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31.
Anthony Dobi Susana B. Bravo Bryan Veeren Beatriz Paradela-Dobarro Ezequiel Álvarez Olivier Meilhac 《Free radical research》2019,53(2):150-169
Advanced glycation end-products (AGEs) trigger multiple metabolic disorders in the vessel wall that may in turn lead to endothelial dysfunction. The molecular mechanisms by which AGEs generate these effects are not completely understood. Oxidative stress plays a key role in the development of deleterious effects that occur in endothelium during diabetes. Our main objectives were to further understand how AGEs contribute to reactive oxygen species (ROS) overproduction in endothelial cells and to evaluate the protective effect of an antioxidant plant extract. The human endothelial cell line EA.hy926 was treated with native or modified bovine serum albumin (respectively BSA and BSA-AGEs). To monitor free radicals formation, we used H2DCF-DA, dihydroethidium (DHE), DAF-FM-DA and MitoSOX Red dyes. To investigate potential sources of ROS, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and mitochondrial inhibitors were used. The regulation of different types of ROS by the polyphenol-rich extract from the medicinal plant Doratoxylon apetalum was also studied for a therapeutic perspective. BSA-AGEs exhibited not only less antioxidant properties than BSA, but also pro-oxidant effects. The degree of albumin glycoxidation directly influenced oxidative stress through a possible communication between NADPH oxidase and mitochondria. D. apetalum significantly decreased intracellular hydrogen peroxide and superoxide anions mainly detected by H2DCF-DA and DHE respectively. Our results suggest that BSA-AGEs promote a marked oxidative stress mediated at least by NADPH oxidase and mitochondria. D. apetalum plant extract appeared to be an effective antioxidant compound to protect endothelial cells. 相似文献
32.
Fernanda Sousa Susana Jus Anita Erbel Vanja Kokol Artur Cavaco-Paulo G.M. Gubitz 《Enzyme and microbial technology》2007,40(7):1772-1781
A novel protease produced by Bacillus cereus grown on wool as carbon and nitrogen source was purified. B. cereus protease is a neutral metalloprotease with a molecular mass of 45.6 kDa. The optimum activity was at 45 °C and pH 7.0. The substrate specificity was assessed using oxidized insulin B-chain and synthetic peptide substrates. The cleavage of the insulin B-chain was determined to be Asn3, Leu6, His10-Leu11, Ala14, Glu21, after 12 h incubation. Among the peptide substrates, the enzyme did not exhibit activity towards ester substrates; with p-nitroanilide, the kinetic data indicate that aliphatic and aromatic amino acids were the preferred residues at the P1 position. For furylacryloyl peptides substrates, which are typical substrates for thermolysin, the enzyme exhibited high hydrolytic activity with a Km values of 0.858 and 2.363 mM for N-(3-[2-Furyl]acryloyl)-Ala-Phe amide and N-(3-[2-Furyl]acryloyl)-Gly-Leu amide, respectively. The purified protease hydrolysed proteins substrates such as azocasein, azocoll, keratin azure and wool. 相似文献
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35.
Structure-function relationship in cyclodextrin glycosyltransferase from Bacillus circulans DF 9R 总被引:1,自引:0,他引:1
Cyclodextrin glycosyltransferases (CGTases E.C.2.4.1.19) catalyze cyclomaltooligosaccharides (cyclodextrins) production, an important industrial process. We herein report structural features of Bacillus circulans DF 9R cyclodextrin glycosyltransferase including its sequence and several aspects of enzyme structure-function relationship. Protein ethoxyformylation, under our experimental conditions, indicated that only one out of the 13 enzyme histidines was modified leading to a drastic drop in cyclizing and hydrolytic activity. Besides, tryptic digestion of the 14C ethoxyformylated protein and studies of the peptide mixture showed that histidine 233 is the most reactive histidine residue. This is the first cyclodextrin glycosyltransferase with a known primary structure and a glutamine instead of glycine residue at position 179 in the highly conserved −6 subsite, shown to be involved in substrate binding. The presence of glycine at that position was considered as a requirement for such binding following the induced-fit mechanism already proposed. Moreover, the enzyme has all the features previously described for an α- or α/β-cyclodextrin producer. 相似文献
36.
Background
The extracellular enzyme cyclodextrin glucanotransferase (CGTase) synthesizes cyclic malto-oligosaccharides called cyclodextrins (CDs) from starch and related α-1,4-glucans. CGTases are produced by a variety of bacteria, mainly Bacillus species, by submerged culture in complex medium. CGTases differ in the amount and types of CDs produced. In addition, CGTase production is highly dependent on the strain, medium composition and culture conditions. Therefore we undertook this study with a newly isolated strain of Bacillus circulans. 相似文献37.
38.
Jeemeng Lao Ai Oikawa Jennifer R. Bromley Peter McInerney Anongpat Suttangkakul Andreia M. Smith‐Moritz Hector Plahar Tsan‐Yu Chiu Susana M. González Fernández‐Niño Berit Ebert Fan Yang Katy M. Christiansen Sara F. Hansen Solomon Stonebloom Paul D. Adams Pamela C. Ronald Nathan J. Hillson Masood Z. Hadi Miguel E. Vega‐Sánchez Dominique Loqué Henrik V. Scheller Joshua L. Heazlewood 《The Plant journal : for cell and molecular biology》2014,79(3):517-529
The glycosyltransferases (GTs) are an important and functionally diverse family of enzymes involved in glycan and glycoside biosynthesis. Plants have evolved large families of GTs which undertake the array of glycosylation reactions that occur during plant development and growth. Based on the Carbohydrate‐Active enZymes (CAZy) database, the genome of the reference plant Arabidopsis thaliana codes for over 450 GTs, while the rice genome (Oryza sativa) contains over 600 members. Collectively, GTs from these reference plants can be classified into over 40 distinct GT families. Although these enzymes are involved in many important plant specific processes such as cell‐wall and secondary metabolite biosynthesis, few have been functionally characterized. We have sought to develop a plant GTs clone resource that will enable functional genomic approaches to be undertaken by the plant research community. In total, 403 (88%) of CAZy defined Arabidopsis GTs have been cloned, while 96 (15%) of the GTs coded by rice have been cloned. The collection resulted in the update of a number of Arabidopsis GT gene models. The clones represent full‐length coding sequences without termination codons and are Gateway® compatible. To demonstrate the utility of this JBEI GT Collection, a set of efficient particle bombardment plasmids (pBullet) was also constructed with markers for the endomembrane. The utility of the pBullet collection was demonstrated by localizing all members of the Arabidopsis GT14 family to the Golgi apparatus or the endoplasmic reticulum (ER). Updates to these resources are available at the JBEI GT Collection website http://www.addgene.org/ . 相似文献
39.
Nitrogen fixation and assimilation in nodules and roots were studied in soybean (Glycine max L.) exposed to different levels of aluminium (Al) stress (0, 50, 200 and 500 μM). Al at 500 μM induced oxidative stress,
which became evident from an increase in lipid peroxidation accompanied by a concomitant decline in antioxidant enzyme activities
and leghaemoglobin breakdown. Consequently, there was also a reduction in nitrogenase activity. However, the leghaemoglobin
levels and nitrogenase activity were unexpectedly found to be higher in nodules when the plants were treated with 200 μM Al.
Of the enzymes involved in nitrogen assimilation, the activity of glutamate dehydrogenase-NADH was reduced in nodules under
Al stress, but it was significantly higher in roots at 500 μM Al as compared to that in the control. In nodules, the glutamine
synthetase/glutamate synthase-NADH pathway, assayed in terms of activity and expression of both the enzymes, was inhibited
at >50 μM Al; but in roots this inhibitory effect was apparent only at 500 μM Al. No significant changes in ammonium and protein
contents were recorded in the nodules or roots when the plants were treated with 50 μM Al. However, Al at ≥200 μM significantly
increased the ammonium levels and decreased the protein content in the nodules. But these contrasting effects on ammonium
and protein contents due to Al stress were observed in the roots only at 500 μM Al. The results suggest that the effect of
Al stress on nitrogen assimilation is more conspicuous in nodules than that in the roots of soybean plants. 相似文献
40.
Mafalda C. O. Figueiredo Susana A. L. Lobo Sara H. Sousa Fábio P. Pereira Judy D. Wall Lígia S. Nobre Lígia M. Saraiva 《Journal of bacteriology》2013,195(11):2684-2690
Desulfovibrio species are Gram-negative anaerobic sulfate-reducing bacteria that colonize the human gut. Recently, Desulfovibrio spp. have been implicated in gastrointestinal diseases and shown to stimulate the epithelial immune response, leading to increased production of inflammatory cytokines by macrophages. Activated macrophages are key cells of the immune system that impose nitrosative stress during phagocytosis. Hence, we have analyzed the in vitro and in vivo responses of Desulfovibrio vulgaris Hildenborough to nitric oxide (NO) and the role of the hybrid cluster proteins (HCP1 and HCP2) and rubredoxin oxygen oxidoreductases (ROO1 and ROO2) in NO protection. Among the four genes, hcp2 was the gene most highly induced by NO, and the hcp2 transposon mutant exhibited the lowest viability under conditions of NO stress. Studies in murine macrophages revealed that D. vulgaris survives incubation with these phagocytes and triggers NO production at levels similar to those stimulated by the cytokine gamma interferon (IFN-γ). Furthermore, D. vulgaris
hcp and roo mutants exhibited reduced viability when incubated with macrophages, revealing that these gene products contribute to the survival of D. vulgaris during macrophage infection. 相似文献