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).     

OPTIMIZATION OF CULTURE CONDITIONS FOR ENUMERATION OF H2S BACTERIA IN THE FLESH OF SEAFISH

H₂S bacteria of seafish flesh are weakly halophilic and require on average 1.68% NaCl according to statistical studies. Enumeration is optimal on PCA-H₂S(a PCA medium supplemented with sulfur sources and increased NaCl concentrations) incubated at 25C. Total aerobic bacteria can be counted simultaneously on this medium. The proportion of H₂S bacteria relative to total aerobic bacteria increased slightly during prolonged storage of the fish, but was highly variable. Models relating H₂S bacterial counts to spoilage of fish are sigmoidal and showed that when the count exceeds 10,000 CFU/g, whole or filleted fish stored in ice at 0C are unfit for consumption. Shewanella putrefaciens accounted for 69% of the H₂S bacteria at the fifth day of storage and 100% at the fifteenth.     

Tyrosine Hydroxylase Phosphorylation in Bovine Adrenal Chromaffin Cells: The Role of Intracellular Ca2+ in the Histamine H1 Receptor-Stimulated Phosphorylation of Ser8, Ser19, Ser31, and Ser40

Abstract: Tyrosine hydroxylase (TOH), the rate-limiting enzyme in catecholamine biosynthesis, is regulated by phosphorylation. Activation of histaminergic H₁ receptors on cultured bovine adrenal chromaffin cells stimulated a rapid increase in TOH phosphorylation (within 5 s) that was sustained for at least 5 min. The initial increase in TOH phosphorylation (up to 1 min) was essentially unchanged by the removal of extracellular Ca²⁺. In contrast, the H₁-mediated response was abolished by preloading the cells with BAPTA acetoxymethyl ester (50 µ M ) and significantly reduced by prior exposure to caffeine (10 m M for 10 min) to deplete intracellular Ca²⁺. Trypticphosphopeptide analysis by HPLC revealed that the H₁ response in the presence or absence of extracellular Ca²⁺ resulted in a major increase in the phosphorylation of Ser¹⁹ with smaller increases in that of Ser⁴⁰ and Ser³¹. In contrast, although a brief stimulation with nicotine (30 µ M for 60 s) also resulted in a major increase in Ser¹⁹ phosphorylation, this response was abolished in the absence of extracellular Ca²⁺. These data indicate that the mobilization of intracellular Ca²⁺ plays a crucial role in supporting H₁-mediated TOH phosphorylation and may thus have a potentially important role in regulating catecholamine synthesis.     

Ca2+/Calmodulin-Mediated Regulation of Agonist-Induced Sequestration of Gq Protein-Coupled Histamine H1 Receptors in Human U373 MG Astrocytoma Cells

Abstract: We investigated the regulation by intracellular Ca²⁺ of agonist-induced sequestration of G_q protein-coupled histamine H₁ receptors in human U373 MG astrocytoma cells. Histamine-induced sequestration of H₁ receptors from the cell surface membrane was detected as the loss of [³H]mepyramine binding sites on intact cells accessible to the hydrophilic H₁-receptor antagonist pirdonium. The changes in the pirdonium-sensitive binding of [³H]mepyramine were mirrored by changes in the subcellular distribution of H₁ receptors detected by sucrose density gradient centrifugation. The histamine-induced sequestration of H₁ receptors did not occur in hypertonic medium, in which clathrin-mediated endocytosis is known to be inhibited, but was significantly accelerated in the absence of extracellular Ca²⁺ or in the presence of the calmodulin antagonists W-7 and calmidazolium. Inhibitors of protein kinase C (H-7 and GF109203X), Ca²⁺/calmodulin-dependent protein kinase II (KN-62), or protein phosphatase 2B (FK506) did not alter the time course of H₁-receptor sequestration. These results provide the first evidence that agonist-induced, clathrin-mediated sequestration of G_q protein-coupled receptors is transiently inhibited by Ca²⁺/calmodulin, with the result that receptors remain on the cell surface membrane during the early stage of agonist stimulation.     

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.     

Most probable number enumeration of H2-utilizing acetogenic bacteria from the digestive tract of animals and man

Abstract A method is proposed that allows the enrichment and most probable number estimation of H₂/CO₂-utilizing acetogenic bacteria. It is based on the difference in acetate production for serial dilutions incubated under either a test H₂/CO₂ (4:1), or a control N₂/CO₂ (4:1) headspace atmosphere. A nutritionally non-selective medium was used, containing bromoethane-sulfonic acid as inhibitor of methanogenic archaea and 10% pre-incubated clarified rumen fluid. Acetogenic bacteria were enumerated in rumen and hindgut contents of animals and in human feces. They ranged from below 10² to above 10⁸ per gram wet weight gut content and their population levels were the highest in the absence of methanogenesis. The method described therein should prove useful to better understand the diversity and ecological importance of dominant gut acetogens.     

Hydrogen Peroxide Induces a Long-Lasting Inhibition of the Ca2+-Dependent Glutamate Release in Cerebrocortical Synaptosomes Without Interfering with Cytosolic Ca2+

Abstract: We studied the action of H₂O₂ on the exocytosis of glutamate by cerebrocortical synaptosomes. The treatment of synaptosomes with H₂O₂ (50–150 µ M ) for a few minutes results in a long-lasting depression of the Ca²⁺-dependent exocytosis of glutamate, induced by KCl or by the K⁺-channel inhibitor 4-aminopyridine. The energy state of synaptosomes, as judged by the level of phosphocreatine and the ATP/ADP ratio, was not affected by H₂O₂, although a transient decrease was observed after the treatment. H₂O₂ did not promote peroxidation, as judged by the formation of malondialdehyde. In indo-1-loaded synaptosomes, the treatment with H₂O₂ did not modify significantly the KCl-induced increase of [Ca²⁺]_i. H₂O₂ inhibited exocytosis also when the latter was induced by increasing [Ca²⁺]_i with the Ca²⁺ ionophore ionomycin. The effects of H₂O₂ were unchanged in the presence of superoxide dismutase and the presence of the Fe³⁺ chelator deferoxamine. These results appear to indicate that H₂O₂, apparently without damaging the synaptosomes, induces a long-lasting inhibition of the exocytosis of glutamate by acting directly on the exocytotic process.     

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.     

Effects of NH+4-N and NO+3-N on growth and metabolism of Sphagnum magellanicum

Photosynthetic CO₂-fixation, chlorophyll content, growth rate and nitrate reductase activity were used to examine the influence of NH⁺₄-N and NO⁻₃-N on Sphagnum magellanicum cultivated under defined conditions in phytotrons. NO⁻₃-concentrations up to 322 μ M were found to be favourable. Increased NH⁺₄ concentrations, however, resulted in growth inhibition and decreased chlorophyll content at concentrations ≧ 255 μ M ; e.g. 600 μ M NH⁺₄ caused a 20% reduction of nitrate reductase activity and net photosynthesis. For raised bog Sphagna an improved standard nutrient solution is proposed with the following ion concentrations (μ M ): 55 Na⁺; 17 K⁺; 95 NH⁺₄; 22 Ca²⁺; 22 Mg²⁺; 2 Fe³⁺; 20 Cl⁻; 100 NO⁻₃; 57 SO^2-₄; 7.4 H₂PO⁻₄; trace elements: A-Z solution (Hoagland) 50 μl 1000 ml⁻¹; pH 5.8.     

Ferric iron-reducing Shewanella putrefaciens and N2-fixing Bradyrhizobium japonicum with uptake hydrogenase are unable to oxidize atmospheric H2

Abstract Bradyrhizobium japonicum and Shewanella putrefaciens were unable to oxidize hydrogen at atmospheric concentrations (0.55 ppmv), neither in suspension nor when added to sterile soil. The K _m-value of S. putrefaciens for H₂ (39 ppmv in gas phase, 0.22 μM in aqueous phase), using Fe(III) as electron acceptor, showed a 4–5-fold higher affinity for H₂ than that of B. japonicum (1200 ppmv; 0.84 μM) or other hydrogen-oxidizing bacteria. However, the V _max (4.54 fmol H₂ h⁻¹ cell ⁻¹) and threshold (> 0.5 ppmv; 0.35 nM) of S. putrefaciens and the V _max (7.19 fmol H₂ h⁻¹ cell⁻¹) and threshold (> 0.5 ppmv; 0.35 nM) of B. japonicum were in the same order of magnitude as data for Knallgas bacteria from relevant literature. To enable hydrogen oxidation in soil the soil-samples with S. putrefaciens even had to be supplemented with Fe(III). Fresh soil, on the other hand, oxidized hydrogen very efficiently below atmospheric mixing ratios, demonstrating that there must be other oxidation activities in soil.     

The regulation of Mn2+ and Cu2+ uptake in cells of the yeast Candida utilis grown in continuous culture

Abstract Transport of Mn²⁺ was repressed in Candida utilis cells grown in continuous culture in high-Mn²⁺ (100 μM Mn²⁺) medium as compared to cells grown in basic (0.45 μM Mn²⁺) and low-Mn²⁺ (< 0.05 μM Mn²⁺) media. In contrast, no repression of Cu²⁺ uptake occurred in high-Cu²⁺-grown (25 μM Cu²⁺) cells as compared to cells grown in basic medium (0.54 μM Cu²⁺). Cu²⁺-limited cells did not hyperaccumulate Cu²⁺ and there was not significant difference in initial uptake rates for all 3 Cu²⁺ conditions. Mn²⁺ uptake appears to be regulated by a mechanism sensitive to the external Mn²⁺ concentration, whereas Cu²⁺ transport is not governed in this way by the external Cu²⁺.     

Early Events in Free Radical-Mediated Damage of Isolated Nerve Terminals: Effects of Peroxides on Membrane Potential and Intracellular Na+ and Ca2+ Concentrations

Abstract: The effects of peroxides were investigated on the membrane potential, intracellular Na⁺ ([Na⁺]_i) and intracellular Ca²⁺ ([Ca²⁺]_i) concentrations, and basal glutamate release of synaptosomes. Both H₂O₂ and the organic cumene hydroperoxide produced a slow and continuous depolarization, parallel to an increase of [Na⁺]_i over an incubation period of 15 min. A steady rise of the [Ca²⁺]_i due to peroxides was also observed that was external Ca²⁺ dependent and detected only at an inwardly directed Ca²⁺ gradient of the plasma membrane. These changes did not correlate with lipid peroxidation, which was elicited by cumene hydroperoxide but not by H₂O₂. Resting release of glutamate remained unchanged during the first 15 min of incubation in the presence of peroxides. These alterations may indicate early dysfunctions in the sequence of events occurring in the nerve terminals in response to oxidative stress.     

H2 and CO2 production from methanol or formaldehyde by the methanogenic bacterium strain Gö1 treated with 2-bromoethanesulfonic acid

Abstract In cell suspensions of the methanogenic bacterium strain Gö1 or Methanosarcina barkeri H₂ formation from methanol in the presence of 2-bromoethanesulfonic acid (BES) was strictly dependent on sodium ions; apparent K _S for Na⁺, 1.3±0.3 mM.H₂ formation was inhibited by the uncoupler tetrachlorosalicylanilide (TCS), but this inhibition could be temporarily overcome, when a sodium pulse (100 mM) was given to the cell suspension. On the other hand, H₂ formation from formaldehyde in the presence of BES (rate: 300 nmol H₂/h·mg protein as compared to 25 nmol H₂/h·mg protein from methanol) was not sodium-dependent, not TCS-sensitive and not inhibited by addition of monensin. H₂ formation was accompanied by CO₂ formation in stoichiometric amounts, 3 H₂:1 CO₂ for methanol and 2 H₂:1 CO₂ for formaldehyde oxidation.     

Differentiated U937 cells and human monocytes exhibit a differential production of extracellular oxygen species: O2•− excretion versus H2O2 diffusion

Abstract The nature and the localization of the oxidative response triggered by different stimuli in either differentiated U937 cells and peripheral blood-derived human monocytes was investigated using luminometric and cytofluorometric techniques. Differentiated U937 cells essentially produced extracellular superoxide anion (O₂^•−), whatever the stimulus used. Monocytes, however, responded to Salmonella typhimurium , phorbol esters, and opsonized zymosan by an intracellular, an extracellular, and both an intra- and extracellular production of oxygen species, respectively. Furthermore, H₂O₂ but not O₂^•− was detected in the extracellular oxidative response of monocytes. Using differentiated U937 cells, luminol was found to be as efficient as lucigenin in the detection of extracellular O₂^•−, providing sufficient concentrations of extracellular horseradish peroxidase were present. However, both azide and histidine inhibited the lucigenin-enhanced chemiluminescence, suggesting an initial and transient production of singlet oxygen differentiated U937 cells. Taken together these results strongly suggest that, when stimulated, differentiated U937 cells directly excrete O₂^•− in the extracellular medium while, within monocytes, O₂^•− is rapidly dismutated in H₂O₂ which can eventually diffuse outside the cell. Such differences in the oxidative response between the two cell types could be explained by the lack of total closure of the phagosome, only observed in differentiated U937 cells.     

Ascorbate-Induced Oxidative Inactivation of Zn2+-Glycerophosphocholine Cholinephosphodiesterase

Abstract: Zn²⁺-glycerophosphocholine cholinephosphodiesterase, responsible for the conversion of glycerophosphocholine into glycerol and phosphocholine, was inactivated during incubation with ascorbic acid at 38°C. The inclusion of copper ions or Fe²⁺ accelerated the ascorbate-induced inactivation, with Cu²⁺ or Cu⁺ being much more effective than Fe²⁺, suggestive of ascorbate-mediated oxidation. Dehydroascorbic acid had no effect on the phosphodiesterase, but H₂O₂ inactivated the enzyme in a concentration-dependent manner. Also, the enzyme was inactivated partially by a superoxide anion-generating system but not an HOCl generator. In support of involvement of H₂O₂ in the ascorbate action, catalase and superoxide dismutase expressed a complete and a partial protection, respectively. However, hydroxy radical scavengers such as mannitol, benzoate, or dimethyl sulfoxide were incapable of preventing the ascorbate action, excluding the participation of extraneous ^•OH. Although p -nitrophenylphosphocholine exhibited a modest protection against the ascorbate action, a remarkable protection was expressed by amino acids, especially by histidine. In addition, imidazole, an electron donor, showed a partial protection. Separately, when Cu²⁺-induced inactivation of the phosphodiesterase was compared with the ascorbate-mediated one, the protection and pH studies indicate that the mechanism for the ascorbate action is different from that for the Cu²⁺ action. Here, it is proposed that Zn²⁺-glycerophosphocholine cholinephosphodiesterase is one of brain membrane proteins susceptible to oxidative inactivation.     

Cysteine-induced H2S emission, ATP depletion, and membrane electrical responses in oat coleoptiles

Incubation of oat ( Avena sativa L. cv. Victory) coleoptile segments in 4 m M L-cysteine reduced the tissue ATP level to 42 nmol ( g fresh weight)⁻¹ (35% of normal) over a 2 h period. Emissions of H₂S accompanied this depletion in ATP suggesting an H₂S production by desulfhydration of cysteine similar to that reported in other plants. Additions of exogenous H₂S to the sections also caused ATP depletion. Aminooxyacetate, an inhibitor of cysteine desulfhydrase (EC 4.4.1.1), eliminated the cysteine-induced H₂S emission and the ATP depletion. Prolonged exposure to cysteine depressed the electrical polarity of the cell membrane from – 116 mV to –85 mV. That and other electrical responses appear to reflect a reduced capacity for ATP-dependent H⁺ extrusion. These effects should be taken into account whenever cysteine is used in physiological experiments.     

Inhibitory Effect of Omeprazole, a Specific Inhibitor of H+, K+-ATPase, on Spicule Formation in Sea Urchin Embryos and in Cultured Micromere-Derived Cells.

Embryos kept with omeprazole, a specific H⁺, K⁺-ATPase inhibitor, in a period of development between the mesenchyme blastula and the pluteus corresponding stage became abnormal plutei having quite small spicules, somewhat poor pluteus arms and apparently normal archenterons. In micro-mere-derived cells, kept with omeprazole at pH 8.2 in a period between 15 and 40 hr of culture at 20°C, omeprazole strongly inhibited spicule formation but did not block the outgrowth of pseudopodial cables, in which spicule rods were to be formed. These indicate that omeprazole probably exerts no obvious inhibitory effects other than spicule rods formation. Omeprazole-sensitive H⁺, K⁺-ATPase, an H⁺pump, seems to be indispensable for CaCO₃ deposition (formation of spicule rod) in these spicule forming cells. H⁺, produced in overall reaction for CaCO₃ formation: Ca²⁺+ CO₂+H₂O°CaCO₃+2H⁺, is probably released from the cells by this H⁺pump and hence, this reaction tends to go to CaCO₃ production to form spicule rods. Omeprazole, known to become effective following its conversion to a specific inhibitor of H⁺, K⁺-ATPase at acidic pH, is able to inhibit formation of spicule rod at alkaline pH in sea water. This is probably due to an acidification of sea water near the cell surface by H⁺ejection in H⁺, K⁺-ATPase reaction.     

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.     

Relative importance of Na+ and Cl– in NaCl-induced antioxidant systems in roots of rice seedlings

The present study examined the response of antioxidant systems to NaCl stress and the relative importance of Na⁺ and Cl^– in NaCl-induced antioxidant systems in roots of rice seedlings. NaCl treatment caused an increase in the activities of ascorbate peroxidase (APX) and glutathione reductase (GR) in roots of rice seedlings, but had no effect on the activities of superoxide dismutase (SOD) and catalase (CAT). There were detectable differences in APX and GR isoenzymes between control and NaCl-treated roots. Levels of activity for SOD and CAT isoenzymes did not change in NaCl-stressed roots compared with the control roots. NaCl treatment produced an increase in H₂O₂, ascorbate (AsA), dehydro-ascorbate (DHA), reduced glutathione (GSH), and oxidized glutathione (GSSG) levels. Treatment with 50 m M Na-gluconate (whose anion is not permeable to membrane) led to a similar Na⁺ level in roots to that with 100 m M NaCl. It was found that treatment with 50 m M Na-gluconate affected H₂O₂, AsA, and DHA levels, APX and GR activities, OsAPX and OsGR mRNA induction in the same way as 100 m M NaCl. These observed changes seem to be mediated by Na⁺ toxicity and not by Cl^– toxicity. On the other hand, it was found that NaCl, but not Na-gluconate and NaNO₃, caused an increase in GSH and GSSG levels, indicating that Cl^–, rather than Na⁺, is responsible for the NaCl-increased GSH and GSSG levels in roots of rice seedlings.     

Excretion of ions (Cd2+, Li+, Na+ and Cl−) by Tamarix aphylla

Excretion of minerals by the NaCl-resistant and comparatively cadmium-resistant tree Tamarix aphylla (L.) Karst, was investigated. Cd²⁺ was excreted by plants exposed for 1–10 days to 9 or 45 μ M Cd²⁺ solutions. Excretion of this toxic ion increased considerably with time but was less than 5% of the quantities that had been accumulated in the shoots. Excretion of Na⁺ and Cl⁻ was positively correlated with NaCl concentration (1.5, 10, 50 m M ) of the medium. The Na⁺/Cl⁻ ratios of the excrete were positively correlated with the concentration of the treatment solution. Ca²⁺ excretion decreased with increasing NaCl concentrations of the solution. Excretion of K⁺ and Mg²⁺ was only little affected by NaCl. Excretion of Li⁺ occurred whenever this element was supplied in the uptake solution; daily excretion rates of Li⁺ increased with time. The ecological significance of excretion is discussed in relation to the low selectivity of the mechanism in T. aphylla .