Protein carbonylation is a key biomarker of oxidative stress involved in numerous diseases, making its accurate detection essential for understanding pathological mechanisms, developing diagnostic and therapeutic tools. The conventional 2,4-dinitrophenylhydrazine spectrophotometric probe and the more recent fluorimetric 4-hydrazino-7-nitrobenzofurazan (NBD-H) assay presents several drawbacks, including multiple washing steps. To overcome these limitations a series of N-substituted NBD probes was developed by modulating the steric and electronic properties of the hydrazone linkage. Here, we describe the synthesis and evaluation of three novel NBD-derived probes bearing methyl, ethyl, benzyl and pentyl substituents on the hydrazine moiety. Three of them formed stable hydrazone adducts with oxidized bovine serum albumin in human plasma taken as models of oxidative stress induced protein carbonyls. The data revealed that the new NBD-probes (i) exhibited stable fluorescence over time (six-fold improvement in stability), (ii) do not react with free low-molecular-weight carbonyl species, (iii) allowed to directly quantify protein carbonyls in human plasma without prior protein precipitation, (iv) offered lower limits of detection (<0.2 nmol/mg protein) under competitive conditions. Taken together, the new probes could provide a robust and simplified approach for protein carbonyl analysis in complex biological media, compatible with fluorescence microplate readers.

Barium stannate titanate (BaTi0.89Sn0.11O3, BTS11) is one of the reported lead-free materials that exhibit high dielectric and piezoelectric properties near ambient temperature. In this work, BTS11 powders were elaborated via a soft chemistry method at very low temperatures using two synthesized approaches combining sol-gel and hydrothermal processes. In the first approach, the effect of the barium precursor on the purity and the crystallinity of the BTS11 powders were studied. In contrast, in the second approach, the influence of the hydrothermal temperature on the BTS11 proprieties was analyzed. As a result, pure and uniform fine powders were obtained at 180 °C. The appropriate mass fraction of BTS11, which leads to high suspension stability in water, is below 3%. These eco-friendly and pure piezoelectric powders can be used without further heat-treatment in several applications such as lead-free piezoelectric decontamination of organic pollutants and photocatalyst, hydrogen generation and as fillers in flexible nanocomposites nano-generators for energy harvesting and energy storage.

The reaction pathways were investigated by which a fungoid chitosan (CsG) may protect against photooxidative decay of model solutions and a sulphite-free white wine. Samples containing CsG were dark incubated for 2 days before exposure to fluorescent lighting for up to 21 days in the presence of wine like (+)-catechin and/or iron doses. In both systems CsG at winemaking doses significantly reduced the photoproduction of acetaldehyde and, to a better extent, glyoxylic acid, two key reactive aldehydes implicated in wine oxidative spoilage. After 21 days, CsG was two-fold more effective than sulphur dioxide in preventing glyoxylic acid formation and minimizing the browning of white wine. Among the antioxidant mechanisms involved in CsG protective effect, iron chelation, and hydrogen peroxide quenching were demonstrated. Besides, the previously unreported tartrate displacement from the [iron(III)-tartrate] complex was revealed as an additional inhibitory mechanism of CsG under photo-Fenton oxidation conditions.

The efficacy against oxidative degradation in model and sulphite-free white wines of two commercial, insoluble chitosans (one being approved for winemaking) were investigated by electron paramagnetic resonance (EPR). Both compounds at various doses significantly inhibited the formation of alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone (4-POBN)-1-hydroxyethyl adducts under normal wine storage conditions. Pre-incubation with 2 g/L chitosan followed by filtration had a better effect than adding 50 mg/L sulphur dioxide to the experimental Chardonnay wine on the release of 4-POBN adducts after 6 days of incubation with 100 mu M iron(II). In a relevant photo-oxidative system acetaldehyde formation was significantly reduced after 6 days of incubation. Parallel EPR tests were performed to assess the importance of metal chelation (iron and copper) versus direct scavenging of hydroxyl radicals on the effect of chitosan. The present data support the potentiality of using biocompatible chitosan as a healthier complement and/or alternative to sulphur dioxide against white wine oxidative spoilage.

Many studies have shown that flavan-3-ols inhibit mammalian alpha-amylases but the published IC50 and K-i values vary up to a thousand times. We therefore tested the effects of 6 pure flavan-3-ols-abundant in green tea-on the activity of pure porcine pancreatic alpha-amylase (PPA) under steady-state kinetic conditions. We used both amylose and maltopentaose as substrates, along with spectrophotometry and chromatography as analytical tools, respectively. A Docking approach was also used to probe the interaction between PPA and each flavan-3-ol. The results showed that the 6 flavan-3-ols inhibit amylose hydrolysis with K-i comprised between 7 and 34 mu M, according to a mixed inhibition profile for gallocatechin gallate, and a competitive inhibition profile for the 5 other flavanols. Only the galloyl-containing flavan-3-ols inhibited the maltopentaose hydrolysis with a K-i of about 30 mu M according to a noncompetitive profile. We conclude that dietary flavan-3-ols could inhibit starch digestion nonnegligibly. The results of the docking trials were concordant with the kinetic data and have noticeably revealed that the cis-flavan-3-ols epigallocatechin gallate and epicatechin gallate bind similarly to PPA, involving pi-stacking with Trp59.

Background: Diabetes mellitus is the most common disease in the world. One of the approaches therapy used to treat diabetes is inhibition of α -glucosidase activity by natural inhibitors from the plant. Objective: Our work comes to highlight, in vitro, the effect of phenolic extracts of some Algerian Saharan Atlas medicinal plants on the activity of α-glucosidase. Methods: The ethyl acetate extracts were prepared from hydro-methanolic (80:20) extracts of studied plants. The Folin-Ciocalteu method was used to determine the total phenolic content of the extracts. The α-glucosidase activity was determined by a microplate spectrophotometer at 405 nm using paranitrophenol α-D-glucopyranose as substrate. Results: Among the plants tested Arthrophytum scoparium, Helianthemum lippii, Equisetum arvense and Cistus villosus exhibited a strong α-glucosidase inhibitory activity. According to IC50 values, Arthrophytum scoparium exerted the highest inhibitory activity against α-glucosidase (IC50= 5.02 µM) while a weak inhibitory activity was observed with Equisetum arvense (IC50 =21.88 µM). The kinetic study revealed a noncompetitive type inhibition for Arthrophytum scoparium (KI = 4.16 µM) and Helianthemum lippii (KI = 1.46 µM) extracts whereas a full mixed type inhibition (KI = 18.17 µM; KIS = 3.98 µM) and competitive type inhibition (KI = 25 µM) were observed for Cistus villosus and Equisetum arvense, respectively. Conclusion: The results suggest that these plants represent a potential source to develop new antidiabetic drugs for controlling postprandial blood sugar. Keywords: Atlas algerian plants, diabetes mellitus, inhibition, kinetic, phenolic extracts, α-glucosidase.

A novel series of enediynes possessing pentafluorophenylsulfoxide have been developed. The innovative compounds possess antiproliferative activity against a broad panel of human cancer cells originating from breast, blood, lung, kidney, colon, prostate, pancreas or skin with IC50 ranging from 0.6 to 3.4 μM. The antiproliferative activity of enediynes in darkness is associated to their ability to compromise microtubule network. In addition, exposure to UV leads to double-stranded DNA cleavage caused by the newly synthesized molecules reducing further their IC50 in nanomolar range against human tumor cells, including chemo-resistant pancreatic cancer cells. Taken together, the examined data demonstrate that enediynes possessing pentafluorosulfoxide are promising molecules in the cancer therapy.

The metabolic effects of an oral supplementation with a Curcuma longa extract, at a dose nutritionally relevant with common human use, on hepatic metabolism in rats fed a high fructose and saturated fatty acid (HFS) diet was evaluated. High-resolution magic-angle spinning NMR and GC/MS in combination with multivariate analysis have been employed to characterize the NMR metabolite profiles and fatty acid composition of liver tissue respectively. The results showed a clear discrimination between HFS groups and controls involving metabolites such as glucose, glycogen, amino acids, acetate, choline, lysophosphatidylcholine, phosphatidylethanolamine, and β-hydroxybutyrate as well as an increase of MUFAs and a decrease of n-6 and n-3 PUFAs. Although the administration of CL did not counteract deleterious effects of the HFS diet, some metabolites, namely some n-6 PUFA and n-3 PUFA, and betaine were found to increase significantly in liver samples from rats having received extract of curcuma compared to those fed the HFS diet alone. This result suggests that curcuminoids may affect the transmethylation pathway and/or osmotic regulation. CL extract supplementation in rats appears to increase some of the natural defences preventing the development of fatty liver by acting on the choline metabolism to increase fat export from the liver.

ETHNOPHARMACOLOGICAL RELEVANCE:According to Saharian traditional medicine, Anvillea radiata Coss. & Dur. (Asteraceae) has been valued for treating a variety of ailments such as gastro-intestinal, liver and pulmonary diseases, and has gained awareness for its beneficial effect on postprandial hyperglycemia. However, to best of our knowledge, no detailed study of the antidiabetic curative effects of this plant has been conducted yet. AIM OF THE STUDY:To determine the hypoglycemic and antidiabetic effect of dietary supplementation with Anvillea radiata extracts on high-fat-diet (HFD)-induced obesity and insulin resistance in C57BL/6J mice in relation with antioxidant, anti-inflammatory, pancreatic beta-cells and skeletal muscle protection, and digestive enzyme inhibiting properties. MATERIALS AND METHODS:Six extracts (water soluble and organic) from aerial parts of the plant were analyzed phytochemically (total phenolic and flavonoid content) and screened for in vitro superoxide (by chemiluminescence) and hydroxyl radical (by electron paramagnetic resonance spin-trapping) scavenging, antioxidant (DPPH, TRAP and ORAC assays), xanthine oxidase, metal chelating, α-amylase and α-glucosidase inhibitory property, and protective effects on copper-induced lipoprotein oxidation. Then selected hydroalcoholic and aqueous extracts were assessed for toxicity in normal human lung fibroblasts and A549 cancer cells using FMCA and MTT assays. Two water-soluble extracts having the best overall properties were assessed for their (i) protective effect at 1-15µg/mL on metabolic activity of rat insulinoma-derived INS-1 cells exposed to hyperglycemic medium, and (ii) acute hypoglycemic effect on 16-weeks HFD-induced diabetic mice. Then diabetic mice were administered HFD supplemented by extracts (up to 150mg/kg/day) for 12 additional weeks using standard diet as control and the antidiabetic drug, metformin (150mg/kg), as positive control. Then the antidiabetic, anti-inflammatory and antioxidant activity of extracts were determined. RESULTS:Of the highly efficient polyphenolics-enriched hydroalcoholic and ethyl acetate extracts, the lyophilized aqueous (AQL) and butanol extracts were not toxic in cells (≤ 400µg/mL) or when given orally in normal mice (≤ 2000mg/kg), exerted a dose-dependent hypoglycemic action in diabetic mice, which was maximal at the dose of 150mg/kg. Upon administering this dose for 12 weeks, both extracts significantly ameliorated body weight control capacity, recovery of plasma glucose and insulin level, reduced oxidative stress in blood, myocardial and skeletal muscles, and improved hyperlipidemic and inflammatory status. Moreover, diabetes-related complications were optimally ameliorated by oral therapy based on halved doses (75mg/kg) of a mixture of AQL and metformin. CONCLUSIONS:Current investigation supports the traditional medicinal usage of Anvillea radiata and suggests that both readily accessible and low-cost bio-extracts have the potency to develop an antihyperglycemic, antihyperlipidemic and protective agent against beta-cells and muscle dysfunction at doses compatible with the common practices of indigenous people for the management of metabolic disorders.

Myeloperoxidase (MPO) is a key enzyme derived from leukocytes which is associated with the initiation and progression of many inflammatory diseases. Increased levels of MPO may contribute to cellular dysfunction and tissues injury by producing highly reactive oxidants such as hypochlorous acid (HOCl). Myeloperoxidase-generated HOCl is therefore considered as a relevant biomarker of oxidative stress-related damage and its quantitation is of great importance to the study of disease progression.In this context, the current study describes a rapid, sensitive and homogeneous fluorescence-based method for detecting the MPO chlorination activity in biological samples. This assay utilizes 7-hydroxy-2-oxo-2H-chromene-8-carbaldehyde oxime as a selective probe for HOCl detection, and is adapted to 96-well microplates to allow high-throughput quantitation of active MPO.The ability of the method to monitor HOCl release was further investigated in hyperglycemic streptozotocin-treated diabetic rats. The data proved that the present assay has a reliable performance when quantitating the active MPO in the plasma of diabetic animals, a feature of inflammatory disease found concomitant with an elevation of protein carbonyls levels and lipid peroxidation and which was negatively correlated with the ratio of reduced-to-oxidized glutathione.

The broad range of applications of cerium oxide (CeO2) nanoparticles (nano-CeO2) has attracted industrial interest, resulting in greater exposures to humans and environmental systems in the coming years. Their health effects and potential biological impacts need to be determined for risk assessment. The aims of this study were to gain insights into the molecular mechanisms underlying the genotoxic effects of nano-CeO2 in relation with their physicochemical properties. Primary human dermal fibroblasts were exposed to environmentally relevant doses of nano-CeO2 (mean diameter, 7 nm; dose range, 6 x 10(-5)-6 x 10(-3) g/l corresponding to a concentration range of 0.22-22 mu M) and DNA damages at the chromosome level were evaluated by genetic toxicology tests and compared to that induced in cells exposed to micro-CeO2 particles (mean diameter, 320 nm) under the same conditions. For this purpose, cytokinesis-blocked micronucleus assay in association with immunofluorescence staining of centromere protein A in micronuclei were used to distinguish between induction of structural or numerical chromosome changes (i.e. clastogenicity or aneuploidy). The results provide the first evidence of a genotoxic effect of nano-CeO2, (while not significant with micro-CeO2) by a clastogenic mechanism. The implication of oxidative mechanisms in this genotoxic effect was investigated by (i) assessing the impact of catalase, a hydrogen peroxide inhibitor, and (ii) by measuring lipid peroxidation and glutathione status and their reversal by application of N-acetylcysteine, a precusor of glutathione synthesis in cells. The data are consistent with the implication of free radical-related mechanisms in the nano-CeO2-induced clastogenic effect, that can be modulated by inhibition of cellular hydrogen peroxide release.

The biochemistry of Kombucha fermentation was analyzed using green and black teas (GTK and BTK). Sucrose disappeared linearly in Kombucha mediums during the two week fermentation period for GTK (2.3g.day−1.L−1) but only during the first week for BTK (5.0g.d−1.L−1). The produced glucose and fructose formed disappeared faster in BTK than in GTK and in both fermentation mediums glucose was preferred to fructose as the carbon source. Ethanol, acetate equivalents and cellulose linearly increased during the fermentation (0.17, 0.35 and 0.50g.d−1.L−1 in GTK, and 0.15, 0.46 and 0.63g.d−1.L−1 in BTK, respectively). Likewise, the disappearance of glucose+fructose in Kombucha first linearly increased during the first stages of fermentation before it dropped at day 9 for GTK and at day 12 for BTK, suggesting a change of the carbon source. The protein fraction (<0.5g/L) transiently increased during the fermentation of teas and so did a 60-kDa protein band in SDS-PAGE. In both GTK and BTK, only slight changes were observed in total phenolics and in the main tea flavanols. Nevertheless, the theaflavins moderately increased (more than 50% at day 15) and the thearubigins markedly decreased (more than 2-fold at day 15). The Kombucha beverage was able to strongly inhibit starch hydrolysis by porcine pancreatic alpha-amylase and the inhibition potency increased during fermentation progress. The active compounds were suspected to be monomeric and/or oligomeric phenolic compounds. After drinking a bowl of Kombucha beverage, the concentration of these phenolics could be sufficient for inhibiting, to a certain extent, pancreatic alpha-amylase in the small intestine, with a possible impact on starch digestion and on net absorbed glucose.

We have developed a rapid nonradioisotope chemiluminescent assay adapted to high-throughput screening experiments, to evaluate glucose uptake activity in cultured cells. For chemiluminescence quantification of 2-deoxyglucose, we used a luminol oxidation reaction after an enzymatic dephosphorylation of 2-deoxyglucose-6-phosphate. All reactions were performed at 37 degrees C by consecutive addition of reagents, and the assay is able to quantify 2DG in picomole per well. To confirm the reliability of this method, we have evaluated the dose effect of insulin. GLUT4 inhibitors and insulin-sensitizing agent on 2DG uptake into 3T3-L1 cells. The results obtained with the assay for 2DG uptake in vitro in the absence or presence of insulin stimulation, were similar to those obtained by the previous radioisotopic and enzymatic methods. We have also used this assay to evaluate the effect of various reactive carbonyl and oxygen species on insulin-stimulated 2DG-uptake into adipocytes. All reactive carbonyl species tested decreased insulin-stimulated glucose uptake in a time- and dose-dependent manner without affecting basal glucose uptake in 3T3-L1 cells. 4-hydroxynonenal was found to be the most potent in the impairment of glucose uptake. This new enzymatic chemiluminescent assay is rapid and useful for measurement of 2DG uptake in insulin-responsive in cultured cells. (C) 2012 Elsevier Masson SAS. All rights reserved.

The synthesis and characterization of oxidized bis-thioketal-trispiro dinitroxide biradicals that orient the nitroxides in a rigid, approximately orthogonal geometry are reported. The biradicals show better performance as polarizing agents in dynamic nuclear polarization (DNP) NMR experiments as compared to biradicals lacking the constrained geometry. In addition, the biradicals display improved solubility in aqueous media due to the presence of polar sulfoxides. The results suggest that the orientation of the radicals is not dramatically affected by the oxidation state of the sulfur atoms in the biradical, and we conclude that a biradical polarizing agent containing a mixture of oxidation states can be used for improved solubility without a loss in performance.

The mycotoxin aflatoxin B1 (AFB1) is a carcinogenic food contaminant which is metabolically activated by epoxydation. The metabolism of mycotoxins via the mercapturate metabolic pathway was shown, in general, to lead to their detoxication. Mercapturic acids thus formed (S-substitued-N-acetyl-l-cysteines) may be accumulated in the kidney and either excreted in the urine or desacetylated by Acylase 1 (ACY1) to yield cysteine S-conjugates. To be toxic, the N-acetyl-l-cysteine-S-conjugates first have to undergo deacetylation by ACY 1. The specificity and rate of mercapturic acid deacetylation may determine the toxicity, however the exact deacetylation processes involved are not well known. The aim of this study was to investigate the role of ACY1 in the toxicity of some bioactive epoxides from Aflatoxin B1. We characterized the kinetic parameters of porcine kidney and human recombinant aminoacylase-1 towards some aromatic and aliphatic-derived mercapturates analogue of mycotoxin–mercapturic acids and 3,4-epoxyprecocene, a bioactive epoxide derivated from aflatoxin. The deacetylation of mercapturated substrates was followed both by reverse phase HPLC and by TNBS method. Catalytic activity was discussed in a structure–function relationship. Ours results indicate for the first time that aminoacylase-1 could play an important role in deacetylating mercapturate metabolites of aflatoxin analogues and this process may be in relation with their cyto- and nephrotoxicity in human.

The p53-transcriptional target TP53INP1 is a potent stress-response protein promoting p53 activity. We previously showed that ectopic overexpression of TP53INP1 facilitates cell cycle arrest as well as cell death. Here we report a study investigating cell death in mice deficient for TP53INP1. Surprisingly, we found enhanced stress-induced apoptosis in TP53INP1-deficient cells. This observation is underpinned in different cell types in vivo (thymocytes) and in vitro (thymocytes and MEFs), following different types of injury inducing either p53-dependent or -independent cell death. Nevertheless, absence of TP53INP1 is unable to overcome impaired cell death of p53-deficient thymocytes. Stress-induced ROS production is enhanced in the absence of TP53INP1, and antioxidant NAC complementation abolishes increased sensitivity to apoptosis of TP53INP1-deficient cells. Furthermore, antioxidant defenses are defective in TP53INP1-deficient mice in correlation with ROS dysregulation. Finally, we show that autophagy is reduced in TP53INP1-deficient cells both at the basal level and upon stress. Altogether, these data show that impaired ROS regulation in TP53INP1-deficient cells is responsible for their sensitivity to induced apoptosis. In addition, they suggest that this sensitivity could rely on a defect of autophagy. Therefore, these data emphasize the role of TP53INP1 in protection against cell injury.

Numerous reactive oxygen species (ROS) and reactive carbonyl species (RCS) issuing from lipid and sugar oxidation are known to damage a large number of proteins leading to enzyme inhibition and alteration of cellular functions. Whereas studies in literature only focus on the reactivity of one or two of these compounds, we aimed at comparing in the same conditions of incubations (4 and 24 h at 37 °C) the effects of both various RCS (4-hydroxynonenal, 4-hydroxyhexenal, acrolein, methylglyoxal, glyoxal, malondialdehyde) and ROS (H2O2, AAPH) on the activity of key enzymes involved in cellular oxidative stress: superoxide dismutase (Cu,Zn-SOD), glutathione peroxidase (GPx), glutathione S-transferase (GST) and glucose-6-phosphate dehydrogenase (G6PDH). This was realized both in vitro on purified proteins and MIAPaCa-2 cells. Incubation of these enzymes with RCS resulted in a significant time- and concentration-dependent inhibition for both pure enzymes and in cell lysates. Among all RCS and ROS, hydroxynonenal (HNE) was observed as the most toxic for all studied enzymes except for SOD and is followed by hydrogen peroxide. At 100 μM, HNE resulted in a 50% reduction of GPx, 56% of GST, 65% of G6PDH, and only 10% of Cu,Zn-SOD. Meanwhile it seems that concentrations used in our study are closer to biological conditions for ROS than for RCS. H2O2 and AAPH-induced peroxyl radicals may be probably more toxic towards the studied enzymes in vivo.

Sulfate reducers have developed a multifaceted adaptative strategy to survive against oxidative stresses. Along with this oxidative stress response, we recently characterized an elegant reversible disulfide bond-dependent protective mechanism in the pyruvate:ferredoxin oxidoreductase (PFOR) of various Desulfovibrio species. Here, we searched for thiol redox systems involved in this mechanism. Using thiol fluorescent labeling, we show that glutathione is not the major thiol/disulfide balance-controlling compound in four different Desulfovibrio species and that no other plentiful low molecular weight thiol can be detected. Enzymatic analyses of two thioredoxins (Trxs) and three thioredoxin reductases allow us to propose the existence of two independent Trx systems in Desulfovibrio vulgaris Hildenborough (DvH). The TR1/Trx1 system corresponds to the typical bacterial Trx system. We measured a TR1 apparent Km value for Trx1 of 8.9 μm. Moreover, our results showed that activity of TR1 was NADPH-dependent. The second system named TR3/Trx3 corresponds to an unconventional Trx system as TR3 used preferentially NADH (Km for NADPH, 743 μm; Km for NADH, 5.6 μm), and Trx3 was unable to reduce insulin. The Km value of TR3 for Trx3 was 1.12 μm. In vitro experiments demonstrated that the TR1/Trx1 system was the only one able to reactivate the oxygen-protected form of Desulfovibrio africanus PFOR. Moreover, ex vivo pulldown assays using the mutant Trx1C33S as bait allowed us to capture PFOR from the DvH extract. Altogether, these data demonstrate that PFOR is a new target for Trx1, which is probably involved in the protective switch mechanism of the enzyme.

Proprotein convertases (PCs) are a family of serine proteases that are involved in the post-translational processing and activation of a wide range of regulatory proteins. The upstream role of PCs in the control of many physiological and pathological processes generates a growing interest in understanding their regulation. Here, we demonstrate that the serine protease inhibitor plasminogen activator inhibitor 1 (PAI-1) forms an SDS-stable complex with the PC furin, which leads to the inhibition of the intra-Golgi activity of furin. It is known that elevated PAI-1 plasma levels are correlated with the occurrence of the metabolic syndrome and type 2 diabetes, and we show that PAI-1 reduces the furin-dependent maturation and activity of the insulin receptor and ADAM17: two proteins involved in the onset of these metabolic disorders. In addition to demonstrating that PAI-1 is an intracellular inhibitor of furin, this study also provides arguments in favor of an active role for PAI-1 in the development of metabolic disorders.