Regioselective synthesis of plant (iso)flavone glycosides in Escherichia coli

The flavonoids genistein, biochanin A, luteolin, quercetin, and kaempferol are plant natural products with potentially useful pharmacological and nutraceutical activities. These natural products usually exist in plants as glycosides, and their glycosylation has a remarkable influence on their pharmacokinetic properties. The glycosyltransferases UGT71G1 and UGT73C8 from Medicago truncatula are excellent reagents for the regioselective glycosylation of (iso)flavonoids in Escherichia coli grown in Terrific broth. Ten to 20 mg/L of either genistein or biochanin A 7-O-glucoside was produced after feeding genistein or biochanin A to E. coli expressing UGT71G1, and similar levels of luteolin 4’-O- and 7-O-glucosides were produced after feeding luteolin to cultures expressing UGT73C8. For the production of kaempferol 3-O-glucoside or quercetin 3-O-glucoside, the Phe148Val or Tyr202Ala mutants of UGT71G1 were employed. Ten to 16 mg/L of either kaempferol 3-O- or quercetin 3-O-glucosides were produced on feeding kaempferol or quercetin to E. coli expressing these enzymes. More than 90% of the glucoside products were released to the medium, facilitating their isolation

S-nitrosothiols regulate nitric oxide production and storage in plants through the nitrogen assimilation pathway

Nitrogen assimilation plays a vital role in plant metabolism. Assimilation of nitrate, the primary source of nitrogen in soil, is linked to the generation of the redox signal nitric oxide (NO). An important mechanism by which NO regulates plant development and stress responses is through S-nitrosylation, that is, covalent attachment of NO to cysteine residues to form S-nitrosothiols (SNO). Despite the importance of nitrogen assimilation and NO signalling, it remains largely unknown how these pathways are interconnected. Here we show that SNO signalling suppresses both nitrate uptake and reduction by transporters and reductases, respectively, to fine tune nitrate homeostasis. Moreover, NO derived from nitrate assimilation suppresses the redox enzyme S-nitrosoglutathione Reductase 1 (GSNOR1) by S-nitrosylation, preventing scavenging of S-nitrosoglutathione, a major cellular bio-reservoir of NO. Hence, our data demonstrates that (S)NO controls its own generation and scavenging by modulating nitrate assimilation and GSNOR1 activity.5540

Functional and informatics analysis enables glycosyltransferase activity prediction

The elucidation and prediction of how changes in a protein result in altered activities and selectivities remain a major challenge in chemistry. Two hurdles have prevented accurate family-wide models: obtaining (i) diverse datasets and (ii) suitable parameter frameworks that encapsulate activities in large sets. Here, we show that a relatively small but broad activity dataset is sufficient to train algorithms for functional prediction over the entire glycosyltransferase superfamily 1 (GT1) of the plant Arabidopsis thaliana. Whereas sequence analysis alone failed for GT1 substrate utilization patterns, our chemical–bioinformatic model, GT-Predict, succeeded by coupling physicochemical features with isozyme-recognition patterns over the family. GT-Predict identified GT1 biocatalysts for novel substrates and enabled functional annotation of uncharacterized GT1s. Finally, analyses of GT-Predict decision pathways revealed structural modulators of substrate recognition, thus providing information on mechanisms. This multifaceted approach to enzyme prediction may guide the streamlined utilization (and design) of biocatalysts and the discovery of other family-wide protein functions

Neuronal nitric oxide synthase is heterogeneously distributed in equine myofibers and highly expressed in endurance trained horses

Mammalian skeletal muscle expresses splice variants of neuronal nitric oxide synthase (nNOS). Skeletal muscles have a metabolically heterogeneous population of myofibers, and fiber composition in equine skeletal muscle is correlated with athletic ability in endurance events. In this study, we investigated whether nNOS expression in equine skeletal muscle is related to fiber type and endurance training. Biopsy samples obtained from the gluteus medius of sedentary- (SH) and endurance-trained (TH) horses were examined for the electrophoretic mobility of myosin heavy chain (MHC) and NOS activity. Serial tissue cross-sections were stained for myosin ATPase and nicotinamide adenine dinucleotide (NADH) reductase, and also immunostained for nNOS. The gluteus medius of TH had higher levels of nNOS expression and activity when compared to muscle from SH. In SH, nNOS was restricted to the subsarcolemmal area while in TH nNOS was also present at cytoplasmic sites. A splice variant of nNOS was heterogeneously distributed among the different myofibers, its expression being higher in fast-oxidative-glycolytic type IIA fibers than in fast-glycolytic type IIX fibers and absent in slow-twitch type I fibers. Trained horses had a significantly higher relative content of type IIA fibers, a greater oxidative capacity, and a lower percentage of type IIX fibers when compared with SH. The differences in muscle fiber typing between the 2 groups of horses reflected alterations that probably resulted from the endurance-training program. Overall, these results show that nNOS is differentially expressed and localized in the gluteus medius according to the fiber type and the athletic conditioning of the horses.691465

Nitrite as the major source of nitric oxide production by Arabidopsis thaliana in response to Pseudomonas syringae

The origin of nitric oxide (,NO) in plants is unclear and an 'NO synthase (NOS)-like enzyme and nitrate reductase (NR) are claimed as potential sources. Here we used wild-type and NR-defective double mutant plants to investigate 'NO production in Arabidopsis thaliana in response to Pseudomonas syringae pv maculicola. NOS activity increased substantially in leaves inoculated with P. syringae. However, electron paramagnetic resonance experiments showed a much higher 'NO formation that was dependent on nitrite and mitochondrial electron transport rather than on arginine or nitrate. Overall, these results indicate that NOS, NR and a mitochondrial-dependent nitrite-reducing activity cooperate to produce 'NO during A. thaliana-P. syringae interaction. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.579173814382

Decreased arginine and nitrite levels in nitrate reductase-deficient Arabidopsis thaliana plants impair nitric oxide synthesis and the hypersensitive response to Pseudomonas syringae

Nitric oxide ((center dot)NO) produced in plants is implicated in defense responses against pathogens. (center dot)NO synthesis in such conditions has been attributed to a nitric oxide synthase (NOS)-like enzyme and, more recently, to a mitochondrial-dependent NO(2)(-)-reducing activity. In this work, we used an NR-deficient double mutant (nia1 nia2) of Arabidopsis thaliana that is deficient in endogenous NO(2)(-) to analyse the hypersensitive response (HR) against an avirulent strain of Pseudomonas syringae pv. maculicola (Psm). The inoculation of Psm into nia1 nia2 A. thaliana caused leaf chlorosis whereas the HR was induced in wild-type plants. (center dot)NO production in situ was substantially increased in wild-type but not in nia1 nia2 leaves following inoculation of Psm, as measured with the fluorescent (center dot)NO indicator 4,5-diaminofluorescein diacetate. However, the infiltration of L-arginine or NO(2)(-) into nia1 nia2 leaves triggered (center dot)NO production in situ. Moreover, co-infiltration of NO(2)(-) and Psm restored the HR in the leaves of nia1 nia2 plants. The total content of free amino acids, particularly L-arginine, was much lower in nia1 nia2 leaves compared to wild-type leaves. Overall, these results suggest that the HR is affected in NR-deficient plants because these plants lack L-arginine and NO(2)(-), two important endogenous substrates for (center dot)NO synthesis. (c) 2006 Elsevier Ireland Ltd. All rights reserved.1711344

In vitro studies of anticandidal activity of goniothalamin enantiomers

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Aims: The antifungal activity of (R)-goniothalamin (1) and (S)-goniothalamin (ent-1) was evaluated against six Candida species. The in vitro effect of these compounds on yeast adhesion to human buccal epithelial cells (BEC) and Candida albicans and C. dubliniensis biofilms progression were also investigated. Methods and Results: Yeast susceptibility was evaluated by broth microdilution assay and showed that ent-1 exhibited higher potency against all fungal clinical isolated when compared to compound 1. Compounds 1 and ent-1 were as potent as fluconazole in inhibiting the adhesion of C. albicans and C. dubliniensis to BEC. XTT-reducing assay and scanning electron microscopy revealed that 1 and ent-1 were twice as potent as fluconazole in the inhibition of yeast biofilms progression. Conclusions: Our findings indicate that compounds 1 and ent-1 are potent anticandidal agents. Significance and Impact of the Study: This study highlights goniothalamin enantiomers as promising lead compounds for the design of new antifungal with inhibitory activity on yeast adhesion and biofilm progression.107412791286Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Biological Activities of Eco-Friendly Synthesized Hantzsch Adducts

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fourteen Hantzsch adducts with different substituents at the C-4 position were synthesized through multicomponent reactions by using citric or lactic acid as catalysts. To the best of our knowledge, this is the first report on the synthesis of such a class of compounds based on multicomponent reactions catalyzed by non-toxic organic acids. The potential to scavenge reactive nitrogen/oxygen species (RNS/ROS) and the ability to inhibit cancer cell growth were then investigated. Among the synthesized compounds, adduct 15 was the most promising free radical scavenger, while adduct 20 was shown to have a wider spectrum of action on the cancer cells studied. These results highlight Hantzsch adducts as lead compounds for obtaining new free radical scavengers and anticancer agents.96889896Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Free radical scavenging and antiproliferative properties of Biginelli adducts

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)A series of Biginelli adducts bearing different substituents at C-4 position were synthesized by using p-sulfonic acid calix[4] arene as a catalyst. The in vitro potential to scavenge reactive nitrogen/oxygen species (RNS and ROS) and the ability to inhibit cancer cells growth were then investigated. Four adducts were found to be potent scavengers of 2,2-diphenyl-1-picrylhydrazyl (RNS) and/or superoxide anion (ROS) radicals. The antiproliferative activity against cancer cells was disclosed for the first time for 16 monastrol analogs. The capacity of all compounds to inhibit cancer cells growth was dependent on the histological origin of cells, except for BA24, which was highly active against all cell lines. BA20 and BA33 were as potent as the reference drug doxorubicin against adriamycin-resistant ovarian and prostate cancer cells, respectively. These results highlight some monastrol analogs as lead compounds for the design of new free radical scavengers and anticancer agents. (C) 2012 Elsevier Ltd. All rights reserved.20826452650Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES