnophionans, lower than other amphibians. The high rates of nitrogen (especially urea) excretion suggests that this fossorial animal accumulates urea like other burrowing amphibians. Accepted: 27 June 2000     

Oxidative reactions in axenically seedling roots of olive (Olea europaea, L.)

The production of reactive oxygen species (ROS) plays important roles in the life cycle and in the stress response and defence mechanisms of plants. Various enzyme systems are involved in the formation of ROS in the apoplast, including plasmalemma NADPH oxidase and apoplastic peroxidases. The production of O ₂ ^·? and apoplastic peroxidase and exogenous NADH oxidation activities are all strongly dependent on the age of roots??the younger the root, the greater the activity. Apoplastic production of ROS is shown in the root by using specific histochemical probes, this ROS production is growing zone dependent. In the present study, using olive seedlings, differences were also observed between cultivars, especially in O ₂ ^·? production by the Verdial cultivar which was well above that of other cultivars studied. In all the cultivars, treatment of roots with methyl jasmonate (MeJA) or methyl salicylate (MeSA) increased O ₂ ^·? production. Similar results were observed for peroxidase activity, but not for the oxidation of exogenous NADH which was either unaffected (MeJA) or even partially inhibited (MeSA). A conclusion was that MeJA or MeSA induced apoplastic production of ROS does not use exogenous NADH. Treatment with diphenylene iodonium (DPI) reduced the formation of O ₂ ^·? , but affected neither peroxidase nor NADH oxidation activities. Cyanide inhibited O ₂ ^·? production and peroxidase and NADH oxidation activities. Treatment with MnCl₂ had a strong stimulatory effect on peroxidase and NADH oxidation activities, but much less on O ₂ ^·? production. Finally, azide greatly reduced all activities, but especially O ₂ ^·? production. Together, these results indicate a relationship between oxidative activities and the processes of root growth, and that those activities are also dependent on the cultivar, as well as an involvement of peroxidases and plasmalemma NADPH oxidase in apoplast ROS production which is sensitive to DPI, azide, and cyanide but relatively insensitive to MnCl₂, while exogenous NADH oxidation is linked to peroxidase activity.     

Role of extracellular peroxidase in the superoxide production by wheat root cells

Summary Extracellular peroxidase has been shown to contribute to superoxide production in wounded wheat (Triticum aestivum L. cv. Ljuba) root cells. The superoxide-synthesizing system of root cells was considerably inhibited by KCN and NaN₃ and activated by MnCl₂ and H₂O₂. Treatment of roots with salicylic acid and a range of di- and tri-carbonic acids (malic, citric, malonic, fumaric, and succinic acids) stimulated superoxide production in both root cells and extracellular solution. The H₂O₂-stimulated superoxide production in the extracellular solution was much higher when roots were preincubated with salicylic or succinic acid. Exogenous acids enhanced peroxidase activity in the extracellular solution. Pretreatment of root cells with the detergents trypsin and sodium dodecyl sulfate had similar effects on the peroxidase activity. Significant inhibition of both superoxide production and peroxidase activity by diphenylene iodonium suggests that the specificity of the latter as an inhibitor of NADPH oxidase is doubtful. Results obtained indicate that extracellular peroxidase is involved in the superoxide production in wheat root cells. The mobile form of peroxidase can be readily secreted to the apoplastic solution and serve as an emergency enzyme involved in plant wound response.Abbreviations DPI diphenylene iodonium - ECS extracellular solution - ROS reactive oxygen species - SA salicylic acid     

Copper-mediated oxidative burst in Nicotiana tabacum L. cv. Bright Yellow 2 cell suspension cultures

Summary.  In cell suspension cultures of Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) a rapid and concentration-dependent accumulation of H₂O₂ is induced by excess concentrations of copper (up to 100 μM). This specific and early response towards copper stress was shown to be extracellular. Addition of 300 U of catalase per ml decreased the level of H₂O₂. Superoxide dismutase (5 U/ml) induced an increase in H₂O₂ production by 22.2%. This indicates that at least part of the H₂O₂ is produced by dismutation of superoxide. Pretreatment of the cell cultures with the NAD(P)H oxidase inhibitors diphenylene iodonium (2 and 10 μM) and quinacrine (1 and 5 mM) prevented the generation of H₂O₂ under copper stress for 90%. The influence of the pH on the H₂O₂ production revealed the possible involvement of cell-wall-dependent peroxidases in the generation of reactive oxygen species after copper stress. Received May 20, 2002; accepted July 26, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Plant Physiology, Department of Biology, University of Antwerp (RUCA), Groenenborgerlaan 171, 2020 Antwerp, Belgium.     

Nitric oxide retards xanthine oxidase-mediated superoxide anion generation in Phalaenopsis flower: an implication of NO in the senescence and oxidative stress regulation

Senescence is a developmentally regulated and highly ordered sequence of events. Senescence leads to abscission of plant organs and eventually leads to death of a plant or part of it. Present study revealed that Phalaenopsis flower undergo senescence due to over activation of O₂ ^·−generating xanthine oxidase (XO), which consequently increases the concentrations of O₂ ^·− leading to enhanced oxidative damage and disturbed cellular redox environment as indicated by increased lipid peroxidation and DHA/AsA + DHA ratio, respectively. While activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and non-specific peroxidase (POD) were enhanced in sepals and petals of old flower, activities of catalase (CAT) and glutathione reductase (GR) were decreased. Exogenous application of nitric oxide (NO) retarded H₂O₂-induced senescence of Phalaenopsis flower by downregulating activity of XO and concentrations of O₂ ^·−, H₂O₂ and malondialdehyde (MDA, an index of lipid peroxidation). Exogenous application of NO also downregulated SOD activity and upregulated antioxidant enzymes involved in the detoxification of H₂O₂ (CAT and APX), and in the regulation of redox couples viz, monodehydroascorbate reductase (MDHAR) and GR, together with the modulation in non-protein thiol status and DHA/AsA + DHA ratio.     

Oxygen uptake does not increase linearly at high power outputs during incremental exercise test in humans

A group of 12 healthy non-smoking men [mean age 22.3 (SD 1.1) years], performed an incremental exercise test. The test started at 30 W, followed by increases in power output (P) of 30 W every 3 min, until exhaustion. Blood samples were taken from an antecubital vein for determination of plasma concentration lactate [La⁻]_pl and acid-base balance variables. Below the lactate threshold (LT) defined in this study as the highest P above which a sustained increase in [La⁻]_pl was observed (at least 0.5 mmol · l⁻¹ within 3 min), the pulmonary oxygen uptake (V˙O₂) measured breath-by-breath, showed a linear relationship with P. However, at P above LT [in this study 135 (SD 30) W] there was an additional accumulating increase in V˙O₂ above that expected from the increase in P alone. The magnitude of this effect was illustrated by the difference in the final P observed at maximal oxygen uptake (V˙O_2max) during the incremental exercise test (P _max,obs at V˙O_2max) and the expected power output at V˙O_2max(P _max,exp at V˙O_2max) predicted from the linear V˙O₂-P relationship derived from the data collected below LT. The P _max,obs at V˙O_2max amounting to 270 (SD 19) W was 65.1 (SD 35) W (19%) lower (P<0.01) than the P _max,exp at V˙O_{2max .} The mean value of V˙O_2max reached at P _max,obs amounted to 3555 (SD 226) ml · min⁻¹ which was 572 (SD 269) ml · min⁻¹ higher (P<0.01) than the V˙O₂ expected at this P, calculated from the linear relationship between V˙O₂ and P derived from the data collected below LT. This fall in locomotory efficiency expressed by the additional increase in V˙O₂, amounting to 572 (SD 269) ml O₂ · min⁻¹, was accompanied by a significant increase in [La⁻]_pl amounting to 7.04 (SD 2.2) mmol · l⁻¹, a significant increase in blood hydrogen ion concentration ([H⁺]_b) to 7.4 (SD 3) nmol · l⁻¹ and a significant fall in blood bicarbonate concentration to 5.78 (SD 1.7) mmol · l⁻¹, in relation to the values measured at the P of the LT. We also correlated the individual values of the additional V˙O₂ with the increases (Δ) in variables [La⁻]_pl and Δ[H⁺]_b. The Δ values for [La⁻]_pl and Δ[H⁺]_b were expressed as the differences between values reached at the P _max,obs at V˙O_2max and the values at LT. No significant correlations between the additional V˙O₂ and Δ[La⁻]_pl on [H⁺]_b were found. In conclusion, when performing an incremental exercise test, exceeding P corresponding to LT was accompanied by a significant additional increase in V˙O₂ above that expected from the linear relationship between V˙O₂ and P occurring at lower P. However, the magnitude of the additional increase in V˙O₂ did not correlate with the magnitude of the increases in [La⁻]_pl and [H⁺]_b reached in the final stages of the incremental test. Accepted: 30 October 1997     

Detection of the change point in oxygen uptake during an incremental exercise test using recursive residuals: relationship to the plasma lactate accumulation and blood acid base balance

The purpose of this study was to develop a method to determine the power output at which oxygen uptake (V˙O₂) during an incremental exercise test begins to rise non-linearly. A group of 26 healthy non-smoking men [mean age 22.1 (SD 1.4) years, body mass 73.6 (SD 7.4) kg, height 179.4 (SD 7.5) cm, maximal oxygen uptake (V˙O_2max) 3.726 (SD 0.363) l · min⁻¹], experienced in laboratory tests, were the subjects in this study. They performed an incremental exercise test on a cycle ergometer at a pedalling rate of 70 rev · min⁻¹. The test started at a power output of 30 W, followed by increases amounting to 30 W every 3 min. At 5 min prior to the first exercise intensity, at the end of each stage of exercise protocol, blood samples (1 ml each) were taken from an antecubital vein. The samples were analysed for plasma lactate concentration [La]_pl, partial pressure of O₂ and CO₂ and hydrogen ion concentration [H⁺]_b. The lactate threshold (LT) in this study was defined as the highest power output above which [La⁻]_pl showed a sustained increase of more than 0.5 mmol · l⁻¹ · step⁻¹. The V˙O₂ was measured breath-by-breath. In the analysis of the change point (CP) of V˙O₂ during the incremental exercise test, a two-phase model was assumed for the 3rd-min-data of each step of the test: X _i =at _i +b+ɛ _i for i=1,2,…,T, and E(X _i )>at _i +b for i =T+1,…,n, where X ₁, … , X _n are independent and ɛ _i ∼N(0,σ²). In the first phase, a linear relationship between V˙O₂ and power output was assumed, whereas in the second phase an additional increase in V˙O₂ above the values expected from the linear model was allowed. The power output at which the first phase ended was called the change point in oxygen uptake (CP-V˙O₂). The identification of the model consisted of two steps: testing for the existence of CP and estimating its location. Both procedures were based on suitably normalised recursive residuals. We showed that in 25 out of 26 subjects it was possible to determine the CP-O₂ as described in our model. The power output at CP-V˙O₂ amounted to 136.8 (SD 31.3) W. It was only 11 W – non significantly – higher than the power output corresponding to LT. The V˙O₂ at CP-V˙O₂ amounted to 1.828 (SD 0.356) l · min⁻¹ was [48.9 (SD 7.9)% V˙O_{2 max} ]. The [La⁻]_pl at CP-V˙O₂, amounting to 2.57 (SD 0.69) mmol · l⁻¹ was significantly elevated (P<0.01) above the resting level [1.85 (SD 0.46) mmol · l⁻¹], however the [H⁺]_b at CP-V˙O₂ amounting to 45.1 (SD 3.0) nmol · l⁻¹, was not significantly different from the values at rest which amounted to 44.14 (SD 2.79) nmol · l⁻¹. An increase of power output of 30 W above CP-V˙O₂ was accompanied by a significant increase in [H⁺]_b above the resting level (P=0.03). Accepted: 25 March 1998     

Superoxide production and oxygen consumption in endothelium-intact and -denuded artery stimulated by angiotensin II

Arteries stimulated by angiotensin II (AII) to contract do not display the expected augmentation of O₂ consumption seen with other cardiovascular contractile agonists. We tested the hypothesis that superoxide (O₂⁻) or other reactive oxidant species generated by AII played a role in the paradoxical O₂ consumption response in porcine carotid artery, with or without an intact endothelium. Endothelium-denuded arteries were incubated with either 1 μM diphenylene iodonium (DPI), an inhibitor of NAD(P)H oxidase, 300 u/ml superoxide dismutase (SOD), a scavenger of O₂⁻, or 20 U/ml catalase, an enzyme which promotes conversion of O₂⁻ (scavenged in the form of H₂O₂) to O₂. DPI treatment resulted in the expected increase in O₂ consumption upon contractile activation with AII challenge (1.05± 0.23 μmol/g/min; n = 6, p < .01), as did treatment with SOD (0.67± 0.20 μmol/g/min; n = 4, p < .05). Catalase incubation resulted in a burst of O₂ generation upon AII challenge (1.30 ± 0.21 μmol/g/min; n = 10, p < .001). In endothelium-intact arteries, O₂ consumption was again not augmented with AII challenge; instead, a burst of O₂ production was observed (0.66 ± 0.22 μmol/g/min; n = 9, p < .05), which was not affected further by addition of catalase. Thus, the absence of apparent augmentation of O₂ consumption during contractile activation of endothelium-denuded arteries was attributed to simultaneous NAD(P)H oxidase-dependent production of O₂⁻, and attendant H₂O₂ and O₂ generation which either and masked the detection of O₂ consumed or suppressed mitochondrial uptake of O₂, or both. An intact endothelium was required to manifest the burst of O₂ generation with AII stimulation under normal conditions. (Mol Cell Biochem xxx: 235–239, 2005)     

Succinate is the controller of O<Stack><Subscript>2</Subscript><Superscript>−</Superscript></Stack>/H<Subscript>2</Subscript>O<Subscript>2</Subscript> release at mitochondrial complex I : negative modulation by malate,positive by cyanide

Mitochondrial production of H₂O₂ is low with NAD substrates (glutamate/pyruvate, 3 and 2 mM) (G/P) and increases over ten times upon further addition of succinate, with the formation of a sigmoidal curve (semimaximal value at 290 μM, maximal H₂O₂ production at 600 μM succinate). Malate counteracts rapidly the succinate induced increased H₂O₂ release and moves the succinate dependent H₂O₂ production curve to the right. Nitric oxide (NO) and carbon monoxide (CO) are cytochrome c oxidase inhibitors which increase mitochondrial ROS production. Cyanide (CN⁻) was used to mimic NO and CO. In the presence of G/P and succinate (300 μM), CN⁻ progressively increased the H₂O₂ release rate, starting at 1.5 μM. The succinate dependent H₂O₂ production curve was moved to the left by 30 μM CN⁻. The V_max was little modified. We conclude that succinate is the controller of mitochondrial H₂O₂ production, modulated by malate and CN⁻. We propose that succinate promotes an interaction between Complex II and Complex I, which activates O₂⁻ production.     

Chitosan-induced programmed cell death in plants

Chitosan, CN⁻, or H₂O₂ caused the death of epidermal cells (EC) in the epidermis of pea leaves that was detected by monitoring the destruction of cell nuclei; chitosan induced chromatin condensation and marginalization followed by the destruction of EC nuclei and subsequent internucleosomal DNA fragmentation. Chitosan did not affect stoma guard cells (GC). Anaerobic conditions prevented the chitosan-induced destruction of EC nuclei. The antioxidants nitroblue tetrazolium or mannitol suppressed the effects of chitosan, H₂O₂, or chitosan + H₂O₂ on EC. H₂O₂ formation in EC and GC mitochondria that was determined from 2′,7′-dichlorofluorescein fluorescence was inhibited by CN⁻ and the protonophoric uncoupler carbonyl cyanide m-chlorophenylhydrazone but was stimulated by these agents in GC chloroplasts. The alternative oxidase inhibitors propyl gallate and salicylhydroxamate prevented chitosan- but not CN⁻-induced destruction of EC nuclei; the plasma membrane NADPH oxidase inhibitors diphenylene iodonium and quinacrine abolished chitosan- but not CN⁻-induced destruction of EC nuclei. The mitochondrial protein synthesis inhibitor lincomycin removed the destructive effect of chitosan or H₂O₂ on EC nuclei. The effect of cycloheximide, an inhibitor of protein synthesis in the cytoplasm, was insignificant; however, it was enhanced if cycloheximide was added in combination with lincomycin. The autophagy inhibitor 3-methyladenine removed the chitosan effect but exerted no influence on the effect of H₂O₂ as an inducer of EC death. The internucleosome DNA fragmentation in conjunction with the data on the 3-methyladenine effect provides evidence that chitosan induces programmed cell death that follows a combined scenario including apoptosis and autophagy. Based on the results of an inhibitor assay, chitosan-induced EC death involves reactive oxygen species generated by the NADPH oxidase of the plasma membrane.