1,8-Naphthalimide–Cu(ІІ) ensemble based turn-on fluorescent probe for the detection of thiols in organic aqueous media

A 1,8-naphthalimide–Cu(II) ensemble was rationally designed and synthesized as a new turn-on fluorescent probe utilizing the ‘chemosensing ensemble’ method for detections of thiols (Cys, Hcy and GSH) with high selectivity over other α-amino acids at pH 7.4 in organic aqueous media (EtOH/HEPES, v/v = 9:1). The recognition mechanism was attributed to the remove Cu(II) from the 1,8-naphthalimide–Cu(II) ensemble by thiols and the release of flurescence of ligand 1. Remarkable fluorescence enhancements were therefore observed in the sensing process of thiols by the 1,8-naphthalimide–Cu(II) ensemble. Furthermore, the 1,8-naphthalimide–Cu(II) ensemble was successfully applied to the fluorescence imaging of thiols in CHO cells with high sensitivity and selectivity.     

Antifungal potentiality of mycogenic silver nanoparticles capped with chitosan produced by endophytic Amesia atrobrunnea

This research reports the fabrication of silver nanoparticles (AgNPs) from endophytic fungus, Amesia atrobrunnea isolated from Ziziphus spina-christi (L.). Influencing factors for instance, thermal degree of incubation, media, pH, and silver nitrate (AgNO₃) molarity were optimized. Then, the AgNPs were encapsulated with chitosan (Ch-AgNPs) under microwave heating at 650 W for 90 s. Characterization of nanoparticles was performed via UV–visible (UV–vis) spectrophotometer, Fourier-transform infrared spectrophotometer (FTIR), zeta potential using dynamic-light scattering (DLS), and field-emission-scanning electron microscope (FE-SEM). Anti-fungal activity of Ch-AgNPs at (50, 25, 12.5, 6.25 mg/L) was tested against Fusarium oxysporum, Curvularia lunata, and Aspergillus niger using the mycelial growth inhibition method (MGI). Results indicated that Czapek-dox broth (CDB) with 1 mM AgNO₃, an acidic pH, and a temperature of 25–30 °C were the optimum for AgNPs synthesis. (UV–vis) showed the highest peak at 435 nm, whereas Ch-AgNPs showed one peak for AgNPs at 405 nm and another peak for chitosan at 230 nm. FTIR analysis confirmed that the capping agent chitosan was successfully incorporated and interacted with the AgNPs through amide functionalities. Z-potential was −19.7 mV for AgNPs and 38.9 mV for Ch-AgNPs, which confirmed the significant stability enhancement after capping. FES-SEM showed spherical AgNPs and a reduction in the nanoparticle size to 44.65 nm after capping with chitosan. The highest mycelial growth reduction using fabricated Ch-AgNPs was 93% for C. lunata followed by 77% for A. niger and 66% F. oxysporum at (50 mg/L). Biosynthesis of AgNPs using A. atrobrunnea cell-free extract was successful. Capping with chitosan exhibited antifungal activity against fungal pathogens.     

Study of the interaction of human serum albumin with Alstonia scholaris leaf extract-mediated silver nanoparticles having bactericidal property

This study investigates the green synthesis of AgNPs from 1 mM aqueous AgNO₃ using 10% leaf extract of Alstonia scholaris (Chhatim) for its wide antibacterial and medicinal properties. The synthesized AgNPs were duly characterized by UV–vis (UV–vis) spectrophotometry, dynamic light scattering, field emission scanning electron microscopy, transmission electron microscopy, energy-dispersive analysis of X-rays spectroscopy, and fourier transform infrared spectrophotometry. Their antibacterial property was tested against Escherichia coli (ATCC 25922), and minimum inhibitory concentrations of 0.08 nM of AgNPs were obtained, which suggests improved therapeutic efficacy. We report the interaction of human serum albumin (HSA) with this nanoparticle, and this interaction was studied by UV–vis, fluorescence, and circular dichroism spectroscopies and zeta potential measurement at room temperature. It was found that the AgNPs form a complex with HSA, which may cause the slightest change in the conformation of HSA. The calculated values of Stern-Volmer quenching constant, binding constant, and binding distance were 1.82 × 10⁷ M⁻¹, 1.58 × 10⁷ M⁻¹, and 3.68 nm, respectively. Therefore, in future, the present study may provide useful information to design a better antibacterial compound by using green synthesized nanoparticles with fewer side effects.     

Oxidation of the diphosphoinositol polyphosphate phosphohydrolase-like Nudix hydrolase Aps from Drosophila melanogaster induces thermolability—A possible regulatory switch?

Unlike mammalian cells, Drosophila melanogaster contains only a single member of the diphosphoinositol polyphosphate phosphohydrolase subfamily of the Nudix hydrolases, suggesting that functional specialisation has not occurred in this organism. In order to evaluate its function, Aps was cloned and characterized. It hydrolyses a range of (di)nucleoside polyphosphates, the most efficient being guanosine 5′-tetraphosphate (K_m = 11 μM, k_cat = 0.79 s^?1). However, it shows a 5-fold preference for the hydrolysis of diphosphoinositol pentakisphosphate (PP-InsP₅, K_m = 0.07 μM, k_cat = 0.024 s^?1). Assayed at 26 °C, Aps had an alkaline pH optimum and required a divalent ion: Mg²⁺ (10–20 mM) or Mn²⁺ (1 mM) were preferred for nucleotide hydrolysis and Mg²⁺ (0.5–1 mM) or Co²⁺ (1–100 μM) for PP-InsP₅ hydrolysis. GFP-fusions showed that Aps was predominantly cytoplasmic, with some nuclear localization. In the absence of dithiothreitol Aps was heat labile, rapidly losing activity even at 36 °C, while in the presence of dithiothreitol, Aps was heat stable, surviving for 5 min at 76 °C. Heat lability was restored by H₂O₂ and mass spectrometric analysis suggested that this was due to reversible dimerisation involving two inter-molecular disulphides between Cys23 and Cys25. Aps expression was highest in embryos and declined throughout development. The ratio of PP-InsP₅ to inositol hexakisphosphate also decreased throughout development, with the highest level of PP-InsP₅ found in embryos. These data suggest that the redox state of Aps may play a role in controlling its activity by altering its stability, something that could be important for regulating PP-InsP₅ during development.     

Determination of asiatic acid in beagle dog plasma after oral administration of Centella asiatica extract by precolumn derivatization RP-HPLC

A novel precolumn derivatization reversed-phase high-performance liquid chromatography (RP-HPLC) method with UV–vis detection for the quantitative determination of total concentration of asiatic acid (AA) in beagle dog plasma is described. AA was extracted with n-hexane-dichloromethane-2-propanol (20:10:1, v/v/v) from plasma, which had been hydrolyzed by acid and derivatized with p-Toluidine. Chromatographic separation was achieved on a C₁₈ column using gradient elution in a water–methanol system. Detection was set at UV wavelength of 248 nm. A calibration curve ranging from 0.01 to 1.5 μg/mL was shown to be linear, and the lower limit of quantification (LLOQ) was 0.01 μg/mL. The intra- and inter-day precisions which were determined by three different concentrations (0.05, 0.2 and 0.8 μg/mL) ranged from 4.4% to 13.1% and 4.6% to 14.2%, respectively. Mean extraction recoveries were no less than 65% for AA and ursolic acid (IS). Plasma samples containing asiatic acid were stable for 30 days at ?20 °C. The method was successfully applied to a pharmacokinetic study in beagle dogs after oral administration of Centella asiatica extract, and the main pharmacokinetic parameters obtained were: T_1/2, 4.29 h; T_max, 2.70 h; C_max, 0.74 μg/mL; AUC_0–t and AUC_0–∞, 3.74 and 3.82 μg h/mL, respectively.     

Study of the Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase by site-directed mutagenesis,spectroscopic, and computational methods

The Cys-His bridge as electron transfer conduit in the enzymatic catalysis of nitrite to nitric oxide by nitrite reductase from Sinorhizobium meliloti 2011 (SmNir) was evaluated by site-directed mutagenesis, steady state kinetic studies, UV–vis and EPR spectroscopic measurements as well as computational calculations. The kinetic, structural and spectroscopic properties of the His171Asp (H171D) and Cys172Asp (C172D) SmNir variants were compared with the wild type enzyme. Molecular properties of H171D and C172D indicate that these point mutations have not visible effects on the quaternary structure of SmNir. Both variants are catalytically incompetent using the physiological electron donor pseudoazurin, though C172D presents catalytic activity with the artificial electron donor methyl viologen (k_cat = 3.9(4) s^? 1) lower than that of wt SmNir (k_cat = 240(50) s^? 1). QM/MM calculations indicate that the lack of activity of H171D may be ascribed to the N^δ1H…OC hydrogen bond that partially shortcuts the T1–T2 bridging Cys-His covalent pathway. The role of the N^δ1H…OC hydrogen bond in the pH-dependent catalytic activity of wt SmNir is also analyzed by monitoring the T1 and T2 oxidation states at the end of the catalytic reaction of wt SmNir at pH 6 and 10 by UV–vis and EPR spectroscopies. These data provide insight into how changes in Cys-His bridge interrupts the electron transfer between T1 and T2 and how the pH-dependent catalytic activity of the enzyme are related to pH-dependent structural modifications of the T1–T2 bridging chemical pathway.     

Inhibition of carbonic anhydrases from the extremophilic bacteria Sulfurihydrogenibium yellostonense (SspCA) and S. azorense (SazCA) with a new series of sulfonamides incorporating aroylhydrazone-, [1,2,4]triazolo[3,4-b][1,3,4]thiadiazinyl- or 2-(cyanophenylmethylene)-1,3,4-thiadiazol-3(2H)-yl moieties

A series of new sulfonamides was prepared starting from 2-oxo-N′-(4-sulfamoylphenyl)-propanehydrazonoyl chloride, a sulfanilamide derivative, which was reacted with aroylhydrazides, amines, or thiols. A library of derivatives incorporating aroylhydrazone, [1,2,4]triazolo[3,4-b][1,3,4]thiadiazinyl- or 2-(cyanophenyl-methylene)-1,3,4-thiadiazol-3(2H)-yl moieties was thus synthesized. The new compounds were investigated as inhibitors of four α-carbonic anhydrases (CAs, EC 4.2.1.1), the human (h) isoforms hCA I and II, and the bacterial ones recently isolated from the extremophilic bacteria Sulfurihydrogenibium yellostonense (SspCA) and Sulfurihydrogenibium azorense (SazCA). Low nanomolar activity was observed against hCA II (K_Is of 0.56–17.1 nM) whereas hCA I was less inhibited by these compounds (K_Is of 86.4 nM–32.8 μM). The bacterial CAs were also effectively inhibited by these derivatives (K_Is in the range of 0.77–234 nM against SazCA, and of 6.2–89.1 against SspCA, respectively), with several low nanomolar/subnanomolar inhibitors detected against both of them. As SspCA and SazCA are among the most thermostable and catalytically active CAs, it is of interest to find modulators of their activity for potential biotechnologic applications.     

Cassava (Manihot esculenta Crantz of cv. KU50) peroxidase and its potential for the detection of some thiol compounds based on the inhibitory effect of 3,3′,5,5′-tetramethylbenzidine oxidation

Cassava peroxidase (CSP) isolated from cassava leaves (Manihot esculenta Crantz of cv. KU50) was purified by DEAE and concanavalin-A column chromatography. CSP was a haem-containing cationic glycoprotein with the molecular weight of 38 and 44 kDa determined by MALDI-TOF-MS. Its kinetic catalysis in the presence and absence of some thiols were investigated and compared to those of horseradish and soybean peroxidases by using urea hydrogen peroxide (UHP) and 3,3′,5,5′-tetramethylbenzidine (TMB) as substrates. Inhibitory effects of some pesticides containing thiol groups such as thiosemicabazide, thiourea and thiophanate-methyl on TMB oxidation were graphically demonstrated their ability to be either competitive or noncompetitive inhibitors, depending on their properties. The degrees of inhibition were expressed as K_i and IC₅₀ values. The applicable range for the detection of thiosemicabazide in solution was found to be in the range of 10–100 μM, whereas those of thiourea and thiophanate-methyl were 40–400 μM and 10–460 μM, respectively. On the contrary, the inhibitory effects of some phenolic pollutants; 4-nitrophenol, 4-phenylphenol and pentachlorophenol on TMB oxidation were not significantly observed and the phenomenon was discussed.     

Limited survey on aflatoxin contamination in rice

Aflatoxins (AFS) are toxic and carcinogenic fungal metabolites. Aflatoxin B1 is the most toxic and has been classified as a Group I carcinogen by the International Agency for Research on Cancer (IARC). Samples of imported rice were analyzed for their AFS content. Finley ground rice subsamples were extracted with water/methanol (100:150 v/v) followed by purification with Immunoaffinity columns (IAC). AFS purified from extracts were determined with RP-HPLC-FLD using post column electrochemical derivatization with a Kobra Cell. Concentrations of aflatoxin B1 and total AFS in test rice samples were ≤0.123 and ≤2.58 µg/kg, respectively. Tween 80 improved recoveries (86 and 106%) of aflatoxin B1 and aflatoxin G1 from brown rice. Recoveries of Aflatoxin B2 and aflatoxin G2 were substantially reduced (non-detected to 27%) by Tween 80 used in IAC cleanup of brown rice extracts. Visible dense growth of Aspergillus parasiticus (food isolate) occurred at 25 °C but higher aflatoxin B1amounts (23.9–39.3 µg/kg) accumulated when the mold grew at 37 °C in rice seeds stored for three weeks. It could be concluded that levels of aflatoxin B1 and total AFS in rice samples were within the permissible amounts of the EU and other international legislations.     

S-Nitrosylation of STIM1 by Neuronal Nitric Oxide Synthase Inhibits Store-Operated Ca2 + Entry

Store-operated Ca^2 + entry (SOCE) mediated by stromal interacting molecule-1 (STIM1) and Orai1 represents a major route of Ca^2 + entry in mammalian cells and is initiated by STIM1 oligomerization in the endoplasmic or sarcoplasmic reticulum. However, the effects of nitric oxide (NO) on STIM1 function are unknown. Neuronal NO synthase is located in the sarcoplasmic reticulum of cardiomyocytes. Here, we show that STIM1 is susceptible to S-nitrosylation. Neuronal NO synthase deficiency or inhibition enhanced Ca^2 + release-activated Ca^2 + channel current (I_CRAC) and SOCE in cardiomyocytes. Consistently, NO donor S-nitrosoglutathione inhibited STIM1 puncta formation and I_CRAC in HEK293 cells, but this effect was absent in cells expressing the Cys49Ser/Cys56Ser STIM1 double mutant. Furthermore, NO donors caused Cys49- and Cys56-specific structural changes associated with reduced protein backbone mobility, increased thermal stability and suppressed Ca²⁺ depletion-dependent oligomerization of the luminal Ca^2 +-sensing region of STIM1. Collectively, our data show that S-nitrosylation of STIM1 suppresses oligomerization via enhanced luminal domain stability and rigidity and inhibits SOCE in cardiomyocytes.     

Thiol metabolism play significant role during cadmium detoxification by Ceratophyllum demersum L.

In the present study, the level of thiols and activity of related enzymes were investigated in coontail (Ceratophyllum demersum L.) plants to analyze their role in combating the stress caused upon exposure to cadmium (Cd; 0–10 μM) for a duration up to 7 d. Plants showed the maximum accumulation of 1293 μg Cd g^?1 dw after 7 d at 10 μM. Significant increases in the level of total non-protein thiols (NP-SH) including phytochelatins (PCs) as well as upstream metabolites of the PC biosynthetic pathway, cysteine and glutathione (GSH) were observed. In addition, significant increases in the activities of cysteine synthase (CS), glutathione-S-transferase (GST), glutathione reductase (GR), as well as in vitro activation of phytochelatin synthase (PCS), were noticed in response to Cd. In conclusion, under Cd stress, plants adapted to a new metabolic equilibrium of thiols through coordinated synthesis and consumption to combat Cd toxicity and to accumulate it.     

Coumarinyl-substituted sulfonamides strongly inhibit several human carbonic anhydrase isoforms: solution and crystallographic investigations

We investigated a series of coumarinyl-substituted aromatic sulfonamides as inhibitors of four carbonic anhydrase (CA, EC 4.2.1.1) isoforms with medical applications, the cytosolic hCA I, and II, and the transmembrane, tumor-associated hCA IX and XII. Compounds incorporating 7-methoxy-coumarin-4-yl-acetamide-tails and benzenesulfonamide and benzene-1,3-disulfonamide scaffolds showed medium potency inhibition of hCA I (K_Is of 73–131 nM), effective hCA II inhibition (K_Is of 9.1–36 nM) and less effective hCA IX and XII inhibition (K_Is of 55–128 nM). Only one compound, the derivatized 4-amino-6-trifluoromethyl-benzene-1,3-disulfonamide with the coumarinyl tail, showed effective inhibition of the transmembrane isoforms, with K_Is of 5.9–14.2 nM, although it was less effective as hCA I and II inhibitor (K_Is of 36–120 nM). An X-ray crystal structure of hCA II in complex with 4-(7-methoxy-coumarin-4-yl-acetamido)-benzenesulfonamide (K_I of 9.1 nM against hCA II) showed the intact inhibitor coordinated to the zinc ion from the enzyme active site by the sulfonamide moiety, and participating in a edge-to-face stacking with Phe131, in addition to other hydrophobic and hydrophilic interactions with water molecules and amino acid residues from the active site. Thus, sulfonamides incorporating coumarin rings have a distinct inhibition mechanism compared to the coumarins, and may lead to compounds with interesting inhibition profiles against various α-CAs found in mammals or parasites, such as Plasmodium falciparum.     

Photochemical NO release from nitrosyl RuII complexes with C-bound imidazoles

The series of nitrosyl complexes trans-[Ru(NH₃)₄L(NO)]Cl₃, L = caffeine, theophylline, imidazole and benzoimidazole in position trans to NO were prepared and their photochemical properties studied. All complexes showed nitric oxide (NO) release under light irradiation at 330–440 nm. Quantum yields for [Ru(NH₃)₄L(H₂O)]³⁺ formation (?_Ru(III)) were sensitive to the natures of L, λ_irr and pH. The major product of the irradiation of trans-[Ru(NH₃)₄L(NO⁺)]³⁺ is the trans-[Ru^III(NH₃)₄L(Cl)]²⁺ and NO as suggested by UV–Vis, electrochemical, and FTIR techniques.     

NAD-based inhibitors with anticancer potential

Three classes of novel inhibitors of inosine monophosphate dehydrogenase have been prepared and their anti-proliferative properties were evaluated against several cancer cell lines.(1) Mycophenolic adenine dinucleotide analogues (8–13) containing a substituent at the C2 of adenine ring were found to be potent inhibitors of IMPDH (K_i’s in range of 0.6–82 nM) and sub-μM inhibitors of leukemic K562 cell proliferation. (2) Mycophenolic adenosine (d and l) esters (20 and 21) showed a potent inhibition of IMPDH2 (K_i = 102 and K_i = 231 nM, respectively) and inhibition of K562 cell growth (IC₅₀ = 0.5 and IC₅₀ = 1.6 μM). These compounds serve both as inhibitors of the enzyme and as a depot form of mycophenolic acid. The corresponding amide analogue 22, also a potent inhibitor of IMPDH (K_i = 84 nM), did not inhibit cancer cell proliferation. (3) Mycophenolic-(l)- and (d)-valine adenine di-amide derivatives 25 (K_i = 9 nM) and 28 (K_i = 3 nM) were found to be very potent enzymatically, but did not inhibit proliferation of cancer cells.     

Two new pseudohalide-bridged Cu(II) complexes with a hydrazone ligand: Syntheses,crystal structures and magnetic studies

Two new pseudohalide-bridged copper(II) complexes [{Cu(PBH)(μ_1,1-CNO)}₂] (1) and {Cu(PBH)(μ_1,5-NCNCN)}_n (2) (where HPBH = 2-pyridinecarboxaldehyde benzoyl hydrazone) have been synthesised and characterised by elemental analysis, CV, IR and UV–Vis spectral studies. The tridentate hydrazone pro-ligand (HPBH) was obtained by the condensation of benzhydrazide and pyridine-2-carboxaldehyde. Structures of both complexes have been established by X-ray crystallography which shows that 1 is a μ_1,1-CNO^?-bridged dimer whereas 2 is a μ_1,5-dca-bridged (dca = dicyanamide) linear polynuclear structure. Variable temperature magnetic susceptibility studies indicate weak antiferromagnetic interactions with J values ?0.50 cm^?1 and ?0.10 cm^?1 for 1 and 2, respectively.     

Cloning,overexpression, purification,and site-directed mutagenesis of xylitol-2-dehydrogenase from Candida albicans

Xylitol-2-dehydrogenase from Candida albicans was cloned and overexpressed in Escherichia coli. The purified recombinant XDH has an apparent molecular weight of 40 kDa which belongs to the medium chain alcohol dehydrogenase family and exclusively uses NAD⁺ as a cofactor. The recombinant caXDH has a K_M of 8.8 mM and 37.7 μM using the substrate xylitol and NAD⁺, respectively, and its catalytic efficiency is 53,200 min^?1 mM^?1. Following site-directed mutagenesis, one of the engineered caXDHs with six mutations at Ser95Cys, Ser98Cys, Tyr101Cys, Asp206Ala, Ile207Arg, and Phe208Ser shifted its cofactor dependence from NAD⁺ to NADP⁺ in which the K_M and k_cat/K_M towards NADP⁺ are 119 μM and 26,200 min^?1 mM^?1, respectively.     

Hydrolysis of the amide bond in histidine- and methionine-containing dipeptides promoted by pyrazine and pyridazine palladium(II)-aqua dimers: Comparative study with platinum(II) analogues

Two dinuclear palladium(II) complexes, [{Pd(en)Cl}₂(μ-pz)](NO₃)₂ and [{Pd(en)Cl}₂(μ-pydz)](NO₃)₂, have been synthesized and characterized by elemental microanalysis and spectroscopic (¹H and ¹³C NMR, IR and UV–vis) techniques (en is ethylenediamine; pz is pyrazine and pydz is pyridazine). The square planar geometry of palladium(II) metal centers in these complexes has been predicted by DFT calculations. The chlorido complexes were converted into the corresponding aqua complexes, [{Pd(en)(H₂O)}₂(μ-pz)]⁴⁺ and [{Pd(en)(H₂O)}₂(μ-pydz)]⁴⁺, and their reactions with N-acetylated l-histidylglycine (Ac–l–His–Gly) and l-methionylglycine (Ac–l–Met–Gly) were studied by ¹H NMR spectroscopy. The palladium(II)-aqua complexes and dipeptides were reacted in 1:1 M ratio, and all reactions performed in the pH range 2.0 < pH < 2.5 in D₂O solvent and at 37 °C. In the reactions of these complexes with Ac–l–His–Gly and Ac–l–Met–Gly dipeptides, the hydrolysis of the amide bonds involving the carboxylic group of both histidine and methionine amino acids occurs. The catalytic activities of the palladium(II)-aqua complexes were compared with those previously reported in the literature for the analogues platinum(II)-aqua complexes, [{Pt(en)(H₂O)}₂(μ-pz)]⁴⁺ and [{Pt(en)(H₂O)}₂(μ-pydz)]⁴⁺.     

Hemoglobin glutathionylation can occur through cysteine sulfenic acid intermediate: Electrospray ionization LTQ-Orbitrap hybrid mass spectrometry studies

Glutathionylated hemoglobin (Hb-SSG) is now recognized as a promising biomarker of systemic oxidative stress. Aim of this study is to gain a mechanistic insight into its formation. The ability of GSSG to form Hb-SSG through a thiol-disulfide exchange mechanism was firstly examined. For this purpose, GSSG (ranging from 0.23 to 230 μmol/g Hb, 15 μM–15 mM final concentrations) was incubated with 1 mM Hb and the relative content of Hb-SSG determined by direct infusion mass spectrometry (Orbitrap as analyzer). No detectable Hb-SSG was observed at a GSSG concentration range found in physiopathological conditions (0.13–0.23 μmol/g Hb). To reach a detectable Hb-SSG signal, the GSSG concentration was raised to 2.3 μmol/g Hb (0.5% relative abundance). The relative content of Hb-GSSG dose-dependently increased to 6% and 11% at 77 and 153 μmol/g Hb, respectively. The second step was to demonstrate whether Hb-SSG is formed through a sulfenic acid intermediate, a well-recognized mechanism of S-protein glutathionylation. Cys β93 sulfenic acid was found to be formed by oxidizing Hb with 1 mM H₂O₂, as demonstrated by direct infusion and LC–ESI-MS/MS experiments and using dimedone as derivatazing agent. When H₂O₂-treated Hb was incubated with physiological concentrations of GSH (9 μmol/g Hb), the corresponding Hb-SSG form was detected, reaching 15% of relative abundance. In summary, we here demonstrate that Hb glutathionylation can occur through a Cys sulfenic acid intermediate which is formed in oxidizing conditions. Hb glutathionylation is also mediated by a thiol-disulfide transfer mechanism, but this requires a concentration of GSSG which is far to be achieved in physiopathological conditions.     

Potent BRAF kinase inhibitors based on 2,4,5-trisubstituted imidazole with naphthyl and benzothiophene 4-substituents

The RAS–RAF–MEK–ERK pathway is hyperactivated in 30% of human cancers. BRAF is a serine–threonine kinase, belonging to this pathway that is mutated with high frequency in human melanoma and other cancers thus BRAF is an important therapeutic target in melanoma. We have designed inhibitors of BRAF based on 2,4,5-trisubstituted imidazoles with naphthyl and benzothiophene-4-substituents. Two compounds were discovered to be potent BRAF inhibitors: 1-(6-{2-[4-(2-dimethylamino-ethoxy)phenyl]-5-(pyridin-4-yl)-1H-imidazol-4-yl} benzo[b]thiophen-3-yl)-2,2,2-trifluoroethanol (1i) with BRAF IC₅₀ = 190 nM and with cellular GI₅₀ = 2100 nM, and 6-{2-[4-(2-dimethylamino-ethoxy)-phenyl]-5-pyridin-4-yl-3H-imidazol-4-yl}-naphthalen-1-ol (1q) with IC₅₀ = 9 nM and GI₅₀ = 220 nM.     

Catalytic properties of a GH10 endo-β-1,4-xylanase from Streptomyces thermocarboxydus HY-15 isolated from the gut of Eisenia fetida

A novel GH10 endo-β-1,4-xylanase (XylG) gene from Streptomyces thermocarboxydus HY-15, which was isolated from the gut of Eisenia fetida, was cloned, over-expressed, and characterized. The XylG gene (1182 bp) encoded a polypeptide of 393 amino acids with a deduced molecular mass of 43,962 Da and a calculated pI of 6.74. The primary structure of XylG was 69% similar to that of Thermobifida fusca YX endo-β-1,4-xylanase. It was most active at pH 6.0 and 55 °C. The susceptibilities of xylans to XylG were as follows: oat spelt xylan > birchwood xylan > beechwood xylan. The XylG also showed high activity (474 IU/mg) toward p-nitrophenylcellobioside. Moreover, at pH 6.0 and 50 °C, the V_max and K_m values of the XylG were 127 IU/mg and 2.51 mg/ml, respectively, for oat spelt xylan and 782 IU/mg and 5.26 mM, respectively, for p-nitrophenylcellobioside. A homology model indicated that XylG folded to form a (β/α)₈-barrel with two catalytic residues of an acid/base (Glu181) and a nucleophile (Glu289). The formation of a disulfide bond between Cys321 and Cys327 were predicted by homology modeling.