Lactate metabolism in the muscle of the crab Chasmagnathus granulatus during hypoxia and post-hypoxia recovery

The present study showed that the lactate/glucose ratio in the hemolymph of Chasmagnathus granulatus maintained in normoxia (controls) was 4.9, suggesting that lactate is an important substrate for this crab. Periods of hypoxia are part of the biological cycle of this crab, and lactate is the main end product of anaerobiosis in this crab. Our hypothesis was that this lactate would be, therefore, used by gluconeogenic pathway or can be oxidized or excreted to the aquatic medium during hypoxia and post-hypoxia periods in C. granulatus. The concentrations of hemolymphatic lactate in animals in normoxia are high, and are used as an energy substrate. In hypoxia, muscle gluconeogenesis and excretion of lactate to the aquatic medium would contribute significantly in regulating the concentration of circulating lactate. Utilization of these pathways would serve the objective of maintaining the acid-base equilibrium of the organism. Muscle gluconeogenesis participates, during the recovery process, in metabolizing the lactate produced during the period of hypoxia. Lactate excretion to the external medium, was one of the strategies used to decrease the higher hemolymphatic lactate levels. However, oxidation of lactate in the muscle is not a main strategy used by this crab to metabolize lactate in the recovery periods.     

Regulation of alternative oxidase activity during phosphate deficiency in bean roots (Phaseolus vulgaris)

Cyanide-resistant respiration was studied in mitochondria isolated from the roots of bean plants ( Phaseolus vulgaris L. cv. Złota Saxa) grown hydroponically up to 16 days on a phosphate-sufficient (+P, control) or phosphate-deficient (−P) medium. Western blotting indicated that the alternative oxidase (AOX) was present only in its reduced (active) form, both in phosphate-sufficient and phosphate-deficient roots, but in the latter, the amount of AOX protein was greater. Addition of pyruvate to the isolation, washing and reaction media made mitochondria from +P roots cyanide-insensitive, similar to mitochondria from −P roots. The doubled activity of NAD-malic enzyme (NAD-ME) in −P compared with +P root mitochondria may suggest increased pyruvate production in −P mitochondria. Lower cytochrome c oxidase (COX) activity and no uncoupler effect on respiration indicated limited cytochrome chain activity in −P mitochondria. In −P mitochondria, the oxygen uptake decreased and the level of Q reduction increased from 60 to 80%. With no pyruvate present (AOX not fully activated), inhibition of the cytochrome pathway resulted in an increased level of the ratio of reduced ubiquinone (Qr) to total ubiquinone (Qt) (Qr/Qt) in +P mitochondria, but did not change Qr/Qt in −P mitochondria. When pyruvate was present, the kinetics for AOX were similar in mitochondria from −P and +P roots. It is suggested that AOX participation in −P respiration may provide an acclimation to phosphate deficiency. Stabilization of the ubiquinone reduction level by AOX might prevent the harmful effect of an increased formation of reactive oxygen species.     

Role of sugars and organic acids in regulating the concentration and activity of the alternative oxidase in Poa annua roots

Detached roots of Poa annua were used to study alternative oxidase protein expression upon the addition of sucrose, glucose, fructose, inositol, mannitol, citrate or malate, at a concentration of 1 or 10 mM for 24 h. After 24 h the capacity of cytochrome c oxidase was decreased equally in all treatments. Only citrate induced the expression of the alternative oxidase, especially at a concentration of 1 mM (15-fold). The activity of the alternative pathway (measured with the (18)O-fractionation technique) was not affected by the addition of sucrose for 24 h as compared with time zero. However, after the addition of citrate or mannitol the activity of the alternative pathway decreased to almost zero. The discrepancy between the large increase in alternative oxidase protein concentration when citrate was applied and the concomitant decrease in alternative pathway activity is discussed.     

The expression of alternative oxidase and uncoupling protein during fruit ripening in mango

The expression of alternative oxidase (Aox) and uncoupling proteins (Ucp) was investigated during ripening in mango (Mangifera indica) and compared with the expression of peroxisomal thiolase, a previously described ripening marker in mango. The multigene family for the Aox in mango was expressed differentially during ripening. Abundance of Aox message and protein both peaked at the ripe stage. Expression of the single gene for the Ucp peaked at the turning stage and the protein abundance peaked at the ripe stage. Proteins of the cytochrome chain peaked at the mature stage of ripening. The pattern of protein accumulation suggested that increases in cytochrome chain components played an important role in facilitating the climacteric burst of respiration and that the Aox and Ucp may play a role in post-climacteric senescent processes. Because both message and protein for the Aox and Ucp increased in a similar pattern, it suggests that their expression is not controlled in a reciprocal manner but may be active simultaneously.     

Oxygen concentration regulates EGF-induced proliferation and EGF-receptor down regulation

Reducing oxygen from 20% to 2.5% increases EGF-induced DNA synthesis and cell proliferation in cultures of human diploid fibroblasts. Reducing oxygen also changes the pattern of EGF binding to the cell surface. The loss of surface binding that follows EGF attachment to cells in 20% oxygen does not occur in 2.5% oxygen.     

Regulation of alternative oxidase gene expression in soybean

Soybean (Glycine max cv. Stevens) suspension cells were used to investigate the expression of the alternative oxidase (Aox) multigene family. Suspension cells displayed very high rates of cyanide-insensitive respiration, but Aox3 was the only isoform detected in untreated cells. Incubation with antimycin A, citrate, salicylic acid or at low temperature (10 °C) specifically induced the accumulation of the Aox1 isoform. Aox2 was not observed under any conditions in the cells. Increases in Aox1 protein correlated with increases in Aox1 mRNA. Treatment of soybean cotyledons with norflurazon also induced expression of Aox1. Reactive oxygen species (ROS) were detected upon incubation of cells with antimycin, salicylic acid or at low temperature, but not during incubation with citrate. Aox1 induction by citrate, but not by antimycin, was prevented by including the protein kinase inhibitor staurosporine in the medium. The results suggest that multiple pathways exist in soybean to regulate expression of Aox genes and that Aox1 specifically is induced by a variety of stress and metabolic conditions via at least two independent signal transduction pathways.     

cDNA cloning and expression analysis of a putative alternative oxidase HsAOX1 from wild barley (Hordeum spontaneum)

A novel alternative oxidase (AOX1) gene, designated HsAOX1 (GenBank accession number JF440341) was cloned by RT-PCR from wild barley (Hordeum spontaneum). The full length of HsAOX1 is 1115 bp with an open reading frame of 987 bp, encoding a protein of 328 amino acids with molecular weight of 36.89 kDa and a theoretical isoelectric point of 6.81. As found in other plant AOX1 proteins, sequence alignment showed that HsAOX1 had conserved metal binding and hydrophobic ??-helix regions and had high homology to other AOX1 in plants. The expression analysis by semi-quantitative RT-PCR revealed that HsAOX1 was induced in response to cold stress, H₂O₂ treatment, SA, antimycin A and KCN. These results showed that HsAOX1 functions not only during inhibition of cytochrome electron transport but also during oxidative stresses, thus suggesting a role of HsAOX1 in preventing the generation of free radicals by the mitochondrial electron transport chain. The cloning and characterization of the HsAOX1 gene will be useful for further studies of biological roles of HsAOX1 in plants.     

Endosomal NADPH oxidase regulates c-Src activation following hypoxia/reoxygenation injury

c-Src has been shown to activate NF-kappaB (nuclear factor kappaB) following H/R (hypoxia/reoxygenation) by acting as a redox-dependent IkappaBalpha (inhibitory kappaB) tyrosine kinase. In the present study, we have investigated the redox-dependent mechanism of c-Src activation following H/R injury and found that ROS (reactive oxygen species) generated by endosomal Noxs (NADPH oxidases) are critical for this process. Endocytosis following H/R was required for the activation of endosomal Noxs, c-Src activation, and the ability of c-Src to tyrosine-phosphorylate IkappaBalpha. Quenching intra-endosomal ROS during reoxygenation inhibited c-Src activation without affecting c-Src recruitment from the plasma membrane to endosomes. However, siRNA (small interfering RNA)-mediated knockdown of Rac1 prevented c-Src recruitment into the endosomal compartment following H/R. Given that Rac1 is a known activator of Nox1 and Nox2, we investigated whether these two proteins were required for c-Src activation in Nox-deficient primary fibroblasts. Findings from these studies suggest that both Nox1 and Nox2 participate in the initial redox activation of c-Src following H/R. In summary, our results suggest that Rac1-dependent Noxs play a critical role in activating c-Src following H/R injury. This signalling pathway may be a useful therapeutic target for ischaemia/reperfusion-related diseases.     

Oxygen control prevents denitrifiers and barley plant roots from directly competing for nitrate

Abstract Two denitrifying bacteria ( Pseudomonas chlororaphis and P. aureofaciens ) and a plant (barley, Hordeum vulgare ) were used to study the effect of O₂ concentration on denitrification and NO₃⁻ uptake by roots under well-defined aeration conditions. Bacterial cells in the early stationary phase were kept in a chemostat vessel with vigorous stirring and thus a uniform O₂ concentration in the solution. Both Pseudomonads lacked N₂O reductase and so total denitrification could be directly measured as N₂O production.
Denitrification decreased to 6–13% of the anaerobic rate at 0.01% O₂ saturation (0.14 μM O₂) and was totally inhibited at 0.04% O₂ saturation (0.56 μM O₂). In this well-mixed system denitrification was 10-times more oxygen sensitive than stated in earlier reports. Uptake of nitrate by plants was measured in the same system under light. The NO₃⁻ uptake rate decreased gradually from a maximum in 21% O₂-saturated medium (air saturated) to zero at 1.6% O₂ saturation (22.4 μM O₂). Owing to the very different non-overlapping oxygen requirements of the two processes, direct competition for nitrate between plant roots and denitrifying bacteria cannot occur.     

Aldehyde oxidase in roots, leaves and seeds of barley (Hordeum vulgare L.)

Aldehyde oxidase (AO, EC 1.2.3.1) proteins in leaves, roots and seeds of barley (Hordeum vulgare L.) plants were studied. Differences in substrate specificity and mobility in native PAGE between AO proteins extracted from roots, leaves and seeds have been observed. Four clear bands of AO reacting proteins were detected in barley plants capable of oxidizing a number of aliphatic and aromatic aldehydes such as indole-3-aldehyde, acetaldehyde, heptaldehyde, and benzaldehyde. Mouse polyclonal antibodies raised against purified maize AO cross-reacted with barley AO proteins extracted from roots, leaves and seeds. At least three different AO proteins were detected in roots on the basis of their mobility during PAGE after native Western blot analysis while in leaves and seeds only one polypeptide cross-reacted with the antibody. SDS-immunoblot analysis showed marked differences in molecular weight between subunits of the AO bands extracted from roots, leaves and seeds. Two distinct subunit bands were observed in roots, with relatively close molecular weights (160 kDa and 145 kDa), while a single subunit with a molecular weight of 150 kDa was observed in leaf and seed extracts.Menadione, a specific and potent inhibitor of animal AO did not affect barley AO proteins. Root and leaf AO differed in their thermostability and susceptibility to exogenous tungstate. The AO proteins in plants may be a group of enzymes with different substrate specificity, tissue distribution and presumably fulfilling different metabolic roles in each plant organ.     

Genomic structure and expression of alternative oxidase genes in legumes

Mitochondria isolated from chickpea (Cicer arietinum) possess substantial alternative oxidase (AOX) activity, even in non‐stressed plants, and one or two AOX protein bands were detected immunologically, depending on the organ. Four different AOX isoforms were identified in the chickpea genome: CaAOX1 and CaAOX2A, B and D. CaAOX2A was the most highly expressed form and was strongly expressed in photosynthetic tissues, whereas CaAOX2D was found in all organs examined. These results are very similar to those of previous studies with soybean and siratro. Searches of available databases showed that this pattern of AOX genes and their expression was common to at least 16 different legume species. The evolution of the legume AOX gene family is discussed, as is the in vivo impact of an inherently high AOX capacity in legumes on growth and responses to environmental stresses.     

Oxygen concentration regulates NO-dependent relaxation of aortic smooth muscles

Nitric oxide (NO) functions as an endothelium-derived relaxation factor and regulates vascular resistance. Recent studies in this laboratory (Arch. Biochem. Biophys. 323, 27–32, 1995) revealed that the lifetime of NO significantly increased at physiologically low levels of oxygen concentrations and, hence, this gaseous radical strongly inhibited mitochondrial electron transport for a fairly long duration at low oxygen concentrations. The present work describes the effect of oxygen concentration on NO-induced relaxation and guanylate cyclase (GC) activity of endothelium-denuded aorta of the rat. Both NO and 2,2′-hydroxynitrosohydrazono)bis-ethanamine (NOC18), an NO donor, induced the relaxa-tion of endothelium-denuded helical segments of rat aorta which were contracted by norepinephrine. NO-dependent relaxation of arterial specimens was enhanced by lowering oxygen concentration in the medium with concomitant increase in their cGMP levels. Anoxia induced the relaxation of the aorta by some NO-enhanceable and methylene blue-insensitive mechanism. These results suggested that local concentrations of oxygen might play important roles in the regulation of NO-dependent GC activity and vascular tonus of resistance arteries.     

Oxygen concentration regulates NO-dependent relaxation of aortic smooth muscles

Nitric oxide (NO) functions as an endothelium-derived relaxation factor and regulates vascular resistance. Recent studies in this laboratory (Arch. Biochem. Biophys. 323, 27-32, 1995) revealed that the lifetime of NO significantly increased at physiologically low levels of oxygen concentrations and, hence, this gaseous radical strongly inhibited mitochondrial electron transport for a fairly long duration at low oxygen concentrations. The present work describes the effect of oxygen concentration on NO-induced relaxation and guanylate cyclase (GC) activity of endothelium-denuded aorta of the rat. Both NO and 2,2'-hydroxynitrosohydrazono)bis-ethanamine (NOC18), an NO donor, induced the relaxa-tion of endothelium-denuded helical segments of rat aorta which were contracted by norepinephrine. NO-dependent relaxation of arterial specimens was enhanced by lowering oxygen concentration in the medium with concomitant increase in their cGMP levels. Anoxia induced the relaxation of the aorta by some NO-enhanceable and methylene blue-insensitive mechanism. These results suggested that local concentrations of oxygen might play important roles in the regulation of NO-dependent GC activity and vascular tonus of resistance arteries.     

Tissue-specific expression of the alternative oxidase in soybean and siratro

Alternative oxidase activity (cyanide-insensitive respiration) was measured in mitochondria from the shoots, roots, and nodules of soybean (Glycine max L.) and siratro (Macroptilium atropurpureum) plants. Activity was highest in the shoots and lowest in the nodules. Alternative oxidase activity was associated with one (roots) or two (shoots) proteins between 30 and 35 kilodaltons that were detected by western blotting with a monoclonal antibody against Sauromatum guttatum alternative oxidase. No such protein was detected in nodule mitochondria. Measurements of oxygen uptake by isolated soybean root and nodule cells in the presence of cyanide and salicylhydroxamic acid indicated that alternative oxidase activity was confined to the uninfected cortex cells of the nodule. Immunoprecipitation of translation products of mRNA isolated from soybean shoots revealed a major band at 43 kilodaltons that is assumed to be the precursor of an alternative oxidase protein. This band was not seen when mRNA from nodules was treated in the same fashion. The results indicate that tissue-specific expression of the alternative oxidase occurs in soybean and siratro.     

Aldehyde oxidase isoforms and subunit composition in roots of barley as affected by ammonium and nitrate

Aldehyde oxidase (AO; EC 1.2.3.1) isoforms in roots of barley plants grown on ammonium or nitrate as nitrogen sources were studied. Roots of ammonium-grown barley plants exhibited considerable levels of AO2, AO3, and AO4 activities after native PAGE. Significantly lower AO2 and AO3 activity bands were observed in roots of plants grown on nitrate. When abscisic aldehyde was used as a substrate a strong response of the AO2 band was observed as well as a faint reaction of the AO3 band, but no activity of AO4 was observed using this substrate. The 160 and 145 kDa polypeptides were detected in ammonium grown plants. Root extracts of nitrate-fed plants revealed only a minor 145 kDa protein band and none of the 160 kDa subunit was detected. The assembly of the AO3 heterodimer requires the simultaneous presence of 160 and 145 kDa subunits. Subunit analysis of AO2 and AO4 revealed homodimeric composition of 160 and 145 kDa, respectively. Western blot analysis revealed changing AO subunits levels during germination and plant development. Differential expression of AO subunits (160 and 145 kDa) and subsequent formation of isoforms, which differ in substrate specificity, distribution and fulfil different enzymatic reactions, may constitute an important regulatory mechanism in the plant.     

Oxygen transport during steady-state submaximal exercise in chronic hypoxia

Arterial O2 delivery during short-term submaximal exercise falls on arrival at high altitude but thereafter remains constant. As arterial O2 content increases with acclimatization, blood flow falls. We evaluated several factors that could influence O2 delivery during more prolonged submaximal exercise after acclimatization at 4,300 m. Seven men (23 +/- 2 yr) performed 45 min of steady-state submaximal exercise at sea level (barometric pressure 751 Torr), on acute ascent to 4,300 m (barometric pressure 463 Torr), and after 21 days of residence at altitude. The O2 uptake (VO2) was constant during exercise, 51 +/- 1% of maximal VO2 at sea level, and 65 +/- 2% VO2 at 4,300 m. After acclimatization, exercise cardiac output decreased 25 +/- 3% compared with arrival and leg blood flow decreased 18 +/- 3% (P less than 0.05), with no change in the percentage of cardiac output to the leg. Hemoglobin concentration and arterial O2 saturation increased, but total body and leg O2 delivery remained unchanged. After acclimatization, a reduction in plasma volume was offset by an increase in erythrocyte volume, and total blood volume did not change. Mean systemic arterial pressure, systemic vascular resistance, and leg vascular resistance were all greater after acclimatization (P less than 0.05). Mean plasma norepinephrine levels also increased during exercise in a parallel fashion with increased vascular resistance. Thus we conclude that both total body and leg O2 delivery decrease after arrival at 4,300 m and remain unchanged with acclimatization as a result of a parallel fall in both cardiac output and leg blood flow and an increase in arterial O2 content.(ABSTRACT TRUNCATED AT 250 WORDS)