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.     

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.     

HISTAMINE-SENSITIVE ADENYLATE CYCLASE IN HYPOTHALAMUS OF RAT BRAIN: H1 AND H2 RECEPTORS

Abstract— In in vitro experiments on rat hypothalamic homogenates the effects of biogenic amines such as histamine (HA), noradrenaline (NA), dopamine (DA), serotonin (5-HT) and drugs such as isoprenaline (ISP), 2-(2-pyridyl)ethylamine (H₂ stimulant—H_ls), 4-methyl-histamine (H₂ stimulant H_2s), mepyramine (H₁ antagonistp H_la), cimetidine (H₂ antagonist—H_2a) were tested on adenylate cyclase activity. HA possessed a powerful stimulating effect on hypothalamic adenylate cyclase activity, higher than that shown by the other substances.
The stimulating effect of HA was greatest in hypothalamic tissue from male rats, while tissue from females showed only a modest stimulation. H_2s, induced a greater stimulation of adenylate cyclase than H_ls. On the other hand, the H_2a inhibited HA stimulation to a greater extent than the H_la, H_la and H_2a, when used together, completely inhibited the HA stimulation. HA may have a neurotrans-mitter role in the hypothalamus, and in this area there appears to be a mixed population of H₁ and H₂ receptors, with a majority of H₂ receptors.     

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.     

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.     

Microbial H2/CO2 acetogenesis in animal guts: nature and nutritional significance

Abstract The intestinal tract of invertebrate and vertebrate animals, including man, is an anoxic habitat wherein microbial formation of acetate from H₂+ CO₂ is often a major H₂-consuming reaction. This paper will discuss the magnitude and microbiology of H₂/CO₂ acetogenesis in animal guts, its impact on host animal nutrition, competition for H₂ between anaerobic microbes, and the global significance of intestinal H₂/CO₂ acetogenesis.     

Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley—powdery mildew interaction

Active oxygen species (AOS) are believed to have important roles in plants in general and in plant—pathogen interactions in particular. They are believed to be involved in signal transduction, cell wall reinforcement, hypersensitive response (HR) and phytoalexin production, and to have direct antimicrobial effects. Since current methods are inadequate for localizing AOS in intact plant tissue, most studies have been conducted using cell suspension culture/elicitors systems. 3,3-diaminobenzidine (DAB) polymerizes instantly and locally as soon as it comes into contact with H₂O₂ in the presence of peroxidase, and it was found that, by allowing the leaf to take up this substrate, in-vivo and in-situ detection of H₂O₂ can be made at subcellular levels. This method was successfully used to detect H₂O₂ in developing papillae and surrounding haloes (cell wall appositions) and whole cells of barley leaves interacting with the powdery mildew fungus. Thus, H₂O₂ can be detected in the epidermal cell wall subjacent to the primary germ tube from 6 h after inoculation, and subjacent to the appressorium from 15 h. The earliest time point for observation of H₂O₂ in relation to epidermal cells undergoing HR is 15 h after inoculation, first appearing in the zones of attachment to the mesophyll cells underneath, and eventually in the entire epidermal cell. Furthermore, it was observed that proteins in papillae and HR cells are cross-linked, a process believed to be fuelled by H₂O₂. This cross-linking reinforces the apposition, presumably assisting the arrest of the pathogen.     

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.     

Phosphate Induces Rapid H2O2 Generation in Soybean Suspension Cells

Abstract: Involvement of reactive oxygen species has been implicated in plant defence against pathogens. We report here a novel pathway of H₂O₂ generation induced by the addition of phosphate in soybean ( Glycine max L.) cell suspension cultures. This H₂O₂ generation was initiated shortly after the addition of phosphate, and lasted only approximately one hour, as opposed to several hours observed during an attack by an avirulent strain of the bacterial pathogen Pseudomonas syringae pv. glycinea (Psg). In addition, when cell cultures were treated with both phosphate and the avirulent pathogen, two distinct oxidative burst events were observed. In contrast to DPI-sensitive Psg -induced H₂O₂ generation, phosphate-induced H₂O₂ generation was insensitive to this NADPH oxidase inhibitor. This suggests that an NADPH oxidase-independent pathway may be involved in the phosphate-induced H₂O₂ accumulation, which could be involved in sensing of phosphate availability in the environment.     

Acetogenesis from H2 and CO2 by methane- and non-methane-producing human colonic bacterial communities

Abstract: The purpose of the study was to define the potential for reductive acetogenesis of colonic microflora from six non-methane- and four methane-excreting human subjects in relation to numbers of the different H₂-utilizing microorganisms. Faecal bacterial suspensions were incubated in the presence of NaH¹³CO₃ and under a gas phase composed of either 100% N₂ (control) or 80% H₂–20% N₂. The effects of a specific methanogenesis inhibitor or of sulfate supplementation were also determined. Quantitative nuclear magnetic resonance showed the presence of both single- and double-labelled acetate in all incubations under hydrogen. H₂/CO₂-acetogenesis appears to be a quantitatively important activity only in the presence of very low numbers of methanogens. Inhibition of methanogenesis induced a large increase in ¹³CO₂ incorporation into acetate in CH₄-producing samples. These results showed that methanogens can efficiently outcompete acetogens in human colonic contents. In contrast, no clear-cut competition for H₂ between acetogenesis and dissimilatory sulfate-reduction could be demonstrated. A slight reduction of the acetogenic activity was only observed at the highest sulfate addition (100 mM).     

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.     

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).     

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.     

Production and detoxification of H2O2 in lettuce plants exposed to selenium

Selenium is considered an essential element for animals. Despite that it has not been demonstrated to be essential for higher plants, it has been attributed with a protective role against reactive oxygen species in plants subjected to stress. In this study, lettuce plants ( Lactuca sativa cv. Philipus) received different application rates (5, 10, 20, 40, 60, 80 and 120 μM) of selenite or selenate, with the aim of testing the effect of Se on the production and detoxification of H₂O₂ in non-stressed plants. The results indicate that the form selenate is less toxic than selenite; that is, the plants tolerated and responded positively to this element, and even increasing in growth up to a rate of 40 μM for the form selenate. On the contrary, the application of selenite triggered a higher foliar concentration of H₂O₂ and a higher induction of lipid peroxidation [malondialdehyde content and lipoxygenase activity] in comparison to that observed after the selenate application. Also, the plants treated with selenate induced higher increases in enzymes that detoxify H₂O₂, especially ascorbate peroxidase and glutathione (GSH) peroxidase, as well as an increase in the foliar concentration of antioxidant compounds such as ascorbate and GSH. These data indicate that an application of selenate at low rates can be used to prevent the induction in plants of the antioxidant system, thereby improving stress resistance.     

Translocation of precytochrome C2 into intracytoplasmic membrane vesicles of Rhodobacter capsulatus requires a peripheral membrane protein

Rhodobacter capsulatus is a member of the group α-purple bacteria which are closely related to the ancestral endosymbiont that gave rise to mitochondria. It has therefore been hypothesized that the molecular mechanisms governing protein export in α-purple bacteria have been conserved during the evolution of mitochondria. To enable analysis of protein export in α-purple bacteria we describe here the development of a homologous cell-free synthesis/export system consisting entirely of components of R. capsulatus. Translocation of precytochrome C₂ into intracytoplasmic membrane vesicles of this organism was found to require the proton-motive force and proceed at a significantly higher efficiency when membranes were present during protein synthesis. Furthermore, we show that, in this cell-free system, translocation depends on a preparation of peripheral membrane proteins Which do not possess detectable SecA- and SecB-like actvities.     

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.