A multishear microfluidic device for quantitative analysis of calcium dynamics in osteoblasts

Reactive oxygen species (ROS) are an important factor in the development of skin lesions in diabetes. A new antioxidant, hydrogen, can selectively neutralize hydroxyl radicals (OH) and peroxynitrite (ONOO⁻) in cell-free systems, whereas it seldom reacts with other ROS. Fibroblasts are a key component of skin. In the present study, we investigated the protective effects of hydrogen-rich medium on human skin fibroblasts (HSFs) under oxidative stress. Confocal microscopy was used to assay both the intracellular superoxide anion () concentration and the mitochondrial membrane potential (ΔΨ). Cell viability was determined using the Cell Counting Kit-8 (CCK-8). The concentrations of cellular malonaldehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), 8-hydroxy-2′-deoxyguanosine (8-OHdG) and 3-nitrotyrosine (3-NT) were also measured. The results revealed that both mannitol and high glucose could cause oxidative stress in HSFs. Interestingly, the use of a hydrogen-rich medium significantly reduced the level of intracellular , stabilized the ΔΨ and attenuated production of MDA, 8-OHdG and 3-NT which efficiently enhanced the antioxidative defense system and protected the HSFs from subsequent oxidative stress damage. In other words, hydrogen decreased the excessive generation of intracellular and elevated the cellular antioxidative defense. Based on our results, hydrogen may have applications in the treatment of skin diseases caused by diabetes.     

A comparative study of antioxidative activities of cell-wall polysaccharides

Oxidative burst in plants is elicited by biotic and abiotic stressors. Analogously to some monosaccharides which act as intracellular antioxidants, cell-wall polysaccharides may be in charge of buffering free-radical production in the extracellular compartment under pronounced prooxidative settings. Although a wide range of plant polysaccharides have been examined for their antioxidative properties, this usually has not been done in a coherent and comparative manner and against biologically relevant reactive species. Here we show that different cell-wall polysaccharides, cellulose, pectin, d-galacto-d-mannan, arabinogalactan, and xylan, exhibit distinctive antioxidative activities against the hydroxyl radical (OH)-generating Fenton reaction and superoxide. We found, using an EPR spin-trapping method, that the main carriers of ‘anti-Fenton’ activity in the plant cell wall are pectin and xylan. They most likely act by binding metal ions in such a manner to allow the Fenton reaction, after which they scavenge OH. Such a mode of action is preferred by cells resulting in a safe degradation of H₂O₂. On the other hand, the polysaccharides examined showed similar superoxide scavenging capacities. We propose that plants may employ different antioxidative characteristics of polysaccharides to regulate their redox status by modifying the composition of the cell wall.     

Trichloroacetic acid dehalogenation by reductive radicals

Advanced oxidation processes, using either UVC/H₂O₂ or UVC/K₂S₂O₈, both in the presence of H₂CO₂ or CH₃OH are very efficient in mineralizing aqueous solutions of trichloroacetic acid (TCAA) leaving no toxic residues. The main reaction initiating TCAA depletion is its reduction by the radicals or CH₂OH to yield radicals and Cl⁻ anions. Further thermal reactions of lead to the formation of CO₂ and HCl. Molecular oxygen competes with TCAA for and CH₂OH radicals. However, in experiments under continuous irradiation of initially air-saturated solutions in closed reactors, the dissolved molecular oxygen concentration was depleted to low enough levels to favor the reaction of the reducing radicals with TCAA. A general reaction mechanism is proposed and discussed. The reaction between superoxide radical anions and TCAA was found to be of low efficiency.     

Redox chemistry of copper complexes of 6,7-dicyanodipyridoquinoxaline: A pulse radiolysis study

A series of copper (II) complexes having the general formula Cu(phen)_n(dicnq)_2−nCl₂ (n = 0,1,2) (1,10-phenanthroline (phen) and/or 6,7-dicyanodipyridoquinoxaline (dicnq) were synthesized and characterized by optical, elemental analysis and IR. The reactions of oxidizing (OH) and reducing () radicals with these complexes were studied by pulse radiolysis. Their absorption spectra have bands in the UV region (?350 nm) consisting of an intense π → π∗ transition due to the ligands (ε ∼ 10⁵ dm³ mol⁻¹ cm⁻¹) and weak MLCT (dπ → π∗) band in the visible region and are non-luminescent. The OH radical reacts with all complexes at diffusion controlled rates and reacts by addition to the ligands and in the case OH adduct of Cu(dicnq)₂Cl₂, an intramolecular charge transfer followed deprotonation resulting in Cu(I) complex was noticed. The rates of reaction of with Cu(II) complexes are high (k ≈ 10¹⁰ dm³ mol⁻¹ s⁻¹) and the transient spectra show absorption maximum at 440 nm indicating reduction of Cu(II) to Cu(I).     

On the mechanism of formation and spectral properties of radical anions generated by the reduction of -[Re(CO)3(5-nitro-1,10-phenanthroline)] and -[Re(CO)3(3,4,7,8-tetramethyl-1,10-phenanthroline)] pendants in poly-4-vinylpyridine polymers

The electrochemical reduction in aprotic media of -[Re^I(CO)₃L]⁺ pendants in poly-4-vinylpyridine polymers is compared to that of [Re^I(CO)₃L]⁺ complexes (L = 5-nitro-1,10-phenanthroline and 3,4,7,8-tetramethyl-1,10-phenanthroline). The UV-Vis absorption spectra of the reduced radical anions of 5-nitro-1,10-phenanthroline (NO₂-phen) and 3,4,7,8-tetramethyl-1,10-phenanthroline (tmphen) were obtained by spectro-electrochemistry of [Re^I(CO)₃(NO₂-phen)(CH₃CN)]⁺ and [Re^I(CO)₃(tmphen)(CH₃CN)]⁺, respectively. Similar spectra were obtained for the radical anions -phen and tmphen⁻ after pulse radiolysis experiments with -[Re^I(CO)₃L]⁺-containing polymers. The analysis of the time-resolved difference spectra was performed using “multivariate curve resolution” (MCR) techniques. Unlike , CH₂OH radicals were unable to reduce tmphen ligands. The reaction of and/or CH₂OH with -[Re^I(CO)₃(NO₂-phen)]⁺-containing polymers generates -[Re^I(CO)₃(-phen)] pendants which after disproportionation give rise to products with λ_max = 380 nm. The kinetic behavior of -[Re^I(CO)₃(-phen)] pendants under different experimental conditions is discussed.     

Dinuclear gold(III) pyrazolato complexes - Synthesis, structural characterization and transformation to their trinuclear gold(I) and gold(I/III) analogues

The reaction of AuCl₃py with Na(pz∗) (pz∗ = pyrazolato, or substituted pyrazolato anion) yields stable dinuclear [cis-Au^IIICl₂(μ-pz∗)]₂ complexes. In the presence of a base, the latter undergo reduction with concomitant transformation of the dinuclear -structure to trinuclear Au^I, Au^III (containing trans Au^IIICl₂-centres) and species.     

Kinetics of reduction of tyrosine phenoxyl radicals by glutathione

Modification of tyrosine (TyrOH) is used as a marker of oxidative and nitrosative stress. 3,3′-Dityrosine formation, in particular, reflects oxidative damage and results from the combination of two tyrosyl phenoxyl radicals (TyrO). This reaction is in competition with reductive processes in the cell which ‘repair’ tyrosyl radicals: possible reductants include thiols and ascorbate. In this study, a rate constant of 2 × 10⁶ M⁻¹ s⁻¹ was estimated for the reaction between tyrosyl radicals and glutathione (GSH) at pH 7.15, generating the radicals by pulse radiolysis and monitoring the tyrosyl radical by kinetic spectrophotometry. Earlier measurements have suggested that this ‘repair’ reaction could be an equilibrium, and to investigate this possibility the reduction (electrode) potential of the (TyrO,H⁺/TyrOH) couple was reinvestigated by observing the fast redox equilibrium with the indicator 2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonate). Extrapolation of the reduction potential of TyrO measured at pH 9–11 indicated the mid-point reduction potential of the tyrosyl radical at pH 7, E_m7(TyrO,H⁺/TyrOH) = 0.93 ± 0.02 V. This is close to the reported reduction potential of the glutathione thiyl radical, E_m7 = 0.94 ± 0.03 V, confirming the ‘repair’ equilibrium constant is of the order of unity and suggesting that efficient reduction of TyrO by GSH might require removal of thiyl radicals to move the equilibrium in the direction of repair. Loss of thiyl radicals, facilitating repair of TyrO, can arise either via conjugation of thiyl with thiol/thiolate or oxygen, or unimolecular transformation, the latter important at low concentrations of thiols and oxygen.     

Comparison of antioxidant abilities of magnolol and honokiol to scavenge radicals and to protect DNA

The antioxidant properties of magnolol and honokiol were evaluated in the experimental systems of reducing ONOO⁻ and ¹O₂, bleaching β-carotene in linoleic acid (LH) emulsion, and trapping 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS⁺) and 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH), and then were applied to inhibit the oxidation of DNA induced by Cu²⁺/glutathione (GSH) and 2,2′-azobis(2-amidinopropane hydrochloride) (AAPH). Magnolol and honokiol were active to reduce ONOO⁻ and ¹O₂. Honokiol showed a little higher activity to protect LH and to inhibit Cu²⁺/GSH-induced oxidation of DNA than magnolol. In addition, honokiol exhibited higher activities to trap ABTS⁺ and DPPH than magnolol. In particular, honokiol trapped 2.5 radicals while magnolol only trapped 1.8 radicals in protecting DNA against AAPH-induced oxidation. The obtained results suggested that low antioxidant ability of magnolol may be related to the intramolecular hydrogen bond formed between di-ortho-hydroxyl groups, which hindered the hydrogen atom in hydroxyl group to be abstracted by radicals. Therefore, the antioxidant capacity of magnolol was lower than that of honokiol.     

Reactive oxygen and nitrogen species regulate inducible nitric oxide synthase function shifting the balance of nitric oxide and superoxide production

Inducible NOS (iNOS) is induced in diseases associated with inflammation and oxidative stress, and questions remain regarding its regulation. We demonstrate that reactive oxygen/nitrogen species (ROS/RNS) dose-dependently regulate iNOS function. Tetrahydrobiopterin (BH₄)-replete iNOS was exposed to increasing concentrations of ROS/RNS and activity was measured with and without subsequent BH₄ addition. Peroxynitrite (ONOO⁻) produced the greatest change in NO generation rate, ∼95% decrease, and BH₄ only partially restored this loss of activity. Superoxide () greatly decreased NO generation, however, BH₄ addition restored this activity. Hydroxyl radical (OH) mildly decreases NO generation in a BH₄-dependent manner. iNOS was resistant to H₂O₂ with only slightly decreased NO generation with up to millimolar concentrations. In contrast to the inhibition of NO generation, ROS enhanced production from iNOS, while ONOO⁻ had the opposite effect. Thus, ROS promote reversible iNOS uncoupling, while ONOO⁻ induces irreversible enzyme inactivation and decreases both NO and production.     

Formation of an observable intermediate during the reduction of [Co(NH3)5CN] by CRR(OH) radicals

CR¹R²OH, Rⁱ = CH₃ or H, react with the complex [Co^III(NH₃)₅CN]²⁺ to form an observable intermediate probably via bonding to the nitrogen of the cyanide. This intermediate isomerizes to form a second intermediate. The second intermediate decomposes into Co²⁺(aq), 5NH₄⁺, CN⁻ and R¹R²CO. The plausible structures of the intermediates are discussed. The radicals CH³, CH₂CHO, , and are considerably less reactive towards this complex, the formation of intermediates in their presence is not observed.     

Half-sandwich Mo(III) complexes with asymmetric diazadiene ligands

The asymmetric 1,4-diazadiene ligands R^∗NCHCHNR^∗ [R^∗ = (S)-CH(CH₃)Ph], , and 2,2′-bis(4-ethyloxazoline), as-ox, have been used to generate half-sandwich Mo^III derivatives by addition to Cp₂Mo₂Cl₄. Ligand affords a mononuclear, paramagnetic 17-electron product, , whereas as-ox leads to the isolation of a dinuclear compound where only one molecule of ligand has been added per two Mo atoms, Cp₂Mo₂Cl₄(as-ox). In the presence of free as-ox, this compound coexists with the paramagnetic mononuclear complex in solution. Both products are capable of controlling the radical polymerization of styrene under typical atom transfer radical polymerization (ATRP) conditions. However, the tacticity of the resulting polystyrene does not differ from that given by conventional free radical polymerization.     

The influence of the cation of quaternized chitosans on antioxidant activity

Various quaternized chitosans (QCSs) were synthesized according to previous method. Their reducing power and antioxidant potency against hydroxyl radicals (OH) and hydrogen peroxide (H₂O₂) were explored by the established systems in vitro. The QCSs exhibited markedly antioxidant activity, especially TCEDMCS, whose IC₅₀ on hydroxyl radicals was 0.235 mg/mL. They showed 65–80% scavenging effect on hydrogen peroxide at a dose of 0.5 mg/mL. Generally, the antioxidant activity decreased in the order TCEDMCS > TBEDMCS > EDMCS > PDMCS > IBDMCS > Chitosan. Furthermore, the order of their OH and H₂O₂ scavenging activity was consistent with the electronegativity of different substituted groups in the QCSs. The QCSs showed much stronger antioxidant activity than that of chitosan may be due to the positive charge density of the nitrogen atoms in QCSs strengthened by the substituted groups.     

A glycyl free radical as the precursor in the synthesis of carbon monoxide and cyanide by the [FeFe]-hydrogenase maturase HydG

HydG uses tyrosine to synthesize the CN⁻/CO ligands of [FeFe]-hydrogenase active site. We have mutated two of the [4Fe-4S]-cluster cysteine ligands of the HydG C-terminal domain (CTD) to serine. The double mutant can still synthesize CN⁻ but not CO. In a mutant lacking the CTD both CN⁻ and CO synthesis are abolished. Like in ThiH, the initial steps of CN⁻ synthesis are carried out in the TIM-barrel domain of HydG but some component(s) of the CTD are later needed. The mutants indicate that CO synthesis is metal-based and occurs in the CTD. We postulate that CN⁻/CO synthesis is initiated by H₂N-CH-. Fragmentation of this radical into H₂N-CH₂ and CO₂ or H₂CNH and provides plausible precursors for CN⁻/CO synthesis.     

Anionic effects on the formation of tridentate 4′-chloro-2,2′:6′,2″-terpyridine copper(II) complexes having 1:1 or 1:2 ratio of metal and ligand and their crystal symmetry

A facile synthetic procedure has been used to prepare one five-coordinate and four six-coordinate copper(II) complexes of 4′-chloro-2,2′:6′,2″-terpyridine (tpyCl) ligand with different counterions (, , , , and ) in high yields. They are formulated as [Cu(tpyCl-κ³N,N′,N′′)(SO₄-κO)(H₂O-κO)] · 2H₂O (1), trans-[Cu(tpyCl-κ³N,N′,N″)(NO₃-κO)₂(H₂O-κO)] (2), [Cu(tpyCl-κ³N,N′,N″)₂](BF₄)₂ (3), [Cu(tpyCl-κ³N,N′,N″)₂](PF₆)₂ (4) and [Cu(tpyCl-κ³N,N′,N″)₂](ClO₄)₂ (5) and versatile interactions in supramolecular level including coordinative bonding, O-H?O, O-H?Cl, C-H?F, and C-H?Cl hydrogen bonding, π-π stacking play essential roles in forming different frameworks of 1-5. It is concluded that the difference of coordination abilities of the counterions used and the experimental conditions codominate the resulting complexes with 1:1 or 1:2 ratio of metal and ligand.     

Density-functional analysis of the electronic structure of tris-bipyridyl Ru(II) sensitisers

Excited state transitions and energies of a series of [Ru(bpy)₃]²⁺ type complexes incorporating the ligand, 4,4′-bis-phosphonato(methyl)-2,2′-bipyridine (dmpbpy) was investigated, and the influence of this organometallic ligand on the electronic structure of the complexes was examined using Time-Dependent Density Functional Theory (TD-DFT). Experimental data and the theoretical TD-DFT calculations were presented to support the effect of non-equivalent ligand substitution on spectral and molecular orbital (MO) energy properties on this class of tris-chelate surface sensitisers. For the series of complexes studied, it was identified that the lowest lying LUMO states were consistently found to reside on the ligand 2,2′-bipyridine (bpy) for gas phase calculations. As an implication of this, it was suggested that this could impact the effectiveness of these complexes as surface sensitisers in PEC cell applications such as the dye-sensitised solar cell (DSC) due to the lower probability of the excited state electron residing on a ligand anchored to the semiconductor substrate. However, further calculations in a solvation medium showed that the electron withdrawing nature of PO₃H₂ on dmpbpy saw the lowest lying LUMO states are populated on dmpbpy. This inhomogeneous distribution of electron density across non-equivalent ligands may have implications for further ‘spectral tuning’ of surface sensitisers. Despite the TD-DFT gas phase calculations not being corrected for solvent/media effects, the three longest wavelength bands associated with known charge transfer phenomena were identified. The symmetry allowed _MLCT in the visible region was assigned as a  ← dπ transition, the mid-UV spectrum _LC was assigned  ← π in origin. Whilst the near-UV shoulder on the blue side of _MLCT showed dσ ← dπ and π∗ ← dπ transitional character and was tentatively described as _MC/MLCT. UV-Vis absorption spectra calculated for solvated analogues containing dmpbpy indicated that the low energy transitions associated with the MLCT are subject to bathochromic shift due to solvent polarity by 0.062 eV (500 cm⁻¹) compared with the gas phase calculations, which is more highly correlated to the observed experimental transitions.     

New applications of ruthenium solar cell sensitizers N3 and N719 as luminescence turn-on anion sensors

Optical sensing of F⁻, Cl⁻, Br⁻, I⁻, OAc⁻, , , and by cis-dithiocyanatobis(2,2′-bipyridyl-4,4′-dicarboxylic acid)ruthenium(II) (N3) and bis(tetrabutylammonium) cis-dithiocyanatobis(2,2′-bipyridine-4-COOH,4′-COO⁻)ruthenium(II) (N719) have been investigated in dimethyl sulfoxide (DMSO), by means of UV-Vis absorption and emission spectrophotometric titrations. Additions of F⁻, OAc⁻, and in DMSO solution caused obvious UV-Vis spectral changes with appearance of several isosbestic points, and remarkable emission enhancements along with large blue shifts in emission bands. The values of F⁻-induced emission intensity enhancement factor (emission quantum yield enhancement factor), I/I₀ (φ/φ₀), were found to be 40 (86) and 38 (58) for N3 and N719, respectively. No obvious spectral changes were observed upon addition of Cl⁻, Br⁻, I⁻, and in DMSO solutions. Luminescent F⁻ sensing in DMSO/H₂O (4:1, v/v) has also been demonstrated to be operative with a luminescence enhancement factor of 12, indicating that N3 is very potential for practical application as fluorescent anion sensor in aqueous solution. An interaction mechanism of anion-induced deprotonation of N3 and N719 was confirmed, and the deprotonation reaction equilibrium constants of N3 and N719 were derived as well.     

The uremic solute indoxyl sulfate acts as an antioxidant against superoxide anion radicals under normal-physiological conditions

The effect of the uremic solute indoxyl sulfate (IS) on scavenging superoxide anion radicals () generated from both the xanthine/xanthine oxidase (X/XO) system and activated neutrophils was investigated by electron paramagnetic resonance spectroscopy, combined with 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO). The findings show that the presence of normal-physiological serum concentrations of IS (0.1-10 μM) resulted in decreased formation of EMPO-superoxide adduct without affecting XO activity. Furthermore, IS showed scavenging activity against cell-derived generated from activated neutrophils. In addition, IS also eliminated hydroxyl radicals. These findings suggest that IS acts as a novel endogenous antioxidant under normal-physiological conditions.     

Photocatalysis of chloroform degradation by μ-dichlorotetrachlorodipalladate(II)

Unlike other chlorometallate complexes that catalyze the photodecomposition of haloalkanes through photodissociation of a chlorine atom, both and catalyze chloroform decomposition through a process that appears to involve C-H bond breakage from an excited state association complex with chloroform. This would account for the greatly retarded rate of decomposition in CDCl₃ and for the generation of CCl₄ as a side product. In chloroform, and are in slow equilibrium with each other. The rate for the conversion of - in chloroform at 23 °C obeys the expression (0.03 M⁻¹ s⁻¹) [][Cl⁻]. The equilibrium constant, K = [][Cl⁻]²/[]², was estimated to be 3 × 10⁻³ M in CHCl₃.     

Highly hydroxylated or γ-cyclodextrin-bicapped water-soluble derivative of fullerene: The antioxidant ability assessed by electron spin resonance method and β-carotene bleaching assay

Antioxidant ability of the water-soluble derivative of fullerene (C60), prepared by high-degree hydroxylation [C60-(OH)₃₂·8H₂O] or C60/γ-cyclodextrin (1:2 mol/mol) clathrate formation [C60/(γ-CD)₂], was assessed by electron spin resonance method and β-carotene bleaching assay. These C60 derivatives have an ability to diminish a 1:2:2:1 quartet ESR spectrum attributed to hydroxyl radicals (OH) as shown by DMPO-spin trap/ESR method. Meanwhile, a singlet radical-signal different from OH-attributed signals increased in a manner dependent on concentrations of C60-(OH)₃₂·8H₂O. This might suggest that C60-(OH)₃₂·8H₂O scavenges OH owing to dehydrogenation of C60-(OH)₃₂·8H₂O, and is simultaneously oxidized to a stable radical species, which may be a dehydrogenated fullerenol radical (C60-O). Furthermore, these water-soluble derivatives of C60 suppressed fading of yellowish color characteristic of intact β-carotene in β-carotene bleaching assay. Antioxidant abilities of these derivatives were assessed as retention of yellowish color (viz absorbance at 470 nm) for 180 min. Namely, β-carotene-attributed chromaticity (% relative absorbance at 470 nm compared with the control) after 180 min was 69% for C60-(OH)₃₂·8H₂O (400 μM: C60-eq.), and 32% for C60/(γ-CD)₂ (400 μM: C60-eq.), whereas it was 6% for l(+)-ascorbic acid (400 μM) which is hydrophilic, and 85% for (±)-α-tocopherol (400 μM) which is lipophilic, respectively. Thus C60-(OH)₃₂·8H₂O and C60/(γ-CD)₂ can scavenge OH, and have a distinct antioxidative activity in the aqueous system containing linoleic acid which is abundantly contained in the cell membrane together with other unsaturated lipids. These C60 derivatives have a potential to protect the cell membrane from oxidative stress due to OH.