Biological oxidation of hydrogen in soils flushed with a mixture of H2, CO2, O2 and N2

Abstract A stainless steel cylinder filled with soil was flushed upstream with a H₂/CO₂/air mixture. The consequence was a strong enrichment of the aerobic, autotrophic hydrogen-oxidising microflora, which reached densities enabling them to oxidize 84.5 ml H₂· dm⁻²· h⁻¹ in the first 25-cm layer. H₂ concentration profiles, hydrogen uptake activity and cell numbers correlated well with each other. Most of the organisms isolated were dinitrogen fixers. Thus, soils containing hydrogen-oxidising bacteria may act as a biological shield between H₂-rich environments and air, and may be utilized as biofilters, e.g., in the waste-processing industry.     

Effect of H2 evolution on 15N2 fixation, C2H2 reduction and relative efficiency of leguminous symbionts

The (C₂H₄+ H_2(C2H₂))/¹⁵N₂ ratios of 15 clover- Rhizobium symbionts. soybean, and black medick symbionts were measured. Relative efficiency based on the C2H4 production and on ¹⁵N₂ incorporation were compared, and in most symbionts there was little difference between the two measures of relative efficiency. Total measurable electron flux through nitrogenase during acetylene reduction and ¹⁵N₂ incorporation were nearly equal for most symbionts studied. The relative efficiency and the (C₂H₄+ H_2(C2H₂))/¹⁵N₂ ratio showed an inverse correlation. Use of this ratio appears preferable to use of the ratio of C2H2 reduction/N₂ reduction. Some evolution of H₂ was observed in the presence of C₂H₂.     

Relationship between C2H2 reduction, H2 evolution and 15N2 fixation in root nodules of pea (Pisum sativum)

The quantitative relationship between C₂H₂ reduction, H₂ evolution and ¹⁵N₂ fixation was investigated in excised root nodules from pea plants ( Pisum sativum L. cv. Bodil) grown under controlled conditions. The C₂H₂/N₂ conversion factor varied from 3.31 to 5.12 between the 32nd and the 67th day after planting. After correction for H₂ evolution in air, the factor (C₂H₂-H₂)/N₂ decreased to values near the theoretical value 3, or in one case to a value significantly ( P < 0.05) below 3. The proportion of the total electron flow through nitrogenase, which is not wasted in H₂ production but used for N₂ reduction, is often stated as the relative efficiency (1-H₂/C₂H₂). This factor varied significantly ( P < 0.05) during the growth period. The actual allocation of electrons to H₂ and N₂, expressed as the H₂/N₂ ratio, was independent of plant age, however. This discrepancy and the observation that the (C₂H₂-H₂)/N₂ conversion factor tended to be lower than 3, suggests that the C₂H₂reduction assay underestimates the total electron flow through nitrogenase.     

K+ATP channel opening prevents succinate-dependent H2O2 generation by plant mitochondria

The role of a recently identified K⁺_ATP channel in preventing H₂O₂ formation was examined in isolated pea stem mitochondria. The succinate-dependent H₂O₂ formation was progressively inhibited, when mitochondria were resuspended in media containing increasing concentration of KCl (from 0.05 to 0.15  M ). This inhibition was linked to a partial dissipation of the transmembrane electrical potential (ΔΨ) induced by KCl. Conversely, the malate plus glutamate-dependent H₂O₂ formation was not influenced. The succinate-sustained H₂O₂ generation was also unaffected by nigericin (a H⁺/K⁺ exchanger), but completely prevented by valinomycin (a K⁺ ionophore). In addition, cyclosporin A (a K⁺_ATP channel opener) inhibited this H₂O₂ formation, while ATP (an inhibitor of the channel opening) slightly increased it. The inhibitory effect of ATP was strongly stimulated in the presence of atractylate (an inhibitor of the adenine nucleotide translocase), thus suggesting that the receptor for ATP on the K⁺ channel faces the intermembrane space. Finally, the succinate-dependent H₂O₂ formation was partially prevented by phenylarsine oxide (a thiol oxidant).     

The oxidative stress response in Enterococcus faecalis: relationship between H2O2 tolerance and H2O2 stress proteins

The hydrogen peroxide (H₂O₂) stress response in Enterococcus faecalis ATCC19433 was investigated. A 2·4 mmol l⁻¹ H₂O₂ pretreatment conferred protection against a lethal concentration (45 mmol l⁻¹) of this agent. The relatively high concentrations of H₂O₂ used for adaptation and challenge treatments in Ent. faecalis emphasised the strong resistance towards oxidative stress in this species. Various stresses (NaCl, heat, ethanol, acidity and alkalinity) induced weak or strong H₂O₂ cross-protection. This paper describes the involvement of protein synthesis in the active response to lethal dose of H₂O₂, in addition to the impressive enhancement of synthesis of five H₂O₂ stress proteins. Combined results suggest that these proteins might play an important role in the H₂O₂ tolerance response.     

Tracer studies of gas circulation in Nuphar: 18O2 and 14CO2 transport

The objective of this study was to investigate the behaviour of different legumes against salinity and water stress, thus trying to discover simultaneous adaptations to both stresses. The nitrogen fixation, transpiration, predawn leaf water potential, and stomatal response of Medicago sativa L. (cvs. Tierra de Campos and Aragon), Trifolium repens L. (cv. Aberystwyth S-184) and T. brachycalycinum Katzn. et Morley (= T. subterraneum L. cv. Clare) were compared at three levels of stress (0.05, 0.3 and 0.5 MPa of either NaCl or polyethylene glycol 6000) in nutrient solution. The plants were stressed for three days and then returned to control nutrient solution. The changes in the parameters analyzed were dependent on the proportion of stress treatments and the nature of the species, always being greater in plants from PEG than from NaCl solutions. Transfer of lucerne and subclover plants from NaCl at 0.05 MPa to a non-saline medium resulted in an increase of nitrogen fixation above the level of the non-salinized control plants, especially significant in lucerne. Analysis of possible inorganic impurities in commercial PEG suggest that such type of impurities are not responsible for the toxic effects reported. Plant damage resulting from PEG treatment was apparently due to penetrations of PEG (as determined qualitatively by using the tetraiodinebismuthic acid technique) or low-molecular organic impurities into the plant. – The results are discussed as part of the adaptation of the different species to salinity and water stress. The best performance was given by "Tierra de Campos".     

Generation of H2O2 in Brain Mitochondria

Generation of H2O2 by rat brain mitochondria using succinate and glycerol-1-phosphate as substrates has been demonstrated. Earlier workers were unable to detect this activity in sucrose-Tris buffer. We found that this was due to a lag in the expression of activity in sucrose medium. Using phosphate buffer (50 mM), good rates are now obtained. Generation of H2O2 by rat brain mitochondria required the presence of antimycin A and was dependent on the substrates succinate and glycerol-1-phosphate. Low rates were obtained with NAD+-linked substrates and none with choline, glutamate, and NADH. The Km and Vmax values for H2O2 generation were considerably lower than the corresponding values for the respective dehydrogenase activity, measured by dye reduction. Oxygen-radical scavengers inhibited H2O2 generation, suggesting oxygen radical involvement. Depletion of ubiquinone from mitochondria resulted in loss of H2O2 generation. Reconstitution of such depleted particles with ubiquinone restored the capacity to generate H2O2 in a concentration-dependent manner. Levels of H2O2 production were found to be maximal in cerebellum. Brain mitochondria from rabbit, hamster, mouse, and guinea pig also have the capacity to generate H2O2 on oxidation of glycerol-1-phosphate.     

N2 fixation by Acacia species increases under elevated atmospheric CO2

In the present study the effect of elevated CO₂ on growth and nitrogen fixation of seven Australian Acacia species was investigated. Two species from semi‐arid environments in central Australia (Acacia aneura and A. tetragonophylla) and five species from temperate south‐eastern Australia (Acacia irrorata, A. mearnsii, A. dealbata, A. implexa and A. melanoxylon) were grown for up to 148 d in controlled greenhouse conditions at either ambient (350 µmol mol^?1) or elevated (700 µmol mol^?1) CO₂ concentrations. After establishment of nodules, the plants were completely dependent on symbiotic nitrogen fixation. Six out of seven species had greater relative growth rates and lower whole plant nitrogen concentrations under elevated versus normal CO₂. Enhanced growth resulted in an increase in the amount of nitrogen fixed symbiotically for five of the species. In general, this was the consequence of lower whole‐plant nitrogen concentrations, which equate to a larger plant and greater nodule mass for a given amount of nitrogen. Since the average amount of nitrogen fixed per unit nodule mass was unaltered by atmospheric CO₂, more nitrogen could be fixed for a given amount of plant nitrogen. For three of the species, elevated CO₂ increased the rate of nitrogen fixation per unit nodule mass and time, but this was completely offset by a reduction in nodule mass per unit plant mass.     

Elevated CO2 stimulates associative N2 fixation in a C3 plant of the Chesapeake Bay wetland

In this study, the response of N₂ fixation to elevated CO₂ was measured in Scirpus olneyi, a C₃ sedge, and Spartina patens, a C₄ grass, using acetylene reduction assay and ¹⁵N₂ gas feeding. Field plants grown in PVC tubes (25 cm long, 10 cm internal diameter) were used. Exposure to elevated CO₂ significantly (P < 0·05) caused a 35% increase in nitrogenase activity and 73% increase in ¹⁵N incorporated by Scirpus olneyi. In Spartina patens, elevated CO₂ (660 ± 1 μ mol mol ^{− 1}) increased nitrogenase activity and ¹⁵N incorporation by 13 and 23%, respectively. Estimates showed that the rate of N₂ fixation in Scirpus olneyi under elevated CO₂ was 611 ± 75 ng ¹⁵N fixed plant ^{− 1} h ^{− 1} compared with 367 ± 46 ng ¹⁵N fixed plant ^{− 1} h ^{− 1} in ambient CO₂ plants. In Spartina patens, however, the rate of N₂ fixation was 12·5 ± 1·1 versus 9·8 ± 1·3 ng ¹⁵N fixed plant ^{− 1} h ^{− 1} for elevated and ambient CO₂, respectively. Heterotrophic non-symbiotic N₂ fixation in plant-free marsh sediment also increased significantly (P < 0·05) with elevated CO₂. The proportional increase in ¹⁵N₂ fixation correlated with the relative stimulation of photosynthesis, in that N₂ fixation was high in the C₃ plant in which photosynthesis was also high, and lower in the C₄ plant in which photosynthesis was relatively less stimulated by growth in elevated CO₂. These results are consistent with the hypothesis that carbon fixation in C₃ species, stimulated by rising CO₂, is likely to provide additional carbon to endophytic and below-ground microbial processes.     

Subdivision of equine Tf into H1 and H2

Subdivision of equine TfH into two variants, designated H1 (faster) and H2 (slower), has been accomplished by high voltage, thin layer polyacrylamide gel electrophoresis at pH 7.9. Transferrin H1 and H2 have been shown to be controlled by codominant alleles and gene frequencies of the Tf alleles have been determined in the Australian Thoroughbred, Standardbred. Quarter Horse and Arabian Horse breeds.     

Plant responses to H2S and SO2 fumigation. II. Differences in metabolism of H2S and SO2 in spinach

Fumigation of spinach (Spinacia oleracea L. cvs Estivato and Monosa) with H₂S or SO, for 1 to 6 days resulted in accumulation of sulfhydryl (SH) compounds in the shoots of both H₂S- and SO₂-exposed plants. The sulfate concentration in shoots of SO₂-exposed plants increased linearly with time. SH accumulation showed saturation kinetics as a function of time as well as H₂S concentration, ascribed to the internal H₂S concentration in the plant and the availability of substrates for glutathione synthesis, respectively. SH compounds accumulated more at lower exposure temperatures, whereas sulfate accumulation was more pronounced at higher temperatures. These results are discussed in relation to the possible foliar uptake of H₂S and SO₂, the temperature dependence of uptake and the water solubility of these gases. The possibility of SO₂-induced H₂S emission rather than sulfate accumulation as a source for SH accumulation is also discussed. Cessation of fumigation resulted in a decrease in SH compounds and sulfate content that could be accounted for by sulfur metabolism and growth, respectively.     

B1 and B2 Bradykinin Receptors Encoded by Distinct mRNAs

Abstract: Bradykinin receptors have been subdivided into at least two major pharmacological subtypes, B₁ and B₂. The cDNAs encoding functional B₂ receptors have recently been cloned, but no molecular information exists at present on the B₁ receptor. In this article, we describe experiments examining the possible relationship between the mRNAs encoding the B₁ and B₂ types of receptor. We showed previously that the Human fibroblast cell line W138 expresses both B₁ and B₂ receptors. In this report, we describe oocyte expression experiments showing that the B₁ receptor in W138 human fibroblast cells is encoded by a distinct mRNA ∼2 kb shorter than that encoding the B₂ receptor. We have used an antisense approach in conjunction with the oocyte expression system to demonstrate that the two messages differ in sequence at several locations throughout the length of the B₂ sequence. Taken together with the mixed pharmacology exhibited in some expression systems by the cloned mouse receptor, the data indicate that B₁-type pharmacology may arise from two independent molecular mechanisms.     

P2X2, P2X2–2 and P2X5 receptor subunit expression and function in rat thoracolumbar sympathetic neurons

The present study investigated the pharmacological properties of excitatory P2X receptors and P2X(2) and P2X(5) receptor subunit expression in rat-cultured thoracolumbar sympathetic neurons. In patch-clamp recordings, ATP (3-1000 microM; applied for 1 s) induced inward currents in a concentration-dependent manner. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS; 30 microM) counteracted the ATP response. In contrast to ATP, alpha,beta-meATP (30 microM; for 1 s) was virtually ineffective. Prolonged application of ATP (100 microM; 10 s) induced receptor desensitization in a significant proportion of sympathetic neurons in a manner typical for P2X(2-2) splice variant-mediated responses. Using single-cell RT-PCR, P2X(2), P2X(2-2) and P2X(5) mRNA expression was detectable in individual tyrosine hydroxylase-positive neurons; coexpression of both P2X(2) isoforms was not observed. Laser scanning microscopy revealed both P2X(2) and P2X(5) immunoreactivity in virtually every TH-positive neuron. P2X(2) immunoreactivity was largely distributed over the cell body, whereas P2X(5) immunoreactivity was most distinctly located close to the nucleus. In summary, the present study demonstrates the expression of P2X(2), P2X(2-2) and P2X(5) receptor subunits in rat thoracolumbar neurons. The functional data in conjunction with a preferential membranous localization of P2X(2)/P2X(2-2) compared with P2X(5) suggest that the excitatory P2X responses are mediated by P2X(2) and P2X(2-2) receptors. Apparently there exist two types of P2X(2) receptor-bearing sympathetic neurons: one major population expressing the unspliced isoform and another minor population expressing the P2X(2-2) splice variant.     

Stomatal acclimation over a subambient to elevated CO2 gradient in a C3/C4 grassland

An investigation to determine whether stomatal acclimation to [CO₂] occurred in C₃/C₄ grassland plants grown across a range of [CO₂] (200–550 µmol mol^?1) in the field was carried out. Acclimation was assessed by measuring the response of stomatal conductance (g_s) to a range of intercellular CO₂ (a g_s–C_i curve) at each growth [CO₂] in the third and fourth growing seasons of the treatment. The g_s–C_i response curves for Solanum dimidiatum (C₃ perennial forb) differed significantly across [CO₂] treatments, suggesting that stomatal acclimation had occurred. Evidence of non–linear stomatal acclimation to [CO₂] in this species was also found as maximum g_s (g_smax; g_s measured at the lowest C_i) increased with decreasing growth [CO₂] only below 400 µmol mol^?1. The substantial increase in g_s at subambient [CO₂] for S. dimidiatum was weakly correlated with the maximum velocity of carboxylation (V_cmax; r² = 0·27) and was not associated with CO₂ saturated photosynthesis (A_max). The response of g_s to C_i did not vary with growth [CO₂] in Bromus japonicus (C₃ annual grass) or Bothriochloa ischaemum (C₄ perennial grass), suggesting that stomatal acclimation had not occurred in these species. Stomatal density, which increased with rising [CO₂] in both C₃ species, was not correlated with g_s. Larger stomatal size at subambient [CO₂], however, may be associated with stomatal acclimation in S. dimidiatum. Incorporating stomatal acclimation into modelling studies could improve the ability to predict changes in ecosystem water fluxes and water availability with rising CO₂ and to understand their magnitudes relative to the past.     

Cadmium-induced subcellular accumulation of O2·− and H2O2 in pea leaves

Cadmium is a toxic metal that produces disturbances in plant antioxidant defences giving rise to oxidative stress. The effect of this metal on H₂O₂ and O₂^·? production was studied in leaves from pea plants growth for 2 weeks with 50 µm Cd, by histochemistry with diaminobenzidine (DAB) and nitroblue tetrazolium (NBT), respectively. The subcellular localization of these reactive oxygen species (ROS) was studied by cytochemistry with CeCl₃ and Mn/DAB staining for H₂O₂ and O₂^·?, respectively, followed by electron microscopy observation. In leaves from pea plants grown with 50 µm CdCl₂ a rise of six times in the H₂O₂ content took place in comparison with control plants, and the accumulation of H₂O₂ was observed mainly in the plasma membrane of transfer, mesophyll and epidermal cells, as well as in the tonoplast of bundle sheath cells. In mesophyll cells a small accumulation of H₂O₂ was observed in mitochondria and peroxisomes. Experiments with inhibitors suggested that the main source of H₂O₂ could be a NADPH oxidase. The subcellular localization of O₂^·? production was demonstrated in the tonoplast of bundle sheath cells, and plasma membrane from mesophyll cells. The Cd‐induced production of the ROS, H₂O₂ and O₂^·?, could be attributed to the phytotoxic effect of Cd, but lower levels of ROS could function as signal molecules in the induction of defence genes against Cd toxicity. Treatment of leaves from Cd‐grown plants with different effectors and inhibitors showed that ROS production was regulated by different processes involving protein phosphatases, Ca²⁺ channels, and cGMP.