Involvement of prostaglandin E receptor EP3 subtype in duodenal bicarbonate secretion in rats

We investigated the involvement of prostaglandin E (PGE) receptor subtype EP3 in the regulatory mechanism of duodenal HCO₃⁻ secretion in rats. A proximal duodenal loop or a chambered stomach was perfused with saline, and HCO₃⁻ secretion was measured using a pH-stat method and by adding 2 mM HCl. Mucosal acidification was achieved through 10 min of exposure to 10 mM HCl in the duodenum or 100 mM HCl in the stomach. Various EP agonists or the EP4 antagonist were given i.v., while the EP1 or EP3 antagonist was given s.c. or i.d., respectively. Sulprostone (EP1/EP3 agonists) stimulated duodenal HCO₃⁻ secretion in a dose-dependent manner, and this response was inhibited by AE5-599 (EP3 antagonist) but not AE3-208 (EP4 antagonist). AE1-329 (EP4 agonist) also increased duodenal HCO₃⁻ secretion, and this action was inhibited by AE3-208 but not AE5-599. The response to PGE₂ or acidification in the duodenum was partially attenuated by AE5-599 or AE3-208 alone but completely abolished by the combined administration. Duodenal damage caused by mucosal perfusion with 150 mM HCl for 4 h was worsened by pretreatment with AE5-599 and AE3-208 as well as indomethacin and further aggravated by co-administration of these antagonists. Neither the EP3 nor EP4 antagonist had any effect on the gastric response induced by PGE₂ or acidification. These results clearly demonstrate the involvement of EP3 receptors, in addition to EP4 receptors, in the regulation of duodenal HCO₃⁻ secretion as well as the maintenance of the mucosal integrity of the duodenum against acid injury.     

Effects of anions, acetazolamide, thiocyanate and amiloride on fluid secretion by the Malpighian tubules of Locusta migratoria L.

The effect of Cl⁻, HCO₃⁻, Br⁻, acetazolamide, thiocyanate and amiloride on urine formation in Locusta migratoria Malpighian tubules have been determined. The rate of fluid secretion depends markedly on the concentration of Cl⁻ in the bathing solution with concentrations less than 90 mM resulting in reduced fluid secretion. Substitution of Br⁻ for Cl⁻ had no significant effect on the rate of the fluid secretion. Replacement of NaHCO₃ with NaCl in Hepes buffered Ringer solution reduced the rate of urine production by 23%. Fluid secretions were reduced in the presence of 10⁻⁴–10⁻² M acetazolamide, a carbonic anhydrase inhibitor. The combined effect of acetazolamide in the absence of HCO₃⁻ appears to be additive. A 1 mM concentration of thiocyanate, an ionic inhibitor, reduced fluid secretion by 35%. Amiloride interferes with the electrogenic entry of Na⁺ into the cell and a 1 mM solution reduced fluid secretion by 94% with secretion completely inhibited in 80% of the tubules tested.     

Characterization of the particulate nitrite oxidase and its component activities from the chemoautotroph Nitrobacter agilis

1. Difference spectra, at room and liquid N₂ temperatures, of S₂O₄²⁻-, and NO₂⁻-reduced intact cells and cell-free preparations of Nitrobacter agilis demonstrated the presence of cytochromes of the c- and a-types. Reduction of cytochromes by succinate, and to a limited extent, by NADPH also occurred, provided KCN (0.1 mM) was also present.

2. A particulate, heat-labile nitrite oxidase having an absolute requirement for O₂ was prepared from N. agilis cells using sonic oscillation and differential centrifugation. The particles also possessed NADH oxidase, succinoxidase, formate oxidase and traces of NADPH oxidase activity. The stoichiometry of the nitrite oxidase reaction approached the theoretical value of 2 moles of NO₂⁻ consumed per mole of O₂ consumed. The pH optimum of the nitrite oxidase system shifted to progressively more alkaline values as the NO₂⁻ concentration was increased, changing from a pH value of 6.8 at 0.6 mM KNO₂ to pH 8.0 at 0.01 M KNO₂ with apparent K_m's of 0.2 and 1.2 mM NO₂⁻, respectively. Computations of the HNO₂ concentrations present under the above conditions showed an approx. 500-fold greater affinity for HNO₂ which was independent of pH, suggesting the involvement of HNO₂ as both a substrate and an inhibitor (at higher concentrations) of the nitrite oxidase system. The marked inhibition by NaN₃, NaCN and Na₂S, as well the light-reversible inhibition by CO, indicated the presence of cytochrome oxidase which was subsequently characterized. NO₂⁻ proved to be a competitive inhibitor of the nitrite oxidase system.

3. The particulate preparation also possessed a heat-labile nitrite-cytochrome c reductase activity which was energy independent and routinely measured under anaerobic conditions. As in the case of nitrite oxidase, the affinity of the enzyme for NO₃⁻ increased as the pH was lowered, but the pH optimum remained unaffected. In terms of calculated HNO₂ concentration an approximately constant K_m of about 0.2 μM was estimated at the several pH's examined. The inhibition by NO₃⁻ was shown to be competitive. The marked sensitivity of the reductase to several metal-binding agents implicated a metal component in the electron transport chain at the site prior to cytochrome c.

4. The membrane-like composition of the nitrite oxidase system is indicated.     

Inhibition of the reoxidation of the secondary electron acceptor of Photosystem II by bicarbonate depletion

In bicarbonate-depleted chloroplasts, the chlorophyll a fluorescence decayed with a halftime of about 150 ms after the third flash, and appreciably faster after the first and second flash of a series of flashes given after a dark period. After the fourth to twentieth flashes, the decay was also slow. After addition of bicarbonate, the decay was fast after all the flashes of the sequence. This indicates that the bicarbonate depletion inhibits the reoxidation of the secondary acceptor R²⁻ by the plastoquinone pool; R is the secondary electron acceptor of pigment system II, as it accepts electrons from the reduced form of the primary electron acceptor (Q⁻). This conclusion is consistent with the measurements of the DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea)-induced chlorophyll a fluorescence after a series of flashes in the presence and the absence of bicarbonate, if it is assumed that DCMU not only causes reduction of Q if added in the state QR⁻, but also if added in the state QR²⁻.     

Rb transport in Leishmania infantum promastigotes under various in vitro culture conditions

The survival of Leishmania, which encounter drastic changes of environment during their life-cycle, requires regulation and control of ionic concentrations within the cell. We analysed the influence of growth stage, ionic composition of the medium, heat and acidic stress on ⁸⁶Rb⁺ influx in L. infantum promastigetes. Proliferating promastigotes exibited faster and higher ⁸⁶Rb⁺ uptake than stationary cells. Cl⁻ anion did not have any effect, but in the presence of physiological concentration of HCO₃⁻, ⁸⁶Rb⁺ uptake was significantly increased. This enhancing effect was only partially inhibited by N,N′-dicyclohexylcarbodiimide (DCCD), a blocker of ion-translocating ATPases. ⁸⁶Rb⁺ influx was abolished by N-ethylmaleimide (NEM), indicating a major contribution of plasma membrane transporters. Heat shock and acidic shock notably decreased ⁸⁶Rb⁺ influx. Our data provide indirect evidence that an energy-dependent system which brings K⁺ in, such as K⁺/H⁺-ATPase evidenced by Jiang et al. (1994), is active in Leishmania in different environments. Mechanism(s) other than ion-translocating ATPase occur, at least in the presence of HCO₃⁻, and their contribution to K⁺ pathways varies in different environmental conditions.     

Ca-binding and Ca-sensitizing functions of cardiac native tropomyosin, troponin, and tropomyosin

Procedures are described by which troponin and tropomyosin can be isolated from cardiac muscle rapidly, with minimal damage by oxidation. Cardiac relaxing proteins inhibit actomyosin ATPase activity in the presence of ethyleneglycoltetraacetic acid (EGTA), and permit graded stimulation by Ca²⁺. This stimulation is independent of preexisting inhibition, and greater than that obtained with skeletal proteins. Characteristics of Scatchard plots for Ca²⁺ binding suggest that troponin contains one class of sites which interact at high fractional occupancy. Interaction appears to be enhanced by tropomyosin. Mean values for the estimated maximum affinity and capacity of six canine cardiac troponin preparations were: 4.92·10⁶ M⁻¹, and 21.58·10⁻⁶ moles·g⁻¹. Values for skeletal troponin were not significantly different. Native tropomyosin bound about half as much Ca²⁺ per g, with maximum affinity the same as troponin. Pure tropomyosin bound no Ca²⁺. Cardiac and skeletal proteins differ in that the former are much more labile, and more readily influenced by ions and drugs.     

Nitrite effect on nitrous oxide emission from denitrifying activated sludge

Laboratory-scale experiments were conducted to examine the N₂O emission during the denitrification process. For each of the 6 runs carried out, synthetic effluent was fed in a 10 l batch mixed liquor to investigate the effect of nitrite on N₂O emission and Helium was continuously bubbled through the reactor at constant rate (0.12 l/min) to favour N₂O transfer and detection. An increasing COD/NO₃⁻-N influent ratio from 3 to 7 was firstly applied (runs 1–3). Secondly, NO₂⁻ pulse additions were performed during run 4 and 5 (10 and 20 mg N/l, respectively). Finally, the reactor was fed with influent containing both NO₂⁻ and NO₃⁻. We showed that N₂O emission was detected shortly after NO₂⁻ accumulation, few minutes after the substrate feeding. The highest emission occurred at the lower COD/NO₃⁻-N ratio (=3) and at the higher NO₂⁻ addition (20 mg N/l). In addition, the higher nitrogen conversion to N₂O gas (14.4%) was obtained with an influent containing initially both NO₂⁻ and NO₃⁻. Our results suggest a direct effect of the NO₂⁻ concentration on the N₂O emission. We have also confirmed the inhibitory effect of NO₂⁻ concentration on N₂O reduction.     

Resonance Raman studies of amaylose — iodine complexes

Resonance Raman measurements have been performed with solutions of iodine-complexed synthetic amyloses (DP 25–200), malto-oligomers (DP 3–18, and -cylodextrin. Interest was focused on the minimum chain length for helical complex formation and a possible preferred length for the polyiodine chain. Four fundamental vibrations are observed at 164, 112, 52 and 24 cm⁻¹. The 112 cm⁻¹ Raman line was shown to arise both from free I₃⁻ (enhanced at 363.8 nm excitation) and from bound iodine (relatively most intense at 457.9 nm excitation). The main signal of complexed iodine at 164 cm⁻¹is enhanced at an excitation wavelength close to the long wavelength absorption maximum. This signal is observed firt with malto-octaose and -cyclodextrin. The less intense signals at 52 and 24⁻¹ are only detected at DP 15 and higher. Raman spectra give no evidence for a preferred length of the polyiodine chain. Significantly identical Raman spectra are obtained when using different molar ratios of I₂/KI solution or I₂ solution initially free of I⁻ ions. The results are discussed in view of previous assignments of the Raman lines to I₂⁻, I₃⁻/I₂, and I₅⁻ subunits. Our findings are incompatible with I₃⁻ units as the only bound species. They are compatible with both I₃⁻/I₂ and I₃⁻ subunits under certain conditions. In the case of I₂ solution used for complexation we favour the polyiodine chain model proposed previously by Cramer^35,36. The I₃⁻ ions formed could function mainly as chain initiators, as has been suggested by Cesàro and Brant³⁰.     

Thermoluminescence as a probe of photosystem II. The redox and protonation states of the secondary acceptor quinone and the O2-evolving enzyme

The thermoluminescence band observed in chloroplasts after flash excitation at ambient temperatures has recently been identified as being due to recombination of the electron on the semiquinone form of the secondary plastoquinone acceptor, Q_B, with positive charges on the oxygen-evolving enzyme, S₂ and S₃ (Rutherford, A.W., Crofts, A.R. and Inoue, Y. (1982) Biochim. Biophys. Acta 682, 457–465). Further investigation of this thermoluminescence confirms this assignment and provides information on the function of PS II. The following data are reported: (1) Washing of chloroplasts with ferricyanide lowers the concentration of Q_B⁻ in the dark and predictable changes in the extent of the thermoluminescence band are observed. (2) The thermoluminescence intensity arising from S₂Q_B⁻ is approximately one half of that arising from S₃Q_B⁻. (3) Preflash treatment followed by dark adaptation results in changes in the intensity of the thermoluminescence band recorded after a series of flashes. These changes can be explained according to the above assignments for the origin of the thermoluminescence and if Q_B⁻ provides an important source of deactivating electrons for the S states. Computer simulations of the preflash data are reported using the above assumptions. Previously unexplained data already in the literature (Läufer, A. and Inoue, Y. (1980) Photobiochem. Photobiophys. 1, 339–346) can be satisfactorily explained and are simulated using the above assumptions. (4) Lowering the pH to pH 5.5 results in a shift of the S₂Q_B⁻ thermoluminescence band to higher temperatures while that arising from S₃Q_B⁻ does not shift. This effect is interpreted as indicating that Q_B⁻ is protonated and the S₂ to S₃ reaction involves deprotonation while the S₁ to S₂ reaction does not.     

The quenching of chlorophyll a fluorescence as a consequence of the transport of inorganic carbon by the cyanobacterium Synechococcus UTEX 625

The chlorophyll a fluorescence yield of the cyanobacterium Synechococcus UTEX 625 decreased upon the initiation of inorganic carbon transport. The fluorescence yield recovered upon the depletion of inorganic carbon from the medium or upon the addition of DCMU. The inhibition of photosynthetic CO₂ fixation by iodoacetamide did not prevent this reduction of fluorescence yield. Similar results were obtained for both Na⁺-stimulated HCO₃⁻ transport and for the transport (presumably of CO₂) that is stimulated by carbonic anhydrase. A transient lowering of the fluorescence yield was also observed when cell suspensions were pulsed with CO₂. In cells not inhibited with iodoacetamide, a very close quantitative relationship existed between the net rate of O₂ evolution and the maximum extent of fluorescence quenching seen as a function of the inorganic carbon concentration. The fluorescence quenching, however, was not due to CO₂ fixation but rather to the transport of inorganic carbon or the accumulation of the internal pool of inorganic carbon. If quenching is due to the latter it is not surprising that the extent of quenching corresponds to the maximum rate of photosynthesis as the rate of photosynthesis also depends on the size of the internal pool. The results with DCMU suggest that the quenching is Q quenching and transport must provide a mechanism for the oxidation of Q other than CO₂ fixation.     

HCO3-对不同基因型水稻根生长及养分吸收分配的影响

采用营养液培养法,研究了缺Zn敏感(IR26)和耐性水稻品种(IR8192-31-2)根生长和养分吸收受HCO₃^-影响的差异.结果表明,HCO₃^-(20mmol·L^-1)严重抑制敏感品种根系生长,特别是在低Zn或缺Zn条件下,而对耐性品种影响很小,在低Zn水平下,对根系生长甚至有轻微的促进作用.HCO₃^-不仅抑制敏感品种对Zn的吸收和分配,而且也抑制对Cu、Mn和Fe的吸收,表明HCO₃^-对Zn的吸收无专性抑制作用,HCO₃^-抑制敏感品种根系生长可能是其诱发缺Zn的最初作用.NCO₃^-处理下,耐性品种上位叶和下位叶Zn浓度以及比率高于敏感品种,表明耐性品种将Zn从下位叶向上位叶转运的效率高,Zn在植物体内的转运能力可能是耐性品种适应石灰性土壤缺Zn的主要机制之一.     

Perchlorate and nitrate reductase activity in the perchlorate-respiring bacterium perclace

The perchlorate (ClO₄⁻)-respiring organism, strain perc1ace, can grow using nitrate (NO₃⁻) as a terminal electron acceptor. In resting cell suspensions, NO₃⁻ grown cells reduced ClO₄⁻, and ClO₄⁻ grown cells reduced NO₃⁻. Activity assays showed that nitrate reductase (NR) activity was 1.31 μmol min⁻¹ (mg protein)⁻¹ in ClO₄⁻ grown cells, and perchlorate reductase (PR) activity was 4.24 μmol min⁻¹ (mg protein)⁻¹ in NO₃⁻ grown cells. PR activity was detected within the periplasmic space, with activities as high as 14 μmol min⁻¹ (mg protein)⁻¹. The NR had a pH optimum of 9.0 while the PR had an optimum of 8.0. This study suggests that separate terminal reductases are present in strain perclace to reduce NO₃⁻ and ClO₄⁻.     

Bipyridylium quaternary salts and related compounds. V. Pulse radiolysis studies of the reaction of paraquat radical with oxygen. Implications for the mode of action of bipyridyl herbicides

1. Rate constants for reduction of paraquat ion (1,1′-dimethyl-4,4′-bipyridy-lium, PQ²⁺) to paraquat radical (PQ⁺·) by e⁻_aq and CO₂⁻· have been measured by pulse radiolysis. Reduction by e⁻_aq is diffusion controlled (k = 8.4·10¹⁰ M⁻¹·s⁻¹) and reduction by CO₂⁻· is also very fast k = 1.5·10¹⁰ M⁻¹·s⁻¹).

2. The reaction of paraquat radical with oxygen has been analysed to give rate constants of 7.7·10⁸ M⁻¹·s⁻¹ and 6.5·10⁸ M⁻¹·s⁻¹ for the reactions of paraquat radical with O₂ and O₂⁻·, respectively. The similarity in these rate constants is in marked contrast to the difference in redox potentials of O₂ and O₂⁻· (− 0.59 V and + 1.12 V, respectively).

3. These rate constants, together with that for the self-reaction of O₂⁻·, have been used to calculate the steady-state concentration of O₂⁻· under conditions thought to apply at the site of reduction of paraquat in the plant cell. On the basis of these calculations the decay of O₂⁻· appears to be governed almost entirely by its self-reaction, and the concentration 5 μm away from the thylakoid is still 90% of that at the thylakoid itself. Thus, O₂⁻· persists long enough to diffuse as far as the chloroplast envelope and tonoplast, which are the first structures to be damaged by paraquat treatment. O₂⁻· is therefore sufficiently long-lived to be a candidate for the phytotoxic product formed by paraquat in plants.     

Nitrogen mineralisation and plant nitrogen acquisition in a nitrogen-limited calcareous grassland

A field study measured the rate of soil mineral N supply and its effects on plant biomass and N accumulation in a 13-year-old, naturally regenerating, calcareous grassland. Gross rates of N mineralisation (2 μg g⁻¹ day⁻¹, i.e. 0.69 kg ha⁻¹ day⁻¹), assessed using ¹⁵N pool dilution, were at the lower end of the range previously reported for grasslands. Weekly additions of liquid N fertiliser ([NH₄]₂SO₄, NH₄NO₃ or KNO₃) and, to a lesser extent the addition of water, increased plant growth substantially, demonstrating that the primary constraint to plant growth was low N availability. In plants that had received NO₃⁻, the activity of the inducible enzyme nitrate reductase in shoots initially increased in proportion to the amount of NO₃⁻ supplied. However, as above-ground herbage accumulated, nitrate reductase activity declined to similar low levels in all treatments, despite the continuance of the constant NO₃⁻ additions. The decline in NR specific activity reflected declining tissue NO₃⁻ concentrations, although total plant NRA may have remained constant during the period of study. The study has shown that plant growth is limited by low N mineralisation rates and indeed the soil is a sink for much added N. Low water availability provides an additional constraint on N mineralisation in this calcareous grassland soil. Any disturbances in the N cycle which increase the availability of mineral N will result in a substantial increase in plant growth within this ecosystem.     

Mass spectral evidence for carbonate-anion-radical-induced posttranslational modification of tryptophan to kynurenine in human Cu, Zn superoxide dismutase

Previously, we showed that oxidation of tryptophan-32 (Trp-32) residue was crucial for H₂O₂/bicarbonate (HCO₃⁻)-dependent covalent aggregation of human Cu,Zn SOD1 (hSOD1). The carbonate anion radical (CO₃⁻)-induced oxidation of Trp-32 to kynurenine-type oxidation products was proposed to cause the aggregation of hSOD1. Here we used the matrix-assisted laser desorption ionization–time of flight mass spectroscopy, high-performance liquid chromatography–electrospray ionization mass spectroscopy, and liquid chromatography mass spectroscopy methods to characterize products. Results show that a peptide region (31–36) of hSOD1 containing the Trp-32 residue (VWGSIK) is oxidatively modified to the N-formylkynurenine (NFK)- and kynurenine (Kyn)-containing peptides (V(NFK)GSIK) and (V(Kyn)GSIK) during HCO⁻-dependent peroxidase activity of hSOD1. Also, UV photolysis of a cobalt complex that generates authentic CO₃⁻ radical induced a similar product profile from hSOD1. Similar products were obtained using a synthetic peptide with the same amino acid sequence (i.e., VWGSIK). We propose a mechanism involving a tryptophanyl radical for CO₃⁻-induced oxidation of Trp-32 residue (VWGSIK) in hSOD1 to V(NFK)GSIK and V(Kyn)GSIK.     

Oxidative phosphorylation coupled to oxygen uptake and nitrate reduction in Micrococcus denitrificans

A procedure is described for preparing particles from cells of Micrococcus denitrificans which were broken osmotically after treatment with lysozyme.

1. 1. The preparations catalysed ATP synthesis coupled to O₂ uptake or NO₃⁻ reduction. With NADH or succinate as the electron donors the P:O ratios were about 1.5 and 0.5, respectively; and the P:NO₃⁻ ratios were about 0.9 and 0.06, respectively.

2. 2. Addition of ADP or P_i to the reaction mixture increased the rates of NADH-dependent O₂ uptake and NO₃⁻ reduction. Addition of 1 mM 2,4-dinitrophenol, which inhibited phosphorylation by 50–60%, increased the basal rates of electron transport.

3. 3. Evidence derived from spectrophotometry and from the differential inhibition by antimycin A of O₂ and NO₃⁻ reduction leads to the conclusion that the nitrate reductase interacted with the respiratory chain in the region of the b-type cytochrome, and that the c-type cytochrome present was not involved in the reduction of NO₃⁻ to NO₂⁻.

Crystal and microparticle effects on MDCK cell superoxide production: oxalate-specific mitochondrial membrane potential changes

We have previously shown that crystals of calcium oxalate (COM) elicit a superoxide (O₂⁻) response from mitochondria. We have now investigated: (i) if other microparticles can elicit the same response, (ii) if processing of crystals is involved, and (iii) at what level of mitochondrial function oxalate acts. O₂⁻ was measured in digitonin-permeabilized MDCK cells by lucigenin (10 μM) chemiluminescence. [¹⁴C]-COM dissociation was examined with or without EDTA and employing alternative chelators. Whereas mitochondrial O₂⁻ in COM-treated cells was three- to fourfold enhanced compared to controls, other particulates (uric acid, zymosan, and latex beads) either did not increase O₂⁻ or were much less effective (hydroxyapatite +50%, p < 0.01), with all at 28 μg/cm². Free oxalate (750 μM), at the level released from COM with EDTA (1 mM), increased O₂⁻ (+50%, p < 0.01). Omitting EDTA abrogated this signal, which was restored completely by EGTA and partially by ascorbate, but not by desferrioxamine or citrate. Omission of phosphate abrogated O₂⁻, implicating phosphate-dependent mitochondrial dicarboxylate transport. COM caused a time-related increase in the mitochondrial membrane potential (Δψ_m) measured using TMRM fluorescence and confocal microscopy. Application of COM to Fura 2-loaded cells induced rapid, large-amplitude cytosolic Ca²⁺ transients, which were inhibited by thapsigargin, indicating that COM induces release of Ca²⁺ from internal stores. Thus, COM-induced mitochondrial O₂⁻ requires the release of free oxalate and contributes to a synergistic response. Intracellular dissociation of COM and the mitochondrial dicarboxylate transporter are important in O₂⁻ production, which is probably regulated by Δψ_m.     

Reactions of the ferri-ferrocytochrome-c system with superoxide/oxygen and CO2/CO2 studied by fast pulse radiolysis

The reduction of ferricytochrome c by O₂⁻ and CO₂⁻ was studied in the pH range 6.6–9.2 and Arrhenius as well as Eyring parameters were derived from the rate constants and their temperature dependence. Ionic effects on the rate indicate that the redox process proceeds through a multiply-positively charged interaction site on cytochrome c. It is shown that the reaction with O₂⁻ and correspondingly with O₂ of ferrocytochrome c) is by a factor of approx. 10³ slower than warranted by factors such as redox potential. Evidence is adduced to support the view that this slowness is connected with the role of water in the interaction between O₂⁻/O₂ and ferri-ferrocytochrome c in the positively charged interaction site on cytochrome c in which water molecules are specifically involved in maintaining the local structure of cytochrome c and participate in the process of electron equivalent transfer.     

Examination of the Inhibitory Effect of Norathyriol in Formylmethionyl-Leucyl-Phenylalanine-Induced Respiratory Burst in Rat Neutrophils

Norathyriol, aglycone of a xanthone C-glycoside mangiferin isolated from Tripterospermum lanceolatum, concentration dependently inhibited the formylmethionyl-leucyl-phenylalanine (fMLP)-induced superoxide anion (O₂^˙−) generation and O₂ consumption in rat neutrophils. In cell-free oxygen radical generating system, norathyriol inhibited the O₂^˙− generation during dihydroxyfumaric acid (DHF) autoxidation and in hypoxanthine-xanthine oxidase system. fMLP-induced transient elevation of [Ca²⁺]_i and the formation of inositol trisphosphate (IP₃) were significantly inhibited by norathyriol (30 μM) (about 30 and 46% inhibition, respectively). Norathyriol concentration dependently suppressed the neutrophil cytosolic phospholipase C (PLC). In contrast with the marked attenuation of fMLP-induced protein tyrosine phosphorylation (about 70% inhibition at 10 μM norathyriol), norathyriol only slightly modulated the phospholipase D (PLD) activity as determined by the formation of phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt). Norathyriol did not modulate the intracellular cyclic AMP level. In the presence of NADPH, the phorbol 12-myristate 13-acetate (PMA)-activated particulate NADPH oxidase activity was suppressed by norathyriol in a concentration-dependent manner and the inhibition was noncompetitive with respect to NADPH. Norathyriol inhibited the iodonitrotetrazolium violet (INT) reduction in arachidonic acid (AA)-activated cell-free NADPH oxidase system at the same concentration range as those used in the suppression of PMA-activated particulate NADPH oxidase activity. Taken together, these results suggest that the scavenging ability of norathyriol contributes to the reduction of generated O₂^˙−, however, the inhibition of O₂^˙− generation from neutrophils by norathyriol is attributed to the blockade of PLC pathway, the attenuation of protein tyrosine phosphorylation, and to the suppression of NADPH oxidase through the interruption of electrons transport.     

Fluorescence yield kinetics in the microsecond-range in Chlorella pyrenoidosa and spinach chloroplasts in the presence of hydroxylamine

The kinetics of fluorescence yield inChlorella pyrenoidosa and spinach chloroplasts were studied in the time range of 0.5 μs to several hundreds of microseconds in the presence of hydroxylamine. Fluorescence was excited with a just-saturating xenon flash with a halfwidth of 13 μs (λ = 420 nm). The fast rise of the fluorescence yield which was limited by the rate of light influx, was, in the presence of 10⁻³–10⁻² M hydroxylamine, replaced by a slow component which had a half risetime of 25 μs in essence independent of light intensity. This slow fluorescence yield increase reflects a dark reaction on the watersplitting side of Photosystem II. Simultaneous oxygen evolution measurements suggested that a fast fluorescence component is only present in organisms with intact O₂-evolving system, whereas a slow rise predominantly occurs in organisms with the watersplitting system irreversibly inhibited by hydroxylamine.

The results can be explained by the following hypotheses: (a) The primary donor of Photosystem II in its oxidized state, P⁺, is a fluorescence quencher. (b) Hydroxylamine prevents the secondary electron donor Z from reducing the oxidized reaction center pigment P⁺ rapidly. This inhibition is dependent on hydroxylamine concentration and is complete at a concentration of 10⁻² M. (c) A second donor (not transporting electrons from water) transfers electrons to P⁺ with a half time of roughly 25 μs.