Higher serum CCL17 may be a promising predictor of acute exacerbations in chronic hypersensitivity pneumonitis

Background

Recent research has suggested that the Th1 and Th2 chemokine/cytokine axis contributes to the development of chronic hypersensitivity pneumonitis (HP). Acute exacerbations (AE) are significant factors in the prognosis of chronic HP. Little is known, however, about these biomarkers in association with AE in chronic HP patients.

Methods

Fifty-six patients with chronic HP were evaluated, including 14 patients during episodes of AE. Th1 mediators (C-X-C chemokine ligand [CXCL]10 and interferon [IFN]-γ), Th2 mediators (C-C chemokine ligand [CCL]17, interleukin-4, and interleukin-13), and pro-fibrotic mediator (transforming growth factor [TGF]-β) were measured to evaluate the mediators as predictors of AE. C-C chemokine receptor (CCR)4 (receptor for CCL17)-positive lymphocytes were quantified in lung specimens.

Results

Serum CCL17 levels at baseline independently predicted the first episode of AE (HR, 72.0; 95% CI, 5.03-1030.23; p = 0.002). AE was significantly more frequent in the higher-CCL17 group (≥285 pg/ml) than in the lower-CCL17 group (<285 pg/ml) (log-rank test, p = 0.0006; 1-year incidence: higher CCL17 vs. lower CCL17, 14.3% vs. 0.0%). Serum CCL17 levels and CCR4-positive cells during episodes of AE were increased from the baseline (p = 0.01 and 0.031).

Conclusions

Higher serum concentrations of CCL17 at baseline may be predictive of AE in patients with chronic HP, and CCL17 may contribute to the pathology of AE by inducing the accumulation of CCR4-positive lymphocytes in the lungs.     

A novel arsenate reductase from the bacterium Thermus thermophilus HB27: Its role in arsenic detoxification

Microorganisms living in arsenic-rich geothermal environments act on arsenic with different biochemical strategies, but the molecular mechanisms responsible for the resistance to the harmful effects of the metalloid have only partially been examined. In this study, we investigated the mechanisms of arsenic resistance in the thermophilic bacterium Thermus thermophilus HB27. This strain, originally isolated from a Japanese hot spring, exhibited tolerance to concentrations of arsenate and arsenite up to 20 mM and 15 mM, respectively; it owns in its genome a putative chromosomal arsenate reductase (TtarsC) gene encoding a protein homologous to the one well characterized from the plasmid pI258 of the Gram + bacterium Staphylococcus aureus. Differently from the majority of microorganisms, TtarsC is part of an operon including genes not related to arsenic resistance; qRT-PCR showed that its expression was four-fold increased when arsenate was added to the growth medium. The gene cloning and expression in Escherichia coli, followed by purification of the recombinant protein, proved that TtArsC was indeed a thioredoxin-coupled arsenate reductase with a k_cat/K_M value of 1.2 × 10⁴ M^− 1 s^− 1. It also exhibited weak phosphatase activity with a k_cat/K_M value of 2.7 × 10^− 4 M^− 1 s^− 1. The catalytic role of the first cysteine (Cys7) was ascertained by site-directed mutagenesis. These results identify TtArsC as an important component in the arsenic resistance in T. thermophilus giving the first structural–functional characterization of a thermophilic arsenate reductase.     

Cyanobacterial cytochrome cM: Probing its role as electron donor for CuA of cytochrome c oxidase

It is well known that efficient functioning of photosynthetic (PET) and respiratory electron transport (RET) in cyanobacteria requires the presence of either cytochrome c₆ (Cytc₆) or plastocyanin (PC). By contrast, the interaction of an additional redox carrier, cytochrome c_M (Cytc_M), with either PET or RET is still under discussion. Here, we focus on the (putative) role of Cytc_M in cyanobacterial respiration. It is demonstrated that genes encoding the main terminal oxidase (cytochrome c oxidase, COX) and cytochrome c_M are found in all 44 totally or partially sequenced cyanobacteria (except one strain). In order to check whether Cytc_M can act as electron donor to COX, we investigated the intermolecular electron transfer kinetics between Cytc_M and the soluble Cu_A domain (i.e. the donor binding and electron entry site) of subunit II of COX. Both proteins from Synechocystis PCC6803 were expressed heterologously in E. coli. The forward and the reverse electron transfer reactions were studied yielding apparent bimolecular rate constants of (2.4 ± 0.1) × 10⁵ M^− 1 s^− 1 and (9.6 ± 0.4) × 10³ M^− 1 s^− 1 (5 mM phosphate buffer, pH 7, 50 mM KCl). A comparative analysis with Cytc₆ and PC demonstrates that Cytc_M functions as electron donor to Cu_A as efficiently as Cytc₆ but more efficient than PC. Furthermore, we demonstrate the association of Cytc_M with the cytoplasmic and thylakoid membrane fractions by immunobloting and discuss the potential role of Cytc_M as electron donor for COX under stress conditions.     

Kinetic studies of the reduction of hexaaquacobalt(III) perchlorate by a cobaloxime, [Co(dmgBF2)2(H2O)2]

The reaction of with Co(dmgBF₂)₂(H₂O)₂ in 1.0 M HClO₄/LiClO₄ was found to be first-order in both reactants and the [H⁺] dependence of the second-order rate constant is given by k_2obs = b/[H⁺], b at 25 °C is 9.23 ± 0.14 × 10² s⁻¹. The [H⁺] dependence at lower temperatures shows some saturation effect that allowed an estimate of the hydrolysis constant for as K_a = 9.5 × 10⁻³ M at 10 and 15 °C. Marcus theory and the known self-exchange rate constant for Co(OH₂)₅OH^2+/+ were used to estimate an electron self-exchange rate constant of k₂₂ = 1.7 × 10⁻⁴ M⁻¹ s⁻¹ for .     

The effect of binding of spider-derived antimicrobial peptides, oxyopinins, on lipid membranes

Oxyopinins (Oxki1 and Oxki2) are antimicrobial peptides isolated from the crude venom of the wolf spider Oxyopes kitabensis. The effect of oxyopinins on lipid bilayers was investigated using high-sensitivity titration calorimetry and ³¹P solid-state NMR spectroscopy. High-sensitivity titration calorimetry experiments showed that the binding of oxyopinins was exothermic, and the binding enthalpies (ΔH) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) small unilamellar vesicles (SUVs) were − 18.1 kcal/mol and − 15.0 kcal/mol for Oxki1 and Oxki2, respectively, and peptide partition coefficient (K_p) was found to be 3.9 × 10³ M^− 1. ³¹P NMR spectra of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) membranes in the presence of oxyopinins indicated that they induced a positive curvature in lipid bilayers. The induced positive curvature was stronger in the presence of Oxki2 than in the presence of Oxki1. ³¹P NMR spectra of phosphaditylcholine (PC) membranes in the presence of Oxki2 showed that Oxki2 produced micellization of membranes at low peptide concentrations, but unsaturated PC membranes or acidic phospholipids prevented micellization from occurring. Furthermore, ³¹P NMR spectra using membrane lipids from E. coli suggested that Oxki1 was more disruptive to bacterial membranes than Oxki2. These results strongly correlate to the known biological activity of the oxyopinins.     

Role of Phe113 at the distal side of the heme domain of an oxygen-sensor (Ec DOS) in the characterization of the heme environment

The heme-based oxygen-sensor enzyme from Escherichia coli (Ec DOS) is a heme-regulated phosphodiesterase with activity on cyclic-di-GMP and is composed of an N-terminal heme-bound sensor domain with the PAS structure and a C-terminal functional domain. The activity of Ec DOS is substantially enhanced by the binding of O₂ to the Fe(II)-protoporphyrin IX complex [Fe(II) complex] in the sensor domain. The binding of O₂ to the Fe(II) complex changes the structure of the sensor domain, and this altered structure becomes a signal that is transduced to the functional domain to trigger catalysis. The first step in intra-molecular signal transduction is the binding of O₂ to the Fe(II) complex, and detailed elucidation of this molecular mechanism is thus worthy of exploration. The X-ray crystal structure reveals that Phe113 is located close to the O₂ molecule bound to the Fe(II) complex in the sensor domain. Here, we found that the O₂ association rate constants (>200 × 10⁻³ μM⁻¹ s⁻¹: F113L; 26 × 10⁻³ μM⁻¹ s⁻¹: F113Y) of the Fe(II) complexes of Phe113 mutants were markedly different from that (51 × 10⁻³ μM⁻¹ s⁻¹) of the wild-type enzyme, and auto-oxidation rates (0.00068 min⁻¹: F113L; 0.039 min⁻¹: F113Y) of the Phe113 mutants also differed greatly from that (0.0062 min⁻¹) of the wild-type enzyme. We thus suggest that Phe113, residing near the O₂ molecule, has a critical role in optimizing the Fe(II)-O₂ complex for effective regulation of catalysis by the oxygen-sensor enzyme. Interactions of CO and cyanide anion with the mutant proteins were also studied.     

The human xenobiotic-metabolizing enzyme arylamine N-acetyltransferase 1 (NAT1) is irreversibly inhibited by inorganic (Hg) and organic mercury (CH3Hg): Mechanism and kinetics

Human arylamine N-acetyltransferase 1 (NAT1) is a xenobiotic-metabolizing enzyme that biotransforms aromatic amine chemicals. We show here that biologically-relevant concentrations of inorganic (Hg²⁺) and organic (CH₃Hg⁺) mercury inhibit the biotransformation functions of NAT1. Both compounds react irreversibly with the active-site cysteine of NAT1 (half-maximal inhibitory concentration (IC₅₀) = 250 nM and k_inact = 1.4 × 10⁴ M⁻¹ s⁻¹ for Hg²⁺ and IC₅₀ = 1.4 μM and k_inact = 2 × 10² M⁻¹ s⁻¹ for CH₃Hg⁺). Exposure of lung epithelial cells led to the inhibition of cellular NAT1 (IC₅₀ = 3 and 20 μM for Hg²⁺ and CH₃Hg⁺, respectively). Our data suggest that exposure to mercury may affect the biotransformation of aromatic amines by NAT1.     

Screening the bioactive compounds in aqueous extract of Coptidis rhizoma which specifically bind to rabbit lung tissues β2-adrenoceptor using an affinity chromatographic selection method

A receptor affinity chromatographic selection method was developed for screening the bioactive compounds binding to β₂-adrenoceptor (β₂-AR) in Coptidis rhizome. The bioactive compounds were analyzed by molecular recognition with a β₂-AR affinity column. The retention compounds eluted from the β₂-AR column were separated online with reverse-phase high-performance liquid chromatography by column switching technology, and identified by a coupled ion-trap mass spectrometer. Four compounds were screened as the bioactive compounds of Coptidis rhizome and identified as 2,9,10-trimethoxy-3-hydroxyl-protoberberine (jateorhizine), 2,3-methylenedioxy-9-methoxy-protoberberine, 2,3,9,10-tetramethoxy-protoberberine (palmatine) and 2,3-methylenedioxy-9,10-dimethoxy-protoberberine (berberine). The association constants of jatrorrhizine, palmatine and berberine to the β₂-AR were determined by the zonal elution method with standards. Berberine and palmatine had only one type of binding site on the immobilized β₂-AR. Their association constants were (2.28 ± 0.11) × 10⁴/M and (3.00 ± 0.10) × 10⁴/M, respectively. Jatrorrhizine had at least two type of binding sites on the immobilized β₂-AR, and the corresponding association constants were (2.20 ± 0.09) × 10⁻⁴/M and (6.78 ± 0.001) × 10⁵/M.     

Responses of Gmelina arborea, a tropical deciduous tree species, to elevated atmospheric CO2: Growth, biomass productivity and carbon sequestration efficacy

The photosynthetic response of trees to rising CO₂ concentrations largely depends on source-sink relations, in addition to differences in responsiveness by species, genotype, and functional group. Previous studies on elevated CO₂ responses in trees have either doubled the gas concentration (>700 μmol mol⁻¹) or used single large addition of CO₂ (500-600 μmol mol⁻¹). In this study, Gmelina arborea, a fast growing tropical deciduous tree species, was selected to determine the photosynthetic efficiency, growth response and overall source-sink relations under near elevated atmospheric CO₂ concentration (460 μmol mol⁻¹). Net photosynthetic rate of Gmelina was ∼30% higher in plants grown in elevated CO₂ compared with ambient CO₂-grown plants. The elevated CO₂ concentration also had significant effect on photochemical and biochemical capacities evidenced by changes in F_V/F_M, ABS/CSm, ET₀/CSm and RuBPcase activity. The study also revealed that elevated CO₂ conditions significantly increased absolute growth rate, above ground biomass and carbon sequestration potential in Gmelina which sequestered ∼2100 g tree⁻¹ carbon after 120 days of treatment when compared to ambient CO₂-grown plants. Our data indicate that young Gmelina could accumulate significant biomass and escape acclimatory down-regulation of photosynthesis due to high source-sink capacity even with an increase of 100 μmol mol⁻¹ CO₂.     

Probing hydrogen peroxide oxidation kinetics of wild-type Synechocystis catalase-peroxidase (KatG) and selected variants

Catalase-peroxidases (KatGs) are unique bifunctional heme peroxidases that exhibit peroxidase and substantial catalase activities. Nevertheless, the reaction pathway of hydrogen peroxide dismutation, including the electronic structure of the redox intermediate that actually oxidizes H₂O₂, is not clearly defined. Several mutant proteins with diminished overall catalase but wild-type-like peroxidase activity have been described in the last years. However, understanding of decrease in overall catalatic activity needs discrimination between reduction and oxidation reactions of hydrogen peroxide. Here, by using sequential-mixing stopped-flow spectroscopy, we have investigated the kinetics of the transition of KatG compound I (produced by peroxoacetic acid) to its ferric state by trapping the latter as cyanide complex. Apparent bimolecular rate constants (pH 6.5, 20 °C) for wild-type KatG and the variants Trp122Phe (lacks KatG-typical distal adduct), Asp152Ser (controls substrate access to the heme cavity) and Glu253Gln (channel entrance) are reported to be 1.2 × 10⁴ M^− 1 s^− 1, 30 M^− 1 s^− 1, 3.4 × 10³ M^− 1 s^− 1, and 8.6 × 10³ M^− 1 s^− 1, respectively. These findings are discussed with respect to steady-state kinetic data and proposed reaction mechanism(s) for KatG. Assets and drawbacks of the presented method are discussed.     

Kinetic and equilibrium studies of acrylonitrile binding to cytochrome c peroxidase and oxidation of acrylonitrile by cytochrome c peroxidase compound I

Ferric heme proteins bind weakly basic ligands and the binding affinity is often pH dependent due to protonation of the ligand as well as the protein. In an effort to find a small, neutral ligand without significant acid/base properties to probe ligand binding reactions in ferric heme proteins we were led to consider the organonitriles. Although organonitriles are known to bind to transition metals, we have been unable to find any prior studies of nitrile binding to heme proteins. In this communication we report on the equilibrium and kinetic properties of acrylonitrile binding to cytochrome c peroxidase (CcP) as well as the oxidation of acrylonitrile by CcP compound I. Acrylonitrile binding to CcP is independent of pH between pH 4 and 8. The association and dissociation rate constants are 0.32 ± 0.16 M⁻¹ s⁻¹ and 0.34 ± 0.15 s⁻¹, respectively, and the independently measured equilibrium dissociation constant for the complex is 1.1 ± 0.2 M. We have demonstrated for the first time that acrylonitrile can bind to a ferric heme protein. The binding mechanism appears to be a simple, one-step association of the ligand with the heme iron. We have also demonstrated that CcP can catalyze the oxidation of acrylonitrile, most likely to 2-cyanoethylene oxide in a “peroxygenase”-type reaction, with rates that are similar to rat liver microsomal cytochrome P450-catalyzed oxidation of acrylonitrile in the monooxygenase reaction. CcP compound I oxidizes acrylonitrile with a maximum turnover number of 0.61 min⁻¹ at pH 6.0.     

A novel bifunctional peptidic aspartic protease inhibitor inhibits chitinase A from Serratia marcescens: Kinetic analysis of inhibition and binding affinity

Background

Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides and antiasthmatics. The majority of chitinase inhibitors reported are natural products like argifin, argifin linear fragments, argadin, allosamidin and disulfide-cyclized peptides. Here, we report a novel peptidic inhibitor API (Aspartic Protease Inhibitor), isolated from Bacillus licheniformis that inhibits chitinase A (ChiA) from Serratia marcescens.

Methods

The binding affinity of API with ChiA and type of inhibition was determined by the inhibition kinetics assays. Fluorescence and CD spectroscopic analysis and chemical modification of API with different affinity reagents elucidated the mechanism of binding of API with ChiA.

Results and conclusions

The peptide has an amino acid sequence N-Ile¹-Cys²-Glu³-Ala⁴-Glu⁵-His⁶-Lys⁷-Trp⁸-Gly⁹-Asp¹⁰-Tyr¹¹-Leu¹²-Asp¹³-C. The ChiA–API kinetic interactions reveal noncompetitive, irreversible and tight binding nature of API with I₅₀ = 600 nM and K_i = 510 nM in the presence of chromogenic substrate p-nitrophenyl-N,N′-diacetyl-β-chitobioside[p-NP-(GlcNAc)₂]. The inhibition progress curves show a two-step slow tight binding inhibition mechanism with the rate constant k₅ = 8.7 ± 1 × 10^− 3 s^− 1 and k₆ = 7.3 ± 0.6 × 10^− 5 s^− 1. CD-spectra and tryptophanyl fluorescence analysis of ChiA incubated with increasing API concentrations confirms conformational changes in enzyme structure which may be due to irreversible denaturation of enzyme upon binding of API. Chemical modifications by WRK abolished the anti-chitinase activity of API and revealed the involvement of carboxyl groups in the enzyme inactivation. Abolished isoindole fluorescence of OPTA-labeled ChiA demonstrates the irreversible denaturation of ChiA upon incubation with API for prolonged time and distortion of active site of the enzyme.

General significance

The data provide useful information that could lead to the generation of drug-like, natural product-based chitinase inhibitors.     

趋化因子CCL11-糖胺聚糖-趋化因子受体CCR3的相互作用研究

目的 探究趋化因子CCL11与受体CCR3、糖胺聚糖（glycosaminoglycans，GAGs）相互作用过程及机制，为深入阐明CCL11-GAGs-CCR3相互作用关系提供理论参考。方法 利用基因工程技术，构建筛选了CCR3-EGFP单分子表达水平的CHO稳转细胞系，利用全内反射荧光成像（total internal reflection fluorescence，TIRF）与等温滴定量热（isothermal titration calorimetry，ITC）技术研究了不同体外溶液条件下GAGs与CCL11的相互作用，并利用趋化实验及活细胞单分子成像实验考察了GAGs-CCL11对CCR3-EGFP稳转细胞趋化行为的调控及CCR3-EGFP在细胞膜上聚集状态的影响。结果 随着硫酸软骨素链长度的增加，其与CCL11结合放热增多，表明其相互作用力增强，其促进CCL11聚集作用增强。单分子荧光成像技术结合趋化试验研究发现，不同种类及不同比例的GAGs均会影响CCL11与CCR3的相互作用，GAGs的加入，抑制了CCL11对CCR3-EGFP稳转细胞的趋化效应及促CCR3-EGFP聚集的能力，且随着硫酸软骨素分子质量的增加，抑制作用显著增强。结论 GAGs的存在可以显著调控CCL11的聚集状态，进而影响其与受体CCR3的相互作用，本研究为进一步阐明CCL11-GAGs-CCR3相互作用关系提供了一定的实验基础。     

The sedimentation properties of ferritins. New insights and analysis of methods of nanoparticle preparation

Background

Ferritin exhibits complex behavior in the ultracentrifuge due to variability in iron core size among molecules. A comprehensive study was undertaken to develop procedures for obtaining more uniform cores and assessing their homogeneity.

Methods

Analytical ultracentrifugation was used to measure the mineral core size distributions obtained by adding iron under high- and low-flux conditions to horse spleen (apoHoSF) and human H-chain (apoHuHF) apoferritins.

Results

More uniform core sizes are obtained with the homopolymer human H-chain ferritin than with the heteropolymer horse spleen HoSF protein in which subpopulations of HoSF molecules with varying iron content are observed. A binomial probability distribution of H- and L-subunits among protein shells qualitatively accounts for the observed subpopulations. The addition of Fe²⁺ to apoHuHF produces iron core particle size diameters from 3.8 ± 0.3 to 6.2 ± 0.3 nm. Diameters from 3.4 ± 0.6 to 6.5 ± 0.6 nm are obtained with natural HoSF after sucrose gradient fractionation. The change in the sedimentation coefficient as iron accumulates in ferritin suggests that the protein shell contracts ∼ 10% to a more compact structure, a finding consistent with published electron micrographs. The physicochemical parameters for apoHoSF (15%/85% H/L subunits) are M = 484,120 g/mol, ν? = 0.735 mL/g, s_20,w = 17.0 S and D_20,w = 3.21 × 10⁻⁷ cm²/s; and for apoHuHF M = 506,266 g/mol, ν? = 0.724 mL/g, s_20,w = 18.3 S and D_20,w = 3.18 × 10⁻⁷ cm²/s.

Significance

The methods presented here should prove useful in the synthesis of size controlled nanoparticles of other minerals.     

Structural and thermodynamic characterization of metal ion binding in Streptococcus suis Dpr

The use of protein cages for the creation of novel inorganic nanomaterials has attracted considerable attention in recent years. Ferritins are among the most commonly used protein cages in nanoscience. Accordingly, the binding of various metals to ferritins has been studied extensively. Dps (DNA-binding protein from starved cells)-like proteins belong to the ferritin superfamily. In contrast to ferritins, Dps-like proteins form 12-mers instead of 24-mers, have a different ferroxidase center, and are able to store a smaller amount of iron atoms in a hollow cavity (up to ∼ 500, instead of the ∼ 4500 iron atoms found in ferritins). With the exception of iron, the binding of other metal cations to Dps proteins has not been studied in detail. Here, the binding of six divalent metal ions (Zn²⁺, Mn²⁺, Ni²⁺, Co²⁺, Cu²⁺, and Mg²⁺) to Streptococcus suisDps-like peroxide resistance protein (SsDpr) was characterized by X-ray crystallography and isothermal titration calorimetry (ITC). All metal cations, except for Mg²⁺, were found to bind to the ferroxidase center similarly to Fe²⁺, with moderate affinity (binding constants between 0.1 × 10⁵ M^− 1 and 5 × 10⁵ M^− 1). The stoichiometry of binding, as deduced by ITC data, suggested the presence of a dication ferroxidase site. No other metal binding sites were identified in the protein. The results presented here demonstrate the ability of SsDpr to bind various metals as substitutes for iron and will help in better understanding protein-metal interactions in the Dps family of proteins as potential metal nanocontainers.     

Inhibition studies with anions and small molecules of two novel β-carbonic anhydrases from the bacterial pathogen Salmonella enterica serovar Typhimurium

Two new β-carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogen Salmonella enterica serovar Typhimurium, stCA 1 and stCA 2, were characterized kinetically. The two enzymes possess appreciable activity as catalysts for the hydration of CO₂ to bicarbonate, with k_cat of 0.79 × 10⁶ s⁻¹ and 1.0 × 10⁶ s⁻¹, and k_cat/K_m of 5.2 × 10⁷ M⁻¹ s⁻¹ and of 8.3 × 10⁷ M⁻¹ s⁻¹, respectively. A large number of simple/complex inorganic anions as well as other small molecules (sulfamide, sulfamic acid, phenylboronic acid, phenylarsonic acid, dialkyldithiocarbamates) showed interesting inhibitory properties towards the two new enzymes, with several low micromolar inhibitors discovered. As many strains of S. enterica show extensive resistance to classical antibiotics, inhibition of the β-CAs investigated here may be useful for developing lead compounds for novel types of antibacterials.     

Binding of ureas and amides to a Cu(II) terpyridine complex in methanol

The binding of seven ureas and amides to Cu(terpy)²⁺ in methanol has been studied by EPR, NMR and UV-Vis spectroscopy. The association constants range from 6.5 M⁻¹ for acetamide to 45.3 M⁻¹ for propyleneurea and correlate with the gas phase basicity of ligands. In methanol/water mixtures the affinity drops rapidly on increase in water content to 20 vol.%, but the binding of propyleneurea is detectable with K = 4.1 M⁻¹ even in pure aqueous medium.     

Electronic effects on the interactions of complexes [Ru(phen)2(p-L)] (L=MOPIP, HPIP, and NPIP) with DNA

In order to explore the electronic effects of Ru(II) complexes binding to DNA, a series of Ru(II) complexes [Ru(phen)₂ (p-MOPIP)]²⁺ (1), [Ru(phen)₂ (p-HPIP)]²⁺ (2), and [Ru(phen)₂(p-NPIP)]²⁺ (3) were synthesized and characterized by elementary, ¹H NMR, and ES-MS analysis. The binding properties of these complexes to CT-DNA were investigated with spectroscopic methods and viscosity experiments. Furthermore, the computations for these complexes applying the density functional theory (DFT) method have also been performed. The results show that all of these complexes can well bind to DNA in intercalation mode and DNA-binding affinity of these complexes is greatly influenced by electronic effects of intercalating ligands. The intrinsic binding constants for 1, 2, and 3 are 0.20, 0.69, and 1.56 × 10⁵ M⁻¹, respectively. This order is in accordance with that of the electron-withdrawing ability of substituent [-OR < -OH < -NO₂]. Such a trend in electronic effects of Ru(II) complexes binding to DNA can be reasonably explained by the DFT calculations.     

Applicability of new spin trap agent, 2-diphenylphosphinoyl-2-methyl-3,4-dihydro-2H-pyrrole N-oxide, in biological system

Electron spin resonance using spin-trapping is a useful technique for detecting direct reactive oxygen species, such as superoxide (). However, the widely used spin trap 2,2-dimethyl-3,4-dihydro-2H-pyrrole N-oxide (DMPO) has several fundamental limitations in terms of half-life and stability. Recently, the new spin trap 2-diphenylphosphinoyl-2-methyl-3,4-dihydro-2H-pyrrole N-oxide (DPhPMPO) was developed by us. We evaluated the biological applicability of DPhPMPO to analyze in both cell-free and cellular systems. DPhPMPO had a larger rate constant for and formed more stable spin adducts for than DMPO in the xanthine/xanthine oxidase (X/XO) system. In the phorbol myristate acetate-activated neutrophil system, the detection potential of DPhPMPO for was significantly higher than that of DMPO (k_DMPO = 13.95 M⁻¹ s⁻¹, k_DPhPMPO = 42.4 M⁻¹ s⁻¹). These results indicated that DPhPMPO is a potentially good candidate for trapping in a biological system.     

Recombinant glucose oxidase from Penicillium amagasakiense for efficient bioelectrochemical applications in physiological conditions

GOX is the most widely used enzyme for the development of electrochemical glucose biosensors and biofuel cell in physiological conditions. The present work describes the production of a recombinant glucose oxidase from Penicillium amagasakiense (yGOXpenag) displaying a more efficient glucose catalysis (k_cat/K_M(glucose) = 93 μM⁻¹ s⁻¹) than the native GOX from Aspergillus niger (nGOXaspng), which is the most industrially used (k_cat/K_M(glucose) = 27 μM⁻¹ s⁻¹). Expression in Pichia pastoris allowed easy production and purification of the recombinant active enzyme, without overglycosylation. Its biotechnological interest was further evaluated by measuring kinetics of ferrocinium-methanol (FM_ox) reduction, which is commonly used for electron transfer to the electrode surface. Despite their homologies in sequence and structure, pH-dependant FM_ox reduction was different between the two enzymes. At physiological pH and temperature, we observed that electron transfer to the redox mediator is also more efficient for yGOXpenag than for nGOXaspng(k_cat/K_M(FM_ox) = 27 μM⁻¹ s⁻¹ and 17 μM⁻¹ s⁻¹ respectively). In our model system, the catalytic current observed in the presence of blood glucose concentration (5 mM) was two times higher with yGOXpenag than with nGOXaspng. All our results indicated that yGOXpenag is a better candidate for industrial development of efficient bioelectrochemical devices used in physiological conditions.