Tyrosinase‐catalyzed oxidation of resveratrol produces a highly reactive ortho‐quinone: Implications for melanocyte toxicity

trans‐Resveratrol (3,5,4′‐trihydroxy‐trans‐stilbene, RES), a naturally occurring polyphenol, has recently attracted increased interest as a health‐beneficial agent. However, based on its p‐substituted phenol structure, RES is expected to be a substrate for tyrosinase and to produce a toxic o‐quinone metabolite. The results of this study demonstrate that the oxidation of RES by tyrosinase produces 4‐(3′,5′‐dihydroxy‐trans‐styrenyl)‐1,2‐benzoquinone (RES‐quinone), which decays rapidly to an oligomeric product (RES‐oligomer). RES‐quinone was identified after reduction to its corresponding catechol, known as piceatannol. RES‐quinone reacts with N‐acetylcysteine, a small thiol, to form a diadduct and a triadduct, which were identified by NMR and MS analyses. The production of a triadduct is not common for o‐quinones, suggesting a high reactivity of RES‐quinone. RES‐quinone also binds to bovine serum albumin through its cysteine residue. RES‐oligomer can oxidize GSH to GSSG, indicating its pro‐oxidant activity. These results suggest that RES could be cytotoxic to melanocytes due to the binding of RES‐quinone to thiol proteins.     

Cross-genome map based dissection of a nitrogen use efficiency ortho-metaQTL in bread wheat unravels concerted cereal genome evolution

Monitoring nitrogen use efficiency (NUE) in plants is becoming essential to maintain yield while reducing fertilizer usage. Optimized NUE application in major crops is essential for long-term sustainability of agriculture production. Here, we report the precise identification of 11 major chromosomal regions controlling NUE in wheat that co-localise with key developmental genes such as Ppd (photoperiod sensitivity), Vrn (vernalization requirement), Rht (reduced height) and can be considered as robust markers from a molecular breeding perspective. Physical mapping, sequencing, annotation and candidate gene validation of an NUE metaQTL on wheat chromosome 3B allowed us to propose that a glutamate synthase (GoGAT) gene that is conserved structurally and functionally at orthologous positions in rice, sorghum and maize genomes may contribute to NUE in wheat and other cereals. We propose an evolutionary model for the NUE locus in cereals from a common ancestral region, involving species specific shuffling events such as gene deletion, inversion, transposition and the invasion of repetitive elements.     

Applications of β-gal-III isozyme from Bacillus coagulans RCS3, in lactose hydrolysis

Bacillus coagulans RCS3 isolated from hot water springs secreted five isozymes i.e. β-gal I-V of β-galactosidase. β-gal III isozyme was purified using DEAE cellulose and Sephadex G 100 column chromatography. Its molecular weight characterization showed a single band at 315 kD in Native PAGE, while two subunits of 50.1 and 53.7 kD in SDS PAGE. β-Gal III had pH optima in the range of 6-7 and temperature optima at 65 °C. It preferred nitro-aryl-β-d-galactoside as substrate having K_m of 4.16 mM with ONPG. More than 85% and 80% hydrolysis of lactose (1-5%, w/v) was recorded within 48 h of incubation at 55 °C and 50 °C respectively and pH range of 6-7. About 78-86% hydrolysis of lactose in various brands of standardized milk was recorded at incubation temperature of 50 °C. These results marked the applications of β-gal III in processing of milk/whey industry.     

苯酚的生物降解基因组成及其调控机制

苯酚的生物降解受多组分的降解基因控制,形成邻位和间位等不同的代谢途径.结合部分实验结果,介绍了邻位和间位代谢系统的基因组成及其作用机制等方面的研究进展,讨论了调节基因在苯酚降解菌中的作用和不同因素对苯酚降解的影响.     

Enzymology of the degradation of (di)chlorobenzenes by Xanthobacter flavus 14p1

Xanthobacter flavus 14p1 used 1,4-dichlorobenzene as the sole source of carbon and energy but did not grow on other (chloro)aromatic compounds. 1,4-Dichlorobenzene was attacked by a chlorobenzene dioxygenase, and the intermediate chlorocatechol was metabolized by the modified ortho pathway. All enzymes necessary to convert 1,4-dichlorobenzene to 3-oxoadipate showed a low substrate specificity and also accepted the respective intermediates of chlorobenzene or 1,3-dichlorobenzene degradation. Of the three compounds chlorobenzene, 1,4-dichlorobenzene, and 1,3-dichlorobenzene, the latter was the most toxic for X. flavus 14p1. Furthermore, 1,3-dichlorobenzene did not induce chlorocatechol 1,2-dioxygenase activity of the organism. Chlorobenzene, however, induced chlorocatechol 1,2-dioxygenase, dienelactone hydrolase, and maleylacetate reductase activities. As demonstrated by chloride release, also chlorobenzene dioxygenase, chlorobenzene cis-dihydrodiol dehydrogenase, and chloromuconate cycloisomerase activities were present in chlorobenzene-induced cells, but chlorobenzene failed to support growth. Presumably a toxic compound was formed from one of the intermediates. Received: 10 June 1996 / Revision received: 23 December 1996 / Accepted: 18 January 1997     

Plasmid-mediated degradation of o- and p-phthalate by Pseudomonas fluorescens

A Pseudomonas fluorescens strain SKP3 capable of utilizing both phthalic acid and terephthalic acid as sole source of carbon and energy was isolated by enrichment technique. Phthalic acid, terephthalic acid and protocatechuic acid were easily oxidized by both phthalate-grown and glucose-grown cells without a lag period. Phthalic acid is metabolized through the ortho cleavage pathway and terephthalic acid through the meta cleavage pathway and the enzymes of the two pathways are constitutive in nature. A large plasmid of approximately 140kb in size was found to be involved in the degradation of phthalates. The catabolic plasmid pSKL was transferable to different hosts.     

Catabolic pathways and cellular responses of Pseudomonas putida P8 during growth on benzoate with a proteomics approach

The catabolic pathways and cellular responses of Pseudomonas putida P8 during growth on benzoate were studied through proteomics approach. Two-dimensional gel electrophoresis (2-DE) gel profiles of P. putida cells grown on 100 and 800 mg/L benzoate were quantitatively compared using threshold criteria and statistical tools. Protein spots of interest were identified through database searching based on peptide mass fingerprints (PMFs) obtained using matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Eight catabolic enzymes involved in both the ortho-cleavage (CatB, PcaI, and PcaF) and the meta-cleavage (DmpC, DmpD, DmpE, DmpF, and DmpG) pathways for benzoate biodegradation were identified in P. putida grown on 800 mg/L of benzoate while no meta-cleavage pathway enzymes were observed in the 2-DE gel profiles of P. putida grown on 100 mg/L of benzoate. The activation of both the ortho- and the meta-cleavage pathways in P. putida P8 grown on high benzoate concentration was confirmed directly at the protein level. In addition, another 28 differentially expressed proteins were also identified, including proteins involved in (i) detoxification and stress response (AhpC, ATPase-like ATP-binding region, putative DNA-binding stress protein, SodB and catalase/peroxidase HPI); (ii) carbohydrate, amino acid/protein and energy metabolism (isocitrate dehydrogenase, SucC, SucD, AcnB, GabD, ArcA, ArgI, Efp and periplasmic binding proteins of several ABC-transporters); and (iii) cell envelope and cell division (bacterial surface antigen family protein and MinD). Based on the data obtained, physiological changes of P. putida in response to growth on benzoate at different concentrations were discussed.     

Distribution of SH-SY5Y human neuroblastoma cells in the cell cycle following exposure to organophosphorus compounds

The current study investigated the relationship of the cell cycle phase (as G(0)/G(1), S, and G(2)/M) and cytotoxicity (as sub-G(1) DNA) to determine whether alterations in cell replication were associated with organophosphate (OP) compound induced cytotoxicity. Results demonstrated that, overall, OP compound--induced cell cycle changes were variable and depended on the OP compound, exposure concentration, and temporal relationship to cytotoxicity. Noncytotoxic OP compound treatments substantially decreased the percentage of cells in S phase of the cell cycle when compared to controls. A corresponding increase was seen in the percent of cells in G(0)/G(1) phase of the cell cycle. In the precytotoxic interval of exposure, most cytotoxic OP compound treatments substantially decreased the percentage of cells in G(2)/M phase of the cell cycle. Corresponding increases were seen primarily in G(0)/G(1) phase cells. Effects on cells in S stage of the cell cycle varied with the OP compound. In the during cytotoxic interval of exposure, most cytotoxic OP compound treatments substantially increased the percentage of cells in S phase of the cell cycle. A corresponding decrease in the percent of cells in G(0)/G(1) stage of the cell cycle was observed. Furthermore, treatments either increased or decreased the percentage of cells in G(2)/M phase of the cell cycle when compared to controls, with decreases more likely with the most cytotoxic OP compound exposures. Overall, the in vitro data suggest that exposure to OP compounds can alter the cell cycle status of SH-SY5Y neuroblastoma cells depending on compound, concentration, and interval from initial exposure. Changes in cell cycle, however, did not differentiate between OP compounds that are known for their ability to acutely inhibit acetylcholinesterase versus those inducing type I and type II delayed neurotoxicity.     

Development and validation of a spectrofluorimetric method for the quantification of ceftriaxone in pharmaceutical formulations and plasma

We describe the development and validation of a new, simple, sensitive and cost‐effective method for the determination of ceftriaxone in commercial formulations and spiked human plasma. The method proposes the conversion of ceftriaxone into a fluorescent product by reacting with ortho‐phthalaldehyde (OPA) in the presence of sulfite at room temperature. The reaction medium is buffered to pH 10 using borate buffer. The derivatized reaction product is highly fluorescent and exhibits maximum fluorescence intensity at λ_em = 386 nm after excitation at λ_ex = 324 nm. The experimental parameters affecting progress of the derivatization reaction were carefully studied and optimized. Under optimum experimental conditions, the method has an excellent correlation coefficient of 0.9984 with a broad linear range of 0.4?20 µg/mL. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 1.30 × 10^?3 and 3.90 × 10^?3 µg/mL, respectively. The interference effects of common excipients on the quantification of drug were investigated and no interference effect was observed. The proposed method has been successfully applied to the determination of ceftriaxone in pharmaceutical formulations and spiked human plasma samples. The method has been validated statistically through percent recovery studies using standard addition and by comparison with a reference HPLC method. The developed method exhibits excellent inter‐ and intraday precision. Copyright © 2013 John Wiley & Sons, Ltd.