Binding of the inhibitor NH3 to the oxygen-evolving apparatus of spinach chloroplasts

Experiments are described on flash-induced luminescence of isolated spinach chloroplasts after addition of NH₄Cl. The results indicate a binding of NH₃, presumably in competition with water, in the oxidation states S₂ and S₃, i.e. the states reached upon illumination of dark-adapted material with one and two flashes, respectively. In the initial state S₁, no binding of NH₃ occurs. In state S₂ the binding of ammonia is rapid (half-time about 0.5 s) and rapidly reversible; in state S₃ the binding is slower (half-time about 10 s) and slowly reversible. NH₃ bound to S₄ prevents the oxidation of water. NH₃ bound to S₂ decreases the rate of the back reaction of reduced primary acceptor (Q⁻), indicating a charge stabilization, i.e. a decrease in the redox potential of S₂ due to interaction with ammonia. In Tris-washed chloroplasts, the stability of the positive charge generated in a flash is much smaller than in normal chloroplasts and not increased by NH₃. On the basis of these observations it is postulated that, in the absence of NH₃, states S₂ and S₃ are stabilized by manganese-coordinated, bound water.     

Hematoxylin-Eosin Staining of OsO4-Fixed Epon- Embedded Tissue; Prestaining Oxidation by Acidified H2O2

Successful application of hematoxylin-eosin staining to 0.5-1 μ sections of OsO₄-fixed Epon-embedded mammalian tissue is made possible by first treating the sections for approximately 1 min at 25-30 C with 10% H₂O₂ acidified with 0.1 or 0.01 N H₂SO₄ to pH 3.2. Subsequent steps are: washing; drying; Hams hematoxylin at 50 C, 1-2 min; washing; drying; 0.2-0.3% NH₄OH in 70% ethanol, 3-5 sec, drying at 50 C; 5% aqueous eosin for 3 & 45 sec at 25-30 C, washing; drying; clearing in xylene and mounting in resin. The use of acidified H₂O₂ prevents the staining of Epon and permits the characteristic staining picture to be obtained. Sections were attached to glass slides without adhesive and processed horizontally on a rack. Slides should be well drained and blotted before each drying step, to prevent formation of precipitate on the section.     

Staining of Oligodendroglia with Silver Ammino Tungstate in Unembedded and Embedded Material

Specimens of brain or spinal cord fixed in formalin, Cajal's formol-bromide, or Koenig, Groat and Windle's formalin-acacia can be used to stain oligodendrocytes in frozen, in paraffin, or in celloidin sections. The sections are soaked 3-5 min in 0.02% acetic acid, pH 3.4, then rinsed 2-3 sec in 3% H₂O₂ and transferred to a silver bath prepared as follows: Mix equal parts of 10% AgNO₃ and 10% Na₂WO₄, and dissolve the precipitate with concentrated NH₄OH; avoid an excess of ammonia. Silver at room temperature for 15-20 sec, develop in 1% formalin, dehydrate, and mount. For embedded material, prepare a mixture consisting of 1 part of 10% aqueous Aerosol MA and 4 parts of 10% Aerosol OT in 95% alcohol. Add 5 drops of this mixture to each 50 ml of dilute acetic acid and 3% H₂O₂; 5 drops to each 20 ml of the silver bath.     

Effects of carbonyl cyanide m-chlorophenylhydrazone and hydroxylamine on the Photosystem II electron exchange mechanism in 3-(3,4-dichlorophenyl)-1,1-dimethylurea treated algae and chloroplasts

The effects of NH₂OH and carbonyl cyanide m-chlorophenylhydrazone (CCCP) on 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated algae and chloroplasts were studied. In the presence of DCMU, the photochemically separated charges can only disappear through a recombination back reaction; both substances induce an irreversible reduction of the donor side and after sufficient illumination their action in the presence of DCMU leads to the formation of a permanent fluorescent state.

In the DCMU + CCCP system, a fast fluorescence induction curve is observed. The fluorescence yield is brought to its maximum by two flashes. The luminescence emission is strongly inhibited and most centers reach their permanent fluorescent state after one flash.

In the DCMU + NH₂OH system, a slow fluorescence rise is observed and several saturating flashes are needed for the fluorescence yield to reach its maximum. The exhaustion of the NH₂OH oxidizing capacity and the complete transformation to a permanent fluorescent state also require a large number of flashes.

The reduction pathway catalyzed by CCCP appears to be a good competitor to the back reaction, while NH₂OH seems to be a relatively inefficient donor.

In addition the action of NH₂OH and CCCP on fluorescence suggests that the donor side influences the quenching properties of Photosystem II centers. A possible mechanism is proposed.     

Mechanism of hydroxyl radical scavenging by rebamipide: identification of mono-hydroxylated rebamipide as a major reaction product

Rebamipide, an antiulcer agent, is known as a potent hydroxyl radical (_•OH) scavenger. In the present study, we further characterized the scavenging effect of rebamipide against _•OH generated by ultraviolet (UV) irradiation of hydrogen peroxide (H₂O₂), and identified the reaction products to elucidate the mechanism of the reaction. Scavenging effect of rebamipide was accessed by ESR using DMPO as a _•OH-trapping agent after UVB exposure (305 nm) to H₂O₂ for 1 min in the presence of rebamipide. The signal intensity of _•OH adduct of DMPO (DMPO-OH) was markedly reduced by rebamipide in a concentration-dependent fashion as well as by dimethyl sulfoxide and glutathione as reference radical scavengers. Their second order rate constant values were 5.62 × 10₁₀, 8.16 × 10₉ and 1.65 × 10₁₀ M_-1 s_-1, respectively. As the rebamipide absorption spectrum disappeared during the reaction, a new spectrum grew due to generation of rather specific reaction product. The reaction product was characterized by LC-MS/MS and NMR measurements. Finally, a hydroxylated rebamipide at the 3-position of the 2(1H)-quinolinone nucleus was newly identified as the major product exclusively formed in the reaction between rebamipide and the _•OH generated by UVB/H₂O₂. Specific formation of this product explained the molecular characteristics of rebamipide as a potential _•OH scavenger.     

Influence of different site symmetries of Eu centers on the luminescence properties of Anderson-based compounds

Two compounds, [Eu(H₂O)₇][Al(OH)₆Mo₆O₁₈] · 4H₂O (1) and {(C₂H₅NO₂)₂[Eu(H₂O)₅]}[Al(OH)₆Mo₆O₁₈] · 10H₂O (2), have been synthesized by conventional solution method and determined by single-crystal X-ray diffraction. Compound 1 shows a 1D chain structure built up of alternating Anderson-type polyanions [Al(OH)₆Mo₆O₁₈]³⁻ and hydrated rare-earth ions Eu³⁺. Compound 2 displays a 3D supramolecular network structure containing 1D sandglass-like channels along c axis, which were occupied by repetitive array of (H₂O)₈ clusters. Extensive hydrogen bonds play an important role in the formation of the 3D structures of 1 and 2. Luminescence measurements reveal that 1 and 2 exhibit intense red and orange fluorescent emission at room temperature, respectively. Origin of the distinct emission can be assigned to the different site symmetries of Eu³⁺ centers in the two compounds. These results are consistent with the crystal structures of the two compounds.     

A Modification of the Thiocholine Method for the Demonstration of Cholinesterases

A modification of the thiocholine technique for cholinesterases is described, in which mounted sections are dehydrated for 2 min in each ascending grade of alcohol before development of CuS by using alcoholic (NH₄)₂S. This reagent is prepared by first saturating half-concentrated (NH₄)₂OH with H₂S; 3 ml of this is diluted with 22 ml of ethyl alcohol, then saturated with CuS. With these modifications better definition of sites of cholinesterase activity is achieved.     

An investigation of water splitting in the Kok scheme of photosynthetic oxygen evolution

The involvement of OH bond breaking in the 4 dark reactions of the Kok scheme of photosynthetic oxygen evolution was investigated using Chlorella and spinach chloroplasts. When the photosynthetic material was suspended in a ²H₂O based medium, the reaction rates in all 4 cases were only slightly reduced as compared to the rates observed in an H₂O based medium. This was evidence that these rate processes were probably not limited by the breaking of an OH bond. Observations were also made on the yields of O₂ from dark adapted Chlorella subjected to a sequence of brief saturating light flashes. The oscillating flash yield sequence observed with algae suspended in ²H₂O showed greater damping of the oscillations than when the algae were suspended in H₂O. A computer fit of the Kok model to these results revealed a slightly higher proportion of misses, (i.e. absorbed quanta that do not drive photochemistry) in the ²H₂O case.     

Qualitative Analysis by Thin-Layer Chromatography of Some Common Dyes Used in Biological Staining

Thin-layer chromatography will resolve impurities in commercial dyes, and will do so much faster than paper chromatography. Solvent systems consisting of (a) n-propanol: n-butanol: NH₄OH (conc.): H₂O—4:4:1:1; (b) n-propanol: NH₄OH (conc.): H₂O—8:1:1 on silica gel G plates; and (c) n-propanol: NH₄OH (conc.): H₂O-7:2:1 on Adsorbosil plates were found to be the most effective. Dyes studied were azure A, azure B, azure C, methylene blue, toluidine blue O, thionin, pyronin B, pyronin Y, methyl green, crystal violet amido black 10B and buffalo black (NBR).     

The deactivation of Photosystem II in Chlorella as a second-order process

The kinetics of deactivation of the S₃ state in Chlorella have been observed under a variety of conditions. The S₃ state appears to decline in a dark period coming after a sequence of 30 saturating flashes in a second-order reaction, the rate constant of which is 0.132/[S*₃] s⁻¹ and which involves an electron donor, D₁, of concentration 1.25[S*₃] where [S*₃] is the concentration of the S₃ state when the oxygen yield of the light flashes is constant. If a 1 min period of 650 nm illumination is employed after the sequence of flashes, the subsequent S₃ state deactivation kinetics are more complex. There is an initial phase of S₃ state deactivation, accounting for about 35% of the original S₃ state, which is complete in less than 100 ms. The remaining 65% of the S₃ state appears to deactivate in a second-order reaction, the rate constant of which is 1.36/[S*₃] s⁻¹ and which involves an electron donor of initial concentration 0.58[S*₃]. If a 1 min period of 710 nm illumination comes after the 30 flashes, at least 98% of the S₃ state deactivates according to first-order kinetics. It is shown that this can be explained using a second-order model if there is an electron donor present of which the concentration is large compared with [S*₃]. However, S₃ state deactivation observed after 5 min of dark and two saturating flashes can be described neither by a first-order model nor a second-order model. Deactivation of the S₂ state after a 5 min dark period and one saturating flash follows second-order kinetics with a rate constant of 0.2/[S*₃] s⁻¹ and appears to involve an electron donor of initial concentration 1.3[S*₃]. Arguments are presented which tend to rule out the primary electron acceptor to Photosystem II as being any of the electron donors but it appears quite possible that the large plastoquinone pool is involved.     

外源H2O2对盐碱混合胁迫下裸燕麦幼苗生长和抗性生理的影响

为探讨信号分子过氧化氢（H₂O₂）增强裸燕麦盐碱耐性的作用及其生理机制,以裸燕麦品种‘定莜6号’为材料,在日光温室内用珍珠岩培养幼苗至三叶一心期时叶面喷施0.01 mmol·L^-1 H₂O₂的同时根部浇灌75 mmol·L^-1盐碱混合溶液（NaCl：Na₂SO₄：NaHCO₃：Na₂CO₃=12：8：9：1）或添加H₂O₂淬灭剂二甲基硫脲（DMTU）,研究对幼苗生长及叶片光合色素含量、活性氧代谢和渗透调节物质积累的影响。结果表明：喷施H₂O₂能够缓解盐碱混合胁迫对裸燕麦幼苗生长的抑制,提高幼苗根长、株高和植株干重及叶片叶绿素a、叶绿素b、总叶绿素、类胡萝卜素含量和超氧化物歧化酶、过氧化物酶、过氧化氢酶、抗坏血酸过氧化物酶活性,降低超氧阴离子、H₂O₂、丙二醛、抗坏血酸、谷胱甘肽和游离氨基酸含量,促进抗氧化物质类黄酮、总酚和原花青素及渗透调节物质可溶性蛋白质、可溶性糖和脯氨酸积累。添加DMTU部分或完全逆转了H₂O₂的上述作用。采用隶属函数综合评价显示,喷施H₂O₂提高了盐碱混合胁迫下裸燕麦幼苗的综合评价值D,添加DMTU完全逆转了H₂O₂对D值的提升作用。表明外源H₂O₂通过参与活性氧代谢和渗透调节物质积累等生理代谢调控缓解盐碱混合胁迫诱导的氧化伤害和生长抑制,从而提高裸燕麦对盐碱胁迫的适应能力。     

Systematic synthesis, structural characterization, and reactivity studies of vanadium(V)–citrate anions [VO2(C6H6O7)]2, isolated from aqueous solutions in the presence of different cations

The aqueous chemistry of vanadium with physiologically relevant ligands constitutes a subject of burgeoning research, extending from bacterial metalloenzymic functions to human-health physiology. Vanadium, in the form of VCl₃ and V₂O_5, reacted expediently with citric acid, in a 1:2 molar ratio in water at pH4, and, in the presence of various cations, afforded crystalline materials bearing the general formula (Cat)₂[V₂O₄(C₆H₆O₇)₂]·nH₂O (A) (Cat⁺=Na⁺, NH₄ ⁺, n=2; Me₄N⁺, K⁺, n=4). Exploration of the reactivity of A toward H₂O₂ yielded the peroxo-containing complexes (Cat)₂[V₂O₂(O₂)₂(C₆H₆O₇)₂]·2H₂O (B) (Cat⁺=K⁺, NH₄ ⁺). Both classes of compounds were characterized analytically and spectroscopically. The X-ray structures of complexes A and B emphasize the exceptional stability of the dimeric rhombic unit V^V ₂O₂, which is retained upon H₂O₂ reaction, and the preserved mode of coordination of the citrate ligand as a doubly deprotonated moiety. In these complexes, typical six and eight coordination numbers were observed for the Na⁺ and K⁺ counter-ions, respectively. The variety of synthetic approaches leading to A, along with the stepwise and direct assembly and isolation of peroxo-compounds (B), denotes the significance of reaction pathways and intermediates in vanadium(III–V)–citrate synthetic chemistry. Hence, a systematic investigation of reactivity modes in aqueous vanadium–citrate systems emerges as a crucial tool for the establishment of chemical interconnectivity among low MW complex species, potentially participating in the intricate biodistribution of that metal ion in biological fluids.     

Limiting steps in Photosystem II and water decomposition in Chlorella and spinach chloroplasts

1. 1. By varying the redox potential of a chloroplast suspension, we obtained new evidence for an equilibrium between states S₀ and S₁ in the model of Kok, B., Forbush, B. and McGloin, N. (1970, Photochem. Photobiol. 11, 457–475). The mid-point potential of the S₀ to S₁ couple is close to that for the pool of the electron acceptor of System II, A to A⁻.

2. 2. The limiting steps between two consecutive photoreactions of System II in Chlorella and spinach chloroplasts, have been studied.

2.1. (a) The limiting step from S₁ to S₂ (noted γ₁ (Δt)) is not exponential. Its temperature coefficient becomes greater as the reaction proceeds. The shape of the kinetics is an intrinsic property of each center. Chloroplasts fixed with 2% glutaraldehyde, show simple first order kinetics.

2.2. (b) The limiting step from S₀ to S₁ (γ₀ (Δt)) exhibits the same characteristics as γ₁ (Δt)).

2.3. (c) The limiting step from S₂ to S₃ (γ₂ (Δt)) shows sigmoidal kinetics; two reactions are involved. One of the reactions exhibits the same properties as γ₀ (Δt) and γ₁ (Δt).

2.4. (d) The limiting step from S₃ to S₀ (γ₃ (Δt)) is a first order reaction, two times slower than the other transitions. This reaction is interpretated in terms of oxygen release.

3. 3. We also studied the limiting steps in the presence of low concentrations (50 μM) of hydroxylamine. The results favor the binding of two molecules of hydroxylamine to every photochemical center.

Inhibitory effects of carrier-immobilized synthetic histo-blood group A-, B-, H-, and SiaLe-oligosaccharides on H2O2 generation by human polymorphonuclear leukocytes

Carbohydrate-bearing polymers of biologically inert design are versatile tools to delineate functional aspects of oligosaccharides. Binding of synthetic N-substituted polyacrylamide (PAA) conjugates of histo-blood group (A_di, A_tri, B_di, B_tri, H_di, SiaLe^a, and SiaLe^x) to human polymorphonuclear leukocytes (PMNs), and effects on H₂O₂ generation elicited by different agonists such as digitonin, N-formyl-Met-Leu-Phe (FMLP) and the galactoside-specific lectin from Viscum album L. (VAA) were assessed. PMNs expressed binding sites for blood group-related neoglycoconjugates in the range of N10⁶–10⁷/cell with K_D-values in the μM range. Treatment of PMNs (2×10⁶ cells/ml) with PAA-probes (50 μg/ml) for 5 min did not activate the “respiratory burst”. However, it led to suppression (range 20–70%) of H₂O₂ generation of cells in the presence of elicitors. In detail, the FMLP-induced response was significantly decreased by A_di, A_tri, B_tri, H_di, SiaLe^a, and SiaLe^x conjugates, whereas for digitonin one only by A_di, A_tri, B_tri. All the seven tested PAA-probes were found to inhibit significantly VAA-mediated release of H₂O₂ from PMNs. In this case, interference can take place already, at the stage of initial binding, especially for B- and H-epitopes, but less prominently for A- and SiaLe-epitopes. These results support the notion that PAA-immobilized histo-blood group oligosaccharides can serve as effector molecules with the ability to reduce the H₂O₂-generation of PMNs, warranting further studies on the involved reaction pathway.     

Coffee drinking increases levels of urinary hydrogen peroxide detected in healthy human volunteers

Freshly-voided human urine contains significant concentrations of hydrogen peroxide (H₂O₂). This H₂O₂ appears to arise in whole or in part by superoxide-dependent autoxidation of urinary biomolecules. Since instant coffee also contains high levels of H₂O₂, we examined the effect of coffee drinking on urinary levels of H₂O₂. Studies on healthy human volunteers showed that coffee drinking is rapidly and reproducibly followed by increased levels of H₂O₂ detectable in the urine for up to 2 h after drinking the coffee. The levels of H₂O₂ detected in urine suggest that exposure of human tissues to H₂O₂ may be greater than is commonly supposed. It is possible that H₂O₂ in urine could act as an antibacterial agent, and that H₂O₂ is involved in the regulation of glomerular function.     

Non-oxygen-forming pathways of hydrogen peroxide degradation by bovine liver catalase at low hydrogen peroxide fluxes

Heme catalases are considered to degrade two molecules of H₂O₂ to two molecules of H₂O and one molecule of O₂ employing the catalatic cycle. We here studied the catalytic behaviour of bovine liver catalase at low fluxes of H₂O₂ (relative to catalase concentration), adjusted by H₂O₂-generating systems. At a ratio of a H₂O₂ flux (given in μM/min^- 1) to catalase concentration (given in μM) of 10 min^- 1 and above, H₂O₂ degradation occurred via the catalatic cycle. At lower ratios, however, H₂O₂ degradation proceeded with increasingly diminished production of O₂. At a ratio of 1 min^- 1, O₂ formation could no longer be observed, although the enzyme still degraded H₂O₂. These results strongly suggest that at low physiological H₂O₂ fluxes H₂O₂ is preferentially metabolised reductively to H₂O, without release of O₂. The pathways involved in the reductive metabolism of H₂O₂ are presumably those previously reported as inactivation and reactivation pathways. They start from compound I and are operative at low and high H₂O₂ fluxes but kinetically outcompete the reaction of compound I with H₂O₂ at low H₂O₂ production rates. In the absence of NADPH, the reducing equivalents for the reductive metabolism of H₂O₂ are most likely provided by the protein moiety of the enzyme. In the presence of NADPH, they are at least in part provided by the coenzyme.     

Oxidant-mediated AA release from astrocytes involves cPLA(2) and iPLA(2)

Excessive generation of reactive oxygen species (ROS) in the central nervous system (CNS) is a leading cause of neuronal injury. Despite yet unknown mechanisms, oxidant compounds such as H₂O₂ have been shown to stimulate the release of arachidonic acid (AA) in a number of cell systems. In this study, H₂O₂ and menadione, a compound known to release H₂O₂ intracellularly, were used to examine the phospholipases A₂ (PLA₂) responsible for AA release from primary murine astrocytes. Both H₂O₂ and menadione dose-dependently stimulated AA release, and the release mediated by H₂O₂ was completely inhibited by catalase. H₂O₂ also stimulated phosphorylation of extracellular signal-regulated kinases (ERK1/2) and cytosolic phospholipase A₂ (cPLA₂). However, complete inhibition of cPLA₂ phosphorylation by U0126, an inhibitor for mitogen-activated protein kinase kinase (MEK) and GF109203x, a nonselective PKC inhibitor preferring the conventional and novel isoforms, only reduced H₂O₂-stimulated AA release by 50%. MAFP, a selective, active, site-directed, irreversible inhibitor of both cPLA₂ and the Ca²⁺-independent iPLA₂, nearly completely inhibited H₂O₂-mediated AA release; but, HELSS, a potent irreversible inhibitor of iPLA₂, only inhibited H₂O₂-mediated AA release by 40%. Along with the observation that H₂O₂-mediated AA release was only partially inhibited upon chelating intracellular Ca²⁺ by BAPTA, these results indicate the involvement of both cPLA₂ and iPLA₂ in H₂O₂-mediated AA release in murine astrocytes.     

生境对茶叶品质和产量影响的光合生理机制

为了探明茶叶产量和品质形成的特殊生境及其光合生理机制,在中国科学院长沙农业环境观测站利用3种间种乔木模式(S₁:桂花树-茶树, S₂:乐昌含笑-茶树, S₃:桂花树-乐昌含笑-茶树)与纯茶园(CK)建成了4种典型生境,比较研究了这些特殊生境下茶叶产量与品质形成的光合生理生态特性.结果表明: 生境S₁、S₂、S₃显著降低了茶树叶片温度(TL)、光合有效辐射通量(PAR),叶片净光合速率(P_n)、蒸腾速率(T_r)和气孔导度(g_s)的日均值,显著降低了茶叶茶多酚总含量.生境S₁、S₂、S₃显著提高了叶室相对湿度(RHS)、茶叶氨基酸总含量,显著提高了茶叶产量和品质,并且S₃>S₁>S₂>CK,其中生境S₁和S₃的茶叶适宜加工成高档绿茶和名优绿茶.综合各指标,生境S₃是茶园优质高产的理想间种模式.     

Structural organization of the oxidizing side of photosystem II. Exogenous reductants reduce and destroy the Mn-complex in photosystems II membranes depleted of the 17 and 23 kDa polypeptides

Removal of 23 and 17 kDa water-soluble polypeptides from PS II membranes causes a marked decrease in oxygen-evolution activity, exposes the oxidizing side of PS II to exogenous reductants (Ghanotakis, D.F., Babcock, G.T. and Yocum, C.F. (1984) Biochim. Biophys. Acta 765, 388–398) and alters a high-affinity binding site for Ca²⁺ in the oxygen-evolving complex (Ghanotakis, D.F., Topper, J.N., Babcock, G.T. and Yocum, C.F. (1984) FEBS Lett. 170, 169–173). We have examined further the state of the functional Mn complex in PS II membranes from which the 17 and 23 kDa species have been removed by high-salt treatment. These membranes contain a structurally altered Mn complex which is sensitive to destruction by low concentrations of NH₂OH which cannot, in native PS II membranes, cause extraction of functional Mn. In addition to NH₂OH, a wide range of other small (H₂O₂, NH₂NH₂, Fe²⁺) and bulky (benzidine, hydroquinone) electron donors extract Mn (up to 80%) from the polypeptide-depleted PS II preparations. This extraction is due to reduction of the functional Mn complex since light, which would generate higher oxidation states within the Mn complex, prevents Mn release by reductants. Release of Mn by reductants does not extract the 33 kDa water-soluble protein implicated in Mn binding to the oxidizing side of PS II, although the protein can be partially or totally extracted from Mn-depleted preparations by exposure to high ionic strength or to high (0.8 M) concentrations of Tris. We view our results as evidence for a shield around the Mn complex of the oxygen-evolving complex comprised of the 33 kDa polypeptide along with the 23 and 17 kDa proteins and tightly bound Ca²⁺.