Different effects of SNP and GSNO on mitochondrial O<Stack><Subscript>2</Subscript><Superscript>.−</Superscript></Stack>/H<Subscript>2</Subscript>O<Subscript>2</Subscript> production

Sodium Nitroprusside (SNP) and S-Nitrosoglutathione (GSNO) differently affect mitochondrial H₂O₂ release at Complex-I. mM SNP increases while GSNO decreases the release induced by succinate alone or added on top of NAD-linked substrates. Stimulation likely depends on Nitric Oxide ( ^. NO) (released by SNP but not by GSNO) inhibiting cytochrome oxidase and mitochondrial respiration. Preincubations with SNP or high GSNO (10 mM plus DTE to increases its ^. NO release) induces an inhibition of the succinate dependent H₂O₂ production consistent with a ^. NO dependent covalent modification. However maximal inhibition of the succinate dependent H₂O₂ release is obtained in the presence of low GSNO (20–100 μM), but not with SNP. This inhibition appears independent of ^. NO release since μM GSNO does not affect mitochondrial respiration, or the H₂O₂ detection systems and its effect is very rapid. Inhibition may be partly due to an increased removal of O₂^.− since GSNO chemically competes with NBT and cytochrome C in O₂^.− detection.     

On the mechanism of apoplastic H<Subscript>2</Subscript>O<Subscript>2</Subscript> production during lignin formation and elicitation in cultured spruce cells—peroxidases after elicitation

A cell culture of Picea abies (L.) Karst. was used for studies of H₂O₂ generation during constitutive extracellular lignin formation and after elicitation by cell wall fragments of a pathogenic fungus, Heterobasidium parviporum. Stable, micromolar levels of H₂O₂ were present in the culture medium during lignin formation. Elicitation induced a burst of H₂O₂, peaking at ca. 90 min after elicitation. Of exogenous reducing substrates that may be responsible for the synthesis of H₂O₂ from O₂, NADH stimulated H₂O₂ production irrespective of elicitation. Cysteine (Cys) and glutathione (GSH) partially scavenged the constitutive H₂O₂, but usually increased or prolonged elicitor-induced H₂O₂ formation. Culture medium peroxidases were not able to generate H₂O₂ in vitro with Cys or GSH as reductants. These thiols, however, generated H₂O₂ non-enzymically at pH 4.5. [³⁵S]Sulphate feeding to spruce cells showed that endogenous sulphur-containing compounds (including GSH, GSSG and cysteic acid) existed in the culture medium. The apoplastic levels of these were, however, undetectable by the monobromobimane method suggesting that their contribution to apoplastic H₂O₂ formation is probably minor. Azide, an inhibitor of haem-containing enzymes, slightly inhibited constitutive H₂O₂ generation but strongly delayed the elicitor-induced H₂O₂ accumulation. Diphenylene iodonium, an inhibitor of flavin-containing enzymes, efficiently inhibited H₂O₂ production irrespective of elicitation. Elicitation led to downregulation of the expression of several peroxidase genes, and peroxidase activity in the culture medium was slightly reduced. Expression of three other peroxidase genes and a respiratory burst oxidase homologue (rboh) gene were upregulated. These data suggest that both peroxidases and rboh may contribute to H₂O₂ generation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Spectroscopic Behavior and Biological Activity of K<Subscript>2</Subscript>[VO(O<Subscript>2</Subscript>)NTA]·2H<Subscript>2</Subscript>O

The dihydrated potassium salt of the complex anion [VO(O₂)NTA]²⁻ (NTA = nitrilotriacetate anion, [N(CH₂-COO)₃]³⁻) was thoroughly characterized by electronic and vibrational (infrared and Raman) spectroscopies. The bioactivity of the complex on the cell proliferation was tested on three cell lines in culture (UMR106 rat osteosarcoma-derived cells, Caco-2 derived from a human colon adenocarcinoma, and RAW 264.7, a macrophage murine cell line).     

Succinate is the controller of O<Stack><Subscript>2</Subscript><Superscript>−</Superscript></Stack>/H<Subscript>2</Subscript>O<Subscript>2</Subscript> release at mitochondrial complex I : negative modulation by malate,positive by cyanide

Mitochondrial production of H₂O₂ is low with NAD substrates (glutamate/pyruvate, 3 and 2 mM) (G/P) and increases over ten times upon further addition of succinate, with the formation of a sigmoidal curve (semimaximal value at 290 μM, maximal H₂O₂ production at 600 μM succinate). Malate counteracts rapidly the succinate induced increased H₂O₂ release and moves the succinate dependent H₂O₂ production curve to the right. Nitric oxide (NO) and carbon monoxide (CO) are cytochrome c oxidase inhibitors which increase mitochondrial ROS production. Cyanide (CN⁻) was used to mimic NO and CO. In the presence of G/P and succinate (300 μM), CN⁻ progressively increased the H₂O₂ release rate, starting at 1.5 μM. The succinate dependent H₂O₂ production curve was moved to the left by 30 μM CN⁻. The V_max was little modified. We conclude that succinate is the controller of mitochondrial H₂O₂ production, modulated by malate and CN⁻. We propose that succinate promotes an interaction between Complex II and Complex I, which activates O₂⁻ production.     

A theoretical study on the reaction mechanism of O<Subscript>2</Subscript> with C<Subscript>4</Subscript>H<Subscript>9</Subscript>• radical

Ab initio calculations have been performed using the complete basis set model (CBS-QB3) to study the reaction mechanism of butane radical (C₄H₉•) with oxygen (O₂). On the calculated potential energy surface, the addition of O₂ to C₄H₉• forms three intermediates barrierlessly, which can undergo subsequent isomerization or decomposition reaction leading to various products: HOO• + C₄H₈, C₂H₅• + CH₂CHOOH, OH• + C₃H₇CHO, OH• + cycle-C₄H₈O, CH₃• + CH₃CHCHOOH, CH₂OOH• + C₃H₆. Five pathways are supposed in this study. After taking into account the reaction barrier and enthalpy, the most possible reaction pathway is C₄H₉• + O₂ → IM1 → TS5 → IM3 → TS6 → IM4 → TS7 → OH• + cycle-C₄H₈O.     

Insight into π-hole interactions containing the inorganic heterocyclic compounds S<Subscript>2</Subscript>N<Subscript>2</Subscript>/SN<Subscript>2</Subscript>P<Subscript>2</Subscript>

Similar to σ-hole interactions, the π-hole interaction has attracted much attention in recent years. According to the positive electrostatic potentials above and below the surface of inorganic heterocyclic compounds S₂N₂ and three SN₂P₂ isomers (heterocyclic compounds 1–4), and the negative electrostatic potential outside the X atom of XH₃ (X = N, P, As), S₂N₂/SN₂P₂?XH₃ (X = N, P, As) complexes were constructed and optimized at the MP2/aug-cc-pVTZ level. The X atom of XH₃ (X = N, P, As) is almost perpendicular to the ring of the heterocyclic compounds. The π-hole interaction energy becomes greater as the trend goes from 1?XH₃ to 4?XH₃. These π-hole interactions are weak and belong to “closed-shell” noncovalent interactions. According to the energy decomposition analysis, of the three attractive terms, the dispersion energy contributes more than the electrostatic energy. The polarization effect also plays an important role in the formation of π-hole complexes, with the contrasting phenomena of decreasing electronic density in the π-hole region and increasing electric density outside the X atom of XH₃ (X = N, P, As).

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Graphical abstract Computed density difference plots for the complexes 3?NH ₃ (a ₁), 3?PH ₃ (b ₁), 3?AsH ₃ (c ₁) and electron density shifts for the complexes 3?NH ₃ (a ₂), 3?PH ₃ (b ₂),3?AsH ₃ (c ₂) on the 0.001 a.u. contour

     

Presence of Ornithine in the Urate-binding α<Subscript>1</Subscript>—<Subscript>2</Subscript>Globulin

THE urate-binding α₁–α₂ globulin has been isolated from human plasma in a highly purified state¹. The protein was purified by DEAE-‘Sephadex’, ammonium sulphate precipitation and semi-preparative Polyacrylamide gel electrophoresis. The urate-binding α₁–α₂ globulin is a rod-shaped glycoprotein, containing 12.1% carbohydrate, with an isoelectric point of 4.6 and a molecular weight of 67,000 ± 4,000. Amino-acid analysis indicated an unknown basic compound which appeared as an extra peak just in front of lysine¹. To identify this compound, high voltage paper electrophoresis has been carried out on a plate electrophoresis apparatus in pyridine-acetate buffer pH 3.5. A spot separated out corresponding to ornithine. Amino-acid analysis on a BC-200 automatic analyser (Bio-Cal Instruments Co., West Germany), with a 54 cm column at 55° C and with 0.35 M sodium citrate buffer, pH 5.28, as elution buffer at a flow-rate of 150 ml./h, showed that ornithine was present. The presence of ornithine in the protein hydrolysate was also verified by gas chromatography/mass spectrometry².     

Facet shapes and thermo-stabilities of H<Subscript>2</Subscript>SO<Subscript>4</Subscript>•HNO<Subscript>3</Subscript> hydrates involved in polar stratospheric clouds

The nucleation, ice crystal shapes and thermodynamic stability of polar stratospheric clouds particles are interesting concerns owing to their implication in the ozone layer destruction. Some of these particles are formed by conformers of H₂O, HNO₃, and H₂SO₄. We carried out calculations using density functional theory (DFT) to obtain optimized structures. Several stable trimers are achieved —divided in two groups, one with HNO₃ moiety, second with H₂SO₄ moiety— after pre-optimization at B3LYP/6-31G and subsequently optimization at B3LYP/aug-cc-pVTZ level of theory. For both most stable conformers five H₂O molecules are added to their optimized trimers to calculate hydrated geometries. The OH stretching harmonic frequencies are provided for all aggregates. The zero-point energy correction (ZEPC), relative electronic energies (?E), relative reaction Gibbs free energies ?(?G)_k-relative, and cooling constant (K _cooling ) are reported at three temperatures: 188 K, 195 K, and 210 K. Shapes given in our calculations are compared with various experimental shapes as well as comparisons with their thermo-stabilities.

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Graphical Abstract Facet shapes and thermo-stabilities of H₂SO₄?HNO₃ hydrates involved in polar stratospheric clouds. IR spectrum of WNS-1+5W structure and its circular facet

     

The neural γ<Subscript>2</Subscript>α<Subscript>1</Subscript>β<Subscript>2</Subscript>α<Subscript>1</Subscript>β<Subscript>2</Subscript> gamma amino butyric acid ion channel receptor: structural analysis of the effects of the ivermectin molecule and disulfide bridges

While ~30% of the human genome encodes membrane proteins, only a handful of structures of membrane proteins have been resolved to high resolution. Here, we studied the structure of a member of the Cys-loop ligand gated ion channel protein superfamily of receptors, human type A γ₂α₁β₂α₁β₂ gamma amino butyric acid receptor complex in a lipid bilayer environment. Studying the correlation between the structure and function of the gamma amino butyric acid receptor may enhance our understanding of the molecular basis of ion channel dysfunctions linked with epilepsy, ataxia, migraine, schizophrenia and other neurodegenerative diseases. The structure of human γ₂α₁β₂α₁β₂ has been modeled based on the X-ray structure of the Caenorhabditis elegans glutamate-gated chloride channel via homology modeling. The template provided the first inhibitory channel structure for the Cys-loop superfamily of ligand-gated ion channels. The only available template structure before this glutamate-gated chloride channel was a cation selective channel which had very low sequence identity with gamma aminobutyric acid receptor. Here, our aim was to study the effect of structural corrections originating from modeling on a more reliable template structure. The homology model was analyzed for structural properties via a 100 ns molecular dynamics (MD) study. Due to the structural shifts and the removal of an open channel potentiator molecule, ivermectin, from the template structure, helical packing changes were observed in the transmembrane segment. Namely removal of ivermectin molecule caused a closure around the Leu 9 position along the ion channel. In terms of the structural shifts, there are three potential disulfide bridges between the M1 and M3 helices of the γ₂ and 2 α₁ subunits in the model. The effect of these disulfide bridges was investigated via monitoring the differences in root mean square fluctuations (RMSF) of individual amino acids and principal component analysis of the MD trajectory of the two homology models—one with the disulfide bridge and one with protonated Cys residues. In all subunit types, RMSF of the transmembrane domain helices are reduced in the presence of disulfide bridges. Additionally, loop A, loop F and loop C fluctuations were affected in the extracellular domain. In cross-correlation analysis of the trajectory, the two model structures displayed different coupling in between the M2–M3 linker region, protruding from the membrane, and the β1-β2/D loop and cys-loop regions in the extracellular domain. Correlations of the C loop, which collapses directly over the bound ligand molecule, were also affected by differences in the packing of transmembrane helices. Finally, more localized correlations were observed in the transmembrane helices when disulfide bridges were present in the model. The differences observed in this study suggest that dynamic coupling at the interface of extracellular and ion channel domains differs from the coupling introduced by disulfide bridges in the transmembrane region. We hope that this hypothesis will be tested experimentally in the near future.     

Three-dimensional structure of the large cytoplasmic H<Subscript>4</Subscript>–H<Subscript>5</Subscript> loop of Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase deduced by restraint-based comparative modeling shows only one ATP binding site

Homology modeling of the complete structure of the large cytoplasmic loop between the fourth and fifth transmembrane segments (H₄–H₅ loop) of the subunit of Na⁺/K⁺-ATPase is reported. The deduced amino acid sequence shows high sequence identity and homology to the Ca²⁺-ATPase (32.8% identity and 53.3% similarity in our alignment), whose tertiary structure has been solved recently at 2.6-Å resolution by X-ray crystallography. This high homology allowed the construction of a model structure using the MODELLER program. Refinement was achieved through interactive visual and algorithmic analysis and minimization with the TRIPOS force field included in the SYBYL/MAXIMIN2 module. The docking of ATP as a substrate into the active site of the model was explored with the AUTODOCK program followed by molecular mechanics optimization of the most interesting complexes. Thus, the docking of ATP into the resulting model of the H₄–H₅ loop gave evidence for the existence of one ATP binding site only. We were able to specify Cys⁵⁴⁹, Phe⁵⁴⁸, Glu⁵⁰⁵, Lys⁵⁰¹, Gln⁴⁸², Lys⁴⁸⁰, Ser⁴⁷⁷, Phe⁴⁷⁵ and Glu⁴⁴⁶ as parts of the ATP binding site with Lys⁵⁰¹ located in the depth of the positively charged binding pocket.Electronic Supplementary Material available.     

Synthesis,characterization, and studies on DNA binding of the complex Fe(Sal<Subscript>2</Subscript>dienNO<Subscript>3</Subscript>·H<Subscript>2</Subscript>O)

A new metal complex, Fe(Sal₂dienNO₃·H₂O) (where Sal is salicylaldehyde and dien is diethylenetriamine), has been synthesized and characterized. The interactions between the Fe(III) complex and calf thymus DNA has been investigated using UV and fluorescence spectra, viscosity, thermal denaturation, and molecular modeling. The cleavage reaction on plasmid DNA has been monitored by agarose gel electrophoresis. The experimental results show that the mode of binding of the complex to DNA is classical intercalation and the complex can cleave pBR322 DNA.     

Kinetic analysis of recombinant mammalian α<Subscript>1</Subscript> and α<Subscript>1</Subscript>β glycine receptor channels

To analyze the influence of the beta-subunit on the kinetic properties of GlyR channel currents, alpha(1)-subunits and alpha(1)beta-subunits were transiently expressed in HEK 293 cells. A piezo dimorph was used for fast application of glycine to outside-out patches. The rise time of activation was dose dependent for both receptors and decreased with increasing glycine concentrations. Subunit composition had no effect on the time course of activation. Coexpression of alpha(1)- and beta-subunits resulted in a significantly lower EC(50) and a reduced slope of the dose-response curve of glycine compared with expression of alpha(1)-subunits alone. For both receptor subtypes, the time course of desensitization was concentration dependent. Desensitization was best fitted with a single time constant at 10-30 micro M, with two at 0.1 mM, and at saturating concentrations (0.3-3 mM) with three time constants. Desensitization of homomeric alpha(1)-receptor channels was significantly slower than that of alpha(1)beta-receptor channels. The time course of current decay after the end of glycine pulses was tested at different pulse durations of 1 mM glycine. It was best fitted with two time constants for both alpha(1) and alpha(1)beta GlyR channels, and increased significantly with increasing pulse duration.     

Effects of Prostaglandins F<Subscript>2α</Subscript>, E<Subscript>2</Subscript> and E<Subscript>1</Subscript> on Fertility in Mice

PROSTAGLANDIN (PG) F_2αhas antifertility effects in many species^1–3 but there are conflicting suggestions as to its mechanism of action. For example, it may cause the degeneration of the corpus luteum by decreasing blood flow in the uteroovarian vein⁴; alternatively, its action may be due to a hypersecretion of luteinizing hormone (LH) by the pituitary^3,5. I have investigated the effects of PGF_2α, E₂ and E₁ on pregnancy in mice and examined the mechanism of action of PGF_2α.     

On the generation of OH<Superscript>·</Superscript> radical species from H<Subscript>2</Subscript>O<Subscript>2</Subscript> by Cu(I) amyloid beta peptide model complexes: a DFT investigation

According to different studies, the interaction between amyloid β-peptide (Aβ) and copper ions could yield radical oxygen species production, in particular the highly toxic hydroxyl radical OH^· that is suspected to contribute to Alzheimer’s disease pathogenesis. Despite intensive experimental and computational studies, the nature of the interaction between copper and Aβ peptide, as well as the redox reactivity of the system, are still matter of debate. It was proposed that in Cu(II) → Cu(I) reduction the complex Cu(II)–Aβ could follow a multi-step conformational change with redox active intermediates that may be responsible for OH^· radical production from H₂O₂ through a Fenton-like process. The purpose of this work is to evaluate, using ab initio Density Functional Theory computations, the reactivity of different Cu(I)–Aβ coordination modes proposed in the literature, in terms of OH^· production. For each coordination model, we considered the corresponding H₂O₂ adduct and performed a potential energy surface scan along the reaction coordinate of O–O bond dissociation of the peroxide, resulting in the production of OH^· radical, obtaining reaction profiles for the evaluation of the energetic of the process. This procedure allowed us to confirm the hypothesis according to which the most populated Cu(I)–Aβ two-histidine coordination is not able to perform efficiently H₂O₂ reduction, while a less populated three-coordinated form would be responsible for the OH^· production. We show that coordination modes featuring a third nitrogen containing electron-donor ligand (an imidazole ring of an histidine residue is slightly favored over the N-terminal amine group) are more active towards H₂O₂ reduction.     

Pellino-1 Protects Periodontal Ligament Stem Cells Against H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Apoptosis via Activation of NF-κB Signaling

To determine the protective effects of Pellino-1 against H₂O₂-induced apoptosis in periodontal ligament stem cells (PDLSC). We demonstrated that H₂O₂ decreases PDLSC viability by 40 and 50% with the concentrations of 400 and 500 μM, respectively, with an observed downregulation of Pellino-1 mRNA and protein; we further concluded that overexpression of Pellino-1 significantly lowers 8-hydroxy-2′-deoxyguanosine levels by 10% and upregulates superoxide dismutase 1, glutathione peroxidase levels, and catalase mRNA levels by 200, 40, and 250%, respectively. More importantly, we found that overexpression of Pellino-1 inhibited H₂O₂-induced cellular apoptosis through the activation of the NF-κB signaling pathway. Pellino-1 may be critically important for cell survival in the presence of oxidative elements; activation of the NF-κB signaling cascade was required for the overexpression of Pellino-1 to protect the cells from H₂O₂-induced apoptosis.     

Indirubin-3-Oxime Prevents H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Neuronal Apoptosis via Concurrently Inhibiting GSK3β and the ERK Pathway

Oxidative stress-induced neuronal apoptosis plays an important role in many neurodegenerative disorders. In this study, we have shown that indirubin-3-oxime, a derivative of indirubin originally designed for leukemia therapy, could prevent hydrogen peroxide (H₂O₂)-induced apoptosis in both SH-SY5Y cells and primary cerebellar granule neurons. H₂O₂ exposure led to the increased activities of glycogen synthase kinase 3β (GSK3β) and extracellular signal-regulated kinase (ERK) in SH-SY5Y cells. Indirubin-3-oxime treatment significantly reversed the altered activity of both the PI3-K/Akt/GSK3β cascade and the ERK pathway induced by H₂O₂. In addition, both GSK3β and mitogen-activated protein kinase inhibitors significantly prevented H₂O₂-induced neuronal apoptosis. Moreover, specific inhibitors of the phosphoinositide 3-kinase (PI3-K) abolished the neuroprotective effects of indirubin-3-oxime against H₂O₂-induced neuronal apoptosis. These results strongly suggest that indirubin-3-oxime prevents H₂O₂-induced apoptosis via concurrent inhibiting GSK3β and the ERK pathway in SH-SY5Y cells, providing support for the use of indirubin-3-oxime to treat neurodegenerative disorders caused or exacerbated by oxidative stress.     

Process contribution evaluation for COD removal and energy production from molasses wastewater in a BioH<Subscript>2</Subscript>–BioCH<Subscript>4</Subscript>–MFC-integrated system

In this study, a three-stage-integrated process using the hydrogenic process (BioH₂), methanogenic process (BioCH₄), and a microbial fuel cell (MFC) was operated using molasses wastewater. The contribution of individual processes to chemical oxygen demand (COD) removal and energy production was evaluated. The three-stage integration system was operated at molasses of 20 g-COD L^?1, and each process achieved hydrogen production rate of 1.1 ± 0.24 L-H₂ L^?1 day^?1, methane production rate of 311 ± 18.94 mL-CH₄ L^?1 day^?1, and production rate per electrode surface area of 10.8 ± 1.4 g m^?2 day^?1. The three-stage integration system generated energy production of 32.32 kJ g-COD^?1 and achieved COD removal of 98 %. The contribution of BioH₂, BioCH₄, and the MFC reactor was 20.8, 72.2, and, 7.0 % of the total COD removal, and 18.7, 81.2, and 0.16 % of the total energy production, respectively. The continuous stirred-tank reactor BioH₂ at HRT of 1 day, up-flow anaerobic sludge blanket BioCH₄ at HRT of 2 days, and MFC reactor at HRT of 3 days were decided in 1:2:3 ratios of working volume under hydraulic retention time consideration. This integration system can be applied to various configurations depending on target wastewater inputs, and it is expected to enhance energy recovery and reduce environmental impact of the final effluent.     

An optical material for the detection of trace S<Subscript>2</Subscript>O<Subscript>3</Subscript><Superscript>2−</Superscript> in milk based on a copper complex

A novel S₂O₃ ^2? luminescent sensor (Cu²⁺-p-CPIP) was developed and the presence of S₂O₃ ^2? caused an obvious fluorescence enhancement at 420 nm upon excitation at 330 nm, which could be distinguished with the naked eye under a UV lamp. Remarkably, the compound exhibited excellent selective and sensitive response to S₂O₃ ^2? over other common anions with a micromolar limit of detection (0.442 μM) in DMSO/H₂O (v/v, 1:1) buffer. The absorbance intensity and the color of Cu²⁺-p -CPIP solution changed gradually with the increase of S₂O₃ ^2? concentration. The proposed method was applied to the determination of S₂O₃ ^2? in milk samples and the recoveries were 97.5–105%. The preparation of Cu²⁺-p -CPIP exhibited the quick, simple and facile advantages. The results showed that Cu²⁺-p -CPIP can be a good candidate for simple, rapid and sensitive colorimetric detection of S₂O₃ ^2? in aqueous solution.     

Hydrogen-bonding behavior of various conformations of the HNO<Subscript>3</Subscript>…(CH<Subscript>3</Subscript>OH)<Subscript>2</Subscript> ternary system

Nine minima were found on the intermolecular potential energy surface for the ternary system HNO₃(CH₃OH)₂ at the MP2/aug-cc-pVDZ level of theory. The cooperative effect, which is a measure of the hydrogen-bonding strength, was probed in these nine conformations of HNO₃…(CH₃OH)₂. The results are discussed here in terms of structures, energetics, infrared vibrational frequencies, and topological parameters. The cooperative effect was observed to be an important contributor to the total interaction energies of the cyclic conformers of HNO₃…(CH₃OH)₂, meaning that it cannot be neglected in simulations in which the pair-additive potential is applied.

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Graphical abstract The H-bonding behavior of various conformations of the HNO₃(CH₃OH)₂ trimer was investigated